Acer saccharum subsp. saccharum) and its only subspecies in tropical America (Acer saccharum subsp. skutchii) using nuclear and chloroplast data. The studied populations span a geographic range from Maine, USA (46°N), to El Progreso, Guatemala (15°N). We examined genetic subdivisions, explored the locations of ancestral haplotypes, analyzed genetic data to explore the presence of a single or multiple glacial refugia, and tested whether genetic lineages are temporally consistent with a Pleistocene or older divergence.Geological events in the latter Cenozoic have influenced the distribution, abundance and genetic structure of tree populations in temperate and tropical North America. The biogeographical history of temperate vegetation that spans large ranges of latitude is complex, involving multiple latitudinal shifts that might have occurred via different migration routes. We determined the regional structuring of genetic variation of sugar maple . Splits during the Pleistocene separated the rest of the phylogroups. The most frequent and widespread haplotype occurred in half of the sites . Our data also suggested that multiple Pleistocene refugia , but not western Mexico , contributed to post-glacial northward expansion of ranges. Current southern Mexican and Guatemalan populations have reduced population sizes, genetic bottlenecks and tend toward homozygosity, as indicated using nuclear and chloroplast markers.The divergence of western Mexican populations from the rest of the sugar maples likely resulted from orographic and volcanic barriers to gene flow. Past connectivity among populations in the southeastern USA and eastern Mexico and Guatemala possible occurred through gene flow during the Pleistocene. The time to the most common ancestor values revealed that populations from the Midwest and Northeast USA represented different haplotype lineages, indicating major divergence of haplotypes lineages before the Last Glacial Maximum and suggesting the existence of multiple glacial refugia.The online version of this article (doi:10.1186/s12862-015-0518-7) contains supplementary material, which is available to authorized users. Carya illinoinensis, Liquidambar styraciflua, Fagus grandifolia) include higher diversity in southern, never-glaciated areas, and lower diversity in northern areas where glaciated landmasses existed [Acer rubrum, A. saccharinum, Quercus rubra) that survived in multiple refugia closer to the ice margin [Pinus chiapensis, Picea chihuahuana, Pseudotsuga menziesii, Fagus grandifolia var. mexicana) also show genetic differentiation related to the history of migration and isolation during glacial and interglacial periods [Geological events have affected the distributions and levels of gene flow among populations of North American tree species. Repeated glacial and interglacial periods promoted range shifts of a number of tree species during late Pliocene and the Quaternary –4. Studi existed –11. Furte margin –12. Popu periods –16. Addi periods , 18.Periods of connectivity and disjunction among North American and Central American refugial areas and their consequences on the genetic structure of the temperate tree populations are not known for most species. Limited geographical sampling reduces the capacity to reconstruct historical refugia and identify the spatial location of genetic breaks in temperate tree species at a continental level. Assessing large-scale relationships of populations should elucidate the importance of range expansions and contractions in producing the current distribution patterns of species, as well as generate hypotheses regarding the genetic structure and diversity of temperate tree lineages affected by glaciation in the latter part of the Cenozoic.Acer saccharum (sugar maple) is a widespread temperate tree species. Study of the genetics of this species has the potential to elucidate a late Pliocene and Quaternary history that may be shared by other elements of the North America flora. The species has a continuous distribution from southern Quebec to the southeastern USA, and then is disjunct in distribution between the eastern USA, Mexico, and Guatemala [A. saccharum underwent northward geographic expansions from a single continuous ice-free refugium in the Southern USA about 21,000 years B.P., around the beginning of the retreat of the Laurentide ice sheet [Acer rubrum likely existed north of their Pleistocene pollen-based range limits, but, it is not yet known if the same applies to A. saccharum [Acer saccharum exhibits some genetic differentiation in southeastern Canada and northeastern USA, but less genetic variation than other temperate trees or shrubs [uatemala . Fossil ce sheet . The evice sheet . Nonethece sheet . In addir shrubs . In addir shrubs , 25. TheWe investigated patterns of genetic variation and structure in extant populations of sugar maples in the tropics and explored their genetic relationships with populations in the temperate United States. We used these data to infer their evolutionary history and large-scale connectivity. We quantified within- and among-population genetic variation across the species range using chloroplast sequences and microsatellite loci. We hypothesized that ancestral haplotypes have persisted in tropical populations in unglaciated areas that have changed little over long periods, and that these populations have harbored higher genetic diversity. Next, we estimated divergence times of haplotypes among populations and considered whether observed genetic subdivisions were temporally consistent with the late Pliocene and Quaternary geologic events. Finally, we tested the hypotheses of expanding/contracting populations and the presence of a single refugium versus multiple refugia during episodes of glaciation. Based on our study, we propose the location of more than one continuous ice-age refugium in North America, as well as potential migration routes in relation to the complex phylogeographical patterns of North and Central American hardwood forests.Acer saccharum subsp. saccharum was conducted in old-growth forests of USA. Sites where the species identity was doubted by botanists were not sampled. Sampling in Mexico and Guatemala included all known populations where A. saccharum subsp. skutchii has been recorded [Saccharodendron (Rafinesque) Murray, section Acer, characterized by apetalous flowers and connate calyces. Acer saccharum subsp. skutchii represents the only member of the series distributed in Mexico and Guatemala [Sampling of recorded . These suatemala . Both suuatemala .The sampled populations of sugar maple spanned a geographic range from Maine, USA (46°N), to El Progreso, Guatemala 15°N). Fresh foliage was sampled from 233 adult individuals from 16 native populations . The forward primer was fluorescently labeled using 6-FAM or HEX for MAP10  on the 5- end for detection on an ABI 3130XL .Six microsatellite loci were amplified using primer pairs and polymerase chain reaction (PCR), the protocol described by Pandey et al. . Six outm dNTP, 10 μm of each primer, 1.5 mm MgCl2, and 1 U HotStarTaq polymerase . PCR programs consisted of an initial denaturation at 95 °C for 15 min, followed by 30 cycles of 45 s at 94 °C, 45 s at appropriate annealing temperature, and 45 s at 72 °C, and a final extension of 15 min at 72 °C. Annealing temperatures were determined using a gradient PCR and were as follows: 52 °C (MAP9 and MAP33), 55 °C (MAP10), 55.8 °C (MAP34), 58 °C (MAP40), 48 °C (MAP46). Two microliters of PCR product were added to 10.8 μL of Hi-Di Formamide  and 0.2 μL ROX 400 HD size standard and run on an ABI 3130XL automated sequencer. The allele size calling  was done using GeneMapper v4.1 , and the binning  was done with Autobin [PCR and fragment analysis were carried out as follows. PCR amplifications of 20 μL aliquots were performed using 20 ng of genomic DNA , 0.2 m Autobin . MICRO-C Autobin .ndhF-rpl32R intergenic spacer in the small single-copy region and psbJ-petA intergenic spacer in the large single-copy region from the chloroplast were amplified [et al. [m of each primer, 0.2 mm dNTP, 1.5 mm MgCl2, and 1 U HotStarTaq polymerase . PCR programs consisted of an initial denaturation at 95 °C for 15 min, followed by 35 cycles of 1 min at 95 °C, annealing at 52 °C for 1 min, followed by a ramp of 0.3 °C/s to 65 °C, and 4 min at 65 °C, and a final extension of 10 min at 66 °C. PCR products were purified using the solid phase reversible immobilization technique and sequenced in both directions using BigDye Terminator v3.1  on an ABI-PRISM 3730XL. Sequence chromatograms were visually inspected and edited in Sequencher v4.1 , alignment was done in MUSCLE [http://www.ebi.ac.uk), and manually edited in McClade v4.08 [The mplified . Both remplified . PCR was [et al.  with somn MUSCLE  using thHE) and observed heterozygosity (HO) per locus, as well as the private allelic richness of each population were calculated in the R package “adegenet” [FIS) was estimated for each population, and differences among populations were compared using Mann-Whitney U-tests.Deviations from Hardy-Weinberg Equilibrium (HWE) and linkage disequilibrium were assessed in GENEPOP v4.1 . The tesdegenet” . The inbHE), and on the number of alleles and sample size (HEQ) were compared, based on the assumption that the allele diversity is reduced faster than the heterozygosity. The estimates were calculated under the infinite allele model (IAM) and stepwise-mutation model (SMM) with 1000 iterations, and the allele frequency graphical mode. Significance was tested using the Wilcoxon test with the Bonferroni correction, all implemented in BOTTLENECK v1.2.02 [k) to the range in allele size (r) is calculated (M-ratio), where k is reduced faster than r. Thus, a reduced M-ratio indicates a population having been through a bottleneck. The significance of the ratio was evaluated using the critical value (Mc) under the two-phase mutation model (TPM), with the average size of non one-step mutations = 2.8, as determined by [M-ratio and Mc were estimated using M_P_VAL and Critical_M software, respectively [Each population was evaluated for evidence of population bottlenecks. Tests were done using the approaches described by Cornuet and Luikart  and Garz v1.2.02 . Under t v1.2.02 , the meamined by , and θ vectively .S), number of haplotypes (h), haplotype diversity (Hd), average per site pairwise nucleotide diversity (π), and the relationship between polymorphic sites and alleles sampled (θw). The parameters were estimated using DnaSP v5.10.01 [Genetic variation was evaluated for each population and region. The variation was given by number of polymorphic sites  [Significant genetic structure was determined using two different population-based measures. Pairwise genetic differentiation analysis was performed using FST  and DestST [Dest  with a p2.9.3.2) , where t2.9.3.2) .FST and the RST. Permuted values of RST larger than FST indicate a contribution of stepwise mutations to differentiation among populations. Tests were performed in SPAGeDi v1.3 [RST was used to account for the mutational distances among alleles under the stepwise mutation model [RST is analogous to FST, but it is based on allele size rather than allele identity.Patterns of structure were also assessed by permuting allele sizes among alleles and the calculation of the eDi v1.3  using 10on model . RST is Dest [Dest is determined by mutation and migration rates and number of populations, and it is independent of population size [GST.A genetic differentiation estimate was calculated. The estimator Dest  was implDest . Dest ision size . It is ma priori assumptions of population subdivision, a Bayesian clustering analysis was used. The analysis was run under an admixture model with correlated allele frequencies for 10 million iterations after a burn-in period of 100,000 iterations for each value of K, and 10 replicates per run. Population structure was inferred using a range of K from one to six. The analysis was performed in STRUCTURE v2.3.3 [K based on the likelihood scores. The analysis estimates coancestry coefficients for individuals assigned to each of K populations.To detect the number of genetically differentiated populations without E v2.3.3 . The EvaE v2.3.3  was implE v2.3.3  to estima priori structure parameters, identifies groups that are geographically homogeneous and genetically differentiated. The most likely number of groups (K) was determined using 10,000 iterations and 10 repetitions for K values from two to eight groups. The most likely structure was defined by the maximum value of ΦCT (the proportion of genetic variation among groups of populations) that did not include any groups of a single population [A spatial analysis of molecular variance (SAMOVA) was conducted to identify groups of related populations based on geographical and genetic distances . This appulation . The anapulation .The relationships among chloroplast haplotypes were also assessed using statistical-parsimony networks implemented in R Statistical Software with the package PEGAS , 52.The genetic structure was further analyzed with the Monmonier maximum difference algorithm to confirm the significance of structure recovered with previous metrics. The algorithm was used to locate areas of maximum genetic distance within a geometric network of geo-referenced points constructed with a Delauney triangulation.Dest [ST [ST estimator was computed using SPADS v1.0 [Two different bootstrapped genetic distance matrices were used, one using nuclear microsatellite data, Dest , and othDest [ST . MatriceADS v1.0  and visuADS v1.0  to genernaSP. Outgroup sequences were generated for Dipteronia sinensis, and Acer glabrum var. neomexicanum and downloaded from GenBank for A. buergerianum subsp. ningpoense (KF753631.1). Dipteronia and Acer are sister clades (subfamily Hippocastanoideae) [Acer-Dipteronia) with Jukes-Cantor correction. The resulting estimate  was divided by twice the divergence time estimated for Acer  of parameter estimates and convergence and mixing of runs were assessed using Tracer v1.5 [The relationships among chloroplast haplotypes were assessed with a phylogenetic analysis based on Bayesian inference in BEAST v1.7.5 . The chlnoideae) . The besnoideae) , 58. The14.6 Ma, ) to obtacer v1.5 . The rescer v1.5 . A maximcer v1.5  and examcer v1.5 .a priori hypotheses of Pleistocene population structure tested were the single refugium null model and the multiple glacial refuge model. The gene-tree population-tree approach was applied, following Knowles [Ne) until total coalescence. Five arbitrary Ne were tested . The amount of discordance between the gene trees and the hypothesized populations trees was measured by the s statistic [s value for the haplotype tree was first obtained and then, the distribution of simulated s values for each population tree was analyzed and graphed.Coalescent simulations were used to test whether patterns of population differentiation are consistent with the single or multiple refuge models. The two  Knowles  and impl Knowles . Haplotytatistic . The s vs value for the haplotype tree falls outside of the 95 % confidence interval for that model [The single refugium hypothesis assumes that extant populations of sugar maple are derived from a refugial population, with expansion beginning as the glaciers retreat. The divergence time of populations was estimated to early, middle and most recent Pleistocene glacial events. The null model of fragmentation of a single ancestral population is rejected if the at model .Under the multiple glacial refuge model, the extant population structure of haplotypes results from isolation and divergence within four hypothesized glacial refugia. Glacial refugia were represented by assemblages of populations from: a) West Mexico, b) Midwest USA c) Southeast USA – East Mexico – Guatemala, d) Northeast USA. We tested whether populations a and b were isolated since early Pleistocene  or before ; and a recent split of c and d groups at 6250 generations / 250,000 ybp, or 3500 generations / 140,000 ybp or 500 generations / 20,000 ybp.FIS per population ranged from -0.47 to 0.395. Two populations in Mexico showed a tendency toward increased homozygosity , whereas among the USA sites, only populations from Vermont, Ohio, and Tennessee did not exhibit this homozygosity pattern  and the populations in USA .A total of 17 alleles were detected at the six microsatellite loci. The total number of observed alleles varied from two (MAP40) to seven (MAP34), and allelic richness ranged from 1.5 to 2.1. Private alleles were present in one locus (MAP34) in the Tamaulipas, Chiapas, Jalisco, and Maine populations. Departures from the HWE were found in two populations from western Mexico and four from the USA   in any of the populations and under any models. These results were not consistent, however, with the distribution of allele frequency. Eight populations  displayed a mode shift, indicating that a bottleneck had occurred. In contrast, the normal L-shape distribution of allele frequencies indicated that eight populations had not experienced a recent bottleneck. Under the mutation-drift equilibrium scenario, the rarest allele class was more frequent, forming an L-shape graph [Genetic signatures of population bottlenecks were detected, although there was disagreement between the different tests used. The pe graph . After apsbJ-petA and the ndhF-rpl32R intergenic spacer were 729 and 770 nucleotides long, respectively. The total number of haplotypes in the concatenated sequences was 34, and diversity Hd = 0.8609. The spacer ndhF-rpl32R had a haplotype diversity Hd = 0.667, and nucleotide diversity π = 0.00295 [psbJ-petA had Hd = 0.620 and π = 0.00168.A number of haplotypes were recovered from two chloroplast regions. Chloroplast sequences of the  0.00295 ; and theHd from 0-1). A site in Guatemala had only one haplotype, whereas larger numbers of haplotypes occurred in the southeastern and midwestern USA (Table Haplotype diversity was variable among populations (SA Table . NucleotSA Table .Table 2NFST) values ranged from -0.0703 to 0.5082 indicating differentiation among populations .There was variation in genetic structure across the 16 populations. The pairwise θ  and Chiapas (south) in Mexico , and Chiapas (south)  = -1158.03, inferred that the most likely number of clusters (K) was two . Higher levels of K included single-population groups with only a slight increase in the index  consisted of one group composed by the populations from Guatemala, Mexico , the Southeast USA, and Ohio. A second group included only the populations from western Mexico , and the Northeast and the Midwest USA populations each composed the third and fourth group.The optimal number of groups indicated by SAMOVA was Acer populations was 34. Haplotype “AA” was the most frequent and widespread, occurring in half of the sites   Fig. . The secio) Fig. . “E” andCT = 0.545 P = 0.001).Geographic boundaries detected by the Monmonier algorithm supported the separation of midwestern USA and western Mexican sites. The bootstrap support of the segments ranged from 46–55 % Fig. , whereasBoundaries were also indicated using nuclear data. Strong genetic change occurred among populations in Mexico and USA and between Mexican and Guatemalan populations, suggesting current limits to gene flow (bootstrap support 41–58 %)  received high support (0.98) Fig. . Haplotys statistic from the gene tree was s = 14. The observed value of s = 14 was not within the 95 % confidence interval of the simulated distribution under any of the different times of population splitting and for all Ne values.The single refugium null model was not supported by the data. The Slatkin and Maddison’s s = 14 falls within the 95 % confidence interval  and the midwestern USA had the highest genetic differentiation; their haplotype lineages diverged, respectively, during the Pliocene and early Pleistocene. Further differentiation of northeastern USA haplotype lineages was dated to the pre-Illinoian glacial episode (Pleistocene). Population in the southeastern USA, eastern Mexico, and Guatemala displayed signals of past connectivity and lack genetic differentiation. In addition, most populations in the tropics had increased homozygosity and lower genetic diversity, possible as a result of bottlenecks lasting many generations. Haplotype data suggested the presence of multiple glacial refugia; the tropical populations likely acted as refugia during the Quaternary glacial and interglacial events, while two other refugia were maintained in the Midwest and north of the Appalachian Mountains.Acer saccharum in populations from Canada, possibly a result of genetic drift [Low levels of genetic diversity from nuclear DNA data were observed in all populations. A greater tendency toward homozygosity occurs in maples from western and southern Mexico compared to maples Vermont, Ohio, and Tennessee. The trend could be the result of a genetic bottleneck in southern Mexico. The reduced genetic variation is also present in the Guatemalan populations. The increased homozygosity in southern tropical populations indicates that populations at the most southern ends of the range of this species should be the most vulnerable to new selection pressures. Populations in southern Mexico and Guatemala are also the most threatened by anthropogenic disturbance . Consistic drift –25. Neveic drift . In addiet al. [Chloroplast gene diversity in most maple populations was variable. High levels of nucleotide and haplotype diversity were distributed in the southern and the midwestern regions of the USA. High nucleotide diversity (π) also was also observed in the southern population of Chiapas, Mexico. Nevertheless, populations in tropical regions contained the lowest haplotype diversity. Gunter et al.  found thet al. ; no signet al. . Rates oThe genetic structure recovered with nuclear and chloroplast DNA indicates general patterns of connectivity among populations. Nuclear data suggest current gene flow and larger effective population sizes in southeastern and northeast USA, whereas the opposite is true in the tropical populations, having disjunct and geographically fragmented populations. Chloroplast data indicate that very little gene flow has occurred among the midwestern sites and the rest of the populations in the USA since some distant time in the past.Haplotypes from more southern regions may be considered ancestral, which is suggested by high frequency and distribution of haplotypes in southeastern USA and south Mexico and Guatemala. Haplotype lineages in western Mexico  were the first to diverge, during the Pliocene. Eastern Mexican and Guatemalan lineages had a time for the most common ancestor similar to the northeastern USA populations, dated to the pre-Illinoian glacial period. The haplotypes from the Midwest, supported as a lineage by Bayesian inference, have survived isolated since the early Pleistocene (when ice-sheets began to grow), with possible gene migrations from more central (Ohio) or southern (Alabama) ice-free areas. Samples from Iowa, Missouri, as well as sequences of other chloroplast regions, may help to clarify the routes of migration for the midwestern populations.Dest and FST measures. Bayesian clustering analysis did not differentiate the western Mexico populations, whereas Dest and FST gave strong support for these populations as a different group. Similarly, the population in Chiapas was assigned to the USA cluster with STRUCTURE, but it was differentiated using Dest and FST. The Chiapas population might represent an outlier, possible due to the high levels of homozygosity and reduced population sizes. Caution is needed in making inferences, however, because the accuracy of the assignments in STRUCTURE is affected when there are insufficient microsatellite markers [The results from the STRUCTURE analysis suggested that the genotyped individuals fall into two general clusters. Thus, there is one cluster from the tropics, which also includes Alabama, and one cluster composed of USA sites, but excludes the Southeast USA. These results contrasted with  markers .Dest and FST supported differentiation for nuclear DNA and SAMOVA for chloroplast DNA. Their isolation thus appears to be old and is supported by the time for the most common ancestor estimates for haplotypes in this study. Our data further suggest very limited present gene flow  from Alabama to the populations in the Midwest.Populations in midwestern USA and western Mexico were highly differentiated from the rest of the sites under study. Liquidambar styraciflua in the southeastern USA and Mexico [Fagus grandifolia and Pinus chiapensis in Mexico [The nuclear DNA also indicates lack of gene flow at the present time between the northern Mexico and southern USA populations. Nonetheless, the signal of past connectivity among the Southeast USA, East Mexico, and Guatemalan populations is still present, as indicated by the low chloroplast differentiation among these populations. Similar signals of past connectivity have been identified between populations of d Mexico . Populatn Mexico , 15.Acer saccharum could have become established in western Mexico during the Pliocene or late Miocene, when the temperatures were low. The presence of other temperate tree genera  during the Pliocene in central Mexico is supported by the presence of macrofossils [Liquidambar and Podocarpus matudae [Populations of ofossils . In addi matudae –70.The uplift of the Trans-Mexico Volcanic Belt, a major mountain barrier stretching from west to east across the country, isolated western areas of Mexico in the late Miocene. This mountain belt was characterized by major volcanic activity during the late Pliocene and the Quaternary, mainly along the western side , 72. TheAcer in cloud forests. Even though the Trans-Mexico Volcanic Belt reaches eastern Mexico, it had reduced volcanic activity along its eastern side [Glacial periods during the Pleistocene could have favored species expansions with continuous gene flow through eastern–southern Mexico to Guatemala, which then ended after the last glacial maximum . Pollen from cores of the Basin of Mexico indicates three glacial advances during 30,000–25,000 years B.P., 12,000 years B.P., and during late Holocene , 75. A rern side  and, togPinus and Juglans [Acer during the Pleistocene. Another small maple refugium should have occurred in Ohio, based on the diversity of haplotypes. The Ohio population also shared a larger proportion of the most abundant haplotype with the southeastern and tropical sites, and a smaller proportion of haplotypes from the northeastern site. Maples in the Northeast might have expanded from a refugium in the northern area of the Appalachian Mountains. During the last glaciations, ice-free areas existed in the Pennsylvania region, from where the maples could have migrated following deglaciation [in situ glacial survival of maples [Acer rubrum and Fagus grandifolia could also have persisted in periglacial areas in Ohio, expanding following deglaciation and making a more important contribution to species expansion than other populations in the southeastern USA [Smilax also supports the hypothesis that the Midwest served as a refugium [Small maple refugia may have existed in the Midwest USA and north of the Appalachian Mountains. A population in the Midwest might have persisted through the glaciations from the beginning of Pleistocene, experienced possible gene flow from the sites in the south (Alabama), but remained isolated from the more eastern sites . The Mississippi River Valley is hypothesized to have increased genetic differentiation to the east and the west of the river in  Juglans , and theaciation . The lowf maples , 78, 79.f maples . On the f maples . This batern USA , 12. Thutern USA . Our restern USA . Recent refugium . Moreoverefugium , have alHabitat fragmentation may have resulted from increased dry and warm conditions that followed the last glacial maximum. In addition, anthropogenic habitat transformation has affected the cloud forests containing sugar maples in Mexico and Guatemala . ReducedReduced genetic variation currently is present in the most southern tropical maple populations, as indicated by nuclear and chloroplast data. Haplotype diversity and distribution indicate past connectivity among populations from the Southeast USA, East Mexico, and Guatemala. Thus, gene flow and species expansion through eastern–southern Mexico to Guatemala was likely favored during glacial periods of stable humidity during the Quaternary. Earlier, western Mexican  populations diverged from the rest of the sugar maples during the Pliocene. Volcanic activity during late Pliocene and Quaternary and topographic conditions in western Mexico probably promoted this early differentiation. The time for the most common ancestor estimates denote that midwestern USA populations have been different lineages since the early Pleistocene and that northeastern USA lineages diverged during the pre-Illinoian glaciation, long before the Last Glacial Maximum. Small maple refugia may have existed in the midwestern USA and north of the Appalachian Mountains, and expansion may have occurred from such refuges during interglacial periods. Thus, we suggest that geological events in the Pliocene were major determinants of the current genetic structure of sugar maple populations in the area of West Mexico, whereas the geological events that occurred during the Quaternary explain the genetic structure of sugar maples in East Mexico and the USA. This study supports the notion of multiple glacial refugia for temperate hardwood forests in North and Central America and highlights the importance of connectivity among temperate forests in the USA and those in Central America.The chloroplast DNA data set supporting the results of this article is available in GenBank, with the accession numbers KT933356–KT933397.


Colletotrichum fructicola is found in all five continents and is capable of causing severe diseases in a number of economically important plants such as avocado, fig, cocoa, pear, and tea-oil trees. However, almost nothing is known about its patterns of genetic variation and epidemiology on any of its host plant species. Here we analyzed 167 isolates of C. fructicola obtained from the leaves of tea-oil tree Camellia oleifera at 15 plantations in seven Chinese provinces. Multilocus sequence typing was conducted for all isolates based on DNA sequences at fragments of four genes: the internal transcribed spacers of the nuclear ribosomal RNA gene cluster (539 bp), calmodulin (633 bp), glutamine synthetase (711 bp), and glyceraldehyde-3-phosphate dehydrogenase (190 bp), yielding 3.52%, 0.63%, 8.44%, and 7.89% of single nucleotide polymorphic sites and resulting in 15, 5, 12 and 11 alleles respectively at the four gene fragments in the total sample. The combined allelic information from all four loci identified 53 multilocus genotypes with the most frequent represented by 21 isolates distributed in eight tea-oil plantations in three provinces, consistent with long-distance clonal dispersal. However, despite evidence for clonal dispersal, statistically significant genetic differentiation among geographic populations was detected. In addition, while no evidence of recombination was found within any of the four gene fragments, signatures of recombination were found among the four gene fragments in most geographic populations, consistent with sexual mating of this species in nature. Our study provides the first insights into the population genetics and epidemiology of the important plant fungal pathogen C. fructicola.The filamentous fungus  Domestication of natural ecosystems and the expanding trade of goods and services have accelerated the emergence and spread of pathogens ,2. In agColletotrichum contains over 60 recognized species and likely many more cryptic (still to be described) species [Colletotrichum gloeosporioides species complex (CGSC) [The ascomycete fungal genus  species –7. The g species ,7,8. Spe species . In the x (CGSC) . The mosx (CGSC) ,12.Colletotrichum fructicola is a recently described species within CGSC [C. fructicola is very similar to or indistinguishable from others within CGSC and molecular markers are needed in order to separate it from other closely related species [C. fructicola has been found to cause diseases in many plants, including fruit rot in Persea americana and Coffea arabica as well as leaf anthracnose in Malus domestica, Fragaria × ananassa, Ficus edulis, Limonium sinuatum, Pyrus pyrifolia, Dioscorea alata, Dioscorea rotundata, Tetragastris panamensis, and Camellia sinensis [C. fructicola is geographically broadly distributed—it has been reported on all five continents, throughout the Americas, Western and Eastern Asia, Western Europe, Western Africa, and Australia [hin CGSC . Morphol species ,11. Despsinensis ,11. In australia ,11. HoweCamellia oleifera [C. oleifera is native to China and is cultivated in many parts of southern China [C. oleifera has a deep taproot system and can grow in a diversity of niches, including steep hills with relatively poor nutrients [C. oleifera for both food (i.e. oil) production and environmental protection (e.g. preventing soil erosion) [One of the major host plants of CGSC in China is the tea-oil tree oleifera ,12. C. orn China ,21. Thisutrients –22. As aC. oleifera over the last few decades has also attracted increasing attention of plant pathologists to infectious diseases on this plant. While historical records are not available, recent estimates suggested that up to 40% of C. oleifera yield were lost in some plantations due to CGSC anthracnose [C. oleifera plants, resulting in premature leaf and fruit rot and drop as well as wilting of the leaves and buds. In southern China where most of this species is artificially cultivated, anthracnose can occur from early April to late October, peaking in August [C. oleifera, relative little is known about its molecular epidemiology, including the causative agent(s), its potential dispersal pattern, the relationships among geographic populations, and its modes of reproduction in nature.The expanding cultivation of hracnose ,12. Stran August ,12. HoweC. gloeosporioides species complex, MGGA has helped define the 22 phylogenetic species within this species complex [ITS, CL, GS, and GD) to analyze strains of CGSC from anthracnose-infected leaves of tea-oil trees from 15 plantations in 7 southern Chinese provinces   and mult complex . Here werovinces .C. fructicola. The genotype data at the four loci for the 167 isolates were analyzed to address the following specific issues, (i) evidence for clonality and recombination within individual populations , (ii) the extent to which clones are spread among the geographic populations of C. fructicola in southern China, and (iii) the genetic relationships among the geographic populations, including potential evidence of genetic isolation by geographic distance.Among the 199 isolates of CGSC that we obtained, 167 were found belonging to ucticola ,32, thusC. oleifera infected leaves with symptoms of anthracnose were obtained, including 50 leaves from each of the 15 tea-oil tree plantations in seven provinces in southern China  and agarose gel electrophoresis. The DNA samples were diluted to 0.5 μg/ml and used in PCR reactions in order to amplify: (i) the internal transcribed spacer (ITS) regions of the nuclear ribosomal RNA gene cluster; (ii) calmodulin (CL); (iii) glutamine synthetase (GS); and (iv) glyceraldehyde-3-phosphate dehydrogenase (GD). These markers have been used in previous studies and shown to be effective for discriminating strains and species within CGSC [The total genomic DNA was isolated from each strain using a protocol described previously . The quahin CGSC ,8,11. Eahin CGSC  and the hin CGSC ,11. The Colletotrichum gloeosporioides species complex (CGSC). However, as shown in previous studies [ITS, CL and GD) were found in all the type or epitype strains representing the 22 known species within CGSC [ITS; KJ131771-KJ131969 for GD; KJ131970-KJ132168 for GS; and KJ132169-KJ132367 for CAL gene fragments. To determine the species identity of the 199 isolates that we obtained in this study, we first used BLASTN analyses of the sequences at each individual locus against GenBank database to investigate whether the isolates belonged to the  studies ,8,11, se studies ,11 and uhin CGSC ,11, our hin CGSC ,11 were hin CGSC ,35. The hin CGSC ,37, follC. fructicola while the remaining 32 isolates belonged to 3–4 other species , their dN/dS ratios were calculated using the MEGA software [Among the 199 CGSC isolates, 167 belonged to cies See  below. TAlEx 6.5  was usedsoftware ,37.In natural environments, all microbes can reproduce asexually through budding, fission, and for filamentous fungi, hyphal extension and/or asexual sporulation. Baring mutation, asexually reproduced progeny would have an identical genotype as the parental cell. The clonally derived progeny cells could disperse to different ecological niches and geographic locations. However, mutations could accumulate during asexual reproduction and form clonal lineages. Indeed, evidence for such clones and clonal lineages has been found in all microbial species examined so far, including many fungal pathogens of animals and plants , 39, 40.IA or a modified measure of IA called rd which is a standardized IA by the number of loci) [IA is random recombination while that for phylogenetic incompatibility is strict asexual reproduction and clonality. The underlying principles, methods of calculations and interpretations of the results for both tests are described in the Multilocus program manual [Recombination shuffles alleles and generates novel genotypes. At the population level, a signature of recombination is the lack of strong association among alleles at different loci. Thus, to identify whether there was recombination in natural microbial populations, two complementary statistical measures have often been used: phylogenetic incompatibility  and the of loci) . These tm manual . Here weC. fructicola, we first examined whether there is evidence for recombination within each of the four sequenced gene fragments using the total sample of 167 isolates. Both the index of association and phylogenetic incompatibility tests were conducted.In natural populations, recombination may occur among nucleotide sites within individual DNA fragments ,43. If sC. fructicola: (i) individual geographic populations; (ii) the total sample that included all 167 isolates; and (iii) the clone-corrected total sample of 53 unique genotypes. The third sample type was analyzed to examine, after excluding an obvious clonal component (i.e. isolates sharing the same genotype), whether the signatures of recombination would be more evident. For the analyses of individual geographic populations, only the eight populations  and total genetic differentiation based on the analysis of molecular variance (AMOVA) were calculated [ST values were used to identify major axes separating the geographic populations. The statistical significance of the FST and AMOVA results was obtained by comparing the distribution of the observed values to the expected distribution based on re-sampled data (1000 times in our study) while assuming no differentiation. During re-sampling to generate the expected distribution, the subpopulation sizes were held constant but individuals were reassigned to different subpopulations to simulate gene flow to generate genetically undifferentiated subpopulations. Furthermore, the relationships between geographic distance and genetic distance among geographic populations were examined using the Mantel test implemented in the GenAlEx 6.4 program [We analyzed the genetic relationships among populations of AlEx 6.4  was usedlculated ,45. Prin program . If obviIn addition to the above pre-defined, geography-based analyses, we also used STRUCTURE 2.3.4 to identify the number of genetically distinct clusters (K) in our sample and to assign individual isolates to specific clusters (K) ,46. STRUC. fructicola than other plantations and whether they could potentially be the centers of C. fructicola diversification. To examine the possibilities, we employed two tests. In the first, we compared the number of alleles, the number of private alleles, and the allelic diversities using the Wilcoxon signed-rank test (a non-parametric paired rank order test) between these two plantations and the remaining 10 plantations each with a sample size greater than 5 . In the second test, we used the DIYABC program [C. fructicola populations in tea-oil tree plantations. In the first scenario, the HNTJL and JXCB populations of C. fructicola served as ancestral populations and were the first two to diverge and the other 10 geographic populations subsequently diverged from them. In the second scenario, all samples of C. fructicola from the 12 plantations diverged at the same time. Default parameters  were used to generate the simulated data for posterior probability calculations of the two scenarios. 1000 simulations were done to help derive statistical significance of the difference between the two scenarios.As described above, two (HNTJL and JXCB) of the 15 sampled plantations were tea-oil tree breeding centers. We were interested in whether these two plantations also had higher genetic diversities of the fungal pathogen  program  to compaC. fructicola and its close relatives within the C. gloeosporioides species complex were isolated from the 750 leaf samples (each leaf was from a different tea-oil tree), representing a 26.5% overall success rate. However, the success rates varied widely among the geographic samples , C. siamense (19 isolates), C. gloeosporioides (6 isolates), and C. camelliae (2 isolates). However, five isolates  did not have clear phylogenetic affiliations with any of the 22 known species  are shown in Following the procedure recommended by Prihastuti et al.  and Weir species . The geoC. siamense, C. gloeosporioides, and C. camelliae, C. fructicola.Due to the small sample sizes of the three species . Of the DNA fragments from the three protein-coding genes, the CL gene fragment (633 bp) contained four polymorphic sites (= 0.63% polymorphic sites); the GS gene fragment (711 bp) contained 60 polymorphic sites (= 8.44% polymorphic sites); and the GD gene fragment (190 bp) contained 15 polymorphic sites (= 7.89% polymorphic sites). The total number of polymorphic sites for the combined 2073 bp was 98 (= 4.73% polymorphic sites). Among the four gene fragments, the highest mean nucleotide diversity was found in the GD fragment , followed by GS , ITS , and CL . The mean diversity index at the 98 polymorphic nucleotide sites for the combined gene sequences was 0.044±0.009 (n = 98).The four sequenced gene fragments differed in their number and percentages of polymorphic nucleotide sites within C. fructicola are 15, 5, 12, and 11 respectively for the ITS, CL, GS, and GD gene fragments. The mean allelic diversity among the four loci for each of the 12 populations with sample sizes greater than 5 is presented in Treating each gene fragment as an individual locus, the allele numbers at each of the four loci in the total sample of Interestingly, none of the polymorphisms caused amino acid substitutions (data not shown). All of the observed polymorphic sites in the three protein-coding genes were either in intronic regions or caused synonymous substitutions. The results suggested strong purifying selection at the three protein-coding loci, consistent with their house-keeping functions and essential physiological roles within cells. Similarly, the nucleotide variations observed in the sequenced ITS fragments were found in either ITS1 or ITS2 and no polymorphism was found in the 5.8S rRNA gene located between the ITS1 and ITS2 regions. These results also suggest that the mutations observed here are likely neutral, not involved in geographic or ecological adaptations of the strains.C. fructicola are presented in C. fructicola were obtained and showed no polymorphism, each of the other 14 geographic populations had polymorphism in at least one locus. The largest number of alleles at one locus within an individual plantation was seven, found in two populations  the average number of alleles per locus , (ii) the highest number of alleles at an individual locus , and (iii) the total number of genotypes . However, there was no statistically significant correlation between sample size and the number of private alleles among the 15 geographic populations .The summary data for the average number of alleles per locus, the range of allele numbers among the four loci, the total number of private alleles  at the four loci, and the number of genotypes within each geographic population of C. fructicola . This result is consistent with our phylogenetic analysis results. Specifically, in the phylogenetic analyses using the maximum parsimony approach, a single most parsimonious tree was obtained from each gene fragment, suggesting no parallel mutation or recombination within individual genes in our sample. Our results differ from many investigations in bacteria where signatures of intra-genic recombination are commonly observed ,27,43. HC. fructicola from tea-oil trees in southern China. Specifically, three pieces of evidence suggested evidence of clonality. First, 21 of the 53 multilocus genotypes were represented by two or more isolates each, with most of these genotypes representing isolates from both the same and different plantations and geographic areas. Second, our phylogenetic compatibility tests indicated that all of our samples had some levels of phylogenetic compatibility  different genotypes and the chances of finding any two strains with an identical genotype would be extremely small. Thus, we believe that the clonal genotypes observed here reflect at least partly a clonal component of C. fructicola reproduction in nature.Due to the lack of evidence for intra-genic recombination, we used allelic information based on all SNPs at each locus to investigate signatures of recombination between the four gene fragments for the above sample types. Population genetic tests suggested evidence for both clonality and recombination in natural populations of tibility . Third, tibility . We woulC. fructicola samples. For example, all individual subpopulations failed to reject the null hypothesis of random recombination . Similarly, all except the Tianjiling population in Hunan (HNTJL), showed evidence of phylogenetic incompatibility , with a Pearson’s correlation coefficient of 0.321  of the 12 plantations each with a sample size greater than five indicated that 86% of the observed genetic variations were found within individual plantations and 14% were found among the plantations. The pairwise population Fof 0.321 .Aside from these three geographic populations being genetically very different, several other population pairs also showed some levels of differentiation among each other . Indeed,C. fructicola isolates based on four loci as shown in In the STRUCTURE analysis, the information at all 98 polymorphic nucleotide sites for all 167 isolates was included. Our results indicated that the highest-likelihood value and the mode of ΔK index distribution were observed when K = 2 . The K =C. fructicola and potentially serve as the distribution centers of the C. fructicola pathogen genetic variation. In the first set of tests, the two populations of C. fructicola from the tea-oil tree breeding centers HNTJL and JXCB did not show statistically significant higher number of alleles or higher allelic diversities than other populations (C. fructicola samples from the two tea-oil tree breeding centers diverged earlier than those from other plantations) was more likely than the second scenario   . Our resC. fructicola, represented by 167 isolates distributed in all 15 plantations. The 167 isolates belonged to 53 multilocus sequence types, among which 21 were each represented by two or more isolates. Eighteen of the 21 genotypes were distributed in at least two provinces, consistent with clonal dispersals among plantations and geographic populations. However, our analyses also identified evidence for recombination in all regional and local populations, consistent with sexual reproduction of this organism in nature. Though the majority of genetic variation was found within local populations, geographic populations showed evidence for significant genetic differentiation. Specifically, the northernmost (HBSZ), easternmost (FJLY) and southernmost (HNWZS) populations were significant different from each other and from other geographic populations. Furthermore, there is genetic evidence that the two tea-oil tree breeding centers might have contributed to C. fructicola pathogen diversity in commercial tea-oil tree plantations. Below we discuss the limitations of our results and the implications of our study on the epidemiology and management of anthracnose diseases on tea-oil trees.In this study, we obtained and analyzed the patterns of genetic variation among isolates and populations of CGSC from tea-oil tree leaves with anthracnose syndromes in southern China. A total of 199 isolates belonging to the CGSC were obtained from 15 tea-oil tree plantations in seven provinces, representing the broad geographic distribution of tea-oil tree plantations in China. The relatively low isolation rates of CGSC among many of our samples may be caused by several factors, including the stringent disinfection procedure used to kill leaf surface microbes, the different severity of leaf infection , the application of fungicides in some of the plantations, the delay between field sampling and lab isolation, and/or the presence of other pathogens causing similar symptoms as CGSC. Sequence analyses of four gene fragments identified that the 199 isolates clustered with strains of four known species. However, five of the isolates did not have clear species affiliations. The dominant species was C. fructicolca . Our result differs from those reported previously on the causative agents of anthracnose on tea-oil trees. Previous studies have reported C. gloeosporioides as the causal agent [C. gloeosporioides sensu stricto. This result is similar to what was recently reported for anthracnose causal agents of tropical fruits [C. gloeosporioides sensu stricto and two strains were identified as belonging to C. fructicola [C. gloeosporioides species complex. Interestingly, five of the 199 isolates had no clear phylogenetic affiliations with the 22 known species [Following criteria established previously ,11, our al agent ,12. Howeal agent , includial agent . As a real agent  for our C. fructicola from tea-oil trees in southern China. Of the four gene fragments, GS was found to contain the highest proportion of polymorphic nucleotide sites, followed by GD, ITS, and CD. The total number of alleles per locus ranged from 5 to 15 in our C. fructicola sample. Interestingly, despite the extensive polymorphisms within each locus, we found no evidence of heterozygosity within any of the isolates at these loci, consistent with the haploid status of this species in nature. These results support previous conclusions that these four loci are excellent markers for identifying genotypes of C. fructicola.Our analyses identified that the four analyzed gene fragments were polymorphic in most local populations of C. fructicola. The results here are similar to most fungal populations in nature [Glomerella cingulata and the 22 known phylogenetic species within CGSC defined based on gene sequences. For example, we don’t know whether each phylogenetic species within CGSC has a distinct corresponding sexual state in nature. At present, only three of the 22 phylogenetic species have been reported to have corresponding teleomorphs in nature: C. fructicola (corresponding to Glomerella cingulata var. minor); C. kahawae (corresponding to Glomerella cingulata var. migrans); and C. camelliae (corresponding to Glomerella cingulata f. sp. camelliae) [C. fructicola, consistent with the expectation of mating and sexual reproduction in nature for this species [As expected, evidence for both clonality and recombination were found for most geographic populations of n nature ,39,51,52melliae) ,31,32. Imelliae) ,29,39,53 species ,32. Our C. fructicola from tea-oil tree leaves. Among the 66 pairwise population comparisons, 41 rejected the null hypothesis of no significant genetic differences while the remaining 25 failed to reject the null hypothesis. Some of the 25 pairwise comparisons that failed to reject the null hypothesis were likely due to their small sample sizes and not a true lack of differences. For example, samples from JXCB-B and HNYX had only six isolates each. Even though the FST value was relatively high . Taken together, the observed genetic differentiation among geographic populations in our study was likely an underestimate of the true genetic differentiations among these populations in nature.Our study identified a statistically significant contribution of geographic separation to the overall observed genetic variation within at 0.16, , the p vpothesis . AlternaC. fructicola. Instead, local selection and adaptation as well as local clonal expansion might have been important factors. A recent study of geographic populations of another anthracnose-causing fungal pathogen Colletotrichum truncatum from chili peppers in China also identified a significant contribution of the north-south divide to the observed genetic variations [The observed genetic differentiation among geographic populations could be due to (i) gene flow barriers, (ii) adaptations to local ecological niches, (iii) differential clonal expansions of different genotypes at different locations, or (iv) combinations of the aforementioned three factors. The wide geographic distributions of many multilocus genotypes suggest that geographic barriers were unlikely solely responsible for the genetic differentiation among geographic populations of riations . Given triations ,38,40,53C. fructicola populations from tea-oil trees are not known. However, abiotic factors such as temperature and biotic factors such as host trees as well as populations of C. fructicola from other host plants in the vicinity of the sampled tea-oil tree plantations could have all contributed to genetic differentiations. For example, other trees as well as crops such as peanuts, watermelon, and chili peppers are often grown in or around the tea-oil tree plantations and some of these plants can also suffer anthracnose diseases. At present, their causative agents and epidemiology are largely unknown. It’s entirely possible that cross-infection and recombination can occur between pathogens from different host plants, contributing to the genetic differences among the plantations. In addition, latitudinal differences could have contributed to adaptations to different temperatures between the southern-most and northern-most populations here, as was suggested for C. truncatum [At present, the specific ecological factors that might have contributed to the genetic differentiation among local runcatum . For exaPrevious studies of plant fungal pathogens suggested that the centers of host origin and/or host diversity were often associated with greater genetic diversity of fungal pathogens, including greater allelic diversity, higher genotypic diversity, and/or more private alleles ,38,40,53C. fructicola genetic diversity in tea-oil trees is the populations of C. fructicola from crops and trees in fields and forests adjacent to the tea-oil tree plantations. Among the 15 tea-oil tree plantations, the Wuzhishan plantation from Hainan Island was by far the youngest (only four years old at the time of sampling in 2012). The tea-oil trees in this plantation were imported as seedlings from the Changbu plantation in Jiangxi province (JXCB) in south-central China. Interestingly, despite evidence for clonal genotype dispersal among other geographic areas, none of the five C. fructicola isolates from this plantation had a genotype identical to those from the Changbu plantation in Jiangxi (or any other population in Mainland China). Instead, all five isolates from the HNWZS plantation had the same allele at the GD locus and this allele was not found in Changbu, Jiangxi  from all 15 plantations analyzed here belonged to one large interbreeding population. The seemingly contradictory results were likely due to the different assumptions and methods of the two analyses (i.e. STRUCTURE and AMOVA) [C. fructicola from tea-oil tree may contain two cryptic species. The analyses of more strains from these plantations and additional phenotypic and genotypic characterizations should help reveal the taxonomic relationships between these two clusters.Despite the observed geographic differentiation among local populations, STRUCTURE analyses showed that most d AMOVA) –47. For C. fructicola on tea-oil tree leaves in southern China. Our results provide a foundation from which to further explore the epidemiology and management strategies of tea-oil tree anthracnose in southern China. However, additional information will be needed in order to develop effective strategies for managing anthracnose in tea-oil tree plantations. For example, one essential piece of information would be the specificity of host-pathogen interaction between the cultivars of tea-oil plants and species of CGSC and genotypes of C. fructicola. Hundreds of tea-oil tree cultivars have been officially registered [This study revealed high genetic variation, clonal genotype dispersion, limited but unambiguous evidence for recombination within and among geographic populations of gistered . HoweverC. fructicola from other tissues and organs of tea-oil plants as well as from other species of plants. If similar genotypes were found among C. fructicola samples from other plants growing nearby, a more holistic approach would be needed to take into account of the life histories of different hosts in order to develop effective strategies for controlling anthracnose. Our study provides a foundation from which to investigate these and other related issues.Similarly, very little is known about the patterns of genetic variation among samples of S1 FigNumbers in parenthesis indicate the number of strains with that sequence type from the specific plantations.(TIFF)Click here for additional data file.S2 FigNumbers in parenthesis indicate the number of strains with that sequence type from the specific plantation.(TIFF)Click here for additional data file.S3 FigNumbers in parenthesis indicate the number of strains with that sequence type from the specific plantation.(TIFF)Click here for additional data file.S4 FigNumbers in parenthesis indicate the number of strains with that sequence type from the specific plantation.(TIFF)Click here for additional data file.S5 Fig(TIFF)Click here for additional data file.S6 Fig(A) When the sample sizes were six at each of the two populations as we currently have; and (B) When the sample sizes were doubled to 12 each.(TIFF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableHighlighted samples were excluded from population genetic analyses. For each isolate, the string of capital letters indicates its geographic location corresponding to those in (DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Ruditapes decussatus is commercially important in southwestern Europe, suffering from population decline and hybridization with exotic Manila clam (R. philippinarum). Previous studies with intronic markers showed a genetic subdivision of the species in three races . However, detailed population genetic studies to help management of the main production areas in the southwest of Europe are missing. We have analyzed eight Atlantic and two Mediterranean populations from the Spanish coasts using 14 microsatellites and six intronic markers. Microsatellites confirmed the Atlantic and West Mediterranean races detected with introns and showed that genetic variability was higher in Mediterranean than in Atlantic populations. Both marker types showed that genetic differentiation of Atlantic populations was low and indicated that populations could be managed at the regional level in the case of Cantabrian and Gulf of Cadiz areas, but not in the case of Rias Baixas and the Mediterranean. This study shows the interest of including different types of markers in studies of genetic population structure of marine organisms.The clam  Ruditapes decussatus is an infaunal bivalve that lives in sandy‐muddy bottoms of estuaries, lagoons, and coastal flats along the Mediterranean Sea and the northeast Atlantic, from Senegal to Norway (Fischer‐Piette and Métivier www.fao.org). Commercial exploitation is carried out through traditional methods based on collection by hand during low tides, or from small boats using rakes. The high prices that clams can reach in the market have led to intensive exploitation in several areas, and natural European populations have declined in many of the places where the species used to be common in the past. R. decussatus is facing also the spread of the Manila clam (Ruditapes philippinarum), which was introduced in Europe in the early 1970s to respond to the increasing demand of clams and to some limitations of carpet‐shell clams to cover this demand  Walne .R. decussatus have appeared along the last 25 years . Borsa et al. , with high values at some loci (FST > 0.2). Differences between populations within each of the three races were also significant. Finally, the mtDNA marker showed a phylogenetic break located at the transition from the W Mediterranean Sea to the Adriatic and Aegean seas, which was corroborated by sequencing the most variable iRFLP markers.In another study, Cordero et al.  analyzedIn the present paper, we report the results of a study of 10 carpet‐shell clam populations from the coasts of Spain with the same iRFLP markers used by Cordero et al.  and withR. decussatus. For this purpose, we scored microsatell‐ites and introns in the same samples. Microsatellites are usually considered strictly neutral markers, because the overwhelming majority is located in noncoding intergenic genome regions . Genotyping of Tbp was based on its length polymorphism and was carried out by running the PCR products in a 2% agarose gel and photographed under UV light exposure. Genotyping of the five remaining markers was carried out by scoring their restriction fragment length polymorphism (RFLP) in 1.5% agarose gels under UV light. Specific conditions are explained in detail in Cordero et al. Six nuclear intronic regions described in previous studies  were genotyped as described in Borrell et al. . IndividFstat v. 2.9.3 , number of alleles (introns), and allelic richness (microsatellites) with R software  at each locus and population were measured with the sts Rice . All thests Rice .Null allele frequencies at introns were calculated with the program INEst  was calculated. Moreover, an overall analysis of genetic differentiation was carried out through hierarchical FST’ statistics with the software GenoDive, v. 2.0b23  of contiguous K  and each kind of marker with the software Obstruct  and the geographic distance (km) as the coastline distance between sample locations.To test for isolation by distance Slatkin , a ManteFST values with the program BayeScan v. 2.1  and from 0.260 to 0.486 (Ho). One private allele at low frequency was found at marker Tbp in Eo. No significant differences in allele number or expected heterozygosity among localities were detected .Allele frequencies and other statistics are given in Appendix FIS were always positive and varied between 0.060 and 1.000. Ech deviated in eight localities and Srp54 in five. Null allele estimates for Ech resulted in frequencies higher than 0.2 at all localities except Del (0.064), while no null alleles were detected for locus Srp54 at those localities with significant deviations. Therefore, we considered null alleles only at Ech.Two intronic loci showed positive significant deviations from HWE after sequential Bonferroni correction Appendix . SignifiHo) and 0.633–0.736 (He). Private alleles (1–7) were present in all localities, except at Pon, Vil and Rio. They were always at low frequencies (<2.5%), except one locus at Mur . The Friedman test detected significant differences in allelic richness  and expected heterozygosity  among localities. A post hoc analysis showed significant differences between Cam and Mur for allelic richness and between Mur and three localities  for expected heterozygosity.Genetic variation statistics by locus, locality, and overall are collected in Appendix FIS ≥ 0.258). Results from Micro‐Checker analysis suggested the presence of null alleles in the four cases (frequency 0.112–0.227), but only one of them showed null allele frequencies over 0.200 (RdATC‐238 in Red). Of the tests performed to detect linkage disequilibrium only, the comparisons involving RdATC‐022 and RdATC‐199 were significant after sequential Bonferroni correction. This suggests that these loci might be closely linked and that they cannot be treated as independent variables.Deviations from HWE were found in only four cases after sequential Bonferroni correction: the locus RdATC‐238 departing in two localities and the loci RdATC‐022 and RdATC‐219 in one. Deviations were always caused by a heterozygote deficit , indicating the existence of genetic differentiation among samples. Values of FST across markers (Table Ubc) to 0.194 (Trdmt), and FST’ ranged from 0.016 to 0.337. FST for Atlantic populations alone was 0.051.The overall multilocus estimate for FST  showed a significant result , and in this case, the FST value (0.025) was relatively high despite their proximity. The highest pairwise FST values were obtained when comparing the Mediterranean populations with the Atlantic.Pairwise multilocus ST Table  ranged fFSC = 0.033, P < 0.01). When considering the Mediterranean and three groups in the Atlantic (Cantabrian Sea/Rías Baixas/Gulf of Cadiz), or only these three groups, the percentage of variation within group was, in practical terms, zero (FSC = −0.018). The percentage of variation among groups was significant irrespective of whether the Mediterranean sample was included or not .Hierarchical analyses of molecular variance Table  revealedThe neighbor‐joining tree based on Nei genetic distances Fig. A showed K). Overall Obstruct R2 values for K = 2–4 varied between 0.91 and 0.98 and were highly significant (P < 0.001), indicating strong correlation between inferred ancestries and predefined populations and therefore strong population structure. The values of ln P , indicating the existence of genetic differentiation among samples. The FST values per locus . Standardized values (FST’) ranged from 0.013 to 0.261  and Mediterranean  regions separately, its value dropped by more than 50% but it was still significant (P < 0.001).The analysis of genetic differentiation was carried out using 13 loci. Locus RdATC‐022, which showed linkage disequilibrium with RdATC‐199, was ignored. The global multilocus estimate for FST . Nevertheless, this grouping also showed a significant within‐groups component . When Atlantic samples were further subdivided (Cantabrian/Rías Baixas/Gulf of Cadiz), the among‐groups and within‐groups components dropped . FCT and FCT’ in this case were 0.015 and 0.043, respectively.Different regional groups were tested using an AMOVA Table . The higThe neighbor‐joining tree Fig.  clearly K. Obstruct analysis showed high overall R2 values (R2 ≥ 0.97). The maximum value for the “estimated likelihood of K” was observed for K = 3, but K values of 2 and 4 showed similar values  . However, for microsatellites, the Mantel test indicated that the degree of genetic differentiation increased significantly with distance  indicating support for an isolation by distance (IBD) model .No significant correlation was observed between geographic distance and the test statistic for an IBD model for intronic markers (75) Fig. . Correladel Fig. . When thFST for these loci were the lowest recorded. Values of alpha were negative in all significant cases. Results were similar when only Atlantic populations were analyzed.The test was carried out separately for introns and microsatellites because the different mutation rates that characterize the two types of markers could bias the result if they were pooled  in 10 populations of o et al.  for otheFST outliers for the two types of markers by the method of Foll and Gaggiotti  as compared to the Atlantic.Measures of genetic variability  were lower at introns than at microsatellites, a result that could be anticipated from the specific molecular nature of both types of markers. For introns, variation in o et al.  for the o et al.  in theiry et al.  also repFST, was 0.029 for microsatellites and 0.088 for introns. These values are not directly comparable due to strong differences in the levels of variability and mutation rates for each type of marker (Hedrick FST measure (FST’) proposed by Hedrick  were higher than for microsatellites , but the difference of mean FST’ between introns and microsatellites was not statistically significant.Genetic differentiation among populations, measured as  Hedrick , 2005. T Hedrick  to hieran Tables , 4. AverThe pattern of population differentiation differed between markers. While microsatellites fitted an isolation by distance model in the total area and in the Atlantic, introns did not. A potential interpretation of these results is that introns have lower power than microsatellites to detect an IBD pattern due to the low number of alleles per locus and the lower number of loci scored. Alternatively, the IBD detected at microsatellites could be a spurious result of the subdivided genetic structure of clam populations Meirmans .i.e., historical subdivision and gene flow restrictions) are responsible for the genetic differentiation of clam populations from the two marine basins. This differentiation is usually explained in the framework of sea level changes caused by Pleistocene glaciations   , which is the effective number of migrants , we have distinguished three regions that are genetically different: Cantabrian Sea, Rias Baixas, and the Gulf of Cadiz. It remains to be seen whether the genetic differentiation detected reflects a genetic discontinuity between the three Atlantic regions investigated here, or whether there is a continuous genetic variation along the Atlantic coasts. This will require a more regularly spaced population sampling as well as including the coasts of Portugal in it.Atlantic populations showed also significant genetic differentiation. Here, significant  Slatkin , we obtaTrdmt and, to a lower extent, by locus Srp54  across the whole area of study and a lack of significant differentiation among the three clam races (FCT). This pattern contrasted with the pattern presented by the remaining 4 loci, which was characterized by significant, usually high, differentiation between races. Cordero et al.  should be considered a separate management unit. However, this will not be clearly established until more populations within each ria, other rias, and nearby areas in Northwest Spain are characterized in more detail. This is an urgent task as they contribute the majority of the Spanish clam production.Our results have rendered the most complete characterization to date of wild exploited populations of None declared.


Gaucho, N = 352; Baiano, N = 148) with different histories of geographic and ethnic colonization. Of the total sample, four markers were found to be significantly associated with skin color, but only two  were consistently associated with MI in both samples (Gaucho and Baiano). Therefore, only these 2 SNPs should be preliminarily considered to have forensic significance because they consistently showed the association independently of the admixture level of the populations studied. We do not discard that the other two markers (HERC2 rs1129038 and TYR rs1126809) might be also relevant to admixed samples, but additional studies are necessary to confirm the real importance of these markers for skin pigmentation. Finally, our study shows associations of some SNPs with MI in a modern Brazilian admixed sample, with possible applications in forensic genetics. Some classical genetic markers in Euro-North American populations are not associated with MI in our sample. Our results point out the relevance of considering population differences in selecting an appropriate set of SNPs as phenotype predictors in forensic practice.The understanding of the complex genotype-phenotype architecture of human pigmentation has clear implications for the evolutionary history of humans, as well as for medical and forensic practices. Although dozens of genes have previously been associated with human skin color, knowledge about this trait remains incomplete. In particular, studies focusing on populations outside the European-North American axis are rare, and, until now, admixed populations have seldom been considered. The present study was designed to help fill this gap. Our objective was to evaluate possible associations of 18 single nucleotide polymorphisms (SNPs), located within nine genes, and one pseudogene with the Melanin Index (MI) in two admixed Brazilian populations ( In humans and other animals, pigmentation results from melanin synthesis, which occurs in melanocytes stored in vesicles known as melanosomes. Melanocytes are found in the epidermis, hair bulb, and iris, and are directly responsible for the pigmentation of these organs and structures The human pigmentation pathway is regulated by a network controlled by many genes, as well as by environmental, mechanical, and epigenetic factors. For instance, Quillen et al. Homo sapiens from Africa to other continents A considerable number of genetic studies involving normal and pathological variation in human pigmentation have emerged recently SLC24A5 and SLC45A2, others, such as OPRM1 and EGFR, have also contributed to differences in pigmentation between Native Americans and Europeans. Moreover, Norton et al. SLC24A5, SLC45A2, and TYR have a predominant role in the evolution of lighter skin color in Europeans, but not in East Asians. This indicates recent convergent evolution of lighter pigmentation phenotypes, and is suggestive the importance of natural selection in this process. Quillen and Shriver Additionally, these studies documented the extensive diversity of pigmentation genotype-phenotype architecture across human populations. For example, Quillen et al. As noted above, several studies have indicated different populations have different genetic backgrounds for pigmentation genes. Therefore, caution should be exercised when extrapolating results from one population to another. The gene pools of contemporary admixed Brazilian populations present varying levels of contribution from continental ancestral groups  reflected in the striking differences that have been reported among them http://www.ucl.ac.uk/silva/candela). As a whole, the CANDELA project recruited 7,500 subjects over 18 years of age from five Latin America countries . For this investigation, we worked with all available samples to date from persons born in Rio Grande do Sul (RS) (n = 352), the southernmost state of Brazil, and from those born in the state of Bahia (BA) (n = 148). Our goal was to evaluate possible associations between 18 SNPs and MI in two populations with contrasting ancestry formation.This study recruited a subset of volunteers from the 1,600 Brazilian participants in the Consortium for the Analysis of the Diversity and Evolution of Latin America , whereas African heritage is more visible in BA with respect to both phenotypic and cultural aspects. Apart from these widely recognized characteristics, other historical, demographic, and genetic particularities can distinguish these populations The above-indicated states have distinct colonial histories: RS is characterized by more substantial European colonization  is used to refer to anyone born in RS, and not only to those born in the Pampean region of this state. In the present study, we employ this term to refer to any volunteer from RS, whereas the term Baiano is used to identify individuals from BA.The traditional This project was approved by the Research Ethics Committees of the Universidade Federal do Rio Grande do Sul, Hospital de Clínicas de Porto Alegre, and the Universidade Estadual do Sudoeste da Bahia . All subjects signed an informed consent form approved by the above-mentioned ethics committees, in accordance with the Declaration of Helsinki.The CANDELA protocol involved the acquisition of data on physical appearance, including a quantitative measure of skin color using the Derma spectrometer , which provides the MI. In humans, values ranging from 20 to 100 reflect fairer to darker skin tones. MI was measured on the proximal medial portion of both arms to generate a single average value. This location was chosen because it captures the constitutive skin pigmentation , with potentially little effect of sun exposure www.lgcgenomics.com). Eighteen SNPs were investigated: rs1524668 (ADAM17), rs4785763 , rs6058017 (ASIP), rs1129038 (HERC2), rs1805009, rs1805008, and rs1805007 (MC1R), rs1800407, rs1800401, and rs1800414 (OCA2), rs1426654 , rs6867641, rs26722, and rs16891982 , rs3750965 and rs3829241 (TPCN2), and rs1042602 and rs1126809 (located at the TYR gene). These polymorphisms were chosen because previous studies have linked them either directly or indirectly with the human pigmentation pathway  between the Gaucho and Baiano samples. The assumptions of normality and homoscedasticity were tested using Kolmogorov-Smirnov and Levene's tests.Allele frequencies were estimated by gene counting. Agreement of genotype frequencies with Hardy-Weinberg equilibrium (HWE) was tested using the “Utility programs for analysis of genetic linkage” http://www.cs.rpi.edu/~drinep/HGDPAIMS/WORLD_5000_INFAIMs.txt). Allele frequencies in Native Americans are available for a fraction of these markers Population structure due to admixture is a well-known confounding factor in association studies. To minimize this problem, the proportion of African, European and Amerindian ancestry was estimated using the 40 Ancestry Informative Markers (AIMs) studied in all CANDELA samples. Paschou et al. Gaucho vs. Baiano) as an additional covariate in the total sample analysis to assess whether the results were robust to this additional known level of population structure. A Bonferroni-corrected p value threshold of 0.0028 was considered statistically significant for ANOVA tests.ANOVA  were employed to test for the association of genetic polymorphisms with the melanin index (MI) by comparing these values across genotypes; Sidák-corrected multiple comparisons were performed when appropriate. The proportions of African and European ancestries were included in the model as covariates. Note that covariates should be included in the ANOVA only if they are associated with both dependent (melanin index) and independent (genotype) variables. We also included the group label  years differed significantly from the Baiano mean age  (p<0.001). The mean MI of the total sample was 32.90 (range 22.04–76.40), while Gaucho and Baiano showed average MI values of 31.54 (22.04–76.40) and 36.68 (23.72–65.01), respectively. These results indicate that the Baiano group presents a darker skin color compared with the Gaucho group (p<0.001).Since the assumptions for the ANOVA and t-tests were not satisfied for age and MI, we used 1/MI and 1/age transformations. Non-parametric methods were used when this transformation was not enough to satisfy those assumptions. The mean age for the total sample (N = 500) was 25 (range 18–85) years, 168 (33.6%) being males and 332 (66.4%) females. The Gaucho sample, genotype frequencies were in accordance with those expected by HWE, except for SLC24A5 rs1426654 , which showed a lower number of heterozygotes than expected. For the Baiano group, only the genotype frequencies of SLC45A2 rs16891982 were not in HWE , also due to a lower than expected number of heterozygotes. Allele and genotype frequencies were statistically different between the 2 populations for ASIP rs6058017, HERC2 rs1129038, SLC24A5 rs1426654, and SLC45A2 rs16891982 , as well as Native American ancestry (p = 0.017). The percentage mean values are: Gaucho: European, 84.86; African, 6.99; and Native American, 8.15; Baiano: 61.47, 29.91, and 8.62, respectively ; and African  proportions; and a weak correlation between 1/MI and Native-American ancestry . However, only the African and European ancestries were associated with both genotypes and the melanin index, as opposed to the Native American ancestry. Sex was not associated with MI (p = 0.817) and a negligible correlation  was observed between MI and age. Therefore, in the ANOVA analysis of the association between the 18 genetic markers and MI, we used European and African proportions as covariates to adjust for population stratification.We also observed a moderate correlation between 1/MI and European , and for the Baiano subsample (N = 148). The results are presented in SLC24A5 rs1426654 (p<0.001) and SLC45A2 rs16891982 (p<0.001); and HERC2 rs1129038 (p = 0.001), and TYR rs1126809 (p = 0.001). But when the Gaucho and Baiano populations were considered separately, only two of these SNPs (SLC24A5 rs1426654 and SLC45A2 rs16891982) remained significantly associated with MI (p<0.001). The inclusion of the group label (Gaucho vs. Baiano) as an additional covariate in the combined population analysis did not change our results (data not shown).Three sets of ANOVA analyses were performed to evaluate the association between MI and genotypes for the 18 markers: for the total sample (N = 500), for the HERC2 rs1129038, SLC45A2 rs16891982 and TYR rs1126809, the respective alleles A, C, and A, present a dominant effect  seem to behave relatively consistently as skin color predictors in admixed samples. HERC2 rs1129038 and TYR rs1126809 were significantly associated with MI in the total sample analyses only. We do not discard the possibility that these two markers might also be relevant, but additional studies are necessary to confirm their real importance for skin pigmentation. It is possible that their association in Gaucho and Baiano disappeared due to small sample size when these populations were considered separately. On the other hand, some classical markers associated with skin pigmentation in Europeans and others populations , indicating the possibility that they are population-specific. Therefore, they should be avoided for general inferences in forensic DNA phenotyping. This notable absence of association between classical skin pigmentation loci (including ASIP and MC1R) and skin color also was noted by Beleza et al. SLC24A5 rs1426654 and SLC45A2 rs16891982 with this trait.Our results showed that just two polymorphisms  were significantly associated with MI in all analyses. Two other markers showed suggestive results (HERC2 rs1129038 and TYR rs1126809), indicating the need for additional studies for confirmation. An interesting finding is that while some polymorphisms seem to behave consistently as skin color predictors, others seem to be population-specific, indicating that genotype-phenotype relationships found in one population cannot be uncritically extrapolated to another.The main implication of our study is the establishment of some genetic markers as skin color predictors in admixed populations. Two SNPs (File S1Gaucho (N = 352) and Baiano (N = 148) samples. Table S3, Mean and range values for the ancestry proportion (%) in the Baiano and Gaucho samples.Table S1, Characteristics of the SNPs studied. Table S2, Genotype and allele frequencies for 18 SNPs in the (DOCX)Click here for additional data file.