Evidence for Multiple Genetic Forms with Similar Eyeless Phenotypes in the Blind Cavefish, Astyanax mexicanus

Thomas E. Dowling, David P. Martasian and William R. Jeffery

*Department of Biology, Arizona State University;
{dagger}Bodega Marine Laboratory, University of California;
{ddagger}Department of Biology, University of Maryland


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
A diverse group of animals has adapted to caves and lost their eyes and pigmentation, but little is known about how these animals and their striking phenotypes have evolved. The teleost Astyanax mexicanus consists of an eyed epigean form (surface fish) and at least 29 different populations of eyeless hypogean forms (cavefish). Current alternative hypotheses suggest that adaptation to cave environments may have occurred either once or multiple times during the evolutionary history of this species. If the latter is true, the unique phenotypes of different cave-dwelling populations may result from convergence of form, and different genetic changes and developmental processes may have similar morphological consequences. Here we report an analysis of variation in the mitochondrial NADH dehydrogenase 2 (ND2) gene among different surface fish and cavefish populations. The results identify a minimum of two genetically distinctive cavefish lineages with similar eyeless phenotypes. The distinction between these divergent forms is supported by differences in the number of rib-bearing thoracic vertebrae in their axial skeletons. The geographic distribution of ND2 haplotypes is consistent with roles for multiple founder events and introgressive hybridization in the evolution of cave-related phenotypes. The existence of multiple genetic lineages makes A. mexicanus an excellent model to study convergence and the genes and developmental pathways involved in the evolution of the eye and pigment degeneration.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Many hypogean (subterranean) animals are known for their troglomorphic characters, including the reduction of eyes and pigmentation, but the evolution of these striking morphologies is still unresolved (Culver, Kane, and Fong 1995Citation ; Romero 1985Citation ). Major issues in the evolution of cave animals include the process of cave colonization and the mechanisms responsible for the appearance of troglomorphic features. Highly restricted geographic ranges and, for the most part, lack of genetic studies of hypogean species and their epigean (surface dwelling) ancestors hamper most evolutionary studies of hypogean animals. A notable exception is the teleost Astyanax mexicanus, which exhibits an epigean form and about 30 different conspecific hypogean forms (Mitchell, Russell, and Elliot 1977Citation ). Hypogean A. mexicanus populations exhibit degenerate eyes, which are sunken into the orbits and covered by a flap of skin, reduction or loss of melanin pigmentation, an expanded gustatory system, and other troglomorphic traits (Schemmel 1967, 1974Citation ; Wilkens 1988Citation ; Jeffery and Martasian 1998Citation ; Jeffery et al. 2000Citation ; Jeffery 2001Citation ).

The epigean form of A. mexicanus is widely distributed in northeastern México and southern Texas. The first hypogean A. mexicanus populations were discovered in La Cueva Chica (Chica cavefish), La Cueva de los Sabinos (Los Sabinos cavefish), and La Cueva de El Pachón (Pachón cavefish) in the Sierra de El Abra (fig. 1 ), a limestone escarpment in Tamaulipas and San Luis Potosí, México (Mitchell, Russell, and Elliot 1977Citation ), and initially described as three different species. Breeding, electrophoretic, and karyotypic studies now support the contention that the epigean and hypogean forms are the same species (Wilkens 1971Citation ; Avise and Selander 1972Citation ; Kirby, Thompson, and Hubbs 1977Citation ). Since the first cavefish populations were discovered in the Sierra de El Abra region, 26 additional hypogean populations have been reported (e.g., La Cueva de la Curva or Curva cavefish; El Sótano de la Tinaja or Tinaja cavefish), the majority from caves in an extensive valley paralleling the western slope of the escarpment (fig. 1 ; Mitchell, Russell, and Elliot 1977Citation ). Geographically isolated hypogean populations have also been discovered in the Sierra de Guatemala to the north and in the Micos region to the west of the Sierra de El Abra (fig. 1 ; Wilkens and Burns 1972Citation ; Mitchell, Russell, and Elliot 1977Citation ). Some hypogean populations, including the Chica cavefish and the cavefish population from La Cueva del Río Subterraneo (Subterraneo cavefish) in the Micos region (fig. 1 ), contain mixtures of eyed, intermediate, and eyeless individuals resulting from introgression with epigean forms (Avise and Selander 1972Citation ; Mitchell, Russell, and Elliot 1977Citation ; Romero 1983Citation ). More recently, an additional hypogean Astyanax population has also been reported in Gruta de las Granadas, Guerrero, Mexico, outside the range of A. mexicanus (Espinasa, Rivas-Manzano, and Expinosa Pérez 2001Citation ). The Guerrero cavefish were probably derived from epigean Astyanax aeneus, a broadly distributed epigean species inhabiting southern México and Central America.



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Fig. 1.—Sketch map of the Sierra de El Abra region in Tamaulipas and San Luis Pótosi, México, showing locations of caves and hypogean and epigean sampling sites. Filled circles: locations of hypogean populations sampled. A. La Cueva Chica (Chica cavefish). B. La Cueva de la Curva (Curva cavefish). C. El Sótano de la Tinaja (Tinaja cavefish). D. La Cueva de los Sabinos (Los Sabinos cavefish). E. La Cueva de El Pachón (Pachón cavefish). F. La Cueva del Río Subterráneo (Subterráneo cavefish). Open circles: locations of other known hypogean populations. Roman numerals: locations of sampled epigean populations. I. Río Frio. II. Río Comandante. III. Nacimiento del Río Mante. IV. Arroyo Lagarto. V. Río Puerco. VI. Arroyo Los Sabinos. VII. Arroyo San Felipe. VIII. Nacimiento del Río Choy. IX. Río Tampaón. X. Río Naranjo. XI. Arroyo Micos. Additional epigean sampling sites outside this area are not shown. Shaded areas represent uplands, whereas unshaded areas represent lowlands and valleys. Major streams are represented by fine lines. The insert indicates the approximate location of the sketched area in northeastern Mexico. Adapted from Jeffery & Martasian (1998)Citation

 
Two hypotheses have been advanced to explain how hypogean A. mexicanus populations were established. There was either a single founder event with subsequent dispersal between the cave systems or there were two or more founder events that resulted in morphologically convergent troglomorphic populations derived from different epigean ancestors. The single origin of cave populations in the Sierra de El Abra is supported by low levels of variability at 17 different allozyme loci in the Chica, Pachón, and Los Sabinos cavefish (Avise and Selander 1972Citation ). These data led Avise and Selander (1972)Citation to conclude that genotypic (and presumably phenotypic) variation among these hypogean populations resulted from stochastic variation associated with small population sizes. Subsequent mark and recapture studies, however, suggested that some cavefish populations are larger than previously appreciated (Mitchell, Russell, and Elliot 1977Citation ), reducing the impact of genetic drift. Regardless, the cavefish populations inhabiting large contiguous cave systems (e.g., Los Sabinos and Tinaja cavefish; Mitchell, Russell, and Elliot 1977Citation ) may have originated from a single invasion. More recent studies using RAPD markers suggested that the Chica, Pachón, Tinaja, and Curva cavefish populations are more closely related to each other than to nearby surface fish (Espinasa and Borowsky 2001Citation ), although bootstrap support for this relationship was not robust. The hypothesis of multiple, independent origins is supported by genetic crosses between Los Sabinos and Pachón cavefish which exhibit complementation of eye phenotypes in the F1 generation (Wilkens 1971Citation ) and the existence of isolated hypogean populations where gene flow might be impeded by geographic barriers (Wilkens and Burns 1972Citation ; Mitchell, Russell, and Elliot 1977Citation ).

Given the recent progress in developmental biology of A. mexicanus (Jeffery and Martasian 1998Citation ; Jeffery et al. 2000Citation ; Yamamoto and Jeffery 2000Citation ; Jeffery 2001Citation ; Strickler, Yamamoto, and Jeffery 2001Citation ), it has become more important than ever to understand the evolution of the hypogean forms. Here we describe an analysis of variation in the mitochondrial NADH dehydrogenase 2 (ND2) gene and morphological studies among hypogean and epigean populations of A. mexicanus, which identify a minimum of two genetically distinct hypogean lineages. The results are consistent with roles for several independent origins or introgressive hybridization (or both) in the evolution of hypogean A. mexicanus and their troglomorphic phenotypes.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Animals
Epigean and hypogean A. mexicanus were collected from the sites shown in figure 1 using baited traps, hand-held nets, and seines. Additional populations were obtained from the following localities (sample labels in parentheses): epigean A. mexicanus, Phantom Cave, Texas (TEX1 and 2) and Cuatro Ciénegas, Coahuila, México (CC1); A. aeneus, Río Mezcalapa, Tabasco, México (MU1 and 2), Río Jamapa, Veracruz, México (RJ1), Río Arenal, Costa Rica (RA1–3), and Río San Carlos, Costa Rica (RSC1–3). The Peruvian outgroup species Astyanax bimaculatus was purchased from a pet store (That Fish Place, Lancaster, Pa.). Some A. mexicanus individuals were brought back to the laboratory to establish breeding populations (under the auspices of Mexican Permit Number 040396-213-03), whereas others were captured, fin-clipped, and released. A small portion of the caudal fin was excised and placed in 95% ethanol saturated with EDTA for subsequent DNA extraction.

Characterization and Analysis of DNA Variation
DNA was extracted from tail-fin clips by standard phenol-chloroform extraction procedures (Davis, Dibner, and Batty 1986Citation , pp. 320–323). Single-stranded conformational polymorphisms (SSCPs) (Dowling et al. 1996Citation ; Sunnucks et al. 2000Citation ) were screened on 6% native polyacrylamide gels using the primers ND2-Acave (5'-CGCCACAATCCTCAACGG-3') and ND2-Ccave (5'-TGGCGGTTGATGAGTATG-3'). At least one strand of one representative of each SSCP mobility variant on each gel was sequenced to verify haplotype, either manually (Perkin-Elmer cycle sequencing kit) or using an ABI 377 automated sequencer. This procedure resulted in several sequences from most haplotypes, representing multiple populations (e.g., the most common haplotype, A1, was sequenced in 17 individuals from 12 populations). SSCP variants were identified by a two-character code, with the letter indicating the lineage and the number denoting the allele within that lineage. Allele numbers were assigned in order of discovery and do not reflect levels of divergence.

The entire ND2 gene was characterized from individuals representative of each SSCP allele, A. mexicanus from Texas and northern México, several samples of A. aeneus, and the outgroup, A. bimaculatus. Sequences were obtained from one strand each of two separate amplification products generated with the primer pairs ND2-Bcave (5'-AAGCTATTGGGCCCATACCC-3')-ND2-Ccave and ND2-Dcave (5'-CACCATTTGCCCTTCTCATA-3') and ND2-E (5'-TTCTACTTAAAGCTTTGAAGGC-3') using methods described above. The ND2 sequences have been deposited in GenBank under accession numbers (AF441132AF441164).

Population genetic analyses of SSCP alleles were performed using Arlequin 2.0 (Schneider, Roessli, and Excoffier 2000Citation ). Standard measures of diversity (e.g., gene and nucleotide diversities, average number of differences, theta) were calculated for each population (reviewed in Nei 1987Citation , pp. 254–286) and levels of divergence among populations quantified by AMOVA (Excoffier, Smouse, and Quattro 1992Citation ). The number of alleles was also tabulated for each sample and corrected by dividing by sample size. Jukes-Cantor distances among haplotypes were calculated using MEGA2 (Kumar et al. 2001Citation ), distances among populations generated with REAP (McElroy et al. 1992Citation ), and similarities visualized using the Neighbor-Joining method as implemented in MEGA2. Geographic structure of SSCP variation was also assessed using nested clade analysis (reviewed in Templeton 2001Citation ). Clade structure was determined using the program TCS 1.13 (Clement, Posada, and Crandall 2000Citation ) and significance tested using GeoDis 2.0 (Posada, Crandall, and Templeton 2000Citation ). Phylogenetic trees of SSCP alleles were generated by PAUP* (Swofford 1998Citation ) through heuristic search with 25 random addition sequence replicates, with no root specified. Topologies from sequences of the entire ND2 gene were recovered as above, except that A. bimaculatus was used as the outgroup. Jukes-Cantor distances were also calculated from full gene sequences and clustered using the Neighbor-Joining algorithm as implemented in PAUP* (Swofford 1998Citation ). Support for specific nodes of topologies obtained through parsimony and Neighbor-Joining analyses of complete gene sequences was examined by bootstrap resampling (1,000 replicates for each approach).

Staining and Analysis of Axial Skeletons
Larval and adult fishes were fixed in formalin for 1–4 days. The specimens were double stained for cartilage and bone by the Alcian Blue-Alizarin Red method (Wassersug 1976Citation ). The number of rib-bearing thoracic vertebrae was counted in cleared whole-mount specimens.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
To characterize the genetic distinctiveness of different A. mexicanus forms, we examined sequence variation in a mitochondrial gene, ND2, from 364 individuals representing 6 hypogean and 11 epigean populations (fig. 1 , table 1 ). Analysis of SSCPs in a 306-bp fragment of ND2 identified 26 variable positions and 20 haplotypes. Of the variable positions, 20 (77%), 4 (15%), and 2 (8%) of the changes occurred in the third, first, and second positions, respectively. All changes but one were transitions, and six resulted in amino acid substitutions.


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Table 1 Numbers and Distribution of SSCP Haplotypes

 
Estimates of population genetic parameters from SSCP fragments are provided in table 2 . On an average, epigean populations exhibited 3.6 haplotypes (range 2–6) and gene diversity of 0.45 (range 0.24–0.75). Estimated nucleotide diversity was 0.0020 (range 0.0008–0.0055), and the average pairwise comparison yielded 0.61 differences (range 0.24–1.68). These values were generally higher than estimates from hypogean populations, with only two of the six hypogean samples (Tinaja and Subterráneo) exhibiting more than one haplotype (table 2 ). Tests of variability (measured by number of alleles corrected for sample size and theta) between epigean and hypogean populations indicated significantly reduced variability in hypogean populations relative to epigean samples (SPSS for Windows, version 10.0.7, Mann-Whitney U tests, P = 0.004 and 0.006, respectively).


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Table 2 Standard Measures of Genetic Variation for Each Population (Labeled as Indicated in Fig. 1)

 
Phylogenetic analysis of SSCP variants identified two distinct lineages (A and B, fig. 2 ), differing by a minimum of seven substitutions (ca. 3.5% sequence divergence). Lineages A and B contained 16 and 4 haplotypes, respectively, with most haplotypes within lineages differing by one or two substitutions. The 11 epigean populations sampled exhibited only A lineage haplotypes (table 1 ), with the majority of individuals sampled exhibiting A1 (59.9%), A3 (14.9%), A5 (8.1%), or A9 (9.5%). Haplotype A1 was widely distributed, found in all but one epigean sample. Hypogean samples exhibited a mixture of seven lineage A and B haplotypes, five of which were unique to cavefish samples. All 47 individuals in the Chica and Pachón samples (localities A and E, respectively) exhibited haplotype A1. In a mixed sample of epigean, intermediate, and hypogean morphotypes from La Cueva del Río Subterráneo (locality F), all 10 individuals with the cave morphotype possessed a unique haplotype (A15) that differed from A1 by one change. The 9 and 18 individuals from this cave with intermediate and epigean morphotypes exhibited haplotypes A1, A15, or A5. Lineage B haplotypes (B1, B2, B3, and B4) were only found in the remaining three caves (localities B–D, fig. 1 ), and all individuals sampled from these localities exhibited lineage B haplotypes.



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Fig. 2.—One of 12 minimum length topologies (drawn to scale) of SSCP haplotypes in A. mexicanus (length = 28 steps, consistency index [CI] = 0.857, retention index [RI] = 0.939, uninformative characters excluded from calculations but included in branch lengths of figure). Clades provided by TCS are identified by squares and labeled as described in text. The shaded circle represents an inferred ancestral haplotype. Labels for alleles as provided in table 1

 
The geographic distribution of variation was assessed by AMOVA, with samples divided into northern (I–IV, E, fig. 1 ) and southern (V–XI, A–D, fig. 1 ) tributaries. This approach identified significant differences among populations (FST = 0.88, P < 0.001). This result was largely attributable to variation among populations within tributaries (FSC = 0.87, P < 0.001), with no significant difference between these two tributaries (FCT = 0.03, P = 0.303). Clustering of distances (fig. 3 ) indicated that there was little genetic differentiation among southern epigean populations, whereas northern surface populations (especially III) were more divergent. Hypogean populations were distinct from geographically adjacent epigean populations, especially samples B–D, potentially reducing levels of among-tributary variation detected. Reanalysis of these data excluding cave populations resulted in a reduction of variation among populations (FST = 0.53, P < 0.001) but an increase in the portion because of differences among tributaries (FCT = 0.17, P = 0.018), identifying moderate restrictions to gene exchange among northern and southern tributaries.



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Fig. 3.—Neighbor-Joining topology of epigean and hypogean populations. Labels as provided in figure 1 .

 
Nested clade analysis (reviewed in Templeton 2001Citation ) was performed to examine the geographic structure of haplotypic variation. Four one-step clades were identified (fig. 2 ). Three of these (1-1, 1-2, and 1-3) are primarily found in epigean samples and fall in lineage A (defined above) with the fourth (1-4 identical to lineage B) found only in three caves. Clades 1-1 and 1-2 were represented by a few, rare haplotypes that were not associated with geography. Analyses of the remaining two one-step clades (1-3 and 1-4) indicated that some haplotypes were associated with specific geographic localities (P < 0.001 for both clades), with patterns of significance resulting from isolation by distance for both clades. Analysis of the total cladogram also identified significant association of haplotypes (P < 0.001) with geographic locations; however, the lack of samples throughout the drainage precludes discrimination between fragmentation and isolation by distance as the responsible factor.

To place the level of ND2 haplotype divergence between lineages A and B in phylogenetic perspective, the following samples were examined: A. mexicanus from northern México (outside the Sierra de El Abra region) and Texas, populations of a closely related species, A. aeneus from Veracruz and Tabasco, México (Obregón-Barboza, Contreras-Balderas, and de Lourdes Lozano-Vilano 1994Citation ) and Costa Rica (Bussing 1998Citation , pp. 79–85), and a Peruvian outgroup species (A. bimaculatus). Sequence for the entire ND2 gene was obtained from these individuals and representatives of each SSCP haplotype. Of the 1,056 positions, 122 were variable in the ingroup taxa. Distribution of variation was similar to that of the SSCP fragment with 94 (77%), 21 (17%), and 7 (6%) polymorphic third, first, and second positions, respectively.

Parsimony and Neighbor-Joining analysis (fig. 4 ) yielded similar results, differing only in the placement of the root. Lineage A haplotypes from the Sierra de El Abra and Micos regions (Chica, Subterráneo, and Pachón cavefish and local epigean populations) formed a monophyletic group (bootstrap value >86%), with epigean samples from northern México and Texas, a close sister group (bootstrap value of >90%). The closest relatives of this lineage were haplotypes of A. aeneus from Veracruz and Tabasco, México (bootstrap value of >98%) but exclusive of A. aeneus from Costa Rica. The ND2 haplotypes from lineage B formed another monophyletic group (bootstrap value of >99%), which was devoid of epigean haplotypes and divergent from the other A. mexicanus lineage (ca. 3.5% sequence divergence). This level of divergence was comparable to that between A. aeneus from Costa Rica and the widespread A. mexicanus lineage, producing a trichotomy between these three lineages.



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Fig. 4.—One of the nine phylogenetic trees obtained from analysis of sequence from the entire ND2 gene (length = 166 steps, CI = 0.687, RI = 0.891, uninformative characters excluded from calculations but included in branch lengths of figure). Topology is drawn to scale except for branches involving the outgroup, A. bimaculatus. Numbers on branches represent results from bootstrap analysis for both parsimony and Neighbor-Joining analyses (before and after the slash, respectively). Labels for A and B lineages as provided in table 1

 
During the course of our studies we observed differences in body length between lineage A and B cavefish, with the latter showing anteroposterior compression. To investigate the morphological basis of body compression, we examined axial skeletons in various epigean and hypogean populations (fig. 5 , table 3 ). Skeletal preparations showed that all the sampled epigean A. mexicanus, as well as A. aeneus and A. bimaculatus, have 12 rib-bearing thoracic vertebrae. Most Chica and Pachón lineage A cavefish also have 12 thoracic vertebrae; however, the three types of lineage B cavefish usually exhibit only 11 thoracic vertebrae. The Subterraneo lineage A cavefish exhibited a mixture of axial skeletons with 11 or 12 rib-bearing thoracic vertebrae. A likelihood ratio test indicated that A. mexicanus from lineage A exhibited significantly more rib-bearing vertebrae than those of lineage B (SPSS for Windows, version 10.0.7, G = 99.2, P < 0.001), indicating that mtDNA haplotype lineage is associated with vertebrae number.



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Fig. 5.—Representative axial skeletons of A. mexicanus lineage A (A) and lineage B (B). The arrowheads indicate the number of thoracic, rib-bearing vertebrae in lineage A (12) and B (11). The numbers indicate every fifth vertebra in the axial skeleton

 

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Table 3 Number of Thoracic Vertebrae in Adult Epigean and Hypogean Astyanax Populations

 

    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Sequence analysis of the mitochondrial gene ND2 identified considerable variation in A. mexicanus. Epigean populations were generally more variable than hypogean populations. The only hypogean populations with more than one haplotype were the Subterráneo and Tinaja cavefish. The existence of more than one haplotype at La Cueva del Río Subterráneo may result from ongoing hybridization between epigean and hypogean forms (e.g., Avise and Selander 1972Citation ; Mitchell, Russell, and Elliot 1977Citation ; Romero 1983Citation ; Langecker, Wilkens, and Junge 1991Citation ). El Sótano de la Tinaja hosts the largest hypogean population of those studied (Mitchell, Russell, and Elliot 1977Citation ), possibly allowing it to maintain more variability than other smaller populations.

This outcome is consistent with the results of a previous allozyme survey. In a survey of 17 loci, Avise and Selander (1972)Citation found variation to be high in six epigean samples (average heterozygosity of 11.2%) with three hypogean samples having considerably lower variation (less than 7.7%). They attributed this reduction in levels of within-population variation to genetic drift in small cave populations. Genetic distances among samples were also low, leading to the conclusion that hypogean and epigean forms are conspecific.

Our examination of the geographic distribution of mtDNA variation indicated that there was considerable structure among populations. These differences were largely caused by isolation by distance of surface and cave populations and could not be attributed to any specific geographic partition. Phylogenetic analysis of ND2 haplotypes identified a minimum of two distinct genetic lineages in A. mexicanus: lineage A, consisting of the Chica, Pachón, and Subterráneo cavefish and closely related epigean populations and lineage B, consisting of the Los Sabinos, Tinaja, and Curva cavefish populations, with no closely related epigean counterparts. The lineage A and B haplotypes are more divergent from each other than they are from those of another species, A. aeneus from southern México and Costa Rica. These results indicate that the phenotypes shared by A and B lineage cavefish, including reduction of eyes and pigmentation, exist within a background of relatively high genetic divergence.

The ND2 haplotype data are supported by morphological and biochemical differences between lineage A and B cavefish populations. As shown here, most lineage A cavefish have 12 rib-bearing thoracic vertebrae, apparently the ancestral state in A. mexicanus, whereas lineage B cavefish are compressed along their anteroposterior axes and usually have only 11 rib-bearing thoracic vertebrae. Reduction of body size has been proposed as a troglomorphic character in fishes (Romero and Paulson 2001Citation ), but this is the first time that quantitative changes in the axial skeleton have been linked to this feature. The Los Sabinos, Tinaja, and Curva cavefish are lightly pigmented because of the presence of vestigial melanocytes, whereas melanin-producing chromophores are absent in the albinistic Pachón cavefish (Wilkens 1988Citation ; Jeffery 2001Citation ). Although all cavefish show enhanced numbers of gustatory organs relative to epigean fish, the number of taste buds is much greater in Pachón than in Los Sabinos cavefish (Schemmel 1967Citation ; Mitchell, Russell, and Elliot 1977Citation ). Finally, the eye regulatory genes Pax6 and Prox1 exhibit slightly different expression patterns in the presumptive optic regions of Pachón and lineage B cavefish embryos (Jeffery et al. 2000Citation ; Strickler, Yamamoto, and Jeffery 2001Citation ). These properties suggest that distinct morphological and biochemical differences are present in lineage A and B cave populations.

The molecular and morphological data imply that lineage B cavefish either were colonized by epigean A. mexicanus long ago, permitting accumulation of relatively extensive nucleotide substitutions in the ND2 gene, regression of the axial skeleton, and the appearance of other troglomorphic features, or were established more recently by a unique epigean lineage that is extinct or no longer occupies the region. Given the extent of our sampling in surface waters in the Sierra de El Abra and surrounding regions in northern and southern México, the former alternative seems more likely; on the other hand, ND2 haplotypes exhibited by lineage A cavefish are identical (Chica and Pachón) or nearly identical (Subterráneo) to adjacent epigean localities, possibly indicating a more recent origin for these hypogean populations. In support of this interpretation, the axial skeleton of lineage A (table 3 ) and the eyes and pigmentation of Chica and Subterráneo cavefish are less regressed than those of other cavefish populations (Mitchell, Russell, and Elliot 1977Citation ). However, the high degree of eye regression and complete absence of body pigmentation in Pachón cavefish conflicts with this interpretation (Mitchell, Russell, and Elliot 1977Citation ; Wilkens 1988Citation ; Jeffery and Martasian 1998Citation ; Jeffery 2001Citation ). Discrimination between these alternatives will require a detailed phylogeographic analysis (reviewed in Avise 2000Citation ) of this complex group, with the latter alternative supported if more extensive sampling identifies epigean lineages similar to the unusual lineage B cave haplotype.

It is also possible that lineage A hypogean populations could be old and share a common origin with B lineage cavefish, with their mtDNAs more recently transferred from epigean populations through introgressive hybridization (e.g., Dowling and Hoeh 1991Citation ; Gerber, Tibbets, and Dowling 2001Citation ). The Chica population contains putative hybrid individuals with intermediate eye and pigment morphologies thought to be derived by periodic introgression with epigean fishes, which enter La Cueva Chica via a connection with the nearby Río Tampaón (Mitchell, Russell, and Elliot 1977Citation ; Romero 1983Citation , fig. 1 ). In contrast to La Cueva Chica, La Cueva de El Pachón is a former spring resurgence isolated from surface drainage in the valley below, and with no obvious access route for epigean fish (Mitchell, Russell, and Elliot 1977Citation ). Avise and Selander (1972)Citation and Mitchell, Russell, and Elliot (1977)Citation failed to observe hybrids in La Cueva de El Pachón. However, Langecker, Wilkens, and Junge (1991)Citation reported hybrids in this cave in 1986–1988. We did not see hybrids in La Cueva de El Pachón in 1996–2000, and every fish we have captured there has regressed eyes and is devoid of body pigmentation. Introgression with local epigean fishes at La Cueva de El Pachón would have affected our haplotype results, unless the hybrids observed by earlier investigators had been expunged from the population. If hybridization does not account for our results, then it is possible that Pachón cavefish have evolved more recently than lineage B cavefish and are undergoing troglomorphic evolution more rapidly than other cavefish populations.

ND2 data from the Micos region also suggest that hybridization may not readily account for the origin of the unique A15 haplotype of Subterráneo cavefish (table 1 ). An intermittent stream containing epigean fish sinks into La Cueva del Río Subterráneo during the rainy season, and cave pools near the entrance contain fish of mixed forms (Wilkens and Burns 1972Citation ; Mitchell, Russell, and Elliot 1977Citation ). In this cave, hypogean fishes are located in pools distant from the entrance and exhibit haplotype A15, which was not found in epigean populations collected in the intermittent stream immediately outside the cave (N = 40, table 1 ). Although our sample of intermediate forms is small, six of nine individuals exhibited the diagnostic haplotype A15, suggesting that hybridization may typically involve hypogean females and epigean males, counter to the direction necessary for replacement of hypogean mtDNA haplotypes. The potential impact of introgressive hybridization from epigean forms into lineage A cavefish populations must be provided by a future examination of variation in nuclear gene loci.

Our results are consistent with two scenarios for the origin of Astyanax cavefish. First, divergent ND2 haplotypes present in lineage B cavefish populations and to a lesser degree in the Subterráneo cavefish are consistent with multiple independent origins. Second, some of the haplotype data could also be explained by variation in the level of introgressive hybridization among certain hypogean populations and their proximate epigean populations. In these cases, the influence of allelic variation from surface populations also would generate distinctive genetic lineages, creating significant consequences for the evolution of troglomorphic features. The importance of genetic diversity within different cavefish populations was demonstrated by crosses between Pachón and Los Sabinos cavefish, which resulted in F1 progeny with more extensive optic development than either parent, suggesting that mutations in different genes are involved in eye degeneration (Wilkens 1971Citation ).

Considering the wide distribution of A. mexicanus and the large number of reported hypogean populations (Mitchell, Russell, and Elliot 1977Citation ), it is likely that many different genetic combinations exist in natural populations. This diversity of genetic backgrounds and the ability to routinely propagate and manipulate the embryos of this species in the laboratory (Jeffery and Martasian 1998Citation ; Yamamoto and Jeffery 2000Citation ; Jeffery 2001Citation ) make A. mexicanus a valuable model for studying the evolution of eye and pigment degeneration.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank Catherine Beard, Brenna Bemis, Deidre Heyser, Kelly Hornaday, Clark Hubbs, David Jeffery, Dino Rossi, Allen Strickler, Yoshiyuki Yamamoto, and Meredith Yeager for assistance in fish collecting and Deidre Heyser and Kelly Hornaday for technical assistance. Dean Hendrickson provided samples from Cuatro Ciénegas, México and Texas. W. L. Minckley and Aldemaro Romero provided critical comments and helpful discussion. This research was supported by NSF grants to T.E.D. (DEB 9220683) and W.R.J. (DEB 9726561 and IBN-0110275).


    Footnotes
 
Keith Crandall, Reviewing Editor

Keywords: Astyanax mexicanus population structure mtDNA ND2 Back

Abbreviation: ND2, NADH dehydrogenase 2. Back

Address for correspondence and reprints: Thomas E. Dowling, Department of Biology, Arizona State University, Tempe, Arizona 85287-1501. thomas.dowling{at}asu.edu . Back


    References
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

    Avise J. C., 2000 Phylogeography: the history and formation of species Harvard University Press, Cambridge, Mass

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Accepted for publication November 13, 2001.