The relationship between imagery and mental representations induced through perception has been the subject of philosophical discussion since antiquity and of vigorous scientific debate in the last century. The relatively recent advent of functional neuroimaging has allowed neuroscientists to look for brain-based evidence for or against the argument that perceptual processes underlie mental imagery. Recent investigations of imagery in many new domains and the parallel development of new meta-analytic techniques now afford us a clearer picture of the relationship between the neural processes underlying imagery and perception, and indeed between imagery and other cognitive processes. This meta-analysis surveyed 65 studies investigating modality-specific imagery in auditory, tactile, motor, gustatory, olfactory, and three visual sub-domains: form, color and motion. Activation likelihood estimate (ALE) analyses of activation foci reported within- and across sensorimotor modalities were conducted. The results indicate that modality-specific imagery activations generally overlap with—but are not confined to—corresponding somatosensory processing and motor execution areas, and suggest that there is a core network of brain regions recruited during imagery, regardless of task. These findings have important implications for investigations of imagery and theories of cognitive processes, such as perceptually-based representational systems. Perception describes our immediate environment. Imagery, in contrast, affords us a description of past, future and hypothetical environments. Imagery and perception are thus two sides of the same coin: Perception relates to mental states induced by the transduction of energy external to the organism into neural representations, and imagery relates to internally-generated mental states driven by representations encoded in memory. Various forms of mental imagery have been implicated in a wide array of cognitive processes, from language comprehension  . Iterative searches within the citations among candidate imagery studies located additional candidate imagery studies with the intention of creating a comprehensive list of studies explicitly examining imagery or imagery-like tasks. For purposes of this study, imagery-like tasks were defined as those for which the retrieval of perceptual information from long term memory was required. These tasks were framed as perceptual knowledge retrieval by study authors and extensively cited or were cited by explicit studies of imagery. As discussed above, perception-based theories of knowledge representations are explicitly rooted in imagery Paivio, , and a lImagery vs. low-level baseline contrasts were categorized with respect to one of eight modality conditions: Auditory, Tactile, Motor, Olfactory, Gustatory, Visual-Form, Visual-Color, and Visual-Motion. Modality categorizations were generally straightforward to determine , though classification of visual imagery subtypes required careful consideration of the task, stimuli and baseline contrasts used. One study  or within-modality , no particular baseline task dominated any modality. Implicit or resting-baselines were used in approximately 40% of studies, nearly all of which employed tasks requiring no overt response on the part of the participant. The remaining studies employed somewhat more complex baseline tasks generally designed to account for attention or response processes  under the assumption of cognitive subtraction. Direct contrasts between perception- and imagery-related activity tend to show reduced activity in primary perceptual areas for imagery relative to perception . Correction for multiple comparisons was performed using a false-discovery rate (FDR) threshold of pN < 0.05. GingerALE reports the number of voxels meeting the selected FDR threshold within each ALE map. Except where noted, a cluster size threshold, equal to the FDR rate times the number of suprathreshold voxels, was applied to each map (hereafter extent-thresholded clusters). For example, if 1000 voxels reached a FDR threshold of 0.05, then the expected number of false positives within that ALE map would be 50. A cluster size threshold of 50 in this example ensures that no extent-thresholded cluster would consist entirely of false positives. Because the number of FDR-significant voxels varied by modality, this approach resulted in different cluster thresholds across modalities. It should be noted, however, that imagery-related clusters were analyzed independently and with respect to sensorimotor ROIs rather than with each other (see below). Thus, these differences had little to bear on the results that follow, other than to increase the confidence with which conclusions can be drawn about the meaningfulness of any given extent-thresholded cluster for that analysis.Concordance among imagery vs. baseline activation foci reported across the neuroimaging literature was analyzed using a widely used activation likelihood estimate (ALE) meta-analytic approach  for each ROI are indicated in each modality analysis. Multiple atlases were necessitated by the fact that no single atlas contained ROI definitions corresponding to all modalities included in the present analysis. In some cases, different atlases contained different definitions of the same region. When a given anatomical region was defined in exactly one atlas, that definition was taken as the ROI; when multiple atlases defined the same region, the intersection  was taken as the ROI. This atlas-based approach was intended to arrive at a set of ROIs that are easily reproducible and for which there should be general agreement are representative of the corresponding sensorimotor cortices.http://afni.nimh.nih.gov/afni/download). Briefly, 3dClustSim calculates cluster size threshold (k) for false positive (noise-only) clusters at specified uncorrected alpha level. Though the ALE analyses used FDR corrected alpha thresholds, the equivalent voxel-wise alpha threshold for each ALE map is available in the GingerALE output. 3dClustSim carries out a user-specified number of Monte Carlo simulations of random noise activations at a particular voxel-wise alpha level within a masked brain volume. Ten thousand such simulations for each ALE map were used for this study. The number of simulations in which clusters of various sizes appear within the volumetric mask is tallied among these simulations. These data are then used to calculate size thresholds across a range of probability values for that region. For example, in a specified volume using a voxel-wise alpha of 0.001, if clusters of size 32 mm3 or greater appear in 50 of 10,000 iterations by chance, this correspond to a p < 0.05 cluster-level significance threshold. In other words, within the specified volume using a voxel-wise alpha of 0.001, clusters exceeding 32 mm3 are unlikely to occur by chance. To be clear, the cluster thresholds calculated using 3dClustSim was used to calculate an overlap criterion for each ROI, and not as an additional ALE cluster thresholding step. To the author's knowledge, no previous meta-analysis of neuroimaging data has attempted to qualify overlap between ALE clusters and a priori ROIs. However, the cluster size threshold approach is widely used to test statistical significance of clusters in conventional ROI analyses. That is, size thresholding is often used to determine whether a cluster of a particular size occurring within a given ROI is statistically significant. The present analysis had identical requirements, thus it was deemed to be an appropriate metric of overlap significance. A benefit of this approach when considering different ROIs is that it naturally takes into account differences in ROI extents: Larger sensorimotor ROIs require correspondingly greater overlap with imagery clusters for the overlaps to reach statistical significance.The degree of overlap was assessed for each ROI by determining whether the number of voxels in the extent-thresholded ALE clusters overlapping with a given ROI reached an overlap criterion. The overlap criterion was set independently for each ROI using 3dClustSim . Nonetheless, the following results do not speak to whether one imagery modality recruits a particular region more than any other imagery. Inter-modal contrasts were not performed for two reasons: First, such contrasts address the question, not of what regions are implicated in a particular type of imagery, but what regions are implicated more for that type of imagery than any other. Networks defined by such contrasts would thus be more exclusive, and reducing the usefulness of these analyses to those interested in a non-comparative description of imagery for a particular modality. Second, there is a practical problem imposed by the disparity between the frequencies with which imagery in each sensorimotor modality has been investigated. This disparity would plausibly skew any such comparisons and generate networks driven by a single modality. When analyses are restricted to within-modality, however, differences with respect to numbers of studies are not problematic: a coherent network can be identified from relatively few studies, provided they are mutually consistent. Though modality-specific activations are not explicitly contrasted, crossmodal overlap between clusters is noted where it occurs.The results are presented in order of generality. The first analysis identifies those regions consistently active relative to baseline in neuroimaging studies of imagery across all modalities. The eight subsequent analyses identify regions consistently active relative to baseline in modality-specific imagery for each of 5 sensorimotor modalities and 3 subtypes of visual imagery. All coordinates are reported in MNI standard space.pN < 0.01 (FDR corrected) and a minimum cluster size threshold of 800 mm3 was used for the general imagery analysis. One thousand hundred and three foci from 84 contrasts involving 915 participants contributed toward these results. Nine primarily left-lateralized clusters reached the significance threshold  for both baseline conditions, though no cluster extent threshold was used . The conjunction analysis found nine clusters, primarily in bilateral dorsal parietal and left inferior frontal regions that were active across all imagery modalities for all baseline conditions  and highlight commonalities among them . Imagery vs. baseline contrasts in the ALE analyses involved two broad classes of low-level baseline tasks: The resting state baseline tasks are assumed to be homogeneous across the 33 contrasts that used them. The non-resting state baseline tasks used across the remaining 50 contrasts were more varied, typically involving passive perceptual control conditions  or foil trials. Because ALE is sensitive to activation consistencies, it was plausible that baseline-related (rather than strictly imagery-related) networks may emerge in the ALE statistics. This concern was conservatively addressed by a conjunction analysis of resting-baseline vs. non-resting-baseline studies. The significance threshold was maintained at ns Table . These r3. Ninety-three foci from 11 experiments involving 127 participants contributed toward these results. For the purposes of this analysis, primary auditory cortex was defined by the AAL template definition of Heschl's Gyrus within the MRIcroN software package (http://www.mccauslandcenter.sc.edu/mricro/mricron/index.html). Ten clusters were reliably associated with auditory imagery at a statistical threshold of pN < 0.05 (FDR corrected)   with premotor cortex. The posterior-most motor imagery cluster, centered at , did overlap substantially with the tactile imagery ROI. The overlapping region was centered at . The 3dClustSim simulations determined that the volume of this overlapping region corresponded to a cluster size corrected threshold of p < 0.001 within the primary somatosensory cortex ROI.Minimum cluster size threshold in the ALE analysis of motor imagery studies was set at 712 mm3. Forty-nine foci from four experiments involving 44 participants contributed toward these results. For the purposes of this analysis, primary somatosensory cortex was defined by the union of the Brodmann area 1, 2, and 3 definitions within the MRIcroN software package. Three left-lateralized clusters were reliably associated with motor imagery at a statistical threshold of pN < 0.05 (FDR corrected) , which overlapped entirely with primary somatosensory cortex. The 3dClustSim simulations determined that the volume of this overlapping region corresponded to a cluster size corrected threshold of p < 0.001 within the primary somatosensory cortex ROI. No tactile imagery ALE cluster overlapped with the primary motor cortex ROI.Minimum cluster size threshold in the ALE analysis of tactile imagery studies was set at 88 mm3. Note that this cluster size threshold was smaller than the GingerALE-recommended minimum threshold for this dataset. The 3dClustSim analysis determined that 45 mm3 clusters of size would occur by chance within the gustatory ROI with a probability of 0.05. This reduced cluster threshold permitted the detection of clusters that would reach corrected-level significance in the ROI. Fifty-three foci from five experiments involving 63 participants contributed toward these results. For the purposes of this analysis, gustatory cortex was defined by the AAL template definition of bilateral frontal operculum and anterior bilateral insula . The ALE analysis of all gustatory imagery studies found nine clusters that were reliably associated with gustatory imagery , was overlapped completely by the gustatory cortex definition. The 3dClustSim simulations determined that the volume of this overlapping region corresponded to a cluster size corrected threshold of p < 0.05 within the primary gustatory cortex ROI.Minimum cluster size threshold in the ALE analysis of gustatory imagery studies was set at 45 mmry Table . There w3. Fifty-one foci from five experiments involving 80 participants contributed toward these results. Olfactory cortex was defined by the AAL template definition of bilateral piriform cortex. The ALE analysis of all olfactory imagery studies found four clusters that were reliably associated with olfactory imagery , between olfactory cortex and the third largest cluster centered at . The 3dClustSim simulations determined that the volume of this overlapping region corresponded to a cluster size corrected threshold of p < 0.001 within the primary olfactory cortex ROI.Minimum cluster size threshold in the ALE analysis of olfactory imagery studies was set at 136 mmry Table . There wtypes of visual information. Importantly, a number of functionally specialized brain regions contain neurons that are preferentially tuned to different aspects of visual input: The lateral occipital complex (LOC) is specialized for shape processing , between LOC and the smallest cluster centered at . The 3dClustSim simulations determined that the volume of this overlapping region corresponded to a cluster size corrected threshold of p < 0.001 within the LOC ROI. Because the visual ROIs were adjacent to one another, the overlap of the visual form clusters with the color and motion ROIs was additionally assessed. The cluster overlapping with LOC additionally overlapped the visual color ROI definition by 126 mm3, which corresponded to a cluster size corrected threshold of p < 0.005 within the visual color ROI. No visual form ALE cluster overlapped with the visual color ROI.Minimum cluster size threshold in the ALE analysis of visual form imagery studies was set at 2384 mm3. Eighty-one foci from seven experiments involving 76 participants contributed toward these results. For the purposes of this analysis, V4 was defined by the Juelich Histological Atlas definition of left and right V4. The ALE analysis of all visual color imagery studies found 4 left-lateralized clusters that were reliably associated with visual color imagery , was found between V4 and the largest cluster centered at . The 3dClustSim simulations determined that the volume of this overlapping region corresponded to a cluster size corrected threshold of p < 0.05 within the V4 ROI. No visual color ALE cluster overlapped with either the visual form or visual motion ROIs.Minimum cluster size threshold in the ALE analysis of visual color imagery was set at 192 mm3. One hundred and ten foci from 10 experiments involving 97 participants contributed toward these results. For the purposes of this analysis, V5 was defined by the intersection of the Juelich Histological Atlas definition of left and right V5 with the reverse inference map generated by Neurosynth . The 3dClustSim simulations determined that the volume of both overlapping regions corresponded to a cluster size corrected threshold of p < 0.001 within the V5 ROI. The cluster overlapping with right V5 additionally overlapped the visual form ROI definition by 262 mm3, which corresponded to a cluster size corrected threshold of p < 0.001 within the visual form ROI. The form and motion imagery clusters did not overlap within either of the ROIs, however there was a 244 mm3 overlap between form and motion imagery clusters centered in the right superior parietal lobule . The overlap between form and motion imagery activations in BA 7 is notable in light of the implication of this region in the integration of visual and motor information , but were primarily left-lateralized. It was noted earlier that perceptually-based representational theories assume that multisensory imagery underlies semantic retrieval. Others have suggested that the default-mode network, a well-defined network of brain regions more active during periods of rest than under cognitive load, may arise in part out of introspective processes, including imagery  manipulations of imagery representations.One challenge for this interpretation concerns the failure to show recruitment of primary sensorimotor perceptual cortices for the auditory and motor modalities. The ALE analyses showed imagery in these modalities does reliably recruit posterior superior temporal gyrus (STG) and premotor cortex, respectively. These results are consistent with Kosslyn et al.  review fThough the question of whether visual imagery, in the general sense, recruits early visual cortex has been extensively studied , but seldom acts on an object without also touching it. Similarly, an asymmetrical relationship existed among the three visual modality subtypes: First, form imagery clusters additionally overlapped the color ROI, but not Finally, and perhaps most importantly for investigations of perceptually-grounded representations, in no modality were imagery clusters restricted to brain regions immediately involved in perception. Those clusters that did overlap with primary somatosensory regions generally extended beyond these areas. In contrast to perception or imagery-based accounts of knowledge representations, amodal models of semantic memory assume concept knowledge is maintained as an abstraction bearing no connection to perceptual processing  unimodal convergence zones (Binder and Desai, Though neuroscientific studies of imagery have proliferated over the last decade, not all forms of imagery have been investigated to the same extent—imagery of the chemical senses and tactile imagery appear to be relatively underrepresented. Some of the questions posed here may not be adequately answerable without further study in these imagery domains. Similarly, the present review omits studies of imagery in other domains, such as emotional, temporal or spatial imagery, which may be more abstract forms of meta-imagery involving the integration of multiple modalities or function as representational primitives.Finally, these results are generally consistent with the assumption that mental imagery underlies representational knowledge, though the matter is far from resolved. These considerations point toward a need for further investigation in the imagery domain. These efforts will help relate cognitive processes to one another and to arrive at a fully grounded model of cognitive processing.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Rhinella crucifer group of toads were defined under morphological criteria alone. Previous data suggest limited correspondence of these species to mitochondrial lineages, and morphological intergradation at transitions between forms suggests hybridization. Here we extensively sampled populations throughout the geographic distribution of the group and analyzed mitochondrial and nuclear sequence data to delimit genetic units using tree–based and allele frequency–based approaches.Delimiting genetic units is useful to enhance taxonomic discovery and is often the first step toward understanding evolutionary mechanisms generating diversification. The six species within the These approaches yielded complementary results, with allele frequency-based methods performing unexpectedly well given the limited number of loci examined. Both mitochondrial and nuclear markers supported a genetic structure of five units within the group, with three of the inferred units distributed within its main range, while two other units occur in separate isolates. The inferred units are mostly discordant with currently described forms: unequivocal association exists for only two of the six species in the group. Genetic evidence for hybridization exists for two pairs of units, with clear cyto–nuclear allele mixing observed in one case.Rhinella crucifer group. Correspondence between genetically distinguishable units and the currently recognized species is only possible for Rhinella henseli and R. inopina. The recognition of other species relies on the reassessment of the geographic range of R. crucifer, the examination of the type series of R. ornata for hybrids, and on the use of additional markers to verify the genetic distinctiveness of R. abei. We state that R. pombali should not remain a valid species since its description appears to be based on hybrids, and that the name R. pombali should be considered a synonym of both R. crucifer and R. ornata. The fifth inferred but undescribed genetic unit may represent a new species. Our results underscore the potential of the R. crucifer species group to contribute to a better understanding of diversification processes and hybridization patterns in the Neotropics, and provide the basis for future evolutionary and taxonomic studies.Our results confirmed that current taxonomy does not represent evolutionary units in the  Molecular data in taxonomic studies has profoundly impacted the field by bringing fresh perspectives on discussions about species concepts and delimitation,2 harnesa priori information. Assignment results consistently recovered five genetic breaks and six demes could be delimited based on the nuclear data alone , which suggests that additional data (more nuclear markers) will likely uncover a nuclear genetic structure consistent with that of mtDNA for the eastern regions of the states of Paraná and Santa Catarina.Clusters defined by assignment analyses of nuclear markers correspond well to the mtDNA topology; of the six inferred demes, C Figure. SubstruHemidactylus fasciatus for each K, calculated ΔK values[We used allele trees and population assignment analyses to assess genetic structure in the nuclear sequence data. We constructed trees for each nuclear marker using maximum likelihood with the same parameters applied to the mtDNA data. For population assignment analysis we used the algorithm implemented in the program STRUCTURE 2.3 coding pK values with StrK values and checa priori information. Because of low levels of haplotype sharing in the mitochondrial data, we coded well-supported major clades as alleles . We performed hierarchal FCA runs, starting with the complete dataset and removing individuals forming divergent groups systematically until groups were no longer separable.We combined phased nuclear sequences and the mitochondrial data  in a three-dimensional factorial correspondence analyses  using GThe authors have no financial or other competing interest to declare.MTCT, KRZ, CFBH and JA designed the study and collected the samples in the field. MTCT produced the molecular data, performed the analysis and drafted the paper. JA, KZ and CFBH revised the manuscript. All authors approved the final manuscript.Nuclear gene genealogies. Numbers before clades indicate support values .Click here for fileIndividuals samples in this study. Columns indicate voucher or tissue number, localities (with codes), sequenced mtDNA fragments, haplotypes, mitochondrial clade, nuclear alleles and individual codes for the STRUCTURE analysis.Click here for file


Erigeron breviscapus (Vant.) Hand.-Mazz. is an important, widely used Chinese herb with scutellarin, 1,5-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid and erigoster B being its major active compounds. We aimed to resolve the influence of biotic and abiotic factors on the concentrations of these compounds and to determine appropriate cultivation methods to improve the yields of the four compounds in this herb.In order to detect the major genetic and natural environmental factors affecting the yields of these four compounds, we applied AFLP markers to investigate the population genetic differentiation and HPLC to measure the concentrations of four major active compounds among 23 wild populations which were located across almost the entire distribution of this species in China. The meteorological data including annual average temperature, annual average precipitation and annual average hours of sunshine were collected. The relationships among the concentrations of four compounds and environmental factors and genetic differentiation were studied.Low intraspecific genetic differentiation is detected, and there is no obvious correlation between the genetic differentiation and the contents of the chemical compounds. We investigated the correlation between the concentrationsof four compounds  and environmental factors. Concentrations of two compounds  were correlated with environmental factors. The concentration of 1,5-dicaffeoylquinic acid is positively correlated with latitude, and is negatively correlated with the annual average temperature. The concentration of 3,5-dicaffeoylquinic acid is positively correlated with annual average precipitation. Therefore, changing cultivation conditions may significantly improve the yields of these two compounds. We found the concentration of scutellarin positively correlated with that of erigoster B and 3,5-dicaffeoylquinic acid, respectively. We inferred that the synthesis of these two pairs of compounds may share similar triggering mechanism as they synthesized in a common pathway. Erigeron L. (Asteraceae: Tribe Astereae) includes 390 species worldwide Erigeron are herbs or rarely subshrubs, usually perennial, rarely annual or biennial. Thirty-five Erigeron species are found in China (13 species are endemic to China), most of which are distributed in western China. Erigeron breviscapus (Vant.) Hand.-Mazz. is an important herb in traditional Chinese medicine and has been used to treat cardiovascular and cerebral vessel diseases Erigeron breviscapus is diploid The genus E. breviscapus have been subject of extensive studies for its medicinal value. A series of flavonoids and other phenols from this herb have been proven to be effective in treating cerebral infarction, digestive disorders and heart diseases O-β-D-glucuronide, is one of the major flavonoid glucuronides isolated from this species. This compound can inhibit [3H]-LSD binding to the serotonin-7 receptor (5-HT7 receptor) E. breviscapus, have similar bioactivities with that of scutellarin. They have antioxidant properties, vessel-dilation activity and certain inhibitory effects on adenosine diphosphate (ADP) induced platelet aggregation in rats. Since E. breviscapus has profound medicinal functions, the demand for it has been increased rapidly. Consequently, finding methods to raise its yield is urgently needed, and thus we focused our study on ways to increase the concentration of scutellarin, 1,5-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid and erigoster B in E. breviscapus.The chemical components of Hypericum brasiliense particularly increases the levels of phenolic compounds, and plants that are kept at a constant low or high temperature have higher levels of phenolic compounds. In Hypericum sp. (St. John's wort), higher temperatures can cause higher concentrations of pseudohypericin and hypericin in its shoot tissues E. breviscapus, previous studies have been resolved the relationships between the content of phenols and water stress 2 concentration E. breviscapus. Since E. breviscapus is usually cultivated in outdoor farmlands rather than in greenhouses, investigating the linkage between natural environmental factors and the content of phenols will supply basic information for choosing optimal cultivation area and techiniques. Therefore, we gathered data for environmental factors including annual average temperature, annual average precipitation, annual average hours of sunshine from the collecting sites to detect effects of related factors on the four major compounds. Although E. breviscapus is perennial herb, the above-ground portion dies during autumn, and new shoots germinate in spring. The compounds of this herb are mainly isolated from newly developed shoot, which is primarily affected by environmental factors of the related growing season. We thus only included the annual average value in our analyses. The relationship between amounts of medicinal compounds and the population's geographic distribution was ignored in the previous researches on E. breviscapus. However, this relationship is crucial for choosing optimal cultivation locations, and thus the relationships between the main distribution information  and the four major compounds was also analyzed.These four major chemical compounds are phenols, which are important plant secondary metabolites that have various functions in adapting to both micro- and macro-environments, such as water stress, temperature stress, UV light and disease resistance E. breviscapus. We investigate the relationship between genetic and chemical differentiation of four compounds in order to reveal the potential high yield lineages. Multiple kinds of molecular markers have been applied in similar studies to resolve intraspecific genetic differentiation. Amplified fragment length polymorphisms (AFLPs) is an effective way to investigate population genetic structure and diversity and has become one of the most widely used molecular markers to study genetic differentiation since Vos E. breviscapus, and we used high-performance liquid chromatography (HPLC) to measure the percentage of scutellarin, 1,5-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid and erigoster B in each population. By analyzing relationships among population genetic differentiation, contents of the four chemical compounds and the environmental factors mentioned above, the main purpose of this study was to resolve the influence of biotic and abiotic factors on the concentrations of these compounds and to seek appropriate measures to improve the yields of this crucial medicinal herb.In addition to environmental factors, the composition and content of chemical compounds are closely correlated with the genetic differentiation of the related organism Erigeron breviscapus is not currently a protected species in China, therefore, no specific permission was required to carry out our fieldwork.All specimens were collected in locations for which specific permission was not required. None of these locations were protected areas or private lands. E. breviscapus . For genetic analyses, 15 individuals from each population were sampled, with individuals at least 30 m apart.Twenty-three populations were sampled across the distribution of viscapus . Ten to http://cdc.cma.gov.cn/index.jsp) and local meteorological stations   was used. The column was kept at 30°C and the flow rate was 1 ml/min. The diode array detector was set at 330 nm. Methanol (MeOH), acetonitrile (MeCN) and H2O (0.5% formic acid) were used in the mobile phase. The gradient elution was 0–30 min: 15% MeOH +11.5% MeCN +73.5% H2O (0.5% formic acid); 30–40 min: 15% MeOH +15% MeCN +70% H2O (0.5% formic acid); 40–55 min: 45% MeCN +55% H2O (0.5% formic acid); 55–60 min: 100% MeCN.In order to reduce individual variation, the individual specimens were dried out under the room temperature, and then individuals from each population were pooled together. Each dried sample was ground into powder and weighed (5 g), and extracted with aqueous acetone (50%) in an ultrasonic bath for 40 min. The extracts were purified for HPLC analyses using filters . HPLC analyses were carried out using an Agilent 1200 liquid chromatography system, equipped with a quaternary solvent delivery system, an autosampler and a photodiode array detector . An Agilent ZORBAX SB-CE. breviscapus according to their chemical concentration data indicated in Principal component analysis (PCA) was applied to examine the relative distribution of different populations of Genomic DNA was extracted from silica-gel-dried leaves using the cetyltrimethylammonium bromide (CTAB) method EcoRI and MseI restriction enzymes and buffer 4  at 37°C for 3 h. At 22°C for 3 h, the EcoRI and MseI adapters were ligated to the digested DNA fragments subsequently with T4 DNA ligase . PCR pre-amplification was performed using a total volume of 20 µl reaction mixture containing 5 µl of the ligated product, 1.0 µl MseI primer and 1.0 µl EcoRI primer (75 ng/µl), 2.0 µl 10×PCR buffer , 1.6 µl dNTPs , 0.2 µl r Taq DNA polymerase , 1.6 µl MgCl2  and 7.6 µl double deionized water. The pre-amplification cycles started with 5 min denaturation at 65°C, followed by 30 cycles of 30 s at 94°C, 30 s at 56°C, and 1 min at 72°C, and ended with a final extension step at 72°C for 5 min. The subsequent selective-amplification was conducted using a total volume of 20 µl reaction mixture containing 0.6 µl of the pre-amplified product, 1.0 µl MseI selective primer and 1.0 µl EcoRI selective primer (75 ng/µl), 2.0 µl 10×PCR buffer , 1.6 µl dNTPs , 0.2 µl r Taq DNA polymerase , 1.6 µl MgCl2  and 12 µl double deionized water. The selective-amplification cycles began with 2 min denaturation at 94°C, followed by 13 cycles of 30 s at 94°C, 30 s at 65°C (decreasing by 0.7°C per cycle), and 1 min at 72°C, subsequently accompanied by 23 cycles of 30 s at 94°C, 30 s at 56°C, and 1 min at 72°C, and ended with a final extension step at 72°C for 5 min.The AFLP technique was performed using a slightly modified version of the protocol of Vos et al. EcoRI + ACC/MseI + CAG, EcoRI + ACG/MseI + CAG, EcoRI + ACA/MseI + CAC, labeled with FAM. The fragment analyses were performed with ABI PRISM 3730xl Avant Genetic Analyzer .A total of 23 primer combinations, which were previously used to study taxa in Asteraceae The fragments, 60–450 base pairs (bp) in length, were scored as present (1) or absent (0) in the software Genemapper 4.0 . For all of the primer pairs, the selection of markers for scoring involved two steps. First, the peaks which exceeded 300 relative fluorescent units (RFU) were coded as present, while ones below this criterion were considered as absent. Second, the AFLP binary matrices of these primer pairs were checked for any mistakes or ambiguities by comparing with the original files of graphs, and corrected for further analysis. Poor amplifications occurred only with 4 individuals, two from population B, one from population O, one from population U, and these samples were excluded from the further analysis in order to reduce artificial bias. For further data analyses, the original AFLP data matrix was converted into an input file for ARLEQUIN software using AFLPDAT HT), the genotypic diversity within populations (HS), based on Nei's GST) was calculated as follows: GST = (HT - HS)/HT, in POPGENE ver. 1.31. The effective number of migrants per generation, an indirect estimate of the gene flow between any pair-wise populations, was estimated as: Nm = 0.5 (1 - GST)/GSTTo estimate genetic variation, POPGENE ver. 1.31 The unweighted pair group method with averages (UPGMA) clustering analysis, based on the Nei's unbiased genetic distance FST) matrix was calculated as the other matrix. Then, the relationship between the population genetic pairwise distance (FST) matrix and the population chemical pairwise distance matrix was computed using a Mantel test with 10,000 permutations in ARLEQUIN ver. 3.11. We used the same methods mentioned above to calculate the relationship between chemical pairwise distance and geographic distance . We also tested the relationship between the population genetic pairwise distance (FST) and the geographic distance  using Mantel test.To eliminate the effects of different scales of measurement, the chemical data were standardized using the Standardization Module in NTSYSPC ver. 2.11c software The concentrations of the four chemical components measured by HPLC and the environmental data gathered from meteorological stations and field work were combined together and analyzed by correlation test in XLSTAT ver. 2011 software, in order to detect the correlation among them.The concentration of scutellarin varied from 0.5804% to 2.1946% among 23 populations, erigoster B ranged from 0.0665% to 0.6401%, 3,5-dicaffeoylquinic acid ranged from 0.2726% to 1.5320% and 1,5-dicaffeoylquinic acid varied from 0.0519% to 0.2673% . Based or = 0.068, P = 0.235), which further supported this conclusion.The PCA distribution plot of samples suggested that the chemical characteristics differentiated populations from each other, but no obvious cluster was detected among the 23 populations . InteresHT) was 0.134, while the average Nei's genetic diversity within population (HS) was 0.102. Analysis of Molecular Variance (AMOVA) based on all populations as one group indicated that majority of genetic variation  occurred within populations, while the variation among populations was 23.22% (P = 0.000). The coefficient of gene differentiation (GST) was 0.241, which was similar to the result obtained with the AMOVA. These results indicated more genetic variation was within populations, which suggested the differentiation among populations was not significant.A total of 521 AFLP fragments were obtained for the three primer combinations used, with 504 (PPB = 96.74%) polymorphic bands. With the assumption of Hardy-Weinberg equilibrium, the total Nei's genetic diversity Nm suggested a high level of inter-population gene flow (Nm = 1.575), which means there were 1.575 migrants per generation. According to Wright Nm <1, local populations tend to differentiate, whereas there will be little differentiation among populations and migration is more important than genetic drift, if Nm ≥1. The high value of Nm in this species indicated that gene flow among populations is quite frequent, which caused low intraspecific differentiations.The result of FST) and geographic distance , i.e. the genetically close populations are distributed close to one another.To further detect the population structure and find potential clusters, the UPGMA tree was calculated and the tree with bootstrap values (>50%) is shown in The relationship between genetic differentiation and chemical differentiationFST) and chemical pairwise distance  showed that population genetic differentiation did not correlate with the concentration of the chemical components. However, the molecular markers indicated weak genetic differentiation among E. breviscapus populations. Meanwhile, the PCA plot suggested no obvious clusters based on the four chemical components, which indicated that the chemical differentiation among populations had no clear clustering pattern  diverges from the 4-coumaroyl-CoA. Therefore, according to the positive relationship between the scutellarin and erigoster B as well as 3,5-dicaffeoylquinic acid, we hypothesize that these two pairs of compounds may share the same stimulus-triggering mechanism that enhance the production of the reactions in the shikimate pathway and phenylpropanoid metabolism, i.e. these compounds may have similar ecological adaptation functions in E. breviscapus.Interestingly, the correlation test revealed a positive relationship between two pairs of the four chemical compounds investigated. The concentration of scutellarin was inferred to be positively related to concentrations of erigoster B and 3,5-dicaffeoylquinic acid, respectively. As discussed in the introduction, scutellarin is a flavonoid, whereas erigoster B and 3,5-dicaffeoylquinic acid are part of the conjugated hydroxycinnamate class, and their chemical structures are shown in E. breviscapus were detected, and the genetic differentiation was largely unrelated to the chemical differentiation. It is thus hard to select high quality germplasm resources according to genetic lineage. Appropriate cultivation conditions should be applied to increase the yields of related compounds efficiently. The concentrations of 3,5-dicaffeoylquinic acid and 1,5-dicaffeoylquinic acid could be increased through decreasing the cultivation temperature and increasing the moisture. The climate of high latitude regions can enhance the yield of 1,5-dicaffeoylquinic acid significantly, and thus it can be considered a preferred cultivation areas for E. breviscapus. Furthermore, the positive correlation between the concentration of scutellarin and that of erigoster B as well as 3,5-dicaffeoylquinic acid maybe attributable to the same stimulus that induces the shared parts of their synthesis pathway, whose mechanisms need more thorough examination.In sum, low genetic and successive chemical differentiations among populations of


Dysoxylum malabaricum (white cedar) is an economically important tree species, endemic to the Western Ghats, India, one of the world's eight most important biodiversity hotspots. As D. malabaricum is under pressure of disturbance and fragmentation together with overharvesting, conservation efforts are required in this species. In this study, range-wide genetic structure of twelve D. malabaricum populations was evaluated to assess the impact of human activities on genetic diversity and infer the species’ evolutionary history, using both nuclear and chloroplast (cp) DNA simple sequence repeats (SSR). As genetic diversity and population structure did not differ among seedling, juvenile and adult age classes, reproductive success among the old-growth trees and long distance seed dispersal by hornbills were suggested to contribute to maintain genetic diversity. The fixation index (FIS) was significantly correlated with latitude, with a higher level of inbreeding in the northern populations, possibly reflecting a more severe ecosystem disturbance in those populations. Both nuclear and cpSSRs revealed northern and southern genetic groups with some discordance of their distributions; however, they did not correlate with any of the two geographic gaps known as genetic barriers to animals. Approximate Bayesian computation-based inference from nuclear SSRs suggested that population divergence occurred before the last glacial maximum. Finally we discussed the implications of these results, in particular the presence of a clear pattern of historical genetic subdivision, on conservation policies.The impact of fragmentation by human activities on genetic diversity of forest trees is an important concern in forest conservation, especially in tropical forests.  Forest fragmentation caused by changes in human land use is of primary concern for sustainability and conservation biology in terrestrial ecosystems across the Earth  that runs along the entire west coast of peninsular India Fig. . Togetheus Raman . Althougus Raman , the heaus Raman , and it us Raman , assessmus Raman . As the us Raman , a wide-Unfortunately only a handful of studies have examined genetic variation of plant species in the Western Ghats  is a large canopy tree found in evergreen and semievergreen forests of 200–1200 m altitude in the Western Ghats. It grows to a height of 30–40 m or more, and 3–4 m girth  under the Indian National Threat Assessment using the same criteria as the IUCN . It therefore seems that anthropogenic changes create a cascade of events at the local scale, which in turn could affect wide-scale genetic patterns. In light of this, it is important to evaluate genetic diversity at the species level and the historical connectivity among populations. Regarding the genetic connectivity and forest fragmentation, D. malabaricum is insect pollinated, and seeds are dispersed mainly via the Malabar grey hornbill (Ocyceros griseus) and occasionally by other large birds such as imperial pigeons and wood pigeons , there was a significant increase in relatedness among juveniles within the shortest distance class (<100 m). This was interpreted as the first signs of increased inbreeding due to fragmentation. Although gene flow between neighboring forest patches did occur, and the maximum distance of pollination detected was 23.6 km, it is not known whether this is enough to maintain genetic connectivity and population genetic structure at a larger scale still needs to be analyzed.A study on fine-scale spatial genetic structure (SGS) and paternity analysis by Ismail et al.  in the CD. malabaricum across its distribution range and discuss implications for conservation efforts in Western Ghats. More specifically we (1) compare population genetic structure at biparentally inherited nSSRs (dispersed through pollen and seeds) and maternally inherited cpSSR (dispersed only through seeds), (2) assess the impact of the Palghat and Shencottah gaps on genetic structure, (3) infer population demographic history by approximate Bayesian computation (ABC) approach and finally, (4) based on these results offer some suggestions for conservation and management strategies.In the present study, we used 11 species-specific nuclear SSR  markers  in 6.0 μL mixtures containing 1.2 μL of 1–10 ng of genomic DNA, 3.0 μL of Multiplex PCR master mix buffer, 1.2 μL of H2O, and 0.6 μL of primer mix (with the concentration of each primer pair adjusted to 1–2 μmol/L). Samples were amplified by a DNA thermal cycler  using the following program: initiation of hot-start DNA polymerase and denaturation at 95°C for 15 min; 32 cycles of 95°C for 30 sec, 57°C for 30 sec, and 72°C for 30 sec; and a final 30 min extension step at 72°C. PCR products were loaded on a MegaBACE1000  and genotyped using the fragment profiler v. 1.2 software . In cases where amplification and/or genotyping failed, the procedure was tried one more time to avoid missing data. Three genotype data sets were created; one for all individuals, one for adults only and one for seedlings and juveniles only. These three data sets were analyzed separately in order to allow comparisons between age classes. For this purpose, the data set containing adults was reduced to retain only the populations where juvenile and seedling data was also available.DNA was extracted using a modified CTAB protocol  method, which detects unexpected homozygosity patterns. The genetic diversity parameters within each population were evaluated by determining the gene diversity  using the computer program FSTAT 2.9.3  were adjusted applying a sequential Bonferroni correction ) and the natural logarithms of direct minimum geographic distance among populations. The genetic relationships among populations were evaluated by generating a Neighbor-joining (NJ) tree based on the DA genetic distances , assuming that each cluster is in Hardy–Weinberg and linkage equilibria. Here, K = 1 through K = 15 were investigated under the correlated allele frequencies model by running 100,000 iterations of each K, with a burn-in length of 100,000 iterations, and averaging the results over 20 runs. Data on sampling location was used in the LOCPRIOR function, which can further assist the clustering. To help determine the optimal K, ΔK was calculated as described by Evanno et al. , haplotype richness based on eight haploid individuals .The haplotype frequencies over populations were visualized on a map using the “Pies on map” function in Genetic Studio Dyer . The genRST . Pairwise RST values were calculated in Arlequin 3.1 and IBD was evaluated with the two matrices of RST/(1 − RST) and the natural logarithms of geographical distance in GenAlEx. The standardized values of G’ST were also calculated. An NJ tree based on the (δμ)2 genetic distances  and multidistance class (ω) criteria by the method of Smouse et al.  was assumed at t3 in each scenario. Importantly, the models assume that there is no migration among populations under any of the three scenarios.Recently, ABC has emerged as a powerful and flexible approach to estimate demographic and historical parameters and to quantitatively compare alternative scenarios  and the number of alleles (A) were used as summary statistics for each of the three populations, and for population pairs, FST was also used. One million simulations were performed for each scenario, and the most likely scenario was evaluated by comparing posterior probabilities with the logistic regression method. The goodness-of-fit of the three scenarios were also assessed by a principal component analysis (PCA) using the option “model checking” in DIYABC.As DIYABC requires that one population traces back as the ancestral population, PopA was chosen in all scenarios, although the level of genetic diversity at SSR loci was almost similar in all populations. This arbitrary choice had no impact on the results, as additional analyses using different ancestral populations produced consistent results. The change in number of repeats followed a generalized stepwise mutation model  value did not deviate significantly from zero at any locus in any population. However, significant positive overall values (P < 0.05) were detected in Jadegadde (2), Navangere (4), Sarekoppa (6) and Coorg (8) populations when the overall values were calculated. The FIS was higher in the northern populations and the correlation with latitude was significant (P < 0.05). Genotype disequilibrium was significant at only one out of the 55 locus pairs (P < 0.05). In the BOTTLENECK analysis, a significant HE excess (P < 0.05) was detected in the northernmost populations, Yakambi (1) population under the IAM, while significant HE deficits were detected in Navangere (4) and Sholayar (10) populations under the TPM (P < 0.05).As only a small amount of null alleles was detected and the all loci , we usedall loci . The fixFST and G’ST, were 0.09 and 0.33, respectively. Although IBD was clear ) increased progressively up to K = 8 where it started to plateau  indicating the presence of a phylogeographic structure. The level of population differentiation obtained with G’ST was 0.87, which is higher than the value detected with nSSRs (G’ST = 0.33). Overall, the genetic structure in the cpSSR was clearly different from the one detected in nSSRs, but was in line with the nSSR in that there was no relationship with the two geographical gaps , NB (PopB), NC (PopC), ND (PopD), N1 (Pop1) and the ancestral N2 (Pop2), respectively,  in each population and HE, A and FST for all possible combinations of population pairs did not differ significantly from simulated values based on parameters values drawn from the posterior distributions for scenario 3. In the PCA, the observed data (large yellow dot) is among the values obtained from the posterior distribution (large blue dots), the small dots corresponding to the prior distribution. This indicates a good fit of the posterior distribution based on scenario 3 to the data  Table . The medctively, . The medstimated . The medthe data .D. malabaricum in the Western Ghats using both nuclear and cpSSRs. Firstly, as regeneration was poor, potentially because of overharvesting or newly generated niche competition induced by human land use, we aimed to compare genetic diversity among age classes, expecting lower genetic diversity in younger individuals. However, no difference was found between them. This pattern was also detected in a fine-scale genetic study of D. malabaricum in the southern part of the Western Ghats  are concentrated to the north, whereas most of the least disturbed populations (category A) are found in the south (Table FIS from 0 in this study), a recent study on parentage and kinship analysis of D. malabaricum in a fragmented agro-forestry landscape  and by the observation of a smaller number of adult trees in this population (<20 adult trees) than in other northern marginal populations  .In the present study we investigated genetic diversity and population structure across the range of th Table . Even inThree and two genetic clusters were observed with nSSRs and cpSSR, respectively, but notably, the Palghat and Shenocottah gaps did not appear to be reproductive barriers for either genome. The distributions of the clusters were not congruent, for nuclear and cp markers and an explanation could be that population demographic history differs between the genomes, reflecting differences in inheritance and dispersal modes. The cpSSR revealed a clear geographical gradient in distribution of ordered haplotypes, which is likely informative although only one locus was examined.D. malabaricum. Estimating generation time remains difficult in species with long reproductive spans like forest trees  , in which an admixture-like pattern was detected by the STRUCTURE analysis. However, the ABC analysis suggested that the most likely scenario was a simple population split into four population groups. The divergence time of the four groups was estimated to 1760 generations ago, or 44,000 years ago when we assume a generation time of 25 years for G’ST = 0.33) suggests that there may be some gene flow, although the clear STRUCTURE results suggest that recent gene flow may not be too important. Therefore, estimates of divergence time and effective population sizes will likely be biased downwards and upwards, respectively. However, the main results of pre-LGM divergence would not be changed even when gene flow is taken into account as ignoring gene flow would lead to underestimating the divergence time.The finding that an instant split into four groups fit the data better than a model with three groups, where two of them merged to found a fourth, admixed population is in itself interesting. This suggests that the Agumbe population is a unit of its own, rather than an admixed population. This interpretation is supported by a recent study where ancestral polymorphism was explicitly taken into account in the case of freshwater fish  and its secondary impact (niche competition in the ecosystem) as reflected in the imbalance between age classes are still serious concerns for the long-time survival of the species. Especially, evidence of inbreeding in the northern populations, which are also among the most disturbed ones, and of a recent bottleneck in the Yakambi population, may be the first signals of unsuccessful regeneration due to human activities. Our study hence suggests that conservation priority should be given to these northern populations and it is recommended to start efforts to evaluate regeneration dynamics in this region. This would entail fine-scale analysis of genetic structure among age classes. Our data also suggest the presence of long distance seed dispersal at least within the two groups detected with cpSSR. Given the importance of long-distance dispersal in D. malabaricum, it is desirable to maintain populations of its seed disperser, the Malabar grey hornbill. Luckily, this species has recently been listed by the IUCN as “Least concern” (BirdLife International D. malabaricum.The geographic patterns of genetic diversity of D. malabaricum. Based on the present study, we suggest that D. malabaricum should be managed as four separate units, according to the clusters found with nSSRs. Although the population structure of cpSSR was not congruent with this, priority should be given to the results of the nSSRs for two reasons; the inference is based on a larger number of independent loci, and it revealed more fine-scale diversity which might be lost if the species were to be managed as only two units. Therefore, the minimum ambition should be to conserve one population each from the four clusters as a representative for their respective genetic diversity. The status of the Agumbe population as a separate unit is not fully settled but should it be an admixed population, it still harbors genetic diversity from all the three other known lineages and thus specific conservation efforts would be justified. Although currently large-scale transplantation or restoration of individuals may not be needed in order to mitigate inbreeding over most of the species’ range, the proposed conservation units would be informative for adaptive management of this natural resources. Moreover, it is required to evaluate the ecological dynamics of not only the target species but also the whole ecosystem associated to it and to design a more practical approach of ecosystem management in this biodiversity hotspot.Wide-scale genetic structure studies have provided useful information on conservation units and seed zones and guidelines for restoration or plantation efforts in forest conservation and tree breeding programs of many tree species, especially economically important ones (Bucci and Vendramin DIYABC input file and genotype data have been uploaded to DRYAD doi: 10.5061/dryad.g2b10.Prior distributions of parameters used in DIYABC and distribution of cp haplotypes over the twelve populations and have been uploaded online as supporting information.


The control of bovine tuberculosis (bTB) is at a critical point in the last stage of eradication in livestock. Wildlife species recently have emerged infected with TB in Europe, particularly ungulates in the Iberian Peninsula. Epidemiological information regarding TB in wild ungulates including affected species, prevalence, associated risk factors and appropriate diagnostic methods need to be obtained in these countries.Sus scrofa) red deer (Cervus elaphus), roe deer (Capraelus capraelus), fallow deer (Dama dama), Spanish ibex (Capra pyrenaica hispanica) and mouflon (Ovis musimon), in Spain to assess the seroprevalence against Mycobacterium bovis or cross-reacting members of the Mycobcaterium tuberculosis complex (MTBC), and to provide information on associated risk factors. Previously, two in-house indirect enzyme linked immunosorbent assays (bPPD-ELISA and MPB83-ELISA) were developed using known TB status sera. Positive reference sera were selected from infected animals confirmed by culture. The M. bovis isolates belonged to spoligotypes SB0121, SB0120, SB0295, SB0265 and SB0134. Two hundred and two out of 1367  animals presented antibodies against M. bovis by both bPPD-ELISA and MPB83-ELISA. Significantly higher TB seroprevalence was observed in wild boar compared to the other species analyzed. Interestingly, seropositivity against M. bovis was not found in any out of 460 Spanish ibex analyzed. The logistic regression model for wild boar indicated that the seropositivity to M. bovis was associated with age, location and year of sampling, while the only risk factor associated with M. bovis seroprevalence in red deer and fallow deer was the age. The seroprevalence observed indicates a widespread exposure to MTBC in several wild artiodactyl species in southern Spain, which may have important implications not only for conservation but also for animal and public health.A cross-sectional study was carried out on wild artiodactyl species, including Eurasian wild boar ( Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex (MTBC), is an infectious disease worldwide extended in a large rank of hosts including humans, livestock and wildlife Tuberculosis (TB) caused by Sus scrofa) and red deer (Cervus elaphus), act as true TB wildlife reservoirs in the Mediterranean ecosystem In Spain, the control of the disease in wildlife is a critical point in the last stages of the eradication programs of bTB M. bovis purified protein derivative (bPPD) is the most frequently antigen used for serological diagnosis of TB, different methods using other antigenic proteins, such as MPB83, MPB70, ESAT-6 and CFP10, and combinations of them, have been recently developed. In this sense, multiantigen print immunoassay (MAPIA) and lateral-flow-based rapid test (RT) have been shown as useful diagnosis tools in multiple host species Post-mortem diagnosis of TB in hunter-harvested wild ungulates is usually based on direct methods, including TB-like lesion recording, histopathological analyses and mycobacterial culture M. bovis in wild artiodactyls from southern Spain and (2) to provide information on the risk factors associated with this infection. To achieve this goal, two in-house ELISAs (bPPD-ELISA and MPB83-ELISA) were developed to detect antibodies against M. bovis. The ELISAs were validated with sera obtained from wild ungulate species of known microbiological status (positive controls) and sera from wild ungulate species from TB-free areas (negative controls).The aims of the present study were (1) to analyze seroprevalence against M. bovis isolates were confirmed as spoligotypes SB0121 , SB0120 (2 wild boar and 1 red deer), SB0295 (3 wild boar), SB0265 (1 wild boar) and SB0134 (1 red deer)  were considered positive to TB antibodies . Ninety-ed deer) .M. bovis ranged from 52.3% in wild boar to 0.0% in Spanish ibex. Significantly higher TB seroprevalence was observed in wild boar compared to the other species analyzed (P<0.001) and in fallow deer compared to Spanish ibex (P = 0.016). The logistic regression model indicated that seropositivity to M. bovis in wild boar was associated with age class, location and year of sampling, while the only risk factor associated with M. bovis seroprevalence in both red deer and fallow deer was the age class  and negative (n = 56) control samples, the Se results for bPPD-ELISA and MPB83-ELISA were 72.7% and 100%, respectively, while Sp for both ELISAs was 100%. Using the reference  and field  samples, the Kappa values for agreement between bPPD-ELISA and MPB83-ELISA were 0.788 and 0.697, respectively.Good Kappa values for agreement between ELISAs were obtained using both reference and field samples. Although both ELISAs showed 100% Sp, the Se obtained for bPPD-ELISA was lower (72.7%) compared to MPB83-ELISA (100%). A large number of field sera (58) showed positive results by MPB83-ELISA but negative by bPPD-ELISA, seven of them could be confirmed by mycobacterial culture. The lower Se of the bPPD-ELISA or the early detection of antibodies using the MPB83-ELISA, could explain the differences between tests M. bovis spoligotypes among wildlife and domestic species in southern Spain. SB0121 was the most often spoligotype isolated (67%), which is in accordance with the previously reported in both wildlife and livestock in the Iberian Peninsula P<0.05, Chi square test) in the frequency of spoligotypes isolated were observed between the present study and those previously published Our results confirm the current circulation of the same M. bovis isolation and, therefore, the seroprevalence could have been underestimated, the results obtained confirm that TB infection is common in some wild artiodactyl populations in southern Spain P<0.05, Chi square test) to those reported in countries as France, Italy and Portugal P>0.05, Chi square test). High TB prevalence and increased densities in the lasts decades The frequency of bTB antibodies ranged between 8.0% and 13.3% using the bPPD-ELISA and MPB83-ELISA, respectively. Even though TB seropositivity (7.5%) was determined only from samples positive by both ELISAs and by P<0.05, Chi square test) to those reported in these areas High differences in the prevalence of antibodies against MTBC have been reported in red deer (ranging between 0% and 50%) and fallow deer (ranging between 3% and 27%) among European countries, with the highest levels usually obtained in Spain M. avium complex (MAC). Infections with environmental mycobacteria locally occur in wild ungulates from southern Spain, but at lower prevalence than MTBC infections M. avium paratuberculosis are not common in Iberian red deer One additional point to consider is the possible interference of ELISA results by cross reaction of environmental mycobacteria or of mycobacteria belonging to the M. bovis in this species in the study area. Susceptibility to M. bovis infection was previously shown in domestic sheep from areas with high environmental contamination M. bovis in both domestic sheep and mouflon has been recently reported in Spain Seropositive mouflon were detected in three different regions, which suggest circulation of M. bovis has been widely studied in domestic goats due to their high susceptibility to infection Caprinae. In the present study, no seropositivity against TB was detected in the 460 Spanish ibex analyzed. In the same way, Cubero et al. (2002) Although caprine tuberculosis caused by TB in roe deer has been reported only sporadically in European countries, with prevalences ranging between 0% and 3% M. bovis antibodies. The results were in agreement with those previously reported Odoicoleus virginianus) The higher seroprevalence in adult wild boar, red deer and fallow deer, likely reflects their greater probability of exposure and the lifelong persistence of The risk of being a seropositive wild boar significantly increased in the provinces of Córdoba and Seville, which is in accordance with the higher geographical distribution of bTB in cattle M. bovis is widespread among wild artiodactyl populations in Andalusia, especially in the wild boar. The results suggest that different species could act in the maintenance and inter-specific transmission of TB in the study area, and should, therefore, be included in eradication programs in Spain.In conclusion, the results obtained using reference sera showed that the ELISA tests are potentially useful for the diagnosis and large scale monitoring of TB infection in wild artiodactyls This study did not involve purposeful killing of animals. Samples from dead animals were collected from individuals legally hunted during the hunting seasons. No ethical approval was deemed necessary. Blood samples from captive Spanish ibexes were collected in compliance with the Ethical Principles in Animal Research in the Wildlife Breeding Centers, which depend on the Autonomous Government of Andalusia. The collection of this material did not require the approval of an ethics committee because are considered among routine procedures and the samples were collected and stored before the design of this study. Thus, protocols, amendments and other resources were done according to the guidelines approved by the Autonomous government of Andalusia.g for 15 minutes. Serum or fluid exudate samples were stored at −20°C until analysis.A total of 1367 samples from wild ungulates were collected in 46 hunting estates from Andalusia, , southern Spain, between 2006 and 2010 . Blood oAge was determined by tooth examination in wild boar, red deer, fallow deer and mouflon, and by horn segment counts in Spanish ibex M. bovis spoligotypes were identified using the standardized membrane with 43 spacers as previously described M. bovis.A total of 26 animals  which presented gross lesions compatible with bTB were tested by mycobacterial isolation. Selected animals were sampled in two different TB endemic areas of the Provinces of Córdoba and Jaén   during tM. bovis has been isolated during the last decade) in different regions of Spain. Negative reference sera were collected in previous studies by Dr. Gortázar The negative reference sera (n = 56) used in the study were obtained from 15 red deer, 15 fallow deer, 11 Spanish ibex and 15 wild boar from TB-free areas (areas where no M. bovis by using two in-house enzyme linked assays (ELISA) using the protocol previously described Serum samples were tested for the presence of antibodies against In addition, a total of 1147 sera, including all positive bPPD-ELISA and 1038 negative bPPD-ELISA sera were tested by MPB83-ELISA. Unfortunately, 220 of the samples seronegative to bPPD-ELISA could not be tested using MPB83-ELISA due to limited sample volume. The antigen MPB83  was diluted in carbonate/bicarbonate at a final concentration of 0.5 µg/ml and was coated in Nunc-Maxisorp™ 96-well plates . Antigen-coated plates were incubated overnight at 4°C. The plate blockade as well as sera and conjugated-proteins dilutions were performed as described above for bPPD-ELISA.2SO4. The Optical Density at 450 nm (OD450) was then read in a plate-spectrophotometer. For each sample, the ΔOD450 was calculated as sample OD450 minus background OD450 (unspecific absorbance in wells where antigen had not been added). A sample was classified as positive when the ΔOD450 was higher than the cut-off point, calculated as the mean of background OD450+3×standard deviation. In the present study, positive samples to one of the ELISAs were considered undetermined samples, and were included as negative sera to determine the seroprevalence to bTB. Therefore, a serum sample was considered seropositive to M. bovis when showed positive results to both ELISAs or presented lesions that were culture-positive to M. bovis.The reaction was revealed with soluble TMB  and stopped 20 minutes later with 0.5 M HRelative sensitivity (Se) and specificity (Sp) values were calculated for bPPD-ELISA and MPB83-ELISA from the reference sera. The optimal cut-off that resulted in the maximum sum of the Se and Sp values were determined by receiver operating characteristic (ROC) analysis. The Kappa value for the agreement between ELISAs was calculated using the procedure described by Kraemer (1982) vs. female), years of sampling , and locations  with serological results were analyzed using Fisher's exact test. Factors showing a P-value<0.25 were further scrutinized for associations using Pearson correlation coefficient (r) to avoid collinearity problems. Finally, a multiple logistic-regression model was performed as described by Hosmer and Lemeshow (1989) P<0.05). The statistical analyses were performed using SPSS v15.0 software .Differences in ELISA performance between species  were tested using the Post-hoc Tukey test. Due to the absence of seropositivity in Spanish ibex or to the limited number of roe deer and mouflon tested, the associated risk factors were not analyzed for these species. In order to avoid potential bias associated to co-linearity, wild boar and cervids  were tested separately. Associations between independent variables such as age classes , sexes (male


Plants do it. Animals do it. People do it, too. During meiosis—the cell division that creates eggs and sperm—all these organisms mix the genetic material inherited from their mother and father. This mixing is called recombination, and it results from crossover events, where pairs of chromosomes swap genetic material. Evolutionary biologists have long known that recombination seems to increase genetic diversity. That is, high rates of recombination in a species are correlated with high rates of genetic diversity between individuals in that species.how recombination leads to increased diversity. Do crossover events predominantly cause mutations, or is their primary effect to break up linkage between genes? Genes that are linked—near each other on the same chromosome—are more likely to be inherited together. By breaking up linkage, recombination makes it easier for natural selection to target individual genes while avoiding the potentially disadvantageous effect of simultaneously reducing diversity at neighboring genes (a phenomenon known as “Hill-Robertson interference”).What has been less clear is between species—at those points. They found no increase in divergence, leading to the conclusion that the association between diversity and recombination was not predominantly caused by mutations. However, these analyses suffered from the implicit assumption that recombination rates were the same in related species. Recently, a number of studies have found that this is not always the case; even in closely related species, local recombination rates can vary greatly, raising questions about work that has not taken this variability into account and leaving room for the mutation hypothesis to resurface.To assess whether recombination causes mutations, researchers have previously looked at recombination rates at certain points on the same chromosome of closely related species and assessed whether there was increased genetic divergence—genetic variation PLOS Biology, Suzanne McGaugh, Mohamed Noor, and colleagues at Duke University and the University of Wisconsin-Madison settle the question, at least for fruit flies. Using relatives of the well-known Drosophila melanogaster, the researchers compared the local genetic variation within and between species  at regions of the flies' chromosomes that they knew to have similar recombination rates.In this issue of D. pseudoobscura and D. miranda, local recombination rates vary throughout the genome. For this experiment, McGaugh and colleagues created fine-scale maps of recombination rates for both species, covering 43 percent of the D. pseudoobscura genome, and 31 percent of D. miranda. Using these maps, they located regions of the genomes that showed similar rates of recombination over several generations. To determine whether recombination was associated with genetic divergence between the two species, as would be expected if mutation were playing a role in increasing genetic diversity near recombination sites, they modeled the relationship between recombination and divergence. They also modeled the relationship between local recombination in D. pseudoobscura and local increases in genetic diversity (within-species variation).In both divergence. However, within the D. pseudoobscura genome, the local rate of recombination was correlated with overall genetic diversity. They also documented local effects that appear to be due to natural selection: in the immediate vicinity of non-synonymous substitutions—mutations that alter the amino acid sequence of a protein—they documented “hitchhiking” of non-coding genetic diversity, which would be expected if evolutionary selective sweeps had fixed the primary adaptive mutation.The team's findings were unambiguous: the rate of recombination in a region had no relationship to the genetic In addition, the team made ultrafine-scale maps of a subset of the regions covered by the fine-scale maps. These provided a more detailed view of recombination rates in the two species than had been seen before. Using the ultrafine-scale maps, the researchers saw even greater local variations in recombination rates than showed up on the fine-scale maps. For example, in one region, where the fine-scale map had shown a range of 4.4–5.6 cM/Mb (centimorgans per megabase), the ultrafine-scale version revealed a much wider range of 3.5–21.1 cM/Mb. They conclude that recombination rates in different regions of a genome may vary considerably more than previously realized, and that recombination rates measured using fine-scale maps are likely crude averages of more wildly varying rates over even smaller regions.Drosophila lack several proteins normally considered crucial to meiotic crossover and DNA double-strand break repair in other organisms.The researchers note that there is much left to learn about how recombination rates vary at extremely fine scales within species, and also how recombination patterns vary between species and taxa. They note that further study is needed into the mechanisms that determine the distribution of recombination events, especially as McGaugh SE, Heil CSS, Manzano-Winkler B, Loewe L, Goldstein S, et al. (2012) Recombination Modulates How Selection Affects Linked Sites in Drosophila. doi:10.1371/journal.pbio.1001422


COI gene, we investigated the balance between mutation and migration as drivers of genetic diversity in two Branchipodopsis fairy shrimp species  endemic to remote temporary rock pool clusters at the summit of isolated mountaintops in central South Africa. We showed that both species colonized the region almost simultaneously c. 0.8 My ago, but exhibit contrasting patterns of regional genetic diversity and demographic history. The haplotype network of the common B. cf. wolfi showed clear evidence of 11 long distance dispersal events (up to 140 km) with five haplotypes that are shared among distant inselbergs, as well as some more spatially isolated derivates. Similar patterns were not observed for B. drakensbergensis presumably since this rarer species experienced a genetic bottleneck. We conclude that the observed genetic patterns reflect rare historic colonization events rather than frequent ongoing gene flow. Moreover, the high regional haplotype diversity combined with a high degree of haplotype endemicity indicates that evolutionary- (mutation) and ecological (migration) processes in this system operate on similar time scales.Recent findings suggest a convergence of time scales between ecological and evolutionary processes which is usually explained in terms of rapid micro evolution resulting in evolution on ecological time scales. A similar convergence, however, can also emerge when slow ecological processes take place on evolutionary time scales. A good example of such a slow ecological process is the colonization of remote aquatic habitats by passively dispersed zooplankton. Using variation at the protein coding mitochondrial  Inselbergs are isolated rocky outcrops rising abruptly from the landscape. Often occuring as monoliths, they are characterized by contrasting and often more extreme environmental conditions compared to the surrounding plains Branchipodopsis Sars 1998 (Crustacea: Anostraca) are typical inhabitants of rock pool habitats on the top of inselbergs In Southern Africa, fairy shrimp of the genus Recent studies suggest a convergence of time scales between ecological and evolutionary processes which is usually explained in terms of rapid micro evolution resulting in evolution on ecological time scales COI or cox1) to investigate the frequency of LDD in two fairy shrimp: the locally common Branchipodopsis cf. wolfi Daday, 1910 and the rarer Branchipodopsis drakensbergensis Hamer and Appleton, 1996; both of which are endemic to remote rock pool habitats in central South Africa and often occur as mixed populations. Since both species are absent from aquatic habitats in the surrounding lowland areas, colonization and exchange of individuals between inselbergs, which are usually separated by tens of kilometers, implies LDD.Here we use partial sequences of the mitochondrial protein coding cytochrome oxidase subunit 1 gene . B. cf. wolfi represents a taxon which is morphologically very similar to the B. wolfi a widespread species in Southern Africa COI gene from confirmed B. wolfi populations from Botswana (Genbank Accession No. GU171358). Based on accepted molecular thresholds indicative of species level differentiation in fairy shrimp at the COI gene (10–6%) this taxon should probably be considered a separate species awaiting formal description Xenopus laevis), water flow through eroded channels and wind Although conclusive evidence is currently unavailable, preliminary investigations including morphological analyses and analyses of species distributions along main environmental gradients revealed no indications for environmental or temporal niche segregation Based on the current distribution of genealogical lineages, topology of haplotype networks, partitioning of genetic variation and isolation by distance analyses, we discuss the demographic history of the species in the area and the frequency of dispersal at different spatial scales. Finally, to assess how the time scale of a slow ecological process such as LDD measures up to the time scale of an evolutionary process such as mutation, we compare mutation - and migration rates.We specifically test the following hypotheses:Since previous work demonstrated frequent passive dispersal and gene flow within pool clusters We anticipate similar rates of LDD for both species, given that the eggs are morphologically similar and are likely to share the same dispersal vectors.Since avian vectors are scarce in this area, if wind dispersal is important, LDD events could be constrained by the dominant wind direction.Due to the isolated nature of inselberg habitats we expect that migration rates among inselbergs will be very low, possibly of the same order of magnitude as mutation rates in the studied gene, illustrating a convergence of the time scales on which these two main drivers of  genetic diversity operate.All samples were collected on private land with explicit permission of the respective landowners. Collection and export permits were granted by the Free State Province Department of Tourism, Environmental and Economic affairs: permit no.: HK/P1/07375/001. Collected animals were anaesthetized in carbonized water before transfer to ethanol.2) and a southeastern (31.7 km2) which can be considered separate inselbergs. While most inselbergs in the region only house a small number of pools near the summit (0–10), the northwestern plateau of the Korannaberg (Korannaberg North) is the only inselberg that boasts several spatially isolated clusters of up to 36 pools. Despite the much larger area, rock pools are very rare on the southeastern plateau (Korannaberg South) probably due to the scarcity of large exposed sandstone rock ledges.The landscape in the east of South Africa's Free State province is well known for the presence of inselbergs rising up from the wide open grasslands . These sth until August 14th 2008 in the dry season. Populations were sampled at three spatially hierarchical scales: (a) populations on different inselbergs (b) populations from different pool clusters on the same inselberg and (c) populations within a single pool cluster.Fairy shrimp resting eggs were collected during a field survey that took place from July 27In order to investigate genetic variation among inselbergs, two populations were sampled on each inselberg. To investigate variation among pool clusters, four pool clusters present on Korannaberg North were used since this is the only inselberg that harbors multiple pool clusters. Here, again, two populations were sampled in each cluster. Finally, to assess within-cluster variation, nine populations were sampled in one specific cluster (cluster A on Korannaberg North). This pool cluster is the largest in the region containing 36 large pool basins and comprises the deepest and presumably oldest pools Branchipodopsis resting eggs were isolated from the sediment under a stereomicroscope and stored in 100% ethanol. Genomic DNA was extracted for multiple individuals from each population making use of the HotSHOT protocol Dry sediment containing resting eggs was collected from pool basins using a shovel and a fine brush and transferred to plastic ziplock bags. c oxidase subunit I (COI gene) was sequenced for up to 16 individuals per population using polymerase chain reaction (PCR). However, due to occasional low densities of viable eggs in pool sediment and failed PCR reactions the number of individuals included in our analyses was lower for some populations  and reverse primer BranCOIR1: (5′-AATCAGAAGAGGTGTTGATAGAGG-3′) proved to be most effective amplifying a fragment of 485 bp (excluding primers). Sequences were obtained making use of the ABI Prism Big Dye Terminator sequencing protocol and the ABI Prism BigDye Terminator Cycle Sequencing Reaction Kit (PE Applied Biosystems). PCR fragments were sequenced with the above-mentioned forward and reverse primers. Sequences were aligned and trimmed in BioEDIT A fragment of the protein-coding mitochondrial cytochrome ulations . PCR cycIn order to remove anomalies and export consensus sequences based on the two complementary electropherograms obtained for each individual, SeqScape© software v. 2.5.0 (Applied Biosystems) was used. Haplotype networks for each of the two species were created in Network 4.0 In order to visualize the degree of haplotype endemicity and haplotype sharing among sites we used pie charts to plot the distribution of different haplotypes on a map of the study area. Both at the level of pools, pool clusters and inselbergs, haplotype endemicity was calculated as the percentage of haplotypes unique to that specific locality.Given the fact that dispersal rates are low enough, information about the route and the direction of historic long distance dispersal events can be derived from two sources: network topology and patterns of haplotype sharing. In star-shaped networks with one clear central haplotype linked to a specific locality, this can be considered the source of the initial colonization. In contrast, in the case of haplotype sharing, which reflects more recent migrations, directionality can be deduced when haplotypes in the source habitat are more closely related to the shared haplotype than the destination. If this difference was not clear, directionality could not be determined.B. cf wolfi seems entirely restricted to the sampled range, and that it is unlikely that B. drakensbergensis has colonized the Korannaberg multiple times from independent sources  and nucleotide diversity (π) were calculated in Arlequin 3.5.1.2 for each pool, each pool cluster and each inselberg. Since dominant winds consistently blow from the north and to a lesser extent from the northwest http://R-project.org). Independent variables in these analyses included altitude (m above sea level), longitude and latitude , habitat size (pool surface area in m2), habitat duration (basin depth in cm). In rock pools habitat duration is primarily determined by depth of the pool basin hence we can use this variable as a proxy Analysis of molecular variance (AMOVA) For analyses using inselbergs as units, the frequencies of haplotypes found on each inselberg were used to calculate indices. A similar procedure was followed for analyses using pool clusters unites. Analyses using pool clusters as independent units were only performed for Korannaberg North since this is the only inselberg which harbors multiple pool clusters. Analyses at the level of individual populations were performed for a reduced dataset containing only the populations in the intensively sampled pool cluster A on Korannaberg North.MIsolation by distance (IBD) analyzes were performed using the web application IBDW From the haplotype networks, it is possible to count both the total number of mutation events (Mu) as well as the number of haplotypes (HR) that originated through these mutation events. Haplotype networks, however, also typically contain missing haplotypes. Assuming that new haplotypes are generated solely in a single step fashion, these virtual haplotypes represent those that disappeared in the course of time as a result of genetic drift. As a result, comparing the total number of mutation events (Mu) to the number of haplotypes (HR) provides a means to assess the relative role of genetic drift and the ratio HR/Mu can be used as a measure of the effect of genetic drift on the total remaining diversity since the onset of diversification.r) can be assessed by counting the number of haplotypes shared among inselbergs added to the number of haplotypes derived from haplotypes that occur on other inselbergs. Due to genetic drift, however, an unknown number of historical migration events between inselbergs remain unnoticed. Yet, because genetic drift has the same effect on genetic diversity irrespective of how the diversity originated (mutation or migration), the number of total expected migration events (me) in the course of history calculated, based on the number of detected migrations corrected for those lost by drift using the equation me = mr × (Mu/Hr). This value of me can then be used to estimate the rate of migration over time, if the start of the genetic diversification can be dated using a molecular clock.Additionally, for each species, the total number of migration events  were shared among distant inselbergs indicative of long distance dispersal. Two  were shared among two pool clusters while one  was shared among three pool clusters on Korannaberg North. Three abundant haplotypes were shared among respectively, eight , and five  of the populations sampled in cluster A on Korannaberg North. 76% of the haplotypes were exclusive to specific inselbergs, 67% were specific to pool clusters while 52% were restricted to a single population , no haplotypes were shared among inselbergs. Two haplotypes  were shared among two pool clusters as well as among two populations in cluster A on Korannaberg North. 80% were exclusive to pool clusters while 70% of the haplotypes were population specific . For B. drakensbergensis Fu's Fs indicated a marginally significant demographic contraction . Using the most abundant haplotype , whereas the average age between all haplotypes was estimated at 308 ky (440–237 ky). For B. drakensbergensis a virtual root of the network was taken as half the distance between the two most distant haplotypes as we could not identify a clear root to the network. This dated the origin of the diversification within B. drakensbergensis at 876 ky B.P. (1 252–674 ky B.P.). The average age between all haplotypes was estimated at 800 ky (1 143–615 ky).The test of population expansion or contraction (Fu's Fs) was inconclusive for B. drakensbergensis, and limited number of populations (n = 6) resulting in limited statistical power, the remaining analyses are only presented for B. cf. wolfi. The AMOVA results indicated that the genetic variation was dominantly structured among inselbergs (43%) and, unexpectedly, among populations (42%) while 15.2% of the remaining variation was distributed among pool clusters. No significant relations between habitat characteristics and any of the calculated diversity indices  could be confirmed at the level of individual inselbergs, pool clusters or populations within a pool cluster. No significant isolation by distance patterns were detected among pool clusters on the same inselberg  or among pools in the same pool cluster . Among inselbergs the correlation between geographic distance (LOG transformed) and genetic similarity could be considered marginally significant .Due to the restricted geographic distribution (two inselbergs) of B. cf. wolfi with a haplotype richness of 21, the number of mutational steps (Mu) in the network amounted to 36, resulting in a Hr/Mu ratio of 0.58 indicating that 58% of the genetic diversity was lost due to genetic drift. A total of 11 residual migration events was detected, yielding a total expected historical number of migration events of 19. Given that the onset of diversification was estimated at 826 ky B.P., this gives an average long-distance among inselbergs rate of migration of one individual every 43.5 ky.For B. drakensbergensis, a haplotype richness of 10 and 39 mutational steps, results in a Hr/Mu ratio of 0.256. A single residual migration event among inselbergs was detected, yielding an estimated grand total of one migrant every 225 ky, on average. Mutation rates for the sequenced gene fragment according to molecular clocks, in turn, range from 0.7 to 1.3 mutations per ky or one mutation every 80–150 ky.For B. drakensbergensis, was represented by a small number of distantly related lineages surviving in relict populations in a restricted range. The common B. cf. wolfi, on the other hand, exhibited higher haplotype diversity, a more stable demographic history and a wider geographic distribution.In this study, the genetic structure of two fairy shrimp species endemic to isolated rock pool clusters at the top of inselbergs in central South Africa was investigated. The rare Branchinecta coloradensis, which inhabits pools in the Rocky Mountains (USA), for instance, high levels of allozymic differentiation were observed between populations in different valleys (5–10 km apart) while on local scales (0–100 m) populations were genetically similar Branchipodopsis wolfiCurrent results based on mitochondrial sequence data suggest that, for rock pool inhabiting fairy shrimp, most genetic variation seems to be partitioned at the regional scale between inselbergs and to a lesser extent between different pool clusters. To some extent, this is consistent with conclusions of earlier studies describing allozymic variation The high levels of genetic diversity and the presence of many unique haplotypes linked to specific localities, found on the limited geographic scale of this study system is remarkable when compared to other species of large branchiopods in temperate and Mediterranean regions B. cf. wolfi, the abundance of the two most common haplotypes , whereas B. drakensbergensis most likely experienced a demographic bottleneck. Although we cannot provide conclusive answers at this point, differences in niche requirements, dispersal capacity, or a competitive disadvantage could explain why, from the two species, only B. drakensbergensis experienced a bottleneck and why this species is currently only found on a subset of the inselbergs and in a subset of the rock pools inhabited by B. cf. wolfi. Differences in dispersal capacity, however, do not provide a satisfactory explanation since the dormant eggs, which represent the dispersing life stages, are apparently identical in both species Although the diversification in both species in the area started nearly simultaneously around 800 ky B.P., both the topology of the haplotype networks as well as tests of population expansion and contraction suggest that the species have a different demographic history. The demographic history of Bostrychia hagedash) no other birds have been observed visiting rock pools in the area. On the other hand, the area is characterized by very strong winds which were shown to facilitate short distance dispersal within pool clusters, we considered the possibility of long distance dispersal mediated by wind B. cf. wolfi). Genetic diversity is therefore more a function of population size and the associated rate of genetic drift, rather than of the rate of immigration. This result, however, does not rule out that some of the LDD events detected in this study could be mediated by wind, since these events are often associated with extreme events such as storms and whirlwinds rather than standard conditions Being small and inconspicuous rock pool habitats are not very attractive to bird life, compared to larger wetlands and lakes. With exception of some small song birds and a single observation of a visiting ibis  analyses. No significant IBD could be demonstrated in this study. Only at the largest (between inselberg) scale a marginally significant trend points to the possible presence of IBD. Absence of IBD is usually explained in terms of frequent dispersal or strong founder effects B. cf. wolfi, and nearly five times smaller in B. drakensbergensis. These calculated migration rates are relatively coarse estimates, but at the very least they indicate that effective long distance migration events are extremely rare. Initially, we expected similar migration rates for both species, given that the eggs are likely to share dispersal vectors. The much lower estimates for B. drakensbergensis may be due to the fact that it underwent a demographic contraction and significantly departed from mutation-drift equilibrium, and because the species is so rare in the region. Our data thus suggest that occasional haplotype sharing results from very rare effective dispersal events of a stochastic nature. These findings are relevant in the light of the evolving metacommunities concept Based on our estimates of the rate of effective dispersal over time, it appears that migration rates were of the same order of magnitude as mutation rates in Table S1Overview of the number of haplotypes (No. H) and percentage of exclusive haplotypes  present at the level of inselbergs, pool clusters and populations.(DOCX)Click here for additional data file.