Oreochromis niloticus) is a globally significant aquaculture species rapidly gaining status as a farmed commodity. In West Africa, wild Nile tilapia genetic resources are abundant yet knowledge of fine-scale population structure and patterns of natural genetic variation are limited. Coinciding with this is a burgeoning growth in tilapia aquaculture in Ghana and other countries within the region underpinned by locally available genetic resources. Using 192 single nucleotide polymorphism (SNP) markers this study conducted a genetic survey of Nile tilapia throughout West Africa, sampling 23 wild populations across eight countries , representing the major catchments of the Volta, Niger, Senegal and Gambia River basins. A pattern of isolation-by-distance and significant spatial genetic structure was identified throughout West Africa , which largely corresponds to major river basins and, to a lesser extent, sub-basins. Two populations from the Gambia River , one from the western Niger River (Lake Sélingué) and one from the upper Red Volta River (Kongoussi) showed markedly lower levels of diversity and high genetic differentiation compared to all other populations, suggesting genetically isolated populations occurring across the region. Genetic structure within the Volta Basin did not always follow the pattern expected for sub-river basins. This study identifies clear genetic structuring and differentiation amongst West African Nile tilapia populations, which concur with broad patterns found in previous studies. In addition, we provide new evidence for fine-scale genetic structuring within the Volta Basin and previously unidentified genetic differences of populations in Gambia. The 192 SNP marker suite used in this study is a useful tool for differentiating tilapia populations and we recommend incorporating this marker suite into future population screening of O. niloticus. Our results form the basis of a solid platform for future research on wild tilapia genetic resources in West Africa, and the identification of potentially valuable germplasm for use in ongoing breeding programs for aquaculture.Nile tilapia ( Oreochromines are a sub-family of the Cichlidae family of fishes native to Africa and parts of the Levant that have large commercial significance for aquaculture and capture fisheries1. The Nile tilapia, Oreochromis niloticus, has a broad natural distribution spanning from the Nile River basin southwards through the Eastern and Western Rift Valley lakes in East Africa, and westwards through the basins of Lake Chad, Niger, Benue, Volta, Gambia and Senegal rivers1. The species has been well studied from a taxonomic perspective. A complex sub-species structure and distribution of O. niloticus has been documented, especially across East Africa where a network of geographically and biologically diverse lakes have shaped its evolution. Seven sub-species of O. niloticus have been identified based on traditional taxonomic methods; all except one – O. niloticus niloticus - are distributed amongst the Rift Valley lakes of East Africa1.The O. niloticus5, which can be generalized into three main groups: (i) Sudano-Sahelian populations, covering West Africa, (ii) Ethiopian Rift valley populations and (iii) Nile drainage and Kenyan Rift Valley populations6. A hierarchical pattern of genetic differentiation has been observed in O. niloticus, whereby the effects of major paleo-geographic and climatic events are the predominant factor at the macro-geographic scale, and present-day river systems are influential on genetic connectivity and patterns of genetic structure at smaller geographic scales5. The same authors have also suggested limited dispersal capabilities combined with non-random mating caused by social behaviours may also influence small scale population structure. Other studies indicate that isolated and previously undescribed O. niloticus populations in Kenya can show strong genetic differentiation from other nearby populations, and may constitute new wild genetic resources with potential importance for exploitation in commercial aquaculture activities7. Furthermore, studies of patterns of mitochondrial DNA variation have revealed additional complexities amongst O. niloticus populations, with evidence of historical hybridization occurring between closely related O. aureus8 and O. leucosticus9 species occurring in the western and eastern regions of Africa, respectively.Molecular studies have identified clear differentiation between west and east African populations of O. niloticus in East Africa, relatively few studies have focused on populations throughout West Africa with high resolution. This is despite West Africa being the largest ichthyoregion naturally occupied by O. niloticus. Earlier studies have indicated genetic differentiation at broad scales (i.e. 1000’s of km)5. However, spatial coverage of these studies is limited and consequently knowledge of finer scale patterns of genetic structure of O. niloticus in West Africa remains uncertain.Although much attention has been given to the population characterization and genetic structure of Oreochromis niloticus is by far the most prominent amongst tilapia species produced by aquaculture, being the most farmed tropical fish species globally. Approximately 4.1 million tonnes of Nile tilapia were produced by aquaculture in 2017, having a value of US$7.6 billion10. West African O. niloticus populations have made a significant contribution towards the development of farmed genetic resources that underpin much of global production. For example, at least four of the eight founder populations (and likely to be five) of the highly successful Genetically Improved Farmed Tilapia (GIFT) strain of O. niloticus were either sourced directly from wild populations in Ghana and Senegal or cultured populations derived from stocks of Ghanaian origin11. The GIFT strain has undergone continuous selective breeding in Southeast Asia since the early 1990’s, and has been widely recognized as having substantial impact on tilapia aquaculture development across the region13. Additionally, a selective breeding program based on locally sourced wild O. niloticus is rapidly gaining popularity by aquaculture farmers in Ghana and bringing economic benefits and boosting primary productivity for the country16. Such successes demonstrate the potential value that can be derived from these wild genetic resources provided the appropriate management and technological approaches are applied.O. niloticus is similarly increasing17. The historic popularity of Nile tilapia as a capture fishery and for aquaculture production has seen its repeated translocation and deliberate introduction to many places where it was not originally found. The genetic origin of such introductions are often vague or unknown, yet may have an important impact on the genetic composition of wild populations.Tilapia aquaculture is poised for rapid growth throughout Sub-Saharan Africa in the coming years. As tilapia aquaculture increases throughout Sub-Saharan Africa, an impetus to better understand the distribution of wild genetic resources and patterns of population structure throughout the natural distribution of The advantages of investigating molecular genetic characteristics of wild Nile tilapia are two-fold. Firstly, understanding wild population structure can assist the management or safeguarding of wild genetic resources, particularly in the context of the potential impacts caused by aquaculture escapes. Secondly, the identification of genetically distinct and diverse wild populations is of potential benefit to aquaculture breeding programs, which require a broad genetic diversity in their founder populations to achieve sustained productivity improvements and avoid problems of inbreeding accumulation. By providing insights to both the management and use of tilapia genetic resources, molecular genetic data offers better understanding in a climate where large-scale aquaculture development in West Africa is growing and proper resource usage is critical.This study presents the opportunity to understand patterns of genetic structure and factors affecting diversity across the region, to better document the natural Nile tilapia genetic resources in West Africa, and further build on previous research to understand the potential macro or micro geographic factors influencing their diversity. Using single nucleotide polymorphism (SNP) markers, we studied 23 populations across five river basins in West Africa to assess spatial patterns of genetic diversity and genetic differentiation of indigenous wild Nile tilapia populations. The specific aims of this study are to: (1) Characterize genetic diversity of Nile tilapia populations throughout West Africa using SNP markers; (2) Quantify the relative differences of genetic structure in Nile tilapia populations within versus among watershed basins throughout West Africa; (3) Explore the genetic characteristics of Nile tilapia populations not recognized as part of its natural distribution but are known locally to be sources of the species; and (4) Identify potential factors contributing to patterns of genetic diversity and differentiation in West African Nile tilapia populations. We anticipate this will provide a clearer insight towards what populations may be of potential value for conservation or further use in ongoing aquaculture expansion plans for the region.O. niloticus populations were sampled from across eight countries  of West Africa, representing the major catchments of the Volta, Niger, Senegal and Gambia River basins  as per the manufacturer’s instructions, and stored at −20 °C until later processing. DNA extraction was conducted at the laboratory of the International Centre for Research and Development on Livestock in Subhumid Zones (CIRDES), Burkina Faso. Samples were standardized to a DNA concentration of 50 ng ulO. niloticus18. Genotyping was performed in a 192 SNP multiplex assay using the Golden Gate Assay (Illumina), and deployed on a BeadXpress platform using Veracode technology. A total of 15 SNPs on the assay were located on chromosomes/linkage groups where there are known associations with sex determination  in Nile tilapia20, however, these SNPs used were not located in the vicinity of these sex-linked regions (data not shown) and therefore have minimal risk of biasing results of the study. A minimum call rate of 0.8 was used to score genotypes. A full list of the SNP markers used, the linkage group according to Guyon et al.21 and the dbSNP accession number of each SNP are given in the Supplementary Materials section.Multi-locus genotypes were generated from 192 SNP markers, selected from a suite of 384 SNP markers developed for adegenet22. Allelic richness of each population was calculated using the R package hierfstat23 based on a minimum sample size of 15 individuals, which accounts for differences in sample size and genotype failure. Tests for Hardy-Weinberg Equilibrium (HWE) were performed at each locus for each population using the HWE.test.genind function of adegenet, based on the Chi-squared statistic. P-values of each test were adjusted within population according to the false discovery rate method of Benjamini and Hochberg24 using the p.adjust function in R25, to an equivalent significance level of α = 0.05.Number of polymorphic loci, observed heterozygosity and expected heterozygosity were calculated for each population using the R package 26, as implemented in NeESTIMATOR v2.0127. A random mating model was applied, using a PCRIT  of 0.05.Effective population size and its 95% confidence interval were estimated for each population using the linkage disequilibrium method of WaplesF-statistics  were estimated using ARLECORE, the console version of ARLEQUIN v3.528, to determine the proportion of genetic variation partitioned within and among various levels of population sub-structure . Pairwise FST between populations were also calculated using ARLECORE, with the significance of pairwise values tested using a non-parametric Monte Carlo approach (1000 permutations). A Bonferroni correction was applied to P-values using the p.adjust function in R25 to control the likelihood of type I errors associated with multiple comparisons. To investigate the significance of major geographical factors such as river basins (watersheds) on genetic structuring in Nile tilapia, an hierarchical Analysis of Molecular Variation (AMOVA) was implemented using ARLEQUIN. Several models were tested based on different groupings of populations representing reasonable explanatory drivers of genetic differentiation. The aim here is to explore what groupings could better explain the observed genetic variation when partitioned amongst groups (FCT), amongst populations within groups (FSC) and amongst populations (FST). The population were grouped by (1) major river basins; (2) major river basins and sub-basins (where present); and (3) climatic zones.Wright’s FST) was done based on 10 000 permutations using the mantel.randtest function in adegenet. Geographic distances used are straight line distances between population sample sites calculated from longitude and latitude coordinates using the earth.dist function of the R package fossil29.To test if genetic differentiation patterns followed an isolation-by-distance model of population divergence, Mantel’s test for correlation between geographic distances (km) and genetic distances  of population allelic frequencies30. This method allows the analysis of both variability in allele frequencies and spatial autocorrelation amongst populations. sPCA was conducted using the spca function of adegenet22. Spatial information was incorporated into the analysis based on a Gabriel graph connection network of population sample sites. Monte-Carlo tests for the presence of significant global and local spatial structure were performed using the global.rtest and local.rtest functions of adegenet, respectively, based on 9999 permutations. Here, global structure refers to positive spatial autocorrelation (i.e. populations closer to each other are more similar) and local structure refers to negative spatial autocorrelation (closer populations are more dissimilar). Because of the potential sampling biases in allele frequencies caused by the presence of rare variants, the dataset was trimmed to use only those loci that had a minor allele frequency (MAF) greater than 0.01 across all individuals.Spatial patterns of genetic variability in K) that conform to expectations of Hardy-Weinberg equilibrium and linkage equilibrium between loci32. An admixture model with correlated allele frequencies was chosen to estimate the group membership probability (Q) of each individual for K = 2 to K = 20. For each level of K ten replicates of 300 000 Markov-Chain Monte Carlo iterations were run after a 150 000 iteration burn-in period. Default parameters were used otherwise. To identify the most likely number of clusters the Evanno method33 was implemented using STRUCTURE HARVESTER34. CLUMPP35 was used to align clusters across replicate runs and calculate a mean Q estimate for each individual, which were visualized using DISTRUCT36.The Bayesian clustering approach implemented in the software STRUCTURE v2.3 was used to partition genotype data according a predefined number of groups (37. DAPC creates synthetic variables (discriminant functions) that attempt to maximize differences between groups or genetic clusters whilst minimizing allele frequency variance within a cluster. It has a major advantage in that no population genetic assumptions (such as Hardy-Weinberg expectations) are required to describe patterns of genetic variation across individuals and clusters. Prior definitions of groups are required for DAPC, which were done using the find.clusters function of adegenet to implement a k-means clustering algorithm. The function also generates a Bayesian Information Criterion (BIC) for each value of k, which was then used to identify the optimal value of k. Ideally the BIC will decrease until an optimal k is reached and then subsequently increase for greater values of k. DAPC was performed for values of k increasing from 2 to 20 using the dapc function of adegenet, retaining 75 principal components and 6 discriminant functions for each value of k. The trimmed dataset containing loci with a MAF greater than 0.01 was also used for this analysis, for the same reasons described earlier.An alternative approach to identifying genetically distinct clusters is through Discriminant Analysis of Principal Components (DAPC)A total of 192 SNPs were genotyped for all 1001 samples. Four samples were removed from the final dataset due to genotyping failure. Eight SNPs were monomorphic and seven SNPs had ≥15% missing genotypes, and were subsequently removed from the final dataset. A total of 177 SNP markers were used for the final analyses, unless otherwise stated. Fifty four SNP markers had a minor allele frequency (MAF) < 0.01.s), a measure of the average number of alleles per locus taking into account potential biases due to differences in sample sizes, ranged from 1.30 to 1.62. Observed heterozygosity was very similar to expected heterozygosity in all populations when averaged across loci (Table P < 0.05), with the exception of Nawuni in northern Ghana (5.2 percent loci not in HWE) and the Afife Reservoir (15.1 percent).Genetic diversity was lowest in the Kudang, Walekounda (both located in the Gambia River basin), Lake Sélingué (western Niger basin) and Kongoussi (Red/White Volta basin) populations. Populations that showed the greatest level of genetic variation were from Malanville (eastern Niger basin) and two populations from the southern region of Ghana (Site VL2 from Lake Volta and the Afife Reservoir in southern Ghana). Total number of alleles within each population ranged from 257 to 345, which translated to an overall percentage of polymorphic loci by population ranging from 33.8 to 79.7 percent. Allelic richness (RNe) was in general positively correlated with genetic diversity measures. Over 50 percent of populations sampled had Ne in excess of 100 and none below 50. An exception to this pattern was the Kongoussi population in northern Burkina Faso, which showed the greatest Ne (351.6) yet had comparatively low heterozygosity and allelic richness (Table Effective population sizes (ss Table . Full dep < 0.001), with 14.4% of overall genotypic variation attributable to differences among populations (i.e. FST.) when no hierarchical sub-groupings were considered other than the individual and population level (Table FCT) accounts for up to 13.2 per cent of overall variation , two populations from the Gambia River (GKu and GWa), and two populations from the Oti River in the Volta Basin (VOt and VPj), which showed no significant differences to each other after Bonferroni correction. Clear patterns of genetic differentiation are observed between the Gambia River populations and all other populations sampled  and geographic distances , indicating isolation-by-distance is a moderate influence on genetic differentiation at the regional scale . Consistent with this, sPCA revealed significant global spatial structure (p = 0.03) and non-significant local structure (p = 0.97) overall, indicating the genetic similarity of populations are positively correlated with their geographic proximity to each other. Within the Volta Basin, however, a significant yet much weaker relationship between genetic and geographic distances was present , indicating isolation-by-distance is a relatively minor factor shaping genetic differentiation amongst populations. Patterns of genetic differences and similarity across West Africa are evident when the lagged scores of the first three principal components of the sPCA are spatially plotted  for the Bayesian approach was four, based on the Delta K method33, although a secondary peak in Delta K is also seen for k = 13 which could indicate a more fine scale genetic structuring. Changes in BIC showed that an optimum value of k to use for DAPC is likely to fall between 10 and 15. Detailed changes in BIC and Delta K, and the comparison between DAPC and STRUCTURE results can be found in the Supplementary Materials. Clustering at the lowest level (k = 2) shows a distinction between the Volta Basin populations and all others, particularly for the DAPC analysis  increases, individual clustering follows closely according to river basin. Within the Volta Basin, four genetic clusters can be observed and can be described as a Lake Volta cluster , an Oti River cluster, a Black Volta cluster and a Red/White Volta cluster. The relative differences amongst clusters identified with DAPC is visualized for k = 10 in Fig. Bayesian (STRUCTURE) and multivariate (DAPC) based approaches revealed highly consistent patterns of individual genetic clustering for differing values of f k Fig. . The mossis Fig. . As the O. niloticus across West Africa can generally be explained by river basins, and to some extent, sub-basins, with gene flow amongst populations following patterns of isolation-by-distance. However, genetic similarity in populations that are over 1400 km apart connected by the Niger River  provides evidence that sufficiently high gene flow along a large, continuous waterway can occur over genetically relevant timescales. In contrast, within a much shorter distance around the Red and White Volta River regions we see genetic heterogeneity among several populations , indicating population differences can persist across relatively nearby geographies.The multiple analyses conducted in this study all corroborate a pattern of spatial genetic structure and differentiation of Nile tilapia populations across West Africa. The broad scale isolation-by-distance patterns are consistent with the significant spatial correlations of population allele frequencies identified by sPCA. Genetic differentiation of 8, who observed O. niloticus populations from Senegal to be most divergent from other West African populations, we observe a genetic similarity between populations from Senegal and those from Upper and mid-Niger River. This is consistent with the belief that following the last pluvial, around 12,000 to 7,500 years ago, the Senegal and Gambia Rivers were recolonized by freshwater fauna from the Niger Basin38. Similarly, the Black Volta and the Pendjari Rivers are likely to have been tributaries of the Niger River in the past38, which may explain the moderate genetic differentiation observed among sampling sites covering the three main tributaries of the Volta Basin.Our study reveals additional population complexity that has not been documented previously. The most divergent populations amongst those sampled were from Lake Sélingué in the Niger Basin and from the Gambia River, which showed strong differentiation to all other populations. Unlike Rognon & Guyomard18 and in two selectively bred tilapia populations39, albeit using SNP arrays not necessarily optimized for informativeness in West Africa. Lower genetic diversity in the Gambia River and the far western region of the Niger River is consistent with patterns often seen at a species range limit or isolated populations. Rognon & Gouyomard8 found decreased enzyme diversity in Nile tilapia samples from the Senegal River and cited founder effects at the margins of the distribution as the likely explanation. Founder effects may explain the pattern of genetic differentiation and reduced diversity identified in the Gambia River. In contrast, however, we found relatively high levels of SNP diversity in the Senegal River population, which may indicate this region is supporting larger, more stable populations compared to the nearby Gambia River. This is corroborated by differences in Ne, which in the absence of gene flow would lead to differing rates of diversity loss through genetic drift. Alternatively, the Senegal River may have had more frequent, intermittent historical connectivity to the Niger Basin40, possibly modulating the impacts of genetic drift on diversity loss. Two populations at Lake Sélingué in the Upper Niger River, and Kongoussi in the Upper Red Volta River also show a substantially lower percentage of polymorphic loci than others. This may be a result of their geographic location in the upper reaches of their respective river systems causing relative isolation. Consequently, limited gene flow between other populations or increasing exposure to seasonal fluctuations and intermittence in water flow could explain a likely increase the rate of genetic diversity loss caused by genetic drift.We find genetic diversity using SNPs to be similar across the majority of populations sampled, with the exception of several populations exhibiting lower genetic diversity than others. Most populations showed greater SNP heterozygosity than previously reported for the Volta Basin41. Lake Volta, created after the completion of the Akosombo Dam in 1965, is the largest man-made lake in the world by surface area and spans 8,502 km2. Despite such vast areas and the likely enormous expansion of Nile tilapia populations throughout the lake after its formation we detect no evidence for departures from Hardy-Weinberg Equilibrium (HWE) and little genetic differentiation amongst any Lake Volta populations, with some over 400 km apart. This could mean that there has either been a limited founder effect on Nile tilapia populations as a result of the formation of Lake Volta and that genetic structuring of these populations has not changed. Alternatively, any genetic differentiation prior to or post Akosombo Dam construction has been homogenized by sufficient gene flow across the lake and equilibrium of allele frequencies has been reached. Across all sites, only the population from the Afife Reservoir exhibited notable HWE departures. This site is located adjacent to the mouth of the Volta River, in the Kloukpa River basin, and is not thought to part of the natural distribution of Nile tilapia1. A departure from HWE coupled with moderate genetic differentiation and relatively low effective population size seen in the Afife Reservoir is a possible indication of founder effects due to being recently established, likely from nearby Volta River populations.Rapid population expansions as a result of the new water bodies created by dams can potentially have pronounced effect on the genetic properties of a population5. Our study identifies a greater overall FST amongst populations within the Sudano-Sahelian region than previous estimates based on microsatellite markers (RST = 0.09)5. This could be due to a greater sampling coverage, particularly the inclusion of populations from the Gambia River, which are highly differentiated from all other populations sampled. This, however, may also be due to differences in expected heterozygosity derived from highly polymorphic microsatellite markers compared to dimorphic SNP markers, which can influence the upper bounds of possible FST values that can be obtained42. Our study presents the first estimate of population diversity based on SNP markers in West Africa, providing an important baseline as SNP increasingly become the marker of choice for population studies and for commercial aquaculture breeding programs.Previous population genetic studies of Nile tilapia have identified major breaks across its natural distribution, delineating three macro-geographic groups: (i) Sudano-Sahelian populations, covering West Africa, (ii) Ethiopian Rift valley populations and (iii) Nile drainage and Kenyan Rift Valley populations43, contrasting to temperatures that rarely extend beyond 21 to 34 °C in southern Ghana44, which highlights the vastly different thermal amplitudes within the natural range of O. niloticus. Although hierarchical AMOVA based on grouping of populations according to their climatic zone did not explain as much variation as groupings based on river basins, the presence of pockets of genetically isolated populations throughout the Sahel region identified in this study, such as Lake Sélingué and Kongoussi, are of particular interest in this context. Notably, the population at Kongoussi may likely experience fluctuations in environmental conditions substantially greater than the populations in the main rivers due to low water exchange, extended periods of zero rainfall and its shallowness across that region. Genetically unique populations that are potentially valuable genetic resources have been recently discovered in other regions of Nile tilapia’s natural distribution, notably in East Africa7. Results such as this provide a basis for more in-depth investigation into whether genetically distinct populations represent potential genetic resources for future utilization in commercial breeding programs. Future genetic studies would be well placed to target greater sampling intensities and finer-scale geographic coverage around areas where genetically distinct populations are present. Complementary to this, research focusing on understanding whether such differentiation confers to any physiological differences at the population level would be especially valuable.Climatic fluctuations in the Sahel region can be dramatic throughout the year, especially compared to the tropical regions of southern Ghana, which may exert greater selective pressure for commercially significant traits such temperature and salinity tolerance. Temperature ranges from 12 to 47 °C throughout the year in Burkina Faso48. Knowledge of local population genetics developed through studies such as this (possibly utilising more targeted SNP genotyping tools) have an important role to detect, analyse and help manage the potential the impacts of likely escapes. Recent work identifying species-specific SNP markers among closely related tilapia species would also be of value where geographical overlaps occur, and there is potential for hybridization49.In addressing questions of genetic diversity and genetic structuring of West African Nile tilapia populations, the potential implications on the growing tilapia aquaculture industry throughout the region must be considered. We show there are high levels of genetic diversity amongst populations located across most major water basins, and identify some populations that are highly differentiated from others, indicating the presence of genetically unique populations. Our results suggest there is ample genetic diversity throughout wild Nile tilapia populations in West Africa to support the establishment and long-term development of selective breeding programs for aquaculture in the region. The consequence, however, of a large aquaculture industry based on selectively bred, domesticated populations, is that widespread use and distribution of these animals is inevitable. This may have potential impacts on natural populations when farmed animals unintentionally escape, and is a growing issue in other parts of Africa8. In addition, this study provides new evidence for fine-scale genetic structuring within the Volta Basin and for further genetically differentiated populations in Gambia. Spatial genetic patterns and individual clustering methods indicate genetic structure can be largely explained by major rivers and sub-basins throughout the region. The 192 SNP marker suite used in this study is an effective tool for differentiating tilapia populations and we recommend incorporating this marker suite into future population screening of O.niloticus, potentially in conjunction with other species-specific SNPs. Our results form the basis of a solid platform for future research of wild tilapia genetic resources in West Africa, and the identification of potentially valuable germplasm for use in aquaculture breeding programs.This study identifies clear genetic structuring and differentiation amongst West African Nile tilapia populations, which concur with broad patterns found in previous studiesDataset 1Supplementary Info 2Supplementary Info 3


Plasmodium falciparum malaria remains a major health challenge in Nigeria despite the global decline of its incidence and mortality rates. Although significant progress has been made in preventing the transmission of P. falciparum and controlling the spread of the infection, there is much to be done in the area of proper monitoring, surveillance of the parasite, investigating the population dynamics and drug resistance profiling of the parasite as these are important to its eventual eradication. Polymorphic loci of msp1, msp2 and/or glurp genes or microsatellites have been traditionally used to characterize P. falciparum population structure in various parts of Nigeria. The lack of standardization in the interpretation of results, as well as the inability of these methods to distinguish closely related parasites, remains a limitation of these techniques. Conversely, the recently developed 24 single nucleotide polymorphism (SNP)-based molecular barcode assay has the possibility of differentiating between closely related parasites and offer additional information in determining the population diversity of P. falciparum within and between parasite populations. This study is therefore aimed at defining the population diversity of P. falciparum in and between two localities in Nigeria using the SNPs barcode technique.msp2 genotyping was used to investigate both intra and inter population diversity of the parasite population in two urban cities of Nigeria.The 24-SNP high-resolution melt (HRM) barcode assay and msp2 analyses showed 21% and 19.4% polygenomic infections in Enugu and Ibadan, respectively. Low levels of genetic diversity (π) of 0.328 and 0.318 were observed in Enugu and Ibadan parasite populations, respectively, while the FST value of 0.02 (p = 0.055) was obtained when the genetic divergence of both populations was considered.Based on SNP barcode analysis, polygenomic malaria infections were observed in 17.9% and 13.5% of population from Enugu and Ibadan, respectively, while P. falciparum population diversity. This study also showed that P. falciparum populations in Enugu and Ibadan had a degree of intra-population diversity, but very low divergence between the population. A low degree of polygenomic infections were also observed in the two parasite populations unlike previous years. This maybe as a result of the effect of artemisinin-based combination therapy (ACT), long-lasting insecticide-treated nets (LLITNs) and intermittent preventive treatments in the study populations.The 24-SNP barcode assay was effective in analysing  Plasmodium falciparum is still a major health challenge in Nigeria despite the global decline of its incidence and mortality rate [Malaria caused by ity rate . Malariaity rate . Even wiity rate . As suchity rate .msp1), merozoite surface protein 2 (msp2) and glutamine rich protein (glurp) to establish the population features of P. falciparum in various parts of Nigeria is well documented [P. falciparum, have also been used in characterizing P. falciparum field isolates with good success [The use of molecular markers such as the merozoite surface protein 1  analysis [P. falciparum and has the ability to differentiate between closely related parasites. SNP genotyping approaches have also been used to evaluate interventions, pursue malaria elimination and monitor malaria transmission [P. falciparum barcode also uses separate probes for each assay, allowing better peak differentiation for more accurate SNPs genotyping, which makes it different from other HRM applications. The gradual increase in temperature during the melting stages of the HRM allows for the detection of slight genetic differences like SNPs. A major advantage the HRM SNP barcode has over the use of molecular polymorphic markers such as msp1, msp2 and glurp is the ability to detect changes in population structure such as decline or increase in transmission rate or relatedness of isolates based on identity by descent. However, this technique is not as effective in differentiating reinfection/recrudescence as in the polymorphic markers i.e., msp1 and msp2. Also, HRM is relatively easy to use, simple, flexible, low cost, non-destructive in nature, very sensitive and specific [The malaria barcode  is a comanalysis  is more smission , 11. HRMsmission . HRM is smission . The melspecific .P. falciparum parasites in Senegal [P. falciparum in Panama [P. falciparum obtained from blood samples of children with uncomplicated malaria, to have an insight to the population structure of the parasite.The 24-SNPs barcode assay can be used to detect changes in parasite population features by tracking parasite genotypes over time and through routes of vector and human migration . For ins Senegal , interro Senegal  and inven Panama . HoweverSamples used in this study were obtained from two major urban cities, Ibadan and Enugu in Nigeria Fig. . Ibadan This study was part of a larger drug efficacy study investigating the effect of artemether-lumefantrine (AL) and artesunate-amodiaquine (AA) in the treatment of uncomplicated malaria infection. Two drops of finger prick blood was blotted on 3 mm Whatmann filter paper  before treatment and during follow up on days 3, 7, 14, 21 and 28. However, DNA was extracted from dried blood spot on filter papers obtained before treatment only and was used for this parasite diversity study.DNA was extracted from dried blood impregnated filter paper using Qiagen DNA extraction kit and according to the manufacturer’s protocol. Briefly, one-quarter of the dried blood spot was used for extraction and DNA content was eluted in a final volume of 60 μl with buffer AE (Qiagen DNA extraction kit).msp2 gene was amplified by nested PCR following protocols described by Happi and others [msp2 alone were used for characterization of parasite population structure in patients’ samples because previous studies from Nigeria have shown that it is the most reliable marker for studying P. falciparum population diversity in the country [The polymorphic block 3 of the d others . PCR rea country , 15–18. P. falciparum HB3 clone genomic DNA (MR4 stock number MRA-155) was used as control. A pre-amplification step was then performed on samples with parasite DNA concentration lower than 0.1 ng/µl [We initially quantified parasite genomic DNA by qPCR against a standard curve of known concentrations. All quantifications were done in triplicates and .1 ng/µl . All sam.1 ng/µl .The HRM assay was run on the Roche LightCycler 480 Real-Time PCR System using the manufacturer’s settings and the cycling conditions as stated by Daniels and others [Prior to analysis of the raw barcode data, proper clean up measures were taken to ensure that only high quality data were considered for further analysis. Two (2) major parameters were used to clean the data, these were: (i) only ATCG, N (mixed infection) or X (missing SNP) should be present in the barcode data (ii) and samples with barcodes containing more than 4 missing SNPs (X) were excluded from the analysis.Polygenomic infections was established in the parasite population by examining the number of heterozygous SNPs (N) in each sample assayed. It is popular knowledge that the human blood-stage malaria parasites is haploid, therefore, a monogenomic infection should have only one allele at each SNP locus while a polygenomic infection is expected to carry multiple alleles. Therefore, in categorizing the parasite population as polygenomic, a minimum threshold value of at least two (2) heterozygous SNPs (N) was utilized as defined by Sisya and others . This isMinor allelic frequency (MAF) was computed according to methods earlier described by Baniecki et al. . BrieflyThe population diversity (π) was calculated as described by Baniecki et al. . BarcodeST) for all pairs of populations. The online software, COIL  was used to determine the FST for both populations.The population divergence was measured by calculating the fixation index (F) was performed with the online program, ClustVis (https://biit.cs.ut.ee/clustvis/) on each of the parasite populations (Ibadan or Enugu) separately as well with both populations together.Principal component analysis  were run on HRM to achieve a full 24 SNP barcode. Coverage for 23 out of the 24 barcode assays were observed, as one was faulty, i.e., all samples and controls assayed for that SNP were negative. Omitting this, the overall data still included a rather high (25%) negative (X) genotyping calls. Therefore, data cleaning was essential for obtaining useful data for population diversity analysis. Overall, 37 and 28 samples from Ibadan and Enugu, respectively, produced barcodes with sufficient data quality and were considered for further statistical analyses, giving us a total of 65 samples.msp2 genotyping technique, from which 74 samples were successfully analysed. Analysis showed that 19.4% (7 of 36) and 21% (8 of 38) of the samples obtained from Ibadan and Enugu, respectively were polygenomic. The complexity of infection (COI) using msp2 polymorphic marker were 0.73 and 0.52 in Enugu and Ibadan, respectively. Based on analysed SNP barcode data, 17.9% (5 of 28) and 13.5% (5 of 37) of samples from Enugu and Ibadan, respectively were polygenomic.A total of 100 samples were tested using the The minor allelic frequency (MAF) values for samples obtained from both populations showed that these populations are highly diverse. In both populations, only two SNPs had MAF values lower than 0.1: Enugu: assay 8 and 24, Table .Table 12The intra population diversity values for Enugu and Ibadan were 0.328 and 0.318, respectively. The calculated π statistic shows that these populations are slightly diverse.ST) value of 0.02 (p = 0.0549) suggests that both populations are not significantly genetically diverse from each other. Thus, confirming the similarity between the MAF values of the two populations.The population divergence . When the PCA was performed on each parasite population, the points showed more divergence with small clustering of the parasites in each study site Fig. a, b espeP. falciparum samples in Nigeria. The HRM technique has been used to evaluate parasite population dynamics of P. falciparum in several parts of the world [P. falciparum in samples obtained from representative parts of the country has served as an indicator for transmission patterns of the parasite within the country and influenced various government control and intervention measures such as adoption of ACTs for malaria treatment, rapid diagnosis of all malaria cases, wider distribution of LLIN and quality monitoring of malaria cases to ensure the progressive control of malaria in Nigeria [This study present the very first application of the 24-SNPs molecular barcode in effectively measuring inter and intra population diversity for he world , 10, 20.he world . The knohe world . High cohe world . Over thP. falciparum transmission within and between various parts of the country. For instance, Engelbrecht et al. [msp2 genotyping technique, reported polyclonal infections at 80% from samples obtained in the Northern region of Nigeria. Previous studies in Ibadan using msp2 reports 89–100% polygenomic infections in samples obtained prior to treatment [Previous molecular epidemiological studies showed high levels of polyclonal infections especially in malaria hyper endemic parts of the country, thus indicating high t et al.  using threatment , 25. Howreatment , 26.msp2 analysis in this study for both populations (Enugu: 0.73 and Ibadan: 0.52) suggest a low genetic diversity. This shift to monogenomic infections may be due to present use of ACT in the country, which has gametocide activity as well as capability to reduce parasite load rapidly. Thus, causing a reduction in the transmission rates and coupled with other malaria control efforts such as use of LLIN and IPT for children and pregnant women in the country.Observations in this study from the two parasite populations also revealed a similar trend of higher percent of monogenomic infections. The complexity of infection values obtained by msp2 [HRM has been reported to be a better genotyping technique over the use of length polymorphic markers such as msp2 , howevermsp1, msp2, and glurp is that the HRM is a more robust genotyping technique having the ability to resolve two closely related samples [A major advantage the HRM technique has over other genotyping techniques such as  samples . This isP. falciparum clones originally present in these regions. The high prevalence of monoclonal infections observed in this study as well as other parameters determined in this study further supports evidence of the low genetic diversity of P. falciparum in these locations.Parasite population diversity parameters such as the AMAF and barcode π provide further insight to the nature of parasite diversity within Enugu and Ibadan. Baniecki et al.  had suggP. falciparum population between Ibadan and Enugu was investigated by computing population divergence features such as fixation index (FST) and principal component analysis (PCA). When SNP barcode data of two populations are analysed the FST value ranges from 1 (high divergence) to 0 (no divergence) [ST value of 0.02 between Ibadan and Enugu parasite populations was observed. This suggests that there is low genetic variation in circulating P. falciparum parasites of both populations. Although these two regions are over 500 km away from one another, they are by no means isolated thus encouraging circulation of same parasite population. The lack of genetic variance observed in this study could be due to constant and ease of travel between the two cities, similar epidemiological conditions in both areas and/or similar selective pressure from the various control and management measures within the country at large. A recent study has shown that great geographical distance, even at the continental level, does not necessarily result in high level of genetic divergence between populations [P. falciparum in Enugu and Ibadan. A similar clustering patterns were observed between the parasite populations  . In thisulations . The PCAons Fig. .Based on the observations of low genetic divergence between Enugu and Ibadan populations as well as the low intra population diversity in each population, its suggested that HRM technique, although may be more expensive can be used to investigate the current trend of population diversity and divergence in other parts of the country. This would provide sufficient information about the composition of parasite populations across Nigeria and significantly influence controls measure and intervention for the spread of malaria.msp2 and HRM techniques were to a large extent, monogenomic. This may suggest that the current malaria transmission rate in each of these regions is also low. The HRM method has proven to be a good genotyping tool and can reliably resolve single nucleotide changes. Furthermore, this 24-SNP barcode may be a useful tool for general surveillance, as continued cyclical screening of malaria-positive samples in regions across Nigeria will be invaluable for tracking major mutations and population-level trends. It can also provide data that will help in tracking malaria transmission and parasite evolution. Elucidating these parasites and population level differences using the 24-SNP barcode can inform clinicians’ treatment decisions, equipping them to recommend different sources of treatment with information beyond individual patient data and symptoms.In summary, results from this study showed that the two populations, Enugu and Ibadan, had a low degree of intra-population diversity, as samples analysed using both P. falciparum population diversity and divergence in Nigeria. Future work is to utilize this assay to build on this characterisation of parasite populations in regions of varying endemicity across the nation. Also, reports from other studies has shown that this technique can be used to monitor changes in parasite population diversity and clonality over time, transmission patterns, and emerging drug resistance which were not considered in this study and could be included in future studies in Nigeria. By building a more complete snapshot of P. falciparum infection, policy makers, drug developers, and healthcare professionals can be empowered to take informed action to further the goal of malaria elimination in Nigeria.This work demonstrates, that the 24-SNP HRM barcode can effectively be used to monitor


Goats are bred worldwide and present in a wide variety of production environments. Local breeds, which are well adapted to a range of agro-ecological conditions, contribute to ensuring the sustainability of livestock farming in marginal and difficult areas in both developed and developing countries. Compared to other livestock species, goats have been domesticated in a single region and subject to a limited amount of hybridization between breeds, thus they represent one of the best species for the study of genetic diversity and adaptation.http://www.goatgenome.org) was created in 2012 with the general goal of increasing the range of genomic tools and publicly available information for the goat. In 2013, the 50 K goat single nucleotide polymorphism (SNP) panel was developed , the African Goat Improvement Network (AGIN), which is a group resulting from the USAID Feed the Future (FtF), the USDA Livestock Improvement Project, the European Union sponsored, 3SR—Sustainable Solutions for Small ruminants and NEXTGEN projects.To collect, store and standardize data from many different sources, input tools and a relational database were developed and made accessible to all consortium partners. Provision of data was regulated by a data transfer agreement and uploading was facilitated through a user-friendly graphical interface that allowed users to drag and drop files to the server. A special focus was placed on security to ensure that the data were not compromised in any way.The first phase of submission and study, to which the series of AdaptMap papers in GSE refer to, accepted data until November 2015. At that point, the AdaptMap raw dataset included 4653 animals sampled from across the world and represented 148 populations, 35 countries and five continents.Figures The Illumina GoatSNP50 BeadChip  that incAfter populating the database, quality control was performed on the whole set using the Plink 1.9 software . IndividThe dataset included 12 crossbred populations: admixed Boer, Galla x Saanen, Matabele cross, admixed Mubende, Old Irish Goat cross, Saanen x ANB, Saanen x Creole, admixed Small East African, Small East African x Alpine, Small East African x Galla, Small East African x Saanen, and Small East African x Toggenburg. These populations were excluded from the analyses for the study of diversity and selection signatures but they do represent an interesting resource for further investigation on adaptation and performances. Similarly, six populations  were represented by fewer than three animals; for this reason, they were not considered in the study on diversity. Breeds with large populations from multiple locations  were split to account for the area of origin. Populations with more than 50 individuals were randomly thinned to 50.The general scope of the AdaptMap project was to investigate diversity in the goat with a focus on the impact of domestication, adaptation associated with local environmental conditions, and adaptation in response to selection for production systems. The ultimate goal is to enable sustainable breeding by leveraging the use of genomic information. Towards these aims, study groups were organized to make best use of the available genotypic information, and by analytical themes. The first phase of the project addressed by the AdaptMap consortium focused on population genetics analyses and population history, selection signatures, landscape genomics, visible genetic profiles, and the identification of a panel of parentage SNPs. The series of papers published here provide the first reports of the major results from these investigations.Among the major results obtained, perhaps the most striking was the corroboration of prior observations and results, and of our hypothesis that close associations exist between the genomes of goat breeds and geographical measures.On a large scale, the studied populations could be partitioned quite distinctly into groups according to continental origin. Three major gene pools were clearly identified, corresponding to goats from Europe, Africa and western Asia. Within these three pools, the patterns of variation were consistent with measures of “distance” in geography, human history and common animal husbandry practices. Specifically, the greatest genetic variability was observed for populations from western Asia, near to the putative center of domestication, with relatively little differentiation among breeds within these regions. Within the other two gene pools, patterns of variation obeyed geographical rules, both on “macro” and “micro” levels. On the macro level, both overall genetic variation and similarity with the western Asian populations tended to decrease for populations sampled from regions located further from western Asia.On the micro level, the variation observed was consistent with expectations associated with recognized paths of human migration out of the Fertile Crescent region. Comparing the European and African gene pools, European breeds tended to be more discreet, which is consistent with the practice of creating breeds through active human-controlled genetic isolation, rather than allowing breeds to form as a result of climatic influence and geographic barriers. Greater gene flow among populations was observed in Africa. Within both these pools, the genetic impacts of particular instances of geographic isolation were clearly observed, inasmuch as populations from Iceland, Madagascar and the Canary and Balearic Islands tended to have longer and more numerous runs of homozygosity. Various putative “hotspots” of runs of homozygosity were observed, and occasionally these hotspots were unique across continental groups. Geography-based signatures of selection were also identified, the strongest of which was associated with mean annual temperature. Some of the signatures of selection observed correspond to signatures that were reported previously in independent analyses of goats.Finally, a highly informative panel for parentage assessment was developed to assist breeding in goat populations worldwide.AdaptMap has now completed the first phase of its planned research but the cooperation generated from the shared objectives, collaborations and large accumulated data offer new opportunities to explore the goat genome. The discovery of additional geographical and ecological differences, individual mutations for traits, disease susceptibility and phenotypic differences will help to provide clues into the amazing adaptability of the goat worldwide and its importance to livestock production.


Swertia perennis(Gentianaceae) is a perennial diploid and clonal plant species that is discontinuously distributed in peat bogs in the mountains of Europe, Asia and North America as well as in the lowlands of Europe. The current geographical dispersion of S. perennis is probably the result of quaternary climatic changes that have played an important role in determining the distribution of Swertia and other plant and animal species.trnLF region and trnH-psbA spacer) to elucidate the phylogeography of S. perennis in Europe. Plants were collected from 28 populations in different locations in the lowlands and mountainous areas of Europe . cDNA was analysed to detect the genetic relationship between specimens from different locations.In this study we used molecular techniques and combined data from chloroplast DNA markers (s were identified across the dataset. They were characterised by a high level of genetic variability but showed a lack of phylogeographical structure. This pattern may be the result of repeated recolonization and expansion from several areas. Such genetic differentiation may also be attributed to the relatively long-term isolation of S. perennis in Pleistocene refugia in Europe, which resulted in independent separation of different cpDNA phylogenetic lineages and variation in the nucleotide composition of cpDNA.A total of 20 haplotypeS. perennis existed in Europe. This lack of structure may also indicate a high level of gene flow in times when the landscape and fen systems were not fragmented in numerous geographically-isolated populations. This makes it difficult to speculate about the relationships between Asiatic and European plant populations and the origin and distribution of this species in Europe. Today, it seems to be restricted due to the occurrence of plants which clearly reflects the genetic variability from the ancient period.The lack of strong phylogeographical structure makes it impossible to indicate the centre of haplotype diversity; however, refugia located in the Carpathians, Sudetes or Alps are the most probable sites where  Ranunculus pygmaeus, which probably migrated to the valleys of the Alps from source populations in Siberia via the Carpathians  . Howeveraxifraga  and Salialix sp. , no studPopulations that are dispersed in scattered localities are susceptible to negative effects that range from their individual population history to possible fluctuations in their size . Genetic drift and founder or bottleneck effects may also influence the genetic structure of populations that are often genetically isolated . The resSwertia perennis, in the Polish lowlands, Carpathians, Sudetes, Bohemian Forest and Alps. The combination of samples from various localities together with cpDNA markers should permit the identification of different haplotype lineages across the 1,300 km disjunct distribution range in Europe. This study also aimed to assess whether (like in other temperate mountain species) e.g., from Central Asia) may be a valid explanation for the present distribution of S. perennis, and compared the genetic relationships between plant populations growing in geographically-isolated sites in natural or semi-natural patches in the landscape  organism that flourishes in July or August (Bombus and Vespidae). The plants develop up to 50 winged seeds in ovate fruit capsules (S. perennis) were strongly reduced in number and size due to direct destruction of wetlands, drainage and fertilisation , according to the manufacturer’s protocol. Dried plant leaves were previously frozen using liquid nitrogen and disrupted from using Mixer Mill MM400 . The quality and quantity of the DNA was determined using 1% TBE agarose gel.S. perennis: the trn L-trnF Intergenic Spacer, trnL Intron and the trnH(GUG) –psbA spacer. All three chloroplast DNA regions are widely used for phylogenetic studies at all taxonomic levels –psbA with trnH(GUG) and psbA primers  with the following parameters: 8 min at 95 °C, followed by 30 cycles of 45 s at 95 °C, 45 s at annealing temperature  and 1 min at 72 °C, followed by a final extension step of 10 min at 72 °C. Prior to sequencing, PCR products were purified using GeneMATRIX PCR/ DNA Clean Up Purification Kit . Sequencing, post-reaction purification and reading were done by Genomed  using an ABI 377XL Automated DNA Sequencer . All sequences are available in GenBank (accession numbers - trnH(GUG) –psbA spacer: KY798346 –KY798347, KY817321 –KY817346; trnL-trnF Intergenic Spacer, trnL Intron: KY798346, KY798347, KY798348, KY906142 –KY906166). All molecular analyses was done at Department of Botany and Plant Ecology Wrocław University of Environmental and Life Sciences.In similar as c levels . The trnradicted . Additioon e.g., . The trn primers  and trnH primers . The DNAS. perennis populations. Maximum parsimony were conducted using PAUP* 4.0b10 . Arlequin 3.5.1.2 –psbA cpDNA locus also revealed a variation in length among individuals as well as the studied sites. A total of 19 sites with insertions or deletions  analyses were congruent with Bayesian interference (BI) analyses. The concatenate sequence data were more informative than single trees based on the trnLF and trnH–psbA regions, and the results are presented in The aligned lengths of the 33 sequences of the S. perennis within Europe, and no congruent groups were identified using TCS software . The dataset did not reveal any distinct regions of software . Severalsoftware . In cladS. perennis possessed high levels of plastid DNA diversity (HD = 0.841) and nucleotide diversity (π = 0.00154). Analysis of molecular variance (AMOVA) indicated a high degree of differentiation among individuals from populations (Fst = 0.649) and a high level of differentiation (64.93) among groups of populations . The findings also illustrate cpDNA variation between individuals from different S. perennis populations in Europe, with numerous unique haplotypes dispersed across this geographical area. A clear phylogeographic structure of S. perennis was not detected in Europe, and the results illustrate distinct structural differences between geographical haplotype lineages and the complicated nature of preglacial and postglacial plant dispersal . They could also have arisen before they spread to their current localities, from where they dispersed and mutated. Plants collected from Hala Cebulowa and Hala Miziowa were situated approximately 500 m apart, and their haplotypes differed with regards to trnH–psbA length and nucleotide variation in the trnH–psbA and trnLF regions. Similarly, the haplotypes detected in plants from Karkonosze populations (Sudetes mountains) were located about one km from each other (Kocioł Wielkiego Stawu–Kocioł Małego Stawu and Kocioł Łomniczki–Kopa) and they also differed in terms of their trnH–psbA and trnLF length or nucleotide variation. Other haplotypes were specific only for the sites or localities in which they were grown and did not form groups of similar sequences  and biological processes, the results of which can be observed using genetic analyses. However, it is difficult to define the same rules for all plant species, particularly with respect to plant migration. A good example is the history of ispersal . The obsispersal  and 3. Tequences . CloselyS. perennis is found in European mountains, NorthernThe genetic diversity of cpDNA and the lack of phylogeographical structure may be attributed to the range formation history, its shifting during the ice age and the emergence of a group of boreal mountain plants . During Eurasia and North America , as well as in isolated boreal .S. perennis seems to be a residual effect after more homogenous distribution. It is also possible that cpDNA mutates rapidly, seed production or dispersal does not occur or is scarce and gene flow is scattered or nonexistant. At the contact zones between migrating fronts, significant haplotype mixing has occurred, as all populations are colonising new territories. It is possible that the migration of S. perennis occurred in a similar way or in several phases. Mountain plants could have survived the last Wistulian glaciation; however, after the end of the cold period, new plants (possibly from Siberia) may have expanded to their present European territory. This may explain the difference in cpDNA nucleotide composition in individuals from distinct populations of S. perennis. It is also possible that the species both survived glaciations in situ and remigrated several times, therefore broad-fronted repeated recolonization and glacial survival can shape genetic variation (Similar haplotypes identified in this study may also form part of a residual lineage of haplotypes that colonised peat bog areas in mountains. The five most similar haplotypes (17, 18, 19, 26 and 28, presented on ariation .S. perennis in the study areas of the European range may be the result of numerous overlapping factors, such as multidirectional gene flow in the dispersal history, long-distance dispersal during postglacial recolonization and survival in several detached refugia (In conclusion, the non-existent phylogeographical structure of  refugia . The low refugia . It is a10.7717/peerj.5512/supp-1Supplemental Information 1Click here for additional data file.


Adaptation can occur with or without genome‐wide differentiation. If adaptive loci are linked to traits involved in reproductive isolation, genome‐wide divergence is likely, and speciation is possible. However, adaptation can also lead to phenotypic differentiation without genome‐wide divergence if levels of ongoing gene flow are high. Here, we use the replicated occurrence of melanism in lava flow lizards to assess the relationship between local adaptation and genome‐wide differentiation. We compare patterns of phenotypic and genomic divergence among lava flow and nonlava populations for three lizard species and three lava flows in the Chihuahuan Desert. We find that local phenotypic adaptation (melanism) is not typically accompanied by genome‐wide differentiation. Specifically, lava populations do not generally exhibit greater divergence from nonlava populations than expected by geography alone, regardless of whether the lava formation is 5,000 or 760,000 years old. We also infer that gene flow between lava and nonlava populations is ongoing in all lava populations surveyed. Recent work in the isolation by environment and ecological speciation literature suggests that environmentally driven genome‐wide differentiation is common in nature. However, local adaptation may often simply be local adaptation rather than an early stage of ecological speciation. There are a number of ways that geographic and ecological factors can lead to genome‐wide differentiation across populations. A common pattern is isolation by distance (IBD) Wright, , wherebyOf course, adaptation does not always lead to genome‐wide differentiation, especially when locally adaptive phenotypes have a simple genetic basis and no consequences for reproductive isolation. While examples of IBA and IBE are increasingly common in the literature  and 26 S. cowlesi (CZLF‐Sceloporus). We included additional phenotypic data of S. cowlesi at CZLF from a previous study , 18 C. collaris from the Oscura Mountains (OS), seven S. cowlesi from Tularosa (TU), and six S. cowlesi from Rita Site (RI). At Aden Afton Lava Flow (AALF), we quantified the dorsal coloration of eight U. ornatus (AALF‐Urosaurus). Surrounding the Aden Afton Lava Flow, we quantified the dorsal coloration of seven U. ornatus from Corralitos Ranch Road (CR) and six from the Organ Mountains (OM). At the PALF, we quantified dorsal coloration in 14 C. collaris (PALF‐Crotaphytus) and five S. cowlesi (PALF‐Sceloporus). Surrounding the PALF, we quantified the dorsal coloration of 23 C. collaris from Elephant Butte (EB), five C. collaris from Big Gyp Mountain (BG), five S. cowlesi from Mesa Camp (MC), and five S. cowlesi from Bosque del Apache  according to Brattstrom . To convert the RGB values to a color space meaningful for most vertebrate visual systems, we used properties of color and brightness opponency  using the enzymes SphI and MluCI. We followed Peterson et al.  to demultiplex individuals, remove low‐quality reads, create individual contigs, and to create a de novo reference assembly. We demultiplexed individuals while allowing for one mismatch per barcode. After demultiplexing, we separated individuals by species and continued the pipeline on each species independently. We filtered out reads that were low quality (Phred score <20), were contaminated by bacteria, had anomalous runs of a single base (more than 50% of the read), or did not contain the restriction enzyme cut site . After filtering, we removed individuals that did not have enough reads to continue . Next, we masked repetitive elements known from vertebrate metazoans using RepeatMasker  and clustered reads into contigs for each individual using CD‐hit  to add read groups to the alignment and GATK .Before analyzing our data, we filtered our reads to ensure only high‐quality loci. We followed the RADToolKit pipeline , had a minor allele frequency >0.05, and were present in at least 80% of individuals. We plotted the eigenvalues of the covariance matrix in R to visualize population structure in each of the population trios.First, to understand basic population structure, we visualized genetic data for each species using a principal components analysis (PCA). We used the program ngsCovar to create a covariance matrix for each individual's genotypes , had a minor allele frequency >0.05, and had a genotype posterior probability cutoff of at least 0.95. We calculated pairwise distances from field‐collected longitude and latitude data using the R package raster . FST values can reflect processes of divergence, processes of migration, or combinations therein . Using ∂a∂i, we created joint site frequency spectra for each pair of populations. We ran four models of demographic scenarios to model how alleles might change with the formation of the lava flow and ongoing migration. We ran a neutral model with no divergence, a model with divergence but no migration, a model with divergence and symmetric present‐day migration, and a model with divergence and asymmetric present‐day migration .Finally, to determine whether gene flow might contribute to observed patterns of genetic differentiation between lava flow and nonlava flow populations, we calculated 3Sceloporus . Specifically, only CZLF‐Crotaphytus and CZLF‐Sceloporus  from 28 individuals. For S. cowlesi, sequences from CZLF‐Sceloporus fell below our quality thresholds and thus were excluded from further analysis. Using only individuals from the PALF‐Sceloporus population trio, we assembled a new de novo reference assembly and aligned reads for analysis as above. We recovered 748,818 high‐quality sites on 10,426 contigs with a median coverage of 14.85X (range: 6.7X–141.1X) for those 12 S. cowlesi. For U. ornatus, we recovered 1,460,625 high‐quality sites on 20,051 contigs with a median coverage of 20.5X (range: 5.8X–45.9X) from 13 individuals. Raw data are archived on the Short Read Archive (BioProject Accession PRJNA417310), and scripts to reproduce this workflow are available at https://github.com/alexkrohn/LavaFlowLizards.For our genomic analyses, we recovered 262,338,151 reads from two runs of sequencing. After filtering reads for quality and removing individuals with too much missing data, we recovered different numbers of sites and contigs per species. For Crotaphytus collaris and S. cowlesi populations showed little structuring by population or habitat, while U. ornatus populations appeared to cluster separately. However, PCA does not assess whether patterns of genetic variation are better explained by IBD or IBE. Our ngsDist and Mantel analysis indicated that genetic differentiation within population trios often followed patterns of IBD, but not IBE  as one would expect under IBD . All of our population trios did, however, show signals of spatial autocorrelation . Given that partial Mantel tests have high false‐positive rates with spatially autocorrelated datasets, but not inflated false‐negative rates, our negative results seem robust. The positive association between genetic distance and environmental distance in the CZLF‐Crotaphytus population trio may be a false‐positive , but AIC values were too close (>10) to determine whether migration was symmetric or asymmetric. Finally, in S. cowlesi, the best model predicted migration between PALF‐Sceloporus and one nonlava flow population (PALF – MC) but could not distinguish whether migration is symmetric or asymmetric. Thus, regardless of levels of phenotypic divergence between lava flow and nonlava flow populations, migration is ongoing across populations within species.Demographic modeling revealed that migration between lava flow and nonlava flow populations occurs often, and regardless of phenotypic differentiation. es Table . Demograon Table . There w4C. collaris and S. cowlesi from the CZLF  and U. ornatus from the Aden Afton Lava Flow  were melanistic, indicating strong phenotypic divergence from nonlava flow populations. Despite what was reported in earlier literature  were not significantly darker than surrounding nonlava flow populations. The phenotypic patterns themselves are intriguing: the only lava flow lizards that were not melanistic were those from the oldest lava flow, where the substrate is less homogenously dark. However, our most important finding was that, irrespective of phenotypic differentiation, the lava populations typically did not exhibit more divergence from nonlava conspecifics than expected based on geography. Most comparisons also revealed that migration occurs between lava flow and nonlava flow populations. Thus, we find some evidence of local adaptation, but little evidence of the accompanying genetic isolation often reported in the IBE and IBA literature.We demonstrated that genome‐wide differentiation does not accompany local adaptation in three species of lava flow lizards in the Chihuahua Desert. Consistent with previous literature Lewis, , 1951, wFST < 0.15; Hartle & Clark, U. ornatus), genetic differentiation between lava and nonlava populations was comparable to background differentiation. Moreover, we did not uncover a signal of genetic divergence accumulating or decaying with lava flow age. For example, in Crotaphytus, FST between lava and nonlava populations was comparably low regardless of whether the lava flow was 5,200 or 760,000 years old. Using partial Mantel tests, only one comparison (CZLF‐C. collaris) showed significant genetic differentiation due to the environment, and even in this comparison, the magnitude of genetic differentiation between habitats was small . In most cases, overall divergence between lava and nonlava populations was low  can make it difficult to distinguish between demographic scenarios, even with thousands of loci (Pinho & Hey, C. collaris at PALF (Krohn & Rosenblum, S. cowlesi (Hardwick et al., S. cowlesi populations, despite phenotypic differentiation. Without a link between the genetic mechanisms of melanism and reproductive isolation, gene flow could easily persist even with selection for background‐matched lizards.There are alternative reasons why lava flow lizards may not show genome‐wide differentiation with phenotypic differentiation. Melanism has a genetic basis in many animal species (van't Hof, Our replicated results show that regardless of the species studied or the age of the lava flow, locally adapted populations show ongoing gene flow and no more genome‐wide differentiation than expected by geographic distance. Clearly, adaptation does not always lead to genome‐wide divergence, or speciation. Given the similarity of our results across populations, it is possible that selection–migration balance without genome‐wide differentiation may be very common Hendry, . HoweverNone declared.A.R. Krohn designed the experiment, conducted the field and laboratory work, analyzed the data, and wrote the manuscript. E.T. Diepeveen contributed to fieldwork and laboratory work. K. Bi contributed to data analysis. E.B. Rosenblum contributed to the experimental design and manuscript preparation. Click here for additional data file. Click here for additional data file.


Mesoamerica is a remarkable region with a high geological and ecological complexity. Within northern Mesoamerica, the biotic province of the Sierra Madre del Sur (SMS) in southwestern Mexico harbors exceptionally high avian endemism and diversity. Herein, we searched for spatially and temporally concordant phylogeographic patterns, in four bird genera from three distinct avian orders co-distributed across Mesoamerica and investigated their causes through hypothesis testing regarding historical processes. Selected species include endemic and differentiated populations across the montane forests of Mesoamerica, and particularly within the SMS.We gathered mitochondrial DNA sequences for at least one locus from 177 individuals across all species. We assessed genetic structure, demographic history, and defined a framework for the coalescent simulations used in biogeographic hypothesis testing temporal and spatial co-variance. Our analyses suggested shared phylogeographic breaks in areas corresponding to the SMS populations, and between the main montane systems in Mesoamerica, with the Central Valley of Oaxaca and the Nicaragua Depression being the most frequently shared breaks among analyzed taxa. Nevertheless, dating analyses and divergence patterns observed were consistent with the hypothesis of broad vicariance across Mesoamerica derived from mechanisms operating at distinct times across taxa in the SMS.Our study provides a framework for understanding the evolutionary origins and historical factors enhancing speciation in well-defined regions within Mesoamerica, indicating that the evolutionary history of extant biota inhabiting montane forests is complex and often idiosyncratic. Comparison of phylogeographic patterns across co-distributed species provides valuable insights on the probable congruence of processes that may have driven intraspecific diversification in particular regions –4. SeverSituated at the northernmost Neotropics, Mesoamerica possess one of the highest levels of endemism and species diversity, but also one of the most globally threatened biota and high rates of deforestation , 21. The2, is located along the Mexican Pacific slope  [2 to a neighboring country due to habitat continuity, [The Sierra Madre del Sur (SMS) biogeographic province is included within the Mesoamerican highlands, in the Mexican Transition Zone . This i5–102°W) . The reg5–102°W) ), 15 , and 54tinuity, . Furthertinuity, –48, the tinuity, , 49.FigIsolated montane forest patches are generally thought to act as promoters for divergence between populations of several species, which have served as models for the understanding of speciation processes in island-like settings –54. ThesHerein, we compared phylogeographical structure patterns among four co-distributed and not closely-related Neotropical bird taxa with well-differentiated populations restricted to the SMS. We tested the following biogeographic hypotheses in an ABC framework: (a) a southern origin  followed by a northward expansion; (b) a Mexican origin and range expansion towards southern Central America; and (c) a northern Central American origin with north and southward dispersal. We also aimed to determine whether phylogeographic structure and divergence times in each taxon are congruent among taxa, therefore revealing patterns of simultaneous diversification. Specifically, we used mitochondrial datasets in a comparative phylogeography framework to test: (1) The presence of geographically-structured genetic variation; (2) the existence of spatially-congruent genetic variation across analyzed species; (3) if biogeographic history and similar diversification scenarios support vicariance, dispersal or population admixture. In addition, we also examined temporal patterns of diversification through hABC simulations, testing for either synchronous or multiple pulses for diversification in bird assemblages across the SMS region.All of our phylogenetic and genetic assignment analyses supported previous findings on the genetic structure for all of the taxa Fig. . GeneticAulacorhynchus. Two of these, SMS (restricted to the SMS highlands) and EMx-NCA  are differentiated by 27 mutational steps  from the SMS to be grouped in the TMVB clade, which also includes samples from localities of Jalisco and Michoacán. This paraphyletic relationship was recovered in all analyses, receiving strong support from both bootstrap (> 80) and Bayesian posterior probabilities  and E. eximia , has been described to be disjunct [Finally, in disjunct , 68, 69.FCT) were not significant, whereas fixation indices among populations within groups (FSC) were. Genetic diversity varied among datasets. Overall, we obtained high haplotype diversity (Hd > 0.5) and low nucleotide diversity  from southern Panama northwards across the lowlands and narrowest region in Central America (Panama Channel Fault Zone) started during the Pliocene at 4.3 Myr (95% CI: 4–4.8 Myr), followed by successive northward expansion and further vicariance of populations, and the final divergence between eastern (EMx-NCA) and southwestern (SMS) Mexican populations (t2) about 1.3 Myr (95% CI: 420 kyr – 2.5 Myr) during the Pleistocene. When the lineages of Guerrero and Oaxaca were considered as distinct populations  event is dated around 3.3 Myr (95% CI: 2.5–6.1 Myr), while the most recent divergence time (t1) occurred about 204 kyr (95% CI: 12.8–730 kyr).Even when divergence dates varied, analyses showed that divergence between populations occurred during the Pleistocene, mainly within the last 2 Myr. The best-supported scenario for four population groups in Chlorospingus when six groups are considered  of Mexican and Central American lineages during the Pliocene at 5.8 Myr (95% CI: 2.5–9.5 Myr (Table t2) between NCA and SCA at 1.3 Myr  at 2 Myr (95% CI: 1.1–5.3 Myr) between Mexican and Central American lineages, a divergence event (t2) estimated to occur after the Nicaragua Depression formation between Central American populations at 1 Myr (95% CI: 288 kyr – 2.1 Myr), and a final divergence event (t1) in the SMS lineage originating Guerrero and Oaxaca populations during a interglacial period in the pre-Illinoian stage at 656 kyr  population through the Isthmus of Tehuantepec at t4 (~ 1.3 Myr); followed by vicariance events driven by the effect of Mexican highlands at t3 (~ 426 kyr) between SMOc and an hypothetical ancestral population that at t2 (~ 260 kyr) is fragmented into the TMVB and SMS populations  2.7 Myr (95% CI: 2.1–3.4 Myr), in time (t3) and before major climatic oscillations in the mid-Pleistocene, the ancestral Eupherusa lineage splits into two hypothetical ancestral lineages, one that further splits at t2 (~ 1.3 Myr) giving rise to the populations in eastern Mexico and Central America (E. eximia–E.nigriventris), and the second which splits into populations in the SMS (E. poliocerca-E. cyanophrys) at 716 kyr  of an ancestral Eupherusa lineage into two ancestral populations. One diverged into eastern Mexican and Central American populations at t2 (~ 1.2 Myr), whereas the other splits into southwestern Mexican populations at 688 kyr  and the highest mode model-averaged posterior estimate of the hyperparameter Ω = 0.1415 , in comparison with posterior probability of two (PPΨ = 2 = 0.282) or three (PPΨ = 3 = 0.275) divergence events Aulacorhynchus and Chlorospingus, obtaining high support for a single divergence event (PPΨ = 1 = 0.6749), along with a mode value of Ω consistent with synchronous divergence (Ω = 0.000475) and an Ω = 0, which is included within the 95% HDP intervals , we found low support for a single divergence event, suggesting two divergence events across these taxa (PPΨ = 2 = 0.5436) and a Ω mode of 0.05 consistent with asynchronous divergence  of Ψ = 1 = 0.4536) and Ω = 0.0022, also indicating support for synchronous diversification between these taxa occurring at ~ 1.06 Myr , and a Ω hyperparameter value = 0  across the Nicaraguan Depression lowlands between Costa Rica and Nicaragua occurring ~ 0.924 Myr  with distinct natural histories but which inhabit roughly the same regions and wide elevational range along the humid montane forests of Mesoamerica, present highly similar phylogeographic structure, including a significant split between the highlands in Guerrero and Oaxaca within the SMS, which may be expected due to discontinuous distribution ranges, as well as to their presumably reduced dispersal abilities. The pattern observed in Cardellina and Eupherusa do not fully agree with the expectation that the degree of isolation between montane ranges is responsible for shaping the genetic differentiation between populations, as has been reported in previous studies of montane birds . Th. ThAulacpatterns .Aulacorhynchus and Chlorospingus, between Chiapas and Mexican-Central American lineages. This has been also supported by other bird species [Chlorospingus) evidencing that populations are clearly sorted within Oaxaca across its Central Valley, and more importantly morphological variation of these populations has also been documented as in E. eximia nelsoni [Our results, as several others from the Neotropics see , 76, 77), 7776, 7 species , plants  species , rodents species , 35, ins species  and a sn species . Moreove nelsoni . Isolati nelsoni , 77, 80) nelsoni , lizards nelsoni , 82, fro nelsoni  and even nelsoni .Eupherusa). The overall structures in the haplotype networks resemble an expected pattern for ancient divergence among the sky-islands where reciprocally monophyletic groups occur on each sky-island with no mixing (see Aulacorhynchus and Chlorospingus), and is consistent with estimated divergence and the gradual cooling occurring in the Gelasian stage of late Pliocene-early Pleistocene [Eupherusa it is difficult to discern specific events for each divergence estimate due to wide confidence intervals. Cardellina is an exception which resembles a star-shaped network of a post glacial sky-island divergence [The patterns observed in the haplotype networks mostly reveal a consistent match between haplogroups and subspecies, as well as species boundaries  among populations within groups were significant, as in Aulacorhynchus, Chlorospingus and Cardellina. This pattern suggests the existence of further structured populations in at least one of the defined clusters, as supported by haplotype networks  and low nucleotide diversity (π) for the majority of analyzed groups within each taxon. These patterns have been attributed to population growth following demographic bottlenecks enhancing retention of novel mutations , 87, whie Tables , 4. Desprding to  these pording to .Cardellina analyses revealed.Our results highlight the importance of Pliocene-Pleistocene events in promoting the intra-specific genetic structure in the analyzed species, as reported in other studies. Divergence between SCA and northern populations is concordant with the formation of the Nicaragua Depression , furthAulacorhynchus, Chlorospingus, and Cardellina, along with their respective pairwise FST comparisons, support the hypothesis of sequential northward population range expansions from Central America with ancestral populations being split by vicariant events, such as fracture and changes in altitudinal ranges of montane forests linked to climatic oscillations, therefore promoting geographical isolation. Although our analyses support a southern origin, the pattern observed in Eupherusa + Thalurania ridgwayi suggest alternative scenarios as also probable. Based on previous evidence supporting T. ridgwayi as a member of the Eupherusa genus [T. ridgwayi in the diversification scenario comparisons, and we were unable to discern if diversification according to the best-supported scenario occurred from two potential regions that may have served as refugia , or whether SMS (E. poliocerca-E. cyanoprhys) and Central American lineages of Eupherusa come from a migration event from northwestern Mexico. Further studies should focus on the reconstruction of ancestral areas of distribution to clarify this issue .Overall, dates from the DIYABC analyses suggest more recent diversification times than our BEAST analyses, thus highlighting the effect that distinct assumed mutation rates have an effect of temporal frame estimation ; neverthAulacorhynchus and Chlorospingus) in the SMS region across the Río Verde drainage lowland barrier, with a mean divergence date of 0.9339 Myr, which 95% HDP confidence intervals falls within the estimated divergence dates between the same populations. When Eupherusa population pair (cyanophrys-poliocerca) was added, estimations supported two diversification events, thus implying that even when the three taxa share their overall distributional ranges in the SMS, they do not fully share a community-wide historical pattern across the landscape.Even when species ranges overlap across their distributional areas, some divergence time estimation frames are not congruent with each other, therefore, asynchrony in diversification within Mesoamerica could be depicted as a result of Pleistocene climatic oscillations which promoted rapid pulses of diversification and speciation rates across multiple lineages , 18, 19.C. rubra lineage the observed strong signature of expansion in TMVB and SMS populations due to Pleistocene conditions and forest migrations resulted in the admixture of two isolated lineages; our results reveal that demographic dynamics for other species persisted in isolated regions throughout the Pleistocene allowing the evolution of high endemism, which could be an overall result of high effective population sizes.Even when we do not fully out rule any orogenic effect over the analyzed species, major changes to population structure should be promoted by the onset of climatic oscillations in the Pleistocene, which have been invoked to explain patterns of genetic differentiation in the increasing rates of speciation and diversification in Neotropical biota, given the evidence of a downward altitudinal migration of the forest line which resulted in a montane forest-like dominated landscape in the lowlands with further retreat when temperatures raised –101. AltGenetic endemism as a result of isolation of intraspecific lineages, as seen within the SMS should be considered in conservation measures given that they preserve historical components and maintain the species capability of adaptation , 66.Our survey provides a framework for understanding the complex geographic history of Mesoamerica and its role in promoting lineage diversification in birds. From our analyses we recover multiple independently evolving lineages restricted to montane areas across the region, our results also highlight that congruence may be difficult to occur given idiosyncratic life histories of species; nevertheless, co-distributed taxa will share more common history than a random scenario could predict. Montane regions within Mesoamerica are strong drivers of lineage diversification, and as such our results point into major areas of great genetic diversity and endemism, such as isolated highlands of the SMS in the Mexican states of Oaxaca and Guerrero. Given ancient development of major montane systems, climatic oscillations and orogeny derived conditions were key drivers of diversification between these lineages as splits were calculated to occur mainly within the Pleistocene.Aulacorhynchus), the Red and Red-faced Warbler (genus Cardellina), Bush-tanagers (genus Chlorospingus), and hummingbirds . These taxa include at least one differentiated population restricted to the SMS for which we have previous genetic and morphometric information [We selected four resident and co-distributed avian taxa from the montane forests of Mesoamerica, all of which represent different orders and families , as well as life histories. Selected taxa include Toucanets , and conducted analyses using alignments of concatenated mtDNA loci  the examination of the standard deviation of split frequencies ; and b) by verification of parameter estimates in TRACER v1.6 [Aulacorhynchus albivitta [Chlorospingus flavopectus phaeocephalus [Cardellina versicolor [Thalurania ridgwayi [For each taxon, we retrieved mitochondrial DNA (mtDNA) sequences deposited in GenBank  to visualize the relationships among haplotypes by constructing networks using the median-joining algorithm [We used NETWORK 4.6.1.1  from 2 to 10, following possible sub structuring of populations within the main montane ranges in Mesoamerica. We performed 10 independent runs of 10,000 iterations each, a thinning value of 1000 and a 10% burn-in. Best results were selected according to highest posterior probabilities.To explicitly test for phylogeographic structure in each taxon avoiding a priori criteria to delineate populations, we used a Bayesian model-based clustering algorithm, as implemented in GENELAND 4.0.3 , 121, whAulacorhynchus were divided into four population groups (K = 4): 1) Sierra Madre del Sur (SMS) samples from the Mexican states Guerrero and Oaxaca, 2) eastern Mexico and north Central America (EMx-NCA), 3) Costa Rica and northern Panama (CR-NP), and 4) southern Panama (SP); for Chlorospingus, we obtained K = 6: 1) SMS (Guerrero-Oaxaca), 2) northern Chiapas , 3) northeastern Oaxaca and Sierra Madre Oriental (SMO), 4) Los Tuxtlas Massif (TM), 5) northern Central America (NCA), and 6) southern Central America (SCA). Samples of Cardellina were divided into K = 4: 1) Trans Mexican Volcanic Belt (TMVB), 2) SMS, 3) Sierra Madre Occidental (SMOc), and 4) northern Central America (NCA-C. versicolor). Finally, the Eupherusa-Thalurania samples, where GENELAND clustering followed current species delimitation: 1) E. cyanophrys , 2) E. poliocerca (Guerrero-Oaxaca), 3) E. eximia , 4) E. nigriventris (Costa Rica-Panama), and 5) Thalurania rigdwayi .Genetic structure was assessed through hierarchical analysis of molecular variance (AMOVA) using pairwise differences, based on the number of clusters (K) obtained by GENELAND for each taxon. Following these results, samples of FST fixation index [FST tests was assessed using 1000 permutations. To test for evidence of recent demographic changes in the selected species, we estimated historical demographic dynamics through the calculation of Fu’s FS statistic [p <  0.02 in the case of FS statistic) was calculated through 1000 simulations. All analyses were conducted in ARLEQUIN 3.1 [We also assessed genetic diversity of each population group within each species through the estimation of haplotype diversity (Hd), and nucleotide diversity (π). Genetic divergence between groups was measured using pairwise on index , 123, inon index . Signifitatistic  and Tajitatistic  neutraliQUIN 3.1 .8 generations sampling every 1000 steps, using a Yule speciation process [http://tree.bio.ed.ac.uk/software/figtree/).We used a Bayesian MCMC-based approach to calculate divergence times among haplogroups within each species independently using BEAST v1.8.4 . For eac process , 130 wit process , and (B) process –134 for  process . Adjustm process , 136. Ea process  against  process . Node ag process , and treAulacorhynchus and Chlorospingus), we contrasted the same scenarios considering those clades as different populations in the analyses. All competing scenarios were eliminated in successive rounds, where the preferred hypotheses were the ones with highest posterior probabilities.We tested for different hypotheses of population divergence and admixture using an approximate Bayesian computation (ABC) approach in DIYABC 2.1.0 . We condAulacorhynchus, Chlorospingus, and Cardellina; and two for Eupherusa-Thalurania  The posterior probabilities of competing scenarios were computed using a logistic regression on the 1% of simulated datasets closest to the observed data. The selected scenario was that with highest probability value and a non-overlapping 95% confidence interval. For the best supported scenario, we performed a model-checking procedure using a Principal Components Analysis (PCA) on test statistics to visualize the fit between simulated and observed datasets. Confidence on the chosen scenario was assessed though the calculation of type I and type II error rate, from 500 simulated pseudo-observed datasets (PODs) generated with the data of the best-supported scenario [From the selection process described above, we tested three final models for or birds –145, we scenario , 147. Poscenario . Divergescenario , 148.We tested whether genetic differentiation occurred simultaneously between the main geographical barriers in the analyzed clusters by performing a hierarchical approximate Bayesian computation (hABC) analysis as implemented in MSBAYES . Populat6 data sets were simulated under the specified multi-taxon model using prior distribution for the demographic parameters. The prior of the maximum possible number of divergence events (Ψ) was set to be equal to the number of lineage pairs tested. For the last stage of the MSBAYES analyses, we used the acceptance/rejection algorithm to approximate the posterior distribution for the hyper parameters that characterize the degree of variability of demographic and temporal parameters given the empirical data . We used Ω estimates to evaluate the support for each hypothesis: synchronous diversification is expected when Ω ≤ 0.01; asynchronous diversification if Ω > 0.01 [E(τ) estimates  to absolute time (Tdiv), using the equation Tdiv = E(τ) × (θAVE/μ) × g, where g is the generation time in years, and θAVE is the mean of the upper θ prior [We used jModelTest , 150 to Ω > 0.01 , 152. To θ prior , and μ i θ prior , and an  θ prior , 148.Additional file 1: Genbank accession numbers. Samples obtained for the realization of analyses in this study.Additional file 2: Marginal likelihoods for sequence evolution. Marginal likelihoods estimation for sequence fit to a strict or relaxed clock and comparison of evolutionary rate assumptions through Bayes factors (BFs).Additional file 3: Hypothetical scenarios of diversification of 4 bird taxa in Mesoamerica. Scenarios tested with ABC for Aulacorhynchus, Chlorospingus, Cardellina, and Eupherusa-Thalurania with confidence scenario choice values (Type I and II errors).Additional file 4: Posterior parameter estimates for the best supported scenarios. Population and divergence time estimates obtained with ABC for Aulacorhynchus, Chlorospingus, Cardellina, and Eupherusa-Thalurania. (DOCX 33 kb)Additional file 5: Final tested hypothetical scenarios of diversification of 4 bird taxa in Mesoamerica. Competing demographic scenarios of Aulacorhynchus, Chlorospingus, Cardellina, and Eupherusa-Thalurania with posterior probabilities and model checking.