Bactris gasipaes Kunth) has had a central place in the livelihoods of people in the Americas since pre-Columbian times, notably for its edible fruits and multi-purpose wood. The botanical taxon includes both domesticated and wild varieties. Domesticated var gasipaes is believed to derive from one or more of the three wild types of var. chichagui identified today, although the exact dynamics and location of the domestication are still uncertain. Drawing on a combination of molecular and phenotypic diversity data, modeling of past climate suitability and existing literature, we present an integrated hypothesis about peach palm’s domestication. We support a single initial domestication event in south western Amazonia, giving rise to var. chichagui type 3, the putative incipient domesticate. We argue that subsequent dispersal by humans across western Amazonia, and possibly into Central America allowed for secondary domestication events through hybridization with resident wild populations, and differential human selection pressures, resulting in the diversity of present-day landraces. The high phenotypic diversity in the Ecuadorian and northern Peruvian Amazon suggest that human selection of different traits was particularly intense there. While acknowledging the need for further data collection, we believe that our results contribute new insights and tools to understand domestication and dispersal patterns of this important native staple, as well as to plan for its conservation.Peach palm ( Bactris gasipaes Kunth) is the only palm species with domesticated populations in the Neotropics [Bactris genus [gasipaes and all wild populations (previously identified as different species) into var. chichagui. Within var. chichagui three types were proposed, based on fruit morphological traits [gasipaes may be derived from var. chichagui type 3, possibly through hybridization with type 1 [Peach palm ..Bactris h type 1  but withThe earliest archaeological evidence of crop domestication in South America is from western Ecuador 12,000 years ago, and concerns a now abandoned squash species . EvidencThree main hypotheses about the locations of peach palm domestication exist. Patiño Rodriguez  pointed et al [Drawing on the results of a genetic and morpho-biochemical characterization of a continent-wide peach palm collection, the purpose of this study is to contribute to the debate on the origin and dispersal of peach palm in the neotropics. We explore the possibility of a fourth hypothesis, being the conjunction of the second and third hypotheses mentioned above; more specifically, we suggest a primary domestication event in southwestern Amazonia followed by secondary domestication at different sites of peach palm’s current distribution coinciding with the location of some of the currently described landraces. Hernandez-Ugalde et al  argued tet al , which—aet al . We postet al ), and thet al ,27. Baseex situ collections, i.e. that of CATIE , in Turrialba, Costa Rica and that of INIA  in Iquitos, Peru, as well as from collections in commercial farms in Colombia; samples were obtained/collected between 2009 and 2010. Material transfer agreements were signed with the genebanks of CATIE and INIA for their respective samples and permission was obtained by the owners of the farms from which the samples from Colombia were collected; only domesticated specimens were collected on-farm.Leaf material was collected from 87 individual plants coming from two important B. gasipaes var. chichagui. These individuals , and the interindividual fixation index (Fis), were calculated and a Mantel test was conducted between the samples’ geographical distances and their genetic distances , searchiMorphological and biochemical traits were measured (see B. gasipaes var. gasipaes) and 55 to the wild form (B. gasiapes var. chichagui). Additional presence points to those of the present study were collected from a variety of sources, including published papers [We constructed a dataset containing 221 presence points of which 166 pertained to the cultivated form  values and comparing these with a geographical null model . To thischichagui by drawing a convex hull, extended with a 3% buffer of the largest hull axis, around all presence points in the environmental space determined by a the two first axes of a PCA based on the bioclimatic variables  of the Last Glacial Maximum (LGM)  while foWe constructed maps to visualize the spatial distribution of genetic diversity of peach palm in representative sample sites across Central and South America. Neutral molecular data are very useful for the identification of genetic diversity hotspots. Here we considerd the following parameters: allelic richness, observed heterozygocity, and the richness of locally common alleles. Locally common alleles (LCAs) are DNA sequences that occur at relatively high frequency (here >5%) in only a limited portion of the total distribution area of a species (here <25%). An interesting feature of LCA richness is that it can be indicative of the level of historic and/or ongoing genetic isolation of populations, and hence can be informative about the location of potential past refugia ,43. Diveet al [et al [To increase the geographical coverage of the genetic diversity patterns in peach palm, we performed a similar replication-bootstrapping exercise for allelic richness and locally common alleles based on the molecular marker dataset of Hernández et al  which inet al  for furtl [et al  with ourex situ conservation sites where they are held. Nonetheless, Fisher’s F test and Student’s t-test showed that coefficients of variation calculated on the basis of all individuals in each collection are homoscedastic  and not significantly different between collections , suggesting that the differences in the environment and/or management of the two collections did not significantly affect the extent of the phenotypic variation observed here. To further neutralize possible differences in phenotypic variation between the collections, we standardized the results obtained from the above replication-bootstrapping exercise for each collection separately. We finally merged the rasters of phenotypic variation of the collections with the same procedure described above for the genetic parameters. All calculations and spatial analyses were performed in R [We applied a similar replication-bootstrapping procedure to our dataset of morphological and biochemical characteristics of peach palm fruits and calculated the average coefficient of variation across all parameters . The procedure was applied separately on the subset of samples conserved in CATIE and those conserved in INIA, in order to avoid joining samples whose phenotypic variation may have been affected by different environmental conditions at the med in R ; all mapmed in R .We identified priority areas for on-farm conservation of peach palm based on the geographical intersect between areas for which the ensemble of distribution models returned high suitability scores during present and future (2050s) climate conditions, and areas holding the highest levels of allelic richness. Each of the layers here considered was restricted to the values above the third quartile of their density distributions. Similarly, we considered the geographical intersect between areas for which suitability scores under current and future (2050) climate conditions were high, and areas above the third quantile of the distribution of phenotypic variation (using the dataset resulting from the bootstrapping exercise described earlier), in order to look into priority areas for conservation of peach palm’s morphological and biochemical diversity.After removing the three putatively wild samples from our dataset, only 80 of the samples submitted to genetic analyses and 66 of the samples submitted to morphological and biochemical analyses yielded meaningful results. All analysed loci were polymorphic with a total number of 68 alleles; level of polymorphisms per locus ranged from 4 (locus BG-24) to 12 alleles (locus BG-1 and BG-63) with an average of 7.5 alleles per locus. Expected heterozygosity (He) varied between 0.636 (locus BG-17) to 0.881 (locus BG-3). The extent of reduction in observed heterozygosity was used to quantify the level of genetic differentiation between the individuals at each locus, through F statistics . The differentiation among individuals at different loci ranged from 0.037 to 0.455 (maximum at locus BG-17)  [Bactris. Another area of congruence is the northern Peruvian Amazon, where the highest levels of phenotypic diversity of our peach palm dataset was observed, confirming earlier observations [Our results suggest an overall tendency to higher levels of genetic diversity in South American, and particularly western Amazonia, compared to Central American samples, corroborating the hypothesis of South America as being the region of the species’ earliest domestication ,20,21,48acao L.) . While nrvations . In thisrvations ) were forvations . Such ceOn the other hand, the combination of high genetic and phenotypic diversity in these landraces might additionally relate to longstanding indigenous management practices as suggested by ethnobiological studies in the Huaorani indigenous group from the Ecuadoran Amazon, another hotspot of both genetic and phenotypic peach palm diversity . The Huagasipaes) and wild (var. chichagui) populations, while in cacao such a distinction is much less straightforward [The apparent compatibility of diversity patterns in peach palm and cacao is likely due the fact that they have similar habitat requirements and their distributions may have been influenced by early humans in similar ways. Both species are very characteristic elements of homegardens in Amazonia ,53. Howetforward . This haDrawing on the present results, and building on previous findings reported in literature, in what follows we present a hypothesis of how peach palm may have evolved from a wild species into the cultivated landraces known today across Latin America. While acknowledging that our dataset is too small to unmistakingly validate this hypothesis, we hope that it can inspire future research aimed at furthering our understanding of the temporal and spatial dynamic of this important Amazonian crop.gasipaes clearly did not exist at the time of the last glacial period; therefore, explaining the congruence between genetic diversity patterns in the domesticated variety and LGM refugia requires a closer look at peach palm’s wild types, particularly types 1 and 3 which are considered to have been involved in the domestication process [et al suggested that the distribution patterns of wild peach palm were likely to have been strongly influenced not only by the last glacial period, but also by geological events during the Pliocene and Miocene, possibly leading to genetic differentiation of isolated palm populations [Domesticated var.  process –11,20. Hulations . While tHuman occupation of South America may have started as early as 22,000 BP  and humaRegardless of the actual location were it took place, it is likely that this initial domestication event resulted in the emergence of the wild type 3, as an incipient domesticate from a type 1 progenitor , which wet al [gasipaes was spread (late Holocene). Clement et al [Cristo-Araújo et al  advocatent et al  similarlB. gasipaes var. gasipaes through continued human selection and hybridization with (progenitor) type 1, a hypothesis on which many authors share consensus [Our model assumes that the incipiently domesticated type 3 formed the basis of secondary domestication processes leading to onsensus ,17,61,62onsensus ,63. Intronsensus ,64. The chichagui observation as far north as the Japurá River  and west (Amazonian Ecuador and northern Peru) at the time secondary domestication processes were initiated in these places. There is even the possibility that it still occurs in these areas but that botanical collections have missed it until now. For example, the Brazilian RADAM collection contains a potential var. er  which ier  may similarly be explained by human dispersal across the mountains , in a siet al  that will [et al  may be dl [et al ,68.et al [Bertholletia excelsa), another Amazonian species important for non-timber forest products, from southwestern source populations may have coincided with the expansion of horticulturalist societies towards the northeast. The fact that the distributions of both peach palm and Brazil nut in central and eastern Amazonia are concentrated along the main rivers and their tributaries [In southwestern Amazonia, hybridization between one or more populations of incipiently domesticated type 3 trees and type1 populations that may have survived in the putative LGM refugium in the Cuzco-Madre de Dios region may have given rise to the Pará landrace. This is supported by the finding that wild peach palm samples (likely type 3) from Acre, Brazil  were genetically very similar to samples from the Pará landrace ,69. A siet al  have simbutaries ,63 and sbutaries ,63, whicbutaries  supportsOne of the the best ways to test our tentative domestication model would be to genetically characterize naturally occurring type 1 and type 3 populations across their ranges (on both sides of the Andes), and most importantly in each of the convergence areas where (i) high diversity levels have been observed in domesticated peach palm and (ii) suitable habitat conditions prevailed during the LGM. This would then allow for a more systematic comparison of the genetic makeup of local wild and domesticated populations, and a better evaluation of the putative association between genetically differentiated populations of type 1 and potential LGM refugia. The preferential use of chloroplast DNA markers in particular would be beneficial since nuclear markers are biparentally inherited and hence are subject to recombination. On the contrary, chloroplast DNA is almost always maternally inherited, which avoids the issue of recombination and is more appropriate for identifying the location of the domestication event(s) that gave rise to peach palm’s landraces.ex situ field collections [Suitability modeling of peach palm under different climate change scenarios suggests a positive future. This finding is in line with a recent continent-wide assessment of the relation between species range sizes and their resilience to climate change in the Americas. The authors found that species with wide distributions, such as peach palm, are generally habitat generalists and therefore better armed to deal with a changing climate . Similarlections –75 altholections . While tlections  such as lections , that maIn conclusion, our preliminary results highlight the usefulness of spatial analyses for making molecular data more explicit and meaningful within the broader context of geography, landscapes, climate dynamics, and human history and culture. Further validation of the initial hypotheses presented here for peach palm is needed, requiring the assemblage of larger genetic and phenotypic datasets at continental level (of both wild and cultivated populations), the use of standardized molecular markers (both nuclear and chloroplast) and protocols across different datasets and regions to facilitate aggregate analyses.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 TableBactris gasipaes var. gasipaes accessions from CATIE, INIA and those collected on-farm in Colombia.Allele number, average allele frequency, observed and expected heterozygosity, and the fixation index Fis were measured on (DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.S3 TextModels considered and calibration methods.(DOCX)Click here for additional data file.


Loxosceles rufescens  is an ideal example: native to the Mediterranean, the species has dispersed worldwide via cohabitation with humans. A previous study revealed considerable molecular diversity, suggesting cryptic species, but relationships among lineages did not correspond to geographic location.Understanding the evolutionary history of morphologically cryptic species complexes is difficult, and made even more challenging when geographic distributions have been modified by human-mediated dispersal. This situation is common in the Mediterranean Basin where, aside from the environmental heterogeneity of the region, protracted human presence has obscured the biogeographic processes that shaped current diversity. c oxidase subunit I identified 11 different evolutionary lineages, presenting two contrasting phylogeographic patterns: (1) lineages with well-structured populations in Morocco and Iberia, and (2) lineages lacking geographic structure across the Mediterranean Basin. Dating analyses placed main diversification events in the Pleistocene, and multiple Pleistocene refugia, identified using ecological niche modeling (ENM), are compatible with allopatric differentiation of lineages. Human-mediated transportation appears to have complicated the current biogeography of this medically important and synanthropic spider.Delimitation analyses on cytochrome L. rufescens in the Mediterranean Basin, with emphasis on the origins of mtDNA diversity. We found support for the hypothesis that northern Africa was the center of origin for L. rufescens, and that current genetic diversity originated in allopatry, likely promoted by successive glaciations during the Pleistocene. We corroborated the scenario of multiple refugia within the Mediterranean, principally in northern Africa, combining results from eight atmosphere–ocean general circulation models (AOGCMs) with two different refugium-delimitation methodologies. ENM results were useful for providing general views of putative refugia, with fine-scale details depending on the level of stringency applied for agreement among models.We integrated ecological niche models with phylogeographic analyses to elucidate the evolutionary history of The online version of this article (doi:10.1186/s12862-014-0195-y) contains supplementary material, which is available to authorized users. The Mediterranean Basin was placed among 25 world biodiversity ‘hotspots for conservation priority’ based on high levels of endemism and rapid loss of natural areas . Humans Several factors  promoted development of this diversity at different temporal and geographic scales, such as the Messinian Salinity Crisis and the onset of a Mediterranean-type climate ~3.2 Ma . GlaciatDifferent approaches have been used to delimit glacial refugia in the Mediterranean. Traditionally, paleoecological evidence -17 and cLoxosceles spider species coexist in the Mediterranean: L. rufescens (Dufour 1820) and the Tunisian L. mrazig Ribera and Planas 2009. Loxosceles rufescens originated in the Mediterranean [L. rufescens, suggesting cryptic speciation. The morphological simplicity within Loxosceles makes traditional species delimitation “singularly difficult” , species ,34. TherL. rufescens, and elucidate evolutionary processes that promoted this diversity via a combination of phylogeographic and ENM approaches. Our working hypothesis is that current mtDNA diversity was generated allopatrically in glacial refugia across the Mediterranean, and that L. rufescens biogeography was since obfuscated by human activity. This hypothesis offers three opportunities for testing: (1) divergence times should coincide with periods of repeated glaciations (~2.6 – 0.02 My), (2) multiple putative refugia should have existed to provide areas of origin for distinct evolutionary lineages, and (3) widespread lineages should show no spatial structure within the Mediterranean Basin.In this contribution, we examine mtDNA diversity within L. rufescens populations across the Mediterranean Basin to replicate and complement previous sampling [We sampled sampling  and to ic oxidase subunit I (cox1) using LINF and GAYAR primers [Taq polymerase (Promega) or Biotools Pfu DNA Polymerase (Biotools). PCR products were cycle-sequenced in both directions using the same PCR primers and the BigDye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems). Sequences were derived from these products in an ABI 3700 automated sequencer at the Serveis Científico-Tècnics of the Universitat de Barcelona and in Macrogen, Inc. . Raw sequences were edited and assembled with GENEIOUS v.4.6.5 [Loxosceles cox1, we mostly used Loxosceles-specific primers; we also translated sequences into amino-acids, and checked for stop-codons.We included at least one individual from each locality in molecular analyses. Total genomic DNA was extracted using SPEEDTOOLS Tissue DNA Extraction Kit (BIOTOOLS) following manufacturer’s protocols. We amplified a portion of the cytochrome  primers , produci primers  and C1-N primers  internal v.4.6.5 . To avoi v.4.6.5  for LoxoSequences were aligned unambiguously in GENEIOUS using ClustalW  with defcox1 haplotype. ML analyses were conducted in RAxML v.7.4.2 [We used maximum likelihood (ML) and Bayesian inference (BI) to infer phylogenetic relationships from a dataset containing one representative of each  v.7.4.2  with the v.7.4.2 . We appl v.7.4.2  with two v.7.4.2  until ef v.7.4.2  using a  v.7.4.2  after re v.7.4.2  and usedN > 10), we applied two neutrality tests: Fu’s FS [R2 [Genetic p-distances between and within lineages see  were cal Fu’s FS  and R2 [s FS [R2  in DnaSPs FS [R2 . We asseL. rufescens, but genetic distances between lineages are high and different lineages are often sympatric at micro-scales , the, theL. r-scales ,55,56), ,56, L. r-scales , haplotype richness (HR), and rarity index (R) [in situ history, so identification of this pattern may indicate regions of origin for the different lineages [We investigated how mtDNA diversity is distributed across geography within ollowing ; this mendex (R) , in R [6ndex (R)  using culineages . We explWe analyzed the geographic structure of each lineage using a Mantel test implemented in the Isolation By Distance Web Service v.3.23 . GeograpL. rufescens lineage, we explored two divergent rates. First, we used a Loxosceles-specific molecular rate obtained using fossil and island ages as calibration points [L. rufescens fall somewhere between these end points. Rates were incorporated as priors under a normal distribution with mean 0.095 ± 0.001 and 0.0199 ± 0.001, respectively. Dating analyses were conducted in BEAST [Since we lacked reliable calibration points within the n points . Second,n points ). As thein BEAST , using ain BEAST  and a YuL. rufescens were calibrated within a region that we hypothesized was ‘sampled’ by the species over its relevant history; in other words, a region the species had been able to deem suitable/unsuitable , which provided an estimate of the dispersal capability of the species. We excluded areas that we were unable to sample, or where closely-related species occur . These steps left a calibration area comprising Morocco, the Iberian Peninsula, Balearic Islands, Sardinia, Sicily, continental Italy, Greece and adjacent islands, Crete, and Tunisia. After model calibration in this area, we projected results to the entirety of the Mediterranean Basin, within a bounding rectangle of 48.2–26.7° latitude and −14.8–41.2° longitude , interse et al. . To calcWe obtained climate data from eight coupled atmosphere–ocean general circulation model (AOGCMs) simulations: Community Climate System Model (CCSM), Centre National de Recherches Météorologiques (CNRM), Consortium for Small-scale Modelling (COSMOS), Goddard Institute for Space Studies (GISS), Institute Pierre Simon Laplace (IPSL), Model for Interdisciplinary Research on Climate (MIROC), Max-Planck Institut für Meterologie (MPI), and Meteorological Research Institute (MRI). These AOGCMs were derived from the multi-model ensemble in the Coupled Model Intercomparison Project Phase 5 (CMIP5)  and the We downloaded monthly simulation outputs for annual precipitation and mean, maximum, and minimum temperatures from the pre-industrial experiment, which characterized current climatic conditions. Past conditions were characterized using paleoclimate simulations for the mid-Holocene (~6 Ka) and Last Glacial Maximum  from each AOGCM, with the exception of GISS and COSMOS, which lacked mid-Holocene outputs. To produce climate scenarios at resolutions relevant to the spatial scale of species’ distributions, we downscaled climate layers to 0.5° resolution using a standard change-factor approach : (1) forFor each AOGCM, we performed a principal components analysis in R  on the 1We used a subset (130 records) of occurrence data associated with samples employed in the genetic analyses. These localities were obtained directly from fieldwork in natural areas by EP and others (see Acknowledgments), and have precise latitude/longitude coordinates derived from GPS measurements. To consider the potential biasing effects introduced by spatial autocorrelation, such that spatially-clumped points would over-represent certain environments, we calculated spatial lags in environmental data using Geostatistical Analyst in ArcMap v.10 , and subsampled the records to create 10 replicate datasets using a script in R  written L. rufescens, but we acknowledge that ensemble-modeling approaches may shed additional light on model-dependent results .M) may be encountered, leading to situations of extrapolation. To identify these regions, we used a script in R [L. rufescens.When transferring models temporally or spatially, conditions outside the range of climatic values in the calibration region  approaches . PartialE = 1% . We perfL. rufescens and lack constraining data for the earlier half of the epoch.We identified possible refugia as areas that remained continuously suitable from the LGM to the present. Because glaciations were common throughout the Pleistocene, with glacial and interglacial conditions recurring in nearly-regular cycles with similar amplitudes (at least since 800 Ka) , we assusensu Rull [We identified refugia using two approaches: approach one (M1) required AOGCMs to agree on suitable area for each time slice, applying four different thresholds . This resulted in four different suitability maps for the LGM, the mid-Holocene, and the present-day. The intersection of the three time slices was taken as the final refugial area, which created four different possible refugial scenarios Figure a-d. The nsu Rull ).Figure New sequences obtained during this study were deposited in GenBank with accession numbers KJ560560 - KJ560863 . In both cases, B Figure . Clade AClade B comprised five lineages (B1-B5), all well-supported . B5 placed as sister to the remaining lineages of clade B, and lineage B1 was sister to a well-supported clade  comprising B2-B4, but this latter relationship was not well supported . B2 placed as sister to B3 and B4 . B3 was composed exclusively of Iberian Peninsula individuals; the remaining lineages included individuals from different Mediterranean regions.Fs test for demographic expansion was negative and significant in all cases except lineage B3. The R2 test was low and significant for the two lineages with higher sample sizes (A6 and B5) in the left tail. As a whole, these results suggest a recent demographic expansion for all lineages except for B3.Genetic p-distances ranged from 1.5-7.8% between the various lineages . Clusters identified  were mostly congruent across methods; in all three, seven clusters were delimited. Slight differences between approaches appeared in terms of detecting singletons: in total, we recovered 14 entities (clusters plus singletons) using the ultrametric tree obtained with BEAST , while the remaining two analyses delimited 11 entities . Differences occurred in lineages B5, A4, and A6, wherein GMYC analyses using the BEAST tree split each of these lineages into two clusters  and haplotype richness (HR); however, they exhibited higher values for the rarity index (R) in southern Morocco. Analyses of individual lineages showed clear geographic patterns for lineage B2 and B4, wherein higher values for all diversity statistics were obtained in the Iberian Peninsula and Balearic Islands, respectively. Lineages A6 and B5 showed no clear geographic pattern, with the highest values in various isolated regions. Different parametrizations for this test produced similar patterns (results not shown). That is, similar results were obtained when the number of individuals was reduced to three , except within lineage B2, where high values for all statistics characterized the region around Greece. The Mantel test showed weak correlations between genetic and geographic distance matrices for lineages A6, B2, B4, and B5 (r < 0.24), with tests not significant except for lineage B5  with the Parachtes rate.Divergence time estimates differed depending on the rate used in calibration  were also consistent across AOGCMs, covering desert areas (e.g. the Sahara and Sinai Peninsula) and some regions around the Black Sea; these regions were not considered in our analyses.Models from individual AOGCMs were predictive of independent suites of occurrence points, with all models statistically significant in partial ROC tests  retained suitable conditions, although reduced in extent, but extensive regions of the Sahara, which were largely unsuitable in the present and mid-Holocene, were identified as suitable during the LGM under most AOGCMs. Regions environmentally outside those represented within Results from individual AOGCMs were similar, with the exception of IPSL. Because IPSL uses a different vegetation model from other AOGCMs , we ran refugial delimitation analyses with and without IPSL. Putative refugia were congruent across the two methodologies and with and without IPSL Figure a-e. WhenL. rufescens across the Mediterranean Basin, as reported previously by Duncan et al. [We document high mtDNA genetic diversity in n et al. , underscn et al. . Thus, in et al. , and theL. rufescens, and is, in fact, highly heterogeneous .Four lineages A1-A4) are distributed along the western slopes of the Atlas Mountains .Biogeographic patterns within clade B are more complex. The exception is lineage B3, where all individuals are found in the Iberian Peninsula. In lineages B2, B4 and B5, individuals with the most common haplotype are widespread across the Mediterranean, as in A6, although no clear pattern emerges that links genetic diversity with geography , which lends further support to a northern African origin for these lineages and for L. rufescens as a whole. This region has been postulated as a climatic refugium for various animals and plants in light of its complex orography  and climatic stability , with current distributional and genetic patterns most likely the result of population mixing through human-mediated transportation. Although some of these divergent lineages now occur in sympatry, they likely originated in allopatry, given the dominance of this speciation mechanism for the genus ,34 and t, A6, B, Genetic diversity patterns should help in elucidating the origins of these lineages, assuming that refugial areas harbor higher genetic diversity (but see ). Such iLoxosceles rufescens in the Mediterranean Basin based on mtDNA data. Genetic diversity was not distributed uniformly, and we found two contrasting phylogeographic patterns: (1) the southern region of Morocco holds several lineages with well-structured populations, (2) whereas lineages distributed across the broader Mediterranean Basin generally lack geographic structure. By combining results from eight AOGCMs with two different refugium-delimitation methodologies, we corroborated the scenario of multiple refugia within the Mediterranean, principally in northern Africa. ENMs were useful for providing general views of putative refugia, with fine-scale details depending on the level of stringency applied for agreement among models. Although refugial delimitation remains challenging, by combining ENM with phylogeographic approaches, we found support for the hypothesis that northern Africa was the center of origin for L. rufescens, that current genetic diversity probably originated in allopatry and was promoted by successive glaciations during the Pleistocene, and that protracted human activities impacted the current distributional patterns of L. rufescens within the Mediterranean Basin.In this study, we delimited 11 evolutionary lineages within http://purl.org/phylo/treebase/phylows/study/TB2:S15925. Information on geographic location and Genbank accession number for the 310 individuals included in this study is available in Additional file The data sets supporting the phylogenetic results of this article are available on the TreeBase repository, ID: 15925,


Collections from Hawaii to the Eastern Indian Ocean comprise one large, undifferentiated population. This pattern of limited genetic subdivision across reefs of the central Indo-Pacific has been observed in a number of large-range reef fishes. Conversely, small-range fishes are often deeply structured over the same area. These findings demonstrate population connectivity differences among species at biogeographic and evolutionary timescales, which likely translates into differences in dispersal ability at ecological and demographic timescales. While interspecific differences in population connectivity complicate the design of management strategies, the integration of multiscale connectivity patterns into marine resource planning will help ensure long-term ecosystem stability by preserving functionally diverse communities.Recent reports of localized larval recruitment in predominately small-range fishes are countered by studies that show high genetic connectivity across large oceanic distances. This discrepancy may result from the different timescales over which genetic and demographic processes operate or rather may indicate regular long-distance dispersal in some species. Here, we contribute an analysis of mtDNA cytochrome  The recent dramatic decline of marine ecosystems –3 has leMost near-shore marine species exhibit an early pelagic larval phase  andRecent reports emphasizing the influence of species ecology on dispersal and connectivity –30 may oChaetodon vagabundus), direct tests of larval dispersal have only been applied to fishes with small geographic ranges  that are restricted to either the tropical West Pacific [Whether employing induced otolith tags or multi locus parentage assignment, direct larval tracking has consistently revealed unexpectedly high levels of local larval retention , 35–38.  Pacific , 36, 37, Pacific . Convers Pacific , indicatDispersal ability is thought to play an important role in establishing and maintaining large geographic ranges  across a range that stretches from the east coast of Africa to Hawaii and Pitcairn Island in the Central Pacific [ST =−0.006, P = .752 [To further investigate the relationship between species range size and patterns of population connectivity, we assessed mitochondrial DNA (mtDNA) diversity in the Brown Surgeonfish ( Pacific . The Bro Pacific –62. The  Pacific ). A prevP = .752 ), a pattSimilar phylogeographic comparisons have provided valuable insights into species’ life history , 66, ecoN = 281 [N = 279; b was amplified using the heavy strand primer (5’-GTGACTTGAAAAACCACCGTTG-3’) from [2, 0.26 µM of each primer, 50 nM dNTPs, 1.0 units Taq DNA polymerase and 2.0 µL of 10x PCR buffer  in 20 µL total volume. PCR thermal cycling consisted of an initial denaturing step at 94° C for 1 min, followed by 35 cycles of 94, 55, and 72°C for 30 s each, with a final extension of 72°C for 10 min. Sequencing reactions with fluorescently labeled dideoxy terminators were analyzed on an ABI 3100 automated sequencer  in the EPSCoR genetic analysis facility at the Hawaii Institute of Marine Biology. Only rare and questionable haplotypes were sequenced in both directions. Newly derived unique haplotypes have been deposited in Gen Bank (accession numbers HQ157717–157797). Resulting sequences were aligned in Mafft 6.62 [Tissue collections from Hawaii (N = 281 ) were suN = 279; , and a 63’) from  and ligh3’) from . In briefft 6.62 .h) and nucleotide (π) diversities for each collection site were calculated in Arlequin 3.11 [T-test. A statistical parsimony network of haplotypes was constructed using TCS 1.2.1 [Haplotype  framework (Arlequin 3.11) to identify maximally differentiated groupings without a priori assumptions of group identity. To ensure validity of the maximally differentiated groupings, the simulated annealing process was repeated 100 times from a different initial partition of samples into K groups. The configuration with the largest, statistically significant estimate of among group differentiations (Φct) is retained as the best sample grouping. Samova was run with values of K = 2 to 10 to identify the most likely number of populations. Because Samova cannot be run for K = 1, a separate AMOVA analysis was conducted with all collections combined into a single group. Deviations from random expectations were tested with 20,000 permutations. Patterns of pairwise genetic differentiation among individual sampling sites (Φst) were estimated in Arlequin with the mutational model of Tamura and Nei [odeltest [Dest . Samova and Nei , which wodeltest , 87. We n 13 see ).amova-defined populations.A Mantel test with 10,000 simulations was used to test for an isolation-by-distance (IBD) signature . TosF [rlequin with 90,000 simulated samples. Fu [sF is particularly sensitive to deviations from a constant population size, with population expansion resulting in a significant, negative sF. If there was evidence of population expansion, we estimated ancestral and contemporary female effective population sizes (Nef) from the equation: θ = Nef2 μt, with μ being the estimated annual fragment mutation rate and t is the estimated generation time. Estimates of pre- and post expansion θ were derived from the sudden expansion model of the mismatch distributions. Population ages in years were estimated from the population age parameter (τ), with τ = 2 μT, where T is the time since the most recent bottleneck. We provisionally applied a generation time of 5 years for the Brown Surgeonfish [−8 per year [We tested for a signature of population expansion with Fu’s Fs  and by cFs  in Arleqples. Fu  noted thgeonfish  and a wiper year .b sequencing revealed 110 closely related haplotypes  is similar to other peripheral collections  but is significantly lower than central Indo-Pacific collections .Cytochrome The statistical parsimony network demonstrates both the high diversity and low differentiation of haplotypes collected from sites distributed across two ocean basins . Abundanamova identified three maximally differentiated groupings, with significant population differentiation occurring only at eastern and western edges of the range  dropped incrementally with the addition of K > 3 populations (data not shown) indicating a lack of further population subdivision. Pairwise Φst and Dest highlight both the isolation of Moorea as well as a pattern of increasing divergence in the Indian Ocean with distance from the IPB . Pairwise estimates of Φst within the Samova-defined central Indo-Pacific population were near zero. Nonetheless, there was a clear IBD signature across this broad region  even though Christmas Island, located in the western Indian Ocean, was only marginally different from Hawaiian collections  and was genetically indistinguishable from Kiritibati, Fiji, and Palau.S the IPB . Both thamova populations. While a significant deviation between simulated and observed mismatch distributions was observed only in the Moorean collection , Fu’s sF was significantly negative in all three populations , with sGenetic evidence of postglacial population expansion is common in reef fishes and indicates broad ecosystem level responses to global climate change . These pLutjanus kasmira), which differs only in having an additional genetic break between Moorea and the Marquesas [The population structure of the Brown Surgeonfish is remarkably similar to the widely distributed Bluestriped Snapper (arquesas . NotablyAcanthurus triostegus) revealed little to no genetic subdivision within the Central and West Pacific [Naso unicornis) from the West Indian Ocean to French Polynesia in the South Pacific [Aulostomus chinensis,) from West Australia to Panama [Gymnothorax flavimarginatus) from East Africa to the East Pacific [The lack of pronounced mtDNA genetic structure across the central Indo-Pacific in both the Brown Surgeonfish and Bluestriped Snapper has been observed in a growing number of widely distributed reef fishes. Of the six published genetic surveys of reef fishes with geographic ranges extending from Africa to the East Pacific , all but the Convict Surgeonfish , Pyntropyge , 107), Tlostomus ), Unicorus [Naso , 108), Mnothorax ), and atLutjanus ) and Suranthurus , 109) haFor widely distributed species, genetic homogeneity across much of the Indo-Pacific is largely concordant with biogeographic provinces and barriers, previously defined based on species distributions. For example, the region stretching from the eastern Indian Ocean to the Central Pacific is recognized by biogeographers as the Indo-Polynesian province –112. HobPlectropomus leopardus) is restricted to reefs from West Australia to Fiji, and a survey of mtDNA control region diversity indicated deep genetic partitions, with a minimum of six highly differentiated populations  and the restricted-range Wrasse,  revealed concordant morphological and genetic differentiation, and indicates evolutionary level partitions among the closely linked reefs of the West Pacific [Amphiprion [Chrysiptera [Dascyllus [Pomacentrus [Epinephelus [Plectropomus [This pattern of widely distributed reef fishes successfully bridging long-distances contrasts starkly with the remarkably complex patterns of population structure commonly observed in smaller range fishes. The leopard coral grouper ( to 0.94 ). Likewi Pacific . Small rphiprion –117; genysiptera , 118; geascyllus , 119; genephelus , 114; geFor species occurring within the central Indo-Pacific, broad genetic homogeneity should be facilitated by stepping stone gene flow across the regions relatively abundant reefs. Indeed, dispersal can be accomplished across this vast region without having to traverse more than 800 km of open ocean , yet eveIt has been noted that mtDNA markers may not accurately reflect population histories, due to either direct selection on the marker or adjacent DNA segments . HoweverHow do these findings translate into policies for ecosystem-level management of reef communities? The debate continues over the extent to which genetic connectivity relates to demographic connectivity . FurtherWhile interspecific differences in realized dispersal would appear to complicate the development of effective marine reserves, reserve design may be simplified by focusing on the species that show the finest scale of genetic isolation , 131. Em


Fs neutrality tests) to evaluate potential genetic pool populations with highly suitable areas for two parapatric endangered species of tuco-tucos (Ctenomys minutus and C. lami). Our results demonstrated that both species were largely influenced by vegetation and soil variables at a landscape scale and inhabit a highly specific niche. Ctenomys minutus was also influenced by the variable altitude; the species was associated with low altitudes (sea level). Our model of genetic data associated with environmental suitability indicate that the genetic pool data were associated with highly suitable areas for C. minutus. This pattern was not evident for C. lami, but this outcome could be a consequence of the restricted range of the species. The preservation of species requires not only detailed knowledge of their natural history and genetic structure but also information on the availability of suitable areas where species can survive, and such knowledge can aid significantly in conservation planning. This finding reinforces the use of these two techniques for planning conservation actions.Conservation of small mammals requires knowledge of the genetically and ecologically meaningful spatial scales at which species respond to habitat modifications. Conservation strategies can be improved through the use of ecological niche models and genetic data to classify areas of high environmental suitability. In this study, we applied a Maxent model integrated with genetic information (nucleotide diversity, haplotype diversity and Fu's  Ctenomys inhabit the southern part of the Neotropical region from the extreme south up to southern Peru, including the entire Patagonian region and showing a wide latitudinal variation. The range of the genus extends from sea level to 4,000 m in the Andean region Ctenomys) have attracted special interest in terms of speciation and evolution by virtue of their patchy distributions, low vagility, territoriality, and extensive karyotypic variation Subterranean rodents of the genus Ctenomys minutus, which inhabits sandy fields and dunes and has a range extending from Jaguaruna Beach in the state of Santa Catarina to the town of São José do Norte in the state of Rio Grande do Sul. Ctenomys lami is an endemic species inhabiting a sandy region named ‘Coxilha das Lombas’ along a narrow line of old dunes that extends from north of Guaiba Lake to the northwestern sandbanks of Barros Lake C. minutus and C. lami is also present. It is probable that this zone was formed due to habitat alterations. A wide humid zone once separated both species and represented a barrier Ctenomys lami is cited as vulnerable in the Red List of the International Union for Conservation of Nature C. minutus is listed as data deficient. However, given the current state of information about tuco-tucos in Brazil, Fernandes et al. In the coastal plain of southern Brazil, the species with the widest geographic distribution is Ecological niche models (ENMs), based on the use of multiple strategies in conservation plans, are an important tool for determining the distribution of threatened species for conservation purposes Ctenomys minutus and C. lami inhabit the coastal plain of southern Brazil. Several studies have addressed the degradation of this coastal environment, identifying both natural and human-induced factors contributing to long- and short-term change C. lami and C. minutus based on the following goals:In this study, we applied ENMs integrated with genetic information to evaluate potential genetic pool populations with highly suitable potential areas. We developed maximum-entropy  and C. lami (n = 29), each representing an established population of these tuco-tucos, to generate the ENMs ), soil, vegetation, and altitude) and five variables for C. lami . To avoid information loss, we included all variables represented in the PCAs from both species to generate models for the two species. The models were constructed with a total of nine variables because equal variables were selected for the two species.We used 19 Worldclim bioclimatic variables obtained through interpolated data from derived rainfall and temperature, with a resolution of 2.5 arc-minutes (five kilometers); one altitude variable http://www.cs.princeton.edu/~schapire/maxent/). Maxent estimates the ecological niche of a species by determining the distribution of maximum entropy (ME), subject to the constraint that the expected value of each environmental variable under this estimated distribution matches its empirical average. The technique was designed as a machine learning algorithm −5. We produced maps of the potential distribution of the species using the logistic output format The models were constructed with Maxent version 3.3.3k C. minutus and Lopes and Freitas C. lami, where a detailed description of the genetic data can be obtained. For C. minutus, a total of 30 localities were sampled across the entire distribution of the species, and seven principal clades were highlighted in the Bayesian phylogenetic tree. We focused on these seven main genetic haplogroups described by Lopes et al. C. lami, 28 locations were sampled, and the data are presented by the authors based on the four karyotypic blocks proposed for the species  sequences corresponding to the cytochrome oxidase I gene and the hypervariable control region. The data were obtained from Lopes et al. C. minutus was characterized by examining 340 individuals over the entire distributional range and using information from cytochrome oxidase I gene and control region sequences. For C. lami, a total of 178 specimens were sampled, using information from the same mitochondrial DNA markers. Our analyses were based primarily on three different genetic parameters obtained from genetic data estimated by these authors ijπ is the proportion of nucleotide differences between the ith and jth types of DNA sequences and ix and jx are the respective frequencies of these sequences. Nucleotide diversity is a measure of genetic variation. The second parameter, haplotype diversity (Hd), is a measure of the uniqueness of a particular haplotype in a given population. Hd is defined by the equationix is the (relative) haplotype frequency of each haplotype in the sample and N is the sample size. Haplotype diversity is given for each sample. The third parameter, Fu's Fs, is the probability of observing a random sample with a number of alleles equal to or smaller than the observed value given the observed level of diversity and the assumption that all of the alleles are selectively neutral. If we call this probability Ŝ, thenFs would be expected from a recent population expansion or from genetic hitchhiking. A positive value of Fs is evidence for a deficiency of alleles, as would be expected from a recent population bottleneck or from overdominant selection C. minutus and with each karyotypic block for C. lami.Briefly, the geographical genetic structure of C. minutus, whereas C. lami presented a very narrow distribution probability , indicating that they inhabit a highly specific landscape niche.Both species were strongly influenced by vegetation and soil variables. Model performance, defined as the area under the curve, was highly discriminative for both species , and block C showed the highest value of π; however, the same pattern was not followed by the Hd values, which were higher in blocks B and D. The Fs values were not significant for any of the analyzed blocks , as would be expected from a recent population expansion or from genetic hitchhiking  at present or through history; however, this factor is necessary for the actual presence of species Ctenomys lami appears to be restricted to the region of Coxilha das Lombas and showed a potential distribution at low to medium levels of occurrence beyond the limits of this region. The species was shown to be associated with local vegetation and soil type and to be geographically limited by the presence of wetlands and geographical inaccessibility, factors that characterize this region.This is the first study to use ENM methods to test parameters that influence the occurrence of the genus Ctenomys minutus was restricted to the coastal plain of southern Brazil, including the region's first line of dunes and extending to sandy fields. This species shows a sympatric zone with another tuco-tuco species (C. flamarioni) in a range of approximately 15 km C. minutus inhabits the first line of dunes. Below the sympatric zone, C. minutus occupies only the regions of sandy fields, although the ME model indicates that it can potentially occur up to the line of dunes. The reason for this apparent discrepancy is that near the southern limits of this sympatric zone, the first line of dunes is already occupied by C. flamarioni up to the southern boundary of Rio Grande do Sul state. This finding is, most likely, related to the historical patterns of occupation by these species. Ctenomys minutus shows a pattern of north-south occupation extending from Santa Catarina to Rio Grande do Sul state C. flamarioni shows a pattern of occupancy from south to north C. minutus. According to Fornel et al. C. minutus that inhabit sand fields and those that inhabit dunes, environmental characteristics such as soil hardness produce morphological adaptations in tuco-tucos. These authors found some variation among different populations belonging to different habitats in cranial shape, and these intraspecific phenotypic differences appear to arise as a combination of selection and drift acting as diversifying forces. However, according to our model of environmental suitability, this discontinuity may not inhibit free dispersal by specimens from different habitats. Therefore, these different environments may also be associated with distinct levels of environmental suitability, although we could not measure this difference in our models.The distribution of C. minutus appears to show higher environmental plasticity than C. lami. The models proposed for these two species showed a low overlap of areas of high suitability. This characteristic appears to be defined primarily by the presence of the Coxilha das Lombas region and the geographical inaccessibility of this region. A hybrid zone between C. lami and C. minutus is present in the northern portion of this coxilha. This zone has been the subject of previous study. According to Gava and Freitas Although they are morphologically similar, both species studied here occupy distinct areas of the coastal plain. According to the ME models, C. lami and C. minutus was strongly influenced by soil and vegetation. C. minutus was also influenced by altitude, as it was associated with low altitudes (sea level). According to Kubiak C. minutus is associated with areas with higher vegetation and biomass, which strengthens the evidence for the importance of this variable to the species. Given that tuco-tucos are exclusively subterranean, the use of variables that are directly linked to the species niche demonstrated to be extremely important in building the models, regardless of soil and vegetation variables, might have suffered from their degree of generalization. It is recommended that the choice of predictors in ENMs should consider the ecological relevancy of the predictors to the target species Moreover, edaphic conditions can be very important for the definition of a species' fundamental niche C. lami showed that there are differences in the levels of genetic diversity (π and Hd) between populations, although the suitability is currently the same for all of the blocks. Lopes and Freitas Our analyses of the genetic data associated with the ME model for C. minutus. In the current study, these haplogroups, represented by three genetic parameters, were associated with areas of high environmental suitability. This pattern may result from the occurrence of higher genetic diversity in the areas of higher suitability for the species. According to Lopes et al. d, thus other extrinsic  or intrinsic  factors could be affecting the pattern of genetic diversity and structure.Lopes et al. Fs index that was statistically significant for C. minutus was from the Barros Lake haplogroup, which was associated with a highly suitable area. As this pattern was not found for C. lami or for the other haplogroups of C. minutus, it is not possible to support our hypothesis that highly suitable areas favor population expansion or genetic hitchhiking, as indicated by a significant negative value of the Fs index.The only value of the C. lami are the progressive urbanization and human settlement in its territory, associated, for example, with agricultural activities C. minutus is threatened by land speculation associated with urban constructions along the coast and on the shores of the lagoon and by the fragmentation of the dune habitat. In the south, silviculture involving Pinus sp. directly affects the populations of the species . These areas are in neighboring locations in the northeastern portion of the distribution. However, none of the southern populations are located in protected areas. Two state conservation units (Parque de Itapuã and Refugio de Vida Silvestre Banhado dos Pachecos) are located within the range of C. lami, but only parts of these areas overlap the distribution of the species. Over the past year, extensive field work has been conducted to search for C. lami, but few populations have been found outside these two units .The preservation of species requires not only detailed knowledge of their natural history and genetic structure but also information on the availability of suitable areas where species can survive; such knowledge can aid significantly in conservation planning. Both species analyzed here have suffered intense pressure from the fragmentation and reduction of their habitats. This process results in the death or isolation of individuals because these species are extremely niche-specific. The principal threats to C. minutus. This result supports the use of these two techniques to plan conservation actions. This pattern was not evident for C. lami, perhaps due to the restricted range of the species. Despite the current wide use of molecular markers to estimate genetic variation at different geographical scales and originating from different evolutionary processes Our models of genetic data associated with environmental suitability indicated that genetic pool data were associated with highly suitable areas for Figure S1Input points given to the software (Maxent).(TIF)Click here for additional data file.


Vaccinium myrtillus L.) fruits are perceived by consumers to be a health-promoting food, conferred by their high content of phenolic compounds and carotenoids. The health properties of these molecules have raised commercial interest in these berries globally, and caught the attention of breeders. Bilberry is a wild crop and represents an important source of genetic variability for blueberry breeding. Our study presents information related to the genetic diversity and structure of bilberry populations derived from Northern Europe, providing new knowledge for the development of genetic studies and for breeding purposes.Bilberry ( Vaccinium myrtillus) is one of the most important wild crops growing in the forests of Northern European countries, noted for its nutritional properties and its beneficial effects on human health. Assessment of the genetic diversity of wild bilberry germplasm is needed for efforts such as in situ conservation, on-farm management and development of plant breeding programmes. However, to date, only a few local  genetic studies of this species have been performed. We therefore conducted a study of genetic variability within 32 individual samples collected from different locations in Iceland, Norway, Sweden, Finland and Germany, and analysed genetic diversity among geographic groups. Four selected inter-simple sequence repeat primers allowed the amplification of 127 polymorphic loci which, based on analysis of variance, made it possible to identify 85 % of the genetic diversity within studied bilberry populations, being in agreement with the mixed-mating system of bilberry. Significant correlations were obtained between geographic and genetic distances for the entire set of samples. The analyses also highlighted the presence of a north–south genetic gradient, which is in accordance with recent findings on phenotypic traits of bilberry.The success of plant breeding in the coming years will be associated with access to new sources of variation, which will include landraces and wild relatives of crop species. In order to access the reservoir of favourable alleles within wild germplasm, knowledge about the genetic diversity and the population structure of wild species is needed. Bilberry ( The success of plant breeding over the past century has been associated with a narrowing of the available genetic diversity within elite germplasm of species. New sources of variation include landraces and wild relatives of crop species, and although exploiting wild relatives as a source of novel alleles is challenging, it has provided notable successes in crop improvement . Most crVaccinium is a genus of ∼450 plant species in the family Ericaceae that are widely distributed in the Northern Hemisphere and also in the mountains of tropical Asia and Central and South America , which results in evident morphological differences. Regarding domestication and commercial fruit crop production, the most important species are V. corymbosum (highbush blueberry), V. virgatum (rabbit-eye blueberry), V. angustifolium (lowbush blueberry), V. macrocarpon (cranberry) and V. vitis-idaea (lingonberry). The genus also contains the wild V. myrtillus (bilberry) and a number of other currently non-cultivated Vaccinium species that show great potential as new berry crops .Thirty-two individual bilberry samples derived from different seeds collected at different latitudes in several Nordic countries were included in this study Table , Fig. 1:in vitro-cultured bilberry shoot tips, using the EZNA™ SP Plant DNA Mini Kit  following the manufacturer's instructions. The concentration of DNA was estimated with the NanoDrop N-1000 spectrophotometer  at 260 nm. Fifteen primers representing di-, tri-, tetra- and pentamer repeats, previously used to characterize other Vaccinium species , 0.3 µM of each primer, 200 μM dNTPs, 0.8 U of DyNAzyme™ II DNA Polymerase (Finnzyme). The thermal profile consisted of 10 min at 94 °C, followed by 31 cycles of 1 min at 94 °C, 1 min at 46.5 °C and 2 min at 72 °C and a final extension at 72 °C for 10 min. The PCR reaction was purified using sodium acetate–ethanol DNA precipitation. One microlitre of the purified sample was analysed on a capillary electrophoresis system, Agilent 2100 Bioanalyzer with DNA7500 kit  according to the manufacturer's instructions. Each primer–clone sample combination was repeated at least two times and only replicated bands were included in the analyses. Fragments of similar size across individuals were assumed to be homologous.Genomic DNA was isolated from shoot tips of actively growing  species , were cop|] and p is the ratio of genotypes sharing the band. Moreover, the binomial matrix was used to produce the input matrix following GenAlex version 6.1 software manual instructions (I) calculated as I = −1 × (p × ln(p) + q × ln(q)), where p and q are the estimated allele frequencies.The amplification product sizes were scored using 2100 Expert Software (Agilent Technologies). The results were transformed into a binomial matrix as present (1) or absent (0) for each marker. Since the ISSR marker is dominant, we assumed that each band represented the single bi-allelic locus . Band paructions  and analAnalysis of molecular variance (AMOVA) was used to partition the total genetic variance into ‘within-populations' or ‘among-populations’ levels. The software GenAlex was used to generate a matrix of pairwise genetic distances between individuals and to calculate the following variance components: degrees of freedom, sum of squares, mean sum of squares, estimated variance and conversion of estimated variances to percentage of total variance. The number of permutations for significance testing was set at 9999. Canonical correspondence analysis (CCA) was done using Past software v. 2.17c , has beer for each distance class using two different options. The autocorrelation coefficient (r) is similar to Moran's I .Spatial analysis was conducted using the genetic spatial autocorrelation (SA) (oran's I  and rangr) between geographical distance and genetic relatedness under fixed bearing angles (degrees north of due east). For each sector, r is calculated from each distance pair weighted by the cosine of its angle with respect to the angle of the centre arc of the respective sector. Geographical locations were imported with associated genetic data. PASSAGE calculates a distance matrix from genetic data and geographical co-ordinates and an angular matrix from geographical co-ordinates. The correlation was calculated for each 10° sector, from 0° to 180°, and the significance was estimated using 999 randomizations.Directional autocorrelation analysis was carried out by testing for the direction of maximum genetic correlation using the bearing procedure implemented in PASSAGE version 2 . The beaK value from 1 to 11. Each run consisted of a burn-in period of 100 000 steps, followed by 1 000 000 Markov Chain Monte Carlo replicates, assuming an admixture model and correlated allele frequencies. No prior information was used to define the clusters. The most likely K was chosen comparing the average estimates of the likelihood of the data, ln(Pr(X|K)), for each value of K  detected by the UBC-857 primer and the smallest number (n = 25) by the UBC-873 primer . The average number of loci per primer was 31.75, with the highest number of loci  at distance classes 0–100 evidencing non-random spatial genetic structure within populations. Moreover, the positive correlation decreases until 600 km (r = 0.028). Figure r values. The bearing analysis from PASSAGE indicated the strongest correlation occurred along a north–south axis , while the weakest occurred in the east–west direction .In the correlogram that resulted from SA analysis, the K genotypes  of each group in each gene pool. Considering the mean value for each geographical group of genotypes (Q), six different patterns of genetic makeup were evidenced. The first type of pattern included two Norwegian groups (N4 and N7) out of three with the highest Q in gene pool 7 . Finnish populations represented two distinct patterns with the highest Q value in gene pool 5 for L and M groups  and in gene pool 2 for P and S groups . The IS1 population had the maximum Q value in gene pool 4 (0.59) and the K group in gene pool 1. Finally, individuals from Iceland (IS2), Norway (N2) and Sweden (R) had similar Q values in gene pools 3, 6 and 7 . A similar high level of diversity in the same genus (80.4 %) was reported in highbush and rabbit-eye blueberry  and lowbVaccinium species such as lingonberry  (to H.H.) and by Nordic Innovation Centre—New Nordic Food project ‘Bilberry: Towards functional food markets’ (2007–10) (to H.H. and L.J.).The study was funded by the L.Z. performed the DNA analyses and the ISSR data scoring and was involved in writing and editing; L.P. performed all the statistical analyses and was involved in writing and editing; L.J. and H.H. provided contribution to the concept and the design of the work. All authors read and approved the final manuscript.None declared.


Frankliniella occidentalis, using mitochondrial and microsatellite markers. This study was conducted in eight localities from four climate regions in China, where F. occidentalis was introduced in the year 2000 and had lower genetic diversity than its native populations. We also tested the level of genetic differentiation in these introduced populations. The genetic diversity of the samples at different years in the same locality was not significantly different from each other in most localities. FST and STRUCTURE analysis also showed that most temporal population comparisons from the same sites were not significantly differentiated. Our results showed that the invasive populations of F. occidentalis in China can maintain temporal stability in genetic composition at an early phase of establishment despite having lower genetic diversity than in their native range.Many species can successfully colonize new areas despite their propagules having low genetic variation. We assessed whether the decreased genetic diversity could result in temporal fluctuations of genetic parameters of the new populations of an invasive species, western flower thrips,  There is mounting evidence that species can successfully colonize new areas disjunct from their original habitat despite their propagules having low genetic variation12478Frankliniella occidentalis (Pergande), is the most economically important pest within the insect order ThysanopteraF. occidentalis can reduce yields of many vegetable crops  and ornamental flowers, damage cosmetic appearance, vector plant diseases (e.g. tomato spotted wilt virus), impose quarantine risks that negatively impact trade1111F. occidentalis is endemic to western North America from Mexico to AlaskaF. occidentalis around the world and now it is found on every continent but AntarcticaF. occidentalis has rapidly invaded and become established in many provinces in almost every climate region in China16F. occidentalis’s native range, two habitat-specific phylogenetic lineages  and cool/moist (CM) type) with allopatric distribution have been observedet al. (2010)F. occidentalis populations in China is lower than that in its native rangeF. occidentalis in China provides an opportunity to study the temporal genetic dynamics of recently introduced populations with lower genetic diversity. Such knowledge is crucial for the management of this species and for understanding the factors associated with invasion success.The invasive western flower thrips, edet al. 1020 and F. occidentalis populations using mitochondrial and nuclear markers. We aimed to determine whether the genetic diversity, population size and the allele frequencies of the invasive populations changed and analyzed which factors might account for the temporal genetic dynamics.In this study, we used a large sampling scheme including eight localities distributed in four climate regimes in China to investigate the post-established dynamics of F. occidentalis adult females were sampled from May 2009 to August 2012  and JQ09, JQ11, JQ12 and HRB12 having the lowest (Nh = 2). The haplotype diversity ranged from 0.053 (JQ11) to 0.667 (QD12)  were simultaneously found in this study (Hap4 and Hap6 correspond to CM form and other five haplotypes correspond to HD form)  . Two F. HD form) . However . Two F.ame site .HE) per population and year ranged between 0.504 in JQ11 to 0.645 in KM12 (AR), calculated based on the minimum number of nine individuals, ranged from 3.564 in JQ11 to 5.001 in DL09 with an average value of 4.503 across all the localities. A total of 24 pairwise tests in allelic richness (AR) among years within the same locality yielded only three significant differences . Generally speaking, the genetic diversity indices among years within each locality did not change significantly.A total of 260 alleles were observed from 24 loci across all populations and years. The number of alleles per locus ranged from 2 (WFT24) to 28 (WFT25) with a mean of 10.8. The unbiased expected heterozygosity  as the case in Yang et al. (2012)The ifferent . Pairwisifferent . The sixifferent . AMOVA different . Pairwisand SY12 . This aland SY12 . One hypand SY12 . The K =and SY12 . The Bayg et al. 1220. GENF. occidentalis in China in order to gain insight into the temporal genetic dynamics of invasive species during their post-establishment phase. The genetic composition of all but one of these populations  was maintained over the two- to four-year study period (tens of generations) despite having lower genetic variation than the native America populations.We assessed changes in genetic composition of eight recently introduced populations of AR) in JQ and the unbiased expected heterozygosity (uHE) in JQ and QD. FST and STRUCTURE analysis also showed that most among-years comparisons from the same sites were not significantly differentiated. Furthermore, the hierarchical analysis of molecular variance (AMOVA) showed much closer genetic relationships among temporal samples from the same populations compared to samples from different populations. Generally speaking, we detected no changes in estimates of genetic diversity and temporal genetic structure in these localities over the 2–4 year periods spanning tens of generations. As discussed earlier, the genetic diversity of F. occidentalis in China was lower than that in its native range in America18Aphis nerii) in the southern United StatesGlossina fuscipes, were stable over 8–12 generations in UgandaDysaphis plantaginea, also showed weak temporal structure in French apple orchardsBemisia tabaci in ChinaAedes japonicas in Northeastern AmericaRhagoletis completa also showed greater temporal variability than native populationsB. tabaci populations in Queensland, AustraliaThe present study revealed that the among-years genetic variation in the same locality was not significantly different from each other at most localities, except for the allelic richness of F. occidentalis, these programs did not completely eradicate the migration and gene flow in these populations. Hence, the gain of neutral genetic variation through the gene flow might balance the loss through genetic drift.There is evidence for a slight population genetic bottleneck as the genetic diversity was certainly reduced in the introduced populations of F. occidentalis adapted to the host plants on which they were maintained in only a few generations showed better reproductive performance than on other plants10F. occidentalis can feed on over 250 different plants in 62 different families10F. occidentalis could be an explanation for its rapid adaptation and maintenance of the genetic composition in China. Several other insects are also thought to have rapidly adapted to their specific habitats either by the stress-induced modification of the genome or by phenotypic evolution during the post-establishment period. Australia was colonized by Drosophila buzzatii some 600–700 generations ago. Although microsatellite DNA is usually located in the non-coding region, two of 15 microsatellites studied in this species were subjected to recent selection, one exhibiting local adaptation in different populations and the other balancing selection and these two loci may be in linkage disequilibrium with functional genes under selectionet al. (2012)Aedes albopictus across 15 degrees of latitude in the United States. In addition, the beetle Longitarsus jacobaeae at high-elevation sites has adapted to the cooler conditions by life-history changesF. occidentalis across different regions.Moreover, we cannot completely rule out the role of natural selection for the maintenance of the genetic composition of the studied populations. Local adaptation might result in the enhanced fitness and hence contributed to the maintenance of the genetic composition. It was shown that the benefits of local adaptation could balance against the inbreeding cost that could develop in part owing to the isolating effect of local adaptation itself F. occidentalis in genetic composition was only estimated over a short time period. Whether the genetic composition and their biological and ecological characteristics of these populations will remain stable or not in the subsequent years should be monitored.Our results suggest that the populations can maintain their genetic composition over a short time in their early phase of establishment despite having lower genetic diversity than their native range. However, the polymorphism level and number of the loci employed could potentially limit our ability to detect changes in genetic diversity at specific regions across the genome. Genomic data may provide more information on the changes of genetic diversity as any relevant change at the nucleotide level will not be picked up with our markers. The sensitivity for the detection of potential changes in genetic diversity and structure could also be increased with more temporal sample points and greater numbers of individuals screened. In addition, the actual levels of genetic diversity associated directly with population fitness traits or adaptive characters such as growth rate, behavior etc. cannot be directly evaluated with our approach of assaying microsatellite loci, which represent neutral genetic loci. Hence, temporal genetic stability of these populations should be interpreted cautiously since more data  should be used to verify our conclusion. Furthermore, temporal variation of F. occidentalis adult females were sampled from May 2009 to August 2012 Samples were then genotyped at 25 microsatellite loci developed for 08et al. 122043. PNh), nucleotide diversity (π) and haplotype diversity (Hd) for each population were assessed using DNASP v5Mitochondrial DNA sequences were manually checked and assembled with CodonCode Aligner 3.6.1 . No double peaks, frameshifts or stop codons were present in any of the sequences, suggesting that our sequences represent mtDNA and are not nuclear mitochondrial pseudogenes (numts)MICRO-CHECKER 2.2.3 was used to detect genotyping errors of microsatellite data due to null alleles, stuttering, or allele dropout using 1000 randomizationsNA), observed heterozygosity (HO), unbiased expected heterozygosity (uHE) and mean number of alleles (Na) were assessed using GenAlEx 6.41AR) was assessed with FSTAT 2.9.3.2The genetic diversity indices such as total alleles per locus ΔK statistic of Evanno et al. (2005)We then used STRUCTURE 2.3.3How to cite this article: Yang, X.-M. et al. Temporal Genetic Dynamics of an Invasive Species, Frankliniella occidentalis (Pergande), in an Early Phase of Establishment. Sci. Rep.5, 11877; doi: 10.1038/srep11877 (2015).


Anopheles gambiae and Anopheles coluzzii - the major malaria vectors in sub-Saharan Africa - is localized into large “islands” toward the centromeres of chromosome-X and the two autosomes. Linkage disequilibrium between these genomic islands was first detected between species-specific polymorphisms within ribosomal DNA genes (IGS-rDNA) on the X-chromosome and a single variant at position 702 of intron 1 (Int-1702) of the para Voltage-Gated Sodium Channel (VGSC) gene on chromosome arm 2 L. Intron-1 sequence data from West and Central Africa revealed two clearly distinct and species-specific haplogroups, each characterized by very low polymorphism, which has been attributed to a selective sweep. The aim of this study was to analyse Int-1 sequence diversity in A. gambiae and A. coluzzii populations from the Far-West of their range, in order to assess whether this selective-sweep signature could persist in a zone of high interspecific hybridization.Genomic differentiation between A. coluzzii, 31 A. gambiae, and 12 hybrids from The Gambia and Guinea Bissau, located within the Far-West geographical region, and in 53 A. gambiae s.l. samples from the rest of the range.A 531 bp region of VGSC Int-1 was sequenced in 21 A. coluzzii confirms previous evidence of a macro-geographic subdivision into a West and a Central African genetic cluster, and reveals a possible genetic distinction of A. coluzzii populations from the Far-West.Far-West samples exhibit dramatic Int-1 polymorphism, far higher within each country than observed throughout the rest of the species range. Moreover, patterning of haplotypes within A. coluzzii, with subsequent extensive gene flow with secondarily-colonizing A. gambiae, potentially yielding a novel insight on the dynamic processes impacting genetic divergence of these key malaria vectors.The results suggest a relaxation of selective pressures acting across the VGSC gene region in the hybrid zone. Genetic differentiation in the Far-West could be attributable to a founder effect within The online version of this article (doi:10.1186/s12936-014-0522-1) contains supplementary material, which is available to authorized users. Anopheles gambiae complex represent a valuable model for studies of ecological speciation (‘speciation with gene flow’) 2, 200 μMet al. . However, the close physical proximity of kdr and Int-1 would make recombination between resistant and susceptible kdr alleles and their linked Int-1 polymorphisms highly unlikely over a short timescale. Moreover, in A. coluzzii, as the samples analyzed came from populations where kdr-alleles were either absent or present at moderate to low frequencies , which became fixed in westward sympatric areas after the splitting of the two species.In West and Central Africa, Int-1 genetic differentiation between s Figure . Across uggested ,23. Sincic areas ,39. Indemutation . It is pt Africa ,42 [but on, 6.3% ,39. Recescenario ,16, subsA. gambiae and A. coluzzii is found - as indicated by the lower Fst observed in this region (0.14) as opposed to the rest of the range (0.51-0.69)  into A. gambiae is observed. These data are consistent with previous studies showing weak association between chromosome-X and −2 centromeric regions and occurrence of asymmetric introgression from A. coluzzii into A. gambiae in the westernmost extreme of their range [In the Far-West region, a strong reduction of inter-specific genetic divergence between egion 0.1 as opposir range -21. Furtir range  and founir range . However‘speciation island’ hypothesis [A. gambiae and A. coluzzii and promote a higher rate of gene-flow in the Far-West region. This hypothesis, however, is in contrast with data from other West and Central African areas, where introgression from A. gambiae to A. coluzzii of a kdr-related genomic portion in linkage with Int-1 does not produce an increase in hybridization rates [There are contrasting possible explanations for the remarkable Int-1 polymorphism observed in the Far-West region. Under the pothesis  relaxation rates . Alternaon rates ,16 and tA. coluzzii across its range provides some hints on further intra-specific geographical patterns (Figure A. coluzzii populations colonizing this region in the past. This last point merits further investigation through a multi-locus approach at a wider genome scale to shed light on the genetic characteristics of source populations originating the A. gambiae/A. coluzzii hybrid zone.Finally, the frequency and distribution of Int-1 haplotypes within s Figure . In fact