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Abstract

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

RAPD markers and sequences of chloroplast DNA (cpDNA) atpB-rbcL intergenic spacers were used to characterize the pattern of genetic variation and the phylogenetic relationships of the relict populations of Alsophila spinulosa located in Jian Feng Ling (JFL) and Diao Luo Shan (DLS), Hainan, and Tang Lang Shan (TLS), Ding Hu Shan (DHS), and Da Xi Shan (DXS), Guangdong, of southern China. 28 random primers generated 118 bands, out of which 26 (22.03%) were polymorphic loci, distinguishing 17 different RAPD phenotypes. Percentage of polymorphic loci, Shannon phenotypic diversity and Nei's gene diversity comprehensively indicated that JFL possessed the highest diversity, TLS and DHS in intermediate and DLS or DXS the least; the corresponding values of the population appeared correlated with the population size. Differentiation was detected among populations of A. spinulosa (1−Hpop/Hsp=0.7453, GST=0.7763, and ϕst=0.8145). AMOVA showed that 47.44% of the variance was partitioned among regions (Hainan and Guangdong), 34.01% attributed among populations within regions, whereas only 18.55% occurring within populations. Low level of intra-specific diversity was maintained in A. spinulosa with Shannon diversity and gene diversity merely 0.0560 and 0.0590, repectively. Sequence length of atpB-rbcL intergenic spacer varied from 724 bp to 730 bp. Base composition was with A+T content between 63.17% and 63.70%. 13 haplotypes of atpB-rbcL noncoding spacers were identified. UPGMA dendrogram of RAPD phenotypes, principal components analysis based on RAPD patterns, minimum spanning network and neighbour-joining (NJ) tree established on atpB-rbcL haplotypes consistently suggested the geographical subdivision of populations of A. spinulosa between Hainan and Guangdong. Breeding system and conservation strategy of A. spinulosa was discussed based on the information of population genetic structure and variation.

Alsophila spinulosa (Hook) Tryon, belonging to family Cyatheaceae, is a relict fern species with tree-like trunk. Historically, it had been flourishing during the Jurassic of Mesozoic (180 million years ago), however, after being damaged by glaciation, its distribution dwindled drastically (Tryon 1970). As a species with long evolutionary history, A. spinulosa preserves particular biological characteristics, such as: (1) its spore life-span is short, normally only surviving for 7 to 8 days, (2) the biocycle from spore germination to the formation of young sporophyte takes as long as one year and within this period the prothallus is prone to be injured by adverse environmental factors due to lack of protection structure; (3) it requires relatively high temperature, humidity and illumination conditions for spore germination, gametophyte formation and embryogenesis; (4) both its root system and transfusion tissue are still primitive (Tryon and Lugardon 1990; Cheng et al. 1990). Therefore, A. spinulosa adapts poorly to the present environment and climate, merely restricting its presence to warm, humid and shady niches at low latitudes. Wild populations and individuals of A. spinulosa are extremely spare in China and the species has been regarded as NPC (National Protection Categories) 1-V in the Red List (Fu 1991). During the past decade, A. spinulosa has been increasingly threatened by local economic exploitation and human impact, some populations are already extinct in southern China. Urgent measures must be taken to save this endangered plant.

Information of genetic variability within and among populations of threatened species is proved to be critical to management and conservation because population genetic investigations provide necessary guidance to interpreting the present status and future prognosis of the concerned species (Ellstrand and Elam 1993; Palacios and Gonzalez-Candelas 1997; Drummond et al. 2000). Over the years, detection of genetic diversity has progressed to assay molecular DNA variation. Random amplified polymorphic DNA (RAPD) is one of the widely used tools for the investigation of genetic diversity. Compared with other methods for detecting DNA polymorphisms, this technique has potential advantages for the investigation of rare plants. It is relatively inexpensive and technically straightforward for conducting experiments and does not require prior knowledge of the genome (Rosseto et al. 1995). The amplified fragments provide a large number of polymorphic loci, which are especially useful for studies of species with low genetic variation (Dawson et al. 1993). One of the shortcomings of RAPD analysis is the lack of reproducibility between reactions, which has been attributed to PCR artefacts. This problem can be reduced or solved through pilot studies to optimize amplification variables (Hadrys et al. 1992). RAPD markers have another drawback in being inherited in a dominant manner, thereby making a direct interpretation of individual genotypes problematic. This difficulty has been circumvented by Stewart and Excoffier (1996) and RAPD markers have been successfully used to clarify the genetic structure of plant populations (Palacios and Gonzalez-Candelas 1997; Bauert et al. 1998; Allnutt et al. 1999; Roman et al. 2001; Lacerda et al. 2001).

On the other hand, recently, gene genealogies and coalescence theory develop powerful methodologies to investigate population genetic structure (Castelloe and Templeton 1994). To conduct a phylogenetic analysis of populations using DNA sequence data would be a more informative approach for surveying population differentiation (Nei and Kumar 2000). Chloroplast DNA (cpDNA) noncoding spacers have been frequently utilized for such investigations on plants (Lu et al. 2002; Huang et al. 2001). Their uniparental inheritance, nearly neutral and fast evolution are well suited to reconstruct intraspecific phylogeographical patterns (Ferris et al. 1998). In addition, technically, DNA sequencing can avoid length homoplasies which usually occur when using restriction fragment length polymorphism (RFLP) and PCR-based fingerprinting methods (Chiang et al. 2001)

In this study, RAPD markers and cpDNA atpB-rbcL intergenic spacer sequences were used to characterize the genetic structure and phylogenetic pattern of 5 relict populations of A. spinulosa distributed in Hainan province and Guangdong province of southern China. The purpose of this investigation is: (i) to assess the level of genetic diversity and its hierarchical apportionment; (ii) to determine whether geographical differentiation occurs among these populations at interregion level; and (iii) to tentatively discuss the conservation management of A. spinulosa.

MATERIAL AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

Plant material

Five natural, relict populations of A. spinulosa were sampled, located in Jian Feng Ling (JFL) and Diao Luo Shan (DLS), Hainan province; and Tang Lang Shan (TLS), Ding Hu Shan (DHS), and Da Xi Shan (DXS), Guangdong province of southern China. Leaves were collected individually from total 60 plants, including 16 of JFL (population size 100), 10 of DLS (population size 15), 14 of TLS (population size 57), 9 of DHS (population size 25), and 11 of DXS (population size 11). Individuals grow on humid and sunny slopes, by streams or in ravines. Young and healthy leaves were randomly sampled from individuals with intervals of at least 5 m and immediately preserved in silica gel. All samples were stored at −20°C until DNA extraction was carried out.

DNA extraction

DNA was extracted from leaves according to a modified CTAB protocol (Su et al. 1998). DNA concentration and purity were determined by measuring UV absorption using a Pharmacia 2000 UV/Visible spectrophotometer. DNA intactness was also checked through 0.8% agarose gel electrophoresis.

RAPD amplification and examination

RAPD was performed in a 25 μl total volume containing 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.0 mM MgCl2, 0.2 mM of each d NTP, 0.5 unit of Taq DNA polymerase, 5 ng of primer, 35 ng of template DNA and DNA-free water. Each reaction mixture was overlaid with 20 μl of PCR grade paraffin oil. The reaction was performed on a Perkin Elmer 2400 thermocycler programmed for an initial melting step at 94oC for 200 s, followed by 40 cycles each at 94oC for 60 s, 36oC for 60 s and 72oC for 120 s and ending with an extension step at 72oC for 10 min. A negative control reaction in which DNA was omitted was included with each run in order to verify the absence of contamination. Amplification products were electrophoresed on 1.5% agarose gel and then photographed under UV light.

Primer evaluation

In RAPD studies, some primers gave more reliable results than others depending on the plants. A pilot experiment was conducted to evaluate the suitability of the primers with A. spinulosa based on the clarity of the profiles obtained. 60 decamer random oligonucleotide primers purchased from Shanghai Songon Company, China, were tested to find 28 primers which gave reproducible banding patterns. Only these 28 primers (Table 1) were chosen for the analysis of the whole sample set.

Table 1.  Random oligonucleotide primers and their sequences used for RAPD analysis.
PrimersSequences (5′[RIGHTWARDS ARROW]3′)PrimersSequences (5′[RIGHTWARDS ARROW]3′)
S12CCTT GACG CAS151TCAG GGAG GT
S29GGGTAACG CCS152TTAT CGCC CC
S46ACCT GAAC GGS155ACGC ACAA CC
S61TTCG AGCC AGS165TGTT CCAC GC
S64CCGC ATCT ACS283ACAG CCTG CT
S65GATG ACCG CCS286AAGG CTCA CC
S85CTGA GACG GAS291AGAC GATG GG
S90AGGG CCGT CTS390TGGG AGAT GG
S91TGCC CGTC GTS482TCTG TCGG TC
S97ACGA CCGA CAS492AGTA GGGC AC
S99GTCA GGGC AAS501TGCG GGTCCT
S106ACGC ATCG CAS506GTCT ACGG CA
S134TGCT GCAG GTS507ACTG GCCT GA
S145TCAG GGAG GTS512ACAG GTGC GT

PCR amplification of cpDNA atpB-rbcL noncoding spacer

PCR was performed in a reaction volume of 100 μl using 50 mM KCl, 10 mM Tris-HCl, 1.5 mM MgCl2, 0.1% Triton X-100, 200 μM of each dNTP, 50 ng template DNA, 2U Taq polymerase, 40 pmol of each primer. Primers of Chiang et al. (1998) were used to amplify atpB-rbcL noncoding spacer of cpDNA. Primer1: 5′-ACATCKARTACKGGACCAATAA-3′, Primer2: 5′-AACACCAGCTTTRAATCCAA-3′. Primers were synthesized by Shanghai Bioasia Biotech Ltd., China. The thermocycling profile consisted of 3 min at 94oC, 30 cycles of 40 s at 94oC, 50 s at 50oC, 80 s at 72oC and an additional extension for 7 min at 72oC. The size of PCR products was determined by agarose electrophoresis.

DNA cloning and sequencing

PCR products were purified by electrophoresis on a 1.0% low melting agarose gel. The desired DNA band was cut and recovered using UNIQ-10 kit (Shanghai Bioengineering Ltd., China). Purified PCR product was ligated to a pMD18-T vector and then was used to transform competent E. coli cells DH-5α. Positive clone was identified by PCR. Purified plasmid DNA was sequenced in both directions by standard methods on an ABI 377 automated sequencer. Primers M13F and M13R located on pMD18-T vector were utilized for sequence determination.

Data analysis

Presence (1)/absence (0) of each scorable band was determined for all detected individuals and was recorded in a binary data matrix of different RAPD phenotypes. Percentage of polymorphic loci was generated.

Genetic diversity was measured using Shannon's diversity index H=−Σpilnpi where pi is the frequence of a given RAPD fragment (Lewontin 1973). H was calculated for two levels: the average diversity within population (Hpop) and the diversity within species (Hsp).

Gene diversity was estimated using the PopGen 32 software package (Yeh and Yang 1999) which gave the values of number of allele, number of effective allele, and gene diversity within species and populations.

A matrix of pairwise distances between individuals was calculated using Euclidean squared distance measures by NTSYSpc2 software package (Rohlf 1993). The resultant distance matrix was subjected to AMOVA analysis using WinAMOVA version 1.55 software (Excoffier 1992). The variance components within and among populations (regions) and ϕ statistics (ϕst) were computed. The variance components were tested statistically by non-parametric randomization tests using 3,000 permutations.

A cluster analysis based on Jaccard similarity coefficient (Jaccard 1908) matrix was performed using UPGMA method and a dendrogram was constructed in order to visualize the relationships among single individuals. Principal components analysis (PCA) of RAPD phenotypes was conducted using NTSYSpc2 software package (Rohlf 1993) to extract multidimensional relationships among the populations.

Sequences of the determined atpB-rbcL noncoding spacer of cpDNA were registered to Genbank with accession numbers of AY304397-AY304416. Sequences were aligned with the program CLUSTAL X (Thompson et al. 1997). Length variation and nucleotide composition were calculated using BioEdit (Hall 1999). Neighbour-joining analysis by calculating Kimura 2-parameter distance was conducted using PHYLIP (Felsenstein 1995). The reliablitity of the clades reconstructed was tested by bootstrapping with 1000 replicates. Minimum spanning network was constructed with the aid of the MINSPNET (Excoffier and Smouse 1994).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

Analysis of genetic variation with RAPD

28 RAPD primers used to analyse the genomic DNAs of total 60 individuals from five different populations of A. spinulosa generated 118 clear and reproducible bands, varying in size from 300 bp to 3000 bp. The number of bands per primer ranged from 1 to 8 with an average of 4.21 bands/primer. Seven out of 28 (25%) random primers detected 26 polymorphic loci, giving the percentage of polymorphic loci as 22.03% (Table 2). In TLS, only 2 out of the 28 primers, S64 and S97, detected 4 and 2 polymorphic sites, respectively. For DXS, 2 primers, S97 and S512, amplified 2 and 1 polymorphic fragments separately. In DHS, 1, 1, 2, and 2 polymorphic bands were detected by primers S64, S97, S145 and S12, respectively. As for JFL, 1, 1, 6 and 2 polymorphic band(s) were yielded using primers S64, S97, S145 and S506, respectively. Finally, in DLS 2 polymorphic bands were detected by primer S145. The percentage of polymorphic loci varied among populations with the highest value in JFL (9.09%) and the lowest in DLS (1.96%) (Table 2).

Table 2.  Percentage of polymorphic loci revealed by 28 random primers in five A. spinulosa populations.
PopulationsNo. of samplesNo. of lociNo. of polymorphic lociPercentage of polymorphic sites
Tang Lang Shan1410160.0594
Da Xi Shan1110430.0288
Ding Hu Shan910660.0566
Diao Luo Shan1010220.0196
Jian Feng Ling16110100.0909
Total60118260.2203

A total of 17 different RAPD phenotypes were distinguished by the 28 random primers in the five sampled populations. 2, 2, 3, 3 and 7 phenotypes were found in DLS, DHS, TLS, DXS and JFL populations, respectively. No RAPD phenotype was shared among the populations, suggesting a high degree of population differentiation.

Shannon diversity indices based on RAPD banding patterns, ranging from 0 to 0.283, revealed the highest diversity in JFL (0.0276), intermediate in DHS (0.0207), TLS (0.0089) and DLS (0.0066) and the lowest in DXS (0.0044). The diversity within populations on average was 0.0136, whereas the average diversity within species was 0.0560. This study detected that most of the variation from the total diversity occurred among populations (1−Hpop/Hsp=75.71%), compared with the diversity within populations (24.29%) (Table 3).

Table 3.  Phenotypic diversity estimated by Shannon's index and its partition within and among populations of A. spinulosa.
Tang Lang ShanDa Xi ShanDing Hu ShanDiao LuoShanJian Feng LingDiversity within populations (Hpop)Diversity within species (Hsp)Hpop/Hsp(Hsp−Hpop)/Hsp
0.00890.00440.02070.00660.02760.01360.05600.24290.7571

Results of Nei's gene diversity demonstrated that JFL possessed the highest diversity (0.0220), followed in order by the TLS (0.0153), DHS (0.0152), DXS (0.0127) and DLS (0.0069). The GST value was 0.7763 (Table 4), suggesting significant gene differentiation among populations, which was in agreement with the result of Shannon diversity.

Table 4.  Nei's gene diversity and its partition within and among populations of A. spinulosa.
PopulationsNumber of allelesNumber of effective allelesGene diversity (H)Gene diversity within populations (HS)Gene diversity among populations (DST)Total gene diversity (HT)Gene differentiation coefficient (GST)
Tang Lang Shan1.05941.02630.01530.01320.04580.05900.7763
Da Xi Shan1.02881.02880.0127    
Ding Hu Shan1.05081.02180.0152    
Diao Luo Shan1.01691.01190.0069    
Jian Feng Ling1.09091.03250.0220    

Hierachical analysis of phenotypic diversity was performed to investigate the partitioning of the RAPD variation within and among populations using AMOVA procedure with the modification proposed for RAPD data by Stewart and Excoffier (1996). Significant ϕst value among populations (ϕst=0.8145, p<0.001) was uncovered, suggesting highly phenotypic differentiation. Then, five sampled populations were defined as two groups (TLS/DHS/DXS vs JFL/DLS) according to their geographical locations (Guangdong vs Hainan) for further dissection. The results demonstrated that most of the genetic diversity (47.44%) was attributable to the variance among regions, 34.01% partitioned among populations within regions, whereas only 18.55% genetic diversity occurring within populations (Table 5).

Table 5.  AMOVA analysis for the partition of RAPD variation of A. spinulosa populations.
Source of variationDfSum of squaresVariance components% Total variancep-value
Among regions191.5882.560130.4744<0.001
Among populations within regions387.9391.835540.3401<0.001
Within populations5555.0561.001030.1855=0.1036
Total59234.5835.39670  

Cluster analysis of the 17 different RAPD phenotypes based on Jaccard's similarity coefficient using UPGMA method led to the identification of two major clades: TLS, DXS and DHS populations distributing in Guangdong form a group; whereas JFL and DLS populations from Hainan constitute the other. (Fig. 1). The result was supported by PCA (principle components analysis) based on RAPD phenotypic data. The three-dimensional representation established from the first three principle components differentiated two groups: one consisting of Hainan populations (JFL/DLS) and the other consisting of Guangdong populations (TLS/DXS/DHS) (Fig. 2).

image

Figure 1. UPGMA tree showing the relationships among the 17 different RAPD phenotypes obtained in Alsophila spinulosa. The Jaccard similarity coefficient was used.

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image

Figure 2. Plot of principal components analysis based on RAPD phenotypic characters obtained in Alsophila spinulosa.

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Gene genealogies of cpDNA haplotypes

Sequences of atpB-rbcL intergeic spacer of cpDNA were also determined for conducting a phylogenetic analysis of populations of A. spinulosa. Sequence length varied from 724 bp to 730 bp, with a consensus length of 731 bp; 46 sites (6.3%) were variable. Nucleotides A and T are common in the chloroplast sequence, with contents between 63.17% and 63.70%, which is consistent with the nucleotide composition of most noncoding regions. In total, 13 haplotypes of cpDNA atpB-rbcL spacers were identified in A. spinulosa. Both species, A. podophylla and A. denticulata, were utilized as outgroups since molecular phylogeny of family Cyatheaceae indicates that subgenus Gymnosphaera to which Alsophila podophylla and Alsophila denticulata belong is dichotomized as sister group of subgenus Alsophila which contains A. spinulosa (Wang et al. 2003). Figure 3 shows a minimum spanning network reconstructed on mutational changes between cpDNA haplotypes. Haplotypes of JFL and DLS firstly separated from those identified in TLS, DHS, and DLS, suggesting that A. spinulosa may be subdivided into two geographical groups: Hainan populations and Guangdong populations. Within Hainan region, 2 haplotypes, JFL02 and JFL03, coalesced to JFL01 (=JFL04=JFL05=DLS01) with 3 mutations. As for Guangdong region, 4 haplotypes (DHS01, DHS03, TLS04 and DXS03) coalesced to TLS01 (=TLS02=DXS04) with 1 to 3 mutations, this branch and haplotype DXS01 again coalesced to TLS05 (=DHS02) with 1 mutation each; on another branch haplotypes TLS03 and TLS06 coalesced to DXS02 (=DXS05) with 1 and 4 mutation(s), respectively.

image

Figure 3. Minimum spanning network relating haplotypes of atpB-rbcL spacer of cpDNA found in populations of Alsophila spinulosa. A. podophylla (AP) and A. denticulate (AD) were used to root the tree. Major links between haplotypes are represented as thick lines. Other possible link is given as thin line. Numbers in circles on links between haplotypes indicate the number of mutational differences between haplotypes.

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A neighbour-joining (NJ) tree was also constructed based on sequences of haplotypes of atpB-rbcL intergenic spacer of cpDNA (Fig. 4). Haplotypes of A. spinulosa formed a monophyletic group within which two major clades were identified. One consisted of populations from Hainan, and the other corresponded to populations from Guangdong (bootstrap values=100%). Two clades formed sister groups to each other. Of the Guangdong clade, except for TLS03, TLS06, DXS02 and DXS05, most of the rest haplotypes coalesced near the tip of the tree; as for Hainan clade, JFL02, JFL03 and DLS01 coalesced exactly at the tip of the tree, suggesting the relatively recent occurrences of the coalescence events. In addition, sequences of the same populations were never grouped into a monophyletic clade. Consequently, the branches from different locations were highly mixed, indicating great amount of gene flow between them.

image

Figure 4. Neighbour-joining tree of Alsophila spinulosa, rooted using Alsophila podophylla and Alsophila denticulata as outgroups, based on sequences of haplotypes of the atpB-rbcL intergenic spacer of cpDNA. Numbers above branches indicate the bootstrap values of 1000 replicates.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

Genetic diversity and structure

Compared with seed plants, relatively few reports on genetic diversity in pteridophytes have been conducted. Schneller et al. (1998) employed the RAPD technique to analyse the genetic variation in natural populations of the apomictic fern Dryopteris remota. The molecular method exhibited a greater power for resolving genetic variability in this species, compared to research using allozymes that revealed very little variation. Influenced by the Quaternary glaciers, ancestral populations of A. spinulosa were forced to retreat into refugia and survived in the tropical and subtropical montane zone. Thus, they provide important materials for investigating fern genetic population structure. This study demonstrated that RAPDs combined with sequence data of cpDNA atpB-rbcL noncoding spacers also yield substantial information for genetic variation and differentiation of relict populations of A. spinulosa.

Results of percentage of polymorphic loci, Shannon phenotypic diversity and Nei's gene diversity comprehensively indicated that JFL possessed the highest diversity, TLS and DHS in the intermediate and DLS (or DXS) the least; the corresponding values of the population appeared correlated with the population size. Large population exhibiting greater genetic diversity compared to smaller populations was also demonstrated in other researches (Cosner and Crawford 1994; Keiper and McConchie 2000). Moreover, JFL and DHS were within protected environments, where the habitat had been stable; in contrast, the populations of DLS, TLS and DXS were small (no greater than 100 m) as a result of the land uses that surrounded them. Loveless and Hamrick (1984) suggested that small population shows lower genetic variation primarily due to the effects of genetic drift.

Differentiation was detected among populations of A. spinulosa (1−Hpop/Hsp=0.7453, GST=0.7763 and ϕst=0.8145). AMOVA showed that 47.44% of the variance was partitioned among regions (Hainan and Guangdong), 34.01% attributed among populations within regions, whereas only 18.55% occurring within populations. Similar population structure has also been revealed in other fern populations (Schneller and Holderegger 1996; Keiper and McConchie 2000; Pryvor et al. 2001). Geographical subdivision of populations of A. spinulosa between Hainan and Guangdong was also supported by UPGMA dendrogram of RAPD phenotypes, principal components analysis based on RAPD patterns, minimum spanning network and NJ tree established on cpDNA atpB-rbcL haplotypes (Fig. 1–4). Distribution of A. spinulosa in China is restricted to the area between 18.5°N to 30.5°N. In this area, during the late Tertiary and the early Quaternary, Hainan was separated from China mainland due to crustal movement and sea transgression. Within the late Pleistocene, global sea dropping possibly made Hainan linked to mainland again, but with the advent of following warm period in the Holocene Hainan renewedly became isolated (Xing et al. 1995). Since then, migrations of individuals of A. spinulosa between Hainan and Guangdong were obstructed by Qiongzhou strait with a width of 20–40 km. Even though A. spinulosa produces very small, wind or water-dispersed spores, spore-dispersal across the ocean was hardly achieved, because of their weak vitality (e.g. loss of vitality around 8 days; Cheng et al. 1990). Changes of environmental factors, such as humidity, temperature and illumination, were also lethal to germinating spores. Therefore, the lack of effective gene flow due to vicariant event might be among the main reasons to facilitate interregional population differentiation of A. spinulosa.

A very low level of intra-specific genetic diversity was maintained in A. spinulosa. Within species diversity was merely 0.0560 measured by Shannon's index, whereas 0.0590 estimated by Nei's gene diversity. The results showed that the intra-species diversity of A. spinulosa at most achieved 20% of that of other pteridophytes (Camacho and Liston 2001). Since A. spinulosa had been damaged by glaciation and only survived in the tropical and subtropical refugia, both bottleneck effect and founder effect were probably accountable for the low level of genetic diversity within species. Because of genetic drift and lack of effective gene flow among fragmented populations, the population genetic variation were reduced. The same phenomenon have also been noticed in other relict plants (Mosseler et al. 1993; Bauert et al. 1998).

Breeding system

Gene flow, the genetic structure of population and the evolutionary potential of a species are significantly influenced by plant breeding systems (Loveless and Hamrick 1984; Korpelainen 1995). Although the breeding system of A. spinulosa remains unknown, the bisexual condition, the synchronous maturation of male and female gametes and the reproductive investigations of other species and hybrids in the genus of Alsophila indicate that A. spinulosa have the potential to self-fertilize (Cheng et al. 1990; Conant 1990). Moreover, it is known that fertilization between prothallia of A. spinulosa highly depends on water, through which the sperm swim from the antheridium to the archegonium (Cheng et al. 1990). This prerequisite further hinders the chance of fusion of sperm and egg from different gametophytes. Low level of genetic diversity as well as high degree of population differentiation mentioned above also suggest that the most probable breeding system of A. spinulosa is one that is dominanted by inbreeding, since inbreeding lead to an increase in homozygosity by inheriting identical alleles from genetically similar parents (Pages and Holmes 1998).

Population conservation

This research provides some insight into the genetic structure and variation of A. spinulosa populations, which meet the basic requirements for conservation management. Given the high level of population differentiation and low level of genetic diversity of the species, to retain existing diversity, more reservation regions or sites should be established in order to conserve as many populations as possible if in situ preservation measures are devised. In this study, the establishment of national reserve have proven to be an effective approach for the persistence of A. spinulosa through maintaining stable habitats such as JFL site. On the other hand, introduction of individuals carrying novel genes to depleted populations may partially restore the genetic diversity (Butler et al. 1994). However, care must be taken when choosing such a measure since the introduction of genotypes that have evolved under widely different selective regiments may reduce the overall population fitness, i.e. outbreeding depression (Hamrick et al. 1991; Keiper and McConchie 2000). Yet, upon outcrossing, populations with a long history of inbreeding may exhibit significant heterosis. Therefore, introduction of individuals among populations still has implications for the conservation of A. spinulosa.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

We thank Prof. Wang Bo-Sun at Dept of Biology, School of Life Sciences, Zhongshan (Sun Yat-sen University), who kindly identified plant materials. Financial support was received from National Natural Science Foundation of China (30170101) and Natural Science Foundation of Guangdong Province, China (011125).

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  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References
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