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. 2023 Feb 1;132(1):15–28. doi: 10.1093/aob/mcad022

Deciphering complex reticulate evolution of Asian Buddleja (Scrophulariaceae): insights into the taxonomy and speciation of polyploid taxa in the Sino-Himalayan region

Fengmao Yang 1,2,#, Jia Ge 3,4,#,✉, Yongjie Guo 5,6, Richard Olmstead 7, Weibang Sun 8,9,✉
PMCID: PMC10550280  PMID: 36722368

Abstract

Background and Aims

Species of the genus Buddleja in Asia are mainly distributed in the Sino-Himalayan region and form a challenging taxonomic group, with extensive hybridization and polyploidization. A phylogenetic approach to unravelling the history of reticulation in this lineage will deepen our understanding of the speciation in biodiversity hotspots.

Methods

For this study, we obtained 80 accessions representing all the species in the Asian Buddleja clade, and the ploidy level of each taxon was determined by flow cytometry analyses. Whole plastid genomes, nuclear ribosomal DNA, single nucleotide polymorphisms and a large number of low-copy nuclear genes assembled from genome skimming data were used to investigate the reticulate evolutionary history of Asian Buddleja. Complex cytonuclear conflicts were detected through a comparison of plastid and species trees. Gene tree incongruence was also analysed to detect any reticulate events in the history of this lineage.

Key Results

Six hybridization events were detected, which are able to explain the cytonuclear conflict in Asian Buddleja. Furthermore, PhyloNet analysis combining species ploidy data indicated several allopolyploid speciation events. A strongly supported species tree inferred from a large number of low-copy nuclear genes not only corrected some earlier misinterpretations, but also indicated that there are many Asian Buddleja species that have been lumped mistakenly. Divergent time estimation shows two periods of rapid diversification (8–10 and 0–3 Mya) in the Asian Buddleja clade, which might coincide with the final uplift of the Hengduan Mountains and Quaternary climate fluctuations, respectively.

Conclusions

This study presents a well-supported phylogenetic backbone for the Asian Buddleja species, elucidates their complex and reticulate evolutionary history and suggests that tectonic activity, climate fluctuations, polyploidization and hybridization together promoted the diversification of this lineage.

Keywords: Buddleja, phylogenomics, reticulate evolution, polyploidy, plastid genome, low-copy nuclear gene

INTRODUCTION

Reticulation in evolution can occur as a result of hybridization, introgression or lateral gene transfer (Mallet et al., 2016; Suvorov et al., 2022) and is believed to be one of the main driving forces in the diversification of angiosperms (Mallet et al., 2016; Debray et al., 2022; Suvorov et al., 2022). Hybridization often occurs in lineages that have undergone recent radiations when their habitats undergo dramatic change, such as during climatic fluctuations and anthropogenic disturbance (Rieseberg and Willis, 2007; Abbott et al., 2013; Estep et al., 2014). Allopolyploids arise from the integration of distinct parental chromosome sets (Van de Peer et al., 2017), which will lead to a highly dynamic genome (Pontes et al., 2004; Zhou et al., 2011) and might help plants to survive and thrive in precarious environmental conditions (Estep et al., 2014; Soltis et al., 2014; Edgeloe et al., 2022). Hybridization can also result in adaptive introgression, allowing species to adapt to new environments (Owens et al., 2016; Ma et al., 2019; Oziolor et al., 2019). Given that 25 % of all plant species are thought to have been involved in interspecific hybridization (Mallet, 2005), the construction of phylogenetic networks is particularly important for understanding the evolutionary history of plant species, especially that of recently radiated taxa (Mallet et al., 2016; Goulet et al., 2017).

Polyploidy (either allopolyploidy or autopolyploidy) is prevalent in angiosperms (Van de Peer et al., 2017). Polyploids, or plants that have undergone whole-genome duplications (WGDs), were once considered to be ‘evolutionary dead ends’ or ‘evolutionary noise’, because WGDs were thought to have only limited long-term evolutionary potential (Stebbins, 1950). Indeed, one study based on phylogenetic approaches has shown that polyploids have higher extinction rates and lower speciation rates than their diploid relatives (Mayrose et al., 2011). However, many recent studies have demonstrated that polyploidy is positively correlated with species adaptation and diversification (Levin and Soltis, 2018; Ren et al., 2018; Han et al., 2020), and WGD is now recognized as a major evolutionary force in plants (Soltis et al., 2014; Van de Peer et al., 2017; Wu et al., 2020).

Phylogenetic study of neopolyploids has proved to be challenging (Rothfels, 2021), because many polyploids arise from hybridization (allopolyploids; Funk and Omland, 2003; Rieseberg and Willis, 2007; Barker et al., 2016). An allopolyploid typically exhibits reproductive isolation from its parents, and allopolyploidy is generally considered to be a common mode of speciation (Ramsey and Schemske, 2002; Rieseberg and Willis, 2007; Abbott et al., 2013). Despite advances in the use of genomic data to resolve reticulate evolution in allopolyploid species (Guo et al., 2019; Jia et al., 2022), building a comprehensive evolutionary history for large taxonomic groups remains difficult (Diaz-Perez et al., 2018; Rothfels, 2021; Debray et al., 2022; Suissa et al., 2022).

Buddleja L. (Scrophulariaceae) are typically shrubs or small trees (Norman, 2000). Plants in this genus are known as butterfly bushes owing to their attractiveness to butterflies (Stuart, 2006) and are widely cultivated and important components in horticulture and human culture (Fig. 1; Tallent-Halsell and Watt, 2009). Some species (e.g. Buddleja davidii; Tallent-Halsell and Watt, 2009) have escaped cultivation and have become problematic and invasive in natural areas. In China, the genus is known as ‘Zui Yu Cao’, and the leaves of certain species (e.g. B. lindleyana and B. curviflora; Houghton, 1984) are used in fishing owing to their toxicity to fish. Some species have culinary applications and are used as medicines (e.g. B. officinalis, B. asiatica, B. davidii and B. lindleyana; Houghton, 1984; Li et al., 2020; Yan XX et al., 2021).

Fig. 1.

Fig. 1.

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Photographs of Asian Buddleja taxa: (1) B. colvilei; (2) B. sessilifolia; (3) B. forrestii; (4) B. macrostachya; (5) B. nivea; (6) B. myriantha; (7) B. candida; (8) B. albiflora; (9) B. fallowiana; (10) B. davidii; (11) B. alternifolia; (12) B. tsetangensis; (13) B. jinsixiaensis; (14) B. caryopteridifolia; (15) B. crispa; (16) B. curviflora; (17) B. japonica; (18) B. lindleyana; (19) B. lindleyana (GJ68); (20) B. yunnanensis; (21) B. subcapitata; (22) B. officinalis; (23) B. paniculate; (24) B. delavayi; (25) B. microstachya; (26) B. sp. 1; (27) B. brachystachya; (28) B. asiatica; (29) B. asiatica = B. subserrata; and (30) B. bhutanica.

The genus Buddleja comprises ~90 species in the tropical, subtropical and warm-temperate areas of Africa, Asia and North and South America (Norman, 2000; Chau et al., 2017). The Asian Buddleja clade is well supported as being monophyletic (Chau et al., 2017). In descriptive taxonomy, this is a notoriously difficult group of species, which is reflected in the frequent changes to species delimitation in the group (Marquand, 1930; Leeuwenberg, 1979; Li, 1982, 1988; Bao, 1983; Zhang et al., 2014; Ge et al., 2018) and controversial taxonomic systems (Bentham, 1846; Marquand, 1930; Leeuwenberg, 1979; Li, 1982; Li and Leeuwenberg, 1996; Norman, 2000; Oxelman, 2004; Chau et al., 2017). The Flora of China, in addition to several other studies, currently list 27 species in the Asian Buddleja clade (Li and Leeuwenberg, 1996; Norman, 2000; Liu and Peng, 2004, 2006; Zhang et al., 2014; Zhu et al., 2014; Ge et al., 2018). The Sino-Himalayan region of Southeast Asia is the centre of diversity for Asian Buddleja, harbouring 25 of the 27 Asian Buddleja species (all except for B. curviflora and B. japonica; Wu et al., 2010). The tectonic activity and climate fluctuations that took place in the Sino-Himalayan region during the Miocene are believed to have played a crucial role in the diversification of plant species in this region (Ding et al., 2020). However, whether the diversification of the Asian Buddleja is related to those palaeoclimatic and geological events has not yet been investigated.

Asian Buddleja species show a high proportion of polyploid species, and different ploidy levels are observed, including diploids, tetraploids, hexaploids, dodecaploids, 16-ploids and 24-ploids (2n = 38, 76, 114, 228, 300 and 456; Chen et al., 2007). Polyploidy might facilitate the adaptation of Buddleja to an alpine environment and promote niche diversification and speciation in the genus in the Sino-Himalayan region (Chen et al., 2007).

Interspecies hybridization is common in Buddleja, owing to overlaps in distribution, flowering period and pollinators between species (Liao et al., 2021). Twenty-five natural hybrids of Buddleja have been inferred based on morphological characteristics, 19 from the Neotropics and six from the Old World taxa (Norman, 2000). Two natural Asian Buddleja hybrids have been confirmed with both morphological and molecular evidence (Liao et al., 2015, 2021). It is thought that hybridization might promote speciation via allopolyploid speciation or via ‘adaptive introgression’ allowing the plants to adapt to new ecological niches (Abbott et al., 2013). Given that polyploidy, hybridization and cytonuclear conflicts are common in Asian Buddleja (Chen et al., 2007; Chau et al., 2017), events leading to reticulation might play an important role in the diversification of this lineage. Morphological continuity, low sequence differentiation and hybridization or polyploidization between the newly diverged lineages can exacerbate the difficulties facing taxonomic and polygenetic research (Stoughton et al., 2018). Previous studies, although revealing the phylogenetic relationships between Buddleja species worldwide, failed to cover all Asian species and did not explain the observed cytonuclear conflicts (Chau et al., 2017, 2018). More informative molecular sequences and extensive sampling are urgently needed to illustrate the phylogenetic structure and complex reticulate evolutionary history in this lineage.

We used a large number of low-copy nuclear (LCN) genes, single nucleotide polymorphisms (SNPs), nuclear ribosomal DNA (nrDNA) sequences and whole plastid genomes assembled from data generated by genome skimming technology to illustrate the phylogenetic relationships and evolutionary history of Asian Buddleja species. The reticulate relationships in this lineage were highlighted initially because of cytonuclear conflicts and were confirmed with gene tree incongruence and Bayesian clustering. The aims of the present study were as follows: (1) to reconstruct a robust phylogenetic backbone for the Asian Buddleja clade and lay the foundations for future species delimitation in this lineage; (2) to explore the reticulate evolutionary history of Asian Buddleja; and (3) to infer the evolutionary history of the Asian Buddleja lineage and its potential associations with tectonic activity and climatic fluctuations.

MATERIALS AND METHODS

Taxon sampling, DNA extraction and sequencing

A total of 80 accessions (Supplementary data Table S1), including data from 64 newly sequenced accessions and 16 sequences already available from GenBank, were included in this study. Our samples represented 32 taxa, including 27 species, three hybrids and two undescribed species. All voucher specimens are listed in the Supplementary data (Table S1).

Total DNA was extracted from silica gel-dried leaf tissues using a cetyltrimethylammonium bromide (CTAB) method. Purified DNA was fragmented, and short insert (500 bp) libraries were constructed according to the manufacturer’s instructions on an Illumina HiSeq X Ten platform, and were then sequenced on an Illumina HiSeq platform with a read length of 300–500 bp, by a commercial service (Beijing Ori-Gene Science and Technology).

Flow cytometry

Flow cytometry analyses were carried out at the Laboratory of Molecular Biology of Germplasm Bank of Wild Species in Southwest China following the protocol described by Doležel et al. (2007). Thirty-three leaf samples of 29 Buddleja taxa were collected (one or two samples of each of the 26 species and one sample of each of the three hybrids; Supplementary data Table S2). About 0.5 cm2 of fresh young leaf tissue was chopped with a razor blade in a Petri dish containing 0.8 mL ice-cold MGb buffer. The resulting solutions were subsequently filtered through 40 µm nylon mesh to obtain the cell nuclei; 50 µL of propidium iodide solution (1 mg/mL) and 5.0 µL of RNAse (100 µg/mL) were added to each sample, and the samples were then stored in the dark for 0.5–1 h. The nuclear DNA content was measured on a flow cytometer using the DNA 2C-values of Zea mays L. and Solanum lycopersicum L. as the internal standards. The number of nuclei was normalized to 10 000 per sample using the fluorescently labelled propidium iodide in each experiment, the cross-validation (CV) % was controlled to within 5 %, and the nuclei were surveyed by BD FACSCalibur. The relative nuclear DNA content of each plant sample was then determined by comparison with the peak positions of the nuclei from the internal standards. The ploidy level was determined based on the ratio of G1 peak positions of the diploid B. asiatica and tetraploid B. davidii nuclei.

Sequence assembly, annotation and alignment

The paired-end reads were filtered using fastp v.0.20.1 (Chen et al., 2018) with the default parameters. The plastid genomes were assembled using the GetOrganelle pipeline v.1.7.1 (Jin et al., 2020) with the recommended parameters for embryophyte plant plastome assembly (https://github.com/Kinggerm/GetOrganelle). Annotation of plastids was performed using the plastid genome annotator (PGA; Qu et al., 2019), and the recommended Amborella trichopoda plastome genome was selected as a reference. The results were aligned with five published plastid genomes from Asian Buddleja species (Ge et al., 2018) using MAFFT v.7.3.08 (Katoh and Standley, 2013), and the annotations were checked manually in Geneious v.9.0.2 (Biomatters, Auckland, New Zealand). The coding sequences (CDS) regions were extracted from each plastid using Geneious and aligned using MAFFT. The nrDNA sequences were assembled using GetOrganelle with the recommended parameters for plant nuclear ribosomal RNA assembly (https://github.com/Kinggerm/GetOrganelle). The assembled nrDNA sequences were aligned and checked manually in Geneious. Aligned whole plastid sequences and CDS regions were trimmed using Gblocks (Talavera and Castresana, 2007) in PhyloSuite (Zhang et al., 2020) with the default parameters. However, owing to the uneven quality of the nrDNA assembly, aligned nrDNA sequences were trimmed using Gblocks with half gape position allowed (-b5 = h).

Phylogenetic analyses were implemented using whole plastid sequences and nrDNA sequences using RAxML v.8.2.12 with 1000 bootstraps, and with the ‘GAMMAI’ substitution model, as indicated by Abadi et al. (2019).

LCN gene construction and discovery of nuclear variation

Given that there is only one complete published genome within the genus Buddleja to date, the LCN genes were identified following the methods described by Ma et al. (2021). The protein-coding genes of Buddleja alternifolia (Ma et al., 2021) and Tectona grandis (a woody member of the Lamiaceae; Zhao et al., 2019) were analysed with Orthofinder (Emms and Kelly, 2015) to identify the orthologous gene clusters. The HybPiper pipeline v.1.3.1 (Johnson et al., 2016) was used with the default settings for targeting genes. Gene sequences were aligned using MAFFT and converted to codon alignments using pal2nal (Suyama et al., 2006). Aligned codons were trimmed using trimAl (Capella-Gutiérrez et al., 2009). The gene trees were constructed using IQtree v.1.6.12 (Nguyen et al., 2014) with 1000 bootstrap replicates. Species trees were inferred using ASTRAL-III v.5.7.1 (Zhang et al., 2018) based on multiple gene trees. Conflicts between plastid, nrDNA and species trees were examined using phytools (Revell, 2012) in R.

We used BWA v.07.17 (Li and Durbin, 2009) to make an index for the genome of B. alternifolia and used BWA-MEM with the default parameters to map the filtered reads to the reference genome. Variant detection was carried out using the genome analysis toolkit GATK4 (McKenna et al., 2010) following the best practices workflow for variant discovery (DePristo et al., 2011). Hard filters were implemented on the raw SNP dataset with the following filter parameters; (1) SNPs with read depth >200 or <5; (2) SNPs with missing rate of >80 %; (3) MAF >0.05; and (4) non-biallelic SNPs.

Hybrid analysis

To reduce the computational burden and to increase the accuracy of speculation, 32 samples with relatively high sequencing quality and low missing sequence rate (Supplementary data Table S2) were chosen to form a sub-dataset. Buddleja caryopteridifolia, B. myriantha and B. jinsixiaensis were excluded, owing to the very high rate of missing sequences in the gene matrix. Finally, 23 species and a hybrid plant were selected to simulate the reticulate evolutionary history of Asian Buddleja. PhyloNet (Than et al., 2008) was used to infer possible hybrid events with the InferNetwork_MPL geneTreeList function and the parameters ‘-x 6 -b 50’. The optimal number of hybridization events was estimated by searching the global optimum of the likelihood (Cao et al., 2019). The optimum phylogenetic networks were visualized in Dendroscope (Huson et al., 2007). A Bayesian clustering analysis was also performed using Admixture (Alexander et al., 2009) with the same samples as those used in the PhyloNet analysis. We tested numbers of clusters from two to seven, with the optimal number of clusters estimated via the lowest cross-validation error rate. We used the package ‘Pophelper’ (Francis, 2017) in R v.3.6.3 (R Core Team, 2018) to visualize the Admixture results.

Molecular dating

Given that the homogeneity of chloroplast sequences is much higher than that of LCN genes and that multiple published chloroplast genomes could provide more options for a calibration point, the concatenated plastid CDS regions from the non-redundant dataset were used to estimate the divergence time of Asian Buddleja. Divergence time was estimated in BEAST v.1.10 (Drummond and Rambaut, 2007). Two calibration points were chosen from TimeTree (http://timetree.org/). The root of the time tree was constrained to 71 Mya, with a normal distribution and s.d. of 10 Mya. The ancestral node of the Scrophulariaceae samples selected in this study was constrained to 44 Mya, with a normal distribution and s.d. of 10 Mya. The BEAST analyses were performed using an uncorrelated log normal relaxed clock with a Yule tree prior, a random starting tree and ‘Gamma + Invariant Sites’ as the model of sequence evolution. The Markov chain Monte Carlo (MCMC) analysis was run for 200 million generations, sampling every 1000 generations, and the first 20 million samples were discarded as burn-in. Convergence of the MCMC runs was checked using Tracer v.1.6. Tree Annotator v.1.8.0 (Drummond et al., 2012) was used to summarize the set of post-burn-in trees and their parameters to produce a maximum clade credibility chronogram showing the mean divergence time estimates with 95 % highest posterior density (HPD) intervals. Figtree v.1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/) was used for image drawing of the time tree. Lineage-through-time (LTT) plots were drawn using the APE package (Paradis et al., 2004) in R.

RESULTS

Determination of ploidy diversity using flow cytometry

The levels of ploidy of the 33 samples were determined. Fifteen samples representing B. alternifolia, B. asiatica (GJ1 & GJ34), B. caryopteridifolia, B. crispa, B. curviflora, B. jinsixiaensis, B. lindleyana (GJ5 & GJ68), B. officinalis, B. paniculata, B. tsetangensis, B. yunnanensis, B. crispa × B. paniculata and B. × wardii were considered to be diploids. The rest were presumed to be polyploids, including seven tetraploids (B. brachystachya, B. candida, B. davidii, B. fallowiana, B. macrostachya, B. myriantha and B. sessilifolia), five hexaploids (B. albiflora, B. delavayi, B. forrestii, B. sp. 1 and B. sp. 1 × delavayi), one 12-ploid (B. nivea) and one 24-ploid (B. colvilei); the B. microstachya samples were found to consist of both tetraploid and hexaploid samples, and the B. macrostachya samples were found to consist of both hexaploid and 12-ploid samples. The ploidy levels of eight Asian Buddleja species and hybrids were determined using flow cytometry and are reported here for the first time. Tetraploid is a new ploidy level for B. brachystachya, and the ploidy levels determined for the remaining species are consistent with those published previously. The available cytological data are shown in the Supplementary data (Table S2).

Nuclear and plastid gene assembly and SNP calling

The number of clean reads for genome skimming data ranged from 6.6 million (B. myriantha GJ37) to 38.7 million (B. yunnanensis GJ75) with an average of 18.6 million (Supplementary data Table S3). In order to prevent the bias caused by the uneven sample depth in LCN gene assembly, ten resequenced samples downloaded from GenBank were reduced to 20.0 million reads.

A total of 10 791 LCN genes were discovered using OrthoFinder. The number of genes recovered for each sample varied from 9240 (B. jinsixiaensis) to 10 763 (B. davidii). After trimming away those with a maximum missing rate >30 %, 10 429 LCN genes were used to construct the ASTAL species tree. The nrDNA sequence of B. bhutanica GJ42 was discarded owing to its short and fragmented sequences. The trimmed nrDNA data matrix comprised 8724 characters, of which 1666 were parsimony-informative sites. Consistent with previous research (Ge et al., 2018), the plastomes of Buddleja showed typical quadripartite architecture (Supplementary data Fig. S1). The trimmed whole plastome data matrix comprised 158 290 characters, of which 1605 were parsimony-informative sites. After filtering, a total of 87 039 SNPs were obtained from the 32 high-quality samples.

Phylogenetic reconstruction

The phylogenetic structure of the ASTRAL species tree is generally in accordance with that of the nrDNA tree (Fig. 2B). There are, however, complex conflicts between the plastid and species trees (Fig. 2A).

Fig. 2.

Fig. 2.

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Tanglegram of the ASTRAL-III species tree and (A) plastid tree or (B) nuclear ribosomal DNA (nrDNA) tree. The two ASTRAL species trees are identical topologically, but rotated at some nodes to match up with the plastid or nrDNA trees. Different colour blocks represent clades with obvious cytonuclear conflict.

Phylogenetic reconstruction based on the plastid dataset indicated that Buddleja asiatica, B. bhutanica and five polyploid species (B. sessilifolia, B. colvilei, B. macrostachya, B. myriantha and B. candida) composed plastid clade 1, and the remaining species composed plastid clade 2 [bootstrap support (BS) = 100 %; Fig. 2A]. The plastid phylogeny suggested that B. alternifolia is polyphyletic, because the three B. alternifolia samples (RE121, RE123 and RE126) sampled in Sichuan clustered together with B. subcapitata and B. caryopteridifolia, while the remaining samples formed a clade with B. jinsixiaensis (Fig. 2A). In addition, the B. crispa complex (B. crispa and B. caryopteridifolia; Leeuwenberg, 1979) was also revealed to be polyphyletic in the plastid phylogeny, with GJ31 and GJ57 being far apart from the other two samples of B. crispa (GJ35 and GJ56).

The species tree inferred from the LCN genes strongly (bootstrap support BS = 100 %) supported three clades in Asian Buddleja: ASTRAL clade 1 included B. asiatica and B. bhutanica; ASTRAL clade 2 included ten polyploid species with mainly Himalayan–Hengduan Mountains distribution (Chen et al., 2007; Wu et al., 2010); and ASTRAL clade 3 included the remaining species (Fig. 3). A notable conflict between the plastid tree and the species tree is visible in the cases of the five polyploid species (B. forrestii, B. nivea, B. albiflora, B. fallowiana and B. davidii), which formed a clade together with another five polyploid species in the species tree, but nested within plastid clade 2 (Fig. 2A). The species tree also supported the monophyly of both the B. alternifolia complex and B. crispa complex, which did not appear as clades in the plastid tree (Fig. 2A). Although the nrDNA tree shared a similar topography to the species tree, the position of B. nivea was in dramatic conflict (Fig. 2B).

Fig. 3.

Fig. 3.

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ASTRAL-III species tree. The ploidy of each species is indicated by the coloured circles.

Network and gene flow analysis

A sub-dataset of 32 samples, including 23 species and one hybrid, was used to process the PhyloNet analysis and the Bayesian clustering (Supplementary data Table S3). Up to six hybridization events among the clades of Buddleja were examined in PhyloNet. Six reticulate evolutionary events proved to be the best scenario, based on the global optimum of the likelihood. The best two values of K (the number of ancestral populations) in the Bayesian clustering analysis, as indicated by CV error values, were two and three. A reticulation event and the mixture of two genetic backgrounds are clearly visible in B. paniculata × B. crispa GJ20, confirming its hybrid origin. PhyloNet analysis also suggested three hybridization events (Fig. 4) in five polyploid species (B. forrestii, B. nivea, B. albiflora, B. fallowiana and B. davidii), in which there were clear conflicts between the plastid and nrDNA trees (Fig. 2A). Buddleja forrestii might have originated as a hybrid between B. sessilifolia and the ancestor of another four species. In addition, four polyploids (Buddleja delavayi, B. microstachya, B. brachystachya, and B. sp. 1) in clade 3 of the species tree contained two reticulation events, and the Bayesian clustering results also supported admixture. The B. crispa complex is likely to have received gene flow from the B. alternifolia cluster, in addition to a ‘ghost introgression’ (donors of gene flow might be extinct or unsampled).

Fig. 4.

Fig. 4.

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Best-supported species networks inferred with PhyloNet for the 32 samples, and best two scenarios from Bayesian clustering analysis inferred from Admixture with the same samples.

Molecular dating

Divergence time estimates based on the CDS region of the plastid indicated that the divergence time of the two clusters in the plastid tree was 14.2 Mya (95 % HPD: 8.44–21.57 Mya). The LTT plots suggested that the Asian Buddleja clade experienced two rapid diversifications, at 8–10 and 0–3 Mya (Fig. 5). The topography of the plastid tree cannot reflect that of the real species tree, owing to the reticulation; however, this does not affect the estimated dating of diversification in Buddleja.

Fig. 5.

Fig. 5.

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BEAST analysis of divergence times based on plastid data, with trends in global climate change over 30 Mya depicted (red line) and lineage-through-time plot for this taxon (purple line).

DISCUSSION

The performance of the different phylogenetic trees

In this study, we reconstructed the phylogenetic relationships within Asian Buddleja using biparental (nrDNA sequences and LCN genes) and maternal (whole plastid) sequences. Phylogenetic structures inferred using these different sets of sequences have unique advantages and potential biases (Álvarez and Wendel, 2003; Nieto Feliner and Rosselló, 2007; Gitzendanner et al., 2018). A comprehensive assessment of all phylogenetic trees should allow us to have a relatively accurate understanding of the evolutionary history of Asian Buddleja.

The phylogenetic structure resulting from analysis of the nrDNA data showed the same three-cluster structure as the ASTRAL species tree (Fig. 2B). However, conspicuous differences in the length of the nrDNA sequence assembly, owing to uneven sequencing quality, caused bias in the phylogenetic relationships at the species level, such as in the cases of B. fallowiana and B. microstachya. In addition, the unexpected position of B. nivea might be caused by the short length of the nrDNA assembly of the two samples (GJ26 and GJ64), given that B. nivea grouped with many polyploids in both the plastid and species trees (Fig. 2A).

Owing to the lack of genomic resources in the genus Buddleja, methods designed to identify orthologous genes were not able to avoid paralogous genes in this lineage (Xiong et al., 2022). The presence of paralogous genes can lead to problems in inference of a species tree (Fitch, 1970; Cheon et al., 2020; Kapli et al., 2020). Recent studies (Smith and Hahn, 2021a; Yan Z et al., 2021) have suggested that, in certain circumstances, species tree inference in the presence of paralogues is as accurate as phylogenetic analyses using orthologues. Many approaches can also reduce the adverse effects of paralogous genes on the construction of a species tree, such as using quartet-based gene tree methods (e.g. ASTRAL; Yan Z et al., 2021) and increasing the number of loci used in phylogenetic inference (Smith and Hahn, 2021b). In the present study, we used a large number of LCN genes and the coalescence method (ASTRAL-III) to infer the best possible species tree of Asian Buddleja.

Implications for species delimitation of Asian Buddleja

Species delimitation of Buddleja in Asia is notoriously difficult (Li, 1982) owing to the transitional traits between species (e.g. the B. crispa complex) and huge variation within species (e.g. B. davidii). Moreover, hybridization creates many individuals with transitional morphology (Liao et al., 2015), which results in conflict among the different classification systems (Leeuwenberg, 1979; Li, 1982). Through extensive sampling and multiple sequence construction, the present study yielded a strong phylogenetic backbone for this lineage, allowing us to provide evidence for the delimitation of certain species.

Three samples of B. asiatica (GJ34, GJ40 and GJ41) collected in Nepal and Tibet formed a sister group to the remaining samples of B. bhutanica and B. asiatica, implying that these specimens exhibit high genetic differentiation from the other Buddleja specimens in this clade (Fig. 3). Through morphological comparison (Supplementary data Table S4; Fig. S2) and examination of the original descriptions and type specimens (997787 BM! and 521826 BM!), these samples with Himalayan distribution might refer to Buddleja subserrata (Hamilton, 1825), a synonym of B. asiatica, and suggests that B. subserrata might be recognized as a distinct species.

Based on our phylogenetic reconstructions, the B. crispa complex includes at least three species: species 1 includes GJ35 and GJ56 (B. crispa); species 2 includes GJ31 (B. caryopteridifolia); and species 3 includes GJ57, which is morphologically different from B. caryopteridifolia (Supplementary data Table S5; Fig. S3), which suggests that it might be a distinct species. Buddleja crispa is widely distributed and is prone to hybridization with other species (Liao et al., 2015, 2021), resulting in morphological continuity. Thus, 15 species and many varieties were reduced to synonyms (Leeuwenberg, 1979). Our study not only confirmed the species position of B. caryopteridifolia but also implied that there are synonyms that might have been mistakenly incorporated into B. crispa.

Buddleja officinalis and B. paniculata are considered morphologically similar to each other and are easily confused. Buddleja paniculata typically has a white corolla, with the corolla tube being both shorter and thinner than that typical of the lilac B. officinalis (specimen numbers 263011 A!, 276688 GZU!, 6968182 BR! and 1096401 K!; Fig. 1; Leeuwenberg, 1979; Li, 1992). Both species are known locally as ‘Mi Meng Hua’ in Chinese (Yang Fengmao, personal observation). The Chinese name ‘mun-chua’ (another common name of ‘Mi Meng Hua’) is mentioned in the original description of B. officinalis (Maximowicz, 1880), whereas B. paniculata was first introduced as having the Chinese name ‘Hou Yao Zui Yu Cao’ in 1982 (Li, 1982). Flora Yunnanica (Bao, 1983) lists only B. officinalis, and most ‘Mi Meng Hua’ plants sampled in Yunnan have been identified as B. officinalis (e.g. Liao et al., 2015; Yan XX et al., 2021; Yang et al., 2023). However, molecular and morphological comparisons (Supplementary data Table S6; Fig. S4) suggest to us that the ‘Mi Meng Hua’, widely distributed throughout Yunnan, is in fact B. paniculata (‘Hou Yao Zui Yu Cai’ in Chinese).

The B. lindleyana sample GJ68 exhibits large morphological and molecular differences from other samples (GJ5 and GJ6) of B. lindleyana: it has distinctly serrated leaves [Fig. 1 (19)] and was once treated as a variety B. lindleyana var. sinuatodentata (Marquand, 1930). Our study reveals that it might be a distinct taxonomic unit that needs further study.

The specimen (0022547 KUN!) of GJ18 was identified as a new species, Buddleja adenocarpa B. S. Sun, in 1960, and Leeuwenberg reidentified it as B. brachystachya. Our study showed that GJ18 did not cluster with the B. brachystachya samples collected around the type locality, and therefore supported it as a distinct species. Careful comparison and further verification should be carried out in the future.

Reticulate evolutionary history of Asian Buddleja

Hybridization in extant species of Asian Buddleja has been documented and studied extensively (Leeuwenberg, 1979; Liao et al., 2015, 2021). The complex and deep cytonuclear conflicts revealed in the present study indicated that allopolyploidy, hybridization and introgression might have been present throughout the evolutionary history of Asian Buddleja.

Five polyploid species (B. forrestii, B. nivea, B. albiflora, B. fallowiana and B. davidii) formed a monophyletic group with another five polyploid species in the ASTRAL species tree and the nrDNA tree (with the exception of B. nivea; Fig. 2B) but were nested with the diploid species in the plastid tree (Fig. 2A). Cytonuclear conflicts in these polyploid species might indicate allopolyploid speciation, which is common in the formation of polyploidy (Morales-Briones et al., 2018). The B. crispa complex and B. alternifolia each clustered as monophyletic groups in the ASTRAL species tree but were separated as polyphyletic groups in the plastid tree. Buddleja crispa is known to be involved in hybridization events with B. alternifolia (Liao et al., 2021) and with B. paniculata (Liao et al., 2015). Although most of the modern hybrids examined were F1s, the extensive contact and hybridization throughout the history of these species might have contributed to plastid capture in those lineages.

PhyloNet analysis verified the hybrid of B. paniculata and B. crispa, which was previously regarded mistakenly as a hybrid of B. officinalis and B. crispa (Liao et al., 2015) owing to the misidentification of B. paniculata. The present study revealed ancestral introgression in the B. crispa complex, which might explain the cytonuclear discordance in this complex. Although gene flow from B. alternifolia to B. crispa was detected, we are unable to explain the polyphyletic nature of B. alternifolia in the plastid tree, particularly the unexpected position of three samples (RE120, RE121 and RE126) in Sichuan. This might be attributable to the fact that the species that originally caused the chloroplast capture has become extinct or remains unsampled (Li et al., 2022) or it may have occurred long ago, with an ancestor of the B. crispa complex or B. subcapitata being involved in the hybridization that led to plastic transfer. Six polyploid species (B. forrestii, B. nivea, B. albiflora, B. fallowiana, B. davidii and B. candida) were shown to have undergone complex hybridization and genetic introgression (Fig. 4), which could explain the cytonuclear discordance in five of the species, although not that in B. candida (Fig. 2).

The origin of the hexaploid species B. forrestii might be a result of allopolyploidy, because one of its putative progenitors is tetraploid (B. sessilifolia). An allopolyploid origin of B. forrestii would explain why it grouped together with B. sessilifolia in the species tree (and is morphologically similar to B. sessilifolia; Fig. 1) but is widely separated from it in the plastid tree. Reticulate phylogenetic analysis indicated that hybridization and allopolyploidy might have played an important role in the diversification of the Asian Buddleja.

History of diversification in Buddleja

Our results indicated that there were two stages of rapid diversification in the Asian Buddleja lineage (Fig. 5). The first stage occurred ~8–10 Mya, which might correspond to the last uplift in Hengduan Mountains and the intensification of the Asian monsoon (Favre et al., 2015; Yang et al., 2021). The second stage of rapid diversification might have occurred as a result of the Quaternary climate fluctuations (2.6 Mya; Clark et al., 2009), which caused the radiation of many species in the Himalayas–Hengduan Mountains (Muellner-Riehl, 2019; Zhang et al., 2021).

Extensive plateau uplift in the Miocene (5–15 Mya) intensified the summer monsoons, increasing the precipitation and erosion through river incision, leading to greater topographic relief (Herman et al., 2013). Moreover, a remarkable increase in the intensity of silicate weathering at ~7–9 Mya, induced by the enhanced monsoons, caused massive CO2 consumption and fast global cooling (Yang et al., 2021). This series of processes has not only accelerated the evolution of the biodiversity in the Himalayas–Hengduan Mountains (Ding et al., 2020; Xu et al., 2020), but also that of the monsoonal forests in South China (Kong et al., 2022). The effect of climate modifications during the Quaternary ice age (0.1–2.6 Mya; Clark et al., 2009) caused steep ecological gradients in mountainous areas (Wu et al., 2022). At this time, rapid species radiation occurred in many mountainous areas, including the Himalayas, the Hengduan mountains, the Andes and the mountains of New Zealand (Hughes and Atchison, 2015). Buddleja Ser. Curviflorae Marq. comprises three species and has a disjunct distribution: B. lindleyana is found mainly on the Chinese mainland, whereas B. curviflora and B. japonica are found in Taiwan and Japan. The inferred time of divergence of B. lindleyana and the ancestor of B. curviflora and B. japonica was ~7.35 Mya (95 % HPD: 4.17–11.59 Mya). If this is correct, a Late Miocene landbridge across the East China Sea (~5.0–7.0 Mya; Kimura, 2003) would have allowed the common ancestor of B. curviflora and B. japonica to migrate from the Chinese mainland to Japan. Similar divergence times between other species with disjunct distributions in China and Japan have been found in Euptelea (Eupteleaceae; 6.39 Mya; Cao et al., 2020) and Deinanthe (Hydrangeaceae; 7.1 Mya; Sakaguchi et al., 2021). The inferred divergence time of B. curviflora from B. japonica was ~5.70 Mya (95 % HPD: 2.97–9.46 Mya), shortly after the formation of Taiwan Island (~6.5 Mya; Huang, 2017).

Our study suggests that a combination of tectonic activity, climate change, extensive hybridization and polyploidization might have contributed to the diversification of the Asian Buddleja.

SUPPLEMENTARY DATA

Supplementary data are available online at https://academic.oup.com/aob and consist of the following. Table S1: sample and sequence information. Table S2: ploidy levels of Asian Buddleja species determined by flow cytometry and according to previous studies. Table S3: number of sample sequences and selection of subset samples in the PhyloNet analysis. Table S4: differences in morphological characters between Buddleja asiatica and Buddleja subserrata. Table S5: differences in morphological characters between Buddleja caryopteridifolia and sample GJ57. Table S6: differences in morphological characters between Buddleja officinalis and Buddleja paniculata. Fig. S1: the structure of the Buddleja chloroplast. Fig. S2: morphological comparison between Buddleja asiatica and Buddleja subserrata. Fig. S3: morphological comparison between the specimen GJ57 and Buddleja caryopteridifolia GJ31. Fig. S4: morphological comparison between Buddleja officinalis and Buddleja paniculata.

mcad022_suppl_Supplementary_Material
Click here for additional data file. (13.8MB, doc)

ACKNOWLEDGEMENTS

We thank Ende Liu for sharing specimens and useful information; Gao Chen, Rongli Liao, Cheng Liu, Jidong Ya, Jing Yang, Zhi Chen, Congjia Li, Lingyun Tang and Wenyun Chen (Kunming Institute of Botany), Xing Zhang (Yunnan Forestry Technological College), Weichang Gong (Honghe University), Renbin Zhu (Xishuangbanna Tropical Botanical Garden) and Chuchun Chien (National Sun Yat-sen University Herbarium, Taiwan) for assistance with sample collection; Rengang Zhang and Wei Gu for assistance with data analysis; Dechang Meng, Wenguang Wang, Zhi Xie, Jiaming Song, Xuexue Wu and Instagram blogger ‘Gary Long and Trewithen Garden’ for providing pictures; Wenguang Sun (Yunnan Normal University) for flow cytometry data analysis; and the herbaria KUN, PE and K, Kunming Botanical Garden for providing plant material or DNA from their collections.

Contributor Information

Fengmao Yang, Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, China; Yunnan Key Laboratory for Integrative Conservation of Plant Species with Extremely Small Populations, Kunming Institute of Botany, Chinese Academy of Sciences (CAS), Kunming 650201, Yunnan, China.

Jia Ge, Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, China; Yunnan Key Laboratory for Integrative Conservation of Plant Species with Extremely Small Populations, Kunming Institute of Botany, Chinese Academy of Sciences (CAS), Kunming 650201, Yunnan, China.

Yongjie Guo, Germplasm Bank of Wild Species of China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, China; University of Chinese Academy of Sciences, Beijing 100049, China.

Richard Olmstead, Department of Biology and Burke Museum, University of Washington, Seattle, WA 98195, USA.

Weibang Sun, Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, China; Yunnan Key Laboratory for Integrative Conservation of Plant Species with Extremely Small Populations, Kunming Institute of Botany, Chinese Academy of Sciences (CAS), Kunming 650201, Yunnan, China.

CONFLICT OF INTEREST

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

FUNDING

This work was supported by Yunnan Fundamental Research Projects (grant number 202001AT070097) and the National Natural Science Foundation of China (grant numbers 32071653, 30970192, 31770240 and 31400478).

DATA AVAILABILITY

The data that support the findings of this study can be found in online repositories. The names of the repository and accession number can be found below: https://db.cngb.org/search/project/CNP0003159/.

LITERATURE CITED

  1. Abadi S, Azouri D, Pupko T, Mayrose I.. 2019. Model selection may not be a mandatory step for phylogeny reconstruction. Nature Communications 10: 934. doi: 10.1038/s41467-019.08822.w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Abbott R, Albach D, Ansell S, et al. 2013. Hybridization and speciation. Journal of Evolutionary Biology 26: 229–246. doi: 10.1111/j.1420-9101.2012.02599.x. [DOI] [PubMed] [Google Scholar]
  3. Alexander DH, Novembre J, Lange K.. 2009. Fast model-based estimation of ancestry in unrelated individuals. Genome Research 19: 1655–1664. doi: 10.1101/gr.094052.109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Álvarez I, Wendel JF.. 2003. Ribosomal ITS sequences and plant phylogenetic inference. Molecular Phylogenetics and Evolution 29: 417–434. doi: 10.1016/s1055-7903(03)00208-2. [DOI] [PubMed] [Google Scholar]
  5. Bao SY. 1983. Buddleja Linn. In: Wu CY, ed. Flora Yunnanica, Vol. 3. Beijing: Science Press, 454–473. [Google Scholar]
  6. Barker MS, Arrigo N, Baniaga AE, Li Z, Levin DA.. 2016. On the relative abundance of autopolyploids and allopolyploids. New Phytologist 210: 391–398. [DOI] [PubMed] [Google Scholar]
  7. Bentham G. 1846. Scrophulariaceae. In: De Candolle A, ed. Prodromus systematis naturalis regni vegetabilis pars X, Vol. 7. Paris: Victor Masson, 432–447. [Google Scholar]
  8. Cao YN, Zhu SS, Chen J, et al. 2020. Genomic insights into historical population dynamics, local adaptation, and climate change vulnerability of the East Asian Tertiary relict Euptelea (Eupteleaceae). Evolutionary Applications 13: 2038–2055. doi: 10.1111/eva.12960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cao Z, Liu X, Ogilvie HA, Yan Z, Nakhleh L.. 2019. Practical aspects of phylogenetic network analysis using PhyloNet. bioRxiv: 746362. [Google Scholar]
  10. Capella-Gutiérrez S, Silla-Martínez JM, Gabaldón T.. 2009. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 25: 1972–1973. doi: 10.1093/bioinformatics/btp348. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chau JH, O’Leary N, Sun WB, Olmstead RG.. 2017. Phylogenetic relationships in tribe Buddlejeae (Scrophulariaceae) based on multiple nuclear and plastid markers. Botanical Journal of the Linnean Society 184: 137–166. doi: 10.1093/botlinnean/box018. [DOI] [Google Scholar]
  12. Chau JH, Rahfeldt WA, Olmstead RG.. 2018. Comparison of taxon-specific versus general locus sets for targeted sequence capture in plant phylogenomics. Applications in Plant Sciences 6: e1032. doi: 10.1002/aps3.1032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Chen G, Sun WB, Sun H.. 2007. Ploidy variation in Buddleja L. (Buddlejaceae) in the Sino-Himalayan region and its biogeographical implications. Botanical Journal of the Linnean Society 154: 305–312. doi: 10.1111/j.1095-8339.2007.00650.x. [DOI] [Google Scholar]
  14. Chen SF, Zhou YQ, Chen YR, Gu J.. 2018. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34: i884–i890. doi: 10.1093/bioinformatics/bty560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Cheon S, Zhang JZ, Park C.. 2020. Is phylotranscriptomics as reliable as phylogenomics? Molecular Biology and Evolution 37: 3672–3683. doi: 10.1093/molbev/msaa181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Clark PU, Dyke AS, Shakun JD, et al. 2009. The Last Glacial Maximum. Science 325: 710–714. doi: 10.1126/science.1172873. [DOI] [PubMed] [Google Scholar]
  17. Debray K, Le Paslier MC, Berard A, et al. 2022. Unveiling the patterns of reticulated evolutionary processes with phylogenomics: hybridization and polyploidy in the genus Rosa. Systematic Biology 71: 547–569. doi: 10.1093/sysbio/syab064. [DOI] [PubMed] [Google Scholar]
  18. DePristo MA, Banks E, Poplin R, et al. 2011. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nature Genetics 43: 491–498. doi: 10.1038/ng.806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Diaz-Perez A, Lopez-Alvarez D, Sancho R, Catalan P.. 2018. Reconstructing the origins and the biogeography of species’ genomes in the highly reticulate allopolyploid-rich model grass genus Brachypodium using minimum evolution, coalescence and maximum likelihood approaches. Molecular Phylogenetics and Evolution 127: 256–271. doi: 10.1016/j.ympev.2018.06.003. [DOI] [PubMed] [Google Scholar]
  20. Ding WN, Ree RH, Spicer RA, Xing YW.. 2020. Ancient orogenic and monsoon-driven assembly of the world’s richest temperate alpine flora. Science 369: 578–581. doi: 10.1126/science.abb4484. [DOI] [PubMed] [Google Scholar]
  21. Doležel J, Greilhuber J, Suda J.. 2007. Estimation of nuclear DNA content in plants using flow cytometry. Nature Protocols 2: 2233–2244. doi: 10.1038/nprot.2007.310. [DOI] [PubMed] [Google Scholar]
  22. Drummond AJ, Rambaut A.. 2007. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evolutionary Biology 7: 214. doi: 10.1186/1471-2148-7-214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Drummond AJ, Suchard MA, Xie D, Rambaut A.. 2012. Bayesian phylogenetics with BEAUti and the BEAST 1.7. Molecular Biology and Evolution 29: 1969–1973. doi: 10.1093/molbev/mss075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Edgeloe JM, Severn-Ellis AA, Bayer PE, et al. 2022. Extensive polyploid clonality was a successful strategy for seagrass to expand into a newly submerged environment. Proceedings of the Royal Society B: Biological Sciences 289: 20220538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Emms DM, Kelly S.. 2015. OrthoFinder: solving fundamental biases in whole genome comparisons dramatically improves orthogroup inference accuracy. Genome Biology 16: 157. doi: 10.1186/s13059-015-0721-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Estep MC, McKain MR, Vela Diaz D, et al. 2014. Allopolyploidy, diversification, and the Miocene grassland expansion. Proceedings of the National Academy of Sciences of the United States of America 111: 15149–15154. doi: 10.1073/pnas.1404177111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Favre A, Päckert M, Pauls SU, et al. 2015. The role of the uplift of the Qinghai-Tibetan Plateau for the evolution of Tibetan biotas. Biological Reviews 90: 236–253. doi: 10.1111/brv.12107. [DOI] [PubMed] [Google Scholar]
  28. Fitch WM. 1970. Distinguishing homologous from analogous proteins. Systematic Zoology 19: 99–113. [PubMed] [Google Scholar]
  29. Francis RM. 2017. Pophelper: an R package and web app to analyse and visualize population structure. Molecular Ecology Resources 17: 27–32. doi: 10.1111/1755-0998.12509. [DOI] [PubMed] [Google Scholar]
  30. Funk DJ, Omland KE.. 2003. Species-level paraphyly and polyphyly: frequency, causes, and consequences, with insights from animal mitochondrial DNA. Annual Review of Ecology, Evolution, and Systematics 34: 397–423. doi: 10.1146/annurev.ecolsys.34.011802.132421. [DOI] [Google Scholar]
  31. Ge J, Cai L, Bi GQ, Chen G, Sun WB.. 2018. Characterization of the complete chloroplast genomes of Buddleja colvilei and B. sessilifolia: Implications for the taxonomy of Buddleja L. Molecules 23: 1248. doi: 10.3390/molecules23061248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Gitzendanner MA, Soltis PS, Yi TS, Li DZ, Soltis DE.. 2018. Plastome phylogenetics: 30 years of inferences into plant evolution. In: Chaw, SM, Jansen, RK, eds. Advances in Botanical Research, vol. 85. Academic Press, 293–313. [Google Scholar]
  33. Goulet BE, Roda F, Hopkins R.. 2017. Hybridization in plants: old ideas, new techniques. Plant Physiology 173: 65–78. doi: 10.1104/pp.16.01340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Guo Z-H, Ma P-F, Yang G-Q, et al. 2019. Genome sequences provide insights into the reticulate origin and unique traits of woody bamboos. Molecular Plant 12: 1353–1365. doi: 10.1016/j.molp.2019.05.009. [DOI] [PubMed] [Google Scholar]
  35. Hamilton DDF. 1825. Scrophularinae. In: Don D, ed. Prodromus florae Nepalensis. London: Veneunt apud J. Gale, 92. [Google Scholar]
  36. Han T-S, Zheng Q-J, Onstein RE, et al. 2020. Polyploidy promotes species diversification of Allium through ecological shifts. New Phytologist 22: 571–583. [DOI] [PubMed] [Google Scholar]
  37. Herman F, Seward D, Valla PG, et al. 2013. Worldwide acceleration of mountain erosion under a cooling climate. Nature 504: 423–426. doi: 10.1038/nature12877. [DOI] [PubMed] [Google Scholar]
  38. Houghton PJ. 1984. Ethnopharmacology of some Buddleja species. Journal of Ethnopharmacology 11: 293–308. doi: 10.1016/0378-8741(84)90075-8. [DOI] [PubMed] [Google Scholar]
  39. Huang CY. 2017. Geological ages of Taiwan stratigraphy and tectonic events. Scientia Sinica Terrae 47: 394. [Google Scholar]
  40. Hughes CE, Atchison GW.. 2015. The ubiquity of alpine plant radiations: from the Andes to the Hengduan Mountains. New Phytologist 207: 275–282. doi: 10.1111/nph.13230. [DOI] [PubMed] [Google Scholar]
  41. Huson DH, Richter DC, Rausch C, Dezulian T, Franz M, Rupp R.. 2007. Dendroscope: an interactive viewer for large phylogenetic trees. BMC Bioinformatics 8: 460. doi: 10.1186/1471-2105-8-460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Jia K-H, Wang Z-X, Wang LX, et al. 2022. SubPhaser: a robust allopolyploid subgenome phasing method based on subgenome-specific k-mers. New Phytologist 235: 801–809. doi: 10.1111/nph.18173. [DOI] [PubMed] [Google Scholar]
  43. Jin J-J, Yu W-B, Yang J-B, et al. 2020. GetOrganelle: a fast and versatile toolkit for accurate de novo assembly of organelle genomes. Genome Biology 21: 241. doi: 10.1186/s13059-020-02154-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Johnson MG, Gardner EM, Liu Y, et al. 2016. HybPiper: extracting coding sequence and introns for phylogenetics from high-throughput sequencing reads using target enrichment. Applications in Plant Sciences 4: 1600016. doi: 10.3732/apps.1600016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Kapli P, Yang Z, Telford MJ.. 2020. Phylogenetic tree building in the genomic age. Nature Reviews Genetics 21: 428–444. doi: 10.1038/s41576-020-0233-0. [DOI] [PubMed] [Google Scholar]
  46. Katoh K, Standley DM.. 2013. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular Biology and Evolution 30: 772–780. doi: 10.1093/molbev/mst010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Kimura M. 2003. Land connections between Eurasian continent and Japanese Islands-related to human migration. Migration and Diffusion 4: 14–33. [Google Scholar]
  48. Kong HH, Condamine FL, Yang LH, et al. 2022. Phylogenomic and macroevolutionary evidence for an explosive radiation of a plant genus in the Miocene. Systematic Biology 71: 589–609. doi: 10.1093/sysbio/syab06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Leeuwenberg AJM. 1979. The Loganiaceae of Africa XVIII Buddleja L. II revision of the African and Asiatic species. Mededelingen Landbouwhogeschool Wageningen 79: 1–163. [Google Scholar]
  50. Levin DA, Soltis DE.. 2018. Factors promoting polyploid persistence and diversification and limiting diploid speciation during the K–Pg interlude. Current Opinion in Plant Biology 42: 1–7. doi: 10.1016/j.pbi.2017.09.010. [DOI] [PubMed] [Google Scholar]
  51. Li H, Durbin R.. 2009. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25: 1754–1760. doi: 10.1093/bioinformatics/btp324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Li JL, Zhang YJ, Ruhsam M, et al. 2022. Seeing through the hedge: phylogenomics of Thuja (Cupressaceae) reveals prominent incomplete lineage sorting and ancient introgression for Tertiary relict flora. Cladistics 38: 187–203. doi: 10.1111/cla.12491. [DOI] [PubMed] [Google Scholar]
  53. Li PT. 1982. Study on the genus Buddleja L. of China. Acta Botanica Yunnanica 4: 227–274. [Google Scholar]
  54. Li PT. 1988. Notes on the Chinese Euphorbiaceae and Loganiaceae. Journal of South China Agricultural University 9: 49–53. [Google Scholar]
  55. Li PT. 1992. Loganiaceae. In: Wu CY, ed. Flora Reipublicae Popularis Sinicae, Vol. 61. Beijing: Science Press, 265–309. [Google Scholar]
  56. Li PT, Leeuwenberg AJM.. 1996. Buddleja. In: Wu CY, Raven PH, eds. Flora of China, Vol. 15. Beijing: Science Press, 329–337. [Google Scholar]
  57. Li S, Zhang Y, Guo YJ, Yang LX, Wang YH.. 2020. Monpa, memory, and change: an ethnobotanical study of plant use in Mêdog County, South-east Tibet, China. Journal of Ethnobiology and Ethnomedicine 16: 5. doi: 10.1186/s13002-020-0355-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Liao RL, Ma YP, Gong WC, et al. 2015. Natural hybridization and asymmetric introgression at the distribution margin of two Buddleja species with a large overlap. BMC Plant Biology 15: 146. doi: 10.1186/s12870-015-0539-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Liao RL, Sun WB, Ma YB.. 2021. Natural hybridization between two butterfly bushes in Tibet: dominance of F1 hybrids promotes strong reproductive isolation. BMC Plant Biology 21: 133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Liu ED, Peng H.. 2004. Buddleja subcapitata (Buddlejaceae), a new species from SW Sichuan, China. Annales Botanici Fennici 41: 467–469. [Google Scholar]
  61. Liu ED, Peng H.. 2006. Buddleja microstachya (Buddlejaceae), a new species from SW Yunnan, China. Annales Botanici Fennici 43: 463–465. [Google Scholar]
  62. Ma YP, Wariss HM, Liao RL, et al. 2021. Genome-wide analysis of butterfly bush (Buddleja alternifolia) in three uplands provides insights into biogeography, demography and speciation. New Phytologist 232: 1463–1476. doi: 10.1111/nph.17637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Ma YZ, Wang J, Hu QJ, et al. 2019. Ancient introgression drives adaptation to cooler and drier mountain habitats in a cypress species complex. Communications Biology 2: 213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Mallet J. 2005. Hybridization as an invasion of the genome. Trends in Ecology & Evolution 20: 229–237. doi: 10.1016/j.tree.2005.02.010. [DOI] [PubMed] [Google Scholar]
  65. Mallet J, Besansky N, Hahn MW.. 2016. How reticulated are species? Bioessays 38: 140–149. doi: 10.1002/bies.201500149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Marquand CVB. 1930. Revision of the Old World species of Buddleja. Kew Bulletin of Miscellaneous Information 5: 177–208. [Google Scholar]
  67. Maximowicz C. 1880. Buddleja L. Bulletin de l’Academie Imperiale des Sciences de St-Pétersbourg, sér 26: 496–497. [Google Scholar]
  68. Mayrose I, Zhan SH, Rothfels CJ, et al. 2011. Recently formed polyploid plants diversify at lower rates. Science 333: 1257. doi: 10.1126/science.1207205. [DOI] [PubMed] [Google Scholar]
  69. McKenna A, Hanna M, Banks E, et al. 2010. The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Research 20: 1297–1303. doi: 10.1101/gr.107524.110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Morales-Briones DF, Liston A, Tank DC.. 2018. Phylogenomic analyses reveal a deep history of hybridization and polyploidy in the Neotropical genus Lachemilla (Rosaceae). New Phytologist 218: 1668–1684. doi: 10.1111/nph.15099. [DOI] [PubMed] [Google Scholar]
  71. Muellner-Riehl AN. 2019. Mountains as evolutionary arenas: patterns, emerging approaches, paradigm shifts, and their implications for plant phylogeographic research in the Tibeto-Himalayan region. Frontiers in Plant Science 10: 195. doi: 10.3389/fpls.2019.00195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. Nguyen LT, Schmidt HA, von Haeseler A, Minh BQ.. 2014. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Molecular Biology and Evolution 32: 268–274. doi: 10.1093/molbev/msu300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  73. Nieto Feliner G, Rosselló JA.. 2007. Better the devil you know? Guidelines for insightful utilization of nrDNA ITS in species-level evolutionary studies in plants. Molecular Phylogenetics and Evolution 44: 911–919. doi: 10.1016/j.ympev.2007.01.013. [DOI] [PubMed] [Google Scholar]
  74. Norman EM. 2000. Buddlejaceae. Flora Neotropica Monograph. New York: The New York Botanical Garden Press. [Google Scholar]
  75. Owens GL, Baute GJ, Rieseberg LH.. 2016. Revisiting a classic case of introgression: hybridization and gene flow in Californian sunflowers. Molecular Ecology 25: 2630–2643. doi: 10.1111/mec.13569. [DOI] [PubMed] [Google Scholar]
  76. Oxelman B, Kornhall P, Norman EM.. 2004. Buddlejaceae. In: Kubitzki K, ed. The families and genera of vascular plants, VII: flowering plants, dicotyledons, Lamiales (except Acanthaceae including Avicenniaceae). Berlin: Springer-Verlag, 39–44. [Google Scholar]
  77. Oziolor EM, Reid NM, Yair S, et al. 2019. Adaptive introgression enables evolutionary rescue from extreme environmental pollution. Science 364: 455–457. doi: 10.1126/science.aav4155. [DOI] [PubMed] [Google Scholar]
  78. Paradis E, Claude J, Strimmer K.. 2004. APE: analyses of phylogenetics and evolution in R language. Bioinformatics 20: 289–290. doi: 10.1093/bioinformatics/btg412. [DOI] [PubMed] [Google Scholar]
  79. Pontes O, Neves N, Silva M, et al. 2004. Chromosomal locus rearrangements are a rapid response to formation of the allotetraploid Arabidopsis suecica genome. Proceedings of the National Academy of Sciences of the United States of America 101: 18240–18245. doi: 10.1073/pnas.0407258102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  80. Qu XJ, Moore MJ, Li DZ, Yi TS.. 2019. PGA: a software package for rapid, accurate, and flexible batch annotation of plastomes. Plant Methods 15: 50. doi: 10.1186/s13007-019-0435-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  81. R Core Team. 2018. R: a language and environment for statistical computing. Vienna: R Foundation for Statistical Computing. https://www.R-project.org [Google Scholar]
  82. Ramsey J, Schemske DW.. 2002. Neopolyploidy in flowering plants. Annual Review of Ecology, Evolution, and Systematics 33: 589–639. [Google Scholar]
  83. Ren R, Wang HF, Guo CC, et al. 2018. Widespread whole genome duplications contribute to genome complexity and species diversity in angiosperms. Molecular Plant 11: 414–428. doi: 10.1016/j.molp.2018.01.002. [DOI] [PubMed] [Google Scholar]
  84. Revell LJ. 2012. phytools: an R package for phylogenetic comparative biology (and other things). Methods in Ecology and Evolution 3: 217–223. [Google Scholar]
  85. Rieseberg LH, Willis JH.. 2007. Plant speciation. Science 317: 910–914. doi: 10.1126/science.1137729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  86. Rothfels CJ. 2021. Polyploid phylogenetics. New Phytologist 230: 66–72. doi: 10.1111/nph.17105. [DOI] [PubMed] [Google Scholar]
  87. Sakaguchi S, Asaoka Y, Takahashi D, et al. 2021. Inferring historical survivals of climate relicts: the effects of climate changes, geography, and population-specific factors on herbaceous hydrangeas. Heredity 126: 615–629. doi: 10.1038/s41437-020-00396-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  88. Smith ML, Hahn MW.. 2021a. The frequency and topology of pseudoorthologs. Systematic Biology 71: 649–659. doi: 10.1093/sysbio/syab097. [DOI] [PubMed] [Google Scholar]
  89. Smith ML, Hahn MW.. 2021b. New approaches for inferring phylogenies in the presence of paralogs. Trends in Genetics 37: 174–187. doi: 10.1016/j.tig.2020.08.012. [DOI] [PubMed] [Google Scholar]
  90. Soltis DE, Visger CJ, Soltis PS.. 2014. The polyploidy revolution then…and now: Stebbins revisited. American Journal of Botany 101: 1057–1078. doi: 10.3732/ajb.1400178. [DOI] [PubMed] [Google Scholar]
  91. Stebbins GL. 1950. Variation and evolution in plants. New York: Columbia University Press. [Google Scholar]
  92. Stoughton TR, Kriebel R, Jolles DD, O’Quinn RL.. 2018. Next-generation lineage discovery: a case study of tuberous Claytonia L. American Journal of Botany 105: 536–548. doi: 10.1002/ajb2.1061. [DOI] [PubMed] [Google Scholar]
  93. Stuart DD. 2006. Buddlejas. Portland: Timber Press. [Google Scholar]
  94. Suissa JS, Kinosian SP, Schafran PW, Bolin JF, Taylor WC, Zimmer EA.. 2022. Homoploid hybrids, allopolyploids, and high ploidy levels characterize the evolutionary history of a western North American quillwort (Isoetes) complex. Molecular Phylogenetics and Evolution 166: 107332. doi: 10.1016/j.ympev.2021.107332. [DOI] [PubMed] [Google Scholar]
  95. Suvorov A, Scornavacca C, Fujimoto MS, et al. 2022. Deep ancestral introgression shapes evolutionary history of dragonflies and damselflies. Systematic Biology 71: 526–546. doi: 10.1093/sysbio/syab063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  96. Suyama M, Torrents D, Bork P.. 2006. PAL2NAL: robust conversion of protein sequence alignments into the corresponding codon alignments. Nucleic Acids Research 34: W609–W612. doi: 10.1093/nar/gkl315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  97. Talavera G, Castresana J.. 2007. Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments. Systematic Biology 56: 564–577. doi: 10.1080/10635150701472164. [DOI] [PubMed] [Google Scholar]
  98. Tallent-Halsell NG, Watt MS.. 2009. The invasive Buddleja davidii (butterfly bush). Botanical Review 75: 292–325. doi: 10.1007/s12229-009-9033-0. [DOI] [Google Scholar]
  99. Than C, Ruths D, Nakhleh L.. 2008. PhyloNet: a software package for analyzing and reconstructing reticulate evolutionary relationships. BMC Bioinformatics 9: 322. doi: 10.1186/1471-2105-9-322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  100. Van de Peer Y, Mizrachi E, Marchal K.. 2017. The evolutionary significance of polyploidy. Nature Reviews Genetics 18: 411–424. doi: 10.1038/nrg.2017.26. [DOI] [PubMed] [Google Scholar]
  101. Wu SD, Han BC, Jiao YN.. 2020. Genetic contribution of paleopolyploidy to adaptive evolution in angiosperms. Molecular Plant 13: 59–71. doi: 10.1016/j.molp.2019.10.012. [DOI] [PubMed] [Google Scholar]
  102. Wu SD, Wang Y, Wang ZF, Shrestha N, Liu JQ.. 2022. Species divergence with gene flow and hybrid speciation on the Qinghai–Tibet Plateau. New Phytologist 234: 392–404. doi: 10.1111/nph.17956. [DOI] [PubMed] [Google Scholar]
  103. Wu ZY, Sun H, Zhou ZK, Li DZ, Peng H.. 2010. Floristics of seed plants from China. Beijing: Science Press, 83–89. [Google Scholar]
  104. Xiong HF, Wang DY, Shao C, et al. 2022. Species tree estimation and the impact of gene loss following whole-genome duplication. Systematic Biology. 71: 1348–1361. doi: 10.1093/sysbio/syac040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  105. Xu W, Dong W-J, Fu T-T, et al. 2020. Herpetological phylogeographic analyses support a Miocene focal point of Himalayan uplift and biological diversification. National Science Review 8: nwaa263. doi: 10.1093/nsr/nwaa263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  106. Yan XX, Hong LY, Pei SJ, et al. 2021. A natural yellow colorant from Buddleja officinalis for dyeing hemp fabric. Industrial Crops and Products 171: 113968. doi: 10.1016/j.indcrop.2021.113968. [DOI] [Google Scholar]
  107. Yan Z, Smith ML, Du P, Hahn MW, Nakhleh L.. 2021. Species tree inference methods intended to deal with incomplete lineage sorting are robust to the presence of paralogs. Systematic Biology 71: 367–381. doi: 10.1093/sysbio/syab056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  108. Yang M-J, Luo S-H, Guo K, Liu Y, Li S-H.. 2023. Chemical investigation of Buddleja officinalis leaves and localization of defensive triterpenoids to its glandular trichomes. Fitoterapia 164: 105379. doi: 10.1016/j.fitote.2022.105379. [DOI] [PubMed] [Google Scholar]
  109. Yang YB, Ye CC, Galy A, et al. 2021. Monsoon-enhanced silicate weathering as a new atmospheric CO2 consumption mechanism contributing to fast Late Miocene global cooling. Paleoceanography and Paleoclimatology 36: e2020PA004008. [Google Scholar]
  110. Zhang C, Rabiee M, Sayyari E, Mirarab S.. 2018. ASTRAL-III: polynomial time species tree reconstruction from partially resolved gene trees. BMC Bioinformatics 19: 153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  111. Zhang D, Gao FL, Jakovlić I, et al. 2020. PhyloSuite: an integrated and scalable desktop platform for streamlined molecular sequence data management and evolutionary phylogenetics studies. Molecular Ecology Resources 20: 348–355. doi: 10.1111/1755-0998.13096. [DOI] [PubMed] [Google Scholar]
  112. Zhang X, Chen G, Gong WC, Sun WB.. 2014. Buddleja caryopteridifolia (Scrophulariaceae), a species to be recognized based on morphology, floral scent and AFLP data. Phytotaxa 161: 181–193. doi: 10.11646/phytotaxa.161.3.1. [DOI] [Google Scholar]
  113. Zhang X, Landis JB, Sun YX, et al. 2021. Macroevolutionary pattern of Saussurea (Asteraceae) provides insights into the drivers of radiating diversification. Proceedings of the Royal Society B: Biological Sciences 288: 20211575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  114. Zhao DY, Hamilton JP, Bhat WW, et al. 2019. A chromosomal-scale genome assembly of Tectona grandis reveals the importance of tandem gene duplication and enables discovery of genes in natural product biosynthetic pathways. GigaScience 8: giz005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  115. Zhou R, Moshgabadi N, Adams KL.. 2011. Extensive changes to alternative splicing patterns following allopolyploidy in natural and resynthesized polyploids. Proceedings of the National Academy of Sciences of the United States of America 108: 16122–16127. doi: 10.1073/pnas.1109551108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  116. Zhu RB, Kang B, Cheng JM, Zhang BQ, Zhao XY.. 2014. Buddleja jinsixiaensis (Scrophulariaceae), a new species from Shaanxi, China. Phytotaxa 159: 291–294. doi: 10.11646/phytotaxa.159.4.6. [DOI] [Google Scholar]

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Supplementary Materials

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Data Availability Statement

The data that support the findings of this study can be found in online repositories. The names of the repository and accession number can be found below: https://db.cngb.org/search/project/CNP0003159/.

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