Conservation Genetic Studies Research Articles

"The Reference Genome Of The Kidnapper Ant, Polyergus Mexicanus".

Polyergus kidnapper ants are widely distributed, but relatively uncommon, throughout the Holarctic, spanning an elevational range from sea level to over 3000 m. These species are well known for their obligate social parasitism with various Formica ant species, which they kidnap in dramatic, highly coordinated raids. Kidnapped Formica larvae and pupae become integrated into the Polyergus colony where they develop into adults and perform nearly all of the necessary colony tasks for the benefit of their captors. In California, Polyergus mexicanus is the most widely distributed Polyergus, but recent evidence has identified substantial genetic polymorphism within this species, including genetically divergent lineages associated with the use of different Formica host species. Given its unique behavior and genetic diversity, Polyergus mexicanus plays a critical role in maintaining ecosystem balance by influencing the population dynamics and genetic diversity of its host ant species, Formica, highlighting its conservation value and importance in the context of biodiversity preservation. Here, we present a high-quality genome assembly of P. mexicanus from a sample collected in Plumas County, CA, USA, in the foothills of the central Sierra Nevada. This genome assembly consists of 364 scaffolds spanning 252.31Mb, with contig N50 of 481,250kb, scaffold N50 of 10.36Mb, and BUSCO completeness of 95.4%. We also assembled the genome of the Wolbachia endosymbiont of P. mexicanus - a single, circular contig spanning 1.23Mb. These genome sequences provide essential resources for future studies of conservation genetics, population genetics, speciation, and behavioral ecology in this charismatic social insect.

Developing Strain-Specific Simple Sequence Repeat (SSR) Markers for Chlorella sorokiniana.

Chlorella sorokiniana green microalga offers many environmentally friendly applications, including wastewater treatment, biofertilizers, animal feed, and biofuel production. Different strains of C. sorokiniana have unique properties that may suit one application but not another. There is a need to distinguish between the many available strains of C. sorokiniana to choose the one that best fits the application. Consequently, our research goal was to develop strain-specific simple sequence repeat (SSR) markers to differentiate between the different strains. Seventeen markers spanning ten out of the twelve chromosomes of the C. sorokiniana genome were developed and validated on eight different strains from culture collections and our lab, and were then analyzed by fragment analysis. The results demonstrate the potential of these polymorphic markers to detect the genetic differences between the strains of C. sorokiniana, and to serve as useful tools for the intra-species population genetic analysis and conservation genetics studies of C. sorokiniana.

ONeSAMP 3.0: estimation of effective population size via single nucleotide polymorphism data from one population.

The genetic effective size (Ne) is arguably one of the most important characteristics of a population as it impacts the rate of loss of genetic diversity. Methods that estimate Ne are important in population and conservation genetic studies as they quantify the risk of a population being inbred or lacking genetic diversity. Yet there are very few methods that can estimate the Ne from data from a single population and without extensive information about the genetics of the population, such as a linkage map, or a reference genome of the species of interest. We present ONeSAMP 3.0, an algorithm for estimating Ne from single nucleotide polymorphism data collected from a single population sample using approximate Bayesian computation and local linear regression. We demonstrate the utility of this approach using simulated Wright-Fisher populations, and empirical data from five endangered Channel Island fox (Urocyon littoralis) populations to evaluate the performance of ONeSAMP 3.0 compared to a commonly used Ne estimator. Our results show that ONeSAMP 3.0 is broadly applicable to natural populations and is flexible enough that future versions could easily include summary statistics appropriate for a suite of biological and sampling conditions. ONeSAMP 3.0 is publicly available under the GNU General Public License at https://github.com/AaronHong1024/ONeSAMP_3.

Tidings from the Tides–De novo transcriptome assembly of the endemic estuarine bivalve Villorita cyprinoides

The Indian black clam Villorita cyprinoides Gray, 1825, is an economically valuable estuarine bivalve that faces challenges from multiple stressors and anthropogenic pressures. However, limited genomic resources have hindered molecular investigations into the impact of these stressors on clam populations. Here, we have generated the first transcriptomic reference datasets for V. cyprinoides to address this knowledge gap. A total of 25,040,592 and 22,486,217 million Illumina paired-end reads generated from two individuals were assembled using Trinity and rnaSPAdes. From the 47,607 transcripts identified as Coding Domain Sequences, 37,487 returned positive BLAST hits against six different databases. Additionally, a total of 14,063 Single Sequence Repeats were identified using GMATA. This study significantly enhances the genetic understanding of V. cyprinoides, a potential candidate for aquaculture that supports the livelihoods of many people dependent on small-scale fisheries. The data generated provides insights into broader genealogical connections within the family Cyrenidae through comparative transcriptomics. Furthermore, this transcriptional profile serves as baseline data for future studies in toxicological and conservation genetics.

Development of mitochondrial DNA cytochrome c oxidase subunit I primer sets to construct DNA barcoding library using next-generation sequencing.

Insects are one of the most diverse eukaryotic groups on the planet, with one million or more species present, including those yet undescribed. The DNA barcoding system has been developed, which has aided in the identification of cryptic species and undescribed species. The mitochondrial cytochrome c oxidase I region (mtDNA COI) has been utilised for the barcoding analysis of insect taxa. Thereafter, next-generation sequencing (NGS) technology has been developed, allowing for rapid acquisition of massive amounts of sequence data for genetic analyses. Although NGS-based PCR primers designed to amplify the mtDNA COI region have been developed, their target regions were only a part of COI region and/or there were taxonomic bias for PCR amplification. As the mtDNA COI region is a traditional DNA marker for the DNA barcoding system, modified primers for this region would greatly contribute to taxonomic studies. In this study, we redesigned previously developed PCR primer sets that targetted the mtDNA COI barcoding region to improve amplification efficiency and to enable us to conduct sequencing analysis on NGS. As a result, the redesigned primer sets achieved a high success rate (> 85%) for species examined in this study, covering four insect orders (Coleoptera, Lepidoptera, Orthoptera and Odonata). Thus, by combining the primers with developed primer sets for 12S or 16S rRNA regions, we can conduct more detailed taxonomic, phylogeographic and conservation genetic studies using NGS.

Genome Assembly of Pyrocephalus nanus: A Step Toward the Genetic Conservation of the Endangered Little Vermilion Flycatcher of the Galapagos Islands.

Incredibly powerful whole genome studies of conservation genetics, evolution, and biogeography become possible for non-model organisms when reference genomes are available. Here, we report the sequence and assembly of the whole genome of the little vermilion flycatcher (Pyrocephalus nanus; family Tyrannidae), which is an endemic, endangered, and declining species of the Galapagos Islands. Using PacBio HiFi reads to assemble long contigs and Hi-C reads for scaffolding, we assembled a genome of 1.07 Gb comprising 267 contigs in 152 scaffolds, scaffold N50 74 M, contig N50 17.8 M, with 98.9% assigned to candidate chromosomal sequences and 99.72% of the BUSCO passeriformes 10,844 single-copy orthologs present. In addition, we used the novel HiFiMiTie pipeline to fully assemble and verify all portions of the mitochondrial genome from HiFi reads, obtaining a mitogenome of 17,151 bases, containing 13 protein-coding genes, 22 tRNAs, 2 rRNAs, two control regions, and a unique structure of control region duplication and repeats. These genomes will be a critical tool for much-needed studies of phylogenetics, population genetics, biogeography, and conservation genetics of Pyrocephalus and related genera. This genome and other studies that use it will be able to provide recommendations for conservation management, taxonomic improvement, and to understand the evolution and diversification of this genus within the Galapagos Islands.

Development of microsatellite markers for the endangered butterfly Luehdorfia japonica Leech, 1889 (Lepidoptera: Papilionidae)

ABSTRACTThe endangered butterfly species Luehdorfia japonica Leech, 1889 (Lepidoptera, Papilionidae) is endemic to the central and western parts of Honshu, Japan. This species inhabits deciduous forests and coppices, but areas of optimal habitats are decreasing due to the development of land and the abandonment of regular coppice management. We developed 17 microsatellite loci for L. japonica based on de novo genome sequence data and found that 16 of these loci exhibited polymorphisms in 34 individuals of L. japonica. In addition, polymorphisms of 15 of these microsatellite loci were observed in two individuals of L. puziloi. The number of alleles and the expected heterozygosity per locus in L. japonica were 2–11 and 0.11–0.83, respectively. A principal coordinate analysis based on this genetic information revealed genetic differentiation both within and among geographic populations of L. japonica. Thus, these microsatellite loci could potentially be useful for future conservation genetic studies, including monitoring the genetic diversity and population structure of this endangered butterfly species.

Genomic data revealed inbreeding despite a geographically connected stable effective population size since the Holocene in the protected Formosan Long-Arm Scarab beetle, Cheirotonus formosanus.

Biodiversity conservation is a top priority in the face of global environmental change, and the practical restoration of biodiversity has emerged as a key objective. Nevertheless, the question of how to effectively contribute to biodiversity restoration and identify suitable systems for such efforts continues to present major challenges. By using genome-wide SNP data, our study revealed that populations from different mountain ranges of the Formosan Long-Arm Scarab beetle, a flagship species that receives strict protection, exhibited a single genetic cluster with no subdivision. Additionally, our result implied an association between the demographic history and historical fluctuations in climate and environmental conditions. Furthermore, we showed that, despite a stable and moderately sized effective population over recent history, all the individuals we studied exhibited signs of genetic inbreeding. We argued that the current practice of protecting the species as one evolutionarily significant unit remains the best conservation plan and that recent habitat change may have led to the pattern of significant inbreeding. We closed by emphasizing the importance of conservation genetic studies in guiding policy decisions and highlighting the potential of genomic data for identifying ideal empirical systems for genetic rescue, or assisted gene flow studies.

Characterization of Eighty-Eight Single-Nucleotide Polymorphism Markers in the Manila Clam Ruditapes philippinarum Based on High-Resolution Melting (HRM) Analysis.

Single-nucleotide polymorphisms (SNPs) are the most commonly used DNA markers in population genetic studies. We used the Illumina HiSeq4000 platform to develop single-nucleotide polymorphism (SNP) markers for Manila clam Ruditapes philippinarum using restriction site-associated DNA sequencing (RAD-seq) genotyping. Eighty-eight SNP markers were successfully developed by using high-resolution melting (HRM) analysis, with a success rate of 44%. SNP markers were analyzed for genetic diversity in two clam populations. The observed heterozygosity per locus ranged from 0 to 0.9515, while the expected heterozygosity per locus ranged from 0.0629 to 0.4997. The value of FIS was estimated to be from -0.9643 to 1.0000. The global Fst value was 0.1248 (p < 0.001). After Bonferroni correction, 15 loci deviated significantly from the Hardy-Weinberg equilibrium (p < 0.0006). These SNP markers provide a valuable resource for population and conservation genetics studies in this commercially important species.

Genetic variation in Loudetia simplex supports the presence of ancient grasslands in Madagascar

Societal Impact StatementRecognizing Loudetia‐dominated grasslands were widespread prior to human colonization highlights that open ecosystems were and continue to be an important component of Madagascar's biodiversity. A better understanding of the plant species that form grassland ecosystems is necessary for effective land management strategies that support livelihoods, but substantial financial and logistical barriers exist to implementing conservation genetic studies using contemporary genomic tools. Some challenges for population genetic analyses of non‐model polyploids lacking reference genomes can be ameliorated by developing computational resources that leverage a cost‐effective data generation strategy that requires no prior genetic knowledge of the target species. This may benefit conservation programs with small operating budgets while reducing uncertainty compared to status quo microsatellite assays.Summary The extent of Madagascar's grasslands prior to human colonization is unresolved. We used population genetic analyses of a broadly dominant C4 fire‐adapted grass, Loudetia simplex, as a proxy for estimating grassland change through time. We carefully examined the utility of target‐enrichment data for population genetics to make recommendations for conservation genetics. We explored the potential of estimating individual ploidy levels from target‐enrichment data and how assumptions about ploidy could affect analyses. We developed a novel bioinformatic pipeline to estimate ploidy and genotypes from target‐enrichment data. We estimated standard population genetic summary statistics in addition to species trees and population structure. Extended Bayesian skyline plots provided estimates of population size through time for empirical and simulated data. All Malagasy L. simplex individuals sampled in this study formed a clade and possibly indicated an ancestral Central Highland distribution of 800 m in altitude and above. Demographic models suggested grassland expansions occurred prior to the Last Interglacial Period and supported extensive grasslands prior to human colonization. Though there are limitations to target‐enrichment data for population genetic studies, we find that analyses of population structure are reliable. Genetic variation in L. simplex supports widespread grasslands in Madagascar prior to the more recent periods of notable paleoclimatic change. However, the methods explored here could not differentiate between paleoclimatic change near the Last Glacial Maximum and anthropogenic effects. Target‐enrichment data can be a valuable tool for analyses of population structure in the absence a reference genome.

Genetic resources of macroalgae: Development of an efficient method using microsatellite markers in non-model organisms

Red and brown seaweeds are species with high ecological and economic importance. Here we report the feasibility of cost-effective molecular marker development in 6 species from different phyla. Microsatellite markers of two brown seaweed species Alaria esculenta and Pylaiella littoralis, and of four red seaweed species Calliblepharis jubata, Gracilaria gracilis, Gracilaria dura and Palmaria palmata were identified and characterized in genomic sequences obtained using Double-Digest Restriction site Associated DNA (ddRAD). A total of 64,623,186 reads were generated from two runs of multiplexed Illumina Miseq sequencing, from which 30,636 reads containing microsatellite motifs including 15,443 where primer pairs could be designed. Five hundred seventy-six primers pairs were selected for amplification trials to determine levels of polymorphism. Of these 576 loci, 338 could be amplified and 142 of the 338 loci were polymorphic. A total of 28 usable polymorphic markers were developed in A. esculenta, 18 in P. littoralis, 11 in C. jubata, 14 in G. gracilis, 21 in G. dura and 13 in P. palmata. The overall number of alleles per locus ranged from 2 to 22. These 105 new microsatellite markers will be useful for further studies of population genetics, breeding programs and conservation genetics of these species. Compared with traditional approaches, our study yielded thousands of microsatellite loci for six different species in a short time and with affordable costs. This study, based on the use of ddRADseq, provided preliminary data about the genetic structure and reproduction mode of these six non-model species based on a small number of individuals from two geographically-distant populations and on the genetic structure and reproduction mode of two non-model species, i.e. detection of clonality for two red algae, C. jubata and G. dura and detection of highly genetically divergent populations corresponding probably to different cryptic species under the name P. littoralis.

The First Mitochondrial Control Region Dataset: Critically Endangered Freshwater Turtles in Malaysia, Orlitia borneensis and Batagur borneoensis

The Malaysian Giant Turtles and Painted Terrapin mitochondrial control region has the first data deposited to the GenBank database. This dataset describes phylogenetic tree relationships between the genera Orlitia and Batagur. Orlitia borneensis and Batagur borneoensis are freshwater turtles listed as critically endangered on the International Union for Conservation of Nature (IUCN) Red List and among 24 species of turtles in Malaysia. Since both species are critically endangered, the data provided here can be used for future conservation genetics studies in order to protect the species from being driven to extinction. A sample of each species was collected aseptically from adult O. borneensis (male) and B. borneoensis (female) in captivity at Bukit Paloh, Kuala Berang, Terengganu. The data for this study can be found in the GenBank database with accession numbers OQ571740 and OQ571740.

Testing the efficacy of different molecular tools for parasite conservation genetics: a case study using horsehair worms (Phylum: Nematomorpha).

In recent years, parasite conservation has become a globally significant issue. Because of this, there is a need for standardized methods for inferring population status and possible cryptic diversity. However, given the lack of molecular data for some groups, it is challenging to establish procedures for genetic diversity estimation. Therefore, universal tools, such as double-digest restriction-site-associated DNA sequencing (ddRADseq), could be useful when conducting conservation genetic studies on rarely studied parasites. Here, we generated a ddRADseq dataset that includes all 3 described Taiwanese horsehair worms (Phylum: Nematomorpha), possibly one of the most understudied animal groups. Additionally, we produced data for a fragment of the cytochrome c oxidase subunit I (COXI) for the said species. We used the COXI dataset in combination with previously published sequences of the same locus for inferring the effective population size (Ne) trends and possible population genetic structure.We found that a larger and geographically broader sample size combined with more sequenced loci resulted in a better estimation of changes in Ne. We were able to detect demographic changes associated with Pleistocene events in all the species. Furthermore, the ddRADseq dataset for Chordodes formosanus did not reveal a genetic structure based on geography, implying a great dispersal ability, possibly due to its hosts. We showed that different molecular tools can be used to reveal genetic structure and demographic history at different historical times and geographical scales, which can help with conservation genetic studies in rarely studied parasites.

Population genetic assessment of Viburnum japonicum in China using ddRAD-seq.

Viburnum japonicum is a rare plant species and endemic to the coastal region of Eastern Asia with extremely small populations. Within mainland China, this species can be only found in narrow habitats of the northeast coastal islands of Zhejiang Province. However, there are scarce conservation genetic studies on V. japonicum, which has limited the effective conservation and management of this rare species. Here, 51 individuals in four natural populations covering the Chinese geographic range of the species were sampled to assess the genetic diversity and population structure. A total of 445,060 high-quality single nucleotide polymorphisms (SNPs) were identified using double digest restriction-site associated sequencing (ddRAD-seq). The overall average values of observed heterozygosity (Ho), expected heterozygosity (He), and average nucleotide diversity (π), were 0.2207, 0.2595, and 0.2741, respectively. The DFS-2 population exhibited the highest level of genetic diversity among all the populations. Genetic differentiation between populations was moderate (F ST = 0.1425), and there was selfing between populations (F IS = 0.1390, S = 24.52%). Of the total genetic variation, 52.9% was found among populations through AMOVA analysis. The Mantel test (r = 0.982, p = 0.030) combined with analyses of the Maximum Likelihood (ML) phylogenetic tree, ADMIXTURE, and principal component analysis (PCA), revealed that populations of V. japonicum were genetically segregated and significantly correlated with their geographical distribution. Our study demonstrated that V. japonicum maintained a medium level of genetic diversity and differentiation with a strong population structure, and the results were mainly affected by its island distribution pattern and self-crossing characteristics. These results provide insights into the genetic diversity and population history of V. japonicum, critical information for conserving and sustainably developing its genetic resources.

The Complete Mitochondrial Genome and Phylogenetic Analyses of To Chicken in Vietnam.

Indigenous chicken breeds have both cultural significance and economic value since they possess unique genetic characteristics that enable them to adapt to the local environment and contribute to biodiversity, food security, and sustainable agriculture in Vietnam. To (Tò in Vietnamese) chicken, a Vietnamese indigenous chicken breed, is popularly raised in Thai Binh province; however, little known is about the genetic diversity of this breed. In this study, we sequenced the complete mitochondrial genome of To chicken for a better understanding of the diversity and origin of the breed. The results of sequencing showed that the mitochondrial genome of To chicken spans a total length of 16,784 base pairs and comprises one non-coding control region (known as the displacement-loop (D-loop) region), two ribosomal RNA genes, 13 protein-coding genes, and 22 transfer RNA genes. The phylogenetic tree analyses and estimated genetic distances based on 31 complete mitochondrial genome sequences indicated that To chicken has a close genetic distance with the Laotian native chicken breed, Lv'erwu breed in China, and Nicobari black and Kadaknath breeds in India. The result of the current study might be important for conservation, breeding, and further genetic studies of To chicken.

Phylogenomics and phylogeography of Menispermum (Menispermaceae).

Phylogenomics have been widely used to resolve ambiguous and controversial evolutionary relationships among plant species and genera, and the identification of unique indels in plastomes may even help to understand the evolution of some plant families. Menispermum L. (Menispermaceae) consists of three species, M. dauricum DC., M. canadense L., and M. mexicanum Rose, which are disjuncly distributed among East Asia, Eastern North America and Mexico. Taxonomists continue to debate whether M. mexicanum is a distinct species, a variety of M. dauricum, or simply a synonym of M. canadense. To date, no molecular systematics studies have included this doubtful species in phylogenetic analyses. In this study, we examined phylogenomics and phylogeography of Menispermum across its entire range using 29 whole plastomes of Menispermaceae and 18 ITS1&ITS2 sequences of Menispermeae. We reconstructed interspecific relationships of Menispermum and explored plastome evolution in Menispermaceae, revealing several genomic hotspot regions for the family. Phylogenetic and network analyses based on whole plastome and ITS1&ITS2 sequences show that Menispermum clusters into two clades with high support values, Clade A (M. dauricum) and Clade B (M. canadense + M. mexicanum). However, M. mexicanum is nested within M. canadense and, as a result, we support that M. mexicanum is a synonym of M. canadense. We also identified important molecular variations in the plastomes of Menispermaceae. Several indels and consequently premature terminations of genes occur in Menispermaceae. A total of 54 regions were identified as the most highly variable plastome regions, with nucleotide diversity (Pi) values > 0.05, including two coding genes (matK, ycf1), four introns (trnK intron, rpl16 intron, rps16 intron, ndhA intron), and 48 intergenic spacer (IGS) regions. Of these, four informative hotspot regions (trnH-psbA, ndhF-rpl32, trnK-rps16, and trnP-psaJ) should be especially useful for future studies of phylogeny, phylogeography and conservation genetics of Menispermaceae.

An integrative study of species distribution modelling and conservation genetics: Magnolia in Hispaniola

On the island of Hispaniola, five endemic species of Magnolia occur, all of which are threatened with extinction. Little is known about their distribution and genetic health, hampering targeted conservation actions. The objective of this study is to assess the potential distribution and the genetic health of the Magnolias of Hispaniola, to inform concrete guidelines for effective conservation management. Using species distribution modelling (SDM), we predict habitat suitability for the Magnolias of Hispaniola by analysing 21 variables, describing climate and landscape features, on 635 occurrences. We genotyped 417 individuals using 16 microsatellite markers, to test for genetic structure and degree of inbreeding. The SDM and genetic data confirm the recognition of the four studied Magnolia species. The known individuals of the three Dominican Magnolias are structured into five populations which show ample genetic diversity and little inbreeding overall. For conservation management, we propose to focus on exploration using the SDM results, and protection and reinforcement using the genetic and occurrence data. The genetic results guide prioritization of species and populations. The SDM results guide spatial prioritization. Installing and/or protecting habitat corridors between populations, starting with the two species with the lowest genetic diversity and relatively nearby populations, is recommended as a durable conservation strategy. Meanwhile, reinforcement efforts can be undertaken to artificially increase gene flow for which we appoint sink and source population pairs, using the genetic data.

Phylogenetic Study of Philippine Pigs (Sus scrofa L.) from Ifugao and Kalinga Based on Mitochondrial DNA D-loop Analysis

Conservation genetic studies for Philippine pigs are essential to sustain global biodiversity. To study the phylogenetic relationship of Philippine pigs, 79 samples were collected from Ifugao (n = 41) and Kalinga (n = 38) provinces located in the Cordillera Administrative Region, northern Philippines. Mitochondrial DNA control region analysis was undertaken to investigate the phylogenetic relationship between the pig populations from the two provinces and other Asian and European pig breeds. Fourteen (14) haplotypes (H1–H14) were detected in the Ifugao and Kalinga samples. Several haplotypes – specifically, H1, H4, H9, H12, and H14 – formed an independent clade with the Type I Lanyu haplotypes, which are known to be unique in Taiwan pigs. The remaining haplotypes (H2, H3, H5–H8, H10, H11, and H13) clustered with the haplotypes of other Asian pig breeds within the major Asian clade. These results suggest the close relationship between Kalinga and Ifugao pigs and Type I Lanyu pigs. The existence of duplicate motifs also revealed that the native pigs from Ifugao and Kalinga belong to a distinct genetic lineage that clustered with other Philippine pig samples and separate from other Asian pigs, whereas the other samples may represent a recent introgression of modern lineages.

Molecular Identification of Captive Snakes from their Shed Skin Using Cyt b &amp; 12s rRNA Markers

Aims: To successfully utilize non-invasive shed skin samples for species identification of captive snake species of Tamil Nadu and create genetic repository.&#x0D; Study Design: The experiment was designed to apply the ammonium acetate method of DNA extraction from shed skin. Two mitochondrial markers were used to ascertain identification of species.&#x0D; Place and Duration of Study: Advanced Institute for Wildlife Conservation (AIWC), Tamil Nadu Forest Department, Vandalur, Chennai, Tamil Nadu. The samples were collected between May and October 2019 from Arignar Anna Zoological Park, Vandalur, Guindy National Park, Chennai, and Amirthi Zoological Park, Vellore, Tamil Nadu.&#x0D; Methodology: We collected fresh shed skin from 8 different snake species from captivities of zoos and dried between 48 to 72 hours to remove moisture. Independent DNA isolations were performed for each sample. The DNA isolated samples were quantified using Nanodrop Spectrophotometer for concentration. Independent PCR amplification of mitochondrial regions of cytochrome b and 12S rRNA were performed and agarose gel electrophoresis was carried out. PCR products were subjected to sanger sequencing using genetic analyzer.&#x0D; Results: The DNA concentration from all 8 different snake species ranged between 250 to 1600 ng/µL and average quality ratios A260/280 of 1.85 and A260/230 of 2.10. Both the mitochondrial gene regions cytochrome b and 12S rRNA showed specificity in species amplification with NCBI BLAST result ranging from 99-100%. Phylogenetic trees using maximum-likelihood method classified closely related species under the same clade, with a bootstrap support of 60-100%. Genetic distances of snake species ranged from 0.148-0.457 in cytochrome b region and 0.148-0.457 in 12S region.&#x0D; Conclusion: Shed skin is often overlooked from utilization for species identification. In this study, DNA from shed skin of 8 captive snakes is extracted and amplified using Cyt b and 12s mitochondrial markers. Individual phylogenetic trees are constructed for each marker to find relatedness of different snake species with one another. This work is an initiation of genetic repository creation of captive snake species of Tamil Nadu and could be effectively employed in conservation and population genetic studies of snakes.

Phylogeographic Structuring of the Kuroshio-Type Prawn Macrobrachium japonicum (Decapoda: Palaemonidae) in Taiwan and Ryukyu Islands

Oceanic currents, biological traits, and geological evidence from historical climate change have shaped the distribution of genetic diversity and demographic history in Macrobrachium japonicum. In this study, we investigated the phylogeographic structures and demographic history of M. japonicum in Taiwan and the Ryukyu Islands by the partial mitochondrial COI gene. The topology of the haplotype minimum-spanning network revealed a star-like structure, which suggested a recent range expansion. With the apparent lack of strong dispersal barriers to gene flow between ocean basins, amphidromous prawns, as marine organisms with larvae of high dispersal abilities, are generally expected to show genetic homogeneity. Divergence data suggested that M. japonicum populations diverged 0.462 ± 0.164 myr. Complex patterns of asymmetrical gene flow indicate that variation in dispersal ability is affected by the dominant Kuroshio Current systems of the Pacific Ocean. Analysis of its demographic history revealed that M. japonicum underwent a decline in size following a recent population expansion based on ABC analysis. Bayesian skyline plot analyses of all populations indicated that the expansion of M. japonicum was estimated to have occurred in 175 Kya and that the effective population size rapidly increased by approximately 50 Kya. The results of this study may offer both practical and theoretical value for the further study of conservation genetics, management, and sustainable resource utilization.