Search

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. more...

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 F1female 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. more...

Understanding population demography and dispersal of species at risk is integral for evaluating population viability, identifying causes of decline, and assessing the effectiveness of recovery actions. In pond-breeding amphibians, juvenile survival and dispersal are key components linked to population and metapopulation stability but little is known about this life stage. We use mark-recapture methods to estimate juvenile daily apparent survival, dispersal distance, and initial dispersal orientation during summer and fall dispersal of endangered Ambystoma jeffersonianum and their unisexual dependents (Ambystoma laterale – jeffersonianum). Over four years (2015-2018), 1018 juveniles (612 bisexual, 406 unisexual) were marked and 192 (19%) were recaptured at least once. Total captures varied widely between years, with the highest number of captures (88% of all individuals) occurring in 2017. Cormack-Jolly-Seber estimates of daily apparent survival were low in all years (0.76-0.95) but was higher for unisexuals than bisexuals. The majority of juveniles (71%) did not disperse further than 10-40 m after which movement appeared to cease. While most juveniles remained close to their natal pond, at least 2% of juveniles in 2017 travelled further than 100 m. Dispersal orientation varied by year and there was no difference in either dispersal orientation or distance between bisexual and unisexual individuals. This work is the first to estimate and compare juvenile survival and dispersal of sympatric A. jeffersonianum and A. laterale – jeffersonianum individuals, the results of which help inform population viability assessment and increase our understanding of juvenile dispersal dynamics and habitat use. more...

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. more...

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. more...

The behavioral phenotypes of hybrids vary in degree of similarity to their parent species. Unisexual salamanders (Ambystoma laterale sp.), the result of ancient hybridization, contain nuclear DNA of multiple sperm-host species whose habitat preferences differ from one another. We radio tracked unisexual salamanders from four vernal pools to quantify migration distances and post-breeding habitat selection and compared these to published accounts for Blue-Spotted Salamanders (A. laterale) and Jefferson Salamanders (Ambystoma jeffersonianum). Unisexual salamanders used sites with higher numbers of small mammal burrows, lower substrate temperatures, and lower cover by forest floor vegetation than available sites, similar to the sperm-hosts. Unisexual salamanders also migrated distances within the range reported for these sperm-hosts. Even so, individual migration distances were context specific. We implore managers to use caution when designating management zones around breeding pools by considering ... more...

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. more...

Polyploid animals have independently evolved from diploids in diverse taxa across the tree of life. We review a few polyploid animal species or biotypes where recently developed molecular and cytogenetic methods have significantly improved our understanding of their genetics, reproduction and evolution. Mitochondrial sequences that target the maternal ancestor of a polyploid show that polyploids may have single (e.g. unisexual salamanders in the genus Ambystoma) or multiple (e.g. parthenogenetic polyploid lizards in the genus Aspidoscelis) origins. Microsatellites are nuclear markers that can be used to analyze genetic recombinations, reproductive modes (e.g. Ambystoma) and recombination events (e.g. polyploid frogs such as Pelophylax esculentus). Hom(e)ologous chromosomes and rare intergenomic exchanges in allopolyploids have been distinguished by applying genome-specific fluorescent probes to chromosome spreads. Polyploids arise, and are maintained, through perturbations of the ‘normal' meiotic program that would include pre-meiotic chromosome replication and genomic integrity of homologs. When possible, asexual, unisexual and bisexual polyploid species or biotypes interact with diploid relatives, and genes are passed from diploid to polyploid gene pools, which increase genetic diversity and ultimately evolutionary flexibility in the polyploid. When diploid relatives do not exist, polyploids can interact with another polyploid (e.g. species of African Clawed Frogs in the genus Xenopus). Some polyploid fish (e.g. salmonids) and frogs (Xenopus) represent independent lineages whose ancestors experienced whole genome duplication events. Some tetraploid frogs (P. esculentus) and fish (Squaliusalburnoides) may be in the process of becoming independent species, but diploid and triploid forms of these ‘species' continue to genetically interact with the comparatively few tetraploid populations. Genetic and genomic interaction between polyploids and diploids is a complex and dynamic process that likely plays a crucial role for the evolution and persistence of polyploid animals. See also other articles in this themed issue. more...

The chromosomal localization of 45S ribosomal RNA genes in Ambystoma jeffersonianum was determined by fluorescence in situ hybridization with 18S rDNA fragment as a probe (FISH-rDNA). Our results revealed the presence of rDNA polymorphism among A. jeffersonianum populations in terms of number, location and FISH signal intensity on the chromosomes. Nine rDNA cytotypes were found in ten geographically isolated populations and most of them contained derivative rDNA sites. Our preliminary study provides strong indication of karyotypic diversification of A. Jeffersonianum that is demonstrated by intraspecific variation of 45S rDNA cytotypes. rDNA cytotype polymorphism has been described in many other caudate amphibians. We predict that habitat isolation, low dispersal ability and decline of effective population size could facilitate the fixation and accumulation of variable rDNA cytotypes during their chromosome evolution. more...

The evolutionary longevity of unisexual salamanders in the genus Ambystoma may be attributed to their flexible reproductive system and meiotic intergenomic interactions. More than 20 different unisexual genomic combinations have been found and all the unisexuals live with at least one of the sexual species A. laterale, A. jeffersonianum, A. texanum, and A. tigrinum. Most unisexuals rely on A. laterale orA. jeffersonianum as sperm donors. Intergenomic exchanges were previously reported in A. jeffersonianum-dependent unisexual populations from southern Ontario and are believed to be an important meiotic mechanism that provides genetic diversity. The situations of intergenomic exchanges in many of A. laterale-dependent unisexual populations, however, remain unknown. In this study we collected specimens from populations where unisexuals use A. laterale as sperm donors, including mainly triploid A. 2 laterale – jeffersonianum (or LLJ), and employed genomic in situ hybridization (GISH) to examine the intergenomic exchanges. Five patterns of intergenomic exchanges were detected. Intergenomic exchanges are less frequent and lack association among populations in A. laterale-dependent than in A. jeffersonianum- dependent unisexual populations, but more recombined homeologues were observed in LLJ unisexuals. Our observations show that the patterns and frequencies of intergenomic exchanges are different when unisexuals use different sexual species as sperm donors. We propose a few possible mechanisms that may account for these different observations. more...

We report two types of aneutriploids in unisexual salamanders Ambystomalaterale-2jeffersonianum (LJJ) and Ambystoma 2 laterale-jeffersonianum (LLJ). One karyotype has 3n = 42: L27 (L8–); J15 (J8p+), and we suggest that it was induced by homoeologous pairing after premeiotic endomitosis followed by an unequal L8;J8 segregation. The second karyotype has 3n = 43: L14 (L10q); J29 (J12+), which can be explained by meiotic nondisjunction followed by unbalanced segregation. These two rare aneutriploids demonstrate two different types of meiotic errors that might help to explain the high mortality observed in this complex. Case one also indicates that contemporary intergenomic exchanges and homoeologous recombinations may occur after a premeiotic chromosome doubling event. Our study provides additional evidence for the extremely flexible reproduction of unisexual Ambystoma. more...

Intergenomic interactions that include homoeologous recombinations and intergenomic translocations are commonly observed in plant allopolyploids. Homoeologous recombinations have recently been documented in unisexual salamanders in the genus Ambystoma and revealed exchanged chromosomal segments between A. laterale and A.jeffersonianum genomes in individual unisexuals. We discovered intergenomic translocations in two widespread unisexual triploids A.laterale – 2 jeffersonianum (or LJJ) and its tetraploid derivative A.laterale – 3 jeffersonianum (or LJJJ) by genomic in situ hybridization (GISH). Two different types of intergenomic translocations were observed in two unisexual populations and one contained novel chromosomes generated by an intergenomic reciprocal translocation. We also observed chromosome deletions in several individuals and these chromosome fragmentations were all derived from the A. jeffersonianum genome. These observed intergenomic reciprocal translocations are believed to be caused by non-homologous pairing during meiosis followed by breakage-rejoining events. Genomes of unisexual Ambystoma undergo complicated structural changes that include various intergenomic exchanges that offer unisexuals genetic and phenotypic complexity to escape their evolutionary demise. Unisexual Ambystoma have persisted as natural nuclear genomic hybrids for about four million years. These unisexuals provide a vertebrate model system to examine the interaction of distinct genomes and to evaluate the corresponding genetic, developmental and evolutionary implications of intergenomic exchanges. Intergenomic translocations and homoeologous recombinations appear to be frequent chromosome reconstruction events among unisexual Ambystoma. more...

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. more...

Unisexual salamanders in the genus Ambystoma live in obligate sympatry with the sexual species Ambystoma laterale or Ambystoma jeffersonianum, from which they are not easily distinguished. Because the sexual species are protected in many parts of their range, accurate identification of sexual and unisexual individuals is required for conservation purposes. Unisexual individuals are currently identified using isozyme electrophoresis, which requires sacrificing the animal. Here we present a nonlethal method of identification for members of the A. laterale–jeffersonianum part of the Ambystoma complex utilizing genome-specific microsatellites with or without the addition of flow cytometry. more...

The most recent estimate of the number of extantamphibian species is 4800 (Glaw & Kohler, 1998) withapproximately 90% of these being anurans (frogs), thevast majority of which are the so-called ‘advanced’or neobatrachian frogs (Ruvinsky & Maxson, 1996). Neobatrachia includes all living hyloids (i.e. bufonids,centrolenids, hylids, leptodactylids, myobatrachids,pseudids, sooglosids) and ranoids (i.e. ranids, mantel-lids, microhylids, hyperolids). The designation of thesuborder Neobatrachia, originally based on morpho-logical evidence (Reig, 1958), is supported by recentmolecular work (Hedges & Maxson, 1993; Hillis more...

We used 16S rRNA, 12S rRNA, and cytochrome-b sequence to investigate the history of the "30-chromosome" Hyla, a diverse assemblage of neotropical treefrogs. Three aspects of these frogs were examined: (1) phylogenetic relationships among constituent species groups, among the species of one of these groups (Hyla leucophyllata group), and among populations of Hyla leucophyllata; (2) the apparent age of cladogenetic events; and (3) the phylogeography of H. leucophyllata. Mixed success in resolving the phylogeny is not because of a lack of character variation; levels of genetic divergence are high and suggest pre-Pleistocene diversification, even among populations. Close temporal proximity of ancient cladogenetic events might make resolution of the topology difficult using any character set. At the population level, current geographic proximity is a poor predictor of phylogenetic affinity. A long history of dispersal and colonization may complicate, or even preclude, the accurate recovery of the history of this species in the Amazon Basin. It remains to be seen whether the patterns found here will prove common among neotropical frogs. more...

Premetamorphic northern leopard frogs (Rana pipiens) and green frogs (Rana clamitans) were evaluated at seven wetland sites, four of which were within apple orchards, to determine if environmental conditions associated with orchard management in southern Ontario, Canada, affected frog early development. Synchronous with breeding events (May–July), embryos and tadpoles were exposed, in situ and in the laboratory, to study site pond water for 2 to 3 weeks. Six pesticides commonly applied in apple orchards (Guthion® 50WP, Imidan® 50WP, Thiodan® 50WP, Dithane® DG, Nova® 40W, and Basudin® 500EC) and technical grade diazinon were also evaluated for direct toxicity to green frogs using continuous and discontinuous toxicity tests. Embryos and larvae exhibited poor hatching success and survival at some orchard sites during in situ assays, but no specific water source produced consistently poor developmental success. Reduced tadpole growth occurred at several study sites, showing no clear distinction between reference and orchard sites. Despite the collection of substantial environmental data, only surface water temperature appeared to be correlated with growth rates. Basudin 500EC, technical grade diazinon, and Dithane DG caused mortality, deformities, or growth inhibition during early development at environmentally relevant concentrations ( more...

Northern leopard frogs (Rana pipiens) and green frogs (Rana clamitans) were evaluated at eight wetland sites, four of which were within apple orchards, to determine if environmental changes associated with orchard management affected measured biological parameters. Size, age, genetic variation, condition indices, levels of circulating steroid hormones, 7-ethoxyresorufin-O-deethylase activity (EROD), and organochlorine and organophosphorus residues in breeding males sampled at pond sites in orchards were compared to the same parameters measured in breeding males from reference sites. Also, the size and physiological condition of young-of-the-year captured in orchard and reference ponds were compared. No evidence of a reduction in genetic variation was found in populations of either species at any sites, but unexpectedly high average heterozygosity values (0.191–0.282) in concert with low overall fixation indices (0.012–0.059) in adults of both species did suggest that pond populations were interacting with neighboring populations in nonorchard habitats. Few significant differences in levels of circulating steroid hormones or condition indices of breeding males were found among sites. Significant EROD induction in male green frogs collected from one orchard site during one sampling event was the only indication that a metabolic challenge due to presence of cytochrome P450-inducing toxicants may have existed, whereas elevated concentrations of organochlorines (dichlorodiphenyltrichloroethane [DDT]- or endosulfan-related) in green frog tissues suggested that frogs at three orchard sites were taking up pesticides. Significant differences in size of equivalent-age male and juvenile leopard frogs and green frogs occupying different study sites suggested that suboptimal habitat characteristics existed at one or two of the four orchard sites. However, site-specific habitat deficiencies could not be related to orchard study sites in general, and, thus, wetlands in apple orchards appeared to provide viable breeding habitat for both northern leopard frogs and green frogs. more...

Patterns and levels of allozyme variation among populations of Amazonian frogs were used to test the riverine barrier hypothesis of species differentiation. Two frog species were sampled from each of the two main forest habitats on both banks of the Jurua' River in the southwestern Brazilian Amazon Basin at various points along its course to contrast different barrier strengths. Scarthyla ostinodactyla and Scinax rubra were sampled from flooded forest (varzea), and Physalaemus petersi and Epipedobates femoralis from non-flooded forest (terra firme). All species showed high levels of within-population genetic variation. Average Nei's (1978) and Rogers' (1972) genetic distances between sampled sites for all species were high indicating substantial among-population differentiation. The observation of low gene flow between sampled sites within species was further substantiated with Slatkin's (1993) A([ analyses. Randomization tests suggested that there was some population structure at a few assayed polymorphic loci that was consistent with the riverine barrier hypothesis. However, it was apparent from the raw allozyme frequency data that these results were largely driven by substantial differentiation at one or a few collecting localities rather than by basin-wide patterns of riverine differentiation. Phenograms using genetic distance matrices supported this interpretation. Patterns of geographic variation are probably more consistent with the idea of this region being a zone of secondary contact. more...

This paper presents the results of a bioclimatic and spatial analysis of reptiles and amphibians native to Ontario. Using a large database of known observations and a new spatially explicit climate model of the entire province, climatic domains of 43 species and subspecies were derived and subsequently mapped. A useful aspect of the analysis is that it not only explicitly summarizes what is currently known about species occurrences, it also identifies those parts of the province which have a similar climate. This will assist in designing field survey and monitoring programs and ecological restoration activities. By overlaying each of the 43 maps in a Geographic Information System, regions of the province with potentially large species gradients were identified. These areas are currently the focus of ongoing field studies. more...

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. more...

A new tetraploid species of sand frog, genus Tomopterna, is described from the eastern Cape. On the basis of mtDNA sequences, allozymes and advertisement calls, it appears to have arisen as a hybrid between the adjacent diploid populations of T. delalandii and T. cryptotis. The hybridization event is estimated to have occurred 1,5 MVA. Excluding Xenopus, this is the only allotetraploid member of an advanced frog family that is known. more...

Chromosomes from all 17 species of native Jamaican Eleutherodactylus as well as introduced E. johnstonei were subjected to computer-assisted analyses. Diploid chromosome numbers of 24, 26, 28, 30 and 32 were found and no two species had identical karyotypes. Karyotypic data were superimposed on a phylogeny derived from allozyme and immunological data in order to assess karyotypic changes that occurred in lineages of Jamaican Eleutherodactylus. Chromosome number changes have occurred at least nine times on the island and have involved both fission and fusion mutational events. C-bands and the sites of secondary constrictions varied and provide very little phylogenetic information. In most instances, karyotypically determined interspecific evolutionary relationships corresponded with the molecular data. The combination of karyological analyses and molecular data clarified lineages which involved convergent chromosome numbers or extremely divergent karyotypes. Karyotypic changes in Jamaican Eleutherodactylus are best explained by chromosome fission, fusion, translocations and inversions which arose in isolated demes and have been fixed through inbreeding and genetic drift. Rates of karyotypic evolution among Jamaican Eleutherodactylus are much faster than previous published rates for frogs. Karyotypic evolution appears to be dictated by behavioural factors and effective population sizes irrespective of taxonomic groupings. more...

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. more...

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 (~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. more...

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. more...

Polyploid hybrid salamanders of the genus Ambystoma tend to produce unreduced eggs. At higher temperatures (15°C), the sperm often contributes genetically, leading to tetraploid offspring from triploid mothers and pentaploid offspring from tetraploid mothers. At lower temperatures (5°C), the sperm nucleus usually is lost, and the eggs develop gynogenetically. We examined the cytology of fertilization at 5°C in order to determine the nature of the temperature-dependent nuclear loss. As in other urodeles, fertilization is polyspermic. The sperm nuclei decondense to form pronuclei, and one sperm pronucleus associates with the egg pronucleus. At metaphase, a clump of chromatin appears along with the chromosomes at the metaphase plate. This chromatin clump fails to segregate at anaphase and is left at the site of the cleavage furrow. The chromatin clump is likely the discarded sperm pronucleus, so its loss is due to its failure to condense properly into chromosomes. © 1992 Wiley-Liss, Inc. more...

Although bisexual reproduction has considerable evolutionary benefits, several all-female vertebrates exist. Unisexual salamanders in the genus Ambystoma are common around the Great Lakes region in eastern North America. They originated from a hybridization event that involved a female that shared a common ancestor with Ambystoma barbouri 2.4 to 3.9 million years ago but, unexpectedly, A. barbouri nuclear genomes were unknown in unisexuals. Unisexual salamanders steal sperm from donors of normally bisexual species, so their reproductive mode is described as kleptogenesis. Most known unisexuals are polyploid and they all possess at least one A. laterale genome. One or more other genomes are taken from sperm donors that may include A. jeffersonianum, A. laterale, A. texanum and A. tigrinum. We examined unisexual adults and larvae in a southern Ohio pond where unisexual individuals coexist with male A. barbouri. This population provided an opportunity to test hypotheses pertaining to the role of A. barbouri in the evolution of the disparate cytoplasmic and nuclear genomes in unisexual salamanders. Microsatellite DNA loci, mitochondrial DNA sequences and genomic in situ hybridization were used to identify the genomic constitution of individuals. A. barbouri was found to be an acceptable sperm donor for unisexuals but only contributed genomes in ploidy-elevated individuals. In the absence of A. jeffersonianum, this Ohio population is likely experiencing a recent switch in sperm donors from A. jeffersonianum to A. barbouri and demonstrates the evolutionary flexibility and dynamics of kleptogenesis. more...

Enzyme electrophoresis of three species of Pleurodema (38 specimens) collected from six sites in northwestern Argentina resolved 44 allozymes for 15 loci. Pleurodema cinerea and P. borellii, which are morphologically similar, are believed to be reproductively isolated by habitat differences. Although the genetic distance was smallest between the two P. borellii populations, P. borellii was not genetically distant from P. cinerea (average values for Nei's and Rogers' intersite genetic distance indices (D) were 0.155 and 0.229, respectively). Pleurodema tucumana was genetically distant from both P. cinerea and P. borellii (average Nei's D = 1.918; average Rogers' D = 0.799). A comparison of mating calls also suggested that P. cinerea and P. borellii are very similar to each other. The calls of P. borellii and P. cinerea differed only in average pulse rate (100.4 and 59.9 pulses per second, respectively) but this difference may have been temperature related. No conclusive evidence was found in this study to indicate that P. cinerea and P. borellii are distinct species. more...

Calls produced by hybrids resulting from laboratory crosses of tetraploid Hyla versicolor females and either diploid Hyla chrysoscelis (type I) or Hyla arborea (type II) males were induced through manipulation of environmental conditions. Type I hybrids produced trilled calls similar in note repetition to H. versicolor, but more similar in dominant frequency to H. chrysoscelis. Mean duration was shorter than in both parent calls. Type II hybrids produced calls which were longer in duration and lower in note repetition rate than H. versicolor, but shorter in duration and higher in note repetition rate than H. arborea. Dominant frequency of type II hybrids was lower than in H. arborea but not significantly different than in H. versicolor. Hybrid calls were not strictly intermediate, and may provide information regarding parental relationships. more...

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. more...

Four salamander species of the genus Ambystoma hybridize in the Great Lakes region of eastern North America. The hybrids are mostly polyploid and virtually all-female. Basic chromosomal morphology and C-banding patterns of Ambystoma laterale, A. jeffersonianum, A. texanum, and A. tigrinum tigrinum were examined in an attempt to find some markers that would be useful to recognize genomic constitution of the hybrids. Several minor morphological differences were found among the karyotypes of the four species, but none were of sufficient magnitude to unambiguously assign genomic content in a hybrid. There was no evidence of sexually dimorphic bands in any of the species.Key words: chromosomes, Ambystoma, C-bands, hybridization, amphibia. more...

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. more...

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. more...

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. more...

Rivers have been suggested to have played an important role in shaping present-day patterns of ecological and genetic variation among Amazonian species and communities. Recent molecular studies have provided mixed support for the hypothesis that large lowland Amazonian rivers have functioned as significant impediments to gene flow among populations of neotropical species. To date, no study has systematically evaluated the impact that riverine barriers might have on structuring whole Amazonian communities. Our analyses of the phylogeography of frogs and small mammals indicate that a putative riverine barrier (the Juruá River) does not relate to present-day patterns of community similarity and species richness. Rather, our results imply a significant impact of the Andean orogenic axis and associated thrust-and-fold lowland dynamics in shaping patterns of biotic diversity along the Juruá. Combined results of this and other studies significantly weaken the postulated role of rivers as major drivers of Amazonian diversification. more...

Mitochondrial DNA cytochrome b sequence data from a dart–poison frog, Epipedobates femoralis , were used to test two hypotheses of Amazonian diversification: the riverine barrier and the ridge hypotheses. Samples were derived from sites located on both banks of the Rio Jurua and on both sides of the Iquitos Arch in western Amazonia. The phylogeographic structure was inconsistent with predictions of the riverine barrier hypothesis. Haplotypes from opposite river banks did not form monophyletic clades in any of our phylogenetic analyses, nor was the topology within major clades consistent with the riverine hypothesis. Further, the greatest differentiation between paired sites on opposite banks was not at the river mouth where the strongest barrier to gene flow was predicted to occur. The results instead were consistent with the hypothesis that ancient ridges (arches), no longer evident on the landscape, have shaped the phylogeographic relationships of Amazonian taxa. Two robustly supported clades map onto opposite sides of the Iquitos Arch. The mean haplotypic divergence between the two clades, in excess of 12%, suggests that this cladogenic event dates to between five and 15 million years ago. These estimates span a period of major orogenesis in western South America and presumably the formation of these ancient ridges. more...