The aim of this study was to obtain unbiased estimates of the diversity parameters, the population history, and the degree of admixture in Cika cattle which represents the local admixed breeds at risk of extinction undergoing challenging conservation programs. Genetic analyses were performed on the genome-wide Single Nucleotide Polymorphism (SNP) Illumina Bovine SNP50 array data of 76 Cika animals and 531 animals from 14 reference populations. To obtain unbiased estimates we used short haplotypes spanning four markers instead of single SNPs to avoid an ascertainment bias of the BovineSNP50 array. Genome-wide haplotypes combined with partial pedigree and type trait classification show the potential to improve identification of purebred animals with a low degree of admixture. Phylogenetic analyses demonstrated unique genetic identity of Cika animals. Genetic distance matrix presented by rooted Neighbour-Net suggested long and broad phylogenetic connection between Cika and Pinzgauer. Unsupervised clustering performed by the admixture analysis and two-dimensional presentation of the genetic distances between individuals also suggest Cika is a distinct breed despite being similar in appearance to Pinzgauer. Animals identified as the most purebred could be used as a nucleus for a recovery of the native genetic background in the current admixed population. The results show that local well-adapted strains, which have never been intensively managed and differentiated into specific breeds, exhibit large haplotype diversity. They suggest a conservation and recovery approach that does not rely exclusively on the search for the original native genetic background but rather on the identification and removal of common introgressed haplotypes would be more powerful. Successful implementation of such an approach should be based on combining phenotype, pedigree, and genome-wide haplotype data of the breed of interest and a spectrum of reference breeds which potentially have had direct or indirect historical contribution to the genetic makeup of the breed of interest. After domestication in the near east cattle became widespread in Europe along the Danubian and Mediterranean route . During th century , HE, and AR of Cika in comparison with the other 14 breeds are shown in nA) and the mean number of alleles (mA) per haplotype block ranged from 26,053 and 5.24 in HRP to 44,416 and 8.93 in IMB, respectively, while Cika  was in the middle of these extremes. Cika also showed lower nA and mA compared to API . IMB showed the highest number of private  and rare alleles  while BBV had the lowest values . The discrepancy between these two extremes is enormous, i.e., the number of private and rare alleles in the highly selected BBV breed is 8.27 and 5.84 times lower, respectively. The second Buša breed (HRB) showed lower but still very high npA (875) and nrA (801). However, Cika showed the largest difference between npA (237) and nrA (422). Among all 15 breeds only in two Bušas (HRB and IMB) and N’Dama were HO lower than HE. Only IMB (0.743) and PMT (0.738) showed higher HO compared to Cika (0.737). Allelic richness, a diversity measure not influenced by sample size, was the highest in HRB (7.771) and the lowest in HRP (5.101). Cika had allelic richness (6.695) similar to API (6.492), HRI (6.735) and PMT (7.141), which are the three geographically closest breeds. Allelic richness showed highest correlation with mA (0.90) and HE (0.88) but lowest with HO[SNP] (0.53).The number of the common alleles  but DEST showed better diversification (from 0.008 to 0.318). The highest average DEST values were observed among NDA from Africa, autochthonous HRP and highly selected RHF as well as BBV when compared with other breeds (0.174–0.263). The lowest average genetic differentiations were observed for Bušas, PMT and Cika when compared with other breeds (0.076–0.097). Pairwise DEST value between Cika and API (0.046) was similar to the one between Cika and IMB (0.045) as well as between API and HRB (0.055). The lowest DEST value when comparing Cika with other breeds was between Cika and HRB (0.022) followed by Cika and PMT (0.033) differentiation. The lowest DEST when comparing all breeds was between HRB and IMB (0.008), which demonstrates the low genetic differentiation of Buša.Pairwise population differentiations are shown as DA-distances were plotted as a Neighbour-Net (AR = 7.141) doesn’t show clear clustering in this design and has a higher genetic similarity to Alpine breeds. The Neighbour-Net graph suggested relatively long-term and close co-evolution of both Braunvieh populations (OBV and BBV). Similarly close but shorter co-evolution is suggested for DFV and FGV. On the contrary, Cika and Pinzgauer showed relatively long but wide phylogenetic connection  methods implemented in the pulation , shows hpulation . This reMix runs . Most coMix runs .DPS distances in the two dimensional presentation (2D). The distribution of the larger set with 607 individuals of 15 breeds is shown in Admixture analysis of the reduced set detected the lowest cross-validation error at K = 13. Admixture results for this K value in the larger set with 76 Cika animals . The dataset included Cika and four breeds  with possible direct contribution to the Cika gene pool as identified by genome-wide IBS and IBD matrix. In the three parts of the Cika cattle, like many other local breeds, was under pressure with the introduction of cosmopolitan breeds and was near extinction due to the continuous upgrading with Pinzgauer as well as crossing with Fleckvieh and replacement with Braunvieh. When Cika population was included in the conservation program, doubts have been raised about the uniqueness of the breed and the level of the admixture. The Cika herd book had records on mostly admixed animals with known pedigree going back a number of generations and with relatively accurate estimated proportions of foreign genes from Pinzgauer and Red Holstein. In 2002, potentially pure Cika animals from the mountain area were included in the program as 100% pure Cika based only on type traits classification even though their pedigree was unknown. However, this increased population size but also doubts in the breeding community about the uniqueness of the population.These conditions partly resemble a curious situation that arose from the restoration and conservation of the Austrian Murbodner (AMB) cattle breed . OriginaHO was obtained for IMB (0.743), while HO estimated from the SNP genotypes (HO[SNP]) was comparably high in RHF, SIC, PMT and IMB (0.342–0.344). The high HO[SNP] estimated in RHF sample is in conflict with a relatively low haplotype diversity and could be due to the ascertainment bias. Supporting this result is also the low correlation (0.53) between haplotype allelic richness and HO[SNP]  Cika only as well as to build a knowledge base for later practical conservation work on similar populations. Ability of the methods implemented in the Cika and HRB animals, which were identified as highly inbred by the genome-wide IBD matrix, were outliers in the 2D space, far away from their breed clusters .These ouindicine or African taurine ancestry into Balkan cattle breeds (Buša or Podolic) this possibility exists. Furthermore, Balkan cattle population is located closer to current Anatolian cattle that carried both indicine and African taurine ancestry [TreeMix procedure support results of [TreeMix analyses. In addition to the close relationship between Cika and API, TreeMix suggests relatively recent mixture with DFV. This could explain higher genetic diversity of current Cika population. On average, the neutral diversity estimators put Cika in the position between the Balkan breeds (Buša and Podolic) and the Alpine breeds, which corresponds with the geographical location of Slovenia as well as with admixed status of Cika.According to the expected and observed haplotype heterozygosity, Balkan breeds were more diverse than Alpine breeds and dual-purpose breeds were more diverse than highly selected single-purpose breeds. Large current and past effective population size as well as virtual absence of systematic artificial selection combined with proximity to major centres of domestication in the Fertile Crescent could be main reasons for higher diversity in Buša strains ,14. Evenancestry  which cosults of  and suggDEST values observed between RHF and other breeds  and PMT (0.033) despite remarkably different breed characteristics . Furthermore, DEST value to Buša strain from Albania (0.045) is also lower then to Austrian Pinzgauer (0.046) despite relatively long geographical distance. These low DEST values to both Buša strains and PMT suggest either low genetic differentiation of all four subpopulations or ancient genetic relationships through past trades which is possible given the geographical proximity. The lowest Admixture suggest low differentiation as being more relevant. Despite the verifiable evidence of the direct introgression from Pinzgauer to Cika, it should be considered that the DEST matrices were prepared with only potentially pure Cika animals. These animals show relatively low genetic differentiation to Pinzgauer (DEST = 0.046). The key question is which part of relatively high genetic diversity in Cika is caused by admixture and which part represents original neutral diversity of the population under relatively low artificial selection.The high average ttleneck . Cika shr breeds  and the nA, mA, npA, nrA, and AR . This is due to sharing of 24% rare allele with API, i.e., otherwise private alleles of API or SIC were classified as rare due to sharing. Taken together, these patterns suggest that even the sample of 26 most pure Cika is still partly admixed. Then again, unsupervised clustering performed by Admixture and 2D presentation of DPS-distances identified Cika as a breed worth preserving. Even though there are recognisable proportions of Alpine (mainly API and DFV) and southern  contributions in most Cika animals, both unsupervised approaches identified Cika as a population with a unique genetic identity. Comparison of the clustering based on the initial set of 76 Cika animals . Appropriate inclusion of progenies from these individuals (via regular or target mating) into the nucleus could effectively remove exogenous haplotypes and avoid increase in inbreeding. Likewise missing and wrong information in the incomplete pedigree records could be efficiently improved and corrected based on the molecular composition of a breed. Generally, the advantage of using molecular information increased with the increase in SNP density. In scenarios evaluated by  the SNP Populations of livestock animals are characterised by several introgression events since domestication. In cattle, some of these events are known as secondary domestication, mostly due to the influence of wild aurochs males on domestic dam populations . These aEach population is dynamic with a different identity and should be considered on an individual basis, but the following overall conclusions can be drawn from the results of this study. Combined phenotype, pedigree, and genome-wide information can be used to detect the “purest” individuals to be used as the nucleus for recovery of the native genetic background of the admixed domestic population. The ascertainment bias introduced in the construction of SNP arrays must be taken into consideration while estimating diversity and phylogeny parameters as well as for the removal of introgressed genetic material. The successful implementation of a conservation and recovery approach will benefit from a design which includes the most complete spectrum of possible source haplotypes.S1 FigA) The ML dendrogram of the relationships between 15 sampled populations rooted by African taurine N’Dama cattle. B) ML tree of 15 cattle populations assuming one migration event. C-E) Three variants of ML tree of 15 cattle populations assuming two migration events. F-G) Two variants of ML tree of 15 cattle populations assuming three migration events.(PDF)Click here for additional data file.S2 Fig(a) and a reduced dataset with 26 Cika animals (b). RHF, Red Holstein; FGV, Franken Gelbvieh; DFV, German Fleckvieh; MWF, Murnau—Werdenfelser; BBV, Braunvieh; OBV, Original Braunvieh; API, Pinzgauer; SIC, Cika; HRI, Istrian cattle; HRP, Slavonian—Syrmian Podolic; PMT, Piedmontese; RMG, Romagnola; HRB, Croatian Buša; IMB, Illyrian Mountain Buša; NDA, N’Dama.A dataset with all 76 Cika animals (PDF)Click here for additional data file.S3 FigDPSdistance below global average were connected by thin grey line. A) Highlighting a set of 55 pure Cika animals recognised by merged information of haplotype, pedigree data and type traits classification, B) a set of 47 pure Cika animals recognised only by pedigree data were highlighted, C) a set of 58 pure Cika animals recognised only by type traits classification. RHF, Red Holstein; FGV, Franken Gelbvieh; DFV, German Fleckvieh; API, Pinzgauer; SIC, Cika; SIC-F, inbreed SIC animal; SIC*FGV*DFV, SIC related to FGV and DFV; SIC*API(I), SIC related to particular API animal; SIC*API(P), SIC related to API population; SIC-F*RHF, inbreed SIC animal related to RHF; SIC*RHF, SIC related to RHF.Animals with (PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Phlebotomus and Sergentomyia using the EF-1α and Cyt b genotype regions to describe the sand fly fauna and genetic aspects collected at war-torn sites of the Khuzestan boundary between Iraq and Iran.Our investigation uses nucleotide variations of the genera D) and neutral allele frequency (Fu’s Fs) tests to measure the effect of randomly evolving DNA sequence on the genetic diversity of sand fly populations in response to habitat fragmentation and landscape modification.All sand fly species were characterized using molecular genetics. The field work was conducted in six districts including 24 locations in remote areas for three years at the peak of sand fly activity during cutaneous leishmaniasis (CL) transmission seasons. The distribution of CL vectors was determined based on the climatic regionalization using the kriging method in ArcGIS model. DNA of sand fly pools were screened via polymerase chain reaction (PCR) using neutrality . The haplotypes of Cyt b from the subgenus Sergentomyia and P. papatasi revealed more heterogeneity (Tajima’s D > +2) than P. alexandri (D > +1), which suggests widespread heteroplasmic mitochondrial DNA mutations in the same mtDNA gene among different sand fly species. Subgenus  Sintonius exhibited greater fitness (D = 0) and  no evidence of selection. The sequence of the nuclear gene EF-1α indicated similar nucleotide differences, as observed for the Cyt b gene, in all sand fly species, but lower levels of polymorphisms (D > +1) were observed compared with the mitochondrial Cyt b gene (D > +2) in the subgenus  Sergentomyia.Among the 1213 specimens, ten species were identified based on morphology. The non-native species Phlebotomus and Sergentomyia population gene pools due to recent anthropogenic influence. A phylogenetic analysis showed that the closely related species are positioned in monophyletic clades, except for the subgenus Sergentomyia and P. sergenti, and highlights the importance of haplotype variations for the development of adaptability.Our findings describe random nucleotide diversity in the  Leishmania spp. through their geographical distribution due to landscape fragmentation, migration, environmental and climatic oscillations. Certain fundamental criteria support the notion that sand flies are potential vectors: the capacity of sand flies to use their midgut receptor to attach a specific Leishmania species, behavioral or genetic diversification, vector-parasite coevolution and the adaptive manipulation of their interactions ..SergentoP. sergenti to be in a separate clade of the Cyt b region  and mitochondrial (Cyt b) gene markers. Certain sand fly species, such as P. papatasi and P. alexandri, featured a greater number of different haplotypes than the other sand fly species, which is related to specific geographical distances .Accurate identification of sand fly species based on molecular analyses is a valuable approach for determining the epidemiological aspects and distribution of natural populations in unexamined areas. In our investigation, using phylogenetic analysis, molecular characterization was performed to identify the population structure of the sand flies that were collected along the Khuzestan borders, which distinctly revealed the previously unrecorded species ion Fig. . This stP. sergenti in new areas where no record exists implies expanding distribution of the vectors and their adaptation to local habitat conditions or environmental modifications along the Khuzestan borders (Table L. tropica had been isolated from human ulcers at the same locations in the Dashte-Azadegan district (Susangerd and Howeyzeh) [S. christophersi was found as a new record. However, we could not locate S. christophersi from the western borders of Khuzestan Province or west of Shush city  . The twooweyzeh)  and S. city Fig. , maybe JP. papatasi are k1 = 13.726 for purines and k2 = 9.879 for pyrimidines  rate is lower in P. alexandri sequences (R = 1.18) for the Cyt b gene, perhaps due to postmutation processes from the high mutation rate of methylated cytosines to thymine in the P. papatasi population. However, heterogeneity manifestations exhibit a selective advantage for survival and fitness in the sand fly population sequences along the Khuzestan borders (Table The transition/transversion (ts/tv) rate ratios for es Table . FurtherEF-1α gene in P. papatasi, P. alexandri and the subgenus  Sintonius was less than three, and the quality of the remainder was insufficient to evaluate the DNA fragments after resequencing. Perhaps this phenomenon is due to the presence of multiple loci or two copies in these species as a result of gene duplication events [Sergentomyia is R = 3.31. Tajima’s D value of the nuclear gene (EF-1α) indicated low levels of polymorphisms  compared with the mitochondrial Cyt b gene  (Table EF-1α) and the low number of nucleotide substitutions between the species of a single subgenus, which are homogenized by gene conversion [EF-1α gene is multi copy number, this may be there are significant differences between the studied individuals and not an actual difference.The number of interpretable sequences for the n events . Therefonversion . The EF-D test for the Cyt b gene of P. papatasi (D > +2) revealed higher nucleotide diversity than that observed for P. alexandri and the genus  Sergentomyia (more mutations or high polymorphisms between pairs than the number of segregating sites), different haplotypes of P. papatasi and P. alexandri were positioned in their own clades with similar rates of haplotype diversity  . The positive Tajima’s D value in Khorramshahr and Abadan could be due to a relative population decrease, subdivision or recent bottlenecking among P. papatasi, P. alexandri and Sergentomyia spp. , P. alexandri (Fu’s Fs  = 3.568) and Sergentomyia spp. (Fu’s Fs  = 0.913) [Phlebotomus and Sergentomyia and this may result from selection pressure (the Z-test was P < 0.05 and dN/dS ratios were biased toward synonymous mutations among closely related species of sand flies) induced by environmental conditions. Pursuant to our molecular analyses, a selective sweep/hitch-hiking may have occurred in the subgenus Sintonius (S. clydei and S. tiberiadis) due to a population expansion or older bottlenecking, the negative values of Fu’s Fs genotype (Fu’s Fs = -0.463) indicating excess of low frequency haplotypes/alleles and beneficial mutation. We deduced that a balancing selection occurred due to a habitat modification (Iraq-Iran war). However, Tajima's D value of the Cyt b gene regions indicates an equivalent number of pairwise nucleotide differences and segregating sites (D = 0) for the subgenus Sintonius, which indicates that the population was evolving as a per mutation-drift equilibrium and therefore, increasing the fitness of the Sintonius spp. The observed demographic changes between the populations of Sintonius spp. and other sand fly species can arise from random fluctuation of neutral mutation which likely goes up or down through genetic drift and demographic events. A decrease in genetic and phenotypic variation or conservation biology  is possible for sand fly species due to human impact [Although the data of Tajima’s ly) Fig. . In spitp. Table . Positivn impact . Human ePhlebotomus spp. and the remainder of the genus Sergentomyia (except Sintonius), with considerable random mutations and low genetic variation at a geographical location along the Khuzestan borders . Perhaps both, mitochondrial and nuclear genes, are involved in perfect harmony with balancing selection under imposed pressures from nature and human intervention. A GC-biased gene conversion may have occurred due to the presence of GC-rich isochores regions in the nuclear EF-1α gene in comparison with the mitochondrial Cyt b gene. However, the observed corresponding selection could be expected for housekeeping genes, as a fundamental rule underlying molecular mechanisms in establishing proper conditions for life.Consistent with our molecular results, we observed the rapid expansion of nucleotide differences among sequences for Considering landscape modifications in the attacked areas after military operations of war, increased genetic homogeneity or accumulation of similar set of alleles was expected. The high rate of allele frequency and polymorphisms among the sand fly sequences found herein may be considered as an effect of genetic drift associated with habitat fragmentation and a reduction in genetic variation (extrinsic selection).The warfare between Iraq and Iran has ruined the natural habitat of phlebotomine sand flies as anthropogenic factors , 36 and EF-1α gene revealed less genetic structuring between the P. papatasi, P. alexandri and Sergentomyia populations than for the Cyt b gene across the Khuzestan borders. In contrast, an empirical examination of the Cyt b gene indicated higher genetic variation in the populations of P. papatasi, P. alexandri and a number of species of the genus Sergentomyia. All closely related sand fly species, except certain species of the subgenus Sergentomyia, produced a higher bootstrap likelihood (> 70 %) of developing reproductive isolation  and nuclear gene (EF-1α) was performed using the phylogeny of Larrossius spp., but no consistent data were generated [P. papatasi and P. alexandri, from one location patch to another, denotes not only for physical eligibility, but also for population dynamics [Phylogenetic analysis and molecular identification of the on Figs. , 5. The ade Fig. . Furthermutation . A compaenerated . The disdynamics , 39 due We drew more attention to the effect of the combination of human activities and new environmental effects at the Iran-Iraq borders on vector genotypes as well as the selection and emergence of existing genotypes. The neutrality and molecular analyses suggest that our findings provide compelling evidence of randomly evolving DNA sequences from both mitochondrial and nuclear DNA haplotypes to support the fitness and survival of wild-caught sand flies with neutral mutation. Further investigation of different sand fly species of the old and new world is necessary to determine the evolution, behavior and vector competence of unknown or non-native sand fly species due to various environmental conditions and in different regions around the world, which have not yet been studied.Cyt b, cytochrome b; EF-1α, elongation factor 1alpha; HD, haplotype diversity; mtDNA, mitochondrial DNA; PCA, principal component analysis; ts/tv, transition/transversion; ZCL, Zoonotic Cutaneous Leishmaniasis.CIM, clustering integration method; CL, cutaneous leishmaniasis;


Animal breeding programs have been very successful in improving the mean levels of traits through selection. However, in recent decades, reducing the variability of trait levels between individuals has become a highly desirable objective. Reaching this objective through genetic selection requires that there is genetic variation in the variability of trait levels, a phenomenon known as genetic heterogeneity of environmental  variance. The aim of our study was to investigate the potential for genetic improvement of uniformity of harvest weight and body size traits  in the genetically improved farmed tilapia (GIFT) strain. In order to quantify the genetic variation in uniformity of traits and estimate the genetic correlations between level and variance of the traits, double hierarchical generalized linear models were applied to individual trait values.Our results showed substantial genetic variation in uniformity of all analyzed traits, with genetic coefficients of variation for residual variance ranging from 39 to 58 %. Genetic correlation between trait level and variance was strongly positive for harvest weight (0.60 ± 0.09), moderate and positive for body depth (0.37 ± 0.13), but not significantly different from 0 for body length and width.Our results on the genetic variation in uniformity of harvest weight and body size traits show good prospects for the genetic improvement of uniformity in the GIFT strain. A high and positive genetic correlation was estimated between level and variance of harvest weight, which suggests that selection for heavier fish will also result in more variation in harvest weight. Simultaneous improvement of harvest weight and its uniformity will thus require index selection.The online version of this article (doi:10.1186/s12711-016-0218-9) contains supplementary material, which is available to authorized users. In animal breeding, particular attention is paid to improving the mean level of traits through selection and this has been successful for many breeding programs. One such successful example is the genetically improved farmed tilapia (GIFT) project, which was led at WorldFish  and resuAlthough good management during the grow-out phase can help reduce the CV, as noted by Ponzoni et al. , its aveAn alternative approach to management procedures for reducing the variability of a trait is selective breeding. Selection for more uniform individuals requires that the variability of the trait itself has a genetic component i.e. that there is genetic variation, which is also known as genetic heterogeneity of environmental  variance , 17. In The main objective of our study was to investigate the potential for genetic improvement of uniformity of harvest weight and body size traits in the GIFT strain. For this purpose, we analyzed within-family variance of harvest weight, body length, depth, and width, by applying a DHGLM to individual trait values . To quanWe used data that were obtained from an experiment that was specifically designed to estimate indirect genetic effects (IGE) for growth rate in the GIFT strain . This exTo produce families, the GIFT breeding program uses a nested-mating design, where one male is mated to two females. For this work, we used the same mating scheme to produce the experimental fish, and thus two full-sib families were obtained from each father. Each full-sib family contributed 80 offspring to the experiment. Fry that belonged to the same full-sib family were nursed together and separately from other families. During the grow-out phase, fish were kept in groups. Before placing each fish in a group, they were individually identified with a PIT (Passive Integrated Transporter) tag. Following the optimal design for the estimation of IGE , familieThe groups were kept in net-cages that were placed in earthen ponds in rows and columns. For each batch, two ponds were available. Due to the small number of fish available for batch 2010, only one pond was used. The groups for each block were distributed randomly and as evenly as possible over both ponds. Thus, the 55 groups of a block were split into 27 groups for pond 1, and 28 groups for pond 2.During the grow-out phase, fish were fed with commercial dry pellets containing 32 % of protein; the amount of pellets (3 to 5 % of average live weight) and feeding frequency (twice a day) were the same as for the GIFT selective breeding population. However, because the fish were kept in net-cages rather than in communal rearing, the feeding strategy differed from that in the standard GIFT program. Rather than spreading the food over the entire surface of a pond, it was placed in the corner of each net-cage so that the fish could express their competitive tendency see . More deFish were harvested 5 to 8 months after the grow-out period, when the average weight ranged from 200 to 250 g. At harvest, the following traits and parameters were recorded: live body weight (g), body measurements , tag number, sex, pond, and net-cage label. The age at harvest of each fish was computed from the recorded spawning and harvesting dates . Over thIdeally, each group should contain 16 individuals at harvest. However, due to mortality, some groups contained very few individuals, and a threshold was set for group and family size. Thus, groups that contained less than seven individuals in total or less than three fish per family were discarded, which reduced the number of groups to 446. With two families in each group, 892 family-by-group combinations and 6090 individual records were available for each trait. Table The environmental component in the phenotypic variation of a trait can be measured either on the same individual for which repeated observations are available or on the individuals belonging to the same family . In our Sire and dam, group, kin, and social maternal effect were included as random effects. A group effect was included to account for non-heritable indirect effects, which create a non-genetic covariance among individuals within the same group . If thisei is the residual for individual i. Due to linearization, a bivariate DHGLM can then be used:Lee and Nelder  developedividual , 43. As ^i2/1-hi . Insteadi et al. , which rTo facilitate interpretation in the Results section, the group effect for trait level is presented as he trait , and, \dve model , 46. Wheent}σAV2 , 46.Estimated genetic parameters for levels of harvest weight, body length, depth, and width are in Table Heritabilities of harvest weight and body width were similar (0.25 ± 0.05), while heritabilities of body length and body depth were a little higher (~0.30 ± 0.05). The group effect explained ~15 % of the phenotypic variance for length and depth, and 27 % for width. The kin effect explained ~10 % of the phenotypic variance for all three body size traits.Estimated genetic parameters for the variance of harvest weight, body length, depth, and width are in Table Estimated genetic correlations between level and variance for harvest weight and body size traits are in Table In this study, we used a DHGLM to estimate genetic variation in uniformity of harvest weight and three body size traits, i.e. length, depth, and width. Our results showed substantial genetic variation in uniformity of all analyzed traits, with Estimated heritability for individual harvest weight was moderate 0.25 ± 0.04), which is similar to results from previous studies on Nile tilapia 5 ± 0.04,, 48. To Heritabilities for individual body size traits were also moderate (0.25 to 0.32), which provide opportunities to improve body size traits in Nile tilapia. Body size traits could become traits of interest in future breeding programs since selection for heavier fish may lead to body shapes that deviate from the natural shape, the latter being favored by consumers , 49, 50.Variance components that are estimated using the exponential model, as in this study, are independent of the scale of the trait, and thus, are comparable across traits and species , 30. We Regardless of the underlying model, comparison of additive genetic parameters for uniformity across different studies can also be done by using the genetic coefficient of variation for residual variance (t}GCVVe) , 46. \do2 to 0.6 . For unin et al.  and Sae-n et al. , respectVVe only , 27, 46.The estimated level and variance for harvest weight could be influenced by the non-normal distribution of harvest weight. In data on aquaculture species, skewness is not unusual , 53. A sOur results imply that the observed variation in harvest weight in the GIFT strain could be reduced by selective breeding. However, selection for more uniform fish may result in a trade-off on improvement of harvest weight. The genetic correlation between level and variance of harvest weight is high and positive, 0.60 Table , which mTo maximize profit, not only uniformity of weight but also uniformity of size, may play an important role in fish farming, especially for markets where fish are sold as whole. The magnitudes of ns Table  is expecIn our analyses we used a sire and dam model, which fits the additive genetic mid-parent mean, while the Mendelian sampling deviation is part of the residual. This can potentially inflate the size of the estimated genetic variation in residual variance in case of heterogeneous Mendelian sampling variation, which is then confounded with the genetic part of the residual variance . A MendeIn aquaculture species, maternal common environmental effects can have an important role in explaining differences among families. These effects can be included in the estimation of genetic parameters as non-genetic effects that account for covariances between full-sibs due to a shared environment. In this study, maternal common environmental effects were excluded from the models because of convergence problems, which arose when those effects were included. The same issue was observed in other studies that used the same dataset and for which the results showed confounding of maternal common environmental effects and direct genetic effects , 34. TheA recent study on birth weight of mice treated environmental variability as a maternal trait, and found a positive response to selection . In an eIn Table S2  was lower than the values found in previous studies on the GIFT strain where fish were communally reared (48 to 59 %) , 50. ThuAlthough studies on the genetic heterogeneity of environmental variance date as far back as 1942 , selectiWe studied the genetic variance of the residual variability. However, the total phenotypic variability also depends on other factors , as showAn interesting property of the specific design of our experiment is that it allows the simultaneous study of uniformity and social effects such as group and kin effects in our study and indirect genetic effects, which were analyzed in other studies on the same data , 34. HowOur study revealed substantial genetic variation in uniformity of harvest weight and body size traits, which opens promising prospects for the genetic improvement of uniformity by selective breeding of the GIFT strain. The genetic correlation between level and variance of harvest weight was high and positive, which indicates that selection for heavier fish may also result in more variation in harvest weight. Simultaneous improvement of harvest weight and its uniformity will thus require index selection.


Ondatra zibethicus, is a semiaquatic rodent native to North America that has become a highly successful invader across Europe, Asia, and South America. It can inflict ecological and economic damage on wetland systems outside of its native range. Anecdotal evidence suggests that, in the early 1900s, a population of muskrats was introduced to the Isles of Shoals archipelago, located within the Gulf of Maine, for the purposes of fur harvest. However, because muskrats are native to the northeastern coast of North America, their presence on the Isles of Shoals could be interpreted as part of the native range of the species, potentially obscuring management planning and biogeographic inferences. To investigate their introduced status and identify a historic source population, muskrats from Appledore Island of the Isles of Shoals, and from the adjacent mainland of Maine and New Hampshire, were compared for mitochondrial cytochrome b sequences and allele frequencies at eight microsatellite loci. Appledore Island muskrats consistently exhibited reduced genetic diversity compared with mainland populations, and displayed signatures of a historic bottleneck. The distribution of mitochondrial haplotypes is suggestive of a New Hampshire source population. The data presented here are consistent with a human-mediated introduction that took place in the early 1900s. This scenario is further supported by the zooarchaeological record and island biogeographic patterns. This is the first genetic study of an introduced muskrat population within US borders and of any island muskrat population, and provides an important contrast with other studies of introduced muskrat populations worldwide.The muskrat,  Questions regarding the origin, distribution, and maintenance of insular fauna have been central to the study of evolutionary biology since the time of Charles Darwin and Alfred Russell Wallace. Island systems are frequently touted as natural laboratories for the study of fundamental ecological and evolutionary processes The colonization of islands, with or without human mediation, generally involves only a small subsample of the mainland source population. Information concerning initial founding conditions is typically unknown and traditional methods, such as the use of historical or zooarchaeological records, often fail to provide comprehensive resolution. Because morphological evidence of endemism can be ambiguous Ondatra zibethicus inhabiting the Isles of Shoals archipelago in the Gulf of Maine  and New Hampshire (NH) . During This study contrasts two hypotheses of natural colonization that would produce distinctive genetic patterns  with the anecdotally informed but otherwise untested hypothesis of a human-mediated muskrat introduction in the early 1900s. It aims to illustrate the usefulness of a combined mitochondrial and microsatellite DNA approach and provide a framework of multiple complementary statistics for the detection of a cryptically introduced population. Through this approach, we compare expectations under each hypothesis, including timing of bottleneck, endemism of haplotypes, degree of gene flow, and levels of genetic diversity and differentiation, to distinguish the most likely scenario of muskrat colonization. For example, if muskrats colonized naturally via a land bridge, there would be no signatures of a recent bottleneck, very high levels of genetic differentiation with the mainland, and we would likely find at least one mitochondrial haplotype endemic to AP due to an early divergence followed by isolation over several thousand years. Conversely, recurring natural colonization events would be reflected in low genetic differentiation and a high maintenance of genetic diversity due to continual gene flow. A human-mediated colonization in the 1900s would display different signatures than either natural colonization mechanism, primarily differing in the timing of the population bottleneck, absence of an endemic haplotype, and magnitude of gene flow and differentiation with the mainland.This study was approved by the Cornell University Institutional Animal Care & Use Committee, and operated under IACUC General Operation of the Mammalogy Collection at Cornell University protocol #2006-0156. Samples of AP muskrats were collected on private property owned by the Shoals Marine Laboratory of Cornell University, under Maine Department of Inland Fisheries and Wildlife, Wildlife Scientific Collection permit #2011-349. All AP samples were collected under strict adherence to American Society of Mammalogy guidelines Muskrats were non-lethally sampled via tail clips (<1 cm) on Appledore Island (AP), Isles of Shoals, ME, from June to July 2011 using Havahart live-traps . MuskratMembers of state trapping associations donated tissue samples (stored in absolute ethanol) from the mainland  and a pre-existing cytochrome b gene sequence, Genbank Accession #AF119277 Taq polymerase, 1 µL PCR buffer, 2 mM MgCl2, 0.2 µM of each primer  and 0.2 mM dNTP. PCR reactions included an initial denaturation at 94°C for 5 minutes, followed by 35 cycles of 94°C for 15 seconds, 50°C for 15 seconds, and 72°C for 1 minute, with a final extension of 72°C for 5 minutes. PCR products were cleaned using ExoSAP-IT and CleanSEQ (Agencourt), and amplified using a Big Dye Cycle Sequencing kit (Applied Biosystems). Sequencing was performed using an Applied Biosystems 3730 DNA Analyzer at the Life Sciences Core Laboratories Center at Cornell University (http://cores.lifesciences.cornell.edu/brcinfo/).A DNeasy Blood & Tissue Kit (Qiagen) was used to extract DNA from a total of 107 tissue samples. Mitochondrial DNA sequences of the cytochrome Oz06, Oz08, Oz22, Oz27, Oz30, Oz41, Oz43, Oz44; http://cores.lifesciences.cornell.edu/brcinfo/).Eight primer pairs were used to PCR amplify autosomal microsatellite loci . Tajima's D r, was calculated to determine the goodness of fit of the data for each expected distribution Amplification resulted in 872 base pairs of cytochrome jModelTest Oz22 was found to have a high frequency of null alleles and was removed from further analysis. Allele frequencies and number, pairwise differentiation (FST), and observed (Ho) and expected (He) heterozygosity were calculated using MSA v.4.05 ST. The measure RhoST is similar to FST, but takes allele size into account and has been suggested to be a more accurate estimate of population differentiation under microsatellite evolution conditions Microsatellite alleles were sized and analyzed using GENEMAPPER v.4 (Applied Biosystems). MICRO-CHECKER v.2.2.3 Population structure was assessed using the Bayesian clustering method STRUCTURE v.2.3 M-ratio. The software BOTTLENECK v.1.2.02 e relative to Heq , is a signal of a recent decrease in effective population size. One thousand independent simulations were run assuming an infinite alleles model (IAM) We implemented two approaches in evaluating past bottlenecks, as commonly used in conservation genetic studies: BOTTLENECK and M-ratio method as implemented in M P VAL M-ratio is defined as M = k/r, the mean ratio of the number of alleles (k) to the size range of those alleles (r) per locus, for a sample of microsatellite loci. The significance of this ratio was determined by comparing the observed M-ratio to a simulated population with the same mutational model and sample size in the program CRITICAL M c, which was set at the lower 5% tail of the distribution. An observed M-ratio that falls below this Mc constitutes strong evidence that a bottleneck has occurred.The c: sp (frequency of single step mutations), Δg (the size of non one-step changes) and θ ( = 4Neμ). The value of sp = 0.88 was taken from c can be sensitive to Δg and Θ, values for Δg were varied between 2.8  parameters is necessary for the calculation of Mb sequence in the 79 individuals analyzed, resulting in 11 unique cytochrome b haplotypes, A–K  corresponded to a model for expected values of demographic stability, as suggested by the small value of r = 0.06 . Converstability .The TCS statistical parsimony network revealed relationships among haplotypes identical to those portrayed by a neighbor-joining tree . DespiteOz22, and this locus was not included in further analyses. Ninety-four alleles were detected at 7 microsatellite loci for 85 muskrats. No significant deviation from Hardy-Weinberg was found (P >0.05 for each locus across each population), and there was likewise no evidence of linkage disequilibrium . The AP population exhibited the fewest number of alleles per locus, lowest He and Ho, and lacked private alleles (P<0.0001). Little pairwise differentiation between ME and NH was found, with FST = 0.028   . Values 001)  M-ratio analyses and simulations produced significant evidence for a historical bottleneck. Estimations of Ne for the current island population drawn from M-ratio values were below the critical simulated value, Mc, when Δg = 2.8 , despite the presence of human artifacts and even Norway rat bones several hundred years earlier The genetic data reported here are congruent with anecdotes suggesting that humans introduced muskrats in the early 1900s for the purposes of fur harvest. Although on their own, these data are not sufficient to unequivocally warrant an “introduced” status, several other lines of independent evidence support such a designation. Morphologically, AP muskrats have slightly narrower crania than mainland muskrats Comparison with other insular mammal populations of land bridge islands in the northern Gulf of ME also supports a human-mediated introduction. From a source pool of ∼33 species of mammals native to the state of ME, only muskrats are present on AP. Crowell Crowell b sequences and microsatellite data of AP muskrats revealed consistently lower levels of genetic diversity as compared to the mainland source. Both ME and NH exhibited levels of heterozygosity comparable to those found for muskrats inhabiting nearby regions of NY, Quebec and New Brunswick, with Ho = 0.68–0.83 o was 0.54 for the AP muskrats.Founder events involve a subsample of the individuals from a source population, resulting in decreased genetic variation and increased divergence between source and founder populations ST values.As evidenced from the lack of phylogeographic grouping within the haplotype genealogies and the STRUCTURE results, muskrats in ME and NH show no evidence of geographic structure, presumably reflecting ongoing gene flow. The Bayesian clustering analysis performed using STRUCTURE suggests that AP is a cluster distinct from the mainland regions, discrediting the hypothesis of recent gene flow that would result from continued migration by swimming. This was confirmed by significant, elevated pairwise Fss and D values for a fur seal population that underwent a known bottleneck in the 19th century due to overhunting. They reported values for Fs of 4.423 and D of 1.35, although neither was significant. These values, derived from a species with a known historic bottleneck, are similar to those reported here for muskrats: Fs = 5.007, D = 1.35. The mismatch distribution for AP resulted in a rejection of a constant population size, as interpreted from a high raggedness statistic Both mitochondrial and microsatellite DNA markers were used to assess evidence for a recent bottleneck associated with a human-mediated colonization of AP. Using mitochondrial markers, we found that Fu's FThe program BOTTLENECK produced ambiguous results. During a founder event, rare alleles are more likely to be lost than common variants e. Heterozygosity excess is a transient signature only detectable for 0.2–4.0(Ne) generations after a bottleneck event eq. Indeed, populations with an explosive growth rate can still experience overall reductions in genetic diversity but not display heterozygosity excess due to a bottleneck The power of BOTTLENECK's analyses is largely dependent on the generation time of the focal species and the size of the effective founding population, NM-ratios, allele frequency shifts, and heterozygosity excess can provide windows into population decline and recovery across different timescales and thus provide a method of comparison for hypotheses of bottleneck timing. Heterozygosity excess and allele frequency distributions recover faster than the M-ratio M-ratio juxtaposed with non-significant heterozygosity excess and allele frequency shifts— this is exactly the result reported here. Conversely, a recent bottleneck, such as if muskrats were introduced very recently in 1990, would show significant signatures in both slowly and rapidly recovering measures A combined approach that includes analyzing Mitochondrial DNA revealed 11 distinct haplotypes found in 79 muskrats, and only 2, F and J, on AP. From haplotypes alone, it is possible that the AP muskrats are either (1) solely from the NH coastline, where both haplotypes F and J co-occur; or (2) from both ME and NH, with the island's F lineage coming from ME (given the high frequency of the F haplotype in ME). The coastal area of NH is the only region sampled that contains the 2 haplotypes also present on AP, making it the most probable source population . In addition, this coastal area is a major shipping, fishing, and naval shipyard , with high levels of boat traffic to the Isles of Shoals. Although we recognize that the distinction between ME and NH is politically and economically, rather than biologically, meaningful, retaining such a distinction between state-lines with different human dynamics  allows us to glean further insight into the human dimension of the muskrat colonization event.An intentional introduction in the early 1900s is consistent with the genetic data presented here. In her observations on the Isles of Shoals' natural history, Celia Thaxter presciently contemplated “how the [Norway] rats came here first is not known; probably some old ship imported them” Figure S1Map of Appledore Island and muskrat sampling locations. Red circles are haplotype J, blue circles are haplotype F, and purple circles are microsatellite data only. Sampling was conducted in thoroughly surveyed muskrat habitat; areas of the island that did not yield samples were occupied by highly aggressive nesting seabirds (which are a significant deterrent to muskrats), lacked appropriate vegetation, or were not in close proximity to fresh water. The results of our habitat use surveys were congruent with those of a 1984 study (TIF)Click here for additional data file.Figure S2Neighbor-joining tree of all haplotypes (872 base pairs of cytochrome b) using HKY85+I parameters, made in PAUP* v.4. Letters correspond to haplotypes, with specimen IDs in parentheses. Geographic information for each specimen can be found in (TIF)Click here for additional data file.Figure S3ΔK plot for STRUCTURE data. The value of ΔK peaks strongly at K = 2 (341.2816), declines abruptly to K = 3 (3.6114), and continues to decline for values K = 4 and greater.(TIF)Click here for additional data file.Table S1List of all specimens used in this study, their mitochondrial haplotype, and their geographic coordinates. * indicates specimen only yielded microsatellite data. See (DOCX)Click here for additional data file.Table S2ST for microsatellite DNA at seven loci.Results of pairwise Rho Mainland is ME and NH combined as a single population.(DOCX)Click here for additional data file.Table S3Results of sign and Wilcoxon sign-rank tests from BOTTLENECK simulations under an infinite alleles model (IAM), step-wise mutation model (SMM), and two-phase mutation model (TPM). A significant P value indicates excess heterozygosity, indicative of a bottleneck. *For TPM, we report only 10% SMM values as illustrative of our TPM results. All results for TPM regardless of proportion of SMM in the model  were P>0.05 and non-significant. a = P<0.05, b = P<0.01, c = P<0.005.(DOCX)Click here for additional data file.


Technology Identification of candidate genes associated with complex diseases by genetic testing, linkage studies and GWAS approach resulted in relevant genetic panels specific for each of many CDs. However diagnostic value of these panels remains obscure for the most of known CDs so far. Individual genetic data contributing to human health as well as to the onset of a CD are quickly accumulated in Individual DNA -data bank, which could be interpreted as Individual Gene-Pass (IGP).Advances in human genome studies resulted in identification of numerous genes which, if mutated, provoke monogenic disorders of different kind or can contribute to a number of complex diseases (CD). While basic problems of monogenic disorders are already solved the genetic impact into CDs seems to be illusive so far in the most cases and remains a cumber-stone of currant PPPM. Associated gene studies already substantiated the issue of Gene-pass of Reproductive Health (GPRH). It consists of two mandatory parts of genetic testing results. The first - carriers testing in spouses for sever monogenic diseases. The second - the panels of candidate genes associated with preeclampsia, endometriosis, trombophilia, habitual abortions, diabetes, nerve tube defects chromosome non - disjunction, placenta insufficiency, as well as some pharmacogenetic tests elaborated for pregnant women. GPRH at use in our institute will be demonstrated. The genetic data together with relevant laboratory tests extrapolated to individual case history after sophisticated bioinformatic analysis (MDR test) are used for prospective individual risk evaluation.In spite on huge progress of whole genome sequencing it will never substitute the clinically important IGP incorporating only health meaningful genes and associations Suggested GPRH is the only one of many other IGPs already existing de facto. Genetic data supplemented with epigenetic information contributing to regulation of associated gene expression should be considered the next important step towards System Genetic Level of CD studies for PPPM purposes.


Establishment of conservation priorities for primates is a particular concern in the island archipelagos of Southeast Asia, where rates of habitat destruction are among the highest in the world. Conservation programs require knowledge of taxonomic diversity to ensure success. The Philippine tarsier is a flagship species that promotes environmental awareness and a thriving ecotourism economy in the Philippines. However, assessment of its conservation status has been impeded by taxonomic uncertainty, a paucity of field studies, and a lack of vouchered specimens and genetic samples available for study in biodiversity repositories. Consequently, conservation priorities are unclear. In this study we use mitochondrial and nuclear DNA to empirically infer geographic partitioning of genetic variation and to identify evolutionarily distinct lineages for conservation action. The distribution of Philippine tarsier genetic diversity is neither congruent with expectations based on biogeographical patterns documented in other Philippine vertebrates, nor does it agree with the most recent Philippine tarsier taxonomic arrangement. We identify three principal evolutionary lineages that do not correspond to the currently recognized subspecies, highlight the discovery of a novel cryptic and range-restricted subcenter of genetic variation in an unanticipated part of the archipelago, and identify additional geographically structured genetic variation that should be the focus of future studies and conservation action. Conservation of this flagship species necessitates establishment of protected areas and targeted conservation programs within the range of each genetically distinct variant of the Philippine tarsier. Biodiversity-rich tropical forests are being degraded worldwide, and the pace of forest destruction is exceptionally rapid in insular Southeast Asia Traditionally, taxonomy and conservation have been inextricably linked and most conservation strategies have targeted formally named taxonomic units: species or subspecies Despite being the focus of a disproportionate number of intensely focused studies, multiple lines of evidence suggest that numerous primate taxa await discovery and formal taxonomic description. For example, between the 1975 and 1999 nocturnal primate species diversity grew worldwide by 2.85-fold increase In the Philippines, many morphologically indistinguishable yet genetically differentiated “cryptic” species We assessed the conservation genetics of Philippine tarsier to determine (1) how many distinct evolutionary lineages can be identified, (2) whether genetic structure conforms to biogeographical predictions and/or (3) expectations derived from current taxonomy (three subspecies). Due to limited natural history data or consensus from other sources of information , we argue that genetic data should be used to distinguish evolutionarily distinct tarsier population groups as objective, empirically defined conservation priorities.T. syrichta was nondestructively sampled (ear and tail-tip biopsies) from throughout as much of the species' range as feasible  Genetic material from 5′–TCACAACGTCTTGCTCAACC–3′ and Tars12s_AJB.R2, 5′–TTGAGGAGGGTGACGGGCGG–3′); ND2 ; CytB . We used the following thermal profiles for the mitochondrial fragment amplification: 1 min at 95°C followed by 38 cycles of 94°C for 30 sec, 30 sec at the primer specific annealing temperature , and 72°C for 1 min, and a final extension phase at 72°C for 10 min.Genomic DNA was extracted from tissues following Fujita's guanidine thiocyanate method Amplified products were visualized on 1.5% agarose gels and PCR products were purified with 1 µL of a 20% dilution of ExoSAP-IT  on the following thermal profile: 30 min at 37°, followed by 15 min at 80°. Cycle sequencing reactions were run using ABI Prism BigDye Terminator chemistry , and purified with Sephadex Medium  in Centri-Sep 96 spin plates . Purified products were analyzed using an ABI Prism 3130xl Genetic Analyzer (Applied Biosystems). Sequence contigs were assembled and edited using Sequencher 4.8 .Sequencing proved highly problematic for our many degraded tissue samples and approximately half our samples would not amplify for any targeted gene regions. Of the half that could be amplified for >2 target regions, initial sequence alignments were produced in Muscle Tarsius syrichta across the southern Philippines. Each locality was represented by 1–27 individuals (mean = 3.88). DNA was extracted using QIAGEN DNeasy extraction kits and we used nine microsatellite makers previously developed for Tarsius syrichta: T5, T6, T22, T34, T35, T43, T69 Tarsius spp.: T42, T50 We sampled a total of 66 individuals from 17 localities of Partitioned Bayesian analyses of the combined dataset were conducted in MrBayes v3.1.2 Partitioned maximum likelihood (ML) analyses were conducted in RAxMLHPC v7.0 To generate an ultrametric phylogeny for our Generalized Mixed Yule-Coalescent (GMYC) analysis, we ran a strict clock divergence time analysis of the mtDNA data, partitioning the data by gene, and fixing the mean substitution rate to 1.0 in BEAST v1.7.5 Tarsius syrichta spans the Mindanao PAIC in the southern Philippines. As an initial starting point, our a priori hypothesis of population structure, given our understanding that all these islands are part of the same faunal region T. syrichta samples, as well as infer the probabilities of individuals belonging to each of the estimated populations. We considered individuals with q values between 0.10 and 0.90 to be admixed a priori knowledge of inter-locus relationships, analyses were run under the correlated allele frequency model and the admixture ancestry model K, to range from one  to seven . We ran each analysis for 10 million iterations, with a burn-in of 500,000. The rate of change of K (ΔK) was evaluated in Structure Harvester (Web v0.6.93) Distruct v1.1  eastern and (b) western subclades; substantial genetic divergence was found among groups  Bohad hoc ΔK test of the results of Structure analyses of microsatellite loci preferred a K of two , suggesting an initial estimate of only two distinct populations as defined by our nuclear data alone. All individuals were assigned to one of these two genetic clusters with q values above 0.90. With the exception of populations from Dinagat Island and the northeastern peninsula of Mindanao Island .We anticipate future studies will further refine the known distribution of the Dinagat-Caraga tarsier and extend its range to nearby Siargao Island. Additionally it is not clear if the Surigao del Norte  samples identified here occur naturally on Mindanao or are the result of Dinagat and/or Siargao island animals confiscated from smugglers by regional wildlife enforcement and subsequently released on Mindanao , prioritization of populations for conservation action follows the recognition of named taxonomic units, i.e., species and subspecies. In the face of differing taxonomic arrangements, biogeographic expectations, and differing geographic patterns of nuclear versus mitochondrial genetic variation, how should the Philippine tarsier be prioritized for implementation of applied conservation measures? Currently, one tarsier sanctuary has been established on Bohol Island and another is under consideration for construction on Leyte. Would these two, and only these two efforts adequately conserve genetic components of Philippine tarsier diversity? We argue that they would not. Would establishment of conservation programs based on current tarsier taxonomy  properly conserve the genetic variation we have documented? Similarly, we argue that such an approach would fail to conserve the genetic variation elucidated here.Tarsius syrichta. Hill (1955) recognized T. s. syrichta/carbonarius/fraterculus as a poorly defined subspecies, perhaps synonymous, with T. s. syrichta. Museum specimen variation seemed insignificant to Niemitz (1984), but an inadequate basis for judgment, according to Musser, and Dagosto (1987).” Our study sheds some light on the uncertainty of Brandon-Jones et al. T. syrichtaBrandon-Jones et al. A number of genetic and social system phenomena could conceivably account for the differences in lineage-specific support we have inferred between mitochondrial and nuclear loci. Possibilities include persistence of genetic polymorphism, lineage sorting, nuclear gene flow between distinct mitochondrial lineages, sex-biased dispersal, mitochondrial gene sweeps, or an undocumented Philippine tarsier mating system. Given the present sampling, we are unable to distinguish between these possibilities, which may provide intriguing questions for future research. However, from a practical and applied conservation perspective, we argue that a single, clearly optimal, and objective solution results from the combination of patterns observed here in mitochondrial and nuclear gene loci.combined results of our variable sources of information that likely will have the best chance of maximally preserving genetic variation in the Philippine tarsier. As such, we advocate a multi-tiered conservation program involving conservation programs and protected areas in the ranges of each distinctive population lineages identified here, minimally including (1) Bohol-Samar-Leyte, (2) Dinagat-Caraga, and (3) Mindanao. Until a comprehensive taxonomic study using multiple lines of evidence  can be undertaken, we find that the existing evidence provides an inadequate basis for distinguishing between taxonomic alternatives. Instead, we emphasize that the archipelago's tarsier populations are partitioned into at least the three genetic variants, which we have empirically defined here for practical conservation purposes. We caution primatologists from taking taxonomic action until multiple lines of evidence converge on a meaningful solution Given our variable inferences, we advocate a combined, cumulative approach towards identification and recognition of tarsier evolutionary lineages for conservation planning. Thus, we argue that in order to maximally preserve genetic variation, it is the Appendix S1Review of Philippine tarsier taxonomy.(PDF)Click here for additional data file.Appendix S2Vouchers, locality data, GPS coordinates, and Genbank numbers for included samples.(PDF)Click here for additional data file.


Oryza rufipogon Griff.), the progenitor of Asian cultivated rice (O. sativa L.), is endangered due to habitat loss. The objectives of this research were to evaluate the genetic diversity of wild rice species in isolated populations and to develop a core collection of representative genotypes for ex situ conservation. We collected 885 wild rice accessions from eight geographically distinct regions and transplanted these accessions in a protected conservation garden over a period of almost two decades. We evaluated these accessions for 13 morphological or phenological traits and genotyped them for 36 DNA markers evenly distributed on the 12 chromosomes. The coefficient of variation of quantitative traits was 0.56 and ranged from 0.37 to 1.06. SSR markers detected 206 different alleles with an average of 6 alleles per locus. The mean polymorphism information content (PIC) was 0.64 in all populations, indicating that the marker loci have a high level of polymorphism and genetic diversity in all populations. Phylogenetic analyses based on morphological and molecular data revealed remarkable differences in the genetic diversity of common wild rice populations. The results showed that the Zengcheng, Gaozhou, and Suixi populations possess higher levels of genetic diversity, whereas the Huilai and Boluo populations have lower levels of genetic diversity than do the other populations. Based on their genetic distance, 130 accessions were selected as a core collection that retained over 90% of the alleles at the 36 marker loci. This genetically diverse core collection will be a useful resource for genomic studies of rice and for initiatives aimed at developing rice with improved agronomic traits.Common wild rice ( Oryza rufipogon Griff., also known as common wild rice, is the progenitor of Asian cultivated rice (Oryza sativa L). Wild rice is widely distributed in the tropics and subtropics of Asia. In China, wild rice is distributed from 19 to 28°N in eight provinces, i.e., Hainan, Guangdong, Guangxi, Yunnan, Fujian, Taiwan, Hunan, and Jiangxi. Many populations of genetically diverse wild rice are present in these provinces [rovinces . It is erovinces .The natural habitats of wild rice species have been shrinking due to urbanization ,12. ConsMorphological data are valuable for assessing and comparing diversity patterns within and among populations . MoleculGlycine max (soybean), Arachis hypogaea (peanut), and Sesamum indicum (sesame) crops [Developing a core collection is an efficient approach for exploring, characterizing, and capturing the genetic diversity of large populations. Over 5,000,000 accessions of crop species are held in gene banks around the world . A core e) crops ,24–26. Ce) crops . Genetice) crops ,27–29.ex situ conservation; and 3) ensured that the core collection retained the genetic diversity of the entire population.Previous collections of common wild rice were limited to individual populations from a few ecological regions –32. Howein situ and ex situ conservation of wild plants. All participants in this project have provided consent for publication of images , and three qualitative traits  were evaluated at maturity following the protocol developed by the International Rice Research Institute (IRRI). These qualitative and quantitative traits, which have a large impact on rice yield and significantly affect the genetic diversity of the germplasm ,20, were2, 0.01% glutin). The PCR profile was 94°C for 5 minutes followed by 30 cycles of 94°C for 45 s, 55°C for 45 s, and 72°C for 45 s, and a final extension at 72°C for 5 minutes. PCR products were separated by electrophoresis on a 6% polyacrylamide denaturing gel and displayed by silver nitrate staining [Molecular markers with high levels of genetic diversity were selected from our previous study of genetic diversity in a core collection of cultivated rice . From thstaining . A DNA lstaining .Phenotypic and genotypic data were used to estimate genetic diversity and construct the core collection. Dendrograms of SSR markers and morphological traits were analyzed by NTSYSpc version 2.10 and SPSS17.0 software, respectively ,38. Box PilnPi , and accounts for the abundance or richness of a species [Pi2) /(2n - 1)  to estimate the average genetic diversity per locus within an individual population [P2i−P2iP2j, Pi and Pj are the population frequency of the ith and jth allele) was estimated using PICcale software . PIC is a measure of a marker’s ability to detect polymorphisms in a population, based on the number of alleles detected and their frequency distribution; hence, it provides an estimate of the discriminating power of a marker [Packing diagrams illustrate the genetic diversity in populations detected using SSR loci , and wer species ,16. Nei'pulation . The pola marker .To assess variation in the morphological traits and molecular data, the analysis of variance (ANOVA) and analysis of molecular variance (AMOVA) were performed using SPSS17.0  and ARLEhttp://ibi.zju.edu.cn/software/qga/) and Venn analysis (http://bioinfogp.cnb.csic.es/tools/venny/) was performed to compare this core collection with the one based on genetic distance.Three strategies were used to select wild rice accessions for long-term conservation . (1) Strt-test (X2). If the p-value was greater than 0.05, then the difference between the core and entire population was considered to be non-significant. PCA was used to identify the major sources of variation between the accessions selected as the core collection and the eight populations. If the core sets were distributed uniformly in the PCA distribution graph, the core collections were considered to maintain a high level of genetic diversity and to be representative of the whole germplasm.Furthermore, genetic diversity parameters, including number of alleles, na, ne, h, I, and PIC, were used to estimate the representativeness of the core collections. These genetic diversity parameters were compared between each population and its core collection using Student's Box plots in The coefficient of variation (CV) is a useful statistic for comparing the variability of a morphological trait among populations. The highest variability among populations was observed for the number of secondary branches (CV = 8.00), while seed setting percentage (CV = 0.03) exhibited the lowest variability. The BL population has the highest mean CV for the ten quantitative traits, due to the particularly high CV of the number of secondary branches. Although the CV of awn length and number of spikelets per panicle was higher than that of the number of secondary branches in the QH population, a comparative analysis revealed that the CV of the number of secondary branches was significantly higher than that of the seven remaining traits . We perfex situ in Guangzhou. The Mantel test showed a significant correlation  between flowering rate and distance north of the equator  over the eight populations. Polymorphic alleles (292), total alleles (318), PPB (91.82%), na (1.910), and PIC (0.777) exhibited the highest level in the ZC population than in the other populations . The ne,Moreover, we conducted a combined analysis of morphological and molecular data, and the results were nearly consistent with the separate analyses of morphological and molecular data  based on phenotypic and genotypic data . PCA revAMOVA revealed that molecular variation within populations (80.68%) was higher than among populations (19.32%) . We deteTo develop a core collection, we constructed dendrograms based on separate and combined analyses of phenotypic and genotypic data. The dendrograms based on separate analyses revealed a greater genetic distance than did those based on the combined analysis of data. To validate that the accessions were representative of the entire population, the accessions with a high genetic distance (3 for phenotypic and 0.76 for genotypic data) were selected as the core sets from phenotypic and genotypic dendrograms . FurtherX2, the means, and standard errors for the five genetic parameters shown in Genetic diversity parameters were observed to estimate the coverage of core collections constructed from the entire common wild rice germplasm. The whole set of 36 SSR markers detected an average of 206.5 and 195.5 alleles in each population and core collection, respectively. Core collections retained 94.7% of alleles found in the whole germplasm. Alleles in the core collections were present in between 91.5 and 99.5% of the eight populations, and the coverage was above 90% of the alleles detected in the whole germplasm. The average number of alleles ranged from 1.338 for the RM201 marker to 1.969 for the RM413 marker, with an average of 1.806. PIC ranged from 0.235 for the RM201 marker to 0.754 for the RM283 marker, with an average of 0.640 . To examWe subjected both SSR markers and morphological data to genetic diversity analysis and established core collections from eight wild rice populations. We detected high levels of genetic diversity in common wild rice populations from Guangdong province. For example, the ZC population had the highest total number of alleles, polymorphic alleles, percentage of polymorphic bands, average number of alleles, and polymorphism information content, while the GZ and SX populations had higher effective numbers of alleles, Nei's gene diversity, and Shannon's information index than the other populations. The high number of effective alleles indicates the importance of alleles in the ZC, GZ, and SX populations. These three populations from Guangdong province had higher levels of genetic diversity than the other populations. These results are in agreement with earlier studies ,13–15. TMorphological and molecular data can be analyzed separately or in combination to determine genetic diversity based on genetic distance and hierarchical cluster analysis ,48. HereOryza sativa was developed in China [Three methods are commonly used to develop core collections, and each of these methods has advantages and disadvantages ,34,51. Tin China . Furtherin China .In this study, we used the UPGMA and genetic distance to develop the core collection. We validated the percentage of alleles retained, the genetic diversity, and the PCA between the core and entire collection to ensure the genetic diversity of the core collection. Common wild rice has some excellent traits, such as those exhibited by the 4w-102 and 4w-103 accessions, and these accessions were not selected when using the first method described above .An ideal core collection encompasses the maximum genetic diversity of the entire germplasm with minimum repetitiveness. Various methods are used to construct a core collection, including phenotypic, isozyme, protein, SNP, and DNA marker data. However, there is no universally accepted method for constructing a core collection; all methods have advantages and disadvantages. Phenotypic or genotypic traits have been recognized as useful parameters for developing core collections ,55. TherThe sampling proportion of a core collection is important for selecting a suitable sampling percentage to obtain the maximum genetic diversity and the maximum range of geographical types. Usually, 5–30% of the sampling percentage is selected from the entire germplasm ,56. In tGeographical isolation is one of the barriers that blocks the introgression of wild species and generates differentiation after long-term adaptive evolution ,58. All Ex situ conservation provides germplasm that breeders can use to improve elite cultivars. Although there are hundreds of natural populations of wild rice, conservation strategies favor the ex situ conservation of common wild rice. Evaluating genetic diversity is extremely important for the ex situ conservation of wild plants [ex situ conservation is the best strategy for preserving the genetic diversity of common wild rice. We have conserved (in complete isolation) accessions of common wild rice from three provinces of China in our wild rice germplasm garden . We found that all of the conserved populations maintained a high level of genetic diversity in the last two decades. However, Xie et al. revealed that wild rice lost genetic diversity during ex situ conservation. Thus, in situ conservation is also required to preserve the genetic diversity of common wild rice [d plants ,60. Consild rice .ex situ. Our strategy was highly successful in selecting representative accessions from the entire population based on phenotypic and genotypic data. In addition, we showed that individual analysis of morphological and molecular data is more effective than combined analysis of these data when constructing a core collection of wild rice. Furthermore, it is better to construct a core collection of wild rice based on UPGMA and genetic distance than on core collection construction software alone. The core collection not only represents the mean and variances but also the range of variables of the entire population. Therefore, morphological and genotypic data can be used to construct a highly representative core collection. To avoid inter- and intra-population introgression of wild rice, it is critical that the common wild rice germplasm be conserved (both ex situ and in situ) as core collections.In conclusion, we have assembled a core collection of common wild rice with abundant morphological and genetic diversity from different ecological regions of China and conserved this collection S1 FileEx situ and in situ conservation of wild rice in China . UPGMA dendrograms of the common wild rice populations from eight districts/counties based on combined phenotypic and genotypic data . Dendrograms denoting UPGMA clustering analyses of the common wild rice populations from eight districts/counties based on phenotypic and genotypic data . Conservation of the core collection of common wild rice . Venn diagram representing the overlapping of core collections developed by QGA software and genetic distance .(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.


Rhododendron protistum var. giganteum, which is a highly endangered species with only two known endemic populations in a small area in the southern part of Yunnan Province in China. We detected moderate to high genetic diversity at the species level, but low genetic differentiation between the two extant populations. These results suggest that some rare and endangered species are able to maintain high levels of genetic diversity even at small population sizes.This is the first study of the genetic diversity and structure of the big tree rhododendron,  Rhododendron protistum var. giganteum, is a highly endangered species with only two known endemic populations in a small area in the southern part of Yunnan Province in China. Unfortunately, limited information is available regarding the population genetics of this species. Therefore, we conducted amplified fragment length polymorphism (AFLP) analysis to characterize the genetic diversity and variation of this species within and between remaining populations. Twelve primer combinations of AFLP produced 447 unambiguous and repetitious bands. Among these bands, 298 (66.67 %) were polymorphic. We found high genetic diversity at the species level  and low genetic differentiation (Gst = 0.110) between the two populations. Gene flow between populations (Nm) was relatively high at 4.065. Analysis of molecular variance results revealed that 22 % of the genetic variation was partitioned between populations and 78 % of the genetic variation was within populations. The presence of moderate to high genetic diversity and low genetic differentiation in the two populations can be explained by life history traits, pollen dispersal and high gene flow (Nm = 4.065). Bayesian structure and principal coordinate analysis revealed that 56 sampled trees were clustered into two groups. Our results suggest that some rare and endangered species are able to maintain high levels of genetic diversity even at small population sizes. These results will assist with the design of conservation and management programmes, such as in situ and ex situ conservation, seed collection for germplasm conservation and reintroduction.Comprehensive studies on the genetic diversity and structure of endangered species are urgently needed to promote effective conservation and management activities. The big tree rhododendron,  Genetic diversity is one aspect of biological diversity that is extremely important for conservation strategies . In geneRhododendron protistum var. giganteum, exhibits a very limited distribution, with only two populations found in the Gaoligong Mountains of northwestern Yunnan Province in China  markers because this technique has been successfully employed in other studies that evaluated the genetic diversity of other Rhododendron is the largest woody plant genus in the Ericaceae family with at least 1025 species and one of the most common woody plants that are distributed across the northern temperate zone, tropical southeast Asia and northeastern Australia  was double-digested using restriction endonucleases EcoRI (5 U) and MseI (5 U)  in a total reaction volume of 20 μL for 4 h at 37 °C. This reaction was deactivated at 70 °C for 20 min. The digested DNA (4 µL) was added to 16 µL of the ligation mixture with 50 pmol MseI adaptor, 5 pmol EcoRI adaptor and 2 U T4 DNA ligase (New England Biolabs). This mixture was incubated at 16 °C for 16 h, and then deactivated at 65 °C for 10 min.2+-free medium) and 1.5 U DNA Taq polymerase  to yield a total volume of 25 µL. Pre-selective polymerase chain reaction (PCR) amplification profiles are listed as follows: 94 °C for 3 min; 30 cycles of 30 s denaturing at 94 °C, 30 s annealing at 56 °C and 60 s elongation at 72 °C; and holding at 72 °C for 5 min. The pre-selective PCR products were then diluted 10-fold, and the template was used for selective amplifications.The ligated DNA (2 µL) was pre-selected using 33 ng of the primers for MseI and EcoRI adaptors Table  with 5 n2+-free medium) and 1.5 U DNA Taq polymerase (Transgen Biotech). The selective PCR amplification profile was obtained by denaturation at 94 °C for 30 s, annealing at 65 °C for 30 s, temperature decrease by 0.9 °C/cycle and extension at 72 °C for 1 min for 12 cycles; followed by 94 °C for 30 s, 56 °C for 30 s, 72 °C for 1 min for 23 cycles and 72 °C for 5 min.An initial screening was performed using two individuals from each area by using 64 primer combinations for selective amplifications. A total of 12 primer combinations (Table Amplified DNA products were mixed with 98 % formamide loading buffer (10 µL), heated at 95 °C for 7 min and immediately cooled in an ice bath for 30 min. The products were resolved by 6 % polyacrylamide gel electrophoresis in 0.5× Tris–borate–EDTA buffer by using a 100-bp DNA ladder marker (Transgen Biotech) for 4 h at 1500 V and then stained with 0.1 % silver nitrate.Ne), Nei's genetic diversity (h), Shannon's information index (I), level of gene flow (Nm), total gene diversity (Ht), variability within populations (Hs) and coefficient of genetic differentiation (Gst) were calculated using POPGENE version 1.32 , respectively, to construct a binary matrix. The binary matrix was edited using GenALEx version 6.4.1 . The perion 1.32  with manAnalysis of molecular variance (AMOVA) was conducted to calculate the partitioning of genetic variation between and within the two populations by using GenALEx version 6.4.1 .K ranging from 2 to 20, a burn-in of 1 × 105 iterations and 1 × 105 subsequent Markov Chain Monte Carlo steps. The combination of admixture and correlated allele frequency models was also analysed. The second-order rate of change in the log probability of the data with respect to the number of clusters (ΔK) was also used to estimate the probable likely number of genetic clusters  were polymorphic. The total number of fragments of each primer combination ranged from 29 (M-CAA/E-AGC) to 50 (M-CAC/E-AGG) with an average of 37.25. Percentage polymorphism varied from 48.28 to 80.00 % with an average of 67.10 % per primer combination (Table h) and Shannon's information index (I), in the combined data matrix of all 12 primers, were 0.240 and 0.358, respectively. The genetic diversities within species (Ht) and within populations (Hs) were 0.238 and 0.212, respectively (Table Gst) was 0.110. Based on the Gst value, the level of gene flow (Nm) was estimated as 4.065 (Nm > 1). These results indicated high gene flow and low differentiation between extant populations.Nei's gene diversity  showed a higher genetic diversity level than the DHT population   at the species level than other critically endangered tree species such as Metrosideros bartlettii (PPL = 44 %)   (Ostrya rehderiana (PPL = 29.90 %) (Litsea szemaois (PPL = 80.8 %)  and gene flow (Nm) between the two extant populations were 0.110 and 4.065, respectively. Long distance dispersal of pollen and/or seeds results in low genetic differentiation and high gene flow between populations of the same species  and birds .This study was financially supported by grants F.Q.W., S.K.S., Y.H.W., W.B.S. initiated and designed the research; S.K.S. and W.B.S. obtained funding for the study; S.K.S., F.Q.W. and X.J.Z. collected materials and performed the experiments, F.Q.W. and S.K.S. analysed the data and wrote the paper. All authors read, edited and agreed to submit the manuscript.None declared.


Leishmania (L.) killicki (syn. L. tropica), which causes cutaneous leishmaniasis in Maghreb, was recently described in this region and identified as a subpopulation of L. tropica. The present genetic analysis was conducted to explore the spatio-temporal distribution of L. killicki (syn. L. tropica) and its transmission dynamics. To better understand the evolution of this parasite, its population structure was then compared with that of L. tropica populations from Morocco. In total 198 samples including 85 L. killicki (syn. L. tropica)  and 113 L. tropica specimens  were tested. Theses samples were composed of 168 Leishmania strains isolated from human skin lesions, 27 DNA samples from human skin lesion biopsies, two DNA samples from Ctenodactylus gundi bone marrow and one DNA sample from a Phlebotomus sergenti female. The sample was analyzed by using MultiLocus Enzyme Electrophoresis (MLEE) and MultiLocus Microsatellite Typing (MLMT) approaches. Analysis of the MLMT data support the hypothesis that L. killicki (syn. L. tropica) belongs to the L. tropica complex, despite its strong genetic differentiation, and that it emerged from this taxon by a founder effect. Moreover, it revealed a strong structuring in L. killicki (syn. L. tropica) between Tunisia and Algeria and within the different Tunisian regions, suggesting low dispersion of L. killicki (syn. L. tropica) in space and time. Comparison of the L. tropica (exclusively from Morocco) and L. killicki (syn. L. tropica) population structures revealed distinct genetic organizations, reflecting different epidemiological cycles. Leishmania killicki (syn. L. tropica) was discovered in 1986. Few studies have been conducted on this parasite exclusively described in Maghreb. Consequently, many elements on its epidemiology, transmission, population structure and dynamics remain unknown.L. tropica populations from Morocco using Multilocus Enzyme Electrophoresis (MLEE) and MultiLocus Microsatellite Typing (MLMT) typing. MLMT data support the hypothesis that L. killicki (syn. L. tropica) belongs to the L. tropica complex despite the strong genetic differentiation between them. Despite the probable recent divergence between L. killicki (syn. L. tropica) and L. tropica, they seem to evolve differently. Indeed, L. killicki (syn. L. tropica) appears slightly polymorphic and highly structured in space and time, while L. tropica was genetically heterogeneous, slightly structured geographically and temporally. The different population structures revealed distinct genetic organizations, reflecting different epidemiological cycles. Several parameters could explain these opposite epidemiological and genetic patterns such as ecosystems, vectors and reservoirs.To better understand the evolution of this parasite, its population structure has been compared with that of  Leishmania species that cycle between their phlebotomine sandfly vectors and mammalian reservoir hosts  and from Algeria )    . This re < 0.05) .Leishmania parasites, many taxa, such as L. killicki (syn. L. tropica), are still not completely characterized. The main objective of this study was to understand the epidemiology and transmission dynamics of L. killicki (syn. L. tropica) by analyzing its population structure and by comparing the genetic patterns of L. killicki (syn. L. tropica) and L. tropica populations in Maghreb.Despite a great knowledge on L. killicki (syn. L. tropica) and L. tropica revealed a strong genetic differentiation associated with a lower genetic polymorphism within L. killicki (syn. L. tropica). Furthermore, the NJ tree showed that L. killicki (syn. L. tropica) creates a homogeneous and monophyletic cluster within the L. tropica complex. These data support the recently obtained MultiLocus Sequence Typing (MLST) results [L. killicki (syn. L. tropica) emerged from L. tropica by a founder effect. The strong genetic differentiation indicates an independent evolution and an absence of gene flow between the two taxa after the founder event. The geographic distance and the ecological barriers between Morocco  and Tunisia, Libya and Algeria  samples) as well as the different transmission cycles can explain this diversification. Maghreb countries are essentially separated by mountains and the Sahara desert that could prevent the circulation and migration of Leishmania vectors and reservoirs. Furthermore, L. killicki (syn. L. tropica) transmission cycle is most probably zoonotic [L. tropica appears to be both zoonotic or anthroponotic [The comparison of  results  suggestizoonotic , 15, wheoponotic .L. killicki (syn. L. tropica) samples from Tunisia and Algeria revealed also a differentiation within this taxon, but lower than the one detected with L. tropica. This result supports the idea that L. killicki (syn. L. tropica) spread recently and may be still spreading between the different countries after the founder event. It is not known yet where the L. tropica subpopulation emerged to generate L. killicki (syn. L. tropica), but the number of reported cases suggests Tunisia. Despite the low sample size from Algeria, we detected a strong and significant genetic differentiation between the population from Tataouine and the samples from Algeria and a low genetic differentiation between the Gafsa and Algerian populations. These results seem to indicate a more recent diversification between the Gafsa and Algerian populations, supporting the hypothesis of a recent L. killicki (syn L. tropica) dispersion from Gafsa to Algeria. Conversely, the only isolate from Libya is genetically closer to the Tataouine than to the Gafsa population. This pattern is in agreement with the geographical distances/characteristics of these regions. Indeed, the mountains in the Gafsa area, where the probable reservoir(s) of L. killicki (syn L. tropica) live(s), belong to the Atlas Mountain chains, while mountains in the Tataouine region are connected to the Libyan mountains.The comparison of L. killicki (syn. L. tropica) populations from Gafsa and Tataouine, despite their low genetic diversity indices, they show a strong and significant genetic differentiation with a lower genetic diversity among the Tataouine samples. These data suggest that the Tataouine population is more recent and that these two populations are genetically isolated. The presence of geographical barriers separating the South West and South East of Tunisia  could explain this structuring.Concerning the L. killicki (syn. L. tropica) in Tunisia shows a low circulation of genotypes between the different populations not only in space, but also in time within a region. Based on this observation and because most isolates were from infected humans, we can hypothesize that L. killicki (syn. L. tropica) mainly circulates in the reservoir host C. gundi and humans are accidentally infected. This is in agreement with the zoonotic character of L. killicki (syn. L. tropica) compared to L. tropica, which is known to be an anthropozoonotic or zoonotic pathogen, according to the infection focus [Analysis of the spatio-temporal evolution of on focus .L. tropica from Morocco and L. killicki (syn. L. tropica) from Tunisia revealed that the population structures of these two taxa are different. Indeed, L. killicki (syn. L. tropica) populations from Tunisia showed an important genetic differentiation and differences in terms of genetic diversity, whereas the L. tropica populations from Morocco were genetically more homogeneous and only slightly differentiated. These data suggest that L. killicki (syn. L. tropica) poorly disperses (except for rare migration events from a region to another) compared to L. tropica from Morocco. This finding might reflect different ecological patterns, such as epidemiological cycles, infection of the reservoirs or vector behavior.Comparison of L. killicki (syn. L. tropica) population genetics allowed exploring the evolutionary history of this parasite and highlighting its different genetic patterns compared to L. tropica. Despite the probable recent divergence between these taxa, they seem to evolve differently in terms of epidemiological cycle and thus transmission dynamics. Particularly, this study supports the hypothesis of a zoonotic transmission cycle for L. killicki (syn. L. tropica). Our data also suggest that Gafsa could be the historical focus of L. killicki (syn. L. tropica), although the sample size from the other regions was too small to firmly validate this hypothesis.To conclude, this detailed study on P. sergenti vector populations in Tunisia and their susceptibility to L. killicki (syn. L. tropica) and the parasite biology in C. gundi to better understand the transmission cycle of this parasite.It is now essential to study the L. killicki (syn. L. tropica) should be still considered a L. tropica subpopulation, our analyses indicate that in the future, this taxon position may have to be reconsidered.Although for the moment, S1 Fig(TIF)Click here for additional data file.S1 Table(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.S3 Table(XLS)Click here for additional data file.