Repetitive DNA Sequences and Evolution of ZZ/ZW Sex Chromosomes in Characidium (Teleostei: Characiformes)

Characidium constitutes an interesting model for cytogenetic studies, since a large degree of karyotype variation has been detected in this group, like the presence/absence of sex and supernumerary chromosomes and variable distribution of repetitive sequences in different species/populations. In this study, we performed a comparative cytogenetic analysis in 13 Characidium species collected at different South American river basins in order to investigate the karyotype diversification in this group. Chromosome analyses involved the karyotype characterization, cytogenetic mapping of repetitive DNA sequences and cross-species chromosome painting using a W-specific probe obtained in a previous study from Characidium gomesi. Our results evidenced a conserved diploid chromosome number of 2n = 50, and almost all the species exhibited homeologous ZZ/ZW sex chromosomes in different stages of differentiation, except C. cf. zebra, C. tenue, C. xavante and C. stigmosum. Notably, some ZZ/ZW sex chromosomes showed 5S and/or 18S rDNA clusters, while no U2 snDNA sites could be detected in the sex chromosomes, being restricted to a single chromosome pair in almost all the analyzed species. In addition, the species Characidium sp. aff. C. vidali showed B chromosomes with an inter-individual variation of 1 to 4 supernumerary chromosomes per cell. Notably, these B chromosomes share sequences with the W-specific probe, providing insights about their origin. Results presented here further confirm the extensive karyotype diversity within Characidium in contrast with a conserved diploid chromosome number. Such chromosome differences seem to constitute a significant reproductive barrier, since several sympatric Characidium species had been described during the last few years and no interespecific hybrids were found.     

Single Origin of Sex Chromosomes and Multiple Origins of B Chromosomes in Fish Genus Characidium

Chromosome painting with DNA probes obtained from supernumerary (B) and sex chromosomes in three species of fish genus Characidium (C. gomesi, C. pterostictum and C. oiticicai) showed a close resemblance in repetitive DNA content between B and sex chromosomes in C. gomesi and C. pterostictum. This suggests an intraspecific origin for B chromosomes in these two species, probably deriving from sex chromosomes. In C. oiticicai, however, a DNA probe obtained from its B chromosome hybridized with the B but not with the A chromosomes, suggesting that the B chromosome in this species could have arisen interspecifically, although this hypothesis needs further investigation. A molecular phylogenetic analysis performed on nine Characidium species, with two mtDNA genes, showed that the presence of heteromorphic sex chromosomes in these species is a derived condition, and that their origin could have been unique, a conclusion also supported by interspecific chromosome painting with a CgW probe derived from the W chromosome in C. gomesi. Summing up, our results indicate that whereas heteromorphic sex chromosomes in the genus Characidium appear to have had a common and unique origin, B chromosomes may have had independent origins in different species. Our results also show that molecular phylogenetic analysis is an excellent complement for cytogenetic studies by unveiling the direction of evolutionary chromosome changes.     

Chromosome painting of Z and W sex chromosomes in Characidium (Characiformes, Crenuchidae)

Some species of the genus Characidium have heteromorphic ZZ/ZW sex chromosomes with a totally heterochromatic W chromosome. Methods for chromosome microdissection associated with chromosome painting have become important tools for cytogenetic studies in Neotropical fish. In Characidium cf. fasciatum, the Z chromosome contains a pericentromeric heterochromatin block, whereas the W chromosome is completely heterochromatic. Therefore, a probe was produced from the W chromosome through microdissection and degenerate oligonucleotide-primed polymerase chain reaction amplification. FISH was performed using the W probe on the chromosomes of specimens of this species. This revealed expressive marks in the pericentromeric region of the Z chromosome as well as a completely painted W chromosome. When applying the same probe on chromosome preparations of C. cf. gomesi and Characidium sp., a pattern similar to C. cf. fasciatum was found, while C. cf. zebra, C. cf. lagosantense and Crenuchus spilurus species showed no hybridization signals. Structural changes in the chromosomes of an ancestral sexual system in the group that includes the species C. cf. gomesi, C. cf. fasciatum and Characidium sp., could have contributed to the process of speciation and could represent a causal mechanism of chromosomal diversification in this group. The heterochromatinization process possibly began in homomorphic and homologous chromosomes of an ancestral form, and this process could have given rise to the current patterns found in the species with sex chromosome heteromorphism.     

Chromosomal divergence and maintenance of sympatric Characidium fish species (Crenuchidae, Characidiinae)

Cytogenetic studies were performed in two syntopic species of Characidium, C. lauroi and Characidium sp. cf. C. alipioi, from Ribeir?o Grande, Paraíba do Sul river basin. Both species have diploid number 2n=50 chromosomes, but differ in chromosome shape, C-banding pattern and location of nucleolar organizing regions. In Characidium sp. cf. C. alipioi a new type of ZW sex chromosome system composed of equal sized metacentric chromosomes is reported for the first time in the genus Characidium. Species of Characidium with a sex chromosome system form a monophyletic group. Variations in this system are interpreted as resulting from geographic isolation among allopatric species.     

Sex chromosome composition revealed in Characidium fishes (Characiformes: Crenuchidae) by molecular cytogenetic methods

The W chromosome of the fishes Characidium cf. fasciatum, Characidium sp. and Characidium cf. gomesi is heterochromatic, as is usually seen in most Characidium species. Samples of W-chromatin were collected by mechanical microdissection and amplified by DOP-PCR (degenerate oligonucleotide-primed polymerase chain reaction), to be used as painting probes (DCg and CgW) and for sequence analysis. FISH (fluorescence in situ hybridization) with DCg probe painted the whole W chromosome, the pericentromeric region of Z chromosomes and the terminal region of B chromosomes. DOP-PCR-generated fragments were cloned, sequenced and tested by in situ hybridization, but only CgW4 produced positive hybridization signals. Clone sequence analysis recovered seven distinct sequences, of which six did not reveal any similarity to other known sequences in the GenBank or GIRI databases. Only CgW9 clone sequence was recognized as probably derived from a Helitron-transposon similar to that found in the genome of the zebrafish Danio rerio. Our results show that the composition of Characidium’s W chromosome does seem rich in repetitive sequences as well as other W chromosomes found in several species with a ZW sex-determining mechanism.     

Cytonuclear equilibrium following interspecific introgression in a turtle lacking sex chromosomes

When reproductive barriers break down, interspecific hybridization can lead to gene flow between evolutionarily distinct species. Studying the fate of these introgressing elements can offer valuable insights into the factors contributing to reproductive isolation. We have identified a population of false map turtles (Graptemys pseudogeographica) that hybridized historically with the common map turtle (Graptemys geographica), but were subsequently isolated from interbreeding for several generations by unique geological events. Although many studies conclude that genic interactions involving sex chromosomes impact the introgression of mitochondrial or nuclear genomes, Graptemys turtles have environmental sex determination, and thus introgression can be explored while controlling for the effects of sex‐specific heterogameity. We identified and sequenced a species‐specific mitochondrial control region marker, as well as two nuclear markers (ODC and HNFAL), in turtles from across the ranges of these species. We found both nuclear and mitochondrial introgression in our study population, and present evidence consistent with the proposed time range of reproductive contact and isolation. We also report an absence of cytonuclear or linkage disequilibrium among markers, indicating that some important pre‐ and postzygotic barriers to gene flow that characterize other systems are absent in Graptemys. Finally, we show that Graptemys turtles have a complex molecular evolutionary history, and that leaks in reproductive barriers probably occur frequently. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 106 , 405–417.     

The effectiveness of pre‐ and post‐zygotic barriers in avoiding hybridization between two snapdragons (Antirrhinum L.: Plantaginaceae)

Reproductive barriers play an important role in the maintenance of species boundaries. However, to date, few studies have provided a detailed analysis of reproductive isolation barriers between species or examined their importance in maintaining species identity. This is the first detailed study into pre‐ and post‐zygotic reproductive isolation barriers in Antirrhinum, based on a mixed population with two species that rarely co‐occur. The study revealed that pollinator constancy and preference and poor hybrid seed viability were the most important reproductive isolating mechanisms. Reproductive isolation was practically complete by both pre‐ and post‐zygotic barriers. Average pre‐zygotic isolation was greater than post‐zygotic isolation, in accordance with the trend observed in flowering plants in which reproductive isolation is principally caused by pre‐zygotic mechanisms. However, average post‐zygotic isolation was also high, in contrast to what was expected among Antirrhinum spp. This case highlights the importance of quantifying the reproductive isolation barriers thoroughly to understand how and why species boundaries are maintained. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176 , 159–172.     

Diversification of a ZZ/ZW sex chromosome system in Characidium fish (Crenuchidae, Characiformes)

Karyotype and other chromosomal markers of Characidium cf. gomesi were analyzed using conventional (Giemsa-staining, Ag-NOR and C-banding) and molecular (Fluorescent in situ hybridization (FISH) with 18S and 5S rDNA biotinylated probes) techniques. Both sexes had invariably diploid chromosome number 2n = 50 while karyotypes of males and females differed. That of male consisted of 32 metacentric + 18 submetacentric chromosomes and that of female consisted 31 metacentric + 18 submetacentric + 1 subtelocentric chromosomes. The Z chromosome was medium-sized metacentric, while W was highly heterochromatinized subtelocentric element. NORs as revealed by Ag-staining were situated at 2–7 telomeric regions while FISH with 18S probes showed consistently 10 signals at telomeric regions. FISH with 5S rDNA probe showed constantly signals at one metacentric pair. Distribution of centromeric heterochromatin was mostly in all chromosome pairs, besides some telomeric sites. The common origin of the sex chromosome system of ZZ/ZW type in the karyotypes of other representatives of the genus analyzed so far might be hypothesized based on biogeography and partial phylogeny of the group.     

Morphometric analysis of floral variation in the Pyrola picta species complex (Ericaceae): interpretation and implications for ecological and phylogenetic differentiation

The Pyrola picta species complex of western North America comprises four species (P. picta, P. dentata, P. aphylla and P. crypta) that grow sympatrically in some parts of their collective ranges, have remarkably similar flowers and share pollinators. These species do not exhibit the genetic signatures typical of random or heterospecific mating, but instead show genetic divergence patterns indicating that they maintain surprising levels of reproductive isolation. To better understand how species boundaries are maintained, the current study uses statistical ordination analyses to determine whether species isolation across shared geographical ranges might be achieved through subtle differences in floral characters among species. The possible contribution of differences in flowering phenology (e.g. temporal reproductive isolation) to reproductive isolation was also evaluated for the small subset of populations in which two or more species occur in direct sympatry. Among species in the P. picta complex, there are both phylogenetic and geographical trends in some floral characteristics, whereas other characters do not covary with either geography or species identity. In several sympatric populations, differences in flowering phenology among species suggest that timing plays a major role in non‐random (i.e. mainly conspecific) mating. The conclusions of this study are that reproductive isolation in the P. picta species complex is reinforced by differences in the timing of floral maturation and the morphologies of androecium and floral display characters. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 00 , 000–000.     

Patterns of hybridization in a multispecies hybrid zone in the Ranunculus cantoniensis complex (Ranunculaceae)

Simultaneous hybridizations among three or more parental taxa occur in nature, but these have rarely been analysed. The present study investigates the natural hybridization of diploid members of the Ranunculus cantoniensis complex over a microgeographical area in south‐western China. Sequence information from maternally inherited plastid DNA (trnQ‐rps16 and rpL32‐trnL) and biparentally inherited nuclear ribosomal DNA was used to identify hybrids, and these were further confirmed by a dramatic reduction in pollen viability. The populations from the contact zone contained individuals that had more than two nuclear ribosomal internal transcribed spacer (nrITS) types; in addition, a network analysis revealed the presence of hard conflicts between the nuclear and plastid DNA data. These results prove that R. trigonus and R. silerifolius var. dolicanthus have relatively little reproductive isolation from each other, leading to a high gene flow rate in contact populations. The four investigated species can hybridize with each other in this contact zone, forming seven hybrid types. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174 , 227–239.     

Differentiated ZZ/ZW sex chromosomes in Apareiodon ibitiensis (Teleostei,Parodontidae): cytotaxonomy and biogeography

Conventional and molecular chromosomal analyses were carried out on three populations of Apareiodon ibitiensis sampled from the hydrographic basins of the São Francisco River and Upper Paraná River (Brazil). The results reveal a conserved diploid number (2n = 54 chromosomes), a karyotype formula consisting of 50 m‐sm + 4st and a ZZ/ZW sex chromosome system that has not been previously identified for the species. C‐banding analysis with propidium iodide staining revealed centromeric and terminal bands located in the chromosomes of the specimens from the three populations and allowed the identification of heteromorphism of heterochromatin regions in the Z and W chromosomes. The number of 18S sites located through fluorescent in situ hybridization (FISH) varied between the populations of the São Francisco and Upper Paraná Rivers. The location of 5S rDNA sites proved comparable in one pair of metacentric chromosomes. Thus, the present study proposes a ZZ/ZW sex chromosome system for A. ibitiensis among the Parodontidae, and a hypothesis is presented regarding possible W chromosome differentiation stages in this species through DNA accumulation, showing geographical variations for this characteristic, possibly as a consequence of geographical reproductive isolation.     

Species delimitation in Neotropical fishes of the genus Characidium (Teleostei,Characiformes)

Neotropical darters of the genus Characidium have a complex systematic history with several examples of sympatry throughout their distribution range in Neotropical freshwaters. Although various species within the genus have been used as models to investigate chromosomal evolution and biogeography, species boundaries and relationships still remain uncertain. Here, we use mitochondrial DNA sequences to perform species delimitation analyses within Characidium and test previous hypotheses of species richness within the Characidium zebra complex and among sympatric morphotypes of C. alipioi. Results indicate high genetic distances within tested species complexes and revealed the presence of strongly supported lineages such as the large C. lauroi group from southeastern Brazil. This suggests that the evolutionary history of these groups may be correlated with biogeographic history. Analyses also reveal that three geographically isolated populations of C. zebra represent a single species, leading us to reject prior hypothesis of multiple species. Species delimitation using mitochondrial data strongly supports the presence of two sympatric species within C. alipioi in southeastern Brazil despite limited morphological variation and conserved chromosomal patterns. These results provide a framework to further the study of systematics and evolution within Characidium.     

Satellite DNA and chromosomes in Neotropical fishes: methods,applications and perspectives

Constitutive heterochromatin represents a substantial portion of the eukaryote genome, and it is mainly composed of tandemly repeated DNA sequences, such as satellite DNAs, which are also enriched by other dispersed repeated elements, including transposons. Studies on the organization, structure, composition and in situ localization of satellite DNAs have led to consistent advances in the understanding of the genome evolution of species, with a particular focus on heterochromatic domains, the diversification of heteromorphic sex chromosomes and the origin and maintenance of B chromosomes. Satellite DNAs can be chromosome specific or species specific, or they can characterize different species from a genus, family or even representatives of a given order. In some cases, the presence of these repeated elements in members of a single clade has enabled inferences of a phylogenetic nature. Genomic DNA restriction, using specific enzymes, is the most frequently used method for isolating satellite DNAs. Recent methods such as C₀t–1 DNA and chromosome microdissection, however, have proven to be efficient alternatives for the study of this class of DNA. Neotropical ichthyofauna is extremely rich and diverse enabling multiple approaches with regard to the differentiation and evolution of the genome. Genome components of some species and genera have been isolated, mapped and correlated with possible functions and structures of the chromosomes. The 5SHindIII‐DNA satellite DNA, which is specific to Hoplias malabaricus of the Erythrinidae family, has an exclusively centromeric location. The As51 satellite DNA, which is closely correlated with the genome diversification of some species from the genus Astyanax, has also been used to infer relationships between species. In the Prochilodontidae family, two repetitive DNA sequences were mapped on the chromosomes, and the SATH 1 satellite DNA is associated with the origin of heterochromatic B chromosomes in Prochilodus lineatus. Among species of the genus Characidium and the Parodontidae family, amplifications of satellite DNAs have demonstrated that these sequences are related to the differentiation of heteromorphic sex chromosomes. The possible elimination of satellite DNA units could explain the genome compaction that occurs among some species of Neotropical Tetraodontiformes. These topics are discussed in the present review, showing the importance of satellite DNA analysis in the differentiation and karyotype evolution of Actinopterygii.     

Cytosystematics of the Simulium tuberosum group (Diptera: Simuliidae) in Thailand

The polytene chromosomes of 3347 larvae of the Simulium tuberosum group in Asia were analysed, representing the largest ever cytogenetic study of black flies in the Oriental Region. Band‐by‐band comparisons, relative to the established standard chromosome map for the subgenus Simulium, revealed 17 cytogenetically distinct taxa in Thailand, plus an 18th in China. Six of these taxa correspond to morphologically described species (S. doipuiense, S. rufibasis, S. setsukoae, S. tani, S. yuphae and S. weji). Recognition of the 18 taxa is based largely on unique inversions, either fixed or sex linked, primarily in the long arm of chromosome III. The greatest cytological diversity was discovered in the S. tani lineage, with ten cytoforms. This marked chromosomal diversification within S. tani is based largely on two inversions that have assumed different roles over evolutionary time, variously functioning in different combinations as fixed inversions, sex‐linked inversions and autosomal polymorphisms. Shared unique chromosomal features, relative to the subgeneric standard chromosome map, allowed evolutionary relationships among the cytotaxa to be inferred. Fluctuations in climate during the Pleistocene might have promoted differentiation of the Southeast Asian S. tuberosum group in isolated refugia such as mountains. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 155 , 289–315.     

Genomic landscape of reproductive isolation in Lucania killifish: The role of sex loci and salinity

Adaptation to different environments can directly and indirectly generate reproductive isolation between species. Bluefin killifish (Lucania goodei) and rainwater killifish (L. parva) are sister species that have diverged across a salinity gradient and are reproductively isolated by habitat, behavioural, extrinsic and intrinsic post‐zygotic isolation. We asked if salinity adaptation contributes indirectly to other forms of reproductive isolation via linked selection and hypothesized that low recombination regions, such as sex chromosomes or chromosomal rearrangements, might facilitate this process. We conducted QTL mapping in backcrosses between L. parva and L. goodei to explore the genetic architecture of salinity tolerance, behavioural isolation and intrinsic isolation. We mapped traits relative to a chromosome that has undergone a centric fusion in L. parva (relative to L. goodei). We found that the sex locus appears to be male determining (XX‐XY), was located on the fused chromosome and was implicated in intrinsic isolation. QTL associated with salinity tolerance were spread across the genome and did not overly co‐localize with regions associated with behavioural or intrinsic isolation. This preliminary analysis of the genetic architecture of reproductive isolation between Lucania species does not support the hypothesis that divergent natural selection for salinity tolerance led to behavioural and intrinsic isolation as a by‐product. Combined with previous studies in this system, our work suggests that adaptation as a function of salinity contributes to habitat isolation and that reinforcement may have contributed to the evolution of behavioural isolation instead, possibly facilitated by linkage between behavioural isolation and intrinsic isolation loci on the fused chromosome.     

The evolution of sex pheromones in an ecologically diverse genus of flies

In theory, pheromones important in specific mate recognition should evolve via large shifts in composition (saltational changes) at speciation events. However, where other mechanisms exist to ensure reproductive isolation, no such selection for rapid divergence is expected. In Bactrocera fruit flies (Diptera: Tephritidae), males produce volatile chemicals to attract females for mating. Bactrocera species exhibit great ecological diversity, with a wide range of geographical locations and host plants used. They also have other mechanisms, including temporal and behavioural differences, which ensure reproductive isolation. Therefore, we predicted that their sex pheromones would not exhibit rapid divergence at speciation events. In the present study, we tested this idea by combining data on male sex pheromone composition for 19 species of Bactrocera with a phylogeny constructed from DNA sequence data. Analyses of the combined data revealed positive correlations between pheromone differences and nucleotide divergence between species, and between the number of pheromone changes along the phylogeny and the branch lengths associated with these changes. These results suggest a gradual rather than saltational mode of evolution. However, remarkable differences in sex pheromones composition exist, even between closely-related species. It appears therefore that the mode of evolution of sex pheromones in Bactrocera is best described by rapid saltational changes associated with speciation, followed by gradual divergence thereafter. Furthermore, species that do not overlap ecologically are just as different pheromonally as species that do. Thus, large changes in pheromone composition appear to be achieved, even in cases where other mechanisms to ensure reproductive isolation exist. We suggest that these differences are closely associated with rapid changes in host plant use, which is a characteristic feature of Bactrocera speciation. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 97 , 594–603.     

Chromosome evolution in pseudoxyrhophiine snakes from Madagascar: a wide range of karyotypic variability

In this first cytogenetic survey on the lamprophiid snake subfamily Pseudoxyrhophiinae, we studied the karyology of ten snake species belonging to seven genera from Madagascar (Compsophis, Leioheterodon, Liophidium, Lycodryas, Madagascarophis, Phisalixella and Thamnosophis) using standard and banding methods. Our results show a wide range of different karyotypes ranging from 2n = 34 to 2n = 46 elements (FN from 40 to 48), with nucleolus organizer regions (NORs) on one (plesiomorphic) or two (derived/apomorphic) microchromosome pairs, and W chromosome at early or advanced states of diversification from the Z chromosome. The observed W chromosome variations further support the most accepted hypothesis that W differentiation from the Z chromosome occurred by progressive steps. We also propose an evolutionary scenario for the observed high karyotype diversity in this group of snakes, suggesting that it is derived from a putative primitive pseudoxyrhophiine karyotype with 2n = 46, similar to that of Leioheterodon geayi, via a series of centric fusions and inversions among macrochromosomes and translocations of micro‐ either to micro‐ or to macrochromosomes. This primitive Pseudoxyrhophiinae karyotype might have derived from a putative Lamprophiidae ancestor with 2n = 48, by means of a translocation of a micro‐ to a macrochromosome. In turn, the karyotype of this lamprophiid common ancestor may have derived from the assumed primitive snake karyotype (2n = 36 chromosomes, with 16 biarmed macro‐ and 20 microchromosomes) by a series of centric fissions and one inversion. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 112 , 450–460.     

Ancestral and neo-sex chromosomes contribute to population divergence in a dioecious plant

Empirical evidence from several animal groups suggests sex chromosomes disproportionately contribute to reproductive isolation. This effect may be enhanced when sex chromosomes are associated with turnover of sex determination systems resulting from structural rearrangements to the chromosomes. We investigated these predictions in the dioecious plant Rumex hastatulus, which is composed of populations of two different sex chromosome cytotypes caused by an X-autosome fusion. Using population genomic analyses, we investigated the demographic history of R. hastatulus and explored the contributions of ancestral and neo-sex chromosomes to population genetic divergence. Our study revealed that the cytotypes represent genetically divergent populations with evidence for historical but not contemporary gene flow between them. In agreement with classical predictions, we found that the ancestral X chromosome was disproportionately divergent compared with the rest of the genome. Excess differentiation was also observed on the Y chromosome, even when we used measures of differentiation that control for differences in effective population size. Our estimates of the timing of the origin of neo-sex chromosomes in R. hastatulus are coincident with cessation of gene flow, suggesting that the chromosomal fusion event that gave rise to the origin of the XYY cytotype may have also contributed to reproductive isolation.     

Multiple-chromosome sex systems in the darkling beetles Blaps gigas and Blaps gibba (Coleoptera, Tenebrionidae)

We have studied mitotic and meiotic chromosomes in the males of two species of Blaps: B. gigas and B. gibba. Karyological characteristics such as the occurrence of a multivalent configuration at diakinesis and two types of metaphase-II spreads support the notion that multiple-chromosome sex systems involving five chromosomes in B. gigas and eight chromosomes in B. gibba have developed in these species. Results obtained by means of silver staining and C-banding techniques suggest that the complex sex systems occurring in B. gigas and B. gibba may have originated from exchanges of terminal ribosomal genes among the Y chromosome and some autosomes.     

ANOLIS SEX CHROMOSOMES ARE DERIVED FROM A SINGLE ANCESTRAL PAIR

To explain the frequency and distribution of heteromorphic sex chromosomes in the lizard genus Anolis, we compared the relative roles of sex chromosome conservation versus turnover of sex‐determining mechanisms. We used model‐based comparative methods to reconstruct karyotype evolution and the presence of heteromorphic sex chromosomes onto a newly generated Anolis phylogeny. We found that heteromorphic sex chromosomes evolved multiple times in the genus. Fluorescent in situ hybridization (FISH) of repetitive DNA showed variable rates of Y chromosome degeneration among Anolis species and identified previously undetected, homomorphic sex chromosomes in two species. We confirmed homology of sex chromosomes in the genus by performing FISH of an X‐linked bacterial artificial chromosome (BAC) and quantitative PCR of X‐linked genes in multiple Anolis species sampled across the phylogeny. Taken together, these results are consistent with long‐term conservation of sex chromosomes in the group. Our results pave the way to address additional questions related to Anolis sex chromosome evolution and describe a conceptual framework that can be used to evaluate the origins and evolution of heteromorphic sex chromosomes in other clades.