• central and adjacent southwestern China;
  • cpDNA;
  • phylogeography;
  • Primula ovalifolia Franch


  1. Top of page
  2. Abstract
  3. 1 Material and methods
  4. 2 Results
  5. 3 Discussion
  6. Acknowledgements 
  7. References
  8. Supporting Information

Abstract  To investigate the mechanisms responsible for the high diversity in central and adjacent southwestern China, we inferred the phylogeographic history of Primula ovalifolia from chloroplast DNA data. One hundred and thirty five individuals from 13 natural populations (including one Primula tardiflora population) were analyzed. A total of 23 haplotypes were identified. Most of them were exclusive to a single population. Strong phylogeography structure was detected, with NST (0.936) significantly higher than GST (0.784). Phylogenetic analysis showed that all haplotypes were clustered into three lineages (clades I, II, and III). High genetic diversity was revealed, possibly due to the interglacial contraction and glacial expansion. The three identified lineages may have undergone different historical processes after the mid-Pleistocene, due to their early divergent time. Multiple refugia were inferred for the three lineages. Pleistocene climatic oscillations, combined with the complex local topography, were responsible for the strong phylogeographic pattern of P. ovalifolia. Our analysis supports the hypothesis that the central and adjacent southwestern region of China was an important refugium for the survival, persistence, and further speciation of most East Asia flora, which has led to high species diversity in this region.

The Quaternary climatic oscillations had profound effects on the geographical distribution of many species (Webb & Bartein, 1992). Most flora and fauna taxa migrated southward (in the Northern Hemisphere) and survived in refugia during glacial periods as the ice-sheets advanced, then expanded northward during interglacial periods when the ice retreated (Hewitt, 1996), while cold-adapted taxa would survive in refugia during interglacials (Stewart et al., 2010). In recent years, many phylogeographic studies have explored the evolutionary consequences of climatic fluctuations and complex local topology in China. Most of them were focused on the Qinghai–Tibet Plateau and its surrounding area, an important region for temperate species as a refugium during the Quaternary glacial period (Zhang et al., 2005; Gao et al., 2007; Yang et al., 2008; Tian et al., 2009; Wang et al., 2011), and some in southern (Yuan et al., 2008; Song et al., 2009; Tian et al., 2010) and eastern China (Li et al., 2008; Gong et al., 2008; Qiu et al., 2009b). Although these areas have been intensively studied, very few investigations have been carried out in central and adjacent southwestern (C & SW) China (Wang & Ge, 2006; Zhou et al., 2010).

As part of a global biodiversity hotspot (south-central China; see Myers et al., 2000), C & SW China has unique topography, with many high and steep mountains, deep valleys, basins, and rivers (Ying, 2001). This topographically complex region was likely to provide stable habitats for many species during the ice ages, where species could survive in different refugia, and probably led to the new generation lineages (Hewitt, 2000). In accordance with this hypothesis, there are 6390 recognized plant species in central China (excluding those in southwestern China), of which 4035 are endemic (Ying, 2001). Thus, the central China region has been defined as one of the three Chinese plant diversity hotspots; the adjacent southwest is part of another Chinese hotspot, the Hengduan Mountains (Ying, 2001). This region is also considered to be an important glacial refugium because many primitive temperate and tertiary relict flora species are concentrated here (Ying et al., 1979; Wang, 1992). Molecular data can provide useful information for deducing the evolutionary history of particular species, and are especially useful for species without a fossil record (Comes & Kadereit, 1998). The current paucity of information for C & SW China limits our understanding of the origin of the high diversity in this region.

Comes & Kadereit (1998) suggested that herbs may provide the best model systems for investigating the role of the Quaternary climatic changes in driving diversification and speciation, as they have far more life cycles within a given time period than long-lived trees, and thus may have responded more quickly to environmental change in Quaternary timescales. Primula ovalifolia Franch. is an alpine, perennial herb species of the family Primulaceae, primarily distributed in central and adjacent southwestern China (Hubei, W Hunan, E Chongqing, E Guizhou, Sichuan, and NE Yunnan provinces), around the Sichuan Basin. Primula ovalifolia mainly grows in shaded habitats in broad-leaved forests and ravines, at altitudes between 600 and 2500 m (Hu, 1990). This species has distinctive veins that are prominently raised, alveolate abaxially, and impressed adaxially. Primula tardiflora C. M. Hu, a superficially similar species, was once treated as a subspecies of P. ovalifolia (Hu, 1990), but was later considered an independent species (Hu & Kelso, 1996). In an earlier study, Nan et al. (2002) found no genetic differentiation among five sampled P. ovalifolia populations across Sichuan and Hubei Provinces by inter-simple sequence repeat markers.

In this study, we carried out a phylogeographic analysis of P. ovalifolia using chloroplast DNA sequence data trying to: trace the evolutionary history of P. ovalifolia; explore possible mechanisms responsible for the current genetic structure of this species; and examine the taxonomic relationship of P. ovalifolia and P. tardiflora. In addition, we aimed to obtain molecular evidence to aid our understanding of the formation of the central and adjacent southwestern China biodiversity hotspot and test the hypothesis that this region was an important Quaternary glacial refugium for the flora of East Asia.

1 Material and methods

  1. Top of page
  2. Abstract
  3. 1 Material and methods
  4. 2 Results
  5. 3 Discussion
  6. Acknowledgements 
  7. References
  8. Supporting Information

1.1 Population sampling

Leaf samples were collected from a total of 125 Primula ovalifolia individuals from 12 natural populations (Table 1). One population (10 individuals) of P. tardiflora was also sampled to examine its relationship with P. ovalifolia. These populations nearly covered the entire geographical distribution of the species. Within each population, 10–24 individuals were randomly sampled, spaced at least 10 m apart. Fresh leaves were immediately dried and stored in silica gel until DNA extraction.

Table 1.  Sample location, haplotype, and genetic diversity of populations of Primula ovalifolia and Primula tardiflora
Population codeLocation (All in China)Alt. (m)Sample sizeWatterson's estimate, θNucleotide diversity, πHaplotype diversity, HdHaplotype
SZSangzi, Hunan1200180.000 480.000 730.660H15, H16, H18, H19, H20
YCYichang, Hubei114310000H21
FJSFanjinshan, Guizhou197010000H14
YLYiliang, Yunnan1952100.000 290.000 170.200H22, H23
JFSJinfoshan, Chongqing170010000H17
PZPengzhou, Sichuan160010000H3
DJYDujiangyan, Sichuan114010000H1
DYDayi, Sichuan1400100.000 440.000 480.689H2, H8, H9
EMEmeishan, Sichuan1699100.000 150.000 080.200H12, H13
HYHongya, Sichuan156610000H6
BX1Baoxing, Sichuan1800 9000H6
BX2Baoxing, Sichuan2400 80.000 790.000 630.607H4, H5, H7
P. tardiflora Emeishan, Sichuan2060100.000 290.000 290.356H10 , H11
Total  135 0.002 600.002 280.916 

1.2 DNA extraction, amplification, and sequencing

Total genomic DNA was extracted using a modified CTAB protocol (Doyle, 1991). We used three chloroplast DNA (cpDNA) fragment sequences to explore the phylogeographic patterns of Primula ovalifolia: the trnT-L intergenic spacer; a trnL-F fragment comprising the trnL intron and intergenic spacer between the trnL and trnF genes; and the rpsl6 intron. These fragments (designated trnT-L, trnL-F, and rps16) were amplified using the primer pairs “a” and “b”, “c” and “f” (Taberlet et al., 1991), and rpsF and rpsR2 (Oxelman et al., 1997), respectively. The polymerase chain reaction products were purified using a QIAquick gel extraction kit (Qiagen, Valencia, CA, USA). Direct sequencing was carried out using a cycle sequencing ready-reaction kit (Perkin Elmer, Foster City, CA, USA) following the manufacturer's instructions, and the products were analyzed using an ABI 377 DNA sequencer (Applied Biosystems, Foster City, CA, USA). The primers used in the sequencing reactions were the same as those used for the amplifications.

1.3 Data analysis

The DNA sequences of the three cpDNA regions were combined, aligned by ClustalX (Thompson et al., 1997) and adjusted manually. Genetic diversity parameters (Hd, θ, and π) (Watterson, 1975; Nei, 1987) were calculated for each population and at the species level by DnaSP version 4.50 (Rozas et al., 2003). The average gene diversity within populations (Hs) and total gene diversity (HT) were estimated by Permut (Pons & Petit, 1996).

To assess the contribution of mutational differences between haplotypes to population differentiation, GST and NST values were estimated by Permut (Pons & Petit, 1996). GST only considers haplotype frequencies, whereas NST considers both haplotype frequencies and their genetic divergence. NST > GST usually indicates the presence of phylogeographic structure, that is, the more frequent occurrence of closely related haplotypes in the same area than less closely related haplotypes (Pons & Petit, 1996). Differentiation within populations, between populations within groups (according to geographical location of the samples and the results of the genealogical analysis), and between groups were calculated by amova (Excoffier et al., 1992) using the program Arlequin version 3 (Excoffier et al., 2005).

Phylogenetic relationships between P. ovalifolia cpDNA haplotypes were assessed under maximum parsimony (MP) by paup* version 4.0 beta 10 (Swofford, 2002) with P. tridentifera Chen & C. M. Hu as the outgroup. Full heuristic tree searches were carried out with 1000 replicates of “random” sequence entries, with the tree bisection–reconnection branch swapping and MulTrees options selected. Branch support was assessed by bootstrap analysis with 1000 replicates of the full heuristic searches using the same settings. In addition, the genealogical degree of relatedness among P. ovalifolia cpDNA haplotypes was estimated with 95% statistical parsimony criteria by TCS version 1.21 (Clement et al., 2000). In this analysis, indels and inversions were treated as single mutation events and coded as substitutions. Length variations in mononucleotide repeats were excluded from parsimony and network analyses.

The phylogeographic history of P. ovalifolia was evaluated by nested clade analysis (NCA) (Templeton et al., 1987), based on an unrooted cpDNA haplotype network (estimated by the TCS program), using Geodis version 2.6 (Posada et al., 2000) to infer historical and ongoing processes affecting the populations with an updated version of Templeton's inference key (available at The network showed all linkages with >95% probability of being most parsimonious.

Mismatch distribution analysis was used to test the hypothesis that sudden expansion of the populations’ range has occurred (Rogers & Harpending, 1992; Harpending, 1994), by calculating the raggedness index (r), which indicates the smoothness of a mismatch distribution (Harpending et al., 1993). Under the population growth model, r values are expected to be low, and the values we obtained were tested for deviation from a constant population size model by simulations implemented in DnaSP. This program was also used to estimate Tajima's D (Tajima, 1989) and Fu and Li's D* and F* parameters (Fu & Li, 1993), which were used to test the hypothesis that all mutations were selectively neutral and wondering if there was recent population expansion existed in the species (Kimura, 1983).

To further elucidate the demographic history of P. ovalifolia, a Bayesian skyline plot (Drummond et al., 2005) was constructed by Beast 1.4.8 (Drummond & Rambaut, 2007), based on the Bayesian Markov chain Monte Carlo approach, to estimate population size fluctuations over time. Chains were run for 20 million generations under a GTR substitution model. The evolutionary model was selected using the Akaike Information Criterion by Modeltest version 3.7 (Posada & Crandall, 1998). The first 10% of runs were discarded as “burn-in”. The time since the most recent common ancestor (TMRCA) was also estimated by Beast 1.4.8. We used a substitution mutation rate of 1.52 × 10−9 per nucleotide per year, as proposed by Yamane et al. (2006) for non-coding chloroplast regions, to estimate TMRCA in years. A relaxed (uncorrelated log-normal) molecular clock was assumed. The results were summarized and checked by Tracer 1.5 (Drummond & Rambaut, 2007); the effective sample size value of all parameters was greater than 200, which was considered as a sufficient sampling level.

2 Results

  1. Top of page
  2. Abstract
  3. 1 Material and methods
  4. 2 Results
  5. 3 Discussion
  6. Acknowledgements 
  7. References
  8. Supporting Information

2.1 Haplotype variation

Sequences of trnT-L, trnL-F, and rps16 were deposited in the GenBank database under the accession numbers HQ439138–HQ439149, HQ439126–HQ439137, and HQ439116–HQ439125, respectively. The combined cpDNA sequences of Primula ovalifolia varied in length from 2402 to 2450 bp, and were aligned with a consensus length of 2468 bp. All analyses in this study were based on this combined sequence. A total of 23 haplotypes were identified based on 40 variations (34 nucleotide mutations and six indels) between the cpDNA sequences. The distribution of haplotypes H1–H23 among the 12 sampled P. ovalifolia populations and one P. tardiflora population is shown in Fig. 1, and the haplotype frequencies at each locality are presented in Table 1. Most haplotype is unique to a single population except haplotype H6, which is shared by populations HY and BX1. Seven populations are fixed for a single different haplotype (including populations YC, FJS, JFS, PZ, DJY, HY, and BX1). The remaining populations are polymorphic with 2–5 haplotypes. In these populations, population SZ possesses five haplotypes (H15, H16, H18, H19, and H20). High genetic diversity was found at the species level, with Hd= 0.916, π= 0.002 28, and θ= 0.002 60. The average gene diversity within populations (Hs) was 0.213 and the total gene diversity (HT) 0.987. At the population level, the highest haplotype diversity (Hd= 0.660) and nucleotide diversity (π= 0.000 73, θ= 0.000 48) were found in population SZ. The second highest genetic diversity was found in population BX2 (π=0.000 63 and θ= 0.000 79) (Table 1).


Figure 1. Sites where Primula ovalifolia was sampled in central and adjacent southwestern China. The geographical distribution of the haplotypes, based on combined data from three chloroplast regions for 125 individuals of P. ovalifolia and P. tardiflora, is shown. Different patterns were assigned for each haplotype according to the legend on the left side of the figure. The circumferences of the circles are proportional to the haplotype frequencies. BX1, Baoxing, Sichuan; BX2, Baoxing, Sichuan; DJY, Dujiangyan, Sichuan; DY, Dayi, Sichuan; EM, Emeishan, Sichuan; FJS, Fanjinshan, Guizhou; HY, Hongya, Sichuan; JFS, Jinfoshan, Chongqing; PZ, Pengzhou, Sichuan; SZ, Sangzi, Hunan; YC, Yichang, Hubei; YL, Yiliang, Yunnan.

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2.2 Phylogenetic and genealogical relationships of cpDNA haplotypes

Four equally parsimonious trees with 56 steps (consistency index = 0.982; retention index = 0.981) were generated from the MP analysis of the 23 identified cpDNA haplotypes. The strict consensus tree is shown in Fig. 2A. Haplotypes from the same or adjacent areas were grouped into clades, representing three main lineages, designated Clades I, II, and III, with 77%, 95%, and 66% bootstrap support, respectively. Haplotypes of P. tardiflora (H10 and H11) formed a separate clade (Clade II) with those from the P. ovalifolia population EM (H12 and H13), which is located in the same region (Emei Mountain, southwest of the Sichuan Basin). Clade III contained haplotypes H1–H9, sampled from the central Sichuan (west of the Sichuan Basin). All other haplotypes sampled from central China and regions in close proximity (east and south of the Sichuan Basin) (including Hubei, Hunan, E Chongqing, E Guizhou, and NE Yunnan provinces) comprised Clade I. However, the relationships between these three lineages were not fully resolved in the MP tree. The haplotype relationships within each clade were also uncertain with weak bootstrap support. According to the results of the unrooted network analysis (Fig. 2: B), a total of 23 haplotypes were clustered into three major groups, largely consistent with the MP tree (Fig. 2: A). No obvious ancestral-like haplotype was found at the center of the whole network. The three separated groups were connected with each other by a lot of uncollected or extinct haplotypes.


Figure 2. A, Strict consensus of four equally parsimonious trees obtained from the analysis of the 23 chloroplast DNA Primula ovalifolia haplotypes. Bootstrap values (>50%) based on 1000 replicates are shown under the branches and the time since the most recent common ancestor of each node is shown above the branch with a line point at the node in unit of Mya. BX1, Baoxing, Sichuan; BX2, Baoxing, Sichuan; DJY, Dujiangyan, Sichuan; DY, Dayi, Sichuan; EM, Emeishan, Sichuan; FJS, Fanjinshan, Guizhou; HY, Hongya, Sichuan; JFS, Jinfoshan, Chongqing; PZ, Pengzhou, Sichuan; SZ, Sangzi, Hunan; YC, Yichang, Hubei; YL, Yiliang, Yunnan. B, Unrooted 95% plausible TCS network (Clement et al., 2000). The small open circles indicate hypothetical missing haplotypes, and each line represents one mutational step. The three different colors in most parsimonious tree and TCS haplotype network indicate the three major identified clades.

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2.3 Population genetic structure

The three lineages that we identified have distinct geographic distributions with no shared haplotypes. Each population contained unique haplotypes, except populations HY and BX1, which shared one haplotype (Fig. 1). High genetic differentiation was found in this species (GST = 0.784), and amova revealed that 98.48% of the total genetic variation was partitioned among populations and only 1.52% of the variation within populations (Table 2). These results indicate that haplotypes are geographically structured across the range of P. ovalifolia. In addition, NST (0.936) was significantly higher than GST (0.784) (P < 0.01), indicating strong phylogeographic structure in this species.

Table 2.  Hierarchical amova of samples of Primula ovalifolia based on nucleotide sequences
Source of variation d.f. Sum of squaresVariance componentsPercentage of variation (%)Fixation index
  1. d.f., degrees of freedom; FCT, correlation of haplotypes within groups relative to total; FSC, correlation within populations relative to groups; FST, correlation within population relative to total; NA, not applicable. *P < 0.01.

Species level
Among populations 122648.02121.30398.48 F ST= 0.985*
Within populations12240.0750.328 1.52NA
Divided into three groups
Among groups  12209.05932.72688.66 F SC= 0.922*
Among populations within groups 11438.9633.85810.45 F ST= 0.991*
Within populations12240.0750.328 0.89 F CT= 0.887*

2.4 Phylogeography and historical demography analysis

Coalescent analyses carried out by Beast showed that the TMRCA for all P. ovalifolia haplotypes was ca. 2.44 Mya (Table S1). The TMRCA of each lineage is also shown in Table S1.

The nested diagram was also reconstructed from cpDNA haplotypes (Fig. 3). Null hypothesis of no association between haplotype distribution and geographical location was rejected for five subclades (1-5, 1-9, 2-1, 2-4, and 3-2) and for the total cladogram from the NCA (see Table 3). The NCA suggested that restricted gene flow with isolation by distance was the major historical process in subclades 1-5 and 3-2. No inference was made for subclade 1-9 (inconclusive outcome). However, past gradual range expansion and subsequent fragmentation or a past larger range followed by extinction in intermediate areas appear to have played important roles in shaping the present-day distribution of other P. ovalifolia haplotypes (subclades 2-1, 2-4 and for the total cladogram).


Figure 3. Resulting set of nesting clades for all 23 Primula ovalifolia chloroplast DNA haplotypes identified. Nested haplotype clades with an increasing number of steps are enclosed in rectangles and the number of each nesting level and individual clade are indicated.

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Table 3.  Inference chain on the results of geographical distance analysis from Fig. 3
CladeChain of inferenceInference
1-51-2-3-4 NORestricted gene flow with isolation by distance (restricted dispersal by distance in non-sexual species)
1-9 Inconclusive outcome
2-11-2-3-5-6-13-14-21 NOPast gradual range expansion followed by fragmentation or a past larger range followed by extinction in intermediate areas
2-41-2-3-5-6-13-21 NOPast gradual range expansion followed by fragmentation or a past larger range followed by extinction in intermediate areas
3-21-2-3-4 NORestricted gene flow with isolation by distance (restricted dispersal by distance in non-sexual species)
Total cladogram1-2-3-5-6-13-21 NOPast gradual range expansion followed by fragmentation or a past larger range followed by extinction in intermediate areas

The observed multimodal mismatch distribution and the low Harpending's raggedness index (r= 0.015, P = 0.112) (Fig. 4) for P. ovalifolia cpDNA haplotypes do not fit a model of sudden expansion (Rogers & Harpending, 1992). This was also supported by non-significant values of all neutrality test parameters (Tajima's D=–0.1119, P > 0.10; Fu and Li's D*= 1.263, P > 0.10 and F*= 0.835, P > 0.10). These findings indicate that no population expansion of this species has occurred in recent times. The historical temporal fluctuation in effective population size inferred by the Bayesian skyline plot is shown in Fig. S1. Population size was stable for a long time and has evidently grown since approximately 0.5 Mya. This expansion continued for approximately 0.4 My through the penultimate glacial cycle of the Quaternary period. However, population size seems to have gently grown again during the last 50 000 years, which includes the last glacial maximum period.


Figure 4. Mismatch distribution established for Primula ovalifolia. The thin line represents the expected (Exp) mismatch distribution of a stationary population, and the dotted line represents the observed (Obs) mismatch distribution from segregating sites of the aligned sequences of trnT-L, trnL-F and rps16 of the P. ovalifolia chloroplast DNA.

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3 Discussion

  1. Top of page
  2. Abstract
  3. 1 Material and methods
  4. 2 Results
  5. 3 Discussion
  6. Acknowledgements 
  7. References
  8. Supporting Information

3.1 Historical demography

High genetic diversity was found in Primula ovalifolia (HT= 0.987), indicating that this species has a long evolutionary history of limited gene exchange among populations (Chiang et al., 2006; Varvio et al., 1986). This was corroborated by the TMRCA estimations. The TMRCA of all P. ovalifolia haplotypes could be tracked back to 2.44 Mya, coinciding with the early Pleistocene. It is difficult to trace the origin of P. ovalifolia due to the unresolved haplotype relationships. However, strong phylogeographic structure was found (NST > GST, P < 0.01), suggesting that closely related haplotypes occurred in the same geographic area. All populations were clustered into three main lineages (clades I, II, and III) (Fig. 2), which were disjunctly distributed in central and adjacent southwestern China around the Sichuan Basin. There are two scenarios that could have led to this disjunct distribution: long-distance dispersal to suitable habitats; or vicariance due to range contraction or fragmentation of a previously wider distribution (Collevatti et al., 2009). Chloroplast DNA is maternally inherited, so the cpDNA data only reflect the gene flow by seed dispersal in angiosperms (Petit et al., 2003). There are no long-distance dispersal mechanisms for P. ovalifolia seeds, and long-distance dispersal of its seeds has never been reported. Given the high species genetic diversity and interpopulation differentiation inferred from the acquired cpDNA data, the latter scenario seems a more likely explanation of the disjunctive distribution of this species. Thus, we assume that P. ovalifolia, which mainly grows in shaded habitats in temperate broad-leaved forests, may have had a broad range in central and southwestern China during the Quaternary (Yang et al., 1989; Xu et al., 1973; Guo, 1974).

Severe climatic oscillations in the Pleistocene epoch significantly influenced the distribution, diversity, and speciation of most species (Hewitt, 1996; Klicka & Zink, 1997; Avise & Walker, 1998). Many recent phylogeographic studies of alpine plants and birds in China have provided molecular evidence to support this hypothesis (Song et al., 2009; Li et al., 2009; Qiu et al., 2009a; Wang et al., 2010). According to the structure of the haplotype network (Fig. 3), these three main lineages of P. ovalifolia were connected by many missing haplotypes, indicating that there was a massive extinction of ancient haplotypes of this species in the Quaternary. During cooler glacial periods, P. ovalifolia could have shifted to lower elevations, expanding around the Basin, as did the temperate forests, with occasional gene exchange between fragment populations. After the glacial period, P. ovalifolia may quickly respond to the changed environment and migrate back to higher elevations for existing in the “refugia” (Qian & Ricklefs, 2000; Harrison et al., 2001).

The early divergence time (Table S1) meant that the three major lineages had distinct evolutionary histories during the Quaternary. Clade I (located west of the Sichuan Basin) probably survived in higher elevation refugia in the warm stage (interglacial), but shifted to lower elevations with gradual expansion during the glacial periods. Populations BX1 and HY shared an ancient haplotype, with more haplotypes found in population BX2, indicating that the Baoxing–Hongya region would be an important past refugium for this lineage. In clade III (from the eastern and southern Sichuan Basin), populations were separated from each other by distances exceeding 100 km. The haplotype network showed that H18 is an ancient haplotype, with all others being derived from it. Given the high polymorphism (Table 1) and ancient haplotype, the Hunan Sangzi region would be an important past interglacial refugium for clade III lineage. Following the rapid uplift of the Qinghai–Tibet Plateau, there was large-scale tectonic movement in central China, which created several mountainous regions between the Sichuan Basin and the Yunnan–Guizhou Plateau (Zheng & Li, 1990; Wang, 1991; Li et al., 2001). These mountain ranges probably isolated lineages in different locations and resulted in barrier of gene flow, leading to relatively high interpopulation differentiation (Table 2). The historical process of the clade II lineage is partly discussed above. The Emei Mountain region with its complicated topography was little affected by climate oscillation (Jiang & Wu, 1998; Shi et al., 1999). Many species might migrate to this region to survive the glacial period or interglacial period, leading to four forest vegetations living in this mountain (Zhuang, 1998).

Multiple glacial refugia during the Quaternary have also been suggested for two other plant species, Cathaya argyrophylla Chun & Kuang (Pinaceae) (Wang & Ge, 2006) and Saruma henryi Oliv. (Aristolochiaceae) (Zhou et al., 2010), which are also mainly distributed in central China. Some other plants and animals are thought to have had glacial refugia in central China and adjacent regions (Qiu et al., 2009a; Li et al., 2009). Several high mountain ranges, especially the Qinling Mountains, prevented cooler weather from the north reaching central China and kept the climatic conditions warm and moist for a long period of time (Ying et al., 1979; Sun, 2002), which was ideal for distribution and diversification of species.

In conclusion, P. ovalifolia had a complex evolutionary history during the Quaternary. Pleistocene climatic oscillations, combined with the complex local topography, were responsible for the strong phylogeographic pattern of P. ovalifolia. Our study provides further information to explain the high diversity of this region. As multiple refugia were inferred, our study supports the hypothesis that the central and adjacent southwestern China region was an important refugium for East Asia flora during the Quaternary. The stable environment and complex local topography best account for the extant species richness and abundance of primitive temperate species in this region. However, our understanding of historical biogeographic events in central and adjacent southwestern China is still incomplete, and more phylogeographic studies are needed to explore the ice age legacy in this region.

3.2 Relationship between Primula tardiflora and Primula ovalifolia

Primula tardiflora has similar morphology to P. ovalifolia, and is confined to a small area in the Emei Mountains. It can be distinguished by the attenuate base of leaf blade, sparsely pubescent veins, and blooming phenology (it flowers approximately 1 month later than P. ovalifolia). The parsimony tree and unrooted haplotype network analyses both showed that the cpDNA haplotypes (H10 and H11) of P. tardiflora were nested within those of P. ovalifolia, which revealed that P. tardiflora is genetically closed to the population EM of P. ovalifolia.

Both lineages of P. tardiflora and population EM of P. ovalifolia were sampled from Emei Mountain, located southwest of the Sichuan Basin. Emei Mountain was formed during the Himalayan orogeny in the mid-Tertiary, and became complicated after the Quaternary tectonic movement (Hu, 1964; Gu & Li, 2006). The vertical distribution vegetation (Li, 1984) indicates that there may be different habits in different altitudes on the Emei Mountain. Although both grow under the broad-leaved forest, the P. tardiflora lineage is found above 2000 m, higher than P. ovalifolia. Our data suggest that the genetic variability between P. tardiflora and P. ovalifolia seems not sufficient to support their status as two distinct species. However, we are aware of the fact that the present result was only derived from cpDNA data. As P. tardiflora has unique haplotypes and morphological and phenological features, it might represent an ideal example of ecological speciation. We therefore consider the species delimitation of P. tardiflora as an unresolved issue, and await future sampling of highly polymorphic nuclear markers (Hey et al., 2003; Duminil et al., 2006).


  1. Top of page
  2. Abstract
  3. 1 Material and methods
  4. 2 Results
  5. 3 Discussion
  6. Acknowledgements 
  7. References
  8. Supporting Information

We are grateful to two reviewers for very helpful comments, and to Da-Hai ZHU and Lian-Cheng TIAN for assistance in collecting plant materials. This study was financially supported by the National Natural Science Foundation of China (Grant Nos. 31070189 and 31170205) and a Special Research Program of Shanghai Chenshan Botanical Garden.


  1. Top of page
  2. Abstract
  3. 1 Material and methods
  4. 2 Results
  5. 3 Discussion
  6. Acknowledgements 
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. 1 Material and methods
  4. 2 Results
  5. 3 Discussion
  6. Acknowledgements 
  7. References
  8. Supporting Information

Fig. S1. Bayesian skyline plot representing the historical demographic trends of extant lineages of Primula ovalifolia.

Table S1. Time to most recent common ancestor (TMRCA) for each lineage (in million of years before present) given as mean/median with 95&percnt; highest posterior density interval in parentheses.

JSE_204_sm_suppmatS1.doc31KSupporting info item
JSE_204_sm_suppmatS2.jpg169KSupporting info item

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