In the Pecos River, Texas, and New Mexico

GENETIC DIFFERENTIATION AMONG SUBPOPULATIONS OF THREE GAMBUSIA

SPECIES (PISCES: POECILIMAE) IN THE PECOS RIVER, TEXAS, AND NEW MEXICO

A Thesis Submitted to the Faculty of

Baylor University

In Partial Fulfillment of the

Requirements for the Degree

Master of Science

Elisabeth Milstead

Waco, Texas August, 1980 ABSTRACT

Heterogeneity among subpopulations of three species of Gambusia was determined by electrophoretic analysis of allelic frequencies. The analysis included (1) all populations of G. nobilis, an endemic of the Pecos River drainage of New Mexico and Texas which occurs only in four

widely separated spring systems; (2) five populations of G. affinis, a

widely distributed species with a more continuous distribution in the

Pecos drainage; and (3) four populations of G. geiseri, an endemic from

the east side of the Edwards Plateau which has been introduced into three springs of the Pecos drainage on the west side of the Plateau. The average FsT values (Wright's standardized variance in allelic frequency) for Pecos River populations within the range of G. nobilis were 0.755 for G. nobilis, 0.160 for G. affinis, and 0.278 for G. geiseri. The relatively high value for G. geiseri compared to G. affinis may result from founders effect. Even when populations of G. geiseri and G. affinis

from the opposite side of the Edwards Plateau from the Pecos are consid-

ered, the heterogeneity of G. nobilis remains higher than that of the

other two. The same result remains when a population of a second sub-

species of G. affinis is included in the analysis. The average FsT value

for G. nobilis is, in fact, higher than that previously reported for any species. In addition, of the three species of Gambusia, G. nobilis

showed the highest divergence on the basis of overall genetic identity (Nei's measure, 16-17 loci). The data support the classical concept that

gene flow exerts a dedifferentiating effect on subpopulations. The un-

iii usually high genetic variance observed for G. nobilis may be characteristic of fishes restricted to desert springs.

IV TABLE OF CONTENTS

ABSTRACT ...... iii

LIST OF TABLES ...... VI

LIST OF FIGURES ...... VII

ACKNOWLEDGEMENTS ...... VIII

INTRODUCTION ...... 1

, DISTRIBUTION AND HISTORY OF GAMBUSIA IN THE PECOS RIVER ...... 14

MATERIALS AND METHODS ...... 8

RESULTS ...... 11

DISCUSSION ...... 20

LITERATURE CITED ...... 27

APPENDIX ...... 32 LIST OF TABLES

Table Page 1. Number of alleles per locus in three species of Gambusia ... 9 2. Allelic frequencies at four loci in four populations of G. nobilis ...... 12

3. Allelic frequencies at eight loci in five populations of G. affinis ...... 13 4. Allelic frequencies at five loci in four populations of G. geiseri ...... 14 5. F and heterogeneity Chi-square values for polymorphic loci inST various combinations of populations of G. nobilis ...... 15 6. F and heterogeneity Chi-square values for polymorphic loci inINST various combinations of populations of G. affinis ...... 16 7. F and heterogeneity Chi-square values for polymorphic loci inINST various combinations of populations of G. geiseri ...... 17

8. Average FsT values for various fish populations ...... 23

vI LIST OF FIGURES

Figure Page

1. Localities IN the PECOS RIVER DRAINAGE 5

VII ACKNOWLEDGEMENTS

I would like to thank Drs. Anthony A. Echelle, Robert C. Gardner, and O. T. Hayward for the time and effort spent in serving on my committee and critically reviewing the manuscripts. I am especially grateful to Dr. Echelle for his advice and help during all phases of this study. Alice Echelle and D. Allen Rutherford offered invaluable assistance in making collections and executing the laboratory work.

David Hillis and Doyle Mosier also assisted in making collections. Dr.

John Patton, Dr. Clark Hubbs, Ken Asbury, Jim Duke, Mike W. Smith, and

Mike Dean provided information and discussion on various aspects of this study. Jim Johnson of the U. S. Fish and Wildlife Service in Albuquerque was instrumental in the initiation of this study.

Financial assistance was provided by the U. S. Fish and Wildlife

Service, contract No. 14-16-0002-79-133, Sigma Xi, and the Baylor

University Graduate School. The bulk of this manuscript was prepared while I was a student at the University of Oklahoma Biological Station.

VIII INTRODUCTION

Fishes in desert river systems of North America often exhibit a dichotomy regarding opportunity for gene flow between subpopulations. Some species occur only in small, relatively stenothermal, springfed habitats which are discontinuously distributed. The various populations are therefore separated by large areas that are uninhabitable by the species.

In such cases, the populations may have been effectively isolated from each other since pluvial periods of the Pleistocene (e.g., Miller, 1948).

Other species thrive in situations that show great fluctuations in physi- cal and chemical features of the environment. Consequently, species of the latter group are more continuously distributed in tributaries and major streams, and should exhibit relatively high levels of gene flow between subpopulations. Good examples of this dichotomy include poeciliid fishes of the genus Gambusia in the Chihuahuan desert of the United States and Mexico (Hubbs and Springer, 1957). In this paper I compare electrophoretically detected genetic heterogeneity in two species of Gambusia in the Pecos River system of Texas and New Mexico--one of which, G. nobilis, is a spring dweller, while the other,

G. affinis, occurs in the same spring systems and yet is also widely distributed in the Pecos River and its major tributaries. I also present an analysis of genetic heterogeneity in G. geiseri, an introduced (Hubbs,

1980) spring-dweller of the Pecos River system. Recently, there has been a number of reports of genetic heterogeneity among subpopulations of stream dwelling fishes. These include studies by

1 2

Avise and Felley (1979) and Avise and Smith (1974) on L2pomis macrochirus,

Echelle et al. (1975, 1976) on two species of Etheostoma, Koehn (1970) on catostomids, Koehn et al. (1971) on Notropis stramineus, Merritt et al. (1978) on Rhinichthys cataractae, and Ryman et al. (1979) on Salmo trutta. Studies of populational variation in Gambusia consist of brief

treatment of G. affinis in small local areas of central Texas and South Carolina, respectively, by Yardley and Hubbs (1976) and Yardley et al. (1974). Studies dealing with genetic variation in desert fishes include Turner's (1974) work on desert pupfishes and Koehn's (1970) studies of esterase variation in catostomids. However, Turner's study was not directly concerned with intraspecific populational variation, and Koehn's dealt only with esterase heterogeneity. To date there have been no

comparisons of the genetic structure of desert species which exhibit the

above described dichotomy in opportunity for gene flow.

Contrary to the conclusions of Ehrlich and Raven (1969) and Endler (1977), it is intuitively apparent that gene flow should be a major cohesive evolutionary force contributing a dedifferentiating effect among subpopulations of a species. However, there have been few empirical

tests in which proper controls were employed. In a brief review, Jackson

and Pounds (1979) were able to cite only four studies with some controls (Metter and Pauken, 1969; Rees, 1970; Jackson, 1973; and Echelle et al., 1976), all of which support the concept that gene flow has a strong dedifferentiating effect on subpopulations of species. Thus, I began my study with the following prior hypotheses: (1) the strongly isolated

populations of G. nobilis should dhow the greatest degree of differen-

tiation; (2) because G. geiseri in the Pecos River represents a series of 3 recently introduced populations of a species with a highly restricted natural distribution, the various populations of G. seiseri should dhow the lowest level of differentiation; (3) even in the absence of natural selection (Rohlf and Schnell, 1971), differentiation in continuously distributed organisms may be expected to increase with geographic distance as a function of the "isolation by distance" phenomenon (Wright, 1943).

Thus, subpopulations of G. affinis should dhow intermediate levels of differentiation within that segment of the Pecos River encompassed by the range of G. nobilis, i.e., greater differentiation than G. geiseri, lower than G. nobilis. DISTRIBUTION AND HISTORY OF GAMBUSIA IN THE PECOS RIVER

G. nobilis, which is endemic to the Pecos River, presently occurs only in four spring systems (Fig. 1): springs and gypsum sinkholes on the Bitter Lake National Wildlife Refuge, near Roswell, Chaves Co., New

Mexico; Blue Spring near Whites City, Eddy Co., New Mexico; a spring system near Balmorhea, in Reeves and Jeff Davis counties, Texas; and a springfed segment of Leon Creek near Fort Stockton, Pecos Co., Texas. At present, the locations that support G. nobilis also support G. affinis, and two of those localities, Balmorhea area and Leon Creek, support recently introduced populations of G. geiseri. G. geiseri also occurs with G. affinis in Independence Creek, a springfed stream of the lower Pecos in Terrell Co., south of Sheffield, Pecos Co., Texas. In these systems G. nobilis and G. geiseri predominate in waters that provide some measure of environmental stability, while G. affinis predominates in unstable, eurythermal habitats such as shallow, open pools (Echelle and Echelle, 1979). Consequently, G. nobilis and G. geiseri tend to be more abundant near spring headwaters, while G. affinis is the most abundant in tailwater habitats (Bednarz, 1979; Hubbs et al., 1978; Echelle and

Echelle, 1979).

G. affinis is widely distributed at low elevations in the Pecos River drainage, and it seems well adapted to invade and occupy temporary waters such as pools from brief flooding. Consequently, although two of the spring systems included in this study, Balmorhea area and Leon Creek, are isolated from the Pecos River proper except during infrequent floods,

4 5

Figure 1. Localities in the Pecos River drainage. 1 = Bitter Lake National Wildlife Refuge (=BLNWR), 2 = Blue Spring, 3 = Balmo±hea area, 4 = Lake Leon, 5 = Leon Creek, 6 = Independence Creek. am. • win ••••• ••• • • elm • smogs w•••• •••• ml•• ••••■••••1 • a. • IN. • ••• • •■•• •Im• mo•••••• • • • • • • ••• •■••••■ i NEW i .1 • mi. MD •■••••■•••■•■• um • •■••••• ••• • •=. • .• • ■••••••••• ••■ • MEXICO ii

S. CANADIAN R.

PECOS R.

BRAZOS 1 R.

i 2 i • ,■•■•• am• •••• •■■ • •••• ••• • •••■••••• • wa• .__.-.-.. •••• • •••• • ••• i i *TEXAS 3 4!"1" RIO GRANDE

100 KM 6

G. affinis is more continuously distributed among spring systems than are G. nobilis and G. geiseri. Excepting a single specimen of G. nobilis from the Pecos River near Fort Sumner, New Mexico (Michael Hatch, pers. comm.), there are no records of either G. nobilis or G. geiseri from the Pecos River or its main tributaries.

The three species with which this study is concerned have had quite different histories in the Pecos River. Because of their present distri- butions and the apparent competitive superiority of G. affinis in non- spring waters of the Pecos River system, it appears likely that, as suggested by Hubbs and Springer (1957), occurrence of G. nobilis in the Pecos River area predates that of G. affinis. Hubbs and Springer (1957) suggested that G. nobilis or its ancestor may have once been widespread in the Pecos and that, during the Pleistocene, there would have been alternations between expansion of the populations into streams during wet periods and contraction into springs during dry periods. These alternations would have corresponded with periods of, respectively, free gene flow and restricted gene flow, The invasion of the Pecos area by G. affinis or its ancestor would have caused more intense restriction of gene flow among spring populations of G. nobilis as a result of competition between the latter species and intervening stream populations of the former species. The Pecos River apparently supports two subspecies of G. affinis,

G. a. speciosa in the lower reaches and G. a. affinis in the upper reaches. The two subspecies may intergrade somewhere in the Pecos, but, based on dorsal ray counts there is no sharply defined intergrade zone (Peden, 1970).

G. a. speciosa occurs in the Rio Grande southward into Mexico (Peden, 1970); thus, it probably invaded the Pecos area from the south. G. a. 7

affinis occurs from south and central Texas northward to Indiana and east-

ward to Alabama (Rosen and Bailey, 1963, in part; however, these workers did not recognize G. a. speciosa as distinct from G. a. affinis). G. geiseri is native to the closely associated Comal and San Marcos Rivers near San Marcos, Hays Co., Texas. Because the species was absent

in early collections from areas where it now is common, Hubbs and Springer

(1957) and Hubbs (1980) concluded that presence of G. geiseri in the Rio Grande drainage, including the Pecos, was a result of introductions for

mosquito control, possibly in the 1920's. During a short period in the 1920's the name G. affinis was mistakenly applied to G. geiseri. Conse- quently, at a time when the literature recommended G. affinis for mosquito

control, G. affinis was thought to occur only in the vicinity of San Marcos (Hubbs, 1980). MATERIALS AND METHODS Collections of G. nobilis were made during June of 1979 from the following four localities: Sago Spring on the Bitter Lake National Wild-

life Refuge (BLNWR) near Roswell, Chaves Co., New Mexico; Blue Spring near

Black River Village, Eddy Co., New Mexico; Phantom Lake Spring, 13 km SW

of Balmorhea, Reeves Co., Texas; and Diamond-Y spring, a tributary of Leon

Creek, 16 km N of Fort Stockton, Pecos Co., Texas. G. affinis was collected

from three localities in June of 1979: "Unit 15" on BLNWR; the environmental studies pond on Baylor University campus, Waco, McLennan Co., Texas;

and Blue Spring, New Mexico. Additional collections of G. affinis were

made on 15 February 1980 from the Lake Leon outflow canal (a part of the Leon Creek drainage), 11 km W of Ft. Stockton, and from Independence Creek, 33 km S of Sheffield, Pecos Co., Texas. Samples of G. geiseri were taken in June of 1979 from Phantom Lake Spring, Diamond-Y Spring, and the San Marcos River, at San Marcos, Hays Co., Texas. An additional sample of

G. geiseri was taken from Independence Creek on 15 February 1980. All

collections were made by seine and transported to the lab on dry ice.

Horizontal starch gel electrophoresis was done essentially as described

by Selander et al. (1971), Don Buth (pers. comm.), and Siciliano and Shaw

(1976). Stains, buffer systems, and number of alleles detected for each

locus for each species are shown in Table 1. All gels were run for six

hours at 50 milliamperes. Single locus heterogeneity among populations of each species was examined for each locus at which the frequency of the common allele was

8 Table 1. Number of alleles per locus in three species of Gambusia,

Enzyme Locus Species G. nobilis G. affinis G. geiseri

a Aspartate aminotransferase AAT-A one two three Creatine kinase a CK-A one one one a Creatine kinase CK-B one two one a Esterase EST-B one --- two Glucose phosphate isomerase a GPI-A three two one a Glucose phosphate isomerase GPI-B two two two a Glutamate dehydrogenase GDH one one one Isocitrate dehydrogenase b IDH-A two two four Isocitrate dehydrogenase b IDH-B one two two Lactate dehydrogenase b LDH-A one two b one Lactate dehydrogenase LDH-B one three two b Lactate dehydrogenase LDH-C one one . one Malate dehydrogenase b MDH-A one one one b Malate dehydrogenase MDH-B one two one B Malate DEHYDROGENASE MDH-C one three one Phosphoglucomutase a PGM-B three two two Superoxide DISMUTA,SE A SOD-D one one one a. Buffer system = continuous tris-versene borate buffer, pH 8.0; 25mg NAD added to gel. Buffer system = continuous tris-citrate buffer, pH 7.0. 10

0.95 or less in at least one population. Wright's (1965) standardized genetic variance was used as follows

FST =

2 Where a = unstandardized variance of allelic frequency and p = mean allelic frequency across populations. The commonest allele at each locus was used in these computations. To test the significance of interpopulational heterogeneity in allelic frequencies, a heterogeneity Chi-square test

(Workman and Niswander, 1970) was used k 2 - X ..-211z(orf fi.) j=1 Pj where N is the number of individuals per population, o is the variance Pj of the frequency of the jth allele, ij is the mean of the frequencies of the jth allele, and k is the number of alleles at each locus. For the Chi-square testsequalsample sizes of N = 21 were obtained for all populations of G. nobilis and G. affinis and N = 19 for all populations of G. geiseri by using the data for the first 19 or 21 specimens examined for allozymic variation. Agreement with Hardy-Weinberg expectations was examined for each sample by computing the following Chi-square value for each locus observecIAL ex 2 X 2 = 11(l- pected) where Hi is the proportion of heterozygotes (Li and Horvitz, 1953).

Allelic frequencies for all loci were used to calculate Nei's (1972) measure of genetic distance for each pair of populations of each species. RESULTS

Allelic frequencies for the polymorphic loci are shown in Tables 2-4 for each of the three species. Seventeen loci were included in the analyses for G. nobilis and G. geiseri, but EST had to be omitted for G. affinis due to inconsistent scoring. Only MDH-C for the Bitter Lakes population had a statistically significant deviation from Hardy-Weinberg expectations for genotypic frequencies (P40.05). At the 0.05 level, at least one "Type 1" error could be expected to occur among the 76 computations that I made (Sokal and Rohlf, 1969); thus, the observed deviation may have occurred by chance alone. The portion of the observed variance in allelic frequencies that may be accounted for by sampling error was estimated by Pi/2N where and are the mean allelic frequencies across all populations and N is the total number of specimens (Cavalli-Sforza and Bodmer, 1971; Wright, 1951). The maximum error variance was 0.006 for G. nobilis and G. affinis and 0.007 for G. Leiseri. Sampling variance at single loci ranged from

2-44 (5 = 3.3) of observed variance (FsT values, Tables 5-7) in G. nobilis, 3-264 (7c . 17.24) in G. geiseri, 2-25% (31 = 11.5%) in G. affinis. The small size of these percentages indicates that the sample sizes used for this study were adequate for interspecific comparisons of heterogeneity, especially since the computation of error variance was based on mean allelic frequencies for subsets of the original samples which

were smaller than the average sample sizes used in calculating the FsT

measure of heterogeneity.

11 Table 2. Allelic frequencies at four loci in four populations of G. nobilis. BLNWL = Bitter Lake National Wildlife Refuge. Numbers in parentheses = sample sizes. Alleles are given nu- meric designations that correspond to relative electrophoretic mobilities.

Locus Allele BLNWR Blue Sp. Balmorhea Leon Ck. (22) (22) (21) (144)

GPI-A 84 0.977 1.000 0.262 0.826 100 0.023 - - 0.174 73 - - 0.738 - GPI-B 100 0.955 0.477 1.000 1.000 67 0.045 0.523 - - ra IDH-A 100 1.000 1.000 0.500 1.000 88 - - 0.500 - PGM-B 100 0.977 1.000 0.024 1.000 87 - 0.976 - 111 0.023 - - - Table 3. Allelic frequencies at elght loci in five populations of G. affinis.

Independence Locus Allele BLNWR Blue Sp. Leon Ck. Ck. Waco (22) (34) (41) (24) (21)

GPI-A 100 - - 0.738 - 73 1.000 1.000 1.000 0.262 1.000 GPI-B 100 1.000 0.765 0.671 0.524 0.952 67 - 0.235 0.329 0.476 0.048 IDH-A 100 0.139 - - 0.652 88 0.861 1.000 1.000 0.348 1.000 IDH-B 100 1.000 0.853 1.000 0.978 1.000 87 0.147 0.022 LDH-A 98 1.000 1.000 0.890 0.543 1.000 100 0.110 0.457 LDH-B 75 0.977 0.809 1.000 0.935 1.000 100 0.023 62 0.191 0.065

MDH-C 85 0.690 0.985 1.000 1.000 ' 0.976 100 0.310 0.024 68 0.015 PGM-B 100 0.850 0.926 0.951 0.717 0.929 87 0.150 0.074 0.049 0.283 0.071 Table 4. Allelic frequencies at five loci in four populations of G. geiseri.

Independence Locus Allele Balmorhea Leon Ck. Ck. San Marcos R. (36) (35) (35) (19)

AAT-A 82 0.292 0.871 1.000 0.947 100 0.667 0.129 - 0.053 114 0.041 EST-B 100 0.917 1.000 1.000 1.000 86 0.083 - - - GPI-B 100 1.000 1.000 0.985 0.895 67 - 0.015 0.105 IDH-A 81 0.944 1.000 0.985 0.947 88 0.056 _ - 100 _ - 0.01$ _ 109 - - - 0.053 PGM-B 100 1.000 1.000 0.574 0.737 87 - - 0.426 0.263 15

Table 5. F,T and heterogeneity Chi-square values for 16olymorphic loci in various combinations of populations of G. nobilis.

Populations excluded Locus F Chi-Square df ST

None GPI-A 0.664 45.110 6 <.005 GPI-B 0.533 22.286 3 <.005 IDH-A 0.571 24.000 3 c.005 PGM-B 1.252 55.126 6 <.005 Phantom Lake GPI-A 0.146 7.904 2 <.025 Spring GPI-B 0.547 22.838 2 <.005 Blue Spring GPI-A 0.641 50.988 2 <.005 and BLNWR IDH-A 0.677 28.000 1 <.005 PGM-B 1.906 80.063 1 <.005 16

Table 6. F and heterogeneity Chi-square values for STpolymorphic loci in various combinations of populations of G. affinis.

Populations excluded Locus F Chi-Square df ST

None GPI-A 0.866 36.363 4 <.005 GPI-B 0.228 10.457 4 <.05 IDH-A 0.599 24.708 4 c.005 IDH-B 0.125 7.052 4 NS LDH-A 0.390 15.462 4 < .005 LDH-B 0.122 5.411 8 NS MDH-C 0.279 12.526 4 < .025 PGM-B 0.084 4.529 4 NS

Independence GPI-B 0.188 9.891 2 <.01 Ck. and IDH-A 0.146 6.122 2 4.05 Waco LDH-A 0.114 4.120 2 NS LDH-B 0.164 7.275 4 NS MDH-C 0.316 14.529 2 < .005 PGM-B 0.033 3.606 2 NS 17

Table 7. FsT and heterogeneity Chi-square values for polymorphic loci in various combinations of populations of G. geiseri.

Populations excluded Locus F Chi-Square df ST

None AAT-A 0.622 30.429 3 <.005 EST-B 0.085 4.098 3 NS GPI-B 0.088 4.098 3 NS IDH-A 0.026 6.104 6 NS PGM-B 0.309 11.566 3 <.01

San Marcos AAT-A 0.707 35.199 2 <:.005 R. EST-B 0.085 4.135 2 NS IDH-A 0.036 4.135 2 NS PGM-B 0.497 18.610 2 4.005

Independence AAT-A 0.689 38.661 1 4.005 Ck. and San EST-B 0.088 4.211 1 < .05 Marcos R. IDH-A 0.058 4.211 1 < .05 18

F values for G. nobilis were consistently high when all four ST populations were used in the computations (Table 5). Much of this heterogeneity was due to strong differentiation of the Phantom Lake

Spring population. When the latter population was removed from consider- ation, the number of polymorphic loci among populations decreased from four to two and the average FST value decreased from 0.755 to 0.347.

However, even the latter value is higher than any of the FST values for various sets of populations of G. geiseri and G. affinis. F values for G. affinis were lower than those for G. nobilis, even ST when two populations (Waco and Independence Creek) far outside the range of G. nobilis are included, and the difference is even more striking if only those G. affinis from within the range of G. nobilis are considered (Table 6). The difference in heterogeneity remains marked when only those populations of G. affinis and G. nobilis taken from the same aquatic systems are considered (BLNWR, Blue Spring, Leon Creek); this gives average FST values of, respectively, 0.160 and 0.347 for the two species.

Average FST values for G. geiseri (Table 7) were approximately the same as those for G. affinis. Comparing G. .geiseri and G. nobilis from the two spring systems where they were taken together (Balmorhea and Leon

Creek) gives average FST values of 0.278 and 1.071, respectively. Relative levels of heterogeneity among the three species of Gambusia can also be expressed as percent of polymorphic loci showing significant (P(0.05) heterogeneity. This is valid because sample sizes were similar in these computations (21 in G. nobilis and G. affinis, 19 in G. geiseri). Considering all populations of each species, these percentages were 100% (4 loci), 61, (8 loci), and 40% (5 loci), respectively, for G. nobilis, 19

G. affinis and G. geiseri. Thus, again, the G. nobilis populations are more heterogeneous than either G. affinis or G. geiseri. Nei's (1972) genetic identity between all pairwise combinations of G. nobilis samples ranged from 0.876-0.998 (R . 0.940), while those values for G. affinis and G. geiseri were, respectively, 0.901-0.995 (R = 0.958) and 0.959-0.997 (R= 0.982). As noted above for single loci, G. nobilis from Phantom Lake Spring was the most differentiated of the four populations of the species. Genetic identity between the Phantom

Lake Spring population and the other three populations of G. nobilis was markedly lower than that among the latter three-0.876-0.901 (R = 0.891) and 0.981-0.998 (R =0.988), respectively. The former value was also lower than the comparable value (0.905-0.919; R = 0.911) between the most differentiated population of G. affinis (i.e., Independence Creek) and the other Pecos River populations. Interestingly, samples of G. nobilis and G. affinis which were taken from the same spring systems showed the same average level of genetic identity (R = 0.998). Genetic identity among the three G. geiseri populations in the Pecos River system (0.959- 0.988; R . 0.976) was similar to that of the other two species. DISCUSSION

The role of gene flow as a major cohesive force in the maintenance of similarity among different populations of the same species was questioned by Ehrlich and Raven (1969) and, more recently, by Endler (1977). The classical concept maintains that, given enough time, isolatedpopulations of a species will diverge from one another, and that gene flow will have a dedifferentiating effect (Jackson and Pounds, 1979; Mayr, 1963). Ehrlich and Raven (1969) suggested that natural selection is the primary force determining degree of differentiation among populations and that gene flow plays only a minor role. Other workers (e.g., Gould and Johnston, 1972), on the other hand, emphasize that while natural selection is certainly a major force in populational differentiation, it is "notoriously hard to prove that selection has operated, since most of its gross results can also be produced by nonselective agencies."

Jackson and Pounds (1979) suggested that the intent of Ehrlich and Raven (1969) was to provide new perspective by playing devil's advocate. However, Jackson and Pounds concluded that, rather than stimulating empirical tests, Ehrlich and Raven's conclusion has been accepted uncriti- cally to the point of creating a "new orthodoxy" that attributes little importance to gene flow as a cohesive, or dedifferentiating, force. The latter concept has received theoretical support from Endler's (1977) analysis which includes mathematical models. Jackson and Pounds (1979) concluded that the new orthodoxy "is based on a recognition of the efficacy of [natural] selection, on simplistic models, and on opinion, not on studies in nature." 20 21

In general my data seem more consistent with the classical concept

than with the new orthodoxy. The former concept predicts a close relationship between opportunity for gene flow and degree of differentiation, while the latter predicts that the two may vary independently.

Pecos River populations of G. nobilis show much greater heterogeneity in allelic frequencies than do either the more continuously distributed

G. affinis or the recently introduced G. geiseri. The marked difference holds regardless of whether the computations of variance in G. affinis and G. geiseri include only populations occurring alongside G. nobilis or if they include populations far outside the range of G. nobilis. Although springs in the Pecos River drainage were larger and more numerous in the past, even in recorded history (Brune, 1975), G. nobilis probably has been effectively excluded from the Pecos River proper for hundreds or thousands of years as a result of competition with the highly abundant G. affinis population. As Echelle et al. (1976) demonstrated for darter populations in the Red River drainage of Oklahoma, competition in downstream areas with a better adapted congeneric species may be a major factor restricting gene flow among headwater populations. Extensive collections by W. J. Koster in the Pecos drainage of New Mexico in the

1940's and 1950's produced only a single specimen from the Pecos River proper (Michael Hatch, pers. comm.). Excepting that one specimen, G. nobilis has never been taken from the Pecos River itself (Echelle and Echelle, 1979). Interaction between G. nobilis and G. affinis in the Pecos River system probably began at least during the middle Pleistocene when (Thomas, 1972; Leonard and Frye, 1975) the Pecos River captured the headwaters of the ancestral Brazos River. This event would have brought

G. affinis into the Pecos if the species was not already there as a result of earlier geologic events (see Echelle and Echelle, 1978, for a partial review of the ichthyogeography of the Pecos River). Hubbs and Springer (1957) suggested that geographic isolation as a result of competition with

G. affinis may have contributed to the evolution of the various species of the Gambusia nobilis group. This could also explain the high level of genetic heterogeneity within G. nobilis itself.

Amount of gene flow among populations of a species would be proportional to the integral, through time, of instantaneous opportunities for gene flow. For the four present populations of G. nobilis this probably amounts to little or no gene flow for many, perhaps hundreds or thousands, of generations. Even if an occasional stray from one population entered the Pecos River proper it would be unlikely to find its way into one of the other spring systems. This degree of isolation is surely greater than that for any other species of fish listed in Table 8. Correspondingly, the average variance in allelic frequencies of G. nobilis is higher than that reported for any other species of fish (Table 8), and, to my knowledge, it is the highest reported for any organism (e.g., see

Selander and Kaufman, 1975). The extreme degree of heterogeneity in G. nobilis may be characteristic of many other desert populations of spring- dwelling fishes.

The high level of heterogeneity at single loci in G. nobilis apparently has not been accompanied by genetic divergence usually associated with the species level. Genetic identity between the most divergent population of G. nobilis (Phantom Lake Spring) and the other three (0.876-0.901) was well within the range of that (Avise, 1974) for conspecific populations.

The more continuously distributed G. affinis had much lower heterogeneity in the Pecos drainage than does G. nobilis. Since only 23

Table 8. Average FST values for various fish populations.

Area Number of Species Covered Loci F Source ST

Astyanax Rio Tampaon 10 0.386a Avise and Selander mexicanus region 1972 Tamaulipas, Mexico Rhinichthys Connecticut R., 6 0.139a Merritt et al. cataractae MA, CT, NH 1978 Notropis Kansas R., 1 0.015a Koehn et AL. stramineus KS 1971 Lepomis Savannah R., 3 0.563 Avise and Felley macrochirus SC 1979 Santee-Cooper 3 0.158 R., SC Etheostoma Several major 3 0.502a Echelle et al. radiosum tributaries 1975 of Red R., OK and AR Etheostoma Clear Boggy 2 0.463 Echelle et al. spec tabile R., OK 1976 Washita R., 2 0.046 OK a F values computed from data presented by the authors. ST populations in tributaries were sampled, the observed variance may

represent the upper bound for G. affinis in the Pecos. Interestingly,

the observed variance for G. affinis remained lower than that of G. nobilis even when two samples well outside the range of G. nobilis were included.

The Independence Creek sample, which is within the Pecos drainage, con-

tributed much more heterogeneity to the G. affinis samples than did the sample from Waco, Texas, which is across the Edwards Plateau from the Pecos.

On the basis of morphology, the latter population and populations from

the upper and middle Pecos are members of the same subspecies, G. a. affinis, while the former shows strong similarity to the southern subspecies, G. a.

speciosa (Peden, 1970). Thus, heterogeneity in the Pecos apparently is

partly a result of subspecific differentiation. The high homogeneity among the G. a. affinis samples on the two sides of the Edwards Plateau may be heightened by interdrainage introductions for malarial control

(Koster, 1957) and headwater transfers during wet seasons. G. affinis from eastern populations has been introduced over much of the state of

New Mexico (Koster, 1957), and the species has been observed on the divide

between the upper Pecos River and the eastward flowing (Fig. 1) South

Canadian River in temporary pools left by flooding between the two drain- ages (Jim Duke, pers. comm.). G. affinis seems well adapted to quickly invade recently flooded areas, an ability that would increase the proba- bility of headwater transfer.

Heterogeneity in allelic frequencies of G. geiseri was higher, relative

to that of G. affinis, than expected on the basis of gene flow. Because they apparently arose as a result of introductions by man, populations of

G. geiseri in the Pecos River system can be thought of as recently estab- lished (less than 100 years ago) peripheral isolates of the species. Despite the short period of isolation, heterogeneity among subpopulations of G. geiseri in the Pecos was comparable to that of G. affinis over a large

geographic area and in which a large amount of time was available for divergence. Conceivably, the observed heterogeneity in G. geiseri is a result of founders effect. Small samples of G. geiseri may have been introduced into a given area in the first place, and only a small subset

of those might have found their way to the headspring situations favored

by the species.

Gene frequencies in local populations result from four forces: natural selection, genetic drift, mutation, and migration ( = gene flow).

However, for most populations the question of whether subpopulations will diverge from each other or evolve as a unit seems to depend only on the relative magnitudes of natural selection and gene flow. This follows from

theoretical arguments by Crow and Maruyama (1971) and others (e.g., Spieth,

1974) which demonstrate that, in general, interpopulational exchanges of

only one individual per generation are sufficient to overcome the differentiating effects of genetic drift and mutation. Thus, for selectively neutral

traits, gene flow is of paramount importance in interpopulational differentiation: as Spieth (1974) noted for the effects of gene flow, " . . . the

distinction between absolutely none and almost none is enormous." However,

for spring-dwelling fishes in desert environments, such as G. nobilis,

gene flow among subpopulations may indeed be zero for many generations.

Thus, the role of drift and mutations cannot be discounted and may be

significant.

The point made by Ehrlich and Raven (1969) is that the rate of

migration among subpopulations may be so low for many, if not most,

species that the effects of gene flow, or lack of same, are negligible 26

compared to the effects of natural selection in both the differentiation of and in the "cohesiveness" of subpopulations. My results for Gambusia nobilis and G. affinis and the information on other fishes (Table 8) show a positive relationship between opportunities for gene flow and degree of interpopulational differentiation. Thus, regardless of whether observed differentiation is caused by natural selection or by random events (genetic drift and mutation), the available data appear more consistent with predictions based on the classical concept of the role of gene flow than on those based on the "new orthodoxy."

As emphasized by Jackson and Pounds (1979) there are few adequately controlled tests of the role of gene flow. My comparison of G. affinis and G. nobilis from the same spring systems was meant to satisfy some of the requirements for controls. However, the two species show some segregation by microhabitat. Thus, it is possible that the different levels of genetic heterogeneity result from differential intensities of natural selection on subpopulations of the two species. This could cause the observed differences in degree of differentiation within the two species. On the other hand, there seems to be no reason why selection pressures for alternative alleles in subpopulations of G. nobilis should be greater than those pressures acting on the other species in Table 8.

At the same time, as emphasized above, the unusually high level of genetic heterogeneity in G. nobilis is correlated with extremely small opportunity for gene flow. Thus, gene flow appears to be a major factor in populational differentiation among Gambusia species in the Pecos River system. LITERATURE CITED

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27 28

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Appendix A. Nei's genetic identities for all combinations of four populations of G. nobilis.

Populations

Populations Blue Sp. Balmothea Leon Ck.

BLNWR 0.986 0.896 0.998 Blue Sp. 0.876 0.981 Balmothea 0.901

32 33

Appendix B. Nei's genetic identities for all combinations of five populations of G. affinis.

Populations

Independence Populations Blue Sp. Leon Ck. Ck. Waco

BLNWR 0.984 0.984 0.905 0.993 Blue Sp. 0.995 0.909 0.995 Leon Ck. 0.919 0.994 Independence Ck. 0.901 34

Appendix C. Rei's genetic identities for all combinations of four populations of G. geiseri.

Populations

Populations Leon Ck. Independence Ck. San Marcos R.

Balmothea 0.981 0.959 0.970

Leon Ck. 0.988 0.995

Independence Ck. 0.997