Alfaro cultratus, Poecilia gillii, and Xenophallus umbratilis) that we predicted might display congruent responses due to co-occurrence in identical freshwater drainages. Spatial analyses recovered different subdivisions of genetic structure for each species, despite shared finer-scale breaks in northwestern Costa Rica . Isolation-with-migration models estimated incongruent timelines of among-region divergences, with A. cultratus and Xenophallus populations diverging over Miocene–mid-Pleistocene while P. gillii populations diverged over mid-late Pleistocene. Approximate Bayesian computation also lent substantial support to multiple discrete divergences over a model of simultaneous divergence across shared spatial breaks . Thus, the data support phylogeographic pseudoincongruence consistent with the multiple-response hypothesis. Model comparisons also indicated incongruence in historical demography, for example, support for intraspecific late Pleistocene population growth was unique to P. gillii, despite evidence for finer-scale population expansions in the other taxa. Empirical tests for phylogeographic congruence indicate that multiple evolutionary responses to historical events have shaped the population structure of freshwater species codistributed within the complex landscapes in/around the Nicaraguan depression. Recent community assembly through different routes , and intrinsic ecological differences among species, has likely contributed to the unique phylogeographical patterns displayed by these Neotropical fishes.A central goal of comparative phylogeography is determining whether codistributed species experienced (1) concerted evolutionary responses to past geological and climatic events, indicated by congruent spatial and temporal patterns (“concerted-response hypothesis”); (2) independent responses, indicated by spatial incongruence (“independent-response hypothesis”); or (3) multiple responses (“multiple-response hypothesis”), indicated by spatial congruence but temporal incongruence (“pseudocongruence”) or spatial and temporal incongruence (“pseudoincongruence”). We tested these competing hypotheses using DNA sequence data from three livebearing fish species codistributed in the Nicaraguan depression of Central America ( Ne, effective population size) across multiple codistributed species, comparative studies provide critical assessments of phylogeographical congruence, forming a basis for historical inferences  isthmus is famous worldwide as an example of a land-bridge formation that has shaped continental Neotropical biotas by facilitating widespread dispersals, speciation, and extinctions in North and South America  , and molly, P. gillii (Kner 1863). Within Xenophallus, Jones and Johnson , with analyses of existing data from P. gillii and Xenophallus populations codistributed in the ND, we test two predictions of the concerted-response hypothesis, against the independent- and multiple-response hypotheses. These predictions include (1) that these fishes should exhibit congruent spatial-genetic structuring and (2) that spatial subdivisions should be temporally congruent, due to co-occurrence in drainages correlated with LCA geomorphology , the allopatric sister species to A. cultratus, from five sites in Honduras. Specimens were preserved in 95% ethanol in the field. We augmented data from these samples with published mtDNA cytochrome b (cytb) sequences from A. cultratus ), nucleotide diversity (π), and Watterson's (θw (per site) for each locality and species using DnaSP. We calculated the same summary statistics in DnaSP for each population group . We modeled K = 2–10 groups, drawing from 100 initial conditions, and noted fixation index (ΦCT) trends. Second, we identified genetic barriers among populations using BARRIER 2.2  performed in ARLEQUIN (1000 nonparametric permutations). When faced with isolation-by-distance, SAMOVA and Monmonier's algorithm are more likely to misidentify populations and genetic barriers between them ] and natural log-transformed geographic distance between localities using standard regression, and Mantel tests  searches in GARLI 0.97 (Zwickl We compared phylogenetic relationships (haplotype gene trees) and nodal support among cyt7 Zwickl . Likelih7 Zwickl  selected7 Zwickl , and par7 Zwickl .t) among adjacent population groups from BARRIER. While we were mainly interested in estimating t, IMa2 also estimates population migration rates  and sizes of current  and ancestral populations (θA) using Hey and Nielsen's  ≥10% update rates for t, (2) appropriate chain-swapping rates, and (3) runs converging on similar parameter estimates. Fossil data and species-specific substitution rates were unavailable to us, thus we specified uniform mutation rate (μ) priors spanning lower and upper per-lineage mutation rates published for teleost fish protein-coding mtDNA, 1.7 × 10−9 and 1.4 × 10−8 substitutions/site/year (subs/site/year) [refs. in Waters and Burridge (t estimates were converted to absolute time (Tdiv) using the equation Tdiv = t/μk (where k = sequence length), assuming species generation times equivalent to 1 year/generation  the standard 2% rate  for vertebrate mtDNA  estimated for trout species mtDNA  independently while accounting for coalescent gene-tree stochasticity . We generated hyper-posteriors for the mtDNA, representing 1000 random draws from the joint posterior distribution, by comparing the observed versus simulated summary statistics vectors using the pipeline's standard rejection/acceptance algorithm. Posterior estimates of the number of discrete co-divergences (Ψ) were obtained via polychotomous regression; posterior estimates of population divergence time  and Ω (dispersion index representing the ratio of variance to the mean divergence times across Y taxon pairs) were obtained by local linear regression  to compare the level of posterior support for simultaneous versus nonsimultaneous divergence , using Jeffreys' . Bayes factor calculations accounted for prior support for each hypothesis. To explore our data, we conducted multiple msBayes runs across a range of prior values  to evaluate the effects of the prior on the models, and we conducted Bayes factor hypotheses testing using each model.We further tested the temporal congruence of shared genealogical breaks within northwestern Costa Rica (see Results) using hierarchical approximate Bayesian computation as implemented in the bioinformatics pipeline, MTML-msBayes  showed that marginal relaxed clock standard deviations (“ucld.stdev” parameter) clumped at zero, indicating highly clock-like data. Therefore, we conducted Bayesian skyline model runs using strict clocks . We specified uniform priors spanning teleost mtDNA mutation rates (see IMa2 methods) and substitution models selected by DT-ModSel , and their 95% highest posterior densities (HPD) using TRACER 1.5  and was compared to alternatives using log Bayes factors (log10B10) calculated in TRACER . To distinguish population expansions from purifying or positive natural selection, we tested for neutrality using similar simulations of Fay and Wu's  and taxa would provide strong evidence for congruent past population dynamics.We qualitatively evaluated historical demographic congruence by comparing estimates of past population size fluctuations through time captured using the Bayesian skyline plot method , π (range = 0.0004–0.0076), and θw (range = 0.733–5.586) varied from low to moderate, but were much higher in A. cultratus and P. gillii than X. umbratilis .Among localities, mtDNA genetic diversity was highly spatially variable: to 25.92 . When avbratilis . MtDNA gher taxa . Cytb vaA. cultratus groups , six P. gillii groups , and seven Xenophallus groups , indicating differing numbers and positions of spatial subdivisions between homogeneous populations within species  and southeastern groups (SEG) in the lowlands between Rio Frio and Rio Pocosol flanked by Tenorio and Arenal volcanoes  occurred just east of Rio Pocosol, dividing Frio and Pocosol rivers subpopulations (FRPOG) from a southeastern group (SEG) of lower San Juan and Rio Tortuguero samples  between Sabalo and Frio rivers formed a northwestern group (NWG) of westernmost Lake Nicaragua-tributary subpopulations . In Xenophallus  divided from the other groups just northwest of the active Arenal volcano within the Guanacaste Cordillera highlands  but not the other species . Likewise, Xenophallus genetic and ln-geographic distances showed a positive regression relationship not recovered in A. cultratus or P. gillii (Mantel tests supported isolation-by-distance in . gillii .A. cultratus showed nonsignificant genetic structuring among San Juan–back-arc drainage groups (AMOVA model 1) and A. cultratus and P. gillii had nonsignificant structuring among San Juan tributaries (AMOVA model 2). However, P. gillii were significantly differentiated between San Juan and back-arc drainage groups. In contrast, Xenophallus AMOVA 2 indicated San Juan tributaries were distinct from one another, but that San Juan–back-arc drainage structuring was nonsignificant . Clades III and IV had star-like networks with ancestral Sapoa, San Carlos, and Sixaola drainage haplotypes possibly indicating recent population expansions in these regions, and we estimated a San Carlos origin for the network root (haplotype 1). Poecilia gillii , and small well-supported San Juan and back-arc drainage subclades . Haplotype 24 (San Carlos) was ancestral, sister to all other P. gillii haplotypes; however, haplotype 62 (Parismina) was the network root and showed a star-like pattern consistent with recent expansion. While the SEG harbored most P. gillii alleles, haplotypes 13 and 38–41 were confined to the NWG group. Xenophallus differed from the other species in having four well-supported, nonallopatric clades mostly isolated in drainage basins. The Xenophallus cytb topology contrasted deep (6.0%) divergence of San Carlos (clade I) haplotypes , from all others, against shallow intradrainage variation  and divergence times (t) in most runs, indicated by likelihood surface peaks  and Xenophallus (UG–LSJTOG: Tdiv range = 13.731–2.334 Ma) than P. gillii. All divergences within P. gillii ranged over mid-late Pleistocene, including the FRPOG–SEG population pair (Tdiv range = 0.130–0.0221 Ma). A similar pattern of incongruence arose when comparing all Tdiv estimates together. Whereas we estimated nonzero migration rates in A. cultratus, posterior m distributions peaked at the lower limit of resolution or 95% HPDs included zero in P. gillii and Xenophallus, indicating no ongoing gene flow.Coalescent-based dating analyses in IMa2 yielded reliable estimates of BARRIER population group sizes , though prior settings and results for all models can be found in Ψ (mean = 2.291) and Ω  parameter estimates indicated that a model of multiple discrete divergences, rather than simultaneous divergence, was supported by the data  and continuous divergence (Ψ = 3 vs. Ψ < 3) respectively. Based on a Bayes factor of 1.535 for Ω > 0.01 vs. Ω < 0.01, dispersion index Ω also indicated evidence against a simultaneous divergence model; however, the weight of the evidence was marginal, suggesting a potentially weaker ability of Ω to reject simultaneous divergence for our data. Modal divergence time estimates across the three population pairs from MTML-msBayes were similar to Tdiv estimates from IMa2 falling mostly within the Pliocene–Pleistocene  and positive R2 statistics  was supported by a positive and significant R2   Wakeley . While o Wakeley . Moreoveyes Fig. . Thus, dOrthogeomys cherriei pocket gophers found haplotypes were up to 1.5% diverged in the Costa Rican Central Cordillera , a broader range of elevations  and thermal environments , and a much larger geographic range  than the other species  than Xenophallus, which is endemic to the study area  and genetic diversity  within Rio Tortuguero (Given we used the same locus (similar mutation rates) to compare the phylogeographies of closely related species with overlapping ranges, inadequate phylogenetic signal cannot account for the broad-scale pattern of spatial incongruence among  Bussing . Alfaro  Bussing  and is u Bussing . These d Bussing . This ises Figs. C, S4, anes Figs.  consisteophallus . Thus, lrtuguero .m ≠ 0, as in all of our pilot IMa2 runs and several of our long runs). If marginal distributions of m parameters include 0, gene flow can be rejected in favor of incomplete lineage sorting; otherwise, there is sufficient information to resolve migration as influencing the data. In IMa2 we inferred discordant migration rates between regions among taxa, with nonzero migration between A. cultratus groups while other results suggested no ongoing gene flow . Moreover, we expect that samples from demes with higher m should be more polymorphic than those from demes with lower m (Wakeley and Aliacar A. cultratus (higher π and Hd) and Xenophallus  nonzero peak m estimates between P. gillii population groups  may simply indicate a trivial number of migrants relative to overall population size , teasing these processes apart is difficult. However, nonequilibrium statistical phylogeography tools such as IMa2 that jointly estimate demographic parameters while modeling coalescent and mutational stochasticity, implicitly test whether alleles shared between populations reflect gene flow versus incomplete lineage sorting  provides a weaker basis for making inferences about past population dynamics than rejecting the null model would. However, size-constancy rather than bottleneck-expansions or other growth trends still appears to be the most likely historical demographic scenario for Xenophallus based on a parsimonious interpretation of Bayes factors (Table Xenophallus.In addition to temporally and spatially incongruent phylogeographic histories, we find evidence for incongruent patterns of historical-demographic fluctuations over recent timescales among ND livebearers Figs. , 4. Our rs Table , S4 and rs Table  also supNotwithstanding the many points of incongruence among our focal taxa, we have established that these species share genetic breaks just north of present-day Lake Arenal in northwestern Costa Rica, in between two large Rio San Juan tributaries, the Frio and San Carlos rivers Figs. , 3. ConsIn summary, through multiple empirical tests for congruence, our study has demonstrated that spatially and temporally incongruent phylogeographic and demographic patterns are evident in three species of livebearing fishes that are codependent upon freshwater habitats within the Nicaraguan depression landscape. The majority of our results point to multiple evolutionary responses among these taxa, and we have statistically shown that these corresponded to multiple historical dispersal and vicariance events, possibly suggesting waves of dispersion through the area. Despite overall pseudoincongruence supporting a “multiple-response hypothesis”, however, landscape history appears to have promoted commonalities of phylogeographical structuring, albeit over fine spatial scales. More nuclear loci and expanded spatial sampling covering the entire species ranges are necessary to better tease apart the exact histories responsible for the varying evolutionary trajectories in these taxa. However, a comparative perspective has afforded us a view of the lower Central American freshwater fish assemblage that has provided insights into historical as well as ecological influences on population structure, and which permits drawing several future predictions. First, additional studies of individual taxa similarly confined to these freshwater habitats of the Nicaraguan depression should show similar phylogeographic patterns, although it is likely that even further evidence for a multiple-response scenario will be uncovered. And, secondly, we predict that additional comparative studies will yield many new insights into the relative roles of concerted, independent, and multiple responses in shaping the assembly and diversification of species rich and endemic Central American ecosystems.


Theileria, is a common disease of livestock in Oman. It is a major constraint to the development of the livestock industry due to a high rate of morbidity and mortality in both cattle and sheep. Since little is currently known about the genetic diversity of the parasites causing theileriosis in Oman, the present study was designed to address this issue with specific regard to T. annulata in cattle.Theileriosis, caused by a number of species within the genus Theileria annulata population. Ten genetic markers (micro- and mini-satellites) representing all four chromosomes of T. annulata were applied to these samples using a combination of PCR amplification and fragment analysis. The resultant genetic data was analysed to provide a first insight into the structure of the T. annulata population in Oman.Blood samples were collected from cattle from four geographically distinct regions in Oman for genetic analysis of the ST values being less than 0.03. Slightly higher FST values  were detected when the data for T. annulata parasites in Oman was compared with that previously generated for Turkey and Tunisia.We applied ten micro- and mini-satellite markers to a total of 310 samples obtained from different regions . A high degree of allelic diversity was observed among the four parasite populations. Expected heterozygosity for each site ranged from 0.816 to 0.854. A high multiplicity of infection was observed in individual hosts, with an average of 3.3 to 3.4 alleles per locus, in samples derived from Batinah, Dhofar and Sharqia regions. In samples from Dhira region, an average of 2.9 alleles per locus was observed. Mild but statistically significant linkage disequilibrium between pairs of markers was observed in populations from three of the four regions. In contrast, when the analysis was performed at farm level, no significant linkage disequilibrium was observed. Finally, no significant genetic differentiation was seen between the four populations, with most pair-wise FT. annulata samples representing four geographical regions in Oman revealed a high level of genetic diversity in the parasite population. There was little evidence of genetic differentiation between parasites from different regions, and a high level of genetic diversity was maintained within each sub-population. These findings are consistent with a high parasite transmission rate and frequent movement of animals between different regions in Oman.Genetic analyses of  Theileria annulata, which is transmitted by ticks of the genus Hyalomma and infects cattle. In addition, T. lestoquardi and T. ovis are the common Theileria species associated with theileriosis in small ruminants within Oman [1-∑p2] where n is the number of isolates and p the frequency of each different allele at a locus ) followed by Batinah (n = 58 [74.4%]), Sharqia (n = 26 [72.2%]) and Dhira (n = 68 [56.7%]).The screening of cattle blood samples (n = 447) using 18S rRNA gene primers, revealed a total of 323 (72.3%) T. annulata isolates from the four governorates were succesfully typed using ten micro- and mini-satellite markers. Each marker was found to be highly polymorphic in samples from all four regions, with the overall number of alleles per locus ranging from 16, for TS15 to 51 for TS6  was calculated using the predominant allele dataset for each marker and averaged across all ten loci. The average heterozygosity within each region was generally high, ranging from 0.816 within Dhofar to 0.854 in Batinah.More than one allele per locus was common in all field samples, therefore multi-locus genotypes (MLGs) were constructed, based on the predominant allele present at each locus, and used to generate the dataset for population genetic analysis. The MLG dataset  was usedT. annulata populations in the different regions comprised a single panmictic population with a high degree of genetic exchange, the level of linkage equilibrium of alleles at pairs of loci was measured using the standard index of association (ISA). When the samples from the four regions were treated as a single population (i.e. Oman), an ISA value of 0.019 and a VD value (1.4595) greater than L (1.29) was obtained, indicating LD  [ST = 0.033 and θ = 0.04) was evident among the four sites in Oman when considered together and similarly, a low level of differentiation was indicated when the populations from each region were compared with each other in a pair-wise manner  , 26 weree manner .ST estimated for the three populations , demonstrated moderate differentiation between parasite populations between Oman and Tunisia (G’ST = 0.063 and θ = 0.063) and a slightly higher level of differentiation between Oman and Turkey (G’ST = 0.102 and θ = 0.103)  .The low level of differentiation between parasite populations across four regions in Oman was supported by PCoA analysis  as no clT. annulata in three countries by partitioning variation among and within parasite sub-populations. Most of the genetic variation (93%) was contained within sub-populations with only 7% explained by differences between sub-populations. This suggests the occurrence of a high rate of cross-mating and recombination within the parasite population as a whole.Analysis of molecular variance (AMOVA) was used to assess the population structure of T.annulata isolates, with a complete set of data, was found to carry multiple genotypes, as several alleles were evident at one or more loci. The mean number of alleles for the ten loci was calculated for each isolate to provide an index value that represented the multiplicity of infection. A summary of multiplicity of infection with respect to the area of isolation is presented in Each of 231 (75%) T. annulata isolates in Oman were collected from cattle between 4 and 180 months of age, around 86% of which were female. The majority of cattle were indigenous breeds (71%). Analysis of co-variance (ANCOVA) indicated that there is no apparent association of host variables with multiplicity of infection.Of the 231 isolates examined, 131 (58%) had complete data relating to host phenotype . T. annulata in Oman. We used a panel of polymorphic micro- and mini-satellite loci, among T. annulata in four sites in Oman, allowing the inference of genetic parameters driven by neutral processes to define the population genetic structure. The analysis revealed high diversity at each genomic locus, evidence of LD between loci, a high prevalence of multiple genotypes within infected animals and a low level of differentiation between parasites in the four sampling sites.The work reported in this paper is the first detailed genetic analysis of the He index for each site ranged from 0.816 to 0.854. A similar level of diversity was seen among T. annulata in other endemic countries [T. parva (He ranging between 0.503 and 0.675) in Zambia [e): i.e. the proportion of the population that contributes to the evolution of genetic diversity [e, so the above observation implies that the effective population size of T. annulata in Oman is large. However, other factors can contribute to diversity, such as migration and gene flow [There were extremely high estimates of genetic diversity within populations from the four sites, both in terms of allelic diversity at each locus studied and in the numbers of co-existing genetically distinct organisms within infected animals. There was a high allelic diversity for the ten loci in all sites, the overall n Zambia , althougiversity . Theoretene flow .T. annulata and T.parva in other countries [The mean numbers of genotypes detected per infected animal ranged between 2.9 and 3.4 genotypes. The number of parasite genotypes found within individual hosts may reflect the transmission intensity and abundance of vector (ticks). Thus, the relatively low multiplicity of infection in Dhira compared to the other three sites, may be linked to variation in tick abundance/species and incidence of parasite infection in ticks, however it is possible that confounding factors such as differences in regional sampling or unknown host-related factors may also explain this finding. Unfortunately, there is no information on abundance and seasonality of ticks in different regions in Oman. Only 2.1% of the variability in multiplicity of infection can be explained by bovine host variables recorded . In addition, samples from all sites were collected from apparently healthy animals. The high multiplicity of genotypes among the asymptomatic animals examined in the present study is in accordance with other surveys of clinical cases of ountries . This isountries  where thT. annulata in the vector. Thus, co-uptake of piroplasm/ gametocytes representing distinct genotypes by the feeding tick may result in cross- fertilisation and the formation of heterozygotes following syngamy. Genetic recombination then occurs in the tick gut leading to the generation of novel genotypes [T. annulata in Oman provides estimates for effective number of clones (ne) of 3.35 and inbreeding of 0.3 (f = 1/ne) [Mixed genotypes in the bovine host can be considered a pre-requisite for cross-mating and recombination of enotypes , 33. Assenotypes . Such anenotypes , 35. Ext = 1/ne) , 35. ThiT. annulata populations in Oman. LD can be influenced by diverse factors, other than the extent of inbreeding; including the recombination rate, the local parasite effective population size and population differentiation, caused by subpopulation structure [Despite the observed extensive genetic diversity, a high level of LD was detected in three of the four tructure . HoweverTheileria parasites can occur among infected animals, and it may be hypothesised that the extent of clustering depends on epidemiological factors such as the density of ticks, parasite infectivity and genotype-specific immune responses of the host. Currently, no information is available on the extent of transmission in the four sites. However, vertebrate hosts in close proximity can often carry related parasite genotypes that result from crossing, recombination and asexual amplification in a single infected vector [Theileria, this will be true for ticks that fed on the same infected animal. It has been shown that a small number of genetically related parasites can generate significant linkage disequilibrium [T. annulata populations in Oman may not be related to a clonal pattern of population structure. This is evident by the high diversity of MLGs among isolates in each region implying, as indicated above, a high degree of cross-mating and sexual recombination. Indeed the MLG diversity and estimate of the index of association , and Oman and Turkey (FST = 0.102). Parasite genotypes in Oman appear to be more closely related to those in Tunisia than Turkey. This suggests that despite ecological and epidemiological barriers between the parasite populations, there is likely to have been slightly more mixing between these populations than between Oman and Turkey; this may be related to the frequency of movement of animals [Very low levels of genetic differentiation were detected between ulations . This co animals  since, iFree movement of cattle over a large distance is not a feature in Dhira and Sharqia in Oman. Here animals are kept exclusively on farms, although movement of cattle from one farm to another is not an uncommon practice. It would be of interest to examine parasites in neighboring countries in the region, where theileriosis is also a major problem, to determine how closely related these poplations are, and whether control measures are implemented seperately or if a regional policy can and should be adopted.In summary, analysis of ten putative neutral markers among four populations of T. annulata in geographically separated regions in Oman revealed a high level of genetic diversity with limited differentiation between regional populations. Broadly, the parasite population appears to be panmictic and, similar to previous studies in Theileria, a degree of LD was observed in some of the examined populations. The mild LD observed may be caused by a number of factors, including the over-sampling of related genotypes. A high proportion of the examined animals carried distinct multi-locus genotypes, suggesting a high rate of outcrossing in the tick vector. However, little is known of the interactions among different genotypes and the factors that modulate their transmissibility to the tick. Further investigation of the temporal dynamics of parasite genotypes within infected herds will likely provide a better understanding of complexities of the population structure of T. annulata as a whole.S1 Table(DOCX)Click here for additional data file.


Gopherus flavomarginatus) is the first species of extirpated megafauna to be repatriated into the United States. In September 2006, 30 individuals were translocated from Arizona to New Mexico with the long-term objective of restoring wild populations via captive propagation. We evaluated mtDNA sequences and allelic diversity among 11 microsatellite loci from the captive population and archived samples collected from wild individuals in Durango, Mexico (n = 28). Both populations exhibited very low genetic diversity and the captive population captured roughly 97.5% of the total wild diversity, making it a promising founder population. Genetic screening of other captive animals (n = 26) potentially suitable for reintroduction uncovered multiple hybrid G. flavomarginatus×G. polyphemus, which were ineligible for repatriation; only three of these individuals were verified as purebred G. flavomarginatus. We used these genetic data to inform mate pairing, reduce the potential for inbreeding and to monitor the maintenance of genetic diversity in the captive population. After six years of successful propagation, we analyzed the parentage of 241 hatchlings to assess the maintenance of genetic diversity. Not all adults contributed equally to successive generations. Most yearly cohorts of hatchlings failed to capture the diversity of the parental population. However, overlapping generations of tortoises helped to alleviate genetic loss because the entire six-year cohort of hatchlings contained the allelic diversity of the parental population. Polyandry and sperm storage occurred in the captives and future management strategies must consider such events.The Bolson tortoise ( Gopherus flavomarginatus) provided the opportunity to “rewild” the Chihuahuan Desert of the United States with an extant, but locally extirpated species. The IUCN Red List In 2005, Donlan et al. Mycoplasma, a causative agent for upper respiratory tract disease (URTD) in related species of Gopherus. In September 2006, 30 individuals were translocated; one tortoise was not located and later confirmed deceased. The 26 disease-free adults were divided between two larger enclosures of 3.5 ha each and the four Mycoplasma antibody-positive adults  went to the Living Desert Zoo and Gardens State Park near Carlsbad, New Mexico In 2006, the Turner Endangered Species Fund (TESF) established a translocated captive-breeding population of Bolson tortoises within the species’ prehistoric range in a semi-natural setting in the Chihuahuan Desert of New Mexico Small populations are more susceptible to inbreeding than large populations because of randomly decreasing heterozygosity of individuals. This can lead to a reduction of fitness G. flavomarginatus may augment genetic diversity within the captive population. Thus, we tested additional captive animals in the USA for their potential contributions. We pursue the following six goals: 1) evaluate the natural genetic structure of the wild populations; 2) assess diversity of existing captive population; 3) identify additional individuals eligible for a captive breeding program; 4) assign captive individuals for breeding based on genetic relatedness; 5) assess the genetic structure of the hatchlings after successive years of breeding; and 6) make recommendations for future maintenance of the genetic diversity of the captive population.Genetic analyses of the founding individuals (referred to as the Appleton Ranch population) as well as on samples collected from the wild in Durango, Mexico facilitate the estimation of genetic diversity, which, in turn, informs captive breeding decisions for the population in New Mexico. Targeted introductions of tortoises in zoos and private collections into the breeding program of The University of Arizona Institutional Care and Use Committee (IACUC) approved all tortoise-handling protocols (IACUC Control no. 09-138).The captive population of Bolson tortoises originally from the Appleton Ranch was presumed to consist of multiple founders and their offspring. Unfortunately, the exact genealogy of the population was unknown. Thus, we considered it a population of closely related individuals, including parents and offspring. When translocated to New Mexico, all individuals were considered to be ‘adult’, even though tortoises ranged in size from 214 to 375 mm MCL and individual fecundity was expected to vary as a function of size class, social status, and other characteristics Adult Bolson tortoises were kept in outdoor enclosures on the Armendaris Ranch  or the Living Desert Zoo and Garden State Park (LDZG) in Carlsbad, NM . Initially, the population on the Armendaris existed in two groups that were held in two ∼3.4 ha pens surrounded by 0.6 m high perimeter fencing and located about 3.2 km apart. In the spring of 2011, the “Cedar Tank” enclosure was enlarged to ∼6.5 ha, and the “Deep Well” population was integrated into the Cedar Tank population by early summer of 2011  or fall of 2011 .The reproductive status of the Armendaris Ranch tortoises was determined by using a combination of radiography and ultra-sonography. Gravid females were isolated until they were ready to nest (as determined by behavior and/or radiography). In some cases, the tortoises laid their eggs in natural nests, which we excavated and transported to incubators. In other cases, gravid tortoises were moved to the incubator room and induced to lay eggs by administering oxytocin (1 U/kg). In both cases, eggs were labeled with a unique number that allowed tracking of their mothers. In two cases, a nest was discovered and protected in place in the tortoise enclosure. The mother of one nest was known but the other was not known. A further nine hatchlings, whose parentage was unknown, were discovered.personal communication). Thus, both LDZG males had access to both LDZG females since at least 2006. LDZG eggs were collected either after zoo visitors reported tortoise nesting or when zoo staff noticed disturbed soil. In general, the identity of the mother was unknown. Eggs were transported to a set of incubators located at the Zoo’s veterinary clinic and incubated until hatching.The four individuals  at the LDZG have shared an exhibit since they were moved to Carlsbad in 2006. Historically, these four individuals (housed as two pairs) occupied adjacent pens while they were part of the Appleton group in Arizona, but the fence between them may have been breached . For comparison to wild populations, we obtained 28 archived samples collected from tortoises in their current range from Durango, Mexico We extracted genomic DNA from either whole blood, salvaged red blood cells, lymphatic fluid, muscle tissue  or egg membrane (seven unhatched eggs) using a modified protocol for the BioSprint 96 DNA Blood Kit and the BioSprint 96 robotic magnetic-particle purification system . We quantified recovered DNA using a BioTEK Synergy HT  and diluted working stocks to 5 ng/µl for polymerase chain reaction (PCR). Fragment analysis and DNA sequencing was performed by the University of Arizona Genetics Core.ND3, arginine tRNA, ND4L, and part of ND4) using primers Nap2 and New Gly G. flavomarginatus, we designed a new internal sequencing primer Int615 (TATGTAAACCAAAACAATTATG).We amplified an ∼1500 base pair (bp) portion of mitochondrial DNA /microsatellite loci had been characterized specifically for We assessed the STR dataset for evidence of null alleles, large allele dropout and scoring error due to stuttering using Micro-Checker v.2.2.3 IS) for each locus in each sample group were calculated using GENEPOP and genetic distances were calculated among groups using pairwise FSTWe used ARLEQUIN v.3.11 We performed assignment testing using WHICHRUN v.4.1 We used BOTTLENECK â) ST/(1−FST) using pairs of individuals instead of populations We used SPAGeDi v.1.4 For kinship analysis, we used the assumed or potential parent/hatchling assignments. We used Cervus v.3.0.3 G. flavomarginatus had the same mtDNA haplotype, including those from Appleton Ranch (n = 31), Durango, Mexico (n = 28), and the El Paso Zoo (n = 18). All tortoises were from the same matriline (GenBank Accession no. DQ649408.1).All individuals of G. flavomarginatus  but because of increased stuttering at the longer repeats we could not always confidently distinguish between scores of 39/40 or 40/40 and thus excluded this locus from the interpretation when parental assignment depended on this differentiation The utility of these data for kinship analyses was dependent on the variability of the genetic markers as well as the relatedness of individuals in the population. P(ID) was determined for the 30 individuals translocated to New Mexico from the Appleton population and each subsequent clutch from 2007 to 2012. Applying a cut-off value of P(ID) <0.001–0.0001, as in wildlife forensic cases , this analysis suggested that Test56, Goag06, GP102, GP61 and ROM06 were the most powerful loci to distinguish individuals and resolve relationships.ST = 0.330 and 0.312, respectively). In contrast, the Appleton and Durango populations hardly differed (FST = 0.025). The assignment tests only assigned 65% of Appleton and Durango samples to their respective populations, with the second most likely population being the other. In contrast, 100% of the El Paso samples assigned to that population. Each population exhibited fairly low genetic diversity based on the STRs with the Appleton population averaging 0.282 (s.d. ±0.157), Durango 0.320 (s.d. ±0.175), and El Paso 0.288 (s.d. ±0.170). A significant excess in heterozygosity occurred in captive and wild populations of G. flavomarginatus, suggestive of a population bottleneck .The El Paso Zoo population exhibited very large genetic differentiation from populations at the Appleton Ranch and Durango, Mexico . Three individuals from private collections were resolved as being full-blood G. flavomarginatus .Our examination of the tortoise population at the El Paso Zoo suggested that these animals were hybrids between G. flavomarginatus to assist in captive breeding (p = 0.01) to about half of the hatchlings  also support that the species has reduced genetic diversity. Genetic differentiation estimates among populations of other congeners in natural settings range from 0.061 to 0.37 G. flavomarginatus might owe to an extreme population bottleneck caused by range reduction. However, a pattern can also result from a perpetually small population size.The dearth of genetic diversity in our mtDNA results suggests that wild Bolson tortoises have experienced tremendous reductions in population size since at least the last glacial maximum. This is consistent with a recent study by Urena-Aranda and de los Monteros richness  and the With these genetic data, we have confirmed that maintaining the Appleton Ranch founding individuals as a single breeding population is an appropriate grouping based on natural evolutionary significant units (ESUs) G. flavomarginatus×G. polyphemus hybrids. These results are consistent with an unpublished genetic analysis performed on the same individuals in 2003 at the Center for Conservation and Research at the Henry Doorly Zoo . The original owner of this ‘founder’ population once resided in Las Cruces, NM. As recounted by the owner’s daughter, one tortoise was picked up along a road in Chihuahua, Mexico and a sister brought another tortoise after a trip to Florida. No other animals were acquired . These animals successfully reproduced and offspring may have been passed to other people throughout the years. One offspring is likely the feral individual we genotyped from New Mexico. Notwithstanding, these hybrids are ineligible for repatriation and therefore will not be integrated into our breeding program.Our analyses indicate that tortoises at the El Paso Zoo are G. flavomarginatus is encouraging. These tortoises indicate that others may exist in zoos or private collections. While introducing any new genetic material to the breeding population would be beneficial, we can evaluate the relative contribution of each new individual by assessing their relatedness to the Appleton Ranch population and then prioritize management actions accordingly. Genetic analyses indicate that privately owned “Pancha”  of captive-bred Bolson tortoises exhibits less genetic diversity than the parental population . Very feObservations of nesting yield precise data on the number of females contributing but the contribution of males to hatchling cohorts has more uncertainty. The four individuals housed together at the LDZG exemplify the problem. Although contributing over 88 offspring  to the breeding program over the six years, the two males differ in their contribution of offspring . Male “CMycoplasma agassizii, the causative agent for upper respiratory disease (URTD) in related Gopherus species. In addition, when initially translocated from Arizona, the tortoises had been maintained in essentially one large outdoor pen for the previous 30+ years, with the exception of the four that ended up at the LDZG. These tortoises exhibit social behavior and had breed successfully in the past Although we identify the best potential mating pairs . Fortunately for the Bolson tortoise, captive breeding has already proven successful. Despite over-representation of some individuals to the breeding pool, we take advantage of this knowledge to inform management strategies. For example, offspring from LDZG may be used for an experimental soft-release of juvenile tortoises to outdoor pens because potential risk of mortality would only affect redundant genotypes. We also plan to assess natural nesting success in outdoor pens at the Armendaris Ranch by allowing genetically over-represented females to nest without moving their eggs to incubators. At the same time, we will collect eggs from under-represented females or females who exhibit greater genetic differentiation from the rest of the population and incubate them ex situ to ensure their contribution to the population. Finally, we hope to pair new pure We base our recommendations on maintaining the maximum amount of genetic variation in the rewilding population of Bolson tortoises. Management of this program has challenges on many levels, including animal behavior, health, diet, and habitat, among others. Notwithstanding, genetic data can inform a captive breeding program at its onset to reduce the problem of inbreeding


Leishmania donovani complex, has been responsible for devastating VL epidemics in the Sudan. Multilocus microsatellite and sequence typing studies can provide valuable insights into the molecular epidemiology of leishmaniasis, when applied at local scales. Here we present population genetic data for a large panel of strains and clones collected in endemic Sudan between 1993 and 2001.Visceral leishmaniasis (VL), caused by the members of the Genetic diversity was evaluated at fourteen microsatellite markers and eleven nuclear sequence loci across 124 strains and clones.ST (microsatellite) and KST (sequence) indicated small but significant shifts among the allelic repertoires of circulating strains year on year. Furthermore, we noted the co-occurrence of human and canine L. donovani strains in three of the six clusters defined. Finally, we identified widespread deficit in heterozygosity in all four years tested but strong deviation from inter-locus linkage equilibrium in two years.Microsatellite data defined six genetic subpopulations with which the nuclear sequence data were broadly congruent. Pairwise estimates of FL. donovani in Sudan, and minor population structuring between years is characteristic of entrenched, endemic disease transmission. Seasonality in vector abundance and transmission may, to an extent, explain the shallow temporal clines in allelic frequency that we observed. Genetically similar canine and human strains highlight the role of dogs as important local reservoirs of visceral leishmaniasis.Significant genetic diversity is present among The online version of this article (doi:10.1186/s13071-014-0496-4) contains supplementary material, which is available to authorized users. Leishmania donovani complex. The L. donovani complex is distributed throughout Asia, North Africa, Latin America and Southern Europe, affecting mostly vulnerable and neglected populations. Infection is spread via the bite of haematophagous phlebotomine sand fly species, while the role of non-human reservoir hosts varies from region to region  calculated in MICROSAT  [r) in FSTAT 2.9.3.2 [FIS , per locus per population, also in FSTAT 2.9.3.2. Tests for population-level deviation from Hardy-Weinberg allele frequencies were calculated in Arlequin v3.5 and associated significance levels for p values derived after sequential Bonferroni correction to minimise the likelihood of Type 1 errors. Linkage disequilibrium was defined via the Index of Association and calculated exclusively from biological clones in two populations.Population-level analyses of microsatellite data were undertaken exclusively on Sudanese tor (θw) ) and testor (θw) . Secondltor (θw) . Finally 2.9.3.2  and FIS asat-Ch24, asat-Ch35, fh-Ch24, gp63-mspC) [cytb5R-Ch22II (LinJ.22.0590), 4 hypothetical protein-coding genes  and 2 pseudogenes (LinJ.11.0280 and LinJ.36.0350). PCR reactions to amplify these targets were undertaken in a final volume of 25 μl, comprising: 2.5 μl 10× buffer, 0.05 mM MgCl2, 2.5 μl 0.8 mM dNTPs, 12.5 pmol of each primer, 1.25 U Taq polymerase and 25 ng genomic DNA. Amplification conditions were: 30 cycles at 95°C for 1 min, 54–62°C  and analysed in an ABI PRISM™ 3730 DNA sequencer (Applied Biosystems), according to the manufacturer’s instructions. Sequences were inspected and edited visually in Chromas Lite (Copyright © 2005 Technelysium Pty Ltd) and assembled using ClustalW [L. major MHOM/IL/80/Friedlin and L. infantum MCAN/ES/98/LLM-877 were obtained from www.genedb.org. Haplotypes for both coding and non-coding sequence datasets were scanned for mosaic breakpoints in RDP [Direct DNA sequences were generated from the PCR amplification products of eleven MLST targets. Targets included four housekeeping genes previously identified as suitable markers: 63-mspC) ,30, and ClustalW  in BioEdClustalW . HaplotyClustalW . MLST daPhylogenetic and population genetic analysis of various sequence and population sets were undertaken. Phylogenies were inferred using Maximum-Likelihood (ML) implemented in PhyML (4 substitution rate categories) . The besKST. KST compares the expected number of nucleotide differences between a pair of sequences within one population with a pair taken across all populations [Population genetic differentiation between strains from years 1997, 1998, 1999 and 2001 was calculated from sequence haplotypes in DNAsp  using KSulations . StatistL. donovani from Barbar El Fugara, Sudan, were found alongside strains from humans in four populations  across several different sampling years .For population level analyses of the microsatellite dataset we focused on only four well represented years from which samples were available: 1993, 1997, 1998 & 2001. Samples from the latter three time points were isolated uniquely from the village of Barbar El Fugara in the hyper-endemic Atbara River region of Eastern Sudan. Estimates for allelic richness over this time period show few radical differences between populations year-on-year . Both coding and non-coding regions were scanned for evidence of mosaic breakpoints that might be associated with homologous recombination, which would also potentially disrupt phylogenetic signal in subsequent trees, but no evidence for such events was uncovered. The ML topology derived from coding loci revealed substantial genetic diversity, but little bootstrap support, as one might expect between samples from the same species across a restricted area   Table .Table 4L. donovani in Sudan [Visceral leishmaniasis in Sudan is a major and on-going public health problem . Moleculin Sudan ,4, althoin Sudan ). Our hiin Sudan , howeverTrypanosoma cruzi discrete typing unit I, a related trypanosomatid, two samples taken 20 years apart from the same geographic focus can be identical at 48 microsatellite loci [The stability of genetic diversity in parasite populations in space is frequently used to infer patterns of regional and global parasite spread (e.g. ). Temporite loci . There aL. donovani strains. Like previous authors, we were able to detect reduced heterozygosity in the populations studied [L. donovani from two populations revealed strong evidence for predominant clonality, despite consistently high values for FIS. Furthermore, sequence data showed no evidence for the mosaics that one might expect to accompany recombination. It is important to state that, although our data do not confirm the presence of genetic exchange, we cannot rule out the occurrence of some recombination/inbreeding, as suggested by Rougeron et al. [Leishmania infantum in Turkey [As well as the defining patterns of parasite genetic diversification in the Atbara River region, a secondary goal was to evaluate evidence for genetic exchange among Sudanese  studied . However studied ,20,21. An et al. . We noten Turkey , while mn Turkey .Leishmania have now demonstrated the power that such approaches have to reveal the extent and mechanism of genetic exchange in natural populations [Leishmania species [L. donovani transmission were coincident with overlapping L. donovani genotypes, indicating that dogs may have an important role in sustaining human VL in Sudan, which deserves further investigation.Rapid, low cost, high-resolution genotyping strategies have an important role in elucidating the molecular epidemiology of visceral leishmaniasis, especially where the burden of the disease is felt the most. Population genomic studies of ulations . It has  species ,20,21; e species . Althoug


Lissotriton helveticus), we found that they formed a panmictic population, which evidences sexual compatibility between the two phenotypes. The high gene flow could be understood as an adaptation to unstable habitats in which phenotypic plasticity is favored over the fixation of developmental alternatives. This makes then possible the persistence of a polyphenism: only metamorphosis could be maintained in case of occasional drying whereas paedomorphosis could offer specific advantages in organisms remaining in water.Paedomorphosis and metamorphosis are two major developmental processes that characterize the evolution of complex life cycles in many lineages. Whereas these processes were fixed in some taxa, they remained facultative in others, with alternative phenotypes expressed in the same populations. From a genetic perspective, it is still unknown whether such phenotypes form a single population or whether they show some patterns of isolation in syntopy. This has deep implications for understanding the evolution of the phenotypes, i.e. towards their persistence or their fixation and speciation. Newts and salamanders are excellent models to test this hypothesis because they exhibit both developmental processes in their populations: the aquatic paedomorphs retain gills, whereas the metamorphs are able to colonize land. Using microsatellite data of coexisting paedomorphic and metamorphic palmate newts ( Polymorphisms, the presence of discrete intraspecific variation, are widespread in a large variety of organisms36Phenotypic variation can be achieved through heterochronic processes, i.e. changes in the rate or timing of developmental events relative to the same events in the ancestors101318Facultative paedomorphic populations exist in a large number of species of newts and salamanders2326Lissotriton helveticus) as a model species.In this context, the aim of this study was to test two alternative scenarios of the evolution of facultative paedomorphosis. These hypotheses are based on the existence or absence of genetic isolation between the two phenotypes. First, the behavioral specializations of each morph could result in their isolation. Second, the absence of sexual isolation could allow the species to subsist in a temporary environment. Our hypothesis is that it is the second scenario which is the most likely one because catastrophes lead to the extinction of paedomorphs in natural populationsE ranged from 0.27 to 0.76 and was lower than HO in all cases . A total of 53 different alleles were identified: 42 were present in the individuals of the two phenotypes , whereas 11 were found only in one of the two phenotypes. The number of private alleles was higher in the metamorphs  than in the paedomorphs  and there were significant differences between morphs . H LOSITAN .IS) were low and negative in all loci in the global population and also both in the metamorphs  and paedomorphs . Genetic differences between phenotypes (sub-populations) measured by FST for each locus separately were low, ranging from −0.011 to 0.004 . In a metapopulation formed by a network of ponds in which the number of individuals fluctuate over time, random changes in allele frequencies can lead to increased heterozygosity following a population decline2629Variation among individuals in reproductive success, population bottlenecks, and local extinction contribute to the development of fine-scale genetic subdivision by increasing the rate of stochastic fluctuation in allele frequencies at a given location42In conclusion, the evolution of polyphenisms such as facultative paedomorphosis depends on the existence or absence of behavioral or ecological isolation between alternative phenotypes. In our study, we have evidenced the coexistence of two phenotypes in the same habitat in an evolutionary scenario that predicts the persistence of facultative paedomorphosis as a polyphenism. The lack of inbreeding and the presence of a heterozygosity excess could be understood as a strategy which is adaptive under environmental change, with each phenotype displaying higher performance in contrasted habitats but constrained by the temporal heterogeneity of their habitat.n = 24 for each group). The males had also several secondary sexual traits, such as a tail filament. Immature newts never show these sexual traits2) from the tail fin. This part of the tail regenerates fast and such sampling has no detrimental effects on newtsWe captured 96 palmate newts  in the same pond . The adulthood of each individual was established by the presence of a developed cloaca, i.e. well swollen in both sexes and with groves in femaleset al.DNA was extracted from the tail tip using Qiagen DNeasy_Blood & Tissue Kits according to the protocol. Genetic analysis was based on the study of ten microsatellite loci  previously described by Drechsler A), and expected (HE) and observed (HO) heterozygosity and allelic richness (AR) were obtained for each morph  using FSTAT v. 2.9.3–Weinberg equilibrium by GENEPOP v.4.2ST-outlier detection methodAll analyses were based on a sample size of 96 individuals (48 paedomorphs and 48 metamorphs). Potential genotyping errors such as presence of null alleles were analysed using MICRO-CHECKER v. 2.2.3ST values were calculated to analyze the differentiation among phenotypes in allele frequencies, and FIS to estimate the fixation index using FSTAT v2.9.3ST significance by 10,000 permutations, and the genetic variability was evaluated within and among the two phenotypes with a molecular analysis of variance (AMOVA). STRUCTURE v2.2The overall and pairwise Fet al.STRUCTURE HARVESTER v0.6.94 was used to infer the K using the Evanno methodThe significance of differences in allelic richness and number of private alleles between two phenotypes was tested using the Wilcoxon signed-ranks test by STATISTICA 10. This test is appropriate because homologous loci were used in the two phenotypeseThe capture permit of newts was issued by DREAL Languedoc-Roussillon, following approval by the Conseil National de la Protection de la Nature (decree 2013274-0001). All manipulations were carried out in accordance with all relevant guidelines and regulations. They were approved by the animal ethical committee of the institution where was carried out the study, i.e. the University of Liège (authorization 1613). All newts were released in their capture location after sampling, following the recommendation of the capture permit.How to cite this article: Oromi, N. et al. High gene flow between alternative morphs and the evolutionary persistence of facultative paedomorphosis. Sci. Rep.6, 32046; doi: 10.1038/srep32046 (2016).


HS, HeS, π and h), inbreeding coefficient (FIS), allelic richness (AR) and differentiation among populations (FST) for both nuclear and chloroplast genomes. In addition, we used phylogenetic generalized least squares (pGLS) to account for phylogenetic independence on predictor variables and verify the robustness of the results from significant GLMMs. In general, GLMM revealed more significant relationships among LHTs and genetic patterns than pGLS. After accounting for phylogenetic independence , FST for nuclear microsatellites was significantly related to pollination mode, mating system and habitat. Plants specifically with outcrossing mating system had lower FST. Moreover, AR was significantly related to pollination mode and geographical range and HeS for nuclear dominant markers was significantly related to habitat. Our findings showed that different results might be retrieved when phylogenetic non-independence is taken into account and that LHTs and ecological attributes affect substantially the genetic pattern in Neotropical plants, hence may drive key evolutionary processes in plants.We studied genetic diversity and differentiation patterns in Neotropical plants to address effects of life history traits (LHT) and ecological attributes based on an exhaustive literature survey. We used generalized linear mixed models (GLMMs) to test the effects as fixed and random factors of growth form, pollination and dispersal modes, mating and breeding systems, geographical range and habitat on patterns of genetic diversity ( The search for patterns in evolutionary ecology has been extensively discussed in literature as a central problem in ecology  ,9,16–18,Besides the mixed structure of the data, traditional reviews have also ignored the phylogenetic relationship among the species but see ,17). Con. Con17])Actually, it is necessary to first test for phylogenetic signal in the studied traits and if they are statistically significant then apply phylogenetically based methods for comparisons and hypotheses testing ,25 , only the significant relationships from GLMMs could suffer phylogenetic effects. Then, the robustness of significant GLMM relationships (and not non-significant ones) should be tested in the light of phylogenetic methods as pGLS , inbreeding (FIS), allelic richness (AR) and differentiation among populations (FST), for both nuclear and chloroplast genomes in Neotropical plants. In addition, we used phylogenetic generalized least squares (pGLS) to account for phylogenetic relationships and verify the robustness of the results found by significant GLMMs.Because of their underrepresentation in most review studies and their importance for conservation, here, we focused on Neotropical plants addressing the effects of life-history traits, geographical range and habitat on patterns in genetic diversity and differentiation based on a literature survey. We used GLMMs to test the effects of growth form, pollination and dispersal mode, mating system , breeding system , geographical range and habitat on patterns of genetic diversity , using Web of Science platform (ISI hereafter), and Scopus Search (http://www.elsevier.com/online-tools/scopus). The survey was performed considering the availability of publications in both databases from 1945 (first register in ISI) to December 2013. We used the most frequent keywords in the area: "population genetics", "phylogeography", "mating system", "reproductive system", “breeding system”, and "genetic structure", combined with (AND) "Neotropical trees" and "Neotropical plants". Additional keywords such as “pollen dispersal” and “gene flow” retrieved no additional articles. We excluded phylogenetic studies, reviews and development of molecular markers such as microsatellite primer development or SNPs discovery. We included only Angiospermae due to the low number of Gymnospermae species in the Neotropics . We considered as Neotropics the region comprising the Neotropical Floristic Kingdom  x (1 + FIS) - 2[(1/FSTm) - 1]}/[(1/FSTm) - 1], was higher than 1 for all species except one, indicating higher contribution of pollen than seed to historical gene flow, which may explains our non significant results.We found no effect of dispersal mode on genetic differentiation at nuclear microsatellites, despite the non-significant phylogenetic signal in our data. Our findings agree with previous predictions that the general higher rates of pollen flow compared to seed flow could obscure the effects of seed dispersal mode on genetic differentiation at nuclear molecular markers ,53. Indeposed by , i.e.  and pollination mode for allelic richness (AR) for nuclear microsatellites. Ecological attributes also explained genetic differentiation for nuclear microsatellites. For chloroplast genome, studies published so far using CAPs, sequencing or microsatellites show no significant patterns associated to LHT or ecological attributes in genetic differentiation neither in genetic diversity (h and π). Genetic diversity for nuclear genome (HeS and He) could not be explained by any LHT or geographical range. However, habitat was significantly related to genetic diversity overall population (HeS) at nuclear dominant markers. We expected that trees have significant higher genetic diversity and allelic richness, and lower FST and FIS than the other growth forms. However, we found no significant effect of growth form in any genetic parameter analyzed. Nevertheless, plants with outcrossing mating system had lower FST.Our findings show that LHTs affect substantially the pattern of genetic diversity in Neotropical plants, hence may drive key evolutionary processes along with ecological features. Considering phylogenetic non-independence, pollination mode and mating system were the LHT with significant effects on genetic differentiation  the effect of phylogenetic non-independence. Spurious relationships may thus arise from conventional statistical analysis by inflating type I error when not accounted for such non-independence. We acknowledge the limitations of our results due to biased taxonomical sampling in the data set and the low number of studied species for some LHT. However, this is a first attempt to find patterns in genetic diversity and genetic differentiation for Neotropical plants using a large data set from Neotropical plants.S1 Fig(TIFF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 TableFST, genetic differentiation among populations; HeS, mean genetic diversity among populations. Note that both genetic parameters are calculated for dominant , isozymes and microsatellite makers. Significant values are denoted in bold and grey-shaded.(DOCX)Click here for additional data file.S6 TableHe, genetic diversity within population; FIS, inbreeding coefficient; AR, allelic richness. Note that both genetic parameters are calculated for isozymes and microsatellite makers. Significant values are denoted in bold and grey-shaded.(DOCX)Click here for additional data file.S7 Tableh, haplotype diversity π, nucleotide diversity. Significant values are denoted in bold.(DOCX)Click here for additional data file.S8 TableFST, genetic differentiation among populations; h, haplotype diversity; π, nucleotide diversity. Significant values are denoted in bold and grey-shaded.(DOCX)Click here for additional data file.S9 TableFST, genetic differentiation among populations; HeS, mean genetic diversity among populations, He, genetic diversity within population; FIS, inbreeding coefficient. SE, standard error.Significant values are denoted in bold. (DOCX)Click here for additional data file.S10 TableFST, genetic differentiation among populations; HeS, mean genetic diversity among populations, He, genetic diversity within population; FIS, inbreeding coefficient. SE, standard error.Significant values are denoted in bold. (DOCX)Click here for additional data file.S11 TableFST, genetic differentiation among populations; HeS, mean genetic diversity among populations, He, genetic diversity within population; FIS, inbreeding coefficient. SE, standard error.Significant values are denoted in bold. (DOCX)Click here for additional data file.


Tripterygion delaisi) from an open coastline in the Mediterranean Sea. Parentage analysis revealed a decrease in dispersal success in the focal area over 1 km distance and approximately 6.5% of the juveniles were identified as self-recruits. Sibship reconstruction analysis found that, in general, full siblings did not recruit together to the same location, and that the largest distance between recruitment locations was much higher (11.5 km) than found for parent–offspring pairs (1.2 km). Direct measurements of dispersal are essential to understanding connectivity patterns in different marine habitats, and show the degree of self-replenishment and sustainability of populations of marine organisms. We demonstrate that sibship reconstruction allows direct measurements of dispersal and family structure in marine species while being more easily applied in those species for which the collection of the parental population is difficult or unfeasible.Connectivity is crucial for the persistence and resilience of marine species, the establishment of networks of marine protected areas and the delineation of fishery management units. In the marine environment, understanding connectivity is still a major challenge, due to the technical difficulties of tracking larvae. Recently, parentage analysis has provided a means to address this question effectively. To be effective, this method requires limited adult movement and extensive sampling of parents, which is often not possible for marine species. An alternative approach that is less sensitive to constraints in parental movement and sampling could be the reconstruction of sibships. Here, we directly measure connectivity and larval dispersal in a temperate marine ecosystem through both analytical approaches. We use data from 178 single nucleotide polymorphism markers to perform parentage and sibship reconstruction of the black-faced blenny ( Connectivity counteracts population structuring, and is crucial for the persistence and resilience of many species. This has been emphasized in conservation policies, including the design of networks of marine protected areas ,2. HowevOne solution to overcome this problem is the use of parentage analysis to study dispersal in species with a relatively stationary adult phase . Such anThe successful application of parentage analysis to the study of larval dispersal requires sampling of a sufficiently large proportion of the parental population for the successful encounter of parent–offspring pairs . Owing tIn the marine environment, the collection of samples from a large fraction of the population can be extremely difficult and time-consuming. Performing sibship analysis has the advantage that only one generation needs to be sampled, whereas parentage analysis requires sampling of both the adult and offspring generations. When extensive adult collection is not feasible, for example in species with high adult motility, sibship reconstruction can be an informative and suitable alternative approach for the estimation of dispersal.Tripterygion delaisi (the black-faced blenny) [In this study, we compare the usefulness of sibship and parentage analyses in deciphering patterns of dispersal and connectivity in the marine environment using a small rocky shore fish,  blenny) , as a mo2.(a)T. delaisi) is distributed throughout the Mediterranean Sea and the northeastern Atlantic Ocean [a). These males protect a small territory, which is referred to as their nest, against predators and uncoloured sneaker males [The black-faced blenny . It is centred on a rocky shore of approximately 2 km in length, surrounded by sandy bottom habitat that extends for many kilometres in both directions, but especially towards the southwest (c), and could act as a dispersal barrier [The study area is on an open Mediterranean coastline of northeastern Spain, near the port of Blanes b. It is outhwest c, and co barrier .c) was performed and 796 territorial males were sampled from their nesting sites. Additionally, 150 camouflaged adults of unknown sex were sampled. Fish were caught with small nets, body length measured and a small tissue sample taken from the dorsal fin before release.A total of 946 adult fish were sampled on scuba with fish nets between April and July 2010. Exhaustive searching for complete coverage of all nests in the 2 km section of coastline . Four of these locations are found to the northeast of Blanes, where, after a large sandy bottom stretch, the rocky shore continues providing habitat for T. delaisi. Southwest of Blanes, juveniles were sampled at all the available sites with rocky habitat (b).A total of 627 juveniles were collected on scuba between the months of July and September 2010. Juveniles were sampled in the Blanes area, as well as at seven external locations, spanning a distance of 48.2 km, between Arenys and Tossa de Mar b. Four o habitat b.(b)T. delaisi, using 96.96 Dynamic SNP Genotyping Array on an EP1 Genotyping System (Fluidigm Corporation) [enepop v. 4.2 [et al. [All 1573 samples were genotyped at 192 SNP markers , developed for oration) . Deviatip v. 4.2  and loci [et al. .Two kinship analyses were performed: parentage and sibship. Parent–offspring matches were established using the software CERVUS , which aCOLONY  was alsoK = 1–3), the values over 10 runs were averaged with 100 000 iterations discarded as burn-in and 200 000 iterations retained. Genetic structuring between juveniles from Blanes and the other sampling locations was evaluated with FST calculated using Genepop v. 4.2 [fields [In order to identify any potential genetic structure within the samples, a Bayesian clustering analysis was performed with STRUCTURE v. 2.3.4 . The anap v. 4.2 . Geograp [fields .(c)The body length of all juveniles was measured, and the lapilli otoliths were extracted and mounted on microscope slides. The age of individuals was determined in order to establish the corresponding dates of hatch and settlement, as well as pelagic larval duration (PLD), following Raventós & Macpherson .Tripterygion larvae are always distributed along inshore waters (less than 2.5 km from shore) [The main documented superficial currents in this region of the Mediterranean Sea do not reach the coastal area, and therefore do not affect near shore species . Howeverm shore) , we compm shore) . The win3.A total of 25 parent–offspring pairs were identified, all in the Blanes area . The samT. delaisi adults do not disperse and males territorially guard one nest, even half siblings will come from either the same nest  or close nests . In either case, these 21 identified pairs are clearly siblings and can be assumed to have dispersed from approximately the same location. The geographical distances between siblings were larger than those between parent–offspring pairs. The maximum distance between siblings was 11.5 km, with 24% of sibling pairs separated by more than 10 km and 38% dispersing less than 1 km from each other . As ch other . Even ifT. delaisi from our study area, as the STRUCTURE analysis identified K = 1 to be the most probable number of genetic clusters  when combining all samples. The same result was encountered when adults and juveniles were analysed separately (data not shown). Furthermore, the FST estimate between juveniles collected in Blanes and those from the other sampling locations was low (FST = 0.0005) and not significantly different from zero.We found no population structure within ρ = 0.265, p = 0.22; night component versus distance: Spearman's ρ = −0.177, p = 0.42). The average day-time wind component was −1.675 ± 0.175 and the average wind direction was 170 ± 3.9, which means the wind blows away from the coast during the day. The average wind component at night was 0.411 ± 0.128 and the average wind direction was 260 ± 9.3, which means the wind blows in a southwesterly direction at night. When comparing the wind component with the dispersal direction of self-recruits, we found no correlation with the day-time wind component, but a nearly significant correlation at night . There were 13 self-recruits that moved in a southwesterly direction from their natal nest and 11 self-recruits that dispersed towards the northeast. One recruit was found on the same rock as its parental nest. The dispersal direction was highly correlated with the location of the parental nest within the sampling area . We find northeastern dispersal to be of shorter distance . Furthermore, no juvenile collected northeast of Blanes was an offspring of any of the fathers in the Blanes area, which suggests that few larvae disperse great distances to the northeast.Dispersal distance did not correlate with the wind variable, which included both wind direction and strength, and was calculated for both day- and night-time  was similar. Saenz-Agudelo et al. [Here, we demonstrate how two genetic techniques for determining kin relationships, parentage and sibship reconstruction analyses, can be used to elucidate fine-scale patterns of larval dispersal for a rocky reef fish in an open-coast temperate ecosystem. Self-recruitment, which is the percentage of larvae that settle back to the natal location, in marine fishes has long been debated , but empo et al.  argue tho et al. . TemperaT. delaisi, as we detected a low proportion of self-recruits, with an average PLD of 18 days, and a similar proportion was estimated for a coral reef species with a PLD of just several days [T. delaisi is a minimum estimate, because sneaker males may also contribute to the progeny. We sampled all the territorial males observed in the study area and tried to capture sneaker males, but were largely unsuccessful, as they are camouflaged and quite difficult to see. However, sneaker males are rarely observed to participate in mating, suggesting generally low reproductive output for this behavioural strategy [Pelagic larval duration has been suggested to have a large influence on the rate of self-recruitment ,29. Howeral days . Neverthstrategy  and a miAmphyprion polymnus were found not to be influenced by physical processes, such as currents [T. delaisi, we found no significant correlation between the dispersal of self-recruits and the component wind variable, probably due to the lack of a pattern of directional dispersal. None of the juveniles collected in the sampling areas northeast of Blanes were offspring of the Blanes parents, which could be due to the large population size. For those recruits that dispersed southwestward, the coastline is formed by sandy beaches with no available rocky reef habitat for approximately 100 km, suggesting little successful dispersal occurring beyond this area. It was previously reported that the population of Blanes is genetically distinct from that of the next analysed locality approximately 250 km to the southwest, with a low number of juveniles assigned to distant locations [Large-scale investigations that integrate population genetics and oceanographic modelling have shown that connectivity can be influenced, or even dominated, by physical processes –44. Howecurrents . For T. ocations . Howeverocations , suggestT. delaisi revealed limited successful dispersal and only short-distance dispersal events in the focal study area. Identified dispersal events declined precipitously more than 1 km from the natal site, as previously found in reef fish species [T. delaisi population in the study area was found to be one genetic unit connected by high gene flow, consistent with previous studies [Parentage analysis in  species ,38. The  studies ,45.T. delaisi sibling pairs was much larger than the maximum distance between parent–offspring pairs (The high connectivity found in population structure analyses was reflected in the sibling pairs identified. Sibship analysis has previously been used to successfully elucidate family structure and mating strategies in several other fish species –48. Hereng pairs . NonetheT. delaisi sibling pair encountered at the same recruitment location. Otolith analysis found that both siblings hatched within 2 days of each other and spent 19 days in the pelagic larval phase, indicating that they probably left the nest at the same time and recruited together. Even though many groups of juveniles were collected simultaneously from the same locations, only this one pair of siblings was identified as having recruited together. This indicates that the majority of full siblings do not recruit to the same location, suggesting a high degree of larval mixing and recruitment heterogeneity [Combining kin relationship estimation with otolith analysis, it is also possible to retrace whether siblings recruited together. Indeed, a recent study on a coral reef fish provided evidence that siblings can travel together throughout the entire pelagic larval phase . Nonetheogeneity .T. delaisi, the parent–offspring comparisons indicated that larvae tend to move short distances along the coast. By contrast, the sibling pairs identified showed that recruitment can be at locations quite distant from their natal site, and from each other, reflecting the high degree of connectivity inferred from population structure analyses.In this study, by selecting to work with a species for which parentage analysis is possible due to the sedentary, territorial behaviour of adults, we can compare the results of the two kinship methods. With parentage analysis, the direction of dispersal events is known, which cannot be accomplished with sibship reconstruction as the location of origin of the siblings is generally not known. Nonetheless, in our study there was no clear pattern of dispersal direction, even though parentage analysis was performed. With sibship reconstruction, which does not require sedentary life history or sampling of parents, the dispersal range of the species is estimated, and inference regarding family structure and dispersal behaviour can be derived. For The information provided by these two methods can be complementary, and provides a more complete picture of dispersal dynamics and population connectivity than either one alone. The direct measurement of dispersal distance and direction by applying parentage analysis is dramatically increasing our knowledge of larval dispersal in marine organisms. However, for many species, including those of conservation concern, sampling a large proportion of the parental population is difficult or unfeasible, or the life history may make it difficult to infer the origin of dispersal events. In such cases, sibship reconstruction can be more easily applied, as only one generation needs be sampled, to elucidate dispersal patterns and structure in marine species.