Pan troglodytes ellioti and P. t. trogolodytes. Their ranges converge in central Cameroon, which is a geographically, climatically and environmentally complex region that presents an unparalleled opportunity to examine the roles of rivers and/or environmental variation in influencing the evolution of chimpanzee populations.The mechanisms that underlie the diversification of tropical animals remain poorly understood, but new approaches that combine geo-spatial modeling with spatially explicit genetic data are providing fresh insights on this topic. Data about the diversification of tropical mammals remain particularly sparse, and vanishingly few opportunities exist to study endangered large mammals that increasingly exist only in isolated pockets. The chimpanzees of Cameroon represent a unique opportunity to examine the mechanisms that promote genetic differentiation in tropical mammals because the region is home to two chimpanzee subspecies: P. t. troglodytes, whereas environmental variation was found to play a much larger role in shaping patterns of genetic differentiation in P. t. ellioti.We analyzed microsatellite genotypes and mtDNA HVRI sequencing data from wild chimpanzees sampled at a fine geographic scale across Cameroon and eastern Nigeria using a spatially explicit approach based upon Generalized Dissimilarity Modeling. Both the Sanaga River and environmental variation were found to contribute to driving separation of the subspecies. The importance of environmental variation differed among subspecies. Gene-environment associations were weak in We found that both the Sanaga River and environmental variation likely play a role in shaping patterns of chimpanzee genetic diversity. Future studies using single nucleotide polymorphism (SNP) data are necessary to further understand how rivers and environmental variation contribute to shaping patterns of genetic variation in chimpanzees.The online version of this article (doi:10.1186/s12862-014-0274-0) contains supplementary material, which is available to authorized users. The Gulf of Guinea and the Congo River Basin biomes Figure  collectiTropical Africa is also noted for exhibiting a complex forest history that has undergone considerable change along with the Earth’s climate, and the behavioral ecology of African primates often reflects a shared history with their habitats -7. This Mandrillus leucophaeus/M. sphinx, Cercopithecus erythrotis/C. cephus, C. nictitans martini/C. n. nictitans, C. pogonias pogonias/C. p. grayi, P. t. ellioti/P. t. troglodytes and possibly, Gorilla gorilla diehli/G. g. gorilla [Pan troglodytes) from bonobos (P. paniscus) [P. t. ellioti and P. t. troglodytes [Rivers have also been proposed to play an important role in delimiting the distribution of many species ,3,10. Fo gorilla -18. The aniscus) , and theaniscus) ,19. Chananiscus) . The Sanglodytes -23, but glodytes . And altglodytes ,23,25.et al. [Until very recently, the role of ecological gradients in driving the diversification of tropical taxa has been under-appreciated. Ecological gradients are zones of transition between habitat types that display marked differences in ecological variables  across their range. These gradients have been linked with driving adaptive variation in several taxa in different parts of the world, for example, suture zones in Australia  and moreet al.  as an ‘eet al. , reptileet al.  and birdet al. ,32. CompP. t. troglodytes occurs south of the Sanaga River, while P. t. ellioti occurs north. P. t. ellioti may be further subdivided into two additional populations: a population that occurs in forested regions of western Cameroon, P. t. ellioti (Rainforest), and a second population that occupies central Cameroon, P. t. ellioti (Ecotone) [Recent analyses suggest that the two subspecies of chimpanzees present in Cameroon may be divided into genetically distinct populations: Ecotone) . While iEcotone) , and allEcotone) . This suThe available genetic data for wild chimpanzees sampled across this region consist of 21 autosomal microsatellite loci and the HVRI region of the mtDNA . Since ti) the diversity and distribution of alleles; (ii) the dates of divergence between populations; (iii) the location of barriers to gene flow; and (iv) the demographic characteristics of populations (e.g. population size and population growth). This study is an extension of a complimentary paper [ii and iv) of chimpanzees in Cameroon and Nigeria. That study found evidence that positive selection likely plays a role in shaping patterns of chimpanzee genetic diversity, and that this may be the results of landscape variation. This study is the next step, and focuses on using a spatially explicit modeling technique, Generalized Dissimilarity Modeling (GDM) [i) and the location of barriers to gene flow (iii).Table ry paper  which long (GDM) , to quanFST) and mtDNA data against straight-line geographic distance, topographic, climatic and vegetation variables assumed to contribute to chimpanzee habitat ecology and biogeographic boundaries. We ran models using: (i) geographic distance between sample locations only; (ii) environmental variables only ; (iii) environmental variables and geographic distance between sample locations, and (iv) environmental variables, distance and riverine barrier layers (‘complete barrier’ layers and resistance surfaces). The percent of genetic variation accounted for by the microsatellite genotype data and the mtDNA data for three different classes of predictor variables are shown in Table The data set used for analysis was used in a previous study on the population genetic structure of chimpanzees . The genFST) between sample locations at autosomal microsatellites are shown in Additional file FST and predictor variables was then interpolated across the study area using the GDM method, which revealed that geographic distance alone accounted for 4% and environmental variation alone accounted for 10% of the observed genetic variation  population . In western Cameroon and eastern Nigeria elevational gradients were once again observed to account for the highest proportion of variation  data, for example, will allow for quantifying these associations, and provide improved spatial and temporal resolution to disentangle the relative role of Pleistocene refugia in generating genetic diversity. The evolutionary impacts of the environmental gradient in Cameroon have been examined in a small number of taxa ,31,32, bMandrillus leucophaeus/M. sphinx, Cercopithecus erythrotis/C. cephus, C. nictitans martini/C. n. nictitans, and C. pogonias pogonias/C. p. grayi [In addition to being an important region for chimpanzees, there are also a number of other primate species and subspecies that are presumably influenced by the Sanaga River, including: p. grayi ,13-15,21p. grayi , it is iThere are many spatially explicit genetic analysis methods that can interpolate a population’s genetic structure across a study region ,40-45. RFST) or morphological diversity among populations. The relationships between these types of variables are often non-linear, such as pairwise genetic distances, which are scaled between 0–1, while environmental variables may increase or decrease beyond this scale. GDM’s use of non-linear regression algorithms is especially appropriate for understanding the complex influences that landscape features have on shaping patterns of genetic diversity [There are a variety of spatial modeling techniques, which include simple regression methods, such as spatially auto-correlated principal components analysis , and raniversity .We used DNA from non-invasively collected chimpanzee hair and fecal samples from a previous study . All samFST values between sample locations using Arlequin version 3.5 [For the microsatellite data set, we calculated pairwise sion 3.5 . Each pasion 3.5  indices sion 3.5 ,23,58,59sion 3.5 . We usedsion 3.5 ,60, and We obtained environmental data layers from several remote sensing platforms to characterize the habitats available to chimpanzees across the study region. These layers are described in Additional file P. t. ellioti  and P. t. troglodytes . We used a logistic distribution of values from the ENM to establish a threshold in which all cells with a value of less than 10% suitability were excluded from the training mask used as a background for all GDMs, based on a visual comparison of the maps. We excluded these cells to accurately estimate the known range of chimpanzees in the region [In order to run the model as accurately as possible, we restricted all spatial analysis to areas within Cameroon and eastern Nigeria where chimpanzees are likely to occur at present time. We used an ecological niche model (ENM) of chimpanzees specifically created for chimpanzees in the study region  that wase region . By excle region . The spahttp://www.r-project.org) using the topography, vegetation and climate data layers. We clipped each data layer to the extent of the estimated chimpanzee distribution mask. This revealed that several of these variables were highly cross-correlated  a cost-effective route between locations and (ii) a matrix of pairwise values representing the incurred cost of travel between locations [We also used resistance surfaces to understand how various landscape features affect gene flow between populations by using an approach that is rooted in circuit theory and implemented in the program CIRCUITSCAPE . CIRCUITocations ,70. CIRCocations , and hasocations ,69,71. Tocations , and theocations ,72. In tk-nearest neighbors interpolation [We used GDM  to quantpolation . We ran polation . For eacP. t. ellioti) or south of the Sanaga River (P. t. troglodytes). The three-population grouping included sample locations south of the Sanaga River (P. t. troglodytes) and we further subdivided presence points north of the Sanaga River into two groups, P. t. ellioti (Rainforest) and P. t. ellioti (Ecotone). We only ran the GDM for the P. t. troglodytes group once, as this group was composed of identical sampling locations. The training masks for all GDM’s using the two- and three-population groups were obtained from the MAXENT models of corresponding groups [We ran additional GDM’s for individual population subsets for two- and three-population models as identified by a previous study . In the g groups .


Cyanobacterial oligopeptides comprise a wide range of bioactive and/or toxic compounds. While current research is strongly focused on exploring new oligopeptide variants and their bioactive properties, the biological role of these compounds remains elusive. Oligopeptides production abilities show a remarkably patchy distribution among conspecific strains. This observation has prompted alternative approaches to unveil their adaptive value, based on the use of cellular oligopeptide compositions as biomarkers of intraspecific subpopulations or chemotypes in freshwater cyanobacteria. Studies addressing the diversity, distribution, and dynamics of chemotypes in natural systems have provided important insights into the structure and ecology of cyanobacterial populations and the adaptive value of oligopeptides. This review presents an overview of the fundamentals of this emerging approach and its most relevant findings, and discusses our current understanding of the role of oligopeptides in the ecology of cyanobacteria. Cyanobacteria are among the oldest and most successful forms of life still present on Earth. Ancient cyanobacteria are believed to have been instrumental in the transition of the atmosphere to oxic conditions. Today, these phototrophic prokaryotes are present in almost every habitat on Earth, including terrestrial, brackish, marine, and freshwater environments. They can be found in extreme environments ranging from arid desert areas and polar regions, to the inside of rocks (endolithic), or as symbionts with other organisms. The remarkable adaptability of cyanobacteria is attributable, in part, to the production of a wide repertoire of secondary metabolites with diverse bioactive activities, which likely contributed to the colonization of such a range of ecological niches. The term secondary metabolism is generally used to refer to metabolic pathways that are not directly involved in the growth, development or reproduction of the organism. In the case of cyanobacteria, secondary metabolites present a vast chemical diversity and are widespread across cyanobacterial taxonomic units. A growing number of these compounds has been isolated and characterized from cultured strains and field samples. Their discovery has been driven by the growing interest toward their potential pharmacological applications or, alternatively, their toxic effects on human and animal health. Resulting from this dual interest, we perceive in the literature a somewhat artificial separation of these metabolites into cyanotoxins and other bioactive compounds. This dichotomy has largely influenced the perspective by which the study of cyanobacterial secondary metabolites has been approached in the last decades. Exceptions notwithstanding, much of the research effort has targeted these compounds toward their potential pharmacological uses, e.g., as antibacterial, antiviral, anticoagulant or anticancer compounds ,2,3,4,5,Synechococcus and Prochlorococcus populations were shown to subdivide into distinct ecotypes with different niche preferences ) changes ,47. A re changes .In addition, single NPRS gene clusters can be responsible for the synthesis of multiple chemical variants . A significant part of this structural variability is achieved by relaxation of substrate specificities, resulting in variable incorporation of amino acids at some residues and thereby leading to the existence of multiple chemical variants synthesized by the same enzyme complex ,50. SubscpcBA) [vice versa [Oligopeptides are widespread in at least four of the five cyanobacterial taxonomic orders or subsections . HowevercpcBA) ,62, or tcpcBA) ,63. Simice versa ,20,64,65The rapid development of bioinformatic tools has contributed to the increased discovery of new microbial secondary metabolites in the last years e.g., ,67,68). ,68. 67,6Instead, Matrix Assisted Laser Desorption/Ionization–Time of Flight Mass Spectrometry (MALDI-TOF MS) has become the technique of choice for chemotyping applications. MALDI-TOF MS enables a rapid determination of intracellular constituents from fresh biomass. As a result, this technique has been increasingly used for the analysis of taxon-specific microbial metabolite patterns for the rapid identification of infective or pathogenic bacterial taxa ,71. Simii.e., co-crystallization). A high energy beam laser is then focused on the sample and ions are thereby produced. The co-crystallizing compound partially absorbs the energy of the ionizing laser, allowing for a soft ionization that maintains the integrity of the molecules during analysis and producing easily identifiable singly-charged ions. Ions are accelerated in an electromagnetic field and guided through a drift-free area, where they travel at different velocities depending on their respective mass/charge ratios. Analyte separation is thereby achieved. Accelerated molecules eventually collide against a detector that provides a resolved mass spectrum. MALDI-TOF mass spectrometry consists in the ionization, separation and detection of analytes. A small amount of fresh cell biomass  is mixed with a co-crystallizing matrix. Most commonly used matrices are low weight, organic, aromatic acids, usually 2,5-dihydroxy benzoic acid (DHB) or α-cyano-4-hydroxycinnamic acid (CHCA), that are dissolved in a mixture of solvents like water, ethanol and acetonitrile, and acidified by a strong acid, usually trifluoracetic acid . Upon soA commonly highlighted limitation of MALDI-TOF MS is that analysis only yields qualitative (or semi-quantitative) results, as peptides differ in their ionization due to differences in amino acid compositions and hydrophobicity. For example, peptides containing arginine in their structure show preferential ionization . SimilarOligopeptides are suitable biomarkers of cyanobacterial subpopulations, as their synthesis at the individual level is determined by the presence or absence of the respective gene clusters. Given the patchy distribution of these clusters among co-existing strains, cyanobacterial cells can present any combination of these clusters and thereby exhibit distinctive qualitative oligopeptide compositions, which delineate different chemotypes ,59. CyanMicrocystis and Radiocystis. In particular, nutrient deprivation and high light resulted in the disappearance of minor microcystin variants  from the peptide fingerprints, presumably due to physiologically induced reductions in oligopeptide synthesis. Alternatively, several aeruginosin, cyanopeptolin, and microcystin variants could only be detected at high cell densities, remaining otherwise undetectable under low cell concentrations [Several studies have evaluated the stability of oligopeptide patterns as reliable subpopulation markers under a range of environmental conditions. Whereas these studies report overall stability of peptide fingerprints, in some cases, nutrient limitation, high irradiance ,81 and ctrations . Difficutrations ,83, althtrations  and intrtrations  were shotrations ,86. Thestrations ,81,82. Hi.e., chemotypes display identical oligopeptide profiles, [i.e., chemotypes share resembling, but not necessarily identical peptide compositions, [Perhaps due to such inconsistencies, existing studies using oligopeptides as subpopulation markers often apply heterogeneous criteria for chemotype delineation, each of which offers a dissimilar trade-off between resolution and stability. Criteria based on (1) single oligopeptide classes ; (2) “srofiles, ,63); andsitions, ,65) coexsitions, , whereassitions,  and aminsitions,  result isitions,  and refeChemotype delineation based on individual peptide variants, however, reveals a picture of high complexity. The extent of structural variability throughout oligopeptide classes could be enormous. Microcystins present over 100 structural variants, whereas other oligopeptides may bear even more structural variability. Cyanopeptolins, for instance, display variable amino acids at all positions, except for two highly conserved residues. One could hence argue that the potential structural diversity of oligopeptides, considering combinatorial incorporation of amino acids and modifications at variable residues across peptide classes, can give rise to up to hundreds, if not thousands of individual oligopeptides, and, thus, may lead to a virtually endless number of chemotypes. However, field studies have shown that naturally-occurring chemotypes delineate intraspecific linages that present unique ecological features ,20 and, Planktothrix chemotypes in a Norwegian lake for over 30 years [Research addressing the composition and dynamics of cyanobacterial oligopeptide chemotypes has revealed that natural populations exhibit a mosaic structure of coexisting strains with different oligopeptide production abilities. Chemotype assemblages showed to be stable over for long periods of time in their respective ecosystem, as evidenced by the continuous presence of four different 30 years . In contAs a result of the different chemical profiles among coexisting strains, the phenology of individual chemotypes dynamically affects the properties of the whole-population with regard to average oligopeptide contents , includiMicrocystis, is well described. Toward the end of the blooming season colonies settle down to the sediments, where they spend the winter in the so-called overwintering phase. Toward spring, benthic colonies regain buoyancy and are recruited back into the water column, giving rise to the summer pelagic population, thereby closing the cycle [Microcystis is traditionally supposed to be triggered by physical factors , chemotype segregation among benthic and pelagic habitats indicates that reinvasion might be more complex than previously described and suggests that recruitment might operate as a selective process that shapes strain compositions in benthic inocula. In fact, promoted recruitment of microcystin-producing strains has been reported in genus Microcystis [Tracking individual oligopeptide-based subpopulations in their natural habitat revealed that cyanobacterial chemotypes delineate subpopulations that interact differently with their environment ,20. The he cycle . In thishe cycle ). Therefrocystis ,96, suppOver the course of the season, the temporal evolution of the benthic composition of chemotypes reflects with fair fidelity the major shifts occurring in the overlaying water column . This inIn light of these considerations, we believe that the ecological relevance of the subspecific level in cyanobacteria as operating basis for biological process is indisputable. Therefore, revisiting the ecology and life-cycles of cyanobacteria under perspectives that recognize the intraspecific dimension of their populations is needed to increase our understanding on how the interplay of ecological processes dynamically shape the populations along their life-cycle at its different stages. Moreover, addressing differential behaviors among chemotypes in their environment constitutes a promising way to identify the selective forces driving their dynamics and thereby unravel the biological role of oligopeptides in the producing organisms.The notion that chemotypes represent ecologically relevant units raises important research questions: Which factors drive the succession of chemotypes in nature? What is the adaptive value of oligopeptides in the ecology of chemotypes? Furthermore, what forces are behind the subdivision of cyanobacterial populations into distinct chemotypes? Do chemotypes represent discrete evolutionary units? If so, what is their evolutionary history? In our opinion, these questions delineate current frontiers of knowledge on the topic. The adaptive value of cyanobacterial oligopeptides remains, despite intensive research, still ambiguous. A number of biological functions have been proposed, including their role as defense compounds against grazers ,100,101,Traditional approaches to address this issue typically focus on individual peptides to investigate their effects on the producing organisms or their competitors. Alternatively, cyanobacterial chemotyping constitutes a complementary approach based on the study of oligopeptide-based subpopulations to infer a general biological role of these compounds. Combined interpretations of the findings stemming from these approaches have contributed to the formulation of novel hypotheses on the adaptive value of oligopeptides that are consistent with observations at different organizational levels . Chemotypical subpopulations in their environment can be controlled either by bottom-up or top-down mechanisms. Bottom-up mechanisms are mediated by resource competition among subpopulations. However, although a role of oligopeptides as chelating agents putatively enhancing nutrient uptake has been proposed ,109, attAlternative notions for the existence of top-down mechanisms controlling cyanobacterial populations are not new. However, grazing and competition with other phytoplankton have been typically pointed out as their possible drivers. In fact, putative roles of oligopeptides as grazing deterrents ,99,100 oPlanktothrix mutants unable to synthesize microcystins, anabaenopeptins and microviridins, respectively, displayed higher susceptibility to chytrid infection, compared to the wild-type strain [Other proposed forms of top-down control refer to parasitic interactions. Parasites in aquatic ecosystems have traditionally been neglected, mostly due to methodological limitations. At present, however, host-parasite interactions are increasingly regarded as fundamental in the functioning of the ecosystem and considered important driving forces of ecological and evolutionary processes, such as species succession, population dynamics or gene flows ,113,114.e strain . These fPlanktothrix populations into distinct chemotypes. Under this scenario, individual chemotypes are considered evolutionary units that co-evolve with chytrid parasites. Narrow host range and high chemotype specificity of chytrid fungi are consistent with co-evolutionary models, whereas chemotype subdivision is also compatible with predicted host diversification as a strategy to resist rapidly evolving parasites. Interestingly, further studies exploring the relationship between cyanobacteria and their fungal parasites reveal that chytrid strains present chemotype-specific host preferences . The faci.e., domains mediating for the activation and incorporation of amino acids into the growing peptide chain), which are responsible for amino acid hypervariability during biosynthesis. This suggests that domains responsible for substrate promiscuity might be under positive selection and hence, the synthesis of new oligopeptide variants might be promoted, thereby further contributing to the diversifying potential of oligopeptide repertoires. In cyanobacteria, predicted host diversification might be reflected, first, at the population level by the co-existence of chemotypical subpopulations with dissimilar susceptibility to parasites. Rather than maintaining an ideal genotype, cyanobacteria may profit from preserving an array of chemotypes to prevent parasites from exploiting the whole population efficiently. The dynamic composition of chemotypes might therefore respond to top-down pressures exerted by parasites or, alternatively, other pathogens . Secondly, signs of diversification are also evident at the peptide level. It is somewhat notorious that during their evolutionary history, the vast pool of peptide congeners has not been reduced to a few efficient ones. Instead, oligopeptide synthesis is characterized by widespread structural ambiguities that result in the production of a wide array of peptide variants, which in fact exhibit different inhibitory properties ,56,57. ADifferences in oligopeptide profiles arguably imply dissimilar fitness costs among coexisting subpopulations, which likely result from energy burdens, but also from antagonistic pleiotropy associated with oligopeptide synthesis . IntraspWhile some aspects of this novel hypothesis definitely remain to be experimentally demonstrated, we believe that the coherence between the available pieces of evidence (at both the population and molecular levels), and the theoretical framework explaining the emergence of intraspecific chemotypes is too striking to be overlooked and deserves serious consideration in future research.The use of oligopeptides as cyanobacterial markers is still in its infancy, but has strongly contributed to tackle the chemical variability among co-existing cyanobacterial strains. High throughput analytical techniques like MALDI-TOF MS allow the rapid analysis of qualitative oligopeptide compositions at the individual level and, thereby, enable the delimitation of chemotypical subpopulations. Oligopeptides proofed to be in general suitable subpopulation markers, given their constitutive synthesis and their steady intracellular concentrations. The analysis of chemotypical subpopulations under natural conditions has revealed that cyanobacterial populations are comprised by a stable mosaic of coexisting chemotypes that are subject to marked seasonal dynamics. Close examinations of the succession of subpopulations in aquatic ecosystems show that chemotypes interact differently with their environment and present different ecological traits. The existence of ecologically functional units below the species level stresses the relevance of the subspecific dimension as the basis on which biological processes operate and shape cyanobacterial populations. Current research in this direction is focused on the identification of the selective forces governing the dynamics of chemotypes in nature, to disentangle the biological role of oligopeptides and to interpret cyanotoxin occurrence in an ecological context. Recent findings stemming from chemotyping approaches strongly suggest that cyanobacterial peptides are involved in defense mechanisms against fungal parasites and, most likely, other pathogens. Cyanobacterial highly diversified oligopeptide repertoires and population subdivision into distinct chemotypes have been proposed as adaptive strategies of cyanobacteria to resist rapidly evolving parasites. Whereas this novel hypothesis on their biological role remains to be experimentally confirmed and will likely shape future research directions, the use of oligopeptides as biomarkers exemplifies how much we can learn by leaving behind traditional notions considering the species as the ecologically relevant unit.


Evolutionary developmental genetics has traditionally been conducted by two groups: Molecular evolutionists who emphasize divergence between species or higher taxa, and quantitative geneticists who study variation within species. Neither approach really comes to grips with the complexities of evolutionary transitions, particularly in light of the realization from genome-wide association studies that most complex traits fit an infinitesimal architecture, being influenced by thousands of loci. This paper discusses robustness, plasticity and lability, phenomena that we argue potentiate major evolutionary changes and provide a bridge between the conceptual treatments of macro- and micro-evolution. We offer cryptic genetic variation and conditional neutrality as mechanisms by which standing genetic variation can lead to developmental system drift and, sheltered within canalized processes, may facilitate developmental transitions and the evolution of novelty. Synthesis of the two dominant perspectives will require recognition that adaptation, divergence, drift and stability all depend on similar underlying quantitative genetic processes—processes that cannot be fully observed in continuously varying visible traits. Drosophila melanogaster and Caenorhabditis elegans, attempting to bridge the gap between micro- and macro-evolution [This paper offers a quantitative genetics perspective on evolution and development. The thoughts have been shaped by our post-doctoral experiences two decades apart, working with volution ,2. We stIn order to place all of this in the broader perspective of evolutionary genetics, we present One of the more remarkable features of developmental systems is their robustness to perturbation . At the A corollary to robustness is plasticity, namely the capacity to develop different, but predictable, morphs under different growth conditions. Whether it is social insect castes, fish adopting different morphs and behaviors, or plants following a so-called norm of reaction along a cline of geographic variation, species have the ability to modulate what is normal. Often times, two or more morphs are themselves robust, indicating that stable developmental systems can exist in the same species side-by-side, posing a considerable challenge for genetic modeling of complex trait evolution . Ecologifushi tarazu pair-rule segmentation gene in Drosophila, they see that there is lability in the precise array of transcription factor binding sites across species [Although developmental systems are robust, they also demonstrate lability, the capacity to evolve rapidly. Adaptive radiations, for example of the Lake Malawi cichlids , illustr species .These twin properties of robustness and lability pose challenges for the standard genetic model of evolution, which posits that stabilizing selection maintains limited variability in populations, and that this is punctuated by periods of directional selection when a species explores a novel ecological niche. Micro-evolution concerns itself with the quantitative and population genetic processes that shape variation within a species, and it is generally assumed that macro-evolutionary trends are due to extrapolation of the same processes across time, supplemented by mechanisms that promote speciation . The rolWe certainly do not mean to imply that micro-evolutionary processes are irrelevant to macro-evolution. Rather, we think that the importance of standing variation is if anything under-appreciated . On the How does natural genetic variation produce differences among individuals, and how does it facilitate the evolution of populations? This is the business of the fields of quantitative genetics and population genetics, both of which predate modern experimental methods in sequencing and molecular genetics by decades. Important early work in these fields established empirical and theoretical foundations to evolutionary biology under the assumption that natural genetic variation was comprised of near-infinite numbers of micro-mutations. In his challenge to evolutionary biologists, Rockman  points oDrosophila, C. elegans, mice, Arabidopsis, Mimulus, sticklebacks, yeast, bacteria, humans, and many other systems [myostatin underlie selection for muscle mass in cattle [When adaptive alleles have been identified in natural populations, they are more likely to have been discovered in candidate-gene approaches than in unbiased surveys of the genome ,25. Most systems  may not  systems . Even as systems ; within n cattle , just asn cattle .The pervasiveness of polymorphisms of small effect is clearly illustrated in another maize experiment, the famous long-term response to selection. Initiated in 1896, the experiment selects on oil content, protein content and other traits and has yielded new strains with extreme values compared to the progenitors; it continues today and shows little sign of slowing . Only a Drosophila has produced a staggering literature on clinal variation, in which populations show local adaptation via shifts in allele frequency along environmental gradients [Newly arising quantitative trait alleles with tiny effect sizes have very little potential to contribute to adaptive evolution. This is because the birth of a new mutation occurs at the lowest possible frequency in a population, and with only a weakly beneficial effect an allele is more likely to be lost by chance than to become established. However, even as much of the theory explored by population genetic models relies on the premise that adaptation proceeds from new mutations, much of the empirical data from natural populations demonstrate evolutionary responses to standing genetic variation. For example, several decades of research in radients . In humaradients ,41. Stanradients ,43. Howeradients . As suchIf the infinitesimal model predominates, and most traits are highly polygenic, arising from the minute contributions of many loci, then by logical extension pleiotropy is extremely pervasive.  or a mutant allele affects two or more traits  is not strictly relevant. All that matters is whether a mutation affects more than one component of fitness , since this is what natural selection sees . For exaOne form of genotype-by-environment interaction is the phenomenon of conditional neutrality. Here, an allele segregating in a natural population will exhibit an effect on phenotype in one environment but no effect in another. This pattern has been commonly described in field studies investigating the genetic basis of local adaptation , and proex vivo perturbation by exposure to cytokines of immune stimulants such as lipopolysaccharide. It is now thought that the environment of a cell, which can include the microbiome, metabolic state, and exposure to pathogens or toxins, establishes a wide network of condition-specific regulation of gene activity. Similarly, the landscape of eQTL has been shown to differ between the normal colon and colorectal cancer. Here, an enrichment of novel mutations in the regulatory elements that contain binding sites for four different transcription factors which are themselves induced in the cancer cells, provides a direct mechanism for the condition dependence of genetic variation that only becomes relevant as disease progresses [Direct experimental evidence for the pervasive nature of condition-dependent genetic effects comes from response-eQTL studies. First described in nematodes, this is the observation that genetic polymorphisms that associate with gene expression may only have their effect in a certain environment . Specifiogresses . It is pogresses .Ubx contributed to genetic assimilation of the bithorax phenocopy, demonstrating how standard quantitative genetic processes can explain what to many was a very surprising phenomenon: That release of cryptic genetic variation can potentiate adaptive evolution. Recently, new work has challenged conventional ideas about cryptic variation [Conditional neutrality can also occur as a function of an interaction between genotypes at two or more loci. By definition, this is statistical epistasis, measured as a departure from additive effects across loci in a population. Although epistasis probably does not contribute substantially to standing phenotypic variation , geneticariation . In the ariation . The allTabby mutant mice do not just have fewer secondary vibrissae than wild-type ones , but much higher variance (between 12 and 20), tell us that perturbation can affect not just the mean, but robustness about the mean. Unfortunately, there have been few follow-up studies of a similar nature. Gibson and van Helden [Drosophila increases in the presence of a Ubx mutation, and the variance for body weight in humans is greater in individuals with the high-BMI allele at the FTO locus [Canalization is evolved robustness . The notn Helden ,67 showeTO locus . Even thTO locus ,70. ThisTO locus , and oneTO locus .et al. [TDH3 gene in yeast seems to have been more influenced by selection against expression noise than transcript abundance per se.Furthermore, we and others have consistently shown that there are vast pools of genetic variation affecting the cryptic phenotypes that are exposed by genetic or environmental perturbation. This includes homeotic and cancer-related phenotypes , embryoget al.  showed tAll of this is consistent with the notion that under persistent stabilizing selection, there is a tendency for genetic systems to become more robust. What is needed to prove that canalization is more than an obscure theoretical notion, are more field studies demonstrating that variance changes under different environmental or genetic circumstances. We suspect that systematic meta-analysis of thousands of published “common garden experiments” would in fact reveal substantial signals of condition-dependent variability. Even better would be comparisons of sister-species in different ecological niches, or of ancestor-progeny species pairs. For now, canalization, like Wright’s shifting balance theory , remainsC. elegans. The 25,000 known nematode species (and likely millions of existing species) exhibit a simple body plan and consistent wormlike appearance, but their genetic diversity places their last common ancestor as more ancient than that shared by humans and lampreys [The shared evolutionary history of all living systems is an essential justification for the use of model organisms: Studying gene function in flies and mice can provide insight into their human orthologs, for example. However, conservation of sequence identity does not always indicate conservation of function. The phenomenon of developmental system drift occurs when developmental and morphological traits remain relatively static across evolutionary time, but the genetic underpinnings that encode them, including the functions and network interactions of conserved genes, diverge . This islampreys .C. elegans and WNT signaling in P. pacificus, nematode species that are 250 million years diverged. The molecular changes to these cell fate specifications include rewiring of the WNT pathway, acquisition of novel protein domains, and retention of conserved domains, indicating that both signal transduction lability and protein modularity can participate in developmental system drift [C. elegans and C. briggsae are near-identical in development and morphology, but orthologous genes show different molecular functions, often including differences in expression [C. elegans strains do show heritable differences in embryonic pathway function, as evidenced by differences in embryonic lethality following perturbation of critical developmental genes [How can genes, genic functions and genetic interactions change so dramatically over evolutionary time while producing a relatively static developmental output? Experimental investigations into nematode biology have begun to characterize the different mechanisms by which similar traits are achieved. For example, vulva formation occurs by EGF/RAS signaling in em drift . At apprpression . Althougpression . Divergeal genes . While eNatural genetic variation is the central focus of evolutionary and quantitative geneticists. However, within the paradigm of most investigations into developmental mechanisms, natural genetic variation becomes genetic background effects. Developmental geneticists often experience the perils of background effects, so virtually all forward- and reverse-genetic experiments are performed in common, isogenic strains. However, while this practice affords careful control of the focal perturbation, it also limits the reach of inference. Since the penetrance of single-gene mutations can vary dramatically across different genetic backgrounds, observations in a single strain may not be representative . MoreoveThe current accessibility of genotyping and sequencing technologies has turned the heads of many developmental biologists towards natural genetic variation, and we are extremely enthusiastic about the promise of such research programs. What we have argued here—that the robustness and lability of developmental systems arise from pervasive cryptic genetic variation, which may explain macro-evolutionary transitions and can be parsed at the micro-evolutionary scale by quantitative genetic methods—is simultaneously relevant to our understanding of mechanisms of development. The implications for understanding complex trait evolution are extensive, and include recognition that there is ample standing variation to suppress the deleterious effects of otherwise deleterious new mutations, that standing and cryptic variation should facilitate rapid response to environmental (including climate) change, and that highly buffered pathways allow tinkering of regulatory mechanisms that may contribute to the emergence of novelty. At the very least, rather than just treating alleles as statistical effects, quantitative genetics needs to be aware of the nature of developmental mechanisms, and molecular evolution needs to model the trajectory of genetic divergence.


Ciona robusta  and C. intestinalis , globally distributed and sympatric in Europe. By gathering new mitochondrial sequences that were merged with published datasets, we analysed genetic patterns in different regions, with a focus on 1) their sympatric range and 2) allopatric populations in N and S America and southern Europe. In the sympatric range, the two species display contrasting genetic diversity patterns, with low polymorphism in C. robusta supporting the prevalent view of its recent introduction. In the E Pacific, several genetic traits support the non-native status of C. robusta. However, in the NE Pacific, this appraisal requires a complex scenario of introduction and should be further examined supported by extensive sampling efforts in the NW Pacific (putative native range). For C. intestinalis, Bayesian analysis suggested a natural amphi-North Atlantic distribution, casting doubt on its non-native status in the NW Atlantic. This study shows that both natural and human-mediated dispersal have influenced genetic patterns at broad scales; this interaction lessens our ability to confidently ascertain native vs. non-native status of populations, particularly of those species that are globally distributed.Human-mediated dispersal interplays with natural processes and complicates understanding of the biogeographical history of species. This is exemplified by two invasive tunicates,  For centuries, species have been intentionally or accidentally transported beyond their native range by human activities  as distinct species. The two species were synonymized in 1985 under the name Ciona intestinalis1011C. intestinalis type A and type B. The taxonomic status of these two taxa has been recently re-evaluated: C. intestinalis type A was assigned to C. robusta described by Hoshino and Tokioka in 1967C. intestinalis type B to C. intestinalis , sensu Millar1516C. robusta and C. intestinalis, respectively, and 3) genetic and genomic studies showed their strong evolutionary divergence, estimated to have occurred ca. 3–5 My BP1218These issues are exemplified by the class Ascidiacea, in which more than 1600 species  were described only after 1950 and several have a worldwide distributionC. robusta is, at least partly, explained by its tolerance to a larger temperature range compared to C. intestinalis23The two species are distributed along temperate and warm-temperate coasts20C. robusta is generally assumed to be native to the NW Pacific, where it was described, and has been reported as an introduced species in the northern and southern hemisphere: in the Atlantic, Mediterranean Sea, Oceania, and North and South Pacific oceans , the uncertainty of the native vs. native status in some regions  and their status as model species in evolutionary and developmental biologyConsidering the re-classification of the two species, the recent report of new introductions . They are both sessile as adults, with a short life cycle involving broadcast spawning for external fertilization, and with a non-feeding larva providing a short planktonic phase202233343536C. robusta is presumed to be native to the NW Pacific, but a recent introduction in the English Channel (in the 2000s) and an older (19th to mid-20th century) introduction in the NE Pacific and Mediterranean Sea, while the age of the establishment in the SE Pacific is unclear. C. intestinalis is considered native to the NE Atlantic, while its status in the NW Atlantic is debated. Our analyses are based on patterns of genetic diversity and population structuring, with the assumption that both natural expansion and human-mediated contemporary dispersal could have influenced the observed patterns of genetic diversity. In the case of C. intestinalis, Bayesian analysis with an Isolation with Migration Model (IMM) is used to contrast alternative scenarios for the expansion of C. intestinalis in the North Atlantic.Our second objective was to use the additional genetic information to assess the native vs. non-native status of the study species in the global regions sampled. The current understanding of the biogeographical status of the various populations is detailed, with references, in the et al.C. robusta and 1140 C. intestinalis individuals were examined on COX3-ND1 sequences of 580 and 529 bp respectively. A subset of 501 individuals of C. robusta and 683 individuals of C. intestinalis sampled for this study  and 147 haplotypes (114 segregating sites) for C. robusta (N = 714) and C. intestinalis (N = 1140) respectively. As expected, the divergence was clearly higher between (14.4%) than within species .Genetic diversity indices obtained using our COX3-ND1 dataset with additional data from Zhan C. robusta at regional level, all Pacific areas sampled were similar  = 0.129 ± 0.153) level. These differences of population genetic diversity are significant between NE Pacific and EC , SE Pacific and EC (P = 0.003) but not between the NE and SE Pacific ranges (P = 0.289). Similar statistical tests could not be reliably carried out with the NW Pacific as two populations only were examined in this region. Note that large differences were observed between the two Mediterranean populations studied (Hd(mean) = 0.093 ± 0.132) indicating the need for further sampling in this region to investigate possible regional trends. The haplotypic richness, which makes correction for sampling effort, is also higher in Pacific ranges  compared to EC (6.41) (Regarding genetic diversity indices for C (6.41) . The lowC (6.41)  in whichC (6.41) . Only prC (6.41) .C. intestinalis, the NE Atlantic showed almost twice the haplotypic diversity observed in the NW Atlantic  = 0.839 ± 0.081) than elsewhere: the North Sea (Hd(mean) = 0.608 ± 0.067, P < 0.01, Mann-Whitney rank sum test) and the NW Atlantic (Hd(mean) = 0.482 ± 0.188, P < 0.001). However, Hd was not significantly different between North Sea and the NW Atlantic (P = 0.162). The high molecular diversity of the EC populations is illustrated by the haplotypic frequencies map  and 255 haplotypes (227 segregating sites over 1313 bp) for C. robusta and C. intestinalis respectively. Genetic diversity indices (Hd: haplotypic diversity and π: nucleotide diversity) are detailed in C. robusta, as shown with COX3-ND1 only, the populations in the EC displayed the lowest haplotype diversity  = 0.265 ± 0.232) compared to populations of other geographical areas, in particular SE Pacific populations (Hd(mean) = 0.897 ± 0.066), where the highest population genetic diversity indices were found. This low molecular diversity in the EC is well illustrated by the haplotype network  and explained by a large proportion of private haplotypes in this region. Compared to COX3-ND1, the concatenated dataset displayed more clearly the presence of three clusters . The hierarchical AMOVA showed a large regional effect . The other regional groups clearly distinguished by the nMDS plot are also noticeable in the network . A similar biogeographic break has been described in the SE Pacific at 30°S–33°SCT = 0.020, P = 0.198). In Europe, hierarchical AMOVA also did not show genetic differences between EC and Mediterranean Sea .For  network  and the  network . For ins network . InteresC. intestinalis, the overall genetic differentiation was significant among populations (ϕST) and the hierarchical AMOVA showed a significant effect of geographical areas (ϕCT) and of populations within areas (ϕSC)  and of populations within areas . Altogether, the outcome of the hierarchical genetic variance analyses is consistent with the network analysis showing no particular geographical partitioning of the three clusters (For as (ϕSC) . Excludiclusters . Pairwisclusters  showed tclusters . The NW clusters  more simC. robusta, the hierarchical AMOVA confirmed a large regional effect (ϕCT)  . SimilarT-values . As for C. intestinalis to evaluate the time of divergence between NW Atlantic and NE Atlantic are provided in Results of the Isolation with Migration Model (IMM) used for Ciona robusta and C. intestinalis. Altogether, C. robusta displayed lower genetic diversity at population and regional levels, but marked spatial genetic structure at these two scales. In contrast, C. intestinalis showed high genetic diversity and a more homogenous genetic structure in the N Atlantic. These contrasting patterns are even more pronounced in the sympatric area (i.e. EC): very low genetic diversity was observed in C. robusta, while C. intestinalis encompassed the whole genetic diversity observed at the species level in our study.Whatever the dataset, contrasting patterns were observed in C. robusta compared to C. intestinalis at a species level. They analyzed few individuals and localities of the two species , but at the genome level (i.e. 852 nuclear loci isolated from full transcriptomes). We here confirm this global pattern with a mitochondrial marker, using a wider geographical coverage and more extensive population sampling, with the addition of three geographic regions: SE Pacific, NW Pacific and EC. mtDNA markers can be sensitive to selective effectsC. robusta (type A) in the NE Pacific and C. intestinalis (type B) in the NW Atlantic: Mann-Whitney rank sum tests on data provided in et al.P = 0.005) and allelic richness (P = 0.018), both lower in C. robusta as compared to C. intestinalis.Roux and coauthorsC. intestinalis was more pronounced than in C. robusta, or that a stronger bottleneck occurred in C.robusta than in C. intestinalis. This last hypothesis is potentially realistic given several glacial and interglacial episodes since the divergence of the two species, which severely impacted the distribution ranges of other marine species414243Tajima’s D was significantly more negative in C. intestinalis B [C. intestinalis] than in C. intestinalis A [C. robusta]”. They however did not get statistical support for a demographic bottleneck based on various statistics , and hypothesized that this result might reflect a lack of statistical power, in particular stemming from insufficient sampling. Dedicated demographic studies, using nuclear markers and including a substantial sampling effort, notably in its Asian range, are needed to investigate in detail the cause of reduced genetic diversity in C. robusta. Decreased polymorphism of C. robusta as compared to C. intestinalis is nevertheless a consistent finding across the few studies carried out so far  regions of relatively high genetic diversity  and ii) one region of lower genetic diversity (EC). The Mediterranean Sea is not classified as only two populations displaying much contrasting features were available. Pacific populations are genetically distinct from European areas  haplotypes, weaker genetic structure, lower genetic diversity  and lack of concordance between gene genealogies and the geographical distribution of the haplotypes. NE Pacific populations, showing high diversity and private haplotypes, do not fit particularly well with these characteristics. In addition an important genetic discontinuity separates populations located north and south of the well-known biogeographic break at Point Conception  numerous and repeated introduction events from genetically diversified sources and 2) two distinct and independent introduction events, in the northern and southern part of this region, from at least two genetically differentiated sources, and subsequent gene flow between the two regions. This is not an exceptional scenario in marine invertebrates, as illustrated by the introduction of Carcinus maenas in the NW Atlantic which involved the establishment of two lineages from two independent introduction eventsCan these genetic patterns cast light on the putative native (in the NW Pacific) and non-native or cryptogenic (in the NE and SE Pacific) status of nception . This biC. robusta. And yet no genetic structure was found between populations located on both sides of this boundary . This finding supports the supposed non-native status of C. robusta in this region  and most of them shared several haplotypes including the dominant Ca1 . The status of this species was accordingly classified as cryptogenic in NW Atlantic, its presence on both temperate coasts being attributable with certainty to neither natural nor anthropogenic dispersal. As an alternative to anthropogenic dispersal, on-going natural dispersal across the Atlantic seems unlikely for organisms such as C. intestinalis which have a brief planktonic phaseLittorina littorea6C. intestinalis, the NW Atlantic population displays similarities to EC populations, with four haplotypes shared by populations on both N Atlantic coasts at medium and high frequencies, one being dominant in all populations  or European (94%) region, and private haplotypes are often considered characteristic of long-term population establishmentC. intestinalis, we observed high genetic diversity on both sides of the N Atlantic that was spread over the same two major haplotypic clusters in both mitochondrial datasets  showed a different pattern: they exhibited the lowest genetic diversity and were all included in the same haplotypic cluster. Such a pattern has already been described in several marine invertebrates and algae in the NE Atlantic . As for the numerous cryptogenic species presenting similar complex patternsC. intestinalis in the NW Atlantic.Note that natural isolation between the two populations does not exclude additional contemporary migrations. It is thus possible that both historical population splitting and human-mediated introductions have occurred in C. robusta in the NE Atlantic of reduced genetic diversity associated with recent introduction. This phenomenon is not commonly observed in marine invaders, presumably because the effects of founder events are short lived in the face of repeated introductions and high propagule loads. The low diversity observed in C. robusta in the EC is thus expected to disappear relatively quickly with new cryptic introductions, a hypothesis testable by temporal genetic monitoring. The data presented here also provide new insights regarding the native vs. non-native status of the two study species in various parts of their ranges. Our genetic data support a non-native status of C. robusta in the SE Pacific, particularly regarding the absence of genetic differences across the well-known biogeographic break at 30–33°S. However, our data do question the non-native status of C. robusta in the NE Pacific and of C. intestinalis in the NW Atlantic. Analyses of Arctic populations of Ciona sp. to assess the relationship of C. intestinalis populations on the two sides of the N Atlantic and additional studies in the NW Pacific range of C. robusta are priorities for further advancing the enquiries reported here. This study emphasizes the need for caution in the interpretation of global genetic patterns in species in which natural and human-mediated dispersal have interplayed for some time.Despite the complexity of reconstructing the eco-evolutionary history of cosmopolitan and cryptogenic species that have possibly been exploiting human-made habitats and vectors for a long time, this study showed that mtDNA-based studies can be helpful in documenting contrasting features between congeners at a global scale. In the EC, although the two congeners display very similar biological features and occupy the same localities, they show contrasting genetic features, including notable differences in genetic diversity; this study highlighted the importance of population demographic events and human-mediated dispersal in determining genetic patterns, apparently over-riding the influence of life-history traits. Comparison between geographical regions revealed an apparent example in Ciona robusta and C. intestinalis populations were sampled in 25 sites of the EC , two in the putative native range, the NW Pacific (Japan), one in the NE Pacific and two in the Mediterranean Sea. Note that to our knowledge, this study is the first to sample a substantial number of individuals from the putative native range of C. robusta to carry out population genetic analyses. C. intestinalis individuals were collected in 25 marinas in the EC, three locations in the Skagerrak (North Sea region) and one in NW Atlantic (North America). Sampling was generally realized under floating pontoons, either using SCUBA diving or by collection from the surface from pontoon floats, hanging ropes and the underside of buoys. Around 24 individuals were sampled in each population with few exceptions , cytochrome oxidase subunit 3 - NADH dehydrogenase subunit I (COX3-ND1) and cytochrome oxidase subunit I (COI), were chosen to allow comparisons with previous datasets21et al.C. robusta and 530 bp for C. intestinalis). Two overlapping peaks at position 434 bp for C. intestinalis were observed on the forward sequence but not on the reverse sequence. As we did not have access to the chromatographs obtained by Zhan and coauthors, we chose to exclude this position from the sequence analyses to avoid adding false haplotypes.For a detailed comparison of the structure between Pacific and Atlantic coasts of North Americawww.fluxus-engineering.com). Analyses of genetic structure were carried out with ARLEQUIN v.3.5ST) were carried out based on 10,000 random permutations. To picture the genetic distances between all study populations, a non-metric multi dimensional scaling (nMDS) was done with pairwise ϕST values, using the PRIMER 6 + softwareCT) and within (ϕSC) geographic areas, a hierarchical analysis of molecular variance (AMOVA) was done.For each species and each population, the number of haplotypes (Nh), the number of polymorphic sites (S), the haplotype diversity (Hd) and the nucleotide diversity (π) were computed using DnaSPet al.21C. robusta and 1313 bp for C. intestinalis. Note that similar results (in terms of genetic diversity and structure) were obtained independently when the two loci were analyzed separately (data not shown). Note that for AMOVA analyses for C. intestinalis, to avoid over-representation of the EC populations, the AMOVA was conducted with a random subsampling of 6 populations and was repeated with several other random subsamples of the populations without changing the results.The same analyses were also done using a subset of the sampling obtained for the two mitochondrial loci  and in SW Pacific  prevented us carrying out similar analyses for C. robusta in the Pacific.Considering the cryptogenic status of How to cite this article: Bouchemousse, S. et al. Contrasting global genetic patterns in two biologically similar, widespread and invasive Ciona species . Sci. Rep.6, 24875; doi: 10.1038/srep24875 (2016).


This study examines the psychometric properties of the Malay version of the Montgomery-Ǻsberg Depression Rating Scale (MADRS-BM).n = 50) and without depression (n = 100) completed the self-rated version of the Montgomery-Ǻsberg Depression Rating Scale (MADRS-S), the Malay versions of the MADRS-BM, the Beck Depression Inventory-II (BDI-II-M), the General Health Questionnaire-12 (GHQ-12), and the Snaith-Hamilton Pleasure Scale (SHAPS-M).A total of 150 participants with (With respect to dimensionality of the MADRS-BM, we obtained one factor solution. With respect to reliability, we found that internal consistency was satisfactory. The scale demonstrated excellent parallel form reliability. The one-week test-retest reliability was good. With respect to validity, positive correlations between the MADRS-BM, BDI-II-M, and the GHQ and negative correlation between the MADRS-BM and SHAPS-M provide initial evidence of MADRS-BM’s concurrent validity. After adjusting for age, gender, ethnicity, educational level, and marital status, individuals with depression significantly reported higher MADRS-BM scores than did individuals without depression. Hence, there is additional evidence for concurrent validity of the MADRS-BM. Cut-off score of 4 distinguished individuals with depression from individuals without depression with a sensitivity of 78 % and a specificity of 86 %.The MADRS-BM demonstrated promising psychometric properties in terms of dimensionality, reliability, and validity that generally justifies its use in routine clinical practice in Malaysia. To study treatment efficacy, researchers often rely on the use of clinician-rated instruments . Clinicino depressive symptoms) to 3 (worst depressive symptoms). Possible score ranges from 0 to 27, with higher scores indicating greater symptom severity. The MADRS-S has been found to have a high degree of concordance with the clinician-rated MADRS and demonstrates adequate reliability  [The MADRS is a popular scale because of its high inter-rater reliability and high sensitivity to detect changes in treatment effects . Due to  = 0.78) .Although in Malaysia, there are a few scientific attempts devoted to validate depression scales such as the Malay versions of the Beck Depression Index (BDI) , Beck DeThe Malay versions of the BDI, BDI-II, and DASS are multidimensional scales. Specific items from these multidimensional scales could not yield a theoretically sound composite score , reducinWe obtained permission from the original author of the MADRS, Stuart M. Montgomery, for conducting this study. A copy of permission letter was sent to the editor of this journal. The scale was translated from English to Malay in parallel by two bilingual clinical psychologists, whereas a bilingual language expert performed the back-translation. Discrepancies between the original version and the back translation were resolved through discussion and adjustments were made, where necessary. In Stage 1, we finalized the initial version of the MADRS-BM with an expert panel of psychiatrists and family physicians.We pilot-tested the initial version of the MADRS-BM using 10 native Malay-speaking nurses to identify any flaws in terms of wording. We noted any words that were considered unsuitable or inappropriate. The scale was also reviewed by a psychiatric consultant, who has vast experience in clinical research, to ensure satisfactory face, semantic, criterion, and conceptual equivalences. In Stage 2, we redefined the Malay version of the MADRS-BM.The study was conducted from September until December 2013 at Psychiatric Outpatient Clinic, University Malaya Medical Centre. The study protocol was approved by the Medical Ethics Committee (MEC) of the University Malaya Medical Centre. For the purposes of the study, we recruited individuals with and without depression. Criteria to select individuals with depression include: (a) subjects who were diagnosed with major depressive disorder ,(b) subParticipants were invited to provide their socio-demographic information such as age, gender, ethnic group, marital status, educational level, religion, and employment status.no depressive symptoms) to 3 (worst depressive symptoms). Higher scores indicate greater depression. As demonstrated in previous study, the scale demonstrated high internal consistency  and split-half reliability  [The BDI-II-M is a 21-item self-report measure of depression based on a 2-week time period . Partici = 0.84) .definitely disagree) to 4 (definitely agree) [The SHAPS-M is a 14-item self-report measure of hedonic experience encompassing interest/pastimes, social interaction, sensory experience, and food/drink. Participants rated items based on a 4-point Likert scale ranging from 1 (y agree) . Lower sy agree) .always) to 3 (never) for positive items and ranging from 3  to 0 (never) for negative items. Higher scores indicate greater symptom severity. As shown in previous study, the scale has good internal consistency  [The Malay version of the GHQ-12 is a 12-item self-report measure of current mental health. Participants rated items based on a 4-point Likert scale ranging from 0 ( = 0.85) .no depressive symptoms) to 3 (worst depressive symptoms). Higher scores indicate greater symptom severity. As demonstrated in previous study, the scale has good parallel form reliability (ICC = 0.78) and adequate reliability  [The English version of the MADRS-S is a 9-item self-report measure of depression. Participants rated items on a 4-point Likert scale ranging from 0 . Baseline characteristics pertaining to participants were computed using descriptive statistics. To establish dimensionality of the MADRS-BM, we performed principal component analysis. We used Cronbach’s alpha to provide an indication of internal consistency. We also assessed the homogeneity of the scales by calculating correlation coefficients between items and total scores, if an item was deleted. To examine the parallel form reliability between the MADRS-BM and MADRS, and the one week test-retest reliability of the MADRS-BM, we calculated the ICCs. In establishing concurrent validity, we examined correlations between the MADRS-BM and other measures  with Spearman’s test. To examine whether individuals with and without depression would differ significantly in terms of the MADRS-BM scores, we performed analysis of covariance (ANCOVA), while controlling for age, gender, ethnicity, , marital status, and educational level. The optimal MADRS-BM cut off score for individuals with depression was determined on the co-ordinate points as indicated in the receiver operating characteristic (ROC) analysis; we then obtained the rates of sensitivity and specificityTable p < .01) and the Kaiser-Meyer-Olkin measure of sampling adequacy for the MADRS-BM was 0.93, indicating that the sampling adequacy was meritorious [Bartlett’s test of sphericity was significant (itorious . A singlp < .01). The scale demonstrated good one-week test-retest reliability .The MADRS-BM exhibited good internal consistency . All the items had corrected item-total correlations that were 0.7 or above. Removal of items, if any, would not increase the alpha value (see Table p < .01) and the GHQ (p < .01) scores, but the scale was significantly and negatively correlated with the SHAPS-M (p < .01). Therefore concurrent validity of the MADRS-BM was established  significantly reported higher MADRS-BM scores than did individuals without depression   Table . Our finThe area under the receiver operating characteristic curve  was 0.91 (95 % CI = 0.86-0.96). The optimal cut-off score to distinguish individuals with depression from individual without depression was ≥ 4 with a sensitivity of 78 % and a specificity of 86 %.Our current findings show that the MADRS-BM has good internal consistency with an alpha value of 0.70. This result is comparable to the properties of the clinician-rated MADRS  . Also coWe also examined the concurrent validity of the the MADRS-BM by linking the MADRS-BM with the BDI-II-M, GHQ-12, and SHAPS-M. Positive correlations between the MADRS-BM, BDI-II-M, and GHQ and negative correlation between the MADRS-BM and SHAPS-M provide initial evidence of MADRS-BM’s concurrent validity. Additional evidence for concurrent validity of the MADRS-BM was reported. After adjusting for some socio-demographic information, individuals with depression significantly reported higher MADRS-BM scores as compared to individuals without depression.In this study, the cut off score for the MADRS-BM was 4, which is lower than the recommended score of 5, as suggested by the original MADRS. One possible explanation is that the current version of the MADRS-BM is a self-rated scale—participants tend to underrate or underestimate their symptoms. Even though the cut off score was lower than that of the MADRS, the MADRS-BM’s sensitivity was greater than that of the MADRS.A few limitations of this study warrant consideration. Firstly, given the cross-sectional nature of this study, we were unable to rule out the causal factors of depression. Likewise, we were unable to assess the predictive validity of the MADRS-BM. Secondly, our sample was recruited from an outpatient clinic in a tertiary hospital using convenience sampling; thus we raised concern over generalizability as one possible limitation. Lastly, some clinical features such as the severity of depression and the types of antidepressants being used by the patients were not documented in the current study. The presence of such clinical features could affect the MADRS-BM scores as reported by participants.In spite of these limitations, the MADRS-BM demonstrated promising psychometric properties in terms of dimensionality, reliability, and validity that generally justifies its use in routine clinical practice in Malaysia. In order to further establish its psychometric properties, future diagnostic studies using the standards for reporting of diagnostic accuracy (STARD) criteria are recommended.This is a requirement for online studies made by the local ethics committee.


The stone marten is a widely distributed mustelid in the Palaearctic region that exhibits variable habitat preferences in different parts of its range. The species is a Holocene immigrant from southwest Asia which, according to fossil remains, followed the expansion of the Neolithic farming cultures into Europe and possibly colonized the Iberian Peninsula during the Early Neolithic . However, the population genetic structure and historical biogeography of this generalist carnivore remains essentially unknown. In this study we have combined mitochondrial DNA (mtDNA) sequencing (621 bp) and microsatellite genotyping (23 polymorphic markers) to infer the population genetic structure of the stone marten within the Iberian Peninsula. The mtDNA data revealed low haplotype and nucleotide diversities and a lack of phylogeographic structure, most likely due to a recent colonization of the Iberian Peninsula by a few mtDNA lineages during the Early Neolithic. The microsatellite data set was analysed with a) spatial and non-spatial Bayesian individual-based clustering (IBC) approaches , and b) multivariate methods [discriminant analysis of principal components (DAPC) and spatial principal component analysis (sPCA)]. Additionally, because isolation by distance (IBD) is a common spatial genetic pattern in mobile and continuously distributed species and it may represent a challenge to the performance of the above methods, the microsatellite data set was tested for its presence. Overall, the genetic structure of the stone marten in the Iberian Peninsula was characterized by a NE-SW spatial pattern of IBD, and this may explain the observed disagreement between clustering solutions obtained by the different IBC methods. However, there was significant indication for contemporary genetic structuring, albeit weak, into at least three different subpopulations. The detected subdivision could be attributed to the influence of the rivers Ebro, Tagus and Guadiana, suggesting that main watercourses in the Iberian Peninsula may act as semi-permeable barriers to gene flow in stone martens. To our knowledge, this is the first phylogeographic and population genetic study of the species at a broad regional scale. We also wanted to make the case for the importance and benefits of using and comparing multiple different clustering and multivariate methods in spatial genetic analyses of mobile and continuously distributed species. In continuous populations, genetic structure vary from a single identifiable group of individuals, with gene flow only restricted by the distance among them, to an unknown number of subpopulations separated by barriers to gene exchange ,2. SpeciMartes foina, Erxleben, 1777) is one of the most widely distributed mustelids in Eurasia )The phylogeography of the stone marten in the Iberian Peninsula is compatible with a single recent colonization of the region, possibly in the early Holocene, followed by population expansion sometime later. In turn, its contemporary genetic structure is characterized by a significant pattern of IBD and an apparent differentiation of the populations at the extreme of an NE-SW axis. To our knowledge, this is the first phylogeographic and population genetic study of the stone marten at broad regional scale. This case study also shows the usefulness of comparing and integrating multiple different techniques in the analysis of spatial genetic structure of mobile and continuously distributed species. Comprehensive analyses and tests of isolation by distance and clinal variation are an essential complement to clustering methods aiming to identify distinct genetic units, in particular due to the difficulties of the latter approaches in dealing with populations under IBD.The genetic breaks detected in the Iberian stone marten population seemed largely related to the major river basins in the peninsula. However, linear barriers are only one of the multiple ways of how landscapes can influence gene flow . TherefoS1 Table(XLSX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.


Aeonium is a well‐known example for plant species radiation on oceanic archipelagos. However, while allopatric speciation among islands is documented for this genus, the role of intra‐island speciation due to population divergence by topographical isolation or ecological heterogeneity has not yet been addressed. The aim of this study was to investigate intraspecific genetic structures and to identify spatial and ecological drivers of genetic population differentiation on the island scale. We analyzed inter simple sequence repeat variation within two island‐endemic Aeonium species of La Palma: one widespread generalist that covers a large variety of different habitat types (Ae. davidbramwellii) and one narrow ecological specialist (Ae. nobile), in order to assess evolutionary potentials on this island. Gene pool differentiation and genetic diversity patterns were associated with major landscape structures in both species, with phylogeographic implications. However, overall levels of genetic differentiation were low. For the generalist species, outlier loci detection and loci–environment correlation approaches indicated moderate signatures of divergent selection pressures linked to temperature and precipitation variables, while the specialist species missed such patterns. Our data point to incipient differentiation among populations, emphasizing that ecological heterogeneity and topographical structuring within the small scales of an island can foster evolutionary processes. Very likely, such processes have contributed to the radiation of Aeonium on the Canary Islands. There is also support for different evolutionary mechanisms between generalist and specialist species.The Crassulacean genus  Species radiations on oceanic archipelagos provide illustrative showcases of evolutionary patterns  than Tenerife and its three precursors  and has a less complex geological history  and a spatially and ecologically more specialized species (Ae. nobile).Species that cope with a broad range of environmental settings, The following two hypotheses were tested:Aeonium populations are reflected in intraspecific genetic structuring.The topographical heterogeneity and the related spatial and ecological isolation of Ae. davidbramwellii) shows a higher genetic population differentiation compared to the specialist species (Ae. nobile).The generalist and wide‐spread species  is the northwesternmost and second youngest island of the Canarian archipelago  and light (0.02–0.04 mg) seeds, suggesting some suitability for wind dispersal  praeger , adapted to the Fastprep tissue homogenizer  and the Kingfisher magnetic particle processor : Leaf tissues (ca. 200 mg) were homogenized in 200 We applied ISSR (Inter‐Simple Sequence Repeat) markers with anchored primers to generate genetic fingerprints, a method that has been proven useful for population genetic analyses of nonmodel species due its capacity to generate highly polymorphic data with high reproducibility and cost efficiency . If available, annealing temperatures were taken from the literature; otherwise, gradient PCRs  were conducted and fingerprint patterns were compared to infer the best conditions for the primers, respectively. The same selection of nine markers turned out to be optimal for both species for the main study (see Table S2).μL reaction volume using the KAPA3G Plant PCR Kit : each reaction contained 1× KAPA Plant PCR buffer, 0.5× KAPA Plant PCR Enhancer, 0.3 μmol/L fluorescently labeled ISSR primer (see Table S2), and 0.25 U KAPA3G Plant DNA Polymerase. The PCR profile consisted of an initial denaturation step at 95°C for 3 min, followed by 40 PCR cycles , and a final extension step at 72°C for 5 min. Amplified ISSR fragments were mixed with the MapMarker size standard  and separated on a capillary electrophoresis system  using a protocol for long DNA fragments as recommended by the manufacturer.Selected ISSR markers (see Table S2) were amplified from genomic DNA in a 12.5 genemarker 1.95 . The suggested binning was checked and carefully corrected by hand for each preliminary locus, before exporting the peak height tables. These were again processed manually, by specifying thresholds for minimum peak heights for each locus, based on the frequency distributions of its peak heights, respectively, as well as on thresholds for minimum peak number and minimum mean peak height for individual samples. Loci and samples that did not reach the respective thresholds, as well as monomorphic and uninformative loci, were stringently discarded, resulting in very conservative assignments of presences/absences of single ISSR fragments for the final binary matrices of our two study species, respectively.Electropherograms were processed and analyzed with Ht, as well as percentage of polymorphic loci PLP and Nei's gene diversity He within populations. Additionally, allelic diversity A and number of alleles unique to a population  were calculated with genalex 6.5  ranging from 1 to 11 for Ae. davidbramwellii and from 1 to 10 for Ae. nobile were tested under the admixture setting. We used the independent allele frequency model to avoid overestimation of gene pool differentiation, but allowed for inclusion of the population origin as prior information in the models . Second, DAPC  were calculated with 10,000 permutations in AFLP‐SURV, using the Bayesian method with nonuniform prior allele frequency distribution and assuming Hardy–Weinberg equilibrium . Linearized FST values were then correlated with logarithmized (log10) pairwise geographical distances  patterns, pairwise differentiation indices . The software generates a null sampling distribution of FST estimates based on neutral expectations and then compares these simulated data with the sampled data. We employed 100,000 iterations for our analyses, using the two recommended algorithm extensions “Force mean FST” and “Neutral mean FST.”2/a) was calculated with the Area Solar Radiation tool in arcinfo 10.0, based on the 2 m × 2 m DEM, and values were extracted as area averages of 25 m radius circles around population coordinates, respectively.We tested the full sets of loci for possible associations to eight environmental variables, which we assumed to state strong and putatively evolutionary relevant ecological gradients among the populations of our two study species (see headline of Table 2 for tested variables). MAT  and mean annual precipitation were interpolated using data collected from 214 and 288 meteorological stations for the Canary Islands, respectively  to correct for background population structure in the LFMM analyses. Of the 10 runs, the run with the lowest Deviance Information Criterion was selected for each variable, respectively. βadasam tests logistic regression models to identify possible loci–environment associations  into account and was provided as P‐values of their Χ2‐tests  and univariate Local Indicators of Spatial Association , the distribution of their allelic variants was geographically displayed in relation to the associated environmental variable suggested by the correlative methods. Additionally, the spatial clustering of their allelic variants was quantified by overall spatial autocorrelation , and population differentiation was moderate with ΦST = 0.104 (P < 0.0001).The ISSR genotyping for structure analysis revealed that K = 3 was the most likely number of genetic clusters within Ae. davidbramwellii, although K = 2 and K = 4 also showed high likelihoods, and Evanno's ΔK had its highest value at K = 2 . Genetic structure was dominated by one cluster which determined major parts of the genotypes of all individuals .We found no clear IBD pattern among populations of A, PLP, and He were found in populations J_d and H_d, both located within or near the Caldera de Taburiente and Barranco de las Angustias, followed by the two differentiated populations outside this region, A_d and C_d, and by I_d, also from the Barranco de las Angustias (Table DW), followed by H_d, A_d, C_d, and I_d. G_d from the lower Barranco de las Angustias revealed low amounts of rare alleles  indication of environmental dependence for loci UBC809_292  and UBT4_333 (mean precipitation from June to August and from May to October). However, while UBC809_292 was also suggested as outlier by both bayescan (PP = 0.79) and mcheza , UBT4_333 was detected only by mcheza .The scan for non‐neutral genetic variation resulted in indications for divergent selection on at least two loci, although there was inconsistency among the results. I = 0.007, P = 0.110 and Moran's I = −0.045, P = 0.440, respectively). However, a clustered pattern of the dominant allele of UBC809_292 in high elevations  on the western side of La Palma was obvious , and overall population differentiation was a bit lower .For structure resulted in K = 3 as the most likely subdividing genetic structure in Ae. nobile, with K = 2 and K = 4 showed high likelihoods as well and ΔK peaking at K = 2 . All populations and individuals were dominated by one genetic cluster .The correlation between pairwise geographical distances and pairwise genetic differentiation between populations revealed the absence of an IBD pattern  in H_n, followed by the two other populations from within the Barranco de las Angustias (E_n and G_n) and by populations C_n . bayescan found no outlier loci under the same settings that were applied for Ae. davidbramwellii. mcheza suggested 11 loci to show variation caused by divergent selection, which, however, were only poorly supported by the two correlative methods used: LFMM found only few correlations with very low significance , and similarly, the correlations indicated by βadasam did not reach the significance levels obtained for Ae. davidbramwellii and were only partially congruent with the outlier suggestions of mcheza. The most significant correlation (P = 0.001) was found between the allelic variation in locus UBC809_562  and mean precipitation from June to August. However, the spatial distribution of this variation was highly inconsistent and did not show a recognizable relationship to the environmental variable (not shown).Signatures of selective forces on genetic variation in Ae. davidbramwellii; 3. the differing niche widths of Ae. davidbramwellii versus Ae. nobile did not reflect in differences in the degree of genetic structuring; however, in the generalist species Ae. davidbramwellii signatures of selection were more distinct.The project aimed to identify population genetic patterns related to geographical and ecological heterogeneity on the western Canary Island of La Palma, to make inferences on the evolutionary potential on the island scale and for potential conservational implications. Our main findings are as follows: 1. Genetic structuring within both species is low although effects of island topography and range patterns are obvious; 2. there are indications of correlations between allelic patterns and environmental heterogeneity in temperature and precipitation variables for Ae. davidbramwellii and Ae. nobile may be due to the following (nonexclusive) reasons:The weak to moderate overall genetic population structures in ΦST values but largely missing IBD patterns speak against common gene flow between adjacent populations, but rather suggest random (including long‐distance) dispersal events within recent timescales and limited gene flow after the establishment of populations  and a slightly older origin for Ae. nobile . Many other Aeonium species show higher divergence estimates. Geologically young oceanic islands like La Palma generally can be assumed to host more young species and lineages with lower levels of population divergence  and Tenerife (Ht = 0.229)   and Solanum lidii on Gran Canaria (Ht = 0.207) observed with AFLP data  spreading over the island, but this is somewhat speculative. DNA sequence‐based phylogeographical studies and estimations of demographic histories may help to make more definite statements.Third, it is also possible that the low overall genetic diversity and differentiation values are due to catastrophic throwbacks of range expansions and bottleneck events in the past of Ae. davidbramwellii and Ae. nobile, compared to a majority of Canary Island endemics , showed patterns of isolated evolution as well. This indicates a general role of topography in promoting population differentiation, which has already been shown for other plant species on the island scale  was generally high in the Barranco de las Angustias, only population H_n in the lowest part of the Barranco showed a large number of private alleles. This population additionally was differentiated in the clustering analyses. Populations in the upper parts of the Barranco (G_n and E_n) together with F_n, located slightly behind the northern ridge of the Barranco, made up an own group, pointing to an early separation from H_n. In particular, the genetically very poor populations in the north  give rise to the assumption that the northern part of the distribution range is the result of recent colonization by Ae. nobile. However, the highly differentiated position and high diversity values of population C_n suggest a relatively early dispersal event, so that C_n established before other populations in its surrounding and had time to evolve. This supports the idea of rather stochastic dispersal events from the Barranco de las Angustias.Despite being more restricted in its distribution than Ae. nobile, which is congruent with the geomorphological features created by the Cumbre Nueva landslide. Over a large area south of the Barranco de las Angustias, comparably mild and low structured slopes impede the occurrence of Ae. nobile. Additionally, the prevailing agriculture, dense settlement, and infrastructure probably pose problems for this species here. However, the low differentiation of the southern populations speaks against early vicariance due to the landslide but more for colonization from the north after this event. The recent volcanic origin of southern areas of La Palma  during the summer months (from May to October and from June to August), variables which constitute strong environmental gradients among the analyzed populations of this species .While many indications of non‐neutral allele distribution in our study were of low significance or were detected only by one or two of the applied approaches, some loci in Ae. davidbramwellii and its CAM‐based physiology  and lower genetic diversity in species occurring in small and intermediate population sizes  compared to species with large populations .In the literature, specialist species are expected to have smaller and more isolated populations than related generalist species; thus, they would be more affected by genetic drift due to meta‐population dynamics, fostering higher variation among populations . Thus, one could have expected lower population differentiation from the general model described above. Additionally, it is younger than Ae. nobile (Kondraskov et al. Ae. davidbramwellii is also characterized by a much stronger environmental heterogeneity and large‐scale topographical subdivision than that of Ae. nobile. We thus suppose that for the moderate population differentiation within Ae. davidbramwellii, the effects of a rather continuous distribution, larger population sizes, and less evolutionary age were compensated by the effects of differentiating selection pressures and strong landscape structuring. In contrast, the similar differentiation level in Ae. nobile might have been dominated by drift effects. In fact, although only marginally significant, a potential IBD pattern was more discernible in Ae. davidbramwellii, pointing to higher gene flow between neighboring populations and lower importance of drift. Also, consistent signals of differentiating selection were found only in the generalist Ae. davidbramwellii, supporting the hypothesized evolutionary effects of strong environmental heterogeneity within its range. In the sampled range of Ae. nobile, ecological gradients are limited in strength, missing divergent selection regimes among populations of this species which provides a likely explanation for the low association of allelic patterns and ecological variables. Furthermore, the only potential large‐scale landscape barrier for gene flow is the region of the ancient Cumbre Nueva landslide. This emphasizes that besides population continuity and population sizes, landscape factors and the strength of environmental heterogeneity should be considered in studies of generalist–specialist patterns, at least on small spatial scales like single islands.Contrastingly, our study revealed no significant differences in overall population structures and total genetic diversity between the generalist and the specialist species. Ae. davidbramwellii during our fieldwork on La Palma, which was not true for Ae. nobile. Especially in populations in the Caldera de Taburiente and the Barranco de las Angustias, individuals showed exceptionally strong branching, whereas in the southern regions of La Palma, branching in this species was rather uncommon. This is a further indication of divergent selection pressures, although not reflected in our genetic data.It also has to be mentioned that we observed considerable regional phenotypic variation in Ae. davidbramwellii and some of its populations, while the specialized Ae. nobile actually seems to be in an “evolutionary dead end,” without great prospects of further intraspecific niche divergence and adaptive differentiation.It has been shown that plant speciation can occur by adaptive specialization of certain populations of generalist species, leading to sympatric pairs of sister species with largely different ecological niche breadths (Grossenbacher et al. However, some of the drawn conclusions might have limitations due to the following two reasons: First, future studies should include more populations and a denser representation of all regions of La Palma. We included many populations from the Barranco de las Angustias region which potentially might have biased our observations of disproportionally high local genetic variation. Further on, larger sampling sizes may allow for more distinct and significant results according to genetic variation estimates within and among populations. Second, although ISSR analyses have proven to be reliable approaches for plant genotyping and population genetic studies (see, e.g., Nybom Aeonium, have not taken place yet. Although not definite, our study revealed basic patterns within the two analyzed species and gives additional insights into species evolution on small oceanic islands.Nevertheless, investigations of intraspecific variation and population structures, and therefore of recent eco‐evolutionary processes and potential drivers for species divergence in None declared.Figure S1. Assessment of the most likely number of clusters of structure runs using the method of Evanno et al. (Aeonium davidbramwellii.o et al.  for AeonFigure S2. Correlation of pair‐wise geographic distance and pair‐wise linearised FST between populations of Aeonium davidbramwellii: Mantel's R = 0.270, P = 0.082.Figure S3. Assessment of the most likely number of clusters of structure runs using the method of Evanno et al. (Aeonium nobile.o et al.  for AeonFigure S4. Correlation of pair‐wise geographic distance and pair‐wise linearised FST between populations of Aeonium nobile: Mantel's R = −0.071, P = 0.397.Click here for additional data file.Table S1. Plate layout and instrument settings for DNA purification via BindIT 3.1 KingFisher software.Table S2. Tested primers for the ISSR analyses, their sequences and PCR annealing temperatures.Table S3. Combined results of outlier loci detection methods for Aeonium nobile.Click here for additional data file.