Admixture is known to greatly impact the genetic landscape of a population and, while genetic variation underlying human phenotypes has been shown to differ among populations, studies on admixed subjects are still scarce. Latin American populations are the result of complex demographic history, such as 2 or 3-way admixing events, bottlenecks and/or expansions, and adaptive events unique to the American continent. To explore the impact of these events on the genetic structure of Latino populations, we evaluated the following haplotype features: linkage disequilibrium, shared identity by descent segments, runs of homozygosity, and extended haplotype homozygosity (integrated haplotype score) in Latinos represented in the 1000 Genome Project along with array data from 171 Brazilians sampled in the South and Southeast regions of Brazil. We found that linkage disequilibrium decay relates to the amount of American and African ancestry. The extent of identity by descent sharing positively correlates with historical effective population sizes, which we found to be steady or growing, except for Puerto Ricans and Colombians. Long runs of homozygosity, a particular instance of autozygosity, was only enriched in Peruvians and Native Americans. We used simulations to account for random sampling and linkage disequilibrium to filter positive selection indexes and found 244 unique markers under selection, 26 of which are common to 2 or more populations. Some markers exhibiting positive selection signals had estimated time to the most recent common ancestor consistent with human adaptation to the American continent. In conclusion, Latino populations present highly divergent haplotype characteristics that impact genetic architecture and underlie complex phenotypes. Latin Americans inhabit continental Latin America and the Caribbean and are the largest ethnic minority in the United States . DespiteThe admixture was extended by 2 incremental pulses of migration: the trans-Atlantic slave trade (from the 16th to the 19th century) and post-Colonial migrations (19th and 20th centuries). A major influx of European, Middle Eastern, Japanese, and Chinese migrants formed the current Latino populations, along with the extant indigenous people and sub-Saharan Africans and/or their descendants .These demographic events largely impact haplotype structure . ConsistAllelic association studies, the most common strategy for establishing genotype-phenotype links, also rely on haplotype structure, since flanking alleles are usually used as markers for a core causal region. This nonrandom statistical association between alleles, called linkage disequilibrium (LD), therefore, greatly impacts association studies. Admixture events can create LD (admixture LD) between all loci with divergent allele frequencies in genomic regions of different source populations, thus allowing to map genes related to a given trait . AdmixtuMoreover, extended haplotypes inherited from a recent common ancestor, known as identical by descent (IBD), reflect genetic relationships between individuals and can be used to identify fine-scale population structure  is necessary to comprehend how admixture impacts the genetic structure of admixed populations as well as to assist in genetic studies’ methodological design. Our study, therefore, aims to expand the characterization of admixed Latin American populations in terms of haplotype structure. In order to accomplish this goal, we (1) quantified LD in Latino samples and compared it with other populations around the world; (2) quantified identity by descent (IBD) segments in the Latin American samples and compared them with reference populations; (3) characterized ancestry components and described the genetic structure in the samples; (4) quantified ROH segment sharing; as well as (5) evaluated EHH (iHS) among admixed Latin American populations. We sought to evaluate populations with different admixture histories and therefore our dataset consists of Hispanic populations of the This study followed the principles of the Declaration of Helsinki and Brazilian ethical guidelines for biomedical research involving humans. All participants signed an informed consent form. Ethics approval was obtained from the local Research Ethics Committees under the following protocols: CAAE35316314.9.1001.5404, CAAE0413.0.146.000-09, 0241.0.146.000-05, CAAE12112913.3.0000.5404, 25000.142907/2013-07, and 25000.142907/2013-07.N = 31, noncarriers of the sickle cell mutation), (2) “High-density microarray technique in the assessment of copy number variation in congenital defects of complex inheritance: oral clefts as a model” (N = 20) (N = 94), and (4) the Joinville Stroke Registry (N = 26). Geographically, the sampling process occurred in 2 Brazilian cities: Campinas , and Joinville   “Assessment of copy number variations in the susceptibility to stroke in patients with sickle cell anemia” (s South) .n = 10) and Maya (n = 6), Mexico; Quechua (n = 2), Peru; and Aymara (n = 25), Bolivia.We also made use of publicly available data from the 1000 Genomes Project , along wGenomic DNA was extracted from the peripheral blood of each Brazilian participant using the QIAamp DNA Blood Midi Kit  and genotyped by 2 platforms: Affymetrix Genome-Wide SNP Arrays 6.0 and 5.0 . While preparing the DNA samples, we rigorously followed the manufacturer’s instructions. The files containing scanned images were examined with Genotyping Console software v4.1.3, using the default settings. We applied the following genotyping algorithms: BRLMM  for the . Subject QC: each sample was checked for discordance concerning sex registration, genotype call rate, and the presence of duplicated or related samples. We also removed known regions of long-range LD in human populations (Marker QC: we removed markers with high missing genotyping rates (>0.05), low minor allele frequency (< 0.05), and those that deviated from the Hardy–Weinberg equilibrium . After going through these filters, one Brazilian sample was removed and the final genotyping rate was 99.94%. Merging datasets: only autosomal SNPs were kept in our analysis.The raw genotype data underwent quality control (QC) using PLINK software (v1.07 and v1.9)  and 2,876 (exonic) markers. The average marker distance is 14,507 bp  averaged across the 10 replicates to choose the best K value. Output files (Q-matrices) from replicated runs for the best K value were analyzed with the CLUMPP v1.1.2  approach implemented in the FineStructure software. In this model-based approach, the posterior probability of a population’s configuration is inferred using a Markov chain Monte Carlo (MCMC) implementation. The parameters convergence to the posterior distribution can be confirmed by comparing population memberships between 2 runs initialized with different random seeds. FineStructure then creates a Maximum a Posteriori set of populations as MCMC state and imposes a tree structure on them.We also applied the concept of “super-individuals” built-in FineStructure. The approach allows some individual samples to be grouped. This approach allows the investigation of substructure details without additional computational costs. To refine the substructure among Brazilian samples, we grouped all other samples into one “super-individual” and proceeded to the FineStructure analysis anew.To assess diversity in Latino populations, we used haplotypes inferred in the phasing step to calculate the haplotype diversity parameter proposed by r2) for SNPs in a 100-kb window. SNP pairs were divided into 1 kb bins and 2r was averaged within each bin.We compared LD in the Latino populations to the populations of European, African, and Latin American ancestry. LD decay was estimated using PLINK as a function of LD by physical distance in kb. We filtered out only markers failing QC, thus keeping 196,749. We calculated the pairwise squared correlation coefficient  comparable to low-coverage WGS data (other populations in the dataset), we set the parameters following the guidelines in Ne (merge-ibd-gaps script) to remove breaks and short gaps in IBD segments resulting from haplotype phase and genotype errors. We assumed a 30-year generation time.We used the IBDNe software to estimate historical Ne  in the LNe  to accouThe integrated haplotype score was proposed by To generate empirical cut-off values for iHS, we performed iHS calculations on neutrally evolving individuals drawn from simulations. The demographic model used here is closely related to that of −8 crossing-over events per chromosome per base per generation and mutation rate of 1.5 × 10−8 mutations per chromosome per base position per generation. Markers used in the downstream analysis are the neutral mutations generated that were still segregating as polymorphisms (did not reach fixation have a frequency greater than 0.05).Using the forward simulation implemented in the SLiM 3.6 software , we perfThe simulation starts at 2.15 million years before the present to allow for a burn-in period to reach mutation-drift equilibrium. The simulation is then divided into 10 runs each of them beginning in the Out-Of-Africa (OOA) migration event. We assumed an effective population size of 10,000 individuals, and a reduction in size to 2,000 starting 50,000 years ago (reflecting the OOA event), followed by a recovery to 10,000 individuals 5,000 years ago , we assumed that the putatively selected haplotype reached its breakpoints. We then retrieve the recombination distance (r) from the GRCh37 genetic map and input it into the above formula for each locus. When the genetic distance was greater than 1.1 cM between recombination breaks , we skipped TMRCA calculation, because an allele selected so recently would require a selection coefficient well above 5%, which is unrealistic  was inferred assuming a star-shaped phylogeny, constant population size, and panmixia for putatively selected alleles as described in After QC, 561 samples from Latin America , along with 1,165 worldwide samples were analyzed.K) from 2 up to 12, we selected K = 6 as the lowest CVE  to 1,335 kb (NAM), see Nonetheless, Peruvians and Native Americans had an enrichment in long ROH, as depicted in To further understand the IBD and LD patterns and their relation to the population history, we estimated the effective population size of the Latin American populations . Our estWe pooled the iHS values results from the 125 groups generated by neutral simulations. Each group underwent iHS analysis with the same parameters used for the empirical data. After removing variants with MAF < 0.05 as advised by selscan authors , we endeWe used the iHS scores obtained from simulations (matched by the number of interrogated markers) to get empirical cut-offs for each Latino population. The iHS cut-off values and the number of remaining markers for each population were: −4.594/3.294 for Peruvians (134 markers remained); −4.749/3.382 for Mexicans (97 markers remained); −5.022/3.826 for Colombians (28 markers remained); −4.711/4.124 for Puerto Ricans (12 markers remained); and −4.783/4.214 for Brazilians (2 markers remained), for comparison between simulated and empirical data, see NCDN (ERC1 (WWOX (TRIM69 (PDE11A (KITLG (TNKS (TRPV5 (TF (EPHB6 (IGSF11 (KALRN (ALDH1A2) and immunoglobulin (IGSF5) families, a gene implicated in face shape variation (GLI3), as well as genes playing roles in the central nervous system  were also found as candidates of selection. Importantly, all the 273 signals (accounting for markers that were common to different populations) were resultant of a putative event of positive selection on the derived allele . We provide the list of intergenic markers putatively under selection, along with the nearest gene and allele TMRCA in Of the total of 244 markers resilient to comparison with simulations (26 of which were common to 2 or more populations), 142 were intronic (58%), 93 were intergenic (38%), and 9 were exonic (4%). From the total 119 genes (19 were common to 2 or more Latino populations) presenting signals that overcome simulation generated thresholds , a few wLCT, SLC24A5, and OCA2, presented preliminary signals of selection in Latino populations but were not strong enough to overcome thresholds from simulated data.Noteworthy, genes that are hallmarks of adaptation in humans, such as We also sought to estimate the allele ages of the relevant selection signals by using the approach of et al. analyzed Hispanic populations  comprehensively regarding demographic histories and the impact of isolation and consanguinity on haplotype features, such as LD  and most arrivals occurred in the 16th and 17th centuries , althougAnother difference in Spanish America is that large centers were densely populated by the time the Spanish arrived, e.g. Tenochtitlan was larger than most, if not all, European cities and was among the largest cities in the globe at the time, possibly reaching 300,000 inhabitants . SimilarWe observed that the amount of the Native American contribution readily translated to the LD patterns in the current Latino populations , a findiThese differences between Puerto Ricans and Brazilians, nonetheless, account for a minimal fraction of the LD pattern, if any. We found a smaller average haplotype size and higher genetic diversity in Brazilians. FineStructure and ADMIXTURE analyses are in agreement with these findings and corroborate previously published studies . However, by comparing iHS values obtained from neutrally evolving simulation data for the same number of tests in each population, the remaining signals drop to 0.84% (244) of the original candidates. This confirms that long-range LD and random sampling can yield extended homozygosity and bias positive selection inspection. Our approach could partially counter these effects, since there is still less confidence in iHS estimates from small-sized samples, such as Mexicans (64), Peruvians (85), and Colombians (94). Nonetheless, the finding of Peruvians baring the most robust signals agrees with the hypothesis that signals are originating from recent adaptation, given the greater amount of Native American ancestry in this population.While reducing the number of iHS signals to a more reasonable and reliable set of markers, the simulation approach used to produce cut-offs may be too rigorous, since Brazilians had a single region overcoming the thresholds, as well as signals in genes well-described as targets of selection were discarded after comparing to simulated individuals.et al. estimate that the TMRCA values as calculated by this method are about half the true TMRCA . The above dataset represents the combined individuals in binary pedigree (bed/bim/fam) format, as well as VCF, and transposed pedigree (TPED) formats. Code used in analyses is available at https://github.com/soulsacross/Cruz-and-Ananina-2022 (last accessed May 9 2022).The complete dataset, including Brazilian samples, can be found at G3 online.jkac111_Supplemental_MaterialClick here for additional data file.jkac111_Supplemental_Figure_S1Click here for additional data file.jkac111_Supplemental_Figure_S2Click here for additional data file.jkac111_Supplemental_Figure_S3Click here for additional data file.jkac111_Supplemental_Figure_S4Click here for additional data file.jkac111_Supplemental_Figure_S5Click here for additional data file.jkac111_Supplemental_Figure_S6Click here for additional data file.jkac111_Supplemental_Table_S1Click here for additional data file.jkac111_Supplemental_Table_S2Click here for additional data file.jkac111_Supplemental_Table_S3Click here for additional data file.jkac111_Supplemental_Table_S4Click here for additional data file.


Chamaedorea tepejilote in Costa Rica. We found that populations of C. tepejilote have moderate to high nuclear simple sequence repeat (SSR) genetic diversity, likely due to large population sizes and its outcrossing mating system. Habitat loss and fragmentation may have contributed to increased genetic structure within slopes. High-elevation mountain ranges appeared to be a significant barrier for gene flow among populations in the Caribbean and Pacific slopes; however, ranges are permeable through low-elevation passes. In contrast, most populations had a single distinct cpDNA haplotype, supporting the hypothesis of several isolated populations that experienced decline that likely resulted in eroded cytoplasmic genetic diversity within populations. The haplotype network and Bayesian analysis linked populations in the Caribbean and the southern Pacific coast, suggesting that gene flow between Pacific and Caribbean populations may have occurred through the southern extreme of the Talamanca Mountain range in Panama, a colonization pathway not previously suggested for LCA plants. This is one of the first phylogeographic studies conducted on tropical palms in the LCA region and the first in the genus Chamaedorea, which sheds light on possible gene flow and dispersal patterns of C. tepejilote in Costa Rica. Our results also highlight the importance of mountain ranges on shaping gene flow patterns of Neotropical plants.Gene flow connects populations and is necessary to sustain effective population sizes, and genetic diversity. In the Lower Central American (LCA) region, the complex topographic and climatic history have produced a wide variety of habitats resulting in high biodiversity. Phylogeographic studies of plants from this area are scarce, and to date none have been conducted on palms. We used SSR and chloroplast DNA (cpDNA) markers to study the genetic diversity and structure of populations of the understory palm  Chamaedorea. It gives insight on gene flow and dispersal patterns of Chamaedorea tepejilotein Costa Rica. Chamaedorea tepejilotepopulations contain moderate to high nuclear genetic diversity and very minimal chloroplast DNA diversity. High-elevation mountain ranges appear to be a barrier for gene flow between populations on the Caribbean and Pacific slopes of Costa Rica, although low-elevation passages are permeable. Gene flow between Pacific and Caribbean populations may have occurred through Panama, a colonization pathway not previously considered for LCA plants.This is one of the first phylogeographic studies on tropical palms in Lower Central America (LCA) and the first in the genus  The importance of gene flow on the magnitude and distribution of genetic diversity of plant species is widely recognized . Gene flThe Talamanca Mountain range in the Lower Central American region (LCA) is a biologically and topographically complex area and several studies recognize it as a world biodiversity hotspot for plants , and theThe few phylogeographic studies conducted on plants from the LCA have primarily focused on tree species but see , speciesChamaedorea is the largest palm genus in the Neotropics , and describe phylogeographic patterns using maternally inherited chloroplast DNA (cpDNA) markers. We propose the following hypotheses: (i) previous research on C. tepejilote populations in southern Mexico has demonstrated that gene flow frequently occurs over great distances . Consequently, we hypothesize relatively high haplotype diversity and low phylogeographic structure in lowland populations, with structure determined primarily by high mountain ranges acting as barriers to gene flow. In contrast, if populations retreated into multiple refugia, we expect to find low haplotypic diversity within populations and high levels of genetic structure due to isolation and drift , greenish to yellow, scented flowers, dry-powdery pollen and black fruits at maturity . Silica was replaced at least twice before DNA extraction to ensure proper tissue dehydration. Nuclear and plastid DNA were extracted using the modified CTAB protocol from We collected leaf tissue from 15–22 individuals in each of 13 populations in Costa Rica ; Fig. 1BC. tepejilote we used 10 SSR loci developed exclusively for C. tepejilote and used the sequences and PCR amplification protocols detailed in To analyse the genetic diversity and structure of adegenet , using Pacific and Caribbean slopes as the regional strata. The AMOVA analysis implemented in the poppr package was used, using 9999 permutations to test the significance of phi-statistics. Pairwise GST values were used to construct an unrooted tree based on the neighbour-joining (NJ) algorithm using the ape (GST and Euclidean distances (5000 permutations). We assessed the significance of all statistics by bootstrapping samples 1000 times among populations. To identify possible geographic barriers to gene flow, we used the Monmonier algorithm  implemented in adegenet. We retained 70 PCs as suggested by cross-validation with 999 replicates.Genetic diversity and structure statistics were estimated using the adegenet , poppr , an elongation period of 1 min at 72 °C and a final elongation step (72 °C/10 min). Amplification was checked on agarose gels. PCR products were cleaned and sequenced in forward and reverse directions at Macrogen .To describe the phylogeographic structure of rps16–trnQ and ndhG–ndhI and realigned them using MAFFT again. Sequences were imported into DNAsp version 6.12.01 and molecular and haplotype diversity was estimated. We also extracted haplotypes into a new Nexus file, which was imported into PopArt v. 1.7 to construct a median-joining haplotype network and plot haplotype frequency onto a map of Costa Rica. We also used DNAsp version 6.12.01 to estimate within and among population divergence using the GST estimate of Nei and the NST estimate proposed by D and Fu’s F, to test for neutrality and demographic change. Haplotypes were also analysed by means of an AMOVA using Arlequin 3.5 , filtered duplicate locations and registries without location information. We also eliminated records that were clearly outside the described distribution of the species and most likely represent registry errors (e.g. samples in the Atlantic or Africa). We also performed a spatial thinning to keep only samples that were separated by at least 5 km from each other. To determine the most suitable habitat we initially used the 19 environmental variables available from WorldClim v. 1.4 data set with a 30 arcmin resolution and eliminated variables 8, 9, 10 and 11 which were strongly correlated among them (r > 0.9). We modelled species distribution using the Maxent algorithm, which is based on presence–absence data  values. Model suitability predictions were visualized using the clog-clog transformation.To determine biogeographic barriers of the study species according to topography, we used ecological niche modelling (ENM) as implemented in the HO = 0.655 ± 0.071 and HE = 0.645 ± 0.062, respectively. Diversity estimates were consistently high across all populations , while all other inbreeding coefficients were not statistically different from zero.All 10 SSR loci were polymorphic with an average allelic richness of 5.80 ± 0.243 alleles. The mean observed and expected heterozygosities were P < 0.001) using SSR markers. The AMOVA found significant genetic structure among Pacific and Caribbean slopes P < 0.001; seeP < 0.001; seeP = 0.333). If we estimated the distance between populations taking elevation into account, the Mantel correlation coefficient increased in magnitude, however it was still not statistically significant We found significant genetic structure among all populations K = 5 [see], which assigned individuals into a similar configuration as that suggested by DAPC and the NJ tree [see]. The Monmonier’s algorithm found a barrier separating Pacific from Caribbean populations , while all the Caribbean populations  formed another group, and MTV as the most distant population on DAPC space , indicatn slopes , but theulations . The barrps16–trnQ and the ndhG–ndhI intergenic spacers were 902 bp and 1066 bp long, respectively. Concatenated sequences had 10 segregating sites and nucleotide diversity was indels as variants. Haplotype diversity or the probability that two randomly chosen molecules are different was D = 0.38517 (P > 0.10) and Fu’s F = 0.012 (P > 0.10), which suggests neutrality and no evidence of recent demographic changes. Nearly all populations were composed of a single haplotype (except in ROD and BRI) and the haplotype network . The predicted distribution model showed that in range . In CostC. tepejilote, as expected for a widely distributed and obligately outcrossing dioecious plant species. We found significant genetic structure among populations of this palm species in Costa Rica, with mountain ranges acting as predominant barriers to gene flow, likely due to the inability of palms to withstand environmental conditions at high elevations. In contrast, plastid markers had almost no within population genetic diversity but a significant phylogeographic structure, congruent with the hypothesis that populations may have resided into multiple refugia, experiencing isolation and drift. Our analyses revealed that haplotypes found on the southern Caribbean and southern Pacific coasts are closely related, indicating that gene flow may have occurred through the southernmost extent of the Talamanca Mountain range; a dispersal pattern not previously proposed for LCA plants. This is the first phylogeographic study on tropical palms in the LCA, a region with high palm diversity.We found moderate to high levels of microsatellite genetic diversity in the palm C. tepejilote, using SSR markers, as we originally hypothesized for a widespread outcrossing species. Genetic diversity of palms has been predominantly studied in commercial or edible species such as oil-producing palms or date palms (e.g. C. ernesti-augusti an average heterozygosity of HE = 0.67 (CI 95 % ± 0.06) and HO = 0.39 (CI 95 % ± 0.049); while Acrocomia aculeata, found average heterozygosity values  similar to those found in this study for C. tepejilote. The heterozygosity levels estimated in our study for C. tepejilote match those expected for a widespread outcrossing species and are higher than those observed for short-lived perennials , indicating that when populations were isolated, drift and mutation were more likely to influence structure than gene flow  Delta K value estimated by the Evanno method. (B) Mean (± SD) of the estimated log of the probability of the data given K (Ln Pr(X|K)) estimated by the STRUCTURE program for 15 replicate runs for each K value. Red dots indicate the optimal K selected by each method.Figure S2. Neighbour-joining tree based on pairwise GST values among 12 populations of Chamaedorea tepejilote (Arecaceae) in Costa Rica. See K = 5 cluster assignment by STRUCTURE in Table S1. Analysis of molecular variance (AMOVA) for SSR markers for populations of Chamaedorea tepejilote (Arecaceae) in Costa Rica, grouped in two regions: Pacific and Caribbean slopes.Table S2. Analysis of molecular variance (AMOVA) using cpDNA haplotypes for populations of Chamaedorea tepejilote (Arecaceae) in Costa Rica, grouped into two regions: Pacific and Caribbean slopes.plac060_suppl_Supplementary_Figure_S1Click here for additional data file.plac060_suppl_Supplementary_Figure_S2Click here for additional data file.plac060_suppl_Supplementary_Table_S1Click here for additional data file.plac060_suppl_Supplementary_Table_S2Click here for additional data file.


Candidatus Nitrosopelagicus brevis”) populations in the Pacific (Hawaii Ocean Time-series [HOT]) and Atlantic (Bermuda Atlantic Time Series [BATS]) Oceans sampled over 2 years. We observed higher nucleotide diversity in “Ca. N. brevis” at HOT, driven by a higher rate of homologous recombination. In contrast, “Ca. N. brevis” at BATS featured a more open pangenome with a larger set of genes that were specific to BATS, suggesting a history of dynamic gene gain and loss events. Furthermore, we identified highly differentiated genes that were regulatory in function, some of which exhibited evidence of recent selective sweeps. These findings indicate that different modes of genetic diversification likely incur specific adaptive advantages depending on the selective pressures that they are under. Within-population diversity generated by the environment-specific strategies of genetic diversification is likely key to the ecological success of “Ca. N. brevis.”Some marine microbes are seemingly “ubiquitous,” thriving across a wide range of environmental conditions. While the increased depth in metagenomic sequencing has led to a growing body of research on within-population heterogeneity in environmental microbial populations, there have been fewer systematic comparisons and characterizations of population-level genetic diversity over broader expanses of time and space. Here, we investigated the factors that govern the diversification of ubiquitous microbial taxa found within and between ocean basins. Specifically, we use mapped metagenomic paired reads to examine the genetic diversity of ammonia-oxidizing archaeal (“IMPORTANCE Ammonia-oxidizing archaea (AOA) are one of the most abundant chemolithoautotrophic microbes in the marine water column and are major contributors to global carbon and nitrogen cycling. Despite their ecological importance and geographical pervasiveness, there have been limited systematic comparisons and characterizations of their population-level genetic diversity over time and space. Here, we use metagenomic time series from two ocean observatories to address the fundamental questions of how abiotic and biotic factors shape the population-level genetic diversity and how natural microbial populations adapt across diverse habitats. We show that the marine AOA “Candidatus Nitrosopelagicus brevis” in different ocean basins exhibits distinct modes of genetic diversification in response to their selective regimes shaped by nutrient availability and patterns of environmental fluctuations. Our findings specific to “Ca. N. brevis” have broader implications, particularly in understanding the population-level responses to the changing climate and predicting its impact on biogeochemical cycles. Prochlorococcus ) . An rMAG belonging to the “Ca. Nitrosopelagicus brevis” species (rMAG_Nbrevis) was the second most abundant rMAG (after Prochlorococcus) across all samples and the most abundant in the samples below the euphotic zone (BEZ samples) at both sites (https://doi.org/10.6084/m9.figshare.19357964 for the relative abundances of the top six most abundant rMAGs across samples), with relative abundances ranging between 5.7 and 84.1%. rMAG_Nbrevis was 1.13 Mbp, smaller than the reference genome of strain CN25 (1.23 Mbp) and missing the two putative genomic islands identified previously (Ca. N. brevis” MAGs to feature high codon usage bias (CUB), and we estimated “Ca. N. brevis” to have the shortest minimal doubling time (3.5 ± 2.2 h)  out of the eight most frequently detected populations . This is surprising and inconsistent with the findings from previous culture studies of “Ca. N. brevis” C25 and U25, with reported doubling times of 7 days  members (https://doi.org/10.6084/m9.figshare.19357970). Our subsequent analyses focused on the populations of rMAG_Nbrevis in the BEZ samples because of their high abundance  through10.1128/msystems.01477-21.3TABLE S1Table S1, CSV file, 0.03 MB.Sample metadata. All samples used in this study and the metadata  are shown. Download Copyright © 2022 Hwang and Girguis.2022Hwang and Girguis.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/msystems.01477-21.4TABLE S2N50, broad taxonomic assignments, and ocean specificity) of dereplicated rMAGs that were used to delineate genetically defined populations in this study are shown. Download Table S2, CSV file, 0.03 MB.rMAG metadata. Quality and statistics : 74.8% ± 1.5% of the SNVs were synonymous, 15.8% ± 0.9% were nonsynonymous, and 5.0% ± 0.3% were intergenic. Such a low ratio of nonsynonymous to synonymous SNVs (pN/pS ratio) (0.068 [±0.007]) signifies that these populations have undergone purifying selection  at both sites and a medium level of nucleotide diversity  (eN) for rMAG_Nbrevis to be ~2.3 × 109 based on the nucleotide diversity  of nonconserved third codon positions . This is within the same order of magnitude as what has been described (eN = ~1.5 × 109) for the prochlorococcal ecotype using single-cell genomes (eN = ~2.6 × 109) for the most abundant Prochlorococcus rMAG in our data set (Cyanobacteriia_123_1).We detected polymorphisms (or single nucleotide variants [SNVs]) in 5.28% ± 1.1% of the sites across rMAG_Nbrevis. SNVs were spread evenly across the genome . In addition, we observed only minor fluctuations in nucleotide diversity in “Ca. N. brevis” populations, with no statistically significant difference in variance between the sites (P > 0.05 [by an F test]) over the 2-year sampling period (https://doi.org/10.6084/m9.figshare.19358003). Statistically significant differences (P < 0.05 [by Welch’s t test]) in nucleotide diversity between the sites were observed in 35 out of 75 non-“Ca. N. brevis” rMAGs that were present at both sites. The majority (31 out of 35) of the populations were more diverse at HOT than at BATS.“g period . As estag period , nucleotCa. N. brevis” populations at both sites, as signified by the decrease in the r2 metric of linkage with increasing distance between SNVs   for each population varied significantly between different taxa and were highly negatively correlated with nucleotide diversity  (Ca. N. brevis” to be 24.6 (±4.1), approaching levels similar to those of previously described quasisexual microbial populations of Prochlorococcus , we detected statistically significant differences in gamma/mu values (P < 0.05 [by Welch’s t test]) between the sites (Ca. N. brevis” populations at HOT (26.7 ± 3.45) than for those at BATS (22.2 ± 3.48) and higher recombination rates for the Prochlorococcus populations at BATS (52.5 ± 24.1) than for those at HOT (31.8 ± 15.0). This suggests the possibility of site-specific environmental influences on the recombination rate of populations of the same species.Homologous recombination plays an important role in generating and maintaining the genetic diversity of microbial populations. In order to estimate the rate of recombination, we first examined the linkage of SNVs across the genome. Using the paired-end reads 2× 150 bp) with a median insert size of ~250 bp, we calculated the linkage of SNV pairs that were found up to 420 bp apart. A decay in linkage disequilibrium, which is characteristic of recombination, was observed in the “een SNVs . We alsoeen SNVs . This paeen SNVs , in popuer (HGT) , and in  species . The ave< 1E−10) . We estirococcus , Synechohococcus , and Vib   and found that “Ca. N. brevis” featured a level of pangenome openness similar to those of Prochlorococcus and SAR324, while SAR11 exhibited a much more closed pangenome. Using the 43 “Ca. N. brevis” MAGs binned (>98% average nucleotide identity [ANI] with each other), we evaluated the “Ca. N. brevis” pangenome across HOT and BATS and examined the differential coverage of genes between the BEZ samples of the two sites. The pangenome consisted of 3,316 genes, with 3,099 of these being sufficiently long for read mapping. We removed 258 genes that were mapped at a coverage higher than three times the average coverage of rMAG_Nbrevis in the corresponding sample, as they likely represented genes shared with other species or binning errors. The coverage of pangenome genes relative to the average genome coverage varied significantly among genes in a sample, indicating within-population variation in gene content across multiple strains . In addition, we found distinct gene contents between sites; we identified 616 genes that were differentially abundant between the sites   than in HOT BEZ (γ = 0.18) . In order to confirm that this finding is likely not an artifact of the higher completeness of “Ca. N. brevis” MAGs binned from BATS, we compared the completenesses of the 43 BEZ “Ca. N. brevis” MAGs included in the pangenome analysis and found that those binned from HOT featured statistically higher completeness (P < 0.01 [by Welch’s t test]) , suggesting that the difference in the actual pangenome sizes between the two sites may be even larger than that observed in this metagenomic analysis.We observed a high variation in gene content among the “he sites  (adjuste[ANOVA]) . Of the BATS see . Levels https://doi.org/10.6084/m9.figshare.19358042) was approximately 7-fold more abundant at BATS, while the phosphonate transport system PhnCDE (https://doi.org/10.6084/m9.figshare.19358042). In contrast, the low-affinity and high-velocity inorganic phosphate transport (Pit) (https://doi.org/10.6084/m9.figshare.19358042). Interestingly, the two phosphate systems (PstSABC and Pit) of differing affinity levels were found to substitute for each other at the same location in the genome, adjacent to a phoU gene . Furthermore, we identified phylogenetic diversity in the Pit system (a phosphate transport regulator [DUF47] family protein and pit)  detected in “Ca. N. brevis” MAGs assembled from HOT. This indicates that this locus  may be undergoing frequent substitutions of gene cassettes involved in phosphate transport. The diversity of phosphate transport systems was previously identified by Qin et al. (pit) specific to HOT, for which we could calculate the r2 and nucleotide diversity values in the context of the rest of the genome. We found significantly higher linkage and lower nucleotide diversity values for these two genes than for the rest of the genome, suggesting the possibility of a recent selective sweep and/or a high level of purifying selection acting on these loci   system wn et al. ; howeverese loci .10.1128/msystems.01477-21.1TEXT S1Text S1, DOCX file, 0.01 MB.Supplemental results on ocean-specific genes. Download Copyright © 2022 Hwang and Girguis.2022Hwang and Girguis.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Ca. N. brevis” populations differ between HOT and BATS in the shared gene content, we calculated the fixation indices (STF) of each shared gene in rMAG_Nbrevis between the two sites. The average STF values across all shared genes between the HOT and BATS populations were relatively low, at 3.8% ± 3.2%, indicating that the alleles of most genes were dispersed between the two populations. However, we identified 14 genes with statistically high STF values ranging up to 44.2%  . We identified eight nonsynonymous substitutions in this phoU sequence at BATS and observed that these substitutions were enriched in or near the two putative pockets (STF genes involved in transcription regulation were DNA/RNA-binding protein AlbA (AlbA), a winged-helix-turn-helix DNA-binding protein (WHTH), a putative nitrogen regulatory protein (PII), an HTH_45 domain-containing protein (HTH), and a putative transcription regulator (TR). Additionally, another large fraction of the high-STF genes consisted of housekeeping genes, such as 30S ribosomal protein S5 (RpS5), DNA-directed RNA polymerase subunit K (RpoK), asparagine synthetase (ASNS), and threonine-tRNA ligase (TARS), and genes involved in transport, such as MIP channel protein (MIP) and sodium-dependent bicarbonate transport domain-containing protein (SBT). The phoU gene at BATS had significantly lower nucleotide diversity and higher mean r2 values, suggesting higher selective pressure on phoU at BATS than at HOT , both of which were located near phoU . Using consensus SNVs in neutral third positions in the codon, we estimated that “Ca. N. brevis” populations at the two sites diverged at least hundreds of thousands of years ago (see https://doi.org/10.6084/m9.figshare.19358066 for more details).In order to understand how “d test]) . Notably pockets . Other hn at HOT . Similar10.1128/msystems.01477-21.2TEXT S2Text S2, DOCX file, 0.01 MB.Supplemental results on the time of divergence between HOT and BATS populations. Download Copyright © 2022 Hwang and Girguis.2022Hwang and Girguis.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the https://doi.org/10.6084/m9.figshare.19358069). In order to quantify the differences in allele frequencies between “Ca. N. brevis” populations in different samples, we calculated the STF scores between every pair of samples in alleles that were sequenced with a coverage of >20× and within 2 standard deviations from the mean. We observed higher STF values between HOT samples than between BATS samples  . Using the pairwise mean STF as a distance metric, we conducted a principal-coordinate analysis (PCoA) and identified clustering of “Ca. N. brevis” populations at HOT samples with increasing temperature   . The temlation]) . Other gCa. N. brevis,” one of the most abundant archaeal species, with key ecological functions in global carbon and nitrogen cycling. We contextualize our findings with other abundant members of the community and found that some microbial populations exhibit more genetic differentiation between environments. For instance, among the microbial populations surveyed in the BEZ of HOT and BATS (P < 0.01 [by a t test]) in both nucleotide diversity and pN/pS ratios between the sites, while no significant differences were detected in the other bacterial populations, Gammaproteobacteria_112_0, SAR324_101_1, and Acidimicrobiia_57_1. Future research should investigate the relationship between the genetic diversity of microbial populations and their ecological roles  were observed. Furthermore, we show population structure clustering with temperature at HOT and identified alleles with frequencies that are highly correlated with temperature. We therefore propose that the higher genetic diversity at HOT is linked to the fluctuating conditions in the below-euphotic zone and that it may provide the standing variation needed for resilience against environmental instability at HOT. Correlations between in situ temperatures and sequence heterogeneity, as well as temperature-correlated allele frequency trajectories, have also been observed in marine SAR11 populations , investigated by Kashtan et al. (https://doi.org/10.6084/m9.figshare.19358069). Kashtan et al. . Interestingly, in contrast to Prochlorococcus, for which basin-specific genes were organized into genomic islands that are recombined , and metaBAT2  and BATS  previously reported by Biller et al. . The sammetaBAT2 , and themetaBAT2 . The quametaBAT2 .Ca. N. brevis” based on an ANI threshold of >97% using fastANI  was calculated for all pairs of SNVs with at least 20 pairs of connecting reads and normalized by rarefying to 20 read pairs using inStrain (eN) of “Ca. N. brevis” were estimated by calculating πneutral in nonconserved third codon positions as previously described by Kashtan et al. )  of 10−10 mutations per bp per generation  from each sample were mapped with Bowtie2 in sensitive mode . Genome- profile  and wereinStrain . The recinStrain  for all inStrain . The lown et al.  In shorteutral))  was usedneration , 85. NotCa. N. brevis” populations found in the BEZ habitat. We chose to focus on the BEZ samples because of the consistently high abundances of “Ca. N. brevis” at this sampling depth over the entire sampling period at both sites. For pangenome construction, we used Roary v3.13.0   value for each “Ca. N. brevis” gene was calculated between two metagenomic samples using a method previously described by Crits-Christoph et al.   using the pcoa function in R with cailliez correction. The lower bound for the time of divergence between the HOT and BATS backbone populations was estimated by first identifying the SNVs where the consensus alleles of the two populations differ  using inStrain compare   in inten/phyre2)  and visu compare . We usedn et al.  to calcuAll statistical tests were performed using R v4.0.2  and visuPRJNA385855. rMAG_Nbrevis binned in this study is available in Metagenomic reads and contigs used in this study can be found under NCBI BioProject accession number 10.1128/msystems.01477-21.5DATA SET S1Data Set S1, TXT file, 1.1 MB.Fasta file of rMAG_Nbrevis. Download Copyright © 2022 Hwang and Girguis.2022Hwang and Girguis.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the


Galemys pyrenaicus). For this, we used the ddRADseq technique, which generated 1421 SNPs. The heterozygosity values of the analyzed individuals were among the lowest recorded for mammals, ranging from 26 to 91 SNPs/Mb. Furthermore, the individuals from one of the localities, highly isolated due to strong barriers, presented extremely high inbreeding coefficients, with values above 0.7. Under this scenario of low genetic diversity and elevated inbreeding levels, some individuals appeared to be almost genetically identical. We used different methods and simulations to determine if genetic identification and parentage analysis were possible in this population. Only one of the methods, which does not assume population homogeneity, was able to identify all individuals correctly. Therefore, genetically impoverished populations pose a great methodological challenge for their genetic study. However, these populations are of primary scientific and conservation interest, so it is essential to characterize them genetically and improve genomic methodologies for their research.Recently, populations of various species with very low genetic diversity have been discovered. Some of these persist in the long term, but others could face extinction due to accelerated loss of fitness. In this work, we characterize 45 individuals of one of these populations, belonging to the Iberian desman ( Loss of genetic diversity may have detrimental effects on both population fitness and viability in the long term by restricting evolutionary potential3. In addition, inbreeding, caused by mating between close relatives, can lead to inbreeding depression, reducing fitness and population growth in the short term4. Both of these factors significantly increase the probability of extinction of populations.Some of the main threats that can affect the viability of a population include habitat loss and fragmentation, which, often, lead to the isolation of small populations and, consequently, reduced genetic variation and inbreedingPhocoena sinus), with 105 SNPs/Mb5, and the Iberian lynx (Lynx pardinus), with 102 SNPs/Mb6. In a few other species, the heterozygosity is yet another order of magnitude smaller, such as in the Island fox , with just 14 SNPs/Mb found in an individual from San Nicolas Island7. How these species have reached these extraordinarily low levels of genetic diversity and how this may be aggravated by consanguinity and inbreeding depression is still unclear11. To address these questions, genomic analyses of populations with extremely low genetic diversity are essential.Indeed, a growing number of studies based on complete genomes have recently revealed very low genetic diversity in certain mammalian species of conservation concern. In some of these, heterozygosity is as low as ~ 100 SNPs/Mb (heterozygous sites or SNPs per mega-base), one order of magnitude below that of species with large populations. These include species such as the vaquita , also known as the Pyrenean desman, is a small semi-aquatic mammal that inhabits clean rivers in mountains of the northern half of the Iberian Peninsula13. In recent years, the species has suffered significant population declines for reasons that are still being analyzed, but loss and fragmentation of the riparian habitat appears to be one of the most critical factors14. Previous double digest restriction site-associated DNA (ddRAD) and whole-genome sequencing studies of the species revealed that the Iberian desman has exceptionally low heterozygosity levels16. The lowest levels were recorded in the Pyrenees, where heterozygosity ranged from 12 to 116 SNPs/Mb, covering the two lowest orders of magnitude reported in mammals. Studies based on kinship networks revealed important connectivity problems for the species due to physical barriers, such as dams, which are leading to populations with very high inbreeding levels in the upper parts of rivers18. Ecological barriers, including the desiccation of rivers, the presence of predators such as the American mink, and contamination coming from urban areas14, can also isolate populations.The Iberian desman  technique, which is popular in population genomic studies due to the ease of obtaining data from a large number of individuals19, and assembled the reads using the recently sequenced Iberian desman genome15. We determined the heterozygosity rate and the inbreeding coefficient for 45 individuals to describe the genetic diversity of this population in detail and to understand whether genetic diversity and inbreeding were homogeneous in this area or whether these values were more extreme in some particular localities. We also tested the resolution power of the SNPs obtained by ddRADseq to genotype individuals in these populations of extremely low genetic variability and showed that most of the methods tested were unable to perform a correct individualization. Solving these methodological problems is crucial to address critical conservation problems in populations of species with extremely low genetic diversity.Here, we analyze a population of Iberian desman from the western Pyrenees, where it was previously shown, using relatively few individuals, that the species exhibits shallow genetic diversity throughout the area20.The population studied is located in the provinces of Gipuzkoa and Navarra (Spain), in the north of the Iberian Peninsula Fig. . We usedNo animal was specifically captured for this study, as they had been captured for previous works to monitor these populations. Therefore, this study did not require ethics approval by a specific committee.19 with some modifications. Each library was made in groups of 24 samples, and samples with low sequence yield were repeated in subsequent experiments, as indicated in Supplementary Table DNA was extracted using the DNEasy Blood and Tissue Kit (QIAGEN). Genomic libraries were constructed following the ddRADseq protocolStacks 2.60 package21. First, the process_radtags program was used to separate reads belonging to different individuals according to the barcodes using the recovery option (-r) and to filter out reads with a quality score limit (-s) of 10. After this step, reads from samples sequenced in different libraries were combined. This set of filtered reads is available at Dryad (see Data Availability section). Then, the reads were mapped to the reference genome of the Iberian desman15 with BWA v0.7.1722 using the mem algorithm. Subsequently, SAMtools v1.923 was used to produce BAM alignments where reads with a minimum mapping quality (-q) of 20 were kept. The mapped reads were processed using Gstacks from the Stacks package with the SNP calling model snp and both alpha thresholds for discovering SNPs and for calling genotypes of 0.01. Using the populations program of the same package, the sequences were saved in FASTA format after selecting loci with a minimum proportion of called individuals (r) of 0.9  and a minor allele frequency (MAF) of 0. Using the FASTA sequences of the loci, heterozygosity was estimated for each specimen as the number of heterozygous positions divided by the total sequenced length of the loci. The SNP dataset of all samples containing the first SNP from each locus was obtained with r = 0.9 and MAF = 0.025 and it was filtered for linkage disequilibrium (r2 > 0.8) with PLINK v1.90b6.2224. These SNPs were saved in PLINK and VCF formats for further analyses.The sequences obtained were filtered and assembled using the 16.The sex of all individuals was determined bioinformatically by mapping the reads against a Y-chromosome loci database as in previous work25 using the admixture model, correlated allele frequencies, and a number of populations (K) ranging from 1 to 6. A total of 1,000,000 iterations were run with a burn-in of 100,000. For each K value, 10 independent runs were performed and summarized with CLUMPP26. The optimal value of K was estimated with the method of Evanno et al.27, as implemented in STRUCTURE HARVESTER28.Population structure was analyzed with STRUCTURE 2.3.429.The principal component analysis (PCA) was performed with the KING v.2.2.5 program29. KING uses a method to infer the kinship coefficient between pairs of individuals which does not require allele frequency information. Specifically, we used the KING-robust method, which is not affected by population structure, with the kinship option. Negative kinship values and pairs with a flag error of 0 or 0.5, corresponding to unrelated individuals, were not used. The kinship coefficients were doubled to convert them to relatedness coefficients.We calculated the kinship coefficients between pairs of Iberian desmans with the KING program31. We used the dyadml estimator32, which was previously shown to be the most adequate for SNPs derived from ddRADseq data18, and the full nine states identity-by-descent (IBD) model, which takes inbreeding into account. Confidence intervals (95%) were calculated with 100 bootstraps, and only relatedness values with confidence intervals that did not overlap 0 were used. Using the same model, we estimated the inbreeding coefficient for each Iberian desman. The inbreeding coefficient was also estimated with PLINK24, which estimates this value based on the observed versus expected number of homozygous genotypes. It should be taken into account that these two estimates of the inbreeding coefficient, based on SNP frequencies without genomic position information, measure relatively recent inbreeding events and do not reflect past inbreeding, as do measures based on runs of homozygosity estimated from whole genomes15.The relatedness coefficients were also calculated using the RELATED programAs the relatedness coefficients were obtained from specimens with very low genetic diversity and very high inbreeding levels, we tested the accuracy of the estimates in this special context. For this, we used the reads of duplicate samples from the same individual that were repeated in different runs to increase their coverage  were used to estimate relatedness coefficients and the individualization was performed considering that duplicate samples have a relatedness coefficient of 1GetCrosses18. Each pedigree had founders from different areas , as a consequence of purging of recessive strongly deleterious mutations in the past46. However, inbreeding should also be taken into account in these populations because it can have a negative impact when weakly and mildly deleterious variants, very difficult to purge, become homozygous47. Consequently, inbreeding may be a critical factor for predicting the fate of these low-diversity populations.Heterozygosity was unusually low in all specimens , with values of 0.65 in some individuals48; and grey wolves (Canis lupus) of the highly inbred population on Isle Royale, with values of 0.8149. The presence of highly industrialized areas and large human population nuclei, such as Ordizia and Beasain, downstream from the Amundarain-Zaldibia and Aiaiturrieta-Ataun desman populations, could have played a role in isolating these individuals from the other populations. Specifically, the channeling of the rivers and the destruction of the riparian habitat, together with water pollution around these urban areas, could create ecological barriers to dispersal and favor the inbreeding of the isolated populations. No close relationships were found between individuals from this area and the rest (Fig. 52. Early knowledge of this problem and an action aimed at promoting dispersal from genetically healthier nearby populations to these strongly inbred populations, whether natural or assisted, could have helped to reverse this situation.Indeed, the inbreeding coefficients determined for the Iberian desmans in this area were of great interest as they varied widely among the different localities Fig. . In the ing Fig. . These vest Fig. , so eachThere are other isolated desman populations of the Iberian desman with very low genetic diversity that could follow a similar extinction trajectory. Studies should be promoted to characterize not only their demographic status, but also the heterozygosity and inbreeding levels of these populations, in order to evaluate their conservation status and take the necessary measures to ensure their long-term viability.Supplementary Information.


Margaritifera margaritifera in the Iberian Peninsula. A total of 130 individuals were collected from 26 locations belonging to 16 basins. We obtained 31,692 SNPs through Genotyping by Sequencing (GBS) and used this dataset to infer population structure. Genetic diversity given as observed heterozygosity was low. Pairwise FST comparisons revealed low levels of genetic differentiation among geographically close populations. Up to 3 major genetic lineages were determined: Atlantic, Cantabrian and Douro. This structure suggests a close co-evolutionary process with brown trout , the primordial fish host of this mussel in the studied area. Some sub-basins showed some genetic structuring, whereas in others no intrapopulation differentiation was found. Our results confirm that genetic conservation units do not match individual basins, and that knowledge about the genetic structure is necessary before planning recovery plans that may involve relocation or restocking. The same reasoning should be applied to strictly freshwater species that are sessile or have restricted dispersal abilities and are currently imperiled worldwide.Effective conservation actions to counteract the current decline of populations and species require a deep knowledge on their genetic structure. We used Single Nucleotide Polymorphisms (SNPs) to infer the population structure of the highly threatened freshwater pearl mussel  Indeed, in the last fifteen years, the use of SNPs in population genetics has been developed as an extensively used genomic tool to assess patterns of genetic variation in populations. Advantages of SNPs include the higher number of analyzed loci (thousands or hundreds of thousands), which results in a more extensive screening of the genome, a lower genotyping error, a higher reproducibility among laboratories and more precise estimates of diversity due to the high mutation rate of microsatellites, which may lead to underestimate heterozygosity3. However, challenges in the use of SNPs still remain, such as the requirement of a large number of markers to obtain robust estimations of genetic variation among populations, when compared to multi-allelic microsatellites that, as an advantage over SNPs, provide higher levels of polymorphism, a faster evolutionary rate and low levels of ascertainment bias4. Consequently, although the use of SNPs has become increasingly more popular, microsatellites should not be disregarded. In fact, both kind of markers may be considered as complementary in Evolutionary Biology, as both may respond to different issues. For example, while adaptive processes cannot be assessed by the use of neutral markers such as microsatellites (located in non-coding regions), some SNPs (located in coding and non-coding regions) may be under selection, providing information about demographic (drift) and functional (selection) processes. However, microsatellites may perform more efficiently in relatedness and parentage analyses as a consequence of being multi-allelic markers (5 and references therein). Although SNPs and microsatellites may be equally valid and comparable in the analysis of the neutral genetic variation, the one analyzed in this paper, some studies comparing SNPs and microsatellites-based approaches with the same sampling set have advocated for the use of SNPs to attain more reliable inferences of patterns of genetic structure and diversity in different aquatic organisms, such as the brown trout6. Therefore, the analysis of SNPs, the markers used in this study, may be considered an efficient genomic tool to investigate evolutionary patterns and to make grounded decisions regarding conservation priorities for the most threatened fauna (e.g. Iberian lynx:7).The understanding of genetic structure and diversity patterns of highly endangered organisms is crucial to guarantee the implementation of effective conservation actions that counteract the current decline of species and populations, ensuring their long-term preservation. The application of Single Nucleotide Polymorphisms (SNPs) to address evolutionary and conservation issues has become widely extended in non-model organisms9. The freshwater pearl mussel Margaritifera margaritifera L. is a threatened species listed as critically endangered in Europe and included in the European Habitats Directive under Annexes II and V, and in Appendix 9. It has an Holarctic distribution, with populations in rivers on both sides of the Atlantic Ocean, ranging from the USA and Canada and from Russia and Scandinavian countries to the Iberian Peninsula9, that constitutes the southern limit of its distribution range. Here it is mainly located along several hydrological basins from the northwestern quadrant, from Asturias in the Cantabrian coast to the Douro river basin in the southern edge, and one isolated population in the Tagus Basin10  and the brown trout  being the preferred hosts8. Some of the healthiest M. margaritifera populations are located in rivers with massive salmon populations, but in most areas where the species is distributed, salmon populations are extinct or near extinction, and glochidia infect Salmo trutta populations more frequently or even exclusively11. In this sense, a co-evolutionary relationship between freshwater mussels and their fish host  has been established, although some authors consider it to be more a symbiosis-commensalism than a parasitism relationship12.The life cycle of M. margaritifera is poor. Indeed, during the last century, its European populations have suffered an estimated decline of around 90% because of habitat loss and fragmentation, overexploitation for pearl extraction, pollution and the introduction of invasive species13. The decline of host fish  in most of the rivers inhabited by the freshwater pearl mussel, especially in southern European regions15, could also have contributed to the decline of M. margaritifera populations and must be considered in the future trend of the species16. In the Iberian Peninsula, populations of this species have an additional constraint, as southern regions might be more susceptible to climatic change associated to a stronger decrease in precipitation and an increase of more unstable hydrological conditions than in northern latitudes, which has been reported as a potential extinction risk for M. margaritifera16.The conservation status of 18. Relocation, restocking, and reintroduction actions pose questions about the genetic compatibility between populations as well as genetic-based adaptations to local environments that have been poorly addressed18, besides ethical issues regarding the anthropogenically-mediated mixture of distinct evolutionary lineages. Thus, the long-term preservation of this highly endangered species can greatly benefit from genomic studies19, since the analysis of SNPs can act as a powerful tool for describing the genomic variation of the freshwater pearl mussel at a wider genome scale, which is imperative for the effective implementation of conservation actions as those described above.Because of the continuous threats to populations, several conservation actions have been taken in the last decades that aim to reduce or revert the species decline. These include habitat protection and restoration, host fish restocking, specimens’ relocation and captive breeding for restocking or reintroductionsM. margaritifera in the Iberian Peninsula, most of them focusing on populations from the Spanish northwestern region of Galicia21. Nevertheless, the whole distribution range of Iberian M. margaritifera has not been analyzed to date. These previous studies based on microsatellite markers have shown populations with a high degree of structuring and reduced genetic diversity20. High degrees of population structure have also been identified in central and northern European populations of freshwater pearl mussels, but in this case the degree of genetic diversity was variable, often with higher values23. Contrary to these genetic patterns, North American populations of M. margaritifera are characterized by high levels of genetic diversity and low degree of genetic differentiation, with a probable single panmictic population, and its conservation status has been considered as relatively safe in comparison with European populations25. Studies based on SNP markers for Iberian M. margaritifera populations have not been performed yet. Only two SNP-based studies for Margaritifera were performed in a restricted area of North America , as well as one study in the unionid genus Cyprogenia, endemic to North America27.Several population-scale studies have tried to understand the genetic structure and diversity of M. margaritifera in the Iberian Peninsula through the analysis of its genetic structure based on SNPs; (2) to estimate the genetic diversity of these evolutionary units; (3) to determine the factors that better explain their genetic structure and diversity; (4) to define conservation units for conservation purposes; and finally, (5) to propose how the information on genetic patterns and evolutionary units should be incorporated in conservation and management plans.The aims of this study were (1) to identify the evolutionary units of M. margaritifera were collected from 26 locations at 16 hydrological units, corresponding to coastal Cantabrian and Atlantic basins and sub-basins belonging to the large Douro watershed , following the manufacturer’s instructions. For some samples, DNA concentration rendered too low for library construction and an ethanol precipitation protocol was used to increase it. The final concentration of all samples was determined using the Qubit® 2.0 Fluorometer and DNA fragmentation was verified in a 1% agarose gel. Samples were subjected to a paired-end Genotyping by Sequencing (GBS) protocol , which was performed at LGC Genomics, GmbH, Germany. DNA was fragmented using the restriction enzyme MslI, libraries were constructed and sequenced using Illumina NextSeq with a read length of 150 base pairs (bp).28 and MultiQC29 and all data was included in the subsequent analyses. The process_radtags program of STACKS v.2.530 was used to truncate all reads to the same length (135 bp) and to discard reads with low quality scores and uncalled bases, using the default settings for the window size (0.15 of the read length) and the base quality threshold (10 Phred score). The Stacks software only cuts the final end of the reads, therefore, Trimmomatic v.0.3631 was used to eliminate the first 5 bases in all sequences. The final length of reads was 130 bp. All sequences passed quality standards to be included in the subsequent analyses after process_radtags analysis (Phred score > 30).Sequence quality of reads was assessed using FastQC 0.1130 was used to build a catalog of loci. A subset of individuals covering all sampled sub-basins was used in the construction of the catalog; these individuals are represented in Table 31. The program populations included in the software STACKS v.2.530 was used to test the number of genotyped loci and variant sites. For the subset of individuals used for the catalog, the parameters that maximized the number of SNPs were M = 1, m = 4 and n = 2, and the resulting catalog was used in downstream analyses.The de novo pipeline from the STACKS v.2.5 software32 with a word length of 10 and a sequence identity threshold of 0.98. The paired pre-processed sequences (those truncated to 130 bp) of all individuals were aligned to this catalog using BWA-MEM from bwa v. 0.7.1733 with default parameters. We sorted the output alignments and removed unmapped reads using Samtools v1.1034. We used the software Freebayes v.1.3.135 to carry out the SNP-calling based on haplotypes for variant detection and considering the cleaned catalog as reference to genotype SNPs, discarding reads and bases with low quality, and without using Hardy–Weinberg equilibrium priors . The output from Freebayes (.vcf file) was subjected to different filters in order to (1) eliminate indels; (2) remove genotypes with a depth of coverage (DP) lower than 4; (3) remove sites with excess of heterozygosity when pooling all individuals; (4) remove sites with more than 60% missing data and, finally, (5) eliminate a minimum allele frequency below 1%. These filters were applied using a combination of options from VCFtools v0.1.1536 and BCFtools v1.634. The final dataset comprised 31,692 SNPs with 32.79% missing data and 119 individuals from the initial 130.Given the possibility that forward and reverse sequences of the same DNA fragment were treated as different loci, similar reads within the catalog were clustered using CD-HIT-EST from the CD-HIT v. 4.8.1 softwarepopulations of STACKS v. 2.5430 was used to estimate genetic diversity statistics for each population, using the final vcf file : number of private alleles, number of polymorphic sites, nucleotide diversity, and FIS values (inbreeding coefficient). Significance for FIS values was estimated in Arlequin v.3.5 using 10,000 permutations tests37.Genetic diversity was evaluated by analyzing the expected and observed heterozygosity at different hierarchical levels  using custom scripts. The program ST values for all population pairs using the Weir and Cockerham's estimator38 in the Arlequin v. 3.5 software37. The corresponding significance  were computed with 10,000 permutations and were adjusted for multiple comparisons by using Bonferroni  corrections. To investigate population structure at different hierarchical levels we performed an Analysis of Molecular Variance (AMOVA)37. More specifically, we tested three distinct grouping schemes for which, in decrescent order of stratification, we considered as "populations" in the context of AMOVA analyses: (1) within individualized sampling locations considered as independent ; (2) among sampling locations grouped within individualized hydrological sub-basins; and (3) among individualized sub-basins . Significance was tested with 10,000 permutations.Genetic structure was inferred by calculating pairwise F39 and ADMIXTURE v1.340, testing values of K from 1 to 16 (number of sub-basins) and performing 100 independent runs for each value of K. For sNMF, we identified the most plausible K value as the one with the lowest cross-entropy. For ADMIXTURE, we identified the best K value as the one with the lowest fivefold cross-validation error and for 2 ≤ K ≤ 3, we assessed similarity across the 100 replicates using the Greedy algorithm implemented in CLUMPP v1.1.241.Finally, to investigate population structure using individual-based methods, we determined the ancestry proportions from specified number of clusters (K) using sNMF v.2.0 from the R package LEAFor river basins having more than two sampling locations , approximate geographic distances between sampling locations were measured by hand (in km), following the natural meandering of rivers, using the Path tool available in Google Earth Pro version 7.3.3.7786 (© Google LLC). Insurmountable barriers for fish  were mapped along with the drawing of pathways.ST/(1-FST) estimates  and their respective pairwise FIS values, either positive or negative, were always close to zero for all populations and non-significant for all of the locations  and most of the remaining populations, and between the Porcia Cantabrain basin and other Cantabrian (Eo and Ouro) and Atlantic basins (Tambre and Ulla). However, Alberche samples had a relatively high proportion of missing data in comparison to other populations, therefore this result has to be taken with caution. The higher FST values were found between pairwise comparisons of either Esva and Esqueiro Cantabrian basins with several Douro basin populations, as well as between the Tâmega sub-basin (Douro) and Atlantic-Cantabrian populations , statistically significant at both location and basin/sub-basin, especially those involving the Douro lineage with the remaining populations belonging to the Atlantic and Cantabrian lineages, as well as the Tagus Basin (Alberche). These results indicate a high differentiation of Douro populations  among individuals within locations, indicating overall limited genetic differentiation, there was an important percentage of variation attributed to differences between hydrological sub-basins   Fig. . Within hem Fig. ; however8. Highly threatened populations that show considerable genetic diversity depletions are often subjected to recovery plans which include conservation measures like the injection of genetic novelty, through the introduction of specimens from other populations, and the increase of population size to minimize stochastic threats42. However, genetic data has seldom been considered in M. margaritifera conservation actions, even though it is a highly threatened species with multiple captive breeding and restocking programs throughout Europe43.Conservation units from a genetic point of view should be regarded as having a common ancestor and evolutionary history, as well as enough genetic diversity, so that adaptation to local conditions and adaptability to future changes are adequately balanced21. However, in these studies the Ulla (Arnego) and Eo populations  exhibited low values of genetic diversity, which was not the case in the present study. These inconsistencies may be associated with the lower number of loci analyzed in microsatellites studies when compared to SNP datasets, hence SNPs reflect genome-wide genetic diversity of populations more accurately than microsatellites6. The overall low genetic diversity values found for M. margaritifera are commonly found in other endangered freshwater organisms, such as Australian freshwater crocodiles and Mexican golden trout45 and have also been estimated for North American M. margaritifera24 and M. hembeli26, suggesting a strong relation between the loss of genetic diversity and the poor conservation status of these species. The low genetic diversity values found in the Iberian populations have been explained by strong genetic drift effects due to population bottlenecks21. Populations with low effective population sizes face stronger effects of genetic drift, leading to a random loss or fixation of alleles, and consequently resulting in the loss of genetic diversity42. Nevertheless, some of the largest Iberian populations (census data) such as that of the Narcea Cantabrian river showed the lowest diversity, whereas some of the populations with the smallest census data such as that from the Neiva small Atlantic river basin showed higher diversity values. Although genetic diversity was low, no evidence of significant inbreeding was found for any M. margaritifera populations . This was an unexpected result considering the high autofecundation described for some of the Iberian populations20, which is a common strategy in freshwater mussels when environmental conditions are not optimal or when population sizes are small46 and may be a methodological artifact.Our SNP data corroborate previous studies based on microsatellites that found low genetic diversity in Iberian hydrological basinsM. margaritifera populations, which has been associated to the effect of genetic drift and founder effects in isolated populations, ecological differentiation due to different selective pressures, or to the human impact that have led to habitat fragmentation and decreasing of gene flow23. Contrary to these findings, mitochondrial markers (COI and 16S rRNA) or allozyme data have not supported such levels of population differentiation47, probably because of slower evolutionary rates of these markers relative to microsatellites. Therefore, our results confirm and complement previous studies based on microsatellites and show a strong differentiation between populations from different basins and sub-basins, but variable levels of differentiation within sub-basins. Even so, several pairs of populations from the Atlantic and Cantabrian coasts show very little differentiation, suggesting they may have been separated recently . Our results concerning the Douro River system and the small independent Atlantic basins to the north, such as the Neiva river, indicate that gene flow between these populations was maintained until more recently than between Atlantic and Cantabrian populations. The low or neglectable differentiation within the same river or sub-basin suggests that gene flow was effective across locations. On the other hand, the higher differentiation detected between populations from either banks of the river Douro suggests that the main course of this river constituted an effective barrier to gene flow. This may be related to migratory paths and behavior of host fish in the past.Previous studies based on microsatellites showed a strong structure among European M. margaritifera populations, higher genetic differentiation and low levels of genetic diversity are expected due to the presence of suboptimal or atypical habitat conditions in comparison to core populations49. The geographical position of the Iberian Peninsula, located under the influence of the Mediterranean climate, constitutes the southern limit of several European freshwater organisms, which exhibit such genetic patterns . It is hypothesized that these species could not extend their distribution to Northern Africa due to either climatic (more arid Mediterranean climate conditions52) or biogeographical  constraints; nevertheless, in the Iberian Peninsula, these species are often restricted to sparse areas that are ecologically adequate, probably leading to local adaptation processes.In peripheral distribution ranges, as is the case of the Iberian M. margaritifera populations in our study reflects, among other factors, the dispersal abilities of mussel species and their fish hosts55. A strong relation between the degree of genetic distinctiveness and the fish host species has been confirmed for M. margaritifera: the genetic structure is more intensely marked when the fish host is Salmo trutta (trout) rather than Salmo salar , due to the higher dispersal ability of the Atlantic salmon57. In the Iberian Peninsula, the strong decline of the Atlantic salmon15 often left the trout as the only available host for most M. margaritifera populations. In addition, the genetic structure of the Iberian brown trout is in part congruent with our results for the genetic structure of Iberian M. margaritifera, implying some degree of co-evolution between the two species: three main trout lineages  have been recognized and associated to the role of Iberian glacial refuges in the Pleistocene59. The Atlantic lineage actually includes the populations closer to the lower course of the Douro river, apart from populations from Galician and Cantabrian coastal basins, effectively splitting the Douro basin trout populations in two separate lineages , which are urgently needed for M. margaritifera populations to prevent imminent extinction. Considering the results of genetic structure overall, at least three different conservation units could be identified in the Iberian Peninsula for M. margaritifera, based on the three large genetic lineages: Atlantic, Cantabrian and western Douro. However, due to the singularity of each independent population within the three genetic lineages and the low genetic diversity found in most of them, any planned conservation actions must take in consideration the specific genetic patterns of each population. Our study showed that, in some cases, several tributaries share the same genetic identity and can be considered a single population, but in other cases different genetic backgrounds are identified within the same river. This emphasizes the importance of not generalizing results and accurately characterizing each population before planning conservation actions. We can therefore identify conservation units for M. margaritifera in the Iberian Peninsula at different scales: a large single metapopulation that includes all the species distribution and that had the potential to maintain gene flow to different degrees through river continuity or geologic events; regional units , sub-basin (e.g. Tâmega sub-basin), river  or location (e.g. Paiva). The identification of these discrete evolutionary units is crucial for species conservation, as it helps to define management units to best protect genetic diversity without eroding genetic structure67.Genetic structure and genetic diversity are gaining more importance in the development of more effective conservation plans, as both are considered key components of biological diversityM. margaritifera has been the object of much conservation interest in Europe for the last two decades68. Several in-situ and ex-situ conservation actions have been proposed and implemented throughout the continent, including the Iberian Peninsula13. Captive breeding programs may be implemented to restock or reintroduce the species in a certain river system70 and have been extensively implemented through LIFE projects. Restocking and reintroduction may also be achieved via translocation within or between river systems71. The translocation of specimens within a river system is a common action to bring together the few specimens left in certain patches and, in this way, increase the probability of fertilization and glochidia production, or to avoid the negative impact of local negative pressures, such as bridge construction. A river or stream is often used as a basic conservation unit for freshwater mussels. However, our results suggest that the adequate unit to minimize genetic mixture may be a whole sub-basin/basin (as is the case of the Tâmega sub-basin). In other cases, some genetic structure is present within the same river, such as in the Paiva and Narcea rivers, which may lead to a narrower definition of conservation unit. In these cases, translocations may be difficult to plan to preserve the original gene pools, as it requires in depth study of the species genetic structure along the river.It can also be argued that under the current situation where most populations are isolated, reduced or both, translocating mussels from different areas of the same river systems may contribute to increase genetic diversity, much like the free movement of hosts would do in undisturbed conditions, and thus favoring the conservation prospects of the species. In this sense, translocations between rivers, sub-basins or even basins, in theory, would potentially benefit the species ability to cope with changes. However, balancing the maintenance of evolutionary genetic identity and gene flow across river systems is a delicate process that must be carefully planned case-by-case in a framework that considers all possible outputs. Furthermore, possible habitat adaptations of specific genetic profiles may also have evolved. Discarding this possibility may jeopardize altogether the success of a translocation or reintroduction program. The inclusion of more individuals per population or different analytical methodologies such as whole genome resequencing could help reducing the limitations from our data set, such as the presence of missing data or the number of individuals (two in Porcia) and allow taking sound conservation decisions.M. margaritifera populations in the Iberian Peninsula shed some light on relevant issues in planning and development of effective management decisions, including restocking and translocations. Indeed, an integrative conservation approach combining genetic and ecological knowledge about the species is urgently needed for the long-term preservation of the freshwater pearl mussel.The understanding of the genetic structure and diversity of Supplementary Information.


Cairina moschata) provide unique genetic resources patterned by both tropical environmental conditions and human activities, the evaluation of their genetic diversity and population structure will shade light on the mechanism of their remarkable adaptive capacities. We therefore analyzed the variation in mtDNA cytochrome b and nuclear DNA CYP2U1 sequences of 378 Nigerian Muscovy ducks (comprising of 287 de novo and 91 downloaded) plus 80 published sequences of Muscovy ducks from India. The results showed high haplotype diversity (0.800 ± 0.023) among Nigerian Muscovy duck populations with 91 distinct haplotypes for the nuclear DNA CYP2U1 gene but low (0.266 ± 0.033) for cytochrome b with 31 haplotypes. The median-joining networks of both markers grouped Nigerian Muscovy ducks into two; the first group consisting of only Nigerian Muscovy duck populations, and the second group Nigerian with Indian populations. Neutrality test results indicated that Nigerian populations experienced recent population expansion and/or genetic hitchhiking. A geographic signal was absent in line with previously studied poultry species in Nigeria. The most prominent haplotype dominated across all regions in Nigeria, which may be due to extensive genetic intermixing except for the Indian population . This indicated low genetic differentiation between and within Nigerian Muscovy duck as revealed by the suitability of the nuclear DNA CYP2U1 gene.The domestic Muscovy duck ( Cairina moschata) is indigenous to the tropical regions of Central and South America  and the University Ethical Review Committee, University of Ilorin, Nigeria (UERC/ASN/2021/2161).n = 7 males; n = 9 females), Kano State , Taraba State , Oyo State , Ekiti State , and Bayelsa State . Our sample collection considered genetically unrelated domestic Muscovy ducks with geographical and ecological perspectives from the six states in Nigeria. This included random sampling of at least two matured birds per flock from domestic Muscovy ducks raised on free-range scavenging system with a distance of at least 500 m apart. Information on the pedigree of the birds were obtained from their owners before blood collection. Blood samples were collected from each bird through their wing venipuncture with the assistance of trained veterinary personnel. Blood samples were preserved in 95% ethanol at room temperature thereafter transported to the laboratory and stored at 4 °C for instant use, or −80 °C for extended storage.We sampled a total of 287 domestic Muscovy ducks  from farmer’s flocks in six different States in Nigeria as follows ; Sokoto Genomic DNA was extracted from whole blood using the standard phenol/chloroform method . We exam2, 0.2mM dNTPs, 0.2 µM each primer, 1.5 U Takara Taq DNA polymerase, and approximately 30 ng genomic DNA. The amplification conditions: 95 °C for 5 min, 30 cycles at 94 °C for 30 s, 54 °C for 45 s, 1 min for 72 °C and a final extension of 72 °C for 5 min (Two overlapping primers were used to amplify 940 bp of cytochrome b: forward for the first set DUKCYB1L (5′-ATCTTTCGCCCTATCCATCC-3′) and reverse DUKCYB2R (5′-TTTGGTTTACAAGACCAATGTTTT-3′), and the second, P105 (5′-GCCTCCTGCTAGCCATACAC-3′) and P106 (5′-TACGGCGGGAAAGAGAAATA-3′) . The cytor 5 min . After, In addition, 747 bp fragment of nuclear DNA CYP2U1 was amplified using forward primer P103 (5′-GTTATTTGGTTATGCATATCGTG-3′) and reverse primer P104 (5′-GAGACGGTTGGCGTATATGG-3′) . SimilarL08385; Anas platyrhynchos cytochrome P (450 2U1XM_013099877) using SeqMan Pro of Lasergene 7.1.0 . Further, 149 cytochrome b sequences of Muscovy ducks (India (n = 78) KX985658–KX985735; Nigeria (n = 71) MZ330001–MZ330071) and 159 nuclear DNA CYP2U1 sequences (India (n = 80) KX985578–KX985657; Nigeria (n = 79) MZ383446–MZ383524) from India and Nigeria in previous studies (de novo [GenBank: MZ383528–MZ383776] and 149 from public database) and 396 nuclear DNA CYP2U1 sequences (237 de novo (GenBank: MZ383202–MZ383439) and 159 from public database) (The 248 cytochrome b and 237 nuclear DNA CYP2U1 sequences were mapped to the reference sequences of cytochrome b (GenBank accession no.  studies  were minatabase)  were donatabase)  in MEGA atabase) .All the sequences were compared, and haplotypes identified using DnaSP 5.10.1. FurtherKM886937) and 396 Muscovy duck nuclear DNA CYP2U1 sequences. Both networks were constructed using the median-joining (MJ) algorithm (www.fluxus-engineering.com). The Median networks with all possible short trees were simplified by running the maximum parsimony (MP) calculation option to eliminate superfluous nodes and links.We investigated the association of Nigerian Muscovy duck with other Muscovy duck samples retrieved from public database . MEGA velgorithm  implemenD  and 71 from the GenBank) constitute 31 distinct haplotypes  newly generated in this study together with other samples from Nigeria (n = 16), India (n = 11), the USA (n = 1), and China (n = 1) retrieved from the GenBank (KM886937) was used to root the phylogenetic tree. The phylogenetic tree showed significant clustering of Nigerian Muscovy duck together with Indian, USA, and Chinese Muscovy. A few minor clustering was also noticed within the major clade  and −25.209 (P < 0.05), respectively  was observed between Oyo and Niger populations (FST) were observed between Taraba and the six populations except Ekiti, Niger and Sokoto, Bayelsa and Kano, Oyo and Ekiti. Negative FST values is effectively seen as zero values. This indicates absence of genetic subdivision between the populations. The negative FST may possibly be product of sampling size and low haplotype diversity. The regression analysis results showed a negative correlation between geographic distance and genetic distance , suggesting the possibility of “absence” or “insignificant change” in genetic distance associated with increase in geographic distance  among populations, de novo [GenBank: MZ383202–MZ383439] and 79 from the GenBank) constitute 91 distinct haplotypes  and −5.228 (P < 0.05), respectively  was observed between Ekiti and Sokoto populations (FST) were observed between Taraba and the six populations except Ekiti. The negative FST may possibly be product of sampling size and low haplotype diversity. The regression analysis results  also showed consistency with cytochrome b gene  among populations, In this study, the suitability of the nuclear DNA CYP2U1 gene was confirmed by assessment of variations in the genetic diversity and populations structure of Nigerian domestic Muscovy ducks in addition to the conventional mtDNA cytochrome b. High genetic diversity was revealed among Nigerian Muscovy duck sampling sites with 91 distinct haplotypes for the nuclear DNA CYP2U1 gene but low for cytochrome b with 31 haplotypes  and S5. D and Fu’s Fs tests ; cytochrome b and H1 (ABO14); CYP2U1) comprises individuals across all eight different locations in Nigeria. This suggested that one group of the Muscovy duck present in Nigeria descended from a single domestication. The most prominent haplotypes (H4 (ADE15); cytochrome b and H1 (ABO14); CYP2U1) with the highest frequencies exhibited a star-like pattern ; Fig. 1BFST distance between most Nigerian Muscovy duck populations being studied. The regression analysis results showed a negative correlation between genetic and geographical distance in the two genes  revealed two groups  in Nigerian Muscovy duck populations: the first group consisting of only Nigerian Muscovy duck sampling sites while the second group consists of the Nigerian and the India Muscovy duck populations. Neutrality test results indicated that all Nigerian populations experienced recent population expansion and/or genetic hitchhiking. This result suggested that the genetic structure could be explained by founder effect. The absence of geographic signal in Nigerian Muscovy duck sampling sites suggests extensive genetic intermixing consistent with previously studied poultry species in Nigeria. Further, the suitability of the nuclear DNA CYP2U1 gene in the analysis of genetic diversity was confirmed.10.7717/peerj.13236/supp-1Supplemental Information 1The 72 CYTB sequences were selected from haplotypes in Click here for additional data file.10.7717/peerj.13236/supp-2Supplemental Information 2The 168 CYP2U1 sequences were selected from haplotypes in Click here for additional data file.10.7717/peerj.13236/supp-3Supplemental Information 3(a) Nigerian Muscovy duck mismatch distribution – CYTB (b) Nigerian Muscovy duck Population expansion mismatch distribution – CYTB (c) Nigerian Muscovy duck Mismatch distribution - CYP2U1 (d) Nigerian Muscovy duck Population expansion mismatch distribution - CYP2U1. With 1000 bootstrap replication and pairwise difference, using ARLEQUIN v3.5 .Click here for additional data file.10.7717/peerj.13236/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.13236/supp-5Supplemental Information 5n = 5), Sokoto (n = 15), Ekiti (n = 42), Kano (n = 57), Bayelsa (n = 62), Oyo (n = 66) and Kwara (n = 57), Niger (n = 14). 2 North ; Central ; West ; South (Bayelsa) 3 remaining populations include all the populations listed above except the mentioned population.1 Overall is Nigerian Muscovy duck populations which were as follows (number of samples in parentheses): Taraba , Sokoto (n = 8), Ekiti (n = 36), Kano (n = 52), Bayelsa (n = 67), Oyo (n = 71) and Kwara (n = 64), Niger (n = 15). 2 North ; Central ; West ; South (Bayelsa) 3 remaining populations include all the populations listed above except the mentioned population.1 Overall is Nigerian Muscovy duck populations which were as follows (number of samples in parentheses): Taraba  and geographical distance (*100 km) in CYP2U1 of Nigerian Muscovy populations. ST) and geographical distance (*100 km) in Cytochrome b of Nigerian Muscovy populationsClick here for additional data file.10.7717/peerj.13236/supp-8Supplemental Information 8Click here for additional data file.