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Ann Bot. Jan 2008; 101(1): 111–124.
Published online Nov 3, 2007. doi:  10.1093/aob/mcm267
PMCID: PMC2701833

Phylogeny and Biogeography of the Genus Ainsliaea (Asteraceae) in the Sino-Japanese Region based on Nuclear rDNA and Plastid DNA Sequence Data


Background and Aims

The flora of the Sino-Japanese plant region of eastern Asia is distinctively rich compared with other floristic regions in the world. However, knowledge of its floristic evolution is fairly limited. The genus Ainsliaea is endemic to and distributed throughout the Sino-Japanese region. Its interspecific phylogenetic relationships have not been resolved. The aim is to provide insight into floristic evolution in eastern Asia on the basis of a molecular phylogenetic analysis of Ainsliaea species.


Cladistic analyses of the sequences of two nuclear (ITS, ETS) and one plastid (ndhF) regions were carried out individually and using the combined data from the three markers.

Key Results

Phylogenetic analyses of three DNA regions confirmed that Ainsliaea is composed of three major clades that correspond to species distributions. Evolution of the three lineages was estimated to have occurred around 1·1 MYA during the early Pleistocene.


The results suggest that Ainsliaea species evolved allopatrically and that the descendants were isolated in the eastern (between SE China and Japan, through Taiwan and the Ryukyu Islands) and western (Yunnan Province and its surrounding areas, including the Himalayas, the temperate region of Southeast Asia, and Sichuan Province) sides of the Sino-Japanese region. The results suggest that two distinct lineages of Ainsliaea have independently evolved in environmentally heterogeneous regions within the Sino-Japanese region. These regions have maintained rich and original floras due to their diverse climates and topographies.

Key words: Ainsliaea, ETS, ITS, ndhF, phylogeography, rheophytes, Sino-Japanese region


The genus Ainsliaea DC. (Asteraceae) comprises about 40 species of perennial herbs widely distributed in the Sino-Japanese region (Mabberley, 1998). Ainsliaea belongs to tribe Mutisieae. The most closely related genera are Pertya Sch. Bip. and Myripnois Bunge, based on a molecular phylogeny of Mutisieae using the plastid ndhF gene (Kim et al., 2002). Ainsliaea can be distinguished from other related genera by the pappus and flower heads; the pappus has plumose bristles and mostly three (one or five in some species) florets per flower head, whereas related genera have a scabrous pappus and one to more than ten floret(s) per head (Kitamura, 1981). The infrageneric classification of the genus recognizes three sections (Beauverd, 1909; Tseng, 1996) based on leaf arrangements on the stem and stem branching patterns: section Ainsliaea (Scaposae sensu Beauverd) has radical leaves and clustered scapes; section Aggregatae Beauverd has cauline leaves; section Frondosae Beauverd has branching aerial stems. Aggregatae and Ainsliaea do not have branching stems. However, this taxonomic subdivision has been controversial (e.g. Koyama, 1995; Peng et al., 1998), partly due to the uniform reproductive characteristics and geographically continuous distribution of the genus. Most Ainsliaea species grow in the forest understorey or in rocky areas with humid and wet conditions, but some are confined to river- and streambeds where they experience strong water flow during floods.

The Sino-Japanese region covers over 5000 km in an east–west direction. The flora is rich in endemics (Takhtajan, 1969, 1986; Good, 1964; Wu and Wu, 1996; Qian, 2002). The floristic richness of the Sino-Japanese region is greater than that of the North American floristic region, even though these two regions have similar climates and floristic components (Qian and Ricklefs, 1999, 2000; Qian, 2002, 2004). Wu and Wu (1996) proposed the Sino-Japanese region as a floristic kingdom and recognized two environmentally heterogeneous subkingdoms: the Sino-Himalayan Forest subkingdom and the Sino-Japanese Forest subkingdom. South-west (SW) China is representative of the Sino-Himalayan subkingdom. Almost half of the total plant species in China are distributed in SW China with more than one-third of its species being endemic to this region (Wu, 1980; Wu and Wu, 1996). The floral richness of this region is attributed to its diverse climate and topography, and vegetation varies from tropical to boreal forest (Axelrod et al., 1998; Qian, 2004; Xiang et al., 2004). The characteristics of this region were created by the uplifting of the Himalayas and the Tibetan Plateau in the late Palaeocene (about 55 MYA) to the early Pleistocene (Committee of Chinese Academy of Sciences for Physical Geography of China, 1984). Moreover, the development of the Himalayas brought a large amount of rainfall to the region by forming a monsoon system in East Asia (Sun and Wang, 2005). Such a diverse geology and climate has significantly affected the evolutionary history of plants.

The eastern edge of the Sino-Japanese region, ranging from south-east (SE) China to mainland Japan and the Korean Peninsula through Taiwan and the Ryukyu Islands, is included in the Sino-Japanese Forest subkingdom. This region is also characterized by a remarkably rich flora and high rainfall due to the south-eastern monsoon. It encompasses >120 continental islands (Takhtajan, 1969, 1986). The biota of these islands experienced dramatic distribution changes attributed to topographical changes during the Quaternary climatic oscillations. A land-bridge once existed between SE China and mainland Japan through the Ryukyu Islands and Taiwan during glacial periods, but was fragmented during inter- and post-glacial periods (Kizaki and Oshiro, 1977, 1980; Ujiie, 1990; Kimura, 1996, 2000). The Ainsliaea species in this region may have evolved allopatrically due to the topogeographical events of the Quaternary.

Ainsliaea is endemic to this floristic region and is mainly distributed in the corridor-shaped area from the Himalayas to Japan and the Korean Peninsula, through SW to SE China, Taiwan and the Ryukyu Islands (Fig. 1). Quaternary climatic oscillations led to repeated migration, adaptation and extinction of plant and animal species; the present genetic structure of populations, species and communities was mainly formed during the Pleistocene (Bennett, 1997; Hewitt, 2000). Previous molecular phylogenetic studies have revealed evolutionary patterns sculpted by the Quaternary climatic oscillations (e.g. Xiang et al., 2000, 2004; Wen, 2001; Xiang and Soltis, 2001). However, most of the studies addressed eastern Asian–eastern North American biogeography, and few phylogeographic studies have embraced the whole Sino-Japanese region. A recent phylogenetic study on varieties of Spiraea japonica, from SW and SE China to mainland Japan, found an allopatric evolutionary history in eastern and western areas of the Sino-Japanese region (Zhang et al., 2006). However, this study analysed only 14 samples of the species across the wide distribution range and did not cover the entire Sino-Japanese region. Because Ainsliaea is widely distributed in the Sino-Japanese region, migration and evolution of Ainsliaea species may have occurred separately in several parts of this lengthy corridor. Reconstruction of Ainsliaea phylogenetic relationships and estimation of divergence times between outgroups and the major clades of the genus would provide a better insight into the floristic evolution in the Sino-Japanese region during the Quaternary.

Fig. 1.
Distribution of the genus Ainsliaea. Only A. latifolia and its close relatives are found in the temperate area of south-east Asia. Four taxa (A cordifolia, A. acerifolia var. acerifolia, A. acerifolia var. subapoda and A. apiculata) are shared between ...

One of the remarkable aspects of Ainsliaea is the occurrence of ‘rheophyte’ species (van Steenis, 1981, 1987). Rheophytes are adapted to river-bank environments in which plants are periodically covered with floodwater after heavy rains and they generally have narrow leaves. In the region from SE China to the Ryukyu Islands, at least four species (A. walkeri and A. trinervis in SE China, A. oblonga and A. linearis in the Ryukyu Islands) are adapted to this type of environment and exhibit this distinctive morphology. Because of the unusual ecology of these species, they are restricted to river banks. These rheophyte species are distributed in places that are characterized by high annual rainfall, e.g. A. linearis on Yakushima Island of the Ryukyus, where annual rainfall ranges from 3000 to 7000 mm year−1 (Takahara and Matsumoto, 2002). Plant genera that include several rheophyte species rarely occur in temperate regions (van Steenis, 1981, 1987). The occurrence of four rheophyte species of Ainsliaea is quite unusual. The unique suite of morphological features characteristic of rheophytes may be an adaptation to the rich environment and high annual rainfall in the region from SE China to the Ryukyu Islands. A molecular phylogenetic analysis of these species would contribute to our understanding of the evolution of rheophytes, i.e. whether they arose convergently after allopatric diversification to adapt to their rheophitic environments or were derived from a common ancestral rheophytic species. For example, two rheophytes, A. walkeri from SE China and A. linearis from Yakushima Island, were estimated to be the same or closely related species based on their similar morphology (Kitamura, 1981); however, their phylogenetic relationships and evolutionary history are still uncertain. Phylogenetic analysis of Ainsliaea would clarify the taxonomic situation and would provide evolutionary insights.

The aim of the present research was to resolve interspecific phylogenetic relationships and to estimate the divergence time of Ainsliaea by analysing three DNA sequence datasets. The nuclear ribosomal internal transcribed spacer (ITS) and nuclear ribosomal external transcribed spacer (ETS) regions have been shown to be useful sources of information for resolving the interspecific phylogenies in Asteraceae (Baldwin, 1992, 1993; Kim and Jansen, 1994; Sang et al., 1994, 1995; Clevinger and Panero, 2000; Linder et al., 2000), and the plastid ndhF region has been used previously to reconstruct the phylogeny of Asteraceae (Kim and Jansen, 1995; Kim et al., 2002, 2005).


Taxonomic sampling

Twenty-eight species and six varieties of Ainsliaea, four species of Pertya and Diaspananthus uniflorus (all Asteraceae) were sampled. The broadly distributed species and their subspecies or varieties (A. henryi, A. macroclinidioides and A. macroclinidioides var. okinawensis) were sampled from several points to examine their monophyly. Diaspananthus, a monotypic genus endemic to Japan, is almost identical to Ainsliaea except for floral characteristics (D. uniflorus has only one floret per flower head); however, a previous phylogenetic analysis of plastid DNA sequences for Mutisieae (Kim et al., 2002) did not include this genus. Pertya and Diaspananthus were used as outgroups in this study. Samples of Ainsliaea and putative outgroups were collected from their natural habitats, cultivated stocks at botanical gardens and from herbarium specimens. The taxa, GenBank accession numbers and voucher information are listed in Table 1.

Table 1.
Materials used in the present study

DNA extraction, amplification and sequencing

Silica gel-dried or fresh leaf material was frozen in liquid nitrogen and ground to a powder. After the polysaccharides were removed from this powder with Hepes buffer (pH 8·0; Setoguchi and Ohba, 1995), total DNA was extracted using the 2× CTAB method (Doyle and Doyle, 1987). The extracted DNA was dissolved in 100 µL of TE buffer and used for polymerase chain reaction (PCR). The primers used to amplify ITS1 and ITS2, the 3′ end of ETS and the 3′ end of the plastid ndhF gene are listed in Table 2, with primer sequences and their original references. PCR was conducted in a total reaction volume of 25 µL containing 18·5 µL of autoclaved ion-exchanged water, 0·2 mm dNTP mixture, 2·0 mm 10× ExTaq Buffer (Takara ExTaq), 0·625 U Takara ExTaq (Takara Bio, Ohtsu, Japan), 0·2 µm of each primer, and 1·25 µL of DNA. PCR was performed in 35 cycles with the following conditions for each region: ITS1 and ITS2 (1 min at 94 °C, 1 min at 48 °C, 1 min at 72 °C), ETS and ndhF (1 min at 94 °C, 1 min at 56 °C, 1 min at 72 °C).

Table 2.
Primer sequences used for PCR and cycle sequencing

The PCR products were sequenced in both directions using the standard methods of the BigDyeTM Deoxy Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA) using the same primers as above on an ABI 3100 Genetic Analyser (Applied Biosystems).

Sequence alignment and phylogenetic analysis

ITS sequences were determined for 37 taxa, ETS sequences for 36 taxa and ndhF sequences for 33 taxa. All sequences have been deposited in a DNA database (DDBJ/EMBL/GenBank; accession numbers AB288427-AB288547). Sequence data were edited and assembled using AutoAssembler™ (Applied Biosystems). Parsimony analyses were performed for four data sets: ITS alone, ETS alone, ndhF alone and a combined dataset of all three DNA regions (ITS + ETS + ndhF). Phylogenetic analyses were conducted using PAUP* 4.0b10 (Swofford, 2002). Parsimony analyses were performed with heuristic searches of 1000 replicates of random stepwise addition using tree bisection and reconnection (TBR) branch swapping. The gaps caused by mononucleotide repeat units were removed from consideration in the phylogenetic analysis, because homology can be highly uncertain for these repeated nucleotides (Kelchner, 2000). All characters and character state transformations were equally weighted. The consistency index (CI) excluding uninformative characters and retention index (RI) (Kluge and Farris, 1969, Farris, 1989) were calculated. Statistical support was evaluated through nonparametric bootstrap analysis (Felsenstein, 1985) with 1000 replicates using NNI branch swapping for each data set and decay indices (DI; Bremer, 1988, 1994) with the Parsimony Ratchet Analyses using PAUP (PRAP; Muller, 2004). An incongruence length difference (ILD) test (Farris et al., 1995) was conducted to investigate character congruence between taxonomically equivalent ITS, ETS and ndhF partitions.

Relative rate tests (Sarich and Wilson, 1973; Wu and Li, 1985) were used to examine the heterogeneity of the ITS and ETS sequence divergence rates between major clades. The divergence rate of ndhF was not calculated due to the small number of substitutions. Pertya robusta was used as the reference outgroup taxon in substitution rate comparisons. Sequence divergences for the tests were calculated only from substitutions based on the method of Jukes and Cantor (1969), implemented in RRTree (Relative-Rate Tests between groups of sequences on a phylogenetic tree; Robinson-Rechavi and Huchon, 2000).

The estimation of divergence time for selected lineages was calculated as T = DA/2λ (Nei, 1987). For ITS, the rate of nucleotide substitutions per site per year (λ) of Eupatorium was used, which has been calculated as 1·95 × 10−8 substitutions site−1 year−1 (Schmidt and Schilling, 2000) as an example of perennial herbs in Asteraceae in temperate regions.


Sequencing comparison

DNA site variation for each of the three regions and the combined data (ETS + ITS + ndhF), as well as tree statistics, are shown in Table 3. Within Ainsliaea, the nucleotide sequence length of the three regions was 646–650 bp for the ITS region, 475–481 for the ETS region and 947 for the ndhF region. The percentage of potentially parsimony-informative sites in the ITS region was 17·15 % from 649 characters. The ETS data matrix contained proportionally more informative sites (19·50 %) from 481 characters than the other data sets. The ndhF sequences included only 36 potentially parsimony-informative sites (3·91 %) from 947 characters. The number of indels was small. These indels were excluded from the analyses because they did not affect any results of the phylogenetic analyses. There were two indels which distinguish Ainsliaea and Diaspananthus from Pertya in the ITS and ETS. Within Ainsliaea species, two and one indels were found in ITS and ETS, respectively. In ndhF, there was one indel which distinguishes Diaspananthus from the others and no indels were found within Ainsliaea.

Table 3.
DNA site variation and tree statistics of Ainsliaea species for the three data sets used in the cladistic analyses presented in this study

Phylogenetic analysis

Of the most parsimonious ITS trees 3323 were recovered; the strict consensus is shown in Fig. 2 with bootstrap support and DI. The most parsimonious trees had a length of 316 steps (CI 0·56, RI 0·83; Table 3).

Fig. 2.
Strict consensus of the 3323 most parsimonious trees from ITS sequences for 37 taxa and five outgroups. Tree length = 316. Numbers above and below branches indicate bootstrap support and decay index, respectively. Branches with broken lines are only in ...

Ainsliaea was paraphyletic, with Diaspananthus uniflorus falling in the ingroup, whereas the four species of Pertya were strongly supported as a monophyletic group (bootstrap value of 100 %, DI of 14). The infrageneric taxonomy recognizing three sections (Ainsliaea, Aggregatae and Frondosae) was inconsistent with the molecular phylogenetic trees. The ITS data supported three major clades of Ainsliaea, which were correlated with regional distributions.

Clade A was strongly supported (bootstrap 95 %, DI 2). All of the species in this clade are distributed from SE China to Japan through Taiwan and the Ryukyu Islands. Within this clade, A. macroclinidioides (distributed in SE China and Taiwan) did not form a monophyletic group and the Taiwanese species was placed within a polytomy of Japanese–Taiwanese species along with A. macroclinidioides var. okinawensis. Monophyly was also not supported for A. macroclinidioides var. okinawensis from Okinawa and Ishigaki Island of the Ryukyu Islands. The four rheophytic species, A. linearis, A. oblonga, A. trinervis and A. walkeri (indicated by * in Fig. 2) were included in a monophyletic group with ten OTUs (operational taxonomic units) of inland taxa (bootstrap 100 %, DI 9). The Chinese and Japanese rheophytes were divided into two clades, and the two Chinese rheophytes did not form a clade.

Clade B was weakly supported (bootstrap 66 %, DI 2). The species in this clade also showed a regional pattern, ranging between SW China and the Himalayas, except the outgroup Diaspananthus uniflorus, which is endemic to Japan. Within this clade, A. henryi from China and Sumatra was monophyletic, but the sample from Taiwan did not fall with the other samples, making A. henryi paraphyletic, albeit with low bootstrap support (<50 %).

Clade C was strongly supported (bootstrap 100 %, DI 6) and comprised three species (A. glabra, A. lancifolia and A. sutchuenensis) mainly distributed in Sichuan Province of South China. Ainsliaea sutchuenensis and A. lancifolia were strongly supported as sister species.

Parsimony analysis based on ETS sequence data resulted in 88 equally parsimonious trees of 258 steps, CI 0·63 and RI 0·84. The consensus of the 88 most parsimonious trees is shown in Fig. 3. The ETS tree had some incongruence with the ITS tree. Four species of Pertya were strongly supported as outgroups (bootstrap 100 %, DI 17). In the ETS tree, Diaspananthus was weakly supported as sister to Ainsliaea (bootstrap 54 %, DI 1). The three traditional sections of Ainsliaea were again inconsistent with the ETS phylogeny. Three species from Sichuan (clade C) formed a strongly supported clade (bootstrap 100 %, DI 12), as in the ITS trees. However, the other two major regional clades, A and B, were only weakly supported. Clade B, which included species distributed from SW China to the Himalayas, was poorly supported (bootstrap <50 %). Samples of the widely distributed A. henryi from China, Sumatra and Taiwan were included in a polytomy with A. yunnanensis, A. lijiangensis and A. fulvipes. Clade A, comprising species ranging from SE China to mainland Japan through Taiwan and the Ryukyu Islands, was weakly supported (bootstrap 60 %, DI 1). Within this clade, the Japanese and Korean endemic taxa A. cordifolia, A. acerifolia var. acerifolia, A. acerifolia var. subapoda and A. dissecta comprised a monophyletic group (bootstrap 78 %, DI 3). The four species of rheophytes fell in a monophyletic group with inland taxa. As in the ITS tree, they were polyphyletic in the ETS tree.

Fig. 3.
Strict consensus of the 88 most parsimonious trees from the ETS sequences for 36 taxa and five outgroups. Tree length = 258. The four rheophyte species are indicated by *. The broadly distributed taxa A. macroclinidioides var. okinawensis, A. macroclinidioides ...

The most parsimonious trees based on ndhF did not resolve the relationships among Ainsliaea species well because the number of potentially parsimony-informative characters was only 3·9 %, the lowest among the three DNA regions. The phylogenetic analysis resulted in 6876 most parsimonious trees of 76 steps, CI 0·78 and RI 0·88. The consensus tree is shown in Fig. 4. In this case, monophyly of the genus Ainsliaea was weakly supported (bootstrap 69 %, DI 1), whereas the three clades (A, B and C) found in the ITS and ETS phylogenies formed a polytomy.

Fig. 4.
Strict consensus of the 6876 most parsimonious trees from ndhF sequences for 33 taxa and five outgroups. Tree length = 76. Upper-case letters A, B and C represent the three major clades shown in the ITS and ETS trees. The three rheophyte species are indicated ...

Although the ILD test identified incongruence between the ITS and ETS (P < 0·001), between the three regions (P < 0·001), the combined tree from the three data sets is shown because the topologies of the ITS, ETS and ndhF trees did not conflict with respect to the major clades, and the combined data resolved the relationships among the major lineages of Ainsliaea and the outgroups (Fig. 5). The combined data set of 2076 bp generated 367 most parsimonious trees of 668 steps, CI 0·58 and RI 0·79. In the strict consensus tree, Ainsliaea taxa formed a monophyletic group with moderate support (bootstrap 74 %, DI 4), and Diaspananthus was strongly supported as sister (bootstrap 100 %, DI 38). The three major clades of Ainsliaea were supported, with bootstrap support and DI, respectively, of 94 % and 5 for clade A, 86 % and 3 for clade B, and 100 % and 28 for clade C.

Fig. 5.
One of the 367 most parsimonious trees from the combined ITS, ETS and ndhF sequence data for 32 taxa and five outgroups. Tree length = 668. The branch length represents the number of the base substitutions. The numbers above branches indicate the bootstrap ...

Relative rate test

There was no significant difference in substitution rates between clades A and B + C for the alignments of 649 ITS or 480 ETS bp. The mean substitution rates between the two lineages were similar (ITS: KA = 0·0575319, KB+C = 0·058341, ΔK = 0·000814284 ± 0·00673774, ETS: KA = 0·115124, KB+C = 0·107004, ΔK = 0·00812007 ± 0·00672482). Therefore, clade A (SE China to mainland Japan) and clade B + C (SW China and the surrounding areas) do not significantly differ in their mutation rates, and a molecular clock was not rejected.

Estimation of divergence time

The estimated divergence times among the major clades and outgroups were calculated based on the ITS sequence data. The estimated divergence time between Pertya and the other clades was 2·16 ± 0·36 MYA, i.e. from the end of Tertiary to the early Pleistocene. As for the three major clades of Ainsliaea, because the mean number of base substitutions between them was almost the same (29–34), their molecular divergence time converged at around 1·1 MYA in the early Pleistocene. The monophyletic group ranging from SE China to mainland Japan through the Ryukyu Islands and Taiwan (clade A), which encompassed the four rheophytes (the clade indicated by # in Fig. 2), was estimated to have diverged at 0·31 ± 0·12 MYA in the middle Pleistocene.


Utility of different DNA regions for resolving interspecific phylogeny

Some topological conflicts were found between the ITS and ETS trees, whereas ndhF provided less information for phylogenetic relationships in Ainsliaea. The ITS trees indicated that the supposed outgroup Diaspananthus uniflorus was placed within the ingroup; however, D. uniflorus was placed as sister to Ainsliaea in the ETS and combined phylogenetic trees. Moreover, there were some inconsistencies in interspecific relationships within clades A and B. This incongruence between the ITS and ETS trees indicates that there may have been an uneven rate of concerted evolution (gene conversion) within the Ainsliaea lineage, even though these two regions are adjacent in the nrDNA and are usually treated as parts of a single molecule with the same evolutionary history (Baldwin and Markos, 1998; Alvarez and Wendel, 2003). The results of the ILD test also indicated incongruence.

The topological conflict between the ITS and ETS trees may have been caused by introgression, which could be attributed to frequent hybridization among closely related species within the lineage (Okuyama et al., 2005). Watanabe et al. (1992) reported hybridization between Ainsliaea species in Japan and suggested that genetic assimilation was occurring. The internal structure of clade B was weak and the bootstrap value for each branch was low. Considering the number of species in a fairly limited range in Yunnan Province and the surrounding areas, it is likely that recombination and introgression have occurred in some species during their evolution. Therefore, the effect of introgression among species could lead to the phylogenetic inconsistencies seen here.

Although there were some inconsistencies between the two regions, the ITS and ETS trees are consistent in having three major clades (A, B and C, in Figs 2 and and3).3). The bootstrap support for these major clades was higher in the ITS tree than in the ETS tree, although the ETS tree had a higher percentage of potentially parsimony-informative sites than the ITS tree. The present results differ from previous studies in which ETS was the more informative region for resolving interspecific phylogenetic relationships (Baldwin and Markos, 1998; Acevedo-Rosas et al., 2004).

In contrast, analysis of ndhF resulted in uninformative trees and the three major clades seen in the ITS and ETS trees formed a polytomy. This may have been the result of fewer informative site changes, ascribed to a slow substitution rate in ndhF. However, each clade in the ndhF tree could be included in one of the major clades (A, B or C) of the ITS and ETS trees, and the major branches of the three data sets were not in conflict with each other. Therefore, the phylogeographical analysis in this study depended on the combined phylogenetic analyses of the three data sets.

Phylogeographical analysis of the Sino-Japanese region

The genus Ainsliaea is composed of three major clades, which correspond to species distributions within the Sino-Japanese region; (A) the eastern region from SE China to Japan, through Taiwan and the Ryukyu Islands, (B) the western region covering Yunnan and its surrounding areas, including the Himalayas; and (C) Sichuan (Fig. 1). Although the origin and past distributions of the three groups remain uncertain, it is plausible that Ainsliaea taxa evolved allopatrically and that their descendants have been isolated on the eastern (clade A) and western (clades B and C) sides of the Sino-Japanese corridor. The east and west regions are generally thought to possess rich floras with high endemism; SW China (including Sichuan) possesses especially high diversity (Axelrod et al., 1998; Qian, 2004; Xiang et al., 2004), and the region from SE China to Japan through Taiwan and the Ryukyu Islands also encompasses a rich flora (Takhtajan, 1986; Wu and Wu, 1996). Floristic studies (Wu, 1979; Wu and Wu, 1996) suggest that the Sino-Japanese plant region should be divided into two subkingdoms: the Sino-Himalayan Forest subkingdom and the Sino-Japanese Forest subkingdom. The present phylogenetic findings are highly geographically structured and recognize each area as an independent evolutionary unit in the Sino-Japanese region that is consistent with a floristic unit, the eastern and western divisions. Moreover, these phylogeographical units agree with the infraspecific relationships in Spiraea japonica, with two clades corresponding to the eastern and western divisions (Zhang et al., 2006).

Origin of the three lineages of Ainsliaea was estimated at around 1·1 MYA, or the early Pleistocene. In this era, the uplift of the Himalayas occurred (Committee of Chinese Academy of Sciences for Physical Geography of China, 1984). The present moist monsoon climate of the Sino-Japanese region was formed by uplift of the Himalayan Range as a result of the collision of Eurasia, the Indian subcontinent and the Burma–Malaya Geoblock (Zhang et al., 1984; Sengor and Natal'in, 1996), estimated to have occurred during the late Pliocene (Fort, 1996) or the late Pliocene and Pleistocene (Hsü, 1978). Therefore, the present findings suggest that topogeographical and/or palaeoclimatic barriers sculpted the phylogeographical lineages in the early Pleistocene. The climatic and topographical changes may have led to allopatric speciation in Ainsliaea in three geographic areas. Previous studies indicated the presence of varied vegetation, from tropical to frigid forests, and these rich environments would have supplied refugia during Quaternary climatic oscillations, harbouring species diversity and acting as important centres for survival, speciation and evolution (Axelrod et al., 1998; Qian, 2004; Xiang et al., 2004). The three regions occupied by clades A, B and C might represent refugia, and the plants occurring there could have been sources for later speciation and evolution in each area.

On the western side of the Sino-Japanese region, Ainsliaea species have diverged in two regions in SW China, i.e. Sichuan (clade C) and the adjacent area represented by Yunnan (clade B). Most of the species included in these two clades are restricted to a fairly narrow area of Sichuan and Yunnan. Several factors could have contributed to allopatric speciation: the presence of effective topographic barriers (e.g. splitting the ranges of ancestral species, preventing gene flow or enhancing the founder effect), strong sexual selection (e.g. leading to divergence in mate recognition systems), ecological specialization (e.g. limiting population distribution and prompting divergence), low dispersal rates (e.g. reducing gene flow among populations) and bottlenecks in population size facilitating genetic peak shifts (Barraclough et al., 1998; Schluter, 2001; Turelli et al., 2001). In SW China, topographic complexity can be ascribed to the uplift of the Himalaya-Tibetan massif from the late Paleocene to the early Pleistocene. The Himalayan movements during the early Pleistocene created a mosaic of plateaux, mountains, basins and gorges in this small area of Yunnan and its surrounding areas (Committee of Chinese Academy of Sciences for Physical Geography of China, 1984). We propose this environmental complexity restricted the movement of Ainsliaea species due to the geographic barriers, causing limited gene flow among populations. The fractured populations with restricted gene flow and higher selection pressure led to species differentiation by adaptation to local environments. In fact, species in clade C are from Emei Mountain, which is part of a large mountain chain and is extraordinarily rich in endemics. Considering that the three major clades diverged at around 1·1 MYA and speciated through the Pleistocene, the present results support the idea that complex topography and varied environments, linked with the continual movements of the Himalayas, functioned as major factors leading to species differentiation in this area.

The present results indicate that clade A is geographically restricted and suggest that this area would have been a centre of diversification for the genus Ainsliaea. The estimated divergence time between the species of SE China and the Ryukyu Islands (Fig. 5, represented by #) was about 0·31 MYA, i.e. the middle Pleistocene. A similar divergence time, approx. 0·25 MYA, was also estimated for Cardiandra (Hydrangeaceae) species with similar geographic distribution (Setoguchi et al., 2006). During this period, the Ryukyu Islands and Taiwan repeatedly formed a land-bridge from SE China to mainland Japan (Kizaki and Oshiro, 1977, 1980; Ujiie, 1990; Kimura, 1996, 2000). The phytogeographical structure of some plant taxa and traces of introgressive hybridization between allopatric species in the Ryukyu Islands have also been reported (e.g. Setoguchi and Watanabe, 2000; Chiang et al., 2001; Hiramatsu et al., 2001). The present distribution of Ainsliaea species is likely to have been sculpted by these historical geographic events, and allopatric speciation due to insular isolation might have been promoted in this region.

Among the species on the Ryukyu Islands, including the rheophytes A. linearis and A. oblonga, little differentiation in the DNA sequences was found (0–2 bp out of 649 bp of ITS), whereas they are morphologically very different (e.g. Watanabe et al., 1992), possibly indicating rapid diversification. Genetic polymorphism was also found within A. macroclinidioides var. okinawensis located on Okinawa and Ishigaki Islands. The genetic distances among A. macroclinidioides var. okinawensis populations were larger than those among other species from the Ryukyu Islands. This might be attributable to isolation on islands and subsequent adaptive radiation to their environment following migration over land-bridges in the Quaternary glacial periods.

Zhang et al. (2006) pointed out several distinct geographic and population characteristics and a slower divergence rate in SE China (as comparedwith SW China): fewer geographical barriers, weaker divergent selection pressure, higher gene flow and fewer bottleneck events. However, the relative rate tests suggest almost the same divergence rates in the eastern (SE China to Japan) and western (SW China and its surrounding areas) sides of the Sino-Japanese region for Ainsliaea. Moreover, it was found that topographical events including repeated transgression and regression significantly accelerated species differentiation through immigration and isolation in this region of numerous continental islands. Therefore, it is conceivable that the region from SE China to mainland Japan through Taiwan and the Ryukyu Islands had functioned as a floristic unit maintaining original lineages during the topographical and climatic changes throughout the Quaternary.

Circumscription and taxonomy of Ainsliaea

The monophyly of the genus Ainsliaea was supported based on analyses of ETS, ndhF and the combined data (Figs 3, 4 and 5, respectively), whereas Diaspananthus uniflorus was embedded in Ainsliaea in the ITS analysis (Fig. 2). The classification of the two genera is solely based on floral characters: the genus Ainsliaea has three florets (occasionally one or five), whereas Diaspananthus has one floret and Diaspananthus is sometimes included in Ainsliaea (Mabberley, 1998). The phylogenetic relationships between Ainsliaea and Diaspananthus should be further examined using larger DNA sequence data sets.

The three sections proposed by Beauverd (1909) were not supported by the present molecular analyses. The present results indicate that the genus consists of three monophyletic groups, corresponding to geographical distributions of species, not vegetative morphological characteristics. These three groups should be recognized as infrageneric taxonomic units, but no particular morphological characteristics have been identified for each clade. The chromosome number is still uncertain in many species, and microstructural characters such as pollen features have not been observed. Based on the three lineages found in this study, further analyses of comparative morphology are needed to suggest an infrageneric system for the genus.

The present study also suggests species boundaries with wide distributions. Ainsliaea henryi (samples from China, Sumatra and Taiwan) was not monophyletic in clade B. For A. macroclinidioides and its relatives in clade A, disagreement over the traditional classification and genetic structure was again observed and further analysis is necessary to reconsider their classification. In contrast, A. fragrans and A. fragrans var. integrifolia, disjuncts in SE China and mainland Japan, were monophyletic with little sequence differentiation. These two taxa are distinguished by differences in leaf pubescence, but this characteristic also varies among habitats even within the same population (personal observations in China and Japan). Therefore, the present molecular phylogenetic analysis supports the monophyly of A. fragrans among allopatric localities in eastern Asia, whereas A. henryi and A. macroclinioides are not monophyletic, and further taxonomic studies are needed to clarify their circumscription.

Diversification of rheophyte species

The phylogenetic relationships of the four rheophyte species were inferred. These grow along river banks that are occasionally flooded by violent river flow. Ainsliaea linearis and A. oblonga are distributed on Yakushima and Okinawa Islands of Japan, respectively, and A. trinervis and A. walkeri are found in SE China. These four species are included in a clade with ten OTUs of inland taxa. The Japanese A. linearis and A. oblonga are monophyletic, whereas the Chinese A. trinervis and A. walkeri are polyphyletic. Almost no base substitutions were seen between A. linearis and A. oblonga, whereas there were ten substitutions in ITS and ETS between Japanese and Chinese species, and five substitutions were found between the two Chinese species. Kitamura (1981) speculated that the Japanese A. linearis and the Chinese A. walkeri, which are morphologically similar, might be the same species. We agree with the existing classification recognizing A. walkeri and A. linearis as independent species, because the present analysis suggests that there is no correlation between morphological similarity and genetic relatedness. Differentiation of ITS sequences, with 15 substitutions, indicates that they have experienced different evolutionary processes. The morphological similarity was not caused by genealogy but represents convergent evolution for the peculiar rheophytic environment.

The two rheophytes in SE China may have evolved independently, whereas it appears that the Japanese A. linearis and A. oblonga are derived from a single ancestor. These findings suggest that allopatric evolution of these rheophyte species, both in mainland China and the Ryukyu Islands, was accomplished by adapting to the rheophytic environment (river banks subject to periodic flooding). The estimated divergence time of approx. 0·31 MYA in the mid-Pleistocene suggests that river-bank adaptations may have occurred during the drastic climatic oscillations and land-bridge formation. van Steenis (1981, 1987) suggested that most rheophytic plants are endemics confined to particular areas. Moreover, migration (gene flow) between river systems is thought to be fairly limited (Liao and Hsiao, 1998). Therefore, the independent evolution of rheophytic Ainsliaea species is due to adaptation to the environment on the eastern edge of the Sino-Japanese region, where annual rainfall is high (e.g. 3000–7000 mm on Yakushima Island; Takahara and Matsumoto, 2002).

Conservation aspects

SE China and the Ryukyu Islands are characterized by high annual rainfall and a large number of mountain streams. Rheophytic Ainsliaea species have adapted to such mountain stream environments over a relatively short time period. However, these environments have rapidly declined due to the construction of dams and electricity-generating stations in these areas. Rheophytic plants cannot breed away from their riparian (inland) habitats, because heavy water flow is necessary for excluding competitors. Once a dam is constructed and river flow is controlled, seasonal flooding does not occur, leading to a loss of natural populations and genetic diversity. The region from SE China to the Ryukyu Islands is a centre of species differentiation in the Sino-Japanese region. The mountain stream environment contributes to the biodiversity of this region, and conservation of the mountain stream environment is needed to sustain the unique plants of this region.


We thank Mr Chien-I. Huang, Academia Sinica, Taiwan, and Ms Min Deng and Mr Yongsheng Yi, Kunming Institute of Botany, China, for supporting plant collections in the field. This study was supported by Grants-in-Aid for Scientific Research (#13575011 and 15405014) from the Japan Ministry of Education, Culture, Science, Sports, and Technology.


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