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Genetic diversity of Calocedrus macrolepis (Cupressaceae) in southwestern China
Biochemical Systematics and Ecology 32 (2004) 797–807 www.elsevier.com/locate/biochemsyseco
Genetic diversity of Calocedrus macrolepis (Cupressaceae) in southwestern China De-Lian Wang a, Zhong-Chao Li a, Gang Hao a, Tzen-Yuh Chiang b, Xue-Jun Ge a, a
South China Institute of Botany, Chinese Academy of Sciences, Guangzhou 510650, People’s Republic of China b Department of Biology, Cheng-Kung University, Tainan, 701, Taiwan Received 28 June 2003; accepted 20 December 2003
Abstract Genetic diversity and differentiation among five populations of Calocedrus macrolepis in southwestern China were investigated using inter-simple sequence repeats (ISSR) markers. Low genetic diversity was revealed, with mean percentage of polymorphic loci at P ¼ 26:9% and average expected heterozygosity of HE ¼ 0:1114. Low levels of genetic differentiation among populations were detected based on Nei’s genetic diversity analysis (4.2%), Shannon’s diversity index (2.7%), and AMOVA (4.1%). Pairwise genetic identity (I ) values among populations ranged from 0.9899 to 0.9964, with a mean of 0.9932. There was no correlation between genetic and geographic distance among the populations studied. Possible genetic bottlenecks during Quaternary glaciations coupled with effects of genetic drift that shaped scattered and small populations were postulated to be the main reasons for the low genetic diversity in Calocedrus macrolepis. # 2004 Elsevier Ltd. All rights reserved. Keywords: Calocedrus macrolepis; Cupressaceae; Genetic variation; ISSR
1. Introduction To estimate the level and distribution of genetic variation in endangered species is a primary objective of conservation genetics (Fritsch and Rieseberg, 1996). Genetic variation at intraspecific level is a prerequisite for future adaptive change
Corresponding author. Tel.: +86-20-8523-2051; fax: 86-20-8523-2831. E-mail address: [email protected] (X.-J. Ge).
0305-1978/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2003.12.003
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or evolution and has profound implications for species conservation (Schaal et al., 1991) and future breeding programs. Loss of genetic diversity usually has deleterious effects on species fitness and may threaten the survival of populations (Malone et al., 2003; Reed, 2003). Genetic diversity of a species is determined by many evolutionary factors, such as dispersal, life history characters, and demographic history. Correlations between genetic diversity and various attributes of species have been examined based on a vast amount of data of genetic diversity in natural plant populations (Hamrick and Godt, 1989). It was found that gymnosperms (mostly conifers) maintain relatively high levels of genetic variation within populations and display little genetic differentiation among populations (Hamrick et al., 1992). However, relatively low genetic diversity or high level of genetic differentiation has been recorded within a number of coniferous taxa, such as Pinus resinosa (Simon et al., 1986), Abies bracteata (Ledig, 1987), and Cathaya argyrophylla (Ge et al., 1996). The genus Calocedrus Kurz consists of two species and one variety of evergreen trees with amphi-north Pacific distribution in western North America and eastern Asia. Calocedrus decurrens (Torr.) Florin occurs in western USA and Mexico, whereas C. macrolepis Kurz is endemic to southwestern China and the adjacent regions of Myanmar and Vietnam. In China, C. macrolepis is distributed in Guizhou and Yunnan Provinces at elevations ranging from 300 to 2000 m and can be found sporadically in Guangxi and Hainan Provinces. C. macrolepis Kurz var. formosana (Florin) Cheng et L. K. Fu grows in northern and central Taiwan (Li, 1975; Fu et al., 1999). Calocedrus has been considered as representing an ancient lineage. This gymnosperm genus was widespread in the temperate according to fossil evidence of European Tertiary (Kracek, 1999) and Asiatic Oligocene to Pliocene sediments (WGCPC, 1978; Liu et al., 1996). Calocedrus macrolepis is an element of evergreen subtropical forests in mountainous areas. Its DBH (diameter at breast height) of the trunk can reach 60–80 cm, and the height 15–25 m (rarely 30) m on favorable sites. The wind-pollinated species is monoecious, with male and female cones occurring on different branches (Fu et al., 1999). With two subapical, unequal wings, the light and small seeds of C. macrolepis can be disseminated by wind for a long distance. Populations of C. macrolepis are dramatically shrinking in China due to heavy deforestation and extensive habitat loss in past decades, although regeneration is sound in places with ample light, such as habitats along streams or at forest edges. Calocedrus macrolepis has been classified as a vulnerable species according to the criteria of IUCN Red List (Farjon, 2001), and has been listed as a species under serious threat in China (Fu, 1995) and Vietnam (WCMC, 1997). Previous studies have mostly focused on ecology (Ning et al., 1997; Cheng et al., 2001) and cytology (Li, 1998) of C. macrolepis. However, its intraspecific genetic variation remains unknown, despite the level of genetic diversity, and the way in which it is maintained among populations is of major concern in conservation biology (Maki, 2003). The state of variability within and between populations can be estimated by applications of both biometrical and molecular procedures. However, it is only the procedures of molecular genetics that allow a measure of population structure at the level of the gene and genome to be gained (Hayward and
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Hamilton, 1997). Among various molecular tools, inter-simple sequence repeats (ISSR) has been widely used for population genetic study recently (Gupta et al., 1994; Tsumura et al., 1996). ISSR fingerprinting has demonstrated a hypervariable nature of the markers and its potential power for population studies (Culley and Wolfe, 2001). Technically, ISSR fingerprinting is more reproducible than RAPD amplification due to the longer SSR-based primers, thus enabling higher-stringency DNA amplifications (Wolfe et al., 1998). In this study, we assess the extent of genetic variation within and between populations of C. macrolepis in China with ISSR markers and formulate recommendations for conservation and breeding strategies of this endangered conifer based on the observed genetic structure.
2. Materials and methods 2.1. Sample collection and PCR amplification Twigs of 121 individuals of C. macrolepis were collected from five populations in Guizhou and Yunnan Provinces in southwestern China (Fig. 1, Table 1). Trees ca. 5 m apart were randomly sampled within each population. Twigs were dried with v silica gel and stored at 4 C prior to DNA extraction. Genomic DNA was extracted using a CTAB method described by Doyle (1991). 100 primers (University of British Columbia primer set 9) were first screened for PCR amplification. Ten ISSR primers (UBC # 807, 808, 811, 812, 835, 836, 842, 857, 874 and 881) that generated clear, reproducible banding patterns were chosen for final analysis. Polymerase chain reactions (PCR) and gel electrophoresis were carried out as described in Ge et al. (2003).
Fig. 1. Map showing locations of the population of C. macrolepis sampled.
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Table 1 Populations of Calocedrus macrolepis for ISSR analysis Population Anning, Yunnan Lincang, Yunnan Yuanjiang, Yunnan Danzhai, Guizhou Libo, Guizhou
Pop. Code AN LC YJ DZ LB
Altitude (m)
Latitude (N)
Longitude (E)
Sample size
1600 1200 1600 900 960
24 60 v 23 50 v 23 30 v 26 160 v 25 140
102 200 v 100 90 v 101 500 v 107 590 v 107 540
30 30 30 19 12
v
v
2.2. Data analysis Only bands that could be unambiguously scored across all the population samples were used in the analysis. ISSR profiles were scored for each individual as present (1) or absent (0) of a specific band. A set of measures of intra- and interpopulation genetic statistics were generated using the program POPGENE 1.31 (Yeh et al., 1999) including: Nei’s (1973) gene diversity, the percentage of polymorphic loci (P), expected heterozygosity (HE), total genetic diversity (HT), genetic diversity within population (HS) and the relative magnitude of genetic differentiation among populations (GST ¼ HT HS =HT ). Gene flow was estimated using the formula: Nm ¼ 0:25ð1 GST Þ=GST . Nei’s (1972) genetic identity (I ) and genetic distance (D) were calculated for all pairwise combinations of populations. The POPGENE software package was also used to calculate Shannon’s index of phenotypic diversity for ISSR diploid data according to Ho ¼ Rpi log2 pi (Lewinton, 1972), in which pi is the frequency of a given ISSR fragment. Ho was calculated at two levels: the average diversity within populations (Hpop), and the total diversity (Hsp). The proportion of diversity among populations was estimated as (Hsp HpopÞ=Hsp. Components of variance partitioned into within and between populations were also estimated using AMOVA. Input data files for the AMOVA v. 1.55 program (Excoffier et al., 1992) were generated using AMOVA-PREP (Miller, 1998). The number of permutations for significant testing was set at 1000 for analysis. AMOVA variance components were used as estimates of the genetic diversity within and between populations. A UPGMA (unweighted pair-group method using arithmetic average) dendrogram was constructed based on the matrix of Nei’s genetic distance using the SAHN—clustering and TREE programs from NTSYS-pc 2.0 (Rohlf, 1998). In order to test for a correlation between genetic (D) and geographical distances (in km) among populations, a Mantel test was performed using Tools for Population Genetic Analysis (TFPGA; Miller, 1997) (computing 1000 permutations). 3. Results 3.1. The ISSR profile and within-population variability A total of 78 different ISSR bands were scored, corresponding to an average of 7.8 bands per primer. Among the 78 loci, 21 were polymorphic at the species level
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Table 2 Genetic variability within populations of Calocedrus macrolepis shown by ISSR. P, percentage of polymorphic loci; HE, expected heterozygosity. Ho: Shannon’s diversity. Standard deviations are in parentheses Population
P (%)
HE
Ho
AN LC YJ DZ LB Mean
26.92 26.92 26.92 26.92 26.92 26.92
0.104 (0.179) 0.108 (0.182) 0.111 (0.188) 0.118 (0.197) 0.116 (0.194) 0.1114 (0.0057)
0.154 (0.260) 0.158 (0.265) 0.161 (0.271) 0.169 (0.282) 0.167 (0.279) 0.1618 (0.0062)
(Table 2). All of these 21 loci are polymorphic in all populations, only different on their frequencies. The percentage of polymorphic loci (P) is 26.9% for all five populations. Assuming Hardy–Weinberg equilibrium, the average gene diversity was estimated to be 0.1114 within populations (HE), and 0.116 at the species level (HT). The Shannon indices (Ho) ranged from 0.154 to 0.169, with an average of 0:1618 0:0062 at population level (Hpop) and 0.1662 at species level (Hsp). 3.2. Between-population diversity The coefficient of overall genetic differentiation between populations (GST) was 0.042 as estimated by partitioning of the total gene diversity. The Shannon’s diversity index analysis partitioned 2.7% of the total variation between populations. Pairwise genetic identity values (I ) among populations ranged from 0.9899 to 0.9964 with a mean of 0.9932. Genetic distances (D) between populations ranged from 0.0036 to 0.0101, with a mean of 0.0069 (Table 3). The level of gene flow (Nm) was estimated to be 5.7 individuals per generation between populations. According to AMOVA analysis, there were significant (P < 0:001) genetic differences among the five populations of C. macrolepis. Of the total genetic diversity, 4.1% was attributable to among-populations and the rest (95.9%) to differences within populations (Table 4).
Table 3 Geographical distance and Nei’s (1972) genetic distance Population
AN
LC
YJ
DZ
LB
AN LC YJ DZ LB
– 0.0067 0.0090 0.0101 0.0062
266 – 0.0080 0.0081 0.0049
175 173 – 0.0061 0.0059
585 843 694 – 0.0036
565 807 648 115 –
Geographical distance (above diagonal) and Nei’s genetic distance (below diagonal).
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Table 4 AMOVA for five populations of Calocedrus macrolepis Source of variation
d.f.
Sum of squares
Mean squares
Variance component
% total variance
P-value
Among populations Within populations
4 116
39.5732 571.7491
9.893 4.929
0.2101 4.9289
4.1 95.9
< 0.001 < 0.001
Fig. 2. UPGMA dendrogram based on Nei’s (1972) genetic distance.
The correlation coefficient (r) between genetic and geographical distance using Mantel’s test for all populations was 0.0823 (P ¼ 0:6750). The UPGMA tree based on ISSR (Fig. 2) revealed a similar result in that the genetic distances among the populations do not show a spatial pattern corresponding to their geographic locations (Fig. 1). 4. Discussion Most conifers have high levels of genetic diversity and low levels of differentiation among populations, as measured by allozymes (Hamrick et al., 1992). However, due to the complex effects of historical factors such as speciation process and Quaternary glaciation, it is difficult to make an a priori prediction of the levels of genetic diversity in endangered species (Maki, 2003). When compared to other species (cf. Hamrick et al., 1992), total genetic diversity of C. macrolepis (HT: 0.116) is lower than that of the mean genetic diversity both for gymnosperms (0.169) and for long-lived woody perennial species (0.177). Compared to most Cupressaceae species (Table 5), the genetic variation in C. macrolepis is lower than that in the congeneric C. decurrens (HE ¼ 0:18; Harry, 1984), but slightly higher than that in Thuja occidentalis (Perry et al., 1990), T. plicata (Yeh, 1988) and Austrocedrus chilensis (Ferreyra et al., 1996).
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Table 5 Summary of genetic variation for some Cupressaceae species. P, percentage of polymorphic loci; HE, expected heterozygosity; Ho, Shannon’s diversity Species
P (%)
HE
Austrocedrus chilensis Calocedrus macrolepis C. decurrens Chamaecyparis formosensis C. nootkatensis C. taiwancensis C. thyoides Cupressus sempervirens Fitzroya cupressoides Juniperus communis J. coreana J. rigida J. thurifera Pilgerodendron uviferum Thuja occidentalis T. plicata
41
0.071
26.92
0.116
– 55.2
0.18
50 77.8 50.0 82.6
0.171
Ho
Genetic differentiation (%)
Marker used Reference
–
allozyme
Ferreyra et al., 1996
0.1662
2.71–4.2
ISSR
Present study
– 0.329
– 15.1
allozyme RAPD
Harry, 1984 Hwang et al., 2001
13.9 14.7 9.4 7.3
allozyme RAPD allozyme allozyme
14.4
RAPD
Ritland et al., 2001 Hwang et al., 2001 Kuser et al., 1997 Raddi and Suemer, 1999 Allnut et al., 1999
0.429 0.145 0.350
–
0.547
83.5
–
–
AFLP
54.6 72.7 98 35.7
0.199 0.224 – 0.712
11.8 17.4 38.90 15.9–18.6
allozyme allozyme RAPD RAPD
Van der Merwe et al., 2000 Huh and Huh, 2000 Huh and Huh, 2000 Jime´nez et al., 2003 Allnutt et al., 2003
50 26.3
0.094 0.04
1.6 3.3
allozyme allozyme
Perry et al., 1990 Yeh, 1988
– 0.730
Genetic diversity maintained in a plant species is influenced by specific characteristics of the species (Hamrick and Godt, 1989), as well as by its evolutionary history. The low level of genetic variation in C. macrolepis may be attributed to both its evolutionary history and its small isolated populations. The genus Calocedrus has an eastern Asia and North American disjunct distribution pattern. The isolation of most disjunct taxa in eastern Asia and North America may have occurred during the global climatic cooling throughout the late Tertiary and Quaternary (Xiang et al., 2000). Fossil evidence (Kracek, 1999) and the disjunct distribution of C. macrolepis var. formosana in Taiwan indicate that the ancestor of C. macrolepis was much more widespread in the Tertiary than at present. The suitable climate in southwestern China may have allowed the survival of certain forest taxa since the Mesozoic Era (Li, 1953; Walker, 1986). At least since the Oligocene, Calocedrus has lived in this area (WGCPC, 1978; Liu et al., 1996). Therefore, this area may represent one of refuge for gymnosperms (Contreras-Medina and Vega, 2002). Through the periodical and repeated range contraction, this species may have experienced severe bottlenecks and subsequent genetic drift that led to loss of genetic diversity. Although C. macrolepis is locally abundant in the subtropical eastern Asia, it consists of scattered and small fragmented populations in southwestern China due
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to the impacts of human activities. The vulnerable species displays a severe decline in the extent of occurrence and/or quality of habitat, together with the effects of exploitation. Although geographic range was considered a good predictor of the levels of genetic variation in plant populations (Hamrick and Godt, 1989), widespread species occurring in small and disjunct populations may not fit this pattern (Premoli et al., 2001). Fragmentation and isolation of populations can result in reduced genetic variation by genetic drift (Sherwin and Moritz, 2000), as detected in Pilgerodendron uviferum (Cupressaceae), a species widespread in temperate South America but consisting of scattered and small populations (Premoli et al., 2001). Positive correlations between genetic variability and population size have been reported for a number of plant species (Fischer et al., 2000). Sustained restrictions in population size are the main reason for loss of genetic diversity that will reduce the ability to evolve in response to ever-present environmental change and increase the risk of extinction (Frankham et al., 2002). The level of genetic differentiation is low among these five populations of C. macrolepis. This finding is in agreement with the conclusion of Hamrick et al. (1992). In most woody species, a mean GST value of 0.073 was recorded for 121 species of gymnosperm examined based on allozyme analyses (Hamrick et al., 1992). Even compared to genetic differentiation estimates of other Cupressaceae species, the values recorded for C. macrolepis are relatively low (Table 5), while slightly higher than that of Thuja occidentalis (1.6%; Perry et al., 1990) and T. plicata (3.3%; Yeh, 1988). Low level of genetic differentiation in gymnosperms is usually attributed to the frequent wind-pollination and breeding systems that promote outcrossing (Bennett et al., 2000). In addition, the seeds of C. macrolepis with membranous wings are also helpful for its long distance dispersal, as indicated by the high Nm values (=5.70) between populations. It should be mentioned that although our samples of five populations were collected from the main distribution area, some distribution areas, such as Taiwan, Northern Myanmar and NE Vietnam were not yet sampled, which may to some extent lead to biased estimates of genetic diversity of the species as a whole to a certain extent. This is critical for understanding the entirety of genetic diversity of C. macrolepis since the Taiwan populations have been recognized as a variety (C. macrolepis Kurz var. formosana (Florin) Cheng et L. K. Fu) (Cheng and Fu, 1978) or species (C. formosana (Florin) Florin) (Florin, 1956). The ultimate goals of conservation are to ensure sustainable survival of populations and to preserve their evolutionary potential. The estimates of genetic diversity and genetic differentiation provide a basis for implementing efficient and practical conservation programs for C. macrolepis. As the differences among populations of C. macrolepis in southwestern China are in allele frequencies rather than in gene composition, loss of populations at certain locations may not cause immediate loss in genetic diversity but more damage may occur in terms of long-term genetic consequences due to reduced number of populations and smaller population size. Replacement of the lost populations at certain locations can be achieved by transplanting seedlings from long-established populations.
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Acknowledgements We thank Dr Yong-Ming Yuan and Dr Li-Bing Zhang for helpful comments and providing references. Thanks are also due to two anonymous reviewers for their critical reviews and suggestions. This study was financially supported by the Knowledge Innovation Key Project (KSCX2-SW-104) and Field Frontiers Project (Director Foundation of SCIB) of the Chinese Academy Sciences.
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Genetic diversity of Calocedrus macrolepis (Cupressaceae) in southwestern China De-Lian Wang a, Zhong-Chao Li a, Gang Hao a, Tzen-Yuh Chiang b, Xue-Jun Ge a, a
South China Institute of Botany, Chinese Academy of Sciences, Guangzhou 510650, People’s Republic of China b Department of Biology, Cheng-Kung University, Tainan, 701, Taiwan Received 28 June 2003; accepted 20 December 2003
Abstract Genetic diversity and differentiation among five populations of Calocedrus macrolepis in southwestern China were investigated using inter-simple sequence repeats (ISSR) markers. Low genetic diversity was revealed, with mean percentage of polymorphic loci at P ¼ 26:9% and average expected heterozygosity of HE ¼ 0:1114. Low levels of genetic differentiation among populations were detected based on Nei’s genetic diversity analysis (4.2%), Shannon’s diversity index (2.7%), and AMOVA (4.1%). Pairwise genetic identity (I ) values among populations ranged from 0.9899 to 0.9964, with a mean of 0.9932. There was no correlation between genetic and geographic distance among the populations studied. Possible genetic bottlenecks during Quaternary glaciations coupled with effects of genetic drift that shaped scattered and small populations were postulated to be the main reasons for the low genetic diversity in Calocedrus macrolepis. # 2004 Elsevier Ltd. All rights reserved. Keywords: Calocedrus macrolepis; Cupressaceae; Genetic variation; ISSR
1. Introduction To estimate the level and distribution of genetic variation in endangered species is a primary objective of conservation genetics (Fritsch and Rieseberg, 1996). Genetic variation at intraspecific level is a prerequisite for future adaptive change
Corresponding author. Tel.: +86-20-8523-2051; fax: 86-20-8523-2831. E-mail address: [email protected] (X.-J. Ge).
0305-1978/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2003.12.003
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or evolution and has profound implications for species conservation (Schaal et al., 1991) and future breeding programs. Loss of genetic diversity usually has deleterious effects on species fitness and may threaten the survival of populations (Malone et al., 2003; Reed, 2003). Genetic diversity of a species is determined by many evolutionary factors, such as dispersal, life history characters, and demographic history. Correlations between genetic diversity and various attributes of species have been examined based on a vast amount of data of genetic diversity in natural plant populations (Hamrick and Godt, 1989). It was found that gymnosperms (mostly conifers) maintain relatively high levels of genetic variation within populations and display little genetic differentiation among populations (Hamrick et al., 1992). However, relatively low genetic diversity or high level of genetic differentiation has been recorded within a number of coniferous taxa, such as Pinus resinosa (Simon et al., 1986), Abies bracteata (Ledig, 1987), and Cathaya argyrophylla (Ge et al., 1996). The genus Calocedrus Kurz consists of two species and one variety of evergreen trees with amphi-north Pacific distribution in western North America and eastern Asia. Calocedrus decurrens (Torr.) Florin occurs in western USA and Mexico, whereas C. macrolepis Kurz is endemic to southwestern China and the adjacent regions of Myanmar and Vietnam. In China, C. macrolepis is distributed in Guizhou and Yunnan Provinces at elevations ranging from 300 to 2000 m and can be found sporadically in Guangxi and Hainan Provinces. C. macrolepis Kurz var. formosana (Florin) Cheng et L. K. Fu grows in northern and central Taiwan (Li, 1975; Fu et al., 1999). Calocedrus has been considered as representing an ancient lineage. This gymnosperm genus was widespread in the temperate according to fossil evidence of European Tertiary (Kracek, 1999) and Asiatic Oligocene to Pliocene sediments (WGCPC, 1978; Liu et al., 1996). Calocedrus macrolepis is an element of evergreen subtropical forests in mountainous areas. Its DBH (diameter at breast height) of the trunk can reach 60–80 cm, and the height 15–25 m (rarely 30) m on favorable sites. The wind-pollinated species is monoecious, with male and female cones occurring on different branches (Fu et al., 1999). With two subapical, unequal wings, the light and small seeds of C. macrolepis can be disseminated by wind for a long distance. Populations of C. macrolepis are dramatically shrinking in China due to heavy deforestation and extensive habitat loss in past decades, although regeneration is sound in places with ample light, such as habitats along streams or at forest edges. Calocedrus macrolepis has been classified as a vulnerable species according to the criteria of IUCN Red List (Farjon, 2001), and has been listed as a species under serious threat in China (Fu, 1995) and Vietnam (WCMC, 1997). Previous studies have mostly focused on ecology (Ning et al., 1997; Cheng et al., 2001) and cytology (Li, 1998) of C. macrolepis. However, its intraspecific genetic variation remains unknown, despite the level of genetic diversity, and the way in which it is maintained among populations is of major concern in conservation biology (Maki, 2003). The state of variability within and between populations can be estimated by applications of both biometrical and molecular procedures. However, it is only the procedures of molecular genetics that allow a measure of population structure at the level of the gene and genome to be gained (Hayward and
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Hamilton, 1997). Among various molecular tools, inter-simple sequence repeats (ISSR) has been widely used for population genetic study recently (Gupta et al., 1994; Tsumura et al., 1996). ISSR fingerprinting has demonstrated a hypervariable nature of the markers and its potential power for population studies (Culley and Wolfe, 2001). Technically, ISSR fingerprinting is more reproducible than RAPD amplification due to the longer SSR-based primers, thus enabling higher-stringency DNA amplifications (Wolfe et al., 1998). In this study, we assess the extent of genetic variation within and between populations of C. macrolepis in China with ISSR markers and formulate recommendations for conservation and breeding strategies of this endangered conifer based on the observed genetic structure.
2. Materials and methods 2.1. Sample collection and PCR amplification Twigs of 121 individuals of C. macrolepis were collected from five populations in Guizhou and Yunnan Provinces in southwestern China (Fig. 1, Table 1). Trees ca. 5 m apart were randomly sampled within each population. Twigs were dried with v silica gel and stored at 4 C prior to DNA extraction. Genomic DNA was extracted using a CTAB method described by Doyle (1991). 100 primers (University of British Columbia primer set 9) were first screened for PCR amplification. Ten ISSR primers (UBC # 807, 808, 811, 812, 835, 836, 842, 857, 874 and 881) that generated clear, reproducible banding patterns were chosen for final analysis. Polymerase chain reactions (PCR) and gel electrophoresis were carried out as described in Ge et al. (2003).
Fig. 1. Map showing locations of the population of C. macrolepis sampled.
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Table 1 Populations of Calocedrus macrolepis for ISSR analysis Population Anning, Yunnan Lincang, Yunnan Yuanjiang, Yunnan Danzhai, Guizhou Libo, Guizhou
Pop. Code AN LC YJ DZ LB
Altitude (m)
Latitude (N)
Longitude (E)
Sample size
1600 1200 1600 900 960
24 60 v 23 50 v 23 30 v 26 160 v 25 140
102 200 v 100 90 v 101 500 v 107 590 v 107 540
30 30 30 19 12
v
v
2.2. Data analysis Only bands that could be unambiguously scored across all the population samples were used in the analysis. ISSR profiles were scored for each individual as present (1) or absent (0) of a specific band. A set of measures of intra- and interpopulation genetic statistics were generated using the program POPGENE 1.31 (Yeh et al., 1999) including: Nei’s (1973) gene diversity, the percentage of polymorphic loci (P), expected heterozygosity (HE), total genetic diversity (HT), genetic diversity within population (HS) and the relative magnitude of genetic differentiation among populations (GST ¼ HT HS =HT ). Gene flow was estimated using the formula: Nm ¼ 0:25ð1 GST Þ=GST . Nei’s (1972) genetic identity (I ) and genetic distance (D) were calculated for all pairwise combinations of populations. The POPGENE software package was also used to calculate Shannon’s index of phenotypic diversity for ISSR diploid data according to Ho ¼ Rpi log2 pi (Lewinton, 1972), in which pi is the frequency of a given ISSR fragment. Ho was calculated at two levels: the average diversity within populations (Hpop), and the total diversity (Hsp). The proportion of diversity among populations was estimated as (Hsp HpopÞ=Hsp. Components of variance partitioned into within and between populations were also estimated using AMOVA. Input data files for the AMOVA v. 1.55 program (Excoffier et al., 1992) were generated using AMOVA-PREP (Miller, 1998). The number of permutations for significant testing was set at 1000 for analysis. AMOVA variance components were used as estimates of the genetic diversity within and between populations. A UPGMA (unweighted pair-group method using arithmetic average) dendrogram was constructed based on the matrix of Nei’s genetic distance using the SAHN—clustering and TREE programs from NTSYS-pc 2.0 (Rohlf, 1998). In order to test for a correlation between genetic (D) and geographical distances (in km) among populations, a Mantel test was performed using Tools for Population Genetic Analysis (TFPGA; Miller, 1997) (computing 1000 permutations). 3. Results 3.1. The ISSR profile and within-population variability A total of 78 different ISSR bands were scored, corresponding to an average of 7.8 bands per primer. Among the 78 loci, 21 were polymorphic at the species level
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Table 2 Genetic variability within populations of Calocedrus macrolepis shown by ISSR. P, percentage of polymorphic loci; HE, expected heterozygosity. Ho: Shannon’s diversity. Standard deviations are in parentheses Population
P (%)
HE
Ho
AN LC YJ DZ LB Mean
26.92 26.92 26.92 26.92 26.92 26.92
0.104 (0.179) 0.108 (0.182) 0.111 (0.188) 0.118 (0.197) 0.116 (0.194) 0.1114 (0.0057)
0.154 (0.260) 0.158 (0.265) 0.161 (0.271) 0.169 (0.282) 0.167 (0.279) 0.1618 (0.0062)
(Table 2). All of these 21 loci are polymorphic in all populations, only different on their frequencies. The percentage of polymorphic loci (P) is 26.9% for all five populations. Assuming Hardy–Weinberg equilibrium, the average gene diversity was estimated to be 0.1114 within populations (HE), and 0.116 at the species level (HT). The Shannon indices (Ho) ranged from 0.154 to 0.169, with an average of 0:1618 0:0062 at population level (Hpop) and 0.1662 at species level (Hsp). 3.2. Between-population diversity The coefficient of overall genetic differentiation between populations (GST) was 0.042 as estimated by partitioning of the total gene diversity. The Shannon’s diversity index analysis partitioned 2.7% of the total variation between populations. Pairwise genetic identity values (I ) among populations ranged from 0.9899 to 0.9964 with a mean of 0.9932. Genetic distances (D) between populations ranged from 0.0036 to 0.0101, with a mean of 0.0069 (Table 3). The level of gene flow (Nm) was estimated to be 5.7 individuals per generation between populations. According to AMOVA analysis, there were significant (P < 0:001) genetic differences among the five populations of C. macrolepis. Of the total genetic diversity, 4.1% was attributable to among-populations and the rest (95.9%) to differences within populations (Table 4).
Table 3 Geographical distance and Nei’s (1972) genetic distance Population
AN
LC
YJ
DZ
LB
AN LC YJ DZ LB
– 0.0067 0.0090 0.0101 0.0062
266 – 0.0080 0.0081 0.0049
175 173 – 0.0061 0.0059
585 843 694 – 0.0036
565 807 648 115 –
Geographical distance (above diagonal) and Nei’s genetic distance (below diagonal).
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Table 4 AMOVA for five populations of Calocedrus macrolepis Source of variation
d.f.
Sum of squares
Mean squares
Variance component
% total variance
P-value
Among populations Within populations
4 116
39.5732 571.7491
9.893 4.929
0.2101 4.9289
4.1 95.9
< 0.001 < 0.001
Fig. 2. UPGMA dendrogram based on Nei’s (1972) genetic distance.
The correlation coefficient (r) between genetic and geographical distance using Mantel’s test for all populations was 0.0823 (P ¼ 0:6750). The UPGMA tree based on ISSR (Fig. 2) revealed a similar result in that the genetic distances among the populations do not show a spatial pattern corresponding to their geographic locations (Fig. 1). 4. Discussion Most conifers have high levels of genetic diversity and low levels of differentiation among populations, as measured by allozymes (Hamrick et al., 1992). However, due to the complex effects of historical factors such as speciation process and Quaternary glaciation, it is difficult to make an a priori prediction of the levels of genetic diversity in endangered species (Maki, 2003). When compared to other species (cf. Hamrick et al., 1992), total genetic diversity of C. macrolepis (HT: 0.116) is lower than that of the mean genetic diversity both for gymnosperms (0.169) and for long-lived woody perennial species (0.177). Compared to most Cupressaceae species (Table 5), the genetic variation in C. macrolepis is lower than that in the congeneric C. decurrens (HE ¼ 0:18; Harry, 1984), but slightly higher than that in Thuja occidentalis (Perry et al., 1990), T. plicata (Yeh, 1988) and Austrocedrus chilensis (Ferreyra et al., 1996).
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Table 5 Summary of genetic variation for some Cupressaceae species. P, percentage of polymorphic loci; HE, expected heterozygosity; Ho, Shannon’s diversity Species
P (%)
HE
Austrocedrus chilensis Calocedrus macrolepis C. decurrens Chamaecyparis formosensis C. nootkatensis C. taiwancensis C. thyoides Cupressus sempervirens Fitzroya cupressoides Juniperus communis J. coreana J. rigida J. thurifera Pilgerodendron uviferum Thuja occidentalis T. plicata
41
0.071
26.92
0.116
– 55.2
0.18
50 77.8 50.0 82.6
0.171
Ho
Genetic differentiation (%)
Marker used Reference
–
allozyme
Ferreyra et al., 1996
0.1662
2.71–4.2
ISSR
Present study
– 0.329
– 15.1
allozyme RAPD
Harry, 1984 Hwang et al., 2001
13.9 14.7 9.4 7.3
allozyme RAPD allozyme allozyme
14.4
RAPD
Ritland et al., 2001 Hwang et al., 2001 Kuser et al., 1997 Raddi and Suemer, 1999 Allnut et al., 1999
0.429 0.145 0.350
–
0.547
83.5
–
–
AFLP
54.6 72.7 98 35.7
0.199 0.224 – 0.712
11.8 17.4 38.90 15.9–18.6
allozyme allozyme RAPD RAPD
Van der Merwe et al., 2000 Huh and Huh, 2000 Huh and Huh, 2000 Jime´nez et al., 2003 Allnutt et al., 2003
50 26.3
0.094 0.04
1.6 3.3
allozyme allozyme
Perry et al., 1990 Yeh, 1988
– 0.730
Genetic diversity maintained in a plant species is influenced by specific characteristics of the species (Hamrick and Godt, 1989), as well as by its evolutionary history. The low level of genetic variation in C. macrolepis may be attributed to both its evolutionary history and its small isolated populations. The genus Calocedrus has an eastern Asia and North American disjunct distribution pattern. The isolation of most disjunct taxa in eastern Asia and North America may have occurred during the global climatic cooling throughout the late Tertiary and Quaternary (Xiang et al., 2000). Fossil evidence (Kracek, 1999) and the disjunct distribution of C. macrolepis var. formosana in Taiwan indicate that the ancestor of C. macrolepis was much more widespread in the Tertiary than at present. The suitable climate in southwestern China may have allowed the survival of certain forest taxa since the Mesozoic Era (Li, 1953; Walker, 1986). At least since the Oligocene, Calocedrus has lived in this area (WGCPC, 1978; Liu et al., 1996). Therefore, this area may represent one of refuge for gymnosperms (Contreras-Medina and Vega, 2002). Through the periodical and repeated range contraction, this species may have experienced severe bottlenecks and subsequent genetic drift that led to loss of genetic diversity. Although C. macrolepis is locally abundant in the subtropical eastern Asia, it consists of scattered and small fragmented populations in southwestern China due
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to the impacts of human activities. The vulnerable species displays a severe decline in the extent of occurrence and/or quality of habitat, together with the effects of exploitation. Although geographic range was considered a good predictor of the levels of genetic variation in plant populations (Hamrick and Godt, 1989), widespread species occurring in small and disjunct populations may not fit this pattern (Premoli et al., 2001). Fragmentation and isolation of populations can result in reduced genetic variation by genetic drift (Sherwin and Moritz, 2000), as detected in Pilgerodendron uviferum (Cupressaceae), a species widespread in temperate South America but consisting of scattered and small populations (Premoli et al., 2001). Positive correlations between genetic variability and population size have been reported for a number of plant species (Fischer et al., 2000). Sustained restrictions in population size are the main reason for loss of genetic diversity that will reduce the ability to evolve in response to ever-present environmental change and increase the risk of extinction (Frankham et al., 2002). The level of genetic differentiation is low among these five populations of C. macrolepis. This finding is in agreement with the conclusion of Hamrick et al. (1992). In most woody species, a mean GST value of 0.073 was recorded for 121 species of gymnosperm examined based on allozyme analyses (Hamrick et al., 1992). Even compared to genetic differentiation estimates of other Cupressaceae species, the values recorded for C. macrolepis are relatively low (Table 5), while slightly higher than that of Thuja occidentalis (1.6%; Perry et al., 1990) and T. plicata (3.3%; Yeh, 1988). Low level of genetic differentiation in gymnosperms is usually attributed to the frequent wind-pollination and breeding systems that promote outcrossing (Bennett et al., 2000). In addition, the seeds of C. macrolepis with membranous wings are also helpful for its long distance dispersal, as indicated by the high Nm values (=5.70) between populations. It should be mentioned that although our samples of five populations were collected from the main distribution area, some distribution areas, such as Taiwan, Northern Myanmar and NE Vietnam were not yet sampled, which may to some extent lead to biased estimates of genetic diversity of the species as a whole to a certain extent. This is critical for understanding the entirety of genetic diversity of C. macrolepis since the Taiwan populations have been recognized as a variety (C. macrolepis Kurz var. formosana (Florin) Cheng et L. K. Fu) (Cheng and Fu, 1978) or species (C. formosana (Florin) Florin) (Florin, 1956). The ultimate goals of conservation are to ensure sustainable survival of populations and to preserve their evolutionary potential. The estimates of genetic diversity and genetic differentiation provide a basis for implementing efficient and practical conservation programs for C. macrolepis. As the differences among populations of C. macrolepis in southwestern China are in allele frequencies rather than in gene composition, loss of populations at certain locations may not cause immediate loss in genetic diversity but more damage may occur in terms of long-term genetic consequences due to reduced number of populations and smaller population size. Replacement of the lost populations at certain locations can be achieved by transplanting seedlings from long-established populations.
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Acknowledgements We thank Dr Yong-Ming Yuan and Dr Li-Bing Zhang for helpful comments and providing references. Thanks are also due to two anonymous reviewers for their critical reviews and suggestions. This study was financially supported by the Knowledge Innovation Key Project (KSCX2-SW-104) and Field Frontiers Project (Director Foundation of SCIB) of the Chinese Academy Sciences.
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