Genetic Diversity and Geographical Differentiation of Dipteronia Oliv

中国科技论文在线 http://www.paper.edu.cn

Biochemical Systematics and Ecology 35 (2007) 593e599

Genetic diversity and geographical differentiation of Dipteronia Oliv. (Aceraceae) endemic to China as revealed by AFLP analysis

Juan Yang a, Zeng-Qiang Qian a, Zhan-Lin Liu a, Shan Li b, Gen-Lou Sun c, Gui-Fang Zhao a,*

a Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Science, Northwest University, No. 229, Northern Taibai Road, Xi’an, Shaanxi 710069, PR China b College of Life Sciences and Technology, Tongji University, Shanghai, PR China c Department of Biology, Saint Mary’s University, Halifax, Nova Scotia B3H 3C3, Canada Received 3 November 2006; accepted 25 March 2007

Abstract

The genus Dipteronia Oliv. endemic to central and southern China consists of two species, Dipteronia sinensis Oliv. and Dipteronia dyeriana Henry, both of them are rare and endangered. AFLP markers were used to characterize the genetic diversity and geographical differentiation of the genus. Eight out of 32 PstI þ 3/MseI þ 3 selective primer combinations screened were applied to the analysis on 142 individuals of 17 D. sinensis and 4 D. dyeriana populations, respectively. A total of 324 fragments with 316 polymorphic were ampliﬁed. The proportion of polymorphic loci (PPB) was 97.53%. The Nei’s gene diversity in D. sinensis and D. dyeriana was 0.3319 and 0.3047, respectively. About 43.6% (GST ¼ 0.4356) of the genetic variation occurred among the populations, indicating a relatively high genetic differentiation among the populations. Cluster analysis grouped the 21 populations into two groups according to their species delimitation. The populations of D. sinensis were further divided into three subgroups corresponding to their geographical distributions. Correlation analysis revealed a signiﬁcant correlation (p < 0.05) between geographical distance and genetic distance of these populations, suggesting that the relatively high genetic differentiation among the populations of D. sinensis might be caused by geographical isolation. Ó 2007 Elsevier Ltd. All rights reserved.

Keywords: Ampliﬁed fragment length polymorphism (AFLP); Dipteronia Oliv.; Genetic diversity; Geographical differentiation; Correlation analysis

1. Introduction

The genus Dipteronia Oliv. (Aceraceae) is endemic to China and consists of only two species, Dipteronia sinensis Oliv. and Dipteronia dyeriana Henry. Both of the species are diploid (2n ¼ 18) trees distributed in the broadleaved

* Corresponding author. Tel.: þ86 29 88305207; fax: þ86 29 88303572. E-mail address: [email protected] (G.-F. Zhao).

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deciduous forests in central and southern China, occurring along mountain streams at altitudes from 1000 to 2400 m above the sea level (Amy and Steven, 2001). These two species are rare and endangered, have only small and sparsely distributed populations, and have been classified as Chinese Rare and Endangered Plant Species. Dipteronia is one of the ancient relic woody genera in the floristic region of North Temperate Zone. Fossil records showed that species of this genus were in the Tertiary widely distributed in both Asia and North America (Amy and Steven, 2001). The Qua- ternary glaciation has been suggested as the causal reason of the extinction of this genus in North America, because during the glaciation the northern part of North America was mostly covered by continental icecaps (Liu, 1997; Xia, 1997). On the contrary, the Quaternary glaciers in China were mainly developed in a few mountains and no major icecap was formed (Li, 1975, 1998). Therefore, many Tertiary relict plants like Dipteronia could survive in areas of central and southern China, although their general distribution has been significantly fragmented by the glaciation. The genetic structure of these relict species are influenced by the glaciation and subsequent population fluctuation caused by secondary dispersal events. So far, most studies on Dipteronia have focused on its morphology and taxonomy (Zhao et al., 1998; Tian et al., 2001; Qi et al., 2001; Li et al., 2003). The genetic structures of the extant populations have never been studied, although a general understanding of population’s genetic structure is crucial for a successful protection of biodiversity of these species. Amplified Fragment Length Polymorphism (AFLP) (Vos et al., 1995) represents a powerful, highly reproducible, PCR-based DNA-fingerprinting technique for studying the origin and complexity of a species. Because a large number of polymorphic loci can be investigated in a single experiment, the AFLP technique has become one of the major methods of choice for studies of genetic diversity, particularly in species where markers requiring genomic sequence information are not available. The AFLP technique has been used successfully in plant evolution and population genetic studies, especially for endangered species (e.g., Travis et al., 1996; Palacios and Gonzalez-Candelas, 1999; Drummond et al., 2000; Schmidt and Jenssen, 2000). In this study, AFLP has been used to investigate the genetic diversity of the two rare and endangered species of Dipteronia. The aims of the study are: (1) to determine the level of genetic diversity and the patterns of genetic variation and differentiation within and among the extant populations of Dipteronia; (2) to characterize genetic structure and intra-species genetic polymorphism of them; and (3) to determine whether the genetic distances among the populations are correlated with their geographical distribution patterns. The results may help us to un- derstand the cause of their endangered state, and to provide basic information to enable us develop a scientific strategy for the efficient conservation of these species.

2. Materials and methods

2.1. Plant materials

One hundred and forty-two individuals from twenty-one populations were sampled across nine provinces in China, including Shaanxi, Henan, Gansu, Sichuan, Chongqing, Hubei, Hunan, Guizhou, and Yunnan (Fig. 1 and Table 1). The sampling strategy was designed to cover the whole distribution range of the genus as widely as possible. The individuals at least 10 m apart were sampled. Young leaves were collected, dried in a plastic bag with silica gel, transported to the laboratory and stored in a 80 C freezer until use.

2.2. Extraction of genomic DNA

Total genomic DNA was extracted from dried leaves using a modiﬁed CTAB method (Wang and Fang, 1998; Yang et al., 2005). Approximately 0.5 g leaves were ground in a mortar pretreated in a 80 C freezer, with small quantities of PVP and L-ascorbic acid added. Then the powder was transferred into a 1.5 ml Eppendorf tube with 700 mlof extraction buffer (100 mM pH 8.0 TriseHCl, 1.4 M NaCl, 20 mM pH 8.0 EDTA, 2% CTAB, 1% PVP, 1% b-mercap- toethanol), and incubated at 65 C for 90 min with a shake every 10 min. An equal volume of chloroform/isoamyl alcohol (24:1) was added and mixed for 10 min. The mixture was then centrifuged at 12,000 g for 10 min. This pro- cedure was repeated twice. The supernatant was precipitated with a double volume of cold 100% ethanol, and centrifuged at 12,000 g for 10 min. The precipitate was washed twice with 70% ethanol and air-dried. Then, the DNA extract was suspended in 200 ml 0.1 TE buffer (10 mM pH 8.0 TriseHCl; 1 mM EDTA, pH 8.0). RNase (10 mg/ ml) was added to the DNA solution and kept at 20 C for long-term storage or at 4 C for immediate use. 中国科技论文在线 http://www.paper.edu.cn

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Fig. 1. Sampling locations for all 21 Dipteronia populations (ﬁlled circle, D. sinensis; ﬁlled square, D. dyeriana).

2.3. Digestion and ligation

Digestion and ligation were done concurrently in a 20 ml reaction system including 200 ng template DNA, 4 U MseI, 4 U PstI, 50 pM MseI adaptor and 5 pM PstI adaptor, 2.5 ml10 Reaction buffer, 2.5 ml 10 mM/l ATP, 3 U T4 DNA ligase and 9 ml ddH2O. It was then incubated at 37 C for 5 h, 8 C for 4 h and overnight at 4 C.

Table 1 Sampling information and analytic results of all 21 Dipteronia Oliv. populations by using Popgen 32 Software Taxa Population Sample size Altitude (m) Longitude Latitude PPB (%) H Hsh D. sinensis 1 TBS 10 1154 107460 33250 41.76 0.1643 0.2400 2 TJS 4 1603 107480 33520 37.35 0.1520 0.2213 3 BMG 3 1346 107480 33340 27.47 0.1178 0.1694 4 NX 3 1616 108200 33450 50.00 0.1981 0.2907 5 XLS 9 1526 106000 34210 48.15 0.2051 0.2941 6 CDZ 13 1468 102420 30280 74.69 0.2778 0.4104 7 LDG 10 1950 102450 30290 58.64 0.2335 0.3407 8 JZX 6 1479 108430 32030 61.11 0.2528 0.3662 9 ZPH 8 1388 110320 30020 56.48 0.2253 0.3282 10 HPS 5 1500 110310 30010 25.00 0.1016 0.1471 11 JCS 9 870 110350 31240 50.00 0.1999 0.2900 12 WJG 8 811 110330 31240 57.41 0.2420 0.3483 13 LMH 7 1297 110290 31190 62.96 0.2774 0.3962 14 ZCG 9 1735 110550 31050 71.30 0.3024 0.4351 15 QTW 7 1685 110550 31030 71.30 0.2812 0.4121 16 SRS 7 1138 112160 33430 66.05 0.2813 0.4051 17 YJ 3 1098 108420 27590 11.11 0.0403 0.0605 RDS 21 **** **** **** 84.57 0.3310 0.4841 Total 121 **** **** **** 88.58 0.3319 0.4880 D. dyeriana 18 WSH 5 2217 104240 23370 56.79 0.2277 0.3316 19 MZ 5 1902 103230 23240 39.51 0.1607 0.2333 20 PB 5 2019 103520 23010 57.72 0.2422 0.3494 21 HLT 6 1923 102540 25020 54.63 0.1870 0.2822 Total 21 **** **** **** 78.09 0.3047 0.4450 Dipteronia Total 142 **** **** **** 97.53 0.3618 0.5350 中国科技论文在线 http://www.paper.edu.cn

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2.4. Preselective ampliﬁcation

Preselective PCR amplification was performed in a volume of 25 ml containing: 2 ml of DNA solution from the digestion and ligation reaction, 0.5 ml of each PstI (50 ng/ml) and MseI (50 ng/ml) preselective primer, 0.4 ml dNTP 2þ (10 mM/l), 2.5 ml10 PCR buffer (with Mg ), 1 U Taq polymerase and 18.6 ml ddH2O. The preselective PCR con- dition was 2 min at 94 C; 30 cycles of 30 s at 94 C, 30 s at 56 C, 80 s at 72 C; and finally 5 min at 72 C. The amplified product was diluted 1:15 with 0.1 TE.

2.5. Selective ampliﬁcation

Out of 32 PstI þ 3/MseI þ 3 primer combinations screened, 8 best combinations (P-GAA/M-CAA, P-GAA/ M-CAC, P-GAA/M-CAG, P-GAG/M-CAA, P-GAG/M-CAA, P-GAG/M-CAC, P-GAG/M-CTG, P-GAG/M-CTT) were selected to use in this study. Selective PCR amplification was performed in a 25 ml containing 2 ml of diluted preselective amplification product, 2.5 ml10 PCR buffer (with Mg2þ), 0.3 ml dNTP (10 mM/L), 1 ml PstI selective primer (5 ng/ml), 1 ml MseI selective primer (30 ng/ml) labeled with FAM, 0.6 U Taq polymerase and 17.7 ml ddH2 O. The selective PCR reactions were performed with the following profile: 2 min at 94 C, 12 cycles of 30 s at 94 C, 30 s at 65 C (minus;0.7 C per cycle), 80 s at 72 C; then 23 cycles of 30 s at 94 C, 30 s at 55 C, 80 s at 72 C and with a final 5 min at 72 C.

2.6. Electrophoresis

Prior to electrophoresis, 2 ml of selective ampliﬁcation product was added to 2 ml loading buffer mix (98% form- amide (v/v), 10 mM EDTA, 0.25% xylene cyanol (w/v), 0.25% bromophenol blue (w/v)), heated at 95 C for 5 min and placed on ice. One microlitre was then immediately loaded on a 4% denaturating polyacrylamide gel. Electropho- resis was performed at a constant voltage (1680 V) at 51 C for 4 h using an automated DNA sequencer (Model 377, PE Applied Biosystems).

2.7. Data analysis

Data were collected and analyzed using the Genescan Analysis Software (PE Applied Biosystems). The AFLP bands within the readable range between 50 bp and 500 bp were scored as binary characters (1 for the presence and 0 for absence of a band for a particular size). Since the number of populations for D. dyeriana is low, a random D. sinensis sample (RDS) of a size identical with that of D. dyeriana was created, in order to efﬁciently compare the genetic diversity between the two speceis. The genetic diversities within genus, species, and population were evaluated with the percentage of polymorphic loci (PPB), Nei’s gene diversity index (H) and Shannon diversity index (Hsh), and Nei’s genetic differentiation index among populations (GST) using Popgen32 Software (Ver.1.31, Yeh et al., 1999). AMOVA (Analysis of Molecular Var- iance) (Excofﬁer et al., 1992) Software was used to calculate variance components within and between species, and genetic differentiation index between species (¢ST). The Rogers’ (1972) genetic distances among populations were estimated using TFPGA Software (Tools for Population Genetic Analysis) (Miller, 1997). UPGMA (unweighted pair-group method with arithmetic averaging) analysis was performed using TFPGA Software. The latitudes and longitudes of all the studied populations were interpreted into geographical distances using Ma- pinfo 8.0 Program. Mantel’s test (Mantel, 1967; Sokal, 1979) between geographic and genetic distances was analyzed using the software Mantel-Struct (MP Miller, Department of Biological Sciences, Box 5640, Northern Arizona Uni- versity, AZ 86011-5640).

3. Results

3.1. Genetic diversity

A total of 324 AFLP bands were obtained from the 142 individuals of 21 Dipteronia populations with the 8 PstI þ 3/MseI þ 3 selective primer pair combinations. Of the loci observed 316 (97.53%) were polymorphic in the 中国科技论文在线 http://www.paper.edu.cn

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whole data set. At species level, the PPB values for D. sinensis and D. dyeriana were 88.58% and 78.09%, respectively, and within RDS was 84.57% (Table 1). The proportion of polymorphic loci (PPB), Nei’s gene diversity index (H) and Shannon diversity index (Hsh) within populations displayed similar values in both D. sinensis and D. dyeriana (Table 1). In the 17 populations of D. sinensis, the highest PPB value (74.69%) was detected in population CDZ, and the lowest (11.11%) in population YJ. While in the four populations of D. dyeriana, the highest PPB value was 57.72 and the lowest one was 39.51%. The Nei’s gene diversity in D. sinensis ranged from 0.0403 (YJ) to 0.3024 (ZCG) and from 0.1607 (MZ) to 0.2422 (PB) in D. dyeriana .The Hsh value ranged from 0.0605 to 0.4351, and from 0.2333 to 0.3494 for D. sinensis and D. dyeriana, respectively.

3.2. Genetic structure

AMOVA analysis showed that a significantly high level of genetic differentiation occurred between D. sinensis and D. dyeriana (¢ST ¼ 0.3904). About 44% of total genetic variation was detected among populations within the genus (GST ¼ 0.4356). The genetic differentiation among populations (GST)ofD. sinensis was 0.3678, while that among populations of D. dyeriana was 0.3266. The genetic distances among all 21 populations of Dipteronia were calculated using TFPGA Software. The results showed that the average genetic distance (GD) value was 0.2424, the lowest GD was 0.0571 (CDZ vs. LDG) and the highest GD was 0.5709 (BMG vs.MZ). Among D. sinensis populations, the lowest GD was 0.0571 (CDZ vs. LDG) and the highest one was 0.3691 (CDZ vs.YJ). While among D. dyeriana populations, the lowest value was 0.1141 (WSH vs. PB) and the highest one was 0.3371 (MZ vs.PB). The mean GD value among D. sinensis was 0.1759 and the value among D. dyeriana was 0.1825. The cluster analysis using UPGMA method clearly separated the D. sinensis populations from the D. dyeriana populations (Fig. 2). A significant correlation was detected between the geographical and genetic distances among the populations of D. sinensis (p < 0.05), while there is no significant correlation (p > 0.05) among D. dyeriana’s populations.

4. Discussion

4.1. Genetic diversity of Dipteronia

This study revealed a relatively high level of genetic diversity in Dipteronia (H ¼ 0.3618, Hsh ¼ 0.5350). At the species level, the genetic diversity of D. sinensis (H ¼ 0.3319, Hsh ¼ 0.4880) and RDS (H ¼ 0.3310, Hsh ¼ 0.4841) was higher than that of D. dyeriana (H ¼ 0.3047, Hsh ¼ 0.4450). The relatively high level of genetic diversity detected in these two rare and endangered species was not surprising. Previous studies also showed that the

Fig. 2. Dendrogram of all 21 Dipteronia Oliv. populations based on Rogers’ genetic distance (UPGMA method). D. sinensis and D. dyeriana are accessions clustered into two separate groups. 中国科技论文在线 http://www.paper.edu.cn

598 J. Yang et al. / Biochemical Systematics and Ecology 35 (2007) 593e599

rare and endangered species of distinct types can maintain high levels of genetic diversities (Hickey et al., 1991; Swen- son et al., 1995; Li and Jin, 2006; Wang et al., 2005, 2006). The genetic diversity in a random D. sinensis sample of a size identical with that of D. dyeriana (21 individuals) was higher than the genetic diversity within D. dyeriana, which indicated that the relatively low level of genetic diversity of D. dyeriana was not mainly due to its small population sizes. D. dyeriana is only distributed in the south- east region of China’s Yunnan Province, while D. sinensis has a wide distribution range. A higher level of genetic diversity detected in D. sinensis than in D. dyeriana was consistent with the recognized viewpoint that the genetic diversity within eurychoric species is higher than that within stenochoric species (Qian and Ma, 1994).

4.2. Genetic differentiation of Dipteronia

Genetic analyses showed a relatively high level of genetic differentiation between D. sinensis and D. dyeriana (¢ST ¼ 0.3904). Cluster analysis showed that D. sinensis and D. dyeriana accessions were well separated into two distinct groups. The observed pattern simply reflected the distinct species relationship. Previous studies on morphology, and ITS and trnLeF sequence data also suggested distinct positions of these two species (Xu, 1998; Tian et al., 2002; Tian and Li, 2004). In addition, the distant geographical isolation may also have led to the dramatic differentiation between D. sinensis and D. dyeriana, which are geographically distributed in different regions. Cluster analysis further divided the 17 populations of D. sinensis into three subgroups. Population YJ formed a distinct subgroup, the five populations (TJS, BMG, XLS, TBS and NX) formed a second subgroup, while the rest 11 populations formed the third subgroup. The results corresponded well with their geographical distributions. A study of Alpert et al. (1993) on the inter-populational genetic differentiation of Fragaria chiloensis showed a significant correlation between inter-populational genetic and spatial distances. Botanga et al. (2002) also revealed a significant linear correlation between genetic and geographical distances in Striga asiatica (L.). In this study, the result of the correlation analysis (p < 0.05) suggested that the populations of D. sinensis might be isolated by geographical distances, because low gene flow (Nm ¼ 0.8593, Nm ¼ (1 GST)/4GST) could not counteract with population divergence caused by genetic drift.

Acknowledgement

The authors want to thank Dr. Yong-Ming Yuan (Institut de Botanique, Universite´ de Neuchaˆtel, Switzerland) for the revision of the manuscript. This project was co-supported by the National Natural Science Foundation of China (30270154) and the Program for Changjiang Scholar and Innovative Research Team in University (PCSIRT).

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