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Hybrids between the critically endangered Siamese crocodile ( Crocodylus siamensis ) and least-concern saltwater crocodile ( C. porosus ) in captive populations represent a serious challenge for conservation and reintroduction programs due to the impact of anthropogenic activities. A previous study used microsatellite and mitochondrial DNA data to establish the criteria for identifying species and their hybrids; however, the results may have been influenced by biased allelic frequencies and genetic drift within the examined population. To overcome these limitations and identify the true signals of selection, alternative DNA markers and a diverse set of populations should be employed. Therefore, this study used DArT sequencing to identify genome-wide single nucleotide polymorphisms (SNPs) in both species and confirm the genetic scenario of the parental species and their hybrids. A population of saltwater crocodiles from Australia was used to compare the distribution of species-diagnostic SNPs. Different analytical approaches were compared to diagnose the level of hybridization when an admixture was present, wherein three individuals had potential backcrossing. Approximately 17.00-26.00% of loci were conserved between the Siamese and saltwater crocodile genomes. Species-diagnostic SNP loci for Siamese and saltwater crocodiles were identified as 8051 loci and 1288 loci, respectively. To validate the species-diagnostic SNP loci, a PCR-based approach was used by selecting 20 SNP loci for PCR primer design, among which 3 loci were successfully able to differentiate the actual species and different hybridization levels. Mitochondrial and nuclear genetic information, including microsatellite genotyping and species-diagnostic DNA markers, were combined as a novel method that can compensate for the limitations of each method. This method enables conservation prioritization before release into the wild, thereby ensuring sustainable genetic integrity for long-term species survival through reintroduction and management programs.

Microsatellites are polymorphic and cost-effective. Optimizing reduced microsatellite panels using heuristic algorithms eases budget constraints in genetic diversity and population genetic assessments. Microsatellite marker efficiency is strongly associated with its polymorphism and is quantified as the polymorphic information content ( PIC ). Nevertheless, marker selection cannot rely solely on PIC . In this study, the ant colony optimization (ACO) algorithm, a widely recognized optimization method, was adopted to create an enhanced selection scheme for refining microsatellite marker panels, called the PIC -ACO selection scheme. The algorithm was fine-tuned and validated using extensive datasets of chicken ( Gallus gallus ) and Chinese gorals ( Naemorhedus griseus ) from our previous studies. In contrast to basic optimization algorithms that stochastically initialize potential outputs, our selection algorithm utilizes the PIC values of markers to prime the ACO process. This increases the global solution discovery speed while reducing the likelihood of becoming trapped in local solutions. This process facilitated the acquisition of a cost-efficient and optimized microsatellite marker panel for studying genetic diversity and population genetic datasets. The established microsatellite efficiency metrics such as PIC , allele richness, and heterozygosity were correlated with the actual effectiveness of the microsatellite marker panel. This approach could substantially reduce budgetary barriers to population genetic assessments, breeding, and conservation programs.

Populations of Siamese crocodiles ( Crocodylus siamensis ) have severely declined because of hunting and habitat fragmentation, necessitating a reintroduction plan involving commercial captive-bred populations. However, hybridization between Siamese and saltwater crocodiles ( C. porosus ) has occurred in captivity. Siamese crocodiles commonly have post-occipital scutes (P.O.) with 4-6 scales, but 2-6 P.O. scales were found in captives on Thai farms. Here, the genetic diversity and population structure of Siamese crocodiles with large P.O. variations and saltwater crocodiles were analyzed using mitochondrial DNA D-loop and microsatellite genotyping. Possible crocodile hybrids or phenotypic variations were ascertained by comparison with our previous library from the Siam Crocodile Bioresource Project. Siamese crocodiles with <4 P.O. scales in a row exhibit normal species-level phenotypic variation. This evidence encourages the revised description of Siamese crocodiles. Moreover, the STRUCTURE plot revealed large distinct gene pools, suggesting crocodiles in each farm were derived from distinct lineages. However, combining both genetic approaches provides evidence of introgression for several individual crocodiles, suggesting possible hybridization between Siamese and saltwater crocodiles. We proposed a schematic protocol with patterns observed in phenotypic and molecular data to screen hybrids. Identifying non-hybrid and hybrid individuals is important for long-term in situ/ex situ conservation.