Haplophilus subterraneus show a high proportion of individuals with morphological anomalies, suggesting high levels of developmental instability. The broad geographic distribution of this phenomenon seems to exclude local environmental causes, but the source of instability is still to be identified. The goal of the present study was to collect quantitative data on the occurrence of phenodeviants in different populations, along with data on the patterns of genetic variation within and between populations, in order to investigate possible association between developmental instability and genetic features. In a sample of 11 populations of H. subterraneus, distributed in western and central Europe, we looked for phenodeviants, in particular with respect to trunk morphology, and studied genetic variation through the genotyping of microsatellite loci. Overall, no support was found to the idea that developmental instability in H. subterraneus is related to a specific patterns of genetic variation, including inbreeding estimates. We identified a major genetic partition that subdivides French populations from the others, and a low divergence among northwestern areas, which are possibly related to the post-glacial recolonization from southern refugia and/or to recent anthropogenic soil displacements. A weak correlation between individual number of leg bearing segments and the occurrence of trunk anomalies seems to support a trade-off between these two developmental traits. These results, complemented by preliminary data on developmental stability in two related species, suggest that the phenomenon has not a simple taxonomic distribution, while it exhibits an apparent localization in central and eastern Europe.European populations of the geophilomorph centipede  Developmental stability is the property of an organism to buffer random perturbations of the developmental process. The higher the level of developmental stability, the closer the features of the expressed phenotype to that of the target phenotype, i.e. the phenotype specified by the genetic makeup of the organism and the environmental conditions during its development [developmental instability indicates a deficiency in resisting developmental disturbances [elopment –2. The aurbances .fluctuating bilateral asymmetries [fluctuating translational asymmetries [frequency of segmental phenodeviants [The exact target phenotype is generally unknown, but body symmetries can be exploited to quantify developmental instability by measuring deviations from the expected symmetry. Random non heritable deviations from bilateral symmetry, or mmetries , have bemmetries ,6. Howevmmetries . In segmmmetries , which pmmetries ,9, or stdeviants ).Developmental instability can have different causes, as for instance the individual genetic makeup at specific loci, the level of stochasticity of certain developmental processes and a variety of stressful environmental factors , and difHaplophilus subterraneus   revealed a high proportion of individuals with naturally occurring morphological anomalies of several kinds [The study of a Polish population of the geophilomorph centipede al kinds . The speal kinds –15, stroA preliminary survey on the geographical distribution of the phenomenon  showed tH. subterraneus at a wider geographical scale, we conducted a targeted collection campaign through central and western Europe. We collected quantitative data on the occurrence of phenodeviants in each population, and on the pattern of genetic variation within and between populations. A first inspection of the morphological data of this campaign, supplemented with data from other collections in central and western Europe, south Britain and Jutland confirmed the occurrence of high level of developmental instability for all European populations [In order to further investigate the occurrence of morphological anomalies in ulations .H. subterraneus individuals and population.Here we analyse the geographic distribution of anomalies as indicators of developmental instability, examine the patterns of genetic variation as indicators of demographic events such as bottlenecks (and inbreeding), and study the possible association between morphological and genetic features of Haplophilus subterraneus is an active predator inhabiting litter and soil, where it feeds on other invertebrates. H. subterraneus is distributed in the central and western Europe, from the Pyrenees to southern British Isles and western Germany. It usually occurs in natural woodland sites, but it is also commonly found in synanthropic sites like urban parks and cemeteries [As most geophilomorph centipede species, meteries . Isolatemeteries . For furmeteries .Haplophilus subterraneus, distributed in western and central Europe, from southern France to central Poland, that is, across the whole natural range of the species and beyond its east border  were fixed in 100% ethanol and within minutes cut transversally in two pieces: the head plus a few anterior trunk segments (about eight) were stored in 100% ethanol, while the rest of trunk was placed in 75% ethanol. The reason for this procedure is that the optimal solution for preserving material aimed at DNA extraction and purification, i.e. absolute ethanol, is unsuitable for preservation of material destined to an accurate morphological inspection, as this produces extended coiling and contractions in the body, followed by its stiffening. On the other hand, preliminary tests on DNA purifications from samples preserved in 75% ethanol showed a high level of DNA degradation, that was successfully prevented by the use of pure ethanol. For the Poznań population, genotyped and morphologically inspected individuals form to two distinct (non-overlapping) samples, and animals were stored in ethanol entire (not cut). Overall, 1018 specimens were used for morphometric analyses, including the search for morphological anomalies, while genetic analysis was carried out on a partial subsample of 200 specimens .For further details on sampling sites and animal collection see . All speThe trunk of geophilomorph centipedes comprises one anterior segment bearing a pair of poisonous maxillipedes (forcipular segment), a series of variable length of leg-bearing segments, and a terminal apodous ano-genital region of uncertain segmental composition, but reputed segmentally invariant across species, sexes and individuals ,21. ThusAll specimens were examined by ML with a stereoscopic light microscope up to 80x magnification. The aberrant morphologies recorded in the sample have been classified on the basis of Leśniewska et al.’s  taxonomyTA, 0: absence, 1: presence); ii) a ranked categorical variable that gives a score (0–3) based on the ‘seriousness’ of trunk anomalies ; iii) a binary categorical variable that records the presence of appendage anomalies . Examples of morphological anomalies are reported in Here we have considered separately trunk anomalies, affecting structures of the body to the exclusion of appendages, and appendages anomalies. In order to compare the frequency of morphological anomalies across populations, three categorical variables referable to single specimens were devised: i) a binary categorical variable that records the presence of trunk anomalies  and the population of origin, sex, and number of segments  were analysed using a generalized linear model  in two different data sets. The first data set included all available individuals , where more than half are from the Poznań population. The second data set (n≈480) included only a randomly selected subset of 60 (out of 598) individuals from Poznań, in order to have approximately similar sample sizes from each locality. Considering that the different sampling area could be also considered as a random extraction of European populations, we also tested the binary response variables TA and AA using the population of origin as a random effect in a generalized linear mixed model .TA and AA: binary response variable; TAS: categorical variable) and genetic indicators of inbreeding  was analysed by simple parametric (Pearson) and non parametric (Spearman) correlation analyses. Only ten points were available for these analyses, as the population of Jarret, with only three genotyped individuals, was not included.The association between population variables related to developmental anomalies  and genetic indices , as implemented in the software Genetix 4.05 , and theAll the basic statistics elaborated from genetic and morphological data are reported in There are significant differences between populations for LBS , with the populations of Jarret and Diksmuide presenting significantly extreme values .TA and TAS means  are strongly correlated .The frequency of trunk phenodeviants varies between 7% and 25%, while the frequency of appendage phenodeviants varies between 11% and 37%. As expected, being based on the same kind of anomalies, is is significantly larger than 0 only in the city park of Poznań.Most of the populations show a relatively high level of genetic variation, with expected heterozygosity larger than 50%. Lower values are observed in France  and the Netherlands (0.33). These three populations show also slightly lower number of alleles. The inbreeding coefficient Fst = 0.29 . When TAS is analysed, only the effects of the population of origin and its interaction with the number of segment remain significant . No significant effect of these predictor variables on AA was recorded. Analysing the reduced and more balanced data set, the interaction between population and number of segments was significant both in the analysis of TA and TAS , and the effect of the population of origin was significant only for TAS (P = 0.009). Again, AA did not show any significant response. Considering the population of origin as a random effect in a linear generalized mixed model, the population of origin and the interaction between population of origin and number of segment had significant effects on TA both in the complete data set  and in the reduced data set . No significant effect on AA was recorded.Significant association between inbreeding and the presence/absence of anomalies were not identified, independently on the inbreeding estimator used and the kind and severity of the morphological anomaly. On the contrary, the population of origin, the number of segments, and their interaction had significant effects on e, k and Fis) produced non-significant results.Both parametric and non-parametric correlations between population frequencies of anomalies and all measures of genetic variation  or outbreeding depression  are the cause of the morphological defects observed in this centipede.Overall, our investigation on the geographic distribution of the occurrence of anomalies, complemented by the check for correlations between the incidence of anomalies and genetic variation patterns, provide no support to the idea that developmental instability recorded in the European populations of the centipede TA and TAS), the means of the variable of appendage anomalies (AA) do not differ significantly among populations. This adds to other arguments on the questionable value of appendage anomalies as indicators of developmental stability. In fact, while trunk anomalies are likely congenital, the scarce information on the regenerative potential and the morphology of healing in geophilomorph appendages [Unlike the variables of trunk anomalies , which H. subterrraneus seems to reflect demographic changes due to the glaciation events. In fact, the much higher level of genetic structure observed in France compared to the northeastern European populations, and the major genetic partition of the samples in southern and northern populations, can be related to the fact that southern samples come from the more stable distribution range of this species, continuously occupied also during the glaciations due to favourable climatic conditions, whereas northern populations reflects a more recent history of post-glacial colonization accompanied by a genetic divergence from the refuge(s).As observed in many other species –38, the An alternative, or complementary, explanation for the lower structure in northern areas is that human displacement of garden soils in recent years (<200 years ago) prevented population differentiation. This hypothesis, compatible with the synanthropic habits of the species, could also explain why, on the average, northern populations have similar or slightly higher heterozygosities compared to the French groups, which is in general not expected in recolonized areas compared to refugia . PassiveThe repetition of serially homologous structures along the main body axis is a common form of modularity, found in the body architecture of many multicellular eukaryote taxa. Several studies have explored how natural selection may have promoted the evolution of a modular body organization, and the potential of repetitive body units in taxa diversification . HoweverH. subterraneus. This correlation concurs with the observation that most of the teratological cases so far recorded in the whole Geophilomorpha are found in the taxon Himantariidae [H. subterraneus belongs. These observations might suggest the idea of a trade-off between the number of body modules and developmental stability, as if the length of a segmental series, in addition or in combination with other factors, could affect the developmental precision of its phenotypic expression. This may seem at odds with the geophilomorph relatively homonomous  trunk segmental pattern, as a relatively simple morphological design can be supposed to be associated to equally simple developmental processes of segmental patterning and differentiation. However, measures of morphological complexity of the trunk segmental structures [We found a significant, although not strong, positive correlation between individual number of leg bearing segments and the occurrence of trunk anomalies in tariidae , charactructures , mapped ructures , show thHowever specifically aimed studies, extended to other segmented (or modular) taxa, are needed to assesses effectiveness and/or generality of this possible trade-off.H. subterraneus sufficiently sampled and reasonably well inspected, which show high frequencies of abnormal morphologies, a comparably high frequency of abnormalities characterizes also a population of the closely related species H. souletinus, from the Pyrenees [Stigmatogaster gracilis, from Italy, revealed only appendage anomalies (in 29.6% of the specimens), but no one trunk anomaly. Because of the doubtful value of appendage anomalies as indicators of developmental stability (see above), there is therefore no compelling evidence of high levels of developmental instability in this species. This result is noteworthy, as the most recent geophilomorph phylogeny [S. gracilis as the sister species of H. subterraneus, with H. souletinus branching out basally with respect the other two species. Thus, the phenomenon seems to have a taxonomic distribution that cannot be circumscribed to a single clade, while, irrespective of the phylogeny, although not caused by strictly local factors, it presents an apparent localization in central and eastern Europe. Further studies will be necessary to understand the causes of this developmental instability and its taxonomic and geographic patterns.Finally, we note that the taxonomic boundaries of this phenomenon cannot easily be defined. Similarly to all populations of Pyrenees . Howeverhylogeny  resolvesS1 Dataset(XLS)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


Epinephelus merra (Serranidae), Halichoeres trimaculatus (Labridae), and Holothuria atra (Holothuriidae). The two fish species, E. merra and Ha. trimaculatus, exhibited low genetic structuring and high amounts of gene flow, whereas the sea cucumber Ho. atra displayed high genetic partitioning and predominantly westward gene flow. The idiosyncratic patterns observed among these species indicate that patterns of connectivity in Fiji are likely determined by a combination of oceanographic and ecological characteristics. Our data indicate that in the cases of species with high connectivity, other factors such as representation or political availability may dictate where reserves are placed. In low connectivity species, ensuring upstream and downstream connections is critical.Coral reef fish serve as food sources to coastal communities worldwide, yet are vulnerable to mounting anthropogenic pressures like overfishing and climate change. Marine reserve networks have become important tools for mitigating these pressures, and one of the most critical factors in determining their spatial design is the degree of connectivity among different populations of species prioritized for protection. To help inform the spatial design of an expanded reserve network in Fiji, we used rapidly evolving mitochondrial genes to investigate connectivity patterns of three coral reef species targeted by fisheries in Fiji:  Coral reefs are some of the planet’s most complex and diverse ecosystems, providing nearly US$ 30 billion in goods and services to global economies annually, through tourism, fisheries, and coastal protection23One tool commonly used to mitigate global reef degradation is the creation of marine protected areas (MPAs) – spatially explicit areas of ocean where human activities are regulated or prohibited. One subset of MPAs are no-take marine reserves, wherein direct harvesting of marine resources is prohibited both spatially and temporally. When effectively implemented, no-take marine reserves are important tools for conservation and fisheries management. They provide a refuge for exploited species, allow for fish and invertebrate stocks to recover, reproduce, and reseed adjacent unprotected areas through larval export and adult movement, while they protecting existing habitat from further degradation567Networks of reserves confer additional benefits, protecting a greater diversity of habitats and promoting the stability of meta-populations9111113Species’ behaviour, locomotion, life history, and habitat all play into how organisms interact with their physical environment, and thus influence connectivity. The larvae of many reef fishes possess an array of sensory modalities and the physiological capacity for directional swimming that allows for non-random settlement15161718202122In 2005, the Fiji government set the goal of protecting 30% of its marine waters by 2020Epinephelus merra (Serranidae), the three-spot wrasse Halichoeres trimaculatus (Labridae), and the black sea cucumber Holothuria atra (Holothuriidae).Previous work on connectivity among reefs in Fiji revealed asymmetrical gene flow along an east-west gradient for three of five coral reef fish species studied, potentially indicating that the Bligh Waters – a fast-moving current that bisects the main islands – could be facilitating larval transport for multiple taxa within Fiji29E. merra, 376 bp of the same locus from 91 individuals of Ha. trimaculatus, and 376 bp of the COI gene from 40 individuals of Ho. atra , while Ho. atra COI sequences were considerably less genetically diverse , within-group (FSC), and within-population (FST) genetic differentiation  fit into one of three haplotypes .MIGRATE analyses resolved high levels of migration but with complex patterns of directionality among populations. For received . These ps a sink . AdditioHo. atra sequences showed that three of the five highest migration values were from the eastern island of Vanuabalavu out to more western sites. The highest estimated median number of migrants per generation was from Vanuabalavu to Taveuni (672.3), while the lowest estimated number of migrants per generation was from Malevu to Vanuabalavu , and positive for Ho. atra (0.71194). All values were non-significant .Tajima’s D values were negative for both E. merra R2 = 0.001, Ha. trimaculatus R2 = 0.005, Ho. atra R2 = 0.010), indicating that none of these species’ patterns of genetic differentiation can be explained by geographic distance between populations.Finally, the Mantel test revealed negative or flat levels of correlation between pairwise measures genetic differentiation and geographic distance in all three species , suggesting that there are many other factors besides this species’ larval potential to drift great distances that determine larval dispersal and consequent connectivity.In contrast, the sea cucumber Ho. atra haplotype, accounting for almost 58% of samples, is found in all four sampled locations, suggesting an overall trend of connectivity across the region despite observed differences in the degree of connectivity in each pair of sites. The boom-bust demographic cycle of Ho. atraHo. atra’s positive and non-significant Tajima’s D, which indicates that the sea cucumber populations studied may have undergone recent demographic contractionThe most frequent E. merra and Ha. trimaculatus were complex and occasionally contradictory – for instance, Vanua Levu exported more migrants than it received from other locations in E. merra, whereas in Ha. trimaculatus the opposite was true. Both fish species also exhibited higher numbers of migrants coming from the western locality of Naviti and settling in the eastern island of Vanuabalavu than vice versa, indicating that western populations could be seeding eastern localities in spite of the prevailing westward-moving oceanic currents. Ho. atra shows a different pattern, in which the larvae seem to follow the oceanic currents resulting in the eastern island of Vanuabalavu exporting higher numbers of larval migrants to the western localities than vice versa. This concurs with previous studies showing unidirectional patterns of east-to-west gene flow in several taxaHo. atra’s east to west pattern of gene flow is also supported by the haplotype diversities of each location, as the western population of Naviti, in the Yasawa islands, has the highest intra-population genetic diversity while Vanuabalavu, which lies farthest to the east in the Lau group, has the lowest. This suggests that larvae spawned in Nagigi, Taveuni, Vanuabalavu and other unsampled populations may potentially recruit more often to reefs in the western Yasawa Islands, promoting high genetic diversity. These contrasting patterns of gene flow observed between the fishes and the sea cucumber could be due to demographic stochasticity, differences in reproductive mode or timing, or seasonal variation in the flow of the Bligh Waters, which is common in other intra-pelagic currentsResults of the MIGRATE analyses were complex and idiosyncratic among all three species studied . Gene flet al.The dearth of literature on the inter- and intra-annual physical oceanography of the Fijian archipelago makes it difficult to assess the role of variation in currents on the data presented here. Genetic connectivity studies conducted in regions with better-studied oceanography have shown both inter-annual and intra-annual oceanographic features to be responsible for population connectivity or structure. Galarza E. merra and Ha. trimaculatus were strongly negative and while non-significant, they may indicate that both species may have undergone rapid population expansion in recent evolutionary history. Because the program MIGRATE assumes stable population dynamics, migration estimates can be strongly influenced by population expansion or contraction. Thus, the results of gene flow in the fish species should be taken with caution, particularly in the case of directionality. While the Tajima’s D value for Ho. atra was slightly positive, potentially indicating that this species has undergone a recent population bottleneck, the p-value for this estimate was much larger than those of the fishes and therefore likely does not have a significant effect on MIGRATE analysesThe Tajima’s D values for both E. merra at two locations (n = 10 and 12), and for Ho. atra at three locations . High amounts of nucleotide and haplotype diversity, like those found in E. merra, can sometimes be a relic of low sample sizes - however, high genetic diversity is a very common finding in marine fishes, and studies with very large sample sizes often observe high haplotype diversities as well33Ha. trimaculatus displayed almost identical measures of haplotype and nucleotide diversity, while having larger sample sizes than E. merra at nearly every location. Our power analyses show that with the sample sizes used for E. merra and Ha. trimaculatus, the diverse mtDNA control region confers low statistical power in detecting very weak levels of differentiation (ΦST of 0.01 or below), thus limiting our study’s ability to rule out a type II error within this range. However, the magnitude of differentiation below 0.01 is so low that even if our study did miss populations exhibiting partitioning at these levels, the conclusions drawn would be effectively the same – that both fish species exhibit high amounts of connectivity between their populationsAnother caveat of this study is its low sample sizes for Ho. atra, on the other hand, this study observed very low haplotype diversities across all locations, even in Vanuabalavu where sample sizes were high (n = 18). While low sample sizes understandably lead to low observed genetic diversity, it is also likely that this species’ reproductive ecology  has contributed to higher local densities of genetically identical individuals than in exclusively sexually reproducing species. Thus, while results for this species should certainly be taken with caution, it is possible that low sample sizes in this study may be capturing an adequate amount of the gene pool at each location.In ST values than nuclear DNA4748Critiques for mtDNA-based phylogeographic studies often concern the variable rate of mutation observed between these markers and others – mtDNA often reach higher FHo. atraAdditionally, use of markers that have been used for previous phylogeography studies allows for easier comparison of results across taxa and locationsE. merra and Ha. trimaculatus populations. Instead, each region sampled should be considered equally important for protection, as each node in these meta-populations contributes ecologically and economically significant amounts of migrants to the others. This finding provides managers with greater flexibility in conservation prioritization, as they can focus on areas that have strong local support for conservation, or areas like the Vatu-i-Ra seascape (between Viti Levu and Vanua Levu), which have been identified as having exceptional habitat qualityThe high connectivity observed among populations of the fish species suggests that prioritizing one region over another for the placement of marine reserves in Fiji is not necessary for the conservation and persistence of Ho. atra, some populations are well connected to each other and others are isolated, which necessitates a more nuanced spatial management scenario. For instance, since the Vanua Levu and Taveuni populations are genetically differentiated, it is recommended that they be treated as separate management areas to preserve their genetic diversity, despite the fact that both lie within Fiji’s Northern Division. Additionally, since the Vanuabalavu population in the Lau archipelago to the east likely provides larval recruits and thereby genetic diversity to western reefs, this region should be a conservation priority for the management of the species. Upstream areas, such as Vanuabalavu for Ho. atra and Naviti for the fish, should be considered priorities for monitoring, as the health of downstream populations is largely reliant on the well-being of upstream areas.Conversely, with E. merra and Ha. trimaculatus that exhibit high amounts of connectivity within the country, but for those like Ho. atra with low connectivity and high genetic partitioning among populations, management at the provincial or local scale may be required to ensure the persistence of isolated populations.Ultimately, our data suggest management at the larger Division or Provincial scale may be appropriate for species like Epinephelus merra and Halichoeres trimaculatus are both widespread Indo-Pacific coral reef fishes, and are commonly targeted by fishermen in Fiji56E. merra is typically caught by spear or by hook and line, and is most commonly used for subsistence and occasionally sold in fish markets. Ha. trimaculatus is caught primarily via net, and is more often used for subsistence or fed to livestock, depending on their size at harvest. Both species are found in protected seaward reefs and lagoonal areas, and both have small home ranges, rarely moving more than 1 km away from their adult home reefs58Holothuria atra, known as the black sea cucumber or the lollyfish, is a major commercial fisheries target throughout the Indo-Pacific60Ho. atra can reproduce both sexually via broadcast spawning, and asexually by fissioning into anterior and posterior parts and then regenerating a full body from each part3862Ho. atra’s larval duration lasts approximately 18–25 days53The study protocol was approved by the Columbia University Animal Care Committee (protocol no. AC-AAAF6300) and followed the laws of the Republic of Fiji and with permission of the traditional marine resource owners.We sampled at six locations across Fiji . Study sHo. atra were collected by hand  gene using primers designed byE. merraKT353114-KT353195, Ha. trimaculatusKT329098-KT329188, Ho. atraKT378456-KT378495.Genomic DNA was extracted from gill clips and dermal samples using a DNeasy Tissue Kit . For fish samples, we amplified the mitochondrial control region by PCR using the primers CR-A and CR-EΦST measures  were calculated for each species in ARLEQUIN 3.5To investigate the amount of genetic differentiation occurring between sampled populations, pairwise E. merra, each of the 6 sampling localities was initially treated as a separate entity, and when no significant differences were detected between the villages of Donicali and Naracivo , the data were pooled into island-level assemblages and reanalysed. Extremely low sample sizes for Ha. trimaculatus and Ho. atra in Donicali  would have made individual locality results unreliable, so these two species were grouped at the island-level at the outset. Further, if no differentiation was detected between the island-level assemblages for each species, analyses were repeated with populations grouped by Division to investigate whether Fiji’s large-scale political boundaries are appropriate for the management of biological populations.To test whether regional-level genetic structuring was present, analyses of molecular variance (AMOVAs) were also performed in ARLEQUIN. For We calculated magnitude and direction of gene flow at the island-level using MIGRATE-nTo test whether any observed genetic differences could be due to geographic distance versus phylogeographic barriers, a Mantel test was also performed for each species using Isolation By Distance 3.23How to cite this article: Eastwood, E. K. et al. Population Connectivity Measures of Fishery-Targeted Coral Reef Species to Inform Marine Reserve Network Design in Fiji. Sci. Rep. 6, 19318; doi: 10.1038/srep19318 (2016).


Pseudococcus viburni. An extensive survey was completed using DNA barcoding methods to identify Chilean mealybugs to the species level. Moreover, a fine-scale study of Ps. viburni genetic diversity and population structure was carried out, genotyping 529 Ps. viburni individuals with 21 microsatellite markers. Samples from 16 localities were analyzed using Bayesian and spatially-explicit methods and the genetic dataset was confronted to host-plant, management and environmental data. Chilean crops were found to be infested by Ps. viburni, Pseudococcus meridionalis, Pseudococcus longispinus and Planococcus citri, with Ps. viburni and Ps. meridionalis showing contrasting distribution and host-plant preference patterns. Ps. viburni samples presented low genetic diversity levels but high genetic differentiation. While no significant genetic variance could be assigned to host-plant or management strategy, climate and geography were found to correlate significantly with genetic differentiation levels. The genetic characterization of Ps. viburni within Chile will contribute to future studies tracing back the origin and improving the management of this worldwide invader.The present study aimed to characterize the distribution of mealybug species along Chilean agro-ecosystems and to determine the relative impact of host plant, management strategy, geography and micro-environment on shaping the distribution and genetic structure of the obscure mealybug  Pseudococcus viburni (Signoret) has expanded across the globe and is now present in over 60 countries46Scale insects are worldwide-distributed agricultural pests that cause major economic losses of billions of dollars every year135The spatial distribution of agro-ecosystems is a key factor determining the species composition and population genetic structure of agricultural pests11et al.Pseudococcus viburni (Signoret) to be the most common mealybug species in Chilean vineyards, followed by Pseudococcus meridionalis Prado and Pseudococcus cribata González. In contrast with previous studies though, no Pseudococcus longispinus (Targioni Tozzetti) or Planococcus ficus (Signoret) were found, which could result from the limited area being covered (two regions) and the fact that only vineyards were sampledNational production of grapes and fruit orchards in Chile has significantly increased during the last decade1718Ps. viburni genetic diversity and population connectivity was carried out using microsatellite markers. A comprehensive analysis of Ps. viburni samples allowed us to determine the relative impact of host plant, management strategy, geography and micro-environment on shaping the distribution and genetic structure of the pest.The present study aimed at characterizing the diversity and distribution of mealybug species infesting Chilean agro-ecosystems, including both vineyards and deciduous fruit crops. In order to achieve this goal, we completed an extensive survey between Atacama and the Northern part of Biobío (covering most cultivated areas in Chile) and using DNA barcoding methods to identify mealybug samples to the species level. Moreover, a fine-scale study of et al.A total of 38 sampling sites were surveyed along Chile in 2012 and 2013 . Sample Ps. viburni (see results section) and kept for further analysis using the 21 polymorphic microsatellite markers developed by Correa et al.Ps. viburni populations. Individual PCRs were performed in a total volume of 10 μl, with cycling conditions as in Correa et al.TM size standard. Allelic profiles were obtained for each individual using the GenemarkerTM v1.75 software (SoftGenetics LLC).Out of the initial 38 sampling sites, a total of 16 samples were identified as O) and expected (HE) heterozygosity were computed using the software GeneClass v2http://www.ccmar.ualg.pt/maree/software.php?soft=sarich). Deviations from Hardy-Weinberg Equilibrium (HWE) were tested for each locus and sampling locality using the software Genepop v4.2Ps. viburni were analyzed using the software BOTTLENECK v.1.2.02For each sampling locality, mean number of alleles, observed . Sampling localities were grouped with the neighbor-joining algorithm and the pairwise genetic chord distancehttp://bioinformatics.org/populations/).Pairwise Genetic clusters were then inferred by minimizing deviation from HWE and linkage disequilibrium (LD) with the Bayesian clustering approach implemented in STRUCTURE35Isolation by distance (IBD) was tested by performing a linear regression of the genetic distances (FST or DST) and the geographical distances (Km) and obtaining the Pearson’s correlation coefficient. Matrices of genetic distance and geographical distance were subjected to a Mantel test with 10,000 permutations in Arlequin v.3.5Ps. viburni, temperature and precipitation variables were recovered to 1-km spatial resolution from WorldClim version 1.4 (http://www.worldclim.org)Several analyses of molecular variance (AMOVA) were performed in order to evaluate the importance of non-genetic categorical factors on the distribution of genetic diversity. In particular, sample sites were grouped according to (i) administrative region , (ii) type of agricultural management , and (iii) host plant . The significance for the AMOVAs was obtained through non-parametric permutation procedures  as implemented in Arlequin v3.5Planococcus citri, Pseudococcus longispinus, Pseudococcus meridionalis and Pseudococcus viburni). Ps. viburni was the most widespread pest, being found on vineyards, apple trees, plum trees or pear trees in six out of seven administrative regions sampled: Atacama (AT), Coquimbo (CQ), Valparaíso (VL), Metropolitana (MT), O’Higgins (OH) and Maule (ML)  ; Fig. 1.Ps. viburni samples showed an overall mean number of alleles of 3.06, with values ranging from 2.29 (ML1) to 3.62 (VL3). The overall mean allelic richness was 2.97 (N = 20 genotypes), with MT2 presenting the highest (3.52) and AT1 the lowest (2.20) values  values . After c) values . FrequenPs. viburni samples showed significant differentiation levels after Fisher’s exact tests (P < 0.05). The results obtained using the distance-based neighbor-joining approach showed a clear support for the separation of AT1 and CQ1 from the remaining samples  to 0.350 (between AT1 and ML3) . All pai samples . Despite samples , 2b. Fin samples .2 = 0.79; P < 0.0001) or DST values  when considering all the 16 sample sites, indicating the presence of IBD. When the IBD test was performed without the two most distant localities (AT1 and CQ1), a weaker but still significant pattern was also observed both using FST  or DST values .AMOVA results showed that a significant 7.73% of the total genetic variability can be explained by administrative regions . A significant positive correlation was found between geographical distance and either FST  algorithm , which showed a lower AICc value than both the Delaunay triangulation  or the Relative Neighbor Graph   . The SoIks = 30) . A graphks = 30) .No significant genetic variance could be assigned to differences among organic and conventional management strategies  or among host plants  . As for Ps. viburni, Ps. meridionalis, Ps. cribata, Ps. longispinus and Pl. ficus, but the only molecular study performed up to date could not recover the last two speciesPs. longispinus and Pl. citri infesting Chilean crops. Therefore, previous reports of Pl. ficus could correspond in fact to Pl. citri, given the difficulties of morphology-based identification of mealybugs. The main Pseudococcus species infesting Chilean fruit crops was the obscure mealybug Ps. viburni, followed by Ps. longispinus and Ps. meridionalis.The distribution of insect pest species across agricultural ecosystems can be shaped by multiple factors, ranging from specific associations with the cultivated plant, environmental gradients or the use of pesticides. The study of these multiple factors, together with an accurate discrimination between mealybug species, are determinant for an adequate adjustment of management strategies. Previous morphology-based surveys of Chilean mealybugs have found Ps. viburni and Ps. longispinus were recovered from vineyards and fruit orchards. Nevertheless, while Ps. viburni occurred mainly in vineyards around the central regions of Chile  and became less frequent south of O’Higgins, Ps. longispinus showed an opposite pattern and was more abundant in Apple orchards south of that region. These different spatial distribution and host-plant preference patterns are in agreement with previous studies on the biology and systematics of PseudococcidaePs. viburni belongs to the grape mealybug (Ps. maritimus) species complex, while the long-tailed mealybug (Ps. longispinus) and related species are commonly found on fruit orchards  was found to be highly structured geographically, and the authors relate this fact to the limited dispersal capacity of the insect and the patchy distribution of the obligate hostP. viburni.Climate differences might correlate with geographical distances, so that spurious genetic-climate correlations could result from an isolation by distance pattern. AMOVA, IBD and sPCA analyses all support the presence of reduced connectivity between Ps. viburni is an important contribution to design effective control strategies for pest management both in Chile and elsewhere. Even though the exact center of origin for the obscure mealybug remains unclear, South America has been suggested as the most likely native area. Therefore, the genetic characterization of Ps. viburni within Chile will contribute to future studies tracing back the origin and improving the management of this worldwide invader .The identification of well-differentiated groups within How to cite this article: Correa, M. C. G. et al. Mealybug species from Chilean agricultural landscapes and main factors influencing the genetic structure of Pseudococcus viburni. Sci. Rep.5, 16483; doi: 10.1038/srep16483 (2015).


Eleutherococcus species, Eleutherococcus senticosus and E. sessiliflorus. Both species are distributed in northern China, regions that are generally considered to be sensitive to climatic fluctuations. These regions once hosted temperate forest, but this temperate forest was replaced by tundra and taiga forest during the Last Glacial Maximum (LGM), according to pollen records. Using three chloroplast DNA fragments, we assessed the genetic structure of 20 and 9 natural populations of E. senticosus and E. sessiliflorus, respectively. Extremely contrasting genetic patterns were found between the two species; E. sessiliflorus had little genetic variation, whereas E. senticosus had considerably higher levels of genetic variation (15 haplotypes). We speculated that a recent severe bottleneck may have resulted in the extremely low genetic diversity in E. sessiliflorus. In E. senticosus, populations in Northeast China (NEC) harbored all of the haplotypes found in this species and included private haplotypes. The populations in NEC had higher levels of genetic diversity than did those from North China (NC). Therefore, we suggest that both the NC and NEC regions can sustain LGM refugia and that lineage admixture from multiple refugia took place after the LGM elevated the local genetic diversity in NEC. In NEC, multiple genetic hot spots were found in the Changbai Mountains and the Xiaoxing'an Range, which implied that multiple locations in NEC may sustain LGM refugia, even in the Xiaoxing'an Range.Climate oscillations are the key factors to understand the patterns in modern biodiversity. East Asia harbors the most diverse temperate flora, largely because an extensive terrestrial ice cap was absent during repeated Pleistocene glaciation–interglacial cycles. Comparing the demographic histories of species that are codistributed and are close relatives may provide insight into how the process of climate change influences species ranges. In this study, we compared the spatial genetic structure and demographic histories of two coexisting  Comparative phylogeography of codistributed species seeks to understand general patterns in the evolutionary history of communities , whereas temperate forests probably retreated to the South, below 30°N  sequence data and ecological niche modeling (ENM) to compare the demographic histories of the two shrub species. Three specific questions were asked: (1) Do the two codistributed species have similar levels of genetic diversity? (2) What are the differences in the population expansion patterns between the two codistributed species? (3) Where are the LGM refugia for the two species? Did these species occupy a single refugium or multiple refugia?sus Fig. A and B arus Fig. C and D hrus Fig.   were used for amplification in all sampled individuals of E. senticosus and E. sessiliflorus. Polymerase chain reaction (PCR) was performed in 40 μL, containing 10–20 ng of template DNA, 1 × buffer, 200 mmol/L each dNTP, 2.0 mmol/L MgCl2, 0.1 mmol/L each primer, and 2 units of Taq . Our PCR procedure comprised an initial denaturation at 95°C for 5 min, followed by 35 cycles of 1 min at 94°C, 50 sec at 54°C, and 1.5 min at 72°C, with a final extension at 72°C for 8 min. PCR products were sequenced at the Beijing Branch of Shanghai Majorbio Bio‐pharm Technology Co., Ltd. All of the cpDNA haplotypes were deposited in GenBank with Accession Nos. KU378054–KU378101.Total genomic DNA was extracted using a plant genomic kit . To check for polymorphisms, 13 commonly used cpDNA fragments were initially chosen for amplification in 8 individuals from 4 different populations  with the Clustal module. Each indel was treated as a single site in all the following analysis. The genetic diversity parameters  (Nei θ) (Watterson RS) and the contributions to total gene diversity of each population (CT) were calculated with the method described by Petit et al. (https://www6.bordeaux-aquitaine.inra.fr/biogeco_eng/Scientific-Production/Computer-software/Contrib-Permut/Contrib). Rarefaction size was set to be the same as the smallest population . The rarefaction analyses partitioned the contribution of CT to CS and CD, with CS representing intrapopulation diversity and CD representing interpopulation diversity. Population differentiation  was also calculated by Permut 1.0  Nei  and (θ) t et al.  in ContrdN) and the synonymous substitution rate (dS) between sequence pairs of the matK gene from each haplotype in the two species. We used the Nei–Gojobori model /SQRT(Var(dS) + Var(dN)) was computed, wherein the variance of the difference was calculated using the bootstrap method (500 replicates).Potential signals of selection in chloroplast sequences were tested by comparing the relative abundance of the nonsynonymous substitution rate  version 2.0 software  was calculated each time with a user‐defined group number (K). We obtained a series of FCT values for group numbers (K) from 2 to 8. The optimal number of groups was determined by the highest FCT value.To assess the spatial genetic structure of populations, values of the fixation index 7 was used and recorded 104 trees. Because the two studied species shared no haplotypes, we treated E. sessiliflorus as an outgroup for the haplotype tree of E. senticosus. The convergence of MCMC simulations was determined by the ESS value in Tracer 1.5 (Rambaut and Drummond http://tree.bio.ed.ac.uk/software/figtree/).To infer the phylogenetic relationships between haplotypes in both species, we used Bayesian inference in Beast 1.8.0 (Drummond and Rambaut t) was estimated using the relationship τ = 2ut = 2kμt, where μ is the substitution rate per site per year (s/s/y) and k is the length of the sequence under study. An estimator of the substitution rate for angiosperm species (2.0 × 10−9 s/s/y) was used in this study , Global Biodiversity Information Facility (http://www.gbif.org/), and Tropicos (http://www.tropicos.org/). To mitigate potential effects of spatial sampling biases, we employed the R package spThin   mean diurnal temperature range, (2) temperature seasonality, (3) maximum temperature of the warmest month, (4) minimum temperature of the coldest month, (5) annual precipitation, (6) precipitation of the driest month, and (7) precipitation seasonality.We used maximum entropy modeling (Maxent 3.3.3k) . Paleocoast lines and the paleoclimate surfaces of the exposed seafloor area (assuming a sea level depression of 130 m) were estimated using seafloor topography data (ETOPO1) produced by the National Geophysical Data Center of the National Oceanic and Atmospheric Administration . We validated our models through choosing a cross‐validation set at 50 replicates using the mean area under the curve (AUC) of the receiver‐operating characteristics (ROC).For LGM prediction, we used climate data from the Model for Interdisciplinary Research on Climate (MIROC), provided by the Paleoclimate Modelling Intercomparison Project . The only haplotype (H16) of E. sessiliflorus was at least 17 mutational steps from those of E. senticosus  were shared in NC. Among the NEC populations, private haplotypes occurred in populations BS (H8 and H10), DS (H11), and CH (H14) in the Changbai Mountains and KDQ (H15) in the Xiaoxing'an Range. Most populations harbored chloroplast polymorphisms, except for 3 populations, including SHB in the Yanshan Mountains, PQG in the Taihang Mountains, and QS in the Changbai Mountains, which were fixed with single haplotypes.In E. senticosus, with Hd = 0.835, π = 0.00086, and θ = 0.00110. The average allele richness within populations (RS) was 2.413, and the total allele richness (RT) was 3.148 when the rarefaction size was set to 4. Population BS in the Changbai Mountains had the highest levels of haplotype diversity (RS = 2.367 and Hd = 0.889), with nucleotide diversity π = 0.00124 and θ = 0.00146. Population CH in the Changbai Mountains had the most abundant haplotypes , with RS = 2.269, Hd = 0.867, π = 0.00100, and θ = 0.00081 . Z‐tests of (dN−dS) of each haplotype sequence pairs were not significantly different .The Mantel test for isolation by distance (IBD) failed to detect a significant correlation between the genetic and geographic distance with 999 permutations (GST = 0.278 and NST = 0.151). The SAMOVA analysis also indicated a low population genetic structure. Among the predefined number of groups (K = 2–8), FCT values were low: the highest value was 0.261 when K = 2 , with population QS as one group and the remaining populations as the other group.Extremely low population genetic differentiation was found . H5 was present in 30% of the individuals and 65% of the populations. Moreover, other haplotypes were also geographically widespread despite the few individuals, such as H9, with 19 individuals found in 8 populations; H3, with 16 individuals found in 7 populations; and H6, with 28 individuals found in 7 populations  contained H3, H4, H5, H6, H7, H9, H11, and H14; the remaining haplotypes constituted the other clade (CP2). This tree was consistent with the haplotype network . According to the relationship τ = 2ut = 2kμt, where k is 2666 bp, the expansion time (t) estimated for E. senticosus was 0.20 Mya (95% HPD: 0.04–0.59). In addition, another indicator, Tajima's D = −0.576 (P = 0.314), did not significantly deviate from a neutral pattern, suggesting that there was no significant signal of a recent spatial expansion.Significant spatial expansion was detected for E. senticosus and E. sessiliflorus, with AUC values of 0.971 ± 0.011 and 0.983 ± 0.015 (mean ± SD), respectively. Under the present climate, the distribution of E. senticosus was predicted to occur in the NEC region, the NC region, and the Korean Peninsula with high probability  in NEC      .Although Eleutherococcus species, all of the sampled locations of E. sessiliflorus, except for DQG and BSh, overlapped with those of E. senticosus. Both species are congeneric shrubs with similar seeds and pollen dispersal systems, generation times, and species ranges. The mating systems were both outcrossing‐dominated and self‐compatible . However, the 61 individuals from 9 populations of E. sessiliflorus are scattered across the species range. The scattered sample strategy should have characterized the important genetic variation of E. sessiliflorus. We propose that two possible scenarios may have generated the extremely low genetic variation in E. sessiliflorus: (1) a severe bottleneck, which resulted in a low extant effective population size  in matK were not significant and thus do not permit the rejection of the hypothesis that these sequences have evolved neutrally.In addition to the same sequencing size sampled in both Hordeum gussoneanum, based on both DNA sequencing and microsatellite length variation, might be due to a severe and relatively recent bottleneck that erased all of the chloroplast variation. Although the total absence of genetic variation throughout the range of each species has not been commonly found in publications, extremely low regional genetic variation, especially in the northern portion of the range of a species, is often observed in temperate species  was lower than that in NEC (RS = 1.707), although this difference was not significant (P = 0.405). The CT value in NC was negative, except for populations SHB and WLS, whereas 6 populations in the Changbai Mountains had positive values , NC should have a higher potential to sustain LGM populations because NC is located in the South and should therefore have more suitable habitats  are ignored by the current simulations due to the low resolution of ancient climate data. Hence, we include ENM results in our study, but considering the low resolution and significant flaws, we do not think it provides reliable supporting information for understanding the contrasting genetic patterns between the two codistributed Eleutherococcus species.Although the two codistributed LGM Fig. C and D. LGM Fig. . Such daLGM Fig. . Thus, sLGM Fig. , 2015. AE. senticosus and E. sessiliflorus are closely related and overlap in their ranges, contrasting genetic patterns were detected based on cpDNA sequencing and coherent sampling strategies of these two congeneric species. E. sessiliflorus had no genetic variation across all of the sampled loci, whereas E. senticosus had considerably higher levels of genetic diversity. We infer that a recent severe bottleneck likely occurred in E. sessiliflorus. For E. senticosus, relatively higher genetic diversity was found in the Changbai Mountains than in NC. We suggest that both NC and the Changbai Mountains may have sustained LGM refugia. Lineage admixture from separate LGM refugia may have contributed to the high genetic variation in the Changbai Mountains. There were signals indicating that the Xiaoxing'an Range may have had cryptic LGM refugia. Confirmation of this possibility will require more evidence. Large amounts of nuclear genetic markers will be needed to accurately infer the origin of the contrasting genetic patterns between these codistributed and closely related species.Although E. senticosus, as predicted by ENM. The Bo Sea was converted to land during the LGM due to the lowering of the sea level. Analysis of samples from the Korean Peninsula would be beneficial for inferring demographic history in future studies. Currently, however, we believe that the unsampled status of the Korean Peninsula and the Bo Sea will not diminish the importance of the conclusions that are made in this paper. Both the Korean Peninsula and Bo Sea refugia may contribute to the extant local gene pool of the Changbai Mountains. Some private haplotypes in the Changbai Mountains may have originated from the two above unsampled LGM refugia. However, no IBD pattern was recovered. Populations with high genetic diversity were located at medium to high latitudes . This location is considerably further north than would have been possible to admix in the region containing the three potential southern refugia  of each haplotype sequence pairsClick here for additional data file.


Passiflora spp.), molecular markers have only recently been utilized in genetic studies of this genus. In addition, both basic genetic researches related to population studies and pre-breeding programs of passion fruit remain scarce for most Passiflora species. Considering the number of Passiflora species and the increasing use of these species as a resource for ornamental, medicinal, and food purposes, the aims of this review are the following: (i) to present the current condition of the passion fruit crop; (ii) to quantify the applications and effects of using molecular markers in studies of Passiflora; (iii) to present the contributions of genetic engineering for passion fruit culture; and (iv) to discuss the progress and perspectives of this research. Thus, the present review aims to summarize and discuss the relationship between historical and current progress on the culture, breeding, and molecular genetics of passion fruit.Despite the ecological and economic importance of passion fruit ( Passifloraceae Juss. ex DC. belongs to the order Violales, class Magnoliopsida, and phylum Magnoliophyta. Although records of plants that belong to this family have been in existence since 1553, there have been disagreements regarding the number of genera and species in the family since the first literary record of a plant in this group was written in Colombia .ssiflora ,8,9,10. P. edulis (with 105 accessions) and P. alata (with 65 accessions) [The vast majority of passion fruit accessions are maintained in Brazilian institutions, highlighted by the collections and germplasm banks maintained by Embrapa , the Instituto Agronômico de Campinas (IAC), and the Instituto Agronômico do Paraná (IAPAR) as well as the Universidade Estadual Paulista (UNESP), Universidade Estadual do Norte Fluminense (UENF), Universidade Federal do Rio de Janeiro (UFRJ), and Escola Superior de Agricultura Luiz de Queiroz (ESALQ/USP) . The curessions) ,59.Passiflora genetic breeding programs [Passiflora has been neglected during cultivar generation [The discrepancy between the natural variability inherent to the genus and the reduced variability represented in BAGs should be remedied as quickly as possible because it hinders the establishment and progression of programs . Similarneration . Thus, aneration .Passiflora breeding programs prioritized production  and not necessarily the other aspects of genetic diversity, such as resistance, ornamentation, and the production of potential medicinal compounds.The above conditions are justified, at least partially, because the first studies in the 1980s ,71 that P. caerulea, P. cincinnata, P. coccinea, Passiflora gilberti, P. laurifolia, P. setacea, and P. suberosa), self-compatibility , and variability in the season of production compared with commercial species (P. setacea and P. coccinea) [P. caerulea, P. cincinnata, P. coccinea, and P. setacea, interspecific hybrids are being developed and characterized [In addition to the potential for commercialization because of the natural beauty of their flowers and the taste of their fruits, wild passion fruit species have desirable characteristics for the yellow passion fruit, including resistance to pathogens and pests (occinea) ,54. For cterized ,54,72 ancterized ,63.According to these studies, the genetic base of the yellow passion fruit is restricted with respect to disease resistance and tolerance. In fact, the authors favor the use of genetic resources from wild species in breeding programs. Several authors have emphasized research on the description, characterization, and use of germplasm as a priority for passion fruit production to obtain more productive passion fruit cultivars with increased resistance to diseases ,73. The Passiflora and to the various stages of breeding programs.Although the realities of existing cultivars, such as low variability and susceptibility to disease, still endure for cultured passion fruit, an increasing number of studies devoted to the characterization of resistance in wild species and commercial accessions are being performed ,34,74,75The use of molecular markers for genetic characterization projects with a variety of objectives for a range of organisms is routine in many laboratories. Although molecular biology techniques have become prevalent in recent decades, leading to a reduction in the cost of most molecular markers, the availability of resources and background information are still determining factors in the choice of methods and markers to be used. In recent decades, the increase in the number of molecular markers associated with the growth of related fields of study, such as population genetics, genetic breeding, statistics, and bioinformatics, has generated immense progress in the knowledge applicable to conservation and biodiversity manipulation.Passiflora genus. These studies range from the use of dominant markers to the development and employment of co-dominant markers and single-nucleotide polymorphisms (SNPs). Passiflora. A simple quantitative evaluation of published papers, the data from which are shown in Passiflora research began approximately 15 years ago with the use of the random amplified polymorphic DNA (RAPD) markers to study the diversity of 52 accessions from 14 Passiflora species [Genetic studies associated with molecular markers are becoming standard for the  species .Passiflora , and most of the research has focused on Passiflora species or accessions with previously established commercial interest. Research on ecological justifications and aims coupled with species conservation have not been performed, although brief discussions on the characterization and ex situ conservation have been published.Despite the availability of approximately 4000 fragments of DNA and RNA sequences from database , geneticMost molecular genetic studies  are based on the use of dominant markers. Although dominant markers are useful for genetically distinguishing species (for tests to confirm hybrids and the direction of convergent and divergent breeding), they have limitations, such as low repeatability and suboptimal genetic information profiles (homozygote and heterozygote specimens cannot be distinguished), that are highly relevant for population studies.P. edulis [P. edulis indicated low genetic variability [Despite the limitations inherent in the use of dominant markers, genetic diversity estimates based on wild and commercial accessions have contributed to the understanding of the genetic variability of passion fruit. In this context, RAPD and AFLP markers have been used to demonstrate high genetic variability among accessions of . edulis ,79 as weiability ,80. The iability , and theiability .P. cincinnata, P. nitida, and P. setacea [P. nitida [P. setacea [The use of RAPD markers has also contributed to estimates of diversity in wild species of passion fruit, such as  setacea ,63,81. IPassiflora in 2010 [et al. [Results on the characterization of genetic variability and the identification of potential regions related to resistance genes, phylogenetically conserved in plants, were first published for the genus  in 2010  , and the preliminary results confirm the efficiency of these markers for the characterization of genetic variability in commercial and wild species of passion fruit . RGA marP. alata .Passiflora using co-dominant markers began in 2005 and were mainly limited to the development of microsatellite markers for the species P. edulis [P. alata [P. cincinnata [P. setacea [P. contracta [Passiflora indicated low levels of polymorphism in these genomic regions, at least for the species evaluated  collected in 60 commercial plantations [P. edulis [P. alata [Research that characterizes genetic diversity in wild populations by microsatellite markers remains unpublished, although reports on evaluations of germplasm materials in Colombia  and Brazet al.  in a stum et al.  in a stus et al. ,98 have tellites ,88,89,90ntations . Ten mic. edulis  and sevePassiflora accessions were obtained with SSR markers in 2013 [et al. [The first successful estimates of genetic diversity in  in 2013 ,94. Howe [et al. , where 1Mangifera indica [Olea europaea [Zea mays L. [Solanum lycopersicum [The variation in the genetic diversity estimated in published papers, primarily those analyzing commercial species, is due to not only the specific characteristics of the genotypes evaluated but also the effects of the different statistical methodologies employed by the authors. Methodological influences on the results of diversity estimates have been reported for a variety of species, including a indica , Olea eueuropaea , Zea may mays L. , and Solpersicum . Differepersicum . In the P. alata) [Passiflora species.Research projects conducted as part of a joint venture between the Universidade Estadual de São Paulo  and Centre National de Ressources Génomiques Végétales  during the past five years have introduced the first SNP identifications in commercial passion fruit species  . HoweverCowpea aphid-borne mosaic virus) and the bacterial spot disease (caused by Xanthomonas axonopodis pv. passiflorae), for the which chemical control measures are not effective and resistant cultivars are not available, a growing number of advances in the use of genetic engineering have been observed in recent decades [Considering the diversity of species and intra- and interspecific genetic variability available for use in breeding programs of passion fruit, genetic engineering as a methodological strategy to increase favorable characteristics for passion fruit culture has been specifically employed to search for disease resistance . With re decades ,107,108.P. edulis) were transformed from fragments of genes obtained from viral isolates known to cause PWD. These studies were initiated based on a pathogen-derived resistance (PDR) approach that likely relied on the induction of post-transcriptional gene silencing (PTGS).Studies attempting to produce transgenic passion fruit have been conducted since the late 1990s and early 2000s at the Universidade Federal de Viçosa (UFV), Minas Gerais, Brazil, and Universidade de São Paulo (USP), São Paulo, Brazil; the first results were published in 2005  and 2006NIb gene and the 5' region of the CP gene derived from a viral isolate of CABMV was used by Alfenas et al. [CP gene was used by Trevisan et al. [NIb and CP genes were isolated. It is probable that the high variability in the nucleotide sequence of the NIb gene was the main factor contributing to this result. Subsequently, the transgenic plants (R0) were self-crossed to generate homozygous lines (R1), and a plant of this line was resistant to seven viral isolates [1 lineage, contributes substantially to the spectrum of resistance observed [An untranslatable construct consisting of two-thirds of the s et al. . In turnn et al. . In bothn et al.  was restisolates . The autobserved ,109.et al. [1 and R2, both from transgenic plants characterized by Trevisan et al. [2 plants selected by the authors for future studies.In addition, one of the transgenic plants produced by Trevisan et al.  was immuet al.  identifin et al. , as welln et al. . New croP. edulis for resistance to diseases and herbicides through the introduction of a baculovirus anti-apoptotic gene (p35 gene) [attA) [P. edulis transformed with p35 gene sequences showed no resistance to CABMV but showed increased tolerance to bacterial spot disease and glufosinate herbicide when compared to non-transgenic plants [P. edulis transformed with the attA sequence of the gene showed no leaf lesions after inoculation with X. axonopodis pv. Passiflorae, indicating possible resistance to the pathogen. Initial tests attempting to obtain transgenic plants were conducted with P. alata [P. alata. Similar to the work conducted with P. edulis, the transformations achieved in P. alata were conducted using Agrobacterium tumefaciens and a fragment of the CP gene CABMV.Although in smaller proportions or in less advanced stages, studies devoted to obtaining transgenic plants were also conducted with 35 gene)  and the ) [attA) . Plants c plants . Ten plaP. alata . These eIn conjunction, the progress observed with the studies dedicated to the production of transgenic passion fruits indicates that, at least for PWD, it is possible that resistant plants will soon become available for cultivation and/or for use in breeding programs dedicated to the production of resistant cultivars with higher productivity.The contributions of molecular biology, and especially the use of the molecular markers, in (pre-) breeding programs may be didactically separated into at least six research modes: (i) the direction of convergent and divergent crossings leading the initial stages of breeding; (ii) the confirmation of intra- and interspecies hybrids with a reduction in the occurrence of escapes; (iii) the contribution of retro-crossings with a decrease in the time necessary for the re-composition of the genome; (iv) the genetic mapping and identification of quantitative trait loci (QTLs) that are useful for the subsequent introgression of genes into commercial species and cultivated varieties; (v) the prospection and characterization of resistance genes, both by mapping strategies and through the use of analog markers of resistance genes; and (vi) the identification and protection of hybrids and cultivars. Coupled with the effective use of markers, these joint activities contribute to the establishment of an assisted selection program because all of the information provided enables the efficient use of the available germplasm.P. edulis [P. setacea [P. cincinnata [P. edulis.In addition to their contribution to understanding the diversity of populations or groups of regional accessions, estimates of passion fruit genetic diversity have enabled the identification of converging and diverging crosses that can contribute to different stages of a (pre-) breeding program. For commercial species, such as . edulis , as well setacea  and P. cncinnata , preferrncinnata ,58 can aThe absence of agronomic characterizations for most accessions in the aforementioned studies is the greatest challenge to introducing information from genetic diversity estimates into current breeding programs. Difficulties in the joint use of data from phenotype and genotype characterizations  have been reported for different cultures and are an aspect of the challenge inherent in studies of natural populations . For culPassiflora species of agronomic interest associated with fruit production was published in 2008 [Different molecular markers, such as RAPD and microsatellites, are highly efficient and user-friendly techniques that can be used to confirm intra- and interspecies hybrids. The first study to employ molecular markers to confirm hybrids from 17 interspecies crosses of 14  in 2008  and was  in 2008 ,113 baseP. setacea into the cultivars GA-2, AR-1, and EC2-O of P. edulis [ P. edulis) in hybrids while maintaining the resistance of the wild ancestor (P. setacea) [Molecular markers also contribute to the characterization and selection of specimens with a higher genomic contribution from the recurrent parent ,114. TheP. edulis using RAPD markers. The population used to construct the first map helped to identify the first QRLs linked to the genus Passiflora using AFLP markers [et al. [X. axonopodis pv. passiflorae.One of the most thorough passion fruit studies, at least with regard to molecular markers within the context of breeding, consists of the construction of genetic maps and identification of regions associated with characteristics of interest . Early genetic mapping of passion fruit species began in 2000, and the first maps were published in 2002 . These m markers . The qua [et al.  explainePassiflora map was the use of microsatellite markers for the map construction in addition to AFLP [P. alata was published in 2013 [The use of map integration strategies to construct a single map (rather than two maps representing ascendants)  was a la to AFLP . An impo in 2013 . The use in 2013 .Research dedicated to the construction of physical maps has also been conducted . PhysicaThe advances in the genetic mapping of passion fruit associated with the reduction of costs of next-generation sequencing (NGS) should enable genetic characterizations based on genotyping-by-sequencing (GBS), at least for commercial species, in both the short and medium term. In this context, alternative methodologies that reduce costs and enable GBS, such as the use of restriction enzymes to reduce genome complexity , should Molecular markers are also used in different stages: after cultivars are obtained and distributed , when paternity is determined (and thus the culture is protected), and when monitoring cultivar genetic purity, regardless of whether the cultivars are clonal or seminal . When leConsidering the challenges of breeding passion fruit, as well as the available knowledge and conservation of the genetic variability of this genus, we believe that the following research activities should be prioritized: (i) the identification of new accessions to increase the representative breadth of germplasm banks, especially for wild species; (ii) the measurement of population genetics estimates that enable a true understanding of the genetic diversity and structure of passion fruit; (iii) phenotypic characterizations that contribute to both the useful evaluations of the germplasm for interspecific crosses and studies of the association between phenotypic and molecular data; (iv) research efforts devoted to identifying genomic regions associated with resistance to diseases; and (v) the potential use of transgenic strains previously evaluated in crosses targeted in breeding programs.Passiflora are only in the initial stages. Coupled with a large gap in group population genetic research, the increasing availability of microsatellite markers for Passiflora species and the use of cross-amplification strategies to popularize marker use should increase the number of research studies. Further research should be performed to identify and characterize microsatellite loci because the current results indicate low allelic diversity among SSR loci. In this context, the use of strategies for sequencing and genotyping on a large scale will be important.The recent research on the diversity of wild passion fruit species and the increasing use of molecular markers, particularly co-dominant markers, have made knowledge from population studies available and are promising for further genetic research on passion fruit. Although highly relevant for conservation and breeding activities, population studies of Passiflora via molecular markers, strategies for the exploration and conservation of germplasm collections are also necessary to enable morpho-agronomic characterizations to be performed efficiently to increase the understanding of both neutral biological diversity and diversity that is subject to natural selection. In this way, the interactions involving the accessions present in banks, germplasm collections, and elite materials used in breeding programs will be enhanced.In parallel with the population estimates that must be performed for the genus Similar to the expected growth in microsatellite marker usage in population studies, recent genomic investigations of commercial passion fruit species have developed and characterized SNP markers. These studies should be promoted because they will encourage different types of research, as has occurred with the saturation of genetic maps, the identification of QTLs, and the exploitation of mapping by association. Collections, germplasms, or elite accessions can be employed in such a strategy, and thus, a detailed map of the genomic regions of interest can be achieved. Moreover, the development and use of large-scale genotyping should allow for the use of strategies for genome-wide selection (or simply genomic selection), enhancing the association of genetic diversity data with characteristics of agronomic interest. In addition, the use of GBS strategies should help to reduce the costs and time required for germplasm characterization and the selection of passion fruit genotypes.


Panthera onca), pumas (Puma concolor), and ocelots . We assessed levels of genetic diversity, defined potential genetic clusters, and examined gene flow for the three target species on a countrywide scale using a combination of individual- and population-based analyses. Wild felids in Belize showed moderate levels of genetic variation, with jaguars having the lowest diversity estimates , followed by pumas , and ocelots . We observed low to moderate levels of genetic differentiation for all three target species, with jaguars showing the lowest degree of genetic subdivision across the country, followed by ocelots and pumas. Although levels of genetic diversity and gene flow were still fairly high, we detected evidence of fine-scale genetic subdivision, indicating that levels of genetic connectivity for wild felids in Belize are likely to decrease if habitat loss and fragmentation continue at the current rate. Our study demonstrates the value of understanding fine-scale patterns of gene flow in multiple co-occurring felid species of conservation concern, which is vital for wildlife movement corridor planning and prioritizing future conservation and management efforts within human-impacted landscapes.With increasing anthropogenic impact and landscape change, terrestrial carnivore populations are becoming more fragmented. Thus, it is crucial to genetically monitor wild carnivores and quantify changes in genetic diversity and gene flow in response to these threats. This study combined the use of scat detector dogs and molecular scatology to conduct the first genetic study on wild populations of multiple Neotropical felids coexisting across a fragmented landscape in Belize, Central America. We analyzed data from 14 polymorphic microsatellite loci in 1053 scat samples collected from wild jaguars ( Carnivores are particularly vulnerable to loss and fragmentation of natural habitats because of their space and prey requirements, occurrence at low densities, and dependence on forest and/or habitats that provide sufficient cover , 2. LandPanthera onca), the largest Neotropical felid, which co-occurs with several other felids , has been extirpated from more than half of its historic range during the last 100 years, and its distribution continues to contract, mainly due to severe deforestation and habitat fragmentation, direct persecution, and loss of main prey species . Co-occurring pumas and ocelots face similar challenges to their survival. Pumas have also disappeared from large portions of their historic range and are listed as a ‘Least Concern’ species under IUCN and as an Appendix I  and an Appendix II (remaining subspecies) species under CITES ) )  in combi[L_max]) . We condon 0.3.4  in R, veon 0.3.4 .Of 1053 scat samples collected, 530 (50%) were successfully identified to species and individual. At the individual level, we detected 65 jaguars , 54 pumas  and 30 ocelots  . In totaAR, H = 13.19, P = 0.002; AP,H = 7.52, P = 0.023; He, H = 4.55, P = 0.103), and highest for ocelots followed by pumas and jaguars  or ocelots . Pumas had significantly lower allelic richness estimates at the northern site compared with most other regions , but did not differ for other diversity estimates . FIS values for all target species and geographical regions were positive and at low to moderate levels, ranging from 0.00 to 0.08 for jaguars, 0.01 to 0.12 for pumas, and 0.04 to 0.14 for ocelots (P < 0.000) and F98 (P = 0.011) for pumas, and loci FCA391 (P = 0.001), FCA275 (P < 0.000), and FCA741 (P = 0.011) for ocelots significantly deviated from Hardy-Weinberg expectations. Significant linkage disequilibrium after sequential Bonferroni correction (P ≤ 5.00E-04) was only detected among one pair of loci (FCA096 and FCA441) in jaguars.Countrywide genetic diversity estimates were different among species , and north-central and south-central (FST = 0.050) sites. For pumas, moderate genetic differentiation was detected among most regions. FST values were highest between north and south (FST = 0.131), followed by south-central and south (FST = 0.107), central and south (FST = 0.100), north and south-central (FST = 0.084), and north- and south-central (FST = 0.061) sites. Pairwise G’ST estimates indicated moderate to high levels of genetic differentiation for all three target species across several study sites . More than half of the jaguars (56%) detected in the central region were assigned to the southern genetic cluster , whereas the rest had partial membership in the northern and southern genetic cluster. South-central jaguars from the Cockscomb Basin Wildlife Sanctuary were fully assigned to their region . Jaguars detected in southern Belize were primarily (75%) of admixed ancestry . Finally, our analysis applying spatially-explicit Bayesian clustering in GENELAND resulted in K = 3 as the most probable number of genetic clusters but no new spatial groups were defined (Q = 0.71) and southern group (mean Q = 0.87), with several jaguar individuals in central Belize  forming a cline of admixed ancestry. Southern jaguars exhibited lower levels of genetic admixture in comparison to the north, which was particularly evident at Cockscomb Basin Wildlife Sanctuary, where most jaguars (~ 70%) had strong membership assignments (Q > 0.90), thus very low levels of admixed ancestry.Non-spatial Bayesian clustering analysis in STRUCTURE indicated no genetic structure for jaguars with quencies , S1A Figanalyses , S1B Fig defined . The bouQ > 0.7) into a northern cluster with one disperser (Q = 0.17) and two admixed individuals (Q = 0.46 and 0.49)  with the exception of one admixed individual. Using spatially-explicit Bayesian clustering in GENELAND, we identified four genetic clusters and one ghost population for pumas, corresponding to our main a priori geographical regions within Belize, including the north (100% residents with mean Q = 1), north-central , central and central-south  and south   , S2B Fig = 0.24) .K = 1) (Q > 0.95) to this site.For ocelots, Bayesian clustering analysis in STRUCTURE (admixture and LOCPRIOR models) did not reveal any population subdivision (K = 1) , S3 Fig.K = 1) . While tr = 0.010, P = 0.510, r = 0.099, P = 0.100; females, r = -0.201, P = 0.200) between-individual genetic and geographic distances, verifying that geographic distance is not driving genetic differentiation at this spatial scale. Significantly positive relationships between genetic and geographic distance were detected for pumas . This suggested that gene flow in female pumas is spatially limited and may be one of the factors driving genetic differentiation in pumas in Belize. In ocelots, we found a significant, but weak signature of IBD , indicating that geographic distance potentially has a small effect on gene flow. However, sample sizes in female jaguars and male ocelots were low , thus careful interpretation of results is essential.Examination of IBD patterns in jaguars showed no significant correlation  and an x-intercept of r at ~ 20 km  and an x-intercept at ~ 68 km , and an x-intercept of r at ~ 23 km  among the three target species, with highest mean relatedness coefficients for jaguars , followed by pumas , and ocelots . Mean relatedness coefficients within sites were slightly higher for jaguars , followed by pumas , and ocelots . However, mean relatedness coefficients within sites were not significantly different among species . In all three species, within-site relatedness was significantly higher than among-site relatedness . However, a skewed sex-ratio towards males in jaguars and females in ocelots may introduce bias into the analysis.Relatedness among sites was significantly different  for jaguars. The three animals were located, and originally came from, respectively: (1) the central from the south, (2) the central-south from the south, and (3) the south from the north sites. For pumas, we detected a total of four first-generation migrants. The animals were located, and originally came from respectively: (1) north from the north-central, (2) north-central from the north, (3) central from the north, and (4) south from the south-central. For ocelots, we identified two first-generation migrants. One ocelot was located in the northern cluster (Fireburn Nature Reserve) and genetically assigned to the southern cluster. The other ocelot originally came from the southern cluster (Cockscomb Basin Wildlife Sanctuary) and was signed to the northern cluster . All firContemporary and bidirectional migration rates estimated between all sampling sites in BayesAss+ suggested an average migration rate of 0.07 (range 0.02–0.18) for jaguars across Belize , Table 6Preserving and restoring genetic connectivity in anthropogenically altered and fragmented landscapes is a key aspect of conservation e.g., , 64). Co. Co64]).He = 0.54), the south (He = 0.44) for pumas, and northern Belize for ocelots (He = 0.61). In comparison with other genetic studies, using Fca microsatellite markers ),s e.g., , 80). Si. Si2; F s e.g., , 82). Wh. Wh2; F s e.g., , 72, 83), 832; F s , 852; F s , 912; F s e.g., , 92–95).–95.2; F s ,, 96,9, 9For ocelots, non-spatial Bayesian clustering analysis rendered support for no or low levels of genetic differentiation. Furthermore, autocorrelation analysis suggested a large spatial extent of genetic structure (~ 84 km) in a mesocarnivore, indicating that gene flow for ocelots across this landscape may be relatively high. Given ocelots’ smaller body size and their tolerance to human activity to some extent  they maThis study directly compared patterns of fine-scale genetic connectivity in multiple Neotropical felids. For jaguars and pumas, which differ in size across regions of coexistence, jaguars are the larger, dominating species . We obseGene flow may be also driven by various other ecological factors, including the degree to which habitat use and human disturbance limit movement in wide-ranging species such as jaguars and pumas. Former studies described pumas as opportunistic in their habitat use since they are known to use a wider variety of habitats in comparison to jaguars e.g., , 96). In. In96]).With increasing anthropogenic impact and landscape change, it is crucial to genetically monitor wild felid populations. Understanding levels of fine-scale gene flow and genetic structure patterns of multiple co-occurring felid species reveal behavioral and other factors that influence gene flow across the landscape, which is vital for planning and prioritizing future conservation and management efforts on a countrywide scale. This study provides comprehensive baseline conservation genetic data and demonstrates that noninvasive genetic sampling is an efficient research approach to simultaneously assess levels of genetic diversity and differentiation of multiple Neotropical felid species in the wild. We found that genetic diversity for wild felids in Belize is moderate and that levels of genetic connectivity within the country are moderate to high. Although Belize has a high proportion of forest cover (~ 62.7%) and protected areas (~ 36%) compared to other Mesoamerican countries, we believe that levels of genetic connectivity are likely to decrease if habitat loss and fragmentation continue at the current rate. Despite the dispersal capabilities of the two large felids, our study detected evidence for fine-scale genetic differentiation particularly between northern and southern sites, indicating that the more-developed and human-dominated areas adjacent to north-central and central Belize may be subtlety-affecting movements of felids. Consequently, we recommend prioritizing countrywide conservation and management efforts for wild felids, such as maintaining and enhancing biological connectivity among the national protected area system within Belize through strengthening formerly identified wildlife movement corridors  , 121. ThS1 FigK value for the admixture model (a) without and (b) with adding sampling locations as prior (LOCPRIOR) was chosen based on posterior probability (mean LnP(K)) and delta K (ΔK) for each K value.The optimal (DOCX)Click here for additional data file.S2 FigK value for the admixture model (a) without and (b) with adding sampling locations as prior (LOCPRIOR) was chosen based on posterior probability (mean LnP(K)) and delta K (ΔK) for each K value.The optimal (DOCX)Click here for additional data file.S3 FigK value for the admixture model (a) without and (b) with adding sampling locations as prior (LOCPRIOR) was chosen based on posterior probability (mean LnP(K)) and delta K (ΔK) for each K value.The optimal (DOCX)Click here for additional data file.S4 FigPanthera onca in BelizeSpatial autocorrelogram for   showing the genetic correlation coefficient (r) as a function of geographic distance across spatial distance classes. Dashed red lines represent upper (U) and lower (L) bounds of the null distribution based on 9,999 random permutations. Error bars represent 95% confidence intervals about r based on 999 bootstraps.(DOCX)Click here for additional data file.S5 FigPuma concolor in BelizeSpatial autocorrelogram for   showing the genetic correlation coefficient (r) as a function of geographic distance across spatial distance classes. Dashed red lines represent upper (U) and lower (L) bounds of the null distribution based on 9,999 random permutations. Error bars represent 95% confidence intervals about r based on 999 bootstraps.(DOCX)Click here for additional data file.S6 FigLeopardus pardalis in BelizeSpatial autocorrelogram for   showing the genetic correlation coefficient (r) as a function of geographic distance across spatial distance classes. Dashed red lines represent upper (U) and lower (L) bounds of the null distribution based on 9,999 random permutations. Error bars represent 95% confidence intervals about r based on 999 bootstraps.(DOCX)Click here for additional data file.S1 Materials and Methods(DOCX)Click here for additional data file.