GENETIC VARIATION AND PJ NEW WORLD CICHLIDS

A thesis presented by Irving L. Kornfield to The Graduate School in partial fulfillment or the requirements for the degree of Master of Arts in Ecolozy and

State University of New York at Stony Brook October, 1972

STATE UNIVERSITY OF NEW YORK

AT STONY BROOK

THE GRADUATE SCHOOL

IR7I1G L. KO=FIELD

We, the dissertation committee for the above candidate for the I.A. degree, hereby recommend acceptance of the dissertation.

Richard K. Koehn (Dept. of Ecology and Evolution)

DOUGLAS J. FUTUYMA (DEPT. OF ECOLOGY AND EVOLUTION)

GEORGE C. Williwds (DEPT. OF ECOLOGY AND EVOLUTION)

The dissertation is accepted by the Graduate School.

DATED :

1 HERBERT deisenger, Dean GRADUATE SCHOOL TABLE OF CONTENTS

PAGE

Abstract ......

Acknowledgments ......

List of Figures ...... List of Tables ......

Introduction ......

Material and Methods ...... Results ......

Discussion ......

Appendix ......

Bibliography ...... ABSTRACT

Genetic variability was sCudieC rineteen electro- phoretic loci in two undescribed er-lc ,Achlids from Cuatro Cienegas, Coahuila, Mexico er(2. in Cichlosoma staTIg21 211. from the Rio Grande in Texas. The two endemic species differ markedly in their pharyngeal dentition and general ecologies. Only the Lactate Dehydro- genase was polymorphic in 130 endemic cichlids sampled from several lagunas. C. cyanogutt m was monomorphic at all loci examined in a sample of 38 individuals from the Rio Grande. The low proportion of polymorphic loci (=.055) and low average number of heterozygous loci per individual (=.003) differ markedly from all previous estimates. The data are not consistent with , the founder effect, or low environmental variability as explanations for low . No difference in isoenzyme mobility was observed between the two endemic cichlids (Rogers coefficient of genetic similarity, S = 1.0). C. cyanoguttatum exhibited identical mobility with the Cuatro Cienegas cichlids at more than half the loci examined (S = 0.582). The signifi- cant evolutionary divergence • that has occurred in certain morphological features of the endemic Cuatro Cienegas species. suggests that variability and similarity estimates based upon isoenzyme loci do not validly reflect genetic events associated with speciation. ACKNOWLEDGMENTS

I would like to thank Mr. Gary L. Mr. K. Thompson, and Dr. Charles Waltace for the collec- tion of Rio Grande cichlids essential to this study, and Mr. Richard Stone for assistance during the collection of endemic cichlids in Mexico. A great debt is owed to Sr. Pepe Lugo, resident naturalist and entrepreneur, who cont'nues to provide invaluable assistance to all biological investigations in the Cuatro Cienegas Basin. Gratitude is expressed to Mr. John Kishpaugh, Dr. W. L. Minckley, Dr. Dennis Powers, and Dr. F. James Rohlf for assistance and advice in helping to solve many problems generated during the progress of this study. Appreciation is extended to Dr. George C. Williams for many interesting discussions and helpful suggestions. Dr. Douglas J. Futuyma's interest in the progress of this study was only surpassed by his numerous perceptive observations. I am grateful for his continued concern. Dr. Richard K. Koehn has, from the beginning, provided the ideas, tools, patience and enthusiasm necessary for the successful completion of this study and for my personal development. His uncompromising integrity and genuine interest has made it a pleasure to be his student. I express my deep appreciation to my wonderful wife, Tori, for her patience and understanding throughout the course of this study. This work was supported in part by research grants

NSF (GB-25343) and USPHS (GM-28963) to Dr. Koehn and a ( SUNY Faculty/ Research Fellowship to Dr. Futuyma.

0-VAA, S OA:

C,- US9H S CO-Y -E-E v LIST OF FIGURES

FIGURE PAGE

SAMPLE LOCALITIES OF C. CYANOGUTTATUM AT MISSION, TEXAS, AND ACHLOSOMA sm. from CUATRO CIENEGAS, MEXICO

2 SAMPLING LOCALITIES WITHIN THE CUATRO CIENEGAS BASIN

3 External morphology and pharyngeal BONES of Cichlosoma spp ......

4 Zymograms of MONOMORPHIC ISOENZYMES

Zymograms OF VARIABLE ISOENZYMES ..... 6 Serum protein electroplerograms 11 A LIST OF TABLES

TABLE PAGE

Electrophoretic and staining methods .... Estimated genetic variability in the Cuatro Cienegas cichlids ...... INTRODUCTION

, Numerous studies on a wide dive- or organisms have appeared characterizing many isoenzymes as polymorphic.

Many reports have inferred Atirly high levels of overall genomic variability in natural populations (e.g. Lewontin and Hubby, 1966: Selander et al, 1970; Ayala et al, 1972). . These findings provide a basis from which to investigate major problems of , notably the rela- tionship of variability to immediate and long-term survival of a species.

Since most studied enzyme polymorphisms cannot be maintained by alone (Tobari and Kojima, 1972), a variety of genetic mechanisms have been proposed to explain the continued existence of this variation. Selective neutrality of isoenzymes has repeatedly been suggested on theoretical grounds (Kimura, 1968; King and Jukes, 1969; King, 1972). There is presently, however, an overwhelming body of evidence implicating the role of in the maintenance of isoenzyme variability. While the selective agents responsible, or the selective mechanisms by which these may operate are not generally known (see tulmer, 1971; Koehn and Mitton, 1972) both population studies and biochemical observations have empirically demonstrated selective maintenance of enzyme variation

(Koehn 1969; Merritt, 1972). Given different of , environmental heterogeneity (or some component

thereof) can act as a factor in the maintenance of genetic

variability. (Levene, 1953; Levins and MacArthur, 1966; Prout, 19 ; Levins, 1968). From this theoretical basis,

Koehn and Mitton (1972) demonstrated identical patterns of

isoenzyme variation in two sympatric marine bivalves along an environmental gradient, suggesting an optimal response

of different organisms to environmental heterogeneity.

Powell (1971), in a laboratory study of Drosophilia willistoni over fifteen generations, demonstrated higher

average heterozygosity and number of alleles per locus in populations maintained in heterogeneous environments.

Variation in the environment of an organism is (particular-

ly ia the aLscuLv of humeubLaLit. muchunisms) apparently

reflected by the presence and continued existence of • beneficial adaptations responding to those varying environ-

mental parameters. If an environmental signal is invariant

over time (e.g. constant temperature), an with a

selective advantage will eventually become fixed. The

• contrasting case, however, is more complex. That is, if the environmental signal is not constant, the genetic

strategy adopted at a responding locus could be fixation

or , depending upon the relative duration of

alternate states of the signal (see Levins, 1968, for discussion). Electrophoretic comparisons of proteins between species (e.g. Hubby and Throckmorto 4)5, 1968; Selander et al, 1969; Johnston and Selander, 1971;

Rockwood et al, 1971; Smith trid Koehn, 1971; Turner, 1972; Weber et al, 1972) have proviged insights into phyletic relationships and genetic reorganization during SPECIATION. Although electrophoretic compari- sons have been successfully EMPLOYED AT the genus level, the systematic utility beyond congeneric comparisons is limited by an increasing probability of non-genetic identity (i.e., equivalent net charge but different composition). The extent of genomic reorgani- zation following speciation suggested by Hubby and ;, 4,‘ 1...a.rInen■uoj Jufwaypii-t-ka, tutu a cidnUer el al (1969) in Mus, remains controversial. Critical questions concerning genetic events during speciation remain to be answered. Some freshwater fish taxa are characterized by an ususually rapid proliferation of specialized species. "Explosive speciation" characterizes hundreds of cichlid species endemic to the Great Rift Valley lakes of Africa. Although a traditional view of the mechanics of their speciation is generally held (see Mayr, 1963), the complex reproductive habits (Baerends and Baerends-Van Roon, 1950) and rapidity of speciation within limited geographic areas (Greenwood, 1965) present an extremely unusual biological / situation that suggests numerous unexamined questions of

evolutionary significance (see Greenwood, 1956; 1959;

1964; Fryer and Iles, 1969). Extreme modification of dentition and cranial morphology is commonly observed

among the various trophic tAes (Greenwood, 1959; 1964).

Additionally these fishes exhibit a high degree of intra- specific variation in just "...those characters of the

animal which were utilized as a basis for specific

differentiation by systematists...." (Fryer, 1959). This

• study deals with Cichlasoma cyaglosuttatum, (Baird and Girard) which occurs throughout the major rivers of Texas

and northern Mexico (Blair et al, 1957; Anonymous, 1967),

• and three endemic undescribed species of Cichlosoma restricted to waters of the Cuatro Cienegas Basin,

Coahuila, Mexico (Taylor and Minckley, 1966). While

river environments can generally be considered variable

with regard to annual temperature variation, suspended

matter, foods, etc., thermal springs of Cuatro Cienegas

provide environments invariant in temperature and ionic content (Minckley, pers. comm.). The most abundant and widely distributed of the endemic species possesses very

fine pharyngeal teeth and light supporting musculature,

and feeds primarily upon vegetation and algae. Occurring

with less abundance and more limited distribution, is a

form characterized by thick heavy pharyngeal teeth used

primarily in feeding upon snails. On cursory external

examination the two forms are indistinguishable. The distribution and abundance of a third endemic form is very restricted. It is presumably predatory in habit, and differs markedly in shape and dentition from the other two types. It has an elongated form and very strong jaws (Taylor and Minckley, 1966). The occurrence of these endemic fishes within a very small geographic area is essentially identical to the previously described situation of the African Lake cichlids, but on a much reduced scale.

All three species are presumed descendants of the ancestral

Rio Grande Cichlid, C. cyanoguttatum. In laboratory studies of the two common Cuatro Cienegas forms, the dentition does not change with growth, nor have natural hybrids been produced between species (Minckley , pers. comm.).____ Examina- c,vel 3,000 field pcCimunb 017 CuaLru Cienegus cichlids did not reveal any hybrids or individuals possess- ing intermediate dentition (Minckley, pers. comm.). It should be noted that assignment of taxonomic status and a formal description of these forms has not yet appeared in the literature but will here be referred toad int. as species.

Preliminary studies on genetic variation in the three endemic Cichlosoma species has been performed by Koehn and

Greenwood (unpubl.). Their results from serum electropho- resus indicated the absence of protein variation either within or among the three endemic species. These observa- tions were inconsistent with previous estimates of enzyme variability, 'and electrophoretically demonstrated dif- ferences between species. In this st- L7dy, the number of individuals and loci examined was greatly expanded to more generally characterize levels of genetic variability, 1 and to search for possible interspecific differences.

Specifically, the following questions were addressed:

(1) What is the magnitude of genetic variability of these cichlid species, and how does this compare to previously reported levels in other organisms?; (2) Is the level of isoiyme variability in the Rio Grande Cichlid, C. cyano- guttatum, significantly different from the Cuatro Cienegas cichlids?; and (3) What is the degree of genetic identity among all cichlids tested and how does this compare to previous reports for other orvanisms? Materials and Methods

Forty specimens of C. cyanoguttotim were collected in October 1971 from the Rio Grande near MislAon, Texas (Fig. 1), and shipped live to Stony Brook, N. Y. 1 One-hundred-fourteen specimens of the algae-eating species and forty-four specimens of the snail-eating species were collected by hook and line in July 1971 from four locali- ties within the basin of Cuatro Cienegas (Figs. 1 and 2, and Appendix). External morphology ad pharyngeal teeth of the two common Cuatro Cienegas species and the Rio Grande Cichlid are illustrated in Fig. 3. Thirty-eight specimens of C. cyanoguttatum were electrophoresed, while a variable number of Cuatro Cienegas cichlids were tested for each enzyme. Tissue samples showed high staining activity even after nine months of storage. Serum samples were obtained by the method of Koehn (1969). The liver and a small piece of vntral skeletal muscle were excised from each specimen and placed in centrifuge tubes containing an equal volume of deionized water. In field sampling, tubes were cooled on ice, and returned to a local freezer. The tissue samples were later stored at -60°C. Dissected fish were tagged and preserved in 7 percent formaldehyde and later transferred to 40 percent isopropanol. Tissue samples were prepared for electrophoresis by ultrasonic cell disruption for approximately ten seconds. Homogenates were centrifuged at 0°C for twenty minutes at 15,000g arid the supernatant used immediately for electrophoresis. Blood samples were electrophoresed in 7 percent acrylamide gel with an EC474 Vertical Gel Electrophoresis Apparatus* for three hours at 400 volts. The running gel contained 7 grams Cyanogum-41*, 0.1 gm ammonium persulfate, and 0.1 ml TMED (N,N,N ,N', -tetramethylethylenediamine) in 100m1 of 0.38 M Tris-HC1, pH 8.9, solution. The electrode buffer was 0.1 Tris-Glycine, pH 8.3. Following electrophoresis, gels were stained for ten minutes with .2 percent Amido Black 10B in a solvent of a 5:5:1 mixture of methanol, water, and acetic acid, respectively. Gels were destained in a 5 percent acetic acid solution. Isoenzyme separatiJn of liver and muscle extracts was performed by horizontal starch-gel electrophoresis as described by Koehn and Rasmussen (1967). Electro- phoretic buffers were identical for all species (see Table 1 for buffer systems and staining references). Starch gels were incubated at 37°C in the appropriate staining solution, photographed, and fixed in a 5:5:1 mixture of methanol, water and acetic acid.

* E-C Apparatus Corp., Philadelphia, Pa. Electrophoretic Results

Typical zymograms for all isoenzyme:; are depicted in Figure 4. Although some variability was observed (see below), snail and algae-eating species exhibited identical banding at all loci. No breeding experiments have been performed with these fish and therefore postu- lated loci are based upon previous studies of electro- phoretic patterns in other organisms In the absence of discrete alternate , the interpretation of some complex patterns is quite confusing. Since some loci produce multiple electrophoretic bands (see esterases), the number of bands observed may suggest more loci than are actually represented. For this reason, in the case of multiple banding, a minimum and maximum estimate of the number of loci present is provided.

Acid Phosl22_a_i_ ta_s_s (3.1.3.2) Two bands were observed in all individuals of the Cuatro Cienegas cichlids (Ni.,123). Electrophoretic patterns of Cichlasoma cyanoguttatum acid phosphatase were also two banded. The mobility of the slower band was less in C. cyanoguttatum than the slow band of the Cuatro Cienegas cichlids, while the faster was equal. The two bands are interpreted as the products of two independent loci. Amino ikeptidase (3.4.1.2) A single.electrophoretic band was observed in both the Cuatro Cienegas (N . 89) and Rio Grande cichlids, but the relative mobility was greater in C. cyanoguttatum.

THERE WAS SOME SLIGHT differential migration in banding among individuals, however no was observed for which a heterozygote basis could be postulated. There was no statistical association between a particular mobility and species, sex, or locality of collection. The single band suggests only a single locus.

Esterase (3.1.1.1) Electrophoresis revealed two distinct five banded patterns in the Cuatro Cienepas cichlids (N = 123). ThrA bands (1 3 4; Fig. 5) were common to both phenotypes, but individuals exhibited either bands SB and 2A (Type I) or 2B and SA (Type II). The Type II phenotype was in highest frequency, (84%), but there was no association between phenotype and SPECIES, sex, or size.

If the Type II phenotype is assumed to represent a homozygote, and the alternate Type I phenotype a hetero- zygote, there is good fit to an expected Hardy-Weinberg 2 distribution (X = .38, p>0.5). However, the probabil- (1) ity of a random fit to an expected Hardy-Weinberg distribu- tion is >0.6 for any combination of numbers partitioned into two classes totalling 123. Type I does not exhibit any additional bands normally characteristic of a hetero- zygote. Additionally, esterase activity has been shown to be extremely variable and under the control of various modifiers (Oki et al, 1966; Wpnenoff, 1972). C. cyEllgaLl im exhibited a single invariant pheno- type consisting of three bands corresponding in electro- phoretic position to bands 1, 3, and 5B of the Cuatro Cienegas cichlids (Fig. 5). Since there is no experi- mental genetic basis for the interpretation of these observations, the number of loci expressed could vary from a conservative estimate of two to a liberal estimatz; of four.

General Protein of Muscle Extracts Three identical bands were observed in all specimens of C. cyanoguttatum and Cuatro Cienegas cichlids (N = 110). Additional invariant bands of very low staining intensity were noted, however these were not consistently observed. The three bands are interpreted as the products of either two or three independent loci.

Glutamate Oxaloacetate Transaminase (2.6.1.1) All samples exhibited a four banded pattern. The • mobility of C. cyanoguttatum was identical to the Cuatro Cienegas species (N = 89). The slowest electrophoretic band showed markedly stronger activity than the others, which decreased in activity anodally. It has been our experience with this enzyme in fish th5t it is very sensitive to freezing and thawing. It i3 quite possible that the large number of bands was an artifact of storage and therefore, only one locuA is postulated.

Lactate ppjademealL-1 (1.1.1.28)

The Cuatro Cienegas cichlids exhibi.tcd a polymorphism at a single LDH locus (N = 138). Although a five banded heterozygote would be expected for a functionally tetra- meric isoenzyme, usually only three or four bands were observed (Fig. 5). Because of restricted subunit assembly (Whitt and Horowitz, 1970) a smaller number of bands is commonly observed in fish. The frequency of the fast of two alternate alleles was .97+ .01. This did not fit a Hardy-Weinberg distribution (X2 =5.30) probably owing to the occurrence (1) of a single slow homozygote. Heterozygotes showed no statistical association with species, locality, sex or size. C. cyanoguttatum exhibited a single uniform band slower in electrophoretic mobility than both postulated alleles of the Cuatro Cienegas cichlids.

Malate Retlydranne (1.1.1.37) Two invariant bands were observed in both Cuatro Cienegas (N = 120) and Rio Grande fishes. The mobility of the faster band was the same in both, while the slower band was relatively faster in the Cuatro Cienegas cichlids. It is assumed that two loci are represented here.

1 Phosphoglucomutase (2.7.5.1) A single band was observed in both C. cyanoguttatum and the Cuatro Cienegas cichlids (M = 118). Although activity and band width varied slightly among samples, no significant differences could be noted among any individuals. A single locus is postulated for PGM.

Tetrazolium Oxidase (/ . ? . ? . ?) One invariant electrophoretic band was observed in all Cuatro Cienegas (N = 123) and C. Exanclarttatum individuals. The C. spp. banding was faster than the mobility of C. cyanoguttatum. Infrequently additional slower bands of markedly less activity were seen in the Cuatro Cienegas individuals.

Xanthine ilehydroanale (1.2.3.2) One invariant band was observed in all samples of C. cyanoguttum and Cuatro Cienegas cichlids (N = 129), but the relative mobility of the latter cichlids was faster.

Serum proteins • Eight bands were generally observed in acrylamide electrophoresis of Cuatro Cienegas cichlid blood samples (Fig. 6). Serum proteins from C. syanoouttatum specimens were not run. There was variation among elcctrophoretic separations in the total number of distinguishable bands (5-10), but this was presumab0 due to different periods of electrophoresis, staining, and storage. The only variation noted in the Cuatro Cienegas cichlids (N = 120) was the occasional absence of one dark-staining band (Fig. 6). This absence is not believc-d to be an artifact of dilution of the sample since some samples lacked these bands but exhibited dark staining in all others. There was no association between presence/absence of bands and species. A minimum of four or a maximum of seven serum protein loci are postulated to be exhibited with this stain. // I TWO criteria have generally been employed to define , "The more (-0Y% RUC14; 0,E CO1144E 1_1)9 TL balanced polymorphism f the—frequaity—bf the commonest allele is equal to or less than 0.95,.while the more liberal criterion permits frequencies of up to 0.99 (see Ayala et al, 1972; Richmond, 1972). Under either criterion, the level of variability in these cichlids was extremely low. The proportion of polymorphic loci ranged from 0.000 to 0.055, relative to a cut-off frequency of 0.95 and 0.99, respectively. Similarly, heterozygous loci/individual ranged from 0.00 to 0.30 percent (Table 2). ' As noted earlier, in the absence of experimental verification genetic assertions about these species are inferential. However, the almost total absence of allelic variants would, even if breeding had been done, make it

IMPOSSIBLE TO ESTABLISH THE number of loci involved in the observed electrophoretic pattbrns. •Since virtually all loci possessed only one allele, electrophoretic mobility . comparisons between species was very direct. Rogers' coefficient (S) of genetic similarity (Selander, 1972) was employed to quantify the degree of interspecific isoenzyme congruence. Although the ESTIMATE CAN be computed in two ways electrophoretic bands with unequal mobility are

ASSUMED to be the products of divergent loci (rather than different alleles at the same locus). THE two endemic Cuatro Cienegas species exhibited identical electrophoretic phenotypes at all loci (S 1.0). Genetic similarity between the Cuatro Cienegas CICHLIDS and the congeneric Cichlosoma cyanoguttatum was 0.582. Discussion

Cichosoma sxanoguttatum inhabits a fairly variable aquatic environment. Water temperatures in the Rio Grande near Mission, Texas seasonally range from 14.2°C (Hubbs, 1951) to over 25°C. Periodic spring flooding conditions would be expected to dramatically alter other physical characteristics of the water mass such as nutrient content and suspended matter. The Cuatro Cienegas cichlid species, at the other extreme, inhabit a virtually constant environ- ment. Thermal springs provide nearly constant temperature and mineralogical regimens for much of the aquatic in Cuatro Cienegas (Minckley and Cole, 1968; Minckley, 1969;

, 91-gs fri41. Ao" vs and MinCklAy ; PrArq_ ('nmr.). Mnct rnrseic 1v. -r and spring fed from the bottom, so water mixture is continuous, and no stratification can develop. However, some ponds in the Cuatro Cienegas basin are fairly shallow, and possibily subject to the effects of air temperature variation and seasonal water runoff. Although hydro-morphometric properties vary within the basin, annual variation in water temperature and ionic content is additionally buffered by generally low ratios of surface area to volume in most ponds and lakes. Thermal tolerance in fishes may remain extremely broad even after long period of constant temperature (e.g. in Cyprinondon as shown by Brown and Feldmuth, 1971). However, freshwater snails and other invertebrates from Cuatro Cienegas are quite sensitive to temperature change (Minckley, Pers. Comm.). Inferentially, the evolution n1(.1 continued existence of a number of temperature-sensitive gastropods provides further support for the environmental constancy of the endemic Mexican species. A number of studies have reported variable, but unexpectedly high levels of isoenzyme polymorphism and associated heterozygosity for organisms generally inhabit- ing variable environments (see Selander et al 1970 and Selander and Johnson, 1972, for general review). In the genus Drosophila, estimates vary dramatically interspecifically, but most studies on a wide range of organisms report polymorphisms at greater than 25 percent of examined loci with greater than 10 percent of the loci being heterozygous per individual. Recently, a few studies have reported extremely low levels of genetic polymorphism at isoenzyme loci in a variety of natural populations (see Johnson and Selander, 1971; Serov, 1972; Webster et al, 1972). In low variability populations, consideration should be given to population structuring and the consequent probability of stochastic events. If these species exist with small effective population size, either presently or past, sampling events such as or the founder effect might reasonably explain low levels of genetic variability. In such cases, low variability estimates are interesting, but they are important only if populations in general are not large panmictic breeding units. For example, the low levels of observed variability in cove dwelling populations of the teleost Asyanax are probably due to genetic drift (Avise and Selander, 1972). ft is possible in fact, that many recent observations showing low variability are the results of historical sampling accidents (see Webster et al, 1972 for general discussion). In the Cuatro Cienegas species, population sizes are of an order (approximately 100 - 1000) where stochastic events could conceivably play a role. However, in the absence of demonstrable differences between isolated populations of either the same or different species stochastic processes cannot reasonably explain the observed homoiygosity. That is, ail genetic variability could not have been eliminated via sampling accidents, or the observed morphological differentiation between the species would not have occurred. Population sizes of C. cyanoguttatum appear to be quite large (G. L. Powell, pers. comm.), and the limited number of specimens electrophoretically tested constitute a small selected sample of fishes collected over a twenty mile range along the Rio Grande. No empirical . data on abundance or population structuring are available. • In view of the absence of extensive field observations and limited sample size employed in this study, it does not seem appropriate to characterize the entire species as electrophoretically monomorphic. Since C. cyanoguttatum inhabits a variable environ- ment but is electrophoretically monomorphic, support is denied for the environmental k-esponse strategy suggested by recent studies on other organisms (see Koehn, 1969). Fishes inhabiting variable environments would be expected to be genetically variable as an to fluctuating selection pressures. A variety of possible explanations exist to explain the observed monomorphism, but it is unlikely that population structuring or environmental response are primary causes.. For instance, it is pos3ible that the ancestoral C. sylimmit.tatum may have had little genetic variability when it originally invaded North America, though it would appear that sufficient time has elapsed for the accumulation of beneficial . Secondly, it is possible that C. cyanoguttatum views its environment as constant, in contrast to our own perceptions of its habitat. However, we would not expect all loci to respond with exactly the same strategy. Thirdly, the loci we have sampled may not typify the entire . In view of the widely held assumption that 20-30 studied loci may adequately estimate variability in the entire genome of a species, this last point deserves special attention. For example, electrophoretic studies on genetic reorganization following speciation have been much debated (see Selander et al, 1969; Nei, 1971 for general discussion), but it is generally felt that this technique provides a valid reflection of genetic differences between species. Studies of genetic similarity between subspecies and sibling species have indicateil an association between overall electrophoretic similarity and level of evolutionary divergence (Hubby and Throckmorton 1965; 1965; 1968; Rockwood et al 1971). Estimated genetic identity. of = 0.582 between C. cyanoguttatum and the Cuatro Cienegas cichlids is well within the range reported for sibling pairs. The absolute electrophoretic identity between the endemic algae-eating and snail-eating cichlid species stands in sharp contast to all previous observations between species pairs. There is evidence from core samples that inundation of the Cuatro Cienegas Basin from the outside last occurred approximately 30,000 years before present (Minckley, Etn. comm.). The endemic cichlids may have become established at that time, although speciation could have taken place more recently. If selection were strong enough, mutant alleles would never have reached appreciable frequencies in these populations. However, regardless of the mechanisms proposed to explain this monomorphism, our isoenzyme data 1 suggest all endemic forms are conspecific. This is obviously incompatible with the observed morphological differentiation and all other observations to date that strongly support reproductive isolation between the endemic Cuatro Cienegas cichlids. It has been commonly ossumej in studies of intra-specific genetic variabflity and r-specific genetic differentiation, that isoenzyme loci adequately reflect genetic events characteristic of the entire genome. However, loci studied here do not reflect the genomic differentiation that occurred during their speciation. In other words, speciation in this case did not involve random genetic reorgLnization, but rather reorganization of restricted portions of-the genome. It is possible that the electrophoretic idelgtity of these endemic fishes is a reflection of parallel adaptation to identical physical environments that presently exist, or that may have existed in the past (see Koehn and Mitton 1972).

• If our interpretations are correct, isoenzyme loci may not always accurately reflect evolutionary events involving the total genome. Because cif the widespread evidence that enzyme polymorphism is adaptive, electrophoretic characterization of variability may reflect to a large part the nature of current environments of a species rather than its evolutionary history. APPENDIX

LOCATION

Texas-1 Drainage canal a few meters from the Rio Grande river in Hiladg county near Mission, Texas.

Mexico-1 El Mtyjarral East. Irool:l.r border. Approximately 50-70 meters long, 15-20 meters wide. Ave7age depth 0.3-0.6 meters. Sampling done on northwest corner.

Mexico-2 Pond approximately 3/4 kilometers northeast of Rancho Orozco. Almost circular in outline, diameter approx- imately 25 meters. Maximum depth 5 meters. A small stream flows out the southeast corner.

Mexico-3 Pond approximately 1/2 kolometer northeast of Laguna Tio Candido. There was a series of small ponds in this area roughly in a line. This was the first one encountered coming from Tio Candido. Roughly circular in outline, the diameter Was approximate- ly 5 meters. The pond is drained by a small channel on the north bank.

Mexico-4 Pond approximately 3 kilometers south- east down road from Rancho Orozco, then northeast approximately 1/2 kilometer. A very small pond approximate- ly 4 meters diameter. Depth less than one meter. FIGURE 1. Sample localities of C. cyanoguttatum at Mission, Texas, and Cichlosoma spp. from Cuatro Cienegas, Mexico. 1000 980 102°

28° COAHUILA TEXAS

- Cuatro Cienegas 7

rso 0 50 ; NUEVO LEDN scale of mlles • (1,20 100° 98° FIGURE 2. Sampling localities within the Cuatro Cienegas Basin (Adapted from Minckley, 1969) N

0 CuroCienegas '

5 5 scale of kilometers FIGURE 3. External morphology and pharyngeal bones of Cichlasoma snluuttatum (A), endemic algae- feeding Cuatro Cienegas cichlid (B), endemic • snail-feeding Cuatro Cienegas cichlid (C). Infrapharyngeal (below) and superpharyngeal bones (above) are at the right of each species. a .. „ t • "NO `4 v,ic o..."""••••, ••$ 44, .*

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•", r -IrreiTorf:7.4witilisledimeswasmoiswir •-tellIkiteloollalMININISMOS11011111r FIGURE 4. Zymograms of monomorphic isoenzymes. Observed electrophoretic phenotypes for 10 monomorphic protein systems of

C. symagm a (left) and Mexican cichlid (right). Banding was identical for both Cuatro Cienegas species. _G 1 TT+D.REVIS.'‘TR... 0)(A ID 1 , A6;c Ph0spk04 Aøiino. pepti_akse 4C O3TT TRANS AMINITSG GcKerk Pro-i.e■n C=D

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3. Q;F4CFIO?# VIM

'TYPE I 'TYPEIR RID GRALITE A 13 13 g■ 6SZAW)L. 6-1041.11) TABLE 1. Electr-ophoretic and staining methods

2 _ENZYME TISSUE' BUFFER STAIN REFERENCE SYSTEM

3 Acid Phosphatase TCW Allen & Weremink (1971) . LiOH Lewis (1970) Aminopeptidase 4 Esterase LiOH Shaw & Prasad (1970) General Protein LiOH Amido Black • Glutamate oxaloacetate transaminase LiOH Shaw & Prasad (1970) 5 Glyceraldehyde-3-phosphate dehydrogenase LiOH Shaw & Prasad (1970) 6 dehydrogenase . LiOH Shaw & Prasad (1970) Lactate 5 Malate dehydrogenase TCW Zee et al (rg70) 7 Phosoglucomutase TCW Shows et al (1969) , 8 Tetrazolium oxidase TB Wright & Shaw (19( 9) Xanthine dehydrogenase TB Yen & Glassman (19(5)

• 1 - L = liver, M = muscle 2 - LiOH: Discontinuous pH 8.4 LiOH buffer of Selander et al (1969) 7 TB: Tris-borate EDTA pH 8.7 buffer of Whitt and HoioVftz (1970) TCW: Tris-citrate pH 6.9 buffer of Whitt (1970) 3 - stain modification: 0.1 M Tris-malate pH 5.0 buffer. 4 - stain modification: 0.5 M Phosphate pH 7.0 buffer. 5 - stain modification: 0.5 M Tris-HC1 pH 7.0 buffer. 6 - stain modification: no NaCN added to reaction mixture. 7 - stain modification: no KCN added, addition of .5mg Glucose 1,6 diphosphate to reaction mixture. 8 - stain modification: 0.1 M Phosphate pH 7.0 buffer. White bands appear when stained for glycerol-3-phosphate dehydrogenase. TABLE 2. Estimated genetic variability in the Cuatro Cienegas cichlids.

Polymorphism Estimated loci criterion minimum - maximum percent loci polymorphic 0. 0 0.0 Conservative* percent loci heterozygous per individual 0.0 0.0

percent loci polymorphic 5.5 4.2 Liberal** percent loci heterozygous per individual 0.3 0.2

* p < .95; ** p < .99. See text for discussion.