Macrotermes gilvus (Hagen), to elucidate the spatiotemporal dynamics of dispersal routes of terrestrial fauna in Pleistocene Southeast Asia. We sampled 213 termite colonies from 66 localities throughout the region. Independently inherited microsatellites and mtDNA markers were used to infer the phylogeographic framework of M. gilvus. Discrete phylogeographic analysis and molecular dating based on fossil calibration were used to infer the dynamics of M. gilvus dispersal in time and space across Southeast Asia. We found that the termite dispersal events were consistently dated within the Pleistocene time frame. The dispersal pattern was multidirectional, radiating eastwards and southwards out of Indochina, which was identified as the origin for dispersal events. We found no direct dispersal events between Sumatra and Borneo despite the presence of a terrestrial connection between them during the Pleistocene. Instead, central Java served as an important link allowing termite colonies to be established in Borneo and Sumatra. Our findings support the hypothesis of a north-south dispersal corridor in Southeast Asia and suggest the presence of alternative dispersal routes across Sundaland during the Pleistocene. For the first time, we also propose that a west-east dispersal through over-water rafting likely occurred across the Pleistocene South China Sea. We found at least two independent entry routes for terrestrial species to infiltrate Sumatra and Borneo at different times.Dispersal of soil-dwelling organisms via the repeatedly exposed Sunda shelf through much of the Pleistocene in Southeast Asia has not been studied extensively, especially for invertebrates. Here we investigated the phylogeography of an endemic termite species,  Southeast Asia (SEA) harbours 20–25% of the planet’s plant and animal species and is thus a highly biodiverse region –2. SEA tSeveral hypotheses regarding the size of open vegetation dispersal corridors in Sundaland have been proposed. Heaney  first poMacrotermes gilvus (Hagen). Termites are ideal subjects for biogeography and evolutionary studies because they are easily sampled, feed and live in a broad range of landscapes and are sensitive to environmental disturbances. Furthermore, colony maturation is slow and dispersal of reproductive alates is short ranged  option in microsat  for a linear relationship using IBDws 3.15 [Multiple measures of genetic diversity  revealed 50 haplotypes with 98 variable sites from 141 individuals (out of 160) that produced amplifiable gene fragments for both the COII and 16S rRNA genes . SeventyM. gilvus , West Java (n = 3), and one sample (V7) from Vietnam were excluded due to small sample size or poor amplification quality. A NJ analysis of individual termite genotypes based on the proportion of shared allele (Dps’) and kinship coefficient (Dkf’) genetic distances produced concordant topologies that support the population geographic subdivision observed in the mtDNA analysis  of 0.31 . Furthermore, assessment of invariant loci (monomorphic loci) revealed that the Malay Peninsula, Singapore, and North Sumatra populations shared a monomorphic locus MG3, whereas Riau, West Sumatra, Central Java, and East Java were monomorphic at locus MG2 , indicating that the populations were not experiencing recent bottleneck effect  displayed an inconsistent pattern and often associated with significant departures from HWE within the respective population. This suggests that either the presence of null alleles or a significant LD could be the result of local sampling effect in the respective populations rather than non-random association of alleles between the loci. There were no scoring errors due to stutters or large allelic drop out detected from this analysis. All populations in this study displayed a relatively high Garza-Williamson index  was remarkably high at 0.831 (P < 0.0001), indicating profound genetic differentiation among sites. Unexpectedly, geographically distant population pairs such as Thailand and the Philippines as well as Vietnam and Java displayed close genetic association with no significant genetic differentiation  despite the absence of any obvious geophysical barriers separating them. Similarly, North Sumatran termites were genetically isolated from the Riau and West Sumatran termites, with a high ΦST > 0.930; P < 0.0001. These conditions further suggest that the genetic association between sites was not restricted to contemporary processes but rather was influenced by past complex historical processes that took place in SEA.Pairwise genetic differentiation between population pairs broadly supported the observed phylogeographic structure of FST comparisons also revealed significant genetic differentiation except for three geographically proximate pairs: West Sumatra-Riau, East Java-Central Java, and East Java-Madura . Similar to the mtDNA findings, unexpectedly high pairwise FST/RST estimates were found among population subdivisions within Borneo and Sumatra  and microsatellite analysis .For microsatellites, all pairwise a-Madura . Genetic Sumatra . The Manstructure  supported the partitioning of the termite individuals into seven distinct clusters  showed significant probability of being a migrant (P < 0.001) that may have had recent ancestry in the Thailand-Philippines cluster; the probabilities were 0.005 and 0.769 that the individual came directly from this group or had a single ‘parent’ from this population, respectively.An assignment test based on the Bayesian clustering of multilocus microsatellite genotypic data as implemented in the program  ΔK = 7,  as obserbeast  revealed a pattern of historical migration of M. gilvus among study sites across SEA. Thailand was supported as the root location for all lineages across SEA with the highest root probability at 95% Highest Posterior Density (HPD) at 0.2076, and the root height of the current dataset estimated at 1.83 Ma (95% HPD: 1.20–2.56 Ma), 1.21 Ma (1.58–0.73 Ma), and 0.70 Ma (0.47–1.01 Ma) based on the 0.9% Myr–1, 1.5% Myr–1, and 2.28% Myr–1 of mtDNA substitution rates, respectively. In the absence of independent criteria to select between the different time estimates, the age estimate using the 1.5% Myr–1 divergence rate was selected as a rough median to illustrate the time dynamics of M. gilvus divergence, but the estimates obtained with the other calibration ages are also shown  . EastwarMacrotermes at 21.5 Ma (11.6–38.2 Ma) and 14.4 Ma (7.4–26.0 Ma), respectively. These results are consistent with the previous estimates given by Nobre et al [Macrotermes gilvus clade was estimated to have diverged at 2.67 Ma (1.2–5.32 Ma) .Fossil calibration dated the origin of the fungus-growing termites and the divergence of the genus re et al  of 31.0 5.32 Ma)  from M. us clade  is generM. gilvus populations in SEA. Eight phylogeographic groups were identified in this study based on phylogenetic relationships, genetic structure, and molecular diversity indices at both markers: Thailand, Vietnam, the Malay Peninsula/Singapore/North Sumatra, Riau/West Sumatra, Java, Southwest Borneo (B1), Northwest Borneo (B2), and the Philippines. The global FST/RST value at 0.34/0.64 was highly significant (P < 0.0001), signaling a substantive genetic differentiation among the populations as a whole, and most of the pairwise comparisons displayed a pronounced level of genetic differences (> 0.25). Recent gene flow and haplotype sharing between the phylogeographic groups were rare, which corroborates this finding. Nevertheless, the genetic similarity and haplotype sharing between North Sumatra and the Malay Peninsula (MP3/SU) suggests possibilities for retention of ancestral traits and/or recent gene sharing across these two populations.The patterns of spatial structure resolved from mtDNA and microsatellite data were mostly consistent in displaying a highly structured r2 < 0.22), suggesting that the genetic differentiation between populations was not a simple function of genetic isolation-by-distance but rather a complex interaction of other historical processes followed by an isolation mechanism. This is plausible given the complexity of geological perturbations taking place surrounding SEA in the past, including repeated sea level fluctuations [HE from microsatellite data along with neutral expectation following mtDNA’s Tajima’s D and Fu’s Fs tests indicate that each studied population is demographically stable, mixing randomly, and potentially in mutation-drift equilibrium. The average low inbreeding coefficient in all populations corroborates the occurrence of random mating and potentially reflects a substantial effective population size.Regression of genetic distance against geographical distance revealed a significant positive isolation-by-distance pattern for both mtDNA and microsatellite data. This indicates that the present day South China Sea may in fact act as an effective barrier to dispersal among the insular termites  and the termites from mainland SEA. However, the correlation coefficient values from both molecular markers were weak  1.09–0.42 Ma (early Pleistocene)–migration of termites due east and south from Indochina to establish the Java and the Philippines lineages, respectively, 2) 0.45–0.17 Ma (mid Pleistocene)–establishment of the Malay Peninsula and Southwest Borneo lineages from Vietnam and Central Java, respectively, and 3) 0.06–0.02 Ma (late Pleistocene)–independent colonization of Sumatra from the Malay Peninsula and West Java, respectively. The establishment of the Javanese lineage from Thailand , after its dispersal to Java 0.67 Ma (95% HPD: 0.33–1.04) (Homo erectus) from Asia through Sundaland into Java well into the Pleistocene [We identified at least three major range expansions of Thailand  providesThailand . Followi33–1.04) , illustristocene .structure [M. gilvus population from the Philippines is genetically similar to the Thailand and Vietnam populations as compared to other regions  overcame a wide 300+ km Miocene oceanic gap between Africa and Madagascar. Since evidence for a direct land connection between Indochina and the Philippines is still lacking, under this scenario, over water dispersal mechanism would be most likely explanation for M. gilvus to have dispersed across these two regions.Schutze et al  reportedsibility . Besidese genera , 10. Thee et al.  show thaM. gilvus to disperse across these two regions during the Pleistocene. Instead, central Java displayed remarkable habitat stability , serving as a central link to the south of the Equator and radiating out to establish termite lineages in Southwest Borneo, West Sumatra, and East Java. Our findings support the hypothesis of a minimal savanna corridor [We found no evidence of effective migration events between Sumatra and Borneo. The high genetic divergence between the two regions is congruent with results of other studies on speciation in neotenic net-winged beetles  populaticorridor  that woucorridor  that wouM. gilvus occurred soon after the catastrophic super-eruption of Toba approximately 73.5 ka in northern Sumatra [Based on the termite dispersal events, we suggest two alternative routes of entry by which the termite could have colonized insular Sumatra as inferred from the discrete phylogeographic analysis. The first route involves a northern incursion from the southwest region of the Malay Peninsula and Singapore across the Malacca Straits anytime between 60 and 20 ka. The timing of the dispersal events suggests that colonization of  Sumatra . Such a  Sumatra –73. The  Sumatra –75 sugge Sumatra . The timth– 6th century AD , ou, outh– 6An unexpected genetic break was observed between B1 and B2 in Borneo despite the absence of contemporary geophysical barriers separating them. Genetic isolation-by-distance is possible, but we observed contrasting scenario based on the genetic affinity shared between the Malay Peninsula and North Sumatra termites, which are separated by a similar geographical distance as seen between B1 and B2 (~150 km). Alternatively, unfavorable soil condition between the two habitats may play a role as barriers to dispersal but this feature remains to be investigated. It is also important to note that B1 and B2 termites displayed different evolutionary origins, and the time dynamics of their colonization events were hundreds of thousands years apart. This finding suggests multiple points of entry for ancient terrestrial fauna to colonize Borneo at different time points during the Pleistocene glacial phase.M. gilvus in this region. Our findings support several existing hypotheses of dispersal corridors in SEA and additionally suggest an alternative dispersal routes for terrestrial fauna to disperse across Sundaland during the Pleistocene. Despite several attempts to simulate the vegetation distribution at the last glacial maximum based on climatic/vegetation modelling , evideng Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Tablest, 2nd and 3rd codon positions of COII gene respectively. Partitioning scheme with best AIC score is highlighted in bold.Numbers in bracket represents 1(DOCX)Click here for additional data file.S5 TableBest partitioning scheme for Bayesian inference is shown in bold. Italicized values indicate number of parameters used to calculate the bayes factor.(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S9 Table(DOCX)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.S1 Appendix(DOCX)Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.


Coffea canephora have been introduced in several tropical countries with potential for crop development. In Ecuador, the species has been cultivated since the mid-20th century. However, little is known about the diversity and genetic structure of introduced germplasm. This paper provides an overview of the genetic and phenotypic diversity of C. canephora in Ecuador and some proposals for implementing a breeding program. Twelve SSR markers were used to analyze 1491 plants of C. canephora grown in different living collections in Ecuador, compared to 29 genotypes representing the main genetic and geographic diversity groups identified within the species. Results indicated that most of the genotypes introduced are of Congolese origin, with accessions from both main subgroups, SG1 and SG2. Some genotypes were classed as hybrids between both subgroups. Substantial phenotypic diversity was also found, and correlations were observed with genetic diversity. Ecuadorian Robusta coffee displays wide genetic diversity and we propose some ways of improving Robusta in Ecuador. A breeding program could be based on three operations: the choice of elite clones, the introduction of new material from other countries , and the creation of new hybrid material using genotypes from the different diversity groups.Genetic resources of Coffea canephora is originated from the lowland tropical forests of Africa, which stretch from Guinea to Uganda, and its cultivation is recent (end of the 19th century). Robusta coffee fields are now widely found in all lowland intertropical regions of Africa, America, and Asia [ C. canephora was first described at molecular level in the 1980s [ C. canephora diversity has been recently proposed [ C. canephora breeding programs.he 1980s . Those she 1980s , 8, 9. Aproposed . This co C. canephora genetic resources were first introduced in the mid-20th century [In Ecuador, century , 12; the C. canephora genetic material came from the “Tropical Agricultural Research and Higher Education Center (CATIE),” Costa Rica, in 1951, 1964, 1972, 1977, and 1986. They corresponded to the “Robusta” type (putative SG2) and all were planted at the Pichilingue Tropical Research Station (EETP) of the National Institute of Agricultural Research of Ecuador (INIAP), with the first C. canephora plantations appearing in 1952 in Los Ríos province, from where they were gradually extended to several coastal provinces and toward the north of Ecuadorian Amazonia [Ecuadorian historical records show that the first introductions ofAmazonia .Later, in 1987 and 2006, genetic material of the “Conilon” type (putative SG1) was imported from Brazil. Additionally, unofficial sources report introductions of genetic material imported as seeds from Vietnam and Indonesia 2009, 2010), as well as Robusta from Brazil (2010). Using seeds for genetic material transfers for a self-incompatible tree cannot ensure its genetic origin since crosses between genotypes from different genetic groups are likely to occur within germplasm collections [, as well C. canephora breeding program was developed. However, a first group of elite material “clones” was identified by INIAP in 1998, based on yield and morphological traits. To date, these clonal C. canephora materials have been recommended for commercial planting under the conditions of northern Ecuadorian Amazonia. A recent study on the phenotypic characterization of C. canephora accessions planted in the living genebank collection, located in the EETP of the INIAP, showed a high variability between and within these accessions [ C. canephora breeding program in Ecuador.During the second half of the 20th century, a nonspecificcessions . Consequ C. canephora in Ecuador,provide an overview of the genetic and phenotypic diversity and conservation of C. canephora breeding program.address the bases for implementing aThis paper will C. canephora clonal plants collected during 2011, 2012, and 2013. In 2011, one hundred thirty-seven leaf samples were collected from two fields. The first field is the Dublinsa-Denisse Farm, nearby Isidro Ayora in Guayas province that has a living collection of C. canephora collected from different locations at the Amazonian region of Ecuador. The second field is located at the EETP research station, nearby Quevedo city, Los Rios province. In 2012, two hundred leaf samples from 12 clonal accessions and 154 leaf samples from seven clonal accessions and a polyclonal mix were collected at the Dublinsa-Denisse Farm and the EETP, respectively. In 2013, one thousand leaf samples from 7 clones were sampled at the INIAP's Amazonian Central Station (EECA).In all, 1491 leaf samples were taken from differentThe summary of the plant material used for genotypic analysis across the different years is shown in Tables Genomic DNA extractions were performed according to Cubry et al. . The 149GT.In 2011, PCR reactions ran as described in Cubry et al. . PCR proμL total volume) containing 2.5 μL of PCR buffer , 2.5 μL of dNTP , 0.25 μL of MgCl2, 0.2 μL of 10 μM forward primer with an M13 tail at the 5′-end (5′-CACGACGTTGTAAAACGAC-3′), 0.25 μL of 10 μM reverse primer, 0.25 μL of fluorescently labeled M13-tail , 0.1 U of Taq DNA polymerase , 5 μL of template DNA (5 ng/μL), and 14 μL of H20. The PCR conditions were as follows: an initial denaturation at 94°C for 5 min; 30 cycles at 94°C for 45 s, 55°C for 45 s, and 72°C for 1 min; and a final extension at 72°C for 10 min. PCR products were pooled in a solution B containing 2 μL of 6-FAM, 2 μL of VIC, 2.5 μL of NED, and 3.5 μL of PET of A's solutions. From this solution B, a volume of 4 μL was taken and added to 10 μL of Hi-Di formamide and 0.12 μL of GeneScan 600 LIZ size standard and analyzed on an ABI 3500xL Genetic Analyzer . Alleles were scored using GeneMapper v4.1 software .In 2012 and 2013, PCR reactions were performed according to De Bellis et al. . In a soData were obtained separately in 2011, 2012 and 2013 for each sampling year, from which merged three matrices. Standard controls of known genotypes and duplicates samples together with genotyping database  were used to help us to pool/shift alleles to ensure good coherence of the genotyping data. From the raw data, were removed the 48 duplicates and the samples with more than 17% of missing data  for further analysis. The number of alleles detected per marker was recorded and compared to those detected in Leroy et al. . We compA first diversity tree was drawn up using data from the 2011 sampling operation in Ecuador. Diversity from Ecuador was evaluated in relation to the global diversity of the species.A global analysis, using data from the three sampling years, was performed to identify unique genotypes for future conservation and for breeding programs. In all, 1168 samples were kept for analysis, based on missing values . A Neighbor-Joining (NJ) tree was built on the whole data removing pairs of data with more than 70% of missing values. To take into account the noise due to highly repeated genotypes, the max-length subtree procedure was used to eliminate redundancy and to identify the number of unique individuals without loss in the number of alleles. A Principal Coordinates Analysis (PCoA) was used to construct a good image of the diversity between genotypes.Specific analyses were performed at the DUBLINSA and EECA stations to check the homogeneity and the diversity of the materials introduced.Phenotypic analysis was performed at the EETP collection, using data from 2010 to 2012. The evaluation was carried out in 256 plants, corresponding to 16 accessions. Each accession contained different number of individual (between 12 and 20). A phenotypic dendrogram was performed by UPGMA clustering method using the Euclidean distance.Genotyping data were available for 146 plants. Tree diversity was determined for molecular data, and their relationship with phenotypic data generated by Plaza et al.  was stud dudi.pca function from the ade4 R package [ s.corcircle function and the representation of individuals for the first two components was drawn using the s.class function.Phenotypic data were measured between 2010 and 2012 for all the trees planted in 2007. During this time, the traits observed were the following: plant height (PH), stem diameter (SD), number of branches (NB), number of nodes per branch (NN), and internode length (IL). Outlier data were removed whenever found and replaced the missing values. PCA analyses were performed using the package . Five prTwo types of analyses were performed on these data. A clustering tree was first built on 249 plants using the dissimilar Euclidian distance evaluated by average linkage for the 17 traits observed across the three years. Another analysis was performed using a Principal Components Analysis (PCA) on five vigor traits  across three years observed on the 146 trees analyzed for genetic diversity. C. canephora.The results on the clonal trials at the EETP and Dublinsa revealed great diversity between “clones,” and also a high level of diversity within “clones,” which was more surprising. The global genetic analysis indicated the Congolese origin for all the genotypes. They were classed as SG1 or SG2 genotypes, with some hybrids between these two Congolese subgroups. This result can be explained by considering the origin of plants introduced in Ecuador: the SG1 genotypes could have come from Brazil (Conilon genotypes are known to be from the SG1 group) and the SG2 genotypes from CATIE (from the Congo basin). A global analysis was performed in 2015, based on the results obtained for the 1168 accessions, by considering all the different genotypic classes revealed for each year in each plot. A global diversity tree was established using these 1168 accessions (figure not shown). After eliminating redundancy, the 138 unique genotypes (including 29 controls) were observed in the diversity tree (figure not shown). The PCoA analysis performed 138 unique genotypes, most of these genotypes were included in the SG1 and SG2 diversity groups, and six genotypes could be considered as hybrids between the groups .A diversity tree was constructed considering the 146 plants from the 16 different origins with data available for molecular diversity. Twelve diverse groups were identified within the 146 (from 154) plants analyzed in 2012 .Per origin analysis: COF1 p2 was different from COF1 p10; COF3 p2, 5, 8, 18, and 19 were genetically similar; p7 was different; COF4 p7 and 9 were similar; p15 was different. COF 5 p6, 15, 16, 17, and 19 were similar; NP2024 p 7, 10, 15, and 17 were similar; NP2044 p6, 16, and 17 were similar; NP3018 p 8 and 19 were probably different, since the origin was very variable.Global genetic diversity of the 24 “elite” clones: COF1 p10, COF3 p2, 5, 8, 18, and 19, and COF4 p7 and 9 were similar; COF5 p6, 15, 16, 17, and 19 and NP2024 p7, 10, 15, and 17 were similar; NP2044 p6, 16, and 17 were similar; COF4 p15 and COF3 p7 were similar; COF1 p2 and NP3018 p19 were almost identical; their similarity has to be verified.On the other hand, a phenotypic characterization of this collection was reported by Plaza et al.  showing The genetic diversity of the selected clones was therefore very low, since only 5 different groups of genotypes (six if including NP3018) were present in the selected clones. As regards the global diversity of the Pichilingue collection, almost half of this diversity was not present in the selected clones. The diversity of the Conilon/SG1 group was not present in the selected clones from this collection. C. canephora collection . We also observed mixtures between genotypes in both clonal trials. The CONERBO and POLICLON genotypes which belonged to Conilon type can be considered as plants from a mix of seeds, introduced from Brazil in the 80s; these origins present high genetic diversity. Regarding the EETP collection, it should be noted that many plants, with different labels, were quite similar from a genetic point of view. We also had to consider that the Conilon exhibited wide diversity, possibly due to its environmental share of diversity or due to their seed origin. C. canephora diversity was carried out, using a core collection approach [In 2014, a global analysis ofapproach . The comAnother finding was the small number of intergroup hybrids between the SG1 and SG2 diversity groups. This low level of hybridization can be explained by the history of the introduced material. Both introductions were composed of genotypes from a single subgroup (SG2 from CATIE and SG1 from Brazil). The accessions were planted in one location (EETP) and then transferred to Manabí, Santo Domingo de los Tsáchilas, and Morona Santiago provinces, mainly by cuttings. Thus, few hybrids can be found between groups in the Ecuadorian collections.Nevertheless, high diversity was observed within each diversity group (SG1 and SG2). For SG2, this diversity was related to the large number of accessions that were introduced, and previous studies  confirmeThe phenotypic characterization carried out by Plaza et al.  and our  C. canephora for evaluating their future adaptive potential in different geographical regions of Ecuador. Differences between phenotypic and genetic information have also been found in other crops [In our view, a combined analysis of phenotypic and molecular marker results is crucial for a better understanding of evolutionary changes in this introduced species; this would allow a better analysis of variation patterns withiner crops –22. C. canephora, a program of reciprocal recurrent selection was conducted using the hybrid vigor observed between genotypes of different origins [ C. canephora in Ecuador, with optimum use of the existing diversity and improved management of the existing material, based on the results presented. This strategy for the implementation of a C. canephora breeding program should also be of interest for other countries where coffee genotypes have been introduced in recent decades.Lastly, this study was intended to identify diversity and ways of using it to increase the production of Robusta coffee in Ecuador. In Ivory Coast, considering the high genetic diversity found in origins , 23, 24.To complete genotypic analyses with phenotypic data to increase knowledge on the accessions in the field for their vigor, productivity, and stress and disease toleranceTo reorganize the collections based on the genetic diversity observed in our studies to avoid duplications and identifying unique genotypesTo implement a breeding program by selecting the best genotypes for traits of interest  As a first step, we propose the following actions, using the diversity existing in Ecuador:To introduce new genetic material from diverse groups  that are not present in Ecuador and test them under Ecuadorian conditionsTo establish a breeding program based on new hybrids obtained from crosses between genotypes from different diversity groups, adapted to the edaphoclimatic conditions in Ecuador  or seed gardens (for hybrids)) As a second step, we propose the following actions, by increasing genetic diversity:Over the long term, hybrid selection might be the optimum breeding method, as seeds are more suitable for distribution to growers, and the nurseries are easier for farmers to manage. C. canephora genotypes introduced in Ecuador are of Congolese origin, containing accessions from both subgroups, SG1 and SG2.The present research concluded that Ecuadorian Robusta coffee displays a wide genetic diversity between clones and also a high level of diversity within clones. This research confirms that most of the


Genetic diversity confers adaptive capacity to populations under changing conditions but its role in mediating impacts of climate change remains unresolved for most ecosystems. This lack of knowledge is particularly acute for foundation species, where impacts may cascade throughout entire ecosystems. We combined population genetics with eco-physiological and ecological field experiments to explore relationships among latitudinal patterns in genetic diversity, physiology and resilience of a kelp ecosystem to climate stress. A subsequent ‘natural experiment’ illustrated the possible influence of latitudinal patterns of genetic diversity on ecosystem vulnerability to an extreme climatic perturbation (marine heatwave). There were strong relationships between physiological versatility, ecological resilience and genetic diversity of kelp forests across latitudes, and genetic diversity consistently outperformed other explanatory variables in contributing to the response of kelp forests to the marine heatwave. Population performance and vulnerability to a severe climatic event were thus strongly related to latitudinal patterns in genetic diversity, with the heatwave extirpating forests with low genetic diversity. Where foundation species control ecological structure and function, impacts of climatic stress can cascade through the ecosystem and, consequently, genetic diversity could contribute to ecosystem vulnerability to climate change. Genetic variation among individuals within a population provides a mechanistic basis for plasticity and adaptability, such that a multiplicity of genotypes (genetic diversity) provides a greater range of possible functional responses , and thus a higher probability that a population will resist, or recover from, a perturbation 3. Despite an advanced conceptual understanding of the implied positive relationships among genetic diversity, physiological versatility and ecological resilience, empirical evidence for their existence is lacking from natural populations, and strongly biased towards experiments on model organisms and clonal plants4. Knowledge about the role of genetic diversity in underpinning species performance and ecosystem vulnerability is, however, critical to successfully mitigate impacts of pressures, such as population over-exploitation, pollution, invasive species and global warming.A core tenet of evolutionary theory is that the ability of species to adapt and persist through changing environments is contingent on latent functional responses suited to new conditions7. The mechanisms that translate the physical forcing of climate change into biological changes are, however, poorly understood, creating major uncertainty about future ecological scenarios9, and limiting our ability to predict impacts9 and implement mitigation strategies, such as targeted conservation and rehabilitation11. A critical knowledge gap relates to the response of species to impending changes, and the role of genetic factors in mediating population persistence through latent functional responses12. That is, the resilience of a population to climatic stress might depend on possessing sufficient genetic variation to allow a range of responses to stressors, some of which will promote population persistence.Increasing temperatures have already impacted most ecosystems on the planet12, because of their critical influence on community organization and ecosystem functioning. Indeed, the importance of foundation species in mediating climate stress for associated biodiversity is likely to increase in a warmer future13. Where populations of foundation species have reduced potential to respond to environmental stress, resilience might be compromised14 and, if perturbations are severe, entire populations could perish with impacts cascading through the ecosystem16. Knowledge of the role of genetic diversity in determining the response of foundation species to climatic stress is, therefore, particularly critical for assessing the overall vulnerability of an ecosystem, and to ensure successful conservation and management strategies18.Our lack of understanding of how genetic diversity mediates population response to climate stress is particularly acute for ‘foundation species’ 19 with eco-physiological and ecological field experiments14 to examine relationships between latitudinal patterns in genetic diversity, physiological versatility and ecological resilience of kelp forests, one of the most important foundation species of temperate marine habitats globally21. We also documented subsequent responses of kelp (Ecklonia radiata) forests to an extreme climatic perturbation (a marine heatwave), thus demonstrating through a ‘natural experiment’, the possible influence of latitudinal patterns in genetic diversity on ecosystem vulnerability to climatic stress.Here, we combined population geneticsEcklonia radiata) along a latitudinal gradient in Western Australia14  and number of alleles , across southwestern Australia , explained little variation in impact and was only selected (with genetic diversity) in one of the five top performing combinations of predictors , such observations remain critical as a foundation for subsequent detailed experiments to pin-point processes. Moreover, because other studies have found limited outcrossing to strongly reduce kelp sporophyte fecundity and fitness28, and linked kelp survival and regrowth after canopy loss to variation in photo-physiology30, there exists a plausible mechanistic link between genetic diversity, physiological performance and canopy persistence in the face of disturbance. This link should underpin future studies designed to definitively demonstrate causality.Current understanding of how genetic diversity mediates ecosystem vulnerability to climatic stress has a strong theoretical basis, but evidence from natural populations is rare. Here, we provided empirical evidence for strong covariation between functional responses and population vulnerability to environmental stress, and latitudinal patterns in genetic diversity of kelp. These insights were only possible through a unique combination of independent broad scale field studies, controlled manipulations, and a ‘natural disturbance experiment’ affecting an entire coastline. Given the opportunistic and correlative nature of events and data collections, our study was not designed to tease apart the exact causal mechanisms linking latitudinal patterns of genetic variation, physiological and ecological performance, and population impact. Moreover, identifying underlying causes of these patterns is challenging, because latitudinal gradients often integrate multiple mechanisms, many of which are not mutually exclusive, and can covary themselves. However, even if significant association between predictors and responses do not prove cause-effect relationships 31, from surviving ‘seed banks’ of microscopic stages that can persist for many months under unfavorable conditions32, or from macroscopic sporophytes resisting the perturbation29. Nevertheless, our experiments prior to the heatwave revealed systematic differences in the physiology and capacity for recovery and regrowth across the 12 kelp forests, matching the latitudinal patterns of genetic diversity. This is consistent with the hypothesis that the initially observed differences in impact of the heatwave were not caused by an external disturbance alone . Nevertheless, subsequent trajectories of recovery could have been influenced by post-disturbance forces such as Allee effects33, or concurrent changes in consumer pressure34. Almost six years have now elapsed without any signs of recovery in the most heavily impacted populations25. No macroscopic sporophytes have been observed . Because ‘seedbank’ longevity is thought to be less than one year32, and new zoospores would have to arrive against flow of the predominant current15, it seems unlikely these kelp forests will recover in the near future.Kelp forests are generally robust to disturbances, as canopies can recover through recruitment of new gametophytes38, (ii) geometric constraints on the distribution of genotypes (a ‘mid-domain effect’39), (iii) selection for a narrow subset of stress-tolerant genotypes at low latitudes41, or (iv) historical extinction or colonization events43. These mechanisms are not mutually exclusive and might occur simultaneously. For example, selection for stress tolerant genotypes and restricted connectivity are both likely to be important where a population is contracting from deteriorating environmental conditions at its range edge . Teasing apart the mechanism behind the observed patterns of genetic diversity is beyond the scope and capacity of the present study, however, given that the response of low latitude kelp forests to the marine heatwave (extirpation) was opposite to what one would expect for stress-tolerant genotypes (persistence), it is unlikely that selection was the primary mechanism driving the observed patterns of genetic diversity44.Several mechanisms could explain the differences in genetic diversity among kelp forests along the latitudinal gradient and have implications for interpreting the response of kelp forests to the heatwave. These include (i) reduced connectivity or smaller effective population sizes in marginal populationsWe propose that latitudinal variation in genetic diversity may have played a role in mediating the response of kelp forests to the marine heatwave. First, high genetic diversity coincided with slightly more benign (cooler) pre-heatwave conditions, possibly protecting these kelp forests against change Fig. , variabi36 rather than prior selection for thermal stress . Indeed, in the wake of the population impacts observed here, it is conceivable that genotypic frequencies, or population tolerances46, now reflect selection, with only tolerant genotypes surviving . Although this study cannot establish causal links it strongly suggests that genetic diversity may at least partially explain the dramatic differences in observed population impacts.We suggest that low diversity populations represent a subset of genotypes created via processes such as low connectivity and effective population sizesess Fig. , where t10. It is therefore plausible that impending environmental changes will cause substantial ecological changes in temperate marine ecosystems, and that patterns of genetic diversity could play a role in mediating the manifestations of these impacts. Importantly, our data provides an empirical example of how functional responses and vulnerability to environmental stress might be anticipated by mapping population level genetic diversity. This result forms the basis for future work establishing general genetic diversity relationships that are independent of latitude and may transcend geographic settings and stressors. Establishing such predictive relationships of genetic diversity for conferring physiological versatility and ecological resilience is particularly critical for foundation species, because they exert an essential influence on biodiversity and energy flow across multiple trophic levels13. Moreover, establishing such relationships is vital to assessing the vulnerability of populations9, especially where genetic diversity might already be low35 or exclusively maintained in refugia48, where future exploitation37 or climatic forcing is predicted to be greatest49 and where normal mechanisms of recovery might be compromised . Loss of genetic diversity could drive widespread loss of physiological versatility and ecological resilience, with flow-on effects cascading through to critical, and potentially irreversible, changes to ecosystem structure and functioning.The relationships among genetic, physiological, and ecological population-level responses of kelp forests occurred over a temperature gradient of similar magnitude to projected sea temperature increases in the upcoming 50–100 yearsEcklonia radiata is the only true Laminarian kelp along this coastline so the term “kelp” refers to Ecklonia radiata throughout this manuscript. However, Ecklonia radiata forms both monospecific52 and mixed forests with fucoids53 and here we sampled monospecific forests or, for canopy removal experiments, those comprised of at least 50% Ecklonia radiata cover.We examined potential drivers of ecosystem vulnerability to climate stress by comparing baseline datasets that compared latitudinal patterns in genetic diversity, physiology and resilience, to the response of kelp forest ecosystems to a natural climatic event. Ecklonia radiata) along a latitudinal gradient in Western Australia14, Fig. 14. The response of the same kelp forests to the heatwave which occurred in 201126, was measured after the event in 201354. Baseline genetic diversity was estimated using kelp plants collected along the same latitudinal gradient and locations, but slightly different sites (a few km apart) in 200619. Genetic diversity was thus matched to each exact kelp forest using its strong relationship with latitude , were measured in 12 kelp forests (de Table . Given te) and number of alleles (Na), two of the most commonly reported diversity metrics e.g.4, using 6 proven microsatellite markers, in 12 populations, 32 kelps per population55. Although these are neutral markers that do not enable firm inferences about the effects of genetic diversity on selection or adaptation, there is evidence that neutral marker diversity can be positively correlated with key demographic parameters of population performance, such as reproductive success56 and survival of juveniles and adults59. Importantly, these markers can demonstrate key attributes of breeding systems, such as gene flow and the level of population isolation, factors which correlate with fitness and evolutionary potential60. Indeed, in Western Australia E. radiata populations are characterized by strong isolation by distance driven by poleward flowing boundary currents62.Genetic diversity was measured as expected heterozygosity  and maximum electron transfer rate (ETRmax) of photosystem two (PSII) were measured by Pulse Amplitude Modulated [PAM] fluorometry after 15 minutes of dark-adaption14. While these measurements were instantaneous, the treatment responses integrated the capacity for long-term (80 days) acclimation to canopy loss. Photosynthesis is a fundamental metabolic process in seaweeds and variation in photosynthetic performance, including quantum efficiency, has been linked to differences in kelp survival and kelp canopy recovery in the face of perturbation, presumably as a consequence of differences in tolerance to excessive light and photoinhibition63. Moreover, photosynthetic performance has been directly linked to climatic perturbation in both juvenile64 and adult66E. radiata.Physiological versatility was measured as the coefficient of variation (standard error divided by the mean) in photosynthetic performance (see below for details) of kelp recruits (8–10 kelps per population) 80 days after experimental kelp canopy removal14). Our measurements of kelp canopy re-growth do not differentiate between micro- and macroscopic kelp recruits surviving in the understory, and new recruitment from surrounding adults. This measure of resilience, therefore, integrates elements of both resistance to, and recovery from, disturbances.Ecological resilience of kelp forests was measured as kelp canopy re-growth two years after experimental kelp canopy removal  showed no visible changes to kelp cover in the period prior to the heatwave67, T. Wernberg & S. Bennett personal observation.Population impacts in response to an extreme natural climatic perturbation – a marine heatwave appendix  - were ae and Na), physiological versatility, resilience and heatwave impact were tested with linear mixed models using R v.3.2.2. For these analyses, the nlme package was used to model data with ‘Region’ included as a random factor to account for underlying spatial autocorrelation among sites (n = 12). The r2 from the linear mixed model was calculated using the sem.model.fits function based on the marginal r2 formula of Nakagawa and Schielzeth68. This marginal r2 quantifies the proportion of total variability that is explained by the fixed effects terms in the model.To allow better direct visual comparisons of all response variables across latitudes, the magnitude of response in each population characteristic was scaled as a percentage of the range across all 12 kelp forests. However, the range of raw values for each population characteristic are given in the Fig. 69. Instead of using a proxy, we used a range of measured site characteristics that are widely known to influence kelp forests  distances between samples, these analyses are analogous to standard multiple regressions70, where significance is obtained through a randomization test , thus avoiding assumptions about normal distribution of data and residuals . To identify which predictor combinations best explained variation in kelp forest responses to the heatwave, we used a model selection procedure with an exhaustive search among all predictor combinations and a distance-based analogue to the Akaike’s Information Criterion, modified to accommodate small sample sizes relative to the number of predictor variables (AICc). This procedure identified the most parsimonious (lowest AICc) predictor subsets see70, for, technical, details, while also taking co-linearity among predictors  into account71. To be conservative, we interpreted these analyses qualitatively because 12 sites  provides low power to quantitatively partition variation among sets of multiple predictor variables.Distance-based linear modeling and redundancy analysis was used to identify the relative strength of relationships between population impacts following the heatwave, and seven potential physical and biological predictor variables appendix . BecauseSupplementary Information


Egernia cunninghami), a philopatric species distributed along Australia's Great Dividing Range, and assessed evidence of localized adaptation. Analysis of 4,274 SNPs from 94 individuals sampled at four localities spanning 500 km and 4° of latitude revealed strong genetic structuring at neutral loci (mean FST ± SD = 0.603 ± 0.237) among the localities. Putatively neutral SNPs and those under divergent selection yielded contrasting spatial patterns, with the latter identifying two genetically distinct clusters. Given low genetic connectivity of the four localities, we suggest that the natural movement rate of this species is insufficient to keep pace with spatial shifts to its climate envelope, irrespective of habitat availability. In addition, our finding of localized adaptation highlights the risk of outbreeding depression should the translocation of individuals be adopted as a conservation management strategy.Establishing corridors of connecting habitat has become a mainstay conservation strategy to maintain gene flow and facilitate climate‐driven range shifts. Yet, little attention has been given to ascertaining the extent to which corridors will benefit philopatric species, which might exhibit localized adaptation. Measures of genetic connectivity and adaptive genetic variation across species’ ranges can help fill this knowledge gap. Here, we characterized the spatial genetic structure of Cunningham's skink ( This connects the northern Rocky Mountains of the USA and Canada  as a model philopatric species to describe spatial patterns of neutral and adaptive genetic variation along the GDR. Reptiles in general have received less attention in the climate change literature, despite being relatively more sensitive to climate change than other vertebrate taxa, such as birds and mammals  Is genetic structure at neutral loci a good proxy for adaptive variation? We discuss the implications of our results for mitigating negative impacts of climate change on this species.Our main goals are to assess the levels of gene flow and evidence for selection among four populations of Cunningham's skink that are separated by ~100–500 km. Specifically, we ask: (1) Is localized philopatry in 22.1Tail‐tip muscle tissue samples of Cunningham's skink were collected from 94 individuals at four localities across the GDR within New South Wales, Australia Figure . We sampIndividuals were captured using Elliott live traps and by hand. Captured individuals were measured and released at the point of capture after a small terminal portion of the tail was removed. The exact locations where samples were taken were marked using a global positioning system (GPS) unit (Garmin GPSmap 62). Tissue samples were kept in 97% ethanol at room temperature prior to laboratory analysis. Lizard capturing and handling followed Macquarie University Animal Ethics Committee recommendations (ARA 2013/015) and was licensed by the Office of Environment & Heritage, NSW National Parks and Wildlife Service (SL101164).2.2™ Blood & Tissue Genomic DNA Extraction Mini‐Prep Kit; Epoch Life Science, Inc.) following manufacturer's protocols. SNP discovery and genotyping were performed at Diversity Arrays Technology Pty. Ltd.  using standard DartSeq™ protocol . Discovered SNPs were called only if they were present in both homozygous and heterozygous forms.Genomic DNA was extracted from tissue using a commercially available spin‐column kit (GenCatchhttp://dx.doi.org/10.5061/dryad.ko1kj) and Diversity Array Technology Pty. Ltd., Canberra, Australia (Report‐DEgs14‐1547).To ensure the quality of individual samples, all duplicate markers and those with minor allele frequencies <5% (MAF < 0.05) across all populations were removed. We also removed markers with average read depth <10 or >45 for both alleles; individuals genotyped at <100% call rate (CR: proportion of genotyped SNPs); <94% reproducibility and two or more SNPs that occurred on one sequence read to avoid physical linkage. DArT sequencing returned a total of 81,732 SNPs, of which 4,274 were retained for downstream analyses after secondary filtering. DNA sequences and statistics  are accessible from the Dryad Digital Repository (2.3FST) among regional groups  higher than the mean were considered candidates for divergent selection and those with FST significantly less than the mean among groups were considered candidates for balancing selection. Finally, we ran LOSITAN  and observed (Ho) heterozygosity  on neutral loci using Arlequin 3.5  values  at each sampling locality, and computed levels of expected  for putatively neutral SNPs and those under divergent selection using three methods, (1) Bayesian clustering, (2) Discriminant analysis of principal component (DAPC), and (3) Neighborhood‐joining phylogenetic tree. The Bayesian clustering approach implemented in STRUCTURE  loci were identified as candidates for divergent selection, 36 (0.84%) as under balancing selection, and the rest as putatively neutral. Fifty‐four of the loci under divergent selection were common to all three methods . Given that false positives are often associated with the outlier loci, we conservatively considered only these 54 loci for downstream analyses of adaptive genetic structure. Alignment of the trimmed sequences of the 54 SNPs (Table S2) to the list of nonredundant nucleotides in the UNI‐PROT/SWISS‐PROT and NCBI database provided a total of four hits with an 3.2p < .01) from HWE, but only 115 (2.8%) remained significant after adjusting for FDR at α = .01. Expected heterozygosity (He) varied across localities, ranging from 0.056 ± 0.002 at Sydney to 0.202 ± 0.03 at Armidale (mean ± SE). In general, He was not significantly different from the corresponding observed heterozygosity (Ho), suggesting that the observed deviation from HWE was an artifact of sampling. Fixation index (FIS) was small and nonsignificant, except for samples from Sydney  that were a combination of individuals from two isolated sites: Barrenjoey headland  in the Kur‐ring‐gai Chase National Park and Box Head  in Bouddi National Park (Table FST) was high and significantly different from zero in each case (p < .01), ranging from 0.126 (Bathurst vs. Crookwell) to 0.742 (Sydney vs. Crookwell) and averaging 0.542 over all populations and loci  deviated significantly  and those from Bathurst and Crookwell as overlapping clusters. For SNPs under divergent selection, two clusters were identified—Armidale as a singleton and Sydney, Bathurst, and Crookwell as the other  in southeastern Australia revealed strong genetic partitioning and signatures of selection. Genetic structure at neutral SNPs and those under divergent selection yielded contrasting spatial patterns, showing that in this species, neutral genetic variation is not necessarily a good proxy for adaptive variation. At neutral loci, the genetic distances between each of the four localities analyzed were high, with a relatively high proportion of unique alleles at Armidale. SNPs putatively under divergent selection clustered into two groups .FST ≥ 0.644). This level of genetic partitioning is substantially above the FST level of 0.35, which is approximately the point at which the spread of advantageous alleles across a species’ range is prevented   , similar spatial patterns at neutral and adaptive genetic variations have been reported  is unlikely to allow for spread of favorable alleles  (Symula, Keogh, & Cannatella, Varanus varius, have also been identified, with divergences estimated to have occurred during the Pleistocene (Smissen et al., Strong genetic structuring in Cunningham's skink complements similar findings reported in phylogeographic studies of other lizards, mammals, birds, and invertebrates inhabiting the GDR (Chapple et al., We provide the first genetic evidence for different selection pressures along the GDR for a vertebrate. The presence of divergent selection raises some concerns for alternative management strategies. Translocation is often proposed as a means to rescue species from potential extinction, particularly where the current range becomes unsuitable and there is little or no overlap between this and areas projected to be suitable in the future. Initiating gene flow raises the potential risk of outbreeding depression (Frankham et al., Unless climate change forces unprecedented levels of movement in Cunningham's skink, our data suggest that gene flow will be insufficient to spread advantageous alleles in the future. Knowledge of areas where divergent selection is associated with differences in climate can potentially be used to select areas for assisted migration purposes. However, studies on genetic structure based on neutral markers, such as those using microsatellites, may not be informative in this respect, because, as we have shown, patterns of variation at selected parts of the genome may not be concordant (Hemmer‐Hansen et al., None declared. Click here for additional data file.


Heat-shock factor-1 (HSF-1) is an important transcription factor that regulates pathogenesis of many human diseases through its extensive transcriptional regulation. Especially, it shows pleiotropic effects in human cancer, and hence it has recently received increased attention of cancer researchers. After myriad investigations on HSF-1, the field has advanced to the phase where there is consensus that finding a potent and selective pharmacological inhibitor for this transcription factor will be a major break-through in the treatment of various human cancers. Presently, all reported inhibitors have their limitations, made evident at different stages of clinical trials. This brief account summarizes the advances with tested natural products as HSF-1 inhibitors and highlights the necessity of phytochemistry in this endeavor of discovering a potent pharmacological HSF-1 inhibitor. Xenohormesis)  . Howeverormesis) , 3. Its The pathophysiology of carcinogenesis is regulated by many factors, primarily by the oncogenes. There are also many additional factors, which are not yet classified as oncogenes, that modulate the overall genesis and growth of cancer , 5. HSF-Activation of HSF-1 is not only involved in carcinogenesis but it also enhances cancer cell survival post-chemotherapy or radiotherapy. For example, UV or doxorubicin-induced cancer cell death was found to be enhanced by inhibiting with HSF-1 , 12. HSFin vitro and in small animal models have shown promising results in increasing drug sensitivity and avoiding cancer relapse. Although there is no clinical investigation on inhibition of HSF-1 and demonstration of enhanced efficacy of cancer treatment, many preclinical studies, including our recent study , which can target HSF-1 mRNA, sequence cloned expressions vectors have been successfully developed and demonstrated to knockdown HSF-1 in different cell types , 22. Typin vitro human cells that it could inhibit HSPs expression by inhibiting their transcription factor HSF-1. Triptolide, an active ingredient in the Tripterygium wilfordii, sometimes called thunder god vine but more properly translated thunder duke vine, was found to be a HSF-1 inhibitor  and its derivatives were proved to be inhibitor of HSF-1 transcriptional activity.Other large quantity screening of compounds for potential inhibitors are undergoing. Recently an unbiased cell-based phenotypic screen was conducted to identify inhibitors of HSF-1. Among the 200,000 small molecules 4,6-disubstituted pyrimidine 1 with IC50 value of 2.00 µM was found to be good inhibitor . Howeveran HSF-1 . In thisN-allylamino-17-demethoxygeldanamycin, 17AAG) was developed with so much hope but before it was commercialized and clinical trials were stopped . HSF-1 is released/untethered from the complex upon phosphorylation of Hsp90, and it becomes active by forming HSF-1 trimers. Therefore, inhibiting the phosphorylation of HSP90 could prevent the trimer formation. This approach has been attempted with novel pharmacological agents . Tanespi stopped . HSF-1 fThe approaches, used thus far, have been useful in determining mechanism but are not very clinically applicable. Unfortunately, drug(s) that selectively inhibit HSF-1 with high-binding activity have not been developed. Importantly, studies to date support efforts to identify and tailor novel drugs that specifically target HSF-1. There is an urgent need to explore natural product(s) for highly efficient and specific inhibition of HSF-1 and its transcriptional activity, or secondary targets. The use of pharmacological agents to inhibit HSF-1 is very clinically relevant, since the delivery of a single dose of a well-characterized and safe pharmacological agent is far more feasible and often safer than using genetic approaches.in vitro assays and successful demonstration of retardation of tumor growth in small animal models, each of the inhibitors listed above has limitations in translation into a drug for clinical use. As HSF-1 is important for both cancer and normal cells under stress conditions, systemic inhibition of HSF-1 could exert not only anticancer activity but also toxicity to normal cells. Therefore, for its use in cancer treatment, it is important to identify cancer cell specific inhibitors to minimize the cytotoxic effects on normal cells. This requires either fine-tuning of existing compounds by altering the functional groups/motifs by synthetic approaches or discovering and isolating new natural product(s) that can overcome the existing challenges. Such a potential pharmacological agent for HSF-1 inhibition could be identified from plants or plant-derived metabolites by screening a vast number of compounds. It is a potential opportunity for phytochemists to discover and isolate natural product(s) as a reliable inhibitor of HSF-1 to improve the lives of cancer patients. There are some compounds that have been identified as potential leads. A terpenoid, cantharidin , higher Carvacrol in stressed Oregano x majoricum, and so on [refer the Table 1 in the reference (There are also multiple evidences that stress can produce bioactive compounds in plants, and these compounds have been proven to offer anti-carcinogenic effect in small animal models and human, and therefore to be useful in cancer therapy as dietary supplements. Examples of such compounds are increase of curcumin content in stressed eference . Contrareference . This paAll authors contributed intellectually and in writing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Xanthoceras sorbifolium Bunge has great potential for producing biodiesel. In order to select and evaluate appropriate germplasm to produce biodiesel, we analyzed the genetic diversity of Xanthoceras sorbifolium Bunge germplasm based on morphological traits and simple sequence repeats (SSRs) in this study. Fifty-six germplasm samples were evaluated using nine morphological traits and 23 SSR loci. Significant differences among germplasms were observed in eight morphological characters. The SSR markers analysis showed high genetic diversity among the germplasms. All SSRs had polymorphisms, and we detected 77 alleles in total. The number of alleles at each locus ranged from two to six, averaging 3.35 per marker. The polymorphic information content values ranged from 0.36 to 0.61, averaging 0.49. Expected heterozygosity, observed heterozygosity, and Shannon’s information index calculations detected large genetic variations among germplasms. The high average number of alleles per locus and the allelic diversity observed in the set of genotypes analyzed indicated that the genetic base of this species is relatively wide. Thus, microsatellite markers can be used to efficiently distinguish Xanthoceras sorbifolium Bunge germplasms and assess their genetic diversity. Hundred-grain weight and lateral diameter were positively correlated with monounsaturated fatty acids and depended on genotype. These results suggest that seeds with higher hundred-grain weight and lateral diameter could be more suitable to produce biodiesel. Our data will lay a foundation for selecting appropriate germplasm to produce biodiesel based on seed phenotype and will contribute to the conservation and management of this important plant genetic resource. Xanthoceras sorbifolium Bunge (Family Sapindaceae) is a small tree that produces edible fruit and seeds with high oil content. The plants are long lived (up to 1000 years) and tolerant of drought, low temperature, alkaline soils, and low fertility. Because of high monounsaturated fatty acid contents, the oil from this plant has considerable potential for producing biodiesel  werSeed phenotype, kernel oil contents, and FAME composition were examined by analysis of variance (ANOVA). The distance matrices were based on the Gower general similarity coefficient . ClusterThe polymorphic bands in the SSR marker analysis were scored as either present (1) or absent (0). The alleles were coded alphabetically  in order of decreasing size. The number of alleles per locus (Na), the number of effective alleles per locus (Ne), the Shannon index, observed heterozygosity (Ho), and expected heterozygosity (He) were calculated using POPGENE 32 software . PolymorXanthoceras sorbifolium Bunge germplasms were determined  and monounsaturated fatty acids (r = 0.348**), but negatively correlated with polyunsaturated fatty acids (r = −0.367**). Lateral diameter was positively correlated with saturated fatty acids (r = 0.336*) and monounsaturated fatty acids (r = 0.339**), but negatively correlated with polyunsaturated fatty acids (r = −0.367**).A partial correlation analysis was conducted to study the relationship between seed phenotype and oil characteristics . The resXanthoceras sorbifolium Bunge germplasms  .Variations in phenotypic traits are based on the variation and interactions at the genotypic level of the plant as well as the environmental pressure on the plant . AlthougData on the relationships between genotypes help solve problems in breeding programs and germplasm resource management . Many tyXanthoceras sorbifolium Bunge [Xanthoceras sorbifolium Bunge germplasms, and 23 SSR polymorphic markers were highly informative. The proportion of polymorphic loci that obtained in this study (100.00%) was exceeded the proportions in previous RAPD and ISSR studies [Xanthoceras sorbifolium Bunge and identifying its germplasm.Molecular techniques based on DNA markers, such as RAPD and ISSR, have been used to characterize genetic diversity in um Bunge –13, but  studies –13. The is study 0.00% was studies  and has Our Mantel test analysis detected a significantly positive correlation between phenotypic and genetic distances among the 56 germplasms , suggestXanthoceras sorbifolium Bunge germplasm to achieve higher production of higher quality biodiesel. Our data will lay the foundation for selecting excellent germplasm to produce biodiesel based on seed phenotype, regardless of the environment.Our results help understand the relationships between germplasm characters and genotype and will improve the Xanthoceras sorbifolium Bunge germplasms. The large average number of alleles per locus and allelic diversity in the set of genotypes analyzed indicate that the genetic spectrum was relatively wide. Our results show that SSR markers are a useful tool to explore Xanthoceras sorbifolium Bunge diversity. Hundred-grain weight and lateral diameter were positively correlated with monounsaturated fatty acids, and were dependent on genotype. These results suggest that seeds with higher 100-grain weight and lateral diameter values could be more suitable to produce biodiesel.Our data showed significant variations in the morphological traits and microsatellite DNA polymorphisms among 56 S1 TableThis is the raw data of morphological traits.(XLSX)Click here for additional data file.S1 Folder(ZIP)Click here for additional data file.


Astacus astacus) displays a complex historical and contemporary genetic status in Europe. The species divergence has been shaped by geological events (i.e. Pleistocene glaciations) and humanly induced impacts  on its populations due to species commercial value and its niche degradation. Until now, limited genetic information has been procured for the Balkan area and especially for the southernmost distribution of this species (i.e. Greece). It is well known that the rich habitat diversity of the Balkan Peninsula offers suitable conditions for genetically diversified populations. Thus, the present manuscript revisits the phylogenetic relationships of the noble crayfish in Europe and identifies the genetic make-up and the biogeographical patterns of the species in its southern range limit.The noble crayfish  were used in order to elucidate the genetic structure and diversity of the noble crayfish in Europe. Two of the six European haplotypic lineages, were found exclusively in Greece. These two lineages exhibited greater haplotypic richness when compared with the rest four  while they showed high genetic diversity. Divergence time analysis identified that the majority of this divergence was captured through Pleistocene, suggesting a southern glacial refugium . Furthermore, six microsatellite markers were used in order to define the factors affecting the genetic structure and demographic history of the species in Greece. The population structure analysis revealed six to nine genetic clusters and eight putative genetic barriers. Evidence of bottleneck effects in the last ~5000 years  is also afforded. Findings from several other research fields  have been utilized to perceive the genetic make-up of the noble crayfish.A. astacus. Such refugia have served as centres of expansion to northern regions. Recent history of the noble crayfish in southern Balkans reveals the influence of environmental  and anthropogenic factors.The southernmost part of Balkans has played a major role as a glacial refugium for The online version of this article (doi:10.1186/s12862-017-0971-6) contains supplementary material, which is available to authorized users. Quaternary climatic oscillations have influenced the flora e.g. –3) and f and f3])More specifically, Greece with its complex geographic landscape , coupledAstacus astacus). It is a well-established European freshwater crustacean and its distribution expands from Norway (North end) to Greece (South end) and from France (West end) to Russia (East end) [An example of obligate freshwater species that could serve as a “model organism” for tackling historical events and biogeographical processes is the noble crayfish (ast end) . It playast end) . The speast end) ) and it ast end) , 3. Sexuast end) , 20. Femast end) . The estast end) , anecdotast end)  or even ast end) ) estimatast end)  and is cast end) .The economic and cultural value of the noble crayfish is well known in the Central and Northern Europe . At the Translocations may affect noble crayfish by influencing its range expansion, gene flow and gene pool, as identified by several studies using various molecular markers , 17. It In the past, increasing attention was paid upon the genetic structure of the noble crayfish populations using allozymes . It should be noted, there are a few studies incorporating Greek crayfish to their genetic analysis, but they all refer to the stone crayfish, rrentium , 35. Therrentium  and refeAstacus astacus populations in the southernmost Balkan Peninsula, which has not been included in previous assessments [A. astacus in Europe and the genetic structure of the species in its southernmost distribution range) extensive sampling in the continental Greece was performed in an exhaustive manner. The genetic structure of the species is meant to be utilized as a baseline for management and conservation activities.The lack of genetic information on the noble crayfish of Greece, coupled with the particular geographic features of the region, initiated the present study. This study largely focuses on essments , 29 althessments , microsaTwo hundred eighty four potential noble crayfish sites have been investigated in continental Greece. The selection of the potential sites was inferred from the bibliography  a partial 16S sequence using 1471 and 1472 primers , and 2) The dataset used for the analysis comprised 37 randomly chosen noble crayfish  collected from 21 sites of Greece  values, densities plots and trace logs for each parameter were visualized in Tracer v. 1.5 [6 generations (10%) were discarded as burn-in. ESS values for all parameters were >486, larger than the threshold value of 200 identified by Tracer v. 1.5. The best fit tree was found using the Maximum clade credibility tree option in the Target tree type implemented in TreeAnnotator v. 1.8. Graphical representation of the relation of the number of haplotypes and samples with the phylogroups, were implemented in R v. 3.1.2 [Phylogenetic inference was performed using a strict clock model and a coalescent tree prior in Beast v. 1.8 . The Marr v. 1.5 . The firv. 3.1.2 . Pairwisv. 3.1.2 .Astacus astacus. In order to estimate divergence time, different mutation rates were taken into account: 1) mitochondrial Arthropod substitution rate , and 2)on years . Both apon years , 35.n < 6); Table The analysis comprised 278 samples from 16 sites [i.e. from the 21 sampled sites, 5 sites were excluded from the subsequent analysis due to low sampling size . Allele scoring was facilitated by using the same individuals for each locus as a reference sample between gels, in combination with the molecular genetic marker . Scoring accuracy was increased by independent genotyping of the samples by two researchers.Microsatellite analysis was performed in a semi-automated Li-COR® 4200 DNA Analyzer , using 6.5% acrylamide sequencing gels (Li-COR®KB Plus™). Data was genotyped in Saga4 repetitions). Mean number of alleles per locus, allele frequencies and observed, expected and unbiased expected Nei’s heterozygosity [4 generations, 20 batches and 5000 iterations per batch. Significance was assessed following the sequential Bonferroni procedure [IT, FIS and FST) were computed in FSTAT v. 2.9.3.2 [STCalc v. 2.2 [ST (104 permutations).Genotyping errors were assessed using Micro-Checker v. 2.2.3  , RST and geographic distances among sites were analysed using Mantel test with 104 randomizations, available at Isolation By Distance Web Service v. 3.23 . Geogra3 (IBDWS .5 steps and 9 x 104 thinning were used. The generation time was considered as per Pianka [2 = (α + ω)/2, where α is the age at maturation and ω the longevity. Since the longevity of the species is debated (see Background) two approaches were used: 1) the sexual maturity of the species (mean 3.5 years) and 2) the most probable longevity of 7.5 years (generation time 5.5). MsVar analysis was performed utilizing Aristotle University of Thessaloniki, Scientific Computing Office, IT Center, Institutional Computer Cluster resources . 50% of each chain was discarded as burn-in. The convergence among MCMC runs was assessed visually and via the Gelman & Rubin [Effective population size was estimated using ONeSAMP , which rr Pianka , using t & Rubin  and Broo & Rubin  statisti & Rubin . Point e & Rubin . Hedge’s & Rubin .The relative probability of the demographic event was assessed using approximate Bayes Factors (BF). A generation time of 5.5 for the noble crayfish was used in the analysis. Different levels of BF were considered as in Salmona et al. . BFs wer2 = 1.51, df = 165, p > 0.05 and COI: χ2 = 9.26, df = 207, p > 0.05). The substitution saturation test showed no significant saturation on either gene, since the index of substitution saturation (Iss for 16S = 0.20 and Iss for COI = 0.025) was lower than the critical value (Iss.c for 16S = 0.685 and Iss.c for COI = 0.686), with T = 41.104, df = 333, p < 0.001 for 16S and T = 73.571, df = 349, p < 0.001 for COI.The full dataset comprised 66 concatenated haplotypes derived from the combination of 53 unique haplotypes for COI and 17 unique haplotypes for 16S. In COI alignment no gaps were present, whereas in 16S alignment one or two gaps were observed. The aligned final dataset had a total length of 684 bp, with 334 bp for 16S and 350 bp for COI. The best fit model generated for 16S was HKY + I  and for The tree inferred from the Bayesian phylogenetic analysis revealed six distinct genetic clades Fig. . PhylogeAll newly identified haplotypes were unique to Greece, and represent 36.4% of the total number of haplotypes (24 out of 66 haplotypes). The percentage of the phylogroup-specific haplotypes divided by the number of individuals (V) was greater than 58.06% in both phylogenetic lineages G1 and G2. In contrast, the percentage V for phylogroups G3 to G6 was between 4.87% and 16.32%  from the remaining groups of Europe (G3 to G6) at 1.765 ± 0.0036 MYA  due to their negative values (between −0.271 in LOG and −0.033 in AA2). Maximum value of FIS was recorded at AA1  to 5.57 (KAS) and the mean observed heterozygosity ranged from 0.19 (KRN) to 0.77 (KAS and DOX) (Table ST = 0.4 and RST = 0.39), with the estimated values of FST generally greater than RST for most of the loci  to 0.7499 (KRN-SKR) and RST from −0.0325 (KLV-NEO) to 0.8778 (KRI-KRN) . The overall genetic heterogeneity was high (FThe inferred number of clusters (K) was nine for the analysis using Structure software  agreed with the occurrence of all barriers  and present log10(N0) population sizes  among the genetic clusters ranged from 4.97 to 5.51 (Table 10(N0) varied between 0.5 and 1.42  (Table 10(N0/N1)) of the genetic clusters decreased by 14 to 20%   in Europe  is in accordance with the wide geographic range of the species in this area [A. astacus may be biased by the distribution of the sites within each haplogroup and the number of individuals per haplogroup in the Balkan Peninsula. These findings may corroborate the taxonomical division of the noble crayfish, proposed by Karaman [A. astacus astacus, A. a. colchicus and A. a. balcanicus. Based on our results, it is impelling to suggest that the Greek phylogroups may represent the subspecies A. a. balcanicus. Haplotype groups G3 to G6  and obsn Europe . The pren Europe . Neverthn Europe , 29 and n Europe , 91).The phylogroups of the noble crayfish diverged during Early and Middle Pleistocene. Specifically, the estimated average divergence times were between ca. 0.3 and 1.8 MYA Table . In the The noble crayfish in Greece is confined in the upper parts of river systems, with a limited, scattered, discontinuous and grouped distribution  . Struct < 0.723 , 29 corrST and RST values between sites from different river catchments . Information on the status of the species (Table The identified nine clusters (K = 9) revealed a relation between them and the geographic landscape of Greece Figs.  and 5. Fes Table  discloseA. astacus is prohibited for less than 7 cm TL , while the minimum allowed fishing length is 10 cm TL . Similar phenomena have also been observed in Norway [Person(s) involved in translocation of the noble crayfish in Greece had not followed good conservation practices, such as similarities or dissimilarities of habitats, riverine systems or genetic make-up of the species  insinuates a severe decrease in the population size during the Holocene last ~5000 years), resulting in small effective population sizes , it is difficult to distinguish climate-induced variations from human-mediated influence on riverine systems of Greece , 110. HiA. astacus populations from Southern Balkans (Greece) is high and they form a distinct group when compared with their European counterparts. The divergence time analysis implied a potential differentiation of the species during the Early and Middle Pleistocene (between ca. 0.3 and 1.8 MYA). During that period, several Pleistocene climatic oscillations and geological processes corroborate the inferred divergence. Therefore, we consider that the area of the Southern Balkans may have played an important role in shaping the genetic make-up of A. astacus populations . Microsatellite analysis focusing on the noble crayfish from its southernmost distribution  disclosed six to nine distinct clusters. The combination of the inferred genetic clusters with the genetic barriers and the status of each sampled site  unraveled the influence of several factors. Specifically, it was deduced that the landscape of the region (mountains and river systems) and human related interventions (mostly translocations) were the main factors affecting the genetic structure of the noble crayfish. Finally, it was figured out that during mid to late Holocene, a severe decrease in the effective population size of the Greek noble crayfish took place. During that time period, several climatic, geological and anthropogenic processes occurred influencing the distribution of noble crayfish in the southernmost Balkan Peninsula (i.e. Greece). It is concluded that the noble crayfish has been primarily influenced by physical processes (climate and geology), while recently, anthropogenic implications (mainly translocations by humans and niche degradation) may have affected its genetic and geographic structure.The noble crayfish revealed a high genetic diversity, with its southernmost range limit playing a crucial role. Mitochondrial analysis (16S and COI) inferred six phylogroups; two of them (corresponding to the southernmost distribution of the species) were identified for the first time. Genetic analyses revealed that the genetic diversity of Additional file 1:List of sequences used in the mitochondrial analysis. A detailed table with the information of each sequence used is given. The information comprises the name of the site, country of origin, haplotype, GenBank Accession numbers and bibliographic references. (DOC 362 kb)Additional file 2:The modified conditions used for the PCR and amplification of each microsatellite loci are given. (DOC 32 kb)Additional file 3:Starting values  for hyperpriors (log(N0), log(N1), log(Θ) and log(T)) for each independent MCMC run. (DOC 30 kb)Additional file 4:Pairwise within and between genetic distances of the haplotype groups (G1 to G6) for COI and 16S. (DOC 56 kb)Additional file 5:P-value for Hardy-Weinberg equilibrium test are listed for the microsatellite loci and the sampling sites. (DOC 140 kb)Genetic diversity of the microsatellite loci and sampling sites. Table with the number of alleles, allelic richness, number of private alleles, expected heterozygosity, unbiased expected Nei’s heterozygosity, observed heterozygosity, inbreeding coefficient and exact Additional file 6:IT, FIS and FST) and stepwise mutation model (RST). (DOC 30 kb)Fixation indices for all microsatellite loci. Values of the fixation indices for all microsatellite loci based on infinite allele model Estimated pairwise FAdditional file 8:Inference of number of clusters. The file contains graphical representations of a) DeltaK for each K (1 to 16) produced by Structure Harvester and b) Bayesian information criterion (BIC) for every number of clusters, using DAPC. (DOC 108 kb)Additional file 9:Assignment probability (K between 6 and 9) and assignment probability per cluster, as identified by DAPC analysis. (DOC 35 kb)Additional file 10:Graphical representations of the DAPC scatterplots of the first two principal components for K between 6 and 8. (DOC 325 kb)Additional file 11:ST, FST/(1-FST) or RST) for all sites. (DOC 637 kb)Mantel test correlation between geographical distance (kilometers) and genetic distances  sixteen sampling stations, all microsatellites loci and the FST, c) sixteen sampling stations, each microsatellite locus  and FST, d) nine genetic clusters (of the Structure software), all microsatellites loci and the genetic distance DCE, and e) nine genetic clusters (of the Structure software), all microsatellites loci and the FST. (DOC 10235 kb)Graphical representation of genetic barriers (1 to 10) based on: a) sixteen sampling stations, all microsatellites loci and the genetic distance DAdditional file 13:Map of the sampled noble crayfish in Greece (Copyright © 2014 Esri and its licensors. All rights reserved) where abbreviation of the sampling sites, names of major rivers, mountains and sierra, type of genetic markers used  and elevation are given. (DOC 2355 kb)Additional file 14:Point estimates with their upper Confidence Interval (CI) for all parameters (log(N0), log(N1), log(Θ) and log(T)) and every genetic cluster for a generation time of 3.5 and 5.5. (DOC 48 kb)Additional file 15:Posteriors and priors densities plots for the past (N1) and present (N0) population sizes of each genetic cluster (cluster 1 to 9) and the global data set . (DOC 859 kb)Additional file 16:Posteriors and priors densities plots for time since the population decline (T) for each genetic cluster (cluster 1 to 9) and the global data set . (DOC 674 kb)Additional file 17:The relative probability of different hypotheses was assessed using approximate Bayes Factors (BF). A generation time of 5.5 for the noble crayfish was used in the analysis. Hypotheses were examined for the past 12,000 and 7000 years. (DOC 2096 kb)


Solea senegalensis and Solea aegyptiaca. The relationship between genetic composition at nuclear loci and individual body shape variation was studied in four populations sampled across the hybrid zone located in northern Tunisia. A strong correlation between genetic and phenotypic variation was observed among all individuals but not within populations, including the two most admixed ones. Morphological convergence between parental species was observed close to the contact zone. Nevertheless, the samples taken closest to the hybrid zone also displayed deviant segregation of genotypes and phenotypes, as well as transgressive phenotypes. In these samples, deviant body shape variation could be partly attributed to a reduced condition index, and the distorted genetic composition was most likely due to missing allelic combinations. These results were interpreted as an indication of hybrid breakdown, which likely contributes to postmating reproductive isolation between the two species.Hybrid zones provide natural experiments where new combinations of genotypes and phenotypes are produced. Studying the reshuffling of genotypes and remodeling of phenotypes in these zones is of particular interest to document the building of reproductive isolation and the possible emergence of transgressive phenotypes that can be a source of evolutionary novelties. Here, we specifically investigate the morphological variation patterns associated with introgressive hybridization between two species of sole,  These zones have been shown to act as semipermeable barriers to gene flow that selectively filter introgression by preventing genomic regions involved in reproductive isolation to be exchanged among species , Bizerte Lagoon (n = 47), Gulf of Tunis (n = 15), and Kerkennah Islands (n = 14)  Figure , these l2.2GH2, Am2B‐2, CK6‐2, and Met1) that were previously used to distinguish S. senegalensis and S. aegyptiaca outside the hybrid zone by Ouanes et al. (Whole genomic DNA (40 ng/μl) was extracted from fin clips using Qiagen DNeasy blood and tissue kit. Four EPIC loci  based on group centroids as implemented in Genetix 4.05.2  to capture the multidimensional variation associated with body shape , while specimens sampled in Kerkennah Islands were located on the negative part of this axis (S. aegyptiaca side of the hybrid zone)  based on genetic data clearly separated the two samples from the periphery of the contact zone on the first axis, which explained most 50.3%) of the variation contained in the dataset. Tabarka specimens were positioned on the positive side of the first axis (% of the ca Table . The sec3.2p = .12), indicating the absence of residual allometric effects after Procrustes transformation. The first axis of the CDA explained 60.9% of body shape variation and separated the four samples according to their geographic position relative to the center of the hybrid zone  from the two peripheral ones  . Finally, performing the same Procrustes analysis within each samples separately revealed no significant correlation between genotype and phenotype variation   obtained from genetic and morphometric data analysis showed a high correlation between shape and genetic variation (m) Figure b. In the) Figure a. When p) Figure a and 4a.) Figure a reveale4S. senegalensis and S. aegyptiaca is in good agreement with previous results by Ouanes et al. . The fact that morphological variation closely parallels genetic variation along the first axis in both analyses is expected under a simple dilution of additive genetic effects. Therefore, our data support that at least part of the geographic variation in body shape is explained by the neutral introgression of additive morphological traits along a gradient of admixture. Nevertheless, we found no significant correlation between morphological and genetic variation within samples, including those from the inner part of the hybrid zone. This was expected as most variation occurs between parental populations. Moreover, we only used a few genetic markers that are extremely unlikely to be linked with the loci controlling shape variation.Our analysis of genetic variation across the hybrid zone between s et al. . Here, uS. senegalensis population introgressed by S. aegyptiaca alleles, whereas it is the opposite for the Gulf of Tunis. Phenotypic effects affecting all admixed genotypes in the same direction and contrasting with the absence of such effects in both parental populations could concern some fitness‐related trait reflecting the general performance of individuals, such as the condition factor. Interestingly, the deformation grid on the positive part of axis 2 shows a less convex shape of the ventral part in the inner samples which could reflect a lower condition of hybrid individuals compared to those from peripheral samples. In parallel, we also detected a signal of genetic distortion on axis 2 of the genetic correspondence analysis. This suggests the existence of preferential allelic combinations that cannot simply result from the neutral dilution of the parental genomes in introgressive crosses. Although we only used four neutral markers for genotyping, these selective effects can be likely captured due to genomewide association because of the existence of numerous genetic incompatibilities between highly differentiated  genomes. The observed genetic distortions possibly reflect selective elimination of certain allelic combinations from the admixed populations, which may due to some form of hybrid breakdown. Such distortions in the wild closely reflect segregation distortions commonly observed in experimental crosses between divergent species (Casellas et al., Our analysis reveals a different signal of variation along the second axis of the morphological space. On this axis, the two inner populations are clearly similar to each other while being differentiated from the parental outer samples which are themselves not differentiated along axis 2. This second pattern deviates from the previously mentioned mechanism of morphological convergence as it cannot be explained by additivity of the morphological QTLs alone. Thus, it suggests the existence of more complex epistatic and pleiotropic effects due to hybridization between divergent genomes. This could be due for instance to a mechanism of hybrid breakdown acting on different types of hybrid pedigree, as the population of Bizerte Lagoon is a aegyptiaca alleles inside the senegalensis background generates a positive transgression in the Bizerte sample and the opposite is true for the Gulf of Tunis sample.Finally, a more classical pattern consistent with phenotypic transgression due to QTLs with antagonistic effects is evidenced along axis 3 of the morphospace. This effect is expected to occur in opposite directions on both sides of a hybrid zone as exemplified in Table To conclude, our study of the relationship between genetic composition and morphological shape highlighted mainly two key findings. First, when considering all four population samples, a significant correlation between genetic and phenotypic data was observed. This is expected because of the global linkage generated inside a tension zone maintained by equilibrium between counter‐selection of recombinant genotypes and gene flow from parental species. Second, deviant segregation of genotypes and phenotypes evidenced by transgressive positions of the most introgressed inner samples along axes 2 and 3 of the multivariate analyses could be interpreted both in terms of signature of hybrid breakdown and recombination of QTLs with antagonistic effects. Altogether, these results call for a genomewide assessment of the architecture of gene exchange between these two hybridizing species of sole. Such approach would be useful to specify what proportion of the genome can still be exchanged neutrally between species, and what are the fitness consequences of interspecific gene flow in recombinant genotypes.None declared.