Cycas revoluta and C. taitungensis, species that constitute the section Asiorientales and represent a unique, basal lineage of the Laurasian genus Cycas. Fossil evidence indicates divergence of the section from the rest of Cycas at least 30 million years ago. Geographically, C. taitungensis is limited to Taiwan whereas C. revoluta is found in the Ryukyu Archipelago and on mainland China.This study addresses the apportionment of genetic diversity between atpB and rbcL genes of cpDNA were reconstructed. Phylogenetic analyses revealed paraphyly of both loci in the two species and also in the section Asiorientales. The lack of reciprocal monophyly between these long isolated sections is likely due to persistent shared ancestral polymorphisms. Molecular dating estimated that mt- and cp DNA lineages coalesced to the most recent common ancestors (TMRCA) about 327 (mt) and 204 MYA (cp), corresponding with the divergence of cycad sections in the Mesozoic.The phylogenies of ribosomal ITS region of mtDNA and the intergenic spacer between Fates of newly derived mutations of cycads follow Klopfstein et al.'s surfing model where the majority of new mutations do not spread geographically and remain at low frequencies or are eventually lost by genetic drift. Only successful 'surfing mutations' reach very high frequencies and occupy a large portion of a species range. These mutations exist as dominant cytotypes across populations and species. Geographical subdivision is lacking in both species, even though recurrent gene flow by both pollen and seed is severely limited. In total, the contrasting levels between historical and ongoing gene flow, large population sizes, a long lifespan, and slow mutation rates in both organelle DNAs have all likely contributed to the unusually long duration of paraphyly in cycads. The analysis of geographical structure and phylogeographical pattern has been a major focus in population genetics and molecular ecology . PopulatCoalescence studies provide information on the genealogical processes (backwards in time) that have led to the current apportionment of genetic variation within populations or species. Coalescence-based phylogeography uses haplotype networks, which trace the mutational relationships among alleles within or among species in a geographical context , to infeCycas revoluta Thunb. and C. taitungensis Shen et al. constitute the section Asiorientales which is a basal lineage of this Laurasian genus . Des. Des20]]Today, islands of the Ryukyu archipelago are isolated from each other. In the past, sea level oscillated during periodic glacial cycles (Milankovitch cycles) -29. DuriatpB and rbcL genes of cpDNA were used to trace phylogeographical relationships in C. revoluta and C. taitungensis. Organelle DNAs are maternally inherited in cycads; they disperse only via seeds . Most cistances ,37. The istances . In natuistances ,39. Oceaistances . Many plinghamia , MicheliMichelia , KandeliKandelia , Trema . Con. Con53]]ity [cf. ].et al. ..54].Nm values, deduced from Fst values, are much greater than one  and 1.87 to 10.25 (cpDNA) , contributing to the lack of genetic differentiation between populations, as indicated by Fst values of 0.0056 (cpDNA) and 0.021 (mtDNA) .C. revoluta and C. taitungensis, respectively.Like other island species, cycad populations were colonized via founders from neighboring islands or the mainland. Today, deep water channels hinder seed dispersal among islands and most seed dispersal occurs only within islands. However, during the glacial maxima [cf. ], sea leAsiorientales and its sisters. Such lack of reciprocal monophyly between taxa may result from selection, e.g., the S locus of Brassica .Cycas given that these are neutral loci and thus not under strong selection. Discerning between hybridization and lineage sorting can be difficult , as Nm values . The shThe evolution of these two cycad species is complex, affected by range expansion and contraction, fluctuations in population size, widespread historical and current restricted gene flow during complex glacial cycles as well as low rates of molecular evolution in organelle DNA. All of these processes result in a pattern of genetic similarity among populations, a lack of reciprocal monophyly, and long coalescent times.Cycas section Asiorientales ever since the split from its sisters some 30 million years ago. Large population sizes of the constituting species C. revoluta and C. taitungensis contributed to the lack of reciprocal monophyly betwen Cycas sections. Providing limited current seed dispersal across oceans, unexpected low levels of geographical subdivision are attributable to the sharing of common haplotypes among island populations. Demographic expansion and contraction with fluctuating population sizes, widespread historical vs. current restricted gene flow during complex glacial cycles, and low rates of molecular evolution in organelle DNA all have to be aroused to explain such population structuring.In summary, paraphyly at both organelle DNAs has been long maintained in Cycas revoluta and C. taitungensis in Taiwan, Fukien, and Ryukyus  and C. thouarsii R. Brown (Section Rumphiae) , were crocedure . The atpL spacer  or mtDNAL spacer ], dNTP aPCR products were purified by electrophoresis in 1.0% agarose gel using 1 × TAE buffer. The gel was stained with ethidium bromide and the desired DNA band was excised and eluted using QIAquick Gel Kit (QIAGEN). For all individuals, purified amplicons were ligated to a pGEM-T easy vector system (Promega). Five clones were randomly selected and purified using the Plasmid Mini Kit (QIAGEN). Purified cloned ampicons were sequenced in both directions using BigDye chemistry (Perkin Elmer) on ABI 377A automated sequencer (Applied Biosystems). Primers for sequence determination were T7-promoter and SP6-promoter located on p-GEM-T easy Vector termination site. As no within-individual variation was detected for either DNA marker, direct sequencing with eluted PCR products was also conducted to all individuals.Nucleotide sequences were aligned with the program BlastAlign . After tThe hypothesis of a molecular clock was tested by relative rate tests ,91. The Summary statistics such as the number of segregating sites (S), and the average number of pairwise difference (k) between haplotypes were determined. The level of genetic diversity within populations was quantified by measures of nucleotide divergence, θ  and φ 9, using DC. taitungensis and C. revoluta split for both mt- and cpDNAs. To estimate divergence between lineages a well-supported rate of evolution is required. In seed plants, evolutionary rates are estimated at 1.01 × 10-9 and 4.50 × 10-10 substitutions per site per year for synonymous sites of cp- and mt-DNAs, respectively . Th. ThC. ta.ed.ac.uk. We usedR v. 1.3 . PosteriWe used the simulation program IM ,103 to iYCC and KHH carried out the molecular genetic studies and the statistical analysis, and participated in the sequence alignment. TYC, TWH and BAS designed the research and drafted the manuscript. SJM, SH, and XJG took part in material sampling. All authors read and approved the final manuscript.


Aegla neuquensis (Aeglidae: Decopoda). This taxon occurs in two large river systems in the Patagonian Steppe, from the foothills of the Andes Mountains east to the Atlantic Ocean.Multiple loci and population genetic methods were employed to study the phylogeographic history of the Patagonian freshwater crab A nuclear phylogeny and multilocus nested clade phylogeographic analysis detected a fragmentation event between the Negro and Chico-Chubut river systems. This event occurred approximately 137 thousand years ago. An isolation-with-migration analysis and maximum-likelihood estimates of gene flow showed asymmetrical exchange of genetic material between these two river systems exclusively in their headwaters. We used information theory to determine the best-fit demographic history between these two river systems under an isolation-with-migration model. The best-fit model suggests that the Negro and the ancestral populations have the same effective population sizes; whereas the Chico-Chubut population is smaller and shows that gene flow from the Chico-Chubut into the Negro is four times higher than in the reverse direction. Much of the Chico-Chubut system appears to have only been recently colonized while the Negro populations appear to have been in place for most of the evolutionary history of this taxon.Due to mitochondrial introgression, three nuclear loci provided different phylogeographic resolution than the three mitochondrial genes for an ancient fragmentation event observed in the nuclear phylogeny. However, the mitochondrial locus provided greater resolution on more recent evolutionary events. Our study, therefore, demonstrates the need to include both nuclear and mitochondrial loci for a more complete understanding of evolutionary histories and associated phylogeographic events. Our results suggest that gene flow between these systems, before and after fragmentation was through periodic paleolakes that formed in the headwaters region. Fragmentation between the Negro and Chico-Chubut systems was driven by the disappearance of these paleolakes during the Patagonian Glaciation. Aegla neuqeunsis (Aeglidae: Decopoda).Phylogeographic inference is a powerful tool for understanding an organism’s evolutionary history Aegla neuquensis is found in two major river systems (Negro and Chico-Chubut) in the Argentinian Steppe  during the last several glacial cycles  pe Fig1: . The NegAegla neuquensis in general and formed paleolakes near the headwaters specifically. However we predict little impact of glaciation on this taxon because most of the distribution of Aegla neuquensis occurs beyond the limits of glacial ice. In these ice-free regions, we expect to recover stable demographic histories and genetic structuring between rivers and populations. In contrast we predict glacial cycles will have significantly impacted the headwaters region of this taxon. In this region we expect to recover the genetic signature of historical fragmentation when no paleolakes were present and when there were paleolakes in the region gene flow between the Negro and Chico-Chubut systems. Because the signature of previous glacial cycles is likely to be overwritten by later events, we expect these events to date to recent cycles Five major glacial events occurred in the region over the last four million years Aegla crabs makes them excellent candidates for phylogeographyic study. Aegla species in general occur in freshwater habitats throughout the Patagonian region Aegla, including A. neuquensis ideal subjects of phylogeographic study The natural history of Aegla neuquensis. Comparing patterns and process across multiple markers will allow us to make rigorous inferences We employ three nuclear and one mitochondrial (three genes) locus and a variety of population genetic approaches to recover phylogeographic patterns and processes in Mitochondrial and nuclear haplotype diversity was high. We recovered 146 unique haplotypes from 295 individuals in our combined mtDNA data set . Thus, there is no evidence that the mitochondrial data are experiencing natural selection that might obscure phylogeographic signal. The Vaca and Telsen populations clustered with the outgroup taxa and are more genetically distant from the remaining ingroup samples  was 235 ka  from the Chico-Chubut into the Negro system (0.0404) greater than the reverse (0.0118). The estimated ancestral (qa) population (where θ = 4 Neμ) size was the same as the Negro system (8.3701), while the Chico-Chubut has the smallest population size (1.8135). The time of population divergence between these two river systems was 137 ka. The geometric mean of the mutation rate across loci was 4.008839×10−5/combined loci/generation. The 95% posterior probability distribution (not shown) for time (t) does not include zero, supporting the hypothesis of historical isolation  with post isolation gene flow. Each of the three IMa runs converged on nearly identical parameter estimates and had relatively high ESS values . Thus, we conclude that these analyses were well sampled and converging on correct values and that we were justified in combining the three runs for the model selection procedure.The highest ranked demographic model using Akaike Information Criterion (AIC) was ABADE  was generally high except those of Bayas, Covunco and Chubut, but nucleotide diversity (θπ) was relatively low, especially in the populations from the Chico-Chubut River , and pairwise STФ values were large and significant (P<0.05) among all populations, excluding those between Bocatoma and Chimpay, Bocatoma and Cullin, Bocatoma and Pichileufu, Piedra del Aguila and La Rinconada, and Los Altares and Ameghino (STФ = 0.708−0.988) were the largest, followed by those between the Neuquen-Negro River and Limay River (0.503–0.986), while those between the Limay River and Chico-Chubut River (0.566–0.927) were relatively small. Total genetic diversity as measured by theta was much greater in Negro system versus that found in the Chico-Chubut system (Population genetic diversity as indicated by haplotype diversity (ut River . These oAmeghino . Genetict system .P<0.05) and negative for Bayas, Sarmiento (not shown) and Chico-Chubut River  and Negro and Chico-Chubut systems  population was equal in size to the current size of the Negro population and that both are larger than the Chico-Chubut population. These relative population sizes are consistent with the patterns of haplotype diversity. The isolation-with-migration model suggests that gene flow between systems is asymmetrical, with up to four times as many individuals moving into the Chico-Chubut system from the Negro system as occurred in the opposite direction.et al.The fragmentation event between the Negro and Chico-Chubut systems occurred roughly 137 ka. Of the five major climatic events outlined by Ruzzante Our estimate of the timing of this fragmentation event could be much older than 137 ka because mitochondrial introgression (see below) occurred after the two systems and the three nuclear loci diverged. Therefore our estimate of 137 ka could be biased when we combined these two genomes into a single analysis. Observation of reciprocal monophyly in nuclear loci over a relatively recent period further suggests that our divergence estimate is biased by mitochondrial introgression, however, the extremely low historical effective population size (a population bottleneck) observed in the Chico-Chubut system suggests that monophyly could have occurred over short evolutionary time.Aegla neuquensis but are in fact more closely related to other Aegla taxa. These rivers are geographically isolated from other systems and have not been connected to rivers that contain other A. neuquensis. This evolutionarily independent group was treated as a new taxon in our analyses and will be formally described in a forthcoming work.Samples from the Vaca and Telsen Rivers are morphologically similar to Post fragmentation histories of these two systems were different. First, the Negro system appears to have been demographically stable and has maintained a greater amount of genetic diversity throughout its history. In contrast, the Chico-Chubut system has relatively lower levels of diversity even after a period of demographic expansion over the last several thousand years. In addition, the most recent common ancestor is younger in the Chico-Chubut system than in the Negro system.Gene flow recovered using MIGRATE suggests very different patterns in these two systems and within individual rivers. Downstream gene flow in both mitochondrial and nuclear markers is extensive within most of the Negro system, with very little movement upstream. Conversely, gene flow is bidirectional between populations in the headwaters of the Limay River. Gene flow in the Chico River is exclusively upstream. Bidirectional flow is seen in the Chubut River, with the Los Altares population serving as the primary source of emigrants within this river. Both systems exhibited restricted gene flow. Differences in gene flow appear consistent with the overall patterns of genetic diversity and demographic history, in the following manner. First, the stable demographic history and larger genetic diversity of the Negro system is characteristic of populations at equilibrium and therefore not colonizing new habitats The best demographic model detected some gene flow between systems after they were fragmented. This gene flow would have occurred within the last 137 ka. Only during the last glacial maximum (23–25 ka) would there have been the opportunity for individuals to move between systems via another paleolake. Our best model suggests that the majority of individuals moved south from the headwaters of the Neuquen River into the Chico River, though some appear to have dispersed in the reverse direction.Patagonian freshwater systems are unique and geographically diverse. Andean orogeny (16–23 million years ago [Ma]) played a major role in shaping these systems primarily due to the mountain range’s relative proximity to Pacific and Atlantic coasts . PacificA. neuquensis (this paper) and A. alacalufiA. alacalufiA. alacalufi persisted in the relatively ice-free western islands, whereas much of the mainland distribution was recently colonized. Xu et al.A. neuquensis. In this taxon, only a small portion of the total distribution was directly impacted by glacial cycles Glacial cycles and geography played fundamental but different roles in the evolutionary history of Aegla neuquensis. Nearly all phylogeographic studies that have employed both nuclear and mitochondrial data have observed greater resolution in mitochondrial loci, including relatively deeper levels of genetic divergence in the mitochondrial versus the nuclear loci Aegla neuquensis, the opposite pattern was observed, though within rivers the mitochondrial locus was more informative. Multiple analyses of independent nuclear loci suggest that populations of Aegla neuquensis in the Chico-Chubut system are evolutionarily independent from those in the Limay-Negro system. Phylogenetic analysis of our mitochondrial locus recovered a non-monophyletic Limay system with Chico-Chubut haplotypes embedded within it. Additionally, and again contrary to theoretical expectations, genetic diversity was greater (between Limay-Negro and Chico-Chubut) in the nuclear loci than in the mitochondrial locus. Why are there conflicting signals between genomes?Mitochondrial loci have long been the standard marker for phylogenetic and phylogeographic analyses because they are more likely to recover recent evolutionary history Aegla taxon and the breeding system of Aegla suggests that dispersal would be much greater in males than in females. Furthermore, our gene flow analysis suggests similar local patterns of gene flow for both nuclear and mitochondrial data sets.Several alternative explanations have been proposed to explain conflicting signals between genomes The most likely explanation for the contradictory signal between genomes is interspecific mtDNA introgression with restricted nuclear gene flow. This phenomenon of “mitochondrial introgression” is observed when the mitochondrial genome of one species occurs against a predominant nuclear background of another species Aegla neuquensis was possible because we employed multiple analytical methods and loci from both nuclear and mitochondrial genomes A detailed understanding of the patterns and processes that shaped the evolutionary history of A. neuquensis, an area approximately 3.4×105 km2  that represents each locality and major branches of mtDNA phylogenetic tree. Nuclear fragments were amplified with newly designed primers EF1AX1 (5′ GCTGAGCGTGAACGTGGTATCAC 3′) and EF1XR (5′ GTTTGTGTTGACCAGAATAAAC 3′), primers EF1F4 and EF1XR0 http://dx.doi.org/10.5061/dryad.93js1254.DNA extraction, PCR amplification, and sequencing of three mitochondrial genes  are described in a previous study A. neuquensis and sequences of outgroup species  and Bayesian methods. The mitochondrial and nuclear data consisted of haplotypes of concatenated sequences from Multilocus nested clade phylogeographic analysis (NCPA) was used to test for historical and demographic events that are geographically congruent across two or more loci A, theta of descendent population one θ1, theta of descendent population two θ2, and two measures of gene flow (m1 and m2) between the two daughter populations. In addition, the time of the split between daughter populations were estimated using IMa. The best models were determined using a chi-square distribution with and without Bonferroni correction. Akaike Information Criterion (AIC) was also employed to rank the models (see t) was converted to years before present using a rate of 0.118 substitutions/site/million years that was estimated from the closely related A. alacalufit by the geometric mean of the mutation rate across loci per generation Demographic history between Chubut (Chico and Chubut) and Negro (Limay and Neuquen) river systems, using nDNA and mtDNA, was examined using an isolation-with-migration analysis implemented in IMa Hd), nucleotide diversity, current genetic diversity (θπ) and historical genetic diversity (θW), were assessed using DnaSP 5.0 STФ index and population structure was measured with an analysis of molecular variance (AMOVA), as implemented in Population genetic diversities, as indicated by haplotype diversity  as implemented in BEAST 1.6.1 and visualized in Tracer 1.5 les Data  and S2 ales Data .Figure S1Plots of time of most-recent-common-ancestor for individual loci from three independent runs and from the three runs combined. Analyzed using Extended Bayesian skyline plots (EBSP) as implemented in BEAST 1.6.1 and visualized in Tracer 1.5. Each analysis was run for 50 million generations with every 1000 generations retained. The first 10 million generations (20%) were removed as burnin. Analyses were run with a coalescent tree and relaxed uncorrelated lognormal clock priors. We employed the mtDNA mutation rate of 0.118 substitutions/site/million years (DOC)Click here for additional data file.Data File S1Beast input file (.xml) for Extended Bayesian Skyline Plot analysis of the Negro River system.(PDF)Click here for additional data file.Data File S2Beast input file (.xml) for Extended Bayesian Skyline Plot analysis of the Chico-Chubut River system.(PDF)Click here for additional data file.


Pseudopleuronectes americanus), a formerly abundant fish, using 11 microsatellite markers . The effective number of breeders for 1–2 years was remarkably small, with point estimates ranging from 65–289 individuals. Excess homozygosity was detected at 10 loci in all bays (FIS = 0.169–0.283) and individuals exhibited high average internal relatedness . These both indicate that inbreeding is very common in all bays, after testing for and ruling out alternative explanations such as technical and sampling artifacts. This study demonstrates that even historically common marine fish can be prone to inbreeding, a factor that should be considered in fisheries management and conservation plans.In contrast to freshwater fish it is presumed that marine fish are unlikely to spawn with close relatives due to the dilution effect of large breeding populations and their propensity for movement and reproductive mixing. Inbreeding is therefore not typically a focal concern of marine fish management. We measured the effective number of breeders in 6 New York estuaries for winter flounder ( McNeely et al. Pleuronectes platessa) in the North Sea and, after ruling out alternative hypotheses, concluded that inbreeding was responsible for this pattern. Despite having a relatively large census population size, plaice tend to spawn in their natal area and have high variance in reproductive success, increasing the probability that spawning pairs or groups will contain related individuals. Despite this remarkable finding, there have been few follow-up studies of inbreeding in marine fish, even though heterozygote deficiencies have been detected in many other species, including redfin culter (Culter erythropterus) Engraulis encrasicolus) Sebastes melanops) Coregonus lavaretus) Recent studies have shown that marine fish populations can be more structured than traditionally thought Pseudopleuronectes americanus) is a demersal flatfish that was once very common in the inshore bays and estuaries of the Northwest Atlantic and supported very large commercial and recreational fisheries The winter flounder  landings have reached record low levels, and despite management there is little evidence of recovery Winter flounder began declining in the late 1980s and the age structure of many populations has become truncated, with a low proportion of the remaining fish older than 5 years Necessary permits for sampling and handling fish were obtained from responsible agencies and authorities  Permit #GATE-2007-SCI-0021 and GATE-2011-SCI-0014 for Jamaica Bay and a permission from the Town of East Hampton Trustees for Napeague Harbor). All winter flounder were sacrificed by being flash frozen on dry ice in the field as approved in our IACUC protocol .Young-of-the-year (YOY) winter flounder were collected with a 1 m beam trawl every two weeks from June to October in 2010 and May to October in 2011. Samples were collected in 6 bays : Jamaica2, 0.12 mM of each dNTPs, 0.16 µM of the reverse primer and the fluorescently labeled M13 primer and 0.04 µM of the species specific forward primer and 1 unit Taq polymerase in a final reaction volume of 10 µl. Thermal cycling profiles were 4 minutes at 94°C, 30 cycles of 94°C for 15 seconds, primer specific annealing temperature for 15 seconds, 72°C for 45 seconds and 5 cycles of 94°C, 15 seconds at 53°C and 45 seconds at 72°C with a final extension for 10 minutes at 72°C. Locus-specific annealing temperatures (Ta) are given in Genomic DNA was extracted from fin clips (0.015–0.035 mg of tissue) using the Qiagen Blood and Tissue extraction kit . Eleven microsatellite loci were amplified using PCR primers that were either used for previous winter flounder microsatellite studies −1)/4 Expected and observed heterozygosity ST for several temporal groups. We first tested all samples caught in 2010 (N = 115) against those caught in 2011 (N = 99). We then pooled samples caught in the early sampling season (May–July) and the late sampling season (August–October) and calculated pairwise FST for the following four temporal groups: early 2010 (N = 89), late 2010 (N = 26), early 2011 (N = 70) and late 2011 (N = 29). The program Structuretructure was run using the admixture and the non-admixture model both using and not using a priori information regarding the sampling location. We simulated K = 2–15 for 10 independent runs each to determine convergence with a burn-in period of 15,000 Markov chain Monte Carlo (MCMC) steps followed by 350,000 MCMC steps. This approach is capable of detecting if there were strongly differentiated groups . To test for the possibility of genetically distinct groups of winter flounder spawning in the same bay at different times we calculated pairwise Fb) was estimated for each bay and LI using the linkage disequilibrium method as implemented in LDNecrit = 0.02. This method estimates the effective number of breeders based on a single sample by using the small level of linkage of alleles that occurs due to sampling error during recombination. Since we used young-of-the year individuals sampled over 1–2 years, the parameter we estimated is the number of breeders that effectively produced the sampled cohorts, not the effective population size Ne for the whole generation ottleneckThe effective number of breeders  level, the inbreeding coefficient Ficrochecker did not detect evidence of stuttering or large allelelic drop out at any locus. While Microchecker did suggest the presence of null alleles due to strong deviations from HWE, none of the homozygotes exhibited an additional allele after being re-amplified at lower annealing temperatures (N = 8 homozygotes per locus). All individuals were successfully amplified and genotyped at all loci, indicating that there were no individuals that were homozygous for null alleles .Eleven microsatellite loci were amplified in 267 individuals sampled in 6 LI bays . Pairwise FST values between bays were consistently significant (p<0.05) for Napeague Harbor (except compared to Hempstead Bay) and Shinnecock Bay . Even after removing Napeague Harbor, the most divergent and distant site, the IBD pattern was still significant . Temporal pairwise FST calculated between the years 2010 and 2011 was 0.005 (p = 0.4); pairwise FST-values for comparison between the early and late sampling season ranged between 0.005 and 0.010 (p = 0.3–0.6). Bayesian clustering implemented in Structure failed to detect population structure from K = 2–15 for the entire sample set. Additionally, analysis of pairwise relatedness of all sampled individuals showed that less than 6% were related at a level of half-siblings or higher (r>0.25).The global Fcock Bay , though cock Bay  was alsoIS values over all loci were significantly different from zero and positive in all 6 bays, ranging from 0.169–0.283  in all bays. Many individuals also exhibited very high internal relatedness and individuals' genotypes exhibited a high proportion of homozygous loci. These patterns could not be explained by an artificial inflation of homozygosity resulting from technical issues. Large allelic dropout produces a pattern skewed towards homozygotes for small alleles, which did not occur in the LI winter flounder. If null alleles were at high enough frequencies at all loci to drive these patterns, we should have observed many null homozygotes (blanks for certain loci) when we had none. We would probably have also been able to amplify null alleles in homozygotes by lowering temperatures, but this did not occur. Inadvertent sampling of closely related individuals can also generate HWE deviations of this nature, but few (∼6%) of the sampled young-of-the-year fish exhibited high relatedness (r>0.25) ruling out a Wahlund effect due to sampling of closely related individuals. A Wahlund effect due to undetected population structure is also unlikely, as no cryptic genetic structure within any of the bays was detected using Structure.Young of the year winter flounder living in New York estuaries exhibit relatively high genetic diversity in terms of microsatellite allelic richness, yet the low m-ratios observed suggest that rare alleles may have been lost within individual sample locations. Genetic diversity was weakly geographically partitioned between some of the bays, with distance between sites playing a small but significant role in driving this structure. All bays were out of HWE due to large excesses of homozygotes across 10 of 11 loci, leading to high inbreeding coefficients (FCanis lupus) Cervus elaphus) Ancistrocerus antilope) Salvelinus fontinalis) Engraulis encrasicolus) Coregonus lavaretus lavaretus) Sebastes melanops) The most likely explanation for these large deviations in HWE is that inbreeding is occurring in LI winter flounder. Similar evidence for inbreeding has been documented in a wide variety of terrestrial and freshwater animals  are needed to assess genetic differentiation between winter flounder in these bays.We propose the inbreeding observed in LI populations may be due a confluence of a small spawning population in each bay and a propensity of these fish to spawn in their natal estuary. It has generally been assumed that marine fish exhibit panmictic population structure and do not require management at the sub-population level We suggest that relatively few mature adults are contributing to each generation, resulting in a high proportion of related fish spawning with each other. Like any broadcast spawning species it is probable that there is large variation in reproductive success in this species due to high larval and pre-recruit mortality Danio rerio) in a laboratory setting. They confirmed that inbreeding has a much stronger effect when combined with the exposure to a chemical stressor, resulting in lower embryo viability and few offspring. Western populations of winter flounder on LI, particularly those in Jamaica Bay, are exposed to anthropogenic habitat degradation, particularly large amounts of municipal sewage effluent which has been shown to contain estrogenic compounds Inbreeding could directly contribute to the failure of some marine fish to recover from exploitation as it has been linked to lower survival and reproduction rates and lower resistance to disease and environmental stress Marine fish have historically been managed without an underlying concern that populations could be reduced to the point where they would become vulnerable to local extinction and processes that reduce their genetic diversity. We show that the effective number of breeders is so low in some parts of the winter flounder range that it is able to be estimated using genetic approaches that work most effectively for small populations. Although we observe only weak evidence of a genetic bottleneck at this stage, the number of spawning adults is sufficiently low for inbreeding to be occurring. Given similar findings in genetic studies of North Sea plaice


Sea ice is believed to be a major factor shaping gene flow for polar marine organisms, but it remains unclear to what extent it represents a true barrier to dispersal for arctic cetaceans. Bowhead whales are highly adapted to polar sea ice and were targeted by commercial whalers throughout Arctic and subarctic seas for at least four centuries, resulting in severe reductions in most areas. Both changing ice conditions and reductions due to whaling may have affected geographic distribution and genetic diversity throughout their range, but little is known about range-wide genetic structure or whether it differed in the past. This study represents the first examination of genetic diversity and differentiation across all five putative stocks, including Baffin Bay-Davis Strait, Hudson Bay-Foxe Basin, Bering-Beaufort-Chukchi, Okhotsk, and Spitsbergen. We also utilized ancient specimens from Prince Regent Inlet (PRI) in the Canadian Arctic and compared them with modern stocks. Results from analysis of molecular variance and demographic simulations are consistent with recent and high gene flow between Atlantic and Pacific stocks in the recent past. Significant genetic differences between ancient and modern populations suggest PRI harbored unique maternal lineages in the past that have been recently lost, possibly due to loss of habitat during the Little Ice Age and/or whaling. Unexpectedly, samples from this location show a closer genetic relationship with modern Pacific stocks than Atlantic, supporting high gene flow between the central Canadian Arctic and Beaufort Sea over the past millennium despite extremely heavy ice cover over much of this period. Balaena mysticetus). Ongoing dramatic declines in sea-ice extent will likely affect genetic exchange rates in bowhead whales as well as other arctic marine mammals such as beluga and walruses  and Pacific  populations has yet to be fully elucidated. Interchange between the Atlantic and Pacific Oceans through the central Canadian Arctic was likely possible during the warmer conditions of the early and mid-Holocene, but climatic reconstructions indicate a rapid increase in ice cover around 3 kya that excluded bowhead whales from the central channels of the archipelago . A shortHistorical and ancient samples represent a valuable but underutilized source of information about marine mammal responses to both climate change and whaling over long periods. Genetic data from such samples have been used to test hypotheses about population response to climate shifts and hunting in many terrestrial species e.g., , but havThe extensive archeological and stranded remains of bowhead whales across the Arctic provide an opportunity to better understand the factors shaping genetic connectivity in bowhead populations. In this study, we used ancient and modern bowhead control region sequences to compare genetic diversity and population differentiation between: (1) all putative modern management stocks, including sequences from Spitsbergen samples aged 30–3,000 years old ; and (2)Cassiope tetragona), caribou bone (Rangifer tarandus), and willow  from the house assemblages of which these samples are a part bracket the occupation of the features between 500 and 800 ybp.Samples of preserved baleen and bone were collected from archeological sites on Somerset Island (western side of PRI) as described in The location of this site on the western side of PRI/Gulf of Boothia is on the summering ground of what would today be considered part of the BBDS stock. However, satellite tracking data also indicate that the area is also used by animals from Foxe Basin . Bowhead whales only visit PRI, which is characterized by heavy ice cover, for about 2 months per year. Solid fast ice coverage in the Canadian Arctic Archipelago during fall, winter, and spring forces all cetaceans to move out into open water or to areas with mobile pack ice . This foAll extractions were performed in dedicated ancient DNA facilities at the American Museum of Natural History. No modern whale DNA had been extracted and no amplifications had taken place within this facility. All samples were stored in separate airtight plastic bags until use to prevent cross-contamination. Samples were pretreated to remove potential surface contaminants as described in 2O. Baleen was subsampled following the protocol of 20 in order to reduce the EDTA concentration. To extract DNA from the bone pellet, samples were incubated with 0.5-mL Lifton's buffer and 35 uL of 20 mg/mL proteinase K at 56°C for 50 h. Extraction was completed using standard phenol/chloroform purification and ethanol precipitation procedures  to generate ∼0.1g of bone powder. Bone powder was then treated to remove any remaining contaminants by soaking in 10% Clorox for 20 min followed by a rinse in sterile Hocedures .Amplification conditions are given in Successful amplification products were sequenced in both directions using fluorescence-labeled dideoxy terminators on an ABI 3700 High-throughput Capillary DNA Sequencer (Applied Biosystems). To authenticate sequences, a subset (∼15%) of samples with unique haplotypes were re-extracted, amplified, and sequenced in both directions in an entirely separate facility , and scored blind relative to the original sequences.N = 176) and Disko Bay, West Greenland and Pangnirtung, Canada  collected between 1997 and 2006. Total cellular DNA was extracted from bowhead skin samples using different techniques. Earlier samples (before 2000) were extracted using the methods described in Modern D-loop data were generated from biopsy samples collected from areas of Northern Canada and West Greenland including Pelly Bay, Repulse Bay, and Igloolik  (N = 24) (N = 38)  BCB Seas (N = 394) ; (2) Okh(N = 24) ; and (3)(N = 38) .ΦST statistic is a more appropriate measure of differentiation than frequency-based FST (which does not take into account molecular distances between haplotypes) , and species identity was determined using the NCBI database (BLAST), as well as a diagnostic character approach for species delimitation . Haplotylotypes) . We assery model . In addim = 0.1, m = 0.01, m = 0.001). In brief, we modeled the following scenarios  and whaling histories. We used the program BayesSSC . Six duplicated sequences were found (4 in HBFB and 2 in BBDS), and removed from the mtDNA sequence set. For ancient samples, no sequence differences were found between original sequences and samples that were re-extracted and sequenced in an independent facility. Haplotype, nucleotide, and sequence diversity were high for all sample sets examined, with the exception of Okhotsk Sea. Haplotype diversity was significantly higher in PRI  and BCB (0.892−0.935) compared with Canada-Greenland (0.785−0.848) and Okhotsk (0.484−0.775), based on coalescent analyses performed in DNAsp  of these private haplotypes were singletons in the dataset. The most frequently observed haplotype was the same for all sample sets, and the second most frequently observed haplotype was the same for five of the six sample sets (with Okhotsk being the exception).ΦST = −0.002, P > 0.1)  comparison a. No difparisons . The anaparisons  was applThe optimal minimum spanning network  shows thΦST values for three pairwise comparisons across 10,000 simulations (m = 0.1) is shown in ΦST values  interval for 3A, 3B, 3C, and 3D at m = 0.1. For the BCB-PRI comparison, the observed value of ΦST falls within the 95% HPD interval for all scenarios that use m = 0.1 with the exception of 2C. For the comparison between PRI and BBDS alone or Canada-Greenland, the observed ΦST falls within the 95% HPD interval for 1A, 2A, 2B, 1C, 3C, and 3D at m = 0.1. Based on these results, the following scenarios can be excluded : 1A, 2A, 3A, 1B, 2B, 3B, 1C, 2C, 1D, and 2D. In other words, for m = 0.1, the only scenarios that are not excluded are 3C (contemporary gene flow with PRI as a separate population) and 3D . For lower migration rates (m = 0.01 and m = 0.001), all scenarios are excluded , we grouped these two stocks into a single population (Canada-Greenland) for the majority of scenarios. The distribution of T values . For theOur analysis of the five putative populations of bowhead whales, including data from ancient samples from two locations (PRI and Spitsbergen), represents the first genetic comparison across the entire range of the species and illustrates the utility of ancient specimens in reconstructing the history of genetic exchange in exploited marine mammals. The results indicate that Arctic sea ice has not acted as a strong barrier to migration between the Atlantic and Pacific over the late Holocene as previously assumed, and that genetic diversity has been lost from eastern Canada in the period between ∼500 ybp and the present.Measures of modern diversity are similar to those reported in earlier studies of population-level diversity in the control region of bowhead whales e.g., . HoweverEubalaena australis) population, and estimated pre-exploitation richness at 100–150 haplotypes. We observed 62 haplotypes in the modern dataset alone, without any correction for undersampling or sequence length, which would increase the number of estimated haplotypes.The high number of mitochondrial haplotypes observed across bowhead whale populations may not be atypical for baleen whales. The relatively high number of unique or population-specific haplotypes among PRI samples (roughly one-fifth of PRI haplotypes are unique to that sample set), as well as the high divergence of several of these haplotypes compared with modern samples , suggestFst = 0.009) between Barrow, Alaska (n = 231) and Igloolik, Canada (n = 37) estimated using 21 microsatellite loci , and unWithin ocean basins, we did not observe any consistent relationship between ice barriers and population differentiation. Although BBDS and HBFB have previously been considered separate management stocks based on the assumption that persistent ice in Fury and Hecla Strait would prevent movement between them, we found no significant differentiation between them. These results are consistent with observed movement patterns from satellite telemetry data, but need to be tested with additional markers, which was not possible in the scope of this study.Severe population reductions due to whaling may have altered signals of genetic diversity and connectivity between bowhead populations, making it difficult to reconstruct pre-whaling patterns. Data from ancient PRI samples can provide insights into how population structure differed in the past. PRI is located in the summering ground of the modern Canada-Greenland population, but has been connected to the Beaufort Sea (BCB population) via the Northwest Passages intermittently over the Holocene. The data presented here show that the genetic difference between PRI samples from 500 to 800 ybp and modern Canada-Greenland samples is unexpectedly larger than the difference between these ancient samples and modern BCB samples.If PRI whales were the forbearers of modern Canada-Greenland, as geography would suggest, what could cause such strong genetic differentiation between them? One possibility is that the population bottleneck during the height of whaling caused a dramatic reshuffling of haplotype frequencies between the two sampling time points. However, simulations show that even an extreme (∼71–99%) reduction in the Canada-Greenland population does not explain the degree of genetic differentiation between the two. An alternative explanation, which is supported by simulation results (Scenario C) is that PRI samples represent a genetically unique population or set of maternal lineages based around site fidelity to a summering ground on the western side of the Inlet. Recent observations suggest that PRI is a summering area for female whales with calves and juveniles that move from Foxe Basin in the early summer , as wellΦST values are consistent with PRI as an independent population (Scenario 3C), simulation results support high exchange between PRI and BCB, and did not exclude full panmixia between them (Scenario 3D). These findings support the idea that exchange across the Canadian Arctic was occurring during the late Holocene, prior to the recent decrease in sea ice that has permitted overlap in range between Atlantic and Pacific populations  and the large number of bowhead individuals recovered from the PaJs-2 locality  suggestThe strong differentiation between PRI and modern populations based on AMOVA results contrasts with overall lack of differentiation between the other set of ancient samples, Spitsbergen, with modern populations (with the exception of the comparison with Okhotsk). No differentiation was observed between BCB and Spitsbergen, corroborating earlier findings by What might have caused the disappearance of PRI haplotypes from the modern populations of bowhead whales? Both changing climate at the end of the Holocene and whaling (and perhaps an interaction between the two) may have played a role. The most obvious and significant source of mortality for bowhead whales between 500 ybp and the present was commercial whaling, which eliminated a large part of the population . By far,Two other explanations for the disappearance of PRI haplotypes are possible : (1) Thule Inuit whaling; and (2) ice entrapment. Extensive archeological surveys of Thule sites resulted in minimum estimates of 1,830–2,745 whales from Somerset Island alone . These nThese results highlight the complex interplay of factors – including climate history, behavioral ecology, and past exploitation – that shape population genetic patterns in polar marine mammals. In contrast with our initial hypothesis that Pacific and Atlantic populations were last connected during the mid-Holocene e.g., , the genWe have attempted to infer a complex demographic history from limited genetic data, and additional genetic information and ancient samples from throughout the region will be needed to further test these hypotheses. Nevertheless, the unique set of maternal lineages found in the central Canadian Arctic (PRI) and the unexpected relationships between this area and other modern populations demonstrate the value of ancient samples in better understanding the role of climatic history and human hunting in shaping genetic diversity and structure in arctic species. Additional ancient samples from across the range of the bowhead whale and integrated data from SNPs and microsatellites would advance analyses beyond the rough estimates possible using mitochondrial data alone. Such studies would allow an unprecedented evaluation of both natural and anthropogenic impacts on genetic variability in a key indicator species in the rapidly changing Arctic.


Sylvia atricapilla) is related to individual differences in migratory behavior. Our 3-year study revealed a positive correlation between individual migratory origins, denoted via isotope (δ2H) values, and genetic distances. Genetic diversity and admixture differed not only across a recently established migratory polymorphism with NW- and SW-migrating birds but also across δ2H clusters within the same migratory route. Our results suggest assortment based on individual migratory origins which would facilitate evolutionary responses. We scrutinized arrival times and microhabitat choice as potential mechanisms mediating between individual variation in migratory behavior and assortment. We found significant support that microhabitat choice, rather than timing of arrival, is associated with individual variation in migratory origins. Moreover, examining genetic diversity across the migratory divide, we found migrants following the NW route to be genetically more distinct from each other compared with migrants following the traditional SW route. Our study suggests that migratory behavior shapes population genetic structure in blackcaps not only across the migratory divide but also on an individual level independent of the divide. Thus, within-population variation in migratory behavior might play an important role in translating environmental change into genetic change.In migratory birds, traits such as orientation and distance are known to have a strong genetic background, and they often exhibit considerable within-population variation. How this variation relates to evolutionary responses to ongoing selection is unknown because the underlying mechanisms that translate environmental changes into population genetic changes are unclear. We show that within-population genetic structure in southern German blackcaps ( In several migratory species, hybrid zones between recently diverged taxa are located on migratory divides, which are areas where populations with different migratory behavior meet  can affect the genetic structure of populations and the ability of migratory species to evolve in response to changing selective pressures  in southern Germany. Contingent upon our findings, we then analyse whether the observed genetic structure of the population is explicable by temporal and spatial isolation in arrival times and microhabitat choice of territorial blackcaps. Earlier work showed that migratory orientation and distance in blackcaps, a species that migrates alone, are not learned but have a strong genetic component (Berthold δ2H) values, a proxy for the birds' wintering area  to directionally assign blackcaps to either one of the two main wintering origins of southern German blackcaps. Those origins were northwestern (NW) migrants wintering in Great Britain or south-western (SW) migrants wintering in the Mediterranean  and perform an uninformed cluster analysis to portion individuals based on δ2H similarities (δ2H clusters). We then partitioned the overall genetic diversity in our dataset on the basis of variation in blackcap tissue δ2H values. Assuming that individual differences in migratory behavior contribute to population genetic structuring, we predicted (1) pairwise genetic distances to increase with pairwise geographical distances between wintering areas and (2) pairwise genetic distances within δ2H clusters to be lower than the mean pairwise genetic distance across the whole study population. We (3) examined whether spatio-temporal isolation of blackcaps contributed to the observed genetic structure of sympatric blackcaps. To our knowledge, this is the first study that investigates population genetic structure in relation to migratory behavior on an individual level.Unlike previous studies  using the comparative equilibration method described in detail by Wassenaar and Hobson  in 2006 and in Freiburg (48°00′N 07°51′E) in 2007 and 2008. The Freiburg site was chosen because of logistical reasons and was also surveyed for habitat analyses in 2010 and 2011 (see below). In each year, we captured birds from mid-March on, when the first migrants arrived from their wintering quarters to mate on their breeding grounds, and caught birds every day from early morning to noon until the end of April. Blackcaps were caught within an area of 50 ha in a deciduous forest with mist nets using tape recordings of their song as a decoy. Each morning we intensively patrolled for singing activity and captured all newly singing males as well as nonsinging individuals within this area. Because of this procedure, we considered the first day a bird was caught as a proxy for arrival date and we calculated a dayscore as the difference in arrival relative to the start of the field season for statistical analysis (days from 15th March). Our proxy for arrival date reflects accurately arrival in the study area. However, we cannot exclude that an individual had already arrived a few days before in an area outside the study area. All individuals were marked with a standard aluminum ring , sexed, aged . Individual δ2H values entered our analyses as a continuous variable and were used as a proxy for (north–south) geographical distance between wintering origins . We used the following microsatellites for the genetic analysis: Syl1, Syl2, Syl3, Syl4, Syl5, Syl6, Syl7, Syl8, Syl9, Syl10 , and/or was accompanied by a female. The margins of a territory were either defined as a 20 m radius from the center where the male was spotted most of the time or they were inferred from male's behavioral patterns . The territory size estimated by both measures did not differ .δ2H values and (2) use a generalized additive model (GAM) to fit a smooth response surface of δ2H values over the limits of the ordination biplot. We include the latter procedure to additionally test for a combined nonlinear association between the habitat ordination axes (“habitat space”) and individual δ2H values.In mid June, each territory was surveyed for its vegetation structure by recording the variables described in http://hpgl.stanford.edu/projects/microsat). The following genetic distance measures were calculated in our study: Reynolds Theta  and (2) loglikelihood comparisons between the full model and the reduced model (excluding δ2H as a fixed effect).Individual genetic distance matrices based on the 10 microsatellites were calculated using the software package microsat . Respective clusters were then analyzed for deviations of their mean within-cluster genetic distance from the overall mean genetic distance to ask whether the cluster-intern genetic structure was more homogenous than that of the overall population structure of the dataset . This was tested by permutating the composition of respective clusters, while keeping cluster sizes constant. We analyzed between-cluster differences in individual genetic diversity using two different measures: mean individual heterozygosity and mean d²  between δ2H clusters were calculated with FSTAT , the stepwise mutation model (SMM), and the two-phase model . Studies on avian microsatellite evolution suggest that the SMM/TPM models are most appropriate  an evaluation of the most likely k for our dataset (without a priori information) using STRUCTURE and the Δk criterion  as the residuals of the highest assignment score to the random assignment value  and (2) the overall percentage of each simulated genetic cluster within the respective δ2H clusters. These two simple measures allow a proportionate comparison of the genetic clusters within the δ2H clusters.A Bayesian admixture model analysis was conducted on population structure using the software STRUCTURE 2.3  and the individual pairwise δ2H distances in claw stable isotope values . The mixed model incorporating individuals as a random structure also revealed a significant effect of δ2H distance on genetic distance . The relatively low correlation coefficient is typical for individual pairwise comparisons illustrating substantial genetic and geographical variation in overwintering locations in our blackcap population. Similar results were found when we (1) based the test on alternative genetic distance measures , (2) excluded potential long-distance migrants from our dataset , and (3) partitioned our dataset according to study year and/or site . We (2) repeated the test on 500 randomly chosen subsets comprising 100 individuals from our dataset, respectively. Here, we found that 80% of all 500 Mantel tests were significant (P < 0.05) and 93% were marginally significant (P < 0.1) with a mean test statistic comparable to our original results (r ± SE=0.13 ± 0.002). Isotope distances were not related to individual distances in arrival times of birds on their breeding grounds  suggesting that individual birds from distinct wintering regions did not differ strongly in arrival times.We found a significant positive correlation between the individual pairwise genetic distance  and two δ2H clusters containing birds presumably from more southerly wintering quarters (S-migrants), separated according to their approximate distance to the breeding grounds .To analyse the relation of migratory origin and the underlying population structure, we partitioned the election : one δ2Hδ2H cluster 3 vs. δ2H cluster 1 and 2, δ2H clusters tended to differ in their mean within-genetic distance from the mean within-genetic distance obtained by permutation of the entire population. The northerly migrants were significantly more distant from each other than the southerly wintering migrants which were less distant from each other compared with the permutation mean  compared with the other δ2H clusters . The inbreeding coefficient (FIS) was higher for the N migrants and not different among the two S-migrating clusters . No heterozygote excess in either δ2H cluster was found when testing under the assumptions of the SMM (P > 0.80), whereas there was a significant heterozygote excess found under TPM and IAM assumptions , indicating no clear evidence for a recent bottleneck in any cluster.A particularly interesting result was that the mean individual genetic distances among birds overwintering at northern latitudes were higher compared with birds that overwintered south of the breeding grounds (k = 2 genetic clusters yielded no significant differences in admixture among the three δ2H clusters (r) did not differ between the δ2H clusters , nor did the overall proportions of each genetic cluster in the δ2H clusters . However, when simulating k = 3, we found significant differences in Δpr and overall proportions of genetic clusters between δ2H clusters , indicating a more confident assignment of individuals from that δ2H cluster to either of the three simulated populations. We also found marginal differences in Δpr scores between δ2H clusters 1 and 2 . The percentage represented by the third genetic cluster within each of the δ2H clusters increased from 14% in cluster 1 to 21% in cluster 2, to 36% in cluster 3 , and presence of nettles (Urtica dioica), and PC3 was mainly affected by disturbance and density in the crown layer and presence of nettles  in the birds' distribution . Five of nettles .δ2H values  and a marginal relation with PC3 . However, assuming a nonlinear relation, the generalized additive model fitted onto the first two PCs indicated a significant association between individual δ2H values and the main individual variation in occupancy of the habitat space  than to birds wintering at more distant locations. Initially, it seems that the overall correlation (r = 0.13) in the pairwise comparisons among 195 individuals was weak. However, this only shows that there is substantial genetic variation within blackcap populations which is consistent with other studies on migratory birds  and yellow-rumped warblers (Setophaga coronata) complex in the absence of strong assortative mating  and significantly lower individual genetic diversity, denoted as overall heterozygosity and mean d², compared with the two clusters of SW migrants that follow the traditional south-western route. These findings are consistent with the new route having evolved only recently, involving far fewer individuals than the SW route, and document thus for the first time that the genetic structure of the NW-migrating population deviates from that of SW-migrating populations.Our study on blackcaps differing in wintering origins found significant differences in genetic diversity along the migratory polymorphism in southern Germany. Migrants following the recently established northwestern route  and significantly lower genetic diversity (heterozygosity) for the NW migrants. However, NW migrants were on average genetically more distinct from each other than SW migrants , which did not differ genetically between isotope clusters  were on average assigned with higher confidence to one of the three simulated genetic populations, that is, they had a higher probability of pertaining to a specific genetic cluster, as denoted in higher residual assignment coefficients (Δpr). Moreover, the within-cluster fractions of the third genetic population decreased along the N-S gradient from 36% in δ2H cluster 3 to 14% in δ2H cluster 1, a result which is strongly confirmed by examining the extremes of the δ2H spectrum and the correlation between assignment scores and individual δ2H values  the translation of environmental change into adaptive population genetic change (e.g., Pulido and Berthold


Dipteryx alata is a native fruit tree species of the cerrado (Brazilian savanna) that has great economic potential because of its multiple uses. Knowledge of how the genetic variability of this species is organized within and among populations would be useful for genetic conservation and breeding programs. We used nine simple sequence repeat (SSR) primers developed for Dipteryx odorata to evaluate the genetic structure of three populations of D. alata located in central Brazil based on a leaf sample analysis from 101 adults. The outcrossing rate was evaluated using 300 open-pollinated offspring from 25 seed-trees. Pollen dispersal was measured by parentage analysis. We used spatial genetic structure (SGS) to test the minimal distance for harvesting seeds in conservation and breeding programs. Our data indicate that the populations studied had a high degree of genetic diversity  Dipteryx alata and Caryocar brasiliense have been destroyed and continue to be neglected (The cerrado (Brazilian savanna) is the second largest Brazilian biome and a hotspot of global biodiversity . Large peglected . CurrentD. alata, locally known as “baru”, has a wide range of applications, e.g., as human and animal food, a substrate for the pharmaceutical industry and construction wood , have been used in natural population studies as they are codominant and highly polymorphic when compared with other classes of markers. SSR markers have been widely used to address several questions in conservation and population genetics, such as population structure, intra-population spatial genetic structure, contemporary pollen and seed dispersal and mating systems , 4.30 μL of forward and reverse primers (1 μM), 0.2 μL of Taq DNA polymerase ; 1.3 μL of BSA (2.5 μg/ mL) and 1.34 μL of ultrapure water. Amplifications were done using an MJ Research PT-100 thermal cycler adjusted to the following conditions: 5 min at 94 °C, 30 cycles of 1 min at 95 °C, 1 min at the specific temperature for each primer , obtained from CENARGEN-Embrapa .DNA was extracted from the leaves of adult trees and offspring by using the CTAB method described by h primer , 1 min aA), the mean observed (Ho) and expected (He) heterozygosities, and the fixation indices for each population (fi) were estimated by using the program FSTAT 2.9.3 . Since most microsatellite mutations involve the addition or subtraction of a small number of repeat units according to a stepwise mutation model, population differentiation was also assessed by Slatkin's corrected RST coefficient  among populations was estimated indirectly, using the island model proposed by FST) was substituted by θp, RST and n) was calculated as The historic gene flow (tm) by the maximum likelihood method, 2) single outcrossing rate (ts), 3) the outcrossing rate between related individuals rp) and 5) the selfing correlation (rs) for the population. The standard error of the estimates was obtained through 10,000 bootstraps in which the resampling units were the families.The program MLTR 3.2 , which iNull alleles were detected by comparing seed-tree genotypes with their offspring in the MS population using the program MLTR. Null alleles, rather than mistyping, were considered to be present when a homozygous seed-tree mismatched its offspring genotype. In the presence of null alleles the homozygous seed-tree genotype was corrected to a heterozygous form  and the offspring genotypes were corrected through an expected Mendelian segregation ratio of 1:1, as described by Ne(reference) is the minimum effective sample size of seeds to be collected  and 2) Ne(v) is the variance effective size for a single seed-tree progeny of size n (n = 100), estimated byThe minimal number of seed-trees Fs is the fixation index in offspring and Θxy is the average coancestry coefficient within families, estimated as the half of the relatedness coefficient, Fa is the fixation index in the adult population and s is the selfing rate, where Paternity analysis of each seed was done by maximum-likelihood paternity assignment using the program CERVUS 3.0 (xy) between all pairs of adult trees based on Spatial genetic structure (SGS) within the sampled populations was studied using the estimate of the average coancestry coefficient (θfi) can be corrected by eliminating the Wahlund effect through the application of a relationship for F-statistics described in FIT and FST by fi and θxyi, respectively, such that the corrected intrapopulation fixation index becomes ith population.In the presence of SGS, the fixation index values within populations are over-estimated due to the Wahlund effect . This inibed in  . For thiD. alata  amplified visible products and were polymorphic in D. alata . ExclusiD. alata . All locThe mean observed . Dominant pollen donors were present in the 81 seeds originated by outcrossing with seven trees contributing with 62% of the pollen.Analysis of the spatial genetic structure showed that populations MG and GO had significant coancestry coefficient estimates  and management units (MUs) in order to achieve adaptive evolutionary conservation (AEC) objectives in each population (ex situ conservation strategies and restoration projects should be guided by the levels of genetic diversity and local adaptation so as to avoid problems related to maladaptation (Our data support the existence of high genetic diversity within and among populations of Two out of three populations showed significant levels of SGS that generally result in a high fixation index. We suggest that in order to mitigate the effects of SGS when establishing a minimum distance between individuals, future studies should sample all individuals of the same ontogenetic stage in a fixed area; this approach will provide a better understanding of SGS and its influence on genetic structure. The presence of SGS suggests that seed harvesting for genetic conservation and breeding programs needs to be done at a minimum distance of 196 m between seed-trees in order to avoid related seed-parents.D. alata in the Brazilian cerrado. Nevertheless, the high reproductive dominance indicates that the pollinators are probably not playing their role because of the reduced numbers of reproductive trees and isolation. The low number of effective pollen donors and the expected high number of full-sibs per seed-tree should be taken into account when harvesting seeds for breeding and conservation programs. In this case, to maintain stable population inbreeding levels in ex situ conservation programs, at least 18 seed-trees with 100 seeds each are required in order to retain a reference effective size of 50.The estimated outcrossing rate suggested the presence of a mixed mating system that is already leading to higher levels of inbreeding. We found evidence of long distance gene flow via pollen that may reduce genetic drift in the small fragmented populations of


Squalidus argentatus (Sauvage and Dabry de Thiersant 1874) is a small-sized freshwater fish which is distributed in Mainland China, Hainan Island and Taiwan. The populations of S. argentatus have dropped sharply probably due to overharvesting and water pollution recently. Eleven polymorphic microsatellite markers were developed for the cyprinid fish S. argentatus. These new markers were tested on 43 individuals collected from Yangtze River and Qiantang River. The number of alleles, observed and expected heterozygosity per locus, in two populations ranged from 3 to 14, from 0.333 to 0.954 and from 0.480 to 0.928, respectively. Only two loci are significantly deviated from Hardy–Weinberg expectations due to the heterozygote deficiency. No significant linkage disequilibrium was detected between the pairwise comparisons of these loci. These polymorphic microsatellite loci will enable us to study the genetic variation, population structure, and conservation genetics of this species in the future. Squalidus argentatus (Sauvage and Dabry de Thiersant 1874)  consists of a group of small-sized freshwater fishes widely found in the lower or middle reaches of Eastern Asia, including Mainland China, Hainan Island and Taiwan. There are at least seven valid species of Squalidus found in mainland China and two endemic species reported from Taiwan [S. argentatus is the only species commonly found in above mentioned areas [S. argentatus has experienced a dramatic decrease probably owing to over-fishing and environmental destruction. In order to develop effective strategies for fishery management and conservation, information, such as the population genetic structure and genetic diversity and divergence of S. argentatus, is important. DNA-based genetic analyses can yield valuable information for assessing levels of variation and population genetic structure in freshwater fishes. Because of their high level of polymorphism and co-dominant inheritance in Mendelian fashion, microsatellites have been widely used as DNA markers in the studies of population structure and conservation genetics of freshwater fishes [S. argentatus, have been conducted, there is no report available on molecular markers isolation for this species. The microsatellite markers identified will allow the undertaking of further genetic studies on S. argentatus and provide valuable genetic information to assist the management and culture of this resource.Genus ed areas . Within r fishes . Althougr fishes  of S. arSqualidus argentatus. In brief, total genomic DNA was extracted from muscle tissue or fins preserved in 95% ethanol, by proteinase K digestion at 55 °C. DNA was purified by traditional phenol-chloroform protocol and ethanol precipitation [2, 0.5 U Taq polymerase (Promega), and 5 pmols of one RAPD primer. Several RAPD primers were used to amplify fragments from the target species’ genome in separate reactions. Reactions of PCR amplification were conducted in a thermal cycler (Eppendorf Mastercycler) using the following conditions: initial denaturing at 94 °C for 3 min; 40 cycles of 94 °C denaturing for 50 s, 37 °C annealing for 1 min, 72 °C extension for 1 min; and 72 °C for 10 min. RAPD-PCR products were size-selected to preferentially obtain small fragments (500–1200 bp). Approximately 100 ng of PCR product was ligated into a pMD 19-T vector (Takara) according to the manufacturer’s instructions, and the ligation mixture was transformed into Escherichia coli DH5α competent cells. Clones were screened of the library for microsatellites by PCR with both vector primers plus a microsatellite specific primer (GT and GA repeats) [In the present study, we developed 11 polymorphic microsatellite loci isolated using PCR-based isolation of microsatellite arrays (PIMA) of pitation . The PCRpitation –9. It tapitation . Amplifirepeats) .2, and 0.12 μM of each primer. PCR reactions were as follows: 94 °C for 4 min followed by 40 cycles at 94 °C for 30 s, 30 s–50 s at primer-specific annealing temperature . Both strands of the DNA insert were sequenced. Sequencing of positive insert PCRs was conducted by Sangon Biotec (Shanghai) Co., Ltd. with an ABI PRISM 3730 sequencer using the BigDye Terminator kit (Applied Biosystems) and by using vector primers. Primer pairs were newly designed for eleven microsatellite sequences with the program PRIMER 3 software . Prelimiperature , 72 °C fHO) and expected (HE) heterozygosities were calculated. All loci were tested for fitness to the Hardy–Weinberg equilibrium (HWE), and all pairwise combinations of loci were tested for linkage disequilibrium. All these parameters and tests were computed using ARLEQUIN 3.5 [Allele frequency, observed  and 0.333–0.857 (average of 0.668), respectively, in Yangtze River. The HE and HO ranged from 0.717–0.928 (average of 0.830) and 0.476–0.954 (average of 0.747), respectively, in Qiantang River. One locus in Yangtze River (MISA10) and in Qiantang River (MISA11) is significantly deviated from Hardy–Weinberg equilibrium after Bonferroni’s correction (P < 0.001) due to the heterozygote deficiency, respectively (P < 0.05), but for stuttering and allelic dropout tests, no evidences were found in all loci (P > 0.05). After applying sequential Bonferroni corrections to compensate for multiple statistical tests, no evidence of genotypic linkage disequilibrium at any pair of loci was found. Microsatellite makers described here should be useful to monitor population dynamics and to determine dispersal patterns and genetic diversity within and between populations of this species. These polymorphic microsatellite loci will be used to investigate population structure and gene flow among S. argentatus populations and are expected to provide a better understanding about the ecology of the species, and consequently a basis for a sustainable management framework in the future.A total of 43 ectively . These dSqualidus argentatus has experienced a dramatic decrease in the last decade. Therefore, there is an urgent need for the development and application of microsatellites to provide an effective tool for investigation of genetic variation and population structure in S. argentatus. In conclusion, we report, these 11 polymorphic microsatellite loci presented in this study were the first set of microsatellite markers designed specifically for S. argentatus, and provides the ground work for further studies on the genetic structure, gene flow, conservation management and molecular evolution of this vulnerable species.The wild resource of


Symphodus melops) is a temperate fish species, inhabiting the coasts of Europe, that show strong indications of current as well as historical (ice-age) range shifts towards the north. Nine neutral microsatellite DNA markers were screened to study genetic signatures and spatial population structure over the entire geographic and thermal gradient of the species from Portugal to Norway. A major genetic break (FST  = 0.159 average among pairs) was identified between Scandinavian and more southern populations, with a marked reduction (30% or more) in levels of genetic variability in Scandinavia. The break is probably related to bottleneck(s) associated with post-glacial colonization of the Scandinavian coasts, and indicates a lack of present gene flow across the North Sea. The lack of gene flow can most likely be attributed to the species’ need for rocky substrate for nesting and a relatively short pelagic larval phase, limiting dispersal by ocean currents. These findings demonstrate that long-distance dispersal may be severely limited in the corkwing wrasse, and that successful range-shifts following present climate change may be problematic for this and other species with limited dispersal abilities, even in the seemingly continuous marine environment.One mechanism by which marine organisms may respond to climate shifts is range shifts. The corkwing wrasse ( Currently, climate change is one of the major factors reducing the abundance of many marine species, and increasing the likelihood of local extinction in the oceans In order to predict potential future impact on marine organisms due to the current global climate change, we may study the consequences of earlier, large-scale climate shifts, i.e. those associated with the most recent glacial period. During glacial phases, the sea surface temperatures along western Europe dropped markedly, and the majority of warm temperate organisms must have moved south, either into the Mediterranean, along the West-African shores, or in glacial refugia in warm-water pockets closer to the glaciated regions The generally wider distribution ranges occupied by marine species compared to terrestrial species has been suggested to produce a buffering effect against rapid fluctuation in the environmental conditions, lowering the risk of extinction in the former group Range shifts, population bottlenecks, and expansions are demographic processes that may lead to particular genetic signatures of extant populations Symphodus melops, Linnaeus, 1758) is a rocky shore marine fish suspected to have shifted northwards in abundance over the last few decades. In its southern range (“rear edge”) it is now nearly extinct in the Mediterranean Corkwing wrasse . Briefly, the coastline in the south is fully exposed to the Atlantic Ocean with very high tidal amplitude and with strong coastal currents and waves, and temperature is at the maximum of the species tolerance. In the north (Scandinavia) temperature is at the cold tolerance limit The study area spanned across the species distribution range from southern Portugal (Algarve) to Scandinavia cf. . The areS. melops were collected using various gears: beach seine in Norway and Sweden http://www.lovdata.no. Accessed 2013 Jun 3.); Sweden: Djurskyddslagen §13 ; UK: Animal Welfare Act 2006 (http://www.legislation.gov.uk. Accessed 2013 Jun 3.)). Spear fishing  was carried out by divers with a permit (#246/2000). All sampling and handling of fish were performed by experienced personnel , and were carried out explicitly for this study. The species is not protected by European law or by any of the countries where and when sampling was performed , and no special permits are required for sampling the species either for research or commercially. Whole sacrificed fish were immediately frozen and stored (either at −20C or −70C on dry ice) until reaching laboratory facilities, where tissue was taken and put into 96% ethanol prior to DNA extraction.Samples of icrochecker software DNA was extracted using the Viogene Inc. extraction kit . Microsatellite DNA fragments were separated using a 16 capillary ABI 3170 automated sequencer. We applied Qiagen 5 Taq DNA Polymerase for the PCR reactions, using the supplied self-adjusting magnesium 10× buffer. PCR analyses were based on nine microsatellite DNA loci, using published protocols Hs within samples; HT for the total FISgenepop software  were used to judge “significance” of estimated FST-values, using the FDR approach to control for type I statistical errors in multiple tests. Temporal stability of spatial structure was tested for with AMOVA analyses of spatial samples and temporal replicates, using Arlequin ver. 3.5 Genetic differences among localities were quantified by DCE) 2DCE distance has been suggested to achieve better estimates of tree topology compared to other methods, regardless of mutation model, IAM or SMM 2 has been proposed to estimate branch lengths. The tree topology was therefore determined by a neighbour-joining (NJ) tree using the matrix of DCE distances with PHYLIP DCE tree topology and determining the branch lengths from Goldstein et al.’s δµ2 matrix The phylogenetic relationship among populations was examined by constructing a hybrid tree following Angers and Bernatchez In order to clarify possible dispersal and/or colonization routes into Scandinavia, two additional analyses were conducted. Firstly, we performed an assignment tests  and the particle-tracking model conducted are similar to the model systems described by Vikebø et al. FIS estimates and P-values varied among loci within samples as well as among samples at the same locus  to 0.912 (SMC8), while the total number of alleles spanned from a  = 13 (SMA107) to 90 (SMB11) , as compared to the Scandinavian ones . With the exception of two singletons (at SMA11 and SMD110), no alleles were observed in the Scandinavian samples that were not also found in samples to the south. Viewed as a function of latitude, these differences among samples in genetic variability measures may be seen as an abrupt decline in variability (heterozugosity) at around 57 to 58 °N , and accompanied by an equally abrupt increase in genetic differentiation  differed among loci and averages over sample sites ranged from  (SMB11) . Geneticon FST; . The PCAon FST;  identifion FST; , is a doon FST; . Below wFST  = 0.085, P<0.001 cf. FST-estimates  among the three southernmost samples from the Iberian Peninsula , whereas samples from France and the UK  for the most part were differentiated from each other , as were samples from Scandinavia  . AMOVA analyses  bootstrap support . Each clAssignment tests revealed that more than 92% of individuals from the four Scandinavian samples were assigned to the northern UK samples , rather than to the southern baseline group (PL and RO) cf. . We alsoOceanographic drift simulations  show thaFST values . Such a decline is expected in a severe bottleneck over a brief time period, or of less severe reductions in effective population size over a longer period. Robalo et al. The presumed Scandinavian bottleneck was also reflected in the phylogenetic tree , whose mOn a finer geographic scale, genetic divergence among samples within regions (France-UK and Scandinavia), is expressed by significant pairwise allele frequency differences  and highFST  = 0.159, approx: On the basis of the findings reported herein and those by Robalo et al. Taurulus bubalisS. melops showed a marked reduction in levels of genetic variability, contrasting with T. bubalis which displayed homogenous high levels throughout its range. Almada et al. T. bubalis had an older origin than the one predicted for corkwing wrasse The finding of higher genetic divergence across open water, (i.e. discontinuous habitat) than along rocky shoreline, has been documented for several other coastal marine fish with limited pelagic larval stage and specific habitat requirements  from their southern conspecifics, has important implications for our understanding of the colonization processes and range shift mechanisms following global climate changes. The present isolation of Scandinavian populations implies that the historical range-shift associated with its post-glacial colonization was a unique event, most likely made possible by the presence of suitable shorelines along Doggerland that temporally acted as a migration path from UK to the Skagerrak shores. Following the subsequent submergence of Doggerland the species was apparently deprived of this northward path, and migration to Skagerrak ceased. As judged by the reduced genetic variability in present corkwing wrasse populations in the Skagerrak, the number of successful migrants may anyway have been limited. This scenario implies that species’ range-shifts may be a complicated event, even in the oceans, whose continuous expanse have traditionally been regarded as highly conductive to animal dispersal