DOI: 10.5061/dryad.z34tmpgbs
title: Evidence for speciation underground in diving beetles (Dytiscidae) from a subterranean archipelago

description: Most subterranean animals are assumed to have evolved from surface
 ancestors following colonisation of a cave system, however very few
 studies have raised the possibility of ‘subterranean speciation’ in
 underground habitats (i.e. obligate cave-dwelling organisms (troglobionts)
 descended from troglobiotic ancestors). Numerous endemic subterranean
 diving beetle species from spatially-discrete calcrete aquifers in Western
 Australia (stygobionts) have evolved independently from surface ancestors;
 however, several cases of sympatric sister species raises the possibility
 of subterranean speciation. We tested this hypothesis using vision
 (phototransduction) genes that are evolving under neutral processes in
 subterranean species and purifying selection in surface species. Using
 sequence data from 32 subterranean and five surface species in the genus
 Paroster (Dytiscidae), we identified deleterious mutations in: long
 wavelength opsin (lwop), arrestin 1 (arr1), and arrestin 2 (arr2) shared
 by a sympatric sister-species triplet, arr1 shared by a sympatric
 sister-species pair, and lwop and arr2 shared among closely related
 species in adjacent calcrete aquifers. In all cases, a common ancestor
 possessed the function-altering mutations, implying they were already
 adapted to aphotic environments. Our study represents one of the first
 confirmed cases of subterranean speciation in cave insects. The assessment
 of genes undergoing pseudogenisation provides a novel way of testing modes
 of speciation and the history of diversification in blind cave animals.
description: Sequence data of three
 phototransduction genes (<i>arr1, arr2, </i>and<i>
 lwop</i>) were obtained from targeted sequencing of exons from nine
 phototransduction genes derived from parallel studies of 10 stygobiotic
 and two surface <i>Paroster</i> species (Tierney et al. 2015,
 2018; Langille 2020). The study
 used RNA baits, designed from transcriptome data from the surface species
 <i>Paroster nigroadumbratus</i> (Tierney et al. 2015), to
 enrich for <i>arr1</i> (1128 bp), <i>arr2</i>
 (1194 bp) and <i>lwop</i> (1235 bp) exons, targeting the
 entire genes. The enriched fragment libraries (followed standard
 library preparation from Meyer and Kircher 2010, and MYbaits targeted
 protocol following the MYbaits user manual v2 and Langille 2020) of each
 exon were sequenced using an Illumina MiSeq and 150 bp paired-end reads.
 Sequence data from <i>lwop</i> were also obtained from a
 Geneious blast search of whole-genome shotgun libraries, sequenced using
 an Illumina MiSeq, for a sympatric sister species triplet from the Sturt
 Meadows calcrete (Hyde et al. 2018):  <i>P.
 macrosturtensis</i> (14.6 million sequences), <i>P.
 mesosturtensis </i>(1.0 million sequences), and <i>P.
 microsturtensis </i>(4.6 million sequences). The dytiscid sequence
 data obtained via targeted exon capture were assembled,<i>
 </i>using a combination of bowtie2 (Langmead and Salberg 2012) and
 MIRA4 (Chevreux et al. 1999), and aligned to the reference sequence using
 Geneious v.10.2.6 (Kearse et al. 2012), and the plugin ClustalW (Larkin et
 al. 2007). Comparative sequence analyses of
 the <i>lwop</i>, <i>arr1</i> and
 <i>arr2</i> exon data revealed shared deleterious mutations
 (frameshift and/or stop codons) among taxa (see results) and we further
 targeted the exons containing these mutations from additional
 <i>Paroster</i> species using PCR-amplification and Sanger
 sequencing analyses (see Table S2 for details of primers; Supplementary
 Information 1 for detailed PCR-amplification protocol; Table S3 for list
 of species with successfully/not successfully sequenced data). Sequencing
 was performed using a Prism BigDye Terminator Cycle sequencing kit (PE
 Applied Biosystems) with 10 mL reaction volumes according to the
 manufacturer’s protocol, with reaction products purified using a
 Multiscreen 384 vacuum well SEQ plate (Millipore Sigma). Reactions were
 capillary sequenced by the Australian Genome Research Facility (Adelaide,
 Australia).
description: Missing bases are represented by 'N'.