Biodiversity Science ›› 2019, Vol. 27 ›› Issue (5): 543-556.doi: 10.17520/biods.2018214

• Original Papers • Previous Article     Next Article

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
  • Yao Meng E-mail:yaom@pku.edu.cn

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

Fig. 1

Map of sampling area in Shiqu and Xinlong counties of Ganzi Tibetan Autonomous Prefecture, Sichuan"

Table 1

Summary of species identification by DNA barcoding and fecal morphology"

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

Table 2

Number of prey MOTUs assigned to various taxonomic levels"

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

Table 3

Summary of prey MOTUs of seven carnivores identified by molecular dietary analysis. Species with the identical sequence identity are separated by “/”, and species with ≥ 1% difference in sequence identity are separated by “|”."

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

Fig. 2

Dietary compositions by prey order identified in the diets of different carnivores. %RO indicates the relative frequency of occurrence of different food MOTUs."

Fig. 3

Dietary overlap among carnivores"

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