生物多样性 ›› 2019, Vol. 27 ›› Issue (5): 543-556.doi: 10.17520/biods.2018214

• 研究报告 • 上一篇    下一篇

基于粪便DNA及宏条形码技术的食肉动物快速调查及食性分析

邵昕宁1, 2, 宋大昭3, 黄巧雯3, 李晟1, 2, 姚蒙1, 2, *()   

  1. 1 北京大学生命科学学院, 北京 100871
    2 北京大学生态研究中心, 北京 100871
    3中国猫科动物保护联盟, 北京 101011
  • 收稿日期:2018-08-03 接受日期:2019-01-25 出版日期:2019-05-20
  • 通讯作者: 姚蒙 E-mail:yaom@pku.edu.cn

Fast surveys and molecular diet analysis of carnivores based on fecal DNA and metabarcoding

Shao Xinning1, 2, Song Dazhao3, Huang Qiaowen3, Li Sheng1, 2, Yao Meng1, 2, *()   

  1. 1 School of Life Sciences, Peking University, Beijing 100871
    2 Institute of Ecology, Peking University, Beijing 100871
    3 Chinese Felid Conservation Alliance, Beijing 101011
  • Received:2018-08-03 Accepted:2019-01-25 Online:2019-05-20
  • Contact: Yao Meng E-mail:yaom@pku.edu.cn

在陆地生态系统中, 大型食肉动物对于稳定食物网结构和生态系统功能有重要作用。在世界范围内, 由于栖息地丧失和破碎化、猎杀、人类活动干扰以及病原体的传播, 大型食肉动物生存正面临严重威胁, 多种食肉动物地理分布范围及种群数量大幅度缩减。如何有效保护大型食肉动物物种多样性及种群已经成为世界关注的焦点问题和保护生物学的重要研究方向。川西高原地处我国西南山地与青藏高原东缘交界地带, 属于世界生物多样性热点地区, 是世界大型食肉动物物种最丰富的地区之一, 而日益增强的人类活动可能会加剧对当地动植物资源的破坏, 进而威胁野生食肉动物的生存。获得准确的物种多样性信息及食肉动物食性数据有助于深入了解该地区生态系统结构及食物网关系, 对研究物种共存机制及生物多样性保护有重要意义。本研究通过从四川甘孜藏族自治州新龙县和石渠县野外采集的食肉动物粪便样品中提取DNA, 利用DNA条形码进行物种鉴定, 快速获得该地区食肉动物物种构成信息。38份粪便样品经鉴定来自于7种食肉动物, 分别为5种大型食肉动物(狼Canis lupus、棕熊Ursus arctos、豹Panthera pardus、雪豹P. unica、狗Canis lupus familiaris)和2种中小型食肉动物(豹猫Prionailurus bengalensis、赤狐Vulpes vulpes)。进一步利用高通量测序和宏条形码技术对7种食肉动物粪便中的食物DNA进行精准食性分析, 得到包含19种哺乳类、8种鸟类和1种鱼类共计28个不同的食物分子可操作分类单元(molecular operational taxonomic unit, MOTU)。结果显示, 狼、狗、棕熊最主要的食物来源为偶蹄目动物, 其中取食频率最高的物种为家牦牛(Bos grunniens); 而豹猫和赤狐食物中小型哺乳动物如啮齿目和兔形目占重要比例, 其中高原松田鼠(Neodon irene)和高原鼠兔(Ochotona curzoniae)被取食频率最高。豹和雪豹的食物分别为偶蹄目的中华斑羚(Naemorhedus griseus)和岩羊(Pseudois nayaur)。本研究显示了粪便DNA及宏条形码技术在食肉动物多样性快速调查及高通量精确食性分析中的应用前景, 并为此类研究提供了技术路线的有力借鉴。

关键词: 生物多样性编目, 兽类调查, 非损伤性取样, DNA条形码, 食物网

Large carnivores play an important role in the regulation of food-web structure and ecosystem functioning. However, large carnivores face serious threats that have caused declines in their populations and geographic ranges due to habitat loss and degradation, hunting, human disturbance and pathogen transmission. Conservation of large carnivore species richness and population size has become a pressing issue and an important research focus of conservation biology. The western Sichuan Plateau, located at the intersection of the mountains of southwest China and the eastern margin of the Tibetan Plateau, is a global biodiversity hotspot and has high carnivore species richness. However, increasing human activities may exacerbate the destruction of local flora and fauna, thereby threatening the survival of wild carnivores. Information on species composition and dietary habits can improve our understanding of the structure and function of the ecosystem and food-web relationships in the study area. In addition, species composition and dietary habits are of great significance for understanding multi-species coexistence mechanisms and preserving biodiversity. This study collected carnivore fecal samples from Xinlong and Shiqu counties in the Ganzi Tibetan Autonomous Prefecture, Sichuan Province. DNA was then extracted from the samples and the species was identified based on DNA sequences and DNA barcoding techniques. Seven carnivores were identified, including five large carnivores (Canis lupus, Ursus arctos, Panthera pardus, P. uncia and Canis lupus familiaris) and two medium and small-sized carnivores (Prionailurus bengalensis and Vulpes vulpes). Using fecal DNA, high-throughput sequencing and metabarcoding, we conducted diet analysis for the seven carnivores and found 28 different food molecular operational taxonomic units (MOTUs), including 19 mammals, eight birds and one fish species. The predominant prey categories of wolves, dogs and brown bears were ungulates. The domestic yak (Bos grunniens) was the most frequently identified prey species. Small mammals such as rodents and lagomorphs accounted for a significant proportion in the diets of leopard cats and red foxes, The most frequent prey of this category of carnivore were the Chinese scrub vole (Neodon irene) and plateau pika (Ochotona curzoniae). In addition, leopards and snow leopards mainly fed on the Chinese goral (Naemorhedus griseus) and blue sheep (Pseudois nayaur), respectively. Our study highlights the utility of fecal DNA and metabarcoding technique in fast carnivore surveys and high-throughput diet analysis, and provides a technical reference and guidance for future biodiversity surveys and food-web studies.

Key words: biodiversity inventory, mammalian survey, non-invasive sampling, DNA barcoding, food-web

图1

四川省甘孜藏族自治州石渠县及新龙县采样地点示意图"

表1

物种分子鉴定结果及形态物种预判正确率"

物种
Species
样品数
No. of sample
物种预判
样品数
No. of morphologically identified sample
预判正确
样品数
No. of correctly identified sample
预判正确率
Accuracy of morphological identification (%)
Canis lupus 8 4 1 25.0
Canis lupus familiaris 6 6 1 16.7
豹猫 Prionailurus bengalensis 6 4 4 100
赤狐 Vulpes vulpes 3 3 0 0
棕熊 Ursus arctos 3 3 0 0
Panthera pardus 1 1 1 100
雪豹 Panthera uncia 1 1 1 100
合计 Total 28 22 8 36.4

表2

食物分子可操作分类单元(MOTU)中不同分类级别的数目及比例"

分类等级
Taxonomic level

Species

Genus

Family

Order
总计Total
MOTU数目 MOTU no. 17 8 2 1 28
比例 Percentage (%) 60.7 28.6 7.1 3.6

表3

7种食肉动物食物分子可操作分类单元(MOTU)分子鉴定结果。最佳匹配物种结果中斜线(/)用于分隔序列一致度相同的物种, 竖线(|)用于分隔一致度相差 ≥ 1%的物种。"

食物 MOTU
Food MOTU
在不同食肉动物样品中的出现频次
Occurrence frequency in different carnivore samples
GenBank 最佳匹配物种
Best match species in GenBank
拉丁学名
Scientific
name
MOTU
名称
MOTU name

Wolf

Dog
豹猫
Leopard cat
棕熊
Brown bear
赤狐
Red
fox

Leopard
雪豹
Snow leopard
中文名
Chinese
name
拉丁名
Scientific
name
最高一致度
Best identity (%)
序列号
Accession
no.
N = 8 N = 6 N = 6 N = 3 N = 3 N = 1 N = 1
偶蹄目 Artiodactyla
Bos grunniens 家牦牛 6 4 6 1 0 0 0 家牦牛 Bos grunniens 100 KX232527.1
Elaphodus cephalophus 毛冠鹿 2 2 3 0 0 0 0 毛冠鹿 Elaphodus cephalophus 99 DQ873526.1
Sus scrofa 野猪 2 2 3 0 0 0 0 野猪 Sus scrofa 100 KX886757.1
Pseudois nayaur 岩羊 2 0 0 0 1 0 1 岩羊 Pseudois nayaur 100 KP998469.1
Moschus 麝属 2 0 1 0 0 0 0 马麝/林麝 Moschus chrysogaster/
Moschus berezovskii
100 KP684123.1/
AY184425.1
Przewalskium albirostris 白唇鹿 1 0 0 1 0 0 0 白唇鹿 Przewalskium albirostris 100 JN632690.1
Naemorhedus
griseus
中华斑羚 0 0 0 0 0 1 0 中华斑羚 Naemorhedus griseus 100 JN632664.1
奇蹄目 Perissodactyla
Equus caballus 1 0 0 0 0 0 0 Equus caballus 100 KU575247.1
兔形目 Lagomorpha
Ochotona curzoniae 高原鼠兔 2 3 5 0 2 0 0 高原鼠兔 Ochotona curzoniae 100 KM225732.1
Lepus oiostolus 高原兔 1 0 6 0 0 0 0 高原兔 Lepus oiostolus 99 AY745187.1
啮齿目 Rodentia
Neodon irene 高原松田鼠 4 1 5 0 2 0 0 高原松田鼠 Neodon irene 100 HQ416908.1
Apodemus 姬鼠属 2 0 5 0 0 0 0 大耳姬鼠|
高山姬鼠
Apodemus latronum|
Apodemus chevrier
100|
99
HQ333256.1|
HQ896683.1
Marmota
himalayana
喜马拉雅
旱獭
2 0 0 0 0 0 0 喜马拉雅
旱獭
Marmota himalayana 100 JX069958.1
Niviventer
excelsior
川西白腹鼠 0 1 1 0 0 0 0 川西白腹鼠 Niviventer excelsior 99 JQ927552.1
Trogopterus
xanthipes
复齿鼯鼠 0 0 1 0 0 0 0 复齿鼯鼠 Trogopterus xanthipes 97 AY227546.1
Petaurista 鼯鼠属 0 0 1 0 0 0 0 红背鼯鼠/
霜背大鼯鼠|
红白鼯鼠
Petaurista petaurista/
Petaurista philippensis|
Petaurista alborufus
98|
97
KP973556.1/ KP973555.1| AY227541.1
Eozapus
setchuanus
林跳鼠 0 0 0 0 1 0 0 林跳鼠 Eozapus setchuanus 98 KJ648495.1
食物MOTU
Food MOTU
在不同食肉动物样品中的出现频次
Occurrence frequency in different carnivore samples
GenBank最佳匹配物种
Best match species in GenBank
拉丁学名
Scientific
name
MOTU
名称
MOTU name

Wolf

Dog
豹猫
Leopard cat
棕熊
Brown bear
赤狐
Red
fox

Leopard
雪豹
Snow leopard
中文名
Chinese
name
拉丁名
Scientific
name
最高一致度
Best identity (%)
序列号
Accession
no.
N = 8 N = 6 N = 6 N = 3 N = 3 N = 1 N = 1
食肉目 Carnivora
Arctonyx albogularis 猪獾 0 0 0 2 0 0 0 猪獾 Arctonyx albogularis 100 HM106329.1
鼩形目 Soricomorpha
Sorex 鼩鼱属 0 0 1 0 0 0 0 纹背鼩鼱/
小纹背鼩鼱|
长爪鼩鼱/
普通鼩鼱
Sorex cylindricauda/
Sorex bedfordiae|
Sorex unguiculatus/
Sorex araneus
99|
98
KF696672.1/
GU981054.1| KX754508.1/
KT210896.1
鲤形目 Cypriniformes
Cyprinidae 鲤科 2 1 0 1 0 0 0 汪氏近红鲌/
厚颌鲂
Ancherythroculter wangi/
Megalobrama pellegrini
100 MG783573.1/
JX242529.1
鸡形目 Galliformes
Crossoptilon crossoptilon 白马鸡 1 1 0 0 1 0 0 白马鸡 Crossoptilon crossoptilon 100 KP259808.1
Gallus gallus 家鸡 1 0 0 0 0 0 0 原鸡 Gallus gallus 100 KX987152.1
Perdix 山鹑属 0 0 0 0 1 0 0 斑翅山鹑/
高原山鹑/
灰山鹑
Perdix dauurica/
Perdix hodgsoniae
/
Perdix perdix
100 KY411596.1/
KF027440.1/
KF781322.1
雁形目 Anseriformes
Anas 鸭属 0 1 0 0 0 0 0 绿头鸭/
琵嘴鸭/
绿翅鸭/
斑嘴鸭/
针尾鸭
Anas platyrhynchos/
Anas clypeata/
Anas crecca/
Anas poecilorhyncha/
Anas acuta
100 KX592536.1/
KT345702.1/
KC771255.1/
KC466567.1/
KF312717.1
雀形目 Passeriformes
Phylloscopus 1 柳莺属1 0 0 1 0 0 0 0 暗绿柳莺|
淡眉柳莺
Phylloscopus trochiloides|
Phylloscopus humei
99|
98
KP267717.1|
KP267716.1
Phylloscopus 2 柳莺属2 0 1 0 0 0 0 0 褐柳莺/
黄腰柳莺/
乌嘴柳莺
Phylloscopus fuscatus/
Phylloscopus proregulus/
Phylloscopus maculipennis
100 JF505332.1/
AY635103.1 /AY635100.1
Muscicapidae 1 鸫科 0 0 1 0 0 0 0 鹊鸲/
北红尾鸲
Copsychus saularis/
Phoenicurus auroreus
99 KU058637.1/ KF997864.1/
KF997863.1
Passeriformes 雀形目 0 0 1 0 0 0 0 家燕/
红喉歌鸲
Hirundo rustica/
Luscinia calliope
96 KX398931.1/
HQ690246.1

图2

目水平上不同食肉动物食物组成。%RO为不同食物分子可操作分类单元(MOTU)出现的相对频率。"

图3

食肉动物食性重叠情况"

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