Genetic diversity and population structure
Of the 34 regions surveyed in the chloroplast genomes, rpl32-trnL intergenic spacer was previously proven to be suitable for the population-level phylogenetic studies , and thus has been successfully applied in phylogeography analyses of plants [18, 19]. In comparison to mean estimate of cpDNA diversity (h
= 0.67) detected by various cpDNA markers in 170 plant species , our study showed that C. taliensis possessed an abundant variation in the chloroplast rpl32-trnL intergenic with cpDNA diversity (h) of 0.841. In addition, levels of total haplotype diversity and overall nucleotide diversity within natural populations of C. taliensis at rpl32-trnL (h = 0.841; π = 0.00314) (Table 3) were higher than the three cultivated populations of C. taliensis (h =0.610, π = 0.00225), indicating the reduction of genetic diversity during the domestication . However, nucleotide diversity within C. taliensis in this study appears as high as that of nine cultivated populations of C. sinensis var. assamica from Yunnan, China (h =0.728, π = 0.00469) .
By using rpl32-trnL intergenic spacer and PAL gene fragment as markers, we estimated genetic structure of C. taliensis populations across its distribution range in China. cpDNA data suggest that the differentiation among the C. taliensis populations was rather high (G
ST = 0.988; N
ST = 0.989), placing it among the surveyed plant species with the highest cpDNA differentiation . Partitioning of genetic variability showed that, on average, merely 1.25% of cpDNA variation was distributed within C. taliensis populations and up to 98.75% among populations (Table 6A). In comparison with the above-described cpDNA data, it is of interest to uncover that nrDNA PAL data showed a contrastingly different genetic structure of C. taliensis populations. Genetic differentiation (G
ST = 0.222, N
ST = 0.301) shows that, on average, up to 69.9-77.8% of nrDNA variation was partitioned within C. taliensis populations and merely 22.2-30.1% among populations. The estimates were slightly higher than the mean value of Gst of 0.184 for the other 77 angiosperms species . In this study, we found that N
ST was significantly higher than G
ST, suggesting that pairs of different nrDNA haplotypes from the same population have more similar sequence than pairs of different haplotypes from markedly different populations. AMOVA analysis further revealed that the majority of PAL nucleotide diversity (77.49%, P < 0.001) was significantly attributable to the variation within populations (Table 6B). The lack of genetic differentiation in the nuclear genes probably results from ancestral polymorphisms maintained by a larger effective population size, or high dispersal possibilities of nuclear genes . As for the wild tea tree of C. taliensis, it is likely that smaller effective population size of organelle DNA than nuclear DNA results in strong genetic drift and high levels of population differentiation [4, 5]. Because of the nature of cpDNA maternal inheritance in angiosperms, seed dispersal often plays an important role in shaping population genetic structure of maternally inherited cpDNA. C. taliensis usually generates heavy nut fruit with short-distance seed dispersal, and thus, rather limited abilities of seed dispersal among populations might lead to the observation of high cpDNA population subdivision.
Demographic history of C. Taliensis
NCPA in this study indicates that restricted gene flow and effects of the past fragmentation appear to be of significance in together shaping the observed patterns of chlorotype variation in C. taliensis. Allopatric fragmentation was apparently detected in clades 1–1 and 2–3 of the chlorotype network. The most likely explanation is that the species has recently suffered the degradation and fragmentation of natural habitats in consequence of recent human overexploitation to subtropical forests. Our field surveys particularly found that, driven by market incentives, a number of natural populations of C. taliensis have been seriously destroyed and thus become endangered in small effective population sizes caused by over-picking of organic leaves from natural populations of C. taliensis (Gao and Liu, unpublished data). Moreover, the range of C. taliensis in China covers western region of Yungui Plateau, which is adjacent to the southeast of Qinghai-Tibetan Plateau with an average elevation of approximately 4,500 m above sea level, the largest and highest plateau in the world . The extremely complex topography and climates were formed during the uplift of the Plateau especially in the southeastern region of Qinghai-Tibetan and Yungui plateaus. As a result, significant increase in geological and ecological diversity has largely enhanced rapid divergence and speciation in small and isolated populations . In addition to the recently fragmented habitats as a result of human destruction, the estimated range of separation times of 0.24-3.12 MYA among clades in this study post-dates the most recent uplift the Tibetan Plateau around 3.4 MYA [28, 29]. The species may have experienced habitat fragmentation possibly as a result of the uplift of Qinghai-Tibetan Plateau and subsequent larger-scale drainage. The past fragmentation may have resulted in the observed chlorotype structure of C. taliensis, although there is a lack of direct evidence to strongly support such an association of geographical patterns with the unspecified historical events. NCPA also detected the restricted gene flow in Clade 1–4 which included individuals from a total of eleven populations. It is true that the C. taliensis plants often produce heavy nut fruits with short-distance seed dispersal and thus the gene flow is fairly restricted among populations. The seriously fragmented habitats together with their endangered status indeed have largely accelerated the restricted gene flow among the small surviving natural populations detected in the species. However, one important characteristics of chlorotypes distribution was that Chlorotype C1 was found in the seven populations resided in the Lancang River region without exception. Such a geographical distribution of Chlorotype C1 suggests that the Lancang River might have provided northwards or southwards natural corridors for the long-distance dispersal of C. taliensis in China.
Hybridization is expected to have served as a possible driver of the observed patterns of chlorotypes. Geographic distribution of C. taliensis mostly covers the extensively growing range of C. sinensis. As previously documented in Baiying Mountains, Yunnan Province, hybrid zone which consists of a number of populations, called as “Er’Ga’Zi Tea”, was found to have formed between these two species (Chong-ren Yang, personal communications). However, the extent and effects of hybridization which might affect levels genetic variation and patterns of geographic population structure of C. taliensis remains largely unsettled and stays to be further studied.
It is our discovery that populations with geographical proximity did not share closely related geographical chlorotypes. For example, although TCB and TCD populations were geographically close with only 10 km distance apart, they were fixed for the distinct chlorotypes of C5 and C6, respectively. Such a pattern of cpDNA variation may come from incomplete lineage sorting of polymorphisms. As a kind of stochastic process randomly allocating ancestral polymorphisms into different populations or species, lineage sorting has been proven to be a major factor for the lack of associations between genealogical relationships of haplotypes and their geographical distributions . Another possible explanation is that the past and recent habitat fragmentation of ancestral populations has led to the observed patterns of chlorotype structure in the species.
Isolation by distance can be tested through both the correlation of genetic and geographical distances and the nested clade phylogeographic analysis . In this study, Mantel tests exhibited a significant correlation between genetic and geographical distances of PAL haplotypes, supporting the isolation by distance model across the study populations in C. taliensis. Moreover, the total cladogram of PAL haplotype network also showed the evidence of restricted gene flow with isolation by distance. Overall, both the correlation of genetic and geographical distances and NCPA together demonstrated that the population genetic structure of the species fitted the model of isolation by distance. Nevertheless, Mantel’s tests failed to detect significant correlations between genealogical relationships of chlorotypes and geographical distances. The discrepancy between the nrDNA PAL and cpDNA phylogeography of C. taliensis could result from different transmission mechanisms of nuclear and organelle genes, and/or their different tempos of lineage sorting through drift .
Implications for the germplasm conservation
As the most popular non-alcoholic beverage throughout the world, a large number of tea germplasms have been collected and ex situ preserved in China, Japan, India and Kenya . Unlike cultivated tea varieties, their wild relatives have cold tolerance and are resistant to common diseases infecting cultivated tea tree, and thus they constitute valuable gene resources for local and international tea tree improvement programs in the future. Although efforts have been made to preserve cultivated tea germplasms, it is vital that more attention to be paid to the conservation of their wild relatives has been largely neglected so far. Knowledge of genetic variation between and within populations of rare and endangered species is extremely useful for making appropriate management strategies directed towards their conservation . Of these wild species, C. taliensis is one of the most important wild relatives of the cultivated tea and is subject to increasing threats as a result of the overexploitation and deforestation. The uncovered genetic profile presented here not only helps to gain important insights into genetic structure of C. taliensis populations, but also has critical implications for taking appropriate strategies of the conservation and germplasm management.
Comprehensive understanding of regional genetic structure of C. taliensis in this study is required to design an appropriate conservation scheme. In view of abundant genetic diversity resided within C. taliensis populations, an appropriate strategy for both germplasm sampling and developing in situ conservation of those populations with a higher variation on behalf of different geographical regions is needed. In order to capture the considerable genetic variation harbored among populations, ex situ germplasm collection should have sufficient sample size from each population. Since at least 22% genetic diversity of PAL nucleotide diversity distributed among populations, germplasm collections should be sampled from extensive geographic origins. The observation that the majority of PAL variation was distributed within populations of this species is instructive for adopting a plan of involving fewer populations but more individuals within populations. Apparently, the populations with abundant haplotype diversity, such as XM, YJ, GM, YD, ZY, CY, and NE (Table 5), may be more attractive for both in situ conservation and ex situ germplasm collections. The populations with the unique PAL haplotypes, such as CNB, JC, TCH, XM, YD and YJ, should be given conservation priority (Table 5). The observed chlorotype structure showed an allopatric fragmentation in C. taliensis such as between Clades 1–1 and 2–3. Notably, three chlorotypes (C3, C11 and C10) distributed in the westernmost range of C. taliensis, while other two (C2 and C12) was found in eastern populations of the species (Figure 3), further implying that both germplasm sampling and setting in situ conservation localities should take different geographical origins and the observed chlorotype structure into consideration.
Considering that the most remnant populations of C. taliensis are turning into smaller as a result of human destruction, however, it is quite possible that the process of habitat fragmentation will lead to a loss of genetic diversity by dramatically increasing mating opportunities between relatives within small populations. The C. taliensis populations may have suffered habitat fragmentation due to either the uplift of Tibetan Plateau or recent deforestation, and thus brought about the observed chlorotype structure in C. taliensis. However, NCPA suggest that restricted gene flow/seed dispersal may have resulted in smaller effective population sizes of the outcrossing plant species due to the recently fragmented habitats and long-distance colonization. Consequently, conservation and restoration genetics should concentrate on the maintenance of historically significant processes such as strong gene flow/seed dispersal as well as large effective population size in the species.