Allele-specific marker generation and linkage mapping on the Xiphophorus sex chromosomes.

There is great interest in the sex chromosomes of Xiphophorus fishes because both WY/YY and XX/XY sex-determining mechanisms function in these species, with at least one taxon possessing all three types of sex chromosomes, and because in certain interspecific hybrids melanoma arises as a consequence of inheritance of the sex-linked macromelanophore determining locus (MDL). Representational difference analysis (RDA) has been used to clone two sequences from the sex-determining region of X. maculatus, including a cholinergic receptor, nicotinic, delta polypeptide (CHRND) orthologue. Allele-specific assays for these sequences, as well as for the sex-linked XMRK1 and XMRK2 genes, were developed to distinguish W, X, and Y chromosomes derived from a X. maculatus (XX/XY) strain and a X. helleri (WY/YY) strain. Linkage mapping localized these markers to linkage group (LG) 24. No recombinants were observed between XMRK2 and MDL, confirming a role for XMRK2 in macromelanophore development. Although the master sex-determining (SD) locus certainly resides on Xiphophorus LG 24, autosomal loci are probably involved in sex determination as well, as indicated by the abnormal sex ratios in the backcross hybrids that contrast theoretical predictions based on LG 24 genotyping. Marker development and allelic discrimination on the Xiphophorus sex chromosomes should prove highly useful for studies that utilize this genus as an animal model.

A microsatellite genetic linkage map for Xiphophorus.

Interspecies hybrids between distinct species of the genus Xiphophorus are often used in varied research investigations to identify genomic regions associated with the inheritance of complex traits. There are 24 described Xiphophorus species and a greater number of pedigreed strains; thus, the number of potential interspecies hybrid cross combinations is quite large. Previously, select Xiphophorus experimental crosses have been shown to exhibit differing characteristics between parental species and among the hybrid fishes derived from crossing them, such as widely differing susceptibilities to chemical or physical agents. For instance, genomic regions harboring tumor suppressor and oncogenes have been identified via linkage association of these loci with a small set of established genetic markers. The power of this experimental strategy is related to the number of genetic markers available in the Xiphophorus interspecies cross of interest. Thus, we have undertaken the task of expanding the suite of easily scored markers by characterization of Xiphophorus microsatellite sequences. Using a cross between Xiphophorus maculatus and X. andersi, we report a linkage map predominantly composed of microsatellite markers. All 24 acrocentric chromosome sets of Xiphophorus are represented in the assembled linkage map with an average intergenomic distance of 7.5 cM. Since both male and female F1 hybrids were used to produce backcross progeny, these recombination rates were compared between "male" and "female" maps. Although several genomic regions exhibit differences in map length, male- and female-derived maps are similar. Thus Xiphophorus, in contrast to zebrafish, Danio rerio, and several other vertebrate species, does not show sex-specific differences in recombination. The microsatellite markers we report can be easily adapted to any Xiphophorus interspecies and some intraspecies crosses, and thus provide a means to directly compare results derived from independent experiments.

Xiphophorus interspecies hybrids as genetic models of induced neoplasia.

Fishes of the genus Xiphophorus (platyfishes and swordtails) are small, internally fertilizing, livebearing, and derived from freshwater habitats in Mexico, Guatemala, Belize, and Honduras. Scientists have used these fishes in cancer research studies for more than 70 yr. The genus is presently composed of 22 species that are quite divergent in their external morphology. Most cancer studies using Xiphophorus use hybrids, which can be easily produced by artificial insemination. Phenotypic traits, such as macromelanophore pigment patterns, are often drastically altered as a result of lack of gene regulation within hybrid fishes. These fish can develop large exophytic melanomas as a result of upregulated expression of these pigment patterns. Because backcross hybrid fish are susceptible to the development of melanoma and other neoplasms, they can be subjected to potentially deleterious chemical and physical agents. It is thus possible to use gene mapping and cloning methodologies to identify and characterize oncogenes and tumor suppressors implicated in spontaneous or induced neoplasia. This article reviews the history of cancer research using Xiphophorus and recent developments regarding DNA repair capabilities, mapping, and cloning of candidate genes involved in neoplastic phenotypes. The particular genetic complexity of melanoma in these fishes is analyzed and reviewed.

MNU induction of neoplasia in a platyfish model.

Interspecific hybrid crosses between members of the fish genus Xiphophorus have been used for over 70 years to study the genetic aspects of melanoma formation. In the well-established "Gordon-Kosswig" cross, the platyfish X. maculatus is outcrossed to the swordtail X. helleri, and the resulting backcross segregants spontaneously develop melanoma. We recently produced a distinct cross between X. maculatus and another platyfish species, X. couchianus. X. maculatus strain Jp 163 A is homozygous for several X-linked pigment pattern genes, including the Spotted dorsal (Sd), Dorsal red (Dr), and Anal fin spot (Af). Af is a sex-limited trait, coding exclusively for melanophores distributed on the modified anal fin or "gonopodium" in the adult male fish. Within F1 and BC1 hybrids (to X. couchianus), the Sd pigment pattern is phenotypically suppressed, whereas Dr and Af are enhanced. We exposed BC1 hybrids to the direct-acting carcinogen N-methyl-N-nitrosourea (MNU). Treatment led to the development of schwannomas, fibrosarcomas, and retinoblastomas. In addition, numerous MNU-treated males that inherited Af developed a pronounced melanotic phenotype, with melanin-containing cells oftentimes totally covering the gonopodium and extending further to grow within the ventral regions of the fish. Genetic linkage analysis of the BC1 hybrids revealed a significant (p < 0.01) association between CDKN2X genotype and the phenotypic degree of melanization. Such an association is consistent with a locus within linkage group V playing a role in the development of melanosis and delineates three genetic preconditions and a carcinogenic scheme resulting in melanosis of the ventral regions of hybrid fish. The overall study further alludes to the potential of using Xiphophorus fish to study carcinogenic mechanisms for tumors other than melanoma (schwannoma, fibrosarcoma, and retinoblastoma) and should enable extensive pathologic and molecular genetic studies of derived neoplastic abnormalities.

Characterization of the CDKN2A and ARF genes in UV-induced melanocytic hyperplasias and melanomas of an opossum (Monodelphis domestica).

We examined the involvement of the cyclin-dependent kinase inhibitor 2A (CDKN2A) locus in the pathogenesis of ultraviolet (UV) radiation-induced melanomas in an opossum (Monodelphis domestica) melanoma model in which suckling young were exposed to UVB to produce melanocytic lesions. Monodelphis CDKN2A and alternated reading frame (ARF) cDNAs were cloned and sequenced, and the expression patterns of these genes were determined by reverse transcription-polymerase chain reaction in normal tissues, 39 primary melanocytic skin lesions, and two tumor-derived cell lines, one nonmetastatic and one metastatic. Primary melanocytic lesions, including hyperplasias, benign melanomas, melanomas metastatic to lymph nodes, and melanomas metastatic to nodes and additional visceral organs, were categorized accordingly as types I-IV. Levels of CDKN2A transcripts were most abundant in type III tumor samples and the metastatic cell line but absent in the nonmetastatic cell line. ARF transcripts were expressed in all tumors and cell lines. A UV-signature mutation was detected with the wild-type allele at the CDKN2A locus in type II and III primary tumor samples and in the nonmetastatic cell line. Interestingly, in the metastatic cell line, only the mutant allele was present and expressed. These data suggest dynamic changes in the expression and/or structure of the CDKN2A and ARF genes represent one molecular defect associated with the etiology of melanoma formation and progression in the Monodelphis model system.

Genetic analysis of susceptibility to spontaneous and UV-induced carcinogenesis in Xiphophorus hybrid fish.

Xiphophorus interspecies hybrids provide genetically controlled models of tumor formation. Spontaneous melanomas form in first-generation backcross (BC(1)) hybrids produced from backcrossing F(1) hybrids derived from the platyfish X. maculatus Jp 163 A and the swordtail X. helleri to the X. helleri parental strain (the Gordon-Kosswig hybrid cross). Nodular melanomas originate in the dorsal fin from cells constituting the spotted dorsal (Sd) pigment pattern. A parallel genetic cross, with X. maculatus Jp 163 B, exhibits the spotted side (Sp) pigment pattern instead of Sd, and produces BC(1) hybrids exhibiting a much lower frequency of spontaneous melanoma formation. These hybrids are susceptible to melanoma development if irradiated with UV light as fry. Other hybrids involving these two strains of X. maculatus and different swordtail and platyfish backcross parents also have been investigated as potential tumor models, and show differing susceptibilities to UV-induced and spontaneous melanomas. Genotyping of individual BC(1) hybrids from several Xiphophorus crosses has implicated a locus, CDKN2X (a Xiphophorus homologue of the mammalian CDKN2 gene family, residing on Xiphophorus linkage group V), in enhancing pigmentation and the susceptibility to spontaneous and UV-induced melanoma formation in BC(1) hybrids from some crosses, but not others. Homozygosity for X. helleri and X. couchianus CDKN2X alleles in BC(1) hybrids can predispose individuals to melanoma, but this susceptibility is modified in other crosses depending both on the contributing sex-linked pigment pattern locus from X. maculatus (Sd or Sp), and the genetic constitution of the backcross parent. Xiphophorus BC(1) hybrids constitute unique genetic models offering the potential to analyze the contributions of specific genes to spontaneous and induced tumor formation in different, but comparable genetic backgrounds.

Genetic analysis of neoplasia induced by N-nitroso-N-methylurea in Xiphophorus hybrid fish.

Interspecific crosses within the genus Xiphophorus have historically been used to study the genetic aspects of melanoma formation. Melanomas typically occur as a result of deregulation of polymorphic, naturally occurring macromelanophore pigment patterns. Hybrid crosses also have been used to study the inducibility of melanoma by physical sources (such as UV light) and chemicals (such as N-methyl-N-nitrosourea, MNU). We previously defined a genomic region that is implicated in fish melanomagenesis and identified a candidate tumor suppressor gene (CDKN2X) within this genomic area. Highly significant associations between BC(1)-hybrid CDKN2X genotypes and UV-induced melanoma formation exist in a backcross produced from 2 inbred parental lines. However, when BC(1) hybrids are exposed to MNU as the tumor induction agent, a significant association between inheritance of CDKN2X alleles and tumor development is not observed. These data suggest there is mechanistic and genetic heterogeneity in melanomas derived from different etiologies within BC(1) hybrid fish.

A proposed classification scheme for Xiphophorus melanomas based on histopathologic analyses.

We studied the histopathologic characteristics of melanomas induced in the Xiphophorus model. This fish model has been used for several decades to study the molecular and genetic mechanisms underlying its susceptibility to melanoma induction. Numerous distinct interspecific hybrid crosses currently are being used in research on carcinogenesis. We previously reported that tumors were induced in such hybrid crosses after treatment with N-methyl-N-nitrosourea or UV radiation. In this report, we describe the histopathologic features of Xiphophorus melanomas and propose a new classification system. We suggest that melanomas in these fishes can be classified as follows: melanocytic melanomas; melanophorous-macromelanophorous polymorphic melanomas; spindle cell type melanomas; epithelioid cell melanomas; and amelanotic melanomas. The new classification of Xiphophorus melanomas should allow correlations between histopathologic characteristics and carcinogen treatment, and between histopathologic characteristics and the genetic background of the hybrid fish.

Xiphophorus genetic linkage map: beginnings of comparative gene mapping in fishes.

The explosive expansion of gene maps of mouse and man has provided strong support for hypotheses first advanced from comparing fish and mammalian genomes that the vertebrate genome was derived from multiple ancestral tetraploidizations with subsequent preferential translocations among paralogous chromosomes. At least two genome duplication events have become widely accepted in lineages leading to vertebrates, and a third has been proposed either before, or after, divergence of fishes and tetrapods. Cytogenetic and comparative gene mapping studies suggest that teleost gene maps have diverged more slowly from gene arrangements in the vertebrate ancestor than have those of mammals. The recent assembly of extensive maps of >100 genes in three fish species, medaka (Beloniformes), Xiphophorus swordtails and platyfishes (Cyprinodontiformes), and zebrafish (Cypriniformes) and the development of less extensive maps in several other fish orders provides the first salient opportunity to assess homology of most or all chromosomes among fishes.

Overexpression of a fish CDKN2 gene in a hereditary melanoma model.

The fish genus Xiphophorus provides a vertebrate model useful in etiological studies of cancer. Hybrid fish can spontaneously develop melanomas deriving from the inheritance of melanistic pigment patterns and the simultaneous absence of proper genetic regulation. A cyclin-dependent kinase inhibitor gene, termed CDKN2X, was mapped to a genomic region that is implicated in fish melanoma tumor suppression. The related human tumor suppressor locus CDKN2A (P16, INK4A, MTS1) is deleted, mutated or transcriptionally repressed through methylation of cytosine bases within the 5' CpG island in a variety of neoplasms, including melanoma. The fish CDKN2X locus harbors a CpG island within its promoter and first exon, analogous in location to CpG islands in human CDKN2A and CDKN2B loci. The methylation state of individual CpG dinucleotides was investigated in genomic DNA derived from control tissues and melanomas within the CDKN2X 5' CpG island. The studied genomic area was found to be virtually unmethylated in all tested tissues including melanomas. In addition, RNA expression studies of the fish CDKN2X locus revealed that it is significantly overexpressed in melanoma, in contrast to what has been reported for the human CDKN2A locus in melanoma. Such overexpression may be a consequence of the pronounced upregulation of the Xmrk-2 receptor tyrosine kinase oncogene reported in several Xiphophorus melanoma models.

Comparative structure and characterization of a CDKN2 gene in a Xiphophorus fish melanoma model.

We have cloned, sequenced, and characterized the RNA expression properties of a fish CDKN2 gene from Xiphophorus helleri and X. maculatus. This gene, termed CDKN2X, shows a high degree of amino acid sequence similarity to members of the mammalian CDKN2 gene family, which includes the tumor suppressor loci CDKN2A (P16) and CDKN2B (P15). Comparative sequence analysis suggests that fish CDKN2X is similarly related to all four mammalian gene family members, and may represent a descendant of an ancestral prototypic CDKN2 gene. CDKN2X was mapped to a region on autosomal Xiphophorus linkage group V (LG V) known to contain the DIFF gene that acts as a tumor suppressor of melanoma formation in X. helleri/X. maculatus backcross hybrids. Thus, CDKN2X may be a candidate for the tumor suppressor DIFF gene. Here we have sequenced CDKN2X in both Xiphophorus species and have characterized its expression in normal and melanotic tissues within control and backcross hybrid fish. A simultaneous expressional analysis of the Xmrk-2 tyrosine kinase receptor gene, which is strongly implicated in melanomagenesis in this system, was also performed. RT - PCR analyses revealed that both genes were highly expressed in melanomas. For CDKN2X, this result contrasts numerous findings in human tumors including human melanoma in which either CDKN2A (P16) deactivation or LOH was observed.

Mapping of tyrosine kinase gene family members in a Xiphophorus melanoma model.

Xiphophorus fish have been the subject of intensive genetic research for more than 60 yr, primarily because of the availability of a number of interspecific hybrids that are malignant melanoma models with apparently simple oncogene and tumor suppressor gene determinants. The gene map of Xiphophorus is one of the most extensive among nonhuman vertebrates, with about 100 genes assigned to at least 20 independently assorting linkage groups (LGs), as well as more than 250 anonymous DNA sequence markers, providing coverage for most of the genome for genetic mapping studies. This characteristic has resulted in the mapping of a tumor suppressor locus, DIFF, which is one of two genetic determinants of melanoma formation in the best-studied hybrid melanoma, the Gordon-Kosswig melanoma model. The other gene responsible for melanoma formation in this model is a sex-linked tyrosine kinase gene related to EGFR and called Xiphophorus melanoma receptor kinase (Xmrk). The cellular oncogene homologues of the non-receptor tyrosine kinase family orthologous toyes and fyn have also been found to be overexpressed in malignant melanomas of Xiphophorus and may be involved in tumor progression. We report here the map location of a Xiphophorus yes gene, YES1, in LG VI, closest to the EGFR gene and the assignment of a fyn gene homologue to newly designated LG XV, linked to the gene for cytosolic alpha-galactosidase. We also confirmed that an EGFR-related sequence (EGFRL1) that we previously assigned to Xiphophorus LG VI by cross-hybridization to a viral erbB probe was the EGFR orthologue. Our results suggest that the presence of expressed duplicates of members of the tyrosine kinase gene family in teleost fishes may increase the potential number of targets in oncogenic cascades in fish tumor models.

Identification and mapping of two divergent, unlinked major histocompatibility complex class II B genes in Xiphophorus fishes.

We have isolated two major histocompatibility complex (MHC) class II B genes from the inbred fish strain Xiphophorus maculatus Jp 163 A. We mapped one of these genes, designated here as DXB, to linkage group III, linked to a malic enzyme locus, also syntenic with human and mouse MHC. Comparison of genomic and cDNA clones shows the gene consists of six exons and five introns. The encoded beta1 domain has three amino acids deleted and a cytoplasmic tail nine amino acids longer than in other teleost class II beta chains, more similar to HLA-DRB, clawed frog Xela-F3, and nurse shark Gici-B. Key residues for disulfide bonds, glycosylation, and interaction with alpha chains are conserved. These same features are also present in a swordtail (Xiphophorus helleri) genomic DXB PCR clone. A second type of class II B clone was amplified by PCR from X. maculatus and found to be orthologous to class II genes identified in other fishes. This DAB-like gene is 63% identical to the X. maculatus DXB sequence in the conserved beta2-encoding exon and was mapped to new unassigned linkage group LG U24. The DXB gene, then, represents an unlinked duplicated locus not previously identified in teleosts.

Localization of a CDKN2 gene in linkage group V of Xiphophorus fishes defines it as a candidate for the DIFF tumor suppressor.

The Xiphophorus hybrid melanoma model represents one of the earliest reported cases of genetically regulated tumor susceptibility. Melanoma formation in Xiphophorus hybrids may be explained by the inheritance of two genes: a sex-linked oncogene, Xmrk, and a putative tumor suppressor locus, termed DIFF, located in Linkage Group V (LG V). Several genetic mapping procedures were used to produce a new Xiphophorus LG V map with 20 loci. All markers, particularly a recently cloned Xiphophorus CDKN2 gene family member, called CDKN2X, were tested for associations of genotype with degree of macromelanophore pigment pattern modification and susceptibility to melanoma formation in backcross hybrids of seven genetic types, involving 1,110 fish and three pigment patterns. Highly significant associations of CDKN2X genotypes with such phenotypic effects suggests that this gene is a strong candidate for the classically defined DIFF tumor suppressor gene. Because published results have documented the involvement of the CDKN2A (p16, MTS1, and INK4A) tumor suppressor gene in human melanoma formation, the possibility of CDKN2 genes acting as tumor suppressors in both man and Xiphophorus is likely.

Cloning and comparative sequence analysis of TP53 in Xiphophorus fish hybrid melanoma models.

We have cloned and sequenced the p53-encoding cDNA of green swordtail (X. helleri) and southern platyfish (X. maculatus). These two fish species are often used to produce hybrids that develop melanomas after genetic crossing. Computer translation of derived cDNA sequences revealed that p53 polypeptides from these two species are virtually identical, exhibiting only two conservative amino acid substitutions. TP53 mRNA expression was detected in virtually all tissues tested. Comparison of these fish p53 polypeptide sequences with those of other vertebrates, including other fishes, amphibians, and mammals, revealed that conservation is especially high in several previously defined protein domains. In addition, sequencing of the 3' TP53 genomic region of X. maculatus reveals similarity to the human TP53 locus in overall organization. Knowledge of the Xiphophorus TP53 sequences will allow assessment of mutational alterations within tumors generated from numerous fish genetic crosses.

A CDKN2-like polymorphism in Xiphophorus LG V is associated with UV-B-induced melanoma formation in platyfish-swordtail hybrids.

The genetic basis of spontaneous melanoma formation in spotted dorsal (Sd) Xiphophorus platyfish-swordtail hybrids has been studied for decades, and is adequately explained by a two-gene inheritance model involving a sex-linked oncogene, Xmrk, and an autosomal tumor suppressor, DIFF. The Xmrk oncogene encodes a receptor tyrosine kinase related to EGFR; the nature of the DIFF tumor suppressor gene is unknown. We analyzed the gentic basis of UV-B-induced melanoma formation in closely related, spotted side platyfish-swordtail hybrids, which carry a different sex-linked pigment pattern locus, Sp. We UV-irradiated spotted side Xiphophorus platyfish-swordtail backcross hybrids to induce melanomas at frequencies 6-fold higher than occur spontaneously in unirradiated control animals. To identify genetic determinants of melanoma susceptibility in this UV-inducible Xiphophorus model, we genotyped individual animals from control and UV-irradiated experimental regimes using allozyme and DNA restriction fragment length polymorphisms and tested for joint segregation of genetic markers with pigmentation phenotype and UV-induced melanoma formation. Joint segregation results show linkage of a CDKN2-like DNA polymorphism with UV-B-induced melanoma formation in these hybrids. The CDKN2-like polymorphism maps to Xiphophorus linkage group V and exhibits recombination fractions with ES1 and MDH2 allozyme markers consistent with previous localization of the DIFF tumor suppressor locus. Our results indicate that the CDKN2-like sequence we have cloned and mapped is a candidate for the DIFF tumor suppressor gene.

Nonmammalian models for sunlight carcinogenesis: genetic analysis of melanoma formation in Xiphophorus hybrid fish.

Genetic hybrids of Xiphophorus fishes have been used for decades to study heritable melanoma formation. In these models, overexpression of pigmentation patterns from melanin-producing pigment cells can lead to genetically regulated melanoma formation in backcross hybrids. In the best studied of these models, the Gordon-Kosswig hybrid melanoma, tumors form spontaneously in all individuals of a subset of backcross hybrids between the platyfish Xiphophorus maculatus Jp 163 A and the swordtail species Xiphophorus helleri. Backcross hybrids susceptible to melanoma formation inherit a sex-linked oncogene, Xmrk, associated with the spotted dorsal (Sd) pigment pattern and have lost both copies of an autosomal gene, DIFF, from the X. maculatus parent. Spontaneous melanoma formation conforms to simple, two-gene Mendelian inheritance in which DIFF behaves as a recessive tumor suppressor gene. Recently, Xiphophorus hybrids in which melanomas can be induced by UV and near-UV visible light exposure have been described. We report here results of genetic linkage analysis of one of these Xiphophorus light-inducible hybrid melanoma models, in backcross hybrids between the two platyfish species X. maculatus Jp 163 B and Xiphophorus couchianus. Our linkage results provide the first estimate of recombination between the tumor suppressor locus, DIFF, and glycerate-2-dehydrogenase (GLYDH) in Xiphophorus linkage group V. Also, they demonstrate that DIFF regulates hyperplasia of spotted side (Sp) pigment cells in this hybrid model, analogous to its regulation of hyperplasia of Sd pigment cells in the "classical" Gordon-Kosswig hybrid. Joint segregation analyses of melanoma-bearing fish indicate that segregation of DIFF is genetically linked to melanoma induction by 405 nm light in this model but that induction of melanomas by UV wavelengths apparently does not depend on segregation of the DIFF locus.

Genetic mapping in Xiphophorus hybrid fish: assignment of 43 AP-PCR/RAPD and isozyme markers to multipoint linkage groups.

The combined use of the arbitrarily primed polymerase chain reaction [AP-PCR, also known as random amplification of polymorphic DNA (RAPD)] and isozyme mapping resulted in the production of 87 potential marker loci, enabling an overall expansion within the genetic map of the fish genus Xiphophorus. Use of DNA sequencing-style acrylamide gels and carefully controlled conditions of amplification and silver staining allowed exceptional resolution and reproducibility of AP-PCR/RAPD generated markers. Linkage analysis of AP-PCR/RAPD and isozyme markers resulted in the addition of 16 new markers to Xiphophorus linkage groups (LGs) I, II, III, V, IX, X, XII, and XIV. Addition of 5 AP-PCR/RAPD markers to linkage group U6 containing the Tailspot pigment pattern locus (P) and designation of eight new unassigned linkage groups with 22 markers was also accomplished. Genetic linkage data allowed inference of the existence of a novel pigment pattern modifier locus. Expansion of the Xiphophorus gene map by linkage analysis of AP-PCR/RAPD markers in conjunction with isozyme polymorphisms should lead to the rapid saturation of genetic linkage groups such as LG V, which will probably be instrumental to cloning the Diff tumor suppressor gene locus.

Stable association of a pigmentation allele with an oncogene: nonhybrid melanomas in Xiphophorus variatus.

Sex-linked genes in several species of the fish genus Xiphophorus cause macromelanophore pigmentation patterns on the flanks of the fish. Some, but not all, of these patterns can develop into melanomas. The tumorigenic alleles are tightly linked to a supernumerary oncogene sequence, Xmrk. The data show that the association of Xmrk with two of the tumorigenic alleles of X. variatus, P2 and Li, holds over a broad geographic area. From the distribution of the fish and the geology of the area, it is probable that this association is older than the late Tertiary. The persistence of this association suggests that Xmrk confers some benefit on P2-and Li-bearing individuals to offset the deleterious effect. The nature of this benefit remains unknown.