Diversity is widely known to fuel adaptation and evolutionary processes and increase robustness at the population, species and ecosystem levels. The Neo-Darwinian paradigm proposes that the diversity of biological entities is the consequence of genetic changes arising spontaneously and randomly, without regard for their usefulness. However, a growing body of evidence demonstrates that the evolutionary process has shaped mechanisms, such as horizontal gene transfer mechanisms, meiosis and the adaptive immune system, which has resulted in the regulated generation of diversity among populations. Though their origins are unrelated, these diversity generator (DG) mechanisms share common functional properties. They (i) contribute to the great unpredictability of the composition and/or behavior of biological systems, (ii) favor robustness and collectivism among populations and (iii) operate mainly by manipulating the systems that control the interaction of living beings with their environment. The definition proposed here for DGs is based on these properties and can be used to identify them according to function. Interestingly, prokaryotic DGs appear to be mainly reactive, as they generate diversity in response to environmental stress. They are involved in the widely described Red Queen/arms race/Cairnsian dynamic. The emergence of multicellular organisms harboring K selection traits  has led to the acquisition of a new class of DGs that act anticipatively to stress pressures and generate a distinct dynamic called the “White Queen” here. The existence of DGs leads to the view of evolution as a more “intelligent” and Lamarckian-like process. Their repeated selection during evolution could be a neglected example of convergent evolution and suggests that some parts of the evolutionary process are tightly constrained by ecological factors, such as the population size, the generation time and the intensity of selective pressure. The ubiquity of DGs also suggests that regulated auto-generation of diversity is a fundamental property of life. In reference textbooks, the extremely wide diversity of biological entities is traditionally presented as the consequence of genetic changes that occur spontaneously and randomly. However, a growing number of studies in multiple fields now challenge this paradigm of diversity, to the point that it is no longer indefensible. This article attempts to summarize current knowledge about the sources and consequences of genetic and phenotypic diversity in living systems.As we will see, individual diversity makes ecosystems, species and populations more robust to environmental stress and can favor the emergence of complex collective behaviors such as collectivism. This demonstrates that diversity not only constitutes the fuel for the evolutionary process, but can also confer new functions and better adaptive capacity at all levels of biological complexity. The magnitude of these fitness gains suggests that partial control of the generation of diversity may be beneficial. In keeping with this assumption, numerous examples of tightly regulated mechanisms generating individual genetic and phenotypic diversity have been described among prokaryotes, parasites and complex multicellular organisms. These mechanisms seem to play a key role in the evolution of biological systems as well as in the dynamics of the host–pathogen relationship. Their ubiquity suggests that they are indispensable to adaptation to environmental stress, and that regulated auto-generation of diversity must be considered as a fundamental trait of biological systems. Based on these observations, the present article proposes a new conceptual framework for thinking about the diversity and organization of biological systems. It has three goals in particular: (i) to provide a functional definition of the acquired mechanisms contributing to generate diversity, based on their shared properties, (ii) to identify the factors defining their dynamics, and finally (iii) to discuss the complex question of how and why they were selected and stabilized during evolution.7 species  suggest that there could be 109 or more prokaryotic species worldwide   encoding essential metabolic and informational functions, but often carry unique, strain-specific sequences  serving in the adaptation to local environmental pressures . The siz strains  but a re strains . This fl genome)  and fuzz species  in micro species .•Second, the phenotypic diversity of living forms is greater than their genotypic diversity. Prokaryotes  and prot•Third, genetically identical individuals can express considerable variation in behaviors, such as social, sexual, anti-predatory responses, and can show task and diet specialization . Alternatively, high species diversity also implies higher chances of having species that efficiently fulfill functions required by their environment (known as the selection probability effect or sampling effect). A large number of species may imply a certain level of functional redundancy: the loss of one species has a smaller effect in a diverse system than in a species-poor one , cellular age, asymmetric division, cell-cell interactions or epigenetic modifications . In other terms, under this paradigm, genetic accidents are considered as the most important source of diversity and adaptation to environmental stress conditions. However, most spontaneous genetic mutations appear to be neutral. A few are detrimental and beneficial mutations seem to be very rare. This has led to Muller’s ratchet concept  and degeneracy  .•Now here, you see, it takes all the running you can do to keep in the same place” in Lewis Carroll’s “Through The Looking-Glass” (1871), such dynamics are often called the “Red Queen” in evolutionary biology because competitors must constantly evolve to maintain their fitness  in the evolution of archaeal and bacterial genomes . BacteriEscherichia coli  was previously proposed in In summary, recent advances in microbiology and genetics have led to the conclusion that genetic variation in prokaryotes is not only a “by-product of nature’s imperfections” but is mainly due to HGT mechanisms. It has been estimated  that HGTexchange . As genein vivo infection has highlighted the within-host evolution ability of various bacterial pathogens  strains that display an increased mutation rate, usually as a result of a loss of functionality of DNA repair systems , allowing it to escape from the adaptive immune response –encoding genes from a genomic repertoire of ∼2000 different silent genes and pseudogenes mainly located in subtelomeres. This location favors ectopic recombination between VSGs, increasing the diversity of VSGs. Comparison of the VSG expression profile in distinct mice infected with the same T. brucei strain demonstrates that each infection presents a distinct sequence of VSGs  is a diverse family of hypervariable proteins that are inserted into the surface of P. falciparum-infected erythrocytes. PfEMP1 mediates binding of infected erythrocytes to the endothelial lining of blood vessels as a strategy to avoid clearance by the spleen and is a major target of host humoral adaptive immunity. PfEMP1 is encoded by a large multi-gene family called var. Each parasite genome contains ∼60 var genes but many millions of rearranged var gene sequences are produced by recombination every 48-h life cycle in infected individuals  that form the mucus facilitating parasite penetration through the snail epidermis. Histone modifications of the SmPoMuc promoters generate SmPoMuc transcription polymorphism leading to important phenotypic diversity among the parasite population and favoring success of the infection  is decoupled from sexuality. Like HGT in prokaryotes, sexuality occurs principally in reaction to stressful environmental conditions . In contThe mechanisms underlying phenotypic variability not associated with evident individual fitness gains have attracted little interest from evolutionary theorists or ecologists. Some of those mechanisms, however, appear to be very interesting when analyzed at the population level.11, 1015 and 1018 distinct B cell receptors, αβT-cell receptors and γδT-cell receptors, respectively  during evolution are still the subject of hot debate . These K characteristics drastically reduce the probability that accidental or reactive genetic variations in germinal cells will lead to rapid phenotypic adaptation of metazoan populations. I hypothesize that anticipative DGs have been favored by natural selection in several phyla of higher metazoans because they can overcome these limitations. As a tribute to Leigh Van Valen and Lewis Carroll, I propose to call the dynamic generated by anticipative DGs the “White Queen” dynamic, in reference to the White Queen’s famous quote in “Through the Looking-Glass” (1871): “Sometimes I’ve believed as many as six impossible things before breakfast”. This metaphor seems particularly appropriate as anticipative DG activities are based on random phenotypic diversification that is not adaptive at individual level (impossible things) and anticipative (before breakfast) .As previously noted, the activity of some DGs appears to be induced by environmental stress. These reactive DGs participate in the widely described Red Queen/arm race/Cairnsian dynamic. Interestingly, other DG mechanisms, such as DGs acquired by pathogens, seem to act constitutively to allow the pathogen to escape the host adaptive immune response and achieve long-term persistence in the host. Yet other DGs, such as obligatory sexual reproduction, generation of an adaptive immune repertoire and neuronal mosaicism appear to be regulated independently of environmental conditions and can be considered as anticipative DGs. Such anticipative DGs appear mainly in metazoans, suggesting that they are selected in response to particular constraints. In addition to limitations of gene exchange by HGT, most higher metazoans present K selection traits such as a long reproductive life cycle and a small population size (compared to bacteria)  and memorization of past experience. For example, in prokaryotes, CRISPR-Cas systems participate in the response to foreign genetic elements such as phages and provide a form of acquired immunity. Their action leads to the integration of new sequences in the prokaryotic genome. Thus, CRISPR-Cas systems diversify prokaryotic populations and customize individuals by integrating part of their immune history at the genome level. In mammals, as previously discussed  theory, interacting organisms may have an evolutionary incentive to help each other if they share genes, and the magnitude of this incentive is thought to increase with the degree of relatedness between them. As infection is generally due to a limited number of microbial pathogens, we can reasonably hypothesize that DGs associated with microbial pathogens act within a population composed of closely related individuals. Thus, their selection could be explained from an inclusive fitness selection perspective. However, DGs associated with multicellular eukaryotes, such as meiosis mechanisms and the mechanisms favoring diversity of the adaptive immune response or of the neural network, act clearly within populations composed of unrelated individuals. To explain their selection, we need to use the multi-level (or group selection) theory, which postulates that natural selection not only acts on individuals but can act (simultaneously) on multiple levels of biological organization, such as cells, individuals, groups or species. Therefore, processes that are disadvantageous at one level can be positively selected for a benefit conferred at a higher level of the biological hierarchy.Based on William D. Hamilton’s the sum of the genetic information of the host and its microbiota”the macroscopic host and its synergistic interdependence with bacteria, archaea, fungi, and numerous other microbial and eukaryotic species including algal symbionts”Animals and plants can no longer be considered individuals.” By redefining the individual as a consortium of unrelated agents, the holobiont theory postulates that cooperation between unrelated agents is a proven fact. As previously discussed by myself and others  of biological complexity, it is not really surprising that evolution processes have favored the selection of specific mechanisms to regulate the emergence of diversity among populations. Interestingly, the existence of DGs profoundly changes the dynamics of evolution. In contrast to the classical Neo-Darwinian paradigm where diversity is generated passively by accidental events, living entities also appear able to actively generate diversity in reaction to stress (Red Queen/Cairnsian dynamic) and/or anticipatively at certain points in time (White Queen dynamic) . As discussed previously, the predominance of the Red Queen and White Queen dynamics seems to depend on the r/K selection traits of organisms. Under this new vision, evolution is conceived as a more “intelligent” and Lamarckian-like process than is generally accepted. Though genetic diversity due to DGs remains random, it is regulated in time and space. DG activities are generally focalized on particular sets of genes over a short period of time, as observed with phase variation in bacteria  By acting on the genome of germinal cells, some DGs, such as HGT and meiosis mechanisms, directly affect diversification and speciation by producing new alleles. If the effects of DGs are not inheritable, as in the case of phenotypic diversity generated by the AIS and neuron mosaicism that are limited to a fraction of somatic cells and thus do not affect germinal cell lines, DGs can favor genetic robustness and thus preserve rare alleles and help maintain genetic diversity in populations that also favor the speciation process. (ii) DG activities can also lead to an increasing complexity of social organization inside biological systems. It is therefore likely that they participated in the emergence of complex systems such as eukaryotes, multicellular organisms and societies. Each of these systems can be viewed as a consortium of cooperative agents . By incrauto-generation of diversity can be viewed as a fundamental property of biological systems. From this perspective, the actual lack of biodiversity observed in a great number of ecosystems  Biodiversity should no longer be seen only as a “characteristic of life” and the result of accidental processes but as indispensable to the survival of life and actively generated by the latter. Thus, osystems  should bpothesis ] shared Finally, from a theoretical point of view, diversity, self-organization  and unpredictability have always been considered as characteristics of biological systems that are fundamental though difficult to reconcile . RecogniA theoretical scientific framework must satisfy Popper’s refutability criteria by presenting testable predictions. In this article, I have tried to synthesize current knowledge about the mechanisms of diversity generation. In opposition to the classical Neo-Darwinian paradigm presenting diversity exclusively as the result of accidental processes, I propose that a large part of genetic and phenotypic diversity is the consequence of regulated mechanisms that I call Diversity Generators (DGs). I propose two experimentally refutable predictions about DGs:•DGs are indispensable to the survival of living entities, and therefore all natural biological entities must have at least one mechanism that meets the definition of a DG. To my knowledge, HGT mechanisms have been described in all bacteria and Archaea and the large majority of eukaryotic organisms display meiotic/sexual reproduction. The rare metazoans, like Rotifera, that have lost sexual meiosis and remain diversified seem to present high levels of HGT  the acquisition of specialized constitutive DGs by pathogens permanently exposed to a host adaptive immune response, and (ii) the acquisition of anticipative DGs by complex multicellular organisms to compensate for their K selection traits, such as a long reproductive life cycle and a small population size.The theory of evolution constitutes the conceptual foundation of modern biology and consequently of the life sciences. Unfortunately, its impact on the organization of human societies remains negligible. For example, education and fundamental research are subject to an increasing number of evaluation criteria. Although these controls were initially developed to optimize education and fundamental research, they also serve to standardize them. Is it reasonable to homogenize the intellectual formation of individuals and research activities, while diversity is a source of robustness, synergy and complexity in all living systems? More worryingly, global population growth will require sustained food production during the 21st century. However, the industrialization of agriculture over the past 50 years has led to a dramatic fall in the diversity of agricultural products. Plants and animals have been intensively selected for strength and productivity. While this strategy led to good results over the short term, it is reasonable to doubt the ability of standardized populations to resist future climate changes that will likely lead to the emergence of new pathogens. Must I remind the reader that a particular genotype/phenotype is optimally adapted only to a given set of environmental conditions? There are no phenotypes that perform perfectly in all situations. Thus, there is an urgent need to reconsider the importance of diversity within populations, both from a theoretical and a practical point of view.The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Canis lupus signatus), using standard molecular markers. A combination of paternal and maternal molecular markers was used to investigate the genetic composition, genetic differentiation and genetic relationship of native Portuguese dogs and the Iberian wolf. A total of 196 unrelated dogs, including breed and village dogs from Portugal, and other dogs from Spain and North Africa, and 56 Iberian wolves (wild and captive) were analyzed for nuclear markers, namely Y chromosome SNPs, Y chromosome STR loci, autosomal STR loci, and a mitochondrial fragment of the control region I. Our data reveal new variants for the molecular markers and confirm significant genetic differentiation between Iberian wolf and native domestic dogs from Portugal. Based on our sampling, no signs of recent introgression between the two subspecies were detected. Y chromosome data do not reveal genetic differentiation among the analyzed dog breeds, suggesting they share the same patrilineal origin. Moreover, the genetic distinctiveness of the Iberian wolf from other wolf populations is further confirmed with the description of new mtDNA variants for this endemism. Our research also discloses new molecular markers for wolf and dog subspecies assignment, which might become particularly relevant in the case of forensic or noninvasive genetic studies. The Iberian wolf represents a relic of the once widespread wolf population in Europe and our study reveals that it is a reservoir of unique genetic diversity of the grey wolf, Canis lupus. These results stress the need for conservation plans that will guarantee the sustainability of this threatened top predator in Iberia.This study investigates the gene pool of Portuguese autochthonous dog breeds and their wild counterpart, the Iberian wolf subspecies ( Blood, hairs or tissue samples were collected for nine Portuguese native dog breeds at dog shows, breeding kennels, and distinct locations in historical breed regions, and for other breeds in such geographically neighboring regions as Spain and Northwest Africa (Morocco) (Table 2.1.2n = 42) and Spain (n = 2); and 12 live captive specimens kept at the Iberian Wolf Recovery Centre . Wild specimens' samples were either tissue or blood , obtained from the wolf tissue bank (SMLM) managed by the Portuguese Institute for the Conservation of Nature and Forest (ICNF); blood from live captive animals was collected by a veterinarian. Samples included 11 females, 44 males, and one specimen of undetermined sex and were collected between 1987 and 2008  were genotyped in a set of 122 dogs and 52 wolves: AHT121  and based on GenBank sequences (Accession numbers: DQ973626‐DQ973805). The haplotype 1 sequence from isolate y20 from Canis lupus familiaris was used as reference for SNaPshot primer design . PCR product sizes ranged from 130 to 492 bp  599, 619, and 873 at the amplicon Ydog_28  and nt 66 and 146 at the amplicon Ydog_G (part 1)  according to the labeling scheme of Natanaelsson et al.  Table S.2.2.2.2PCR amplifications were carried out individually for each Y chromosome fragment using the primer pairs described in Table 2.2.2.3® SNaPshot™ Multiplex Kit (Applied Biosystems) according to the manufacturer's protocol. Two separate multiplex reactions were prepared: one included pooled PCR products and extension primers for loci Ydog_21, Ydog_28, Ydog_G (part 1) and Ydog_B (part 2) (SNPs position here named Ydog_B), covering seven dog‐specific SNPs; and the other multiplex included the PCR products and extension primers for the remaining loci mentioned in Table This step consisted of a multiplex reaction for single‐base primer extension using the ABI PRISM2.2.2.4In the search for new polymorphisms, sequencing reactions were performed for each locus in dogs and wolves. PCR products were sequenced on both directions by BigDye Terminator v1.1 Sequencing Kit and analyzed on an automated fluorescence‐based ABI PRISM 3130 Genetic Analyser (Applied Biosystems).2.2.2.5Four Y chromosome microsatellite , loci—990‐35, MS41A, MS41B, and MS34TTR .2.32.3.1FST value were calculated in GenAlEx 6.5b  iterations; burn‐in period of 10,000 interactions] were set to run using the ‘admixture’ model and assuming that allele frequencies are correlated among populations for a K = 2. The STRUCTURE PLOT software , and the level of genetic divergence between the dog and wolf subspecies was determined through the fixation index PhiPT for Y‐STRs alone and in combination with Y‐SNPs. p‐values were estimated after 9,999 permutations.Regarding Y‐STRs, we estimated haplotype diversities and analyzed molecular variance (AMOVA) using GenAlEx v6.501 (http://www.fluxus-engineering.com) using only the polymorphic loci. The Y‐STR loci were weighted inversely to their variance, and considering the much lower rate of substitutions relative to autosomal STR mutations; SNP loci were weighted as follows: transitions w = 10 and transversions w = 30. We first constructed a reduced‐median network and then applied a median‐joining analysis to create the final networks.A combined analysis of Y chromosome SNP and STR markers was performed with the software NETWORK 5 , and all sequences were collapsed to unique haplotypes using Fabox DNA Collapser (http://users-birc.au.dk/biopv/php/fabox/).The software Sequencher 4.8  was used to edit and assemble the mtDNA sequence trace chromatograms. Sequence alignments were verified by eye and edited using MacClade 4.08 (http://www.ub.edu/dnasp/)  were calculated for each population separately. The extent of divergence among these two populations and gene flow was also computed and statistically assessed. Because there are some indels in the sequence alignment, gaps were treated as a fifth character.Analyses at the geographic scale of the Iberian Peninsula were performed considering 420 bp of the mtDNA fragment sequenced. Genetic diversity and differentiation analyses were performed with DnaSP v5.10.01 (http://www.huntmountainsoftware.com/html/rarefaction.html).To test if differences in haplotype richness between wolves and dogs found in Iberia were due to unbalanced sampling, the total number of haplotypes for each population was estimated using a rarefaction resampling method as implemented in the software Analytic Rarefaction 2.0 . Sequences generated in this study that had no exact match to sequences deposited in public nucleotide databases were submitted to GenBank.To assess how Iberian wolves are related to other gray wolves and to dogs native to the Iberian Peninsula and neighboring areas, additional mtDNA control region I sequences of wolves, native dog breeds, and village dogs from Iberia and North Africa were retrieved from GenBank . The program jModelTest v.0.1.1 (http://computing.bio.cam.ac.uk/software.html) was used to select the model of evolution that best explains the nucleotide variation within the data set, excluding the outgroup and using the Akaike information criterion (AIC). The software MrBayes 3.1.2 (http://mrbayes.sourceforge.net/) was used to perform the BI analyses and two independent analyses, starting from different random trees, were run in parallel for 107 generations, with four simultaneous Markov chains, sampling every 100th generation and discarding as burn‐in the first 25% of samples. As convergence diagnostics, we used the average standard deviation of split frequencies (<0.01) and inspected the plot of the log likelihood values (no increasing or decreasing tendency over time).For worldwide comparisons, sequences were trimmed to 230 bp, a mtDNA control region fragment common to wolf sequences across many studies .The absolute number of nucleotide differences among haplotypes was determined using the software PAUP version 4.0b10 . The partitioning analysis using the Bayesian clustering procedure clearly shows a sharp separation between dogs, , and the Iberian wolf  and wolves , did not reveal additional polymorphic sites (GenBank accession numbers GQ366706‐GQ366793 and KT967955‐KT967970). For female samples, no PCR products were obtained for any locus confirming the Y chromosome specificity of the designed primers.Sequence data, for the studied loci for a subset of dogs  and 27 Iberian wolves  and another including Iberian wolves (IbWolf HG) , whereas the IbWolf HG comprises four lineages: YSNP H9, YSNP H32, YSNP H33, and YSNP H34, following the nomenclature of Ding et al.  and 0.052 (for Iberian village dogs and African dogs). These values were not significant .Regarding STRs alone, a total of 25 haplotypes are described for the studied dogs Table B. The IbSE for the Iberian wolf is smaller compared to the dog population and is 0.41 ± 0.04. The PhiPT pairwise value between dogs and Iberian wolves of this study is 0.27 (p = .00).The Iberian wolf displays seven Y‐STR haplotypes, three of these are shared with dogs and four are exclusive to the Iberian wolf Table B. The HDIbAfrDog HG and the IbWolf HG, 25 and 11 haplotypes were disclosed, respectively .The combination of 10 informative Y chromosome loci (four STRs plus six SNPs) allowed the identification of 36 haplotypes. Within the y Figure A,B. The Canis data available from Ding et al.  and dogs (mostly sampled in southwest Asia and the Middle East) , and dogs from Africa  and East Asia . The YSNP H9 haplotype differs from YSNP 10B, another haplotype shared between wolves and dogs, by a single SNP (locus B)  Figure A. In parB) Table A.YSNP H1  is the most common haplotype among dogs worldwide—1 Figure A, also f3.3YSNP H32, H33, and H34—segregate in the network among haplogroups HG1 & HG3, HG9, and HG23 where several unsampled nodes were previously detected by Brown et al. , wH‐1B (1.8%), wH‐1C (5.4%), wH‐2 5.3%), wH‐3 (1.8%), wH‐4 (1.8%), and wH‐5 (1.8%), where wH‐1A is the most frequent haplotype found throughout Portugal and Spain  and were submitted to GenBank , the Iberian wolf sequences generated in this study cluster with other wolf sequences forming clades with high node support (posterior probability ≥0.90), except for: haplotype wH‐2 in the 230 bp worldwide analysis that forms a group with low support with other wolf sequences; and haplotype wH‐5 (shared with dogs) that forms a group with high node support with other dog sequences (dog haplogroup A) (posterior probability 0.94)  and 0.32 ± 0.080, respectively. The difference in nucleotide diversity (π) for dogs and wolves found in Iberia is even higher, 0.014 ± 0.0006 (mean ± SD) and 0.002 ± 0.0008, respectively.Concerning dogs and wolves found in Iberia for which we have haplotype frequency information , the rarefaction analysis revealed that haplotype richness was approximately 2.5 times higher for dogs compared to the wolves (18.8 vs. 7). Haplotype diversity (HD) for the Iberian dogs was also approximately three times higher than the value computed for Iberian wolves, 0.92 ± 0.008 , where the FST value between the two subspecies is 0.486.The statistical test indicates that Iberian dogs and wolves are strongly genetically differentiated .The Y chromosome data provide an update to the most parsimonious phylogenetic tree presented by Ding et al.  with theThe studied Iberian dogs belong to HG1, one of the haplogroups universally shared by dogs  depending on the set of autosomal markers used , is necessary for the reconstruction of the history of Canis subspecies in Iberia and for investigating associated population evolutionary processes.Y chromosome molecular markers disclose a genetic distinctiveness of the Iberian wolf from other wolves and from domestic dogs. Four Y‐SNP markers are diagnostic and can segregate Iberian wolves from male dog samples. This result obtained from contemporaneous samples does not support a local domestication event for dogs in Iberia, as the genetic makeup of local dogs is apparently made of a myriad of Y chromosome lineages (including Y‐STRs) that came from elsewhere. In any case, breeds have a very recent origin when compared to the domestication of dogs, and the haplotypes that are currently present in native breeds may not be representative of the haplotypes that were present in early dog subspecies. Further investigation using historical and ancient samples and high‐resolution markers  was collected in the year 2000 within the known wolf range in the north of Portugal (Serra da Cabreira), from a deceased animal with a typical wolf phenotype, showing no external signs of admixture with domestic dog. Probably due to poor sample preservation and thus DNA degradation, we were not able to generate microsatellite data for this sample, neither autosomal nor Y‐linked microsatellites. Nevertheless, this sample exhibits a wolf Y‐SNP profile: wolf‐typical nucleotides were detected at the described diagnostic loci. Being a male wolf, for this sample to belong to a first‐generation hybrid, it could have only resulted from the cross between a female dog and a male wolf, which is considered rare. The first record of this kind of hybrid in Europe dates from 2008–2009 in Latvia, a single specimen, which was reported in Hindrikson et al. . Given t4.3We have shown, using both nuclear and mitochondrial data, that the Iberian wolf displays greater diversity than previously reported, with new maternal and paternal haplotypes disclosed.Canis lineages that did not diverge after the domestication of dogs. Although hybridization has been recorded in Iberia, the fact that in our dataset the percentage of dog–wolf mtDNA haplotype sharing in this area is roughly half of what was found elsewhere supports the conclusion that hybridization in Iberia, more specifically in Portugal (as most wolf samples are from this region), is a rare event. If disturbance of wolf habitats in Iberia continues, due to both habitat loss and/or fragmentation, in addition to an increase in the feral dog population, which largely overlaps with the wolf range (Álvares, We hypothesize that mtDNA haplotypes shared between wolves and dogs may represent relic common ancestral Álvares, , the genWolves found in Iberia represent a reservoir of unique genetic and ecomorphologic variants of the gray wolf. Therefore, according to Hofreiter and Barnes'  proposalNone declared.JX845621‐JX845625. Y chromosome DNA sequences: GenBank accession numbers GQ366706‐GQ366793 and KT967955‐KT967970.Mitochondrial DNA sequences: GenBank accession numbers  Click here for additional data file.


While the functionality and healthy food value of red rice have increased its popularity, such that market demand for it is expected to rise, most strains suffer from low grain yield. To perform diversity and population structure analyses of red rice germplasm, therefore, becomes essential for improving yields for commercial production. In this study, fifty red rice germplasm from the Bangladesh Rice Research Institute (BRRI) genebank were characterized both morphologically and genetically using fifty simple sequence repeat (SSR) markers. Overall, 162 alleles were detected by the markers with the detected allele number varying from two to seven. Additionally, 22 unique alleles were identified for use as a germplasm diagnostic tool. The highest and lowest polymorphic information content (PIC) indices were 0.75 and 0.04 found in markers RM282 and RM304, respectively, and genetic diversity was moderate, varying from 0.05 to 0.78 (average: 0.35). While phylogenetic cluster analysis of the fifteen distance-based agro-morphological traits divided the germplasm into five clusters , a similar SSR analysis yielded only three major groups , and a model-based population structure analysis yielded four . Both principal component and neighbors joining tree analysis from the population structure method showed the tested germplasm as highly diverse in structure. Moreover, an analysis of molecular variance (AMOVA), as well as a pairwise FST analysis, both indicated significant differentiation (ranging from 0.108 to 0.207) among all pairs of populations, suggesting that all four population structure groups differed significantly. Populations A and D were the most differentiated from each other by FST. Findings from this study suggest that the diverse germplasm and polymorphic trait-linked SSR markers of red rice are suitable for the detection of economically desirable trait loci/genes for use in future molecular breeding programs. Rice is the main food for more than half of the world’s population and is the staple food for the people of Bangladesh, constituting over 91% of the food grain production, and providing 62% of the caloric as well 46% of the protein intake in the average daily diet [Bangladesh has three major rice crops, Aman, Aus, and Boro, which are grown during three overlapping seasons and with a large number of varieties suitable for various agro-ecological and climatic niches . The AusModern breeding techniques could improve the yield of red rice to overcome this problem. Successful breeding for crop improvement, however, depends on genetic variability in the parents , such thVery few studies, however, have investigated the genetic diversity of Aus rice in Bangladesh –8. WhileClustering a large number of germplasms represents a simple and effective process for assessing the genetic variability of germplasm , along wCultivars developed using natural populations and germplasm can have unique target characteristics for attaining high yields and stress tolerance. Analyzing genetic structures, as well as identifying allelic variation among and/or within a population, represents an important approach for the design of a molecular breeding program . AnalyziThis study uses red rice to perform a diversity analysis using both morphological and molecular approaches. In general, red (and black) rice are staple foods that an increasing number of consumers are demanding due to their documented health-promoting benefits . These iCommercial availability of red rice in markets is limited, however, by a lack of improved varieties at the producer level. Few farmers cultivate this rice using local varieties, due to low yields, long durations, and susceptibility to lodging . MoreoveRice germplasm, including Aus, Aman and Boro rice cultivated throughout Bangladesh, have been collected and conserved by the Bangladesh Rice Research Institute (BRRI) genebank. To date, approximately 8,200 germplasm have been preserved by the BRRI genebank, of which 1,500 are Aus rice, with more than 30% of those considered red rice. For this study, we cultivated fifty red rice germplasm—collected by BRRI from the sites noted in All experiments described here were conducted at the Bangladesh Rice Research Institute in Gazipur, Bangladesh during the March to June Aus rice growing season in 2015. Fifty red rice germplasm were seeded directly under upland conditions in two 5 m x 3 m plot replicated trials. Production practices, including fertilizers, intercultural operations, pest control, and grain harvesting, were conducted according to standard methods.2) analysis [Fifteen agro-morphological traits—leaf area index, culm diameter, effective tiller number, days to flowering, plant height, days to maturity, five panicles weight, filled grains per panicle, unfilled grains per panicle, grain length, grain breadth, 1000 grain weight, length-breadth ratio, and yield per hill—were recorded. Genetic diversity was worked out for the principal component analysis  and Mahaanalysis . All mulWe used fifty well-distributed SSRs for the diversity analysis; position (cM), repeat motifs, and chromosomal positions for the SSR markers can be found in the rice genome database . Most ofThe band-size for each of the markers was scored using the AlphaEaseFC 4.0 software. Using PowerMarker version 3.25 , summaryPopulation STRUCTURE for germplasm was determined using STRUCTURE, (version 2.3.4) , 55. The2 statistic, the fifty red rice germplasm with fifteen agro-morphological traits grouped into five clusters   demonstrated significant variation among fifty red rice germplasm for all fifteen agro-morphological traits. Using PCA, we observed that the first axis mainly accounted for the variation in the germplasm (27.89%) followed by second axis (18.41%). Our results also showed that the first six components with vector values >1 accounted for 80.08% of total variation, with components PCA3, PCA4, and PCA5 contributing 11.26%, 9.77%, and 6.08%, respectively. Using the first two components, , V) see . The clu2, respectively.Average intra- and inter-cluster distance for the entire germplasm based on phenotypic traits was determined. Considerable variation occurred in intra-cluster distances (ranging from 0.65 in cluster II to 1.24 in cluster III), indicating wider diversity prevailing among germplasm within clusters. The germplasm under cluster III (1.24) was the most heterogeneous; germplasm under cluster II (0.65) was comparatively homogenous or less diverse. Inter-cluster distances ranged from 3.70 to 16.31, ranging from a maximum in clusters IV and III (16.31), clusters III and I (14.98), and clusters V and III (12.23), indicating a wider genetic diversity among the germplasm between these groups. In contrast, we observed the lowest inter-cluster distance between clusters I and IV (3.70) and clusters I and V (4.49), indicating a narrower, genetically close, diversity. In general, the germplasm comprising clusters IV and III were more diverse than those of clusters I and IV. Overall, cluster IV had the highest inter-cluster distances compared to other clusters, indicating wider genetic germplasm diversity within cluster IV relative to others.The germplasm of fifty red rice populations collected by BRRI from several locations in Bangladesh were amplified using fifty SSR markers . All of The study found 22 unique alleles detected in all tested germplasm . ClusterBayesian analysis using STRUCTURE characterized the population structure for tested red rice germplasm. The LnP(D) as well Evanno’s ΔK values identified four genetically distinct populations , 0.4226 (B), 0.3742 (D), and 0.3534 (C), with an average 0.4125, thus indicating a high population structure. We calculated specific FST values for populations A, B, C, and D, using STRUCTURE software. The average distances  between the individuals in the same cluster were 0.2144 for A, 0.2831 for B, 0.3017 for C, and 0.4317 for D. The largest genetic (net nucleotide) distance (0.2240) was observed between A and D, and the lowest genetic distance was observed between A and C.Both a neighbor-joining (NJ) tree and principal component analysis (PCA), based on population derived from the structure analysis, were also performed. Both NJ tree and PCA established the results obtained from structure analysis. The model-based population groups (A–D) were distinctly separated from each other . In the The four populations obtained from structural analysis were subsequently exposed to AMOVA in order to determine the variation across and within populations. While a 14% variance was observed across populations, for individuals, 80% variance among and 6% within was observed . The paiIn general, the results from FST analysis and AMOVA both agreed with results obtained using the phylogenetic tree-based similarity coefficient distribution as well as the structure analysis, with all of these aforementioned approaches confirming the presence of both a statistically moderate genetic diversity and a high level of population structure. This represents a critical step for carrying out any future association mapping (AM) analysis.In this study, we analyzed the diversity (both morphology and molecular) and population structure of fifty kinds of red rice that grow in different locations of Bangladesh in order to begin to lay a foundation for future yield-improvement rice breeding programs . Based o2), culm diameter (mm), filled grains per panicle, and yield/hill (gm) were observed in cluster III. Cluster III also showed the lowest mean for 1000 grain weight (g). Cluster II showed the highest cluster means for the panicle length trait. The performance mean of the different clusters for the characteristics studied demonstrated that traits for dwarf stature, short growth duration, lower leaf area index (cm2), culm diameter (mm), panicle length (cm), filled grains per panicle, unfilled grains per panicle, and yield/hill (g) were clubbed in cluster IV along with the trait for maximum number of effective tiller.Traits for culm diameter, plant height, panicle length, five panicles weight, and filled grains per panicle led the most to genetic diversity . The hig2 statistic, resonates with Siddique et al. Siddique, Khalequzzaman, Islam, Ahamed and Rashid [2 statistics. The clustering pattern in this study demonstrated that the germplasm constellated in the same cluster, albeit without originating from the same geographic area. Chakravorty, Ghosh and Sahu [Comparing these results to other research, the five clusters here, grouped using the Mahalanobis Dd Rashid , who repand Sahu  evaluateet al. [Correlation analysis of genotypic and phenotypic traits can provide important information among study crop species or different groups of crop accessions . Researcet al. , who meaet al.  suggest et al. , 65.et al. [Using SSR marker profiling to identify the genetic polymorphism available among the tested red rice germplasm, the measured average number of alleles per locus (3.24) compares with Islam , who stuet al.  also detet al. , as wellet al.  and 0.05et al. . Ahmed, et al. , howeveret al. , lower PIdentification of unique alleles can have great importance for breeding purposes , 69. In et al. [et al. [In the present study, genetic diversity ranged from 0.05 and 0.68, averaging 0.35 , with a et al. , where aet al.  reported [et al.  reported [et al. .Neighbor-joining tree analysis showed all fifty red rice germplasm separated into three major groups, with group II divided into further sub-groups. While the majority of the germplasm (82%) were in group II, and group III contained only the Surjamukhi and Noyantara germplasm, group II also had several sub-groups .et al. [Population structure analysis displayed four populations A, B, C and D. The highest number of germplasm were grouped in population C, with only four—Honumanjata, Kharojamri, Khatomala and Laithajhora—in group D. Both AMOVA and FST analyses confirmed the population structure and its statistical significance Tables  and 4. Tet al.  similarlGroup I derived from STRUCTURE analysis showed higher yield than other groups, with group IV showing the lowest yield. Most of the population of group IV are short and have a lower growth phase that limits the yield and yield-contributing traits.In general, care should be taken when selecting germplasm for any breeding program attempting yield enhancement. Cluster analysis from the agro-morphological features grouped all the germplasm into five groups. Significant (at both p ≤ 0.05 and p ≤ 0.01) differences were found in all fifteen agro-morphological traits, which confirms the presence of diversification among the fifty red rice germplasm. From molecular analysis using SSR markers, three groups were found, while population structure analysis showed all tested germplasm constructed as four populations, each significantly differentiated and with their variation confirmed by AMOVA.From the findings of the population structure analysis, in particular, large genetic differences were also observed between two populations (group A and D), suggesting that these could be crossed to improve grain yield. As such, the germplasm, as well as the highly polymorphic SSR markers identified in this study, have the potential to facilitate a red rice breeding program for enhanced yield. Based on these findings, we are currently developing such a program to facilitate more desirable, novel trait-linked markers usable for not only the mapping of QTLs/genes against a diversity of abiotic and biotic stresses but also the breeding of socially and economically desirable traits in red rice. The findings of this study provide a solid foundation for such further work in the effort to increase the yields of this increasingly popular and both medically and nutritively beneficial red rice.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.


Sabatieria, abundantly present in reduced cold-seep sediments. Samples originated from four Eastern Mediterranean cold seeps, separated by hundreds of kilometers, and one seep in the Southeast Atlantic.There is a general lack of information on the dispersal and genetic structuring for populations of small-sized deep-water taxa, including free-living nematodes which inhabit and dominate the seafloor sediments. This is also true for unique and scattered deep-sea habitats such as cold seeps. Given the limited dispersal capacity of marine nematodes, genetic differentiation between such geographically isolated habitat patches is expected to be high. Against this background, we examined genetic variation in both mitochondrial (COI) and nuclear  DNA markers of 333 individuals of the genus ST = 0.149). The haplotype network of COI contained 19 haplotypes with the most abundant haplotype (52% of the specimens) shared between all four seeps. The number of private haplotypes was high (15), but the number of mutations between haplotypes was low (1–8). These results indicate intermediary gene flow among the Mediterranean Sabatieria populations with no evidence of long-term barriers to gene flow.Individuals from the Mediterranean and Atlantic were divided into two separate but closely-related species clades. Within the Eastern Mediterranean, all specimens belonged to a single species, but with a strong population genetic structure  contains supplementary material, which is available to authorized users. The deep sea is one of the largest ecosystems on earth, covering two-thirds of the planet and containing a substantial proportion of the earth’s biodiversity. This ecosystem has long been considered a vast and continuous environment with stable biotic, physicochemical and hydrographical conditions  which enWhile our knowledge on the identity, diversity, and distribution of extant species continues to grow, insights on the evolutionary processes that are at the heart of this diversity in the deep sea are lagging behind. Genetic divergence in the marine environment may result from contemporary and historical processes related to the different oceanic basins , changesNext to hydrothermal vents, cold seeps are among the most widely spread deep-sea reducing environments and are characterized by high levels of methane and sulphide, the latter being toxic to most metazoans. Associated fauna therefore need to be adapted to the complexity of the presence of biogenic structures, geochemical conditions and microbial processes. Active seeps have been reported all over the world, even in the Arctic and Antarctic polar regions , 22, andHalomonhystera disjuncta, are known to be ovoviviparous [Nematodes are among the most numerous multicellular taxa on earth, successfully adapted to nearly every ecosystem, but many species are biologically still poorly understood. Different species feed on materials as varied as algae, fungi, small animals, faecal matter and dead organisms . Most neviparous , 28. TheCompared to more common deep-sea environments, most cold seeps support highly productive ecosystems characterized by poor nematode species richness, high biomass and elevated dominance of a few genera able to withstand the high concentrations of toxic hydrogen sulphide e.g., , 29–33).–33.29–33Sabatieria species, S. mortenseni, in one cold seep in the Atlantic and four cold seeps in the Eastern Mediterranean Sea to investigate the genetic diversity patterns within and between reduced cold-seep environments.Here, we investigate the genetic structure and connectivity in species of heterogeneously distributed cold seeps in the deep sea. We sampled the dominant Sabatieria is one of the most common nematode genera in marine benthic habitats, especially in muddy and muddy sand sediments [Sabatieria species are considered valid [Sabatieria species appear to be divided in five distinct subgroups according to morphological differences related to the type and distribution of precloacal supplements, characteristics of the gubernaculum and apophysis, the number of turns of the amphideal fovea, and the cephalic setae [pulchra group, which consists of six species, including S. mortenseni. Species of this group are distinguished by the disposition of the precloacal supplements, which are usually conspicuous and relatively few in numbers (5–9), and by the characteristic gubernaculum median pieces and short paired cervical setae [S. mortenseni has been found in reduced sediments of shallow-water sites in Brazil, USA, Antarctica [The genus ediments , and is ediments –48, inclediments , 49, 50.ediments . Many ofediments , 53. Cured valid  and are ed valid –57). Sabic setae . The speal setae . S. morttarctica –61 and ttarctica . It is atarctica . Based oSabatieria specimens were collected at five cold seeps, one located in the Atlantic Ocean and four in the Eastern Mediterranean area  and MSM13/3 – MSM13/4 campaign of the RV Maria S. Merian (November–December 2009) for the Mediterranean seeps, and during the Guineco cruise (2008) onboard RV Meteor for the Atlantic REGAB site : 45; PM(2B): 42; PM: 62; Cheops: 112; Amon MV: 115; Amsterdam MV: 108; REGAB: 34) were photographed using a Leica DMR microscope equipped with a Leica DFC 420 camera to serve as a morphological reference. Individuals examined here were assigned to Sabatieria mortenseni based on the number of turns of the amphideal fovea . In this way, 618 Sabatieria specimens were collected from the Mediterranean seeps, and 41 from samples of the REGAB seep. DNA was extracted by adding 1.5 μl proteinase K (10 mg/ml) to each WLB-stored specimen, followed by incubation for 1 h at 65 °C and 10 min at 95 °C, and finally centrifugation for 1 min at 14000 rpm prior to usage.The cold seeps investigated in this study were the subject of international projects, and were thoroughly investigated during multiple campaigns. Therefore, samples for both morphological and molecular research were available for these particular reduced environments. Samples were collected during MEDECO Leg 2 of RV tudy: 3, : 2.5; [6tudy: 3, : 2.5–3; tudy: 3, : 2.25–2.rding to . Multiplrding to  to assurc oxidase subunit 1 (COI) gene were amplified using the forward primer JB3 (5′-TTTTTTGGGCATCCTGAGGTTTAT-3′) [For the polymerase chain reaction (PCR), 1 μl of DNA template was used. In this study, 420 base pairs (bp) of the mitochondrial cytochrome TTAT-3′)  and the TTAT-3′) . StandarAfter identifying the different COI haplotypes, DNA from each haplotype was used to amplify two nuclear DNA gene regions: the 18S region of the small ribosomal subunit and the D2D3 region of the conserved 28S large ribosomal subunit. 752 bp of the D2D3 region was amplified with the forward primer D2A (5′-ACAAGTACCGTGAGGGAAAGTTG-3′) and the reverse primer D3B (5′-TCCTCGGAAGGAACCAGCTACTA-3′) , while ±Praeacanthonchus punctatus, Ptycholaimellus carinatus and Odontophora setosa, all from the same subclassis (Chromadoria) as Sabatieria, were added to the dataset as outgroup . The same strategy was followed for the two nuclear markers with the exception that a Tamura-Nei + G substitution model [Odontophora setosa and Praeacanthonchus punctatus) as well as published sequences extracted from GenBank . For D2D3, there were no Ptycholaimellus sequences available. The unique haplotypes found in this study have been submitted to GenBank and are available under accession numbers LT703318-LT703405. Representative DNA samples were deposited in the MoMentuM database of CeMoFe (Centre for Molecular Phylogeny and Evolution), UGent.COI sequences were checked and trimmed for further phylogenetic analysis in DNASTAR Lasergene Seqman Pro . Primer on model  was usedSabatieria COI sequence variation across the different seeps. Population pairwise ΦST-statistics were calculated  to determine which seeps were significantly differentiated from each other. Because the HKY model given by jModelTest is not available in ARLEQUIN, the more inclusive Tamura-Nei (TrN) model [ST and other relevant statistics. Significance levels of the obtained p-values were corrected according to the sequential Bonferroni method [h) [D [Fs [D and Fu’s Fs reflect an excess of rare polymorphisms in a population, which indicates either positive selection or an increase in population size [Analysis of Molecular Variance (AMOVA) was performed in ARLEQUIN version 3.5.1.2  and usedN) model  with gami method . Next toi method  and haplthod [h) , 87, andd [h) [D  and Fu’s) [D [Fs  neutraliion size . These thttp://popart.otago.ac.nz).To test whether genetic divergence between populations increased with geographic distance , geograSequence alignment yielded a COI fragment of 420 bp long. This fragment contained 135 conserved and 264 variable positions, 193 of which were parsimony informative and 69 of which were singletons. Both the NJ and ML tree topology of COI haplotypes showed two well-supported clades . The D2D3 fragment was 752 bp long with 206 conservative and 531 variable positions, of which 155 parsimony informative and 373 singletons. The NJ and ML analysis of both markers showed congruent tree topologies which were similar to the topology of the COI tree, with two well-supported clades . A Blast search of its 18S sequence identified it as Terschellingia longicaudata (95–98% similarity), a species that is abundantly present at Amon MV. This haplotype was therefore excluded from further population genetic analyses.The 18S fragment was 942 bp long, contained 678 conserved and 262 variable positions, 98 of which were parsimony informative and 163 of which were singletons. Two insertions of a single bp were noted in two outgroup species , divided evenly over all four cold seeps from the Eastern Mediterranean area Table . A fragmThe TCS network further indicated the dominance of haplotype A, which was found in all four seeps Fig. . Two groh) was higher in Amsterdam MV (0.773 ± 0.022) than in the other seeps (between 0.576 ± 0.045 and 0.616 ± 0.052), while nucleotide diversity (π) was an order of magnitude lower in Cheops than in the three other seeps , attributing 15% of the genetic variability in the dataset to differences between locations  and significantly different from zero after Bonferroni correction  and log-transformed  distances. The same was true for the three seeps in the southern part of the East Mediterranean  when analyzed separately . Tajima’s D was only significantly negative for Cheops . None of Fu’s Fs were significantly different from zero. Also the analysis of the whole dataset as one group did not yield significant results .Haplotype diversity . Also forth Sea . Bik andorth Sea  found evorth Sea  revealedribed in   was abundantly present in all reduced sediments sampled . The species was also present in the surrounding oxidized sediments, albeit in much lower abundances , indicating its preference for hypoxic or anoxic conditions. These data suggest a widespread distribution in the area under study. However, the extent of genetic similarity between Sabatieria mortenseni from the non-reduced environment and the cold seeps is not clear, since specimens from the non-reduced environment were preserved in formalin.For st values suggest that suspension and transport by currents are not enough to establish extensive gene flow between the Sabatieria populations of the different cold seeps. Instead, several haplotypes are shared between seeps, the number of mutations between haplotypes is low and private haplotypes are present in each cold seep, all indicating that intermediate gene flow is occurring without long-term barriers to dispersal between cold seeps . The prThe East Mediterranean is one of two sub-basins of the Mediterranean Sea and hosts a complex array of current systems, including deep-water formation in the Adriatic Sea , cyclonSabatieria populations of the East Mediterranean cold seeps was observed (see Mantel test results). This was also evident from the COI phylogenetic tree where populations did not cluster according to their geographic location . Given ion Fig. . Insteadion Fig. . Such a D was only significantly negative for Cheops MV. Instead, the two haplotype groups were separated by 2 or 3 mutations suggesting recent admixture between previously isolated populations , historical events often leave their mark on current species distributions. On an evolutionary timescale, the Mediterranean deep-sea environment and its fauna are relatively young compared to that of other oceans. Our results show a significant population genetic structuring between the cold seeps within the Eastern Mediterranean sea which may reflect past isolation and (re-) colonization events. The haplotype network for COI consisted of a few central widespread and abundant haplotypes (A and B) surrounded by multiple private haplotypes . Ts e.g., , 111) an anD was S. mortenseni may result from a strong adaptation to the particular local conditions at the different cold seep habitats. Such a scenario of differentiation by adaptation seems plausible considering the environmental heterogeneity typically found in chemosynthetic habitats [Sabatieria in the East Mediterranean.Finally, the genetic structure present in habitats . In thishabitats ), leadinSabatieria mortenseni from seeps located in different ocean basins: genetic evidence pointed towards two independently evolving lineages, one in the Eastern Mediterranean and one in the Southeast Atlantic region. Yet, no differences in diagnostic morphological features were observed and morphometric characters overlapped with those of S. mortenseni reported in [Paracanthonchus gynodiporata living on macroalgae were genetically homogeneous across more than 1000 km Brazilian coastline but showed substantial phenotypic plasticity [Halomonhystera disjuncta from the Håkon Mosby mud volcano is genetically distinct from the intertidal Halomonhystera disjuncta [Sabatieria from the continental shelf in Antarctica [S. mortenseni from [S. mortenseni reported from the literature . Numeroorted in , 116 andorted in  that domorted in , 39, 118asticity . Crypticisjuncta  and two tarctica . The cureni from , 74, speSabatieria populations may not have been completed yet. As a reference, cryptic species found in the shallow-water nematode Litoditis marina are at least 6.5 Myr old [Sabatieria species are known to withstand fluctuations in environmental chemistry as they regularly are the dominant genus in various reduced ecosystems .–33.SabatSabatieria mortenseni inhabiting cold seeps from the bathyal East Mediterranean Sea and Atlantic Ocean are genetically structured. The presence of shared haplotypes and the low number of mutations between haplotypes indicate that barriers to dispersal between these cold seep populations are relatively recent. The low abundances of S. mortenseni in the surrounding sediment together with water currents in the area may form part of a source-sink system facilitating gene flow between distant populations. Our results further show the presence of cryptic species within S. mortenseni from seeps located in different ocean basins: genetic evidence pointed towards two independently evolving lineages, one in the Eastern Mediterranean and one in the Southeast Atlantic region but no differences in diagnostic morphological features were observed. Our results expand our knowledge on genetic diversity in marine nematodes and confirm that a single nematode species can be widely distributed despite the lack of pelagic larval stages.The populations of the marine nematode


The lack of such a quantitative assay has limited the identification and development of STAT3 DNA-binding domain inhibitors. Here, we report a modified DNA-binding ELISA to incorporate recombinant STAT3 protein to evaluate small molecules that prevent STAT3-DNA binding. The concomitant use of the ELISA and fluorescence polarization assay enables the classification of direct STAT3 inhibitors by their site of action. Our data provide further support that niclosamide inhibits STAT3 through interaction with the DNA-binding domain. Furthermore, the ELISA can support medicinal chemistry efforts by identifying DNA-binding domain inhibitors and allowing the determination of an IC50 value, supporting the ranking of inhibitors and development of structure-activity relationships. Therefore, we propose a tandem evaluation approach to identify small molecules that target the SH2 domain or the DNA-binding domain of STAT3, which allows for quantitative evaluation of candidate STAT3 inhibitors.In many forms of cancer the signal transducer and activator of transcription 3 (STAT3) transcription factor remains constitutively active, driving cancer survival and progression. The critical role of STAT3 in tumorigenesis has prompted a campaign of drug discovery programs to identify small molecules that disrupt the function of STAT3, with more recent efforts focusing on direct STAT3 inhibition. There are two target binding sites for direct STAT3 inhibitors: the SH2 dimerization domain and the DNA-binding domain. An  Signal transducer and activator of transcription 3 (STAT3) is a member of the STAT family of transcription factors that regulate many cellular processes including cell growth and survival, and immune response –3. The ain vitro screening assay, hindering the drug discovery process. It has been proposed that niclosamide, an anthelmintic drug, inhibits the ability of STAT3 to bind DNA [A number of strategies have been examined to inhibit STAT3 activation, such as targeting the activating JAK2 kinase; however, these broad-spectrum approaches often result in off-target effects , 14, 15.bind DNA , and thebind DNA   assay, developed by Schust and Burg [et al., have demonstrated that unphosphorylated STAT3 (uSTAT3) is capable of binding its corresponding DNA sequence using a protein electrophoretic mobility shift assay (PEMSA), supporting the use of recombinant STAT3 for the evaluation of DBD inhibition; however, small molecule inhibitors were not evaluated via this method [in vitro DNA-binding STAT3 ELISA as part of a tandem evaluation approach to elucidate and quantitatively assess small molecule inhibitors that target either the SH2 dimerization domain or the DBD of STAT3.The cell-based assays used to evaluate STAT3 inhibition with small molecules include the STAT3-dependent dual luciferase reporter assay , 28, eleand Burg , allows s method . In thisvia cysteine alkylation [50 of 7.39 ± 0.95μM in the FP assay . The average OD recorded for the recommended 5μg total protein of HepG2 nuclear extracts was 0.793, equating to an average STAT3-DNA bound content of 0.21μg Figure , althoug50 of 1.27 ± 0.38μM  and no STAT3-inhibitor co-crystal structures are present, suggesting that the resolution of the STAT3 crystal structure is not a trivial endeavor.Stattic alkylates cysteine residues in the DBD of STAT3 , 35, resID: 4E68 , and 1BGID: 4E68 ) of the 50 would result in too few cells remaining to produce the required 20μg of total protein in the nuclear extracts. Although stattic demonstrated inhibition of STAT3-DNA binding in the ELISA using recombinant STAT3, no inhibition was observed in the ELISA using nuclear extracts, possibly due to the non-specific cysteine alkylation of other cellular proteins. S3I-1757 has previously displayed high micromolar activity in cells [50 could be determined from the MTT assay (data not shown). Therefore, S3I-1757 was not advanced forward for the evaluation of STAT3 inhibition by ELISA in HeLa cells. A18 induced a ~55% reduction in STAT3-DNA binding at its MTT derived EC50 of 12μM while A26 displayed no significant activity at the corresponding concentration of 6μM. Results reported for a STAT3 EMSA in H1299 cells is consistent with a 50% reduction of STAT3-DNA binding for A18 at 12μM and no reduction in binding for A26 at 6μM [in vitro ELISA using recombinant STAT3, but activity in the cell-based ELISA supports a mode of action other than direct STAT3 inhibition  can be established. Furthermore, the data obtained from the ELISA using nuclear extracts from HeLa cells provides initial support that STAT3 inhibitors targeting the DBD are more efficient than those targeting the SH2 domain in cellular systems. In this study we have established an approach for identifying DBD inhibitors of STAT3 and our data strongly supports that niclosamide targets the DBD of STAT3. Although niclosamide was not designed or developed as a STAT3 inhibitor, it may provide a structural basis for the development of more selective STAT3 inhibitors targeting the DBD.Taken together, our data support that the FP assay and ELISA using recombinant STAT3 can serve as an effective quantitative approach to identify potent direct inhibitors of STAT3 acting at the SH2 dimerization domain or the DBD Figure . The useHuman cervical carcinoma cells (HeLa) were obtained from ATCC and were verified by the vendor. Cells were grown in Eagle's Minimum Essential Medium (EMEM) supplemented with 10% heat inactivated FBS. Compounds S3I-1757 (AOBIOUS), stattic (ApexBio Tech LLC), and niclosamide  were purchased. Compounds A18 and A26 were synthesized according to previously reported methods , 43.2 (Genscript) was added to each assay well at a concentration of 10 nM. Final well volumes were 30μL. Following incubation with fluorescent peptide for 30 minutes at room temperature with mild agitation, plates were examined using FP Fluorescein Dual module with excitation filter FITC FP 480 and emission filter FITC FP P-pol535 and S-pol535. Data was expressed as percent control using the equation %C = (mPdrug-mPfree)/(mPSTAT3-mPfree)*100 where mP is the value for FP measurement.Fluorescence polarization (FP) assay was conducted as previously reported , 30. BriMeasurement of STAT3 binding to DNA was performed using a TransAM STAT3 ELISA kit (Active Motif). The TransAM STAT3 ELISA kit provides oligonucleotides containing the STAT3 consensus binding sequence immobilized on the ELISA plates. The kit was utilized according to product directions with the replacement of cellular nuclear extracts with recombinant STAT3 protein. Full-length, GST-tagged recombinant human STAT3 protein (abcam) was incorporated into the TransAM STAT3 ELISA kit in increasing amounts (0.01μg–1.0μg). To each well to be used, 30μL of Complete Binding Buffer was added. To the sample wells was added recombinant STAT3 diluted in 20μL of Complete Lysis Buffer and the plate was incubated at room temperature with mild agitation for 1 hour. A blank well containing 20μL of Complete Lysis Buffer was also included. Following the 1 hour incubation, wells were washed 3 times with 200μL of 1X Wash Buffer and then incubated with STAT3 antibody (1:1000 dilution) in 100uL of 1X Antibody Binding Buffer for 1 hour without agitation. Wells were washed 3 times with 200μL of 1X Wash Buffer and incubated with HRP-conjugated antibody (1:1000 dilution) in 100uL of 1X Antibody Binding Buffer for 1 hour without agitation. Finally, wells were washed 4 times with 200μL of 1X Wash Buffer and then developed using room-temperature Developing Solution for 15 minutes. Stop Solution was added and the plate was immediately read for absorbance at 450 nm with reference wavelength at 655 nm on a microplate reader.via the previously mentioned method. Data was normalized and reported as percent of control.Full-length, GST-tagged human STAT3 protein and various concentrations of S3I-1757, A18, A26, stattic, or niclosamide were incubated in Complete Lysis Buffer at a final volume of 20μL containing 0.08μg recombinant protein for 1 hour with mild agitation. Following incubation with STAT3 inhibitors, samples were added to corresponding ELISA wells with 30μL of Complete Binding Buffer and inhibition of STAT3 protein binding to the DNA consensus sequence was determined HeLa cells were plated in 96-well plates at a density of 5,000 cells/well in 100μL of media and were incubated for 24 hours. Media was then aspirated and replaced with 100μL of media containing various concentrations of compounds ranging from 500μM-0.0019μM. Cells were exposed to compounds for 24 hours, following which the media was aspirated and 50μL MTT solution diluted in media was added to each well at a final concentration of 1 mg/mL. MTT was exposed to cells for 4 hours, aspirated, and then 100μL of DMSO was added to each well. Plates were agitated for 10 minutes prior to reading absorbance at 540 nm in a microplate reader. Data was normalized and reported as percent of control.via Bradford assay.Nuclear extracts were prepared using a nuclear extraction kit (Signosis) according to product directions. Briefly, 100 mm dishes of cells were washed with 1X PBS and then 1 mL of 1X Buffer I was added and dishes were rocked on ice for 10 minutes. Cells were released from the dish using a sterile scraper and centrifuged at 1200rpm for 5 minutes at 4°C. The supernatant was discarded and the pellets were resuspended in 1X Buffer II and shaken on ice for 2 hours. Samples were centrifuged at 1200rpm for 5 minutes at 4°C and the resulting supernatants containing the nuclear extracts were placed in new microcentrifuge tubes. Protein concentrations of samples were determined 50 value. The dose-response of niclosamide was similarly prepared at concentrations ranging from 1.0μM-0.0675μM. Following incubation with compounds, cellular nuclear extracts were prepared.HeLa cells were plated at 1 million cells per 100 mm dish and incubated for approximately 72 hours, resulting in serum-starved confluent plates. Plates were then washed with PBS and fresh media was added containing concentrations of A18, A26, stattic, or niclosamide for 24 hours. Cells were treated with compound concentrations derived from their 24 hour MTT assay ECMeasurement of STAT3 binding to DNA was performed using the TransAM STAT3 ELISA kit. Sample wells contained 20μg of nuclear extracts from treated HeLa cells diluted in 20μL of Complete Lysis Buffer. The ELISA was then performed as described in the recombinant ELISA section. Data was normalized and reported as percent of control.50 and EC50 curves was normalized, and error bars represent the standard error of the mean (SEM). Experiments were repeated in triplicate.Statistical analysis was performed using GraphPad Prism 5.0, data for IC


Ammodytes tobianus and Hyperoplus lanceolatus). Specifically, we assessed both their population genetic and gut microbial composition variation and investigated not only which environmental parameters correlate with the observed variation, but whether host genome also correlates with microbiome variation.Deciphering the mechanisms governing population genetic divergence and local adaptation across heterogeneous environments is a central theme in marine ecology and conservation. While population divergence and ecological adaptive potential are classically viewed at the genetic level, it has recently been argued that their microbiomes may also contribute to population genetic divergence. We explored whether this might be plausible along the well-described environmental gradient of the Baltic Sea in two species of sand lance  contains supplementary material, which is available to authorized users. A major current focus within marine ecology and conservation is to improve our understanding of the mechanisms governing population genetic divergence and local adaptation across heterogeneous environments. Although gene flow may hamper local adaptation, genetic outlier loci across environmental gradients in several marine fishes imply possible local adaptation despite low overall levels of population genetic divergence –3. In a Although the debate about adaptation to different environments is classically viewed at the genetic level, it has recently been argued that an organism’s associated microbiome might also play a role . This arWe explored the potential of a host genomic-microbial approach by assessing both the population genetic and gut microbial variation in two sand lance species along the well-described environmental gradient in the Baltic Sea. The Baltic Sea is a semi-enclosed brackish water basin in Northern Europe, which changes from a nearly limnetic to an almost fully marine environment. Fish species, such as herring , 3, 11, Five species of sand lances (fishes of the family Ammodytidae) occur at high abundances in the Northeast Atlantic and adjacent waters. Sand lances are known to be closely associated with specific soft substrates  and charAmmodytes tobianus and Hyperoplus lanceolatus, along the North Sea–Baltic Sea environmental gradient. We used the data to firstly estimate the levels of population differentiation across this gradient. Secondly, we tested if the observed population genetic structure correlated with environmental factors, including salinity and sea surface temperature (SST), as well as the relative bacterial composition in the water at sampling sites. Thirdly, we characterized each species’ gut microbiome, its inter-specific variation, and its relationship with both environmental parameters as well as host genomic divergence. Lastly, in light of our findings, we discuss the future potential of how a hologenomic approach may add significantly to our understanding of marine species’ ecological adaptive potential.We generated genome-wide SNP data and gut microbiome taxonomic composition data for two ecologically and economically important sand lance species, A. tobianus and H. lanceolatus were collected during May through September 2015 from 12 and 15 different sites, respectively  and ideEcoT221, which has a six-base pair (bp) recognition sequence, and fragments in the size range from 200 to 380 bps were used as the basis for the GBS libraries.Whole-cell genomic DNA was extracted using the KingFisher™ Duo Prime Purification System  following the manufacturer’s protocol for the KingFisher™ Cell and Tissue DNA purification kit . The DNA concentration was estimated using a Qubit™ 2.0 fluorometer . The fragment size range of DNA extractions was estimated for a subset of DNA extractions using an Agilent 4200 TapeStation™ (Agilent Inc.). DNA extractions were subsequently cleaned using the ZR-96 Genomic DNA Clean & Concentrator™  following the manufacturer’s protocol. Population genomic data were generated from the DNA extracts following the GBS approach originally developed by Elshire et al.  at the IGBS libraries were sequenced at the Institute of Biotechnology commercial service as single-end 64 bp using an Illumina HiSeq2000™ . Subsequent analytical steps were conducted separately for each sand lance species. Details of data quality filtering and SNP calling are described in Additional file enoDive v.2.0 to estimate overall and pairwise levels of genetic divergence as FST. We assumed an infinite alleles model ).Ammodytes tobianus in the Baltic Sea—mainly between Køge Bugt and Åland in the Western to Inner Baltic Sea—seems to belong to two genetically differentiated populations. We suggest that this observation may be attributed to either spatial or temporal segregation. Spatial segregation would occur if different breeding stocks left their spawning areas and mixed at sampling sites outside the spawning season. As we did not sample during the spawning season, we cannot exclude this option. We find spatial segregation unlikely, however, given the sand lances’ residential behavior and limited dispersal capacity [A. tobianus may indeed consist of two distinct but often sympatric spawning types, an autumn and a spring spawning contingent [A. tobianus in the Baltic Sea consists of two spawning types [A. tobianus in the Southern Baltic Sea in the Gulf of Gdansk and based on vertebral counts assigned the individuals of this area to the autumn-spawning component of the stock. While we did not find significant differences in vertebral counts in our fishes that would support a hypothesis of different spawning types (data not shown), we hypothesize that the two genetic A. tobianus Baltic populations that we detected here may nonetheless represent two sympatric spawning types. In order to gain certainty about co-occurrence of different spawning types in the Baltic Sea, future studies will have to sample individuals during the respective spawning season (autumn and spring) and investigate the presence of ripe gonads in adult individuals. The presence of different spawning types in the same habitat is also known from other Baltic species such as herring [capacity , 21. Anocapacity . Populatntingent  that arentingent . These sntingent . As earlng types . More reng types  investig herring , 56. OurA. tobianus and H. lanceolatus, we found elevated levels of genetic divergence at loci that correlated with SST and salinity . Fishes have evolved physiological mechanisms that differ fundamentally between high- and low-saline environments in order to maintain an internal salinity at 9 PSU [Oreochromis mossambicus), a euryhaline cichlid, led [As a geologically young sea that has undergone extreme changes in environmental conditions in the last 8000 years, the Baltic Sea has served as a popular model to assess divergence along an environmental gradient in marine organisms . Our stuat 9 PSU . Divergeat 9 PSU  argued tlid, led  to inferlid, led . The obsWe incorporated data of gut microbiome composition from a subset of specimen in each sand lance species to explore associations between host genomics and environment upon the gut microbiome composition. At a basic level, the sand lance gut microbial community composition was comparable to that reported in other fishes –70. MembA. tobianus compared to H. lanceolatus. Although gut microbial diversity is likely correlated with diet [A. tobianus. However, inclusion of diet analysis and additional spatio-temporal replicates in future studies will help us better understand the processes shaping this pattern.Microbial diversity estimates in both sand lance species varied substantially among individuals. The microbial diversity was overall higher in ith diet , 74, we ith diet , whereasNumerous studies across a wide range of organisms, from ants  over fisA. tobianus from a subset of sites, in addition to samples from other fish species from a geographically intermediate site. As expected, the intra-specific gut microbial composition was more similar within species compared to that among species  suggests at least partial bacterial uptake from the environment. In our comparison of relative abundance changes of major bacterial taxa between fish guts and surrounding water along the North Sea– Baltic Sea environmental gradient, Gammaproteobacteria showed identical relative abundance trends in guts and water. This implies that the relative abundance of Gammaproteobacteria in sand lance guts may be driven by their relative abundance in the surrounding environment. In Alphaproteobacteria and Actinobacteria on the other hand, gut-bacterial relative abundance trends were converse to those of the surrounding water, suggesting that relative abundances in these taxa might depend more strongly on host species.We assessed the potential differences among fish species and their gut microbial community composition in order to gain insights into possible drivers of the gut microbiome composition. Our analysis indicated non-random differences in gut microbiome composition among different fish species. However, our analysis did not account for variation in microbial communities among sampling sites. Consequently, we cannot attribute the observed differences to key and specific variables, such as spatial heterogeneity, fish age, or prey. In order to account for this caveat, we visualized the gut microbiome composition of A. tobianus to include—besides SST and salinity—the proportional abundance of major microbial taxa in the gut microbial samples. The relative abundance of Actinobacteria, Alphaproteobacteria, and Gammaproteobacteria correlated significantly with nearly all detected outlier loci. Actinobacteria are known to be part of the gut, mucosa, and skin in fishes, while Proteobacteria are thought to be the dominant phylum in the guts of many fishes. Among the Proteobacteria, Gammaproteobacteria typically break down and ferment complex sugars and provide particularly important digestive roles [Last, we expanded our environmental dataset in the population-genomic outlier analyses of ve roles . The resHowever, our study design prevented us from identifying functional aspects, e.g., how salinity and SST correlated with the microbial community in the water column in the Baltic. Controlled laboratory experiments would be needed in future studies to differentiate between correlations of environmental bacteria and an organism’s gut microbiome alone and/or with additional environmental parameters, ultimately fully linking the system together. The combination of in situ work and common garden experiments seems particularly promising for moving beyond the detection of correlations and to assess the causal roles of microbiome and environmental factors in shaping the adaptive potential of wild vertebrate species. However, taken together, our results support the notion that the gut microbial community composition is, in part, a function of the host’s genomic background.It has recently been suggested that in order to cope with rapidly changing environmental conditions, organisms may rely on high phenomic plasticity conferred by their gut bacterial symbionts . ChangesA. tobianus in the Baltic Sea exists as two genetic stocks co-occurring in the same habitat. This sparks interest in adapting future fishery management measures. Third, the gut microbial communities of sand lances are not a mere reflection of environmental microbes, but rather the fishes seem to excerpt some degree of internal control and selection.In this study, we take a new approach by using population genomic and 16S gut microbial data to shed light onto the population genetic divergence of two closely related non-model fish species along an environmental gradient. Three major findings emerge from our study: First, the Baltic Sea harbors unique genetic populations of sand lances that are differentiated from the North Sea. We discovered that genomic regions showed elevated divergence not only as a potential response to salinity- and SST-related natural selection, but that these regions also correlate with the relative bacterial composition of the water. This could hint at a potential influence of environmental microbes on the adaptive genetic divergence of these marine fishes. Second, we confirmed that Recent insights into the extent of microbial influence on an organism’s well-being and fitness suggest that the potential of individuals, populations, and species to genetically adapt to changing environments may not be governed by the interactions of these entities with the environment alone. Rather, bacteria seem to partake in forming the adaptive potential of many organisms by composing part of their holobiome. In this sense, population genomic studies aimed at understanding a species’ local adaptive potential will miss part of the story when not considering bacterial communities.We are only beginning to understand how the interplay among hosts, their microbes, and the environment regulates ecological adaptation. One can imagine that in the future, as genomic technologies become cheaper and more readily applicable, our study may serve as a benchmark to design yet more rigorous approaches to learn what enables an organism to survive and adapt in a rapidly changing environment. The combination of in situ work and common garden experiments seems particularly promising for moving beyond the detection of correlations and to assess the causal roles of bacterial and environmental factors in shaping the adaptive potential of wild vertebrate species.Additional file 1:Supplementary Material. (DOCX 13909 kb)


Ozotoceros bezoarticus) is close to beingclassified as ‘globally threatened’, with the largest population occurring inthe Brazilian Pantanal. Since capture is stressful to these animals,non-invasive sampling methods such as the use of feces can provide reliablesources of DNA. The aim of this study was to use fecal samples to evaluate thegenetic variability of the Brazilian Pantanal population of pampas deer. Sixheterologous microsatellite markers were used to screen 142 stool specimens.Seventy-four deer were identified, of which 50 adults were used to determine thegenetic characteristics of the population. The Pantanal population showed highgenetic diversity . This is the first investigation to characterize a SouthAmerican deer species using fecal DNA and demonstrates the usefulness andefficiency of this approach, as well as the feasibility of obtaining informationthat could not have been easily obtained by traditional DNA sampling. Ourfindings suggest that management strategies for this species may be much moreeffective if applied now when the population still shows high geneticvariability.The pampas deer ( Population genetic studies of wild populations of species of the family Cervidae havebecome feasible with the advent of fecal DNA analysis . The fecOzotocerus bezoarticus) is a medium sized (20-40kg) Neotropical cervid with marked variation in body size among individuals andpopulations ,according to the manufacturer's recommendations.The stool sample collection area included the Nhumirim  and Alegria farms, located in the central region of theBrazilian Pantanal, known as Nhecolândia  . Deer weO. b. leucogaster captured in the study area. The markerswere selected because they showed cross-amplification with other Neotropicaldeer species, while seven of them had proven transferability to five species of Mazama , 120 μM of dNTPs, 0.4 U of Taq polymerase(Invitrogen), 45 ng of genomic DNA and 0.08 mM of each primer. The sametemperature cycles were used for all primers, with variation only in theannealing temperature, as follows: 94 °C for 5 min, 94 °C for 1 min, 50-59 °Cfor 1 min and 2.6 μL of ultrapure water. The remainder of the 11 μL reaction volume wascompleted with DNA sample. The amplicons were then run on ABI 3100 and ABI 3130automatic sequencers and assigned genotypes using the program GeneMarker v.2.4.2(SoftGenetics LLC).To individualize the 142 fecal samples collected, fluorescent labeling of the PCRproducts was done by adding fluorescence  to one of each primerpair. The reaction had a final volume of 11 μL, containing 0.5 μM of eachprimer, 1X buffer, 0.3 μL of MgCle.g.To overcome problems such as allelic dropout and false alleles , the reaHE) and observed heterozygosity (HO), and analyses of linkage disequilibrium were calculated using theprogram Genepop 1.2 (PID), which corresponds to the probability of two random individualsexhibiting the same genotype (Q), which corresponds to the force with which a locusexcludes an individual from being the mother of an offspring (Weir 1996), wereestimated using the program Identity 1.0 , expected  and were used forfecal sample individualization.All pampas deer loci tested showed successful cross-amplification and werepolymorphic since more than two alleles per locus were evident in blood samplesfrom the 13 females studied .Six locAt least one microsatellite locus was amplified from the 142 fecal samplesanalyzed, but the six selected loci were amplified in only 40.8% of the samples.Considering our selection criteria (sample genotyping for more than three loci),only 131 samples were used in the sample individualization step. The latteranalysis allowed us to identify 74 deer during the study period.2 =0.0082 and p = 0.86). The smallest amplified fragment  showed 76.8% successful amplification in the142 samples tested, while the largest fragment  was successfully amplified in 69% of samples. The locus with the greatestsuccess in amplification (93.7%) was RT01 (220-230 bp).The six loci used showed an amplified microsatellite fragment size of 112-310 bp. There wTableS1) in which there were no overlappinggenerations, only locus CA71 showed Hardy-Weinberg disequilibrium (E) was 0.75(ID) obtained for the six loci was 1.74 × 10−8,while the mean probability of paternity exclusion (Q) was >99%.Based on the analysis of 50 samples from adult deer  with the use ofmicrosatellite markers we conducted the first population genetic study on a largesample of pampas deer in the Brazilian Pantanal, in Mato Grosso do Sul, home to thelargest population of this species.PID) was close to 99%, indicating that the six loci were more than adequate toindividualize the fecal samples and that the individualization of 74 deer based onthese loci was reliable. Furthermore, the low value for the probability of paternityexclusion for all the loci showed that they formed a set of appropriate codominantmarkers for paternity/maternity and genetic studies in pampas deer populations.The six loci studied were amplified in at least 69% of the samples, more thanrequired for deer individualization analysis, for which at least three loci persample were amplified, as recommended by The amplification of at least one microsatellite locus in all the samples and 5-6loci in ~70% of the samples analyzed indicated a high rate of success for fecal DNA.These results indicate that the use of fresh fecal samples can enhance the successof studies using fecal DNA samples . In contE = 0.75) for the Pantanal population (n = 50) was slightly lowerthan that reported  , suggesting that the genetic variability ofthe Pantanal population is slightly lower than previously described for the sametype of genetic marker. The high genetic diversity observed in this population ofpampas deer was also observed in a study of dominant nuclear markers of randomamplified polymorphic DNA (RAPD) , to establish and improve grazing pastures(The high genetic diversity reported here strongly supports the relevance of thisot flood . Anotherpastures. These epastures.Based on the set of nuclear microsatellite markers evaluated here, further researchshould be done to compare the genetic diversity of different populations of pampasdeer and to determine whether recent structuring has occurred in differentlocations. New studies should also analyze whether the structuring observed usingmitochondrial markers for populations in Brazil (