Rhodnius nasutus, a vector of the etiological agent Trypanosoma cruzi, is one of the epidemiologically most relevant triatomine species of the Brazilian Caatinga, where it often colonizes rural peridomestic structures such as chicken coops and occasionally invades houses. Historical colonization and determination of its genetic diversity and population structure may provide new information towards the improvement of vector control in the region. In this paper we present thoughtful analyses considering the phylogeography and demographic history of R. nasutus in the Caatinga.R. nasutus specimens were collected from Copernicia prunifera palm trees in eight geographic localities within the Brazilian Caatinga biome, sequenced for 595-bp fragment of the mitochondrial cytochrome b gene (cyt b) and genotyped for eight microsatellite loci. Sixteen haplotypes were detected in the cyt b sequences, two of which were shared among different localities. Molecular diversity indices exhibited low diversity levels and a haplotype network revealed low divergence among R. nasutus sequences, with two central haplotypes shared by five of the eight populations analyzed. The demographic model that better represented R. nasutus population dynamics was the exponential growth model. Results of the microsatellite data analyses indicated that the entire population is comprised of four highly differentiated groups, with no obvious contemporary geographic barriers that could explain the population substructure detected. A complex pattern of migration was observed, in which a western Caatinga population seems to be the source of emigrants to the eastern populations.A total of 157 R. nasutus that inhabit C. prunifera palms do not comprise a species complex. The species went through a population expansion at 12–10 ka, during the Holocene, which coincides with end of the largest dry season in South America. It colonized the Caatinga in a process that occurred from west to east in the region. R. nasutus is presently facing an important ecological impact caused by the continuous deforestation of C. prunifera palms in northeast Brazil. We hypothesize that this ecological disturbance might contribute to an increase in the events of invasion and colonization of human habitations. Trypanosoma cruzi. Although new community-based ecosystem management  initiatives have been implemented, vector control based on insecticide-spraying of households remains one of the most effective strategies to diminish parasite transmission to humans. However, this strategy is not sustainable where native triatomine species are capable of colonizing peridomestic structures and invading human dwellings. The application of molecular markers with the potential of recovering both historical and contemporary information on vector population structure and diversity can improve the understanding of vector dissemination and thus contribute to the development of better disease control strategies. In this study we analyzed Rhodnius nasutus populations endemic to the Brazilian Caatinga biome using two sets of markers: a fragment of the mitochondrial gene cytochrome b and eight nuclear microsatellite loci. The information generated and described herein is important as it may contribute to the advancement of our understanding of Chagas disease vector ecology and phylogeography.Chagas disease is endemic to Latin America and the Caribbean and it is estimated that 6–7 million people are infected with the etiological agent  Trypanosoma cruzi  and transmitted primarily through the feces of infected triatomine bugs  [Chagas disease is caused by the protozoan uviidae) . Endemicuviidae) .T. cruzi-infected triatomines continuously invade houses in both rural and urban areas maintaining the risk of transmission [Although curable when treated early with antiparasitics in the acute phase, there is no vaccine available and treatment of chronic patients only reduces serum parasite detection, but not cardiac clinical complications . Therefosmission , 8.Rhodnius nasutus Stål, 1859 is one of these species, frequently reported in bird and mammal nests on the crown of Copernicia prunifera (Carnaúba) palm trees in the vicinity of human habitations. It is often naturally infected with the T. cruzi parasite and is capable of invading rural and urban houses and colonizing peridomestic structures [Attalea speciosa, Mauritia flexuosa, Syagrus oleracea and Acrocomia intumescens palm trees [R. nasutus has also been reported to occur in Licania rigida (Oiticica) dicotyledon trees typical of northeast Brazil [The Caatinga and the Cerrado biomes together, harbor most of the triatomine species diversity in Brazil . Of the ructures , 11. It lm trees , 13. Intt Brazil , 14.R. nasutus population structure in the Caatinga biome and estimation of present gene flow may contribute to a better understanding of the species' historical colonization process as well as provide new information towards the improvement of vector control in the region. In this paper we present the phylogeography and demographic history of this Chagas disease vector in the Caatinga inferred with mtDNA (cyt b) and nuclear (microsatellites) markers.Determination of Rhodnius specimens were collected after the inspection of 53 Copernicia prunifera palms in eight geographic localities within the Brazilian Caatinga biome  and were carried out following Gurgel-Gonçalves et al. [Rhodnius nasutus specimens were identified morphologically based on Lent and Wygodzinsky [Insect field captures were approved by the s et al. . Brieflyodzinsky  and confodzinsky . Sequencet al. [Rhodnius robustus I-V, R. prolixus, R. neglectus, R. nasutus and R. barretti) and analyzed to confirm species identity based on genetic distances using Mega v. 5 [Rhodnius sequences available in the GenBank public database through the Basic Local Alignment Search Tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi), considering the following parameters: percentage of identity and coverage, e-value, and also Bayesian phylogenetic analyses.DNA extraction was performed using the Promega Wizard Genomic DNA extraction kit  following the manufacturers recommendations. A 682-bp fragment of the cyt b gene was PCR-amplified as described in Monteiro et al. . Amplicoet al. ). Both Dega v. 5 . TaxonomR. nasutus and the other members of the ‘robustus lineage’ was inferred in BEAST v. 1.8 [Rhodnius barretti [9 generations, sampling every 20,000 generations. Convergence of parameters and proper mixing were confirmed through the calculation of effective sample sizes (ESS) in Tracer v. 1.6 [4 were considered appropriate [A Bayesian phylogenetic cyt b tree based on the 147 samples of morphologically-identified T v. 1.8 . Rhodniubarretti  was usedbarretti . We testbarretti . Three ir v. 1.6 , and ESSropriate .Rhodnius sp.”. Suggested divergence rate for triatomines of 1.1 to 1.8% per Myr was used as a prior with normal distribution [Gene tree and species tree were reconstructed with *BEAST . Informaribution .R. nasutus cyt b haplotypes. Since samples from Jaguaruana were collected in 2004 and all other samples in 2010, we tested whether this could have an impact in our study by carrying out an analysis of variance (AMOVA) grouping samples according to the collection date with Arlequin v. 3.5 [Molecular indices of haplotype diversity (Hd) and nucleotide diversity (π) were computed in DnaSP v. 5  for eachn v. 3.5 .FST comparisons [2 to evaluate the fit of data to theoretical models of distribution of genetic distances under different demographic scenarios was calculated in the R environment [Levels of genetic differentiation among populations were determined with Wright’s pairwise parisons  in Arleqparisons . Correctparisons  under 5%ironment . Deviatiironment  and Tajiironment  tests inironment . Signifiironment .R. nasutus. The best model was selected through the comparison of marginal likelihood, calculated with the path sampling algorithm [6 MCMC chain length and 60 steps, based on Bayes Factors (BF) [7 generations with the first 2 x 106 discarded as burn-in and parameter values sampled every 2 x 103 generations) were analyzed with Tracer v. 1.6 [4 were considered appropriate [R. nasutus demographic history to paleoclimatic events estimated through oxygen isotope marine paleotemperature records [We also used Beast v. 2.4  to reconlgorithm  under 10ors (BF) . In all r v. 1.6 . Convergropriate . Becauseropriate . We matc records , used asTaqGold DNA polymerase, 2 mM of each dNTP, 1.5 mM MgCl2, 10 mM of magnesium free Buffer 10X , 5 pmol of each primer and approximately 10 ng of extracted DNA. PCR conditions were as follows: 1 min at 95°C, 30 cycles of 30 s at 94°C, 30 s at Ta °C and 45 s at 72°C, and 72°C for 30 min; Ta is the annealing temperature for each locus  [All PCR products were run with a size standard GS500 LIZ on an ABI 3730xl Genetic Analyzer, and allele fragment lengths quantified using the Peak Scanner software v. 1.0 . Because these primers are heterologous (designed to amplify microsatellite loci of other species) –46, the SHA) as a measure of gene diversity were performed with GenAlEx v. 6.5 [Microsatellite genotypes were screened for likely scoring errors, allele dropout, and presence of null alleles with Micro-Checker v. 2.2 . Number x v. 6.5 . Deviatix v. 6.5 .FST) between sampling localities and inbreeding coefficients (FIS) were carried out with Arlequin v. 3.5 software [FST was non-significant were considered as belonging to the same population. Mutual index (SHUA) [Genetic differentiation between populations (pairwise software . Samplinx (SHUA)  were alsx (SHUA) .admixture model due to the lack of information about the ancestry of the field-collected specimens, with correlated allele frequencies, which means that these frequencies in different populations are likely to be similar as a consequence of migration or shared ancestry [5 iterations and 106 Markov Chain Monte Carlo (MCMC) generations, respectively. Ten independent runs were performed for each value of K (for 2–8), as suggested by Pritchard et al. [The Bayesian clustering program Structure v. 2.3  was usedancestry . Burn-ind et al. . The mosd et al. .The Ewens–Watterson neutrality test , which rR. nasutus. Basically, this analysis reconstructs a minimum-spanning tree based on the pairwise FST matrix and calculates three different parameters: (a) degree, which is defined as the number of connections a node has in the network, summarizing which populations are exchanging migrants; (b) clustering, which measures the number of subpopulations within a population; and (c) betweenness, which is the number of shortest paths between populations, reflecting areas of intense gene flow. These measures allow identifying “source” and “sink” populations (i.e. those with the highest degree and betweenness) [We also constructed a population network with EDENetwork v. 2.1  to detereenness) .FST results) representing different patterns of migration. Migrate-n analysis was performed with a single long cold chain and four hot swapping chains using a static heating scheme , with two independent runs, sampling at every 100th step for a total of 2 x 105 recorded steps (burn-in = 3 x 104 steps). The 10 migration models were compared based on their marginal likelihood and probability using thermodynamic integration with Bezier approximation (implemented in Migrate-n) according to Beerli and Palczewski [To investigate the patterns of gene flow between the populations defined by Structure we performed a Bayesian approach implemented in Migrate-n v. 3.2 . Parametlczewski . The plolczewski  using Milczewski . The miglczewski .FST values) [Isolation by distance was estimated for cyt b and microsatellite data through linear regression analyses of dependence, comparing the logarithms of genetic distances  , and geo values) . The sigC. prunifera palm trees investigated (62%) were infested with Rhodnius specimens. Higher infestation percentages were detected in deforested areas  and thus were discarded from the analysis. Therefore, our dataset included 147 cyt b sequences of 595-bp long from Caatinga .R. nasutus “reference” sequence (JX273155) generated by our group [R. neglectus is not known to occur [Pairwise comparison of the sequences obtained with those available in GenBank revealed 99–100% identity with another ur group . This seto occur .Rhodnius specimens collected formed a monophyletic and well-supported clade with the R. nasutus reference sequence (PP = 1.0), corroborating further their taxonomic identity. A bayesian coalescent gene tree disclosed the short branches (i.e. low sequence divergence) among R. nasutus samples. Although posterior probabilities for R. nasutus clades were low (PP < 0.9), it is noticeable that only the R. nasutus sequences from Carnaúba dos Dantas and Sousa clustered in separate clades without the presence of at least one sequence from another locality. Sequences from the other six localities clustered together in other three different clades.Hasegawa-Kishino-Yano (HKY ) was selST = 0.18, P = 0.98). Therefore, these samples were included in further analyses. Molecular divergence of R. nasutus cyt b sequences varied between 0%-0.8%  unique haplotypes.AMOVA analysis between Jaguaruana and the other populations did not indicate any sampling effect of collecting at different times as genetic differences between these groups were not significant , neutrality tests, mismatch distribution and BSP analyses were performed with the entire dataset. Mismatch distribution analysis (2 for population growth model is 0.95 (p = 0.008) while for constant population size model is 0.72 (p = 0.160)). Neutrality tests did not indicate significant departures from neutrality . Bayesian analyses of population growth revealed that marginal likelihood for constant population was -1240.82 and for exponential growth was -1228.27. Thus, the demographic model with the best fit was the exponential growth model (BF = 25.11).Considering that all analysis  exhibitsDemographic history of the species  depicts R. nasutus specimens collected in the Caatinga biome.Eight microsatellite loci  were anaThe number of alleles per locus ranged from 1 (R8) to 14 (List14-064). Since the R8 locus was monomorphic for all individuals, it was excluded from the analysis.The basic statistics for the microsatellite data is summarized in R. nasutus specimens from Jaguaruana. In addition, positive FIS values were significant in Group 2, which comprised Jaguaruana specimens.Among 155 individuals, 111 had unique genotypes: ALT+CAM– 22; PAR– 18; PIR– 8; CAR– 17; JAG– 25; STA– 9; SOU—15. There are two shared genotypes between CAR and JAG, one genotype shared between STA and JAG, one shared between PAR and PIR, and another shared between PAR and SOU. The highest number of shared genotypes was observed among R. nasutus in Altos (ALT), Campo Maior (CAM), Parnaíba (PAR), and Piracuruca (PIR). Group 2 is the most numerous and genetically variable and contains samples from Jaguaruana (JAG) and Carnaúba dos Dantas (CAR). Group 3 includes specimens from Serra Talhada (STA), and Group 4 is composed of specimens from Sousa (SOU). These groups were in accordance with the pairwise FST comparisons among sequences from different localities. Pairwise FST values between sampling localities  describing genetic variation in population was shown in Group 1 (0.955). Group 1 also displayed the highest number of private alleles (10) over all loci. The other groups had a smaller number of private alleles: four private alleles were found in Groups 2 and 4, and only two private alleles were found in Group 3 , 3–4  and 1–2 . The lowest values of FST were obtained for groups 1 and 3, although the lowest values of SHUA were indicated between groups 2 and 3. Also, relatively low values of FST and SHUA were found between groups 1 and 4 .Genetic differentiation based on the  program  exhibiteR. nasutus specimens from Group 1. One-tailed Wilcoxon sign-rank test for heterozygote excess indicated that each group was in mutation-drift equilibrium for all mutation models evaluated: IAM, TPM, and SMM (P > 0.05).Results of the Ewens-Watterson test indicated neutral evolution of all loci in all groups, but in Group 1, in which neutrality was rejected in locus L43 as observed homozygosity values were lower than expected . BecauseEDENetwork analysis revealed strong connections between Groups 1 and 3 and 1 and 4. Groups 2 and 3 were also connected. We did not observe any indication of substructuring within groups (Clustering = 0). Groups 1 and 3 were identified as possible source-sink populations based on the network analysis. These groups exhibited the highest connection values  and betweenness, which indicate that they are exchanging migrants with other groups .P) for the 10 different models representing various scenarios of migration in Migrate-n revealed support for the 7th model  than among populations within groups  or between groups . The latter non-significant value of AMOVA statistic thus indicates gene flow between west and east groups.Two of seven microsatellite loci  had complex repetition motifs that did not fit a SMM. Although List14-064 and List14-021 were reported with the same dinucleotide repeat motifs [th model . Migratin” model  revealed2 = 0.05, P = 0.258). However, a weak correlation was observed between microsatellite-based FST distances and geographic distances , suggesting that distance contributes ~29% to the shaping of genetic diversity [Linear regression analysis  did not iversity .R. nasutus specimens collected from C. prunifera palm trees throughout most of the Caatinga biome. The pattern of genetic diversity found with the analyses of seven microsatellite loci and cytb sequences suggests that the colonization of the Caatinga by R. nasutus took place from west to east through the region . Therefore, we can hypothesize that restricted gene flow in the Caatinga region might result from human modifications, since the main sylvatic ecotope of this species, C. prunifera palm trees, has been reduced significantly due to continuous deforestation [C. prunifera palm trees are necessary to adequately address this hypothesis.The genetic structure revealed by microsatellite analyses Figs  and 5 ins >0.25;  well aborgentina , 65. Alt species  for a diestation . Biodiveestation . Future R. nasutus groups from eastern Caatinga  evidenced by the low number of alleles present (< 5) in the majority of the loci analyzed, and low heterozygosity levels. Reduced diversity commonly occurs in endemic species which have low effective population sizes caused by habitat fragmentation [FIS = 0.122, P = 0.018).Reduced genetic diversity was detected in the three entation . In thisentation . Indeed,SHA (0.955), further indicating its high variability. Two loci display heterozygote excess (L43 and List14-021), which might result from isolate-breaking (when two previously isolated populations are mixed [Although two loci are monomorphic (List14-025 and R31), Group 1 from western Caatinga presents the highest heterozygosity levels. Moreover, this group has the highest Shannon’s allele information index re mixed ). It is R. nasutus populations analyzed. However, this result should be interpreted with caution, since the small sample size of some locations could prevent proper testing of this hypothesis [Evidence for departures from neutrality and bottleneck events were not detected in the pothesis . Moreovepothesis , (ii) arpothesis , or  and human activity.Unidirectional gene flow may be explained by a decrease in capacity at the source habitat and availability of expansion potential at the target area . The nicFST comparisons corroborate the latter hypothesis, as 24 out of 28 values are high (> 0.6) and significant , which could indicate high gene flow among populations or reflect retention of ancestral polymorphism . The remnificant .Populations can become structured quickly during a range expansion event as a consequence of the colonization of newly available patchy areas by pioneering groups with reduced population sizes . In the R. nasutus expanded from west to east throughout the Caatinga in the last 12–10 ka. Molecular diversity indices showed high haplotype diversity (Hd) and low nucleotide diversity (π), which indicates that despite the high number of different haplotypes per population, they only differ by 1–5 nucleotides. This observation is consistent with expansion of source populations with small effective size [R. nasutus is the exponential growth (BF = 25.11), evidenced by a 5-fold increase in the species population size in the Holocene  [The demographic history of l epochs . Based o4–12 ka) .R. nasutus went through a population expansion 12–10 ka, during the Holocene. This expansion is consistent with global climate changes , thus corroborate the hypothesis of an isolate-breaking effect.The genetic structure of bly Figs  and 4. Cbly Figs . AlthougIncongruent results may also be explained by different evolutionary mechanisms  and intrinsic biological peculiarities of triatomines . Poor accordance between population genetic structures inferred from mitochondrial and microsatellite markers have also been reported for various animal groups, including mammals , amphibiR. nasutus was able to recently disperse within the Caatinga in response to favorable climatic events and environmental conditions. The species is presently facing an important ecological impact caused by the continuous deforestation of C. prunifera palm trees. Perhaps this ecological disturbance is contributing to the observation that R. nasutus now colonizes other species of palms , which raises the risk of T. cruzi transmission to humans.umescens , 13) and. rigida ). These wellings , 11, 86.R. nasutus to increase its population size when environmental conditions are favorable. Therefore, closer attention should be paid to the distribution of R. nasutus, as it can maintain T. cruzi transmission within the Caatinga. Given the limitations inherent to insecticide spraying such as continuous re-infestation of insecticide-treated households by abundant native vectors, molecular studies focusing on the distribution patterns and demographic trends of sylvatic triatomines should be conducted in order to further improve the effectiveness of Chagas disease control.Demographic history data indicates the capacity of S1 Table(DOCX)Click here for additional data file.S2 TableN–number of sequences analyzed.(DOCX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 Fig(JPG)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.


NPR2, HINT2, SPAG8, INSR), development of limbs and skeleton, and tail formation , embryonic development of tendons, bones and cartilages , regulation of body temperature (TRPM8), body weight and height variation (DIS3L2), control of lipogenesis and intracellular transport of long-chain fatty acids (FABP3), the occurrence and morphology of horns (RXFP2), and response to heat stress (DNAJC18). Our findings suggest that Ethiopian fat-tail sheep represent a uniquely admixed but distinct genepool that presents an important resource for understanding the genetic control of skeletal growth, fat metabolism and associated physiological processes.Variations in body weight and in the distribution of body fat are associated with feed availability, thermoregulation, and energy reserve. Ethiopia is characterized by distinct agro-ecological and human ethnic farmer diversity of ancient origin, which have impacted on the variation of its indigenous livestock. Here, we investigate autosomal genome-wide profiles of 11 Ethiopian indigenous sheep populations using the Illumina Ovine 50 K SNP BeadChip assay. Sheep from the Caribbean, Europe, Middle East, China, and western, northern and southern Africa were included to address globally, the genetic variation and history of Ethiopian populations. Population relationship and structure analysis separated Ethiopian indigenous fat-tail sheep from their North African and Middle Eastern counterparts. It indicates two main genetic backgrounds and supports two distinct genetic histories for African fat-tail sheep. Within Ethiopian sheep, our results show that the short fat-tail sheep do not represent a monophyletic group. Four genetic backgrounds are present in Ethiopian indigenous sheep but at different proportions among the fat-rump and the long fat-tail sheep from western and southern Ethiopia. The Ethiopian fat-rump sheep share a genetic background with Sudanese thin-tail sheep. Genome-wide selection signature analysis identified eight putative candidate regions spanning genes influencing growth traits and fat deposition ( Several candidate genes with possible links to fat-tail development, i.e., HOXA11, BMP2, PPP1CC, SP3, SP9, WDR92, PROKR1, and ETAA1, were identified using genome scans that contrasted fat- and thin-tail Chinese sheep  genetic relationships and structure within and between Ethiopian indigenous sheep of different fat-tail morphotypes alongside other sheep populations and breeds from the Caribbean, European, Middle East, China and Africa, and (ii) candidate genome regions and genes associated with tail morphology, fat deposition and possible eco-climatic adaptation in African indigenous sheep. For the latter, 11 Ethiopian indigenous sheep of different fat-tail morphotypes and two populations of thin-tail sheep from Sudan were analyzed.www.mn-net.com) following the manufacturers protocol. All 166 genomic DNA samples were genotyped using the Ovine 50 K SNP BeadChip assay. The assay includes 54,240 SNPs composed of 52,413 autosomal, 1449 X-chromosome and 378 mitochondrial SNPs, respectively.The sampling strategy targeted breeds of indigenous sheep from different geographic regions in Ethiopia Table . Geographttp://www.sheephapmap.org/hapmap.php, Supplementary Table Ovine 50 K SNP BeadChip genotypes of Caribbean, European, Middle East and Chinese, as well as western, northern and southern African sheep, respectively were obtained from the Sheep HapMap database , expected (He), and observed (Ho) heterozygosity and inbreeding coefficient (F) were estimated for each population and across all populations using PLINK v1.9, to evaluate the levels of genetic diversity present in Ethiopian and Sudanese sheep, respectively.The proportion of polymorphic SNPs (K) and proportions of genome ancestry that was shared among the study populations.Principal component analysis (PCA) were performed using PLINK v1.9 to investigate the genetic structure and relationships among the studied breeds based on genetic correlations between individuals. A graphical display of the first two principal components (PC1 and PC2) was generated using GENESIS  within and between Ethiopian and Sudanese populations, we used the maximum likelihood tree-based approach implemented in TreeMix  were to determine if A was derived from the admixture of populations B and C; a significantly negative value of the f3-statistics would suggest population A is admixed. The f4-statistics   were to test the validity of hierarchical clustering pattern in four-population trees. Significant deviations of the f4-statistics from zero for the three possible topologies of the four-population trees would provide evidence of gene flow between the populations tested. A significantly positive Z-score indicates gene flow between populations that are related to either A and C or B and D while a significantly negative Z-score indicates gene flow between populations that are related to A and D or B and C. Standard errors were estimated using blocks of 500 SNPs.The FST, hapFLK and Rsb.For this analysis, we separated 12 of the 13 Ethiopian and Sudanese populations into four genetic groups based on the population clusters revealed by PCA. The four population groups included, western  and southern  long fat-tail, and fat-rump  sheep from Ethiopia and thin-tail sheep  from Sudan. One short fat-tail sheep  was included with the fat-rump sheep and the other (Gafera), which appeared to be genetically distinct, was dropped from further analysis. Equal numbers of samples were chosen at random to represent each genetic group. Three comparisons which contrasted the fat-rump (E1), western (E2) and southern (E3) long fat-tail sheep with the thin-tail sheep (S) from Sudan were performed. The selection signature analysis involved three approaches, FST analysis using the HIERFSTAT package . The hapFLK statistic was then computed as the average value across 40 expectation maximization (EM) runs to fit the LD model. The P-values were obtained by running a python script “Scaling_chi2_hapFLK.py” available at (https://forge-dga.jouy.inra.fr/documents/588) which fits a chi-squared distribution to the empirical distribution. As with the FST calculations, the hapFLK statistics were also standardized using the formula:The P-values was based on the null distribution of empirical values  ≥ 3 −0, 5)]), where Φ(x) represents the Gaussian cumulative distribution function , the PRsb can be interpreted as –Log10 , where P is the two-sided P-value associated with the neutral hypothesis. For each comparison, SNPs that exhibited PRsb ≥ 3  were taken to be under selection  and OMIM databases (http://www.ncbi.nlm.nih.gov/omim/) and a review of literature.Candidate regions that overlapped between Pn, He, Ho, and F, as indicators of within-breed genetic diversity, are shown in Table Pn, He, and Ho were observed in Bonga while the highest values were observed in Molale-Menz, Hammari and Kabashi, and Arabo, respectively.The average values of The PCA plot incorporating the global populations and which was constructed using a sample size of five animals that were selected at random per population, is shown in Figure To obtain a clearer picture of the variation within the fat-tail sheep, we performed the PCA excluding the thin-tail sheep Figure . PC1 sepTo further illustrate the distribution of genetic variation among East African populations, we performed the PCA with only the Ethiopian and Sudanese thin-tail sheep Figure . PC1 sepK = 9 suggesting this to be the most optimal number of clusters explaining the variation in this dataset  and the West African Djallonke show a distinct but common genetic ancestry with the Ethiopian and Sudanese sheep for 3 ≤ K ≤ 6.Admixture analysis on the global dataset, separates the study populations following their geographic origins Figure . The croK) were tested with Admixture on the East African dataset. The lowest cross-validation error suggests K = 4  accounted for by the model covariance matrix (W) was used to identify the information contribution of each migration vector added to the tree. Up to 15 possible migration vertices were computed. The first eight migration edges (gene flow) accounted for more than half of the total model significance explained by the f statistic, with the first migration edge having a f value of 0.51. We therefore chose m = 8 as the best predictive value for the migration model. Vectors from 9 to 15 resulted in only small incremental changes in the f value  were observed between Hammari and Kabashi (thin-tails) and Arabo and Kefis (fat-rump) , and long fat-tail from western (E2) and southern (E3) Ethiopia, respectively. The two short fat-tail sheep  analyzed here were separated from each other  were used to define candidate regions under selection.The Admixture, TreeMix and PCA Figures  revealedr Figure  with Molr Figure . For selr Figure , which p*S comparison, the fat-rump sheep were differentiated from the thin-tail in 23 candidate regions that overlapped between at least two selection signature methods and which spanned 86 genes  comparison   Figure , Table 5y Figure , Table 5*S comparison include embryonic skeletal system morphogenesis , response to cold (GO:0009409), innervation (GO:0060384), stem cell maintenance (GO:0019827) and positive regulation of cell adhesion (GO:0045785). The top GO terms associated with the E2*S candidate genes include cellular response to heat (GO:0034605), lipid binding (GO:0008289), magnesium ion binding (GO:0000287) and response to gamma radiation (GO:0000287). The GO terms for the genes from the E3*S comparison included skin development (GO:0043588), regulation of actin cytoskeleton reorganization (GO:2000249) and wound healing (GO:0042060).We performed gene ontology (GO) enrichment analysis for the candidate genes revealed in each pairwise comparison . The FST approach detects signatures arising from an increase or decrease in allele frequency differentiation between populations/breeds, hapFLK detects the same but based on increase/decrease in haplotype frequency differentiation between populations while accounting for hierarchical population structure , sperm associated antigen 8 (SPAG8) and natriuretic peptide receptor 2 (NPR2). Previous studies reported these genes to be associated with birth and carcass weights, and fat depth, respectively, in cattle , which might be responsible for fat accumulation in adipose tissues. Angiopoietin-like 8 (ANGPTL8), when induced by insulin receptor (INSR), inhibits lipolysis and controls post-prandial fat storage in white adipose tissue and directs fatty acids to adipose tissue for storage during the fed state  gene was present in the region identified on Oar6. This gene is expressed in cartilage, where collagen II is a major component, as well as in embryonic bone and tendon, suggesting that it could be a major procollagen processing enzyme in musculoskeletal tissues  and ALX homeobox 4 (ALX4) were identified on the candidate region on Oar11 and Oar15, respectively. Mutations in the former result in overgrowth of caudal spinal cord and tail vertebrae in mice  with functions that could possibly be related to tail formation. Bone Morphogenetic Protein 4 (BMP4) was revealed by Rsb and FST to be on a candidate region on Oar7 and it has been implicated in tail formation  expression has been associated with back-fat thickness in sheep  is an essential protein for the optimal functioning of PPARG. Despite this association, our analysis did not reveal any signals spanning PPARG, but two of our methods (Rsb and FST) revealed a signature on Oar20 that spanned the PPARD gene, a paralogue to PPARG.Our enrichment analysis for the E1*S), we identified a cluster of genes  that are associated with the GO biological process, response to cold. This is not surprising considering that three out of the four E1 populations are living at a high altitude and therefore in a relatively cold habitat. Indeed, Adreno receptor Beta 3 (ADRB3) plays a major role in energy metabolism and regulation of lipolysis and homeostasis  included HOXC6, SULF2, WNT11, and HOXB9. WNT11 was identified by ZFST on Oar15 while HOXC6 and HOXB9 were revealed by hapFLK on Oar3 and Oar13, respectively. The WNT gene family and the T gene have been implicated in vertebral development in laboratory mice  were present in a candidate region on Oar13. These, along with other paralogs , formed a cluster of functional genes related to the GO term, lipid binding functional process  were sampled. High fecundity and prolificacy is a common reproductive trait preferred by farmers in the Bonga sheep (field observations by the last author). This may explain the occurrence of the CIB4 and PRKAA1 in a candidate region in the E2*S comparison. The CIB4 gene was suggested to be linked, in some way, to high fecundity in the small Tail Han sheep  resulted in 36 genes that occurred in candidate regions that were revealed by at least two methods used to detect selection signatures. Fatty acid binding proteins FABP3 and FABP1 found on candidate regions on Oar2 and Oar3, respectively are the genes that relate most closely to fat deposition. SREBF1 along with PPARG are the main transcription factors controlling lipogenesis in adipose tissue and skeletal muscle   occurred in the candidate region on Oar3; it is among the genes that are reported to be associated with insulin release and sensitivity, and obesity in humans  has been associated with horn morphology  were common between E1*S and E2*S comparisons. On Oar2, three genes were identified within the overlapping candidate region, i.e., CLTA which is associated with prion protein deposition in sheep  are associated with fat deposition in sheep as herein discussed for each of the three comparisons.Twelve genes  was in a candidate region that overlapped between the E1*S and E3*S comparisons. DIS3 like 3'-5' exoribonuclease 2 (DIS3L2) has also been identified, among genes involved in cancer, cellular function and maintenance, and neurological disease, in a candidate region under selection in cattle  were in candidate regions that overlapped between the E2*S and E3*S comparisons. Among these, DnaJ heat shock protein family (HSP40) member C18 (DNAJC18) and spermatogenesis associated 24 (SPATA24) on Oar5 were reported among genes involved in heat stress tolerance and male reproductive function, respectively, in East African Shorthorn Zebu cattle  in Ethiopian indigenous sheep. The genotypes of most of the individuals analyzed were made up of at least two genetic backgrounds which could be accounted for by some level of current or historical admixture between populations. Selection signature analysis identified several putative candidate regions spanning genes relating to skeletal structure and morphology, fat deposition and possibly adaptation to environmental selection pressures. Our results indicate that Ethiopian indigenous sheep could be a valuable animal genetic resource that can be used to understand genetic mechanisms associated with body fat metabolism and distribution. This is especially important because fat deposits are a crucial component of adaptive physiology and excessive fat deposition in adipose tissue can result in obesity and overweight, and energy metabolism disorders in humans.https://www.animalgenome.org/repository/pub/NOTT2018.0423/).Genotypic data of 160 animals representing eleven Ethiopian and two Sudanese sheep populations are deposited and available at  and provided critical inputs on data analysis and in writing the manuscript. FA, MA, and MOA supported the sampling and genotyping of Najdi, Omani and Libyan sheep. AK and AyA lead and coordinated the sampling of Ethiopian sheep HM lead and coordinate the sampling of Sudanese sheep. All authors contributed to the interpretation of the results based on their knowledge on local indigenous sheep genetic resources of their respective countries. All the authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Barbus  have allopatric distributions in the Tyrrhenian and Adriatic basins of Italy. Barbus plebejus inhabits the mid‐ to northern Adriatic basins, while B. tyberinus is widespread in all central‐northern basins draining into the Tyrrhenian Sea. For basins in Southern Italy draining into the southern parts of these seas, there remains a knowledge gap on their barbel populations due to no previous genetic and morphological studies, despite their apparent biogeographic isolation. Correspondingly, this study quantified the presence and distribution of barbels in the Adriatic and Tyrrhenian basins of Southern Italy through genetic and morphological analyses of 197 fish sampled across eight populations. Testing of how local isolation has influenced the evolution and persistence of these populations was completed by examining sequence variation at two mitochondrial loci (cytochrome b and D‐loop) and performing geometric morphometric analyses of body shape, plus measuring 11 morphometric and meristic characters. Phylogenetic and morphological analyses revealed the presence of two genetically distinct lineages that differed significantly from adjacent B. tyberinus and B. plebejus populations. These two new taxa, here described as SI1 and SI2 Barbus lineages, are highly structured and reflect a complex mosaic biogeographic pattern that is strongly associated with the underlying hydrographical scenarios of the basins. The geographic isolation of these basins thus has high evolutionary importance that has to be considered for maintaining endemism.The Italian peninsula is a biodiversity hotspot, with its freshwater fish fauna characterized by high levels of local endemism. Two endemic fluvio‐lacustrine fishes of the genus  In Southern Italy, apparent biogeographic isolation may have influenced barbel evolutionary structure. Through genetic and morphological analyses, we quantified the presence and distribution of barbels in Southern Italy describing two new taxa, highly structured and reflecting a complex mosaic biogeographic pattern that is strongly associated with the underlying hydrographical scenarios of the basins. The Adriatic sites were in the Aterno‐Pescara (A1) basin that represents the first Adriatic drainage in AC district, and the Sangro (A2), Biferno (A3), Fortore (A4) up to Ofanto (A5) basins , the fish were attributed to a species according to their phenotypic characteristics  gene  using a 3730XL DNA Sequencer. All new haplotypes generated in this study were deposited in the GenBank database .Total genomic DNA was extracted from all individuals using a proteinase K digestion, followed by sodium chloride extraction and ethanol precipitation  was used as an outgroup. The Cyt b and D‐loop sequences of Barbus species available in GenBank were included in the cyt b and D‐loop phylogenetic inferences: B. barbus, B. plebejus, and B. tyberinus  were also added . To test the isolation between populations (within and between Tyrrhenian and Adriatic basins), population genetic differentiation was calculated using the fixation index ΦST  by permuting haplotypes between populations 3,024 times, as implemented in Arlequin v 3.5.A minimum spanning network was created from the multiple D‐loop sequences alignment produced in this study using a statistical parsimony criterion as implemented in PopART v 1.7 software  were scored and seven new haplotypes detected  as detailed in Table b sequences separated out the all fluvio‐lacustrine and rheophilic Barbus  species well, but as they did not clearly resolve the evolutionary relationships, they showed unresolved polytomy  and “South Italy 2” (SI2) Barbus lineages. In the D‐loop phylogenetic tree, the haplotypes recorded in Vomano River  barbel were 3.9% and 3.6%, respectively. It is noteworthy that SI Barbus lineages were more similar to B. plebejus (1.5%–1.8%) than to B. tyberinus (2.1%–2.4%) and that the inter‐lineage uncorrected p‐distance between SI1 and SI2 Barbus lineages (1.7%) was in a middle position  were linked by more than 13 mutational steps and revealed some distinct patterns. The SI1 Barbus lineage (N = 60) was composed by five new haplotypes that were connected by up to seven mutational steps, with the most frequent BSI01 positioned in the middle of the radiation  showed a larger number of haplotypes , with the two most frequent haplotypes (BSI201 and BSI202) separated by four mutational steps  of 0.001 and 0.003, and haplotype diversity (H) of 0.61 and 0.78, respectively. The mismatch distribution analyses do not support a sudden expansion model for both lineages , as they revealed multiwave trends , whereas the SI2 Barbus lineage was present both in the three middle Tyrrhenian basins  and in the two most southern Adriatic basins . Among the AC district barbel populations, only the A5, T2, and T3 populations were dominated by the BSI201 haplotype . Variations along the CV1 (54%) were mainly associated with the eye diameter, the depth of the posterior body, and the shape of the caudal fin; those along the CV2 (22%) were mainly associated with the overall fish body shape. The SI1 and SI2 Barbus lineages were partially separated from each other along both axes, as also indicated by the Mahalanobis distance value (MD = 3.27). Comparisons with the other two Italian Barbus species revealed the SI1 Barbus lineage had a higher overlapping position with B. tyberinus (MD = 3.26) than with B. plebejus (MD = 3.59). The SI2 Barbus lineage was more separated from both B. tyberinus (MD = 3.58) and B. plebejus (MD = 4.01). Both SI Barbus lineages showed the highest Mahalanobis distance values against B. barbus , and, in the case of SI2 Barbus lineage, a complete separation with the exotic B. barbus was observed in the CVA plot.The geometric morphometric analyses of the CVA plot revealed there was partial visual separation in body shape morphology in the two SI s Figure . This wap < .05) between the SI1 and SI2 Barbus lineages for all the analyzed traits, except for the number of dorsal fin branched rays and the number of scales on the lateral line. Both lineages had values of the latter character that were not statistically different from B. tyberinus (p > .05). Moreover, no significant differences were recorded between SI1 Barbus lineage and B. tyberinus for any of the morphometric traits (p > .05), except for the height of the third dorsal fin ray (p < .05). The SI2 Barbus lineage was not statistically different from B. plebejus (p > .05), both for all the morphometric traits and for the number of dorsal fin branched rays.The ANOVA results Table  and Tukep > .05), the geometric morphometric analyses of the CVA plot indicated some visual separation  from those attributed to the SI2 Barbus lineage . The minimum Mahalanobis distance (MD = 3.95) was recorded between the T2 and T3 populations, belonging to two contiguous Tyrrhenian basins, while the maximum value (MD = 10.50) was found between T1 and A2 populations  confirms the complex mosaic pattern recorded across the north and central Italian peninsula, where the allopatric origins and dispersion routes of the species have been primarily influenced by distinct historical events  and Italian bleak . These basins were not part of the paleo‐Po expansion . Within this restricted area, the SI1 Barbus lineage had high levels of genetic variability and was thus highly structured. These results suggest that climatic, hydrological, and geological factors probably shaped their local isolation and did not result in dispersion events via temporary connections  and the southern Adriatic basins  between Barbus species in future. If this happens, it is likely to remain undetected along this complex of cryptic species and will potentially lead to the loss of local endemism. Consequently, these results highlight the necessity for any fish and fishery management programmes in this region to recognize the inherently high conservation value of these endemic barbels and avoid undesirable mixing with other barbels through, for example, fish stocking exercises.In conclusion, within the hydrogeographic units of the AC district of Southern Italy, there is high genetic structure in the barbel populations that can be related to the isolation of the basins, resulting in very limited gene flow between them. The limitation in dispersion was due to minimal river capture events in the upstream part of the basins that, due to their typically Mediterranean regime, are characterized by low discharge, and thus, the fish were unable to mix due to insurmountable geographical barriers. Consequently, the AC district can be considered as unique in relation to the biogeography of their endemic barbel populations, with their geographic and hydrological isolation from basins further north being important in this. These results emphasize that, across this district, the evolutionary processes of the endemic barbels have favoured a mosaic pattern, although it is suggested that this requires further work by use of an enlarged dataset, including studies on other freshwater taxa. Although we recorded a limited presence of None declared.SZ, ML, SQ, and AC conceived the ideas and designed the methodology; ML and AC collected the data; SQ analyzed the photographs and performed morphological analysis; IV and VDS worked in laboratory and analyzed the data; and all authors contributed critically to the drafts and gave final approval for publication. Click here for additional data file.


Predictions about the consequences of a small population size on genetic and deleterious variation are fundamental to population genetics. As small populations are more affected by genetic drift, purifying selection acting against deleterious alleles is predicted to be less efficient, therefore increasing the risk of inbreeding depression. However, the extent to which small populations are subjected to genetic drift depends on the nature and time frame in which the bottleneck occurs. Domesticated species are an excellent model to investigate the consequences of population bottlenecks on genetic and deleterious variation in small populations. This is because their history is dominated by known bottlenecks associated with domestication, breed formation and intense selective breeding. Here, we use whole‐genome sequencing data from 97 chickens representing 39 traditional fancy breeds to directly examine the consequences of two types of bottlenecks for deleterious variation: the severe domestication bottleneck and the recent population decline accompanying breed formation. We find that recently bottlenecked populations have a higher proportion of deleterious variants relative to populations that have been kept at small population sizes since domestication. We also observe that long tracts of homozygous genotypes (runs of homozygosity) are proportionally more enriched in deleterious variants than the rest of the genome. This enrichment is particularly evident in recently bottlenecked populations, suggesting that homozygosity of these variants is likely to occur due to genetic drift and recent inbreeding. Our results indicate that the timing and nature of population bottlenecks can substantially shape the deleterious variation landscape in small populations. HoweverIn small populations, the mutation–selection balance is challenged by population contractions, which reduce the efficacy of purifying selection to remove harmful mutations Ohta, . As a reGenetic drift, or the random fluctuations in the number and frequency of alleles, is mostly responsible for the deleterious genetic landscape in small populations. However, as studies in plant and animal species have suggested, the extent to which small populations are subjected to genetic drift considerably varies depending on the nature and time frame in which the bottleneck occurs    .DNA of 97 individuals from 39 traditional chicken breeds from the Netherlands was used for whole‐genome sequencing on an Illumina HiSeq 3,000. Four samples from each of the known living dy Table . Based o) Figure , includi) Figure  using th) Figure , indel r2.2Gallus species excluded) were investigated through a principal component analysis (PCA) performed on the filtered vcf files in PLINK v.1.9  were used as an outgroup to predict the ancestral and derived allelic state of all polymorphic sites. A variant was categorized as ancestral if the three wild samples had the same genotype . The 11,706,316 identified variants were extracted from each sample with a minimum average genome coverage >10× and classified as homozygous ancestral, heterozygous or homozygous derived.Sequencing data of the three wild 2.7Gallus gallus annotation database (release 90). Protein‐coding variants were defined based on their SIFT score as synonymous, nonsynonymous tolerated (SIFT score ≥0.05), and nonsynonymous deleterious (SIFT score <0.05). We also catalogued mutations that disrupt the generation of a fully functional protein either by introducing a stop codon or by truncating the protein reading frame as loss of function (LoF) variants. To be more confident on the detection of deleterious variants, we implemented the approach developed by . Variants were considered truly deleterious if SIFT score <0.05 and GERP score >1.0.Variant annotation was performed with the Variant Effect Predictor (VEP) , whereas under the alternative model differences in the number of homozygous derived genotypes are expected between large fowls and neo‐bantams (plf≠pnb). The log‐likelihood values of both the null and alternative models were used to calculate the likelihood ratio test (LRT) as, Λ=-2loglikelihoodnull-loglikelihoodalternative.Following  spectrum was calculated for synonymous, tolerated and deleterious variants, considering only bi‐allelic SNPs. We then generated a histogram with 10 bins  starting from a very low (0–0.10) to a very high (0.90–1.0) derived allele frequency.2.11Lg is the total length of the genome, LROH is the total length of ROHs, i is the individual, and j is the ROH class jò representing small, medium, long and any ROH, respectively  of 39 traditional chicken breeds Table . On aver3.1Genome‐wide autosomal heterozygosity and ROHs were used to investigate the extent and nature of genetic variation in our populations. Whole‐genome heterozygosity ranged from 12.2 to 40.8 SNPs/10‐kb Table . On avereN). According to our results, the chicken ancestral population size remained stable up to approximately 10,000 years, after which it dropped from an initial eN of 106 to 104–103  mutations. Of the initial set of deleterious variants, 1,674 were classified as deleterious by both SIFT and GERP++. The efficacy of selection at removing deleterious variants from a population was investigated by looking at the distribution of the derived allele frequency (DAF) spectrum. The frequency spectrum showed more rare (DAF <0.1) derived alleles than nearly fixed or fixed deleterious alleles  to formally test for individual differences between neo‐bantams and large fowls. According to the likelihood ratio test, the number of homozygous derived genotypes was not significantly different between large fowl and neo‐bantam counterparts for deleterious  and LoF  variants  and nonsynonymous tolerated  variants. We also investigated in each of the three management groups the total genetic burden resulting from the accumulation of deleterious mutations  was lower for deleterious and LoF variants relative to putatively neutral ones (synonymous and nonsynonymous tolerated) Figure . Comparets Table . Signifi3.4Pearson's correlation: 0.844, p‐value: <2.2e‐16) and nondamaging sites . As expected, the number of homozygotes occurring outside ROHs decreased with the fraction of the genome in any ROH, as the genome simply contains fewer ROH‐free regions. A negative correlation between homozygous sites and genome not covered by ROHs confirmed our expectations for damaging    Figure b and non4http://edepot.wur.nl/424249). Even though (slightly) different breeds were grouped into the same population, we expect potential bias to be minimal, as already shown in Bortoluzzi et al. , which is the direct consequence of the threatened population size of most of these breeds  is an important evolutionary factor in determining the level of genetic variability and the effectiveness of purifying selection at removing harmful mutations  (Bosse et al., In a recent study on commercial chicken lines, Derks et al.  observed5In this study, we showed that the timing and nature of a population bottleneck can substantially shape the deleterious variation landscape in small populations. In particular, we showed that populations kept at small size for long period of time since the bottleneck have a reduced burden of deleterious alleles compared with recently bottlenecked populations. The reduced deleterious burden in these populations, which is also linked to a reduced number and total length of ROHs across the genome, is likely responsible for their genetic success.According to our study, facilitating purging of deleterious mutations through inbreeding avoidance should be at the core of future breeding and conservation programmes in small populations (de Cara, Villanueva, Toro, & Fernandez,  Click here for additional data file. Click here for additional data file. Click here for additional data file.


In addition to pharmacogenomic alleles related to increased toxicity risk, we also have evidence that alleles related to dosage and metabolism have large frequency differences between the two populations, which are associated with their specific ancestries. Using these findings, we have developed and validated an inexpensive allele-specific PCR assay to test for the presence of such population-enriched pharmacogenomic SNPs in Colombia. These results serve as an example of how population-centered approaches to pharmacogenomics can help to realize the promise of precision medicine in resource-limited settings.While genomic approaches to precision medicine hold great promise, they remain prohibitively expensive for developing countries. The precision public health paradigm, whereby healthcare decisions are made at the level of populations as opposed to individuals, provides one way for the genomics revolution to directly impact health outcomes in the developing world. Genomic approaches to precision public health require a deep understanding of local population genomics, which is still missing for many developing countries. We are investigating the population genomics of genetic variants that mediate drug response in an effort to inform healthcare decisions in Colombia. Our work focuses on two neighboring populations with distinct ancestry profiles: Antioquia and Chocó. Antioquia has primarily European genetic ancestry followed by Native American and African components, whereas Chocó shows mainly African ancestry with lower levels of Native American and European admixture. We performed a survey of the global distribution of pharmacogenomic variants followed by a more focused study of pharmacogenomic allele frequency differences between the two Colombian populations. Worldwide, we found pharmacogenomic variants to have both unusually high minor allele frequencies and high levels of population differentiation. A number of these pharmacogenomic variants also show anomalous effect allele frequencies within and between the two Colombian populations, and these differences were found to be associated with their distinct genetic ancestry profiles. For example, the C allele of the single nucleotide polymorphism (SNP) rs4149056 [Solute Carrier Organic Anion Transporter Family Member 1B1 (SLCO1B1) The precision medicine approach to healthcare entails a customized model whereby medical decisions and treatments are specifically tailored to individual patients . CurrentA recently articulated alternative to the precision medicine model is referred to as precision public health . The focColombia is home to a highly diverse, multi-ethnic society. The modern population of Colombia is made up of individuals with genetic ancestry contributions from ancestral source populations in Africa, the Americas, and Europe . ColombiPrevious studies have shown that the frequencies of pharmacogenomic variants can vary across populations with divergent genetic ancestries. This includes variation in pharmacogenomic variant allele frequencies among distantly related populations worldwide  as well 1 – is currently working to develop genomic diagnoses that are tailored to the local population, and members of the ChocoGen Research Project2 are exploring the connections between genetic ancestry and health disparities in the understudied Colombian population of Chocó. Here, these two groups have joined forces in an effort to (i) discover pharmacogenomic variants with special relevance for these two Colombian populations and (ii) develop cost-effective and rapid pharmacogenomic assays for those variants, which can be readily deployed in resource-limited settings.Colombia’s first clinical genomics laboratory – GenomaCES from Universidad CES in Antioquia3) (Pharmacogenomic single nucleotide polymorphisms (pharmaSNPs), i.e., human genetic variants associated with specific drug responses, were mined from the Pharmacogenomic Knowledgebase (PharmGKB3) . PharmGK4]. All of the pharmaSNPs from PharmGKB were found to be present in 1000 Genomes Project phase 3 variant calls. Genome sequence variation for the Colombian population of Chocó was characterized as part of the ChocoGen Research Project5 as previously described (Data on human genome sequence variation were taken from the phase 3 data release of the 1000 Genomes Project . For theescribed .ST) for a genome-wide set of n = 28,137,656 pruned SNPs and for the set of n = 1995 pharmaSNPs using the program PLINK (r2 threshold of 0.5 with a sliding window of 50 nt and a step size of 5 nt. MAF (p) values for each SNP were calculated across all populations as: ST values for each SNP were calculated among populations as: σ2 is the observed MAF variation. Pairwise genomic distances were computed as 1-identity-by-state/Hamming distances between genomes using the PLINK distance command with the –distance-matrix option. The resulting high-dimensional pairwise genomic distance matrix was projected in two dimensions using multi-dimensional scaling (MDS) method implemented in the base package of the R statistical language  and fixation index (Fam PLINK . Linkagelanguage . The proclusters .f) between Antioquia (ANT) and Chocó (CHO) were measured as (1) the log-transformed ratio of the population-specific allele frequencies log2(fANT/fCHO) and (2) as the population-specific allele frequency difference Δ = fANT/fCHO. These two effect allele difference metrics were plotted orthogonally and the Euclidean distance from the origin was calculated for each pharmaSNP to yield a composite difference.The differences in pharmaSNP effect allele frequencies , and β quantifies the strength of the association. The significance of the ancestry association is measured as the P-value obtained from a t-test, where βt = β/SE.The influence of genetic ancestry on pharmaSNP genotype frequencies was measured via ancestry association analysis. To do this, individuals’ genetic ancestry fractions – African, European, and Native American – inferred using ADMIXTURE with the genome-wide SNP set, were regressed against their individual pharmaSNP genotypes. The strength of the resulting ancestry × pharmaSNP associations were quantified using a linear regression model: Q ≥ 30 (Whole exome sequence (WES) analysis was conducted on a cohort of 132 de-identified patients characterized for the purposes of genetic testing by the GenomaCES laboratory . The stuQ ≥ 30 . SequencQ ≥ 30 , and varQ ≥ 30 . The resQ ≥ 30 . Manual Q ≥ 30 .TM Taq DNA Polymerase kit, with 25 μL final reagent volume, on the Bio-Rad thermocycler . PCR products were visualized and scored as homozygous non-effect allele, heterozygous, or homozygous effect allele using electrophoresis performed with 2.5% agarose gels stained with ethidium bromide (10 μL) with a running time of 60 min at 70 V in 1X TBE buffer. UV light was used to visualize the gel-separated PCR products.The identity of pharmaSNP allelic variants was assayed in the same 132 patients using custom-designed allele-specific PCR assays following the Web-based Allele-Specific PCR (WASP) primer design protocol . Both th6) provides a catalog of pharmaSNPs together with information regarding their known impacts on drug response. PharmGKB categorizes pharmaSNPs with respect to their specific effects on drug efficacy, dosage, or toxicity/adverse drug reactions as well as the level of evidence for their role in drug response: (1) high, (2) moderate, (3) low, or (4) preliminary. We mined the PharmGKB database for pharmaSNPs across all four evidence levels, yielding a total of 1995 SNPs genome-wide.We operationally define pharmaSNPs as human nucleotide variants that are known to affect how individuals respond to medications. The Pharmacogenomics Knowledgebase (PharmGKBST = 0.07), a measure of between-population differentiation, for global populations compared to genome-wide SNPs  population groups characterized as part of the 1000 Genomes Project . Levels ect data .Given the high levels of variation and between-population discrimination shown by pharmaSNPs, we also evaluated the extent to which they carry information about genetic ancestry and admixture, particularly for the Colombian populations of Antioquia and Chocó. Pairwise genomic distances were computed for the Colombian populations together with a set of global reference populations from Africa, the Americas, and Europe, using both pharmaSNPs and the genome-wide SNP set. Pairwise genomic distances computed using both sets of SNPs were used to reconstruct the evolutionary relationships among human populations worldwide. The results for the genome-wide  and pharK = 3 ancestry components, genome-wide SNPs clearly distinguish the reference African, European, and Native American populations, and characterize the Colombian populations of Antioquia and Chocó as distinct mixtures of all three ancestries . PharmaSNP effect allele frequency differences between Antioquia and Chocó were measured in two ways – (1) as the log transformed ratio of allele frequencies Antioquia/Chocó and (2) as the allele frequency differences between Antioquia and Chocó – in order to capture both high relative differences at low allele frequencies and high absolute differences at high allele frequencies  is found at higher frequency in Chocó and is positively correlated with African ancestry and negatively correlated with both European and Native American ancestry  shows a similar pattern with higher frequency in Chocó, a positive correlation with African ancestry, and negative correlations with both European and Native American ancestry  is found in higher frequency in Antioquia, showing a negative correlation with African ancestry and a positive correlation with European ancestry  observations of population-specific allele frequencies in Colombia along with related ancestry-associations, (ii) pharmacogenomic associations with drugs that are widely prescribed in Colombia and used to treat common conditions, and (iii) pharmacogenomic associations with drugs for which GenomaCES investigators have anecdotal information from collaborating physicians that pharmacogenomic tests would be of use to the local population, based on their observations of anomalous drug responses in their patients. It should be noted that the population and clinical criteria are not mutually exclusive; indeed, physicians’ observations of anomalous drug responses in their patient populations are almost certainly related to the population-specific allele frequencies of the relevant pharmaSNPs.∗5), located with an exon of the SLCO1B1 protein coding gene on the short arm of chromosome 12, is shown in An example of an allele-specific PCR assay developed for the simvastatin-associated pharmaSNP rs4149056 (SLCO1B1∗5) variant detection assay, we then ran it on a cohort of 132 de-identified patients from the GenomaCES laboratory, all of whom have exome sequences available for confirmatory analysis. The results of the allele-specific PCR and exome analyses are highly similar; taking the exome results as the ground truth against which to compare the PCR assay yields an overall accuracy of 97.7% for this test (∗2) and rs1057910 (CYP2C9∗3) – were tested on the same patient set and confirmed via exome sequence analysis. These two allele-specific PCR genotyping assays show even higher accuracies of 98.5 and 100%, respectively. We calculated a number of additional performance metrics for all three of these tests, breaking down each assay into its three constituent genotypes, the results of which are shown in Having confirmed the accuracy of the rs4149056  build the local capacity needed to perform such tests and (ii) develop genomic assays that are specifically tailored to the needs of Colombian populations. To these ends, Universidad CES has invested substantially in the development of local capacity in genomic medicine via the establishment of GenomaCES, which is Colombia’s first homegrown genomic medicine laboratory. As we have shown here, GenomaCES is working to develop inexpensive and rapid pharmacogenetic genotyping tests based on relatively simple allele-specific PCR assays. Developing local tests of this kind can help to ensure that variants of specific relevance to the country are prioritized for testing and to avoid the prohibitively high costs of commercially available tests and/or kits.All datasets generated for this study are included in the manuscript and/or the AV-A, IJ, and JG conceived of and designed the study. SN, AM, EN, LR, AC, KO’N, SV-G, and CM-R performed all data analysis. AM, SV-G, WJ-Á, and IT performed laboratory assays. MM-R, AV-A, IJ, and JG supervised and managed all aspects of the project in Colombia and the United States. MM-R and JG acquired study subject samples. SN, LR, and IJ prepared figures and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Nothobranchius spp.) are adapted to seasonally desiccating habitats , surviving dry periods as dormant eggs. Given their peculiar life history, geographic aspects of their diversity uniquely combine patterns typical for freshwater taxa (river basin structure and elevation gradient) and terrestrial animals . However, our current knowledge on fine-scale inter-specific and intra-specific genetic diversity of African annual fish is limited to a single, particularly dry region of their distribution . Using a widespread annual killifish from coastal Tanzania and Kenya, we tested whether the same pattern of genetic divergence pertains to a wet equatorial region in the centre of Nothobranchius distribution.African annual killifishes  gene  and 10 nuclear microsatellite markers . We found five lineages with a clear phylogeographic structure but frequent secondary contact. Mitochondrial lineages were largely congruent with main population genetic clusters identified on microsatellite markers. In the upper Wami basin, populations are isolated as a putative Nothobranchius prognathus, but include also a population from a periphery of the middle Ruvu basin. Other four lineages  coexisted in secondary contact zones, but possessed clear spatial pattern. Main river channels did not form apparent barriers to dispersal. The most widespread lineage had strong signal of recent population expansion.In populations of Nothobranchius species from a wet part of the genus distribution  is not constrained by main river channels and closely related lineages frequently coexist in secondary contact zones. We also demonstrate contemporary connection between the Ruvu and Rufiji river basins. Our data do not provide genetic support for existence of recently described cryptic species from N. melanospilus complex, but cannot resolve this issue.We conclude that dispersal of a  Ephemeral habitats with patchy distribution are common but represent challenging environment to dispersal and colonization. While some species have evolved specialised stage to overcome dispersal limitation, others have responded by strong population resilience and possess stages resistant to shifting environmental conditions. For example, small cladocerans often produce resting stages that are dispersed by wind and, consequently, many cladoceran species have very large ranges and weak genetic structure (e.g. ). In conTemporary freshwater pools are common in highly seasonal environments, including African savanna. Their specialised fauna includes widespread invertebrate species with specific dispersal stage (e.g. crustaceans and aquatic insects) as well as specialised killifish species that are extremely poor dispersers , 4. In ANothobranchius inhabit ephemeral freshwater pools developed on vertisol (dark cracking clay) soils [Nothobranchius distribution covers extensive region from relatively dry subtropical areas with a single rainy season in the north and south across humid equatorial areas with two rainy seasons a year. Nothobranchius diversification follows allopatric scenario [Nothobranchius range (Mozambique) harbours highly geographically structured populations, with important roles of genetic drift and dispersal limitation [Nothobranchius lineages [Annual killifish are adapted to ephemeral pools in Africa and Neotropics by possessing a resilient developmental stage. After habitat desiccation, their embryos survive in dry pool sediment in the form of diapausing eggs , 7. Withy) soils . Nothobrscenario , with isscenario , 12. Themitation –15. In tNothobranchius genetic diversity from dry subtropical region pertain to wet equatorial region of African savanna. Specifically, we investigated population genetic pattern in Nothobranchius melanospilus species group, geographically widespread and locally common killifish in lowland East Africa [Nothobranchius melanospilus  and two recently described cryptic species; Nothobranchius prognathus Costa 2019 and Nothobranchius kwalensis Costa 2019 from peripheral parts of the N. melanospilus range [Nothobranchius fishes [COI) gene and 10 nuclear microsatellite markers to examine the role of river basins, river channels and elevational gradient in structuring N. melanospilus species group.In the present study, we tested whether the patterns of t Africa . This spus range . The spePfeffer, 96 and tws fishes . Using 2Nothobranchius melanospilus is the most commonly recorded species of the genus. It is found in natural temporary pools, swamps and small temporary streams as well as in ricefields, man-made ditches and culverts [culverts . The speculverts , 17. Geoculverts , 20, 21.N. melanospilus, on the basis of combination of morphometric characters and female colouration. Based on that study [N. melanospilus sensu stricto is distributed only south of the Wami basin (and on Zanzibar Island). The upper Wami basin populations were described as Nothobranchius prognathus Costa 2019 and populations from southeastern Kenya as Nothobranchius kwalensis Costa 2019. Hence, our study putatively concerns to a complex of three closely related species of the N. melanospilus group. Our samples cover the entire range of the species group, except for Zanzibar Island and the lower Pangani basin.A recent taxonomic work used museum specimens  to formaat study , N. melaN. melanospilus species group distribution was modified by East African Rift tectonics that formed the Ruvu and Rufiji throughs [N. melanospilus species, is located on a shallow shelf near the mouths of the Ruvu and Wami rivers and was likely linked to mainland populations until 12,000 years ago. Small coastal rivers between the Ruvu and Rufiji (e.g. the Luhule and Mbezi rivers) are separated from the Ruvu and Rufiji by Pugu and Mtoti hills, forming so-called Mbezi Triangle with endemic Nothobranchius species [The region of throughs . The two species , 16. The species .Nothobranchius, female N. melanospilus species group are readily recognised from other Nothobranchius species by their unique dark spots on the body [N. kwalensis from the Ramisi basin . All field sampling and export procedures followed regulations of Tanzania, with permits and research associateship issued by Sokoine University of Agriculture in Morogoro .Specimens from most populations were collected during a dedicated field trip in May and June 2017, using dip and seine nets. Fish were identified in the field; unlike in other Tanzanian the body . Most fiCOI gene was amplified using primers TRNYF1 (AGG GAG TTA CAA TCC ACC ACT ATT T) and TRNSR1 (ATG GGG GTT CAA TTC CTT CCT TT), alternatively, and a forward primer COI852F (CTT TAT TGT TTG AGC CCA CCA CA) [b gene, but none of the 7 tested primers  following a standard protocol. Full details of the genotyping methods, primer sequences, microsatellite multiplexing, and PCR protocols are presented in Additional file  CCA CA)  for a seWe used a set of 10 microsatellite loci in four multiplex PCR sets  approach. We used PartitionFinder v. 2.1.1 [Nothobranchius guentheri was used as an outgroup. Bayesian analysis was performed by Markov Chain Monte Carlo (MCMC) simulation using MrBayes 3.2.6 [http://tree.bio.ed. ac.uk/software/figtree).Phylogenetic relationships within v. 2.1.1  to seleces 3.2.6 . Two indes 3.2.6  and the http://qgis.org). Diversity estimates, i.e. number of polymorphic sites (Np), number of haplotypes (Nh), haplotype diversity (Hd), nucleotide diversity , the average number of nucleotide differences (k) and Watterson’s estimate of θ (θ = 4Ne*μ) were calculated using DnaSP v. 5.10.01 [All sequences were geo-referenced and the geographical distribution of lineages was plotted onto map using QGIS 2.18  at each locus and population was estimated in FreeNA . The meap < 0.05. Pairs of loci were significantly linked only in one or two populations and the microsatellite loci can be considered to be unlinked.Deviations from linkage and Hardy-Weinberg equilibrium (HWE) for each locus and population were detected in Genepop 4.0.10 , 32. LinO) and unbiased expected heterozygosity according to Nei (1978) (HE) in GENETIX 4.05.2 [O, HE, AR) populations with < 8 sampled individuals were not used .Genetic variability was estimated by calculating observed heterozygosity  against ln-distance between sample sites. Mantel tests were used to test the correlation between matrices of genetic differentiation and geographical distances between sampling sites by 1000 permutations in Genepop 4.0.10 [To quantify genetic differentiation between populations, we computed pairwise estimators of ribed in  and implribed in , as therp 4.0.10 .6 iterations, following a burn-in period of 105 iterations. We used admixture ancestry model and correlated allele frequencies model (with λ = 1). The output of STRUCTURE analysis was post-processed in CLUMPAK software [To investigate the spatial genetic structure among individuals, we used STRUCTURE 2.3.4 . The indsoftware  to identsoftware  and a prCOI sequences (657 bp). Their phylogenetic analysis revealed five main lineages, some of which possessed a finer substructure , from pools within the upper Wami River basin (Tendigo swamp). In addition, Wami lineage pertained to one pool in an isolated part of the middle Ruvu basin (population T15). This haplogroup was distributed at the highest elevation (246–435 masl compared to < 167 masl in other lineages), but had low statistical support , we detected 30 different haplotypes of ure Fig. . Only thure Fig. . This liCOI variability and historical demography are shown in Table Demographic history was analysed separately for geographically two most widespread mitochondrial lineages – Ruvu (blue) and Wami (orange). Unimodal pattern of mismatch distribution graphs  showed deviance from HWE, when calculated over all loci  for populations with at least 8 individuals are shown in Table N = 8). The lowest intra-population genetic variability  was detected in populations T57, T31 and T15 located at relatively isolated pools. In contrast, the highest intra-population genetic diversity  was found in populations from the floodplain of lower parts of the major rivers  and in population T14 in the middle reach of the Ruvu  pairwise FST = 0.113 ± 0.07. The pairwise FST values were significantly different from zero in 95.83% of population pairs; only five of 120 pairwise FST were not significant . The pattern of isolation-by-distance showed weak but significant association between geographical and genetic distances ), the ΔK criterion , and a pNothobranchius melanospilus species group were clearly structured, with geographically adjacent lineages sometimes coexisting within a single population. Clustering to mitochondrial lineages had a good correspondence in nuclear microsatellite data. The most widespread lineage  was found in all sampled regions except of the Wami basin. It coexisted with Rufiji (red) lineage in the lower Rufiji basin and Mbezi (green) lineage in the lower Ruvu. Its broad distribution agrees with a clear signature of recent expansion indicated by neutrality indices. Despite their coexistence, the three common lineages  had their apparent centres of distribution that are indicative of potential refugial persistence in the lower Ruvu basin, lower Rufiji basin and smaller coastal rivers between the Ruvu and Rufiji, including Mbezi Triangle.Populations of N. prognathus [N. prognathus should be considered as a valid species.Three upper Wami basin populations formed a separate cluster on mitochondrial and nuclear markers, apparently consistent with a recent elevation of the populations from this region to a specific level, as ognathus . In our N. melanospilus-species group. This region is terra typica of N. kwalensis, a newly described cryptic species that can only be unambiguously diagnosed for female specimens [N. melanospilus haplotype (Ruvu) and morphologically, fish were typical N. melanospilus. Despite its relative distinctness, there was no statistical support to separate this haplotype. Clearly, more individuals and markers are needed to resolve validity of N. kwalensis. Our sampling  were completed prior to the description of N. kwalensis and N. prognathus as separate species and we have no other specimens to further elaborate on our tentative findings. We acknowledge that this leaves the question of validity of N. kwalensis, and its geographical distribution, unresolved. Given the presence of the Ruvu lineage of N. melanospilus in coastal Kenya, it is possible that N. melanospilus sensu stricto rather than N. kwalensis may inhabit coastal plains of the northern Tanzania.A single divergent haplotype has been discovered in coastal Kenya . Validity of this haplotype has been confirmed by repeated analysis (including new DNA sampling from voucher specimen). Morphological inspection of the voucher individual  confirmed that it belongs to pecimens , which wN. melanospilus species group populations have been separated into at least five refugia that are consistent with divisions into the main river basins in the region. The lineage from at least one refugium (Ruvu) undergoes recent expansion and coexists with at least two other N. melanospilus lineages. Importantly, the only mitochondrial lineage (Rufiji) with a significant support for its genetic distinctiveness from other N. melanospilus-species-group lineages coexist widely with the Ruvu lineage and nuclear markers did not indicate the lack of panmixia. The fact that two lineages that were formally described to represent cryptic species of the complex – from the Wami (N. prognathus) and Ramisi  – are less distinct than the Rufiji lineage apparently supports the arguments of Wildekamp [N. melanospilus sensu stricto and the two putative cryptic species as normal intraspecific variation commonly seen in this [Nothobranchius species [Regardless the taxonomic implications, we conclude that current ildekamp , who reg in this  and many species , 40. We He, Ho and AR estimates), suggesting that their distinct population genetic signatures arose from genetic drift due to either population bottlenecks or founder effects.Intra-specific structure derived from microsatellite markers was largely congruent with mitochondrial data. Given a low number of individuals available, we have not genotyped Kenyan populations on microsatellite markers. Several populations that were not differentiated on mitochondrial data formed separate clusters at finer genetic substructuring. Those populations were typically genetically depauperated . Other adjacent populations had non-significant FST values, especially in the lower Ruvu  and, unexpectedly, between Ruvu (T14) and Rufiji (T17) populations . This corroborates that dispersal in equatorial region of coastal Tanzania might be more suitable for frequent dispersal across main river channel and among adjacent populations. The region experiences much longer duration of the wet phase, with two rainy seasons each year and a longer duration of habitat inundation compared to a single rainy season in subtropical Mozambique [Nothobranchius fishes experience variable climatic and ecological challenges that may affect their dispersal, diversification and coexistence in local killifish assemblages [The populations were principally structured by their respective river basins. Main channels of large rivers did not constitute apparent barriers to dispersal in f Africa . For exazambique  with veralue 0.00. Other azambique . It demoemblages .Nothobranchius melanospilus species group are largely congruent with phylogeographic patterns of other cyprinodontid fishes. For example, Rivulus cylindraceus from Cuba has wide distribution, with two haplogroups coexisting in some adjacent drainages and highly divergent haplogroups in isolated edges of the range [Fundulus olivaceus from midwestern and southern USA, distinctive haplogroups coexist in secondary contacts across drainages, while in sympatric Fundulus notatus four mitochondrial haplogroups are strictly isolated by respective river basins [Fundulus heteroclitus from Atlantic coast of North America combines latitudinal isolation-by-distance pattern with a division into two sharply separated clades [Our data on he range . In Fundr basins . Finallyd clades .Austrolebias is also not structured by main river channels [Austrolebias bellottii species group is widespread throughout the lower Paraná/La Plata and Uruguay basins, in a situation very similar to the N. melanospilus species group distribution pattern. A phylogeographic pattern of A. bellottii demonstrates repeated vicariance and dispersal events resulting in broad coexistence of major haplogroups across major river basins [N. melanospilus is also present) and mainland [Within annual killifish, genetic variability of Neotropical annual killifish from the genus channels . Especiachannels . For exar basins . Lowlandr basins  and climr basins , 51, incmainland .N. melanospilus lineages was shaped by repeated marine regression and transgressions in the Quaternary [Hydrocynus tanzaniae, with lineages of Middle Pleistocene-dated divergence between the Ruvu and Rufiji basins [Rhinogobius duospilus, in Hong Kong streams and Iberian cyprinid, Squalius valentinus, whose limited dispersal capabilities resulted in a clear intra-specific signature of Quaternary climatic oscillations [We propose that distribution of aternary –51, tranaternary  as well aternary . While si basins , as wellllations , 56.Nothobranchius fishes typically live in ephemeral pools [Nothobranchius populations are finely structured in small, short-existing pools in dry inland region of southern Mozambique [Nothobranchius fishes occurring in extensive marshes and semi-permanent streams [Nothobranchius fishes are also present in the elevated part of equatorial East Africa (> 800 masl) and it remains to be tested how local populations are structured there. In that region, local topography and geographic history do not support as frequent dispersal as in coastal equatorial regions [al pools . Howeverzambique , 42 wherzambique . In contzambique , 10, 44, streams , 16, 57.Distribution of genetic lineages of annual fishes from a wet part of the genus distribution  appears not to be constrained by dispersal limits posed by main river channels and closely related lineages frequently coexist in secondary contact zones. Annual fishes are promising research system for understanding links between ecological and evolutionary processes , 58 and Additional file 1: Protocols for genotyping of microsatellites and mitochondrial DNA. Table S1. Used microsatellite loci. Table S2. Primer sequences and marker features in GRZ strain of N. furzeriAdditional file 2: Additional figures supplementing results. Figure S1. Bayesian reconstruction of mitochondrial phylogeny of the N. melanospilus species complex based on 83 ingroup and 1 outgroup haplotypes of the 657 bp fragment of mitochondrial gene COI, including identity of individual samples. Bayesian inference posterior probabilities from MrBayes 3.2.6 are shown for each node. Figure S2. Correlation between Ln (distance) and linearized pairwise FSTENA values (FST/(1 − FST)) tested by the Mantel tests (1000 permutations) analysed in GENEPOP. Figure S3. Mismatch distribution for two widespread mtDNA lineages. Dashed lines connect observed values and solid lines show the expected distribution under a demographic expansion modelAdditional file 3: Additional table supplementing results. Table S3. Pairwise FST estimates based on 10 microsatellite lociAdditional file 4: Evaluation of 20 runs in STRUCTURE 2.3.4  for each number of presumable clusters from K = 1 to K = 10. Figure S2. Likelihood (ln Pr(X|K)) of models in STRUCTURE for increasing number of hypothetical populations (K). Figure S3. Estimation of the best K division using the ΔK criterion according to Evanno et al. (2005). The values indicate relative increase of credibility depending on the number of K.