The qualification of orthology is a significant challenge when developing large, multiloci phylogenetic data sets from assembled transcripts. Transcriptome assemblies have various attributes, such as fragmentation, frameshifts and mis-indexing, which pose problems to automated methods of orthology assessment. Here, we identify a set of orthologous single-copy genes from transcriptome assemblies for the land snails and slugs (Eupulmonata) using a thorough approach to orthology determination involving manual alignment curation, gene tree assessment and sequencing from genomic DNA. We qualified the orthology of 500 nuclear, protein-coding genes from the transcriptome assemblies of 21 eupulmonate species to produce the most complete phylogenetic data matrix for a major molluscan lineage to date, both in terms of taxon and character completeness. Exon capture targeting 490 of the 500 genes (those with at least one exon textgreater120 bp) from 22 species of Australian Camaenidae successfully captured sequences of 2825 exons (representing all targeted genes), with only a 3.7% reduction in the data matrix due to the presence of putative paralogs or pseudogenes. The automated pipeline Agalma retrieved the majority of the manually qualified 500 single-copy gene set and identified a further 375 putative single-copy genes, although it failed to account for fragmented transcripts resulting in lower data matrix completeness when considering the original 500 genes. This could potentially explain the minor inconsistencies we observed in the supported topologies for the 21 eupulmonate species between the manually curated and ‘Agalma-equivalent’ data set (sharing 458 genes). Overall, our study confirms the utility of the 500 gene set to resolve phylogenetic relationships at a range of evolutionary depths and highlights the importance of addressing fragmentation at the homolog alignment stage for probe design.

Museums hold most of the world’s most valuable biological specimens and tissues collected, including type material that is often decades or even centuries old. Unfortunately, traditional museum collection and storage methods were not designed to preserve the nucleic acids held within the material, often reducing its potential viability and value for many genetic applications. High-throughput sequencing technologies and associated applications offer new opportunities for obtaining sequence data from museum samples. In particular, target sequence capture offers a promising approach for recovering large numbers of orthologous loci from relatively small amounts of starting material. In the present study, we test the utility of target sequence capture for obtaining data from museum-held material from a speciose mammalian genus: the horseshoe bats (Rhinolophidae: Chiroptera). We designed a ‘bait’ for capturing > 3600 genes and applied this to 10 species of horseshoe bat that had been collected between 93 and 7 years ago and preserved using a range of methods. We found that the mean recovery rate per species was approximately 89% of target genes with partial sequence coverage, ranging from 3024 to 3186 genes recovered. On average, we recovered 1206 genes with ≥ 90% sequence coverage, per species. Our findings provide good support for the application of large-scale bait capture across congeneric species spanning approximately 15 Myr of evolution. On the other hand, we observed no clear association between the success of capture and the phylogenetic distance from the bait model, although sample sizes precluded a formal test.