Origin of Gila Seminuda (Teleostei: Cyprinidae) Through

Home , Gila (fish)

Proc. Nati. Acad. Sci. USA Vol. 89, pp. 2747-2751, April 1992 Evolution Origin of Gila seminuda (Teleostei: Cyprinidae) through introgressive hybridization: Implications for evolution and conservation (speciation/morphological variation/genetic variation/mtDNA)

BRUCE D. DEMARAIS*, THOMAS E. DOWLING*, MICHAEL E. DOUGLAS*, W. L. MINCKLEY*, AND PAUL C. MARSHt *Department of Zoology, Arizona State University, Tempe, AZ 85287-1501; and tCenter for Environmental Studies, Arizona State University, Tempe, AZ 85287-3211 Communicated by Wyatt W. Anderson, December 23, 1991

ABSTRACT Morphological and genetic characters from southwestern United States display a complicated array of cyprinid fishes of the genus Gila were examined to assess a phenotypes, certain ones of which seem correlated with hypothesized hybrid origin of Gila seminuda from the Virgin differences in habitat (13, 14). These phenotypes have long River, Arizona-Nevada-Utah. The presumed parents, Gia puzzled ichthyologists. Enlarged fins, streamlined bodies, robusta robusta and Gba elegans, are clearly differentiated shallow caudal peduncles, and high numbers of reduced, from one another based on morphology, allozymes, and embedded scales are typical oftaxa in large, turbulent rivers. mtDNA haplotypes. G. seminuda is morphologically interme- More than one species may occur sympatrically in the largest diate and polymorphic at allozyme loci diagnic for the streams, where macrohabitats are sharply demarcated. Small parental species. Restriction endonuclease analysis of mtDNA streams usually support a single phenotype, with small fins, showed G. seminuda nearly identical to G. elegans. These thick body, deep caudal peduncle, and low numbers of large, results support an origin of the bisexual taxon G. seminuda overlapping scales. Moderate-sized rivers may be occupied through introgressive hybridization. The Gila population in the by fish of intermediate morphology. Moapa River, Nevada, also appears to be of hybrid origin and Historically, most hypotheses to explain this habitat- is considered a distinctive population of G. seminuda. Inter- related pattern of morphological variation in Gila have specific hybridization is potentially an important mode of stressed local ecotypic/ecophenotypic responses to specific evolution among western North American fishes, and valid environments (15, 16). More recently, hybridization between species of hybrid origin may exist in other groups as well. distinct species has been forwarded to explain morphologi- Consideration of this mode of evolution argues for the need to cally intermediate populations (17). Smith et al. (18) hypoth- conserve entire species complexes. esized that selective forces strong enough to maintain sepa- rate taxa in large rivers were insufficient to do so in moderate- Interspecific hybridization is potentially an important mode sized streams. In the latter, selection was thought to shape of evolution in plants and animals. Botanists have long phenotypes to resemble mosaics ofattributes from large-river recognized its role in producing new taxonomic entities and taxa, perhaps assisted by genetic variation incorporated via in incorporating genetic variation into existing taxa (1, 2). hybridization from several distinct morphs. Zoologists have shown less interest in this phenomenon, Smith et al. (18) suggested that the nominal Gila seminuda primarily because introgressive hybridization seems compar- Cope and Yarrow (19) from the Virgin River, Nevada- atively uncommon in animals, and hybrid taxa are rare. Arizona-Utah (long considered Gila robusta seminuda), Whether or not hybrid speciation plays a significant role in originated through hybridization between G. robusta robusta animal evolution remains an open question. Most animal and Gila elegans. G. seminuda is the only species ofits genus evolutionists believe it does not (3). However, we argue in ever collected in the Virgin River, a moderate-sized tributary our discussion that hybridization may have played an impor- of the much larger Colorado River, where G. r. robusta, G. tant role in the evolution offishes in western North America. elegans, and Gila cypha occur sympatrically. Among vertebrates, fishes exhibit the greatest number of We report results of a test in which the putative hybrid proposed hybrid taxa. Yet, most are unisexuals (reviewed in origin of G. seminuda was examined by using morphological ref. 4), although some bisexual forms have been proposed and genetic characters. Taken together, our data provide (5-9). Morphological intermediacy between two presumed strong support for the origin of G. seminuda through intro- parental species fostered the latter suggestions. However, gressive hybridization involving G. robusta and G. elegans. when allozymes and/or mtDNA were evaluated, most putative examples have been either strongly questioned or refuted (e.g., see refs. 10-12). Clearly, morphological intermediacy MATERIALS AND METHODS alone is insufficient to adequately test hypotheses of hybrid Preserved and frozen materials were obtained for G. elegans, origin. G. seminuda, G. r. robusta (three populations), and a stock Morphology has nevertheless remained the yardstick with from the Moapa River, Nevada, previously identified as G. which to measure species, and, in fact, most species are robusta (hereafter referred to as MRN). Sample sizes used for described on a traditional morphological basis. Yet, mor- morphologic, allozymic, and mtDNA analyses, respectively, phology presents difficulties in separating genetic and non- are as follows: G. elegans, 23, 20, and 4; G. seminuda, 24, 17, genetic components. For example, cyprinid fishes of the and 4; G. r. robusta (Bill Williams River basin, Arizona), 26, genus Gila that inhabit the Colorado River basin of the 20, and 3; G. r. robusta (Salt River basin, Arizona), 26, 20, and 3; G. r. robusta (Verde River, Arizona), 18, 19, and 4; The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: MRN, Gila robusta stock from Moapa River, Ne- in accordance with 18 U.S.C. §1734 solely to indicate this fact. vada; PC, principal component.

2747 Downloaded by guest on September 26, 2021 2748 Evolution: DeMarais et al. Proc. Natl. Acad. Sci. USA 89 (1992)

MRN, 26, 20, and 3. Samples of G. seminuda used for -0.30- mtDNA analyses came from the upper [St. George, Utah (n = 2)] and lower [Littlefield, Arizona (n = 2)] Virgin River. For mtDNA, an additional three MRN and 14 G. seminuda -0.35- (six from the upper and eight from the lower Virgin River) were analyzed with two diagnostic enzymes. Morphological -0.40- data for 25 artificially produced F1 hybrids (hereafter referred to as hybrids) between female G. elegans and male G. r. robusta are presented for comparative purposes. -0.45- Samples of G. elegans were from captive populations at Dexter National Fish Hatchery, New Mexico. The original C' -0.50 stock was derived from artificial spawning of 11 adults from Lake Mohave, Arizona-Nevada (20), and the fish were a composite of F2 progeny from nonassisted spawning in -0.55- hatchery ponds. Preserved hybrids were also provided by Dexter National Fish Hatchery. Specimens of G. seminuda, -0.60- MRN, and G. r. robusta were collected from wild popula- EE tions; locality data are available from the authors. Measurements by digital or dial calipers (nearest 0.1 mm) -0.65- of 24 morphometric variables were made on preserved spec- E-6E E-5. imens housed at Arizona State University. Methods were I those ofHubbs and Lagler (21) as modified by DeMarais (22). -7.5 -7.0 -6.5 -6 .0 -5.5 -5.0 All computations were made on an IBM 3090 using the Pci Statistical Analysis System (SAS) (23). Variation among samples was assessed by principal component (PC) analysis FIG. 1. Plot of scores on PC1 and sheared component 2 (H2). sheared to reduce the effects of overall size on shape Sample designations: G. r. robusta (B, Bill Williams; V, Verde; and varia- S, Salt), G. seminuda (squares), MRN (triangles), G. elegans (E), (G. tion (24). Components were derived from the covariance elegans x G. r. robusta)Fj hybrids (H). Only the outermost speci- matrix of log1o-transformed morphometric variables and mens for each sample are shown, connected by polygons to encom- were sheared by locality. pass all other individuals. Sample sizes are given in the text. For analysis of allozymic variation, muscle and liver samples stored at -80'C were homogenized in distilled water. tiated among taxa (Fig. 1), with length and depth of caudal Gene products representing 30 presumptive loci (ACP-A, peduncle contributing most strongly to separations. Relative mAH-A, sAH-A, AK-A, ADH-A, sAP-A, mAAT-A, sAAT-A, to G. r. robusta, G. elegans had a longer and shallower caudal CBP-J, CBP-2, CK-A, EST-I, EST-2, FH-A, mIDHP-A, peduncle. Hybrids were morphologically intermediate. Al- sIDHP-A, LDH-A, LDH-B, mMDH-A, sMDH-A, sMDH-B, though distinct, G. seminuda exhibited H2 scores that over- mMEP-A, sMEP-A, PEPB, PEPD, PEPA, PEPS, PGM-A, lapped those of known hybrids. MRN and populations of G. PK-A, sSOD-A) were resolved by electrophoresis of homoge- r. robusta exhibited considerable overlap, with only the nates through 12% starch gels (25). Locus nomenclature scores for MRN approaching those of G. seminuda. MRN, G. followed that of Shaklee et al. (26). Buffer conditions, EC seminuda, and hybrids were slightly differentiated from G. r. numbers, and tissue sources are available from the authors. robusta and G. elegans on H3 and H4 (data not shown), mtDNA was isolated from heart, liver, and gonads (when components that accounted for <2.0%o of total variation. available) dissected from specimens stored at -80°C. Meth- Clustering of distances generated from mean H2, H3, and ods for isolation and analysis were as described by Dowling H4 scores summarized morphological relationships among et al. (27). mtDNAs were characterized by digestion with the and between taxa (Fig. 2A). Populations of G. r. robusta and following 6-base-recognizing restriction endonucleases: G. elegans were most divergent. MRN was most closely BamHI, Bcl I, Bgl II, BstEII, EcoRI, HindIII, Nco I, Nde I, linked to G. r. robusta. Hybrids and G. seminuda were Nhe I, Pvu II, Sac I, Xba I, and Xho I. similar, the distance between them only slightly greater than Relationships among taxa were estimated independently those separating populations of G. r. robusta. Hybrids, for each set of characters. For morphology, mean scores on however, were more similar to G. elegans, while G. semin- sheared principal components 2, 3, and 4 for each population uda more closely resembled G. r. robusta. were used to calculate average taxonomic distances between Allozymes. Levels of allozyme variation were low, both all pairwise combinations of taxa (28). Relationships were within and between taxa. Maximum genetic distances oc- visualized by using the FITCH algorithm of PHYLIP (29), a curred between G. elegans and G. r. robusta (Fig. 2B), least-squares-based method that makes no assumption con- largely due to fixed or nearly fixed differences at two loci. All cerning equality of evolutionary rates. For allozymes, mod- G. elegans were homozygous for a fast allele at both CK-A ified Rogers genetic distances were calculated from allele and CBP-1. All G. r. robusta (representing three populations) frequencies (30), and relationships were visualized by using had only an alternative, slow CK-A allele. Salt River G. r. the DISTANCE WAGNER algorithm of BIosYs-1 (31). Estimates robusta were fixed for a slow CBP-J allele, while Bill of sequence divergence among mtDNAs were calculated Williams and Verde River populations possessed the slow from fragment comparisons (32), and relationships were allele along with the fast (G. elegans-type) allele at low envisioned by FITCH. frequency (12% and 5%, respectively). MRN and G. seminuda were intermediate to G. r. robusta and G. elegans (Fig. 2B) due to the presence of both alleles RESULTS at CK-A and CBP-J. MRN possessed a higher proportion of Morphology. All nominal taxa were readily differentiated the fast allele at both loci (68% and 60%o, respectively), by PC analysis (Fig. 1). The first PC (PC1) and sheared PC2 clustering it with G. elegans, while G. seminuda aligned with (H2), respectively, accounted for 93.6% and 4.6% of the total G. r. robusta due to lower proportions (29o and 47%) ofthese sample variation. PC1 was interpreted as a general size factor same alleles. All possible combinations of genotypes (i.e., and did not contribute to intersample differentiation. In heterozygotes and alternative homozygotes) for the two contrast, H2 was a size-free shape component that differen- marker loci were present. Based on G tests, both loci were in Downloaded by guest on September 26, 2021 Evolution: DeMarais et al. Proc. Natl. Acad. Sci. USA 89 (1992) 2749

A. Morphology B. Albzymes C. mtDNA

robusta robusta robusta LSalt MRN Littlefieldi seminuda

WFD I elegans slogans 0.00 0.05 0.10 0.00 0.10 0.20 0.0 1.0 2.0

FIG. 2. Dendrograms (rooted at the midpoint) derived from morphology (A), allozymes (B), and mtDNA (C), as described in the text.

Hardy-Weinberg equilibrium in MRN and G. seminuda, and would have to possess the entire range of variation and then multilocus tests failed to detect significant association of randomly give rise to populations exhibiting different mean alleles. phenotypes with only a fraction of the original variance. mtDNA. Limited mtDNA differentiation (0.0-0.2% se- While morphological intermediates such as G. seminuda quence divergence) was present within and between popu- might be expected under such a scenario, the existence of a lations of G. r. robusta. The mtDNA of G. elegans was highly variable ancestor is more difficult to envision because distinct from that of G. r. robusta (3.2-3.3% sequence modern species generally do not possess such high levels of divergence) but similar to those of MRN and G. seminuda morphological variation. (0.2-0.4%). As in G. r. robusta, limited variation was present Patterns of allozyme variation are consistent with differ- within and between samples of G. seminuda and MRN ential sorting of alleles among lineages, which would require (0.3-0.5%). The mtDNAs of G. elegans, G. seminuda, and only that an ancestral form be polymorphic at the two MRN clustered together, as did those of the various G. r. diagnostic loci. Thus, MRN and G. seminuda could have robusta (Fig. 2C). mtDNAs were isolated from an additional retained the ancestral condition, while G. r. robusta and G. 14 G. seminuda (from two localities) and 3 MRN to further elegans became fixed for alternative alleles. test for G. r. robusta-like mtDNA in the Virgin River system. Because of its haploid and strict maternal mode of inher- Cleavage with two restriction enzymes diagnostic for G. r. itance without recombination, differential sorting ofancestral robusta and G. elegans mtDNA (Bcl I and HindIII) yielded polymorphism is especially likely for mtDNA (36). Thus, only typical G. elegans fragment patterns in all individuals. similarity of G. seminuda, MRN, and G. elegans mtDNAs could result from chance inheritance of similar haplotypes DISCUSSION from a polymorphic ancestor that possessed restriction sites currently diagnostic for G. r. robusta and G. elegans. Our Wagner (33) described three steps in the study ofhybrid taxa: data, however, are inconsistent with this hypothesis. Esti- (i) detection of hybrids, (ii) test of postulated hybridity, and mates of sequence divergence between mtDNAs of G. ele- (iii) phylogenetic placement. G. seminuda was recognized gans and G. r. robusta were 8-fold greater than those among early as a morphological link between G. elegans and G. r. G. seminuda, MRN, and G. elegans (0.4% or less) and 16-fold robusta. Ellis (34) and Miller (15) thought it an intergrade greater than those among populations of G. r. robusta (0.2% between G. elegans and G. r. robusta (then considered or less). Therefore, MRN and G. seminuda apparently share subspecies), or an intermediate subspecies. Carl L. Hubbs with G. elegans an mtDNA ancestor not shared with G. r. speculated in 1938 that Gila from the Virgin River drainage robusta. (specifically the Moapa River) were of hybrid origin (35); Consideration of one data set in light of the other empha- Smith et al. (18) were the first to actually suggest the hybrid sizes additional inconsistencies. For example, if virtually hypothesis in print. Thus, Wagner's step 1 has been well identical mtDNAs of G. seminuda, MRN, and G. elegans established. This paper provides a test of hybrid origin (step reflect true relationships, then morphological convergence of 2), while phylogenetic analysis (step 3) remains to be exam- G. seminuda and especially MRN toward a G. r. robusta ined. phenotype, or divergence of G. elegans from a G. r. robusta- Morphological, allozymic, and mtDNA data are congruent like ancestor, would have to have been recent and extremely with respect to the divergence and distinctiveness of the rapid. However, available evidence suggests that this was not putative parental species, G. elegans and G. r. robusta. the case. Most modern species of western cyprinoid fishes However; our analyses produced discordant estimates of were well differentiated by the Pliocene (37), and late'Mi- relatedness for G. seminuda and MRN relative to G. elegans ocene fossils in Arizona have been referred to G. r. robusta and G. r. robusta (Fig. 2). Two alternative hypotheses could (38). Some of these had skeletal features reminiscent of G. explain these conflicting results: (i) expression of ancestral elegans and/or G. cypha, suggesting that morphological polymorphisms, and (ii) hybrid origin. divergence of Colorado River basin Gila was already under Ancestral Polymorphism vs. Hybrid Origin. As populations way >6 million years ago. Alternatively, if morphology diverge, relationships may become obscured by the stochas- accurately reflects among-taxa relationships, then mtDNA tic nature of inheritance. If this were the case in Gila, data become difficult to interpret. For example, between the discordance among inferred relationships across character morphologically similar G. r. robusta and MRN, both rapid types could reflect insufficient time for ancestral polymor- divergence of mtDNAs and unreasonable convergence of phisms to become fixed (i.e., incomplete lineage sorting) and MRN haplotypes toward those of G. elegans would be for new diagnostic characters to evolve. necessary to create the observed relationships. Given the magnitude ofmorphological differences between The strongest argument against the ancestral polymor- G. elegans and G. r. robusta, stochastic inheritance from a phism hypothesis is the general agreement of the three polymorphic ancestor seems unlikely. An ancestral form character sets. It seems unlikely that a stochastic process Downloaded by guest on September 26, 2021 2750 Evolution: DeMarais et al. Proc. Natl. Acad. Sci. USA 89 (1992) would produce coincidental sorting of unique features in persisted. These may have had a selective advantage over morphology, allozymes, and mtDNA to form G. r. robusta parental forms since interspecific gene flow increases genetic and G. elegans, as well as the morphological and allozymic variability faster than mutation alone, thereby allowing rapid intermediacy of MRN and G. seminuda. responses to changing environments (1, 51, 52). Morphology, allozymes, and mtDNA are all consistent Before human perturbations, both G. elegans and G. r. with past hybridization between G. elegans and G. r. ro- robusta were widely distributed in the Colorado basin. The busta. Various features of body shape place G. seminuda former lived in mainstem rivers and their largest tributaries, almost exactly intermediate between G. elegans and G. r. and the latter occurred syntopically, while occupying smaller robusta (Fig. 1) and similar to artificially produced hybrids of streams as well (53). Opportunities for hybridization were that same parentage, while MRN, although clearly G. r. thus abundant, and the origin of G. seminuda/MRN through robusta-like, tends toward G. elegans. Allozymes provide such an event would be predicted in intermediate-sized the same pattern of intermediacy. MRN and G. seminuda tributaries during regional drought or other natural catastro- exhibit a mixture of alleles diagnostic for G. elegans and G. phes. r. robusta in the Colorado River basin below Grand Canyon No physical barrier is known that would have impeded (B.D.D., unpublished data). Results for G. elegans are, continual gene flow or actual migration of either G. elegans however, based on only a single population of this rare or G. r. robusta into the Virgin River. Yet, G. seminuda has species. established and Unlike morphology or allozymes, mtDNA is nonrecombi- continues to maintain independence from nant, and strict maternal inheritance prevents its expression these taxa. It has stabilized morphologically, maintained as a mosaic of characters within individuals. The mtDNA of Hardy-Weinberg equilibrium, and sustained a distinct and only one of the hybridizing species may be present within unique distribution parapatric to both parents for the >100 hybrid zones, depending on the direction and extent of years since it came to the attention of ichthyologists. introgression (e.g., see refs. 39-41), or both mtDNAs may The taxonomic status of stabilized hybrid derivatives such persist long after the disappearance of one parental taxon as G. seminuda will likely remain a point of contention. (e.g., see refs. 42 and 43). By itself, the presence of only G. However, current relegation of G. seminuda to a subspecies elegans-like mtDNA in G. seminuda and MRN has little of G. r. robusta is inappropriate, if for no other reason than meaning; however, the strong contrast between morpholog- arbitrariness; it could just as easily be considered a subspe- ical and mtDNA characteristics in these forms (such that G. cies ofG. elegans. More importantly, subspecific designation r. robusta-like or intermediate morphologies are coupled with obscures both the evolutionary origin and independence ofG. G. elegans-like mtDNAs) is compelling evidence for their seminuda as a distinct lineage. Because G. seminuda exhibits hybrid origin. phenotypic, genetic, and geographic integrity, its reelevation Hybridization and Evolution. Acquisition of ecological di- to specific rank is warranted. vergence and attainment ofreproductive isolation are the two We thus conclude that G. seminuda and MRN comprise a major problems that must be overcome before hybrid speci- distinct species, originating through Pleistocene or post- ation can successfully occur (3). If either fails, recombinant Pleistocene hybridization between G. elegans and a local phenotypes are swamped out of existence via gene flow with form of G. robusta and promoted by habitat changes brought parental forms. Hybrid recombinants generally show ecolog- on by regional aridification. ical divergence from both parental forms and seem most Conservation Implications. Demonstration of hybrid origin suited for hybridized habitats (1). In fact, the presence of for G. seminuda and MRN has specific ramifications for their such a suitable habitat has been argued as the primary limiting conservation status. When G. seminuda was listed as endan- factor for hybrid speciation (ref. 44 and references therein). gered (54), MRN was considered an "undescribed" subspe- Modern examples of widespread hybridization between cies of G. robusta and specifically excluded from protection. fish species can usually be traced to stocking of alien species However, information presented here indicates a close rela- and/or anthropogenic habitat alterations. Interspecific (and tionship between these forms. Therefore, the listing of G. even intergeneric) hybridization is common (45, 46), with seminuda should be amended to include the Moapa River most such incidents resulting only in first-generation hybrids population. or local and transitory hybrid swarms. Evidence for hybrid origin also has general implications for The early discovery and description of G. seminuda (19) conservation of the entire group. Most Gila native to the preclude the possibility of introductions and/or man-induced Colorado River basin are threatened with extinction, with habitat change as promoting their hybrid origin. Further- several members listed or candidates for listing as endan- more, neither G. r. robusta nor G. elegans has ever been gered (G. cypha, G. elegans, G. r. robusta, Gila robusta collected from the Virgin River system (47). LaRivers (16) jordani, G. seminuda). The tremendous morphological di- noted G. elegans in the lower Virgin River, but this may have versity of this group is paralleled by its genetic complexity, been speculation since it was common in the adjacent main- and we anticipate that hybridization played an important role stem Colorado River before impoundment of Lake Mead and in generating and maintaining this complexity. While the persisted in the reservoir until the 1960s (ref. 48; W.L.M., general validity of this hypothesis remains to be tested, it still unpublished data). requires serious consideration, particularly when recovery Natural phenomena surely had consequences similar to programs are implemented and further studies are designed. those of human perturbations (45) and perhaps with far If hybridization is indeed an important mode of evolution in broader implications, such as bursts of speciation (44). Drain- western fishes, then protection ofdistinct forms coupled with age transfers through stream capture or tectonism forced an active bias against suspected hybrids could prove detri- allopatric faunas into contact, and progressive desertification mental to the entire complex. Not only could valid species of shrank aquatic habitats more than enough to hinder ecolog- hybrid origin (such as G. seminuda) be eliminated, but a ical segregation (49). Dramatic environmental change has valuable mode of evolution could be truncated. Given the been the rule in western North America throughout the potential impact of such a program, research emphasis should Tertiary period (50), and repeated cycles of isolation and instead be placed on determining the extent of hybridization sympatry must have been common during the evolutionary and its effect on the evolution of these fishes. Meanwhile, it history of modern western fishes. Differentiating taxa, re- would be prudent to protect the entire complex so that peatedly forced into contact, almost certainly exchanged valuable genetic variation distributed throughout the basin genetic material, and some stocks of hybrid origin must have will not be lost forever. Downloaded by guest on September 26, 2021 Evolution: DeMarais et A Proc. Natl. Acad. Sci. USA 89 (1992) 2751

We thank A. A. Echelle (Oklahoma State University) for com- (1990) in Molecular Systematics, eds. Hillis, D. M. & Moritz, ments and critical review, J. C. Avise (University of Georgia) for C. (Sinauer, Sunderland, MA), pp. 45-126. helpful discussion, G. G. Scoppettone [United States Fish and 26. Shaklee, J. B., Allendorf, F. W., Morizot, D. C. & Whitt, Wildlife Service (USFWS), Reno, Nevada] for providing specimens G. S. (1990) Trans. Am. Fish. Soc. 119, 2-15. from the Moapa River, and B. L. Jensen (USFWS, Dexter, New 27. Dowling, T. E., Moritz, C. & Palmer, J. D. (1990) in Molecular Mexico) for the specimens from Dexter National Fish Hatchery. Our Systematics, eds. Hillis, D. M. & Moritz, C. (Sinauer, Sunder- work with listed taxa was under federal subpermits PRT 676811 and land, MA), pp. 250-317. 704930 from USFWS, accompanied by comparable authorizations 28. Sneath, P. H. A. & Sokal, R. R. (1973) Numerical Taxonomy from the states of Arizona, Nevada, and Utah. 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