1

INTRODUCTION

EVOLUTION, CONSERVATION AND CANE

TOADS IN AUSTRALIA

2

CONTEMPORARY EVOLUTION IN CONSERVATION

There is an increasing realisation amongst biologists that evolution can

occur rapidly – on time scales generally associated with “ecological time”

(Stockwell et al. 2003; Thompson 1998). It is interesting that biologists have

only recently incorporated the concept of “contemporary evolution” into their

worldview. We have known since before the modern synthesis that, per

generation, the response to selection (R) in a trait is equal to the heritability of

that trait (h2) multiplied by the strength of selection (S) operating in that

generation (Lynch and Walsh 1998):

R = h2S

This apparently simple equation tells us much about the nature of adaptation,

not the least of which is that it can happen rapidly; dependent upon high

heritability and strong selection acting within the range of variability for the

trait in question.

We have also had, for many years, empirical evidence that under the

right conditions evolution precedes rapidly from strong selection in natural

populations (e.g. Kettlewell 1973). Why then, has it taken so long to appreciate

the potential importance of evolution acting along timescales that are of

relevance to our everyday existence? This is a question for a science historian –

which I am not – but perhaps one of Gould’s (1991) creeping fox-terrier clones

is to blame: Someone (perhaps Darwin himself, who as it turns out, was

occasionally confused about the difference between microevolution and

3

speciation), told us that evolution takes a long time, and we have been

repeating this mantra to ourselves and our students ever since.

Nevertheless, contemporary evolution has slapped us in the face until

we have had no choice but to pay it some attention. Predictably in hindsight,

contemporary evolution first became apparent in areas of our lives that we care

deeply about; our health and our food. The green revolution and its attendant

barrage of pesticides was, on reflection, a thoroughly unintended experiment in

evolution. Pesticides, that were initially incredibly effective became

increasingly less so as pests rapidly evolved resistance (Palumbi 2002).

Similarly, the discovery of antibiotics seemed set to save us from the

scourge of disease (well, most of them anyway). Over time, our magic bullets

have often come to resemble peashooters; many pathogens evolving either flak

jackets or the simple ability to dodge (Ewald 1994). Two of the biggest human-

killers on the planet – malaria and HIV – have achieved this position of prestige

precisely because of their ability to evolve at such speed that our attacks

continually hit nothing but air (Gardner et al. 2002; Palumbi 2002).

We have no choice but to acknowledge evolution when it foils our plans,

but we often fail to appreciate that it can also be useful. Domestication is, of

course, evolution by human selection (blessed are the pigeon fanciers, for they

shall convince Darwin of the truth). And recently, medical and agricultural

researchers are realising that evolutionary problems require evolutionary

solutions – “evolutionarily stable strategies” are the new weapons in the wars

4

against Malaria, HIV and pesticide resistance (Ewald 1994; Palumbi 2002;

Rausher 2001).

But let us step aside from directly human-oriented concerns and apply a

worldview of contemporary evolution to conservation. There is little doubt that

we are living in a time of accelerated extinction (Chapin et al. 2000; May et al.

1995). Species are going extinct at higher than normal rates due to the activities

of our species. Like all species, we interact with the world and, as a

consequence, modify it. Unlike most species however, our modifications are

large and global in their reach. In the dry language of science however, our

impacts are nothing more than “environmental change”, and a geological

perspective tells us that environmental change is not a new phenomenon;

continents have moved, climate has changed, land bridges have allowed mass

invasions, and mountains have come and gone (Morrison and Morrison 1991).

Ultimately, species either evolve through environmental change or they go

extinct: Even Raup’s (1993) extinction through bad luck will almost always be

the result of an environmental change. The activities of humans are causing

rapid, global environmental modifications; the rapidity and size of these

changes are causing an increase in the extinction rate.

Understanding how to reduce or reverse the effect of human-mediated

impacts on species and communities has become the province of conservation

biology. Conservation biologists know that they are fighting an uphill battle

and are well aware that the resources do not come close to matching the extent

of the problem. Strategies and priorities thus need to be effective and as

5

efficient as possible. It is in this desperate, outnumbered rearguard that

contemporary evolution is a mostly ignored, but potentially powerful ally.

It is a property of living systems that they evolve – they are inherently

dynamic and we know that evolution can happen in ecological time.

Evolutionary time is, like all time, relative; in the case of evolution, time is

relative to the generation time of the organism. If an environmental change

occurs along a time scale that is not too rapid, a population can mount an

adaptive response. In other words, if the per generation strength of selection

(S) is not too large there is a good chance of evolution instead of extinction.

Evolved adaptation is (almost by definition) the best “strategy” for the long-

term persistence of a species; from a conservation perspective, whether or not a

species will adapt to a change is more important than whether it suffers short-

term impact.

For conservationists, overwhelmed by the sheer number of species

potentially at risk from human activities, the contemporary evolution

perspective suggests that the problem may be slightly smaller than it otherwise

looks. An ability to evolve around environmental change may see many

species rescue themselves. The challenge for conservation biologists is to

understand which species are likely to exhibit rapid evolution and which

categories of environmental change are likely to encourage evolution rather

than extinction.

Organisms with short generation times relative to the pace of change will

have evolution on their side. Similarly, environmental changes that are

6

relatively slow and cause selection on traits that are unlikely to have been

previously important for fitness will encourage evolution. Conservation

biologists need to start understanding where the boundaries lie in these

continuums: When does an environmental change become too rapid to expect

an evolved response from an impacted species? When does the generation time

of an organism become too long to preclude adaptive response to an

instantaneous change of a given magnitude? These kinds of questions are not

necessarily simple to answer but in answering them we can have a profound

impact on the size of the conservation battle. Perhaps less species are in need of

“rescuing” than we currently believe and perhaps some environmental changes

are less important than others.

This thesis represents an attempt at a small piece of this puzzle. In the

pages that follow, I examine the potential for Australian snake species (which

have relatively long generation times of 1-3 years) to adapt to a strong and

instantaneous shift in their selective environment; the invasion of a toxic prey

item.

7

SNAKES AND TOADS IN AUSTRALIA

“To others who scent a ‘nigger in the woodpile’ and suggest the possibility that the toad

will, in turn, itself become a pest, we can point to the fact that nearly 100 years have

elapsed since it was first introduced into Barbados, and there it has no black marks

against its character. Experience with it in other West Indian islands and in Hawaii,

certainly points to the fact that no serious harm is likely to eventuate through its

introduction into Queensland” R W Mungomery upon returning to Australia

from Hawaii with 101 toads in 1935.

Australia has a particularly diverse reptile fauna, among which we count

around 140 native, terrestrial snake species (Cogger 2000). Thirty-three of these

species are typhlopids – mostly blind, burrowing eaters of ant and termite eggs.

The remaining terrestrial species belong to the colubrid, pythonid and elapid

families and eat vertebrate prey. Of these, the colubrids (11 species) are

relatively recent colonisers of the continent, probably arriving from south-east

Asia during the glaciations of the early Pleistocene (2 mya, Greer 1997). The

pythons (15 species) and elapids (81 species) have been present much longer, at

least since the early Miocene (ca. 20 mya, Shine 1991a).

Australia’s snakes (even the newer ones!) have a long history on the

Australian continent. This venerable history contrasts garishly against the

history of Bufo marinus. The species was variously referred to as the “giant

toad” or “marine toad” by authors of the early part of this century (e.g.

8

Kinghorn 1938), however it is now known through most of its introduced,

English-speaking range as the “cane toad” (Lever 2001) – a fitting epithet for a

species that has benefited so profoundly from the sugar-cane industry.

Originally from South America, the global sugar industry, in a welter of small,

poorly thought out decisions, introduced cane toads throughout much of the

Caribbean and Pacific (Easteal 1981). It was the sugar industry that brought

toads to Australia in 1935; in simple wooden crates, 101 toads were shipped

from Hawaii to Gordonvale in north Queensland where, it was hoped, they

were to save the sugar farmers from crop-ruining beetle pests (Mungomery

1935). The plan was that the toads were to eat their way through the cane

beetle populations, leaving farmers with little beetle-filled faeces and healthy

crops.

The plan wasn’t a notable success and by the 1940s toads were made

entirely redundant by the development of pesticides (Low 1999). By then

unfortunately, farmers (eager to ride toads to bigger yields) had distributed

them along the Queensland coast and the national toad population was already

well beyond control (Sabath et al. 1981). Toads have done well in Australia;

they now occupy well over 1 million square kilometres of the continent and are

continuing to expand their range every year (Sutherst et al. 1995).

Cane toads are a member of the Bufonidae; a family that had never

before occupied Australia. Thus they were a novel prey item for Australian

predators (including snakes) which had coevolved with Australia’s relatively

non-toxic anuran fauna (Erspamer et al. 1984). Bufonids produce a family of

9

toxins known as bufodienolides (or bufogenins); extremely potent cardiac

toxins similar to the digitalis toxins of plants but unique to toads (Chen and

Kovarikova 1967). Bufodienolides were investigated for possible use as

pharmaceuticals in the 1960s only to be rejected because they produced

excessive side effects and had a very narrow therapeutic window; increasing

doses (among other effects) reduce the heart’s ability to synchronise muscle

contractions and consequently lead to fibrillation (Chen and Kovarikova 1967).

In cane toads, this toxin is synthesised and stored primarily in the large

parotoid glands located above the shoulder. There is little doubt that this well-

placed chemical arsenal has had a massive impact on naïve Australian

predators who die attempting to consume toads (Burnett 1997; Covacevich and

Archer 1975; Oakwood 2003; Rayward 1974 and Chapter 1, this thesis).

Natural selection comes in many guises, perhaps the most obvious of

which is outright death. When a toad kills a predator, that predator has had its

reproductive potential severely curbed; it has been selected against. Toads thus

represent a change in the Australian environment and a new agent for

“natural” selection. Furthermore, they also represent an instantaneous

environmental change; toads rapidly colonise an area so are usually either

absent, or present in large numbers. As a model system within which to

explore the potential for populations to adapt rapidly, toads and Australian

snakes may have a lot to tell us…

10

THESIS SYNOPSIS

This introduction is a loose tour of my reasons for embarking on this line

of research. It has, of course, been written after the fact, but I feel that all but

the broadest reasons were somewhere in the back of my head at the outset.

Perhaps neglected as an original motivation however, was the long-running

observation by myself and others that in most areas with toads, some species of

snakes that should be present simply are not. I wanted to know more.

Chapter one is a necessity chapter. The sad fact is that no-one has

unequivocally proven an impact by toads on any element of the Australian

fauna. This is not because it hasn’t happened, but because it is almost

impossible to prove. Proving a population reduction in uncommon, difficult to

find organisms with populations that fluctuate wildly across seasons and years

is tough. I was well aware that I could spend a decade proving an impact on

one species and I was not willing to do this. Chapter one thus examines a

mechanism of impact and shows that the mechanism works. The assumption, of

course, is that the mechanism translates into an effect, or that a car in gear with

the engine on is likely to move.

Chapter two is an interest chapter. Following the finding of ridiculous

levels of resistance to toad toxin in keelbacks, I was interested to see if they

really could eat toads all day long, every day and not experience some effect.

Chapter three also began as an interest chapter but quickly became

important, and served to remind me that it is best to understand how selection

11

is operating before one goes out to look for its effects. The initial observation

came as I was weighing dried parotoid glands and skin during initial toxin

extractions. I noticed that parotoid weight became a larger proportion of total

skin weight as toad size increased. I immediately concluded that larger snakes

were at greater risk of poisoning from toads. It took me some time to realise

that snake allometry was also important and that my initial conclusion was, in

fact, wrong.

Chapters four, five and six represent the question that I was really

interested in from the beginning – are snakes evolving around the impact of

toads? With perfect hindsight, I know that I didn’t spend enough time

examining prey preference. A quick change in prey preference is probably the

best and most likely adaptation to the presence of a lethally toxic prey. This is

one of those ideas that is appallingly obvious after you have worked it out but,

for whatever reason, is a little opaque beforehand. In hindsight, I suspect that

this is the most effective and likely adaptation for predators faced with the

arrival of cane toads. Further work will have the benefit of this insight.

Chapters seven and eight share the same data and similar analyses but

proved to be impossible to merge. Concatenation seemed more sensible.

Chapter seven examines the effect of time since colonisation on the aspects of

toad morphology that mediate their impact on predators. The result is a

pattern for which I can only guess at the mechanism. The pattern is, however,

interesting and important because it indicates that toads become a less

dangerous meal through time.

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Chapter eight is a chapter about potential, and seems a fitting place to

end. Spatial and temporal analyses of environmental impact and species

vulnerability could allow the identification of significant times and places for

adaptation to occur. The potential application for such information is extremely

large and I haven’t even begun to point out its scope.

CHAPTER 1

ASSESSING THE POTENTIAL IMPACT OF

CANE TOADS (BUFO MARINUS) ON

AUSTRALIAN SNAKES.*

* Published as: Phillips, B L, Brown, G P and Shine, R, 2003. Assessing the potential impact of cane toads (Bufo marinus) on Australian snakes. Conservation Biology 17: 1738-1747.

13

14

ABSTRACT

Cane toads (Bufo marinus) are large, highly toxic anurans introduced into

Australia in 1935. Anecdotal reports suggest that the invasion of toads into an area is

followed by dramatic declines in the abundance of terrestrial native frog-eating

predators, but quantitative studies have been restricted to non-predator taxa or aquatic

predators and have generally reported minimal impacts. Will toads substantially affect

Australian snakes? Based on geographic distributions and dietary composition, I

identified 49 snake taxa as potentially at risk from toads. The impact of these feral prey

also depends on the snakes’ ability to survive after ingesting toad toxins. Based on

decrements in locomotor (swimming) performance after ingesting toxin, I estimate the

LD50 of toad toxins for 10 of the “at risk” snake species. Most species exhibited similar

and low ability to tolerate toad toxins. Based on head-widths relative to sizes of toads, I

calculate that 7 of the 10 taxa could easily ingest a fatal dose of toxin in a single meal.

The exceptions were two colubrid taxa (keelbacks, Tropidonophis mairii and slatey-

greysnakes, Stegonotus cucullatus) with much higher resistance (up to 85-fold) to

toad toxins and one elapid (swampsnake, Hemiaspis signata) with low resistance but

a small relative head size (and thus, low maximum prey size). Overall, my analysis

suggests that cane toads threaten populations of approximately 30% of Australia’s

terrestrial snake species.

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INTRODUCTION

One of the most significant threatening processes for biodiversity

worldwide concerns anthropogenic shifts in geographic distributions of

organisms, with natural ecosystems in many parts of the world being invaded

by non-native plants and animals (Mack et al. 2000; Williamson 1996). Many

such invasions are likely to cause only minor and localized ecological

disruption, but some feral animals cause massive degradation and in some

cases widespread extinction of the local fauna and flora (Fritts and Rodda 1998;

Ogutu-Ohwayo 1999). The processes and outcomes of ecological invasion vary

considerably among systems, but potentially one of the most powerful effects

involves the invasion of a toxic species into a fauna with no previous exposure

to such toxins (Brodie and Brodie 1999a). In such cases native predators may be

unable to tolerate the novel toxin and thus die in large numbers as they first

encounter the invader.

Although there has been extensive research on the ecological impacts of

invading organisms, some major questions have attracted much less attention

than others. In particular, it may often be true that the most important impact

of a toxic feral taxon will be on predators, yet research on changes in the

abundance of predators is fraught with logistical obstacles. Because they are

relatively rare and mobile, simply quantifying the abundance of many

predators, let alone detecting impacts on their abundances, poses a formidable

problem. In the face of such challenges researchers have tended to focus on the

16

impacts of invading species on smaller more abundant organisms – typically

potential prey or competitors. There may thus be a real danger that studies will

accumulate showing no (or minor) negative impacts from the invading

organism, and the weight of negative evidence will encourage wildlife

managers to afford less priority to potential impacts of the invasion. This is a

dangerous path to follow in the absence of information on the effects of the

invading taxon on predators. I believe that the spread of cane toads in

Australia reveals exactly this scenario.

The cane toad (Bufo marinus, Bufonidae) is a large (up to 230 mm body

length) anuran native to South and Central America (Zug and Zug 1979).

Toads were introduced widely throughout the Pacific, primarily as an agent for

biological control by the sugar industry (Lever 2001); they were introduced into

Australia in 1935 (Lever 2001). Since then, they have spread from their initial

release points in eastern Queensland (Qld) to encompass more than 863,000 km2

(50% of Qld: Sabath et al. 1981; Sutherst et al. 1995). Cane toads now extend

into northern New South Wales (NSW) and the Northern Territory (NT) and

are predicted to further increase their range, primarily throughout coastal and

near-coastal regions of tropical Australia, to encompass an area of

approximately 2 million km2 (Sutherst et al. 1995 Figure 1).

These amphibians can reach astounding densities in suitable habitat (up

to 2138 individuals/ha: Freeland 1986). In addition, the toad possesses a

formidable chemical defence system – all life-history stages are toxic (Crossland

1998; Crossland and Alford 1998; Flier et al. 1980; Lawler and Hero 1997). The

17

active principles of the toxin (bufogenins) are extremely powerful (Chen and

Kovarikova 1967) and unique to toads (Daly et al. 1987). Toads are not native to

Australia (Lutz 1971) and therefore are both novel and toxic to Australian

predators.

18

Figure 1. The approximate current and predicted distribution of the cane toad in Australia. Predicted distribution is shown under current climatic and 2030 global warming scenarios (after Sutherst et al. 1995).

Approximate Current Distribution

Predicted Distribution (current climate)

Predicted Distribution (2030 climate)

800km

19

Although intuition suggests that the toad invasion may have a major

ecological impact in Australia, there has been limited study of this topic. The

first actual impact to be noticed was that toads were becoming significant

predators of the European honey bee (Apis mellifera), causing some economic

loss for apiarists (Goodacre 1947; Hewitt 1956). It was not until the early 1960s

however that anecdotal reports of population declines in native species became

apparent: Breeden (1963) reported observations of declines in snakes, monitors

(Varanus spp.), frilled lizards (Chlamydosaurus kingii) and quolls (a marsupial

carnivore, Dasyurus spp.) following the appearance of toads. This was followed

by observations of declines in snakes, monitors and birds following the arrival

of toads in south-eastern Queensland and northern New South Wales (Pockley

1965; Rayward 1974). Covacevich and Archer (1975) provided further evidence

for the potential impact of toads on predators by collecting numerous anecdotal

reports of terrestrial predators (snakes, monitors, and marsupial carnivores)

dying as a consequence of attempting to ingest toads.

Quantitative data on interactions between native species and toads have

become available in the ensuing years (e.g., Catling et al. 1999; Crossland 1998;

Crossland 2000; Crossland 2001; Crossland and Alford 1998; Crossland and

Azevedo-Ramos 1999; Freeland and Kerin 1990; Lawler and Hero 1997;

Williamson 1999a). Primarily for logistical reasons, these studies have focused

on interactions between toads and relatively small, abundant native organisms

(primarily fish, frogs, and aquatic invertebrates). Several of these studies have

concluded that the ecological impact of toads may be less extreme than might

20

be supposed from intuition or public concern (e.g., Catling et al. 1999; Freeland

and Kerin 1990; Williamson 1999a). Despite all these studies, however, there

are no published quantitative analyses of the potential or realized effects of

cane toads on the subset of native taxa identified anecdotally more than 30

years ago as most likely to be at risk: the terrestrial predators of frogs (The one

possible exception being Burnett 1997, which focussed on Varanids and

Marsupial predators, although this study too relied on anecdotal information).

Even if competition between toads and small vertebrates is minor and their role

as predators on invertebrates is modest, they might still impose a massive

ecological impact if they kill a high proportion of the anurophagous predators

that attempt to ingest them.

Despite the fact that snakes represent the largest vertebrate group likely

to be affected, there are almost no data describing interactions between toads

and native snakes. Knowledge in this area comes entirely from single

observations (Covacevich and Archer 1975; Ingram and Covacevich 1990; Shine

1991c), typically of the nature of “an individual of species x survived and an

individual of species y died after eating a toad.” Snakes are potentially at

considerable risk from toads because many Australian snakes prey upon frogs

(Shine 1991a) and, unlike birds or mammals, have few options for prey capture.

They must use their mouths to capture and consume the toad entire and, hence,

cannot avoid direct exposure to toxins in the toad’s body.

Thus, there is a critical need to evaluate the severity of the probable

impact of cane toads on Australian snakes. To do so, we require information on

21

two separate topics: (1) how many Australian snake species are potentially

vulnerable to toads, based on their geographic distributions and dietary habits

(i.e., how many species eat frogs and live in areas that toads will occupy); and

(2) how many Australian snake species can tolerate a quantity of toxins

equivalent to ingesting a small toad?

To answer these questions, I reviewed published information on

distributions and dietary habits of Australian snakes and tested the ability of 10

at risk snake taxa to tolerate toad toxins.

22

METHODS

The number of snake species potentially at risk

To identify which Australian snake species might potentially be affected

by the invasion of the toad, I used ecoclimatic predictions of the likely eventual

distribution of toads within Australia (Sutherst et al. 1995) and published and

unpublished data on the dietary composition (Shine 1991a and refs in Greer

1997; J. Webb & G. Brown, pers. comm.) and geographic distribution (Cogger

2000) of Australian snakes.

Sutherst et al. (1995) generated two maps of the likely final distribution

of cane toads in Australia, one under the present climate and one under a

conservative 2030 climate change scenario. The latter method produced a

slightly larger predicted distribution of the toad. I used both maps for my

analysis (Fig. 1) but note that even the larger potential range might be a

conservative estimate because adaptation by the toad or lack of competition

from congeners may increase its range outside the ecoclimatic envelope of its

native range (used by Sutherst et al. (1995) to generate predictions for the

Australian invasion). Thus, my estimates of the snake species affected may be

conservative.

As well as species recorded as eating frogs, I also included some species

that are likely to consume frogs but for which detailed dietary information is

not available. For these species, I assessed their likelihood of consuming frogs

based on dietary habits of their congeners. For each species of snake recorded

23

or likely to include frogs in their diet, I estimated the proportion of the species’

range likely to overlap with that of the toad. Multiplying this percentage by the

proportion of frogs in the diet of each species yielded an index (between 0–100)

of the potential impact from the toad (Table 1).

Snake’s tolerance to toad toxins

I tested 10 species of native snake for their susceptibility to toad toxin.

Because populations of snakes that are sympatric with toads might have

already adapted to this novel prey type, snakes were collected from areas

where toads were either absent or had been present for <15 years. Table 2 lists

the species studied, with information on their body sizes and localities of

collection. The study taxa included four species of snakes from the family

Colubridae, one species from the Pythonidae, and four from the Elapidae.

These species were chosen because they were all identified as “at risk” and

were sufficiently common at my study sites to enable collection. Animals that

were obviously ill, in poor condition, or contained large prey items were

excluded from the study.

I obtained toad toxin from skins of 78 freshly killed cane toads collected

from the Lismore area (northern NSW). Toads were killed by freezing. I made

a single extraction of toad toxin for the entire study to remove among-toad

variance in toxicity and accurately control dosing. I measured freshly killed

toads for snout/urostyle length, head-width, and mass. I then removed the

dorsal skin (from the back of the head to the knees) including the parotoid

24

glands. This was allowed to dry at room temperature over several days. Each

skin was weighed. I then blended the dried skins with 10x v/w of 40% ethanol.

This mixture was strained and the solids discarded. The resulting liquid was

allowed to evaporate to 50% of its initial volume at room temperature. I

recorded the final volume and then dispensed the extract into 25 mL containers

and froze it. Bufogenins are stable, partially water soluble compounds with a

very high evaporation temperature (Meyer and Linde 1971). My crude extract

thus contains the bufogenins although it is possible that some were lost due to

saturation (see Discussion).

I tested the resistance of individual snakes to toad toxin using the

decrement in swimming speed following a dose of toxin (Methodology

modified from that of Brodie and Brodie 1990). Each snake was encouraged to

swim around a circular pool, 3 m in diameter. The circumference of the pool

was divided into quarters. I recorded snakes’ speeds (with an electronic

stopwatch) as the time to traverse a single quarter. Before dosing, I subjected

each snake to two swimming trials one hour apart. In each trial I recorded a

snake’s time over eight quarters of the pool. The fastest speed from each trial

was taken and the resulting times averaged over the two trials. This yielded an

estimate of maximum swimming speed before dosing (b). I also measured the

snake’s mass, snout-vent length (SVL) and head-width at this time.

The following day I gave each snake a specific dose of toxin through a

feeding tube attached to a syringe or calibrated micropipette. I inserted the

tube into the snake’s stomach to a depth of 30–50% of its SVL. Swimming trials

25

commenced one hour after dosing. I swam each snake twice: one hour post-

dose and two hours post-dose. Maximum swimming speed was calculated as

before to yield an estimate of maximum swimming speed after dosing (a). I

then calculated the percent reduction in swim speed (%redn) following dosing

for each snake (%redn = 100 x (1-b/a)). Experiments on neonate snakes have

confirmed that reduction in swimming speed following this methodology is

due to the toad toxin and not the carrier fluid (Chapter 5, this thesis).

Because I collected most of the data in the field, temperature could not be

rigorously controlled across trials. I kept temperature differences between

before/after trials within 2ºC by running the post-dose trial at a time when the

water temperature was similar to that of the pre-dose trial. Although

maximum speed may vary with temperature, the repeatability of speed assays

in snakes has been shown to be consistent across temperatures (Brodie and

Russell 1999). Thus, I expect the percentage reduction measure to be unaffected

by temperature differences across sets of before/after trials. All animals were

adjusted to the water temperature for a minimum of 30 minutes before trials

commenced by placing animals in plastic boxes floated in the pool.

I subjected each species to a range of toxin doses, with the exact range

based on the effects observed. A weak or zero effect in a trial meant that the

dose was doubled for the next snake, a lethal effect meant that the next dose

was quartered. To minimize mortality, I initially tested snakes within each

species on low doses. Each snake was tested once only. Where sample size

permitted, I tested multiple snakes at each dosage level. Dosage rates were

26

calculated on a volume to mass ratio for each individual (0.002 mL/g of body

mass). Different dosages were achieved by dilution of the original toxin extract.

I used six initial dilution levels (0.025x, 0.05x, 0.1x, 0.2x, 0.5x and 1x) with some

species later given intermediate doses. Higher doses were achieved by

successively increasing the dose per mass of undiluted extract (thus 2x = 0.004

mL/g, 4x = 0.008 mL/g, etc).

This process yielded data on reduction in speed as a function of dose for

each species. In all cases there was a strong positive relationship between dose

and percentage reduction in speed, within the range of doses that elicited an

effect. Percent reduction scores were transformed according to the following

formula modified from that of Brodie et al. (2002):

y’ = ln(2/y-1)

where y is the proportional reduction in speed (%redn/100). There were three

instances where the proportion reduction was <0. Because these values do not

transform correctly they were entered as a proportional reduction of 0.01 for the

purposes of transformation (following Brodie et al. (2002)). This transformation

makes it simple to estimate the dose giving a 100% reduction in speed (the

LD50, y = 1). The reason for this is that when y = 1, y’ = 0. Thus the LD50 is the

X-intercept of the regression of y’ on dose which can be estimated as −α /β ,

where α is the intercept and β is the gradient of the line. Least-squares

regressions of y’ on dose were conducted for each species and LD50 estimates

made.

27

I performed analysis of covariance on transformed mean proportional

speed reduction data with species as the factor and dose as the covariate to test

for differences in resistance between species. Because of the large discrepancy

in doses between some species, the data violated the assumptions of ANCOVA.

I thus performed the ANCOVA on the two natural groups of resistance (low

and high) to test for differences within each group. To test whether different

species required different doses to achieve a similar decrement in swimming

speed across all species, I performed an ANOVA with species as the factor and

dose as the dependent variable. I conducted this analysis on all individual data

where the decrement in speed was >20%. Selecting the data in this way

ensured that I was only comparing doses within the effect range for each

species.

A snake species’ vulnerability to toads will be determined not only by

the amount of toxin that it can tolerate, but also by the size of anurans that it

consumes relative to its own body mass. A snake that eats only very small

toads might thus be able to survive ingestion, whereas a snake that takes larger

prey relative to its own body size might exceed the lethal dose. Because snakes

are gape-limited predators, a snake’s head size offers an index of the maximum

size of prey that it can consume (Shine 1991d). I thus calculated the head width

of a toad large enough to contain a potentially lethal dose of toxin for an

average-sized specimen of each snake species, and compared that prey size to

the head-width of this average snake.

28

For species with sufficient data I calculated the LD50 (in terms of absolute

dose) for a snake of average body size. I then converted this dose into the

equivalent mass of toad skin and used unpublished data on the relationship

between toad body size and skin mass to calculate the size of toad that would

constitute this LD50. To compare this potentially lethal minimum toad size to

the size of toad that a given snake species could physically ingest, I calculated

the average mass and gape width for each snake species. I then divided the

LD50 toad size (expressed as toad head-width) by the mean snake head-width to

provide an index of lethal prey size relative to the snake’s physical ability to

ingest a prey item of that size. That is, the head-width of a toad of size

sufficient to provide the LD50 to an average sized snake was expressed as a

percentage of mean gape width for snakes of each species. Percentages of

<100% mean that the snake could easily ingest a lethal-sized toad, whereas

higher values make it increasingly unlikely that the snake could ingest a toad

large enough to kill it.

29

RESULTS

The number of snake species potentially at risk from toads

Analysis of distribution and dietary preference of Australian snakes

suggests that 49 species are potentially at risk from the invasion of the cane

toad (Table 1). Of these, 26 are likely to have their range totally encompassed

by that of the toad (under predicted 2030 climate change) and three have

already had their range totally encompassed by that of the toad. Nine of the “at

risk” species are already recognized as being threatened either on a federal or

state level ( Cogger et al. 1993). Thus, the toad invasion constitutes a potential

threat to 70% of the Australian colubrid snakes (7 of 10 species), 40% of the

pythons (6 of 15), and 41% of the elapids (36 of 87). These at risk taxa include 9

of the 38 terrestrial species identified as being of most concern in terms of

conservation status (Cogger et al. 1993).

Snakes’ tolerance to toad toxins

For most species of snake that I tested, the percent reduction in

locomotor performance was highly associated with survival after ingestion of

toxin: most animals with 100% reduction in swim speeds died 1–2 hours after

dosing. Animals with <100% reduction generally recovered over the course of

8–24 hours. Common blacksnakes (Pseudechis porphyriacus) were an exception

to this generality, with two (of four) individuals given a 0.3x dose exhibiting

swim-speed reductions of only 36% and 65%, but dying 8–24 hours later. For

30

the purposes of analysis, these individuals were scored as showing a 100%

reduction in speed.

31

Table 1. Australian snake species potentially affected by the invasion of the

cane toad.

Column 1 lists the percentage of frogs in the diet of each species. Columns 2-4 give the percentage of the species’ range encompassed by the toad currently and under the predicted distribution of toads (under present climate and a 2030 predicted climatic scenario). The index of potential impact is based on the proportion of frogs in the diet and the predicted percent overlap with toads (under 2030 climate scenario). The last column states the conservation status of individual species as listed in the action plan for Australian reptiles (Cogger et al. 1993, V = vulnerable, R = rare or insufficiently known). “?” represents an unknown quantity. “%?” represents a species for which frogs constitute a portion of the diet but for which a percentage was unobtainable. * The conservation status of this species refers to a South Australian population that is unlikely to come into contact with toads.

Species Frogs in Potential impact Conservationdiet(%) Current Potential Potential index status

(current climate) (2030 Climate)BoidaeAntaresia childreni 33 70 100 100 33Antaresia maculosus 6 95 100 100 6Antaresia stimsoni 8 7 10 10 0.8Morelia spilota 1 43 55 64 0.64Morelia carinata ? 0 100 100 ? RMorelia oenpelliensis ? 20 100 100 ? R

ColubridaeBoiga irregularis 6 65 91 100 6Dendrelaphis calligastra 50 100 100 100 50Dendrelaphis punctulatus 78 63 87 95 74.1Enhydris polylepis 30 83 100 100 30Stegonotus cucculatus 50 79 100 100 50Stegonotus parvus ? ? 100 100 ? RTropidonophis mairii 97 70 100 100 97

ElapidaeAcanthopis antarcticus 6 43 51 58 3.48 RAcanthopis praelongus 27 65 100 100 27Cacophis churchilli ? 100 100 100 ?Cacophis squamulosus 6 63 100 100 6Demansia papuensis ? 45 100 100 ?Demansia psammophis 7 16 21 23 1.61Demansia simplex ? 0 100 100 ?Demansia vestigiata 27 87 100 100 27Denisonia devisii 88 45 55 60 52.8Denisonia maculata 95 100 100 100 95 VDrysdalia coronata 53 0 38 88 46.64Drysdalia coronoides 5 0 0 16 0.8Echiopsis atriceps ? 0 100 100 ? VEchiopsis curta 31 0 14 32 9.92 VElapognathus minor 66 0 0 50 33 VHemiaspis damelii 95 60 80 100 95Hemiaspis signata 22 75 92 100 22Hoplocephalus bitorquatus 77 72 83 94 72.38 VHoplocephalus stephensi 11 50 75 100 11 VNotechis ater %? 0 22 56 ? V*Notechis scutatus 92 5 5 20 18.4Pseudechis australis 20 20 31 32 6.4Pseudechis colletti 25 77 77 77 19.25Psuedechis guttatus 40 64 86 100 40Pseudechis papuanus ? ? 100 100 ?Pseudechis porphyriacus 60 32 41 53 31.8Pseudonaja affinis 2 0 14 36 0.72Pseudonaja guttata 41 44 47 47 19.27Pseudonaja nuchalis 4 19 27 27 1.08Pseudonaja textilis 9 48 52 57 5.13Rhinoplocephalus incredibilis ? ? 100 100 ?Rhinoplocephalus nigrescens 1 61 69 77 0.77Rhinoplocephalus pallidiceps 6 45 100 100 6Suta ordensis ? 0 100 100 ?Suta suta 3 24 30 31 0.93Tropidechis carinatus 41 71 86 100 41

Percent Overlap

32

In all snake species tested, a higher dose (mL toxin/g) resulted in a

greater reduction in locomotor performance (Fig. 2). The estimated LD50 was

approximately 55 times higher for Tropidonophis and 22 times higher for

Stegonotus, than for the other eight taxa I tested (Table 2, Fig. 1; x-axis is ln-

transformed in this figure). The LD50 for Tropidonophis was 85 times higher

than the lowest LD50 estimate – that for Enhydris.

The data clearly indicate two groups of taxa – those with high resistance

(Tropidonophis and Stegonotus) and those with low resistance (all others). I

performed ANCOVA separately on these two groups with species as the factor,

dose as the covariate, and transformed proportional speed reduction data as the

dependant variable. In both cases there was no significant interaction between

species and dose factors (high, F1,13 = 0.019, p = 0.89; low, F6,14 = 2.02, p = 0.13).

After removing the interaction term, both analyses suggested significant

differences between species in resistance (high, F1,14 = 24.58, p = 0.0002; low, F6,20

= 3.17, p = 0.023). In the high group Tropidonophis was significantly more

resistant than Stegonotus. In the low group, this result appeared to be driven by

Hemiaspis which was slightly more resistant than other species although

Fisher’s PLSD gave only one significant pair wise comparison (Hemiaspis vs.

Pseudonaja, p = 0.02). After excluding percent reductions <20% there was no

significant difference in the percent reduction scores between species (F8,91 =

1.81, p = 0.085). The dose required to achieve these similar reduction scores

differed significantly among species (Fig. 3; F8,91 = 84.05, p < 0.0001). Fisher’s

PLSD confirmed that this effect was due entirely to Tropidonophis and

33

Stegonotus, which both required significantly higher doses than other species (p

< 0.02 in all cases), with Tropidonophis being significantly higher than Stegonotus

(p < 0.0001).

34

Figure 2. Percentage reduction in speed as a consequence of toad toxin dose for 10 species of Australian snake. The x-axis, is ln(100 x dose) where dose is expressed as a concentration of toxin extract administered at a rate of 0.002mL/g. The upper graph (a) shows data for elapid and pythonid species; the lower graph (b) shows data for colubrid species. Plotted points represent the mean value for all individuals tested at each dosage level (error bars omitted for clarity).

-20

0

20

40

60

80

100

0 1 2 3 4 5 6 7 8

Ln (100 x dose)

Boiga

Tropidonophis

Stegonotus

Enhydris

Dendrelaphis

-20

0

20

40

60

80

100R

educ

tion

in s

peed

(%)

0 1 2 3 4 5 6 7 8

Antaresia

Pseudonaja

Pseudechis

Hemiaspis

Acanthophis

(a) Elapids + Python

(b) Colubrids

35

0.000

0.005

0.010

0.015

0.020

0.025

0.030

Mea

n do

se (m

L/g

)

Acan

thop

his

Hem

iasp

is

Pseu

dech

is

Pseu

dona

ja

Anta

resia

Den

drel

aphi

s

Enhy

dris

Steg

onot

us

Trop

idon

ophi

s

Species

Figure 3. The average dose required to cause a reduction in speed greater than 20% for nine species of Australian snake. Mean dose is expressed as millilitres of toxin extract per gram of snake weight. Error bars represent 2 standard errors and are too small to visualize at this scale for all species except Stegonotus and Tropidonophis.

36

Table 2. The snake species examined, collection localities, morphological statistics, and results of toxin trials.

Total n refers to the number of individuals tested for toxin resistance (numbers for morphological measures were different in some instances). Numbers in parentheses represent standard errors. Gape width is the distance across the head at the hinge of the jaw. The LD50 for each species is expressed as (1) the dose of toxin per body mass of snake (μL/g), (2) the absolute dose based on the weight of an average individual, expressed in milligrams of dried toad skin equivalent, and (3) as the percentage of the average snake’s head-width that a toad’s head width, whose size is sufficient to provide the absolute dose, represents (see text for details).

Species Location total n SVL (mm) Weight (g) Gape width (mm)

Dose (mL/g) Absolute (mg) Percent of GapeAntaresia childreni Humpty Doo, NT 5 892.5 (33) 227.6 (34) 15.5 (0.6) 0.816 19.25 64.44Boiga irregularis Casino, NSW 1 1210 (-) 299 (-) 21.9 (-) - - -Dendrelaphis punctulatus Lismore, NSW 10 984 (122) 218 (61) 19.7 (2.9) 0.744 17.51 48.83Enhydris polylepis Humpty Doo, NT 20 611 (19) 113 (12) 13.2 (0.3) 0.448 10.56 65.17Stegonotus cucullatus Humpty Doo, NT 18 1041 (45) 303 (37) 18.6 (0.8) 15.016 353.49 111.32Tropidonophis mairii Humpty Doo, NT 27 550 (20) 84 (8) 12.5 (0.5) 37.992 894.35 185.54Acanthophis praelongus Humpty Doo, NT 20 418.5 (22) 117.7 (20) 20.1 (1.1) 0.774 18.24 48.91Hemiaspis signata Casino, NSW 3 455 (7) 32 (2) 9.4 (0.1) 0.768 18.07 107.7Pseudechis porphyriacus Casino, NSW 28 900 (41) 347 (51) 22.0 (0.8) 0.692 16.29 42.99Pseudonaja textilis Lismore, NSW 3 1225 (115) 592 (220) 24.9 (1.6) 0.572 13.46 36.58

LD50

31

37

DISCUSSION

Cane toads have been spreading rapidly through Australia for more than

60 years, and warnings of their possible ecological impact on the native fauna

have been voiced throughout that period (Lever 2001). Despite the clear

inference from anecdotal reports that terrestrial predators were the component

of Australian ecosystems most likely to be affected by the toads’ arrival,

research on toad impacts has been dominated by studies of the effects of toads

on potential prey items, competitors and aquatic predators. Several such

studies have suggested that toad impacts are likely to be less severe than had

been predicted, and these results have been interpreted to mean that toads may

pose less of a conservation disaster than anticipated by ecological doom-sayers

(e.g. Freeland and Kerin 1990). Unfortunately, this conclusion is misleading: a

lack of effect at lower trophic levels tells us nothing about potential impacts on

predators, the component of the fauna most likely to be affected.

Why have previous workers focused on taxa other than terrestrial

predators, despite anecdotal reports of major mortality events in native

terrestrial predators (snakes, monitors, marsupial carnivores) following toad

arrival? Logistical difficulties in quantifying the abundance of large vertebrate

predators are the most likely reason, especially when combined with high

levels of stochasticity in resources and thus, predator populations, in many

Australian habitats (Flannery 1994). Even if we cannot measure abundances

accurately in the field, however, we can study the vulnerability of predators in

38

a laboratory setting to assess the likely result of encounters between a predator

and a toad. The clear result from my analysis is that the invasion of cane toads

is likely to have caused, and will continue to cause, massive mortality among

snakes in Australia.

The methods I used to estimate vulnerability, based on geographic

distributions and dietary composition, are crude and subject to several sources

of error (most leading to a conservative bias). Notably, there is still uncertainty

about the eventual distribution of cane toads within Australia, and we do not

know how a given proportion of amphibian prey items within a particular

snake species’ diet will translate into prey preferences within specific

populations or among individuals within any given population. Obviously the

impact of toads will be different if a 50% utilization of anuran prey is due to

50% of individual snakes eating only frogs whereas the other individuals do not

attempt to consume this prey type, as opposed to the (more probable) scenario

where all individuals within the population prey upon anurans as well as other

prey.

Nonetheless, my analysis indicates that a high proportion of Australian

snake species are potentially at risk from toads (Table 1). Although it is

possible that habitat differences will reduce contact with toads for some species,

the fact that the toad is an extreme generalist in Australia and can be found in

most habitats (Lever 2001) suggest that this factor will be relatively

unimportant. My calculations probably underestimate vulnerability for many

of the taxa listed as feeding on frogs only infrequently. Low percentages of

39

anurans in the diet do not necessarily equate to low potential impact for two

reasons. Firstly cane toads typically attain higher population densities than

most native frogs; thus, any individual snake prepared to attack an anuran prey

item is likely to encounter a toad. Secondly many snake species exhibit

ontogenetic and or sex-based shifts in prey preference, such that certain

size/sex classes within a population consume a higher proportion of anurans

than do other size/sex classes. For example, both Pseudonaja textilis and Boiga

irregularis display an ontogenetic shift from ectothermic to endothermic prey

(Savidge 1988; Shine 1989; Shine 1991c). Sexual divergence in prey composition

has been recorded in A. praelongus, P. porphyriacus, and B. irregularis and is

likely to be widespread across many snake species (Shine 1991b, Pearson &

Shine 2002). In all these cases, the percentage of frogs in the diet (averaged

across all individuals) may lead to an underestimate of the likely impact of

toads on a population.

My laboratory studies on the effects of toad toxins on snake locomotor

speeds are also likely to have underestimated the severity of effects from

ingesting entire toads. Firstly I only extracted toxin from the dorsal skin of

toads. Toxin that is present in the ventral skin and internal organs was not

included in the extraction. Secondly the extraction process is unlikely to have

been 100% efficient and some toxin will have been lost. Therefore the actual

lethal dose in terms of toad size is likely to be even lower than those listed in

Table 3. It is also important to note that many snakes will take multiple prey

40

items. My calculations are limited to the effect of a single prey item. Once

again I am underestimating the potential impact on an individual snake.

Nevertheless, it appears that most species of snakes can easily ingest a

single toad large enough to be fatal (Table 2). This result reflects the facts that:

(1) most of the snakes I tested were severely affected even by small amounts of

toad toxins; (2) even small cane toads contain considerable toxin; and (3) most

snakes can swallow prey items that are relatively large compared to their own

body mass. In this respect, broad-headed snakes (such as Acanthophis and

Hoplocephalus) are at higher risk than relatively small-headed taxa.

Interestingly, the slightly higher resistance of Hemiaspis coupled with it’s small

relative head-width suggest that this species will be less affected by toads (LD50

as % of gape width = 107%). Nevertheless, the clear result from these analyses

is that most of the snake species that I tested are likely to be at substantial risk

when cane toads invade their habitat.

Most of the snake species I tested exhibited low (and relatively similar)

tolerance to the toxins of the cane toad (Table 2). The most striking exception in

this respect was the keelback T. mairii. This species has been reported

previously to ingest toads without ill effects (Covacevich and Archer 1975), but

other authors have reported that keelbacks sometimes died after eating toads

(Ingram and Covacevich 1990; Shine 1991c). A survey of wild-caught keelbacks

indicated that toads constituted only a small proportion of prey items inside

alimentary tracts of these snakes (Shine 1991c). My data support the notion that

T. mairii is extremely resistant to toad toxin and, hence, that individuals of this

41

species are unlikely to die as a consequence of ingesting a toad. The only other

snake species reported to tolerate ingestion of toads is the treesnake,

Dendrelaphis punctulatus (Covacevich and Archer 1975), but my data argue

against this possibility; several individuals died after relatively small doses

(Table 2).

Although both keelbacks and slatey-greysnakes are predicted to survive

the ingestion of a toad (Table 2), this does not mean these species are capable of

eating toads on a regular basis. Physiological costs associated with neutralizing

the toxin may entirely negate any energetic benefit associated with the

consumption of the prey. Alternatively, the toxin may have a chronic

cumulative effect. In the laboratory, keelbacks maintained exclusively on a diet

of toads lost condition and died (Shine 1991c). It is also important to remember

that my methodology removed among-toad variance in toxicity. It is entirely

likely that some toads are more toxic than others and thus the outcome of an

individual encounter may vary from predictions made here.

Both of the snake species that show high levels of resistance to toad toxin

are colubrids. This family is believed to be a recent (post mid-Miocene but

probably as late as the Pleistocene) invader of the Australian continent (Cogger

and Heatwole 1981; Greer 1997). Recent ancestors of Australian colubrids are

likely to have been sympatric with Bufo in south-east Asia, raising the

possibility that some Australian colubrids may be pre-adapted to bufonid

toxins. Tropidonophis spp. in south-east Asia prey on native bufonids (Malnate

and Underwood 1988), and a genus closely related to Stegonotus in central

42

China contains at least one species that preys on toads (McDowell 1972; Pope

1935). The three other colubrids I tested (Enhydris polylepis, Boiga irregularis and

Dendrelaphis punctulatus) showed low levels of resistance, dismissing the

possibility of a familial-level divergence in resistance to toad toxins.

Most of the snake species I tested showed a similar response to toad

toxin on a dose per unit mass basis (Fig. 3, Table 2). This result is made more

striking by the fact that snakes tested cover a broad phylogenetic span (three

families). It thus seems likely that most Australian snake species will show a

similar low level of resistance. However the significance of this common and

low resistance level must be assessed in relation to the behaviour, ecology, and

morphology of each snake species. Specific factors important in the interaction

include foraging behaviour, habitat preference and the ability of snakes to learn

or acquire resistance. Further research is currently underway to assess these

factors – particularly the possibility of an adaptive response.

The maximum relative prey size of each species does appear to mediate

the impact of toads. For example Hemiaspis signata, despite exhibiting a similar

LD50 estimate to susceptible species, is less likely to be affected because

individuals can ingest only small toads relative to their body mass (Table 3).

Acanthophis praelongus on the other hand is capable of eating much larger toads

than is required to provide a lethal dose, and is thus predicted to be badly

affected.

My data suggest that many species of Australian snake are likely to be

adversely affected by the invasion of the cane toad. The exact magnitude of the

43

effect will depend on factors specific to each species and whether or not

populations can mount an effective adaptive response. However it seems

prudent to treat the invasion of the cane toad as a serious threat to many

populations of frog-eating snakes. Some of the species listed in Table 1 are

already regarded as threatened and it would be wise for wildlife managers to

give serious consideration to the impact of the cane toad on these species in

particular. More generally, we should not allow logistical impediments to

discourage work on the components of natural systems most likely to be

affected by alien organisms.

ACKNOWLEDGEMENTS

I am extremely grateful to J. Hayter and E. Bateman for encouragement

and invaluable assistance with the collection of animals. S. Hahn provided

helpful advice regarding the extraction of toad toxins. G. Brown collected the

Enhydris dataset and R. Shine offered statistical advice and reviewed an earlier

version of the manuscript. J. Webb kindly provided access to an unpublished

manuscript. I would also like to thank the staff at Beatrice Hill research farm

for their generous hospitality. Funding for this project was provided by grants

from the Australian Research Council and The Royal Zoological Society of New

South Wales.

44

CHAPTER 2

SUBLETHAL COSTS ASSOCIATED WITH THE

CONSUMPTION OF TOXIC PREY BY

AUSTRALIAN KEELBACK SNAKES

45

ABSTRACT

The value to a predator of a prey item depends not only on the nutritional

content of the prey but also on accessory costs such as the time, effort and risk needed to

overpower and consume the prey, and any negative consequences of toxins in the prey.

Because snakes take relatively large prey, such sublethal costs associated with poor prey

choice are likely to have direct fitness consequences. I examine the costs to Australian

keelback snakes (Tropidonophis mairii) associated with consuming cane toads (Bufo

marinus). Cane toads are an introduced species in Australia and are highly toxic;

keelbacks are one of the only species of Australian snake that can consume toads without

dying. Nonetheless, snakes took longer to consume toads than native frogs and toad

toxin reduced snake locomotor performance for up to six hours after ingestion. These

effects are likely to increase a snake’s vulnerability to predation. Although I found no

significant nutritional cost associated with the consumption of toads the additional risks

from eating this prey type make toads less “profitable” than native frogs. Snakes may

therefore be under selection to delete such items from their diets.

46

INTRODUCTION

Research into the nutritional aspects of prey choice has a long history,

spurred on by optimal foraging theory and its attendant controversies (Perry

and Pianka 1997). As optimal foraging theory matured however, it became

clear that factors other than nutritional benefit were also important in the

evolution of optimal foraging behaviour. For example, processing time is also

important (Schoener 1971) as are any other factors that reduce the predator’s

benefit from consuming that type of prey (e.g. Demott and Moxter 1991;

Downes 2001). In short, a predator’s choice of prey potentially has subtler

implications for fitness than are captured by the examination of calories alone.

The act of prey acquisition often carries with it many potential risks and this

risk-taking is likely to have fitness consequences. Interestingly, theoreticians

have incorporated subtler risks such as predation and competition (e.g. Brown

1999; Houston et al. 1993) but empirical work has lagged behind (Perry and

Pianka 1997).

The riskiness of prey acquisition is perhaps nowhere better

demonstrated than by snakes, which tend to eat large prey and are relatively

defenceless during the ingestion phase (Cundall and Greene 2000).

Additionally, many snakes consume toxic prey – usually amphibians – many of

which contain toxic skin secretions (Daly et al. 1987; Duellman and Trueb 1994).

Snakes thus face not only the obvious risks associated with subduing large prey

and being vulnerable to predation while ingesting this prey but they also often

risk being poisoned. Specialised diets and venom may well be evolutionary

47

responses to these risks (Greene 1997) and hence testament to their importance

to snake fitness.

Much of the literature dealing with the costs of toxic prey revolves

around herbivores and the costs associated with the ingestion of phytotoxins

(e.g. Agrawal and Klein 2000; Demott and Moxter 1991; Guglielmo et al. 1996).

In these systems optimal choices are often inferred to depend almost solely

upon the nutritional value of the food and the energetic cost of dealing with the

toxin. Fitness consequences associated with other aspects of the organism’s

environment, such as predation, are often ignored (Bernays and Graham 1988;

Dicke 2000). Few studies then, examine the potential consequences of toxic

prey choice from both a nutritional and ecological perspective.

If we are to understand foraging behaviour, then optimal foraging

should be defined as that which maximises lifetime fitness (Perry and Pianka

1997). As such, factors other than nutrition alone must be considered. Snakes,

offer ideal study organisms in which to examine this problem – poor choice of

prey not only has nutritional consequences but may also increase a snake’s

chance of injury or predation. In this paper I examine implications of prey

choice in the Australian keelback snake (Tropidonophis mairii) in terms of risk as

well as energy.

48

METHODS

Study species

The keelback is a small, crepuscular, non-venomous colubrid that feeds

almost exclusively on anurans (Cogger 2000; Shine 1991c). It is the only

Australian snake known to be regularly capable of ingesting the introduced

cane toad (Bufo marinus) without dying (Phillips et al. 2003). Cane toads are

highly toxic and highly successful invaders of the Australian continent, having

colonised more than 1 million square kilometres since their introduction in 1935

(Lever 2001; Sutherst et al. 1995). Once toads become established they can reach

astounding densities, often becoming the most common anuran species in an

area (Freeland 1986). Because of this, toads are often the most common prey

available to anurophagous snakes such as the keelback. In this study, I evaluate

the consequences to keelbacks of consuming cane toads instead of native prey

(rocket frogs, Litoria nasuta). Rocket frogs are a common, relatively non-toxic

species (Erspamer et al. 1984) that figure prominently in the natural diet of the

keelback (Shine 1991c).

I tested three possible non-lethal effects associated with ingesting toads

vs frogs. First, I tested for non-lethal performance effects and the duration of

such effects, using locomotor performance as an indicator of performance.

Second, I tested the possibility that toads require greater handling time

(consumption time). Third, I tested the possibility that toads make “poorer”

meals from an energetic perspective.

49

Effects of toxin on locomotor performance

I tested the effect of toxin on the locomotor performance of 12 neonate

keelbacks hatched in the laboratory as part of an on ongoing study into the

ecology of this species. Neonate keelbacks are likely to be at the highest risk

from toads because small snakes tend to eat relatively larger prey items and

hence consume relatively larger doses of toxin (Chapter 3). Additionally,

young keelbacks suffer extremely high mortality in the field, probably as a

result of predation (G Brown pers. comm.). I assigned three keelbacks to each

of four treatment groups, each receiving different doses of toxin. Toxin was

extracted from the skin of 78 toads. Details of the toxin extraction procedure

can be found in chapter 1.

Locomotor performance was assayed using a methodology modified

from that of Brodie and Brodie (1990) and used extensively in chapter 5.

Individuals were swum along a 2m swimming trough and were timed with an

electronic stopwatch over three consecutive 50cm segments of the trough.

Animals were encouraged to swim by tapping them on the tail. Water

temperature was maintained at 23±1oC.

A swimming trial consisted of two consecutive laps of the trough. This

yielded 6 measurements of swim speed over 50cm of which only the fastest was

retained. All animals (1-2 days post hatching) were initially subjected to three

swim-trials one hour apart. This yielded three maximum sprint speed times

50

that were averaged to generate the pre-dose estimate of maximum swim speed

(expressed as the time taken to cover 50cm, b).

On the following day, snakes were given a specific dose of toxin or

control solution. The doses were either 25μL, 50μL or 100μL of toxin or 100μL

of control solution for each treatment group. These doses all fall within the

range that a snake would experience from ingesting a single toad (Phillips et al.

2003). Control solution was prepared in a manner identical to the toxin

extraction but without the addition of dried skin. Dosing was achieved by use

of a micropipette attached to a thin rubber feeding tube. The tube was inserted

into the stomach to a depth of 5cm from the snout before toxin was expelled.

Animals were observed for 1 minute following this procedure to ensure the

toxin was not regurgitated. Several swim trials were then undertaken for each

individual. Locomotor performance was assessed 30min, 1.5hrs, 2.5hrs, 6hrs

and 18hrs post dosing. Once again, only the fastest speeds for each trial were

taken. This yielded two time measurements, which were then averaged to give

post-dose swim speed (again, expressed as time, a) for each time post dose. The

percentage reduction in swim speed (%redn) was calculated from these times

using the formula, %redn = 100 x (1-(b/a)).

Consumption time and energetic benefit

Adult keelbacks were captured by hand at Tyto Wetlands, 1 km south

west of Ingham, Qld in February 2003. Snakes were housed individually in

plastic tubs in a field laboratory maintained at 27oC. Each animal was given a

51

pile of straw for a retreat site and a container of water, which was available at

all times throughout the experiment. A total of 21 snakes were collected. After

seven days of acclimation, each snake was weighed, given an identification

number and randomly allocated to one of two treatment groups. One

treatment group was offered frogs (Litoria nasuta) for prey (10 snakes) and the

other group was offered cane toads (Bufo marinus, 11 snakes).

Snakes were offered prey on three separate occasions separated by three

days. Prey items were left in each snake’s enclosure for 24 hours. At the end of

this period the cage was carefully searched for prey items and I recorded

whether or not the prey item was eaten. I attempted to keep the relative

amount of food per snake similar between the two groups. This was achieved

by feeding each snake approximately the same relative prey mass (prey mass

divided by snake mass) each feeding session. The toads I collected weighed

less than the frogs on average, so often multiple toads were given to a snake to

reach the equivalent mass of a single frog.

Where possible I observed snakes consuming prey and scored the prey

orientation (head first or legs first) and the time it took a snake to consume a

prey item (from initial grab until the snake’s mouth was completely closed with

the prey item in the throat).

After the three feeding opportunities, no snake was offered food. To

allow digestion of food and passage of faeces, I waited one week after the

conclusion of feeding before weighing all the snakes again. During this period,

all snakes were offered water as before.

52

I compared toad and frog treatments using ANCOVA with change in

snake mass as the dependent variable and total prey mass as the covariate. I

also compared consumption time between the treatments (with relative prey

mass as a covariate) to determine whether frogs were consumed faster than

toads.

53

RESULTS

Effects of toxin on locomotor performance

All doses of toxin given to neonate keelbacks elicited a reduction in

locomotor performance; an effect that decayed with time (Fig. 1). Repeated

measures analysis of variance with %redn as the dependent variable revealed a

strong interaction between dose level and time (Wilks’ lambda; ≈F12,13.5 = 5.96, p

= 0.0013). This effect remained even after the control group was excluded from

the analysis as expected if all the time series converge on a similar value (see

Fig. 1 at time 18 hrs).

Therefore, I chose to perform an ANOVA at each time interval and use

Dunnet’s post hoc test to compare %redn for each treatment against that of the

control. This analysis showed significant differences between the two highest

dose categories and the control for the first three post-dose trials (0-2.5 hrs). At

six hours post-dose, only the high dose category exhibited a significant

difference and by 18 hours post-dose none of the treatment groups showed

locomotor decrements significantly different from those of the control group

(Fig. 1).

54

-25

0

25

50

75

100

Mea

n re

duct

ion

in sp

eed

(%)

0 5 10 15 20

Hours post dose

Control

Low dose (25uL)

Medium dose (50uL)

High dose (100uL)

-25

0

25

50

75

100

Mea

n re

duct

ion

in sp

eed

(%)

0 5 10 15 20

Hours post dose

Figure 1. Effect of toxin on locomotor performance in neonate keelback snakes at three dosage levels over 18 hours. For clarity, error bars (one standard error) are shown only for the high dose and control treatments (errors were similar across all treatments). Closed symbols represent treatment groups where the percentage reduction in locomotor performance was significantly different from that of the control (Dunnet’s post hoc test).

55

Consumption time

Two snakes from the frog prey treatment died before the end of the

feeding experiment for reasons unrelated to the experiment. Thus my final

sample size was eight animals in the frog treatment and eleven in the toad

treatment.

One individual took > 15 minutes to consume a large toad, after which it

appeared unable to move (not responding to a touch on the tail) for > 3 hours.

While this individual demonstrates the potential difficulties associated with

eating toads the data point was removed from further analyses because it was

an extreme outlier and may bias the analysis. ANCOVA of consumption time

by prey type and prey orientation (relative prey mass as the covariate) revealed

no significant interaction terms (p > 0.1 in all cases). After removal of

interaction terms I found a significant association between relative prey mass

and consumption time (F1,13 = 47.66, p < 0.0001) and a significant difference in

consumption times between the two prey types (F1,13 = 8.48, p = 0.012) with

toads taking longer to be consumed (Figure 2). Prey orientation, however, had

no significant influence on consumption time (F1,13 = 0.05, p = 0.83).

Additionally, no significant difference was observed in prey orientation

between the two prey types (% head first: toads, 60%; frogs 40%; χ2 = 0.805, p =

0.37).

Energetic benefit

56

At the conclusion of feeding trials, all animals were weighed and their

change in mass over the course of the experiment was calculated. All animals

fed frogs gained mass whereas four of the eleven individuals maintained

exclusively on toads lost mass. I used ANCOVA with change in snake weight

as the dependent variable. Prey type, initial snake weight, and total prey

weight were used as independent variables. In this model, there were no

significant interaction terms (p > 0.08 in all cases). Following the removal of

interaction terms, prey type had no significant effect on weight change in the

snakes (F1,15 = 0.001, p = 0.98) although both initial snake weight and total prey

weight had a strongly significant effect on weight change (p < 0.0005 in both

cases).

57

0

250

500

750

1000

1250

Con

sum

ptio

n Ti

me

(s)

0 5 10 15 20

Relative Prey Mass

Toads

Frogs

Figure 2. Consumption time against relative prey size for keelback snakes consuming two prey types – toads and frogs. Relative prey mass = the mass of prey divided by the mass of predator multiplied by 100. The single extreme consumption time value (time >1000 s) for an individual consuming a toad was excluded from the statistical analysis.

58

DISCUSSION

The keelbacks in this study demonstrated significant sublethal costs

associated with the consumption of toads. Despite the apparent lack of

energetic cost associated with the consumption of toads, these toxic anurans

took longer to consume, and the toxin in toad skin inhibited snake locomotion

for up to six hours.

Keelbacks are unusual among Australian reptiles in having a very high

resistance to toad toxin. For example, keelbacks are more than 70 times more

resistant to toad toxin than are many other Australian snakes (Phillips et al.

2003). Thus they are one of the few taxa that are able to consume toads without

dying as a result. My results also suggest that keelbacks can derive energetic

benefit from the consumption of toads. Although there may be a differential in

energy benefit between frogs and toads, it is not striking and was not detectable

with my small sample sizes.

Although keelbacks are capable of eating toads and can derive

significant energy benefit from them, they do experience costs associated with

toad ingestion. First, consuming a toad reduces the keelback’s locomotor

performance. The extent of this cost is dose related and will thus depend upon

the relative size of the toad eaten. Some doses reduced snake mobility for at

least 6 hours. The biological relevance of these doses is evidenced by the

observation of one snake being unable to move for more than three hours

following the ingestion of a large toad during the feeding trials. Such

59

hampered locomotor performance potentially has serious implications for

predator avoidance, particularly for an active, fast moving snake such as

Tropidonophis mairii.

Also, the keelbacks in this study took longer to consume a toad than to

consume frogs of equal size. Higher consumption time is likely to require

greater energy expenditure and more importantly, will increase a snake’s

susceptibility to predation. The higher consumption times for toads may be

due to several possible factors. First, toads have drier skin than frogs so more

salivary lubrication is necessary for ingestion of toads. Second, toads inflate

their lungs when grasped by a snake (thereby making ingestion more difficult),

whereas Litoria nasuta does not. Other species of Australian frog (e.g. Cyclorana

spp.) do inflate their bodies (Williams et al. 2000) and it would thus be possible

to determine whether the increased consumption time was due to this aspect

alone. Third, ingestion time may be extended because keelbacks affected by

toad toxin exhibit muscular weakness and lowered performance. The rapid

effect of toad toxin may affect the efficiency of ingestion by snakes.

These subtler costs (whatever their mechanism) are likely to have fitness

consequences. There are two reasons for this: (1) Snakes run a high risk of

injury in the act of subduing prey. Because snakes are limbless, all prey

acquisition and handling must be initiated with their head (Cundall and Greene

2000). Because the head contains all the sensory organs it is the most vulnerable

part of the snake. Despite this vulnerability, snakes often consume relatively

large prey, so the risk of injury whilst subduing prey is high. Given these

60

circumstances, rapid subdual of prey is important (probably the reason many

species have evolved venom or prey specialisation). Prey that take longer to

subdue are more likely to cause an injury. (2) To avoid predators, most snakes

depend upon crypsis and flight (Greene 1997). Catching and subduing prey

will often rob an individual of the advantage of crypsis. After prey has been

subdued, the process of ingestion is relatively slow (snakes eat large meals) and

while ingesting prey, snakes are unable to move effectively and are unable to

defend themselves. Rapid ingestion of prey thus minimises the chance of

predation. Additionally, prey that are sufficiently toxic to impair locomotor

performance clearly reduce a snake’s ability to flee.

Even in the absence of a direct energetic consequence of eating toads,

these subtler effects are likely to have an impact on snake fitness. These “risk

consequences” thus represent an additional evolutionary hurdle to any snake

population utilising toads as a food resource. While relatively obvious in

snakes, such subtle effects may be very common in predator/prey interactions

generally and should not be overlooked. Even in the absence of obvious energy

differentials between prey types, “risk consequences” may be driving the

evolution of prey choice.

ACKNOWLEDGEMENTS

I would like to thank Virginia McGrath, David Fouche, Michael Kearney,

Luke Shoo, Gavin Bedford, James Smith and Kath Nash for their assistance in

61

the field. I am also grateful to Thomas Madsen and Greg Brown for providing

logistical support. Richard Shine and two anonymous referees improved

earlier drafts. This research was supported by grants from the Norman

Wettenhall Foundation and the Linnean Society of NSW (to BLP) and a grant

from the Australian Research Council (to RS).

62

CHAPTER 3

ALLOMETRY AND SELECTION IN A NOVEL

PREDATOR-PREY SYSTEM: AUSTRALIAN

SNAKES AND THE INVADING CANE TOAD

63

ABSTRACT

Because many organismal traits vary with body size, interactions between

species can be affected by the respective body sizes of the participants. I focus on a novel

predator-prey system involving an introduced, highly toxic anuran (the cane toad, Bufo

marinus) and native Australian snakes. The chance of a snake dying after ingesting a

toad depends on the size of the snake and the size of the toad, and ultimately reflects the

effect of four allometries: (1) Physiological tolerance (the rate that physiological

tolerance to toad toxin changes with snake size); (2) swallowing ability (the rate that

maximal ingestible toad size (i.e. snake head size) increases with snake body size); (3)

prey size (the rate that prey size taken by snakes increases with snake head size) and (4)

toad toxicity (the rate that toxicity increases with toad size). I measured these

allometries, and combined them to estimate the rate at which a snake’s resistance

changes with toad toxicity. The parotoid glands (and thus, toxicity) of toads increased

disproportionately with toad size (i.e., relative to body size, larger toads were more

toxic) but simultaneously, head size relative to body size (and thus, maximal ingestible

prey size relative to predator size) declined with increasing body size in snakes. Thus,

these two allometries tended to cancel each other out. Physiological tolerance to toxins

did not vary with snake body size. The end result was that across snake species, mean

adult body size did not affect vulnerability. Within species, however, smaller predators

were more vulnerable, because the intraspecific rate of decrease in relative head size of

snakes was steeper than the rate of increase in toxicity of toads. Thus, toad invasion

may cause disproportionate mortality of juvenile snakes, and adults of the sex with

smaller mean adult body sizes.

64

INTRODUCTION

An organism's body size profoundly influences a multitude of

biologically significant traits, ranging from metabolic rates to habitat use,

modes of locomotion and life history characteristics (Calder 1984; Schmidt-

Nielsen 1984). These kinds of effects are seen within as well as among species,

although often less dramatically in intraspecific comparisons because of the

smaller size range combined with developmental constraints (Schmidt-Nielsen

1984). Nonetheless, ontogenetic increases in body size will influence the way

an organism interacts with its environment, modifying aspects such as

reproductive capacity, competitive ability, optimal choice of food and shelter

and the risk of predation (Gould 1966). In consequence, evolutionarily

significant attributes of the life history (e.g., rates of mortality and reproductive

success) often are closely linked to body size (Calder 1984; Stearns 1992).

Commonly, the important issue is not absolute body size, but an organism's

size relative to that of other organisms. Predator-prey interactions offer some of

the clearest examples of situations where fitness consequences depend upon the

respective body sizes of the two participants. For example, a predator's ability

to locate, capture, kill and ingest a prey item will obviously depend not only

upon its own body size, but also on its size relative to that of the prey item.

Encounters between predators and highly toxic prey offer an

opportunity to examine the multiple allometries affecting the outcome of

predator/prey interactions. Information on this topic is of special value when

65

the interaction involves an invasive species that causes massive disruption to

natural ecosystems. The invasion of the cane toad (Bufo marinus) in Australia

provides such a case. Cane toads were introduced into Australia in 1935 in a

failed attempt at biological control of agricultural pests (Lever 2001). They have

spread their range rapidly since that time, and eventually will occupy almost

one third of the continent (Sutherst et al. 1995). Toads are highly toxic,

especially to Australian predators with no evolutionary history of exposure to

their toxins (Chen and Kovarikova 1967; Lutz 1971). Many Australian native

predators die if they attempt to ingest a toad (Covacevich and Archer 1975;

Phillips et al. 2003). Thus, toads represent a novel selective force for these

predators and a massive management problem for conservation agencies (e.g.

van Dam et al. 2002). At least 30% of Australia’s terrestrial snake species are at

risk from these toxic invaders, and most snakes are likely to die if they ingest a

toad (Phillips et al. 2003). Australian snakes are thus facing severe population

declines in the presence of toads.

How does body size influence the interaction between snakes and toads?

Toads and snakes offer an excellent opportunity to study the effects of body

size (allometry) on predator-prey interactions because: (1) toads are a strong

novel selective force on snakes; (2) both snakes and toads span a large range in

body sizes (as is true for ectotherms in general: Bonnet et al. 1998; Pough 1980);

(3) the interaction is relatively simple to quantify, because snakes are gape-

limited predators that must swallow an entire prey item rather than tearing it

apart; and (4) the timing and extent of the toad invasion in Australia is well

66

documented (Lever 2001). In this paper I describe allometric variation in toad

toxicity, snake resistance to toad toxin, and snake head sizes. Using these data,

I examine the ways in which the respective body sizes of prey and predator

influence snake vulnerability, and hence the selection pressures that toad

invasion imposes on body sizes of snakes.

67

MATERIALS AND METHODS

Analysis framework

All my analyses concern allometry. Relationships between log-

transformed morphological variables should yield simple ratio relationships

depending upon the units of measurement for each variable (Calder 1984;

Schmidt-Nielsen 1984). Thus, null slopes are obtained by assuming that there is

no change in the shape of an organism through ontogeny (i.e. isometry). Under

this assumption and with both variables log-transformed, plotting mass (a

cubic variable) against length (a linear variable) should yield a slope of three.

Similarly, a length variable plotted against another length variable should yield

a slope of one.

Ultimately, I aim for an understanding of how snake resistance changes

with toad toxicity. In calculus terms, I want to calculate:

δ(snake resistance)δ(toad toxicity )

This allometry can be viewed as the product of a number of contributing

allometries where numerators and denominators cancel out, such that:

δ(snake resistance)δ(toad toxicity )

=δ(snake resistance)

δ(snake length)×

δ(snake length)δ(snake head size)

×δ(snake head size)

δ(toad size)×

δ(toad size)δ(toad toxicity )

Thus, to understand the impacts of allometry on the interaction between

toads and snakes, we need information on four kinds of allometries:

68

1. The allometry of snake resistance against snake length

δ(snake resistance)δ(snake length)

⎛

⎝ ⎜

⎞

⎠ ⎟ .

Does a snake’s ability to deal with toad toxin increase or decrease in proportion

to the snake’s size? The absolute quantity of toxin required to kill a snake

presumably will increase with increasing body size, but the snake's

vulnerability will depend upon the slope of this relationship. For example, if

larger snakes can withstand higher toxin doses relative to their size, then a

smaller predator may be at higher risk from ingesting a toad.

2. The allometry of snake length against head size

δ(snake length)δ(snake head size)

⎛

⎝ ⎜

⎞

⎠ ⎟ .

Do larger snakes have heads that are smaller (relative to body length) than do

smaller snakes? Ontogenetic decreases in relative head size occur in many

kinds of animal, but are especially important in snakes because these animals

are gape-limited predators. That is, gape size determines maximum ingestible

prey size (Arnold 1993; Cundall and Greene 2000). Thus, deviations from

isometry between a snake's body size (which influences the absolute amount of

toxin that would be needed to kill the snake) and its gape size (which influences

maximum prey size, and thus the absolute amount of toxin that can be

ingested) may have important implications for the toad/snake interaction.

Thus, for example, a decrease in relative head size in larger snakes might

render them less likely to ingest a fatal toxin dose.

69

3. The allometry of snake head size against toad size

δ(snake head size)δ(toad size)

⎛

⎝ ⎜

⎞

⎠ ⎟ .

This allometry represents the interaction between predator and prey. If snakes

of all sizes only consumed prey items that were the biggest they could

physically ingest, this relationship should be approximately isometric. Usually,

however, snakes eat smaller prey as well as occasional maximal-sized items

(Arnold 1993). For example, many of the prey encountered by very large

snakes might be well below their maximal swallowing capacity, whereas this is

less likely for smaller conspecifics. If this is the case then smaller snakes are

taking relatively larger meals (and hence more toxin) relative to their own body

size.

4. The allometry of toad size against toad toxicity

δ(toad size)δ(toad toxicity )

⎛

⎝ ⎜

⎞

⎠ ⎟ .

Large toads presumably contain more toxin than do small toads, but the rate at

which the body size of a toad increases relative to its toxin content has strong

implications for a predator that may consume small toads when young and

large toads when mature.

Whether or not a snake dies after eating a toad will depend upon all of

these allometries, so that we need to combine information on all of these aspects

to understand the effect of snake body size on the level of risk imposed by

toads. This approach can be used to investigate allometries at two levels:

70

intraspecific (are smaller or larger individuals within a given snake population

more at risk?) and interspecific (are species with smaller or larger mean adult

body sizes more at risk?). Below, we explain the methods by which we

estimated each of the component allometries.

71

Data collection

All the snake species I examined have a substantial range overlap with

toads and feed largely on anurans. Additionally, all the species I examined

(with the exception of keelbacks, Tropidonophis mairii) have similar, low

resistance to toad toxin (Phillips et al. 2003) and as such, are predicted to be

badly affected by the presence of toads. Keelbacks are unusual among

Australian snakes in having a very high resistance to toad toxin (Phillips et al.

2003).

1. The allometry of snake resistance to toad toxin

δ(snake resistance)δ(snake length)

⎛

⎝ ⎜

⎞

⎠ ⎟ .

To calculate this allometry we would, ideally, give the same absolute

dose of toxin to snakes across a range of body lengths and measure the effect.

Unfortunately, the range of toad-toxin doses that elicit measurable but non-

lethal effects on snakes is extremely narrow (Phillips et al. 2003). Thus, I opted

to give each snake a size-adjusted dose and then determine if any residual

correlation existed between response to the toxin and body size. Such a

correlation would indicate a deviation from isometry in snake resistance.

Additionally, because I expected resistance to scale with body mass rather than

length, I gave snakes a mass-adjusted dose rather than a length-adjusted dose.

The allometry of resistance against mass can be converted to resistance against

length by multiplication with the allometry of mass on length, i.e.:

δ(snake resistance)δ(snake length)

=δ(snake resistance)

δ(snake mass)×

δ(snake mass)δ(snake length)

72

Thus, for the intraspecific comparison, I first examined the allometry of

resistance against mass and then measured the allometry of mass on length to

arrive at an estimate of the allometry of resistance on length.

Intraspecific allometry of snake resistance to toad toxin.

Toxin was obtained from skins of 78 freshly killed cane toads collected

from the Lismore area (northern NSW) and killed by freezing. A single

extraction of toad toxin was made for the entire study, to remove among-toad

variance in toxicity and accurately control dosing. The dorsal skin (from the

back of the head to the knees) including the parotoid glands was removed and

dried at room temperature over several days. The dried dorsal skin and

parotoid glands were weighed and then blended with 10x v/w of 40% ethanol.

This mixture was strained and the solids discarded. The resulting liquid was

allowed to evaporate to 50% of its initial volume at room temperature. The

final volume was recorded and then aliquoted into 25ml containers and frozen.

I used this extract to assess toxin resistance of two species of snake: the

keelback, Tropidonophis mairii (Colubridae) and the red-bellied blacksnake,

Pseudechis porphyriacus (Elapidae). These two species span the entire range of

toxin resistance among Australian snakes with P. porphyriacus having the

common, low resistance and T. mairii having very high resistance (Phillips et al.

2003). Tropidonophis mairii were collected from the Adelaide River floodplain 60

km east of Darwin, Northern Territory and P. porphyriacus were collected from

various localities within New South Wales and Queensland. Within each

73

species, each snake was given the same dose of toad toxin extract relative to

body mass. However, the dose differed between species and was calculated

from earlier trials (Phillips et al. 2003) to maximise my ability to detect variation

between individuals in resistance whilst minimising mortality. Thus, T. mairii

were given 24μL/g, and P. porphyriacus 0.4μL/g body mass. To quantify

physiological tolerance, I measured locomotor speeds of each snake before and

after receiving this standard dose of toad toxin (see Chapter 1 for details of

these methods). Because each snake was given the same dose of toxin relative

to its body mass, we can expect that there will be no relationship between body

mass and response to toxin (i.e., % locomotor decrement). To look for any such

relationship (which would indicate a deviation from isometry), I regressed

locomotor decrement against mass for each species.

Intraspecific allometry of snake mass relative to body length.

I obtained mass and length data for four species of snake including the

two species that were tested for toxin resistance (above). The additional species

were the swampsnake (Hemiaspis signata) and the green treesnake (Dendrelaphis

punctulata). These data come from snakes collected in various localities in

Queensland and New South Wales (including the snakes used in the toxin

resistance trials). I used reduced major axis regression (Sokal and Rohlf 1995)

to calculate the slope coefficients of mass against snout-vent length (SVL) for

each species. The observed relationship from the data was tested against the

74

null expectation (slope = 3) using simple t-tests, with standard errors taken

from the appropriate least squares regression.

Interspecific allometry of snake resistance to toad toxin.

Data from Phillips et al. (2003) strongly suggest that most Australian

snakes have similar levels of mass-adjusted resistance to toad toxin. The

species tested by Phillips et al (2003) spanned a broad range of body sizes (420-

1225mm SVL) so for the purposes of the interspecific comparison I assume that

resistance scales isometrically with body length.

2. The allometry of snake length against head size

δ(snake length)δ(snake head size)

⎛

⎝ ⎜

⎞

⎠ ⎟ .

I measured body and head size for preserved museum specimens of five

species of snake: two colubrids (keelbacks T. mairii, green treesnakes D.

punctulatus) and three elapids (swampsnakes H. signata, rough-scaledsnakes

Tropidechis carinata, red-bellied blacksnakes P. porphyriacus). All specimens

were measured for snout-vent length (SVL), head-width (HW; across head,

where supraoculars meet parietals) and jaw length (JL; jaw hinge to tip of

snout).

Within each species, I assessed allometry in head/body dimensions by

regressing log-transformed SVL against head size variables, using RMA

regression (null slope = 1).

75

Because allometry of traits within a species may differ from patterns

across taxa, I also examined relative head dimensions interspecifically, across 30

species of Australian snakes from four families. For this analysis I used

published data on mean SVL and head length (head width data were not

available, Shine 1991b); head width data were unavailable. These data were

plotted and allometric coefficients calculated as for the intraspecific analyses.

3. The allometry of snake head size against toad size

δ(snake head size)δ(toad size)

⎛

⎝ ⎜

⎞

⎠ ⎟ .

For the calculations that follow, I assume that this relationship is

isometric (i.e. slope = 1). While it is reasonable to assume that maximum

ingestible prey size scales isometrically with head size, actual prey size taken by

snakes is likely to deviate from isometry. This is simply because most snakes

continue to eat small items throughout life, incorporating larger items into their

diet as they grow (reviewed in Arnold 1993). Thus, the maximum slope of this

allometry will be approximately isometric (i.e. = 1) but the average slope is likely

to show strong positive allometry (i.e. >1) as snake head size increases faster

than average prey size. The implications of this assumption and likely bias are

detailed below (Discussion).

4. The allometry of toad size against toad toxicity

δ(toad size)δ(toad toxicity )

⎛

⎝ ⎜

⎞

⎠ ⎟ .

76

Most toxin in the skin of toads is stored in the large parotoid glands

located above the shoulders (Meyer and Linde 1971). Thus, we can use the size

of the parotoids as an index of the amount of toxin carried by a toad. The

allometry of parotoid size should offer a reasonable surrogate measure of the

allometry of toad toxicity.

I measured all toads in the collection of the Queensland Museum (QM)

to obtain a broad overview of the allometry of toad toxicity across the majority

of their Australian distribution. All toads were measured for snout-ischium

length (SIL), head width (measured across the head at the tympanum), parotoid

length (from above and behind the eye to the most distal point), and parotoid

width (back of shoulders to most distal point). Both head width and SIL were

used as size variables; SIL because it is a common measure of size and head

width because this is the measure that influences the ingestibility of a toad by a

snake. Morphometric variables were log-transformed prior to analysis. I used

Reduced Major Axis (RMA) regression to calculate slope coefficients (null slope

= 1 in all cases).

5. Combined allometry of snake resistance on toad toxicity

δ(snake resistance)δ(toad toxicity )

⎛

⎝ ⎜

⎞

⎠ ⎟ .

Although I identified five different allometric relationships relevant to

the overall relationship between snake body size and vulnerability to toad toxin

(above), my empirical data revealed that one of these did not depart from

isometry (see below). This was snake resistance to toad toxin, which was

77

isometric with mass within and among species. Additionally, we have

assumed that the relationship between snake head width and toad size is

isometric (above). Thus to examine deviations from isometry, we only need to

consider the effects of the allometry of (1) Snake mass on snake length, (2) snake

length on snake head size and (3) toad size on toxicity. That is (see Figure 1):

δ(snake resistance)δ(toad toxicity )

∝δ(snake mass)

δ(snake length)×

δ(snake length)δ(snake head size)

×δ(toad size)

δ(toad toxicity )

The product of the null values for each of these allometries produces a

null value for this allometry equal to three. For each of these component

allometries, I generated an estimate of the slope and the standard error around

that slope. I used a randomisation procedure to arrive at a value of the product

of these three estimates and the associated error. To do this I drew 10,000

random samples from each one of the distributions described by the allometry

estimate and its standard error. This resulted in 10,000 sets of three samples for

which I calculated the product. The resulting distribution estimated both the

product and the standard error (the standard deviation of my sample

distribution) around my estimate. This analysis was conducted in Excel using a

purpose-written procedure.

78

Figure 1. Schematic representation of the calculation of null and observed slopes, and the final allometry - representing the change in snake resistance with changes in toad size. Data shown are for the red-bellied blacksnake (Pseudechis porphyriacus). Dotted lines represent the null slope in all instances and the solid line represents the RMA regression slope.

5

5.5

6

6.5

7

7.5

Ln(s

nake

leng

th)

2.5 3

3.5 4

Ln(snake head length)

3

4

5

6

7

8

Ln(s

nake

mas

s)

6

6.5 7

7.5

Ln(snake length)

2

3

4

5

6

Ln(to

ad le

ngth

)

5 6 7 8 9

Ln(toad parotoid gland length)

Null slope 3.00 x 1.00 x 1.00 = 3.00

RMA slope 3.05 x 1.64 x 0.84 = 4.20

68

79

RESULTS

1. The allometry of snake resistance to toad toxin

δ(snake resistance)δ(snake length)

=δ(snake resistance)

δ(snake mass)×

δ(snake mass)δ(snake length)

⎛

⎝ ⎜

⎞

⎠ ⎟ .

I found no significant correlation between mass-adjusted toxin resistance

and mass for either snake species that I tested (Table 1). Because there was no

significant correlation, ordinary least squares regression was used to estimate

slopes (RMA regression on uncorrelated variables will always yield a slope of

approximately one and thus give a misleading impression of a relationship

(Sokal and Rohlf 1995)). Therefore, for further analysis I have assumed that

resistance to toxin scales isometrically with body mass for all species.

Body mass, however, deviated from isometry in three of the four species

that I tested (Table 2). Both the colubrids exhibited positive allometry for mass

whereas one of the elapids (Hemiaspis) showed strong negative allometry.

Pseudechis showed no evidence of a deviation from isometry. I was unable to

obtain data for Tropidechis and in further calculations I assume isometry for

mass in this species, noting that this assumption does have implications on

further calculations for this species.

2. The allometry of snake length against head size

δ(snake length)δ(snake head size)

⎛

⎝ ⎜

⎞

⎠ ⎟ .

All five snake species exhibited strong positive allometry for body size

(SVL) against head size variables. This relationship was true irrespective of the

80

head size variable considered (Table 3). Thus, head size relative to body size

decreases in larger specimens within each of these species (i.e., body size

increases disproportionately with an increase in head size). Thus, maximal

ingestible prey size relative to snake body size should decline in larger snakes,

even though maximum prey size increases in absolute terms. Hence, all else

being equal, larger snakes would be less capable of ingesting a fatal dose of

toad toxin. The interspecific comparison showed the same pattern as seen

within the species I tested; that is, smaller species had relatively larger heads

and thus, the relationship between body size and head size showed positive

allometry, although of a lesser magnitude to that of the intraspecific

comparison (Table 3).

81

Table 1. Results of the regression of toxin resistance scores against body

mass for two species of Australian snake.

“OLS Coefficient” refers to the ordinary least squares regression coefficient. Neither regression yielded a statistically significant coefficient.

Table 2. RMA regressions of snake mass on snake length.

The null value in this case is three. + or – denote positive and negative allometry respectively, 0 represents no significant deviation from isometry. The number of symbols in each case indicates the statistical significance of each deviation from isometry – one symbol represents p<0.05, two symbols represents p<0.01 and three symbols indicates p<0.001.

Species n OLS Coefficient s.e. r2 pElapidaePseudechis 30 0.012 0.0081 0.079 0.13ColubridaeTropidonophis 24 -0.051 0.0793 0.018 0.53

n Coefficient s.e. r2 AllometryElapidaePseudechis 65 3.05 0.084 0.95 0Hemiaspis 48 2.57 0.091 0.94 ---ColubridaeDendrelaphis 14 3.71 0.325 0.9 +Tropidonophis 104 3.20 0.075 0.94 ++

Snake Mass/SVL

82

Table 3. Results of RMA regressions of SVL (y-axis) against head size variables (x-axis) within five species of Australian snake

and across 30 species of Australian snake.

Allometry coefficients (slope), standard errors and r2 values are given. The ‘Allometry’ column represents the direction and significance of any deviation from isometry. + denotes positive allometry, 0 represents no significant deviation from isometry. The number of symbols in each case indicates the statistical significance of each deviation from isometry – one symbol represents p<0.05, two symbols represents p<0.01 and three symbols indicates p<0.001.

Species n Coefficient s.e. r2 Allometry Coefficient s.e. r2 AllometryElapidaeHemiaspis 156 1.53 0.032 0.91 +++ 1.61 0.042 0.89 +++Pseudechis 107 1.64 0.042 0.93 +++ 1.67 0.04 0.93 +++Tropidechis 135 1.29 0.028 0.94 +++ 1.56 0.042 0.9 +++

ColubridaeDendrelaphis 275 1.44 0.024 0.92 +++ 1.63 0.037 0.86 +++Tropidonophis 143 1.54 0.031 0.94 +++ 1.77 0.045 0.91 +++

Across Species 30 1.16 0.087 0.84 +

Head Length Head Width

72

83

3. The allometry of toad size against toad toxicity

δ(toad size)δ(toad toxicity )

⎛

⎝ ⎜

⎞

⎠ ⎟ .

Measurements of 157 preserved toads revealed significant negative

allometry for body size against parotoid size (Table 4). This negative allometry

of body size was seen regardless of whether parotoid length or parotoid width

was used as the indicator of toad parotoid size and whether SIL or head width

was used as an indicator of toad size (Table 4). Thus, because parotoid size

increases faster than body size, toads become disproportionately more toxic

with increasing body size. This result is not simply the result of changes in skin

morphology with age, as parotoid weight also increases disproportionately

with skin weight (Phillips, unpub. data).

4. Combined allometry of snake resistance on toad toxicity

δ(snake resistance)δ(toad toxicity )

⎛

⎝ ⎜

⎞

⎠ ⎟

Because resistance to toxin scales isometrically with snake body size

(above), we can assess the allometry of selection by toads on snakes based

simply on the remaining three allometric coefficients: Body mass on body

length in snakes, head-width on parotoid size in toads, and body length on

head size in snakes (see Figure 1). Multiplying these coefficients yields a net

allometry that can be compared to a null value of three, to assess the overall

allometry of snake resistance to toads. These overall allometric coefficients

were significantly greater than 3 in all species tested (Table 5). This result

reflects the fact that relative head sizes (and hence, maximal ingestible prey

sizes relative to predator body size) declined with increasing body size, and did

84

so at a faster rate than toad toxicity increased with toad body size. In contrast,

the equivalent interspecific analysis did not deviate from isometry (Table 5),

suggesting that vulnerability to toad ingestion was not affected by a species'

mean adult body size.

85

Table 4. Reduced Major Axis coefficients for regressions of head width and snout-ischium length (SIL) against parotoid size for

cane toads from throughout Queensland.

- denotes negative allometry, 0 represents no significant deviation from isometry. The number of symbols in each case indicates the statistical significance of each deviation from isometry – one symbol represents p<0.05, two symbols represents p<0.01 and three symbols indicates p<0.001.

74

n Coefficient s.e. r2 allometry Coefficient s.e. r2 allometryParotoid Length 157 0.84 0.010 0.98 --- 0.83 0.009 0.98 ---Parotoid Width 157 0.79 0.010 0.97 --- 0.81 0.011 0.98 ---

Head WidthSnout-Ischium Length

86

Table 5. The allometry of the relative toxicity of toads intraspecifically (within each of for seven species of Australian snakes)

and interspecifically (across 30 species of Australian snakes).

Relative resistance allometry was calculated from the product of RMA coefficients of snake mass on length, snake body size on head size and toad body size on parotoid size in toads (from tables 2-4). See text for explanation. The null value for relative resistance allometry thus calculated is three. The last column represents the direction and significance of allometry. + denotes positive allometry, 0 represents isometry. The number of symbols in each case indicates the significance of each deviation from isometry – one symbol represents p<0.05, two symbols represents p<0.01 and three symbols indicates p<0.001. Allometries indicated with an asterix are missing the error variance of the mass/length regression and so the standard error has been underestimated. # represents an assumed value.

Species Coefficient s.e. Coefficient s.e. Coefficient s.e. Coefficient s.e. AllometryElapidaeHemiaspis 2.57 0.091 1.53 0.032 0.84 0.010 3.31 0.141 +Pseudechis 3.05 0.084 1.64 0.042 0.84 0.010 4.21 0.167 +++Tropidechis 3# - 1.29 0.028 0.84 0.010 3.25 0.027 +++*

ColubridaeDendrelaphis 3.71 0.325 1.44 0.024 0.84 0.010 4.48 0.408 +++Tropidonophis 3.20 0.075 1.54 0.031 0.84 0.010 4.14 0.137 +++

Across Species 3# - 1.16 0.087 0.84 0.010 2.92 0.074 0*

Relative Resistance of SnakesSnake mass/length Toad size/toxicitySnake length/head size

75

87

DISCUSSION

Cane toads have had, and will continue to have, a massive impact on

Australia’s native snakes (Phillips et al. 2003). More than 30% of Australia’s

terrestrial snake fauna is at risk and many snakes are likely to die if they ingest

a moderate-sized cane toad. My results indicate that the body size of a snake

plays a significant role in determining the likelihood that it will ingest a fatal

dose of toad toxin. Because a snake's vulnerability often does not scale

isometrically with its body size, the ecological impact of toads is likely to fall

disproportionately upon certain body sizes, and thus upon certain age and/or

sex classes within the predator population. Such age-selective or sex-selective

mortality patterns may have substantial implications for the nature and

magnitude of ecological effects imposed by invading toads. For the species

tested here, small individuals are predicted to be the most heavily impacted by

toads. In my interspecific comparisons, however, the two major allometries

cancel each other out, such that the impact of toads is likely to be independent

of mean adult body size of a species. Overall, my analyses suggest that the

impact of toads will not be influenced by the average body size of a species, but

will be greater on smaller animals within a given predator population.

One of the allometries critical to this conclusion was assumed rather than

measured: the rate at which snake head size increases with prey size. The

assumption of isometry in this case is highly conservative, because in practice,

many snakes continue to take small prey items as they grow (Arnold 1993). The

true value of this allometry will thus be greater than 1.0 and probably

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substantially so. If we allow the coefficient of snake head size on toad size to be

greater than one (as would be the case if a snake continued to eat small prey

items as it grew) the final calculation of snake resistance on toad toxicity would

have an even stronger positive allometry. The assumption in this case has thus

led to a very conservative bias.

It is important to note some other caveats associated with my

conclusions.

1. My data are derived from very broad-scale samples, and may have

combined areas with locally heterogeneous allometries. Intensive

sampling would be needed to assess this possibility.

2. I have relied on museum samples and have thus used many individuals

to span the size range within a species. We cannot be sure that such

patterns (e.g. in relative head size) are simply the result of individual

growth trajectories unless we follow single individuals through

ontogeny (Gould 1966). The patterns observed here could be the

consequence of selection through ontogeny. In fact, it is possible that the

patterns we observe here are a consequence of selection imposed by the

presence of toads – almost all the snakes in the Queensland museum

have come from toad-exposed localities. Further research comparing

snakes from toad-exposed and toad-naïve areas would be necessary to

address this possibility.

3. Many snake species exhibit ontogenetic shifts in prey preference, such

that certain size classes within a population consume a higher proportion

89

of anurans than do other size classes. For example, the Australian

snakes Pseudonaja textilis and Boiga irregularis display an ontogenetic shift

from ectothermic to endothermic prey (Savidge 1988; Shine 1989; Shine

1991c). Such a shift would reinforce the effects of the positive allometry

for snake resistance shown here: that is, small snakes face stronger

selection from toads.

4. Gape-limitation sets an upper but not lower limit to prey sizes, so that

larger snakes often consume prey that are much smaller than their

maximum ingestible prey size (Miller and Mushinsky 1990; Shine 1991d).

Larger snakes thus have a wider choice of prey options and smaller

conspecifics will tend to eat meals that are closer to their maximum

ingestible prey size. Again, this effect will increase the effect of the

resistance on toxicity allometry described here.

5. The measure of toad toxicity used here assumes that the toxicity of toad

toxin does not change through ontogeny. No published information

exists on this issue, and I am forced to assume that toxin composition

remains approximately the same throughout a toad’s lifetime.

Nevertheless, my analyses suggest that selection in this novel predator-

prey system is not distributed equally by size class within a predator species.

Specifically, selection is likely to be more intense against smaller (younger)

individuals within a population. Such a bias may influence rates of recruitment

and mortality, and may eventually lead to a slow population decline because

90

large adults survive but there is minimal recruitment to replace them.

Additionally, differential selection against smaller individuals may translate

into a significant sex bias in vulnerability. Most anuran-eating Australian

snakes exhibit sexual size dimorphism, with the male up to 22% larger than the

female in some species and up to 32% smaller in others (Shine 1994).

Obviously, sex biased mortality also can have strong implications for the

demographic stability and long-term viability of a population. More generally,

it is important to consider the implications of allometry when considering

interactions between species. Whether the interaction is being studied from the

perspective of co-evolution, the impact of invasive species or competition

ecology, the allometry of relevant interacting traits will influence the intensity

of the interaction throughout an organism’s lifetime.

ACKNOWLEDGEMENTS

I would like to thank Greg Brown and the staff at Beatrice Hill research

station for their generous hospitality. Ian Jenkins and Virginia McGrath

provided logistical support and help in the field for which I am very grateful. I

would also like to thank Andrew Amey, Heather Janetski and Patrick Couper

(at the QM) for access to specimens and assistance. Richard Shine, Daniel

Warner, Greg Brown and Jon Webb made helpful comments on a previous

draft. Funding for this work was provided by grants from the Royal Zoological

Society of NSW, the Linnean Society of NSW, The Norman Wettenhall

91

Foundation, the University of Sydney and a grant from the Australian Research

Council.

92

CHAPTER 4

ADAPTING TO AN INVASIVE SPECIES: TOXIC

CANE TOADS INDUCE MORPHOLOGICAL

CHANGE IN AUSTRALIAN SNAKES

93

ABSTRACT

The arrival of invasive species can devastate natural ecosystems, but the long-

term effects of invasion are less clear. If native organisms can adapt to the presence of

the invader, the severity of impact will decline with time. In Australia, invasive cane

toads (Bufo marinus) are highly toxic to most snakes that attempt to eat them.

Because snakes are gape-limited predators with strong negative allometry for head size,

maximum relative prey mass (and thus, the probability of eating a toad large enough to

be fatal) decreases with an increase in snake body size (small snakes eat larger prey

relative to their own body mass) and increases with relatively larger head size (snakes

with relatively larger heads can consume larger prey). Thus, the arrival of toads should

exert selection on snake morphology, favouring an increase in mean body size and a

decrease in relative head size. I tested these predictions with data from preserved

museum specimens of four species of Australian snakes, collected over a period of > 80

years. GIS layers provided data on the duration of toad-exposure for each snake

population, as well as environmental variables (latitude, precipitation, temperature). I

used a model-selection approach to assess whether or not snake morphology has changed

through time due to exposure to toads. As predicted, two toad-vulnerable species

(Pseudechis porphyriacus and Dendrelaphis punctulatus) showed a steady

reduction in gape size and a steady increase in minimum body length with time since

exposure to toads. In contrast, two species at low risk from toads (Hemiaspis signata

and Tropidonophis mairii) showed no consistent change in these morphological traits

as a function of the duration of toad exposure. These results provide strong evidence of

adaptive changes in native predators as a result of the invasion of toxic prey.

94

INTRODUCTION

Human-induced environmental change is the greatest threat to global

biodiversity. Such processes include global climate change, invasive species,

habitat removal, over-harvesting, and altered biogeochemical cycles (Chapin et

al. 2000; Diamond 1989; Novacek and Cleland 2001). These changes have

caused many extinctions (local and global) and will lead to many more, but

whenever the impact is non-random (i.e. selective), there is the potential for

adaptive evolution. Under the right circumstances, adaptive evolution can

happen very rapidly in wild populations. Such “contemporary evolution”

(sensu Stockwell et al. 2003) occurs as a consequence of selection during natural

events (e.g. Grant and Grant 2002; Higgie et al. 2000; Reznick et al. 1996).

Importantly however, it has also been documented from “unnatural” (human-

mediated) events. The classic example of industrial melanism in peppered

moths is the most celebrated case (Grant 1999; Kettlewell 1973), however there

is also clear evidence of adaptive evolution in populations as a consequence of

overfishing (Olsen et al. 2004), global warming (Bradshaw and Holzapfel 2001)

and heavy-metal pollution (Macnair 1987).

These studies highlight the importance of examining the potential for

adaptive change in impacted populations. Doing so can clarify both the nature

of the impact and the response of the affected population. Clearly, a population

exhibiting an adaptive response is more likely to persist in the face of an

environmental change than one that fails to adapt. Invasive species are of

particular interest in this respect, because they constitute a major threat to

95

global biodiversity (Diamond 1989; IUCN 2001; Williamson 1999b). Although

invasive species have caused extinctions (e.g. Ogutu-Ohwayo 1999), they may

also exert non-random selection upon impacted species such that the native

organisms can adapt to the presence of the invader. Although much

evolutionary research has been directed towards invasive species themselves

and how they adapt to new environments, much less research has been

conducted on counter-adaptations by native species (D'Antonio and Kark 2002;

Lee 2002).

Many species of Australian snake have been severely impacted by the

invasion of highly toxic cane toads (Bufo marinus), a conservation problem that

also offers an ideal situation to explore the possibility of an adaptive response

by natives to an invader. Cane toads were introduced into Australia in 1935.

Since then they have spread throughout large areas of Queensland and have

entered the Northern Territory and New South Wales, currently occupying a

range of approximately 1 million square kilometres (Lever 2001). The

ecological impact of toads on the native fauna has been poorly elucidated,

mainly due to logistical difficulties and a lack of baseline data for comparison

(van Dam et al. 2002). Nevertheless, there is a clear inference that the invasion

of the toad has had a massive impact on species of Australian snakes. Toads

are highly toxic and most Australian snakes attempting to eat toads will die. A

recent study suggests that 49 species of snake are potentially impacted by the

toad and that the majority of these species are poorly equipped to deal with a

likely dose of toad toxin (Phillips et al. 2003).

96

Snakes however, are gape-limited predators and thus their ability to

poison themselves by consuming a toad depends upon their head size relative

to their body mass. Thus, within any given population, a snake with a small

head relative to its body mass will be at less risk of ingesting a toad large

enough to kill it, than will a conspecific capable of ingesting a relatively larger

toad. At an intraspecific level, two major factors influence the size of a snake’s

head relative to body mass: the snake’s absolute size (because smaller

individuals have relatively larger heads, as is generally true in most kinds of

organisms: Calder 1984) and relative head size (because even at the same body

length, some individuals will have larger heads than will others). Thus, the risk

of a snake consuming a toad large enough to be fatal will depend upon snake

body size and relative head size (Chapter 3). Accordingly, we can expect that

the arrival of toads will impose selection on the morphology of snakes,

favouring individuals with larger-than-average body sizes and smaller-than-

average relative head sizes.

In this study, I examine morphological variation in four species of

Australian snakes. Two of these taxa (one colubrid, one elapid) are predicted to

face little to no impact from toads, either because they are too small to ingest a

fatal dose or because they have high physiological resistance to toad toxins.

The other two taxa (again, one colubrid and one elapid) are predicted to be

much more vulnerable to toad invasion. I examine variation in body size and

relative head size with reference to environmental variables and time since

exposure to toads. I predict that mean body sizes and/or relative head sizes

97

will have changed through time since toad arrival in the toad-vulnerable

species, but not in the other taxa.

98

METHODS

Study species and collection of morphological data

To ensure phylogenetic independence, I selected distantly related taxa

within each of my two categories (vulnerable and non-vulnerable species). All

four study species feed primarily or exclusively on anurans, and are widely

distributed through the parts of Queensland invaded by cane toads since 1935.

The two highly vulnerable taxa comprised red-bellied blacksnakes (Pseudechis

porphyriacus, Elapidae, n = 99) and green treesnakes (Dendrelaphis punctulatus,

Colubridae, n = 242). Like most Australian snake species, these snakes are

highly susceptible to toad toxins, and will die if they ingest even a relatively

small toad. To consume a fatal dose of toxin, Pseudechis needs only to consume

a toad whose head width is 43% of its own and Dendrelaphis needs only

consume a toad whose head width is 49% of its own (Chapter 1). The two

relatively non-vulnerable species were swampsnakes (Hemiaspis signata,

Elapidae, n = 158) and keelbacks (Tropidonophis mairii, Colubridae, n = 124).

These two taxa are less likely to be severely impacted by cane toads. Hemiaspis

is a relatively small species with an unusually small head, and hence consumes

only very small anurans even relative to its own body mass; to consume a fatal

dose it needs to consume a toad that is 108% of its head-width (Chapter 1). In

contrast, Tropidonophis is a larger species with a normal-sized head, but is

unusual in displaying a high physiological tolerance for toad toxins; to

consume a fatal dose, this species needs to consume a toad that is 185% of its

99

head-width and so feeds readily on toads without severe ill-effects (Covacevich

and Archer 1975; Chapters 1 and 2).

I collected morphological data from preserved specimens held at the

Queensland Museum. Toads have populated more than 60% of Queensland

and all sampled animals came from areas where toads have colonised. I

selected these taxa based on their wide phylogenetic separation, their

abundance, and their differential vulnerability to toads. Each specimen was

measured for snout-vent length (SVL), head length (HL, base of jaw to tip of

snout), head width, (HW, across the head at the junction of supraoculars and

parietals) and gape width (GW, across head at last supralabial). Snout-vent

length was measured to the nearest centimetre with a flexible tape, and all head

measurements were taken to the nearest 0.1mm using dial callipers. Data on

the date and locality of collection for each specimen were taken from museum

registers.

Collection of climatic data

To minimise the chance of a spurious correlation as a consequence of

spatial autocorrelation and to increase my chance of detecting an effect by

reducing error variances, I included the effect of climate and latitude on snake

morphology in my analyses. I derived two climatic layers for Australia using

the program ANUCLIM (Hutchinson et al. 1999) and a digital elevation model

of Australia with 5km grid cells. Snake locality data were laid over the

resultant climate grids in ARCVIEW. The climatic data for each locality were

100

extracted using the ARCVIEW extension BIOCLIMav (Moussalli 2003). I used

two climatic variables that are likely to influence snake morphology: annual

mean temperature (AMT) and annual precipitation (AP).

Collection of data on duration of exposure to toads

More than 2000 records of toad locality and date were available from the

Queensland Museum and from the dataset collected by Floyd et al. (1981).

Sabath et al. (1981) and Easteal et al. (1985) used the latter dataset to map the

spread of toads in Australia, however the results were hand drawn maps of the

toad distribution at five yearly intervals. Improvements in mapping tools since

then (i.e. GIS) allowed me to create a single digital map of far greater accuracy,

which can be used to provide information on the toad expansion at yearly

intervals.

I used linear interpolation of locality dates in ARCVIEW to derive a layer

describing the arrival date of toads. To do this I plotted toad locality data and

identified the earliest record of toads at each site by cumulatively stepping

through the dataset at two-yearly intervals beginning at 1935 (the year that

toads were released in Queensland). Minimum area polygons were drawn

around records selected at each step and records with a later date inside each of

these polygons were deleted. Following this process I used a linear spline to

create a surface describing the timing of toad arrival throughout Queensland.

The resulting surface is shown in Figure 1.

101

Following the derivation of this surface, snake locality records were

plotted and the year of toad arrival at each site was extracted using a spatial

join. For each measured snake I subtracted the year of toad arrival (from the

GIS layer) from the collection year (from the Queensland Museum database) to

yield exposure time (ET) – that is, the number of years a population of snakes

had been exposed to toads at the time a snake was collected. Negative values

for ET (populations that were toad-naïve at the time of collection) were

converted to zero values.

102

Figure 1. GIS layer describing the timing of the cane toad invasion in Queensland, Australia. The extreme western edge of the distribution follows the extent of distribution records in Queensland and may not accurately reflect the actual invasion extent. Data from Floyd et al. (1981) and the specimen register of the Queensland Museum.

103

Data analysis

My primary interest lay in determining the effect of the presence of toads

on body size and relative head size in snakes. However, other variables

doubtless also influence snake morphology and thus, I needed to incorporate

them into the analyses to reduce spurious correlations and so that I could focus

on the residual variance – that potentially explicable by the time since toad

arrival. I predicted that latitude, annual mean temperature, and annual

precipitation may all influence snake morphology and so I included these,

along with exposure time, as variables in a multiple regression. However,

climatic and latitudinal variables were correlated to varying extents so for each

species I calculated the first two principal components of climatic and

latitudinal variables (PC1 and PC2) and used these as independent variables in

my analysis. Two analyses were run for each species. The first used snake

snout-vent length (SVL) as the dependent variable and the second used snake

head-size. Head size (HS) was calculated as the first principal component of

the three head size variables I measured (HL, HW and GW). The multiple

regression for snake head-size also included snake body size (SVL) as a fixed

independent variable as we are only interested in changes in relative head size.

In all cases, correlations between independent variables in the multiple

regression were low. I log-transformed all morphometric variables and the

exposure time variable was mean-centred ( y'= y − y ) prior to analysis (principal

components are already mean centred). Mean-centring (such that the new

mean is zero) ensures that estimated coefficients are informative even in the

104

presence of interactions; this method also reduces colinearity between variables

and their interaction terms (Jaccard and Turrisi 2003).

With three independent, non-fixed variables I had 7 combinations of

primary variables that could produce a model (ignoring interaction terms).

Because I had no a priori knowledge about how each variable would affect

snake morphology and because the total number of models was small, I ran

each of these combinations as a full model and deleted interaction terms if p-

values indicated they were not significant (i.e., p > 0.05). To make model

exploration and interpretation tractable, I only considered first-order

interactions. For each combination of primary variables I thus derived the most

parsimonious reduced model and I calculated the Akaike information criterion

(AIC) value and Akaike weight (wi) for this model. I collected the best set of

models for each species and each independent variable based upon these AIC

values, with models <2 units from the best model (i.e. Δi < 2) retained within the

best set (Burnham and Anderson 2001). All statistical analysis was performed

in JMP (v5).

Some of the models thus selected contained interaction terms. My

primary interest was whether exposure time to toads was an important

influence on snake morphology and, if so, the direction of the effect. The

presence of interaction terms complicates the interpretation of main effects

because the partial coefficient for the main effect of interest depends on the

values of other variables. Mean-centring causes the main effect coefficient to be

calculated for the mean value of interacting variables. However, in all models

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with interaction terms affecting exposure time, I also calculated a range of

coefficients for exposure time using values for the interacting variables that

were two standard deviations above and below their mean.

RESULTS

Principal components

Principal components of climate and latitude for each species are shown

in Table 1. Although results varied between specific datasets, PC1 appears to

capture most of the variation due to latitude with PC2 accounting for most of

the residual variation in precipitation and temperature. PC1 and PC2

accounted for between 88-97% of the variance in the three input variables.

Snake Body Size (SVL)

My models of snout-vent length suggest that, in most species, this trait

was influenced by all three of the independent variables that were used (Table

2). At least one independent variable had a significant effect on snake body size

for all species examined except Tropidonophis. Models for this species exhibited

low Akaike weights and very low r2 values indicating that all the independent

variables explained negligible variance in body size for this species.

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Time since exposure to toads appeared in the best model set for all

species but only indicated a significant effect in Pseudechis and Dendrelaphis. In

both these cases, the effect of ET on snake body size was positive indicating that

these species – the two most vulnerable to toads – increase in average size with

increasing exposure time. An interaction between exposure time and PC2 acts

as a modifier to the partial coefficient of ET for Pseudechis but within the bounds

defined by 0 ± 2σPC2 the partial coefficient did not change sign, remaining

positive.

Across all four species, the predicted impact from toads (i.e. the relative

size of toad that each species would need to consume to ingest a lethal dose, see

methods) was highly negatively correlated with the mean coefficient for the

effect of ET on SVL (r = -0.89, n = 4, n.s.; or r = -1.00, n = 3, p = 0.005 if Pseudechis

is excluded because of the interaction between ET and PC1; Figure 2). This

indicates that the predicted level of impact from toads predicts the rate of

response in body size.

Snake Relative Head-Size

Exposure time and PC1 contributed significantly to variation in snake

relative head-size (Table 3). Models for Tropidonophis again showed very low

Akaike weights (none of the eight models could be considered notably better

than the others) suggesting that none of the independent variables explained

much of the variation in relative head-size in this species. In the remaining

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three species, ET had a significant negative effect on relative head-size. For

Hemiaspis however, this negative effect became positive at low values of PC1, as

a consequence of the interaction between these terms. Thus we are left with an

unequivocally negative effect for only two species, Pseudechis and Dendrelaphis,

the two facing the highest impact from toads. For these species, relative head

size decreases with time since exposure to toads.

Across all species, the predicted impact from toads was highly correlated

with the coefficient for the effect of ET on relative head-size (r = 0.73, n = 4, n.s.;

or r = 0.99, n = 3, p = 0.055 if Hemiaspis is excluded due to the interaction

between ET and PC2; Figure 2) indicating that the relative impact of toads also

affects the rate of response in relative head-size.

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Table 1. Description of the first two principal components of climatic and latitudinal variables for each snake species.

Principal components were constructed from three raw variables; Annual Mean Temperature (AMT), Annual Precipitation (APrecip) and Decimal Latitude (DecLat). Cumulative percent describes the cumulative percentage of the total variance captured by the principal components.

Species Principal Eigenvalue Cumulativecomponent percent AMT Aprecip DecLat

Hemiaspis PC1 1.88 62.69 0.49 0.57 -0.66PC2 0.81 89.83 0.79 -0.60 0.07

Pseudechis PC1 2.14 71.35 0.55 0.58 -0.60PC2 0.53 88.86 0.80 -0.58 0.17

Dendrelaphis PC1 2.03 67.63 0.66 0.32 -0.68PC2 0.89 97.15 -0.28 0.94 0.18

Tropidonophis PC1 1.99 66.17 0.66 0.30 -0.69PC2 0.92 96.71 -0.29 0.95 0.13

Eigenvectors

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Table 2: Parameter estimates for best model sets for multiple regression analyses of body size (SVL) in four species of

Australian snake.

Three independent variables were used: time since exposure to toads (ET) and two principal components (PC1 and PC2) incorporating data on latitude, annual mean temperature and annual precipitation. Parameter estimates significantly different from zero are shown in bold. wi is the Akaike weight of each model and Δi refers to the change in AIC value from the best model.

Species r2 ² i w i Intercept ET ET Range PC1 PC2 InteractionsHemiaspis 0.054 0.00 0.46 0.002 -0.089

0.058 1.38 0.23 0.002 -0.016 -0.0900.054 1.96 0.17 0.003 0.002 -0.029 -0.092

Pseudechis 0.196 0 0.49 0.007 0.016 0.004-0.028 -0.092 ET*PC2, 0.00830.153 0.85 0.32 0.001 0.014

Dendrelaphis 0.077 0.00 0.64 -0.003 0.006 0.0480.077 1.95 0.24 -0.003 0.006 0.048 0.007

Tropidonophis 0.014 0.00 0.27 0.007 -0.0420.023 0.85 0.18 0.006 -0.004 -0.0490.006 0.85 0.18 0.005 -0.0030.000 1.53 0.13 0.006 0.0080.014 1.97 0.10 0.007 -0.042 0.008

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Table 3: Parameter estimates for best model sets for multiple regression analyses of head size in four species of Australian

snake.

Snake body size (SVL) is included as a fixed independent variable as we are only concerned with relative head size. Three independent variables were used: time since exposure to toads (ET) and two principal components (PC1 and PC2) incorporating data on latitude, annual mean temperature and annual precipitation. Parameter estimates significantly different from zero are shown in bold. wi is the Akaike weight of each model and Δi refers to the change in AIC value from the best model.

Species r2 ² i w i Intercept SVL ET ET Range PC1 PC2 InteractionsHemiaspis 0.9402 0.00 0.71 -0.295 5.267 -0.006 -0.017-0.006 0.030 ET*PC1, 0.0042

0.9402 1.97 0.26 -0.293 5.263 -0.006 -0.016-0.006 0.031 -0.007 ET*PC1, 0.0041

Pseudechis 0.9569 0.00 0.26 0.040 3.040 -0.005 -0.0810.9556 0.46 0.20 0.041 3.049 -0.0050.9575 0.83 0.17 0.033 3.034 -0.007 0.036 -0.0870.956 1.69 0.11 0.039 3.045 -0.007 0.0300.9548 1.91 0.10 0.042 -0.079

Dendrelaphis 0.9233 0.00 0.77 0.016 3.937 -0.005 -0.194 0.012 PC1*PC2, 0.0526

Tropidonophis 0.9485 0.00 0.19 -0.049 3.201 0.043 0.034 PC1*PC2, -0.06550.9455 0.07 0.18 -0.051 3.1750.9464 0.35 0.16 -0.052 3.187 0.0350.9491 0.56 0.14 -0.033 3.193 0.002 -0.013-0.017 0.046 0.007 ET*PC1, -0.00560.9477 1.55 0.09 -0.001 3.176 0.001 -0.012-0.016 0.012 ET*PC1, -0.00500.9467 1.67 0.08 -0.050 3.193 0.002 0.0390.9457 1.76 0.08 -0.049 3.178 0.0010.9456 1.99 0.07 -0.051 3.175 0.011

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Pseudechis

Dendralaphis

Hemiaspis

Tropidonophis-0.005

0

0.005

0.01

0.015

0.02

Bod

y si

ze E

T pa

ram

eter

0 50

100

150

200

Pseudechis

Dendralaphis

Hemiaspis

Tropidonophis

-0.008

-0.006

-0.004

-0.002

0

0.002

Hea

d si

ze E

T pa

ram

eter

0 50

100

150

200

Predicted impact

Figure 2. Parameter estimates describing the rate of change in body size and head size for each snake species, plotted against the predicted impact from toads (from Chapter 1). See text for statistical tests.

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DISCUSSION

The results of my modelling strongly support the prediction that

Australian snakes will display morphological adaptations that reduce their

vulnerability to cane toads. The duration of exposure to toads was significantly

associated with changes in mean body size and relative head size in the two

snake species that were identified (from previous work) as being extremely

vulnerable to toads. Importantly, the changes occurring since toads arrived

were in the directions predicted by my hypothesis of size-dependent

vulnerability (i.e., mean body sizes have increased, and relative head sizes have

decreased). In contrast, the two taxa that were identified as being less

vulnerable to toads, showed fewer (or no) significant changes in morphology

associated with the presence of these toxic anurans. My modelling suggested

that exposure to toads may influence head-size for one of these species, but the

exact nature of any such effect remains obscure. There was much less

ambiguity about associations between morphology and the duration of

exposure to toads in the two toad-vulnerable species.

Furthermore, the rate of change in morphology as a consequence of

exposure to toads appears to be linearly related to the predicted level of impact

for each species. This is exactly what we would expect if the rate of response to

toads was driven by the strength of selection imposed by toads, all else being

equal.

In the two toad-vulnerable species (Dendrelaphis and Pseudechis), there is

a significant increase in mean body size in populations sympatric with toads.

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Because small individuals face a much higher risk of fatal poisoning by toads

(Chapter 3), these shifts through time likely result from an ongoing loss of small

individuals from populations exposed to toads. At its simplest, this effect may

be the product of consistently high mortality rates among juvenile snakes in

each generation, such that the population structure in toad-exposed areas is

shifted towards larger, older animals. This would imply strong selection

against small body size, but not necessarily longer-term adaptation. Another

possibility is that the presence of toads elicits a developmental response (such

as increased growth rate through increased food availability) leading to fewer

small individuals. The final possibility is that toads have exerted significant

selection on life-history tactics of the snakes, such that populations in toad-

exposed areas now produce larger (and presumably, fewer) offspring per

clutch, or the young snakes (independent of changes food availability) grow

more rapidly to a size at which they become less vulnerable to toads.

Similar ambiguity in interpretation also occurs with the causal processes

responsible for changes in relative head size. Both the “vulnerable” species,

(Dendrelaphis and Pseudechis), as predicted, showed a significant decrease in

head size associated with time since exposure to toads. This could be due either

to an ongoing impact and an adaptive response to that impact or, alternatively,

to developmental changes in head growth associated with dietary change

subsequent to the arrival of toads. Although early studies reported that relative

head sizes in snakes were not developmentally plastic with respect to

temperature (Arnold and Peterson 1989; Forsman 1996), recent studies provide

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evidence that relative head sizes in snakes can shift as a consequence of

differences in mean prey size (Bonnet et al. 2001; Queraz-Regal and King 1998).

Although I have no direct data to distinguish between these two scenarios

(selection versus plasticity), my data argue against an indirect environmental

effect. Because the effect of exposure time across species is strongly related to

the likelihood of a species ingesting a toad large enough to kill, we can be

confident that the observed effect is a consequence of a direct interaction

between toads and snakes. In other words, the morphological effect is not

driven by changes in prey abundance, prey size or other indirect environmental

effects. Therefore, the morphological changes must be a consequence of (and

probably also a response to) selection against small bodies and large heads.

If we accept that the morphological changes observed are a consequence

of selection then the obvious corollary is whether this selection is resulting in

evolutionary change. Although there are a number of reasons why populations

might not respond to selection (Merila et al. 2001), the simplest is that there is

insufficient heritable variation at the traits of interest. To be confident that the

observed effect here is an evolved response to selection we would need to

understand the heritability of head size and body size (particularly offspring

size) in our species. While these estimates were not available, it is generally

accepted that there is almost always heritable variance available in populations,

particularly for life-history and morphological traits (although see Hoffmann et

al. 2003 for an exception; Roff 1997). Certainly, recent work by Sinervo and

Doughty (1996) showed a very high heritability (0.62) for egg size (i.e. offspring

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size) in a species of lizard, and egg size in birds typically shows high levels of

heritability also (>0.5, Christians 2002). Morphological traits (e.g. head-size),

tend to have heritabilities of around 0.4 (Roff 1997). It seems very unlikely that

snake populations are not responding to this selection due to a lack of heritable

variation.

Additionally, the relatively short generation time of these snake species

snakes (<3 years, Shine 1978) allows more than 20 generations to have elapsed

since initial exposure to toads in some areas. These facts suggest that offspring-

size and head-size are likely to have had both the time and lability to exhibit

evolved change. Further research on possible life-history shifts in toad-exposed

predator populations would be of great interest.

The general approach outlined here, using a combination of museum

time-series and spatial data, could be used to assess morphological change in

any species provided that it is adequately represented in collections and the

spatial timing of the change can be mapped. This highlights not only the

relative ease with which impacts of and responses to environmental change can

be assessed when the relevant data are available, but also the importance of

museums as storehouses for specimen series that can be used to examine

temporal processes.

While the snake species examined here exhibited morphological change

in response to impact from toads, there is also reason to believe that other traits

may be under selection simultaneously. Traits such as prey preference (the

tendency of a snake to eat toads), resistance to toxin and habitat choice are all

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likely to be under selection and may be showing similar adaptive responses.

Chapters five and six assess these possibilities.

The data presented here indicate an adaptive response by a population

impacted by an invasive species. As such these results can be added to a

growing list of studies suggesting rapid adaptation associated with

environmental change. Furthermore, this study demonstrates adaptive change

in response to impacts from an invasive species; it is one of the first studies to

do so (the only other we are aware of being Kiesecker and Blaustein 1997).

Clearly, the potential for impacted populations to adapt needs to be considered

when assessing long-term impacts of environmental change. Assessing the

possibility and extent of an impact associated with an environmental change is

a useful first step (e.g. Thomas et al. 2004), but the next logical step is to assess

the potential for impacted species to adapt. Without such information, we

cannot predict the long-term consequences of environmental change.

ACKNOWLEDGEMENTS

I would like to thank Patrick Couper, Andrew Amey and Heather

Janetski at the Queensland Museum for access to specimens and many cups of

tea. I am also grateful for the GIS advice and technical assistance provided by

Adnan Moussalli and Michael Kearney. Earlier drafts were improved by

comments from Richard Shine, Ary Hoffmann and Greg Brown. The

Australian Research Council provided funding.

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CHAPTER 5

ASSESSING THE POTENTIAL FOR AN

EVOLUTIONARY RESPONSE TO RAPID

ENVIRONMENTAL CHANGE: INVASIVE

TOADS AND AN AUSTRALIAN SNAKE*

* Published as: Phillips B L, Brown G P and Shine, R, 2004. Assessing the potential for an evolutionary response to rapid environmental change: Invasive toads and an Australian snake. Evolutionary Ecology Research 6:

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ABSTRACT

Extinctions are ultimately caused by a change in an organism’s environment.

Species that can adapt are more likely to persist indefinitely in the face of such changes.

I argue that an understanding of the factors encouraging and/or limiting the potential

for adaptation is an important consideration in assessing the long-term outcomes of

environmental change. Such an approach suggests a cohesive way of assessing the

potential for an impact and the long-term consequences of a particular environmental

change. I illustrate this approach with a case study of a native Australian snake (the

keelback, Tropidonophis mairii) faced with the invasion of an extremely toxic prey

item (the cane toad, Bufo marinus). I examine the likely strength of selection, the

heritability of toxin resistance and the likelihood of trade-offs or pre-adaptation. I assess

an internal trade-off (between toxin resistance and locomotor performance) and an

external trade-off (between resistance to the toxin of toads and a native prey species,

Litoria dahlii). My analysis reveals weak selection, high heritability and no trade-offs

in resistance to toad toxin, suggesting that keelbacks are capable of mounting a rapid

adaptive response to invasion by the cane toad.

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INTRODUCTION

Rapid environmental change is currently the biggest threat to global

biodiversity. Such changes include global climate change, invasive species,

habitat removal, altered biogeochemical cycles and others less ubiquitous

(Chapin et al. 2000; Novacek and Cleland 2001). These changes have led to

many extinctions (local and global) and will lead to many more. However,

environmental change is not a new phenomenon – the history of the earth

reveals that many major changes have occurred in the past. Continents have

moved, climate has changed and previously isolated communities have been

thrust together (Morrison and Morrison 1991). What makes anthropogenic

environmental change unusual is its rapidity and ubiquity (Chapin et al. 2000).

Whether a species will persist or go extinct in the face of rapid

environmental change will largely depend upon the species’ ability to adapt to

the change. While the question of whether or not a species will go extinct in the

short term is important, whether or not it will adapt should be the ultimate

concern. Adapting to the new environment is clearly the best “strategy” for

long-term persistence of a species.

This perspective suggests an approach to assessing the potential long-

term impact of an environmental change. Whether a species is likely to adapt

to a given change will depend on several factors including:

1. The per generation strength of selection imposed by the change

(excessively high selection may lead to extinction, whereas lower levels

of selection will encourage adaptive evolution)

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2. The ability of the population to respond to the selective force (i.e. does

the population have sufficient heritable variation at the characters under

selection?)

3. Whether the population has other constraints limiting adaptive potential

(e.g. trade-offs, long generation-time relative to the pace of change).

Invasive species offer an excellent opportunity to study the evolutionary

implications of rapid environmental change for several reasons. First, the

timing and spatial pattern of the invasion is often well documented. Second,

invaders often have a large impact on native species and the mechanism of

impact is often well understood. Finally, invasions of species can happen

naturally in ecosystems and thus it is expected that some capacity to adapt to

them should exist.

In this paper, I describe a case study in which I assess the likelihood of a

rapid adaptive response by a native species to an invader. To do so I assess the

heritability of an adaptive trait and the likelihood of trade-offs (both intrinsic

and extrinsic) acting against adaptive change. This approach is logistically

simpler than documenting short term impacts, and can clarify probable long-

term effects of the invader on the native species.

A case study: cane toads and Australian snakes

The history of the cane toad in Australia represents an excellent example

of the invasion of a dangerous prey item. Cane toads (Bufo marinus) were

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introduced into Australia in 1935. Since then they have spread throughout

large areas of Queensland and have entered the Northern Territory and New

South Wales, currently occupying a range of approximately 1 million square

kilometres (Lever 2001). The exact impact of toads on the native fauna has been

poorly elucidated, mainly due to logistical difficulties and a lack of baseline

data for comparison (van Dam et al. 2002). Nevertheless, there is a very clear

inference that the invasion of the toad has had a massive impact on species of

Australian snakes. A recent study suggests that 49 species of snake are

potentially impacted by the toad and that the majority of these species are

poorly equipped to deal with a likely dose of toad toxin (Phillips et al. 2003).

The main active principal of toad toxin is a class of steroid-derived

compounds known as bufogenins (or bufodienolides, Chen and Kovarikova

1967), unique to toads and biochemically very different from the active peptides

which constitute the main defensive secretions of Australian frogs (Daly and

Witkop 1971; Erspamer et al. 1984; Erspamer et al. 1966). Bufogenins are

extremely toxic, exerting strong cardiac effects. Thus with the arrival of the

cane toad, Australian snakes were faced with a novel and extremely powerful

toxin in potential prey items.

Australian frog-eating snakes are thus under selective pressure to adapt

to the presence of the toad. Four possible adaptive solutions are identifiable

(Brodie and Brodie 1999a):

1. Populations can increase resistance to toad toxin,

2. populations can evolve to avoid/exclude toads as a prey item,

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3. populations can evolve modified habitat preferences (spatial and/or

temporal) such that exposure to toads is reduced and/or

4. populations can evolve shifts in morphology (particularly relative gape

width) that reduce ingestible prey size and hence, exposure to lethal

doses of toxin.

None of these solutions are mutually exclusive and the presence of toads

is likely to drive populations towards all four. Whether each solution is

achievable will depend upon the magnitude of response required (the strength

of selection), the heritable variance for each relevant trait and the presence or

absence of trade-offs or pre-adaptations. However, any one of these solutions,

on its own, may allow a population to persist with toads.

This paper explores the possibility of adaptation by increased resistance

to toad toxin in one species of snake (the keelback, Tropidonophis mairii). In

doing so I examine the strength of selection, the heritable variation and the

possibility of trade-offs or pre-adaptation as a result of co-evolution with

dangerous native prey (Dahl’s aquatic frog, Litoria dahlii).

A relevant pre-adaptation to toad toxin could exist through previous,

long-term exposure to other toxic amphibians. Although the effects of native

frog toxins on Australian snakes are poorly known, most Australian frogs

contain skin toxins, yet many are still eaten by frog-eating snakes (Greer 1997).

One exception to this generality is Litoria dahlii. This frog is extremely toxic to

most sympatric snake species and is abundant in floodplains of tropical

Australia (Madsen and Shine 1994). The only snake species found capable of

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consistently surviving the ingestion of Litoria dahlii was the keelback,

Tropidonophis mairii (Madsen and Shine 1994). This species is also extremely

resistant to the toxin of the cane toad (Phillips et al. 2003).

Examining this system (T. mairii, L. dahlii and B. marinus) allows us to ask

the specific question: Are keelbacks likely to adapt to the presence of toads? To

assess the potential for adaptive response in toxin resistance I:

1. calculate the heritability of toxin resistance (heritability of a trait is the

proportion of the variation in the trait that is directly heritable and thus

gives an indication of the “ability” of the trait to respond to selection),

2. examine possible trade-offs (strong trade-offs may constrain adaptive

options) and

3. examine the possibility of pre-adaptation through co-evolution with

native prey (pre-adaptation potentially reduces the strength of selection).

In making these assessments and using T.mairii as a model, I am also

able to address several questions of general relevance to the adaptive solutions

available to Australian snakes. Firstly, Tropidonophis is highly resistant to both

L. dahlii and Bufo toxins. This raises the possibility that resistance to one toxin

confers resistance to the other (i.e. pre-adaptation to Bufo through co-evolution

with L. dahlii). If this is the case, we might expect a lowered impact on snakes in

areas where L. dahlii is abundant due to similar selective forces imposed by both

toads and L. dahlii.

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Secondly, the relationship between resistance to the two toxins may be

the reverse of that suggested above: adaptation to one toxin may reduce

resistance to the other. The presence of such a trade-off would restrict adaptive

options for Australian snakes faced with the selective force of the presence of

toads — adapting to one dangerous prey would reduce their ability to tolerate

the other dangerous prey taxon.

Thirdly, adaptation to either prey may entail decreased performance in

other traits related to fitness. One such trait might be decreased locomotor

performance associated with higher levels of resistance to the toxin. Brodie and

Brodie (1999b) argue that there is a strong trade-off between resistance to

tetrodotoxin and locomotor performance in garter snakes (i.e., faster snakes are

disproportionately affected by toxin).

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METHODS

Toxin Extraction

I extracted toxin from freshly killed L. dahlii and B. marinus by removing

the dorsal skin (front of parotoids to knees in B. marinus and back of tympanum

to knees in L. dahlii) and drying it at room temperature before weighing. Dried

skins were cut into pieces and placed in a blender with 10 times v/w of 40%

ethanol. Skins were rehydrated for 2 – 3 h before blending. The resulting

liquid was strained and then reduced to 50% of initial volume by evaporation at

room temperature. These preparations were stored at 4°C between use. A

control solution was created using 40% Ethanol evaporated to 50% of its

original volume at room temperature.

Collection of snakes

Gravid female keelbacks were collected by hand near Humpty Doo, in

the Northern Territory. Females were measured and weighed and kept in

captivity until they oviposited (usually within 1 week of capture). At the time

of collection, toads were not yet present in this area; the invasion front was

approximately 300km south and west of Humpty Doo.

Newly laid eggs were measured and weighed and placed on a mixture

of vermiculite and water in a plastic bag to incubate. Thirteen clutches were

split into four incubation treatments — two hydric regimes (wet=1:1 ratio of

vermiculite to water by mass, dry=2:1 ratio) were run orthogonally with two

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temperature regimes (high variation, mean 23.4°C, variance 9.4 and low

variation, mean 23.5°C, variance 5.1). The remaining 13 clutches were incubated

under conditions identical to the wet substrate/low temperature variance

treatment.

Toxin Resistance Assay

Resistance to toxin in newly-hatched keelback snakes was assayed using

post-dose reduction in locomotor performance, a methodology modified from

that of Brodie and Brodie (1990). Individuals were swum along a 2m

swimming trough and were timed with an electronic stopwatch over three

consecutive 50cm segments of the trough with a stopwatch. Animals were

encouraged to swim by tapping them on the tail. Water temperature was

maintained at 23±1oC.

All individuals were weighed before testing to the nearest 0.1 g on a

digital scale. A swimming trial consisted of two consecutive laps of the trough.

This yielded six measurements of swim speed over 50cm of which only the

fastest was retained. All animals (1-2 days post hatching) were initially

subjected to three swim-trials one hour apart. This yielded three maximum

sprint speed times which were averaged to generate the pre-dose estimate of

maximum swim speed (b).

On the following day, snakes were given a specific dose of toxin or

control solution. Dosing was achieved by use of a micropipette attached to a

thin rubber feeding tube. The tube was inserted into the stomach to a depth of

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5cm from the snout before toxin was expelled. Animals were observed for 1

minute following this procedure to ensure the toxin was not regurgitated. Two

swim trials were then undertaken for each individual 30min and 90min post

dosing. A third trial was not run due to the increased possibility of recovery by

this time. Once again only the fastest speed for each trial was taken. This

yielded two time measurements which were averaged to give post-dose swim

speed (a). The percentage reduction in swim speed (%redn) was calculated

from these times using the formula, %redn = 100 x (1-(b/a)).

Locomotor effects

Several experiments were performed. The first two were to determine

whether the administration of toxin caused a reduction in swim speed. For

toad toxin I was also able to assess the effect of increasing dosages on the

decrement in swim speed. Due to limited toxin extract I did not assess this

factor for L. dahlii toxin.

For L. dahlii toxin, two clutches of keelbacks (previously untested, single

incubation treatment, 19 individuals) were used. Each clutch was split into two

groups: Group 1 was given 50μL of L. dahlii toxin and group 2 was given the

same volume of control solution in the manner described above. The post-dose

decrement in swim speed for each individual was recorded. A one factor

ANOVA was used to determine the effects of toxin vs control on the decrement

in swim speed.

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For toad toxin, five clutches of keelbacks (previously untested, single

incubation treatment, 24 individuals) were split by clutch into four groups.

Each group was given either 100μL of control solution, 25, 50 or 100μL of toad

toxin. Decrement in swim speed was assessed as previously described. A one

factor ANOVA was used to assess the effect of dose level on the decrement in

swim speed.

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Toxin resistance trade-offs: Litoria dahlii versus Bufo marinus

To assess the correlation between responses to toad toxin and responses

to L. dahlii toxin within individual snakes I used 15 clutches (from both the

mixed and single incubation treatments) and tested all 115 individuals for

resistance to both toad and L. dahlii toxins. On the day following pre-dose

swimming trials, neonates were tested with 50μL of toad toxin. On the fourth

day following dosing with toad toxin, animals were given 50μL of L. dahlii toxin

and tested in the same manner as for toad toxin.

The order of dosing was not staggered because my first priority was to

develop a large dataset on toad resistance for the heritability analysis (see

below). However I did conduct a small experiment to determine whether being

previously tested for toad toxin affected L. dahlii resistance measures. In this

experiment I split four clutches (18 individuals) into two groups. One group

was tested first for resistance to toad toxin and then for resistance to L.dahlii

toxin. The second group had the testing order reversed. In both groups, four

days elapsed between each test. Data from the individuals tested for toad toxin

resistance first was also used in the heritability analysis (see below).

Data Analysis

Heritability

I measured locomotor decrements of 167 individuals representing 24

clutches (including individuals assessed for resistance to L. dahlii toxin tradeoffs

and toad toxin locomotor effects) to estimate heritability using a full-sib design.

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Full-sib designs are unable to control for the effect of a common maternal

environment, potentially inflating estimates of heritability. Of the 24 clutches

used for heritability analysis, 13 had been incubated under varying conditions

(see above). This process effectively minimised the common incubation

environment of clutches and allowed me to explicitly test the effect of

incubation treatment on toxin resistance. Variance components under the full-

sib design were estimated by restricted maximum likelihood (REML) in SPSS.

REML is the best technique for generating unbiased estimates of variance

parameters with an unbalanced design (Shaw 1987). I used a jacknife approach

(iteratively removing one family) to estimate heritability and its standard error.

Both neonate mass and maternal mass may influence resistance to toad toxin.

Before calculation of heritability, these factors were removed from toxin

resistance data by taking the residuals of a multiple regression of both neonate

mass and maternal mass on %redn.

Locomotor Trade-offs

To assess the possibility of a trade-off between toxin resistance and

locomotor performance, I regressed pre-dose speeds against post-dose speeds

for 15 clutches (115 individuals) tested with both toxins. The gradient of the

line in each case was assessed against null expectations under the following

model:

A = mB + c

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where A is the post-dose speed, B is the pre-dose speed, m is the effect of the

toxin (null: equal to the average proportion of original speed for each toxin) and

c is a constant. This method for inferring the presence of a trade-off differs from

that used previously for similar data (Brodie and Brodie 1999b), with the crucial

difference being in construction of the null slope. My approach is more

conservative, because the null slope under my model will always be less than

the null of 1 assumed by Brodie and Brodie (1999b). Furthermore, the test is

made more conservative because individual mass could not be removed from

the analysis without compromising the logic of calculating the null. The

inclusion of mass will bias the slope upwards, resulting in conservative

inference under this analysis.

A randomisation test was performed to assess the significance of any

deviation of m from that expected under the null hypothesis (Manly 1991). In

this case, a trade-off is evidenced by a slope lower than the null. The effect of

individual mass on response to the standard dose of toxin could not be

removed without sacrificing the ability to calculate a null slope. The effect of

individual mass is likely to bias the observed slope in a positive direction

making this a conservative test for the presence of a trade-off in this system. To

determine the power of the data to detect a deviation from the null slope, the

randomised distribution was advanced by successive decreases of 0.05. For

each of these increments the overlap of the new distribution with non-

significant values of the test distribution was calculated (β). The power was

calculated as 1-β for each increment (Sokal and Rohlf 1995).

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Toxin resistance trade-offs: Litoria dahlii versus Bufo marinus

Because both toxins were tested on each individual in this experiment,

there is no need to adjust post-dose times by pre-dose times. In fact doing so

would cause the measures for the two toxins to become correlated through the

effect of the common pre-dose speed. Thus post-dose times (a) for each toxin

for each individual were compared. Because post-dose speed is likely to be

correlated with both pre-dose speed and snake mass, I removed their effect by

taking the residuals of a multiple regression of pre-dose speed and snake mass

on post-dose speed for each toxin. The Pearson product-moment correlation

coefficient was calculated to determine the strength of the correlation between

L. dahlii and toad toxin post-dose times after correcting for pre-dose speed and

snake mass. A randomisation test (Manly 1991) was utilised to test the

significance of the observed correlation and to assess the power of the data to

detect a correlation of various strengths. The value of the observed correlation

was compared with that of 1999 randomised sets of the data. For non-

significant results, power was assessed as above.

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RESULTS

The effect of toxins

The toad skin extract amounted to 144.7mg of skin per ml of final extract.

The extract of L. dahlii skin equated to a final concentration of 119.0 mg per ml

of final extract. Three snakes of 208 tested died after I gave them a dose of toxin.

Whether this was due to the toxin or simply handling stress is not discernable.

Most animals that were tested recovered over the course of 8 to 24 hours

(Chapter 2).

The mean masses of snakes did not differ significantly between

treatment groups in the L. dahlii toxin experiment (F1,17=0.90 p=0.35). The dose

of toxin (equating to 5.95 mg of dried L. dahlii skin) gave an average reduction

in speed of 27.9% in the toxin group as opposed to a 9.4% increase in speed in

the control group (F1,17=5.88, p=0.026).

A similar pattern was observed for toad toxin. The percentage reduction

increased with increasing dose of toxin (Fig. 1). The difference between doses

was significant overall (F3,20=16.003 p<0.0001). Fisher’s PLSD revealed

significant differences between all dosage levels and the control (p<0.037 in all

cases) and significant differences between each dosage level (p<0.023 in all

cases) except for the 50/100μL comparison (p=0.091). 50μL of toxin gave an

average reduction of 66.1% compared to a reduction of 19.5% in the control

group.

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Figure 1. Percentage reduction in swimming speed in neonate keelbacks across a range of doses of toad toxin. Different individuals were tested at each dosage level. Error bars represent standard errors.

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75

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100u

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Heritability

Multiple regression revealed significant effects of both individual

(neonatal) mass and maternal mass on toxin resistance (mass, p=0.0025;

mother’s mass, p=0.0187; R2=0.058). The residuals from this analysis were used

in subsequent analyses.

A total of 83 eggs hatched in the temperature/moisture experiment.

Residual toxin resistance of the hatchlings was not significantly affected by the

interaction between temperature and moisture treatments (F1,79 = 1.378, p=0.24).

After removal of this factor, I detected no significant effect on residual toxin

resistance of either temperature or moisture treatments (temperature, F1,80=0.94,

p=0.33; moisture, F1,80=0.009, p=0.93).

Restricted maximum likelihood provided an estimate of between-clutch

variance of 43.85 compared with a within-clutch variance of 148.36. This

yielded an estimation of 0.456 for full-sib heritability. Jacknifing provided a

standard error of 0.0488 for this heritability estimate. This high heritability does

indicate partial pseudoreplication in the design of my locomotor experiments

(above), which treated siblings as independent in statistical analyses. As such

these results (particularly for the experiment on the effects of L. dahlii toxin,

which only used two families but for which the heritability is unknown) should

be interpreted with caution.

Locomotor trade-offs

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The average %redn across all snakes tested with 50uL of toad toxin was

62.5%. The null slope of after-speeds on before-speeds was thus 0.375. The

observed slope was 0.325 and was not significantly less than the null (p=0.101).

Power analysis suggested that my data had >95% power to detect a negative

deviation from the null of 0.16 and had >50% power to detect a deviation of

0.08. In comparison, the average percentage reduction across all animals tested

with L. dahlii toxin was 30.8%. In this case, the observed slope of 0.4 was

significantly less than the null of 0.69 (p<0.0005, Figure 2).

Toxin Trade-offs

Testing snakes for resistance to Bufo toxin before testing them for

resistance to L. dahlii toxin had no significant effect on the estimate of resistance

to L. dahlii toxin (F1,16=0.084, p=0.78). Including data from this experiment, a

total of 115 neonate keelbacks were tested for their resistance to both L. dahlii

and toad toxin. Of these, three snakes were unable to swim after dosing with

toad toxin. Because I was unable to record a time for these animals they were

deleted from the analysis rather than being assigned a large but arbitrary time.

Multiple regression revealed significant effects of pre-dose speed and snake

mass on post-dose speed for L. dahlii toxin (pre-dose speed p<0.0001; mass

p=0.0002). For toad toxin however, only pre-dose speed was significant (pre-

dose speed p=0.0045; mass p=0.5689). Using the residuals from these

regressions the product-moment correlation coefficient was calculated and

compared with the null hypothesis of no correlation (H0:r=0, Ha: r≠0). The

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observed correlation for the data was r=0.044 (Fig. 3). The significance of this r-

value was compared with the distribution of r-values obtained from 1999

randomisations of the dataset. 31.35% of random r-values were greater than

that observed, equating to a two-tailed probability of p=0.628. Because this is a

non-significant result it is important to assess the power of the dataset to detect

various levels of r. The power analysis suggested that the data had >95%

power to detect an r-value ≥ 0.35 and >53% power to detect r-values ≥ 0.2.

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Figure 2. Scatterplot of pre-dose vs post-dose swim speeds for snakes given; A) B. marinus toxin and, B) L. dahlii toxin. Null and observed slopes are plotted. The observed slope is significantly smaller only for snakes given L. dahlii toxin p<0.0005.

0

5

10

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20

25

30

0 10 20 30 40

Before Speed (cm/s)

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Aft

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(cm

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Null

A. Bufo

B. L. dahlii

Observed

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Figure 3. Scatterplot post-dose speeds for L. dahlii toxin and B. marinus toxin after correcting for pre-dose speed and body mass.

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resi

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L. dahlii residuals

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DISCUSSION

The effect of toxins

My results show firstly that the methodology provides a sensitive and

non-lethal assay of the effect of toxin on snakes. Both toxins elicited a reduction

in speed in hatchling keelbacks relative to a control dose. For toad toxin at

least, this reduction strongly depended on dosage. Small differences in dosage

rate caused detectable changes in post-dose speed even with relatively small

sample sizes, suggesting that this methodology will be suitable for assaying

variation in response to a standard dose of toxin.

A very similar methodology has been used to assay tetrodotoxin

resistance in several genera of North American snakes, yielding similar results.

That is, increased doses elicited greater reduction in locomotor speed

(Motychak et al. 1999). This same pattern has also been shown for nine other

species of Australian snake exposed to toad toxin (Phillips et al. 2003). While

the mechanism contributing to the decrease in speed is likely different between

toad toxins (action primarily cardiac), tetrodotoxin (a neurotoxin) and L. dahlii

toxins (action under investigation) the basic effect appears to be simply that a

sick animal swims more slowly than a healthy animal. On this basis, it seems

likely that the assay will also be useful in detecting variation in resistance to

most toxins.

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Keelbacks are more resistant to toad toxin than are any of the other

Australian snakes studied to date (9 species across 3 families). It is unlikely that

an individual would be able to ingest a large enough volume of cane toad to

acquire a lethal dose (Phillips et al. 2003) although some instances of keelbacks

apparently dying following the ingestion of a toad have been observed (Ingram

and Covacevich 1990, Phillips, pers. obs.). Irrespective, individuals ingesting

toads are likely to incur short-term locomotor deficits. The long-term effects of

a diet of toads are unknown but there may well be serious fitness costs (Chapter

2) and, in one study, keelbacks maintained exclusively on toads sickened and

died (Shine 1991c). Thus we can expect the imminent presence of toads to exert

at least mild selection on keelbacks.

Heritability

Full-sib heritability estimates also include portions of dominance and

environmental variance and are thus likely to overestimate heritability

(Falconer and Mackay 1996). I have no information on the contribution of

dominance variance and am forced to make the common assumption that it is

small. I was able to remove variance attributable to maternal mass however,

and incubation treatments appeared to have little influence on toxin resistance.

This result suggests that covariance due to common environment will be

minimal in my estimate.

Additionally, it is likely that many of my full-sib groups are, in fact, half-

sib groups. Multiple paternity is common in snakes; multiple matings are often

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observed (including in T. mairii) and molecular data often reveal multiple

paternity (e.g. Barry et al. 1992; McCracken et al. 1999). The effect of these

cryptic half-sib groups is to give an under-estimate of heritability under a full-

sib analysis (Brodie and Garland 1993), making my estimate conservative.

Despite these qualifications, my estimate of heritability for resistance to

toad toxin (0.456 ± 0.1) suggests relatively high levels of heritable variation for

this trait in this population. Heritability estimates for physiological traits are

generally around 0.3 (Roff 1997). Lack of a heritable basis to variation is thus

unlikely to be a major impediment to adaptive change in this trait.

Locomotor trade-offs

Locomotor trade-offs were not detected for response to toad toxin, but I

found a strong trade-off between locomotor performance and resistance to L.

dahlii toxin. This strong trade-off in locomotor performance for L. dahlii toxin

contrasts with the minimal or non-existent trade-off detected for toad toxin.

The Humpty Doo keelback population was naïve to toads at the time of testing

but has been exposed to L. dahlii for many generations. It is tempting to

speculate that selection for increased resistance to L. dahlii toxin has resulted in

resistance levels being driven upwards by selection to the point where they are

balanced by trade-offs. The lack of trade-off for toad resistance may be due

simply to the fact that resistance to toads is not yet under directional selection

in this population.

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Toxin trade-offs

The power analysis suggests that the data have excellent power to detect

reasonable levels of correlation between responses to the two toxins. My results

thus clearly indicate a very poor correlation (if any) between an individual

snakes’s response to toad and L. dahlii toxins. Given that the action of the two

toxins is likely to be different this result is not altogether surprising. However,

it suggests that selection for resistance to L. dahlii toxin will not pre-adapt a

population for resistance to toad toxin. The lack of correlation also suggests

that there is likely to be no trade-off between resistance to these two toxins.

That is, selection for increased resistance to one toxin does not equate to

reduced ability to deal with the other toxin, at least in keelbacks. In this system

at least, snakes appear free to evolve resistance to toad toxin without sacrificing

resistance to the toxins of native prey.

While pre-adaptation in terms of resistance to toad toxin is unlikely,

sympatry with an extremely toxic prey item may cause selection for other traits

that will pre-adapt a population to the invasion of the toad. Most important of

these is a change in prey preference or foraging tactics – the presence of L. dahlii

may have lead to the evolution of “fussiness” in attack and feeding responses.

Such evolved predator tactics may reduce the impact of the toad or allow a

more rapid recovery following the toad invasion. Further research is required

to assess these possibilities.

The potential for adaptation

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Humpty Doo keelbacks exhibit significant and relatively high heritability

for resistance to toad toxin. Thus, in the absence of trade-offs or constraints,

toxin resistance is likely to respond to selection. I found no evidence for either

a trade-off or pre-adaptation due to the presence of the native frog, Litoria dahlii.

Thus, snakes sympatric with L. dahlii are unlikely to have either an advantage

or disadvantage in terms of an adaptive increase in toxin resistance.

Additionally, in keelbacks there appears to be no intrinsic trade-off between

locomotor performance and resistance to toad toxin suggesting no impediment

to adaptive change through this factor either.

Thus I find evidence for heritable variation, probable mild selection and

no obvious trade-offs for resistance to toad toxin in keelbacks. The clear

inference is that this species is capable of adaptively responding to toad

invasion by increasing toxin resistance. The relatively short generation time of

keelbacks (<18 mo: Brown and Shine 2002) suggests that rapid adaptive

response will be possible in this species. In keeping with this prediction,

keelbacks (but not most other frog-eating snake species) remain abundant in

areas that toads have occupied for > 50 years.

While rapid adaptive response is possible for toxin resistance, it remains

possible that one or more of the other three traits listed (Introduction) may be

more labile and respond more rapidly to the presence of toads. The current

results simply mean that an adaptive response is possible in keelbacks. To

persist through environmental change, one adaptive solution is all that is

necessary.

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My approach, using experimental data, is a level of abstraction away

from direct measurement of population-level impact but clarifies the likelihood

of an adaptive response. Also, by examining factors that may be influential

(e.g. the presence of a native, toxic frog) we can make a broader level of

inference, beyond the particular study species. For example the lack of

relationship between resistance to the two toxins in the keelback suggests that a

similar lack of relationship is likely in other species (i.e. the toxins are very

different). Thus the presence of the toxic native is likely to have little bearing

on resistance to toad toxin for other snake species in the community.

Examining the problem of environmental change in this manner places

individual studies within the broader unifying framework of evolutionary

theory. Ultimately, this should allow meaningful comparisons to be made

across species and systems. Such a unified approach is necessary for furthered

understanding of the impacts of invasive species and the consequences of

environmental change generally (Caughley 1994; D'Antonio and Kark 2002).

ACKNOWLEDGEMENTS

I am grateful to the staff at Beatrice Hills farm for their generous hospitality.

Greg Brown collected morphological data for mothers and set up the incubation

treatments. Richard Shine, Greg Brown, Stuart Pimm and two anonymous

referees provided comments on an earlier draft. I thank Megan Higgie and

Mark Blows for advice on REML.

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133

CHAPTER 6

AN INVASIVE SPECIES CAUSES RAPID

ADAPTIVE CHANGE IN AN IMPACTED

NATIVE SPECIES: BLACKSNAKES AND CANE

TOADS IN AUSTRALIA

134

ABSTRACT

Rapid environmental change as a consequence of human activities has led to a

heightened extinction rate. It is increasingly apparent however, that some species are

able to adapt to these anthropogenic modifications. Additionally, the pace of change

varies with the specific impact considered, such that some categories of environmental

change may be more likely to elicit an evolved response in impacted populations.

Invasive species represent an instantaneous environmental change (i.e. they are either

present or absent). Understanding the potential for species to adapt to such a

modification potentially sheds light on the likelihood of species adapting to more gradual

changes (such as global climate warming). Here I examine Australian blacksnakes

(Pseudechis porphyriacus) to determine whether they have mounted an adaptive

response to the invasion of a lethally toxic prey item, the cane toad (Bufo marinus).

Snakes from toad-exposed localities showed increased resistance to toad toxin and a

decreased preference for toads as prey. In separate laboratory experiments I was unable

to teach naïve snakes to avoid toxic prey, nor was I able to increase snake resistance to

toad toxin through repeated sub-lethal doses. These observations and experiments

strongly suggest that black snakes have exhibited an evolved response to the presence of

toads. As toads only arrived in Australia in 1935, these evolved responses are rapid,

occurring in less than 23 snake generations.

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INTRODUCTION

The current biodiversity crisis and high species extinction rate is a

consequence of rapid environmental change, mediated by human activities

(Ehrlich 1995). Overharvesting, invasive species and altered climate are all

examples of significant environmental change. Because such modifications are

usually directional, we can expect non-random impact on species affected by a

specific change. Non-random impact, of course, equates to Darwinian

selection. Therefore, many of the environmental changes mediated by humans

exert strong selection on affected species. It is increasingly apparent that

evolutionary responses to strong selection can occur rapidly, on time scales

traditionally thought of as “ecological” (Hendry and Kinnison 1999; Stockwell

et al. 2003; Thompson 1998).

Understanding which species are likely to adapt to a given change

provides valuable information for the setting of conservation priorities.

Additionally, some categories of environmental change may facilitate adaptive

responses by impacted species. In short, understanding the potential for

evolution to affect the outcomes of anthropogenic impact allows us to further

refine conservation priorities and strategies (Ashley et al. 2003).

Invasive species are a major concern to conservationists (Mack et al.

2000). They are second only to climate change in ubiquity and there are several

examples of invasive species driving native species to extinction (e.g. Ogutu-

Ohwayo 1999). The arrival of an invasive species represents a much more

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abrupt environmental change than do more gradual processes, such as global

warming. Thus, from an evolutionary perspective, invasive species often

represent an instantaneous and strong change in the selective environment. If

species can adapt to such instantaneous change, it seems more likely that they

can also respond to relatively slow changes (such as climate warming). What is

the possibility that native species can adapt to an invader? Obviously this will

depend, among other things, upon the strength of the selection pressure and

(given the instantaneous nature of the change) the amount of heritable variation

at traits mediating the impact in native species. Here I examine the possibility

of an adaptive response by an Australian snake to the invasion of a toxic prey

item, the cane toad.

Toads were introduced into Australia in 1935 (Lever 2001). They are

highly toxic and the principal toxin is unique to toads (Chen and Kovarikova

1967). As Australia has no native species of bufonids (Cogger 2000; Lutz 1971)

the arrival of toads presents a highly toxic potential prey item to a naïve

predator fauna. Australian snakes, in particular, have faced massive impacts in

the presence of toads. More than 49 species of snake have the potential to be

impacted, and almost all of these are poorly equipped to survive a likely dose

of toad toxin (Phillips et al. 2003). The arrival of toads thus imposes selection

on at least three traits: physiological resistance to toad toxin, prey preference

(the tendency to eat toads) and the morphology of impacted snake species

(relatively small-headed snakes are less likely to be capable of consuming a

toxic dose: Phillips and Shine submitted-b).

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One native species facing a high impact from toads is the red-bellied

blacksnake (Pseudechis porphyriacus). This relatively large elapid feeds primarily

on frogs and has very low resistance to toad toxin (Phillips et al. 2003).

Anecdotal reports indicate massive declines in black snake populations

following the arrival of toads (Covacevich and Archer 1975; Fearn 2003; Phillips

and Fitzgerald 2004; Rayward 1974). The current distribution of the black snake

includes areas of sympatry and allopatry with toads. Here I compare toad-

naïve and toad-exposed populations of black snakes to examine the possibility

that these snakes display an adaptive response to the presence of toads.

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METHODS

Comparing toad-exposed and toad-naïve populations

1) Prey Preference.

Twelve black snakes were collected from each of the following

categories: (1) populations exposed to toads for 40-60 years (Childers and

Agnes Waters, Qld) and, (2) toad-naïve snakes from two populations; one

immediately adjacent to the expanding toad front (Casino, NSW) and one

approximately 300km from the front (Macquarie Marshes, NSW). After a

minimum of 3 weeks in captivity, each snake was offered a frog (Limnodynastes

peronii – a widespread species, sympatric with snakes at all collection localities)

and a toad in random order, three days apart. Prey items were offered to the

snakes freshly killed, to eliminate behavioural differences between the prey and

so I could remove the parotoid glands from the toads; not doing so would likely

have resulted in the death of snakes during the course of the experiment. In

each case the snakes were left undisturbed for 24 hours, after which I recorded

whether or not the prey item had been eaten. Differences in numbers of prey

consumed between toad-exposed and toad-naïve localities were compared

using Fisher’s exact test.

2) Toxin resistance.

Fourteen snakes from toad-exposed localities and 24 from toad-naïve

localities were tested for resistance to toad toxin. These snakes included fifteen

139

tested previously for prey preference (5 from toad-exposed areas and 10 from

toad-naïve areas) and additional animals collected from a range of localities

within each category. Toad-exposed snakes came from a range of populations

representing exposure times of 5-60 years.

Resistance to toad toxin was assayed in a manner identical to that

reported in Phillips et al. (2003), using the same toxin extract. Toad toxin was

obtained from skins of freshly killed cane toads collected from the Lismore area

(northern NSW). A single extraction of toad toxin was made for the entire

study, to remove among-toad variance in toxicity and to accurately control

dosing. The resistance of individual snakes to toad toxin was assayed using the

decrement in swimming speed following a dose of toxin (methodology

modified from Brodie and Brodie 1990). A large percentage reduction (%redn)

in swimming speed indicates a lower resistance to toxin than a smaller

reduction in swimming speed.

Each snake was given a dose of 80μg of toad skin per gram of body mass

(a dose previously calculated to be non-lethal but provide measurable

reductions in speed: Chapter 1). Dosing was achieved with a feeding tube

attached to a syringe or calibrated micropipette, inserted into the snake’s

stomach to a depth of 30% of its snout-vent length.

Differences in %redn were compared between exposed and naïve

populations using a t-test. Additionally, I examined the relationship between

%redn and time since exposure to toads in toad-exposed populations by simple

linear regression.

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Learning and acquired resistance experiments

Observed differences in resistance and prey preference either could be

acquired during a snake’s lifetime, or be the result of changes in gene frequency

due to adaptive evolution. Following the observation of differences between

exposed and naïve populations, I exposed captive snakes to toad toxins to

evaluate the possibility of either a learnt response or acquired resistance in

naïve black snakes.

1) Learning.

Sixteen snakes were collected from toad-naïve areas. Snakes were kept

in captivity for a month before the learning trial commenced. Each snake was

offered two prey types, three times each in random order. I used laboratory

mice and a lizard (Eulamprus tympanum, Scincidae; an allopatric and hence

novel species to all my snakes) as my two prey types. Prey items were offered

to each snake dead and one at a time. Snakes were left undisturbed for 24

hours, after which I recorded whether or not the prey item had been eaten.

Successive feedings were four days apart. Following these six feeding events, a

prey item was introduced that contained a high but sublethal dose of toad toxin

(120μg of toad skin per g of body mass, 65% of LD50; a dose that reduces a

snake’s locomotor ability by more than 50% for >24 hours). Eight snakes

received a toxin-laced lizard and eight snakes received a toxin-laced mouse.

Following the consumption of this prey item, I repeated the previous feeding

141

schedule, recording the number of prey consumed. At the conclusion of the

trials I counted the number of prey items of each type that were consumed

before and after the dose of toxin and used repeated measure ANOVA to assess

differences in these scores.

142

2) Acquired resistance.

Twenty snakes from toad-naïve populations (including those used in the

learning trials) were assessed for the possibility of acquired resistance. Every

five days, I administered either a dose of toxin or a dose of water to each snake.

This was repeated four times so that half the snakes received four doses of toxin

and half received four doses of water. Dosing method was identical to that

described for assessment of differences in resistance to toxin between exposed

and naïve populations (dosing rate: 60μg per gram of body weight, dose = 32%

of the LD50). Four weeks after the last dose, all snakes were assessed for

resistance to toxin in a manner identical to that used to compare exposed and

naïve populations. Following the calculation of %redn scores, I compared the

toxin and control groups and also compared all snakes in this experiment with

snakes assessed for resistance from naïve localities. This allowed me to test

whether repeated doses of toxin increase resistance and also whether a single

dose of toxin (sixteen snakes had received at least one dose of toxin in the

learning experiment) could increase resistance.

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RESULTS

Comparing toad-exposed and toad-naïve populations

1) Prey preference.

All the snakes, from each exposure category, ate the frog that was

offered to them (Figure 1). Exactly half (i.e. six) of the snakes from toad-naïve

populations consumed a toad, and no toads were consumed by snakes from

toad-exposed populations – a significant difference (χ2 = 5.6, df = 1, p = 0.014;

Figure 1).

2) Toxin resistance.

A significant difference between exposed and naïve populations was

detected with naïve populations exhibiting higher %redn and thus, lower

resistance to toad toxin (exposed mean = 16%, naïve mean = 32%; two-tailed t-

test, unequal variances; t = 3.112, df = 24, p = 0.005, Figure 2). Additionally,

%redn scores decreased (and hence level of resistance increased) with exposure

time (F1,12 = 7.72, p = 0.017, Figure 3). Importantly, the y-intercept of the

regression of %redn on exposure time yielded a value of 34%, a very similar

value to the mean %redn of naïve populations (32%).

144

0

25

50

75

100

Per

cent

age

eate

n

yes no

Exposure to toads

Toads eaten (%)

Frogs eaten (%)

Figure 1. The percentage of snakes from toad-exposed and toad-naïve populations willing to eat a toad or a frog. Twelve snakes from each exposure category were used. No snake from a toad-exposed locality would consume a toad. Error bar represents one standard error.

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-25

0

25

50

75

100

Red

uctio

n in

spe

ed (%

)

Exposure category

Naive Exposed

Figure 2. Resistance to toad toxin in toad-exposed and toad-naïve populations. Open circles represent individual snakes, cross bars represent the mean for each category. A large percentage reduction in speed indicates low resistance to toxin. Hence snakes from toad-exposed populations exhibited higher resistance to toad toxin.

-20

0

20

40

60

Red

uctio

n in

spe

ed (%

)

0 10 20 30 40 50 60 70

Exposure time (years)

Figure 3. Resistance to toad toxin as a function of the time a population has been exposed to toads. A large percentage represents a low resistance. Hence resistance to toad toxin increases with increasing exposure time.

146

Learning and acquired resistance experiments

1) Learning.

To compare numbers of prey eaten before and after the administration of

toxin required a repeated measures ANOVA with prey type and toxin/control

(treatment) as orthogonal factors and number of prey eaten (before and after

administration of the treatment) as the dependent variables.

This analysis revealed that snakes fed a toxic prey item showed no inclination

to avoid the prey item in further feeding opportunities (F1,27 = 0.007, p = 0.93,

Figure 4). This effect was independent of prey type (interaction, F1,26 = 0.1678, p

= 0.69) and suggests that a black snake surviving an encounter with a toad is

unlikely to avoid toads in the future.

2) Acquired resistance.

After four doses of toxin over the period of a month there was no change

in the level of resistance exhibited by snakes when compared with a control

group (F1,18 = 2.95, p = 0.10) and mean %redn in swimming speed was higher in

the toxin-exposed group (42%), than the control group (22%); that is, snakes

given several doses of toad toxin tended to exhibit lower rather than higher

resistance to toxin. Because most of the snakes involved in this experiment had

previously been exposed to a single dose of toxin during the learning

experiment, it is possible that this single dose changed their resistance. To

assess this possibility I compared the resistance of all the snakes in this

experiment (n=20, mean %redn = 32.16) to the resistance of all snakes

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previously tested from naïve populations (n=24, mean %redn = 32.42).

ANOVA revealed no significant difference in %redn between these two

samples either (F1,42 = 0.0016, p = 0.97).

0

1

2

3

4

Mea

n nu

mbe

r of p

rey

take

n

Before After

Toxin

Control

Figure 4. Can snakes learn to avoid toxic prey? The number of prey taken by snakes before and after exposure to a toxic prey item. Two prey types were used, only one of which was laced with toxin for each snake (see methods).

148

DISCUSSION

My results show differences between blacksnakes from toad-exposed

versus toad-naïve populations in their physiological resistance to toad toxin

and their willingness to eat toads. Importantly, both of these differences are in

an adaptive direction; that is, we see an increased resistance to toxin and

lowered preference for consuming toads in toad-exposed populations. These

changes either could be plastic changes (acquired within an individual snake’s

lifetime) or evolved changes (a genetically-coded response to the strong

selection imposed by toads). To discriminate between these possibilities, I

attempted to elicit acquired responses in toad-naïve captive snakes. However, I

found no evidence that snakes can learn to avoid a toxic prey item, nor that

they can acquire physiological resistance to toad toxin. My inability to elicit

acquired responses in either of these two traits suggests that the differences

observed between toad-exposed and toad-naïve populations are due to

adaptation rather than phenotypic plasticity.

The interpretation of an evolved response depends on the degree to

which my acquired resistance and learning experiments mimic reality. In

designing these experiments I operated under the premise that most snakes will

be lucky to survive an encounter with a large toad (Phillips et al. 2003, Seabrook

and Fitzgerald unpub. data, Shine unpub. data). That is, a black snake that eats

a large toad is likely to die. The window of sub-lethal toxin effect is relatively

narrow (Phillips et al. 2003), such that few toads will be large enough to cause

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illness but small enough to be non-lethal. Because it is unlikely, therefore, that

an individual snake will have several chances to learn avoidance, my learning

experiment was based on a single noxious encounter. The fact that black snakes

did not learn to avoid toxic prey is surprising given that Burghardt et al. (1973)

and Terrick et al. (1995) elicited learnt aversion in gartersnakes (Thamnophis

spp.) after a single toxic encounter. Gartersnakes are often sympatric with toxic

amphibians and may have evolved this learning capacity as a response to toxic

prey (Brodie and Brodie 1999a). In contrast, blacksnakes are not known to be

sympatric with any naturally-occurring, dangerously toxic prey and thus may

have been under little or no selection to learn avoidance. It remains possible,

however, that some cue specific to toads could increase a snake’s tendency to

learn avoidance (much as aposematic colouration appears to enhance learned

avoidance in gartersnakes (Terrick et al. 1995)). If this is the case, learning

could occur but my experiment wouldn’t elicit it. Given that Australian snakes

have no evolutionary history with toads, or their toxins, and toads are not

aposematically coloured, it seems unlikely (but not impossible) that a toad-

specific cue would increase learning ability. Nevertheless, if we assume that

naïve blacksnakes are unable to learn avoidance despite a near-lethal encounter,

the observation of strong differences in prey preference between toad-naïve and

toad-exposed populations implies an evolved response. Whether this response

is a congenital disposition to avoid toads or an evolved ability to learn from a

single noxious encounter remains to be seen. Prey-preference has a highly

heritable basis in gartersnakes (Arnold 1981; Arnold 1992) and so it seems likely

150

that this trait will also have high heritability in blacksnakes. Additionally, it is

important to note that there is variation for the tendency to eat toads in naïve

populations of blacksnakes (only half of the naïve snakes consumed a toad, Fig.

1). The heritability of learning ability, however, has never been measured and I

detected little variation in this trait in naïve populations of black snakes

(although sample sizes were relatively small). Further work exploring the basis

of the change in prey preference would be enlightening.

While most snakes probably only get a single chance with a large toad, it

is possible that they could consume several small toads at different times with

minimal ill effect, and acquire an increased level of resistance through an

immune or other physiological response. The acquired resistance experiment

thus exposed snakes to four sub-lethal doses. One month after these dosings,

toxin-exposed snakes were no better equipped to deal with toad toxin. Again,

this result suggests that the differences between exposed and naïve populations

are probably evolved rather than acquired.

In light of the prey-preference results (no snake from toad-exposed areas

consumed a toad), it superficially seems paradoxical that I also detected

evidence of selection on toxin resistance. This difference may be the result of

historically strong selection when toads first arrived and toad-avoidance had

yet to become fixed (or nearly fixed) in the population. Alternatively, if the

prey preference result reflects an evolved ability to learn avoidance of toxic

prey, there may be ongoing selection on toxin resistance. Additionally, spatial

and temporal variation in relative prey abundances and/or levels of snake

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preadaptation, would also lead to concurrent evolution in resistance and prey

preference (Brodie and Brodie 1999a; Gomulkiewicz et al. 2000).

Recent work also documents a reduction in the relative head size of

blacksnakes (and hence their ability to eat large prey items) as a consequence of

exposure to toads (Chapter 4). Thus it appears that blacksnakes show adaptive

change in multiple traits in response to the presence of toads. Given that the

generation time of blacksnakes is approximately 3 years (Shine 1978) and that

toads have only been present in Australia for <70 years, these adaptive changes

are rapid (occurring in < 23 generations).

The current study is one of the first to demonstrate adaptation by a

native species in response to an impact of conservation concern from an

invasive. As such it places invasive species into a growing list of environmental

changes to which adaptive response has been demonstrated (Stockwell et al.

2003). That some species at least are capable of mounting such a rapid adaptive

response to an instantaneous change in the environment suggests that other,

more gradual changes (such as global warming) may also elicit adaptive

responses rather than extinction. This result highlights the importance of

considering the potential for adaptation when predicting the long-term impact

of environmental change and also highlights the need to maximise the adaptive

potential of managed species through the maintenance of large, genetically

diverse populations.

ACKNOWLEDGEMENTS

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This study would not have been possible without the assistance of many

people who helped with the collection of snakes (a difficult undertaking in

toad-exposed areas). Foremost among these is Ian Jenkins with additional help

from Eric Bateman, Julie Dickson, David Fouche, Richard Ghamroui, Jeff

Hayter, Andrew Hugall, Ray Jones, Michael Kearney, Amanda Lane, Clare

Morrison, Adnan Moussalli, Luke Shoo, Devi Stuart-Fox, Eric Vanderduys and

Michael Wall. Steve Phillips and Jai Thomas assisted with husbandry and

maintenance. Richard Shine and Michael Wall reviewed and improved an

earlier version of this chapter. Funding was provided by grants from the

Australian Research Council, The Royal Zoological Society of NSW, The Royal

Linnean Society of NSW and the Norman Wettenhall Foundation.

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CHAPTER 7

THE MORPHOLOGY, AND HENCE IMPACT, OF

AN INVASIVE SPECIES (THE CANE TOAD,

BUFO MARINUS) CHANGES WITH TIME SINCE

COLONISATION.

154

ABSTRACT

It is increasingly apparent that the success of a particular invasive species may

be related to its phenotypic lability. Despite often strong founder effects and

concomitant reductions in genetic diversity, many successful invasive species still

exhibit adaptive change in response to their new environment. Successful invaders are

also a major global conservation problem. To understand the likely long-term impacts

of a particular invader it is critical that long-term changes in the invader’s phenotype

are assessed, as these changes may well influence the level of impact the invader has on

native species. Here I examine morphological change, as a consequence of time since

colonisation and other spatial factors, in the cane toad (Bufo marinus). Cane toads are

highly toxic and have had a major impact on Australian native predators since they

were introduced in 1935; naïve predators die attempting to consume them. The amount

of toxin that a predator is exposed to depends upon both the body size of the toad and

also the relative toxicity of the toad (here measured by the relative size of the toad’s

parotoid glands). Using multiple regression and a model-selection approach, I

discovered that both toad size and relative toxicity decrease with time since colonisation.

This shows first, that toads, like many other successful invasives, exhibit high

phenotypic lability. Additionally, this result strongly suggests that the impact from

toads on predators decreases as a consequence of time since colonisation.

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INTRODUCTION

Invasive species are increasingly being used as model systems within

which to study adaptation and plasticity (Lee 2002). Paradoxically, many

invasive species appear to exhibit adaptive responses to new environments

despite what superficially appear to be strong founder effects and concomitant

reductions in genetic diversity. Given that successful invaders represent a

biased sample of all potential invaders, it seems increasingly likely that the

adaptive potential of invaders is strongly linked to their probability of success

in a new environment (Kolbe et al. 2004; Parker et al. 2003; Simons 2003;

Stockwell et al. 2003; Thompson 1998). Thus, understanding the evolutionary

processes underlying successful invasions is of both broad theoretical and

practical interest.

Invasive species also often have large impacts on native communities

and, given their ubiquity, are regarded as a major threat to global biodiversity

(Mack et al. 2000; Williamson 1996). Thus, examining change in invasive

species may also give us insights into the long-term impact of a particular

invader: by examining change in traits that mediate an invader’s impact we can

understand how the level of impact changes through time.

Cane toads (Bufo marinus) are extremely successful invaders throughout

the Caribbean and Pacific, having successfully invaded more than 20 countries

to date (Lever 2001). Their colonisation history from their native range in South

America is very well documented (Easteal 1981): by the time toads were

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brought to Australia in 1935, they had already undergone several founder

events and genetic diversity as measured at allozyme and microsatellite

markers was much reduced (Estoup et al. 2001). They are a large, toxic anuran,

and since their initial release they have spread to occupy more than one million

square kilometres of the Australian continent (Lever 2001). Among other

potential impacts, toads are known to massively impact native terrestrial

predators, which are naïve to toad toxin and die attempting to ingest them

(Burnett 1997; Covacevich and Archer 1975; Oakwood 2003; Phillips et al. 2003;

Smith and Phillips submitted). The dose of toxin that a predator will be

exposed to in an interaction (and hence the risk of death to the predator)

depends upon two factors: the body size and the relative toxicity of the toad.

Large toads contain greater quantities toxin than small toads and even at the

same body size, some individuals will be more toxic than others (Chapter 3).

While size can be measured directly, relative toxicity may be more complicated.

Conveniently, most toxin in the skin of toads is stored in the large parotoid

glands located above the shoulders (Meyer and Linde 1971). Thus, we can use

the size of the parotoids as an index of the amount of toxin carried by a toad.

Here I examine changes in these two aspects of toad morphology, body

size and relative parotoid size, as a consequence of time since colonisation.

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METHODS

Collection of morphological data

I measured all of the 140 cane toads present in the collection of the

Queensland Museum. This specimen series represented animals collected since

1935 (the year of toad arrival). Each individual was measured for snout-

ischium length (SIL), parotoid gland length (PL) and parotoid gland width

(PW). Information on collection locality and date of collection was also taken

from the museum database.

Collection of data on time since colonisation

More than 2000 records of toad locality and date were available from the

Queensland Museum and from the dataset collected by Floyd et al. (1981).

Sabath et al. (1981) and Easteal et al. (1985) used the latter dataset to map the

spread of toads in Australia, however the results were hand drawn maps of the

toad distribution at five yearly intervals. Improvements in mapping tools since

then (i.e. GIS) allowed me to create a single digital map, of far greater accuracy,

which can be used to provide information on the toad expansion at yearly

intervals. To do this I used linear interpolation of locality dates in ARCVIEW to

derive a layer describing the arrival date of toads; details of the process can be

found in Chapter 4.

Following the derivation of this surface, the Queensland Museum toad

locality records were plotted and the year of toad arrival at each site was

158

extracted. For each measured toad I subtracted the year of toad arrival (from

the GIS layer) from collection year (from the Queensland Museum database) to

yield time since colonisation (TSC) – that is, the number of years a population of

toads had been present in an area at the time a toad was collected.

Collection of climatic data

Because my data has a spatial component, I attempted to account for as

many spatially varying factors as possible to reduce the potential for a spurious

correlation and to reduce error variances. In addition to Latitude (DecLat, from

the Queensland Museum database), I derived several climatic layers for

Australia using the program ANUCLIM (Hutchinson et al. 1999) and a digital

elevation model of Australia with 0.05º grid cells. Toad locality data were laid

over the resultant climate grids in ARCVIEW and I extracted the climatic data

for each locality using the ARCVIEW extension BIOCLIMav (Moussalli 2003). I

used several climatic variables that are likely to influence toad morphology:

annual mean temperature (AMT), minimum temperature of the coldest period

(MinTempCP), annual precipitation (APrecip), precipitation seasonality

(PrecipSeas), moisture index seasonality (MoisIndSeas) and annual mean

humidity at 3pm (AMHumid).

Data analysis

I examined the effect of TSC on toad morphology using a model

selection approach. By testing the relative information content of all possible

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models, I determined whether TSC was an important factor (i.e. was it present

in the best model/s) and also selected the most parsimonious model (i.e. the

model that explained the most variance with the least number of factors)

describing toad morphology.

Because many of the climatic variables were correlated to varying

degrees, and to reduce the number of factors, I calculated the first three

principal components of climatic and latitude variables. Two analyses were

run: The first used toad snout-ischium length (SIL) as the dependent variable

and the second used toad parotoid size. Parotoid size (PS) was calculated as the

first principal component of the two parotoid size variables I measured (PL and

PW). The multiple regression for toad parotoid size also included toad body

size (SIL) as a fixed independent variable as I was only interested in changes in

relative parotoid size. I log-transformed all variables prior to the calculation of

principal components and the TSC variable was mean-centred ( y'= y − y ) prior

to analysis. Mean-centring (such that the new mean is zero) ensures that

estimated coefficients are informative even in the presence of interactions; this

method also reduces colinearity between variables and their interaction terms

(Jaccard and Turrisi 2003). Mean centring was not necessary for principal

components because their mean was already zero.

With four independent, non-fixed variables I had 15 combinations of

primary variables that could produce a model (ignoring interaction terms). To

make model exploration and interpretation tractable I only examined first order

interactions between factors. Each of the 15 combinations was run as a full

160

model and I deleted interaction terms if p-values indicated they were not

significant (i.e., p > 0.05). For each combination of primary variables I thus

derived the most parsimonious reduced model and I calculated the Akaike

information criterion (AIC) value for this model. I collected the best set of

models for each species and each independent variable based upon these AIC

values, with models <2 units from the best model (i.e. Δi < 2) retained within the

best set (Burnham and Anderson 2001). All statistical analyses were performed

in JMP (v5).

Some of the models thus selected contained interaction terms. My

primary interest was whether time had an important influence on toad

morphology and, if so, the direction of the effect. The presence of interaction

terms complicates the interpretation of main effects because the partial

coefficient for the main effect of interest depends on the values of other

variables. Mean-centring causes the main effect coefficient to be calculated for

the mean value of interacting variables. However, in all models with

interaction terms affecting the coefficient of TSC, I also calculated a range of

coefficients using values for the interacting variables that were two standard

deviations above and below their mean.

161

162

RESULTS

Calculation of multivariate model components

The first three principal components of climatic and latitude variables

accounted for almost 95% of the variation in these seven factors (Table 1).

Eigenvectors indicate that PCClimLat1 is principally a latitude/temperature

axis and captures most of the latitudinal variation in the environmental factors.

PCClimLat2 appears to be principally a precipitation axis, capturing the

resultant variation in humidity and moisture index seasonality. PCClimLat3 is

more difficult to interpret but may be capturing altitudinal variation in the

environmental variables.

For toad parotoid size (PS), the first principal component of PW and PL

captured more than 99% of the variation in both these variables (reflecting their

strong correlation) with equal loadings on both.

Toad snout-ischium length

The single best model describing toad SIL included all but one of the

independent variables and accounted for more than 20% of the variation in toad

SIL. Time since colonisation (TSC) has a negative effect on toad SIL in this

model, however its effect is modified by an interaction with PCClimLat2 – the

precipitation axis: At high values of PCClimLat2 (approx. two standard

deviations above the mean) the negative effect of TSC is reduced or reversed.

163

Functionally, this means that toad body size decreases less rapidly after

colonisation in wetter (higher precipitation) areas than in drier areas.

Table 1: Results of principal component analysis of climate and latitude

variables for cane toad records from the Queensland Museum.

Abbreviations are as follows: AMT, Annual mean temperature; Aprecip, Annual precipitation; PrecipSeas, Precipitation seasonality; MoisIndSeas, Moisture index seasonality; MinTempCP, Minimum temperature of the coldest period; AMHumid, Annual mean humidity at 3pm; DecLat, Decimal latitude. Eigenvector weights > 0.3 are shown in bold.

Table 2: Parameter estimates for the best models describing toad body size

(SIL) and relative parotoid size as a function of climate and latitude

(PCClimLat1, 2 and 3) and time since colonisation (TSC).

Principal components:PCClimLat1 PCClimLat2 PCClimLat3

Eigenvalue 3.96 2.40 0.27Cumulative percent 56.57 90.88 94.68

Eigenvectors Ln(AMT) 0.463 -0.082 0.610Ln(Aprecip) 0.130 0.612 -0.097Ln(PrecipSeas) 0.471 -0.147 -0.393Ln(MoisIndexSeas) 0.359 -0.400 -0.195Ln(MinTempCP) 0.433 0.254 0.457Ln(AMHumid) 0.088 0.610 -0.222Ln(DecLat) -0.471 -0.017 0.410

Factor Snout-ischium length Parotoid size

Intercept -0.0157 -0.0011

Snout-ischium length - 2.5853PCClimLat1 -0.0491 0.0275PCClimLat2 0.0874 0.0220PCClimLat3 - -0.1182Time since colonisation -1.5584 -0.4617

PC1 x PC2 - -0.0314PC2 x TSC 0.5273 -

r2 0.204 0.985TSC range -3.18 - 0.07 -

164

Coefficients significantly different from zero are shown in bold. For brevity, PCClimLat has been further abbreviated to PC for interaction terms and time since colonisation has been abbreviated to TSC. TSC range is the partial coefficient calculated for TSC based on values of interacting variables two standard deviations from the mean.

Toad parotoid size

The single best model describing variation in relative parotoid size was

also complex, involving all independent variables (Table 2). In this model time

since colonisation had a negative effect on relative parotoid size, unmodified by

interactions.

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DISCUSSION

My results show a significant effect of climate, latitude and time since

colonisation on toad morphology. Interestingly, both snout-ischium length

(SIL) and relative parotoid size models indicate that time since colonisation

(TSC) has a significant effect on toad morphology, independent of other

spatially varying factors. Time since colonisation appears to be associated with

a reduction in both overall size of toads and in the relative size of their parotoid

glands. That toad morphology should be so strongly influenced by recent

colonisation history is an important result and a reminder of the importance of

recent history in invasion events.

For toad body size, my model contained an interaction that modified the

magnitude (and eventual direction) of the effect of TSC. In extremely high

rainfall areas (around two standard deviations above average), the negative

effect of TSC on toad size is reduced or reversed.

Why do large, big-glanded individuals become less common through

time? There are two possible reasons why this may be so and they are not

mutually exclusive. First, toads may change their environment through time

(e.g. by depleting food resources) such that attaining large size and maintaining

costly structures (which poison glands presumably are) becomes increasingly

difficult. Freeland (1986) found that toad densities and body condition were

lower in long-established populations compared with newly established

populations. Interestingly however, the change in body condition was not

166

associated with a difference in the size of fat bodies, indicating that lack of food

was not necessarily driving the change. Unfortunately, Freeland’s (1986) study

was conducted over a single year, so it is possible that local variation in

conditions in that year contributed to the patterns he observed. Certainly,

studies by Alford and colleagues (1995) in the same areas a few years later

reported the opposite pattern in density.

The second possible explanation for changes in toad morphology with

TSC is that toads may exhibit adaptive change to a new environment (e.g.

reduced predation pressure, following the extirpation of predator populations)

where large body sizes and poison glands confer little or no selective

advantage. There are certainly many instances documenting apparent

adaptation by invaders to a novel environment (e.g. Grosholz and Ruiz 2003;

Losos et al. 1997; Simberloff et al. 2000). While my data are unable to

unequivocally support either scenario, they do show that Australian toads are

morphologically labile. This lability may be an important factor in their success

as an invader. In fact it may be that most successful invaders are successful

partly because they exhibit phenotypic lability in response to new

environments (Lee 2002; Parker et al. 2003).

Irrespective of the exact mechanism causing morphological change in

toads, either scenario assumes a cost associated with producing a large body or

a large parotoid gland. Areas of high precipitation are undoubtedly favourable

environments for cane toads (given that their native range encompasses the

Amazon Basin) and it seems logical that physiological costs associated with

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developing a large body or large glands will be minimised in favourable

environments. This reasoning probably explains the presence of an interaction

between TSC and precipitation in my SIL model.

My data also suggest that toad body size and relative parotoid size

exhibit latitudinal clines (PCClimLat1 is primarily a latitude/temperature axis).

Interestingly, the models for body size indicate an increase in toad body size

with increasing latitude/decreasing temperature, in line with Bergmann’s rule

for endotherms and against the general pattern in body size clines for

ectotherms (although only insects, squamate reptiles and turtles have been

examined in any detail, Ashton and Feldman 2003; Mousseau 1997).

Additionally, there is no support for an interaction between time since

colonisation and latitude/temperature. If the cline in toad body sizes was an

evolved effect, as has been demonstrated for latitudinal clines in Drosophila

body size (Gilchrist et al. 2004; Huey et al. 2000), we would expect an

interaction between TSC and PCClimLat1. The absence of this interaction

suggests that the cline in toad body sizes shown here is a consequence of

developmental plasticity rather than evolution, as the cline is present

irrespective of the length of time since colonisation.

Because the quantity of toxin carried by a toad is a factor of the toad’s

body size and relative parotoid size, reductions in these traits will translate into

a reduced impact on predators. Thus, the change in morphology associated

with TSC indicates that, except in the wettest areas of the toad’s current

distribution, the level of impact imposed by toads on predators will decrease

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with time since colonisation. Whether this level of impact remains lowered will

depend upon the exact mechanism driving the morphological change.

Nevertheless, my results show the importance of considering the possibility of

rapid phenotypic change in an invader when assessing the long-term impact of

the invader on a native community. It is increasingly becoming apparent that

successful invaders are successful partly because they exhibit phenotypic

lability in response to a new environment. If this is true, then examining rapid

phenotypic change at traits influencing an invader’s impact on natives is an

important prerequisite to understanding the long-term impact of an invader.

ACKNOWLEDGEMENTS

I thank Patrick Couper, Andrew Amey and Heather Janetski at the

Queensland museum for access to specimens, entertaining discussion and cups

of tea. Richard Shine provided useful discussion and comments on an earlier

draft. Funding was provided by the Australian Research Council and the

Norman Wettenhall Foundation.

169

CHAPTER 8

SPATIAL AND TEMPORAL VARIATION IN THE

MORPHOLOGY (AND THUS, PREDICTED

IMPACT) OF THE INVASIVE CANE TOAD

(BUFO MARINUS) IN AUSTRALIA

170

ABSTRACT

The impact of an invasive species is unlikely to be uniform in space or time, due

to variation in key traits of the invader (e.g. morphology, physiology, behaviour) as well

as in resilience of the local ecosystem. The low genetic diversity typical of invasive

species suggests that much of the variation in an invading taxon (and thus, in its

ecological impact) is likely to be generated by the environment and recent colonisation

history. I use a model-selection approach to describe effects of the environment and

colonisation history on key morphological traits of an invader (the cane toad, Bufo

marinus). These “key traits” (body size and relative toxicity) mediate the impact of

toads on Australian native predators, which often die as a consequence of ingesting a

fatal dose of toad toxin. Measurements of museum specimens collected over > 60 years

and across the state of Queensland show that seasonal variation in toad body size (in

turn, due to seasonal recruitment) effectively swamps much of the spatial variance in

this trait. However, relative toxicity of toads (measured by size of the poison glands

relative to body size) showed strong spatial variation and little seasonal variation.

Thus, the risk to a native predator ingesting a toad will vary on both spatial and

temporal scales. For native predators capable of eating a wide range of toad sizes (e.g.,

quolls, varanid lizards), seasonal variation in overall toad size is likely to be the most

significant predictor of risk (and thus, ecological impact of toads). In contrast, gape-

limited predators restricted to a specific range of toad sizes (such as snakes) will be most

strongly affected by the relative toxicity of toads. Gape-limited predators will thus

experience strong spatial variation in risk from toad consumption.

171

INTRODUCTION

Invasive species are a major threat to global biodiversity (IUCN 2001;

Mack et al. 2000). Human-assisted transport and environmental change has

broken down biogeographical barriers in many parts of the world leading to

species invasions and irrevocable changes to native communities. Although

some invaders have little impact on natural ecosystems, other taxa exert

varying levels of impact, and this process can lead to local or global extinction

(Ogutu-Ohwayo 1999; Williamson 1996).

Clearly, the magnitude of impact of an invasive species will be different

not only for different native species, but also may vary among populations of

any given species. Even if we restrict analysis to interactions between a single

predator-prey species-pair (for example), the magnitude of impact will be far

from constant through space and time. For example, some populations of

native taxa may be more or less vulnerable to the threat posed by the invader;

and similarly, populations of the invader may differ in traits (such as body size

or toxicity) that determine the intensity of their effect on the native system.

Such spatial and temporal variance in traits of the invader may be generated

either by plasticity or local adaptation; and if invading populations vary in such

ways, this variation needs to be considered by ecologists and conservation

biologists attempting to understand or manage the system (Parker et al. 2003).

The combined impact of founder effects and drift at initial small

population sizes is expected to leave invaders with low levels of genetic

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diversity (Allendorf and Lundquist 2003). Although this attribute (among

others) has encouraged researchers to utilise invasive species as model systems

in evolution (Lee 2002), it also suggests that much of the variation within a

population of invaders is likely to be of environmental (rather than genetic)

origin. Hence, much of the potential variance in impact from an invader is also

likely to be generated by the local environment. Understanding how the

environment affects an invader and how this flows on to affect impacted native

species has the potential to clarify much of the spatial and temporal variation in

the impact of invasive species.

For any invading taxon, the effect of the environment on key traits may

depend not only on the environment itself, but also on how long the invader

has been present in an area (Chapter 7). A longer duration of time in a given

area may allow the invading population to adapt; and also, may allow time for

the invader to modify the environment (e.g., by depressing resource levels).

Thus, any analysis of the effect of the environment on an invasive species

should incorporate information on the duration of time for which the invader

has been present. Factors such as distributional range (Elton 1958), resource

availability and coadaptation with native communities (Thompson 1998) may

all be strongly influenced by recent history in invasive species.

The above ideas suggest the following approach to assessing the spatial

and temporal variation in impact by an invading taxon:

1. First, we need to understand the mechanism of impact, in order to

identify what traits of the invader (and possibly the native species)

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mediate that impact. These traits may be behavioural, morphological,

and/or physiological. The important challenge is to identify traits for

which changes are likely to modify the level of impact.

2. These traits need to be sampled at different locations and at different

times. At each location, environmental variables that may influence the

trait (including colonisation history) should also be sampled.

3. The effects of the environment and time on each trait should be modelled

so that the resulting model can be mapped onto the area of interest.

Below I describe a case study examining temporal and spatial variation

in predicted impact from an invasive species as a consequence of

environmental factors, including colonisation history.

Case study: Australian predators and the invasive cane toad

Cane toads are large, toxic anurans native to Central and South America.

Introduced into Australia in 1935, toads have spread throughout large areas of

Queensland and have recently entered the Northern Territory and New South

Wales. This invasive taxon currently occupies a range of more than one million

square kilometres within Australia (Lever 2001). Toads reach extremely high

densities in suitable habitat (densities >2000 per hectare have been recorded,

Freeland 1986) and have three types of potential impact on Australian native

species (Freeland 1987): 1) predation on small animals, 2) competition for food

and/or shelter resources and, 3) because they are extremely toxic and the toxin

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is novel to Australian predators, toads are likely to kill most native predators

that attempt to eat them.

Despite these three possibilities, the ecological impact of toads on the

native Australian fauna has been poorly elucidated, mainly due to logistical

difficulties and a lack of baseline data for comparison (van Dam et al. 2002).

Nevertheless, there is mounting evidence that the bufonid invasion has severely

impacted populations of native predators that attempt to eat toads and die as a

consequence of ingesting the toxin. Specifically, evidence is accumulating that

toads have had a severe impact on native snakes (Covacevich and Archer 1975;

Fearn 2003; Phillips et al. 2003; Phillips and Fitzgerald 2004; Webb et al. in

press), varanid lizards (Burnett 1997; Smith and Phillips submitted) and quolls

(a medium sized marsupial carnivore, Burnett 1997; Oakwood 2003; van Dam et

al. 2002) all of which die attempting to eat toads. It is this mode of impact that I

deal with in the current analysis.

For a predator consuming a toad, the dose of toxin will depend upon

two factors: the body size of the toad (bigger toads carry more toxin, Phillips

and Shine submitted-b), and the relative toxicity of the toad (even at the same

size, some toads will be more toxic than others: Chapter 3). These factors will

be of different importance to different kinds of predator taxa. Some species

(e.g. quolls and large varanids) are capable of capturing and consuming toads

of a very wide range of body sizes. This is because either they are not gape-

limited (quolls) or they are partially gape-limited but so large that they are able

to ingest even the biggest toad (large varanids). For these species, toad size will

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be the most important variable determining the probability of the predator's

survival after it has ingested a toad. For smaller species of gape-limited

predators, however, such as most snakes and small varanid lizards, the size

range of toads that can be ingested is limited by the predator's ability to

swallow large prey (Arnold 1993; Shine 1991d). Thus, such predators will

capture and consume only relatively small toads. For these predators, maximal

toad body size within a population will be irrelevant to risk; instead, the

vulnerability of such taxa will strongly depend upon the relative toxicity of the

toad (i.e., its toxicity relative to body size). Thus we need to examine two traits

of cane toads, variation in body size and variation in relative toxicity. Body size

can be measured directly, using snout-ischium length (SIL) as a measure.

Relative toxicity is more difficult to assess but conveniently, most toxin in the

skin of toads is stored in the large parotoid glands located above the shoulders

(Meyer and Linde 1971). Thus, we can use the size of the parotoids as an index

of the amount of toxin carried by a toad.

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METHODS

Collection of morphological data

I measured the 140 cane toads present in the collection of the Queensland

Museum. This specimen series represented animals collected since 1935 (the

year of toad arrival). Each individual was measured for snout-ischium length

(SIL), parotoid gland length (PL) and parotoid gland width (PW). Information

on collection locality and date of collection were also taken from the museum

database.

Collection of climatic data

I used several climatic variables that plausibly might influence toad

morphology: annual mean temperature (AMT), minimum temperature of the

coldest period (MinTempCP), annual precipitation (APrecip), precipitation

seasonality (PrecipSeas), moisture index seasonality (MoisIndSeas) and annual

mean humidity at 1500 h (AMHumid). Each of these variables was obtained

from climate layers I derived using the program ANUCLIM (Hutchinson et al.

1999) and a digital elevation model of Australia with 0.05º grid cells. Toad

locality data were laid over the resultant climate grids in ARCVIEW. I

extracted climatic data for each locality using the ARCVIEW extension

BIOCLIMav (Moussalli 2003).

Collection of data on time since colonisation

177

More than 2000 records of toad locality and date were available from the

Queensland Museum and from the dataset collected by Floyd et al. (1981).

These records were used to interpolate a map surface describing the arrival date

of toads throughout Queensland (see Phillips and Shine submitted-a for details,

Fig. 1). Following the derivation of this surface, the Queensland Museum toad

locality records were plotted and the year of toad arrival at each site was

extracted. For each measured toad I subtracted the year of toad arrival (from

the GIS layer) from the collection year (from the Queensland Museum

database) to yield time since colonisation (TSC) – that is, the number of years a

population of toads had been present in an area at the time a toad was

collected.

Data analysis

My primary aim was to derive a model that would capture as much of

the effect of the environment on toad morphology as possible. For this

purpose, I adopted a model selection approach. By testing the relative

information content of all possible models, I selected the most parsimonious

model (i.e. the model that explained the most variance with the least number of

factors) describing environmental variation in toad morphology.

Once I selected the best model I used it to map predicted body size and

relative parotoid size within the toad’s distribution. I predicted that collection

month (the month in which a toad was collected), latitude, annual mean

temperature, annual precipitation, minimum temperature of the coldest period,

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precipitation seasonality, moisture index seasonality and annual mean

humidity may all influence toad morphology. Because many of the climatic

variables were correlated to varying degrees, and to reduce the number of

factors, I calculated the first three principal components of climatic and latitude

variables. These principal components were used, along with TSC and

collection month (CM) as independent variables in a multiple regression. I

predicted that parotoid size and body size might vary throughout the year,

hence the inclusion of CM. However if morphology does vary with CM, it is

likely to be a quadratic relationship, therefore I also included a CM2 term. Two

analyses were run for each species. The first used toad snout-ischium length

(SIL) as the dependent variable and the second used toad parotoid size.

Parotoid size (PS) was calculated as the first principal component of the two

parotoid size variables I measured (PL and PW). The multiple regression for

toad parotoid size also included toad body size (SIL) as a fixed independent

variable as I was only interested in changes in relative parotoid size. I log-

transformed all variables prior to the calculation of principal components.

Those variables not involved in principal components (TSC and CM) were

mean-centred ( y'= y − y ) prior to analysis. Mean-centring (such that the new

mean is zero) ensures that estimated coefficients are informative even in the

presence of interactions; this method also reduces colinearity between variables

and their interaction terms (Jaccard and Turrisi 2003).

With six independent, non-fixed variables I had 62 combinations of

primary variables that could produce a model (ignoring interaction terms). To

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make model exploration and interpretation tractable I only examined first order

interactions between factors. Each of these 62 combinations was run as a full

model and I deleted interaction terms if p-values indicated they were not

significant (i.e., p > 0.05). For each combination of primary variables I thus

derived the most parsimonious reduced model and I calculated the Akaike

information criterion (AIC) value for this model. I collected the best set of

models for each species and each independent variable based upon these AIC

values, with models <2 units from the best model (i.e. Δi < 2) retained within the

best set (Burnham and Anderson 2001). All statistical analyses were performed

in JMP (v5).

Following the derivation of the best model, I reconstructed this model in

Arcview, using the component factors, to map environmental variation in toad

morphology within the toad’s Queensland range. Because collection month

was an important factor in both models (see results) I calculated each model for

each month (1-12). I then averaged the results across the 12 months (to describe

the spatial variation in morphology) and also calculated the coefficient of

variation (to describe the relative amount of seasonal variation). Because the

relative parotoid size model also included toad body size (SIL) as a factor I

calculated all maps for the relative parotoid size of a 60mm toad.

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Figure 1. GIS layer describing the timing (by year) of the cane toad invasion in Queensland, Australia. The extreme western edge of the distribution follows the extent of distribution records in Queensland and may not accurately reflect the actual invasion extent. Data from Floyd et al. (1981) and the specimen register of the Queensland Museum.

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RESULTS

Calculation of multivariate model components

The first three principal components of climatic and latitude variables

accounted for almost 95% of the variation in these seven factors (Table 1).

Eigenvectors indicate that PCClimLat1 is principally a latitude/temperature

axis and captures most of the latitudinal variation in the environmental factors.

PCClimLat2 appears to be principally a precipitation axis, capturing the

resultant variation in humidity and moisture index seasonality. PCClimLat3 is

more difficult to interpret but may be capturing altitudinal variation in the

environmental variables.

For toad parotoid size (PS), the first principal component of PW and PL

captured more than 99% of the variation in both these variables (reflecting their

strong correlation) with equal loadings on both.

Toad snout-ischium length

The best model describing spatial and temporal variation in toad body

size included all six independent variables and accounted for more than 38% of

the variation in toad SIL. However I was unable to exclude the possibility of a

second model in which PCClimLat3 was absent (Table 2).

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Table 1: Results of principal component analysis of climate and latitude

variables for cane toad records from the Queensland Museum.

Abbreviations are as follows: AMT, Annual mean temperature; Aprecip, Annual precipitation; PrecipSeas, Precipitation seasonality; MoisIndSeas, Moisture index seasonality; MinTempCP, Minimum temperature of the coldest period; AMHumid, Annual mean humidity at 3pm; DecLat, Decimal latitude. Eigenvector weights > 0.3 are shown in bold.

Table 2: Parameter estimates for the best models describing toad body size

(SIL) and relative parotoid size as a function of climate (PCClimLat1, 2 and

3), time since colonisation (TSC) and collection month (CM).

Principal components:PCClimLat1 PCClimLat2 PCClimLat3

Eigenvalue 3.96 2.40 0.27Cumulative percent 56.57 90.88 94.68

Eigenvectors Ln(AMT) 0.463 -0.082 0.610Ln(Aprecip) 0.130 0.612 -0.097Ln(PrecipSeas) 0.471 -0.147 -0.393Ln(MoisIndexSeas) 0.359 -0.400 -0.195Ln(MinTempCP) 0.433 0.254 0.457Ln(AMHumid) 0.088 0.610 -0.222Ln(DecLat) -0.471 -0.017 0.410

Factors Parotoid sizeModel 1 Model 2 Model 1

Intercept -0.183 -0.146 -0.009

SIL - - 2.529PCClimLat1 -0.050 -0.052 0.039PCClimLat2 0.161 0.125 0.049PCClimLat3 -0.195 - -0.131TSC -1.290 -1.547 -0.370CM -0.001 0.005 0.009CM2 0.013 0.011 -

PC1*PC2 -0.064 - -0.044PC1*CM 0.015 0.018 -PC1*TSC - - 0.176PC2*TSC 1.013 0.735 0.279PC2*CM -0.031 -0.029 -PC3*CM - - 0.024

Δi 0 1.926 0

r2 0.387 0.355 0.987

Snout-ischium length

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Coefficients significantly different from zero are shown in bold. For brevity, PCClimLat has been further abbreviated to PC for interaction terms. Δi represents the difference in AIC value from the best model.

184

Toad parotoid size

The single best model describing variation in relative parotoid size was

also complex, involving six independent variables (Table 2). All the climatic

variables and both TSC and CM were present, although CM2 was absent. The

single best model accounted for 98.7% of the variation in absolute parotoid size.

However, almost all of this variance (98%) was explained simply by the size

variable, SIL (i.e., bigger toads have bigger parotoid glands). Thus, my model

explained 0.7% of the remaining 2% of variance. Hence, factors other than SIL

(i.e. climatic and spatial factors) accounted for 35% of the residual variation in

parotoid size (i.e. relative parotoid size).

Mapping spatial variation in toad size and relative parotoid size

In both cases, my models explained approximately 35% of the variation

in the variable of interest (toad size or relative parotoid size). However, in both

cases the model is complex, with several interaction terms making

interpretation difficult. To examine the spatial pattern of body size and relative

parotoid size variation, I translated my best models into GIS layers derived

from the appropriate independent variables.

a) Toad body size.

Despite significant spatial variation in average toad size, seasonal

variation was even greater (Fig. 2). A high coefficient of variation across most

of Queensland points to strong seasonal fluctuations in average body size,

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particularly in the north. This most likely reflects a massive, seasonal influx of

young toads each year, due to seasonal recruitment. The model predicts that

average body size of toads will be highest in the Wet Tropics and south-east

Queensland, with low to moderate levels of seasonal variation in those areas.

b) Relative parotoid size.

The spatial pattern in predicted relative parotoid size was complex but

did not exhibit the massive seasonality apparent in the SIL model (Fig. 3). High

seasonal variation was restricted to the Wet Tropics and small areas of south-

east Queensland. Toads with relatively large parotoids were predicted to be

present towards the north of the state with pockets of large-glanded individuals

present in the Wet Tropics, mid-east and south-eastern Queensland. The model

suggests that large-glanded individuals are present in some of the areas that

have been occupied by toads for long periods, despite an overall trend for

relative parotoid size to decrease with time since colonisation (Chapter 7 and cf.

Figs 1 and 3).

186

A)

B)

187

Figure 2. Maps of the spatial and temporal variation in toad body size (SIL), as predicted by climatic and temporal data. A) Mean SIL across months and, B) the coefficient of variation for SIL across months.

A)

B)

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Figure 3. Maps of the spatial and temporal variation in toad relative parotoid size, as predicted by climatic and temporal data. A) Mean relative parotoid size across months and, B) the coefficient of variation for relative parotoid size across months.

189

DISCUSSION

My results show a significant effect of climate, collection month and time

since colonisation on toad morphology. Because the resultant models are

complex, mapping them across collection months enabled a better

understanding of the results than simply examining coefficients. Additionally,

mapping the models also provides a description of the spatial and temporal

variation in my “key traits”. Doing so revealed that while spatial variation in

toad body size (SIL) appears to be influenced by climate and time since

colonisation, collection month has an overwhelming effect on predicted average

SIL. Seasonal variation in SIL is thus likely to swamp the spatial effect of

climate, particularly in north and north-west Queensland. In these areas, for a

predator to which toad SIL is an important factor influencing the likelihood of

death by poisoning (i.e. quolls and large varanids), seasonal variation in toad

size is likely to be a much more important factor than any spatial variation in

SIL.

Conversely, relative parotoid size, although influenced by collection

month, appears to exhibit variation that is primarily spatial and is affected by

both time since colonisation and climatic variables. For predators constrained

by prey size (and thus for which relative parotoid size determines predator

vulnerability, e.g. snakes and small varanids), there appears to be meaningful

spatial variation in the potential impact imposed by toads. Some of this

190

variation can be explained by climatic variables and some of it can be explained

by time since colonisation.

Interestingly, my models suggest that time since colonisation (TSC) has

had a significant effect on toad morphology, in terms of both SIL and relative

parotoid size. In areas where toads have been for a long period, the animals

tend to be relatively small and to have relatively small parotoid glands. My

maps of toad morphology represent an extrapolation from the original dataset,

in that TSC is currently greater than 66 years in many areas although it rarely

exceeded 55 years in the dataset. The extrapolation of the negative trend in

relative parotoid size with TSC is reflected in predicted values of relative

parotoid size that are rarely positive in my map. The strong effect of recent

colonisation history on toad morphology is a powerful reminder of the

importance of history in landscape-level patterns, and thus of the need to

incorporate, wherever possible, recent history into landscape-level models.

Although spatiotemporal projection of my SIL model predicted high

seasonal variation in toad body size, some areas are expected to maintain large

average body sizes throughout the year (the Wet Tropics and south-east

Queensland). These large body size areas included parts of the state where

toads were first introduced (Figs 1 and 2). Superficially, the prediction of large

body sizes in some areas long after initial colonisation appears inconsistent

with the overall negative effect (i.e. partial coefficient) of TSC (see also Chapter

7). Mapping also revealed that some of the areas with the largest relative

parotoid size are areas where toads were first introduced (the same anomalous

191

areas as for variation in SIL, Figs 1 and 3). This is another apparent

contradiction of the overall prediction of a reduction in parotoid size with

increasing time since colonisation (based simply on the partial coefficient for

TSC).

These apparent anomalies can be explained by two facts. First, my

model predicts shifts in the effect of TSC with precipitation and latitude (i.e.

interactions). For SIL, the effect of TSC will be negative except in areas of high

precipitation; for relative parotoid size both high precipitation and low

latitude/high temperature will change the effect of TSC in a positive direction.

These complexities reduce and sometimes reverse overall trends of toad body

size relative to time since colonisation. Second, toads were not introduced to

climatically random areas of Queensland. They were first introduced into

major sugar cane growing districts – i.e. areas with high precipitation and

warm temperatures. Thus, the areas where toads were first introduced are the

same areas where my model suggests that time since colonisation should cause

little or no decrease in either body size or relative parotoid size.

My best model describing toad size accounted for 38% of the variation in

toad body size. Toad age presumably accounts for a large portion of the

additional variation in toad size and was not explicit in my model. For relative

parotoid size, my best model accounted for 35% of the variation. Other factors

such as water pH or the presence or absence of predators and competitors are

known to affect amphibian morphology (Relyea 2001; van Buskirk 2002) and

data on such topics were unobtainable in the current study. Further

192

investigation into the factors affecting the relative toxicity of toads would be of

great interest, to further tease apart the processes generating spatial and

temporal variation in morphological traits of this invading species.

Nevertheless, my models do indicate significant variation in relative

parotoid size due to climate and colonisation history, and thus allow me to

identify spatial variation in the likely intensity of selection that toads impose on

native predators. This is especially true for gape-limited predators that are

restricted to a specific size range of toads. Because of the spatial variation in

relative toxicity of toads, two identical predators in different areas feeding on

same-sized toads will have different chances of ingesting a fatal dose of toxin.

My model demonstrates that at least some of this difference in predator

vulnerability can be related to differences in climate and time since

colonisation. Further work is necessary to elucidate other environmental

factors affecting toad parotoid size. An improved understanding of the factors

influencing parotoid size will allow the identification of areas where impact

from toads is weakest and it is in these areas that impacted native populations

have the highest chance of survival and eventual adaptation.

The approach that I have adopted in this analysis is potentially

applicable to many invasive species systems. Identifying traits mediating the

impact on natives and then quantifying spatial variation in those traits provides

a tool for predicting the level of impact and how it varies in time and space.

Both conservation biologists and managers can use such information in their

attempts to mitigate impacts of invasion. Such analyses may also prove useful

193

to ecologists and evolutionary biologists utilising invasive species systems to

answer theoretical questions.

ACKNOWLEDGEMENTS.

I thank Patrick Couper, Andrew Amey and Heather Janetski at the Queensland

museum for access to specimens, entertaining discussion and cups of tea.

Michael Kearney provided helpful advice on analyses and along with Michael

Wall and Richard Shine, provided a constructive review of an earlier draft. I

thank the Australian Research Council and Norman Wettenhall Foundation for

financial support.

194

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