Evidence for speciation underground in diving beetles (Dytiscidae) from a subterranean archipelago

Sequence data of three phototransduction genes (arr1, arr2, and lwop) were obtained from targeted sequencing of exons from nine phototransduction genes derived from parallel studies of 10 stygobiotic and two surface Paroster species (Tierney et al. 2015, 2018; Langille 2020). The study used RNA baits, designed from transcriptome data from the surface species Paroster nigroadumbratus (Tierney et al. 2015), to enrich for arr1 (1128 bp), arr2 (1194 bp) and lwop (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 lwop 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): P. macrosturtensis (14.6 million sequences), P. mesosturtensis (1.0 million sequences), and P. microsturtensis (4.6 million sequences). The dytiscid sequence data obtained via targeted exon capture were assembled, 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 lwop, arr1 and arr2 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 Paroster 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).

本研究获取了3个光转导基因（phototransduction genes）arr1、arr2及lwop的序列数据，这些数据源自对9个光转导基因外显子的靶向测序，相关基因来自针对10种穴居水生Paroster属物种与2种地表生Paroster属物种的平行研究（Tierney等，2015、2018；Langille，2020）。本研究基于地表生物种Paroster nigroadumbratus的转录组数据设计RNA探针（Tierney等，2015），用于富集arr1（1128 bp）、arr2（1194 bp）及lwop（1235 bp）的外显子，覆盖目标基因的全长区域。每个外显子的富集片段文库均采用Illumina MiSeq平台进行150 bp双端测序；文库构建遵循Meyer与Kircher（2010）的标准文库制备流程，MYbaits靶向捕获流程则参照MYbaits用户手册v2及Langille（2020）的方案。此外，lwop的序列数据还可通过Geneious BLAST搜索获取，该搜索针对Sturt Meadows钙结核地层的同域姊妹物种种组（Hyde等，2018）的全基因组鸟枪法文库开展，此类文库同样采用Illumina MiSeq平台测序，涉及的物种包括P. macrosturtensis（1460万条序列）、P. mesosturtensis（100万条序列）及P. microsturtensis（460万条序列）。本研究通过靶向外显子捕获获得的龙虱科序列数据，结合bowtie2（Langmead & Salberg，2012）与MIRA4（Chevreux等，1999）进行组装，并使用Geneious v.10.2.6（Kearse等，2012）及其ClustalW插件（Larkin等，2007）将组装序列比对至参考序列。对lwop、arr1及arr2外显子数据的比较序列分析显示，各分类群间存在共有的有害突变（移码突变和/或终止密码子突变）（详见结果部分）；本研究进一步通过PCR扩增与Sanger测序，对额外Paroster属物种中携带此类突变的外显子开展靶向测序（引物详情见表S2；详细PCR扩增方案见补充信息1；测序成功/失败的物种列表见表S3）。桑格测序采用Prism BigDye Terminator循环测序试剂盒（PE Applied Biosystems），反应体系为10 mL，实验流程严格遵循制造商说明书；反应产物通过Multiscreen 384孔真空SEQ板（Millipore Sigma）进行纯化。测序反应由澳大利亚基因组研究设施（澳大利亚阿德莱德）完成毛细管电泳测序。