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exogenous diamines on the interaction between ectomy- Genetic Diversity of the Relict Plant Taiwania cryptomerioides Hayata
(Cupressaceae) in Mainland China
By ZHONG-CHAO LI1), XIAO-LAN WANG2) and XUE-JUN GE1),*) Abstract
repeats (ISSR). In comparison with other coniferous The genetic diversity and differentiation of five popu- species, T. cryptomerioides from mainland China pos- lations of Taiwania cryptomerioides Hayata in mainland sesses little genetic variation, particularly at the level of China were investigated using inter-simple sequence individual populations (the percentage of polymorphicloci, Nei’s gene diversity and Shannon’s indices of diver- sity at the species and population levels are 38.02 %, ) South China Botanical Garden, The Chinese Academy of 0.1326, 0.1986 and 9.27 %, 0.035, 0.0518 respectively).
Sciences, Guangzhou 510650, P. R. China.
In contrast, the level of population differentiation is ) Center for Functional Genomics and Microarray, Guangzhou much higher (G : 0.7269; Shannon’s genetic differentia- θ B: 0.668; AMOVA genetic differen- ) Corresponding author: Dr. XUE-JUN GE. South China Botanical tiation: 72.37 %). The genetic divergence of pairs of pop- Garden, The Chinese Academy of Sciences, Guangzhou 510650,P. R. China. Tel: +86-20-3725 2551; Fax: +86-20-3725-2831. E- ulations was not significantly correlated with the geo- mail address: gexuejun@mail.sysu.edu.cn, xjge@scbg.ac.cn graphical distance separating them. Current patterns of genetic variation were related to biogeographic history Information about the genetic structure of a rare and the small population size. On the basis of these species such as this, as well as data on its overall level findings, we discuss the development of conservation of genetic diversity, are particularly important for strategies for this endangered species.
species conservation. Genetic analyses can provide valu-able insights into the processes influencing extinction Key words: Taiwania cryptomerioides; genetic diversity, ISSR,China, relict species, conservation (CLARKE and YOUNG, 2000) and, increasingly, geneticdata have been used to define conservation managementunits and to predict changes in population structure and Introduction
dynamics (NEWTON et al., 1999). However, in comparison Like most of the genera within the Cupressaceae that with the rarities from Europe and North America, few evolved since the Jurassic period, Taiwania, one of the detailed studies of genetic variation have been under- so-called “living fossils”, is considered to be an ancient taken on rare plants in China. Subtropical mainland genus in evolutionary terms with its origin dating back Asia was one of the most important refugia for plants to the Tertiary. According to the fossil evidence, e.g. Tai- during the Pleistocene glaciations because it was not wania schaeferi Schloem.-Jäg. discovered in Klein- covered by ice sheets. Many species that became extinct saubernitz (Germany) (WALTHER, 1999), Spitsbergen elsewhere survived in this region, but in a state of isola- (Norway) and Alaska (LePage BA, unpublished data), tion and in small populations. Strong genetic differenti- this genus was widespread in what are now the temper- ation has been discovered in some relict conifer species ate regions of the northern hemisphere (FLORIN, 1963).
in China, for example Cathaya argyrophylla (GE et al., Today, Taiwania, a monotypic genus containing only 1998), Glyptostrobus pensilis (LI and XIA, 2005), Metase- Taiwania cryptomerioides Hayata, has a scattered dis- quoia glyptostroboides (LI et al., 2005) and Ametotaxus tribution. Its distributional range covers mountain argotaenia (GE et al., 2005). The genetic diversity of areas on the border between Burma and China (south- T. cryptomerioides from Taiwan has been assessed previ- western part of Yunnan province), the Hoang Lien Son ously by allozyme (LIN et al., 1993), ISSR and ITS mountain range in northern Vietnam, and Taiwan sequence analysis (CHANG, 2005). A low genetic differen- (FARJON, 2002; FARJON and GARCIA, 2003). Taiwan is an tiation (G : 5.2 % based on allozyme data, LIN et al., important refuge for T. cryptomerioides: it grows on the 1993; Φ : 15.26 % using ISSR, CHANG, 2005) was found Central Mountain at the altitudes of 1600–2600 m, in the Taiwanese populations. Nevertheless, the where the largest population and greatest timber yield intraspecific genetic variation across the distributional in the world are found (HUANG, 1983). In addition, there are still a few individuals of T. cryptomerioides in main- Among various molecular tools, the inter-simple land China: in the remote Lichuan mountains in Hubei sequence repeats (ISSR) method has been widely used Province, Leishan mountains in Guizhou Province, and for studies of population genetics, because the repeats Gutian-Pingnan mountains in Fujian Province. These are highly variable, and the method is economical in areas are regarded as having acted as key refugia for terms of time, money, and labor (GUPTA et al., 1994; relict species during Pleistocene glaciations (WU, 1980; ZIETKIEWICZ et al., 1994; TSUMURA et al., 1996; WOLFE WANG and LIU, 1994). The populations in mainland and LISTON, 1998). ISSRs have also been used to deter- China have only been widely known to botanists since mine the genetic diversity of species of conservation con- the early decades of the 20th century, and have some- cern (ESSELMAN et al., 1999). Technically, the ISSR reac- times been regarded as comprising a different species, tion is more specific than RAPD amplification due to the namely T. flousiana Gaussen. Due to human overex- longer SSR-based primers (WOLFE and LISTON, 1998).
ploitation in the past century, the conservation status of One limitation of the ISSR technique is that the bands this species is “vulnerable” according to the IUCN Red are scored as dominant markers and that genetic diver- List criteria (FARJON, 2001) and it is a protected species sity estimates are based on diallelic characters, thus the level of genetic diversity may be underestimated. The T. cryptomerioides is a component of evergreen primary objective of this study was to use ISSR markers broadleaf forests in mountainous areas. It occurs at an to investigate genetic variation within and between the altitude of 500–2800 m in “coniferous, broad-leaved, or extant T. cryptomerioides populations in mainland mixed evergreen valley forests on acid, red, or brown China. We wished to determine whether these popula- soils in warm or warm temperate regions with high tions are strongly differentiated, as is the case for the summer and autumn rainfall but drier winters, usually other relict conifer species mentioned above. The result scattered and associated with Chamaecyparis formosen- of this investigation could assist in the development of sis, C. obtusa var. formosana, Cunninghamia lanceolata, conservation strategies, by identifying those populations Pinus wallichiana, or Tsuga dumosa, but sometimes of particular importance in terms of their genetic char- forming pure stands” (FU et al., 1999). It can grow as tall as 70 meters and is therefore known as the ‘King ofAsian Conifers’. This diploid species (2n = 22, LI et al., Material and Methods
2000) is wind-pollinated and monoecious, with male andfemale cones occurring on different branches (FU et al., 1999). With two subapical, unequal wings, seeds of A total of 104 individuals of Taiwania cryptomerioides T. cryptomerioides are light and small, and can be car- was studied, representing four extant wild populations in mainland China and one plantation population (TC) Table 1. – Genetic variability within populations of Taiwania cryptomerioides revealed by ISSR (N: sample size per population; P: percentage of polymorphic loci; H : Nei’s gene diversity; Hpop: Shannon indices).
(Table 1, Fig. 1). Among the four wild populations, many were carried out in a PTC-200 thermal cycler (MJ individuals are several hundred years old with a DBH research) following the method of GE and SUN (1999).
greater than 80 cm; their ecological and phytocoenologi- One hundred primers, each 15–23 nucleotides long cal features have been described previously (Table 1; (UBC primer set # 9, Biotechnology Laboratory, Univer- QIU et al., 1984; LIU et al., 1987; GE, 1995; FENG et al., sity of British Columbia), were screened for the produc- 1998; YANG and JIANG, 1999). Individuals were sampled tion of a high proportion of polymorphic and repro- at random, regardless of their size and age. Young, ducible banding. Fifteen primers (UBC # 807, 808, 811, healthy leaves were collected in the field, and dried in 826, 827, 828, 835, 836, 840, 846, 849, 855, 866, 880, and silica gel prior to DNA extraction. Total DNA was 886) fulfilled these requirements. PCR products were extracted using the CTAB method described by DOYLE electrophoresed on 2.0 % agarose gels buffered with 0.5 x (1991) then dissolved in 100 µL of TE buffer. DNA con- TBE. A 100 bp DNA Ladder (New England Biolabs) was centration was determined by comparison to uncut used as a size marker. After staining with ethidium bro- lambda DNA on 1% agarose gels; the average DNA con- mide, the fragments were identified using image analy- centration was about 20–30 ng/µl.
sis software for gel documentation (LabWorks SoftwareVersion 3.0; UVP, Upland, CA 91786, USA). PCR amplification was carried out in a volume of 20 µl; the reaction comprised 20 ng of template DNA, Only bands that could be unambiguously interpreted 10mM Tris-HCl (pH 8.0), 50mM KCl, 0.1% Triton x 100, across all the population samples were used in this 2.5mM MgCl , 0.1mM dNTPs, 2 % formamide, 0.2 µM study. ISSR profiles were scored as discrete characters primer and 1.5 units of Taq polymerase. Amplifications (presence or absence of amplified products) for each Figure 1. – The sampling locations of Taiwania cryptomerioides Hayata.
individual. The resulting data matrix was analyzed were 0.0518 at the population level (Hpop) and 0.1986 using POPGENE v. 1.31 (YEH et al., 1999) to estimate at the species level (Hsp), respectively (Table 1). The cul- genetic diversity parameters: the percentage of polymor- tivated TC population exhibited the lowest genetic phic loci (P) and expected heterozygosity (H ). At the diversity. Among the four wild populations, population species level, genetic diversity measures (H : total gene GP had the greatest level of variability (P: 20.31%, H : diversity; G : coefficient of gene differentiation) and the 0.0806, Hpop: 0.1179); population LS had the lowest level of gene flow (Nm) were measured using Nei’s level of variability (P: 3.65%, H : 0.0121, Hpop: 0.0186) (1973) gene diversity statistics. Genetic diversity was (Table 1). The average polymorphism of the four wild also estimated using Shannon’s information statistic = -Σpi log p where p is the fre- The coefficient of genetic differentiation between pop- ulations (G ) was 0.7269, estimated by partitioning the two levels: the average diversity within the populations total gene diversity. The Shannon’s diversity index (Hpop), and the total diversity (Hsp). Then the propor- analysis partitioned 73.92 % of the total variation tion of diversity between the populations was estimated between populations, in broad agreement with the as: (Hsp-Hpop)/Hsp. result of genetic differentiation analysis. The level of In addition, an analysis of molecular variance gene flow (Nm) was estimated to be 0.0939. The AMOVA (AMOVA; EXCOFFIER et al., 1992) was used to estimate analysis provided corroborating evidence for the genetic variance components for ISSR phenotypes, partitioning structure obtained from Nei’s genetic diversity statistics the variation between populations and between individ- and Shannon’s diversity estimate. There were highly uals. AMOVA analysis was performed using the Arle- significant (P < 0.001) genetic differences between the quin 2.000 program (SCHNEIDER et al., 2000). To test the five populations of T. cryptomerioides. Of the total mole- presence of isolation by distance, a Mantel test between cular variance, 72.37 % was attributable to between-pop- the pairwise genetic distance and geographic distance ulation diversity and the rest (27.63 %) to differences from each population pair was applied using Mantel’s between individuals within populations (Table 2). A sim- test in the software TFPGA (MILLER, 1997) (1000 per- ilar result was obtained from the Hickory calculation: θ B mutations). Pairwise genetic distances (Φ ) were got was 0.668 for the f free model, which had the smallest from AMOVA analysis, which are analogous to tradition- Genetic distances (Φ ) between populations of An alternative Bayesian method, allowing direct esti- T. cryptomerioides were calculated by Arlequin 2.000 (SCHNEIDER et al., 2000) (Table 3). The highest distance ous knowledge of F , was employed to estimate θ B value was 0.9011 between TC and LS, and the lowest (analogous to F ) using Hickory 1.0 (HOLSINGER and was 0.5037 between LS and GP. Geographically TC and LEWIS, 2003). Using the default sampling parameters GS are the closest pair of sites (269 km), however, the (burn-in = 5 000, sample = 25 000, thin = 5), θ B was cal- genetic distance between them, 0.7608, was not the low- culated under four different models. The first was a full est of all the pairs. The result of a Mantel test with 1000 model, in which both θ B and f (i.e., F , the inbreeding permutations revealed that the genetic divergence of coefficient) were estimated. Two alternative models populations was not significantly correlated with geo- assumed either θ B or f equal to zero. Finally, because graphical distance (Mantel test, r = -0.3124, P = 0.2170). estimates of f based on dominant markers are usuallystrongly biased (especially at low sample sizes, i.e.,n < 10), HICKORY was used to construct a model in Discussion
which f was allowed to vary. The deviance information Low level of genetic diversity and strong genetic differen- criterion (DIC) was employed to determine which model was the best fit for the data: lower values indicate a bet- Correlations between genetic diversity and various attributes of different species have been examined based Table 3. – The pairwise genetic distance (ΦST) (below diagonal) In this study, 73 of the 192 electrophoretic bands and geographic distance (km) (above diagonal).
(38.02 %) were polymorphic within the populations. Thepercentages of polymorphic loci (P) for a single popula-tion ranged from 1.56 % (TC) to 20.31% (GP), with anaverage of 9.27 %. The average genetic diversity wasestimated to be 0.0350 at the population level (H ) and 0.1282 at the species level (H ). The Shannon indices Table 2. – Analysis of molecular variance (AMOVA) for five populations of Taiwania cryptomerioidesd.f.: degrees of freedom; SS: sum of squares; MS: expected mean squares.
a Significance tests after 1000 permutations.
on a vast amount of data relating to natural plant popu- pensilis (LI and XIA, 2005), Metasequoia glyptostroboides lations (HAMRICK and GODT, 1989; NYBOM, 2004). Most (LI et al., 2005), Cupressus chengiana (HAO et al., 2006), conifers have high levels of genetic diversity and low Torreya jackki (LI and JIN, 2007) and Abies ziyuanensis levels of differentiation between populations (HAMRICK (TANG et al., 2007) (Table 4). Since these species are et al., 1992). Examples include Fitzroya cupressoides characterized by their relict status, biogeographic histo- (Hpop: 0.547, Φ : 0.1438; ALLNUTT et al., 1999), Junipe- ry may play an important role in determining their rus rigida, J. coreana (H : 0.224 and 0.199; G : 0.173 genetic diversity. T. cryptomerioides is one of the repre- and 0.118, respectively; HUH and HUH, 2000), Cedrus sentatives of relict gymnosperms from the Tertiary peri- atlantica (Nei’s gene diversity within population, H : od that are found in China. Fossil evidence indicates 0.194; F : 0.148; θ B: 0.178; RENAU-MORATA et al., 2005) that the ancestor of T. cryptomerioides was much more and Juniperus phoenicea (H : 0.148, H : 0.130, G : widespread during the Tertiary than its current distrib- 0.12; MELONI et al., 2006). Similar genetic diversity and ution suggests (WALTHER, 1999). Accordingly, a reason- genetic differentiation have also been reported for the able hypothesis is that the modern range of T. cryptome- Taiwanese populations of T. cryptomerioides (LIN et al., rioides was the result of population fragmentation and 1993; CHANG, 2005). In this study, however, ISSR poly- contraction after the Quaternary glacial cycles. The morphisms revealed a low level of genetic diversity in remnant populations contained only part of the genetic the mainland populations of T. cryptomerioides, with an variation present before the range reduction. Genetic average of only 11.19% of ISSR bands being polymorphic drift and inbreeding may have further decreased the in the four wild populations studied. In addition, strong genetic diversity and increased genetic differentiation in population differentiation was demonstrated based on the small, isolated populations of T. cryptomerioides in Nei’s gene diversity, Shannon’s information statistic, AMOVA and Bayesian estimates. About 70 % of the total In addition to historical influences, the present small genetic variation was partitioned between the popula- population size of T. cryptomerioides may be important.
tions of T. cryptomerioides studied in mainland China.
According to the literature there are currently about The low genetic diversity within populations and high 6000–7000 T. cryptomerioides individuals in mainland genetic differentiation among populations we observed China (HU et al., 1995); populations LC and GP are com- in T. cryptomerioides are similar to that of many relict- posed of only a dozen individuals (GE, 1995; YANG et al., ual endangered conifer species in mainland China.
2006). This suggests that this species is experiencing These include Cathaya argyrophylla (GE et al., 1998), erosion of its genetic diversity. In this study, only four- Ametotaxus argotaenia (GE et al., 2005), Glyptostrobus teen electrophoretic bands (8 %) with a frequency lower Table 4. – Our results compared with those of relict gymnosperm species endemic to China.
P: percentage of polymorphic loci; H : Nei’s gene diversity; Hpop: Shannon indices; G : the coefficient of gene differentiation.
than 30 % were observed, suggesting a possible stochas- ies, which can be used to determine a species’ natural tic process of genetic drift, which could result in the loss range (BEEBEE and ROWE, 2004). Further study is need- of low-frequency alleles in the populations (BROYLES, ed to screen out the mtDNA fragments with intraspecific polymorphic variation in this species.
The genetic structure of plant populations reflects the interactions of various evolutionary processes. Low lev- Conservation Implications
els of genetic differentiation in gymnosperms are usual-ly attributed to wind-pollination and breeding systems The maintenance of genetic diversity is crucial to the that promote outcrossing. T. cryptomerioides is dioe- survival of organisms, because it allows them to evolve cious, wind pollinated and has light seeds that are usu- and adapt to changing environmental conditions. The ally dispersed by wind (about 1000 seeds in 1.01 g; high level of genetic differentiation between the studied populations of Taiwania cryptomerioides indicates that HUNG and CHANG, 1990). It seems likely, therefore, that considerable gene flow should take place between popu- a considerable amount of the overall genetic variation of lations. For Taiwan populations of T. cryptomerioides, the species in mainland China would be lost if manage- little genetic differentiation is apparent, thus support- ment concentrated only on the remaining large popula- tions. Therefore, several populations throughout the geographic proximity of the populations in Taiwan, the entire range should be considered for conservation. At populations in mainland China are separated from each present three populations in mainland China (GS, LS other by several hundred kilometers (Table 3). This and LC) are protected in situ. Despite being situated extreme isolation makes gene flow negligible, resulting within a protected area, population LC has been severe- in the lack of a correlation between genetic distance and ly affected by land reclamation, and should therefore be geographical distance found in this study. For example, accorded a high priority for future conservation action.
the genetic distance between populations that are rela- As for ex situ conservation in botanical gardens, further tively close geographically (i.e., populations TC and GS) reduction of genetic variability by genetic drift should be was no lower than between geographically more distant avoided, therefore one additional measure could be arti- ones. The present locations of T. cryptomerioides in ficial gene flow between populations, i.e., artificial intro- mainland China coincide with the key refugia for other ductions should include as many populations as possi- relict species during the Pleistocene glaciations (W 1980; WANG and LIU, 1994). For example, at the north-ern limit of this species, Lichuan (LC) in Hubei Acknowledgments
Province, there are many relict species, including We thank Mr. GUO-SHENG HE, YONG-FU YU and YONG- Davidia involucrata, Ginkgo biloba, Keteleeria fortunei, MING YUAN for their help in collecting samples. This work Taxus chinensis, Tetracentron sinensis, Emmenopterys was supported by the National Natural Science Founda- henryi and Bretschneidera sinensis. The famous relic plant Metasequoia glyptostroboides was also discoveredin this region in the 1940s (see http://www.metase- References
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Genetic Variation Within Two Sympatric Spotted Gum Eucalypts Exceeds
Between Taxa Variation
By J. W. OCHIENG1),4),*), M. SHEPHERD1), P. R. BAVERSTOCK1), G. NIKLES2), D. J. LEE3) and R. J. HENRY1) Abstract
implications for LD mapping, we investigated the level Population substructure and hybridization, among of molecular divergence between the two species at two other factors, have the potential to cause erroneous sympatric locations separated by 300 kilometres. Very associations in linkage disequilibrium (LD) mapping.
few individuals of intermediate morphology were identi- Two closely related spotted gum eucalypts, Corymbia fied, despite the two species occurring only metres variegata and C. henryi (Myrtaceae) occur in sympatry apart. Analysis of genetic structure using 12 microsatel- in the east coast of Australia and potentially interbreed.
lite loci showed that genetic differentiation between pop- They are morphologically similar but are distinguished ulations of the same species at different locations as separate species based on capsule and foliage size. To = 0.07 for both species; p = 0.0001) was significantly determine whether they hybridize in nature and its higher than that observed between species at each loca-tion (mean F Bunyaville respectively; p = 0.0001; all Mann-Whitney ) Centre for Plant Conservation Genetics, Southern Cross Uni- versity, P. O. Box 157 Lismore, NSW, 2480, Australia.
U-test p ≤ 0.01). No species-specific alleles or significant allele frequency differences were detected within a site, ) Department of Primary Industries and Fisheries, 80 Meiers Road, Indooroopilly, Queensland, Australia.
suggesting recurr#ent local gene flow between the two 3) Department of Primary Industries and Fisheries, LB 16 Fraser species. The lack of significant allele frequency differ- Road, Gympie 4570, Queensland, Australia.
ences implies no population stratification along taxo- 4) Permanent address: Faculties of Agriculture & veterinary nomic lines. This suggested that there is little concern Medicine, University of Nairobi, P. O. Box 30197 Nairobi, for cryptic hybridization when sampling from sites of *) Communicating author: JOEL W. OCHIENG. Centre for Plant Conservation Genetics, Southern Cross University, P. O. Box 57 Key words: panmixia, hybridization, gene flow, association Lismore, NSW 2480 Australia. Tel: +61 2 6620 3961; Fax: mapping, population structure, admixture, reproductive isola- +61 2 6622 2080. E-mail: jochieng@uonbi.ac.ke

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