Echinococcus granulosussensu stricto complex were investigated based on sequencing of mitochondrial DNA (mtDNA). Total 81 isolates of hydatid cyst collected from ungulate animals from different geographical areas of North India were identified by sequencing of cytochrome c oxidase subunit1 (coxi) gene. Three genotypes belonging to E. granulosussensu stricto complex were identified . Further the nucleotide sequences (retrieved from GenBank) for the coxi gene from seven populations of E. granulosussensu stricto complex covering 6 continents, were compared with sequences of isolates analysed in this study. Molecular diversity indices represent overall high mitochondrial DNA diversity for these populations, but low nucleotide diversity between haplotypes. The neutrality tests were used to analyze signatures of historical demographic events. The Tajima’s D test and Fu’s FS test showed negative value, indicating deviations from neutrality and both suggested recent population expansion for the populations. Pairwise fixation index was significant for pairwise comparison of different populations , indicating genetic differentiation among populations. Based on the findings of the present study and those from earlier studies, we hypothesize that demographic expansion occurred in E. granulosus after the introduction of founder haplotype particular by anthropogenic movements. The genetic diversity and population genetics of the  Echinococcous granulosus, is a global public health problem that infects the human and ungulate animals [E. granulosus involves dogs and other carnivores as definitive host and livestock as intermediate host [Cystic echinococcosis, an infection with the metacestode of the dog tapeworm  animals . The lifate host . Eggs shate host . E. granulosus is a complex of distinct strains with different host affinities. To date, 10 different genotypes have been described by molecular genetic analysis and the genotypic variation closely follows the biological and phenotypic characteristic of the parasite. These 10 genotypes/strains are named on the basis of their ideal intermediate host. These include sheep strain (G1), Tasmanian sheep strain (G2), buffalo strain (G3), horse strain (G4), cattle strain (G5), camel strain (G6), pig strain (G7), cervid strain (G8), pig/human strain (G9) and Fenno-Scandian cervid strain (G10) [E. granulosus genotype should be split into different species: E. granulosussensu stricto (genotypes G1-G3), E. equinus (genotype G4), E. ortleppi (genotype G5), E. canadensis (genotype G6-G10) and E. felidis (lion strain) [E. granulosus reflects the differences in life cycle pattern and host range; thus the knowledge of genetic diversity and population genetics of this parasite is of immense public health importance for formulation of control strategies for hydatidosis. In Western India, 4 different genotypes  were reported in different intermediate hosts such as cattle, buffaloes, pigs and sheep [E. granulosus and found the G1 and G3 genotype by cox1 gene sequencing, an extensive study on the genotypes of parasite involving large number of isolates and covering large geographical endemic areas is lacking in this region. strain G, cattle  strain G, pig str strain G, cervid  strain G and Fennnd sheep  from LudE. granulosus and acts as an important genetic marker to study the population genetic structure of this parasite as it is haploid, non recombining, multicopy, rapidly evolving and maternally inherited. The rate of evolution of mitochondrial genome appears to be exceeding from that of single-copy fraction of nuclear genome. The high rate may be due to an elevated rate of mutation in mitochondrial DNA, because of this high rate of evolution, mitochondrial DNA is likely to be useful for high-resolution analysis of the evolutionary process [E. granulosus by sequencing of mitochondrial cox1 gene. The results were further compared with nucleotide sequences retrieved from GenBank from other geographical regions to study the genetic variability and population genetics of this parasite. Mitochondrial DNA has shown to be useful in differentiation of all the genotypes of  process . The aimThe study was approved by the Institutional Ethics Review Committees of the Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, Sher-i- Kashmir Institute of Medical sciences (SKIMS), Srinagar, Kashmir and Indira Gandhi Medical College and Hospital (IGMC), Shimla, India. In this study hydatid cysts were collected from animals during post-mortem examination following inspection by the veterinary officers at the respective slaughter houses with their due consent. No experimentation was done on animals, therefore no approval from Institutional animal ethics committee was required.Hydatid cysts were collected randomly during 2010-2012 from 4 different geographical areas in North India. The numbers depend upon the availability of the sample. Cyst samples were obtained from 74 freshly slaughtered and heavily infected sheep from slaughter houses, falls under the jurisdictions of the municipal corporation of Chandigarh (n=66), Shimla, Himachal Pradesh (n= 3) and Srinagar, Jammu and Kashmir (n=5). Permission was obtained from these slaughter houses to use these hydatid cysts. Seven hydatid cysts were kindly received from Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, North India. These cysts were isolated from sheep (n=2), buffaloes (n=2), goats (n=2) and cow (n=1). Thus, in total isolates from 81 animals were analyzed. The cysts were transported to the Department of Parasitology, Post Graduate Institute of Medical Education and Research (PGIMER), under refrigerated conditions for further processing. Intact hydatid cysts were separated and washed with distilled water. Cyst from each animal was considered as an isolate.cox1 gene was performed by using primers and PCR conditions as described previously [ Cyst samples were washed thrice in PBS to remove ethanol and genomic DNA was extracted from each sample by QIAamp DNA mini kit , according to the manufacturer’s instructions. For molecular identification, PCR amplification of the eviously  with mineviously . To avoiE. granulosussensu stricto population deposited in the GenBank from India and other South Asian, East Asian, European, Middle East, African, South American and Australian countries were retrieved from the National Center for Biotechnology (http://www.ncbi.nlm.nih.gov) and compared with sequences of isolates used in the present study , haplotypes number (h), haplotype diversity (Hd) and nucleotide diversity (π) and average number of pairwise nucleotide differences within population (K), were estimated using DnaSP 4.5 Software . The neu The pairwise genetic difference was estimated for all populations by calculating Wright’s F-statistics (Fst) and gene flow (Nm) was calculated by population genetics package Arlequin 3.1 . In addicox1 gene with JB3/JB4.5 primer yielded PCR product of 446 bp. Nucleotide sequences of all the 81 isolates from North India analyzed in the present study were aligned with reference sequences of each genotype within E. granulosus retrieved from GenBank. Total 3 genotypes of E. granulosus were found: G3 genotype (n=58), G1 genotype (n=22) and G2 genotype (n=1). The various sequences of the haplotypes found in this study, were deposited in GenBank with accession numbers JX854022-34 and KC422644-45. Sequences of these isolates along with those retrieved from GenBank were used to construct a phylogenic tree (The amplification of nic tree . Among 3cox1 sequences estimated by TCS software detected two common haplotypes (SA1 and SA23)  . The SA2 Among 369 sequences , total 73 haplotypes were found: 20 in South Asia, 14 in East Asia, 17 in Europe, 25 in Middle East, 10 in Africa, 13 in South America, 5 in Australia . Along tcox1 gene from India and other countries  and average number of nucleotide substitutions per site between all these populations (Dxy) varied from 1.33 and 0.00391 (East Asia and South America) to 2.8 and 0.00821 respectively . PairwisE. granulosus namely G1 (sheep strain), G2 (Tasmanian sheep strain), G3  and G5 genotypes (cattle strain) in livestock in Maharashtra and adjoining areas in Western India. The predominant genotype was found to be G3 (63%) present in all species of livestock followed by the G5 (19.56%), the G1 (13%) and the G2 genotype (4.34%). In earlier reports from Ludhiana (North India), only 2 genotypes, buffalo strain (G3) and common sheep strain (G1) were found to infect the livestock [E. granulosus [Pednekar et al.  have repivestock . In our , the G1 % and theE. granulosus were found to infect the livestock: G1, G2 and G3 genotypes. In concordant to the earlier studies from livestock  in India, the G3 genotype (71.8%) was found as predominant genotype. The second most common genotype was the G1 genotype in 27.16 % isolates followed by the G2 genotype in only one isolate from Srinagar, Kashmir (North India) which was similar to the findings in Eastern India [In the present study, 3 genotypes of rn India . Furtherrn India , 87.5% irn India , 77.4% irn India , 71.59% rn India  and 55.8rn India . This daE. granulosus cox1 gene sequences for the G2 genotype, registered in GenBank or international database are infact the microvariants of G3 genotype. This discrepancy could be due to some cox1 gene sequences for G2 genotype deposited in GenBank which were used as reference ones [E. granulosus on the basis of sequencing of cox1 gene. In the present study the G2 genotype was clustered in Clade II. The large numbers of nce ones . These rnce ones  who had E. granulosus [cox1 gene as a promising candidate for revealing the population genetics of E granulosus. In the present study, E. granulosussensu stricto populations from wide geographical areas were analyzed to examine the parasite genetic diversity. For this, sequences of only E. granulosussensu stricto complex (G1-G3 genotypes) were retrieved from GenBank/EMBL/DDBJ International Databases as there is scarcity of data in GenBank for other genotypes and also in the present study, E. granulosussensu stricto complex was found as predominant species in livestock.To date, very few studies have explored in depth population genetic structure of anulosus –33. ThesE. granulosussensu stricto (SA1 and SA23) are widely distributed in different geographical area. Interestingly, the nucleotide sequence of this haplotype (SA23) was 100% identical to previously described as predominant haplotype in Europe (EG1: JF513058) [ In the present study, haplotype network has shown that two predominant haplotypes of F513058) , China aF513058) , Iran anF513058) . The hapDespite the wide distributional range, the estimation of inter-population comparison  also support low to high level of genetic differentiation between these populations. The populations of Europe, Middle East and South America showed low divergence and shared the common haplotypes. The Europe and Middle East populations are highly closely related to each other which were suggested by a very low and non-significant Fst value. Gene flow (Nm) was also found to be very high between these two populations. The South Asian population was the most differentiated with very low gene flow among other populations. This could be related to the presence of G3 as predominant genotype in this population as demonstrated in dendrogram and haplotype network. In the present study, in spite of high haplotype diversity, low nucleotide diversity values suggested small differences between haplotypes. This was also demonstrated by the haplotype network, which represents mostly single nucleotide differences between majorities of haplotypes. In the present study, the combination of high haplotype and low nucleotide diversity can be a signature of a rapid population expansion from a small effective population size as observed in the earlier study . A numbeE. granulosussensu stricto should be compared in different endemic areas to know about the ancestral origin and process of worldwide dispersal of this parasite.The Interpretation of demographic expansion correlates well to the widely observed patterns of domestication of sheep which started around 12,500 B.C. The various genetic and archaeological evidences suggest that domestication of sheep occurred first in Southwest Asia (Middle East) and then spread successfully into Europe and Africa, and the rest of Asia . The resE. granulosus in ungulate animals in North India. The present study reveals a high genetic diversity within populations of E. granulosus and but relatively low to high level of genetic differentiation among the populations. The observed patterns of genetic diversity within and between the populations are likely caused by population expansion after the introduction of founder haplotype. Finally, present study supports the earlier report that it is important to correlate molecular epidemiology with evolutionary biology so that population genetics and phylogenetic analyses are able to confer a considerable added value in the characterization of strains and species of pathogens [In conclusion, molecular analysis demonstrates the presence of G3 as predominant genotype followed by the G1 and G2 genotype of athogens .


Squalidus argentatus samples from thirteen localities within mainland China and Taiwan were conducted for biogeographic studies, as their dispersal strictly depends on geological evolution of the landmasses. A total of 95 haplotypes were genotyped for mtDNA cyt b gene in 160 specimens from nine river systems. Relatively high levels of haplotype diversity (h = 0.984) and low levels of nucleotide diversity (π = 0.020) were detected in S. argentatus. Two major phylogenetic haplotype groups, A and B, were revealed via phylogenetic analysis. The degree of intergroup divergence (3.96%) indicates that these groups diverged about 4.55 myr (million years) ago. Haplotype network and population analyses indicated significant genetic structure (FST = 0.775), largely concordant with the geographical location of the populations. According to SAMOVA analysis, we divided these populations into four units: Yangtze-Pearl, Qiantang-Minjiang, Jiulong-Beijiang and Taiwan groups. Mismatch distribution analysis, neutrality tests and Bayesian skyline plots indicated a significant population expansion for lineage A and B, approximately dated 0.35 and 0.04 myr ago, respectively. We found strong geographical organization of the haplotype clades across different geographic scales that can be explained by episodes of dispersal and population expansion followed by population fragmentation and restricted gene flow.Phylogeographical analyses on  The Cyprinidae represent one of the most diverse freshwater fish groups and are a major component of the primary freshwater fish fauna of Africa, Eurasia and North America, comprising more than 220 genera and 2400 species . Among tSqualidus Sauvage and Dabry consists of a group of small-sized freshwater fishes broadly distributed in the lower and middle reaches of eastern Asia, including Korea, Japan, Vietnam, Hainan Island, Taiwan and most major drainages of mainland China [Squalidus, S. argentatus is the most extensively distributed both in China and Taiwan, where S. argentatus is restricted to the Tamsui River  [Squalidus argentatus is one of the few cyprinid species distributed across the Taiwan Strait in addition to Hemibarbus labeo, Sinibrama macrops, Pseudorasbora parva and Distoechodon tumirostris. Population genetics regarding the trans-strait distribution of H. labeo and S. macrops have been addressed [H. labeo and S. macrops of northern Taiwan are genetically associated with their conspecificities in the Qiantang River rather than those in the Minjiang River that connected to rivers of northern Taiwan during marine regression [S. argentatus, with similar trans-strait distribution could provide more substantial evidence to infer the dispersal pattern of freshwater fishes of Taiwan. In the present study, we collected S. argentatus of four geographic sub-districts based on Li’s hypothesis in our study: (1) Pearl River sub-district ; (2) ZheMin sub-district ; (3) Taiwan sub-district ; and (4) Kiang-Husi Sub-district  b sequences of S. argentatus were performed. We thus elucidated the evolutionary relationships within S. argentatus, particularly focused on (1) describing the population genetic diversity; (2) discussing relationships between phylogenetic structures and geographic characters; (3) demonstrating the possible dispersal history between southern China and Taiwan; and (4) applying the interpretation of Management Units (MUs) [S. argentatus.Taiwan is a continental shelf island (sensu) , which esensu) ,15. Durisensu) ,17. The sensu) . Squalidddressed ,19. Unexgression ,19. A clgression ,17. Addiand SK);  Taiwan sts (MUs) ,21 for tb generated from 160 tissue samples of Squalidus argentatus were aligned and a total of 95 haplotypes were found. Alignment of all cyt b gene sequences revealed a total of 190 variable sites, among which 102 variable characters were singleton and 88 characters were parsimony-informative. Cyt b sequences were globally G deficient (15.0%), which is characteristic of the mitochondrial genome [b-based studies on a variety of fishes, including cyprinids [Sequences of 1,140 bp of cyt l genome ; the othyprinids .h) within each population are presented in h) among the 160 samples was estimated to be 0.984, and the mean nucleotide diversity was 0.02063 , and haplotype diversity . This result indicates the presence of phylogeographic structure, since the closely related haplotypes would be detected more frequently than those less closely related haplotypes in the same area [The existence of a phylogeographic structure was tested following Pons and Petit  by calcuame area .h = 0.986; lineage B: π = 0.00534, h = 0.870).The NJ mtDNA gene tree indicated two strongly supported lineages  after Bonferroni correction. Most of the pairwise FST values were significant with a P value < 0.05 . According to four geographic districts, (1) Pearl River sub-district (populations SG and LZ); (2) ZheMin sub-district ; (3) Taiwan sub-district (population SD); and (4) Kiang-Husi Sub-district , AMOVA analysis showed that most variances occurred among populations within geographic district (P < 0.001), contributing mainly to total variance . In the AMOVA, most of the molecular variance (75.74%) was attributable to variations between lineages, 11.40% attributable to variations among populations within groups, and 12.86% of the molecular variance related to variations within the same population. The SAMOVA revealed increasing FCT values when the numbers of groups increased . The best partitioning of the genetic diversity by SAMOVA was obtained when samples were divided into four units : (i) Yangtze-Pearl group: Yangtze River and Xijiang River (LZ) in the tributaries of the Pearl River ; (ii) Qiantang-Minjiang group: the river systems between Qiantang River and Minjiang River ; (iii) Jiulong-Beijiang group: three rivers in lineage B, including Jiulong River, Hanjiang River and Beijiang River (SG) in the tributaries of the Pearl River ; and (iv) Taiwan group: Taiwan Island (population SD) .Pairwise e < 0.05 . Differevariance . The restion SD) . ReducedFs = −36.677, P < 0.000), although Tajima was not significant  . This outcome was also supported by the low Harpending’s Raggedness index . Demographic analyses showed evidence of range expansions in two lineages. Tajima’s D and Fu’s tests were both significantly negative for two lineages, indicating that this species experienced a demographic expansion event under a neutral model. To characterize the expansion pattern further, a model of sudden demographic growth was fitted to the pairwise sequence mismatch distribution  . Fu’s te growing . The modribution . In eachS. argentatus. Skyline plots for S. argentatus indicate that recent population expansion began approximately 0.35 myr ago. Analyses of lineage A and lineage B samples both detected an initial population expansion starting at 0.35 and 0.04 myr ago, respectively. The lineage B exhibited a smaller effective population size compared to the lineage A, both before and after expansion  for each of the Bayesian skyline analyses was greater than 200, suggesting that the 10 million generations were sufficient to determine the demographic history for xpansion .We used 1.05%/myr and a strict clock model implemented in BEAST to estimate the TMRCAs of different lineages and times of their separation . The anaSqualidus argentatus populations. Grant and Bowen [b sequences is similar to that of the other species [Nucleotide and haplotype diversities can provide information in the history of nd Bowen  interprend Bowen . Compare species ,8–11. Ge species . At the  species , it is n species .S. argentatus has a high level of genetic structuring, which is similar to many other widespread freshwater fishes observed in this region of southern China, such as Zacco platypus [Opsariichthys bidens [Glyptothorax [Hemibarbus labeo [FST = 0.775). Populations from different regions are characterized by a unique haplotype, which reflects the effects of geographic isolation [Our results show that platypus ,10, Opsas bidens –11, Glyptothorax  and Hemius labeo . Geneticsolation . The larS. argentatus in South China is supported by pairwise genetic distances between groups (3.96%). Owing to the discrete geographical patterns, it can be inferred that the population genetic structure of S. argentatus in South China may have been significantly influenced by geological processes in the past. Allopatric populations will occupy recognizable, deeply-separated branches with time in an intraspecific gene tree. The amount of genetic differentiation may reflect mutations accumulated after the separation of populations and/or effects of lineage sorting from a polymorphic ancestral gene pool. Overall, lineage A and B could be associated with some degree of geographic structuring according to their distribution in South China. However, the Nanling and WuYi Mountains would have probably played a key role in geographically isolating representatives of lineage A and B, as barriers blocking dispersals between populations on either slope. The Nanling Mountains are the watershed of the Yangtze and Pearl River systems [S. argentatus populations distributed in Xijiang (LZ) are grouped into lineage A, and the population in Beijiang (SG) grouped into lineage B. However, Xijiang and Beijiang are the tributaries of the Pearl River system. The Xijiang population is more closely related to populations in the Yangtze and Minjiang Rivers than to the Beijiang population in terms of genetic structure. Close inter-relationships between populations from the Xijiang and Yangtze River are a common pattern observed in several cyprinids distributed in neighboring tributaries of these two river systems [Zacco platypus and Opsariichthys bidens [The existence of two monophyletic groups of  systems . The S.  systems ,30. This systems , connects bidens .Evolution and dispersal of freshwater fishes are closely related to paleogeography and especially to the history of basin connections, as a consequence of the geological development of landscapes. According to SAMOVA results, we divided the populations of the present study in mainland China into three groups, including the Yangtze-Pearl, Qiantang-Minjiang and Jiulong-Beijiang groups. The mountainous region, known as the WuYi Mountains, may play a critical role in blocking possible dispersal between drainages on both slopes. This is well supported by the phylogenetic and population genetic analyses that divide the western-slope drainage, the Yangtze River (Yangtze-Pearl group), and the eastern-slope drainage, the Minjing River (Qiantang-Minjiang group), into two separated clades .Hemibarbus medius, Puntius semifasciolatus and Sinibrama melrosei, while the Minjiang River is the southernmost boundary of Sinibrama macrops and Sarcocheilichthys parvus [The population collected from the Minjiang and Mulan Rivers (belonging to the lineage A) presented a genetic composition that was completely different from that of the Jiulong River (belonging to the lineage B), despite the short distance. This phylogenetic pattern is also observed in cyprinid fishes among which the Minjing and Jiulong Rivers serve as distribution boundaries. The Jiulong River is the northernmost border of s parvus . Geologis parvus ,33. ThisVaricorhinus barbatulus, [Formosania lacustre, [Cobitis sinensis, [S. argentatus in Taiwan. Squalidus argentatus, H. labeo and S. macrops in Taiwan are all only distributed in the Tamsui River. Our phylogeographic pattern of S. argentatus coincides with H. labeo and S. macrops, with a sister group in the Qiantang River instead of the Minjiang River [S. argentatus is limited to the Tamsui River in northern Taiwan and has failed to spread further, dispersal from mainland China is hypothesized to be a recent event.The origin of the freshwater fish fauna of Taiwan, based on faunal similarity, suggested two (north and south) routes which connect Taiwan and mainland China, assuming the dispersal of freshwater species via land bridges during marine regression ,17,34–36acustre, ; Cobitisinensis, ) indicatng River ,19. SincO. bidens populations in the same region. Concordant results for the geographical divergence support a common history for these co-distributed freshwater taxa [G. fokiensis; [O. bidens; [H. labeo; [The BEAST analysis estimated an approximate age of 4.55 ± 1.0 myr for all haplotypes in South China, a time close to the divergence of ter taxa . Geologikiensis; ; O. bide bidens; ; H. labe. labeo; ).b in other freshwater fishes in southern China [S. argentatus were lineage-specific and depended predominantly on the colonization history and geography of the two evolutionarily independent clades. Furthermore, the Bayesian skyline plots indicated that the population size increased, particularly around 0.30 myr ago in lineage A. Estimation of population expansion time for lineage A is much older than that of lineage B. Demographic expansions into these river systems in lineage B may have occurred more recently, and this is concordant with the molecular dating from mtDNA markers. This expansion started approximately 0.05 myr ago in the late Pleistocene period in lineage B.In our analysis, both the neutrality tests and the mismatch distribution analysis, indicated population expansion in both lineage A and B. In fact, Tajima’s D and Fu’s Fs tests significantly rejected the selective-neutrality hypothesis, as well as smooth unimodal mismatch distributions for two lineages. This is in agreement with the observation reported for cyt rn China ,11,12. TS. argentatus, support the idea that future conservation plans should be aimed at managing the populations of both lineages independently and, furthermore, be considered as two ESUs. For the different MUs, we suggest that protection is required synchronously because of their genetic uniqueness. We suggest that four regions identified by SAMOVA should be regarded as different MUs, conforming to the major zoological regions of mainland China and Taiwan.According to the model proposed by Moritz , evolutiSqualidus argentatus were collected from 13 populations in 9 drainages, including 12 populations in Mainland China and one population in Taiwan  with primers SRCD1 (5′-CTCGGATTTTAACCGAGACC-3′) and H15915 (5′-CTCCGATCTCCGGATTACAAGAC-3′) [Taq polymerase . Reactions of PCR amplification were conducted in a thermal cycler (Eppendorf Mastercycler) using the following conditions: one cycle of denaturation at 95 °C for 4 min, 30 cycles of denaturation at 94 °C for 45 s, annealing at 48 °C for 1 min 15 s, and extension at 72 °C for 1 min 30 s, followed by 72 °C extension for 10 min and 4 °C for storage. PCR products were purified by electrophoresis in a 1.0% agarose gel using 1X TAE buffer. The gel was stained with ethidium bromide and the desired DNA band was cut and eluted using the Agarose Gel Purification Kit . Products of the cycle sequencing reactions were conducted by Sangon Biotec (Shanghai) Co., Ltd. with an ABI PRISM 3730 sequencer using the BigDye Terminator kit (Applied Biosystems). The primers used for sequencing were the same as those for PCR amplification. All sequences have been deposited in GenBank under the following inclusive accession numbers: JQ421136-JQ421295.The entire cytochrome AGAC-3′) . Each 10b gene of mtDNA sequences obtained were aligned with the program CLUSTAL X 1.81 [h) [The entire cyt L X 1.81  and then1.81 [h) , nucleot1.81 [h)  and nucl1.81 [h)  using th1.81 [h) .The sequence data was analyzed with the Neighbour-joining (NJ) method using Kimura 2-parameter distance method with MEGA 4 . NeighboGST and NST was carried out using DnaSP 5.0 [The estimates of geographic spread were compared with those obtained by the phylogeographic differentiation test of Pons and Petit . As an onaSP 5.0 .S. argentatus several methods were used including, Tajima’s D statistics [R2 [FS-test [i.e., mismatch distribution), estimated exponential growth rate (g) with program LAMARC [R2, Fu’s FS and Tajima’s D test indicate recent population expansions under assumptions of neutrality [R2, Fu’s FS and Tajima’s D values was evaluated using the coalescent algorithm implemented in DnaSP 5.0 [S. argentatus was explored using mismatch analysis [b mitochondrial sequences. This method is based on the premise that compared with constant population size, population growth or decline leaves distinctive signatures in DNA sequences. If the cyt b locus examined here is neutral and has been transmitted under equilibrium conditions, then a multimodal distribution of haplotypes should result. Alternately a unimodal distribution  could indicate non-equilibrium conditions, especially population expansion. To compare observed distributions with those expected under the expansion model, we calculated the sum of square deviation (SSD) and the Harpending’s raggedness index [6 generations. Burn-in and plots for each analysis were visualized using Tracer version 1.5 [To infer population demographic history of atistics , Ramos-Otics [R2  and Fu’s[FS-test  of neutrm LAMARC , and Baym LAMARC . Departuutrality –49. SignnaSP 5.0 , comparianalysis  of cyt bss index . In ordess index . This cosion 1.5 .FST values and analysis of molecular variance (AMOVA) were used to assess the population configuration and the geographical pattern of population subdivision, as implemented by Arlequin Version 3.5 [FST values among sites were calculated and assessed for significance by comparison with 10,000 permutations of data. For the hierarchical analysis, populations were grouped according to the geographical districts. For the AMOVA hierarchical analysis, populations were grouped according to main lineages (N = 2) and four sub-districts (N = 4): (1) Pearl River sub-district (populations SG and LZ); (2) ZheMin sub-district ; (3) Taiwan sub-district (population SD); and (4) Kiang-Husi Sub-district . Statistical significance of differentiation at the three levels was quantified and tested using Arlequin Version 3.5 [FCT statistic. This analysis also delineates genetic barriers between the inferred groups. We performed these analyses based on 500 simulated annealing steps, and compared maximum indicators of differentiation  when the program was instructed to identify K = 2 through K = 6 partitions of the sampling area for each sampling area within each analysis. Mantel tests [Pairwise sion 3.5 . Pairwission 3.5 . The prosion 3.5  was alsoel tests , implemeel tests  with 100b gene of cyprinids and hence to obtain absolute values of TMRCA. All the analyses were performed using the HKY model of nucleotide substitution. Adequate sampling and convergence to the stationary distribution was checked using TRACER Version 1.5 [A Bayesian analysis of combined data was also used to estimate the divergence times of the major lineages to the most recent common ancestor (TMRCA) by software BEAST version 1.5.3 . Based osion 1.5 . PosteriS. argentatus has a high level of genetic structure among geographical populations. A strong phylogeographic structure due to restricted gene flow among geographic populations was identified in this species, suggesting that the WuYi and Nanling Mountains have formed barriers for this species. We divided all populations of S. argentatus into four groups, consisting of Yangtze-Pearl group, Qiantang-Min group, Jiulong-Beijiang group and Taiwan group. According to the network, there are at least two major migratory routes in mainland China, one from the Yangtze-Pearl group to the Jiulong-Beijiang group and the other to the Qiantang-Min group. The population in Taiwan came from the Qiantang River in mainland China. Demographic analyses implied a population expansion occurred during the recent history of the species. Conforming to the major phylogeographical regions of mainland China and Taiwan, four Management Units have been identified and should be considered for future management projects.Our study showed that


Agabus bipustulatus along several parallel altitudinal gradients in northern Scandinavia. This water beetle is well known for its remarkable morphological variation associated with mountain regions throughout the western Palaearctic. Two morphological ecotypes are recognised: a montane type with reduced flight muscles and a lowland type with fully developed muscles. Using a multilocus survey of allozyme variation and a morphological analysis with landmark-based morphometrics, across thirty-three populations and seven altitudinal gradients, we studied the local adaptive process of gene flow and selection in detail. Populations were sampled at three different elevations: below, at and above the tree line. The results indicate that the levels of divergence observed between ecotypes in morphology and allele frequencies at α-Glycerophosphate dehydrogenase relative to those shown by neutral molecular markers reflects local diversifying selection in situ. Four main lines of evidence are shown here: (1) A repeated morphological pattern of differentiation is observed across all altitudinal transects, with high reclassification probabilities. (2) Allele and genotype frequencies at the α-Gpdh locus are strongly correlated with altitude, in sharp contrast to the presumable neutral markers. (3) Genetic differentiation is two to three times higher among populations across the tree line than among populations at or below. (4) Genetic differentiation between ecotypes within independent mountain areas is reflected by different sets of allozymes.The repeated occurrence of habitat-specific polyphyletic evolved ecotypes throughout the ranges of widely distributed species implies that multiple, independent and parallel selection events have taken place. Ecological transitions across altitudinal gradients over short geographical distances are often associated with variation in habitat-related fitness, these patterns suggest the action of strong selective forces. Genetic markers will therefore contribute differently to differences between ecotypes in local hybrid zones. Here we have studied the adaptive divergence between ecotypes of the water beetle  Gene flow is traditionally seen as the key factor that holds gene pools of local populations together, homogenising the genetic variation of interbreeding populations and opposing the effects of drift and local selection Agabus bipustulatus  one of the most common water beetles in the western Palearctic. It shows a remarkable morphological and geographical variation strongly associated with high altitude mountain regions α-Glycerophosphate dehydrogenase locus (α-Gpdh), a major regulating enzyme in the metabolic pathway generating energy for flight A. bipustulatus complex also confirmed that the associations between the α-Gpdh genotypes and altitude are observed across the entire west Palaearctic region In general, ideal systems for exploring the potential role of diversifying selection for ecotypic divergence are found where common environmental factors, varying across some gradient, repeatedly lead to the existence of similar phenotypic forms in independent geographic localities A. bipustulatus an ideal system to study local environmental adaptation in the absence of gene barriers These repeated and parallel distributions of morphological and enzyme phenotypes make A. bipustulatus is sufficiently strong to impede local gene flow across several independent parallel environmental gradients in Northern Scandinavia. Specifically, we analysed patterns of variation in α-Gpdh and morphological traits as well as in putatively neutral allozyme markers to assess genetic differentiation among populations across one longitudinal and seven altitudinal environmental gradients. These gradients span over three distinct climatic zones ranging from boreal through subalpine to alpine.The purpose of this study was to analyse whether habitat-specific selection in in situ divergent selection is important for creating and maintaining ecotypic differentiation in the presence of countervailing gene flow in A. bipustulatus, we expect (i) sharp changes in morphological and α-Gpdh phenotypes, i.e. the traits subject to selection, across all tree lines. Since selectively neutral loci should show random patterns of variation among populations, especially given the potential for migration among populations, then if selection is imposing reproductive isolation (ii) the strongest differentiation in neutral markers should be observed between populations above and below the tree line both within and between mountains. Furthermore, individuals of different ecotypes within mountains should be less differentiated than individuals of the same ecotype from different mountains. (iii) If this is occurring independently in each location, the combination of neutral loci reflecting this differentiation should be specific to each mountain site.If Agabus bipustulatus is a dark medium-sized water beetle with a Palearctic distribution. The lowland ecotype is found in many types of habitats. In the boreal region, it prefers smaller streams, and springs and pools with little vegetation and stony bottoms. Above the tree line, the montane ecotype is most common in shallow lakes with no fish, chiefly with stony bottoms and some marginal vegetation Agabus bipustulatus has three larval instars The timing of male accessory gland enlargement indicates that the mating season is between May and September A. bipustulatus are scarce but have shown that lowland forms with fully developed flight muscles disperse over distances up to several hundred meters A. bipustulatus is migratory as suggested by Southwood Water beetles have two major means of dispersal. For short-distance dispersal (i.e. within a pond/lake), they swim or, if dispersal occurs along running water, passively drift with the current. The second mean of dispersal, flying, is mainly used for longer distances (i.e. between ponds/lakes). Main period for long-distance dispersal occurs in late summer A total of 1279 individuals from 33 populations were sampled within seven altitudinal transects and one reference latitudinal transect in the Swedish Lapland during September and early October, 1998 , Table 1Betula pubescens ssp. czerepanovii. It constitutes a major ecosystem shift between boreal and alpine zones The single latitudinal transect begins c. 100 km to the east of the mountain region and runs eastward towards the Baltic Sea coast for c. 200 km. This lowland region (LR) transect contains three sampled areas (named A-C), of which A has three populations and B and C have two each . The sevpartial warps) and nonaffine (uniform) shape components. These components can be used to calculate differences between morphology within and between ecotypes or the associations between shape and other parameters ) Geometric morphometrics utilise the spatial covariation of homologous landmarks to calculate affine  Rigrelative warps) are only used to describe the shape variation and were not included in any statistical analysis 100Gpdh allele and altitude in each sex were analysed using a Partial least square (PLS) analyses. This multivariate modelling analysis describes the relationship between sets of dependent  and predictor variables . The analyses were carried out separately for the two sexes in the SIMCA-S version 6.01 program Morphological variation within the total sample was analysed with a Principle Component Analysis (PCA) of the partial warps and the uniform components in the Relative warps program v 1.20 partial warp scores), that a selected specimen of an ecotype belongs to a particular group . We thus use the over-all shape variation to estimate the number of montane ecotypes present at the different altitude levels rather than a visual determination. This analysis is done with a discriminate function analysis (DFA) and subsequent classification analysis. We expect to find specimens that are “misplaced” between ecotypes along the altitudinal gradient as also observed by Eriksson Distribution pattern of the ecotypes were calculated by the posterior probability, based on the overall shape (Ak), Esterase (Est-1 & Est-2), α-Glycerophosphate dehydrogenase (α-Gpdh), Hexokinase (Hk), Isocitrate dehydrogenase (Idh), Malate dehydrogenase (Mdh), Malic enzyme (Me), Mannose reductase (Mnr) and Triosephosphate isomerase (Tpi). Staining recipes and gel buffers are modified from those of Shaw & Prasad et al.Starch gel electrophoresis was used to measure genetic variation. A total of 14 enzyme systems were screened, of which nine coded for ten scorable loci; Adenylate kinase  overlap or lie between the confidence intervals of FIP calculated over loci FIP were estimated from 5000 bootstrap randomisations. Per locus confidence intervals of FIP were calculated from jackknife re-sampling. Genetic diversity of each locus over population was calculated using Nei's unbiased estimator Prior to analyses of population structure, we tested whether the assayed loci could be assumed to behave in a selectively neutral manner. Neutrality can be assumed if the confidence intervals of FST calculations between populations at different elevation levels in mountain regions.In order to access the genetic differentiation of neutral markers between populations above and below the tree line, both within and between mountains, we utilise a three-level hierarchical analyses of gene frequencies. The strength of the population differentiation was estimated via pairwise Fip is the correlation of genes within Individuals relative to Population, Fpa the correlation of genes within Population relative to Areas, Fat the correlation of genes within Areas relative to Total, Fia the correlation of genes within Individuals relative to Areas, Fpt the correlation of genes within Populations relative to Total, and Fit the correlation of genes within Individuals relative to Total. The analysis was carried out using SAS v8.1 software with the “NESTED” procedure. Ratios of sums were used to obtain per locus and overall estimations Mdh and Mnr loci were excluded from this analysis since scores from the Mdh locus in Braket-XIV and from the Mnr locus in Gieravardo and Kråkberget-IV were not considered to be reliable.The relative levels of differentiation within and among altitudinal gradients in the mountain region  were estimated from three-level hierarchical analyses of gene frequencies FST at their respective elevations; calculated groups are MAxMA, MTxMT, MBxMB, MAxMT, MBxMT, and MAxMB. Population differentiation is expected to be higher among populations above the tree line (MAxMA) than among populations below and at the tree line , since the populations of flying forms should be more connected than the populations of non-flying forms. For similar reasons, genetic differentiation between MBxMT elevation levels should be lower than between MAxMT and MAxMB within mountains. Estimates were carried out according to Weir and Cockerham To examine levels of genetic differentiation between elevations in the mountain region, population samples were designated as above (MA), at (MT) and below (MB) the tree line . Geneticα-Gpdh loci) and independently for the mountain areas F to J where the population sample-size made the comparison statistically meaningful  of allele frequencies with the PCAGEN software aningful . The purA. bipustulatus lowland and mountain ecotypes  of FIP that overlapped the C. I. over loci. The α-Gpdh locus has two alleles named 100 and 108. The 100/100α-Gpdh genotype is significantly more frequent in populations above than at or below the tree line in the altitudinal gradient . The frequency of the 108/100α-Gpdh genotype, on the other hand, decreases successively from below, to at and above the tree line (H) of the α-Gpdh locus can be roughly divided into three categories: 1) genetic diversity within populations below the tree line (MB) and in the lowland (LR) varies between H = 0.4–0.5; 2) populations at the tree line (MT) H = 0.2–0.3 and 3) populations above the tree line (MA) H<0.1. However, exceptions exist; Rörmyrberget-III in the lowland has a genetic diversity of H = 0.32, and a few populations at the tree line in area G, I and J have a genetic diversity of 0.39 to 0.50, and no differences were observed across the tree line in area F and from the tree line to above in area E. Genotypic frequencies for each sampled population is given All loci surveyed, except ree line . No signGpdh100 allele per population, were estimated with the Partial least square (PLS) analysis for both sexes independently. The analyses resulted in one significant (R1) component for each sex, with a correlation of 0.63 for females and 0.52 for males  of the PLS models for the female and male analyses was 36.4% and 14.3%, respectively. The highest loading values were observed for the number of copies of the α-Gpdh100 allele and altitude in both sexes.The relationship between morphological variation , altitude, CS (size) and the number of copies of the α-or males . The sigThe genetic diversity over all populations was normally distributed and varied from 0.153 to 0.404 with an overall mean and standard deviation (SD) of 0.261±.07. Below the tree line, genetic diversity was similar in both the LR and MR. At the tree line, genetic diversity was similar in both the LR and MB regions. The diversity increased slightly at the tree line and then decreased moderately above it but these differences are not significant .Fip values per population , with a mean and standard deviation of 0.203±0.12.Significant positive deviations in genetic expectations under random mating were observed within 55% of the populations, as seen in the pulation . The FiFpa = 0.080 and Fpt = 0.075, respectively , at (MT) and above (MA) the tree line. These calculations showed that the mean pairwise Fst values between the elevation classes are higher between than within mountain areas. For elevation classes within mountain areas, the genetic differentiation increases from Fst = 0.026, between MB and MT, to Fst = 0.071 between MT and MA  with a global Fst value of 0.097  of the population structure, which explained 53% of the total genetic variation  separating the two ecotypes in the PCA analyses of mountains F to J are, in descending order: Area A. bipustulatus lowland and montane ecotypes in morphology and α-Gpdh genotype reflects independent local adaptive differentiation in situ across altitudinal gradients via four main lines of evidence: (1) Repeated patterns of morphological differentiation are observed in all independent gradients across the tree line, with high reclassification probabilities, at elevation levels . Minor admixing of ecotypes occurs at all altitudinal levels. (2) The allelic and genotypic variation at the α-Gpdh locus is strongly correlated with altitude and habitats across the tree line, a pattern not observed in molecular markers deemed as neutral. (3) Molecular markers indicate that the strongest genetic differentiation is observed between ecotypes and that this differentiation is more pronounced between mountain areas than within, implying that gene flow may be impeded as a result of local adaptation. (4) The genetic differentiation between ecotypes, estimated from the enzyme systems, is reflected through different sets of loci in each independent mountain area. Evidently, the observed ecological transitions within A. bipustulatus across altitudinal gradients take place over short distances and are likely to be associated with variation in habitat-related fitness. Drotz et al.Gpdh variation in A. bipustulatus across the entire western Palaearctic region. The smaller scale process documented here could thus shed new light on the high morphological and taxonomic diversity within this species that has been discussed for more than 120 years Here we will argue that the divergence between The interpretation of the morphological and genetic adaptation across the tree line will depend on whether the dispersal potential of the ecotypes exceeds the realised gene flow Similar to the results of Eriksson A. bipustulatus. If hydrolysis begins after oviposition we would observe more montane type specimens below the tree lines in the mountain region, since more specimens should be able to fly. Our results indicate that the hydrolysis starts prior or during the pupal stage. Dispersal of the mountain ecotype should be more or less restricted to short dispersal events within lakes and/or downstream movements within water systems. These assumptions are supported by the morphological data for e.g. Atoklinten-II (H5), Atoklinten-IV (H7) and Braket-XIV (J2) which consist of individuals of extreme mountain type, high number of copies of the α-Gpdh100 allele and come from different mountain ridges above the tree line  is more similar to the lowland type morphological and genetically than to the mountain type  up to the tree line (MT) than between areas  and below the tree line (MB) in the mountain region (MR) are of similar magnitude implying similar levels of connectedness . Gene flen areas . Geneticree line . Howeverree line . The adaree line . The selAquarius remigis, Fst values of 0.46 are reported among streams H = 0.058 and 0.234, respectively Fst values have also been reported between flying and non-flying carabid species, Fst = 0.003–0.16 and 0.26–0.27, respectively Studies of wing dimorphic species or comparisons between flying and non-flying species have often shown extensive genetic differentiation between populations or species. Within the flightless stream dwelling waterstrider A. bipustulatus. Thus the A. bipustulatus complex emerges as a promising example of non-allopatric evolution of in situ reproductive isolation In conclusion, there is ample evidence that adaptive divergence maintains local ecotypic differentiation in spite of ongoing gene flow in


Theileria parva, is an economically important disease in Africa. It is a major constraint to the development of the livestock industry in some parts of eastern, central and southern Africa. In Zambia, theileriosis causes losses of up to 10,000 cattle annually.Theileriosis, caused by Theileria parva from Isoka and Petauke districts in Zambia. Microsatellite analysis was then performed on all Theileria parva positive samples for PCR using a panel of 9 microsatellite markers. Microsatellite data was analyzed using microsatellite toolkit, GenAlEx ver. 6, Fstat ver. 2.9.3.2, and LIAN computer softwares.Cattle blood samples were collected for genetic analysis of Theileria parva from a total of 61 samples (33 from Isoka and 28 from Petauke) using a panel of 9 microsatellite markers encompassing the 4 chromosomes of Theileria parva. Wright’s F index (FST = 0.178) showed significant differentiation between the Isoka and Petauke populations. Linkage disequilibrium was observed when populations from both districts were treated as a single population. When analyzed separately, linkage disequilibrium was observed in Kanyelele and Kalembe areas in Isoka district, Isoka district overall and in Petauke district. Petauke district had a higher multiplicity of infection than Isoka district.The combined percentage of positive samples in both districts determined by PCR using the p104 gene primers was 54.9% , while in each district, it was 44.8% (95% CI: 34.8 – 54.8%) and 76.1% (95% CI = 63.9 – 88.4%) for Isoka and Petauke districts, respectively. We analyzed the population genetic structure of Theileria parva from Isoka and Petauke districts showed a low level of genotype exchange between the districts, but a high level of genetic diversity within each district population, implying genetic and geographic sub-structuring between the districts. The sub-structuring observed, along with the lack of panmixia in the populations, could have been due to low transmission levels at the time of sampling. However, the Isoka population was less diverse than the Petauke population.Population genetic analyses of  Theileria parva (T. parva), is transmitted by the 3-host tick Rhipicephalus appendiculatus. The. The7]. T. parva is mainly haploid during its life cycle, except for the polyploidy schizont stage and the diploid zygote and kinete stages [T. parva male and female gametes in the tick gut fuse to form short-lived diploid zygotes which then develop into motile kinetes [T. parva are used to infect the tick vector [e stages . Briefly kinetes  and migr kinetes ,12. The  kinetes ,13. The  kinetes ,13. Genek vector .Trypanosoma cruzi, Toxoplasma gondii and Cryptosporidium hominis) [Cryptosporidium parvum) [Crytosporiduim parvum) [Trypanosoma brucei and Plasmodium falciparum population structures have shown dependence on host specificity and transmission intensity [Studies on the population structures of various protozoan parasite species -21 have hominis) -24, panm parvum)  populatintensity ,27. In gT. parva in Uganda reported a mixture of genotypes in many isolates and linkage disequilibrium (LD) in 3 populations isolated from different areas [Micro- and mini-satellite markers have been used to genotype several species of apicomplexan parasites and their vectors, revealing different population structures ,21,26-30nt areas . When munt areas .T. parva populations has practical implications in disease control and prevention. For instance, populations with a high degree of genetic diversity arise when high levels of recombination occur. This information is very important in vaccine development as it is easier to develop a vaccine against a clonal pathogen than a highly diverse pathogen. A panel of 9 polymorphic micro-satellite markers was used to genotype T. parva positive cattle blood DNA in order to answer the following questions: (i) what is the population structure of the T. parva in eastern and northern Zambia? (ii) Does gene flow occur between T. parva populations sampled from each area? (iii) Do the T. parva populations from the sampling areas consist of a single or multiple distinct populations? To our knowledge, this is the first study on the population genetics of T. parva in Zambia.A crucial and important aspect in population genetic studies is to determine the effect of random and non-random mating on the population structures of disease-causing agents and consequently on the epidemiology of the diseases . InformaThis study received ethical clearance for collection of animal blood from the Biomedical Research Ethics Committee, University of Zambia and from the Department of Veterinary and Livestock Development, Eastern Province, Zambia. The recombinant DNA experiments were approved by Hokkaido University.T. parva parasite laboratory stocks were used for the initial marker screening to determine which markers amplified more isolates and were sufficiently polymorphic for use in the genotyping of field samples from Isoka and Petauke districts: Onderstepoort , Serengeti , Muguga , Nyakizu , Entebbe , Katumba , Kiambu Z464/C12 and B8 , Katete B2  and Buffalo Z5E5 (both origin and year of isolation unknown). These stocks were also used as Genescan control samples during the analysis of field samples.About 10 mL of whole blood samples (n=142) were collected in heparinized tubes from indigenous and mixed breeds of cattle from Kanyelele (n = 62) and Kalembe n=34) areas in Isoka district (n=96) of the northern province (NP) and from Saukani area in Petauke district (n=46) of the eastern province (EP) , 0.25 μM of each primer and distilled water. The PCR conditions were as follows: denaturation at 95°C for 5 minutes; 35 cycles at 95°C for 15 seconds, 50 - 58°C for 60 seconds and 72°C for 1 minute, followed by a final extension step of 5 minutes at 72°C. The amplified products were observed on 1.5% ethidium bromide pre-stained agarose gels to determine the success of PCR amplification. To achieve high genotyping resolution of field samples, microsatellite PCR products were denatured and then capillary electrophoresed in an ABI 3130 genetic analyzer . DNA fragment sizes were analyzed relative to the ROX-labeled GS600 LIZ size standard (Applied Biosystems) using Gene Mapper software (Applied Biosystems). This facilitated the resolution of multiple products with 1 base pair (bp) difference in a single reaction. Multiple products from a single PCR reaction indicated the presence of mixed genotypes. The output data from the genetic analyzer were provided as the area under the peak of each allele (quantitative measurement), with the predominant allele possessing the greatest peak area. In this way, the predominant allele at each locus was identified for each sample, and this data was combined to generate a multi-locus genotype (MLG) representing the most abundant genotype in each sample. Only the alleles with the prescribed base pair range were used to generate the MLG and samples from the same area were electrophoresed and genescaned on the same plate. Separate PCRs were also carried out for different regions.PCR was performed using primers Table  designedhttp://animalgenomics.ucd.i.e./sdepark/ms-toolkit/) was used for the similarity comparison of the MLGs [http://www.anu.edu.au/BoZo/GenAIEx/) [STAT computer package version 2.9.3.2 was used to calculate estimates of F statistics for population genetic analysis (http://www2.unil.ch/popgen/softwares/fstat.htm). LIAN (http://adenine.biz.fh-weihenstephan.de/lian/) was used to test the null hypothesis of linkage equilibrium by calculating a quantification of linkage equilibrium/linkage disequilibrium called the standardized index of association (IAS) [D) which was then compared to the variance expected (VE) for LE. Monte Carlo (MC) computer simulation was used to test the null hypothesis that VD = VE. The computer software calculates a 95% confidence limit L (LMC). When VD is greater than L, the null hypothesis of LE is discarded.An allele sharing co-efficient  in Excelthe MLGs . PrincipenAIEx/) . FSTAT con (IAS) . The staThe majority of the samples in this study possessed several alleles at one or more loci, representing a mixed infection. The mean number of alleles for the nine loci in each sample was calculated and this index value represented the multiplicity of infection within each sample. The overall mean for the index values for each sample was then calculated to provide the multiplicity of infection for each region.T. parva positive percentage of 54.9%  from both districts. The percentage of positive samples within each district was 44.8% (95% CI: 34.8–54.8%)  and 76.1%  for Isoka and Petauke (Saukani area), respectively.The screening of cattle blood samples (n=142) using the p104 gene primers showed a combined T. parva, was used to genotype 61 T. parva positive samples, that is 33  from Isoka district and 28 from Petauke district (Saukani area). Seven markers that either failed to produce signals on gel electrophoresis or exhibited reduced polymorphism were excluded from the final analysis  to 12 (MS71) for Isoka and 4 (MS57 and MS75) to 12 (MS53) for Petauke  and mean number of genotypes per locus for Kanyelele and Kalembe areas in Isoka were 0.675 and 5.67, and 0.503 and 3.67, respectively, with the population from Kanyelele being more diverse than that from Kalembe. The overall estimated heterozygosity (He) and mean number of genotypes/locus for Isoka district was 0.619 and 6.78, respectively  was 0.049, implying moderate differentiation between the 2 populations . The IAS measures the association between alleles at pairs of loci, with IAS values close to 0 or negative indicating panmixia and those significantly greater than 0 indicating non-panmixia [D) between the data and that predicted for panmixia (VE) and L, the 95% confidence interval for VD relative to the null hypothesis, were calculated in order to test the hypothesis of panmixia. Using this analytical method, when the VD value exceeds the L value, LD is indicated and the null hypothesis of panmixia is discarded. When the VD is less than L, LE is indicated and the null hypothesis of panmixia is accepted. When Isoka and Petauke samples were treated as a single population, a IAS value of 0.078 (p< 0.001) and a VD value greater than the value of L was obtained, suggesting LD  and the VD value was greater than the L Value, indicating a state of LD. The IAS values for Kanyelele and Kalembe populations within Isoka were 0.075 (p< 0.001) and 0.243 (p< 0.001), respectively, and both areas showed a VD value greater than the L value, suggesting LD  and a VD value greater than the L value indicating LD were obtained for the Petauke population.In order to determine whether the parasite populations within the two districts comprised a single panmictic population with a high degree of genetic exchange, the level of LE of the alleles at pairs of loci was measured using the standard index of association  and from Saukani area in Petauke district (EP) of Zambia, which are approximately 600 km (straight line distance) apart and are separated by the North Luangwa, South Luangwa and Lukusuzi National Parks  observed between the population from Isoka and Petauke districts. Furthermore, a state of LD with a IAS value greater than 0 was also observed when the Isoka and Petauke populations were treated as a single population  was observed between the 2 populations  and a state of LD was also observed. This state of LD in Isoka  and Petauke might be due to the lack of mixing or movement of animals resulting in a lack of gene flow between the populations that can likely be attributed to the relative isolation of the sample areas from each other and the resulting restricted circulation of genotypes. Despite Kanyelele and Kalembe being far apart, animals from the two areas tend to share grazing grounds during the dry period because of the scarcity of pastures and thus it can be hypothesized that the sharing of grazing grounds allows the introduction of genotypes indigenous to one area into another area. However, due to the availability and close proximity of moderate vegetation for animal grazing during the month of May and the preceding months, it is unlikely that animals from Kanyelele and Kalembe areas would have mixed or shared grazing grounds as they will tend to graze pastures closer to their villages. This lack of mixing of animals would have prevented the introduction of genotypes from one area into another and vice-versa, hence the state of restrictive circulation. The tick vector population is also low at this particular time of the year, thus reducing the challenge levels of infection (i.e. low levels of transmission). It can further be hypothesized that the low challenge levels of infection is also likely to give rise to the presence of fewer genotypes of T. parva which may result in the state of LD and non-panmixia observed in the majority of areas/regions. This is in agreement with a report in which Plasmodium falciparum populations showed strong LD, extensive population differentiation and low diversity in regions of low transmission [The population from Kanyelele occupied all four quadrants while that from Kalembe only occupied three quadrants Figure B. Althouns Table . This waon Table . In the smission . The stasmission . A largeThe majority of samples analyzed in this study comprised mixed infections, with Petauke district showing a higher multiplicity of infection than Isoka district Table . Within T. parva population from Isoka district is distinctively different from that of Petauke district, with significant gene diversity in each population. There was no evidence of genetic exchange among the populations from the 2 districts. Additionally, these results show that the populations from Kanyelele and Kalembe areas in Isoka district comprised similar genotypes although panmixia of parasites between the 2 areas could not be demonstrated. The sub-structuring and population structure of the T. parva populations observed in each district could be due to the restrictive circulation of parasites as a result of the lack of mixing of animals from different regions and/or the low transmission levels of T. parva in the respective areas within each district, which may be due to the reduced population of tick vectors at the time of sampling.The results of this study indicate that the The authors declare no competing interest.WM performed the molecular genetic analyses, data analysis, and statistical analysis and drafted the manuscript. BN was involved in field collection and helped to draft the manuscript. RN purified DNA from the parasite reference stocks and helped in editing the manuscript. MS participated in analyzing data and drafting the manuscript. TK and KI participated in drafting the manuscript. NI helped in collecting the samples and editing the manuscript. CS and HS helped to conceive the study, participate in its design and assisted in obtaining funding. All the authors read and approved the final manuscript.


The identification of isolation signatures is fundamental to better understand the genetic structure of human populations and to test the relations between cultural factors and genetic variation. However, with current approaches, it is not possible to distinguish between the consequences of long-term isolation and the effects of reduced sample size, selection and differential gene flow. To overcome these limitations, we have integrated the analysis of classical genetic diversity measures with a Bayesian method to estimate gene flow and have carried out simulations based on the coalescent. Combining these approaches, we first tested whether the relatively short history of cultural and geographical isolation of four “linguistic islands” of the Eastern Alps  had left detectable signatures in their genetic structure. We then compared our findings to previous studies of European population isolates. Finally, we explored the importance of demographic and cultural factors in shaping genetic diversity among the groups under study. A combination of small initial effective size and continued genetic isolation from surrounding populations seems to provide a coherent explanation for the diversity observed among Sauris, Sappada and Timau, which was found to be substantially greater than in other groups of European isolated populations. Simulations of micro-evolutionary scenarios indicate that ethnicity might have been important in increasing genetic diversity among these culturally related and spatially close populations. Homo sapiens and the great number of opportunities human populations had to meet and admix have limited the overall impact of genetic isolation in many instances Identifying signatures of genetic isolation is more challenging in humans than in most other animal species. In fact, the relatively young evolutionary age of Unfortunately, our current knowledge of genetic isolation in human populations is incomplete. This depends not only on an inadequate sampling of candidate populations and insufficient coverage for important regions, but also on the difficulties in detecting unambiguous signatures of genetic isolation. In contrast to the methodological advancements achieved in the study of isolation in natural populations e.g. , currentThe above-mentioned limitations are even more evident when using unilinearly transmitted polymorphisms, due to the fact that they behave as single loci in evolutionary terms. Nevertheless, these genetic systems continue to represent today an important tool to study geographically and/or culturally isolated populations. In fact, differently from most autosomal loci, there is a relative abundance of data for comparison, both for cosmopolitan and admixed or small and remote groups. Furthermore, they are cheaper than panels of autosomal SNPs and less affected by ascertainment bias. It is also worth noting that unilinear markers provide a potential data basis for the application of some methods which are now being increasingly used in human population genetics The present study aims to test whether a short history of cultural and geographical isolation may have left detectable genetic signatures in some European populations and, in a wider perspective, to assess the importance of demographic history and cultural factors in shaping genetic diversity across linguistic and/or geographic isolates on a continental scale. In order to overcome the limits of current approaches in detecting genetic isolation in human populations, we integrated classical genetic diversity measures with estimates of gene flow under an isolation with migration model. Combining these approaches, we first analyzed the genetic variation of mitochondrial DNA (mtDNA) polymorphisms in four German-speaking linguistic isolates from the Eastern Italian Alps . In order to put our results into a broader context, we built a large dataset which comprises both geographical and/or linguistic isolates and open populations from different parts of the European continent. In this way, we were able to detect converging signatures of genetic isolation in three of the groups under study, Sappada, Sauris and Timau. We then extended our study to the investigation of Y chromosome polymorphisms and we used coalescent simulations in order to explore the role of effective size and gene flow in determining the diversity observed among cultural and geographical isolates from the Italian Alps.Our overall dataset comprises both unstudied populations and groups which have been analyzed in the course of previous research. The former include three linguistic islands of the Eastern Italian Alps  and a Cimbrian group from the Eastern pre-Alps (Lessinia) . SappadaDespite a certain degree of cultural exchange with the surrounding neo-latin groups, these ethno-linguistic isolates have maintained a common cultural background and traditions Data produced in the course of this study were combined with results available in literature and online databases Istituto Italiano di Antropologia. An appropriate informed consent with a withdrawal option was signed by all donors, and all their data were anonymized according to the “Decreto Legislativo della Repubblica Italiana, n° 196/2003”.The research project was approved by the institutional review board of the Buccal swabs were collected from a total of 193 individuals, comprising a sample of 40 from Lessinia, 59 from Sappada, 48 from Sauris and 46 from Timau. Donors were selected only if they were unrelated to other donors at grandparent level and with known-family origin. DNA was extracted using a modified “salting-out” procedure and HVR-1 and HVR-2 were amplified by PCR . Amplified DNA was purified using a High Pure PCR Product Purification Kit , sequenced and compared with the Cambridge Reference Sequence rCRS Haplotype diversity (HD) and its standard error were calculated according to Nei 1987 HThe IMa2 software, which applies the Isolation with Migration model, was used to estimate gene flow between populations http://genfaculty.rutgers.edu/hey/software#IMa2). We performed 2*106 MCMC steps with burn-in period of 106, geometric heating  and 80 Metropolis-coupled chains. mtDNA sequences were assumed to mutate under the Hasegawa-Kishino-Yano (HKY) mutation model −5 ) calculated according to the rates reported in Soares et al., 2009 In order to allow comparisons among gene flow estimates, IMa2 runs were performed with priors which were kept constant for all population pairs. Uniform priors were used for the estimation of effective population size (q = 0–6000) and splitting time (t = 0–2.7), whereas an exponential prior (mean = 0.2) for gene flow (m) was adopted  and then slowly expand (growth rate = 0.017). We used a uniform distribution for splitting times (32–48 generations), with an unequal gene flow between source and sink populations (0.0001 from source to sink and 0.001 in the opposite direction). The three scenarios for mtDNA were set as follows : 1) Sink population effective size = 100–300, gene flow between sink populations = 0–0.005; 2) Sink population effective size = 100–300, gene flow between sink populations = 0.015–0.02; 3). Sink population effective size = 700–900, gene flow between sink populations = 0–0.005. For Y chromosome, we used the same values of effective size but halved gene flow in order to account for the effects of patrilocality in the model. We simulated 104 genealogies for each scenario for both mtDNA (333 bp) and Y chromosome (5 STRs) using mutation-rate estimates for HVR-1 by Soares et al., 2009 We generated random genealogies for three evolutionary scenarios with different effective population sizes and gene flow rates using the Fastsimcoal software A total of 87 different haplotypes were observed in the four populations sampled using HVR-1, HVR-2 and 17 SNPs. They were first assigned to 12 main haplogroups  and, then, further classified into 48 sub-haplogroups see  accordinComparing our results  to availThe multi-dimensional scaling plot based on Fst values for both hypervariable regions see  highlighWe replicated the analyses of intra- and inter-population genetic diversity using a dataset which was limited to HVR-1. However, the set contained a larger number of populations (46 vs 20 for the HVR-1/HVR-2 dataset), that included 14 European linguistic and/or geographic isolates. The reduced HD of Sappada, Sauris and Timau is reconfirmed . IntriguAs a more direct test of genetic isolation, we estimated incoming and outgoing gene flow between the populations that show robust signatures of isolation  and a neighbor or a Central Western European population. Due to the lack of HVR-2 sequences for neighbors, these analyses were performed using HVR-1 data only.We further analysed the genetic diversity among populations carrying out an analysis of the molecular variance using both mtDNA and Y chromosome STRs see  and S6. Sappada, Sauris and Timau showed a value of among-population molecular variance which was three times higher for mtDNA and two times for Y chromosome. Interval estimates obtained for these populations (from 0.090 to 0.136) and other linguistic isolates (from 0.006 to 0.055) using a jackknife procedure do not overlap for mtDNA. Regarding Y chromosome, only the comparison between Albanian Aromuns from Dukasi and Andon Poci produced a value of among-group diversity (0.204) which is comparable to what we observed in German speaking linguistic islands from the Eastern Alps (from 0.187 to 0.261).We first modeled a micro-evolutionary scenario for mtDNA and Y chromosome diversity in Sappada, Sauris and Timau fitting the historical knowledge regarding the splitting time and effective population size. As implied by the “local ethnicity” hypothesis (see below), we assumed an extremely low gene flow among populations. We, then, defined another two scenarios with varying degrees of gene flow and effective population size. Finally, we compared the 95% confidence intervals of distributions obtained for each scenario with observed Fst values see .The observed value of among population diversity  falls clearly within the range of the distributions expected under the “small effective size and low gene flow” scenario for both mtDNA and Y chromosome polymorphisms . FurtherThe other two hypotheses do not seem to be as well supported from simulations. Neither the “moderate effective size and low gene flow” nor the “small effective size and high gene flow” distributions of values encompass the observed Y chromosome Fst. For mtDNA, they are both compatible with the observations. However, the two alternative scenarios receive less support from the distribution of simulations that fall within different ranges of values around the observed Fst value , while lThe so called “Linguistic islands” of the Alps, small groups surrounded by communities that speak a distinct language Interestingly, we were unable to detect any signatures of population expansion in Sappada, Sauris and Timau. However, this evidence is based on the analysis of gene pool of extant populations, so our results do not contradict a scenario in which the signatures of a population expansion could have been erased by a subsequent genetic drift event see . In our However, as discussed in the http://lifesci.rutgers.edu/%7Eheylab/ProgramsandData/Programs/IM/Introduction_to_IM_and_IMa_3_5_2007.pdf ). Secondly, we extended the analysis to a wide spectrum of populations, including neighbors and a reference European population. In this way, we were able to appreciate the different ratios between incoming and outgoing gene flow in populations with a different demographic history. Thirdly and finally, we adopted very stringent criteria for the validation of results (see http://lifesci.rutgers.edu/%7Eheylab/ProgramsandData/Programs/IMa2/Using_IMa2_8_24_2011.pdf). In fact, priors set up for pairs formed by linguistic isolates and neighbors or reference population were found to be unfit for other population pairs, e.g. between isolates or between open populations. Even following these strict rules, however, we were able to detect coherent signatures of a substantially lower incoming gene flow in Sappada, Sauris and Timau compared to open neighboring groups. The difference was even more evident when the latter were replaced by a wide reference Central-Western European population. These results provide support to an unambiguous definition of Sappada, Sauris and Timau communities as genetic isolates, likely due to the combined effect of linguistic and geographical barriers to gene flow.For all the reasons discussed above, we decided to go one step further and apply a method for gene flow estimates based on Bayesian theory. This approach has been so far scantily adopted in human population genetics studies ults see  and keptThere is a general consensus concerning the substantial homogeneity of the genetic structure of European populations relative to what can be observed in other continents All these processes have generated geographically-separated groups, even though they have remained often close to each other. While in most cases, they have maintained their original cultural traits, their level of genetic diversity remains to be established. To this purpose, we compared German speaking populations from the Eastern Alps with linguistic (Aromuns) and geo-linguistic isolates . The results of Amova show a greater within-group diversity for Y chromosome than for mtDNA, which is a likely effect of patrilocality. However, the main finding regards the high differentiation among Sappada, Sauris and Timau for both mtDNA and Y chromosome polymorphisms, both in absolute and comparative terms. How can we explain this result? The most obvious and likely reason could be that Sappada, Sauris and Timau were founded by small groups, as suggested by historical sources Such a strong territoriality in defining ethnic identities and boundaries, which we name “local ethnicity”, may have played a role in marriage strategies, decreasing the genetic exchange among the three linguistic islands. Accordingly, a high level of endogamy has been observed in Sauris in biodemographic studies which cover a time period from the mid eighteenth to the mid nineteenth century To test this hypothesis, we used a heuristic approach based on coalescent simulations in a Bayesian framework. The high and statistically significant Fst values observed for Sappada, Sauris and Timau well fit the scenario modeled according to the “local ethnicity” hypothesis. Neither increasing the effective size nor assuming a higher gene flow, were we able to observe a comparable congruence between observed and simulated data. This suggests that a combination of small initial effective size with continued genetic isolation from surrounding populations and a reduced gene flow among communities may provide a worthwhile working hypothesis for the diversity observed among the linguistic islands of the Eastern Alps.In this paper, we have attempted to overcome some of the limitations of current approaches regarding the study of genetic isolation in human populations using unilinear polymorphisms. Undoubtedly, there is room for further improvement. By increasing the resolution (e.g. sequencing the entire mtDNA molecule) or, even better, exploiting the greater potential of evolutionarily independent loci  could help produce narrower estimates of gene flow and demographic parameters, and overcome the difficulties encountered when applying the IM method to populations with very different demographic histories. Similarly, our simulations could be seen as a first step towards the application of more complex and realistic scenarios. Even with these caveats, however, complementing classical measures of genetic diversity with Bayesian estimates of gene flow and simulations of micro-evolutionary models seems to be a suitable strategy to better understand genetic isolation and its relations with demographic and cultural factors in human populations.File S1Mitochondrial DNA and Y Chromosome raw data of the populations under study.(XLS)Click here for additional data file.Table S1mtDNA HVR-1 (16033–16365 np) and HVR-2 (73–340 np) genetic diversity and demographic parameter estimates in 20 European populations.(DOC)Click here for additional data file.Table S2mtDNA HVR-1 (16033–16365 np) genetic diversity and demographic parameter estimates in 46 European populations.(DOC)Click here for additional data file.Table S3Haplogroup frequency distribution in populations under study.(DOC)Click here for additional data file.Table S4Estimates of effective population size, gene flow and splitting time for all runs performed.(DOC)Click here for additional data file.Table S5Measures of MCMC mixing behavior.(DOC)Click here for additional data file.Table S6List of populations used for the analysis of Y chromosome STRs.(DOC)Click here for additional data file.Figure S1Posterior distributions of parameter estimates. Parameter abbreviations: q1, q2 and qa , T (splitting time), m1>2 (gene flow rate per haplotype from populations 2 to 1) and m2>1 (gene flow rate per haplotype from populations 1 to 2). A) Sappada vs Cadore; B) Sappada vs Central-Western Europe; C) Sauris vs Udine; D) Sauris vs Central-Western Europe; E) Timau vs Udine; F) Timau vs Central-Western Europe.(PDF)Click here for additional data file.Figure S2Evolutionary topology used for the simulations of the three scenarios.(PDF)Click here for additional data file.Figure S3Nucleotide pairwise mismatch distribution for the populations under study.(PDF)Click here for additional data file.Figure S4Fst value distributions of simulated scenarios. The gene flow distributions of the local ethnicity scenario with original (red line) and modified values (green lines) are shown in frames A and B whereas the effective size settings in frames C and D. Frames E and F show the percentual decrease of Fst modal values obtained with increasing values of gene flow and effective size.(PDF)Click here for additional data file.