Take one step backward to move forward: Assessment of genetic diversity and population structure of captive Asian woolly-necked storks (Ciconia episcopus).

The fragmentation of habitats and hunting have impacted the Asian woolly-necked stork (Ciconia episcopus), leading to a serious risk of extinction in Thailand. Programs of active captive breeding, together with careful genetic monitoring, can play an important role in facilitating the creation of source populations with genetic variability to aid the recovery of endangered species. Here, the genetic diversity and population structure of 86 Asian woolly-necked storks from three captive breeding programs [Khao Kheow Open Zoo (KKOZ) comprising 68 individuals, Nakhon Ratchasima Zoo (NRZ) comprising 16 individuals, and Dusit Zoo (DSZ) comprising 2 individuals] were analyzed using 13 microsatellite loci, to aid effective conservation management. Inbreeding and an extremely low effective population size (Ne) were found in the KKOZ population, suggesting that deleterious genetic issues had resulted from multiple generations held in captivity. By contrast, a recent demographic bottleneck was observed in the population at NRZ, where the ratio of Ne to abundance (N) was greater than 1. Clustering analysis also showed that one subdivision of the KKOZ population shared allelic variability with the NRZ population. This suggests that genetic drift, with a possible recent and mixed origin, occurred in the initial NRZ population, indicating historical transfer between captivities. These captive stork populations require improved genetic variability and a greater population size, which could be achieved by choosing low-related individuals for future transfers to increase the adaptive potential of reintroduced populations. Forward-in-time simulations such as those described herein constitute the first step in establishing an appropriate source population using a scientifically managed perspective for an in situ and ex situ conservation program in Thailand.

Diversity of PBI-DdeI satellite DNA in snakes correlates with rapid independent evolution and different functional roles.

To better understand PBI-DdeI satellite DNA located in the centromeric region of python, molecular evolution analysis was conducted on 40 snake species. A ladder-like pattern of DNA bands with repetition of the 194-210 bp monomer was observed in 15 species using PCR. Molecular cloning was performed to obtain 97 AT-rich monomer sequences. Phylogenetic and network analyses showed three PBI-DdeI subfamilies with sequences grouped in species-specific clusters, suggesting rapid evolution. Slow evolution was found in eight species with shared PBI-DdeI sequences, suggesting recent species diversification, allowing PBI-DdeI no time to diverge, with limited homogenization and fixation processes. Quantitative real-time PCR showed large differences in copy number between Python bivittatus and other snakes, consistent with repeat scanning of whole genome sequences. Copy numbers were significantly higher in female Naja kaouthia than in males, concurring with chromosomal distribution of PBI-DdeI specifically localized to female W chromosomes. PBI-DdeI might act as an evolutionary driver with several repeats to promote W chromosome differentiation and heterochromatinization in N. kaouthia. Analysis revealed PBI-DdeI with a reduced copy number, compared to P. bivittatus, in most snakes studied, and it is possible that it subsequently dispersed and amplified on W chromosomes with different functional roles in N. kaouthia.

Dynamics of telomere length in captive Siamese cobra (Naja kaouthia) related to age and sex.

Telomeres comprise tandem repeated DNA sequences that protect the ends of chromosomes from deterioration or fusion with neighboring chromosomes, and their lengths might vary with sex and age. Here, age- and sex-related telomere lengths in male and female captive Siamese cobras (Naja kaouthia) were investigated using quantitative real-time polymerase chain reaction based on cross-sectional data. A negative correlation was shown between telomere length and body size in males but not in females. Age-related sex differences were also recorded. Juvenile female snakes have shorter telomeres relative to males at up to 5 years of age, while body size also rapidly increases during this period. This suggests that an accelerated increase in telomere length of female cobra results from sex hormone stimulation to telomerase activity, reflecting sexually dimorphic phenotypic traits. This might also result from amplification of telomeric repeats on sex chromosomes. By contrast, female Siamese cobras older than 5 years had longer telomeres than males. Diverse sex hormone levels and oxidative stress parameters between sexes may affect telomere length.

Chromosome map of the Siamese cobra: did partial synteny of sex chromosomes in the amniote represent "a hypothetical ancestral super-sex chromosome" or random distribution?

BACKGROUND: Unlike the chromosome constitution of most snakes (2n=36), the cobra karyotype shows a diploid chromosome number of 38 with a highly heterochromatic W chromosome and a large morphologically different chromosome 2. To investigate the process of sex chromosome differentiation and evolution between cobras, most snakes, and other amniotes, we constructed a chromosome map of the Siamese cobra (Naja kaouthia) with 43 bacterial artificial chromosomes (BACs) derived from the chicken and zebra finch libraries using the fluorescence in situ hybridization (FISH) technique, and compared it with those of the chicken, the zebra finch, and other amniotes. RESULTS: We produced a detailed chromosome map of the Siamese cobra genome, focusing on chromosome 2 and sex chromosomes. Synteny of the Siamese cobra chromosome 2 (NKA2) and NKAZ were highly conserved among snakes and other squamate reptiles, except for intrachromosomal rearrangements occurring in NKA2. Interestingly, twelve BACs that had partial homology with sex chromosomes of several amniotes were mapped on the heterochromatic NKAW as hybridization signals such as repeat sequences. Sequence analysis showed that most of these BACs contained high proportions of transposable elements. In addition, hybridization signals of telomeric repeat (TTAGGG)n and six microsatellite repeat motifs ((AAGG)8, (AGAT)8, (AAAC)8, (ACAG)8, (AATC)8, and (AAAAT)6) were observed on NKAW, and most of these were also found on other amniote sex chromosomes. CONCLUSIONS: The frequent amplification of repeats might involve heterochromatinization and promote sex chromosome differentiation in the Siamese cobra W sex chromosome. Repeat sequences are also shared among amniote sex chromosomes, which supports the hypothesis of an ancestral super-sex chromosome with overlaps of partial syntenies. Alternatively, amplification of microsatellite repeat motifs could have occurred independently in each lineage, representing convergent sex chromosomal differentiation among amniote sex chromosomes.

A novel method of caenophidian snake sex identification using molecular markers based on two gametologous genes.

Sex identification provides important information for ecological and evolutionary studies, as well as benefiting snake conservation management. Traditional methods such as cloacal probing or cloacal popping are counterproductive for sex identification concerning very small species, resulting in difficulties in the management of their breeding programs. In this study, the nucleotide sequences of gametologous genes (CTNNB1 and WAC genes) were used for the development of molecular sexing markers in caenophidian snakes. Two candidate markers were developed with the two primer sets, and successfully amplified by a single band on the agarose gel in male (ZZ) and two bands, differing in fragment sizes, in female (ZW) of 16 caenophidian snakes for CTNNB1 and 12 caenophidian snakes for WAC. Another candidate marker was developed with the primer set to amplify the specific sequence for CTNNB1W homolog, and the PCR products were successfully obtained in a female-specific 250-bp DNA bands. The three candidate PCR sexing markers provide a simple sex identification method based on the amplification of gametologous genes, and they can be used to facilitate effective caenophidian snake conservation and management programs.