Establishing species boundaries in Bornean geckos

Species delimitation using mitochondrial DNA (mtDNA) remains an important and accessible approach for discovering and delimiting species. However, delimiting species with a single locus (e.g. DNA barcoding) is biased towards overestimating species diversity. The highly diverse gecko genus Cyrtodactylus is one such group where delimitation using mtDNA remains the paradigm. In this study, we use genomic data to test putative species boundaries established using mtDNA within three recognized species of Cyrtodactylus on the island of Borneo. We predict that multilocus genomic data will estimate fewer species than mtDNA, which could have important ramifications for the species diversity within the genus. We aim to 1) investigate the correspondence between species delimitations using mtDNA and genomic data; 2) infer species trees for each target species; and 3) quantify gene flow and identify migration patterns to assess population connectivity. We show mtDNA approaches overestimate species d..., To generate genomic data, we conducted double digest restriction-site associated DNA sequencing (ddRADseq). We double-digested each sample using the digestion enzymes SbfI and MspI in CutSmart Buffer (New England Biolabs) for 7 hours at 37º C. For fragment purification, we used Sera-Mag SpeedBeads. We then ligated eight distinct barcodes to the cut sites of the fragmented DNA and subsequently size-selected (between 415 and 515 bp after accounting for adapter length) each library on a Blue Pippin Prep size fractionator (Sage Science). For the final library amplification, we used Phusion Hi-Fidelity DNA Polymerase and Illumina's indexed primers. We determine the concentration and size distribution of each indexed pool using an Agilent 2200 TapeStation. Lastly, we sent the quantified pools to QB3-Berkeley Genomics, UC Berkeley for qPCR to determine sequenceable library concentrations before multiplexing equimolar amounts of each pool for sequencing on two Illumina HiSeq 4000 lane (51-bp, s..., , # Establishing species boundaries in Bornean geckos

[https://doi.org/10.5061/dryad.w0vt4b90w](https://doi.org/10.5061/dryad.w0vt4b90w)

## Description of the data and file structure

Fieldwork was conducted in Borneo in the Malaysian state of Sarawak between 2014 and 2018 (Permits: NPW.907.4.4.(Jld.14)-79; (119)JHS/NCCD/600-7/2/107).

To delimit species using mtDNA, we assembled and aligned NADH dehydrogenase subunit 2 (ND2) data. We generated an assembly for *Cyrtodactylus* species in Borneo, comprising two evolutionarily distinct clades referred to herein as the small-bodied (*Cyrtodactylus cavernicolus, C. miriensis, C. pubisulcus, C. hantu*) and large-bodied (*C. consobrinus, C. hutan, C. kapitensis, C. malayanus*) clades. Sample sizes for the target species are as follows: *C. miriensis* = 19, *C. pubisulcus* = 20, and *C. consobrinus* = 9. We used the assembly to apply commonly used species delimitation methods (BPTP; MPTP; GMYC; ASAP) and calculate pairwise-distances (p-distance...