African Journal of Biotechnology
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Potentilla acaulis Linn. (Rosaceae) is an important companion species in central Asia steppe. However, no information was so far detected about genetic diversity of this species. In recent years, effects of habitat fragmentation have become central issues in conservation genetics. In order to evaluate the genetic structure and to measure the effects of isolation caused by habitat fragmentation, the randomly amplified polymorphic DNA (RAPD) data were generated and analyzed from 110 samples collected from seven sites of local populations of P. acaulis distributed in northern China. Eleven RAPD primers produced a total of 61 unambiguous bands, of which 51 bands (83.6%) were polymorphic. A high level of genetic diversity was recognized within the populations of P. acaulis: 34.4 to 68.9% of polymorphic bands observed in the given population. Analysis of molecular variance (AMOVA) showed that, genetic variability was greater within populations (83.4%) than among populations within regions (12.0%) or among regions (4.6%) investigated in this study. In addition, a low degree of genetic differentiation (ΦST= 0.17) was detected among all populations, which indicated that isolation had weak effects on genetic structure. The statistical analysis also revealed that, the genetic distances of P. acaulis among different populations were not significantly related with their geographic distances. Therefore, P. acaulis should be treated as a separate species that needs more attention from a conservation point of view and it should be considered as a conservation strategy to increasing gene exchange among isolated populations.
Key words: Potentilla acaulis Linn. (Rosaceae), steppe, habitat fragmentation, genetic diversity.
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Habitat fragmentation associated with land use and grazing activities has long been concerned among ecologists and agriculturists. Some studies have indicated that, land use is the main factor of habitat deterioration and fragmentation, while these degraded tendencies are accelerated in recent years (Du et al., 2004; League and Veblen, 2006; Li et al., 2007; Lioubimtseva and Henebry, 2009). However, in most of the problems involved in habitat fragmentation, the population isolation has become ineluctable and urgent especially for the grassland conservation. Isolated plant populations have a great potential in the loss of genetic diversity and suffer much from related injurious effects on fitness (Reed and Frankham, 2003; Pluess and Stöcklin, 2004). Consequently, genetic effects induced by habitat fragmentation have been widely studied in conservation genetics (Washitani et al., 2005). With the increasing pressure of cultivating cropland and grazing livestock in pastoral area, enlarged amounts of endemic species are enlarged and considered to encounter dual menaces from animals and human. Moreover, corresponding strategy often set up priority on the conservation of species which occupy the larger part of the population distribution on their own territory. In recent years, the research in respect to genetic structure of central Asian steppe species was only focused on the studies about some perennial shrubs from desert lowlands (Xu et al., 2003; Ge et al., 2005; Sheng et al., 2005) or two naturally fragmented endemic herbs of the uplands of Tibet (Chen et al., 2005; Xia et al., 2005). In our study, an explorative research was carried out on the northern China endemic Potentilla acaulis, which originally grows in semiarid or sandy land, hilly region and rocky upland habitats. Therefore, it has becomes an important companion species of steppe to soil and water conservation in degraded grassland. There were mainly three objectives in our study on P. acaulis: (1) genetic diversity distribution of intra- or inter- populations; (2) influence degree of fragmentation on population genetic traits and the correlation between genetic distance and geographical distance; (3) setting up the basis of conservation strategies.
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Plant and sample sites
P. acaulis, as a perennial, xerophilous, tufted frutescent and stoloniferous herbaceous plant species with digitate ternate compound leaves, is an important species of central Asia steppe and mainly distributes in sandy arid and semiarid steppe. Inflorescences are abundantly produced from April to May and seeds maturation is completed during June, even in drought years, each tuft 0 to 20 bearing branches and each bearing branch grew out 2 to 5 flowers (Liu et al., 2007). The plants are anemophilous or anthophilous but self-compatible and their seeds have no special dispersal adaptations and germinate with slight testa restrictive dormancy (Zhao et al., 2010). The sample sites are located on the National Grassland Ecosystem Field Station (NGEFS) in northern China (41°46'05.15"N, 115°40'44.45"E), which lies in the 1384 m altitude and has a total annual precipitation of 300 to 400 mm. Detailed information about the vegetation were listed in the study by Huang et al. (2007). In the study sites, we observed that the density of crawling P. acaulis on the individuals ranged about 30 to 185 ramets/m2 and the average height of ramets was below 3 cm.
Collection of plant materials
Seven 1×1 km plots were established within a 14×18 km P. acaulis distribution area (Figure 1). In the study area, a total of 110 individuals of P. acaulis were randomly selected from the seven plots. The locations of all the individuals of P. acaulis were determined by a survey laser instrument (Criterion 300, laser technology). Within the seven plots investigated, 110 individuals of P. acaulis were identified. The shapes and dimensions of those measured and P. acaulis vegetation within each individual were mapped on graph papers. P. acaulis leaf samples were collected from all those selected from the seven plots investigated. Then, the leaf samples were desiccated using silica gel and stored at room temperature before analysis.
DNA extraction and random amplified polymorphic DNA (RAPD) analysis
Total DNA of each sample was extracted from about 20 mg of silica-gel-dried P. acaulis leaves with a standard kit (TaKaRa; universal genomic DNA extraction kit, Tokyo, Japan). Sixty primers were screened for polymorphism, readability and reproducibility (Sangon, random primer kit, Shanghai, China). Eleven primers were selected out as the final analysis primers (Table 1). DNA was amplified in reaction volumes of 25 μl containing 1 μl DNA (10 ng/μl), 2 μl of primer (Invitrogen), 2 μl of each dNTP (TaKaRa), 2 μl 10×buffer (TaKaRa), 2 μl Taq polymerase (0.5 U/μl, TaKaRa) and 16 μl H2O. Polymerase chain reaction (PCR) of all samples was simultaneously carried out in a thermocycler (PTC-0200, Bio-Rad; MJ, California, USA). The thermocycler was programmed for one cycle of 4 min at 94°C, followed by 36 cycles of 30 s at 94°C, 45 s at 38°C and 120 s at 72°C with a final cycle of 7 min at 72°C. DNA fragments were determined by the electrophoresis in 1% agarose (Sigma, St. Louis, MO) gels with a tris-acetate-ethylenediaminetetraacetic acid (TAE) buffer system at 110 V for 2 h and stained with ethidium bromide. DNA bands were then visualized using ultraviolet (UV) light (Gel Doc 2000, Bio-Rad), only bands in the range between 240 and 2000 bp were scored.
Statistical analysis
Reliable and reproducible random amplified polymorphic DNA (RAPD) bands were converted into a raw data matrix with 1 (present) or 0 (absent). The weak and poorly defined bands with no reproducibility were ignored in the analysis and only polymorphic bands were used in the further statistical analysis. Because RAPDs are dominant markers, the standard measure of genetic diversity can only be estimated when the populations are presumed to abide by Hardy-Weinberg equilibrium. When we did not explicitly test whether the study populations abided by the above equilibrium, we based the analysis on simple measures of the multivariate diversity, which required no assumptions on the genetic structure. Similarities were calculated using Jaccard’s coefficient (Jaccard, 1908) as shown below:
Sij = a/(a+b+c) (1)
Where Sij is the similarity between two individuals; i and j, refers to shared bands, b refers to bands exclusive to sample; i, and c refers to bands exclusive to sample j. Values were transformed to a distance measure by subtracting them from one (Legendre and Legendre, 1998). The distance matrix was used to calculate mean distance among individuals, as well as within and among populations. Jaccard’s genetic distance was also used in ordination. A principal coordinate analysis (PCoA) of all samples was carried out on square root transformed distances as these were supposed to have metric properties (Legendre and Legendre, 1998). Genetic diversity was estimated by the Shannon index (H) (Lewontin, 1972):
Where k is the number of RAPD bands produced with the respective primer and pi is the frequency of the i-th fragment. The polymorphism information content (PIC) for each RAPD marker was calculated with the formula described by Roldán-Ruiz et al (2000):
PICi = 2fi(1-fi) (3)
Where PICi is the polymorphic information content of marker; i, fi the frequency of the marker bands which were present; 1-fi represents the frequency of marker bands which were absent. Polymorphism information content (PIC) values for dominant marker bands such as RAPD markers have a maximum of 0.5 for fi= 0.5 (De-Riek et al., 2001). The genetic diversities among populations were quantified with the pairwise ФST, calculated using ARLEQUIN version 2.0 (Excoffier et al., 2005). A ФST value was calculated for each population pair, which is an analogous measure to the fixation index (FST) (Excoffier et al., 1992) and its significance is determined based on 1000 permutations. ФST values were also used for indirect determination of the effective number of migrants among populations (Excoffier et al., 1992). Analysis of molecular variance (AMOVA) was based on the nonparametric permutational approach (Excoffier et al., 1992) and on pairwise squared Euclidean distances between RAPD phenotypes. Relationship between genetic distance and geographical distance was tested by Mantel’s test (Mantel, 1967). Simple bivariate correlations were calculated with SPSS ver.13.0 (SPSS, 2005).
Table 1. Primers used in this study and statistics for RAPD marks obtained for P. acaulis populations.
RAPD, Random amplified polymorphic DNA; PIC, polymorphism information content.
Table 2. Genetic variation among populations of P. acaulis estimated with 11 RAPD primers.
RAPD, Random amplified polymorphic DNA;*6 clonal individuals represented in 110 samples (not analyzed).
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Intra-population genetic structure
Random amplified polymorphic DNA (RAPD) analysis of P. acaulis was performed with 11 selected primers, which yielded 61 reliable bands, of which 51 bands were polymorphic (83.61%) (Table 1). The 110 samples represented 104 RAPD phenotypes and 6 clonal samples were found. Among the given populations, 34.43% to 68.85% of all bands present were polymorphic, in which the low figures were found in the south-eastern (Pa6) and high in the north-western (Pa2). The difference in genetic distance among samples from given populations showed a small range to that found by the number of polymorphic bands (0.042 to 0.075) and the Shannon index showed more scattered values between 0.177 (Pa6) and 0.365 (Pa1) (Table 2).
Inter-population genetic differentiation
The principal coordinate analysis provided evidence of an inter-population genetic differentiation of P. acaulis (Figure 2). The first and second axes were equally important and respectively explained 23% and 19% of the total variation among populations. Remote samples from the Pa1, Pa3 and Pa6 were comparatively separated in the ordination space, while samples from the other region overlapped, although Pa7 was isolated from the other populations by cropland (Figure 1). All values in the AMOVA, including Φ statistics for each pairwise comparison, were significant (Table 3). The highest variance was observed within populations (83%, P= 0.0001) and the variances among regions and among populations within given regions are respectively, 5% (P= 0.0002) and 12% (P= 0.0001) (Table 3).
Correlation of geographical and genetic distances
Geographical distance alone could not explain the genetic distance between populations, which was showed by the pairwise values of genetic distances and geographical distances (Table 4). Among all seven populations, correlations between genetic and geographical distances were nearly significant (R= 0.415, P= 0.0598) by the means of Mantel tests. There was no correlation between the pairwise ΦST values and the geographical distances (R= 0.273, P= 0.0762), however, the correlation between ΦST values and genetic distances was highly significant R= 0.884 (P= 0.0006).
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RAPD polymorphisms and genetic diversity within populations
RAPD analysis was found to provide a solution for detecting the genomic diversities and structures in populations of P. acaulis. The 104 different phenotypes among 110 plants on 61 available fragments were investigated by using 11 RAPD primers (Table 1). The result that 6 clonal individuals represented in 110 samples suggested that, P. acaulis was capable of growing and reproducing clonally. To understand the reproduction strategies of P. acaulis, it is essential to determine the relative success in establishment from vegetative propagation and seeds. In plants, the prevalence of vegetative reproduction often resulted in local genetic uniformity, although some clonal species sometimes exhibited a great intra-population genetic differentiation sometimes (Ellstrand and Roose, 1987; Widén et al., 1994). The results of our study showed that, a high proportion of diversity was detected within populations (83.39%) and the remainder part of the genetic diversity was observed among populations (Table 3), which was similar with other reports that estimated for desertification grassland or desert in these areas tended to be lower from northern China and Mongolia mountain endemics (Wesche et al., 2006). The partition levels and partitions of genetic diversity within and among populations in our study were comparable to those obtained in several other studies based on RAPD of long-lived and outcrossing clonal species (Kreher et al., 2000; Nybom and Bartish, 2000; Albert et al., 2004). Propagule recruitment from genets of perennial clonal plants could decrease genetic depletion within populations and there was a kind of specific reproductive strategy in these plants (Price and Marshall, 1999). In our study, the genetic diversity of population revealed a high level. This was probably related to vegetation reproduction of this species, which appeared to prolong generation periods and lowered the renewal ratio of population. As another result, the genotypes of species did not easily lost, which would be helpful for the maintenance of alleles (Auge et al., 2001). However, compared with the non-clonal plants of similar generation length, the clonal plants had less influence to fragmentation (Kudoh et al., 2001).
Table 3. Summary of the analysis of molecular variance (AMOVA) for P. acaulis populations.
Significances based on 9999 permutations. d.f., Degree of freedom.
Table 4. Pairwise population matrix of geographical distance (km, above diagonal) and pairwise ΦST values (below diagonal).
**P= 0.0001; *P= 0.0034 based 9999 permutations.
Genetic differentiation among populations
A relatively high proportion (12%) of genetic differentiation was observed among populations of P. acaulis (Table 3). However, in other studies, the proportion was usually less than 5% for most plant species (Jelinski and Cheliak, 1992; Chen and Song, 1998). Genetic differentiation was known to attribute to the inter-population gene flow, genetic drift and inbreeding (Loveless and Hamrick, 1984). Genetic differentiation identified by RAPD analysis reflected the complex events involved in forms of dispersal (Bohonak, 1999). Isolation of population weakened gene flow, which limited communication of genetic information between populations and the effect of genetic drift or inbreeding would act on random fixation of alleles. Since the range of seed dispersal observed in our P. acaulis population was limited, the gene transfer might be deduced by pollen dispersal (Fore and Guttman, 1999).
Effects of isolation
We found that the genetic structure was partly influenced by geographical distances. Populations were distinct but not fully separated (Figure 2). It must be pointed out that, Pa7 had overlapped with other populations even though Pa7 was separated by croplands. This result confirmed that, geographical isolation did not always affect genetic diversity (Hogbin et al., 1998). According to Wright (1942), a positive correlation was detected between genetic distance and geographical distance when gene flow and genetic drift of populations reached equilibrium (Wright, 1942; Hutchison and Templeton, 1999). Our results of Mantel statistics showed that, genetic distance among populations was not correlated with geographical distance distinctly (R= 0.415, P= 0.0598), which might have resulted from the gene flow was not a dominant factor during genetic differentiation of species, especially in the moment when the species are faced with a strong biological selection against environmental change of habitat (Wright, 1951). The same conclusion was drawn from the research of Agrostis tenuis (Liu and Godt, 1983). Nevertheless, there were also some different reports about relativity between genetic distance and geographical distance in Eucalyptus crucis and Gleditsia triacanthos (Sampson et al., 1988; Schnabel and Hamrick, 1990). ΦST values based on RAPD were deemed to be related to life form, species breeding system and seed dispersal. An empirical value for long-lived perennials was 0.25 and mean value evaluated from species with a mixed breeding system showed 0.27, whereas the value for dispersal of seeds by wind was 0.25 (Nybom and Bartish, 2000). The phenomenon of low level of genetic differentiation (ΦST= 0.17) was caused by one or more factors of life history traits and it was a possible reason why genetic differentiation degree of P. acaulis population was less than other plant species in same area (Wang et al., 2005; Sui et al., 2009).
Conservation implication
Genetic exchange is extremely important to the maintenance of genetic diversity and often relates to the reproductive traits, such as allocation of reproductive biomass, dispersal of pollen and seeds, mature period of propagule (Reed and Frankham, 2003). Increased use of agricultural land and invasion by exotic species had caused the fragmentation and destruction of population habitats, as a result, some populations became smaller and more isolated from each other than in the past (Chen, 2000). The fact that populations of P. acaulis had survived well till today suggests that they were not sensitive to the activities of humans and their live stocks, though the distribution extent of the populations has been partly affected by croplands. In that case, some modes of grassland management might be needed, which increased distribution continuity of population and reserved at least some small population as a kind of biology corridor. The fragmented species with isolated generations ordinarily showed the adaptive traits which effectively maintained the genetic diversity and exchange, such as perennial life forms, amelioration of dispersal capacity or multiform reproductive modes (Nybom and Bartish, 2000). Therefore, further research should necessarily be made on reproduction adaptability and fitness of population, which would be useful in developing a conservation strategy.
Acknowledgements
This work was supported by grants from China Ministry of Science and Technology “973” project no. 2007CB106805. We would like to thank Mr. Shounan Shen, who kindly provided the test site and equipment support. We wish to thank Jin Wang and Li Lin for aid in process of samples collection, Dr. Jing Shi from the University of Tennessee for the advices of previous version of this paper.
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