Investigations into the perplexing interrelationship of the Genus Takifugu Abe, 1949 (Tetraodontiformes, Tetraodontidae)

The phylogenetic relationships within the genus Takifugu Abe, 1949 (Tetraodontiformes, Tetraodontidae) remain unresolved. Because of the use of Takifugu as model organisms, the resolution of these relationships is crucial for the interpretation of evolutionary trends in biology. Pufferfishes of this genus are comprised of a comparatively small number of species and are mainly distributed along the coastal region of the western part of the Sea of Japan and the coastline of China. Mitochondrial gene sequences were employed to test the phylogenetic hypotheses within the genus. Seventeen species of the genus were examined. Molecular phylogenetic trees were constructed using the maximum parsimony, neighbor-joining, maximum likelihood and Bayesian methods. Our hypothesis of internal relationships within the genus differs from previous hypotheses. Our results indicate that (1) the genus Takifugu is a monophyletic assemblage; (2) the genus is divided into 6 subgroups based on the molecular data; and (3) there is low genetic diversity among the species within this genus. In addition, speciation within Takifugu appears to be driven by hybridization and isolation by distribution. Our results also suggested that the taxonomy in the genus should be clarified based on both molecular and morphological data.

铃蟾属(Bombina)的分子系统学研究

本研究采用直接测序的方法，研究了铃蟾属（两栖纲：无尾目：铃蟾科）的 分子系统发育关系及系统学问题。我们测定了我国铃蟾属5 个种共22 个样本的 线粒体DNA 部分片段的序列，包括12S rRNA， 16S rRNA， ND4-tRNALEU 以 及细胞色素b 共四个基因片段，欧洲铃蟾、花铃蟾以及B. pachypus 的相应序列 通过GenBank 获得。利用MrBayes 3.0b4 和 PAUP* 4.0b10 软件对系统发育关系 进行了重建（贝叶斯推断法，最大简约法，最大似然法和邻近法），分析结果表 明，铃蟾属可分为两大支系，一支包括利川铃蟾及中国西南部铃蟾（微蹼铃蟾、 强婚刺铃蟾、大蹼铃蟾）；另一支包括东方铃蟾，花铃蟾、欧洲铃蟾以及Bombina pachypus，从分子水平上支持了铃蟾属内不同亚属的划分。我们的结果拒绝东方 铃蟾起源于大蹼铃蟾的假设，同时表明：相对于东方铃蟾，欧洲铃蟾与花铃蟾间 的关系更近。利川铃蟾的有效地位得到支持，中国西南部3 种铃蟾 (微蹼铃蟾、 强婚刺铃蟾、大蹼铃蟾)在所有系统树中形成一个单系，利川铃蟾与之形成姐妹 群。根据我国西南部3 种铃蟾间的很小的遗传差异，以及以往的形态学研究和核 型研究的证据，我们认为将微蹼铃蟾、强婚刺铃蟾归入大蹼铃蟾可能更为合适。 分化时间估计表明，铃蟾属两个亚属的分化发生在3.08–9.16 MYA，我们推测秦 岭的抬升以及同时期青藏高原的上升是铃蟾属内产生亚属分化的主要原因。

隆肛蛙属Feirana种群的系统学与动物地理学研究

对隆肛蛙属的物种构成进行了订正，建立新属肛刺蛙属Yerana gen. nov.；订正后的隆肛蛙属现仅隶2种， 即隆肛蛙F. quadrana和太行隆肛蛙F. taihangnicus。运用形态学分析探讨了隆肛蛙属物种及种群的形态差异和分类关系，通过分子系统学研究探讨了隆肛蛙属物种及种群的分类和系统发育关系，运用动物地理学方法结合系统发育关系探讨了隆肛蛙属种群的地理分布格局成因与历史过程。主要结果和推论如下： 1．隆肛蛙属物种构成的订正及一新属建立 建立新属肛刺蛙属，将隆肛蛙属中的原叶氏隆肛蛙F. yei归隶新属肛刺蛙属并更名为叶氏肛刺蛙Y. yei，，新属建立的主要依据为：（1）雄性肛部隆起，肛孔下方有两个布满黑刺的大的白色球形隆起，具单咽下内声囊， 第一指具婚刺；（2）形态量度分析表明叶氏肛刺蛙与隆肛蛙和太行隆肛蛙的形态差异远大于后两者之间的差异；（3）叶氏肛刺蛙的分布区与隆肛蛙和太行隆肛蛙的分布区距离较远且呈隔离状态；（4）分子系统学研究资料（Jiang et al.,2005）证明叶氏肛刺蛙与隆肛蛙和太行隆肛蛙非单系发生；叶氏肛刺蛙在第二支中位于基部。因此，隆肛蛙属现仅隶2种，即隆肛蛙和太行隆肛蛙。 2．隆肛蛙属种群形态学研究 对隆肛蛙属中隆肛蛙和太行隆肛蛙的15个地理种群565只标本的28项形态性状进行了测量，运用典型判别分析法对其分析的结果表明：（1）太行隆肛蛙与隆肛蛙形态差异明显，支持其为不同的物种；（2）原隆肛蛙河南伏牛山种群和山西中条山种群应为太行隆肛蛙的地理种群；（3）隆肛蛙不同地理种群之间形态差异明显，其中四川安县种群、陕西周至种群和湖北利川种群与模式产地重庆巫山种群的差异可能达到了亚种或亚种以上分化水平。对隆肛蛙属量度分析的15个种群进行定性形态分析表明其分为三种形态型，对应隆肛蛙、过渡型和太行隆肛蛙，其变异特征主要为内跗褶、雄性肛部隆起及疣粒分布、第五趾外侧缘膜等，这与量度分析结果相似。 3．隆肛蛙属种群分子系统学研究 测定隆肛蛙属Feirana的2种19种群的线粒体12S rRNA和16S rRNA基因片段、ND2基因的DNA序列，比对后共计1953bps。（1）遗传多样性与距离分析：结果表明，隆肛蛙属种群具很高的遗传多样性，19个种群样品表现出19种单倍型（遗传多样性指数Hd＝1.0）； ND2基因的进化信息含量远高于12SrRNA和16SrRNA。隆肛蛙属2种群组内的种群间的遗传距离远小于两种群组间的距离，种群在不同基因上的遗传距离表现的关系与对应的系统树一致。（2）系统发育关系分析：结果表明，不同基因片断基于不同方法构建的隆肛蛙属种群系统发育树结构基本一致，基本表明隆肛蛙属种群为单系发生；它们在系统树中分为两大支，分别对应于隆肛蛙和太行隆肛蛙；支持中条山种群（沁水、历山和济源种群）和伏牛山种群（栾川和内乡种群）为太行隆肛蛙的地理种群，而原隆肛蛙秦岭中东段的部分种群（柞水、宁陕、长安大坝沟种群）也应为太行隆肛蛙的地理种群。（3）亚种分化分析：根据遗传距离分析和系统发育关系分析结果，并考虑形态上的差异情况以及地理分布信息，隆肛蛙所隶种群组可分为2亚种，即隆肛蛙指名亚种F. quadrana quadrana包括四川盆地东缘大巴山东段-巫山-武陵山北麓种群和秦岭中段（周至板房子和长安广货街）种群，他们在系统关系树上聚为一支；安县亚种F. quadrana anxianensis包括四川盆地西缘岷山东麓-龙门山-大巴山和秦岭西段的种群（安县、青川、文县、南江和凤县种群），他们在系统关系树上聚为一支。太行隆肛蛙所隶种群组也可分为2亚种，即太行隆肛蛙指名亚种F. taihangnicus taihangnicus包括中条山的种群（沁水、历山和济源种群）和中东秦岭的部分种群（柞水、长安大坝沟和宁陕种群），他们在系统关系树上聚为一支；太行隆肛蛙伏牛亚种F. taihangnicus funiuensis，为伏牛山地区的种群（栾川和内乡种群），他们在系统关系树上聚为一支。 4．隆肛蛙属种群动物地理学研究 隆肛蛙属19种群的分歧年代分析： 以长江巫山段和黄河三门峡段的形成历史时期为参考点，根据已测隆肛蛙属19种群及其外群包括N. pleski、P. yunnanesis、P. robertingeri、F. limnocharis的1953bps DNA序列构建分子钟，获得各支系的分歧年代。结果表明：①棘蛙族在70Ma左右开始其独立演化历程，这与Roelants et al.（2004）的分析结果～60±15Ma左右开始分化基本一致，后者印证了本文的分子钟。②隆肛蛙属的起始分化年代较早，隆肛蛙和太行隆肛蛙两种群组的最近祖先种群大概在46Ma～50Ma左右；隆肛蛙和太行隆肛蛙种群组内的种群分化年代相对两种群组间晚得多， 隆肛蛙种群组内两亚种分化起始年代约为10Ma左右，而太行隆肛蛙种群组内两亚种分化起始年代约为6Ma。 隆肛蛙属种群分布格局形成过程分析： ①隆肛蛙属的系统关系与地理分布格局密切相关，大部分系统分支分级与地理距离成正比；②隆肛蛙属最近祖先种群的分化中心可能位于秦岭中部地区， 隆肛蛙属的种群分布格局的形成表现为隔离分化与扩散相结合的机制，由隔离分化产生的隆肛蛙祖先种群主要从秦岭中部向西南方向扩散，后隔离分化为两亚种；太行隆肛蛙祖先种群向东北方向扩散也分化为两亚种。 隆肛蛙属种群分布区域地质历史的探讨：本文所建分子钟和种群分化方式印证了该区域的几次主要地质事件，包括岷山-龙门山-西秦岭等地区的快速差异隆起、第四纪冰期等。 The specific composition of the genus Feirana should be revised. A new genus Yerana gen. nov.（Ranidae：Dicroglossinae）was established based on morphological data-set and molecular phylogeny, as a result, only two species F. quadrana and F. taihangnicus are classified into Feirana now. Morphological differences and taxonomy of populations of Feirana were investigated based on morphological and morphometric data; phylogenetic relationships and taxonomy of populations of Feirana were elucidated using molecular data, and then the proceeding of the distribution pattern of populations of Feirana were discussed. The main results and conclusions and proposals were presented as following: 1. Revising of the specific composition of the genus Feirana and establishment of a new genus The new genus Yerana, only containing the type species Y. yei, was established based on the following evidences: (1) In adult male, distinct up-heaved circular vesicle presents around the anal, and under anal there are two white balls on which black spines exist, black horny spines scatter on the upper side of first finger, and internal single subgular vocal sac presents; (2) there is obvious morphometric differences between Yerana and Feirana; (3) Yerana is distributed far from Feirana; (4) evidences of molecular phylogeny（Jiang et al.,2005）suggested that Yerana take a special phylogenetic clade which is different from other genus included in the tribe Paini. As a result, there are only two species in Feirana, i.e., F. quadrana and F. taihangnicus. 2. Morphological research of populations of Feirana Twenty-eight characters of 565 individuals of 15 populations of the genus Feirana were measured, the results of Canonical Discriminant analysis of the morphometric data-set indicated that: (1) there are very prominent differences between the two species F. quadrana and F. taihangnicus. The validity of species F. taihangnicus was approved here; (2) Mt. Funiu population and Mt. Zhongtiao population should belong to the species F. taihangnicus; (3) Obvious differences exist among 12 populations of F. quadrana, the differentiation among Zhouzhi population, Anxian population, Lichuan population, and Wushan population together with the others probably reach sub-specific or specific level. Result of morphological comparison between 15 different populations show that 3 morphological types are recogenized in according with F. quadrana, F. taihangnicus and intergradation, this result conform to the result of morphometric analysis. 3. Molecular phylogenetic study on populaions of Feirana Fragment of 12SrRNA and 16SrRNA genes, and ND2 gene of 19 populations of two species of Feirana were sequenced and aligned, from which 1953 bps were received. (1) analyses of genetic distance and hereditary diversity indicated that: genetic distance between populations in each group were less than distance between two groups of Feirana, 19 haplotypes were recognized from 19 samples of 19 populations, so the hereditary diversity of populations of Feirana was very high (Hd=1.0), phylogenetic information in ND2 gene is more than fragment sequence of 12SrRNA and 16SrRNA genes. (2) Result of molecular phylogeny indicate that the phylogenetic trees constructed using different methods based on different sequence data sets showed the revised genus Feirana is monophyletic since the 19 populations of Feirana were firstly clustered together as one large clade, which was further clustered into two major clades, corresponding to F. quadrana(GroupⅠ) and F. taihangnicus(GroupⅡ), respectively. So populations of Qinshui and Lishan in Mt. Zhongtiao, populations of Luanchuan and Neixiang in Mt. Funiu, and populations of Zhashui, Dabagou of Chang’an and Ningshan in eastern Mt. Qinling should belong to the species F. taihangnicus; (3) Subspecific differentiation. on the basis of genetic distance, phylogenetic trees and geographical distribution, F. quadrana should have two subspecies, i.e., F. quadrana qudadrana, consisting of the populations Guanghuojie of Chang’an and Zhouzhi in Mid-Mt. Qinling, populations in Wushan area and northern Mt. Wuling (Lichuan), and F. qudadrana anxianensis, consisting of the populations in eastern Mt. Ming shan-Mt. Longmen-western Mt. Daba-western Mt. Qinling (Anxian, Qingchuan, Wenxian, Nanjiang and Fengxian); F. taihangnicus should also has two subspecies, i.e., F. taihangnicus taihangnicus, consisting of the populations in Mt. Zhongtiao and eastern Mt. Qinling, and F. taihangnicus funiuensis, consisting of the populations in Mt. Funiu. 4. Zoogeography of populaions of Feirana Analysis for divergent time of 19 populations of Feirana: Using the dates of run-through of Wushan segment of Changjiang River as the time when the population of Lichuan started differentiated from the populations of Wushan and Shennongjia, and the dates of Sanmenxia segment of Yellow River as the time when the populations in Mt. Zhongtiao started differentiated from the population of Dabagou in Chang’an, molecular clock was established using sequences with 1953 bps of 19 populations of Feirana and outgroup including N. pleski, P. yunnanesis, P. robertingeri, F. limnocharis in order to estimate divergent time of all clades. Result of that indicated that: ① the tribe Paini started to evolve independently at about 70Ma when is in consistent with that estimated by Roelants et al.（2004）with result of about ～60±15Ma, they were corroborated by each other, this confirms the validity of this molecular clock; ② divergent time for speciation of Feriana is early, ancestral populations of F. quadrana and F. taihangnicus were found about 46Ma～50Ma; differentiation of populations within species is greatly late to the divergence of the two species, divergent time for F. quadrana is 10Ma and divergent time for F. taihangnicus is 6Ma. Proceeding of distribution pattern of Feirana. Phylogenetic relationships of populations of Feirana matched quite with distribution pattern of them, the relationships among clades showed in phylogenetic trees is direct ratio to geographical distance of them; the estimated date of speciation between two species of Feirana was as early as speciation of Paa yunnanesis and Nanara pleski; middle part of Mt. Qinling is the center of speciation of Feirana, combination of mult-events of dispersal and vicariance are probably the mechanism of speciation of Feirana, F. quadrana colonized the mid-Mt. Qinling and then differentiated into two subspecies in southwest direction, ancestral population of F. taihangnicus colonized the mid-Mt. Qinling and then differentiated into two subspecies in northeast direction. On geological history of the distribution of Feirana. According to molecular clock and speciation model of populations of Feirana, some geological events are confirmed, including special rise of Mt. Minshan- Mt. Longmen-western Mt. Qinling, glacial age.

角蟾科（Megophryidae; Anura）蝌蚪的形态多样性和生态适应

角蟾科（Megophryidae）是以角蟾属（Megophrys Kuhl and Van Hasselt, 1822）为模式属而建立的，隶于无尾目（Anura），变凹型亚目（Anomocoela）。角蟾科包括2 亚科11 属142 种，分布于东洋界，从巴基斯坦、中国西部向东直到菲律宾和苏达群岛；中国有9 属75 种分布于华中和华南地区。角蟾科被认为是原始的两栖动物之一，其分类学、系统学、生态学、动物地理学的研究均深受中外科学家的瞩目。近年来，通过形态学、古生物学、细胞学、生态学、支序系统学的研究，角蟾科的分类与系统学研究取得了较大进展。与成体形态和分子系统学研究结果相比较，蝌蚪的研究存在更多的问题和挑战，尚需深入研究：（1）角蟾科蝌蚪的形态多样性分析；（2）角蟾科的系统发育关系与蝌蚪的演化，以及口漏斗的起源；（3）角蟾科蝌蚪表型分化与栖息环境和觅食行为的适应演化。针对上述问题，本文对角蟾科9 属30 种蝌蚪的形态特征，包括外部宏观形态和口器外部结构特征、口器内部显微结构、唇齿和角质颌的亚显微结构作了深入细致、多层次的比较研究；通过12s rRNA 和cytochrome b 基因构建最大简约树，采用贝叶斯系统发育进行分析，蝌蚪型的演化采用祖先性状的重建方法分析；得到如下结论：1）初步将角蟾科蝌蚪分为4 种类型；并且建立了2 种新的角蟾科蝌蚪类型。A 型：拟髭蟾型蝌蚪，该型蝌蚪包括拟髭蟾属、髭蟾属、齿蟾属和齿突蟾属的物种；B 型：新类型，掌突蟾型蝌蚪，该型蝌蚪在本文中包括掌突蟾属、小臂蟾属的物种；C 型：新类型，短腿蟾型蝌蚪，一种特化类型，该型蝌蚪在本文中仅包括短腿蟾属的物种；D 型：角蟾型蝌蚪，该型蝌蚪在本文中包括无耳蟾属、小口拟角蟾属和异角蟾属的物种。2）对角蟾科的分类进行了修订：（1）支持角蟾科两个亚科的分类系统；（2）角蟾亚科包括拟角蟾属、异角蟾属、无耳蟾属和短腿蟾属；该亚科形态差异小，系统学关系比较复杂，暂不作族级分类的再划分；（3）拟髭蟾亚科分为2 个族：拟髭蟾族，该族物种具有类型A 的蝌蚪，包括4 个属：拟髭蟾属、髭蟾属、齿蟾属、齿突蟾属；掌突蟾族，该族物种具有类型B 的蝌蚪，包括2 个属：掌突蟾属和小臂蟾属。3）结合分子系统进化关系探讨了4 种蝌蚪类型的演化。（1）角蟾科蝌蚪的最近共同祖先来自于一类具有拟髭蟾型蝌蚪性状的蝌蚪；（2）掌突蟾型蝌蚪和角蟾亚科的蝌蚪是由具有拟髭蟾型蝌蚪性状的祖先蝌蚪分别演化而来；（3）短腿蟾型蝌蚪是角蟾型蝌蚪的一种特化类型；（4）外群蝌蚪具有与拟髭蟾型蝌蚪相似的性状，进一步印证了类拟髭蟾型蝌蚪是角蟾科蝌蚪的最近共同祖先的假说；（5）具有口漏斗的蝌蚪类型是由不具口漏斗的蝌蚪类型演化而来，在角蟾科中口漏斗是一种衍生性状。4）分析了角蟾科四种蝌蚪类型与栖息环境的适应演化。（1）角蟾科蝌蚪的口部和体形的变化反映了该科蝌蚪由缓流向类似静水生境的回水凼的渐变式适应，角蟾科蝌蚪的形态显示了多方面的适应变化；（2）随着蝌蚪类型由A 向D的演化，当水速较大时，拟髭蟾型的蝌蚪营流水攀吸型生活方式；当水速递减时，掌突蟾型蝌蚪营流水附着型生活方式；当水速进一步递减时，具有较小口漏斗的短腿蟾型蝌蚪和具有大漏斗的角蟾型蝌蚪营流水浮泳型生活。角蟾科蝌蚪对于水流递减的适应演化说明蝌蚪的生态学适应是具有进化意义的；（3）蝌蚪口器内部结构的分化揭示了蝌蚪和食性的适应关系，蝌蚪以口部的唇齿与角质颌刮取或吞吸水中的物质，然后，通过口乳突有选择地过滤进入口腔中食物。拟髭蟾亚科蝌蚪的唇齿多而窄，唇齿间距宽，颌鞘粗而稀，反映了其植食性为主的特点；它们的舌前乳突一般为指状，在口腔入口处所占面积小，其机械过滤的作用很多被唇齿和角质颌分担了；而角蟾亚科的蝌蚪，其角质颌弱，其舌前乳突一般为匙状，几乎填满了口腔入口处，因此舌前乳突起了主要的机械过滤作用。The family Megophryidae is the largest and most diverse families inArchaeobatrachia, and most of its species occur in India, Pakistan, and eastward intoChina, Southeast Asia, Borneo and the Philippines to the Sunda Islands. Currently thefamily includes 142 species have been grouped into two subfamilies, Megophryinaeand Leptobrachiinae. The mountains of central and southern China are rich in speciesof Megophryidae, 75 species belong to 9 genera and two subfamilies.The family was supposed to be ideal materials of studies in many fields of biology,such as taxonomy, evolution, systematics, ecology, and biogeography. Recently, therehave a great development in taxonomy and systematics of megophryids throughstudied by morphology, paleontology, cytology, ecology, and cladistics. However,larvae of megophryids were generally unknown, although the tadpoles might be veryimportant for above studies.In this paper, we examined the evolutionary scenario of the tadpoles’ morphologyin the context of a phylogenetic framework. Our objectives are (1) to evaluate thedivergence of larval body shape and oral discs in the family Megophryidae, (2) toexplore the evolutionary trends of the larvae in megophryidae, and test if thefunnel-shaped oral disc is apomorphic, and (3) to explore the relationship of the larvalstructure, diet and microhabitat.We examined larval morphology of 30 megophryid species, the larval body shape,oral discs, the buccopharyngeal cavity, and jaw sheaths and denticles of the Chinesemegophryid frogs were re-examined. We constructed a phylogeny of the species on thebasis of published mitochondrial cytochrome b and 16S rRNA gene segments usingpartitioned Bayesian analyses. Furthermore, hypothetical changes of larval morphologywere inferred using parsimony principle on the phylogeny. The results showed that:1) Four tadpole types in Megophryidae. The larval morphological charactersseries in Chinese megophryids fall into four general categories according to the bodyshape and oral discs: (A) Leptobrachiini type, species from genera Leptobrachium,Oreolalax, Scutiger and, Vibrissaphora share this type of tadpoles. (B) Leptolalax type,species of genus Leptolalax have this type of tadpoles. (C) Brachytarsophrys type,species of the genus Brachytarsophrys have this type of tadpoles. (D) Megophryinitype, species of the genera Atympanophrys, Ophryophryne, and Xenophrys share this type of tadpoles. Of which B and C are two novel types.2）Taxonomic implications. The present study leads us to reconsider the generalclassification of tribes attributed to members of Megophryidae. More specifically,concerning the phylogenetic relationships and the two novel tadpole types describedherein, we propose a provisional taxonomy for the family but suggest that further taxasampling of other megophryids be performed to confirm this taxonomic change. TheMegophryidae is composed of two subfamilies (Leptobrachiinae and Megophryinae).The Leptobrachiinae was recogonized the two tribes: (1) tribe Leptobrachiini sensuDubois, corresponding to the tadpole of type A, including four genera, i.e.,Leptobrachium, Oreolalax, Scutiger and, Vibrissaphora; (2) tribe Leptolalaxini,corresponding to the tadpole of novel type B, including two genera, i.e., Leptolalaxand Leptobrachella. However, the relationships among the genera of Megophryinaewere largely unresolved, they recognized no monophyletic groups above the generalevel. A more thorough sampling will likely foster a better taxonomic solution.3) The larval evolutionary scenario in Megophryidae.Type A is characteristicof normal-mouthed with multiple tooth rows, representing the tadpole type of theMRCA of Chinese megophryids. Type B is characteristic of normal-mouthed withreduced tooth rows, prolonging labium, and integumetary glands. Type C ischaracteristic of no labial teeth and smaller umbeliform oral disc. Type D ischaracteristic of no labial teeth, enlarged umbeliform oral disc, representing the tadpoleof the MRCA of subfamily Megophryinae. A previous hypothesis, referring tofunnel-shaped oral discs as an apomorphy, is supported.4) The larval adaptation to habitats in Megophryidae. Tadpoles generallyadhere to substrates using their mouths, and the microhabitat that the tadpoles occupyreflects the degree of adhesion and oral complexity. The morphological changes inmegophryid tadpoles virtually allow a progressive adaptation to a changing habitatfrom faster water to slower water. Within the tadpoles of Type A to type D, the TOTbecomes smaller and smaller, and the oral disc orientates from anteroventral toumbelliform upturned, and eye position orientates from dorsal to lateral, and the trunkis more and more depressed and tail becomes relatively longer and slender. Within therunning water, the normal-mouthed with multiple tooth rows of Leptobrachiini tadpoles are correlated with lotic-suctorial, benthic feeders with anteroventral oraldisc and the largest body. With the water’s velocity decreasing, the lotic-adherentfeeders of Leptolalax tadpoles have tube-shaped labium with reduced tooth rows andintegumetary glands. And then, the smaller umbeliform in Brachytarsophrys tadpolesand the enlarged umbeliform oral disc in the Megophryini tadpoles are inhabitmicrohabitats of non-flowing backwaters of rivers, indicative of adaptive traits oflotic-neustonic surface feeders. The scheme of megophryid tadpoles andmicrohabitats provided the first clear evidence which congruent with the hypothesis ofAltig and Johnston (1989). The ecological divergence plays a general role in thedivergence and evolution of megophrid larvae. There is a definite correlation amongthe buccopharyngeal cavity, diet and feeding mechanisms, the tadpole graze orswallow the food particles, then through papillae which like a sieve and sort out foodparticles to the oesophagus. The tadpole of Leptobrachiinae possess multiple toothrows, wide intertooth distance as well as thick and sparse jaw sheath, these tadpolesinhabit bottom of the streams and graze on epiphyton or major detritus of organicmatter on the substrates, their prelingual papillae like single finger, the mechanicalpurpose of papillae served share in by tooth and jaw. The tadpoles of Megophryinaeoccur near the water surface of small streams and are the filter feeder, their dietincludes plankton and organic debris floating on the water surface, those tadpolepossess weak jaw, their prelingual papillae like spoon, the mechanical purpose ofpapillae served mostly for sieve.

青蟹属的系统进化和中国沿海拟穴青蟹的群体遗传结构研究

为了进一步研究青蟹属系统进化的科学问题，并揭示我国东南沿海青蟹群体遗传结构和群体进化细节信息，本论文主要开展了以下两个方面的研究：（1）基于线粒体12S rRNA、16S rRNA和COI三种基因序列探讨中国东南沿海青蟹的种类归属与青蟹属的系统进化；（2）利用线粒体COI基因标记分析中国东南沿海拟穴青蟹的群体遗传结构。序列特征、遗传距离和系统进化分析结果都表明本文研究的青蟹均为S. paramamosain。NJ、BAYES和ML系统进化树显示S. paramamosain与S. tranquebarica互为姐妹种，S. olivecea应该是4种青蟹中最早分化出来的种类。10个地理群体130只拟穴青蟹的线粒体DNA（mitochondrial DNA，mtDNA）细胞色素氧化酶亚基I（COI）基因序列Mantel检验结果显示群体间的遗传分化程度与地理距离没有显著的相关性。分子进化中性检验结果表明自然选择在分子进化过程中起了重要作用，并暗示该物种在最近经历了一个快速的群体爆发及扩张事件。

Els límits i les relacions entre els peixos acanthopterygii: filogènia molecular de mugilomorpha i atherinomorpha

Sovint, la sistemàtica, basada principalment en caràcters morfològics, no es correspon amb els processos evolutius relacionats amb l'aparició dels grups d'organismes. En l'actualitat, la utilització de les dades moleculars es fa indispensable per a una revisió i millora de la classificació biològica de diversos organismes, com els peixos Acanthopterygii. A la sèrie Mugilomorpha la incongruència entre la taxonomia i la filogènia sorgeix de l'elevada semblança morfològica trobada per part dels seus membres. Pel que fa referència a la sèrie Atherinomorpha, la problemàtica principal resideix en determinar la seva proximitat evolutiva respecte a la sèrie anterior i en establir les relacions filogenètiques dins de la mateixa. Per tant, s'hi ha volgut estimar tant la divergència genètica dins de cada sèrie com inferir les relacions filogenètiques entre ambdues mitjançant la seqüenciació directa del DNA de les regions mitocondrials corresponents al tRNA-Phe, 12S rRNA, COI, cytb, tRNA-Thr, tRNA-Pro i regió control.

Filogeografia e sistemática molecular de répteis de alguns arquipélagos africanos do Atlântico oriental

Os répteis, nomeadamente os lagartos, lagartixas e osgas, constituem um dos grupos de vertebrados com maior sucesso de colonização das ilhas oceânicas. Juntamente com as aves, devem constituir o grupo que naturalmente melhor se disseminou pelas ilhas oceânicas. Os mamíferos e anfíbios que aí possam existir são na sua maioria de introdução antropogénica. Como são bons colonizadores constituem bons modelos para o estudo de fenómenos e padrões de colonização das ilhas sobretudo tendo em conta que possuem ainda baixa dispersão dentro de cada ilha. Neste trabalho utilizamos marcadores do DNA mitocondrial (12S rRNA, 16S rRNA, citocromo b), marcadores do DNA nuclear (c-mos e enolase) assim como marcadores enzimáticos, para estudar os padrões de colonização, as relações entre espécies, a detecção de espécies introduzidas, a importância dos dados moleculares em relação a outro tipo de dados, nos répteis terrestres dos Arquipélagos da Madeira, Selvagens e Cabo Verde, e ilhas do Golfo da Guiné (São Tomé, Príncipe e Annobon). As sequências de DNA quer mitocondrial quer nuclear permitiram revelar a existência de uma estrutura geográfica em Mabuya spp. de São Tomé (de natureza intraespecífica) e de Cabo Verde (interespecífica) bem como em Lacerta dugesii (intraespecífica) do Arquipélago da Madeira. Esta estrutura é mais evidente em Lacerta dugesii, que apresenta haplótipos típicos e exclusivos de cada um dos quatro grupos principais de ilhas (Madeira, Porto Santo, Desertas e Selvagens), sem que se tivessem observado haplótipos comuns a mais do que um grupo de ilhas. Os dados moleculares obtidos permitem ainda inferir os casos de expansões demográficas recentes como no caso das populações de Lacerta dugesii da Madeira e Porto Santo ou pelo contrário indicativas de subdivisão geográfica da população como no Arquipélago das Selvagens. Nesta espécie apenas terá ocorrido um evento de colonização, e os nossos dados não corroboram a possibilidade de introdução nas Ilhas Selvagens mediada pelo homem. Mabuya spp. de Cabo Verde também forma um grupo monofilético, subentendendo a exemplo de L.dugesii um evento de colonização mas bem mais antigo, dando origem a eventos de radiação evolutiva, tendo-se formado novas espécies que por sua vez terão sido actores na colonização entre ilhas. Usando como modelo os Arquipélagos das Canárias e Cabo Verde, o número de eventos de colonização é menor nos escincídeos do que nos geconídeos. As ilhas do Golfo da Guiné parecem introduzir uma excepção à regra. Assim Mabuya spp. do Golfo da Guiné (São Tomé, Príncipe e Annobon) serão resultantes de 4 eventos de colonização, sendo dois responsáveis pelo aparecimento de M. maculilabris (uma forma no Príncipe e outra em São Tomé), M. ozorii (Annobon) e M. affinis (Príncipe). A exemplo de Lacerta dugesii, Mabuya maculilabris apresenta uma forte estruturação geográfica. Fazendo recurso a sequências já publicadas no GenBank, podemos propor um novo arranjo taxonómico no género Mabuya, não se devendo considerar quatro grupos (sensu Mausfeld), mas sim cinco, em que se adiciona um novo grupo que contempla as espécies do Norte de África e Turquia. As osgas em Cabo Verde, a exemplo das Canárias, apresentam grande variabilidade e terão sido resultado de maior número de eventos de colonização do que os Escincídeos. A nossa análise revela que existem em Cabo Verde maior número de grupos geneticamente distintos do género Tarentola, do que havia sido registado anteriormente. Os Hemidactylus também devem ter sido resultantes de mais do que um evento de colonização: um para Hemidactylus bouvieri e um para Hemidactylus brooki da Ilha do Sal. Hemidactylus brooki existente nas restantes ilhas bem como Hemidactylus mabouia são muito provavelmente de introdução antropogénica. No Golfo da Guiné o número de eventos de colonização não é maior nas osgas do que nos Escincídeos, constituindo assim uma excepção à regra, sendo os Hemidactylus resultantes de pelo menos dois eventos de colonização (quatro em Mabuya). Utilizando Lacerta dugesii como modelo, não encontramos qualquer congruência entre dados enzimáticos, morfológicos e moleculares. Com a aplicação de técnicas moleculares foi possível identificar espécies introduzidas como Hemidactylus mabouia na Madeira, Cabo Verde, São Tomé e Príncipe e Annobon bem como Ramphotyphlops braminus em Annobon. Estas espécies caracterizam-se por serem geneticamente homogéneas. Foi ainda possível verificar o estatuto taxonómico das várias espécies. Em Lacerta dugesii as três subespécies não deverão ser omitidas. Em Mabuya de Cabo Verde dever-se–ão manter as espécies consideradas e as relações estabelecidas. Em Tarentola spp. uma nova subespécie de Tarentola gigas deverá ser considerada e alvo de novas investigações. Os restantes grupos obtidos, geneticamente distintos, são em maior número do que havia sido registado, e deverão ser alvo dum estudo exaustivo.Confirmou-se a presença duma Mabuya em Annobon, muito provavelmente Mabuya ozorii, espécie esquecida ou omitida em muitas listas de espécies como na “EMBL Reptile database”. Duas formas de M. maculilabris em São Tomé e Príncipe, deixam transparecer a possibilidade da existência dum complexo de espécies. A análise de dados moleculares permitiu também referir que M. maculilabris não parece ter sido introduzida pelo homem nestas ilhas. Do ponto de vista conservacionista é fundamental monitorizar as espécies introduzidas pois podem levar à extinção de espécies indígenas, e monitorizar a manutenção dos vários grupos geneticamente distintos encontrados, muitos deles com distribuições restritas. Por fim, ao testar o c-mos na filogenia de Lacerta dugesii, podemos dizer que este gene nuclear pode também ser utilizado sob determinadas condições, ao nível intraespecífico. A região controle do DNA mitocondrial revelou-se também adequada na estimativa das relações filogenéticas. Verificou-se que esta estrutura é em Lacerta dugesii, bem menos variável que o gene do citocromo b (também mitocondrial). Mostra ainda uma variação entre populações e apresenta aspectos curiosos relacionados com a sua estrutura no contexto do que é conhecido actualmente dentro dos vertebrados.

Molecular phylogeny of the armored catfish family Callichthyidae (Ostariophysi, Siluriformes)

The family Callichthyidae comprises eight genera of fishes widely distributed across the Neotropical region. In the present study, sequences of the mitochondrial genes 12S rRNA, 16S rRNA, ND4, tRNA(His), and tRNA(Scr) were obtained from 28 callichthyid specimens. The sample included 12 species of Corydoras, three species of Aspidoras, two species of Brochis, Dianema, Lepthoplosternum, and Megalechis, and two local populations of Callichthys and Hoplosternum. Sequences of Nematogenys inermis (Nematogenyidae), Trichomycterus areolatus, and Henonemus punctatus (Trichomycteridae), Astroblepus sp. (Astroblepidae), and Neopleeostomus paranensis, Delturus parahybae, and Hemipsilichthys nimius (Loricariidae) were included as the outgroup. Phylogenetic analyses were performed by using the methods of maximum parsimony and maximum likelihood. The results of almost all analyses were very similar. The family Callichthyidae is monophyletic and comprises two natural groups: the subfamilies Corydoradinae (Aspidoras, Brochis, and Corydoras) and Callichthyinae (Callichthys, Dianema, Hoplosternum, Lepthoplosternum, and Megalechis), as previously demonstrated by morphological studies. The relationships observed within these subfamilies are in several ways different from those previously proposed on the basis of morphological data. Molecular results were compared with the morphologic and cytogenetic data available on the family. (C) 2003 Elsevier B.V. All rights reserved.

Molecular systematics of the armored neotropical catfish subfamily Neoplecostominae (Siluriformes: Loricariidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Morphological and Molecular Variation in the Pumpkin Toadlet, Brachycephalus ephippium (Anura: Brachycephalidae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Análise das relações entre gêneros da subfamília neoplecostominae (Siluriformes: Loricariidae) com base em sequências de DNA

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Filogeografia das espécies de tainha, Mugil liza e M. platanus (Teleostei: Mugiliformes)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Análise filogenética da subfamília Callichthyinae (Teleostei: Siluriformes: Callichthyidae) com base em seqüências de DNA nuclear e mitocondrial

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

A phylogenetic analysis of Pseudopaludicola (Anura) providing evidence of progressive chromosome reduction

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)