Genetic structure and phylogeographic relationships of the Bellamya complex: A nascent aquacultural snail in the Pearl River basin,China

The current genetic structure of a species is determined by both historical demographics and contemporary gene flow among populations. As an economically important snail in China, Bellamya has attracted much attention for its breeding and aquaculture and has provided a good study system for this theoretical structure. Our results showed that high genetic diversity occurred in most populations distributed in the Pearl River basin, except for the population in Nanning, Guangxi Zhuang Autonomous Region. A lack of distinct genealogical branches or geographic clades was found for the Bellamya complex across the Pearl River basin. Nevertheless, low but significant genetic structure and high differentiation were detected between Anshun (in Guizhou Province) and other geographic populations segregated by the Yunnan–Guizhou Plateau, suggesting that geographic barriers exist around this region, while this scenario was not applicable to the populations in Yuxi, Yunnan Province. The demographic history described a relatively stable pattern before the last glacial period, but the effective population sizes have drastically decreased since the late Pleistocene. The lack of genetic structure across the Pearl River basin is mainly attributed to a high level of gene flow by passive migration due to flooding and anthropogenic and animal translocation. The vicariance caused by the uplift of the Yunnan–Guizhou Plateau during the early Pleistocene likely contributed to the divergence of the plateau and plain sublineages in the Pearl River basin. A genetic diversity and phylogeography analysis are valuable to developing further guidance on breeding and conservation strategies for Bellamya germplasm resources.     

基于线粒体Cyt b基因序列的绿鳍马面鲀6个野生群体的遗传结构分析

利用线粒体细胞色素b(cytochrome b,Cyt b)基因分析了我国沿海绿鳍马面鲀(Thamnaconus septentrionalis)6个野生群体(大连、秦皇岛、蓬莱、日照、舟山和汕头)的遗传多样性和群体遗传结构。结果显示,在176个个体915 bp的Cyt b部分序列中共检测到32个变异位点和30个单倍型;6个群体的单倍型多样性为0.883~0.953,核苷酸多样性0.0032~0.0039;群体间固定指数较小,呈中低度分化;AMOVA分析表明,遗传变异主要来源于群体内,群体间遗传分化未达到显著水平。群体历史动态分析表明,绿鳍马面鲀在16.9万~42.2万年前经历种群扩张。总体上,各群体间的遗传分化较小,并未形成相应的地理支系,推测黑潮的输送作用以及绿鳍马面鲀的洄游习性维持了各群体间高强度的基因交流;中更新世中晚期的气候波动对绿鳍马面鲀的种群扩张以及地理分布格局可能具有重要影响。本研究结果加深了人们对于绿鳍马面鲀资源情况的认识,为绿鳍马面鲀资源的保护和合理利用提供了理论依据。     

基于线粒体COⅠ序列的中国前鳞(鱼夋)遗传多样性分析

前鳞(鱼夋)(Liza affinis)是分布于西北太平洋的日本南部及中国台湾海域等的经济鱼类。为了解中国前鳞(鱼夋)的遗传背景,该研究分析了采自中国4省82尾样本COⅠ基因5'端712 bp序列,共发现21个单倍型,遗传多样性(H_d=0.4840±0.0700,π=0.0010±0.0002)较低。根据群体所属的地区、海域、海峡进行分组,通过AMOVA分析,得出组间的Fst值皆小于0.05,P值皆大于0.05,组间变异的比例极低(–0.6%～0.46%),表明中国前鳞(鱼夋)群体间无明显分化,其原因可能是:1)频繁的基因交流。前鳞(鱼夋)的洄游范围较大,且受海流影响,导致不同地区的群体间有着密切的基因交流;2)近期种群扩张事件。中国前鳞(鱼夋)整体的Fu’s F_s值为显著性负值(F_S=–20.3900,P=0),核苷酸错配峰图为明显单峰,单倍型网络图呈星状结构,均表明中国前鳞(鱼夋)群体经历过种群扩张,估算扩张大约发生在13.4199～1.4911万年前,可能是冰期和间冰期的交替中海平面的变化所致。此外,洞头群体的遗传多样性(H_d=0.8080±0.1130,π=0.0021±0.0006)明显高于其他地理群体,建议将其作为优先保护的对象。     

Genetic diversity and population history of the migratory catfishes Pangasianodon hypophthalmus and Pangasius bocourti in the Cambodian Mekong River

ABSTRACT:   Polymerase chain reaction (PCR) restriction fragment length polymorphism analysis of mitochondrial DNA was applied to the genetic structure and evolutionary history of the more ancestral Pangasianodon hypophthalmus ( n  = 82), and the recently speciated catfish Pangasius bocourti ( n  = 90) from the Cambodian Mekong River. Both pangasiids were characterized by a lack of genetic population structure that may result from high levels of contemporary gene flow. Genetic diversity was lower in P. hypophthalmus than in P. bocourti . However, a different evolutionary history was inferred for both species based on genealogical and demographic analyses (mismatch analysis, Tajima's D- and Fu's F _S-tests). The genetic profile of the more ancestral P. hypophthalmus shows indications of a recent population bottleneck, whereas the recently speciated P. bocourti shows signatures of historical population expansion. This study stresses the importance of preserving the migration routes in the Cambodian Mekong basin in order to maintain the genetic diversity and long-term integrity of both species.     

Fine‐scale population structure and connectivity of bottlenose dolphins,Tursiops truncatus,in European waters and implications for conservation

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基于线粒体控制区序列的三疣梭子蟹增殖放流亲蟹遗传多样性研究

为研究山东沿海三疣梭子蟹增殖放流亲蟹群体的遗传多样性状况,实验采用536 bp的线粒体DNA控制区片段作为分子标记,对4个亲蟹群体的遗传多样性和遗传结构进行了分析。结果显示,301个三疣梭子蟹个体共检测到155个单倍型,4个群体的单倍型多样度为0.972 3~0.993 0,核苷酸多样度为0.021 2~0.023 6,表现出丰富的遗传多样性。AMOVA分析和Fst分析结果均显示,4个三疣梭子蟹亲蟹群体间遗传分化微弱,未形成明显的遗传结构,NJ系统树中未出现与各群体相对应的的谱系分支。研究表明,4个增殖放流亲蟹群体的遗传多样性丰富,且其遗传结构与放流海域的野生群体间没有明显分化,种质资源质量较好。此外,群体历史动态分析显示,渤海南部和黄海北部的三疣梭子蟹历史上曾经历过群体扩张事件。     

Spatial and temporal mitochondrial DNA genetic homogeneity of dolphinfish populations (Coryphaena hippurus) in the eastern central Pacific

Dolphinfish (Coryphaena hippurus) samples were collected over four consecutive years from four locations in the eastern central Pacific to evaluate the genetic variation in a 751 bp segment of the mitochondrial NADH subunit 1 (ND1) to test for the presence of genetic population structure. Sequence analyses revealed no significant differences among collections from the same location in the different years sampled nor between locations. Mismatch distributions, estimations of population expansion parameters, and neutrality tests revealed significant fluctuations in population size in coincidence with past glacial and interglacial periods during the late Pleistocene. The low levels of nucleotide diversities and shallow coalescence of mtDNA genealogies observed were coincident with the estimated demographic parameters and neutrality tests, in suggesting the presence of important past population size fluctuations or range expansion. The prevention of the accumulation of deep lineages independently on how it was originated, probably delayed the emergence of a population divergence process which might account for the lack of genetic differences detected.     

基于线粒体COI基因的马科鱼类DNA条形码研究

为明确中国马鲅科(Polynemidae)鱼类的分类地位,测定了中国7省10个地点马鲅科鱼类3属5种33条COI基因5′端序列,结合GenBank下载的5属16种41条同源序列,共分析了6属20种马鲅科鱼类DNA条形码。结果显示,20种鱼类种间平均遗传距离为19.6%,是种内平均遗传距离0.9%的22倍;其中14种形成了单系分支,支持其物种有效性。四指马鲅(Eleutheronema tetradactylum)和多鳞四指马鲅(E.rhadinum)种间遗传距离(16.0%)是种内平均遗传距离(1.2%)的13倍,支持将二者作为2个独立物种的分类处理。多鳞四指马鲅种内遗传距离(2.3%)大于2%,形成了2个自展数据支持率为99%的分支,分支间遗传距离(4.8%)是分支内平均遗传距离(0.1%)的48倍,表明在中国沿海分布的多鳞四指马鲅有可能存在2个亚种或隐藏种。黑斑多指马鲅(Polydactylus sextarius)与马达加斯加多指马鲅(P.malagasyensis)外部形态极为相似,种间遗传距离仅为1.0%,且在分子系统树上镶嵌混杂为一支,仅根据分子数据结果,推测二者可能为同一物种;但由于没有可检视的标本,也不能排除GenBank序列种名注释中形态鉴定出错的可能。马伦氏多指马鲅(P.mullani)与七丝指马鲅(Filimanus heptadactylus)也混为1支,分支内遗传距离仅为0.1%;如果不是GenBank序列种名注释出错,则二者应为同一物种。GenBank序列中来源于北部湾的六丝多指马鲅(P.sexfilis),则可能是黑斑多指马鲅的错误鉴定。     

Why are so many boreal freshwater fishes anadromous? Confronting ‘conventional wisdom’

Suggestions that anadromy predominates in northern high latitudes because boreal marine seas have high primary productivity are questioned. Instead, post‐Pleistocene invasion of far northern fresh waters, which became inhabitable as Pleistocene temperatures ameliorated, via coastal seas, is suggested as a primary cause for northern distributions of widely anadromous fish families. Also, cold temperature preferences may be implicated in determining the ranges of these families, as well as the inhospitability of far northern fresh waters and the lack of ecosystem space to support the huge populations of boreal anadromous fishes. Moreover, there are significant numbers of tropical anadromous fish species.     

基于mtDNA控制区序列的拟平鳅遗传变异和种群分化

采用DNA序列分析方法,分析拟平鳅不同地理种群间的遗传变异和亲缘关系。共检测了12条水系87尾拟平鳅的mtDNA控制区序列,在477 bp片段中有95个核苷酸变异位点,其中39个单个多态位点,56个简约信息位点。系统发育分析(NJ)结果显示,华南西部沿海地区拟平鳅20个单倍型分成3支;分支I由广东西部独立入海水系和珠江水系的种群组成,分支II由广西东部独立入海水系和海南岛昌化江的种群组成,而海南岛的万泉河和南渡江种群聚合成分支III。表明海南岛不同水系的拟平鳅已经发生了遗传分化,一部分与广西的种群相似,另一部分是海南特有的。3个分支内的变异率（0.583%～1.775%）都明显小于各分支间的变异率（4.205%～5.715%）,也远小于各分支与外类群间的变异率（11.825%～13.73%）。说明华南西部沿海和海南岛的拟平鳅已分化成3个在遗传上有明显差异的群。基于现在的地理格局,研究曾经假设华南西部沿海独立水系种群与珠江水系种群的关系较密切,而与海南岛种群的关系较疏远。分子变异分析（AMOVA）表明,大部分的变异（65.04%）分布于地理区内种群间,而地理区间的变异仅占2.24%,与假设不符。表明12条水系拟平鳅mtDNA控制区序列的遗传分化可能主要是由于第三纪水系的隔离,而不是第四纪冰后期海南岛与大陆的分离所致。而第四纪盛冰期低海平面所造成的陆连,使相互分离的水系有机会连接,因此,海南岛昌化江种群与广西沿海独立水系种群关系密切,珠江水系种群保持与广东西部沿海独立水系种群的密切关系。而琼中海峡这个地理屏障形成的时间还不够长久,并没有成为有效的生殖屏蔽。AMOVA分析结果与上述NJ分析中各个分支之内的分子变异非常小的结果一致。     

基于COⅠ基因序列的东、黄海区野生与养殖大黄鱼遗传多样性分析

为研究野生与养殖大黄鱼(Larimichthys crocea)群体的遗传多样性,对大黄鱼8个野生群体及6个养殖群体共336个样本的线粒体COⅠ基因部分序列进行了扩增和测序分析。实验最终获得序列片段长621 bp,总变异位点38个,简约信息位点23个,单变异位点15个,其中野生群体包含38个变异位点,占总变异的100%,养殖群体包含8个变异位点,占总变异的21.05%。在所有样本中共检测出单倍型34个,单倍型多样性为0.587,核苷酸多样性为0.00194,野生及养殖群体单倍型多样性指数分别为0.714~0.952、0.000~0.581。大黄鱼养殖与野生两个组群间的遗传分化指数为0.04982,占总变异的4.98%,差异极显著(P0.01),组群间群体间的变异占1.46%(P0.05),群体内的变异占93.56%(P0.01)。以上结果表明,大黄鱼的遗传变异主要来自于群体内,养殖群体的遗传多样性显著低于野生群体,两者的遗传多样性程度均处于较低水平,养殖群体间或野生群体间不存在显著的遗传分化,而养殖与野生两大组群间存在着显著的遗传分化。此外,通过对群体遗传结构及进化树的分析表明,东、黄海大黄鱼应属于同一地理种群,但两者间存在较低程度的遗传分化现象,黄海的大黄鱼群体遗传多样性高于东海群体。本研究可为大黄鱼种质资源的保护和恢复提供理论依据。     

基于COⅠ基因的中国及邻近海域部分笛鲷属鱼类DNA条形码研究

笛鲷属(Lutjanus)鱼类经济价值高,物种数量多,但外部形态保守,传统分类鉴定困难。为了解中国笛鲷属鱼类的物种多样性状况,测定了中国沿海19个地点11种笛鲷73个样本线粒体COⅠ基因5'端序列,并与从GenBank下载中国及邻近海域69条同源序列进行DNA条形码分析。研究表明,勒氏笛鲷(L. russelli)、金焰笛鲷(L. fulviflamma)、奥氏笛鲷(L. ophuysenii)、约氏笛鲷(L. johni)、蓝点笛鲷(L. rivulatus)、五线笛鲷(L. quinquelineatus)、千年笛鲷(L. sebae)、紫红笛鲷(L. argentimaculatus)、白斑笛鲷(L. bohar)、马拉巴笛鲷(L. malabaricus)、黄笛鲷(L. lutjanus)、印度笛鲷(L. indicus)、画眉笛鲷(L. vitta)、星点笛鲷(L. stellatus)、四带笛鲷(L. kasmira)、驼背笛鲷(L. gibbus)、焦黄笛鲷(L. fulvus)、红鳍笛鲷(L. erythopterus)等18种笛鲷142条COⅠ序列组成18个自展数据支持率(bootstrap)为99%～100%的单系分支,分支间平均遗传距离14.4%(2.9%～21.8%)约为分支内平均遗传距离0.17%(0～0.6%)的85倍,其中10种笛鲷独立成支,支持其物种有效性。勒氏笛鲷分成2小支,小支间平均遗传距离(2.9%)是小支内平均遗传距离(0.5%)的5.8倍,未满足"10×法则",其准确的物种分类地位尚待明确。印度笛鲷与勒氏笛鲷、画眉笛鲷与奥氏笛鲷出现混杂,推测从GenBank下载的印度笛鲷序列KF830898和KF830905可能来自勒氏笛鲷;画眉笛鲷序列KU943888可能来自奥氏笛鲷。中国近海星点笛鲷和蓝点笛鲷混杂且种间遗传距离仅为2.1%,接近一般种内遗传距离2%;红鳍笛鲷和马拉巴笛鲷遗传距离种间(0.3%)与种内(0.2%～0.3%)相当,且形态相似,推测是同一物种,也不排除它们存在种间杂交和为近期分化物种的可能。     

Mitochondrial DNA variation in the East China Sea and Yellow Sea populations of Japanese Spanish mackerel <Emphasis Type="Italic">Scomberomorus niphonius</Emphasis>

Japanese Spanish mackerel Scomberomorus niphonius is a commercially important species in the East China Sea and Yellow Sea, but there is limited knowledge of its genetic population structure. In order to detect its genetic structure, sequence variation of the first hypervariable segment of the control region was analyzed among eight populations of S. niphonius from the East China Sea and Yellow Sea. A total of 119 polymorphic sites were detected in the 505-bp segment of the control region among 134 individuals of S. niphonius, defining 112 haplotypes. Mean haplotype diversity and nucleotide diversity for the eight populations were 0.9963 ± 0.0017 and 0.0236 ± 0.0119, respectively. As expected, analysis of molecular variance detected no significant differences at all hierarchical levels, and most of the conventional population Φ_ST statistics were negative, indicating that no significant population genetic structure exists in the East China Sea and Yellow Sea. Moreover, the exact test of differentiation supported the null hypothesis that S. niphonius within the East China Sea and Yellow Sea constitutes a panmictic mtDNA gene pool. Neutrality tests and mismatch distribution revealed that S. niphonius underwent population expansion in the late Pleistocene. Strong dispersal capacity of larvae and adults, long-distance migrations, and ocean currents in the studied area could be the reasons for genetic homogeneity in this species in the East China Sea and Yellow Sea. Insufficient time to accumulate genetic variation might be another explanation for the lack of genetic structure in the East China Sea and Yellow Sea.     

基于mtDNACytb基因序列的我国不同水系野生鲇种群遗传多样性与种群历史分析

为进一步了解鲇这一广布种的遗传多样性与种群遗传结构,阐明该经济鱼类的种群历史动态,本研究对我国长江(上游、中游)、珠江、福建、海南、辽河地区的野生鲇样本进行mtDNA Cytb基因序列测定与种群遗传多样性和遗传结构分析。结果显示,在所分析的216个序列样本中,共检测出78个单倍型,其中Hap_45在鲇不同群体中分布最为广泛;鲇总群体具有较高的单倍型多样性(Hd=0.948±0.009)和核酸多样性(Pi=0.017 99±0.000 55),暗示了其自然种群数量的相对稳定;单倍型系统发育与网络关系图分析显示,78种单倍型被分为4个分支,且不同单倍型分布相对集中,划分为4个谱系。中性检验与错配分析表明,不同水系野生鲇种群(4个谱系)在约晚更新世时期(0.04~0.05百万年)经历过近期种群扩张事件。     

Stock structure of Japanese flounder inferred from morphological and genetic analyses

ABSTRACT:   Stock structure of Japanese flounder Paralichthys olivaceus has been inferred mainly from either morphological or genetic analyses. However, because the results of both analyses did not always agree with each other, an inclusive conclusion has never been obtained. In this study, the stock structure has been inferred from both morphological and genetic analyses using 722 wild Japanese flounder collected from nine locations along the Japanese coast. The dorsal and anal fin ray counts were larger in the southern than in the northern populations. In total, 1041 bp of mitochondrial NADH dehydrogenase subunit-2 ( ND2 ) and 1830 bp of ND5 sequences were aligned. There are 578 variable sites in the concatenated sequence from the two genes, which defined a total of 490 haplotypes. Both results of morphological and genetic analyses indicated that the western Kyushu group, which included the Nagasaki and Kagoshima populations, was divided from the other seven populations. This is the first report to reveal the heterogeneity of the western Kyushu group based on statistical analysis.     

中国近海路氏双髻鲨群体线粒体DNA控制区序列比较分析

采用线粒体DNA控制区序列比较分析了路氏双髻鲨日照和霞浦群体的遗传结构及遗传多样性现状。研究结果显示:在长度为548 bp的mtDNA控制区序列上,34尾路氏双髻鲨个体仅检测到5个单倍型,两群体均呈现出较低的遗传多样性水平,日照群体的单倍型多样度(h)、核苷酸多样度(π)以及两两序列比较的平均碱基差异数(p)(h=0.6000±0.1305;π=0.0046±0.0031;p=2.5333±1.4856)略高于霞浦群体(h=0.5109±0.0955;π=0.0024±0.0017;p=1.2899±0.8373);邻接关系树显示,5个单倍型明显分为两支,净遗传距离为0.016;群体遗传分化指数为Fst=-0.047(P=0.914),表明两群体之间不存在显著差异,确切P检验结果也表明两群体存在随机交配现象(P=0.731);核苷酸不配对分布呈双峰类型,其中一个峰对应类群内序列差异,另外一个峰对应两个类群序列间差异。研究表明,路氏双髻鲨较小的有效群体导致了较低的遗传多样性,其资源状况已不容乐观。     

基于线粒体COⅠ基因序列的中国沿海缘边银鲈遗传多样性分析#br#

为了解中国沿海缘边银鲈(Gerres limbatus)的遗传背景,分析了9个地理群体140条样本线粒体细胞色素氧化酶Ⅰ(cytochrome oxidase subunitⅠ,COⅠ)基因5′端652 bp序列。结果发现14个变异位点,13个单倍型,其中有9个独有单倍型和4个共享单倍型,整体单倍型和核苷酸多样性都较低(H_d=0.276±0.050,π=0.0005±0.0001)。琼州海峡以东海域的遗传多样性(H_d=0.338~0.456,π=0.0006~0.0011)高于琼州海峡以西北部湾海区(H_d=0~0.295,π=0~0.0005)。单倍型网络图未出现明显谱系结构和地理结构。群体遗传分化系数F_st显示,硇洲群体与其他群体间出现不显著的中低度遗传分化(F_st=0.0773~0.1696,P>0.05),其他群体间分化不明显(F_st=-0.0415~0.0612,P>0.05),总体上群体间分化程度都不高。AMOVA分析显示,遗传变异绝大部分(95%以上)来源于群体内。中性检验结果显示,群体总体的Tajima’s D(-2.2549)和Fu’s F_s(-16.4725)结果均为显著性负值(P<0.01),核苷酸错配图呈现较为明显单峰,单倍型网络图呈典型星状结构,表明缘边银鲈历史上存在过种群扩张,扩张时期约为距今0.1304~0.0435百万年。由于总体单倍型多样性小于0.5,核苷酸多样性小于0.005,推测中国缘边银鲈种群曾经历过较为严重的瓶颈效应。宁德、硇洲群体的核苷酸多样性相对较高,应优先保护。北部湾各群体遗传多样性极低,需要采取措施避免遗传多样性下降。     

中国主要海洋底层鱼类生物学研究的回顾与展望

回顾了中国海洋主要底层经济鱼类资源生物学的研究历史和科研成果,并重点叙述了带鱼、小黄鱼、大黄鱼和绿鳍马面鲀等重要经济鱼类资源生物学研究概况.概述的内容包括地理种群及其产卵群体的鉴别和划分、生活史型的演变、种群和群体结构、种群数量变动、年龄组成和生长特性、摄食习性、性成熟周期、性腺成熟指数(GSI)、产卵群体生殖力、产卵场、索饵场、越冬场及洄游路线、资源量和渔获量、各种作业渔船的调整及其捕捞力量的限制措施等,并展望了中国海洋底层经济鱼类资源生物学研究的前景.     

Genetically structured population and demographic history of the goldlined spinefoot <Emphasis Type="Italic">Siganus guttatus</Emphasis> in the northwestern Pacific

Sequence variation of the mitochondrial DNA was analyzed to examine the genetic structure and demographic history of the goldlined spinefoot Siganus guttatus in the northwestern Pacific. In total, 451 nucleotide sequences spanning from the tRNA^Thr gene to the middle of the control region were determined from 254 specimens collected from five localities; three in the Ryukyu Islands (Okinawa, Miyako, and Ishigaki Islands), Taiwan, and Cebu Island of the Philippines; 73 variable sites and 75 haplotypes were detected. Our results showed restricted gene flow and genetic differentiation among all populations, with the exception of genetic homogeneity between the Miyako and Ishigaki populations; that is, this species will be able to cross between Miyako and Ishigaki (ca. 120 km) by the transport of pelagic larvae, and gene flow between Okinawa and Miyako/Ishigaki Islands (ca. 330–450 km) is restricted. A non-dispersal strategy will lead to restricted gene flow and genetic structuring in S. guttatus. Both the neutrality tests and the mismatch distribution indicated that S. guttatus might have been in populations at demographic equilibrium. This suggests that population range expansion may have been restricted owing to a non-dispersal strategy that may have restrained S. guttatus from demographic expansion after glaciation. The result will be of fundamental importance for resource management of this species.     

Alpine and Apennine barriers determining the differentiation of the rudd (Scardinius erythrophthalmus L.) in the Italian peninsula

Abstract –  A phylogeographic study of rudd Scardinius erythrophthalmus L. (Pisces: cyprinidae) was addressed to determine the differentiation of Italian populations in relation to the presence of mountain barriers, which identify two biogeographic districts (Padano-Venetian, Tuscano-Latium). At this scope, 409 bp long sequences of mtDNa cytochrome b gene were obtained from Italian and central European samples. Italian rudds phylogeny showed low level of divergence which although resulted from the central European haplotypes. The genetic structure of haplotypes in the studied districts revealed a significant recent fragmentation event. The Padano-Venetian populations showed patterns of past range expansion caused by the specie dispersion by river connections occurred during Pleistocene. This pattern provides evidence for (i) the role of Alps as a barrier for rudd dispersion; (ii) a significant genetic structure among the studied districts related to recent isolation events by the Apennine barrier; (iii) the role of Pleistocene sea level variations in determining phylogeography of the Padano-Venetian populations.