Plant Biology ISSN 1435-8603
RESEARCH PAPER Phylogeography of Chinese cherry (Prunus pseudocerasus Lindl.) inferred from chloroplast and nuclear DNA: insights into evolutionary patterns and demographic history T. Chen1, Q. Chen1, Y. Luo1, Z.-L. Huang1, J. Zhang1, H.-R. Tang1,2, D.-M. Pan3 & X.-R. Wang2 1 College of Horticulture, Sichuan Agricultural University, Ya’an, China 2 Institute of Pomology and Olericulture, Sichuan Agricultural University, Chengdu, China 3 College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou, China
Keywords ABSTRACT Chinese cherry (Prunus pseudocerasus Lindl.); Prunus pseudocerasus cpDNA; nrDNA; phylogeography. Chinese cherry ( Lindl.) is a commercially valuable fruit crop in China. In order to obtain new insights into its evolutionary history and provide valu- Correspondence able recommendations for resource conservation, phylogeographic patterns of 26 nat- X.-R. Wang, Institute of Pomology and ural populations (305 total individuals) from six geographic regions were analyzed Olericulture, Sichuan Agricultural University, using chloroplast and nuclear DNA fragments. Low levels of haplotype and nucleotide Chengdu 611130, China. diversity were found in these populations, especially in landrace populations. It is E-mail: [email protected] likely that a combined effect of botanical characteristics impact the effective popula- tion size, such as inbreeding mating system, long life span, as well as vegetative repro- Editor duction. In addition, strong bottleneck effect caused by domestication, together with X. Wang founder effect after dispersal and subsequent demographic expansion, might also accelerate the reduction of the genetic variation in landrace populations. Interestingly, Received: 5 October 2014; Accepted: 10 populations from Longmen Mountain (LMM) and Daliangshan Mountain (DLSM) December 2014 exhibited relatively higher levels of genetic diversity, inferring the two historical genetic diversity centers of the species. Moreover, moderate population subdivision doi:10.1111/plb.12294 was also detected by both chloroplast DNA (GST = 0.215; NST = 0.256) and nuclear DNA (GST = 0.146; NST = 0.342), respectively. We inferred that the episodes of effi- cient gene flow through seed dispersal, together with features of long generation cycle and inbreeding mating system, were likely the main contributors causing the observed phylogeographic patterns. Finally, factors that led to the present demographic pat- terns of populations from these regions and taxonomic varieties were also discussed.
this species inhabit wide eco-geographic regions in China INTRODUCTION (from North China Plain to Yungui Plateau) (Fig. 1). Consid- Rosaceae consists of over 100 genera and 3000 species, includ- erable genetic variation associated with multiple favorable ing many important fruit crops with diverse growth habits characters have been accumulated in these populations, such as (herbaceous, liana, bush and tree forms) and fruit types (pome, intensive pest/disease resistance, outstanding nutritional values, drupe, achene, hip, follicle and capsule) (Hummer & Janick unique flavor and remarkable abiotic adaptation (Huang et al. 2009; Janick 2005; Potter et al. 2007). The members of Rosa- 2013), thereby providing the promising opportunities for ceae such as Fragaria, Rosa, Rubus, and Prunus provide high- cherry breeding (Gutierrez-Pesce et al. 1998; Rugini 2004). value nutritional foods and contribute desirable aesthetic and Phylogeography has recently emerged as a powerful method commercial products. Chinese cherry (Prunus pseudocerasus for understanding population structure and evolutionary his- Lindl.) is an endemic tetraploid species (Iwatsubo et al. 2004; tory of plant species by synthesizing the influence of both his- Oginuma 1988), distributing throughout the temperate and torical and current genetic exchange (Petit et al. 2005; Schaal warm-temperate forests in China (Yu€ 1979). As an important et al. 1998). A large number of reports have witnessed increas- fruit crop of Rosaceae, Chinese cherry has been domesticated ing applications of phylogeography in economically important and cultivated for more than 3000 years (Liu & Liu 1993; Yu€ plants and their wild relatives, such as Oryza rufipogon (Huang 1979). It is a hermaphrodite, perennial woody plant with high et al. 2012), Spartina pectinata (Kim et al. 2013), Medicago sati- levels of inbreeding rate, strong animal or gravity seed dispersal va (Sakiroglu & Brummer 2013), Camellia taliensis (Liu et al. ability as well as long intergeneration period of 3–6 years (Yu€ 2012), Triticum monococcum (Oliveira et al. 2011), Miscanthus 1979). The drupe fruit contains rich nutritional ingredients sinensis (Shimono et al. 2013). However, most of these studies and trace elements, such as vitamins, minerals, fiber and anti- only focused on domesticated annual crops, phylogeographic oxidant compounds for healthy diets, and its flower is also studies of perennial fruit crops were scarcely reported, including endowed with well-known ornamental value (Huang et al. Rosaceae species (Bai et al. 2014; Zong et al. 2014). As for 2013; Yu€ et al. 1986). Both landrace and wild populations of Chinese cherry, although our previous study has provided
Plant Biology 17 (2015) 787–797 © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands 787 Phylogeographic study of Prunus pseudocerasus Chen, Chen, Luo, Huang, Zhang, Tang, Pan & Wang
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Fig. 1. Sampling locations and haplotypes distribution of P. pseudocerasus. A: The geographic distribution of sampled populations. The landrace and wild populations are represented with circles and triangles, respectively. The dotted circles with different colors delimit different geographic regions, as shown in the key. The six geographic regions are: Daliangshan Mountain (DLSM), Sichuan Basin (SCB), Longmen Mountain (LMM), Qinba Mountain (QBM), North China Plain (NCP) and Yungui Plateau (YGP). B: The geographic distribution and frequencies of cpDNA haplotypes. C: The geographic distribution and frequencies of nrDNA haplotypes. Colors represent the different haplotypes, as shown in the key. preliminary information about the genetic variation and popu- markers, elucidate the population structure patterns, and iden- lation structure, based on a single chloroplast DNA (cpDNA) tify hot-spots of diversity to define more accurate conservation fragment (Chen et al. 2013), more data are also critical for fur- criteria; (ii) clarify the usability of ITS polymorphism status in ther understanding the phylogeographic patterns of this species. population genetic studies in the tetraploid species; and (iii) DNA sequences derived from nuclear and cytoplasmic ge- explore the long-term population dynamics that have shaped nomes play a vital role in plant phylogeographic analyses (Soltis the current population structure, determine its demographic & Soltis 1998; Zheng et al. 2008). It was reported that intergenic and evolutionary history, as well as investigate recently colo- spacers of cpDNA such as rpl32-trnL and rps16-trnQ could nized areas throughout its distribution. offer higher levels of variation, thereby frequently used to inves- tigate intraspecific genetic diversity, population genetic struc- ture and phylogeography (Shaw et al. 2005, 2007; Small et al. MATERIALS AND METHODS 2005). In addition, internal transcribed spacer (ITS) region Sampling strategy from nuclear ribosomal DNA (nrDNA) was also widely applied in plant phylogeographic and evolutionary studies (Alvarez & A total of 305 individuals from 26 natural populations were Wendel 2003). Although intra-individual ITS polymorphism collected in six geographic regions (from southwest to north of were found in non-hybrid diploid taxa, even in the same gen- China), representing almost the entire distribution range of the ome (Alvarez & Wendel 2003; Zheng et al. 2008), homogenous species in China (Fig. 1 and Table 1). These populations con- nrDNA arrays within individuals were identified in many sisted of 17 landraces and nine wild populations, with 139 and reports (Bailey et al. 2003; Zheng et al. 2008) and successfully 166 individuals, respectively. All the landraces were primarily used in many species (Wissemann & Ritz 2005; Yang et al. domesticated to best-fit local climatic, soil conditions and the 2012), including Malus (Robinson et al. 2001) and Maloideae taste preferences of the consumers over centuries. For each (Campbell et al. 2007). These markers might provide unprece- population, young and healthy leaves were randomly sampled dented opportunities to investigate genetic structure and popu- from 2 to 49 individual trees, based on available population lation history of species, particularly by means of a combined size and reproductive modes. In order to ensure adequate pop- analysis of biparentally inherited nuclear and maternally inher- ulation coverage, samples were randomly taken from about 50 ited organelle ones (Liu et al. 2012; Petit et al. 2005; Schaal et al. to 1000 m intervals in each population. Leaves were individu- 1998; Turchetto-Zolet et al. 2012; Yuan et al. 2010). ally collected in field and silica-dried instantly for subsequent In this study, a total of 26 populations from six geographical DNA extraction. regions (defined based on geographical characteristics and sub- sequent ecological and climatic types, Fig. 1 and Table 1) were DNA isolation, PCR amplification, and sequencing screened and comprehensively analyzed using biparentally inherited nuclear and maternally inherited organelle markers. Total genomic DNA was isolated according to a CTAB-based We aimed to: (i) assess the levels of genetic diversity and popu- method (Chen et al. 2013). Three chloroplast genome regions lation differentiation with different inherited molecular (trnL-rpl32, trnQ-rps16 and rps16 intron) were amplified and
788 Plant Biology 17 (2015) 787–797 © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands Chen, Chen, Luo, Huang, Zhang, Tang, Pan & Wang Phylogeographic study of Prunus pseudocerasus
Table 1. Sampling locations, haplotypes and genetic diversities.
number haplotype collected latitude (N)/ sample size haplotypes diversity nucleotide diversity symbol locality variety longitude (E) altitude (m) cpDNA (ITS) cpDNA (ITS) cpDNA (ITS) cpDNA (ITS)
Daliangshan Mountain (DLSM) 22 (21) 2 (3) 0.091 (0.592) 0.00039 (0.00560) LXC Zhangmujing,Xichang,SC landrace 27°580/102°060 1801 13 (12) 1 (1) 0.000 (0.000) 0.00000 (0.00000) WXC Dechang,Xichang,SC wild 27°170/102°130 1878 9 (9) 2 (3) 0.222 (0.417) 0.00032 (0.00277) Sichuan Basin (SCB) 90 (90) 9 (10) 0.418 (0.535) 0.00256 (0.00145) LPJ Pujiang,SC landrace 30°090/103°290 571 4 (4) 1 (2) 0.000 (0.667) 0.00000 (0.00095) LJY Jianyang,SC landrace 30°280/104°180 677 2 (2) 1 (2) 0.000 (1.000) 0.00000 (0.00142) LYA Duiyan,Yaan,SC landrace 29°580/102°570 621 18 (17) 2 (2) 0.209 (0.309) 0.00030 (0.00044) LSM Anshun,Shimian,SC landrace 31°250/102°180 1898 10 (10) 1 (2) 0.000 (0.356) 0.00000 (0.00051) LMS Danling,Meishan,SC landrace 30°040/103°260 748 8 (8) 2 (1) 0.571 (0.000) 0.00182 (0.00000) LHY Shuangxi,Hanyuan,SC landrace 29°310/102°380 1214–2003 6 (6) 1 (3) 0.000 (0.733) 0.00000 (0.00123) LFL Fuling,CQ landrace 29°420/107°280 390–638 8 (8) 3 (2) 0.679 (0.571) 0.00225 (0.00081) WEM Puxing,Emei,SC wild 29°570/103°450 3 (3) 1 (1) 0.000 (0.000) 0.00000 (0.00000) WYC Zhougongshan,Yucheng,SC wild 30°010/102°980 688–1432 21 (21) 5 (6) 0.595 (0.657) 0.00220 (0.00222) WSM Xinmin,Shimian,SC wild 29°260/102°100 1243–2325 10 (11) 3 (5) 0.378 (0.764) 0.00137 (0.00383) Longmen Mountain (LMM) 107 (108) 7 (20) 0.572 (0.642) 0.00217 (0.00193) WGX Guixi,Beichan,SC wild 32°000/104°380 726–1014 47 (49) 3 (3) 0.162 (0.476) 0.00058 (0.00070) WTL Taolong,Beichan,SC wild 31°590/104°070 1445–1683 34 (33) 5 (18) 0.720 (0.867) 0.00299 (0.00409) WQC Qingchuan,Guangyuan,SC wild 32°240/104°480 1141–1264 26 (26) 4 (4) 0.662 (0.591) 0.00209 (0.00094) Qinba Mountain (QBM) 25 (25) 4 (4) 0.357 (0.630) 0.00111 (0.00106) LHZ Hanzhong,SX landrace 33°130/106°570 601 8 (8) 1 (3) 0.000 (0.607) 0.00000 (0.00112) LFP Foping,SX landrace 33°310/107°590 1847 4 (4) 1 (3) 0.000 (0.833) 0.00000 (0.00166) LTS Maiji,Tianshui,GS landrace 34°260/105°560 1190 4 (4) 1 (3) 0.000 (0.833) 0.00000 (0.00142) WSN Shirengou,Shangnan,SX wild 33°460/110°850 9 (9) 4 (2) 0.650 (0.500) 0.00223 (0.00071) North China Plain (NCP) 31 (32) 3 (5) 0.127 (0.492) 0.00028 (0.00174) LTH Taihe,AH landrace 39°540/116°250 36 6 (6) 1 (1) 0.000 (0.000) 0.00000 (0.00000) LLY Luoyang,HN landrace 35°080/112°390 206 4 (4) 2 (1) 0.500 (0.000) 0.00072 (0.00000) LZZ Zhengzhou,HN landrace 34°370/113°350 190 2 (2) 1 (1) 0.000 (0.000) 0.00000 (0.00142) LWF Weifang,SD landrace 36°130/119°040 14–212 19 (20) 2 (5) 0.105 (0.632) 0.00020 (0.00259) Yungui Plateau (YGP) 29 (29) 3 (6) 0.316 (0.594) 0.00183 (0.00341) LBJ Hezhang,Bijie,GZ landrace 27°180/105°190 1486 17 (17) 1 (2) 0.000 (0.118) 0.00000 (0.00017) LGY Wudang,Guiyang,GZ landrace 26°390/106°310 1200 7 (7) 3 (5) 0.667 (0.905) 0.00287 (0.00639) WGY Baihuanghu,Guiyang, GZ wild 26°390/106°310 1200 5 (5) 3 (4) 0.700 (0.900) 0.00344 (0.00626) landrace 140 (139) 7 (10) 0.218 (0.554) 0.00060 (0.00136) wild 164 (166) 14 (28) 0.557 (0.674) 0.00209 (0.00312) total 304 (305) 15 (30) 0.419 (0.621) 0.00152 (0.00233)
sequenced (Alvarez & Wendel 2003). Of which, only trnQ- and 90 s at 72 °C were performed in the thermal cycler PTC- rps16 intergenic spacer was chosen for higher amplification 200 (MJ Research). Finally, PCR products were directly quality and polymorphism extent. PCR amplifications and sequenced using the Big Dye Terminator Cycle Sequencing kit sequencing reactions of this region were performed with the (Applied Biosystems, Foster City, CA, USA) on an ABI PRISM primers trnQ: 50-GCG TGG CCA AGY GGT AAG GC-30 and 3730 automatic DNA sequencer (Applied Biosystems, Foster rps16: 50-GTT GCT TTY TAC CCT CAC ATC GTT T-30 (Shaw City, CA, USA) (Beijing Genomics Institute (BGI), Shenzhen). et al. 2007). The flanking ITS1 and ITS2 regions and 5.8S All sequences were deposited in the GenBank database under nrDNA were also amplified and sequenced with the primers accession numbers JQ082198-JQ082212 for trnQ-rps16 and ITS5: 50-GGA AGT AAA AGT CGT AAC AAG G-30 and ITS4: KF241102-KF241133 for the ITS region. 50-TCC TCC GCT ATA TGA TAT GC-30 (Huang et al. 2011). For sequences of low quality, PCR amplifications and sequenc- Data analysis ing reactions were repeated. Both the trnQ-rps16 and ITS fragments were amplified in a All individual consensus sequences were edited using DNAS- 25 ll reaction mixture, containing 20 ng genomic DNA, 2.0 ll TAR5.0 (DNASTAR Inc., Madison, WI, USA) and aligned in of 10 9 reaction buffer (10 mM Tris-HCl, pH 8.0, 50 mM KCl, CLUSTAL_X 1.81 (Thompson et al. 1997). Manual editing was 1.5 mM EDTA), 1.2 ll of MgCl2 (25 mM), 1.4 ll of dNTP mix also performed in Molecular Evolutionary Genetics Analysis (10 mM), 3.5 pM of each primer, and 1.5 U of Taq DNA poly- version 5.0 (MEGA5) (Tamura et al. 2011). Insertions or dele- merase (TIANGEN, Beijing, China). Following one cycle of tions (indels) in the trnQ-rps16 region were treated as substitu- 3 min at 94 °C, 30 PCR cycles of 45 s at 94 °C, 70 s at 53 °C, tions following Caicedo and Schaal (Caicedo & Schaal 2004).
Plant Biology 17 (2015) 787–797 © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands 789 Phylogeographic study of Prunus pseudocerasus Chen, Chen, Luo, Huang, Zhang, Tang, Pan & Wang
ITS pseudogenes were also detected by the presence of indels RESULTS and variation in 5.8S rDNA (Zheng et al. 2008). Molecular diversity indexes, such as number of segregating sites (S), hap- Patterns of variability and genetic diversity in cpDNA and lotype diversity (h) (Nei et al. 1983), nucleotide diversity (p) nrDNA (Jukes & Cantor 1969), and the mean number of pairwise The consensus sequence for the cpDNA regions in \differences (k) were calculated using DNAsp 5.1 (Librado & P. pseudocerasus (304 individuals) consisted of 753 base Rozas 2009). pairs (bp), varying from 693 to 739 bp. 15 haplotypes were Phylogeographic structure and gene genealogy among defined based on 14 polymorphic sites (1.86%), including haplotypes were assessed using median-joining method three substitutions and 11 indels. A 32 bp length indel was (Bandelt et al. 1999) with Network4.2.0.1 (Forster et al. observed between 447–480 bp (Table S1). Haplotype h1 2004) and the statistical parsimony criteria (Crandall et al. and h4 represented 87.0% of the sampled individuals 1994) with the program TCS (Clement et al. 2000). Bayes- (Table S2). Haplotype h1, as the most dominant haplotype ian Analysis of Population Structure version 5.4 (BAPS in the genetic composition of the species, was detected in 5.4) (Caicedo & Schaal 2004) and Neighbor-Joining (NJ) 226 total individuals. The mean haplotype and nucleotide cluster analysis were also employed, and robustness of each diversities were h = 0.410 and p = 1.72 9 10 3, respectively. internal branch in NJ tree was evaluated with 10,000 boot- Haplotype diversity (h) for each of the 26 populations ran- strap replicates (Turchetto-Zolet et al. 2012). The BAPS ged from 0 to 0.720 and nucleotide diversity (p) ranged method uses Markov Chain Monte Carlo (MCMC) simula- from 0 to 3.44 9 10 3. The highest levels of haplotype and tion to cluster different populations or individuals into nucleotide diversity were observed in the population WTL variable groups (K). The K with the highest marginal log- (h = 0.720, p = 2.99 9 10 3) and WQC (h = 0.662, likelihood represents the number of groups that best fits p = 2.09 9 10 3) from Longmen Mountain (LMM), fol- the data (Turchetto-Zolet et al. 2012). Ten algorithm repe- lowed by population WGY (h = 0.700, p = 3.44 9 10 3) titions for each K (1–20) were performed. and LGY (h = 0.667, p = 2.87 9 10 3) from the Yungui Spatial genetic structure of haplotypes was analyzed using Plateau (YGP). Other regions exhibited relatively lower lev- SAMOVA 1.0 (Caicedo & Schaal 2004) (http://web.unife.it/prog- els of haplotype and nucleotide diversity (Table 1). Haplo- etti/genetica/Isabelle/samova. html), and the number of initial type and nucleotide diversities of cpDNA in landrace conditions was set to 100 with 1000 permutations (Dupanloup populations (h = 0.218 and p = 0.6 9 10 3) were much et al. 2002). This program is based on a simulated annealing lower than those in wild populations (h = 0.557 and procedure that maximizes the proportion of the total genetic p = 2.09 9 10 3) (Table 1). variance caused by differences between groups of populations The aligned sequences of the ITS (ITS1 + 5.8S + ITS2) in (F ). It defines groups of populations that are geographically CT 305 samples were 703 bp in length, comprising 227 bp for homogeneous and maximally differentiated from each other. ITS1 and 276 bp for ITS2. All these sequences presented The optimal K is characterized by the highest F defining the CT only one type label without length variation and limited number of groups. Analysis of molecular variance (AMOVA) was heterozygous loci in three regions (ITS1, 5.8S and ITS2). also performed to calculate the patterns of isolation using pair- 32 haplotypes (H1-H32) were identified based on 34 poly- wise F comparisons among populations and geographic ST morphic sites (all substitutions, without indels). The most regions. All the populations were partitioned by taxonomic frequent haplotype H5 (observed 167 times, in 54.75% of varieties (landrace and wild populations), SAMOVA groups and individuals) and H6 (observed 86 times, 28.20%) were geographic regions. The partitions of genetic diversity within observed in 26 and 17 populations, respectively. Each and among populations were analyzed using Arlequin version remaining haplotypes were observed in less than five popu- 3.5.1.2 (Excoffier & Lischer 2010). lations (Fig. 1C and Table S4). Total haplotype and nucleo- Permut version 1.0 (http://www.pierroton.inra.fr/genetics/ tide diversities were h = 0.621 and p = 2.33 9 10 3. labo/Software/ PermutCpSSR/index.html) (Pons & Petit 1996) Haplotype diversity (h) for each of the 26 populations ran- was used to test for phylogeographic structure by comparing ged from 0 to 0.905 and nucleotide diversity (p) ranged G and N , with l0,000 permutations. G considers haplo- ST ST ST from 0 to 6.39 9 10 3. The highest levels of haplotype and type frequencies, while N considers sequence differences ST nucleotide diversity were found in population WGY between haplotypes. An N value that is significantly larger ST (h = 0.900, p = 6.26 9 10 3) and LGY (h = 0.905, than the G value indicates phylogeographic structure among ST p = 6.39 9 10 3) from YGP, followed by WTL (h = 0.867, populations (Turchetto-Zolet et al. 2012). p = 4.09 9 10 3) from LMM (Table 1). Neutrality tests and mismatch distributions were analyzed to Comprehensive analysis of the data from cpDNA and detect possible population expansion events. Under the nrDNA revealed that the LMM region exhibited the highest assumption of neutrality, a population expansion produces a levels of haplotype (h = 0.572 in cpDNA and 0.642 in nrDNA) large significantly negative value of Tajima’s D (Tajima 1989), and nucleotide diversity (p = 2.17 9 10 3 in cpDNA and Fu and Li’s D* and Fu and Li’s F* (Aris-Brosou & Excoffier 1.93 9 10 3 in nrDNA). Moreover, chloro-type h1 and rDNA- 1996; Fu & Li 1993;Tajima 1996), as well as a unimodal mis- type H5 were found in all 26 populations, comprising of 229 match distribution curve. All these tests were performed using and 167 individuals, respectively, followed by chloro-type h4 DNAsp 5.1 (Librado & Rozas 2009), and a total of nine groups and rDNA-type H6. However, chloro-type h4 is absent in the were considered, including the whole samples, two taxonomic North China Plain (NCP) and Daliangshan Mountain (DLSM) varieties (landrace and wild), and six different regions (Table 1). (Fig. 1A).
790 Plant Biology 17 (2015) 787–797 © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands Chen, Chen, Luo, Huang, Zhang, Tang, Pan & Wang Phylogeographic study of Prunus pseudocerasus
A C
B D
Fig. 2. Haplotypes median-joining network of P. pseudocerasus For 32 nrDNA in 26 populations (A) and in six geographic regions (C), for 15 cpDNA haplo- types in 26 populations (B) and in six geographic regions (D). Circle size is proportional to the haplotype frequencies, and colors represent the different popula- tions, as shown in the key. The circles on the network delimit the three lineages for nrDNA and two lineages for cpDNA respectively identified using Bayesian Analysis Population Structure (BAPS) analysis.
AB
Fig. 3. Population structure and group clusters by the Neighbor-Joining (NJ) method of P. pseudocerasus For 15 cpDNA haplotypes (A) and 32 nrDNA haplo- types (B). Different clusters are distributed across each population. Colors represent the different clusters, as shown in the key.
haplotypes were found in LMM region, indicating the extraor- Gene genealogies and population structure dinary roles of this area in the evolution of the species (Fig. 2). Two different network approaches were applied to estimate According to the BAPS and NJ tree, cpDNA and nrDNA gene genealogy among the cpDNA and nrDNA haplotypes. were optimally partitioned into two and three genetically struc- Similar gene genealogies were obtained by both methods, and tured groups, respectively (Fig. 2 and Fig. 3). SAMOVA was only the median-joining network is shown in Fig. 2. The haplo- applied to define groups and identify locations of genetic type networks of both nrDNA (Fig. 2A,C) and cpDNA uniqueness among the 26 populations from six geographic (Fig. 2B,D) presented a shallow gene tree. In nrDNA, the hap- regions (Table 2). For cpDNA, a model with three groups lotype H5 occurred in all geographic populations, and nested (K = 3) displayed the expected largest value of FCT and maxi- as the interior node. In addition, most exterior haplotypes were mal variance (FCT = 0.40822, P = 0.000). Populations from observed in populations from LMM (Fig. 2A,C). In cpDNA YGP formed one group (LGY and WGY), populations from network, haplotype h1 was nested and widespread in all 26 Sichuan Basin (SCB), LMM and Qinba Mountain (QBM) populations from six regions. Haplotype h4, however, shown formed another (WTL, WQC, LFL, and WSN), and the last as the interior node, was nested across ten populations that group was composed of all other regions. In nrDNA, the largest were mostly restricted to LMM (Fig. 2B,D). Taken together, FCT value and a statistically maximal among-group variance seven out of 15 cpDNA haplotypes and 20 out of 32 nrDNA were observed when K = 2(FCT = 0.81549, P = 0.0319). Popu-
Plant Biology 17 (2015) 787–797 © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands 791 Phylogeographic study of Prunus pseudocerasus Chen, Chen, Luo, Huang, Zhang, Tang, Pan & Wang
Table 2. Fixation indices corresponding to groups of populations inferred by SAMOVA for P. pseudocerasus using cpDNA and nrDNA. marker group population groupings FCT P-value cpDNA 2 (LYA,LXC,LPJ,LJY,LSM,LMS,LHY,LFL,LHZ,LFP,LTH,LGY,LBJ,LLY,LZZ,LTS,LWF,WXC,WEM, 0.37642 0.00196 WGY,WSN,WSM,WYC,WGX)(WTL,WQC) 3 (LYA,LXC,LPJ,LJY,LSM,LMS,LHY,LHZ,LFP,LTH,LBJ,LLY,LZZ,LTS,LWF,WXC,WEM,WSM,WYC, 0.40822 0.00000 WGX)(WTL,WQC,LFL,WSN)(WGY,LGY) 4 (LYA,LXC,LPJ,LJY,LSM,LMS,LHY,LHZ,LFP,LTH,LBJ,LLY,LZZ,LTS,LWF,WXC,WEM,WSM,WYC, 0.40000 0.00000 WGX)(WTL,WQC)(LFL,WSN)(WGY,LGY) 5 (LYA,LXC,LPJ,LJY,LSM,LMS,LHY,LHZ,LFP,LTH,LBJ,LLY,LZZ,LTS,LWF,WXC,WEM,WSM,WYC, 0.40472 0.00000 WGX)(WTL)(WQC)(LFL,WSN)(WGY,LGY) 6 (LYA,LXC,LPJ,LJY,LSM,LMS,LHY,LHZ,LFP,LTH,LBJ,LLY,LZZ,LTS,LWF,WXC,WEM,WGX)(WTL, 0.37633 0.00000 WQC)(LFL,WSN)(WGY,LGY)(WSM)(WYC) 7 (LYA,LXC,LPJ,LJY,LSM,LMS,LHY,LHZ,LFP,LTH,LBJ,LZZ,LTS,LWF,WXC,WEM,WGX)(WTL, 0.36705 0.00000 WQC)(LFL,WSN)(WGY,LGY)(LSM)(LMS)(LLY) nrDNA 2 (LYA,LXC,LPJ,LJY,LSM,LMS,LHY,LFL,LHZ,LFP,LTH,LGY,LBJ,LLY,LZZ,LTS,LWF,WEM,WGY, 0.81549 0.03910 WSN,WSM,WTL,WQC,WYC,WGX)(WXC) 3 (LYA,LXC,LPJ,LJY,LSM,LMS,LHY,LFL,LHZ,LFP,LTH,LGY,LBJ,LLY,LZZ,LTS,LWF,WEM,WGY, 0.57356 0.07136 WSN,WSM,WTL,WQC,WGX)(WXC)(WYC) 4 (LYA,LXC,LPJ,LJY,LSM,LMS,LHY,LFL,LHZ,LFP,LTH,LGY,LBJ,LLY,LZZ,LTS,LWF,WEM,WSN, 0.65097 0.02444 WTL,WQC,WYC,WGX)(WXC)(WSM)(WGY) 5 (LYA,LXC,LPJ,LSM,LMS,LHY,LFL,LHZ,LFP,LTH,LBJ,LLY,LZZ,LTS,LWF,WEM,WSN,WSM,WTL, 0.68382 0.00098 WQC,WYC,WGX)(WXC)(LGY)(WGY)(LJY) 6 (LXC,LPJ,LJY,LSM,LMS,LHY,LFL,LHZ,LTH,LGY,LBJ,LTS,LWF,WEM,WGY,WSN,WSM,WTL, 0.51493 0.05083 WQC,WYC,WGX)(WXC)(LLY)(LZZ)(LFP)(LYA) 7 (LYA,LXC,LPJ,LJY,LSM,LMS,LHY,LFL,LHZ,LFP,LTH,LGY,LTS,LWF,WSN,WSM,WTL,WQC, 0.56853 0.00196 WYC,WGX)(WXC)(LLY)(LZZ)(WEM)(LBJ)(WGY)
lations from DLSM formed one group (WXC), and popula- structure. By contrast, for cpDNA, the GST value (0.215, tions from the rest formed another. The remaining groups dis- P = 0.08) was not significantly different from the NST value played a relatively smaller FCT value in both cpDNA and (0.256, P = 0.15). nrDNA (Table 2). AMOVA analysis revealed 24.92% and 31.57% (P < 0.0001) of Demographic history the total variation among populations for cpDNA and nrDNA, respectively (Table 3). When populations were grouped by two Neutrality tests for nrDNA revealed significant negative values taxonomic varieties (landrace and wild), the cpDNA showed in all samples and populations from DLSM and SCB. While low levels of variation between landrace and wild resources for populations from NCP and landrace, significant negative (17.86%, P < 0.0001; Table 3). For nrDNA, most of the values of neutrality tests were detected in both markers variation (69.33%) was revealed within populations, and only (Table 4). The observed mismatch distributions were multi- 0.28% was attributed to differences between the two varieties modal in all six geographic areas as well as in all considering (P < 0.0001; Table 3). When the populations were partitioned populations except the NCP. As for the two taxonomic varie- according to the SAMOVA groups, most of the variation was ties, the mismatch distributions were unimodal for landrace observed among groups (40.82% and 81.55% in cpDNA and populations in both cpNDA and nrDNA haplotypes nrDNA, respectively). Only 0.45% (cpDNA) and 2.39% (Table 4). (nrDNA) of the variance (P < 0.0001; Table 3) was revealed among populations within groups. When the populations were grouped based on geographic regions, most of the variation DISCUSSION was observed within populations (74.76% and 67.56% in ITS polymorphism and its utility in P. pseudocerasus cpDNA and nrDNA, respectively) (Table 3). No matter how groups were defined, based on natural populations, geographic The application of ITS region for phylogeographic patterns regions, or varieties (wild and landrace), the overwhelming and population structure analysis often depends on the absence majority of the variation was observed within populations of intra-individual polymorphism and putative pseudogenes (Table 3). In addition, pairwise Fst values among populations (Yang et al. 2012; Zheng et al. 2008). In the present study, only ranged from 0 to 0.616 for cpDNA and 0 to 0.927 for nrDNA one ITS homologous copy was found within individuals, when (Fig. 4A and Table S5). Among geographic regions, Fst values the flanking ITS1 and ITS2 regions and 5.8S nrDNA were ranged from 0 to 0.196 (cpDNA) and 0.001 to 0.416 (nrDNA) amplified and directly sequenced, implying the homogeneity of respectively (Fig. 4B and Table S6). Non-significant pairwise nrDNA arrays within individuals and the deficiency of intra- Fst values were detected for the majority of natural populations individual ITS polymorphism. This might be the result of or regions. The GST value (0.146, P = 0.03) estimated for the concerted evolution of the ITS locus that eliminated sequence nrDNA data was significantly smaller than the NST value variation among homeologous copies in the tetraploid species (0.342, P = 0.04), indicating a meaningful phylogeographic (Feliner & Rossello 2007). No length variation in the consensus
792 Plant Biology 17 (2015) 787–797 © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands Chen, Chen, Luo, Huang, Zhang, Tang, Pan & Wang Phylogeographic study of Prunus pseudocerasus
Table 3. AMOVA for the 26 P. pseudocerasus populations using nrDNA and cpDNA sequences.
sum. of variance variation source of variation d.f squares components percentage fixation indices (P-value) cpDNA (all locations)
among populations 25 47.469 0.13251 24.92 FST = 0.24922 (0.000) within populations 278 110.972 0.39918 75.08 cpDNA (varieties)
among varieties 1 11.56 0.06085 10.87 FCT = 0.10865 (0.046)
among populations with in varieties 24 35.909 0.10002 17.86 FST = 0.28724 (0.000)
within populations 278 110.972 0.39918 71.28 FSC = 0.20036 (0.000) cpDNA (SAMOVA groups)
among groups 2 37.512 0.27748 40.82 FCT = 0.40822 (0.000)
among populations with in groups 23 9.957 0.00308 0.45 FST = 0.41275 (0.000)
within populations 278 110.972 0.39918 58.37 FSC = 0.00765 (0.000) cpDNA (geographic regions)
among regions 5 13.833 0.01198 2.24 FCT = 0.02243 (0.449)
among populations with in regions 20 33.635 0.12276 22.99 FST = 0.25236 (0.000)
within populations 278 110.972 0.39918 74.76 FSC = 0.23520 (0.000) nrDNA (all locations)
among populations 25 88.733 0.2627 31.57 FST = 0.31571 (0.000) within populations 279 158.864 0.5694 68.43 nrDNA (varieties)
among varieties 1 2.496 0.02329 0.28 FCT = 0.00284 (0.574)
among populations with in varieties 24 86.237 0.27512 33.5 FST = 0.30665 (0.000)
within populations 179 158.864 0.5694 69.33 FSC = 0.32577 (0.000) nrDNA (SAMOVA groups)
among groups 1 51.859 2.89065 81.55 FCT = 0.81549 (0.042)
among populations with in groups 24 36.847 0.08464 2.39 FST = 0.83936 (0.000)
within populations 279 158.864 0.5694 16.06 FSC = 0.12941 (0.000) nrDNA (geographic regions)
among regions 5 32.147 0.05731 6.8 FCT = 0.06800 (0.108)
among populations with in regions 20 56.586 0.2161 25.64 FST = 0.32440 (0.000)
within populations 279 158.864 0.5694 67.56 FSC = 0.27511 (0.000)
AB
Fig. 4. Distance matrix of pairwise FST values calculated by cpDNA (below diagonal) and nrDNA (above diagonal) of P. pseudocerasus Among 26 natural popu- lations (A) and six geographic regions (B). sequences (703 bp among 305 individuals) also implied the investigating phylogeographic patterns and demographic absence of potential non-functional nrDNA copies (pseudoge- history of this species. nes) among the polymorphic individuals. Furthermore, rela- tively large number of polymorphic sites (34 substitutions) and Patterns of genetic diversity haplotypes (32 haplotypes) in the consensus sequence of ITS region indicated the relatively higher genetic variation in In spite of the large collection covering most distribution areas nrDNA, which partitioned the populations into three genetic of the species and rich resources from both landrace and wild groups. All these results suggest the validity of ITS sequence in varieties (17 landrace and nine wild populations), relatively
Plant Biology 17 (2015) 787–797 © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands 793 Phylogeographic study of Prunus pseudocerasus Chen, Chen, Luo, Huang, Zhang, Tang, Pan & Wang
Table 4. Summary of demographic expansion tests in this study. parameter DLSM SCB LMM QBM NCP YGP landrace wild all populations cpDNA Tajima’sD 1.16240 1.27482 0.31851 0.72953 1.73075 0.61183 1.77764* 1.05338 1.35708 Fu & Li (1993)D* 1.57469 0.88967 0.30805 0.87697 2.71050* 1.14719 1.85957 2.17489 1.03925 Fu & Li (1993)F* 1.67803 1.21059 0.36879 0.96618 2.81260* 0.73181 2.17892 2.10159 1.40195 mismatch distribution multimodal multimodal multimodal multimodal unimodal multimodal unimodal multimodal multimodal nrDNA Tajima’sD 2.55632** 2.02174* 1.67469 0.14952 2.0272* 0.47102 1.99716* 1.68418 2.01216* Fu & Li (1993)D* 1.33993 3.70406* 2.21216 0.20284 2.9994* 0.94312 3.71951* 2.51511* 3.71640* Fu & Li (1993)F* 1.95715* 3.68274* 2.40296* 0.21699 3.1606* 0.59469 3.66469* 2.61460* 3.58642* mismatch distribution multimodal multimodal multimodal multimodal unimodal multimodal unimodal multimodal multimodal
Significant values are indicated in bold (*P < 0.05; **P < 0.01). lower levels of haplotype and nucleotide variation for both However, the genetic diversity was not randomly distributed trnQ-rps16 intergenic region (h = 0.419) and ITS (h = 0.621) in space for both cpDNA and nrDNA, and different genetic were observed (Table 1) in comparing to other plants, such as diversity patterns were also observed across the sampling areas. Rhododendron pseudochrysanthum (Huang et al. 2011), Schizol- High levels of genetic diversity as well as many divergent haplo- obium parahyba (Turchetto-Zolet et al. 2012) and Scutellaria types were observed in populations from LMM (WTL and baicalensis (Yuan et al. 2010), as well as the mean cpDNA WQC populations) and YGP region (LGY and WGY popula- diversity (h = 0.67) (Petit et al. 2005). Moreover, much lower tions) (Table 1 and Fig. 1, Fig. 2B and Fig. 3B). These results levels of haplotype and nucleotide diversity were identified in suggested the presence of two potential genetic diversity centers landrace populations (h = 0.218 for cpDNA and 0.554 for of P. pseudocerasus, one in the LMM region and the other in nrDNA) than those in wild populations (h = 0.557 for cpDNA the YGP region. Conversely, low genetic diversity levels were and 0.674 for nrDNA). observed in the populations from the other four areas, espe- Theoretically, the level of genetic variability in populations cially DLSM and NCP region. Whereas populations from the tends to be positively correlated with the effective population DLSM shared the haplotypes h1 (cpDNA) and H26 (nrDNA), size (Nybom 2004). While in Chinese Cherry, selfing is a pre- and populations from NCP region consisted of landraces. dominant mating system (Huang et al. 2008; Yu et al. 2010), which may increase homozygosity but reduce effective popula- Phylogeographic structure and demographic patterns tion size and genetic diversity (Gl emin et al. 2006; Nybom 2004). Furthermore, perennial fruit crops such as P. pseudocer- In the present study, the limited population differentiation and asus, with long lifespan and strong vegetative propagation weak phylogeographic patterns among the six eco-climate habit, often possess a relatively smaller effective population size niche (six geographic regions) were verified, for the majority of than sexually reproducing grain food crops and annual fruit nucleotide diversity of cpNDA (74.76%, P < 0.001) and crops (Cornille et al. 2012; Meyer & Purugganan 2013; Miller nrDNA (67.56%, P < 0.001) were significantly attributed to the & Gross 2011; Qi et al. 2013). Therefore the combined effects variation within populations (Table 2). The variations among of these botanic characteristics might impact the effective pop- regions were only 2.24% and 6.8% for cpDNA and nrDNA, ulation size and finally lead to the lower level of genetic diver- respectively. These results suggested that gene flow among dif- sity of this species. ferent locations was not fragmented by large distance. The geo- The history of domestication and selection may play a major graphic features and climatic factors did not appear to be role in reducing the effective population size, causing a strong critical for this species’ demographic patterns. Similar results bottleneck effect in the landrace populations (Gl emin & Batail- was also reported in other Prunus species based on different lon 2009; Wright et al. 2005). A dramatic reduction of genetic markers, such as P. mahaleb (Garcia et al. 2007), P. avium diversity in landraces was likely due to the selective propagation (Mariette et al. 2010), P. jamasakura (Tsuda et al. 2009), and of some individuals with excellent characters (e.g. larger fruit P. lannesiana (Kato et al. 2011). As for P. pseudocerasus and size, high yield or good taste) in a cultivated setting. Moreover, other Prunus species, the moderate degree of phylogeographic historical processes such as a founder effect after dispersal and patterns was likely attributed to the efficient gene flow through subsequent demographic expansion also given rise to the pres- seed dispersal system, since the seeds of these species could be ence of fixed haplotypes and the lower levels of genetic diversity dispersed for a long distance by birds and other animals (Tsuda in landrace populations. Since the observed unimodal mis- et al. 2009; Kato et al. 2011). match distribution and significant negative values of neutrality Furthermore, long generation times and overlapping genera- tests were revealed in landrace populations for both cpDNA tions were expected to be served as a possible driver for the and nrDNA haplotypes (Table 4), which suggested that the phylogeographic patterns of long-lived woody plants (Morris probable population expansion of landrace were derived from a et al. 2010; Petit et al. 2005). The short history of evolution and more recent establishment. In addition, the clonal propagation, low mutation rate probably acted as the consequences of the long lifespan might also maximize the impact of this founder’s long generation time of these species (Valtuena~ et al. 2012). effect in landrace populations (Miller & Gross 2011). For P. pseudocerasus, combined effects from these two factors
794 Plant Biology 17 (2015) 787–797 © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands Chen, Chen, Luo, Huang, Zhang, Tang, Pan & Wang Phylogeographic study of Prunus pseudocerasus on cpDNA and nrDNA might propose an assumption on what mismatch distribution in NCP region and landrace populations cause the low evolutionary rate and the short evolutionary his- for both cpDNA and nrDNA haplotypes (Table 4). These tory for isolating these populations. This might be another rea- results showed a recent population expansion in landrace pop- son for the low level of population differentiation and weak ulations. It might attribute to long time of domestication, culti- phylogeographic patterns, resulting in the sharing of ancestral vation and artificial selection. Most individuals with excellent genetic polymorphisms. characters were selected from wild populations and propagated Based on sequences variation among P. pseudocerasus popu- (Table 4). For the populations from other regions, the observed lations, low levels of genetic differentiation both in cpDNA mismatch distributions were multimodal for both cpDNA and (GST = 0.215, NST = 0.256) and nrDNA (GST = 0.146, nrDNA haplotypes, revealed the presence of stable population NST = 0.342) were detected, however, a significant phylogeo- in the most regions. graphic structure was verified for nrDNA (GST
Plant Biology 17 (2015) 787–797 © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands 795 Phylogeographic study of Prunus pseudocerasus Chen, Chen, Luo, Huang, Zhang, Tang, Pan & Wang
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