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. 2005 Mar 21;95(7):1145–1151. doi: 10.1093/aob/mci125

Genetic Diversity in Fragmented Populations of Berchemiella wilsonii var. pubipetiolata (Rhamnaceae)

MING KANG 1, MINGXI JIANG 1, HONGWEN HUANG 1,*
PMCID: PMC4246897  PMID: 15781439

Abstract

• Backgroud and Aims Berchemiella wilsonii var. pubipetiolata (Rhamnaceae) is distributed in fragmented habitat patches in eastern China. It is highly endangered because of severe disturbance by anthropogenic activities. Information on genetic variation and structure is critical for developing successful conservation strategies for this species.

• Methods Allozyme variation of population genetic diversity and structure was investigated for a total of 98 individuals sampled from four extant populations using isoelectric focusing in thin-layer polyacrylamide slab gels.

• Key Results Based on 20 loci scored from the nine enzymes examined, a high genetic diversity was detected at both the species and population level, while there was a loss of low frequency alleles (<0·1) in all populations. Most loci showed deviation from Hardy–Weinberg equilibrium due to excess of heterozygotes in all populations, suggesting that selection for heterozygotes has occurred in this species. The genetic diversity was mainly found within populations with a moderate genetic differentiation (FST = 0·13), but the two geographically discontinuous population groups showed significant differences, with F-statistic values of 0·078 for the Zhejiang populations and 0·014 for the Anhui populations, respectively.

• Conclusions It appears most likely that this species has experienced a recent decrease in population size, and genetic drift in small populations has resulted in a loss of alleles occurring at low frequency. The differentiation into two population groups reflects a population genetic consequence that has been influenced by the different land-use in the two regions. Some conservation concerns are discussed together with possible strategies for implementing in situ and ex situ conservation.

Keywords: Berchemiella wilsonii var. pubipetiolata, allozyme, habitat fragmentation, genetic diversity, heterozygote excess, conservation genetics

INTRODUCTION

Habitat loss and fragmentation have become great concerns to conservation geneticists as anthropogenic activities and environmental deterioration have broken large, continuous populations into small and isolated ones. Populations in fragmented habitats are considered more vulnerable to demographic, environmental and genetic stochasticity, and therefore face a higher risk of local extinction (Boyce, 1992; Tilman et al., 1994, Lande, 1999).

Experimental and field investigations have demonstrated that fragmented populations may lose allelic richness or genetic diversity, and have increased population differentiation due to genetic drift and inbreeding depression (Buza et al., 2000; Shea and Furnier, 2002; Tomimatsu and Ohara, 2003). However, many studies based on allozymes have shown that even narrowly restricted species may maintain high levels of diversity (Ranker, 1994; Lewis and Crawford, 1995; Sharma et al., 2003). Young et al. (1993) found increased levels of gene flow among fragmented populations. Britten (1996) suggested that isolated populations might exhibit high heterozygosity due to strong selection pressures. Such a genetic structure was also detected in populations of a narrowly endemic plant established before and after fragmentation (Gonzalez-Astorga and Nunez-Farfan, 2001). The genetic consequences caused by habitat fragmentation are not yet well understood and further investigation is needed (Young et al., 1996). In the present study, an attempt was made to examine the impact of habitat fragmentation on the genetic diversity and population structure of Berchemiella wilsonii var. pubipetiolata, an endangered plant with a narrow distribution in eastern China. Such population genetic information is a prerequisite to understanding the species survival possibility in the short-term, so that an effective conservation strategy for long-term survival can be formulated and implemented.

Berchemiella wilsonii was collected in the western Hubei Province, central China and described by E. H. Wilson in 1907 (Fu and Jin, 1992). Berchemiella wilsonii var. pubipetiolata was first described in 1988 (Qian, 1988). It is a deciduous tree up to 12 m tall which differs clearly from var. wilsonii in having pubescent petioles. Berchemiella wilsonii var. pubipetiolata is only found in two disjunct regions: the north-eastern Dabie Mountain in Anhui Province and the western Tianmu Mountain in Zhejiang Province (Li, 1988; Qian, 1988). Seven populations of the species were previously documented (Qian, 1988) (Fig. 1). However, the populations of B. wilsonii var. pubipetiolata have been severely disturbed by deforestation, road construction, tourism and urbanization in the past two decades. During extensive field surveys conducted in 2001 and 2002, only four sizeable populations were located, including two populations in the north-eastern Dabie Mountain and two populations in the western Tianmu Mountain. The species is a weak competitor and is scattered as a forest understorey tree in lowland drainage basins adjacent to streams and rivers at altitudes between 500 and 1200 m a.s.ll. In Zhejiang Province, B. wilsonii var. pubipetiolata individuals grow either in typical native forest of mixed evergreen and deciduous broad-leaved trees dominated by Liquidambar formosana, Acer davidiana and Cyclobalanopsis gracilis, or in the understorey of cultivated Carya cathayensis plantations. However, in Anhui Province, the populations are largely undisturbed and the dominant companion species usually are native Platycarya strobilacea, Acer davidiana and Cyclobalanopsis gracilis.

Fig. 1.

Fig. 1.

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Map of the historical and extant populations of Berchemiella wilsonii var. pubipetiolata and sampling locations (open circles, extinct sites; filled circles, sampling sites): 1, Tuankou; 2, Maxiao; 3, Jixi; 4, Wanfushan; 5, Majiahe; 6, Jinzai; 7, Qianshan.

Little is know about the biology of the species. During the authors' field survey, mature trees were regularly found to produce hundreds of small hermaphrodite flowers (diameter approx. 4 mm) in May and June and are probably cross-pollinated. The fruits are mauve, long-ellipsoid drupes (approx. 8 mm long and approx. 3 mm in diameter) that ripen in August and are mostly dispersed by gravity. A recent study indicated that the both seed vigour and germination rate of this species were very low (37·8 % and 4 %, respectively) (Dang, 2004).

In this paper, allozyme analysis was used to examine the levels and patterns of genetic diversity in populations of B. wilsonii var. pubipetiolata to assess the effect of habitat fragmentation on population genetic structure of the species. The baseline genetic information should be useful for developing appropriate conservation strategies for B. wilsonii var. pubipetiolata.

MATERIALS AND METHODS

Populations sampled

About 25 plants of Berchemiella wilsonii (Schneid.) Nakai var. pubipetiolata Qian were sampled from four extant populations, excepting the Maxiao population site where some extant plants are not accessible on high mountain cliffs (Fig. 1 and Table 1), including two western Tianmu Mountain in Zhejiang Province (Tuankou population, 30°00′N, 119°03′E; Maxiao population, 30°09′N, 118°52′E) and two other populations in north-eastern Dabie Mountain in Anhui Province (Wanfushan population, 31°03′N, 116°34′E; Majiahe population, 31°05′N, 116°11′E). The Anhui populations are in a regional nature reserve within which logging was prohibited in 1992. Most individuals in Zhejiang had been logged by local farmers, and samples were collected from re-sprouts of the old root stumps. No seedlings or new ramets were found at the sites of the two Zhejiang populations. A few seedlings were observed in the two Anhui populations. In the present study, only adult individuals were exhaustively sampled but separated from each other by at least 5 m to avoid repeated sampling of the same tree.

Table 1.

Genetic diversity of Berchemiella wilsonii var. pubipetiolata within populations

Population
 N
 A
 P
 Ho
 He
Tuankou 25 1·85 (0·67) 70·0 0·580 (0·430) 0·349 (0·243)
Maxiao 18 1·85 (0·67) 70·0 0·592 (0·432) 0·345 (0·235)
Wanfushan 24 1·85 (0·49) 75·0 0·563 (0·452) 0·324 (0·217)
Majiahe 31 1·80 (0·41) 70·0 0·545 (0·431) 0·308 (0·218)
Mean 1·85 71·3 0·590 0·348
Overall 2·10 (0·64) 85·0 0·567 (0·384) 0·378 (0·199)
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Standard errors are shown in parenthesis.

N, sample size; A, mean number of alleles per locus; P, percentage of polymorphic loci; Ho, observed heterozygosity; He, expected heterozygosity.

One-year-old twigs with mature buds were collected in spring 2003 before bud break. A total of 98 individuals was sampled, comprising 25 individuals at Tuankou, 18 at Maxiao, 24 at Wanfushan and 31 at Majiahe. Samples were stored at 4°C until electrophoresis was carried out.

Electrophoresis procedure

Twigs were soaked in water at room temperature (approx. 25°C) for 5 d and enzymes were extracted from the swollen buds. The extraction procedure follows Huang et al. (1994). Eletrophoresis was conducted using an isoelectric focusing polyacrylamide slab gel system (Mulcahy et al., 1981). Of the12 pre-screened enzyme systems, nine that resolved into clear banding patterns were then used for allozyme genetic analysis including acid phosphatase [ACP; EC (Enzyme Commission) 3.1.3.2], NAD(P)H-diaphorase (DIA; EC 1.6.2.2), esterase (EST; EC 3.1.1.–), isocitrate dehydrogenase (IDH; EC 1.1.1.41), malate dehydrogenase (MDH; EC 1.1.1.37), malic enzyme (ME; EC 1.1.1.40), phosphogluconate dehydrogenase (PGD; EC 1.1.1.44), phosphoglucoisomerase (PGI; EC 5.3.1.3) and shikimate dehydrogenase (SKD; EC 1.1.1.25). Gels were stained as described by Weeden and Wendel (1989) with minor modifications. A total of 20 loci was scored: Acp-1, Acp-2, Acp-3, Dia-1, Dia-2, Dia-3, Est-2, Est-3, Est-4, Idh-1, Idh-2, Mdh-1, Mdh-3, Mdh-4, Me, Pgd-1, Pgd-2, Pgi, Skd-1 and Skd-2.

Data analysis

A set of standard measures of genetic diversity were calculated using TFPGA version 1.3 (Miller, 1997), including allele frequencies for each locus, number of alleles per locus (A), percentage polymorphic loci (P, 0·95 criterion), observed heterozygosity (Ho), expected heterozygosity (He). Fixation indices (F) for each locus were estimated and tested for deviation from the Hardy–Weinberg equilibrium following the algorithm recommended by Levene (1949).

TFPGA was also used to determine F-statistics (Weir and Cockerham, 1984) at both the population and the group level. Statistical significance was tested using methods described by Workman and Niswander (1970). To determine the significance of the FST for each locus, the chi-square (χ2) test was used as χ2 = 2NFST(k − 1), with d.f. = (k − 1)(s − 1), where k is the number of alleles and s is the number of populations (Workman and Niswander, 1970). The 95 % confidence intervals of the F-statistics were estimated by 1000 bootstraps over loci for multi-locus, and by jackknifing over populations for the single-locus (Weir, 1990). Chi-square tests for heterozygeneity of allele frequencies across populations were performed using FSTAT version 2.9.1 (Goudet, 2000). A cluster analysis was conducted based on Nei's (1978) unbiased genetic distance/identity using unweighted pair group mean analysis (UPGMA).

RESULTS

A total of 42 alleles were scored in 20 loci, and the allele frequencies are presented in Table 2. Three loci (Dia-1, Mdh-1 and Pgd-1) were monomorphic in all four populations and the other 17 loci were polymorphic in at least one population. Allele frequencies were mostly heterogeneous across populations, except for Mdh-4 and Pgd-2 which were uniformly heterozygous in all four populations. Three alleles are exclusive to the Anhui populations, Dia-3b, Mdh-3b and Skd-1c, while another three alleles are exclusive to the Zhejiang populations, Acp-1c, Acp-3c and locus Skd-1a. Only one allele, Skd-2c, was found uniquely in the Tuankou population. There were no low frequency alleles (<0·1) observed in the Tuankou population, but three in each of the other three populations (Table 2).

Table 2.

Allele frequencies in Berchemiella wilsonii var. pubipetiolata at 20 allozyme loci

Locus
 Allele
 Tuankou
 Maxiao
 Wanfushan
 Majiahe
Acp-1 a 0·440 0·083 0·500 0·500
b 0·400 0·639 0·500 0·500
c 0·160 0·278 0·000 0·000
Acp-2 a 0·340 0·694 1·000 0·984
b 0·660 0·306 0·000 0·016
Acp-3 a 0·500 0·500 0·500 0·500
b 0·260 0·083 0·500 0·500
c 0·240 0·417 0·000 0·000
Dia-1 a 1·000 1·000 1·000 1·000
Dia-2 a 1·000 0·639 0·542 0·629
b 0·000 0·361 0·458 0·371
Dia-3 a 1·000 1·000 0·625 0·871
b 0·000 0·000 0·375 0·129
Est-2 a 0·480 0·500 0·500 0·500
b 0·520 0·500 0·500 0·500
Est-3 a 0·440 0·417 0·479 0·355
b 0·560 0·500 0·500 0·645
c 0·000 0·083 0·021 0·000
Est-4 a 0·300 0·667 0·313 0·597
b 0·700 0·333 0·688 0·403
Idh-1 a 0·500 0·500 0·938 1·000
b 0·500 0·500 0·063 0·000
Idh-2 a 0·500 0·583 0·500 0·516
b 0·500 0·417 0·500 0·484
Mdh-1 a 1·000 1·000 1·000 1·000
Mdh-3 a 1·000 1·000 0·896 0·823
b 0·000 0·000 0·104 0·177
Mdh-4 a 0·500 0·500 0·500 0·500
b 0·500 0·500 0·500 0·500
Me a 0·800 0·500 0·667 0·613
b 0·200 0·500 0·333 0·387
Pgd-1 a 1·000 1·000 1·000 1·000
Pgd-2 a 0·500 0·500 0·500 0·500
b 0·500 0·500 0·500 0·500
Pgi a 0·500 0·333 0·833 0·903
b 0·500 0·667 0·167 0·097
Skd-1 a 0·580 0·500 0·000 0·000
b 0·420 0·500 0·500 0·500
c 0·000 0·000 0·500 0·500
Skd-2 a 0·220 1·000 0·979 0·968
b 0·580 0·000 0·021 0·032
c 0·200 0·000 0·000 0·000
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Genetic diversity parameters within populations are listed in Table 1. The mean number of alleles per locus (A) at population and species level were 1·85 and 2·10, respectively. The overall value of P was 85·0 % for the species, while the average P at the population level was 71·3 %, ranging from 70·0 % for the Tuankou, Maxiao and Majiahe populations to 75·0 % for the Wanfushan population. The mean expected heterozygosity (He) was 0·378, ranging from 0·308 for the Majiahe population to 0·349 for the Tuankou population. However, the observed heterozygosity was very high with an average value of 0·590, ranging from 0·545 at Majiahe to 0·592 at Maxiao population. A significantly high deviation from Hardy–Weinberg equilibrium was found in all populations, reflected by the fact that most values of F indexes are near, or equal, to −1 at 17 polymorphic loci (Table 3). This result indicates that an excess of heterozygotes existed in all populations examined.

Table 3.

Fixation indices (F) and chi-square tests for Hardy–Weinberg equilibrium in four populations of Berchemiella wilsonii var. pubipetiolata

Loci
 Tuanlou
 Maxiao
 Wanfushan
 Majiahe
Acp-1 −0·418** −0·094* −1·000** −1·000**
Acp-2 −0·337NS −0·440* – −0·016NS
Acp-3 −0·600** −0·756** −1·000** −1·000**
Dia-2 – −0·565* −0·846** −0·590**
Dia-3 – – −0·600** −0·148NS
Est-2 −0·923** −1·000** −1·000** −1·000**
Est-3 −0·786** −0·756** −0·923** −0·550**
Est-4 −0·429* −0·500* −0·261NS −0·676**
Idh-1 −1·000** −1·000** −0·067NS –
Idh-2 −1·000** −0·714** −1·000** −0·937**
Mdh-3 – – −0·116NS −0·215NS
Mdh-4 −1·000** −1·000** −1·000** −1·000**
Me −0·250NS −1·000** −0·500* −0·632**
Pgd-2 −1·000** −1·000** −1·000** −1·000**
Pgi −1·000** 0·000NS 1·000** −0·107NS
Skd-1 −0·724** −1·000** −1·000** −1·000**
Skd-2 0·235** – −0·021NS −0·033NS
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–, Not applied.

*

P < 0·05;

**

P < 0·01;

NS

not significant.

Partitioning of population genetic diversity based on the 17 polymorphic isozyme loci revealed that a significant population differentiation occurred in the natural habitat of B. wilsonii var. pubipetiolata, as demonstrated by mean FST = 0·130, suggesting that about 13 % of allozyme diversity occurred among populations (Table 4). Chi-square tests showed that 12 of the 17 polymorphic loci were significantly different in allele frequencies across populations (Table 4). Further examination of the population genetic structures in the two different provinces and testing of the significance of population differentiation showed that Zhejiang populations had a significant FST of 0·078, while the differentiation in Anhui populations was negligible (FST = 0·014) (Table 5). Indirect estimation of gene flow for the overall populations is Nm = 1·68, but Nm = 2·97 for Zhejiang populations and Nm = 17·28 for Anhui populations, respectively.

Table 4.

Wright's F-statistics for Berchemiella wilsonii var. pubipetiolata populations

Locus
 FIS
 FIT
 FST
Acp-1 −0·614 −0·472 0·091**
Acp-2 −0·374 0·161 0·389**
Acp-3 −0·823 −0·639 0·101**
Dia-2 −0·671 −0·434 0·148**
Dia-3 −0·454 −0·144 0·213**
Est-2 −0·981 −0·980 0·000
Est-3 −0·760 −0·737 0·013NS
Est-4 −0·473 −0·312 0·109**
Idh-1 −0·902 −0·362 0·284**
Idh-2 −0·914 −0·905 0·005NS
Mdh-3 −0·177 −0·076 0·086**
Mdh-4 −1·000 −1·000 0·000
Me −0·634 −0·551 0·051*
Pgd-2 −1·000 −1·000 0·000
Pgi −0·173 0·109 0·240**
Skd-1 −0·932 −0·514 0·217**
Skd-2 0·195 0·605 0·509**
Overall† −0·717 (0·059) −0·494 (0·110) 0·130** (0·040)
95 % CI −0·871–0·634 −0·731–0·316 0·057–0·214
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*

P < 0·05;

**

P < 0·01;

NS

not significant.

†

Standard deviations in parentheses.

Table 5.

Wright's F-statistics estimated for each population groups and overall populations of Berchemiella wilsonii var. pubipetiolata

 FIT
 FST
 FIS
 Nm
Overall populations −0·494 0·130** −0·717 1·68
Within groups
    Zejiang group −0·553 0·078* −0·683 2·97
    Anhui group −0·728 0·014NS −0·753 17·28
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*

P < 0·05;

**

P < 0·01;

NS

not significant.

Mean genetic identity among the four populations was 0·905, ranging from I = 0·991 found between the Majiahe and Wanfushan populations, to I = 0·857 between the Tuankou and Wanfushan populations. The UPGMA cluster analysis further revealed two distinct clusters representing the Zhejiang and Anhui groups (Fig. 2).

Fig. 2.

Fig. 2.

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UPGMA dendrogram showing genetic relationship of four populations of Berchemiella wilsonii var. pubipetiolata, based on Nei's unbiased genetic distance (Nei, 1978).

DISCUSSION

Genetic diversity

Many investigations of allozyme variation have demonstrated that small and fragmented plant populations have low levels of genetic diversity (Godt et al., 1997; Jones and Gliddon, 1999; Kollmann et al., 2000; Cheon et al., 2002). This is obviously not the case in B. wilsonii var. pubipetiolata. The values of P and He at the species level (P = 85·0, He = 0·378) were much higher than the average values previously found in other narrowly distributed woody species (P = 61·5, He = 0·165), while A was comparable to values for narrowly distributed species (A = 2·08) (Hamrick et al., 1992). Genetic diversity maintained within a species is considered as to a result of both historical events and recent evolutionary processes (Gonzalez-Astorga et al., 2003). The present genetic diversity retaining in populations of B. wilsonii var. pubipetiolata appears to be related to life history of the species. Being a member of a Tertiary relic genus (Li, 1988), B. wilsonii var. pubipetiolata may retain a high diversity which is in agreement with Witter and Carr (1988) who proposed that older taxa might be more diverse. Additionally, this species has experienced a recent decline in population size and local population extinctions during the past 20 years (Fig. 1); therefore, the current results might just reflect the historical patterns of genetic diversity in B. wilsonii var. pubipetiolata.

Although B. wilsonii var. pubipetiolata had high diversity at both population and species level in the present study, all populations lack rare alleles (<0·1) (Table 2). This may be explained as a result of random genetic drift caused by the recent reduction in population size. In general, low frequency allozyme alleles are far more abundant than alleles at intermediate frequency in undisturbed natural populations (Chakraborty et al., 1980). However, alleles at low frequencies are prone to be lost more rapidly than the loss of overall genetic variation when populations experience a rapid reduction in size (Nei et al., 1975; Leberg, 1992). Thus overall heterozygosity may not decrease substantially unless the population remains small for several generations (Nei et al., 1975; Allendorf, 1986). Such a loss of rare alleles has been reported in the tree Eucalyptus albens (Prober and Brown, 1994), the endangered daisy Rutodosis leptorrhynchoides (Young et al., 1999) and the endemic shrub Brongniartia vazquezii (Gonzalez-Astorga and Nunez-Farfan, 2001). Apparently, habitat fragmentation in natural populations could result in an immediate loss of rare alleles and a reduction of allele richness rather than a reduction of overall genetic heterozygosity. It is suggested that allele composition and richness should be monitored in genetic assessments of newly fragmented populations, as these estimators may be initial signs of deterioration of the population genetic dynamics.

Genetic fixation and excess of heterozygotes

Negative values of fixation index (F) were found at almost all loci across all four populations of B. wilsonii var. pubipetiolata, indicating an extreme excess of heterozygotes. A possible explanation for this pattern is that natural selection might favour heterozygotes that can cope with environment changes in these highly fragmented populations. Two factors are usually involved in driving selection for heterozygotes, environmental stress and inbreeding depression. For instance, Luijten et al. (2000) found excess of heterozygotes in small populations of Arnica montana when population size decreased, suggesting that the heterozygous individuals in small populations are survivors from formerly larger populations which have relatively high fitness. However, inbreeding depression often results in low levels of heterozygosity. Such an example is Piper cernuum (Mariot et al., 2002) in which heterozygote excess was revealed in all but the smallest populations, which was probably attributable to the effects of accentuated genetic drift. Accordingly, the occurrence or distribution of heterozygote excess seems to be species or case specific. In a fragmented population that has become successively smaller, random genetic drift should have a noticeable effect on allele composition and in reducing the proportion of heterozygotes. However, if a small population is the result of a recent and sudden reduction in size, inbreeding may not yet have had an effect on genetic structure. In tree species, heterozygote excess is quite common in adult stages or in old age classes (Bush and Smouse, 1992), and it is most likely that selection favouring heterozygotes and/or favouring against homozygotes is a common phenomenon in natural plant populations. Doligez and Joly (1997) used allozyme analysis for genotyping seeds and adults in a tropical tree Carapa procera, and found excess homozygotes in seeds and excess heterozygotes in adults, suggesting a strong selection for heterozygotes. Similar results have also been detected in other long-lived perennials (Arnica montana, Luijten et al., 2000; Piper cernuum, Mariot et al., 2002; Dioon edule, Gonzalez-Astorga et al., 2003).

Furthermore, in small and fragmented populations, the homozygous seeds produced by inbreeding may have lower germination and higher mortality than heterozygous seeds by outbreeding. Also, homozygous seedlings may have lower vigour than heterozygous ones. A high proportion of heterozygous seedlings has been observed in many plant populations (Bush and Smouse, 1992; Lee et al., 2000). Theoretically, inbreeding will result in an excess of homozygotes, but the proportion may be reduced at different life stages. For example, lethals and sublethals expressed at an early stage would leave more heterozygous individuals to be detected in surviving seeds and result in heterozygote excess (Keller and Waller, 2002). In a parallel study, it was found that the seed vigour of newly collected seeds was only 37·8 %, and the germination rate was only 4·0 % in the natural habitat. The highest germination rate obtained so far was less than 13·0 %, in spite of the fact that various treatments were attempted in experimental conditions (Dang, 2004). It is hypothesized that most homozygous seeds caused by inbreeding were aborted and could not germinate, which suggests that genotyping analysis for seeds and seedlings at different ages should be considered in future.

Genetic differentiation

The value of FST, a measure of the degree of differentiation among populations, was 0·130 for B. wilsonii var. pubipetiolata, revealing that about 87 % of the total genetic diversity resides within populations. This value is slightly higher than the average GST for narrowly long-lived woody species (GST = 0·124) but almost equal to that of species with gravity dispersed seeds (GST = 0·131) (Hamrick et al., 1992). The theory of population genetics predicts that, as a population becomes progressively smaller and more isolated, genetic differentiation caused by genetic drift and inbreeding depression should increase (Templeton et al., 1990). However, the genetic consequences of habitat fragmentation are complicated because of different life-history characteristics and pre-fragmentation abundances (Young et al., 1996). It appears that the moderate genetic differentiation and substantial gene diversity in the populations of B. wilsonii var. pubipetiolata documented in the present study reflect recent fragmentation of habitat and population decline. Although the geographical distance between the two Anhui populations is farther than that between the two Zhejiang populations, a much higher gene flow rate occurred between the former than between the latter, suggesting that seed and pollen dispersal is more efficient between the Anhui populations. This is consistent with the field observations that the numbers of individuals within populations were higher in the Anhui region, and also that re-generating seedlings were found in this region while no seedlings were found in Zhejiang region. Moreover, the population differentiation in Zhejiang and Anhui is significantly different (Table 5), reflecting the different land-use histories in the two regions. In fact, the insignificant FST of 0·014 detected among the Anhui populations could be considered negligible, suggesting a less disturbed habitat or a good recovery of B. wilsonii var. pubipetiolata populations since the area was designated as a nature reserve and logging was prohibited. The fact that some seedlings were observed in the region is promising. The two Zhejiang populations have been disturbed by road construction, agricultural practice and tourist exploitation over the past 50 years, and the habitat deterioration still continues. Berchemiella wilsonii var. pubipetiolata usually grows sparsely under stands of Chinese walnuts, which form the major economic resource for local residents. With increasing demand for walnuts, plantations have replaced the natural habitat of B. wilsonii var. pubipetiolata and local extinction of the species can be expected if no further conservation efforts are made.

Implications for conservation

The present results indicate that B. wilsonii var. pubipetiolata is not genetically depauperate in general. It appears that demographic stochasticity is a more immediate threat to the species than reduction of overall genetic diversity (Lande, 1999), and the main factor responsible for the reduction in population size is anthropogeneic activity. Habitat protection of the Zhejiang populations is particularly urgent since they are located in farmland. Conflicts between conservation and local interests is a difficult issue. There is evidence that tourism has severe impacts on threatened plants (Kelly et al., 2003). With the promotion of ‘ecotourism’ by local government, there have been rapidly increasing numbers of visitors in the Zhejiang region in recent years, the effects of which need to be investigated and monitored. Since most individuals grow on the sides of gullies in scenic sites (except for Majiahe population), B. wilsonii var. pubipetiolata is vulnerable to trampling and destruction by the infrastructure construction associated with tourism.

On the other hand, the genetic threat should not be neglected. The allozyme evidence obtained in the present study reflects the history of the population and the current status of genetic diversity remaining in B. wilsonii var. pubipetiolata. The genetic dynamics in the populations or the long-term survivability are still not understood, although it is suspected that population fragmentation will result in loss of rare alleles. The large percentage of aborted seeds and little seedling recruitment also observed raise great concern for long-term survival and suggests that immediate conservation measures need to be taken. However, the causes of seed abortion need further studies. Further studies should also focus on the reproductive biology of B. wilsonii var. pubipetiolata. Finally, it is suggested that appropriate numbers of seeds should be sampled from all populations in an ex situ conservation programme.

Acknowledgments

This work is supported by KIP Pilot Project of Chinese Academy of Sciences (KSCX2-SE-104) and National Natural Sciences Foundation of China (30470185). We thank Dr Hong Qian and Dr Desmond Layne for their discussion on the manuscript, Mrs Yuanyuan Chen for her laboratory assistance and Mr Haishan Dang for his help in field collection. We also thank Dr Mikael Hedren and another anonymous reviewer for their valuable comments and suggestions.

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