Supportive breeding is one of the last resort conservation strategies to avoid species extinction. Management of captive populations is challenging because several harmful genetic processes need to be avoided. Several recommendations have been proposed to limit these deleterious effects, but empirical assessments of these strategies remain scarce. We investigated the outcome of a genetic management in a supportive breeding for the Houbara Bustard. At the phenotypic level, we found an increase over generations in the mean values of gamete production, body mass and courtship display rate. Using an animal model, we found that phenotypic changes reflected genetic changes as evidenced by an increase in breeding values for all traits. These changes resulted from selection acting on gamete production and to a lesser extent on courtship display. Selection decreased over years for female gametes, emphasizing the effort of managers to increase the contribution of poor breeders to offspring recruited in the captive breeding. Our results shed light on very fast genetic changes in an exemplary captive programme that follows worldwide used recommendations and emphasizes the need of more empirical evidence of the effects of genetic guidelines on the prevention of genetic changes in supportive breeding. Among these ex-situ programmes, supportive breeding is used when the species habitat is still available, but wild populations cannot sustain themselves (e.g. because of overexploitation). The goal of supportive breeding programmes is therefore to increase the effective size of wild populations through release of captive-born individuals , so that testing for trends in these genetic values will inform us on potential genetic changes. New methods even allow assessing the strength of these trends compared with expectations based on drift only   to attract females. Males provide females with nothing, but genes through the semen. Females generally lay clutches from one to four eggs.n = 296) were transferred in Morocco to the Emirates Center for Wildlife Conservation in 1996. Two campaigns for egg collection were conducted in Morocco in 1996–1997 and between 2002 and 2008. In total, 564 chicks from wild-laid eggs were integrated to the captive breeding by 2009 . Food and water were provided daily Courtship display, ejaculate size, number of eggs laid and body mass per year were analysed to assess change in breeding values over time. Measurements of these traits were available for thousands of birds with a known pedigree that reached 74 528 individuals in 2011. Full statistics on pruned pedigrees used in analyses are given in lmer function: body mass ∼ day + day2 + 1|bird identity + 1|year, with day 1 = 1 January).Body mass (±1 g) was measured in both males and females several times per year. Because body mass shows some within-year variation  as described by Lande and Arnold . Becauselm function) because estimates are not affected by the non-normality of data (Lande and Arnold glm function). Age and a squared age were set as fixed factors in the models.We first estimated selection differentials for each year using linear regressions with a normal distribution of errors  than according to generations. Therefore, to assess for temporal trends in selection, we used the meta-analysis implemented in milar to  but inclMCMCglmm package).Phenotypic trends were assessed with a mixed model for each separate trait. Because trait values change with age, we included age and age² effects. The trend was assessed across generation, not years, so that generation was included as a continuous variable and year as a random effect. To account for repeated measurements, we also included individual identity as a random effect. A normal distribution was assumed for body mass and a Poisson distribution for number of eggs, display rate and ejaculate size  and X the design matrix relating phenotypic observations to the vector of fixed effects. For the random effects, a is the vector of additive genetic values, pe the vector of permanent environment effects, and yr the vector of year of measurement effect, with Za, Zpe and Zyr their respective incidence matrices. All random effects are assumed to be normally distributed, and elements of a are assumed to be drawn from where The animal models were run using the Bayesian method . The slope of the regression between average breeding values against generation is stored at each iteration for both actual (reg1) and simulated (reg2) breeding values. The slope of the genetic trend in the captive population is the posterior mode of reg1. The significance of the trend is based on the number of times the trend is superior or inferior to 0, depending on the sign of the posterior mode. The comparison of the genetic trend to expected trend under drift is simply the number of times the trend from reg1 is superior to the trend from reg2.In contrast to traditional REML methods, using regression of breeding values based on posterior distribution allows a conservative estimate of evolutionary trend  for each analysis: (i) a parameter-expanded prior , and (ii) a slightly informative prior , where Vp is the phenotypic variance and r the number of random factors. Note that the prior for VR is  in both cases. Our results were not sensitive to the choice of priors , but not in males , leading to a very large captive population of about 10 000 adult breeders , reared and bred individually for 15 years (∼5 generations) so that impressive pedigree records and precise phenotypic dataset were available. The aim of the Houbara breeding management was fourfold: (i) avoiding inbreeding, (ii) equalizing the representation of each founder line by forming pairs based on their mean kinship, (iii) maintaining high genetic diversity by regularly integrating new founders, from wild-laid eggs, to the captive broodstock and (iv) limiting best breeders to be over-represented in the captive population by preferentially releasing in the wild offspring sired by the most represented breeders (i.e. the genetic dumping strategy).We show here, using recently implemented quantitative genetics tools, evidence that genetic changes can occur despite these strict guidelines, although consequences do not lead inevitably to lower fitness in the Houbara Bustard.We found that global selection gradients were higher for ejaculate size and number of eggs  than for courtship display (0.16) and body mass . Statistically significant selection coefficients of reproductive traits indicate that the best captive breeders did contribute more to the number of recruited offspring, despite the effort produced to equalize the representation of each founder in the captive population.The difficulty to limit contribution of prolific breeders might stem from the so-called growth phase that represents the early stage of captive breeding during which managers have to increase captive population size to rapidly set up a sizeable flock of adult breeders. During this period, demographic goals might have prevailed over genetic ones. In line with this, there is a strong disequilibrium in founder representation, with only 19% of founders representing up to 83.5% of the genetic variability of the captive population in 2007 Lesobre .The difficulty to limit contribution of prolific breeders to the next generation is probably very common in supportive breeding for species with strong reproductive skew. The situation seems difficult to solve because poor breeders will never achieve a great contribution to the next generation. Surprisingly, despite similar issues may be very common in many captive breeding, empirical evidence of such phenomenon is very hard to find in the literature.One solution to improve the contribution of poor breeders from rare genetic lines could be to reduce the growth speed of captive populations to enable managers to better control the contribution of different individuals to the next generation. However, the implementation of such strategy is challenging as threatened species usually suffer from a fast decline in the wild, which may preclude any attempt to decrease the speed of the population's growth in captivity. Breeding rapidly numerous individuals is required to prevent wild-supplemented populations from losing genetic diversity or even from extinction  transferred to the captive breeding will thus be classified as founders (G0). Even under the hypothesis of a massive-biased reinforcement of the wild population, this should result in a smaller difference in life-history traits between the generations (e.g. G0 and G4). However, the speed of these changes could be explained by an acceleration of the response to selection due to genetic correlations. Indeed, all genetic correlations among these traits are positive , suggesting that selection was not relaxed on males even after 13 years of breeding. This could be due to a significantly higher reproductive pressure exerted on males compared with females. As one male's semen can be used to inseminate several females, a male producing large ejaculate was likely to sire multiple offspring from several different females, while poor breeders only produce a sufficient amount of sperm to inseminate one female.Responses to directional selection raise questions about the maintenance of genetic diversity in the captive broodstock. We could expect a depletion of genetic diversity under these conditions. Contrary to the expectations, initial genetic diversity in the ECWP's captive population has been maintained up to 98% in 2007, mainly thanks to the regular addition of new founders and the large effective population size Lesobre .In the Houbara Bustard, we found genetic changes in several life-history traits. However, we would like to discuss the possibility that the consequences of these genetic changes are not necessarily harmful at this stage. Our results show that males with large ejaculate size and high courtship display and more fecund females have been favoured in captivity. In the wild, the species is expected to be under strong sexual selection pressures because of its lek-based mating system  after 50 generations, regardless of initial population size, when the populations were moved to ‘wild’ conditions  decreased reproductive success in the wild by 55% between wild-born offspring sired by wild-born parents and a first generation issued from captive-born parent once released in the wild. Another example suggesting a lower fitness in captive-bred individuals in the wild comes from Heath et al. (Oncorhynchus tshawytscha), unintentional selection for female fecundity resulted in smaller eggs size, which was known to reduce early survival. Heavily supplemented wild populations with captive-born salmons also had reduced egg size, which raises serious concerns about the success of captive breeding and supplementation programmes. It is worthwhile to note that all these studies have been conducted on populations that were not under a strict genetic management, contrary to the Houbara's supportive breeding. To the best of our knowledge, we were not aware of similar studies that were investigating the fitness of released captive-born individuals from supportive breeding programmes following worldwide genetic recommendations, such as the equalization of founder genetic lines.In contrast to the situation in the Houbara captive breeding, a study conducted in h et al.  showing Based on these previous examples, we might expect a fitness reduction in released captive-born Houbaras due to the response to selection in life-history traits. We found in previous experimental studies that more fertile and ‘sexy’ captive males that were able to maintain their courtship activity and sperm quality following an immune challenge sired offspring with a better 1-year survival once released in the wild compared with low-quality males , the concerns could be even more important than in the Houbara programme because the excessively large Houbara's captive population may have facilitated relaxation of selection.To conclude, our results address the question of the success of recognized guidelines for genetic management of captive populations to prevent genetic changes. The answer seems not so straightforward, and definitively, more empirical studies are needed to provide managers with appropriate strategies to preserve, in both captive and supplemented populations, genetic diversity but also genetic quality, as suggested by some authors Wedekind .


G. maculatus  along two biogeographical provinces in the Chilean coast. Extreme levels of genetic diversity and strong phylogeographic structure characterize the species suggesting a low amount of influence of the last glacial cycle over its demography. However, we recognized contrasting patterns of genetic diversity and structure between main biogeographical areas here analyzed. Along the Intermediate Area (38°–41° S) each estuarine population constitutes a different unit. In contrast, Magellanic populations (43°–53° S) exhibited low levels of genetic differentiation. Contrasting patterns of genetic diversity and structure recorded in the species between the analyzed biogeographic areas are consistent with the marked differences in abiotic factors  and to inherent characteristics of the species .Major geologic and climatic changes during the Quaternary exerted a major role in shaping past and contemporary distribution of genetic diversity and structure of aquatic organisms in southern South America. In fact, the northern glacial limit along the Pacific coast, an area of major environmental changes in terms of topography, currents, and water salinity, represents a major biogeographic transition for marine and freshwater species. We used mitochondrial DNA sequences (D-loop) to investigate the consequences of Quaternary glacial cycles over the pattern of genetic diversity and structure of  Until t,000 km3 ,16–17. H,000 km3 . In fact,000 km3 ,21.According to Zemlak , postglaGalaxias maculatus is one of the most widely distributed freshwater fish in the planet and exhibits a Gondwanic distribution. Populations of G. maculatus are currently found in freshwater and marine ecosystems of southern Australia, Tasmania, New Zealand and surrounding islands, and in the temperate latitudes of South America and the Falkland/Malvinas Islands [G. maculatus has been explained by vicariance [G. maculatus includes landlocked and migratory populations in freshwater, estuarine and marine ecosystems [G. maculatus detected a small influence of the last glacial cycles over the genetic diversity of the species in South America. In fact, female populations size in G. maculatus seems to have remained relatively constant until ~ 500 Ka when populations sizes increased exponentially reaching contemporary levels [G. maculatus experienced a severe genetic bottleneck between 1.1 Ma and 0.6 Ma, a period when the Patagonian Ice Sheet reached its maximum northward expansion [The Family Galaxiidae includes ~ 50 species of Gondwanan-distributed fishes commonly found in cool-temperate regions in the Southern Hemisphere. All species inhabit freshwater ecosystems, although, some of them present a migratory life stage with a salt-tolerant larval phase known as whitebait. These diadromous species exhibit considerably greater dispersal capabilities than those that are non-migratory . Among d Islands . The tracariance  and dispcariance  hypothescariance –36. Alonosystems  in two oosystems . Phylogey levels . More rey levels  recognizxpansion ,15. Follxpansion .Galaxias maculatus constitutes a suitable model to infer how historical and contemporary climatic processes have shaped the distribution of the genetic diversity in the species. Here we evaluated the consequences of the Quaternary glacial cycles over the pattern of genetic diversity and structure in a diadromous species with special emphasis in coastal and estuarine populations along two biogeographic marine areas that were differentially affected by ice advances and retreats.The Chilean coast represents an interesting area to evaluate the relative effect of glaciological history, oceanography, and habitat discontinuities for aquatic organisms. The presence of extensive ice sheets during the glacial periods of the Quaternary likely eradicated recurrently most of the suitable freshwater and brackish marine habitats along the Magellanic Province while the Intermediate Area was considerably less impacted. In this context, Galaxias maculatus) as model study, a common freshwater and brackish fish species along the Chilean coast. The species is not protected and is considered as vulnerable in Central Chile and as out of danger in the Magellanic Province. Permission to undertake field works and collect specimens was issued by the Chilean Fisheries Service Director , under the technical memorandum (427/2011). The Instituto de Ecología y Biodiversidad (IEB/12-2011) and Chilean Fisheries Service (SERNAPESCA 427/2011) ethic committees approved sampling protocols and experiments. For this, we compiled with local legislation and the Convention on Biological Diversity.This work was conducted using puye  Moncul River ; 2) Queule River ; 3) Lingue River ; 4) Valdivia River ; 5) Chaihuín River , and 6) Maullín River . Secondly, we included seven localities from the Magellanic Province, between 43° S and 53° S, within the influence of the LGM Patagonian Ice Sheet. Magellanic localities were: 1) Yelcho River , 2) Concoto Island ; 3) Williams Channel, Rivero Island ; 4) María Eugenia Bay ; 5) Tortel, Calén Fjord ; 6) Pascua River, Calén Fjord ; and 7) Strait of Magellan , 1 U Taq (Invitrogen), 17.5 μl of double-distilled water and 5 ng of DNA. Thermal cycling parameters included an initial denaturation step at 94°C for 5 min, followed by 35 cycles at 94°C for 90 sec, 60.7°C for 90 sec, and 72°C for 90 sec, and a final 10 min extension at 72°C. PCR amplification products were purified using QIAquick Gel Extraction Kit (QIAGEN) and sequenced in both directions with an Automatic Sequencer ABI3730 x 1 at Macrogen Inc. .Whole specimens were fixed in ethanol (95%) and DNA was prepared from muscle tissue using a salting-out method . A partik), the number of segregating sites (S), haplotypic diversity (H), the average number of pairwise differences (П), and nucleotide diversity (π) for each locality, for each main area, and for the whole D-loop data set using DnaSP v.5.00.07 [S) for each locality, area, and for the whole data set to estimate whether D-loop sequences in G. maculatus deviate from expectations under a neutral model. Considering the high levels of genetic diversity previously estimated in the species [ST) following Pons and Petit [nn) that measure how often nearest neighbors (in sequence space) of sequences are from the same locality in geographic space [nn analyses was determined through a permutation test using 10,000 iterations. We inferred the spatial genetic structure in the species by estimating the number and composition of groups that were most differentiated based on sequence data with an Analysis of Molecular Variance (AMOVA) following Excoffier et al. [D-Loop sequences were edited using Proseq v. 3.5  and alig.5.00.07 . We perf species ,39,41 wend Petit  in Arleqnd Petit . The staic space . The star et al.  in Arleqr et al.  and the r et al. .G. maculatus using Maximum Parsimony Networks in Hapview (http://www.cibiv.at). To estimate the pattern of demographic history in the species, we compared the distribution of pairwise differences between haplotypes (mismatch distribution) for each locality, main area, and for the whole data set, to the expected distribution under the sudden expansion growth model of Rogers and Harpending [http://beast.bio.ed.ac.uk/Tracer). The uncorrelaed lognormal model was the best fit for D-loop data in G. maculatus. We conducted three independent Bayesian MCMC runs using the GTR + I + G model, previously estimated using MrModeltest v. 2.3 (http://www.abc-se/~nylander), and a specific population level mutational rate estimated for G. maculatus by Salinas [6 generations (sampled every 1000 step), discarding a 10% of the trees as burn-in. The convergence of runs was confirmed with Tracer ensuring a minimum of 1000 effective sampling for each statistics (ESS). The results of the multiple runs were combined using LogCombiner [We constructed genealogical relationships in rpending . This anrpending . This merpending  with TRA Salinas . Two indCombiner . The medWe compared different models of gene flow between the main areas of interest (Intermediate Area and Magellanic) with the software MIGRATE v.3.5 , to testN > S) and from Patagonia to the Intermediate Area (m S > N). We carried out separately several preliminary runs in the M mode  of the software to set the best set of priors that ensure mixing and convergence. Uniform priors were used to estimate effective population size  and splitting time (t = 300), whereas an exponential prior (mean = 1) for gene flow (m) was adopted. We performed 80 x 106 MCMC steps sampling every 100 generations, with a burn-in of 8 x 106. D-loop sequences were assumed to mutate under the HKY mutation model following the author guidelines [Θx/ mx)/2)  using an overall substitution rate per year (m = 1.1 x 10−4) estimated on previous studies [We estimated different demographic indices and evaluated two asymmetric isolation-with-migration models between main areas of interest (Intermediate Area and the Magellanic Province) using IMa2 software ,59: i) fidelines ,59. Fina studies .G. maculatus included 353 individuals and consisted in 926 nucleotide positions. As expected working with a non-coding region, several insertion and deletions were detected and were not considered in further analyses. Sequences were A-T rich (57.1%) compared to G-C content (42.9%). Levels of genetic diversity in the species were extremely high with 230 polymorphic characters (24.83%); and 156 of them (67.82%) were parsimoniously informative. In spite of the high level of genetic polymorphosm recorded in the species, D-loop sequences were not saturated. Haplotype diversity (H) ranged between 0.942 (Lingue River) and 1.000  and nucleotide diversity (π) were very high in most of the localities and ranged between 30.55/0.033 (Chaihuín River) and 10.74/0.011 (Williams Channel). Levels of genetic diversity of G. maculatus were higher in Intermediate Area localities than in Magellanic ones, particular for П and π (П (P = 0.00066) and π (P = 0.00063) in Intermediate Area populations compared to those from the Magellanic Province. Similarly, pairwise NST comparisons detected a high degree of genetic structure between Intermediate Area and Magellanic Province populations  indicate moderate levels of phylogeographic signal in the whole data set. Nevertheless, Snn statistic recognized a higher degree of phylogeographic signal along the Intermediate Area localities (Snn = 0.63) than in Magellanic Province ones (Snn = 0.43). General pattern of genetic structure registered in G. maculatus was supported by AMOVA analyses detecting 7 groups with a maximal difference accounting for 37.98% of the total variance, and only 2.01% was due to within-group variation among localities  and population expansions between the Intermediate Area and the Magellanic Province. Based on these analyses, the trmca of the Intermediate Area occurred ∼ 72 Ka while the tmcra for the Magellanic Province occurred ∼ 48 Ka . SimilarGene flow analyses using MIGRATE and different migration models detected evidence of asymmetrical gene flow from the Magellanic Province to the Intermediate Area. Among the tested models the one from the Magellanic Province to the Intermediate Area received the highest probability . SimilarMolecular-based genetic studies have become pivotal to further understand and unravel how Quaternary glacial cycles affected the distribution and demography of populations, species, and communities ,4,60–65.G. maculatus have been previously included in phylogeographic and demographic studies [G. maculatus harbors extremely high levels of genetic diversity along its distribution in the Chilean Pacific coast. In fact, our results further corroborate previous molecular studies in the species [G. maculatus, expanding for more than 1000 km, showed low levels and even absence of genetic differentiation. Again, major differences in terms of the genetic structure between formerly glaciated and unglaciated areas are adjusted to the predictions of the Expansion-Contraction model. Nevertheless, high levels of genetic diversity, measured as П and π, among Magellanic localities and the absence of dominant broadly distributed haplotypes in this area do not fit with the expectations of recent postglacial expansion from refugial areas at lower latitudes. Moreover, the presence of a markedly differentiated and highly diverse haplogroup (Hap04) in this Province does not support the idea of a recent postglacial recolonization. Such results contrast to those observed in other South American freshwater [Aplochiton . Most of these studies have demonstrated the important role of the last glacial cycle over the demography of Magellanic fishes with the presence of typical star-like haplotype genealogies as a consequence of recent colonization processes associated to strong founder effects. In this context, population genetic patterns in Patagonian terrestrial plants and vertebrates indicate that different processes and directional range shifts have generated a mosaic of phylogeographical patterns that are far more complex than a simple north to south described one [G. maculatus, Zemlak et al. [in situ or periglacial refugia during cooling periods of the Quaternary.Even if coastal and Andean South American samples of  studies ,39, here studies ,39,41, G species ,41,72, p species  recorded species  at the seshwater ,25,73–76eshwater ,68. In feshwater  recordedibed one . In the k et al. ,39 propoG. maculatus larvae have been reported in ichthyological samples along the Magellanic Province [G. maculatus. In contrast, genetic structure along the Intermediate Area suggests low levels of connectivity between river basins. In this context, open sea areas between the coastal rivers here analyzed seem to be preventing the genetic continuity of the species along the Intermediate Area. Such results are quite surprising considering the high dispersal potential described for New Zealand coastal populations of G. maculatus [Low genetic differentiation along the Magellanic Province, a complex geographic and oceanographic area, may be a response of particular characteristics of the species. The southern fjords and channels ecosystems along the Pacific coast of Patagonia are considered as one of the largest estuarine ecosystem in the planet ,78. Subaaculatus . In thisaculatus .G. maculatus agree with the 42°S biogeographic break described along the Chilean coast resulting from different oceanographic, geologic, and climatic processes [G. maculatus.Major differences in terms of genetic diversity and structure recorded in rocesses ,77. Alonrocesses  and fresrocesses . Differerocesses . First, rocesses . Intermerocesses  while throcesses ,85. A corocesses –90. Ther


Pelagia noctiluca (Scyphozoa) in the Southern Tyrrhenian Sea. Homozygote excess was detected at eight loci, and individuals exhibited intra-population relatedness higher than expected by chance in at least three samples. This result was supported by the presence of siblings in at least 5 out 8 samples, 4 of which contained full-sib in addition to half-sib dyads. Having tested and ruled out alternative explanations as null alleles, our results suggest the influence of reproductive and behavioural features in shaping the genetic structure of P. noctiluca, as outcomes of population genetics analyses pointed out. Indeed, the genetic differentiation among populations was globally small but highlighted: a) a spatial genetic patchiness uncorrelated with distance between sampling locations, and b) a significant genetic heterogeneity between samples collected in the same locations in different years. Therefore, despite its extreme dispersal potential, P. noctiluca does not maintain a single homogenous population, but rather these jellyfish appear to have intra-bloom localized recruitment and/or individual cohesiveness, whereby siblings more likely swarm together as a single group and remain close after spawning events. These findings provide the first evidence of family structures and consequent genetic patchiness in a species with highly dispersive potential throughout its whole life cycle, contributing to understanding the patterns of dispersal and connectivity in marine environments.Genetic drift and non-random mating seldom influence species with large breeding populations and high dispersal potential, characterized by unstructured gene pool and panmixia at a scale lower than the minimum dispersal range of individuals. In the present study, a set of nine microsatellite markers was developed and used to investigate the spatio-temporal genetic patterns of the holoplanktonic jellyfish  Short life cycles, conferring the ability to mobilize huge populations that rapidly appear and disappear, allow gelatinous zooplankton to swiftly colonize available ecological spaces planula), that metamorphoses into a benthic asexually reproducing polyp stage, giving rise to planktonic adult medusae through lateral budding (Hydrozoa), strobilation (Scyphozoa) or complete metamorphosis (Cubozoa). The presence of benthic polyps may limit the distribution of pelagic jellyfish in coastal areas with available hard substrates for larval settlement and polyp formation Pelagia noctiluca are holoplanktonic (i.e. do not have a benthic polyp stage), and have a wide distribution in both inshore and offshore waters Many medusozoans have complex life cycles with the succession of three stages: a short-living larva , in order to elucidate the processes behind the genetic composition of each “bloom unit”. Indeed, as already mentioned above, this species shows complex population dynamics that may suggest the driving action of unknown factors. Moreover, even if P. noctiluca is a holopelagic species, from a genetic point of view it may behave as a typical CGP species: it is characterised by high dispersal ability and the individuals of each bloom unit presumably spend together most part of their life, similarly to those belonging to a spatially stable benthic population. This particular habit suggests the possibility that related individuals, born at the same time and in the same area, could later spawn close to each other, producing inbred offspring and a bloom unit structure with high level of relatedness. Considering this hypothesis, the expected genetic patchiness could be further exacerbated by the high mortality rate due to stochastic factors, as unfavourable environmental conditions (i.e. sea storms), or by the high variance in reproduction success due to the external fertilization strategy Previous population genetics and phylogeographic studies focusing on Pelagia noctiluca is not an endangered species and no special permits were needed for sampling. All the animals were released without serious damages after sampling. Cnidarians are also renowned for their high regeneration potential.Pelagia noctiluca (Forsskål 1775), were collected from 4 different locations in the Southern Tyrrhenian Sea, and from one site in the Northern Adriatic Sea  and connected by a superficial eastward current In order to address the species' genetic patterns on both spatial and temporal scale, the sampling was replicated in three consecutive years  for Ustica (UST) and in two consecutive years  for Messina (MES). No temporal replicates were available for the Northern Adriatic Sea (NAD06), Ischia (ISC10), and Lipari (LIP11) locations, respectively sampled only in 2006, 2010 and 2011. The samples were collected by hand-net during bloom events choosing exclusively adults of the same size class, ranging from 7 to 10 cm of bell diameter. For each individual, a small piece of bell margin or oral arm was excised, preserved in 95% ethanol and stored at −20°C until DNA extraction.Total genomic DNA was extracted from the ethanol preserved tissues, following a CTAB-phenol-chloroform based protocol www.ecogenics.ch) using the high-throughput genomic sequencing approach described by Abdelkrim et al. P. noctiluca individuals belonging to the only two populations already available in 2010, Ustica (UST) and Ischia (ISC), were analysed on a Roche 454 GS-FLX platform  using 1/8 run and the GS-FLX titanium reagents. The total 53,066 reads had an average length of 277 base pairs. Of these, 843 contained a microsatellite insert with a tetra- or a trinucleotide of at least 6 repeat units or a dinucleotide of at least 10 repeat units. Suitable primer design was possible for 122 reads, 12 of which were tested in agarose by Ecogenics GmbH Switzerland. In order to complement the set of loci, 10 additional reads were selected from the microsatellite library provided by Ecogenics using the software MSATCOMMANDER 1.0.8-beta http://bioinfo.ut.ee/primer3-0.4.0/) P. noctiluca individuals of a single population (UST10), performing PCRs as follows. The PCR volume of 10 µl contained approximately 50 ng of genomic DNA, 1× Mastermix (RBC Taq Polymerase Kit), 0.5 µM of a fluorescently labelled (6-FAM) universal M13 primer (5′CACGACGTTGTAAAACGAC3′) and the species-specific reverse primer, 0.15 µM of species-specific forward primer with a 5′ M13 tail http://www.macrogen.com) using the internal size standard LIZ 500 (Applied Biosistems). The chromatograms were analysed using GeneMarker v. 2.2.0 (SoftGenetics) and a final panel of nine loci was obtained after excluding loci with low signal, unclear peaks, excessive stuttering and the non-polymorphic ones.Microsatellite sequences were isolated by Ecogenics GmbH Switzerland (http://www.macrogen.com) using the internal size standard LIZ 500 (ABI). Re-extraction and repeated multiplex amplifications were performed on individuals with failed PCRs. In addition, to assess loci amplification and scoring repeatability, 10% of total sampled individuals were randomly re-amplified and alleles sized Two multiplex panels were created with the help of the software Multiplex Manager 1.0 A) was obtained using the software FSTAT v. 2.9.3.2 O; HE) were calculated by GENETIX v. 4.05.2 Levels of polymorphism were calculated for each locus in each population. Number of alleles (Nhttp://genepop.curtin.edu.au/). The corrected significance threshold for multiple tests was set using the Bonferroni procedure Microsatellite loci are informative when they are independent one another and do not produce redundant information, therefore linkage disequilibrium was tested for each pair of loci within each population using Fisher's exact tests by Genepop web version As mentioned, the dataset was carefully checked against inconsistencies due to human mistake, genotyping errors and amplification failure. However, in order to evaluate and minimise the presence of artefacts, the final dataset was analysed with Microchecker v. 2.2.3 IS) using FSTAT IS values were calculated by performing 1000 randomizations of genotypes among samples.In order to verify the effect of the correction for null alleles on HWE, two parallel analyses  were performed with Arlequin ver. 3.5.1.3 IS values and deviation from HWE expectations may be interpreted as an evidence of null alleles presence. Nevertheless, the Hardy-Weinberg disequilibrium (HWD) can be also due to phenomena as Wahlund effect High FThe degree of relatedness between individuals of the same “bloom unit” was firstly investigated using the software ML-Relate Since every method of parental analysis and sibship reconstruction includes a certain level of uncertainty , and setting medium length of the run and high likelihood precision. Due to the lack of prior information about sibship size, the value 0 was set up for known male and female genotypes as well as for known paternal and maternal sibship. Since the program Colony can accommodate null alleles and other stochastic genotyping errors in the analysis, once again two runs were performed for each sampling site, taking or not the presence of null alleles into account. In the runs “without nulls”, the genotyping error was set to the default level (0.005), whereas the runs “with nulls” were performed allowing locus specific genotyping error levels, obtained adding the null alleles frequencies calculated by Microchecker v. 2.2.3 for each locus in each population to the default genotyping error rate. HS and FS dyads frequencies were calculated for both corrected and uncorrected datasets and then compared by means of a two tailed t-test.Family structures were inferred reconstructing extended sibship networks, namely clusters of individuals connected by a chain of HS or FS intermediate individuals. In practice, if the individual A shares a parent with the individual B, and the individual B shares a parent with the individual C, in that case A and C are linked through B and are members of the same extended sibship network, even if they do not have a common parent. Since the extended sibship networks can also include FS, they were represented as FS families  nested into sibship networks. For each sibship network the number of FS families (and the respective number of family members) was reported, when any.ST values and corresponding p-values were calculated with Arlequin ver. 3.5.1.3. Prior to this analysis, in order to estimate the extent of bias possibly introduced by the presence of null alleles, pairwise FST were also calculated using the software FreeNA STST values were obtained using 50,000 bootstrap iterations. FST estimates obtained with and without applying the ENA algorithm were compared by means of a two tailed t-test.To examine patterns of genetic variation among the studied population samples, pairwise FOf 22 initially selected loci, 13 were not polymorphic or showed very low signal, not clear peaks or excessive stuttering and were discarded, while 9 markers were suitable for the present study and resulted to be polymorphic in all population samples . The totMicrochecker did not identify scoring errors associated with stuttering, but suggested the presence of null alleles by analysing strong deviations from HWE . Indeed,Two out of 36 pair-wise locus comparisons revealed significant linkage disequilibrium, after standard Bonferroni adjustment All loci but one  did not globally result in HWE. HWD was due to heterozygote deficiency for all cases, suggesting two alternative explanations: the presence of technical artefacts, such as non-amplifying alleles , or the influence of biological factors IS values generally higher than zero  due to higher r-values obtained taking into account the presence of null alleles  were found in UST10, LIP11, MES12 and NAD06, with respectively 3 FS families nested within 7 sibship networks, 4 FS families nested within 9 sibship networks, 2 FS families nested within 12 sibship networks and 5 FS families nested within 11 sibship networks. All FS families were composed by two individuals. A graphic representation of the family structures found in these 4 populations is shown in ST values were initially calculated with FreeNA to verify if accounting for null alleles could bias this genetic distance estimation  but highly significant (P<0.0001). Inspection of pairwise FST values , and homogeneous with the sample UST11, collected in 2011 at ≈230 Km of distance . Moreover, temporal variation was identified also at the Ustica site .Population pairwise Ftimation . FST valT values  indicatePelagia noctiluca blooming populations in the Southern Tyrrhenian Sea exhibited significant deviation from HWE due to large excess of homozygotes for 8 out of 9 microsatellite loci, leading to high inbreeding coefficients (FIS). Moreover, higher relatedness than expected by chance, between individuals within and among population samples was detected and supported by the presence of half-sib (HS) individuals in at least 5 out of 8 samples, 4 of which (at least) contained full-sib (FS) individuals also. The genetic differentiation among samples was globally small, but it highlighted a spatial and temporal genetic patchiness probably reflecting the influence of reproductive processes, as also suggested by the outcomes of the relatedness analyses.IS values, as supported by the missing recovery of HWE  only between Southern and Northern Atlantic samples, geographically very far from each other (≈10.000 km), whereas no genetic difference was found at a smaller geographic scale. The authors interpreted this result as a suggestion of historical rather than contemporary gene flow. Conversely, Stopar et al. P. noctiluca populations from the Mediterranean Sea and the North-western Atlantic Ocean. These authors could not detect significant genetic differentiation relative to both COI and IST1/ITS2 markers, apart from differences between samples from Northern and Southern Adriatic Sea . Such a short-scale pattern of genetic differentiation is not coherent with an isolation by distance model, and has been interpreted as the outcome of basin-scale hydrodynamic processes reducing the mixing of individuals born in different areas of the Adriatic Sea.Additional evidences of biological factors influencing the P. noctiluca blooms . Conversely, 5 out of 7 pairwise comparisons between the populations from the Southern Tyrrhenian Sea and the one from the Adriatic Sea  did not result statistically significant (P≥0.06934). Moreover, pairwise comparisons among consecutive samples collected in the same locations  in different years showed significant genetic differences, highlighting the lack of temporal stability of the genetic composition of the a blooms . AltogetP. noctiluca can be explained as fine-scale genetic patchiness, which may be generated through processes driving localised temporal variation of numbers and genotypes of recruits Genetic heterogeneity on a small geographic scale, especially when temporal genetic differentiation is stronger than spatial differentiation, is generally attributed to temporal changes in the genetic composition of recruits P. noctiluca population level, together with the presence of half- and full-siblings in several samples, the present study may contribute to clarify the role of family structure in CGP formation. The most probable scenario to explain the temporally unstable genetic patchiness of P. noctiluca populations is the co-occurrence of large variation in the reproductive success of individuals and genetic drift. Indeed, the high intra-population inbreeding level, highlighted by homozygote excess and positive FIS values, combined with the presence of related individuals in several samples could be a symptom of variance in reproductive success generated by environmental, reproductive and behavioural factors. Moreover, the high mortality rate of ephyrae To date, many studies provided evidence of a widespread occurrence of chaotic genetic patchiness (CGP) in several benthic marine species P. noctiluca populations in the Southern Tyrrhenian Sea are seemingly characterised by a spatio-temporal CGP and random genetic drift may represent a leading micro-evolutionary force shaping the genetic structure of this species. Even if several studies have already shown similar patterns in species with high larval dispersal ability Chaotic genetic variability among populations, explained by alternate periods of rarity and abundance and related genetic drift, is in accordance also with the “Flush and Crash” speciation model Table S1Loci potentially affected by null alleles. Results of the Microchecker v. 2.2.3 analysis for each locus and population.(DOCX)Click here for additional data file.Table S2Hardy Weinberg Equilibrium (HWE) p-values. HWE p-values were calculated for the original and the corrected dataset . In bold, significant p-values (α = 0.05). * Results belonging to the dataset corrected with Microchecker v. 2.2.3 are indicated in italic. § For locus Plenoc_16756 no differences are reported due to HWE of all population samples.(DOCX)Click here for additional data file.Table S3Relatedness Monte Carlo Simulation, null alleles accounted for , 1000 iterations.(DOCX)Click here for additional data file.Table S4Relatedness Monte Carlo Simulation, no null alleles accounted for, 1000 iterations.(DOCX)Click here for additional data file.Table S5Pairwise FST values calculated using the program FreeNA. The table S5A shows the results of the analyses performed using the uncorrected dataset. The table S5B is referred to the FST values obtained implementing the ENA correction method. Pairwise FST values below the diagonal grey boxes line, lower and upper limits of the 95% confidence interval above the diagonal.(DOCX)Click here for additional data file.


In Senegal, uncontrolled cross-breeding of cattle breeds and changes in production systems are assumed to lead to an increase of gene flow between populations. This might constitute a relevant threat to livestock improvement. Therewith, this study was carried out to assess the current genetic diversity and the phylogenetic relationships of the four native Senegalese cattle breeds .Genomic DNA was isolated from blood samples of 120 unrelated animals collected from three agro-ecological areas of Senegal according to their phenotypic traits. Genotyping was done using 11 specific highly polymorphic microsatellite makers recommended by Food and Agriculture Organization. The basic measures of genetic variation and phylogenetic trees were computed using bioinformatics’ software.E) per breed was high in general with an overall mean of 0.76±0.04. Generally, the heterozygote deficiency (FIS) of 0.073±0.026 was relatively due to inbreeding among these cattle breeds or the occurrence of population substructure. The high values of allelic and gene diversity showed that Senegalese native cattle breeds represented an important reservoir of genetic variation. The genetic distances and clustering trees concluded that the N’Dama cattle were most distinct among the investigated cattle populations. So, the principal component analyses showed qualitatively that there was an intensive genetic admixture between the Gobra zebu and Maure zebu breeds.A total of 115 alleles were identified with a number of alleles (Na) at one locus ranging from 6 to 16. All loci were polymorphic with a mean polymorphic information content of 0.76. The mean allelic richness (Rs) lay within the narrow range of 5.14 in N’Dama taurine to 6.10 in Gobra zebu. While, the expected heterozygosity (HThe broad genetic diversity in Senegalese cattle breeds will allow for greater opportunities for improvement of productivity and adaptation relative to global changes. For the development of sustainable breeding and crossbreeding programs of Senegalese local breeds, effective management is needed towards genetic selection and transhumance to ensure their long-term survival. Trypanosoma sp.) and are spread in the central part of the country, more or less infested with tsetse flies [Senegal is an oceanic country, located in West African continent. Longtime practiced of breeding, livestock keeping represents an important source of livelihood for 3.5 million of people, and contributes to 35% of primary sector gross domestic product (GDP) and 7% of national GDP . Cattle se flies . The N’Dse flies . There ase flies .et al. [In order to achieve food security, Senegal has developed policies for the improvement of livestock production by bovine artificial insemination. Local cattle are continually crossed with exotic breeds . In the et al.  has usedAs a result, uncontrolled crossings of cattle breeds and changes in production systems are assumed to lead to a very confused genetic structure of local cattle with increasing gene flow between populations. Breed’s characterization is very important for the assessment of genetic diversity, the conservation of genetic resources , and theGobra zebu, Maure zebu, and N’Dama have been the subject of population genetics analyses among studies conducted on a continental scale to retrace indicine and taurine migration across Africa -17. ThisThis study was approved by the Ethics Committee of the Cheikh Anta Diop University of Dakar. Signed consent of all participants was obtained after the study was fully explained.Sampling was carried out from October to December 2013 in three agropastoral regions of Senegal namely Saint-Louis (16°02’00’’N and 16°30’00’’W), Kaolack (14°08’35’’N and 16°05’45’’W) and Kolda (13°01’60’’N and 14°52’00’’W). These regions located in three-agro-ecological areas  represenThe animals were randomly sampled and classified according to the age of the animal and physiological status of females. To ensure the representativeness of the sample with the least possible relation between animals, a maximum of eight animals was sampled by the herd and four by each reproduction flock. Thus, the samples were composed males subjects aged between 13 and 144 months, and females with age between 18 and 192 months. For each of the four local cattle breed , 30 individuals were sampled . Farmers® Systems, Plymouth, United Kingdom) containing EDTA as an anticoagulant and stored at 4°C until further use. During the blood sampling, adequate measures were taken to minimize pain and discomfort.Four milliliters of blood were collected from the jugular vein puncture in vacuum tubes , 0.2 μl of FM13 primer (10 μM), 0.3 μl of R primer (10 μM), 0.1 μl of Qiagen Taq DNA polymerase (5 U/μl) and 0.3 μl of dye M13 (700). The amplifications were carried out in a thermal cycler  using the following conditions: Initial denaturation at 94°C for 3 min, followed by 35 cycles of 30 s at 94°C, 30 s at annealing temperature of 50, 55 or 60°C (according to the microsatellite) and 45 s extension at 72°C, then final extension at 72°C for 8 min ended the reactions. Subsequently, the amplified products were mixed with desmilings 700 (fluorescent dyes varying between 71 and 367 bp according to amplified microsatellite) in simplex rearrangements and were resolved on 6.5% denaturing acrylamide-urea gels using a Li-Cor® automated sequencer  following the manufacturer’s procedures. All gels were analyzed using SAGAGT Generation 2.0 software.Microsatellites were amplified by Li-Cor polymerase chain reaction (PCR) in simplex. The PCR reactions for 12 markers such as BM2113, BM1818, ETH10, ETH225, ETH152, HEL1, HEL9, INRA037, INRA063, MM12, TGLA53 and TGLA122, were performed in a 15 μl reaction volume containing 2.0 μl of DNA template and 13 μl of total PCR mix. The mix composed of 1.6 μl of 10X PCR buffer, 1.6 μl of dNTPs (2.5 mM), 0.8 μl of MgClO), unbiased expected heterozygosity (HE) [S) [IS (f) (amount of inbreeding within a population of Weir and Cockerham [IS) was tested using methods of jackknifing and bootstrapping over loci after 1000 permutations of alleles within a population. The allelic richness of a breed is the measure of the number of allele’s independent of sample size which is estimated per locus (Rt) and population (Rs) using Fstat version 2.9.3.2 [et al. [t-test implemented in STATVIEW version 5.0 [The genetic variability of microsatellite loci and populations was measured by estimating a set of characteristic parameters of genetic polymorphism. Before the estimation of these measures, the presence of null allele across loci was checked using the program Micro-Checker version 2.2.3 . Allele ity (HE) , gene di(HE) [S) , FIS (f)ockerham ) were esockerham  and Fstaockerham . The sig 2.9.3.2 . The pri 2.9.3.2  was appl 2.9.3.2 . The pol [et al. ) was est [et al. . The sigsion 5.0  at a sigExact tests for deviations from the Hardy-Weinberg equilibrium (HWE) were performed for each locus, in each population and for all populations using a Markov Chain Monte Carlo simulation  implemented in Genepop 4 version 4.2.2 . The sigTest of the genotypic linkage disequilibrium was estimated between all pairs of loci using a G statistic (log - likelihood ratio) implemented in Fstat version 2.9.3.2  to test et al. [A were calculated through alleles frequencies to determine the genetic relationships among breeds using Genetix version 4.05.2. Moreover, an unbiased standard genetic distance of Nei [S), was calculated using the GenAlEx software version 6.5.Genetic relationships among breeds were explored by multivariate statistical analysis and phylogenetic reconstruction. Genetic distances of Nei et al.  DA were e of Nei  (DS), waS using the GenAlEx program version 6.5.To condense the genetic variation revealed for the panel of 11 microsatellites loci, a multivariate analysis of microsatellite allele frequencies principal components analysis (PCA) was performed from the covariance matrix Det al. [A) using the unweighted pair group method with arithmetic mean (UPGMA) [C) of Cavalli-Sforza and Edwards [Syncerus caffer  from eight microsatellite data  [Phylogenetic trees were generated using genetic distances that are suitable for numeric data. For this, two dendrograms of populations were constructed first from the distance matrix of Nei et al.  (DA) usi (UPGMA)  and the  Edwards  using th Edwards . We use  TGLA53)  to root  TGLA53) . Dendrog TGLA53) .O and HE values of 0.44 and 0.60, respectively. All genetic makers showed PIC values higher than 0.5 with an average value of 0.76. Three of all loci  presented a positive value of FIS overall populations, which was significantly different from zero (IS) was 0.073 which implied a significant deficit of heterozygotes (p<0.05). The values of Chi-square with their p-values of BM2113 , ETH152 , INRA063  and INRA037  showed that these loci deviated very significantly from HWE in all populations. The test of linkage disequilibrium between different combinations of loci considering all samples showed none significant deviations.The genetic parameters per locus are shown in rom zero . So, theS), it ranged from 5.14 (N’Dama) to 6.10 (Gobra) (S) varied from 0.73 in N’Dama to 0.80 in Gobra  . Therefoin Gobra . The GobIS values indicated that three breeds  presented a significant deficit of heterozygotes (p<0.05). Considering all populations and all loci, a highly significant deviation from HWE was noted .The overall mean values of observed heterozygosity (0.71±0.04) and expected heterozygosity (0.76±0.04) indicated the presence of high level of heterozygosity in native local cattle breeds. The FS) and Nei DA genetic distances for each pair of the four cattle populations  to summarize breed relationships , have revealed that cattle populations are distinguished strongly into two major clades  and gene diversity (HS). The mean allelic richness (Rs) varied from 5.1 in N’Dama to 6.1 in Gobra. MacHugh et al. [et al. [et al. [The four Senegalese cattle breeds and particularly the putative zebu populations displayed considerable levels of genetic diversity as estimated by allelic richness (Rs), expected heterozygosity . Similar results were obtained in Borgou cattle which possessed the highest number of rare alleles among West African and Central zebu and taurine breeds . Within n N’Dama . We spec  per breed varied between 0.73 for the N’Dama and 0.79 for the Gobra zebu. Our results showed a considerable level of heterozygosity among the four cattle breeds. A similar level of heterozygosity was reported in Togolese and Beninese, Mozambican and Cameroonian cattle zebu and taurine breeds [S) ranged from 0.73 (N’Dama) to 0.80 (Gobra). The high values of allelic diversity, expected heterozygosity and gene diversity obtained in this study well confirm that Senegalese local cattle breeds represent an important reservoir of genetic variability and they reflect the absence of selection or organized breeding programs for Senegalese cattle, contrary to highly selected breeds which display lower diversity due small effective population sizes [The mean expected heterozygosity  displayed the lowest within breed variability. Since the N’Dama breed is reared in an isolated breeding area separated by the “Gambia River” from the other cattle production systems, it must be less affected by intensive uncontrolled crossings. Therefore, according to investigations study, none of the herds of Gobra, Maure and Djakoré breeds practiced transhumance toward the Upper Casamance area; and 44% of transhumant N’Dama herds, did not exceed a range of 30 km out of the Kolda Region. Furthermore, the amount of genetic diversity in these breeds was comparable to those reported for other cattle breeds in different regions of Africa ,38,39,43et al.  found alIS) between subpopulations might due mainly by population substructure as regards to the strong difference on null allele frequency across loci under a high level of genetic differentiation [et al. [A significant deficit of heterozygosity (p<0.05) was found in Gobra, Maure and N’Dama breeds. Thus all populations has deviated from HWE. A considerable variance of the deficit . In addition, the high rate of inbreeding affecting these populations could destabilize the level of variability of Gobra and Maure zebu to the benefit of Djakoré population. If measures of rearing and conservation strategies are not promptly taken, the melting of genetic pools of different populations would lead to the disappearance of certain Senegalese local cattle breeds. Because, the loss of diversity linked to the disappearance of a breed is measured by the number of alleles that are specific. Priorities of conservation based on allelic diversity can be established.This study based on polymorphism of microsatellite markers revealed that the Senegalese cattle breeds had a considerable level of genetic diversity. Therefore, hybridization, the major influence on allelic diversity in these populations, tends to increase diversity by bringing together alleles from the two distinct lineages (This work is the first detailed study about the genetic variability and phylogenetic relationships of Senegalese cattle breeds. These breeds are important nutritional and economic resources for Senegalese people, and their high variability makes them suitable candidates for conservation and improvement to disconcert to global changes.Conservation of genetic variability in these populations should be considered by breeders, in the interest of long-term future of the populations in their native tract. To begin with, breed societies/associations need to be created, that will be responsible for registration of these cattle populations as breeds, complete maintenance and improvement of the breed to make it economically sustainable in the transforming agricultural scenario of the country.MS and GJS conceived and supervised the entire study. NPN performed microsatellite genotyping under the supervision of GKD. NPN carried out genetic analysis, drafted and revised the manuscript under the guidance of AS and SN. All authors read and approved the final manuscript.


Papio ursinus sensu lato and reveal a dominant role for increasingly arid landscapes during past glacial cycles in shaping contemporary genetic structure. Recent work on baboons (Papio spp.) supports complex lineage structuring with a dominant pulse of diversification occurring 1-2Ma, and yet the link to palaeoenvironmental change remains largely untested. Phylogeographic reconstruction based on mitochondrial DNA sequence data supports a scenario where chacma baboon populations were likely restricted to refugia during periods of regional cooling and drying through the Late Pleistocene. The two lineages of chacma baboon, ursinus and griseipes, are strongly geographically structured, and demographic reconstruction together with spatial analysis of genetic variation point to possible climate-driven isolating events where baboons may have retreated to more optimum conditions during cooler, drier periods. Our analysis highlights a period of continuous population growth beginning in the Middle to Late Pleistocene in both the ursinus and the PG2 griseipes lineages. All three clades identified in the study then enter a state of declining population size (Nef) through to the Holocene; this is particularly marked in the last 20,000 years, most likely coincident with the Last Glacial Maximum. The pattern recovered here conforms to expectations based on the dynamic regional climate trends in southern Africa through the Pleistocene and provides further support for complex patterns of diversification in the region’s biodiversity.Plio-Pleistocene environmental change influenced the evolutionary history of many animal lineages in Africa, highlighting key roles for both climate and tectonics in the evolution of Africa’s faunal diversity. Here, we explore diversification in the southern African chacma baboon  NectariniaLarge-scale environmental change during the Plio-Pleistocene influenced the evolutionary history of many animal lineages in Africa. This is true for many birds e.g. the olivacea/obscura complex D [2 [68]D [2 [Fs . Tajima’P. cynacephalus and P. kindae (as before); the analysis used uncorrected ‘p’ distances and clade structure was tested with 1000 bootstrap replicates. A number of mtDNA phylogenies of Papio support the phylogenetic placement of ursinus as sister to a clade comprising griseipes, P. kindae and P. cynacephalus. These two species are distributed across south-central and east Africa, with P. kindae present in the Miombo woodlands of Angola, the Democratic Republic of Congo, Zambia and possibly western Tanzania; while P. cynacephalus inhabits savannas and light forest habitats in Somalia, Kenya and Tanzania and southwards to the Zambezi Valley into Zambia, Zimbabwe and across south-central Africa to Angola.To explore our central tenet that chacma baboon populations were repeatedly restricted to refugia during periods of regional climate cycling through the late Pleistocene, we analysed the spatial distribution of genetic variation and inferred more recent demographic change in the PU and PG clades using data from the mitochondrial D-loop. We first used haplotype network analysis to construct intraspecific genealogies for chacma baboons using NeighborNet splits  in SPLITK as defined by their spatial proximity in BAPS v5.2 [K indicated that the best partition for our dataset was K = 5  and t (TMRCA) without migration. Nef estimated without migration was 261000 (95% HPD 143000–428000) for PU and 317000 (95% HPD 172000–608000) for PG  migration between the two chacma lineages following their divergence during the Pleistocene. Migration probabilities from PU into PG and vice versa both have their highest posterior probability at zero migrants per generation . Under o) for PG . Irrespe) for PG , when gl2 sand sea. Along with other depositional coastal landforms in southern Africa, this desert formed during the last 3Myr [Papio. We propose that this arid expansion was a primary driver of diversification of the chacma lineage into two clades that today encircle the south-central Kalahari in both the north-east and south-west. Baboons are not the only arid-tolerant mammals to have experienced significant vicariance during this period. Even the hardy African warthog Phacochoerus spp. which is capable of surviving in extremely arid conditions  experienced genetic isolation during periods of intense aridification in the Pleistocene [A notable gap stands out across the widespread distribution of chacma baboons across southern Africa; their absence from the central Kalahari Desert, located within the southern portion of the much larger 2.5 million kmast 3Myr ,79,79,80ast 3Myr , but it ast 3Myr . Geomorpast 3Myr  and the ast 3Myr  are ableast 3Myr  but are istocene .f through time. The resulting plots reveal an extended period of increasing population size for both the PU and the PG2 lineage  or north western (PG1) griseipes clades. Both PU and PG2 indicate a period of population growth from the mid-Pleistocene. In PU we see population size increase beginning at ~800ka. This trend continues beyond ~460ka, at this point the relative rate appears to increase. This is then followed by a slow down at ~160ka. A period of decline in Nef is suggested at ~90ka, which then slows down in the early Holocene. Within griseipes the BSPs indicated distinct differences in the demographic histories and female effective population sizes for PG1 and PG2. In PG2, the larger of the two griseipes lineages, population size slowly increases from the mid-Pleistocene, expanding from ~600ka to ~180ka. At this point while growth in PU is beginning to plateau, PG2 begins a period of slow decline into the Holocene. Over the same time period PG1 experiences a slow decline from ~600ka, increasing in the rate of decline into the Holocene. Tests for neutrality using the D-loop data set indicate that regional clades in both PU and PG clade are out of mutation-drift equilibrium, which may be explained by regional  demographic shifts such as population expansion. Significant values for Tajima’s D and R2 were found in PU clades 2 and 3 , while overall both PU and PG clades supported significant R2 values (p = 0.05).The recent demographic history of chacma mtDNA lineages were reconstructed using Bayesian skyline plots, where MCMC integration under a coalescent model allows us to explore changes in Ne lineage . Disparif observed in both PU and PG2 from the mid to terminal Pleistocene may be the result of a number of prolonged periods of interglacial warming; this pattern is evident in a number of African taxa e.g. Cape buffalo Syncerus caffer, [The historical population trends inferred by these analyses correspond closely to the dynamic regional climate trends of southern Africa since the mid to late Pleistocene. The persistence of of baboons, indicated by molecular and fossil evidence, after 1.5Ma coincides with the Plio-Pleistocene domination of arid adapted species in the biodiversity of Africa ; this is caffer,  which in caffer,  during M caffer, ,88. Duri caffer,  this cli caffer, ,91, part caffer, ,92. Some caffer,  and like caffer, .f observed in all three chacma clades may reflect a period of lineage divergence. This process needs further exploration with finer-scale sampling of baboons across the landscapes of southern Africa.At the onset of the Holocene, a period of distinct ecological transitions occurred. High-resolution stable carbon and nitrogen isotope records suggest a wetter early Holocene and a later dry period , after 3Splitstree networks for the PG and PU D-loop lineages  indicatePronolagus rupestris shares similarities with the distribution of PU3, where rock rabbit populations are confined to the mountain ranges and abutting regions of the Great Escarpment of South Africa [Papio may have experienced a congruent history to that of Pronolagus rupestris, and represent the dispersal of a relict population that took refuge in the Mediterranean-type habitat of the south-western Cape, later expanding out of this glacial refuge along the coastal Cape Fold Mountains towards the north and east. A similar pattern is reported in rock hyrax Procavia capensis with the distribution of a south-eastern clade similarly confined along the Great Escarpment mountain range [Phylogeographic studies of numerous southern African species reveal patterns of genetic structuring suggestive of repeated use of glacial refugia and subsequent expansion and fragmentation of lineages –103. Theh Africa ; the roch Africa . We propin range . Both ofin range ,106, an griseipes clades is more complicated. Nonetheless, the high diversity in the east along the Kalahari (PG1) mirrors that characterizing the PU2 and PU3 clades and it’s likely that this arid interior also played an important role in structuring diversity. The PG2 clade is centered on a region bounded to its west by the Drakensberg-Maluti Mountains and with dense coastal forests in the north. The high diversity seen at the eastern most boundary of this clade might reflect a comparable colonization event from montane refugia in the Drakensberg analogous to the Kuruman Hills (above). However, interpretation of diversity in both of the PG clades demands caution, given the likelihood of gene flow from further north. It is important to note here that the Early Pleistocene pulse of diversification in Papio that gave rise to the north-eastern PG clade also founded the kindae and cynocephalus lineages [Given our lack of information from the regions directly to their north, reconstructing the possible history of the lineages . ThereafPapio, and have persisted in geographical and genetic isolation since the Early Pleistocene. Furthermore, they each continued to evolve along distinct evolutionary trajectories into the Holocene. Phylogeographic reconstructions indicate that the south-western ursinus group contracted into at least three geographically isolated refugia, likely as a result of aridification, and this metapopulation has only increased its range quite recently as climate ameliorated. In contrast, the griseipes baboons in the north-east likely had a more complicated history, originating from a lineage that later diversified into ‘griseipes’, P. kindae and P. cynocephalus [Papio ursinus (south-west) and Papio griseipes (north-east) might be warranted. However, further studies into the geographic distribution and genetic make-up of these taxa, including foci on nuclear gene regions [Together with recent studies ,34,107 tcephalus . Structucephalus ,40,44, wcephalus  and perscephalus ,109. Witcephalus , 111 we  regions , and theS1 FigIndividual level mixture analysis supports K = 5 as the most likely number of groups in the optimal partition based on the log (ml) values for the size of the 10 best visited partitions.(TIF)Click here for additional data file.S1 TableMap ID numbers correspond to (DOCX)Click here for additional data file.


Contemporary Jews retain a genetic imprint from their Near Eastern ancestry, but obtained substantial genetic components from their neighboring populations during their history. Whether they received any genetic contribution from the Far East remains unknown, but frequent communication with the Chinese has been observed since the Silk Road period. To address this issue, mitochondrial DNA (mtDNA) variation from 55,595 Eurasians are analyzed. The existence of some eastern Eurasian haplotypes in eastern Ashkenazi Jews supports an East Asian genetic contribution, likely from Chinese. Further evidence indicates that this connection can be attributed to a gene flow event that occurred less than 1.4 kilo-years ago (kya), which falls within the time frame of the Silk Road scenario and fits well with historical records and archaeological discoveries. This observed genetic contribution from Chinese to Ashkenazi Jews demonstrates that the historical exchange between Ashkenazim and the Far East was not confined to the cultural sphere but also extended to an exchange of genes. Then, mOur analysis of the mtDNA variation in a total of 23,121 individuals from East Asian populations and Jews reveals that mtDNAs of four Ashkenazi Jewish individuals can be allocated into eastern Eurasian haplogroups A and N9a, suggesting that Ashkenazi Jews received a genetic contribution from East Asia . Intrigu11To achieve further insight into the phylogeographic distribution of M33c, mtDNA variants (mainly from the control region) of an additional 32,474 Eurasian individuals were analyzed, so that the total number of Eurasian mtDNAs considered here was 55,595. As shown in http://eng.molgen.org), confirms that M33c is defined by mutations at positions 3316, 4079, 5894, 8227, 8848, 16111, and 16235. Of note is that five clades within M33c appear respectively characterized by diagnostic coding-region variant(s), and these are named M33c1 to M33c5 here. With the exception of M33c2, all the samples in these clades are from China. The likely origin of M33 in South Asia and the restriction to China of M33c, dating to 10 kya according to the estimation based on whole-mtDNA genome, implies some dispersal from South to East Asia in the immediate postglacial.To shed light on the phylogeny within haplogroup M33c, 11 mtDNAs, covering the widest range of internal variation within the haplogroup, were chosen for whole-mtDNA genome sequencing. In good agreement with the previous result1314Intriguingly, sub-haplogroup M33c2  consists of three different haplotypes . Although there is no control-region variant in the defining motif of M33c2, multiple lines of evidence suggest that the pinpointed 14 Ashkenazi Jewish M33c mtDNAs most likely all belong to this clade: (1) all of the 14 mtDNAs share an identical control-region motif ; (2) theAge estimates for M33c2 are similar whether based either on the whole genome or on the control region alone , and theThe ancient Silk Road was an important transportation hub connecting China and the Mediterranean region from the Han Dynasty (206BC–220AD) onwards, and there are likely to have been Jewish merchants at the eastern end of the Silk Road from the early centuries AD. Moreover, Jewish merchants in Europe, referred to as Radhanites, were involved in trade between west and east as early as the ninth centuryAshkenazi origins are controversialExtensive genetic admixture has been observed in populations residing around the ancient Silk Road region23http://www.phylotree.org/).mtDNA variation (mainly from control region) of 23,121 East Asians and Jews, retrieved from previous studies as well as our unpublished data, were considered and analyzed, with especial attention to pinpointing the eastern Eurasian haplogroups in Jews. Then, additional 32,474 individuals were analyzed to gain further insights into the phylogeographic distribution of M33c, leading the total number of Eurasian mtDNAs considered here to 55,595. The study project was approved by the Ethics Committee at Kunming Institute of Zoology, Chinese Academy of Sciences. Each participant was informed about the study and provided informed consent. All mtDNAs collected and considered in the present study were first allocated to haplogroups, based mainly on their control-region motifs, which were then further confirmed by typing specific coding-region variation according to the PhyloTree . The most parsimonious phylogenetic tree (1329The median-joining network of M33c was constructed manuallytic tree  was recoQ.-P.K. designed the research; Y.-C.L., W.Z. and Y.-G.Y. collected the samples; J.-Y.T., H.-W.W. and Y.-C.L. collected the data; J.-Y.T. and H.-W.W. performed the experiments; J.-Y.T., H.-W.W., Y.-C.L. and Q.-P.K. analyzed data; J.-Y.T., Y.-C.L., J.v.S., M.B.R. and Q.-P.K. wrote the paper.Sample information of populations analyzed in present study


Letharia. Previous molecular work employing a gene genealogical approach circumscribed six species-level lineages within the genus, four putative lineages within the nominal taxon L. columbiana (Nutt.) J.W. Thomson and two sorediate lineages. We show that Bayesian clustering implemented in the program STRUCTURE was generally able to recover the same six putative Letharia lineages. Population assignments were largely consistent across a range of scenarios, including: extensive amounts of missing data, the exclusion of SNPs from variable markers, and inferences based on SNPs from as few as three gene regions. While our study provided additional evidence corroborating the six candidate Letharia species, the equivalence of these genetic clusters with species-level lineages is uncertain due, in part, to limited phylogenetic signal. Furthermore, both the BAPS analysis and the ad hoc ΔK statistic from results of the STRUCTURE analysis suggest that population structure can possibly be captured with fewer genetic groups. Our findings also suggest that uneven sampling across taxa may be responsible for the contrasting inferences of population substructure. Our results consistently supported two distinct sorediate groups, ‘L. lupina’ and L. vulpina, and subtle morphological differences support this distinction. Similarly, the putative apotheciate species ‘L. lucida’ was also consistently supported as a distinct genetic cluster. However, additional studies will be required to elucidate the relationships of other L. columbiana s.l. populations with the two sorediate genetic clusters.The inclusion of molecular data is increasingly an integral part of studies assessing species boundaries. Analyses based on predefined groups may obscure patterns of differentiation, and population assignment tests provide an alternative for identifying population structure and barriers to gene flow. In this study, we apply population assignment tests implemented in the programs STRUCTURE and BAPS to single nucleotide polymorphisms from DNA sequence data generated for three previous studies of the lichenized fungal genus  Our ability to accurately recognize species-level biological diversity is key to generating and testing many ecological and evolutionary hypotheses In many cases, the focus of species delimitation problems exists at the interface of traditional population genetic and phylogenetic analyses Letharia (Parmeliaceae) (Th. Fr.) Zahlbr. Previous research suggests a recent diversification history, characterized by limited genetic variation across the genus and patterns of incomplete lineage sorting in multiple loci Letharia populations comprise a conspicuous component of some epiphytic communities in the montane forests of western North America  J. W. Thomson and L. vulpina (L.) Hue, representing a classic example of a lichen species pair L. vulpina is recognized by the production of asexual reproductive structures – soredia and/or isidioid soredia – which contain both fungal and algal cells  instead   and L. vulpina s.l. [ = L. v. s. str. and L. ‘lupina’]. It should be noted that L. columbiana sensu stricto likely corresponds to one of the four provisional names applied to the fertile candidate species ; however, here we follow the provisional names provided by Kroken and Taylor for consistency Using a phylogenetic species recognition (PSR) approach, Kroken and Taylor Letharia is particularly germane given its conservation status in Fennoscandian countries Letharia using multilocus sequence data generated from individuals collected throughout its range. Specifically, we were interested to find if complementary analytical methods and additional specimen sampling supported the same species-level lineages inferred using PSR approach for species recognition in LethariaUnderstanding species boundaries in LethariaLetharia lineages.Analyses based on predefined groups may obscure patterns of differentiation Letharia specimens were included in the present study , Norway (2 site), Sweden (4 sites), and Turkey (2 sites) Letharia specimens collected across an array of altitudes, substrates, and microenvironments L. vulpina s.l. L. vulpina s.l. specimens bore apothecia in addition to vegetative propagules. Sequences representing the fertile taxon L. columbiana s.l. originated from a total of 29 specimens.Molecular sequence data from a total of 432 nt study . Our datnt study . The 51-Identical markers from independent studies were combined and sequences were aligned using the program MAFFT v.7 We used population assignment tests implemented in the programs STRUCTURE v.2.3.2 Letharia inferred under a PSR approach in K = 6 model (the number of putative species). We also explored population assignments for K values ranging from 1–8; with 10 replicate runs for each K value. Each run consisted of 50,000 burn-in generations, followed by 50,000 iterations using the admixture options. The ΔK method K value were combined using CLUMMP v.1.1.2 K = 6. Admixture analyses across inferred genetic groups were conducted using 500 iterations, with the minimum population size taken into account when admixture is estimated set to five, up to ten reference individuals from each population, and 20 iterations for reference individuals.Using the same data from n = 429). Clustering analyses in BAPS and STRUCTURE were performed as described above. We estimated population clusters from the combined dataset for K values ranging from 1–10; with 10 replicate runs for each K value. We used the ΔK method Letharia specimens (L. ‘lupina’ and L. vulpina) dominated our sampling, and sample sizes of putative lineages within the apotheciate taxon L. columbiana s.l. were disproportionally small. We used two approaches to assess the impact of unbalanced sample sizes in our complete dataset. First, we excluded all L. columbiana s.l. samples and inferred genetic structure only within the sorediate Letharia populations. In the second approach, we reduced the number specimens representing the ‘L. lupina’ and ‘L. vulpina’ groups to 30 individuals each, similar to the number of samples representing L. columbiana s.l. All ‘L. ‘lupina’ and L. vulpina from L. lupina’ and ‘L. vulpina’ groups inferred from the K = 2 model in the STRUCTURE analysis of the sorediate specimens  also failed to converge under a number of scenarios and provided inconsistent results (data not shown). Therefore, phylogenetic relationships were estimated from the combined 15-locus dataset using a total-evidence approach n = 429) using locus-specific model partitions in RAxML v.7.6.3 We attempted to estimate relationships among the six putative lineages defined in DFL. lupina’ and ‘L. vulpina’ clusters in DnaSP, and significance was determined using the coalescent process . Under the assumption of neutrality, negative values are expected in populations that have undergone recent expansion because rare alleles are more numerous than expected, while positive values occur if rare alleles are eliminated from populations due to genetic bottlenecks or diversifying selection L. ‘barbata’, L. ‘gracilis’, L. ‘lucida’, and L. ‘rugosa’ due to small sample sizes and uncertainty of boundaries among groups.We compared the mean pair-wise genetic distances within inferred genetic clusters to a previously identified intra- interspecific threshold in parmelioid lichens of 0.015–0.017 substitutions per site (s/s) for ITS sequences Letharia, potentially representing either paragolous gene copies or sequences of non-Letharia origin . The problematic BA sequences were excluded from subsequent analyses and were deposited in GenBank (accession numbers KJ610524–KJ610702).Sample sizes, alignment lengths, numbers of SNPs, percent missing data, and included loci for each analyzed data set is reported in n = 51) using STRUCTURE and a K = 6 model supported the same six putative species-level lineages previously recognized in Letharia, although in a number of cases individuals showed evidence of admixed ancestry , L. ‘gracilis’ (L. columbiana s.l.), L. ‘rugosa’ (L. columbiana s.l.), and L. vulpina (sorediate); the second representing L. ‘lupina’ (sorediate); and the third included L. ‘lucida’ (L. columbiana s.l.) specimens , the STRUCTURE analysis inferred two distinct clusters .The STRUCTURE analysis of the dataset excluding ectively . Howevera’ group . The ana 2 model . The ΔK le sizes ; Fig. S2 cluster . In contvulpina’ . The K =L. barbata’, ‘L. lupina’, ‘L. lucida’, ‘L. lupina’ and ‘L. vulpina’ – from the K = 6 model were recovered as monophyletic; and only ‘L. barbata’ and ‘L. lucida’ were moderately to strongly supported with 69% and 100% bootstrap values, respectively . Genetic diversity indices  for the two sorediate genetic clusters – L. ‘lupina’ and L. vulpina are summarized in L. ‘lupina’, suggesting demographic growth for L. ‘lupina’ versus stability for L. vulpina  with a diversification history apparently coinciding with climatic fluctuations during the Pleistocene Our data suggest a recent diversification history for lineages . Similarlineages . Althouga prioriLetharia failed to converge to a stable, stationary distribution. This may be the result of excessive splitting of populations or levels of genetic variation that are too low to accurately estimate parameter values. Similarly, relationships in the ML topology were recovered with weak statistical support  reported in Letharia lineages circumscribed using a gene genealogical approach (Due to the recent divergence history of approach . The oveK) K may be difficult, the ad hoc statistic ΔK, based on the rate of change in the log probability of data between successive K values in STRUCTURE runs, has been shown to provide an accurate perspective on the uppermost hierarchical level of structure in some cases K that captures the major structure of the data and seems biologically sensible. Although our results were congruent with the previous hypothesis of species boundaries in Letharia when assuming K = 6, both the BAPS analysis and the ad hoc statistic ΔK from results of the STRUCTURE analysis L. columbiana s.l. and L. vulpina s.l. The ad hoc ΔK approach for assessing results from the STRUCTURE analyses of n = 51) supported three distinct genetic groups  (L. columbiana s.l.) or normalizing sample sizes across groups resulted in varying inferences of K  and sorediate groups (L. lupina and L. vulpina) remain largely ambiguous   or normana s.l.) . We idences of K . While tmbiguous . Regardlmbiguous , but difmbiguous .K method support fewer than six genetic clusters, this study provides corroborating evidence that the six previously circumscribed Letharia species do in fact represent some level of genetically isolated groups K = 6 model, individual assignments were largely consistent across a range of different combinations of data . Similarly, the putative apotheciate species ‘L. lucida’ was also consistently supported as a distinct group in the analyses of normalized sample sizes across groups , based on 10 replicates per K, from the STRUCTURE analysis of the sampled sorediate Letharia specimens. C. Results from the ΔK analysis, following Evanno et al. K).(PDF)Click here for additional data file.Figure S2Likelihood and ΔK plots from the STRUCTURE analysis of the dataset with normalized sample sizes. A. Plot of likelihood values for each K (1–10), based on 10 replicates per K, from the STRUCTURE analysis of the sampled sorediate Letharia specimens. B. Results from the ΔK analysis, following Evanno et al. K).(PDF)Click here for additional data file.Table S1Collection and sequence information for all Letharia specimens included in the present study. GenBank accession numbers for sequences >200 base pairs (bp) are provided for sequences from Altermann Bolded GenBank accession numbers for the ‘BA’ locus represent sequences that were excluded from clustering analyses (see text).(XLSX)Click here for additional data file.