生物多样性 ›› 2016, Vol. 24 ›› Issue (3): 341-350.doi: 10.17520/biods.2015260

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基于DNA条形码技术对浙江省外来入侵福寿螺进行分子鉴定

杨倩倩, 刘苏汶, 茹炜岽, 刘光富, 俞晓平*()   

  1. 浙江省生物计量及检验检疫技术重点实验室, 中国计量学院生命科学学院, 杭州 310018
  • 收稿日期:2015-09-23 接受日期:2016-03-11 出版日期:2016-03-20
  • 通讯作者: 俞晓平 E-mail:yxp@cjlu.edu.cn
  • 基金项目:
    国家863项目(2012AA021601)和浙江省自然科学基金(LQ15C140002)

Molecular identification of invasive golden apple snails in Zhejiang Province based on DNA barcoding

Qianqian Yang, Suwen Liu, Weidong Ru, Guangfu Liu, Xiaoping Yu*()   

  1. Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, College of Life Sciences, China Jiliang University, Hangzhou, 310018
  • Received:2015-09-23 Accepted:2016-03-11 Online:2016-03-20
  • Contact: Yu Xiaoping E-mail:yxp@cjlu.edu.cn

外来入侵福寿螺对我国农业生产和水生生态系统平衡等造成严重危害。2010年, 种类鉴定研究首次揭示我国外来入侵福寿螺包括Pomacea canaliculataP. maculata两个种, 而浙江省仅见P. canaliculata一种报道。P. canaliculataP. maculata种间形态近似, 且受环境、食物源等因素影响, 同种内外壳形态特征多样, 因而基于形态特征进行种类的准确鉴定极为困难。本研究在采集浙江省7个区县的福寿螺样本的基础上, 利用DNA条形码技术扩增了101个不同个体的COI序列, 并从BOLD数据库下载了“P. canaliculata种团”的5个近缘种的55条COI序列用于分析, 其中包括P. lineata, P. dolioidesP. paludosa所有已发表序列, 以及P. canaliculataP. maculata的南美洲样品的序列等。序列相似度比对、DNA条形码间隙和系统发育树等分析表明, COI序列可以实现近缘福寿螺的有效鉴别。待测的浙江省福寿螺样品中, 杭州江干区检测到P. canaliculataP. maculata两种, 而舟山普陀区、绍兴上虞区和新昌县、温州瓯海区及杭州西湖区仅检测到P. canaliculata, 表明P. canaliculata在浙江省具有更广的分布范围。P. canaliculataP. maculata分别形成4种和2种单倍型, 各区县样点分别包含1-3种单倍型, 浙江省各发生地呈现较低的遗传多样性。依据系统发育关系推测, 浙江省分布的P. canaliculataP. maculata分别可能来源于阿根廷和巴西。

关键词: COI, Pomacea canaliculata, P. maculata, 分子鉴定, 遗传多样性, 入侵种

Golden apple snails seriously damage crops and aquatic ecosystem in China. Two invasive apple snail species, Pomacea canaliculata and P. maculata, have been reported in China since 2010. Only the distribution of P. canaliculata was reported in Zhejiang Province. It is difficult to distinguish the two species due to their close morphological characteristics and high diversity of shell morphology, which are influenced by both environmental factors and food types. We collected samples from seven localities from Zhejiang Province and sequenced 101 individuals of mitochondrial COI fragments of the DNA barcode region. We also downloaded 55 sequences of five species of the P. canaliculata group, which included all public sequences of P. lineata, P. dolioides, and P. paludosa, and South American sample sequences of P. canaliculata and P. maculata. Analyses including similarity alignments, DNA barcoding gaps, and phylogenetic relationships, revealed that COI sequences were effective to distinguish apple snail species. We detected P. canaliculata and P. maculata distributed in the Jianggan region of Hangzhou, while only P. canaliculata was distributed in the Putuo region of Zhoushan, the Shanyu and Xinchang regions of Shaoxing, the Ouhai region of Wenzhou and the Xihu regoin of Hangzhou. P. canaliculata revealed a much wider distribution range. P. canaliculata and P. maculata were collapsed into 4 haplotypes and 2 haplotypes, respectively. There were 1 to 3 haplotypes in each locality, which indicated a low genetic diversity. The phylogenetic analyses deduced that P. canaliculata and P. maculata were probably introduced from Argentina and Brazil, respectively.

Key words: COI, Pomacea canaliculata, P. maculata, molecular identification, genetic diversity, invasive species

表1

浙江省福寿螺样品采集及COI序列信息表"

编号
Code
采集地点
Locality
采集环境
Habitat
采集时间
Time
数量
No.
GenBank注册号
GenBank accession no.
ZSPT 舟山普陀区
Putuo, Zhoushan
河道
River
2014年8月
August, 2014
31 KP310330, KP310417, KP310432-KP310435, KP310349- KP310365, KR020946, KR020947, KR020958-KR020961, KR020977, KR020978
SXXC 绍兴新昌县
Xinchang, Shaoxing
稻田
Paddy field
2014年10月
October, 2014
10 KR020982, KR020997, KR021015-KR021020, KP310438, KR106989
SXSY 绍兴上虞区
Shangyu, Shaoxing
稻田
Paddy field
2015年7月
July, 2015
7 KT693024-KT693030
WZOH 温州瓯海区
Ouhai, Wenzhou
稻田
Paddy field
2014年10月
October, 2014
13 KR020970-KR020973, KR020979, KR020981, KR020993, KR021007-KR021010, KR021013, KT692988
NBYY 宁波余姚市
Yuyao, Ningbo
稻田
Paddy field
2014年8月
August, 2014
5 KT852725, KT852726, KT852740-KT852742
HZXH 杭州西湖区
Xihu, Hangzhou
荷塘
Lotus pond
2015年6月
June, 2015
15 KT693009-KT693023
HZACA 杭州江干区
Jianggan, Hangzhou
荷塘
Lotus pond
2015年6月
June, 2015
20 KT692989-KT693008

表2

福寿螺各单倍型的分布数量"

编号
Code
Hap1 Hap2 Hap3 Hap4 Hap5 Hap6
ZSPT 7 24
SXXC 1 9
SXSY 1 6
WZOH 9 2 2
NBYY 5
HZXH 6 9
HZACA 14 5 1
合计 Total 43 2 35 15 5 1

表3

两种福寿螺P. canaliculata和P. maculata各单倍型间的K2P遗传距离(下三角)及碱基差异数(上三角)"

Hap1 Hap2 Hap3 Hap4 Hap5 Hap6
Hap1 27 28 28 60 61
Hap2 0.051 12 1 59 60
Hap3 0.053 0.020 13 57 58
Hap4 0.053 0.002 0.022 58 59
Hap5 0.121 0.120 0.116 0.118 13
Hap6 0.123 0.122 0.118 0.120 0.005

表4

自测单倍型序列在BOLD中的比对相似度及鉴定结果"

查询序列
Query
sequences
BOLD比对种
BOLD species
GenBank序列号
GenBank accession no.
相似度
Similarity
(%)
Hap1 P. canaliculata EF514984 100
EF513983 100
EF514980 100
Hap2 P. canaliculata FJ946822 100
AB433759 100
AB433765 100
Hap3 P. canaliculata AB433771 100
FJ946824 100
FJ946825 99.84
Hap4 P. canaliculata EU528578 100
AB433762 100
AB433770 99.85
Hap5 P. maculata* GU236486 100
GU236487 100
GU236488 100
Hap6 P. maculata* EF514986 99.43
EU528502 99.43
AB433781 99.43

图1

基于ABGD算法计算的DNA条形码间隙及序列分组方案。COI序列两两比对的K2P距离分布的距离直方图(A)、距离排序分布图(B)以及基于先验种内差异的分区结果(C)。"

图2

基于福寿螺COI条形码序列的NJ系统发育树(仅显示置信值>60%的序列)"

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