中国特有两栖动物研究1.山东半岛林蛙的生活史

生活史的研究可以为系统发育研究和珍稀濒危物种的保护和管理对策的制定提供参考资料。林蛙是我国分布比较广泛而复杂多样的一个类群,在山东半岛的昆嵛山和崂山,分别分布有昆嵛林蛙（Rana kunyuensis）和中国林蛙（R．chensinensis）。本文报道我们从1997年到2006年,对这两种林蛙在山东半岛的地理分布、繁殖习性、蝌蚪特征和生长发育以及冬眠等方面的调查和观察研究结果。结果表明昆嵛林蛙和分布于崂山的中国林蛙尽管在栖息地生境方面比较相似,但在生活史方面表现出较大的不同。     

新疆蛙类遗传多样性与系统发育研究

[目的]为了解新疆地区中国林蛙中亚林蛙、中亚侧褶蛙和阿尔泰林蛙的遗传多样性及系统发育。[方法]分别于新疆7个地方采集了4种蛙类共53个个体,对线粒体12S RNA基因进行了扩增和测序。[结果]经过拼接和比对,得到长度为893 bp片段的序列,4种蛙类共定义了21个单倍型,其中,中国林蛙、中亚侧褶蛙和阿尔泰林蛙遗传变异程度高,而中亚林蛙的遗传变异很低。各群体总遗传分化系数Gst为0.367 88,总固定系数Fst为0.871 56,总基因流Nm为0.04。中国林蛙与中亚林蛙之间的遗传距离最大,为0.158,与中亚侧褶蛙之间的遗传距离次之,为0.137,与阿尔泰林蛙之间遗传距离最小,为0.034。聚类分析显示中国林蛙和阿尔泰林蛙聚为一支,中亚林蛙和中亚侧褶蛙聚为一支。[结论]新疆地区的中国林蛙、中亚侧褶蛙和阿尔泰林蛙的遗传多样性丰富,中亚林蛙的遗传多样性较低,各群体遗传分化程度较高。     

寒露林蛙的核型和Ag-NORs研究

采用常规骨髓细胞制片法对产于湖南省双牌县的寒露林蛙的核型及NORs位点进行了观察和分析.结果表明寒露林蛙与分布于我国南方的其他林蛙相同:2n=26,NF=52,为5+8核型模式.寒露林蛙核型为14M+10SM+2ST,无次溢痕,NORs位于第11对染色体长臂的中部.通过比较分析我国2n=26的6种林蛙的核型等细胞遗传学信息,本文结果支持形态分类和分子系统发生的研究结论,认为寒露林蛙是一有效种.     

中国林蛙的分类及分布

目的中国林蛙(Rana chensinensis David,1875)是我国林蛙属分布最为广泛的特有种,在上一世纪对其分类问题多有争议,曾被认为广布于中国、蒙古、俄罗斯、日本和朝鲜半岛等。随着对其分类地位的确定,其分布范围也发生了很大变化。为了保护和可持续利用这一重要资源,本文对其分类研究历史和分布进行了总结。方法通过收集、整理和分析研究文献,结合本实验室多年来掌握的野外资料,进行系统的总结。结果中国林蛙自1875年由戴维(中文名为谭卫道)命名以来,曾被划归欧洲林蛙的亚种,并将多种其他林蛙归并入其下。1981年,经过细胞分类学研究,正式恢复为有效种,随后进一步划分出5个亚种。直至本世纪初,经过进一步的分类学研究,厘清了其与东北林蛙和西北林蛙的关系,从而确定其为仅分布于中国的特有种。在此基础上,对其分布也进行了重新确立。结论中国林蛙隶属于蛙科林蛙属中国林蛙种组,目前没有种下分化和亚种形成,分布于我国华北、华中及其周边地区,分布海拔不高于2500m。     

桓仁林蛙与东北林蛙蝌蚪的形态特征及比较

桓仁林蛙（Rana huanrenensis Liu,Zhang,＆ Liu,1993）为一种与东北林蛙（Rana dybowskii）同域分布的林蛙,但其分布区域较狭窄.通过对其蝌蚪的形态特征描述及测量相关的形态学量度并和东北林蛙比较,发现在体色、体型、尾部形态以及身体各部位量度比值等方面存在显著性差异.桓仁林蛙的蝌蚪外形与流水型的蝌蚪相类似,而东北林蛙的蝌蚪则属于典型的静水型蝌蚪.     

中亚林蛙的核型,C带和银带研究及田野林蛙起源的探讨

中亚林蛙（Ranaasialica）的核型为2n＝26,NF＝52．第2号染色体短臂有一条近端着丝粒区C带。第10号染色体长臂上有一对标准NORs。本文认为,田野林蛙（Ranaarvalis）起源于欧洲,是欧洲林蛙群与亚洲2n＝24的林蛙群间的过渡种类。     

辽宁沈阳发现韩国林蛙

2021年11月在沈阳市辽中区蒲河湿地公园（41°30′55″ N,122°78′30″ E,海拔30 m）采集到2号无尾两栖类标本,经形态特征比较确认为无尾目（Anura）蛙科（Ranidae）蛙属（Rana）物种,基于线粒体16S rRNA基因对蛙属19个物种的亲缘关系及系统发育进行分析,其与韩国及中国山东文登昆嵛山分布的韩国林蛙（R. coreana）遗传距离最近,并在最大似然树中聚为一支,应属种内关系。综合形态分析和系统发育比较,确定采集到的标本为韩国林蛙,系辽宁省两栖动物分布新记录种。本次发现将辽宁省分布的蛙属物种增至5种。     

浙江庆元发现长肢林蛙

2022年10月在浙江省庆元县贤良镇采集到5只林蛙属(Rana)雌性个体,经形态和分子系统发育分析鉴定为长肢林蛙(R. longicrus),是浙江省两栖动物分布新记录种。所采集的5只长肢林蛙个体体长40.4 ~ 55.2 mm,头长大于头宽,肩部上方由数个疣粒隐约组成“八”字形黑斑;背侧褶明显,与胯部相连;四肢背面具黑褐色横纹,后肢前伸贴体时胫跗关节超过吻端,形态特征与地模标本基本一致。基于线粒体16S rRNA基因片段进行系统发育分析表明,该蛙与模式产地中国台湾的长肢林蛙序列聚成单系支,且遗传距离小于0.005%。此前记录的长肢林蛙仅分布于我国台湾、广东、福建、江西等地,长肢林蛙在浙江省庆元县的分布为浙江首次记录,这提示该蛙有更广的分布范围。     

中国林蛙的分子系统关系

测定了 6种林蛙和用作外群的 2种侧褶蛙和 1种陆蛙的线粒体 12SrRNA基因序列 393bp。序列两两对位比较表明内外群间的位点替换率是 7 3%到 2 3 1% ,内群中物种间则为 0 0 %到 9 2 %。依据上述DNA序列 ,用距离法和最大简约法的系统发育分析表明 :①研究的 6种林蛙聚为一支 ,构成单系群 ,并有高的BPs值(90 %以上 )支持 ;② 6种林蛙可以分成 2个姐妹群 ,即中国林蛙、黑龙江林蛙和桓仁林蛙为一组 (BPs >94% ) ,峨眉林蛙、昭觉林蛙和镇海林蛙为一组 (BPs >5 0 % ) ;③昭觉林蛙与镇海林蛙有较近的亲缘关系 ;④中国林蛙的榆中种群与牡丹江种群间的遗传分化似乎达到了种级分化水平。     

内蒙古扎兰屯地区发现中国林蛙（无尾目：蛙科）

2016年4月,采集内蒙古呼伦贝尔市扎兰屯地区的林蛙,经形态学(形态描述、体型大小)和分子生物学鉴定,确定该物种为中国林蛙(Rana chensinensis)。研究结果重新证实了中国林蛙在内蒙古呼伦贝尔地区的分布,扩展了对中国林蛙分布范围的认识。     

Evolutionary relationships of Eastern Palearctic Brown Frogs, genus Rana: paraphyly of the 24-chromosome species group and the significance of chromosome number change

The Eurasian 'brown frogs' are a morphologically conservative assemblage consisting of the European Rana temporaria and a large number of similar species considered to be related. Although the chromosome number of the great majority of Rana species is 26, there are several species of brown frogs (including R. arvalis, R. chensinensis, R. omativentris and R. dybowskii ) which instead have 24 chromosomes. Yet comparative study of isozyme variation at 25 gene loci from these and seven other European, Caucasian, and east Asian species indicates that the 24-chromosome brown frogs are not a monophyletic group. To the exclusion of R. arvalis , the east Asian 24-chromosome species form a distinct clade related to the 26-chromosome species R. temporaria, R. dalmatina, R. japonica , and R. tagai more closely than to R. arvalis. This result is reinforced by the comparative position of the nucleolar organizer region (NOR) in the karyotypes of these frogs, as shown by NOR-banding of metaphase chromosomes. The NOR is on chromosome No. 2 in R. arvalis but on chromosome No. 10 in the other three 24-chromosome species. The systematic positions of the Causasian species A. camerani and R. macrocnemis relative to the other species are unclear except that these frogs are distinct enough to be considered part of an outgroup clade. However, in general, the systematics of the entire brown frog group remains unresolved due to great genetic divergences between species and the evident homoplasy of the brown frog morphotype. The parallel origins of reduced karyotypes among the brown frogs demonstrate that caution should be exercised when judging systematic relationships based upon gross karyology in the absence of more detailed information.     

Allozyme variation among Rana balcanica,R. levantina,and R. ridibunda (Amphibia: Anura)1

The allozyme variation among water frogs of the species R. balcanica, R. levantina and R. ridibunda, all formerly considered as one species (R. ridibunda Pallas, 1771), was studied using horizontal starch gel electrophoresis. Blood samples (N # 63) of frogs were collected from five populations in Greece and Israel. Samples (N = 9) of the hybrid frog R. esculenta collected from a locality in Germany were used as an outgroup for phylogenetic analyses. Fifteen enzymes controlled by twenty presumptive loci were identified. Thirteen loci were polymorphic within or among the studied populations. Genetic differentiation among the species was considerably greater than among populations of the same species. Even at Nestos River where R. ridibunda and R. balcanica occur in the same habitats, individuals could be assigned to either species due to characteristic differences of the genotypes (GPI1). This indicates reproductive isolation among these species. The reconstruction of phylogenetic relationships among the three species based on the allozyme data corroborated the model presented on the basis of bioacoustic data: R. ridibunda and R. balcanica (Nei's genetic distance D = 0.0820) are sibling species pertaining to an Eurasian lineage, whereas R. levantina (distance to the European species D = 0.1780 - 0.1955) together with R. perezi represent an independent afroasian lineage.     

Phylogenetic relationships of the Chinese brown frogs (genus Rana) inferred from partial mitochondrial 12S and 16S rRNA gene sequences

Based on partial sequences of the 12S and 16S ribosomal RNA genes, we estimated phylogenetic relationships among brown frogs of the Rana temporaria group from China. From the phylogenetic trees obtained, we propose to include Rana zhengi in the brown frogs. Monophyly of the brown frogs was not unambiguously supported, but four well-supported clades (A, B, C, and D) always emerged, although relationships among them remained unresolved. Clade A contained brown frogs with 24 chromosomes and was split into two distinct subclades (Subclade A-1: R. chensinensis and R. huanrenensis; Subclade A-2: R. dybowskii). Polytomous relationships among populations of R. chensinensis and R. huanrenensis suggested the necessity of further taxonomic assessment. Rana kunyuensis proved to be the sister group to R. amurensis, and these two species formed Clade B. Clade C was composed of R. omeimontis and R. chaochiaoensis, and Clade D included R. sauteri, which has been placed in other ranid genera. These relationships did not change after adding published data, and monophyly of Subclade A-1, A-2, and other East Asian brown frogs with 24 chromosomes (R. pirica and R. ornativentris) was ascertained, though their relationships were unresolved. Clade C, together with R. japonica and R. longicrus, also formed a monophyletic group. Brown frogs related to Clades A and C were estimated to have dispersed from continental Asia to adjacent regions through multiple events.     

Do male moor frogs (Rana arvalis) lek with kin?

Many amphibian species are known to form leks during breeding season, yet it has seldom been tested which evolutionary forces are likely to act on lek formation in this taxon. We tested the kin selection hypothesis for lek formation by using eight variable microsatellite loci to compare the genetic relationship of 203 males in seven Rana arvalis leks. The results indicate that moor frog males do not lek with kin: their relatedness within leks was not higher than expected by chance. Furthermore, spatially distinct leks within same water bodies could not be distinguished from each other as separate units. These results are not expected if kin selection underlie lek formation. On the basis of these results and general knowledge of anuran breeding biology, we suggest that lek formation in explosively breeding amphibians might have evolved by female choice for breeding aggregations, combined with female choice of habitat. Future work should aim at predicting aggregations based on rules of phonotaxis over different spatial scales, and empirical work should document visitation rates not only for leks of a specific size, but also for different travel distances that visiting females may have had to cover.     

Effects of pH and aluminium on embryonic and early larval stages of Swedish brown frogs Rana arvalis, R. temporaria and R. dalmatina

The effects of pH and aluminium on embryonic and early larval stages of Swedish brown frogs Rana arvalis, R. temporaria and R. dalmatina were tested in laboratory bioassays. In all three species egg mortality and time needed for embryonic development to hatching increased when pH declined, but no significant effects were found on embryonic development when aluminium level was elevated. In R. arvalis and R. temporaria larval mortality was affected by both pH and aluminium. In both species the frequency of occurrence of larval deformities increased in acid water, and there was a synergistic effect of pH and aluminium. In R. arvalis swimming behaviour was disturbed by high levels of aluminium at pH 5. In all three species the frequency of stressed larvae increased when pH was depressed and aluminium concentration elevated, and there was a synergistic effect when both were combined. The three species differed significantly in egg mortality, time needed for embryonic development, larval mortality, larval deformities and larval stress al low pH and high aluminium levels. R. arvalis showed the highest acid tolerance and R. dalmatina was the most sensitive to low pH.     

Taxonomy and evolution of European brown frogs

Phylogenetic relationships within the Euro-Mediterranean stock of the Rana temporaria species group (R. temporaria, R. iberica, R. cameranoi, R. macrocnemis, R. holtzi, R. dalmatina, R. graeca, R. latastei) were studied by (1) morphometric analysis, (2) osteological study and (3) electrophoresis. The three data sets were analysed independently and phenograms and cladograms examined for congruent patterns. The tree resulting from electrophoretic analysis is the most clearly resolved (and being also compatible with the other two) is assumed to represent the most likely phylogeny of the Mediterranean brown frogs. Patterns indicated by electrophoretic data matched the molecular clock-hypothesis. This suggests that the first split of the main stock of Mediterranean brown frogs separated ancestors of Rana temporaria and R. iberica from the others during the Late Pleistocene. A second split occurred separating the ancestors of what are currently the other big-bodied and the small-bodied brown frogs. The relationships between and within the three main stocks are discussed in the context of biogeographical and geological data.     

东北虎源等孢球虫的发现

对东北虎粪便进行虫卵检查,分离获得了自然感染的球虫卵囊。对球虫卵囊的形态和经培养发育后的各阶段的卵囊形态进行了观察。东北虎源球虫卵囊符合等孢属球虫的形态学特征。根据Genbank 上发表的猫等孢球虫18S rDNA 序列(L74671)设计一对特异性引物,利用PCR 技术对东北虎源球虫的18S rDNA 进行了扩增。扩增得到一段360 bp 的DNA 片段,并对该片段进行了序列测定。测序结果和其它17 种原虫的相应序列进行序列同源性比较分析,分析显示东北虎源球虫和其它等孢属球虫单独聚为一类。结合对东北虎源球虫的卵囊形态、卵囊发育情况及序列分析结果,确定其为等孢属球虫。      

一株牛蛙源金黄杆菌的分离鉴定及其致病特性

【背景】美国牛蛙养殖过程中病害问题非常突出,尤其是细菌性病害,其病原种类多、病原菌复杂多样、蔓延速度快、发病死亡率高,一直是牛蛙养殖过程中防控的难点。【目的】确定从患病牛蛙体内分离到的一株细菌NW1203的分类地位和致病性。【方法】无菌操作从牛蛙体内取样划线分离细菌,通过形态观察、生理生化试验、16S rRNA基因序列比对进行种属鉴定,通过人工感染、溶血性试验和病理切片观察分析其致病特性。【结果】经形态和生理生化鉴定及16S rRNA基因序列比对,菌株NW1203为金黄杆菌属细菌,与Chryseobacterium sp. F30的相似性达100%,进化树也显示该菌与金黄杆菌属细菌聚类;溶血性试验表明,菌株NW1203对绵羊、小鼠和牛蛙的血细胞都呈完全溶血;人工感染试验及感染病蛙的组织切片观察显示,菌株NW1203对牛蛙具有较强致病性,可引起牛蛙肝、肾、脾等主要组织严重病变,LD50为4.753×103 CFU/g。【结论】明确了菌株NW1203为牛蛙新病原,为牛蛙疾病的防控提供了理论依据。     

Evidence from peptidomic analysis of skin secretions that the red-legged frogs, Rana aurora draytonii and Rana aurora aurora, are distinct species

The northern red-legged frog Rana aurora aurora and the California red-legged frog Rana aurora draytonii are traditionally classified together in the same species group. Ten peptides with antimicrobial activity were isolated from norepinephrine-stimulated skin secretions of R. aurora draytonii and purified to near homogeneity. The peptides were identified as belonging to the ranatuerin-2 family (two peptides), brevinin-1 family (four peptides), temporin family (three peptides), and a novel peptide, RV-23 (RIGVLLARLPKLFSLFKLMGKKV) that has limited structural similarity to the bee venom peptide, melittin. This distribution of peptides contrasts with that found previously in skin secretions from R. aurora aurora collected under the same conditions and at the same time of year (one ranatuerin-2 peptide, two brevinin-1 peptides, and one temporin peptide). The variation in amino acid sequences between corresponding R. aurora draytonii and R. aurora aurora peptides is comparable with the variation in sequences of orthologs from other members of the Amerana group of New World ranid frogs (Rana boylii, Rana muscosa, and Rana luteiventris). It is proposed, therefore, that the red-legged frogs should be regarded as separate species (R. aurora and R. draytonii) within the Amerana group rather than conspecific subspecies. The data emphasize that amino acid sequences of antimicrobial peptides in skin secretions may be used to infer taxonomic and phylogenetic relationships between species of ranid frogs.     

中国东北地区林蛙属物种的分类学研究(两栖纲:蛙科)

用比较分类学方法和数值分类方法相配合,对我国东北产林蛙属物种多样性和原各命名物种的有效性及与中国林蛙的关系进行了探讨。结果认为：地方名为哈土螨的原中国林蛙的东北居群应属于我国蛙类的一新纪录东北林蛙Ｒ．ｄｙｂｏｗｓｋｉｉ,而Ｒ．ｓｅｍｉｐｌｉｃａｔａ和Ｒ．ｃｈｅｎｓｉｎｅｎｓｉｓ ｃｈａｎｇｂａｉｓｈａｎｅｎｓｉｓ（魏刚等,１９９１ａ）为该种的同物异名,重新界定了东北林蛙和中国林蛙的分布区,提出中国