Leishmania major parasites and transmitted by Phlebotomus papatasi sandflies. Recently, uncommon clinical manifestations of CL have been reported. Moreover, L. donovani parasites that cause Visceral Leishmaniasis (VL) have been isolated from CL lesions of some patients who contracted the disease in Khartoum State, Central Sudan with no history of travelling to VL endemic sites on south-eastern Sudan. Because different clinical manifestations and the parasite behaviour could be related to genetic differentiation, or even sub-structuring within sandfly vector populations, a population genetic study was conducted on P. papatasi populations collected from different localities in Khartoum State known for their uncommon CL cases and characterized by contrasting environmental conditions.Cutaneous Leishmaniasis (CL) is endemic in Sudan. It is caused by P. papatasi samples collected from different localities in Khartoum State, Central Sudan. Populations from Kassala State, Eastern Sudan and Egypt were also included in the analyses as outgroups. The level of genetic diversity and genetic differentiation among natural populations of P. papatasi was determined using FST statistics and Bayesian assignments.A set of seven microsatellite loci was used to investigate the population structure of FST) between the Sudanese and the Egyptian populations. Within the Sudanese P. papatasi populations, one population from Gerif West, Khartoum State, exhibited significant genetic differentiation from all other populations including those collected as near as 22 km.Genetic analyses revealed significant genetic differentiation (P. papatasi population from other Sudanese populations may have important implication for the epidemiology of leishmaniasis in Khartoum State and needs to be further investigated. Primarily, it could be linked to the unique location of Gerif West which is confined by the River Nile and its tributaries that may act as a natural barrier for gene flow between this site and the other rural sites. The observed high migration rates and lack of genetic differentiation among the other P. papatasi populations could be attributed to the continuous human and cattle movement between these localities.The significant genetic differentiation of Gerif West  Phlebotomus in the Old World are vectors of Leishmania parasites, the causative agent of different clinical forms of leishmaniasis [Female sandflies of the genus maniasis . The dismaniasis .Leishmania donovani complex usually invade the macrophages of the liver, spleen and bone marrow causing the severe symptoms of the fatal Visceral Leishmaniasis (VL) [L. major complex which cause Cutaneous Leishmaniasis (CL) invade the subcutaneous reticulo-endothelial system and cause self-healing lesions that leave life-long scars [The parasites within the sis (VL) ,4; whileng scars ,6.Phlebotomus papatasi  is the principal vector of Leishmania major in the Old World [P. papatasi are expected to show some genetic structuring along their geographical range as a result of adaptation to local habitats and limited gene flow [Scopoli, 786 is thene flow . Such geene flow ,11.P. papatasi used a number of molecular markers and provided contrasting results. For example, isoenzyme analyses clearly separated Western Mediterranean P. papatasi populations from those from eastern countries but, predictably, failed to reveal differences between urban and rural sandfly populations in Morocco [cytochrome b (mtDNA cyt b) and sequences from the second internal transcribed spacer of the ribosomal DNA (rDNA ITS2) were both used to study the population structure of P. papatasi populations from North Africa and the Mediterranean sub-region countries [cyt b sequences suggested some level of genetic differentiation among widely separated populations and revealed a pattern of isolation-by distance between populations from Syria, Egypt, Israel/Palestine and Turkey [P. papatasi from 18 countries from North Africa, the Mediterranean sub-region, Saudi Arabia and India, but revealed no clear phylogeographic structure between those populations. However, signs of restricted gene flow were found among populations from Iran, Egypt, Syria, Yemen and Turkey [Previous studies focusing on potential population sub-structuring in  Morocco . Mitochoountries ,14. The d Turkey . The ND4Phlebotomus papatasi populations in countries from the North-African and the Mediterranean sub-regions. This study confirmed the occurrence of highly significant genetic differentiation between some populations [Microsatellite markers combined with Bayesian statistic analysis were recently used to study the population structure of ulations . HoweverP. papatasi populations could not be demonstrated at the local geographical level, despite evidence suggesting that it may occur. For example, Schmidt and Schmidt (1963) observed marked morphometric variations within the Egyptian populations of P. papatasi suggestive of sub-structuring [et al. reported the presence of polymorphisms at many isoenzyme loci among populations of P. papatasi from Egypt but no significant genetic differentiation could be detected [P. papatasi populations at the local scale has not yet been explored using microsatellite markers which, with their higher mutation rates, should be comparatively much more informative than isoenzymes and other sequence loci [Thus, so far, genetic differentiation among ucturing . Using idetected ,18. It m Schmidt 63 observL. major and transmitted by P. papatasi[L. donovani and transmitted by P. orientalis is known to be endemic in eastern and southern parts of Sudan [L. donovani parasites have been isolated from CL lesions of some patients who contracted the disease in Khartoum State, Central Sudan, with no history of travelling to VL endemic sites [Leishmania parasites [In Sudan, CL is caused by  papatasi,21. Befo papatasi. Visceraof Sudan ,23, withof Sudan . Recentlof Sudan . Moreoveic sites . Recent arasites -29 whicharasites .P. papatasi populations in Sudan from broad to local geographical scale. This was done using a set of 5 microsatellite markers, especially developed for P. papatasi[P. perniciosus a related phlebotomine species [Because different clinical manifestations and the parasite behaviour may also be related to genetic differentiation or sub-structuring within sandfly vector populations ,31, we c papatasi, and giv species .P. papatasi populations were collected from different localities characterized by the distribution of the atypical CL cases. The level of genetic diversity and genetic differentiation among natural populations of P. papatasi was determined using FST and Bayesian assignments. Identifying potential factors leading to genetic differentiation and structuring in P. papatasi populations might improve our understanding of the epidemiology of the disease and help develop appropriate control strategies.P. papatasi, which are typically easier to identify morphologically than females in this and other phlebotomine species, were collected from five different localities in Sudan separated by 20 to 440 Km .Sirougia is a well-known endemic focus of CL where previous epidemic occurred beside some of the uncommon CL cases came from the nearby villages . Gerif West is not known to be endemic for cutaneous leishmaniasis. However, some of the uncommon CL and CL due to L. donovani cases were from this site. The fifth Sudan study site was the village of Wad Shariefai (SHR) in Kassala State, Eastern Sudan which is located in the immediate vicinity of the dry savannah habitats of the Tajouj Forest. Wad Shariefai village is a site where three closely-related species belonging to the subgenus Phlebotomus phlebotomus  were found sympatrically. Due to the far location of this Eastern site it is expected that P. papatasi from this site would be genetically different from P. papatasi populations from Khartoum Sate, Central Sudan.A total of 126 male specimens of et al. and Aransay et al. [2 and 0.5U Taq ® DNA polymerase  in 20μL total reaction volume. The thermal cycling conditions were an initial hold at 95°C for 2min, followed by 30 cycles of 94°C for 30s, 55°C for 30s and 72°C for 30s and a final extension at 72°C for 5min. For amplifications involving the primer AA13, a touchdown PCR programme was performed. The programme steps were as mentioned above except that the number of cycles was increased to 35 and the starting annealing temperature was 59°C, which was then decreased by 1°C for the first five cycles. For correct sizing of polymorphic PCR products, the forward primers were labelled with 5’-fluorescent tags. Details of the primers sequences; repeat motif, annealing temperature, and fluorescent labelling are given in Table Samples were processed and DNA was extracted as previously described . Microsay et al. ,33. The Pap1, Pap2, AA13, and AA82, while set B was a mix of Pap4, AA18, and AA24. About 16 samples with different dilutions  were initially genotyped at Eurofins MWG Operon Company, Ebersberg, Germany. According to the results from the first genotyped samples, a suitable dilution factor was chosen and the two sets of sample microplates were prepared, tightly packaged in dry ice and submitted for subsequent genotyping in the aforementioned company.PCR product multiplexing was done according to the amplified allele size and the fluorescent dye labelling of each product in order to create two primer sets. Set A contained a mix of Pap2 which consist of dinucleotide repeats), the allele sizes were expected to differ predominantly by multiples of three nucleotides. Therefore any exceptions were treated as a result of insertions or deletions (indels) of odd numbers of bases in the flanking region.Because the repeat arrays of all the microsatellite loci consist of trinucleotide repeats (except for http://www.microchecker.hull.ac.uk[http://cmpg.unibe.ch/software/arlequin3[FST) between populations and the level of gene flow (Nm) among them, as well as assessing whether patterns of genetic divergence among P. papatasi populations followed an isolation-by-distance pattern. The genotype assignment test implemented in ARLEQUIN programme was used to assign individual sandfly genotypes to their population of origin by computing the log-likelihood of each individual multi-locus genotype in each population sample, assuming that the individual belongs to that population [P <0.05) was obtained with 1000 permutations and 100000 Markov chain steps.The data were screened for the presence of stutters, null alleles and large allele dropout using the software Microchecker version 2.2.3 available at ull.ac.uk. Then tharlequin3. The propulation . The sigP. papatasi populations were further investigated using the Bayesian clustering algorithm implemented in the software STRUCTURE version 2, available at http://pritch.bsd.uchicago.edu[Hidden population sub-structure within icago.edu. The proPap1, Pap2 and Pap4 showed varying degrees of heterozygosity among the different study populations; although the two markers Pap3 and Pap5 did not work well with the Sudanese P. papatasi populations even after several trials with different amplification conditions. Amongst the markers for P. perniciosus, AA13, AA18, AA24 and AA82, were successfully amplified in P. papatasi. However, these markers tended to be less polymorphic than in the original species. The total number of alleles per locus ranged from 1 to 5 with locus AA18 being monomorphic in all populations. In addition, each of the two loci AA13 and AA24 were found to amplify two different polymorphic loci with distinct allele size ranges  which were not found to be in linkage disequilibrium. This suggests that these two loci were the results of duplications to different unlinked regions of the genome; thus they were treated as two additional loci thereby increasing the total number of loci examined to nine  of 0.355 ± 0.226 . Meanwhile, MO population was the most heterogeneous among all the Sudanese populations (40% heterozygosity). The most uniform population in the study was TR with 31.5% heterozygosity  and the GW population was monomorphic for AA13a . Observed heterozygosity varied from 0.000 to 0.960 while expected heterozygosity ranged from 0.039 to 0.660. The observed heterozygosity was higher than the expected value in 25 cases and lower than expected in 13 cases of the comparisons made between FST , ranged from 0.007 to 0.233. Significant FST estimates (FST =0.147-0.233) were observed between all P. papatasi populations from Sudan when compared to those from Egypt (Table P. papatasi populations from GW and SHR (FST =0.033), whereas moderate but non-significant differentiation (0.011-0.014) was observed between SHR and other populations from Khartoum State . Within Khartoum State, significant FST (0.100 to 0.125) estimates were also observed between GW and MO, TR, and SR populations. The populations from MO, TR, and SR showed little genetic differentiation amongst themselves as indicated by low FST values ranging from 0.007 to 0.046. A neighbour-joining tree based on all pair-wise FST estimates confirmed that the GW population, whilst being distinct from all other populations in Sudan is comparatively more similar to SHR than to nearer populations  between linearized estimates of genetic differentiation FST (FST/(1-FST)) and geographical distances (log km) . For example, the total number of alleles per locus for these loci ranged from 4 to 9 in 13 Spanish populations of P. perniciosus with a sample size ranging from 13 to 38 but here in our study allelic richness ranged from 1 to 5. Surprisingly, two primer pairs AA24 and AA13 amplified more than one locus in P. papatasi with distinct allele size-ranges and genetically unlinked, indicating a duplication of the genome in the region of those repeat sequences in P. papatasi. These loci were polymorphic in most P. papatasi populations, which contrasts with previous results in P. perniciosus where locus AA24 was not significantly polymorphic in 12 out of 13 studied populations, and locus AA13 was monomorphic in 5 of them [Here, given the low number of microsatellite loci available for  of them .These vaP. papatasi populations sampled from Central and Eastern Sudan, and Egypt. This result is due to the unexpected significant genetic differentiation found at the local geographical scale between the Sudanese populations that interfered with broader geographical patterns of genetic differentiation. Importantly, the P. papatasi population from GW was significantly genetically differentiated from that of SHR, located 400km away in Eastern Sudan. But the same population (GW) was also significantly genetically differentiated from the other 3 populations from Khartoum State  despite these populations being only 22-27km apart. It is noteworthy that GW locality is isolated by the River Nile and its tributaries from the other collection sites in 3 directions . Furthermore there are no direct routes of transportations between this and other locations in Khartoum State . Previous studies demonstrated the presence of different genetic groups among L. major strains from different locations in the same country which could be related to different P. papatasi subpopulations [Interestingly, no significant pattern of isolation-by-distance was observed amongst the 6 ulations ,44. WhetP. papatasi is known to be capable of flying up to 2km per night in open desert where they can be also be carried by air currents. Moreover, sandflies usually swarm over their host during feeding and usually mating takes place on the host; thus they may be capable of movement along with animal herds [It is well known that populations in close proximity are genetically more similar than distant populations and that high gene flow usually precludes local adaptation . Therefoal herds ,47. It sal herds  may contP. papatasi indicate that this species can withstand extreme environmental conditions and survive even in areas with extreme temperature and aridity [The close genetic similarity among sandfly populations from Khartoum State  and Wad Shariefai (SHR), Eastern Sudan, despite the large geographical distance, could be attributed to the prevailing environmental conditions such as wind speed, humidity and temperatures that may play an important role in sandfly dispersal . Althoug aridity -55. The P. papatasi populations separated by large geographical distances has also been reported in previous studies based on different molecular markers [P. orientalis in Sudan using 30 RAPD markers. In addition, populations from Dinder National Park, South-Eastern Sudan were found to be genetically very similar to population from SR, Khartoum State [P. papatasi populations [Low genetic differentiation between  markers ,56,57. I markers . It is wum State . It has ulations .P. papatasi colony also showed unexpected high degree of heterozygosity compared to the Sudanese field populations, although long colonization and adaptation to laboratory conditions are usually thought to minimize the genetic variability in laboratory populations [P. papatasi colony from Sinai, Egypt even after the 33 generations [This study also revealed significant genetic differentiation and restricted gene flow between the Egyptian EGY population and all the Sudanese populations, which suggests some degree of isolation-by-distance despite the overall lack of a significant correlation between the geographical distance and estimates of genetic differentiation. The Egyptian ulations ,61. Unfoerations .Phlebotomus papatasi population from the other Sudanese populations may have important consequences on the epidemiology of leishmaniases in Khartoum State and warrants further investigation. Such genetic variations could potentially correlate with differences in the vectorial capacity of the sandfly vector to transmit different strains of the parasites or even hybrids. Future studies using a larger number of microsatellite loci, more populations and more specimens per population are also needed to further elucidate patterns of genetic differentiation of P. papatasi in Sudan. This knowledge would improve our understanding of the epidemiology of the disease in Sudan, and ultimately would result in improved disease and vector control programs.The significant differentiation of the GW Phlebotomus Phlebotomus were found sympatrically; EGY: Colony samples from Egypt; HWE: Hardy-Weinberg equilibrium; He: Expected heterozygosity; Ho: Observed heterozygosity; SD: Standard deviation; Wright’s FST: Measure of population differentiation; FIS: Inbreeding coefficient; (Nm): Rate of gene flow among populations.CL: Cutaneous leishmaniasis; VL: Visceral leishmaniasis; MO: Moyleih, a semi-desert area on the west bank of the White Nile; TR: El-Trais, an irrigated area on the west bank of the White Nile; SR: Sirougia village, a green rich irrigated area on the east bank of the River Nile; GW: Gerif West, a residential area on the west bank of the Blue Nile; SHR: Wad Shariefai village, in Kassala State, Eastern Sudan where the three closely-related species belonging to the subgenus The authors declare that they have no competing interests.NK carried out the field and laboratory work, microsatellite analysis and drafted the manuscript. MA, FA and DE helped in designing the field study, interpretation of the results and editing the manuscript. FT helped with the data analyses, interpretation of the results and editing the manuscript. All authors read and approved the final version of the manuscript.


Rana [Lithobates] pipiens] has undergone significant declines particularly in the western United States and Canada. Leopard frog population losses in Nevada are largely due to habitat fragmentation and the introduction of nonnative fish, amphibian, and plant species. Only two populations remain in the Truckee and Carson River watersheds of western Nevada which represents the western boundary of this species range. We used sequence data for an 812 base pair fragment of the mitochondrial NADH dehydrogenase 1 (ND1) gene to support a native origin for western Nevada populations. All frogs had a single haplotype (W07) from the distinct western North America ND1 haplotype clade. Data from seven polymorphic microsatellite loci show that Truckee and Carson River populations are highly differentiated from each other and from leopard frogs collected from eastern Nevada sites. Lack of gene flow among and distinct color morphs among the western Nevada populations likely predates the current geographical isolation. Comparisons with other peripheral L. pipiens populations show western Nevada populations have similar levels of gene diversity despite their contemporary isolation . Restoration of leopard frog populations in these watersheds will be challenging given well-entrenched nonnative bullfrog populations and major changes to the riparian zone over the past century. Declines of once common amphibian species has become a major conservation concern. Contemporary isolation of populations on a species range periphery such as the leopard frog populations in the Truckee and Carson rivers further exacerbate extirpation risk as these populations are likely to have fewer genetic resources to adaptively respond to rapidly changing biotic and abiotic environments.Global amphibian declines suggest a major shift in the amount and quality of habitat for these sensitive taxa. Many species that were once widespread are now experiencing declines either in part of or across their historic range. The northern leopard frog ( Global amphibian declines were first documented in the 1980s and more than 500 amphibian populations were designated “of concern” by 1993  is widely distributed across North America  have also designated the northern leopard frog as a sensitive species. Most recently, the United States Fish and Wildlife Service issued a 90-day finding to list the western populations of northern leopard frogs as threatened under the United States Endangered Species Act . Although it is tempting to predict that individuals with high heterozygosity levels at neutral markers, such as microsatellites, will also have greater relative fitness, we recognize that such an association is far from certain. There is, however, some support for this prediction in the literature  and for neutral markers (N = 21) with clutch size and number of eggs produced by males in the Blue tit (Cyanistes caeruleus) and found a clear relationship between heterozygosity at the neutral markers and reproductive success, but not for microsatellites classified as “functional.” Wetzel et al. ((Passer domesticus) was associated with reproductive performance . These authors interpret the result of significant heterozygosity-fitness correlations as an indication that the effects of heterozygosity may be far more significant than previously thought , Tamukai et al.  are characterized by dark spots on their dorsal side and dorsolateral folds. These frogs are considered medium sized and are typically green or brown  with Leopard frogs were found in the Spring and Lake valleys of eastern Nevada  gallery forests and extensive invasion of tall whitetop (Lepidium latifolium L.) together with the introduction of nonnative amphibian species such as the North American bullfrog (L. catesbeiana). The only extant native population on the Truckee River located for this study was found on a private ranch near Wadsworth, Nevada on the Pyramid Lake Paiute Tribe Reservation, approximately 33 miles northeast of Reno, Nevada, which was also occupied during the Hitchcock survey  resulted in changes to the natural hydrology of the lower river . Derby Dk survey . Fifty-shttp://www.otisbay.com).The Nature Conservancy (TNC) has purchased multiple properties along the Truckee River for restoration activities aimed at returning the river to its natural course and function. TNC bought the 305 acre McR on the lower Truckee River approximately 15 miles east of Reno, Nevada, in 2002. Here, they restored a natural channel meander, created ponds, and backwater habitats. Frogs that developed from egg masses collected from a natural population on the Pyramid Lake Paiute Tribe Reservation and reared in captivity were released on the McR site from 2006 to 2009. The eight ponds at McR were extensively searched during this study; however, only four adult leopard frogs were found at a single pond location  due larghttp://www.epa.gov) resulting from the Comstock Mining era in the 1860s to 1880s. Mercury deposited atmospherically into wetlands has been shown to cause severe deformities and mortality in developing southern leopard frogs   is working with multiple governmental and private agencies to improve the conditions of the Carson River, including a comprehensive stewardship plan encompassing the entire Carson River drainage. TNC has become involved in restoring a portion of the river at their 805 acre property, River Fork Ranch (RFR). Sixty-one frogs were collected along the banks of the river and small side channels at this site  and southern Idaho (N = 2) that were deposited in the museum collection at California State University, Northridge , frogs were held around the waist with hind limbs fully extended to prevent unnecessary struggling that could result in harm to the animal. Weight, snout-to-vent length, sex, and age were recorded for each individual along with Universal Transverse Mercator (UTM) coordinates from a global positioning system or latitude–longitude coordinates from United States Geological Survey 7.5-min series topographical maps of the area. A photograph was taken of each individual frog while they were being held as an additional aid in identification during data entry and in an attempt to have comparable images among frogs later used in the spot and color polymorphism analysis.Visual encounter surveys were conducted along the edges of ponds, streams, and rivers within each of the study sites where Guidelines from Leyse et al.  were useμL reaction volumes containing 1.25 μL of each primer, 5 μL 2× Qiagen master mix with taq polymerase, and 5 μL of 15–20 ng/μL genomic DNA. PCR cycle parameters consisted of an initial denaturation at 95°C for 15 min, followed by 35 cycles of denaturing at 94°C for 30 sec, annealing at 54°C for 90 sec, and a final 90-sec extension at 72°C. PCR product was purified using ExoSAP-IT  and then sequenced in both directions on an ABI 3730 DNA Analyzer, Nevada Genomics Center (http://www.ag.unr.edu/genomics), using the ABI BigDye Terminator Cycle Sequencing Ready Reaction Kit v3.1 .DNA was extracted from toe clippings using a DNeasy Blood and Tissue Extraction Kit  according to the manufacturer's specifications and then quantified using a Labsystems Fluoroskan Ascent fluorometer. An 812 base pair fragment of the mtDNA NADH dehydrogenase subunit 1 (ND1) gene was amplified using polymerase chain reaction (PCR). We used the MB77 forward (Hoffman and Blouin μL Multiplex PCR Master Mix (Qiagen), 0.05 μmol/L of each tailed forward primer and 0.5 μmol/L reverse primers, and 20 ng DNA in a 16 μL reaction volume. The first PCR was a touchdown allowing for complete amplification of the DNA by using varying annealing temperatures. PCR parameters were as follows: 15 min hot start at 94°C, followed by 35 cycles at 95°C for 30 sec, annealing temperature for 90 sec beginning at 65°C and decreasing by 0.3°C each cycle ending at 54.5°C, and 72°C for 30 sec followed by a final extension of 62°C for 30 min. The second PCR was to label the initial PCR product with the M13 fluorescent dye. The second reaction consisted of 0.3 μL of 10 μmol/L M13, 8 μL Multiplex PCR Master Mix (Qiagen), and 0.6 μL of each specific reverse primer in a 16 μL reaction volume. Parameters for the second PCR included a 15 min hot start at 94°C followed by 25 cycles of 94°C for 30 sec, 55°C for 90 sec, 72°C for 30 sec, followed by a final extension of 62°C for 30 min. Cycling conditions for the two prelabeled primer sets in panel B were, a 15-min hot start at 94°C, 33 cycles of 94°C for 30 sec, 57°C for 90 sec, 72°C for 30 sec, and one cycle of 62°C for 32 min. Fragment analysis was completed on a Perkin Elmer Applied Biosystems 3730 Genetic Analyzer  at the Nevada Genomics Center using 1 μL diluted PCR product added to 19 μL of HiDye Formamide with LIZ (500) molecular marker. ABI GeneMapper software (version 3.7) was used to score alleles.Seven nuclear microsatellite loci were used for this study . ForwardNRFR = 61; NFJ = 56). No frogs with kandyohi color variation were found. Photographs for 10 of the frogs sampled from each of two sampling localities (RFR and FJ) were chosen to assess total spot number and average spot size (area and perimeter). Only frogs with spots were included in the spot size analysis . If spots were so close together that they were touching and the complete border around each one was not obvious, those spots were counted as one. Images were chosen based on whether all spots could be accurately counted and clearly distinguishable . The spot size data were analyzed using ImageJ version 1.43 (Rasband We measured (1) area and perimeter size per spot, (2) total spot number per frog, and (3) color variation to assess phenotypic variation within and among the western Nevada populations  version 4.2 was used to edit and align raw sequence data obtained from the ABI 3730 DNA Analyzer. We compared the ND1 haplotypes of frogs collected for this study with haplotype data for FST) (Wright RS), the inbreeding coefficient (FIS) and determine whether linkage disequilibrium among loci was present within populations. We estimated gene diversities  using Microsatellite Toolkit in Excel. For the eastern Nevada samples we assessed allelic diversity, richness and private alleles only as these samples were collected from multiple locations (Hitchcock We used FSTAT 2.9.3.2 Goudet  to test ) Wright , calculak clusters or subpopulations. Individuals sampled from the Truckee and Carson rivers populations and 16 individuals from eastern Nevada were included in this analysis. The underlying assumptions for population modeling in STRUCTURE are that the populations sampled are in HWE and that the loci used to characterize individuals and populations are unlinked and at linkage equilibrium within the populations  method developed by Evanno et al. (k). This method calculates the largest change in the natural logarithm of the probability of the data (logeP[D]) between each pair of k and k-1 for all tests of k.We used the program STRUCTURE  for the Truckee and Carson rivers populations using the linkage disequilibrium method (Hill D*) which is Burrow's composite measure of disequilibrium (Campton r)  among alleles were estimated byWe calculated effective population size ] where S is the sample size  and single-step mutation model (SMM), and the Wilcoxon sign-rank test in the program BOTTLENECK  between the RFR and FJ populations. The same individuals used for total spot number analysis were used for the spot size analysis. A linear regression analysis using the statistical package R was used to test for a significant relationship between frog length and number of spots.An independent We successfully sequenced all but one individual from eastern Nevada. A single mitochondrial haplotype (W07) that had been previously described by Hoffman and Blouin  which isP = 0.00143, indicative adjusted nominal level [5%], based on 700 randomizations). Although average levels of heterozygosity were higher in the FJ population . Allelic richness was similar for both the RFR (RS = 2.24) and FJ (RS = 2.25) populations. The CA population had an average heterozygosity of 0.427 and an average allelic richness of 2.10. Allelic richness and diversity for the eastern samples were higher than the two western Nevada populations. However, because the eastern Nevada sample is a composite of frogs from multiple sampling locations, we could not test whether this difference was significant. Private alleles were found primarily in individuals from the eastern basins , but were also present in the Truckee and Carson rivers populations  and Carson (RFR) rivers populations , whereas the Truckee and Carson River populations were highly differentiated . There were no significant FIS values for either the Truckee or Carson River populations . Two genotype clusters (k = 2) represented the best fit of the data  or the Carson River RFR population  based on 1000 iterations. However, the FJ population had a shifted mode, which can be a more sensitive test of recent genetic bottlenecks  or Carson (P = 0.392) rivers. There was no significant difference between mean number of spots per frog between the FJ (10.1 ± 1.286) and RFR (10.7 ± 1.636) populations . In addition, there was no significant difference between the mean size of the area of the spots  or the perimeter of the spots  between the natural populations.Visual inspection of the color patterns between the frogs sampled on the Truckee and Carson rivers revealed distinct differences. The majority of the RFR frogs sampled  were green and the other 16% were brown with no burnsi or kandyohi color morphs. Of the 56 frogs sampled from the FJ population, 91% (51 of 56) were brown, with the rest being burnsi color morphs with no green color morphs observed. We found no relationship between frog length and number of spots for frogs sampled in either the Truckee  identified in the eastern Nevada frogs sampled by Hoffman and Blouin . Two othThe W07 haplotype is also the most ancestral haplotype in the western ND1 haplotype clade and one of the more common haplotypes found in the western United States as well as western Canada and one population in Ontario (Hoffman and Blouin k = 3) and the private allele analyses, which is not surprising given the geographic separation and the fact that leopard frogs were never widespread in intervening locations in central Nevada. The Carson and Truckee River populations are also highly differentiated , which can be attributed to geographic distance (approximately 90-km straight line distance across various landscape types), and/or contemporary isolation. A high global FST of 0.248 was found among Canadian leopard frog populations   is currently of concern as bullfrogs often inhabit the same ponds that leopard frogs use and also prey upon them in their various life stages BCMELP . DeclineIn the short term, individuals from the Carson River population should be moved to additional locations as a risk-spreading strategy and to preclude losses of genetic variation due to the effects of population isolation. Allendorf and Luikart  suggest Batrachochytrium dendrobatidis (Bd) and infects keratinized epidermal cells of amphibians such as the oral discs of tadpoles  is needed to rule out the presence of unsampled populations. If additional populations are found, a landscape genetics approach could be used to test for gene flow and a metapopulation dynamic (Moore et al.


Ischnuraelegans, which has been the target of manyecological and evolutionary studies. We addressed the following questions: (i)Is the population structure affected by longitudinal or latitudinal gradients?;(ii) Do geographic boundaries limit gene flow?; (iii) Does geographic distanceaffect connectivity and is there a signature of past bottlenecks?; (iv) Is thereevidence of a recent range expansion and (vi) what is the effect of geographyand climatic factors on population structure? We found low to moderate geneticsub-structuring between populations , and an effect of longitude, butnot latitude, on genetic diversity. No significant effects of geographicboundaries (e.g. water bodies) were found. FST-andDest-values increased with geographic distance; however, there was noevidence for recent bottlenecks. Finally, we did not detect any molecularsignatures of range expansions or an effect of geographic suitability, althoughlocal precipitation had a strong effect on genetic differentiation. Thepopulation structure of this small insect has probably been shaped by ecologicalfactors that are correlated with longitudinal gradients, geographic distances,and local precipitation. The relatively weak global population structure andhigh degree of genetic variation within populations suggest that I.elegans has high dispersal ability, which is consistent with thisspecies being an effective and early coloniser of new habitats.Identifying environmental factors that structure intraspecific genetic diversityis of interest for both habitat preservation and biodiversity conservation.Recent advances in statistical and geographical genetics make it possible toinvestigate how environmental factors affect geographic organisation andpopulation structure of molecular genetic diversity within species. Here wepresent a study on a common and wide ranging insect, the blue tailed damselfly The spatial structuring of intraspecific neutral genetic diversity contains importantinformation about both historical and current evolutionary processes. For example,extensive gene flow will constrain divergence by preventing local geneticdifferentiation, whereas reduced dispersal and philopatry are expected to causegenetic subdivision Many species in Europe have wide-ranging geographic distributions and several studieshave demonstrated geographic signatures of within species' genetic diversitye.g. , often eIschnuraelegans . This smalldamselfly species is a well-investigated study system in evolutionary ecology,particularly in terms of mating interactions, sexual selection, female colourpolymorphisms, frequency-dependent selection and sexual conflict I.elegans, we investigated the molecular structure of 22 populations overmost of the western part of this species' geographical range  and investigated if we could find evidence for asignature of past historical bottlenecks. Finally, we evaluated several differentecological scenarios by relating environmental factors and their interactions topopulation specific FST-values of I. elegans, namely therole of range expansion (latitude and longitude), geographic suitability  and climatic suitability .In this study, we investigated the genetic diversity and population structure of acommon and wide-ranging insect, the blue taileddamselflye:s), increased the degree of genetic differentiation of populations.Overall, these results confirm the emerging view that this species is a fast andefficient colonizer of disturbed habitats, and commonly undergoes populationextinctions and re-colonisations The results in this study suggest that this small insect has a weak geneticpopulation structure across a major part of its geographic range, and that thegenetic structure does not seem to be severely affected by large geographicbarriers. Nevertheless, we found that high local precipitation rates (e.g. floodingevents), which presumably reduce the local effective population sizedamselflies were collected for subsequent genetic analysis  were sampled in Spain , Portugal  and Morocco  and kept for molecular analysis. Captured individuals werestored in 90% ethanol in small plastic tubes until DNA extraction.Additional sampling details are given in Adults of the damselfly 2, 1.25 nmoldNTP, 0.5 U Ampli-taq polymerase and10–20 ng template. The conditions were as follows:initial denaturation step of 94°C for 2 minutes, then 35 cycles at 94°Cfor 30 s, touch down from 62–58°C for 30 s, 72°C for30 s followed by 72°C for 10 minutes. Multiplex primer reactions wereperformed for combinations of primers with matching annealing temperatures butdiffering size ranges and dye labels, then mixed with a labelled size standardand electrophoresis was conducted on an ABI PRISM 3730 Genetic Analyzer (AppliedBiosystems). Resulting data were analyzed with GeneMapper 3.0 (AppliedBiosystems) for internal standard and fragment size determination and forallelic designations. The same size standard was used on all samples analyzedfor each marker.To extract DNA, the head of each damselfly was used, to avoid contamination withgut parasites and (or) sperm. Heads were subsequently dried and homogenizedusing a TissueLyser (Qiagen). DNA was extracted from the powder by proteinase Kdigestion followed by a standard phenol/chloroform-isoamylalcohol extractionE), observed heterozygosity(HO), and allelic richness for each locus, using the program FSTATversion 2.9.3 ST (θ). FST was used rather than RSTST-estimate was evaluated by permuting genotypes amongsamples and calculating 95% CIs by bootstrapping over loci (number ofpermutations was set at 1000). This method assumes Hardy-Weinberg equilibriumwithin populations. In the pairwise tests of population differentiation, thenominal statistical significance value of 5/1000 was adjusted for multiplecomparisons using the Bonferroni correction when accounting for multiple testingto minimize type I errors.Genetic diversity within populations was assessed in terms of allele frequencies,expected heterozygosity to one (complete differentiation), and simulations have shown that it is anunbiased estimator of differentiation, and outperforms FST, over arange of sample sizes and for markers with different numbers of alleles(including highly variable microsatellite loci)est across loci.In addition to FI. elegans populations and the four I.graellsii populations, since a NJ tree  did not result in a stronglysupported tree (results not shown). STRUCTURE uses a Bayesian Markov chain MonteCarlo (MCMC) method to find the number of genetic clusters  that, based on thelikelihood of the individuals' genotype to belong to these geneticclusters, minimizes deviations from Hardy–Weinberg equilibrium (HWE) andlinkage disequilibrium (LD)K), ranging from oneto ten; and for each K, 20 iterations were run. In this way,multiple posterior probability values (log likelihood (lnL) values) for eachK were generated, and the most likely Kwas evaluated by the ΔK-method following Evanno et al.Ksand the corresponding variance of lnL of each KWe used the Bayesian statistical framework provided by the program STRUCTUREversion 2.2.3 individuals were also inferred with the R-package K), as well as the assignment ofindividuals to the inferred clusters in a single step, treating the number ofclusters as unknown. The analysis was run to identify the geographic structuresamong populations and barriers to dispersal using (i) all 22 I.elegans populations and (ii) all 22 I. eleganspopulations and the four I. graellsii populations. To determinethe number of genetic clusters, four independent runs were implemented for eachanalysis using 100,000 MCMC iterations with a burn-in period of 20,000 and athinning value of 100 and then the model with the highest posterior probabilitywas chosen. K was set toKmin = 1,Kinitial = 4 and aKmax = 22 or 26 clusters, respectively, whilefiltering for null alleles during the run. It should be noted that the filteringwas just a precautionary option, and that the model did not change when thisoption was not selected. However, this option allowed us to calculate thefrequency of null alleles in our dataset. Consistent with previous results Clusters of To examine the distribution of the genetic variance among the clusters identifiedby GENELAND, an analysis of molecular variance (AMOVA) was conducted usingARLEQUIN version 3.11 ST- and Dest-values, but using FST /(1-FST) and Dest /(1- Dest), respectivelyI. elegans populations . Tostatistically determine if genetic and geographic distances between populationsare significantly correlated, a Mantel test on the genetic and geographic matrixwas performed , using the program Isolation by Distance(Isolde) web service (http://ibdws.sdsu.edu/~ibdws/).Isolation-by-distance, which is defined as a decrease in the genetic similarityamong populations as the geographic distance between them increases [sensu46], was investigated by correlating the pairwise differentiation  and provide information on how geneticallydistinct a population is relative to other populations in the sample. Forexample, under a model of diffusive dispersal following a single colonizationevent, populations furthest from the origin would have the highestFST-values due to the cumulative effects of drift from repeatedfounder events. Population-specific FST-values were related toenvironmental factors using a generalized linear model. We chose this approachas our primary method because it tests multiple variables simultaneously. Assuggested by the authors, we used the reversible jump MCMC method, and 10 pilotruns of a length of 5,000 as burn-in prior to drawing samples from a chain of50,000 in length, separated by a thinning interval of 50. All combinations ofvariables were considered and models were evaluated using estimates of posteriorprobability, the 95% highest probability density interval (HPDI). Theoutput also calculates the cumulative probability for each factor individually,so that the factor importance can be compared easily. GESTE can currently be runwith two factors and their interaction at a time, and we run three differentscenarios.To identify the environmental factors that might determine the genetic populationstructure of http://www.worldclim.org/bioclim).First, we investigated if there was any signature of gradual population expansionusing the factors latitude and longitude in the analysis I. elegans populations andranged from 0.60–0.77 and 0.70–0.83,respectively . All the investigated locicontributed to this population differentiation . The pairwise population differentiation ranged betweenFST = 0.00024 toFST = 0.14. Twenty-eight comparisons of thesewere statistically significant after applying a Bonferroni correction formultiple comparisons . The pairwise population differentiation rangedbetween Dest = −0.0085 toDest = 0.5412 .The main difference between the values was that overall differences increased,in particular the interspecific differences, when using the Destformula (see est) between populations isactually higher than suggested using FST-comparisons alone, andhighlights the need to use the more unbiased estimation of Dest whenevaluating the degree of differentiation between population pairs As mentioned above, we also calculated the Dntiation showed aT-values ; however= 0.5412 . There wmula see  and3. TI. elegans populations revealed a significant longitudinalcline  is given in–10 genetic clusters(K = 1–10) is shownin I.graellsii group and three relatively diffuse genetic groupings ofI. elegansthat fall into a geographic pattern that consistsof (i) northern and central , (ii) western and southern (Spain), and (iii)eastern populations and theseidentified fiveand six clusters, respectively, of which the first five wereidenticalbetween analyses (I.elegans populations plus four I. graellsiipopulations) contained the four I. graellsii populationsinwestern andsouthern Spain and Morocco  with 4.30% among the five groupsand 2.74% among the populations within groups  andDest/(1-Dest), respectively) is correlated with thematrix of geographic distances, we did indeed find that the genetic populationdifferentiation followed an isolation-by-distance pattern  of I. eleganspopulations show a weak signal of a heterozygositydeficiency, suggesting thatthey are not at a mutation–drift equilibrium, but that there has been arecent expansion in population size or a recent influx of rare alleles fromgenetically distinct immigrants. This trend is also supported by the overalllower mean of the heterozygositydeficiency compared to the heterozygosityexcessfor all populations, which was 0.3 and 0.8, respectively.The program BOTTLENECK showed that only one of the populations examined  showed a heterozygosityexcess, while four of the populations showed aheterozygositydeficiency . This su–west) was also moreimportant than latitude (south–north) was further supportedwhen looking at the data fit with just the factors alone, which resulted in aposterior probability of 0.117 and 0.056, respectively. Similarly, neither thedistance to coast or altitude (geographic suitability) was strongly correlatedto the population-specific FST-values. Out of the two variables, themodel including the constant term and distance performed better than the modelcontaining the constant and altitude . In both ofthese aforementioned tests (range expansion and geographic suitability), themodel that only included the constant term had the highest posterior probability, while the model containinglatitude and the constant achieved a much lower posterior probability (0.047).The finding that longitude , and theDest-values (meanDest = 0.12) ofthe pairwise genetic population differentiation, albeit higher, were also low tomoderate. Together these results suggest a relatively high degree of geneticconnectivity across the species' geographic range in Europe, oralternatively, a recent population expansion. Odonates (dragonflies anddamselflies) are thought to be relatively good dispersers, and often leave theirnatal habitat after emergence in the search for new ponds and/or rivers Ischnura are known to begood dispersers, as their presence in remote archipelagos demonstrates Ischnuraeleganshas been described as an opportunisticdamselfly species that is typically found in quite disturbed environments, suchas human-made artificial ponds I.elegans exists in environments that experience strong temporal andspatial heterogeneity, leading to strong fluctuations in local populationdensities, the species experiences large fluctuations in both the strength anddirection of selection. This is probably partly the reason for why localpopulations go extinct at a high rate, i.e. there is high population turnover inthis species. Some of the data in this study  also support the general picture that I. elegans is anopportunistic insect species that rapidly colonises newly created habitats I. elegans.Ischnurahastata, for example, is one interesting species inthis respect, as it has been captured on nets mounted on airplanes and has alsocolonised the Galapagos islands –20individuals, which is lower than the recommended samplesize for stable FST- and Dest-estimates. Despite thisshortcoming, we would like to highlight that the strength of our study laid inthe high number of populations analysed and the large geographic area covered,which allowed us to investigate large scale environmental patterns andclines.FLeucorrhiniacaudalis shows a slightly higher degreeof microsatellite differentiation (FST = 0.130)between populations in Switzerland, and a study on the southern damselflyCoenagrionmercuriale by Watts et al. ST-value of 0.17. The two aforementioned studies covered amuch smaller geographic area than the present study and are both relatively rareand threatened species, unlike I. elegans.TheFST-values for these two rarer species strengthens ourconclusionsthat the more abundant and dispersive speciesI.elegansconsists ofpopulations that are connected by a high degreeof gene flow, even over large geographic areas, or has been recently expandingin the area. A third study by Watts et al. Erythrommaviridulumreports similarly lowFST-values as in our study, and this study was carried out on alarge geographic scale, including samples from the UK, Germany, Netherlands,Belgium and France. Watts et al E. viridulumappears to be capable of relatively longdistance dispersal, even over inhospitable habitat.Erythrommaviridulumis also a species that is common andexpanding northwards, including recent establishment in southern Sweden, and hasthus a similar ecology as I. elegans, compared to theaforementioned rarer species with more fragmented and localized populations.Molecular studies on other odonate species show a higher degree of geneticdifferentiation.For example, a study by Keller et al. e) and concomitant increased potential for genetic drift I. elegans affect gene flow and how this interactswith the selection regimes experienced at the edge of their range. Althoughmicrosatellite loci are not directly under selection, due to the fact that theyare non-coding genes, strong local selection at range limits [c.f. 65]would be expected to lower the effective population sizes and hence increase thepotential for genetic drift Populations that contributed most to significant between-population differenceswere found at the edge of the sampling range . These iI. elegans, it is likely thatenvironmental gradients (e.g. in temperature and precipitation) together withfluctuations in population size (due to stochastic events and habitatfragmentation) are responsible for the heightened genetic differentiation ofperipheral populations relative to the rest of the populations , we were able to detect six clusters . The absence of any significantpattern of isolation-by-distance in their study might indicate a relatively lowdegree of statistic power to detect a geographic signature in their case due tothe small-scaled nature of their study, possibly in combination with the factthat these northern marginal populations might not be in equilibrium The genetic differentiation between distance .Abbott eI. elegans and I.graellsii, which could explain the excess of heterozygositydetected for this population. Apart from this population, there was a slighttrend indicating that four populations showed a heterozygositydeficiency.Nevertheless, although the low power of this result prevents to make any strongstatements, the result could point towards a situation where these populationshave recently expanded in size.Analyses using BOTTLENECK did not provide strong support that any of thepopulations suffer from an excess or deficiency of heterozygosity. The onlypopulation to show a heterozygosity excess was the Spanish population Laxe. Inanother study , we have found that outof all populations examined for Spain, Laxe showed the highest degree ofhybridization between I. elegans, as it enables the rapidcolonisation of new areas and also maintains gene flow between populations. Theability to disperse and colonise novel habitats is particularly important whenthe natal habitat becomes unsuitable, for instance, as a result of habitatdeterioration or due to climate change I.elegans has expanded its range 168 km northwards in the last fewdecades, which is more than double the average distance found for other odonatespecies in the same study I.elegans in the UK further demonstrates that I.elegans has the ability to quickly respond to environmental changesby dispersing to new areas. This suggests that the terrestrial adult phase inodonates plays a crucial role in genetically homogenizing closely as well asquite distantly located populations.The emergence of population bottlenecks is probably counteracted by the highdispersal potential in I.elegans, each of which included two factors  and correlatesthese differentiation values to the environmental factors. The first scenariowas to test if the inclusion of latitude and/or longitude in the model wouldresult in a higher posterior probability than when the model was run withoutthese factors, thereby identifying any signatures of spatial populationprocesses, such as range expansions. A recent range expansion would partlyaccount for the relatively low levels of population differentiation that wedetected in I. elegans, since a recent expansion from a largeancestral population andthe retention of ancestral polymorphisms would beexpected to lower the overall population differentiation I. elegans Click here for additional data file.


Euphaea formosa.Pleistocene glacial oscillations have significantly affected the historical population dynamics of temperate taxa. However, the general effects of recent climatic changes on the evolutionary history and genetic structure of extant subtropical species remain poorly understood. In the present study, phylogeographic and historical demographic analyses based on mitochondrial and nuclear DNA sequences were used. The aim was to investigate whether Pleistocene climatic cycles, paleo-drainages or mountain vicariance of Taiwan shaped the evolutionary diversification of a subtropical gossamer-wing damselfly, E. formosa populations originated in the middle Pleistocene period (0.3 Mya) and consisted of two evolutionarily independent lineages. It is likely that they derived from the Pleistocene paleo-drainages of northern and southern Minjiang, or alternatively by divergence within Taiwan. The ancestral North-central lineage colonized northwestern Taiwan first and maintained a slowly growing population throughout much of the early to middle Pleistocene period. The ancestral widespread lineage reached central-southern Taiwan and experienced a spatial and demographic expansion into eastern Taiwan. This expansion began approximately 30,000 years ago in the Holocene interglacial period. The ancestral southern expansion into eastern Taiwan indicates that the central mountain range (CMR) formed a barrier to east-west expansion. However, E. formosa populations in the three major biogeographic regions  exhibit no significant genetic partitions, suggesting that river drainages and mountains did not form strong geographical barriers against gene flow among extant populations.E. formosa's colonization is associated with its high dispersal ability and larval tolerance to the late Pleistocene dry grasslands. The effect of late Pleistocene climatic changes on the subtropical damselfly's historical demography is lineage-specific, depending predominantly on its colonization history and geography. It is proposed that the Riss and Würm glaciations in the late Pleistocene period had a greater impact on the evolutionary diversification of subtropical insular species than the last glacial maximum (LGM).The present study implies that the antiquity of  Climatic oscillations of the Pleistocene period have significantly affected the population dynamics and genetic structures of extant taxa. The effects of glacial expansions and contractions on genetic characteristics and population demographics depend on the latitude, topography and life history of the species in question ,2. Genet2 located almost 100 km from the Asian mainland  [Apodemus semotus) [Rhinolophus monoceros) [Liocichla steerii) [Nephila pilipes) [A phylogeographic study of Taiwanese terrestrial organisms including higher plants , vertebejnegeri ; rice frnocharis ) and invicularis ) demonstashinai)  and woodsemotus)  display noceros) , montanesteerii)  and woodpilipes) , which gZacco pachycephalus) [Varicorhinus barbatulus) [Candidiopotamon rathbunae) [The Pleistocene river drainage plays a central role in shaping the biogeography and current genetic structures of Taiwanese freshwater fauna. Endemic freshwater vertebrates fall into three major biogeographic regions, the eastern, southern and north-central regions, reflecting the paleo-drainages of Pleistocene land bridges  Figure . Previouephalus) , ray-finbatulus)  and strethbunae)  reveals thbunae)  could reEuphaea formosa Hagen, 1869 (Insecta: Odonata), is a gossamer-wing damselfly species endemic to Taiwan, found throughout the island in the lowlands and fast flowing mountain streams at an altitude of less lower than 1,500 meters. The dispersal ability of adult E. formosa has not been estimated but observations from mark-recapture studies suggest that adult dispersal could be high (>10 km). Therefore, E. formosa is a suitable freshwater insect candidate that should have developed the same genetic signatures observed in more vagile terrestrial species in Taiwan [Aquatic insects have a complex life history comprising an aquatic immature stage constrained by freshwater habitats, and a winged adult stage with dispersal being limited by terrestrial barriers and flying ability . The disn Taiwan -22.COII) and nuclear internal transcribed spacer (ITS) to investigate the phylogeography and demographic history of E. formosa. The aim was to determine whether this endemic damselfly exhibits phylogeographic patterns and a genetic structure similar to terrestrial and freshwater fauna in Taiwan. Furthermore, the historical demography and lineage divergence time of E. formosa was investigated to determine the concordance between the population history and major climatic cycles during the Pleistocene period.The present study uses DNA sequences of mitochondrial cytochrome oxidase II . The pairwise sequence divergence of COII ranged from 0 to 8.2% within E. formosa, and from 4.8 to 9.4% between E. formosa and E. yayeyamana. For ITS, the pairwise sequence divergence ranged from 0 to 1.7% within E. formosa, and from 1.7 to 3.2% between E. formosa and E. yayeyamana. A microsatellite locus with GA repeats (0-8) appeared in the ITS1 region. The estimated ω ratio of COII sequences (0.021 in M0) was much less than one, and the LRT of M1 vs. M2 was insignificant . These results indicate that the M2 selection model did not fit the data significantly better than the M1 neutral model. Therefore, the neutral evolution of COII nucleotide sites in Euphaea damselflies is possible.A total of 500 bp of mitochondrial COII and ITS, respectively. The COII phylogeny was well resolved with strong support at the species level, suggesting a monophyletic E. formosa with respect to sister E. yayeyamana  separated by a deep phylogenetic split. The North-central clade was restricted to northern and central Taiwan, with the exception of two derived southern haplotypes (H41 & H43), suggesting recent long-range dispersal. This clade had a balanced tree topology, suggesting a relatively large population that had been stable for a long time period. In contrast, the widespread clade contained two high frequency haplotypes (H01 & H17) that were widely distributed throughout the island. The widespread clade had a star-like phylogeny, indicating rapid population expansion. The ITS tree suggested a monophyletic E. formosa sister to E. yayeyamana  and TVMef+I (lnL = -3225) models were selected as the best-fitting models concerning sequence evolution of a Figure . This pha Figure . This trs Figure . Within h) of COII ranged from 0 to 1, and the haplotype diversities of ITS ranged from 0.75 to 1  of these two gene regions varied between 0 and 0.028 for COII, and between 0.002 and 0.009 for ITS. Populations with the highest COII nucleotide diversities, representing ancestral sources of extant E. formosa, were present in five major river systems in northern and central Taiwan . These populations corresponded to the two proposed paleo-drainage systems of Taiwan, with the exception of the southern population from the Fongkang river (FG) , indicating recent origins or population bottlenecks. The relatively high h (0.657) and low π (0.003) values within the widespread COII clade suggested rapid population growth from a small ancestral population  and π (0.021), indicated a stable population with a long-term, large population size or admixed populations from separated ancestral lineages. However, ITS exhibited high haplotype diversities and low nucleotide diversities in all populations, suggesting population expansion. Population differentiation (FST) was higher in the North-central clade than the widespread COII clade (Table Nm) in the widespread COII clade was greater than that of the North-central COII clade and ITS estimation. Tajima's D of the widespread COII clade and ITS were negative and significantly different to zero, suggesting past population expansions. Tajima's D of the North-central COII clade was positive, but not significantly different to zero. Fu's FS calculated for the widespread, North-central COII clade and ITS were negative and significantly different from zero, implying past population growth. The AMOVA of COII indicated non-significant (8% of molecular variance) genetic differentiation between east and west groups and among east, south, and North-central groups (Table ITS variance was evident within populations (97%). No significant correlation was detected between pairwise genetic and geographic distances of the widespread COII clade , whereas significantly positive correlations between pairwise genetic and geographic were present in the North-central COII clade and ITS .The haplotype diversities  Figure . HoweverCOII clade exhibited a multimodal mismatch distribution, indicating a relatively stable population throughout time. This finding differs significantly from the simulation of sudden expansion and spatial expansion models    . The estimated mrca Tfor E. formosa and sister species E. yayeyamana was approximately 2.6 Mya in the late Pliocene . The mrca Tfor the origin of E. formosa was approximately 0.3 Mya in the middle Pleistocene period .The North-central ) Figure . The wid) Figure . Howevere Figure . These r) Figure . For the) Figure . During E. formosa and its sister species E. yayeyamana of the southern Ryukyu Archipelago, began diverging approximately 2.6 Mya during the late Pliocene  and 2.5% (ITS). Therefore, these two insular gossamer-wings are distinctive "genetic" and "phylogenetic" species [This study provides the first phylogeographic analysis of endemic freshwater insects in Taiwan. The phylogenetic results suggest that the common ancestors of e Figure , approxiyeyamana . With th species .COII and nuclear ITS gene trees for E. formosa were not fully congruent. In addition to incongruence associated with the gene tree/species tree, the results suggested a possible introgression or incomplete sorting of lineages in the ancestral polymorphism of these two markers. The jointly estimated coalescence time for COII and ITS suggests that the extant E. formosa populations probably originated approximately 0.3 Mya, during the middle Pleistocene period. The existence of a deep phylogenetic split and substantial genetic differentiation between the North-central and widespread COII clades, together with the monophyly of haplotypes from these two lineages, imply that there were at least two periods of colonization on the island. Alternatively, there was a single colonization followed by divergence of the two clades within Taiwan , preceding the colonizing periods for the majority of Taiwanese freshwater fish, which have low migratory capabilities limited by availability of continuous aquatic habitats. Therefore, E. formosa, a vagile aquatic insect present in lowland rivers and streams, exhibits an ancient colonization history older than most freshwater vertebrates but younger than lowland terrestrial animals.Previous estimates of colonizing periods for Taiwanese freshwater fishes are less than one Mya, falling within the time frame of the middle to late Pleistocene. These estimates include minnows (0.06 Mya ), cyprinage/myrs ) and a sE. formosa's colonization could be the past climate and vegetations in the continental land bridges during the Pleistocene glaciations. Paleo-environmental and palynological studies suggest that the climate of exposed land bridges connecting the Asian continent to lowland Taiwan during extended glaciations were cooler and dryer than at present, and the vegetation zones in these continental land bridges were predominantly grasslands [Euphaea damselflies, particularly at the larval stage. Euphaea formosa are damselflies of the only two odonate families known to possess lateral abdominal gill appendages in addition to the usual three caudal gills . Laterathoridae , which wthoridae ,42. The COII or nuclear ITS between east and west groups or among east, south and North-central groups  likely derived from the paleo-drainages of northern Minjiang and adjacent northwestern Taiwan first colonized northern Taiwan early during the Pleistocene period . Recent research investigating an endemic babbler in East Asia suggests a similar late Pleistocene population growth (0.17 Mya), after release from separated glacial refugia around southeast China [E. formosa demonstrates a less drastic demographic expansion trend in terms of magnitude of population growth than many temperate taxa [E. formosa. These results imply that earlier glacial periods such as Riss and Würm during the late Pleistocene period could have had greater impact on the historical demography and levels of genetic diversity in subtropical and tropical species than previously thought. Tropical species are often phylogeographically older than their temperate counterparts, and are likely to experience earlier climatic oscillations in the Pleistocene period [E. formosa.The historical demography of extant species was affected by climatic cycles during the Pleistocene period ,3. Phylost China . E. formgrowth 0.7 Mya, afe period . A relate period  and avaie period  could haE. formosa could be due to the flying ability of the adult and larval tolerance to the periodically dry grassland habitats that characterized Pleistocene land bridges. The extant E. formosa population consists of two phylogenetically independent lineages that either originated from the Pleistocene river drainages of northern and southern Minjiang or re-colonized from two separated refugia. Each lineage has undergone divergent evolutionary trajectories within the island. The present findings revealed the spatial and demographic expansion of the widespread lineage that can be dated to the late Pleistocene period . This population expansion was probably due to the availability of habitats in eastern Taiwan during the warmer Holocene interglacial period. In contrast, the North-central lineage has maintained a relatively stable population during the past one and half million years, possibly because of the stable aquatic habitats of northern Taiwan. The ancestral E. formosa expanded into eastern Taiwan via a southern dispersal route, indicating that the CMR formed a barrier to the east-west dispersal of this damselfly. However, the CMR and Pleistocene river drainages are not strong geographic barriers to current gene flow among E. formosa populations, with genetic structures being shaped by geographic distances within the three biogeographic regions. This study led to the proposal that the Riss and Würm glaciations in the late Pleistocene period had a greater impact on the evolutionary diversification of subtropical insular species than the LGM. Further studies investigating taxa with diverse life history characteristics could elucidate the general effect of recent climatic oscillations on the evolutionary diversification of subtropical species.This study has investigated Pleistocene climatic, geographic and natural history factors underlying the evolutionary diversification of an aquatic insect in Taiwan. The endemic gossamer-wing damselfly originated during the early to middle Pleistocene period, earlier than the late Pleistocene colonization of the majority of Taiwanese freshwater vertebrates. The antiquity and colonization history of E. formosa damselflies are characterized by black bands on the hind wings and distinct red stripes on the thorax . An earlier study comparing external morphological characters of E. formosa and E. yayeyamana demonstrated no distinct differentiation, except that E. yayeyamana is smaller (insular dwarfism) [Euphaea species, six E. yayeyamana individuals were used for the analyses.One hundred and fifty nine d Figure . Voucherwarfism) . This suEuphaea-specific C2-N-3740 (5'-TCA TCT AGT GAG GCT TCA-3') were used to amplify the COII gene. A eukaryote-specific 18SF/28SR primer set was used to amplify ITS1, ITS2 and 5.8S genes [Mastercycler Gradient, Hamburg, Germany). PCR reactions contained a total volume of 50 μl, composed of 1 μl of ProTaq polymerase , 2 μl of 10 mM of each primer, 4 μl of 1 mM dNTPs, 5 μl of ProTaq buffer, 35 μl of ddH2O and 1 μl of extracted DNA (100 to 400 ng/μl). For COII, the PCR profile was as follows: denaturing at 94°C for one minute, 35 cycles of amplification at 94°C for one minute followed by 53°C for one minute and 72°C for one minute, and a final extension at 72°C for 10 minutes. For ITS, the PCR profile was the same as that for COII except the annealing temperature was 52°C. PCR products were purified using a Gel/PCR DNA Fragments Extraction Kit  and cloned into competent cells (dH-5α) using the T&A cloning kit . Positive clones were confirmed by performing PCR reactions using M13F/M13R primers. Plasmid DNA was sequenced in both directions on an ABI PRISM™ 377 automatic sequencer  at the Mission Biotech, Taiwan. Manually inspected and edited DNA sequences from raw chromatograph data were aligned using the Clustal W method in MegAlign . COII sequences were translated into amino acid sequences using a mitochondrial genetic code of Drosophila in MacClade v.4.06 [ITS2 sequences with large indels (20 to 58 bps) were excluded from the analyses. Sequences used in this study were deposited in GenBank (EU603519-EU603681). The sequence alignment and associated phylogenetic trees were submitted to the TreeBASE http://purl.org/phylo/treebase/phylows/study/TB2:S11020.DNA was extracted from insect thoracic muscle using the standard CTAB, pheno-chloroform protocol, followed by ethanol precipitation . The pri8S genes . PCR reae v.4.06  to check7 and 6 × 106 generations for COII and ITS, respectively, with trees being sampled for every 100 generations. MCMC runs were terminated after the average split frequencies went below 0.01 and the Convergence Diagnostic Potential Scale Reduction Factor (PSRF) approached one, suggesting convergence of the two separate runs [COII and ITS respectively) were discarded as burn-in. The remaining trees were imported into PAUP* for constructing a 50% majority consensus tree. A species tree was co-estimated from COII and ITS gene trees using *BEAST [COII was set to the average value found in arthropods  was estimated relative to COII. The relaxed local clock model was used, with estimated rate change counts of 2.0 and 1.3 for COII  and ITS , respectively. The species tree prior was set to the default option of Yule process. The MCMC analyses were run for 5 × 108 generations, with parameters sampled for every 1 × 104 generations and the first 10% of the runs being discarded as burn-in. The convergence of runs was determined by examining likelihood scores through time plot using TRACER v. 1.5 [Phylogenetic analyses were performed using Maximum Parsimony (MP) in PAUP* v.4.0b10 . Parsimoate runs . One quag *BEAST  implemeng *BEAST . The subage/myrs ), and thR v. 1.5 .COII. The non-synonymous versus synonymous substitution ratio (ω: dN/dS) was calculated under model M0 (no site rate heterogeneity) with likelihood tree topologies obtained from the phylogenetic analyses. The Likelihood Ratio Test (LRT) was used to detect the significance between model M1  and model M2 (selection) using chi-square statistics. Haplotype diversity (h), nuclear diversity (π), exact tests of population differentiation, pairwise and overall FST among populations were calculated using DnaSP v.4.0 [FST) and geographic distance (km) of populations using 10,000 permutations and a Reduced Major Axis (RMA) regression in IBD [ST), among populations within groups (ΦSG) and among groups (ΦGT) were calculated. Tajima's D and Fu's FS were computed using ARLEQUIN with 1,000 permutations. Positive values of Tajima's D and Fu's FS indicated possible balancing selection (single locus) or population subdivision (multiple loci), whereas negative values suggested positive selection (single locus) or expansion of population size (multiple loci).A codeml module of PAML  was useSP v.4.0 . Mantel n in IBD . The popn in IBD . PopulatCOII haplotypes using a mismatch distribution analysis with 1,000 bootstrap replications in ARLEQUIN. The analyses were carried out separately for the North-central and widespread clades of COII because of a deep phylogenetic gap between these two lineages (see phylogeny result below). The goodness of fit between observed data and expected demographic models was estimated using the Sum of Square Deviations (SSD). The Harpending's Raggedness index (r) was calculated with 1,000 permutations to test the significance of r between the observed and simulated distributions. A Bayesian coalescence-based framework [E. formosa on the basis of both loci. We computed the posterior distributions of effective population sizes (eNτ), time to the most recent common ancestor (mrcaT) and the multi-locus Extended Bayesian Skyline Plot (EBSP) [COII and ITS were set as unlinked while assuming the same demographic history. The mutation rate setting was the same as in the above *BEAST analyses. The starting population size was set to one, and the scale factor k was set to two. Both gene trees were reconstructed using an uncorrelated relaxed lognormal clock model. The window size for COII and ITS random walk integer was set to two and eight, respectively. The MCMC searches were run for 1 × 108 generations with parameters sampled for every 1 × 104 generations and the first 10% of the runs being discarded as burn-in. Each analysis was repeated several times to optimize the scale factors until there were no suggesting messages in the log file. The Effective Sample Size (ESS) of estimated parameter values was determined using Tracer v. 1.5 [http://www.r-project.org/).The demographic history was inferred from non-recombined ramework  was emplt (EBSP)  using a t (EBSP) . COII anr v. 1.5 , and theCPL designed the study; JPH and CPL carried out the field work; JPH generated the molecular data and conducted all DNA sequence and statistical analyses with input from CPL; CPL and JPH wrote the manuscript. The authors read and approved the final manuscript.Specimen and sequence data. Collecting locality of E. formosa and outgroup specimens, and the summary statistics of the sampled damselfly populations.Click here for file


The Taihang Mountain range of north-central China, the Southern region area of Fujian province, and the Chaoshan plain of Guangdong province are 3 major regions in China well known for their high incidence of esophageal cancer (EC). These areas also exhibit high incidences of gastric cardia cancer (GCC). The ancestors of the Chaoshanese, now the major inhabitants in the Chaoshan plain, were from north-central China. We hypothesized that EC and GCC patients in Chaoshan areas share a common ancestry with Taihang Mountain patients. We analyzed 16 East Asian-specific Y-chromosome biallelic markers  and 6 Y-chromosome short tandem repeat (Y-STR) loci in 72 EC and 48 GCC patients from Chaoshan and 49 EC and 63 GCC patients from the Taihang Mountain range. We also compared data for 32 Chaoshan Hakka people and 24 members of the aboriginal She minority who live near the Chaoshan area. Analysis was by frequency distribution and principal component, correlation and hierarchical cluster analysis of Y-SNP. Chaoshan patients were closely related to Taihang Mountain patients, even though they are geographically distant. Y-STR analysis revealed that the 4 patient groups were more closely related with each other than with other groups. Network analysis of the haplogroup O3a3c1-M117 showed a high degree of patient-specific substructure. We suggest that EC and GCC patients from these 2 areas share a similar patrilineal genetic background, which may play an important role in the genetic factor of EC and GCC in these populations. Esophageal cancer (EC) is one of the most common fatal cancers worldwide. China has geographical “hot spots” of high EC incidence. A well-known region with high risk of EC in China is the Taihang Mountain area between Henan, Hebei, and Shanxi provinces in north-central China, the famous “Asian EC belt” ranging from the Caucasian mountains, across northern Iran, all the way to northern China The geographic features of south-littoral Chaoshan and north-central Taihang Mountain area are distinct, but the incidence of EC and GCC is high within these 2 regions The 3 main populations in Chaoshan area include 2 Han populations – Chaoshanese with Chaoshan dialects and Hakka with Hakka dialects – and one local aboriginal She population. Since the Qing Dynasty (216∼207 BC), the Henan and Shanxi Han people of north-central China migrated into the Chaoshan area in Guangdong province via Fujian province because of war and famine. They gradually became the predominant inhabitants of the Chaoshan area and are called Chaoshanese The Hakka and Chaoshanese populations show the characteristics of their unique cultures Our previous research of Y-chromosome and mtDNA haplogroups concluded that the EC high-risk populations in Taihang Mountain, Fujian Minnan and Guangdong Chaoshan share a similar patrilineal and matrilineal genetic background Y-SNP genotyping revealed the haplogroup frequencies of the Chaoshan EC or GCC patients, Taihang Mountain EC or GCC patients, and Chaoshan Hakka and She populations. The highest haplogroup of Chaoshan patients was O3a3c1-M117, which is the characteristic haplogroup for Northern East Asians . It was Principal component analysis (PCA) involves a mathematical procedure that transforms a number of correlated variables into a  number of uncorrelated variables called principal components. The first principal component accounts for as much of the variability in the data as possible, and each succeeding component accounts for as much of the remaining variability as possible. In the principal-component plot, the smaller the distance of two populations, the closer the genetic relationship is between the two. Y-SNP haplogroup frequencies for the patient groups and high-risk population from the same area were positively correlated, and frequencies for all patient groups were positively correlated with the Fujian and Chaoshan high-risk populations . FrequenTo study the affinity among the 4 patient groups and their relationship with other Han and minority nationalities, we analyzed Y-SNP data by hierarchical cluster analysis with average linkage (between groups). We compared 17 Chinese Han populations , 3 southern minority nationalities , DYS390, DYS391, DYS392, DYS393 and DYS394. We included 6 additional Chinese populations and 3 high-risk populations: Zhejiang ST distance matrix, we constructed an unrooted neighbor-joining tree  Chaoshan patients–72 EC and 48 GCC patients; 2) Taihang Mountain patients–49 EC and 63 GCC patients; and 3) Chaoshan EC low-risk population–24 She people from Chaoshan Fenghuang Mountain and 32 Chaoshan Hakka from Chaoshan Puning county. Disease in all patients was confirmed pathologically. All participants involved in our study were given written informed consents. The study was approved by the ethical review committee of Shantou University Medical College. Genomic DNA was extracted from whole blood by the TIANamp Blood DNA kit (DP318-03) .Y-SNPs were genotyped by Sequenom MassARRAY iPLEX Gold module (Sequenom Inc.) . In the network map, individuals with the same mutations of Y-STRs were in the same node, and one node could generate other nodes due to gradual Y-STR mutation The extent of genetic differentiation of the populations was estimated by the RChaoshanese are descendants of north-central China Han people. North-central Chinese Han began to migrate into southern China beginning in the Qin Dynasty (216 BC). The Han Dynasty (206 BC–220 AD) experienced another 3 waves of large-scale migration into southern China because of the decrease in the native population in this area. Gradually, over 2,000 years, the north-central Chinese Han became the main population – Chaoshanese in the Chaoshan region, called Helao, who directly migrated from north-central China, or Fulao, who first migrated to Fujian Minnan, then to Chaoshan with well-maintained language and customs from north-central China. The Taihang Mountain people in north-central China, Fujian Minnan and Chaoshan areas are well known for their high incidence of EC With the development of diagnostic techniques and improved epidemiology, more GCC cases have been confirmed in these areas. EC and GCC are the 2 most common cancers in these 3 areas. Our previous genetic research showed that high-risk populations in these 3 areas share a common ancestry On Y-SNP principal component analysis, the paternal structure for Chaoshan patients differed from that for Chaoshan Hakka and She populations, although they are in geographic proximity and Chaoshan Hakka are also descendants of north-central Chinese Hans. Chaoshan patients clustered closely with the Fujian and Henan high-risk population and patients, although they are geographically distant. Chaoshan Hakka and She populations clustered together, which agrees with historical records. Chaoshan Hakka mainly inhabit the mountain area, for more gene flow with the She population, who also live in the mountain area. Y-SNP haplotype frequencies were positively correlated among patients, which further supports their close genetic affinity. The results of hierarchical cluster analysis also supported the close genetic affinity among patients and high-risk populations. Phylogenetically, the patient groups were more closely related to each other than with the high-risk population . NetworkRecent genome-wide association studies from China high-risk areas showed significant association of a variant at 10q23 in PLCE1 and both esophageal squamous cell carcinoma and gastric cardia adenocarcinoma, which highlights the common genetic mechanisms that may contribute to the etiology of both cancers We suggest that EC and GCC do not occur at random in high-risk populations but are closely associated with a certain patrilineal background structure and these related patients may inherit a pathogenic genetic structure from their common ancestors.In summary, the patrilineal genetic structure of Chaoshan and Taihang Mountain patients is similar, and patients have closer affinity with each other than with the high-risk populations. The EC and GCC patients share a recent common ancestor. In contrast, the Chaoshan Hakka and She populations have a relatively distant relationship with Chaoshanese people, which may explain in part the high incidence of EC and GCC in Chaoshanese people.Table S1Raw data for individual Y-SNP.(XLS)Click here for additional data file.Table S2Raw data for individual Y-STR.(XLS)Click here for additional data file.