Symbiodinium) may influence the response of symbiotic reef-building corals to a warming climate. We analyzed the genetic structure of the stress-tolerant endosymbiont, Symbiodinium glynninomen nudum (ITS2 - D1), obtained from Pocillopora colonies that dominate eastern Pacific coral communities. Eleven microsatellite loci identified genotypically diverse populations with minimal genetic subdivision throughout the Eastern Tropical Pacific, encompassing 1000’s of square kilometers from mainland Mexico to the Galapagos Islands. The lack of population differentiation over these distances corresponds with extensive regional host connectivity and indicates that Pocillopora larvae, which maternally inherit their symbionts, aid in the dispersal of this symbiont. In contrast to its host, however, subtropical populations of S. glynni in the Gulf of California (Sea of Cortez) were strongly differentiated from populations in tropical eastern Pacific. Selection pressures related to large seasonal fluctuations in temperature and irradiance likely explain this abrupt genetic discontinuity. We infer that S. glynni genotypes harbored by host larvae arriving from more southern locations are rapidly replaced by genotypes adapted to more temperate environments. The strong population structure of S. glynni corresponds with fluctuating environmental conditions and suggests that these genetically diverse populations have the potential to evolve rapidly to changing environments and reveals the importance of environmental extremes in driving microbial eukaryote  speciation in marine ecosystems.The migration and dispersal of stress-tolerant symbiotic dinoflagellates (genus  Symbiodinium are ideal models for population genetic studies since numerous samples may be acquired from various marine invertebrate hosts and habitats over a range of spatial and temporal scales [Marine microrganisms have the potential to disperse great distances and may therefore show little population differentiation over large geographic regions ,2. The gl scales –13. Symbiodinium [Pocillopora can represent 90-100% of the live coral cover in near shore hard bottom habitats and encompass a latitudinal range of both tropical  and subtropical  regions. While approximately eight or nine morphospecies of Pocillopora have been reported from the eastern Pacific [Pocillopora may actually comprise a single genetically definable species designated as type 1 [Reef coral communities in the eastern Pacific prosper under sea surface temperatures (SSTs), nutrient concentrations and irradiance levels that fluctuate widely between seasons . These eiodinium . The mos Pacific , recent  Pacific ,16. Accos type 1 ,16,17.Pocillopora throughout the eastern Pacific may in part be attributed to their associations with the intracellular symbiont, Symbiodinium D1 [Symbiodinium D1 is defined genetically by internal transcribed spacer (ITS) region 2 sequence identity and is referred to here as S. glynninomen nudum . Symbios region ,20. Thiss animal . Like maeratures ,21. As aSymbiodinium offer one mechanism by which some reef corals cope with warm or environmentally unstable conditions [Symbiodinium spp. separated by small distances exhibit significant differences in microsatellite allele diversity and suggest that their effective dispersal is limited to 5 or 10 kilometers  at approximately 24°N. In the first large-scale population genetic examination of a Clade D Symbiodinium spp., eleven microsatellite loci were analyzed and the population genetic structure compared with previously published data on the host.The abundant and widespread populations of eastern Pacific  Pacific  should i Pacific ,31. SampPocillopora spp. symbionts across the eastern Pacific [2) of Pocillopora colonies were collected by SCUBA from six sampling locations and preserved in either a high salt, 20% DMSO buffer [Pocillopora morphospecies were analyzed  was detected were utilized for this study. Nucleic acid extractions were conducted and the dominant resident symbiont was identified by denaturing gradient gel electrophoresis (DGGE) fingerprinting of the partial 5.8S and internal transcribed spacer (ITS) region 2 . SamplesSymbiodinium to determine the degree of allelic polymorphism for these populations [Symbiodinium glynni in the eastern Pacific  was used to overcome biases of assigning populations by location by using the microsatellite data to cluster MLGs based on their genetic similarities irrespective of sample origin. The validity of using Structure with haploid genomes is discussed in the a type 1 , using aa type 1 .Pocillopora morphospecies, Structure analyses were conducted on data from the three locations where sampling was adequate to conduct such a comparison; GoC, BB and OAX. For these analyses, data were grouped according to host morphology with identical MLGs removed within each morphological grouping. The analytical parameters used were identical to those for geography, with the exception that the location prior setting was used to better assist with weak structuring [To investigate the possibility of population differentiation according to ucturing  and the Analyses of molecular variance (AMOVA) were then conducted on geographic populations with regional structuring as defined by Structure and by morphospecies to determine the degree of differentiation. AMOVA’s along with a permutation procedure were performed in GenAlEx  to test -2 day-1) were calculated for each sampling location using data for years 2000 to 2009 obtained from the Giovanni online data system, developed and maintained by NASA Goddard Earth Sciences (GES) Data and Information System (DISC). Approximately 24 km2 around each sampling location was selected and data retrieved from the SeaWiFS (PAR) and MODIS-Aqua (SST) databases. Scatter plots were drawn using monthly averages and standard deviations. To compare the variation, box plots were drawn using annual means, first and third quartiles, and extreme maximum and minimum annual values. Monthly averages for sea surface temperatures  and photosynthetically active radiation  that dominated their ribosomal array ,43,44, crelevant ,19. Thest (sensu ), howevee) per locus ranged from 1.02 to 6.22 . The majority of these samples (n = 51) possessed two alleles at just one locus. Another 24 samples contained two alleles at multiple loci . Multiple alleles were never observed for six or more loci or for the conserved loci D1Sym88 and D1Sym92. (ID)) that two samples with the same MLGs may not have originated from the same clone lineage was exceedingly low for most locations and ranged from 7.55 x 10-4 to 7.76 x 10-7 ((ID) based on data from all locations was 4.09 x 10-9. Therefore, there was a 1 in two hundred and fifty million probability that identical genotypes from separate locations are of independent origins. The probability of identity  or genotypic richness ranged from 0.30 to 1.00 for each location . Values S. glynni MLGs [A pairwise analysis of linkage among loci revealed that ~93% of the pairwise comparisons were unlinked , which strongly suggests frequent sexual recombination among nni MLGs . In genek = 2; ln P(D) = -2351.4; k = 6, at which they stabilize and/or decline  = -2489.7) with no further subdivision of the ETP, even when analyzed in isolation  = -1780.3).Two well supported populations were detected using the Evanno et al. method to process the Structure results . Lastly, AMOVA’s for each location were not significant at p = 0.0001 , as were all pairwise comparisons between morphospecies at each location , which indicates there may be weak geographical differentiation within the ETP between PAN and both BB and OAX, which was not detected by Structure or BAPS . In con palmata ,29, but  palmata ,12,45).Symbiodinium glynni genotypes within a colony persist over time, with individual genotypes present in colonies for at least 9 months [Pocillopora distributed across a reef demonstrated that the dominant genotype can be replaced in adult colonies over time, but the frequency and rate of this turnover remains undetermined [S. glynni genotypes showed limited spatial partitioning among and between locations separated by several kilometers . Across strains .Symbiodinium spp. [Symbiodinium diversity [Symbiodinium glynni genotypes sorted randomly among the damicornis, meandrina, and verrucosa morphospecies comprising Pocillopora type 1 . The laolation  frequently disperse over these distances. Surprisingly, identical MLGs were recovered from locations separated by as much as 1,600 km . ,6. S. glS. glynni genotypes must be strongly influenced by Pocillopora’s mode of symbiont acquisition, and thus affects genetic structuring. Recent genetic analysis [Pocillopora type 1 across the eastern Pacific . Aided e  relative to the other eastern Pacific locations . Strains  of S. glynni from the environment can enter and displace the resident strain in adult Pocillopora colonies [S. glynni’s is not. Environmental conditions that pose no physical barriers to dispersal may explain the strong genetic structure exhibited by the symbiont, while maintaining a confluent host population. Temperature and light gradients are important in the distribution and prevalence of ium spp. ,62,63. Tocations . We propcolonies , a procecolonies . TherefoS. glynni where never found to co-occur within the same colony. However, rapid competition and replacement would likely proceed early in the host’s life, during larval settlement and juvenile growth, and only adult colonies were sampled during the course of this research. Additional sampling of juveniles during the late summer or early fall would assess this possibility. Similarly, sampling near the mouth of the Gulf of California, where environmental conditions transition from subtropical to more tropical, would delineate the geographic boundaries of each population and maximize the chance of finding colonies experiencing competitive displacement. Finally, it is possible that the host microsatellite loci examined for Pocillopora type I were not adequately polymorphic and the discordance between host and symbiont populations is the result of markers with low resolution. However, the loci’s high allelic diversity  and confidence in delimiting individuals (probability of identity = 4.2 x 10-6) in a population with high genotypic diversity (R = 0.88) make this possibility unlikely [If locally adapted genotypes are migrating into adjacent populations, then it may seem unusual that subtropical and tropical genotypes of S. glynni genotypes of the subtropics may have evolved recently during a range expansion from the south following the last glaciation (~10-18 KYA). However, the considerable genetic differentiation between tropical and subtropical populations may possibly reflect a longer period of isolation that began during the more extreme conditions associated with the last inter-glacial period. Fossil records indicate that Pocillopora were extensive throughout the Gulf of California during the late Pleistocene , but whSymbiodinium clades and species [Pocillopora colonies from both populations under controlled laboratory settings and/or reciprocal transplanting is needed to substantiate the relative influence of local adaptation and competition on the genetic structure of S. glynni.While environmental gradients clearly influence the distribution and prevalence of different  species ,62,63, t species ,6 and otPocillopora type 1 and S. glynni to disperse throughout much of the eastern Pacific suggests that the shallow water ecosystems they create may be resilient to local disturbances and capable of recovery by migration of individuals from surrounding sources. However, the lack of gene flow between tropical and subtropical populations of S. glynni indicates that these populations may be differently adapted to fluctuating environmental conditions . Populations in the Gulf of California are relatively small and may be particularly vulnerable to extinction if host populations were lost to severe environmental degradation. Conversely, as the climate continues to change, symbiont populations adapted to variable environmental conditions might be a source of important physiological diversity. While more information is needed to evaluate the relative exchange of the eastern Pacific with the rest of the Pacific, the broad thermal tolerance of S. glynni [Pocillopora whose symbionts are adapted to warmer and more variable environmental conditions. The ability of . glynni  and its Figure S1Plots derived from the method by Evanno et al.2005 using the second order rate of change of the Ln P(D) to determine the appropriate value of K based on runs in Structure. a) Plot of the average Ln P(D) (±SD) over five runs for each value of K. b) Result plot of the second order rate of change showing a clear peak at K = 2, representing the genetic break between the GoC and the ETP. c) Raw data from the ten consecutive Structure runs with five replicates at each K and used for the above analyses.(DOC)Click here for additional data file.Figure S2S. glynni populations showing a lack of population subdivision according to Pocillopora morphospecies in the GoC (a), BB (b) and OAX (c).Structure plots of  Structure plots are arranged in order of increasing K and the data is ordered according to morphology of the colony from which it was collected.(EPS)Click here for additional data file.Table S1Description of microsatellite loci used in this study. Ta = annealing temperature, Ae = effective alleles with standard error in parenthesis. Subscript numbers following the repeat motif indicate the number of repeats in the initial cloned sequence that was used to develop locus primers.(DOC)Click here for additional data file.Table S2S. glynni.Haploid allele frequencies and sample size by location for (DOC)Click here for additional data file.Table S3PT) between S. glynni populations based on Pocillopora morphospecies in the eastern Pacific.A pairwise matrix comparing the genetic relationship  Gulf of California, (b) Banderas Bay and (c) Gulf of Tehuantepec. Significant values  represented in bold.(DOC)Click here for additional data file.Text S1Supplemental materials and methods further describing microsatellite analysis and data analysis.(DOC)Click here for additional data file.


Atractylodes macrocephala Koidz. (Asteraceae) is an economically important Chinese medicinal herb. In this study, 15 polymorphic microsatellite markers were developed from A. macrocephala using the compound microsatellite marker technique. Levels of polymorphism within the 15 markers were assessed using 83 individuals from two wild and two cultivated populations in China. The number of alleles per locus ranged from 2 to 20, with an average of 9.9 alleles. Observed and expected heterozygosities ranged from 0.083 to 1.000 and from 0.097 to 0.938, respectively. These markers will be valuable for germplasm classification and identification, as well as for assessing the genetic diversity and spatial genetic structure among wild and cultivated populations of A. macrocephala. Atractylodes macrocephala Koidz. (Asteraceae) is a perennial herb that is endemic to several provinces in China. The dried rhizome of A. macrocephala, also known as Atractylodes Macrocephala Rhizoma (common name: “Baizhu” in Chinese and “Byakujutsu” in Japenese), is widely used in traditional herbal remedies in Asia [A. macrocephala is now rare and threatened in the wild, and has even disappeared in many locations, e.g., Zhejiang Province [ in Asia ,2. It wa in Asia . The ess in Asia , anti-in in Asia , anti-ca in Asia  and anti in Asia  activityProvince . Three pProvince , JL . These primers were then chosen to test for polymorphism.A. macrocephala  ranged from 2 to 20 (mean: 9.9) among the four populations (Ho) and expected heterozygosities (He) ranged from 0.083 to 1.000 (mean: 0.549) and from 0.097 to 0.938 (mean: 0.657), respectively  were 0.553 (range: 0.083–0.917), 0.603 (range: 0.167–0.958), 0.497 (range: 0.100–1.000) and 0.551 (range: 0.200–0.867), respectively (He) were 0.656 (range: 0.194–0.910), 0.658 (range: 0.223–0.885), 0.655 (range: 0.097–0.923) and 0.660 (range: 0.191–0.938), respectively (Ho) were 0.578 (range: 0.083–0.958) and 0.524 (range: 0.100–1.000), respectively (He) were 0.657 (range: 0.194–0.910) and 0.658 (range: 0.097–0.938), respectively , 6.5 (range: 2–12), 6.5 (range: 2–13) and 6.7 (range: 2–13) for population PA, PJ, JL and MC, respectively . On averectively . The expectively . For theectively . On averectively . The expectively . The resp < 0.05) in three populations , while just three loci significantly deviated from the HWE expectations in the wild population MC (p < 0.001) were similar to the test for Hardy-Weinberg equilibrium. Specifically, null alleles may be present at six loci in population PA , four loci in population PJ , four loci in population JL  and two loci in population MC (Am12 and Am15). The departures from Hardy-Weinberg equilibrium, most of which were caused by heterozygote deficiency, are likely to be due to the presence of null alleles . Alternatively, inbreeding in the cultivated populations and genetic drift among small, fragmented wild populations may explain our observations. Two pairs of loci in population PA  and two pairs in population PJ  were found to have significant LD (p < 0.01), while no significant LD was observed in any loci pairs in the two wild populations. Thus, LD is likely due to different population structure rather than to physical linkage of the markers.Nearly half of the fifteen analyzed loci showed significant deviation from the Hardy-Weinberg equilibrium  blunt-end restriction enzymes for both individuals. Fragments flanked by a microsatellite at one end were amplified from both the HaeIII and SspI DNA libraries, and then the fragments whose size were between 400 and 1000 bp were purified and ligated into PMD18-T vector (Takara), and subsequently transformed into DH5α competent cells (Takara) as described by Yuan et al. [The compound microsatellite marker technique based on a dual-suppression-PCR method was used to develop microsatellite markers for is study . Two indis study . The mic [et al.  except tn et al. .6(AG)n or (TC)6(AC)n compound SSR motifs, of which ninety-one possessed unique sequences with sufficient flanking regions for designing specific primers. A specific primer (IP1) was designed using Primer Premier Version 5.0 [Tm value of IP1 was lower than the compound SSR primer and within 2 °C, because primer with lower Tm value normally determines the specificity of the primer pairs. Finally, the primer pair with highest score given by Primer Premier was chosen.A total of 223 positive clones were obtained and sequenced on an ABI Prism 3730 automated DNA sequencer (Applied Biosystems). One hundred and ninety-four sequences were found to contain (AC)sion 5.0 . BecauseTaq polymerase (Takara), 1.5 μL of 10 × PCR buffer with MgCl2, 1.2 μL of dNTPs (2.5 mM each), 0.1 μL bovine serum albumin , 0.5 μL of each primer (10 μM). PCR amplification conditions were as follows: initial denaturation at 95 °C for 5 min; 35 cycles of 30 s at 95 °C, 30 s at the optimized annealing temperature , as well as observed (Ho) and expected (He) heterozygosities, were calculated by CERVUS version 3.0.3 [The number of observed alleles per locus (on 3.0.3 . Hardy-Won 3.0.3 . The preon 3.0.3 .A. macrocephala showed high polymorphism among and within four populations of A. macrocephala. Thus, these markers are suitable for population genetics studies of this medicinal plant. In our ongoing research, we are applying them to investigate genetic diversity and spatial genetic structure over both cultivated and wild populations of this important medicinal species. Furthermore, we expect that these markers will facilitate future studies on the origin and evolutionary history of cultivated populations, as well as on the efficient conservation and sustainable exploitation of the A. macrocephala germplasms.The 15 novel microsatellite markers that we have developed for


The emergence of next-generation sequencing technologies allowed access to the vast amounts of information that are contained in the human genome. This information has contributed to the understanding of individual and population-based variability and improved the understanding of the evolutionary history of different human groups. However, the genome of a representative of the Amerindian populations had not been previously sequenced. Thus, the genome of an individual from a South American tribe was completely sequenced to further the understanding of the genetic variability of Amerindians. A total of 36.8 giga base pairs (Gbp) were sequenced and aligned with the human genome. These Gbp corresponded to 95.92% of the human genome with an estimated miscall rate of 0.0035 per sequenced bp. The data obtained from the alignment were used for SNP (single-nucleotide) and INDEL (insertion-deletion) calling, which resulted in the identification of 502,017 polymorphisms, of which 32,275 were potentially new high-confidence SNPs and 33,795 new INDELs, specific of South Native American populations. The authenticity of the sample as a member of the South Native American populations was confirmed through the analysis of the uniparental  lineages. The autosomal comparison distinguished the investigated sample from others continental populations and revealed a close relation to the Eastern Asian populations and Aboriginal Australian. Although, the findings did not discard the classical model of America settlement; it brought new insides to the understanding of the human population history. The present study indicates a remarkable genetic variability in human populations that must still be identified and contributes to the understanding of the genetic variability of South Native American populations and of the human populations history. The emergence of next-generation sequencing (NGS) technologies, such as Solexa (Illumina) The Americas were the last continents to be occupied by human populations. According to the most widely accepted model, which is based on anthropological, archaeological, and genetic evidence The literature records at least 15 fully annotated WGSs of individuals from European mate-paired full-slide runs that were performed produced 36.8 Gbp of raw data , of which 435,947 (86.84%) were already described in the Single Nucleotide Polymorphism database (dbSNP) and 66,070  were considered potentially new polymorphisms .The total number of high-confidence SNPs with coverage equal to or higher than 10× was 433,310  and the SNPs found in chromosome Y (Y-DNA) We compared the set of high-confidence SNPs that were found in this study with those obtained in the HapMap project phases 1, 2, and 3 The results of STRUCTURE analyses are represented at With a K value in the range of five to seven, no gene flow with the Native American was found. At this level of analysis, the European (CEU and TSI), Asian , and African  populations were found to form homogeneous groups, although the LWK and MKK populations exhibited a small degree of mixture, mainly with Europeans. As expected, the Mexican (MEX) and Indian (GIH) populations exhibited a high proportion of admixture with the various populations.Two other approaches were used to estimate the genetic differences: discriminant analysis of the principal components (DAPC), which illustrated the differences among the individuals and populations ; and the3 statistics did not reveal any gene flow involving the Native American sample with any other population  presented a similar scheme of contribution for K values higher or equals to 5. They had the same population-structure formed by three major groups: European, African and Native American.The aim of the present study was to describe potential new genetic variations to be further analyzed among South Native American populations through the use of NGS technologies. The specialized literature describes the fully sequenced genomes of samples of other populations, including samples of Japanese The results of the present study contribute to the catalogs of genetic variation that have been organized by various projects, such as the 1,000 Genome The comparison of the findings with the available dbSNP, HapMap and 1,000 genomes datasets showed a high number of potentially new polymorphisms. This result indicated the importance of acquiring information on the genetic variability of human populations through the sequencing of individuals from different ethnic groups, particularly those that have not been previously investigated, such as the Native Americans. This approach is specially important to the study of isolated populations regarding their healthy, such as, the finding of markers associated to complex diseases like diabetes type 2, coronary diseases, hypertension and others.et al.The analyses performed limit the possibility of sequencing errors and sample contamination. This study mapping, genome coverage and SNP detection was similar to previous studies A STRUCTURE analyses was performed to establish the relationship between the investigated sample and samples from several different populations. The high number of SNPs shared with Eastern Asian populations corroborate to the classical model of the America occupation.The sample ancestry origin was through the analysis of the maternal  and paternal (Y-DNA) lineages. The A2 mitochondrial haplogroup is typical of the indigenous populations of the Americas, including the South American indigenous groups, and it is found in approximately 15% of the Amazonian tribes 3 statistic to the 1,000 genomes dataset and no admixture scheme was statistical significant. The data support the conclusion that the investigated sample does not exhibit traits of a recent interethnic mixture and, thus, can be considered an authentic South Native American individual.The introgression of European genes into Native American populations, particularly through the crossings between European males and Amerindian females Although, no evidence of recent admixture was found, the 1,000 genomes STRUCTURE analyses indicated an important steady contribution of the Aboriginal Australian to the South Native American sample was found for K larger than 5. Three scenarios can explain similarities between the Aboriginal and our sample: i) an ancient shared history between the groups The present study identified 32,275 potentially new SNPs with a low error rate. These new SNPs may contribute to the understanding of the composition and genetic history of South Native American populations. The authenticity of the sample was demonstrated through the analysis of the maternal, paternal, and autosomal lineages. In addition, an autosomal analysis was able to distinguish the sample from the other continental populations and showed that it is close to the Eastern Asian populations. Other important finding was the indication of a shared history between our sample and the Australian Aboriginal, although no conclusion may be taken. Although, the findings did not discard the classical model of America settlement; it brought new insides to the understanding of the human population history.Ethical consent was obtained according to the Helsinki Declaration. Ethical approval was obtained from the Brazilian National Committee on Research Ethics (CONEP- Parecer No 1062/2006). A signed informed consent was obtained from the village leaders since most subjects were not literate in Portuguese, but all subjects provided verbal assent to participate. A FUNAI/FUNASA health agent, who helped explain the aims and scope of the study to individuals, accompanied all activities.et al.Life Technologies, CA, USA) according to the manufacturer's protocol. Based on the purpose of the present study, two runs were performed each one in a full slide.The genomic DNA was obtained from an Amazon tribe male healthy individual. Along the years, our group gathered a pool of samples from several Amazon tribes. To guarantee the best ethical behavior regarding our samples and their tribes, the studied sample was selected randomly from our pool of male samples together with Roewer's Amazon samples  following the manufacturer's recommended protocol. After the alignment, the output of the Bioscope software was converted to the BAM format, and the sequencings were paired.The SOLiD platform generated thousands of reads with a length of 50 bp. The results were transferred to the processing server, where both runs were aligned to the reference human genome (NCBI Genome Reference build 37 – HG19/GRCh37 Feb. 2009) using the Bioscope™ v.1.3 platform  that characterize ethnic groups. The mitochondrial mutations were identified aligning the reads from the mitochondrial to the Andrews Reference Genome  To further investigate our sample, it was collect two dataset of bi-allelic SNPs genotype data. The first dataset (dataset A) consists in 220 HapMap samples' and our sample's genotype data for 2,955 SNPs genotyped for all HapMap's samples and identified as high quality in our sample. The 220 samples were selected randomly, including 20 representatives for each population: Japanese in Tokyo, Japan (JPT); Han Chinese in Beijing, China (CHB); Chinese in Metropolitan Denver, Colorado (CHD); Gujarati Indians in Houston, Texas (GIH); Yoruban in Ibadan, Nigeria (YRI); African ancestry in Southwest USA (ASW); Maasai in Kinyawa, Kenya (MKK); Luhya in Webuye (LWK); Utah residents with Northern and Western European ancestry from the CEPH collection (CEU); Tuscan in Italy (TSI); and Mexican ancestry in Los Angeles, California (MEX).The second dataset (Dataset B) consists in 1092 samples of project 1,000 genomes phase 1 3f statistic 3f statistic test if a population 1 is admixed regarding the others two populations ), where 3f values significantly negatives indicates that population 1 is admixed.To infer the genetic distance, the FST was calculated by Arlequin v.3.5 software The admixture analyses were performed using Structure v. 2.3.4 software Figure S1Venn diagram of the shared SNPs among Amerindian sample, CEU, YRI, JPT, and CHB populations. The Venn diagram represents the total set of SNPs that were found in the Amerindian sample and those SNPs that are shared with the genotyped samples of the CEU , YRI , CHB , and JPT  populations. Due to the high similarity between the genotypes of the CHB and JPT populations, their representatives were grouped into a single set (CHB+JPT) that represents the mutations that are shared between the Amerindian sample and the CHB and JPT populations.(EPS)Click here for additional data file.Figure S2Complete population genetic structure analysis of 1,000 genomes project's, Aboriginal Australian and Native South American genotype dataset. The diagram of genetic contribution was obtained using Structure software for models of 2 to 9 subpopulations. The populations were grouped labeled as follow, according to their major continental ancestry: IND ; ABO ; Eastern Asian ; Europeans ; African ; and New Americans . The plot represents the rate of contribution from each subpopulation (colors) to the samples (x axis).(TIF)Click here for additional data file.Table S1Total high-confidence SNPs detected. A complete list of all high-confidence SNPs detected in the sample, including its' chromossomic position relative to hg19 reference genome, the reference and mutant base observed with their relative base coverage and dbSNP identification.(ZIP)Click here for additional data file.


A distance values were used for phylogenetic tree construction with the UPGMA algorithm. The two Tsigai and two Maritza populations were found to be geneticallvery closely related to each other y . The Pleven Black Head population was distinct from the other four. These results could be useful for preserving genes in these breeds, thereby ensuring their preservation in Bulgaria. Herein, genetic relationships among five breeds of Bulgarian sheep were estimated using microsatellite markers. The total number of alleles identified was 226 at the 16 loci examined. D Over the past decade, numerous studies on genetic diversity in domestic livestock , based on the analysis of microsatellite loci, have been carried out worldwide. In earlier studies on sheep genetic structure, use was made of polymorphisms of phenotypic traits  or biochemical markers  . Molecul, viz., lowlands, hills, or mountains, where they are raised, and there is evidence of a high level of genetic diversity  and the south-western Bulgarian (Rodopski Tsigai). These breeds are completely white, thus differing from the original Tsigai populations which are multicolor or black to gray . In BulgThe Patch Faced Maritza and the White Maritza sheep are separately bred in the region north of Plovdiv. These populations are small and could be considered under threat, consisting of only 1,000 heads per breed, with about 500-600 ewes each, distributed on 14 and 15 farms, respectively. Projects targeting the preservation of these breeds commenced only recently, in the late 1990s . Milk yiThere are known differences in body measurements and weights, coloring, and even in performance among the examined breeds. The average body-weights and wither-heights of the breeds under study are summarized in The aim of this study  was to investigate genetic similarities among different breeds of Bulgarian sheep, notably Tsigai, Pleven Blackhead and Maritza, in order to facilitate their rational development, utilization and conservation.Hair samples were taken from randomly selected individuals from different herds .In order to ensure that the animals chosen were unrelated, the wool samples from White Maritza sheep were collected from five , Patch-Faced Maritza from four , Pleven Blackhead from two , Rodopski Tsigai from two  and Staroplaninski Tsigai from two  different herds. The selected two-two flocks from Pleven Blackhead and the two Tsigai breeds were chosen from different parts of Bulgaria so as to avoid genetic similarities.The White Maritza and Patch-Faced Maritza flocks were at least 30 km apart. When pedigree information was available, those 3 to 5-year-old animals with no shared common ancestor for at least two generations were selected. Animals without pedigree information were randomly chosen, with the premise that all were 3 to 5 years old and known by the head of the Maritza Breeder Association, through wool samples certifying to minimal genetic similarities. Numbers of animals studied per breed are presented in A) was estimated to define the genetic differences between populations. Phylogenetic trees were constructed using the Phylip Ver. 3.57c software package . Sixteen microsatellite markers  were selected based on their level of polymorphism, location on different chromosomes and recommendation by one or more of the following organizations: United States Department of Agriculture (USDA); Australian Gene Mapping Web Site; Food and Agricultural Organization (FAO); International Society for Animal Genetics (ISAG). PCR and genotyping protocols followed those of  package .All microsatellite loci were found to be polymorphic, with the number of alleles per locus ranging from 7 (MAF35) to34 (MAF70). Genotyping of the MAF70 locus was repeated due to the high number of alleles, thereby confirming correctness. In all the examined populations, the highest number of alleles was at this very locus. The total number of alleles was 226 at the 16 studied loci. The mean number of alleles per locus ranged from 4.8 (MAF35 and MAF65) to 15.6 (MAF70) . Of all exp) was higher than the observed heterozygosity (Hobs), ranging from 0.580 (MAF65) to 0.867 (MAF70), with a mean of 0.736 among the loc. Hobs ranged from 0.256 (MAF65) to 0.713 (ILSTS11), with a mean of 0.523.Observed and expected heterozygozities are presented in IS fixation index among loci varied from 0.051 (TGLA357) to 0.592 (OarAE119). The mean value of Fis was higher than Fst (0.288 > 0.083), and heterozygote deficiency was detected at all examined loci. The FST suggested that around 8,3% of the total genetic variation was due to variation within studied populations and the remaining 91,7% corresponding to differences among individuals.Genetic structure of the populations was analyzed by Wright's F statistics. The FThe mean number of alleles per population ranged from 6.25 (Rodopski Tsigai) to 8.625 (White Maritza) .Mean observed and expected heterozygosities per population ranged from 0.458 (Staroplaninski Tsigai) to 0.577 (Patch-Faced Maritza), and 0.726 (Staroplaninski Tsigai) to 0.798 (Pleven Blackhead), respectively. All populations had lower than expected levels of heterozygosity. Genetic diversity was highest in Patch-Faced Maritzas. Fis values ranged from 0.246 (Patch-Faced Maritza) to 0.376 (Pleven Blackhead), and were positive in all the populations. The heterozygote deficit was the highest in Pleven Blackhead and the lowest in Patch Faced Maritza among the examined populations .A) values, it was apparent that the two Tsigai (Staroplaninski and Rodopski) and the two Maritza (Patch-Faced and White) populations were genetically the closest among those studied  (From Nei minimum genetic distance (Dctively) . Pleven A values. Genetic distances among Bulgarian populations are apparent. The Tsigai and Maritza populations clustered separately in the tree, with the Pleven Blackhead more closely related to the Maritza sheep breeds than the Tsigais (closest to the Patch Faced Maritza).Recently, the preservation of unique, genetically distinct breeds of domesticated animals, especially indigenous, has received much attention. Knowledge and information on genetic diversity, population structure and genetic relationships between populations are absolute prerequisites for defining and accomplishing effective preservation strategies. This study aimed to characterize the genetic diversity and structure of two Bulgarian Tsigai, two Maritza and the Pleven Blackhead sheep populations by using sixteen microsatellites.For the sampling process, unrelated individuals were selected from various flocks, from different parts of Bulgaria, to so avoid genetic similarities among the animals to be studied. In all, 226 alleles were detected at the 16 loci under consideration. The highest number of alleles was at MAF70 (34). At this locus, 23 alleles were detected in Ujmuqin sheep, and 22 alleles in other European sheep breeds (An analysis of Nei genetic distance between Bulgarian Tsigais, Maritzas and Pleven Black Heads indicated that the two Tsigais and the two Maritzas are closely related, whereas Pleven Backheads are genetically unique. A genetic distance value of 0.05 implies moderate differentiation between two breeds . In our Based on our results, we can affirm that Pleven Blackheads genetically differ from both the Bulgarian Tsigais and Maritza sheep, although they are more closely related to the latter. This provides a certain support to the hypothesis of In summary, the four breeds, two Bulgarian Tsigai and two Maritza, were found to be genetically closely related to each other, although there was a significant distance between the two groups. Pleven Blackhead sheep were found, as a whole, to be genetically distinct, although more closely related to the Maritzas.


Located in the Sudan belt, the Chad Basin forms a remarkable ecosystem, where several unique agricultural and pastoral techniques have been developed. Both from an archaeological and a genetic point of view, this region has been interpreted to be the center of a bidirectional corridor connecting West and East Africa, as well as a meeting point for populations coming from North Africa through the Saharan desert.n = 542) have been high-throughput genotyped for 230 coding region mitochondrial DNA (mtDNA) Single Nucleotide Polymorphisms (mtSNPs) using Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight (MALDI-TOF) mass spectrometry. This set of mtSNPs allowed for much better phylogenetic resolution than previous studies of this geographic region, enabling new insights into its population history. Notable haplogroup (hg) heterogeneity has been observed in the Chad Basin mirroring the different demographic histories of these ethnic groups. As estimated using a Bayesian framework, nomadic populations showed negative growth which was not always correlated to their estimated effective population sizes. Nomads also showed lower diversity values than sedentary groups.Samples from twelve ethnic groups from the Chad Basin , but not their mtSNPs, suggesting a more ancestral ethnogenesis. Whereas the nomadic population showed a higher Mediterranean influence signaled mainly by sub-lineages of M1, R0, U6, and U5, the other populations showed a more consistent sub-Saharan pattern. Although lifestyle may have an influence on diversity patterns and hg composition, analysis of molecular variance has not identified these differences. The present study indicates that analysis of mtSNPs at high resolution could be a fast and extensive approach for screening variation in population studies where labor-intensive techniques such as entire genome sequencing remain unfeasible. The African Sahel together with a more southerly localized zone of savannah forms a clearly distinguishable biome. Also known as the Sudan or Macro-Sudan belt, this region displays some common linguistic features across current linguistic families 2. Such a giant lake, the largest in Africa at the time, with a wealth of food resources undoubtedly attracted long-term human settlements. Lake Chad reached its current size of about 20,000 km2 approximately 3,000 years ago, around the time that the Sahara grew to its present size and became nearly impenetrable for humans Approximately in the middle of the Sudan belt is the Lake Chad Basin, with Lake Chad in its imaginary centre. The Lake Chad Basin forms a remarkable ecosystem, where several unique agricultural and pastoral techniques have been developed Variation in mitochondrial DNA (mtDNA) has demonstrated to be useful for the interpretation of historical and contemporary demographic events around the world, and in particular for reconstructing the evolution and origin of human populations. Most population studies carried out in Africa have been based on analysis of the control region of mtDNA, sometimes complemented with analysis of hg diagnostic coding region sites On the other hand, variation in mtDNA is commonly analyzed using standard sequencing procedures targeting the first and/or the second hypervariable regions (HVS-I/II) or the whole control region (including HVS-I/II). Analysis of coding region mtDNA SNPs using minisequencing is another common approach Oral informed consent was required for the samples, and all of them were anonymized. The study was approved by the Ethical committee of the University of Santiago de Compostela. The study also conforms to the Spanish Law for Biomedical Research (Law 14/2007- 3 of July).n = 441; 80%) were previously reported for the HVS-I segment and selected Restriction Fragment Length Polymorphisms (RPLPs) in Černý et al. n = 47), Kotoko (n = 62), Mafa (n = 57), Masa (n = 41), Buduma (n = 30), Chad Arabs (n = 27), Shuwa Arabs (n = 39), Fali (n = 40), Bongor Fulani (n = 50), Tcheboua Fulani (n = 40), Kanembu (n = 50) and Kanuri (n = 59). DNA extract were then submitted to the laboratory of Santiago de Compostela where the genotyping was carried out.'We analyzed 542 individuals from 12 different ethnic populations sampled around the Lake Chad Basin. Most of these samples , namely, NA10830, NA10831, NA10860, NA10861, NA11984, NA12147 (that were used as positive controls). Assessment of the genotyping quality was carried out by replicating some samples from the Chad Basin in different runs plus six good quality DNA samples from the CEPH  African phylogeny and nomenclature is very complex and has been elaborated during the last decade based on the control region and complete genome sequencing efforts DnaSP v.5 http://www.r-project.org/).Principal Component Analysis was carried out on population hg frequencies and using R , (ii) population growth as g = -(lnθt/lnθpresent day)/t, where g is expressed as the relationship between θ at a time t>0 in the past and θ at the present (t = 0), and (iii) migration rate, defined as M = m/µ, where m is the immigration rate per generation), between the 12 ethnic groups used in the present study and using information from mtSNPs. Estimates were obtained for three independent replicates using a Bayesian framework. The jModelTest v.0.1.1. GTR) model. Estimates from iModelTest were obtained using three replicates using 10 initial chains  and two final chains . Transition:transversion rate was set to 30.Lamarc  For estimation of Theta and migration rate it is possible to get results with one region but they will improve markedly with more; doubling the number of regions nearly doubles the available information. Estimation of growth rate is very poor with less than 3 unlinked regions and particularly benefits from having more.” (http://src.gnu-darwin.org/ports/biology/lamarc/work/lamarc-2.1.2b/doc/html/data_required.html). On the other hand, sample sizes for some of the groups are relatively low and therefore the impact on the different estimates is unpredictable. Last but not least, the neighboring source populations for the Chad Basin are not represented in the mathematical model ; therefore, we are committed to assume a simplistic model were the provenance of the different lineages comes from one of the 12 ethnic groups considered.Some caveats should be considered in regards to the demographic estimates obtained. The mtDNA is in reality a single locus and therefore all the values should be taken with caution: “For all of these computations and in order to account for missing data, failed SNPs were imputed according to known phylogeny. Given the fact that there exists a robust mtDNA worldwide phylogeny based on entire mtDNA genomes , the phylogenetic-based approach for imputation seems more reliable than those based on e.g. metrics for linkage disequilibrium Several diversity indices have been computed for the 12 ethnic groups analyzed in this study. These indices have been obtained both individually for HVS-I and mtSNPs, and in combination for HVS-I plus mtSNPs . With feversus those located in the south  were carried out on the 12 Chad Basin populations analyzed in this study using the following grouping schemes: all of the populations individually, populations grouped by language family, and populations grouped according to their locations in the North or the South of the Chad Basin . Most ofPCA was carried out based on hg frequencies to the maximum level of resolution. The three first components account for a total of ∼40% of the variation, and it shows notable divergence between the different ethnic groups from the Chad Basin. Thus, the first principal component (PC1), which accounts for 15% of the variation, locates Mafa and Kanuri in one side of the plot, and the two Fulani populations in the opposite pole. PC2 (13%) shows also the Mafa in one side of the plot and the Kanembu in the other extreme. PC3 (12%) displays again Mafa in one pole opposed to the Kotoko. There are not unique features in the Mafa that makes this population different to the other ethnic groups, but an accumulative effect of several differences in hg frequencies . Apart from the two Fulani samples, the two Arab ones are proximal in the plot indicating a close maternal phylogenetic relationship. The most distinctive features of the two Arab populations compared to the other Chad populations are the presence of non sub-Saharan lineages, such as R0a or M1 hgs.In agreement with the analysis of the different genetic diversity metrics, it is interesting to note that the nomadic populations are more tightly grouped in the scatter plot than the sedentary ones , mirrori−8 base substitution per nucleotide per year We further estimated the population mutation parameter, θ, which in conjunction with the average mtDNA mutation rate was used to infer the effective population sizes of the different Chad Basin populations assuming a neutral model of molecular evolution . For an There are not unique features that would explain the observed migration rates values , but very poorly correlate for the nucleotide diversity . Thus, one can speculate different demographic scenarios for each population according to their differential diversity values. For instance, nomadic or semi-nomadic populations tend to experience loss of diversity by genetic drift , reducing both haplotype and nucleotide diversities. Provided that the gene flow is negligible for a given effective population size, the HVS-I region of more ancestral nomadic populations would be expected to retain more haplotype diversity (due to the presence of more rare variants) than younger nomadic groups. Thus, although the (semi-)nomadic populations  all have very low nucleotide and haplotype diversity values compared with sedentary populations, the two Arab populations retained higher values of haplotype diversity in the HVS-I segment than the Fulani . This hypothesis is compatible with the fact that nomadic populations as a whole have lower diversity than sedentary groups  variants, whereas the agglomeration of identical sequences into different hgs (possibly suffering from bias in mtSNPs selection) enriches the nucleotide diversity. Therefore, values computed using these different mtDNA segments summarize different aspects of the molecular diversity in populations. In fact, diversity values of HVS-I and mtSNPs for the 12 different ethnic groups analyzed moderately correlate for the haplotype diversity  have to be considered with caution because in reality each human population defined only by ethno-linguistic criteria is composed of an amalgamation of genetic lineages of different ages and origins, and therefore, none has a simple past.Haplogroup patterns vary substantially among the different ethnic groups studied. In some, hg composition seems to correlate well with historical documentation and their known demographic past. Thus, the presence of R0a only in Chad Arabs is expected given the high frequency of this hg in Southern Arabia https://www.policia.es/cgpc/index.htm).The few Eurasian profiles observed in the Chad Basin did not cluster in any particular ethnic group. Their control region segments are not informative from a phylogeographic point of view, and these sequences are broadly distributed around Eurasia. The only exception is the U5b1 HVS-I profile T16189C C16192T C16270T C16320T that is detected mainly in Africa With the exception of a few population studies based on complete genomes Analyses of mtSNPs in combination with sequencing information (control region) has provided new insights regarding population features of the Chad Basin populations versus sedentary populations) can leave different signatures in the two sets of markers.Since the mtSNPs used in the present study were designed to detect major and minor branches of the phylogeny, while the control region variation analyzed previously Ne, population growth, and migration rates. Nomadic populations show signals of negative growth , which not always coincide with those that have higher Ne; in fact, both parameters are just moderately correlated (r2 = 0.59). However, larger sample sizes would be required in order to yield solid figures for all these parameters.We have estimated for the first time different demographic parameters of the Chad Basin populations, including Analysis of mtSNPs have allowed to reveal new phylogeographic features in the Chad populations, not discussed previously It has been previously demonstrated Analyzing mtSNPs to a high level of hg resolution in populations allows a better selection of mtDNAs for further entire genome sequencing or for the design of multiplex mtSNP panels of interest in population, medical, and forensic genetics In conclusion, given that genotyping mtSNPs is straightforward compared with the intense effort demanded by sequencing complete genomes, the present study opens the door to more ambitious pan-African studies that would improve our knowledge on the mtDNA phylogeography in this continent.Table S1mtSNP calling rates and number of phylogenetic inconcistencies in the global dataset. Mutation hits in Soares et al  are also given for the phylogenetic inconsistencies.(XLS)Click here for additional data file.Table S2mtSNP genotypes and control region data for the 542 individuals from the Chad Basin region genotyped.(XLS)Click here for additional data file.Table S3Haplogroup frequencies in all the population samples analyzed at the maximum level of resolution provided by the control region and the mtSNPs.(XLS)Click here for additional data file.


Inbreeding is among the major concerns in management of local livestock populations. The effective population size of these populations tends to be small, which enhances the risk of fitness reduction and extinction. High-density SNP data make it possible to undertake novel approaches in conservation genetics of endangered breeds and wild populations.A total of 97 representative samples of domestic and wild pig populations from the Iberian Peninsula, subjected to different levels of threat with extinction, were genotyped with a 60 K SNP panel. Data analyses based on: (i) allele frequency differences; (ii) linkage disequilibrium and (iii) runs of homozygosity were integrated to study population relationships, inbreeding and demographic history.The domestic pigs analyzed belonged to local Spanish and Portuguese breeds: Iberian ─ including the variants Retinto Iberian, Negro Iberian and Manchado de Jabugo ─, Bisaro and Chato Murciano. The population structure and persistence of phase analysis suggested high genetic relations between Iberian variants, with recent crossbreeding of Manchado de Jabugo with other pig populations. Chato Murciano showed a high frequency of long runs of homozygosity indicating recent inbreeding and reflecting the recent bottleneck reported by historical records. The Chato Murciano and the Manchado de Jabugo breeds presented the lowest effective population sizes in accordance with their status of highly inbred breeds. The Iberian wild boar presented a high frequency of short runs of homozygosity indicating past small population size but no signs of recent inbreeding. The Iberian breed showed higher genetic similarities with Iberian wild boar than the other domestic breeds.High-density SNP data provided a consistent overview of population structure, demographic history and inbreeding of minority breeds and wild pig populations from the Iberian Peninsula. Despite the very different background of the populations used, we found a good agreement between the different analyses. Our results are also in agreement with historical reports and provide insight in the events that shaped the current genetic variation of pig populations from the Iberian Peninsula. The results exposed will aid to design and implement strategies for the future management of endangered minority pig breeds and wild populations. Progressive population decline has called the attention of the conservation management and scientific communities. Both for wild and domesticated populations alike there is a fear that inbreeding may lead to loss of allelic variation and adverse phenotypic consequences . In addiIn addition to these concerns, local populations are often considered to be part of the local culture and history. For instance, local pigs are often linked to local cuisine and the local landscape. The Spanish Iberico or Iberian, and Portuguese Alentejano pigs, for instance, are used to produce highly priced products due to their quality, that in part results from feeding with acorns from sparse Mediterranean oak forests, the so called 'Dehesas’. The wild relative of the pig, the wild boar, on the other hand, plays a significant role in the wildlife of the Iberian Peninsula. It is among the main prey species of an iconic predator from the Iberian Peninsula such as Iberian wolf . MoreoveLocal pig populations, both wild and domestic, have been highly affected by human-induced changes. Local, usually fat, breeds for instance, were affected by changes in consumer preference in the middle of the 20th century when consumers started to avoid high-fat meat. As a result, a relatively small number of highly productive pig breeds progressively replaced and marginalized the traditional breeds. Many breeds became extinct in the past decades, while many other traditional breeds today face near-extinction either through dwindling population numbers or hybridization with highly productive breeds . At the The recent availability of a high-density porcine SNP panel  providesHere we present a comprehensive study in which high-density SNP data from domestic and wild pigs were used to address questions important to conservation genetics. First, we assessed the relationships between pigs by population structure analysis and by investigating the persistence of LD phase. Secondly, patterns of LD in each population were used together with a high-density recombination map to estimate past and present effective population size. Finally, the number and size of ROH were investigated in each individual. The joint analysis of all those parameters allowed us to obtain reliable and consistent data of population structure, inbreeding and demographic history in each population providing valuable insights for future management strategies in pig from the Iberian Peninsula.A total of 97 pigs from domestic and wild autochthonous populations from the Iberian Peninsula were genotyped with the Porcine SNP60 Beadchip [st ≥ 0.22 with all populations except for Bisaro where it was 0.18. The variants of Iberian ─Retinto, Negro Iberian, Manchado de Jabugo and five unclassified Iberian pigs─ showed low Nei’s genetic distances between them (≤ 0.06) and low Fst ( ≤ 0.05), and likewise the two populations of wild boar from Spain and Portugal (0.04 and 0.06 respectively). Among domestic pigs, Iberian variants showed the lowest Nei’s genetic distance to wild boar (0.10 - 0.12). The results from Nei’s genetic distances and the pairwise Fst between populations are detailed in Additional file The Principal Component Analysis (PCA) revealed four main clusters represented by wild boar, Iberian, Bisaro and Chato Murciano Figure . Among pThe Bayesian clustering algorithms implemented in the Structure software assigned all individuals to clusters that coincide to their population of origin, with the exception of one Manchado de Jabugo pig MJ_02) that was placed in the cluster of the other Iberian variants. Pairwise genetic distances between individuals  than in the domestic breeds and showed the lowest average r2 across all genetic distances. Among the domestic breeds, LD was the lowest in Iberian (r2 < 0.2 within 0.2 Mbp). By contrast, Manchado de Jabugo and Chato Murciano had the most pronounced extent of LD at short genetic distances, although LD decreased faster in Chato Murciano than in Manchado de Jabugo for genetic distances higher than 1 Mbp.A total of 24,703 SNPs in wild boar, 29,856 SNPs in Bisaro, 33,454 in Chato Murciano, 27,858 in Iberian and 26,246 in Manchado de Jabugo were used to estimate LD for all SNP pairs less than 3 Mbp apart Table . PairwisThe persistence of LD phase was calculated as the Pearson correlation (r) between SNP pairs in all possible population pairs. Similar to LD, r decreased as the distance between markers increased. This was observed for all pairs of populations, although at different degrees Figure . Bisaro e) implementing the equation r2 = 1/(4Nec +1) [e was observed for the wild boar population (Table e (Ne = 151 ± 84) while Manchado de Jabugo and Chato Murciano had smaller effective population sizes (Ne = 46 ± 50 and 59 ± 31 respectively). Nevertheless, notice the large SD of these estimates.The mean values of LD for all 1 Mb bins across the entire genome were used to estimate the current effective population size (N4Nec +1) . This es4Nec +1) . Large Ne at generation T, where T = 1/2c [e from as few as 5 to 20,000 generations ago . This increase in Ne was not observed in any other population  while the correlation was negative between lengths of ROH and recombination rates .High density SNP analysis can provide information on past and current population demography. LD is largely affected by population history and demography -30, consUnderstanding the relationships among and within populations of livestock is a necessary first step to establish conservation priorities and strategies ,36. PopuThe results obtained from the population structure and persistence of phase analyses indicate closer relationships between Chato Murciano and Bisaro, and between Iberian pigs and wild boar. This observed division seems to reflect the classical separation of pig populations from the Iberian Peninsula into two origins: the Celtic type and the Iberian type pigs . Most ofThe high Pearson correlations for persistence of phase at long genetic distances detected between Manchado de Jabugo and other Iberian variants is typical for subpopulations of the same breed . Furthere and ROH can be used to address major issues in conservation genetics such as effects of genetic drift and inbreeding [e, for instance through exploring the extent of LD [e for conservation of populations, the estimation of this parameter is remarkably complex [e assumes an ideal population that is isolated, without migration, with random mating and with constant linear population growth [e is widely used. The estimation of recent Ne has been computed using linked [e separately for 1 Mb bins containing information of recombination rates in order to obtain more information of demographic history [e[e[e must be treated with care due to the low sample sizes, especially in those populations with a sample size lower than 15 animals (i.e. Manchado de Jabugo and Retinto).The analysis of Nbreeding . The smabreeding . With thnt of LD . Despite complex . The estn growth . Althougg linked ,42 and ug linked  genetic istory [e it may mtory [e[e. AdditioROH have been used to infer the history of consanguinity in human populations ,45 and chttp://dad.fao.org/), this population did not show signs of high levels of consanguinity, likely because of its admixed origin. Thus, the conservation program currently implemented in Manchado de Jabugo is effective and necessary to assure the future viability of this population. What is also evident, however, is that this management strategy has gone at the expense of the historical genetic distinctiveness of the breed. By contrast, Bisaro showed signs of low consanguinity in agreement with its mixed origin and the strict conservation program implemented in this breed [The existence of Chato Murciano pigs with a high number of long ROH shows the importance of recent inbreeding and thus low individual genetic diversity. Indeed, we observed three Chato Murciano pigs that had more than 45% of the genome covered by ROH, but also pigs with much lower percentage. This observation is consistent with known management strategies of this breed  and in ais breed . Althougis breed . This caet al. [et al. [The agreement between our observations and expectations based on historic reports highlights that analyzing the structure of ROH can aid in assessing levels of current consanguinity, and historic events such as bottlenecks in local pig populations. Furthermore, the assessment of ROH at the individual level has practical implications in conservation programs. Animals displaying high levels of ROH, for instance, could be excluded or given lower priority for breeding purposes in endangered populations. However, it must be taken into account that the 60 K SNP panel applied may underestimate the number of small ROH due to ascertainment bias  and may et al.  and Purf [et al.  concludee is more inaccurate than the estimation ancient Ne owing to the increase in the variability of Ne values as the length of the segment used for the estimation increase [e for wild boar tended to be higher than for domestic populations, with important drops in Ne. Moreover, wild boar had a very high number of short ROH and no long ROH. A high number of short ROH has been related with a reduced population size in the past and low inbreeding in recent times [e of wild boar drastically. Moreover, continuous events of formation of subpopulations and migration between them, favored by the lack of geographic barriers across the Iberian Peninsula, and even occasional admixture with domestic pigs, could have avoided high inbreeding in wild boar populations. Genetic signs of migration between subpopulations of Iberian wild boars have been described in Portugal [The study of demographic history provides a better understanding of the current risk of inbreeding, and might facilitate predicting the effects of future changes in effective population size. Despite the fact that estimation of demography implies the simplification of a complex biological reality, estimation of effective population size based on LD and recombination rate provides useful predictions and consistent comparisons between populations ,49. It mincrease . The estnt times . This pant times , that woPortugal .e between 20,000 and 10,000 generations ago exclusively in wild boar that seems to reflect the sharp population decrease during the Last Glacial Maximum [e. Studies using Next Generation sequence data are needed to support and increase the accuracy of past Ne estimations in Iberian pig.The low recombination rate observed in large parts of the porcine genome essentially allows a much wider window in the past effective population size. Assuming a generation interval of approximately 2 years . We obse Maximum . The inc Maximum ,54. Rece Maximum ,54. In t Maximum  and wild Maximum . On one  Maximum . Admixtust values between wild boar and Iberian pigs provide support that Iberian may have been crossbreeding with wild boar until medieval times [et al.[Structure analysis showed that Iberian pigs contributed to the wild boar genetic stock Figure . This faal times . This iss [et al., who dess [et al..e and ROH in Bisaro indicated high genetic diversity of this breed as a result of its mixed origin and the efforts carried out to preserve this breed. We observed that the Iberian breed may represent a good model to assess genetic signs of past demographic events as domestication, being an additional argument for the need to preserve Iberian pig breed and to avoid crossbreeding with other breeds. Previous evidence supporting the Iberian breed as being closely related to wild boar were confirmed and further evidence was provided for recurrent crossbreeding between these populations in the past. The analysis of wild populations from different regions of the Iberian Peninsula indicates natural migrations of wild pigs across the Iberian Peninsula as well as low levels of inbreeding in Iberian wild boar.This study provides a comprehensive picture of demographic history, population structure and inbreeding of wild and domestic pig populations from the Iberian Peninsula as well as their relevance in conservation genetics. The occurrence of ROH in Chato Murciano was very high in some individuals, which may be due to a recent bottleneck and also highlights the lack of a well-designed genetic management program. Manchado de Jabugo showed a relatively high heterozygosity. This is unexpected given the extremely low census population size, and most likely reflects recent admixture with commercial pig breeds observed in this population. Conservation programs need to be maintained and carefully designed in order to avoid further loss of genetic distinctiveness. The study of NA total of 97 unrelated pig samples were collected from populations of the Iberian Peninsula, and genomic DNA was extracted by standard protocols. The study included 18 wild boars (WB) from different regions of Portugal (n = 11) and Spain (n = 7), and 79 domestic pigs. The domestic pigs utilized for the analysis belonged to three local breeds: Iberian ─including a Retinto Iberian variant , a Negro Iberian variant , an unidentified Iberian variant , and Manchado de Jabugo  ─, Bisaro  and Chato Murciano .Sus scrofa build 10.2 and with less than 5% missing genotypes were included in the analysis.High-density SNP genotyping was performed using the Porcine SNP60 Beadchip  designed to genotype 62,163 SNPs , accordist values between populations were calculated using the Power marker software [Structure v2.0 [[.Allele Sharing Distances were calculated using PLINK v1.07 . Nei’s gsoftware . The paisoftware . The admure v2.0  was usedure v2.0  using thure v2.0 . Moreovee v2.0 [.2vs. distance per chromosome and means plot of r2 in each breed vs. each chromosome were made in R environment (http://www.r-project.org/).Markers significantly deviating from Hardy-Weinberg equilibrium (P < 0.001) and with a MAF lower than 0.05 were excluded from LD analysis using PLINK v1.07 . LD (r2)et al.[To calculate the persistence of phase and the time since two breeds diverged we followed the procedure implemented by Badke et al.. Briefly2 = 1/(4Nec +1) → Ne = (1/4c) * (1/r2 -1), where r2 is the LD, c is the marker distance in Morgans between SNP and Ne is the effective population size [c[e.Effective population sizes were calculated in all populations implementing the equation rion size . Addition size [c. This foe, but this assumption may lead to incorrect estimates of Ne. Recombination rate varies considerably across and within porcine chromosomes [et al.[2 for all possible pairs of SNPs included in each bin. Mean values and standard deviations among bins were subsequently used to estimate past and present effective population size. The approximation of past Ne assumes that c is much larger than the mutation rate (~10-8 per locus and generation) [c < 10-6 where not considered for past Ne estimation.Previous authors  tended tomosomes , to an eomosomes . Insteads [et al.. The effs [et al.. Towardseration)  so bins The software PLINK v1.07  was usedNumber of ROH, total length of ROH and the average of ROH length in each animal were calculated for each chromosome and the mean across animals was estimated for each breed. Those ROH longer than 100 Mbp were categorized as long ROH. The percentage of the total genome length affected by ROH in each animal was also inferred.The data sets supporting the results of this article are included within the article .The authors declare that they have no competing interestsJMHM analyzed the data and wrote the manuscript. HJM designed and conceived this study. MAMG critically review the manuscript. MB designed the ROH analysis and supervised the corresponding writing parting of the manuscript. MPE collected samples and helped draft the manuscript. RPMAC and GRV collected the genetic data and participated in the supervision of the study. All authors read and approved the final manuscript.st pairwise  and Nei’s genetic distances  between populations. Table S2: Average r2 value for SNP spaced 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0.FClick here for fileNeighbor-Joining tree of pig populations constructed from individual pairwise genetic distance. WBPOR, wild boar from Portugal; WBSPN, wild boar from Spain; RE, Retinto Iberian; NI, Negro Iberian, IB, Iberian (unidentified variant); MJ, Manchado de Jabugo; BI, Bisaro; CM, Chato Murciano.Click here for filePCA analysis using exclusively Iberian pigs. IB_Unk, Iberian unknown variant; NI, Negro Iberian; RE, Retinto; MJ, Manchado de Jabugo.Click here for fileSTRUCTURE analysis and Evanno method to determine the optimal number of clusters; Membership coefficient of the breeds tested in the four clusters inferred by STRUCTURE software.Click here for filePersistence of phase for intervals of 100 kb ranging from 0 to 10 Mb.Click here for fileEstimated effective population size (Ne) over time: detail of 10,000 generations ago. WB, wild boar; NI, Negro Iberian; RE, Retinto; MJ, Manchado de Jabugo; BI, Bisaro; CM, Chato Murciano.Click here for fileEstimation of the percentage covered by ROH in each population. WB, wild boar; IB, Iberian; MJ, Manchado de Jabugo; BI, Bisaro; CM, Chato Murciano.Click here for file


Dorylus subgenus Anomma) using morphology, mitochondrial DNA sequences, and nuclear microsatellites.Hybridization can have complex effects on evolutionary dynamics in ants because of the combination of haplodiploid sex-determination and eusociality. While hybrid non-reproductive workers have been found in a range of species, examples of gene-flow via hybrid queens and males are rare. We studied hybridization in East African army ants (Dorylus molestus-like" and a "Dorylus wilverthi-like" form. This pattern is best explained by introgression following hybridization between D. molestus and D. wilverthi. Microsatellite data from workers showed that the two morphological forms correspond to two distinct genetic clusters, with a significant proportion of individuals being classified as hybrids.While the mitochondrial phylogeny had a strong geographic signal, different species were not recovered as monophyletic. At our main study site at Kakamega Forest, a mitochondrial haplotype was shared between a "D. molestus and D. wilverthi has occurred, and that mating between the two forms continues to regularly produce hybrid workers. Hybridization is particularly surprising in army ants because workers have control over which males are allowed to mate with a young virgin queen inside the colony.We conclude that hybridization and gene-flow between the two army ant species  While botanists have long accepted that hybridization plays an important role in plant evolution and regularly leads to the emergence of new species , zoologiPogonomyrmex harvester ants, where purebred females become queens and interlineage hybrids become workers [Formica wood ants with two clearly distinct male genepools and hybrid queens [In haplodiploid eusocial animals such as ants, the consequences of hybridization can be strikingly different from those in other organisms. First, a diploid queen that has mated with a heterospecific haploid male will still produce purebred sons via arrhenotokous parthenogenesis, so that hybrid males will only be produced in the F2 generation. Second, potentially negative consequences of hybrid sterility can be mitigated or even avoided when hybrid individuals mostly or exclusively become non-reproductive workers ,9. Hybri workers ,14. Anotd queens . This stDorylus (Anomma) molestus and Dorylus (Anomma) wilverthi, occur in sympatry. African swarm-raiding army ants (the "driver ants") are a prominent feature in afrotropical forests, where they are prime invertebrate predators with colony sizes of over ten million individuals. They form a well-supported clade within the Dorylus subgenus Anomma [In the present study we investigated possible hybridization between different species of African swarm-raiding army ants, with particular focus on Kakamega Forest, Kenya, which is one of the few sites where the two closely related species, s Anomma . Like ins Anomma . Becauses Anomma ,19.Dorylus colonies are headed by a single queen (monogynous), but due to the high queen-mating frequency, most workers in a colony are half-sisters, i.e. they have the same mother queen but different fathers [An interesting consequence of this idiosyncratic reproductive system is that males have to "run the gauntlet of the workers" before they can mate, so that workers have ample opportunities to choose the mates of their queen-sister . If her  fathers .D. wilverthi being essentially restricted to intact rainforest, while D. molestus also occurs in savannah habitats and open agricultural landscapes [Dorylus (Anomma) colonies [D. sjoestedti var. sjoestedti-wilverthi [Previous work showed that the two species at Kakamega occupy clearly distinct ecological niches, with ndscapes -23. Our colonies , and witilverthi .Dorylus workers from Kakamega. Our results allow novel inferences about the evolutionary relevance of hybridization in social insects.Here we use a combination of morphological, mitochondrial and nuclear genetic data to show that hybridization and introgression have occurred historically between different species of swarm-raiding army ants at a large geographic scale in East Africa, and that ongoing hybridization can be detected in D. wilverthi-like workers from Kakamega  and one D. wilverthi population (Kakamega Forest). None of the pair-wise tests for genotypic disequilibrium between microsatellite markers was significant . Pair-wise estimates of genetic differentiation between four analysed populations at nuclear microsatellites (STF) are given in Table ST Fbetween the D. wilverthi-like and the D. molestus populations were slightly but consistently higher than estimates between the different D. molestus populations  than in the D. molestus populations . Interestingly, the D. wilverthi-like population was close to fixation  for a haplotype that was also common in the sympatric D. molestus-like population at Kakamega  and was closely related to the other local D. molestus haplotypes , although a model with five subdivisions had very similar likelihood scores . The four clusters corresponded well to the four populations, although a significant proportion of individuals had reasonably high likelihoods of ancestry in a population other than their source population , STRUCTURE consistently recovered one cluster consisting of the two Kakamega populations and another cluster consisting of D. molestus samples from Tana River and Mt. Kenya, rather than combining the three D. molestus populations. At k = 3, STRUCTURE split the two Kakamega populations, and at k = 5 the Tana River + Mt. Kenya samples generally formed three clusters, while the two Kakamega clusters were retained (results not shown).Replicate runs in STRUCTURE were highly consistent in every analysis. The highest likelihood scores were associated with a model with four subdivisions , and one D. molestus-like worker from Kakamega was assigned to the D. wilverthi-like population. Similarly, two D. molestus workers from Mt. Kenya were assigned to the Tana River population, and six workers from Tana River were assigned to the Mt. Kenya population. No other cross-assignments were found.Results from a GENECLASS2 analysis were fully consistent with the results obtained from STRUCTURE. Five D. molestus and D. wilverthi from allopatric populations, but are also significantly different from "pure" D. molestus and D. wilverthi with intermediate phenotypes for several key characters  species are not generally recovered as monophyletic. Below we argue that this finding is best explained by the combined effects of unrecognized cryptic diversity within D. molestus and a complex history of mitochondrial introgression in the Lake Victoria region. D. molestus haplotypes tend to cluster into several well supported shallow clades, which are connected to other such clades by deeper branches , so that this overall pattern is suggestive of unrecognized cryptic diversity within D. molestus.While mitochondrial haplotypes are clearly structured according to geographic origin Figures  and 4, ds Figure . FurtherD. wilverthi-like form is close to fixation for a haplotype that is also frequent in the D. molestus-like form. This haplotype is nested within the clade of Kakamega D. molestus haplotypes , might ns Table .D. molestus clade. This strong genetic structure at the maternally inherited mitochondrial locus, and a lack thereof at biparentally inherited nuclear loci, stems from the fact that the permanently wingless army ant queens cannot cross water barriers, while the winged males readily do so [Another noteworthy case of apparent mitochondrial introgression was found in the population from Tana River, where haplotypes from both sides of the river tend to cluster in clearly distinct clades Figures  and 4 ally do so .Dorylus (Anomma) species. Under incomplete lineage sorting, the minimum divergence time between sequences is given by the time elapsed since speciation [cytochrome oxidase II haplotypes, which have high mutation rates of ca. 1.5% per million years in insects . On theeciation . In the ts (e.g. ). Given D. molestus and D. wilverthi.The two morphological forms at Kakamega Forest can be clearly distinguished based on the nuclear microsatellite loci. This again shows that species boundaries have not become completely blurred, which would be expected if gene flow had been significant over extended periods of time. On the other hand, despite genetic differentiation among species being consistently higher than between conspecific populations Table , a largeDorylus (Anomma) species. This is one of the few cases of documented gene flow between species of eusocial Hymentoptera ([Dorylus (Anomma) [Dorylus queens and males, to clarify the precise pattern of hybridization and the amount of ongoing gene-flow between the two sympatric forms.This study provides conclusive evidence that introgressive hybridization has occurred between different toptera  ,44. The (Anomma) , might eDorylus (Anomma) army ants, we collected 151 colony samples  for mitochondrial DNA sequencing from 24 localities, representing four morphologically distinguishable species, D. molestus, Dorylus sjoestedti, Dorylus terrificus, and D. wilverthi , the eastern slope of Mt. Kenya , and Lower Tana River . Subsets of these samples were also sequenced for mitochondrial DNA (see Table Samples for microsatellite genotyping (one worker per colony) were collected from four populations at three different sites in Kenya: Kakamega Forest  swarm-raiding army ants . Importantly, D. wilverthi and D. molestus workers are easily distinguished: D. wilverthi workers have the posterior angles of the head uniquely prolonged into a raised, slightly outwardly recurved point  on the petiole , D. wilverthi-like individuals from Kakamega (N = 45 workers from nine colonies), "pure" allopatric D. molestus (N = 157 workers from 31 colonies belonging to 20 populations across the species' range), and "pure" allopatric D. wilverthi (N = 85 workers from 16 colonies belonging to 10 populations across the species' range). Measurements were taken with a MS5 Leica stereomicroscope fitted with an ocular micrometer. Data were analysed following Schöning et al. [We identified samples morphologically using the diagnostic criteria given in the original species descriptions and in . Furtherrding to ,46, and e Figure  and signilverthi ,48. To qg et al. .® (DNeasy®) and MACHEREY-NAGEL (Nucleo Spin®Tissue) kits. A mitochondrial fragment of the cytochrome oxidase II (COII) gene was amplified and sequenced using primers tRNALeu [COII sequence. GenBank accession numbers and details for sequenced samples are given in Table DNA for sequencing was extracted from 1-2 worker legs using standard QIAGEN tRNALeu  and Barb tRNALeu  as has b tRNALeu . SequencDNA for microsatellite genotyping was extracted by boiling 1-2 worker legs in 100 μl of 5% Chelex 100 (Bio-Rad). A total of 202 individuals were genotyped at five microsatellite loci  as has been described previously .Phylogenetic analyses of mitochondrial sequences were conducted in a Bayesian framework using the program MrBayes 3.1.2 ,52, and 6 generations and trees were sampled every 100 generations. Average standard deviations of split frequencies were consistently < 0.01 after 13*106 generations, indicating that the independent runs had converged. Accordingly, the first 130,000 trees from each run were discarded as burn-in. Based on this sample of 140,002 trees from both runs combined, all potential scale reduction factors for model parameters were < 1.02, indicating effective sampling from the posterior probability distribution. A consensus phylogram with posterior probabilities based on both runs was calculated in MrBayes.To assure convergence of Markov Chain Monte Carlo runs in MrBayes we repeated the analysis from two independent starting trees. One cold and three heated chains were run in parallel for 20*10We initially performed three independent runs in GARLI setting the number of generations after which the run is terminated if no new significantly better scoring topology has been found to 30,000, and otherwise using default parameter settings. We then performed a run with 1,000 bootstrap replicates setting the above parameter at 10,000.D. molestus, D. wilverthi and D. terrificus was constructed using a statistical parsimony approach [A mitochondrial haplotype network for all East African haplotypes of approach  implemenapproach . The conaN), expected heterozygosity (sH), allelic richness corrected for sample size (sR), and the inbreeding coefficient IS Fwere estimated for each population using the program FSTAT 2.9.3.2 [IS Fwas tested for significant deviation from zero using 400 randomisations to assess Hardy Weinberg equilibrium. The same program was used to test for genotypic disequilibrium between all pairs of microsatellite loci within each population (Table ST Fas an estimate of genetic differentiation at microsatellite loci between populations and species. FSTAT was also used to calculate a Nand s Rfor mitochondrial haplotypes.The number of alleles per microsatellite locus ( 2.9.3.2 . FIS wasD. molestus and D. wilverthi at Kakamega. First, the clustering method of the program STRUCTURE 2.2 [k, the number of subdivisions, ranging from one to 10, under an admixture model with correlated allele frequencies [6 generations and the first 105 generations were discarded as burn-in. Estimates of hybrid frequencies were based on individual probabilities of having ancestry in populations other than the source population. The comparisons between conspecific populations were used as a control to assure that the analyses had sufficient power in assigning individuals to different clusters given the observed levels of genetic differentiation between populations. More recent and potentially ongoing gene-flow between conspecific populations should generally result in more ambiguous assignments of individuals to populations as compared to genetically isolated heterospecific populations, irrespective of whether these occur in sympatry or not. We used the program DISTRUCT 1.1 [We used two different model-based Bayesian methods to study hybridization between TURE 2.2  was usedquencies . Markov RUCT 1.1  to graphet al. [Second, we used the assignment methods in the program GENECLASS2  to estimet al. . The numThe authors declare that they have no competing interests.DJCK, MKP, CS, JJB designed the study an acquired funding. MKP and CS collected all army ant samples and identified them. DJCK and MKP conducted the molecular genetic studies and analysed the data. CS conducted the morphometric analyses. DJCK wrote the first version of the paper. All authors contributed to and approved the final manuscript.DJCK is the head of the Laboratory of Insect Social Evolution at The Rockefeller University. He is broadly interested in the evolution of social insects, and army ants are his main study systems. MKP is a postdoctoral ecologist at the Department of Animal Ecology and Tropical Biology, University of Würzburg, Germany. He studies the ecology and conservation of tropical rainforest animals. CS is a postdoctoral fellow at the Länderinstitut für Bienenkunde in Hohen Neuendorf, Germany, where he presently works on honeybees. He has a long-standing interest in the ecology and evolution of African army ants. JJB is the director of the Centre for Social Evolution at the University of Copenhagen.