镇海棘螈和大凉疣螈年龄结构研究

通过趾骨切片可以准确鉴定年龄，了解一个物种的最长寿命，也为我们研究确定一个物种的生长特点、性成熟期,以及一个地区一个物种的年龄结构、种群生态(Marnell，1998)和群落生态提供重要信息(Morrison，et a1.，2004)。 本论文使用骨骼鉴龄法对中国浙江省宁波市北仑瑞岩寺林场的镇海棘螈（Echinotriton chinhaiensis）雌性繁群进行了年龄结构研究。结果显示：第一次参加繁殖的年龄为3龄；繁群中数量占优势的是5龄、6龄。而在6龄以后参加繁殖的雌性个体数便开始随着年龄的增大而逐渐减少。参加繁殖的雌性年龄最大个体为8龄。平均年龄为5.13龄。同时对其年龄和头体长、体全长的相关性检验,发现其年龄与头体长和体全长不相关,镇海棘螈雌性的生长方式表现为性成熟后能量主要用于繁殖。 另外,对李子坪大凉疣螈(Tylototriton taliangensis) 雄性繁群进行了年龄结构研究。结果显示：大凉疣螈雄性第一次参加繁殖的年龄为4龄；繁群中数量占优势的是5龄、6龄、7龄。而在7龄以后参加繁殖的雄性个体数便开始随着年龄的增大而逐渐减少。参加繁殖的雄性中年龄最大的个体为10龄。平均年龄为6.7龄。对其年龄和头体长、体全长的相关性检验,发现其年龄与头体长和体全长不相关,大凉疣螈雄性生长特点也表现为性成熟后生长缓慢的特点。 研究材料方面，本文采用野外采样与标本馆标本相结合的方式获得了中国蝾螈科2个重要保护物种繁殖群体的剪（指）趾材料，使得建立于其上的年龄结构工作更加可靠、更加具有代表性。 此外,本论文讨论了镇海棘螈瑞岩寺种群繁殖总量年度间的差异及其产生原因。将1998、1999、2000、2008、2009年镇海棘螈（Echinotriton chinhaiensis） 瑞岩寺种群的繁殖量进行比较，发现虽然雌性平均窝卵数比较稳定，但繁殖总量小于1998、1999、2000年任何一年总产卵量的50%。对2008年镇海棘螈繁殖量大幅下降的原因分析发现, 2007年9、10月影响严重台风的两次强台风、瑞岩寺景区开发等因素可能是造成近年该种群繁殖量大幅下降的原因。而2008年初50年不遇的低温是否影响镇海棘螈的繁殖值得进一步追踪研究。2009年繁殖量较2008年没有明显的增长，可能是由于2007年的台风影响了其繁殖营养的积累。台风的影响可能存在滞后现象,对此有待进一步监测证明。 本研究首次对中国蝾螈科物种进行的年龄结构鉴定，为进一步了解中国蝾螈科动物的种群生态打下了坚实的基础。 Using skeletochronology, we can know the life span of a species, age of reaching sexual mature, and of course age structure, which are vital(Morrison，et a1.，2004). Skeletochronology was performed on Echinotriton chinhaiensis Ruiyansi female population. The result shows that: The oldest individuals were 8 years old and the youngest ones were 3 years old. Individuals of age class 5(39.13%) and 6(21.74%) were most numerous. The number of individuals participated in reproduction decreased with the increase of age after the sixth year. Average age is 5.13 years. There is no correlation between age and body size (SVL and TL). For female chinhai salamander, energy is devoted to reproduction after reaching sexual maturation. While using skeletochronology to study Tylototriton taliangensis Liziping male population, the oldest individuals is 10 years old, and the youngest ones is 4 years old. Individuals of the age class 5, 6, and 7 dominat this population. The number of individuals decrease with the increase of age also after the seventh year. Average age is 6.7 years old in this population. there is also no correlation between age and body size (SVL and TL).It turned out that T. taliangensis tend to grow slowly after reaching sexual maturation. In this thesis, specimens from both wild and museum were used to gain enough toe clipping samples. A big sample size guarantees the reliability of this study. In the meantime, E. chinhaiensis’s annual reproduction of the year 1998, 1999, 2000 ,2008,and 2009 was compared. The result shows there is a huge decline in E. chinhaiensis’s annual reproduction in 2008,even the egg clutch is very stable. After analyzing, it turned out the huge decline in 2008 was probably caused by typhoon in 2007, besides the effect of tourism development and cash crop planting. While the impact of extreme weather of 2008 on reproduction needs further investigation. In the year 2009, there is no obvious increase in annual reproduction. It maybe due to lasting impact of typhoon in 2007. It is the first age-structure study on these two Chinese salamanders. A solid foundation was laid for further population ecology study of these two species.

Biological activities of skin secretions of the salamander Tylototriton verrucosus

Water-soluble skin secretions of salamander Tylototriton venucosus, first described by Anderson in 1871, were studied for their biological and enzymatic activities. They were found to be toxic to mice with an intraperitoneal LD50 of 11.5 mg/kg. Using Sephadex G-75 gel filtration, it was proven that the toxic components of the secretions are proteins with molecular weights ranging from 30,000 to 50,000 Da. The secretions of T. venucosus display a wide spectrum of antimicrobial activities and also contain both proteolytic activity and trypsin inhibitory activity. In contrast, neither hemolytic nor hemorrhagic activities were found. The secretions were determined to have phospholipase A(2) activity; however, no acetylcholine esterase activity was detectable under the assay conditions.

A TAXONOMIC REVIEW OF TYLOTOTRITON VERRUCOSUS ANDERSON (AMPHIBIA, CAUDATA, SALAMANDRIDAE)

Generally it has not been recognized that salamanders of two distinctive color morphs currently are assigned to Tylototriton verrucosus Anderson. One form is uniformly dark brown dorsally, with bright orange coloration confined to the ventral edge of the tail; the other has a dark brown to black dorsal ground color with orange dorsolateral warts, an orange vertebral crest, and orange lateral and medial crests on the head. In addition, the limbs and ventrolateral surfaces of the second form have a variable pattern of orange coloration. The brown form occurs in northeastern India, Nepal, northern Burma, Bhutan, northern Thailand, the type locality in extreme western Yunnan, and perhaps in northern Vietnam. The orange-patterned form occurs only in western Yunnan Province, People's Republic of China. The two forms appear to be allopatric but occur close together in the area of the type locality near the Burma border in western Yunnan. There is no evidence of color intergradation in specimens from this region. Analyses of morphometric and meristic characters, however, suggest the possibility of limited genetic exchange between adjacent populations of brown and orange-patterned forms in western Yunnan. The genetic and taxonomic relationships between the two forms is not fully resolved. However, these two highly distinctive forms obviously have evolved along independent trajectories and merit taxonomic recognition. We therefore propose to restrict the concept of Tylototriton verrucosus to the brown form and designate a neotype for that purpose, and we describe a new species to receive the orange-patterned form.

哀牢髭蟾（Vibrissaphora ailaonica）和红瘰疣螈(Tylototriton verrucosus)的分子系统地理学研究

本研究用线粒体细胞色素b 基因全序列和ND4 基因序列探讨了哀牢髭蟾 （Vibrissaphora ailaonica）分子亲缘地理学和保护遗传学，利用线粒体细胞色素b、 控制区全序列和部分12SrRNA 基因序列研究了分布于中国的红瘰疣螈 （Tylototriton verrucosus）分子亲缘地理学和保护遗传学。 哀牢髭蟾为生活于高海拔生境（大约2000-2600m）的濒危蛙类。各种致危因 素引起了关于对小的、隔离种群长期存活的关注，然而迄今没有关于该物种遗传 多样性的报道。本论文首次研究了分布于中国云南省的哀牢髭蟾的亲缘地理学和 保护遗传学。对采自于9 个种群81 个个体，我们应用线粒体mtDNA cyt b 和 ND4 基因共计1990bp, 获得了51 个单倍型。系统发育分析显示三个分化较深且互为单 系的族群，大致相应于分别被元江和藤条江河谷分开的三个地理单元（元江以东、 金平和藤条江以西地理单元），暗示了长期的地理隔离分化。分子变异等级分析 （AMOVA）显示遗传变异主要为三个地理单元之间（81.68％）和种群内的个体之 间（11.26%）。三个地理单元显著的地理分布暗示在空间和时间上的隔离，这与中 国西南地区在晚中新世到上新世的哀牢山的造山运动和隆升所引起的重要的气候 和古地质变化时间相一致。我们推测低海拔干热河谷可能是三个地理单元之间基 因交流受限的主要生态障碍。根据上述研究结果，我们建议对这三个遗传分化显 著的地理单元，元江以东地区、金平地区、藤条江以西地区作为独立的管理单元 分别加以保护。 本研究通过测定中国横断山区的红瘰疣螈（Tylototriton verrucosus）17个采集 点的123个红瘰疣螈标本的线粒体mtDNA Cyt b、D-loop和12SrRNA三个基因片段 (2347 bp)序列，首次研究了红瘰疣螈在横断山区的群体遗传结构和分子系统地理格 局，并讨论了T. shanjing的物种有效性。基于123个样品定义的49个单倍型的贝叶 斯和NJ系统发育分析表明：(1) T. verrucosus和T. shanjing均未各自构成单系，而是 共同构成一个单系群;(2) 横断山区的红瘰疣螈分为3个线粒体DNA地理单元，即滇 东南、滇中-滇西和片马地理单元，并且地理单元间不存在共享单倍型，说明红瘰 疣螈具有明显的系统地理分布格局。AMOVA分析同样表明3个地理单元之间存在 显著差异，并且分子变异主要发生在3个地理单元间（62.4%）。T. shanjing与T.verrucosus的mtDNA cyt b 序列差异平均值仅为1.1％,明显小于它们与两外群(贵 州疣螈和大凉疣螈)及外群间的遗传距离(6.5-9.9%)。因此，根据T. verrucosus和 T.shanjing的遗传差异以及系统发育分析结果都不支持T. shanjing的物种地位,T. shanjing为T. verrucosus的同物异名，并建议恢复T. verrucosus的中文名红瘰疣螈。 基于上述结果，我们建议将分布于滇东南、滇中－滇西、片马地区的红瘰疣螈作 为三个独立的管理单元分别加以保护。

六种常见两栖类动物皮肤分泌物的生物活性比较

对6种常见两栖类动物皮肤水溶性分泌物的生物学活性与酶学活性进行了研究。其中，红瘰疣螈（Tylototriton verrucosus）、大蹼铃蟾（Bombina maxima）、华西蟾蜍（Bufo andrewsi）皮肤分泌物对小白鼠具有致死毒性，对小白鼠腹腔注射的半数致死剂量分别为11.5、18.8和264mg/kg。而沼哇（Rana nigromaculata）、泽蛙（Rana guentheri）、黑斑蛙（Rana limnocharis）的皮肤分泌物在小白鼠腹腔注射剂量达到500 mg/kg时仍不显示致死毒性。红瘰疣螈、大蹼铃蟾皮肤分泌物的毒性成分为3～60 kDa的多肽与蛋白质。6种两栖类动物皮肤分泌物都具有蛋白酶水解活性与胰蛋白酶蛋白酶抑制活性。红瘰疣螈、大蹼铃蟾和华西蟾蜍皮肤分泌物具有广谱抗菌活性。大蹼铃蟾、华西蟾蜍皮肤分泌物具有肿瘤细胞细胞毒活性。6种皮肤分泌物对哺乳类血液凝固系统无显著影响。仅发现红瘰疣螈皮肤分泌物具有磷脂酶A_2活性。6种皮肤分泌物均无乙酰胆碱脂酶活性。

从线粒体Cyt b基因探讨红瘰疣螈物种地位的有效性

疣螈属的红瘰疣螈(Tylototriton shanjing)和棕黑疣螈(T.verrucosus)的物种界限一直不清楚.测定了来自中国西南地区14个地点的T.shanjing和T.verrucosus共40只标本的线粒体DNA Cyt b基因(753 bp).结果表明:(1)用邻接法、最大简约法和贝叶斯法等3种系统发育分析法分别重建棕黑疣螈种组系统发育树的拓扑结构不支持T.shanjing是单系群;(2)T.shanjing与T.verrucsus的mtDNA Cyt b序列差异平均值仅为1.2%.未达到种级水平.因此,全部T.shanjing样品都属于同一个物种,即T.verrucosus,不支持T.shanjing的物种地位,T.shanjing为T.verrucosus的同物异名,并建议恢复T.verrucosus的中文名红瘰疣螈.根据基于40个样品Cvt b基因序列的系统发育树和遗传变异以及地理分布,这些红瘰疣螈(T.verrucosus)样品聚为3支,即中国西南地区的红瘰疣螈可分为片马、滇中滇西和滇东南3个地理居群.

镇海棘螈繁殖群体生态和遗传结构研究

genetics, such as: population size, reproduction, mating system, growth, development,genetic structure and systematics status; The main results are presented below: The seasonal variation of the operational sexual ratio of this animal was found in the field and the ration always bias the female in the breeding season. Aiming at this character and considering the distance of time and space of both sexual habitat in breeding season, we census female population first by toe-clipping mark-recapture method, then estimated the population size with the definitive sexual ratio. Up to now, this species was found only at the Beilun district of the Ningbo City. The population size of the Ruiyan Temple Forest Park approximates to 369. The status of this population is extremely endangered, so besides protecting this population at the original locality, we also suggested to breed the salamander in fenced locality and to hatch embryos artificially, and send metamorphosed juveniles back to nature. We can transfer some individuals to other similar habitats or breed them under artificial conditions for saving this species from extinction. The early developmental stage of the Chinhai salamander is the same as its relative species, E. andersoni. Their balanceres are poorly developed and disappear very early. Temperature and moisture significantly influence the embryonic development of the Chinhai salamander. The embryonic stage is approx. 29 days under room temperature. The hatchling grows in a logarithmic curve. The larvae stage in water is approx. 58- 88 days. Many factors influence the nomal development, including two aspects of internal and external. Due to these factors, the effective protected measures were presented in detail. The breeding migration of E. chinhaiensis takes place at late March～late April every year. This salamander's hatching rate is high, but the rate of hatchling migrating into water is low. The average effectiveness of all the nest sites is 36.7%. The maternal self-conservation was contrary to the reproductive success of the egg-laying strategy. In the strategy of egg-laying behavior, the first factor selected by the female was its self-conservation, the second is embryonic survival rate, and the last is rate of hatchling survival rate. The oviposition selection is significant for the survival of the larvae. Based on the analysis of the evolutionary process of reproductive behaviors nad egg-laying site selections of all genera of the family Salamandridae, we deduced that perhaps Echinotriton is a transitional type in the evolutionary process from water to land. Due to its location in the adaptive stage in the terrestrial evolution, Echinotriton chinhaiensis's terrestrial nest may be one of important reason that causes this species to be endangered. The genetic deversity analysis shows that although the population size of the Chinhai salamander is quite small compared to other Chinese salamandrid species, the genetic diversity of this population is not reduce remarkably. We explain this phenomena with the polygamy mating system of this species. The result of 4 families' parenthood determinations shows that the parenhood determination can be taken without any paternal information. The "children" of every female include rich genetic information from at least two "fathers". It implies that female Chinhai salamander mates more than once with different males in a breeding season. The molecular evidence, the behavioral observation evidences and the sperm evidence in the female cloaca proved that this species has a polygamy mating system. The kin recognition in the mating of adult salamander was first discussed. The taxonomic status and phylogenetic relationships of 12 species representing 6 genera in the family Salamandridae were studied using DNA fingerprinting. The results showed that the DNA fingerprinting. The results showed that the DNA fingerprinting patterns demonstrated rich genetic diversity and species diversity, and also revealed the taxonomic status and phylogenetic relationshipes of higher taxa to a certain extent. The results are highly consistent with those obtained from the studies based on the morphology, ecology, cytology and molecular biology. The compreshensive analysis indicate that Tylototrition hainanensis and T. wenxianensis should be valid species; Echinotriton should be a valid genus;Tylotortriton is a natural cluster; Tylotortriton asperrimus should be put in Tylototrition rather than in Echinotriton, Hypselotriton and Allomestriton are synonyms of Cynops and Paramesotriton, respectively. There are three main groups in Chinese salamandride: Cynops, Paramesotriton and Pachytrition from the first group, the species of the Tylototriton from the second, and E. chinhaiensis composes the third.