I LLINOIUNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN

PRODUCTION NOTE

University of Illinois atUrbana-Champaign Library

Large-scale Digitization Project, 2007.

S

The Population Biology of Torreya taxifolia:Habitat Evaluation, Fire Ecology, and

Genetic Variability

Mark W. Schwartz and Sharon M. Hermann

Center for BiodiversityTechnical Report 1992(Z)

Illinois Natural History Survey607 E. Peabody Drive

Champaign, Illinois 61820

Tall Timbers, Inc.Route 1, Box 678

Tallahassee, Florida 32312

Prepared forFlorida Game and Freshwater Fish Commission

Nongame Wildlife Section620 S. Meridian Street

Tallahassee, Florida 32399-1600

Project Completion ReportNG89-030

TABLE OF CONTENTSpage

Chapter 1: Species background and hypotheses for.......5the decline of Torreya taxifolia,

species Background ....... . . . . . .. . . . .6

Hypotheses for the Decline........0Changes in the Biotic Environment ...... 10Changes in the Abiotic Environment ..... 13

Discu~ssion *0o ** eg. *.*. *.0.*09 0 0 6 0 o**** o*...21

Chapter 2: The continuing decline of Torreyap iola....2Study.Area and Methods ooo................25Results * ** ** ** ** ** ** .. .. .. .. .. .. .. .. .. .. .30

Chapter 3: Genetic variability in Torreya taxif-olia......4Methods.......................* 0 C 0 0 o49Results . . . . . . . ...... *oe*.........o51

-0L-icmion *.. ~ 0000 00000@55

Management _Recommendations .000000000000.0.60

Chapter 4: The light relations of Tgr .taz'ifgli with ..... 62special emphasis on the relationship to growthand,,disease-

Methods o..............0.0.0.0.0.00.eoo63Light and Growth . .. .. .. .. .. .. .. .. .. .. .64

Measurements'-of photosynthetic rates 0,.65

Light and Growth . .. .. .. .. .. .. .. .. .. .. .69

Measurements of photosynthetic rates ..71.Discussion....... .. . . . . *0* * * * * * * ** . 81

Chapter 5: The foliar fungal associates of Torreya............85ta ifola: pathogenicity and

susceptibility to smokeMethods 0 0 0 ... 0..0.....0..0..0..0..0..0..0..0..0. *8 6

Fungal Isolation 00000-0900960000000690086Pathogenicity Tests *00000....00.0.0.087

Fungal.Susceptibility to Smoke ........ 87Field Tests of Smoke Hypothesis ....... 90

Results............00000000000000 .000000000*91Fungal Isolation and Pathogenicity ....91,Smoke Susceptibility ..... oo............92

Discussion..................000 00000000000098

2

FIGURES

Chapter 1 page1.1 Range Map of Torreya taxifolia ........................... 71.2 Annual Precipitation in Blountstown, FL, 1922-1982 ...... 141.3 Water Temperature Data, Above and Below Lake Seminole ...16

1.4 Air Temperature Data, Blountstown, FL, 1931-1984 ........ 18

Chapter 22.1 Range Map Detailing Study Areas ......................... 262.2 Stem Size Distribution for Census Population ............ 31.2.3 Number of Stems per Individual .......................... 31

2.4 Distribution of T. taxifolia With Respect to Elevation ..342.5 Distribution With Respect to Canopy Cover ............ 342.6 Distribution With Respect to Aspect ..................... 342.7 Apparent Age Distribution of Census Population .......... 362.8 Age Correction Regression ............................... 36

Chapter 33.1 Map of Sampled Populations..............................50BLANK PAGE ............. .. . *****. .* * * . * * .. ************** 533.2 Cluster Diagram of Populations .......................... 58

Chapter 44.1 Comparison of Growth by Light ........................... 704.2 Light Saturation Photosynthesis Curve ................... 734.3 Photosynthetic Rates of Diseased Versus Healthy Needles .744.4 Photosynthetic Rates by Needle Age ...................... 754.5 Light Saturation Curves for Five Closely Related Species.774.6 Photosynthetic Rates Across Relative Humidity, 5 Species.784.7 Photosynthetic Rates Across Temperature, 5 Species ...... 794.8 Rate of Water Loss in Absence of Watering, 5 Species ....804.9 Photosynthetic Rates Across. Water Stress, 5 Species ..... 80

Chapter 55.1 Smoke Residue Deposition Versus Time Treatment .......... 895.2 Effects of Direct Smoke Treatment on Fungal Growth ...... 935.3 Effects of Smoke Residue on Fungal Growth ............... 94

5.4 Effects of Smoke Residue on Spore Germination Rate ...... 95

5.5 Effects of Smoke Residue on Spore concentration ......... 96

5.6 Effects of Smoke Residue on Spore'Growth Rate ........... 97

Chapter 6None

TABLES

Chapter 1 page1.1 Fungal Associates of Torreya taxifolia .................. 91.2 Hypotheses for the Decline of T. taxifolia ............. ll

Chapter 22.1 Census Population ...................................... 282.2 Mean Length of Longest Stem, by Population ............. 332.3 Frequency of Disease Symptoms .......................... 382.4 Growth Fate of T. taxifolia, by Size Class ............. 382.5 Growth Fate of T. taxifolia, by Disease Load ........... 42

Chapter 33.1 Allele Frequencies for Enzymes Studied ................. 523.2 Heterogeneity of Sampled Populations ................... 543.3 Genetic Distance Between Sampled Populations ........... 563.4 Genetic Diversity Within and Between Populations ....... 57

Chapter 44.1 Growth Fate of Trees in Different Canopy Cover Classes .724.2 Disease Incidence of Trees in Canopy Cover Classes ..... 72

Chapter 55.1 Fungi Isolated From T. taxifolia Used in Experiments ...89

Chapter 6None

CHAPTER 1

Species background and hypotheses for

the decline of Torreva taxifolia

During the late 1950's, Torreya taxifolia suffered from a

catastrophic decline, presumably fungal in origin, that decimated

adult populations throughout its range (Godfrey and Kurz 1962).

The cause of this decline remains a mystery; no specific pathogen

has been implicated (Alfieri et al. 1967, USFWS 1986); the

predominant speculation is that any of a combination of several

environmental changes rendered T. taxifolia more susceptible to

native pathogens (USFWS 1986). Four hypotheses for the cause of

the decline of T. taxifolia have been proposed (Alfieri et al.

1967, Toops 1981, Barnes 1985, USFWS 1986). Correlative evidence

to address two of these hypotheses is presented here. In

addition, five new hypotheses for the decline, and evidence to

assess two of these hypotheses, are presented here. Direct

evidence to adequately test any of the hypotheses is mostly

lacking because no healthy individuals remain in the wild, no

undisturbed habitat remains, and the primary pathogen eludes

discovery. With this lack of direct evidence, we are left to

piece together anecdotal and correlative evidence as to the cause

of the dieback.

Providing management to facilitate recovery of T. taxifolia

is also hindered by a lack of sufficient information. The purpose

5

of this chapter is to discuss the relative merits of different

hypotheses proposed to explain the decline. The results presented

here point toward management recommendations despite an inherent

inability for adequate testing of virtually any hypothesis. This

chapter discusses, by example, the difficulty in isolating

pathogen-induced declines in endangered species, and how to

approach management recommendations given this information gap.

SPECIES BACKGROUND

Florida torreya (T. taxifolia, Taxaceae) is an endemic

conifer restricted to ravine slopes along the eastern side of the

Apalachicola River in the central panhandle of Florida (Figure

1.1). This dioecious evergreen tree can grow up to 20m in height

at maturity (Godfrey 1988) and was formerly a common understory

tree in the region (Chapman 1885, Harper 1914, Reinsmith 1934,

Kurz 1838a, 1938b). The ravine habitats which support T.

taxifolia are characterized by a diverse mixture of hardwoods and

conifers, most notably beech (Fagus grandifolia), and magnolia

(Magnolia grandiflora), along with various oaks and hickories

along upper slopes (Platt and Schwartz 1990, Schwartz 1990).

Several factors reduced the abundance of the species prior

to the decline. In the 1800s, T.. taxifolia was cut along the

Apalachicola River for fuel (Chapman 1885, Gray 1889), and for

fenceposts and shingles through the 1900s (Chapman 1885,

Reinsmith 1934, Kurz 1938b, Burke 1978). However, T. axifolia

only became endangered after a catastrophic decline during the

1950s. This decline is thought to have been caused by fungal

Figure 2.2.. The range of Torreya taxifolia, including an outlyingpopulation in the vicinity of Lake Ocheese-e. Habitat featuresdigitized from 1953 and 1955 USDA aerial photographs usingARC/INFO ver 5.0. Tgrreya~tYa x±i.I is restricted within itsrange to floodplain and ravine habitats.

Rangeof Torreya taxifolia

Suitable Habitat*Floodplain

flRavines

Other Land Cover*Towns

SAgriculture

ElForested Uplands

ILa Uswe kemined From USOA kqIW PhonogrrKgy, 1953-105

10 KM

r -r 1

pathogens, although no primary pathogen has been identified

(Godfrey & Kurz 1962, Alfieri et al. 1967, 1984). The apparent

lack of an introduced pest is unusual among catastrophic diebacks

of fungal origin (Manion 1981). By 1962, virtually no adult T.

taxifolia remained in the wild (Godfrey and Kurz 1962). Recent

censuses indicate that approximately 1000 juveniles, and no

adults, remain in the wild (Baker 1982, Chapter 2). Most

individuals in the wild are less than 2 m tall, are sexually

immature, and carry symptoms of foliar diseases (Chapter 2).

Few specifics were recorded about the decline of the Tv

taxifolia. The earliest observation of disease symptoms may have

been in 1938 (Nieland, pers. comm. as cited in Alfieri al.

1967), however, other reports from this decade make no mention of

disease (Reinsmith 1934, Kurz 1938a, 1938b). The next report of

disease came from Torreya State Park in 1955 (USFWS 1986). By the

time the decline was first documented, adult populations had

already been decimated (Godfrey. and Kurz 1962). No information is

available on the rate of the decline, exact symptoms of disease,

or the location of any disease epicenter if one existed.

Symptoms of disease in T. taxifolia at present are needle

spots, needle necrosis and stem cankers (Chapter 2). Early

reports of disease cited low vigor and needle blight (Godfrey &

Kurz 1962). The fungal associates isolated from L. taxifolia do

not include introduced obvious pathogens (Table 1.1). The

recovery plan for T. taxifolia (USFWS 1986) cites the lack of an

introduced pathogen to suggest that environmental changes may

Table 1.1. A list of potential fungal associates of Torreyataxifolia, including species isolated from diseased tissue (A),as well as fungi hypothesized to be in association with T.taxifolia (B) (from Alfieri et al. 1967, 1984).

A) Fungi isolated from Torreya taxifolia

1) Phyllosticta sp. (needle spot)*2) Macrophoma sp. (needle blight)*3) Fusarium sp. (root rot)*4) Pestalotia natens (needle spot)*5) Acremonium sp. (?)*6) Botrosphaeria sp. (needle spot)7) Xvlocoremium flabelliforme (?)*

8) Sclerotium rolfsii (southern blight)9) Rhizoctonia solani (root rot)

10) Sphaeropsis sp. (needle blight)

11) Physalospora sp. (twig and needle blight) ?

B) Fungi hypothesized to be associated with . taxifolia

12) Alternaria sp. (needle spot)13) Diplodia natalensis (twig dieback)14) Pythium sp. (root rot)15) Phytophthora cinnamomi (root rot)

* - tested for pathogenicity with negative results

9

have created plant stress, which in turn may have rendered T.

taxifolia more susceptible to infection by native pathogens. The

native pathogen hypothesis requires additional framework because

these pathogens generally do not reach epidemic proportions

unless the host plants are under stress (Schoeneweiss 1978).

HYPOTHESES FOR THE DECLINE

Four previously proposed hypotheses along with five new

hypotheses are presented (Table 1.2). Very little data can be

brought to bear directly upon many of these hypotheses. Each of

these nine hypotheses is briefly discussed, citing any evidence,

upon which to assess the validity of the hypotheses.

HYPOTHESES RELATED TQ CHANGES IM THE BIOTIC ENVIRONMENT

Al Introduced Fungal Pathogen

Exotic pathogens could invade the range of T. taxifolia in

any of a number of ways. Perhaps the most likely means for

introduction of a pathogen that could have caused die-back in T.

taxifolia was through slash pine plantations in uplands adjacent

to ravines. Conifer plantations have been implicated in diebacks

elsewhere (Struhsaker et al. 1989), and slash pine was heavily

planted during the 1950s in the uplands of the southern end of

the range of T. taxifolia. Aerial photographs indicate that

approximately 36 % of the land within the range of T. taxifolia

is forested uplands (Figure 1); most of these forested uplands

became pine plantations during the 1950s.

The introduced fungus Phytophthora cinnamomi, which causes

little leaf disease in southern pines, has been proposed as a

10

Table 1.2. Nine hypotheses proposed to explain the decline ofTorreya taxifolia and the data related to assessing thesehypotheses.

HYPOTHESIS SOURCE EVIDENCE

Introduced Fungus Alfieri et al. 1967 Anecdotal evidencedoes not support

__________________________hypothesis

Pathogen Vectors Schwartz 1990 Very poor evidence

Water Stress USFWS 1986 Climate recordssupport hypothesisby showing a dryperiods associatedwith time ofdecline.

Microclimatic USFWS 1986 USGS water tempera-Warming Toops 198* ture data do not

support hypothesisas proposed.

Regional Warming new Climate recordshows no warmingtrend, potentialcooling of wintertemperatures. Doesnot support hypo-thesis.

Hydrologic Changes USFWS 1986 No evidence of theeffect of hydro-

____________________logic changes.

Fire Suppression Schwartz 1990 Weak supportiveevidence.

Air Pollution new No evidence.

Fungal Pathogens as new No evidence.an epiphenomenon_________________

11

possible introduced pathogen of T. taxifolia (Alfieri et al.

1967, USFWS 1986). This root-rotting fungus, which is thought to

have been introduced from Australia (Pratt et al. 1973), is known

in northern Florida (Barnard, pers. comm.). Its pathogenicity on

T. taxifolia is untested, but has been suggested to be associated

with the decline (USFWS 1986).

Two pieces of evidence suggest that P. cinnamomi is not

associated with T. taxifolia at the present time. First, roots of

T. taxifolia that were examined by myself and others in the field

appear healthy. Second, Barnard (unpubl. data) examined soil

samples from around the base of 17 diseased T. taxifolia. He

failed to isolate P. cinnamomi, or any other obvious pathogen,

using techniques that had previously been successful in other

north Florida soil samples. No other introduced fungus has been

postulated as a pathogen.

_i1 Changes in Resource use hy Pathogen Vectors

Torreya taxifolia suffers stem damage by deer as antler

rubs. The conversion of uplands to pine plantations in the 1950s

entailed total clearcuts. Earlier harvests of longleaf pine had

been restricted to canopy trees, leaving juveniles to regenerate.

Deer, which are abundant in the region, prefer soft-barked

coniferous trees for antler rubs. The removal of young pines from

the uplands may have increased the use of T. taxifolia by deer.

In turn, wounds inflicted by deer may have increased the rate of

fungal infection and facilitated the spread of disease. While

nearly all larger individuals carry scars from past deer rubs,

12

the lack of a pathogen prevents testing of this hypothesis.

Cl Fungal Pathogens as Epiphenomenon of Decline

There is no guarantee that the symptoms of disease that are

now observed on T. taxifolia bear any direct connection to the

cause of the decline. Some pathogen, not now found on T.

taxifolia, may have swept through the region causing the decline.

If such a pathogen is now missing, or at very low densities, we

would likely not detect its presence.

HYPOTHESES RELATED TO CHANGES IN THE ABIOTIC ENVIRONMENT

DI Water Stress

Barnes (1985) suggests that drought may have stressed T.

taxifolia, making them more susceptible to facultative diseases.

Water stress is known to predispose plants to disease (Yarwood

1959, Parker 1965, Cook 1973, Schoeneweiss 1975, 1978, Griffin

1978) as well as weaken a plant's ability to survive fungal

infection (Ayres 1978, Burdon 1987). Hepting (1963) and

Schoeneweiss (1978) cite numerous examples of secondary pathogens

causing stem cankers in birch, maple, and oak during drought

years. The foliar fungi isolated from T. taxifolia fit the

general description of secondary pathogens (Alfieri et al 1967,

1984, Schwartz 1990). Secondary pathogens have been observed to

become primary pathogens under conditions of plant stress

(Schoeneweiss 1975, 1978).

Weather records from Blountstown show low annual rainfall in

1938 and 1953, years near when disease was recorded in T.

taxifolia (Figure 1.2). Although drought may have contributed to

13

Figure 1.2. Annual precipitation recorded from BlountstownFlorida, adjacent to the natural range of Torreya taxifolia. Thedrought year most closely associated with the time of the declineis highlighted.

•JE

Blountstown Station

1953

1940 1960

Year

1980

Inches

80

70"

601

40

30-192 0

. •- r •.

90-

I

the fungal disease, drought seems insufficient as the sole

explanation for the decline. The droughts of 1938 and 1953 were

not unusual for the period between 1922 and 1982 (Figure 1.2).

Because rainfall varies widely in north Florida, adult trees,

which live an excess of 100 years, must survive several droughts

as severe as those of 1938 and 1953 to reach maturity.

EL Microclimatic Warming

Lake Seminole drains into the Apalachicola River at the

northern limit of T. taxifolia. Construction of the Woodruff dam,

which created Lake Seminole in 1956, approximately coincided with

the decline. The dam-related hypothesis is based on changes in

Apalachicola ravine microclimate and flooding patterns (Toops

1981). This hypothesis proposes that water in Lake Seminole

warms, raising temperatures in the Apalachicola River. The warmer

river, in turn, raises ravine air temperatures in what may

already have been a marginal micro-climate for T. taxifolia

(Toops 1981).

This hypothesis is subject to verification through direct

observation. Water gauge temperature data collected by the United

States Geological Survey (USGS) from above (in the Flint and

Chattahoochee Rivers) and below Lake Seminole showed no

difference over the 25 year period from which data are available

(Figure 1.3). With no change in water temperature, we have no

evidence of changes in ravine microclimate that can be attributed

to Lake Seminole.

15

Figure 1.3. Water temperature records, by calendar date, for theFlint, Chattahoochee and Apalachicola Rivers. The Flint andChattahoochee stations are immediately above Lake Seminole, andrepresent the major inputs to the lake. The Apalachicola Riverstation is immediately below Woodruff Dam. The Flint andChattahoochee Rivers form the Apalachicola River at theirconfluence.

* Apalachicola Staton (below Lake Seminose)

0 Chaftahoochee Station (above lake Seminole)

A Flint Rivr Station (above Lake Seminoie)

30

Degrees(Celsius)

204

10 a

0Jan Mar May Jul

I-

Sep

Month

Data from 1960 - 1985

I

Nov

*

I

fgRejional1 Climatic Warming -

Temperatures in the eastern U.S. were cooler during the 19th

century (Wahl 1968). It is possible that the decline was driven

by a marginally warmer and drier climate during the 20th century

that has made the ravine habitats unsuitable for T. taxifolia.

Weather records from the nearby Blountstown station show no trend

in mean summer temperature during the monitoring period of 1931

through 1982 (Figure 1.4). Although mean winter temperatures

appear to decrease over this period. If climate change did

contribute to the decline, then it most likely has been a long

continuous slow process that predates the catastrophic die-back

of the 1950's.

iHydrgloi -changeHydrologic changes within ravines might account for the

decline (USFWS 1986). Conversion of longleaf pine uplands to

slash pine plantations, during the 1950's, entailed extensive

site preparation in the southern third of the range of T.

taxifolia. Ravine temperatures may have risen as a result of the

highly reflective bare mineral sand exposed from site

preparation. Further, the lack of vegetative cover would have

reduced evapotranspiration and perhaps lowered humidity in the

adjacent ravines (USFWS 1986). Sparse vegetative cover would have

altered runoff flow, and soil disturbance overland sheetf low of

water.

This hypothesis is somewhat unsatisfactory because the

extent of upland clearing varied across the range. Large tracts

17

This page is intentionally blank.

Figure 1.4. Mean maximum and minimum temperatures for summer(June-August) and winter (December-January) months from 1932 to1982 as measured in Blountstown, Florida.

---- Summer, max.

-]--- Summer, min.

---- Winter, max.

-- *- Winter, min.

Year

100

80

60

(1

c%,._

CLE--CO

03

40

201I W--

of uplands adjacent to ravines were not converted to slash pine.

We might, therefore, predict that T. taxifolia found in Torreya

State Park, or in the outlying population along Lake Ocheesee,

would have been less affected by the decline, yet these

individuals were susceptible.

HI iU Pollution

The establishment of pine plantations in the 1950's was

coincidental with the construction of several paper mills in the

north Florida region. The pollutants from these paper mills may

have reduced the pH of local precipitation. There is no direct

information available on how reduced pH may effect disease

inception in T. taxifolia.

11 Fire Suppression

The suppression of fire, which effectively began in the

1950s, could have had a direct effect on the onset and severity

of the T. taxifolia decline. Natural fire frequencies in pineland

habitats of north Florida are estimated to occur at 1-3 year

intervals (Komarek 1964, 1968, Robbins & Myers 1989). Historical

accounts cite the use of near annual fire by both Native

Americans and Europeans in Florida (Tebeau 1980, Pyne 1982). This

frequent fire regime was halted under fire suppression that began

in the 1950s.

Although T. taxifolia grows in a relatively nonflammable

ravine habitat, it could have been affected by fire suppression

in two ways. First, fire suppression allowed upslope portions of

ravines to become more heavily wooded. Thus, lower portions of

19

the slopes became more heavily shaded than prior to fire

suppression. Increased shading may inhibit recovery through

slower growth rates and lower plant vigor (see Chapter 4).

Second, fire may have had an indirect effect on ravine

habitats by way of smoke settlement. Ravine habitats are lower

and cooler than the surrounding uplands. During the evening,

smoke from upland fires settles into such low-lying areas. The

smoke generated from large fires is extensive, and anecdotal

reports cite smoke settling so thick at ground level that one

could not drive a car at night.

Smoke is used as a preservative of meats and other goods

because it inhibits the growth of fungi and bacteria (Parmeter &

Uhrenholt 1975a). Experimental evidence indicates that smoke

residue on fungal growth media decreases spore germination and

mycelial growth of many fungi (Melching et al. 1974, Parmeter &

Uhrenholt 1975a, 1975b, Mihail 1979, Zagory & Parmeter 1984). The

deleterious effect of smoke on Fusarium lateritium, the only

fungus that has a demonstrable pathogenic effect on T. taxifolia,

is particularly pronounced (Zagory & Parmeter 1984). Thus, the

frequent and consistent presence of smoke in ravines may have

served as a chemical defense and fire suppression may have

initiated a fungal outbreak throughout the range (Chapter 5).

The smoke hypothesis may fit well if, in fact, foliar

pathogens are responsible for the decline. Needle infections do

not spread within the vascular tissue of the plant; instead, each

needle requires a separate penetration of the stomata by inoculum

20

(Parmeter pers. comm.). Massive infections of needle tissue may

be required before trees are adversely affected. Fire suppression

in the uplands might have allowed populations of needle pathogens

to explode within the ravines.

DISCUSSION.

Each year new research describes how air pollution, global

warming, habitat degradation, and other human-caused changes in

the environment can effect biodiversity (e.g., Melillo et al.

1990, Warrington and Whittaker 1990, Woodwell 1990). Discerning

exactly how changes in the environment effect species health,

however, can be exceedingly complex (Schoenewiess 1975, Bormann

1990). For example, researchers remain divided over the mechanism

responsible for the decline of forests in New England and

northern Europe (Bormann 1990).

The list of fungal isolates of T. taxifolia presented here

varies somewhat from previous studies (Alfieri et al 1967, USFWS

1986). Three explanations arise for these differences. First,

native trees were used for these experiments, while previous

studies used trees growing in the wild as well as trees in lawns,

at Maclay Gardens and on the campus of the University of Florida

(Alfieri et al 1967, El-Gholl personal communication). These

trees outside the natural range are likely to be vectors of other

horticultural diseases that may not infect wild trees. Second,

Alfieri et al. (1967) list several species only hypothesized to

be in association with L taxifolia. Thus, this list of fungal

isolates may be more accurate, with one exception. Phyllosticta

21

sp. was previously isolated from ascopores on needles in the wild

(Alfieri et al. 1967), and was tested for pathogenicity before

with negative results (El-Gholl 1984). Finally, Alfieri et al

(1967) did not list fungi that they perceived to be secondary

pathogens (El-Gholl, personal communication).

The nine hypotheses for the decline of T taxifolia have

been summarized (Table 1.2). One hypothesis (microclimatic

warming) can be dismissed as a result of the evidence presented

here. Three of the hypotheses (introduced fungus, deer vector and

hydrologic change) may be related to the conversion to plantation

pine during the late 1950s. This conversion focused on the

southern third of the range of T. taxifolia, and thus generates a

prediction of a specific spatial pattern to the spread of

disease. For these hypotheses the disease should have spread from

relative epicenters, or had differential effects across the range

of the species. In contrast, the four remaining hypotheses (water

stress, regional climate change, air pollution and fire

suppression) predict a synchronous decline throughout the range.

Although anecdotal accounts from residents seem to suggest a

synchronous decline, we do not have sufficient historical records

to determine if the disease spread from a single locus or was

immediately widespread.

These nine hypotheses are not mutually exclusive. Further, a

single causative agent of the T. taxifolia decline need not be

isolated in order to develop a plan for the recovery. However,

several of the factors I have described do not lend themselves to

22

management efforts for recovery. Air pollution, regional climatic

change, and drought are phenomenon that cannot be locally

controlled, reducing the ability to manage for on-site recovery.

Past hydrologic changes may affect the future of T. taxifolia in

ways that we can neither predict nor control at this time.

Finally, it would be difficult to control an introduced pathogen

without identification.

In contrast, the deer vector and fire suppression hypotheses

point to management opportunities for on-site recovery. Whether

or not deer act as a vector of disease in T. taxifolia, the

antler rubs increase stem mortality. Deer populations can be

reduced, alternative antler rub species can be replaced in the

uplands, or individual T. taxifolia may be protected using

exclosures. Fire management can be restored to the uplands. Both

of these factors are currently being addressed through management

of local preserves by The Nature Conservancy.

This chapter points to several difficulties with respect to

species declines. Isolating disease agents, and factors that

enhance the virulence of these pathogens, is exceedingly

difficult. Yet, the environment faces an ever widening array of

environmental changes through air pollution, climate change,

continued deforestation, and disruption of natural disturbance

processes. The example of T. taxifolia is illustrative because it

demonstrates how difficult disease assessment can be. This

difficulty in discerning disease agents is enhanced when dealing

with an endangered species.

23

CHAPTER TWO

The continuing Population Decline

of Torreya Taxifolia

Although extinction or near obliteration of populations and

species has been of interest to biologists for centuries,

attempts to closely monitor and assess such processes are common

only during the last few decades. Many of these recent studies

focus on endangered species; demographic data on rare plants is

increasingly utilized as a tool in conservation efforts aimed at

preventing extinction and enhancing existing populations (Davy

and Jeffries 1981),o Anecdotal information may suggest the need

for closer inspection or hint at possible remedies, but

quantified data is needed to accurately assess the health of a

population (Travis and Sutter 1986). This may be especially true

for long-lived species where the population decline may be slow.

In addition, individuals of perennial plants may decline in vigor

long before mortality actually occurs. Woody species provide a

semi-persistent record of individual decline that can be used to

monitor fluctuation at both the individual and population levels.

In the United States there has been documentation of

catastrophic decline or extinction of various woody species. One

of the earliest examples is of a small tree (Franklinjia

alatamaha), endemic to a narrow range in coastal Georgia, that

has not been seen in the wild in over 200 years. Over-collecting

has been suggested as the reason for its disappearance (Harper

24

and Leeds 1937) but neither the cause nor the pattern of decline

was actually documented. Over a much larger geographic scale,

eastern forests of North America witnessed the catastrophic

decline of the chestnut (Castanea dentata) during the first half

of this century. For this once dominant tree species, many

components of the near extinction are understood (cf. Braun

1950). In this example, most ecological considerations focused on

community level observations related to species replacement

(reviewed in Woods and Shanks 1959). At the individual level,

only general remarks were usually reported (cf. Nelson 1930).

Repeated measures on individuals may be necessary to effectively

explore population responses and to document patterns of

continued decline or recovery (Travis and Sutter 1986).

STUDY AREA AND METHODS

The range of T, taxifolia encompasses several private and

public preserves and parks (Figure 2.1). This study focuses

primarily on trees within secured habitat, although one site with

more than 40 individuals is included from outside preserve

boundaries. Each area was systematically searched by multiple

workers to insure that all trees within the sample region are

included in the census.

In 1988, a thorough survey of the ravine systems within the

southern portion of the Apalachicola Bluffs and Ravines Preserve

(ABRP, Figure 2.1) was conducted. Seventy-nine trees were added

to the 25 previously known from this preserve. A repeat census in

1991 relocated 100 of these trees and added six new individuals

25

Figure 2.1. The distribution of TorreyA taxifolia. Highlightedareas show populations included in census or discussed in text.

ABRPBGSWTRSPFLCRLKSM

Apalachicola Bluffs and Ravines Preserve (TNC)Big Sweetwater Preserve (TNC)Torreya State ParkFlat Creek (Private Land)Lake Seminole (U.S. Corps of Engineers)

from a portion of the preserve that had not been previously

censused (Table 2.1). Four trees censused in 1988, but not in

1991 are very difficult to find within a remote area of the

preserve and were excluded from the permanent census population.

In addition, 25 trees from U.S. Army Corps of Engineers land

at Lake Seminole (LKSM) were surveyed and tagged in 1988. This

was the entire population as reported elsewhere (Savage 1983, T.

Patrick,pers. comm.). By 1992, we had added two previously

overlooked individuals to this population (Table 2.1). One of

these new trees had previously been considered a second stem of a

known individual, the other was a newly encountered stem. In mid-

summer 1991, 25 trees from a population along Flat Creek (FLCR)

were also measured. This census population was increased to 31

trees in 1992.

Finally, census information was collected from 27 trees in

three separate ravines along the Big Sweetwater Creek (BGSW,

Figure 2.1) in 1991. One year of growth information is available

for the 16 trees from the BGSW population that were surveyed

prior to bud break in 1991. This census population was expanded

to 43 trees in 1992. The census population represents all trees

encountered along particular stretches of ravine systems within

the preserve. The total population on the BGSW preserve has been

measured to exceed 125 trees by Nature Conservancy volunteers.

A large population from Torreya State Park was excluded from

our study. Trees grown from seed were planted in the park during

the 1960s and 1970s in an attempt to increase the population of

27

Table 2.1. Number of Torreya taxifolia in the census populationduring each year of the study along with the number ofindividuals for which growth history is recorded over thisinterval.

POPULATIONA) Census ABRP 1 BGSW FLCR LKSM TOTAL1988 104 0 0 25 12919912 106 273 253 24 1821992 102 43 31 27 203B) GrowthGrowth in '89,90 98 0 0 23 121Growth in '91 106 16 0 24 146Growth '89-91 894 0 0 23 1115

1 - See text for explanation of site abbreviations.2 - Increase in numbers between years represent

expansions of census areas, not increases in populationsize within census populations.

3 - Most 1991 census data was collected after new growth anddoes not include growth information for 1991.

4 - Attrition in numbers is as a result of mortality.5 - Owing to 1988 census techniques we could not distinguish

between stems that grew and stems that did not grow for 20census trees between 1988 and 1991.

28

T. taxifolia. These plantings were not documented and can no

longer be discerned with certainty from natural stock.

Tree size was characterized by the number and length of

stems, the number of internodes along the main trunk of the

longest stem, and the number of branches along the main trunk for

the longest stem. Habitat was charactiized-by three measures.

Relative elevation above the ravine base was estimated using

slope and distance measurements. Canopy density above T.

taxifolia individuals were compared to the forest as a whole,

using a LICOR light sensor. Four readings were measured above

each tree. Ambient light levels were sampled haphazardly while

traversing between census trees at approximately 10 m intervals.

Aspect of the slope upon which trees were located was recorded by

compass.

Plant vigor was assessed through measures of recent growth

and presence of disease symptoms. New growth during the census

period was recorded as additional internode lengths on primary

stems. The length of new growth during the past three episodes of

terminal bud extension was also measured. Further, the age of

branches was estimated from the number of branch internodes. This

measure was compared to the number of stem internodes above the

branch point to estimate the frequency of terminal bud extension

for each tree under the assumption that lateral branches expand

internodes annually (this assumption will be discussed further

below). Terminal bud extension frequency allows an age correction

factor to be calculated that incorporates the likely frequency of

29

terminal bud extension. The approximate age of the oldest stem on

individuals was estimated by counting the number of internodes

along the trunk and multiplying by a population-wide estimate of

the frequency of terminal bud elongation.

To assess disease status we noted whether plants exhibited

any of several characteristic disease symptoms. Needle spots are

defined as discrete round patches of dead tissue between 1 and 3

mm in diameter. Needle spots were previously considered the most

prominent symptom of foliar disease in the wild (Alfieri et al.

1967). Needle necrosis was defined as dead tissue in a discrete

band laterally across needles and toward the needle tip. Stem

cankers were defined as any scar along the stem that was not

obviously a result of abrasion. These cankers were most

frequently swelling from within the woody tissue in larger stems,

and cracking and swelling of the bark in smaller stems. Stem

scars included all scarring of bark that appeared to be from

abrasions such as deer antler r.ubs or treefalls. The presence or

absence of disease symptoms, physical abrasions, and scarring was

noted for each individual.

RESULTS

The population of Torreya taxifolia is characterized by a

majority of individuals less than 1 m tall (Figure 2.2). The

largest stem for 57% of censused trees was between 25 and 100 cm

long, and the mean stem length was 87.8 cm with 6% of individuals

having stems more than 2 m long (Figure 2.2). The mean length of

the longest stem was not randomly distributed across populations

30

Figure 2.2. The distribution of size classes of the longest stemfor individual TorreyA 4taxifolia. Data are from trees sampledfrom throughout the natural range.

U)00I-

0.0Ez

40

30

20

10

In 0 ) a in O n 0 In 0 n W0 In4 In , 0 O4 U, 0 No4 W). 0 N

,. 0 -N - ,- t 0 4 N 4 U) €0

Length of Longest Stem (cm)

Figure 2.3. The number of stems per individual TorreaA taxifolia.Data are from trees sampled from throughout the natural range.

80

U)00I.-

Sz

60

40

20

1 2 3 4 5 6 7 8 9 10+

Number of Stems

AJ% -

(Table 2.2) and was larger toward the north portion of the range.

This trend appears to be a result of more very small individuals

toward the southern range limit (Table 2.2). This pattern of

increasing size with latitude was repeated within ravine systems

of the southernmost population (ABRP). Most individuals are

multi-stemmed, although the most frequent number of stems per

individual was one (Figure 2.3). Length of longest stem was

positively correlated with stem number, although the relationship

was weak (r2=.19, p<.001). Thus, it is not surprising that

individuals in the more northerly populations have more stems

(Table 2.2).

Torreya taxifolia is most commonly found at low elevations

with respect to ravine slopes (less than 6 meters above the

ravine base), although they may range well upslope (Figure 2.4).

The ravine habitats of T. taxifolia span any where from 12 to 45m

in elevation above constant running streams at the base of each

ravine (Schwartz 1990). The substrate of lower slopes is

characterized by higher soil moisture and higher organic content

than that of upper slopes (Schwartz 1990, unpublished data).

Torreya taxifolia is also generally found under a relatively

closed canopy duringthe summer (Figure 2.5), although this

pattern is descriptive of the habitat in general and does not

imply habitat selectivity in T taxifolia. Measures of

photosynthetically active radiation (PAR) averaged 215 Mmol/m2/s

(~s.d.=284, n=257) adjacent to T_. taxifolia, compared to 254

Mmol/m2/s (s.d. 308, n=293) for random points in the forest

32

0

(U

U)

0

4.)(UH

0p4

.1

p

S

140

00

(U41

U' LALANO

4w1

1%0 %O O O*%* *

r% co t-0 IC

'0

41I

I 001 0C-4*

co N tkHI(q* 0 0 I

41 00 U) *% I

AI

V4A

* 1

to I

-44 Ix I(UI I

4.) I

4JI 1

0 WI

4404.

44U00I

431

0r4~

.~0

04)

~4J

E-~4

0'0.

4J

r-

c4z

:I

*1I

00

00

10011T

CO I CO C-

1I 0

* en >I U

4P IIV4%r~0 I 0 IA

I r .4rZ (1)~I 4.)4.)44P

HI 00

*I 4F4 0~~~~ I-r.4J

LO I $4( U'IC4H0 U)M

4I >I 9H04 4

I lQ0

cl

Figure 2.4. The distribution of Torreya taxifolia across relativeelevation above ravine base. Data are from trees sampledthroughout the natural range. Ravines vary in depth from 12 to45m in relative elevation.

301

0 20

1 10z

01 2 3 4 5 6 7 8 9 1011 1213+

Relative Elevation (m)

Figure 2.5. The distribution of Torreya taxifolia by canopy coverclass. Data are from trees sampled throughout the natural range.Canopy openness was measured as the percent of 100 random pointsthat did not intercept canopy vegetation in overhead 28mm wide-angle photographs taken above individual trees.

80-

0 20.0

in 40- W wuwier

Canopy Openness

Figure 2.6. The distribution of Torreva taxifolia with respect toaspect. Data are from trees found along slopes sampled throughoutthe natural range.

401

S 30

0-

"S 20

| 10z

0

Aspect

during leaf flush in March. These values averaged 25 % of full

sun levels measured during this period (x=1043 gmol/m2/s,

s.d.=547, n=42). Incident levels of light is correlated with

slope position; lower slopes tend to receive lower incident light

levels because of increased shading from uplsope vegetation as

well as an increased angle to the horizon (Schwartz 1990).

Finally, T. taxifolia is distributed widely with respect to

aspect (Figure 2.6).

The distribution of the apparent age of primary stems, as

assessed by the number of internodes, ranges between 1 and 21

years, with a mean of 8.0 (Figure 2.7). True stem age is related

to apparent age by the frequency with which stems expand an

apical bud and grow new internodes along the main stem. We have

observed that stems do not expand apical buds each year. The

relationship between apparent lateral branch age (lateral

internodes) and the number of terminal internodes above that

branch point provide a correction factor for age. This age

correction factor (Figure 2.8) suggests that terminal buds are

expanded approximately every other year under the assumption that

side branches expand leader buds each year. This correction

factor suggests that the mean age of stems is 12.0 (s.d=6.3),

with maximum stem age at 31 years. However, over the three years

of growth encompassed in this study (1989-1991) only 47% of

surviving trees expanded any terminal buds (see below), while

approximately 20% of trees expanded a new terminal internode each

year. This low rate of terminal bud expansion during the census

35

iqgure 2.7. Apparent age distribution of the oldest stems ofindividual Torreva taxifolia found in the Apalachicola Bluffs andRavines Preserve. Apparent age is estimated from counting thenumber of terminal internodes along the main stem.

30

I-

0

z

20

10

Apparent Age

Figure 2.8. The distribution of the number of termanal internodesabove individual branch points plotted against the number ofinternodes along the branch eminating from that branch point.This scatterplot estimates the frequency of terminal budelongation under the assumption that lateral branch buds elongateevery year. Thus, the relationship described by the these pointsprovides a correction factor for determination of the approximateminimum age of individual stems.

I

I0

I

12

10

8-

6-

4.

2

u

*** U

Ue W II

* UI •Ir • .l

* 0

* Ya0.47 + 1.4

U

s5*X r 2 = 0.89

- I -

NUXMs or TRanzL IrTnoo1s

0 2 4 6 8 10U

interval indicates that our corrected stem age estimates are also

low. Thus while we cannot age specific stems, the census

population appears to consist of a mixture of trees whose primary

stem dates from the time of the decline along with trees whose

primary stem is a sprout from an earlier stem that has died since

the onset of the decline.

Several patterns of disease incidence are noteworthy. First,

needle spots are found on nearly all trees, but rarely found to

exceed 5% coverage of needles (Table 2.3). Second, needle

necrosis, which has not previously been reported in the

literature, is widespread at low levels throughout the population

(Table 2.3). Finally, stem cankers appear to increase in more

northerly populations. However, this pattern may simply reflect

the fact that cankers often do not manifest themselves in smaller

stems. Canker occurrence is associated with longer total stem

length (t-test, T=4.43, p<.001). Needle disease incidence also

varied considerably between populations (Table 2.4), but small

population sizes at several sites make it difficult to accurately

test the significance of this variability.

The data from this study record growth and mortality for 111

individuals in two populations (ABRP and LKSM) during three

growing seasons (Table 2.1). During this period 11 individuals

(9.9%.) from these two populations died. All mortality was among

individuals from ABRP and ranging from 17 cm to 101 cm tall

(x=46.9 cm, s.d.= 23.6, n~ll) with two or fewer stems. Of the 117

plants that survived the 1988-1991 census interval, 18 (15.3%)

37

Table 2.3. The percent incidence of disease symptoms and damage

in Torreva taxifolia. Stem cankers are lesions in the outer

cambium visible from the surface. Stem scars are any scar tissue

along stems caused either by deer or any other abrasion. Needle

spots are brown circular spots that are distinct and isolated

from other damage. Needle necrosis are dead ends of needles that

constitute more than one-third the needle area.

SYMPTOM POPULATION PopulationABRP BGSW FLCR LKSX mean

Stem cankers 26.1 46.5 64.5 52.0 40.3

Stem scars 27.2 58.1 6.4 4.0 27.7

Needle DamageSpots >0%

>1%>5%

81.935.17.4

79.132.614.0

90.325.8

0

10052.24.3

85.536.37.3

Necrosis >0% 57.4 44.2 37.5 24.0 45.4>1% 29.8 11.6 0 12.0 17.1>5% 9.6 4.7 0 0 5.7

N 94 43 31 25 193

Table 2.4. Number of trees, by size class, differing in

growth fate from 1989-1991. Values in parentheses indicatepercentage of size class total (row total).

Size class Grew Did not Stem Died Totalgrow died

1989, 19900 - 50 cm 10 (34%) 10 (34%) 7 (24%) 2 (7-%) 29

51 - 100 cm 16 (38%) 17 (40%) 7 (17%) 2 (5%) 42101- 150 cm 6 (38%) 7 (44%) 3 (19%) 0 (0%) 16

151- 200 cm 4 (36%) 7 (64%) 0 (0 %) 0 (0%) 11> 200 cm 1 (14%) 5 (71%) 1 (14%) 0 (0%) 7

N 37 (35%) 46 (44%) 18 (17%) 4 (4%) 105

19910 - 50 cm 8 (14%) 29 (50%) 17 (29%) 4 (7%) 58

51 -100 cm 13 (21%) 38 (62%) 8 (13%) 2 (3%) 61101- 150 cm 3 (15%). 16 (80%) 0 (8%) 1 (5%) 20151- 200 cm 7 (37%) 12 (63%) 0 (0%) 0 (0%) 19>200 cm 4 (44%) 5 (56%) 0 (0%) 0 (0%) 9

N 35 (21%) 100 (60%) 25 (15%) '7 (4%) 167

38

lost their primary stem and decreased in size (Table 2.4).

Likewise, of the 182 census trees that survived between 1991 and

1992, 25 (13.7%) lost their primary stem and decreased in size.

When trees lost their primary stem they decreased an average of

45.2 cm (s.d.= 52.6). In total, 32% of those trees followed over

the entire four-year census interval lost a primary stem during

this period.

Differences in stem lengths between 1988 and 1991, along

with number of internodes on the main trunk, were used to

determine which trees expanded terminal buds during the census

interval. Because the 1991 census was completed prior to bud

break in 1991, the 1988-1991 census interval comprised only two

years of growth. For several plants it was unclear whether a

terminal bud had expanded. It-was difficult to accurately measure

the tops of the taller trees, and many of these questionable

plants are among the larger stems. Between 1991 and 1992 growth

fate was more accurately determined through a comparison of

recent terminal internode lengths to determine if a new internode

was added. The most frequent fate of trees over both census

intervals was no new growth (Table 2.4). Approximately 35% of

trees grew between 1988 and 1990, while 21% of individuals grew

in 1991. Over the census interval not one single tree expanded

terminal buds in all three years, fewer than 20% grew in two of

three years, and only 47% expanded a terminal internode at some

time during the period. The only pattern between size and growth

history is that the majority of stem and individual mortality was

39

among small individuals (Table 2.4). Average length of new

growth among those trees that expanded a terminal bud was 9.60 cm

(s.d.= 5.2). In addition, there was a weak, but significant,

positive linear relationship between the length of the primary

stem and the amount of new growth during both census intervals

(r2 < 0.30, p S .02). Among the 13 trees that lost their primary

stem between 1988 and 1991 and expanded terminal buds on

secondary stems average two-year growth was 13.6 cm (s.d.= 8.7

cm) on the new, shorter primary stems. Combining growth and

mortality results for those trees followed for four years we find

that the mean length of the primary stem decreased by 2.5 cm over

the census interval. This decrease in mean size is accounted for

by height loss associated with stem death, coupled with sparse

gains in height through growth and a slight positive effect in

mean size through differential mortality among small individuals.

Trees that grew in 1991 had fewer stems (x=--2.4, s.d.=l.l,

n=36) than those that did not grow (x=2.7, s.d.=2.0, n=99) or

whose primary stem died (x=3.0, s.d.=2.3, n=25). This pattern was

repeated from the 1989, 1990 growth data. Trees that grew in 1991

also more frequently exhibited stem cankers, although this

pattern is probably a result of the fact that cankers are more

frequently found on larger stems, and that larger stems more

frequently grew in 1991 (Table 2.4). Trees whose stems died

during 1991 routinely exhibited a higher incidence of moderate

levels (>1%, >5%) of needle spots and needle necrosis (Table

2.5). Finally, trees that grew and were free from needle necrosis

40

grew more (x=9.8 cm, s.d.=6.0, n=17) than those that exhibited

needle necrosis (x=6.5 cm, s.d.=3.2, n=19)(T-test, T=2.02,

df=23.6,p=.05), although this pattern did not hold for needle

spots.

G-tests of growth category (Grew, Did not, Stem Died, and

Died) by habitat categories (relative elevation, canopy cover,

and aspect) revealed no significant patterns. Similarly, the

occurrence of stem cankers, needle spots and needle necrosis were

unrelated to habitat variables. Finally, growth fate of trees

prior to 1991 was not related to growth fate during 1991 (G-test

of independence, G=1.78, df=7, p>.5).

DISCUSSION

This work suggests a refinement in the estimate of the

number of extant T. taxifolia. This survey, along with additional

census information collected by Nature Conservancy volunteers on

their preserves account for 240 trees. An unpublished report from

Torreya State Park includes over 200 trees (Bryan, personal

communication). Several smaller populations not included in this

survey account for nearly 100 additional trees. Thus, we can

directly account for over 500 trees. This number exceeds one

estimate of the remaining population (Stalter and Dial 1984).

Most known populations of T. taxifolia are within 2 km of the

Apalachicola River. This study, the Torreya State Park survey,

and Baker and Leonard (1982) censused most ravine habitats within

2 km of the river in the southern half of the range of T.

taxifolia. The northern populations in this study (LKSM, FLCR)

41

Table 2.5. The percentage of trees, classified by growth fate in1991 exhibiting various symptoms disease. Needle spots and needlenecrosis are categorized by the portion of the total greenfoliage affected by the symptom.

Growth fate Disease symptom1991 Stem Stem Needle Needle

canker scar spots necrosis>0% >1% >5% >0% >1% >5%

Grew 44.4 25.0 94.4 30.6 5.6 52.8 19.4 0.0Did not grow 35.4 27.3 76.8 33.3 3.0 39.4 13.1 3.0Stem Died 20.0 24.0 92.0 60.0 24.0 68.0 48.0 28.0

Population 35.0 26.3 83.1 36.9 6.9 46.9 20.0 6.3

42

are included because they contain the only large populations of

trees known from this portion of the range. Based on our

assessment of the thoroughness of recent surveys, along with

anecdotal accounts of populations throughout the range (Baker

pers. comm., Gholson pers. comm.) we estimate that the extant

population is probably between 800 and 1500 individuals, and

almost certainly does not exceed 2000 individuals.

The pattern of larger mean size of individuals toward the

northern range limit may be a result of differences in habitat.

The Cody escarpment, delineating the Pleistocene sandy soils in

the south from the clayey Miocene soils in the north, divides the

range in the Big Sweetwater Preserve. Soil differences across

this escarpment may result in differential abilities of T.

1axifolia to survive and grow. Alternatively, this nonrandom

assortment of stem sizes may be a result of differences in the

geographical distribution of foliar disease, as suggested by

Table 5. One aspect of the demographic pattern of the T

taxifolia population that is not depicted well by these data is

that the trees are less abundant within the appropriate habitat

in the northern portion of the range. This is reflected somewhat

by sample sizes within populations.

The trunks of dead T. taxifolia remain well preserved on the

forest floor. The distribution of these trunks, as well as

anecdotal accounts, suggests a formerly more widespread

distribution with respect to slope position than was observed in

this study. Further, previous accounts of the species suggest an

43

historical distribution that included more trees further up

ravines away from the Apalachicola River (Figure 2.1). Few of

these trees remain. Thus, the decline seems to have been

accompanied by a narrowing of the environmental conditions across

which T. taxifolia may be found.

Also of note with respect to dead trunks on the forest floor

are our personal observations that these trunks were never

associated with live T. taxifolia. Thus, the current population

does not appear to consist of survivors from the decline but

probably grew from seeds germinated during or after the onset of

the decline. This observation is contrary to other reports

implying that the current population consists of stump sprouts

from formerly large individuals (Toops 1981, Savage 1983, USFWS

1986). It is not known how long seeds may lie dormant in the

soil, but a large nut is characteristic of seeds that do not

remain dormant for a long period of time.

Incidence of disease symptoms also varies geographically

across the range. High levels of foliar disease symptoms (needle

spots and necrosis) appear to be more frequent on trees in the

southern half of the range, while cankers are more common on

trees in the northern portion of the range. However,

interpopulation patterns of disease incidence is confounded by

other measures of plant vigor. For instance, stem cankcers are

more readily discernible on larger stems. Therefore, we expect a

higher incidence in populations of larger trees. However, these

results provide evidence that stem cankers are•not associated

44

with decreased plant vigor, as measured through frequency and

amount of new growth on cankered versus uncankered stems, while

symptoms of foliar pathogens are associated with higher rates of

stem death and less growth in the case of needle necrosis.

However, needle spotting does not currently appear as chronic as

suggested previously (Alfieri et al 1967, 1984, USFWS 1986).

The growth data demonstrate that the remaining population is

continuing to decline with 10% mortality, 32% stem mortality and

mean size of trees decreasing by 2.5 cm during the four year

census interval. The mean age of the oldest stems of individuals

is approximately half of what we would expect if the population

represented new, healthy recruitment following the decline in the

1950s. Thus, most individuals have lost their primary stem at

least once since, the decline 30 years ago. Further, the age

estimates for the census population approximate a normal

distribution, rather than being clustered at any single age,

suggesting that continued mortality among primary stems is

inhibiting maturation of the population. However, some of the

larger trees (>2 m) appear to represent first-generation recovery

from the time of the decline.

These census data allow an assessment of the pattern of

decline in T. taxifolia. Individuals that died during this census

period were smaller than average. The average length of a primary

stem that died on a surviving individual was slightly longer than

the average length of all primary stems, leaving a new primary

stem that was shorter than the mean for the population. Although

45

few individual cases of stem mortality can be attributed to any

single factor such as stem girdling, we can speculate that the

the mortality figures results highlight two modes of stem

mortality. First, small, suppressed, and presumably stressed,

plants eventually die when reserves are depleted and they cannot

maintain sufficient physiological activity. Second, older and

longer stems appear to eventually succumb to progressive disease

or fall prey to girdling by abrasions such as deer antler rubs,

leaving smaller stems behind. The fact that trees that grew had

fewer stems than those that did not implies that resprouting

behavior may also be a sign. of stress.

The true measure of whether T. taxifolia is exhibiting

greater than expected levels of mortality lies in a comparison

with other woody species with which it is associated. Stem

mortality was just over 10% for all trees greater than 2 cm in

diameter at breast height during a three-year census of trees in

a mapped plot in the same region (Schwartz 1990). Although these

data are not directly comparable, individual mortality (10%) and

stem mortality (32%) among T. taxifolia appears higher than

expected. This high mortality rate combined with the lack of net

growth insures that the population will continue to shrink both

in numbers and mean size.

There is hope of a recovery, however, from a small subset of

trees (<10) that seem to exhibit few Symptoms of disease and

appear to be approaching maturation. At present, there are four

sexually mature males and no sexually mature females known in the

46

wild. Two trees are large enough that we would predict them to be

sexually mature if they were male,, indicating that these may be

females. Without direct intervention to facilitate growth and

survival, T. taxifolia appears destined to continue its slow

decline toward extinction in the wild. The problem remains as to

what caused the decline and whether the vector still persists in

the current population, if any management intervention can

increase growth and vigor among the remaining individuals, and

whether those few individuals that appear to be doing well are so

because they are resistant or because they are lucky.

47

CHAPTER THREE

Genetic Variability in Torreya taxifolia

As a result of potential imminent extinction, the stinking

cedar was chosen a target species for ex situ conservation by the

Center for Plant Conservation and the Arnold Arboretum. Isozyme

analysis was used to characterize the genetic structure of T.

taxifolia in order to develop a plan for collecting and managing

material for a permanent collection to be housed in botanical

gardens.

Studies of the genetic variation associated with rare

species are important for developing our understanding of the

genetic structure of rare species (e.g., Falk and Holsinger

1990), as well as developing general expectations of levels of

genetic variation in species that differ life history

characteristics (e.g., Hamrick and Godt 1989). Assessing genetic

variability in T. taxifolia will assist in conservation efforts

to save this particular species in several ways. First, measuring

the amount and spatial distribution of inter- versus intra-

population variability will help determine a management strategy

for ex situ conservation. Second, this genetic characterization

of T. taxifolia can potentially identify isozyme markers

associated with disease susceptibility or resistance. Finally,

this study will help to determine whether the on-going decline

has caused a detectable genetic bottleneck.

48

METHODS

Needle tissue was gathered from 189 trees in 17 populations

distributed among six major drainage regions across the range of

T. taxifolia (Figure 3.1). In each population we sampled all

individuals encountered up to a maximum of 31 individuals. Table

1 provides sample sizes from each population. Most (150) trees

sampled for genetic analysis also had cuttings removed for

captive propagation. All sampled trees were individually tagged

for future reference.

The measure of genetic variability used for this study was

starch gel electrophoresis following the techniques of Conkle et

al. (1982). Starch gel electrophoresis allows detection of

allelic variation from needle tissue (Conkle et al 1982). Foliar

samples were processed and analyzed in the USFS Tree Genetics

Laboratory in Berkeley, California in collaboration with Dr. C.

Millar. Twenty loci among 16 enzyme systems were identified

(Table 3.1).

For each drainage region, I present the sample size, mean

number of alleles per locus, Nei's (1978) unbiased estimate of

expected heterozygosity (He), and the ratio of observed

heterozygosity (Ho) to He . Between drainage, and between

population, genetic distances are reported using Nei's unbiased

genetic distance (Nei 1978). The total genetic diversity (Hi) at

each locus was partitioned into four hierarchical components (Nei

1977): variation within populations (Hp), variation among

populations within drainages (Dxd), variation among drainages

49

Figure 3.1. The range of Torreya taxifolia depicting drainageregions in which trees were sampled for genetic analysis. Thesesampling units are: A) Lake Ocheesee, B) U.S. Corps of Engineers,Woodruff Dam, C) Flat Creek, D) Torreya State Park, E) BigSweetwater Creek, F) Apalachicola Bluffs and Ravines Preserve.

within regions (Ddr), and variation among regions (Dr). Regions

described in this hierarchical anlysis are: 1) drainages north of

the Cody escarpment, where relatively clayey soils dominate

(Figure 3.1, Lake Seminole, Flat Creek, Torreya State Park and

Big Sweetwater drainages); 2) drainages south of the Cody

escarpment, with relatively sandy soils (Apalachicola Bluffs

Preserve); and 3) trees from the outlying population not within

any ravine drainage (Lake Ocheesee).

The genetic relationship between populations was further

examined using a cluster analysis of Nei's unbiased genetic

distance. Cluster analysis used the unweighted pair-group method

with arithmetic averaging (UPGMA, Sneath and Sokal 1973) on the

17 populations sampled. All calculations were completed using

BIOSYS-1 ver. 1.6 (Swofford and Selander 1989).

RESULTS

Of the 20 loci sampled, only seven exhibited allelic

variation in the trees sampled, three of which contained

variation at a single tree or in a single population (Table 3.1).

Further, each variable locus contained just two alleles (Table

3.1). This low level of variability resulted in few polymorphic

loci and low heterozygosity (Table 3.2). The mean genetic

distance between populations within drainages (x=0.012,

range=0.000 to 0.044, n=29) is equal to the mean genetic distance

between drainages (x=0.012, range= 0.001 to 0.028, n=15) and

slightly, but not significantly, lower than the mean of genetic

distance between all populations (x=0.016, range=0.000 to 0.062,

51

4)

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10 0 4. 0 0 I

I0 I0b O 0 .4 0 - I

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n=136) (Table 3.3). The mean genetic distance between the

outlying Lake Ocheesee population and all other populations is

relatively high (x=0.018, range= 0.000 to 0.050) (Table 3.3).

However, this estimate of genetic distance is weak because sample

size is small. Only five trees remain in this outlying

population.

Using a hierarchical analysis to partition the total genetic

variation sampled (H.) into variation found at the population,

drainage, and regional levels demonstrates that most (79%)

genetic diversity is at the population level (Hp). Of the

remaining variation, relatively more was partitioned among

populations within drainages (Dpd, 10%) than either between

drainages (Ddr, 6.5%) or between regions (Dr, 4.5%) (Table 3.4).

Supporting the prevoius results, the cluster analysis of Nei's

genetic distance did not discern evidence of structured dif-

ferentiation among populations, drainages, or regions (Figure

3.2).

DISCUSSION

Plants with similar life-history characteristics often have

characteristic levels and distributions of genetic variability

(Hamrick and Godt 1989). Our observation of low genetic diversity

in T. taxifolia, along with this diversity being distributed

within, rather than between, populations fits the general pattern

of the distribution of genetic diversity in obligately

outcrossing, wind-pollinated coniferous trees (Hamrick and Godt

1989). However, genetic variation among conifers with extremely

55

4) 1 P HI 0

II D 'K00 0 000 100 4) 1 f-4 0000 > I II

U4J 0 1 1 IN (4 0%0 001wIrzvo I 001000104'00N01

004to I v-4 IM*1 * 00 0 0 * 1

04 INIV'K 0H0e IHO Irzr'I 'K00001

31 O0N Ia ION I

4-)I '0000 00 1

000 10 0I 0 I

4J I I * ' V- vM0 %DN 0'a14.10 I,-4I000000001

w cI 1-000000000.1I -) 11I

0 v I I 0OW0000WON I

4J I0 N Vc4 0 N00 V-4 0 0 N IOH 4) 1O H 0

000 If-1000 0000 'IG0'L0 0 f4I1

4.)$.M I 1 .0.000' 0 H41 0 00 0 I

fl'O i za LI1 0 00 00000000 1

(0 1 I I ( 'K 000 000 'D~-~0 0 0 I

*0 I I'ONu- O OC

tv I u0I 000 I

~ I I K000~ 00OakI a ') ' 00 - 0 00 0 H

Table 3.4. Estimates of the partitioning of the total genediversity (Ht) for seven polymorphic loci. See text forexplanation of levels of genetic variation.

Locus H D Ddr DrPGI-2 .488 .3 8 .*09 -.011 .061AAT .496 .387 .043 .064 .002SIX .326 .280 .010 .032 .004UGP .046 .043 .005 -.001 -.001IDH .019 .016 .000 .005 -.002MDH-2 .013 .013 .003 -.002 .000PGI-1 .002 .002 .000 .000 .000

Mean .199 .157 .020 .013 .009Percent 78.9 10.1 6.5 4.5

57

Figure 3.2. Cluster Analysis of 17 Torreya taxifolia populationsusing Nei's (1978) unbiased genetic distance.

.05 .04 .03 .02 .01 .00 DrainagePopulation RegionTorreya St Park A D

:Torreya St Park B D

'Big Sweetwater A B

--- Beaverdam Creek F

--- - Ocheesee AI I

SIAspalaga Creek D

--- Big Sweetwater D B

I II II 'No Name Creek A F

Lake Seminole B

- No Name Creek C F

----- ---'No Name Creek B FII

- Kelly Creek F

- Big Sweetwater E E

-- Big Sweetwater B E

--- Big Sweetwater C E

Flat Creek B C

IFlat Creek A C

.05 .04 .03 .02 .01 .00

Farris (1972) "F" = 127.4Percent Standard Deviation = 98.7Cophenetic correlation = -0.521

58

limited distributions, such as torrey pine and, can be variable.

For instance, torrey pine has very low variability (Ledig and

Conkle 1983), while monterey pine has high levels of isozyme

variability (Millar et al. 1988, Moran et al. 1988). Both species

have severly limited distributions and small populations.

This characterization of the genetic diversity in T

taxifolia is consistent with a pattern for a species of tree that

has been subjected to population bottlenecks (Critchfield 1984).

A genetic bottleneck hypothesis is supported by the fact that T.

californica, the only other North American congener, has

relatively high levels of genetic variability (Schwartz and

Millar, unpublished data). The observation that there is little

differentiation between populations suggests that this

hypothesized genetic bottleneck occurred prior to the current

decline. If the depauperate nature of T. taxifolia were a result

of the current decline, we would expect to see few common alleles

and many rare alleles scattered among the individuals sampled,

which is not the case. The observed pattern of genetic variation,

under a hypothesis of a current genetic bottleneck, is only

likely if the inferred isozyme alleles lost as a result of this

decline were tightly linked to characters that were strongly

selected against during the current decline, a scenario which

seems improbable.

The hypothesis of a past genetic bottleneck is supported by

two additional pieces of evidence. First, Taxus floridana, a

small coniferous tree restricted to nearly an identical range as

59

T. taxifolia, also has uncharacteristically low levels of genetic

variability (Schwartz and Millar, unpublished data). Second,

isozyme analysis of a few individuals that have been growing

outside the range of T. taxifolia since before the decline do not

show higher levels of genetic diversity than trees in the wild

(Schwartz, unpublished data). Thus, T. taxifolia probably

experienced at least one other population bottleneck prior to the

current population decline that resulted in a reduction of

genetic variability in the species. This is not to say, however,

that the current decline has not resulted in significant loss of

genetic variation. Given the magnitude of the current decline, it

quite likely has suffered additional loss in genetic variation.

Finally, with the low levels of genetic variation observed

in T. taxifolia, it is improbable that a supposedly neutral

marker, such as an isozyme allele, could be identified that would

identify particularly resistant, or susceptible trees. Indeed,

none of the three rare alleles .sampled in this study were found

in either of the two trees sampled that appear to be healthy and

maturing in the wild. Given that we have not been able to

identify which, if any, of the disease symptoms currently

exhibited in the wild are associated with the primary pathogens

of the decline (Schwartz 1990), testing for the co-occurrence of

alleles and disease symptoms is premature.

Management Recommendations

The adopted strategy for ex situ conservation of T.

taxifolia has been to place one cutting from each of the 150

60

trees sampled at each of four botanical gardens (R. Nicholson.

Personal Communication). These cuttings will then be reared to

maturity. This strategy was adopted to minimize the risk of loss

of any individual genotype. Given the low inter-population

variability of T. taxifolia, it appears unlikely that the

crossing of individuals from different populations will create

outbreeding depression problems, or disrupt locally adapted gene

complexes. Thus, this management course should be maintained.

Further, a few mature trees from four known locations

outside the natural range are currently producing seed. Inclusion

of seed from these botanic garden and yard trees could accelerate

the process of developing a permanent ex situ collection, as well

as assist in developing a population for eventual re-stocking of

native habitat. From the results presented here, it appears

unlikely that inclusion of this genetic stock of unknown origin

would significantly disrupt the ex situ conservation population.

Cultivation from available seed is recommended following genetic

screening of these reproductively active trees.

61

CHAPTER 4

The Light Relations of Torreya taxifolia with Special

Emphasis on the Relationship to Growth and Disease

Light is often a limiting resource for plant growth (e.g.,

Tilman 1988). Low light conditions can create plant stress,

potentially enhancing the ability of pathogens to successfully

attack plants (Manion 1981). One hypothesis that may explain the

lack of recovery in T. taxifolia is that growth may be limited by

chronically low light levels (see Chapter 1). This hypothesis

relies on two conditions. First, that there was a decrease in

light level penetrating to lower ravine slopes associated with

fire suppression in the surrounding uplands and upper ravine

slopes (see Chapter 1). Second, the distribution of the species

has been compressed to lower slope (and hence lower light)

positions; a condition witnessed by the locations of dead stems

on the forest floor as well as current mortality patterns (see

Chapter 2).

Thus, low light may be associated with decreased

survivorship, reduced growth, and increased disease symptoms.

These phenomena relate to the hypothesis that increased shading

through increased densities of hardwoods on upper slopes, as a

result of fire suppression, could have contributed to decreased

vigor and the decline of T. taxifolia (see Chapter 1).

62

In addition, the foliar pathogens causing needle spots and

needle necrosis (see Chapter 2) may inhibit T. *taxifolia's

ability to thrive in the low light conditions in which it

currently grows. More specifically, foliar pathogens may inhibit

maximum photosynthetic rates in T. taxifolia. The relationship

between foliar pathogens and plant vigor is particularly

important in T. taxifolia because a causative agent of the

catastrophic decline has not been ascertained. One hypothesis is

that foliar pathogens were responsible for this decline, and are

responsible for the current lack of recovery in the species (see

Chapter 1). We can begin to test this hypothesis by examining the

effect of foliar pathogens on rates of photosynthetic activity.

Alternatively, several phenomena associated with plant

health can be addressed by studying the light relations of T.

taxifolia. Hypothesized environmental stress, such as increased

ravine temperatures, decreased humidity, and drought, may have

increased plant stress in T. taxifolia, and enhanced disease. An

obvious symptom of this purported plant stress would be a

decreased ability to photosynthesize under stressful conditions.

METHODS

We use two general procedures to'assess the light relations

of T. taxifolia. First, we assess the effect of light levels on

tree growth th through field surveys and experimental manipulation

of the light environment of trees. Second, a portable gas

exchange system (LICOR-6200) was used to measure photosynthesis

in T. taxifolia under varying environmental conditions. For these

63

assessments of physiological response to environment stress we

assume that photosynthetic rate is a measure of plant vigor and

that decreased photosynthetic rates indicate plant stress.

Light and Growth

To assess the relationship between light level and growth a

correlation between recent growth and canopy cover was used. This

data was coupled with an experimental test of whole plant growth

in canopy openings versus growth under closed canopies. Twelve

trees were selected for use in an analysis of recent growth and

light levels. All trees were between 95 cm and 328 cm in height.

Recent growth was measured through three variables: 1) mean

length of the three most recent terminal internodes along the

main stem, 2) maximum terminal internode length of these three

most recent terminal extensions, and 3) the proportion of lateral

branch tips extending new growth in the current year.

These growth values were compared to an estimate of canopy

closure for each tree. Canopy closure was measured using the

frequency of point light intercepts assessed through overhead

photography. Four photos were used to characterize the canopy

over each individual tree: one directly overhead, and three at

450 angles facing due south, east, and west. The northern aspect

was excluded as it is less important in determining the amount of

daily direct sunlight a plant receives. Slides of the canopy were

projected onto a screen containing 100 randomly located points.

The same 100 points were used for all slides. For each slide, the

proportion of points where sky was obstructed by vegetation was

64

recorded. A mean of the four slides was used as a measure of

percent canopy closure. Canopy closure was correlated to each of

the growth measures through linear regression.

The experimental test of the effect of light levels on

growth was begun in the spring of 1991. Twelve trees were chosen

to have canopy gaps opened above them. All trees were located on

the Apalachicola Bluffs and Ravines Preserve (Figure 2.1).

Terminal bud expansion, and the length of the most recent needle

bearing branch internodes along the main stem were measured as

response variables. These response measures were compared to

measures of plant growth produced prior to the canopy opening.

To assess the relationship the presence of disease symptoms

and light level we used a contingency table test (G-test, Sokal

and Rohlf 1981) between visual estimates of percent canopy cover

class and the presence of disease symptoms. Finally, we use a G-

test (Sokal and Rohlf 1981) to examine the potential relationship

between growth fate over the three year census interval (see

Chapter 2) and the various canopy cover classes. This test

provides an additional estimate of the relationship between light

level and growth.

Measurements of Photosynthetic Rates

A series of field tests are described below that attempt to

ascertain the relationship between growth, plant vigor and light

in T. taxifolia. Field measurements of photosynthetic rates were

used to address the hypothesis that the decline in T. taxifolia

was a result of foliar pathogens (Chapter 1). In addition, a

65

series of lab experiments were conducted measuring photosynthetic

capacity of T. taxifolia and closely allied species to test

hypotheses relating environmental change to plant stress that may

have facilitated the decline (Chapter 1). All of these

experiments use the LICOR 6200 closed system infra-red portable

gas analyzer. The LICOR 6200 gas analyzer is a closed-system

device that allows non-destructive sampling of photosynthetic

rates by enclosing branchlets of needles into an airtight one

liter chamber. The LICOR 6200 measures the rate of CO2 drawdown,

along with numerous other parameters (e.g., temperature, light

level, and humidity). Instantaneous rates of photosynthesis and

conductance are then calculated by standard gas-exchange

equations. Photosynthesis, naturally, is dependent on

concentrations of CO2 . All experiments were conducted under CO2

concentrations between 350 and 400 ppm (ambient atmospheric

conditions).

We began by establishing a baseline photosynthetic light

response curve for T. taxifolia. This field test was conducted

using first year needles from 15 plants under natural and

enhanced light conditions. Light levels were varied from a

maximum of 12.2 to 1821 /mols/m2/sec Photosynthetically Active

Radiation (PAR). When possible, natural light was used with shade

cloth applied to reduce incident light. In many cases, however,

natural light was insufficient. In these cases light was enhanced

using a 12v tungsten halogen 100 watt projector lamp with

reflector (Sylvania EFP). No difference in maximum rates of

66

photosynthesis were observed when plants were illuminated with

natural versus artificial light sources.

To determine if the presence of symptoms of foliar pathogens

are related to carbon gain, maximum net photosynthetic and

respiration rates of healthy needles were compared to those

bearing needle spots. The difference between the rates of

photosynthesis and respiration is a measure of relative carbon

gain potential (Fritter and Hay 1981). These measures of

differences in rates of photosynthesis between healthy and

diseased needles were conducted in the field during the summer of

1991. Measurements were collected between 9:00 AM and 4:00 PM,

using either direct solar irradiation, or when necessary, the

lamp described above. In all cases, measurements were recorded

only when light levels exceeded the light saturation point for T.

taxifolia (350 Mmol/m2/sec PAR).

A comparative study of the photosynthetic response rates

across environmental variables for five species within the family

Taxaceae was conducted in order to assess whether T. taxifolia is

particularly sensitive to certain types of environmental stress

(see Chapter 1). We compared species responses to light level,

temperature, humidity, and water stress for the following

species: T. taxifolia, T. californica, T, grandis, T. nucifera,

and Taxus floridana. The four species in thegenus Torreya were

chosen to provide a broad sample of how closely related species

respond to environmental conditions. Taxus floridana was chosen

because it is related at the family level and is completely

67

overlapping in distribution and habitat with Torreya taxifolia.

These tests were performed in the greenhouse and in

controlled environment chambers. Seedlings of T. californica and

Taxus floridana were 2- to 3-years old and had been grown outside

under full sun. The Torreya taxifolia, T. randis, and T.

nucifera were derived from cuttings and grown under greenhouse

conditions for 2 years. All plants were acclimated to greenhouse

conditions for at least one month prior to measurements. All

plants were fertilized weekly with 20-20-20 fertilizer and were

grown in 5 inch clay plots containing a peat:pearlite:sand (1:1:1

vol.) mixture.

In order to maintain similar environmental conditions, and

have control over temperature, measurements were conducted in a

Conviron growth chamber (Controlled Environments Inc., Pembina

ND, USA) with a 12 h photoperiod and day/night temperatures of

220C/180 C. The growth chamber was illuminated by banks of 195 W

Sylvania cool white fluorescent bulbs and 40 W General Electric

incandescent bulbs. Together these lights provided approximately

400 Mmol/m2/sec PAR at the top of the crown. To insure light

saturation during measurements, the 12v 100 W projector lamp

described above was used to supplement light within the growth

chamber. All measurements were conducted at PAR greater than 500

Mmol/m2/sec and between 350 and 400 ppm CO2.

Temperature response curves of net photosynthesis and dark

respiration were conducted at a constant vapor pressure to

eliminate the potential effects of varying moisture availability

68

on stomatal aperture (Fritschen and Gay 1979). Measurements of

light, and drought photosynthetic response curves were conducted

at 200C and 50% relative humidity. The drought study was

initiated by saturating pots with water and measuring

photosynthesis daily for 6 days. Water was withheld for the

duration of the study and the pots were individually weighed just

prior to photosynthetic measurements to determine water loss from

the pots. Photosynthetic response to humidity was determined by

maintaining the appropriate relative humidity (approximately 20%,

50% and 90%) within the leaf chamber at 200C.

RESULTS

Light and Growth

Field tests of light and growth had mixed results. Both

maximum and mean terminal extension length were positively

correlated with light level (Figure 4.1). In contrast, we found

no relationship between the proportion of lateral branch buds

that expanded and light (r=.16,p=.62). The observed relationships

between terminal extension and light are influenced by the

contribution of a single tree with very high light levels.

However, the significance of the light and growth relationships

remain after exclusion of this outlying observation.

One year growth measurements on the 12 trees for which light

levels were increased showed no observable increase in growth

response. The majority (9 of 12) either died or failed to expand

a terminal bud during this first year of the test. Thus, we

recorded no positive growth response of trees to increased light

69

Fiqur 4.1. Average length of terminal bud elongation in the past

three years of TorreyA taxifolia plotted against percent canopy

light transmission (see text for explanation of methods). A)

without Aspalaga tree (outlier), B) all observations

A Torreya16

14

Average 12Terminal

ShootElongation 10

8

6

y = 5.5845 + 0.25749x R^2 * 0.302U

B

0

10 20Percent Canopy Light Transmission

30

BTorreya

AverageTerminal

ShootElongation

10

16

14

12

10

8

610 20 30

Percent Canopy Light40-

Transmission50

iA 1 & % ^L iAd9b - a& a0% M A aA ^ A A^ AP^" -

levels treatments. Likewise, canopy cover class was not strongly

related to three-year survivorship, although slightly more trees

in high light classes died or suffered major stem death than

expected, fewer trees died or suffered major stem death in low

light classes than expected, and more trees grew in low light

classes than expected (Table 4.1). This result also runs counter

to our light limitation hypothesis. Finally, neither the

occurrence of needle spots, needle necrosis, or stem cankers

appear related to canopy cover class (Table 4.2).

Measurements of Photosynthetic Rates

A comparison of light curves reveals that the light

saturation point is at approximately 300 Mmol/m2/sec PAR for both

healthy and diseased needles (Figure 4.2). Healthy needles,

however, have an approximately 20 % higher maximum net

photosynthetic capability than diseased tissue (Figure 4.3). In

addition, the 20% reduction in photosynthetic output of diseased

needles exceeds the total surface area effected by the foliar

pathogens, indicating a disruption of internal leaf function,

rather than a simple reduction in photosynthetically active

tissue. Likewise, we measured the dark respiration rates of

healthy needles from eight trees versus diseased needles from

eight trees. Dark respiration rates were calculated using

multiple measurements (1-4) on each tree. Healthy needles have

lower respiration rates diseased needles (Figure 4.3).

Measurements of productivity by needle age demonstrates that

T. taxifolia retains as much as 90% of maximum net photosynthetic

71

Table 4.1 Variation in survival and growth compared to canopycover class. Chi-square test of association lumped adjacent coverclasses 1, and 2 with 3, 4 with 5, and 6 with 7. Individuals thatdied were lumped with individuals whose major stem died.Coalescing of groups was done to avoid sensitivity of theanalysis as a result of small expected values in accordance withSokal and Rohlf (1981).

Canopy cover classGrowth open closedFate 1 2 3 4 5 6 7 tot

Died 0 1 1 0 2 0 0 4Major Stem Death 0 2 7 5 6 0 0 20Did Not Grow 0 3 8 8 15 1 1 36Grew 1 2 3 2 9 3 2 22

Total 1 8 19 15 32 4 3 82

G=13.49, X(.05,7df)=14.07, .05 > p > .1

Table 4.2 Variation in disease symptom by canopy cover class.

Canopy Cover Classopen closed1 2 3 4 5 6 7 total

CankerYes 0 3 5 6 14 15 20 63No 0 6 13 15 29 18 10 91Total 0 9 18 21 43 33 30 154

Needle SpotYes 1 3 8 6 16 10 11 55No 0 6 13 15 30 28 24 116Total 1 9 21 21 46 38 35 171

Needle NecrosisYes 0 5 18 13 33 18 21 108No 1 4 3 9 14 20 14 65Total 1 9 21 22 47 38 35 173

Needles ChewedYes .0 3 5 8 11 15 12 54No 1 3 8 8 12 18 15 65Total 1 6 13 16 23 33 27 119

All G-tests (within damage category): P>. 25

72

0.

U,.0

'7,0.0

U'.0

0~00

U'o'-a

t4J U'00

U'0

0

Photosynthesis

AMm0lC0 2 /m 2 /2

0 0 0v 0g

~ ENOI I0

01 ((Art.

0 0 :0

*m 01'

* ~ 0

U~0'

0Ct

1b~d.~1U)0'

Figure 4.3. Comparative rates of A) net photosynthesis and B)dark respiration healthy needles versus bundles of needles inwhich some needles exhibit needle spotting.

10 -

8-

6-

4-

2-

0

0

I.09

0

S-= 5.56p<.01

x = 4.42

HEALTHY DISEASED

Dark Respiration Rate

t.1.99, pM.07

A A

HealthyNeedle

DiseasedCondition

0

0

Co

LUzI-z(00I-0z0.

c~4

0'U

0

0

0

-1 -

Uc~4

0Up.40U

IC.2Cu

.5.00

.3

a

soI I

Af

Figure 4.4. Rates of net photosynthesis for needles of varyingage. Measurements collected in A) February, prior to new needleexpansion, and B) Autumn.

Maximum Photosynthetic Rates

Ie0S

890

9

8

7.

6

5

4.

3-

4

Needle Age

September, 1991

5-

4.

3"

* *

I U _

0 1 2 3

Needle Age

F 5 6

0

00

U/)

C

(00o

>.0)

0.

1

0

(0

(0

0

o

2

IM . j" I

I - "W- "-It

capacity in fourth year needles (Figure 4.4). Further, this

pattern is repeatable throughout the growing season (Figure 4.4).

A comparison of greenhouse light response curves among the

five species examined here demonstrates that T. taxifolia has a

mean maximum net photosynthetic rate (8.3 4mol C02/m2/s) slightly

above all but T_ californica (Figure 4.5). In addition, T.

taxifolia reaches light saturation at PAR levels approximately

equivalent to other closely related species (Figure 4.5).

However, light curves for T. taxifolia from the field and in the

greenhouse are different in that maximum net photosynthesis

rates, as well as the light saturation point, are higher in

greenhouse plants (Figure 4.2, 4.5).

Net photosynthesis did not vary between species with respect

to relative humidity (Figure 4.6). The suite of species tested

all maximized rates of photosynthesis at about 20°C but varied in

the shape of their temperature response curves (Figure 4.7). In

particular, Taxus floridana and Torreya nucifera showed

particularly narrow ranges of temperature over which net

photosynthetic rates were high.

Finally, these five species were tested with respect to

drought tolerance. These data are presented as mean net

photosynthesis and mean stomatal conductance by day because the

mean decrease in percent of saturated pot weight did not vary

significantly between species (Figure 4.8). In addition, the

plants used for this experiment were of equivalent size. Thus,

the variable "day" can be used as a proxy for partial water

76

Figure 4.5. Comparative light saturation curves for five closelyrelated species: A) Torreya taxifolia, B) T. califoria )T

nucfera,, D) Ta31 floridaia and E) Torreagrandis.

Torrya l taxifol0.

0.000.0001

00

0

y0

500

0

@00.0 * 0.

10'10

U)

S6CM

04

0

1000- 1500---

2000 0

Taxus fioridana

00

000@0000

~00

OJO

NoF

500 1000 1500

Torreya callfomlca00

~ 00

0 0

I

*0 0 0

10

8

6

.4

-, 0'50 1000 1500 2000

Torreya grandis

0

*@' 00

5I 1000 1500

PAR (gL morn2 Is)

Torroya nucifemr

0

1500 2000

PAR (g mor/m2 Is)

4.

0

00

10 1

2000o

Oc

0%

Z w

0 '1

000 0

0~0~

2000

0.0

10-

8'

Ci 4

0

0

0@

0*0

0 .

0

I/

50 1000.

wI W 0

i- v m w a v 0 v 0 v I w I-

- 1

F

I- I ,

Figure 4.6. A comparison of net photosyntnetic rates acrossvariation in relative humidity for five closely related species:A) IorrYa taxioir.~Ja, B) TI. californica, C) L6.9. ncifer~a, D) Iax3JafJlo.i4dni1 and E) To9rre.ya gani

Torreym taxIfola

I0

11 II

109-

7.6.45.M4.3.2.1.0

109.6.7.6.5.

4..

2..

0.

0 20 40 60 80 100

000

Taxus fioridana

0 20 40 60 s0 100

Torreya cailfomica

40I w I a

80 so 104

1029.8-7.625.4.3.2.

0.0

Torreya grandis

0

S I

U - U - I

20 40 60 80

Relative Humidity(%

Torreya nucifera

0 i

I v I * I 1

40 60 80 100

Relative Humidity (%)

I

0 =

z ,oo

I * I * I * I * I

ON

10 -9-

7.6-

4-3.2-.1I0.

0 2

'I

U)..

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'C

-o0

lo

9.

7.6.5.4.3.2.

10

100

0 20

I I I · I · I

- Onew

F w 5

mr-1

IO

I

4.4

Figure 4.7. A comparison of net photosynthetic rates acrossvariation in temperature for five closely related species: A)Tory taxi'folia, B) I. californica, )T nucifera,, D)Tasfloridana, and E) Iflreya rra~idis.

Tormya taxifolla10

0

I

8.

64

4-

2,

0l

Taxus floridana

0 10 20 30 40 50 0 10 20 30 40 50

1

0 10 20 30 40 50

Temperature ( 0C)

not photosynthesIsa dark rsplratlonI

50

E

N

0

E

E

0 10 20 30 40

Temperature ( OC)

Figure 4.8. A comparison of percent soil moisture through timewithout water for five closely related species.

AISI

6?

100

90

80

70)

Uw

0 1 2 3 4 5 6 7

Day

100-

80-

60-

40-

20"

An-v0 1. 2 3

Day

I I I

4 5 6.

Figure 4.9. A comparison of mean net photosynthetic rate againsttime without water for five closely related species.

-- o-- Torreya taxifolla--- Torreya califomica=- Torreya grandis* Torreya nucifera

-- +-- Taxus floridana

41

I.

a

I I .i ' r r i

I -I I-1

pressure of the soil within pots. These results suggest that net

photosynthetic rates in T_. grandis and T_. nucifera are most

sensitive to water stress, and that T. taxifolia is as robust,

with respect to water stress, as any of the species tested

(Figure 4.9).

DISCUSSION

The field results presented here provide mixed support for

the hypothesis that T. taxifolia is currently stressed by low

light. The correlations of growth with light suggest that T.

taxifolia thrives in high light situations and that growth is

limited by low light. In contrast, preliminary growth response of

trees in experimentally created gaps have failed to support the

conclusion that low ambient light may be limiting growth in the

field. However, we require additional data over time to

adequately test whether our experimentally created gaps effect

growth in the field. Initial results from a greenhouse growth

experiment indicate that light levels below 400 Mmol/m2/sec PAR

significantly limit growth rate (Schwartz et al. unpublished

data). The low light saturation point (350 Mmol/m2 /sec PAR) of T.

taxifolia and the frequency with which they are found under dense

shade (Chapter 2) suggests that the tree can remain suppressed

under fairly dense shade for extended periods. An analysis of

tree-ring cores from long-dead trees would help clarify this

question. A cursory examination of tree cores from dead logs

appear to exhibit groups of wide and narrow annual growth rings

characteristic suppression and release growth patterns (Schwartz

81

personal observation). At the present time, most of the trees in

the census population are found in light environments far below

the light saturation point (Chapter 2, p32). Finally, the results

from the three-year census indicate little relationship between

light level and survivorship.

The data we present on differences in net photosynthetic

rates between healthy and diseased tissue, as well as needles of

differing ages, indicate that a foliar pathogen remains a

plausible candidate for the cause of the decline. However, one

must bear in mind that these results show a correlation between

disease and reduced net photosynthesis and do not prove cause and

effect. Although, the fact that we often measured healthy needlesV

immediately adjacent to diseased needles indicates a highly

localized infection, typical of foliar pathogens. Foliar

pathogens have typically been considered unlikely candidates for

catastrophic declines because they rarely kill the trees that

they infect (Manion 1981). Foliar pathogens can defoliate trees

in a single season,. but this is rarely sufficient to kill a tree.

However, T.taxifolia is different for several reasons.

First, we have shown that needles that bear pathogens have a

reduced capacity for carbon gain, a necessary but insufficient

pattern, if we are to believe that foliar pathogens are

responsible for this decline. This reduction in net

photosynthesis will result in plant' stress if, as we suggest, the

pathogen causes the decreased physiological activity, and is not

merely a secondary effect. Second, most conifers demonstrate a

82

marked decline in photosynthesis with needle age. In contrast,

needles up to four years of age on T. taxifolia are

photosynthetically active. Thus, defoliation of older needles, as

is often observed, would significantly reduce the total

photosynthetic capacity of the plant. Further, these older

needles are not readily replaced. Each year's single flush of

needles represent a small component of the plant's total

photosynthetic tissue. The observation that only about 70 * of

lateral branch buds and less than 30% of terminal branch buds

elongate each year (Chapter 2) indicates that it may take several

years for a defoliated tree to recover a full compliment of green

tissue. Since T. taxifolia is most frequently found as small

suppressed trees beneath a dense forest canopy, most trees are

probably close to the minimum carbon gain levels for survival.

The overstory of many of these ravine forests has been in a

maturation cycle for the past 30-50 years since selective cutting

of hardwoods in the mid-1900's. In a maturing forest there are

likely to be relatively few canopy gaps providing high light

levels for release of suppressed trees. The additional stress of

a single bout of heavy defoliation may have been sufficient to

kill most trees. If these factors are not fully responsible for

the decline, then they are at least likely to help explain why T.,

taxifolia has not recovered from the decline of 30 years ago.

This scenario, however, remains speculative until a foliar

pathogen can be isolated and used to test the foliar pathogen

hypothesis.

83

Our comparison of temperature, relative humidity and drought

response of T. taxifolia to other closely related species reveals

that we have no reason to suspect that T. taxifolia should be

more susceptible to environmental stresses than other similar

species. This is particularly interesting in light of the fact

that Taxus floridana grows over the same range and in the same

habitat as T. taxifolia, yet was not affected by a decline in

population size. Further, the similar response between T.

taxifolia and T. californica with respect to water stress is

somewhat surprising because the climate in the habitat of T.

californica is Mediterranean, and very drought prone. Finally, T.

taxifolia appears to be more tolerant to short-term water stress

than Taxus floridana. We would, again, predict the opposite if T.

taxifolia became susceptible to disease as a result of drought

stress, as has been suggested (USFWS 1986, Chapter 1). Thus, we

find no support for the hypothesis that environmental stress,

namely increased ravine temperature, decreased humidity, or

decreased soil moisture, would have rendered T. taxifolia

particularly susceptible to natural pathogens so as to cause the

catastrophic decline. While all of these stresses may contribute

to limit carbon gain, they do not seem to effect T. taxifolia to

a greater extent than other species, particularly a closely

related potential competitor, Taxus floridana.

84

CHAPTER 5

The Foliar Fungal Associates of Torreya taxifolia:

Pathogenicity and Sensitivity to Smoke

As stated previously (Chapter 1), there is no apparent

virulent introduced pathogen involved in the decline Torreya

taxifolia. The root of the problem in isolating potential

environmental mechanisms for the cause of the decline is in

isolating a pathogen. Here we present data that describe our

attempts to isolate pathogens from T_. taxifolia and establish

pathogenicity. However, all these attempts at establishing

pathogenicity of fungal associates have failed. We have not been

able to determine the cause, proximate or ultimate, for the

decline of T. taxifolia. Unfortunately, these pathogenicity tests

also do not exclude the potential pathogens that we tested from

further consideration. We have isolated several potential

pathogens, and tested them for pathogenicity. However, we cannot

say that these fungi might not behave as pathogens under a

particular set of environmental conditions that we have not

tested.

We have also been testing certain foliar fungal associates

for susceptibility to smoke. Through these tests we can assess

the likelihood of the fire suppression hypothesis for the decline

of T. taxifolia (see Chapter 1). This hypothesis suggests that

85

smoke from frequent fires in the adjacent uplands maintained low

populations of potential pathogens. Fire suppression, in the

1950's, allowed pathogen populations to explode, precipitating a

catastrophic population decline. Fungal associate isolation

strategies, pathogenicity tests, and smoke susceptibility tests

are described below.

METHODS

Fungal Isolation

All needles collected for fungal isolation were from wild-

grown trees. Attempts to isolate fungal pathogens from needle and

stem tissue have been repeated in all seasons over the past three

years. We used needles that exhibited foliar symptoms of disease,

such as needle spots and needle necrosis, as well as those that

appeared healthy. We have sampled needles from trees throughout

the range of the species. Needles of T. taxifolia collected for

fungal isolation were surface sterilized in a 10% bleach

solution, a 50% alcohol solution, a flame sterilization, or any,

and all, combinations of these techniques. Needles were immersed

in sterilizing solution for 1-to 3 minutes. Needles were either

cut or placed on artificial medium whole. Stem tissue was also

collected from wood tissue and surface sterilized in the same

manner as needles. Wood samples were healthy portions of cambium

or heartwood adjacent to cankers. We used several types of

artificial growth medium for our isolation attempts. These

include: Potato Dextrose Agar (PDA), Malt Agar, Water Agar, Corn

Meal Agar, and Water Agar with sterilized straw. By using a

86

variety of techniques we hoped to isolate as many different

potential pathogens as possible.

Pathogenicity Tests

A selection of foliar fungal isolates, derived from the

aforementioned methods, were tested for their pathogenicity on T.

taxifolia. A fungal spore slurry of at least 100,000 spores per

cc was misted onto test plants in a controlled environment

chamber. Plants used for pathogenicity tests were divided into

two groups. One group received full water treatment, while the

other was maintained under water stress conditions. Water stress

was defined as a soil moisture of between 70 and 80 percent of

saturated pot weight. This level of soil moisture was previously

observed to result in a significant decrease in photosynthesis

(see Chapter 4). Plants were maintained in these treatments for a

minimum of two weeks after inoculation.

Fungal Associates Susceptibility to Smoke

Smoke susceptibility in fungal isolates was tested in three

experiments. The first experiment tests the effect of smoke

residue on fungal colony growth. Clean culture plates were

placed, top open, in a closed chamber and subjected to smoke for

varying lengths of time from 0 to 20 minutes. Five replicate

plates for each smoke treatment were then inoculated with a small

fungal colony, not exceeding mm in diameter.. All plates were

placed at room temperature and illuminated by sunlight for the

duration of one week. Colony diameter was measured on a daily

basis throughout the experiment. The experiment ended when the

87

unsmoked plates reached the edge of the culture plate, in

approximately one week.

The second experiment tested the direct effect of smoke on

fungal colony growth. Colonies were begun in replicate culture

plates and allowed to grow to approximately 2 cm diameter. Five

replicate plates with fungal colonies were then smoked as per the

procedure described above. Subsequent colony growth was recorded

as above. Smoke for these treatments was cooled along a 20 ft

length of 6 inch duct pipe. The pipe was cooled by running water.

Thus, we eliminated any direct effect of high temperature on the

fungal colonies during smoke treatment.

A final experiment tested the effect of smoke residue on

fungal spore germination. We used five replicate smoked culture

plates for three time treatments (0, 1, and 5 minutes), as in the

first experiment. After smoke treatment a slurry of fungal spores

was spread over the smoked culture medium on each plate. Spores

were examined after 12 hours to-determine rates of spore

germination.

For each experiment clean glass slides were placed on the

tray at the time of smoke treatment. The glass slides were

scanned in a spectrophotometer (Bausch and Lomb Spectronic 70) at

470 nm to measure percent light transmission after smoke

deposition. Percent light transmission was measured for each

smoke treatment level as determined by time. A linear°i

relationship exists between percent light transmission and time

of smoke treatment (Figure 5.1).

88

.Figure 5.1. Biplot of the relationship between time in smokechamber and density of smoke residue deposited on clean glassslides in chamber. Smoke residue is measured as percent of lighttransmission through a clean glass slide'at 470nm.

100

0ZI

E

cu

90

80

70

60-

50

8

8

10 20 30

Time (Minutes)

Table 5.1 Fungi isolated from Torreya a ~iQ1± in the nativerange.

species Smoke 1st iuoo 1st spore

£lUAr.23 pe x xkcrimnamsp. x- x

------------------------------------

A mixture of pine straw and wiregrass was collected from

mature upland longleaf pine forests within the natural range of

Torreya taxifolia for use as fuels for all experiments. These

fuels are representative of the fuels in natural and managed

fire. We chose the most frequently isolated fungi in our tests of

smoke susceptibility. The fungal species used in each these

experiments are listed in Table 5.1.

Field Tests of Smoke Hypothesis

A field test of the amount of smoke deposited into ravines

through upland fire management was begun in the spring of 1992.

This experiment was delayed as a result of a poor fire season in

1991. Thus, the results from this experiment constitute smoke

from only a single fire treatment, and must be regarded as

preliminary.

Finally, a field test of the smoke hypothesis was begun in

1989. Twenty pairs of trees have been planted in the wild for

testing. One tree from each of these pairs was to have been

smoked each spring. We would then monitor the incidence of new

disease in these trees. As a result of vandalism this experiment

has been temporarily abandoned. We have lost 7 of our 40 plants

to natural causes that appear to be related to transplant shock.

None of these plants exhibited symptoms of disease. An additional

17 plants have been dug up over the two year period of this test.

We recently discovered the culprit and this disruption will now

cease. However, we have lost the bulk of our data from this

experiment through this vandalism. The remaining plants, however,

90

appear healthy and show no symptoms of new disease.

RESULTS

Fungal Isolation and Pathogenicity

During the course of three years we have isolated three new

potential pathogens from T. taxifolia (Table 5.1). Pestalotia

natens is an exceedingly common fungus found on nearly every T.

taxifolia throughout the natural range. This fungus is readily

isolated using PDA. Fusarium sp. was also frequently isolated on

PDA from both needles and cambium tissue. A species from the

genus Acremonium was infrequently isolated on a malt extract

agar. Finally, Botrysphaerium sp. was isolated on corn meal agar.

These four isolates were tested for pathogenicity on both well

watered and moisture stressed plants. All tests were negative.

Our list of fungal isolates varies somewhat from previous

studies (Alfieri et al 1967, USFWS 1986). The explanation for

this appears to lie in the fact that we used native trees for our

experiments, while previous studies used trees growing in the

wild as well as trees in lawns, at Maclay Gardens and on the

campus of the University of Florida (Alfieri et al 1967, El-Gholl

personal communication). These trees outside the natural range

are likely to be vectors of other horticultural diseases that may

not infect wild trees. Thus, we prefer our list of fungal

isolates with one exception. Phyllosticta sp. was isolated from

ascospores on needles in the wild (Alfieri et al 1967). We did

not focus on this species because we could not find the

symptomatic ascospores on trees in the field as described by El

91

Gholl (personal communication), and because it was tested for

pathogenicity before with negative results (El-Gholl 1984).

Smoke Susceptibility

The four fungal isolates used for pathogenicity tests were

also tested for susceptibility to smoke. The results are mixed.

Fusarium sp. and Pestalotia natens are both highly susceptible to

smoke and show decreased fungal growth in response to direct

smoke treatment (Figure 5.2), as well as smoke residue on

previously smoked plates (Figure 5.3). In addition, both of these

species show reduced spore germination (Figure 5.4), reduced

spore concentration (Figure 5.5) and reduced growth in

germinating spores (Figure 5.6) in response to smoke residue on

culture plates. In contrast, Acremonium and Botrysphaerium

demonstrate virtually no response to any level or type of smoke

treatment (Figure 5.2, 5.3).

Field tests of the ability of smoke to inhibit disease in T.

taxifolia have been unfruitful. To date, none of the plants that

remain in our experiment, smoked or unsmoked, shows any

characteristic symptoms of disease (needle spots, needle

necrosis, or stem cankers). In addition, we monitored one managed

fire for smoke deposition in ravines. Smoke residue (collected on

glass slides) exceeded the level expected from our minimum

experimental smoke treatment (95% transmission at 470nm for 1

minute smoke treatment - Figure 5.1) in only 1 of 48 samples.

This single sample was at the highest slope position and adjacent

to the fire line.

92

Figure 5.2. The effects of direct smoke treatment on growth offungal associates of L. taxiolUi. A) Pestalotia natans, B)Acremonium sp. Each point is .the mean of growth observed in fivereplicate petri plates containing PDA agar and stored at roomtemperature.

8

6·

E

.5a

w 1' ' I"

-U--

U

0 min1 min

3 min

5 min

10 min

20 min

30 min

0 1 ~2 3 4 5

day

8

U

I0

C.2 20U

a*. I - -

0

p

U

0 min

1 min

3 min

5 min10 min

20 min

7

day

a

rý

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aa

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0 X - W~M au 4)Fa.

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(wa) *wLip Auoioo

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0

Figure 5.4. Histograms of spore germination failure rate forvarious smoke treatments (0, 1, 3 and 5 minutes). Replicatesterile PDA agar filled petri plates were inoculated with aslurry of spores from A) Fusarium sp. and B) Pestalotia natans.Germination rates were observed following 16 hours of incubationat room temperature.

40

30

GerminationFailure 20

(%)

10

0

20

GerminationFailure lO

(%)

0

A.) Fusarium

0 1 3 5

Smoke Treatment (minutes)

B.) Pestalotia

0 1 3 5Smoke Treatment (minutes)

Figure 5.5. Histograms of spore density for various smoketreatments (0, 1, 3 and 5 minutes). Replicate sterile PDA agarfilled petri plates were inoculated with a slurry of spores fromA) Fusarium sp. and B) Pestalotia natans. Spore density,reflecting differences in rates of spore deterioration, wereobserved following 16 hours of incubation at room temperature.

A.) Fusarlum300

Spore 200Density

(#/tran.)100

0

SporeDensity

(#/tran.)

0 1 3 5

Smoke Treatment (minutes

B.) Pestalotia

0 1 3 5

Smoke Treatment (minutes)

13 fde

)

1

Figure 5.6. Histograms of me4n spore growth rates for varioussmoke treatments (0, 1, 3 and 5 minutes). Replicate sterile PDAagar filled petri plates were inoculated with a slurry of sporesfrom A) Fusarium sp. and B) Pestalotia natans. Spore growth, asmeasured by the length of new mycelial strands were observedfollowing 16 hours of incubation at room temperature.

A.) Fusarium sp.300

200-

Growth(microns)

100-

O00 3 5

Smoke Treatment (minutes)

B.) Pestalotia sp.

Growth(microns

0 1

Smoke Treatment3 5

(minutes)

5

n-20U

n=20 n=20n=20

In,,20I

I

DISCUSSION

The results of our pathogenicity tests are not encouraging.

The failure to confirm pathogenicity in any fungal isolate could

either mean that we have failed to isolate the disease agent, or

that the conditions under which the disease agent becomes

pathogenic are restrictive and were not encountered during our

tests. The fact that none of our experimental plants in the field

have exhibited symptoms of disease supports the contention that

the foliar fungi only become pathogenic under severe plant

stress. Although this lack of disease symptoms in field tests

plants.is also consistent with another hypotheses: the disease

agent that decimated T. taxifolia has been reduced or eliminated

from the natural population and thus most T. taxifolia no longer

encounter the disease agent (see Chapter 1).

Alternatively, the disease agent that caused the population

decline may still infect plants in the wild. If the pathogen

requires the host plant to survive, such as most rust fungi, we

would not be able to isolate it on artificial growth medium.

Thus, we would get no positive pathogenicity tests using the

fungi we did isolate. If this is the case, the isolates from this

study may represent primarily secondary pathogens of little

consequence to plant health. However, it is hopeful to note that

the test plants we used were derived from cuttings of diseased

trees. If a host-specific pathogen is involved, it is likely to

be systemic and would have been transferred to cuttings collected

from the wild (for propagation for ex situ conservation).

98

However, these cuttings do not exhibit the symptoms of disease

observed in the wild. In addition, systemic rust fungi produce.

fruiting bodies on the outside of host plants. We have searched

plants in the wild for such symptoms of systemic infection. While

several fruiting bodies on T. taxifolia were observed in the

course of this work, most were identified as saprophytes that

appear to be derived externally, using the trunk for support of

its fruiting body structure (L. Crane, pers. comm.).

Collectively, these results suggest that the disease that

decimated T_. taxifolia may no longer be found. This hypothesis

requires that disease symptoms currently observed are secondary

pathogens breaking out on plants stressed by low light, low

nutrients or low soil moisture. While most foliar pathogens are

considered secondary infections, our evidence neither strongly

supports, nor rejects, this hypothesis.

Our test of the smoke hypothesis yielded mixed results. Two

of the four fungal associates tested proved sensitive to smoke

while the other two are not. Thus, it is possible that smoke

played a role in reducing foliar pathogen attack, but only if the

attacking agent is a fungus that is susceptible to smoke. Several

other, as yet untested, features must also hold for the smoke

hypothesis to be supported. It remains to be satisfactorily

tested whether smoke from managed fires settles in ravines in

sufficient quantities to maintain reduced populations of

potential pathogens. Further, we do not yet know the frequency of

fire required to maintain reduced fungal populations.

99

CHAPTER 6.

General Conclusions and Recommendations

In many respects the results of this 'research are exactly

what we would have predicted. First, the species may be in

decline for any number of reasons, a subset of which were'

previously reported in the US Fish and Wildlife Service Recovery

Plan (USFWS 1986, Chapter 1). Second, the species is continuing

to decline, as expected of plants with no sexual reproduction

that are also being suppressed beneath a relatively dense, young

closed canopy. Given that the species is exceedingly rare and

that the decline eliminated all mature seed producing trees, this

decline seems, if anything, remarkably slow (Chapter 2). Third,

the species is described by a low amount of genetic variability

(Chapter 3). This is consistent with the pattern of several

ancient species that have become narrowly endemic (Chapter 3).0

Fourth, the photosynthetic physiology of the species

confirms-that canopy shading could be suppressing the recovery of

what appear to be otherwise potentially healthy individuals

(Chapter 4). Fifth, the photosynthetic response of the species to

water and temperature stress does not indicate any special

consideration to these factors as potential contributors to the

demise of the species (Chapter 4). Sixth, the foliar pathogens

100

are associated with a significant decline in photosynthetic

capacity of T. taxifolia, consistent with the hypothesis that

foliar pathogens could become primary disease agents under

appropriate conditions (Chapter 4).

Seventh, we failed to contribute any significant advancement

in the realm of discerning a disease agent (Chapter 5). Finally,

smoke treatment may be important to the health of T. taxifolia.

This, however, depends on two specific factors that are yet to be

established: 1) the disease agent must be one of the foliar fungi

that is susceptible to smoke (although we have shown that these

exist), 2) the amount and frequency of smoke treatment required

to keep populations of the potential pathogen at low levels must

be on the order of smoke intrusion into ravines under natural

fire conditions.

We recommend that future research on the conservation

program for Torreya taxifolia should proceed along four lines.

First, continue efforts to isolate a primary pathogen,

particularly with respect to host-specific rust fungi. Second,

continue experiments that provide canopy openings to further test

if these facilitate recovery of vigorous individuals in the wild.

Third, attempt test plantings of closely related species (in

particular Torreya californica) to assess whether these species

are vulnerable to the diseases of T. taxifolia. This

recommendation is particularly important because of the ex situ

conservation program. We need to establish whether we would

jeopardize T. californica by incidental contact with T.

101

taxifolia. This could be an issue because the US Forest Service

Tree Genetics lab, perhaps the most capable institute to conduct

tissue culture propagation of T. taxifolia in the event that

systemic pathogens are found, is located near the range of T.

californica. As a result of this close proximity with the range

of T. californica, this lab has been heretofore excluded from

housing ex situ specimens of T. taxifolia. Finally, we recommend

additional test plantings to determine how newly planted trees

respond with respect to new disease and growth.

In addition, we make the following management

recommendations. First, monitoring of large populations should

continue on an annual basis. Second, canopy gaps should be

maintained around several of the larger, healthy trees in an

effort to stimulate sexual maturity, particularly among the few

trees that seem like they may be female.

Third, several deer exclosures were erected in 1987 to

protect large stems from deer antler rubs. These have largely

failed. Those that remain may be removed and no further

exclosures need be attempted at this time. While the exclosures

have succeeded by preventing further antler rub damage, they

increase the sphere of influence of woody debris falling from the

canopy. Several trees have been damaged as a result of limbs

hitting a deer exclosure cage and dragging it over the top of the

tree it was meant to protect. Thus, it is not clear which is the

lesser of these two problems. However, on-going stocking of young

pines in the adjacent uplands around trees on preserves in the

102

southern end of the range may help alleviate the pressure for

antler rub resources. Deer antler rubs have not appeared to be as

important in more northerly populations.

Fourth, large-scale restocking of the population with

individuals propagated from cuttings should be delayed until more

is known about the pathogens. Finally, fire management procedures

for uplands adjacent to ravines containing populations of T.

taxifolia should be adjusted in an attempt to maximize smoke

deposition to ravines under the constraint of fire management

safety guidelines.

The prospects for recovery of T. taxifolia in the near

future remain dim. However, with the current coordinated efforts

for ex situ propagation the species is unlikely to suffer

extinction. It is hoped that research beyond this project will

eventually make in situ recovery of T. taxifolia a possibility.

103

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