*Department of Biology, Arizona State University;
Bodega Marine Laboratory, University of California;
Department of Biology, University of Maryland
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Abstract |
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Introduction |
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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 1977
), 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 1971
; Avise and Selander 1972
; Kirby, Thompson, and Hubbs 1977
). 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 1977
). 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 1972
; Mitchell, Russell, and Elliot 1977
). 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 1972
; Mitchell, Russell, and Elliot 1977
; Romero 1983
). 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 2001
). 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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Given the recent progress in developmental biology of A. mexicanus (Jeffery and Martasian 1998
; Jeffery et al. 2000
; Yamamoto and Jeffery 2000
; Jeffery 2001
; Strickler, Yamamoto, and Jeffery 2001
), 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.
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Materials and Methods |
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Characterization and Analysis of DNA Variation
DNA was extracted from tail-fin clips by standard phenol-chloroform extraction procedures (Davis, Dibner, and Batty 1986
, pp. 320323). Single-stranded conformational polymorphisms (SSCPs) (Dowling et al. 1996
; Sunnucks et al. 2000
) 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 2000
). Standard measures of diversity (e.g., gene and nucleotide diversities, average number of differences, theta) were calculated for each population (reviewed in Nei 1987
, pp. 254286) and levels of divergence among populations quantified by AMOVA (Excoffier, Smouse, and Quattro 1992
). 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. 2001
), distances among populations generated with REAP (McElroy et al. 1992
), 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 2001
). Clade structure was determined using the program TCS 1.13 (Clement, Posada, and Crandall 2000
) and significance tested using GeoDis 2.0 (Posada, Crandall, and Templeton 2000
). Phylogenetic trees of SSCP alleles were generated by PAUP* (Swofford 1998
) 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 1998
). 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 14 days. The specimens were double stained for cartilage and bone by the Alcian Blue-Alizarin Red method (Wassersug 1976
). The number of rib-bearing thoracic vertebrae was counted in cleared whole-mount specimens.
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Results |
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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 1994
) and Costa Rica (Bussing 1998
, pp. 7985), 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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Discussion |
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This outcome is consistent with the results of a previous allozyme survey. In a survey of 17 loci, Avise and Selander (1972)
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 2001
), 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 1988
; Jeffery 2001
). 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 1967
; Mitchell, Russell, and Elliot 1977
). 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. 2000
; Strickler, Yamamoto, and Jeffery 2001
). 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 1977
). However, the high degree of eye regression and complete absence of body pigmentation in Pachón cavefish conflicts with this interpretation (Mitchell, Russell, and Elliot 1977
; Wilkens 1988
; Jeffery and Martasian 1998
; Jeffery 2001
). Discrimination between these alternatives will require a detailed phylogeographic analysis (reviewed in Avise 2000
) 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 1991
; Gerber, Tibbets, and Dowling 2001
). 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 1977
; Romero 1983
, 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 1977
). Avise and Selander (1972)
and Mitchell, Russell, and Elliot (1977)
failed to observe hybrids in La Cueva de El Pachón. However, Langecker, Wilkens, and Junge (1991)
reported hybrids in this cave in 19861988. We did not see hybrids in La Cueva de El Pachón in 19962000, 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 1972
; Mitchell, Russell, and Elliot 1977
). 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 1971
).
Considering the wide distribution of A. mexicanus and the large number of reported hypogean populations (Mitchell, Russell, and Elliot 1977
), 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 1998
; Yamamoto and Jeffery 2000
; Jeffery 2001
) make A. mexicanus a valuable model for studying the evolution of eye and pigment degeneration.
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Acknowledgements |
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Footnotes |
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Keywords: Astyanax mexicanus
population structure
mtDNA
ND2
Abbreviation: ND2, NADH dehydrogenase 2.
Address for correspondence and reprints: Thomas E. Dowling, Department of Biology, Arizona State University, Tempe, Arizona 85287-1501. thomas.dowling{at}asu.edu
.
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