Background

Delimiting cryptic species in elasmobranchs is a major challenge in modern taxonomy due the lack of available phenotypic features. Employing stand-alone genetics in splitting a cryptic species may prove problematic for further studies and for implementing conservation management. In this study, we examined mitochondrial DNA and genome-wide nuclear single nucleotide polymorphisms (SNPs) in the brown-banded bambooshark, Chiloscyllium punctatum to evaluate potential cryptic species and the species-population boundary in the group.

Results

Both mtDNA and SNP analyses showed potential delimitation within C. punctatum from the Indo-Australian region and consisted of four operational taxonomic units (OTUs), i.e. those from Indo-Malay region, the west coast of Sumatra, Lesser Sunda region, and the Australian region. Each OTU can be interpreted differently depending on available supporting information, either based on biological, ecological or geographical data. We found that SNP data provided more robust results than mtDNA data in determining the boundary between population and cryptic species.

Conclusion

To split a cryptic species complex and erect new species based purely on the results of genetic analyses is not recommended. The designation of new species needs supportive diagnostic morphological characters that allow for species recognition, as an inability to recognise individuals in the field creates difficulties for future research, management for conservation and fisheries purposes.  Moreover, we recommend that future studies use a comprehensive sampling regime that encompasses the full range of a species complex. This approach would increase the likelihood of identification of operational taxonomic units rather than resulting in an incorrect designation of new species.

Tissue samples from Chiloscyllium punctatum were obtained from 17 localities within the Indo-Australian region.  Samples from Thailand, Malaysia, and Indonesia (Sumatra, Java, Kalimantan, South Sulawesi, and Lombok) were sourced from local fish markets. Samples were from Moreton Bay, eastern Australia, samples from Papua New Guinea and Western Australia were provided by the national fish collection (CSIRO, Hobart).

A total of 34 tissue samples collected from 12 locations during 2017 was analysed with the mtDNA gene marker NADH dehydrogenase subunit 2 (NADH2). Samples were amplified using MyTaq DNA polymerase (Bioline Reagents, London) and primers from Naylor et al.(18) with a modified forward primer: ILEM Chilo2 (5'- AAG GAT CAC TTT GAT AGA GT- 3'), and modified reverse primer: ASNM Chilo (5'- AAC ACT TAG CTG TTA ACT AA- 3').

A total of 148 extracted DNA samples (50–150 ng.µl^-1) were genotyped with the DArT-seq approach by Diversity Arrays Technologies (DArT Pty Ltd, Canberra, Australia). The SNP data were filtered using the R package 'dartR'. Loci with 100% repeatability (repAvg=1.0) and without missing values (call rate=1.0) were retained for subsequent analysis.  Monomorphic loci and secondary SNPs within loci were removed, the latter to reduce the possibility of linked fragments. SNP loci with ≤1% minor allele frequency were removed.

The dataset contains:

• The sequence of the mtDNA ND2 marker of Chiloscyllium punctatum from 34 samples were aligned with 3 reference sequences from genebank (GN2590, GN4616, GN4446) and exported to nexus format using Geneious software. 
•  SNP data of C. punctatum from DArT were filtered using R package ‘dartR’ and converted to nexus file using PGDspider software.
•  Filtered SNP data from DArT (in csv. file)