Comparison of paralog identification methods and their impact on species tree topologies in target capture phylogenomics within the Sindora clade (Detarioideae: Leguminosae).

Abstract

Target capture is a common method of generating high throughput DNA sequencing data for phylogenetic reconstruction of species relationships, for which single copy genes are usually most informative. However, a pervasive problem with target capture is that putatively single copy genes may in fact be paralogs resulting from gene duplication, which are problematic for phylogenetic inference because their evolutionary history may differ from the divergence history of species. Here, we use as a case study a target enrichment dataset of 88 species of Detarioideae (Leguminosae) with a focus on the Sindora clade to examine approaches for handling paralogs, including the built-in paralog handling functions in HybPiper and CAPTUS, plus subsequent steps using Putative Paralog Detection and the tree-based Yang & Smith orthology inference approach. We compare the paralogs flagged using these methods and verify their performance with BLAST mapping against a reference genome sequence of Sindora glabra, and then subsequently compare the species tree topologies produced across these methods. Our comparisons of paralogs flagged across the Sindora clade show that the Putative Paralog Detection pipeline was the most accurate in identifying paralogs in terms of its similarity to the BLAST mapping, followed by the built-in paralog identification function of CAPTUS. However, the results we recovered for the Detarioideae subfamily suggest that the largest differences in species tree topology resulted from the use of paralog-filtered alignments (such as with the Putative Paralog Detection pipeline and the Yang & Smith orthology inference approaches) rather than just by removing the sequences of identified paralogous genes. This was the true for HybPiper-assembled datasets but was not seen in CAPTUS-assembled datasets. In all comparisons, the topological differences caused by different paralog handling methods tended to be confined to clades where processes such as hybridisation and introgression are prevalent. Our study provides a roadmap to establish the best approach to identify, eliminate or separate paralogs in the absence of a chromosomally contiguous reference genome for a study group, and highlights the importance of careful data inspection and processing in addition to understanding the extent of paralogy and paralog characteristics (e.g. sequence divergence between copies) for their study group.