Oreochromis niloticus. The present study aimed at better genetic characterization of these threatened populations. Partial mtDNA sequences of the D-loop region and variations at 16 microsatellite loci were assessed in the three hot spring populations and compared with three other natural populations of O. niloticus in the region. Results obtained indicated that the hot spring populations had mitochondrial and nuclear genetic variability similar to or higher than the large closely related populations. This may be attributed to the perennial nature of the hot springs, which do not depend on rainfall but rather receive permanent water supply from deep aquifers. The study also revealed that gene flow between the three different hot spring populations was sufficiently low thus allowing their differentiation. This differentiation was unexpected considering the very close proximity of the springs to each other. It is possible that the swamp creates a barrier to free movement of fish from one spring to the other thereby diminishing gene flow. Finally, the most surprising and worrying results were that the three hot spring populations are introgressed by mtDNA genes of O. leucostictus, while microsatellite analysis suggested that some nuclear genes may also have crossed the species barrier. It is very likely that the recent intensification of aquaculture activities in the Loboi drainage may be responsible for these introgressions. Taking into account the importance of these new genetic resources, protection and management actions of the Loboi swamp should be accorded top priority to prevent the loss of these spring populations.The need to improve food security in Africa through culture of tilapias has led to transfer of different species from their natural ranges causing negative impacts on wild fish genetic resources. Loboi swamp in Kenya is fed by three hot springs: Lake Bogoria Hotel, Chelaba and Turtle Springs, hosting natural populations of  Tilapias are some of the most important species for fisheries and aquaculture in Africa. This has contributed to their massive transfer not only within Africa, but to other countries around the world Oreochromis niloticus , the Nile tilapia, is the most economically important species, with a wide natural distribution in Africa. Its natural range covers the entire Nilo-Sudanian province , Ethiopian Rift-Valley province, Kivu province, North Tanganyika Province (Ruzizi) and the Northern part of the East African Rift-Valley Within tilapias, O. n. niloticus from West-Africa and River Nile, O. n. baringoensis from Lake Baringo, O. n. sugutae from River Suguta (Kenya), O. n. eduardianus from Lakes Edward, Albert, George and Tanganyika, O. n. vulcani from Lake Turkana, O. n. cancellatus from River Awash, and Lake Tana and O. n. filoa from hot springs in the Awash system. Recently, Nyingi et al.O. niloticus was characterised by 10 private microsatellite alleles and five private mtDNA haplotypes. Both extents of mitochondrial and microsatellite differentiations were in the range of those observed among other naturally occurring discrete populations in the region . These observations indicated that the Lake Bogoria Hotel Spring population was not as earlier hypothesised, introduced from other neighbouring East African natural populations, but represents a previously unknown natural population.Seven subspecies of Nile tilapia have been described based on morphological characteristics This population offers new opportunities for aquaculture due to various adaptations including its ability to survive in relatively high temperatures (approx. 36°C). The fish may therefore have developed hypoxic resistance mechanisms since dissolved oxygen levels are generally low in warm waters. In addition, special adaptations may also be present that regulate sex determination mechanisms known to involve temperature O. niloticus population, the Chelaba Spring that is located close to the Lake Bogoria Hotel Spring and the Turtle Spring located at the border of the papyrus marsh of Loboi Swamp. In order to protect these populations from various anthropogenic and environmental threats, there is an urgent need to characterise their natural genetic diversity as a first step of a future action plan for sustainable management. The Loboi Swamp itself has receded in size by over 60% over a short period of 30 years due to expansion of irrigation via a ditch constructed in 1970 O. niloticus population from Lake Baringo has been introgressed by O. leucostictus from Lake Naivasha. However, even though mtDNA introgression was clearly established, no nuclear introgression was apparent. These introgressions have been attributed to possible introduction of O. leucostictus into Lake Baringo to boost tilapia fisheries caused by the decline of O. niloticus population by unsustainable high fishing pressure. Another possible explanation for this may have been the expansion of aquaculture in the region, which has been a continuous threat to wild populations of the Nile tilapia in Kenya. The human population of Lakes Baringo and Bogoria, mainly composed of small-scale crop farmers, have begun to diversify their livelihoods by construction of earthen ponds along the rivers and streams in the region O. niloticus.In a first study O. niloticus populations from the region: Lake Albert, Lake Turkana, Lake Baringo, River Suguta and O. leucostictus from Lake Naivasha.In order to characterize the three hot spring populations of the Loboi Swamp, we carried out a study of their genetic variation in mtDNA  and microsatellites together with other Oreochromis niloticus is not an endangered or protected species in Kenya. Seven populations . After death, a fragment of muscle or fin tissue was taken from each specimen and immediately preserved in 95% ethanol for later DNA analyses. Voucher whole fish specimens were fixed in 4% formalin and later preserved in 70% ethanol, and are presently curated at the National Museums of Kenya (NMK) in Nairobi. Other voucher specimens whose sequences were obtained from Genbank are curated at the Musée Royal de l′Afrique Centrale (MRAC) in Tervuren, Belgium or at the Institut des Sciences de l′Evolution de Montpellier (ISEM), France .2, 1% SDS). DNA was then extracted from each sample following the protocol described for genomic DNA extraction for PCR-based techniques 5′-ACCCCTAGCTCCCAAAGCTA-3′ (forward) and 5′- CCTGAAGTAGGACCAGATG-3′ (reverse). Amplifications were performed in a final volume of 50 µl containing 0.25 mM MgCl2, 0.2 mM of each dNTP, 1 µM of each primer, 5 µl of 10x buffer and 10 units of Taq polymerase (Promega). PCR reaction conditions were: 3 minutes pre-heating at 93°C followed by 40 cycles of 30 seconds at 93°C, 30 seconds at 62°C and 1 minute at 72°C, with a final elongation phase of 72°C for five minutes.Approximately 50 mg of the sample tissue was sheared into fine pieces before being digested at 55°C overnight using 10 µl proteinase K (10 mM/ml) in 190 ml of an extraction buffer solution , expected heterozygosity (EH) and the inbreeding coefficient (ISF) using GENEPOP version 3.4 STF and associated probabilities were estimated using a Markov chain method following Microsatellite data were checked for scoring errors due to stuttering or large allele dropout and presence of null alleles, using MICRO-CHECKER software O. niloticus and O. leucostictus, in order to visualize differentiation at the species level. Thereafter, individuals from Lake Baringo and the three hot springs were compared to individuals of O. leucostictus in order to observe possible introgression. Finally, genotypes of individuals from the hot springs and the neighbouring Lake Baringo were compared to establish their relationship.A Factorial Correspondence Analysis (FCA) was carried out with GENETIX Version 4.05 O. leucostictus and O. niloticus at microsatellite loci assuming two populations (K = 2). The admixture model with correlated allele frequencies was chosen q(i), the fraction of the genome of an individual i of a given species (O. niloticus or O. leucostictus), inherited from the other species, three different runs were done for 500,000 generations after discarding 200,000 generations as burn-in. The 90% probability intervals around the different q(i) were calculated.A Bayesian cluster approach as implemented in STRUCTURE version 2.3.4 To confirm occurrence of nuclear introgression, we used Bayesian method implemented in NEWHYBRIDS 1.1 O. niloticus) and Lake Naivasha (O. leucostictus), where a single haplotype was present in 20 and five specimens examined respectively. Sample size (n), number of observed haplotypes (Hob), number of polymorphic sites (p), average number of nucleotide differences (k) and nucleotide diversity (π) are presented in n = 23), two in Lake Albert (n = 20), one in River Suguta (n = 9), five in Lake Bogoria Hotel Spring (n = 24). Furthermore, two other haplotypes were only found in hot springs populations.Forty-two new partial D-Loop sequences were obtained during this study (GenBank accession numbers are given in Tab. II). Thirty different haplotypes were identified using these and an additional 58 sequences from GenBank (Tab. II). All populations studied were polymorphic except for River Senegal  has been naturally introgressed with mtDNA from O. aureus. The haplotype observed in the River Senegal sample corresponds to this alien haplotype. Consequently, the network has been rooted using the single haplotype from River Senegal.According to the different values of the corrected Akaike Information Criterion (AIC) obtained with MEGA 5.1, the optimal model of sequence evolution was HKY model O. leucostictus from Lake Naivasha, belongs to O. leucostictus as demonstrated earlier O. niloticus population from Lake Baringo, and considered introgressed, but was not observed in the Lake Bogoria Hotel Spring O. leucostictus mtDNA has also occurred in all the hot spring populations of the Loboi swamp.All other haplotypes observed, except one clustered in a single highly supported group . The outlier haplotype present in Lake Baringo, the three hot springs populations and The 28 remaining haplotypes are divided in three groups. The first one (I), supported by high bootstrap values (99% for ML and MD), is the sister group of the two others (II and III) and is composed of two different haplotypes present only in the hot spring populations. The four haplotypes of group II (bootstrap 97% for ML and 96% for DM) are found only in the three hot spring populations and in Lake Baringo. The other 22 haplotypes composed group III, which was supported by important bootstrap values: 72% for ML and 86% for DM. All the populations studied possess at least one haplotype in this group.O. leucostictus) had the least . The three hot spring populations had mean number of alleles ranging from 8.4±0.6, (Chelaba Spring), 8.6±0.5 (Turtle Spring) to 8.9±0.9 (Lake Bogoria Hotel Spring), and total number of alleles ranging from 134, 138 to143 at Chelaba, Turtle and Lake Bogoria Hotel Springs, respectively. Distribution of alleles within the populations is shown in A total of 197 individuals from seven localities were genotyped at 16 microsatellite loci. All loci were polymorphic in all populations with the number of alleles ranging from nine to 44. Locus UNH154 was the most polymorphic with 44 alleles, while UNH129 was the least polymorphic with nine alleles. Lake Turkana population recorded the highest mean number of alleles , while Lake Naivasha population  ISF values out of the 105 calculated indicated significant heterozygote deficiencies , were relatively higher  than the STF observed between the latter two populations (0.0177). As expected, the highest STF values were observed for interspecific comparisons between O. niloticus and O. leucostictus populations, and ranged between 0.2653 at Chelaba Spring to 0.3522 at River Suguta.Pairwise o 0.3522  and wereAll populations had private microsatellite alleles as shown in O. niloticus and O. leucostictus. Based on the first axis , two main clusters corresponding to the two different species were identified. Within the O. niloticus cluster, two distinct groups were observed which correspond on one hand to populations from Lake Bogoria Hotel Spring, Chelaba Spring, Turtle Spring and Lake Baringo (A), and on the other hand to Lake Turkana and River Suguta populations (B).FCA of all the genotypes observed  highlighO. leucostictus, we carried out another FCA . The fact that mtDNA of the introgressed individuals did not appear related to O. leucostictus, and the absence of any intermediate individuals between these two groups suggested that there has been no nuclear introgression between the two species.Taking into account that some populations have been introgressed by mtDNA from ther FCA  of all tFinally, an FCA analysis including the three hot spring populations and the Lake Baringo populations was run . The indO. niloticus and O. leucostictus. No F1 hybrid genotypes were detected from the analysis; this means there were no individuals with Q values around 0.5  and could be considered as pure (not introgressed). All individuals of O. niloticus from Lake Turkana and River Suguta, and most of those from the hot springs and Lake Baringo could also be considered as pure . Eleven individuals had Q values ≥ 0.01  and this number rises to 30 when considering the 90% probability intervals . Only one individual from Chelaba Spring had a Q value >0.1 and ten when considering the 90% intervals .Further analysis of genotypes from introgressed populations was carried out using STRUCTURE in order to determine the proportions of nuclear genetic admixtures between ound 0.5 . All indO. leucostictus had a posterior probability of being pure O. leucostictus greater or equal to 0.9989. In O. niloticus populations, seven specimens had a posterior probability of being pure O. niloticus inferior to 0.9574, and could be considered as possible hybrids. For all the concerned specimens, the probability to be F2 hybrid backcrossed with O. niloticus was higher than 0.0416  native population of O. niloticus in Loboi Swamp (Lake Bogoria Hotel Spring), Kenya has been documented In a survey of Lake Baringo is considered a modern representation of a much larger middle to upper Pleistocene Lake Kamasia O. leucosticus from Lake Naivasha previously confirmed to be present in Lake Baringo O. niloticus populations of the springs. The microsatellite study and visual observations of fish specimens during their capture confirmed that the spring populations were composed of a single species, O. niloticus. It is therefore evident that some of the fish in each of the hot springs have been introgressed by O. leucostictus mitochondrial genome.The analysis of partial mtDNA sequences of the D-loop region showed that the haplotype of O. leucostictus but none from the Lake Bogoria Hotel Spring. However, it is possible that the authors failed to detect any introgression of the Lake Bogoria Hotel Spring population due to the low number of hot spring specimens available for their study (16 specimens).The first studies of the Loboi swamp spring population i.e. escapees of O. leucostictus specimens or from O. niloticus introgressed beforehand in aquaculture farms within the Loboi swamp drainage, as it was hypothesized, then it is possible that these introgressions may have occurred after 2007.If these introgressions originated from aquaculture practices Indeed, fish-farming activities within the region has been enhanced as a result of funding by Kenyan government through the Economic Stimulus Programme (ESP) introduced in 2009. The aim of the ESP project was to construct 200 fishponds worth 10.6 million euros in 140 constituencies by June 2013, with each constituency receiving 70,000 € O. leucostictus from Lake Naivasha. In case of large amount of introgression, individuals with intermediate genotypes were expected.At a first glance, it appears that these introgressions only concern mitochondrial DNA as earlier concluded in the study of Lake Baringo population O. niloticus and O. esculentus. These two species are suspected to hybridize even though no mitochondrial introgression has so far been observed in samples . Results of this microsatellite study (eight loci) allowed the separation of both species using a Factorial Correspondence Analysis. However, using STRUCTURE, eight individuals (six O. esculentus and two O. niloticus) appeared to have 90% probability intervals that extend more than 30% out of a pure species Q value which suggested that these individuals had an important degree of genetic introgression.Recently STRUCTURE has been used O. leucostictus had Q values ≤0.003 and may be considered as not introgressed by O. niloticus genes  when considering the 90% probability intervals had 10% of their genes or more that may be considered to have originated from O. leucostictus .In the present study, all us genes . At leasO. niloticus populations are genetically more variable than the O. leucostictus population, it is possible that this hybridization pattern was induced by a high genetic variance of the O. niloticus samples from the hot springs and Lake Baringo rather than introgression with O. leucostictus.Results obtained with NEWHYBRIDS are congruent with these findings and indicated that seven individuals (as observed using STRUCTURE) may have been introgressed. Taking into account that O. niloticus cluster, but closer to the O. leucostictus cluster , with the FCA analysis, all the mtDNA introgressed individuals appeared within their respective  cluster . Lastly, cluster . This suO. niloticus populations from the hot springs of Loboi Swamp and Lake Baringo by nuclear O. leucostictus genes.All of these observations are congruent and confirm with more detail the previous conclusions Even if the extent of the hot spring biotopes are small, and consequently the size of the effective populations, native mtDNA diversity was higher in the hot springs populations than in Lake Baringo and River Suguta populations. Nine different haplotypes were found in the hot springs  while three were found in Lake Baringo  or River Suguta . Differences in samples sizes may partially account for the observed differences but when one considers Chelaba Spring on its own with four haplotypes for six individuals or Turtle Spring with five haplotypes for seven individuals, one can conclude that haplotype diversity in the hot spring is high.2. The swamp itself is not expected to be a favourable biotope for fish survival as it is completely covered with macrophytes of which the most dominant are Typha domingensis and Cyperus papyrusThis does not seem to be congruent with the very small size of the biotopes. The hot springs are located within a small swamp of about 1.5 kmSTF values that were all statistically significant between populations  influences sex ratios towards males It was also apparent that the three populations of These populations and the genetic resources they represent need to be conserved. The conservation and management of Loboi swamp system should thus be accorded top priority in order to safeguard its critical genetic resource that has great potential in development of tilapia aquaculture.Figure S1Photograph showing Lake Bogoria Hotel Hot Spring.(TIF)Click here for additional data file.Figure S2Photograph showing Chelaba Hot Spring.(TIF)Click here for additional data file.Figure S3Photograph showing Turtle Hot Spring.(TIF)Click here for additional data file.Table S1Species specific mtDNA identification, membership probability (STRUTURE) and Posterior probability (NewHybrids) associated with the introgressed specimens. Post. Prob, posterior probability; On, Oreochromis niloticus, Ol, Oreochromis leucostictus, CS, Chelaba Spring; BS, Bogoria Hotel Spring; TS, Turtle Spring; BA, Lake Baringo.(DOCX)Click here for additional data file.


The Uyghur population has experienced extensive interaction with European and Eastern Asian populations historically. A set of high-resolution genetic markers could be useful to infer the genetic relationships between the Uyghur population and European and Asian populations. In this study we typed 100 unrelated Uyghur males living in southern Xinjiang at 26 Y-STR loci. Using the high-resolution 26 Y-STR loci system, we investigated genetic and phylogenetic relationship between the Uyghur population and 23 reference European or Asian populations. We found that the Uyghur population exhibited a genetic admixture of Eastern Asian and European populations, and had a slightly closer relationship with the selected European populations than the Eastern Asian populations. We also demonstrated that the 26 Y-STR loci system was potentially useful in forensic sciences because it has a large power of discrimination and rarely exhibits common haplotypes. However, ancestry inference of Uyghur samples could be challenging due to the admixed nature of the population. Uyghurs live primarily in Xinjiang, a province in the far western region of China and crossed by the Silk Road which is an important pathway connecting Eastern Asia with Central Asia and Europe. As a result, Uyghurs have experienced extensive interaction with other Asian and European populations. Modern Uyghurs present an admixture of Eastern and Western anthropological and genetic traits12Y chromosome contains the largest non-recombining block in human genome and can be used to trace the male line of descent5678101112In this study we studied the genetic diversity at 26 Y-STR loci of Uyghurs living in southern Xinjiang and used them to infer genetic relationships between the Uyghur population and different European and Asian populations. In addition, we presented and compared forensic parameters for different Y-STR systems and discussed the potential application of the Y-STR loci system to infer ancestry of DNA donors that are potentially from a admixed population, like Uyghur.The following procedures were in accordance with the humane and ethical research principles and were approved by the Ethical Committee of Institute of Forensic Science, Ministry of Justice, China.A total of 100 samples from unrelated Uyghur males recruited from southern Xinjiang were collected. Informed consent was obtained from all participants. For each individual, there was no consanguineous marriage or intermarriages with other ethnic groups within the latest three generations.23 different populations in Eastern Asia, Central Asian and Europe containing a total of 7696 haplotypes were selected as reference populations . The geoet al.Genomic DNAs were extracted from blood stains using the Chelex-100 method as described by Walsh ® 26Y system  as described in our previous study26 Y-STR loci  were amplified using the Goldeneyei2)/(n-1), where Pi is the relative frequency of the i-th allele and n is the sample size16i) by the relative frequencies of different haplotypes. Haplotype discrimination capacity (DC) was calculated as the ratio of unique haplotypes in the sample. Match probabilities (MP) were calculated as ΣPi2, where Pi is the frequency of the i-th haplotype. To analyze genetic distances between the Uyghur population and the reference populations, the analysis of molecular variance (AMOVA) and multidimensional scaling (MDS) that maximizes variation among populations were performed using YHRD online tools (http://www.yhrd.org) based on pairwise RST values.Allelic and haplotype frequencies were calculated by direct counting. Genetic diversity (GD) of single-marker was calculated using Nei’s formula GD = n (1-ΣPRST using the T-REX web serverA neighbor-joining phylogenetic tree was constructed for the Uyghur and the reference populations based on a distance matrix of http://predictor.ydna.ru/). A Y-DNA haplogroup tree was adopted from International Society of Genetic Geneology to show the distribution of samples among haplogroups.Phylogenetic analysis was also carried out on haplogroup level with individual samples used in this study. Y-DNA haplogroup of each individual sample was predicted using the offline version of Vadim Urasin’s YPredictor (http://www.xlstat.com/en/). The multi-copy marker DYS389 and markers that have null alleles or duplication variants in multiple samples in the Uyghur population or any of the reference populations were excluded from the analysis, resulting the following markers used for the analysis: DYS393, DYS390, DYS439, DYS391, DYS392, DYS458, DYS437, DYS448, Y_GATA_H.Linear discriminant analysis (LDA) was performed on Uyghur, European, Central Asian and Eastern Asian samples using XLSTAT (RST (20212221222324RST = 0.0132) and Dai is the most distantly related to Uyghur (RST = 0.1717). The AMOVA results also show that the Uyghur population is slightly more closely related to the European populations than the Eastern Asian populations (p = 0.074), which is consistent with the studies of Xu et al. using SNP markerset al. using classical markerset al. using mtDNAs2728et al. are in southern Xinjiang, which are less affected by the recent migrations of Han Chinese30The detailed typing results at the 26 Y-STR loci of 100 male individuals of Uyghur from southern Xinjiang are shown in RST , and MDSRST . To avoiRST , the UygRST values  in a single individual (Sample ID 98). In addition to DYS385ab, duplication variants were observed at DYS19 in one individual (Sample ID 9) and at DYS449 in another individual (Sample ID 35). Null alleles were observed at DYS448 in one individual (Sample ID 89), at DYS643 in one individual (Sample ID 5) and at Y_GATA_H4 in another individual (Sample ID 6). The high frequency of null allele at DYS448 was also observed in previous studies using other commercial kits4042404445® system were used to amplify the markers exhibiting the null alleles. All samples showed the same results except for sample 6 at Y_GATA_H4, which exhibited allele type 11. The results suggested that biological null alleles and null alleles caused by technical issues could be, and should be, distinguished using different amplification systems. Intermediate alleles and duplication variants were also validated using AGCU Database Y24 and GFS 24Y kits; the results were not different from the Goldeneye® system.A total of five variant alleles were observed in five samples when amplified using the GoldeneyeMultiplex Y-STR loci systems have extensive forensic applications including patrilineal relationship evaluation, mixture identification and ancestry inference. To evaluate the power of the 26 Y-STR loci system in forensic applications in Uyghur population, we measured forensic parameters of the 26 Y-STR loci system . Out of We also compared the forensic parameters of the 26 Y-STR loci system to four different sets of Y-STR markers – the minimal 9 loci, PowerPlex Y12 loci, Y-filer 17 loci and PowerPlex Y23 loci – that are commonly used in forensic practices . The 26 4849Ancestry informative DNA markers are valuable tools in forensic sciences. For Uyghur samples, ancestry inference could be especially challenging due to the admixed nature of the population. We investigated the power of the Y-STR system in ancestry inference by asking how well it can discriminate Uyghur samples from different Asian and European samples.The Y-DNA haplogroup tree involving individual samples from Uyghur and reference populations revealed no clear separation of the Uyghur samples from the reference samples . The UygLDA was performed on the Uyghur, European, Central Asian and Eastern Asian samples to look for markers that are ancestry-informative. In this study we genotyped 100 Uyghur males at 26 Y-STR loci and demonstrated that the 26 Y-STR loci system is useful in describing genetic variation in a Uyghur population in southern Xinjiang. Forensic parameters of the 26 Y-STR loci system showed that the system has high discriminatory power within the Uyghur population and has potential application in forensic studies. We showed that the Uyghur population from southern Xinjiang is genetically admixed with reference populations in Eastern Asia and Europe, with a slightly closer relationship to the European populations. Due to the admixed nature of Uyghur, it is hard to differentiate Uyghur DNA donors from donors in Asia or Europe based on the available Y-STR information.How to cite this article: Bian, Y. et al. Analysis of genetic admixture in Uyghur using the 26 Y-STR loci system. Sci. Rep.6, 19998; doi: 10.1038/srep19998 (2016).


Pollicipes elegans to discriminate between two primary hypotheses about the origin of disjunct distributions of extra-tropical populations: trans-tropical stepping-stone colonization versus an out-of-the tropics origin.Studying species with disjunct distributions allows biogeographers to evaluate factors controlling species ranges, limits on gene flow, and allopatric speciation. Here, we use phylogeographic and population genetic studies of the barnacle P. elegans.Nucleotide diversity peaked in the centre of the species’ range in samples from El Salvador and was lower in samples from higher latitudes at Mexico and Peru. Haplotypes from El Salvador samples also had a deeper coalescent, or an older time to a most recent common ancestor. A deep phylogeographical break exists between Mexico and all samples taken to the south . Isolation-with-migration analyses showed no significant gene flow between any of the three regions indicating that the difference in genetic differentiation among all three regions is explained primarily by differences in population separation times. Approximate Bayesian Computation model testing found strong support for an out-of-the tropics origin of extra-tropical populations in P. elegans. Sea surface temperature and habitat suitability are likely mechanisms driving decline of populations in tropical regions, causing the disjunct distribution.We found little evidence consistent with a stepping-stone history of trans-tropical colonization, but instead found strong evidence for a tropical origin model for the largely disjunct distribution of The online version of this article (doi:10.1186/s12983-015-0131-z) contains supplementary material, which is available to authorized users. Species with disjunct or geographically discontinuous ranges are important systems for understanding the factors controlling species’ distributions, population connectivity, and the process of allopatric speciation –10. The Because dispersal and vicariance may be discriminated when the ages of both the barrier and the population separations are known , understPollicipes elegans [P. elegans is only known from El Salvador (SAL) and Costa Rica (CR) , P. eleCR) Fig. , but fewtropical , 33 or atropical , a transtropical , 32.Fig.P. elegans could have been formed by either dispersal or vicariance. In a dispersal scenario, populations of P. elegans crossed the tropics in a stepping-stone fashion, from one hemisphere to the other, likely during periods of cooler climate [P. elegans at the center of the species’ range could be relicts of a larger, continuous ancestral distribution. Under this scenario, the disjunct distribution arose primarily from decline of populations in tropical regions  and the persistence of extra-tropical populations at the edge of the distribution.The disjunctions in the geographic range of  climate , 5, 34. P. elegans has high dispersal potential because their larvae spend 25–35 days in the plankton , mtDNA sequences (cytochrome oxidase I) gathered from extra-tropical populations (MEX and PER) showed a large sequence divergence (1.2 %) and near-reciprocal monophyly [50, swimming performance, and respiration) of extra-tropical populations of P. elegans is lower than for larvae from tropical populations, and suggested a potential physiological barrier to gene flow into the tropics. In addition to temperature, habitat availability may also be an important factor influencing the distribution of P. elegans, given that the species only lives on rocky intertidal substrate with moderate to extreme wave action [P. elegans is absent, suggesting the disjunct distribution of P. elegans may be driven largely by temperature or potentially by a combination of factors in different regions  samples from all three regions of the species’ range (including previously unsampled SAL) and a statistical phylogeographic framework to evaluate models for the origins of the disjunct distribution of P. elegans. First, we gathered mtDNA sequence data that included tropical populations at the centre of the species' distribution to describe the range-wide population structure of this species. Then, using a combination of phylogenetic analyses, coalescent population genetic methods, and Approximate Bayesian Computation (ABC) we compared alternative biogeographical histories that varied in population separation order, relative isolation time, and ancestral Ne, based on expectations of either trans-tropical dispersal or out-of-the tropics origin of extra-tropical populations models. If the disjunct distribution is a consequence of recent trans-tropical, stepping-stone dispersal, we expect an overall genetic signature of declining genetic diversity  from onP. elegans are currently most scarce; diversity peaked in SAL, with lower values in MEX and in PER  sequence was obtained for all individuals. Nucleotide diversity was greatest in the central  portion of the species range, where populations of ER Table . ContrasD, Fu’s FS and R2) were significant for all MEX populations, providing evidence of either a recent demographic expansion or a selective sweep in this region  created by many sequences with few pairwise differences, plus a smaller peak due to the presence of a small number of more divergent haplotypes  and SAL (ΦST from 0.358 to 0.507). In contrast, all pairwise ΦST estimates between SAL and PER were low and not significant  when MEX populations were considered as one group and all SAL and PER populations as another group  analysis showed evidence for migration between ) overlapped with the divergence times between MEX and SAL (~150 to 250 ka). However, the divergence time between SAL and PER showed consistently more recent and narrower joint posterior density distributions (~50 to 150 ka)  than either MEX  or PER   and time of divergence (t0 and t1) for the best-fitting model (TOEMS) showed contrasting patterns between the northern and southern portions of the species’ range. In the north, MEX and SAL separated first , from a common ancestor that was 35 % of the historical Ne of SAL populations found with IMa2 . However, in the south, PER separated from SAL far more recently  from a common ancestor in the tropics that was much smaller, only 3 % of the averaged historical Ne over the coalescent of SAL populations . Overall, diagnostic plots of parameters Anc and t suggested little influence of the tolerance level and priors in the posterior density plots  is not consistent with recent dispersal across the tropics: in a trans-tropical dispersal scenario, sequential colonization events should leave behind a signature of declining genetic diversity from north to south or from south to north, similar to what has been observed in many other taxa that have recently expanded their geographical range in one direction over time . Al. AlPollil region .P. elegans supports a scenario involving a more diverse and possibly older and/or larger SAL populations, we used ABC model comparison to investigate which history fits best to the observed data. Our ABC comparisons found that a tropical origin model was a better fit for the data than a trans-tropical dispersal model. Under the best-fitting tropical origin scenario, P. elegans was abundant and widely distributed throughout the tropics, but through the loss of most populations in the tropical region (perhaps coupled with colonization at higher latitudes), the species’ range was sequentially divided into three isolated populations. Differences in estimates of historical Ne across regions and over time detected with ABC also revealed a complex history of changes in population size with respect to population separations. For example, instead of reflecting recent colonization of PER from SAL, the relatively low mtDNA diversity in PER when compared to MEX and SAL, reflects the relatively small current population size of PER and the relatively long period of time that the ancestral population (Anc0 from t1 to t0) spent at a relatively small population size, potentially reflecting the predominance of cold glacial periods over the last 400 ka. In contrast, the high mtDNA diversity of the SAL populations can be attributed to the relatively recent large increase in population size that happened after the separation of PER at t0, a result that is not evident from the mismatch or skyride analyses. However, skyride plots also showed no evidence of recent variation in Ne over time in PER, also consistent with an extinction model rather than a history punctuated by a recent colonization events  may currently regulate the distribution of  the TEP , 63, but the TEP . At the on rates –67, the on rates –70, bothon rates . The inton rates –74.P. elegans. El Niño Southern Oscillation (ENSO) and acute seasonal variation have profoundly impacted population and community dynamics through repeated regional mortality events and extra-tropical colonization events, especially in South America [P. elegans in Lobos de Afuera island, a location 200 km away from the PER2 site of this study [P. elegans colonized rocky shores of central Peru, establishing a local short-lived local fishery [e among PER populations has been considerably smaller than either SAL or MEX  between PER and SAL populations suggest that PER populations could provide an earlier snapshot in the process of speciation between tropical and extra-tropical populations. With near reciprocal monophyly, a future study with multi-locus species delimitation methods would be appropriate to address the presence of cryptic species within P. elegans.Speciation in marine taxa like derstood , 79–81. P. elegans. Instead, our results support the idea that the extra-tropical populations of P. elegans likely developed through a process of population separations that was not dominated by dispersal across uninhabitable regions, but instead best described by a tropical origin model. Further, our data and analyses indicate that for P. elegans, the separation of extra-tropical populations from central populations in the warmest tropical water was achieved in two temporally distinct steps: the isolation of MEX populations followed by a more recent isolation of PER populations, both potentially during cold interglacial climates.Although taxa with disjunct distributions have been the subject of intensive biogeographic research –85, compP. elegans in the eastern Pacific. Drivers of the modern disjunct geographical distribution and isolation of populations of P. elegans are likely explained by habitat availability, ocean circulation, and physiological response to thermal stress. Future studies can address if the distinct evolutionary significant units found in MEX and Southern MEX correspond to a novel example of cryptic speciation along a steep latitudinal thermal gradient and validate our inferences about the demographic history of P. elegans using additional independent nuclear markers.The formation of ephemeral dispersal barriers that arose during sea level fluctuations in the Pleistocene , 86, 87 Pollicipes elegans were collected between 2009 and 2011 from sites chosen to capture variation in populations at the north, south, and centre of the species’ range , 0.3 μM each primer, and approximately 50 ng of genomic DNA. Amplification conditions consisted of 94 °C for 2 min followed by 5 cycles of 94 °C for 30 s, 42 °C for 1:30 min, and 72 °C for 1 min. These first 5 cycles were followed by 35 cycles of 94 °C for 30 s, 49 °C for 1 min, 72 °C for 1 min, with a final incubation step at 72 °C for 5 min. PCR products were purified using QIAquick spin filter columns (QIAgen) and sequenced in both directions on an ABI-3730 sequencer (Applied Biosystems). The sequences were inspected for base-calling errors with the forward and reverse strands aligned using Sequencher 4.7 (Gene Codes Corporation). Sequences were edited using BioEdit [ClustalW [Adult individuals of nge Fig. . A total BioEdit  and easiClustalW . SequencP. elegans. Nucleotide diversity (π), haplotype diversity (H), pairwise differences (k), segregating sites (S), Tajima’s D [FS [Arlequin version 3.5.1.2 [R2 [DnaSP version 5 [SSD), Harpending’s r, and the parameter tau (τ) in Arlequin to evaluate the fit of a sudden demographic expansion. The historical demography at each site was reconstructed by using Bayesian Markov Chain (MCMC) method implemented in Beast version 1.7.2 [−8 substitutions per site per year and perform the analysis. Although Chthamalus barnacles are phylogenetically closer to Pollicipes (~239 Ma) [Chthamalus significantly faster rate could reflect a separation that started earlier than the closure of the panama isthmus, resulting in overestimates of the evolution rate. Therefore, using an average rate across crustacean taxa (i.e. 2 % per My) is a conservative approach and appropriate for this study. The output files were checked in Tracer version 1.5 to ensure that all effective sampling sizes (ESS) values were greater than 200. We considered population size changes as significant when the upper and lower 95 % confidence intervals at the root and the tips did not overlap.Frequency based statistics were calculated to evaluate patterns of genetic diversity and demographic history across the distribution range of jima’s D , and Fu’’s D [FS  were cal 3.5.1.2  with 10,.1.2 [R2  was calcersion 5  with 10,on 1.7.2 . The anaon 1.7.2 , a relaxon 1.7.2 . Each GMon 1.7.2 –102 to c~239 Ma) , we inclMrBayes version 3.1.2 [TreeAnnotator version 1.8.2 [RAxML version 7.04 [PAUP* 4.0b10a [TCS [P. pollicipes (GenBank:HM563669) and P. caboverdensis (GenBank:HM563667) were retrieved from GenBank database and used as outgroups.Phylogenetic relationships were inferred using Bayesian Inference (BI), Maximum Likelihood (ML), and Maximum Parsimony (MP). For the model-based methods, the best sequence partition strategy and best-fitting model of nucleotide substitution was identified with PartitionFinder using the Bayesian Information Criterion (BIC). BI analyses were performed using on 3.1.2 , with fion 1.8.2  from a cion 7.04 . Nodal s 4.0b10a , with he10a [TCS . PartialST, a measure of the proportion of nucleotide diversity within sub-populations relative to the total. We used the substitution model in Arlequin most similar to the model identified with PartitionFinder. Significance was assessed with 10,000 permutations of the data and a Bonferroni correction was used to reduce the chances of obtaining false positives with multiple pairwise comparisons. A Spatial Analysis of Molecular Variance (SAMOVA) was then used to define groups of populations by maximizing the proportion of total genetic variance due to differences between groups of populations in SAMOVA version 1.0 [Genetic structure was characterized with Φsion 1.0 .IMa2 [P. pollicipes [−6 mutations per locus per generation. Initial 8 h IMa2 runs (with a burn-in of 8 h) of forty independent heated chains [We estimated demographic parameters by sampling the coalescent in a Bayesian framework with the program IMa2 , which iIMa2 –111 thatllicipes , 113 wasd chains  were perd chains .Likelihood ratio tests (LRTs) were computed to test the significance of migration (m = 0) between each pair of populations , 110. BeBayesian Serial Simcoal (BSSC) [ST and Tajima’s D could be explained as a result of one of four specific demographic models  approach . Bayesial (BSSC)  was usedels Fig. . Given tels Fig. , a scenaels Fig. , a histoels Fig. .0 and t1, or by extinction of intermediate populations (vicariance)  in the colonized region immediately after the split of populations. Each bottleneck event was assigned a prior exponential distribution centered in 20 % of the modern Ne. The sizes of ancestral populations after population splits were represented by parameters Anc0 and Anc1. This parameter is obtained by modeling the ancestral population size at t0 and t1 to be any number between one and twenty times the size of the modern Ne.In all models, an average of parameter estimates obtained from IMa2 runs were used to define the prior boundaries in Bayesian simulation scenarios Fig. . The splce) Fig. . The modce) Fig.  and Peruce) Fig.  were seqico Fig.  or Peru ico Fig.  and thatabc package [0 and t1) and ancestral Ne (parameters Anc0 and Anc1) were estimated for the best-fitting model and validated using four tolerance levels.Each scenario was tested by modeling 1,000,000 coalescent simulations . We conducted model fitting in R using  package  with fee package , a more  package . Cross v


Leishmania (Leishmania) major is one of the etiological agents responsible for CL in Pakistan. Genetic variability and population structure have been investigated for 66 DNA samples of L. (L.) major isolated from skin biopsy of CL patients.Cutaneous leishmaniasis (CL) is a major and fast increasing public health problem, both among the local Pakistani populations and the Afghan refugees in camps. L. (L.) major, was used to investigate the genetic polymorphisms and population structures of Pakistani L. (L.) major DNA isolated from CL human cases. Their microsatellite profiles were compared to those of 130 previously typed strains of L. (L.) major from various geographical localities.Multilocus microsatellite typing (MLMT), employing 10 independent genetic markers specific to L. (L.) major DNA samples. The data displayed significant microsatellite polymorphisms with rare allelic heterozygosities. A Bayesian model-based approach and phylogenetic analysis inferred two L. (L.) major populations in Pakistan. Thirty-four samples belonged to one population and the remaining 32 L. (L.) major samples grouped together into another population. The two Pakistani L. (L.) major populations formed separate clusters, which differ genetically from the populations of L. (L.) major from Central Asia, Iran, Middle East and Africa.All the markers were polymorphic and fifty-one MLMT profiles were recognized among the 66 L. (L.) major might be related to the existence of different species of sand fly and/or rodent reservoir host in Sindh province, Pakistan. A comprehensive study of the epidemiology of CL including the situation or spreading of reservoirs and sand fly vectors in these foci is, therefore, warranted.The considerable genetic variability of  Leishmania. Cutaneous leishmaniasis (CL) is more widely distributed, with about one-third of cases occurring in each of three epidemiological regions, the Americas, the Mediterranean basin, and western Asia from the Middle East to Central Asia .,16.L. (Lequences  with somL. (L.) major (MHOM/IL/1980/Friedlin) was included for which the microsatellite sizes for the 10 loci had been determined by sequencing. MLMType for each strain was obtained by compiling all alleles at each locus. The microsatellite profiles previously described for 130 strains of L. (L.) major originated from different geographical areas, including Africa, Central Asia, Iran and Middle East [Microsatellite genotyping was carried out using 10 variable microsatellite markers: 4gtg, 27gtg, 36gtg, 39gtg, 45gtg, 1gc, 28at, 71at, 1gaca and 1ca . Fluoresdle East ,14 were L. (L.) major DNA sample analyzed. Population structure was investigated by the STRUCTURE software, which applies a Bayesian model-based clustering approach [Multilocus genotype data consists of the number of repeats in each microsatellite markers for each approach . This alapproach .http://bioinformatics.org/~tryphon/populations/) by applying the proportion of shared alleles distance measure (Dps). Phylogenetic trees were constructed using Neighbour-joining (NJ) method by the help of the software programmes POPULATIONS 1.2.28 and MEGA [Microsatellite-based genetic distances were calculated with the software packages MSA  and POPUand MEGA .He, gene diversity) and observed heterozygosity (Ho) as well as inbreeding co-efficient (Fis) and mean number of alleles were estimated using the Genetic Data Analysis software (http://hydrodictyon.eeb.uconn.edu/people/plewis/software.php). The degree of genetic differentiation and gene flow among populations were assessed by calculating Fst values with corresponding p-values. Fst values higher than 0.25 indicate strong genetic differentiation [Expected , Larkana, Sindh province, Pakistan. The patients were aware that their skin scrapings were needed for diagnosis of the disease using molecular diagnostic methods. Doctors obtained the written consent of the patients. The protocols used were approved by Chandka Medical College, Pakistan.L. (L.) major collected from CL cases in endemic areas of Sindh and Balochistan province, Pakistan. In total, 51 different multilocus microsatellite profiles summarizing the repeat numbers obtained for the 10 microsatellite markers were assigned to the 66 Pakistani L. (L.) major samples tested, of which 43 were unique to individual strains and eight were shared by more than one strain  as well as the inbreeding co-efficient (Fis). The Fis values for 10 markers ranged from −0.0508 to 1. Ho ranged from 0 to 0.1250 and He ranged from 0 to 0.7488. All markers but one indicated a depletion of heterozygotes. The exception was the 27GTG marker, which revealed an excess of heterozygotes (He < Ho) corroborated by negative Fis.Homozygous allele combinations predominated in the samples studied. Table Fst value (0.4329) and P-value 0.0001.Bayesian model-based analysis of the 66 samples using STRUCTURE showed that the optimal number of population was 2  major strains from 20 countries in Central Asia, Africa, Iran and Middle East, STRUCTURE analysis assigned these 196 strains to 8 different populations  major studied so far. The Pakistani population was re-analysed separately by STRUCTURE and split into the two sub-populations described above  major using MEGA assigned the Pakistani samples to the same two clusters  major clusters. The Fst values  major from Pakistan was investigated, compared, and correlated with their geographical sources and prevailing environmental and ecological conditions. The present MLMT analysis revealed considerable genetic variation for the 66 Pakistani L. (L.) major DNA samples presenting 43 individual microsatellite profiles and eight were shared by several samples. This is a quite unexpected result because all the samples studied were from different villages and cities of Larkana, Shahdadkot and Dadu districts of Sindh province, except three that came from Balochistan province. Heterogeneity of Pakistani L. (L.)major is thus much higher as previously suggested when little intra-specific polymorphism was found for the parasites from the same area [Fis, Ho and He values, microsatellite loci were mostly homozygous in the Pakistani sample set. Leishmania species have been considered to be clonal diploid organisms [Fis values are supposed to be negative due to heterozygote accumulation [et al. (2009) [L. (L.) major DNA isolates came from the same area. Thus, the heterozygote deficiency found in the studied samples is unlikely to be due to the Wahlund effect . In our study, 62 strains were amplified at all microsatellite loci and only four strains had one missing locus each , but our data analysis using Micro-Checker software (http://www.microchecker.hull.ac.uk/) showed evidence for a null allele with few microsatellite loci . Therefore, we cannot exclude the presence of heterozygote deficiency could result from null alleles. The high FIS values observed across all polymorphic loci are also likely to be due to inbreeding. Selection may cause under-dominance by decreasing the fitness of heterozygous genotypes and gene conversion could lead to a transition from heterozygous to the homozygous stage [FIS should be expected across our 10 non-coding microsatellite loci. As can be seen in Table FIS values were zero or close to zero suggesting that these loci are in equilibrium. All other loci had high FIS values indicating heterozygote deficiencies. Due to the variance of Fis values across the microsatellite loci, we cannot exclude that selection or gene conversions have contributed to the high FIS values observed in this study. We would, however favor the hypothesis of significant inbreeding present in both Pakistani populations of L. major, as previously reported for different Leishmania parasites [In this study, the diversity and population genetic structure of strains of rganisms  in whichmulation . In this. 2009) . In our us stage . In both009 . In arasites -27.L. (L.) major samples to two populations (POP-A and POP-B). Fstatistics confirmed that these are genetically isolated populations. The two samples from Balochistan belonged to Population A. The Pakistani populations identified in the present study where clearly separated from the populations comprising of L. (L.) major strains from Central Asia, Africa, Iran and Middle East.The Bayesian clustering approach implemented in STRUCTURE as well as the phylogenetic analysis based on genetic distances assigned the 66 Pakistani Phlebotomus papatasi and P. salehi, and three rodent species  are incriminated as vectors and reservoirs, respectively, of L. (L.) major parasites in Pakistan [L. (L.) major in Sindh province, Pakistan has distinct epidemiological and biological characteristics. Variations among the samples of L. (L.) major from the same endemic area leading to assignment to different populations were previously attributed to differences in sand fly vector populations [L. (L.) major suggests that the extant parasites in Pakistan may have been restricted there for a long time, rather than being recently introduced from elsewhere by human or animal reservoir migration. The same scenario was recently obseved for L. (L.) tropica in Morocco [et al. (1991) [L. (L.) major in Sindh province, Pakistan, is.The two Pakistani populations did not correlate with the geographical origin of the parasites that fell into them. Their analysis was, however, hampered owing to the small number (only 2) of DNA samples available from Balochistan province. The geographical overlap between two genetically isolated populations might be due to introduction of parasites from different foci through human or reservoir migrations and vector sandfly habitat expansion. One of the most important risk factors in the increase of CL worldwide has been the migration of people from endemic regions . The occPakistan . It is aulations  and reseulations . Indeed, Morocco  where tw. (1991)  speculatLeishmania DNA. We succeeded in amplifying 10 microsatellite loci from 64 clinical DNA samples. Parasite culture is not easy to perform, especially under field conditions, and often not successful. Therefore, assays that can be carried out directly on clinical materials are of great advantage for surveys including high numbers of isolates. In addition, the direct DNA isolation of Leishmania from clinical samples would avoid the potential selection of special parasites during in vitro cultivation.Our study demonstrated the possibility and usefulness of performing MLMT using skin biopsy materials from patient tissues that contain only small amounts of L. (L.) major in Pakistan by using the MLMT approach. We were able to detect two genetically isolated populations of L. (L.) major in Sindh province, Pakistan. Furthermore, our results corroborated the possibility and/or usefulness of genotyping L. (L.) major directly from clinical samples [To the best of our knowledge, this study is the first one that has investigated the population structure and genetic diversity of  samples ,34. A coThe authors declare that they have no competing interests.MZA designed the study, performed microsatellite typing, conducted data analysis and wrote the manuscript. AMB, FRS, JHB, HK, HU and YH collected the skin biopsy samples and isolated the DNA. RN conducted data analysis. GS participated in coordination and helped to draft the manuscript. YH, HK and KK conceived of the study and critically revised the manuscript. All authors have read and approved the final manuscript.


Antheraea mylitta is an economically important wild silkmoth species distributed across India. A number of morphologically and ethologically well-defined ecotypes are known for this species that differ in their primary food plant specificity. Most of these ecotypes do not interbreed in nature, but are able to produce offspring under captive conditions. Microsatellite markers were developed for A. mylitta, and out of these, ten well-behaved microsatellite loci were used to analyze the population structure of different ecoraces. A total of 154 individual moths belonging to eight different ecoraces, were screened at each locus. Hierarchical analysis of population structure using Analysis of MOlecular VAriance (AMOVA) revealed significant structuring (FST = 0.154) and considerable inbreeding (FIS = 0.505). A significant isolation by distance was also observed. The number of possible population clusters was investigated using distance method, Bayesian algorithm and self organization maps (SOM). The first two methods revealed two distinct clusters, whereas the SOM showed the different ecoraces not to be clearly differentiated. These results suggest that although there is a large degree of phenotypic variation among the different ecoraces of A. mylitta, genetically they are not very different, and the phenotypic differences may largely be a result of their respective ecology.Indian tasar silkmoth,  Antheraea mylitta is a natural fauna of tropical India, distributed in different geographical locations and habitats in this country. Possibly, because of the distinct ecological conditions prevailing in these different localities, several morphological variants, traditionally called ecoraces, have been identified in A. mylittaTerminalia (Family: Combretaceae) species and Shorea robusta (Family: Dipterocarpaceae), and also on a number of secondary food plantsTerminalia and Shorea are quite far phylogenetically. Although both are Rosids, Terminalia belong to order Myrtales and Shorea to MalvalesA. mylitta has its own distinctive colour, and is coarser than Bombyx mori silk. However, the tasar silk has higher tensile strength, elongation, and stress-relaxation values than the silk secreted by the domesticated silkworm B. mori. These properties have made tasar silk as competent and desirable as B. mori silk10Populations of several species are further classified by taxonomists into subspecies, races, demes, clines and so on, of which only cline and deme have non-arbitrary definitions2A. mylitta primarily inhabits forested habitats, it is expected that with the gradual depletion of forest cover due to surge in the human activities the habitat lost its continuity and resulted in geographic isolation. This isolation might have allowed the populations to continue separately for generations, resulting in different ecoraces. There is quite a bit of ambiguity in naming of these ecoraces. As the boundaries between the ecoraces are often fuzzy and the races do not obey the concept of static boundaries, the racial identity seems to be arbitrary at times. Quite surprisingly, two crops of the same population are sometimes named as two different ecoracesSince, A. mylitta ecoraces. Although few studies have been carried out with RFLP12Antheraea assama1819There is a lack of well-characterized molecular markers to study A. mylitta ecoraces have made it difficult for researchers to identify ecorace specific phenotypic markersA. mylitta. With these points in mind, we developed 32 microsatellite markers and screened eight A. mylitta ecoraces collected from different geographical locations across India to obtain insights into the population genetics of these ecoraces  taking all the populations together ranged from 0 (Amysat013) to 0.81 ± 0.12 (Amysat023). The minimum average Ho across all the loci was observed in the population Daba Trivoltine (0.25 ± 0.32) and maximum in Sukinda (0.37 ± 0.34).Considerable variation was observed at all microsatellite loci. Except Bhandara, which was monomorphic at the locus Amysat013, all other populations showed polymorphism at all the 10 loci . Number e) and observed heterozygosities (Ho) for this locus are exactly the same. Generally, the more similar He and Ho are, the higher is the probability that the locus is in HWE. This is supported by the observations in all other loci. A similar exact test21Hardy-Weinberg exact tests were done for all the ten loci and inferences were drawn based on the sequential Bonferroni corrected α , Table 2Exact test for genotypic disequilibrium was performed using Markov chain simulations to identify significant association between pairs of loci. Tests for each population as well as global tests were carried out. As there are 10 loci in all, there can be 45 possible pairs. However, due to lack of polymorphism for Amysat013 in the Bhandara population, only 36 tests could be performed for this population. Out of these 351 total comparisons, significant LD, following sequential Bonferroni correction, could be observed in only three cases, i.e. Loci Amysat023 & Amysat032, Amysat026 & Amysat032 and Amysat023 & Amysat026, all in Sukinda population. In case of the global test, following sequential Bonferroni correction, only three pairs of loci , out of 45 comparisons were found to be under LD. All other loci segregated independently of each other .ST value. FIS values were significant for all loci, except Amysat026. Result of the AMOVA, along with the fixation indices and their bootstrap confidence intervals are shown in IS value was considerably high (0.505), suggesting significant inbreeding within these populations. FST was also observed to be significant (0.154). In accordance with these values, 15.4% of the covariance was assigned to the across populations and 43% to the within population component. About 42% of the covariance could be assigned to the within individuals component.The locus specific fixation indices are shown in ST values ranged from 0.015 (between Sarihana and Daba Bivoltine populations) to 0.339 (between Bhandara and Sukinda populations). All pairwise genetic distance values were significant except the one between Sarihana and Daba Bivoltine populations   suggesting a lack of migration-drift equilibrium  see . The Man.44) see . The Manilibrium .In the BOTTLENECK analysis, none of the populations showed significant excess of simulated gene diversity compared to the observed gene diversity either under IAM or TPM, suggesting lack of recent bottleneck in any of these eight populations.et al.’s DA distance, showed two major clusters  of southern and central India and the second group consisted of remaining six ecoraces  mainly from eastern India .The SOM output layer consisted of a 11 × 6 grid . The mapA. mylitta and used them to study the genetic structure of its different ecoraces. Like most other species studied, we observed dinucleotide microsatellites to be the most abundant, followed by tri- and tetra nucleotide. Among the dinucleotide microsatellites, those with (CA) motifs were the most abundant. CA/GT repeats are generally the most common dinucleotide repeat in a wide variety of vertebrates and arthropods25272930313233343536In this paper, we have characterized the microsatellites of A. assama, a species with a very restricted distribution, has been studied and its populations were found to be reasonably differentiated18B. mori have also been subjected to this kind of analysis324546There have been studies investigating the nature and distribution of genetic variation in wild lepidopterans. However, these have mainly focused on threatened or declining species3839414243A. mylitta revealed considerable genetic differentiation across and within the populations of both the ecoraces. In another work, using RAPD markers, genetic variation among the different ecoraces of this moth were assessedA. mylitta ecoraces.A couple of studies using ISSR markers to analyze the intra-race diversity in RailyA. mylitta for a large part of its distribution. Eight ecoraces distributed as far as 1000 km apart were collected and their population genetics was investigated using ten polymorphic microsatellite loci. There was significant deviation from HWE in some of the loci in a few populations. This can be because of several reasons- 1) There was an overall excess of homozygotes than one would expect just by chance alone as reflected by the high locus specific FIS values as well as its weighted average (0.505) across loci in the AMOVA  The sample sizes for each population was consistently low , and may not represent every genotype adequately, and 3) Presence of null alleles, as was found using MICROCHECKER, which in turn may result in the excess of homozygote in all the loci. Significant association was absent in almost all locus pairs in all the populations. Even globally, i.e., when all the populations were included together, there was significant association between only three of the possible 45 pairs of loci.Here, we have studied the genetic structure of he AMOVA . As has A. mylitta populations in the region under study (FST = 0.154). Although result of the Mantel test was significant , there was an absence of migration-drift equilibrium as reflected by the lack of significant correlation between the residuals of a km-FST regression and km . Excluding the localities with non-significant pairwise FST values, i.e., Daba Bivoltine and Sarihana, either sequentially or together, did not change this pattern, i.e., the km-FST correlation was consistently significant, but the km-residual Mantel was not significant. This suggests a lack of migration-drift equilibrium in the region studied. One may posit from the extent of scatter of individual points in A. mylitta. However, as there are not too many points in the short distance range, this lack of equilibrium can also be an artifact of the sampling regimeAMOVA revealed significant structuring of A. mylitta populations into two groups. One group consisted of one population each from southern and central India . Another consisted of populations from eastern India. To investigate this pattern further, Andhra Local and Bhandara was taken as one group and the rest of the six populations were taken as another and an AMOVA was performed, giving 17% of the covariance assigned to the “Among groups” component and 5% to the “Among populations within groups” component, as compared to the overall “among populations” component of 15.41%, suggesting the possibility of substructuring. This is in full agreement with the STRUCTURE analysis. Remarkably, the two Daba ecoraces, though occurring sympatrically was found to be genetically distinct (FST = 0.06452). This is in line with the previous reportA. mylitta. In the NJ tree 2, (CA)10, (GA)10, (GATA)10, (CAC)7, and (AGC)7, following previously reported protocolRsaI and XmnI restriction enzymes, ligated to double stranded superSNX linkers, hybridized with biotinylated microsatellite oligonucleotides and captured on streptavidin coated magnetic beads. Unhybridized DNA was washed away, and captured DNA was recovered by polymerase chain reaction (PCR) using the single stranded superSNX-F as primer. The PCR products were ligated into the pCR®4-TOPO® TA vector (Invitrogen) and transformed into the XL1-Blue competent cells. Colony PCR was performed to select the amplicons of size 500–1000 bp. In total, 155 of the 240 colonies screened contained inserts of size more than 500 bp, which were sequenced using M13 forward and reverse primers, on ABI Prism 3100 Genetic Analyzer (Applied Biosystems). Among these, 46 had non-redundant microsatellite repeats, out of which a majority were found to harbor dinucleotide repeat motifs (65.2%), followed by tri- (17.4%), tetra- (6.5%) and complex (10.9%) repeat motifs. Among dinucleotide repeat motifs CA was found to be more abundant (60%) followed by GA (30%) and AT (10%). No CG motif was observed. A total of 16 clones were eliminated because the microsatellite sequences did not have sufficient flanking regions and so primer sequences could not be designed for amplification. Therefore, primers were designed for the rest of the 30 genomic microsatellite loci. Out of which, eight loci either did not yield expected product size or produced multiple non-specific bands. After the initial PCR assays, 22 microsatellite loci could be successfully amplified.For the construction of genomic library, DNA was isolated from A. mylitta unigene EST sequences2, 5 pmole of each primers, 10 ng of genomic DNA as template and 0.5 U Taq polymerase (MBI Fermentas). The PCR conditions were initially at 94 °C for 3 minutes as initial denaturation and 35 cycles of: 94 °C denaturation for 30 seconds, appropriate annealing temperature  for 30 seconds and 72 °C extension for 45 seconds and 72 °C for 10 minutes as final extension. Of the 14 EST-SSRs and 30 genomic SSRs, 10 and 22 respectively, gave amplification of exact size (Remaining were with multiple bands) as assessed by 1.5% agarose gel electrophoresis , number of alleles in each polymorphic locus (Ap) (polymorphism cut-off = 99%), expected heterozygosity as Nei’s unbiased estimates of genetic diversity (HE)O) and Weir and Cockerham’s6264For each locus, number of alleles (Exact test for genotypic composite linkage disequilibrium (LD)64ST values and permutation test to estimate their significance was performed in ARLEQUIN 3.5 using 10,000 permutations. Pairwise physical distance in kilometers using the GPS coordinates of the sampling sites was estimated using GEOGRAPHIC DISTANCE MATRIX GENERATORSTST/(1- FST) upon physical distance (km), and physical distance, to check for the existence of migration-drift equilibrium, one of the basic assumptions of IBD48Pairwise FThe BOTTLENECK program77Hierarchical population structure was evaluated with the AMOVA6068STCet al.’s δμ81et al.’s DSWSW, is not very good. Rather, it was shown that the probability of the traditional distance methods in returning the correct tree topology is more. In these studies, the measure DAA was selected as the distance measure to construct a dendrogram for the microsatellite data of these eight populations. Neighbour joining (NJ) tree with 10000 bootstrap resampling of the lociThere are several methods of estimating distances among populations from microsatellite allele frequencies and construct a tree from it. There are traditional distance estimates such as Nei’s standard genetic distance Det al.Phylogenetic clustering method using individual genotype data although widely used suffers from several limitations. The actual tree topology is dependent on the genetic distance used and obtaining a reliable confidence interval with a few number of loci is not easy8990We also analysed the data using unsupervised neural networks in the form self-organizing maps (SOM) or Kohonen maps949798991001011024453The network consists of two layers, -i) the input layer containing as many nodes as there are markers in a dataset, each of which is connected to all the individual genotypes, and ii) the output layer, which contains the neurons arranged in a two dimensional grid of hexagonal lattice. Each of these neurons is represented by weight vectors , whose dimension is same as the number of nodes in the input.During the training of the map, distance between each input vector and all the weight vectors are calculated, and the hexagon closest to the input vector is called its best matching unit (BMU). Following this, all the weight vectors are update so that the BMU moves closer to the input vector in the input layer. The topological neighbours of this neuron are also dragged along. This updating is continued in iterations. At the end of this process, individuals with similar multilocus genotypes will be placed closer on the SOM. This then can be visualized using different techniques. Detailed account of the theoretical background as well as the algorithm to make these maps can be found elsewhere95104Our data consisted of 131 different alleles across the 10 loci, which formed the number of nodes in our input layer. For each of these 131 markers an individual was coded as 1, 0.5, or 0, if it was either homozygote, heterozygote or lacks this marker respectivelyHow to cite this article: Chakraborty, S. et al. Genetic analysis of Indian tasar silkmoth (Antheraea mylitta) populations. Sci. Rep.5, 15728; doi: 10.1038/srep15728 (2015).


Ixodes scapularis is a vector of several human pathogens in the United States, and there is geographical variation in the relative number of persons infected with these pathogens. Geographically isolated populations of I. scapularis have established or are in the process of establishing in southern Canada. Knowledge of the genetic variation within and among these populations may provide insight into their geographical origins in the United States and the potential risk of exposure of Canadians to the different pathogens carried by I. scapularis.Part of the mitochondrial (mt) 16S ribosomal (r) RNA gene was amplified by PCR from 582 ticks collected from southern Canada, and Minnesota and Rhode Island in the United States. Sequence variation was examined in relation to the predicted secondary structure of the gene. Genetic diversity among populations was also determined.I. scapularis from the Canadian Prairie Provinces were found in the Midwest of the United States than in other regions, whereas more of the haplotypes of I. scapularis from the Canadian Central and Atlantic Provinces occurred in the Northeast of the United States. Nonetheless, 58% of I. scapularis were of a haplotype that occurs in the Midwest and Northeast of the United States; thus, their geographical origins could not be determined.DNA sequence analyses revealed 52 haplotypes. Most mutational alterations in DNA sequence occurred at unpaired sites or represented partial compensatory base pair changes that maintained the stability of the secondary structure. Significant genetic variation was detected within and among populations in different geographical regions. A greater proportion of the haplotypes of I. scapularis. There is some evidence to support the hypothesis that some lineages of I. scapularis in the Atlantic and Central Provinces of Canada may be derived from colonizing individuals originating in the Northeast of the United States, whereas those in the Prairie Provinces may be derived from individuals originating in the Midwest of the United States. However, additional genetic markers are needed to test hypotheses concerning the geographical origins of I. scapularis in Canada.There is considerable genetic variation in the mt 16S rRNA gene of The online version of this article (doi:10.1186/s13071-014-0530-6) contains supplementary material, which is available to authorized users. Ixodes scapularis) in North America has continued to expand in the Upper Midwest of the United States [I. scapularis is the principal vector of Borrelia burgdorferi sensu stricto, the causative agent of Lyme disease in North America [Anaplasma phagocytophilum) [Babesia microti) [I. scapularis [Ticks are important vectors of human and animal pathogens ,2. The id States ,5, and id States -10. This America . Blackleophilum) , human amicroti) , and ticmicroti) . The relapularis ,14-18. Uapularis .I. scapularis in the United States is divided into three geographically isolated foci: the Northeast, Midwest, and the South [I. scapularis in several provinces in southern Canada. The first of these Canadian populations established at the Long Point peninsula, which includes the Long Point Provincial Park and adjacent National Wildlife area (Ontario) was described in the early 1970s [I. scapularis larvae and nymphs into Canada from the United States each year during their spring migration [The distribution of he South -23. Therly 1970s , whereasly 1970s ,26. Othely 1970s ,27,28. Iigration .I. scapularis in the United States [I. scapularis populations in southern Canada were founded by colonizing individuals that originated from different populations in the Northeast and Midwest of the United States [I. scapularis in southern Canada had not been reported previously in studies conducted in the United States. These eight haplotypes also represented 27% of all ticks characterized in southern Canada [I. scapularis, based on the DNA sequences of Domains IV and V of the mt 16S rRNA gene, was more extensive than previously thought. Therefore, in the present study, we assessed the extent of the variation in the DNA sequences of the mt 16S rRNA gene for I. scapularis, both within and among populations in southern Canada, and the Midwest and Northeast of the United States. We also examined the phylogeographical relationships of I. scapularis to determine the possible geographical origins of the different tick populations in southern Canada.Studies have shown there are two major lineages or clades of d States -33. Indid States ,30-33. Bd States . The absd States . In addin Canada . This suI. scapularis were collected between 2000 and 2011  DNA of each tick was extracted using the QIAamp DNA Mini Kit™ or the DNeasy Blood & Tissue Kit™ (Qiagen), as described previously ,36. A reet al. [All amplicons were subjected to single-strand conformation polymorphism (SSCP) analysis, a mutation-scanning technique that is highly effective in displaying genetic variation among amplicons (150–450 bp) that differ in DNA sequence by one or more nucleotides . The SSCet al. . BLAST sI. scapularis based on the models of other organisms [I. pacificus (GenBank accession no. AF309008) and several haplotypes of the Southern and American clades of I. scapularis  [Sequences were aligned manually, but then modified according to the predicted secondary structure of Domains IV and V of the mt 16S rRNA gene that was constructed for rganisms . Phylogerganisms . The DNAon nos.) ,32 were h) and nucleotide (π) diversities of I. scapularis within established populations were determined using Arlequin [I. scapularis were included in the analyses. Tests for selective neutrality, Tajima’s D [FS [D and FS values should be approximately zero. Significantly negative D (p <0.05) and FS (p <0.02) values are indicative of populations undergoing expansion, whereas significantly positive values are characteristic of populations undergoing bottlenecks [FST) of genetic differentiation between each pair of populations. The significance of departures of FST values from zero was tested using 1000 permutations. A hierarchical analysis of molecular variance (AMOVA) was conducted using Arlequin to determine if there was genetic structuring within and among populations in different geographical regions. For this analysis, the nine established populations of I. scapularis were divided into four groups based on the province (Canada) or state (United States) in which they were located. A Mantel test was also conducted using Arlequin (1000 permutations) to determine if there was a correlation between genetic (FST) and geographical (km) distances among populations.The haplotype  was also used to determine Chao 2 values, estimators of the expected number of haplotypes in a sample [Rarefaction curves were generated using EstimateS  to estima sample .A minimum spanning network tree depicting the relationships of the haplotypes was produced using TCS version 1.21 . This anFifty-two different SSCP profiles were detected among the 582 amplicons  lower proportion of variable positions  in the 5’ and 3’ regions flanking the hypervariable region, which represented 69.1% of the 408 total nucleotides at the 3’ terminal end of the gene. These two flanking regions contained 17 (40.5%) of the 42 variable nucleotide positions among sequence types.Twenty-six (61.9%) of the 42 mutational changes occurred at unpaired sites  in the predicted secondary structure of the mt 16S rRNA gene, while another nine mutational changes represented partial-compensatory base-pair changes that maintained the secondary structure  differed significantly from zero. A single haplotype (Is-1) was found in all nine I. scapularis populations and comprised 34-63% of the ticks within each population .The haplotype diversities and nucleotide diversities of I. scapularis did not converge on an asymptote, there was no significant difference in haplotype diversity between populations of the two geographical areas, as there was overlap in the 95% confidence intervals of the two curves  of the 52 haplotypes detected among the adventitious ticks and individuals from established populations in the present study were each represented by a single tick, while four haplotypes were detected in western and eastern Canada, and in the Midwest and Northeast of the United States  represented a tertiary node in the haplotype network to six other haplotypes that were also only detected in eastern tick populations.The minimum spanning network tree depicting the relationships among haplotypes of the American clade is shown in Figure I. scapularis individuals collected from southern Canada, and the Midwest (Minnesota) and Northeast (Rhode Island) of the United States. All 52 haplotypes belonged to the American clade, as defined by Qiu et al. [I. scapularis individuals of the Southern clade in northern United States and southern Canada is consistent with the findings of other studies [I. scapularis individuals collected from Camp Riley in Morrison County, Minnesota (14 haplotypes), Trustom Pond and Hazard Island in Washington County, Rhode Island (11 haplotypes), and Long Point Provincial Park, Ontario (13 haplotypes) were two to three times greater than that reported previously in these areas  [I. scapularis from northern United States and southern Canada, based on DNA sequences of the mt 16S rRNA gene, is considerably greater than previously thought. Furthermore, there may be a large number of undetected haplotypes within the sampled areas because the Chao 2 estimate of the total number of expected haplotypes  was greater than the 52 haplotypes detected. This is likely given that 15 (29%) of the haplotypes can be considered as rare because only one tick of each of these haplotypes was detected in the present study.Fifty-two haplotypes of the mt 16S rRNA gene were detected among the 582 u et al. . The lac studies -32,34. T studies ,30-34), ctively) ,32,34; hI. scapularis were low (0.002-0.004), while haplotype diversities were relatively high (0.60-0.79) compared to those of some other species of Ixodes in North America [Ixodes angustus, Ixodes kingi and Ixodes sculptus [I. scapularis. Biological differences among these tick species are likely explanations for the differences in haplotype number. For instance, I. angustus, I. kingi and I. sculptus parasitize primarily small mammals  of the nucleotide variation occurred within the “hypervariable” region of Domain V. Mutational alterations in DNA sequence, both within and among species of arthropod, have been shown previously to be more frequent within this part of the gene than in the flanking regions [I. scapularis at unpaired sites (62%) was also greater than that at sites involved in base pairing (38%) in the secondary structure. This difference may also be associated with reduced structural constraints for mutational alterations at nucleotide positions not involved in base pairing. In addition, substitutions at nucleotide positions involved in base pairing were more often partial compensatory base changes, rather than non-compensatory changes, hence maintaining the integrity of the secondary structure of the mt 16S rRNA gene. This is in agreement with the general patterns of mutational change in rRNA genes [Variation in the DNA sequences among  regions ,53-58. T regions . DNA seqNA genes .I. scapularis conducted in southern Canada [I. scapularis is indicative of rapidly expanding populations [I. scapularis in the Midwest of the United States [s tests suggesting population expansion at these localities.The number of nucleotide differences in DNA sequence among the 52 haplotypes ranged from 1–5 bp; however, this difference increased to 9 bp when an additional 10 haplotypes of the American clade -32,34 wen Canada ,32. Haplulations ,32, whicd States  and soutd States ,9,28. FuI. scapularis into southern Canada have been attributed to the transportation of large numbers of larvae and nymphs from the United States by migratory passerine birds [et al. [I. scapularis between a population in southeastern Manitoba and several populations in southern Ontario and Nova Scotia. Subsequently, Mechai et al. [cox1) among blacklegged ticks from different geographical regions in Canada. It was proposed that I. scapularis populations in different geographical regions of southern Canada may be derived from populations in different regions of the United States and associated with the different routes (flyways) taken by passerines during their spring migration [et al. [I. scapularis and other introduced tick species in different regions of Canada and the different flyways of migratory passerines. Thus, blacklegged ticks introduced into the Prairie Provinces of Canada  may have originated from endemic populations in the Midwest of the United States (Minnesota and Wisconsin), while those introduced into the Central Provinces of Canada (Ontario and Quebec) may have originated from endemic populations primarily in the Northeast of the United States , but also from resident populations in parts of the Midwest [Range expansion and the establishment of geographically isolated populations of ne birds ,29. Howe [et al.  reportedi et al.  reportedigration . Scott e Midwest . Northea Midwest . The lat Midwest ; however Midwest .I. scapularis populations both within and among different geographical regions; however, the presence of several shared haplotypes among populations supports the hypothesis of gene flow among populations. Nonetheless, 26 (79%) of the 33 haplotypes found in the western region (Prairie Provinces and Minnesota) were not found in the eastern region , while 19 of the 26 (73%) haplotypes in the east were not found in Minnesota or the Prairie Provinces. In addition, only 8 (32%) of the 25 haplotypes found in the three populations in Minnesota have been reported previously from the Northeast of the United States [FST values). Statistical analyses of the FST data also showed significant differences in the genetic structure between some populations of I. scapularis. For example, there was a significant difference in the population genetic structure of I. scapularis from Hazard Island and Trustom Pond, two localities in South Kingstown (Rhode Island) separated by a distance of only 5 km. The reason why the genetic structure of these two tick populations differs is unclear. Although fewer haplotypes were detected at Trustom Pond, six of the seven haplotypes in this population were also detected in the population at Hazard Island. In addition, the results of the Chakraborty’s test revealed no significant difference in the number of observed and expected haplotypes for either population. In contrast, there were no significant differences between the populations in Itasca State Park (Minnesota), Pembina Valley Provincial Park and Stanley Trail (Manitoba). There were also no differences between the populations in Itasca State Park and St. Croix State Park, or between those in Camp Ripley and St. Croix State Park (Minnesota). A comparison of the haplotypes among adventitious ticks and those in established populations found in the Prairie Provinces with those in other geographical areas  of the I. scapularis individuals collected in all three regions of southern Canada  were of a haplotype  that also occurs in both the Midwest and Northeast of the United States [cox1; [I. scapularis introductions into southern Canada.The results of the AMOVA test also revealed statistically significant genetic structuring of d States ,32, whicd States ,32. Therd States  or cox1;s [cox1; ), in addI. scapularis was greater than previously demonstrated based on the DNA sequence analyses of the mt 16S rRNA gene. Furthermore, a large number of rare haplotypes may still remain undetected. The results also indicated significant differences in genetic diversity both within and among populations from different geographical regions. There was also a significant positive relationship between the genetic differences between populations and the geographical distances that separated them. There was some evidence to support the hypothesis that I. scapularis in the Prairie Provinces of Canada are derived from individuals introduced from the Midwest of the United States, while those in the Atlantic and Central Provinces are derived from individuals that originated in the Northeast of the United States. However, the geographical origins of a large proportion of I. scapularis found in the different areas of southern Canada could not be inferred because they were of a haplotype that occurs in both the Midwest and Northeast of the United States. Therefore, additional studies are needed to explore other genetic markers that may be useful for understanding the trajectories of spread of I. scapularis and its pathogens on a finer scale.In conclusion, genetic variation within