Conservation Genet Resour DOI 10.1007/s12686-016-0534-y METHODS AND RESOURCES ARTICLE A suite of potentially amplifiable microsatellite loci for ten reptiles of conservation concern from Africa and Asia 1 1 3 2 Kyle J. Shaney • Rich Adams • Nia Kurniawan • Amir Hamidy • 1 1 Eric N. Smith • Todd A. Castoe Received: 25 January 2016 / Accepted: 31 March 2016 Ó Springer Science+Business Media Dordrecht 2016 Abstract Here we document the addition of thousands of be applied to a variety of closely related taxa that are also of potentially amplifiable microsatellite loci (PALs) and conservation and commercial interest. associated primer sequences for public use. We conducted whole-genome shotgun sequencing to obtain ultra-low Keywords Conservation genetics Á Indonesia Á PALs Á coverage, random genomic sampling from ten African and Simple sequence repeats Á Squamate reptiles Asian squamate reptile species (representing ten genera). We used unique genomic processing methods and generated PALs for the following species: Acrochordus granulatus, Introduction Ahaetulla prasina, Cerberus rhynchops, Gonocephalus kuhlii, Ophiophagus hannah, Python bivittatus, Tribolono- The trade of reptiles is now a billion dollar industry tus gracilis, Trimeresurus sabahi (Popeia sabahi), Uro- worldwide and the exploitation of reptiles for meat, pets, mastyx geryi and Varanus exanthematicus. All taxa and skins has led to the decline of numerous species included, as well as other related taxa not included in our globally (Natusch and Lyons 2012; Koch et al. 2013). study, are exploited heavily by the international skin and pet Because reptilian diversity is poorly understood in many trades, yet researchers and conservation agencies currently regions of the world, assessing population health and reg- lack substantial genetic resources for guiding conservation ulating harvest quotas for species of conservation concern and management. Using stringent filtering methods, we can be quite challenging (Shaney et al. in press). generated between 467 and 8641 PALs for each of the ten Exploitation of reptiles is particularly an issue in African species (52,164 PALs total), yielding a rich database of and Asian countries where few resources are available for microsatellite loci and amplification primers for these taxa. conservation and wildlife management initiatives. Many In addition to the ten species targeted in our study, species collected and traded from these regions are often microsatellite markers provided in this database can likely erroneously labeled as ‘‘farmed’’ or ‘‘captive bred’’, despite being wild caught (Koch et al. 2013). Additionally, rare and ‘‘at-risk’’ species are commonly misrepresented and traded under common species designations, and limits on & Kyle J. Shaney [email protected] permitted collections are often greatly exceeded and rarely enforced (Shaney et al. in press). One major hurdle in 1 The Amphibian and Reptile Diversity Research Center and addressing these problems is the inability of regulatory Department of Biology, University of Texas at Arlington, 501 agencies to identify the precise geographic location and S. Nedderman Drive, Arlington, TX 76010, USA 2 population origins, or even the precise species designation, Laboratory of Herpetology, Museum Zoologicum of traded animals. Bogoriense, Research Center for Biology, Indonesian Institute of Sciences-LIPI, Jl. Raya Jakarta Bogor km 46, In recent years, researchers have applied molecular Cibinong, West Java 16911, Indonesia ‘conservation forensic’ approaches that employ population 3 Department of Biology, Universitas Brawijaya, Jl. Veteran, genetic markers to assist in the detection of illegally traded Malang, East Java 65145, Indonesia wildlife (Iyengar 2014). For example, Wasser et al. (2004) 123 Conservation Genet Resour used genotypic data to infer the origin of illegally traded assessment by CITES. Despite the high economic value ivory in Africa, and Ghobrial et al. (2010) utilized and vulnerability of these species, very little data is cur- molecular markers to trace the origins of illegally hunted rently available that can be used for conservation efforts. chimpanzees in Cameroon. These conservation forensic Our aim here was to provide a large database of molecular methods thereby provide important management and markers (microsatellite loci and associated primer pairs) enforcement tools that allow officials to determine the that we have identified through analysis of genome-scale precise identification and origins of harvested animals. sequencing efforts that can be used for population genetic Importantly, population genetic data (such as microsatel- monitoring and molecular forensic identification programs lites) provide an invaluable resource for assessing genetic for these and related taxa. diversity and health of target species, which can be further Beyond application to these ten target species, previous used to inform wildlife management guidelines and con- studies indicate that these markers will be further appli- servation legislation. Such molecular approaches have not cable to other related species of reptiles that are relatively been effectively applied to many reptilian species of con- closely related to the target species sampled. For example, servation concern, in part due to a lack of genetic resources Castoe et al. (2012a, b) tested microsatellite applicability and molecular markers available for reptile species of across closely related coral snake species of the genus interest. Micrurus and showed that nearly half of their tested PALs Here, we document and provide thousands of potentially (using the same methods described here) were readily amplifiable microsatellite loci (PALs) and associated pri- amplifiable across multiple species within the same genus. mer sets designed from whole-genome data for ten squa- Other studies have investigated the application of mate reptile species that represent African and Asian microsatellites across taxa and found that a high proportion lineages commonly collected for the reptile trade and/or are of loci successfully cross-amplified between related species of conservation concern. These species include: Acro- (Alacs et al. 2009) and even related genera (Zucoloto et al. chordus granulatus (Little File Snake, from India through 2006). Therefore, we expect that many PALs provided in southeast Asia, IUCN = Least Concern), Ahaetulla pra- our dataset are likely to work well across multiple related sina (Asian Green Vine Snake, from throughout the Sunda species of interest and have broader utility for a variety of Region of southeast Asia, IUCN = Least Concern), Cer- evolutionary and ecological studies. Additionally, recent berus rhynchops (Bockadam Water Snake/Dog-faced studies have highlighted extensive and rapid evolution of Water Snake, from Australia and southeast Asia, genomic microsatellite composition across squamate lin- IUCN = Least Concern), Gonocephalus kuhlii (Kuhl’s eages (Adams et al. in press; Castoe et al. 2011, 2013). Forest Dragon, from Indonesia, IUCN = Not Assessed), Thus, microsatellite loci obtained from these ten species Ophiophagus hannah (King Cobra, from southeast Asia, will not only provide valuable resources for conservation IUCN = Vulnerable), Python bivittatus (Burmese Python, research, but also contribute to the growing set of genomic from southeast Asia, IUCN = Vulnerable), Tribolonotus surveys of microsatellites for reptiles and vertebrate gen- gracilis (Red-Eyed Crocodile Skink, from Indonesia and omes in general. Papua New Guinea, IUCN = Least Concern), Trimeresu- rus sabahi (or Popeia sabihi, Asian Pitviper, from throughout southeast Asia, IUCN = Least Concern), Uro- Methods mastyx benti (Spiny-tailed Lizards, from north Africa and the Middle-East, IUCN = Least Concern), and Varanus Tissue samples were collected from muscle and/or liver exanthematicus (Savannah Monitor, from central Africa, tissue and DNA was extracted using the Phenol-Choloro- IUCN = Least Concern). These ten species include mul- form-Isoamyl alcohol (PCI) approach. Genomic DNA was tiple ‘‘at-risk’’ taxa and five CITES Appendix II species fragmented using the nebulization method (Sambrook and (C. rhynchops, O. hannah, P. bivittatus, U. benti and V. Russell 2006). Genomic shotgun libraries were constructed exanthematicus)–all of which have been largely over- using the NEBNext Illumina DNA library Prep Kit, which looked by conservation initiatives and wildlife manage- includes end-repairing, poly(A)-tailing, and barcoding the ment programs. These ten species are traded globally and samples for pooled sequencing. Libraries were size-selected are illegally traded in many instances, yet our current using the BluePippen prep for a length of 450–550 bp, and understanding of population viability of these taxa is either size-selected libraries were amplified via PCR. Genomic completely lacking or minimal at best (Knapp 2004; libraries were sequenced on the Illumina MiSeq platform Schoppe 2009; Koch et al. 2013, http://trade.cites.org/). using paired-end reads with a forward read length of The five species mentioned which are not currently listed 300–370 bp and reverse read length of 200–250 bp. by CITES are traded in high volume and are likely expe- Post sequencing, raw genomic reads were computa- riencing similar conservation issues, but have yet to receive tionally overlapped and merged into single, longer reads 123 Conservation Genet Resour Table 1 Sample handling information Genus Voucher Sample details Tissue Geographic origin Lat Long Acrochordus