基于分子宏条形码分析四川卧龙国家级自然保护区雪豹的食性

doi:10.17520/biods.2019101

摘要/Abstract

摘要：

作为中亚和青藏高原山地生态系统中的顶级捕食者, 雪豹(Panthera uncia)对于维持食物网结构和生态系统稳定性有重要作用。了解雪豹的食性组成和变化对于理解其生态系统功能和物种间相互作用有重要意义。以往的雪豹食性分析多基于对其粪便中食物残渣的形态学鉴定, 但准确度受人员经验和主观因素影响较大。邛崃山脉位于雪豹分布区东南缘, 该区域的雪豹种群规模小且相对孤立, 研究匮乏。本研究基于非损伤性取样, 在邛崃山脉的卧龙国家级保护区采集疑似雪豹粪便样品38份, 首先提取粪便DNA, 并扩增线粒体DNA 16S rRNA基因片段进行分子物种鉴定, 确定其中22份为雪豹粪便样品。随后, 利用脊椎动物通用引物和雪豹特异性阻抑引物扩增粪便DNA中的食物成分, 并进行高通量测序, 分析雪豹食性构成。食性分析结果显示岩羊(Pseudois nayaur)是卧龙地区雪豹最主要的食物, 在67%的样品中均有检出。家牦牛(Bos grunniens)在33%的样品中出现, 也在雪豹食性中占较高比例。此外, 鼠兔(Ochotona spp.)和鸟类也在少量样品中发现。可见, 野生猎物是卧龙地区雪豹的主要食物资源; 与大世界大多数其他地区的雪豹食性相同, 野生大型有蹄类是雪豹最重要的食物。然而家畜(牦牛)在卧龙雪豹食谱中有相当高的占比, 显示该区域内可能存在较为严重的由雪豹捕食散养家畜引起的人兽冲突问题。

关键词: 雪豹(Panthera uncia), 非损伤性取样, 高通量测序, 分子条形码, 食性分析, 邛崃山脉

Abstract

As the apex predator of plateau ecosystems in Central Asia and the Qinghai-Tibet Plateau, the snow leopard (Panthera uncia) plays an essential role in maintaining food-web structure and ecosystem stability. Learning the diet composition and dynamics of the snow leopard is important for understanding its role in ecosystem functioning and interspecific interactions. Previous diet analyses of the snow leopard have been based mainly on morphological identification of food debris in the feces, though the accuracy of this practice has been broadly debated. The Qionglai Mountains are located at the southeast edge of the snow leopard range, harboring a small and relatively isolated population of snow leopards that are barely studied. Using non-invasive sampling, we collected 38 putative snow leopard fecal samples in the Wolong National Nature Reserve in the Qionglai Mountains. To identify the fecal origin, we extracted the fecal DNA and amplified the mitochondrial DNA 16S rRNA gene fragment. Twenty-two fecal samples were identified as originating from snow leopards. Subsequently, vertebrate universal primers and a snow leopard-specific blocking oligo were used to amplify the food components in the fecal DNA, and then high-throughput sequencing was performed to analyze the diet composition of snow leopards. The blue sheep (Pseudois nayaur) was detected in 67% of the samples and was found to be the main staple food of snow leopards’ diet. The domestic yak (Bos grunniens) appeared in 33% of the fecal samples, also accounting for a high proportion of the snow leopard diet. In addition, pikas (Ochotona spp.) and birds were found in a small number of fecal samples. Therefore, wild prey was found to be the main food source for snow leopards in Wolong. However, livestock (yak) also accounted for a relatively large proportion of their diet.

Key words: snow leopard (Panthera uncia), non-invasive sampling, high-throughput sequencing, barcoding, diet analysis, Qionglai Mountains

陆琪,胡强,施小刚,金森龙,李晟,姚蒙 (2019) 基于分子宏条形码分析四川卧龙国家级自然保护区雪豹的食性. 生物多样性, 27, 960-969. DOI: 10.17520/biods.2019101.

Qi Lu,Qiang Hu,Xiaogang Shi,Senlong Jin,Sheng Li,Meng Yao (2019) Metabarcoding diet analysis of snow leopards (Panthera uncia) in Wolong National Nature Reserve, Sichuan Province. Biodiversity Science, 27, 960-969. DOI: 10.17520/biods.2019101.

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链接本文: https://www.biodiversity-science.net/CN/10.17520/biods.2019101

https://www.biodiversity-science.net/CN/Y2019/V27/I9/960

图/表 4

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引物 Primer	序列 Sequence (5′-3′)	产物长度 Product length	退火温度 Annealing temperature	参考文献 Reference
16S	F: GAGAAGACCCTATGGAGC	~380 bp	50℃	Xiong et al, 2016
	R: ATAGAAACCGACCTGGAT
12SV5	F: TAGAACAGGCTCCTCTAG	~140 bp	60℃	Riaz et al, 2011
	R: TTAGATACCCCACTATGC
UnciB	CTATGCTTAGCCCTAAACCTAGAT AGTTAGCTCAAACAAAACTAT-C3	-	-	Shehzad et al, 2012a

引物 Primer	序列 Sequence (5′-3′)	产物长度 Product length	退火温度 Annealing temperature	参考文献 Reference
16S	F: GAGAAGACCCTATGGAGC	~380 bp	50℃	Xiong et al, 2016
	R: ATAGAAACCGACCTGGAT
12SV5	F: TAGAACAGGCTCCTCTAG	~140 bp	60℃	Riaz et al, 2011
	R: TTAGATACCCCACTATGC
UnciB	CTATGCTTAGCCCTAAACCTAGAT AGTTAGCTCAAACAAAACTAT-C3	-	-	Shehzad et al, 2012a

研究地区 Study area	样本数 No. of samples	物种鉴定方法 Species identification	食性分析方法 Diet analysis	食物种类(出现频率) Prey taxa (FO) (%)						参考文献 References
研究地区 Study area	样本数 No. of samples	物种鉴定方法 Species identification	食性分析方法 Diet analysis	野生有蹄类 Wild ungulates	中小型哺乳类 Small mammals	鸟类 Birds	家畜 Livestock	植物 Plant	未鉴定 Unidentified items
巴基斯坦 Baltistan, Pakistan	49	DNA	形态学 Morphology	24.5	6.1	4.2	69.3	59.2	32.6	Anwar et al, 2011
蒙古 South Gobi, Mongolia	81	DNA	DNA	79.0	-	1.2	19.8	-	-	Shehzad et al, 2012a
尼泊尔 Manang, Nepal	213	形态学 Morphology	形态学 Morphology	51.6	53.5	1.4	17.8	-	-	Oli et al, 1994
尼泊尔 PinValley, Nepal	51	形态学 Morphology	形态学 Morphology	56.9	3.9	-	23.6	25.5	5.9	Bagchi & Mishra, 2006
尼泊尔 Kibber, Nepal	44	形态学 Morphology	形态学 Morphology	29.6	6.8	15.9	38.5	27.3	19.5	Bagchi & Mishra, 2006
塔吉克斯坦 Murghab, Tajikistan	36	DNA	形态学 Morphology	78.0	45.0	-	-	-	-	Kachel et al, 2016
塔吉克斯坦 Madiyan, Tajikistan	20	DNA	形态学 Morphology	50.0	55.0	-	2.0	-	-	Kachel et al, 2016
印度 Ladakh, India	173	形态学 Morphology	形态学 Morphology	35.2	25.5	4.6	22.1	60.7	-	Chundawat & Rawat, 1994
中国青海 Qinghai, China	206	形态学 Morphology	形态学 Morphology	37.6	44.3	-	6.3	8.0	1.4	Schaller, 1988
中国青海 Qinghai, China	307	形态学 Morphology	形态学 Morphology	36.9	58.9	2.3	9.8	2.9	34.2	Liu et al, 2003
中国青海 Qinghai, China	250	形态学 Morphology	形态学 Morphology	67.6	17.6	-	16.0	18.0	6.4	Li, 2012
中国新疆 Xinjiang, China	16	DNA	形态学 Morphology	50.0	112.5	81.3	156.3	25.0	-	Wang et al, 2014
中国四川 Sichuan, China	18	DNA	DNA	66.7	11.1	11.1	33.3	-	-	本研究 This study

研究地区 Study area	样本数 No. of samples	物种鉴定方法 Species identification	食性分析方法 Diet analysis	食物种类(出现频率) Prey taxa (FO) (%)						参考文献 References
研究地区 Study area	样本数 No. of samples	物种鉴定方法 Species identification	食性分析方法 Diet analysis	野生有蹄类 Wild ungulates	中小型哺乳类 Small mammals	鸟类 Birds	家畜 Livestock	植物 Plant	未鉴定 Unidentified items
巴基斯坦 Baltistan, Pakistan	49	DNA	形态学 Morphology	24.5	6.1	4.2	69.3	59.2	32.6	Anwar et al, 2011
蒙古 South Gobi, Mongolia	81	DNA	DNA	79.0	-	1.2	19.8	-	-	Shehzad et al, 2012a
尼泊尔 Manang, Nepal	213	形态学 Morphology	形态学 Morphology	51.6	53.5	1.4	17.8	-	-	Oli et al, 1994
尼泊尔 PinValley, Nepal	51	形态学 Morphology	形态学 Morphology	56.9	3.9	-	23.6	25.5	5.9	Bagchi & Mishra, 2006
尼泊尔 Kibber, Nepal	44	形态学 Morphology	形态学 Morphology	29.6	6.8	15.9	38.5	27.3	19.5	Bagchi & Mishra, 2006
塔吉克斯坦 Murghab, Tajikistan	36	DNA	形态学 Morphology	78.0	45.0	-	-	-	-	Kachel et al, 2016
塔吉克斯坦 Madiyan, Tajikistan	20	DNA	形态学 Morphology	50.0	55.0	-	2.0	-	-	Kachel et al, 2016
印度 Ladakh, India	173	形态学 Morphology	形态学 Morphology	35.2	25.5	4.6	22.1	60.7	-	Chundawat & Rawat, 1994
中国青海 Qinghai, China	206	形态学 Morphology	形态学 Morphology	37.6	44.3	-	6.3	8.0	1.4	Schaller, 1988
中国青海 Qinghai, China	307	形态学 Morphology	形态学 Morphology	36.9	58.9	2.3	9.8	2.9	34.2	Liu et al, 2003
中国青海 Qinghai, China	250	形态学 Morphology	形态学 Morphology	67.6	17.6	-	16.0	18.0	6.4	Li, 2012
中国新疆 Xinjiang, China	16	DNA	形态学 Morphology	50.0	112.5	81.3	156.3	25.0	-	Wang et al, 2014
中国四川 Sichuan, China	18	DNA	DNA	66.7	11.1	11.1	33.3	-	-	本研究 This study

食物类别 Prey taxa	出现样品数 No. of occurrence	出现频率 FO (%)	相对序列丰度 RRA (%)
岩羊 Pseudois nayaur	12	66.7	57.1
牦牛 Bos grunniens	6	33.3	27.4
高原鼠兔Ochotona curzoniae	1	5.5	0.5
红耳鼠兔Ochotona erythrotis	1	5.5	5.6
鹡鸰科Motacillidae	1	5.5	5.6
雉科 Phasianidae	1	5.5	3.8
总计 Total			100