摘要
中国大鲵(Andrias davidianus)俗称娃娃鱼,隶属有尾目、隐鳃鲵科、大鲵属,是我国特有的濒危有尾两栖类动物,也是世界现存两栖类中体型最大的古老动物,曾广泛分布于我国中南部各省。有尾类是扩散能力较差的脊椎动物,它们对水环境的依赖程度较大,克服不同阻碍的能力也较差,因此它们的分布区的大小在一定程度上反映它们的地质年龄和演化历史,在两栖类动物中分布最广的类群可能是一个最古老的类群。中国大鲵在我国分布跨越不同的气候带和不同的水系,是现存有尾目动物中分布最广的一个物种,这说明了中国大鲵漫长的进化历史和位于两栖类中原始类群的地位。中国大鲵还被认为是研究脊椎动物一些新出现的解剖结构进化速率和方式的模式系统,因此它在研究陆生四足类动物系统演化中具有重要的科学价值。本文从形态解剖学、细胞生物学和分子生物学等方面对中国大鲵在有尾两栖类中的进化地位进行了较系统的研究,主要内容如下：
1.从形态解剖学方面对中国大鲵的骨骼系统、呼吸系统、心血管系统、生殖系统和胚胎发育作进一步的研究,并利用已有研究资料对有尾两栖类动物各个系统的特征进行分析比较,以便能更加准确地了解中国大鲵在有尾两栖类中的系统演化地位。中国大鲵在形态结构上具有很多独特的地方,保留了很多在进化过程中的痕迹。中国大鲵骨骼系统中的原始特征包括犁骨齿列的“ω”状圆弧形,保留相当完整的麦氏软骨,脊椎骨属双凹形,荐椎分化尚不明显,肩带和腰带多软骨,髂骨与荐肋连接位置不固定,且通过软骨相连,这决定了其后肢对身体的支持作用仍很有限。中国大鲵的附肢骨也表现出这种过渡类型的原始性,如其后肢的体位更加类似于鱼类,后肢向后伸展,且在趾间有蹼,适应在水中游动,四肢在水中的使用多于在陆地上。
中国大鲵无明显咽部,呼吸通道和食物通道在口腔处形成交叉。呼吸道仅为短的喉头气管室,喉头和气管的分化不明显,喉头气管室后面直接与肺囊相连,吸气主要是通过口腔抽吸压入空气到肺部,呼气时腹壁肌肉收缩,压迫气体从肺部排出,但是体腔没有分隔,肋骨不完善,肺也没有出现高度肌组织化。所以,中国大鲵的这种呼气能力还是比较原始的。因为生活于水环境下太多的浮力会成为一个问题,而肺的存在会使浮力增加,中国大鲵的肺有部分简化,它通过身体前后摇摆的运动和皱褶的皮肤表面来增加气体交换。中国大鲵个体发育过程中出现带鳃的幼体阶段和用肺呼吸的成体阶段,心血管结构的演变重现了其进化过程中从水生到陆生的这个过程。成体心房中出现了纵隔,但是它的房间隔有很多孔洞,使左右心房的血液能够混合。中国大鲵只有一个心室,但心室内壁具有多为前后延伸的肌肉小梁,能够在一定程度上减少左右心房来的血液的混合。动脉圆锥中有纵向的脊状螺旋瓣,可以大致区分这两类含氧量不同的血液而将它们分送肺循环和体循环。成体有四对动脉弓,与鱼类相似,并且它还保留有颈动脉导管、波氏导管、后主静脉和腹壁静脉。
中国大鲵精巢内各级生精细胞从基底到管腔不遵循精原细胞、初级精母细胞、次级精母细胞、精子细胞和精子的排列方式,而是同步发育的生精细胞成团分布,这与硬骨鱼精细管的生精上皮排列方式相似,是一种比较原始的小叶型精巢。中国大鲵的前肾被精巢替代,精巢通过一些输出小管直接与泌尿系统的中肾管相连,因之中肾管输精兼输尿。大多数的有尾两栖类进行体内受精,而中国大鲵与两栖类中的原始种类(小鲵科Hynobiidae和鳗螈科Sirenidae)一样是进行体外受精的。中国大鲵的卵裂方式与其他两栖类不同,而与辐鳍亚纲的鲟科鱼相似。
2.从细胞生物学的角度对中国大鲵精子超微结构、染色体核型和基因组大小进行了研究。中国大鲵的精子由头部、颈部和尾部三部分构成。与其他有尾两栖类精子结构相比在尾部缺失线粒体,尾部中轴为圆柱形,没有顶体钩。在所有进行体内受精的有尾类动物中,精子的产生与泄殖腔腺相关联,相应的精子颈部较长,而在体外受精的种类精子颈部较短并且没有泄殖腔腺。中国大鲵进行体外受精,精子颈部也较短,但是出现了泄殖腔腺,这说明它处在从体外受精向体内受精过渡的一种中间阶段。
中国大鲵具有2n=60条染色体,整个染色体组由15对大染色体和15对微小染色体组成。大染色体包括3对大型的中部着丝粒染色体,3对亚中部着丝粒染色体,9对端部着丝粒染色体。微小染色体包括9对中部和亚中部着丝粒染色体,6对端部着丝粒染色体。使用流式细胞仪测定了中国大鲵的基因组大小数据(41.66pg2C)。并且利用已有研究资料从染色体数目、形状和基因组大小方面对中国大鲵在有尾两栖中的进化地位进行了研究。
3.从分子生物学角度对中国大鲵的同工酶和线粒体基因进行了研究。对中国大鲵六种组织三种同工酶(LDH,EST,SOD)进行了研究,旨在了解中国大鲵遗传物质的特征,为中国大鲵特殊的演化地位提供生化表型的遗传标志,并探讨了中国大鲵LDH同工酶在两栖类种群中的演化。
利用Genebank上收集到的有尾两栖类和一些代表性脊椎动物的线粒体基因组全序列,主要利用分子生物学实验手段,运用支序系统学与分子进化生物学理论及分析方法展开系统发育的研究。建立起系统发育关系树,在此基础上,阐述现存的种属分类问题,并进一步探讨中国大鲵的起源和在有尾两栖类中的进化地位。
Chinese giant salamander(Andrias davidianus), also known as "baby-fish", which belonged to Caudata, Cryptobrachidae, Andrias Tschudi, is the largest living amphibian in the world, and also our country's rare and endangered endemic species. Due to their low dispersal ability, high dependence on water environment, and poor performance in overcoming various barriers, the range of Caudata's distribution reflects their geological age and evolutionary history to some extent, that is, the species which occurs most widely may be the most ancient. Thus, Chinese giant salamander, the most widely distributed in the living amphibians with tails, used to be located in the central and south China across different climate zones and water areas, showing their primitive taxonomy and long evolutionary history. In addition, Chinese giant salamander is regarded as a model system for the research on the evolutionary rate and pattern of newly-arising vertebrate anatomic structures, and of significant scientific value for the study on phylogeny of terrestrial tetrapoda. This paper provide comprehensive evidence about the evolutionary status of Chinese giant salamander in Caudata from the perspective of morphological anatomy, cytological biology, molecular biology and so on, and the major results are as follows:
1. To know more exactly about the phylogenetic position in Caudata of Chinese giant salamander, further research was carried out on the embryonic development and morphological anatomy about skeletal system, respiratory system, cardiovascular system, and reproductive system, which was compared with systematic characteristics of Caudata in previous studies. Chinese giant salamander possessed many distinctive morphological structures, as well as some evolutionary trace. For instance, primitive characters of skeletal systems were observed, likeω-shaped vomerine tooth, completely reserved Maxwell cartilage, biconcave vertebra, and almost undifferentiated sacral vertebra. In addition, pectoral girdle and pelvic girdle were mostly consist of gristle, and the joint of iliac bone and sacral rib wasn't fixed which were connected through the cartilage, resulting in the limited support to body of posterior limbs. Furthermore, appendage bone of Chinese giant salamander also exhibited primitiveness of transitional type. Posterior limbs with webbed-toes, of which body position resembled that of fish, stretched backward and adapted to moving about in the water. Four limbs of Chinese giant salamander were used more in water, rather than in land.
No obvious pharyngeal was found in Chinese giant salamander, and then respiratory passage, namely short laryngo-tracheal chamber and esophagus intersected at oral cavity. Differentiation between larynx and trachea wasn't clear, and the back of laryngo-tracheal chamber interconnected with lung sac directly. Air was pressed into pulmonary during inspiration by buccal pump, while squeezed out during expiration by the contraction of abdominal wall muscle. The existence of pulmonary would gain buoyant force, while too much buoyant force prove to be a problem for the living in aquatic environment. Pulmonary of Chinese giant salamander was partially simplified, however, gas exchange could be strengthened through wrinkled surface of skin and the movement of body back and forth. Body cavity with no division, defective rid, pulmonary without highly specialized muscle tissue, all contributed to the fact that respiratory function of Chinese giant salamander was fairly original.
Individual development of Chinese giant salamander experienced two stages:larval stage with gill and lung-breathing adult stage. The structural succession of its cardiovascular replayed the evolutionary process from hydrophilous to terricolous. The adult cardiac atrium Mediastinum occurred in was divided by interatrial septum with some holes in it, causing the mix of blood. Only one heart chamber was observed in Chinese giant salamander, however, trabecula existed on the inner wall of heart chamber, most stretching back and forth and capable of reducing the interfusion of blood more or less. Longitudinal carinate spiral valve in the arterial cone could approximately distinguish two kind of blood with different oxygen content, and distributed them into pulmonary circulation and systemic circulation, separately. The adults had four pairs of arterial arcades the same as fish, and also retained carotid duct, Botalli ductus, posterior cardinal vein, and vena epigastrica.
The anterior portion of the kidney was replaced by testis linked with urinary system by mesonephric duct, which served as both seminal duct and urine duct. Despite that the great majority of amphibians with tails conducted in vivo fertilization, Chinese giant salamanders was in vitro fertilization as well as some ancient species of amphibians (Hynobiidae and Sirenidae). In addition, the way of cleavage of Chinese giant salamanders differed with other amphibians, but presented similarity with that of Acipenseridae in ray-finned fishes.
2. Combining previous data, genome size, chromosome number and karyotype, and microstructure and ultrastructure of gonads were studied to determine the phylogenetic position of Chinese giant salamanders.
Genome size (C value) was measured using the flow cytometer. The number and karyotype of chromosomes were examined, and the result indicated that the chromosome number of Chinese giant salamanders was 2n=60. The whole chromosome set was composed of 15 pairs of large chromosomes and 15 pairs of microchromosomes, respectively, Of which, large chromosomes contained 3 pairs of metacentric,3 pairs of submetacentric and 9 pairs of telocentric chromosomes, and microchromosomes included 9 pairs of metacentric and submetacentric chromosomes and 6 pairs of telocentric chromosomes.
Spermatogenic cells of Chinese giant salamanders at different stages developed synchronously and joined together into a mass, resembling the arrangement of seminiferous epithelium of seminiferous tubule in Osteichthyes, but showing great difference from the normal alignment of spermatogenic cells, that is, spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids and spermatzoa lying from base layer to lumen. Testis of Chinese giant salamanders proved to be a microphyll type, comparatively original.
The spermatzoon of Chinese giant salamanders consisted of head, neck piece, and tail. Compared to other amphibians, the spermatzoon of Chinese giant salamanders lacked of acrosomal barb, mitochondria in the tail absent, axial fiber cylinder-shaped. In all the Caudata fertilized in vivo, the generation of sperm was related to cloacal gland, correspondingly, the neck of sperm was long. In contrast, there was no cloacal gland in the species fertilized in vitro, and neck of sperm was short. Chinese giant salamanders conducted in vitro fertilization, neck of sperm was short, but had cloacal gland. All the evidence suggested that Chinese giant salamanders had stayed at the intermediate stage from in vivo fertilization to in vitro fertilization.
3. Isoenzymes and mitochondrion genome of Chinese giant salamander were analyzed using molecular biology methods in this paper. To know more about the genetic characteristics of Chinese giant salamander, study on three isoenzymes (LDH, EST, SOD) in six tissues was conducted, which could serve as genetic markers of biochemical characteristics. At the same time, the development of LDH was also discussed in the population of amphibians.
Based on analysis and comparison of complete sequences of mitochondrion genome derived from Genebank of Caudata and some typical vertebrates, phylogenetic development of Chinese giant salamander was investigated by means of molecular biology experimental methods, under the guidance of cladistics and molecular evolution biology. In addition, phylogenetic tree was constructed to elaborate the issue of species classification, and inquire further into the origin of Chinese giant salamander and its evolutionary status in amphibians.
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