Chloroplast DNA-based genetic variation of Rosa roxburghii in Southwest China:Phylogeography and conservation implications

Rosa roxburghii Tratt.is a well-known commercial horticultural crop in China with nutritional and medicinal value.Wild germplasms of this species are mainly distributed in Southwest China but the population is decreasing due to continuous exploitation,habitat destruction,and fragmentation.Therefore,assessing the genetic diversity and phylogeography is essential for efficient conservation.Herein,two chloroplast intergenic spacers(trnL-trnF and accD-psaI)were investigated in 255 individuals from 29 R.roxburghii populations and 18 haplotypes(H1–H18)were identified.High levels of haplotype diversity(Hd=0.829)and nucleotide diversity(π=1.3×10^(−3))were detected in these populations.Also,the genetic variation representing 86.4%of the total variation was detected by an analysis of molecular variance.A significant correlation was established between genetic divergence and geographic distance by the Mantel test(r=0.204,P=0.04,9999 permutations),suggesting the isolation-by-distance model.A significantly higher Nst than Gst(Nst=0.257,Gst=0.136,P<0.05)indicated the phylogeographic structure of R.roxburghii.Further phylogeographic analysis revealed rapid range expansion in the population,probably between 647073 and 217848 years ago.The primary processes shaping the genetic patterns of the R.roxburghii populations included restricted gene flow with isolation distance within clades 1-8,2-3,and overall,contiguous expansion within clades 1-3 and 3-2,past fragmentation,and/or long-distance colonization within clades 1-9 and 2-2.Conservation priority should be given to the core populations GZ,FQ,DF,DS,xy,AL,LC,PB,and XY in the Yunnan-Guizhou Plateau,NZ and MX in the Qingling-Bashan mountains,and MN in the Hengduan mountains,where an in situ preservation and management strategy should be applied.

Evolutionary history and phylogeography of Scots pine (Pinus sylvestris L.) in Europe based on molecular markers

In this review we summarized recent historical records and molecular studies on evolutionary history and phylogeography of Scots pine with focus on the European highly fragmented distribution area of the species. Fossilized pollen, plant micro- and macrofossil records provided evidences on the large-scale species’ range shifts and demographic changes during the Quaternary. Populations of Scots pine were documented both in the glacial (incl. full glaciation) and interglacial periods. Recolonization of Europe after the glaciation originated from the (Sub) Mediterranean areas like the Balkan Peninsula but also from around the Eastern Alps and the surroundings of the Danube plain. Fennoscandia and northern European Baltic regions were most probably colonized from two main directions, from Western Europe and from the Russian Plain. Modern history of Scots pine was hardly affected by anthropogenic activities that started to strengthen in the Bronze and Iron Age. Along with the fossil records, molecular genetic tools were used to infer the origin and putative history including migration, differentiation and demography of the species. In this paper we compiled the major publications (30) of molecular genetic studies of the past 20?years derived from distinctly inherited organelle genomes (mitochondrial, chloroplast, nuclear) revealed by different marker systems (mtDNA-cox1, -nad1, -nad3, -nad7, ISSR, cpSSR, nSSR, B-SAP, SNP). It is important to consider that different phylogeographic patterns can be drawn by the analysis of different DNA marker types. Accordingly the use of more than one marker simultaneously outlines the most sophisticated phylogeographical pattern on the genetic lineages and can reveal high differentiation of the European distribution. Combined marker systems and markers derived from coding sequences have also been used to detect species’ phylogeographic patterns, but these were rarely applied to Scots pine. Although new molecular techniques can provide higher resolution data for populations, the reviewed results can shape the direction of further studies.

Phylogeography of SARS-CoV-2 pandemic in Spain:a story of multiple introductions,micro-geographic stratification,founder effects,and super-spreaders

Spain has been one of the main global pandemic epicenters for coronavirus disease 2019(COVID-19).Here,we analyzed>41000 genomes(including>26000 high-quality(HQ)genomes)downloaded from the GISAID repository,including 1245(922 HQ)sampled in Spain.The aim of this study was to investigate genome variation of novel severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)and reconstruct phylogeographic and transmission patterns in Spain.Phylogeographic analysis suggested at least 34 independent introductions of SARS-CoV-2 to Spain at the beginning of the outbreak.Six lineages spread very successfully in the country,probably favored by super-spreaders,namely,A2 a4(7.8%),A2 a5(38.4%),A2 a10(2.8%),B3 a(30.1%),and B9(8.7%),which accounted for 87.9% of all genomes in the Spanish database.One distinct feature of the Spanish SARS-Cov-2 genomes was the higher frequency of B lineages(39.3%,mainly B3 a+B9)than found in any other European country.While B3 a,B9,(and an important sub-lineage of A2 a5,namely,A2 a5 c)most likely originated in Spain,the other three haplogroups were imported from other European locations.The B3 a strain may have originated in the Basque Country from a B3 ancestor of uncertain geographic origin,whereas B9 likely emerged in Madrid.The time of the most recent common ancestor(TMRCA)of SARS-CoV-2 suggested that the first coronavirus entered the country around 11 February 2020,as estimated from the TMRCA of B3 a,the first lineage detected in the country.Moreover,earlier claims that the D614 G mutation is associated to higher transmissibility is not consistent with the very high prevalence of COVID-19 in Spain when compared to other countries with lower disease incidence but much higher frequency of this mutation(56.4% in Spain vs.82.4% in rest of Europe).Instead,the data support a major role of genetic drift in modeling the micro-geographic stratification of virus strains across the country as well as the role of SARS-CoV-2 super-spreaders.

Phylogeography and Cryptic Species Diversity of Paramesotriton caudopunctatus Species Group(Salamandridae: Paramesotriton) in Guizhou, China

The Paramesotriton caudopunctatus species group is mainly distributed in the karst mountain ecosystems of Guizhou, China. Although some species have been included in previous phylogenetic studies, the evolutionary relationships and divergence-time of members of this species group as a whole remain unexplored. In this study, we report the sequencing of one protein coding mitochondrial gene fragment(ND2) and one nuclear gene(POMC), and use a combination of phylogenetic analyses and coalescent simulations to explore the cryptic diversity and evolutionary history of the P. caudopunctatus species group. Phylogenetic relationships revealed that the P. caudopunctatus species group is composed of two major groups, i. e., East Clade and Western-South Clade. The divergence-time and ancestral area estimation suggested that the P. caudopunctatus species group likely originated in the Doupeng Mountains in Guizhou, China at 12.34 Ma(95% HPD: 8.30–14.73), and intraspecific divergence began at about 2.17 Ma(95% HPD: 1.39–2.97). This timing coincides with the orogenesis of the Miaoling Mountains during the Late Miocene to early Pleistocene. The delimitation of species in the P. caudopunctatus species group supports the existence of the currently identified species, and consensus was confirmed across methods for the existence of least to two cryptic species within what has been traditionally considered to be P. caudopunctatus species group. This study is of significance for understanding the species formation, dispersal, and diversity of the tailed amphibians in the karst mountains ecosystem of Guizhou and the role of the Miaoling Mountains as a geographical barrier to species dispersal.

Comparative mitogenome phylogeography of two anteater genera (Tamandua and Myrmecophaga;Myrmecophagidae, Xenarthra): Evidence of discrepant evolutionary traits

The species within Xenarthra(sloths,anteaters,and armadillos)are quintessential South American mammals.Of the three groups,Vermilingua(anteaters)contains the fewest extant and paleontological species.Here,we sampled and sequenced the entire mitochondrial genomes(mitogenomes)of two Tamandua species(Tamandua tetradactyla and T.mexicana)(n=74)from Central and South America,as well as Myrmecophaga tridactyla(n=41)from South America.Within Tamandua,we detected three different haplogroups.The oldest(THI)contained many specimens with the T.tetradactyla morphotype(but also several with the T.mexicana morphotype)and originated in southeastern South America(currently Uruguay)before moving towards northern South America,where the THII haplogroup originated.THII primarily contained specimens with the T.mexicana morphotype(but also several with the T.tetradactyla morphotype)and was distributed in Central America,Colombia,and Ecuador.THI and THII yielded a genetic distance of 4%.THII originated in either northern South America or“in situ”in Central America with haplogroup THIII,which consisted of~50%T.mexicana and 50%T.tetradactyla phenotypes.THIII was mostly located in the same areas as THII,i.e.,Central America,Ecuador,and Colombia,though mainly in the latter.The three haplogroups overlapped in Colombia and Ecuador.Thus,T.tetradactyla and T.mexicana were not reciprocally monophyletic.For this reason,we considered that a unique species of Tamandua likely exists,i.e.,T.tetradactyla.In contrast to Tamandua,M.tridactyla did not show different morphotypes throughout its geographical range in the Neotropics.However,two very divergent genetic haplogroups(MHI and MHII),with a genetic distance of~10%,were detected.The basal haplogroup,MHI,originated in northwestern South America,whereas the more geographically derived haplogroup,MHII,overlapped with MHI,but also expanded into central and southern South America.Thus,Tamandua migrated from south to north whereas Myrmecophaga migrated from north to south.Our results also showed that temporal mitochondrial diversification for Tamandua began during the Late Pliocene and Upper Pleistocene,but for Myrmecophaga began during the Late Miocene.Furthermore,both taxa showed elevated levels of mitochondrial genetic diversity.Tamandua showed more evidence of female population expansion than Myrmecophaga.Tamandua experienced population expansion~0.6–0.17 million years ago(Mya),whereas Myrmecophaga showed possible population expansion~0.3–0.2 Mya.However,both taxa experienced a conspicuous female decline in the last 10000–20000 years.Our results also showed little spatial genetic structure for both taxa.However,several analyses revealed higher spatial structure in Tamandua than in Myrmecophaga.Therefore,Tamandua and Myrmecophaga were not subjected to the same biogeographical,geological,or climatological events in shaping their genetic structures.

The Clam Cyclina sinensis (Gmelin) Phylogeography Study with 28S rRNA Gene and Potential of Nuclear rRNA Genes in Genetic Assessments of Molluscs

Intraspecific diversity of molluscan species is usually studied based on maternally inherited mitochondrial DNA,from which only part of the evolutionary history can be reflected.Some nuclear ribosomal RNA genes such as 28S rRNA represent poten-tial candidates that can be easily applied in phylogeography because of lacking intraindividual variation.However,considering their low polymorphism,genetic appraisals on whether and how they can be used in population studies are necessary.Here,we applied a short 28S rRNA to assess genetic patterns of the clam Cyclina sinensis along the coast of China and compared the results with a for-mer study based on COI and ITS-1 analyses.The results revealed the 28S rRNA data set was characterized by an extremely low level of variation,with only seven haplotypes defined for 93 individuals.Haplotype and nucleotide diversity for each population was al-most the lowest when compared with the other two markers.However,the distribution of two dominant haplotypes showed clear geo-graphic patterns,and significant population differentiation was revealed between the East China Sea and the South China Sea.These patterns were highly concordant with findings of the former study that populations of C.sinensis were historically separated by land bridges among sea basins.Our study suggested that although the nuclear rRNAs have shortcomings such as low variation,they have advantages including lack of intraindividual variation and high amplification rates.Applying rRNA genes can enrich the toolbox of nuclear markers in molluscan phylogeographic studies.

Phylogeography and morphometric variation in the Cinnamon Hummingbird complex:Amazilia rutila(Aves:Trochilidae)

Background:The Mesoamerican dominion is a biogeographic area of great interest due to its complex topography and distinctive climatic history.This area has a large diversity of habitats,including tropical deciduous forests,which house a large number of endemic species.Here,we assess phylogeographic pattern,genetic and morphometric variation in the Cinnamon Hummingbird complex Amazilia rutila,which prefers habitats in this region.This resident species is distributed along the Pacific coast from Sinaloa—including the Tres Marías Islands in Mexico to Costa Rica,and from the coastal plain of the Yucatán Peninsula of Mexico south to Belize.Methods:We obtained genetic data from 85 samples of A.rutila,using 4 different molecular markers(mtDNA:ND2,COI;nDNA:ODC,MUSK)on which we performed analyses of population structure(median-joining network,STRU CTU RE,FST,AMOVA),Bayesian and Maximum Likelihood phylogenetic analyses,and divergence time estimates.In order to evaluate the historic suitability of environmental conditions,we constructed projection models using past scenarios(Pleistocene periods),and conducted Bayesian Skyline Plots(BSP)to visualize changes in population sizes over time.To analyze morphometric variation,we took measurements of 5 morphological traits from 210 study skins.We tested for differences between sexes,differences among geographic groups(defined based on genetic results),and used PCA to examine the variation in multivariate space.Results:Using mtDNA,we recovered four main geographic groups:the Pacific coast,the Tres Marías Islands,the Chiapas region,and the Yucatán Peninsula together with Central America.These same groups were recovered by the phylogenetic results based on the multilocus dataset.Demography based on BSP results showed constant population size over time throughout the A.rutila complex and within each geographic group.Ecological niche model projec-tions onto past scenarios revealed no drastic changes in suitable conditions,but revealed some possible refuges.Mor-phometric results showed minor sexual dimorphism in this species and statistically significant differences between geographic groups.The Tres Marías Islands population was the most differentiated,having larger body size than the remaining groups.Conclusions:The best supported evolutionary hypothesis of diversification within this group corresponds to geo-graphic isolation(limited gene flow),differences in current environmental conditions,and historical habitat fragmen-tation promoted by past events(Pleistocene refugia).Four well-defined clades comprise the A.rutila complex,and we assess the importance of a taxonomic reevaluation.Our data suggest that both of A.r.graysoni(Tres Marías Islands)and A.r.rutila(Pacific coast)should be considered full species.The other two strongly supported clades are:(a)the Chiapas group(southern Mexico),and(b)the populations from Yucatán Peninsula and Central America.These clades belong to the corallirostris taxon,which needs to be split and properly named.

Phylogeography of <i>Asparagus schoberioides</i>Kunth (Asparagaceae) in Japan

To describe the phylogeographic structures of Asparagus schoberioides Kunth (Asparagaceae) in Japan, we investigated its nucleotide sequence variations with respect to its geographic distribution pattern. Sequencing of the internal transcribed spacer (ITS) 1 region in 29 samples of A. schoberioides revealed 20 polymorphic nucleotide sites. As a result, the 29 samples of A. schoberioides fell into 15 distinct haplotypes and phylogenetic analyses revealed these haplotypes fell into two major clades, Clade 1 and Clade 2. The haplotypes of Clade 1 were distributed chiefly along the Pacific Ocean side of Japan, while those of Clade 2 occurred mainly along the Japan Sea side. This result suggests that A. schoberioides has migrated via two routes in Japan.

Phylogenetic, phylogeographic and divergence time analysis of Anopheles subpictus species complex using ITS2 and COI sequences

Objective:To address the phylogenetic and phylogeographic relationship between different lineages of Anopheles(An.)subpictus species complex in most parts of the Asian continent by maximum utilization of Internal Transcriber Spacer 2(ITS2)and cytochrome C oxidase I(COI)sequences deposited at the GenBank.Methods:Seventy-five ITS2,210 COI and 26 concatenated sequences available in the NCBI database were used.Phylogenetic analysis was performed using Bayesian likelihood trees,whereas median-joining haplotype networks and time-scale divergence trees were generated for phylogeographic analysis.Genetic diversity indices and genetic differentiation were also calculated.Results:Two genetically divergent molecular forms of An.subpictus species complex corresponding to sibling species A and B are established.Species A evolved around 37-82 million years ago in Sri Lanka,India,and the Netherlands,and species B evolved around 22-79 million years ago in Sri Lanka,India,and Myanmar.Vietnam,Thailand,and Cambodia have two molecular forms:one is phylogenetically similar to species B.Other forms differ from species A and B and evolved recently in the above mentioned countries,Indonesia and the Philippines.Genetic subdivision among Sri Lanka,India,and the Netherlands is almost absent.A substantial genetic differentiation was obtained for some populations due to isolation by large geographical distances.Genetic diversity indices reveal the presence of a long-established stable mosquito population,at mutation-drift equilibrium,regardless of population fluctuations.Conclusions:An.subpictus species complex consists of more than two genetically divergent molecular forms.Species A is highly divergent from the rest.Sri Lanka and India contain only species A and B.

Highly divergent sympatric lineages of Leptotila verreauxi(Aves:Columbidae)suggest a secondary contact area in the Isthmus of Tehuantepec,Mexico

Due to a complex geological and biotic history,the Isthmus of Tehuantepec(IT),has been long recognized as a driver for the evolutionary divergence of numerous lowland and highland taxa.Widely distributed in the lowlands of the American continent,the White-Tipped Dove(Leptotila verreauxi)is a polytypic species with 13 recognized subspecies.Four of these have been recorded in Mexico,and the distribution of three abuts at the IT,suggesting a contact zone.To estimate phylogenetic patterns,divergence times and genetic differentiation,we examined two mt DNA(ND2 and COI)and one n DNA(β-fibint 7)markers.We also used correlative ecological niche models(ENM)to assess whether ecological differences across the IT may have acted as a biogeographical boundary.We estimated paleodistributions during the Middle Holocene,Last Glacial Maximum and Last Interglacial,to evaluate the influence of climate changes on the distribution and demographic changes.Our results showed genetically distinct lineages that diverged approximately 2.5 million years ago.Climatic and ecological factors may have played a dual role in promoting differentiation,but also in the formation of a secondary contact zone in the southern IT.Our ecological niche comparisons indicated that the ecological niche of sympatric lineages at the IT are not identical,suggesting niches divergence;in addition,environmental niche models across the region indicated no abrupt biogeographic barriers,but the presence of regions with low suitability.These results suggest that genetic differentiation originated by a vicariant event probably related to environmental factors,favored the evolution of different ecological niches.Also,the absence of a biogeographic barrier but the presence of less suitable areas in the contact regions,suggest that secondary contact zones may be also maintained by climatic factors for the eastern group,but also by biotic interactions for the western group.

Intraspecific phylogeography of Carchesium polypinum (Peritrichia, Ciliophora) from China, inferred from 18S-ITS1-5.8S ribosomal DNA

Based on the variation of site 34, 46, 241, 305 and 322 in the 18S-ITS1 rDNA sequence, 19 Carchesium polypinum populations collected from eight provinces of China were separated into northern and southern population along the delineation between the Yangtze River and the Pearl River. This geographic distribution pattern of Carchesium polypinum maybe results from two factors: the vicariance resulting from the formation of the delineation between the Pearl River and the Yangtze River accompanied with the uplift of Qinghai-Xizang Plateau, and the different dispersal paths of C. polypinum affected by the climate.

Molecular systematics, population genetics and phylogeography on planarians(Platyhelminths: Turbellaria): A brief review of molecular markers

Due to their characteristics, such as powerful regeneration capability, high susceptibility to xenobiotics, wide distribution and weak dispersion ability, planarians are used as ideal test animals in several research fields, and planarianology has become a more and more active subject. This paper reviews the research progress of planarians from the aspects of molecular phylogeny, molecular taxonomy, population genetics and phylogeography, and also summarizes the corresponding molecular markers applied. Moreover, the future direction and tendency in this field is briefly analyzed and prospected.

Phylogeography of Highly Pathogenic H5 Avian Influenza Viruses in China

The spread of H5 highly pathogenic avian influenza viruses poses serious threats to the poultry industry,wild bird ecology and human health.Circulation of H5 viruses between poultry and wild birds is a significant public health threat in China.Thus,viral migration networks in this region need to be urgently studied.Here,we conducted molecular genetic analyses of the hemagglutinin genes of H5 highly pathogenic avian influenza viruses in multiple hosts from 2000 to 2018 in China.Our aim was to clarify the roles of different hosts in the evolution of H5 viruses.We used a flexible Bayesian statistical framework to simulate viral space diffusion and continuous-time Markov chains to infer the dynamic evolutionary process of spatiotemporal dissemination.Bayesian phylogeographic analysis of H5 viruses showed for the first time that H5 viruses in poultry and wild birds were present in Guangdong Province.Furthermore,Guangdong,Jiangsu,Shanghai and Hunan acted as the epicenters for the spread of various H5 subtypes viruses in poultry,and Henan,Shanghai,Hong Kong and Inner Mongolia acted as epicenters for the spread of various H5 subtypes viruses in wild birds.Thus,H5 viruses exhibited distinct evolutionary dynamics in poultry and wild birds.Our findings extend our understanding of the transmission and spread of highly pathogenic H5 avian influenza viruses in China.

Phylogeography of Gyrodactylus konovalovi(Monogenoidea:Gyrodactylidae) in the Qinling Mountains in Central China

Gyrodactylus konovalovi is an ectoparasite on the Amur minnow(Rhynchocypris lagowskii)that is widely distributed in the cold fresh waters of East Asia.In the present study,the phylogeography of G.konovalovi and the distribution of its host in the Qinling Mountains are examined.A total of 102 parasite specimens was sequenced for the mitochondrial cytochrome oxidase subunit 1(cox1)gene,and 43 haplotypes were obtained.The ratio of substitution sites(dN/dS)was 0.016 and indicated strongly purifying selection.Haplotype diversity(h)and nucleotide diversity(π)of suprapopulations of G.konovalovi varied widely between distinct localities from the Qinling Mountains.Phylogenetic trees based on Bayesian inference(BI),maximum likelihood(ML)and maximum parsimony(MP)methods and network analysis revealed that all haplotypes were consistently well-supported in three different lineages A,B,and C,indicating a significant geographic distribution pattern.There was a significant positive correlation between genetic differentiation(Fst)and geographic distance(P<0.001).The results of mismatch distribution,neutrality test and Bayesian skyline plot analyses showed that lineages A and B underwent population expansion after the Last Glacier Maximum(LGM)during the Late Pleistocene,while the lineage C underwent population contraction during the Middle to Late Pleistocene.Based on the molecular clock calibration,the most common ancestor was estimated to have emerged in the Middle to Late Pleistocene.Our study suggests that a clearly phylogeography of G.konovalovi was shaped by climatic oscillation and geological events,such as orogenesis,drainage capture changes and vicariance,during the Pleistocene in the Qinling Mountains in central China.

Wind-dispersed seeds blur phylogeographic breaks:The complex evolutionary history of Populus lasiocarpa around the Sichuan Basin

The strength of phylogeographic breaks can vary among species in the same area despite being subject to the same geological and climate history due to differences in biological traits.Several important phylogeographic breaks exist around the Sichuan Basin in Southwest China but few studies have focused on wind-dispersed plants.Here,we investigated the phylogeographic patterns and the evolutionary history of Populus lasiocarpa,a wind-pollinated and wind-dispersed tree species with a circum-Sichuan Basin distribution in southwest China.We sequenced and analyzed three plastid DNA fragments(ptDNA) and eight nuclear microsatellites(nSSRs) of 265 individuals of P.lasiocarpa from 21 populations spanning the entire distribution range.Distribution patterns based on nSSR data revealed that there are three genetic groups in P.lasiocarpa.This is consistent with the three phylogeographic breaks(Sichuan Basin,the Kaiyong Line and the 105°E line),where the Sichuan basin acts as the main barrier to gene flow between western and eastern groups.However,the distribution pattern based on ptDNA haplotypes poorly matched the phylogeographic breaks,and wind-dispersed seeds may be one of the main contributing factors.Species distribution modelling suggested a larger potential distribution in the last glacial maximum with a severe bottleneck during the last interglacial.A DIYABC model also suggested a population contraction and expansion for both western and eastern lineages.These results indicate that biological traits are likely to affect the evolutionary history of plants,and that nuclear molecular markers,which experience higher levels of gene flow,might be better indicators of phylogeographic breaks.

Genetic structure and historical demography of Malus sieversii in the Yili Valley and the western mountains of the Junggar Basin, Xinjiang, China

Malus sieversii, a wild progenitor of domesticated apple, is distributed in western Xinjiang of China, eastern part of Kazakhstan and Kyrgyzstan in Central Asia. To well understand the genetic structure and the his- torical demography of this important germplasm resource, we sampled 15 populations with 110 individuals of Malus sieversii from the Yili Valley and the western mountains of the Junggar Basin, Xinjiang, and sequenced two nrDNA fragments for these 110 individuals. Meanwhile, we modeled and compared species distributions under the current and the Last Glacial Maximum climatic conditions. The results showed that populations of M. sieversfi from Xinjiang had low levels of genetic diversity and genetic differentiation. During the LGM period, populations of M. sieversfi had lost their northern distributions in the western mountains of the Junggar Basin. M. sieversfi has ex- perienced a demographic expansion from the south of the Yili Valley to the north of the western mountains of the Junggar Basin during the warm interglacial epochs. Due to the high sensibility of M. sieversii to disturbance, we proposed more attention should be paid to the M. sieversfi populations in the western mountains of the Junggar Basin.

Genetic Diversity and Population Structure for the Conservation of Giant Spiny Frog(Quasipaa spinosa)Using Microsatellite Loci and Mitochondrial DNA

The giant spiny frog （Quasipaa spinosa） is an endangered species with a relatively small distribution limited to southern China and Northern Vietnam. This species is becoming increasingly threatened because of over-exploitation and habitat degradation. This study provides data on the genetic diversity and population genetic structure of the giant spiny frog to facilitate the further development of effective conservation recommendations for this economically important but threatened species. We examined 10 species-specific microsatellite loci and Cyt b genes （562 bp） collected from 13 wild populations across the entire range of this species. Results of 10 microsatellite loci analysis showed a generally high level of genetic diversity. Moreover, the genetic differentiation among all 12 populations was moderate to large （overall Fs7= 0.1057）. A total of 51 haplotypes were identified for Cyt b, which suggests high haplotype nucleotide diversities. Phylogeographic and population structure analyses using both DNA markers suggested that the wild giant spiny frog can be divided into four distinct major clades, i.e., Northern Vietnam, Western China, Central China, and Eastern China. The clades with significant genetic divergence are reproductively isolated, as evidenced by a high number of private alleles and strong incidence of failed amplification in microsatellite loci. Our research, coupled with other studies, suggests that Q. spinosa might be a species complex within which no detectable morphological variation has been revealed. The four phylogenetic clades and some subclades with distinct geographical distribution should be regarded as independent management units for conservation purposes.

Genetic characterization of the entire range of Cycas panzhihuaensis (Cycadaceae)

Cycas panzhihuaensis L.Zhou & S.Y.Yang(Cycadaceae) is an endangered gymnosperm species endemic to the dry-hot valley of the Jinsha River basin in southwest China.Although the wild C.panzhihuaensis population from Panzhihua Cycad Natural Reserve is well protected and its genetic diversity has been well assessed,the genetic characteristics of populations outside the nature reserve,which face larger risks of extinction,remain unknown.Furthermore,the population genetics and historical dynamics of this endemic and endangered species have not been examined across its entire range.In this study,to analyze the genetic diversity,phylogeographical structure and demographic history of C.panzhihuaensis from all its seven known locations,we sequenced and compared molecular data from chloroplastic DNA(psbA-trnH,psbM-trnD,and trnS-trnG),single-copy nuclear genes(PHYP,AC5,HSP70,and AAT) from 61 individuals,as well as 11 nuclear micro satellite loci(SSR) from 102 individuals.We found relatively high genetic diversity within populations and high genetic differentiation among populations of C.panzhihuaensis,which is consistent with the patterns of other Asian inland cycads.Although no significant phylogeographical structure was detected,we found that small and unprotected populations possess higher genetic diversity and more unique haplotypes,which revises our understanding of diversity within this species and deserves due attention.Analysis of demographic dynamics suggest that human activity might be the key threat to C.panzhihuaensis.Based on the genetic characterization of C.panzhihuaensis,we propose several practical guidelines for the conservation of this species,especially for the populations with small sizes.

Intraspecific Relationships and Variation of Two <i>Lefua</i>Species (Balitoridae, Cypriniformes) in the Tokai Region, Honshu, Japan

Two species Lefua echigonia and Lefua sp. 2 of the eight-barbel loach inhabit the Tokai region of Honshu, Japan. We determined sequences of the mitochondrial D-loop region to elucidate intraspecific phylogenetic relationships and variation in these two species. Lefua sp. 2 represented high intraspecific genetic similarity and complicated haplotype network, but three assemblages were recognized, including specimens mainly from Yahagi, Toyo, and Tenryu River systems, respectively, and named Groups 1 to 3. Divergence of Group 1 from the others was marginally supported, but Group 2 was paraphyletic to Group 3, suggesting the existence of two populations, i.e. Yahagi River population and Toyo-Tenryu River population. Lefua echigonia also represented high intraspecific genetic similarity, and two assemblages with slight genetic differentiation were discernible, including specimens from Shizuoka and southeastern Aichi prefectures and those from northwestern Aichi, Gifu, and Mie prefectures, respectively, and named Groups A and B. Star-like relationships of haplotypes suggested the dispersal origin located in eastern Aichi prefecture. The two species are threatened to extinction and thus we proposed evolutionary significant units for conservation.

Genetic structure and demographic history of Cycas chenii(Cycadaceae),an endangered species with extremely small populations

Geological activities and climate oscillations during the Quaternary period profoundly impacted the distribution of species in Southwest China. Some plant species may be harbored in refugia, such as the dry-hot valleys of Southwest China. Cycas chenii X. Gong & W. Zhou, a critically endangered cycad species, which grows under the canopy in subtropical evergreen broad-leaved forests along the upstream drainage area of the Red River, is endemic to this refugium. In this study, 60 individuals of C. chenii collected from six populations were analyzed by sequencing two chloroplast intergenic spacers(cp DNA:psb A-trn H and trn L-trn F) and two nuclear genes(PHYP and RBP-1). Results showed high genetic diversity at the species level, but low within-population genetic diversity and high interpopulation genetic differentiation. A Bayesian phylogenetic tree based on cp DNA showed that five chloroplast haplotypes were clustered into two clades, which corresponds to the division of the western and eastern bank of the Red River. These data indicate a possible role for the Red River as a geographic barrier to gene flow in C. chenii.Based on our findings, we propose appropriate in situ and ex situ conservation strategies for C. chenii.