Historical discourses about the Caribbean often chronicle West African and European influence to the general neglect of indigenous people’s contributions to the contemporary region. Consequently, demographic histories of Caribbean people prior to and after European contact are not well understood. Although archeological evidence suggests that the Lesser Antilles were populated in a series of northward and eastern migratory waves, many questions remain regarding the relationship of the Caribbean migrants to other indigenous people of South and Central America and changes to the demography of indigenous communities post-European contact. To explore these issues, we analyzed mitochondrial DNA and Y-chromosome diversity in 12 unrelated individuals from the First Peoples Community in Arima, Trinidad, and 43 unrelated Garifuna individuals residing in St. Vincent. In this community-sanctioned research, we detected maternal indigenous ancestry in 42% of the participants, with the remainder having haplotypes indicative of African and South Asian maternal ancestry. Analysis of Y-chromosome variation revealed paternal indigenous American ancestry indicated by the presence of haplogroup Q-M3 in 28% of the male participants from both communities, with the remainder possessing either African or European haplogroups. This finding is the first report of indigenous American paternal ancestry among indigenous populations in this region of the Caribbean. Overall, this study illustrates the role of the region’s first peoples in shaping the genetic diversity seen in contemporary Caribbean populations. Bordered by the Atlantic Ocean to in the east, it spans from the southern coast of the Bahamas to the northern coast of South America and the eastern coast of Mexico and Central America . Comprised of well over 700 islands, this region is currently home to an estimated 17 million people [The Caribbean is a vast region encompassing nearly 3 million kmrica Fig . ComprisThe initial presence of human populations within the region has been dated to 7,200 years before present (ybp) based on evidence from the Banwari Trace site in Trinidad –5. WithiAs a result of these different expansions, at least eight different ethnic groups were present in the Caribbean at the time of European contact. These include the Guanahatebay in Cuba, the Macorix of Hispañola, the Ciguayo of Hispañola, the Lucayo  in the Bahamas, the Ciboney of Haiti, Jamaica, and Cuba, the Classic Taíno in the Dominican Republic, Puerto Rico, the Virgin Islands, and the Leeward Islands, and the Kalipuna and the Karina Caribs in Windward islands of the Lesser Antilles ,16. The Indigenous Caribbean peoples were greatly affected by the assimilation, disease, and genocide brought about by European colonization and the Trans-Atlantic slave trade . These cWhile archeological, ethnohistorical, and linguistic data provide crucial perspectives on the peopling and history of the Caribbean Basin, many questions regarding the timing and origins of the initial migrations and the impact of European colonization on indigenous Caribbean communities remain unanswered. In response to these issues, and with local and community consent, we characterized mitochondrial DNA and Y-chromosome variation in two indigenous communities, the Santa Rosa First Peoples’ Community (FPC) in Trinidad and the Garifuna of St. Vincent. The resulting data allowed us to examine the plausibility of different models regarding the purported origins of the initial migrations into the Caribbean Basin and evaluate hypotheses about the genetic relationships between indigenous Caribbean and circum-Caribbean populations. These data were also used to assess how the entry of African, Asian and European peoples into the Caribbean has affected the demography of indigenous populations of the Lesser Antilles.Prior to study commencement, this project was reviewed and approved by the University of Pennsylvania IRB #8 and the University of Notre Dame IRB. In addition, prior to sample collection, we obtained approval from the National Ethics Research Committee, Ministry of Health, Wellness and the Environment, St. Vincent and the Grenadines, and the Ethics Committee, Government of the Republic of Trinidad and Tobago, and the Santa Rosa FPC and St. Vincent Garifuna Community. Each participant also provided written informed consent prior to sample collection and documentation of family history information.th century led several rebellions to defend his community from colonial encroachment [The Santa Rosa FPC is based in Arima, a settlement located in the north-central region of Trinidad, roughly 26 km east of Port of Spain. According to the Trinidadian Central Statistics Office 2011 census, approximately 1,328 people in Trinidad identified as indigenous, 7% of whom live in Arima . The hisoachment . As partoachment . Althougoachment . In 1976oachment ,25,28.th century. During this period, France and Britain agreed to leave St. Vincent, Dominica, St. Lucia, and Tobago as so-called ‘neutral islands’ for the indigenous people of the Caribbean. In reality, while both France and Britain worked to ensure that the other would gain no advantage on the neutral islands, Britain formally seized control of St. Vincent in 1763. British encroachment on indigenous lands resulted in the First Carib War (1772–73), after which the British succeeded in dispossessing indigenous peoples and forcing them into a treaty. Although the Vincentian indigenous groups halted the British military advance at that time, within twenty years, the Second Carib War (1795–96) erupted between these antagonists. As a result of this conflict, Vincentian indigenous peoples were deported, first to the nearby island of Balliceaux, and then to Roatán, an island located off of the coast of Honduras [Like that of their counterparts in Trinidad, the history of the Garifuna of St. Vincent is a chronicle of resistance and survival . The GarHonduras ,29.Prior to its becoming a colony, both the British and the French employed ethnic distinctions to divide indigenous Vincentian peoples and help them annex native lands . AccordiGenetic samples, collected via buccal swabs, were obtained from a total of 88 indigenous individuals in both Trinidad and St. Vincent. Of this total, 65 participants were Garifuna from the Kingstown area and two regions on the northeastern coast of St. Vincent. The remaining 23 samples were obtained from members of the Santa Rosa FPC in Arima, Trinidad. All 88 DNA samples were subjected to genetic analysis. After review of the genealogical data, we removed maternal or paternal relatives from the sample set, leaving 7 Santa Rosa FPC and 25 Garifuna females and 5 Santa Rosa and 18 Garifuna male samples for statistical and phylogenetic analyses. Although these sample sizes are small for standard statistical analyses, both communities are small, demographically speaking, numbering under 2,000 people, including relatives. Consequently, the small study sample sizes reflect the communities from which they were derived. While these sample sizes may affect how robust the statistical and phylogenetic analyses described below, they nonetheless allow for some generalizations about human prehistory in this region of the Caribbean.The mitochondrial DNAs (mtDNAs) from these samples were analyzed by using direct sequencing and single nucleotide polymorphism (SNP) genotyping methods as previously described, with the entire control region (CR) (np 16024–576) being sequenced in each sample –36. The We determined the paternal genetic ancestry of the St. Vincent and Trinidadian male participants by screening the non-recombining region of the Y-chromosome (NRY) for 17 phylogenetically informative biallelic markers (SNPs) that define paternal haplogroups and their major sub-branches  . All marPaternal haplotypes were further defined through the analysis of 17 Y-chromosome short tandem repeats (Y-STRs) that are part of the AmpFℓSTR Y-filer Amplification Kit (ABI). A separate custom multiplex reaction was also used to characterize six additional SNPs  and two additional Y-STRs . PCR products were run with GeneScan 500 LIZ Size Standards and read on an ABI 3130xl Gene Analyzer –36,43.We used Haplogroup Predictor to assignWhen comparing the St. Vincent and Trinidad communities to other Caribbean populations, we used data from the Y-STR loci recommended by the Scientific Working Group on DNA Analysis Methods (SWGDAM) . This seST, a measure of population differentiation due to genetic structure, was estimated based on HVS1 sequences for Vincentian, Trinidadian and comparative populations with Arlequin v.3.11 [ST estimates were subsequently visualized through multi-dimensional scaling (MDS) [To assess the population affinities of the St. Vincent and Trinidadian populations, we compared their mtDNA HVS1 sequences np 16024–16400) to those of several North American , Caribben v.3.11  using thn v.3.11 . The int24–16400 ng (MDS) , were th−7 per nucleotide per year [In addition, we constructed median-joining (MJ) networks with MP post-processing function in Networper year .ST, using Arlequin v.3.11 software [ST estimates were then visualized in a multidimensional scaling (MDS) plot in SPSS [The Y-STR data from St. Vincent and Trinidad were compared to those from published studies of Native American populations –70Table. In thissoftware . The RST in SPSS . In addi in SPSS .-5 mutations per locus per year [MJ network and coalescence analyses ,64were aper year , one shoper year to producFor the two populations, we detected maternal indigenous ancestry in 42% of the individuals . HoweverIn addition to indigenous Caribbean ancestry, both communities exhibited maternal lineages from Africa and, in the case of the Trinidadians, South Asia. Most of the non-indigenous lineages in both communities belonged to African haplogroups L0, L1, L2, and L3. Haplogroup L2 was the most common African lineage among the Vincentians, while L2 and L3 were the most common lineages in FPC Trinidadians. In addition, FPC Trinidadians had mtDNAs belonging to haplogroup M33a, a lineage commonly seen in northeastern India .). Furthermore, these HVS1 sequences were used for all subsequent statistical and phylogenetic analyses.Analysis of mtDNA CR sequences revealed a total of 58 distinct haplotypes in the St. Vincent Garifuna and the FPC Trinidadians. Of these, 23 belonged to indigenous American lineages, 31 to African lineages, and 2 to South Asian lineages . Thus, to clarify the genetic affinities of the indigenous Caribbean samples, the Mesoamerica populations were removed from subsequent network analyses.In the refined A2 MJ network, haplotypes from Trinidad, Puerto Rico, Venezuela, and Brazil comprised the central node of the network . An adjoC in B in B in C in In the refined haplogroup C1 MJ network , VincentCoalescence analysis indicated that the indigenous Caribbean A2 haplotypes were older than those from haplogroup C1, with A2 dating to 8489 (± 7095) and C1 to 2452 (± 1501) ybp, respectively. While the age of C1 is generally consistent with estimate based on our mtDNA data from the Greater Antilles, that of A2 was not . The difG in African, European, and Native American Y-chromosome haplogroups were present in both the Trinidadian and Vincentian populations G in . Within The remaining Y-chromosomes from Trinidad and St. Vincent were of African (E1b1a) or likely European  origin. Overall, more than 80% of the Y-chromosome haplotypes in the indigenous Caribbean communities were non-indigenous to the Americas. The Vincentian samples were comprised of about half African and half European lineages, whereas the Trinidadian samples showed a larger frequency of paternal African (80%) than European (20%) lineages.Analysis of Y-STR variation in the St. Vincent Garifuna and the FPC Trinidadians revealed a total of 30 distinct haplotypes in these populations, based on data from 17 loci . Haplogroup L2 was the most commonly observed African haplogroup within the Vincentians, while L2 and L3 were most common in the Trinidadians. Both of these lineages are frequently observed on other Caribbean islands, representing between 20–30% of the mtDNAs ,77,87,91th century census records of British colonial plantations in Trinidad. As illustrated by the 1813 census, the birthplaces of the majority of enslaved Africans in Trinidad were primarily from regions within the Bight of Biafa, Gold Coast, and the Bight of Benin [These findings are generally concordant with the 19of Benin . Based oth century. A previous analysis of Honduran Garifuna revealed only indigenous haplogroups A2 and C1 and several African haplogroups, but no European maternal lineages [B in With regard to the Vincentian Garifuna, there are several noteworthy differences between this community and the Garifuna that were exiled to Honduras in the 18lineages . Althouglineages . Furtherlineages . In our In addition to indigenous American and African mtDNA haplogroups, two members of the Trinidadian indigenous community also had maternal lineages of South Asian origin that belonged to haplogroup M33a . This fiAnalysis of mtDNA variation of all of the haplotypes observed in the indigenous Caribbean populations illustrates that the indigenous Caribbean groups have similar levels of genetic diversity relative to the comparative populations. However, when African and South Asian haplotypes are removed, mtDNA diversity in the indigenous Caribbean groups decreases considerably. This reduction in mtDNA diversity in indigenous Caribbean populations may be attributed to several factors including the effects of genetic drift, the loss of indigenous lineages in historical times, and possibly endogamy which may have led to the enrichment of certain mtDNA haplotypes in the populations. Furthermore, patterns of genetic diversity differed between the Vincentian Garifuna and the FPC Trinidadians. This difference suggests that both groups possess specific sets of mtDNA haplotypes that are reflective of their unique histories, including the possibility of having different ancestral origins in South America.In the MDS plot containing indigenous Caribbean and comparative circum-Caribbean mtDNA data , both stAnalysis of mtDNA diversity in the indigenous Caribbean and Anglophone Caribbean populations shows that indigenous Caribbean groups are genetically distinct along the maternal line from the greater populace. However, the Vincentian Garifuna show greater affinities with the other island populations than the Dominicans and Trinidadians. This pattern may reflect the dual African and indigenous Caribbean ancestry of the Vincentian Garifuna and their subsequent cultural and genetic exchanges with indigenous Caribbean peoples as documented by historic sources ,77,107. Both indigenous Caribbean communities had founder A2 and C1 haplotypes and these were also observed in Puerto Rican populations ,90. Yet,Coalescence analysis of the Caribbean haplotypes indicates that haplogroup C1 is younger 2861 ybp) than haplogroup A2  in the Lesser Antilles. These dates are generally congruent with archeological data from the Caribbean, which indicate the Lesser Antilles were initially inhabited as early as 7,200 ybp in a series of migrations from circum-Caribbean regions 861 ybp t. HoweverBased on the summary statistics, indigenous Caribbean populations show similar levels of paternal genetic diversity to comparative Caribbean populations, although having distinct sets of Y-STR haplotypes. However, as seen in In addition, the Vincentian and Trinidadian populations lack Q-M3 Y-chromosomes bearing derived SNPs  seen in related haplotypes in indigenous populations from western South America ,111. TheMoreover, our data provide the first evidence of indigenous Y-chromosomes in Caribbean populations from the Lesser Antilles. Recently, Marcheco-Teruel et al. (2015)  identifiPhylogenetic analysis shows that Q-M3 STR haplotypes in the FPC Trinidadian and Vincentian Garifuna differ from each other and have distinct genetic affinities with those from different areas of Central and South America . This result was intriguing because of the small geographic distance between the two indigenous Caribbean groups and the circum-Caribbean populations, and the history of pre-Colombian migration within the region ,117. ThuIndigenous Caribbean peoples are thought to be culturally related to Arawakan speakers of mainland South America, partly due to their having incorporated loan words from Cariban speaking groups ,17. Our th century British plantation censuses [Y-chromosome variation in indigenous Caribbean groups also shows evidence of significant historical gene flow from African and European males. African haplotypes, represented by haplogroup E1b1a, likely entered the indigenous Caribbean communities as a result of the Trans-Atlantic trade in enslaved Africans ,119. E1bcensuses , most ofcensuses .Several predominately European haplogroups were also observed in the indigenous Caribbean communities. Like their African counterparts, European genetic contributions to indigenous Caribbean communities initially began during the colonial period –124. HapTwo other non-native paternal lineages observed in the indigenous Caribbean communities were haplogroups I1 and I2. These haplogroups are ubiquitous in Europe, accounting for nearly 18% of European Y chromosomes . HaplogrAlso noteworthy is the pattern of European paternal ancestry among the indigenous Caribbean communities. In the Vincentian Garifuna, nearly half of the Y-chromosomes were of European ancestry while, in the Trinidadian FPC, less than one-quarter of the Y-chromosomes were. This observation could reflect differences in the histories of the two indigenous communities. When comparing the levels of European paternal ancestry in other Caribbean communities with a strong indigenous Caribbean influence, such as those in Dominica and St. Lucia , indigenConventionally, archeological and linguistic data have been the primary sources of information used to infer details concerning the peopling of the Caribbean Basin and these lines of evidence remain important for contextualizing the genetic record. As indicated by archeological and linguistic data, the diversity in the Caribbean reflects both migration and contact, and also isolation and differentiation, over thousands of years. Based on these data, scholars have hypothesized that Vincentian and Trinidadian indigenous groups would more strongly resemble indigenous groups from northern South American because the Carib/Arawakan expansion took place recently, whereas the Taíno groups in the Greater Antilles would resemble other South American groups, and perhaps also Mesoamerican populations, due to their representing an earlier genetic stratum of human populations involved in the initial peopling process. Genetic data from the Vincentian Garifuna and Trinidad FPC, though limited by sample size, support the South American origin and possible Mesoamerican connections of indigenous Caribbean peoples. Furthermore, our genetic data provide more information about the impact of colonization on indigenous Caribbean peoples. While African and European paternal lineages replaced many of those from indigenous Caribbean groups, there remains evidence of indigenous paternal ancestry in contemporary indigenous Caribbean communities.The current study provides a glimpse of the genetic history of the region’s first peoples. Future work will include more comprehensive analyses of mitogenome sequences and Y-chromosome diversity in indigenous Caribbean groups. In addition to the uni-parental genetic loci, analyses of autosomal variation in these populations will further illuminate genetic history of the region.http://cmpg.unibe.ch/software/arlequin35/Arlequin: http://www.ub.edu/dnasp/DnaSP: http://haplogrep.uibk.ac.at/Haplogrep: http://www.hprg.com/hapest5/Haplogroup Predictor: http://www.fluxus-engineering.com/sharenet.htmNetwork: S1 File(XLSX)Click here for additional data file.


NOS3 and EGLN1 genes previously associated with adaptation to high altitude in 150 ancient human DNA samples from Peruvian high altitude and coastal low altitude sites in a time frame between ~8500–560 BP. We compare the data of 109 successful samples to forward simulations of genetic drift with natural selection and find that selection, rather than drift, explains the gradual frequency changes observed in the highland populations for two of the three SNPs.Archaeological evidence shows that humans began living in the high altitude Andes approximately 12,000 years ago. Andean highlanders are known to have developed the most complex societies of pre-Columbian South America despite challenges to their health and reproductive success resulting from chronic exposure to hypoxia. While the physiological adaptations to this environmental stressor are well studied in contemporary Andean highlanders, the molecular evolutionary processes associated with such adaptations remain unclear. We aim to better understand how humans managed to demographically establish in this harsh environment by addressing a central question: did exposure to hypoxia drive adaptation via natural selection within Andean populations or did an existing phenotype –characterized by reduced susceptibility to hypoxic stress–enable human settlement of the Andes? We genotyped three variable loci within the  Humans residing in the high altitude Central Andes  are directly and indirectly affected by two primary environmental extremes: hypobaric hypoxia and cold. Environments characterized by hypoxic - low concentration of atmospheric oxygen- and cold conditions negatively impact the abundance of resources available to sustain human populations11EGLN1, PRKAA1, NOS2, and NOS3 associated with the hypoxia-inducible transcription factor (HIF) pathway and nitric oxide (NO) regulation6111213th centuries inflicted a massive population decline in the Americas, followed by subsequent gene flow from Europe and Africa1615While prehistoric Andean societies developed behavioral and cultural adaptations to buffer against low biotic productivity and cold56729NOS3: rs1799983; EGLN1: rs1769792, rs1769813) in 150 pre-Columbian individuals dating from ~8,500 to 600 BP. 85 samples derive from archaeological sites in the Peruvian highlands; 65 samples from low altitude coastal sites were included for comparative purposes , an enzyme catalyzing the synthesis of NO and is i.a. regulated by hypoxia4421EGLN1 SNPs  provided the highest ranking signals of selection based on locus specific branch length (LSBL) statistics in Andean populations according to a study screening ~900 k SNPs in genes contributing to pathways with known physiological responses to hypoxia, including the HIF pathway, the renin-angiotensin system, and the globin family of genes23EGLN1 as being under selectionEGLN1 SNPs is less obvious than for NOS3. While polymorphisms in this gene have been associated with lowered hemoglobin concentrations in Tibetan populations, a study on Andean women failed to find an comparable association with a hypoxia-related phenotype6EGLN1 gene in the HIF pathway make it a plausible target for our study.The two NOS3 SNP and one of the EGLN1 SNPs in the ancient highland populations while the ancient coastal populations exhibit constant allele frequencies over time.We obtained high confidence genotypes for all three SNPs from 109 ancient Peruvian individuals positively tested for ancient DNA preservation in previous studies. By applying a forward simulation approach modeling drift and selection on standing variationThe archaeological samples analyzed in this study derived from 14 Peruvian coastal and highland sites cf. , Fig. 1.272829303132NOS3 rs1799983 G-allele increases over the ~7,800 year time period for the ancient high altitude population while the EGLN1 rs1769792 G-allele decreases  and EGLN1 rs1769792  over time cannot be explained by drift alone. All tests performed for the coastal populations and EGLN1 rs1769813 in the highland populations returned no significant results , while they are found in LD for both the ancient coastal  and the modern 1000 Genome PEL population  from Lima which represents a recently admixed population from all over Peru, especially from the highlands. On a global population scale both SNPs are also found to be in LD .To test if the observed frequency changes over time can be explained by genetic drift we calculated simulation test (ST) results . Tests fNOS3 and EGLN1 in the ancient Andean highlands result from natural selection on standing variation and to quantify the power of potential selection by determining the selection coefficient (s), we performed forward simulations of drift plus selection based on Sverrisdóttir et al., allowing uncertainty in ancient allele frequency and population size. We excluded EGLN1 rs1769813 from these simulations because ST and WT statistics revealed no significant frequency changes over time  and the Late Intermediate Period and Late Horizon  or modern Quechua highland population were s = 0.008 (recessive) and s = 0.012 (codominant) respectively , s = 0.008 (codominant), and s = 0.005 (recessive).While neutrality (selection coefficient s = 0) for both SNPs was not rejected for this test when assuming very low population sizes (Ne = 100–1000), models considering selection found higher support for all allelic conditions tested . The selectively . For theTo evaluate if the observed allele frequency changes in the highland populations over time could be explained by population discontinuities we calculated the pairwise genetic distances (FST) between the populations employing the previously published mitochondrial hypervariable region I (HVR) data . All FST27EGLN1 rs1769813 previously reported to be under selection in Andean populations by Bigham et al.et al.EGLN1 rs1769813 SNP observed in Central Andean populations. Interestingly, both EGLN1 SNPs tested were only found to not be in LD for the ancient highland populations, while significant non-random associations for the SNPs were found in the ancient coastal, modern Peruvian, and global populations. A possible explanation for this observation could be that a founding effect and subsequent substructuring following the initial divergence into the coastal and the highland populations led to the loss of LD between both markers, and that subsequent selection acting on the EGLN1 rs1769792 SNP and also more recent admixture between coastal and highland populations after the European contact reestablished LD as seen in the modern Peruvian (PEL) population. To further explore possible scenarios explaining the observation made with regards to changes in LD whole gene data of the ancient specimen for EGLN1 would be necessary.Although the physiological responses to hypobaric hypoxia have been well characterized, the underlying genetic basis remains largely unknown. Because of the recent advances in genomics, and a growing number of studies identifying candidate genes involved in particular pathways with hypothesized roles in high-altitude adaptation, it is now possible to directly test the hypothesis that beneficial traits evolved under selection and thus contributed to the survivability and success of societies living in the high altitude environments of the Andes. Here we analyzed putative natural selection candidate loci with standing variation in global populations in ancient Peruvians. We find evidence that selection indeed affected the genetic diversity of Andean highlanders and was ongoing in the high altitude populations during the timespan examined. We did not detect any frequency changes for NOS3 rs1799983 we determined to be under selection has not been identified to be under selection in previous studies of modern DNANOS3 G-allele, it is not possible to determine the exact adaptive phenotype/physiological trait to which this genotype contributes. A reduced susceptibility to acute hypoxic stress-related pathologies, namely AMS and HAPE, associated with the G-allele can be correlated with an increased fitness and survivability in high altitude environmentsNOS3 G-allele could constitute a reproductive advantage in high altitude populations22The NOS3 rs1799983 and the EGLN1 rs1769792 SNPs between the ancient coastal populations and the modern 1000 Genomes population from Lima (PEL) is best explained by a combination of evolutionary factors. An ancient DNA study recently observed population discontinuity along the late pre-contact coast, suggesting larger migrations from the highlands to the coast occurred after ~850 BP, postdating the youngest coastal population tested hereHowever, to infer natural selection based on temporal differences in allele frequency it is necessary to assume population continuity2829303238401742431128NOS3 rs1799983 in non high altitude South American populations e.g. the in the Amazon, showing both high and low frequencies of the G-allele1743NOS3 frequency differences in low altitude South America can best be explained by founder effects.The ALFRED database demonstrates a high diversity for The evolutionary scenarios that found highest support in our study assumed relatively low initial effective population sizes for the highland populations (Ne = 500–5000). This is consistent with the archaeological record showing that the number of initial settlers in the high altitude Andes was considerably low until the stabilization of resource patches and agroeconomic adaptations allowed humans to sustain larger population sizes139NOS3 rs1799983 and the EGLN1 rs1769792 SNP allows us to address whether the Andes were initially peopled by individuals genetically predisposed with a reduced susceptibility to hypoxic stress–assuming that individuals suffering from symptoms of AMS, HAPE, CMS would have returned to lower altitudes–or whether the descendants of these early pioneers adapted to the environmental stressor over time118EGLN1 SNPs tested in this study have been associated with a hypoxia-related phenotype in Andeans. Additionally, genome wide studies of Andean highlanders did not identify EGLN1 to be a top ranked candidate for hypoxia induced selection in Andean populations. So, even though we identify a signal of selection in one of the EGLN1 SNPs tested we have to remark that the involvement of EGLN1 in the genetic adaptation of Andeans to high altitude stress is not fully supported. The observed selection could result from other environmental factors not considered in this study. An additional factor to be considered in the interpretations of our results is that the demography of the studied populations has not been directly inferred. It is not clear if the observed frequency trajectories have been affected by specific demographic processes . To avoid a potential bias resulting from known population bottlenecks resulting from the European contact16The gradual allele frequency change over time observed for of both the NOS3 rs1799983 and the EGLN1 rs1769792 SNP  are relatively low. For the LCT -13,910*T allele, which is strongly associated with lactase persistence in Europeans, recent studies have reported values ranging from s = 0.008 − 0.079518G6PD gene associated with resistance to malaria in endemic regions has been estimated to range from 0.019–0.049652LCT and G6PD alleles is known to be considerably high , the contribution of alleles associated with hypoxia response to the adapted high altitude phenotype is not completely understood, especially for Andean populations26In the context of other genetic loci identified to be under selection in the more recent history of our species , the selection coefficients we determined for the EGLN1 gene and the two SNPs tested in this study. Even though, we identified selection acting on one of the SNPs, the lack of association for EGLN1 with a hypoxia-related phenotype in Andean populations also means that other selection inducing factors than hypoxia could be causal for the observed allele frequency changes. At this point the involvement of EGLN1 in the adaptation of human Andean populations to hypoxia is not fully supportedIn sum, our study reveals direct evidence for natural selection acting on two of three SNPs associated with the adaptation to high altitude stressors and quantifies the strength of selection acting on the specific alleles. We demonstrate the ability of paleogenetics to verify hypotheses solely deriving from modern population studies and the high potential for cooperation between both fields to increase our understanding of our species’ recent evolution. Increased efforts to link genetic diversity to pathways and phenotypes increasing the survivability and reproductive success of these populations are necessary to finally reveal the processes underlying human adaptation to hypoxia in the AndesAlthough our study is restricted to three SNPs and the statistical power suffers from low sample size, it offers a simple model to test selective response to environmental stressors. The approach we choose for this study can of course only explore frequency changes in standing variation, and does not allow to detect de-novo mutations that could have contributed to the adaptation of the studied populations. Future ancient DNA studies employing genome-wide sequencing strategies will progressively contribute to the understanding of human adaptation to altitude and have the potential to overcome the discussed limitations of this study.All samples analyzed in this study have been part of previous studies investigating mitochondrial and nuclear genetic loci or are currently analyzed in genome wide sequencing projects27282930313225et al.28et al.2940All pre-PCR analyses were carried out in laboratories entirely dedicated to ancient DNA analysis. All analyses were carried out according to the precautions and contamination prevention strategies described in Fehren-Schmitz NOS3 and the two EGLN1 SNPs  we developed a multiplex PCR amplifying three targets  spanning the sequences containing the SNP of interest coupled with a Single Base Extension (SBE) assay to directly type the SNPs in parallel. Primers for the main PCR and the SBE PCR were designed using the Primer Select software . The minisequencing/SBE primers were designed one base contiguous to the polymorphic site of interest in forward or reverse orientation. An additional polymeric-A tail was added to the 5′ end in order to ensure an effective separation of product lengths during electrophoresis. Primer sequences can be found in To determine the genotypes of the To remove excess primers and dNTPs from the previous reaction, the 7.5 ul PCR product of the PCR were first incubated together with 2.5 units (U) rAPid Alkaline Phosphatase , and 1 U Exo I  in a thermocycler for one hour at 37 °C, and following 15 minutes at 75 °C for heat inactivation. Afterwards 1 ul of the purified PCR product was used for the SBE reaction in a final reaction volume of 5 ul consisting of the template, 2.5 ul SNaPshot Ready Reaction Mix , 0.1 uM SB primer and 20 uM (NH4)2SO4 . The ammonium sulfate was added for the suppression of nonspecific peaks in the primer extension reactionTo ensure authenticity of the amplification results and to prevent false genotype calls due to allelic dropout we performed a minimum of four independent amplifications per DNA extract for each genetic marker analyzed. For each individual we generated at least two independent DNA extracts resulting in a minimum of eight amplification results per marker used to call a consensus genotype. Alleles were only called if they were typed in over 50% of the amplifications. Additionally, 70 samples were tested in a second ancient DNA lab at the University of Goettingen (Goa) employing the same protocols as described for the UCSC-HPG. Samples were discarded if results were inconsistent or there was a risk of false allele determination due to allelic dropout,NOS3 and the two EGLN1 SNPs were obtained using GenAlEx 6.5EGLN1 SNPs we calculated D’ and r2 statistics58Allele and genotype frequencies for the NOS3 rs1799983 G and the EGLN1 rs1769792 G-allele and observed in the high altitude time transect can be explained by natural selection and to determine the selection coefficient (s) we used a forward simulation approach based on Wilde et al.et al.et al.np + 1, nq + 1) distribution, where np and nq were equal to the number of the respective ancestral and derived allele to reflect uncertainty in the ancient allele frequencies. For both SNPs we then obtained 10,000 MCMC samples by binomial sampling across generations for each combination of priors and allelic condition tested. We used a forward simulation for drift with selection using equation 3.5 from Maynard SmithTo test if allele frequency changes in the FST values were estimated using the Kimura two-parameter modelFST and derived Slatkin’s distanceTo explore the population genetic relationship between the tested ancient populations using a neutral genetic marker we employed the 388-bp mitochondrial HVR I sequences  reported in previous studies28313240How to cite this article: Fehren-Schmitz, L. and Georges, L. Ancient DNA reveals selection acting on genes associated with hypoxia response in pre-Columbian Peruvian Highlanders in the last 8500 years. Sci. Rep.6, 23485; doi: 10.1038/srep23485 (2016).


Aphis gossypii Glover (Hemiptera: Aphididae) is a serious pest of cotton in northern China. A microsatellite analysis was used to characterize the genetic structure of A. gossypii populations from different geographic, host plant, and seasonal populations in 2014. Among 906 individuals, 507 multilocus genotypes were identified, with genotypic richness values of 0.07–1.00 for the populations. We observed moderate levels of genetic differentiation among geographic populations  and host plant populations . A Mantel test of isolation by distance revealed no significant correlations between Slatkin’s linearized FST and the natural logarithm of geographic distance. A Bayesian analysis of population genetic structures identified three clusters. An analysis of molecular variance revealed significant differences among the three clusters , among seasons , and among host populations . Thus, the A. gossypii populations in northern China exhibit considerable genotypic diversity. Additionally, our findings indicated that the 31 analyzed populations could be classified as one of three host biotypes . There were also clear seasonal effects on population genetic structure diversity among aphids collected from Anyang. Aphis gossypii Glover, is a polyphagous species with a worldwide distribution. Economic losses caused by this aphid result from its feeding on agricultural crops, the production of honeydew secretions on lint , and the fact it serves as a vector for viral diseases  indicated a moderate level of genetic differentiation among geographic populations , 67 (43.8%) exhibited moderate genetic differentiation (pairwise FST 0.05 to < 0.15), and 23 (15.0%) revealed considerable genetic differentiation (pairwise FST 0.15 to < 0.25)  (The genetic structure among 18 geographic populations was examined using pairwise comparisons of multilocus Fulations . Pairwisnificant . Between < 0.25) . The pre1–0.141) .ST with and without the ENA correction. The overall FST value  indicated moderate genetic differentiation among the Anyang populations. Pairwise FST estimates between pairs of Anyang populations indicated 50.5% of population differentiation tests were significant (ST < 0.05), 21 (23.1%) exhibited moderate genetic differentiation (pairwise FST 0.05 to < 0.15), 19 (20.9%) revealed considerable genetic differentiation (pairwise FST 0.15 to < 0.25), and 30 (33.0%) showed extreme genetic differentiation (pairwise FST > 0.25) (The genetic structure among 13 Anyang populations was examined using pairwise comparisons of multilocus Fnificant . Between > 0.25) .ST[ENA] = 0.231; 95% CI: 0.184–0.286)  .ST and the natural logarithm of geographic distance  for all 18 geographic populations  (The Mantel test of isolation by distance (IBD) revealed there was no significant correlation between Slatkin’s linearized Fulations . The pre= 0.187) , which iK (Evanno method) occurred with K = 3  was completed using Nei’s genetic distance pairwise matrix. The PCA results divided the 31 populations into three groups , which wThe proportions of Anyang populations from three hosts  depended on the season. In May, the blue proportion of COW-May was 0.850, the red proportion of CUW-May was 0.994, and the blue and red proportions of ZUW-May were 0.360 and 0.636, respectively. In August, the blue proportion of COW-Aug was 0.986, the red proportion of CUW-Aug was 0.795, and the blue and red proportions of ZUW-May were 0.686 and 0.308, respectively. In September, the blue proportion of COW-Sep was 0.988, the red proportion of CUW-Sep was 0.164, the blue proportion of CUW-Sep was 0.829, and the blue and red proportions of ZUW-May were 0.790 and 0.204, respectively .F = 0.26596, P < 0.0001)    revealed significant differences among the three clusters ( 0.0001) , suggest 0.0029) . Under f0.00381) .A. gossypii aphids in this region is considered to be holocyclic ) using the FreeNA program [A neighbor-joining tree using genetic distance was constructed with PHYLIP v. 3.696  for 500,000 iterations after a burn-in period of 750,000. The optimal K value was determined using the highest ΔK value as described previously [K value was calculated using Structure Harvester v. 0.56.3 [Population structures were analyzed using the Bayesian clustering algorithm of Structure v. 2.3.4 software . The Mareviously . The ΔK . 0.56.3 . Populat. 0.56.3  using Ar. 0.56.3 .S1 Fig(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


D parameters well explained the recorded values for haplotype diversity and average γst. We concluded that current intraspecific gene diversities in crabs inhabiting mangrove forests were affected by population fecundity as well as past demographic history. The results were also discussed in terms of management and conservation of fauna in the Western Indian Ocean mangroves.The spatial distribution and the amount of intraspecific genetic variation of marine organisms are strongly influenced by many biotic and abiotic factors. Comparing biological and genetic data characterizing species living in the same habitat can help to elucidate the processes driving these variation patterns. Here, we present a comparative multispecies population genetic study on seven mangrove crabs co-occurring in the West Indian Ocean characterized by planktotrophic larvae with similar pelagic larval duration. Our main aim was to investigate whether a suite of biological, behavioural and ecological traits could affect genetic diversities of the study species in combination with historical demographic parameters. As possible current explanatory factors, we used the intertidal micro-habitat colonised by adult populations, various parameters of individual and population fecundity, and the timing of larval release. As the genetic marker, we used partial sequences of cytochrome oxidase subunit I gene. Genetic and ecological data were collected by the authors and/or gathered from primary literature. Permutational multiple regression models and ANOVA tests showed that species density and their reproductive output in combination with historical demographic parameters could explain the intraspecific genetic variation indexes across the seven species. In particular, species producing consistently less eggs per spawning event showed higher values of haplotype diversity. Moreover, Tajima’s  The genetic structure of populations in the marine realm is a complex process affected by both biotic and abiotic factors including biological, ecological, and behavioural traits of adults and larval stages , 2. DeveHowever, there are many striking exceptions to the above rule. Molecular tools have recently been used to show that the existence of a long pelagic larval phase is often not sufficient to result in genetically homogeneous populations , 4. A grA useful approach for identifying the historical and current factors that shape the spatial distribution and the degree of genetic variation may come from the comparison of closely related and sympatric species with similar biology sharing the same ecosystem , 18. Defen-masse spawning peaks around the equinoctial spring tides [Scylla, ovigerous females migrate to the shelf where they release their larvae into open oceanic waters, thereby favouring larval export [We postulate the above approach could greatly help us to bridge the knowledge gap regarding the possible factors that shape the degree of intraspecific gene diversity and the population genetic structure in mangrove crabs. Mangroves are tropical and subtropical intertidal forests that colonise protected coastlines, such as creeks, lagoons, and estuaries . They ofng tides . In somel export .In this paper, we present a comparative multispecies study on the intraspecific gene diversities and population genetic structures of seven mangrove crabs co-occurring along the East African coast, characterized by similar PLD (approximately 1 month) but substantially different reproductive patterns. The main aim of this study was to investigate whether a suite of biological, behavioural, and ecological traits could affect the degree of genetic diversity within and among populations of the study species, ultimately affecting their demographic connectivity leading to different scenarios in terms of population genetic structures. As possible explanatory factors, we looked at the intertidal micro-habitat colonised by adult populations, and used various parameters of the individual and population fecundity and timing of the larval release. As a genetic marker, we sequenced the partial region of the cytochrome oxidase subunit I (COI). Genetic analyses were used to trace historical events in the West Indian Ocean that affected gene diversity and to unravel the conservation status of East African mangroves.Uca inversa, Uca hesperiae, Uca occidentalis, Perisesarma guttatum, Neosarmatium africanum, Scylla serrata, and Cardisoma carnifex. They belong to four different Brachyuran families and are all representative species of Western Indian Ocean mangrove fauna. Due to their distinct distribution patterns, not all of these species could be sampled at each location  are common along the whole western Indian Ocean [S. serrata and C. carnifex extend to the whole Pacific region [We selected seven mangrove crab species: location . In partr Island –36, and an Ocean , 37; whic region –40.U. inversa, U. hesperiae and C. carnifex were obtained directly, which formed the original dataset of this paper . The genetic data of the other four species were totally or partially gathered from previous studies in the literature and supplemented with our collected data as detailed below and in U. inversa from six populations distributed from Egypt to Mozambique, 125 individuals of U. hesperiae from six populations distributed from Kenya to Mozambique, and 75 individuals of C. carnifex from five populations distributed from Kenya to Mozambique and Tanzania including Seychelles.Genetic data for U. occidentalis (= U. annulipes) [The population genetic structure of nulipes)  was assenulipes)  with newnulipes)  came fronulipes)  that werPerisesarma guttatum included all the sequences published by Silva and co-workers [P. guttatum covered a geographical gradient from Kenya to Mozambique, including six study areas each with three to five single point locations at close range (i.e. about 50 Km). These populations were merged to form six groups in accordance with our sampling design , muscle tissue was removed from one pereiopod. The tissue was immediately placed in ethanol and the specimen was released into their natural environment. The genomic DNA was extracted using the Puregene Kit (Gentra System) and resuspended in TE buffer or distilled water and stored at -20°C.Sure Clean (Bioline) and then resuspended in water. The sequence reactions were performed with the Big Dye terminator mix  followed by electrophoresis in an ABI Prism automated sequencer . The sequences were corrected manually with Flinch TV 1.4.0 (Geospiza®) and aligned by eye with Bioedit version 7.2.5 [A fragment of the cytochrome oxidase subunit I (COI), corresponding to the barcoding region and consisting of 656 base pairs (bp), was amplified using polymerase chain reaction (PCR) with the following primers: COL6b 5’-acaaatcataaagatatygg-3’  and HCO2on 7.2.5 . Due to h), which indicates the probability that two randomly chosen haplotypes are different in a population [θπ), which indicates the percentage mean number of differences between all pairs of haplotypes in a population [θw:) [θπ > θw indicating a recent population growth and θπ < θw indicating a population bottleneck [Genetic heterogeneity within each population and in the overall population was estimated for each species. The haplotype diversity   based onon [θw:) . The comttleneck .http://www.fluxus-engineering.com/sharenet.htm) to investigate the geographic distribution of haplotypes.A minimum spanning network was built for each species using NETWORK version 4.5.0.1   as impleγst) [γst values were calculated using DnaSP and their significance was tested using Snn statistics [For each species we also calculated the Gammast (γst)  as proxyatistics .χ2 tables, without pooling rare haplotypes, using a Monte Carlo simulation [χ2 values obtained from 20,000 random permutations of the original data.The spatial distribution of haplotypes among populations was also assessed by applying Exact tests  as implemulation  as implemulation . Probabirg [rg significance was tested by a parametric bootstrap approach  under the null hypothesis of population expansion as implement in ARLEQUIN.The historical demographic history of our species was reconstructed applying the mismatch distribution analysis  as implemented in ARLEQUIN. Populations at the equilibrium are expected to show a multimodal distribution of haplotype frequencies, while populations having recently passed through a demographic expansion or a range expansion are predicted to have a unimodal distribution . From mirg , 65, whiτ) under both a demographic or spatial expansion hypothesis by using a generalized non-linear least squares approach [τ/2μ, where μ is the mutation rate per site per year. In our case μ is assumed to be 1.15% per million years for the portunid species S. serrata as calculated by [We also estimated the expansion parameters Tau  was calculated as the volume of a sphere. The eggs were then separated from the pleopods with diluted bleach. They were placed between two transparent sheets and photographed using a Canon EOS digital camera at a distance of 40 cm with a 50 mm 2.8 lens. Egg counting at each embryonic stage in each population for each species was carried out using Image J software after adjustments and calibrations. An error estimation was performed by manually counting the eggs in 10% of all the photographs and comparing with the counts given by the software. A subsample of 50 specimens of each of the tested species was randomly selected to record their carapace width (CW) using Vernier calipers. In addition, CW measurements were performed in 20 specimens of C. carnifex and 50 of S. serrata.For each species, information on taxonomic family, distribution range, preferred mangrove habitat, number of spawning events per year, zone of spawning, and PLD were gathered from the literature. The average density of various East African populations of the selected species was also collected from various references, thus to calculate the average number of eggs produced by each population per unit square. The average number of eggs carried by females of s et al. . We coll2, we calculated the average amount of eggs per m2 for each population, the amount of eggs produced per year by each female, and the amount of eggs produced per year per m2. The above estimates per surface area were corrected by a factor based on the average percentage coverage of the vegetation belts particular to each species in the East African mangrove forests  used as the numerical dependent variables. Average γst values were chosen as a proxy of population subdivision, since they give a more reliable interpretation of genetic differentiation. This parameter actually represents an unbiased estimate of Fst that corrects for errors associated with incomplete sampling of populations and is more suitable for mitochondrial haplotype data [U. occidentalis, for an in-depth discussion about the biotic causes of such diversity , which held a Research Permit issued by the Kenya Marine and Fisheries Research Institute (KMFRI).U. occidentalis that only had 18 haplotypes recorded in more than 400 individuals  since all haplotypes only differed by very few mutations , except in ividuals ; Fig 2. han 0.5% ; Fig 2. utations ; Fig 2.Current and historical genetic diversity values for each population group of the seven studied mangrove crabs are shown in Fst/ Φst comparison results showed no significant differences for U. hesperiae, P. guttatum, N. africanum, S. serrata, and C. carnifex. For U. inversa, the pairwise population comparison results showed significant differences between Mida and Lamu and between Lamu and Ras Dege based on haplotype and nucleotide diversity indexes, as well as between Mida and Inhaca based on only nucleotide diversity values (data not shown). For U. occidentalis, we found the populations of Punta Rosa had differences compared to most other populations sampled by Silva et al. [The AMOVA tests revealed a lack of partitioning of the genetic variation in all the studied species . The paia et al.  and the γst, Exact test of population differentiation and contingency χ2 tables of haplotype frequencies) revealed the occurrence of population structures for some species. This was recorded for N. africanum and S. serrata based on γst values  and U. inversa  based on the Exact test of population differentiation; and for S. serrata  from the contingency χ2 tables of haplotype frequencies.Conversely, the other statistical tests applied to analyze global genetic differentiation among populations  and were differentiated from the more widespread haplotypes by very few mutational steps. The networks of the other three species  were more complex and articulated, especially those of the two sesarmid species that in fact had the highest genetic diversity values; however, no evident association between haplotypes and geography occurred in these species too.The network of phylogeographic relationships among all haplotypes was reconstructed for each species . U. inveThe mismatch distributions calculated for the whole population in each of the seven species had a unimodal distribution , as woulrg, it was not possible to reject the null hypothesis of populations being out of equilibrium in any of the studied species except for C. carnifex . Also within the genus Uca, we found consistent differences in populations of U. hesperiae, U. inversa, and U. occidentalis who produced about 22,400, 28,000 and 81,648 eggs m-2 year-1, respectively. Nevertheless, the PLD of these species was roughly the same  confirming that as big a crab species is as it produces more eggs [-2 at each spawning event was negatively correlated with the haplotype diversity, while the species average density and Tajima’s D parameter were positively correlated to haplotype diversity. Overall, these variables accounted for 99% of its variability  are strongly, and inversely, related to haplotype diversity, accounting for 99% of its variability, , while the species of the other two groups had intermediate haplotype diversity values  or very low diversity . Although less prominent than the haplotype diversity, differences in the nucleotide diversity were also observed, but these only closely matched the above groupings based on reproductive outputs and haplotype diversity. The two sesarmid species, P. guttatum and N. africanum, had the highest nucleotide diversity (about 0.5%); U. inversa, U. hesperiae, S. serrata, and C. carnifex had intermediate diversity (about 0.2%); and U. occidentalis had impressively low diversity (0.03%), which was an order of magnitude lower than the other species.The target species in the present study are representative components of mangrove fauna in the Western Indian Ocean as they are abundant in most mangrove swamps in this region , 92. AllS. serrata was far greater than that of the other species, which could be explained by their physical dimensions and high quality diet. In fact, S. serrata are the largest mangrove crab species in the Indian Ocean and are the top predator feeding on molluscs, crabs, and fish [C. carnifex, P. guttatum, and N. africanum are mainly herbivores [Uca species are filter feeders who eat microalgae and bacteria [S. serrata, the dimension of C. carnifex could explain their high reproductive output, as they have large adult dimensions at around five times the size of the other species. U. occidentalis, the smallest crab species in the study, displayed a combination of high reproductive output (about a third more than the other Uca species) and high population densities (about two to 20 times more than the other Uca species and Sesarmidae), which could explain its large annual egg production per unit area.From a reproductive point of view, the impressive egg production capacity of and fish , 99. In rbivores , 100 andbacteria , 101. SiN. africanum, S. serrata, U. inversa and U. occidentalis based on the population pairwise Fst comparisons, γst, χ2 tables, or Exact test results. These results might indicate the existence of differential dispersal abilities among the seven species, which was also suggested by the diverse shapes of the haplotype networks. However, such differences could have been masked by recent historical demographic events and exchange of individuals based on a stepping stone population model [Regarding population genetic structure, high levels of gene flow were recorded for all the species. However, here was some evidence of weak population structures that emerged for on model .D, Fu’s Fs and R2 tests. Based on the demographic parameters of the mismatch distribution analysis under a demographic or spatial expansion model, we calculated the times at which Western Indian Ocean metapopulations of these species could have expanded. These historical events appeared to have occurred at different times for these diverse species, but all were during the Pleistocene period. This epoch could be significant because the Western Indian Ocean has been impacted by great climatic changes, primarily induced by the closing of the Indonesia Gateway around 3–4 Mya [All the species showed clear signs of recent population bottlenecks or selection events, as demonstrated by mismatch distribution patterns and the significant values from Tajima’s  3–4 Mya , 104 and 3–4 Mya .U. occidentalis was a peculiar case, because it had low haplotype and nucleotide diversity values rarely associated with broadcast spawners [Within our studies species spawners  and thisspawners , 108. Onspawners , and genspawners . This cospawners . This hyU. hesperiae, U. inversa, P. guttatum and N. africanum, exhibited higher haplotype diversity and γst values. This correlation can be possibly explained by the hypothesis that broadcast spawners with similar PLD would have a higher chance of dispersing their larvae over larger distances to find suitable mangrove habitats if the numbers of produced larvae were higher. In fact, it is well known that dispersion and recruitment of mangrove crab larvae are related to stochastic events such as offshore wind stress and to deterministic effects such as tidal cycles [U. hesperiae, U. inversa, P. guttatum and N. africanum, the surviving subset should be generally quite heterogeneous, resulting in a constant increase of the haplotype diversity over time. On the other hand, when few females are contributing to large numbers of larvae per capita such as S. serrata and C. carnifex, at every spawning event the larval pool should be genetically homogenous, and should be independent from stochastic recruitment and even from the next generation of adults.Overall, our data suggested that species characterized by lower reproductive output such as l cycles , 111. Thl cycles . Speciesl cycles . Hence, Finally, our findings indicated that distributional range, taxonomic families, temporal and spatial spawning patterns, and the intertidal level occupied by adult populations did not influence the genetic variability index and gene flow estimation. We cannot completely disregard the effects of these factors, which might not have been detected by our analyses due to the limited number of study species and the use of mtDNA alone. Over the past four decades, mtDNA has been the most common marker in population genetic and phylogeographic studies, mainly for its supposed low recombination and effective neutrality , 4. Receθπ< θw). This is to be expected for populations recently overexploited and drastically reduced in size. S. serrata distributed throughout the East African coast is a commercially important species, which is consumed by the local population and sold to tourists, and are thus very likely to be overexploited [This study sheds new light on the population ecology and dynamics of mangrove crabs, and demonstrates how reproductive output is a crucial biological trait affecting genetic diversity and population structure, even in combination with signatures of recent past events. Apart from the ecological relevance, our findings are important for developing conservation strategies for the management of Western Indian Ocean mangroves . Mangrovxploited . On the xploited , 92, 93.S1 TableFor each species, the number of sequences analyzed in each locality is reported. Population groups correspond to localities as used in the statistical analyses. Sequences were obtained for this paper and from literature (see details in the text).(DOCX)Click here for additional data file.S2 Tableγst and values were used as the numerical dependent variable Click here for additional data file.S3 Table2 parameters, the number of variables involved (No. vars) and the selection of them (Selections). Variable legend: 1, Number of spawning events year -1; 2, Maximum adult dimensions; 3, average population density; 5, amount of eggs m -2: 6, amount of eggs female-1 year-1; 7, amount of eggs year -1 m -2; 8 Larval development duration (in days) and 9, Tajima D.For each model, data shown are the values of AIC and R(DOCX)Click here for additional data file.


Lymnaea stagnalis, using a set of 14 populations from contrasted environments in terms of pesticide and other anthropogenic pressures. The hypothesis of population adaptive divergence was tested on 11 life-history traits, using QST -FST comparisons. Despite strong neutral differentiation (mean FST = 0.291), five adult traits or parameters were found to be under divergent selection. Conversely, two early expressed traits showed a pattern consistent with uniform selection or trait canalization, and four adult traits appeared to evolve neutrally. Divergent selection patterns were mostly consistent with a habitat effect, opposing pond to ditch and channel populations. Comparatively, pesticide and other human pressures had little correspondence with evolutionary patterns, despite hatching rate impairment associated with global anthropogenic pressure. Globally, analyses revealed high genetic variation both at neutral markers and fitness-related traits in a species used as model in ecotoxicology, providing empirical support for the need to account for genetic and evolutionary components of population response in ecological risk assessment.Repeated pesticide contaminations of lentic freshwater systems located within agricultural landscapes may affect population evolution in non-target organisms, especially in species with a fully aquatic life cycle and low dispersal ability. The issue of evolutionary impact of pollutants is therefore conceptually important for ecotoxicologists. The impact of historical exposure to pesticides on genetic divergence was investigated in the freshwater gastropod  Understanding the causes of genetic divergence within species is a central goal in evolutionary biology. Population evolution is also a question of importance in environmental sciences and conservation biology, and, as a consequence, evolutionary principles are increasingly taken into account in the management of biodiversity and ecosystems 3/year in 2006) th century Agriculture is probably one of the main causes of human-induced microevolution by selection, as a direct consequence of genetic resource management, e.g., domestication, selection programs on productive species, or side effects of intensive agricultural practices e.g., molluscs, crustaceans) or opportunities  With respect to human-induced stress, some conditions are expected to increase the risk of genetic change in natural populations. For instance, freshwater lentic habitats located within agricultural landscapes are repeatedly contaminated by complex pesticide mixtures, through various modes of transfer from the treated parcels  or on the contrary, trigger similar adaptive responses among populations .QST-FST approach FST index, and its quantitative analogue QST, respectively. Under a purely additive determinism, if quantitative traits evolve neutrally, they should lead to similar levels of differentiation as those inferred from neutral markers (QST  =  FST). Basically, QST and FST are compared to test the null hypothesis of neutrality. Differences between the two indices indicate selection on the traits, with two possibilities: homogenizing (uniform stabilizing) selection towards a unique fitness optimum is revealed by FST>QST, whereas divergent selection (occurrence of different fitness optima among populations) is reflected by the reverse relationship. This principle has been widely used in the last decade to address population adaptive divergence . Due to its habitat characteristics  and its limited dispersal ability, this species is a good model to test non-intentional evolutionary effects of pesticides. Moreover, L. stagnalis belongs to an ecologically important group, which plays a major role in the transfer of energy across food webs, and can represent up to 20–60% of the total biomass of macro-invertebrates in some freshwater ecosystems In the present study, we applied the ImageJ (http://rsbweb.nih.gov/ij/). When available, pesticide concentration data in surface water near the sampled sites were found to be globally consistent with our classification . Finally, populations were also classified according to four criteria: habitat , pesticide pressure , other anthropogenic pressure , and global environmental pressure, as a combination of pesticide and other anthropogenic pressures (GEP: three levels).Adult individuals were collected from 14 natural populations during reproduction, in the course of a single campaign (5–12 July 2011) . SamplinDNA was chelex extracted from haemolymph or foot tissue from 399 wild-caught adults (14 to 33 snails per population). Neutral genetic variation was assessed at 12 microsatellite loci, i.e., A2, A112, B117 N), allelic richness (RA), expected heterozygosity HE, and observed heterozygosity HO, were calculated with Genetix 4.05.2 F indices stat 2.9.3.2 enepop 4.0.10 L. stagnalis is a self-fertile hermaphroditic organism, the selfing rate was estimated per population and statistically compared to zero using Rmesolony 2.0.3.0 Mean allele number  with a permutation test using Fstat.The effect of environmental factors PP, OAP, GEP, H, and genetic cluster was assessed on population genetics parameters . For a given snail, reproduction was followed during 14 days after the first clutch laid in the laboratory. A total of 228 snails over 399 reproduced after 3 months (57%). Families were characterized at various life-history traits , measured on the laboratory-born progeny (G1) as illustrated on 1 snails were reared at 20±1°C, under a 14L/10D photoperiod.Wild-caught adults were brought to the laboratory and reared under standard conditions at the INRA Experimental Unit U3E , as previously described 1 snails were reared as groups reflecting clutch origin . As individuals grew, rearing conditions were adjusted in terms of water volume, individual density, and food supply (see From hatching to the age of 119 days (roughly corresponding to the end of juvenile growth), Gpply see . Vessels63, and a digital calliper afterwards. From the age of 119 days, three G1 snails per family (n = 492) were individually followed for growth and reproduction. For these 492 snails, size was measured at the age of 177.71±5.41 days, 191.71±5.41 days, 205.7±5.40 days, and 226.51±5.56 days , leading to a total of 12 values per individual. Growth was modelled using the Gompertz's model:tL is the shell height at time t, A the asymptotic shell length, b a scaling factor related to shell height at t = 0, and k reflects the growth rate. Using a preliminary subset of 20 individuals, this model performed better than the von Bertalanffy model (lower AIC), and was thus preferred for further growth analysis. Growth parameters were compared among families and populations, as well as size at hatching and size at 119 days.From hatching to the age of 56 days, individual size (as inferred from shell height) was measured every two weeks on four randomly chosen juveniles per group. From the age of 63 days, all reared snails were individually measured. Measurements were performed with a stereomicroscope fitted with an ocular micrometer until dayOnce clutches started to be observed in all aquaria, the 492 snails followed for growth were isolated in 200 mL plastic vessels and fed weekly with 1 g of organic salad immediately after water renewal. Several female reproductive traits were measured: time to first oviposition under isolation (with a censoring limit of 30 days), ability to lay eggs (as the proportion of reproductive snails per family), number of clutches and eggs laid during two weeks, clutch size (number of eggs per clutch), and clutch hatching rate.lme4) Y ∼ factor1 + factor2 + factor3 + covariate1 + covariate2 +…+ (1|population/family).All traits were analyzed with generalized linear mixed effect models , habitat, and environmental pressure . Family was nested within population, and both were treated as random factors. Global tests were followed by post-hoc pairwise comparison tests . All statistical analyses were performed using R 2.14.0 (R Core Team 2012).Fixed effects were tested by model comparison using a log-likelihood ratio test. Fixed factors were: genetic cluster  Y∼1+ (1| pop/fam)).Where FST, and for sampling error and variation in evolutionary history in QST estimation. The method has lower type 1 error rate and better statistical power than previous tests, and seems particularly suited when neutral differentiation is high L. stagnalisQST - FST to the QST - FST distribution expected under the neutral hypothesis, as derived from FST distribution and variance components QST neutral distribution predicted from the Lewontin-Krakauer distribution. Observed values were considered significant when they fell outside the 95% confidence interval of the neutral distribution Population quantitative divergence was tested using the method of Whitlock and Guillaume FIS-values varied widely across loci and samples, ranging from −0.238 (EMSL41 in BUX) to 1.000  . The selfing rate was significantly different from zero in three populations . Effective population size Ne was significantly larger in sites exposed to high pesticide pressure .On 399 individual genotypes, 44 were discarded because of missing or unreadable data at more than three loci. Overall, the observed number of alleles per locus varied from three to 29, mean allelic richness ranged from 1.1 to 9.6 , and gen in BAA) . Genetic in BAA) . All popSTF-value  = 0.291, 95% CI ), as also reflected by pairwise estimates, which were all significant . Population differentiation was significantly affected by global environmental pressure  with a lower value among populations exposed to the highest level of pressure. With regard to specific pressures, lower differentiation was associated with pesticide as well as with other anthropogenic pressures . Habitat affected all tested parameters, when ponds were compared to ditch and channel populations: pond populations were significantly less variable , more inbred  and much more differentiated than were other populations (FST(pond) = 0.502 vs. FST(ditch + channel) = 0.227, P = 0.002).Population genetic parameters were affected by most environmental factors . Genetic1 traits showed significant heterogeneity at both population and family levels  and hatching rate (significantly lower under OAP1) . Third, global pressure affected b (significantly larger snails at t0 under GEP2), and hatching rate, which was significantly impaired in exposed populations (GEP1 and GEP2) relative to reference ones (GEP0).Compared to aquatic system, human pressure appeared much less effective. First, pesticide pressure had no effect, except a marginal one on ability to lay eggs, found greater in exposed populations (P = 0.069). Second, other anthropogenic pressures affected growth parameter b (significantly larger size at ter OAP1) . InteresHE . Second, a negative correlation was found between population inbreeding and G1 fecundity , whereas homogenizing selection was indicated for two traits , and divergent selection for five traits .Results are summarized on L. stagnalis was addressed under a common garden experiment involving life history traits and a set of natural populations from contrasted environments. Five traits or parameters over 11 were found under divergent selection, despite a strong neutral genetic structure. The occurrence of two genetic clusters (Eastern and Western populations) had only a weak influence on quantitative genetic divergence, suggesting that population level was the adequate scale to draw selection inferences b) were affected by this factor (larger hatchlings to the East). Given that early size was found under uniform selection, it was assumed that the genetic divergence associated to geography did not interfere with selection putatively associated with local environmental pressures.The hypothesis that human activities may induce population adaptive divergence in A) and to reproduction . Conversely, uniform selection was the most likely evolutionary hypothesis for early survival and size . Globally, inferred patterns, i.e., early (late) traits under homogenizing (divergent) selection, are in lines with those found in another freshwater snail All traits found to diverge adaptively were adult traits, related to late expressed growth , habitat was the only factor affecting most traits found under divergent selection. Indeed, pond populations were characterized by larger ultimate size (growth parameter A), lower ability to lay eggs, and longer time to oviposition after isolation, compared to ditch and channel ones.Traditional statistical analyses performed on the GL. stagnalis low dispersion and weak habitat connectivity, pond populations were particularly differentiated  and presented generally lower genetic diversity and higher inbreeding than channel and ditch populations. Moreover, compared to channel populations, pond and ditch populations were also more inbred (P = 0.009). These aquatic systems are likely to imply stronger population isolation, even among ditches (due to frequent drought). A similar influence of habitat was emphasized in another freshwater snail, Physa acutaL. stagnalis, although divergent selection was detected for traits also affected by habitat type, genetic drift is likely to highly contribute to divergence among habitats. This result is in line with the detection of significant drift load in experimental populations occupying outdoor close mesocosms Congruent with Rana temporaria, habitat fragmentation correlates with low genetic diversity and high differentiation, and negatively impacts tadpole body size L. stagnalis, although ultimate body size was greater in ponds, the relation between size and fitness is not necessarily positive, because of possible energetic trade-offs between growth and reproduction In the common frog QST-FST based test concluded to neutral divergence for size at hatching, growth rate (parameter k), and two reproductive traits, clutch size and early fecundity (number of eggs laid during 14 days following first clutch since isolation). On the latter, it seems surprising to find no hint of selection, either uniform or divergent, although this may simply reflect low heritability of fitness traits L. stagnalis , suggesting the possibility of high variation without drastic fitness consequences. Consistently, population growth rate was found to be insensitive to fecundity under experimental conditions . G1 families correspond hence to the hermaphroditic equivalent of isofemale lines, as used for broad-sense heritability estimation QST estimates are potentially biased by genetic non additive sources of variation , and by maternal effects. The latter are likely to operate early in life and tend to recede with age QST-FST comparisons As reported above, homogenizing selection was indicated for early traits, which is expected to reflect the occurrence of a uniform fitness optimum between the studied populations. Galba truncatulaL. stagnalis population phenotypic convergence.Alternatively to the uniform selection hypothesis, the pattern exhibited by early traits would result from trait canalization, as a phylogenetically inherited characteristics 1 fecundity, is consistent with previous findings in L. stagnalisQST is modified by the expected stochastic evolution of slightly deleterious alleles as compared to pure selection-based predictions . Inbreeding depression estimated under selfing relative to random outcrossing (ID) is particularly low in L. stagnalis1 fecundity.Furthermore, the negative correlation observed between population inbreeding and several traits related to fecundity  is in line with the occurrence of reduced fitness in inbred populations, and thus of inbreeding depression in these populations. However, as inbreeding was found significant in most populations and because effective population size was usually small, it is suggested that random genetic drift is actually the main cause of these apparent correlations, through reduced genetic diversity and drift load accumulation caused by population isolation and small size L. stagnalis exposed to cocktails of pesticides Neutral genetic variation was inflated in populations exposed to anthropogenic pressures, including pesticides. This was reflected in terms of genetic diversity, allelic richness, and effective population size, and through lower genetic differentiation. These results are opposed to those found in experimental populations of QST-FST comparisons Besides the hypothesis of pesticide innocuousness to gastropod populations, one explanation may be that sampling did not reflect the effective magnitude of pesticide pressure. However, as the design included areas of intensive agriculture, and due to the close proximity of water bodies and treated agricultural parcels, this seems unrealistic. Alternatively, evolutionary patterns associated with pesticide exposure may have been masked by other environmental characteristics, an idea supported by the significant habitat effect observed on most studied traits. Thus, although the number of populations and families was close to the recommended values 1 from polluted areas, might reflect a specific response of exposed populations to overcome early mortality induced by chemicals (hatching rate: OAP0>OAP1 in G1 and G2), which was unexpected to express under common garden conditions. In the context of evolution under stressful environments, trait expression would be best studied under a gradient of stress conditions, as also recommended for molecular traits and pathways QST has been shown to depend on the experimental environment Third, the apparent lack of effect of pesticide pressure on the studied traits might also result from experimental conditions, which were too benign. For example, the trend for enhanced ability to reproduce observed in the GFinally, the present study also emphasized the occurrence of high genetic variation both at neutral markers and fitness-related traits in a species used as model in ecotoxicology. This finding provides empirical support for the need to account for genetic variation in toxicity testing and, as a perspective, in future procedures of ecological risk assessment Figure S1L. stagnalis1 snails) according to population habitat. individual growth Click here for additional data file.Figure S2L. stagnalis hatching rate as a function of anthropogenic pressure .(TIFF)Click here for additional data file.Table S1L. stagnalis G1 rearing conditions as a function of age.Evolution of (DOCX)Click here for additional data file.Table S2L. stagnalis life history traits.Summary of statistical analyses performed on (DOCX)Click here for additional data file.Table S3L. stagnalis population samples .Population genetic parameters calculated per locus and population on 14 (DOCX)Click here for additional data file.Table S4L. stagnalis populations, on the basis of 12 SSR loci.Population genetic differentiation estimated among 14 (DOCX)Click here for additional data file.Table S51 from 14 L. stagnalis populations used to test patterns of adaptive divergence .Life-history trait/parameter mean-values (SE) estimated in the laboratory-born G(DOCX)Click here for additional data file.