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1~JQ ZV0/J
PHYSIOLOGICAL ECOLOGY, POPULATION GENETIC RESPONSES AND
ASSEMBLAGE STABILITY OF FISHES IN TWO SOUTHWESTERN
INTERMITTENT STREAM SYSTEMS
DISSERTATION
Presented to the Graduate Council of the
University of North Texas in Partial
Fulfillment of the Requirements
For the Degree of
DOCTOR OF PHILOSOPHY
By
C. Jerry Rutledge, B.S., M.A.
Denton, Texas
December, 1991
1~JQ ZV0/J
PHYSIOLOGICAL ECOLOGY, POPULATION GENETIC RESPONSES AND
ASSEMBLAGE STABILITY OF FISHES IN TWO SOUTHWESTERN
INTERMITTENT STREAM SYSTEMS
DISSERTATION
Presented to the Graduate Council of the
University of North Texas in Partial
Fulfillment of the Requirements
For the Degree of
DOCTOR OF PHILOSOPHY
By
C. Jerry Rutledge, B.S., M.A.
Denton, Texas
December, 1991
TIS
Rutledge, C. Jerry, Physiological Ecology, Population
Genetic Responses and Assemblage Stability of Fishes in Two I IIIM I
Southwestern Intermittent Stream Systems. Doctor of
Philosophy (Biology), December, 1991, 179 pp., 18 tables, 5
figures, references cited, 154 titles.
Six sites within the Denton and Hickory Creek
watersheds were sampled over three years to assess the
impact of seasonal intermittent stream conditions on the
ichthyofauna. An integrated approach using field and
laboratory techniques was employed to evaluate the responses
of the fishes.
Critical thermal maxima (CTM) were determined for three
species of fishes at three oxygen tensions. Under hypoxic
conditions, CTMs of Fundulus notatus, Cyprinella lutrensis
and Pimephales vigil ax given surface access were
significantly higher than those of conspecifics without
surface access. With surface access, all species had
significantly lower CTMs under hypoxic conditions than other
conditions. CTMs measured under normoxic and hyperoxic
conditions were not different for any of the test species.
The critical oxygen concentration for C. lutrensis occurred
between 1.2 and 2 mg 1 -1 .
Cyprinella lutrensis and Lythrurus umbratilis were
sampled during alternating periods of flowing and
intermittent stream conditions. Allozyme variability was
assessed for 14 loci in each species. Heterozygote
deficiencies were observed for both species. Temporal
heterogeneity, measured by FST , was higher in C. lutrensis
than in L. umbratilis. Allele frequencies fluctuated
significantly in C. lutrensis but not in L. umbratilis. L.
umbratilis exhibited less genetic variation than C.
lutrensis. Effects of cyclic intermittent stream conditions
on the genomes of the species reflect differences in genetic
strategies of a generalist, C. lutrensis, and a specialist,
L. umbratilis.
In 99 sampling events, 27 species representing 9
families were collected. Longitudinal succession was mainly
by species addition rather than replacement. Data indicated
that flowing stream conditions (including floods)
alternating with seasonal intermittent conditions (including
drought) had minimal effect on dominant species in the
southwestern Elm Fork drainage fish assemblage. Drought
reduced assemblage diversity but had little effect on
evenness. Morisita's indices of similarity suggested
persistence of species relationships through time.
Kendall's W, a measure of concordance in species abundance
through time, was highly significant indicating that
assemblages, overall, were stable.
TABLE OF CONTENTS
Page
LIST OF TABLES v
LIST OF FIGURES vi
Chapter
I. INTRODUCTION 1
Study Sites 6
II. THE EFFECTS OF DISSOLVED OXYGEN AND AQUATIC SURFACE RESPIRATION ON THE CRITICAL THERMAL MAXIMA OF THREE INTERMITTENT-STREAM FISHES 12
Introduction 12 Materials and Methods 14 Results 16 Discussion 22
III. POPULATION GENETIC RESPONSES OF TWO MINNOW SPECIES (CYPRINIDAE) TO SEASONAL INTERMITTENT STREAM CONDITIONS 30
Introduction 30 Materials and Methods 33 Results 34
Cyprinella lutrensis 34 Lythrurus umbrati1 is 49
Discussion 57 Cyprinella lutrensis 57 Lythrurus umbratilis 60
IV. THE IMPACT OF SEASONAL INTERMITTENT STREAM CONDITIONS ON DIVERSITY, LONGITUDINAL SUCCESSION, PERSISTENCE AND STABILITY OF THE FISH ASSEMBLAGE IN A SOUTHWESTERN TEXAS STREAM 63
Introduction .63 Materials and Methods 72
Abiotic Factors 73 Fishes 74 Statistical Analyses 75
iii
Results 79 Abiotic Factors 79 Fishes 96
Discussion 112 Abiotic Factors 113 Fishes 118 Fish Species Diversity 118 Longitudinal Succession 121 Persistence and Stability 125
V. DISCUSSION 133
APPENDICES 139
APPENDIX A 139
APPENDIX B 141 APPENDIX C 143 APPENDIX D 147 APPENDIX E 151 APPENDIX F 153 APPENDIX G 155 APPENDIX H 157
REFERENCES CITED 159
xv
LIST OF TABLES
Page
TABLE 1 19
TABLE 2 23
TABLE 3 36
TABLE 4 42
TABLE 5 44
TABLE 6 50
TABLE 7 53
TABLE 8 55
TABLE 9 84
TABLE 10 86
TABLE 11 87
TABLE 12 91
TABLE 13 94
TABLE 14 . 97
TABLE 15 100
TABLE 16 104
TABLE 17 107
TABLE 18 110
LIST OF FIGURES
Page
FIGURE 1 7
FIGURE 2 20
FIGURE 3 39
FIGURE 4 47
FIGURE 5 . 81
VI
CHAPTER I
INTRODUCTION TO THE STUDY
Distributional limits and population densities of
fishes in lotic ecosystems are determined by both abiotic
and biotic factors. Range of a species within a single
watershed (ecological range) or within one or more drainages
(geographical range) is determined in part by the
physiological tolerances of that species to the suite of
extremes of abiotic factors it encounters. Biotic factors
are equally important in influencing distributional limits
and densities of fishes. For instance, if abiotic factors
in a first order, headwater stream limit fishes to a single,
pioneering species population, its members compete
intraspecifically for space and food (including
cannibalism). Interspecific relationships of that pioneer
population might include competition with invertebrates for
resources, parasitism and predator-prey relationships with
non-piscine predators. Downstream additions of other fishes
increase the complexities of intra- and interspecific
relationships in the assemblage. These abiotic and biotic
factors are not mutually exclusive but work in concert, not
only to influence a single species population but fish
assemblage structure and function as well.
Two general mechanisms have been proposed that might
regulate structure in multispecific ecological communities:
deterministic and stochastic processes (Grossman 1982, and
references therein). Grossman (1982) summarized each of
these mechanisms as follows. Deterministic (or equilibrium)
processes operate in habitats that are environmentally
benign or fluctuate in a predictable or regular manner.
Species within deterininistically regulated assemblages
coexist mainly through biotic interactions (e.g., predator-
prey relationships, resource partitioning or other
competition based phenomena). Both presence and relative
abundances of species are predictable. Stochastic (or
nonequilibrium) processes operate in environmentally
unpredictable habitats which lead to periodic (or
stochastic) variations in resource availability preventing
domination of the assemblage by superior competitors.
Coexistence of assemblage species is not fundamentally
influenced through biological interactions. Grossman et al.
(1982) believed that community regulating mechanisms are
best represented as a continuum with deterministic and
stochastic processes as endpoints.
Before a community can be judged stable, one or more
equilibrium points or limit cycles must exist at which the
system remains when confronted with a disturbing force or to
which it returns if perturbed by the force (Connell & Sousa
1983). Establishment of (or empirical evidence supporting)
assemblage stability then, is prerequisite to ascertaining
whether deterministic, stochastic or some combination of
both are regulating assemblage structure. Moyle & Vondracek
(1985) showed that the fish assemblage in a California creek
was temporally persistent and stable, reflecting
deterministic regulation. Grossman et al.'s (1982) analyses
showed lack of persistence in ranks of species abundances
and ranks of trophic groups for all seasons and concluded
that the fish assemblage of an Indiana creek was probably
regulated by stochastic factors. Matthews (1982) showed
that deterministic factors might limit the number of species
present in individual streams within the physicochemically
benign Ozark watershed, but distribution and abundance of
the complete fauna fit a random model better than a
deterministic model. Heffe (1984) reported that communities
of native and introduced fishes in southwestern streams
appear to be regulated by both biotic and abiotic processes.
These studies suggest that fish assemblages may be regulated
by stochastic or deterministic processes or a combination of
both.
The major objective of this study was to determine the
impact of cyclic and seasonal intermittent conditions
(including flooding and drought) on native, lotic fishes at
the population and assemblage levels in Denton and Hickory
Creek watersheds and at the assemblage level in the
southwestern Elm Fork, of the Trinity River drainage. An
integrated approach using field and laboratory techniques
was employed to evaluate the impact and responses of the
fishes. Specific questions or hypotheses related to this
overall objective are presented in the introduction of each
analytical chapter (II, III and IV). Literature germane to
hypotheses proposed is reviewed in chapter introductions.
Cyclic, seasonal intermittent conditions typically
occur in Denton and Hickory Creeks during the summer and
early fall (Buckner et al. 1989). When intermittent pools
form, water quality variables (e.g., water temperature and
dissolved oxygen concentrations [DOC]) may approach the
physiological limits of endemic fishes (Matthews 1987).
Three common fishes of intermittent pools in these creeks
(Fundulus notatus, Cyprinella lutrensis and Pimephales
vigil ax) were selected to assess upper thermal tolerances in
conjunction with varying DOC in the laboratory. Specific
hypotheses, results and conclusions related to these
experiments are in Chapter II.
Another consequence of cessation of stream flow and
formation of intermittent pools is shrinking habitat.
Habitat diversity, such as riffles, runs and pools,
associated with most small and medium-sized streams is
reduced to a series of relatively shallow, disjunct
longitudinal pools. Reduced water volume results in pool
entrapment, diminished food resources and crowding among
fishes that do not migrate downstream to perennially watered
areas. This phenomenon potentially results in severe
population bottlenecks for most species populations. Two
abundant cyprinids, Cyprinella lutrerisis and Lythrurus
umbratilis, were chosen to assess population genetic
responses to these population bottlenecks. Chapter III
includes hypotheses, results and conclusions of these field
and laboratory population genetic experiments.
In the third analytical study, an attempt was made to
evaluate the impact of cyclic, seasonal intermittent periods
(including disturbances of flooding and drought) on a higher
level of ecosystem organization: the fish assemblage.
Lotic communities consist of various combinations of
populations of producers (e.g., filamentous algae,
phyt©plankton), consumers (aquatic invertebrates, fishes)
and decomposers (bacteria, fungi). Meffe & Minckley (1987)
used the term "assemblage" for fishes as a taxonomic subset
of a "community". Assemblage is used in this study in the
same context. Longitudinal succession of the Denton and
Hickory Creek watersheds fish assemblages are presented in
Chapter IV. Also, physicochemical variables and fish
samples from Denton and Hickory Creeks were pooled and
considered random samples of the larger southwestern Elm
Fork of the Trinity River drainage assemblage. Chapter IV
includes hypotheses, results and conclusions of this field
experiment concerning effects of intermittency on drainage
physicochemical characteristics and on diversity,
persistence and stability of the drainage assemblage.
Study sites
Six study sites in Denton and Hickory Creeks are in the
Elm Fork of the Trinity River drainage. The headwaters of
the Elm Fork are in northeastern Montague County, Texas
(Figure 1). It flows easterly toward Gainesville, Texas,
then southerly toward Dallas, Texas, to its confluence with
the West Fork of the Trinity River in Dallas County. The
Elm Fork drainage basin is in Montague, Cooke, Grayson,
Wise, Denton, Collin, Tarrant, and Dallas Counties, an area
comprising 6,379 km2 (2,460 mi2 ). The western portion of
the basin contains many small cities and towns and the land
is predominantly used for agriculture. Tributaries within
this portion are potentially impacted both from agricultural
practices and wastewater effluents from municipalities.
Denton and Hickory Creeks are within the southwestern
portion of the drainage basin of the Elm Fork. Denton Creek
originates in Montague County within sandy soils of the
Western Crosstimbers, flows across blackland prairies and
becomes a sixth-order stream before its impoundment to form
Lake Grapevine in the Eastern Crosstimbers of southeastern
Denton County (Sellards et al. 1932, Tharp 1939).
Headwaters of Hickory Creek are in blackland prairies of
western Denton County, and this creek attains fourth-order
Figure 1. Western portion of the Elm Fork of the Trinity River drainage showing major tributaries, reservoirs and the six collection sites, (NHC=North Hickory Creek, SHC=South Hickory Creek, OC=Oliver Creek, TC=Trail Creek).
status in the Eastern Crosstimbers prior to entering Lake
Dallas, a reservoir formed by the impoundment of the Elm
Fork. Denton and Hickory Creek watersheds have dendritic
drainage patterns, and historically (before impoundments)
both streams confluenced directly with the Elm Fork.
Oliver Creek is a fifth-order stream originating in
east-central Wise County and flowing southeasterly to its
confluence with Denton Creek in southwestern Denton County
(Figure 1). Trail Creek is a third order tributary of
Denton Creek originating in southeastern Wise County and
flowing easterly into Denton Creek in southwestern Denton
County downstream of the Oliver-Denton Creek confluence.
Headwaters of North (fourth order) and South (third order)
Hickory Creeks are in northwestern Denton County, and their
confluence forms Hickory Creek (fourth order) in
northwestern Denton County. Stream order classification of
these streams is based on Horton (1945) as modified by
Strahler (1954, 1957) during flowing conditions. Stream
order for each creek was determined using Texas county and
USGS topographic maps.
Buckner et al. (1985, 1986, 1989) described current, as
well as historical, flow regimes for both Denton and Hickory
Creeks. Flow data are numerous for Denton Creek but limited
for Hickory Creek. Historical flow regimes for Denton Creek
are based on measurements from a gaging station located at
the FM156 crossing (latitude 33o07'08", longitude
10
97ol7'25"). Plow is affected by discharge from flood-
detention pools of 84 floodwater-retarding structures (e.g.,
small reservoirs, stock "tanks", etc.) with a combined
detention capacity of 64.2 hm3 (52,080 acre-ft). These
structures control runoff from 510 km2 (197 mi2) in the
Denton Creek watershed upstream of this gaging station.
From 1950-80, average discharge was 2.2 m3 s-i (77.4 ft3 s-
1): from 1981-89, after completion of additional
floodwater-retarding structures, average discharge increased
to 4.4 m3 s-1 (158 ft3 s-i). Maximum discharge for periods
of record was 982.7 m3 s-l (34,700 ft3 s-i) on Oct 13, 1981.
For fifty years, 1949-1989, seasonal intermittency occurred
in every year except three: 1966, 1975 and 1986.
Flow regimes for Hickory Creek are based on
measurements from a gaging station located at the FM1830
crossing (latitude 33o09'06", longitude 97°08"30") and are
reported from July 1985 to September 1986, only. Nine
floodwater-retarding structures with a combined detention
capacity of 6.8 hm3 (5,560 acre-ft) affecting runoff from 44
km2 (17 mi2) are located in the basin upstream of this
gaging station. Mean discharge for water year 1986 (October
1985-September 1986) was 3 m3 s-1 (107 ft3 s-i). Maximum
discharge of 294.5 m3 s-i (10,400 ft3 s-1) occurred on May
10, 1986 (outside the period of this study). Intermittent
periods occurred in August, September and October 1985.
11
Woody riparian vegetation reflect both prairie and
crosstimber species at most sites. Prairie trees, bois
d'arc (Madura pomifera) and southern hackberry (Celtis
laevigata), are mainly near prairie margins while more
typical crosstimber species such as elms (Ulmus sp.), pecan
(Carya sp.), white oak (Quercus sp.), and Texas ash
(Fraxinus texensis) extend along these streams as gallery
forests species. Cottonwood (Populus deltoides), sycamore
(Platanus occidental is), black willow (Salix nigra), soap-
berry (Sapindus drummondii) and box-elder (.fleer negundo) are
found along the lengths of most of these streams (species
identified using Shinners [1972]). Substrata of all streams
vary from sand, to sand and gravel in areas where these
streams erode through forested crosstimber areas and include
outcroppings of limestone bedrock in prairie locations, in
addition to sand and gravel. Siltation occurs in deeper
pools and eddies in all streams along their lengths.
CHAPTER II
THE EFFECTS OF DISSOLVED OXYGEN AND AQUATIC SURFACE
RESPIRATION ON THE CRITICAL THERMAL MAXIMUM
OF THREE INTERMITTENT STREAM FISHES
Introduction
Fish that live in intermittent streams have the
potential to avoid the environmental consequences of
intermittency by migrating downstream; however, many become
trapped in pools as water levels recede. In addition to
loss of the aqueous milieu, abiotic factors that limit the
survival of fish within pools include high temperature and
low dissolved oxygen concentration (Tramer 1977). Resident
populations of aquatic organisms which survive have
adaptations (physiological, biochemical and/or behavioral)
to cope with these abiotic challenges. Objectives of
several laboratory studies have included the determination
of seasonal and/or diel upper temperature tolerances of fish
as a measure of physiological adaptation to changes in
environmental temperature (Kowalski et al. 1978, Paladino et
al. 1980, Lee & Rinne 1980, Feminella & Matthews 1984,
Bulger 1984, Ingersoll & Claussen 1984, Bulger & Tremaine
1985, McClanahan et al. 1986). Other studies have addressed
the responses of fish to reduced dissolved oxygen
concentrations (see review of Kramer 1987). Some fish have
12
13
been reported to survive anoxic conditions by respiring
anaerobically (Blazka 1958, Burton & Heath 1980). Many
temperate and tropical water-breathing North American fishes
can utilize the thin layer of water enriched with oxygen at
the air-water interface. This is known as aquatic surface
respiration, ASR (Lewis 1970, Gee et al. 1978, Kramer &
Mehegan 1981, Kramer & McClure 1982, Kramer 1983). Matthews
& Maness (1979) measured the tolerances to high temperature
and hypoxia in four minnow species and demonstrated a
positive relationship between temperature tolerance and fish
densities in the field. Other research (Alabaster &
Welcomme 1962, Weatherly 1970, 1973) has addressed the
effects of dissolved oxygen on temperature tolerance in
three different species of fish (Salmo gairdneri, Rutilus
rutilus, and Carassius auratus). Their main thrust was
physiological, i.e., determining the role that oxygen
tension plays in heat stress and death.
In this study three species of fish, which inhabit
intermittent streams, were selected to evaluate the
interacting effects of dissolved oxygen and aquatic surface
respiration (ASR) on their upper temperature tolerances as
determined by the Critical Thermal Maximum (CTM) method
(Becker & Oenoway 1979, Paladino et al. 1980, Kilgour &
McCauley 1987). Fundulus notatus (blackstripe topminnow), a
cyprinodontid with a superiorly positioned mouth, Cyptrinel la
lutrensis (red shiner), a mid-water column cyprinid with a
14
terminally positioned mouth and Pimephales vigilax (bullhead
minnow), a benthic cyprinid with a subterminally positioned
mouth were chosen as representatives of microhabitats within
north Texan summer intermittent pools. Research was
designed to determine the influence of oxygen availability
and access to the surface (and hence ASR) on temperature
tolerance in these three species. I wished to determine if
CTMs under hypoxic, normoxic or hyperoxic conditions were
different, both intra- and interspecifically, and among fish
with and without access to the air-water surface.
Materials and Methods
During the summer of 1985, the fish were seined from
Denton Creek. They were held at 30®C for a minimum of two
weeks in normoxic, aged tap water under a regulated
photoperiod of LD 12:12. Fish were fed daily with flaked
food, except on test days when food was withheld. Upper
temperature tolerances were determined using the Critical
Thermal Maximum (CTM) technique (Cowles & Bogert 1944, Lowe
& Vance 1955, Hutchison 1961, Cox 1974). A calibrated
digital thermometer was used to measure water temperature to
the nearest O.OloC. In each CTM trial, temperature was
increased at a rate of 1°C per three minutes following the
recommendation of Becker & Genoway (1979) by two circulating
temperature controllers. The endpoint for CTM
15
determinations was defined as first loss of equilibrium with
failure of righting response.
A 76-1 aquarium containing a ten-compartment, plastic
mesh and plexiglass chamber was used in all temperature
tolerance tests. The compartments were 7X9X34 cm (high).
Five compartments were open at the top to allow fish access
to the surface, and five compartments were sealed to prevent
access to the air-water interface. CTMs were determined for
Cyprinella lutrensis at dissolved oxygen concentrations of
1.2, 2, 3, 4, 5, 6, 7, 10 and 12 mg 1-1 . CTMs for Fundulus
notatus and Pimephales vigilax were determined under three
different dissolved oxygen regimes operationally defined as
hypoxic (1.2 mg 1*1), normoxic (7 mg l-i) and hyperoxic (12
mg 1_1). Hypoxic conditions were created by bubbling
gaseous nitrogen through the test chamber water; normoxic
conditions by bubbling air through the water; and hyperoxic
conditions by bubbling gaseous oxygen through the water.
Dissolved oxygen concentrations (DOC) were continuously
monitored with a Rexnard Model 33 DO Meter which was
calibrated before each series of tests using the modified
Winkler method (American Public Health Association 1985).
Oxygen concentrations at each experimental treatment were
stable, and standard deviations for hypoxia and hyperoxia
ranged from 0.05 to 0.35 mg 1-1 , respectively.
Fish were transferred from the holding aquaria to the
CTM chamber, set at 30°C (holding temperature) where testing
16
was initiated immediately to prevent fish from acclimating
to hypoxic or hyperoxic conditions.
Since CTMs were expected to be greater in fish which
had access to the surface, one-tailed independent t tests
were used to compare mean CTMs between fish offered and
denied access to the surface under hypoxic conditions.
Since no differences were expected between mean CTMs of fish
offered and denied access to the surface under normoxic and
hyperoxic conditions, these means were subjected to two-
tailed independent t tests. Finally, since fish generally
have access to the surface, single-factor ANOVAs (and
Duncan's multiple range test, a=0.05) were used to compare
CTMs among fish tested with surface access at the various
dissolved oxygen concentrations. Parametric statistics were
chosen since all CTM distributions were normal (Shapiro
Wilds W, a=0.05). All analyses were conducted by the
Statistical Analytical Systems, 1985 edition.
Statistical analyses were performed according to Zar
(1984) using Statistical Analytical Systems (SAS), 1985
edition.
Results
Behavior of individuals of all three species of fish
was similar during CTM testing. Initially, under normoxic
and hyperoxic conditions, fish were quiescent and positioned
near the bottom mesh of the CTM chambers, As temperature
17
increased, fish began to swim repeatedly between the surface
and bottom of the chamber. Within 2 to 3°C of the CTM
endpoint, frenzied swimming occurred, and fish were observed
jumping from the water.
Under hypoxic conditions, fish of all species moved to
the air-water surface or attempted to move to the surface as
soon as they were placed into the water. Fish with access
to the surface remained at the surface until their CTM
endpoint was reached. Fish prevented from reaching the
surface remained in the upper portion of the water column
near the plexiglass surface barrier until their CTM endpoint
was reached.
Under hypoxic (1.2 mg l - 1) conditions, the CTM
(35.45oC) of Cyprinella lutrensis with access to the surface
was significantly higher (0.01>p>0.005) than the CTM for
conspecifics denied surface access, 32.93°C (Table 1).
There were no significant differences between the CTMs of
surface access and surface denied C. lutrensis under
normoxic (7 mg 1 _ 1) or hyperoxic (12 mg l - 1) conditions.
A highly significant difference was observed among the
mean CTMs in Cyprinel la lutrensis with surface access at
various oxygen concentrations (F=12.63, p
18
this range of oxygen concentrations, the slope relating CTM
and oxygen concentrations equaled 0.0088, which was not
significant (p=0.84 from t test of slope).
Under hypoxic conditions, the mean CTM (37.47°C) of
Fundulus notatus with access to the surface was
significantly greater (0.05>p>0.025, one-way t test) than
the mean CTM (35.98C) of conspecifics denied surface access
(Table 1). The mean CTM of F. notatus given access to the
surface under hypoxic conditions was significantly lower
than those under normoxic and hyperoxic conditions; however,
no significant difference existed between normoxic and
hyperoxic CTMs for this cyprinodontid (Duncan's multiple
range test with a=0.Q5).
The response of Pimephales vigilax to hypoxia,
normoxia, hyperoxia, and surface access was similar to those
of Fundulus notatus and Cyprinel la lutrensis. Mean CTM with
surface access, 33.09oC, was significantly greater
(0.05>p>0.025, independent t test) than the mean CTM without
surface access ,32.08oC, under hypoxic conditions for P.
vigilax (Table 1). There was a highly significant
difference among CTM means measured at the three oxygen
concentrations in P. vigilax with surface access (F=214.25,
pc.OOl). The CTM measured under hypoxia was significantly
lower (Duncan's multiple range test, a=0.05) than the CTM
means under normoxic and hyperoxic conditions (Table 2).
Consistent with results for the other two species, no
19
Table 1. CTMs («C) of each species in hypoxic (1.2 mg 1-1), normoxic (7 rag 1 _ 1) and hyperoxic (12 mg l_i) environments with and without access to the surface. Asterisks indicate whether or not the mean CTM of fish with access to the surface is significantly greater than the mean CTM of fish denied access to the surface (independent t-tests with a=0.05) for each oxygen concentration.
02 (mg 1-1 ) 1.2 7 12
Surface Access Yes No Yes No Yes No
Fundul us notatus CTM (°C) 37.47* 35.98* 41.56 41.42 41.55 41.82 SD (° C) 1.70 1.51 0.21 0.50 0.53 0.39 n 10 10 10 10 10 10
Cyprinella lutrensis CTM ( o c ) 3 5 . 4 5 * 3 2 . 9 3 * 3 9 . 6 5 3 9 . 6 1 3 9 . 1 2 3 9 . 0 6 SD ( o c ) 2 . 1 4 1 . 6 7 0 . 2 3 0 . 3 7 0 . 4 9 0 . 6 6 n 10 10 10 10 10 10
Pimephales vigil ax CTM (oC) 33.09* 32.08* 39.32 39.09 39.16 39.32 SD (oC) 1.23 1.20 0.25 0.49 0.42 0.43 n 10 10 10 10 10 10
20
Figure 2. CTMs (°C, mean ± one standard deviation) of Cyprinella lutrensis with access to the surface over a range of dissolved oxygen concentrations of 1.2 to 12 mg l-i. Sample size was ten for each group.
21
(Oo)lAllO
22
significant difference existed between normoxic and
hyperoxic CTMs for this benthic cyprinid.
Duncan's multiple range test (a=0.05) indicated that
the mean CTMs of these three species under normoxic
conditions with access to the surface were statistically
distinct. Pimephales vigilax had a significantly lower CTM
(39.32°C) than Cyprinella lutrensis (39.65°C) and Fundulus
notatus (41.56oC), while the CTM of C. lutrensis was
significantly lower than that of F. notatus.
Discussion
Fry (1947) stated that temperature and oxygen are major
environmental entities influencing the activities of fish.
Studies in which the rate of oxygen consumption is used to
estimate metabolic rate in relation to increasing
temperature have combined these two factors in determining
the effects of oxygen concentration on metabolic rates of
aquatic and amphibious poikilotherms (Well 1935, Spitzer et
al. 1969, Ultsch et al. 1978, Burton S Heath 1980, Saint-
Paul 1984). These studies indicate that oxygen consumption
becomes dependent on oxygen concentration. Fry (1947)
defined this point as the critical oxygen concentration or
tension. Although metabolic rate and critical oxygen
tension were neither objectives nor measured variables in
this study, this concept of critical oxygen concentration
could be included in upper temperature tolerance
23
Table 2. CTMs (°C, mean ± one standard deviation) of each species given access to the surface in hypoxic (1.2 mg l"i) normoxic (7 tng 1-1) and hyperoxic (12 mg 1-1 ) environments. Statistically distinct means from Duncan's multiple range test (a=0.05) are indicated by the horizontal lines immediately below the means.
0 (mg 1-1 )
Fundulus notatus CTM ( o C ) SD ( o C ) n
Cyprinella lutrensis CTM ( o C ) SD ( o C ) n
Pimephales vigil ax CTM ( o C ) SD ( o C ) n
1.2
37.47 41.56
12
41.55 1.70 0.21 0.53
10 10 10
35.45 39.65 39.12 2.14 0.23 0.49
10 10 10
33.09 39.32 39.16 1.23 0.25 0.42
10 10 10
24
determinations when dissolved oxygen concentration is a
measured variable.
CTMs with surface access were determined for C.
lutrensis at nine oxygen concentrations ranging from 1.2 to
12 mg l-i. Over a range of 2.0 to 12 mg l-i, mean CTMs
differed by less than 1.0®C (Figure 2). This suggests that
temperature tolerance in this species, at least when fish
are given access to the surface, is independent of dissolved
oxygen at concentrations as low as 2 mg l-i. The critical
oxygen concentration for this species for upper temperature
tolerance is somewhere between 1.2 and 2 mg l-i. The
insensitivity of temperature tolerance of C. lutrensis to
oxygen availability observed in these experiments may be
explained by the methodology of these experiments. The
combination of initial bath temperature (30C) and rate of
temperature increase (1C per 3 minutes) caused CTM
endpoints to be reached in less than 30 minutes for most
individuals. It is possible that if temperature increase
rates were lower during CTM trials or if fish were initially
acclimated to a lower temperature (e.g., 20°C), oxygen might
have more time to exert a greater masking effect on CTMs.
Nevertheless, C. lutrensis is a generalist species (Matthews
& Hill 1977, Calhoun et al. 1982, Matthews 1985, King et al.
1985) well-adapted for low-oxygenated, warm, intermittent,
summer pools.
25
Fundulus notatus had the highest CTH of the three
species tested, with or without access to the surface, at
all comparable oxygen regimes. With its superiorly
positioned mouth, flattened head and dorsal body surface;
morphologically, F. notatus is the best adapted of these
three species for aquatic surface respiration. ASR was
accomplished by F. notatus with minimal changes in typical
body orientation, and body position was maintained by fin
movement alone. Under hyperoxic conditions, the CTM of F.
notatus with surface access was 1.5°C higher than that of
fish denied the opportunity to use ASR. Lewis (1970)
reported no sign of distress for F. notatus at 0.0 mg 1-1
sub-surface oxygen and temperatures between 22 and 25C.
The CTMs of F. notatus measured under normoxic and hyperoxic
conditions with access to the surface (41.56° and 41.55oC,
respectively) were the highest of these three species.
Based on these results, 1 would hypothesize that F. notatus
should be the most persistent of these three species, at
least in intermittent summer pools, if the abiotic entities
temperature and dissolved oxygen are the only factors
operating.
The CTMs of Cyprinella lutrensis were intermediate
under all comparable oxygen concentrations. The terminally
positioned mouth of C. lutrensis seemed adequate for ASR
under hypoxic conditions, although this species was unable
to maintain an orientation in the water column for ASR with
26
fin movement alone and was observed to swim constantly in
small circles with its body axis about 45° to the surface.
Even so, under hypoxic conditions, the CTM of C. lutrensis
was significantly increased (2.5oc) in fish having access to
the surface. Matthews & Maness (1979) reported mean
survival times of 75 minutes (±30.8 minutes) for C.
1utrensis in water with 1.2 to 1.5 ppm dissolved oxygen at
25oC. When all three species are compared, C. lutrensis
should persist longer in summer intermittent pools than P.
vigilax but should succumb before F. notatus.
Pimephales vigilax, with its subterminally positioned
mouth appeared to be the least efficient at ASR and had the
lowest CTM at comparable oxygen tensions of the three
species. Even so, its CTM measured with surface access
under hypoxia was significantly greater statistically than
without surface access (33.09« vs. 32.08C); however, this
difference, about 1C, is the smallest increase measured in
these three species. While using ASR, the angle between the
surface of the water and the longitudinal axis of its body
was greater than 45°, and P. vigilax continually swam in
small circles in this orientation. This species is a common
and persistent inhabitant of summer, intermittent pools in
the Denton Creek system (personal observation). P. vigilax
may have other behavioral and/or physiological adaptations
to enhance survival in these environments. In addition to
the previously mentioned responses of fish to oxygen
27
depletion (i.e., behavioral avoidance, aquatic surface
respiration and anaerobiosis), shorter term responses
observed in fish include increased ventilation (Saunder
1962, Gee et al. 1978) and hematocrit by the release of
erythrocytes through spleen contraction (Black 1955).
Saint-Paul (1984) reported increased gill surface area to
body mass as an adaptation to hypoxia in Colossoma
macropomum, as well as, seasonal changes in several
hematological characters (e.g., increased mean corpuscular
hemoglobin concentration and relative erythrocyte count).
Brett (1956) stated that low oxygen may be a possible
cause of, or contributor to, death from high temperatures in
some fish. Oxygen insufficiency was hypothesized to precede
an inactivation of the respiratory center which would then
lead to death. Data supporting Brett's hypothesis were
provided by Weatherly (1970), who measured temperature
tolerance of goldfish, Carassius auratus, over dissolved
oxygen tensions of 10% air saturation to about 30
atmospheres. Weatherly reported that low oxygen reduces
temperature tolerance (or survival times at constant lethal
temperatures), whereas superabundant oxygen (above 100% air
saturation) ameliorates thermal stress. Temperature
tolerance of goldfish was highest at oxygen tensions of
approximately 2 to 4 atmospheres and remained essentially
constant up to 30 atmospheres. Conversely, the CTMs
measured in hyperoxic conditions in trials within my study
28
were not significantly different than those measured in
normoxic conditions for any of the three species that were
studied. This finding may be explained by the observation
that the hyperoxic condition in my study (12 mg l -i) was
considerably less than those used by Weatherly (1970).
Objectives of my study were more ecological than
physiological, i.e., interest was more in the responses to
ecologically possible oxygen concentrations than responses
to unnaturally high tensions of oxygen.
In summary, all three fish had significantly lower CTHs
in hypoxic water than in normoxic or hyperoxic water. CTMs
of all three species in hyperoxic water were not
significantly different than those measured in normoxic
water. Under hypoxic conditions, the CTMs of all species
with surface access was significantly greater than those
denied access. These results suggest that tolerance of high
temperatures is extended more by surface access than by
superabundant oxygen, at least over the range of oxygen
concentrations employed in this research.
If the critical oxygen concentration concept is
extended to include oxygen consumption during CTM
determinations, then the CTM of C. lutrensis is independent
of dissolved oxygen concentration as low as 2 mg 1-1.
Finally, if water temperatures in combination with dissolved
oxygen were to reach lethal levels for these three species
in summer, intermittent pools, these results suggest that
29
they should disappear in the following sequence: P.
vigilax, C. lutrensis, and F. notatus.
CHAPTER III
POPULATION GENETIC RESPONSES OF TWO MINNOW SPECIES
(CYPRINIDAE) TO SEASONAL INTERMITTENT
STREAM CONDITIONS
Introduction
Estimates of genetic variability in fish assessed by
electrophoresis during the past 20 years indicate that
varying degrees of spatial population subdivision exist over
relatively small geographic areas. Population subdivision
has been demonstrated in striped bass, Morone saxatilis
(Morgan et al. 1973); bluegills, Lepomis macrochirus (Avise
& Smith 1974); darters, Etheostoma radiosum (Echelle et al.
1975); red shiners, Cyprinella lutrensis (King et al. 1985);
salmonids (Utter et al. 1973); and mosquitofish, Gambusia
sp. (Smith et al. 1983, Kennedy et al. 1985, McClenaghan et
al. 1985, Zimmerman et al. 1989). Clinal variation in
allelic frequencies has been demonstrated in certain species
(Koehn 1969, 1970, Powers & Place 1978, Baumgartner 1986),
as well. Generally, spatial genetic variation or the
maintenance of clines in these studies has been attributed
to natural selection, gene flow and stochastic processes
determined by demographic properties of the particular
species. While fewer in number, studies of the genetic
structure of fish populations through time or across space
30
31
and time reveal contrasting patterns differing from those
focusing on spatial structure alone. Koehn & Williams
(1978) reported patterns of spatial differentiation for two
enzyme loci that were temporally stable in elver and adult
North American eels, Rnguilla rostratum, but temporal
heterogeneity in allele frequencies at a third locus in
elvers. Temporal stability of clines in populations of the
topminnow, Fundulus heteroclitus, has been substantiated
(Powers & Place 1978), and a similar pattern has been
observed in sea lamprey, Petromyzon marinus, ammocoetes in a
single drainage (Jacobson et al. 1986). In contrast,
Kornfield et al. (1982) demonstrated temporal heterogeneity
in spawning populations of the common herring, Clupea
harengus, and McClenaghan et al. (1985) observed significant
temporal changes in a mosquitofish, Gambusia affinis, from
an impoundment receiving thermal effluent. It is apparent
that such conflicting results on temporal variation warrant
additional investigation. An environment which experiences
extreme perturbations offers an ideal system for such a
study.
The major objective of this study was to ascertain the
impact of seasonal stream intermittency through time on the
genetic structure of populations of two coexisting minnows:
red shiners, Cyprinella lutrensis, and redfin shiners,
Lythrurus umbratilis. Both species have similar
reproductive habits, becoming sexually mature in their
32
second or third summer, spawning over sunfish nests, and
surviving for a maximum of 2 years (Pflieger 1975). Red
shiners exhibit wide physicochemical tolerances (Matthews &
Hill 1977, 1979) and often are most abundant where
environmental conditions are too rigorous for other fish
species (Cross 1967). C. lutrensis exhibits a high degree
of genetic variation which appears to be adaptive for
existing under extreme conditions and for invading new
habitats (Zimmerman & Richmond 1981, King et al. 1985,
Wooten 1984). In this regard, red shiners appear to be a
good example of a 'generalist' species. In contrast, L.
umbratilis is more habitat specific, frequently occurring in
relatively clear streams with moderate current (Pflieger
1975). Each species has a reproductive season ranging from
May to late August or early September (Pflieger 1975,
Matthews & Heins 1984).
This objective spawns several related questions. For
instance, does summer drought with ensuing stream
intermittency result in population bottlenecks manifested by
genetic differentiation of local populations and random
fluctuations in allele frequencies (i.e., population
subdivision). Or do the populations remain as apparent
panmictic units as the result of deterministic processes
such as directional selection and/or gene flow? Do
intermittent pools function as 'genetic refugia' for
founders who determine the zygotic frequencies of subsequent
33
local populations when rewatering of the stream occurs?
Finally, do differences in gene diversity and temporal
genetic variation exist between a habitat specialist, L.
umbratilis, and a generalist, C. lutrensis?
Materials and methods
Cyprinella lutrensis and Lythrurus umbratilis were
sampled from Denton and Hickory Creeks, respectively. A
single collection site on each stream representing a local
population for each species was sampled, by seining, through
each period of continuous flow and intermittency from 1983
to 1986. Effort was made to collect all fish in a pool,
with subsampling for electrophoresis before releasing the
remainder to the site. Each site was a quiet pool with
reduced flow during flowing conditions and a persistent pool
during intermittency. C. lutrensis was sampled seven times,
while L. umbratilis was sampled six times.
Specimens were returned to the laboratory and
maintained in aquaria or frozen (--80°C) until processing.
Tissues were homogenized in distilled water and
electrophoresed according to the methods of Kilpatrick &
Zimmerman (1975) and Bohlin & Zimmerman (1982). Alleles
were designated alphabetically in order of decreasing
mobility. For both species, proteins encoded by 14
structural loci were examined including malate dehydrogenase
(Mdh-1, Mdh-2, Mdh-3), lactate dehydrogenase (Ldh-1, Ldh-2),
34
phosphoglucomutase (Pgm-1, Pgm-2), peptidase L-leucyl-L-
alanine (P-Lla), peptidase L-leucylglycyl-glycine (P-Lgg)
and esterases (Est-1, Est-2, Est-3, Est-4, Est-5).
Genetic differentiation among and within samples for
each species was analyzed using F-statistics according to
Wright (1965) as modified by Nei (1977). Genetic similarity
(S) was calculated for pairwise combinations according to
Rogers (1972). Significance of genotypic frequency
differences among samples was tested for each locus by the
chi-squared test with (k-l)(s-l) degrees of freedom, where k
is the number of alleles at the locus, and s is the number
of populations (Workman & Niswander 1970). Significance of
deviations from expected genotypic proportions predicted by
Hardy-Weinberg equations were calculated for each variable
locus for each sampling period using chi-squared tests with
Levene's (1949) corrections for small samples and pooling.
Heterozygosity (H) and polymorphism (P) were calculated
directly from the data. Statistical significance for all
analyses was determined at a=0.05.
Results
Cyprinella lutrensis
Of the fourteen loci examined from C. lutrensis, five
were monomorphic, including Mdh-1,, Mdh-3, Ldh-2, Pgm-1 and
Est-5. Nine loci were polymorphic (Table 3). Ldh-1, Est-2,
35
Est-3, Est-4 and P-Lla were diallelic; three alleles were
found segregating at the Mdh-2 and Est-1 loci; and four
alleles were found segregating at the Pgm-2 and P-Lgg loci.
Allelic compositions of the variable loci in C. lutrensis
were identical to those reported for the species by King et
al. (1985) and Wooten (1984). Changes in allele frequencies
of polymorphic loci in C. lutrensis occurred in two general
patterns (Figure 3). One pattern, occurring at the Est-1,
Est-2, Est-3, P-Lgg and P-Lla loci, was exemplified by
allele frequencies fluctuating over time in an unpredictable
manner. For example, at the Est-3 locus (Figure 1), A and B
alleles alternated between fixation, loss of one allele, or
maintenance of both alleles at frequencies intermediate
between these extremes. The Est-4, Ldh-1, Mdh-2 and Pgm-2
loci exemplify a second pattern, with the common allele
generally remaining at high frequencies or reaching
fixation.
In one case, a rare allele appeared at the Pgm-2 locus
in C. lutrensis from flowing conditions 1984 (F2'84). A new
allele (B) appeared at the Est-2 locus in the intermittent
conditions in 1985 (I3'85), increased in frequency to 0.304
in flowing conditions in 1986 (F4'85-6), and became the
predominant allele during intermittency in 1986 (14*86),
with a concomitant decrease in the Est-2A allele occurring
(Figure 3).
36
Table 3. Allelic frequencies of nine polymorphic loci in a population of Cyprinella lutrensis. Sampling periods prefixes indicate flowing (F) or intermittent (I) stream conditions.
37
V0 ^
I—I I!
CN CO H Xf CO CM O CTi O
vd xr 0 \ o 00 H
o o o o o
co h r - o h U) o
O CN CO If) H CO o c * o
O O O
o o t o uO CF* O
O O
r - O co o RH U0 CO O H H L> O
o H cn O CN t*-lO CO rH
V0 ^ a \ o U5 CO
co 00 rH CTi o
O O O O O O O o o o o
o o o o o o
H O
t o 0 0 CO S 0 4 l~l w II
t o rH CO o OS O
U> CO cr> o
oo m r - o rH CM LO O O rH 00 O
o o O Ch O CO VP o
CN 00 CO rH CTi O
CM 00 00 rH
XF V0 ^ m 00 H o H R- o
CN CN r ^ H H r -r - cN o
o o o o o o
o o o o o o
o o o o o o
o o o o o O O O O O O O H O H O H O
r j S ™ fcl ^ II
CN t o CO ^ r - co O 00 O
OS H R- CN o\ O
CN 00 O O T r o m o O C S I h O
cr» H o o o \ o a * o o
o o o O O O O O O O O O
O o o o o o • •
H o
CT» H t o H 00
o o
o o o o o o
H O
ri* 03 O 03 ^ p q O Q m O < PQ < OP CO
w 3 o o • J
CN i
X I - d 25
I x ;
J
CN I £ o* 0-i
H CM CO 1 j 1
4-> - P I
4J +-»
CO W w w w W
38
^ co ^ M _ if
o o c m o h CO Tt4 o (N
39
Figure 3. Temporal changes in frequencies of common alleles at four loci in the red shiner, Cyprinella lutrensis. Sampling periods correspond to those in text.
40
EST-1
0.75
n 0 .50
Q 0 . 2 5
1 2 3 4 5 6 7
SAMPLING PERIOD
EST-2
0.50
0 0 .25
1 2 3 4 5 6 7
SAMPLING PERIOD
< 1.00
0.75
0.50
Ui -J Hi - j
41
Measures of temporal genetic variation were similar to
the oscillating patterns of allelic frequencies (Table 4).
Heterozygosity (H), calculated over all loci, ranged from
0.10 to 0.16. H undulated in sine curve fashion with
troughs at F1'83 and F3'84-5 and peaks at I2'84, F4'85-6 and
13*86. Polymorphism, ranging from 0.44 to 0.78, followed a
pattern similar to that of heterozygosity.
Heterozygote deficiencies were found in nearly one-half
of polymorphic loci during each of the sampling periods
(Table 4). Deficiencies were found in 86% of sampling
periods at the Pgm-2 and Est-1 loci and in 43% of sampling
periods at the P-Lla locus. The remaining loci (Mdh-2, Est-
2, Est-3, Est-4, P-Lgg) had deficiencies in at least 14% of
the sampling periods. Concomitant to heterozygote
deficiencies was a high overall Fis (0.226) reflecting fewer
heterozygous individuals than expected during each sampling
period.
Accompanying heterozygote deficiencies were significant
deviations from Hardy-Weinberg genotypic expectations (Table
5). Red shiners from intermittent conditions 1984 (I2'84),
and from flowing conditions 1985 (F3'84-5), had 50% or more
of their polymorphic loci deviating significantly from
expectations. Fish from the remaining sampling periods,
except intermittency 1986 (14'86), deviated significantly
from equilibrium at 14 to 40% of their polymorphic loci. In
42
Table 4. Temporal changes in genetic variability measures, heteozygote deficiencies, FST and XZ tests for heterogeneity of polymorphic loci for a population of Cyprinella lutrensis. Sampling period prefixes indicate flowing (F) or intermittent (I) stream conditions.
43
%
fr« W
fri
00
&4
VD I
ID CO
W
p
0)
o d* >1
o u 4)
+A 0) X
CO
CO Xu
t o 0 0
m i
o o
™ Z 1-4 w
* * * * * * * * * * * * * * H rH -*r rF X}* V0 CM
CM CN c o r - o KO U5 cm as
CM r - 00 CO r - t> r^ oo a\ t—i CO CM 00 U> CO
rH CM CM rH rH
vo m CM Tf 00 m o O CM KO ̂ o o o CM o o m rH VjD
« • « • • • • « • •
o o 0 o 1 1
0 1
o o o o 1
o o
o \ o o \£> i n H i n «H o o r? CO o O rH o o CM rH ^
• • » • • • « « «
o o o o f 1
o o o a o 1
o o
CM o rH o O o i n i > o rH CO o M> t> rH i n
« • « m « « • o o
0 0 o o rH
0 0 o o
x r i o c o m CO CM o O O H O ^ • • • •
o o o o t I
! I t
CO CO o 00 o m o h en ^ o c o • • • •
o o o o o I I
U5 00 h r -• •
o o
* d (U
• H M-J
44
Table 5. Summary of polymorphic loci in Hardy Weinberg equilibrium (E) or A? statistic if not in equilibrium for a population of Cyprinella lutrensis. Sampling period prefixes indicate flowing (F) or intermittent streams condi tion.
45
00
WW WW w CM o w o o
(X4
KO I to CO
46
individuals sampled from intermittent conditions 1986
(I4'86), all polymorphic loci were in equilibrium.
Major shifts in allele frequencies resulted in
significant levels of temporal heterogeneity (Table 4). FST
calculated over time ranged from a low of 0.016 at the Mdh-2
locus to a high of 0.807 at the Est-3 locus, with a mean
temporal FST for all loci of 0.403. Mean Fis and FIT were
0.226 and 0.538, respectively.
Genetic similarity (S) between pairwise comparisons of
temporal samples from C. lutrensis had a distinct pattern
between successive samples (Figure 4). In all cases, fish
sampled from a flowing condition were genetically most
similar to those from the preceding intermittent pool than
to a subsequent intermittent pool. For instance, fish
sampled from flowing water 1984-5 (F3'84-5), were
genetically more similar (S=0.840) to fish sampled from the
preceding intermittent pool 1984 (12'84), than to fish
sampled from the subsequent intermittent period (13*85,
S=0.757). Likewise, fish sampled from flowing conditions
1985-6 (F4'85-6), were genetically more similar (S=0.873) to
fish sampled from the preceding intermittent pool 1985
(12'85), than to fish sampled from the subsequent
intermittent conditions (I3'86, S=0.759). Genetic
similarity for all collection periods was 0.765.
47
Figure 4. Genetic similarity (S) between sequential alternating flowing and intermittent conditions for populations of the red shiner, Cyprinella lutrensis, and the redfin shiner, Lythrurus umbratilis.
48
C. lutrensis 0.807 0.705 0.757 0.759
F83 F84 184 F85 185 F86 186
0.840 0.873
L. umbrati]is 0.909 0.960 0.998
F83 F84 184 F85 185 F86 186
0.959 0.981
49
Lythrurus umbratilis
Fourteen loci were examined from L. umbratilis, and, of
these, eight loci were monomorphic. These included Mdh-1,
Mdh-2, Mdh-3, Ldh-2, Pgm-1, Est-3 and Est-4. Six loci were
polymorphic (Table 6). Ldh-1, Est-1 and P-Lla were
diallelic, and three alleles were found segregating at the
Pgm-2, Est-2 and P-Lgg loci. Temporal changes in these loci
were not as striking as those of C. lutrensis during
comparable periods.
Allelic frequencies of the polymorphic loci in L.
umbratilis remained constant, with the same allele
predominating through all sampling periods. At certain
loci, rare alleles detected during one or more sampling
periods were not found during others (Table 6). For
instance, the Ldh-1B allele occurred at a frequency of 0.056
in the 13'85 sample, but was not detected in other samples.
The Pgm-2B allele occurred at a frequencies of 0.104 and
0.017 only in Fl'83 and F4'85-6, respectively; and the Pgm-
2c allele occurred at a frequency of 0.083 in a single
sample (Fl'83).
Genetic variation in L. umbratilis, measured by mean
heterozygosity (H) and polymorphism (P), was reduced when
compared with that of C. lutrensis (Table 7). H ranged from
0.0 to 0.08 and was slightly higher in fish from flowing
conditions (H=0.08, Fl'83) than in those from intermittent
conditions immediately subsequent to flow (H=0.04, 12'84).
50
Table 6. Allelic frequencies of six polymorphic loci in a population of Lythrurus umbratilis. Sampling periods prefixes indicate flowing (F) or intermittent (I) stream conditions. N equals sample size.
51
vo 00
V o O O o o o o o o o o o o o O o o o o o O O o o o o o o o o o o o O o o o CO o o O o o o o o o o o o o o O o o o II • » « • • • • • • • • • • • • • • • d H rH o H o o o rH o o «H rHI rH o o rH o
VO y-N 00 o 1 co
fx* in II oo d
o O O CO r** o O o O O o o o O O O o o o o O 00 rH O O O O o o o o o O O o o o o O o o o o o o o o o o o o o o o o 00 rH o o o o o o o o o o • « • • • • • • « • • • • • * • « •
rH «H o HI o o o o rH o o rH rH rH a o rH o
rjco™ Cu ~ ii
C
o o o CO CO o o o 00 CO o o CM o\ rH o* o o o rH o 00 o o m 00 VO o o 4J •J *~3 o "d 0* to w w m 1 1 •J X J 04 w w M m 04 04
52
Polymorphism ranged from 0.0 to 0.44 and did not track
heterozygosity.
Of six temporal samples of L. umbratilis, three (Fl'83,
F3'84-5, I3'85) had polymorphic loci with heterozygote
deficiencies (Table 7). Fish from the other three sampling
periods (I2'84, F4'85-6, I4'86) either had no heterozygote
deficiencies or had the common allele fixed at all loci.
All polymorphic loci were deficient in heterozygotes from
samples F3'84-5 and I3'85. Heterozygote deficiencies
occurred at 75% of the polymorphic loci in sample Fl'83.
These heterozygote deficiencies are reflected in a high Fis,
0 . 2 2 0 .
Concomitant to heterozygote deficiencies in these three
samples were significant deviations from Hardy-Weinberg
genotypic expectations (Table 8). Fish from F3'84-5 and
13'85 had 100% of their polymorphic loci in disequilibrium,
while those from Fl'83 had 50% of their polymorphic loci in
disequilibrium. The stability of allele frequencies
resulted in lower levels of temporal heterogeneity in L.
umbratilis (Table 7) when compared to C. lutrensis. FST
calculated over time ranged from 0.047 (Ldh-1 locus) to
0.147 (Est-2 locus), with a mean of 0.121.
Pairwise comparisons of temporal samples reflected a
high degree of genetic similarity between all combinations
(S=0.960±0.026). As sampling progressed from the initial
53
Table 7. Temporal changes in genetic variability measures, heterozygote deficiencies, FST, and X* tests for heterogeneity of polymorphic loci for a population of Lythrurus umbratilis. Sampling periods prefixes indicate flowing (F) or intermittent (I) stream conditions.
54
S i
* « * « * * * *
CM CM CO KO CO CO O CO r-f Cft U5 o \ c o o * r -
55
Table 8. Summary of polymorphic loci in Hardy-Weinberg equilibrium (E) or A? statistic if not in equilibrium for a population of Lythrurus umbratilis. Sampling periods prefixes indicate flowing (F) or intermittent (I) stream conditions.
56
V 0
C O O O O U O O
KO 0 0
^ 1
tm o o
O W O O O V
CO t o
c o
* *
C O
o
It *
o r?
l O O O O C O O
CO CM
* 0
0
• H
C5
• H
i—l
O i
£
fd
c o
c o
m
t
o o
* o
T P
o o o ^ u o
0 4 0 0 b-4 W
rH °°
s i r
£ o o w u o o
*4
• Q
- H
r H
• H
* *
* *
57
period (Fl'83) to the last (I4'86), S values increased from
0.909 to 0.998 (Figure 4).
Discussion
The results of this study on temporal genetic variation
indicate that seasonal summer intermittency impacts the
genetic structure of populations of red shiners and redfin
shiners in distinctly different fashion through time.
Consequently, the degrees of temporal population subdivision
are dissimilar in each species. These differences seem
inextricably linked to the varying life history strategies
of each species.
Cyprinella lutrensis
Habitats within Denton Creek where red shiners are
found are as variable (personal observation) as those
reported in other studies (Pflieger 1975, Matthews & Hill
1977, Anderson et al. 1983, Wooten 1984). Concomitant to
the variable habitats occupied by C. lutrensis are high
levels of spatial heterogeneity reported by other authors
(Calhoun 1981, Wooten 1984, King et al. 1985). For
instance, the study of 20 structural gene loci in 22 red
shiner populations by Wooten (1984) reported H and P values
of 0.089 and 0.415, respectively.
Significant temporal heterogeneity and a concomitant
high FST ( 0 . 4 0 3 ) for seven of nine polymorphic loci average
58
in this study substantiate temporal variation occurs in this
species. Of nine loci, five demonstrated dramatic changes
in allele frequencies corresponding to cycles of alternating
flowing and intermittent conditions during the three year
period. These changes involved shifts from one predominant
allele to another, loss of rare alleles, and appearance of
new alleles. As annual summer intermittency transformed
this stream into a longitudinal series of disjunct pools,
apparent chance entrapment resulted in population
bottlenecks and consequent reduction in effective population
size. If breeding occurred during intermittent stages,
genetic drift would compound the effect of the bottleneck.
Heterozygote deficiencies were found for approximately
one-half of the polymorphic loci within each sampling period
and undoubtedly contributed to cases of significant
departure from expected Hardy-Weinberg genotypic proportions
and high positive Fis values for individual time periods.
Wooten (1984) attributed heterozygote deficiencies in C.
lutrensis to a Wahlund (1928) effect and inbreeding.
Sampling across age classes with differing genotypes might
also contribute to these deficiencies, but the short life
span of red shiners should minimize this effect. A Wahlund
effect, caused by fusion of subpopulations with differing
predominant alleles, seems a plausible explanation for these
heterozygote deficiencies.
59
Population estimates of red shiners were
60
These results, combined with spatial population genetic
studies (Zimmerman & Richmond 1981, Wooten 1984, King et al.
1985), provide substantive evidence that C. lutrensis is a
genetically dynamic species subject to both selective and
stochastic processes as described in the shifting balance
model of Wright (1932). As intermittency proceeds,
population bottlenecks and genetic drift occur. Survivors
have the potential to produce large numbers of offspring and
serve as founders during subsequent flowing conditions.
Mixing of genotypic combinations, through migration, could
produce a continual array of potentially favorable genotypes
capable of increasing in frequency to new adaptive peaks by
selection according to the shifting balance model.
Lythrurus umbratilis
Redfin shiners have a geographical range as extensive
as red shiners (Lee et al. 1980). Since they are found most
commonly in clearer, warmer waters with sluggish flow or
pools within lotic systems (Pflieger 1975; Matthews & Heins
1984), their ecological range appears smaller than red
shiners. These minnows were found most frequently in larger
pools with reduced current in Hickory Creek and were the
most abundant species in the watershed.
Based on the enzyme loci investigated, L. umbratilis is
genetically less variable than C. lutrensis. Interestingly,
the highest value of P observed in L. umbratilis (0.44)
61
equalled the lowest value observed in C. lutrensis. Lower
heterozygosity (H=0.0 to 0.08) may be explained in part by
lower polymorphism and a high percentage of those
polymorphic loci with heterozygote deficiencies. Although
new or rare alleles sometimes appeared and were subsequently
lost at some loci, predominant and common alleles at all
polymorphic loci were temporally stable through alternating
periods of intermittency and flow. While small effective
population sizes (
62
to high S values. This evidence supports the hypothesis
that the population genetic structure of L. umbratilis
reflects specialization for a narrower habitat tolerance.
In summary, two differing patterns of dynamics of
temporal genetic variation were exhibited by C. lutrensis, a
generalist, and L. umbratilis, a specialist. The temporal
and spatial genetic structure of C. lutrensis is subject to
both selective and stochastic processes shaping a highly
variable genome adapted for a variety of habitats.
Ostensibly, L. umbratilis is genetically less variable, and
selection rather than stochastic processes has the greatest
effect on its specialized genome. Stability of allele
frequencies and less population subdivision suggest low
levels of genetic variation accompany its narrower habitat
tolerance.
CHAPTER IV
THE IMPACT OF SEASONAL INTERMITTENCY ON DIVERSITY,
LONGITUDINAL SUCCESSION, PERSISTENCE AND
STABILITY OF THE FISH ASSEMBLAGE
OF A TEXAS STREAM
Introduction
Seasonal intermittency is common in headwater, upper
and midstream reaches in most prairie and desert stream
systems (Stehr & Branson 1938, Paloumpis 1958a, John 1964,
Harrell 1978, Collins et al. 1981, Matthews 1987, 1988).
Also, flooding and drought are common events in these
systems.
During floods Stehr & Branson (1938) found stream beds
scoured and large numbers of juvenile fish were swept
downstream. In contrast, Gerking (1950) reported little
effect of summer flooding on fish populations. Severe
floods during early and mid-summer resulted in poor
reproduction or low survival of young-of-year fishes, except
for late and intermittent spawners (Starrett 1951).
Paloumpis (1958a) suggested that fish populations Creek,
survived in small tributary streams (stream havens) during
floods. In the desert southwest, Harrell (1978) found
habitat alteration and most species associations dissolved
63
64
and new ones formed subsequent to flooding. Floods in
desert streams reduced fish populations by removal of
juveniles (Rinne 1975), and moved channel sediments and
eliminated an endangered fish, Poeciliopsis occidental is
(Collins et al. 1981). Meffe (1984) reported that flooding
removed an exotic fish population (Gambusia affinis) to an
alluvial fan, while only displacing downstream a native fish
population (Poeciliopsis occidentalis). Effects of flooding
on these lotic fish populations depended on both intensity
(including stream bed and/or habitat alteration) and timing
(pre- or post-spawning season).
Many investigators studying effects of drought on
stream fishes have focused on species survival in
intermittent pools. Starrett (1950) noted that the
inability of certain abundant species to withstand oxygen
reduction and crowded conditions in small intermittent pools
prevented their wider distribution. Paloumpis (1958a) found
fish populations surviving drought in havens such as
isolated pools, flood plain ponds, and a larger river. Toth
et al. (1982) listed severe droughts of 1976 and 1978, three
consecutive harsh winters, fish kills and modified habitat
conditions brought about by stream alterations (especially
si 1 tation) as factors responsible for the demise of the
silverjaw minnow. John (1964) found highest mortality rates
for Rhinichthys osculus during the summer dry period, caused
by a combination of reduced habitat, shortage of food and
65
high temperatures. These studies indicate that only the
hardiest species survived diminished habitat and trophic
quality caused by intermittent stream conditions.
Other researchers have studied succession and/or
recolonization rates from watered refugia subsequent to
intermittent stream or drought conditions. Larimore et al.
(1959) reported that drought destroyed fish and invertebrate
populations but that pioneer species (Shelford 1911)
repopulated a stream within three weeks. After two years,
most of the 25 native species had established populations.
Griswold et al. (1982) observed total dewatering and
elimination of resident fish populations during a summer
drought in a channelized portion of a river. They found
that 30 species had recolonized from the Auglaize River
within a year. Matthews (1987) found that fish rapidly
recolonized a prairie stream by movement from permanent
pools. He noted a positive correlation between oxygen
tolerance and the ability of species to colonize. Fishes
typical of headwater faunas (composed of a high proportion
of pioneer species) persist within intermittent pools and
are the first to recolonize intermittent streams during
rewatering. If drought results in total dewatering, these
Pioneer species are the first to repopulate from downstream
refugia during rewatering. These pioneers are followed by
other native species.
66
Studies by Thompson & Hunt (1930) and Kuehne (1962)
corroborated results of Shelford's (1911) classic study of
longitudinal succession in stream fishes that increased
downstream species richness occurs mainly by species
addition rather than species replacement. Other studies
have shown increases in species diversity from headwaters to
downstream reaches (Sheldon 1968, Smith & Powell 1971, Ebert
& Filipek 1988, Meador et al. 1990). Some studies have
found a direct relationship between species diversity and
stream order (Harrel et al. 1967, Whiteside & McNatt 1972,
Lotrich 1973). Sheldon (1968) proposed that species
richness was explained better by depth rather than distance
from headwaters, and Evans & Noble (1979) stated that
diversity, in general, seemed to be more highly correlated
with depth than with longitudinal position. Matthews
(1986a) presented evidence agreeing with Evans & Noble
(1979) that stream orders do not serve as strong organizers
of lotic fish communities. Gelwick (1990) found that
longitudinal succession may better reflect fish assemblages
in pools than in riffles in Battles Branch, Oklahoma.
Horwitz (1978), analyzing records from 15 river systems,
found that species diversity increased from upstream to
downstream primarily by addition of new species with little
replacement of the upstream fauna, and that headwater
diversity was lowest in rivers with the most environmentally
variable headwaters. Schlosser (1987) proposed a conceptual
67
framework that attempts to integrate relative roles of
physical versus biological processes in regulating fish
community structure in warmwater streams. Whether
longitudinal position, depth or stream order was used to
explain changes in fish communities, most of these studies
have concluded that downstream increases in species richness
or diversity are correlated with downstream increases in
habitat diversity and more stable environmental conditions.
Studies measuring responses of fish assemblage
diversity to disturbances include both anthropogenic and
natural perturbations. Bechtel & Copeland (1970) showed
that areas in a bay receiving the greatest amounts of
effluents and toxic materials exhibited the lowest mean
annual diversities. Gorman & Karr (1978) compared natural
and modified streams and found that natural streams
supported fish communities of high species diversity which
were seasonally more stable than the lower-diversity
communities of modified streams. Anderson et al. (1983)
measured higher species diversity upstream of a reservoir
than four locations downstream of the reservoir. Zaret
(1982) reported that predation by an introduced piscivorous
fish caused the local extermination of 13 of 17 native fish
species in an environmentally stable lake system, but caused
no local extermination in an adjacent and relatively less
environmentally stable river system in Panama. Kushlan
(1976) showed a diversity increase with a period of water-
68
level stability in an Everglades marsh compared to typical
diversity decreases occurring with seasonal water-level
instability. Harrell (1978) measured an overall decrease in
diversity of the fishes following a flood, but determined
the pre-flood dominant fishes were community dominants and
flood-prone adapted in a Texas desert stream. In general,
these studies have shown a decrease in fish diversity
following a disturbance, whether it was natural or
anthropogenic.
Connell & Sousa (1983) defined and discussed criteria
to judge persistence and/or stability in natural populations
or communities. Persistence is a qualitative measure and is
the presence/absence of species or persistence of
relationships: stability is a quantitative measure and is
the degree of constancy in numbers of organisms. According
to their criteria, a system must be determined first to be
in equilibrium, then faced with a disturbing force before
these two phenomena can be evaluated. If a system remains
in equilibrium when perturbed, then the community exhibits
resistance; if the community returns to equilibrium after
perturbation, the community exhibits adjustment. Included
within adjustment are elasticity and/or resiliency (the
speed of return to equilibrium) and amplitude (the distance
from which the system is capable of returning). Several
lotic ecosystem studies have utilized Connell & Sousa's
criteria to evaluate perturbations to fish assemblages
69
(Hoyle & Vondracek 1985, Meffe & Hinckley 1987, Matthews et
al. 1988).
Studies evaluating persistence and stability of lotic
fish assemblages are longer-termed studies which include at
least one complete turn-over of assemblage species (Connell
& Sousa 1983). Assemblage studies have included both
permanently watered and intermittent stream systems. Moyle
& Vondracek (1985) found the fishes in a California creek,
to be persistent, deterministic (stable) and highly
structured over a 5-year period including extreme floods.
After comparing samples in an Arkansas watershed across
numerous years, Matthews (1986b) suggested that the fish
fauna was stable (via elasticity) and persistent across
years, seasons and a catastrophic flood. Meffe & Minckley
(1987) reported the fish fauna of an Arizona creek
persistent and stable (via resistance) from 1943-79, a
period including the most intense flooding of the creek on
record. Meffe & Berra (1988) determined the assemblage of
an Ohio creek to be persistent and stable over 9 years
(including regular and major flooding). Ross et al. (1985)
compared fish assemblages in a harsh prairie stream in
Oklahoma with a benign Arkansas stream and found both
assemblages highly persistent. Stability differed in the
two systems, but both assemblages were stable (a drought had
no lasting effect on overall community stability in the
prairie stream). Matthews et al. (1988) concluded that at
70
the level of whole-stream faunas, three different midwestern
streams were stable across survey years and that many
individual locations (within each watershed) had relatively
stable fish assemblages. These studies confirm a high
degree of fish assemblage persistence and stability in small
to medium-sized streams (whether permanent flow or not)
perturbed by either flooding or drought.
Two questions have been raised regarding assessments of
disturbance effects on persistence and stability of lotic
fish assemblages. One concerns geographic scale (Connell &
Sousa 1983, Yant et al. 1984, Ross et al. 1985, and
references therein). The other question concerns
predictable vs. stochastic disturbances (Sousa 1984, Resh et
al. 1988, Matthews 1988). Ross et al. (1985) suggested
assemblage stability and/or persistence studies require
sampling regimes encompassing the whole assemblage or
representative random samples of it. Their reasons include
lack of information on the vagility of many stream fishes
and that assemblage bounds are generally not known. Also,
Yant et al. (1984) and Matthews (1986b) pointed out that
conclusions of community stability/persistence studies of
lotic ecosystems based on a single location could be
spurious. Resh et al. (1988) suggested that drought in
prairie streams, with ensuing physicochemical extremes, are
generally predictable over longer periods and resident
populations of organisms may adapt to the extent that these
71
extremes are not really a disturbance. According to these
authors, conclusions of persistence/stability studies
considering or including these questions in the experimental
design might be more meaningful.
Objectives of this study included several questions.
Are these intermittent stream systems harsh environments and
do means of physicochemical variables differ significantly
among and within sites of different stream order? Do
seasonal intermittent stream conditions affect longitudinal
succession of fishes in two, adjacent watersheds within the
same drainage basin? What are the effects of annual
intermittent stream conditions (including flooding and
drought) on fish assemblage diversity in a portion of a
single drainage basin? Finally, what are the effects of
annual flooding and intermittent stream conditions on fish
assemblage persistence and stability in a portion of a
single drainage basin? By pooling samples from several
collection sites from two watersheds within the basin, the
question of geographic scale might be addressed. Although
intermittent conditions in these streams is cyclic as well
as seasonal, varying degrees of intensity and duration of
intermittent periods (including drought) may make their
classification as predictably disturbed debatable. Field
collections for this study began in February, 1983 and were
concluded in July, 1986.
72
Materials and Methods
Six collection sites (three within the Denton Creek and
three within the Hickory Creek watersheds) were selected
based on the following criteria. First, sites were
representative of that portion (i.e., third, fourth, fifth,
sixth order) of the drainage experiencing seasonal
intermittent stream conditions but not becoming totally dry.
Second, sites were accessible and included diversity of
habitat (e.g., riffles, runs and pools) during typical flow;
and third, stream-bed morphologies were suitable for
seining.
All routine collection sites were within Denton County,
Texas (Figure 1). Denton Creek watershed sites included
Trail Creek upstream of the FM156 crossing (third order);
Oliver Creek near Oliver Creek Road (fifth order); and
Denton Creek upstream of FM407 crossing (sixth order,
approximately 13 km. upstream of headwaters of Lake
Grapevine). Hickory Creek watershed sites included North
Hickory Creek downstream of Plainview County Road crossing
(fourth order); South Hickory Creek upstream of FM156
crossing (third order); and Hickory Creek downstream of
FM1830 crossing (fourth order, approximately 3.2 km.
upstream of headwaters of Lake Lewisville).
The Oliver Creek Road site, Denton Creek watershed, is
somewhat different than all the other sites and requires
additional description. Oliver Creek Road is a low water,
73
concrete crossing at Oliver Creek, its surface parallel to
and constructed about lm above the solid limestone bedrock
of the creek bottom. The road functions as a low
impoundment with a concrete culvert (approximately 0.5m
diameter) at the south stream bank which permits restricted
flow during typical or low-flow conditions. The limestone
bedrock continues upchannel from the road about 50m where a
"sink" occurs. This sink is about lm deeper than the
adjacent bedrock and its limestone bottom is typically
covered with silt deposition up to 10cm deep. Due to these
conditions, this site has more lentic characteristics
(mainly a greatly reduced stream flow) than any other site.
Abiotic Factors
Estimated average percent of shading from tree canopy
(direct observation) was made at each site in areas where
pools persisted during intermittent conditions for all
seasons. Maximum stream width and depth during typical flow
and minimum width and depth of intermittent pools were
measured at each site.
When fishes were sampled, dissolved oxygen
concentration (±1 mg W ) and pH were measured with a Hach
water ecology kit. Conductivity (±5 ymthos cm-i) was
measured with a YSI salinity-conductivity-temperature meter,
and water temperature (±0.5C) was measured with a mercury
thermometer.
74
Fishes
All fish collections were made with a straight seine
(1.2 x 3.6 m with 0.6 cm bar mesh) or bag seine (1.2 x 6.1 m
with 0.6 cm bar mesh) during daylight. A reach of
approximately 100 m was seined at each site. Minimum
seining effort was 0.75 to 1 hour at each site during normal
flow and included all microhabitats. Minimum sampling
effort was approximately 15 minutes during maximum
intermittent periods, including as few as three seine hauls
through a single, shallow pool. Seining direction always
included downstream hauls (Paloumpis 1958b), and upstream
and crosscurrent hauls when possible during typical flow
conditions. Seining in riffles included the kickset method.
During initial intermittent periods, collection sites
were reduced to a few (usually 3 to 4) small pools and a
single larger pool. When intermittent, all pools were
sampled or censused. A single pool typically persisted at
each site toward the end of an intermittent period
immediately before rewatering in the fall and frequently
could be censused rather than sampled. Even though a single
pool might be censused under these conditions, that pool was
considered a random sample of similar pools upchannel and
downchannel. Sampling at all sites was approximately
bimonthly during flowing and monthly during initial
intermittent stream conditions. During the 1984
intermittent period, seining frequency was increased to
75
enhance qualitative and quantitative determination of change
in species' relative abundance and t