Genetic diversities and conservation strategies of Camellia japonica populations between China and Japan*
Li Lin1,2, Ji-yuan LI 2*, Sui NI 1*, Yue CHEN 1,2, Zhenqi Fan 2, Xinlei Li 21Research Institute of Subtropical Forestry，Fuyang 311400，China；
2 Faculty of Life Science and Biotechnology of Ningbo University, Ningbo 315211,China）
Introduction
Genetic diversity, which has been recognized as one of three levels of biological diversity requiring conservation (McNeely et al., 1990), is affected by its breeding system, gene flow, genetic drift, natural selection and many other factors (Schaal et al., 1998). Insular species had been considered as model species to study genetic differentiation for characteristics of geographical isolation, gene flow barrier and small population size (Carlos et al., 2000). Nowadays, speciation processes giving rise to endemic species and lineages on oceanic islands have been widely discussed, leading to alternative models of evolution (Emerson, 2002; Silvertown, 2004; Emerson et al., 2005). Less attention has been paid to populations of species distributed both on continents and oceanic islands, nonetheless populations of the same species distributed in insular and mainland areas can provide key insights into microevolutionary processes underlying recent colonization and early stages of differentiation (Mario et al., 2011). As a general pattern, lower levels of genetic variation are expected in island populations as compared to mainland populations due to founder effects and restricted gene flow (Francisco et al.; 2000). However, Tsumura (1993) reported that the theory was not completely correct, the genetic diversity of insular species was affected by many factors (Boag,1988; Nevo,1978).
Camellia japonica, belonging to Theaceae, is widely distributed in southern China, Japan and the Korean Peninsula (Gao et al., 2005). The plants are evergreen shrubs or small trees (Zhang, 1998), with highly ornamental value, so the Camellia germplasm resources had been exploited for a long time and the population reduced sharply (Liang, 2000). In this study, ISSR technology was used to detect the genetic structure of Camellia japonica distributed in China and Japan, the main purpose being to reveal genetic diversity and genetic differentiation among the thirteen populations and analyzing the influence of insular geographical isolation on the population’s genetic structure. This study can provide the basic information for effective conservation and rational utilization of resources.
Materials and methods
1.1 Sampling
C. japonica samples were randomly collected according to the distance method (Joshi et al, 2000), a total of 390 samples were collected from thirteen populations with 30 individuals within each population in southeastern China and South Japan. Five populations were sampled in Zhejiang Province: Taohua Island (TH), Zhujiajian Island (ZJJ), Shancaidong Temple (SCD) and Huiji Temple (HJ) in Putuo Island and Xiangshan County (XS) in Zhejiang Province; three sampled in Qingdao District of Shandong Province: Changmenyan Island (CMY), Botanical Garden (BG) and Wusi Square (WS); and five sampled in Japan: Kagoshima Island (Kago), Shikoku Island (Shiko-1), Shikoku foot off Cape (Shiko-2), Goto original forest (Goto-1) and Goto windbreaks (Goto-2). Young leaves were collected and dried quickly with silica gel in sealed plastic bags in the field and then stored at -800 C for further use.
Fig.1 13 sampled populations in China and Japan
1.2 DNA extraction and ISSR-PCR amplification
Genomic DNA was extracted using the modified CTAB method (Doyle, 1991). DNA was determined qualitatively and quantitatively in 1% agarose gel buffered with 0.5×TBE. Eighty primers from the Biotechnology Laboratory, University of British Columbia (UBC set no. 9), and another 6 primers from previous reports were initially screened for PCR amplification, and 20 primers (Table 1) that produced clear and reproducible banding patterns were chosen for our final analysis. All the reactions were performed in a volume of 20 uL containing 40 ng template DNA, 2.0 uL 10×Buffer, 1.5 mmol·L-1 Mg2+, 0.2 mmol·L-1 dNTP, 0.6 umol·L -1 primer, l U Taq polymerase, which was determined after comparison and optimization. Amplifications were carried out in GeneAmp 9700 DNA Thermal Cycler (PerkineElmer, USA), programmed for a 5 min initial denaturation at 94C, followed by 40 cycles of 40 s denaturation at 94C, 45 s at 45-62C (depending on the used primer) for annealing and 1.5 min at 72C for elongation, and ended up with 10 min extension at 72C. Amplified products were electrophoresed in 1.5% agrose (containing EB 0.5ug·mL-1) and 0.5×TBE gel at 5V⁄cm for 1.5 h and photographed under ultraviolet by FR-200A.
Table 1 Primers used for ISSR amplification
|
Primers |
Sequence（5'-3'） |
Annealing temperature(℃) |
No. of bands recorded |
No. of Polymorphism Loci |
PPB(%) |
|
UBC810 |
(GA)8T |
54.8 |
11 |
9 |
81.82 |
|
UBC811 |
(GA)8C |
54.8 |
12 |
11 |
91.67 |
|
UBC813 UBC818 |
(CT)8T CA)8G |
51.2 51.2 |
10 9 |
9 9 |
90 100 |
|
UBC824 |
(TC)8G |
54.6 |
11 |
11 |
100 |
|
UBC825 UBC827 UBC834 UBC835 UBC836 |
(AC)8T (AC)8G (AG)8YT (AG)8YC (AG)8YA |
52.2 54.8 53.9 56.2 51.2 |
13 12 7 9 11 |
11 11 6 8 11 |
84.62 91.67 85.71 88.89 100 |
|
UBC841 |
(GA)8YC |
56.2 |
13 |
10 |
76.92 |
|
UBC843 |
(CT)8RA |
54.0 |
13 |
12 |
92.31 |
|
UBC848 UBC853 |
(CA)8RG (TC)8RT |
54.8 51.2 |
10 10 |
9 10 |
90 100 |
|
UBC856 |
(AC)8YA |
56.6 |
10 |
8 |
80 |
|
UBC866 |
(CTA)6 |
61.8 |
12 |
11 |
91.67 |
|
UBC873 |
(GACA)4 |
51.6 |
9 |
8 |
88.89 |
|
UBC880 |
(GGAGA)3 |
53.6 |
11 |
10 |
90.91 |
|
IR43 IR53 |
(GA)8CT (CAA)8 G |
52.9 56.6 |
10 8 |
9 7 |
90 87.50 |
|
Total |
|
|
211 |
190 |
90.05 |
|
Average |
|
|
10.55 |
9.50 |
|
Y=(C, T), R=(A, G)
1.3 Data analysis
Since ISSR markers are dominant, we assumed that each band represented the phenotype at a single biallelic locus (Williams et al., 1990). Each unique band size was designated as a locus for each primer and scored as diallelic (present: l, absent: 0). The binary data matrix was input into POPGENE version 1.32, assuming Hardy-Weinberg equilibrium. Population genetic parameters, the percentage of polymorphic bands (PPB), Nei’s gene diversity (HE), Shannon’s information index (H), the coefficient of gene diferentiation (Gst) and Nei’s genetic identity were calculated using POPGENE (version 1.32). Gene flow was estimated according to the formula：Nm = (1 - Gst)／4Gst (Nei, 1973). Based on Nei’s genetic identity, UPGMA dendrogram was drawn by NTSYSpc (version 2.10) (Rohlf，1994)．
To test a correlation between genetic distances (D) and geographic distances (in km) among populations, a Mantel test was performed using GenAlEx 6.41 software for Population Genetic Analysis (Miller, 1997) (computing 999 permutations).
2 Results
2.1 Genetic diversity of C. japonica
A total of 211 bands were presented from 20 screened primers across all 390 individuals of the thirteen populations, corresponding to an average of 10.55 bands per primer. The size of the ISSR bands fragments varied from 100 bp to 2000 bp, of these bands, 190 were polymorphic, i.e. the percentage of polymorphic bands (PPB) for this species was 90.05%. At the population level, the PPB per population varied from 66.35% to 77.25%, with the average of 71.31%. ISSR profiles of 2 populations with primer UBC841 was given in Fig. 2.
Fig.2 ISSR profiles of Taohua (above) and Zhujiajian (low) population with primer UBC841
M: Molecular marker, 1-60: Sample numbers
As analyzed by the software POPGENE, the Nei’s gene diversity (HE) of the species was 0.3414, and the Shannon’s information Index (H) was 0.5013, whereas, the mean Nei’s gene diversity and the mean Shannon’s information index were estimated to be 0.2688 and 0.3941 among populations, respectively.
Table 2 Genetic diversities for thirteen populations of C. japonica
|
Code |
Sample sizes |
Na |
Ne |
HE |
H |
PPB (%)
|
|
TH ZJJ SCD HJ Xiang CMY BG WS Kago Shiko-2 Shiko-1 Goto-1 Goto-2 Average Species |
30 30 30 30 30 30 30 30 30 30 30 30 30 30 390 |
1.6825 (0.4666) 1.6872 (0.4647) 1.6682 (0.4720) 1.6635 (0.4736) 1.6730 (0.4702) 1.7109 (0.4544) 1.7062 (0.4566) 1.7014 (0.4587) 1.7725 (0.4202) 1.7678 (0.4233) 1.7346 (0.4426) 1.7630 (0.4262) 1.7393 (0.4400) 1.7131(0.0388) 1.9005 (0.3001) |
1.4206 (0.3882) 1.4417 (0.3911) 1.4304 (0.3840) 1.4540 (0.3987) 1.4079 (0.3853) 1.4896 (0.3899) 1.4862 (0.4005) 1.4698 (0.3907) 1.5223 (0.3862) 1.5338 (0.3954) 1.4997 (0.4072) 1.5176 (0.3848) 1.5204 (0.3980) 1.4765（0.0423） 1.6052 (0.3470) |
0.2414 (0.2031) 0.2519 (0.2042) 0.2474 (0.2031) 0.2560 (0.2087) 0.2344 (0.2051) 0.2763 (0.2037) 0.2724 (0.2069) 0.2657 (0.2063) 0.2938 (0.1979) 0.2966 (0.2028) 0.2773 (0.2088) 0.2914 (0.1992) 0.2895 (0.2051) 0.2688(0.0211) 0.3414 (0.1682) |
0.3586 (0.2852) 0.3719 (0.2873) 0.3653 (0.2882) 0.3746 (0.2949) 0.3478 (0.2889) 0.4038 (0.2871) 0.3979 (0.2902) 0.3889 (0.2909) 0.4302 (0.2749) 0.4319 (0.2812) 0.4049 (0.2897) 0.4262 (0.2779) 0.4213 (0.2861) 0.3941(0.0287) 0.5013 (0.2241) |
68.25 68.72 66.82 66.35 67.30 71.09 70.62 70.14 77.25 76.78 73.46 76.30 73.93 71.31 90.05 |
Note: The value of standard deviation in parentheses.
2.2 Genetic differentiation of C. japonica
The coefficient of genetic differentiation among populations (Gst) was 0.2127, indicating 21.27% variation within populations, while 78.73% variation presented among individuals. The genetic distance ranged from 0.0210 to 0.1769 with the average of 0.1082, as shown in Table 3. The level of gene flow (Nm) was estimated to be 0.9255.
According to AMOVA analysis, there were highly significant (P < 0.001) genetic differences among the thirteen populations of C. japonica. The result showed that 22% variation resided among populations, and the rest (78%) among individuals (Table 3). The result was similar to the outcome of POPGENE.
In order to analyze the different effect of insular isolation to genetic differentiation, populations in Zhoushan Islands and in Japan were taken as two analysis units. The Gst value among 4 populations in Zhoushan Islands and 5 populations in Japan were 0.1003 and 0.0888, respectively, but the value among 2 populations in Putuo island was 0.0377, 0.0401 and 0.0355 among Shikoku and Goto populations. Based on the comparison, results showed that the genetic differentiation among populations in a same island were lower, indicating that insular isolation may result in low gene flow.
Based on Nei's genetic identity and genetic distance, the genetic differentiation among populations can be further analyzed (Nei, 1978). The geographical distance between the ZJJ population and the TH population was nearest (L=11.95Km), and the genetic distance was lowest (D=0.0367); the XS population and Shikoku populations had the largest geographical distance (L=1119.92Km), corresponding to the largest genetic distance (D = 0.1581) (Table 4). A significant correlation was found between genetic distance and geographic distance (r = 0. 8154, P <0.05) based on the Mantel test.
Table 3 Genetic differentiation among populations of islands
|
Populations |
HT |
HS |
GST |
|
2 populations in Putuo Island, Zhejiang, China 4 populations in Zhoushan Islands, Zhejiang, China |
0.2616 0.2770 |
0.2517 0.2492 |
0.0377 0.1003 |
|
2 populations in Shikoku, Japan 2 populations in Goto Island, Japan 5 populations in Japan |
0.2989 0.3011 0.3179 |
0.2870 0.2904 0.2897 |
0.0401 0.0355 0.0888 |
|
|
TH |
ZJJ |
HJ |
SCD |
XS |
CMY |
BG |
WS |
Kago |
Shiko-2 |
Shiko-1 |
Goto-1 |
Goto-2 |
|
TH |
**** |
0.9640 |
0.9468 |
0.9405 |
0.9241 |
0.8770 |
0.8731 |
0.8713 |
0.8627 |
0.8570 |
0.8429 |
0.8654 |
0.8574 |
|
ZJJ |
0.0367 |
**** |
0.9559 |
0.9501 |
0.9237 |
0.8868 |
0.8807 |
0.8807 |
0.88684 |
0.8624 |
0.8497 |
0.8747 |
0.8693 |
|
HJ |
0.0547 |
0.0451 |
**** |
0.9793 |
0.9330 |
0.8929 |
0.8964 |
0.8913 |
0.8636 |
0.8609 |
0.8429 |
0.8787 |
0.8672 |
|
SCD |
0.0613 |
0.0512 |
0.0210 |
**** |
0.9315 |
0.8970 |
0.9006 |
0.8951 |
0.8644 |
0.8617 |
0.8455 |
0.8810 |
0.8686 |
|
XS |
0.0789 |
0.0794 |
0.0694 |
0.0709 |
**** |
0.8941 |
0.8989 |
0.8886 |
0.8634 |
0.8515 |
0.8378 |
0.8676 |
0.8545 |
|
CMY |
0.1313 |
0.1201 |
0.1133 |
0.1087 |
0.1119 |
**** |
0.9721 |
0.9687 |
0.8963 |
0.8777 |
0.8703 |
0.8940 |
0.8943 |
|
BG |
0.1357 |
0.1271 |
0.1093 |
0.1047 |
0.1066 |
0.0283 |
**** |
0.9758 |
0.8956 |
0.8744 |
0.8658 |
0.8940 |
0.8857 |
|
WS |
0.1378 |
0.1270 |
0.1151 |
0.1108 |
0.1181 |
0.0318 |
0.0245 |
**** |
0.8921 |
0.8780 |
0.8691 |
0.9014 |
0.9001 |
|
Kago |
0.1477 |
0.1411 |
0.1466 |
0.1457 |
0.1469 |
0.1095 |
0.1102 |
0.1142 |
**** |
0.9666 |
0.9577 |
0.9566 |
0.9523 |
|
Shiko-2 |
0.1543 |
0.1480 |
0.1498 |
0.1489 |
0.1608 |
0.1305 |
0.1342 |
0.1301 |
0.0339 |
**** |
0.9731 |
0.9562 |
0.9538 |
|
Shiko-1 |
0.1709 |
0.1629 |
0.1709 |
0.1679 |
0.1769 |
0.1389 |
0.1441 |
0.1403 |
0.0432 |
0.0272 |
**** |
0.9373 |
0.9397 |
|
Goto-1 |
0.1445 |
0.1339 |
0.1293 |
0.1267 |
0.1420 |
0.1121 |
0.1120 |
0.1038 |
0.0444 |
0.0448 |
0.0647 |
**** |
0.9766 |
|
Goto-2 |
0.1538 |
0.1401 |
0.1425 |
0.1409 |
0.1572 |
0.1117 |
0.1214 |
0.1053 |
0.0489 |
0.0473 |
0.0621 |
0.0237 |
**** |
Table 4 Nei's Unbiased Measures of Genetic Identity and Genetic distance
Nei's genetic identity (above diagonal) and genetic distance (below diagonal)
2.3 Cluster analysis
The estimates of genetic distance among the populations ranged from 0.0210 to 0.1769. The dendrogram using UPGMA based on genetic distance showed that all populations were clustered into 2 groups at the coefficient of 0.14. Group A included 8 populations collected from China, while the other 5 Japanese populations were gathered in Group B. 8 China populations could still be divided into 2 subgroups at the coefficient of 0.90, subgroup ‘a’ contained 5 populations in Zhoushan, subgroup ‘b’ was composed of the other 3 populations in Qingdao.
Fig.3 UPGMA dendrogram for thirteen populations of C. japonica based on Nei’s genetic distance
3 Discussions
3.1 Genetic diversity of C. japonica and the affecting factors
ISSR has been applied in genetic studies of rare and endangered species in recent years (Pei et al., 2000; Yang et al.,1996;Lou et al.,2007). The PPB value of the populations varied from 66.35% to 77.25% with an average of 71.31%, while the PPB value of the species was 90.05%, indicating that the genetic diversity in this species was high. Factors such as recent speciation from a more widespread species, recent changes in distribution or habitat, breeding system, somatic mutations, multiple founder events, or Pleistocene refugia have been used to explain high levels of genetic diversity in plants (Lewis and Crawford, 1995; Maguire and Sedgley, 1997; Ranker, 1994; Zawko et al., 2001). Wendel and Parks (1985) used seeds to analyze allozyme variation in 58 Japanese and two Korean natural populations and found this species maintained higher levels of genetic variability within populations than other woody species (PPB = 66. 2 %, HE =0. 265). It has been suggested that pollen flow is extensive in C. japonica (Chung and Kang, 1996; Oh et al, 1996; Ueno et al, 2000), and a high outcrossing rate has been observed (Wendel and Parks, 1985).
Comparing the domestic populations to the Japanese populations we found that the genetic diversity of China populations is lower, the average value of Na、Ne、HE and h were 1.6741, 1.4762, 0.2683, 0.3915, respectively, and were 1.7607, 1.5426, 0.3023, 0.4401 in the Japan populations.
Zhoushan Islands located in the Northeastern of Zhejiang Mt. Tiantai, elevation 200~500m, single habitat condition, populations with similar genetic background (Leng, 2006). Xiangshan population was small-scale distribution because nearby residents collected wild C. japonica as a rootstock, nowadays there are almost no large areas of Camellia forest, and big Camellia trees are also rare. The individuals of Qingdao populations came from Changmenyan Island. Changmenyan Island is located in the Yellow Sea of 21.3 km from Laoshan, an area of only 0.16 km2, the number of Camellia is small, the main cause was vandalism. In the early 1950s, the island was full of Camellia trees, but now not any longer (Zhou, 1994). Affected by these factors, the populations declined. Genetic diversity has been closely related with population size (Frankham, 1996). Small numbers result in the frequency of inbreeding increasing, genetic drift may lead to the loss of alleles within populations, genetic diversity is decreased (Zhou, 1994). In contrast, Japan populations were less disrupted. In Kagoshima and Shikoku, there are large natural Camellia forests distributed, such as the island of Shikoku foot off Cape. Lush camellias are concentrated in the slope rock joints of humus, the camellia tree trunks are thick, diameter of 10~30 cm, the thickest of 40 cm, and are almost 3~4m high.
3.2 Genetic differentiation and the effect of insular geographical isolation
The genetic structure and affecting factors of island populations are important information to develop effective protection measures (Leberg, 1990). The coefficient of genetic differentiation among populations (Gst) was 0.2127, higher than the value of Zhoushan populations (Gst=0.1003) and Japan populations (0.0888). The result was similar to the study of Lengxin (2006), indicating that insular geographical isolation may play an important part in genetic differentiation. Mantel test (r = 0.8312, P <0.05) also confirmed such a possibility.
Insular geographical isolation hinders gene flow (Mantel, 2003), its role is mainly reflected in breaking the way of the pollen and seed spread. Pollen transfer and seed dispersal determine a plant’s reproductive success, range expansion, and population genetic structure (Luo et al, 2007). Kunitake found Zosterops japonica was the main pollinator in the Japan area (Kunitake et al, 2004). These birds are widely distributed in tropical and subtropical regions, playing an important role in the gene exchange among island populations (Micheneau, etc. 2006). The Putuo Island have been a Buddhist shrine since the Tang dynasty, the Camellia trees symbolize longevity, and were widely transplanted and cultivated (You, 2010), which may also have caused gene exchange among Zhoushan Island populations. The mean genetic distance among Qingdao populations was 0.0284, the coefficient of genetic differentiation among populations was 0.0563, showing that ZW and WS populations might have been transplanted from Changmenyan Island in Qingdao District.
The coefficient of genetic differentiation between Putuo Island populations (SC and HJ) was very low (Gst=0.0377), insect (Luo, etc. 2007) and rodents (Harue, etc. 2006) would promote the gene flow between populations, so the two populations had the nearest relationship，UPGMA confirmed the results. The coefficient of genetic differentiation among Zhoushan Islands populations was 0.1003, implying insular isolation played an important role in genetic differentiation.
3.3 Strategies for conservation
Camellia populations in Japan have high genetic diversity, which was probably closely related to their good protection. At present, the southern islands in Japan still have large areas of natural camellia forests grown in protected circumstances. Zhoushan Islands, Changmenyan Island and some small islands in coastal southeastern China have natural camellia stands, but the situation is quite different, human activities such as digging out, collecting fuelwood, removing graft stocks have resulted in a serious threat to natural camellia resources (Liang, 2000). In order to maintain genetic diversity and resource utilization of C. japonica, natural populations should be protected as in situ conservation areas from human disturbance to facilitate its natural generation; enhance islanders’ awareness of ecological protection and resource conservation; prohibit digging up of Camellia seedlings and picking seeds. Secondly, establishing germplasm resource pools ex-situ to protect genetic resources of the insular populations.
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Acknowledgments
This work was honorably supported by the MOST International Cooperation Program (No. 2011DFA30490), Scientific Research Fund for Forestry Public-interest Program (No.200704028), China Forestry State Administration Project China (No. 2007-4-04), Zhejiang Province and Academy cooperation Forestry Science and Technology Program(No. 2008SY08) and Central Public-interest Scientific Institution Basal Research Fund of China (No. CAFYBB2007020).
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