Loss of Different Inverted Repeat Copies from the Chloroplast Genomes of Pinaceae and Cupressophytes and Influence of Heterotachy on the Evaluation of Gymnosperm Phylogeny

Abstract

The relationships among the extant five gymnosperm groups—gnetophytes, Pinaceae, non-Pinaceae conifers (cupressophytes), Ginkgo, and cycads—remain equivocal. To clarify this issue, we sequenced the chloroplast genomes (cpDNAs) from two cupressophytes, Cephalotaxus wilsoniana and Taiwania cryptomerioides, and 53 common chloroplast protein-coding genes from another three cupressophytes, Agathis dammara, Nageia nagi, and Sciadopitys verticillata, and a non-Cycadaceae cycad, Bowenia serrulata. Comparative analyses of 11 conifer cpDNAs revealed that Pinaceae and cupressophytes each lost a different copy of inverted repeats (IRs), which contrasts with the view that the same IR has been lost in all conifers. Based on our structural finding, the character of an IR loss no longer conflicts with the “gnepines” hypothesis (gnetophytes sister to Pinaceae). Chloroplast phylogenomic analyses of amino acid sequences recovered incongruent topologies using different tree-building methods; however, we demonstrated that high heterotachous genes (genes that have highly different rates in different lineages) contributed to the long-branch attraction (LBA) artifact, resulting in incongruence of phylogenomic estimates. Additionally, amino acid compositions appear more heterogeneous in high than low heterotachous genes among the five gymnosperm groups. Removal of high heterotachous genes alleviated the LBA artifact and yielded congruent and robust tree topologies in which gnetophytes and Pinaceae formed a sister clade to cupressophytes (the gnepines hypothesis) and Ginkgo clustered with cycads. Adding more cupressophyte taxa could not improve the accuracy of chloroplast phylogenomics for the five gymnosperm groups. In contrast, removal of high heterotachous genes from data sets is simple and can increase confidence in evaluating the phylogeny of gymnosperms.

Dynamics of chloroplast genomes in green plants

“…A complete representation can be found in Supplemental Figure S1. IR A regions were lost in Cupressophytes [21,22]. The two-step model in the Pinaceae could also apply to the 424 bp of IR left in C. deodara.…”

“…2B, C) [6]. It also has been shown that different IR copies were lost in the cpDNAs of the Pinaceae and Cupressophytes of gymnosperms [21]. IR B regions were lost in Pinaceae in a two-step model, and only 495 bp were left in P. thunbergii, whereas 1.…”

“…The size of cpDNAs ranges from 118,360 to 184,933 bp, and the number of protein-coding genes from 70 to 99 in all 24 species, which exclude tRNA, rRNA, and unique ORF genes. There are inverted repeats (IRs) in all investigated cpDNAs except C. variabilis and S. punctulatum, and the length and the gene content of IRs increase steadily from green algae to monocots except for C. deodara, which lacks IRs [20,21] (Table 1).…”

“…Their lengths differ from 9589 bp (P. patens) to 27,659 bp (P. alba) with C. deodara and P. thunbergii having exceptional small IRs of only 424 bp and 495 bp because of significant gene losses [21] (Table 1). To illustrate the nature of sequence mobilities, IR copies were aligned from three algae (M. viride, C. vulgaris and C. globosum), three bryophytes (A. formosae, M. polymorpha and P. patens), two pteridophytes (P. nudum and E. arvense), three gymnosperms (G. biloba, P. thunbergii and C. deodara), one eudicot (A. thaliana), and one monocot (O. sativa) (Fig.…”

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

“…Homoplastic mutations occurring along the long external branches can obscure the true phylogenetic signal on the internal branches, then long-branch attraction (LBA) occurs, with the long branches erroneously clustered together45. The LBA artifact was suggested to adversely affect the accuracy of tree reconstruction in many phylogenetic studies67891011. It could be amplified in phylogenomics, thereby resulting in a strongly supported but incorrect phylogeny612.…”