Lactase non-persistent adults may develop symptoms of lactose intolerance when consuming dairy products. In the Americas, there is no evidence of the consumption of these products until the arrival of Europeans. However, several American countries’ dietary guidelines recommend consuming dairy for adequate human nutrition and health promotion. Considering the extensive use of dairy and the complex ancestry of Pan-American admixed populations, we studied the distribution of –13910C > T lactase persistence genotypes and its flanking haplotypes of European origin in 7,428 individuals from several Pan-American admixed populations. We found that the ∗T–13910 allele frequency in Pan-American admixed populations is directly correlated with allele frequency of the European sources. Moreover, we did not observe any overrepresentation of European haplotypes in the –13910C > T flanking region, suggesting no selective pressure after admixture in the Americas. Finally, considering the dominant effect of the ∗T–13910 allele, our results indicate that Pan-American admixed populations are likely to have higher frequency of lactose intolerance, suggesting that general dietary guidelines deserve further evaluation across the continent.In adulthood, the ability to digest lactose, the main sugar present in milk of mammals, is a phenotype (lactase persistence) observed in historically herder populations, mainly Northern Europeans, Eastern Africans, and Middle Eastern nomads. As the  MCM6 gene, located −14 kb upstream of the LCT gene, in its enhancer region, are believed to be responsible for the LP phenotype. The ∗T–13910 allele  is widely distributed in the European population; the allele frequencies vary from 8.9% in Tuscany  to 72.0% among British (England and Scotland), with an average of 50.8% in Europe   and are also associated with the LP phenotype. Among these, the most well-known are –14010G > C (rs145946881), –14009T > G (rs869051967), –13915T > G (rs41380347), and –13907C > G (rs41525747)  . In Asianotypes) , and theceed 10% . Other S1525747) .∗T–13910 allele frequencies reported in ancient Native American samples, the low frequencies observed in Asian peoples and present-day Native Americans suggest that the ∗T–13910 allele was introduced in the Americas after the arrival of Europeans. Moreover, African and Middle Eastern SNPs have been reported at very low frequencies in the Americas. The frequency of ∗C–14010  in Afro-Brazilians from the South is 0.27%  in admixed Brazilians from the North and the Northeast are 0.25 and 0.58%, respectively  and ∗G–13915  in Mestizos from Ecuador are 0.20 and 0.50%, respectively (–13910C > T (rs4988235) substitution of European origin. Thus, the admixture events that led to the formation of current Latin American populations contributed to introducing LP-associated alleles in these groups  analyzed this locus for evidence of positive selection in Pan-American admixed populations . Moreover, we discussed our results in light of the use of dairy products in public health policies for nourishment in Latin American countries.The complex demographic processes involved in the formation of the Americas gave rise to mosaic populations in which ancestry proportions vary among and within the countries, affecting the distribution of Mendelian and complex phenotypes . Conside–13910C > T SNP (rs4988235). In order to keep a higher SNP density for haplotype-based analyses, we organized the genomic information in two datasets: (i) LARGE-PD only and (ii) a merged dataset . During the quality control of these two datasets, we excluded SNPs with significant missing data (>10%), loci with 100% of heterozygosity, non-chromosomal information, A/T–C/G genotypes, and SNPs with minor allele count = 1 using PLINK 1.9  for 259 Afro-Brazilians individuals from the south region of Brazil. The sample included 241 individuals from Curitiba and its metropolitan region (Paraná state) and 18 individuals from the quilombola community of Sertão do Valongo (Santa Catarina state)  . For Cura state) , 2005. Aa state)  or by tha state) .n = 259) was sequenced for a fragment of 594 bp in the MCM6 gene, including the –13910C > T SNP (rs4988235). The fragment was amplified by polymerase chain reaction (PCR) on a Mastercycler EP Gradient S® . The forward and reverse primers used were as follows: 5’-GGCAGGGGTTTGGAACTTTC-3’ and 5’-CTGTTGAATGCTCATACGACCA-3’, respectively. Other reagents and the protocol used are described in The Afro-Brazilians sample (® (Eppendorf) at 37°C for 1 h and 80°C for 15 min. The sequencing was performed using the same primers used in the PCR and BigDye® Terminator Cycle Sequencing Standard v3.1 , according to the instructions of the manufacturer. Sequencing reactions consisted of a first step at 95°C for 1 min, followed by 25 cycles of 95°C for 10 s, 50°C for 5 s, and 60°C for 4 min. The sequencing products were purified using ethanol , resuspended in Hi-Di Formamide (Life Technologies), and, finally, submitted to capillary electrophoresis in a 3500xl Genetic Analyzer Sequencer (Life Technologies).The PCR products were purified using exonuclease I  and alkaline phosphatase  on a Mastercycler EP Gradient S® 3.30 software , which aligns the forward and reverse sequences to a human genome reference sequence (GRCh38) available in NCBI  resources, enabling the evaluation of amplified sequences and the genotype annotation for the –13910C > T SNP  in each population using the –extract and –freq flags of PLINK 1.9. For genotype frequencies, we used the –hardy flag, which also calculates whether the population is in Hardy–Weinberg equilibrium for the given locus.We calculated allelic frequencies for the MCM6 region, it was not possible to perform global or local ancestry analyses for these samples. Instead, we extracted the global ancestry information for Sertão do Valongo individuals from Global and local ancestry inferences were performed for each dataset. For both analyses, we merged each dataset with a reference panel of 848 individuals from four continental ancestries: 206 European (IBS and CEU), 207 African (LWK and YRI), and 207 East Asian (CDX and JPT) individuals from the 1000 Genomes Project, and 228 unadmixed Native American individuals from the Peruvian Genome Project . As the r2 > 0.1) using the PLINK flag –indep-pairwise with the following parameters: 50 10 0.1, and applied a minor allele frequency (MAF) filter of 1%. We inferred global ancestry proportions using ADMIXTURE  without removing linked variants. First, for each dataset, all chromosomes were phased with shapeit4  and the length distribution of derived haplotypes around the –13910C > T SNP in each population in order to identify whether the selection signal observed in the populations of European source is also observed in the admixed Pan-American populations. For this purpose, we used the rehh package . Consequently, we did not observe an overrepresentation of the ∗T–13910 allele compared with the average proportion of European ancestry in any population  and ∗G–13915  LP-associated alleles in low frequencies (0.2%).We explored the geographical distribution of the ion 35%, . Converspulation . Moreove∗T–13910 allele carriers (TT and TC genotypes) as a proxy for the proportion of individuals with this phenotype. The observed genotype frequencies did not differ from those expected under Hardy–Weinberg equilibrium in all Pan-American admixed populations , Southeast–South Brazil , and Cuba (53%), which also have higher proportions of European ancestry  in the MCM6 region, we estimated the proportion of uninterrupted European haplotypes and compared it to the genome-wide proportion of European ancestry for each population. Our results (p = 4.042e-6). This pattern suggests the possibility of neutral evolution of this genomic region after the admixture processes that gave rise to these Pan-American admixed populations.To infer which forces are playing a significant role in the evolution of the  results  revealedMCM6 gene. We observed a high density of small European segments in populations with a lower proportion of European ancestry  had the highest value (80%)  . This frue (80%) . Moreoveue (80%) . All the∗T–13910 allele in ancient and present-day unadmixed Native Americans and with the hypothesis that this allele was probably introduced in the Americas by Europeans since the Colonial period. Furthermore, the identification of the ∗T–14011 and ∗G–13915 alleles in low frequencies in Afro-Brazilians from Curitiba agrees with the frequencies previously reported , demonstrating high positive correlation between the distribution of the derived allele and European ancestries, with no evidence of post-admixture positive selection. Furthermore, in light of the current Latin American nourishment policies, our results represent an important aspect to be considered for the understanding of the relationship between the consumption of dairy products and well-being across the continent.In the present study, we conducted the most geographically extensive analysis of the n = 1,039). However, if we restrict our analyses to countries with more than 30 sampled individuals, one may still observe the patterns described. Second, to maintain a balance between individuals and SNP density, we used two separate datasets for the analyses (LARGE-PD and a merged dataset). However, as the study was focused on the –13910C > T SNP and its flanking haplotypes of European origin, the genomic ancestry would still be the same if inferred with a different set of SNPs, as long as the inference is performed with the same SNP density.We can address two limitations of our study. The first was the uneven representation of the population sampled within the countries: sample sizes varied from 13 individuals (Chile) to more than 1,000 individuals  and Native Americans occurred mostly after the independence of Peru in 1824, about 300 years after the European settlement, resulting in the low European ancestry reported in Peruvian and Afro-Peruvian populations, which range between 14 and 22%  from the 1000 Genomes Project]. Thus, the lack of post-admixture selective pressure observed in our study – which datasets included MXL, CLM , MXL, CLM , and PerMXL, CLM  – agreesIt should be noted that admixture processes could hinder the detection of selection signals due to changes in the allelic frequencies and linkage disequilibrium patterns . Further5 and B12, among others , the “Programa Nacional de Alimentación Complementaria” or PNAC in Chile (created in 1952), the “Programa del Vaso de Leche” or PVL in Peru (created in 1984), and the “Programa de Aquisição de Alimentos” in Brazil (created in 2003) are examples of governmental initiatives . Moreove∗T–13910 allele alone may not be completely predictive of lactose intolerance, especially considering the existence of other alleles of non-European origin associated with the LP phenotype , and Uruguay have an elevated proportion (greater than 50%) of individuals who are likely to be lactase persistent, agreeing with the fact that these countries (except for Cuba) are among the largest consumers of dairy products (excluding butter) per year in the continent: 175.3 and 141.8 kg per capita for Uruguayans and Brazilians, respectively . A lower∗T–13910 allele may be a hint that dairy products are not the best option for dietary guidelines in populations with lower proportions of European ancestry, such as Peruvian and African descendant populations . According to the data discussion above, European-biased policies that include dairy products should be rediscussed and reconsidered. Furthermore, until the role of dairy intake in the pathogenesis of complex diseases is fully understood, alternative milk products and plant-based dairy substitutes would possibly be a better option for the dietary guidelines of America.The reduced frequency of the MZ362598–MZ362856.Newly generated sequences were deposited in the GenBank at NCBI website under the accession numbers The studies involving human participants were reviewed and approved by Brazilian CONEP . The patients/participants provided their written informed consent to participate in this study.MHB, CS, and ViB designed the study. AG and NS performed the Sanger sequencing and the analysis of Afro-Brazilian populations. MHB, MP-E, RL, and IR contributed with samples and reagents. CS, MD, J-YM, RK, KN, SW, MLB, ML-C, HG, OC, CP, ET-S, IM, ED, VR, AL, VT, VaB, HF, CR, AS-S, BS-L, PC-C, WF, GA, HA, and CA-B generated the datasets and were responsible by them. ViB, MHB, AG, NS, GA, MMe, TL, MS, CS, DL, MMa, and TO’C, analyzed the data. AG, NS, MHB, and ViB drafted the manuscript. GAC, MMe, TL, MP-E, TO’C, OC, MD, and ET-S critically edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Spinocerebellar ataxias (SCAs) conform a heterogeneous group of neurodegenerative disorders with autosomal dominant inheritance. Five of the most frequent SCAs are caused by a CAG repeat expansion in the exons of specific genes. The SCAs incidence and the distribution of polymorphic CAG alleles vary among populations and ethnicities. Thus, characterization of the genetic architecture of ethnically diverse populations, which have undergone recent admixture and demographic events, could facilitate the identification of genetic risk factors. Owing to the great ethnic diversity of the Mexican population, this study aimed to analyze the allele frequencies of five SCA microsatellite loci  in eleven Mexican Native American (MNA) populations. Data from the literature were used to compare the allelic distribution of SCA loci with worldwide populations. The SCA loci allelic frequencies evidenced a certain genetic homogeneity in the MNA populations, except for Mayans, who exhibited distinctive genetic profiles. Neither pathological nor large normal alleles were found in MNA populations, except for the SCA2 pre-mutated allele in the Zapotec population. Collectively, our findings demonstrated the contribution of the MNA ancestry in shaping the genetic structure of contemporary Mexican Mestizo populations. Our results also suggest that Native American ancestry has no impact on the origin of SCAs in the Mexican population. Instead, the acquisition of pathological SCA alleles could be associated with European migration. The autosomal dominantly inherited polyglutamine spinocerebellar ataxias (polyQ-SCAs) constitute a group of neuromuscular disorders caused by a CAG trinucleotide repeat expansion in the coding region of different genes ,2,3. TheThe Mexican population presents a complex genetic architecture based on the admixture between Native American inhabitants (>50%), European migrants that arrived from Spain and Southern Europe (>40%), and West Africans brought to Mexico through the trans-Atlantic slave trade (<2%) ; such etThe field work was conducted in nine different states of Mexico, namely Chihuahua (CHH), Guerrero (GRO), Michoacán (MIC), Morelos (MOR), Oaxaca (OAX), San Luis Potosí (SLP), Sonora (SON), Yucatán (YUC), and Veracruz (VER) . Blood sGenomic DNA was isolated from peripheral blood leukocytes with the Gentra Puregene Blood kit , following the recommendations of the manufacturer. DNA samples 15 ng) were amplified using a Verity 96-Well Fast Thermal Cycler , with the reaction conditions and chimeric primers described previously . The res ng were stR values) using Arlequin v.3.5 [stR values were assessed using 10,000 permutations, and then visualized in a multidimensional scaling plot (MDS) using SPSS v.20 [p-values were adjusted for false discovery rates in the R software [F statistics using Genètix v.4.05.2 with 10,000 permutations [The allele frequency was evaluated by analysis of molecular variance (AMOVA) and population differentiation analyses  were included in the database for further analyses. Such a database was built through random sampling so as to avoid bias.Data from the literature were used to compare the allelic distribution of SCA loci between MNA and Mestizo populations with worldwide populations . The stuk = 10 alleles), while Mazahuas and Rarámuris (Tarahumaras) exhibited the lowest diversity . Interestingly, the 31 CAG repeats allele was predominant in 7 out of 11 MNA populations analyzed. With respect to the SCA3 locus, Mayans (k = 13) and Nahuas-GRO (k = 11) exhibited the highest diversity of CAG repeat alleles, with the 23 CAG repeat alleles being the most frequent in all of the ethnicities genotyped. For SCA2, the most frequent allele was 22 CAG repeats in all of the MNA populations, while Mayans (k = 9) showed the highest number of CAG alleles. For SCA6, only 10 CAG alleles were identified in MNA, and Rarámuris exhibited the highest diversity (k = 8). Finally, for SCA7, the 10 CAG repeats allele was the predominant one in all of the 14 MNA analyzed. Of note, Nahuas-HGO were set apart from Nahuas-MOR, considering the SCA2 locus genetic data . Overall, the SCA loci allelic frequencies of the MNA populations were similar to that reported for the contemporary Mexican mestizo population [The CAG repeat allele frequencies of the SCA1, SCA2, SCA3, SCA6, and SCA7 loci were estimated in Mexican Native American (MNA) populations belonging to six linguistic families. The allelic distribution of these microsatellite loci was found to vary to a certain extent between MNA populations . Brieflyyans k =  showed tISF range: 0.123–0.253). Regarding the genetic structure of the SCA loci in MNA populations, we found a remarkable deviation from HW in eight out of eleven populations genotyped  due to hTo provide insight into the origin of SCAs in the Mexican population, we search for the presence of SCA pathological alleles in MNA populations. None of the MNA populations genotyped harbored genetic vulnerability to SCAs, apart from a subject belonging to the Zapotec population, who carried a pre-mutated allele of 35 CAG repeats at the SCA2 locus . As SCA2p-value = 0.946). Although the removal of Nahuas-HGO decreased the variation between populations (1.896%), such differences remained significant . Next, MNA populations were divided according to their spoken language , Oto-Manguean (Zapotecos and Pames), Totonaca-Tepehua (Totonacas), Mixe- Zoquean (Mixes), and Macro-Mayan (Mayas)) and their geographic origin. Neither the linguistic nor the geographic criteria impacted the genetic diversity of MNA populations. data not shown). Next, to explore the diversity patterns of MNA populations in more detail, the SCA loci genetic data were analyzed using a population differentiation test based on STR values (p ≈ 0.039). On the other hand, applying of the language affiliation criterion resulted in a cluster conformed by the Mixe-Zoquean, Oto-Manguean, Totonaca-Tepehuan, Uto-Aztecan, and Zapotec linguistic families, all of which were set up apart from the Macro-Mayan family . Significant differences among MNA populations, as well as between MNA populations, were found  . Then, dT values . MNA popT values A, even wT values B; these T values B. UtilizT values , with din family . MDS plon family .RST values   .Rarámuris being plotted separately  populations. Further analysis of this data enabled us to ascertain the impact of Native American ancestry on the contemporary Mexican population structure, which could provide some clues for the geographic origin of SCA pathologies in Mexico. The SCA loci allelic frequencies found in MNA populations resembled that of the contemporary Mexican population , which iDeparture from the HW equilibrium due to heterozygote deficiency was not an unexpected result in MNA populations, considering the fact that this phenomenon is a signature of inbreeding populations. The MNA genetic architecture seems to be influenced by geographic barriers that keep them isolated . MNA pop35) in the Zapotec group. Thus, the presence of SCA pathologies in the Mexican population, with a high prevalence of SCA2 and SCA7, could be the consequence of founding effects, as previously proposed [It is thought that SCAs prevalence in a given population is driven by the presence of intermediate and/or large normal alleles ,24. Consproposed . NRY linproposed ,27,28,29proposed . Taking proposed , and thaproposed .Currently, diverse MNA populations coexist in the geographically wealthy territory of Mexico. The interplay among MNA populations through time could have tailored their present genetic structure. Thus, characterization of the genetic markers in these populations could broaden the picture about the origin and spread of the SCA expanded alleles. Furthermore, the study of genetic markers with biomedical relevance, such as the SCAs microsatellite loci, could help to trace the origin of these pathologies, as well as to identify the genetic factors relevant for biological processes of the MNA and Mestizo populations of today. Nonetheless, these studies should be reinforced by the analysis of ancestral markers.Yoemem population from six out of eleven MNA populations studied. This finding could be explained by the European ancestry, not necessarily Spanish, acquired by its connection with USA populations [ISF = −0.186) supports this hypothesis. Likewise, evaluation of SCA2 and SCA3 loci failed to provide a clear population differentiation of MNA populations. Noteworthy, analysis of the SCA2 locus showed a certain divergence between Nahua populations , which could be the result of a differential genetic structure between them; Nahuas-GRO exhibited high genetic diversity (k = 4), while Nahuas-MOR showed an endogamy behavior (k = 2). It could be argued that the gene flow of Nahuas-GRO with European and African ancestries [p = 0.028), the genetic distance between them was not considerable (RST = 0.037). Similar results have been previously reported using NRY markers [The genetic distance of MNA populations depicted minor differences between them, exhibiting a subtle percentage of variation (3.29%), which is consistent with other studies . The anaulations . The hetcestries ,35,36,37cestries ; the latcestries ,38,39. Fcestries . Althoug markers . It is w markers ,40,41. Yoemem and Zapotecans, exhibited only a marginal significance, which could be due to the geographic closeness between Yucatan and Oaxaca [Yoemem diaspora to the United States of America could explain, at least in part, the partial genetic segregation from Mayans [Rarámuris were marked. A portion of these differences could be attributed to the linguistic families to which they belong. While Rarámuris belong to Uto-Aztecan, Mayans belong to the Macro-Mayan family [The Mayan population was also segregated from five out of eleven MNA populations genotyped upon analysis of the SCA7 microsatellite locus. The genetic distance between Mayans and Mixes, d Oaxaca . Both thd Oaxaca ,40, whild Oaxaca ,36. On tm Mayans . On the n family . With ren family ,35,37.The limitations of this study are that the SCA loci alleles are not the suitable genetic tool to reconstruct demographic and cultural histories of populations, because they are not neutral markers. Furthermore, differences in the sample size between populations could bias our conclusions.In summary, our findings highlight the importance of analyzing MNA populations in order to better understand the influence of Native American ancestry on modelling the genetic structure of contemporary Mexican Mestizo populations. Furthermore, this study suggests that Native Americans had no influence on the origin of the most prevalent SCAs in Mexico, reinforcing the idea that the introduction of the pathological alleles into Mexico could be associated with European migrations.


Illicium griffithii is an aromatic medicinal tree species that has been listed in the IUCN Red List as an endangered species. Dried seed pods of I. griffithii have a good market potential in the spices and pharmaceutical industries. Fruits are the potential source of shikimic acid and used for the production of oseltamivir (a drug against bird flu). However, in recent years, unscientific harvesting and rampant exploitation of the species has caused a negative and adverse effect on its natural population. Proper knowledge of genetic diversity and population structure is crucial to understand the population dynamics, adaptation, and evolutionary pattern of a particular species for conservation. It was from this view point that the present study was undertaken so as to compare the various types of DNA-based molecular markers namely RAPD, ISSR, DAMD, and SCoT by their efficiency and SPAR approach to evaluate the genetic diversity of I. griffithii as well as to analyze population genetic structure for conservation purpose.ST = 0.30, respectively), revealing a low level of genetic differentiation among the eight sampled populations. I. griffithii with an estimated gene flow value of Nm = 0.761 was significantly low among populations. Clustering pattern obtained with Bayesian structure and PCoA diagram revealed that intermixing of genetic material across populations is only possible when the populations lie close to each other. This is further validated with UPGMA clustering method where a positive correlation of genetic variability with geographical distance among closely related populations could be clearly seen.A total of 250 discernible bands were generated with 246 bands (98.40 %) being polymorphic in nature. All the primers in combination gave a mean polymorphic information content (PIC) of 0.81 and Rp value (resolving power) of 4.32. Nei’s, Gst, and AMOVA analysis showed similar values of genetic differentiation among populations (Gst = 0.396, FI. griffithii from Arunachal Pradesh and Meghalaya of Northeast India. This would further help in understanding the population structure and genetic diversity among other Illicium species in order to formulate effective conservation strategies for the improvement of this endangered taxa.The result aids in the identification, collection, and preservation of diverse germplasm of  Illicium griffithii Hook. F. & Thoms. belonging to the family Illiciaceae is one such important aromatic and medicinal tree species that has been listed in the IUCN Red List  which is an active drug against avian influenza or bird flu [I. griffithii with its poor regeneration in the wild, localized natural growth, high anthropogenic pressure, and intangible efforts for conservation have resulted the species to be categorized as critically endangered species in Meghalaya and endangered in Arunachal Pradesh [ Myanmar . Howeverm 1 to 5 . The seem 1 to 5 . The abo Pradesh , 11. In  Pradesh . DNA-bas Pradesh . Since t Pradesh –22.I. griffithii population in the wild [I. griffithii that actually manifests in the form of morphological or reproductive variations concerning its survivability and evolution has been largely neglected. It was from this view point that the present study was undertaken so as to compare the various types of DNA based molecular markers namely RAPD, ISSR, DAMD, and SCoT by their efficiency and SPAR approach to evaluate the genetic diversity of I. griffithii as well as to analyze population genetic structure for conservation purpose.Previously, several studies have been carried out focusing on the biological factors causing the decline of the wild , 23–25. Plant materials for the study were collected from the natural habitats of Meghalaya and Arunachal Pradesh, India. Collected sites are tabulated below with their GPS readings Table , Fig. 2.2+ plus), 5 u/μL GoTaq DNA Polymerase (Takara), and 10 pmol of primer. Reactions were performed in a thermocycler (Eppendorf) with the settings as given in Table Genomic DNA was isolated from young leaves following the CTAB method  with minAmplified products were separated on 2% agarose gel stained with ethidium bromide in 1× TAE buffer and run for 3 h in an electrophoresis unit at 85 V. DNA bands were visualized and photographed in a gel documentation unit  for final SPAR analysis. Each reaction was performed in two replicates and only clear reproducible bands were scored. Amplified products detected on the gels were recorded in a binary matrix where the fragments of similar size were represented as “1” for present or “0” for absent.Genetic similarity based on Jaccard’s coefficient was calculated using SIMQUAL module and a dendrogram was constructed using the NTSYS version 2.20 software package following the unweighted pair group method with arithmetic mean (UPGMA) option of the SAHN module . As per ST) was applied to reveal differentiation between populations and its significance was evaluated following Wright [K) in the data, a series of analyses were performed from K = 1 through 11, using 25,000 burn-in and 25,000 repetitions, with 10 iterations per K.To analyze the within and among population variations, analysis of molecular variance (AMOVA) at two hierarchical levels was performed using Arlequin version 3.01 . F statig Wright . Geneticg Wright , 35 adapI. griffithii. In the present study, out of 100 primers that were initially screened, a total of 28 primers were finally selected for analysis.The four marker systems  used in the present study revealed significant amount of polymorphism independent of each other at various levels proving the usefulness of the SPAR system used, to analyze diversity studies in A total of 7 RAPD primers that were used for the analysis yielded 73 bands out of which 71 bands were polymorphic in nature (97.26%). The primer OPH-19 produced maximum band number (13) with a polymorphic information content (PIC) value of 0.76. The resolving power  of RAPD primers ranged between 2.59 and 5.49. The amplicon size ranged between 0.2 and 2 kb with an average of 10.42 amplicons per primer . The primer SUNSRK-6 produced maximum band number (12) with a PIC value of 0.88. The resolving power  of the primers ranged between 2.66 and 6.34. The amplicon size ranged between 0.2 and 2 kb with an average of 8.71 amplicons per primer . The primer Oligo 5 produced maximum band number (10) with highest polymorphic information content (PIC) of 0.95. The resolving power  of the primers ranged between 2.45 and 5.60. The amplicon size ranged between 0.2 and 2 kb with an average of 7.75 amplicons per primer Table , Fig. 5bSix SCoT primers were selected for analysis that produced a total of 54 bands out of which 53 bands were polymorphic (98.14%). Three SCoT primers  yielded maximum bands numbers (10) with PIC ranging between 0.76 and 0.84. The resolving power  of the SCoT primers ranged between 3.30 and 4.57. The amplicon size ranged between 0.2 and 1.5 kb with an average of 9 amplicons per primer  was performed using the cumulative dataset in order to give a holistic approach to the study. The study yielded 250 amplicons out of which 246 (98.40 %) were polymorphic in nature Table . The meaI = 0.39 ± 0.27) with the lowest being recorded in BN (Table The Pp% (percentage of polymorphic loci) for a single population ranged from 41.20% (BN) to 76% (B2) with a total value of 98.40%. The samples collected from B2 region have the highest observed number of alleles (Na) being (1.76 ± 0.42) while that of BN (1.41 ± 0.49) have the lowest (Na) Table . For allBN Table . Mean coThis was validated with AMOVA analysis where higher variation 69.37%) within populations as compared to lower variation (30.63%) among populations was recorded with an F statistics (FST = 0.30) , ISSRs/DAMDs (spanning selected repetitive sequences), and SCoTs (spanning the start codon regions) could be considered as suitable markers for more meaningful and holistic approach to the analysis of genetic variability. Several authors have stressed the advantages of using more than one class of molecular markers to estimate genetic diversity of threatened taxa [It was found that each marker system was capable of detecting significant genetic polymorphism among the ds Table . The ranned taxa , 45. Numned taxa , 46–49. ned taxa –53. ThisST = 0.30, respectively), revealing a low level of genetic differentiation among the eight sampled populations of I. griffithii  [I. griffithii across disturbed area of Arunachal Pradesh. The study revealed clear dominance of adult trees and lowest number of seedlings across the study sites indicating poor regeneration of I. griffithii. Fruits on maturity expel their seeds to nearby areas which although are capable of germinating fails to convert into early sapling stage justifying the negative role of disturbance that creates an adverse niche for regeneration of I. griffithii [The value of Nei’s genetic differentiation among populations (Gst) may range from zero to one, with a higher value indicating that a larger number of variations lie among various populations. In the present study, different methods  showed similar values of genetic differentiation among populations  . Insectsination)  where thiffithii . Few seeI. griffithii fruits to meet the increasing demands have negatively affected its natural population. Overharvesting of its wild resources has resulted in a sharp decline of the effective population number and size, with patches of local disappearance. Numerous studies are available which confirm the loss of genetic variation in wild populations due to overharvesting adversely impacting the evolutionary potential of a species to adapt to changing environments [I. griffithii may be attributed to all or some of the aforesaid factors mentioned in the study. Our results corroborate with the findings of other workers where moderate genetic differentiation among populations was observed in contrast to maximum genetic variability retained within populations [Among the eight populations sampled here, BM from Arunachal Pradesh showed relatively lower levels of genetic variation. This is due to its peripheral distribution range from the collected central populations   and micropropagation (and/or in vitro seed germination) can be explored to effectively raise individuals from different populations and reintroduce them into the wild. The present study aids in identification, collection, and prioritization of genetically diverse germplasms of the threatened taxon I. griffithii for improvement and conservation.Scientific approaches for conservation and sustainable utilization of plant resources require accurate assessment of the amount and distribution of genetic variation within and among populations . Molecul


DSB and BOLA-DYA) in Somba cattle is interestingly uncommon in other African breeds, suggesting further investigations to understand its association with specific adaptation to endemic diseases in Benin. Overall, our study provides deeper insights into recent evolutionary processes in the Beninese indigenous cattle and their aptitude for conservation and genetic improvement.The Dwarf Lagune and the Savannah Somba cattle in Benin are typical representatives of the endangered West African indigenous Shorthorn taurine. The Lagune was previously exported to African and European countries and bred as Dahomey cattle, whereas the Somba contributed to the formation of two indigenous hybrids known as Borgou and Pabli cattle. These breeds are affected by demographic, economic, and environmental pressures in local production systems. Considering current and historical genomic data, we applied a formal test of admixture, estimated admixture proportions, and computed genomic inbreeding coefficients to characterize the five breeds. Subsequently, we unraveled the most recent selection signatures using the cross-population extended haplotype homozygosity approach, based on the current and historical genotypes. Results from principal component analyses and high proportion of Lagune ancestry confirm the Lagune origin of the European Dahomey cattle. Moreover, the Dahomey cattle displayed neither indicine nor European taurine (EUT) background, but they shared on average 40% of autozygosity from common ancestors, dated approximately eight generations ago. The Lagune cattle presented inbreeding coefficients larger than 0.13; however, the Somba and the hybrids (Borgou and Pabli) were less inbred (≤0.08). We detected evidence of admixture in the Somba and Lagune cattle, but they exhibited a similar African taurine (AFT) ancestral proportion (≥96%) to historical populations, respectively. A moderate and stable AFT ancestral proportion (62%) was also inferred for less admixed hybrid cattle including the Pabli. In contrast, the current Borgou samples displayed a lower AFT ancestral proportion (47%) than historical samples (63%). Irrespective of the admixture proportions, the hybrid populations displayed more selection signatures related to economic traits  than the taurine. In contrast, the taurine, especially the Somba, presented several regions known to be associated with adaptive traits (immunity and feed efficiency). The identified subregion of bovine leukocyte antigen (BoLA) class IIb (including  Bos taurus) on the continent , and able to survive and produce in harsh environments . The Somhttp://www.dahomey-zwergrind.com). To date, neither scientific study nor census addressed these animals. According to the breeder association, the current population is characterized by a small size (adult body weight between 150 and 300 kg and sacrum height from 80 to 105 cm), short horn, easy calving, and good temperament. These characteristics are similar to those of the original Lagune population in Benin and may indicate that the Dahomey population conserved its purity through the past decades. However, the European climate is different from the one encountered in Benin, i.e., less heat stress, but varying temperatures between winter and summer, and also varying sunlight duration. In addition, the new production environment of the Dahomey cattle implies the reduction of disease infections with potentially improved feeding and housing systems in opposition to the harsh production conditions in Benin (characterized by food and water scarcity and the risk for disease infections). Consequently, this geographical isolation may have altered frequencies for alleles and haplotypes associated with specific genetic features in the Dahomey population.The Lagune cattle from Benin had been exported during the early 20th century (around 1904) to different African countries such as Zaire (current Democratic Republic of Congo), Zambia, Gabon, as well as to Europe where they are known as Dahomey cattle , becauseThe livestock production systems in Benin like in the other African countries have experienced a drastic transformation in the last decades . New chaIn regard to the increasing uncontrolled crossbreeding, two other indigenous hybrid cattle, the Borgou and Pabli, are also endangered in Benin. The Pabli originally reported in the region of Kerou (Northwest Benin) is scarcely described and is considered as extinct by absorption from crossbreeding with Borgou . HoweverTo understand the genetic architecture underlying adaptive and productive abilities of various breeds evolving in challenging environments, selection signature analyses have the potential to detect specific genomic footprints in terms of differences in marker allele frequencies or in haplotypic mosaicism . AccordiIn this study, we aimed to genetically characterize the current endangered cattle breeds in Benin and evaluate the effects of admixture and environmental factors related to late changes in production systems. Specifically, we first assessed the admixture level in the different populations and compared them with historical samples. Second, through selection signature analysis, we investigated genomic regions and biological mechanisms involved or affected by recent natural or artificial selection and admixture in the Beninese cattle breeds. Subsequently, we investigated the genetic differentiation resulting from the isolation of the Dahomey cattle.Hair samples were collected from 449 animals from the four local breeds  in Beninese local farms in 2016 and 2017. The sampling locations were identified ensuring a large coverage of the main geographical distribution of the breeds in the country . In eachThe sampled animals were genotyped with the Illumina BovineSNP50 BeadChip. SNP variants with a call rate <95% and a minor allele frequency (MAF < 0.01) as well as animals with a high percentage of missing genotypes  were filtered out in PLINK . The finThe genetic structure of our samples was first assessed by applying the fastStructure software . The alg9, number of starting centroids in each run (n.start) = 30, and the default parameters for the remaining arguments . The class rates (Rk) are related to an expected length and exponential distribution of HBD segments. They are equivalent to twice the number of generations to the common ancestor. The proportion of the genome belonging to a specific HBD class is defined as “realized autozygosity” in the respective HBD class. Similarly, the genome-wide inbreeding coefficient is estimated as the cumulative fraction of the genome that is HBD in the current population with respect to an ancestral base population. As suggested by the authors of the “RzooRoH” package (k) 2, 4, 8, 16, 32, 64, 128, 256, 512, and 512, respectively.We utilized the “RzooRoH” package  in R to  package , we applBos frontalis, 4-ratio. Likewise, we used the same filtering parameters  in PLINK. The genotype data consisted of 52,364 SNP and 30,228 SNP from 1,018 animals before and after quality control, respectively. All the genotype data exploited in this study are fully described and available in a public repository (see the section “Data Availability Statement”). Before being merged with our samples, the historical genotype data were remapped with the current reference assembly ARS-UCD1.2/bosTau9 (GenBank Bioproject PRJNA391427) and flipped (with the --flip flag in PLINK) to correct for strand inconsistency  in PLINK  and usin3) and the F4-ratio estimation in Admixtools, respectively  indicates that the allele frequencies in population X tend to be intermediate between B and C and indicates admixture in X populations from populations related to B and C. The f4 statistics were used to infer admixture proportions in the Beninese cattle populations (target populations) through the calculation of the F4-ratio or alpha . The estimated p-values were subsequently adjusted  approach  was implemented in the “Rehh” package in R  and submitted to gene ontology (GO) enrichment analysis using the Gene Ontology web-tool  was separated from other Lagune samples called LA_out (n = 25). Forty-two Borgou and 56 Pabli animals (except two outgroups) formed a homogeneous cluster named Adm_cur (n = 98). The remaining Borgou animals were allocated to the BO_cur cluster. The Dahomey and the Somba correspond to the DAH_cur and SO_cur, respectively . The major contribution of autozygosity in LA_cur came from the ancient HBD class with Rk = 128 (40% of the HBD proportion). Recent HBD classes with Rk = 4 and Rk = 8 explained 13.78 and 7.80% of the total autozygosity in LA_cur, respectively, whereas 27.67 and 26.11% of the overall autozygosity in LA_out were derived from the two classes. Tracing back to the oldest ancestors, the fraction of the genome that was HBD in SO_cur was equal to 0.08 and was mainly originated from very ancient HBD classes with Rk = 256  and Rk = 512 . The contribution of recent classes to autozygosity in SO_cur was lower (≤13% of the HBD loci). The estimated genomic inbreeding coefficients were 0.08 for BO_cur and 0.07 for Adm_cur. The HBD class with Rk = 256 was the main source of autozygosity, contributing to 77.94 and 73.09% of the total HBD proportions in BO_cur and Adm_cur, respectively.The genome-wide contributions and genomic inbreeding coefficients with respect to different age-related HBD classes in the Beninese cattle populations are presented in The first and second principal components (PC) from PCA presented a clear separation of the Beninese breeds . Within We repeated the diversity analyses on the extended dataset including additional AFT, EUT, AFZ, Asian indicine (AID), and hybrids. Considering the PCA, the taurine animals (AFT and EUT) were separated from indicine (AFZ and AID) and hybrids samples along the first component, whereas the second component (PC2) showed a separation between the AFT and EUT . Interes3-ratios for DAH_cur, LA_cur, and SO_cur . Considering the historical data, the f3-ratio test resulted in positive values for LA_hist but was negative for SO_hist and BO_hist.The formal test of admixture of three populations resulted in positive fd SO_cur . In cont4-ratio test revealed high proportions  in DAH_cur, LA_cur, and SO_cur, respectively , which is positioned within the WDR87 gene. In total, 9 candidate genes were annotated within the three regions. GO enrichment analysis identified functional enriched terms such as cellular processes  displayed 19 significant SNP for positive selection . Among trocesses . QTL assrocesses .p_adjust = 2.34 × 10−07). This SNP is not positioned in any gene. However, the second most significant SNP (p_adjust = 3.57 × 10−04) is positioned in the vicinity of three genes  on BTA 23. In total, 33 genes were mapped in the candidate regions. GO enrichment analysis of the genes revealed highly significant GO biological processes such as antigen processing and adaptive immune response . Among these SNP, 44 were located in eight candidate regions on BTA2, 3, 5, 16, and 27 (p_adjust = 0.003). This SNP was not in the vicinity of any gene. However, three significant SNP  were positioned within the genes HTRA4, DNAJC11, and TMEM53, respectively. A total of 49 genes were mapped within the genomic candidate regions. GO enrichment analysis revealed several significant GO biological processes including cytoplasmic microtubule organization and inner mitochondrial membrane organization . The segment on BTA3 (101–102 Mb) hosted the largest number of extreme SNP (13 SNP). The candidate regions in Adm_cur included 63 genes. The genes DNAJC11, ENTHD1, and MAST2 hosted significant SNP, whereas others  were mapped at a close distance to the significant SNP. GO enriched terms included biological processes such as pigment biosynthetic process and response to chemicals . The inference of the oldest common ancestor dated from eight generations ago (approximately 24 years ago). The high genomic inbreeding coefficient in Rk = 8  in the Dahomey population may be related to the recent creation of the association in 2001. These results suggest that the founders of the current Dahomey population kept by farmers may have originated from a small number of Dahomey cattle four generations ago (http://www.dahomey-zwergrind.com). However, the low frequency of short ROH segments, resulting in low genomic inbreeding coefficients (<0.1) in very young classes (Rk ≤ 4), indicates a reduction of mating between related individuals in recent generations (https://www.zootierliste.de) may contribute to control inbreeding and to increase genetic diversity in the population currently managed by the Dahomey–Zwergrind breeder association.The results of the population structure analyses are in line with the breed foundation of the different cattle populations in Benin and their divergence from other African and European cattle breeds . First, erations . ExchangThe results of the PCA analyses differentiated historical and current populations as well as populations affected by admixture. The identification of admixed animals (LA_out) from relatively pure Lagune is confirmed by the formal admixture test, the three-population test, and the estimation of admixture proportions. Our findings are in accordance with the increasing crossbreeding in Lagune cattle due to the extension of transhumance as reported by We found that Somba cattle are less affected than Lagune by Zebu introgression. Previous studies observed that its habitat in the hilly region of Atacora protected from Zebu introgression . In comp3 and f4 admixture tests. The lower AFT ancestral proportion in BO_cur compared to the historical population BO_hist confirms the increasing introgression of AFZ in smallholder Borgou herds as indicated by The results from fastStructure corroborate the Somba background of the Beninese crossbreeds. In addition, the hybrid populations (BO_cur and Adm_cur) presented a relatively low genetic proximity to the AID , but they were clustered close to the AFZ (ZFU and ZBO). These results are in agreement with their origin as described by different authors . The idePrevious studies  observedBy contrasting the current and historical cattle populations in the Beninese taurine and crossbreeds, we focus on genomic footprints resulting from recent environmental pressures or herd management. Indeed, environmental and socioeconomic factors have induced diverse changes in cattle management practices in Benin. These include the adoption of cattle mobility in taurine or agropastoralist herds (former sedentary), the increase of herd mobility frequencies and amplitudes, the migration and settlement of several agropastoralists from Sahelian countries or Northern Benin into Southern Benin, and the extension of animal entrustment practices  . DespiteBos gaurus, Bos grunniens, and Bubalus bubalis. p-values to define significant SNP and by defining only regions with at least three significant SNP as candidate regions. The respective candidate regions identified for each cattle population are discussed in the ongoing sections.Our approach to detect a strong signal of homozygosity considering temporal subpopulations within the same breed is similar to the one applied by http://www.dahomey-zwergrind.com). Further investigations including a complete sequencing of the Dahomey cattle and other methods of selection signature analyses may help untangle the genetic divergence between the Dahomey and the Lagune.We were not able to detect any candidate regions for DAH_cur. The Dahomey cattle may have acquired very few complete selective sweeps that are not detectable by the XP-EHH. This result reflects the breeding strategy with only focus on leisure and is in line with its high genomic inbreeding coefficient  overlaps with the subregion of BoLA class IIb (BoLA-DRB3 and BoLA-DQA3)  of the bovine leukocyte antigen (BoLA), also called bovine major histocompatibility complex (MHC), which is determinant in the development of acquired immune responses to diverse parasitic and viral diseases . The sellass IIb . These BLA-DQA3) . In addiLA-DQA3)  associatLA-DQA3) , whereasLA-DQA3) . TherefoLA-DQA3) . Furtherh traits , FSTL4 aoduction  and ZFANoduction . The preRNF220 gene. This gene has been previously identified under selection in West African cattle  overlapping in the two hybrid populations are in line with their common indicine background. The significant SNP mapped in the region from 100 to 101.5 MB on BTA3 were positioned in various genes including the n cattle . In addin cattle . These flk yield . In addilk yield . We alsoectively . The regectively . Moreove in pigs .DNAJC11, a heat shock protein gene, involved in response to heat stress (EN1, involved in growth traits in cattle (ADAM32, ADAM9, HTRA4, and KLHL21 with residual feed intake and immune responses, PLEKHA2 and TNFRSF25 with growth and carcass performances, HTRA4 with milk, and HOOK1 with heat stress (Specific selection signatures detected in BO_cur include the selective sweep 46.5–47.5 Mb on BTA16. This region is of great interest. It presents several significant SNP and overlaps with genomic footprints detected in East African Zebu cattle as well as in a subpopulation of the German dual-purpose black and white cattle . The regt stress . In addit stress . The regt stress . In addin cattle . Furthert stress .TMEM106B gene in the German black pied cattle. In addition, the ARL4A gene was reported in the context of selective sweeps in Australian Holstein (SCIN gene, also mapped in the region, is involved in residual feed intake in cattle (ANKRD52, RNF41, and MYL6 associated with height and carcass traits (COQ10A and RNF41 related to milk trait and calf mortality (SARNP gene is related to animal reproduction (DNAJC14 is involved in heat stress (Regarding the selection signatures in Adm_cur, the region 20–21 Mb on BTA4 in Adm_cur is consistent with the region reported by Holstein  and withHolstein . The latHolstein . The SCIn cattle . The rems traits , and COQortality . In addioduction , while tt stress .Overall, the common and specific candidate regions identified in the hybrid populations confirm selection signatures in African and European crossbreeds . They coIn this study, we confirm that the Dahomey cattle currently bred in Europe are a subpopulation of the Beninese indigenous Lagune breed. The high genomic inbreeding in the Dahomey population is due to its current breeding system. The introduction of new animals from zoological parks can increase the diversity of the Dahomey population. Moreover, the Beninese taurine indigenous Lagune and Somba cattle still conserve a high proportion of AFT ancestry, in comparison to the historical population. The Borgou displays a risk of full genetic replacement by African Zebu. However, we observed the existence of a hybrid population relatively less affected by ongoing indicine introgression and comparable to the historical Borgou.We found no evidence of the negative impact of admixture on the adaptive features in the cattle populations including the crossbreeds, as they all present several genomic footprints involved in immune response, feed efficiency, and heat stress. Moreover, specific candidate regions in the Somba cattle demonstrate selection pressures related to endemic diseases in the habitat areas of the breed. Overall, identified recent selection in Beninese indigenous cattle towards productive traits such as reproduction, milk, and carcass traits favor the improvement of the economic merits of the breeds.


Population genetic studies provide accurate information on population structure, connectivity, and hybridization. These are key elements to identify units for conservation and define wildlife management strategies aimed to maintain and restore biodiversity. The Mediterranean island of Sardinia hosts one of the last autochthonous mouflon populations, descending from the wild Neolithic ancestor. The first mouflon arrived in Sardinia ~ 7000 years ago and thrived across the island until the twentieth century, when anthropogenic factors led to population fragmentation. We analysed the three main allopatric Sardinian mouflon sub-populations, namely: the native sub-populations of Montes Forest and Mount Tonneri, and the reintroduced sub-population of Mount Lerno. We investigated the spatial genetic structure of the Sardinian mouflon based on the parallel analysis of 14 highly polymorphic microsatellite loci and mitochondrial D-loop sequences. The Montes Forest sub-population was found to harbour the ancestral haplotype in the phylogeny of European mouflon. We detected high levels of relatedness in all the sub-populations and a mitochondrial signature of hybridization between the Mount Lerno sub-population and domestic sheep. Our findings provide useful insights to protect such an invaluable genetic heritage from the risk of genetic depletion by promoting controlled inter-population exchange and drawing informed repopulation plans sourcing from genetically pure mouflon stocks. A second wave of domestication occurred 6000 years later and led to the modern sheep: polled with no-shedding wool5. As a consequence, the rearing of the first mouflon-like ovine was abandoned in favour of the new economically valuable one6. The original mouflon-like went back to feral life but gradually disappeared in mainland Europe due to intense hunting and habitat erosion. However, relics of these ancestral populations survived in the harshest mountainous areas of the Mediterranean islands of Sardinia, Corsica, and Cyprus, where they established populations which survived until now8. Since the eighteenth century, Corsican and Sardinian mouflon have been used to repopulate several regions of mainland Europe. During the last century, the Corsican and Sardinian mouflon populations experienced severe size contractions due to over-hunting and habitat erosion. However, the implementation of conservation policies in the last 50 years brought the populations to increase and reach the current number of ~1000 and ~6000 individuals in Corsica and Sardinia, respectively11.Ovine domestication began in the Fertile Crescent around 10,000 years ago (YA) from mouflon-like animals with horns and short-shedding wool13, followed ~3–4000 years later by the ovines of the second wave of domestication. In the twentieth century the competition for grazing land between mouflon and domestic sheep, the intensified hunting pressure, and the more recent use of barbed-wire fences along the roads strongly altered the mouflon natural habitat, resulting in population fragmentation into small, isolated groups often confined to less favourable habitats14. Finally, the outbreak of newly introduced infectious diseases  contributed to further reduce the mouflon population size15.The most credited hypothesis dated the arrival of mouflon in Sardinia around 7000 YA16 , including the areas of Mount Tonneri (Seui), Montes Forest (Orgosolo), and Mount Albo (Lula), and a few managed and protected areas where mouflon has been reintroduced. This range covers only a limited part of the original species distribution27.A better knowledge of the genetic differentiation of wildlife populations and the improved understanding of the selective pressure acting upon them are essential tools for the effective management and conservation of endangered species. Molecular markers like mitochondrial DNA (mtDNA) and microsatellites are commonly used to evaluate the genetic health of a population and to define conservation units representing valuable reservoir of genetic variation. Studies based on genome-wide markers and whole mtDNA sequence have shown low levels of genetic diversity and the occasional occurrence of domestic sheep introgression signals in the Sardinian mouflon populations28. HPG-B is the most common haplogroup among sheep breeds29 and is also shared by the mouflon from Sardinia and Corsica. The Sardinian and Corsican mouflon are classified as European mouflon (Ovis gmelini musimon) along with the reintroduced mouflon populations from mainland Europe, despite the great genetic distance detected between the Corsico-Sardinian and the European mouflon mtDNA lineages8. The Corsico-Sardinian mouflon clade shows an early split from the evolutionary branch originating the domestic sheep HPG-B and the mainland Europe mouflon lineage5. Further, the Sardinian mouflon gene pool harbours the oldest HPG-B haplotype identified so far8. Such findings suggest the need to revise the systematic classification and nomenclature of these species. Until a new nomenclature is established, in this report we choose to identify the individuals from mainland Europe as European mouflon, whereas we will refer to insular individuals as Sardinian or Corsican mouflon, according to their origin.To date, five domestic sheep mitochondrial lineages, defined as haplogroups (HPGs) A, B, C, D, and E, have been identifiedHere, we use mtDNA and microsatellite markers to describe the genetic diversity and population structure of the Sardinian mouflon. We estimate the genetic differentiation among and within three representative sub-populations from the historical range of Sardinian mouflon and evaluate the effect of landscape anthropogenic alterations. Finally, we provide the first data on the spatial genetic structure of mouflon in Sardinia.8 and belonging to the HPG-B, Hpts 2–4 described for the first time in this work, and Hpt-5 (GB# KF228720) previously found in Comisana sheep30.We generated a total of 54 sequences of heterogeneous length ranging from 460 to 1180 bp which were deposited in GenBank (GB# MW727287-340). In order to estimate the genetic variability on the entire available sample, the analyses were carried out on a 439 bp fragment of the hypervariable domain I of the mitochondrial D-loop region. A total of five haplotypes were detected: Hpt-1 (GB# MG489885) previously identified in SardiniaWithin the Montes Forest sub-population three haplotypes separated by one-step mutations were observed, resulting in a haplotype diversity of 0.62 Table . Only onThe components of the total genetic variation were estimated by AMOVA and showed a higher proportion of genetic diversity variation “among populations” (79.3%) than “within population” 20.7%) (Table 0.7% (TabThe median-joining network (MJN) analysis detected two main D-loop haplotype clusters well separated from the outgroup Fig. . A clearTo quantify D-loop divergence within the Sardinian mouflon population (n = 54), pairwise genetic distances between groups were calculated (Supplementary Table P < 0.01) in Montes Forest sub-population (4.17) when compared to Mount Tonneri (2.71) and Mount Lerno (2.54), indicating a higher genetic variability in this sub-population.Fourteen out of sixteen genotyped microsatellite loci were found polymorphic in at least one of the three sub-populations with a mean of four alleles by locus  and for pairwise comparisons, revealing a strong genetic differentiation. The highest genetic distance was recorded between Mount Lerno and Mount Tonneri , whereas the Montes Forest sub-population was found to be almost equally differentiated from the other two .The FThe AMOVA indicated that the within population variation accounted for 0.66% of total variation, whereas differences among populations contributed for 29.57% of the total variation  values of the r frequency distributions inferred by the relatedness analysis were 0.054 (± 0.258) for Montes Forest, 0.351 (± 0.175) for Mount Lerno and 0.264 (± 0.227) for Mount Tonneri sub-populations.P < 0.01) . The proportion of r values >  + 0.5 was 13.1%, 19.2% and 19.6% in Montes Forest, Mount Tonneri and Mount Lerno sub-populations, respectively. One individual sourced from Mount Tonneri recorded the highest r estimates of 0.49 and 0.51 with one individual from Montes Forest and one from Mount Tonneri, respectively.As expected, the presence of highly related individuals, either full siblings or parent-offsprings, was observed   was not affected by allele frequencies lower than 0.02. The Montes Forest sub-population showed the highest Ne mean value (2.8) compared to Mount Tonneri (2.4) and Mount Lerno (2.2) (Table Our results showed that the effective population size (2) Table . The ana32. To this end, knowledge on their spatial distribution, population structure and connectivity are key to draw informed conservation plans35. We analysed 14 highly polymorphic microsatellite loci and D-loop sequences to describe the diversity and structure of the current Sardinian mouflon autochthonous population, providing the first evidence of a clear genetic structuring.Remnant island populations often represent unique genetic reservoirs due to their distinctiveness and should be always prioritised for conservation8. Among the other four, three were novel  while Hpt-5, found in the Mount Lerno population, was previously detected in the Comisana domestic sheep breed30 but never recorded in mouflon. Noticeably, none of the haplotypes detected within Montes Forest and Mount Tonneri sub-populations were found to be shared with domestic sheep. We hypothesize that during the subsequent steps of the translocation process from Mount Albo to Mount Lerno, passing through Capo Figari and the Asinara Island, contact with domestic breeds could have occurred.We detected a total of five mtDNA haplotypes, including Hpt-1 that has been described as the oldest haplotype within the HPG-BST = 0.79).Each sampling site showed a unique haplotype composition, suggesting a minimal matrilineal gene flow, in accordance with the high genetic differentiation detected among populations  and Cypriot (O. g. ophion) mouflons37.Our results highlighted the genetic differences between Corsico-Sardinian and European mouflons also found in previous worksST values and the presence of private alleles (n = 12), probably due to a reduced gene flow. However, caution is required in interpreting these results due to the small sample sizes available38. Previous studies on microsatellite loci40 recommended to sample at least 20–25 individuals to reduce errors and increase accuracy. Nonetheless, the calculation of genetic differentiation indices is still informative despite low sample sizes, as is often the case when studying small, endangered populations.In accordance with the mtDNA-based AMOVA, microsatellite analyses showed high FST ratio was > 2, suggesting higher male-biased natal dispersal. This is expected, as mouflon rams are more prone to disperse during rutting phase, albeit mouflon adopt a polygynous reproductive system with philopatric males and females42.The mitochondrial/microsatellites F44. Furthermore, high relatedness is favoured in small populations where the low number of individuals increases the probability of mating with close relatives.High levels of relatedness were recorded in all sub-populations likely due to inbreeding Fig. . The polST results showed a clear differentiation among the three sub-populations. The presence of two groups detected in Montes Forest sub-population both by PCA and multivariate analysis of relatedness .The low allelic richness found in Mount Lerno and Mount Tonneri sub-populations was mirrored by a very low effective population size. Founder effect and genetic drift due to population contraction are known to cause loss of alleles and decrease of genetic variability50 and other Sardinian macrofauna species such as wild boar and hare52.The low genetic variability currently observed in the Sardinian mouflon population might originate from the fragmentation of an original gene pool followed by long-term isolation. Originally, mouflon was widespread across Sardinia Fig.  but humax sheep hybrid populations, such as the Mount Lerno population. Hybridization of wild populations with their domestic counterparts can lead to the loss of wildtype genetic integrity, outbreeding depression, and loss of adaptive features55. Morphological features are rarely informative to discriminate hybrids from pure mouflons especially when several events of backcrosses occurred55. Conversely, molecular investigations have the power to identify mixed ancestry components even with reduced panels of selected ancestry informative markers and are the only diagnostic tools able to effectively identify hybrids before carrying out restocking actions55. Our study prompts for initiatives to restore the habitat connectivity between Montes Forest and Mount Tonneri and promotes controlled inter-population exchange in order to improve the overall genetic diversity and counteract the effects of population fragmentation57. The connectivity disruption effect has been more severe in the Mount Tonneri sub-population as evidenced by the reduction in population size, Ne and allelic richness, probably due to forest fires. Actions as the implementation of biological corridors such as overpasses would allow continuous exchange of individuals among sub-populations, thus reducing the local extinction rate58. Higher gene flow leads to higher heterozygosity, which is often associated with increased fitness (heterozygote advantage), while the risk of outbreeding depression is overall minimal when the populations involved inhabit the same habitat59. This is the case for Montes Forest and Mount Tonneri sub-populations that originated in the last century from the same original gene pool, are quite close to each other and host identical habitats. The present study represents a first important step towards a deeper understanding of the genetic peculiarities of the native mouflon populations living in Sardinia. Our results highlight the deleterious effects on genetic diversity of habitat changes and fragmentation of the original gene pool. Given the key role of the Sardinian mouflon in the recolonization of continental Europe, such a knowledge is an indispensable prerequisite for the protection of its genetic identity, jeopardised by uncontrolled interaction with domestic sheep breeds.We investigated the diversity and population structure of three Sardinian mouflon sub-populations. Particularly, we highlight the genetic distinctiveness and degree of isolation of the Montes Forest and Mount Tonneri sub-populations. We argue that these sub-populations should be considered as separate management units and preserved from contacts with genetically uncharacterized and known mouflon All the animal procedures were performed in compliance with the ARRIVE guidelines and in strict accordance with the guidelines of the Ethics Committee of Sassari University, Italy, which also approved this study.We collected either peripheral blood (captured mouflon) or muscular tissue samples  from 54 Sardinian mouflon from three distinct sites of the island during monitoring actions carried out from 2010 to 2016. A total of 39 samples  were collected in the historical mouflon range. Other 15 samples were sampled from Mount Lerno (Pattada) site, where mouflons were reintroduced 30 years ago by three subsequent transfers, sourcing from Mount Albo and passing through Capo Figari and the Asinara Island  and the NucleoSpin Tissue XS Kit (Macherey-Nagel), respectively. Procedures were carried out according to the manufacturer's protocols. Sample quality and DNA concentration were determined via spectrophotometry using a ND-8000 . To ensure the sequence accuracy, DNA was sequenced from PCR replicates. DNA samples were used to assay 16 microsatellite loci length and the mitochondrial D-loop region polymorphisms in order to genetically characterize each group and to clarify the genetic relationships among them.2, 0.2 0 mM each dNTP, 0.20 μM each primer, 0.02 mM BSA, 1 × PCR buffer and 2 units Taq DNA Polymerase (Sigma-Aldrich), according to Mereu et al.32. PCR amplifications were performed in a Gradient Thermocycler (Eppendorf) by an initial denaturation of 95 °C for 3 min, followed by 30 cycles of 95 °C for 50 s, 60 °C for 30 s, and 72 °C for 1 min.The primer pair CR1-CR2  was used to amplify the mtDNA D-loop fragment. A standard 50 μL PCR mixture was used, including 200 ng DNA template, 2.5 mM MgClPCR products were sequenced using the same primers on an ABI 3130 Genetic Analyzer (Applied Biosystem). Sequencing reactions were carried out following the manufacturer's recommendations (BigDye Terminator 3.1 Cycle Sequencing Kit—Applied Biosystem) and purified through the SigmaSpin Post—Reaction Clean—UP Columns (Sigma-Aldrich).Raw sequencing data were processed by means of the KB base-calling algorithm implemented in the Sequencing Analysis Software 5.3.1 (Applied Biosystem).60.Sequence alignment and data formatting, including trimming to a core length of 439 bp and resolving the ambiguities (N) in base-calling, were performed using BioEdit61.The amount of genetic variation among the three sub-populations, including the number of polymorphic sites (S) and haplotypes (h), the haplotype diversity (Hd) and nucleotide diversity (π), were estimated using DnaSP 6.10.03ST) were carried out using Arlequin 3.562.The analysis of molecular variance (AMOVA) and the calculation of the coefficient of differentiation , Corsica (n = 2), mainland Europe (n = 3), Anatolia (n = 16) and Asia (n = 10). These sequences were a representative sub-sample of all the haplotypes deposited in GenBank and were selected, one for each haplotype, on the basis of similarity detected at the BLAST analysis. Three O. vignei sequences were also included as outgroup (Supplementary Table http://popart.otago.ac.nz].Genetic relationships among haplotypes were investigated by a median joining network (MJN) on the nucleotide sequence matrix of 91 D-loop sequences. To explore the evolutionary relationships between the Sardinian mouflon and other 63 using 10,000 bootstrap replicates. The Tamura-Nei (TN93)64 model of nucleotide substitution with uniform evolutionary rates among sites was chosen to estimate genetic distance between haplotypes on the basis of the lower Bayesian information criterion (BIC) score.The pairwise genetic distances were estimated with the software MEGA 7.0.14O. aries) and cattle (Bos taurus) obtained from public databases . Markers were amplified by PCR in 25 µl reaction mix containing: 10 μl of DNA (1 ng/μl) as a template, 1X PCR Buffer, 1.5 mM of each dNTP, 0.5 μM of each primer, 1 unit of Taq Polymerase and 1.5 mM MgCl2. PCR amplifications were performed in a Gradient Thermocycler (Eppendorf) by an initial denaturation of 95 °C for 2 min, followed by 35 cycles of 94 °C for 30 s, 48–58 °C for 1 min . Allelic richness and FIS values were calculated using Fstat 2.9.367. Differences between populations in allelic richness were tested using the Fisher exact test method, after assessing for normality of the data and variance homogeneity , while FIS significance was tested by 95% confidence interval with 1000 bootstrap replicates by Fstat 2.9.3. Allele frequency, gene diversity , observed heterozygosity (Ho) and Hardy–Weinberg equilibrium (HWE) deviation analyses by population and locus were calculated using the Fisher exact test68 in a contingency table of arbitrary size, as implemented in Arlequin 3.562. The exact P-values were obtained using a Markov Chain Monte Carlo (MCMC) simulation with 10,000 dememorization steps, 500 batches, and 5000 iterations.Microsatellite products were analysed on an ABI PRISM 3100 DNA Analyzer (Applied Biosystems) and data processed using GeneScan 3.1 and Genotyper 2.5ST) used as inter-population indices to assess the relationships among the three mouflons groups was calculated by permuting the genotypes or individuals between the sub-populations. The significance of the genetic distances was tested by Weir & Cockerham69 as implemented in Arlequin 3.5 .Genetic differentiation  by Arlequin 3.5.70 of the R software71.A principal component analysis (PCA) was performed to investigate the ordinal relationships between populations and individuals, using the ade4 1.7-16 package72 which allows estimation of the rate and direction of recent dispersal. The MCMC method was run for 10,000,000 iterations. Delta values  were left as default73.Recent migration rates among sub-populations were assessed using a Bayesian Markov Chain Monte Carlo (MCMC) analysis implemented in BayesAss 1.374 with the 2x option was estimated using GenAlEx 6.50175 to detect related individuals within the same sub-population. This method assumes values from − 1  to 1 . To calculate pairwise relatedness, we used the allele frequency of the total population of assembled genotypes with 9999 permutations and r frequencies were plotted to evaluate its distribution. Distribution factors such as median and mean were compared by χ2 test and ANOVA test respectively, using Minitab 17. Relatedness estimates higher than 0.5 observed in each population were compared in the whole sample by means of Pearson χ2 test (Minitab 17). To assess the presence of sub-structures within sub-populations a matrix of pairwise relatedness distances between individuals was computed by Genalex 6.501 and a multivariate analysis was performed using the Cluster Variables function implemented in Minitab 17. The results were graphically expressed by a UPGMA tree (Minitab 17).Pairwise relatedness (r) between individuals within populations based on the method of Lynch and Ritland76 assuming that microsatellite mutation followed the Infinite Allele Model (IAM), Stepwise Mutation Model (SMM) or Two-Phase Model (TPM)77. In TPM we used 95% single-step mutational events at 12% variance. To determine the significance in the heterozygosity excess we performed the one-tailed Wilcoxon test. A mode-shift test was carried out to detect a distortion on the expected L-shaped distribution of allele frequency and to determine whether the population is under mutation drift equilibrium (shaped curve) or not (mode shift)78.Bottleneck events were detected from allele frequency data by the BOTTLENECK software79. This method has been proven relatively robust in assessing the effective population size (Ne) using microsatellite data80. As rare alleles could result in biased Ne, we calculated three estimates for Ne by excluding allele frequencies of less than (Pcrit) 0.05, 0.02, or 0.01. The 95% CI was estimated by the parametric method81.We estimated the effective population size using the Linkage disequilibrium method implemented in NeEstimator 2Supplementary Figure S1.Supplementary Figure S2.Supplementary Table S1.Supplementary Table S2.Supplementary Table S3.Supplementary Table S4.Supplementary Table S5.Supplementary Table S6.