Evolution of parthenogenetic reproduction in Caucasian rock lizards: A review.

Despite numerous works devoted to hybrid origin of parthenogenesis in reptiles, the causes of hybridization between different species, resulting in the origin of parthenogenetic forms, remain uncertain. Recent studies demonstrate that sexual species considered parental to parthenogenetic rock lizards (Darevskia spp.) avoid interspecific mating in the secondary overlap areas. A specific combination of environmental factors during last glaciation period was critical for ectotherms, which led to a change in their distribution and sex ratio. Biased population structure (e.g., male bias) and limited available distributional range favored the deviation of reproductive behavior when species switched to interspecific mates. To date, at least 7 diploid parthenogenetic species of rock lizards (Darevskia, Lacertidae) originated through interspecific hybridization in the past. The cytogenetic specifics of meiosis, in particular the weak checkpoints of prophase I, may have allowed the formation of hybrid karyotypes in rock lizards. Hybridization and polyploidization are 2 important evolutionary forces in the genus Darevskia. At present, throughout backcrossing between parthenogenetic and parental species, the triploid and tetraploid hybrid individuals appear annually, but no triploid species found among Darevskia spp. on current stage of evolution. The speciation by hybridization with the long-term stage of diploid parthenogenetic species, non-distorted meiosis, together with the high ecological plasticity of Caucasian rock lizards provide us with a new model for considering the pathways and persistence of the evolution of parthenogenesis in vertebrates.

XX/XY sex chromosomes in a blind lizard (Dibamidae): Towards understanding the evolution of sex determination in squamates.

The reconstruction of the evolutionary history of sex determination in squamate reptiles (lizards and snakes) is complicated by missing data in many lineages, erroneous reports, and often questionable inferences on state homology. Therefore, despite the large effort, the reconstruction of the ancestral sex determination in squamate reptiles is still controversial. With the hope to shed light on this problem, we aspired to identify the sex chromosome gene content in Dibamus deharvengi, the representative of the family Dibamidae, the putative sister clade to all other squamates. Our analyses revealed XX/XY sex-determination system in D. deharvengi: the X chromosome contains genes with homologues scattered across chicken chromosomes 8, 12, 13, 18, 30, and 33, and the Y chromosome seems to largely degenerate. To the best of our knowledge, this combination has never been reported to form sex chromosomes in any amniote lineage. It suggests that the sex chromosomes can represent an apomorphy of a clade including D. deharvengi. Our findings cover an important gap in the knowledge of sex determination in reptiles and further support multiple independent origins of sex chromosomes in this group.

Meiotic synapsis of homeologous chromosomes and mismatch repair protein detection in the parthenogenetic rock lizard Darevskia unisexualis.

Parthenogenetic species of Caucasian rock lizards of the genus Darevksia are important evidence for reticulate evolution and speciation by hybridization in vertebrates. Female-only lineages formed through interspecific hybridization have been discovered in many groups. Nevertheless, critical mechanisms of oogenesis and specifics of meiosis that provide long-term stability of parthenogenetic species are still unknown. Here we report cytogenetic characteristics of somatic karyotypes and meiotic prophase I nuclei in the diploid parthenogenetic species Darevskia unisexualis from the new population "Keti" in Armenia which contains an odd number of chromosomes 2n = 37, instead of the usual 2n = 38. We revealed 36 acrocentric chromosomes and a single metacentric autosomal chromosome, resulting from Robertsonian translocation. Comparative genomic hybridization revealed that chromosome fusion occurred between two chromosomes inherited from the maternal species, similar to another parthenogenetic species D. rostombekowi. To trace the chromosome behaviour in meiosis, we performed an immunocytochemical study of primary oocytes' spread nuclei and studied chromosome synapsis during meiotic prophase I in D. unisexualis based on analysis of synaptonemal complexes (SCs). We found meiotic SC-trivalent composed of one metacentric and two acrocentric chromosomes. We confirmed that the SC was assembled between homeologous chromosomes inherited from two parental species. Immunostaining of the pachytene and diplotene nuclei revealed a mismatch repair protein MLH1 loaded to all autosomal SC bivalents. Possible mechanisms of meiotic recombination between homeologous chromosomes are discussed.

Evolution of the parthenogenetic rock lizard hybrid karyotype: Robertsonian translocation between two maternal chromosomes in Darevskia rostombekowi.

Darevskia rostombekowi, the most outstanding of the seven known parthenogenetic species in the genus Darevskia, is the result of an ancestral cross between two bisexual species Darevskia raddei and Darevskia portschinskii. The chromosomal set of this species includes a unique submetacentric autosomal chromosome; the origin of this chromosome was unresolved as only acrocentric chromosomes are described in the karyotypes of Darevskia genus normally. Here, we applied a suite of molecular cytogenetic techniques, including the mapping of telomeric (TTAGGG) n repeats using fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and whole-chromosome painting (WCP) in both D. rostombekowi and parental (D. portschinskii and D. raddei) species. The obtained results in total suggest that a de novo chromosomal rearrangement via Robertsonian translocation (centric fusion) between two maternal (D. raddei) acrocentric chromosomes of different size was involved in the formation of this unique submetacentric chromosome present in the parthenogenetic species D. rostombekowi. Our findings provide new data in specific and rapid evolutional processes of a unisexual reptile species karyotype.

Genotypic similarities among the parthenogenetic Darevskia rock lizards with different hybrid origins.

BACKGROUND: The majority of parthenogenetic vertebrates derive from hybridization between sexually reproducing species, but the exact number of hybridization events ancestral to currently extant clonal lineages is difficult to determine. Usually, we do not know whether the parental species are able to contribute their genes to the parthenogenetic vertebrate lineages after the initial hybridization. In this paper, we address the hypothesis, whether some genotypes of seven phenotypically distinct parthenogenetic rock lizards (genus Darevskia) could have resulted from back-crosses of parthenogens with their presumed parental species. We also tried to identify, as precise as possible, the ancestral populations of all seven parthenogens. RESULTS: We analysed partial mtDNA sequences and microsatellite genotypes of all seven parthenogens and their presumed ansectral species, sampled across the entire geographic range of parthenogenesis in this group. Our results confirm the previous designation of the parental species, but further specify the maternal populations that are likely ancestral to different parthenogenetic lineages. Contrary to the expectation of independent hybrid origins of the unisexual taxa, we found that genotypes at multiple loci were shared frequently between different parthenogenetic species. The highest proportions of shared genotypes were detected between (i) D. sapphirina and D. bendimahiensis and (ii) D. dahli and D. armeniaca, and less often between other parthenogens. In case (ii), genotypes at the remaining loci were notably distinct. CONCLUSIONS: We suggest that both observations (i-ii) can be explained by two parthenogenetic forms tracing their origin to a single initial hybridization event. In case (ii), however, occasional gene exchange between the unisexual and the parental bisexual species could have taken place after the onset of parthenogenetic reproduction. Indeed, backcrossed polyploid hybrids are relatively frequent in Darevskia, although no direct evidence of recent gene flow has been previously documented. Our results further suggest that parthenogens are losing heterozygosity as a result of allelic conversion, hence their fitness is expected to decline over time as genetic diversity declines. Backcrosses with the parental species could be a rescue mechanism which might prevent this decline, and therefore increase the persistance of unisexual forms.

Cytogenetic mechanisms of unisexuality in rock lizards.

Darevskia rock lizards is a unique complex taxa, including more than thirty species, seven of which are parthenogenetic. In mixed populations of Darevskia lizards, tri- and tetraploid forms can be found. The most important issues in the theory of reticulate evolution of Darevskia lizards are the origin of parthenogenetic species and their taxonomic position. However, there is little data on how meiosis proceeds in these species. The present work reports the complex results of cytogenetics in a diploid parthenogenetic species - D. unisexualis. Here we detail the meiotic prophase I progression and the specific features оf mitotic chromosomes organization. The stages of meiosis prophase I were investigated by immunocytochemical analysis of preparations obtained from isolated primary oocytes of D. unisexualis in comparison with maternal species D. raddei nairensis. It has been shown that in D. unisexualis at the leptotene-zygotene stages the axial elements and the synaptonemal complex (SC) form typical "bouquets". At the pachytene-diplotene stage, 18 autosomal SC-bivalents and thickened asynapted sex Z and w univalents were observed. The presence of SYCP1 protein between the lateral elements of autosomal chromosomes proved the formation of assembled SCs. Comparative genomic hybridization (CGH) on the mitotic metaphase chromosomes of D. unisexualis was carried out using the genomic DNA isolated from the parental species D. raddei nairensis and D. valentini. In the pericentromeric regions of half of the mitotic chromosomes of D. unisexualis, specific regions inherited from maternal species have been found. Following our results, we suggest a model for diploid germ cells formation from diploid oocytes without premeiotic duplication of chromosomes in the oogenesis of diploid parthenogenetic lizards D. unisexualis. Taken as a whole, our findings confirm the hybrid nature of D. unisexualis and shed light on heterozygosity and automixis in diploid parthenogenetic forms.

Natural history of Valentin's rock lizard (Darevskia valentini) in Armenia.

Valentin's rock lizard (Darevskia valentini) is suggested to be the parent for several parthenogenetic species (e.g., D. armeniaca, D. bendimahiensis, D. sapphirina, and D. unisexualis) that evolved through hybridization. Complex evolutionary processes (including reticulate evolution) are occurring within the areas where Valentin's rock lizard coexists with these and other rock lizards. Hence, a detailed biological specification of this species is important for understanding how vertebrates evolve. Valentin's rock lizard is a long-lived (up to 9 years), small diurnal lizard with larger females than males, which is unlike other species of the genus. Their relatively large eggs and early reproduction period, which occurs just after emergence from winter shelters, are adaptations for living in a high elevation climate (higher than 2 000 m a.s.l.). Their body temperatures (31-32 °Ð¡) are comparable to body temperatures of rock lizards living in milder climates, though female body temperature is more dependent on substrate temperature and basking due to their lower activity than that found in males. Population density fluctuates from several individuals to several hundred per hectare and is not affected by parthenogen coexistence, although hybrids do occur in sexually biased populations where males are more common than females. The male home range is larger than that of females, though these home ranges broadly overlap. Prey is not limited in the mountain meadows and Valentin's rock lizards feed on a great variety of arthropods. Infanticide occurs in high-density populations.

Extraordinary centromeres: differences in the meiotic chromosomes of two rock lizards species Darevskia portschinskii and Darevskia raddei.

According to the synthesis of 30 years of multidisciplinary studies, parthenogenetic species of rock lizards of genus Darevskia were formed as a result of different combination patterns of interspecific hybridization of the four bisexual parental species: Darevskia raddei, D. mixta, D. valentini, and D. portschinskii. In particular, D. portschinskii and D. raddei are considered as the parental species for the parthenogenetic species D. rostombekowi. Here for the first time, we present the result of comparative immunocytochemical study of primary spermatocyte nuclei spreads from the leptotene to diplotene stages of meiotic prophase I in two species: D. portschinskii and D. raddei. We observed similar chromosome lengths for both synaptonemal complex (SC) karyotypes as well as a similar number of crossing over sites. However, unexpected differences in the number and distribution of anti-centromere antibody (ACA) foci were detected in the SC structure of bivalents of the two species. In all examined D. portschinskii spermatocyte nuclei, one immunostained centromere focus was detected per SC bivalent. In contrast, in almost every studied D. raddei nuclei we identified three to nine SCs with additional immunostained ACA foci per SC bivalent. Thus, the obtained results allow us to identify species-specific karyotype features, previously not been detected using conventional mitotic chromosome analysis. Presumably the additional centromere foci are result of epigenetic chromatin modifications. We assume that this characteristic of the D. raddei karyotype could represent useful marker for the future studies of parthenogenetic species hybrid karyotypes related to D. raddei.

The phytotelm tadpoles of Microhyla arboricola (Anura: Microhylidae) from Vietnam, with comments on reproductive biology and development.

The reproductive biology of Microhyla arboricola (Microhylidae) was studied in two regions of the southern Annamite Mountains in Vietnam. M. arboricola is an obligate phytotelm-breeder that reproduces in water-filled tree hollows in montane evergreen forests. Clutches are attached above the water level in the hollows and contain 5-37 pigmented, relatively large eggs. Larvae hatch at markedly advanced stages and develop in water until metamorphosis is completed. The developing tadpoles are obligately oophagous and feed on conspecific eggs and embryos. M. arboricola tadpoles differ from tadpoles of pond-dwelling Microhyla species in their external morphology (extremely long tails, dorsolateral position of the eyes, dark pigmentation), digestive tract morphology (large, extensible larval stomach and short intestine), and oral morphology. The larval chondrocranium and hyobranchiumof M. arboricola is described. M. arboricola shares its habitat with other hollow-breeding species of anurans. To date, M. arboricola is the only known arboreal species of the genus Microhyla that has a unique reproductive mode. The ecological niche of this species differs greatly from those occupied by other microhylids of Indochinese Peninsula.

Reticulate Evolution of the Rock Lizards: Meiotic Chromosome Dynamics and Spermatogenesis in Diploid and Triploid Males of the Genus Darevskia.

Knowing whether triploid hybrids resulting from natural hybridization of parthenogenetic and bisexual species are fertile is crucial for understanding the mechanisms of reticulate evolution in rock lizards. Here, using males of the bisexual diploid rock lizard species Darevskia raddei nairensis and Darevskia valentini and a triploid hybrid male Darevskia unisexualis × Darevskia valentini, we performed karyotyping and comparative immunocytochemistry of chromosome synapsis and investigated the distribution of RAD51 and MLH1 foci in spread spermatocyte nuclei in meiotic prophase I. Three chromosome sets were found to occur in cell nuclei in the D. unisexualis × D. valentini hybrid, two originating from a parthenogenetic D. unisexualis female and one from the D. valentini male. Despite this distorted chromosome synapsis and incomplete double-strand breaks repair in meiotic prophase I, the number of mismatch repair foci in the triploid hybrid was enough to pass through both meiotic divisions. The defects in synapsis and repair did not arrest meiosis or spermatogenesis. Numerous abnormal mature spermatids were observed in the testes of the studied hybrid.

A new species of Leptolalax (Anura: Megophryidae) from the western Langbian Plateau, southern Vietnam.

We describe a new species of megophryid frog from Loc Bac forest in the western part of the Langbian Plateau in the southern Annamite Mountains, Vietnam. Leptolalax pyrrhops sp. nov. is distinguished from its congeners by a combination of the following morphological attributes: (1) presence of distinct dark brown/black dorsolateral markings, including blackish spots on flanks and dark canthal and/or temporal streaks; (2) rudimentary webbing on toes; (3) tympanum externally distinct; (4) dorsal skin finely shagreened with numerous small tubercles and pustules; (5) medium size for the genus (30.3-33.9 mm in 2 adult males, 30.8-34.3 mm in 7 females); (6) grey-pinkish to dark brownish-violet chest and belly with numerous whitish speckles, also covering the lateral sides of body; (7) ventrolateral glands small, indistinct, do not form a distinct line; (8) pectoral glands comparatively small, comprising 1-3% of adult SVL; (10) iris bicolored, typically bright orange-red in upper two-thirds, fading to silvery green in lower third. The male advertisement call of the new species is also unique among those Leptolalax species for which calls are known, with a single long 'introductory' note, consisting of 5-12 pulses, followed by of 4-5 predominantly single-pulsed notes, and an average dominant frequency of 1.91-2.23 kHz. From the morphologically similar L. applebyi, L. melicus and L. bidoupensis, Leptolalax pyrrhops sp. nov. can be further distinguished by 13.5%, 13.7% and 10.3% sequence divergence at the 16S rRNA mtDNA gene. At present, the new species is known from montane evergreen forest between 800-1100 m elevation. We suggest the species should be considered as Data Deficient following IUCN's Red List categories. To date our finding represents the southernmost known record of the genus Leptolalax from Vietnam.

Two new species of Kalophrynus Tschudi, 1838 (Anura: Microhylidae) from the Annamite mountains in southern Vietnam.

We describe two new mountain-dwelling microhylid species of the genus Kalophrynus from the southern part of the Annamite Mountains in Vietnam. The two new species differ from all known congeners in morphological characters and mtDNA; phylogenetically, they form a sister clade to the large-bodied K. interlineatus (1009 bp, 16S rRNA gene, mtDNA). Both species share the following characteristics: snout pointed in dorsal and lateral views, slightly sloping in profile; tympanum distinct, smaller than eye in diameter; toe webbing moderate; outer metatarsal tubercle present; light dorsolateral line absent. Kalophrynus cryptophonus sp. nov. from Loc Bao, Lam Dong Province is a small-sized species distinguishing from its congeners by a combination of: SVL 27.9-30.4 mm in males, 23.4 mm in female; canthus rostralis indistinct; males with large sharp conical spines on the skin covering mandible margins and finely asperous nuptial pads on the dorsal surface of fingers I-III; dark ocelli in the inguinal region usually present, small, without a light border; anterior palatal dermal ridge short, restricted to medial part of palate. Kalophrynus honbaensis sp. nov. from Hon Ba, Khanh Hoa Province is a medium-sized Kalophrynus, distinguishing from its congeners by a combination of: SVL 26.7-36.8 mm in males; canthus rostralis distinct; males without distinguishable spines on the mandible margins nor the nuptial pads; dark ocelli in the inguinal region present, large, without a distinct light border, anterior palatal dermal ridge developed, parallel to posterior one. Kalophrynus cryptophonus sp. nov. reproduces in hollow bamboo stems; we describe larval morphology and bioacoustics of this species in relation to phytotelm breeding. A review of the distribution of the genus Kalophrynus in Indochina is provided.

A new species of Kukri Snake (Oligodon Fitzinger, 1826; Squamata: Colubridae) from the Cat Tien National Park, southern Vietnam.

We describe a new species of the genus Oligodon from the lowland forests of Cat Tien National Park, Dong Nai Province, in southern Vietnam. Oligodon cattienensis sp. nov. is distinguished from the remaining Southeast Asian kukri snakes by the combination of the following characters: medium-sized, deeply forked hemipenes without spines, 17-17-15 dorsal scale rows, nasal entire, 2 small postoculars, almost equal in size, 167-178 ventrals, 31-35 subcaudals, 24-35 + 5 large dark-edged vertebral blotches in combination with a yellow-orange or red vertebral stripe between blotches, head pattern including ocular band, temporal bands and elongated chevron, ventrals pink or whitish (reddish in juveniles) in life, some bearing a quadrangular dark blotch on each lateral side, or ventrals being entirely dark. Based on the hemipenial morphology the new species is assigned to the Oligodon cyclurus species group. A comparison table for all Indochinese Oligodon is provided.

A new species of the genus Calotes Cuvier, 1817 (Squamata: Agamidae) from southern Vietnam.

We describe a new species of the agamid genus Calotes Cuvier, 1817 from southern Vietnam, which is most similar to Calotes mystaceus Duméril & Bibron, 1837, but can be distinguished from the latter and its other congeners by genetic and morphological differences. We discuss the current distribution of the new species and its sister species C. mystaceus in Mainland Southeast Asia.