Sliding-window phylogenetic analyses uncover complex interplastomic recombination in the tropical Asian–American disjunct plant genus Hedyosmum (Chloranthaceae)

The chloroplast genomic (i.e. plastomic) sequences have long been used for inferring phylogenetic relationships of green plants. Current major plant classifications (e.g. Angiosperm Phylogeny Group classification, APG IV, 2016; Pteridophyte Phylogeny Group classification, PPG I, 2016) are predominantly based on the plastid phylogenies (Stull et al., 2023). Due to the rapid progress in DNA sequencing technologies along with decreasing costs, phylogenetic analyses using whole plastomes have become a routine practice (Wang et al., 2024). Plastomes have been presumed to be single double-stranded circular DNA molecules that are inherited uniparentally, with maternal inheritance in most angiosperms and paternal inheritance in gymnosperms (Birky, 1995; Dong et al., 2012; Greiner et al., 2015). These characteristics led to the general belief that plastomes are free from or less likely to undergo intermolecular recombination (Walker et al., 2019). Therefore, different plastomic genes or regions, which are assumed to share the same evolutionary trajectory, are often concatenated directly for phylogenetic analyses (Jansen et al., 2007; Moore et al., 2010; Li et al., 2021).

Despite the widespread use of plastomes in phylogenetics, both biparental inheritance and recombination of plastomes – processes that could inadvertently affect inference – have been increasingly detected. The mechanisms that maintain uniparental inheritance, including elimination or degradation of the organelle during male gametophyte development or after pollen mitosis or fertilization, may break down and lead to biparental inheritance (Nagata, 2010). Biparental inheritance of plastomes has been reported in some plant groups, such as Passiflora (Passifloraceae; Hansen et al., 2007; Shrestha et al., 2021), Cicer arietinum (Fabaceae; Kumari et al., 2011), and Actinidia (Actinidiaceae; Li et al., 2013). It is believed that heteroplasmy, that is the mixture of different organelle genomes within a cell or individual, is widespread in both animals and plants (Nagata, 2010; Ramsey & Mandel, 2019; Camus et al., 2022), and c. 20% of angiosperm genera may have undergone biparental inheritance (Zhang & Sodmergen., 2010; Sakamoto & Takami, 2024). The biparental inheritance allows the coexistence of both maternal and paternal plastids in the same offspring cell, creating opportunities for interplastomic recombination. Interspecific plastomic recombination has been created and detected in experimental studies (Medgyesy et al., 1985). However, unlike in the intraplastomic recombination, in which causes (e.g. mediated by short repeat sequences) and consequences (e.g. generating structural variations, such as insertions, deletions, isomers, and concatemers) have been extensively studied (Kolodner & Tewari, 1979; Palmer, 1983; Ogihara et al., 1988; Bendich, 2004; Oldenburg & Bendich, 2004, 2016; Kobayashi et al., 2017), interplastomic recombination, especially historical or natural ones, has rarely been explored, with only a few cases reported (Sullivan et al., 2017; Sancho et al., 2018; Zhu et al., 2018).

If interplastomic recombination happened, particularly from divergent parental plastomes, the traditional viewpoint of a plastome as a single nonsegregated locus should be overthrown (Gonçalves et al., 2019; Doyle, 2022). Different regions of the recombined plastome undergo distinct evolutionary trajectories, which are expected to be more closely related to their respective parental (i.e. paternal or maternal) lineages (Fig. 1; more in the Materials and Methods section). If that is the case, they should not be combined in phylogenetic analyses. However, in most phylogenetic studies, the plastid regions are routinely concatenated into a supermatrix, which is then used for phylogenetic inference. In recent years, an increasing number of studies have detected phylogenetic conflicts within plastomes (Walker et al., 2019; Xiao et al., 2020; Zhang et al., 2020; Liu et al., 2022; Thureborn et al., 2024). Conflicts have been demonstrated among different plastomic regions, genes, and functional gene groups (Liu et al., 2012; Walker et al., 2014; Saarela et al., 2018; Zhang et al., 2020; Yang et al., 2021). These conflicts have usually been attributed to stochastic or systematic errors, such as from methodological deficiencies, molecular evolutionary complexities, or as artifacts of incomplete taxon sampling (Burleigh & Mathews, 2007; Liu et al., 2012; Xue et al., 2024). For example, nonfunctional regions, such as intergenic spacers, evolve faster, which are prone to reach saturation, leading to phylogenetic trees that are inconsistent with those derived from coding regions (Liu et al., 2022; Wang et al., 2022). Additionally, rate heterogeneity among different plastid genes or functional groups and horizontal gene transfer between plastomes and mitogenomes or nuclear genomes are also possible factors associated with intraplastomic phylogenetic discordance (Smith, 2014; Zhang et al., 2020; Daniell et al., 2021). Almost no intraplastomic phylogenetic conflict was definitely or exclusively ascribed to interplastomic recombination (but see Sullivan et al., 2017; Sancho et al., 2018; Zhu et al., 2018). Particularly, it has not been elucidated why and how intraplastomic phylogenetic conflict can be reliably attributed to interplastomic recombination. Neither a clear rationale nor a proper statistical framework has been proposed for detecting or assessing the confidence of interplastomic recombination through phylogenetic analysis.

Chloranthaceae is an early diverged angiosperm family comprising four extant genera – Ascarina, Chloranthus, Hedyosmum, and Sarcandra – and over 70 species (Todzia, 1988; Zhang et al., 2011). Chloranthus and Sarcandra have bisexual flowers, while Ascarina and Hedyosmum have unisexual flowers and are usually dioecious (Todzia, 1988; Kong et al., 2002). Hedyosmum possesses over 40 species, which are distributed in tropical America, except for H. orientale isolated in Southeast Asia (Todzia, 1988). Traditionally, Hedyosmum is classified into two subgenera, that is, Hedyosmum and Tafalla. Previous phylogenetic studies utilizing several plastid fragments and nuclear ribosomal internal transcribed spacer (ITS) sequences demonstrated widespread phylogenetic conflicts in Hedyosmum (Zhang et al., 2015). These conflicts involved even the deepest splits within the genus, in which H. orientale was suggested to be either the sister of all other Hedyosmum species or nested within them, being the sister to subgenus Tafalla (Zhang & Renner, 2003; Antonelli & Sanmartín, 2011; Zhang et al., 2011, 2015). These conflicts were detected only between the plastid and nuclear ITS sequences and are, at least partially, ascribed to possible rampant hybridization or reticulated evolution in Hedyosmum (Zhang et al., 2015). However, whether the phylogenetic conflicts exist within the plastome or between plastid fragments has not yet been explored.

In this study, we first propose a rationale and then present a novel statistical framework for detecting interplastomic recombination based on sliding-window phylogenetic analysis (SWPA). Then, based on 22 plastomic sequences of Chloranthaceae, we investigate whether there are any intraplastomic conflicts within Chloranthaceae, particularly in the largest genus Hedyosmum, which experienced complex reticulate evolution. For any such conflict, we also examine whether it can be attributed to interplastomic recombination and what abiotic settings might have been associated with it. Using our methodology, we indeed find a striking phylogenetic conflict in Hedyosmum that is reliably ascribed to interplastomic recombination; that the complex interplastomic recombination with two or more instances of template shifts happened around the Oligocene–Miocene boundary; and that the involved lineages are now disjunct in tropical Asia and America, possibly as a result of global climate cooling or long-distance dispersal. This study highlights the necessity of examining and separately using different intraplastomic fragments with alternative evolutionary trajectories in phylogenetic analyses. The methodology could be easily applied or extended to detect intermolecular recombination elsewhere, such as in nuclear genomes.

In this study, we presented the rationale and a statistical framework based on SWPA for detecting interplastomic recombination. Using our new methodology, we identified a strongly supported interplastomic recombination event in Chloranthaceae. This event could not be detected by traditional phylogenetic analyses and existing recombination detection methods. The recombinant plastome is shown to result from two or more transitions along two distinct parental DNA templates in replication, with one transition in the LSC region and the other in the IR region. The estimated lineage ages indicate the regions of the recombinant with different evolutionary trajectories diverged from their two donors almost simultaneously at the Oligocene–Miocene boundary, being coincident with the assumption of recombination. The recombinant and the two parental lineages are presently disjunct in tropical Asia and America, perhaps as an outcome of subsequent climate cooling or long-distance dispersal.

None declared.

QZ conceived this study. Y-BL, QZ and X-RY collected the data. P-WL led the data analyses with input from Y-BL, Z-JZ and X-MQ under the guidance of QZ. P-WL drafted the manuscript. QZ led the revision with contributions from AA and WW. P-WL and Y-BL contributed equally to this work.

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