High genetic diversity and demographic history of captive Siamese and Saltwater crocodiles suggest the first step toward the establishment of a breeding and reintroduction program in Thailand

The Siamese crocodile (Crocodylus siamensis) and Saltwater crocodile (C. porosus) are two of the most endangered animals in Thailand. Their numbers have been reduced severely by hunting and habitat fragmentation. A reintroduction plan involving captive-bred populations that are used commercially is important and necessary as a conservation strategy to aid in the recovery of wild populations. Here, the genetic diversity and population structure of 69 individual crocodiles, mostly members of captive populations, were analyzed using both mitochondrial D-loop DNA and microsatellite markers. The overall haplotype diversity was 0.924–0.971 and the mean expected heterozygosity across 22 microsatellite loci was 0.578–0.701 for the two species. This agreed with the star-like shaped topology of the haplotype network, which suggests a high level of genetic diversity. The mean ratio of the number of alleles to the allelic range (M ratio) for the populations of both species was considerably lower than the threshold of 0.68, which was interpreted as indicative of a historical genetic bottleneck. Microsatellite markers provided evidence of introgression for three individual crocodiles, which suggest that hybridization might have occurred between C. siamensis and C. porosus. D-loop sequence analysis detected bi-directional hybridization between male and female individuals of the parent species. Therefore, identification of genetically non-hybrid and hybrid individuals is important for long-term conservation management. Relatedness values were low within the captive populations, which supported their genetic integrity and the viability of a breeding and reintroduction management plan. This work constitutes the first step in establishing an appropriate source population from a scientifically managed perspective for an in situ/ex situ conservation program and reintroduction of crocodile individuals to the wild in Thailand.

[1]  T. Gao,et al.  Genetic Diversity in the mtDNA control region and population structure of Chrysichthys nigrodigitatus from selected Nigerian rivers: Implications for conservation and aquaculture , 2016 .

[2]  N. Muangmai,et al.  Role of Chromosome Changes in Crocodylus Evolution and Diversity , 2015, Genomics & informatics.

[3]  S. Peyachoknagul,et al.  Molecular barcoding of venomous snakes and species-specific multiplex PCR assay to identify snake groups for which antivenom is available in Thailand. , 2015, Genetics and molecular research : GMR.

[4]  M. Russello,et al.  Genetic evidence of hybridization between the critically endangered Cuban crocodile and the American crocodile: implications for population history and in situ/ex situ conservation , 2014, Heredity.

[5]  A. Gandolfi,et al.  Defining conservation units in a stocking-induced genetic melting pot: unraveling native and multiple exotic genetic imprints of recent and historical secondary contact in Adriatic grayling , 2014, Ecology and evolution.

[6]  Koichiro Tamura,et al.  MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. , 2013, Molecular biology and evolution.

[7]  V. Dinets.  Long-Distance Signaling in Crocodylia , 2013, Copeia.

[8]  B. vonHoldt,et al.  STRUCTURE HARVESTER: a website and program for visualizing STRUCTURE output and implementing the Evanno method , 2012, Conservation Genetics Resources.

[9]  Rod Peakall,et al.  GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research—an update , 2012, Bioinform..

[10]  A. Thongpan,et al.  New haplotype of the complete mitochondrial genome of Crocodylus siamensis and its species-specific DNA markers: distinguishing C. siamensis from C. porosus in Thailand , 2012, Molecular Biology Reports.

[11]  Akifumi S. Tanabe,et al.  Kakusan4 and Aminosan: two programs for comparing nonpartitioned, proportional and separate models for combined molecular phylogenetic analyses of multilocus sequence data , 2011, Molecular ecology resources.

[12]  A. J. Crawford,et al.  Evolutionary history of Cuban crocodiles Crocodylus rhombifer and Crocodylus acutus inferred from multilocus markers. , 2011, Journal of experimental zoology. Part A, Ecological genetics and physiology.

[13]  A. Hochkirch,et al.  Ex situ conservation genetics: a review of molecular studies on the genetic consequences of captive breeding programmes for endangered animal species , 2011, Biodiversity and Conservation.

[14]  Jinliang Wang.  coancestry: a program for simulating, estimating and analysing relatedness and inbreeding coefficients , 2011, Molecular ecology resources.

[15]  J. Ragle,et al.  IUCN Red List of Threatened Species , 2010 .

[16]  M. Batzer,et al.  Molecular phylogenetic analyses of genus Crocodylus (Eusuchia, Crocodylia, Crocodylidae) and the taxonomic position of Crocodylus porosus. , 2010, Molecular phylogenetics and evolution.

[17]  L. Excoffier,et al.  Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows , 2010, Molecular ecology resources.

[18]  T. Glenn,et al.  253 Novel polymorphic microsatellites for the saltwater crocodile (Crocodylus porosus) , 2009, Conservation Genetics.

[19]  T. Glenn,et al.  A genetic linkage map for the saltwater crocodile (Crocodylus porosus) , 2009, BMC Genomics.

[20]  Pablo Librado,et al.  DnaSP v5: a software for comprehensive analysis of DNA polymorphism data , 2009, Bioinform..

[21]  J. R. Cedeño-Vázquez,et al.  Hybridization between Crocodylus acutus and Crocodylus moreletii in the Yucatan Peninsula: II. Evidence from microsatellites. , 2008, Journal of experimental zoology. Part A, Ecological genetics and physiology.

[22]  J. R. Cedeño-Vázquez,et al.  Genetic characterization of captive Cuban crocodiles (Crocodylus rhombifer) and evidence of hybridization with the American crocodile (Crocodylus acutus). , 2008, Journal of experimental zoology. Part A, Ecological genetics and physiology.

[23]  J. R. Cedeño-Vázquez,et al.  Hybridization between Crocodylus acutus and Crocodylus moreletii in the Yucatan Peninsula: I. Evidence from mitochondrial DNA and morphology. , 2008, Journal of experimental zoology. Part A, Ecological genetics and physiology.

[24]  A. Caccone,et al.  Molecular assessment of the genetic integrity, distinctiveness and phylogeographic context of the Saltwater crocodile (Crocodylus porosus) on Palau , 2007, Conservation Genetics.

[25]  G. Amato,et al.  The complete mitochondrial genome of salt-water crocodile (Crocodylus porosus) and phylogeny of crocodilians. , 2007, Journal of genetics and genomics = Yi chuan xue bao.

[26]  Guy Mergeai,et al.  Optimization of a reliable, fast, cheap and sensitive silver staining method to detect SSR markers in polyacrylamide gels. , 2006 .

[27]  J. Fa,et al.  Phylogeography of the endemic St. Lucia whiptail lizard Cnemidophorus vanzoi: Conservation genetics at the species boundary , 2006, Conservation Genetics.

[28]  A. Ruíz,et al.  Genetic assessment of the Iberian wolf Canis lupus signatus captive breeding program , 2006, Conservation Genetics.

[29]  R. Frankham.  Genetics and extinction , 2005 .

[30]  G. Evanno,et al.  Detecting the number of clusters of individuals using the software structure: a simulation study , 2005, Molecular ecology.

[31]  M. Batzer,et al.  Low Levels of Nucleotide Diversity in Crocodylus moreletii and Evidence of Hybridization with C. acutus , 2004, Conservation Genetics.

[32]  C. Moritz.  CONSERVATION UNITS AND TRANSLOCATIONS : STRATEGIES FOR CONSERVING EVOLUTIONARY PROCESSES , 2004 .

[33]  F. Allendorf,et al.  Introduction: Population Biology, Evolution, and Control of Invasive Species , 2003 .

[34]  D. Ray,et al.  The crocodilian mitochondrial control region: general structure, conserved sequences, and evolutionary implications. , 2002, The Journal of experimental zoology.

[35]  N. Q. Thang,et al.  Identification of purebred Crocodylus siamensis for reintroduction in Vietnam. , 2002, The Journal of experimental zoology.

[36]  Mark J. Clement,et al.  TCS: estimating gene genealogies , 2002, Proceedings 16th International Parallel and Distributed Processing Symposium.

[37]  F. Allendorf,et al.  The problems with hybrids: setting conservation guidelines , 2001 .

[38]  John P. Huelsenbeck,et al.  MRBAYES: Bayesian inference of phylogenetic trees , 2001, Bioinform..

[39]  A. Pavlícek,et al.  Construction and bootstrap analysis of DNA fingerprinting-based phylogenetic trees with the freeware program FreeTree: application to trichomonad parasites. , 2001, International journal of systematic and evolutionary microbiology.

[40]  J. Garza,et al.  Detection of reduction in population size using data from microsatellite loci , 2001, Molecular ecology.

[41]  P. Donnelly,et al.  Inference of population structure using multilocus genotype data. , 2000, Genetics.

[42]  G. Luikart,et al.  Computer note. BOTTLENECK: a computer program for detecting recent reductions in the effective size using allele frequency data , 1999 .

[43]  A. Storfer,et al.  Gene flow and endangered species translocations: a topic revisited , 1999 .

[44]  Y. Fu,et al.  Statistical tests of neutrality of mutations against population growth, hitchhiking and background selection. , 1997, Genetics.

[45]  J M Cornuet,et al.  Description and power analysis of two tests for detecting recent population bottlenecks from allele frequency data. , 1996, Genetics.

[46]  Kermit Ritland,et al.  Estimators for pairwise relatedness and individual inbreeding coefficients , 1996 .

[47]  W. Li,et al.  Statistical tests of neutrality of mutations. , 1993, Genetics.

[48]  F. Tajima.  Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. , 1989, Genetics.

[49]  Garry L. Martin,et al.  Behavior Modification: What it is and how to do it , 2019, Psychology Teaching Review.

[50]  G. Luikart,et al.  BOTTLENECK : A Computer Program for Detecting Recent Reductions in the Effective Population Size Using Allele Frequency Data , 2017 .

[51]  A. Caccone,et al.  Lineage identification and genealogical relationships among captive Galápagos tortoises. , 2012, Zoo biology.

[52]  Theunis Piersma,et al.  The interplay between habitat availability and population differentiation , 2012 .

[53]  Boyd K. Simpson,et al.  Siamese Crocodile Crocodylus siamensis , 2010 .

[54]  Stephen D. E. Park.  Trypanotolerace in West African cattle and the population genetic effects of selection , 2002 .

[55]  J. Huelsenbeck,et al.  MRBAYES : Bayesian inference of phylogeny , 2001 .

[56]  J. P. Ross,et al.  Crocodiles : status survey and conservation action plan , 1998 .

[57]  R. Lacy.  Clarification of genetic terms and their use in the management of captive populations , 1995 .

[58]  J. Thorbjarnarson.  Crocodiles: An action plan for their conservation , 1992 .

[59]  C. Gans,et al.  The chromosomes of the order Crocodilia. , 1970, Cytogenetics.