Application of RAD-based phylogenetics to complex relationships among variously related taxa in a species flock

• We studied the utility of restriction-site associated DNA (RAD) sequences for phylogenetic analysis.
• We tested data matrices with different parameter settings for orthology search and taxon-coverage for individual loci.
• Topologically different, robust trees in terms of bootstrap values resulted from different data matrices.
• Robust trees were not necessarily obtained from larger data matrices with high proportions of missing data.
• Various data matrices produced with different settings should be used to propose alternative phylogenetic hypotheses.

Restriction site-associated DNA (RAD) sequences from entire genomes can be used to resolve complex phylogenetic problems. However, the processed data matrix varies depending on the strategies used to determine orthologous loci and to filter loci according to the number of taxa with sequence data for the loci, and often contains plenty of missing data. To explore the utility of RAD sequences for elucidating the phylogenetics of variously related species, we conducted RAD sequencing for the Ohomopterus ground beetles and attempted maximum-likelihood phylogenetic analyses using 42 data matrices ranging from 1.6 × 104 to 8.1 × 106 base pairs, with 11–72% missing data. We demonstrate that robust phylogenetic trees, in terms of bootstrap values, do not necessarily result from larger data matrices, as previously suggested. Robust trees for distantly related and closely related taxa resulted from different data matrices, and topologically different robust trees for distantly related taxa resulted from various data matrices. For closely related taxa, moderately large data matrices strongly supported a topology that is incompatible with morphological evidence, possibly due to the effect of introgressive hybridization. Practically, exploring variously prepared data matrices is an effective way to propose important alternative phylogenetic hypotheses for this study group.

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