Paleosuchus trigonatus is one of the smallest living crocodilians. Due to its broad distribution, cryptic behavior, and small home range, the species is well suited for the study of phylogeographic patterns on a continental scale. Additionally, this species is under threat due to habitat loss, trade and harvest, but is considered at low conservation risk by the IUCN. In the present study we test the hypothesis that P. trigonatus is comprised of geographically structured lineages. Phylogenetic reconstructions of the mitochondrial cytochrome b gene and single locus species discovery methods revealed the existence of two well-supported lineages within P. trigonatus—an Amazonian and Guianan lineage. Fossil calibrated divergence of these lineages was estimated to have occurred in the Late Miocene (7.5 Ma). The hypothesis that the Atlantic coast drainages might have been colonized from the southeast or central Amazon is supported by demographic metrics and relatively low genetic diversity of the Coastal and upper Branco populations when compared to the Amazon basin populations. The Amazon basin lineage is structured along an east-west gradient, with a sharp transition in haplotype frequencies to the east and west of the Negro and Madeira rivers. These lineages are already under anthropogenic threat and, therefore, are conservation dependent. Recognition of these lineages will foster discussion of conservation future of P. trigonatus and these lineages.Schneider’s dwarf caiman  The importance of knowing the spatial distribution of genetic diversity of “widely” distributed species cannot be underestimated. Current spatial organization of diversity is a window to the past and, through phylogenetic analysis, provides insights into the history of taxa . Moreovea priori intention during phylogenetic, phylogeographic, and population genetic surveys . These  surveys , species surveys , a new s surveys ).Osteolaemus also comprise three deeply divergent molecular lineages  occupying the Amazon, Madeira-Bolivia and Pantanal basins—and the authors speculate that P. palpebrous could be a complex of cryptic species hidden under the same scientific epithet. Independent of being recognized as distinct species or formally described as such, these crocodilian lineages are the direct outcome of evolution, and thus their recognition and study is necessary to comprehend the evolutionary history of this group. Only by focusing on lineages rather than on described species, can we hope to understand the evolutionary history of the studied group , the sister species of P. palpebrosus, is one of the smallest of living crocodilians  to calculate genetic diversity parameters, infer population genetic structure, and to test if instraspecific lineages within P. trigonatus exist and when they diverged, through the use of phylogenetic reconstruction, single locus species discovery and molecular calibration methods. Cytochrome b is a versatile molecular marker most useful for phylogenetic inference at or below the family level , and FrWe removed one or two tail scutes of adults, subadults or juveniles after capture and marking during nocturnal surveys, stored them in tubes containing 95% ethanol and deposited them in the Universidade Federal do Amazonas animal tissue collection (CTGA) under individual ID tags. We captured and sampled all individuals under license granted by the Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis . ICMBio/IBAMA field collection permits are conditional that collection of organisms be undertaken in accordance with the ethical recommendations of the Conselho Federal de Biologia , Resolution 301 . Access to genetic resources was authorized by permit number 034/2005/CGEN.MH757465–MH757694.We extracted whole genomic DNA using 2% CTAB solution  , with anPaleosuchus trigonatus sequences with the Cytochrome b alignment of Paleosuchus palpebrosus (MH846341–MH846546) and then reduced the dataset to unique haplotypes using the function hapCollapse . We then generated a Bayesian Inference phylogeny using the software BEAST2 , A. sinensis (AF432918), Melanosuchus niger (EU161675), Caiman latirostris (EU161674), C. yacare (JF315314), C. crocodilus (EU161660) and Paleosuchus palpebrosus   . We infeCladeAge  implemenCladeAge . We alsoCladeAge  to estimNavajosuchus mooki as the oldest known Alligatorinae, †Brachychampsa sealeyi as the oldest known Caimaninae, Caiman cf. C. †brevirostris as the oldest known Caiman and Jacarean, and Paleosuchus sp. as the oldest known Paleosuchus fossil record. For the CladeAge net diversification rate parameter, we used the minimum (0.006007672) and maximum (0.023543042) values for Alligatoridae diversification rate estimated by For divergence-time estimation, we used the lognormal relaxed molecular clock , the birWe ran three MCMC simulations of 20,000,000 generations, sampling tree topologies and parameters every 1,800 generations, discarding the first 10% generations as burn-in. We used the software Tracer 1.7  to checkP. trigonatus we used a Bayesian clustering analysis implemented by a priori information for the clusters , with 10 repetitions for each value of K. We assumed clusters with the lowest log  values and highest posterior probability to be the best fit for the data. We used Arlequin 3.5   and default settings of the function. We evaluated the goodness-of-fit by analyzing stress values, where values 0.05 < 0.2 are considered poor and <0.05 excellent. We also plotted the dissimilarity matrix between the drainage basins in a three-dimensional scatterplot created using the R package scatterplot3d   utilizinis (MDS)  in the sis (MDS) . This anerplot3d .b haplotypes, we generated a haplotype network in the software HAPLOVIEWER  after the alignment and manual edition of 230 samples of P. trigonatus from 42 localities. We observed a total of 36 haplotypes with 57 polymorphic sites, where 47 sites were parsimony informative and 10 singletons. Most frequent haplotypes were H8 (68/230), H7 (41/230), and H4 (32/230), representing 61.30% of the dataset. Uncorrected p-distance between haplotypes varied from 0.1 to 3.6%. All haplotypes, polymorphic sites, nucleotide position, and number of individuals per haplotype are listed in We obtained a total of 1,020 bp of the mitochondrial Cytochrome Paleosuchus, three lineages of P. palpebrosus and two lineages of P. trigonatus , while divergence between the Bolivia/Madeira and Amazon/Pantanal lineages was estimated at 3.08 Ma (95% HPD = 1.55–5.42 Ma). The median and 95% HPD intervals of divergence time estimates are summarized in Bayesian timetree recovered a well supported phylogeny; with exception of support for the phylogenetic position of Amazon and Pantanal ues of 1 . The divues of 1  while thK = 4 as the optimal number of clusters of Paleosuchus trigonatus   . Within , p = 1) .Transition between the Guiana and Amazonia lineages occurs in the middle Branco River region, with individuals in the northern section of this region belonging to the Guiana lineage and southern individuals to the Amazonia lineage. We failed to detect co-occurrence of these lineages in any given locality. Of the 43 individuals sampled in the lower Negro River drainages, only four individuals of the central-west cluster were found. In contrast, lower Purus River drainages had only one individual from central-east cluster. Madeira River basin had the most heterogeneous haplotype composition. Upper portions of the Madeira River were almost entirely comprised of central-west haplotypes, whereas the contribution of these haplotypes decreased in downstream localities of the Madeira.Paleosuchus trigonatus in each genetic cluster are shown in the Paleosuchus trigonatus shows high levels of haplotype (0.856 ± 0.015) and nucleotide diversity (0.0119 ± 0.001) considering all samples simultaneously. Analyzing individuals from each cluster separately, the lowest haplotype diversity was found in the upper Branco River (0.116 ± 0.073), and the highest in the central-west cluster (0.748 ± 0.043). This pattern is paralleled by number of polymorphic loci, number of haplotypes and nucleotide diversity. Tajima’s D was significant for Coastal (−1.6670), upper Branco (−1.7282), and central-east (−1.6848) clusters, while Fu’s Fs was significant for upper Branco (−1.746), central-east (−10.680), and central-west (−8.669) clusters. Both Coastal and upper Branco had the lowest Nei’s haplotype diversity and smallest number of haplotypes.General genetic parameters of ST and matrices of Euclidean geographic distance between the sampled localities . Pairwise ΦST shows a very high degree of population structuring between river drainages. All comparisons between the Coastal or upper Branco River and other river drainages show ΦST values greater than 0.9 , a clear dissimilarity between the Coastal or upper Branco River and all other river drainages . The secb gene genealogies of Paleosuchus trigonatus show clear evidence of evolutionarily independent lineages. Formal species discovery analyses identified five principal groups of Paleosuchus, three lineages of P. palpebrosus and two lineages of P. trigonatus. With exception of the phylogenetic support for the position of Amazon and Pantanal P. palpebrosus, posterior probabilities of all lineages are strongly supported. The GMYC single locus species discovery method discovered additional groups which correspond to substructure within the Amazonia lineage; also evident in haplotype network, BAPS clustering and MDS analyses. Fossil calibration recovered the divergence time between the Guiana and Amazonia lineages of 7.5 Ma, situated at the Miocene/Pliocene boundary . Thus, molecular calibrations such as from P. palpebrosus and P. trigonatus estimated from mtDNA datasets is always much older than that estimated from nuDNA. Although at this point speculative, this might be due to introgressive hybridization of diverging lineages marked by their mtDNA genomes as has been observed between P. palpebrosus lineages (Inia dolphins (All previous studies relied on the Alligatorinae-Caimaninae divergence (71 to 66 Ma) of lineages  and Iniadolphins . AlthougP. trigonatus was estimated at 7.5 Ma  and Fu’s Fs . Future studies using both mitochondrial and nuclear DNA genomes will allow us to estimate which processes  favored this divergence, which routes the ancestors might have used to colonize the Amazon and Atlantic coast drainages, and to which extent their distributions overlap.The divergence-time between the Guiana and Amazonia lineages of t 7.5 Ma , node 8,ocodilus  and linepebrosus , often lpebrosus . Althoug derived . FurtherOur results already indicate that a transition zone between the Guiana and Amazonian lineages exists in the region of the middle Branco River. There is evidence that the upper Branco River, the main tributary of the Negro River, flowed SW–NE towards the Caribbean Sea, integrating the Proto-Berbice basin, one of the largest drainage basins in the central portion of the Guiana Shield, during the Late Neogene or Early Quaternary (5.3–0.8 Ma) . This baAlthough Pseudoancistrus brevispinis  until the drainage capture of some of the former Proto-Berbice headwaters (which currently are Branco river headwaters) by the present-day middle and lower sections of the Branco River. This capture event changed the drainage flow in the southward direction, connecting the upper Branco River with the Negro River, and disconnecting the upper Branco from the Proto-Berbice. These events would explain the co-occurence of both lineages (Amazonia and Guiana) in the middle Branco River and the structure between Branco and Atlantic coast populations. Connections between Amazon basin and coastal river drainages were also claimed in order to explain the unexpected structuring of genetic diversity in the catfish vispinis , suggestCaiman crocodilus sampled in localities of the Amazon basin and Atlantic coast drainages paralleling the pattern found in P. trigonatus. While isolation-by-distance was discarded by the authors for C. crocodilus, it appears to have been an important structuring factor for Melanosuchus niger populations sampled across the Amazon basin and Atlantic coast drainages  present, in great majority, haplotypes of the central-eastern cluster detected by BAPS see . On the P. trigonatus of Amazon basin rivers draining the Brazilian and Guiana Shields have central-eastern haplotypes, while rivers of Andean origin have central-western haplotypes. As the Madeira River basin receives input from both the Andes and the Brazilian Shield, this would explain the greater admixture proportion of the central-eastern and central-western haplotype groups in the Jaci-Paraná, Manicoré and Aripuanã rivers.In conclusion, most of the individuals of P. trigonatus, providing yet another example of species with a broad geographic distribution and considered at low conservation risk by the IUCN, that comprise multiple independent lineages. These findings have an important bearing on the future of conservation of P. trigonatus. Lineages and/or cryptic species often are under anthropogenic threat or otherwise conservation dependent, something that would not be evident without the recognition of these lineages are evolutionarily independent entities —a specimen from the private collection of Marcus Elieser Bloch—gives the locality “Süd America”.One of the major problems for the taxonomic revision of ns e.g.,  did not ns e.g., . Bauer &Paleosuchus trigonatus. Since it is probably impossible to track down the type locality of the ZMB 243 specimen based on historical data, we suggest another approach. By the use of ancient DNA techniques  specimens from the type locality or its descendants still exist, and (2) that the lineages are geographically structured, it would be possible to assign the holotype to its topotypical populations with high accuracy by screening several loci (SNPs) generated by Next Generation Sequencing techniques and analyzed by methods such as the Discrimant Analysis of Principal Components (DAPC)  .Paleosuchus trigonatus—Guiana and Amazonia—both strongly supported by species discovery, BAPS clustering and MDS analyses, with the two lineages diverging at the Miocene/Pliocene boundary. The Guiana lineage occupies Atlantic coastal draining rivers, and the upper reaches of the Branco River—an Amazon basin river—upstream of the Caracarai rapids, indicating a recent capture of the upper Branco River by the lower Branco. Within the Amazon basin, haplotype frequencies along the east–west axis sharply transition at the Negro and Madeira rivers, a pattern commonly observed not only in aquatic taxa but also in varzea birds (P. trigonatus and its lineages in particular.Geographically broadly distributed species are in particular need of studies that investigate population structuring and the existence of cryptic species hidden under one binomial epithet. It is only such analyses that will provide solid scientific data on diversity and distribution of these lineages, which, ultimately, underpin conservation efforts and management guidelines. This study provides evidence for the existence of two deeply divergent lineages of ea birds . This st10.7717/peerj.6580/supp-1Table S1Click here for additional data file.10.7717/peerj.6580/supp-2Table S2Click here for additional data file.


STAT3 is an oncoprotein overexpressed in different types of tumors, including prostate cancer (PCa), and its activity is modulated by a variety of post-translational modifications (PTMs). Prostate cancer represents the most common cancer diagnosed in men, and each phase of tumor progression displays specific cellular conditions: inflammation is predominant in tumor’s early stage, whereas oxidative stress is typical of clinically advanced PCa. The aim of this research is to assess the correspondence between the stimulus-specificity of STAT3 PTMs and definite STAT3-mediated transcriptional programs, in order to identify new suitable pharmacological targets for PCa treatment. Experiments were performed on less-aggressive LNCaP and more aggressive DU-145 cell lines, simulating inflammatory and oxidative-stress conditions. Cellular studies confirmed pY705-STAT3 as common denominator of all STAT3-mediated signaling. In addition, acK685-STAT3 was found in response to IL-6, whereas glutC328/542-STAT3 and pS727-STAT3 occurred upon tert-butyl hydroperoxyde (tBHP) treatment. Obtained results also provided evidence of an interplay between STAT3 PTMs and specific protein interactors such as P300 and APE1/Ref-1. In accordance with these outcomes, mRNA levels of STAT3-target genes seemed to follow the differing STAT3 PTMs. These results highlighted the role of STAT3 and its PTMs as drivers in the progression of PCa.  Prostate cancer (PCa) represents one of the leading causes of morbidity and mortality among adult males in Western countries. It is a multifactorial and biologically heterogeneous disease that could progress to an advanced hormone-refractory stage, which is still considered incurable ,2.Prostate cancer is characterized by the dysregulation of several intracellular pathways and its onset and progression are determined by the presence and activation status of the androgen receptor (AR), which plays a key role during all phases of tumor growth. In fact, the AR-signaling axis not only regulates normal prostate cell morphogenesis and differentiation, but is also involved in the transition from androgen-dependent PCa to a more aggressive phenotype . AlthougSeveral in vitro and in vivo studies identify STAT3  as a main player in cellular events responsible for the insurgence, progression ,9 and deSTAT3 is a key signaling protein with different biological effects ranging from cellular growth to differentiation and survival in response to different stimuli such as growth factors  , cytokinun-phosphorylated STAT3 (U-STAT3) can bind to the interferon γ-activated sequence (GAS), either as dimer or as monomer. The U-STAT3 regulates gene expression via its binding to AT-rich DNA sequences and drives chromatin structure remodeling ;STAT3 phosphorylation at S727, rather than Y705, is required for its mitochondrial activity . In partSTAT3 was found associated with a variety of cytosolic structures, including focal adhesions, microtubules and mitotic spindles. Indeed, STAT3 plays an important role during the assembly of cytoskeleton networks, such as the actin filaments and microtubules, thus promoting cell migration and invasion .STAT3 can fulfil its multifaceted biological activities through two main intracellular signaling patterns known as the canonical and the non-canonical pathways of STAT3 . In the The biological consequences of STAT3 regulation and activation are quite complex. Aside from its phosphorylation on Y705, STAT3 presents a wide variety of post-translational modifications (PTMs) that affect STAT3 nuclear function other than its non-genomic activities. In fact, STAT3 was also found phosphorylated on S727, acetylated on K685 and glutathionylated on Cys residues 328 and 542; each PTM is involved in a specific STAT3 activity .The phosphorylation on S727, as well as the S-gluthathionylation on C328 and C542, occurs under oxidative-stress conditions ,24. In pOn the other hand, cytokines stimuli induce STAT3 acetylation, and the balance between the protein acetylation/deacetylation is a molecular switch that controls the expression of inflammatory genes .In recent years, several studies have established that the post-translational modifications of proteins address the flow of molecular information from the extracellular environment to intracellular compartments via the activation of specific signaling pathways. Chemical modifications of key proteins involved in signal transduction could regulate numerous physiological events, such as gene transcription, cell proliferation, differentiation and apoptosis, tissue development and tumor progression . STAT3 rOn the basis of literature data, together with our results from previous studies on prostate cancer FFPE tissues , it is cPCa is a multifactorial disease, usually associated with chronic prostatic inflammation. Sustained inflammatory conditions can lead to generation of free radicals that can be responsible for PCa progression and transformation into a more aggressive form. Chronic inflammation and oxidative stress represent closely connected and related processes that do not exclude each other , but it In order to understand if the stimulus-dependent STAT3 PTMs could be a link between signal transduction and specific cellular conditions, their correspondence with definite STAT3-mediated transcriptional programs should be investigated. Only results from samples treated with tBHP will be reported, but analogous outcomes were also obtained with hydrogen peroxide (data not showed).CRP) representing inflammation  ab19534 antibody dilution 1:1000) [Cells were lysed at 4 °C in RIPA buffer  .Reactive oxygen species (ROS) generated by stressing cells with 75 µM tBHP were quantified using the CellROX Green Flow Cytometry Assay Kit  following the manufacturer’s instructions. Samples were analyzed by a BD Accuri C6 flow cytometer (BD Biosciences).SOD2, BIRC-5, c-MYC, MMP-2 and CRP (RT2-qPCR Primer Assays: Human CRP (NM_000567). Cat.no PPH02632A, Human BIRC5 (NM_001012270) Cat.no PPH00271E, Human MMP2 (NM_001127891) Cat.no PPH00151B and Human SOD2 (NM_000636) Cat.no PPH01716B  were evaluated using the CFX Connect Real-Time PCR Detection System  with a SYBR green fluorophore-based real-time reaction . Expression data were analyzed using CFX Manager Real Time PCR Detection System Software, version 3.1 (BioRad).Total RNA was extracted from cells using TRIzol reagents  in accordance with the manufacturer’s instructions. RNA was quantified spectrophotometrically, and its quality was assessed by 1% agarose gel electrophoresis and staining with ethidium bromide. The reverse transcription was carried out with Super Script II R-Transcriptase . Gene expression was evaluated with specific primers for w/v BSA (Sigma-Aldrich) in PBS. Fixed cells were processed for immunofluorescence staining to detect the localization of pS727-STAT3  using a specific primary antibody diluted in PBS containing 2% w/v BSA for 1 h. Following three washes with PBS added with 0.05% Triton and 2% w/v BSA (PBS-T), cells were incubated for 1 h in the darkness with a Fluorescein isothiocyanate FITC-conjugated secondary antibody . Cell nuclei were counterstained with 100 ng/mL Hoechst 33258 for 15 min. After washing with PBS-T, coverslips were mounted on glass microscopy slides with Duolink Mounting Medium and examined with a confocal fluorescence microscope (ZEISS LSM510META) with magnification using 60x oil immersion objective lens. Images were acquired and analysed by ZEN 215 software .Cultured LNCaP and DU-145 cells were grown on coverslips, and LNCaP were treated with IL-6 or tBHP in the same aforementioned conditions. After the stimulation, cells were washed with PBS, fixed with 4% formaldehyde for 15 min and then rinsed with PBS. Cell were then permeabilized with cold MeOH (−20 °C) for 5 min. After washing three times with PBS, cells were blocked overnight with 3% Mitochondria were stained with MitoTracker Orange CMTMRos  before fixation. MitoTracker 1 mM (DMSO stock solution) was diluted to a final concentration of 190 nM in serum-free culture media in accordance with the manufacturer’s instruction. Cells were incubated for 30 min with the mitochondrial dye and then washed three times in serum-free culture media before fixation and immunofluorescence analysis.Crp:For 5′-GAGTTTGTAATAAATAAC-3′ (TSS-172/-155)Rev 5′-CACTATGTAAATAATTTTC-3′ (TSS-85/-67),Birc5:For 5′-GTTGCAGTGAGCTGAGATC-3′ (TSS-1114/-1095)Rev 5′-GTGCCTCCACTGTCTTTTTC-3′ (TSS-917/937),Mmp2:For 5′-CAAGATGGAGTCGCTCTG-3′ (TSS -650/-633)Rev 5′-GTAAGCCTTAACTTGGCCTC-3′ (TSS-580/599),Sod2:For 5′-GTGGGTGTCCAAGAACTGT-3′ (TSS-748/730)Rev 5′-GTACTCTTGCGCCGTACC-3′ (TSS-643/-626).DNA-protein and protein–protein cross-linked complexes were formed in LNCaP and DU-145 cell lines, with or without stimulation (IL-6/tBHP), using 1% formaldehyde (5 min at 37 °C) according to method described in Eufemi et al. . The immProteins were purified from immunocomplexes and identified by immunoblotting.t-test.The repeatability of results was confirmed by performing all experiments at least three times. The obtained values are presented as mean and standard deviation. Statistical analysis was performed with GraphPad Prisma software using a Student’s


Selfish genetic elements  are genetic segments that can enhance their own transmission at the expense of other genes in the genome, even if this has no or a negative effect on organismal fitness. [vulnerable to selfish behaviour by their parts. Early observations of selfish genetic elements were made almost a century ago, but the topic did not get widespread attention until several decades later. Inspired by the gene-centred views of evolution popularized by George Williams[Richard Dawkins,[Nature in 1980—by Leslie Orgel and Francis Crick[Ford Doolittle and Carmen Sapienza[fitness. –6 Genome Williams and Rich Dawkins, two pape Sapienza respecti Sapienza Though l Sapienza PLOS Genetics.This is a Topic Page article for history of genetics. Already in 1928, Russian geneticist Sergey Gershenson reported the discovery of a driving X chromosome in Drosophila obscura. need not be useful for the plants. They need only be useful to themselves.”—Gunnar ÖstergrenMarcus Rhoades described how chromosomal knobs led to female meiotic drive in maize.[uniparentally inherited mitochondrial genes and biparentally inherited nuclear genes could lead to cytoplasmic male sterility in plants.[Barbara McClintock published a series of papers describing the existence of transposable elements, which are now recognized to be among the most successful selfish genetic elements. [Around the same time, several other examples of selfish genetic elements were reported. For example, the American maize geneticist in maize. Similarln plants. Then, inlements. ,18 The dThe empirical study of selfish genetic elements benefited greatly from the emergence of the so-called gene’s-eye view of evolution in the nineteen sixties and seventies. In contrGeorge Williams' Adaptation and Natural Selection[Richard Dawkins‘ best seller The Selfish Gene , the lack of correlation between genome size and perceived complexity of a species. Both papers attempted to counter the prevailing view of the time that the presence of differential amounts of non-coding DNA and transposable elements is best explained from the perspective of individual fitness, described as the”phenotypic paradigm” by Doolittle and Sapienza. Instead, the authors argued that much of the genetic material in eukaryotic genomes persists, not because of its phenotypic effects, but can be understood from a gene’s-eye view, without invoking individual-level explanations. The two papers led to a series of exchanges in Nature.[In 1980, two high profile papers published back-to-back in l debate. The papen Nature.–27Leda Cosmides and John Tooby wrote a landmark review about the conflict between maternally inherited cytoplasmic genes and biparentally inherited nuclear genes.[Robert Trivers published the first book-length treatment of the topic, a comprehensive piece that remains the go-to source on the topic, the tide was changing. While their role in evolution long remained controversial, in a recent review, a century after their first discovery, William R. Rice concluded that “nothing in genetics makes sense except in the light of genomic conflicts”.[If the Selfish DNA papers marked the beginning of the serious study of selfish genetic elements, the subsequent decades have seen an explosion in theoretical advances and empirical discoveries. ar genes. The papear genes. This papnflicts”.Though selfish genetic elements show a remarkable diversity in the way they promote their own transmission, some generalizations about their biology can be made. In a classic 2001 review, Gregory D.D. Hurst and John H. Werren proposed two ‘rules’ of selfish genetic elements.Mendel’s Law of Segregation, alleles in a sexually reproducing organism have a 50% chance of being passed from parent to offspring. Meiosis is therefore sometimes referred to as “fair”.[Sexual reproduction involves the mixing of genes from two individuals. According to s “fair”.effective population size.[Highly self-fertilizing or asexual genomes are expected to experience less conflict between selfish genetic elements and the rest of the host genome than outcrossing sexual genomes.–33 Thereion size. A reduction size.,37 self-ion size. and B chion size.Red Queen dynamics, which may mask the presence of selfish genetic elements in a population. Hybrid offspring, on the other hand, may inherit a given selfish genetic element, but not the corresponding suppressor and so reveal the phenotypic effect of the selfish genetic element.[The presence of selfish genetic elements can be difficult to detect in natural populations. Instead, their phenotypic consequences often become apparent in hybrids. The first reasons for this is that some selfish genetic elements rapidly sweep to fixation, and the phenotypic effects will therefore not be segregating the in the population. Hybridization events, however, will produce offspring with and without the selfish genetic elements and so reveal their presence. The second reason is that host genomes have evolved mechanisms to suppress the activity of the selfish genetic elements, for example the small RNA administered silencing of transposable elements. The co-e element.,42Some selfish genetic elements manipulate the genetic transmission process to their own advantage, and so end up being overrepresented in the gametes . Such diDrosophila melanogaster is the best studied example, and it involves a nuclear envelope protein Ran-GAP and the X-linked repeat array called Responder (Rsp), where the SD allele of Ran-GAP favors its own transmission only in the presence of a Rspsensitive allele on the homologous chromosome.[sensitive sperm, in a post-meiotic process (hence it is not strictly speaking meiotic drive). Systems like this can have interesting rock-paper-scissors dynamics, oscillating between the SD-RSPinsensitive, SD+-RSPinsensitive and SD+-RSPsensitive haplotypes. The SD-RSPsensitive haplotype is not seen because it essentially commits suicide.Segregation distortion can happen in several ways. When this process occurs during meiosis it is referred to as meiotic drive. Many forms of segregation distortion occur in male gamete formation, where there is differential mortality of spermatids during the process of sperm maturation or spermiogenesis. The Segregation Distorter (SD) in romosome.–48 SD acDrosophila pseudoobscura, for example, is on the X chromosome, and XSR/Y males produce only daughters, whereas females undergo normal meiosis with Mendelian proportions of gametes.[D. pseudoobscura.[When segregation distortion acts on sex chromosomes, they can skew the sex ratio. The SR system in  gametes.,50 Segre gametes.another ioobscura.A phenomenon closely related to segregation distortion is homing endonucleases.–54 TheseBarbara McClintock in the 1940s[Transposable elements (TEs) include a wide variety of DNA sequences that all have the ability to move to new locations in the genome of their host. Transposons do this by a direct cut-and-paste mechanism, whereas retrotransposons need to produce an RNA intermediate to move. TEs were first discovered in maize by the 1940s and theithe 1940s TEs havethe 1940s For examthe 1940s TEs thatDrosophila[Both plant and animal hosts have evolved means for reducing the fitness impact of TEs, both by directly silencing them and by reducing their ability to transpose in the genome. It would appear that hosts in general are fairly tolerant of TEs in their genomes, since a sizable portion (30–80%) of the genome of many animals and plants is TEs.,61 When rosophila and dogsrosophila for examB chromosomes refer to chromosomes that are not required for the viability or fertility of the organism, but exist in addition to the normal (A) set.[ (A) set. They perEyprepocnemis plorans.[B chromosomes were first detected over a century ago. Though t plorans.uniparentally  inherited mitochondrial and biparentally inherited nuclear genes. Indeed, one of the earliest clear statement about the possibility of genomic conflict was made by the English botanist Dan Lewis in reference to the conflict between maternally inherited mitochondrial and biparentally inherited nuclear genes over sex allocation in hermaphroditic plants , but the relatedness at the particular locus that underlies the social behavior.A  Hamilton and thenFollowing Dawkins, a greenbeard is usually defined as a gene, or set of closely linked genes, that has three effects,90:It gives carriers of the gene a phenotypic label, such as a green beard.The carrier is able to recognize other individuals with the same label.The carrier then behaves altruistically towards individuals with the same label .Greenbeards where long thought to be a fun theoretical idea with limited possibility of actually existing in nature. However, since its conception several examples have been identified, including in yeast, slime mThere has been some debate whether greenbeard genes should be considered selfish genetic elements. –96 ConflBill Hamilton[Perhaps one of the clearest ways to see that the process of natural selection does not always have organismal fitness as the sole driver is when selfish genetic elements have their way without restriction. In such cases, selfish elements can, in principle, result in species extinction. This possibility was pointed out already in 1928 by Sergey Gershenson and then Hamilton developespeciation.[Bateson-Dobzhansky-Muller incompatibilities has received particular attention.Selfish genetic elements have been shown to play a role in eciation.,42,98 ThP element in Drosophila.[P element were crossed to females lacking it, the resulting offspring suffered from reduced fitness. However, offspring of the reciprocal cross were normal, as would be expected since piRNAs are maternally inherited. The P element is typically present only in wild strains, and not in lab strains of D. melanogaster, as the latter were collected before the P elements were introduced into the species, probably from a closely related Drosophila species. The P element story is also a good example of how the rapid co-evolution between selfish genetic elements and their silencers can lead to incompatibilities on short evolutionary time scales, as little as within a few decades.An early striking example of hybrid dysgenesis induced by a selfish genetic element was the osophila.,100 If mArabidopsis results in both higher activity of transposable elements[Several other examples of selfish genetic elements causing reproductive isolation have since been demonstrated. Crossing different species of  elements and disr elements both of  elements and in s elementsC-value)—animals vary 7,000 fold and land plants some 2,400-fold—has a long history in biology.[Attempts to understand the extraordinary variation in genome size , around 30,000, but accumulation of transposons means that the genome of the latter is some 100 times larger. Transposable element abundance has also been to shown to cause the unusually large genomes found in salamanders.[Two kinds of selfish genetic elements in particular have been shown to contribute to genome size variation: B chromosomes and transposable elements.,108 The amanders.ENCODE Project published a paper claiming that 80% of the human genome can be assigned a function, a claim interpreted by many as the death of the idea of junk DNA, this debate was reignited.[The presence of an abundance of transposable elements in many eukaryotic genomes was a central theme of the original selfish DNA papers mentioned above . Most people quickly accepted the central message of those papers, that the existence of transposable elements can be explained by selfish selection at the gene level and there is no need to invoke individual level selection. However, the idea that organisms keep transposable elements around as genetic reservoir to “speed up evolution” or for other regulatory functions persists in some quarters. In 2012,eignited.,113A common problem for plant breeders is unwanted self-fertilization. This is particularly a problem when breeders try to cross two different strains to create a new hybrid strain. One way to avoid this is manual emasculation, i.e. physically removing anthers to render the individual male sterile. Cytoplasmic male sterility offers an alternative to this laborious exercise.Breeders While many transposable elements seem to do no good for the host, some transposable elements have been “tamed” by molecular biologists so that the elements can be made to insert and excise at the will of the scientist. Such elements are especially useful for doing genetic manipulations, like inserting foreign DNA into the genomes of a variety of organisms.PiggyBac, a transposable element that can efficiently move between cloning vectors and chromosomes using a "cut and paste" mechanism.[One excellent example of this is echanism. The inveechanism.CRISPR allows the construction of artificial homing endonucleases, where the construct produces guide RNAs that cut the target gene, and homologous flanking sequences then allow insertion of the same construct harboring the Cas9 gene and the guide RNAs. Such gene drives ought to have the ability to rapidly spread in a population (see section below on the theory of gene drives), and one practical application of such a system that has been proposed is to apply it to a pest population, greatly reducing its numbers or even driving it extinct.[Homology directed repair, which would make a perfect copy of the drive, or by Non-homologous end joining, which would produce “resistant” alleles unable to further propagate themselves. When Cas9 is expressed outside of meiosis, it seems like non-homologous end joining predominates, making this the biggest hurdle to practical application of gene drives.[ extinct.This has  extinct. Unfortune drives.Much of the confusion regarding ideas about selfish genetic elements center on the use of language and the way the elements and their evolutionary dynamics are described. The beauDrosophila pseudoobscura[Drosophila mediopunctata.[The mouse t-allele is a classic example of a segregation distorter system that has been modeled in great detail.,120 Hetedoobscura and Y chpunctata. A cruciapunctata.The idea of spreading a gene into a population as a means of population control is actually quite old, and models for the dynamics of introduced compound chromosomes date back to the 1970s. More recDrosophila, have a very different dynamics because of sex, and Brian Charlesworth, Deborah Charlesworth, Charles Langley, John Brookfield and others[Drosophila and other species. What is impressive about all these modeling efforts is how well they fitted empirical data, given that this was decades before discovery of the fact that the host fly has a powerful defense mechanism in the form of piRNAs. Incorporation of host defense along with TE dynamics into evolutionary models of TE regulation is still in its infancy.[To model the dynamics of transposable elements (TEs) within a genome, one has to realize that the elements behave like a population within each genome, and they can jump from one haploid genome to another by horizontal transfer. The mathematics has to describe the rates and dependencies of these transfer events. It was observed early on that the rate of jumping of many TEs varies with copy number, and so the first models simply used an empirical function for the rate of transposition. This had the advantage that it could be measured by experiments in the lab, but it left open the question of why the rate differs among elements and differs with copy number. Stan Sawyer and Daniel L. Hartl fitted mnd others,127,128  infancy.Selfish genetic elements have gone from being seen as genetic oddities with little significance to be considered major players in evolution. We now know that their presence and co-evolution with suppressors can affect a range of features at the genome, phenotype, and population levels. While the influx of whole genome data has done much to bring this shift about, particularly for the study of transposable elements, developments in mathematical modelling has also played a key role. The adaption of CRISPR technologies to engineer gene drive systems for population control has added a new rapidly advancing applied dimension to the study of selfish genetic elements.S1 Text(XML)Click here for additional data file.S2 Text(XML)Click here for additional data file.


Ariocarpus, Echinocactus,Polaskia and Pilosocereus, in four taxa of thegenus Cereus: C. fernambucensis subsp.fernambucensis, C. fernambucensis subsp.sericifer, C. jamacaru and C.insularis. Nine microsatellite loci were amplified inCereus resulting in 35.2% of success in transferability,which is higher than the average rate of 10% reported in the literature forcross-genera transferability in eudicots. The genetic variation in thetransferred markers was sufficient to perform standard clustering analysis,indicating each population as a cohesive genetic cluster. Overall, the amount ofgenetic variation found indicates that the transferred SSR markers might beuseful in large-scale population studies within the genusCereus.The study of transferability of simple sequence repeats (SSR) among closelyrelated species is a well-known strategy in population genetics, howevertransferability among distinct genera is less common. We tested cross-genera SSRamplification in the family Cactaceae using a total of 20 heterologous primerspreviously developed for the genera  Simple sequence repeats (SSR) or microsatellites are, in general, non-coding regionscommonly found in Eukaryote genomes composed of tandemly arranged repeat motifs from1 to 6 base pairs , phylogeAn alternative to overcome time consuming and costly development of a new set of SSRprimers for a target species is to carry out the transferability of SSR primersamong related species  occurring in easternBrazil: C. fernambucensis subsp. fernambucensis,C. fernambucensis subsp. sericifer, C.insularis and C. jamacaru. A previous phylogeneticanalysis based on plastid DNA placed C. jamacaru as a member of apolytomic clade, sister of the monophyletic clade composed by C.fernambucensis and C. insularis  as a positive control in the initial tests. Genomic DNA wasextracted from the radicular tissue using the Qiagen DNeasy Plant Mini Kit . As the samples from localities S82 and S83 are geographically (~34km) and genetically close, sharing the same unique alleles and comprising a cohesivegenetic group, we decided to join the individuals from the two populations in asingle sample, hereinafter referred to as S82/S83.We sampled 122 individuals from representative populations of bucensis ,besidesTaq DNA Polymerase(Promega), 1X Taq Buffer (5X Colorless GoTaq® Flexi Buffer), 0.2 μMdNTPs, and primer and MgCl2 concentrations varying when necessary. Weconsidered a locus successfully transferred when the PCR products were clearlyvisualized in 3% agarose gels and showed a product size compatible with the rangedescribed for that locus. The loci successfully amplified were then genotyped in thetotal sample , effective number of alleles(ne), expected (He) andobserved (Ho) heterozygosities, private alleles, andpercentage of polymorphic loci were estimated using GenAlEx 6.5 software (FIS) per population was calculated usingFSTAT 2.9.3.2 (FST (6 generations of MCMC(25% as burn-in). The K-values tested ranged from 1 to 8. To find the best K we usedΔK statistics , nine  showed positive results in transferability for at least onespecies (TableS2), resulting in 35.16% of success intransferability. Except for mEgR 02, the allele size for all lociwas congruent with the expected size (TableS2). We were not able to amplify five SSR locipreviously transferred to Cereus  (TableS3). This result is likely related to nucleotidedifferences among the flanking regions of the samples used in this work, preventingprimer annealing.From the 20 tested loci  than those reported in the original description (FIS) provided no significantresult . The LD analysis results between polymorphicloci were not statistically significant after Bonferroni correction.The percentage of polymorphic loci ranged from 44.4% (populations S113 and S82/S83)to 77.8% (location S80) . Incontcription . No locutresult . The loc S82/S83 .PrivateFST was 0.44,ranging from 0.12 (Pmac82) to 0.79 (mEgR 02)(TableS5). Clustering analyses have shown somewhat distinct results . However, the results from AMOVA suggestthat the three clusters recovered by STRUCTURE better explain the genetic variationstructuring in our data (C. fernambucensis andC. insularis based on cpDNA and the PHYC gene(C. jamacaru and C.fernambucensis subsp. fernambucensis (S104) deservesadditional investigation, but seems to be a spurious grouping as a result of thereduced number of sampled populations.The FreeNA-corrected estimate of global our data , asfollour data . To inveour data . AlthougThe estimated success in transferability observed in this study (35.16%) was higherthan the average of 10% found in cross-genera transferability studies publishedbetween 1997 and mid-2006 (see A. bravoanus andE. grusonii – four transferred of nine tested) or closelyrelated species (P. machrisii – five transferred of 11 tested). Itis worth highlighting that for those loci from A. bravoanus,E. grusonii, and P. chichipe we had a previousexpectation of positive cross-amplification, as they were formerly transferred forsome Cereus species (TableS6).This expectation was not clearly observed here considering cactus phylogeny (Cereus constitutes an interesting biological model toperform evolutionary studies, and efforts were employed to screen informativemolecular markers in this genus (Cereus species. Furthermore, considering the rate of success intransferability, our results should encourage cactus researchers interested in usingthe increasing number of SSR loci that have been described for representatives ofthis highly diverse and relatively overlooked plant family (e.g., The genus


Astragalus lehmannianus (Fabaceae) is a vulnerable species found in the cold deserts of northwestern China. We aimed to characterize polymorphic microsatellite loci for A. lehmannianus to support future studies of population genetic dynamics and conservation management of the species.A. lehmannianus. Levels of observed and expected heterozygosities were 0.000–1.000 and 0.000–0.827, respectively. Furthermore, two and five of the 12 developed primers were successfully amplified in two congeneric species, A. arpilobus and A. oxyglottis, respectively.We used next‐generation sequencing to detect polymorphic microsatellites. Twenty‐five potential microsatellite loci were identified, 12 of which were polymorphic and present in the three study populations of A. lehmannianus.These newly developed microsatellite markers can be used to determine population diversity and to develop conservation strategies in  Astragalus lehmannianus Bunge (Fabaceae) is a vulnerable perennial herb in China that is distributed only on stationary and semi‐stationary dunes on the southern margin of the Gurbantunggut Desert in Xinjiang Uygur Autonomous Region (XJ), northwestern China  F. J. Herm.  approach from the genome of A. lehmannianus and also tested the transferability of these markers to the congeneric species A. arpilobus Kar. & Kir. and A. oxyglottis Steven ex M. Bieb. These markers will provide useful tools for population genetics and demographic history of current populations of A. lehmannianus and related species. Furthermore, the study of genetic diversity using these markers will contribute to the formulation of conservation strategies for A. lehmannianus.Microsatellite markers developed in A. lehmannianus plants collected from three natural populations in XJ , 0.4 μL of TP‐M13 forward primer, and 1.6 μL of each reverse primer and universal M13 sequencing primer labeled with either HEX or 6‐FAM. All candidate primer pairs were tested by a touchdown PCR protocol in the following procedure: 94°C for 4 min; 10 cycles of 94°C for 40 s, 65–55°C (Δ1°C touchdown per cycle) for 50 s, 72°C for 40 s; 30 cycles of 94°C for 40 s, 55°C for 45 s, 72°C for 1 min; and a final extension at 72°C for 6 min. The amplified products were tested on 1.5% agarose gel and 6% polyacrylamide gel. The size of the amplified fragments was measured with an ABI 3730xL Genetic Analyzer , and allele sizes were scored using the software GeneMarker version 1.51 (Applied Biosystems).The RAD‐Seq libraries were created by Huashibaiao Biotech Company  to generate the simple sequence repeat (SSR) information. Our selections were di‐, tri‐, and tetranucleotide motifs with flanking regions larger than 100 bp and a minimum of 10, six, and four repeats, respectively. A total of 165 primer pairs, which were designed using Primer Premier 5.0  and synthesized by Sangon Biotech , were obtained and tested for amplification. We attached the universal M13 sequence (TGTAAAACGACGGCCAGT) tag to the 5′ end of the forward primer using the method described by Schuelke  to labelA. lehmannianus from different populations  in population G, two to 10 alleles (mean 4.92) in population C, and one to five alleles (mean 2.83) in population J. The levels of mean observed and expected heterozygosities, respectively, were 0.507 (range 0.107–0.893) and 0.566 (range 0.249–0.827) in population G, 0.547 (range 0.094–0.938) and 0.505 (range 0.253–0.792) in population C, and 0.567 (range 0.000–1.000) and 0.497 (range 0.000–0.800) in population J (Table A. lehmannianus was successful for two of the 12 loci (AL42 and AL53) in A. arpilobus and for five of the 12 loci  in A. oxyglottis (Table Cross‐species amplification using the microsatellites developed for A. lehmannianus that can serve as a useful resource for estimation of genetic diversity. These newly developed primers can be used for conservation and restoration strategies of this rare species.Our results provide 12 microsatellite markers for M.Y.W., D.Y.T., and X.J.S. conceived and designed the experiments. M.Y.W. performed the experiments. M.Y.W. and X.J.S. analyzed the data. M.Y.W., D.Y.T., and X.J.S. contributed to manuscript preparation. All authors reviewed and approved the manuscript.


Wastewater treatment contributes to environmental sustainable development indicators such as clean water and sanitation, then, is imperative to improve the mechanisms and process of contaminant removals. The sewage and industrial effluents are the major contributors of pollutants in land and water discharges, and are necessary to enrich the available data for having reference parameters for plant designing and optimization. The physical andchemical assays alone could not be considered sufficient to assess properly the plant performance because complex mixtures demand ecological and biological parameters for a holistic evaluation. Hence, the ecotoxicity and the genetic toxicity measurement become an important tool to complement the conventional water quality parameters, but these parameters are not widely reported in the open access literature. Despite of several studies showed ecotoxicity and the genetic toxicity data, these could be considered not sufficient because the resulted information is derived from single compounds. Considering the scarce data mentioned above this article presents data on the genetic an ecological toxicity of an anaerobic effluent post-treated with ozone and ozone/UV generated by Chaparro et al.  and Chap Further information about the characteristics of this effluent can be found in Chaparro and Pires [3. day and an hydraulic retention time of 25 h. The effluent from this reactor was subjected to ozone and ozone/UV oxidation tests without prior pH adjustment. The pH of the HAIB reactor effluent was close to 8.6. The samples were taken at regular intervals during the experimental period to evaluate the ecotoxicity and the genetic toxicity. Ecotoxicity assays were conducted according to the Brazilian standards [The raw industrial wastewater was obtained from a nd Pires . The pultandards ,5, whichtandards . Table 2Grant`s protocol [The genetic toxicity was performed according to the modified version of the protocol . The GenKruskal–Wallis applied by means of the BioEstat 5.0 software (https://bioestat.software.informer.com/5.0/).Finally, the statistical analysis considered the non-parametric


Francolinus francolinus, Phasianidae) is a medium-sized galliform whose distribution range stretches from Cyprus to the Gulf of Bengal. Six morphologic subspecies are known, with three of which occurring in Pakistan, where the species is heavily hunted and used as pet for chirping competitions. We genotyped 98 samples (feathers) at both the entire mitochondrial DNA Control Region gene and nine microsatellite loci to get a deeper insight into the genetic diversity of the black francolin in Pakistan in order to offer cogent recommendations for its conservation management. We identified several mtDNA lineages that were consistent with the currently described subspecies/taxonomy whose pattern of co-occurrence is compatible with the geological history and the faunal movement routes of the region under study. However, the biparentally inherited microsatellites returned a quite discordant picture of an extensive, sex-biased genetic mixing due to the intensive relocations of already overharvested male individuals for chirping competitions. Our results indicated that the genetic integrity of the black francolin in Pakistan could be seriously at risk and call for monitoring and limiting its trade other than enhancing the public awareness of the importance of local biodiversity resources.The ever-increasing human-mediated wildlife reshuffling is raising concern for the conservation of biodiversity. The loss of biological distinctiveness among regions lessens the genetic diversity and consequently the evolutionary potential of local biotas to tackle present-day global change and human disturbance. This process may be sometimes cryptic unless investigated by means of a molecular approach. In this respect, game birds are a paradigmatic case. The black francolin ( Since the Bronze Age, human activities have been shaping the distribution of a variety of taxa with an increasing movement of faunal and floral assemblages worldwide . In the Francolinus francolinus, Phasianidae) is a medium-sized galliform whose distribution range stretches from Cyprus to the Gulf of Bengal [F. f. bogdanovi, from the South West to the North East, and the endemic F. f. henrici mostly in the North of the country [2 in the different habitats of Mang Game Reserve in Haripur (North Pakistan) [2 in Lehri Nature Park (North Pakistan) [2 recorded for the healthy insular population inhabiting Cyprus [The black francolin  , peakingakistan)  and Lal akistan) , respectg Cyprus .The black francolin has been held in high regard for its delicate flavour since the Classic Age and is presently an important source of bush meat for poor people in Asia , 29. PoaN = 23) with records collected exclusively in the central provinces of Punjab and Balochistan. Moreover, the authors used Random Amplified Polymorphic DNA (RAPD) markers, whose suitability in the investigation of genetic variability has been called into question since Pérez et al. [F. f. asiae, occurs in both North East and South East Pakistan. Despite the relatively large number of samples (N = 75) collected across the entire country, the black francolin was investigated at the mitochondrial DNA only. Uncertainty thus remains regarding the spatial genetic structure of the species.Genetic information is fundamental to plan management strategies for the conservation of a given species. For the black francolin of Pakistan, first data came to the light only recently, with Riaz et al.  suggestiz et al. . Later oz et al.  proved tWe assumed that in the absence of physical barriers, genetic admixture among the three subspecies is likely and no overt genetic structure is expected. To test our hypothesis, we characterized the genetic diversity of the black francolin in Pakistan in order to gain data useful for implementing its sustainable use and long-term protection within an evolutionary adaptive framework . We usedN = 21; Central: N = 55; South: N = 22) from 25 populations ranging from the Indian Ocean across the Indus River valleys to the foothills of the Himalayas. Of these, 75 were already investigated only at the mtDNA by Forcina et al. [N = 6; Musa Khel, N = 3; Mekhtar, N = 2; Quetta, N = 3; Zhoab, N = 2 and Chagai, N = 3: Between 2008 and 2014, 98 samples (feathers) of wild black francolins were collected in Pakistan  and following the manufacturer’s instructions. A 3 mm-long fragment from the base of the quill was employed as in Barbanera et al. . The relGallus gallus) genome. Amplifications (25 μl) were performed in a T100 thermal cycler  with 1 μl of Taq DNA Polymerase , 2.5 μl of 2.5 mM dNTP, 2.5 μL of 10X PCR buffer, 2 μl of 25 mM MgCl2, 1.5 μl of each primer (1 μM) and 10 ng of DNA. PCR thermal profile was as follows: 3 min at 94°C, then 35 cycles of 1 min and 20 s at 94°C, 1 min and 30 s at 60°C and 1 min at 72°C, followed by 7 min at 72°C. When PCR products were visualized by means of a gel electrophoresis, all individuals showing one or two bands were identified as males (homogametic sex) and female (heterogametic sex), respectively. The shorter band  was shared between sexes as opposed to the larger one .The sex of each francolin was inferred using a PCR-based method. Chromo Helicase DNA (CHD) gene of ZZ  and ZW  sexual chromosomes was amplified with primers USP1, USP3, CPE15F and CPE15R , 39 prev2, 5 μl of 10X PCR Gold buffer, 5 μl of 2.5 mM dNTP and 1 μl of AmpliTaq Gold DNA Polymerase , 3 μl of each primer (1 μM) and 20 ng of DNA template. Thermal profile was as follows: 3 min at 94°C, then 30 cycles of 1 min at 94°C, 2 min at 55 °C and 1 min at 72°C followed by 7 min at 72°C. In case of very old feathers, we obtained the entire CR gene using the purified products (Genelute PCR Clean-up Kit) of the first amplification as DNA template in two semi-nested PCRs (snPCRs) [st: 628 bp; 2nd: 699 bp) were amplified for each sample in two reaction tubes by applying the same thermal profile as in the first PCR. Final PCR products were purified as above and sequenced on both DNA strands  at Genechron .The entire mtDNA Control Region (CR) gene (ca. 1170 bp) was amplified using primers PHDL (forward) of Fumihito et al.  and H132(snPCRs) . In the F. francolinus and previously analysed by Forcina et al. [N = 3), India (N = 20), Nepal (N = 7), and Bangladesh (N = 1), thus bearing potential for shedding further light on the biogeography of the black francolin across the entire Indian sub-continent. Despite its shortness, this CR fragment had proven to hold a still fairly high resolution power [A first mtDNA alignment  included 98 sequences from birds sampled in Pakistan and those retrieved from 124 samples  collected across the whole distribution range of a et al.  . Only for the population of Pakistan, we used arlequin to calculate haplotype diversity (h), nucleotide diversity (π), number of pairwise differences (k), and to investigate the partition of the mtDNA diversity  among and within regions  using the ϕst analogous of Wright’s F-statistics [We performed both alignments with alx 1.81  and usedin 3.5.1  to inferin 3.5.1  implemenatistics .Alectoris rufa) genome and previously applied in the black francolin by Forcina et al. [N = 4: haplotypes H20, H21 and H23), Afghanistan (N = 1: H21), India (N = 3: H5 and H6) and Nepal . We used these birds as genetic reference for F. f. bogdanovi  and F. f. asiae  in the microsatellite DNA data computations. All cross-amplifications were carried out as in Barbanera et al. [genescan (Thermo Fisher).All 98 Pakistani samples were genotyped at nine STR loci using primers isolated from either chicken or red-legged partridge  [Arlequin 3.5.1, to: compute the number of alleles per locus, the number of unique alleles and the allelic richness. fstat 2.9.3 [In or average gene diversity) and the inbreeding coefficient (f). We also used genepop 3.4 [HE) and observed (HO) heterozygosity and infer deviations from both Hardy-Weinberg Equilibrium (HWE)  and Linkage Disequilibrium (LD) . The Bonferroni correction [arlequin to investigate the partition of STR diversity using amova and Wright’s F-statistics (10000 permutations) [Nem, effective number of migrants per generation via the private allele method of Slatkin, [The STR panel was investigated using er 2.2.3  to test et 1.3.3  (in term PIDsib) . Analyseat 2.9.3  was usedepop 3.4  to calcurrection  was adoptations)  and to eSlatkin, ) among pstructure 2.3.4 [K) was assumed to range from 1 to 10. All simulations were run with 106 Markov Chain Monte Carlo iterations, after a burn-in period of 105 iterations, and replicated 10 times per each K-value to check for the consistency of likelihood values between runs. To prevent a biased estimation of K, genetic admixture and correlated allele frequencies among subpopulations were included as parameters. The optimal K was selected as described by Evanno et al. [structure harvester 0.6.92 [clumpp 1.1.2 [K and get the estimated membership to the K clusters for both populations and groups of populations. The assignment threshold (Qi) to a given cluster was set to 0.80 [The genetic diversity of Pakistani populations was also inferred by means of a Bayesian clustering approach using re 2.3.4 . The numo et al.  using str 0.6.92 . The propp 1.1.2  was used to 0.80  and indiFinally, another Bayesian clustering analysis was done including only black francolins from Haroon Abad, Alipur, Rakhni, Bait Suvai, and Chakwal  for purpratio of 1.97  while 33 as females (two PCR products), with a male/female sex  of 1.97 .amova: ϕst = 0.11, P < 0.001).The entire CR gene alignment included 66 haplotypes H1-H66: , with 63francolinus and arabistanicus subspecies, the bogdanovi—henrici haplogroup included two clusters. The first, F. f. bogdanovi, comprised 63 individuals from Pakistan  and 5 from South East Iran and Afghanistan. The second, F. f. henrici, included 31 francolins from Pakistan only . Finally, the asiae—melanonotus haplogroup comprised four black francolins from Pakistan  and 19 from India, Nepal and Bangladesh. Overall, F. f. bogdanovi birds were separated from the francolinus—arabistanicus and asiae—melanonotus haplogroups by seven and six mutational steps, respectively, while five mutations stepped away the closest relatives of F. f. bogdanovi and F. f. henrici. Among the 23 Pakistani birds new to this study, 16 and 7 were assigned to F. f. bogdanovi  and F. f. henrici , respectively. The population of Badin included haplotypes from all subspecies occurring in Pakistan , it decreased moving both northwards and southwards; F. f. henrici showed the lowest (23%) and highest (52%) occurrence in Central and North Pakistan, respectively. Finally, F. f. asiae  was discovered in North  and South  Pakistan  with respect to those reported by Forcina et al. . We creairection .http://purl.org/phylo/treebase/phylows/study/TB2:S22601), while GenBank accession codes (LT990153-57) are listed in Entire mtDNA CR alignment and STR genotyping data are available at TreeBASE .The STR panel was powerful in discriminating individuals  pointed to the existence of three genetic groups  assigned to F. f. bogdanovi were the most abundant ones in the Central region, whereas they decreased moving both northwards and southwards  while they occurred in the Central region along with those with F. f. henrici genotypes , while those from Haroon Abad and Rakhni showed their highest assignment values to cluster QII .The second Bayesian clustering analysis was performed only for the black francolin  populations of Haroon Abad, Alipur, Rakhni, Bait Suvai and Chakwal. Results pointed to the occurrence of two genetic groups : birds fLow-vagile species are expected to show well-structured phylogeographic patterns across their distribution ranges . Most gaF. francolinus adaptive diversification [In the present study, when only Pakistan was taken into account, our results delivered a more complex scenario. Genetic analyses did not show a clear spatial pattern of haplogroups in any of the subspecies , with thfication , which, fication , the IndF. f. bogdanovi and F. f. henrici as the most abundant subspecies in Central and North Pakistan, respectively, while confirming the F. f. asiae as likely relict in both the extreme North and South of the country [F. f. asiae presumably entered North Pakistan from the bordering Indian Kashmir, it thrived in the southern part of the country as relict of Indian fauna within the Pakistani range of The Great Rann of Kachchh [henrici-bogdanovi haplogroup were found from Pakistan eastwards across northern India to Nepal along the Ganges River Basin. This result is in agreement with Whistler [F. f. henrici for the same areas, and might suggests a F. f. henrici backward recolonisation along the Ganges River Basin following F. f. asiae local extinction. This assumption also relies on the disclosure of isolated representatives of F. f. asiae in North Pakistan as well as in India both north and south to F. f. henrici distribution  and nuclear  DNA. Accordingly, STR-based gene flow estimates  males, which provide no parental cares, migrate to higher latitude breeding grounds first, mate, and move further in the search of new partners, while females reside in the same location for the whole breeding season [Nevertheless, while also indicating Pakistan , microsaPakistan , in shar females . Among pcubation , 76. Somcubation . However of 1.31 , black fectively , Table 1male one . Converstructure , we woulg season . Howeverg season .To conclude, the partly discordant picture retrieved with different markers might be indicative of different phenomena shaping the genetic diversity of the black francolin populations in Pakistan. While the maternally inherited mitochondrial DNA delivered a composite assemblage of subspecies whose pattern of co-occurrence is compatible with the geological history and the faunal movement routes of the region under study, the biparentally inherited microsatellite portrayed an extensive, male-biased genetic mixing demanding for further explanations. These somehow puzzling results most likely points towards the impact of human-mediated relocations of male black francolins in Pakistan.F. f. asiae in Central Pakistan. Interestingly, the latter is apparently the most commercialized among the black francolin subspecies, as indicated by introductions to US [Different to most of the nearby countries, Pakistan allows hunting of the black francolin. With their bright plumage and calls from prominent posts heard miles away, males are a particularly easy target for hunters. However, living males are also highly demanded and traded as pets for the chirping competitions . Forbiddns to US , mainlanns to US , 81 and,ns to US , thus caa priori the possibility that this unintentional genetic mixing might play some sort of genetic rescue effect on declining populations [ad hoc for university students would help triggering the cultural change needed.Noteworthy, the human-mediated genetic mixing in Pakistani black francolin invoked by Riaz et al.  was ultiulations . Neverthulations –90, thisS1 TableN) of individuals , (iii) sampled tissue, (iv) year(s) of sampling and (v) related CR mtDNA haplotype  including information on locality with (i) latitude (Lat.) and longitude (Long.) data, number Click here for additional data file.S2 TableGenBank accession codes for the mtDNA CR haplotypes  used in the analyses of this study Click here for additional data file.S3 TablePID  and PIDsib  data are provided. Loci are sorted according to the increasing order of their PID and PIDsib single-locus values , and a sequentially multi-loci PID (PIDsib) is reported for each locus. See Forcina et al. [Multi-locus a et al.  for deta(PDF)Click here for additional data file.S4 TableP, probability value for LD test; Chi2, test with relative degrees of freedom (d.f.) (Fisher’s method).Fisher global test for departure from Linkage Disequilibrium (LD) for each pair of loci across all populations. No comparison was significant . Legend: (PDF)Click here for additional data file.S5 TableHO) and expected (HE) heterozygosity as inferred for North, Central, and South Pakistan black francolins. The probability value for HWE test (P) was calculated for each locus. *: significant departure from HWE after application of Bonferroni correction . Mono., monomorphic locus.Observed ((PDF)Click here for additional data file.S6 Tablestructure for (i) all single populations Click here for additional data file.


Ectopleura are similar to other hydrozoans in that they consist of multiple polyps physically connected through continuous epithelia and shared gastrovascular cavity, Ectopleura larynx does not asexually bud polyps indeterminately. Instead, after an initial phase of limited budding in a young colony, E. larynx achieves its large colony size through the aggregation and fusion of sexually  produced polyps. The apparent chimerism within a physiologically integrated colony presents a potential source of conflict between distinct genetic lineages, which may vary in their ability to access the germline. To determine the extent to which the potential for genetic conflict exists, we characterized the types of genetic relationships between polyps within colonies, using a RAD‐Seq approach. Our results indicate that E. larynx colonies are indeed comprised of polyps that are clones and sexually reproduced siblings and offspring, consistent with their life history. In addition, we found that colonies also contain polyps that are genetically unrelated, and that estimates of genome‐wide relatedness suggests a potential for conflict within a colony. Taken together, our data suggest that there are distinct categories of relationships in colonies of E. larynx, likely achieved through a range of processes including budding, regeneration, and fusion of progeny and unrelated polyps, with the possibility for a genetic conflict resolution mechanism. Together these processes contribute to the reevolution of the ecologically important trait of coloniality in E. larynx.Hydrozoans typically develop colonies through asexual budding of polyps. Although colonies of  Ectopleura larynx however cannot bud indeterminately and instead achieves its large size by fusing sexually produced juvenile polyps to the adult colony.Coloniality is a life‐history trait that confers great competitive advantage for space in crowded benthic environments. The method by which hydrozoans (phylum Cnidaria) create large colonies to compete for space is through asexual budding of genetically identical individuals that remain attached to one another through continuous epithelia and shared gastrovascular cavity. The colonial hydrozoan E. larynx colonies represent chimeric collections of individuals, including distantly related polyps. Estimates of genome‐wide levels of relatedness for polyps within and between colonies confirm the potential for genetic conflict, despite a background of low genetic diversity in this system. Our results provide an evaluation of genomic diversity in a system that uniquely uncouples coloniality from strict clonality, and suggests that in certain situations selection for large size takes precedence over selection for genetic homogeneity within an integrated colony, producing colonies containing a complex set of genetic relationships.This process appears contrary to what is predicted by evolutionary theory—genetically chimeric colonies give rise to potential genetic conflict between colony members sharing resources, and thus might be opposed by natural selection. To determine the degree of genetic diversity within a colony and whether or not there is a genetic homogenization process, such as somatic cell parasitism, we used a genomic approach to assess genetic variation within and between colonies. We found that Ectopleura larynx Ellis and Solander, 1786 does not asexually bud polyps indeterminately, but rather achieves large colony size through the aggregation and fusion of sexually produced offspring , especially for benthic, sessile organisms where different colonies may unavoidably come in contact with one another and need to prevent fusion Buss . Indeed,E. larynx, whose ancestors were solitary and lost the ability to form extensive colonies through asexual budding  through the aforementioned fusion of sexually produced juvenile polyps from the local population of colonies, ultimately producing colonies that may contain multiple genetic lineages.It is within this evolutionary context that Petersen . New polE. larynx colonies. Using a RAD‐seq approach, we determined the genetic diversity within and between colonies and characterized the types of genetic relationships  present within colonies of E. larynx. Further, to estimate the potential for genetic conflict, we made genome‐scale estimates of relatedness to serve as a proxy for the probability of matching or mismatching at a genetic conflict locus. This represents one of the first genome‐scale studies of diversity within single colonies of an animal. This unique system, which potentially decouples the effects of coloniality and strict asexual reproduction, will allow us to investigate the effects of coloniality and clonality on the evolution and genetics of a species, as well as the interplay between selection for spatial competition and selection for genetically homogenous colonies.Here, we characterize the extent to which these different processes  contribute to the formation of Ectopleura larynx were collected from eight locations along the coasts of Maine and Northern Ireland. Colonies were removed from the edges of docks or from submerged rocks in shaded subtidal areas. Sex was determined morphologically for each colony and 5–15 polyps per colony were chosen at random from colonies composed of approximately 25–100 polyps  approach  and to enable the resolution of overmerged tags (‐d). These adjustments were chosen to prevent both loss of alleles due to insufficient read depth, and false merging of multiple loci into a single stack.We utilized the Stacks pipeline  and with at least eight reads per site/per individual (‐m 8). For within‐colony investigations of relationships and genetic diversity, only colonies that contained more than four polyps that successfully passed previous filtering steps and had at least 100 associated SNPs were retained, resulting in a dataset containing 19 colonies from two populations in Maine and two in Northern Ireland Tables  and S2. E. larynx both with and without selfing using the R package RClone  or if there were more distant types of relationships present within the colony resulting from fusion, which, to our knowledge, has never been assessed at a genome‐scale for a hydrozoan colony of any species. To do so, we compared genetic distances to those expected under sexual reproduction within a given colony of To further understand the types of relationships present within a colony, we examined patterns of allelic segregation between polyps at all SNPs in the given colony's dataset using custom R scripts. The three genetic relationship categories we discriminated between were: (A) genotypes whose only genetic differences were ones that could be explained solely by sequencing error or somatic mutation within a clonal genotype; (B) genotypes that can be explained by Mendelian segregation of alleles found within the main clonal lineage; and (C) genotypes containing alleles not found in other polyps in the colony, giving possible evidence of gene flow from outside the main clonal lineage. We further divided this last category into whether or not genotypes differed by one or both alleles from the genotypes of other polyps in the colony. Mismatching at both alleles as compared to other polyps was taken as evidence of fusion from unrelated polyps. A detailed description of this approach is presented in Supplemental Methods.Because our data (proportion of sites that differ in a given comparison) may violate assumptions of an ANOVA , we conducted k‐means clustering to test for the presence of distinct clusters that matched the three relationship types we described previously . Assessment of optimal number of clusters was carried out in fviz_nclust function from R package factoextra v.1.05 , defined as the probability that a recipient individual carries an allele identical by descent with an allele sampled randomly from a donor  for all possible pairs of polyps in a collecting location, both within and between colonies.For large SNP datasets, one proxy for estimating relatedness of two individuals is to estimate the average probability of a match between alleles drawn at random from each individual, under the assumption that sequence identity implies identity by descent .To create a larger dataset of independent SNPs for estimating relatedness between colonies, we relaxed some SNP filtering and turned on the –write_single_snp option in the Stacks populations module  between offspring resulting from hypothetical within‐colony sexual reproduction, and then plotted our actual within‐colony, between‐polyp pairwise genetic distance data on the same graphs Fig. . Our hypP‐value for Fisher's exact test of association between two categorical variables is 0.6649). Initial examination of the presence of these distance classes in the sets of loci resulting from our “default” and “conservative” Stacks loci reconstruction runs yielded identical results, so the “default” dataset was not investigated further.Some combination of these relationships/distance classes was present in each given colony Fig. , Fig. S1Visually, the three types of within‐colony relationships described above had distinct, nearly nonoverlapping genetic distances Fig. . A majorThe average Phred score for our data set after quality‐filtering of reads was 37.79, corresponding to a Q‐score, or the probability that a particular base is incorrectly called, of just .00017. The Type I and some Type II genetic distances are greater than our Q‐score error estimates and most are less than published empirical estimates .Next, we examined the loci polymorphic in the other two types of relationships to determine if fusion of polyps from other colonies or from polyps not sampled in this study was responsible for some of the genetic divergence between polyps in these comparisons. In particular, we identified a class of loci where polyps were a different homozygote from the other polyps in the same colony. That is, the divergence in these sites could not be explained as solely as products of Mendelian segregation of the clonal alleles or a single step of somatic mutation or sequencing error from other genotypes in the colony. Given the extremely low probability of somatic mutation occurring twice at a given site Orive , the exit = 1.6338, df = 4748.8, P‐value = 0.1024) or overall location/shape .To rule out the possibility that this pattern of divergence was generated by dropout of one allele in either genotype in a comparison  due to low sequencing depth, we compared the distributions of per‐colony read depth between sites a which this pattern occurred versus all other sites. We found that the shape and location of the distributions of read depths were nearly identical Fig.  and did At each collecting location, the distributions of between‐colony and within‐colony genetic distances are largely overlapping, indicating that there are some within‐colony comparisons that are just as large as between‐colony comparisons, and that there are some extremely similar genotypes present in multiple distinct colonies Fig. . The preE. larynx display low levels of polymorphism and allelic diversity (π) when compared to other location‐level, RADseq‐based estimates of genomic diversity of marine invertebrates, even considering other clonal and/or colonial cnidarians  as calculated using the KING‐robust algorithm . For comparison, for a parent‐offspring or full‐sib relationship, φ is expected to be 0.25, and ranges from 0  for unrelated individuals and 0.5 for monozygotic twins or, in our case, identical clones  probabilities using PLINK  than polyps in different colonies. We found that levels of relatedness were on average higher within colonies than between, but that there were many examples of polyps present in the same colonies that did not share either allele at the few polymorphic sites recovered and therefore may differ at relevant “conflict” loci.E. larynx may possess a mechanism of conflict mediation that homogenizes chimeric polyps, such as somatic‐cell takeover  due to the wider genetic distances involved and the potential involvement of the immune system in preventing wholesale fusion of organisms from occurring , can serve as a proxy for the potential for genetic conflict at such loci, in the absence of knowledge in E. larynx is a climatically mediated genetic bottleneck, potentially at either geological time scales due to glaciation . Genetic relationships within selected colonies of E. larynx not already included in Figure 2.Table S4. Summary of major per‐colony results.Figure S2. Comparison of read‐depth distributions between SNPs where polyps were alternative homozygotes vs. all other SNPs included in the analysis.Figure S3. Histogram of the number of sites at which polyps differ in comparisons between colonies at a given collecting site (orange) or within a colony .Figure S5. Results of different methods for choosing a best k (number of clusters) and the inferred cluster membership for K=2 for the proportion of sites which differ in a within‐colony comparison, for all within‐colony comparisons.Table S5. Number of clusters (K) for the for the proportion of sites which differ in a within‐colony comparison, for all within‐colony comparisons, as inferred by a selection of algorithms employed by the NbClust R package.Table S6A. Between‐colony pairwise Fst values for collecting site ME1.Table S6B. Between‐colony pairwise Fst values for collecting site ME2.Table S6C. Between‐colony pairwise Fst values for collecting site IRE1.Table S6D. Between‐colony pairwise Fst values for collecting site IRE2.Table S7. Complete pairwise calculations of genome‐wide relatedness between polyps in each colony of E. larynx.Click here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.


Different genetic patterns have been demonstrated for narrowly distributed taxa, many of them linking rarity to evolutionary history. Quite a few species in young genera are endemics and have several populations that present low variability, sometimes attributed to geographical isolation or dispersion processes. Assessing the genetic diversity and structure of such species may be important for protecting them and understanding their diversification history. In this study, we used microsatellite markers and plastid sequences to characterize the levels of genetic variation and population structure of two endemic and restricted species that grow in isolated areas on the margin of the distribution of their respective genera. Plastid and nuclear diversities were very low and weakly structured in their populations. Evolutionary scenarios for both species are compatible with open-field expansions during the Pleistocene interglacial periods and genetic variability supports founder effects to explain diversification. At present, both species are suffering from habitat loss and changes in the environment can lead these species towards extinction. Petunia mantiqueirensis and Calibrachoa elegans) that grow in isolated areas on the margin of the distribution of their respective genera. Plastid and nuclear diversities were low and weakly structured in their populations. Evolutionary scenarios for both species are compatible with open-field expansions during the Pleistocene interglacial periods and genetic variability supports founder effects to explain diversification. Both species are suffering from habitat loss and changes in the environment can lead these species towards extinction.In our study, we used microsatellite markers and plastid sequences to characterize the levels of genetic variation and population structure of two endemic and restricted species ( Many rare or narrowly distributed species present low levels of genetic diversity . AdditioEcological and evolutionary processes define the distribution of species across space and time. The climatic changes that occurred during the Quaternary impacted the distribution and genetic diversity of most species through cycles of range contraction and expansion in the northern and southern hemispheres , and, moSome studies have focused on the evolutionary history of species adapted to open areas interspersed among regions of higher altitude in the southern section of the Atlantic Forest . The souPetunia and Calibrachoa share several evolutionary constraints and biological characteristics, many times occurring sympatrically, side by side. Based on a proposed origin in open fields , which is the northernmost distribution of the Petunia genus. This species is annual and herbaceous, with bee-pollinated flowers displaying a purple and short corolla tube . We sampled all known populations and adult individuals found in the same flowering season . Vouchers were deposited at the BHCB herbarium  and the geographical coordinates for all collection sites were obtained with a global positioning system (GPS) unit [see]. We collected three or four young leaves from each adult individual of both species while seeking to minimize damage to the plants; we dried leaves in silica gel and then pulverized them in liquid nitrogen for DNA extraction.These two species are classified as federally endangered according the Brazilian Red List  protocol , 1× buffer (Invitrogen), 0.2 mM each dNTP, 2 mM MgCl2, 0.2 µM of each primer and 10 ng of genomic DNA. The amplification conditions for trnH-psbA and trnS-trnG were conducted as described previously for Petunia species (P. mantiqueirensis as previously described (We estimated the genetic diversity based on the plastid sequences (cpDNA) of intergenic spacers,  species . SequencP. mantiqueirensis were genotyped with 12 microsatellite loci markers (SSR) previously developed for Petunia hybrida . We amplified SSR regions in a final volume reaction of 10 µL containing ~10 ng of genomic DNA as a template, 200 µM of each dNTP (Invitrogen), 1.7 pmol of fluorescently labelled M13(-21) primer, 3.5 pmol of reverse primer, 0.35 pmol of forward primer with a 5′-M13(-21) tail, 2.0 mM MgCl2 (Invitrogen), 0.5 U of Platinum Taq DNA polymerase (Invitrogen), and 1× Platinum Taq reaction buffer (Invitrogen). The PCR conditions were as follows: an initial denaturation at 94 °C for 3 min; 30–35 cycles of 94 °C for 20 s, 48–58 °C for 45 s and 72 °C for 1 min; and a final extension cycle at 72 °C for 10 min.To evaluate current variability in each population and species, individuals of GeneMarker 1.97 software  was utilized to determine the alleles. Additionally, all alleles were visually verified and scored. Repeat motifs, respective annealing temperatures and fluorescent dyes used in each fragment are presented in The forward primers were FAM-, NED-, HEX-, VIC- and PET-labelled. The amplified product was visualized on a 2.5 % ultra-resolution agarose gel stained with 2 µL 0.001 % GelRed™. The amplified DNA was denatured and size-fractionated employing capillary electrophoresis on an Applied Biosystems  with a LIZ (500) molecular size standard (Applied Biosystems). ChromasPro 1.7.5  and haplotype sequences were deposited at GenBank (available at https://www.ncbi.nlm.nih.gov/genbank/) under numbers: C. elegansJQ082471.1, JQ082474.1, KM982182.1, KM982262.1; P. mantiqueirensisAY772921.1, DQ792340.1. DNA sequences were aligned using MEGA7 software  and nucleotide (π) diversity indexes and the number of variable sites were calculated with Arlequin 3.5.1.2 , gene diversity (GD), allelic richness (AR) and inbreeding coefficient (FIS) for each locus. The frequencies of the null alleles, polymorphic index content (PIC), levels of observed (Ho) and expected (He) heterozygosity, and any significant deviations from the Hardy–Weinberg equilibrium (HWE), in order to evaluate the informativeness of the markers, were estimated using Cervus 3.0.3 software   approach (bc 2.1.0 . The prit (time since demographic change in scenarios 2 and 3); scenario 4: Ne4 (current Ne), Nb (Ne during bottleneck), Na4 (pre-bottleneck Ne), t2 and t1 . Demographic prior values are presented in 2 and t1, scenario 4), we first used as reference the maximum the influence of the Last Glacial Maximum  and as the minimum the first record of Araucaria forest expansion in the region . We employed Maxent 3.4.1 , Last Glacial Maximum  and Last Interglacial  conditions. Explanatory variables included a set of 19 bioclimatic Raster layers at a 30 arc-second resolution (ca. 1 km2 at the Equator) from the WorldClim website, version 1.4 (available at: http://www.worldclim.org/). The Raster package (http://www.R-project.org) was used to calculate Pearson’s correlation between variables. For P. mantiqueirensis, four variables  with pairwise Pearson correlation coefficients below 0.7 and with a high percentage of model contribution  were used to reconstruct the taxon distribution. For C. elegans, we employed seven variables .Ecological niche modelling (ENM) was used to compare niche preferences and potential range shifts that the species can suffer from in response to future climatic oscillations. Localities from the two species were obtained from field observations by the authors, SpeciesLink  long (471 bp corresponding to trnH-psbA and 650 bp to trnS-trnG). The sequences did not present any variable site, producing a single haplotype. The GC content was 28.1 % in trnH-psbA and 30.5 % in trnS-trnG. For C. elegans, the sequences of 81 individuals produced a concatenated alignment with 1191 bp (445 bp for trnH-psbA and 746 bp for trnS-trnG), leading to two different haplotypes. The GC content was 27.2 % in trnH-psbA and 30.7 % in trnS-trnG. Haplotype diversity in C. elegans was h = 0.0299 ± 0.0287, and nucleotide diversity was π = 0.004 ± 0.014 %. The frequency of each haplotype was CEh1 = 0.98 and CEh2 = 0.02; CEh2 was observed only at one collection site (Eleg1).The plastid intergenic spacers, P. mantiqueirensis were genotyped using 12 SSR loci, being 10 polymorphic and two monomorphic (PM110 and PM195) for this species. All loci were in linkage equilibrium . The 81 individuals of C. elegans were genotyped based on 14 SSR loci, from which five markers were polymorphic and nine monomorphic . All analyses were conducted based exclusively on polymorphic markers for each species, although the monomorphic loci are suggestive of low genetic variability and high homogeneity in these species as all of them are polymorphic among other Petunia and Calibrachoa species, respectively.The 38 individuals of Petunia mantiqueirensis presented a total of 39 alleles, with an average of 3.9 alleles per locus (2 to 7). The mean PIC value was 0.39 (0.05 to 0.75), and considering all loci and gene diversity, it was, on average, 0.36 (0.06 to 0.80). Allele richness was, on average, 3.33 (2.00 to 7.00). Six loci exhibited a significant deviation from HWE across all samples, three of them had a deficit of heterozygotes and the other three displayed an excess of heterozygotes. Tests for HWE indicated no significant FIS values for all loci and populations and the mean estimated frequency of null alleles was lower than 0.5 %  over all loci was 0.087 (P < 0.05). Five FST pairwise comparisons had significant genetic differentiation, ranging from 0.07 to 0.25 (P < 0.05). The Mant3 had significant differentiation from four other populations , as well as Mant4 vs. Mant1 (FST = 0.25) [see]. With the DAPC analysis, the individuals from a same collection site did not preferentially form groups and individuals from Mant4 and Mant7 were the most differentiated [see].The AMOVA indicated that the percentage of variation within populations (91 %) was higher than among populations (9 %), and the fixation index  and the PIC values for these markers ranged from 0.47 to 0.84 (average 0.63). The gene diversity was, on average, 0.54 (0.45 to 0.73) and allele richness 5.32 (3.06 to 8.42). All loci exhibited a significant departure from HWE across all samples with heterozygote deficits . Moreover, three of them had significant and positive FIS values, whereas one presented a significant and negative FIS value. The estimated frequency of null alleles was lower than 0.5 % in C. elegans (P. mantiqueirensis (FIS) [see]. Eleg2 had the highest average of FIS with one locus presenting significantly positive values. Furthermore, the populations had different numbers of loci with significant deviations from HWE, and all presented a deficit of heterozygotes at least in one locus. All populations also featured private alleles. Note that Eleg2 was the site with the lowest genetic diversity as measured by number of alleles (A) and allele richness (AR) [see]. When we consider the species’ global values  over all loci was 0.281. FST pairwise comparisons had significant and strong genetic differentiation in just the Eleg2 compared to the other sites . With the DAPC, individuals from Eleg2 were completely differentiated from the others, whereas a high superimposition was observed among the other collection sites [see].The AMOVA analysis revealed that the percentage of variation within populations (72 %) was higher than among populations (28 %) and the fixation index (P. mantiqueirensis and C. elegans using the ABC approach reflected that the most likely scenario for both species is that in which the population size in the past was much greater than in the present (C. elegans was very high according to the logistic approach (above 0.98), and a little smaller with the direct approach, above 0.90 (95 % CI = ~0.65–1.0), and extremely high for P. mantiqueirensis above 0.99 and 1.0, respectively. Confidence in the choice of these scenarios were high for the C. elegans and very high for P. mantiqueirensis, in which the predictive errors were 0.16 and 0.0, respectively [see]. The posterior model checking for scenario 2 in P. mantiqueirensis presented a good fit while for C. elegans the fit for one of the summary statistics was not good [see]. The posterior density estimates of the demographic parameters for scenario 2 are found in C. elegans while it is higher in t for P. mantiqueirensis[see]. The estimated time since the population reduction presented a wide confidence interval, the model time for P. mantiqueirensis was older than for C. elegans  and it was not younger than ~5 kya for either  curve’ (AUC)  all testent time , suitabl elegans  were furP. mantiqueirensis and C. elegans, which represent the northern limit of these genera’s distribution. Each one of these species grows in disjunct habitats from the rest of species in their respective genera, in micro-environments located at extremes of elevation  and C. heterophylla .The single haplotype found for Petunia mantiqueirensis features a strict relationship with its bee pollinator (Pseudagaspostemon fluminensis) that forages pollen on plants returning at the same plot between 5 and 13 days after the first visit . The maximum foraging distance for this genus of small and solitary bees is a maximum of 300 m  combined with the oscillations in the size of the species distribution areas based on the past modelling distribution may be a probable explanation for the lack of variation in C. elegans. Despite C. elegans being described as self-incompatible and bee-pollinated  to confirm the endangered status for these two species, iversity  resultiniversity . Our finP. secreta , the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and the Programa de Pós Graduação em Genética e Biologia Molecular da Universidade Federal do Rio Grande do Sul (PPGBM-UFRGS). A.B. was recipient of National Institutes for Science and Technology (INCT) in Ecology, Evolution and Biodiversity Conservation scholarship. G.M. was supported by PNPD-CAPES/PPGBot-UFRGS and C.T. was supported by PNPD-CAPES/PPGBM-UFRGS.A.B. and L.B.F. planned, designed and led the project; A.B., G.M., J.N.F. conducted the experiments; A.B, C.T., A.L.A.S., G.M. and S.L.B. ran the analyses; A.B. and L.B.F. wrote most of the text; L.B.F. provided reagents and equipment to develop the experiments. All authors contributed in the preparation of the study and have commented on and approved the final manuscript.The following additional information is available in the online version of this article—Table S1. Petunia mantiqueirensis and Calibrachoa elegans collection points.Table S2. Characteristics and PCR conditions of the microsatellite loci amplified for Petunia mantiqueirensis and Calibrachoa elegans.Table S3. Prior values (minimum and maximum with uniform distribution) for the parameters employed for the four demographic scenarios  analyses.Table S5. Geographic coordinates of Petunia mantiqueirensis and Calibrachoa elegans occurrence records used in the ENM.Table S6. Characterization of 10 microsatellites analysed on Petunia mantiqueirensis collection sites.Table S7. Pairwise FST values estimated for seven Petunia mantiqueirensis collection sites. (*) Significant values (P < 0.01).Table S8. Characterization of five microsatellites analysed on Calibrachoa elegans collection sites.Table S9. AICc scores and area under the receiver operating characteristic curve (AUC) values of the tested models.Table S10. Data availability: Individual allele per microsatellite loci.Figure S1. Clustering of individuals according to a discriminant analysis of principal components (DAPC) scatterplot performed based on SSR genotypes for: (A) seven populations of Petunia mantiqueirensis and (B) four populations of Calibrachoa elegans.plz037_suppl_Supplementary_MaterialClick here for additional data file.plz037_suppl_Supplementary_Table_S10Click here for additional data file.