中国科学院昆明动物研究所机构知识库

物种多样性及其格局的形成是生物学研究中关注的科学问题，不仅具有重要的理论价值，也有实际的保护意义。中国南方与东南亚地区一衣带水，山水相连，有着丰富且特殊的生物多样性，但该区域的多样性演化研究和保护工作相对滞后。角蟾科是中国南方与东南亚地区的特有类群，在两栖类进化中占据重要地位，被认为是始蛙亚目与新蛙亚目的重要过渡类群之一。角蟾科是原始无尾两栖动物中分化最多的科，形态性状多样性丰富，该科在生物学的诸多领域受到广泛关注，是验证进化假说的理想材料。然而，由于分布广，多样性高，因此，一个全面的物种多样性评估及分子系统发育研究极富挑战，尚存在很多争论有待澄清。其中，角蟾属和掌突蟾属是角蟾科中最为复杂，并且亟待调查研究的两大类群。首先，本研究着眼于角蟾属，通过多基因数据的联合分析和广泛采样，第一次较为全面地构建了角蟾属及其近缘属物种的系统演化。前期的研究主要受到样本量和信息量的限制，存在诸多问题有待解决。首先，关于角蟾属的高阶元分类存在很大争议，争论的焦点是：异角蟾属，无耳蟾属和婆罗蟾属是有效的属，还是角蟾属的同物异名。另外，角蟾属的物种多样性是怎样的，地理分布模式有什么特点，缺乏整体的评估。基于此，在本研究中，我们获得了角蟾属57个已知物种中的45个，采样范围囊括中国南方，东南亚和喜马拉雅山地区。系统发育分析结果揭示角蟾属包含多个主要进化支系，这些支系具有特定的地理结构。结果表明线粒体系统树和核基因系统树存在显著的冲突，冲突的位置在基部。分歧时间推算分析显示，角蟾属主要支系的分化时间与该地区的古地质气候事件吻合；角蟾属的分化过程反应了一些重要的生物地理事件。比如，克拉地峡和中国二三级阶梯的隔离分化作用。所有分析都显示角蟾属是个并系，拟角蟾属，短腿蟾属和婆罗蟾属嵌入到角蟾属的内部。结合系统演化关系，形态差异和地理分布特征，本研究对角蟾属的高阶元分类进行了修订：角蟾属被分为3个属，即从角蟾属中恢复了无耳蟾属和异角蟾属，真正的角蟾属局限分布在巽他古陆。尽管如此，我们也提供了另一种分类选项。本研究还揭示：婆罗蟾属无效，是角蟾属的同物异名。另外，基于分子的物种鉴定结果揭示在角蟾中存在大量未被描述的物种（20个潜在新物种）。现在认知的广布种，比如，白颌异角蟾，其实是多个不同物种的组合。通过增加样本量，形态证据支持了基于分子的物种多样性评估。山区森林的动态历史驱动了角蟾的快速分化。（第二章）接着，为了进一步了解中国南方和东南亚地区特有类群的多样性格局和维持机制，本研究以掌突蟾属为研究对象，通过在中国南方，中南半岛和巽他古陆的密集采样，利用多基因数据的联合分析，构建了该类群的演化历史。首先，掌突蟾属特定栖息于中国南方和东南亚的山区森林，是验证生物地理学假说的理想材料。此外，关于掌突蟾属的物种多样性，以及掌突蟾属与角蟾科其他属的系统发育关系还不清楚，缺乏整合研究。基于此，在本研究中，我们获得了掌突蟾属53个已知物种中的48个。基于分子的物种鉴定结果揭示掌突蟾属存在16个未定种，集中分布在中国南方和中南半岛。系统发育分析很好地解决了掌突蟾属与近缘属的亲缘关系，结果显示掌突蟾属不是单系，小臂蟾属嵌入其内部。掌突蟾属的分化模式复杂，涉及大量同域分布和狭域分布的物种。婆罗洲和越南南部是掌突蟾属物种多样性演化的关键区域；新生代的气候变化在掌突蟾属的起源和快速扩张中起到了驱动作用。生物地理学分析未能拒绝“上游殖民”假说，结果显示掌突蟾属起源于东南亚海岛，然后扩散到亚洲大陆，这与传统的观点不同。本研究表明扩散和隔离作用都影响了掌突蟾的多样性分布格局。另外，结合系统演化关系，形态差异和地理分布特征，我们的结果拒绝了掌突蟾属的亚属划分观点，而且，掌突蟾属应是小臂蟾属的同物异名。（第三章）最后，为了深入了解在角蟾中物种多样性的真实情况以及驱动力，本研究通过整合DNA条形码，形态，生态和基因组证据，对在中国东南分布的异角蟾属支系进行了探讨。这一支系分布局限，形态相对保守，但却有着高的隐存多样性（第二章结果）。基于整合分析，结果表明，该类群起源于约16 Ma，该时间段对应中新世中期气候适宜期。本研究使用了不同的物种鉴定策略。首先，基于DNA条形码数据的物种鉴定方法（贝叶斯基因树，ABGD和bPTP）揭示存在5个未定种。然后，基于GBS数据，BPP的物种鉴定分析支持DNA条形码的结果；Structure分析结果显示未定种与姐妹群之间已经没有基因流。另外，PCA分析发现未定种与姐妹群在形态量度上几乎没有重叠。再者，通过调查发现，角蟾通过多样的分化策略（比如：海拔差异和繁殖时间差异）实现快速分化和共存。因此，整合分类学分析支持DNA条形码的结果，证实了角蟾中高的物种多样性，角蟾低的扩散能力和山区丰富的生态位为该类群的快速分化提供了可能。在本研究中揭示出大量地方特有种，这对于处于快速发展的中国东南地区的多样性保护提供了有益参考。总之，DNA条形码的快速性，基因组数据的精准性以及整合分类学的全面性必将把生物多样性研究推向新的高度。（第四章） 

Understanding the biodiversity and the process and factor that generate its pattern in space and time is important in biology and conservation. Southeast Asia and southern China (SEA-SC) harbor a highly diverse and endemically important flora and fauna that is under increasing threat. An understanding of the property, history and drivers of this diversity is lacking, especially in some of the most diverse and threatened groups. Asian toads of the family Megophryidae Bonaparte, 1850 occur throughout SEA-SC and is a transitional group between archaic and advanced frogs. Its species exhibit much diversity, such as morphology and life history, making the family an ideal study group to test evolutionary hypotheses. However, the wide distribution and high species richness present a challenge when attempting a comprehensive assessment of the diversity, phylogeny and biogeography of this group. Much uncertainty and hypothesis need investigation. Among the species, Megophrys sensu lato and Leptolalax are the two most intractable groups.Firstly, we make the first effort to obtain a comprehensive phylogeny of Asian horned toads, Megophrys sensu lato, and its related allies, using broad sampling and a multilocus gene approach. Megophrys currently includes three groups previously recognized as the genera: Atympanophrys, Xenophrys and Megophrys sensu stricto. The taxonomic status and species composition of the three groups remains controversial due to conflicting phenotypic analyses and insufficient phylogenetic reconstructions; likewise, the position of the monotypic Borneophrys remains uncertain with respect to the horned toads. Further, the diversity of the horned toads themselves remains poorly understood, especially for widespread species. Herein, we evaluate species-level diversity based on 45 of the 57 described species from throughout southern China, Southeast Asia and the Himalayas using Bayesian inference trees and the Generalized Mixed Yule Coalescent (GMYC) approach. We estimate the phylogeny using both mitochondrial and nuclear DNA sequences. Multiple mutually exclusive, geographically cohesive major clades exist in Megophrys sensu lato. Analyses reveal statistically significant mito-nuclear discordance, which occur at some basal nodes. The time-calibrated phylogeny shows that the paleoclimatic changes and tectonic movements seem to have driven the major divergence of Megophrys sensu lato, and the geographic separation among clades reflects several important biogeographic barriers. For example, the Isthmus of Kra acts as a barrier for biodiversity exchange. All analyses resolve paraphyly for horned toads as the genus Ophryophryne, Brachytarsophrys and Borneophrys are embedded in it. We resurrect the genera Atympanophrys and Xenophrys from the synonymy of Megophrys to eliminate paraphyly of Megophrys sensu lato and to account for the morphological, molecular and biogeographic differences among these groups. Notwithstanding, We also provide an alternative option. My study suggests that Borneophrys is junior synonym of Megophrys sensu stricto. In addition, The species delimitation methods indicate the presence of 20 putative undescribed species. Widespread species, such as Xenophrys major, likely have dramatically underestimated species diversity. By increasing sampling, the integration of morphological and molecular evidence validate our preliminary discovery and find some new species. Montane forest dynamics appear to play a significant role in driving diversification of horned toads.Secondly, to understand the role past histories played in creating the biodiversity pattern of endemic lineages in SEA-SC, we reconstruct the evolutionary history of the Asian leaf-litter frog, Leptolalax Dubois 1980, using a multilocus approach and samples from southern China, Indochina and Sundaland. Leptolalax includes forest-dependent species that occur throughout SEA-SC, making it an ideal study group to examine specific biogeographic hypotheses. In addition, the diversity of this genus remains poorly understood, and the phylogenetic relationships among species of Leptolalax and closely related Leptobrachella Smith 1928 remain unclear. Herein, we evaluate species-level diversity based on 48 of the 53 described species from throughout the distribution of Leptolalax. Molecular analyses reveal 16 undescribed species, mostly in southern China and Indochina. Our well-resolved phylogeny based on multiple nuclear DNA markers shows that Leptolalax is not monophyletic with respect to Leptobrachella. The diversification pattern of the group is complex, involving a high degree of sympatry and prevalence of microendemic species. Northern Sundaland (Borneo) and eastern Indochina (Vietnam) appear to have played pivotal roles as geographical centers of diversification, and paleoclimatic changes and tectonic movements seem to have driven the major divergence of clades. Analyses fail to reject an “upstream” colonization hypothesis, and, thus, the genus appears to have originated in Sundaland and then colonized mainland Asia. This is contrary to the traditional view. My results reveal that both vicariance and dispersal are responsible for current distribution patterns in the genus. In addition, our analysis rejects monophyly of the two subgenera of Leptolalax. Based on morphological similarity, phylogenetic cohesiveness and geographic continuity, we assign Leptolalax Dubois 1980 to being a junior synonym of Leptobrachella Smith 1928 owing to priority of publication.Lastly, we explore species-level diversity and its drivers in horned toads by integrating DNA barcoding, morphological, ecological and genomic data. We take the horned toad clade Xenophrys, which is restricted to the mountains of Southeast China, a example. These species have relatively similar appearance and high cryptic diversity (Chapter two result). My results suggest that the group originated at about 16 Ma, which coincides with the Mid-Miocene temperature optimum. We perform multiple species delimitation strategies. At first, species delimitation methods (Bayesian inference, ABGD and bPTP) using DNA barcoding data detect five putative undescribed species. STUCTURE across GBS data suggest significantly reduced gene flow, and the multispecies coalescent approach BPP using multiple loci provides robust evidence for the validation of all undescribed and described species suggested by DNA barcoding. A principal component analysis (PCA) of morphological data revels morphological differences for sister-species. The horned toads appear to have evolved multiple ways, given the fine-scale pattern of geographic and ecological diversity, such as co-occurrence on mountains but with separation by elevation or differences in their reproduction periods. The low dispersal ability of horned toads and rugged montane terrain raise the possibility of localized vicariance and additional unknown species. In conclusion, my integrative taxonomy confirms the existence of many cryptic and microendemic species of Xenophrys, which has important implication for conservation in the rapid changing Southeast China. It highlights the utility of DNA high-speed barcoding, the accuracy of genomic data and the power of integrative taxonomy to discern and document biodiversity.