A New Paralog Removal Pipeline Resolves Conflict between RAD-seq and Enrichment

Abstract

Target enrichment and RAD-seq are well-established high throughput sequencing technologies that have been increasingly used for phylogenomic studies, and the choice between methods is a practical issue for plant systematists studying the evolutionary histories of biodiversity of relatively recent origins. However, few studies have compared the congruence and conflict between results from the two methods within the same group of organisms, especially in plants, where extensive genome duplication events may complicate phylogenomic analyses. Unfortunately, currently widely used pipelines for target enrichment data analysis do not have a vigorous procedure for remove paralogs in Hyb-Seq data. In this study, we employed RAD-seq and Hyb-Seq of Angiosperm 353 genes in phylogenomic and biogeographic studies of Hamamelis (the witch-hazels) and Castanea (chestnuts), two classic examples exhibiting the well-known eastern Asian-eastern North American disjunct distribution. We compared these two methods side by side and developed a new pipeline (PPD) with a more vigorous removal of putative paralogs from Hyb-Seq data. The new pipeline considers both sequence similarity and heterozygous sites at each locus in identification of paralogous. We used our pipeline to construct robust datasets for comparison between methods and downstream analyses on the two genera. Our results demonstrated that the PPD identified many more putative paralogs than the popular method HybPiper. Comparisons of tree topologies and divergence times showed significant differences between data from HybPiper and data from our new PPD pipeline, likely due to the error signals from the paralogous genes undetected by HybPiper, but trimmed by PPD. We found that phylogenies and divergence times estimated from our RAD-seq and Hyb-Seq-PPD were largely congruent. We highlight the importance of removal paralogs in enrichment data, and discuss the merits of RAD-seq and Hyb-Seq. Finally, phylogenetic analyses of RAD-seq and Hyb-Seq resulted in well-resolved species relationships, and revealed ancient introgression in both genera. Biogeographic analyses including fossil data revealed a complicated history of each genus involving multiple intercontinental dispersals and local extinctions in areas outside of the taxas modern ranges in both the Paleogene and Neogene. Our study demonstrates the value of additional steps for filtering paralogous gene content from Angiosperm 353 data, such as our new PPD pipeline described in this study. [RAD-seq, Hyb-Seq, paralogs, Castanea, Hamamelis, eastern Asia-eastern North America disjunction, biogeography, ancient introgression]