Acacia atkinsiana and the widespread, semi-continuously distributed A. ancistrocarpa. Lower intra-population diversity and higher differentiation between populations were seen in A. atkinsiana compared to its widespread congener, A. ancistrocarpa. There was little evidence of geographical influences on population genetic structure in A. ancistrocarpa whereas A. atkinsiana exhibited nuclear genetic structure with isolation by distance, differentiation of near-coastal populations from those in the ranges, and differentiation of peripheral populations from those in the centre of the distribution. These results are consistent with expectations of the effect of geographic range and population distribution on genetic diversity, but indicate that distribution of populations rather than geographic range has influenced the observed genetic structure. The contrasting patterns observed here demonstrate that conservation approaches for species management and ecological restoration need to consider the distribution of populations in geographically restricted species.The influence of geographic range on species persistence has long been of interest and there is a need for a better understanding of the genetic consequences for species with restricted distributions, particularly with the increasing rate of global species extinctions. However, the genetic effects of restricted range are often confounded by the impacts of population distribution. We compared chloroplast and nuclear genetic diversity and differentiation in two acacias, the restricted, patchily distributed  Geographic range is considered to be one of a number of key attributes of plant species that can indicate increased risk of extinction –4. SpeciIn line with population genetic theory, broad comparisons between geographically restricted and widespread species have indicated that in general, geographically localised species tend to have less genetic variation than common, widespread species , 18, 19.The distribution of populations is one factor that is likely to have a significant impact on patterns of genetic diversity both within and between populations. Patchily distributed populations are often smaller than semi-continuous populations, making them more vulnerable to the effects of genetic drift and inbreeding that deplete genetic variation , 26–28. Acacia is a dominant genus in Australian environments, particularly in arid ecosystems such as the Pilbara which is a centre of endemism for Acacia [The broad Pilbara region of north-western Australia provides an ideal opportunity to examine the influence of geographic range and distribution of populations on historical and contemporary patterns of genetic structure and intra-population genetic variation in plants. r Acacia , with mar Acacia , knowledr Acacia –41associet al [Callitris glaucophylla in the Pilbara compared to other areas, are evidence of confinement to microrefugia within the region during multiple glacial cycles. A phylogeographic study of Eucalyptus leucophloia showed genetic signature of the Hamersley Range being a refugium during arid climatic cycles with later expansion into non-range areas [There have been several assessments of genetic structure in Pilbara invertebrates –45 and vet al  suggest ge areas . Analysige areas . Within ge areas . SpeciesAcacia atkinsiana has a restricted distribution and is endemic to the Pilbara region compared to its widespread congener A. ancistrocarpa that is distributed across northern Australia. The restricted Acacia atkinsiana forms dense stands but has a patchy distribution compared to the semi-continuous distribution found in the widespread A. ancistrocarpa [Acacia ancistrocarpa has explosive dehiscence of its seed pods, although seeds are only propelled a very short distance from the plant, with most remaining directly beneath the plant. Therefore, we are able to make a comparison of species with differing geographical distributions and population distributions without the confounding effects of differences in gene flow. We assessed genetic diversity and structure using both nuclear and chloroplast markers to provide complementary understanding of factors influencing contemporary and historical variation within the species. Specifically, we investigated whether the distribution of genetic variation within and among populations of A. atkinsiana and A. ancistrocarpa differed, and whether these differences were associated with geographic range or distribution of populations. Consistent with population genetic theory, we predicted that genetic variation would be lower in the geographically restricted and patchily distributed A. atkinsiana than in the widespread and semi-continuously distributed A. ancistrocarpa. Further, the contrasting population distributions of the species led us to hypothesise that the patchily distributed A. atkinsiana would have greater genetic structure than the semi-continuously distributed A. ancistrocarpa, despite its narrower geographic range.Here, we investigated patterns of genetic diversity and structure in two Pilbara acacias that differ in their range and in the distribution of their populations. trocarpa . Althougtrocarpa  and neitAcacia atkinsiana is endemic to the Pilbara and adjacent Ashburton bioregions, being distributed mainly on the southern Roebourne Plain and in the Hamersley and Chichester Ranges [A. ancistrocarpa is widespread across northern Australia, including in the western, central and eastern Pilbara and the Hamersley and Chichester Ranges [Acacia atkinsiana is usually found in pure stands on water gaining sites, rocky, loamy, stony ground or ironstone hills. It is killed by fire, regenerating from soil-stored seed and there is no evidence of hybridisation with other acacias [A. ancistrocarpa grows mostly on acidic red sandy soils and stony pediments, often near water courses or water gaining sites. It is known to hybridise with A. stellaticeps, A. citrinoviridis, A. arida and A. orthocarpa. Hybrids with the latter three species are rare, and in the case of A. orthocarpa, are sterile [Acacia ancistrocarpa is also generally killed by fire but does have the ability to coppice from root stock. Acacias tend to be generalist insect pollinated and seed dispersal is commonly facilitated by ants or along waterways [Acacia atkinsiana and A. ancistrocarpa are classified to the same taxonomic section (Juliflorae) and their relationship can be seen in a detailed phylogeny of Acacia .r Ranges  Fig 1),,Acacia ar Ranges  Fig 2)..Acacia a acacias . In cont sterile . Acacia aterways , 56. AcaA. atkinsiana and A. ancistrocarpa were sampled within each species’ distributional range across the Pilbara ) than A. atkinsiana (mean distance between sampled populations: 147km (range: 30km-336km)). Phyllode material was collected from 24 plants within each of 16 populations of A. atkinsiana (384 plants) and 21 populations of A. ancistrocarpa (504 plants). Permission for field studies was obtained from The Department of Parks and Wildlife. The field studies did not include any endangered or threatened species.Pure (non-hybrid) populations of ara Figs  and 2. RA. atkinsiana and samples were genotyped at 11 microsatellite loci according to Levy et al. [A. ancistrocarpa and samples were genotyped using seven microsatellite primers previously developed for acacias  was used to score alleles.DNA was extracted from y et al. . A Qiage, 69, 29 , AM136, , 69, 29 ) and fou9 and 26 ). AmplifNon-coding spacer regions known to detect intraspecific variation in the chloroplast genome of Australian species  were trihttp://www.genecodes.com); where possible, forward and reverse sequence data were assembled to create a contiguous sequence. The edited sequences for each chloroplast region were aligned using Clustal W 1.4 [Sequence data was examined for quality using ABI Sequence Scanner v1.0 and edited using Sequencher v5.0  between the corrected and uncorrected FST values, the original data sets were used for all remaining analyses.The program FreeNa  was usedFIS), Shannon diversity index (I), observed (HO) and expected (HE) heterozygosity were calculated. Estimates of genetic diversity within populations and whether these differed between the two species, departures from Hardy Weinberg Equilibrium (HWE), and linkage disequilibrium (LD) were calculated using FSTAT [et al. [For each species, microsatellite variation within each population was measured using allele frequency data, from which average allelic richness (AR), inbreeding coefficient  matrix was compared with a geographical distance matrix (log km) [Genetic divergence between populations was estimated by calculating pairwise in FSTAT . To test(log km) , using a(log km) .KMAX) that maximises the within cluster Hardy-Weinberg and linkage equilibria. TESS incorporates spatial information and accounts for spatial autocorrelation (IBD). The admixture model in this program enables a proportionate membership of each individual to be assigned to each cluster. For each species, TESS was run with admixture (CAR model) using 50,000 sweeps following a burnin of 10,000 for 100 replicate runs of K ranging from 2–16 (A. atkinsiana) or 2–21 (A. ancistrocarpa) and applying a spatial interaction factor of 0.6. For each model, the Deviance Information Criterion (DIC) was obtained for each run and, for each value of KMAX, the mean DIC was plotted against KMAX. The best estimate of KMAX was taken as the beginning of the plateau [Population structure was analysed using an individual based Bayesian assignment approach implemented in TESS v2.3.1 . This pr plateau  and CLUM plateau  was usedvS, hS), total diversity  and population differentiation  were calculated using PERMUT v2.0 [π) was calculated in DnaSP v5.10.01 [D statistic, as implemented in ARLEQUIN v3.1 [For each species, ordered and unordered within-population diversity  was more varied in A. atkinsiana, although departures from HWE were only detected in A. ancistrocarpa . These departures from HWE are most likely due to artefacts such as null alleles rather than biological causes such as inbreeding, as none of the selfing rates calculated using RMES were significantly different to zero. High (above 10%) null allele frequencies were present in all populations for locus AM503, but because AM503 was also the most variable and thus informative locus, and because removal of these null alleles did not alter population differentiation (FST), we retained this locus for all analyses. There was no evidence of LD between any loci in any of the populations examined.Most estimates of microsatellite variation were significantly lower in  species . The inbA. atkinsiana (FST = 0.190) than in A. ancistrocarpa (FST = 0.047). Isolation by distance (IBD) was present in A. atkinsiana , but not in A. ancistrocarpa .Overall population divergence was greater in A. atkinsiana revealed a KMAX of seven. Populations within the Hamersley Ranges and along the coast formed two distinct groups  constituted less than a quarter of the total genetic diversity . Mean nucleotide diversity (π) was 0.007% and Tajima’s D did not deviate from the neutral model of evolution  with more (21) polymorphic sites and haplotypes (19) across 124 individuals  was found within populations  and nucleotide diversity was also greater (π = 0.027%). Tajima’s D did not deviate from the neutral model of evolution  than in A. ancistrocarpa . NST was larger than GST for both species but was not significant  that are likely to be ancestral since they are distributed throughout the range, occupy central positions, and have multiple connections in the network. The less common haplotypes are located at the tips and tend to be geographically localised suggesting they have arisen from the common haplotype and dispersed locally [A. ancistrocarpa with H15 being found at disjunct locations and H10 and H11 being geographically distant to genetically similar haplotypes. This may be the result of high levels of gene flow or, for H15, evidence of homoplasy. The high genetic variation and semi-continuous distribution of populations in A. ancistrocarpa would suggest gene flow, rather than homoplasy, as the most likely explanation for the distribution of H15.Although the patterns of haplotype diversity differed in  locally , 81. ExcA. atkinsiana than A. ancistrocarpa, with higher genetic divergence and IBD. This is also most likely due to the difference in distribution between the two species, as this pattern is consistent with theoretical expectations based on differences in distributions of populations [A. atkinsiana populations would be expected to reduce gene flow across the species’ range in comparison with the semi-continuous distribution of populations in A. ancistrocarpa. However, our findings are in contrast to most studies that find little difference in genetic structure between localised and widespread species [A. ancistrocarpa (263km) is indeed greater than A. atkinsiana (147km), genetic divergence is much lower (FST = 0.047 and 0.190 respectively) consistent with greater connectivity through gene flow among populations. Pollination in acacias tends to be facilitated by insects [A. ancistrocarpa is likely to be due to the influence of distribution of populations.There was greater population genetic structure in ulations , 26–28.  species , 19, not species  suggest, insects  and therA. ancistrocarpa sampled here may be due to dispersal of seed by strong wind gusts or surface water flows associated with the passage of tropical depressions that are common across the region [A. atkinsiana in a similar manner and therefore a similar pattern of population connectivity would be expected. Long distance seed dispersal may also be facilitated by birds since dispersal of seeds by emus across large areas is common and has been recorded for other acacia species in Western Australia [Eucalyptus camaldulensis in the Pilbara [Acacia species studied here has any obvious adaptations for dispersal, and would equally likely have seed dispersed by emus. However, emus are more common on the plains and grassy flats, where A. ancistrocarpa is prevalent, than in the upland ranges where A. atkinsiana is found. Over time, therefore, bird mediated seed dispersal is likely to have been greater in A. ancistrocarpa than in A. atkinsiana.Extensive gene flow throughout the range of e region . Howeverustralia , and sug Pilbara . NeitherA. atkinsiana where there were four haplotypes, with two common haplotypes spread throughout the range, while the two rare haplotypes were highly localised and detected in only one or two populations. In contrast, new mutations will disperse more rapidly and over a greater area in large connected populations maintaining greater diversity and less differentiation [A. ancistrocarpa, where the most common haplotype was found throughout the range, although not present in all populations, and several other haplotypes were shared across populations, some with large geographic disjunctions, along with many haplotypes with localised distributions. Thus, distribution and size of populations of these two acacia species appears to have had a demonstrable effect on the amount and distribution of chloroplast genetic variation.Phylogeographic structure in the chloroplast genome was also consistent with expectations based on population size and distribution. In small isolated populations, new mutations are more likely to be lost through drift than in large populations, but if they are retained and become more prevalent, they are unlikely to spread throughout the species range, remaining in localised areas . This isntiation . This paA. atkinsiana was largely independent of the geographical location of populations. This could be due to the accumulation and retention of ancestral polymorphism [The distribution of the common chloroplast haplotypes in morphism , 84 or hmorphism  and suchmorphism , 85.E. leucophloia [C. glaucophylla [A. ancistrocarpa, while in A. atkinsiana it seemed to be influenced by geographical features. In two regions, Hamersley/Chichester Ranges and near-coastal areas, multiple populations were largely assigned to the same genetic cluster, indicating that frequent gene flow occurs among these populations. Populations located at the periphery of the species range were mostly assigned to distinct clusters suggesting greater isolation, with reduced gene flow and enhanced genetic drift leading to genetic divergence [A. atkinsiana and there are no obvious environmental differences in the populations on the periphery of the Hamersley and Chichester Ranges. The population at CHIC may be further isolated by the Fortescue River that separates it from the other populations within the Hamersley Range. Populations found in the Fortescue River floodplain, namely MTF and KAN, while further away from CHIC than some of the Hamersley populations, have reasonable proportions of admixture with CHIC, suggesting gene flow occurs more frequently along the river floodplain than across it. However, because the Fortescue is a seasonal river, the river floodplain is unlikely to present a complete barrier to dispersal. All populations exhibited some degree of admixture with most clusters, and this pattern supports an explanation of IBD superimposed on a landscape where partial barriers to gene flow are present, generating large clusters made up of several neighbouring populations, with genetically distinct peripheral populations and a degree of admixture throughout the range.Given the lack of genetic studies on plants in the Pilbara, it is opportune to consider the patterns of genetic diversity and differentiation observed in these two species in connection with the landscape they occupy. Previous studies of animals endemic to the Pilbara have shown an affiliation between genetic structure and the geology and physiogeography of the region –45. Genecophloia  and perscophylla . In the vergence . Naturalvergence . HoweverA. atkinsiana is consistent with hypotheses of ranges providing heterogeneous conditions that are most suited to historical persistence [A. atkinsiana and A. ancistrocarpa may be due to greater connectivity among populations than in previously studied Pilbara animal systems.The evidence of greater connectivity among the Hamersley and Chichester Range populations of sistence . Alternasistence . Previousistence , 42, 45 A. ancistrocarpa where broad provenance collection zones are appropriate, seed collections for the restricted A. atkinsiana should be sourced more locally according to the patterns of relatively high genetic differentiations.Our results indicate that geographic range  and the distribution of populations (patchy vs semi-continuous) have had significant impacts on the amount of genetic variation within populations and the population genetic structure of two Pilbara acacias. While genetic diversity was consistent with predictions based on geographic range, as generally observed in comparisons of geographically restricted and widespread species, genetic differentiation was consistent with population distribution with the patchily distributed species showing greater differentiation than the more continuously distributed species. The contrasting patterns observed here demonstrate that conservation approaches for species management and ecological restoration need to consider the distribution of populations in geographically restricted species. For example, our study provides important information about optimal seed sourcing for mine site rehabilitation, a common activity in the iron ore rich Pilbara region. Identification of appropriate seed sources, through knowledge of patterns of genetic diversity and differentiation, that aim to optimise genetic diversity while minimising risk of outbreeding depression  is an imS1 Table(DOCX)Click here for additional data file.


AbstractJohniusgrypotus were collected from seven localities from the Bohai Sea to the East China Sea. A 549 base pair (bp) fragment of the hypervariable region of the mtDNA control region was sequenced to examine genetic diversity and population structure. The populations of J.grypotus showed high haplotype diversity (h) with a range from 0.7500 to 0.9740 and low nucleotide diversity (π) with a range from 0.0024 to 0.0067. Low and non-significant genetic differentiation was estimated among populations except for North Yellow Sea population, which has a significant genetic difference with other populations. The demographic history examined by mismatch distribution analyses and Bayesian skyline plot (BSP) analyses revealed that a sudden population expansion occurred almost 20 to 40 thousand years before. Relatively recent population expansion in the last glacial period, large dispersal of eggs or larvae carried by coastal current, and the homogeneity of living environment may have an important influence on the population genetic pattern.The 137 individuals of  LGM), approximately 20000 years before present . A vast body of evidence from the fossil and pollen records clearly demonstrates that many terrestrial species have undergone large and rapid latitudinal shifts in response to Pleistocene climate change, particularly following the end of the last glacial maximum ( present . The livmtDNA) has been the marker of choice in studying population structure and inferring phylogenetic relationships in animals due to its strict maternal inheritance and absence of recombination in most species . The mitochondrial DNA , the Yellow Sea  and the East China Sea (ZS) during 2005 to 2011 (Table The specimens of PCR) with universal primers of DL-S, 5’-CCCACCACTAACTCCCAAAGC-3’(forward), and DL-M, 5’-GCAACGTTCATATTCTCGGAGGC-3’(reverse). Reactions consisted of 10 × PCR buffer, 2.0mM MgCl2, 200μM of each dNTP, 0.3μM of each primer, 0.15 units of Taq DNA Polymerase (TaKaRa), and 1μL template DNA in a final volume of 25μL. PCR reaction was carried out in an Eppendorf thermal cycler with the following conditions: an initial denaturation for 5min at 94°C, followed by 35 cycles of 45s at 94°C, 45s at 50°C and 1min at 72°C, with a final extension at 72°C for 10min. The amplified products were gel separated and purified using the AxyPrep DNA Gel Extraction Kit. Purified products were sequenced using BigDye  with both forward and reverse primers, and analyzed on the 3730 automated sequencer (ABI).The first hypervariable segment of control region was amplified via polymerase chain reaction (Pro gene to prevent any bias in the estimates of sequence parameters from the control region. Molecular diversity indices such as haplotype diversity (h), nucleotide diversity (π), number of polymorphic sites (S), mean number of pairwise differences (k), transversions and transitions were obtained using the program ARLEQUIN Ver. 3.5 .Sequences were proofread, assembled and aligned using DNASTAR software . The 5’end of the control region fragment was extracted from the sequenced products by deleting partial sequences of the tRNARelationship among haplotypes was constructed using a neighbor-joining method performed in MEGA 5.0  with 100stF  with two groups: one group (NYS) and the other group .Genetic differentiation between pairs of population samples was evaluated with the pairwise fixation index Fst . The sigD test of Tajima  and Fs test of Fu  for each parameter exceeded 200.Changes in effective population size (Ne) across time were inferred using Bayesian skyline analyses, which enable past demographic changes to be inferred from the current patterns of genetic diversity within a population . BEAST vJ.grypotus were mostly showed the high gene diversity and low nucleotide diversity.  and remarkable avoidance of G base (15.33%), which was consistent with the base composition of the most of fishes. Sequence comparison revealed 60 haplotypes that were defined by 57 polymorphic sites with 43 transitions and14 transversions (Genbank accession numbers: MF381080 -MF381139). Total 8 haplotypes were shared among individuals, among which one main common haplotype was respectively shared by 37 individuals . The remaining 52 haplotypes were unique to their geographic populations. Intrapopulation diversity indices indicated that ZS population had the lowest genetic diversity among the seven populations. By contrast, the highest haplotype and nucleotide diversity was observed in HZB population. In summary, the seven populations of Unrooted phylogenetic tree was reconstructed by neighbor-joining analysis using 60 haplotypes with the best nucleotide substitution mode. There were no significant genealogical branches or clusters corresponding to sampling localities Fig. . The minstF values revealed that genetic differences between NYS and other populations were significant, and non-significant pairwise stF values were detected PageBreakPageBreakamong other populations.  and FS test  was negative and highly significant, meant departure from the selective neutrality. The sudden expansion model of mismatch distribution was unimodal and a valid goodness-of-fit was observed between observed and expected distributions  and divergence rate of 5%-10% per site per Myr  suggested one episode of population rapid growth in J.grypotus populations. The time of expansions about mismatch distribution was consistent with the rapid population growth intervals generated by BSP  and nonrecombinant DNA . TherefoPageBreaktrast, low genetic diversity generally tends to lead to population shrink, bottleneck and even extinction when facing the environment pressure. High gene diversity (h) suggests that J.grypotus populations have a strong adaptability of environment. However, the low nucleotide diversity (π), on the other hand, indicates that the populations have gone through bottleneck events and lost genetic information. The recent population expansion is likely to result in enhancing the retention of new mutations and a high genetic diversity that especially behaved in gene diversity because that the accumulation of nucleotide diversity was much slower . Some other fish in the Sciaenidae also show same phenomenon of “high h and low π” in control region, such as Pennahiaargentatus . High level of genetic diversity usually possesses the strong capacity for environmental endurance and for population extension. In congentatus , Larimicolyactis , Collichlucidus  and Miicysmiiuy .PageBreakstF values among populations and non-significant among groups. Pennahiaargentatus, Nibeaalbiflora, Larimichthyspolyactis and Miichthysmiiuy within Sciaenidae along the Chinese coastal waters perform the similar genetic structure pattern with J.grypotus . Many marine organisms have pelagic eggs and larvae that can potentially interconnect distant populations through dispersal on ocean currents . The phylogenetic and minimum spanning network displayed a shallow coalescence and non-significant genealogical branches or units. This was probably caused by the recent expansion after population bottleneck and (or) by the large gene flow among populations. This conclusion was sustained by the relatively low pairwise grypotus . Low gencurrents . The pelPageBreakJ.grypotus could’t pass though the deep sea area of the Yellow Sea, duo to it prefers living, reproducing and migrating in the coastal waters  popultions is quite nearly, the adult of l waters . The genJ.grypotus, received the tremendous influence. With the drop of sea levels in the glacial period, J.grypotus could have incurred population shrinkage and preserved some genetic information. The star-like network with one dominant haplotype suggests that J.grypotus most likely spread from one glacial refugium.Apartg from the life history and marine environmental factors, paleogeological changes and paleoclimactic fluctuations also have important influence on genetic diversity, genetic distribution pattern and effective population size . A series of large glacial-interglacial cycles caused pronounced fall and rise of the sea level in the late Pleistocene period, and glacial maxima at intervals of ~100,000 years over the past ~800,000 years were associated with declines in sea level of 120-140 m . This reChelonhaematocheilus , C.lucidus  interstadial in the last glaciation (J.grypotus and subsequently led to rapid population growth and spatial expansion.Demographic and range expansion must generate the recruitment and habitat extension from the refugia during the interglacial period with the temperature increase, habitat enlargement and food richness. The same scenario also has been found in many studies about molecular phylogeography of marine fish, such as ocheilus , P.argelucidus . Based oaciation . The eleJ.grypotus populations. Early life-history characters suggest relatively strong dispersal potential by eggs and larvae for this species, which may play an important role in large gene flow among populations. Therefore, it is essential to strengthen the management of eggs and larvae protection in the approach of conservation strategy. With overexploited fishery resource and water eco-environment deterioration, J.grypotus and other traditional PageBreakeconomic fish are severely threatened and depletion. The high level of genetic diversity of J.grypotus proves that it is not too late to increase awareness of protection. Our study will lay the foundation for periodic detection of genetic diversity, and provide scientific guidance for the management of fisheries and conservation efforts and for the sustainable development of J.grypotus resource.In conclusions, the glacial-interglacial cycles led to the climatic fluctuations in the Pleistocene have an important influence on abundance and distribution of


Johora singaporensis which is one of the world's 100 most threatened species, and currently inhabits less than 0.01 km2 of five low order hill streams within the highly urbanized island city‐state of Singapore. We compared populations of J. singaporensis with that of the non‐threatened, widespread, abundant, and eurytopic freshwater crab, Parathelphusa maculata, and found surprisingly high congruence between their population genomic histories. Based on 2,617 and 2,470 genome‐wide SNPs mined via the double‐digest restriction‐associated DNA sequencing method for ~90 individuals of J. singaporensis and P. maculata, respectively, the populations are strongly isolated (FST = 0.146–0.371), have low genetic diversity for both species , and show signatures of recent genetic bottlenecks. The most genetically isolated populations for both species are separated from other populations by one of the oldest roads in Singapore. These results suggest that anthropogenic developments may have impacted stream‐dependent species in a uniform manner, regardless of ubiquity, habitat preference, or dispersal modes of the species. While signs of inbreeding were not detected for the critically endangered species, the genetic distinctiveness and low diversity of the populations call for genetic rescue and connecting corridors between the remaining fragments of the natural habitat.Freshwater species often show high levels of endemism and risk of extinction owing to their limited dispersal abilities. This is exemplified by the stenotopic freshwater crab,  The sites—two in Bukit Gombak (Sites 1 & 2), one in Bukit Batok (Site 3) and one in Bukit Timah Nature Reserve (Site 4)—were selected based on the known distribution of J. singaporensis  and captured by hand. Given the threatened status of 2.2DNA was extracted from 20 to 26 samples per site using the phenol–chloroform method. Briefly, leg tissues were rinsed with autoclaved water, air‐dried, pulverized and then incubated at 55°C for 18 h in 800 μl CTAB buffer with 400 μg of proteinase K. The lysate was mixed well and washed with two rounds of 500 μl phenol–chloroform–isoamyl alcohol (25:24:1) followed by precipitation with 100% ethanol and a 70% ethanol wash. The final purified DNA pellet was eluted in ~40 μl molecular grade water, and stored at −20°C for further analyses.c oxidase I (COI) genotyping was performed using the primer pairs LoboF1 and LoboR1 , 5 cycles of 94°C (30 s), 45°C (1 min 30 s) and 72°C (1 min), 30 cycles of 94°C (30 s), 54°C (1 min 30 s) and 72°C (1 min), followed by a final extension of 72°C (5 min) for J. singaporensis; 94°C (1 min), 34 cycles of 94°C (45 s), 48°C (45 s) and 72°C (1 min 30 s), followed by a final extension of 72°C (3 min) for P. maculata. Successfully amplified products were purified using SureClean™ (Bioline) following the manufacturer's instructions, and sequenced using BIGDYE™ v. 3.1 (Applied Biosystems Inc.) on an ABI Avant 3130xl Genetic Analyzer.Cytochrome Prior to ddRAD‐Seq library preparation, the DNA extracts were quantified using a NanoDrop 1000 and assessed for quality on a 1% agarose gel. Of the extracted samples, 94 individuals were selected for ddRAD‐Seq such that each sampling locality was represented by 23 to 24 individuals for each species. The ddRAD‐Seq libraries were prepared as in Tay et al. , with moFor the following analyses, default parameters were used unless stated otherwise.2.3P. maculata and J. singaporensis, respectively. Haplotype (hd) and nucleotide (π) diversities were calculated using DnaSP v. 5 . Statistical parsimony haplotype networks were inferred using the TCS method  to obtain a comparable number of reads to P. maculata, and all subsequent demultiplexed reads were filtered for potential chimeras using a custom bash script  of 5 and 10, and maximum number of mismatches between “stacks” (−M) of 1, 2, 4, 8. Secondary reads were not retained; (3) Loci catalogs were assembled per species from these “stacks” in cstacks, with mismatches allowed (−n) following the mismatch thresholds for −M; (4) Loci were identified by matching “stacks” to the catalogs using sstacks; (5) SNPs were called using populations with minimum “stack” depths (−m) of 10, 20, 30, where at least 80% of individuals per population were required to be present at a locus (−r), and a minor allele frequency (MAF) (−a) filter such that at least three copies of an allele are present. For the J. singaporensis data set, two samples were removed due to low coverage from subsequent analyses. To check for possible contamination despite the data filtering, consensus sequences from the assembled catalogs of loci were matched against the entire NCBI nucleotide database and just the bacterial database alone. BLAST matches of e‐value ≤1 × 10−14 were not significant. Quick assessments for effects of different SNP‐calling parameter sets on the genetic clustering patterns across the samples were made for data sets with mismatch thresholds of 2 and 8  were automated using StrAuto  in diveRsity. Signatures of recent genetic bottlenecks were estimated using the heterozygosity excess method in BOTTLENECK , apart from the P. maculata individual which was overrepresented  to be the most distinct from the other three populations, but also distinct as four populations at K = 4  with none of the 95% confidence intervals containing zero .All three methods of population genetic structure showed similar patterns of highly differentiated populations for both species, with the population at Bukit Timah Site 4) being most distinct from the other three populations. While the Evanno method , which are strong indications of genetic bottlenecks. Significant heterozygote excess was observed in J. singaporensis, where HO = 0.31–0.33 while HE = 0.22–0.25 (Table P. maculata populations were mostly low (29.2 to 229.8) with narrow 95% confidence intervals; however, the present data were inconclusive for Bukit Timah and all populations of J. singaporensis, which had negative lower, and infinite upper bounds . The much larger population size of http://www.nas.gov.sg/archivesonline/maps_building_plans/ for 1839, 1939, 1945 and 1969), and found that the chronology of urban development correlated well with the population genetic differentiation signals from the genome‐wide SNP data. Strong genetic structure is observed for both species between the Bukit Timah population (Site 4) and those at Bukit Gombak and Bukit Batok (Sites 1–3) which could be explained by the isolation of Site 4 when Upper Bukit Timah Road was constructed. This is one of the oldest major roads in Singapore and was already present in some form by 1839 as a north–south connection through the island, and possibly earlier with the founding of Singapore as a British port‐settlement in 1819  have similar genetic divergence  until 1945, before development impacted this area. Dispersal by walking between streams within the higher order lowland stream network (which would presumably be more prone to flooding than the highland/hill streams) would be consistent with the observed intermediacy of the gene pool of the Site 2 population in Bukit Gombak among the sites to the west of Upper Bukit Timah Road (Sites 1–3)  and water quality differs considerably  from those of Singapore's natural forest streams in which freshwater crabs are restricted to  by 1969, whereas Sites 1 and 2 within the Bukit Gombak forest were still not fully separated by major road constructions in 1969.Arguably, population genetic divergence may, to a lesser extent, also be influenced by the crabs’ natural history. orks Ng, . Hence,  Sites 1– have simP. maculata is more than ten‐fold larger): Only seven COI haplotypes were found in >90 individuals for each species across four populations, as such the number of haplotypes per sampled individuals is 3–9× lower than previous reports for other species of endemic freshwater crabs  events with no subsequent exchange between populations. Sampling of historical populations would help us to better understand if and when haplotypes might have been lost through bottleneck events; however, there is little published data , a security area , and other areas designated as green spaces   , although its survivorship rates to maturity are unknown. There might also be yet undiscovered populations given that one population was recently discovered in the eastern part of Bukit Timah Nature Reserve  that are associated with the same habitats. All this work should be preceded by detailed surveys of flora, fauna, and the hydrological conditions in the affected habitats.It appears likely that the subtle differences between the two crab species are likely due to the short time frame and the fast development of Singapore over the last two centuries . COI sequences are available on Genbank (Accession numbers MG010138–MG010329). Additional information is available in the Supplementary material.Demultiplexed, raw Illumina sequencing ddRAD‐Seq data for each sample are available on Dryad ( Click here for additional data file.


DNA sequence (RADseq) data to investigate phylogeographic structure throughout Lake Tanganyika (LT) in two broadly sympatric rocky shore catfish species from independent evolutionary radiations with differing behaviors: the mouthbrooding claroteine, Lophiobagrus cyclurus, and the brood‐parasite mochokid, Synodontis multipunctatus. Our results indicated contrasting patterns between these species, with strong lake‐wide phylogeographic signal in L. cyclurus including a deep divergence between the northern and southern lake basins. Further structuring of these populations was observed across a heterogeneous habitat over much smaller distances. Strong population growth was observed in L. cyclurus sampled from shallow shorelines, suggesting population growth associated with the colonization of new habitats following lake‐level rises. Conversely, S. multipunctatus, which occupies a broader depth range, showed little phylogeographic structure and lower rates of population growth. Our findings suggest that isolation by distance and/or habitat barriers may play a role in the divergence of non‐cichlid fishes in LT, but this effect varies by species.Geographic isolation is suggested to be among the most important processes in the generation of cichlid fish diversity in East Africa's Great Lakes, both through isolation by distance and fluctuating connectivity caused by changing lake levels. However, even broad scale phylogeographic patterns are currently unknown in many non‐cichlid littoral taxa from these systems. To begin to address this, we generated restriction‐site‐associated  In the large East African rift lakes, multiple factors have been implicated in species divergence, for example, ecological adaptation, sexual selection, and allopatric divergence , the oldest African rift lake, contains exceptional species richness with high levels of endemism and multiple independent radiations , Synodontis multipunctatus. L. cyclurus is a mouthbrooder , as with cichlids from this lake, comprise multiple independent radiations , sampled from four main locations around LT , and SNPs were called using GATK UnifiedGenotyper  trees were calculated with the GTR+GAMMA model using RAxML 7.7.8  were run using 100,000 generations as burn‐in before sampling 100,000 generations using a model of admixture with correlated allele frequencies . The package fineRADstructure (v. 0.2) , with no missing data, leading to datasets of 10,380 sites for Lophiobagrus and 13,028 for S. multipunctatus. Samples were assigned to populations using 100,000 iterations as burn‐in before sampling 100,000 iterations. The trees were built using 10,000 iterations and the output visualized using the fineradstructureplot.r and finestructurelibrary.r R scripts (http://cichlid.gurdon.cam.ac.uk/fineRADstructure.html).Population structure was investigated using Bayesian clustering in L. cyclurus and S. multipunctatus datasets was further investigated using principal component analysis (PCA) in the R packages “adegenet” . FST values were calculated using the index of Reich, Thangaraj, Patterson, Price, and Singh . The significance of the D‐statistic (from 0) is usually calculated by computing the standard error of the D‐statistic using a block jackknife approach over linkage groups. This approach was not possible in this case, as in the absence of a reference genome, the order of RAD contigs is unknown, so reliability was assessed by randomly subsampling the dataset 1,000 times including 99%, 95%, 90%, 80%, and 70% of the data and calculating the mean and standard error of the D‐statistic. These analyses were conducted using a modified version of a python script from Rheindt et al. . The analysis was performed using average genetic distance between collection localities as within location samples are pseudoreplicates. The uncorrected P‐distances were calculated using the R package ape  in BEAST . This sample, from Sumbu, nests within the Mpulungu clade, and in the structure, analysis is comprised entirely of a cluster found in all Mpulungu samples  were larger than those between the Zambian populations and the most northerly population  . Genetic distances (p‐distance) in L. cyclurus increased significantly with straight‐line distances  and lake shore distances  around LT. This significant relationship between genetic distance and straight‐line distance remained when only the Zambian samples were considered  despite the low number of pairwise comparisons . The FST value of 0.034  but finds no support for further structure within the northern basin. Support for the separation of the northern and southern basins is lower in the phylogeny built with no missing data ; however, at K = 2, the northern and southern basins cluster separately . ESS values, with the exception of some operators, were above 200 in all analyses with the exception of one h Figure . All S. h Figure . In Buju4L. cyclurus, including within basin differentiation. Conversely, population structure in S. multipunctatus is weak, and although there is some support for population differentiation between the northern and southern basins from the phylogenetic and structure analyses, this pattern is not reflected in the FST values suggesting that it is driven by a small number of SNPs. Such weak structure may be due to its dispersal ability at depth  are consistent with the idea that populations are more stable at sites with steeper shorelines compared to shallow regions. However, there are more missing data in the reconstructions from Kigoma and we cannot discount the possibility that the weak or no population growth seen in L. aquilus‐C228 suggested possible interbreeding between this species and L. cyclurus. Notably, this specimen was collected in Mpulungu, despite its partial assignment to a genetic cluster most common in the Sumbu L. cyclurus population and was identified as L. aquilus based on the key of Bailey and Stewart (L. cyclurus, there was admixture between all populations with the overall patterns reflecting geographic structure. Population subdivision in ancestral species can also influence gene trees and the relative proportion of ABBA/BABA SNPs (Eriksson & Manica, L. aquilus has not been investigated and this species was sampled only from Zambia.The placement in both the phylogenetic and structure analyses of  Stewart . Within 4.1L. cyclurus in particular, fine‐scale population structure warrants further investigation with additional sampling localities required. This study is limited by low sample sizes, and further sampling would allow the timing and extent of gene flow between populations to be investigated plus the identification of habitat barriers for this species. Additional sampling localities in the southern basin of LT may allow the placement of sample C171 to be further understood in a broader geographic context. Further L. aquilus samples from these localities would also allow estimates of the extent of gene flow between these species to be investigated.Investigating diversity patterns both within and between species in biodiversity hotpots allow conclusions to be drawn as to which mechanisms are responsible for such elevated diversity. Here, we report contrasting geographic structure at a broad spatial scale in evolutionary divergent LT catfishes using genomic data. Our study suggests that lake‐level fluctuations have a role in structuring their diversity, although the genomic consequences of these differing histories remain to be studied. We recommend the study of additional species from non‐cichlid radiations with different ecologies to provide a more comprehensive understanding of evolutionary patterns and processes in these systems. Within LT catfishes restricted to the littoral zone, and in Conducted under COSTECH permit: no. 2010‐03‐NA‐2009‐207.http://doi.org/10.5061/dryad.671bsSequence data are accessible on NCBI under SRP126168. Datasets used in the analyses are available on Dryad None declared.All authors contributed to study design and wrote the manuscript. CRP and JJD conducted fieldwork, and CRP and KKD generated and analyzed the RAD data. Click here for additional data file.


Perisoreus canadensis), using the highly variable mitochondrial DNA (mtDNA) control region and microsatellite markers combined with species distribution modeling. We sequenced 914 bp of mtDNA control region for 375 individuals from 37 populations and screened seven loci for 402 individuals from 27 populations across the gray jay range. We used species distribution modeling and a range of phylogeographic analyses  to examine evolutionary history and population genetic structure. MtDNA and microsatellite markers revealed significant genetic differentiation among populations with high concordance between markers. Paleodistribution models supported at least five potential areas of suitable gray jay habitat during the last glacial maximum and revealed distributions similar to the gray jay's contemporary during the last interglacial. Colonization from and prolonged isolation in multiple refugia is evident. Historical climatic fluctuations, the presence of multiple dispersal barriers, and highly restricted gene flow appear to be responsible for strong genetic diversification and differentiation in gray jays.An increasing body of studies of widely distributed, high latitude species shows a variety of refugial locations and population genetic patterns. We examined the effects of glaciations and dispersal barriers on the population genetic patterns of a widely distributed, high latitude, resident corvid, the gray jay ( Sedentary species generally retain patterns of genetic variation longer due to limited dispersal, allowing researchers to make inferences about past historic events . Gray jay contemporary range encompasses a number of purported barriers to dispersal , in addition to previously glaciated  and unglaciated areas . Gray jays display plumage and morphological trait variation across their range , many of the sites included in the study had small sample sizes . Here, we use expanded sampling to include more populations from previously glaciated areas and incorporate more sites from the full distribution of gray jays. In addition, incorporating both mtDNA and microsatellite markers allows us to compare historical (mtDNA) and contemporary (microsatellite) genetic patterns in this species. Based on limited dispersal, patterns of glaciation during the LGM, and present distribution, we predict that gray jays expanded from multiple refugia throughout North America, and will exhibit high levels of genetic divergence between populations separated by barriers to dispersal.Using both mitochondrial DNA and nuclear microsatellite markers, we examine genetic structure and the effect of Pleistocene glaciations and dispersal barriers on genetic variation in this species. A previous study by van Els, Cicero, and Klicka  using mt22.1From 2007 to 2012, we captured gray jays at each sampling site (hereafter referred to as a population) using standard mistnetting techniques with call playback. We limited mistnetting locations to within a 50 km radius and sites contained no obvious barriers to dispersal. Sampling sites were paired in two ways: (1) located in areas that were previously glaciated and unglaciated during the last glacial maximum and (2) on either side of possible barriers to dispersal Figure . Additio2.22.2.12, 200 μmol/L dNTP, 0.4 μmol/L of each primer, and 0.5 units goTaq Flexi taq polymerase (Promega) under the following conditions: one cycle of 94°C for 120 s, 52°C for 45 s, and 72°C for 60 s, 37 cycles of 94°C for 30 s, 52°C for 45 s and 72°C for 60 s and one cycle of 72°C for five min. PCR products were run on a 0.8% agarose gel to confirm DNA amplification.We amplified a section of the mitochondrial DNA control region (CR) using primers L46 SJ  and H1030 JCR 18  or at the University of Lethbridge on a 3130 DNA Analyzer (Applied Biosystems). For in‐house sequencing, we used a shrimp alkaline phosphatase‐exonuclease clean up followed by sequencing and sodium acetate precipitation , 1 μmol/L of each primer (forward and reverse), 0.05 μmol/L of the fluorescent primer (Eurofins MWG Operon) and 0.5 units taq polymerase under the following conditions: one cycle of 94°C for 120 s, T1 for 45 s, and 72°C for 60 s, seven cycles of 94°C for 60 s, T1 for 30 s and 72°C for 45 s, 31 cycles of 94°C for 30 s, T2 for 30 s, and 72°C for 45 s, and one final elongation cycle at 72°C for 5 min  directly into the PCR product, we modified all forward primers by adding an M13 sequence (5′‐CAC GAC GTT GTA AAA CGA C‐3′) to the 5′ end. DNA was amplified in a 10 μl reaction with 1× buffer, 1 mmol/L MgClin Table . PCR pro2.32.3.1mega v 5.0  and nucleotide (π) diversity using arlequin v 3.11  using arlequin v 3.11  and expected (He) heterozygosities, and pairwise FST values  values, thus structure harvester . Data were inspected and occurrences outside of North America, without geo‐referencing, or recorded before 1950 were excluded from the analyses. From the GBIF data, we trained and tested the models using location records from field data, multiple museums, Animal Sound Archive Berlin, Borror Laboratory of Bioacoustics, Macaulay Library Audio Data, USDA Forest Service Lamna Point Count, Point Reyes Bird Observatory Point Counts, Ontario Breeding Bird Atlas 1981–1985 and 2001–2005, and Northwest Territories and Nunavut Bird Checklist. Duplicate records and remaining outliers were removed prior to model‐building.We used species distribution modeling (SDM) to construct a model of current, LGM (~21 ka), and Last Interglacial  gray jay distributions. Geo‐referenced locations were obtained from the Global Biodiversity Information Facility  at 2.5 min and 30 arc‐seconds resolution, LGM bioclimatic data from the Model for Interdisciplinary Research on Climate (MIROC) dataset at 2.5‐min resolution  to test the role of ecological variables on genetic variation. We ran two separate analyses, one for mtDNA genetic variation and a second for microsatellite genetic variation. DbRDA is a multivariate approach to test the effect of multiple predictor variables on one or more response variables  of 0.982, ranging from 0.707 (NNWBC) to 1.000  for NNWBC and AKW to 0.59 for NM and coWA  of gray jay populations was two (mean LnP(K) = −5579.66; ΔK = 115.76; Figure K = 2 analysis was composed of western and remaining boreal‐east populations. Again we found hierarchical structure, although there were fewer clusters within this region compared to the first main cluster. Within this second cluster, Vermont was a single cluster, the remaining boreal‐east populations  clustered into a single cluster, while WA and ceOR clustered together, and coWA and VI clustered into a fourth group , suggesting that the models were reasonable as AUC values above 0.75 are considered “potentially useful” , five main areas have a high probability of suitable gray jay habitat 0.5–0.8): most of Alaska and parts of Beringia, two areas in the southern Rockies, the SE US through Tennessee and Virginia, and the Pacific Coast including parts of Vancouver Island, Washington and Oregon  than geographic distance (r2 = .14) or geographic location (r2 = .13), while glaciation, altitude, and mean temperature were all significant, but accounted for a relatively small portion of the variance. For microsatellite genetic structure, the six variables accounted for a very small portion of variance (0.01–0.02). Similar to mtDNA patterns, precipitation during the coldest quarter was the top predictor of genetic variation among the six we tested . Our results indicate a weak effect of isolation by distance on genetic patterns overall, further suggesting the influence of barriers on genetic structure in gray jays.Our dbRDA models at the individual level found a significant relationship between the six environment variables we examined and both mtDNA and microsatellite genetic structure Table . Similar4FST) between most populations and clustering roughly corresponding to larger mitochondrial haplogroups. Exceptions to this include some splits amongst Borealeast populations, inclusion of AKF and CBC with Pacific Coast groups, and several populations that were difficult to consistently assign to a single cluster, suggesting nuclear genetic admixture between some groups.Geographic structuring and population differentiation suggest different evolutionary histories for gray jays in North America. Gray jays are partitioned into seven geographically distinct mitochondrial groups throughout their range: Pacific Coast; Vancouver Island; Intermountain West; CO‐NM; Utah; Newfoundland; and Boreal‐east. Microsatellite markers support similar breaks with significant differentiation  and fossil data Wetmore,  reinforcST and SAMOVA results based on mtDNA indicate individuals on Vancouver Island were likely isolated in a different refugium from those on the mainland as evident from the distinct sets of haplotypes on Vancouver Island. The Pacific Coast populations have remained relatively isolated from other populations, and SDM shows suitable habitat both on the mainland and Vancouver Island during the LGM and LIG. Other North American taxa show evidence of isolation on the mainland . CO‐NM, UT, and IMW groups are all closely associated with Engelmann and blue spruce, which are highly fragmented in the southern portion of their range  has a similar colonization history in the east. However, west of the Rocky Mountains, black spruce is thought to have colonized only from a southern, Pacific refugium  between mainland populations and the three island populations we sampled: Vancouver Island, Anticosti Island, and Newfoundland. Additionally, haplotype analyses and cluster analyses indicate genetic isolation of all three islands, although Anticosti groups with mainland populations based on haplotype analysis, while clustering analysis did not distinguish Newfoundland from other mainland populations. Similar patterns of genetic isolation for both plant and animal species have been found for Vancouver Island and Newfoundland, though usually with high‐resolution nuclear markers and not organellar DNA. The Salish Sea restricts populations on Vancouver Island , the northern Colorado and Utah populations are highly differentiated for both mitochondrial and nuclear DNA. Two possible reasons are large areas of unsuitable habitat or isolation of peripheral, disjunct populations. The Great Basin to the northwest, Wyoming Basin to the north/northeast and Snake River Basin to the north/northwest all act as barriers to dispersal and gene flow with neighboring populations. The divergence between Colorado and neighboring populations in Utah, but not between Colorado and neighboring populations in New Mexico, has been observed in other taxa  group, unlike some of the other isolated populations, occupies a central portion of the gray jay range, yet they are genetically distinct from surrounding groups for both mitochondrial and nuclear markers. Birds in this area are isolated from adjacent populations by the Columbia Basin/Okanogan Highlands to the west (Pacific populations), Columbia Mountains and Rocky Mountain Trench to the north and Columbia Mountains to the east , and the Snake River Basin to the south (Colorado and Utah). A similar genetic break occurs in mtDNA patterns in Engelmann spruce ). Thus, using control region sequences in this study provides a valuable comparison and complement to previous research.While some studies question using a highly variable marker like control region versus ND2 or cytochrome b for phylogeographic and phylogenetic studies, previous work has shown that this marker can be used to resolve deep splits in evolutionary history among avian species (Barker, Benesh, Vandergon, & Lanyon, Similar haplogroup patterns are found in van Els et al. ; however4.5Gray jay populations are highly differentiated, likely a consequence of limited dispersal for both males and females. Historical and contemporary gene flow is influenced by glaciation, barriers to movement such as large bodies of water and large areas of unsuitable habitat, and peripheral isolation. Additional research could include greater numbers of microsatellite loci or other nuclear markers to further enhance and complete our understanding of gray jay history and contemporary gene flow in North America.Overall our findings provide greater insight into the ecology, evolution, and conservation of boreal organisms. For example, gray jay geographic genetic patterns are similar to those found in spruce species, the conifer genus most commonly associated with preferred gray jay habitats, suggesting a close association between habitat and diversification in this species. Given this parallel, we would recommend future comparative phylogeography research that integrates genetic markers and species distribution modeling for gray jay, spruce, and other codistributed species. Incorporating this integrative approach is important, given that boreal habitats are under threat, as a result of climate change.None declared.KMD designed study, collected data, conducted laboratory work, analyzed and interpreted results, wrote and edited manuscript, and approved submitted manuscript. BAG collected data, analyzed and interpreted results, wrote and edited manuscript, and approved submitted manuscript. TMB designed study, interpreted results, edited manuscript, provided research facilities and funding, and approved submitted manuscript. Click here for additional data file.


Myrciaria dubia (Kunth) McVaugh] is currently an important and promising fruit species grown in the Peruvian Amazon, as well as in Brazil, Colombia, and Bolivia. The species is valued for its high content of fruit-based vitamin C. Large plantations have been established only in the last two decades, and a substantial part of the production is still obtained by collecting fruits from the wild. Domestication of the species is at an early stage; most farmers cultivate the plants without any breeding, or only through a simple mass selection process. The main objective of the study was to characterize morphological and genetic variation within and among cultivated and natural populations of camu-camu in the Peruvian Amazon. In total, we sampled 13 populations: ten wild in the Iquitos region, and three cultivated in the Pucallpa region in the Peruvian Amazon. To assess the genetic diversity using seven microsatellite loci, we analyzed samples from ten individual trees per each population (n = 126). Morphological data was collected from five trees from each population (n = 65). The analysis did not reveal statistically significant differences for most of the morphological descriptors. For wild and cultivated populations, the observed heterozygosity was 0.347 and 0.404 (expected 0.516 and 0.506), and the fixation index was 0.328 and 0.200, respectively. Wild populations could be divided into two groups according to the UPGMA and STRUCTURE analysis. In cultivated populations, their approximate origin was determined. Our findings indicate a high genetic diversity among the populations, but also a high degree of inbreeding within the populations. This can be explained by either the isolation of these populations from each other or the low number of individuals in some populations. This high level of genetic diversity can be explored for the selection of superior individuals for further breeding.Camu-camu [ The Amazonian tropical ecosystem is characterized by a high biodiversity of plant species , of which there are more than 150 edible fruit-bearing species used by the local population. However, only a few of these currently have a significant economic importance [Myrciaria dubia (Kunth) McVaugh], a shrub or small tree from the family Myrtaceae, has become an economically important fruit species in the recent decades  and, based on these sequences, seven specific microsatellite primer pairs were designed  with the following profile: 95°C for 2 min; followed by 30 cycles of 95°C for 1 min, either 51°C (M1) or 57°C (M2) for 90 s, 72°C for 1 min; followed by a hold at 72°C for 5 min. The PCR products were separated by electrophoresis in an ABI PRISM 3500 sequencer . A 1-μL aliquot of PCR product was mixed with 0.5 μL of GeneScan-500 LIZ (Applied Biosystems) and 12 μL of Hi–Di formamide (Applied Biosystems). Allele sizes were determined using GeneMarker version 2.4.0 . A microsatellite locus was treated as missing data after two or more amplification failures. Null allele frequencies were calculated using the Brookfield 1 equation , 19, andlme command, with nested design—random effect tree), Principal Component Analysis . PCA diagrams were visualized using     and STRUTRUCTURE , ΔK (S3 CTURE (K  indicateK = 2), depicted by blue color, is dominated by populations from the Curaray , Tigre (TH), Itaya (IP), and Napo  rivers, while populations PC, Pc, and PM are from the Putumayo River (green color). The cultivated populations from Pucallpa (Y2 and Y3) were assigned to the first genetic cluster  and population Y1 is genetically closely related to the populations from the Putumayo River (The genetic cluster (yo River .K = 3), populations from the Putumayo River  formed a separate genetic cluster. The rest of the populations were divided according to the river system into two groups . The genetic composition of the cultivated populations showed that they were combined from only two areas  (When the populations were divided into three genetic clusters ( rivers) .By division into five genetic clusters, we were able to recognize four geographically distinct groups of populations along different rivers, especially when populations occurring in the Napo, Tigre and Itaya rivers were further divided. The cultivated populations Y2 and Y3 were assigned to the population Itaya–Pelejo. Cultivated population Y1 remains part of the “Putumayo” group .M. dubia in the Peruvian Amazon. It is evident those plants grown in plantations were neither selected nor bred, and that they possess quite similar phenotypical characteristics to wild plants. The first attempt at the cultivation of camu-camu in Peru was in 1980, and this trial was established near Iquitos [The measured morphological characteristics showed low variability within and among populations. The wild and cultivated populations were not found to be significantly different, and we did not detect any differences among the populations. The only significant difference we detected was the number of seeds per fruit, where the cultivated populations had a higher number of seeds per fruit. These results clearly show a low degree of domestication of  Iquitos . Thus, tAccording to our results, the use of morphological descriptors for quantitative and qualitative traits of leaves had only little significance. Greater importance should be placed on the detection of fruit characteristics such as diameter, weight, and number of seeds, the weight of the pulp, and vitamin C content, which was not included in this study. It seems that cultivated populations of camu-camu were not yet highly selected according to fruit morphological traits, and, seemingly, there remains potential to select individuals with larger fruits from wild populations.We are aware of the limitation of our study as the sample size per population was relatively low (8–10 trees per population), so that the present results might be different if the sample size of the populations was increased. This is because the estimated genetic diversity indices are strongly dependent of the accurate estimate of gene frequencies within populations.However, compared to our morphological characteristics findings, the microsatellite loci detected a high level of genetic diversity among and within populations. The expected heterozygosity was higher than the observed heterozygosity, and the fixation index showed high rates. Similar results were also obtained by Rojas et al.  and KoshMyrciaria floribunda, which is native to the wet forests of Central and South America. Allozyme markers were used to determine the genetic diversity. The fixation index was 0.153, which is lower than that reported here for M. dubia. This is likely because allozyme markers are less variable than SSR markers. Nevertheless, allozyme markers were still able to demonstrate higher values of homozygotes in the populations compared to our study. The higher fixation index was again explained by the bottleneck effect and low gene flow, and this confirms the results of our study.Franceschinelli et al.  performeIn the dendrogram, it can be seen that populations were divided into groups depending on the river watershed where they were located. For example, in our case, population Napo-Núñez (NN) is located close to the conflux of the Napo and Amazon rivers. Population Itaya–Pelejo (IP) was located on the Itaya River, which also flows into the Amazon, and this conflux is not far from the conflux of the Napo and Amazon. During the rainy season, some fruits were probably naturally transported from the Itaya River to the tree populations of the Napo River, and genes were mixed. This can be a possible explanation why populations NN and IP belong to the same group, and why population NN is not connected with population NY from the same river. This theory was supported by Rojas et al. , who achM. dubia are primarily distributed by water during floods, and pollen is not transmitted over longer distances. Populations from the second group are closer in proximity, and the rivers are often connected during floods and, thus, enable the transfer of genetic material.STRUCTURE software divided the natural populations into two distinct groups. Populations PM, Pc, and PC make up the first group, lying on the river Putumayo. This river is geographically remote from other populations, and upland tropical rainforest complicates its connection with those populations. This is because the seeds of The cultivated populations can also be divided into two groups according to their origin. The populations Y2 and Y3 originated in the catchment area of the rivers Curaray, Tigre, Napo, and Itaya, and were brought by farmers to the Pucallpa region. STRUCTURE software (K = 5) assigned the origins of these populations closer to Iquitos city, around the river Itaya. The Y1 population was assigned to the other group, and its origin lies in the area of the river Putumayo on the border between Peru and Colombia, flowing into Brazil. Further research will be necessary to confirm this hypothesis.in situ and ex situ, and the most promising genotypes should be stored in germplasm banks. For further studies focused on this species, we propose to also collect plant material from the peripheral areas of its occurrence . Plants from those marginal areas might be more resistant to unfavorable climatic conditions and could allow the cultivation of this species outside of the area of its natural occurrence. It is also recommendable to compare the variability of populations from the Peruvian Amazon and Brazil, to find populations with superior plants for further breeding. The fruit collection from wild populations leads to losses of genetic material because the excessive collection of the fruits reduces the amount of seeds, and thus might reduce the number of plants in the population. These losses might affect the structure of these populations in the future and cause its degradation. Because the demand for M. dubia continues to rise, selection and breeding can allow easier and more efficient cultivation, and thus prevent the destruction of the natural populations.Genetic analysis revealed significant differences between wild populations. These observed differences in genetic diversity should be used for the preservation of wild genotypes M. dubia. From all detected characteristics, only fruit parameters had the tendency to be different. However, those could be possibly explained by different environmental conditions in which the populations were grown, or by the lack of collected data. After evaluation of the fruits, it was found that the cultivated populations chosen for our study have not yet passed through any process of domestication, and had more-or-less the same morphological characteristics as the wild varieties.This study found a low morphological variability within and among wild and cultivated populations of M. dubia.Seven of the eight microsatellite loci developed were polymorphic and showed a high level of genetic diversity within and among populations. Bayesian analyses divided the wild populations into two main groups , and were able to show us the origin of cultivated populations. For this reason, microsatellite primers developed within this study can be recommended for further population-genetic studies of Our research showed that populations located in different watersheds also possessed a different genetic composition. The origin of each population assigned according to each watershed increased the genetic distance between the populations and, thus, overall genetic diversity. Genetic diversity among and within cultivated populations was also relatively high, thanks to the different origin and generative propagation of plants by the use of seeds. Transfer of seeds and seedlings by local inhabitants plays a crucial role in the preservation of this genetic diversity.This diversification could be used in the future breeding of this species. The crossbreeding of the individual trees from different geographical locations that suffer from inbreeding, and are genetically different, could result in a heterosis effect, and the resulting hybrids will possess higher genetic diversity, which might be more suitable for growing in plantations.S1 Fig(TIFF)Click here for additional data file.S2 Fig(JPG)Click here for additional data file.S3 Fig(TIFF)Click here for additional data file.S1 Tablen), mean, median, and standard deviation (SD) of each characteristic. Red-marked data with an asterisk were found to be statistically significant at probability level p = 0.05.Including the number of samples ((DOC)Click here for additional data file.S2 TableHo, observed heterozygosity; He, expected heterozygosity; F, fixation index; Fst, fixation index of a subpopulation relative to the total population.(DOC)Click here for additional data file.


Haemorhous mexicanus) using samples collected before and after a pathogen‐induced selection event. Using ddRADseq, we genotyped over 18,000 SNPs across the genome in native pre‐epizootic western US birds, introduced birds from Hawaii and the eastern United States, post‐epizootic eastern birds, and western birds sampled across a similar time span. We found 14% and 7% reductions in nucleotide diversity, respectively, in Hawaiian and pre‐epizootic eastern birds relative to pre‐epizootic western birds, as well as elevated levels of linkage disequilibrium and other signatures of founder events. Despite finding numerous significant frequency shifts (outlier loci) between pre‐epizootic native and introduced populations, we found no signal of reduced genetic diversity, elevated linkage disequilibrium, or outlier loci as a result of the epizootic. Simulations demonstrate that the proportion of outliers associated with founder events could be explained by genetic drift. This rare view of genetic evolution across time in an invasive species provides direct evidence that demographic shifts like founder events have genetic consequences more widespread across the genome than natural selection.Identifying genomic signatures of natural selection can be challenging against a background of demographic changes such as bottlenecks and population expansions. Here, we disentangle the effects of demography from selection in the House Finch ( All species experience range expansions and contractions through time, but human activities are accelerating the pace of range shifts through alteration of habitats and direct movement of species Hulme, . IntroduIn addition to novel demographic shifts, introduced species can also encounter novel selection regimes in newly colonized habitats. Infectious diseases constitute one of the strongest selective pressures encountered in novel habitats and can have profound impacts on a species by increasing host mortality and decreasing reproductive output , a common bird in both urban and rural environments in North America, has become a model for the study of adaptation to novel environments following introductions and for host‐pathogen coevolution , which precipitated an epizootic event that is now well documented by demographic and genetic studies as well as statistical models of host–pathogen coevolution  subspecies  and the Cassin's Finch  , using the standard blood and tissue protocols as appropriate. Each individual was barcoded and genotyped following a ddRADseq , but differed in SNP filtering. Dataset 1 used a less conservative SNP filter, requiring an individual minimum locus depth of 10 to be included (–m 10), and a locus to only be included if it was present in at least 75% of individuals in half of the populations (–r .75 and –p 11). The second, more conservative filter (dataset 2) required an individual minimum locus depth of 30 (–m 30) and had the same inclusion parameters. Analyses of dataset 2 produced results very similar to those of dataset 1, although sometimes less resolved due to the smaller number of loci, so we only present the results of dataset 1 throughout the rest of the article. Individuals from all time periods were used to build these STACKS catalogs. We also ran some preliminary analyses on datasets with more stringent completeness filters, but found that analyzing these datasets had very little effect on overall results. The final dataset (dataset 3) focused on maximizing high‐quality SNPs from the temporal samples for analyses seeking to identify MG‐mediated selection. This dataset only included individuals from the diachronically sampled subpopulations , but included both time periods. The quality filter required an individual minimum locus depth of 30 (–m 30), and a locus was to be included if it was present in at least 75% of individuals in two of the samples: Pre‐E, Post‐E, Pre‐W, and Post‐W (–r .75 –p 2).With the p < .05) in at least two of the three populations ; or a locus mapped to the Zebra Finch (Taeniopygia guttata) genome version 3.2.4 .We further refined each dataset by removing possible problematic loci that matched any of the following three criteria: the locus contained a SNP with observed heterozygosity greater than 0.75; the locus contained a SNP that was not in Hardy–Weinberg equilibrium (2.5K) that best fit the data and to assign individuals to these clusters. We first tested for structure among all pre‐epizootic individuals, including outgroups . Because variation among higher levels of population structure can mask substructuring ; within the native frontalis population ; within the introduced eastern frontalis population ; and within the introduced Hawaiian frontalis population . To determine the optimal number of clusters (K), we considered the highest ΔK, as recommended by Evanno et al. .We used the r2; Hill & Robertson, Pairwise_linkage_disequilibrium.py available at https://github.com/ajshultz/Rad/). Because sample size can affect measures of LD, for each population we randomly chose eight individuals with less than 50% missing data. First, we calculated r2 between all pairs of SNPs located on the same locus (between 1 and 139 base pairs apart). Second, we compared the mean r2 value between SNPs located on read 1 and read 2 loci from the same DNA fragment . We compared these values to the equivocal number of randomly chosen pairs from the catalog of possible loci. By comparing levels of LD from loci on the same DNA fragment to levels from randomly chosen loci, we could assess whether the decrease in LD observed in the single locus analysis degraded to a level of LD lower than that observed in a single read. Additionally, by confirming that levels of LD were similar among populations with randomly chosen loci, we could ensure that any significant differences observed in loci in a single read we observed among populations were not an artifact of dataset structure .We calculated linkage disequilibrium  samples with datasets 1 and 3; and Pre‐W versus Post‐W (CA) samples with datasets 1 and 3. First, we used the −9 per site per year  twice, once with sampling modeled after the Pre‐W versus Pre‐E comparisons  and once with sampling modeled after the Pre‐W versus Hawaiian comparisons . For all simulations, we calculated the significance of allele frequency differences between populations with a Fisher's exact test, corrected for multiple testing as described above.We simulated datasets to generate expectations for the proportion of outlier loci under the neutral model for an introduction event. We used the program ms Hudson,  to gener33.1de novo assembly generated an average of 12,700 unique loci per individual, of which an average of 3,217 were polymorphic (Table S1). Across the three datasets, when loci were mapped to the Zebra Finch genome with a minimum e‐value of 10−40, 12% of loci consistently mapped more than once and were subsequently dropped from the analysis. Approximately 65% of loci mapped just once to the Zebra Finch genome, and 24% did not map at all. Loci fell evenly across the entire genome , and the number of loci per chromosome was significantly correlated with Zebra Finch chromosome length . After filtering for multiple hits to the Zebra Finch genome and deviations from Hardy–Weinberg equilibrium, dataset 1  contained 2,283 loci and 18,096 SNPs, was 73% complete, and had a mean depth per locus of ~60. Dataset 2  contained 889 loci and 6,877 SNPs, was 67% complete, and had a mean depth of ~68. Dataset 3  contained 2,150 loci and 8,561 SNPs, was 55% complete, and had a mean depth of ~66. Across all datasets, there was a high amount of variability in the amount of data missing in any particular individual , most likely caused by sensitivities of the ddRADseq protocol to tissue degradation and long‐term storage.We obtained a total of 40,151,299 paired‐end 150 base pair reads that passed Illumina's quality filter for 126 individuals . The 3.2frontalis House Finch birds, K = 3 was optimal (Table S2), separating out the native western, introduced Hawaiian, and introduced eastern populations , which separated the Kauai individuals from those sampled on Oahu and Maui , but this result did not appear to contain any biologically useful information, with three clusters equally likely in all individuals .Using all 18,096 SNPs in dataset 1, the Pre‐W population had consistently higher levels of polymorphic sites, private sites, observed heterozygosity, and nucleotide diversity than the Pre‐E population or Hawaiian population Table . These rFST among pre‐epizootic populations were consistent with the STRUCTURE analyses. For dataset 1, the species outgroups were the most divergent from the frontalis individuals , followed by the subspecies outgroup . Populations within the eastern and western regions were the least differentiated , whereas levels of differentiation between eastern and western subpopulations were slightly higher . The Hawaiian population was the most differentiated from the other frontalis populations ; FST was even higher between Kauai and the other two islands within Hawaii (0.099–0.129).Average pairwise p < .0001, Pre‐W vs. Hawaiian Wilcoxon rank‐sum test p < .0001), but we found no difference in LD between Pre‐ and Post‐E populations . There were 375 loci from read 1 and read 2 of the same DNA fragment as calculated by our novel python script . Levels of LD were higher for SNPs on pairs of loci from the same DNA fragment than for SNPs on randomly chosen pairs for all populations . However, there were no differences among paired locus r2 values from different populations .The Pre‐W population exhibited lower levels of LD than either the Pre‐E or Hawaiian populations  with a FDR of 5%. Of these, 136 could be mapped to the Zebra Finch genome . BayeScan detected four outliers from two loci in the Hawaiian versus Pre‐W comparison, of which one was mappable . All loci identified by BayeScan were also detected in outlier analyses reported above.Simulations of founding events under the neutral model had proportions of significant allele frequencies greater than or comparable to those found in our empirical data Table . This ho4Both natural selection and drift due to founder events can lead to genotypic changes in populations, and disentangling the relative contributions of these evolutionary forces can be difficult if one relies solely on contemporary samples from a single time period. Comparisons of the genotypes of populations across time and space enable better deductions of the evolutionary processes that underlie genomic change  produced a larger proportion of allele frequency differences than our observed values, suggesting less extreme founding events in the House Finch.Regions of the genome exhibiting significant differentiation as a result of human introductions could be due either to adaptation to the novel environment or to the genetic effects of a bottleneck Lee, . DespitePeromyscus), we find that signatures of selection are likely obscured in founder events on historical time scales due to genetic drift.Although a moderate bottleneck can explain most or all of the observed differences in allele frequency between native and introduced populations, some shifts may have been driven by selection in the novel environments experienced by the introduced populations. The eastern population has smaller legs and feet than the western population . Small sample size has been shown to inflate values of Tajima's D . We detected loci with shifted allele frequencies as a consequence of the human‐induced founder events and suggest that these shifts are likely more often caused by genetic drift than selection. Despite a favorable scenario for detecting pathogen‐mediated signatures of selection in the eastern United States with temporal sampling , RADseq was unable to detect genome‐wide reductions in diversity or loci with significantly different allele frequencies before and after the epizootic, results likely driven by overall low levels of LD throughout the House Finch genome, as well as the low density of markers generated by ddRADseq. Overall, our study provides a rare direct comparison of temporal and spatial events within the same species and confirms a hypothesis that is rarely tested in side‐by‐side comparisons: That demographic shifts, such as bottlenecks or range expansions, may have more profound and genome‐wide consequences for genomic variation than will selection imposed by a novel pathogen. Therefore, our study suggests that, despite conservation concern with selective events such as epizootics, if populations maintain sufficient effective population sizes to mitigate the effects of genetic drift, there may be few genomic consequences of such events in nature.None declared.http://dx.doi.org/10.5061/dryad.0h2g0.Data available from the Dryad Digital Repository:  Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.