植 物 分 类 与 资 源 学 报 ꎬ36 ( ): 2014 2 209~218 Plant Diversity and Resources : DOI 10.7677/ynzwyj201413085 珍稀濒危植物硬叶兜兰的遗传多样性及遗传结构研究 ∗ 黄家林1ꎬ2ꎬ 李树云1ꎬ 胡 虹1∗∗ 中国科学院昆明植物研究所 云南昆明 中国科学院大学 北京 (1 ꎬ 650201ꎻ2 ꎬ 100049) 摘要: 由于人为采集 走私贩卖以及生境的破坏 分布于中国西南石灰岩地区的野生硬叶兜兰居群受到严 、 ꎬ 重的干扰与威胁 为有效地保护这种珍稀野生植物 本研究采用 和 两种分子标记对 个硬叶 ꎮ ꎬ ISSR SRAP 15 兜兰野生居群进行遗传多样性及遗传结构的研究 结果表明 硬叶兜兰在物种水平上具有较高的遗传多样 ꎮ ꎬ 性 H H 硬叶兜兰居群间存在一定程 (ISSR: PPB=9166%ꎬ e=03839ꎻ SRAP: PPB=9929%ꎬ e=02806)ꎮ 度的遗传分化 G G 可能由于较低的基因流 Nm (ISSR: st=02577ꎻ SRAP: st=02383)ꎬ (ISSR: =07201ꎻ Nm 所致 聚类分析以及主成分分析均把 个居群分成 个主要分支 居群间 SRAP: =07991) ꎮ UPGMA 15 2 ꎮ 的地理距离和海拔差距是引起居群遗传分化的自然因素 ꎮ 关键词: 资源保护 分子标记 遗传多样性 遗传分化 硬叶兜兰 ꎻ ꎻ ꎻ ꎻ 中图分类号: 文献标识码: 文章编号: Q16 A 2095-0845(2014)02-209-10 ISSR and SRAP Markers Reveal Genetic Diversity and Population Structure of an Endangered Slipper Orchid Paphiopedilum ꎬ micranthum Orchidaceae ( ) 1ꎬ2 1 1∗∗ HUANG Jia ̄Lin ꎬ LI Shu ̄Yun ꎬ HU Hong KeyLaboratoryofEconomicPlantsandBiotechnology KunmingInstituteofBotany ChineseAcademyofSciences (1 ꎬ ꎬ ꎬ UniversityofChineseAcademyofSciences Kunming650201ꎬChinaꎻ2 ꎬBeijing100049ꎬChina) Abstract Paphiopedilummicranthum : isanendangeredpinkslipperorchidmainlydistributedinthelimestoneareas of southwestern China. Wildpopulationsofthisspecieshavebeenseriouslythreatenedbyexcessivecollectionsꎬram ̄ pant smuggling for exportꎬ and habitat destruction. We used 15 ISSR markers and 11 SRAP markers to investigate the genetic diversity and structure of15 natural populations. A high degree of diversity was observed at the species H H level (ISSR:PPB=9166%ꎬ e=03839ꎻSRAP:PPB=9929%ꎬ e=02806). Certaindegreeofgeneticdiffer ̄ G G entiation among populations (ISSR: st=02577ꎻ SRAP: st=02383) was detected maybe caused by low gene Nm Nm flow(ISSR: =07201ꎻSRAP: =07991). ConsistentwiththeresultsofPrincipalCoordinateAnalysisꎬthe UPGMA dendrogram analysis divided the 15 populations into two main clades. In addition to geographic distanceꎬ the difference in elevation was another natural factor contributing to this differentiation. Knowledge about genetic di ̄ versityandstructuregainedfromourstudywillbebeneficialforthedevelopmentofreasonableandefficientstrategies to conserve this endangered species. Key words Paphiopedilum micranthum : Conservationꎻ DNA markersꎻ Genetic diversityꎻ Genetic differentiationꎻ Paphiopedilum The genus Pfitz.ꎬ a primitive group within Orchidaceaeꎬ is distributed from southern China ∗Funding:TheBasicResearchProgramofYunnanProvince(2007C001Z) andtheLarge ̄ScaleScientificFacilitiesoftheChineseAcademy ofSciences(2009 ̄LSF ̄GBOWS ̄01) ∗∗AuthorforcorrespondenceꎻE ̄mail:huhong@mailkibaccn Receiveddate: 2013-04-11ꎬ Accepteddate: 2013-09-04 作者简介 黄家林 男 助理研究员 主要从事兰科植物的资源保护与开发利用 : (1974-) ꎬ ꎬ ꎮ E ̄mail:huangjialin@mailkibaccn 植 物 分 类 与 资 源 学 报 第 卷 210 36 al et al to New Guinea in tropical and subtropical Asia (Liu .ꎬ 2007)ꎬ and breeding strategies (Chen .ꎬ et al et al .ꎬ 2009). These slipper orchids are named for 2004ꎻ Liao .ꎬ 2011ꎻ Chung and Choiꎬ 2012). the shape of the deeply saccate lip of their flowers. Clarifying genetic diversity and population Growers prize them for their very great ornamental structure of extant populations not only provides in ̄ valueꎬ particularly because these small plants have sights into the evolutionary and demographic history et al spectacularꎬ large flowers (Cribbꎬ 1998). More of threatened species (Hamrick .ꎬ 1982ꎻ Ham ̄ than 20 000 slipper orchid hybrid grexes have been rick and Godtꎬ1990)ꎬ but also facilitates the design registeredꎬ demonstrating their remarkable rise in of effective conservation and management strategies et al. et al popularity (Zeng ꎬ 2010). Provinces in south ̄ (Jian .ꎬ 2006). Howeverꎬ only a few studies western Chinaꎬ including southeastern Yunnanꎬ have been conducted because of the difficulty associ ̄ northern and western Guangxiꎬ and southwestern ated with collecting population samples for slipper Guizhouꎬ are known as diversity and evolutionary orchidsꎬ including one that used random amplified et al et hotspots for this genus (Luo .ꎬ 2003ꎻ Liu polymorphic DNA (RAPD) markers to examine the al .ꎬ 2010). There are 25 of 80 species grow on lime ̄ genetic diversity within four naturally distributed P micranthum et al stone hills in that region. In addition to threats from populations (Li .ꎬ 2002b). rapid habitat destruction associated with faster eco ̄ Inter ̄simple sequence repeats (ISSR) and se ̄ nomic growth and rural development during the past quence ̄related amplified polymorphism (SRAP) are three decadesꎬ wild slipper orchids are facing de ̄ relatively simple and highly reproducible marker structive collecting pressuresdue to large horticultural technologies that do not rely on prior information a ̄ et al and commercial demandsin China (Luo .ꎬ2003ꎻ bout a DNA sequenceꎬ and which require very little et al Paphiopedi ̄ et al Liu .ꎬ 2009). This vulnerability by starting DNA template (Zietkiewicz .ꎬ 1994ꎻ Li lum species to environmental change can be also and Quirosꎬ 2001). These two systems have proven traced to the limited distribution of many species in to be powerful and efficient techniques for analyses the wild and their occurrence in populationsthat often of population genetics diversityꎬ molecular taxonomic contain only a few plants each (Cribb and McGoughꎬ classificationꎬ and marker ̄assisted breeding in many et al 1997). It is urgent to take proper strategies to con ̄ orchid species (Smith .ꎬ2002ꎻ Wallaceꎬ2003ꎻ et al et al et serve these endangered species. Ding .ꎬ 2008ꎻ George .ꎬ 2009ꎻ Wang Paphiopedilum micranthum al et al is restricted to the .ꎬ 2009ꎻ Cai .ꎬ 2011). We thus apply these limestone hills of southwestern China. In the early two markers to investigate the genetic diversity and P micranthum 1980sꎬ plants were exported from China and deserv ̄ population structure of in southwest ̄ edly received First Class Certificates from the Ameri ̄ ern China. Our main goals were to 1) determine the can Orchid Societyꎬ the Royal Horticultural Societyꎬ level of genetic variation within and among popula ̄ and many other awarding groups because these beau ̄ tionsꎬ 2) characterize the extent of genetic differen ̄ tiful plants have pink flowers that are larger than tiation between populationsꎬ and 3) identify the those previously known (Cribbꎬ 1998). Since thenꎬ causes for this observed differentiation. this species has been cultivated in large quantities et 1 Materials and methods while wild populations have declined sharply (Li al P micranthum 11 Sampling and plant material collection .ꎬ 2002b). To preserve and exploit Paphiopedilum and its allied speciesꎬ multiple studies have been We collected 407 individuals of micranthum carried out including the effects of conservation ef ̄ from 15 wild populations in three prov ̄ et al forts (Liu .ꎬ 2004ꎻ 2006)ꎬ physiological ecolo ̄ inces of southwestern China (Fig1ꎻ Table 1). Be ̄ et al et gy (Chang .ꎬ2011)ꎬ pollination biology (Shi cause this species is rhizomatous and forms clumps 期 et al 2 HUANG Jia ̄Lin .: ISSR and SRAP Markers Reveal Genetic Diversity and Population Structure of 2 11 et al of multiple plants in the wild (Tsi .ꎬ1999)ꎬ we SR reaction mixture (20 μL) contained 50 ng of tried to avoid collecting ramets of only a single geno ̄ template DNAꎬ 8 μL of 2×Taq PCR Master Mix -1 type by randomly selecting individuals that were (01 U of Taq polymerase per μLꎬ 05 mmolL -1 -1 spaced at least3 m apart. Young leaves were harves ̄ dNTPꎬ 20mmolL Tris ̄HClꎬ 100 mmolL KClꎬ -1 ted and dried by silica gel for further DNA isolation. and 3mmolL MgCl2)ꎬ plus 2% formamideꎬ 100 -1 The genomic DNA of each sample was extracted from nmolL primerꎬ and double ̄distilled water. The leaves by the standard CTAB (cetyltrimethyl ammo ̄ PCR program included an initial denaturation at 94℃ nium bromide) method of Doyle and Doyle (1987). for 5minꎻ then40 cycles of94℃ for45sꎬ53℃ for 1minꎬ and 72℃ for 2 minꎻ followed by a final ex ̄ tension at 72 ℃ for 7 min. The ISSR ̄PCR products were separated in 20% agarose gels buffered with 05×TBE. A 100bp DNA ladder (Fermentas) was used as a size marker. After staining with ethidium bromideꎬthe DNAfragmentswere identified by image analysis software for gel documentation (Lab Works Softwareꎬ version 30ꎻ UVPꎬ Uplandꎬ CAꎬ USA). 13 SRAP fingerprinting The SRAP marker analysis was performed as de ̄ Paphiopedilum Fig1 Samplingsitesforwildpopulationsof scribed by Li and Quiros (2001). From 88 arbitrary micranthum insouthwesternChina. Population primer pairsꎬ we selected 11 combinations that showed codesareexplainedwithTable1 distinct polymorphism(Table2).Each SRAP reaction 12 ISSR fingerprinting mixture (20μL) contained 40ng of template DNAꎬ -1 The sequences for our ISSR primers were pro ̄ 8μL of 2 × Taq PCR Master Mixꎬ 100 nmolL vided by the Biotechnology Laboratoryꎬ University of each for forward and reverse primersꎬ and double ̄ British Columbiaꎬ Canada. There were 15 (Table 2) distilled water. Amplification was performed under the from 90 arbitrary primers showing good repetitionꎬ following conditions:94℃/3minꎻfive cycles of 94℃ special bandsꎬ and distinct polymorphism. Each IS ̄ /1minꎬ 35℃/1minꎬ 72℃/1minꎻ then 30 cycles Paphiopedilummicranthum Table1 Samplinginformationfor15 populations Populationcode Location Numberofsamples Elevation/m Longitude(E) Latitude(N) YW WenshanꎬYunnan 36 1550 104°18′42″ 23°10′22″ YG GuangnanꎬYunnan 36 1530 105°04′15″ 24°11′31″ YT TianbaoꎬYunnan 24 1350 104°44′26″ 23°03′41″ YX XichouꎬYunnan 27 1540 104°31′51″ 23°20′13″ YF FadouꎬYunnan 29 1580 104°45′19″ 23°24′11″ YJ JinchangꎬYunnan 24 1490 104°49′17″ 23°07′47″ YM MaguanꎬYunnan 21 1438 104°22′20″ 22°56′48″ XL LeyeꎬGuangxi 23 1020 106°21′14″ 24°48′54″ XN NapoꎬGuangxi 24 1077 105°57′21″ 23°24′29″ XY LinyunꎬGuangxi 19 1050 106°44′13″ 24°34′14″ GC CehengꎬGuizhou 29 1273 105°39′59″ 24°59′22″ GN NidangꎬGuizhou 29 1479 104°48′24″ 24°49′49″ GW WangmoꎬGuizhou 28 1274 106°23′12″ 25°14′54″ GZ ZhaojiaduiꎬGuizhou 30 1375 105°00′23″ 25°01′57″ GB BajieꎬGuizhou 28 1182 104°59′41″ 24°54′39″ 植 物 分 类 与 资 源 学 报 第 卷 212 36 Table2 Sequencesof15ISSRprimersand11SRAP Primers ISSRprimers Sequences SRAP primercombinations Forwardprimer Reverseprimer UBC811 (GA)8C Me1 ̄Em9 TGAGTCCAAACCGGATA GACTGCGTACGAATTCGA UBC814 (CT)8A Me2 ̄Em3 TGAGTCCAAACCGGAGC GACTGCGTACGAATTGAC UBC815 (GA)8G Me3 ̄Em11 TGAGTCCAAACCGGAAT GACTGCGTACGAATTCCA UBC822 (TC)8A Me5 ̄Em2 TGAGTCCAAACCGGAAG GACTGCGTACGAATTTGC UBC823 (TC)8C Me5 ̄Em5 TGAGTCCAAACCGGAAG GACTGCGTACGAATTAAC UBC825 (AC)8T Me5 ̄Em7 TGAGTCCAAACCGGAAG GACTGCGTACGAATTCAA UBC827 (TC)8G Me5 ̄Em9 TGAGTCCAAACCGGAAG GACTGCGTACGAATTCGA UBC834 (AG)8YT Me6 ̄Em3 TGAGTCCAAACCGGTAA GACTGCGTACGAATTGAC UBC835 (AG)8YC Me6 ̄Em4 TGAGTCCAAACCGGTAA GACTGCGTACGAATTTGA UBC844 (CT)8RC Me7 ̄Em10 TGAGTCCAAACCGGTCC GACTGCGTACGAATTCAG UBC845 (CT)8RG Me8 ̄Em3 TGAGTCCAAACCGGTGC GACTGCGTACGAATTGAC UBC853 (TC)8RT UBC857 (AC)8YG UBC859 (CG)8RC UBC873 (GACA)4 G N of 94℃/1minꎬ50℃/1minꎬ72℃/1minꎻand a fi ̄ ( st) and gene flow ( m) (Slatkin and Bartonꎬ nal extension of 72 ℃/10 min. All PCR reactions 1989). To examine the genetic relationship among were run in the ABI2720 Thermal Cycler. The SRAP ̄ populationsꎬ we generated a genetic distance map PCR products were analyzed on 6% non ̄denatured via POPGENE. We also constructed a dendrogram polyacrylamide gels in1×TBE buffer running at 380V per Nei’s (1978) genetic distance methodꎬ using constant voltage for 10h. Afterwardꎬ silver ̄staining the unweighted pair ̄group method of averages (UP ̄ et al. was done as reported by Bassam (1991). GMA) and 1 000 permutations of bootstrappingꎬ To ensure the reliability of the genotypeꎬ nega ̄ with TFPGA version 13 (Millerꎬ 1997). A Mantel tive controls were run at each step to check for exog ̄ test was performed to estimate any correlations between enous contamination for ISSR and SRAP. The exper ̄ the matrices of genetic distances and geographical iment was repeated twiceꎬ and only data from in ̄ distancesꎬ using GenAlEx version 65 (Peakall and tensely stainedꎬ unambiguous bands were used for Smouseꎬ2006). Principal Coordinate Analysis (PCoA) statistical analysis. was also conducted with GenAlEx version 65ꎬ 14 Data analysis based on the calculated Jaccard’s similarity coeffi ̄ Amplified bands were scored according to the cients. Correlations between elevational differences presence (1) or absence (0) of homologous bands and genetic distances were estimated by PASSAGE for all samplesꎬ and were displayed as part of a bi ̄ version 2 (Rosenberg and Andersonꎬ 2011). nary matrix. These data were analyzed by POPGENE 2 Results version 132 (Francis and Yangꎬ 2000) to estimate P micranthum 21 Genetic diversity in populations the degree of genetic diversity in . Some essential diversity parametersꎬ eg.ꎬ percent ̄ A total of 88 different bands were scored from age of polymorphic bands (PPB)ꎬ Shannon’s infor ̄ 15 ISSR primersꎬ of which 81 were polymorphic I mation index (Shannon and Weaverꎬ 1949)ꎬ and (9204%). Our 11 SRAP primer combinations pro ̄ H Nei’s genetic diversity e (Neiꎬ1978)ꎬ were eval ̄ duced 280 bandsꎬ from 100 to 500 bp longꎬ across uated at both the population and species levels. Ge ̄ all 407 individuals. Of thoseꎬ 278 were polymorphic netic differentiation between populations was esti ̄ (9929%). A summary of the ISSR and SRAP data Paphiopedilum micranthum mated by the coefficients for genetic differentiation from each population is 期 et al 2 HUANG Jia ̄Lin .: ISSR and SRAP Markers Reveal Genetic Diversity and Population Structure of 2 13 P presented in Table 3. The ISSR analyses resulted in presented significant genetic variation ( <0001) H I a e value of03839 and an value of05646 at the among and within the 15 populations. Based on ISSR species level. Within each populationꎬ the PPB var ̄ analysisꎬ the main component (69%) within the to ̄ ied from 6489% (YJ and XL) to 8936% (YW). tal molecular variance was attributed to differences H The mean e was 02847ꎬ ranging from 01975 between individuals within populationsꎬ with the re ̄ I (XL) to 03238 (YW). Values for showed similar mainder (31%) coming from among populations trendsꎬ ranging from 03024 (XL) to 04799 (Table 4). This was consistent with the POPGENE H G (YW). The SRAP analyses produced a e value of results ( st = 02577). Similarlyꎬ when the SRAP I 02806 and an of 04359 at the species level. data were analyzedꎬ moderate genetic differentiation G Within each populationꎬ the PPB varied from ( st=02383) was found among populations. The av ̄ H 6254% (XL) to 8269% (YW). Values for e erage number of individualsexchanged between popu ̄ I Nm were 01963 to 02410 (mean 02166)ꎻ for ꎬ lations per generation ( ) was07201based on IS ̄ 02974 to 03701 (mean 03301). These data from SR markers and 07991 when SRAP markers were both ISSR and SRAP showed thatꎬ among the 15 used (Table 3). This indicated that limited pollen P micranthum populationsꎬ the genetic diversity of and seed dispersal occurred among populations. 23 Genetic distances among populations was richest within YW and poorest within XL. 22 Genetic differentiation within and among The Mantel tests showed significant positive cor ̄ populations relations between genetic and geographic distances r P AMOVA analysis with GenAlEx 65 software for both ISSRs ( =0455ꎻ =0001) and SRAPs Paphiopedilummicranthum Table3 Geneticdiversityof basedonISSRandSRAP analyses ISSR SRAP Population H I Nm H I Nm PPB/% e Gst PPB/% e Gst YW 8936 03238 04799 8269 02410 03701 YG 8723 03087 04598 6890 02015 03076 YT 7766 02873 04239 6466 02084 03159 YX 8085 02831 04196 7527 02177 03346 YF 7553 02787 04113 7067 02154 03274 YJ 6489 02314 03448 6360 02053 03106 YM 8723 03220 04767 6714 02040 03095 XL 6489 01975 03024 6254 01963 02974 XN 7551 02602 03907 6432 02199 03289 XY 7766 02851 04219 6926 02136 03301 GC 8191 03026 04468 7138 02227 03384 GN 8723 03177 04694 7491 02273 03479 GW 8298 02838 04255 7986 02362 03650 GZ 8830 03035 04506 7995 02384 03651 GB 8298 02912 04312 6749 02018 03077 Mean 8028 02847 04236 7067 02166 03301 Specieslevel 9166 03839 05646 02577 07201 9929 02806 04359 02383 07991 Table4 AMOVAresultsforgeneticvariancewithinandamongpopulationsbyISSRandSRAP P Markers Sourceofvariation df Sumofsquares Meansquares Variationcomponents Totalvariation/%  ̄value Amongpopulations 14 2425072 173219 5918 31 <0001 ISSR Withinpopulations 392 5086606 12976 12976 69 <0001 Amongpopulations 14 4569319 326380 10857 25 <0001 SRAP Withinpopulations 392 12709875 32423 32423 75 <0001 植 物 分 类 与 资 源 学 报 第 卷 214 36 r P ( =0421ꎻ =0003) (Table 5). To examine fur ̄ included 12 populations from Guizhouꎬ Guangxiꎬ ther why genetic distances formed among these popu ̄ and Yunnan provinces while Cluster II comprised the lationsꎬ we used a Mantel test with PASSAGE to in ̄ XLꎬ GWꎬ and XY populations from Guizhou and vestigate any correlation between elevational differ ̄ Guangxi provinces. The PCoA results of ISSR and ences and genetic distances. Hereꎬ positive correla ̄ SRAP data were shown in Figure 3A and Figure 3Bꎬ tions between those two components were identified respectively. Consistent with the UPGMA dendro ̄ r P from both ISSR analysis ( =04356ꎻ =00006) gramꎬ PCoA also revealed that the 15 populations r P and SRAP analysis ( =03953ꎻ =00025). could be separated into two main clusters. The UPGMA analyses applying both ISSRs 3 Discussion (Fig2A) and SRAPs (Fig2B) clearly resolved 31 Genetic diversity the 15 populations into two major clusters. Cluster I P micranthum Table5 Mantelteststoevaluategeneticdistancesꎬgeographic We collected 407 samples of from distancesꎬandelevationaldifferencesfor15 native 15 populations distributed in southwestern Chi ̄ Paphiopedilummicranthum populations na and analyzed their genetic variation in this study. Geneticdistanceversus Geneticdistanceversus The ISSR and SRAP markers revealed higher genetic Geographicdistance Elevationaldifference Cymbidi ̄ P r P r diversity than those of other orchidsꎬ eg.ꎬ um goeringii Goodyera procera Changnienia amoe ̄ ISSR 0455 0001 0436 0001 ꎬ ꎬ na Cypripedium flavum SRAP 0421 0003 0395 0003 ꎬ and (Table 6). Moreoverꎬ Fig2 A. UPGMAdendrogrambasedonISSRdataꎻB. UPGMAdendrogrambasedonSRAP data Fig3 A. PrincipalCoordinateAnalysisof407individualsꎬbasedonISSRdataꎻ B. PrincipalCoordinateAnalysisof407individualsꎬbasedonSRAP data 期 et al 2 HUANG Jia ̄Lin .: ISSR and SRAP Markers Reveal Genetic Diversity and Population Structure of 2 15 H G Table6 Comparisonsofvaluesforgeneticdiversity(PPBand e) anddifferentiation( st) between Paphiopedilummicranthum andotherorchidspecies H G Species PPB/% e st Markers References Paphiopedilummicranthum 9166 03839 02577 ISSR Thispaper 9929 02806 02383 SRAP Thispaper P micranthum etal 7330 02170 ——— RAPD Li .ꎬ2002b Cypripediumflvum etal 8269 02884 01540 AFLP Cai .ꎬ2008 Cypripediumcalceolus etal 3640 01490 00590 Allozyme Brzosko .ꎬ2011 Cymbidiumgoerigii 8819 02628 02440 ISSR GaoandYangꎬ2006 Goodyeraprocera 9703 02930 03900 RAPD WongandSunꎬ1999 Changnieniaamoena etal 7650 01941 ——— RAPD Li .ꎬ2002a etal Orchidaceae(average) ——— ——— 01870 ——— Forrest .ꎬ2004 P micranthum the degree of diversity for found here ramets and support an apparent plexiform distribution et al was greater than that calculated when RAPD markers (Tsi .ꎬ1999). Although a pollination system has P micranthum were used with four previous populations of that spe ̄ not been reported for ꎬ Cribb and Mc ̄ H et al cies (PPB=733%ꎻ e=0217) (Li .ꎬ 2002b). Gough (1997) has classified it as essentially an out ̄ The primary reason for this discrepancy may lie in breeding species that utilizes insect pollination. the choice of markers. The polymorphism of products Thenꎬ its out ̄crossing strategy might also contribute was higher with ISSRs and SRAPs than with RAP ̄ to its higher diversity comparing with that of inbreed ̄ Dsꎬ thereby providing more information about a par ̄ ing orchid species (Ehlers and Pedersenꎬ 2000). 32 Population genetic structure ticular genome. Moreoverꎬ ISSR and SRAP molecu ̄ lar markers are considered more stable and reliable Based on the assumption of Hardy-Weinberg e ̄ when compared with the RAPD technique (Dirle ̄ quilibriumꎬ we detected moderate genetic differentia ̄ et al et al P micranthum G wanger .ꎬ1998ꎻ Esselman .ꎬ1999ꎻ Gilbert tion among our populations of ( stꎬ et al .ꎬ 1999). Another explanation is that we used 02577 by ISSRꎻ02383ꎬ by SRAP). AMOVA ana ̄ more populations and individual samples than were lysis also showed that 31% (ISSR) and 25% (SRAP) et al involved in an earlier investigation by Li . genetic variation existed among populationsꎬ as well H (2002b). Some genetic parameters ( e) are sensi ̄ as 69% (ISSR) and 75% (SRAP) variation within tive to sample sizeꎬ thus influencing how well those populations (Table 4). This differentiation is higher et al parameters can be estimated (Li .ꎬ 2000). comparing with affinitiveꎬ insect ̄pollinated orchid P micranthum Cypripedium calceolus G Plants of are perennial and herba ̄ speciesꎬ such as ( st=0059ꎻ et al C flavum G ceous. Their decades ̄long life spans may contribute Brzosko .ꎬ 2011) and ( st=0154ꎻ et al to their high genetic diversity (Nybomꎬ 2004). Mo ̄ Cai .ꎬ 2008)ꎬ for which seeds are wind ̄disper ̄ et al. reoverꎬ the breeding mode is an important factor af ̄ sed. Forrest (2004) have reported that orchid G fecting genetic diversity (Hamrickꎬ 1982ꎻ Hamrick st values range from 0012 to 0924 (average and Godtꎬ 1990). This species reproduces by both 0187)ꎬ and that variations in population genetics sexually and asexuallyꎬ which has been considered differentiation are huge between orchid species. As G P micranthum as a strategy to maximize heterozygosity and repro ̄ shown hereꎬ the st values for (02577 et al ductive success (Yan .ꎬ 1999). One capsule of and 02383) were higher than the average of 0187 P micranthum contains more than5000 seedsꎬwhich calculated for other orchid species. can ensure a large gene pool that can provide abun ̄ The genetic structure of a plant species is usu ̄ et al dant heterozygotes (Luo .ꎬ 2003). In additionꎬ ally influenced by factors such as mating system and P micranthum has strong capacity of clonal reproduc ̄ extent of gene flow (Hamrick and Godtꎬ1990). The Nm tionꎬ developing rhizomes that often form numerous theory of population genetics suggests that low 植 物 分 类 与 资 源 学 报 第 卷 216 36 et al values (ie.ꎬ <1) cannot prevent the differentiation lation to another (Godt .ꎬ2005). Hereꎬ howev ̄ between populations that is caused by genetic drift erꎬ we could not identify any such barrierꎬ other (Wrightꎬ 1931ꎻ Hartl and Clarkꎬ 1989). Our study than elevational differenceꎬ between Clusters I and Nm produced values of 07201 (ISSR) and 07991 II. Our Mantel tests detected a remarkable correla ̄ P (SRAP)ꎬ both less than 1ꎬ which aggravated the tion ( <005) between genetic distance and eleva ̄ differentiation between populations. The distribution tion. Many studies have shown that elevation ̄associ ̄ of pollen and seeds is a major determinant of gene ated temperature variations play an important role in flow in natural populations (Li and Chenꎬ 2004). directing genetic diversity and differentiation (Ba ̄ et al Because most orchid species rely primarily on wind yerꎬ 1992ꎻ Li and Chenꎬ 2004ꎻ Jiang .ꎬ 2005). Rhodiola dispersalꎬ their seeds can move across great dis ̄ For exampleꎬ the genetic variation among et al angusta tances (Swamy .ꎬ 2004ꎬ 2007). In contrast to populations at Changbai Mountain increases Paphiopedilum Cypripedium related species of and ꎬ as the temperature decreases due to elevation (Yan P micranthum et al howeverꎬ seeds of are not carried as .ꎬ 1999). Likewiseꎬ apart from geographical far (Zhangꎬ 2012). This might explain why genetic distanceꎬ differences in elevation may be an impor ̄ P micranthum differentiation between populations is relatively high tant factor that divided our popula ̄ in that species. Another factor in the reduction of tions into two genetic clusters. 34 Conservation recommendations gene flow between populations is anthropogenic. Be ̄ cause of their high ornamental valueꎬ most of the ol ̄ For endangered speciesꎬ the goals of conserva ̄ derꎬ flowering plants have been collected from wild tion are to ensure the continued survival of popula ̄ populationsꎬ leaving behind only the younger speci ̄ tions and to maintain their evolutionary potential et al mens (Luo .ꎬ 2003). (Hamrick and Godt 1990ꎻ Wong and Sun 1999). 33 Genetic relationships among populations Given our findingsꎬ individuals of Cluster II com ̄ Both the UPGMA and PCoA evaluations divid ̄ prised the XLꎬ GWꎬ and XY populations from P micranthum ed the 15 geographical populations of Guizhou and Guangxi provinces have relatively low into two clustersꎬ with one comprising 12 popula ̄ genetic variation. Thereforeꎬ we recommend that tions in Yunnan and Guizhou and the other contai ̄ these populations be prioritized for conservation pro ̄ ning two populations in Guanxi plus one in Guizhou. tection. Meanwhileꎬ it is necessary to carry out the ex PCoA revealed high genetic similarity in Cluster Iꎬ  ̄situ conservation and the artificial reproduction of P micranthum whereas Cluster II showed evidence of genetic isola ̄ as soon as possibleꎬ to store up mass tion among its three populations. Results of Mantel seedlings of artificial reproduction prepared for wild r P tests based on data from ISSR ( = 0455ꎬ = re ̄introductionꎬ to renew this endangered species r P 0001) and SRAP ( =0421ꎬ =0003) indicated and to make it thriving. that genetic distance was correlated with geographic 4 Conclusion distanceꎬ a phenomenon that is commonly found in et al endemic and endangered species(Godt .ꎬ2005ꎬ Using ISSR and SRAP molecular markersꎬ we et al P micranthum P micranthum Luan .ꎬ 2006). Although is not determined that plants of exhibit a itself endemic to Chinaꎬ it is considered endangered high degree of genetic diversityꎬ and that genetic dif ̄ and protected there. ferentiation is moderate among natural populations. Genetic differentiation between natural popula ̄ When our sample populations were assigned to two tions is usually related to geographical barriersꎬ such clustersꎬ the one representing sites in Yunnan and as high mountains and riversꎬ which make gene ex ̄ Guizhou showed high diversity while that of the Guan ̄ change almost impossible to achieve from one popu ̄ gxi sites had low diversity. The genetic differentiation 期 et al 2 HUANG Jia ̄Lin .: ISSR and SRAP Markers Reveal Genetic Diversity and Population Structure of 2 17 et al between populations was related to variations in biolog ̄ DirlewangerEꎬPronier Vꎬ Parvery C .ꎬ 1998. Genetic linkage Prunuspersica mapofpeach( L.) usingmorphologicalandmolec ̄ ical characteristicsꎬ such as capacity for seed dispers ̄ Theoretical and Applied Genetics 97 ularmarkers [J]. ꎬ : 888— alꎬ as well as associations with geographical distance 895 and elevational differences. 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