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Comparative analysis of genetic diversity and population genetic structure in Abies chensiensis and Abies fargesii inferred from microsatellite markers
Biochemical Systematics and Ecology 55 (2014) 351e357
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Comparative analysis of genetic diversity and population genetic structure in Abies chensiensis and Abies fargesii inferred from microsatellite markers Xie Wang, Qi-Wei Zhang, Yong-Qing Liufu, Yong-Bin Lu, Ting Zhan, Shao-Qing Tang* Ministry of Education Key Laboratory for Ecology of Rare and Endangered Species and Environmental Protection, School of Life Sciences, Guangxi Normal University, Guilin 541004, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 November 2013 Accepted 17 April 2014 Available online
Abies chensiensis Tieghem and Abies fargesii Franchet are two closely related tree species of Pinaceae endemic to China. A. chensiensis is usually found scattered in small forest fragments, whereas A. fargesii is a dominant member of coniferous forest. To evaluate the genetic effect of fragmentation on A. chensiensis, a total of 24 populations were sampled from the whole distribution of the two species. Seven nuclear microsatellite loci were employed to analyze comparatively the genetic diversity and population genetic differentiation. Both A. chensiensis and A. fargesii have high level within-population genetic diversity and low inter-population genetic differentiation. Low microsatellite differentiation (2.1%) between A. fargesii and A. chensiensis was observed. But microsatellite marker was able to discriminate most populations of these two species. Compared to A. fargesii, A. chensiensi has lower allelic diversity and higher genetic differentiation among populations. It suggested the existence of negative genetic impacts of habitat fragmentation on A. chensiensis. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Abies chensiensis Abies fargesii Genetic diversity Genetic differentiation Microsatellite
1. Introduction Abies chensiensis Tieghem and Abies fargesii Franchet are two closely related tree species of Pinaceae endemic to China (Fu et al., 1999). They have similar geographical distribution; these are found in Qinling Mountains, Bashan Mountains and southeast Tibet Plateau (Zhang et al., 2005; Shi et al., 2009). A. chensiensis is usually found scattered in small forest fragments at elevations from 1500 to 2300 m, whereas A. fargesii is a dominant member of coniferous forests found at elevations between 2100 and 3700 m (Zhang et al., 2005; Shi et al., 2009). Timbers of these two species are used for construction, furniture, and wood pulp (Fu et al., 1999). Because of excessive use, the A. chensiensis forest has been degraded. Recently A. chensiensis is listed as “vulnerable” in the China Species Red List and has been included in the Checklist of State Protection Category II in China (Wang and Xie, 2004). Morphologically, the two species are distinguished by A. chensiensis having bracts of seed cones not exserted, whereas A. fargesii has bract of seed cones exserted with much longer cusps. Wang et al. (2011) had investigated species delimitation and biogeography of these two fir species using mitochondrial (mt) and plastid (pt) DNA sequences (Wang et al., 2011). MtDNA haplotypes showed no obvious species bias in terms of * Corresponding author. E-mail address: [email protected] (S.-Q. Tang). http://dx.doi.org/10.1016/j.bse.2014.04.008 0305-1978/Ó 2014 Elsevier Ltd. All rights reserved.
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relative frequency. In contrast, a high level of ptDNA variation was recorded in both species. Maternally inherited genomes (mt DNA) experience considerably more subdivision than paternally inherited genomes (ptDNA) (Wang et al., 2011). Mode of inheritance appears to have a major effect on the estimates of genetic differentiation (Petit et al., 2005). Nuclear microsatellites (SSRs) have widely used in plant genetics and breeding owing to many desirable genetic attributes including hypervariability, multiallelic nature, codominant inheritance and reproducibility (Kalia et al., 2011). Population genetics theory predicts that fragmentation of continuous population into small, isolated stand may lead to an erosion of genetic variation and increase inter-population genetic divergence due to increased random genetic drift, elevated inbreeding and reduced gene flow (Young et al., 1996). But long-distance pollination and sometimes seed dispersal prevent genetic isolation in many tree species (Kramer et al., 2008). In this study, we used biparentally inherited nuclear microsatellites to examine comparatively the levels of genetic variability and genetic differentiation in A. chensiensis and A. fargesii. The aim is to estimate if there is a negative genetic impact of fragmentation on A. chensiensis. 2. Materials and methods 2.1. Population sampling Nine populations of A. chensiensis and 15 of A. fargesii were sampled, which covered the whole distribution (Table 1, Fig. 1). Six to 52 were collected from each population during June 2011 to October 2012. For each individual, approximately 0.5 g of young leaves were collected and immediately stored in plastic bags containing silica gel prior to transport to the lab. 2.2. DNA extraction and microsatellite genotyping Genomic DNA was extracted from dry leaves according to the modified CTAB method (Doyle, 1987). Genotypes of all samples were determined using 7 nuclear SSR markers: Ach1, Ach2, Afa2, Afa3, Afa4 and Afa5 developed for A. chensiensis and A. fargesii (Zhan et al., 2014) and As09 developed for Abies sachalinensis (Lian et al., 2007). PCR amplification was conducted according to the method as described in Zhan et al. (2014). Fragments were separated by a 6% denaturing polyacrylamide gel. The fragments were visualized by silver staining. 2.3. Data analysis GENEPOP version 4.0 (Raymond and Rousset, 1995) was used to test for conformation to HardyeWeinberg equilibrium (HWE) in each population using the Markov chain method (5000 dememorizations, 100 batches, and 1000 iterations) and Table 1 Collecting information and genetic parameters revealed by 7 SSR loci in 24 populations of A. chenisensis and A. fargesii. Species
Population code
Longitude
Latitude
Sample
(E)
(N)
Size
A
Ae
HO
HE
FIS
A. chensiensis
SNQ SJQ SRQ BTQ HDQ LCQ GEQ LZQ WZQ Average
110 280 800 108 500 1000 112 150 100 111 470 3700 108 270 700 107 540 900 104 200 1200 103 530 4200 103 370 100
31 470 400 32 20 3100 33 440 3000 33 380 1300 33 260 400 33 410 2300 33 330 900 34 70 2800 33 540 3500
25 30 7 6 30 30 26 30 30
8.9 8.0 5.3 5.7 8.7 8.9 10.4 8.7 10.1 8.3
4.8 4.9 4.2 4.1 5.2 4.4 6.5 5.9 6.7 5.2
0.697 0.719 0.653 0.738 0.743 0.629 0.659 0.676 0.652 0.685
0.709 0.761 0.671 0.704 0.742 0.705 0.780 0.773 0.759 0.734
0.037 0.072 0.103 0.043 0.016 0.125 0.174 0.142 0.157 0.097
A. fargesii
SNB SCB HLB XQB YZB PHB HDB LCB LLB HSB STB ZNB XCB THB DLB Average
110 230 5600 109 500 1500 109 200 3900 110 280 3200 108 360 4000 108 290 600 108 270 700 107 540 1000 107 470 4100 106 460 4600 104 100 4200 103 190 4400 104 20 4900 103 340 800 103 410 1400
31 430 2900 31 390 2300 31 590 5900 34 250 2000 33 240 3900 33 280 200 33 260 400 33 410 2300 33 540 2200 33 350 2800 33 400 4300 34 260 5400 34 130 2000 34 540 1200 34 540 2100
29 24 30 28 51 28 18 30 30 30 52 30 44 30 30
10.3 10.3 10.9 8.7 11.0 10.0 8.0 10.0 9.7 9.7 12.6 9.7 11.1 11.1 10.9 10.3
6.5 4.7 6.4 5.8 6.9 5.3 5. 4 6.9 5.7 6.2 7.1 6.3 6.8 6.4 7.2 6.2
0.685 0.661 0.629 0.676 0.650 0.673 0.619 0.676 0.705 0.685 0.678 0.624 0.597 0.633 0.705 0.660
0.736 0.718 0.737 0.755 0.747 0.707 0.751 0.810 0.727 0.717 0.742 0.733 0.731 0.762 0.767 0.743
0.087 0.100 0.163 0.123 0.139 0.066 0.203 0.182 0.048 0.061 0.097 0.165 0.194 0.185 0.098 0.127
A: number of alleles; Ae: number of effective alleles per locus; HO: observed heterozygosity; HE: expected heterozygosity; and FIS: inbreeding coefficient.
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Fig. 1. Sampling localities and distributions of A. chensiensis and A. fargesii in China.
for linkage disequilibrium between pairs of loci in each population using the Markov chain method. MICRO-CHECER version 2.2.3 (van Oosterhout et al., 2004) was used to test locus for the presence of null alleles. GENALEX 6 (Peakall and Smouse, 2006) was used to calculate genetic diversity measures and the observed (HO) and expected (HE) heterozygosities over all loci and populations. FSTAT version 2.9.3 (Goudet, 2001) was used to estimate the inbreeding coefficient (FIS) for each locus. Pairwise FST value between populations was estimated using ARLEQUIN version 3.00 (Excoffier et al., 2005). We used Bayesian analysis implemented in the program STUCTURE 2.1 (Pritchard et al., 2003) to infer population structure and explore population assignments of individuals. The number of populations (K ¼ 1e8, 1e15, 1e24 for A. chensiensis, A. fargesii and both taxa, respectively) was tested and determined assuming no prior information about the number of populations sampled and to which population each individual belonged. MCMC parameters set for a burn-in period of 5 105 and run length of 106 iterations under the model of population admixture and such that the allele frequencies are correlated within population, 20 independent runs and tests for robustness of the applied model were performed to ensure consistent results. To determine the optimal K-value, we used the Evanno et al. (2005) method, in which the number of populations (K) was plotted against DK ¼ mrL00 (K)r/srL(K)rin which the estimated number of populations identified by the largest change in log-likelihood L(K) values between estimated number of populations. After determining the number of clusters, this program was used to assign individuals into respective populations. To detect recent genetic bottlenecks, we used the Wilcoxon’s sign-rank test of heterozygosity excess under the stepwise mutation model (SMM) and the two-phase model (TPM). The TPM comprised 95% single step mutations and 5% multi-step mutations. These analyses were conducted using BOTTLENECK 1.2.0.2 (Piry et al., 1999). Isolation by distance Mantel’s test was used to examine the relationship between genetic and geographical distances employing the program IBD (Bohonak, 2002). The analysis was based on 10,000 randomizations of the data. Confidence limits of the relationship were based on 10,000 bootstrap re-samples of the data. Genetic distances between populations were measured as pairwised FST/(1 FST). Geographic distance was natural-log-transformed for the analyses. 3. Results 3.1. Genetic diversity Examination of the HardyeWeinberg equilibrium (HWE) showed 19 times deviation of 168 tests after Bonferroni adjustments. The significant tests were found in loci AS09 (1 population), Ach2 (3 populations), Afa2 (7 populations) and Afa4 (8 populations). The Micro-Checker software detected possible null alleles at these loci in the populations. These deviations possibly resulted from the presence of null alleles. Significant linkage disequilibrium was detected in 8 out of the total 189 tests (P < 0.05) in A. chensiensis, and 9 out of the total 315 tests in A. fargesii, but not more than 1 population for any pairs of loci. For A. fargesii, the number of alleles per locus ranged from 14 to 28, with an average of 18.4. The observed and expected heterozygosities ranged from 0.225 to 0.916 and from 0.261 to 0.885, respectively (Table 2). For A. chensiensis, the number of
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Table 2 Characteristics of the nuclear microsatellites used for A. chensiensis and A. fargesii. Species
Locus
Size range (bp)
A
HO
HE
FIS
FST
A. chensiensis
Ach1 Ach2 Afa2 Afa3 Afa4 Afa5 As09 Average
170e202 154e204 194e216 124e158 346e360 322e348 236e276
16 22 12 10 8 10 21 14.1
0.894 0.760 0.591 0.345 0.608 0.729 0.868 0.685
0.826 0.872 0.757 0.388 0.673 0.759 0.862 0.734
0.083 0.128 0.218 0.110 0.096 0.040 0.007 0.072
0.076 0.061 0.103 0.085 0.138 0.087 0.066 0.088
A. fargesii
Ach1 Ach2 Afa2 Afa3 Afa4 Afa5 As09 Average
166e208 158e216 192e218 124e158 342e372 320e356 238e282
20 28 14 14 14 16 23 18.4
0.916 0.804 0.613 0.225 0.490 0.689 0.882 0.660
0.885 0.897 0.831 0.261 0.742 0.711 0.871 0.743
0.034 0.104 0.263 0.138 0.340 0.031 0.012 0.118
0.032 0.039 0.043 0.045 0.086 0.068 0.050 0.052
A: number of alleles; HO: observed heterozygosity; HE: expected heterozygosity; FIS: inbreeding coefficient; and FST: differentiation coefficient.
alleles per locus ranged from 8 to 22, with an average of 14.1. The observed and expected heterozygosities ranged from 0.345 to 0.894 and from 0.388 to 0.872, respectively (Table 2). Genetic diversity estimates from seven SSR loci at population level are summarized in Table 1. For A. chensiensis, the number of alleles per locus each population ranged from 5.3 to 10.4, with an average of 8.3. Observed heterozygosity (HO) over all loci ranged from 0.629 to 0.743, and expected heterozygosities (HE) ranged from 0.671 to 0.780 (Table 1). For A. fargesii, the number of alleles per locus each population ranged from 8.0 to 12.6, with an average of 10.3. Observed heterozygosity (HO) over all loci ranged from 0.597 to 0.705, and expected heterozygosities (HE) ranged from 0.707 to 0.810 (Table 1). Using pure stepwise mutation model (SMM) and two-phase mutation model (TPM; 95% SMM and 5% IAM), the Wilcoxon’s test revealed that the two firs have not experienced the bottleneck effect recently. 3.2. Genetic differentiation The overall genetic differentiation among populations of Abies chensiensis was estimated to be 0.0634 (FST), pairwise FST values ranged from 0.025 to 0.133.The overall FST value for A. fargesii was 0.0349, pairwise values ranged from 0.003 to 0.103. AMOVA analysis revealed that most of the genetic variability detected was contained within populations (93.66% for A. chensiensis and 96.51% for A. fargesii). This value was 93.64% when two species were considered together, and only 2.1% of the variation resided between species (Table 3). Estimates of gene flow (Nm) among populations estimated from FST were 3.693 and 6.913 for A. chensiensis and A. fargesii, respectively. High estimated gene flow is consistent with the results that there is not significant correlation between genetic and geographical distances (r ¼ 0.0177, P ¼ 0.5670, for A. chensiensis, and r ¼ 0.1866, P ¼ 0.9670, for A. fargesii). In A. chensiensis, STRUCTURE showed that it reached a peak when K ¼ 5 (DK ¼ 30.10), it means the nine sampled populations may be divided into five genetic clusters. In A. fargesii, the results indicated that it has not an obvious peak (DK < 3.09), which means the best K value is 1. For all individuals of A. chensiensis and A. fargesii, the probability of the data was maximum with K ¼ 2 (DK ¼ 134.03), suggesting that the individuals analyzed can be split into two distinct genetic clusters. All populations from A. chensiensis showed a higher proportion of membership of cluster I than cluster II, while 12 of the 15 populations from A. fargesii showed a higher proportion of membership of cluster II than cluster I (Fig. 2). 4. Discussion 4.1. Genetic diversity Woody plants tend to have more genetic diversity than non-woody species with similar life history traits (Hamrick and Godt, 1996; Nybom, 2004). In this study, high levels of genetic variation were observed in A. fargesii (A ¼ 8.4, HO ¼ 0.660, HE ¼ 0.743) and A. chensiensis (A ¼ 14.1, HO ¼ 0.6850, HE ¼ 0.734). The levels of genetic diversity were similar to other conifer species with a wide geographical range based on nuclear microsatellite, such as Pinus contorta (A ¼ 11.8, HO ¼ 0.46, HE ¼ 0.71) (Thomas et al., 1999), Taxus wallichinan var. mairei (A ¼ 13.2, HO ¼ 0.062e0.708, HE ¼ 0.357e0.777) (Zhang and Zhou, 2013), Picea abies (A ¼ 22, HE ¼ 0.640) (Tollefsrud et al., 2009), and Pseudotsuga menziesii (A ¼ 46.17, HO ¼ 0.783, HE ¼ 0.935) (Krutovsky et al., 2009). They are higher than congeneric species with a narrow and fragmented distribution, such as Abies
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Table 3 AMOVA at the three hierarchical levels considered from A. chensiensis and A. fargesii. Species
Source of variation
d.f.
Sum of squares
Variance components
Percentage of variation
All
Among species Among populations within species Within populations Total
1 22 1372 1395
44.992 210.943 3624.113 3880.049
0.05930 0.12021 2.64148 2.82099
2.10 4.26 93.64
FST ¼ 0.06363a FSC ¼ 0.04353a FCT ¼ 0.02102a
A. chensiensis
Among populations Within populations Total
8 419 427
88.422 1113.879 1202.301
0.18004 2.65842 2.83847
6.34 93.66
FST ¼ 0.06343a
A. fargesii
Among populations Within populations Total
14 953 967
122.521 2510.234 2632.755
0.09533 2.63403 2.72937
3.49 96.51
FST ¼ 0.03493a
a
P < 0.01.
ziyuanensis (A ¼ 2.55, HO ¼ 0.319, HE ¼ 0.435) (Tang et al., 2008), A. fraseri (A ¼ 5.52, HO ¼ 0.341, HE ¼ 0.442) (Potter et al., 2008, 2010) and Abies balasae var. phanerolepis (A ¼ 6.4, HO ¼ 0.303) (Potter et al., 2010). 4.2. Genetic differentiation Trees tend to have less among population differentiation than do herbaceous species (Hamrick and Godt, 1996; Nybom, 2004). The degree of population differentiation (FST ¼ 0.035) was low in the A. fargesii populations analyzed. AMOVA revealed that only 3.49% of the total genetic variation was attributed to differences among populations. The FST value of the A. chensiensis (FST ¼ 0.0634) was similar to that found in A. fargesii. Low genetic differentiation estimated was congruent with that detected in other conifer species based on SSR markers, such as Pinus monticola (FST ¼ 0.057) (Mehes et al., 2009), Pinus strobus (FST ¼ 0.084) (Mehes et al., 2009), P. abies (FST ¼ 0.029) (Tollefsrud et al., 2009), A. fraseri (FST ¼ 0.004) (Potter et al., 2008), and A. balsamea (FST ¼ 0.037, based on isozyme) (Shea and Furnier, 2002). But the estimates were lower than that detected in narrow and isolated distribution conifer species, such as A. ziyuanensis (FST ¼ 0.25), A. balsamea (FST ¼ 0.133) (Potter et al., 2010), T. baccata (FST ¼ 0.227) (Dubreuil et al., 2010), and Taxus wallichiana var. mairer (FST ¼ 0.159) (Zhang and Zhou, 2013). Gene flows estimated from FST among populations were high in A. chensiensis (1.628e9.915) and A. fargesii (2.177e79.714). This is congruent with that no significant relationship between genetic distance and geographic distance was found in both species. Relatively lower pollen dispersal ability in Abies species was found (Li, 1991; Poska and Pidek, 2010). A. fargesii and A. chensiensis are distributed in three isolated mountain areas, Qinling Mt., Bashan Mt. and southeast Tibet Plateau. The dispersal of pollen and seed may cannot across the span among these three isolated distribution area. Climatic oscillations in the Quaternary have played a major role in changing the geographical distribution of plant species (Comes and Kadereit, 1998). The pollen fossil record suggested that in the last ice age, the climate was lower 6e9 C than it is now, Abies species distribution descended to low elevation area (<1000 m) in west Huibei province in China, where Bashan Mt. is located (Li, 1991). A. fargesii and A. chensiensis had a continuous distribution in the area between Bashan Mt. and Qinling Mt. The historic genetic gene flow was formed in the period of the last glaciations. Morphological differences between A. chensiensis and A. fargesii are obvious. They grow in different elevation mountain, with environmental difference. But low genetic differentiation between A. fargesii and A. chensiensis (2.1%) was observed, which was congruent with the results estimated from plastid DNA (0.79%) and mitochondrial DNA (1.19%) (Wang et al., 2011). Low SSR differentiation was also observed in other close related tree species (González-Pérez et al., 2009). But most of individuals of the sampled A. chensiensis populations can be assigned to one genetic cluster and most of individuals of the populations from A. fargesii can be assigned to another cluster, except 3 populations. It suggested that microsatellite marker is able to discriminate most populations of these two species. 4.3. Genetic effects of fragmentation Compared to A. fargesii (FST ¼ 0.0349), higher genetic differentiation among populations in A. chensiensis (FST ¼ 0.0634) was observed. The estimated values of HO (0.6850) and HE (0.734) in A. chensiensis are similar to that (HO ¼ 0.660, HE ¼ 0.743) in A. fargesii. But the average number of alleles per locus each population in A. chensiensis (8.3) is lower than that in A. fargesii (10.3). For microsatellites, allelic diversity is probably more informative than expected heterozygosity for assessment of genetic erosion in populations (Spencer et al., 2000; Kang et al., 2008). The reasonable explanation for relatively lower allelic diversity in A. chensiensis is that it distributed in fragmented stand. Smaller population size and relatively restricted gene flow enhanced genetic drift, resulting in the loss of some rare alleles. The relatively lower allelic diversity and higher genetic differentiation among populations suggested the existence of negative genetic effects of habitat fragmentation on A. chensiensis.
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Fig. 2. Population structure of 24 population of A. chensiensis and A. fargesii (K ¼ 2) prepared using the Structure program.
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Genet. 98, 793e801. Tollefsrud, M.M., Sonstebo, J.H., Brochmann, C., Johnsen, O., Skroppa, T., Vendramin, G.G., 2009. Combined analysis of nuclear and mitochondrial markers provide new insight into the genetic structure of North European Picea abies. Heredity 102, 549e562. van Oosterhout, C., Hutchinson, W.F., Wills, D.P., Shipley, P., 2004. Micro-checker: software for identifying and correcting genotyping errors in microsatellite data. Mol. Ecol. Notes 4, 535e538. Wang, J., Abbott, R.J., Peng, Y.L., Du, F.K., Liu, J.Q., 2011. Species delimitation and biogeography of two fir species (Abies) in central China: cytoplasmic DNA variation. Heredity 107, 362e370. Wang, S., Xie, Y., 2004. China Species Red List. Higher Education Press, Beijing, p. 301. Young, A., Boyle, T., Brown, T., 1996. The population genetic consequences of habitat fragmentation for plants. Trends. Ecol. Evol. 11, 413e418. Zhan, T., Zhang, Q.W., Wang, X., Tang, S.Q., 2014. 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Comparative analysis of genetic diversity and population genetic structure in Abies chensiensis and Abies fargesii inferred from microsatellite markers Xie Wang, Qi-Wei Zhang, Yong-Qing Liufu, Yong-Bin Lu, Ting Zhan, Shao-Qing Tang* Ministry of Education Key Laboratory for Ecology of Rare and Endangered Species and Environmental Protection, School of Life Sciences, Guangxi Normal University, Guilin 541004, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 November 2013 Accepted 17 April 2014 Available online
Abies chensiensis Tieghem and Abies fargesii Franchet are two closely related tree species of Pinaceae endemic to China. A. chensiensis is usually found scattered in small forest fragments, whereas A. fargesii is a dominant member of coniferous forest. To evaluate the genetic effect of fragmentation on A. chensiensis, a total of 24 populations were sampled from the whole distribution of the two species. Seven nuclear microsatellite loci were employed to analyze comparatively the genetic diversity and population genetic differentiation. Both A. chensiensis and A. fargesii have high level within-population genetic diversity and low inter-population genetic differentiation. Low microsatellite differentiation (2.1%) between A. fargesii and A. chensiensis was observed. But microsatellite marker was able to discriminate most populations of these two species. Compared to A. fargesii, A. chensiensi has lower allelic diversity and higher genetic differentiation among populations. It suggested the existence of negative genetic impacts of habitat fragmentation on A. chensiensis. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Abies chensiensis Abies fargesii Genetic diversity Genetic differentiation Microsatellite
1. Introduction Abies chensiensis Tieghem and Abies fargesii Franchet are two closely related tree species of Pinaceae endemic to China (Fu et al., 1999). They have similar geographical distribution; these are found in Qinling Mountains, Bashan Mountains and southeast Tibet Plateau (Zhang et al., 2005; Shi et al., 2009). A. chensiensis is usually found scattered in small forest fragments at elevations from 1500 to 2300 m, whereas A. fargesii is a dominant member of coniferous forests found at elevations between 2100 and 3700 m (Zhang et al., 2005; Shi et al., 2009). Timbers of these two species are used for construction, furniture, and wood pulp (Fu et al., 1999). Because of excessive use, the A. chensiensis forest has been degraded. Recently A. chensiensis is listed as “vulnerable” in the China Species Red List and has been included in the Checklist of State Protection Category II in China (Wang and Xie, 2004). Morphologically, the two species are distinguished by A. chensiensis having bracts of seed cones not exserted, whereas A. fargesii has bract of seed cones exserted with much longer cusps. Wang et al. (2011) had investigated species delimitation and biogeography of these two fir species using mitochondrial (mt) and plastid (pt) DNA sequences (Wang et al., 2011). MtDNA haplotypes showed no obvious species bias in terms of * Corresponding author. E-mail address: [email protected] (S.-Q. Tang). http://dx.doi.org/10.1016/j.bse.2014.04.008 0305-1978/Ó 2014 Elsevier Ltd. All rights reserved.
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relative frequency. In contrast, a high level of ptDNA variation was recorded in both species. Maternally inherited genomes (mt DNA) experience considerably more subdivision than paternally inherited genomes (ptDNA) (Wang et al., 2011). Mode of inheritance appears to have a major effect on the estimates of genetic differentiation (Petit et al., 2005). Nuclear microsatellites (SSRs) have widely used in plant genetics and breeding owing to many desirable genetic attributes including hypervariability, multiallelic nature, codominant inheritance and reproducibility (Kalia et al., 2011). Population genetics theory predicts that fragmentation of continuous population into small, isolated stand may lead to an erosion of genetic variation and increase inter-population genetic divergence due to increased random genetic drift, elevated inbreeding and reduced gene flow (Young et al., 1996). But long-distance pollination and sometimes seed dispersal prevent genetic isolation in many tree species (Kramer et al., 2008). In this study, we used biparentally inherited nuclear microsatellites to examine comparatively the levels of genetic variability and genetic differentiation in A. chensiensis and A. fargesii. The aim is to estimate if there is a negative genetic impact of fragmentation on A. chensiensis. 2. Materials and methods 2.1. Population sampling Nine populations of A. chensiensis and 15 of A. fargesii were sampled, which covered the whole distribution (Table 1, Fig. 1). Six to 52 were collected from each population during June 2011 to October 2012. For each individual, approximately 0.5 g of young leaves were collected and immediately stored in plastic bags containing silica gel prior to transport to the lab. 2.2. DNA extraction and microsatellite genotyping Genomic DNA was extracted from dry leaves according to the modified CTAB method (Doyle, 1987). Genotypes of all samples were determined using 7 nuclear SSR markers: Ach1, Ach2, Afa2, Afa3, Afa4 and Afa5 developed for A. chensiensis and A. fargesii (Zhan et al., 2014) and As09 developed for Abies sachalinensis (Lian et al., 2007). PCR amplification was conducted according to the method as described in Zhan et al. (2014). Fragments were separated by a 6% denaturing polyacrylamide gel. The fragments were visualized by silver staining. 2.3. Data analysis GENEPOP version 4.0 (Raymond and Rousset, 1995) was used to test for conformation to HardyeWeinberg equilibrium (HWE) in each population using the Markov chain method (5000 dememorizations, 100 batches, and 1000 iterations) and Table 1 Collecting information and genetic parameters revealed by 7 SSR loci in 24 populations of A. chenisensis and A. fargesii. Species
Population code
Longitude
Latitude
Sample
(E)
(N)
Size
A
Ae
HO
HE
FIS
A. chensiensis
SNQ SJQ SRQ BTQ HDQ LCQ GEQ LZQ WZQ Average
110 280 800 108 500 1000 112 150 100 111 470 3700 108 270 700 107 540 900 104 200 1200 103 530 4200 103 370 100
31 470 400 32 20 3100 33 440 3000 33 380 1300 33 260 400 33 410 2300 33 330 900 34 70 2800 33 540 3500
25 30 7 6 30 30 26 30 30
8.9 8.0 5.3 5.7 8.7 8.9 10.4 8.7 10.1 8.3
4.8 4.9 4.2 4.1 5.2 4.4 6.5 5.9 6.7 5.2
0.697 0.719 0.653 0.738 0.743 0.629 0.659 0.676 0.652 0.685
0.709 0.761 0.671 0.704 0.742 0.705 0.780 0.773 0.759 0.734
0.037 0.072 0.103 0.043 0.016 0.125 0.174 0.142 0.157 0.097
A. fargesii
SNB SCB HLB XQB YZB PHB HDB LCB LLB HSB STB ZNB XCB THB DLB Average
110 230 5600 109 500 1500 109 200 3900 110 280 3200 108 360 4000 108 290 600 108 270 700 107 540 1000 107 470 4100 106 460 4600 104 100 4200 103 190 4400 104 20 4900 103 340 800 103 410 1400
31 430 2900 31 390 2300 31 590 5900 34 250 2000 33 240 3900 33 280 200 33 260 400 33 410 2300 33 540 2200 33 350 2800 33 400 4300 34 260 5400 34 130 2000 34 540 1200 34 540 2100
29 24 30 28 51 28 18 30 30 30 52 30 44 30 30
10.3 10.3 10.9 8.7 11.0 10.0 8.0 10.0 9.7 9.7 12.6 9.7 11.1 11.1 10.9 10.3
6.5 4.7 6.4 5.8 6.9 5.3 5. 4 6.9 5.7 6.2 7.1 6.3 6.8 6.4 7.2 6.2
0.685 0.661 0.629 0.676 0.650 0.673 0.619 0.676 0.705 0.685 0.678 0.624 0.597 0.633 0.705 0.660
0.736 0.718 0.737 0.755 0.747 0.707 0.751 0.810 0.727 0.717 0.742 0.733 0.731 0.762 0.767 0.743
0.087 0.100 0.163 0.123 0.139 0.066 0.203 0.182 0.048 0.061 0.097 0.165 0.194 0.185 0.098 0.127
A: number of alleles; Ae: number of effective alleles per locus; HO: observed heterozygosity; HE: expected heterozygosity; and FIS: inbreeding coefficient.
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Fig. 1. Sampling localities and distributions of A. chensiensis and A. fargesii in China.
for linkage disequilibrium between pairs of loci in each population using the Markov chain method. MICRO-CHECER version 2.2.3 (van Oosterhout et al., 2004) was used to test locus for the presence of null alleles. GENALEX 6 (Peakall and Smouse, 2006) was used to calculate genetic diversity measures and the observed (HO) and expected (HE) heterozygosities over all loci and populations. FSTAT version 2.9.3 (Goudet, 2001) was used to estimate the inbreeding coefficient (FIS) for each locus. Pairwise FST value between populations was estimated using ARLEQUIN version 3.00 (Excoffier et al., 2005). We used Bayesian analysis implemented in the program STUCTURE 2.1 (Pritchard et al., 2003) to infer population structure and explore population assignments of individuals. The number of populations (K ¼ 1e8, 1e15, 1e24 for A. chensiensis, A. fargesii and both taxa, respectively) was tested and determined assuming no prior information about the number of populations sampled and to which population each individual belonged. MCMC parameters set for a burn-in period of 5 105 and run length of 106 iterations under the model of population admixture and such that the allele frequencies are correlated within population, 20 independent runs and tests for robustness of the applied model were performed to ensure consistent results. To determine the optimal K-value, we used the Evanno et al. (2005) method, in which the number of populations (K) was plotted against DK ¼ mrL00 (K)r/srL(K)rin which the estimated number of populations identified by the largest change in log-likelihood L(K) values between estimated number of populations. After determining the number of clusters, this program was used to assign individuals into respective populations. To detect recent genetic bottlenecks, we used the Wilcoxon’s sign-rank test of heterozygosity excess under the stepwise mutation model (SMM) and the two-phase model (TPM). The TPM comprised 95% single step mutations and 5% multi-step mutations. These analyses were conducted using BOTTLENECK 1.2.0.2 (Piry et al., 1999). Isolation by distance Mantel’s test was used to examine the relationship between genetic and geographical distances employing the program IBD (Bohonak, 2002). The analysis was based on 10,000 randomizations of the data. Confidence limits of the relationship were based on 10,000 bootstrap re-samples of the data. Genetic distances between populations were measured as pairwised FST/(1 FST). Geographic distance was natural-log-transformed for the analyses. 3. Results 3.1. Genetic diversity Examination of the HardyeWeinberg equilibrium (HWE) showed 19 times deviation of 168 tests after Bonferroni adjustments. The significant tests were found in loci AS09 (1 population), Ach2 (3 populations), Afa2 (7 populations) and Afa4 (8 populations). The Micro-Checker software detected possible null alleles at these loci in the populations. These deviations possibly resulted from the presence of null alleles. Significant linkage disequilibrium was detected in 8 out of the total 189 tests (P < 0.05) in A. chensiensis, and 9 out of the total 315 tests in A. fargesii, but not more than 1 population for any pairs of loci. For A. fargesii, the number of alleles per locus ranged from 14 to 28, with an average of 18.4. The observed and expected heterozygosities ranged from 0.225 to 0.916 and from 0.261 to 0.885, respectively (Table 2). For A. chensiensis, the number of
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Table 2 Characteristics of the nuclear microsatellites used for A. chensiensis and A. fargesii. Species
Locus
Size range (bp)
A
HO
HE
FIS
FST
A. chensiensis
Ach1 Ach2 Afa2 Afa3 Afa4 Afa5 As09 Average
170e202 154e204 194e216 124e158 346e360 322e348 236e276
16 22 12 10 8 10 21 14.1
0.894 0.760 0.591 0.345 0.608 0.729 0.868 0.685
0.826 0.872 0.757 0.388 0.673 0.759 0.862 0.734
0.083 0.128 0.218 0.110 0.096 0.040 0.007 0.072
0.076 0.061 0.103 0.085 0.138 0.087 0.066 0.088
A. fargesii
Ach1 Ach2 Afa2 Afa3 Afa4 Afa5 As09 Average
166e208 158e216 192e218 124e158 342e372 320e356 238e282
20 28 14 14 14 16 23 18.4
0.916 0.804 0.613 0.225 0.490 0.689 0.882 0.660
0.885 0.897 0.831 0.261 0.742 0.711 0.871 0.743
0.034 0.104 0.263 0.138 0.340 0.031 0.012 0.118
0.032 0.039 0.043 0.045 0.086 0.068 0.050 0.052
A: number of alleles; HO: observed heterozygosity; HE: expected heterozygosity; FIS: inbreeding coefficient; and FST: differentiation coefficient.
alleles per locus ranged from 8 to 22, with an average of 14.1. The observed and expected heterozygosities ranged from 0.345 to 0.894 and from 0.388 to 0.872, respectively (Table 2). Genetic diversity estimates from seven SSR loci at population level are summarized in Table 1. For A. chensiensis, the number of alleles per locus each population ranged from 5.3 to 10.4, with an average of 8.3. Observed heterozygosity (HO) over all loci ranged from 0.629 to 0.743, and expected heterozygosities (HE) ranged from 0.671 to 0.780 (Table 1). For A. fargesii, the number of alleles per locus each population ranged from 8.0 to 12.6, with an average of 10.3. Observed heterozygosity (HO) over all loci ranged from 0.597 to 0.705, and expected heterozygosities (HE) ranged from 0.707 to 0.810 (Table 1). Using pure stepwise mutation model (SMM) and two-phase mutation model (TPM; 95% SMM and 5% IAM), the Wilcoxon’s test revealed that the two firs have not experienced the bottleneck effect recently. 3.2. Genetic differentiation The overall genetic differentiation among populations of Abies chensiensis was estimated to be 0.0634 (FST), pairwise FST values ranged from 0.025 to 0.133.The overall FST value for A. fargesii was 0.0349, pairwise values ranged from 0.003 to 0.103. AMOVA analysis revealed that most of the genetic variability detected was contained within populations (93.66% for A. chensiensis and 96.51% for A. fargesii). This value was 93.64% when two species were considered together, and only 2.1% of the variation resided between species (Table 3). Estimates of gene flow (Nm) among populations estimated from FST were 3.693 and 6.913 for A. chensiensis and A. fargesii, respectively. High estimated gene flow is consistent with the results that there is not significant correlation between genetic and geographical distances (r ¼ 0.0177, P ¼ 0.5670, for A. chensiensis, and r ¼ 0.1866, P ¼ 0.9670, for A. fargesii). In A. chensiensis, STRUCTURE showed that it reached a peak when K ¼ 5 (DK ¼ 30.10), it means the nine sampled populations may be divided into five genetic clusters. In A. fargesii, the results indicated that it has not an obvious peak (DK < 3.09), which means the best K value is 1. For all individuals of A. chensiensis and A. fargesii, the probability of the data was maximum with K ¼ 2 (DK ¼ 134.03), suggesting that the individuals analyzed can be split into two distinct genetic clusters. All populations from A. chensiensis showed a higher proportion of membership of cluster I than cluster II, while 12 of the 15 populations from A. fargesii showed a higher proportion of membership of cluster II than cluster I (Fig. 2). 4. Discussion 4.1. Genetic diversity Woody plants tend to have more genetic diversity than non-woody species with similar life history traits (Hamrick and Godt, 1996; Nybom, 2004). In this study, high levels of genetic variation were observed in A. fargesii (A ¼ 8.4, HO ¼ 0.660, HE ¼ 0.743) and A. chensiensis (A ¼ 14.1, HO ¼ 0.6850, HE ¼ 0.734). The levels of genetic diversity were similar to other conifer species with a wide geographical range based on nuclear microsatellite, such as Pinus contorta (A ¼ 11.8, HO ¼ 0.46, HE ¼ 0.71) (Thomas et al., 1999), Taxus wallichinan var. mairei (A ¼ 13.2, HO ¼ 0.062e0.708, HE ¼ 0.357e0.777) (Zhang and Zhou, 2013), Picea abies (A ¼ 22, HE ¼ 0.640) (Tollefsrud et al., 2009), and Pseudotsuga menziesii (A ¼ 46.17, HO ¼ 0.783, HE ¼ 0.935) (Krutovsky et al., 2009). They are higher than congeneric species with a narrow and fragmented distribution, such as Abies
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Table 3 AMOVA at the three hierarchical levels considered from A. chensiensis and A. fargesii. Species
Source of variation
d.f.
Sum of squares
Variance components
Percentage of variation
All
Among species Among populations within species Within populations Total
1 22 1372 1395
44.992 210.943 3624.113 3880.049
0.05930 0.12021 2.64148 2.82099
2.10 4.26 93.64
FST ¼ 0.06363a FSC ¼ 0.04353a FCT ¼ 0.02102a
A. chensiensis
Among populations Within populations Total
8 419 427
88.422 1113.879 1202.301
0.18004 2.65842 2.83847
6.34 93.66
FST ¼ 0.06343a
A. fargesii
Among populations Within populations Total
14 953 967
122.521 2510.234 2632.755
0.09533 2.63403 2.72937
3.49 96.51
FST ¼ 0.03493a
a
P < 0.01.
ziyuanensis (A ¼ 2.55, HO ¼ 0.319, HE ¼ 0.435) (Tang et al., 2008), A. fraseri (A ¼ 5.52, HO ¼ 0.341, HE ¼ 0.442) (Potter et al., 2008, 2010) and Abies balasae var. phanerolepis (A ¼ 6.4, HO ¼ 0.303) (Potter et al., 2010). 4.2. Genetic differentiation Trees tend to have less among population differentiation than do herbaceous species (Hamrick and Godt, 1996; Nybom, 2004). The degree of population differentiation (FST ¼ 0.035) was low in the A. fargesii populations analyzed. AMOVA revealed that only 3.49% of the total genetic variation was attributed to differences among populations. The FST value of the A. chensiensis (FST ¼ 0.0634) was similar to that found in A. fargesii. Low genetic differentiation estimated was congruent with that detected in other conifer species based on SSR markers, such as Pinus monticola (FST ¼ 0.057) (Mehes et al., 2009), Pinus strobus (FST ¼ 0.084) (Mehes et al., 2009), P. abies (FST ¼ 0.029) (Tollefsrud et al., 2009), A. fraseri (FST ¼ 0.004) (Potter et al., 2008), and A. balsamea (FST ¼ 0.037, based on isozyme) (Shea and Furnier, 2002). But the estimates were lower than that detected in narrow and isolated distribution conifer species, such as A. ziyuanensis (FST ¼ 0.25), A. balsamea (FST ¼ 0.133) (Potter et al., 2010), T. baccata (FST ¼ 0.227) (Dubreuil et al., 2010), and Taxus wallichiana var. mairer (FST ¼ 0.159) (Zhang and Zhou, 2013). Gene flows estimated from FST among populations were high in A. chensiensis (1.628e9.915) and A. fargesii (2.177e79.714). This is congruent with that no significant relationship between genetic distance and geographic distance was found in both species. Relatively lower pollen dispersal ability in Abies species was found (Li, 1991; Poska and Pidek, 2010). A. fargesii and A. chensiensis are distributed in three isolated mountain areas, Qinling Mt., Bashan Mt. and southeast Tibet Plateau. The dispersal of pollen and seed may cannot across the span among these three isolated distribution area. Climatic oscillations in the Quaternary have played a major role in changing the geographical distribution of plant species (Comes and Kadereit, 1998). The pollen fossil record suggested that in the last ice age, the climate was lower 6e9 C than it is now, Abies species distribution descended to low elevation area (<1000 m) in west Huibei province in China, where Bashan Mt. is located (Li, 1991). A. fargesii and A. chensiensis had a continuous distribution in the area between Bashan Mt. and Qinling Mt. The historic genetic gene flow was formed in the period of the last glaciations. Morphological differences between A. chensiensis and A. fargesii are obvious. They grow in different elevation mountain, with environmental difference. But low genetic differentiation between A. fargesii and A. chensiensis (2.1%) was observed, which was congruent with the results estimated from plastid DNA (0.79%) and mitochondrial DNA (1.19%) (Wang et al., 2011). Low SSR differentiation was also observed in other close related tree species (González-Pérez et al., 2009). But most of individuals of the sampled A. chensiensis populations can be assigned to one genetic cluster and most of individuals of the populations from A. fargesii can be assigned to another cluster, except 3 populations. It suggested that microsatellite marker is able to discriminate most populations of these two species. 4.3. Genetic effects of fragmentation Compared to A. fargesii (FST ¼ 0.0349), higher genetic differentiation among populations in A. chensiensis (FST ¼ 0.0634) was observed. The estimated values of HO (0.6850) and HE (0.734) in A. chensiensis are similar to that (HO ¼ 0.660, HE ¼ 0.743) in A. fargesii. But the average number of alleles per locus each population in A. chensiensis (8.3) is lower than that in A. fargesii (10.3). For microsatellites, allelic diversity is probably more informative than expected heterozygosity for assessment of genetic erosion in populations (Spencer et al., 2000; Kang et al., 2008). The reasonable explanation for relatively lower allelic diversity in A. chensiensis is that it distributed in fragmented stand. Smaller population size and relatively restricted gene flow enhanced genetic drift, resulting in the loss of some rare alleles. The relatively lower allelic diversity and higher genetic differentiation among populations suggested the existence of negative genetic effects of habitat fragmentation on A. chensiensis.
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Fig. 2. Population structure of 24 population of A. chensiensis and A. fargesii (K ¼ 2) prepared using the Structure program.
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Acknowledgments The work was supported by the National Natural Science Foundation of China (31060034 and 30760029) and Foundation of Ministry of Education Key Laboratory for Ecology of Rare and Endangered Species and Environmental Protection (1001Z012). References Bohonak, A., 2002. IBD (isolation by distance): a program for analyses of isolation by distance. J. Hered. 93, 153e154. Comes, H.P., Kadereit, J.W., 1998. The effect of quaternary climatic changes on plant distribution and evolution. Trends Plant Sci. 3, 432e438. Doyle, J.J., 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull. 19, 11e15. Dubreuil, M., Riba, M., González-Martínez, S.C., Vendramin, G.G., Sebastiani, F., Mayol, M., 2010. Genetic effects of chronic habitat fragmentation revisited: strong genetic structure in a temperate tree, Taxus baccata (Taxaceae), with great dispersal capability. Am. J. Bot. 97, 303e310. Evanno, G., Regnaut, S., Goudet, J., 2005. 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