The Journal of Heredity 2002:93(1)
© 2002 The American Genetic Association 93:19-21
Mapping a Cave Fish Genome: Polygenic Systems and Regressive Evolution
From the Cave Biology Research Group, Department of Biology, 1009 Main, New York University, Washington Square, NY 10003 (Borowsky) and Zoology Institute and Zoology Museum of the University of Hamburg, Martin-Luther-King-Platz 3, 20146 Hamburg, Germany (Wilkens).
Address correspondence to Richard Borowsky at the address above or e-mail: rb4{at}scires.nyu.edu.
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Abstract |
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We used random amplified polymorphic DNA (RAPD) fingerprinting to generate anonymous DNA markers in the fish Astyanax mexicanus, a species with both surface and cave populations. Surface individuals are eyed and pigmented; troglobitic forms are blind and depigmented. We hybridized surface fish and Pachon population cave fish and produced a RAPD genomic map 1064 cM in length (about half the total length of the genome) that was used to screen for quantitative trait loci (QTL) for troglomorphic traits. Three QTL for reduced eye size, two for decreased numbers of melanophores, two for condition factor, and the locus for the unifactorial trait, albinism, were mapped. These factors account for an average of 46% of the variance in these traits in the backcross. The results are the first direct demonstration that troglomorphic changes in this population are multifactorial. Two closely linked pairs of QTL were found. Each consisted of a regressive and a constructive trait QTL. These close linkages are unlikely to have occurred by chance (P < .05 for each) and suggest that troglomorphic evolution might be facilitated by pleiotropy or by genetic hitchhiking.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Regressive evolution is the reduction of a trait within a lineage over time. Common examples include loss of vision in obligatory cave organisms (troglobites) and metabolic or structural simplifications in parasites. Although we generally think of regressive evolution as a minor issue in biology, its effects are widespread. Without regressive evolution to prune the phenotype, all species would be encumbered by billion-year-long lists of superannuated traits.
The changes that occur in regressive evolution are no different than those that occur in constructive evolution: allelic frequencies and character states alter over time. The problem is in explaining the process. As Darwin (1859) noted, a role for natural selection in regressive evolution is often difficult to envisage. In fact, a general evolutionary basis for character regression remains unknown to this day.
Troglobitic fishes are classic examples of regressive evolution and were cited by Darwin (1859) in discussing the loss of traits "in disuse." Eighty-five cavefish species from nine different orders have already been described (Weber et al. 1998). All share some degree of eye loss and pigmentation reduction and, in their taxonomic diversity, make ideal models to study both the mechanisms and results of regressive evolution.
There are two principal competing hypotheses to account for regressive evolution, illustrated here by loss of visual function in troglobites. The first is that selection favors eye loss, perhaps for reasons of organismal or neural processing economy. An alternative is that the genes controlling the development of eyes become effectively neutral with the relaxation of selective constraints and are free to accumulate mutations impairing their function. In essence, the positions are selection versus neutralism. These basic possibilities and numerous variants have been discussed with reference to the evolution of troglobites (Culver and Wilkens 2000).
Troglobites also exhibit convergence for constructive changes, like increases in sensory modalities other than vision, or increased metabolic efficiency. In contrast to regression of eyes or pigmentation, constructive changes are more easily accounted for by natural selection. In spite of this difference, it is important to consider both regressive and constructive changes together, for a fuller understanding of the cave adaptation process (Culver et al. 1995:25–26). Because traits evolve in the context of changes in other traits, it is important to understand trait correlations in order to describe evolutionary changes adequately and to construct testable hypotheses about mechanisms.
One previously unexplored approach to the problems of regressive evolution and troglobitic evolution is genetic linkage mapping. Linkage mapping and subsequent quantitative trait loci (QTL) analysis is a direct approach to the enumeration of the genes involved in character evolution, and a plausible first step in their eventual isolation and cloning. Selection, as opposed to drift, predicts patterns in the distribution of eye loss genes within the genome, within developmental pathways, or in the types of nucleotide substitutions involved in alterations of function. Therefore identifying the genes involved will aid in testing alternative hypotheses. In addition, linkage data allow the detection of correlations between loci in genomic position.
The Mexican tetra [Astyanax mexicanus (Filippi, 1853) (= A. fasciatus (Cuvier, 1819)] is particularly useful for studying the genetics of cave adaptations in fishes because it has both cave and surface forms that are fully interfertile. Pachon Cave in northeastern Mexico supports a population of blind Mexican tetras isolated from base-level populations by elevation (Mitchell et al. 1977). Compared to A. mexicanus from nearby surface populations, Pachon cavefish have rudimentary eyes, are albino, and are in better condition (weigh more for a given length). Breeding studies show that albinism is unifactorial in Pachon, while quantitative genetic analysis suggests that melanophore number and eye size are multifactorial, with estimates of three to six genes responsible for the trait changes in the Pachon population (Wilkens 1988).
Here we test the hypothesis of multigenic inheritance directly by mapping. Using random amplified polymorphic DNA (RAPD), we produced a partial genomic map to enumerate and identify quantitative trait loci (QTL) for troglomorphic traits in the Pachon cavefish population. We mapped QTL for eye size, pigmentation loss, and "condition factor," a surrogate for metabolic efficiency. The study was small in scale, but the results provide the first direct evidence that troglomorphic traits are multifactorial. Details of the QTL-linked RAPD markers are given elsewhere (http://homepages.nyu.edu/~rb4/).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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An F1 hybrid female (Pachon x Epigean) was mated to a Pachon male to produce a backcross progeny (BC, N = 111) for analysis. BC animals were raised in the light in community tanks to the age of 6–8 months. At sacrifice, BC progeny were preserved in 70% ethanol, weighed to the nearest milligram, and their standard lengths determined to the nearest 0.1 mm. DNA for RAPD analysis was prepared by proteinase K digestion of fin tissue, followed by phenol and chloroform:IAA extractions.
RAPD
RAPD amplifications and scoring were performed after published procedures (Borowsky et al. 1995; Kazianis et al. 1996) modified as follows: we used short primers (10–11 mers) in primer pair combinations and employed Stoffel fragment Taq for amplification. Amplimers were labeled by direct incorporation of 33P, separated on 4% polyacrylamide sequencing gels under denaturing conditions, and visualized by autoradiography. Bands were scored on the autorads by eye. Marker loci with segregation patterns more probably 3:1 than 1:1 were discarded from analysis. Seventy percent of the segregating RAPD markers were suitable for mapping. An average of 18.3 ± 1.8 RAPD markers with approximate 1:1 segregation ratios were typed with each primer combination.
Quantitative Traits
Eye size. The eye region was dissected carefully under 10x magnification on the right side of the animal and the eyeball rudiment exposed. The eyeball diameter was measured optically and the residual of its size, after regression on standard length of the fish, was the quantitative measure of eye development used in all analyses.
Melanophores. Progeny were scored as albino or nonalbino under 25x magnification. In nonalbino fish, melanophores were counted on standardized areas in three locations on the back, near the dorsal fin.
Condition factor. The measure of condition factor (CF) was the residual of the logarithm of weight after regression of log weight on log length.
Linkage and QTL Analyses
Map Manager XP (Manly 1998) was used to identify and build linkage groups (significance level = 0.001). Each quantitative trait was analyzed using Map Manager QT (version 2.6; Manly 1998; Manly and Cudmore 1997; Manly and Olsen 1999) and its associations with mapped RAPD markers were tested by regression analysis (Links Report module). Markers having apparent associations with the trait were examined serially by interval mapping, starting with the marker with the strongest association (Interval Mapping module). For the first association examined, no cofactors were used. For subsequent associations analyzed, markers with previously established significance were included as cofactors. The serial examination was terminated at the first nonsignificant association. Significance levels of QTL were determined by permuting the data (Churchill and Doerge 1994), as implemented by the Permutation Test module. Permutations for the first association examined were made without control for variation at other marker loci. Subsequent permutations were made controlling for loci previously found to be significant.
QTL Contributions to Trait Variance
Estimates of the variance contributions of individual QTL are biased upward when the power to detect QTL is low (Beavis 1994). We corrected for this bias for each QTL (from Figure 15.8 of Lynch and Walsh 1998:474–476) based on the power of its being detected, estimated from initial estimates of its effect, the number of informative progeny, and estimates of dominance (from published data, Wilkens 1985).
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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One hundred forty-two RAPD loci with segregation ratios close to 1:1 were scored. Of these, 81 coalesced into 27 distinct linkage groups using the standard criterion of LOD > 3.0. The total size of the partial map is 1064 cM. A. mexicanus has a haploid number of 25 and, based only on comparison to zebrafish and platyfish, an expected map size of about 2000 cM (zebrafish = 2200 cM, Postlethwait et al. 1994; platyfish = 1800 cM, Morizot et al. 1991).
We tested for correlations between phenotypic traits within the BC progeny. CF was weakly correlated with melanophore number (r = +0.21, df = 78, P = .05) and more strongly with eye size (r = +0.43, df = 107, P = .01). Trait correlations could reflect developmental constraints, pleiotropy, or coincident locations of QTL. The positive correlation between eye size and CF could also reflect the advantage of larger eyes in social interactions and competition for food, since the fishes were raised in community tanks and in the light.
QTL analysis, as outlined above, revealed seven statistically significant, putative QTL: three for eye size, two for melanophore number, and two for CF. In addition to the mapped QTL, the locus for albinism was found to be part of linkage group lg26. The QTL detected account for 27% of the variance in eye size, 39% in pigmentation, and 19% in CF (Table 1). The single locus for albinism accounts for 100% of the variance in that trait. Thus, of the four troglomorphic traits studied, the factors found account for an average of 46% of the variation. For the QTL alone, this figure is 28%. In accordance with our estimate that the map is half complete, these results are consistent with previous quantitative genetic estimates of about six factors for regression of eye size and three to four for regression in melanophore number in the Pachon population (Wilkens 1988).
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The data reveal two pairs of linked QTL, in linkage groups lg25 and lg4. In lg25 a factor for pigmentation is linked to another for CF, near RAPD locus C8a. In lg4 a factor for eye size is linked to another for CF, between RAPD loci I7a and AB6a. Based on the number of linkage groups and their size, the chance probability of either of these co-occurrences is less than 5%. The chance probability of two is much less than 1%. It is of interest that in both cases the linkage is between QTL for regressive and constructive traits. Determining if these co-occurrences are common phenomena or simply intriguing coincidences must await further work.
Although these apparent linkages might be spurious and derive from the positive correlations between traits seen in the BC progeny, there is little evidence for this interpretation. In lg25 the linkage is between CF and pigmentation, and these traits are not significantly correlated in the progeny (r2 = 0.05). In lg4 the linkage is between CF and eye size and, while the overall correlation of these traits is stronger (r2 = 0.21), the QTL for eye size in lg4 is the weakest of the three detected (5% of the variance versus 8 and 14%). The other two eye size QTL show no apparent linkage to QTL for CF.
Close linkage of regressive and constructive QTL might arise from pleiotropy. For example, if a gene for eye size also influences metabolism, selection on metabolism could also affect the character state of eyes. Pleiotropy is looked upon as a potential means by which seemingly "neutral" changes can be driven by natural selection. Its role in troglomorphic evolution has been reviewed by Culver (1982) and by various authors in Culver (1985).
There is also the possibility that linkage reflects a history of genetic hitchhiking. If, after invasion of the subterranean environment, new mutations occur at random in loci affecting both regressive and constructive traits, some of these might be linked. Given linkage, hitchhiking might then facilitate the increase in frequency of those regressive mutants linked to selected constructive mutants. This hypothesis remains to be tested; like pleiotropy, it predicts the close linkage of troglomorphic factors in troglobite genomes. The two hypotheses can be distinguished because pleiotropy predicts the identity of any troglomorphic factors, while hitchhiking predicts close linkage of regressive and constructive QTL. Hitchhiking is a novel and potentially important mechanism for regressive evolution. If true, it would allow for faster evolutionary change than possible through drift alone. While hitchhiking and pleiotropy may play roles in regressive evolution, neither mechanism excludes an important role for drift of neutral mutations under conditions of relaxed selection, as outlined in the introduction.
Genetic analysis of a single family can reveal only a fraction of the variability in an outbred population. Analysis of other Pachon lines or cave populations of this species would presumably yield different estimates of the total numbers and relative magnitudes of the troglomorphy QTL. These studies are in progress. The present work, however, shows clearly that troglomorphic traits are multigenic and that the QTL exhibit significant linkages in the line analyzed.
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Acknowledgments |
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We thank M. McClelland and J. Welsh for support and advice during the early stages of this study. Technical help was provided by B. Andiak, J. Khlevner, and J. Starobinets. This study was partly funded by a Research Challenge Fund grant from New York University (to R.B.).
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Footnotes |
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Corresponding Editor: Lisa Seeb
Received November 17, 2000
Accepted December 7, 2001
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References |
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