Historical biogeography and genetic status of the enigmatic pig-nosed turtle (Carettochelys insculpta) within the Australo-Papuan region

This dataset was used to examine the phylogeographic genetic structure of an iconic species, the pignosed turtle Carettochelys insculpta, the last remaining member of a once globally widespread family, now restricted to a region with a dynamic and complex geological and geographical history – the Australo-Papuan region. It comprises aligned sequences of mtDNA data (Nd4 and Control Region) and SNP data used for population genetics and phylogenetic reconstruction.

Methods
SNP analyses

Skin tissues and extracted DNA were provided to DArT for processing, sequencing and informative SNP marker identification using DArTseqTM (Kilian et al., 2012). DArT performed a genome complexity reduction technique using double digestion of genomic DNA with two restriction endonucleases PstI (5′- CTGCA|G- 3′) and SphI (5′- GCATG|C- 3′), fragment-size selection and next-generation sequencing on an Illumina HiSeq2500 (CA, USA). Sequences were processed using proprietary DArT analytical pipelines (for full details refer to Georges et al. (2018). Initial filtering was based primarily on average and variance of sequencing depth, average allele counts and call rate across samples. Approximately one third of samples were sequenced twice as technical replicates, with scoring consistency identifying high quality SNP markers with low error rates. We applied further quality control filtering using the R package dartR 2.7.2 (Gruber et al., 2018; Mijangos et al., 2022).  These filters were for reproducibility across technical replicates (< 99%), call rate removing both loci and individuals with > 5% missing data, read depth (< 8x and above > 50x) to remove low coverage SNPs and potential paralogs and by removing all but one of multiple SNPs per locus. Specimens were removed from non-bottlenecked populations with close kinship probabilities (≥ 0.23) assessed using the R package popkin (Ochoa & Storey, 2021). We then filtered on linkage disequilibrium to account for the presence of monomorphic heterozygous loci from a possible gene duplication, resulting in a stringently filtered dataset of 16,002 SNPs.

mtDNA analyses

Nd4 sequence was amplified using primers CiND4-F (5’-CACGATGAGGCAACCAAATAGAAC-3’) and CiND4-R (5’-ATTACTTTTACTTGGAATTGCACCA-3’). Control Region sequence was amplified using primers CiCR-F (5’- CTCTATCCCCAAAGCACTGG-3’) and CiCR-R (5’-TTCTTGTATTTAGGGGTTT-3’). Primer CiND-R was modified from the H-Leu primer developed by Stuart and Parham (2004), and primers CiCR-F and CiCR-R were modified from the LCM15382 and H950g primers developed by Abreu-Grobois et al. (2006). Each was modified to better match the Carettochelys insculpta mitochondrial genome sequence of Nie et al. (2010). Amplification was done in separate 25 µL reactions for each locus, using 50 ng of template DNA, 1 × MyTaq HS Red Mix (Bioline) and 0.4 μM of forward and reverse primer. PCR was performed using an EPGradient Thermal Cycler (Eppendorf Mastercycler Pro S 6325) with cycling conditions for ND4 of 95ºC for 2 min, followed by 35 cycles of 20 s at 95°C, 20 s at 60°C and 20 s at 72°C, and a final extension of 1 min at 72°C. Cycling conditions to amplify the control region fragment differed from the Nd4 protocol with an annealing temperature of 50°C, all other conditions were the same.

Amplified products were purified with ExoSAP-IT PCR Product Cleanup Reagent (Thermo Fisher Scientific, Melbourne); 5 µl of PCR product was combined with 2 µl ExoSAP-IT, incubated at 37°C for 15 min followed by enzyme inactivation at 80°C for 15 min. Sequencing reactions consisted of 1 µl purified PCR product, 0.25 µl BigDye® v3.1 (Applied Biosystems, ThermoFisher Scientific, Melbourne), 1× sequencing buffer, 0.16 µM primer and ddH20 to a total volume of 20 µl. PCR cycling conditions were 96°C for 1 min, followed by 35 cycles of 96°C for 10 s, 50°C for 5 s and 60°C for 3 min. Sequencing reactions were purified using an ethanol/EDTA precipitation method (cms 081527, ThermoFisher Scientific, Melbourne). Sequencing was performed on an ABI 3730xl DNA Analyser at the ACRF Biomolecular Resource Facility within the John Curtin School of Medical Research, Australian National University.

Forward and reverse sequences were aligned, trimmed based on quality, manually edited, and coding sequences checked for unexpected frame shift errors or stop codons via amino acid coding in Geneious Prime 2020.2.2 (Biomatters, Auckland, New Zealand).