DOI: 10.5061/dryad.h18931zp6
title: Genomic analyses of the scorpion mud turtle Kinosternon scorpioides in continental and insular Colombia

description: The turtle genus Kinosternon is widespread with at least
 25 species distributed from Mexico to northern Argentina. The taxonomy of
 this genus is controversial and requires a full revision using both
 morphological and molecular approaches. In this study, we did a genomic
 analysis on the species Kinosternon
 scorpioides distributed in insular and continental Colombia in
 order to define conservation units. Total DNA was extracted from 24 tissue
 samples and RADseq genotyping analysis was done. In addition, the intron
 R35 was amplified and sequenced for a subset of samples. A total
 of 35,507 SNPs combined with 1,047 bp of the intron were used for
 spatiotemporal colonization patterns reconstruction and phylogenetic
 analyses. In addition, the SNPs were used for population structure
 inferences and allele frequency-based analyses.
   According to the long-term reciprocal monophyly,
 significant differences in allele frequencies (Fst = 0.37 - 0.8),
 and evidence of reproductive isolation (no admixture/geneflow), indicate
 long-term divergence between groups (2-8 MYA), possibly due to the effect
 of geographic barriers.   Four Evolutionarily
 Significant Units (ESUs) were defined within our sample.  One ESU
 was represented by the insular subspecies K. scorpioides
 albogulare, found in San Andrés island, and three ESUs were defined for
 the subspecies K. s. scorpioides in continental
 Colombia: one trans-Andean, found in northwestern Colombia (Caribbean
 region) and two cis-Andean, found in eastern and southeastern Colombia in
 the Orinoco and Amazon regions, respectively. Colonization of this species
 occurred from an ancestral area of Central America/Caribbean region (~
 8.43 MYA), establishing current populations in San Andrés Island and then,
 in independents events, on to the Colombian Caribbean, next, in the
 Orinoco, and more recently, in the Amazon. We hypothesize that the
 emergence of the Panamá Isthmus, the final uplift North Eastern Andes and
 Vaupes Arch, were key event leading to the differentiation of these ESUs.
 For management and conservation purposes, each of these ESUs should be
 considered as a separate management unit. A full revision of the taxonomy
 of the genus Kinosternon is warranted. 
description: <strong>Sampling
 locations</strong>  A total of 24 tissue samples were obtained from
 wild <em>Kinosternon scorpioides</em> turtles in six sampling
 locations in three Colombian regions: one in the Colombian Amazon Basin
 (Leticia), two in the Caribbean Coast or Caribe Basin (Cispatá Bay and
 Lorica swamp), three in the Orinoco basin (sierra de la Macarena-Guaviare
 Subasin, Puerto Carreño-Orinoco river floodplain and Tuparro National
 Park-Tomo Subasin) and one in San Andrés Island (Figure 1 and Table
 1).  Turtles were captured at night. Then, following
 the protocol designed by the Instituto de Investigación de Recursos
 Biológicos “Alexander von Humboldt” in Colombia (Vargas-Ramírez, 2017),
 approximately 0.5 cm<sup>3</sup> of tissue was cut out from
 the back foot using a scalpel and stored in 90%
 ethanol.  <strong>DNA extraction, quality control, library
 preparation and sequencing</strong>
  Total DNA was extracted using the QIAamp DNA
 tissue mini kit (QIAGEN) and its quality was evaluated in 0.8% agarose
 gels.  DNA was quantified using nanodrop 2000 spectrophotometer (Thermo
 Scientific) and diluted to a final concentration ranging from 30 to 50
 ng/uL.  Genomic DNA was converted into nextRAD
 genotyping-by-sequencing libraries (SNPsaurus, LLC) as in Russello et al.
 (2015). DNA was first fragmented with Nextera DNA Flex reagent (Illumina,
 Inc), which also ligates short adapter sequences to the ends of the
 fragments. The Nextera reaction was scaled for fragmenting 50 ng of
 genomic DNA, although 75 ng of genomic DNA was used for input to
 compensate for the amount of degraded DNA in the samples and to increase
 fragment sizes. Fragmented DNA was then amplified for 27 cycles at 74
 degrees, with one of the primers matching the adapter and extending 10
 nucleotides into the genomic DNA with the selective sequence GTGTAGAGCC.
 Thus, only fragments starting with a sequence that can be hybridized by
 the selective sequence of the primer were efficiently amplified. The
 nextRAD libraries were sequenced on a HiSeq 4000 with one lane of 150 bp
 reads (University of Oregon).  <strong>RADseq
 genotyping analysis</strong>
  The genotyping
 analysis used custom scripts (SNPsaurus, LLC) that trimmed the reads using
 bbduk (BBMap tools, http://sourceforge.net/projects/bbmap/): bbmap/bbduk.sh in=reads/run_2780/2780_CAAGTGTC-GTAAGGAG_S25_L003_R1_001_subset.fastq.gz out=reads/run_2780/2780_CAAGTGTC-GTAAGGAG_S25_L003_R1_001_t.fastq.gz ktrim=r k=17 hdist=1 mink=8 ref=bbmap/resources/nextera.fa.gz minlen=100 ow=t qtrim=r trimq=10  Next, a de novo reference was created by collecting 10 million reads in total, evenly from the samples, and excluding reads that had counts fewer than 7 or more than 700. The remaining loci were then aligned to each other to identify allelic loci and collapse allelic haplotypes to a single representative.  All reads were mapped to the reference with an alignment identity threshold of .95 using bbmap (BBMap tools). Genotype calling was done using callvariants (BBMap tools) (callvariants.sh list=ref_turtle_rm.txt.align_samples out=turtle_total.vcf ref=ref_turtle.fasta ploidy=2 multisample=t rarity=0.05 minallelefraction=0.05 usebias=f ow=t nopassdot=f minedistmax=5 minedist=5 minavgmapq=15 minreadmapq=15 minstrandratio=0.0 strandedcov=t).  The Variant Call Format (VCF) was filtered to remove alleles with a population frequency of less than 3%. The VCF was used directly as input or for interconversion to other programs formats for subsequent coalescence-based and allele-frequency analyses methods. In addition, phylip and nexus format files were generated with the concatenated dataset of loci for each sample for phylogeographical reconstructions. They were excluded samples that had more than 75% missing data.
description: This is a VCF file.