Allozyme Variation in Natural Populations of Picea asperata

A survey of allozymic alleles and genetic diversity was conducted for ten natural populations of Picea asperata Mast. originating from the mountains of Southwest China. A total of twenty-seven alleles at seventeen loci were observed. Ten of the loci were found monomorphic. Our results showed that the populations sampled were characterized by low genetic diversity (mean H e = 0.096) and a low level of inbreeding (mean F is = 0.005). The UPGMA tree of genetic relationships indicated that there was signiﬁcant differentiation among populations. The coefﬁcient of genetic differentiation among populations, based on F st, equaled 0.311. Such extensive inter-populational differentiation detected in P. asperata could have resulted from allele frequency divergence among populations, particularly, in one population. Introgression from another species, variation in environmental conditions, and differing selection pressures could be some of the factors attributing to signiﬁcant differences among populations. In addition, our results showed that the geographic and genetic distances were not correlated in the populations of P. asperata . Based on the genetic information obtained, we concluded that monitoring appropriate genetic markers may be an effective means of identifying potential genetic changes occurring during forest tree evolution.


Introduction
Genetic diversity provides evolutionary potential for sustainable forest health in the face of a changing environment. Since trees are normally the keystone species of forest ecosystems, their genetic diversity is of a special significance. Genetic variability is also the basis for tree improvement. Thus, the genetic diversity of trees can be viewed as the foundation for forest sustainability and ecosystem stability. Allozymes and molecular markers based on DNA can assist in determining the genetic diversity and structure of populations, in estimating evolutionary genetic processes that maintain genetic diversity, and in the development of genetically sustainable forest management practices (Rajora 1999, Rajora and Mosseler 2001a, b, Mosseler et al. 2003. A large number of papers investigating the pattern and distribution of genetic variation within forest tree species have been published following the fast progress in the development of marker techniques. Unlike in the case of species with wide distribution and outcrossing breeding systems, relatively low genetic diversities (Ledig and Conkle 1983, Dayanandan et al. 1999, White et al. 1999, Aguirre-Plantier et al. 2000 and considerable levels of inbreeding (Fuchs et al. 2003) have been observed in isolated populations of some tree species occupying a limited range of distribution. The presence of such low levels of genetic diversity have confirmed the view of Hamrick et al. (1992) that the genetic variation of endemic species may be reduced through the effects of genetic drift in small populations.
Picea asperata Mast. is one of the most important trees for the production of pulp wood and timber, and it is a prime reforestation species in Southwest China. P. asperata occurs in the alpine and canyon regions of northwestern Sichuan province and in the southeastern Gansu province (100°-105°E, 30°-35°N), both of which are important water self-restraint regions (Wu and Raven 1994). Fossil and pollen records have revealed that P. asperata is a tertiary relic, for which the Qinghai-Tibet Plateau has served as a refuge. During its evolutionary process, P. asperata has been exposed to long-term geographical isolation, and later also to artificial selection (Wu 1959). As a result of deforestation, large areas of the natural distribution range of P. asperata have been destroyed and only few remnant populations are preserved. Consequently, the genetic diversity of P. asperata may have decreased. For the development of effective strategies for sustainable ecosystem-based management and conservation of genetic diversity in P. asperata, it is essential to determine and benchmark the amount and pattern of inherent genetic diversity present in the populations. So far, very little attention has been given to the genetic implications resulting from the loss of late succession tree species and forest types.
In this paper, we report the level and pattern of genetic variability in ten populations of P. asperata in their natural range in Southwest China, as determined based on seventeen allozyme loci coding for eight enzymes. The objective is to provide valuable information for further conservation and breeding programs on P. asperata.

Plant Materials
Seed samples were collected from ten natural populations of P. asperata distributed throughout their native range in Southwest China (Table 1). From each population, 30 mother trees, each separated by a distance of 50 m, were sampled.

Allozyme Analysis
Seeds were germinated until the emerging radicle was 2-5 mm in length. Diploid embryo tissues were excised, and enzyme extracts were made from the mixture of two haploid megagametophytes from each mother tree in 0.2 M phosphate buffer (pH 7.5). Extracts were absorbed onto wicks made from Whatmann filter paper and stored at -70 °C until used for enzyme electrophoresis. Two discontinuous buffer systems and horizontal starch-gel electrophoresis (12%) were employed to resolve allozymes for eight enzyme systems (Table 2). The gels were sliced and stained using the methods of Soltis et al. (1983) and Weeden and Wendel (1989) with some modification. The genotypes of individual trees were inferred from the banding patterns (Weeden and Wendel 1989).

Data Analysis
For each population, genetic diversity parameters were assessed in terms of mean number of alleles per locus (A), effective number of alleles per locus (Ae), percentage of polymorphic loci (99% criterion) (P), expected heterozygosity (He) (Nei 1978), and observed heterozygosity (Ho). Within each population, single-locus genetic structure was investigated by testing for deviations from the Hardy-Weinberg (H-W) equilibrium. The extent and direction of the deviation from the H-W equilibrium within each locus was quantified by calculating the weighted mean of Fis (inbreeding coefficient) across all populations and by testing it for the significance of deviation from zero (Wright 1978).
Genetic differentiation among all ten populations was determined by analyzing the heterogeneity of the allele frequencies, by calculating genetic distances (Nei 1978), and by conducting the hierarchical F-statistics analysis (Wright 1978). The heterogeneity of the allele frequencies over all ten populations was examined using a contingency chi-square test (Workman and Niswander 1970). Gene flow (Nm) was estimated from Nm = 0.25(1 -Fst) / Fst. The genetic relationships among all populations were assessed by estimating Nei's genetic distances for all population pairs (Nei 1978). The distances were used to construct a phylogenetic tree (UPGMA). All of the above calculations were performed using the program POPGENE ver 1.32 (Yeh et al. 1997). To reveal the potential correlation between genetic and geographic distances between populations, Mantel's test was performed on original data.  Table 2. Enzyme and buffer system used in the analysis. Histidine

Allele Frequencies and Allelic Differentiation
Eight enzyme systems were analyzed with seventeen loci showing clear banding patterns: ten loci were monomorphic and seven loci were polymorphic. The numbers of alleles observed at each polymorphic locus ranged from two (Idh-1, Mdh-1, Mdh-3 and Pgd-1) to three (Aat-2, Fdh-2 and Idh-2). A total of 27 alleles were identified among the ten P. asperata populations sampled. The distribution frequencies of the alleles are presented in Table 3. Most of the alleles were well-spread over the populations: nineteen alleles were common to all populations while seven alleles were common to the majority of the populations (at least seven populations). Only the allele Idh-2-C was found to be unique and present in population TB only.  morphism and heterozygosity. The order of the genetic diversity among populations was found to be CP>ZN>TB>DL>LP>XJ>RWG>AB>BX >HS. The highest values of these three variability parameters were found in population CP and the lowest values in population HS.

Population Genetic Structure
Significant (P < 0.05) allele frequency heterogeneity was observed at six of the seven polymorphic loci among the ten populations. Only the locus Aat-2 did not possess significant allele frequency heterogeneity over the populations. The number (and %) of loci showing significant allele frequency heterogeneity within individual populations ranged from 5 (71.4%) to 7 (100.0%) ( Table 4). The genetic analyses revealed a high level of differentiation among populations. The coefficient of hierarchical Fst (Table 5), estimated according to Wright (1978), ranged from 0.067 for locus Aat-2 to 0.529 for locus Idh-2, with the average value equaling 0.311. This showed that 31.1% of the total genetic diversity existed among populations. The overall gene flow (Nm) among populations equaled 0.554, which gives an estimate of the average number of migrants between all studied populations per generation. The observed value indicated that gene exchange between populations is limited.

Genotypic Structure and Departure from Hardy-Weinberg Equilibrium
In individual populations, tests for departures from the Hardy-Weinberg equilibrium showed significant deviations for at least one locus in other populations, except in populations TB and DL. Most of the loci (76%) followed the Hardy-Weinberg equilibrium in the majority of the populations. However, significant departures from the Hardy-Weinberg equilibrium were observed in a number of cases (24%), primarily due to a deficit of heterozygotes. Over the whole sample of P. asperata, a mean Fit value of 0.314 was   (Table 5). These Fis values show that there was no consistent tendency toward heterozygote excess or deficiency within each sampled population, and therefore no indication of inbreeding within populations.

Genetic Relationships
The genetic distances, based on the allele frequencies of the allozyme markers, were calculated for each pair of populations to estimate the extent of their divergence (Table 6). The average genetic distance between populations equaled 0.056. The lowest genetic distance (0.002) was found between populations TB and DL, and the greatest genetic distance (0.203) was found between populations XJ and ZN. Mantel's test showed no significant correlation between genetic and geographical distances among the ten populations of P. asperata.
The UPGMA cluster analyses based on Nei's (1978) unbiased genetic distances was performed to further reveal the genetic relationships among the populations (Fig. 1). The cluster analysis indicated clear differentiation among populations. Three main clusters were evident. The first group consisted of the populations TB, DL, LP, CP, BX, AB, RWG and HS. Within the first group, the populations RWG, HS, and AB were distinct from the others. The populations XJ and ZN differed considerably from the other populations and they were clustered alone as a second and third group, respectively. Table 6. Genetic distances and genetic identities among populations of P. asperata (Nei's unbiased measurements, Nei 1978).

Discussion
Many previous studies have revealed that narrowly restricted species tend to have a lower level of genetic diversity than widespread congeners (Karron 1987, Gitzendanner andSoltis 2000). This result was further confirmed in our study. The number of loci analyzed (17) enabled us to obtain a clear picture of genetic variation in natural populations of P. asperata by means of allozyme markers. The populations of P. asperata possessed a low level of polymorphism and heterozygosity (P = 41.2% and He = 0.096) when compared to Pinus pinaster (He = 0.136, Hamrick et al. 1992; He = 0.158, Salvador et al. 2000). Genetic diversity is generally the result of longterm evolution. The low level of genetic variation present in P. asperata populations may be due to severe contractions in the distribution area and population size during the last glacial period, as it has occurred in a number of conifers (Ledig and Conkle 1983, Hawkins and Sweet 1989, Billington 1991, Shapcott 1991, Gibson et al. 1995.
The evidence for a severe contraction in the distribution of P. asperata is mainly reckoned on the analysis of pollen and fossil records. Fossilized fragments have been found in Tertiary deposits from western Sichuan and northern Yunnan (Fang 1996). This confirms that in the past P. asperata was probably much more widely distributed in the southwestern region in China when compared to its present area of occurrence. The results of pollen analysis suggest that north-temperate conifer forests were restricted to a small refuge at the southeastern edge of the Qinghai-Tibet Plateau during the last ice age. Following the retreat of the glacier and climate warming during postglacial times, P. asperata extended from the glacial refuge toward the north and migrated into the mountainous area (Wang 1992, Liu et al. 2002. As a result of low latitude, the forest areas were probably limited to higher altitudinal areas of the mountains, which isolated those populations from each other. Data from fossil and pollen analyses indicated that P. asperata may have suffered a severe population bottleneck during the glacial time (Fang 1996). As summarized by Luikart and Cornuet (1998), populations, which have experienced a long period of bottleneck, should have a low level of genetic variation and a high rate of inbreeding. Traditional measures of genetic variation, e.g., heterozygosity and allelic diversity, can be used to identify past bottlenecks. In principle, it is possible to detect bottlenecks by testing for transient heterozygosity excess that recently bottlenecked populations exhibit at marker loci Luikart 1996, Luikart et al. 1998). However, in our study on P. asperata, there was no detectable effect of a bottleneck on the extent of variation because of a lack of a reference sample either from the time before the event or from another population known to be non-bottlenecked. Thus, there is no powerful support for the presumption that a genetic bottleneck had accompanied isolation in the case of populations sampled in our study. The populations of P. asperata investigated here may have gone through a genetic bottleneck but not recently enough to be detected. Also, it is possible that the variation pattern of allozymes may not be effective enough to identify a bottleneck, as the same method has failed to detect potentially significant bottleneck in other cases, e.g., in Triturus carnifex (Arntzen 2001).
The genetic variation pattern detected in the present study is relatively consistent with the general observation that woody species, especially conifers, maintain most of their variation within populations (Hamrick et al. 1992, Müller-Starck 1995, Collignon and Favre 2000, Collignon et al. 2002, Kraj 2002. However, the degree of population differentiation (Fst = 0.311) observed in P. asperata was quite large when compared with other conifer species (Petit et al. 1995, Zheng and Ennos 1999, Salvador et al. 2000, Gomez et al. 2003, Prus-Glowacki et al. 2003. However, the significant differences mainly resulted from allele frequency divergence among populations, particularly in population ZN. The UPGMA dendrogram, based on genetic distances, showed that population ZN differed considerably from the other populations and was clustered alone as a group. In fact, the Fst value equaled only 0.178 (instead of 0.311) when population ZN was removed from the analysis. All morphological traits showed that the sampled populations belonged to the same variety. Therefore, the reason for the genetical distinctiveness of population ZN may be introgression from another species, as a result of hybridization or pollen contamination. Hybridi-zation occurs commonly between many Picea species, and that explains differentiation patterns in some other Chinese conifer species as well (Szmidt and Wang 1993). On the other hand, the large genetic variability among populations in Southwest China may be due to a high ecological variability, for example, the topography of the area and the variety of climate, which can create differing selection pressures in the populations and a low level of gene flow. Slatkin (1987) has suggested that about one individual or more exchanged between two populations will prevent different neutral alleles at the same locus from being nearly fixed in the populations. The estimates of gene flow among all sampled populations and among the populations excluding population ZN, Nm = 0.554 and Nm = 1.156 (number of migrants exchanged between populations per generation; Wright 1931), respectively, indicated a quite low rate of gene exchange between population ZN and the other nine populations. The private allele found in population TB with a low frequency might be an exotic gene resulting from pollen contamination. In addition, according to Thomas et al. (1994), disjunctive habitats, and population isolation in general, affect a species' ability to survive and disperse. In the circumstances, the isolated habitats of P. asperata have played an important role in preventing sufficient gene flow, which has consequently lead to a considerable genetic heterogeneity among populations (Wright 1943, Kimura and Weiss 1964, Endler 1977. The lack of a correlation between genetic and geographic distances detected here is consistent with previous isozyme studies that showed significant geographic variation only over a much wider range of the species distribution (Yeh andLayton 1979, Yeh et al. 1985). Yeh et al. (1985) suggested that altitudinal variation may be important at a small scale, but our sampling was not intensive enough to test this aspect. Our results indicated that geographical distance as such was not an important factor influencing the genetic relatedness of populations in P. asperata, and there were other factors which are likely to affect genetic differentiation, e.g., introgression from another species, the existence of mountain barriers and the presence of rivers, such as Min River, Jialing River and Yellow River.
In conclusion, the data presented here demonstrate the level and structure of the genetic variability in the distribution area of P. asperata and, consequently, it could supply information for the management of its genetic resources and contribute to the conservation of the species. Our results demonstrated that allozymes are genetic markers that could be practical in monitoring a defined but a limited set of genetic changes that may have occurred during the process of evolution in tree species. Although isozyme loci are relatively easy to screen and they have a particular value for estimating gene frequencies and heterozygosities, their limited numbers and levels of variation lower their marker value. Therefore, the on-going research on P. asperata based on a wide range of markers covering non-coding regions, including SSR, AFLPs, RAPDs and DNA sequence data, will enable us to obtain a more complete picture of the existing genetic structure of P. asperata, and to create specific conservation strategies for the restoration of the degraded populations.