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Genetic analysis of cavefish reveals molecular convergence in the evolution of albinism Meredith E Protas1, Candace Hersey2, Dawn Kochanek3, Yi Zhou2, Horst Wilkens4, William R Jeffery5, Leonard I Zon2, Richard Borowsky3 & Clifford J Tabin1

The genetic basis of vertebrate morphological evolution has traditionally been very difficult to examine in naturally occurring populations. Here we describe the generation of a genome-wide linkage map to allow quantitative trait analysis of evolutionarily derived morphologies in the Mexican cave tetra, a species that has, in a series of independent caves, repeatedly evolved specialized characteristics adapted to a unique and well-studied ecological environment. We focused on the trait of albinism and discovered that it is linked to Oca2, a known pigmentation gene, in two cave populations. We found different deletions in Oca2 in each population and, using a cell-based assay, showed that both cause loss of function of the corresponding protein, OCA2. Thus, the two cave populations evolved albinism independently, through similar mutational events.

The relatively closed, often nutrient-poor, and lightless environment of caves represents a marked change in ecological conditions to which several entrapped species have adapted. Obligate cave-dwelling ani- mals, called troglobites or troglodytes, are characterized by a remark- able convergence of eye and pigment loss across diverse species such as spiders, isopods, salamanders and fish1.

There are 86 known troglodytic species of fish2. The best studied is the Mexican tetra, identified by some authors as Astyanax mexicanus and others as Astyanax fasciatus; the two names should be considered synonymous in the present context and the species will be referred to herein as Astyanax. This species has 29 cave populations in the karst region of the Sierra de El Abra of northeast Mexico and one additional population in Guerrero (Fig. 1a)3,4. A surface, or river-dwelling, sister population of the cave morph lives in southern Texas and northeastern Mexico and can still interbreed with the cave morph. Phenotypically, the cave and surface morphs are very different; among other char- acteristics, the cave morph has a greater weight per unit length, less pigment, regressed eyes, larger nostrils, more maxillary teeth, more cranial neuromasts and more taste buds, as well as differences in

feeding, schooling and aggressive behaviors (Fig. 1b–d)4,5. Molecular phylogenetic studies indicate that several cave populations indepen- dently evolved these characteristics6–8.

To provide a framework in which to study the genetics of this species, we made a microsatellite linkage map. We have isolated and

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Figure 1 Phenotype and locations of albino cave populations of Astyanax

mexicanus. (a) Map of the area in Mexico where the different cave

populations are found. Dots represent cave populations. Caves with red dots

are Molino, Pachón and Japonés, all of which contain a majority of albino

individuals. Inset map at bottom shows the location of the region within

Mexico. (b) A representative surface fish. (c) A representative Molino cavefish. (d) A representative Pachón cavefish.

Received 20 July; accepted 13 October; published online 11 December 2005; doi:10.1038/ng1700

1Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA. 2Children’s Hospital Stem Cell Program, Department of Hematology/Oncology, Howard Hughes Medical Institute, Children’s Hospital Boston, Boston, Massachusetts 02115, USA. 3Cave Biology Research Group, Department of Biology, New York University, 1009 Main, 100 Washington Square East, New York, New York 10003, USA. 4Zoological Institute and Zoological Museum, University of Hamburg, Martin- Luther-King-Platz 3, 20146 Hamburg, Germany. 5Department of Biology, University of Maryland, College Park, Maryland 20742, USA. Correspondence should be addressed to C.J.T. ([email protected]).

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designed primers corresponding to over 600 microsatellites. Using a backcross from the Molino cave population with 111 progeny, we obtained 35 linkage groups composed of 267 markers, out of 300 markers genotyped, with a coverage of 1,916 cM (Fig. 2 and Supplementary Table 1 online). Astyanax has 25 chromosomes, sug- gesting that with the addition of more markers, some of the linkage groups would collapse. We are also genotyping a larger F2 cross from another cave, Pachón, which should coalesce some of the linkage groups and allow for comparisons between the two cave populations.

We identified a number of statistically significant, quantitative loci for different traits present in the cave form, most of which will be described elsewhere. Here we focus on one such trait, albinism. Previous genetic studies have indicated that albinism in the Pachón cave is caused by a single recessive mutation9,10. In the Molino backcross, albinism mapped to a single locus in linkage group 16 with a LOD score of 17.29 at microsatellite marker 218E, accounting for 49.4% of the variance in this trait (Fig. 3a). A similar analysis of the Pachón F2 cross mapped the locus for albinism to the same location with a LOD score of 17.98 at marker 218E, accounting for 42.6% of the variance in this trait (data not shown). This coincidence of loci responsible for albinism raises the following three possibilities: the two cave populations could have the same mutation in the same gene, different mutations in the same gene or mutations in distinct but closely linked genes. To address the latter possibility, we performed a complementation test between a Molino individual and a Pachón individual, which yielded only albino offspring (Fig. 3b). Thus, albinism in these two cave populations is caused by mutations in the same gene.

To identify the gene responsible for albinism in Astyanax, we genotyped individuals of the Molino backcross for a series of candi- date genes, based on known albinism loci in mouse and humans: tyrosinase (Tyr), tyrosinase-related protein-1 (Tyrp1) and ocular and

cutaneous albinism-2 (Oca2) (Fig. 2). One of these genes, Oca2, mapped to the albino locus and increased the LOD score in the Molino backcross to 68.6 (Fig. 3c), now accounting for 93.1% of the variance of this trait, and the LOD score in the Pachón F2 cross to 60.66, now accounting for 71.6% of the variance of this trait (data not shown). Furthermore, there is a perfect association between the genotype of the Oca2 marker and the phenotype of albinism in all successfully genotyped individuals of both the Molino backcross (105 individuals) and the Pachón F2 cross (215 individuals).

Although the function of Oca2 is unknown, it is the most commonly mutated gene in cases of human albinism11 and is also responsible for pigmentation phenotypes in mouse and medaka12,13. To test whether Oca2 mutations are responsible for albinism in cavefish, and, more importantly, to identify the specific genetic lesions in Oca2 responsible for albinism, we compared the sequence of the Oca2 cDNA in surface, Pachón and Molino individuals (Supplementary Fig. 1 online). We found numerous differences in the Oca2 sequences present in the two cave populations as compared to their surface counterparts (Fig. 4a and Supplementary Fig. 1). The Pachón cave population had three polymorphisms that could affect Oca2 function: two were amino acid changes in conserved residues, and the last was a deletion extending from within intron 23 through most of exon 24, such that the cDNA includes part of intron 23 fused to the last nine base pairs of exon 24 and the 3¢ UTR (Fig. 4a and Supplementary Fig. 1). The Molino cave population had only one major difference: exon 21 was missing (Fig. 4a and Supplementary Fig. 1).

The missing exons observed in the Molino and Pachón Oca2 sequence could, in principle, be explained by either alterations in splicing or deletions of genomic DNA. Amplifying from genomic DNA, we found that in both cases, the observed losses of exonic sequence in the Molino and Pachón Oca2 cDNAs were attributable to genomic deletions (Supplementary Fig. 2 online).

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Figure 2 Microsatellite linkage map of Astyanax mexicanus. The names of the microsatellite markers are to the right of each linkage group and the positions

of the markers (in cM) are on the left. Markers in three genes (Oca2, Tyr and Tyrp1) are labeled in red.

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To test whether the polymorphisms in the cave populations’ Oca2 sequences cause albinism through loss of function of Oca2, we used the melan-p cell line, which is a melanocyte cell line generated from an Oca2-deficient mouse14. We made constructs expressing wild-type surface-fish Oca2 and surface-fish Oca2 modified with each of the cavefish polymorphisms driven by the human ubiquitin promoter. We first transfected the surface-fish Oca2 construct to ascertain whether wild-type Astyanax Oca2 could complement Oca2 function in mouse cells. Indeed, melan-p cells transfected with the surface-fish Oca2 showed high levels of pigmentation (Fig. 4b–d). We then transfected cells with constructs encoding the different amino acid substitutions seen in the Pachón cave. Both point mutations caused rescue of pigmentation (Fig. 4e–j). However, neither the deletion found in the Molino cave nor the deletion found in the Pachón cave caused rescue of pigmentation (Fig. 4k–p). Therefore, the deletions cause loss of function of OCA2, whereas the two point mutations do not drastically affect the function of OCA2 in this cell line, strongly suggesting that the exon 21 deletion is the mutation that causes albinism in the Molino cave population and the exon 24 deletion is the mutation that causes albinism in the Pachón population.

The identification of different inactivating mutations in Oca2 in the Molino and Pachón populations suggests that albinism evolved independently in these two caves, by convergent evolution in the same gene. The independent evolution of Oca2 in the two caves is further supported by analysis of amino acid changes and neutral base changes; these polymorphisms do not group the Molino and Pachón populations together to the exclusion of the surface population (Supplementary Table 2 online).

We also examined another cave popula- tion, Japonés, that contains albino indivi- duals (Fig. 1a). In a complementation test between an albino Japonés individual and an albino Pachón individual, only albino off- spring resulted, suggesting that albinism in the Japonés population also arose through mutations in the Oca2 gene (data not shown). Sequencing of Oca2 from one albino Japonés individual showed that neither the Molino nor the Pachón deletion was present (data not shown), though the two point mutations found in the Pachón cave were found. Together with the complementation data, this result suggests that a third, inde- pendent mutation in the Oca2 gene, perhaps in a regulatory sequence, is responsible for the convergent albinism phenotype in the Japonés cave.

The parallel evolution of loss of pigmenta- tion and loss of eyes within the species Astyanax is mirrored in many other cave dwelling organisms. The most debated issue about cave animals is why this loss of eyes and pigmentation occurs. There are three main theories1,8,9,15. First, the genes respon- sible for structures or pathways that are not advantageous in a dark environment, such as eyes or pigmentation, might accumulate dele- terious mutations and, over time, completely degenerate. Second, it might be advantageous to lose eyes and pigmentation to conserve the energy or space that these structures con-

sume. Third, the genetic changes that cause loss of eye and pigmenta- tion might cause changes in other structures or pathways that might be advantageous in the cave environment.

The identification of the mutations responsible for loss of pigmen- tation in Astyanax allows us to consider the molecular nature of the variation facilitating this change in response to the cave environment. It is notable that Oca2 has repeatedly mutated in the cave populations we examined. It is possible that in the cave environment, loss of Oca2 function is actually advantageous, for some as yet unknown reason. Alternatively, Oca2 might be mutated more often than other pigmen- tation pathway genes in Astyanax simply because Oca2 mutations do not seem to have any deleterious effects aside from loss of pigmenta- tion and problems with vision. It is possible that some of the other pigmentation genes have more pleiotropic effects and that those mutations are not as viable; for example, all of the mutations in zebrafish Tyr that cause complete loss of pigmentation are only semiviable16. A final explanation is that Oca2 is the most frequently targeted pigmentation gene in Astyanax for the same reasons that it seems to be in humans: first, Oca2 presents a very large target size for mutagenesis, being 345 kb in humans, with 24 exons11. Second, human Oca2 maps to a region characterized by repetitive sequences, which are often associated with chromosomal rearrangements and deletions17. Although it is not yet known whether the Astyanax Oca2 locus is similarly characterized by a large size and a large number of associated repeat sequences, the most parsimonious explanation seems to be a combination of a lack of deleterious pleiotropic effects in conjunction with the structure of Oca2 itself. These sorts of features may, in general, predispose certain loci to be targets for evolutionary

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Figure 3 Oca2 is linked to albinism in both the Pachón and Molino cave populations. (a) The albino

locus in the Molino backcross is in linkage group (LG) 16. The LOD score is plotted against the

distance (in cM) across this linkage group. (b) An albino Pachón � Molino hybrid, showing noncomplementation. (c) The marker in Oca2 is located at the albino locus in the Molino backcross

and its inclusion in the analysis greatly increases the LOD score.

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forces effecting morphological change. The existence of at least 86 species of cavefish2, a subset of which show albinism, will allow for further examination of the parallel evolution of this trait in the cave environment.

We have seen that albinism has evolved in two different cave populations through independent changes in the same gene. Other studies have also shown that one gene is responsible for the same morphological change in multiple populations or species: ectodyspla- sin (Eda) in body armor and Pitx1 in pelvic reduction in the three- spine stickleback18,19, ovo (shaven-baby) in trichome loss in different drosophilids20, and MC1R in pigmentation in pocket mice, jaguars and several avian species21,22. Thus, the same morphology often evolves by mutation in the same gene, possibly because it is the most efficient or best way for a certain phenotype to evolve.

Another issue often discussed regarding the evolution of morpho- logical change is whether coding changes or regulatory changes generate new traits. Regulatory mutations have an advantage as agents of morphological change in that they can alter a gene’s activity in a subset of the regions in which it is expressed. Regulatory mutations have been identified or implicated in morphological change in plate armor and pelvic reduction in sticklebacks18,19, wing-spot pigmenta- tion in Drosophila biarmipes23, muscle mass in pigs24 and trichome loss in drosophilids20. Often it is difficult to confirm that a gene has a specific regulatory mutation, as these mutations can span large areas of sequence. In contrast, coding differences are easier to find. It is notable that many of the examples of evolutionary change involving coding mutations that have previously been described, including our example of Oca2 in albinism in Astyanax, involve overall changes in pigmentation21,22. This could be because pleiotropic effects of pig- mentation genes are minimal and usually do not affect the viability of the organism.

We have identified specific genetic lesions responsible for the parallel evolution of albinism in different cave populations of Astya- nax, and found that they represent convergent genetic events in separate populations. The genetic tools we have developed will facilitate further investigation into the molecular bases for the evolu- tion of specialized morphological characteristics in the unique and well-studied cave environment.

METHODS Crosses. We used five crosses in this study: a Molino backcross, a Pachón

F2 cross, a Pachón backcross, a Molino � Pachón complementation cross and a Pachón � Japonés complementation cross (see Supplementary Methods online).

Microsatellite identification. To make a genomic library, we digested genomic

surface-fish DNA with Sau3AI and cloned fragments of 500–700 bp into

BamH1-linearized pBluescriptSK+ (Stratagene). We then electroporated the

library into SURE electroporation-competent cells (Stratagene). A 32P- or

digoxigenin-end-labeled (CA)12 probe was used to hybridize colony lifts and

colonies hybridizing to the probe were sequenced using T3 and T7 primers.

Primers to detect microsatellites that had ten or more repeats were

designed using Primer3. The following tag sequence was added to the 5¢ end of every forward primer: 5¢-CACGACGTTGTAAAACGAC-3¢ (Y.Z. and L.I.Z., unpublished data).

Genotyping and linkage map construction. We genotyped 111 individuals

for 300 microsatellite markers in the Molino backcross and 235 indi-

viduals with 254 microsatellites in the Pachón F2 cross. PCR reactions

were 10 ml in volume and contained 0.1 mM MgCl2, 6 mM Tris-HCl, pH 8.3, 30 mM KCl, 0.006% glycerol, 0.25 mM dNTP mix (Roche),

0.06% Tween, 0.06% Nonidet P-40, 0.25 units of Taq DNA polymerase

(Roche), 5 nM forward primer, 200 nM reverse primer and 200 nM of the

fluorescent tag primer 5¢-CACGACGTTGTAAAACGAC-3¢ labeled with one of two phosphoramidite conjugates, Hex and Fam, using a PCR program

as described25. Each PCR reaction was primed with the specific forward

primer. The majority of cycles used the forward primer in excess—the

fluorescently labeled tag (Y.Z. and L.I.Z., unpublished data). Three

Fam-labeled PCR products and three Hex-labeled PCR products were pooled

and run on an ABI 3700 with GeneScan-500 ROX size standard (Applied

Biosystems). Genotyper software (version 3.6) was used to analyze the

genotyping results. The program JoinMap was used to make the microsatellite

linkage map using the Kosambi mapping function, a jump threshold of 5.0 and

Surface Oca2

Pachón Oca2

Molino Oca2

b c

Figure 4 Different Oca2 exon deletions in the Molino population and

the Pachón population cause loss of function of the OCA2 protein.

(a) Schematics of the surface, Pachón and Molino Oca2 coding regions.

Asterisks in the Pachón Oca2 represent changes in conserved amino acid

residues: red asterisk, methionine to valine; blue asterisk, proline to serine

(see Supplementary Figs. 1 and 2). In the Pachón coding sequence, exon

24 is almost completely deleted. Following exon 23 are additional sequence

(intron 23), the last few amino acids of exon 24, and the 3¢ UTR. The Molino coding sequence is identical to that of the surface-fish Oca2 except

that exon 21 is missing. (b–d) Transfection of the unmodified surface-fish

Oca2 construct results in highly pigmented cells. (e–g) Transfection of the

construct encoding the methionine-to-valine substitution, found in the

Pachón cave, results in pigmented cells, although they are slightly less

pigmented than the cells transfected with the surface-fish sequence.

(h–j) Transfection of the construct encoding the proline-to-serine substitution results in highly pigmented cells. (k–p) Transfection of the

construct with the extra intron 23 sequence and exon 24 deletion (k–m)

or the construct with the exon 21 deletion (n–p) does not rescue

pigmentation. b,e,h,k,n show bright-field pictures of the melan-p cell line

transfected with different constructs; c,f,i,l,o show the corresponding

transfected cells visualized by fluorescence (all constructs were co-injected

with pUB-GFP26); and d,g,j,m,p provide schematics of the constructs

transfected in each experiment.

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a ripple function. For the Molino backcross, 267 of 300 markers were grouped

into 35 linkage groups using a LOD threshold of 4.0.

QTL mapping. We used the interval mapping function of MapQTL26 to

determine the albino locus and determined significant LOD scores using a

permutation test.

Cloning and mapping of albinism candidate genes. We obtained fragments of

Astyanax Tyr, Trp1 and Oca2 by degenerate PCR. For primers and specifics, see

Supplementary Methods. The fragment that we initially amplified by degen-

erate PCR of Oca2 contained exons 11–23. We were able to amplify exon 1 of

Oca2 from the DNA of surface fish by designing primers to the area of exon 1

that is most conserved between zebrafish and fugu. Using a forward primer in

exon 1, 5¢-GAGCCCAGGGTCATCAGG-3¢, we obtained the sequence through exon 11. To obtain the 5¢ and 3¢ ends, we used the SMART Race cDNA kit (Clontech).

Genomic analysis of missing Oca2 exons in Pachón and Molino populations.

We carried out PCR to exons 21 and exons 14 for genomic DNA from all

individuals of the Molino backcross using the following primers: exon 21F,

exon 21R, exon 14F and exon 14R (see Supplementary Table 1 online for all

primer sequences). For the Pachón backcross, we amplified intron 23 and exon

24 to the 3¢ untranslated region using the following primers: intron 23F, intron 23R, exon 24F and 3¢ UTR.

Sequencing of full-length Oca2 coding region for Surface, Molino and

Pachón fish. We extracted RNA from fin clips and whole fish using Trizol

(Invitrogen), and we made cDNA using AMV reverse transcriptase (Roche). To

amplify the full-length cDNA, we used primers from the 5¢ and 3¢ untranslated regions of Oca2. In some cases, we amplified Oca2 in two pieces for ease of

amplification, using a reverse primer in exon 12 and a forward primer in exon

10. We compared Oca2 sequences from five surface fish, five Molino fish and

two Pachón fish.

Making overexpression constructs. We cloned surface, Molino and Pachón

full-length sequences into pGEM-T Easy (Promega). We cloned the missing

exon 21 (Molino) and missing exon 24 plus extra sequence (Pachón) cloned

into the surface-fish construct using MluI and SmaI. pUB-GFP26, a vector that

encodes GFP under the control of the human ubiquitin promoter, was used as

the backbone for the overexpression constructs. pUB-GFP was blunted with

SmaI and MscI, removing the GFP, and pGEM-T Easy surface-fish Oca2,

surface-fish Oca2 minus exon 21 and surface-fish Oca2 minus exon 24 were

blunted with PvuII. The different Oca2 fragments and the pUB fragment were

ligated together (Takara), putting surface-fish Oca2 under the control of the

ubiquitin promoter. To make the point mutations found in the Pachón cave, we

carried out site-directed mutagenesis using the QuikChange site directed

mutagenesis kit (Stratagene) on the pUB-surface Oca2 construct.

Cell line assay. We obtained melan-p cells from D. Bennett’s group and

cultured them as described14. We then transfected the cells on 3-cm plates

using Fugene (Roche) and 1 mg of each construct. The transfections were as follows: pUB-GFP alone26, pUB-GFP with pUB-surface Oca2, pUB-GFP with

pUB-surface minus exon 21, pUB-GFP with pUB surface minus exon 24 and

the proline-to-serine amino acid substitution, pUB-GFP with pUB-surface

Oca2 with the methionine-to-valine substitution, and pUB-GFP with pUB-

surface Oca2 with the proline-to-serine substitution. After transfection, we

waited 4 d for pigmentation to develop.

Accession numbers. GenBank: A. mexicanus microsatellite markers,

BV678703–BV678968; A. mexicanus Oca2, DQ232591.

Note: Supplementary information is available on the Nature Genetics website.

ACKNOWLEDGMENTS This work was supported by US National Science Foundation grant IBN0217178 to R. Borowsky and C.J. Tabin. The authors thank L. Mekios for phenotyping

and fish maintenance, the Bennett group for the use of the melan-p cell line, T. Matsuda for the pUB-GFP construct, R.C. Albertson for critical reading of the manuscript, and C. Peichel, R.V. Pearse II, J.L. Galloway and J. Rivera-Feliciano for help and advice.

COMPETING INTERESTS STATEMENT The authors declare that they have no competing financial interests.

Published online at http://www.nature.com/naturegenetics/

Reprints and permissions information is available online at http://npg.nature.com/

reprintsandpermissions/

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