Biochemical Systematics and Ecology 37 (2009) 579–588

Contents lists available at ScienceDirect

Biochemical Systematics and Ecology

journal homepage: www.elsevier.com/locate/biochemsyseco

High genetic differentiation and variation as revealed by ISSR marker in Pseudotaxus chienii (), an old rare endemic to

Yingjuan Su a, Ting Wang b,*, Puyue Ouyang a a State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, , China b Wuhan Botanical Garden/Wuhan Institute of Botany, Chinese Academy of Sciences, Wuhan 430074, Hubei, China article info abstract

Article history: Pseudotaxus chienii (Taxaceae) is an endangered conifer endemic to China, with Received 15 June 2009 a historically disjunct distribution pattern. Eleven populations sampled throughout its Accepted 10 October 2009 range were examined using inter-simple sequence repeats (ISSR) markers. Twenty primers generated 242 bands, and each detected polymorphic loci, with 73.14% of polymorphic loci Keywords: B overall. The estimate for q was 0.5306, whereas GST and FST values were 0.6146 and Pseudotaxus chienii 0.6401, respectively. 50.62% of the total diversity based on Shannon’s index of phenetic Old rare species diversity was attributed to among populations, which was consistent with the qB esti- Population differentiation Genetic variation mation. Compared with other , remarkable genetic differentiation occurred among B Conservation strategies P. chienii populations. A Mantel test indicated that pairwise values of q were significantly related with geographical distances between populations (r ¼ 0.676, P ¼ 0.001). In terms of the above results, conservation strategies and maintenance plans of P. chienii were recommended. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Life history and ecological characteristics have significant effects on the levels and partitioning of genetic diversity in (Hamrick and Godt, 1996; Allnutt et al., 1999). species with long life spans, widespread distributions, outcrossing breeding systems, and the potential for long-distance gene flow tend to be more genetically diverse and display less interpopulation differentiation, which differs substantially from the genetic patterns detected in herbaceous species with other combinations of life-history traits (Loveless and Hamrick, 1984; Hamrick and Godt, 1996; Allnutt et al., 1999; Hilfiker et al., 2004a). In accordance with this pattern, most studies of coniferous reveal high genetic variability and a relatively low degree of population differentiation (Loveless and Hamrick, 1984; Ledig, 1986; Hamrick et al., 1992). However, other patterns including low genetic diversity and large genetic differentiation (Pinus resinosa; Walter and Epperson, 2001), low genetic diversity and low genetic differentiation (Pinus banksiana; Ye et al., 2002), high genetic variation and moderate genetic differentiation (Araucaria cunninghamii; Pye et al., 2009) as well as high genetic variation and extreme population differentiation (Araucaria bidwillii; Pye and Gadek, 2004) have also been recorded within a number of coniferous taxa. The findings are usually explained by invoking demographic events (e.g., range expansion, fragmentation and bottlenecks) and evolutionary processes (mutation, genetic drift and natural selection) which act in shaping population genetic structure.

* Corresponding author. Tel.: þ86 27 87510677; fax: þ86 27 87510251. E-mail address: [email protected] (T. Wang).

0305-1978/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2009.10.005 580 Y. Su et al. / Biochemical Systematics and Ecology 37 (2009) 579–588

Pseudotaxus chienii (W. C. Cheng) W. C. Cheng, belonging to the yew family (Taxaceae), is an endemic conifer of China. The species is a dioecious woody or small tree up to 4 m, with seeds partly enclosed within a fleshy white aril at maturity (Fu et al., 1999). In China, natural populations of P. chienii have been reported in montane regions of southern , southwestern , northwestern and southern , northern , and northern Guangdong (Fu et al., 1999), ranging from 21Nto29N(Fig. 1). Its populations are usually small and isolated, and are thought to have long been patchily distributed (Fu and Jin, 1992). Plants primarily grow in the understorey of evergreen broad-leaved forests on scarp slopes, along the sides of valleys or on cliffs at altitudes of 800–1500 m. They are usually found on sites with acidic (pH 4.2–4.5), nutrient-rich soils, receiving an annual mean precipitation of between 1800 and 2400 mm (Fu and Jin, 1992; Yang et al., 2005). P. chienii was officially declared a protected species in 1992, and population sizes have been continuously declining over the past decade because of high rates of deforestation and overexploitation (Hu et al., 2003; Yang et al., 2005). During field surveys it was noticed that populations situated at Ruyuan, northern Guangdong have become extinct. In addition to having been directly influenced by anthropogenic activities since its first record in 1947 (Cheng, 1947; Fu and Jin, 1992; Hu et al., 2003; Yang et al., 2005), the distribution of P. chienii has long been naturally disjunct, restricted to scattered locations, showing ecological characteristics of ‘‘old rare species’’ (Huenneke, 1991; Holderegger, 1997; Fu et al., 1999; Hilfiker et al., 2004a). Such species are thought to be better adapted to habitat isolation. They may experience a lower severity of detrimental effects of population genetic processes that are associated with habitat fragmentation or isolation (Hilfiker et al., 2004a,b), which further affect their population genetic composition. In this respect, it is interesting to examine whether or not the current isolated populations of P. chienii still maintain substantial amounts of genetic variation. Inter-simple sequence repeats (ISSR) have been widely applied to the assessment of population genetic diversity and structure in plants (e.g., Camacho and Liston, 2001; Deshpande et al., 2001; Barth et al., 2002; King et al., 2002; Meloni et al., 2006). The technique is less prone to laboratory conditions than other PCR-generated markers such as RAPD (randomly amplified polymorphic DNA) (Nagaoka and Ogihara, 1997; Wolfe et al., 1998) and requires no prior sequence-specific information (Barth et al., 2002). However, for population genetic studies, ISSR markers have been criticized owing to their dominance. To mitigate this problem, Holsinger et al. (2002) developed a Bayesian approach to partition genetic variation with data derived from dominant markers. Moreover, Dawson et al. (1995) noted that the Shannon diversity index is relatively insensitive to the skewing effects caused by the dominance of molecular markers. The aim of this investigation is to assess the levels and distribution of population genetic variation in P. chienii using ISSR markers. Particularly, we examined whether P. chienii is able to attenuate genetic depauperation in its isolated populations due to the fact that they have long been naturally fragmented, and whether Bayesian and other standard statistical approaches give similar estimates of the partition of ISSR diversity among its populations.

Fig. 1. Populations of Pseudotaxus chienii naturally located in Zhejiang, Jiangxi, Hunan and Guangxi Provinces, China. Y. Su et al. / Biochemical Systematics and Ecology 37 (2009) 579–588 581

2. Materials and methods

2.1. material

Samples of P. chienii were collected from eleven natural populations distributed throughout its range (Table 1; Fig. 1). Individual plants grow under dense canopies in montane forests, preferentially on scarp slopes, along the sides of ravines, or on cliffs. Young and healthy were randomly sampled from individuals, with intervals of at least 10 m, and immediately preserved in silica gel. For each population, seven to ten individuals were sampled (Table 1), but in Jiao Zi Shan (JXi) only two surviving plants were found. All samples were stored at 20 C until being processed. Vouchers from populations investi- gated have been deposited at the herbarium of Sun Yat-sen University (SYS), Guangzhou, China.

2.2. DNA extraction

Stored leaves were pulverized in liquid nitrogen and DNA was extracted by the modified CTAB protocol (Su et al., 2005). DNA was quantified spectrophotometrically and samples yielding good quality and high molecular weight DNA were used for experiment.

2.3. ISSR amplification and examination

PCRs contained 10 mM Tris–HCl (pH 9.0), 50 mM KCl, 0.1% Triton X-100, 2.0 mM MgCl2, 0.2 mM each dNTP, 0.3 mM primer, 30 ng genomic DNA, and 1 U Taq DNA polymerase in a total volume of 20 ml. PCR amplification was performed in an MJ-Research PTC-100TM Peltier thermal cycler programmed as follows: 5 min at 94 C, one cycle; 40 s at 94 C, 35 s at 53 C, 70 s at 72 C, for 40 cycles; and 5 min at 72 C. A negative control containing all PCR components except DNA were included in every experiment to test for DNA contamination of the reagents. Eighty ISSR primers (UBC ISSR Primers 802, 805, 808 to 881, 886, 891, 895, and 899, University of British Columbia, Vancouver, Canada) were screened, and twenty primers (Table 2)were chosen for genotyping all 91 samples. A master mix was prepared for each primer to minimize possible inconsistencies among different amplification runs. ISSR reactions were performed at least twice to determine the reproducibility of banding patterns. Amplification products were electrophoretically resolved in 1.8% agarose gel containing ethidium bromide (at a final concentration of 0.5 mg/ml) and photographed under ultraviolet light.

2.4. Data analysis

Data were scored in a binary fashion based on the presence (1) or absence (0) of bands. Only those bands that are clear and reproducible were recorded in the data matrix. Percentage of polymorphic loci, Nei’s (1973) gene diversity and coefficient of gene differentiation, GST, were computed by using the POPGEN32P program (Yeh and Yang, 1999). Shannon’s index of phenotypic diversity was calculated using the equation S ¼ pilnpi, where pi is the frequency of a band (Lewontin, 1973). S was calculated for two levels: the average diversity within populations (Spop) and the diversity within the species as a whole (Ssp). Its 95% confidence interval was estimated by a sampled randomization test (Sokal and Rohlf, 2003). Bayesian inference was conducted using the statistical model established by Holsinger et al. (2002), in which f and qB are analogues to the inbreeding coefficient (FIS) and the fixation index (FST)ofF-statistics, respectively. The posterior distributions of f and qB were approximated numerically through Markov Chain Monte Carlo (MCMC) simulations by running HICKORY v1.0 (Holsinger et al., 2002). We used the ‘‘f-free’’ model option with the default parameter settings (burn-in ¼ 50 000, sample ¼ 250 000, thin ¼ 50) to obtain the point estimates of f and qB as well as their 95% credible intervals. Three replicates were run to ensure that the results were consistent.

Table 1 Origin locations of sampled populations of Pseudotaxus chienii.

Provenance Population location and code Sample no. Geographical coordinate Altitude (m) Zhejiang Da Xi Keng, Zhe Dai Kou, Sui Chang (ZJa) 1–7 118450E28150N 1000 Ju Long Ding, Keng Kou Xiang, Qu Jiang (ZJb) 8–15 118560E28430N 870 Ju Long Ding, Keng Kou Xiang, Qu Jiang (ZJc) 16–23 118560E28430N 950 Shuang Gang Kou, Keng Kou Xiang, Qu Jiang (ZJd) 24–31 118570E28430N 830 Da Yuan Wei, Keng Kou Xiang, Qun Jiang (ZJe) 32–39 118570E28430N 1100 Shui Kou, Feng Yang Shan, Long Quan (ZJf) 40–49 119100E27520N 1100 Lao Ying Yan, Feng Yang Shan, Long Quan (ZJg) 50–59 119110E27520N 1100

Jiangxi Bi Jia Shan, Jing Gang Shan (JXh) 60–69 114100E26350N 1350 Jiao Zi Shan, Jing Gang Shan (JXi) 70–71 114100E26350N 950

Hunan Tian Zi Shan, Zhang Jia Jie (HNj) 72–81 109300E28500N 1050

Guangxi Da Ming Shan, Wu Ming (GXk) 82–91 107330E22360N 1250 582 Y. Su et al. / Biochemical Systematics and Ecology 37 (2009) 579–588

Table 2 Primers used in ISSR analyses of Pseudotaxus chienii (R ¼ A, G; Y ¼ C, T).

Primers Sequences (5’/3’) Primers Sequences (5’/3’) UBC810 GAG AGA GAG AGA GAG AT UBC844 CTC TCT CTC TCT CTC TRC UBC811 GAG AGA GAG AGA GAG AC UBC846 CAC ACA CAC ACA CAC ART UBC813 CTC TCT CTC TCT CTC TT UBC847 CACACA CAC ACA CAC ARC UBC815 CTC TCT CTC TCT CTC TG UBC852 TCT CTC TCT CTC TCT CRA UBC820 GTG TGT GTG TGT GTG TC UBC855 ACACAC ACA CAC ACA CYT UBC822 TCT CTC TCT CTC TCT CA UBC857 ACACACACA CAC ACA CYG UBC825 ACA CAC ACA CAC ACA CT UBC858 TGT GTG TGT GTG TGT GRT UBC827 ACA CAC ACA CAC ACA CG UBC866 CTC CTC CTC CTC CTC CTC UBC835 AGAGAGAGAGAGAGA GYC UBC873 GAC AGA CAG ACA GAC A UBC840 GAG AGA GAG AGA GAG AYT UBC880 GGA GAG GAG AGG AGA

A Euclidean squared distance matrix was calculated in ARLEQUIN 2.0 (Schneider et al., 2000) and used as the input file for an analysis of molecular variance (AMOVA) (Excoffier et al., 1992). Using the same software, a Mantel test (Mantel, 1967)was conducted on the matrices of pairwise qB estimates and geographic distances. Relationships among individuals were estimated by unweighted pair-group mean analysis (UPGMA). The 50% majority- rule consensus UPGMA tree was constructed with the Nei and Li distance (Nei and Li, 1979) from 1000 bootstrap replicates using the program PAUP* version 4.0b4 (Swofford, 1998). Principal components analysis (PCA) of ISSR phenotypes was performed using NTSYSPC2 software package (Rohlf, 1993). Linear regression analyses were carried out with SPSS 13.0 for Windows (SPSS, 2005; Li and Luo, 2005).

3. Results

3.1. Analysis of population genetic variation with ISSR

A pilot experiment was performed to evaluate primer suitability. From an initial screening of 80 primers, 20 (Table 2)were identified that produce clear and reproducible banding patterns. Only reproducible bands obtained from the selected primers were scored to construct data matrices for subsequent statistical analyses. The 20 ISSR primers used to assay the genomic DNAs of 91 individuals from 11 populations of P. chienii generated 242 bands, varying in size from 400 bp to 3000 bp. The number of bands per primer ranged from 8 to 21 with an average of 12.11 bands/primer. No significant relationships were found by linear regression analyses between population size or altitudinal distribution and the genetic diversity statistics including number of polymorphic loci, percentage of polymorphic loci, Nei’s gene diversity, and Shannon’s index. All statistics consistently indicated that population GXk possessed the highest diversity value (Table 3). Shannon’s indexes were not significantly different for populations ZJb, ZJc, ZJd, ZJe, ZJf, ZJg, JXh, and HNj (Table 3).

3.2. Population genetic differentiation of P. chienii

Because prior information about the magnitude of inbreeding in P. chienii was unavailable, a noninformative Beta distribution on [1, 1], equivalent to uniform on [0, 1], was used for the priors. With default sampling parameters (burn-in: 50 000; sample: 250 000; thin: 50), a run of f-free analysis in Hickory yielded an overall estimate for qB ¼ 0.5306 (95% credible interval: 0.4944–0.5621), DIC (Deviance Information Criterion) ¼ 3164.0565. Very similar results, qB ¼ 0.5296 (95% credible

Table 3 Summary statistics revealed by using 20 ISSR primers to detect populations of Pseudotaxus chienii.

Population Number of Number of Percentage of Nei’s gene Shannon’s index loci polymorphic loci polymorphic loci diversity (95% confidence interval) ZJa 170 36 0.2118 0.0757 0.0785 (0.0550, 0.1047) ZJb 176 49 0.2784 0.0956 0.1091 (0.0838, 0.1408) ZJc 180 52 0.2889 0.1045 0.1208 (0.0922, 0.1507) ZJd 182 59 0.3242 0.1129 0.1325 (0.1030, 0.1631) ZJe 177 62 0.3503 0.1128 0.1386 (0.1086, 0.1708) ZJf 173 53 0.3064 0.0892 0.1090 (0.0830, 0.1330) ZJg 178 63 0.3539 0.1271 0.1353 (0.1062, 0.1652) JXh 183 71 0.3880 0.1304 0.1422 (0.1143, 0.1726) JXi 158 21 0.1329 0.0551 0.0665 (0.0411, 0.0918) HNj 175 44 0.2514 0.0781 0.0930 (0.0693, 0.1211) GXk 208 95 0.4567 0.1539 0.1726 (0.1458, 0.2005) Total 242 177 0.7314 0.2118 0.2390 (0.2132, 0.2657) Y. Su et al. / Biochemical Systematics and Ecology 37 (2009) 579–588 583 interval: 0.4917–0.5620) and DIC ¼ 3168.1796, was obtained when running the sampler with shorter Markov chains (burn-in: 5000; sample: 25 000; thin: 5). The results imply that convergence of the Markov chain to its stationary distribution has been achieved efficiently. The degree of population genetic differentiation was also quantified by other parameters. AMOVA indicated that FST ¼ 0.6401; 35.99% of the variation was found within populations, 23.04% was partitioned among populations within provenances, and 40.97% was attributed to differences among provenances (P < 0.001) (Table 4). All pairwise FST values between individual pairs of populations were significant (P < 0.05). Meanwhile, values of GST (Nei, 1973) and the component of diversity among populations (1 Spop/Ssp) based on Shannon’s index were 0.6146 and 0.5062, respectively. Furthermore, pairwise estimates of qB between populations showed a significant relationship with geographic distances (r ¼ 0.676, P ¼ 0.001).

3.3. Cluster and principal components analysis of ISSR data

After surveying the combined data matrix of all 20 primers, it was observed that no identical ISSR pattern was shared among the 91 examined individuals of P. chienii. The 50% majority-rule consensus UPGMA tree indicates that individuals mainly fall into four distinct groups which are supported by high bootstrap values and are significantly correlated with their geographical origins Zhejiang, Jiangxi, Hunan, and Guangxi; individuals originated from different provenances are never clustered together; and Group Guangxi is the most distinct from others (Fig. 2). Group Zhejiang can be further divided into two subgroups; one is composed of populations ZJa, ZJb, ZJc, and ZJd, while the other consists of ZJe, ZJf, and ZJg (Fig. 2). Clusterings of Groups Zhejiang, Jiangxi, Hunan, and Guangxi are also supported by the three-dimensional pattern established from principle components analysis (Fig. 3). The first three principal components (PC1, PC2, and PC3) account for 76.60% (36.78%, 24.29%, and 15.53%, respectively) of the total variance.

4. Discussion

Levels of genetic diversity in plant species are affected by numerous factors including breeding systems, seed dispersal mechanisms, geographic ranges, life forms and natural selection; among which, geographic range possibly plays a major role in the maintenance of genetic variation (Hamrick and Godt, 1989, 1996; Hamrick et al., 1991; Maki, 2003; Meloni et al., 2006). Some studies have revealed that plants with a wider distribution tend to possess larger genetic diversity than congeners with a restricted distribution (Karron, 1987; Hamrick and Godt, 1989; Maki and Horie, 1999; Maki et al., 2002; Gitzendanner and Soltis, 2000); however, exceptions still exist (Gitzendanner and Soltis, 2000; Bekessy et al., 2002). In this study, at the species level, relatively high levels of ISSR variation were detected within P. chienii. As shown in Table 5, based on ISSR data its Shannon diversity is higher than other conifers native to South China and its adjacent regions including Calocedrus macrolepis (Wang et al., 2004), Pinus squamata (Zhang et al., 2005), Amentotaxus yunnanensis (Ge et al., 2005), and Glyptostropus pensilis (Li and Xia, 2005), but lower than Amentotaxus argotaenia (Ge et al., 2005) and A. cunninghamii (Pye et al., 2009). Considering that P. chienii and A. argotaenia each encompass large geographic ranges (5 and 13 provinces in China, respectively) (Fu et al., 1999), and A. cunninghamii also possesses the largest latitudinal distribution among all extant araucarian species (Pye et al., 2009), the hypothesis that a wider-distributed species tends to have larger diversity is lent support here, even though they occur in relatively small, fragmented populations. By using other markers such as RAPDs Allnutt et al. (1999) also recorded high intraspecific variation in Fitzroya cupressoides, a threatened South American conifer, which was historically widely distributed. According to the proximity of nearest populations, current P. chienii populations in Zhejiang may be classified as continuous, and those in Jiangxi, Hunan, and Guangxi as isolated (Table 1; Gapare et al., 2005). In conifers, there is some evidence that isolation negatively correlates with neutral genetic variation (Pinus radiata; Karhu et al., 2006), and peripheral and disjunct populations often possess low level of variation compared to core and continuous ones (e.g., in Pseudotsuga menziesii (Li and Adams, 1989), Pinus contorta (Aitken and Libby, 1994), and Pinus coulteri (Ledig, 2000)), possibly due to genetic drift, inbreeding, reduced rates of gene flow, or directional selection (Hartl and Clark, 1997; Hedrick, 2000; Gapare et al., 2005; Eckstein et al., 2006). In contrast to these findings, for P. chienii similar degrees of diversity were found in the Zhejiang populations (ZJb, ZJc, ZJd, ZJe, ZJf, and ZJg) and peripheral, isolated populations (JXh and HNj) as quantified by Shannon’s diversity (Table 3), even though it is a more sensitive measure of variation (Allnutt et al., 2003). Particularly, population GXk which marks the southwestern tip of the species’ current range (Fig. 1) and is shown as most divergent (Fig. 3)

Table 4 AMOVA of ISSR variation for 11 Pseudotaxus chienii populations in 4 provenances: Zhejiang, Jiangxi, Hunan, and Guangxi.

d.f. Sum of squares Variance Percentage of P F statistics components total variation

Among provenances 3 885.534 13.112 40.97 <0.001 FCT ¼ 0.4097 Among populations within provenances 7 477.113 7.373 23.04 <0.001 FSC ¼ 0.3903 Within populations 80 921.354 11.517 35.99 <0.001 FST ¼ 0.6401 The P-value was calculated by a permutation procedure based on 1023 replicates. 584 Y. Su et al. / Biochemical Systematics and Ecology 37 (2009) 579–588

Fig. 2. 50% majority-rule consensus UPGMA tree calculated for 91 examined individuals of Pseudotaxus chienii with Nei and Li distance (Nei and Li, 1979). Bootstrap values (in percent), determined from 1000 replicates, are indicated below branches. possesses higher levels of variation than the northeastern, continuous Zhejiang populations (Fig. 1; Table 3). The data here implies that as an ‘‘old rare species’’ which has long been naturally fragmented (Fu et al., 1999; Huenneke, 1991; Holderegger, 1997; Hilfiker et al., 2004a), P. chienii seems to be more capable of maintaining its variation in isolated populations even though it cannot be excluded that the population GXk might represent one of the potential past centres of diversity. A number of studies have shown that population genetic variation correlates with plant fitness (e.g., Fischer and Matthies, 1998; Amos Y. Su et al. / Biochemical Systematics and Ecology 37 (2009) 579–588 585

Fig. 3. Principal components analysis (PCA) based on ISSR phenotypic characters obtained in Pseudotaxus chienii. The first three principal components PC1, PC2, and PC3 account for 36.78%, 24.29%, and 15.53% of the total variance, respectively. and Balmford, 2001; Vergeer et al., 2003; Hensen et al., 2005), and a comparative assessment of fitness characteristics such as survival and fertility between isolated and continuous populations of P. chienii may bring interesting results. Compared with population genetic estimates based on ISSR analyses of other conifer species (Table 5), a marked genetic differentiation was detected among populations of P. chienii. Pairwise FST comparisons between populations were all significant (P < 0.05), which further highlighted the substantial degree of population differentiation (Allnutt et al., 2003). According to chloroplast matK genes, it was estimated that this species arose at least 120 million years ago (Cheng et al., 2000). P. chienii also represents a Tertiary relict species with distribution patterns which have long been disjunct. Its current populations usually consist of several to tens of individuals, restricted to scattered locations 2–1400 km apart geographically. Since genetic drift can increase the fixation index by 1/2Ne (Ne refers to the effective size of the populations) over each generation (Conner and Hartl, 2004), both long evolutionary history and small Ne of P. chienii should be the major factors creating population genetic differentiation. Moreover, P. chienii is a dioecious species tending to grow under a dense canopy, so individuals are not closely spaced, which hinders its female cones from being pollinated (e.g., Failure to trap pollen out of moving air). This is equivalent to the reduction of female numbers. Because Ne ¼ 4NmNf/(Nm þ Nf)(Nm and Nf are the number of males and females in the population, respectively) (Hedrick, 2000; Conner and Hartl, 2004), restricting female numbers will further reduce Ne. A pronounced geographical pattern of diversification in P. chienii was revealed using our ISSR data. A Mantel test indicated that there is a significant association between geographical and genetic structure, complying with the theoretical predictions from an ‘‘isolation by distance’’ model (Wright, 1943). Similar results have been observed in populations of conifers by using RAPD markers (Nybom and Bartish, 2000; Allnutt et al., 2003). According to the finding, high degree of genetic differentiation recorded for P. chienii may also be accounted for in terms of the very large geographical range of sampled populations which extend over as much as 1400 km. In addition, both consensus UPGMA tree and PCA pattern (Figs. 2 and 3) demonstrated that P. chienii populations from Zhejiang, Jiangxi, Guangxi, and Hunan formed four separate groups; individuals from different provenances were never intermixed, suggesting a lack of gene flow and few individual migrations among geographically distant populations. Pollination of P. chienii is anemophilous; its seeds are mainly dispersed by birds or small mammals

Table 5 B Shannon’s diversity within species (Ssp), Nei’s (1973) total gene diversity (HT), GST, FST, and q obtained from different conifer species based on ISSR data.

B Species Ssp HT GST FST q Reference Calocedrus macrolepis 0.1662 0.116 0.042 0.041 Wang et al., 2004 Pinus squamata 0.048 0.029 0.024 Zhang et al., 2005 Amentotaxus argotaenia 0.2868 0.1839 0.726 0.707 Ge et al., 2005 Amentotaxus yunnanensis 0.2137 0.1413 0.746 0.595 Ge et al., 2005 Glyptostropus pensilis 0.122 0.5136 0.4416 Li and Xia, 2005 Juniperus phoenicea 0.148 0.120 0.235 Meloni et al., 2006 Araucaria cunninghamii 0.4971 0.1812 Pye et al., 2009 Pseudotaxus chienii 0.2390 0.2118 0.6146 0.6401 0.5306 This study 586 Y. Su et al. / Biochemical Systematics and Ecology 37 (2009) 579–588

(Fu and Jin, 1992; Fu et al., 1999). In general, it has been suggested that wind pollination and animal-mediated seed dispersal may prevent differentiation over fairly large geographical areas by allowing for long-distance gene flow (Loveless and Hamrick, 1984; Delgado et al., 1999; Hilfiker et al., 2004a). However, this seems not to be the case in P. chienii. Understanding patterns of genetic variability has valuable implications for the development of conservation strategies and management plans of rare and endangered plants (Holsinger and Gottlieb, 1991; Allnutt et al., 2003), although its real bearing on field practices remains to be questioned (Schaal and Leverich, 2005). In this study, a high degree of population genetic differentiation has been revealed, suggesting that for the conservation of genetic variability in P. chienii, on-site management should aim to preserve each surviving population, since an extinction of any one of them would lead to a loss of total genetic diversity. Meanwhile, it is worthwhile to note that when trying to prevent genetic erosion of extremely small populations with reduced genetic variability (e.g., population JXi); deliberate transfer of germplasm from different populations should be done with caution. Augmentation of very small populations with individuals from other, larger populations may result in a short-term increase in genetic diversity and fitness, but may also refresh the local pool of detrimental recessive alleles that have been purged in demographic history or result in outbreeding depression (Lynch, 1996; Ennos, 1998; Amos and Balmford, 2001; Schaal and Leverich, 2005). Natural selection tends to create plant populations that are precisely adapted to local habitat, and mixing genotypes from different environments will cause a loss of adaptation; and, in some cases, the result can be disastrous (Chamaecrista fasciculata (Fenster and Galloway, 2000); Anchusa crispa (Quilichini et al., 2001); Schaal and Leverich, 2005)). Besides the investigation of neutral variability in P. chienii using ISSR markers, further information such as variation in quantitative genetic traits related to reproduction and fecundity and their effects on demographic processes is urgently required to ensure sound conservation plans. In the present study, calculated from ISSR data of P. chienii populations, Nei’s (1973) method and AMOVA gave values of B GST ¼ 0.6146 and FST ¼ 0.6401, respectively. Both are substantially larger than Bayesian estimate q ¼ 0.5306. However, if the data were analyzed phenetically, Shannon’s index partitioned 50.62% of the diversity among populations, which is consistent with qB estimate. Our results further indicate that the Shannon diversity index is insensitive to the bias generated by the dominance of molecular markers, such as ISSRs and RAPDs, when used to estimate differentiation among populations (Dawson et al., 1995; Meekins et al., 2001; Holsinger et al., 2002).

Acknowledgments

The authors thank Chunquan Chen and Xiangming Zeng from Jinggangsan Natural Reserve, Bingyang Ding and Xiaofeng Jin from College of Life Sciences, Zhejiang University, and Shaoqing Hu from Hangzhou Botanical Garden, China for assistance with the collection of plant materials. We also thank Dr. Jared L. Strasburg at the Department of Biology, Indiana University for advice and revision of English. This work was supported by grants from National Natural Science Foundation of China (30270153); the ‘‘100 Talent Project’’ of the Chinese Academy of Sciences (0729281F02); and the Open Project of the State Key Laboratory of Biocontrol (2007–01), China.

References

Aitken, S.N., Libby, W.T., 1994. Evolution of the pygmy-forest edaphic subspecies of Pinus contorta across an ecological staircase. Evolution 48, 1009–1019. Allnutt, T.R., Newton, A.C., Lara, A., Premoli, A., Armesto, J.J., Vergara, R., Gardner, M., 1999. Genetic variation in Fitzroya cupressoides (alerce), a threatened South American conifer. Mol. Ecol. 8, 975–987. Allnutt, T.R., Newton, A.C., Premoli, A., Lara, A., 2003. Genetic variation in the threatened South American conifer Pilgerodendron uviferum (Cupressaceae), detected using RAPD markers. Biol. Conserv. 114, 245–253. Amos, W., Balmford, A., 2001. When does conservation genetics matter? Heredity 87, 257–265. Barth, S., Melchinger, A.E., Lubberstedt, T.L., 2002. Genetic diversity in Arabidopsis thaliana L. Heynh. investigated by cleaved amplified polymorphic sequence (CAPS) and inter-simple sequence repeat (ISSR) markers. Mol. Ecol. 11, 495–505. Bekessy, S.A., Allnutt, T.R., Premoli, A.C., Lara, A., Ennos, R.A., Burgman, M.A., Cortes, M., Newton, A.C., 2002. Genetic variation in the vulnerable and endemic Monkey Puzzle tree, detected using RAPDs. Heredity 88, 243–249. Camacho, F.J., Liston, A., 2001. Population structure and genetic diversity of Botrychium pumicola (Ophioglossaceae) based on inter-simple sequence repeats (ISSR). Am. J. Bot. 88, 1065–1070. Cheng, W.C., 1947. New Chinese Trees and . Research notes. In: Dendrological Ser. 1. Forestry Institute, National Central University, Nanking. 1–4. Cheng, Y.C., Nicolson, R.G., Tripp, K., Chaw, S.M., 2000. Phylogeny of Taxaceae and Cephalotaxaceae genera inferred from chloroplast matK gene and nuclear rDNA ITS region. Mol. Phylogenet. Evol. 14, 353–365. Conner, J.K., Hartl, D.L., 2004. A Primer of Ecological Genetics. Sinauer, Sunderland. Dawson, I.K., Simons, A.J., Waugh, R., Powell, W., 1995. Diversity and genetic differentiation among subpopulations of Gliricidia sepium revealed by PCR- based assays. Heredity 74, 10–18. Delgado, P., Pinero, D., Chaos, A., Perez-Nasser, N., Alvarez-Buylla, E.R., 1999. High population differentiation and genetic variation in the endangered Mexican pine Pinus rzedowskii (Pinaceae). Am. J. Bot. 86, 669–676. Deshpande, A.U., Apte, G.S., Bahulikar, R.A., Lagu, M.D., Kulkarni, B.G., Suresh, H.S., Singh, N.P., Rao, M.K.V., Gupta, V.S., Pant, A., Ranjekar, P.K., 2001. Genetic diversity across natural populations of three montane plant species from the Western Ghats, India revealed by intersimple sequence repeats. Mol. Ecol. 10, 2397–2408. Eckstein, R.L., O’neill, R.A., Danihelka, J., Otte, A., Ko¨hler, W., 2006. Genetic structure among and within peripheral and central populations of three endangered floodplain violets. Mol. Ecol. 15, 2367–2379. Ennos, R.A., 1998. Genetic constrains on native woodland restoration. In: Newton, A.C., Ashmole, P. (Eds.), Native Woodland Restoration in Southern Scotland: Principles and Practice. Borders Forest Trust, Ancrum, pp. 27–34. Excoffier, L., Smouse, P.E., Quattro, J.M., 1992. Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics 131, 479–491. Fenster, C.B., Galloway, L.F., 2000. Inbreeding and outbreeding depression in natural populations of Chamaecrista fasciculate (Fabaceae). Conserv. Biol. 14, 1406–1412. Y. Su et al. / Biochemical Systematics and Ecology 37 (2009) 579–588 587

Fischer, M., Matthies, D., 1998. RAPD variation in relation to population size and plant performance in the rare Gentianella germanica (Gentianaceae). Am. J. Bot. 85, 811–819. Fu, L.G., Jin, J.M., 1992. Red List of Endangered Plants in China, vol. 1. Science Press, Beijing. Fu, L.G., Li, N., Mill, R.R., 1999. Taxaceae. In: Wu, Z.Y., Raven, P.H. (Eds.), . Science Press/Missouri Botanical Garden Press, Beijing/St. Louis, pp. 89–98. Gapare, J.W., Aitken, S.N., Ritland, C.E., 2005. Genetic diversity of core and peripheral Sitka spruce (Picea sitchensis (Bong.) Carr) populations: implications for conservation of widespread species. Biol. Conserv. 123, 113–123. Ge, X.J., Zhou, X.L., Li, Z.C., Hsu, T.W., Schaal, B.A., Chiang, T.Y., 2005. Low genetic diversity and significant population structuring in the relict Amentotaxus argotaenia complex (Taxaceae) based on ISSR fingerprinting. J. Plant Res. 118, 415–422. Gitzendanner, M.A., Soltis, P.S., 2000. Patterns of genetic variation in rare and widespread plant congeners. Am. J. Bot. 87, 783–792. Hamrick, J.L., Godt, M.J.W., 1989. Allozyme diversity in plant species. In: Brown, A.H.D., Clegg, M.T., Kahler, A.L., Weir, B.S. (Eds.), Plant Population Genetics, Breeding and Germplasm Resources. Sinauer, Sunderland, pp. 43–63. Hamrick, J.L., Godt, M.J.W., 1996. Effects of life history traits on genetic diversity in plant species. Philos. Trans. R. Soc. Lond. B: Biol. Sci. 351, 1291–1298. Hamrick, J.L., Godt, M.J.W., Murawski, D.A., Loveless, M.D., 1991. Correlation between species traits and allozyme diversity implications for conservation biology. In: Falk, D.A., Holsinger, K.E. (Eds.), Genetics and Conservation of Rare Plants. Oxford University Press, New York, pp. 75–86. Hamrick, J.L., Godt, M.J.W., Shermann-Broyles, S.L., 1992. Factors influencing levels of genetic diversity in woody plant species. New Forests 6, 95–124. Hartl, D.L., Clark, G.C., 1997. Principles of Population Genetics. Sinauer, Sunderland. Hedrick, P.W., 2000. Genetics of Populations. Jones and Bartlett, Sudbury. Hensen, I., Oberprieler, C., Wesche, K., 2005. Genetic structure, population size, and seed production of Pulsatilla vulgaris Mill. (Ranunculaceae) in Central Germany. Flora 200, 3–14. Hilfiker, K., Gugerli, F., Schutz, J., Rotach, P., Holderegger, R., 2004a. Low RAPD variation and female-biased sex ratio indicate genetic drift in small populations of the dioecious conifer baccata in Switzerland. Cons. Genet. 5, 357–365. Hilfiker, K., Holderegger, R., Rotach, P., Gugerli, F., 2004b. Dynamics of genetic variation in Taxus baccata: local versus regional perspectives. Can. J. Bot. 82, 219–227. Holderegger, R., 1997. Recent perspectives in conservation biology of rare plants. Bull. Geobot. Inst. ETH 63, 109–116. Holsinger, K.E., Gottlieb, L.D., 1991. Conservation of rare and endangered plants: principles and prospects. In: Falk, D.A., Holsinger, K.E. (Eds.), Genetics and Conservation of Rare Plants. Oxford University Press, New York, pp. 195–223. Holsinger, K.E., Lewis, P.O., Dey, D.K., 2002. A Bayesian approach to inferring population structure from dominant markers. Mol. Ecol. 11, 1157–1164. Hu, S.Q., Chen, Z.H., Shun, M.J., 2003. A study on resources of Pseudotaxus chienii (Cheng) Cheng. J. Zhejiang Univ. (Agric. Life Sci.) 29, 97–102. Huenneke, L.F., 1991. Ecological implications of genetic variation in plant populations. In: Falk, D.A., Holsinger, K.E. (Eds.), Genetics and Conservation of Rare Plants. Oxford University Press, New York, pp. 31–44. Karhu, A., Vogl, C., Moran, G.F., Bell, J.C., Savolainen, O., 2006. Analysis of microsatellite variation in Pinus radiata reveals effects of genetic drift but no recent bottlenecks. J. Evol. Biol. 19, 167–175. Karron, J.D., 1987. A comparison of levels of genetic polymorphism and self-compatibility in geographically restricted and widespread plant congeners. Evol. Ecol. 1, 47–58. King, R.A., Gornall, R.J., Preston, C.D., Croft, J.M., 2002. Population differentiation of Potamogeton pectinatus in the Baltic Sea with reference to waterfowl dispersal. Mol. Ecol. 11, 1947–1956. Ledig, F.T., 1986. Heterozygosity, heterosis, and fitness in outbreeding plants. In: Soule, M.E. (Ed.), Conservation Biology. Sinauer, Sunderland, pp. 77–104. Ledig, F.T., 2000. Founder effects and genetic structure of Coulter Pine. J. Hered. 91, 307–315. Lewontin, R.C., 1973. The apportionment of human diversity. Evol. Biol. 6, 381–398. Li, P., Adams, W.T., 1989. Range-wide patterns of allozyme variation in Douglas fir (Pseudotsuga menziesii). Can. J. For. Res. 19, 149–161. Li, Z.H., Luo, P., 2005. A Course in Statistical Analyses with SPSS for Windows. Publishing House of Electronics Industry, Beijing. Li, F.G., Xia, N.H., 2005. Population structure and genetic diversity of an endangered species, Glyptostrobus pensilis (Cupressaceae). Bot. Bull. Acad. Sinica 46, 155–162. Loveless, M.D., Hamrick, J.L., 1984. Ecological determinants of genetic structure of plant populations. Annu. Rev. Ecol. Syst. 15, 65–95. Lynch, M., 1996. A quantitative-genetic perspective on conservation issues. In: Avise, J.C., Hamrick, J.L. (Eds.), Conservation Genetics: Case Histories from Nature. Chapman and Hall, New York, pp. 471–501. Maki, M., 2003. Population genetics of threatened wild plants in Japan. J. Plant Res. 116, 169–174. Maki, M., Horie, S., 1999. Random amplified polymorphic DNA (RAPD) markers reveal less genetic variation in the endangered plant Cerastium fisherianum var. molle than in the widespread conspecific C. fisherianum var. fisherianum (Caryophyllaceae). Mol. Ecol. 8, 145–150. Maki, M., Horie, S., Yokoyama, J., 2002. Comparison of genetic diversity between narrowly endemic shrub Menziesia goyozanensis and its widespread congener M. pentandra (Ericaceae). Cons. Genet. 3, 421–425. Mantel, N., 1967. The detection of disease clustering and generalized regression approach. Cancer Res. 27, 209–220. Meekins, J.F., Ballard, H.E., Mccarthy, B.C., 2001. Genetic variation and molecular biogeography of a North American invasive plant species (Alliaria petiolata, Brassicaceae). Int. J. Plant Sci. 162, 161–169. Meloni, M., Perini, D., Filigheddu, R., Binelli, G., 2006. Genetic variation in five Mediterranean populations of Juniperus phoenicea as revealed by inter-simple sequence repeat (ISSR) markers. Ann. Bot. 97, 299–304. Nagaoka, T., Ogihara, Y., 1997. Applicability of inter-simple sequence repeat polymorphisms in wheat for use as DNA markers in comparison to RFLP and RAPD markers. Theor. Appl. Genet. 94, 597–602. Nei, M., 1973. Analysis of gene diversity in subdivided populations. Proc. Natl. Acad. Sci. USA 70, 3321–3323. Nei, M., Li, W.H., 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci. USA 76, 5269–5273. Nybom, H., Bartish, I.V., 2000. Effects of life history traits and sampling strategies on genetic diversity estimates obtained with RAPD markers in plants. Perspect. Plant Ecol. Evol. Syst. 3/2, 93–114. Pye, M.G., Gadek, P.A., 2004. Genetic diversity, differentiation and conservation in Araucaria bidwillii (Araucariaceae), Australia’s Bunya pine. Cons. Genet. 5, 619–629. Pye, M.G., Henwood, M.J., Gadek, P.A., 2009. Differential levels of genetic diversity and divergence among populations of an ancient Australian rainforest conifer, Araucaria cunninghamii. Plant Syst. Evol. 277, 173–185. Quilichini, A., Debussche, M., Thompson, J.D., 2001. Evidence for local outbreeding depression in the Mediterranean island endemic Anchusa crispa Viv. (Boraginaceae). Heredity 87, 190–197. Rohlf, F.J., 1993. NTSYS: Numerical and Multivariate Analysis System, Version 1.8. Exeter Software, New York. Schaal, B.A., Leverich, W.J., 2005. Conservation genetics: theory and practice. Ann. Mo. Bot. Gard. 92, 1–11. Schneider, S., Roessli, D., Excoffier, L., 2000. Arlequin Ver. 2.000: a Software for Population Genetics Data Analysis. Genetics and Biometry Laboratory, University of Geneva, Geneva. Sokal, R.R., Rohlf, F.J., 2003. Biometry. W.H. Freeman and Company, New York. SPSS, 2005. SPSS for Windows. SPSS Inc., Chicago. Su, Y.J., Wang, T., Zheng, B., Jiang, Y., Chen, G.P., Ouyang, P.Y., Sun, Y.F., 2005. Genetic differentiation of relictual populations of Alsophila spinulosa in southern China inferred from cpDNA trnL–F noncoding sequences. Mol. Phylogenet. Evol. 34, 323–333. Swofford, D.L., 1998. PAUP*: Phylogenetic Analysis Using Parsimony (* and Other Methods), Version 4. Sinauer, Sunderland. 588 Y. Su et al. / Biochemical Systematics and Ecology 37 (2009) 579–588

Vergeer, P., Rengelink, R., Copal, A., Ouborg, N.J., 2003. The interacting effects of genetic variation, habitat quality and population size on performance of Succisa pratensis. J. Ecol. 91, 18–26. Walter, R., Epperson, B.K., 2001. Geographic pattern of genetic variation in Pinus resinosa: area of greatest diversity is not the origin of postglacial populations. Mol. Ecol. 10, 103–111. Wang, D.L., Li, Z.C., Hao, G., Chiang, T.Y., Ge, X.J., 2004. Genetic diversity of Calocedrus macrolepis (Cupressaceae) in southwestern China. Biochem. Syst. Ecol. 32, 797–807. Wolfe, A.D., Xiang, Q.Y., Kephart, S.R., 1998. Assessing hybridization in natural populations of Penstemon (Scrophulariaceae) using hypervariable inter-simple sequence repeat (ISSR) bands. Mol. Ecol. 7, 1107–1126. Wright, S., 1943. Isolation by distance. Genetics 28, 114–138. Yang, X., Yu, M.J., Ding, B.Y., Xu, S.X., Ye, L.X., 2005. Population structure and community characteristics of Pseudotaxus chienii in Fengyangshan National Natural Reserve. Chin. J. Appl. Ecol. 16, 1189–1194. Ye, T.Z., Yang, R.C., Yeh, F.C., 2002. Population structure of a lodgepole pine (Pinus contorta) and jack pine (P. banksiana) complex as revealed by random amplified polymorphic DNA. Genome 45, 530–540. Yeh, F.C., Yang, R., 1999. POPGENE Version 1.31. Department of Renewable Resources, University of Alberta, Edmonton. Available from: http://www.ualberta. ca/wfyeh. Zhang, Z.Y., Chen, Y.Y., Li, D.Z., 2005. Detection of low genetic variation in a critically endangered Chinese pine, Pinus squamata, using RAPD and ISSR markers. Biochem. Genet. 43, 239–249. 本文献由“学霸图书馆-文献云下载”收集自网络，仅供学习交流使用。

学霸图书馆（www.xuebalib.com）是一个“整合众多图书馆数据库资源，

提供一站式文献检索和下载服务”的24 小时在线不限IP 图书馆。 图书馆致力于便利、促进学习与科研，提供最强文献下载服务。

图书馆导航：

图书馆首页 文献云下载 图书馆入口 外文数据库大全 疑难文献辅助工具