Chromosomes in the African frog genus Tomopterna (Pyxicephalidae) and probing the origin of tetraploid Tomopterna tandyi.

Speciation by polyploidization has been documented to have independently occurred in 12 families of anuran amphibians. Tomopterna tandyi was described as a South African allotetraploid species of sand frogs in the family Pyxicephalidae. Recent taxonomic revisions and new species descriptions in the genus present problems with respect to the evolution of this tetraploid species. Chromosomes, mitochondrial and nuclear gene sequences, isozymes, and male mating calls were examined for T. tandyi and for diploid species of Tomopterna. Mitochondrial sequences confirmed the diploid species, T. adiastola, to be the maternal ancestor that gave rise to the tetraploid about 5 mya. Nuclear sequences and isozymes reveal a complex reticulation of paternal ancestry that may be explained by occasional hybridization of T. tandyi with diploid species of Tompoterna at various times in sympatric populations. Interspecific diploid to tetraploid gene introgression is suspected to have also occurred in Australian and North American tetraploid species of frogs. Diploid to tetraploid introgression is facilitated through triploid hybrids that are more viable than diploid hybrids and produce unreduced triploid eggs.

Gynogenetic diploids, tetraploids, or octoploids, and a path to polyploidy in anuran amphibians.

Unreduced gametes have been implicated in the evolution of polyploid species of plants and animals and are normally produced by female anuran amphibians. Such eggs may initiate the evolution of polyploid species that have arisen independently in several anuran families. Polyploid females could also produce unreduced eggs that might lead to species with higher ploidy levels, or their eggs may develop gynogenetically to reduce the ploidy level. Diploid Hyla chrysoscelis (2n = 24) and tetraploid H. versicolor (4n = 48) are sibling cryptic species of North American grey treefrogs. Artificial crosses using H. versicolor females and genetically distant diploid males were performed to produce haploid H. versicolor and to assess the production of unreduced eggs in this tetraploid species. Gynogenetic diploid (haploid H. versicolor), allotriploid, gynogenetic tetraploid, allopentaploid, autohexaploid, and gynogenetic octoploid tadpoles were confirmed using chromosome counts from tadpole tail tip squashes. The transformation and survival of different ploidies varied. Gynogenetic diploids transformed but expressed aspects of haploid syndrome and died before or shortly after transformation.

Revisiting the evolution of the North American tetraploid treefrog (Hyla versicolor).

Hyla chrysoscelis and H. versicolor are common treefrogs in eastern North America and are a cryptic diploid-tetraploid species pair. They are morphologically identical but H. versicolor is a tetraploid. They can be identified acoustically by the male's advertisement mating call, which has a pulse repetition rate that has twice as many pulses per second in the diploid species, H. chrysoscelis. We used isozymes, microsatellite DNA alleles, and mitochondrial cytochrome b sequences to test the hypothesis that gene exchange occurs between the diploid and tetraploid species in sympatric populations. Each method provided results that are best explained by occasional hybridization of female H. versicolor and male H. chrysoscelis. We propose that H. versicolor first arose from an autotriploid H. chrysoscelis female that produced unreduced triploid eggs. After H. versicolor became established, genes could be passed from H. chrysoscelis to H. versicolor in sympatric populations when these species hybridize. Their F1 female progeny produce unreduced triploid eggs that are fertilized by haploid H. chrysoscelis sperm to reconstitute H. versicolor. Genes can be passed from diploid H. chrysoscelis to tetraploid H. versicolor in sympatric populations.

A family study to examine clonal diversity in unisexual salamanders (genus Ambystoma).

Unisexual Ambystoma are the oldest known unisexual vertebrates and comprise a lineage of eastern North American all female salamanders that reproduce by stealing sperm from as many as five normally bisexual congeneric species. The sperm may be used to only stimulate egg development by gynogenesis but can be incorporated in the zygote to elevate the ploidy level or to replace one of the female's haploid genomes. This flexible and unique reproductive system, termed kleptogenesis, is investigated using a microsatellite examination of 988 offspring from 14 unisexual mothers. All mothers produced clonal and ploidy-elevated offspring. Genome replacement and multiple paternity are confirmed for the first time in unisexual Ambystoma. Microsatellite mutations were found in all five microsatellite loci and the estimated microsatellite mutation rate varied by locus and by genome. Clonal variation is attributed to the inclusion of sperm donors' haploid genomes for ploidy elevation, genome replacement, mutations, and natural selection.

Polyploidy in Amphibia.

This review summarizes the current status of the known extant genuine polyploid anuran and urodelan species, as well as spontaneously originated and/or experimentally produced amphibian polyploids. The mechanisms by which polyploids can originate, the meiotic pairing configurations, the diploidization processes operating in polyploid genomes, the phenomenon of hybridogenesis, and the relationship between polyploidization and sex chromosome evolution are discussed. The polyploid systems in some important amphibian taxa are described in more detail.

Chromosome banding in Amphibia. XXXI. The neotropical anuran families Centrolenidae and Allophrynidae.

The mitotic chromosomes of 11 species from the anuran families Centrolenidae and Allophrynidae were analyzed by means of conventional staining, banding techniques, and in situ hybridization. The amount, location, and fluorochrome affinities of constitutive heterochromatin, the number and positions of nucleolus organizer regions, and the patterns of telomeric DNA sequences were determined for most of the species. The karyotypes were found to be highly conserved with a low diploid chromosome number of 2n = 20 and morphologically similar chromosomes. The sister group relationship between the Centrolenidae and Allophrynidae (unranked taxon Allocentroleniae) is clearly corroborated by the cytogenetic data. The existence of heteromorphic XY♂/XX♀ sex chromosomes in an initial stage of morphological differentiation was confirmed in Vitreorana antisthenesi. The genome sizes of 4 centrolenid species were determined using flow cytometry. For completeness and for comparative purposes, all previously published cytogenetic data on centrolenids are included.

Identifying parental chromosomes and genomic rearrangements in animal hybrid complexes of species with small genome size using Genomic In Situ Hybridization (GISH).

Genomic In Situ Hybridization (GISH) is a powerful tool to identify and to quantify genomic constituents in allopolyploids, and is mainly based on hybridization of highly and moderate repetitive sequences. In animals, as opposed to plants, GISH has not been widely used in part because there are technical problems in obtaining informative results. Using the allopolyploid Squalius alburnoides Steindachner, 1866 fish complex as a model system, we succeeded in overcoming methodological constraints when dealing with parental species with a small genome size. This hybridogenetic complex has biotypes with different genome compositions and ploidy levels, but parental chromosomes are small, morphologically very similar and therefore cannot be distinguished by conventional cytogenetic approaches. Specimens have a small genome (C-value1.2 pg) with a low level of highly and moderate repetitive sequences, mainly located at pericentromeric chromosome regions. Since it is well known that probe annealing depends on probe concentration and hybridization time to obtain uniform hybridization signals along the chromosome arms, we progressively increased the amount of labeled probes from 100ng up to 1µg and the incubation time from overnight up to 5 days. We also made other smaller improvements. Results showed a clear enhancement of signals with respect to previous data, allowing an accurate and reproducible assignment of the parental genomes in both diploid and triploid fish.It was thus evidenced that high probes' concentrations and long incubation time are the key to obtain, without extra image editing, uniform and reliable hybridization signals in metaphase chromosomes of animal hybrids from species with small genome size.

Time and time again: unisexual salamanders (genus Ambystoma) are the oldest unisexual vertebrates.

BACKGROUND: The age of unisexual salamanders of the genus Ambystoma is contentious. Recent and ancient evolutionary histories of unisexual Ambystoma were proposed by a few separate studies that constructed phylogenies using mitochondrial DNA markers (cytochrome b gene vs. non-coding region). In contrast to other studies showing that unisexual Ambystoma represent the most ancient unisexual vertebrates, a recent study by Robertson et al. suggests that this lineage has a very recent origin of less than 25,000 years ago. RESULTS: We re-examined the phylogenetic relationship of the unisexuals to A. barbouri from various populations using both mitochondrial markers as well as the complete mitochondrial genomes of A. barbouri and a unisexual individual from Kentucky. Lineage dating was conducted using BEAST and MultiDivTime on a complete mitochondrial genome phylogeny. Our results support a monophyletic lineage for unisexual Ambystoma that shares its most recent common ancestor with an A. barbouri lineage from western Kentucky. In contrast to the Robertson et al.'s study, no A. barbouri individual shared an identical or almost identical cytochrome b haplotype with any unisexual. Molecular dating supports an early Pliocene origin for the unisexual linage (approximately 5 million years ago). We propose that a unisexual-like cytochrome b numt (or pseudogene) exists in the controversial A. barbouri individuals from Kentucky, which was likely the cause of an erroneous phylogeny and time estimate in Robertson et al.'s study. CONCLUSION: We reject a recent origin of unisexual Ambystoma and provide strong evidence that unisexual Ambystoma are the most ancient unisexual vertebrates known to exist. The likely presence of an ancient cytochrome b numt in some Kentucky A. barbouri represents a molecular "fossil" reinforcing the hypothesis that these individuals are some of the closest extant relatives to unisexual Ambystoma.

Probing the meiotic mechanism of intergenomic exchanges by genomic in situ hybridization on lampbrush chromosomes of unisexual Ambystoma (Amphibia: Caudata).

The meiotic mechanism of unisexual salamanders in the genus Ambystoma was previously explained by observing lampbrush chromosomes (LBCs). In polyploid unisexual females, a pre-meiotic endomitotic event doubles the chromosome number so that, after meiotic reduction, the mature eggs have the same ploidy as the female. It was assumed that synapses during meiotic I prophase, which result in observed bivalents, join duplicated sister chromosomes. Previous studies also found LBC quadrivalents in some oocytes that could be explained by occasional synapses between homologs. The discovery of widespread intergenomic exchanges among unisexual populations has prompted new speculations on this meiotic mechanism. Synapses that involve homeologous chromosomes may be frequent during meiosis and could be responsible for intergenomic exchanges and the high embryonic mortality of unisexuals. Furthermore, LBC quadrivalents may be established by associations between homeologous rather than homologous chromosomes. The present study investigated these two important aspects pertaining to the mechanism of intergenomic exchanges: the frequency of homeologous synapses and the relationship between homeologous associations and meiotic quadrivalents. We applied genomic in situ hybridization (GISH) on LBCs from oocytes of 14 triploid and two tetraploid unisexual females. Homeologous bivalents were not observed, and all 13 LBC quadrivalents that we found were the result of homologous synapses and were not associated with any homeologous or exchanged LBCs. Intergenomic exchanges were used as markers to compare the same chromosomes at meiotic diplotene and mitotic metaphase stages. We conclude that contemporary intergenomic exchanges are very rare, and no direct link exists between intergenomic exchanges and high embryonic mortality. The actual mechanisms and evolutionary implications of intergenomic exchanges appear to be complicated and difficult to assess. The application of GISH-type molecular cytogenetic techniques will help to improve our understanding of the role that intergenomic interactions play in the persistence of unisexual Ambystoma and other unisexual vertebrates.

The prevalence of genome replacement in unisexual salamanders of the genus Ambystoma (Amphibia, Caudata) revealed by nuclear gene genealogy.

BACKGROUND: Unisexual salamanders of the genus Ambystoma exemplify the most ancient lineage of unisexual vertebrates and demonstrate an extremely flexible reproductive system. Unisexual Ambystoma interact with and incorporate genomes from two to four sexual species (A. laterale, A. jeffersonianum,A. texanum, and A. tigrinum), to generate more than 20 genome compositions or biotypes. Unisexual ploidy levels range from diploid to pentaploid, but all contain at least one A. laterale (L) genome. Replacement of nuclear genomes might be responsible for the evolutionary longevity of unisexual Ambystoma but direct evidence for the prevalence of genome replacement in natural populations is absent. Two major puzzling questions have remained unanswered over the last few decades: 1) is genome replacement a common reproductive method in various unisexual populations and, 2) is there an ancient "L" genome that persists in various unisexual genome compositions. RESULTS: We examined 194 unisexual and 89 A. laterale specimens from 97 localities throughout their range and constructed a genealogy of the "L" genomes using a nuclear DNA marker (L-G1C12) to answer the above questions. Six L-G1C12 haplotypes (A-F) were shared by individuals in various A. laterale and unisexual populations. The general geographical distribution of the haplotypes in unisexual populations conformed to those found in A. laterale, indicating that "L" genomes in unisexuals are obtained from sympatric or nearby populations of A. laterale. CONCLUSION: Our data demonstrate that genome replacement frequently occurs in unisexual Ambystoma across their range, and support previous speculations that genome replacement is an important reproductive mechanism that can enhance their evolutionary longevity. Our results show that there is no ancient "L" genome in the unisexual lineages, and no particular "L" genome is favored in any unisexual individual. The presence of an "L" genome in all unisexuals implies that it is important to the maintenance of unisexuals. Nuclear gene genealogy is a powerful tool to examine the historical interaction between sperm-dependent unisexuals and their sexual sperm donors. This methodology could be applicable to many other unisexual lineages to improve our understanding of their reproduction and their ability to persist.

Genealogical relationships of southern Ontario polyploid unisexual salamanders (genus Ambystoma) inferred from intergenomic exchanges and major rDNA cytotypes.

North American unisexual salamanders in the genus Ambystoma are common around the Great Lakes region of North America. They contain an almost identical mitochondrial genome across their distribution that is unlike that of any of the four species whose genomes may be included in their nuclei. Thus, sequence-based phylogenies of unisexual populations are confusing. We used chromosomal intergenomic exchanges and major rDNA cytotypes as combined cytogenetic markers to tentatively construct a genealogy of unisexual Ambystoma in southern Ontario. We employed GISH and sequential/simultaneous GISH/FISH-rDNA to reveal intergenomic exchanges and rDNA cytotypes in unisexual A. laterale--2 jeffersonianum (LJJ) triploids and their tetraploid derivative A. laterale--3 jeffersonianum (LJJJ). We identified 10 different patterns of intergenomic exchanges from 18 isolated populations and used them as primary cytogenetic markers. Major rDNA cytotypes served as independent and supplementary markers. Our results suggest that current LJJ and LJJJ populations in southern Ontario are likely derived from a few unisexual individuals. Intergenomic exchanges are common phenomena and widely distributed in the salamanders of the A. laterale--A. jeffersonianum unisexual complex. Integration of GISH and FISH can exhibit multiple unrelated chromosomal markers on the same chromosome spread and demonstrate lineage relationships in unisexual populations. Similar methods may be applied for studying the molecular cytogenetics of other unisexuals to improve our understanding of their genealogical relationships and historical dispersal.

Unisexual salamanders (genus Ambystoma) present a new reproductive mode for eukaryotes.

To persist, unisexual and asexual eukaryotes must have reproductive modes that circumvent normal bisexual reproduction. Parthenogenesis, gynogenesis, and hybridogenesis are the modes that have generally been ascribed to various unisexuals. Unisexual Ambystoma are abundant around the Great Lakes region of North America, and have variously been described as having all 3 reproductive modes. Diploid and polyploid unisexuals have nuclear genomes that combine the haploid genomes of 2 to 4 distinct sexual species, but the mtDNA is unlike any of those 4 species and is similar to another species, Ambystoma barbouri. To obtain better resolution of the reproductive mode used by unisexual Ambystoma and to explore the relationship of A. barbouri to the unisexuals, we sequenced the mitochondrial control and highly variable intergenic spacer region of 48 ambystomatids, which included 28 unisexuals, representatives of the 4 sexual species and A. barbouri. The unisexuals have similar sequences over most of their range, and form a close sister group to A. barbouri, with an estimated time of divergence of 2.4-3.9 million years ago. Individuals from the Lake Erie Islands (Kelleys, Pelee, North Bass) have a haplotype that demonstrates an isolation event. We examined highly variable microsatellite loci, and found that the genetic makeup of the unisexuals is highly variable and that unisexual individuals share microsatellite alleles with sexual individuals within populations. Although many progeny from the same female had the same genotype for 5 microsatellite DNA loci, there was no indication that any particular genome is consistently inherited in a clonal fashion in a population. The reproductive mode used by unisexual Ambystoma appears to be unique; we suggest kleptogenesis as a new unisexual reproductive mode that is used by these salamanders.

An unexpected recent ancestor of unisexual Ambystoma.

Previous research has shown that members of the unisexual hybrid complex of the genus Ambystoma possess a mitochondrial genome that is unrelated to their nuclear parental species, but the origin of this mitochondrion has remained unclear. We used a 744-bp fragment of the mitochondrial gene cytochrome b within a comparative phylogenetic framework to infer the maternal ancestor of this unisexual lineage. By examining a broader range of species than has previously been compared, we were able to uncover a recent maternal ancestor to this complex. Unexpectedly, Ambystoma barbouri, a species whose nuclear DNA has not been identified in the unisexuals, was found to be the recent maternal ancestor of the individuals examined through the discovery of a shared mtDNA haplotype between the unisexuals and A. barbouri. Based on a combination of sequence data and glacial patterning, we estimate that the unisexual lineage probably originated less than 25 000 years ago. In addition, all unisexuals examined showed extremely similar mtDNA sequences and the resultant phylogeny was consistent with a single origin for this lineage. These results confirm previous suggestions that the unisexual Ambystoma complex was formed from a hybridization event in which the nuclear DNA of the original maternal species was subsequently lost.

Multi-character perspectives on the evolution of intraspecific differentiation in a neotropical hylid frog.

BACKGROUND: Multi-character empirical studies are important contributions to our understanding of the process of speciation. The relatively conserved morphology of, and importance of the mate recognition system in anurans, combined with phylogenetic tools, provide an opportunity to address predictions about the relative role of each in the process of speciation. We examine the relationship among patterns of variation in morphology, call characters, and 16S gene sequences across seven populations of a neotropical hylid frog, Hyla leucophyllata, to infer their relative importance in predicting the early stages of population differentiation. RESULTS: Multivariate analyses demonstrate that both morphological and call characteristics were significantly variable among populations, characterized by significantly lower intra-population dispersion in call space than morphological space, and significantly greater among-population variation in call structure. We found lack of concordance between a 16S DNA phylogeny of Hyla leucophyllata and the significant population-level differentiation evident in both external morphology and male advertisement call. Comparisons of the reconstructed gene trees to simulated lineages support the notion that variation in call cannot be simply explained by population history. CONCLUSION: Discordance among traits may reflect sampling biases (e.g. single genetic marker effects), or imply a decoupling of evolution of different suites of characters. Diagnostic differences among populations in call structure possibly reflect local selection pressures presented by different heterospecific calling assemblages and may serve as a precursor of species-wide differentiation. Differentiation among populations in morphology may be due to ecophenotypic variation or to diversifying selection on body size directly, or on frequency attributes of calls (mediated by female choice) that show a strong relationship to body size.

A molecular phylogeny of the frog genus Tomopterna in Southern Africa: examining species boundaries with mitochondrial 12S rRNA sequence data.

Frogs of the genus Tomopterna occur throughout sub-Saharan Africa. Previous work has shown that there are seven cryptic species, which occupy diverse habitats from grasslands to deserts. The current paper proposes a phylogeny of Tomopterna based on partial sequences of the mitochondrial 12S rRNA gene. A gene tree for the genus, including all seven named species and three undescribed species which were discovered during the course of this study, is presented.