Backbone phylogeny and evolution of Apioideae (Apiaceae): New insights from phylogenomic analyses of plastome data

Highlights

• Great incongruences were detected in the chloroplast-genome-based and transcriptome-based phylogenies, which mainly appeared among the distally branching clades (Group A).

• At least four cases of chloroplast capture were inferred within Group A.

• Dating analysis suggested the rapid evolution of Group A during the middle to late Miocene, which was interpreted as a response to dramatic climate change at that period.

• This paper explored the correlation between expansion and contraction of IRs and the chloroplast-genome-based phylogeny of Apioideae.

Abstract

Traditional phylogenies inferred from chloroplast DNA fragments have not obtained a well‐resolved evolutionary history for the backbone of Apioideae, the largest subfamily of Apiaceae. In this study, we applied the genome skimming approach of next‐generation sequencing to address whether the lack of resolution at the tip of the Apioideae phylogenetic tree is due to limited information loci or the footprint of ancient radiation. A total of 90 complete chloroplast genomes (including 23 newly sequenced genomes and covering 20 major clades of Apioideae) were analyzed (RAxML and MrBayes) to provide a phylogenomic reconstruction of Apioideae. Dating analysis was also implemented using BEAST to estimate the origin and divergence time of the major clades. As a result, the early divergences of Apioideae have been clarified but the relationship among its distally branching clades (Group A) was only partially resolved, with short internal branches pointing to an ancient radiation scenario. Four major clades, Tordyliinae I, Pimpinelleae I, Apieae and Coriandreae, were hypothesized to have originated from chloroplast capture events induced by early hybridization according to the incongruence between chloroplast-based and nrDNA-based phylogenetic trees. Furthermore, the variable and nested distribution of junction positions of LSC (Large single copy region) and IR_B (inverted repeat region B) in Group A may reflect incomplete lineage sorting within this group, which possibly contributed to the unclear phylogenetic relationships among these clades inferred from plastome data. Molecular clock analysis revealed the chloroplast capture events mainly occurred during the middle to late Miocene, providing a geological and climate context for the evolution of Apioideae.

Introduction

Apioideae is the largest subfamily of Apiaceae (a family that has abundant species and high morphological diversity, Fig. 1), currently comprises about 380 genera and 3200 species, representing 85.2% genera and 83.8% species of Apiaceae (Angiosperm Phylogeny Website, Stevens, updated 2021). Species of this subfamily are distributed worldwide but chiefly in northern temperate regions (Calviño et al., 2016). Apioideae species are well known for the conspicuous umbel arranged flowers (simple or compound umbels), and characteristic fruits consisting of two one-seeded mericarps suspended from a free carpophore. Many species of Apioideae are economically important, as foods (e.g., carrot and parsnip), herbs (e.g., species of Angelica L. and Bupleurum L.), and spices (e.g., coriander, fennel and parsley), as well as some poisonous species (e.g., water hemlock and poison hemlock). As an economically important group, Apioideae have been a particular focus of many botanists, and a series of taxonomic and phylogenetic studies have been performed. Phylogenetic relationships among major clades within Apioideae have historically been a major phylogenetic challenge. Previous molecular phylogenetic studies based on a limited number of loci have greatly advanced our understanding of taxa relationships intra Apioideae. However, major classifications of Apioideae do not agree between molecular phylogenetic and morphological studies, or among different molecular phylogenetic analyses (Calviño et al., 2006, Calviño et al., 2016, Zhou et al., 2009, Downie et al., 2010, Banasiak et al., 2013, Jiménez-Mejías and Vargas, 2015).

A total of 41 major clades within Apioideae have been identified using nuclear ribosomal DNA (nrDNA) internal transcribed spacer (ITS) sequences in conjunction with results inferred from the chloroplast DNA (cpDNA) sequences (Downie et al. 2010). However, the uncertain relationships among the major clades have hindered a better understanding of patterns of Apioideae diversification and the evolution of key traits. Following the studies reviewed earlier, portions of the phylogenetic tree remained unresolved, with weakly supported and conflicting relationships (Downie et al., 1996, Downie et al., 2000a, Downie et al., 2001, Plunkett et al., 1996, Plunkett et al., 1997, Plunkett and Downie, 1999, Lee and Downie, 2000, Calviño et al., 2006, Calviño et al., 2016, Zhou et al., 2009). The ambiguous relationships are mostly associated with the distally branching clades belonging to the apioid superclade, which was firstly defined by Plunkett and Downie (1999) and then extended and summarized by Downie et al. (2010). The high level incongruence between chloroplast and nuclear phylogenies of Apioideae was ubiquitous in the previous studies, which was suggested to be caused by lack of phylogenetic signals in the data (Zhou et al., 2009), or resulting from hybridization and/or introgression events occurring in the early history of these plants (Calviño et al., 2006, Yi et al., 2015). Hybridization and/or introgression events may induce chloroplast capture, which was considered a mechanism for the phylogenetic discordance (Okuyama et al., 2005, Yi et al., 2015, Folk et al., 2017, Liu et al., 2017, Liu et al., 2020, Zhao et al., 2020). The weak phylogenetic signal due to insufficient informative sites and stochastic causes was suggested to be the main reason for the depressed resolution of cpDNA-fragments-based phylogenies.

In recent years, transcriptome or genome-wide have been successfully used for studies on multiple species phylogenies. Wen et al (2020) have extracted 3351 nuclear single copy genes from transcriptome data and used for phylogenetic analysis of the Apioideae. A well-supported species tree was generated with a topology differed from the previous phylogenetic trees, especially for the structure within Group A (consists of several clades belong to apioid superclade, including Pyramidoptereae Boiss., Careae Baill., Pimpinelleae Spreng., Apieae Takht. ex V.M. Vinogr., Sinodielsia Clade, Coriandreae W.D.J. Koch, Tordyliinae Engl., and Selineae Spreng.) (Sprengel, 1820, Koch, 1824, Boissier, 1872, Downie et al., 2000c, Downie et al., 2010, Zhou et al., 2008). The low quartet supports but strong local posterior probabilities in branches within this group were also suggested to reflect incomplete lineage sorting of gene trees and the rapid evolutionary divergence of Group A. This was the first attempt to analyze the subfamily Apioideae with larger dataset, but due to the limited sampling (only 28 species), a comprehensive study would be needed in the future.

Due to their small size, high copy number per cell, moderate nucleotide substitution rates, and freedom from problems of paralogy, chloroplast genome sequences have been widely used for the reconstruction of plant phylogenies (Raubeson and Jansen, 2005, Downie and Jansen, 2015, Zhang et al., 2017). A plastome phylogenomics approach has been successfully applied to resolve many enigmatic relationships within angiosperms (Jansen et al., 2007, Moore et al., 2010, Malé et al., 2014, Yu et al., 2017, Yu et al., 2019, Zhang et al., 2017, Li et al., 2019, Valcárcel and Wen, 2019, Xie et al., 2020, Zeb et al., 2020). Most studies of Apioideae based on chloroplast genomes were focused on the reports of individual species or the comparative analysis of a few species, with several phylogenetic analyses of intra-generic or intra-tribal (Downie and Jansen, 2015, Spooner et al., 2017, Yang et al., 2017, Kang et al., 2019, Li et al., 2020, Wang et al., 2021). For example, the entire plastid phylogeny of Daucus L. was reconstructed by Spooner et al. (2017), producing a well-resolved whole-plastid phylogenetic relationship within the genus. Spooner et al. (2017) found that this phylogenetic relationship was incongruent with that inferred from nuclear orthologous genes and detected mitochondrial/nuclear DNA insertions into the plastids of these species. Yang et al.’s (2017) phylogenetic analysis of four Notopterygium H. de Boissieu species suggested a monophyletic clade of this genus and inconsistent interspecies relationships between the cpDNA and ITS markers. Kang et al. (2019) also conducted a larger phylogenetic analysis based on 36 Apioideae chloroplast genomes (covering 12 major clades) and finally failed to recover Tordyliinae as a monophyletic clade (Kang et al., 2019). It is necessary to perform a comprehensive chloroplast genome analysis on the subfamily Apioideae. We use plastid phylogenomics here to better resolve phylogenetic relationships within Apioideae.

The large-scale evolutionary history of Apioideae remains poorly understood, although previous dating analyses have provided some insights. Most previous studies estimated a stem age of Apioideae between 56.64 (45.18, 73.53) Mya and 65.78 (58.21, 74.31) Mya (Nicolas and Plunkett, 2014, Calviño et al., 2016, Wen et al., 2020). Only a few studies have examined the divergence times among the major clades of the subfamily (Banasiak et al., 2013, Wen et al., 2020). The lack of a robust phylogenetic framework and time tree has hindered development of a full understanding of the diversification of Apioideae.

The major objectives of this study are to: resolve the long-standing contentious phylogenetic relationships among major clades within Apioideae; test the utility of chloroplast genome sequence data to resolve phylogenetic relationships of Apioideae; and explore the temporal diversification patterns of Apioideae with respect to environmental changes. Integrating 23 newly and 67 previously reported chloroplast genomes, this study applies multiple phylogenetic reconstruction methods in combination with appropriate models of sequence evolution to estimate phylogenetic relationship. Firstly, we compared the basic components of these genomes, and the protein-coding sequences (CDS) were extracted for the downstream analyses. The variation of each CDS region was also checked. Then, targeted CDS regions were aligned and trimmed for phylogenetic analysis. Furthermore, the divergence times of the major clades were estimated to explore the possible evolutionary histories of Apioideae.