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During the Quaternary, many organisms underwent major changes in geographical distribution in parallel with glacial–interglacial cycles. Thus, species ranges retreated, fragmented or expanded at different times according to climatic conditions, which, in turn, greatly affected lineage divergence within and between species (Comes & Kadereit, 1998; Hewitt, 2000, 2004; Petit et al., 2003; Alsos et al., 2012). Range expansions often led to secondary contact between lineages that had diverged in isolated refugia during periods of range fragmentation, and this in some cases resulted in hybridization and occasionally the origin of hybrid species via homoploid hybrid speciation or allopolyploidy (Stebbins, 1971, 1984; Comes & Kadereit, 1998; Abbott et al., 2003; Brochmann et al., 2004; Paun et al., 2009). Most studies of these biogeographic and evolutionary events in the Northern Hemisphere have been conducted in Europe and North America, while their occurrence in East Asia is less well known (although see Qiu et al., 2011; Liu et al., 2012a).
Unlike in Europe, West Asia and North America, where large continuous ice-sheets covered northern regions during Quaternary glaciations, most areas of East Asia remained ice-free during these periods (Hultén, 1937; Shi et al., 1987; Wu & Wu, 1996; Hewitt, 2000, 2004; Shi, 2002; Abbott & Brochmann, 2003). Consequently, East Asia was an important refugium for many representatives of the Tertiary flora and fauna (‘Tertiary relics’) and is today a species-rich region of the north temperate biota (Shi et al., 1987; Wu & Wu, 1996; Hewitt, 2000; Zhou et al., 2004). Although East Asia was unaffected by extensive ice cover during the Quaternary, changes in climatic variables such as temperature and aridity, as well as in topography and sea level in the region, had a profound effect on structuring genetic diversity. East Asian organisms also underwent cycles of range shifts as forests retreated southward during glaciations and re-colonized northern regions during interglacials (Qian & Ricklefs, 2000; Harrison et al., 2001; Gao et al., 2007; Gong et al., 2008; Tian et al., 2009; Guan et al., 2010; Qiu et al., 2011). In China, aridity increased during glaciations, particularly between the Qinling Mountains–Huai River (c. 34°N) and the tropical south (≤ 22°N), promoting replacement of subtropical forest by steppe and desert vegetation (Sun & Chen, 1991; Zhou et al., 1991). Fossil pollen evidence indicates that during these periods herbaceous plants associated with dry environments, for example, Artemisia, Ajania, Aster and Filifolium, expanded their ranges in southern China, but retreated northward during interglacials (Wang & Xu, 1985; Tong et al., 1992; Yu et al., 2000; Harrison et al., 2001). It remains unknown, however, how such range changes may have affected the genetic structure of these plants and whether they led to divergence, secondary contact events, and on occasion hybrid speciation as occurred in other parts of the Northern Hemisphere. Here we focus on the demographic and evolutionary history of the Chrysanthemum indicum complex, which is generally associated with semi-arid habitats in China.
Chrysanthemum is a temperate Eurasian genus comprising c. 40 species mainly distributed in eastern Asia (Bremer & Humphries, 1993; Oberprieler et al., 2007; Liu et al., 2012b). According to distribution, morphological and molecular phylogenetic data, the Chinese Chrysanthemum species can be divided into two groups, the Chrysanthemum zawadskii group and the Chrysanthemum indicum group (Lin et al., 2011; Liu et al., 2012b). The former occurs in northern China and comprises taxa with erect stems and large capitula containing white-purple ray florets, while the latter group is distributed from north to south China and comprises taxa with creeping stems and capitula with yellow or white ray florets. Here, we focus on the C. indicum complex, which is considered to comprise six species – C. indicum, Chrysanthemum lavandulifolium, Chrysanthemum potentilloides, Chrysanthemum rhombifolium, Chrysanthemum hypargyrum and Chrysanthemum vestitum (Supporting Information Fig. S1) – although clear morphological gaps between these taxa are often obscured in areas of overlap (Lin et al., 2011; Liu et al., 2012b). The species are herbs mainly found in open and relatively dry habitats, for example, on mountain slopes or at the edge of forests. A previous study on the geographic distribution of cytotypes in the C. indicum complex confirmed that C. rhombifolium and C. lavandulifolium were diploid (2n = 18), C. hypargyrum and C. potentilloides were tetraploid (2n = 36), and C. vestitum was hexaploid (2n = 54), while C. indicum comprised two cytotypes, one diploid and the other tetraploid (Li et al., 2013). In contrast to the narrowly distributed C. rhombifolium, C. hypargyrum and C. potentilloides, for which only single populations are known, C. indicum is a widespread species in central and southern China (Fig. S2). However, whereas tetraploid C. indicum is distributed widely, the diploid form and other taxa that comprise the C. indicum complex occur only in central and northern China (Li et al., 2013). Li et al. (2013) suggested that tetraploid C. indicum may have expanded its range southward during a recent Quaternary glacial period when forests retreated in south China as conditions became drier, and then remained there in dry areas when forests expanded their ranges northward during the current post-glacial period. To test this hypothesis and to examine the evolutionary history of the C. indicum complex in more detail, we surveyed both plastid and nuclear ribosomal internal transcribed spacer (nrITS) DNA sequence variation within and among all recognized members of the complex.
A previous survey of molecular variation within the C. indicum complex (Yang et al., 2006) examined variation for nuclear random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers, and chloroplast SSR markers within and among one population of C. lavandulifolium, two populations of diploid C. indicum and nine populations of tetraploid C. indicum. Although limited in terms of number of taxa and populations studied, this investigation revealed widespread sharing of chloroplast and nuclear markers between members of the complex. It was concluded that the complex probably evolved through cycles of multiple differentiation followed by frequent hybridization and polyploidization (via both autoployploidy and allopolyploidy). In the study reported here, we expanded the analysis of the complex so as to cover all recognized diploid and polyploid members. In addition, we greatly increased the number of populations of C. lavandulifolium and tetraploid C. indicum surveyed to obtain a better understanding of the biogeographic and evolutionary history of the complex.
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We undertook the study reported in this paper to clarify the pattern of divergence within the C. indicum polyploid complex in China, and to test the hypothesis that it underwent a southward expansion during the Pleistocene (Li et al., 2013). In this way we aimed to provide information on the broader issue of how Quaternary climatic oscillations affected the demographic and evolutionary history of plants adapted to semi-arid conditions in South East Asia. Our analysis of chlorotype diversity was successful in resolving two main clades within the C. indicum complex that largely distinguished the northern diploid taxon C. lavandulifolium from all other taxa. Most importantly, our analysis provided strong support for the hypothesis that the C. indicum complex underwent a massive range expansion in southern China during the Pleistocene and that this was attributable to an expansion in the range of the tetraploid form of C. indicum. Our results provided no evidence for diploid and other polyploid members of the complex having undergone similar range expansions during the same period.
Our analyses of chlorotype diversity combined with ITS variation were less successful in terms of clarifying the parentage of polyploid taxa and their origins via autopolyploidy and/or allopolyploidy. However, they confirmed that, although hybridization and interspecific gene flow were widespread in the complex, the different taxa within each ploidy level could be largely distinguished from each other according to chlorotype and/or ITS genotype, thus reinforcing their distinctiveness based previously on differences in morphology (Fig. S1) and genome size (Li et al., 2013).
Ancestral diploid lineages within the C. indicum complex
Our survey of plastid DNA variation revealed the presence of two major chlorotype groups, Hg1 and Hg2, within the C. indicum complex. Whereas diploid C. lavandulifolium, which is distributed in the northern part of the distribution of the complex, contains only Hg1 chlorotypes, all other members of the complex, which are distributed more centrally or in the south of China, contain mainly Hg2 chlorotypes. Thus, Hg1 chlorotypes were not found in the two other diploid members of the complex (diploid C. indicum and C. rhombifolium) nor in the tetraploid C. hypargyrum, and were present only rarely in tetraploid C. potentilloides, hexaploid C. vestitum and tetraploid C. indicum (Table S2). Based on chlorotype evidence and the current geographical distribution of taxa, it is feasible that an ancestral form of the complex diverged early in its evolution to give rise to C. lavandulifolium in northern China, and diploid C. indicum and C. rhombifolium in central China. Because the latter two taxa possess chlorotypes from different Hg2 subgroups, it is feasible that they also diverged from each other early in the evolution of the complex.
Interestingly, although C. lavandulifolium and diploid C. indicum are easily distinguished according to chlorotype, this is not so for ITS type. Our results showed that these two taxa share two nonadditive ITS types (AAT and GAT) and one additive ITS type ((A/G)AT) composed of the AAT and GAT types (Table S4). Only a few individuals of C. lavandulifolium possessed an ITS type ((A/G)(A/G)T) not found in diploid C. indicum. Sharing of ITS types between taxa and the joint possession of an additive ITS type may stem from incomplete lineage sorting and/or hybridization between taxa. Here it is feasible that AAT and GAT were present early in the evolution of the complex, with one possibly originating from the other in isolation by point mutation followed by homogenization of repeats as a result of concerted evolution. This may have occurred during divergence of C. lavandulifolium from diploid C. indicum. The finding that each of these genotypes is now present in both taxa may indicate that on occasion the two taxa have come into contact, hybridized, exchanged ITS types and formed additive ITS types.
An additive ITS type in diploid taxa may be produced by hybridization following a cross between individuals possessing different ITS types, in which case both parental types should be present among segregating offspring. Alternatively, it may be present in hybrid derivatives as a result of recombination between ribosomal DNA (rDNA) repeats containing different ITS sequences, in which case the additive sequence is inherited as a single unit and will not segregate (Fuertes Aguilar & Nieto Feliner, 2003). In both diploid C. indicum and C. lavandulifolium, the additive (A/G)AT type was never found in populations in combination with both putative parental types (AAT and GAT), indicating that it does not segregate and instead contains an intragenomic additive ITS sequence generated by recombination. Another way in which an additive ITS type might be generated in a diploid is through incomplete concerted evolution, resulting in partial homogenization of the parental copies. Although this cannot be ruled out as a cause of the additive ITS types detected in these two diploid species, hybridization is considered to be the more likely cause, certainly of the (A/G)AT additive type, given the sharing of AAT and GAT types by both species.
Whereas C. lavandulifolium and diploid C. indicum are very similar in regard to ITS type, the rare diploid taxon C. rhombifolium contains two ITS types unique to the taxon (GAC and (A/G)AC). Thus, the ITS evidence for this taxon agrees with the chlorotype evidence in indicating that C. rhombifolium diverged in isolation from the other two diploids early in the evolution of the complex and has remained genetically isolated thereafter.
Polyploid lineages within the C. indicum complex
All three tetraploid taxa, that is, the widespread tetraploid C. indicum and the two rare taxa, C. potentilloides and C. hypargyrum, were shown to be polymorphic for chlorotype, while C. indicum and C. potentilloides were also polymorphic for ITS type (Tables S1, S3). The different chlorotypes and ITS types recorded in C. potentilloides were shared with tetraploid C. indicum, indicating a close relationship and possible genetic exchange between these two taxa via introgressive hybridization. By contrast, C. hypargyrum was shown to be fixed for an ITS type (ATT) and almost fixed for a chlorotype (H15) not found in other members of the complex. Hence, this species may have diverged in isolation from other diploid and polyploid taxa and remained largely reproductively isolated from them thereafter.
Tetraploid C. indicum contains 31 of the 40 different chlorotypes detected in the complex and seven of 10 ITS types. The most common chlorotypes in this taxon were H11 (present in 46 individuals), H24 (in 35 individuals) and H37 (in 33 individuals) (Table S2), which represent the ancestral chlorotypes of subgroups Hg2a, Hg2b and Hg2d, respectively (Fig. 2b). The ancestral chlorotype (H34) of subgroup Hg2c was also relatively common in tetraploid C. indicum (present in 14 individuals). Only two of these ancestral chlorotypes were present in diploid members of the complex: H24 was very common in diploid C. indicum, while H37 was common in C. rhombifolium. The fact that the other two ancestral chlorotypes, H11 and H34, commonly found in tetraploid C. indicum, along with the majority of Hg2 tip chlorotypes resolved in this taxon, were not detected in any diploid taxon surveyed raises questions about the origins of these chlorotypes. It is feasible that at an earlier stage in the evolution of the complex they were present in diploid representatives, which have since become extinct, possibly as a result of competitive exclusion by tetraploid C. indicum (see Yang et al., 2006). Alternatively, it is possible that the chlorotypes unique to tetraploid C. indicum originated in this taxon during periods of population isolation and range fragmentation. The chlorotype network (Fig. 2b) shows that many chlorotypes differ from their most closely related chlorotypes by single mutations. This is true even for pairs of ancestral chlorotypes, and indicates that such divergence could have been easily generated during periods of population isolation in tetraploid C. indicum. Either way, the fact that the majority of tetraploid C. indicum populations examined are now polymorphic for chlorotypes shared with other populations of the taxon suggests that during the evolution and spread of tetraploid C. indicum there were frequent opportunities for gene flow to occur between populations.
What was evident for chlorotype variation in tetraploid C. indicum was also apparent, though to a lesser extent, for ITS variation. Thus, some ITS types found in this taxon (GGT and G(A/G)T) were not detected in diploid members of the complex and again sharing of different ITS types was common among populations of the tetraploid (Table 1, Table S4). Thus, our findings for this taxon confirm in broad terms those reported previously by Yang et al. (2006), albeit based on surveys of a different set of molecular markers, and fit a model of multiple differentiation and hybridization/polyploidization cycles that Yang et al. (2006) proposed for the evolutionary history of tetraploid C. indicum.
Based on their results, Yang et al. (2006) further proposed that some populations of tetraploid C. indicum originated by autoploidy while others were more likely to have originated by allopolyploidy. Thus, Yang et al. (2006) considered that a tetraploid population of C. indicum located at Shennongjia Mountain, Hubei Province, probably originated by autopolyploidy from a diploid population of C. indicum occurring nearby, because both populations shared many of the same nuclear and chloroplast markers. By contrast, there was no obvious association between markers present in a different diploid population of C. indicum, from Baohua Mountain, Jiangsu Province, and its nearest tetraploid population of C. indicum. Yang et al. (2006) proposed that this tetraploid population may have originated through allopolyploidy. However, our results failed to confirm an autopolyploid origin of the tetraploid population of C. indicum from Shennongjia Mountain (population 26; Table 1). Instead, they showed this population to be polymorphic for chlorotype, with some individuals possessing a chlorotype not detected in the closest diploid population of C. indicum (population 25). Consequently, at least some individuals in this tetraploid population could not have originated via autopolyploidy from diploid individuals nearby or, if they did, the genetic evidence for such an origin has been erased through subsequent hybridization and capture of another chlorotype.
Interestingly, hexaploid C. vestitum was also highly polymorphic for both chlorotype and ITS type. In this taxon, each of the three populations examined was dominated by a different high-frequency chlorotype, representing a different chlorotype subgroup. This might indicate that each population represents an independent origin of the taxon. Because two of the populations are polymorphic for chlorotype, it seems they would have been subject to interpopulation or interspecies gene flow following their origin (populations 38 and 39). For none of the three populations is it possible to determine accurately the original parentage of plants based on chlorotype and ITS data.
Pleistocene southward range expansion of tetraploid C. indicum
A southward range expansion of tetraploid C. indicum during the Pleistocene was strongly indicated by its unimodal mismatch distribution of chlorotype diversity and significantly negative values obtained for Fu's Fs and Tajima's D. By contrast, no such demographic expansions were indicated for either diploid C. indicum or C. lavandulifolium, while other diploid and tetraploid taxa of the complex currently exist only as single populations. Using a fast cpDNA mutation rate, the range expansion of tetraploid C. indicum was dated to have occurred between 720 and 780 kya ago, which is within the period from 600 to 800 kya when South East Asia was subjected to the effects of the longest and most severe Pleistocene glaciation recorded (Zhou & Li, 1998; Shi, 2002; Zheng et al., 2002). However, using a slow mutation rate the expansion was dated to have occurred between 72 and 78 kya, which is at the time of the last glaciation in the region (Shi, 2002). The climate of South East Asia was further affected by several more glaciations between the largest and the most recent glaciations (Zheng et al., 2002). Overall, our analysis clearly shows that the expansion occurred during the Pleistocene, and possibly during the most recent or the previous large glacial period when a marked increase in environmental aridity occurred and subtropical forests were replaced by steppe and semi-desert vegetation in large parts of southern China (Sun & Chen, 1991; Zhou et al., 1991).
There are many examples of polyploids being geographically more widespread than their diploid ancestors, and it has been argued that this is partly a result of a greater ability to colonize open habitats generated by changes in climate or other factors (Stebbins, 1984; Brochmann et al., 2004). The greater colonizing ability of polyploids has been attributed to several causes. First, allopolyploids contain two or more divergent genomes, which may provide adaptation to different environmental conditions. Secondly, levels of heterozygosity in both allo- and autopolyploids are likely to be greater than in their diploid ancestors (Brochmann et al., 2004), and this might further enable adaptation to a wider range of conditions and promote colonization of new open areas (Wu et al., 2010). Thirdly, polyploids can potentially generate considerable genetic and phenotypic novelty immediately following their origin through a range of diverse genetic and epigenetic mechanisms (Doyle et al., 2008; Soltis & Soltis, 2009; Abbott et al., 2013). Given the presumed greater ability of polyploids to colonize available habitats, it is somewhat surprising that the other polyploid taxa in the complex (C. hypargyrum, C. potentilloides and C. vestitum) were not as successful as tetraploid C. indicum in becoming widespread in central and southern China. A possible reason for this is that these polyploids originated later than tetraploid C. indicum and that suitable habitat for colonization had already been occupied by tetraploid C. indicum before they had a chance to spread. Alternatively, they may have become widespread at one stage, but then experienced a range contraction, in contrast to tetraploid C. indicum.
Comparisons of chlorotype diversity and ITS diversity within tetraploid C. indicum indicated that both types of diversity were greater in a group of centrally located populations (populations 17–23, 26, 27 and 29–35) than in southern populations (populations 1–16). This was particularly true for within-population chlorotype and ITS diversity. The latter finding does not fit the ‘stable rear edge’ model of Hampe & Petit (2005) for a species that historically has experienced cycles of range expansion and contraction in response to Pleistocene cycles of climate change. This model predicts that populations occurring at the rear edge of a species distribution will be of small size and geographically isolated from each other and hence should contain low levels of within-population genetic diversity, but high levels of between-population diversity. Rather, our results suggest that populations of large size were retained in central China during the southern expansion of tetraploid C. indicum, enabling maintenance of high levels of within-population diversity during this period. The fact that central China is also home to a rare diploid species, two rare tetraploid species, and a hexaploid species of the C. indicum complex emphasizes that it is an important region for the long-term conservation of genetic diversity within the complex.
Another question of interest concerns how tetraploid C. indicum has remained in southern China during the current post-glacial period when environmental aridity has decreased and subtropical forest is once again favored by prevailing conditions. It is feasible that tetraploid C. indicum has a particularly broad range of habitat tolerance as a result of its polyploid nature and/or has undergone rapid adaptation to changed conditions in southern China during post-glacial times and that this is partly responsible for it remaining widespread at the present time. It is of interest that plants comprising southern populations of tetraploid C. indicum tend to have more entire leaves than those comprising central populations (Fig. S1). It will be of interest to investigate whether these and other possible differences between centrally located and southern populations may be of adaptive significance. Certainly it would be of value to conduct transplant studies to determine if central and southern plants of tetraploid C. indicum are differentially adapted to present-day, local conditions in central and southern China.