河南省马铁菊头蝠（Rhinolophus ferrumequinum）的研究
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摘要
为了搞清楚河南省马铁菊头蝠的亚种分类地位,以及马铁菊头蝠种内的系统发育关系,本研究从3个方面对河南省的马铁菊头蝠进行了研究:一是形态学分析,来初步探讨该物种在河南省不同区系表型特征上的差异性;二是对马铁菊头蝠的消化系统进行观察,来了解其消化系统的组织结构;三是分子学分析,通过提取其DNA进行RFLP分析最后构建系统树,来评价其地方种群的遗传多样性水平和其之间的亲缘关系。本研究从河南省的四大山系8个地区共采集到马铁菊头蝠标本58只,对其进行了组织解剖学观察;形态学测量分析;参照Genbank中吉林省和云南省马铁菊头蝠的细胞色素b基因序列,利用生物学分析软件进行分子分析,结果如下:
1.应用石蜡常规切片,HE染色,对马铁菊头蝠消化系统各器官的组织结构进行了观察,结果表明:食管粘膜上皮为复层扁平上皮,轻微角质化,前、中、后段的上皮结构没有显著差异,食管腺在前段较多,中、后段较少。胃固有层含有大量的管状腺。小肠粘膜表面有许多环形皱壁,在十二指肠上段粘膜下层,分布有十二指肠腺。大肠粘膜表面光滑,无绒毛,在粘膜下层的结缔组织中有小动脉、静脉和淋巴管。肝内结缔组织多,肝小叶分界较明显,肝血窦发达。胰的小叶间分界不明显。
2.对58只马铁菊头蝠的形态学数据进行主成份分析,再以主成份值进行Q型系统聚类,结果表明,河南省四大山系8个地区的马铁菊头蝠之间在形态上没有明显的地理差异。
3.RFLP分析表明河南省四大山系8个地区的马铁菊头蝠之间无明显差别,只有TaqⅠ产生了两种酶切类型。
4.形态学数据和分子序列构建的系统进化树表明:河南、云南样本之间的遗传分化程度较高,分别属于不同的亚种。河南的马铁菊头蝠与日本的马铁菊头蝠被划分在一个进化单元,应该属于马铁菊头蝠日本亚种(Rhinolophus ferrumequinum Nippon)。云南样本鉴定为马铁菊头蝠尼泊尔亚种(Rhinolophus ferrumequinum tragatus)。
To find out the Rhinolophus ferrumequinum status of Henan and to understand its population,digestive system of Rhinolophus ferrumequinum were studied on paraffin sections stained with HE,morphology characters and taxonomy on some Rhinolophus ferrumequinum populations in Henan province were studied to construct the principal component, the principal component values were studied with the Q-type hierarchical clustering method to analyze the genetic differentiation among populations,genome DNA was extracted adopting refind DNA isolation method.
Fifty-eight Rhinolophus ferrumequinum were studied from eight sampling sites in Henan,China. The results showed that the mucosa of oesophagus is multi-layered and the structure of epithelium has no significant difference in the whole oesophagus.Gland is more in the upper end of the esophagus.Lamina stomach has a large number of tubular gland and thick muscle layer.Mucosal surface of the small intestine has many ring folds.The duodenal,s submucosa in the upper class has duodenal gland.The surface of the colorectal is smooth and has no villus.There are small arteries,veins and lymphatic vessels in the connective tissue of the submucosa.The liver has much connective tissue.The boundary of liver lobule is clear and the liver sinusoidal is developed.The boundary of pancreatic lobule was obscure.And the result of morphology showed that it has no significant difference between eight sampling sites.
RFLP analysis showed that four enzymes yielded the same restriction sites,only one enzymes yielded different restriction sites,fragment could be assigned to two haplotypes.The difference occurred exclusively in Bat cave of Guangshan,which may be considered a region of increased genetic variability in Rhinolophus.ferrumequinum,but the level of variability is low.
We have also found that there is no local specificity of their mtCytb alleles in Henan province.Phylogenetic relationships constructed by susing parsimony,likelihood and neighbor-joining methods reveal that,all R.rerrumequinum formed two distinct clades(R.f.nippen and R.f.tragatus). R.f.nippen formed two distinct clades:one group from Henan province and the other from Japan and Jinlin province,this shows that the group from Henan province belongs to R.f.nippen.
1.应用石蜡常规切片,HE染色,对马铁菊头蝠消化系统各器官的组织结构进行了观察,结果表明:食管粘膜上皮为复层扁平上皮,轻微角质化,前、中、后段的上皮结构没有显著差异,食管腺在前段较多,中、后段较少。胃固有层含有大量的管状腺。小肠粘膜表面有许多环形皱壁,在十二指肠上段粘膜下层,分布有十二指肠腺。大肠粘膜表面光滑,无绒毛,在粘膜下层的结缔组织中有小动脉、静脉和淋巴管。肝内结缔组织多,肝小叶分界较明显,肝血窦发达。胰的小叶间分界不明显。
2.对58只马铁菊头蝠的形态学数据进行主成份分析,再以主成份值进行Q型系统聚类,结果表明,河南省四大山系8个地区的马铁菊头蝠之间在形态上没有明显的地理差异。
3.RFLP分析表明河南省四大山系8个地区的马铁菊头蝠之间无明显差别,只有TaqⅠ产生了两种酶切类型。
4.形态学数据和分子序列构建的系统进化树表明:河南、云南样本之间的遗传分化程度较高,分别属于不同的亚种。河南的马铁菊头蝠与日本的马铁菊头蝠被划分在一个进化单元,应该属于马铁菊头蝠日本亚种(Rhinolophus ferrumequinum Nippon)。云南样本鉴定为马铁菊头蝠尼泊尔亚种(Rhinolophus ferrumequinum tragatus)。
To find out the Rhinolophus ferrumequinum status of Henan and to understand its population,digestive system of Rhinolophus ferrumequinum were studied on paraffin sections stained with HE,morphology characters and taxonomy on some Rhinolophus ferrumequinum populations in Henan province were studied to construct the principal component, the principal component values were studied with the Q-type hierarchical clustering method to analyze the genetic differentiation among populations,genome DNA was extracted adopting refind DNA isolation method.
Fifty-eight Rhinolophus ferrumequinum were studied from eight sampling sites in Henan,China. The results showed that the mucosa of oesophagus is multi-layered and the structure of epithelium has no significant difference in the whole oesophagus.Gland is more in the upper end of the esophagus.Lamina stomach has a large number of tubular gland and thick muscle layer.Mucosal surface of the small intestine has many ring folds.The duodenal,s submucosa in the upper class has duodenal gland.The surface of the colorectal is smooth and has no villus.There are small arteries,veins and lymphatic vessels in the connective tissue of the submucosa.The liver has much connective tissue.The boundary of liver lobule is clear and the liver sinusoidal is developed.The boundary of pancreatic lobule was obscure.And the result of morphology showed that it has no significant difference between eight sampling sites.
RFLP analysis showed that four enzymes yielded the same restriction sites,only one enzymes yielded different restriction sites,fragment could be assigned to two haplotypes.The difference occurred exclusively in Bat cave of Guangshan,which may be considered a region of increased genetic variability in Rhinolophus.ferrumequinum,but the level of variability is low.
We have also found that there is no local specificity of their mtCytb alleles in Henan province.Phylogenetic relationships constructed by susing parsimony,likelihood and neighbor-joining methods reveal that,all R.rerrumequinum formed two distinct clades(R.f.nippen and R.f.tragatus). R.f.nippen formed two distinct clades:one group from Henan province and the other from Japan and Jinlin province,this shows that the group from Henan province belongs to R.f.nippen.
引文
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