Sources of gene tree discordance and their implications for systematics and evolution of a megadiverse Australian plant radiation (subtribe Hakeinae, Proteaceae)

Resolving phylogenetic relationships in the presence of conflicting signal across genes is one of the major challenges of the phylogenomic era. Conflicting signals can emerge from biological processes, such as incomplete lineage sorting or introgression, or have technical origins, such as from misaligned sequences. As such, decisions made in the process of estimating species trees may result in alternative tree topologies and large variation in branch support values with important systematic consequences. Here we compare alternative strategies for alignment cleaning, loci filtering, and phylogenetic estimation in 551 taxa in the Proteaceae subtribe Hakeinae, to explore how these methodological choices affect the estimation of relationships. We found that node support values across gene trees were low and gene discordance was high in the Hakeinae, particularly in lineages from the Temperate Forests biome of southeastern Australia. Higher stringency of alignment cleaning tended to decrease node support and filtering desirable loci tended to increase gene concordance. Cleaning, filtering, and phylogenetic estimation method (short-cut coalescent or concatenation) have significant effects of tree topologies with distinct clusters of similar tree topologies detected in tree space. Of note, when using concatenated approaches, the two largest Hakeinae genera, Hakea and Grevillea, were reciprocally monophyletic. However, using coalescent approaches, we regularly found that Hakea was nested within Grevillea. Our results suggest that widespread gene discordance may be the result of rapid radiation and incomplete lineage sorting, demonstrating the importance of assessing the drivers of discordance to understand phylogenetic relationships.

Methods
In short, This study used 551 sequences for 482 Proteaceae taxa, of which 186 sequences were obtained from an earlier study (Cardillo et al. 2017). These 186 sequences include 151 species of Hakea, four species of Grevillea (G. dimorpha, G. evanescens, G. hookeriana, and G. batrachiodes), and Opisthiolepis heterophylla of the Hakeinae, together with eight Proteaceae outgroup taxa: Lomatia silaifolia, Stenocarpus davallioides, Alloxylon pinnatum, A. flammeum, Telopea speciosissima, Banksia paludosa, B. rufa, and Lambertia formosa. In addition, 368 samples were collected for novel DNA extraction and sequencing, sourced from fresh plant tissue obtained from wild or cultivated plants, or from dried herbarium specimens (see Table S1 for a list of samples). These included 320 species of Grevillea and Finschia chloroxantha from the Hakeinae, and additional Proteaceae outgroups: Stenocarpus milnei, Oreocallis mucronata, Macadamia integrifolia, Adenanthos glabrescens, Lambertia formosa, Isopogon formosus. Samples were sequenced using Anchored Hybrid Enrichment.