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