中国东北鹿亚科动物亚化石的古DNA分子鉴定及系统发育分析

doi:10.19615/j.cnki.1000-3118.200722

摘要/Abstract

摘要：

我国鹿类资源丰富,鹿亚科(Cervinae)动物有7个种在中国有分布。鹿亚科的种内系统发育研究很多,但是各物种之间进化关系仍有待进一步研究,其中从分子水平对古代材料的报道更为鲜见。对采集自黑龙江的两个距今分别为3800和5100 a的古代鹿亚科动物亚化石材料开展了古DNA研究。通过古DNA提取、古DNA双链文库构建、高通量测序及数据分析,得到了两条长度分别为16475 bp (GenBank收录号：MT784751, 序列完整度：99.83%)和16167 bp (GenBank收录号：MT784752, 序列完整度：97.96%)的线粒体基因组序列,对两个样品进行了分子鉴定。结合GenBank中现生鹿亚科动物的线粒体同源序列,构建了系统发育树。结果表明：1) 二代测序DNA片段末端碱基特性分析说明序列来自古代样品;2) 两个样品代表的个体在系统发育树中均与马鹿聚类,在物种归属中被分子鉴定为马鹿(Cervus elaphus); 3) 黑龙江的两个古代样品与现生马鹿阿拉善亚种(C. elaphus alxaicus)亲缘关系最近,而与现生马鹿东北亚种(C. elaphus xanthopygus)亲缘关系较远。结合样品年代信息,说明两例样品代表的黑龙江古代马鹿种群,不是现生马鹿东北亚种的直系母系祖先。

关键词: 黑龙江, 鹿亚科, 古DNA, 分子鉴定, 系统发育分析

Abstract:

The deer resources in China are abundant, with seven species in the sub-family Cervinae distributing in various areas. The intraspecific phylogeny of Cervinae has been widely explored, while the evolutionary relationship among different species requires further efforts, in which only few molecular studies on ancient materials have been performed. In this study, we carried out ancient DNA research on two Cervinae subfossils from northeastern China, dating of 3800 and 5100 aBP. Through ancient DNA extraction, double-stranded sequencing libraries construction, next-generation sequencing and bioinformatics data analysis, we reconstructed two mitochondria sequences with lengths of 16475 bp (GenBank accession number: MT784751, sequence integrity: 99.83%) and 16167 bp (GenBank accession numberh: MT784752, sequence integrity: 97.96%), respectively. Based on the mitochondrial homologous sequences of the extant Cervinae species in GenBank, we constructed a phylogenetic tree. The results show that: 1) both the average length and the C-to-T substitution frequencies at 5’- end of the NGS short reads indicate the data are from ancient specimens; 2) the two ancient individuals clustered with Cervus elaphus in the phylogenetic tree, and were molecularly identified as C. elaphus; 3) the two ancient samples from Heilongjiang are phylogenetically close to the extant C. elaphus alxaicus, but far from the extant C. elaphus xanthopygus. Combining the dates of the samples, we suggest that these two samples represent a population of ancient C. elaphus in Heilongjiang, which was not the direct maternal ancestor of the extant C. elaphus xanthopygus.

Key words: Heilongjiang, China, Cervinae, ancient DNA, molecular identification, phylogenetic analysis

中图分类号:

Q915.876

肖博, 盛桂莲, 袁俊霞, 王斯人, 胡家铭, 陈顺港, 姬海龙, 侯新东, 赖旭龙. 中国东北鹿亚科动物亚化石的古DNA分子鉴定及系统发育分析. 古脊椎动物学报, 2020, 58(4): 328-337.

XIAO Bo, SHENG Gui-Lian, YUAN Jun-Xia, WANG Si-Ren, HU Jia-Ming, CHEN Shun-Gang, JI Hai-Long, HOU Xin-Dong, LAI Xu-Long. Ancient DNA molecular identification and phylogenetic analysis of Cervinae subfossils from Northeast China. Vertebrata Palasiatica, 2020, 58(4): 328-337.

图/表 6

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Sample No.	Collection No.	Morphological species	Tissue type	Carbon dating (aBP)	Unearthed place
CADG522	H52710	Cervus sp.	Metacarpus	3800±30	Zhaodong, Heilongjiang
CADG527	H45190	Cervus sp.	Radius	5100±30	Binxian, Heilongjiang

Sample No.	Collection No.	Morphological species	Tissue type	Carbon dating (aBP)	Unearthed place
CADG522	H52710	Cervus sp.	Metacarpus	3800±30	Zhaodong, Heilongjiang
CADG527	H45190	Cervus sp.	Radius	5100±30	Binxian, Heilongjiang

Subfamily	Genus		Species	Accession No.
鹿亚科 Cervinae	花鹿属 Axis		花鹿 Axis axis	JN632599 NC020680
	花鹿属 Axis		豚鹿 Axis porcinus	NC020681 MF435989
	黇鹿属 Dama		黇鹿 Dama dama	NC024819 JN632630 JN632619 NC020700
	麋鹿属 Elaphurus		麋鹿 Elaphurus davidianus	JN399997 JN632632 NC018358
	鹿属 Cervus		白唇鹿 Cervus albirostris	HM049636 NC016707
			坡鹿 Cervus eldi	KU133959
			水鹿 Cervus unicolor	DQ989636 NC008414 EF035448 NC031835 KY946815
			梅花鹿 Cervus nippon	NC006993 NC013834 NC016178 NC018595 NC008462 NC007179 NC006973
		马鹿 Cervus elaphus	天山亚种 C. elaphus songaricus	HQ191429 NC014703 KJ025072
			甘肃亚种 C. elaphus kansuensis	NC039923 MH513320
			东北亚种 C. elaphus xanthopygus	GU457434 NC013836
			塔里木亚种 C. elaphus yarkandensis	GU457435 NC013840
			阿拉善亚种 C. elaphus alxaicus	KU942399
空齿鹿亚科 Odocoileinae	驼鹿属 Alces		驼鹿 Alces alces	NC020677

Subfamily	Genus		Species	Accession No.
鹿亚科 Cervinae	花鹿属 Axis		花鹿 Axis axis	JN632599 NC020680
	花鹿属 Axis		豚鹿 Axis porcinus	NC020681 MF435989
	黇鹿属 Dama		黇鹿 Dama dama	NC024819 JN632630 JN632619 NC020700
	麋鹿属 Elaphurus		麋鹿 Elaphurus davidianus	JN399997 JN632632 NC018358
	鹿属 Cervus		白唇鹿 Cervus albirostris	HM049636 NC016707
			坡鹿 Cervus eldi	KU133959
			水鹿 Cervus unicolor	DQ989636 NC008414 EF035448 NC031835 KY946815
			梅花鹿 Cervus nippon	NC006993 NC013834 NC016178 NC018595 NC008462 NC007179 NC006973
		马鹿 Cervus elaphus	天山亚种 C. elaphus songaricus	HQ191429 NC014703 KJ025072
			甘肃亚种 C. elaphus kansuensis	NC039923 MH513320
			东北亚种 C. elaphus xanthopygus	GU457434 NC013836
			塔里木亚种 C. elaphus yarkandensis	GU457435 NC013840
			阿拉善亚种 C. elaphus alxaicus	KU942399
空齿鹿亚科 Odocoileinae	驼鹿属 Alces		驼鹿 Alces alces	NC020677

Sample	CADG522				CADG527
Library No.	522-1Y	522-1Y	522-1S	522-2S	527-1Y	527-1Y	527-1S	527-2S
Reference sequence	NC018358	KU942399	KU942399	KU942399	NC018358	KU942399	KU942399	KU942399
Total reads	6807540	6807540	30323142	13921352	11761088	11761088	41246962	4391446
Ratio of reads > 30 bp	0.968	0.968	0.967	0.944	0.975	0.975	0.974	0.926
Mapped reads	564	1372	6665	1530	356	782	2508	135
Unique mapped reads	459	1117	4283	1309	304	558	2015	125
Mapped bp	30267	81857	320240	61212	17531	41869	122203	5763
Mapped sequence length (bp)	10314	16260			7437	14141
and coverage (%)	63.06	98.53			45.47	85.69
Average reads (bp)		73	74	46		75	60	46
Depth			26.59				4.63
Final sequence length (bp)			16475				16167
and ratio (%)			99.83				97.96