Multiple measures could alleviate long-branch attraction in phylogenomic reconstruction of Cupressoideae (Cupressaceae).

Long-branch attraction (LBA) is a major obstacle in phylogenetic reconstruction. The phylogenetic relationships among Juniperus (J), Cupressus (C) and the Hesperocyparis-Callitropsis-Xanthocyparis (HCX) subclades of Cupressoideae are controversial. Our initial analyses of plastid protein-coding gene matrix revealed both J and C with much longer stem branches than those of HCX, so their sister relationships may be attributed to LBA. We used multiple measures including data filtering and modifying, evolutionary model selection and coalescent phylogenetic reconstruction to alleviate the LBA artifact. Data filtering by strictly removing unreliable aligned regions and removing substitution saturation genes and rapidly evolving sites could significantly reduce branch lengths of subclades J and C and recovered a relationship of J (C, HCX). In addition, using coalescent phylogenetic reconstruction could elucidate the LBA artifact and recovered J (C, HCX). However, some valid methods for other taxa were inefficient in alleviating the LBA artifact in J-C-HCX. Different strategies should be carefully considered and justified to reduce LBA in phylogenetic reconstruction of different groups. Three subclades of J-C-HCX were estimated to have experienced ancient rapid divergence within a short period, which could be another major obstacle in resolving relationships. Furthermore, our plastid phylogenomic analyses fully resolved the intergeneric relationships of Cupressoideae.