Hybrid zones are regions where individuals of two species meet and produce hybrid progeny, and are often regarded as natural laboratories to understand the process of species formation. Two microevolutionary processes can take place in hybrid zones, with opposing effects on population differentiation. Hybridization tends to produce genetic homogenization, reducing species differences, whereas the presence of mechanisms of reproductive isolation result in barriers to gene flow, maintaining or increasing differences between taxa.Bufo bufo and B. spinosus, through a combination of molecular  and morphological data in a transect in the northwest of France. The results show largely concordant clines across markers, defining a narrow hybrid zone of ca. 30 km wide. Most hybrids in the centre of the contact zone are classified as F2 or backcrossed individuals, with no individuals assigned to the F1 hybrid class.Here we study a contact zone between two hybridizing toad species, We discuss the implications of these results for our understanding of the evolutionary history of these species. We anticipate that the toad contact zone here described will become an important asset in the study of hybrid zone dynamics and evolutionary biology because of its easy access and the abundance of the species involved.The online version of this article (doi:10.1186/s12983-016-0184-7) contains supplementary material, which is available to authorized users. Speciation is usually accompanied by the gradual accumulation of genetic incompatibilities between diverging gene pools through time . Thus, tA powerful approach to characterize the outcomes of post-divergence hybridization and understand the underlying evolutionary processes is provided by cline theory. Geographically, hybrid zones can be defined as clines in characters that show different frequencies in the parental populations. Therefore, it is possible to study hybrid zones by sampling along a transect perpendicular to the contact zone and examining patterns of variation across multiple independent markers –6. SeverTriturus cristatus/T. marmoratus [Bufo bufo and the Spined toad B. spinosus) has revealed significant geographic concordance with the Triturus hybrid zone [Since the publication of some, now classical synthesis papers on the role of Quaternary Ice Ages in shaping current patterns of genetic diversity and structure across taxa , 12, thermoratus ) as a prrid zone .Bufo bufo species group have resulted in the recognition of four species, two of which were formerly regarded as subspecies of B. bufo [B. spinosus, present in Iberia and North Africa, contacts B. bufo in France, forming an extensive contact zone along a NW-SE line, approximately from Caen to Lyon [B. bufo and B. spinosus in northwestern France. The specific questions that we sought to answer are: i) can clines be observed for morphological, nuclear and mitochondrial markers?, ii) do clines of the different traits coincide? and iii) can the position of the contact zone be linked to features of the landscape? On a broader perspective, our goals include testing: iv) whether this contact zone is a hybrid zone, and v) whether the zone is maintained by selection, with a significant degree of reproductive isolation.Recent molecular studies on the systematics of the  B. bufo –16. One  to Lyon . These s to Lyon , but are to Lyon . We therBufo toads in 17 populations in a transect across the contact zone of B. spinosus and B. bufo in northwestern France (Table B. spinosus – B. bufo hybrid population [B. spinosus from B. bufo [N = 190) and adult females (N = 76) were analyzed separately. For each individual we calculated the probability to belong to B. spinosus (Ps), with the logistic regression formulae derived for this part of France .Table 1Diagnostic SNPs were developed on 16S rRNA (16S) and Cytochrome B (cytb) mitochondrial sequences from , yieldinKASP PCRs for both nuclear and mitochondrial SNPs were carried out in 1536 wells plates with circa 1 ng of template DNA and 1 μl of reaction mix containing KASP v4.0 reaction mix and primers following the manufacturer’s directions. The conditions of the KASP PCR were as follows: 15 min denaturation at 94 °C followed by 10 cycles of 20s at 94 °C denaturation and 60s at 61 °C of annealing and extension and lowering the annealing and extension temperature by 0.6 °C each cycle, followed by 26 cycles of 20s at 94 °C denaturation and 60s at 55 °C of annealing and extension in a hydrocycler. After 36 cycles of this touch down protocol fluorescence was measured on a Pherastar plate reader. PCR was continued and after 39, 42 and 45 cycles fluorescence was measured again to follow the trajectory of all samples. Genotypes were called automatically by the module Kraken™ of LIMS controlling the LGC genomics SNP genotyping line, visually inspected and if necessary manually corrected.o) and expected heterozygosity (He) were calculated with GenePop 4.2 [Twelve microsatellite loci were studied in 300 individuals in four multiplex reactions, following . The numePop 4.2  per locuePop 4.2 . For theePop 4.2 .K values of 1–14, with five replicates per value of K. The optimal clustering scheme was selected through application of Evanno’s criterion [B. spinosus, B. bufo, F1-hybrids, F2-hybrids and backcrosses in either direction. Structure and NewHybrids provide different but complementary information about genetic admixture and for visual inspection we constructed bivariate Structure * NewHybrid plots (see [The software Structure  was usedriterion  as impleriterion . Resultsriterion . Individriterion  to obtaiots (see ).s, Parotoids and Metatarsal tubercle, males and females combined), ii) mitochondrial DNA , iii) the loading on the axes of a principal component analysis derived from the panel of microsatellites  and iv) for the nuclear genes BDNF, POMC, RAG1 and RPL3 . As reference cline models we choose the Structure Q-score of belonging to either parental class, and the NewHybrid posterior probabilities of belonging to a hybrid class versus either of the parental species, derived from the microsatellite data. Clines are considered significantly displaced if the two log-likelihood unit support limits of the cline centre do not overlap with the Structure Q-score (Qb = 1 − Qs). Individual microsatellite loci conveyed little information on species identity (results not shown) and GenoDive [The position, width and shape of geographical clines over the transect were determined with the R package HZAR  from popGenoDive  was usedGenoDive .B. spinosus  and at the northern side of the transect they show states typical for B. bufo . Among males, morphologically intermediate individuals are most frequent in populations 9, 10, 13 and 15  and observed (Ho) heterozygosities and allele size range per population are summarized in Additional file 1: heterozygote deficit) were detected eight times in seven populations, involving different loci and with no correspondence with potential null alleles , expected (Hes Table . The perK) in Structure was K = 2  are shown by populations 10 and 12, with intermediate values for the remaining populations. The average Qb-score for population 10 (Beaulieu) is 0.66  suggesting that it is situated just off the centre of the contact, in the direction of B. bufo . Populations 9, 11 and 12 adjacent to Beaulieu contain genetically mixed individuals with pp- and Q-scores as in Beaulieu, as well as individuals with lower pp- and intermediate Q-scores for which the status as hybrid or a parental is equivocal. The microsatellite plot  for populations 13–15 at the B. bufo side of the transect and intermediate for the central populations 7–12 (201–277 m a.s.l.). For five out of eight characters the position of the cline is similar to that of the reference and for three out of eight also the width is similar  and full reproductive isolation. For amphibian examples in which the extent of natural hybridization in contact zones correlates with divergence times see refs. , 32, 33.2s or backcrosses. While this could represent differences in fertility between hybrid classes, it is also likely that later generation hybrids and backcrosses are being pooled together in the F2 class, since the confidence of assignment to different hybrid classes depends on the number of generations elapsed after secondary contact [B. spinosus at higher altitudes than B. bufo, and a centre close to locality 10 (Beaulieu) in our transect. Nevertheless, it is unclear the extent to which selection falls on the parentals and/or the hybrids and whether environmentally mediated selection plays a role in stabilizing the hybrid zone. The taxonomic status of B. spinosus as a species different from B. bufo is recent [B. spinosus.In the hybrid zone studied here, most hybrid individuals were classified by NewHybrids as F contact  and the  contact , 35. Thu contact ). Initia contact , 17, 19. contact , raised s recent –39 and nBufo species, evidence for hybrids is restricted to a narrow area about 30 km wide. Most hybrids were classified as F2 individuals by NewHybrids, which is often the case in relatively old contact zones where many generations of backcrossing complicate precise assignment of individuals to specific hybrid classes [In general, allopatric populations of the two parental species have lower genetic diversity than populations near the centre of the contact zone. The inspection of allele frequencies shows a clear trend in some loci, with some alleles presenting high frequencies on opposite sides of the contact zone and intermediate frequencies co-occurring in the centre of the contact zone. Genetic homogenization due to hybridization is spatially restricted. This is also apparent upon inspection of Structure and NewHybrids analyses, which show increasing levels of admixture close to the centre of the contact zone. Thus, while we have found evidence for ongoing hybridization between the two  classes , 33, 34.B. bufo - B. spinosus contact pre-dates the LGM. Conversely, under CCSM favourable areas are situated at more southern latitudes, with small fragments in coastal areas (both at the Atlantic and the Mediterranean) and large continuous areas restricted to southern Europe, suggesting that the Bufo hybrid zone formed more recently. We propose a scenario in which B. spinosus reached the Normandy region around 8000 YBP, because the species is absent from the Channel island of Guernsey that became isolated from the continent shortly before that date, but made it to Jersey [B. bufo, with a glacial refugium in the Balkan peninsula, reached the landmass that is currently England, Scotland and Wales, but it didn’t reach the Isle of Man or Ireland. We therefore assert that the species contact is thousands of years old, but further studies are required to reconstruct the species’ postglacial dynamics and to track the spatial and temporal stability of the hybrid zone.Phylogeographic analyses on the basis of two different climate reconstructions for the Last Glacial Maximum (LGM), MIROC and CCSM, lead to drastically different scenarios . Under Mo Jersey . Jersey o Jersey . In para1 hybrids, but sex-biased dispersal or asymmetric genetic incompatibilities among the sexes can produce discordant clines. While data for B. spinosus are scarce, a study on British and Swedish populations of B. bufo found that 93% of females and 96% of males that survived between years returned to the same breeding ponds [w) of the cline can be predicted from a diffusion model as a function of dispersal distance (d) and the length of time since contact (t), as w = 2.51 d √t [B. bufo/B. spinosus averages at 1.32 km in eight studies (range 0.12–3.62 km) [Bufo [1’s, and/or the parentals, given their absence and low frequency at the centre of the zone, respectively.The concordance of mtDNA and nuclear clines is expected when there is selection against Fng ponds , togetheng ponds  suggesti.51 d √t . The max3.62 km) , 44, 45 m) [Bufo . At a loDespite the overall congruence of genetic clines, with similar patterns between nuclear and mitochondrial markers, some clines show smoother transitions between pure populations at both ends of the transect. One remarkable discrepancy is between the sharp species transition documented by the Structure and NewHybrid analyses of the microsatellite data Fig.  versus tB. bufo and B. spinosus will become a prime study system in speciation research.We have characterized the common toad system as a hybrid zone, with narrow and coincident clines revealing barriers to gene flow across species, which opens new and exciting lines of research to further pursue with the help of genomic tools. While the relative roles of intrinsic and extrinsic factors in shaping genetic and morphological clines are still open to further scrutiny, the geographical extent of this contact zone, encompassing France from the northwest to the southeast, allows the comparative study of multiple transects, with great opportunity for the identification of consistently versus locally-introgressed genomic regions and thus for discerning the relative roles of indeterminate drift and selection in the origin and maintenance of species boundaries. Thus, we assert that the hybrid zone between


The critically endangered northern white rhinoceros is believed to be extinct in the wild, with the recent death of the last male leaving only two remaining individuals in captivity. Its extinction would appear inevitable, but the development of advanced cell and reproductive technologies such as cloning by nuclear transfer and the artificial production of gametes via stem cells differentiation offer a second chance for its survival. In this work, we analyzed genome-wide levels of genetic diversity, inbreeding, population history, and demography of the white rhinoceros sequenced from cryopreserved somatic cells, with the goal of informing how genetically valuable individuals could be used in future efforts toward the genetic rescue of the northern white rhinoceros. We present the first sequenced genomes of the northern white rhinoceros, which show relatively high levels of heterozygosity and an average genetic divergence of 0.1% compared with the southern subspecies. The two white rhinoceros subspecies appear to be closely related, with low genetic admixture and a divergent time <80,000 yr ago. Inbreeding, as measured by runs of homozygosity, appears slightly higher in the southern than the northern white rhinoceros. This work demonstrates the value of the northern white rhinoceros cryopreserved genetic material as a potential gene pool for saving this subspecies from extinction. Rhinoceroses as a group are particularly affected, with three of the five extant species listed as critically endangered , one listed as vulnerable (greater one-horned rhinoceros), and only one, the white rhinoceros (Ceratotherium simum), listed as near threatened. Despite continuing threats, including poaching and habitat destruction, the southern population of white rhinoceros known as the southern white rhinoceros  is currently the most abundant rhinoceros in the world, with about 20,000 individuals living primarily in South Africa  is listed as critically endangered and is believed to be extinct in the wild, with the recent death of the last male, Sudan, leaving only two remaining individuals in captivity . The average coverage depth of the NWR and SWR genomes sequenced ranged from 10× to 15×.We sequenced the genomes of nine NWR and four SWR individuals Table . All seqSupplemental Fig. S3). These results may reflect historical gene flow between populations but most likely reflect the effect of low level of genetic differentiation between subspecies, homoplasy, or incomplete lineage sorting. Principal component analysis (PCA) also supported the two populations as distinct clusters .To examine population structure and admixture in the white rhinoceros, we used ADMIXTURE  and EIGEclusters C. PC1 .As a measure of genetic variation, we estimated genome-wide heterozygosity calculated as the proportion of heterozygous sites in the genome of each individual. We found that the mean genome-wide heterozygosity in the NWR was slightly higher (0.0011) than that of the SWR samples studied (0.0009). Both subspecies had relatively high levels of genetic variation compared with other threatened species such as the Tasmanian devil or the Bornean orangutan . We alsoTo infer the divergence time and demographic history of the northern and southern subspecies, we used pairwise sequentially Markovian coalescent (PSMC) . Both poSupplemental Table S3). We assumed a mutation rate of 2.5 × 10−8, which has been estimated for humans  in the two white rhinoceros subspecies, with estimates of Froh slightly higher in the southern subspecies overall , which suggests that the differences in autozygosity may be due to recent inbreeding in the SWR. These estimators, Froh and the low mean size of ROH, in addition to the number of unique SNPs per individual , allowed us to identify NWR cell lines particularly valuable for any attempt of genetic rescue. These samples include individuals KB3731, KB9939, KB6571, and KB5764, which have low levels of autozygosity, a low mean size of ROH, and a high number of unique SNPs.To determine inbreeding in white rhinoceroses, we calculated runs of homozygosity (ROH) by scanning 1-Mbp sliding windows throughout the genomes. Our analysis showed similar levels of autozygosity . We identified all coding SNPs that occurred in these regions according to the generated white rhinoceros genome annotation. This resulted in about 240 SNPs in 28 genes potentially identified under selection . Among those, we found 100 SNPs that were fixed differences in either subspecies. None of the SNPs were fixed in both subspecies, suggesting soft selective sweeps. We then identified the gene ontology categories for each gene using Ensembl, finding the olfactory receptor genes as the most common category, with 11 genes involved in sensory perception of smell potentially under selection  in vitro, or combined with maturation in vivo into germline stem/progenitor cells , and cloBos gaurus . Eight NWR samples were derived from cell lines preserved in the San Diego Zoo Frozen Zoo, and one sample corresponds to a zoo-based individual for which no viable frozen cells exist and only DNA were available. Of the NWR samples, seven individuals were from Sudan and two from Uganda (n) chromosome number of 81, a known variant in white rhinoceroses of the most common 2n = 82 karyotype . Base calls were generated using CASAVA 1.8.2 (Illumina), and the resulting demultiplexed sequence reads were filtered for low quality.Nine NWR and four SWR were sequenced by Cofactor Genomics using Illumina paired-end sequencing to a coverage of 10×–15×. Briefly, Genomic DNA libraries were constructed by shearing genomic DNA to the desired size using the Covaris S2 (Covaris). Following shearing, DNA was end-repaired and A-tailed to prepare for adaptor ligation. Indexed adaptors were ligated to sample DNA, and the adaptor-ligated DNA was then size-selected on a 2% SizeSelect E-Gel (Invitrogen) and amplified by PCR. Library quality was assessed by measuring nanomolar concentration and the fragment size in base pairs. Cluster generation and the following sequencing were performed according to the cluster generation manual and sequencing manual from Illumina  using SAMtools mpileup , and varSupplemental Table S5) and were subsequently included in the analysis.In order to identify scaffolds in the rhinoceros genome corresponding to the X Chromosome, we first attempted to BLAST all scaffolds against the horse X Chromosome. However, a large number of scaffolds contained sequences highly similar to the horse X  (PSMC  was usedsite/yr) . In ordesite/yr) .−8 was also used for inferring scaled population parameters. Likelihood and parameter estimates for each model can be found in the Supplemental Materials.We also estimate demographic history using ∂a∂i by testing a series of four increasingly complex models . We seleFroh). The length distribution of ROH was calculated using R  under accession number PRJNA394025.The data generated from this study have been submitted to the NCBI BioProject (


Hapalochlaena maculosa  lacks a planktonic dispersal phase, yet ranges across Australia's southern coastline. This species’ brief and holobenthic life history suggests gene flow might be limited, leaving distant populations prone to strong genetic divergence. This study used 17,523 genome‐wide SNP loci to investigate genetic structuring and local adaptation patterns of H. maculosa among eight sampling sites along its reported range. Within sites, interrelatedness was very high, consistent with the limited dispersal of this taxon. However, inbreeding coefficients were proportionally lower among sites where substructuring was not detected, suggesting H. maculosa might possess a mechanism for inbreeding avoidance. Genetic divergence was extremely high among all sites, with the greatest divergence observed between both ends of the distribution, Fremantle, WA, and Stanley, TAS. Genetic distances closely followed an isolation by geographic distance pattern. Outlier analyses revealed distinct selection signatures at all sites, with the strongest divergence reported between Fremantle and the other Western Australian sites. Phylogenetic reconstructions using the described sister taxon H. fasciata  further supported that the genetic divergence between distal H. maculosa sites in this study was equivalent to that of between established heterospecifics within this genus. However, it is advocated that taxonomic delineations within this species should be made with caution. These data indicate that H. maculosa forms a clinal species pattern across its geographic range, with gene flow present through allele sharing between adjacent populations. Morphological investigations are recommended for a robust resolution of the taxonomic identity and ecotype boundaries of this species.The southern blue‐ringed octopus,  Most maPrevious molecular studies of the Cephalopoda have revealed that genetic structuring of populations generally mirrors life history traits , population connectivity was predominantly influenced by ocean currents  followed an IBD pattern common to cuttlefish and many terrestrial animals  are the most sedentary of the cephalopods  on the subtropical eastern coast, or environments become too warm on the tropical west coast  of local adaptation in driving of the observed genetic divergence between regions. Finally, this study aimed to resolve the phylogenetic relationships among members of the H. maculosa group across their geographic distribution and to provide insight for the taxonomic identity of the species group.This study used genome‐wide single‐nucleotide polymorphism (SNP) markers to explore the microevolutionary processes shaping the genetic structure of the 22.1H. maculosa group were sourced from eight sampling sites across the southwestern and southern coastlines of Australia ; Rockingham, WA ; Mandurah, WA ; Misery Beach, WA ; Emu Point (Albany), WA ; Gulf St. Vincent, SA ; Port Phillip Bay, VIC ; and Stanley, TAS . Specimens from the Fremantle, Mandurah, Emu Point, and Stanley sites were obtained through the bycatch of commercial fishermen. Samples from the Rockingham and Misery Beach sites were obtained through false‐shelter traps comprised of both 200 mm lengths of 20‐mm‐diameter PVC pipes and concrete cavity traps . Animals were sourced under Western Australia DPaW permit: SF00963, Western Australia Fisheries exemption: 2393, and the Department of Environment and Primary Industries fisheries research permit: RP699.A total of 248 samples from the 2.2DNA was extracted from all tissue samples using a modified CTAB/Chloroform—Isoamyl method  to ensure high‐quality data. Additionally, all SNP loci deviating from Hardy–Weinberg equilibrium (HWE) within sample sites were identified using the software package Arlequin  threshold of 0.02) across all six sites with n > 20 were removed from the library. SNPs were only removed if they were below MAF or HWE thresholds in all six of the larger sampling sites because wide divergences were expected between the distal populations in this study. Accordingly, rare SNPs were still retained if they were informative in at least one of the sites. Finally, SNPs associated with X‐ or Y‐linked chromosomes were screened among the 202 individuals with known sex using the full association test in Plink™ , average read depth of 15.994 (±0.059), and an average repeatability of 0.986 (±0.001). PAV markers for phylogenetic analyses were filtered manually to retain the most informative marker set across all individuals and taxa. PAV loci were removed based on a MAF of <2% among sample sites (n > 20) and technical reproducibility of less than 100%. A total of 22,387 PAV loci were retained for phylogenetic analysis across 250 individuals.A total of 33,230 high‐quality unique SNPs (single SNP per sequence tag) and 39,033 unique PAV loci were resolved by DArTseq™. SNPs were filtered for call rate (>70%) and minor allele frequency , mean nonbiased expected heterozygosity (He), and Wright's inbreeding coefficients (Fis) were calculated through Genetix V4.05.2 . These reduced datasets were more likely to omit informative or possible outlier loci, but minimized the likelihood of containing null alleles that could have affected accurate estimations of Fis within individual sites  individual distance was calculated for each individual pair using the “propShared” command in adegenet , and proportion of private alleles (AP) were calculated for each of the six sites (with n > 20) using custom scripts in Microsoft Excel™. To assess the effective population sizes (NeLD) and sibship structure at sampled locations, a subset of 500 loci was selected from the SNP database to be used in these analyses. These 500 loci were selected for having a minimum MAF of 0.05 within all sites and were then filtered for having the highest call rate, repeatability, and read depth among the remaining loci. Filtering SNPs for these analyses helped ensure many of the simplifying assumptions used in the calculations were met  and internal relatedness (IR) were calculated for all individuals using the R package Jombart, . The per2.5F‐statistics . Hierarchical analysis of molecular variance (AMOVA) among individuals and sample sites in different groupings was calculated in Arlequin  pipeline  with 1,000 permutations using Arlequin  in this study.Genetic differences among the six sites with sample sizes >20 were evaluated using Weir and Cockerham's unbiased Jombart, , was conces Nei,  and thei2.6n > 20, following both a frequency‐based approach in Lositan  using the “propShared” command in Jombart, . These pJombart, . Due to 2.7H. fasciata sister taxa were included as an out‐group. The ML analysis was conducted using the software RAxML v8.2 . In addition, the levels of phylogenetic distance among all pairs of individuals were calculated using the F84 evolutionary model for SNPs and the modified restriction method for PAVs (DNAdist and Restdist respective programs) in the Phyllip v3.695 analysis package  using maximum likelihood (ML) and Bayesian methods. For both analyses, data from the 33.1Ho) ranged from 0.076 to 0.166, and mean nonbiased expected heterozygosity (He) ranged from 0.086 to 0.250 (Table p < .001). Wrights inbreeding coefficients (Fis) ranged from 0.043 to 0.182 after rigorous filtering for null alleles  ranged from 43.0 in the Gulf St. Vincent site to 1,794.1 in the Fremantle site  and DAPC analyses revealed that genotyped individuals primarily formed unique clusters based on broad geographic sampling location . The next highest level of variation was observed when the sampled sites were clustered into the four genetic groups: Fremantle; Mandurah and Emu Point; Gulf St. Vincent; Port Phillip Bay and Stanley. This arrangement accounted for the maximum amount of genetic variation among groupings , while 11.66% of variation was among sites within groups (p < .001) and 53.01% of variation among individuals within sites (p < .001). This grouping arrangement is inconsistent with both geographic distribution and the listed distributions of H. maculosa and WBRO  were jointly identified by both statistical methods at an FDR of 0.01 among the Western Australian sites  as between H. fasciata and all sample sites for the H. maculosa group (range: 0.232–0.377) based on the SNP ML tree reconstruction and F84 genetic distances , along with the highest observed IR (0.732) and proportions of half siblings (0.684). These samples were obtained over a 1‐month period from a commercial fishery that only fished over a ~22 km2 area of relatively homogenous benthic habitat. However, the Stanley site also had the lowest Fis score (0.043 after within‐site HWE filtration) and second largest NeLD estimate (468) observed among sample sites in this study. This, in combination with the observation that inbreeding coefficients were significantly heterogeneous at all sites, suggests that although highly related individuals are likely to occur within close proximity as they do in Stanley, genetic evidence infers that inbreeding might be extremely rare. Both Ho and He were highest at the Fremantle and Mandurah sites, where samples were obtained over ~61 and ~220 km2 areas, respectively, and these sites also yielded the two lowest levels of half‐sibling pairs and IR. The highest values for Fis were observed at the Mandurah site. However, due to the low levels of relatedness and large sampling area for this site, the higher Fis observed there was likely a result of Wahlund effect  with comparatively low, uncorrelated, and significantly heterogeneous inbreeding coefficients throughout the sites sampled in this study suggests that this species group might possess a mechanism for inbreeding avoidance. The low dispersal ability of this taxon, which results in the occurrence of closely related individuals within small areas, could leave populations of this species group particularly prone to inbreeding depression . It is unknown why Gulf St. Vincent had a lower NeLD compared to other sample sites, but it is possible the limited sample size and observed sampling of related individuals over a smaller area might impacted this calculation.Where estimated, effective population sizes were highly variable among sample sites Table . The relFst values among sample sites were very high compared to all comparable studies of population divergence in cephalopods , whereas samples from the Mandurah site were obtained from greater depths (17–28 m). It is possible that the deeper habitats around the Mandurah, WA, act as a barrier to dispersal and gene flow between the Fremantle and Mandurah sites, delineating the genetic groups between these two sites. Results from the AMOVA, DAPC, and NETVIEW analyses all indicated that limited gene flow is present between adjacent sample sites, however support the above results in that animals sampled from the Mandurah site share more genetic similarities with the Emu Point site  than individuals in the adjacent Fremantle site . No outlier loci were identified using BayeScan analysis for the eastern sites at low FDR thresholds, which was possibly due to less pronounced local adaptation in this region and also the sensitivity of BayeScan to large differences in background H. maculosa species along its range, most likely due to the limited dispersal capacity associated with this taxon's holobenthic and brief 7‐month life history  might be useful to compare findings in this study to a tropical congeneric possessing a planktonic larval stage .Together, these findings reveal strong divergence among populations of the 5Hapalochlaena genus and the first genetic assessment of a holobenthic cephalopod across its entire range. These findings strongly indicate that H. maculosa and the WBRO form a single clinal species, following a genetic IBD pattern, common to terrestrial animal taxa  will be useful as a comparison of habitats and life histories, in addition to providing phylogenetic context for the genomic divergence observed here between holobenthic members of the Hapalochlaena genus. Further molecular studies, investigating relatedness and sex‐biased dispersal of H. maculosa at a more localized scale, will be useful for additional insights into the behavior of this cryptic taxon.This study provides the first molecular investigation within the  Wright,  and othe Wright, . There wNone declared.All authors contributed to revisions of the text and the final content of the manuscript. P. Morse and K. Zenger contributed to the experimental design. P. Morse and J. Finn collected genetic samples. M. Meekan provided a research vehicle for fieldwork. S. Kjeldsen and P. Morse performed DNA extractions and analyzed data under guidance from K. Zenger. P. Morse and K. Zenger organized funding for this research. P. Morse was the primary author. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Geranium arabicum/kilimandscharicum complex present intricate morphological variation and have recently been suggested to comprise two new endemic taxa. Here we aim to contribute to a clarification of the taxonomy of these populations by analyzing genetic (AFLP) variation in range-wide high-alpine samples, and we address whether hybridization has contributed to taxonomic problems. We identified only two genetic groups. One corresponded to G. kilimandscharicum, which has been reported as exclusively high-alpine and confined to the eastern Rift mountains in East Africa. The other corresponded to G. arabicum, reported from lower altitudes on the same mountains as well as from a wide altitudinal span in Ethiopia and on the western Rift mountains in East Africa. The four populations analyzed of a recently described species from the Bale Mts in Ethiopia were admixed, indicating that they result from recent long-distance dispersal of G. kilimandscharicum from East Africa followed by hybridization with local G. arabicum in naturally disturbed habitats. Some admixture between the two genetic groups was also inferred on other mountains, supporting earlier suggestions of introgression based on morphology. We did not find support for recognition of the recently suggested new subspecies of G. arabicum in Ethiopia. Interestingly, the high-alpine G. kilimandscharicum lacked clear geographic structuring, suggesting a recent history of colonization of the different mountains or extensive intermountain gene flow.The scattered eastern African high mountains harbor a renowned and highly endemic flora, but the taxonomy and phylogeographic history of many plant groups are still insufficiently known. The high-alpine populations of the  The flora of the scattered African high mountains is renowned in biogeography for its peculiar life forms, many endemics, and close relationships to temperate floras in other parts of the world. Although the tropical afro-alpine flora is quite poor in terms of species number, with only 521 species recognized in a recent enumeration .Erica trimera, [Deschampsia cespitosa, [Carex monostachya, [Lobelia gibberoa,[Trifolium cryptopodium, [Erica arborea,[Koeleria capensis, [Carex spp., [Carduus schimperi, [Recent phylogeographic studies have shown that afro-alpine plant species or species complexes can show more or less distinct genetic structuring corresponding to individual mountains or mountain groups, suggesting a long history of isolation after initial colonization. Severagibberoa,; Trifoliopodium, ). In som arborea,; Koeleriapensis, ) and in ex spp., ; Carduushimperi, ).BecauseGeranium arabicum/kilimandscharicum complex. Typical G. arabicumhas pentagonal leaves and flowers in pairs, whereas G. kilimandscharicum has reniform leaves and solitary flowers. However, morphological intermediates do occur and are thought to result from introgression between the two species [Geranium mode of short-distance dispersal by ejecting their mericarps after explosive curling of the awns [G. kilimandscharicum Engl. as exclusively high-alpine and confined to the eastern Rift mountains in East Africa (Mt Kilimanjaro (type), Mt Meru, Mt Kenya, Aberdare Mts, Mt Elgon; [G. arabicum Forssk., is typified from Yemen and reported as widespread in tropical African highlands between 1000 and 4000 m [G. arabicumusually occurs at lower altitudes  and G. kilimandscharicum usually at higher altitudes  localities where the ground has been disturbed by activities of the giant mole rat or by frost heaving. In addition, a tentative new endemic subspecies of G. arabicum was listed in the Flora of Ethiopia and Eritrea, referred to as subspecies ‘Ash 1711’ based on a collection made in the Bale Mts [arabicum(1300–3650 m). Interestingly, [G. kilimandscharicum. A third subspecies, G. arabicum ssp.latistipulatum (A.Rich.) Kokwaro, has been recognized from low altitudes (1000–3150 m) in Erica forests both in East Africa and Ethiopia. The main leaf lobes of ssp. latistipulatum are deeply pinnatisect into narrowly oblong segments separated by wide sinuses. The other two subspecies have less dissected leaf lobes and narrow sinuses, but ssp. ‘Ash 1711’ differs from ssp. arabicum by having more reniform  leaf outline and wedge-shaped (vs rhombic) outline of the main leaf lobes [Recently, a third species of the complex was tentatively described as endemic to the Bale mountains in ium sp.’ . This spBale Mts . This sustingly,  pointed af lobes .Geranium arabicum/kilimandscharicum complex. Here we aim to contribute to a clarification of the taxonomy of the high-alpine populations of this complex by analyzing genetic (AFLP) variation in nearly range-wide samples, and also to address their phylogeographic history. In particular, we ask whether hybridization has contributed to taxonomic problems in this group. For logistic reasons we were not able to cover the lower-altitude subspecies in the widespread G. arabicum, because our sampling was carried out as part of a larger project restricted to high-alpine areas.Further studies are clearly needed to clarify the variation in the eastern African populations of the G. kilimandscharicum, 10 to G. arabicumssp.arabicum, six to G. arabicum ssp. ‘Ash 1711’, and seven to Geranium sp.sensu [G. arabicum ssp. latistipulatum, which is recorded only from a few subalpine areas in East Africa and in Ethiopia , outside the recorded range of G. kilimandscharicum. In addition, we sampled one population referred to G. arabicum ssp. arabicum from the montane forest zone in Mt Kilimanjaro  or DNeasyTM Plant Mini Kit . Leaf tissue was ground in 2.0 μL tubes with two tungsten carbide beads for 2 min at 15 Hz in a mixer mill , after which 250 μL of lysis buffer was added, vortexed, spun briefly, incubated on a heat block for 10 min at 65°C, and centrifuged at 14000 Hz for 2 min. 200 μL of the lysate was transferred to new tubes and loaded to the robot, which was set to produce a final elution volume of 100 μL.EcoRI-ATG/MseI-CGA, VIC-EcoRI-ACA/MseI-CAC, and NED-EcoRI-AGC/MseI-CTG. For each sample, 2.0 μL 6-FAM, 2.0μL VIC and 3.0μL NED labeled selective PCR products were mixed and added to a master mix of 11.7 μL formamide and 0.3μL GENESCAN ROX 500 internal-lane size standard, denatured at 95°C for 5 min and cooled on ice before run on an ABI3100 sequencer .About 10% of the samples were extracted twice to test for reproducibility of the markersfollowing . AFLP da® version 4.0 .Peaks of low intensity were only scored when unambiguous. Error rate calculation and data cleaning [D; estimated as the average proportion of pairwise differences among genotypes; [P%), and genetic distinctiveness or rarity were scored as present (1) or absent (0) using GeneMappercleaning  were donnotypes; ), propormarkers; ) were camarkers; . Pairwismarkers;  and visuhttp://www.lifeportal.uio.no). Analyses were run with K ranging from 1–10, and for each K, 10 replicate runs with a burn-in period of 200,000 and 1,000,000 iterations were used. We used the R-script STRUCTURE-SUM to summarize the results. Log probability of the data, L(K), as a function of K ranging from 1to 10 and the rate of change in the probability between successive runs, ΔK, were calculated according to [K was estimated according to[K, and DISTRUCT [Bayesian clustering was carried out in STRUCTURE v 2.3.3 . We usedrding to . In addiording to. We usedDISTRUCT  to graphp-distance using SplitsTree4 v. 4.12.6 [FST) pairwise distances for the total dataset and for each of the two genetic groups separately, using Mantel test implemented in GeneAlex 6.5[A Neighbor-Net diagram  was cons. 4.12.6 , and sup. 4.12.6 . Analyse. 4.12.6 . SignifieAlex 6.5, using 1K), showed highest increment from K = 1 to K = 2 , as well as some of the plants from the Ethiopian Bale Mts referred to Geranium sp. The G. arabicum group comprised all populations referred to the widespread G. arabicum , as well as the remaining Bale Mts plants referred to Geranium sp. The plants from the four populations  referred to Geranium sp. were accordingly divided between the two genetic groups, with one complete population (ET0677) and four individual plants from the three other populations placed in the G. arabicumgroup, and with the remaining plants placed in the G. kilimandscharicum group  were polymorphic, were kept after cleaning the data. Reproducibility of the markers was 98.1%. In the STRUCTURE analyses, the change in the log probability of the data, L and higher rarity (DW = 2.68) than the G. kilimandscharicumgenetic group . Very little diversity was recorded in the Mt Kilimanjaro and Mt Kenya populations of G. Kilimandscharicum. We found highly significant (P< 0.001) genetic differentiation among the ten mountains, with the strongest divergence between the Simen Mts and Mt Elgon .The Mt Elgon . We alsoMt Elgon  and withGeranium arabicum/kilimandscharicum complex in the eastern African mountains present quite simple genetic structuring, in spite of their intricate morphological variation which recently led to the description of two new endemic taxa. We identified only two genetic groups, of which one is widespread (G. arabicum), and the other seem to have evolved as a specialized high-alpine ecotype in one group of mountains but not elsewhere (G. kilimandscharicum in eastern East Africa). It appears that the morphological complexity and taxonomic problems in the group have been caused by a combination of four processes: by differentiation into one low-alpine and one high-alpine ecotype in eastern East Africa, by local hybridization between these ecotypes, by at least one episode of long-distance dispersal of the high-alpine ecotype followed by hybridization and establishment of admixed plants in naturally disturbed habitats, and by phenotypic plasticity or parallel evolution in some leaf characters. In the following, we discuss the available evidence for the influence of each of these processes.Using genome-wide genetic markers and range-wide sampling, we have shown that the high-alpine populations of the G. arabicum based on both morphology and AFLP data  and the Ethiopian Simen Mts . Thec groups . Long-diimen Mts .G. arabicum, ssp. ‘Ash 1711’, grouped with ssp. arabicum according to their geographic origin rather than morphology  and in many arctic-alpine plant species [G. arabicum , as a function of K ranging from 1 to 10. b) Mean DeltaK for the rate of change in the probability between successive runs, DeltaK as a function of K, calculated according to [a) Log likelihood of the data,L(rding to . c) Aver(DOCX)Click here for additional data file.S2 FigG. kilimandscharicum and c) G. arabicum genetic groups.a) for the total dataset, b) (DOCX)Click here for additional data file.


DNA barcoding has demonstrated that many discrete phenotypes are in fact genetically distinct (pseudo)cryptic species. Genetically identical, isogenic individuals, however, can also express similarly different phenotypes in response to a trigger condition, e.g. in the environment. This alternative explanation to cryptic speciation often remains untested because it requires considerable effort to reject the hypothesis that the observed underlying genetic homogeneity of the different phenotypes may be trivially caused by too slowly evolving molecular markers.Munida gregaria comprises two discrete ecotypes, gregaria s. str. and subrugosa, which were long regarded as different species due to marked differences in morphological, ecological and behavioral traits. We studied the morphometry and genetics of M. gregaria s. l. and tested (1) whether the phenotypic differences remain stable after continental-scale sampling and inclusion of different life stages, (2) and whether each phenotype is underpinned by a specific genotype.The widespread squat lobster gregaria s. str. and subrugosa individuals from 25 stations encompassing almost entire range in South America were included in morphological and genetic analyses using nine unlinked hypervariable microsatellites and new COI sequences. Results from the PCA and using discriminant functions demonstrated that the morphology of the two forms remains discrete. The mitochondrial data showed a shallow, star-like haplotype network and complete overlap of genetic distances within and among ecotypes. Coalescent-based species delimitation methods, PTP and GMYC, coherently suggested that haplotypes of both ecotypes forms a single species. Although all microsatellite markers possess sufficient genetic variation, AMOVA, PCoA and Bayesian clustering approaches revealed no genetic clusters corresponding to ecotypes or geographic units across the entire South-American distribution. No evidence of isolation-by-distance could be detected for this species in South America.A total number of 219 gregaria s. str. and subrugosa ecotypes form a single, dimorphic species M. gregaria s. l.. Based on adequate geographic coverage and multiple independent polymorphic loci, there is no indication that each phenotype may have a unique genetic basis, leaving phenotypic plasticity or localized genomic islands of speciation as possible explanations.Despite their pronounced bimodal morphologies and different lifestyles, the The online version of this article (doi:10.1186/s12862-016-0836-4) contains supplementary material, which is available to authorized users. Different species have different morphologies and lifestyles, which is commonly taken (but not often tested) to reflect different underlying genotypes. The advent of affordable DNA sequencing and molecular barcoding have served to greatly intensify the crosstalk between molecular and taxonomic disciplines by uncovering a large number of previously overlooked genotypes –3, many However, the popularity and large number of cryptic species currently being discovered have led to an under-appreciation of the notion that sharply distinct morphotypes are not always the consequence of genetic differences but can also be invoked from the same genotype, often called by environmental triggers. The differences between associated morphotypes and lifestyles of ecotypes within the same species can be surprisingly pronounced –11.a posteriori knowledge is rare  is not a conclusive demonstration of the absence of genetic differentiation among ecotypes. In order to show that too slowly evolving markers or other artefacts (e.g. mito-nuclear discordance ) did notia pulex –15), butc aphids , 17, marc aphids , polyembc aphids , inbred osophila , cloned osophila . It is uMunida gregaria sensu lato , which is currently considered to comprise the ecotypes M. gregaria sensu stricto Miers 1881 as well as its junior synonym M. subrugosa Dana, 1852 . For clasmania , wider rostrum basis (RBW) and broader and blunter dactylus of the third maxilliped (DaW)  Fig. . These tDNA was extracted from ethanol-preserved abdominal or cheliped muscle tissue using QIAamp DNA Mini Kit . For mtDNA analysis, a region of the COI gene was amplified using the universal primers HCO2198 and LCO1490  for 96 iD) and nucleotide diversity (π) for each ecotype and all samples using DnaSP 5.10 [M. rutllanti , M. quadrispina , and M. gracilis from our own collection . Pairwise genetic distances were calculated within M. gregaria s. l. (pooled gregaria s. str. and subrugosa) and versus the other three Munida species.DNA polymorphism was examined as haplotype diversity , M. quadrispina , M. rosula  and M. congesta , were added. The substitution model that best fits the data was determined using jModelTest 2.1.5 [The COI dataset was analyzed using coalescent based approaches Poisson tree processes (PTP) model  and the st 2.1.5 , 41.http://species.h-its.org/ptp/). We ran the PTP analysis for 500,000 MCMC generations, with a thinning value of 100 a burn-in of 10%. Outgroup taxa were kept since the MCMC chains did not converge when they were removed.PTP does not require an ultrametric tree, as the transition point between intra- and inter-specific branching rates is identified using directly the number of nucleotide substitution . A maximwww.cran.r-project.org), using the single threshold approach [The GMYC method requires a fully resolved tree with branch lengths estimates, which was obtained using the program BEAST 2.4.3 . We usedapproach , 39.M. gregaria s. l. [Ar) in FSTAT 2.9.3.2 [FST as a function of expected heterozygosity under an island model of migration. The statistical power of this set of microsatellite loci to detect significant genetic differentiation between populations/ecotypes was tested with POWSIM 4.1 [χ2) and Fisher’s exact test analysis. Various levels of differentiation (measured as FST in the range from 0.001 to 0.01) were determined by combining different effective population size (Ne) and times since divergence (t). In addition, POWSIM allows calculating type I error probability, which is the probability of rejecting the null hypothesis of genetic homogeneity although it was true by drawing the alleles directly from the base population (t = 0).In total, 218 individuals were screened for genetic variation at 11 microsatellite loci that were originally designed for ia s. l.   was performed in GENALEX 6.5 [The genetic differentiation among the three major sampling areas, i.e., FM, NCP and TdF . Both conditions were run with the correlated allele frequencies option under the non-admixture model, i.e. under the assumption that there is no gene flow between ecotypes, as well as under an admixture model, i.e. allowing limited introgression between clusters. Twenty runs with 200,000 Markov chain Monte Carlo (MCMC) iterations after a burn-in period of 25,000 steps were carried out for each K. The results were uploaded onto STRUCTURE HARVESTER [K was determined using the ad hoc statistic ΔK [L(K) [K were averaged using the software CLUMPP 1.1.2 [Bayesian assignment tests were used to evaluate the level of genetic clustering. We used STRUCTURE 2.3.4  first wiARVESTER  and K waistic ΔK , as wellΔK [L(K) . ResultsPP 1.1.2  and the PP 1.1.2 .L(K) does not always provide the correct number of clusters and the ΔK statistic cannot evaluate K = 1 or the largest value chosen for K [Since en for K , we alsoen for K , which cFST values for mitochondrial data and pairwise (δμ)2 genetic distance [http://biodiversityinformatics.amnh.org/open_source/gdmg/index.php). Geographical distances were log-transformed to account for two-dimensional habitat distribution [The impact of isolation by distance (IBD) across southern South America on genetic differentiation was estimated by Mantel tests as implemented in IBD Web Service 3.23 . For thidistance  for micrribution , and thegregaria s. str. formed two isolated groups, both of which were clearly distinct from the subrugosa samples. The subrugosa individuals comprise the adult and juvenile sub-groups that overlap in part  it is hard to ascertain whether juvenile and adult subrugosa show statistically significant differences . These sequences were collapsed into 30 different haplotypes, possessing 29 variable (segregating) sites, of which 11 were parsimony-informative. The subrugosa ecotype showed slightly higher genetic diversity than gregaria s. str.  was shared by both ecotypes as well as by all the sampling regions, which differed from the other haplotypes in 1 to 3 mutational steps  compared to gregaria s. str. (0.111 ± 0.065), both of which were very low, reflecting a high degree of allelic sharing between the two ecotypes. Compared to the limited variation among COI sequences, the nine microsatellite loci exhibited broader allelic ranges and orders of magnitude higher allelic variation  in probabilities of close to 100% using both chi-square (χ2) and Fisher’s exact test  and 38 (Mgr60) with an average of 14.9. For each locus the number of alleles, allelic size range, allelic richness as well as observed and expected heterozygosities per population and ecotype are reported ), but K = 2 was indicated by the highest statistic ΔK. This is because the change in log probability does not account for the smallest and largest K. Even under K = 2, each individual possessed a roughly equal probability of being assigned to the first versus the second cluster, which indicates that all individuals belong to one single group .Bayesian cluster analyses with STRUCTURE suggested the best oup Fig. . CongrueThe STRUCTURAMA analysis corroborated the inferences of the STRUCTURE analysis. The sampled individuals all belonged to one group with a posterior probability of 1. Eventually, both Bayesian analyses showed no correlation of our microsatellite data with either ecotypes or geographical units.r = 0.0608, P = 0.229; for microsatellites, r = −0.005, P = 0.508) between genetic and log-transformed geographic distances  is nonetheless not blurred across the South American shelf  juveniles in osa Fig. . The disSince the proposal that phenotype and genotype form two fundamental different levels of biological abstractions , untanglMunida gregaria case. Nonetheless, the lack of differentiation at a single marker is insufficient to extrapolate to the entire genome, especially in view of the different inheritance in the mitochondrial and nuclear genomes [In other cases, however, molecular marker-based examination found no genetic differentiation matching discrete phenotypes, which is exemplified by the present  genomes , 75. A p genomes  but fail genomes  due to c genomes  thus res genomes , 76–79.  genomes –82.M. gregaria s. l. are not caused by different genotypes.However, the shortcomings of previous analyses  were addA common caveat to marker-based population genetic studies in case of no differentiation detected among populations  is that there may be still unsampled isolated regions of differentiation within genome. Such ‘genomic islands of differentiation’ , 84 are M. gregaria s. l. are then strongly suggestive of phenotypic plasticity. The exact nature of a trigger that determines which of the morphotypes will be expressed is unknown at present. In similar examples from parthenogenetic Daphnia and aphids, sharply distinct morphotypes arise from the same genetic background [Daphnia water fleas [Chthamalus fissus [Except for the possibility of ‘genomic islands’ underpinning different phenotypes, the different ecotypes within ckground , 17, 91 ckground , 93, fisckground , 95, turckground  and crocckground . Dramatier fleas –100, bars fissus , 14, 101Munida ecotypes. In its South American distribution, Munida gregaria s. l. occupies extensive latitudinal distribution along both coasts of Patagonia and wide bathymetry , wubrugosa , 105. Siectively , changesime , might  monodon . A clearMagellan .Munida gregaria case.Whether or not the two ecotypes spring from a genetically entirely homogenous background or whether small localized genomic islands associated with each exist, our data have made it abundantly clear that the simple, perhaps too simple, model of a genome-wide 1:1 relationship between the genotype and an associated phenotype (however ill equipped we may be to recognize the latter) does not apply to the A next-generation sequencing approach  with higgregaria s. str. and subrugosa ecotypes is not an artefact due to insufficient genomic and/or geographic sampling or slowly evolving markers. Instead they are likely expressed from a single underlying genotype although two largely identical genotypes with interspersed localized genomic islands of differentiation cannot be fully ruled out without a more complete coverage of the genome. Morphological tests affirmed the boundaries between the two ecotypes were not blurred with continental-scale geographic sampling, and remain stable despite an ontogenetic dimension in the data. These findings corroborate the current taxonomic view of M. gregaria s. l.  as a single, dimorphic species, thus demonstrating a pattern very unlike cryptic speciation commonly found in DNA taxonomy and DNA barcoding studies. Our study also emphasizes the necessity of incorporating complementary nuclear multi-locus markers in studies aiming at taxonomy and genotype-phenotype relationship, in view of the increasing numbers of reported discordance between mtDNA and nuclear DNA. M. gregaria is developing into a model affording deeper insights into the phenotype-genotype relationship, environmental control of ontogeny and ultimately into the process of speciation itself.Based on extensive sampling of the species’ distribution in South America and using nine independent polymorphic nuclear microsatellite loci in addition to new mitochondrial COI sequences, we were able to show that the lack of genetic differentiation between distinct


Fragaria vesca, holds great promise as a model organism. It not only represents the important Rosaceae family that includes economically important species such as apples, pears, peaches and roses, but it also complements the well-known model organism Arabidopsis thaliana in key areas such as perennial life cycle and the development of fleshy fruit. Analysis of wild populations of A. thaliana has shed light on several important developmental pathways controlling, for example, flowering time and plant growth, suggesting that a similar approach using F. vesca might add to our understanding on the development of rosaceous species and perennials in general. As a first step, 298 F. vesca plants were analyzed using microsatellite markers with the primary aim of analyzing population structure and distribution of genetic diversity. Of the 68 markers tested, 56 were polymorphic, with an average of 4.46 alleles per locus. Our analysis partly confirms previous classification of F. vesca subspecies in North America and suggests two groups within the subsp. bracteata. In addition, F. vesca subsp. vesca forms a single global population with evidence that the Icelandic group is a separate cluster from the main Eurasian population.The woodland strawberry,  Fragaria are area-specific or continentally endemic, apart from F. chiloensis and the woodland strawberry, Fragaria vesca L. (2n = 2x = 14). F. vesca has a vast natural distribution throughout the Holarctic [F. vesca is widespread in Iceland [F. vesca in North America: F. vesca subsp. bracteata, F. vesca subsp. vesca, F. vesca subsp. californica, and F. vesca subsp. americana[F. vesca subsp. bracteata might be split into two groups based on plastome sequences, which correspond with geography [F. vesca species and subspecies is shown in All species of olarctic  and the olarctic . On the  Iceland –9, where Iceland  and has  Iceland . Althoug Iceland . Howevereography . The proFragaria genus [F. cascadensis [F. × bifera, a hybrid of F. vesca and F. viridis found in Europe [F. × bringhurstii [F. × ananassa subsp. cuneifolia. The Fragaria genus is one of ninety genera in the Rosaceae [F. × ananassa), apple , pears (Pyrus spp.), peach (Prunus persica), and roses (Rosa spp.) [Twenty-two wild species are recognized in the ia genus –4, inclucadensis . In addin Europe , F. × brghurstii , and F. Rosaceae , a familsa spp.) , which tsa spp.) .F. vesca has repeatedly been proposed as a research model for the Rosaceae [F. vesca is a diploid perennial species with a small, fully sequenced genome , an eff  [Solanum tuberosum L.) [Phaseolus vulgaris L.) [Malus domestica), a perennial crop plant, has not undergone any significant loss in diversity during the last 800 years [Reduced diversity in crop plants compared to wild relatives is well recognized in cotton (utum L.) , the potosum L.) , and thearis L.) . This do00 years . Moreove00 years , 45. Als00 years , and dom00 years . To effeF. virginiana subsp. glauca into F. × ananassa [F. moschata and F. chiloensis genomes in their pedigree [F. vesca and F. × ananassa–with backcrossing and phenotyping taking years or decades in some plant species. However, methods such as marker assisted selection (MAS) or more recent genomic selection [Due to the loss of genetic diversity in crop species, their wild relatives have long been suggested as a potentially valuable source of novel traits . This haananassa , 50 and pedigree . Howeverelection , and novelection , promiseF. vesca is known to possess traits of interest for resistance to both abiotic and biotic stress [Fragaria species have been introgressed into cultivars in strawberry breeding programs [F. vesca with the aim of furthering its use in genetic and genomic research and to shed light on the origin of the Icelandic F. vesca population we undertook a population genomic analysis of 295 F. vesca samples originating from Eurasia and America, using 56 SSR markers.c stress , 4 as wec stress . Novel tprograms , 55. Warprograms  proposedF. vesca. Despite our best efforts we were not able to fully cover the current global distribution of introduced F. vesca, with samples missing from areas such as Hawaii, New Zealand, Australia, southern Africa, Madagascar, the Canary Islands, and the Cape Verde Islands, as well as several South American countries. Additionally, 26 cultivars were included in the study, giving a total number of 298 plants. In total, 232 Eurasian plants were analyzed (not including cultivars and outgroups), including 54 from Iceland, 37 accessions originating from North America, one from South America, and two from Japan. Also, two species other than F. vesca were included as outgroups: F. chinensis from China and F. viridis from Sweden . All sampling was done in accordance with regional laws and regulations governing the collection of plant material for research purposes. Accession numbers for material received from GRIN are listed in Plants or berries were collected from a total of 274 locations in 31 countries and 16 states (US) around the world  with the® . The DNeasy 96 Plant Kit protocol was modified for use with the available equipment. a Universal 320 centrifuge (Hettich GmbH & Co.) with the maximum of 4000 RPM in a Hettich 1460 rotor. The amount of DNA extracted with the DNeasy 96 Plant Kit protocol was measured using NanoDrop 1000 (Thermo Scientific).Genomic DNA was extracted from homogenized young leaf tissue using the DNeasy 96 Plant Kit or DNeasy Plant Mini Kit from QIAGENwww.biometra.de) with a 5 μl reaction volume containing 0.6 ng of genomic DNA in 1X PCR buffer , 3 mM MgCl2, 0.08U of BIOTAQ DNA polymerase (Bioline), 0.8 mM dNTPs, and 0.4 μM of each primer. A modified touchdown PCR protocol was followed, as described by Sato et al. [http://softgenetics.com/).Samples of 300 individuals  were anao et al. . The PCRGenAlEx 6.501 [a) per locus, and both observed (Ho), and expected (He) heterozygosity. For a population-wide analysis GenAlEx 6.501 was used to calculate the average number of alleles per population (Nap), number of effective alleles (Ne), number of private alleles (NPA), observed (Ho) and expected (He) heterozygosity, and the fixation index (F = 1-(Ho/He)). GenAlex was also used for principal coordinate analysis (PCoA) and pairwise population Fst values (FST = 1- (average He/HT)). Presence of null alleles was tested using Freena [PowerMarker 3.25 [PowerMarker was also used to construct an evolutionary distance matrix based on Nei et al.’s DA distance method [SplitsTree 4 [Mega version 5 [Descriptive statistics were calculated using Ex 6.501 , 62 for g Freena . Additioker 3.25 , includie method . A phylotsTree 4 . Mega veersion 5  was usedersion 5  with booStructure 2.3.4 [Structure runs were repeated 5 times for each K from 1–20 for the whole dataset and for each K from 1–10 for the ‘American’ and ‘Eurasian’ data-sets. The MCMC method was run with a burn-in period of 50,000 and 10,000 repetitions. Other settings were by default. Structure Harvester [K) for each dataset, where the average likelihood values K (L(K)) for each run were used to find ΔK, i.e., the rate of change in lnPr(X|K), since the maximum value of L(K) can give an overestimate of clusters [To identify the number of populations and admixtures, the dataset was analyzed using the admixture model of re 2.3.4 –72 and tarvester  was usedclusters .FVES2533 and FVES0634. As this is not consistent with the diploid nature of F. vesca, these markers were excluded from further analysis. Descriptive statistics for each of the remaining 56 markers used are listed in N) was 289.9. The 56 polymorphic markers had numbers of alleles ranging from 2–16 for all samples, with a total of thirteen bi-allelic markers. A total of 250 alleles was observed for the 56 markers, giving a mean number of 4.46 alleles per locus. The observed heterozygosity (Ho) ranged from zero for thirteen markers to 0.938 for marker FVES3440 with a mean Ho of 0.075. The expected heterozygosity (He) ranged from 0.003 for marker FAES0293 to 0.797 for marker FVES0109, with a mean of 0.170. The polymorphic information content (PIC) ranged from 0.003 for marker FAES0293 to 0.78 for marker FVES0109, with a mean of 0.151. The major allele frequency (MAF) ranged from 0.393 for marker FVES0109 to 0.998 for marker FAES0293, with an average of 0.885.Of the 68 markers amplified, 10 were monomorphic and therefore uninformative. In addition, more than two alleles were repeatedly observed per sample for markers F. vesca subsp. americana (ID 28) and two Eurasian F. vesca subsp. vesca (ID numbers 145 and 146), were excluded from the analysis due to a higher than expected number of alleles per marker. In these samples, the average number of alleles per locus was 2.4, 2.4 and 2.5, respectively, indicating that they might be polyploids or the results of mixed samples. In addition two Icelandic samples were omitted from analysis due to a labelling mistake.Of the 298 accessions in the collection, three samples, one F. vesca subsp. vesca showed the highest values for Ne = 1.26, Ho = 0.11 and He = 0.15. The highest frequency of private alleles, NPA = 0.30, was found in the F. vesca subsp. bracteata ‘Rocky Mts’ group, with the ‘Pacific Coast’ group tied at 0.26 with the Eurasian group.Descriptive statistics for each proposed population are listed in Structure admixture results for the whole dataset, including all wild individuals and cultivars (n = 295), suggest that the collection should be split into two sub-populations, with K = 2 (ΔK = 127.56) , one containing all wild samples and the other containing all cultivars, which is in line with the results of the PCoA   . This an 127.56) , while to groups . Anothero groups . Structuthe PCoA . To furt= 28.13) , separat= 28.13) , which eividuals .Structure analysis of the American samples (n = 37) suggests that five clusters (K = 5) is the appropriate number (ΔK = 16.61)   for the = 16.61) , with thl origin . The PCol origin  explaineDA distance was calculated for the groups presented here . The diversity observed for each of the markers was relatively low, with a mean number of alleles of only 4.5. These values were much lower than seen in some recent studies on Rosaceae species such as the mean number of alleles of 18.7 observed in the almond (Prunus dulcis (Mill.)) [F. × ananassa [F. vesca, where 4.9 was the mean number of alleles from 21 microsatellites in fifteen F. vesca samples [F. vesca could also lead to an overestimate of null allele frequencies.In the study presented here a total of 68 EST-SSR markers, of which were 56 polymorphic, were used to analyze a global collection of 295 (Mill.))  and 10.8ananassa , yet sim samples . The que samples  might suHo) found here was 0.075 for the whole collection, considerably lower than the average expected heterozygosity (He) of 0.170. These values are then much lower than values seen, for example, in Prunus sibirica, a highly outcrossing species, with values of Ho = 0.639 and He = 0.774 [Ho and He might be the existence of subpopulations within the global collection, that is the North American subspecies, confirmed here through various means, such as Structure analyses. When values for Ho and He were compared for individual subpopulations suggested here the observed heterozygosity was always lower than the expected heterozygosity, except for the Japanese samples where they were equal  is surprising considering the gynodioecy within the group [F. vesca is a self-compatible species and low levels of observed heterozygosity have previously been reported [e seen here, especially in some groups, e.g. the Icelandic samples, could be the result of a recent bottleneck since expansion leads to a reduced diversity [The mean observed heterozygosity  groups  and American (without American ) groups  with gen94–0.354 . In addi Njuguna  where suenotypes , but difenotypes , 79. No analyses  and the analyses , and in s region , althougp. vesca . These structure . Some ofIt is also important to mention that to maximize the accuracy of genetic distance calculations, the number of samples need to be 100 or more, although this also depends on the polymorphism of the markers used . In manyStructure can detect the most likely number of clusters even if the samples contain low genetic variation [Structure revealed two clusters, suggesting two genetic populations among the genotypes collected in Eurasia  in Iceland has been reported [F. viridis or of F. × bifera in this study.It has been demonstrated that the admixture model implemented by ariation . In the  Eurasia , with th Eurasia , 3 and 4 Eurasia . A phylo Eurasia . One pos50 years . The posreported  but has F. vesca plants and compared them with cultivars of the same species. Through our initial analysis of biogeography and genetic diversity within this worldwide collection we have confirmed the previous classification of F. vesca into subspecies using molecular markers, and we have shown that the cultivars chosen are homogeneous and group together with the Eurasian samples. Our data also divide European subsp. vesca into two groups, one consisting of an Icelandic group and some accessions from southern and central Europe, and another consisting of the rest of the Europe, although not without overlap between groups. The clear divergence between the Icelandic group and the Fennoscandian does not correlate with results for other floral species in Iceland which are related to Nordic groups [F. vesca.The use of populations of crop wild relatives as research models has been suggested as an approach to disseminating genetic pathways of importance to adaptation . For succ groups . We findS1 Fig(A) Whole data set, including cultivars. (B) Eurasian samples without cultivars. (C) American samples only.(TIF)Click here for additional data file.S2 FigF. chinensis and F. viridis. Information about bootstrap values above 50 that are not at the end of branches.The tree is rooted to the two other species used in the study, (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableIncluding marker name, sequence of forward and reverse primers, GenBank accession number (Acc. no.), pattern of repeats (Repeat), chromosome location (Chr.), the linkage position (Linkage), and species name.(DOCX)Click here for additional data file.S3 TableNei’s genetic distance  and pairwise population Fst values  between groups.(DOCX)Click here for additional data file.


Corallium rubrum. In the present study, the spatial and temporal variability of red coral settlers in an underwater cave was investigated by demographic and genetic approaches.Larval settlement and intra-specific interactions during the recruitment phase are crucial in determining the distribution and density of sessile marine populations. Marine caves are confined and stable habitats. As such, they provide a natural laboratory to study the settlement and recruitment processes in sessile invertebrates, including the valuable Mediterranean red coral Sixteen PVC tiles were positioned on the walls and ceiling of the Colombara Cave, Ligurian Sea, and recovered after twenty months. A total of 372 individuals of red coral belonging to two different reproductive events were recorded. Basal diameter, height, and number of polyps were measured, and seven microsatellites loci were used to evaluate the genetic relationships among individuals and the genetic structure.Significant differences in the colonization rate were observed both between the two temporal cohorts and between ceiling and walls. No genetic structuring was observed between cohorts. Overall, high levels of relatedness among individuals were found.C. rubrumindividuals on tiles are highly related at very small spatial scales, suggesting that nearby recruits are likely to be sibs. Self-recruitment and the synchronous settlement of clouds of larvae could be possible explanations for the observed pattern.The results show that  Recovery and resilience of sessile benthic organisms mostly depend on their early life history stages such as dispersal, settlement and recruitment . It has Early life stages  in marine invertebrates such as sponges, ascidians and cnidarians, have been investigated using different tools such as laboratory experiments on larval behaviour , on settUnderwater caves sensu  represenCorallium rubrum L. 1758) is one of the abundant species inhabiting Mediterranean caves due to its preference for dim-light conditions and downwards-facing surfaces . By means of microsatellite loci, the relatedness and the fine spatial genetic structure within and between the two cohorts have been analysed in order to provide additional information on early life characteristics and population dynamics of this species. Understanding red coral recruitment processes is of key importance to unveil the drivers of the population dynamics of the species and hence the potential recovery of the overexploited/threatened populations.The study was conducted inside the Colombara (or Marcante) cave  located on the east coast of the Gulf of San Fruttuoso . The cave, 10 m long, stands from 34 to 39 m depth on a rocky cliff southward oriented. The cave walls host a rich assemblage of sessile invertebrates typical of the sublittoral caves of the northwest Mediterranean Sea  with manField experiments were approved by the Marine Protected Area of Portofino and by the Università Politecnica delle Marche (Authorization n. 3/2011 (n. prot. 449/2-1-5.) and authorization no 4/2012 (Protoc. No 409/2-1-1)).In June 2010, about three weeks before the start of red coral spawning , sixteen2 , the tiles were removed   where the red coral population has the same range of density as that of the cave (425 ± 100 colonies × m2) . Plots wThe size structure of red coral individuals was obtained by analysing the frequency distributions of the basal diameter (for both recruits and juveniles) and of colony height (only for juveniles). The correlations between the parameters  were analysed by Pearson’s coefficient. Moreover, we tested the temporal and spatial variations of the abundance of individuals by two separate one-way ANOVAs based on 9,999 permutations with Cohort  and Position  as factors using PRIMER v6 software program .C. rubrum individuals found on the tiles deployed on the walls, the molecular analysis was carried out only on the individuals occurring on the tiles deployed on the ceiling of the cave.Due to the small number of Total genomic DNA was extracted from each individual (1 to 4 polyps per colony) following the CTAB protocol and purified by standard chloroform procedure. Seven microsatellite loci COR9, COR46, L7, COR58, MIC20, MIC24, MIC26  were ampPID) that two sampled individuals shared identical MLG by chance (a), observed (Ho), and unbiased expected (He) heterozygosities  between recruits and juveniles were tested using a Student’s t-test. For all the analyses, when necessary, a correction for multiple tests was applied with a false discovery rate of 0.05 . The second part of this Bayesian analysis utilized the probabilities that the individuals excluded from their sampling tile originated from one of the other tiles. Thus, individuals that were excluded from their sampling tile when P ≤ 0.05, were assigned to another tile when P ≥ 0.1.An exclusion test, performed in GENECLASS 2.0 , was useNe) for each cohort was inferred using the standard linkage disequilibrium method with The value of effective population size -based exact test , and the act test  was estiK = 1 to K = 10 were inferred by running structure ten times with 1,000,000 repetitions each  under the admixture ancestry model and the assumption of correlated allele frequencies among samples. Due to the presence of null alleles, the clustering analysis was conducted on the original data set, using the option of null alleles coded as recessive alleles described in http://taylor0.biology.ucla.edu/struct_harvest/). Results of all runs were averaged in CLUMPAK server . In fact, of the 372 individuals, 350 were found on the ceiling tiles, and only 22 on the tiles deployed on the walls, corresponding to a significantly higher density on the ceiling than on walls   represented the dominant class . Only thr = 0.4) .r = 0.09, r = 0.03, r = 0.3, The size structure of juveniles showed a more variable trend, with diameter values ranging from 0.15 mm to 8.95 mm with an average value of 2.5 ± 1.5 mm . The numP > 0.05 after FDR correction). No evidence of scoring errors due to stuttering or large allele dropout was found, according to MICROCHECKER. An excess of homozygotes was detected at all loci due to the presence of null alleles. Null allele frequencies ranged from 0.09 (Mic24) to 0.25 (Mic26 and Cor58). Number of alleles ranged from 10 (Mic20) to 23 (Cor9 and Mic26). The expected and observed heterozygosity ranged from 0.29 (Cor46) to 0.81(Cor9) and from 0.13 (Mic26) to 0.64 (Mic20), respectively. The estimators of FIS (f) were positive and ranged from 0.21 (Mic20) to 0.86 (Cor46)  were not included in the dataset. A total of 290 red coral individuals were genotyped. No genotypic disequilibrium was observed between loci  .e = 0.59 ± 0.16 and Ho = 0.29 ± 0.20; mean ± SD) was found. Out of the 290 individuals analysed, 281 different multilocus genotypes (MLGs) were identified. Four MLGs were found twice; one MLG was encountered three times and one four times. Individuals sharing the same MLG always came from the same tile. Tiles where identical MLGs were found were T7, T8 and T10. The individuals sharing the same MLG were between 0.2 cm and 13.65 cm apart . In T8 and T10, one recruit and one juvenile shared a MLG. The unbiased probability of identity (PID) that two sampled individuals share identical MLG by chance was 1.5e−04.Overall, a low genetic variability ; while recruits showed a significantly higher allelic richness than juveniles (PAr = 0.001).Juveniles and recruits did not show significant differences in terms of Ht =1,173, P = 0.001). Pairwise relatedness based on the A high degree of genetic relatedness among individuals was found. The mean observed relatedness was significantly different from the expected relatedness under panmixia  was estimated as 68.7 (95% CI [42.5–116.4]) including all the individuals and as 32.5 for recruits (95% CI [20.6–57.8]) and −1,467.8 for juveniles (95% CI [87.1–∞]). The last negative value is expected when the population is sufficiently large that no notable linkage disequilibrium is induced through genetic drift .FST = 0.008, P = 0.16). The clustering method identified K = 2 gene pools based on Evanno’s delta K statistic (K (ranging from one to 16) did not present a real minimum  indicating that individuals within tiles had a higher genetic relatedness than random pairs of individuals. Within the cave, the autocorrelogram suggested an estimated patch size of less than 40 cm  was higher compared to values found by 2) and in Portofino . While adult colonies dwells on the ceiling and on the walls of the cave, in the current study settlers’ density significantly differed between these two habitats, with higher values on the tiles located on the ceiling of the cave. The only three tiles  where no recruitment was recorded were positioned on the walls of the cave. The causes of these differences are not fully understood yet, but they could be related to several factors. Both larval behaviour and habitat constraints could influence the higher recruitment density observed on the ceiling. Even if C. rubrum larvae could survive at least 16 days  in the plankton  were shared by recruits and juveniles. However, up to now, no evidence of asexual reproduction and/or polyp bail-out were reported in Ho = 0.29 ± 0.20) compared to that previously pointed out in natural populations  are a more likely explanation for the m rubrum . This hym rubrum . Settlerm rubrum  but othebalanced , reprodubalanced , and up balanced , includibalanced . In the balanced .C. rubrum individuals settling in the Colombara cave are highly related at very small spatial scales, and that most larvae recruiting nearby are sibs. Evidence of the processes explaining this pattern cannot be provided, however, self-recruitment and the presence of clouds of larvae that settle all together could be possible explanations. Parentage analyses between adult individuals, both inside and outside the cave, and the recruits would help to disentangle the two processes. Understanding processes acting in the early life history of a species is a challenging but crucial task, with major implications for conservation. In fact, these processes drive the population structure and dynamic of the species, and are essential for the resistance and resilience of populations.The present study provides new insight concerning recruitment processes in red coral populations using a submarine cave as an experimental mesocosm. The main outcome of the study is that 10.7717/peerj.4649/supp-1Figure S1Click here for additional data file.10.7717/peerj.4649/supp-2Figure S2Click here for additional data file.10.7717/peerj.4649/supp-3Figure S3Click here for additional data file.10.7717/peerj.4649/supp-4Supplemental Information 1Genotype of each individual, position of each individual on the tile, identification as juvenile or recruit, n. of polyps, diameter, height.Click here for additional data file.