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. 2022 May 9;130(1):53–64. doi: 10.1093/aob/mcac059

An overlooked dispersal route of Cardueae (Asteraceae) from the Mediterranean to East Asia revealed by phylogenomic and biogeographical analyses of Atractylodes

Maoqin Xia 1,#, Minqi Cai 2,#, Hans Peter Comes 3, Li Zheng 4,5, Tetsuo Ohi-Toma 6, Joongku Lee 7, Zhechen Qi 8, Kamil Konowalik 9, Pan Li 10,✉, Kenneth M Cameron 11, Chengxin Fu 12
PMCID: PMC9295924  PMID: 35533344

Abstract

Background and Aims

The East Asian–Tethyan disjunction pattern and its mechanisms of formation have long been of interest to researchers. Here, we studied the biogeographical history of Asteraceae tribe Cardueae, with a particular focus on the temperate East Asian genus Atractylodes DC., to understand the role of tectonic and climatic events in driving the diversification and disjunctions of the genus.

Methods

A total of 76 samples of Atractylodes from 36 locations were collected for RAD-sequencing. Three single nucleotide polymorphism (SNP) datasets based on different filtering strategies were used for phylogenetic analyses. Molecular dating and ancestral distribution reconstruction were performed using both chloroplast DNA sequences (127 Cardueae samples) and SNP (36 Atractylodes samples) datasets.

Key Results

Six species of Atractylodes were well resolved as individually monophyletic, although some introgression was identified among accessions of A. chinensis, A. lancea and A. koreana. Dispersal of the subtribe Carlininae from the Mediterranean to East Asia occurred after divergence between Atractylodes and Carlina L. + Atractylis L. + Thevenotia DC. at ~31.57 Ma, resulting in an East Asian–Tethyan disjunction. Diversification of Atractylodes in East Asia mainly occurred from the Late Miocene to the Early Pleistocene.

Conclusions

Aridification of Asia and the closure of the Turgai Strait in the Late Oligocene promoted the dispersal of Cardueae from the Mediterranean to East China. Subsequent uplift of the Qinghai–Tibet Plateau as well as changes in Asian monsoon systems resulted in an East Asian–Tethyan disjunction between Atractylodes and Carlina + Atractylis + Thevenotia. In addition, Late Miocene to Quaternary climates and sea level fluctuations played major roles in the diversification of Atractylodes. Through this study of different taxonomic levels using genomic data, we have revealed an overlooked dispersal route between the Mediterranean and far East Asia (Japan/Korea) via Central Asia and East China.

Keywords: East Asian–Tethyan disjunction, Atractylodes, Cardueae, phylogenomics, biogeography

Introduction

The disjunct pattern of plant and animal taxa between East Asia and the Mediterranean is generally known as the ‘East Asian–Tethyan disjunction’. Two hypotheses have been proposed to explain these disjunctions: one is the fragmentation of once widespread Cenozoic palaeotropical evergreen forests (Axelrod, 1975; Wen and Ickert-Bond, 2009; Blondel et al., 2010); the other considers long distance dispersal (LDD) or a combination of dispersal and vicariance (Rodríguez-Sánchez et al., 2009; Zhou et al., 2012; Nie et al., 2013; Chen et al., 2014; Wei et al., 2017; Jiang et al., 2019). Climatic and geological events of the Cenozoic intensively influenced the temporal and spatial evolution of these disjunct floras (Chen et al., 2012). For example, closure of the Turgai Strait provided numerous opportunities for interactions between previously isolated flora within Eurasia (Tiffney and Manchester, 2001). The collision of the Indian subcontinent with Eurasia promoted the uplift of the Qinghai–Tibet Plateau (QTP), and subsequent climate change further contributed to the disjunct distribution of many temperate or tropical plants between East Asia and the Mediterranean region that were once widely distributed across Eurasia (Yin and Harrioson, 2000; Guo et al., 2002, 2008; Aitchison et al., 2007; Royden et al., 2008). Our knowledge of East Asian–Tethyan disjunct plant groups has made great progress in recent years thanks to improved phylogenetic reconstruction, molecular dating techniques and ancestral area reconstruction methods. However, previous research on this disjunction pattern has generally been biased toward tropical taxa (e.g. Pistacia L., Xie et al., 2014; Smilax L., Chen et al., 2014), meaning that alternative routes of migration, including dispersal routes across temperate regions, may have been overlooked.

To consider this hypothesis, we focused on the tribe Cardueae (Asteraceae), which contains ~74 genera and 2500 species (Ackerfield et al., 2020), making it one of the largest tribes in the hyper-diverse family Asteraceae (Susanna and Garcia-Jacas, 2009). Species of Cardueae are distributed across all continents, except Antarctica, with the highest levels of diversity found in the Mediterranean Basin, North Africa and West Asia (Susanna and Garcia-Jacas, 2007). These annual, biennial or perennial herbs, as well as a few shrubs and treelets (Barres et al., 2013; Herrando-Moraira et al., 2019), grow in various habitats such as scrub/grasslands, (semi-)deserts, alpine meadows, temperate deciduous and mixed temperate–boreal forests, and tropical savannahs (Susanna and Garcia-Jacas, 2009; Herrando-Moraira et al., 2019). Given its broad intercontinental distribution, Cardueae represents an ideal model system to investigate the formation and evolutionary history of disjunct floras and the biogeographical connections between them.

Previous molecular biogeographical studies by Barres et al. (2013) suggested that Cardueae originated in West Asia around the Middle Eocene, followed by early diversification in the Mediterranean Basin and Central Asia during the Oligocene–Miocene. Today there exists a classical East Asian–Tethyan disjunction pattern within the subtribe Carlininae (comprising five genera: Atractylodes DC., Atractylis L., Carlina L., Thevenotia DC. and Tugarinovia Iljin), which they proposed to be the result of dispersal via a northern route (i.e. North Caspian Sea–Pamir Mountains–Mongolian Plateau–Korean Peninsula–Japanese archipelago; see Supplementary Data Fig. S1a) from the Mediterranean to Central and East Asia in the Late Eocene–Early Oligocene, followed by vicariance (Barres et al., 2013). However, very few samples of the East Asian genus Atractylodes have been included in earlier phylogeographical studies (only Atractylodes japonica Koidz. ex Kitam.), so the predicted dispersal route may not be entirely accurate. In this study, we explore the evolution and biogeographical history of the tribe Cardueae, with a particular focus on Atractylodes, in order to provide new insights into our understanding of the dispersal/migration routes of the East Asian–Tethyan disjunction taxa in the north temperate zone.

Atractylodes consists of four to seven perennial herb species that are endemic to temperate East Asia (China, Japan, Korea and Russian Far East), where they mainly occur on dry mountain slopes or in temperate forests/grasslands (Shi, 1987; Shi and Greuter, 2011; Peng et al., 2012). Rhizomes of these species are used medicinally (Shiba et al., 2006; Zheng et al., 2012, 2013), and the plants are characterized by undivided to pinnatipartite leaves with spinule or spine-tipped teeth, dioecious flowers with white or purplish-red corolla, and obovoid to ovoid and compressed achenes (Shi, 1987; Shi and Greuter, 2011). Morphological and molecular phylogenetic studies have found that Atractylodes is closely related to Atractylis, Carlina and Thevenotia, which are all native to Eurasia, North Africa and the Irano-Turanian region (Petit et al., 1997; Garcia-Jacas et al., 2002; Peng et al., 2012; Barres et al., 2013; Herrando-Moraira et al., 2019).

There is a great deal of taxonomic controversy within Atractylodes (De Candolle, 1838; Ling, 1935; Hu, 1965; Fu et al, 1981; Shi, 1987; Shi and Greuter, 2011), especially as related to the ‘A. lancea complex’, which includes Atractylodes japonica, A. koreana (Nakai) Kitam., A. lancea (Thunb.) DC. (subsp. lancea and subsp. luotianensis) and A. chinensis (DC.) Koidz. (Peng et al., 2012; Zheng, 2013). Additionally, the possibility of hybridization/introgression has been recognized between A. koreana and A. chinensis, as well as between A. lancea and A. chinensis (Shiba et al., 2006). To provide a well-supported phylogenetic reconstruction of this genus, increased sampling of both taxa and molecular data is required to fully resolve some of these long-standing puzzles.

We have chosen to employ restriction site-associated DNA sequencing (RAD-seq), which can produce tens of thousands of multi-locus sequence data in non-model species at relatively low cost (Baird et al., 2008). RAD-seq is a promising tool to resolve phylogenetic relationships among closely related species that diverged only recently and/or experienced hybridization/introgression (e.g. Eaton and Ree, 2013; Liu et al., 2015; Paun et al., 2015; Zinenko et al., 2016). Moreover, the numerous unlinked loci and single nucleotide polymorphisms (SNPs) identified by RAD-seq have proven useful to differentiate introgression from incomplete lineage sorting (ILS) of ancestral polymorphisms (Kronforst, 2008; Yu et al., 2011). In turn, this enables high-resolution examination of lineage sorting dynamics (Davey et al., 2011) via the ‘ABBA-BABA’ test, which uses Patterson’s D-statistic (Green et al., 2010; Durand et al., 2011), to compare the frequencies of discordant SNP genealogies in a pectinate four-taxon tree (Eaton and Ree, 2013; Streicher et al., 2014).

Through the use of these methods, our aim here is to: (1) generate a well-resolved and well-supported phylogenetic reconstruction of Atractylodes; (2) investigate to what extent unresolved relationships within the A. lancea complex might be due to hybridization/introgression and/or ILS; and (3) infer the formation of an East Asian–Tethyan disjunction and the biogeographical history of the genus.

MATERIALS AND METHODS

Taxon sampling and DNA extraction

To maximize geographical representation, 76 samples of seven Atractylodes species [Atractylodes carlinoides (Hand.-Mazz.) Kitam., A. chinensis, A. japonica, A. koreana, A. lancea (subsp. lancea and subsp. luotianensis), A. macrocephala Koidz. and A. ovata (Thunb.) DC.] from 36 localities were used for phylogenetic RAD-seq analyses (Fig. 1; Supplementary Data Table S1). The samples of A. macrocephala were divided into three groups: (I) two populations (PBD and PBX) of ‘Pingzhu’, which is a local landrace in Pingjiang County, Hunan Province; (II) one wild population (QM) of ‘Qizhu’, which is collected from Qimen County, Anhui Province; and (III) three populations (RC, TM and YC) of ‘Baizhu’, which is the most widely cultivated type (Fig. 1). Samples from China, South Korea and Japan were field-collected between 2011 and 2016, while those from North Korea (A. koreana) and Russia (A. ovata) are from specimens provided by the Korea National Arboretum (KH). Total DNA was extracted using silica gel-dried leaves or herbarium specimens following a modified CTAB procedure (Li et al., 2013).

Fig. 1.

Fig. 1.

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Geographical information of the 36 populations of Atractylodes used for RAD-sequencing (see Supplementary Data Table S1 for precise location information).

RAD-seq library preparation and de novo assembly

Library preparation and sequencing of RAD markers for 76 individuals of Atractylodes (Supplementary Data Table S1) were carried out at the Beijing Genomics Institute (Shenzhen, China) using the restriction enzyme EcoR1. The 90-bp paired-end sequencing of multiplexed libraries was conducted on an Illumina Hiseq 2000; raw reads were de-multiplexed based on the sample-specific barcode sequence and then trimmed to a common length of 82 bp. The raw data are deposited as a BioProject in the Sequence Read Archive (SRA) with accession number SRP107226.

We utilized IPYRAD v0.6.11 (Eaton and Ree, 2013; Eaton, 2014; https://ipyrad.readthedocs.io/en/master/) for quality filtering and de novo clustering of the RAD-seq forward data to produce SNP datasets. Base calls with a Phred quality score <33 were converted to Ns, and any read containing Ns was discarded. A threshold of 0.90 was used for both within- and across-sample clustering. When clustering across samples, loci with a heterozygous site shared by more than 50 % of the samples were discarded as putative paralogues, and loci containing more than 20 SNPs were discarded. We kept only one SNP per RAD-seq locus to create a dataset of unlinked loci. To investigate the influence of different degrees of missing data on phylogenetic analysis, we assembled three data matrices with different minima for sample coverage: (1) data matrix ‘D1’, i.e. the maximum data matrix that includes all loci shared across at least 10 samples; (2) the medium data matrix ‘D2’ that contains all loci shared across at least 30 samples; and (3) the minimum data matrix ‘D3’ that includes all loci shared across at least 45 samples.

Phylogenetic inference based on RAD-seq (SNP) data

Since A. carlinoides has been resolved as the first-diverging species within the genus based on previous phylogenetic studies (Zou et al., 2009; Peng et al., 2012; Zheng, 2013; Wang et al., 2021), it was used as the outgroup to root the trees (phylograms) for the phylogenetic RAD-seq analyses. To reveal the influence of different proportions of missing data on phylogenetic inference, we performed phylogenetic inference based on three SNP datasets (hereafter D1, D2 and D3). Maximum-likelihood (ML) phylogenetic trees were inferred in RAxML v8.2.10 (Stamatakis, 2014) at the CIPRES Science Gateway (Miller et al., 2010). Analyses were performed with random starting trees under the GTR + G nucleotide substitution model; node support values were estimated by performing 1000 bootstrap replicates. The results were visualized in FIGTREE v1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/).

Identification of introgression with D-statistic tests

The role of hybridization/introgression and/or ILS in the divergence of Atractylodes species was measured using Patterson’s D-statistic (Green et al., 2010; Durand et al., 2011; Eaton and Ree, 2013). In brief, given a pectinate four-taxon topology (((P1, P2), P3), O), the genome-wide frequencies of two incongruent allele patterns (‘ABBA’ and ‘BABA’) are expected to be equal if the cause of incongruence is ILS. Alternatively, if the cause of incongruence is introgression between P3 and either P1 or P2, the frequencies of these two patterns are not expected to be equal (Eaton and Ree, 2013).

To evaluate the hypotheses of genetic introgression among A. koreana, A. lancea and A. chinensis presented in Shiba et al. (2006), we used the evobiR package (Streicher et al., 2014) in R v3.3.3 to implement D-statistic tests based on the D2 dataset. To measure gene introgression among different species and/or populations, we assumed three scenarios to satisfy the topology of (((P1, P2), P3), O) as follows: (1) (((C1, C2), L), K), where ‘C’ represents A. chinensis populations (LBT, LGS, LKY, LLB, LTB LTL and LYT), ‘L’ represents A. lancea populations (LMS, LSZ and LTT), and ‘K’ represents an A. koreana population (KRS); (2) (((L1, L2), C), K), where ‘L’, ‘C’ and ‘K’ are as above; and (3) (((C, L), K), J), where ‘C’ and ‘L’ are as above, but ‘K’ represents A. koreana populations (KFC, KKY, KLT and KRS), and ‘J’ represents an A. japonica population (JMR). We used one individual per population, and performed all possible combinations between individuals in the P1, P2 and P3 positions. In total, 168 combinations were tested (Supplementary Data Table S2), and each test was performed with a significance test using the jackknife approach. A significant signal of introgression was indicated by Z-scores >3 (Eaton et al., 2015).

Divergence time estimation based on cpDNA and RAD-seq (SNP) data

For dating analyses of Cardueae, we took advantage of four published chloroplast (cp) DNA markers (matK, ndhF, rbcL, trnL-trnF) from 118 species of Asteraceae and three outgroups (Calyceraceae: Boopis anthemoides Juss., Nastanthus patagonicus Speg.; and Goodeniaceae: Scaevola aemula R. Br.) based on previous phylogenetic studies (Kim and Jansen, 1995; Hidalgo et al., 2006; Susanna et al., 2006, 2011; Anderberg et al., 2007; Jansen et al., 2007; Garcia-Jacas et al., 2008; Panero and Funk, 2008; Gruenstaeudl et al., 2009; Sánchez-Jiménez et al., 2010; Barres et al., 2013; Herrando-Moraira et al., 2019), and sequenced six species of Atractylodes (A. carlinoides, A. macrocephala, A. ovata, A. koreana and A. lancea subsp. luotianensis; Supplementary Data Table S3) for the same cp markers following the methods of Barres et al. (2013). A total of 127 DNA sequences were aligned using MAFFT v7 (Katoh and Standley, 2013) in GENEIOUS R9.1.4 (Kearse et al., 2012), coupled with manual corrections. All newly generated cpDNA sequences were deposited in GenBank (see Table S3).

A two-step estimation approach for divergence times was implemented based on Bayesian analyses in BEAST v1.10.4 (Suchard et al., 2018). First, we estimated the crown age of Atractylodes based on the four cpDNA markers (i.e. matK, ndhF, rbcL, trnL–trnF) of 127 individuals (Supplementary Data Table S3). One secondary dated node and four fossil calibration points were used as node age constraints following Herrando-Moraira et al. (2019): (1) a secondary calibration node of 69.56 ± 3 million years ago (Ma) for constraining the stem age of Asteraceae with a normal distribution (Panero and Crozier, 2016); (2) 47.5 ± 1.1 Ma for the divergence time of subfamilies Carduoideae and Mutisioideae with a lognormal distribution (Barreda et al., 2012); (3) 14 ± 1.1 Ma for the stem age of Carduus–Cirsium with a lognormal distribution (Mai, 1995); (4) 8 ± 1.1 Ma for the stem age of Arctium L. with a lognormal distribution (Mai, 2001; López-Vinyallonga et al., 2009); and (5) 6 ± 1.1 Ma for the stem age of Centaurea L. subgenus Cyanus with a lognormal distribution (Wagenitz, 1955; Ivanov et al., 2007) (Supplementary Data Fig. S1). Second, based on the RAD-seq data, we modified dataset ‘D2’ to generate a fourth SNP matrix (‘D4’), which consisted of 36 Atractylodes samples (e.g. one individual per population). The crown age of Atractylodes inferred from the cpDNA chronogram was then used to calibrate the phylogeny of the genus based on the SNP dataset D4.

For the cpDNA dataset, we first checked whether the concatenated sequences were saturated for substitutions by performing the saturation test in DAMBE v4.0.36 (Xia and Xie, 2001). The results did not indicate a significant signal of saturation. Then we utilized jModelTest2 (Darriba et al., 2012) in CIPRES to determine the appropriate substitution model for each partition (i.e. matK, trnL-trnF: GTR + G; ndhF, rbcL: GTR + I + G) according to the Akaike information criterion (AIC). For each BEAST analysis (cpDNA and D4 dataset), we used an uncorrelated lognormal relaxed clock with two Markov chain Monte Carlo (MCMC) runs of 108 generations, sampling every 103 generations; the initial 10 % of cycles were discarded as burn-in. MCMC samples were inspected in Tracer v1.7.1 (Rambaut et al., 2018) to confirm sampling adequacy and convergence of the chains to a stationary distribution. The resulting maximum clade credibility (MCC) chronograms were visualized in FIGTREE v1.3.1.

Ancestral area reconstructions

Ancestral area reconstructions were performed on BEAST-derived MCC trees from the Cardueae cpDNA dataset and the SNP-derived D4 dataset from Atractylodes, respectively. We coded the distribution of each species according to previous research (Barres et al., 2013; Herrando-Moraira et al., 2019). In total, 16 geographical regions were defined: A, East China; B, Northeast Asia; C, Japan; D, western Mediterranean Basin; E, eastern Mediterranean Basin; F, West Asia; G, Central and North Europe; H, Central Asia; I, Macaronesia; J, North America; K, southern South America; L, Central and South Africa; M, Himalayan range; N, North Africa; O, coastal areas along the Indian Ocean from the Horn of Africa to India; and P, Australia. We estimated ancestral ranges on each MCC tree using Bayesian binary MCMC (BBM) analysis in RASP v4.2 (Yu et al., 2015) under the F81 + G model for 108 generations and sampling every 100 generations. The maximum number of areas per species was set to four.

RESULTS

RAD-seq data matrices

From the 76 individuals of seven Atractylodes species, the Illumina Hiseq 2000 yielded a total of 44.9 Gb of raw data from forward-end sequencing. After quality filtering, we obtained an average of ~9.3 × 106 reads per sample, ranging from ~2.1 × 106 to ~16.0 × 106. Following de novo assembly, we obtained an average of ~0.19 × 106 consensus loci (range: ~0.038 × 106 to 0.35 × 106; see Supplementary Data Table S4). When the values of minimum samples per locus were set to 10, 30 and 45, the resulting datasets, D1, D2 and D3, contained 171 240, 27 311 and 2624 unlinked SNPs and 74.6, 52.8 and 31.31 % missing data, respectively.

Phylogenetic relationships within Atractylodes based on RAD-seq data

Phylogenetic (ML) analyses of RAD-seq data based on datasets D1 and D2 produced almost identical tree topologies with high bootstrap support (BS) values (≥80) for all major clades (Fig. 2A, B). With A. carlinoides used as the outgroup, both phylograms revealed a successive grade of A. macrocephala, A. japonica/A. ovata and A. koreana, plus a clade of A. chinensis + A. lancea. Notably, within A. macrocephala, three well-supported subclades (I–III) can be distinguished (all BS ≥ 97), comprising accessions of (I) the two landrace populations (PBD and PBX) of ‘Pingzhu’; (II) one wild population (QM) of ‘Qizhu’; and (III) three cultivated populations (RC, TM and YC) of ‘Baizhu’. Two populations (JKN and RPK) of A. ovata are not part of a monophyletic clade for that species, but instead are embedded among 13 populations of A. japonica. Neither A. ovata nor A. japonica is strictly monophyletic. Finally, within A. lancea there is a distinct subsp. luotianensis (three individuals of the LTT population) that forms a subclade (BS = 100).

Fig. 2.

Fig. 2.

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Maximum-likelihood (ML) phylograms of Atractylodes generated from three RAD-seq matrices differing in the maximum dataset that includes all loci shared across at least 10 (D1), 30 (D2) and 45 (D3) samples. ML bootstrap support >80 % is reported at nodes. Two samples of A. ovata are underlined. Asterisks on the nodes mean bootstrap support = 100%.

When the tolerance for missing data was further reduced, that is from 52.8 % (dataset D2) to 31.31 % (D3), the number of SNPs decreased dramatically (from 27 311 to 2624). As a consequence, the tree topology of dataset D3 (Fig. 2C) received lower support for most clades (e.g. A. chinensis + A. lancea, BS < 80). In fact, the internal topology of most major clades collapsed, resulting in spurious sister relationships, albeit with low support (e.g. A. macrocephala + A. japonica/A. ovata, BS < 80).

Introgression and hybridization in Atractylodes

Significant signals of introgression were identified among A. chinensis (C), A. lancea (L) and A. koreana (K), with only two of 168 tests non-significant (Table 1, details of each test are available in Supplementary Data Table S2). In scenario 1, we set different populations of A. chinensis as P1 and P2, and A. lancea as P3. All 63 tests identified significant introgression, with 45 tests fitted to the ‘ABBA’ pattern, while 18 had a ‘BABA’ pattern. In scenario 2, different populations of A. lancea were set as P1 and P2, whereas A. chinensis was set to P3. All 21 tests showed significant introgression. The LSZ population of A. lancea had a higher introgression with populations of A. chinensis compared to LTT and LMS. Finally, in scenario 3, 56 tests supported the ‘BABA’ pattern, while 26 tests supported the ‘ABBA’ pattern, and two tests (3-5 and 3-10) were non-significant. Most populations of A. koreana (KFC, KLT and KRS) had introgression with populations of A. chinensis, except KKY5, which showed more gene flow with A. lancea.

Table 1.

Statistics of 168 Patterson’s four-taxon D-statistic tests of Atractylodes

Scenario P1 P2 P3 O Nsig/Ntotal (P < 0.001) D raw statistic SD D Z-scores Taxa involved in introgression
1 C1 C2 L K 63/63 −0.77 to 0.33 0.002–0.006 3.46–285.48 A. chinensis – A. lancea
2 L1 L2 C K 21/21 −0.35 to 0.47 0.003–0.015 22.47–157.85 A. lancea – A. chinensis
3 C L K J 82/84 −0.77 to 0.58 0.002–0.009 1.46–179.63 A. chinensis – A. koreana, A. lancea – A. koreana
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Nsig/Ntotal, number of significant replicates/total test number; D raw statistic, the range of Patterson’s four-taxon D-statistic in each scenario; SD D, the range of standard deviation of the D-statistic; Z-scores, the range of Z-scores in each scenario; C, A. chinensis populations (LBT, LGS, LKY, LLB, LTB, LTL and LYT), with C1 and C2 presenting different populations in each test; L, A. lancea populations (LMS, LSZ and LTT), with L1 and L2 presenting different populations in each test; K, A. koreana populations (KFC, KKY, KLT and KRS), but in scenarios 1 and 2, K was represented by KRS; J, A. japonica population (JMR). See Supplementary Data Table S2 for details of each test.

Molecular dating and ancestral area reconstructions

The BEAST-derived cpDNA chronogram of Cardueae (Supplementary Data Fig. S1b) indicates that this tribe originated at ~44.62 Ma [95 % highest posterior density (HPD): 40.66–47.93 Ma] (node N1) and first diverged at 41.55 Ma (36.87–45.52 Ma; node N2) in the Mid-Eocene. The stem and crown age of the subtribe Carlininae is 40.32 Ma (35.64–44.59 Ma; node N3) and 37.04 Ma (30.52–42.73 Ma; node N4), respectively. The genus Atractylodes split from its sister Atractylis + Carlina + Thevenotia at ~31.57 Ma (24.20–38.32 Ma; node N5). In addition, the crown age of Atractylodes was calculated to be during the Late Miocene, ~10.04 Ma (3.27–20.45 Ma; node N6).

The chronogram based on the SNP dataset D4 (Fig. 3) shows the same topology at the species level as the D1 and D2 phylograms (see Fig. 2A, B). Based on this D4 dataset, the crown age of Atractylodes (node N1; Fig. 3), marking the origin of A. carlinoides, was dated to ~9.64 Ma (7.26–13.30 Ma), which was later than that obtained by cpDNA (see above), but had a much narrower 95 % HPD interval. All subsequent speciation events within the genus occurred in quick succession during the Late Miocene–Early Pliocene (~8.00–4.34 Ma, nodes N2, N4, N8 and N9) and diversification of each species occurred at ~4.18–2.74 Ma (nodes N3, N5, N10, N11 and N12).

Fig. 3.

Fig. 3.

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Chronogram showing the Bayesian consensus tree of 36 Atractylodes samples based on SNP-derived dataset D4 and Bayesian binary MCMC (BBM)-derived reconstruction of biogeographical history. The three distribution areas of Atractylodes used in the Bayes-based biogeographical reconstruction are: A, East China; B, Northeast Asia; and C, Japan. The box with number 1 indicates the secondary calibration point for the crown age of Atractylodes estimated from BEAST analysis of a cpDNA dataset. Nodes N1–N12 show crown ages (95 % highest posterior density) for the divergence time of each major clade. Pie diagrams at internal nodes indicate the relative probabilities for each alternative area.

The exact ancestral area of the tribe Cardueae (node N2; Supplementary Data Fig. S1b) and the subtribe Carlininae (node N4) is uncertain, but is concentrated in the East Mediterranean Basin (E), West Asia (F) and Central Asia (H), i.e. the coast of a Palaeo-Tethys Ocean. Both BBM analyses based on the cpDNA dataset (relative probability of ancestral region: P = 0.91, node N6; Fig. S1b) and the SNP dataset D4 (P = 1.00, node N1; Fig. 3) identified East China (A) as the ancestral area of Atractylodes. Atractylodes carlinoides (P = 1.00, node N1), A. chinensis (P = 0.48, node N11), A. lancea (P = 0.98, node N10) and A. macrocephala (P = 1.00, node N3) began to diverge in this ancestral region, with a dispersal event from East China to Northeast Asia identified in A. chinensis (node N11). Both A. koreana (P = 0.92, node N12) and A. japonica/A. ovata (P = 0.59, node N5) originated in Northeast Asia (B). Individuals of A. japonica/A. ovata appear to have undergone multiple dispersals from Northeast Asia to Japan at the Plio-/Pleistocene boundary (see nodes N6 and N7; Fig. 3).

Discussion

A fully resolved RAD-seq phylogeny of Atractylodes with taxonomic implications

All of our phylogenetic inferences of the RAD-seq data support six main clades in Atractylodes (Fig. 2) corresponding to the species A. carlinoides, A. macrocephala, A. japonica/A. ovata, A. koreana, A. chinensis and A. lancea. Congruent with previous studies based on plastid and nuclear data, A. carlinoides and A. macrocephala are the two earliest diverging taxa in this genus and are morphologically distinct compared to all other species (Peng et al., 2012; Zheng, 2013). Relationships among the other species have always been a challenge to resolve. In particular, the so-called ‘A. lancea’ complex (including A. japonica/A. ovata, A. koreana, A. chinensis and A. lancea) formed an unresolved complex in previous plastid and nuclear (ITS/ETS) analyses (Peng et al., 2012; Zheng, 2013), but are clearly recovered as four monophyletic taxa based on our RAD-seq data (Figs 2 and 3).

There have been long-standing controversies over the classification of Atractylodes (De Candolle, 1838; Ling, 1935; Hu, 1965; Fu et al, 1981; Shi, 1987; Shi and Greuter, 2011). One intensely debated issue is whether A. chinensis should be merged within A. lancea. Our molecular data strongly support the species status of A. chinensis as a sister to A. lancea (Fig. 2). Likewise, Shiba et al. (2006) documented non-overlapping morphological differences between A. chinensis and A. lancea, which further supports their continued recognition as separate species. Another controversial taxon is A. ovata, individuals of which are clearly nested within A. japonica in our SNP-derived phylogenetic trees (Fig. 2). There is no evidence that these two species are reciprocally monophyletic. Thus, given the timing of their publication and in accordance with international rules of nomenclature, A. japonica (Kitamura, 1935) should be treated as a synonym of A. ovata (De Candolle, 1838). Finally, we wish to highlight the unique phylogenetic position and ancient origin of a landrace of A. macrocephala, ‘Pingzhu’. This is part of an interesting diversification and domestication pattern that is seen within A. macrocephala. It splits into three subclades, of which subclade I represents a landrace named ‘Pingzhu’ (populations PBD and PBX), subclade II contains a wild population (QM) named ‘Qizhu’, and subclade III includes the remaining populations (RC, TM and YC) named ‘Baizhu’ and grown in traditional cultivation areas (Fig. 2). The topology, branch lengths and divergence times (Figs 2 and 3) together indicate that A. macrocephala might have been domesticated at least twice, and the only wild population (QM) we sampled here apparently was not involved in the domestication process. We hypothesize that ‘Pingzhu’ may have been domesticated from another ancestral species of Atractylodes, which is now extinct. Therefore, ‘Pingzhu’ might be best treated as a distinct species, rather than as a landrace of A. macrocephala. A more extensive sampling of A. macrocephala, particularly including more wild populations of this endangered species, together with a careful study of morphology and ecology, will be key to future studies of the systematics and domestication history of this important medicinal plant.

In this study, a fraction of the RAD-seq loci contain a different phylogenetic signal; that is, in dataset D3 A. macrocephala is sister to A. japonica (Fig. 2C). However, a large number of loci (datasets D1 and D2) seem to overwhelm the occasionally discordant phylogenetic signals in the data (Fig. 2A, B). It is also clear that when we compared the phylogenetic analyses among the three contrasting RAD-seq data matrices (D1, D2 and D3), the two larger datasets D1 and D2, which included more loci but also more missing data, recovered a larger number of fully supported branches (BS = 100; Fig. 2). In contrast, the smaller dataset D3, which contained fewer loci but also less missing data, produced a tree for which many branches have low support values (BS < 80; Fig. 2). Hence, as the data matrix increases in size (i.e. number of loci), the resolution of clades increases, as does bootstrap support on the branches of the tree (Fig. 2). The same tendency has been observed in many studies of different taxa (e.g. Rubin et al., 2012; Wagner et al., 2013; Hou et al., 2015) and is a good reminder that researchers should always consider alternative data filtering strategies and analyses.

Introgression between Atractylodes chinensis and either A. lancea or A. koreana

Given the small number of loci investigated previously and the relatively recent (Late Miocene–Pliocene) divergence time among species of Atractylodes, we suggest that introgression and/or ILS may have played a major role in the discrepancies that have been observed among phylogenetic reconstructions of this genus (primarily resulting in a lack of resolution). This hypothesis is supported by the findings of Shiba et al. (2006), who showed that A. chinensis and A. koreana share an identical ITS sequence, and have recognized potential hybrids between A. chinensis and A. lancea. Additionally, our D-statistic tests detected instances of significant gene flow between A. chinensis and A. lancea populations (scenarios 1 and 2), and also between A. koreana and either A. chinensis or A. lancea populations (scenario 3; Table 1 and Supplementary Data Table S4). However, in scenario 2, we found population LSZ of A. lancea showed a higher degree of introgression with populations of A. chinensis compared to LTT and LMS, suggesting that introgression was more common in more closely distributed populations. This phenomenon has also been observed in scenario 3: the degree of introgression between A. koreana and A. chinensis was higher than that of A. koreana and A. lancea. These widely occurring examples of introgression may explain why a small number of loci from plastid and nuclear genomes cannot, on their own, resolve conflicting phylogenetic relationships within the ‘A. lancea complex’.

An overlooked dispersal route of Cardueae from the Mediterranean to East Asia

Due to the tribe’s large size and widespread distribution, the origin and diversification history of Cardueae have long been a focus of systematists and biogeographers (Susanna et al., 1995; Susanna et al., 2006; Barres et al., 2013; Huang et al., 2016; Herrando-Moraira et al., 2019; Ackerfield et al., 2020). Our dating analysis revealed that both the tribe Cardueae (44.62 Ma, 95 % HPD: 40.66–47.93 Ma) and the subtribe Carlininae (41.55 Ma, 36.87–45.52 Ma) originated in the Mid-Eocene near the coast of a Palaeo-Tethys Ocean (Supplementary Data Fig. S1b). In our study, although using the same calibration points as Herrando-Moraira et al. (2019), we obtained a much earlier origin dating of tribe Cardueae. The use of different molecular markers was the main reason for the difference in dating times between our study (cpDNA) and Herrando-Moraira et al. (2019) (nuclear conserved orthology loci).

Although most species of Cardueae are concentrated in the Mediterranean and adjacent region, some taxa (e.g. Atractylodes, Synurus Iljin and Tricholepis DC.) spread to East Asia and form an East Asian–Tethyan disjunction pattern. The genus Tricholepis, which belongs to subtribe Centaureinae, probably goes beyond the mountains of Central Asia and reaches Southeast Asia (Myanmar and Thailand) (Herrando-Moraira et al., 2019). It seems that Tricholepis took a southern route along the Himalayas. In contrast, for Atractylodes and Synurus, previous studies proposed a hypothesis that they experienced continuous range expansion from the Tethyan Coast to Far East Asia via the Pamir Mountains and Mongolian Plateau and survived in East Asian Tertiary refuges (Susanna et al., 2011; Barres et al., 2013). With comprehensive sampling of Atractylodes species in this study, we found that Atractylodes originated in more southern regions of East Asia (i.e. East China; Supplementary Data Fig. S1a). Therefore, it is necessary to reconsider whether an overlooked route (in between the southern and northern routes) existed in East Asia for subtribe Carlininae.

We found that Atractylodes separated from the Mediterranean clade of Carlina + Atractylis + Thevenotia at ~30.85 Ma (95 % HPD: 22.71–38.50 Ma; Supplementary Data Fig. S1), but diversified recently at ~9.64 Ma (7.26–13.30 Ma; Fig. 3). The closing of the Turgai Strait in the Late Oligocene undoubtedly promoted the interchange between European and Asian floras, especially the spread of many herbs (e.g. Dontostemon Andrz. ex DC., Scabiosa L. and Tragopogon L.) from the Palaeo-Mediterranean to the East, due to the expansion of an arid climate and resulting habitats in Central Asia (Zhou et al., 2006; Bell et al., 2012; Carlson et al., 2012; Friesen et al., 2016). We agreed with Susanna et al. (2011) and Barres et al. (2013) that the ancestor of Atractylodes experienced stepping-stone dispersal from the Tethyan Coast to Central Asia along the Pamir Mountains. However, instead of going straight to South Korea and Japan through the Mongolian plateau, it headed southward into East China. We speculated that Central Tibet was the migration route for Atractylodes between Central Asia and East China (e.g. Jiang et al., 2019). During this period the great valley in Central Tibet had not risen to near-modern elevations and developed a great deal of xerophytic vegetation (Coleman and Hodges, 1995; Tapponnier et al., 2001; Wang et al., 2008; Ding et al., 2014; Su et al., 2019; Spicer et al., 2020; Li et al., 2021). Furthermore, an east–west arid climatic zone extended through China during the Palaeogene (Sun and Wang, 2005; Guo et al., 2008). These climatic and tectonic changes provided an opportunity for the spread of xerically adapted taxa in East Asia. Subsequently, in the Late Oligocene, the northwest retraction of an arid/semi-arid vegetation belt (Sun and Wang, 2005; Guo et al., 2008) throughout the Palaeogene–Neogene transition and the uplift of the north/northeast parts of the QTP caused a weakening of the Asian winter monsoon and an increase of winter precipitation over East Asia, thus resulting in a more humid climate in this region and increased plant diversity (Su et al., 2018, 2019; Deng et al., 2019; Song et al., 2020; Li et al., 2021). At that time, the ancestral populations of Atractylodes that survived under the humid habitats of East Asia formed an East Asian–Tethyan disjunction with the xerically adapted sister genera (Carlina, Atractylis and Thevenotia) existing in the Mediterranean. In addition, all extant species of Atractylodes grow in relatively xeric microhabitats, such as dry slopes, forest understories or mountain ridges, which might be a plesiomorphic ecological preference tracing back to their ancestors.

BEAST analyses based on the RAD-seq D4 dataset dated the main speciation time within Atractylodes to 9.64–4.34 Ma (nodes N1, N2, N4, N8 and N9; Fig. 3) in the Late Miocene/Early Pliocene. Global cooling and strengthening of the East Asian monsoon during that period are the main hypothesized reasons for the divergence of many temperate plants (Sun and Wang, 2005; Qiu et al., 2011). Again, however, the east–west arid zone redeveloped and almost reached the coast of northern China towards the Pliocene. This arid zone has obstructed gene flow between the northern (northeast China, Korea and Japan) and southern (East China) parts of East Asia (Tiffney and Manchester, 2001; Guo et al., 2008), and may be another promotor of Atractylodes diversification.

Although during the Pleistocene East Asia was not covered by large glaciers compared to Europe and North America, the biological distribution in this region was dramatically influenced by climatic fluctuations (Qian, 1999; Harrison et al., 2001). During the Last Glacial Maximum (and possibly earlier glacial periods), populations of Atractylodes probably retreated to East China and may have occupied distinct refugia compared to those that stayed in Northeast Asia. However, during interglacials/postglacials the northward (re-)expansion of warm-temperate evergreen forest (Harrison et al., 2001) resulted in increasing potential habitats for Atractylodes. These expansions also induced secondary contact of different Atractylodes taxa in northern and southern East Asia, which is confirmed by the existence of hybrids and introgression among A. chinensis, A. lancea and A. koreana in our data (Table 1 and Supplementary Data Table S4).

The sea-level fluctuations brought about by climate changes led to the disappearance/appearance of wide stretches of the continental shelf between the Eurasian continent and Japanese archipelago (across the East China Sea, Taiwan/Ryukyu Islands and Tsushima/Korean Strait), which also provided opportunities for speciation, population fragmentation and vicariant allopatric speciation (Qian and Ricklefs, 2000; Harrison et al., 2001; Park et al., 2006; Qiu et al., 2009, 2011). It is estimated that Japanese A. japonica was first isolated from species in continental Asia at 7.27 Ma (5.53–9.35 Ma, node N4), and underwent multiple dispersal/vicariant events during the Pleistocene (~2.64–1.68 Ma, nodes N6 and N7; Fig. 3). The separation of Japanese islands from Eurasia at the Late Pliocene/Early Pleistocene boundary allowed a once widespread ancestral species to split on either side of this sea-barrier (Ota, 1998; Xu et al., 2009), which led to allopatric vicariance of Northeast Asian and Japanese populations (nodes N6 and N7; Fig. 3). Furthermore, repeated sea-level fluctuations during the Quaternary that affected migration among Northeast Asian and Japanese populations of A. japonica led to geographical separation of a formerly widespread ancestor into isolated populations under scenarios of sea-level rise, but allowed migration between Northeast Asia and the Japanese archipelago during glacial maxima via the exposed continental shelf possibly across the deep tectonic Tsushima/Korean Strait between Korea and South Japan (Lin et al., 2002; Kropf et al., 2006; Qiu et al., 2009).

In summary, we have analysed a large genomic data set for a large set of taxa to reveal an unusual dispersal route from the Mediterranean to far East Asia (Korea/Japan) via Central Asia and East China for one lineage of Cardueae. Our results have implications for the taxonomy of Atractylodes, an important plant in traditional Chinese medicine, and further elucidate the role that hybridization and introgression have played in the evolution of these remarkable East Asian thistles.

SUPPLEMENTARY DATA

Supplementary data are available online at https://academic.oup.com/aob and consist of the following. Table S1. Accession data of the RAD-seq for 76 samples from 36 populations of Atractylodes. Table S2. Details of 168 Patterson’s four-taxon D-statistic tests of Atractylodes. Table S3. GenBank numbers of four chloroplast DNA fragments for the tribe Cardueae. Table S4. Results after filtering and clustering RAD-seq data from 76 samples of Atractylodes using IPYRAD. Figure S1. Distribution ranges of Cardueae species with the dispersal route of Cardueae from the Mediterranean to East Asia and BEAST-derived chronogram of 127 Cardueae samples based on the cpDNA dataset.

mcac059_suppl_Supplementary_Table_S1
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mcac059_suppl_Supplementary_Table_S2
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mcac059_suppl_Supplementary_Table_S3
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mcac059_suppl_Supplementary_Table_S4
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mcac059_suppl_Supplementary_Figure_S1
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mcac059_suppl_Supplementary_Figure_Legend
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ACKNOWLEDGMENTS

We thank the Korea National Arboretum (KH) for providing precious samples from North Korea and Russia. We also thank Dr Goro Kokubugata from the National Museum of Nature and Science, Tokyo, and Dr Joo-Hwan Kim from Gachon University for their help with sample collection, and Dr Luxian Liu from Henan University for his help with data analyses.

Contributor Information

Maoqin Xia, Systematic & Evolutionary Botany and Biodiversity Group, MOE Laboratory of Biosystem Homeostasis and Protection, College of Life Sciences, Zhejiang University, Hangzhou, China.

Minqi Cai, Shanghai Science and Technology Museum, Shanghai, China.

Hans Peter Comes, Department of Biosciences, Salzburg University, Salzburg, Austria.

Li Zheng, Systematic & Evolutionary Botany and Biodiversity Group, MOE Laboratory of Biosystem Homeostasis and Protection, College of Life Sciences, Zhejiang University, Hangzhou, China; Key Laboratory of Jiaxing Second Hospital, Jiaxing, Zhejiang, China.

Tetsuo Ohi-Toma, Nature Fieldwork Center, Okayama University of Science, Okayama, Japan.

Joongku Lee, Department of Environment and Forest Resources, Chungnam National University, Daejeon, South Korea.

Zhechen Qi, College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, China.

Kamil Konowalik, Department of Plant Biology, Institute of Environmental Biology, Wrocław University of Environmental and Life Sciences, Kożuchowska 5b, 51-631, Wroclaw, Poland.

Pan Li, Systematic & Evolutionary Botany and Biodiversity Group, MOE Laboratory of Biosystem Homeostasis and Protection, College of Life Sciences, Zhejiang University, Hangzhou, China.

Kenneth M Cameron, Department of Botany, University of Wisconsin, Madison, WI, USA.

Chengxin Fu, Systematic & Evolutionary Botany and Biodiversity Group, MOE Laboratory of Biosystem Homeostasis and Protection, College of Life Sciences, Zhejiang University, Hangzhou, China.

AUTHOR CONTRIBUTIONS

Maoqin Xia: data analyses and writing initial drafts. Minqi Cai: generated sequence data, data analyses and writing initial drafts. Hans Peter Comes, Kamil Konowalik, Kenneth M. Cameron: review and editing. Li Zheng, Tetsuo Ohi-Toma, Joongku Lee: gathering plant materials and editing. Zhechen Qi, Chengxin Fu: recruited financial support and editing. Pan Li: designed the study, gathering plant materials, data analyses and writing initial drafts.

FUNDING

This work was supported by the National Science Foundation of China (grant numbers 31970225, 31370247), the Zhejiang Provincial Natural Science Foundation (grant numbers 2017C32044, LY19C030007), and the Major Science and Technology Projects of Breeding New Varieties of Agriculture in Zhejiang Province (No. 2021C02074).

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