DNA barcoding, genogeography, and phylogeny of bent-toed geckos in Vietnam with a focus on the Cyrtodactylus irregularis species complex

	DNA barcoding, genogeography, and phylogeny of bent-toed geckos in Vietnam with a focus on the Cyrtodactylus irregularis species complex
其他题名	越南弯脚虎物种及Cyrtodactylus irregularis物种复合体的支系地理学，系统发育及DNA条形码研究
	Nguyen Ngoc Sang
学位类型	博士
导师	张亚平,Robert W. Murphy
	2014-01
学位授予单位	中国科学院研究生院
学位授予地点	北京
学位专业	遗传学
摘要	Vietnam is a biodiversity hot-spot. Many new species of vertebrates were discovered in recent years. For amphibians and reptiles, Vietnam, together with Laos and Cambodia, is home to over 600 species, roughly a quarter of which have been described within the last 15 years. Bent-toed geckos, genus Cyrtodactylus (Reptilia: Gekkonidae), are one of the most speciesrich groups of geckos with about 174 species distributed widely from India and Tibet through Southeast Asia to New Guinea and Australia. Thirty-two species of bent-toed geckos have been recorded from Vietnam. The first two species were described in 1921. Most of the remaining species (28 of 32 species) were described in last decade. The Cyrtodactylus irregularis species complex of bent-toed geckos contains at least 11 species. Ten species occur in central and southern Vietnam, especially in the Central Highlands. Geckos in this species complex are quite similar to one other in their morphology. Research on Vietnamese bent-toed geckos has focused mainly on species descriptions based on morphological data. Hence, there is a need to expand on previous work using molecular data. DNA barcoding quickly identifies a board range of animals by using a short fragment of mitochondrial gene COI. The method compares known and unknown sequences to conveniently identify a large set of specimens by using just a tiny piece of tissue from any part or developmental stage of an animal. Herein, I use this approach and other genetic and phylogenetic methods to access the biodiversity, genogeography, and population genetic structure of Vietnamese bent-toed geckos in general and of the C. irregularis species complex in particular. I study ranges from focusing of all possible Vietnamese bent-toed geckos to populations of C. cattienensis. I also test the efficiency of DNA barcoding tool by testing its accuracy by evaluating nuclear gene sequence data. Chapter 1 is an overview of the geography of Vietnam, the biodiversity of bent-toed geckos, and the application of DNA barcoding to resolving their taxonomy. In chapter 2, I take a comprehensive look into all possible bent-toed geckos (66.7%) in Vietnam plus three taxa from Laos and China. I aim to confirm the taxonomic status of the species by using DNA barcoding with 550 bp from COI gene and phylogenetic methods. Tree-based analyses resolve all sampled species. Kimura 2-parameter genetic distances between species are quite high, averaging 21.0±4.2% and ranging from 4.3% to 28.7%. Intraspecific genetic distances are low, averaging a mere 0.18%. In addition to diagnosing species, the results also identify potentially undescribed taxa, including three from Vietnam, two from Laos and one from China. As a result, I described one of these new taxa, Cyrtodactylus puhuensis sp. nov., from Vietnam as a new species based on both genetics and morphology. Results from this chapter show that DNA barcoding approach not only successfully identifies species of bent-toed gecko, but also discovers new taxa in Vietnam and adjacent areas. Chapter 3 focuses only on the C. irregularis species complex. I include all described species of the group and add new populations to test the taxonomic status of each taxon because species in this group are very similar to one other morphologically. My analysis uses 38 samples from 15 localities and this encompasses all eight described species. COI and nuclear gene RPL35 (580 bp) are sequenced. Phylogenetic analyse of the COI data resolve the relationships of all described species with high support values. The tree also resolves five new lineages. Genetic distances between described species average 17.3±2.8% and range from 8.4% to 23.9%. In comparison, distances for RPL35 are low, averaging 2.1±0.7% only, and ranging from 0.1 to 4.1%. Phylogenies based on RPL35 sequences resolve clades for five described species as well as four undescribed taxa. However, analyses do not support the recognition of C. bugiamapensis and C. ziegleri. These taxa cluster within another, unknown taxon. Nevertheless, because geckos from two distant populations form highly supported clades in both mitochondrial and nuclear trees, and differ morphologically from all other known congeners, they were described as two new species, C. phuocbinhensis sp. nov. and C. taynguyenensis sp. nov. Notwithstanding, DNA barcoding successfully identifies closely related species and discovers new species. Chapter 4 focuses on the C. irregularis species complex but in a wide geographical context by adding new samples from many new localities. Here, I explore cryptic diversity and reconstruct the matrilineal genealogy and phylogeny of the species complex. In addition, I test the accuracy of DNA barcoding by using nuclear DNA. The analyses use 167 samples of the C. irregularis species complex collected from 25 localities in Vietnam. DNA barcoding discovers cryptic diversity in the group by identifying 13 unknown lineages from different areas, as well as all eight described species. Seven matrilines are deeply divergent and analyses identify potential new species. K2P genetic distances between sites average 16.6±4.6%. Two divergent sympatric matrilines (K2P = 10.2%) occur at Ta Kou Mountain. Phylogenetic analyses of nuclear data from RPL35 and RAG1 resolve two major clades, which correspond to the presence or absence of enlarged femoral scale rows and southern and northern regions of the complex’s distribution. Phylogeneies analyses support all described species except for C. bugiamapensis and C. ziegleri. Further, the trees also resolve six of seven potential new species. This chapter reports cryptic diversity from 25 localities in a limited area in Vietnam. Further, the geckos are facing a battle for survival owing to continued habitat destruction by anthropogenic activities. Chapter 5 explores genetic patterns for the Cat Tien bent-toed gecko only, which occurs widely in southern Vietnam. Analyses document dispersal and vicariance events. I aim to understand why coastal populations show a high level of genetic divergence while continental ones do not. Firstly, I use mtDNA data for seven localities from chapter 4 to construct genealogical trees and networks for displaying genetic variation and diversity. I then date the divergence times. Secondly, I associate changes in sea-level with DNA analyses and then develop a hypothesis to explain the results. Inland populations of geckos show low levels of genetic divergence (0.9%) and genetic mixture but coastal populations have high levels of genetic divergence (6.8%). The origin of the species dates to about 30.7 million years ago (Ma). Intraspecific divergences appear to have initiated about 18.4 Ma through Miocene. Three coastal populations split into four matrilines while four inland populations form one lineage. Coastal regions consist of mountains and, thus, I hypothesize that sea level fluctuations drove the pattern. After its formation, the gecko appears to have dispersed widely due to low sea levels during Oligocene. Subsequently, lowland coastal populations were alternately flooded and exposed. These sequential isolations drove Miocene genetic divergences. In contrast, sea level changes did not affect inland geckos; this drove the low level of genetic divergence and mixture. Finally, Chapter 6 pursues another perspective into sympatric matrilines in two widely distributed species: C. cattienensis and C. pseudoquadrivirgatus. How does one population support more than one highly divergent matriline? Are these lineages stable? Do they need reproductive barriers? And how does the natural selection affect the lineages? The sympatric lineages found in two geckos are not sisters and their COI genetic distances are 10.2% and 7.1%, respectively. I hypothesize and demonstrate that sympatric lineages will maintain their frequencies constantly from generation to generation. Although having highly different matrilines, the amino acid sequences are identical in one population and differ only by one amino acid in another. Testing strongly supports that strict negative selection affects and controls divergence in both populations. Hence, sympatric lineages can be occurred and maintained stably in natural population without requirement of reproductive barrier
其他摘要	越南是生物多样性热点地区之一。近年来，大量脊椎动物新种在这里被发现。就两栖类和爬行类而言，越南，老挝和柬埔寨拥有600以上的物种，其中近四分之一是近十五年发现的。弯脚虎，Cyrtodactylus属（爬行纲：壁虎科）是物种丰富度最高的壁虎类生物，拥有174个物种，广泛分布于中国西藏，印度，东南亚，新几内亚和澳大利亚。32个弯脚虎物种在越南有分布。最早的两个物种在1921年被描述，大多数物种在近年来才被描述。 Cyrtodactylus irregularis物种复合体包括至少11个物种，其中10个分布于越南中部和南部，尤其是中央山脉。这一物种复合体的物种形态上极为相似。过去的研究主要基于形态数据进行物种描述，因此有必要利用分子数据进一步进行深入研究。通过使用一小段线粒体COI基因序列，DNA条形码技术可以在一个较大的范围内鉴定动物物种。通过比较已知和未知的序列，仅仅利用一小块组织，这种方法可以快速鉴定大量标本。在此，我使用这种技术及其它遗传学及系统发育分析方法，研究了弯脚虎属，尤其是C. irregularis物种复合体的生物多样性，谱系结构和群体遗传结构。本研究涵盖了越南所有弯脚虎属物种及C. cattienensis物种的所有群体。同时，我通过加入核基因数据检验了DNA条形码的有效性。第一章综述了越南的地理特征，弯脚虎属的生物多样性及DNA条形码技术在分类学中的运用。在第二章中，我基于550bp的COI序列，利用DNA条形码技术和系统发育分析方法，研究了几乎所有越南弯脚虎（66.7%）物种和中国、老挝的三个物种其分类地位的有效性。系统发育树恢复了所有包含的物种。基于Kimura二参数模型获得的物种间遗传距离非常高，最低位4.3%，最高位28.7%，平均为21.0 ±4.2%。种内遗传距离较低，平均仅为0.18%。同时，该结果显示存在潜在的未描述的类群，其中3个来自越南，2个来自老挝，1个来自中国。因此，基于分子和形态数据结果，我将来自越南的一个类群描述为一个新的物种Cyrtodactylus puhuensis sp. nov.。本章的研究结果显示DNA条形码技术不仅成功的确定了弯脚虎属物种，还在越南及其邻近地区发现了新的物种。第三章研究关注于C. irregularis物种复合体。本研究包含了该复合体的所有物种，同时由于该类群物种形态上的相似性，本研究增加了新的群体来进行分类学研究。本研究使用了来自15个地点的38份样品，涵盖了所有八个已被描述的物种，测定了COI和核基因RPL35（580bp）两个片段。基于COI数据的系统发育分析解决了所有已描述物种的系统关系，并具有较高的支持率。同时，系统树还发现了5个新的支系。已描述物种之间遗传距离最低为8.4%，最高为23.9%，平均为17.3±2.8%。相对的，基于RPL35的遗传距离较低，为0.1%到4.1%，平均为2.1±0.7%。基于RPL35序列的系统发育分析结果恢复了5个已描述物种和4个未描述的类群，C. bugiamapensis和C. ziegleri未得到支持，这些物种与其它未描述类群聚在一起。尽管如此，由于来自于两个不同群体的壁虎在线粒体树和核基因树中聚分为两支，并与同属其它物种形态上存在差异，因此被描述为两个新的物种C. phuocbinhensis sp. nov.和C. taynguyenensis sp. nov.。尽管如此，DNA条形码技术成功的鉴定了近缘物种并发现了新的物种。第四章研究C. irregularis物种复合体，并加入了更多样品点，扩展了研究的地理范围。在此，我重建了母系基因谱系树及物种复合体的系统发育树，并研究了隐存多样性。更进一步，本研究使用核基因检验了DNA条形码的精确性。本研究包括了来自于越南C. irregularis物种复合体25个地点的167份样品。DNA条形码技术在此类群中除恢复了8个已描述的物种外，还鉴定出13个未知支系。七个高度分化的母系支系被鉴定为潜在的新物种。地点间K2P平均遗传距离为16.6±4.6%。在Ta Kou山脉发现两个高度分化的支系(K2P = 10.2%)。基于RPL35和RAG1的核基因数据进行的系统发育分析恢复了两个大支，与形态特征（股骨是否具有扩大的鳞片）和地理分布（南部和北部）相对应。除C. bugiamapensis 和 C. ziegleri外，所有已描述的物种的物种均得到系统发育分析的支持。同时，系统发育树支持七个潜在物种中的六个。本章介绍了来自越南的有限的区域的25个采样点的潜在多样性。这一区域的壁虎物种由于人类活动造成的栖息地破坏而面临巨大的威胁。第五章研究了灌泛分布于越南南部的Cat Tien弯脚虎。分析证明了迁移和隔离分化事件的存在。本研究主要目的在于阐明海岸群体高度分化而内陆群体分化较小的原因。首先，我是用第四章中七个地点的线粒体数据重建谱系树和网络图来展示遗传变异与分化，之后估算分歧时间。第二，我联系海平面变化与DNA分析结果，提出解释的假说。内陆群体表现出较低的遗传分化(0.9%)和群体混合现象，而海岸群体遗传分化较高(6.8%)。这一物种起源与30.7个百万年前。种内分化开始于中新世，即18.4个百万年前。三个海岸群体分化为4个支系，而内陆群体只有一个。海岸地区包含很多山脉，因此，我提出假说海平面的波动造成这一模式。在物种形成后，由于渐新世海平面较低而广泛迁移。之后，海岸低地群体受到海平面变化的影响。之后的隔离驱动了中新世的遗传分化。相比之下，海平面变化对内陆群体影响不大，从而造成了遗传分化较低并驱动了群体混合。最后，在第六章深入研究了两个广布物种C. cattienensis和C. pseudoquadrivirgatus高度分化的同域母系支系。一个群体如何能够保留多个高度分化的母系支系？这些支系是否稳定？它们之间是否需要生殖隔离？以及自然选择如何影响这些支系？同域支系在两个壁虎物种中被发现，这两个物种并非姐妹种，并且其遗传距离分别为10.2%和7.1%。我假设同域支系会长期保持其频率。尽管拥有高度分化的母系支系，但群体内氨基酸序列完全一样或只有一个位点不同。检验表明负选择在两个群体中均发挥巨大的作用。在此，本研究证明在自然群体中同域支系可以长期保持稳定而不需要生殖隔离的存在。
语种	英语
文献类型	学位论文
条目标识符	http://ir.kiz.ac.cn/handle/152453/7876
专题	科研部门_分子进化与基因组多样性（张亚平）
作者单位	中国科学院昆明动物研究所
推荐引用方式 GB/T 7714	Nguyen Ngoc Sang. DNA barcoding, genogeography, and phylogeny of bent-toed geckos in Vietnam with a focus on the Cyrtodactylus irregularis species complex[D]. 北京. 中国科学院研究生院,2014.

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