Genetic Differentiation and Population Genetic Structure of the Chinese Endemic Dipteronia Oliv. Revealed by cpDNA and AFLP Data

Dipteronia Oliv. is an endangered genus found in China with two species, D. sinensis and D. dyeriana. Previous morphological, cytogenetic, and molecular studies have suggested that D. dyeriana is a species related to D. sinensis. However, it is unclear how the two species diverged and whether gene flow exists between these two species. Here, we performed a molecular study at the population level to characterize genetic differentiation and decipher the phylogeographic history for Dipteronia species based on newly sequenced chloroplast DNA (cpDNA) and amplified fragment length polymorphisms (AFLP) date retrieved from our previous studies. No haplotype was shared between the two species in the cpDNA network. However, the phylogenetic analysis suggested that a haplotype found in D. sinensis (H4) showed a closer relationship with haplotypes of D. dyeriana. Based on our estimated time of divergence, these two cpDNA haplotype lineages of Dipteronia diverged at about 31.19 Ma. Furthermore, two genetic clusters with asymmetric gene flow were supported based on the structure analysis, which corresponded with the two Dipteronia species, and we also detected a low level of asymmetric gene flow between these two species according to the MIGRATE analysis using AFLP data. During the Last Glacial Maximum (LGM, c.21 kya BP), the genus’ predicted distribution was more or less similar to that at present, which was also supported by the mismatch analyses that showed no population expansion of the two Dipteronia populations after the LGM. The combined cpDNA and AFLP data revealed significant genetic differentiation between the two Dipteronia species with asymmetric gene flow, which can be explained by the varying phylogeographical histories of these two species.


Introduction
The geographical distribution of genetic variation in extant populations has generally resulted from interactions between two fundamental processes: population dynamics in response to past geological or climatic changes and a species' evolutionary ability to respond to natural selection [1].A detailed examination of genetic variation within and between populations can help explain the phylogeographic history of a species in response to past geological and climatic oscillations.Numerous studies have identified glacial refugia for temperate plant species and traced their postglacial recolonization routes [2][3][4][5][6][7].The Quaternary climatic oscillations and the associated regional-scale geological events probably promoted intra-specific divergence due to the survival of populations in different glacial refugia and the subsequent barriers to dispersal during recolonization [1].Recent genetic studies conducted in the Chinese population have confirmed the effects that the Quaternary climate oscillations had on the genetic structure of plants, such as Juniperus przewalskii, Pinus tabulaefrmis, and Rhododendron simsii [8][9][10][11].Climatic oscillations during the Pleistocene resulted in several glacial-interglacial cycles, which caused the expansion and contraction of habitats of, for example, Juniperus przewalskii, Pinus tabulaefrmis, and Rhododendron simsii [4,8,9,11,12].Although no massive ice sheets developed in Central China during glacial periods, the tremendous global climatic changes, together with local climatic changes caused by the Qinghai-Tibet Plateau uplift particularly during Quaternary glaciations, have affected the distribution and evolution of many plant species in this area [8,13].In recent years, chloroplast DNA (cpDNA) markers have been frequently utilized to survey the population genetic structure and phylogeography of plants [14], and to locate refugia and post-glacial recolonization routes [14,15].In addition, the bi-parentally inherited AFLP marker has also been used in genetic mapping [16], estimations of phylogenetic relationships [17], and genetic diversity studies [18].
The genus Dipteronia Oliv.(Aceraceae) is found in China with two extant species, D. sinensis Oliv.and D. dyeriana Henry.Both species are diploid (2n = 18) trees distributed in the broad-leaved deciduous forests in central and southern China, occurring along mountain streams at altitudes of 1000-2400 m above the sea level [19].Two species are deciduous trees or small trees with a height of up to 10-25 m tall and pinnately compound leaves.Individual trees are andromonoecious, having both male and bisexual flowers that are born in paniculate inflorescences.The distinction between D. sinensis and D. dyeriana is mainly based on morphology.For example, the panicles of D. dyeriana are densely yellow-green pubescent, unlike in D. sinensis.The fruit of D. dyeriana is larger than the fruit of D. sinensis [20].Controlled pollination tests revealed that D. dyeriana is self-compatible, but a high bee pollinator activity is generally observed in the field, suggesting that the species is at least partially reproduced by insect-mediated outcrossing.Similar to Acer, the genus Dipteronia has schizocarpic winged fruits (samaras) borne in pairs, but in contrast to Acer, the seeded mericarps or "nutlets" are winged essentially all the way around the seed.These fruits, whose broad wings turn from light green to red with ripening, are apparently dispersed by wind [21].These two species are rare and endangered with a limited population and have been listed in Chinese Rare and Endangered Plant Species [22].Studies have shown that rare or endangered species are essential to the conservation of biodiversity and the restoration and survival of the ecosystem [23].Additionally, Dipteronia is one of the ancient relic woody genera in the floristic region of the Northern Hemisphere and the fossil record shows that species of this genus were once distributed in North America (from the late Paleocene to the early Oligocene) in the Tertiary [19,24,25].The Quaternary glaciation has been suggested as the cause of the extinction of this genus in North America, because during the glaciation, the northern part of North America was mostly covered by continental icecaps [26,27].In contrast, the Quaternary glaciers in China were mainly developed in a few mountains and no major icecaps formed [28,29].Many Tertiary relic plants, such as Dipteronia, could survive in refugia in central and southern China, although their distribution area has been significantly fragmented by the glaciations.Thus far, most studies on Dipteronia have been focused on morphological development and taxonomy [30][31][32][33].Only a few studies have been conducted to research the genetic diversity and genetic structure of Dipteronia [34], in which only the genetic diversity and differentiation of the two Dipteronia sepcies have been described based on AFLP analysis [35].
In the present study, we integrated new cpDNA analysis and previous AFLP data [35] to infer the historical and contemporary divergence, in addition to the genetic diversity, of Dipteronia across multiple individuals and populations covering its natural ranges in China.Specifically, we aimed to determine the amount of sequence divergence and potential interspecific gene flow after divergence between D. sinensis and D. dyeriana, as well as to infer the possible refugia.

Sample Collection
In this study, 18 natural populations distributed throughout each species' range were sampled, including 15 individuals from each population (Figure 1 and Table 1).Individuals of the sampled populations were separated by at least 10 m.In addition, using a closely related species, five Acer miaotaiense P. C. Tsoong individuals were selected as an outgroup.Young leaves were collected, dried in a plastic bag with silica gel, transported to the laboratory, and stored in a −80

Sample Collection
In this study, 18 natural populations distributed throughout each species' range were sampled, including 15 individuals from each population (Figure 1 and Table 1).Individuals of the sampled populations were separated by at least 10 m.In addition, using a closely related species, five Acer miaotaiense P. C. Tsoong individuals were selected as an outgroup.Young leaves were collected, dried in a plastic bag with silica gel, transported to the laboratory, and stored in a −80 °C freezer until DNA extraction.Haplotype colors corresponds to those in Figure 2. The blue and red dotted areas represent the distribution areas of D. sinensis and D. dyeriana, respectively.Population abbreviations correspond to those given in Table 1.

DNA Extraction and PCR Amplification
Total genomic DNA was extracted from dry leaves using a modified CTAB method [36,37], before being kept at −20 • C for long-term storage or 4 • C for immediate use.The initial screening of eight cpDNA markers using a subset of 30 samples from five populations revealed four cpDNA markers (trnV intron, rps18-rpl20, rpl20-rps12, and trnH-psbA), generating genetic variations that were used for subsequent phylogeographcical analyses (Table 2).

Chloroplast Data Analysis
Multiple alignments of the cpDNA sequences were manually performed using ClustalX v1.83 [38] and BioEdit v7.0.4.1 [39].Insertions/deletions (indels) were generally retained so as to increase the number of matching nucleotides in a sequence position.Nucleotide diversity (π) and haplotype diversity (H d ) [40] among the population were calculated using the DnaSP v5.0 [41].Neutrality tests, such as Tajima's D and Fu's Fs, were also calculated with this program.We also calculated within-population diversity (H S ), total diversity (H T ), and population differentiation (G ST ) at the species level.To incorporate the relationships between haplotypes, an estimate of population subdivision for phylogenetically ordered alleles (N ST ) was obtained.A higher N ST than G ST usually indicates the presence of a phylogeographic structure.All aforementioned parameters were calculated using the program HAPLONST [42].A statistical parsimony haplotype network based on the matrix of pairwise differences between haplotypes was obtained with the aid of the TCS 1.06 [43] using the 95% connection probability limit, treating gaps as single evolutionary events.To investigate genetic differentiations between two species and among each species, the analysis of molecular variance (AMOVA) was performed with the program ARLEQUIN ver.3.5 [44], while the significance of variance components was tested with 10,000 permutations.Mismatch distribution analyses were carried out in ARLEQUIN ver.3.5 [44] to infer the historical demographic expansion events within the two species.
The best fitted model of DNA substitutions (HKY + I) was selected using the Akaike information criterion (AIC) method in Modeltest v2.3 [45].The Bayesian Markov Chain Monte Carlo (BMCMC) phylogenetic estimate was inferred using MrBayes v3.1.2[46] with default priors.Acer miaotaiense, which is closely related to Dipteronia [47], was used as an outgroup for creating the root of the Bayesian tree.Bayesian analysis was performed with 20,000,000 generations.Four simultaneous chains were run by sampling every 1000 generations.The first 25% of the total trees were discarded and >50% posterior probability consensus trees from the remaining trees were calculated and combined to a final tree.The divergence times for different Dipteronia cpDNA haplotype lineages were estimated using a Bayesian approach implemented in BEAST v1.5.4 [48] using the HKY + I model.The starting tree was randomly generated.We chose a coalescent tree model, which assumed a constant size for our tree.The mean cpDNA mutation rate reported in a previous study for Acer mono [49] was used for the four concatenated cpDNA non-coding regions to estimate the lineage divergent time in BEAST under the uncorrelated lognormal clock model.Three separate MCMC analyses were run for 20,000,000 generations with sampling at every 2000 generations and the first 25% generations were discarded as 'burn-in'.Tracer v1.5 [50] was used to check the parameters and ensure that all the effective sample size (ESS) values were greater than 200.The final tree was generated using the TreeAnnotator v1.8.2 program and visualized in FigTree v1.4.2.

Analysis of AFLP Datasets
We re-scored and re-analyzed two available AFLP datasets: one mainly contained populations of D. sinensis Oliv.(121 individuals), while the other mainly contained populations of D. dyeriana Henry (21 individuals) [35].These datasets were initially generated and analyzed as part of several larger meta-studies [35].The population structure was detected using the model-based Bayesian algorithm in STRUCTURE v2.3.4 [51].We used the admixture model without prior information on population membership and assumed independent allele frequencies among populations.The number of clusters (K) was set to vary depending on the data set.For each value of K, we conducted 10 independent simulations with a burn-in and run length of 100,000 Markov chain Monte Carlo (MCMC) replications.The number of gene pools was inferred by estimating ∆K and lnP (D) [51][52][53].

Species Distribution Models
Species distribution models (SDMs) were carried out in MAXENT v3.3.1 [57] to predict suitable climate envelopes for D. sinensis at present and during the Last Glacial Maximum (LGM: c.21 kya before present; BP), respectively.In addition to the distribution records in this study (see Table 1), 125 collection records were obtained from the Chinese Virtual Herbarium (http://www.cvh.org.cn)[58] and the National Specimen Information Infrastructure of China (http://www.nsii.org.cn)[59].Based on this total of 145 records, a current distribution model was developed using six bioclimatic data layers (annual mean temperature, annual precipitation, precipitation of wettest, driest, warmest and coldest quarter) available from the WorldClim database (http://www.worldclim.org)[60,61] at a 2.5-arcmin resolution for the present .This restricted bioclimatic dataset avoided including highly correlated variables (data not shown) and thus, prevented potential overfitting [62].This model was then projected onto the paleoclimatic dataset simulated by the COMMUNITY CLIMATE SYSTEM MODEL (CCSM v3.0; http://pmip2.lsce.ipsl.fr/)[63,64] to infer the extent of suitable habitats during the LGM.Preparation of the LGM palaeoclimate layers at a 2.5 arc-min resolution used the same method as a previous study [65].The accuracy of each model prediction was tested by calculating the area under the ROC Curve (AUC) [66], while AUC values > 0.8 indicate accurate simulations for the SDM models.

Chloroplast DNA Polymorphism
In this study, the total alignment length of four regions (trnV intron, rps18-rpl20, rpl20-rps12, and trnH-psbA) was 1789 bp.A total of nine haplotypes were identified based on the total alignment sequence (Figure 1, Tables 1 and 2).There are seven haplotypes in D. sinensis and two haplotypes in D. dyeriana, with no shared haplotype between them.The distribution of nine haplotypes was shown in Figure 1 and the most widespread haplotypes were H1 (in five populations), H5 (in six populations), and H9 (in three populations).All the remainders were unique to a specific population (Figure 1; and Table 1).There were three populations that harbored two haplotypes, while the remaining populations only contained a single cpDNA haplotype (Figure 1; and Table 1).The unrooted network of Dipteronia Oliv.haplotypes was broadly consistent with the strict consensus tree with a star-like genealogical signature.The ancestral-like haplotype H5 lies at the center with other derivative haplotypes connected to it independently.H4 was a haplotype of D. sinensis, which connected to H9 of D. dyeriana (Figure 2).In the phylogeny tree, H4 haplotype belonged to D. yeriana.It was predicted that the H4 haplotype was the ancestral haplotype and became extinct in D.yeriana because it couldn't adapt to the environment.However, it was still reserved in D. sinensis.
Forests 2017, 8, 424 6 of 14 was tested by calculating the area under the ROC Curve (AUC) [66], while AUC values > 0.8 indicate accurate simulations for the SDM models.

Chloroplast DNA Polymorphism
In this study, the total alignment length of four regions (trnV intron, rps18-rpl20, rpl20-rps12, and trnH-psbA) was 1789 bp.A total of nine haplotypes were identified based on the total alignment sequence (Figure 1, Tables 1 and 2).There are seven haplotypes in D. sinensis and two haplotypes in D. dyeriana, with no shared haplotype between them.The distribution of nine haplotypes was shown in Figure 1 and the most widespread haplotypes were H1 (in five populations), H5 (in six populations), and H9 (in three populations).All the remainders were unique to a specific population (Figure 1; and Table 1).There were three populations that harbored two haplotypes, while the remaining populations only contained a single cpDNA haplotype (Figure 1; and Table 1).The unrooted network of Dipteronia Oliv.haplotypes was broadly consistent with the strict consensus tree with a star-like genealogical signature.The ancestral-like haplotype H5 lies at the center with other derivative haplotypes connected to it independently.H4 was a haplotype of D. sinensis, which connected to H9 of D. dyeriana (Figure 2).In the phylogeny tree, H4 haplotype belonged to D. yeriana.It was predicted that the H4 haplotype was the ancestral haplotype and became extinct in D.yeriana because it couldn't adapt to the environment.However, it was still reserved in D. sinensis.

Genetic Diversity and Population Differentiation
For D. sinensis, the cpDNA revealed a low level of haplotype diversity (Hd = 0.260) and nucleotide diversity (π = 0.63 × 10 −3 ).The analysis of genetic diversity in the D. sinensis population showed that the total genetic diversity (HT = 0.812) was much higher than the average within-population diversity (HS = 0.086) (Table 3).Additionally, the GST and NST values were 0.894 and 0.912, respectively.A statistical analysis using a permutation test showed that the NST value was higher than the GST value (p > 0.05), indicating an absence of phylogeographical structure for D. sinensis.With respect to D. dyeriana, a lower haplotype diversity (Hd = 0.395) and nucleotide diversity (π = 0.23 × 10 −3 ) was detected and the total genetic diversity (HT = 0.500) was also much higher than the average within-population diversity (HS = 0) (Table 3).The AMOVA results indicated that 29.92% of the total genetic variation was partitioned between the two species, while 63.78% was found among populations (Table 4).The AMOVA analyses revealed that 67.86% and 100% of the species' total cpDNA variation was distributed among the population for D. sinensis (FST = 0.89448, p < 0.001) and D. dyeriana (FST = 1, p < 0.001), respectively, and low genetic differentiation was found within

Genetic Diversity and Population Differentiation
For D. sinensis, the cpDNA revealed a low level of haplotype diversity (H d = 0.260) and nucleotide diversity (π = 0.63 × 10 −3 ).The analysis of genetic diversity in the D. sinensis population showed that the total genetic diversity (H T = 0.812) was much higher than the average within-population diversity (H S = 0.086) (Table 3).Additionally, the G ST and N ST values were 0.894 and 0.912, respectively.A statistical analysis using a permutation test showed that the N ST value was higher than the G ST value (p > 0.05), indicating an absence of phylogeographical structure for D. sinensis.With respect to D. dyeriana, a lower haplotype diversity (H d = 0.395) and nucleotide diversity (π = 0.23 × 10 −3 ) was detected and the total genetic diversity (H T = 0.500) was also much higher than the average within-population diversity (H S = 0) (Table 3).The AMOVA results indicated that 29.92% of the total genetic variation was partitioned between the two species, while 63.78% was found among populations (Table 4).The AMOVA analyses revealed that 67.86% and 100% of the species' total cpDNA variation was distributed among the population for D. sinensis (F ST = 0.89448, p < 0.001) and D. dyeriana (F ST = 1, p < 0.001), respectively, and low genetic differentiation was found within populations (Table 4).At the species level, neutrality test statistics (Tajima's D and Fu and Li's F') (Table 3) were all non-significant, while the mismatch distributions of D. sinensis were multimodal (Figure 3).As the haplotypes of D. dyeriana contained no more than three haplotypes within the Yunnan province, the mismatch analysis was not conducted.At the species level, neutrality test statistics (Tajima's D and Fu and Li's F') (Table 3) were all non-significant, while the mismatch distributions of D. sinensis were multimodal (Figure 3).As the haplotypes of D. dyeriana contained no more than three haplotypes within the Yunnan province, the mismatch analysis was not conducted.

Phylogeny and Molecular Dating Based on cpDNA Data
In the present study, the phylogeny of cpDNA sequences was analyzed based on the Maximum likelihood (ML) and Bayes method with Acer miaotaiense as an outgroup (Figure 4).The phylogenetic tree derived from different methods produced an identical topology.All haplotypes were clustered

Phylogeny and Molecular Dating Based on cpDNA Data
In the present study, the phylogeny of cpDNA sequences was analyzed based on the Maximum likelihood (ML) and Bayes method with Acer miaotaiense as an outgroup (Figure 4).The phylogenetic tree derived from different methods produced an identical topology.All haplotypes were clustered into two clades with weak support values.Most haplotypes, except for one haplotype (H4), of D. sinensis formed a clade with a high support value, while the H4 haplotype of D.sinensis in the network clustered with the two haplotypes belonging to D. dyeriana.The BEAST-derived cpDNA chronogram indicated that the split time of the two cpDNA clades was dated to 31.2 Ma (Figure 4), with a divergence time for six haplotypes of D. sinensis of 7.76 Ma (95% HPD: 2.63-18.34Ma; Figure 4) and a divergence time for two haplotypes of D. dyeriana of 1.04 Ma (95% HPD: 0.01-3.84Ma; Figure 4).

Population Sturcture and Interspecific Gene Flow Based on AFLP Data
In the Bayesian analysis of population structure, the highest likelihood of the AFLP data was obtained when samples were clustered into two groups (K = 2).All the D. dyeriana were clustered into groups and the populations of D. sinensis showed a mosaic structure, which indicated a low level of hybridization between these two species (Figure 5).After estimating the migration rates between the two species, we detected that the mean migration rate from D. dyeriana to D. sinensis was 0.7325 (0.7054-0.7600; 95% HPD) and the reverse migration rate was 0.4860 (0.4601-0.5130; 95% HPD).

Population Sturcture and Interspecific Gene Flow Based on AFLP Data
In the Bayesian analysis of population structure, the highest likelihood of the AFLP data was obtained when samples were clustered into two groups (K = 2).All the D. dyeriana were clustered into groups and the populations of D. sinensis showed a mosaic structure, which indicated a low level of hybridization between these two species (Figure 5).After estimating the migration rates between the two species, we detected that the mean migration rate from D. dyeriana to D. sinensis was 0.7325 (0.7054-0.7600; 95% HPD) and the reverse migration rate was 0.4860 (0.4601-0.5130; 95% HPD).

Present and Past Ecological Niche Models
The AUC value for the current potential distribution of D. sinensis was relatively high (AUC > 0.9), demonstrating a reliable predictive model performance.The predicted distribution ranges of the species under the current status were identical to its actual distribution ranges (Figure 6).The actual distribution ranges of D. dyeriana were slightly contracted compared to the potential range during the LGM.

Genetic Diversity, Genetic Differentiation, and Glacial Refuge
The total genetic diversity of D. sinensis (HT = 0.812) was higher than the mean total genetic diversity (HT = 0.670) detected in 170 plant species for which cpDNA markers have been used [7].The diversity was also higher compared to that of 13 other seed plants used as maternally inherited markers in China [10].The high genetic diversity detected in D. sinensis might reflect the accumulation of nucleotide mutations over long evolutionary time-scales [67,68].Previous research related to the genetic diversity of Dipteronia based on AFLP and random amplified polymorphic

Present and Past Ecological Niche Models
The AUC value for the current potential distribution of D. sinensis was relatively high (AUC > 0.9), demonstrating a reliable predictive model performance.The predicted distribution ranges of the species under the current status were identical to its actual distribution ranges (Figure 6).The actual distribution ranges of D. dyeriana were slightly contracted compared to the potential range during the LGM.

Present and Past Ecological Niche Models
The AUC value for the current potential distribution of D. sinensis was relatively high (AUC > 0.9), demonstrating a reliable predictive model performance.The predicted distribution ranges of the species under the current status were identical to its actual distribution ranges (Figure 6).The actual distribution ranges of D. dyeriana were slightly contracted compared to the potential range during the LGM.

Genetic Diversity, Genetic Differentiation, and Glacial Refuge
The total genetic diversity of D. sinensis (HT = 0.812) was higher than the mean total genetic diversity (HT = 0.670) detected in 170 plant species for which cpDNA markers have been used [7].The diversity was also higher compared to that of 13 other seed plants used as maternally inherited markers in China [10].The high genetic diversity detected in D. sinensis might reflect the accumulation of nucleotide mutations over long evolutionary time-scales [67,68].Previous research related to the genetic diversity of Dipteronia based on AFLP and random amplified polymorphic DNA (RAPD) markers also indicated that D. sinensis has a higher diversity [34,35].In contrast, the

Genetic Diversity, Genetic Differentiation, and Glacial Refuge
The total genetic diversity of D. sinensis (H T = 0.812) was higher than the mean total genetic diversity (H T = 0.670) detected in 170 plant species for which cpDNA markers have been used [7].
The diversity was also higher compared to that of 13 other seed plants used as maternally inherited markers in China [10].The high genetic diversity detected in D. sinensis might reflect the accumulation of nucleotide mutations over long evolutionary time-scales [67,68].Previous research related to the genetic diversity of Dipteronia based on AFLP and random amplified polymorphic DNA (RAPD) markers also indicated that D. sinensis has a higher diversity [34,35].In contrast, the genetic diversity of D. dyeriana found in this study was low (H T = 0.500), probably due to its limited distributed area and low population sizes.
Based on our data set, high genetic differentiation was detected between these two species and variation was also detected among populations.Physical barriers may have contributed to the marked genetic differentiation among Dipteronia populations.Therefore, geographic isolation may also partly account for the pronounced genetic differentiation.Neighboring populations were usually separated by geographic barriers and anthropogenic facilities, which largely hindered gene flow via seed and pollen dispersal among populations.We detected high genetic differentiation and low gene flow between these two species.
According to the coalescent theory, the ancestral haplotypes are expected to occupy a central position in the network of haplotypes with a high frequency and have a great probability of producing mutational derivatives [69].In this study, H5 was found to be predominant and geographically widespread, which was located in the interior position of genealogies (Figure 2) and is most likely the ancestral haplotype.Suitable habitats in refugia may permit species to persist for long periods.Unique genotypes and high levels of diversity often occur in these locations [2].We inferred that the most likely refugium for D. sinensis was the Qinling-Daba mountains because they have the highest number of haplotypes (H1, H2, H3, and H5), as well as the highest haplotype diversity and nucleotide diversity (Figure 1 and Table 3).Meanwhile, H2 and H3 were specific to LY and LS, which were located in the Qinling mountains.The CD population had a high nucleotide and haplotype diversity, with the specific haplotype H4 belonging to it.This population was located in the west of the Sichuan basin, where there seems to be another potential refugium.The population in the Sichuan basin is surrounded by mountains of Northwest China with no glaciers in the Quaternary glacial.Compared with the same latitude, the significantly higher average temperature of the basin provides a good refuge for the genus Dipteronia Oliv.(Aceraceae).Additionally, the ecological niche modelling (ENM) analyses also indicated that these regions were suitable distribution ranges for these species, which also confirmed these refugia.

Demographic History of Dipteronia
Our phylogenetic and phylogeographic analyses based on cytoplasmic and nuclear (AFLP) data clearly support the monophyly of the genus Dipteronia and provide evidence for the genetic distinctiveness of D. sinensis and D. dyeriana, although asymmetric gene flow was revealed between these two species according to the STRUCTURE and MIGRATE analyses.Based on our cpDNA chronogram, the split time of the main clades was dated to the Oligocene, which indicated a long evolutionary history and independent evolution of the two species.Our molecular dating analyses (Figure 4b) indicate that the onset of haplotype diversification in D. sinensis (c.7.77 Ma, 95% HPD: 2.63-18.33Ma) occurred midway during the Pliocene period.The diversified geological and/or climatic changes had possibly acted as an isolating barrier between regional populations and promoted the diversification of two lineages of D. sinensis.Furthermore, the divergence time of two D. dyeriana haplotypes (H8 and H9) was located in the Quaternary, which indicated that the fluctuant climatic changes promoted the diversified events of D. dyeriana populations.However, there is a lack of fossil records of Dipteronia in China.We speculate that the Dipteronia species may have a complex evolutionary history during the long species trajectory of this endemic genus.
In the present study, no phylogeographic structure was detected for D. sinensis.Additionally, no expansion was detected for these two species, which indicated stable habitats where there are some species after the LGM.The ENM results also supported a rare distribution expansion for Dipteronia (Figure 6).

Recommendations for Species Conservation
In our study, the presence of unique D. sinensis cpDNA haplotypes in two different regions suggested the existence of two separate refugia.These putative refugia have long been recognized as centers of plant diversity and endemism in subtropical China [70,71].Our results supported the hypothesis that some parts of China have been spared from the direct impact of Pleistocene glaciations [72] and have served as potential refugia for temperate deciduous trees [73,74].The genetic data from D. sinensis indicated a pattern of long-term fragmentation and refugia survival, as hypothesized by Harrison et al. [73].Although all the D. sinensis populations in the two proposed glacial refugia areas are currently under legal protection as one of the most endangered plant species in China, routine practices and additional precautions for the conservation of this endangered plant are still awaiting consideration with the support of of genetic information.
To preserve the total genetic diversity, in situ conservation and ex situ conservation are both necessary for this endangered genus.In the current study, each suggested glacial survival area of D. sinensis comprised a set of populations possessing unique haplotypes and these areas should be qualified as different "evolutionary significant units" [75], such as the populations, CD, LY, HH, and LS.These distinct units can be used as a reference source for the ex situ conservation when transplanting individuals between areas or to a new site.In ex situ conservation, samples should be collected especially from those populations harboring unique haplotypes.
Currently, there are only five natural populations of D. dyeriana in southeast Yunnan, and our genetic survey revealed the relatively low genetic diversity of this endangered species, as had been reported in a previous study [35].As the genetic drift or inbreeding might have decreased the within-population gene diversity of D. dyeriana, we recommend recovering the genetic diversity in each population through replanting the seedlings to natural habitats to increase genetic exchange and recombination.Therefore, a broad genetic sample should be preserved by ex situ conservation programs (such as seed banks and botanic gardens).This will potentially allow future reintroductions or population reinforcements, whose success will heavily depend upon the genetic quality of the available ex situ sample [76,77].

Conclusions
In the current study, we performed a molecular study at the population level to characterize genetic differentiation and decipher the phylogeographic history for Dipteronia species based on chloroplast DNA (cpDNA) and amplified fragment length polymorphisms (AFLP) data.The genetic diversity (H T = 0.812) of D. sinensis was higher than that of D. dyerana (H T = 0.500).The combined cpDNA and AFLP data revealed significant genetic differentiation between the two Dipteronia species with asymmetric gene flow, which can be explained by the varying phylogeographical histories of these two species.Based on our estimated time of divergence, the two cpDNA haplotype lineages of Dipteronia diverged at about 31.19Ma.During the Last Glacial Maximum (LGM, c.21 kya BP), the genus' predicted distribution was more or less similar to that at present, which was also supported by the mismatch analyses that showed no population expansion of the two Dipteronia populations after the LGM.

Figure 1 .
Figure 1.The distribution of nine chloroplast DNA haplotypes identified within 18 Dipteronia Oliv.Haplotype colors corresponds to those in Figure 2. The blue and red dotted areas represent the distribution areas of D. sinensis and D. dyeriana, respectively.Population abbreviations correspond to those given in Table1.

Figure 1 .
Figure 1.The distribution of nine chloroplast DNA haplotypes identified within 18 Dipteronia Oliv.Haplotype colors corresponds to those in Figure 2. The blue and red dotted areas represent the distribution areas of D. sinensis and D. dyeriana, respectively.Population abbreviations correspond to those given in Table1.

Figure 2 .
Figure 2. TCS-derived network of genealogical relationships between the nine haplotypes found in Dipteronia Oliv.Missing haplotypes are represented by hatch marks.The sizes of circles are approximately proportional to sample size.

Figure 2 .
Figure 2. TCS-derived network of genealogical relationships between the nine haplotypes found in Dipteronia Oliv.Missing haplotypes are represented by hatch marks.The sizes of circles are approximately proportional to sample size.

Forests 2017, 8 ,
x FOR PEER REVIEW 8 of 14 into two clades with weak support values.Most haplotypes, except for one haplotype (H4), of D. sinensis formed a clade with a high support value, while the H4 haplotype of D.sinensis in the network clustered with the two haplotypes belonging to D. dyeriana.The BEAST-derived cpDNA chronogram indicated that the split time of the two cpDNA clades was dated to 31.2 Ma (Figure4), with a divergence time for six haplotypes of D. sinensis of 7.76 Ma (95% HPD: 2.63-18.34Ma; Figure4) and a divergence time for two haplotypes of D. dyeriana of 1.04 Ma (95% HPD: 0.01-3.84Ma; Figure4).

Figure 4 .
Figure 4. (a) Phylogenetic tree (ML) of the nine chloroplast haplotypes detected in Dipteronia Oliv.with H10 as the outgroup using Acer miaotaiense.(b) Beast-derived chronogram for the haplotypes in Dipteronia Oliv.based on four chloroplast DNA spacer regions.Posterior probabilities above 0.95 are shown above nodes.Error bars around nodes correspond to 95% highest posterior densities of divergence times.H10 marks the outgroup using Acer miaotaiense.

Figure 4 .
Figure 4. (a) Phylogenetic tree (ML) of the nine chloroplast haplotypes detected in Dipteronia Oliv.with H10 as the outgroup using Acer miaotaiense.(b) Beast-derived chronogram for the haplotypes in Dipteronia Oliv.based on four chloroplast DNA spacer regions.Posterior probabilities above 0.95 are shown above nodes.Error bars around nodes correspond to 95% highest posterior densities of divergence times.H10 marks the outgroup using Acer miaotaiense.

Figure 6 .
Figure 6.Potential distribution of D. sinensis and D. dyeriana based on ecological niche modeling using MAXENT; as well as the predicted distribution of the model to the last glacial maximum (c.21 kya) and present.The color gradient from white to the red refers to habitat suitability from low to high.

Figure 5 .
Figure 5. Clustering analysis of AFLP for D. sinensis and D. dyeriana populations using STRUCTURE.

Figure 6 .
Figure 6.Potential distribution of D. sinensis and D. dyeriana based on ecological niche modeling using MAXENT; as well as the predicted distribution of the model to the last glacial maximum (c.21 kya) and present.The color gradient from white to the red refers to habitat suitability from low to high.

Figure 6 .
Figure 6.Potential distribution of D. sinensis and D. dyeriana based on ecological niche modeling using MAXENT; as well as the predicted distribution of the model to the last glacial maximum (c.21 kya) and present.The color gradient from white to the red refers to habitat suitability from low to high.
• C freezer until DNA extraction.

Table 1 .
Details of sample locations (taken from GPS coordinates), sample sizes, and chloroplast DNA haplotype frequencies for 18 populations of Dipteronia Oliv. in China.

Table 1 .
Details of sample locations (taken from GPS coordinates), sample sizes, and chloroplast DNA haplotype frequencies for 18 populations of Dipteronia Oliv. in China.

Table 2 .
Characteristics of the four chloroplast DNA spacer regions of Dipteronia Oliv.

Table 3 .
Genetic diversity, differentiation parameters, and neutrality test in all populations of D. sinensis and D. dyeriana.= nucleotide diversity; H d = haplotype diversity; H T = total diversity; H S = within-population diversity; G ST = population differentiation; N ST = population subdivision for phylogenetically ordered alleles. π

Table 4 .
Different hierarchical types of analysis of molecular variance (AMOVA) of D. sinensis and D. dyeriana.
df = degrees of freedom; F CT = correlation of chloroplast types within groups relative to the total; F SC = correlation within populations relative to groups; F ST = correlation within populations relative to the total; and ** p < 0.001.

Table 3 .
Genetic diversity, differentiation parameters, and neutrality test in all populations of D. sinensis and D. dyeriana.Hd = haplotype diversity; HT = total diversity; HS = within-population diversity; GST = population differentiation; NST = population subdivision for phylogenetically ordered alleles.

Table 4 .
Different hierarchical types of analysis of molecular variance (AMOVA) of D. sinensis and D. dyeriana.
df = degrees of freedom; FCT = correlation of chloroplast types within groups relative to the total; FSC = correlation within populations relative to groups; FST = correlation within populations relative to the total; and ** p < 0.001.