Polioptila californica californica) across its U.S. range. With a dataset of 268 individuals genotyped at 19 microsatellite loci, we analyzed genetic structure across the range using clustering analyses, exact tests for population differentiation, and a pedigree analysis to examine the spatial distribution of first-order relatives throughout the study area. In addition, we developed a habitat suitability model and related percent suitable habitat to genetic diversity indices within aggregations at two spatial scales. We detected a single genetic cluster across the range, with weak genetic structure among recently geographically isolated aggregations in the northern part of the range. The pedigree analysis detected closely related individuals across disparate aggregations and across large geographic distances in the majority of the sampled range, demonstrating that recent long-distance dispersal has occurred within this species. Genetic diversity was independent of suitable habitat at a local 5-km scale, but increased in a non-linear fashion with habitat availability at a broader, 30-km scale. Diversity declined steeply when suitable habitat within 30-km fell below 10%. Together, our results suggest that California gnatcatchers retain genetic connectivity across the majority of the current distribution of coastal sage scrub fragments, with the exception of some outlying aggregations. Connectivity may help support long-term persistence under current conservation and management strategies. However, emerging structure among more remote aggregations and associations between available habitat and genetic diversity also suggest that continued loss of habitat could threaten diversity and connectivity in the future.Habitat loss and fragmentation are primary threats to biodiversity worldwide. We studied the impacts of habitat loss and fragmentation on genetic connectivity and diversity among local aggregations of the California gnatcatcher ( Persistence of rare species in fragmented systems can depend on the extent to which local populations are linked both demographically and genetically by dispersal. Particularly for species with small, non-migratory populations, connectivity can be imperative for the maintenance of genetic diversity through gene flow (which is maintained by movement and successful reproduction), reestablishment after local extinctions, and avoidance of inbreeding depression3. Because habitat fragmentation can impede the dispersal capabilities of even volant species5, the identification of these restrictions to gene flow can be essential for long-term species conservation planning.Habitat loss and fragmentation are key factors that influence population loss and species extinction6. While radio or satellite tracking methods can alleviate some of these biases, transmitter and battery size limitations may make such methods untenable for small species7. Alternatively, estimates of population genetic structure have been used to assess population connectivity and indirectly estimate long distance dispersal9. However, in historically large or well-connected populations, the magnitude and detectability of recent changes to genetic structure can reflect other factors besides movement, such as population size, generation times, historical connectivity and the variability and mutation rates in the chosen genetic markers12. For these reasons, it can be difficult to determine whether a lack of detectable genetic differentiation among fragmented populations reflects high ongoing levels of dispersal and gene flow, or is a retained signal of historically high gene flow that does not yet reflect current levels of dispersal and gene flow15.Dispersal can be directly observed, or estimated indirectly. For example, banding and resighting studies are commonly used to estimate dispersal in small songbirds. However, study area size limitations in resighting or recapture surveys can systematically bias the obtained dispersal distributions, and tend to miss or underestimate long distance dispersal events17. Others incorporate temporal sampling to estimate population genetic structure before and after disturbance19, or compare levels of genetic connectivity in fragmented versus contiguous habitat20, or compare among species with different dispersal abilities21. Multilocus genotypes can also be used to statistically estimate recent migration rates22. However, where there is no detectable signal of genetic subdivision, these methods are of limited use23. In cases with no apparent genetic subdivision, an alternative method to distinguish between high ongoing gene flow versus a retained pattern of historically high gene flow is to estimate kinship among sampled individuals from highly variable genetic markers in a pedigree analysis24. This type of analysis can provide context about recent movement and gene flow by examining the geographic distribution of genetically identified first order relatives (full siblings or parents and offspring)26.Previous studies have used several techniques to help distinguish between historical and emerging contemporary genetic patterns. Some have assessed differences in correlations between historical versus current landscape connectivity patterns and genetic differentiationPolioptila californica californica; hereafter gnatcatcher). The gnatcatcher is a resident, non-migratory songbird with a range extending from Ventura County, California, to northern Baja California, Mexico, and is considered a flagship species for coastal sage scrub conservation in southern California27. It nests and forages almost exclusively in native coastal sage scrub habitat, an estimated 80–90% of which has been lost since European settlement28.We examined population genetic structure and estimated relationships within a federally threatened songbird, the coastal California gnatcatcher . Current estimates of the regional population size range between 1000 to 2000 pairs30. Previous behavioral studies of gnatcatchers in southern California found that birds were resident in breeding territories (average 8.1 ha) and non-breeding home ranges (12.4 ha)31, and that longer distance dispersal occurred mostly by fledglings dispersing away from natal territories32. Information on dispersal distance is limited, but a few mark-resighting efforts observed movements up to 14 km. Limitations to dispersal could make remaining aggregations vulnerable to impacts from habitat fragmentation. Therefore, efforts to determine population status and manage for persistence could be assisted by a better understanding of population connectivity across southern California. The goals of our study were to determine whether habitat fragmentation in the U.S. portion of the range has led to population genetic subdivision, and to estimate the geographic extent of recent dispersal. Results can inform future recovery management and monitoring efforts throughout southern California.The number of gnatcatcher pairs declined dramatically throughout southern California in the 1980s and 1990s, and as a consequence, the subspecies was listed in 1993 as federally threatened under the Endangered Species ActWe genotyped 268 gnatcatchers sampled throughout their U.S. range at 26 variable microsatellite loci Fig. . Seven o33 to further test for population subdivision among 18 regional aggregations, we found evidence for population differentiation at the more northern extent of the study area. This method can be more sensitive at detecting recently formed or weaker levels of genetic structure than clustering algorithms33, and so results could suggest subtle or emerging restrictions to gene flow in response to habitat loss and fragmentation. These results varied between analyses on the full dataset and the dataset excluding close relatives. Because the effects of close relatives on genetic structure analyses is a subject of debate34, we present both results here. Using the full dataset, five northern and fragmented aggregations were supported as distinguishable populations based on allele frequency differences , while all other collection locations from the eastern Los Angeles Basin through southern San Diego County formed a single population .Three different clustering methods provided no evidence for genetic structure in the study area in analyses on the full dataset . Thirty-nine putative first order relative pairs were recovered with a probability > 0.9. Thirty-four of these were consistently recovered in all three runs and retained for distance analysis , relatedness (r) and expected heterozygosity (He) were all highly correlated (r > 0.8). We selected Ar to represent genetic diversity in the modeling. Moran’s I measure of spatial autocorrelation was insignificant (p > 0.05) and we dropped latitude and longitude from our models. The number of samples from a sampling aggregation was not included in the models as it was highly correlated with the availability of suitable habitat within 5-km and 30-km buffers around the midpoint of sampling aggregations. There was no association between genetic diversity and the availability of suitable habitat at the 5-km scale. At the 30-km scale, however, availability of suitable habitat was positively related with Ar and best described by a logarithmic function that accounted for 53% of the variation in Ar  and 0.64 for the validation dataset . We hypothesized that within aggregation genetic diversity would be positively correlated with availability of suitable habitat if suitable habitat was associated with regional population sizes and connectivity. Given that the set of aggregated sampling locations was small (n = 18), we constructed and evaluated simple linear and non-linear models relating genetic diversity to environmental variables, including available suitable habitat within 5-km and 30-km surrounding aggregations, which we calculated from the habitat model. Measures of genetic diversity including allelic richness . Less often, however, is the spatial distribution of these relatives examined to provide additional information on the scale of recent dispersal. As our example indicates, it can be useful in this context.Conservation applications of population genetic data often focus on determining the impacts of anthropogenic changes to habitat availability and spatial connectivity on species genetic structure and diversity. However, both recent and historical population processes are reflected in a species’ genetic structure39. However, others have suggested that gnatcatchers do disperse through urban areas40, and a more recent regional analysis of gnatcatcher occupancy found no relationship between occupancy and patch size41. We recovered genetic signatures that are consistent with high connectivity and gene flow among most aggregations of gnatcatchers. Additional information from the distribution of first order relatives further supports very recent or ongoing gene flow in gnatcatchers across disparate aggregations. These findings suggest that gnatcatchers are able to move among most patches of suitable habitat given the current (or recent) spatial arrangement of coastal sage scrub habitat in southern California and their intrinsic movement and dispersal behavior. The possible exceptions are the outlying aggregations in more northern and isolated areas including Ventura, Palos Verdes, Chino Hills, Coyote Hills, and San Joaquin Hills. Relative pairs detected within three of these five aggregations  were exclusively found within these same aggregations and none shared with other locations, further supporting greater isolation of these locations. There is more urban development and less suitable habitat in the northern portion of the range, which may contribute to the population structure detected there. With the exception of Ventura, signals of differentiation among northern aggregations were lost when close relatives were removed from the dataset; however, this may reflect the reduced sample size and lower statistical power in these reduced datasets rather than non-random sampling in these relatively small aggregations34. The stronger signal of differentiation between Ventura and other sites may reflect both distance and isolation. There appears to be very little suitable habitat connecting Ventura to more southern aggregations based on the habitat model  scale suggests that areas with more suitable habitat likely have larger and more well-connected populations than those with less suitable habitat. While California gnatcatchers move through human modified landscapes, there appears to be a threshold in loss of suitable habitat where diversity within aggregations drops and population structure begins to develop. This was most evident in the northern part of the range where urbanization is highest and the amount of suitable habitat is lower. Lower diversity and greater isolation in the northern part of the study area may have importance as this is also the northern range edge for the species. Patterns of lower diversity at range edges are common among species and expected when habitat is not contiguous47. The total population of gnatcatchers exceeds the lower threshold, and may meet or exceed the upper threshold. This may bode well for continued long-term persistence of gnatcatchers in southern California. However, should habitat become further reduced and fragmented or local aggregations fluctuate dramatically in size, we could also see concomitant reductions in genetic connectivity and local genetic diversity over time. Such changes could be detected by regional population monitoring, which could benefit from coordinated efforts across San Diego, Orange, Western Riverside, San Bernardino and Los Angeles Counties, as birds in these regions form a cohesive genetic unit.Current recommended thresholds for effective population sizes are greater than 100 to avoid inbreeding depression in the short term and greater than 1000 to retain adaptive potential in the long termCampylorhyncus brunneicapillus sandiegensis), which has declined dramatically in abundance and has been impacted by genetic isolation within the same preserve network4. The cactus wren has lower maximum observed dispersal distances (up to 10 km), and requires mature cactus stands for nesting, which are patchy within coastal sage scrub habitat. This greater habitat specificity could contribute to greater observed population structure.We found that investigating the distribution of first order relatives was a useful strategy to help distinguish between recent gene flow and historical connectivity signals in population genetic data. This approach indicated that dispersal in California gnatcatchers may be less limited than previously estimated from banding studies. From a conservation perspective, it is encouraging that gnatcatchers retain genetic similarity and a large effective population size across the majority of their range in southern California. High estimated genetic connectivity and large effective population size are positive indicators that gnatcatchers could persist in southern California under current conservation and management strategies, which rely on a network of preserves. The exceptions appear to be more isolated northern aggregations where suitable habitat surrounding aggregations and genetic diversity within aggregations are lowest. Overall, recent habitat fragmentation seems to have had less impact on genetic connectivity in gnatcatchers than observed in a co-occurring songbird, the coastal cactus wren,  were provided by the San Diego State University Museum of Biodiversity. These muscle tissues provided sufficient quantities of genomic DNA for microsatellite library development. All extractions were performed with the DNA Tissue Extraction Kit (Qiagen), each with 20 μL of dithiothreitol added for a digestion step extended to 48 hours. Extracted samples were quantified on a Nanodrop spectrophotometer (Thermo Scientific) and diluted to a maximum of 50 ng/μL prior to PCR amplification.48, and we found 22 of the loci were variable. We also tested loci from previously-developed libraries, finding successful cross-amplification of three loci originally developed for the cactus wren (Campylorhynchus brunneicapillus sandiegensis)4 and one locus for the southwestern willow flycatcher (Empidonax traillii extimus)49. Samples were genotyped on an ABI 3730 DNA Analyzer using the GS600 size standard (Life Technologies) at Bio Applied Technologies Joint, Inc. in San Diego, CA.A shotgun library from one gnatcatcher was prepared using a Roche 454 Jr, providing 4,336 sequences with microsatellites. Of these, we screened 66 for variation using a three-primer technique50, and exact tests for Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium (LD) among loci were performed in GENEPOP51. Loci with inconsistent amplification or that did not consistently conform to HWE and LD expectations were eliminated from the dataset. Individual genotypes and location data are available as a U.S. Geological Survey Data Release 10.5066/F77D2SBP.We amplified 26 variable loci in three sets using the standard conditions of the Multiplex PCR Kit (Qiagen) with loci combined as indicated in Supplementary Table 52 and STRUCTURE53 to determine the number of gene pools. Both programs use genotypic data to assign individuals to clusters that conform to theoretical expectations for randomly mating populations (maximizing Hardy-Weinberg equilibrium and linkage equilibrium). GENELAND takes geographic relationships into consideration along with individual genotypic data, and can identify recently developed clusters54. Analyses were conducted using the uncorrelated alleles model with admixture, testing for clusters (K) between 1 and 10 with 1 million Markov chain Monte Carlo repetitions (MCMC) and a 20% burnin period. We followed the authors’ recommendations to use the uncorrelated alleles model in cases where isolation by distance is detected52. We explored genetic structure without geographic priors included in STRUCTURE53. We used the correlated alleles model with admixture for Ks between 1 and 10, with a burnin period of 100,000 MCMC steps followed by 1,000,000 additional steps, and 20 repetitions at each K. The top 10 highest likelihood runs at each K were analyzed using STRUCTURE HARVESTER55, which averages and graphs likelihoods across runs. We used CLUMPP56 to average results across runs, and these results were visualized in DISTRUCT57. We also employed an individual, centered principal components analysis (PCA) in the R package Adegenet58 to summarize patterns of genetic diversity among individuals without imposing a population genetic model. We plotted individuals by the first four principal axes to visualize patterns of differentiation (data shown for first 2 axes).We used a suite of individual clustering methods and exact tests for population genetic differentiation to determine genetic structure across the range. We used Bayesian clustering algorithms implemented in GENELAND33 which uses exact tests to determine significant differences in allele frequencies among putative populations. If no difference is found, populations are grouped. This method can be more sensitive at detecting recently formed or weaker levels of genetic structure than clustering algorithms33. We used 18 local aggregations to begin this analysis. These aggregations were defined as local groupings of 5 or more individuals within a maximum distance of 30 km and not separated by urban development. We chose to use a maximum distance of 30 km as it is approximately twice the maximum dispersal distance reported in mark recapture studies42 and the average distance found between first order relative pairs in this study (see results), and so could represent a contact distance between individuals that is relevant for population structuring. Because we were interested in capturing potential impacts of habitat fragmentation, if groups of individuals were separated by expanses of urban development, we retained these as separate aggregations even if the distance among them was less than 30 km. We conducted exact contingency tests for pairwise genetic differentiation between all aggregations in GENEPOP, using Fisher’s method to combine p-values across loci33. To limit the effects of individual loci on the overall test, p-values for individual loci were set to a minimum of 0.0001 prior to combining with Fisher’s method. Aggregations were assumed to be part of the same genetic population if the overall p-value for the exact test was greater than 0.01. We did not use an adjustment for multiple tests as this adjusted p-value is already restrictive. We also calculated pairwise FST among all aggregations in GenAlEx 6.50, and tested for an association between pairwise genetic and geographic distances using a Mantel test for matrix correlation in the program IBD 1.53.As an additional means of defining genetically differentiated populations we used the method of Waples and Gaggiotti38, we repeated clustering and genetic differentiation analyses using reduced datasets with one of each first order relative pairs removed (see below for sibship assignment methods). The removal of close relatives, however, has also been shown to reduce the precision of genetic structure analyses34. Hence, we report observed differences in these results between analyses on these two datasets.Because close relatives might be a source of bias in analyses of population genetic structure24 to identify putative sibship and parent offspring pairs within our dataset based on genetic similarity. For COLONY runs, we assumed an outbreeding model and a monogamous mating system, and coded all individuals as potential offspring. Known males and known females (based on field assignments) were coded as potential fathers and mothers respectively. Based on recent regional population size estimates of 1000–2000 pairs41, we set sampling percentages of parents at 5%. We applied the weak sibship prior, full likelihood model with the ‘very long run’ option (1 × 109 iterations). To examine stationarity, we conducted three independent runs with unique random number generator seeds and compared results.To estimate individual relationships and dispersal distances, we examined the spatial arrangement of genetically detected first order relatives (full siblings or parents and offspring). We used the program COLONY 2.0.6.2To assess accuracy in sibship assignments, we used the simulation module in COLONY to generate and analyze five replicate datasets using empirical allele frequencies and identical run conditions to our dataset. We set population size to 2000 with a 10% sampling rate  and measured the Euclidean and least cost path distances between them to estimate the scale of recent dispersal. Least cost path distances were calculated in ArcGIS 10.2.2 from a friction surface with values calculated as the inverse of a gnatcatcher habitat suitability model (see modeling methods below). We also noted whether relatives were found within the same or among different aggregations.2 model predicting suitable habitat for gnatcatchers60. Mahalanobis D2 measures the similarity between points in a modeled landscape and the multivariate mean for a set of environmental variables calculated at locations where the species is present61. The environmental data set consisted of a grid of climatic, topographic, and vegetation/land use variables for southern California. We used Geographic Information Systems (GIS) software (ESRI 2013) to calculate environmental variables at each point in a grid of points spaced 150 m apart  and only one location per 150-m × 150-m grid cell was used. We used gnatcatcher records compiled by the Carlsbad U.S. Fish and Wildlife Service Office67 to validate the models. The validation dataset included 3,225 spatially distinct gnatcatcher locations collected between 2000 and 2013.We used gnatcatcher occurrences and an environmental dataset for southern California to develop a partitioned Mahalanobis D68 code developed for previous modeling efforts69 to develop a partitioned Mahalanobis D2 habitat model for the gnatcatcher that included spatial subsampling and model averaging. To reduce biasing the model towards environmental conditions at locations with relatively large concentrations of gnatcatchers, we divided the range into six geographic areas: LA-Ventura; Orange; Riverside-San Bernardino; Western Riverside; San Diego Coastal; and San Diego Inland. We bootstrapped the calibration data by limiting each geographic area to 50 or fewer occurrences per iteration and performed a Principal Components Analysis (PCA) on each of 1000 samples69. We produced a final model by averaging the PCA output for all iterations after correcting for sign ambiguity70. We rescaled D2 from 0–1 to create a continuous habitat similarity index (HSI) and calculated an HSI value for each 150-m × 150-m grid cell. An HSI of “0” represents environmental conditions that are least similar to the multivariate mean for the species occurrence dataset used to calibrate the model , while a “1” represents those conditions that are most similar . We applied a threshold of 0.5 to define high and very high suitable habitat to calculate least cost dispersal paths and availability of suitable habitat within aggregations (see below). This HSI threshold is consistent with high and very high suitability classifications that were found to be positively associated with gnatcatcher occupancy in previous modeling efforts71.We modified SAS 9.4Ho) and expected (He) heterozygosity and the fixation index in GenAlEx 6.50173, and allelic richness (Ar), adjusted for sample size, in HP-RARE74. Average pairwise relatedness among individuals within aggregations (r)35 was calculated in GenAlEx. We examined relationships between these diversity indices and geographic location (latitude and longitude), sample size within aggregations (N) and percent suitable habitat surrounding aggregations. To calculate percent suitable habitat, we first mapped the mean center point of each of the 18 defined local aggregations. We extracted the amount of suitable habitat relative to the total land cover in 5-km and 30-km radius buffers around each of these points, and expressed this as percent suitable habitat. These radii were chosen to correspond to the median and mean dispersal distances that we inferred from distances among relatives. We used these dispersal distance estimates to determine areas that could include groups of interacting and potentially interbreeding individuals (populations).We ran correlation tests among the four genetic diversity indices, and for those that were highly correlated (r > 0.7) we selected one diversity index for modeling. Using R software75 we ran correlation tests between independent variables and retained the most biologically relevant variable in the model when two variables were highly correlated (r > 0.7). We used Moran’s I in ArcGIS 10.5 software to test for spatial autocorrelation between genetic diversity measures and environmental correlates. Simple linear and non-linear models of genetic diversity were fit with uncorrelated independent variables. We evaluated model fit based on scatterplots, residual plots, R2, and residual standard errors.We examined genetic diversity indices within 18 regional aggregations of gnatcatchers to determine how these might vary across the distribution and with local habitat availability. For regional aggregations, we calculated observed (e) for the total population supported by clustering methods. Effective population size was estimated in the program NeESTIMATOR 2.0176, using the linkage disequilibrium method, assuming random mating and using a minimum allele frequency of 0.01. Confidence intervals (95%) were obtained by jackknifing over loci. A second estimate of Ne was obtained from sibship assignments using the full likelihood method in COLONY24, assuming random mating, with 95% confidence estimates obtained from bootstrapping.We estimated the effective population sizes (NSupplementary Information


Predicted genetic consequences of asexuality include high intraindividual genetic diversity  and accumulation of deleterious mutations , among others. These consequences have been largely studied in parthenogenetic organisms, but studies on fissiparous species are scarce. Differing from parthenogens, fissiparous organisms inherit part of the soma of the progenitor, including somatic mutations. Thus, in the long term, fissiparous reproduction may also result in genetic mosaicism, besides the presence of the Meselson effect and Muller’s Ratchet. Dugesiidae planarians show outstanding regeneration capabilities, allowing them to naturally reproduce by fission, either strictly or combined with sex (facultative). Therefore, they are an ideal model to analyze the genetic footprint of fissiparous reproduction, both when it is alternated with sex and when it is the only mode of reproduction.Dugesia subtentaculata. We find that most individuals, independently of their reproductive strategy, are mosaics. However, the intraindividual haplotype and nucleotide diversity of fissiparous and facultative individuals is significantly higher than in sexual individuals, with no signs of Muller’s Ratchet. Finally, we also find that this high intraindividual genetic diversity of fissiparous and facultative individuals is composed by different combinations of ancestral and derived haplotypes of the species.In the present study, we generate and analyze intraindividual cloned data of a nuclear and a mitochondrial gene of sexual, fissiparous and facultative wild populations of the species mosaic Meselson effect). Interestingly, the different intraindividual combinations of ancestral and derivate genetic diversity indicate that haplotypes generated during periods of fissiparous reproduction can be also transmitted to the progeny through sexual events, resulting in offspring showing a wide range of genetic diversity and putatively allowing purifying selection to act at both intraindividual and individual level. Further investigations, using Dugesia planarians as model organisms, would be of great value to delve into this new model of genetic evolution by the combination of fission and sex.The intraindividual analyses of genetic diversity point out that fissiparous reproduction leaves a very special genetic footprint in individuals, characterized by mosaicism combined with the Meselson effect (named in the present study as the The online version of this article (10.1186/s12862-019-1440-1) contains supplementary material, which is available to authorized users. The fitness of an individual and its lineage largely depends on the number and viability of the offspring produced during its lifetime. In turn, the genetic background of offspring has a major role in their survival and adaptation, for example, when facing population bottlenecks or in the face of environmental changes , 2. ThusSexual reproduction can generate new allelic combinations in the populations through recombination and outcrossing, which can be either favored by selection or selected against , 4. ThisDiffering from sexual and parthenogenetic individuals, a zygotic bottleneck is absent in fissiparous organisms, and descendants inherit part of the soma of the progenitor, including somatic mutations. This adds a level of complexity since, in the long term, fissiparous individuals are predicted to show high levels of genetic mosaicism , 20, in Schmidtea mediterranea or several Dugesia species are indeed masters of regeneration [Planarians of the family Dugesiidae  show outstanding regeneration capabilities among the metazoans . Speciesneration , 25. Theneration , 27. Neoneration , 29. TheTherefore, besides being usually sexual, planarians can also reproduce asexually either by fission or by parthenogenesis, resulting in a group with an astonishing diversity of reproductive modes. Sexual individuals are simultaneous hermaphrodites  Fig. b. In genSchmidtea involves the alternation of parthenogenesis and sexual reproduction [Dugesia alternate fissiparity with sex  and strictly fissiparous populations  were known [An example of a species showing the whole variety of reproductive strategies  is re known –41. Howere known . A priorD. subtentaculata showing either sexual, fissiparous or putative-facultative reproductive strategies, to investigate the following predictions under an evolutionary framework: (1) the existence of high levels of mosaicism in purely fissiparous individuals as well as the absence of mosaicism in purely sexual individuals, (2) the existence of the Meselson effect and Muller’s ratchet in fissiparous individuals and their absence in purely sexual ones, and (3) if putative-facultative populations prove to be facultative, we would expect to find in their individuals a characteristic genetic pattern different from that in exclusively sexual or fissiparous populations.Here, we analyze the intraindividual genetic diversity, by cloning PCR products of two molecular markers , of individuals coming from a total of 10 natural populations of Our results demonstrate in the first place that individuals from the populations bearing sexual and fissiparous individuals are facultative. Moreover, the data obtained provide evidence for the existence of mosaicism in freshwater planarians accompanied by the Meselson effect in fissiparous and facultative individuals, but with no Muller’s ratchet. Additionally, our results point out that the combination of fissiparous reproduction with occasional sex results in an efficient way of generating high levels of genetic diversity controlled by selection that may act at two different levels , which represents an entirely novel model of genetic evolution among metazoans.D. subtentaculata, 5 sexual, 2 facultative and 3 fissiparous, covering almost the maximum area of distribution of the species, which includes Southern France, the Iberian Peninsula, Mallorca  and Northern Africa  and a fragment of the mitochondrial gene Cytochrome oxidase subunit 1 (Cox1). The TMED9 gene was selected because it is a single copy gene in the species Schmidtea mediterranea containing a long intronic region  [Individual total genomic DNA was extracted from the 32 ethanol-fixed specimens using the commercial reagent DNAzol  by following the manufacturer’s instructions. Two genomic regions were PCR-amplified for all the individuals: a fragment of the nuclear gene  751 bp) , 44. The 751 bp) . The ampAll the PCR products were purified using a vacuum system  and subsequently cloned using an HTP TOPO TA Cloning Kit for sequencing  following the manufacturers’ protocols. At least fifteen colonies per individual were amplified using the universal T3 and T7 primers. The sequencing reactions were run either in an automated sequencer (ABI Prism 3730) by the Unitat de Genòmica of Centres Científics i Tecnològics of the Universitat de Barcelona  or by Macrogen Corporation  using the same universal primers. Complementary strands of DNA were edited and assembled using Geneious version 10 .Schmidtea mediterranea available online in SmedGD datababe [We aligned the sequences obtained for both genes at the nucleotide level using the online software MAFFT version 7 . The alidatababe , 48. TheTaq DNA polymerase (2.28 × 10− 5) implemented in the PCR fidelity calculator web tool provided by Thermo Fisher [In a cloning experiment, DNA polymerases can introduce errors in the sequences during the first PCR , during the cloning PCR (amplification of each clone), or in the sequencing reaction. Errors due to polymerase mistakes during the cloning PCR or due to sequencing errors can be generally detected as double peaks in the chromatograms. However, polymerase mistakes during the first PCR cannot be detected in the chromatograms. To evaluate the impact of polymerase errors in our data, we calculated the average number of mutations per haplotype that could be due to polymerase errors during the first PCR of the cloning process using the error rate of o Fisher . For theo Fisher , and refHD) and nucleotide levels (π) for the two gene fragments and the intraindividual proportion of synonymous mutations (Ks), nonsynonymous mutations (Ka) and the ratio Ka/Ks (Ω). To test for significant differences in the estimates of genetic diversity and selection parameters between individuals depending on their reproductive strategy, we conducted an analysis of variance for each estimated parameter using the program Past3 [p-values obtained in the pairwise comparisons were corrected for multiple testing.We calculated the intraindividual number of different TMED9 alleles and Cox1 haplotypes using the program DnaSP v5 . We alsoam Past3 . We usedAn analysis of molecular variation (AMOVA) was performed to see how the genetic variation was partitioned within the different reproductive strategies. AMOVA was conducted with the software ARLEQUIN 3.5.2  using paWe took two different approaches to analyze the genetic data under an evolutionary framework: phylogenetic inferences and haplotype networks. Phylogenetic reconstructions were used to give directionality to the evolutionary processes, while haplotype networks were used to study the relationship between the alleles . We inferred the haplotype networks for the two genes for each individual and at the species level using the program Network version 4.6 . We firsDugesia species phylogenetically close to D. subtentaculata [D. hepta Pala, Cassu & Vacca, 1981 and D. benazzii Lepori, 1951. The degree of saturation of each alignment was assessed with the software Dambe [p-value< 0.01; Cox1: Iss = 0.129, Iss.c = 0.804, p-value< 0.01). The best substitution model for each analysis was determined using jModelTest2 [We inferred the phylogeny of each gene using Bayesian inference (BI) and maximum likelihood (ML) methods. Two taculata  were usere Dambe  using a re Dambe , 58, whidelTest2 . The BaydelTest2  with twodelTest2 . Two indThe results of previous works showed that sexual populations of Soller, Calobra and Bosque were diploid, while the fissiparous population of SantaFe was triploid , 42. TheAn average of 14 and 12 intraindividual sequences were obtained for the nuclear and the mitochondrial marker, respectively  that showed a minority haplotype highly differentiated from the majority one  and fissiparous individuals with a star-like pattern , and the other way around Fig. .The analysis of molecular variation (AMOVA) showed that the genetic variation in sexual populations was mostly explained by differences between populations  Fig. a. In facThe proportion of nonsynonymous mutations (Ka) for both genes within each individual was extremely low and not significantly different between the individuals of the three reproductive strategies when comparing the mean values obtained per reproductive strategy  yielded the same topology for each molecular marker (the supports for ML only varied to slightly higher supports when the rapid bootstrap strategy with more replicates was used). The sexual populations of Bosque, Calobra and Soller were recovered as the first to differ, forming three monophyletic clades  that were highly differentiated between themselves and from the rest of populations  showed a higher number of nuclear alleles than those expected for their ploidy level, indicating that they are mosaics. Differing from most individuals from fissiparous and facultative populations, the genetic pattern of exclusively sexual individuals was characterized at both nuclear and mitochondrial level by one majority allele  and few derived low frequency alleles . This pattern indicates that the majoritarian allele of each individual was most probably sexually inherited, while the rest of low frequency alleles may be the result of somatic mutations.Taq polymerase (2.28 × 10− 5), we estimated that a 75% of the alleles could bear one artifactual mutation. Thus, we applied a widely accepted correction method to our dataset to minimize this effect. Nevertheless, we found that most of the cloned alleles of the same PCR product were differentiated by many more mutations than those expected by mistakes of the Taq polymerase. This suggests that although certain mutations are probably artificial, most of these low-frequency alleles would have originated due to other factors, such as somatic mutations.It could be possible that some of the low-frequency alleles resulted from polymerase errors during the PCR reaction. Based on the error rate of the The appearence of somatic mutations during the lifespan of individuals has been detected in several sexual organisms, including humans , 63, andIn the case of fissiparous populations, we initially predicted the existence of mosaicism and the Meselson effect within individuals, due to the progressive accumulation of somatic mutations in the neoblasts over generations of fissiparous reproduction. The intraindividual nuclear divergent pattern . If part of this genetic diversity corresponded to paralogs or numts, we would expect that they would be equally present in individuals from sexual populations. Particularly, in individuals from sexual populations derived from fissiparous ones, such as Alte and Artavia. Nevertheless, they are not only absent from sexual populations in general but also from individuals from Alte and Artavia. A second caveat, as previously mentioned for sexual populations, is that some mutations of the alleles and haplotypes of fissiparous and facultative individuals could be due to mistakes in the activity of the polymerase. Nevertheless, they could only explain a minority of the intraindividual genetic diversity detected in these reproductive strategies, since most of these alleles and haplotypes are shared between different individuals or are so highly differentiated that it is extremely improbable that all mutations are a consequence of polymerase mistakes Fig. . Therefomosaic Meselson effect, which we define as the existence of a genetically heterogeneous cell population within the body of an organism, carrying highly divergent alleles in homologous genetic regions. Evidence for the occurrence of this effect in other fissiparous Dugesia species can be found in two studies that were focused on D. sicula and D. japonica [. Moreover, although this is the first time that the mosaic Meselson effect is suggested, it may also occur in other fissiparous metazoans such as star-fish and corals [Importantly, given that the number of highly differentiated nuclear alleles is, for most individuals, higher than its ploidy, we can deduce that these highly differentiated alleles are distributed across different cells, in contrast to what is found when the Meselson effect occurs in parthenogenetic individuals. We propose this variation of the Meselson effect to be referred as the japonica , 68. Mord corals , and in d corals –71. At tInterestingly, the evolutionary analysis revealed that the intraindividual genetic diversity of fissiparous and facultative individuals was characterized by the existence of a mix of ancestral and derived alleles at both the nuclear and mitochondrial levels Fig. . The capConsidering the evidence for the existence of mosaicism combined with the Meselson effect in fissiparous and facultative populations, we also expected to find a high incidence of Muller’s ratchet in individuals from these populations. But, contrary to our expectations, our results suggest that purifying selection is occurring in all individuals analyzed in the present study Fig. .These results point out that intraindividual cell selection could be responsible for the elimination of deleterious mutations during periods of fissiparous reproduction. Processes of intraindividual cell selection are well known to occur in insects and mammals due to different mechanisms . In this case, we would expect that all cells that do not need the expression of those genes (including neoblasts) will be able to carry mutated nonfunctional or less efficient copies, and we would detect a Muller’s ratchet effect. Indirect evidence for the expected result of this test comes from a comparison of genomic and transcriptomic data from a laboratory lineage of 20 years , where tD. subtentaculata are three sexual diploid populations while most populations within the monophyletic Mixed clade are triploid fissiparous or facultative , since they will not result in a loss of genetic diversity. All these reasons could help explain the evolutionary success of the genus Dugesia, with more than 85 species distributed in Africa, Asia and Europe [Schmidtea [Recurva [The evolutionary model based on the combination of fissiparous reproduction with different rates of occasional sex that we have proposed represents a paradigmatic mode of evolution for several reasons. While the somatic genetic diversity may be partially hidden from selection when fission is the only way to reproduce, the recovery of sexuality may expose this genetic diversity to selection in the offspring in different combinations  in fissiparous metazoans, specifically in planarians, an organism of complex tissue architecture. Furthermore, our results point out that the mosaic Meselson effect enables the existence of both ancestral and highly derived genetic diversity within the same individual, a genetic characteristic never described before and very interesting under an evolutionary point of view. Concomitantly, our results provide evidence that this special genetic diversity acquired during periods of fissiparous reproduction can be transmitted to the offspring through sexual events, allowing the generation of progeny with a huge range of genetic diversity and providing a scenario of possible multilevel selection . Further investigations using Additional file 1:Table S1. List of populations used in this study, including information on the reproductive strategy, locality and collectors. Table S2. Sequence, source and annealing temperature of the primers used in this study. Table S3. Results of the ploidy inference by flow cytometry. Table S4. List of the different TMED9 alleles obtained in this study. The individuals showing each haplotype and its corresponding GenBank accession number are indicated. Table S5. List of Cox1 haplotypes, with the individuals showing each haplotype and its corresponding GenBank accession number. Table S6. Genetic diversity and proportion of synonymous and nonsynonymous sites at intraindividual level for the two molecular markers analyzed in the present study. Table S7. Results of the statistical tests used to compare the intraindividual mean levels of genetic diversity and types of mutations between the different reproductive strategies. (PDF 407 kb)Additional file 2:Figure S1. Workflow of the ploidy level estimation using flow cytometry. 1) Cutting the animal in two pieces, 2) cell maceration, 3) DNA staining, 4) fluorescence measurement with a Gallios flow cytometer and 5) unknown-ploidy estimation by comparison with control. Figure S2. Approach used in this study for the correction of the putative artifactual mutations due to polymerase mistakes. In parentheses is indicated the frequency of each haplotype within the individual before and after the correction. Figure S3. Bayesian inference tree of all the alleles of the nuclear gene TMED9. The colors of the terminal branches indicate to which individual each allele belongs. The color of the outer circle indicates the reproductive strategy of each individual. Numbers at the nodes indicate the support values for the Bayesian inference (posterior probability)/the maximum likelihood (bootstrap). Bootstrap values correspond to the maximum likelihood analysis conducted with rapid bootstrap. Support values lower than 0.8 (posterior probability) and 75% (bootstrap) are represented with a -. Scale bar indicates the number of substitutions per site. Figure S4. Bayesian inference tree of all the haplotypes of the mitochondrial gene Cox1. Haplotypes of the same individual are pictured with the same color. Numbers in white indicate the different clades within the Mixed clade. Numbers at the nodes indicate the support values for the Bayesian inference (posterior probability)/the maximum likelihood (bootstrap). Bootstrap values correspond to the maximum likelihood analysis conducted with rapid bootstrap. Support values lower than 0.8 (posterior probability) and 75% (bootstrap) are represented with a -. Scale bar indicates the number of substitutions per site. (PDF 878 kb)


Pouteria sapota is known for its edible fruits that contain unique carotenoids, as well as for its fungitoxic, anti-inflammatory and anti-oxidant activity. However, its genetics is mostly unknown, including aspects about its genetic diversity and domestication process. We did high-throughput sequencing of microsatellite-enriched libraries of P. sapota, generated 5223 contig DNA sequences, 1.8 Mbp, developed 368 microsatellites markers and tested them on 29 individuals from 10 populations  from Mexico, its putative domestication center. Gene ontology BLAST analysis of the DNA sequences containing microsatellites showed potential association to physiological functions. Genetic diversity was slightly higher in cultivated than in the wild gene pool , although modified Garza–Williamson Index and Bottleneck software showed evidence for a reduction in genetic diversity for the cultivated one. Neighbor Joining, 3D Principal Coordinates Analysis and assignment tests grouped most individuals according to their geographic origin but no clear separation was observed between wild or cultivated gene pools due to, perhaps, the existence of several admixed populations. The developed microsatellites have a great potential in genetic population and domestication studies of P. sapota but additional sampling will be necessary to better understand how the domestication process has impacted the genetic diversity of this fruit crop. Pouteria Aublet (Sapotaceae) has 325 species, many of them known for their edible fruit and quality wood. It is a non-monophyletic genus composed of at least three distinct evolutionary lineages [P. sapota ((Jacq.) H.E. Moore & Stearn), commonly known as zapote mamey, mamey or mamey rojo [P. sapota is the tropical and sub-tropical evergreen forests distributed on Mexico’s Atlantic slope from North Veracruz to Tabasco state as well as in Mexico’s Pacific slope from Jalisco to Chiapas, continuing through Central America up to Panama, and from 0–1300 m above sea level [P. sapota have been recognized, mainly by fruit characteristics because wild fruits are significantly smaller than cultivated ones. However, there are no formal studies about the existence of subspecies or botanical varieties. The lowlands from Mexico and Central America are the putative center of domestication of P. sapota [The genus lineages ,2. One iea level ,5. Multi. sapota . Mexico . sapota . In this. sapota . At pres. sapota .P. sapota have been used for multiple medicinal purposes, including the effective control of the fungal skin dermatitis [Pouteria species [P. sapota and to what extend this may have impacted its genetic diversity in populations of vegetatively propagated fruit trees [Currently, there is abundant information about the chemical composition of zapote mamey for its high nutritional value as the red fruits contain unique ketocarotenoids, such as cryptocapsinepoxide  and sapormatitis . In fact species . In contit trees ,15. In fit trees ,17,18,19it trees ,21,22. TPouteria species, and data mining is not an option as there is only one entry of 443 bp (GQ402492.1) for P. sapota in NCBI, GenBank. In absence of sequenced genomes, microsatellite-enrichment of genomic libraries has been shown effective to develop molecular markers for genetic studies [P. sapota; and (2) to analyze, for the first time, the structure and genetic diversity of wild and cultivated populations of P. sapota collected in Southeast of Mexico, the putative domestication center of this species.Currently, there is a lack of molecular tools to study  studies ,24. Micr studies ,26. TherP. sapota microsatellite-enriched libraries of genomic DNA resulted in 5223 assembled contigs with an average length of 345 bp , and an average reads per contig of 4.4 . Until now, there was no information about the genetics of P. sapota. In the sequenced libraries we found 1452 contigs with significant hits in BLAST analysis; 28% of them with similarity to Vitis vinifera, 8% to Populus trichocarpa, 5.5% to Glycine max, and the rest had similarity to other tropical and non-tropical plant species. A total of 685 microsatellites were detected in 379 contigs using the software SSR Finder, 384 primer sets were designed and tested, 96% (368) of these primer sets resulted in effective amplification, and 205 SSR were polymorphic and amplified all 31 samples. Further analysis of SSR-containing contigs of polymorphic loci showed that 55 of the 368 developed SSR markers had significant hits  on BLAST to Gene Ontology (BLAST2GO) analysis  of the contigs containing microsatellites had more than one motif (between 2 and 6), in those cases the distances between repeats within each contig were between 0 and 343 bp. No significant correlation  was observed between the total repeat length (8–32 bp) and the polymorphism of the markers using 31 DNA samples for the 193 loci that had a single microsatellite per contig. The discriminating power of the markers was calculated with unique pattern informative combinations (UPIC) software [Individual contigs often presented more than one microsatellite, and these simple-sequence repeats were separated by one or more basepairs (bp). Apparently, when the distance in bp between two microsatellites within a contig was ≤10 bp , 64%–90% of the markers within that contig were polymorphic. When the distance between microsatellites within a contig was longer, for example 11–343 bp then, ≤50% of the markers derived from those contigs were polymorphic . Only 11software  and the P. sapota from Mexico  of a wild population of masaranduba (Manilkara huberi (Ducke) Standl) from Amazonas using SSRs [HE = 0.951, of 20 wild individuals of sapodilla (M. sapota (L.) P. Royen) from Mexico was estimated using SSRs [HE = 0.59, for three morphotypes of argan (Argania espinosa (L.) Skeels) from Morocco was determined using SSRs [HE = 0.35, for seven populations of Puerto Rican bully (Sideroxylon portoricense subsp. Minutiflorum Pittier) from Mexico was obtained using Inter Simple Sequence Repeats (ISSRs) [P. sapota from Mexico (HE = 0.34). However, we believed this estimation of the genetic diversity in these populations is accurate given the large number of loci (approx. 200) analyzed here, and the accuracy of allele calling (1 bp differences detected) when using capillary electrophoresis.All estimators of genetic diversity were slightly higher in the cultivated than in the wild gene pool of m Mexico . This ism Mexico . At presing SSRs . Heterozing SSRs . Further (ISSRs) . Though P. sapota could suggest that there was no founder effect during the domestication process of this species. However, when the founder effect was evaluated using the modified Garza–Williamson index, it was higher in the wild (0.49) than in the cultivated (0.42) gene pool . Another finding supporting a founder effect is that in 62 SSR loci analyzed, one or more alleles observed in the wild individuals, mainly in JC population, were not detected in cultivated individuals; this included markers stv-pos_1117, stv-pos_1602, stv-pos_1677, stv-pos_2282, stv-pos_2375, stv-pos_2461, stv-pos_2565, stv-pos_2755, stv-pos_2977, stv-pos_3228, stv-pos_3569, and stv-pos_3748  or cultivated . Individuals from populations AA and PJ (both wild), and AT (cultivated) were not clearly grouped by their geographic origin.Neighbor-Joining analysis showed a general clustering pattern according to the geographical origin of the populations. Eight of the 11 populations were clearly discriminated with high bootstrap values . The twoThe clustering pattern showed by the N-J analysis seems to be consistent with that obtained with the three-dimensional principal coordinate analysis (3D PCoA) . The twoP. sapota populations was further assessed on the basis of the assignment tests carried out with STRUCTURE. The Evanno method indicated an ideal K = 3 . The existence of admixed population shown by the STRUCTURE analyses is probably responsible for this result. These admixed populations hindered their grouping in the N-J and 3D PCoA analyses (P. sapota that were as close as 3 km apart (WH and JC) and many others that were between 28 to 112 km apart , it seems that the populations of analyses . In zapokm apart , Table 4P. sapota collected in Mexico. Of these, 20 individuals were collected directly in the field from seven wild populations and nine individuals were collected in traditional home gardens from three Mayan towns of the Yucatan peninsula  and fruit size. Also, we included two individuals from the collection at USDA-ARS Tropical Agriculture Research Station (TARS) in Puerto Rico. DNA was extracted from lyophilized young leaves using DNeasy Plant Maxi kit . The accessions of P. sapota from TARS collection were used to generate separate SSR-enriched libraries following the protocol of Techen [Analysis included 29 individuals of eninsula . The claf Techen , brieflyf Techen . The ligf Techen . Sequencf Techen . After bf Techen , and prif Techen  with strf Techen , BLASTn f Techen . P. sapota listed in A total of 384 primer sets were designed and used to screen 31 DNA samples of HO) and the expected heterozygosity (HE) with a level of polymorphism of 0.05, using ARLEQUIN version 3.5 [Considering the low number of individuals by population, we grouped the individuals collected in Mexico in two gene pools: wild and cultivated. Then, to allow comparisons between gene pools with different sample size, the average number of alleles per locus (A) was calculated using the rarefaction method implemented in HP-Rare version 1 . Also, wsion 3.5 .Founder effect due to domestication was estimated with the modified Garza–Williamson index  ,44, expeFST and a hierarchical AMOVA analysis [Genetic differentiation among wild and cultivated populations was analyzed using analysis . Geneticanalysis . A Manteanalysis ,49 was manalysis .a priori criteria, using each allele as an independent variable. (3) An assignment test of individuals was made with STRUCTURE 2.1 [The genetic relationships were inferred by three procedures. (1) A Neighbor-Joining (N-J) analysis was made using the standard genetic distance of Nei  and the TURE 2.1  using thTURE 2.1  using thTURE 2.1 . Bar graP. sapota, an important Neotropical fruit crop. We showed that the genetic diversity is slightly higher in the cultivated gene pool of P. sapota from Mexico in comparison with the wild populations, even though we found evidence of a bottleneck event in the cultivated gene pool, maybe as a result of the domestication process of this species. As suggested by the genetic structure and the cluster analyses performed in this study, wild and cultivated populations of P. sapota from Mexico show low genetic differentiation, perhaps, because of the existence of high levels of genetic flow favored by the production and propagation methods of zapote mamey practiced by traditional farmers from Mexico [It is very important to know about how the domestication process has impacted the extent and distribution of the genetic diversity in vegetatively propagated fruit trees. This work developed and used, for the first time, SSR codominant molecular markers to study this aspect in m Mexico . It is nPouteria is far from being clearly defined [Pouteria. MegaBlast and BLASTn analysis of the P. sapota DNA against the non-redundant database of GenBank (NCBI) for sequences not harboring microsatellites, showed similarity mainly to V. vinifera followed by P. trichocarpa and G. max. The same trend was observed when blasting microsatellite-containing sequences in BLAST2GO (Blast2GO 2009), which uses a different database. Considering that the genus Pouteria is non-monophyletic [Pouteria (Sapotacea) and Vitaceae is interesting and should be further explored. Finally, studies have shown the feasibility of elaborating testable hypotheses based on the biological function of genes related to polymorphic microsatellites [P. sapota.The SSR reported in this work have a great potential for other uses. For example, given the large number of species in banks of germplasm ; compar defined ; this ge defined , probabl defined  and amon defined , the 368tellites . Some po


Ambrosia trifida (Asteraceae).Plant invasions are increasing globally, and extensive study of the genetic background of the source and invading populations is needed to understand such biological processes. For this reason, chloroplast microsatellite markers were identified to explore the genetic diversity of the noxious weed A. trifida was mined for microsatellite loci, and 15 novel chloroplast primers were identified to assess the genetic diversity of 49 Ambrosia samples. The number of alleles amplified ranged from two to six, with an average of 3.2 alleles per locus. Shannon's information index varied from 0.305 and 1.467, expected heterozygosity ranged from 0.178 to 0.645, and the polymorphism information content value ranged from 0.211 to 0.675 (average 0.428). The cross‐species transferability of the 15 microsatellite loci was also evaluated in four related Ambrosia species .The complete chloroplast genome of A. trifida and related species.The novel chloroplast microsatellite markers developed in the current study demonstrate substantial cross‐species transferability and will be helpful in future genetic diversity studies of  Ambrosia trifida L.) belongs to the Asteraceae family  together with 40–50 other ragweed (Ambrosia L.) species . Both species are noxious weeds and interfere with the growth development and establishment of various crops , the amplifications were repeated using fluorescently labeled primers applying the PCR protocol described above using all 49 Ambrosia samples. PCR products were analyzed on an ABI 3730XL DNA sequencer  using a GeneScan 500 LIZ Size Standard (Applied Biosystems). The scoring of electropherograms was carried out with Geneious Prime . All primers amplified successfully across the test individuals and 10 proved to be polymorphic, whereas five gave monomorphic patterns  in North America and in the Rhône‐Alpes region (Genton et al., A. trifida population, the markers developed here should be suitable for further studies investigating the origin of invasive populations in Europe and studying the dynamics of invasion and modes of dispersal. Due to the high rate of cross‐amplification, the developed polymorphic cpSSR primers will likely be useful in intra‐ or interspecific genetic studies of the genus Ambrosia.We used the recently sequenced plastid genome of


Apium graveolens L.) is one of the most economically important vegetables worldwide, but genetic and genomic resources supporting celery molecular breeding are quite limited, thus few studies on celery have been conducted so far. In this study we made use of simple sequence repeat (SSR) markers generated from previous celery transcriptome sequencing and attempted to detect the genetic diversity and relationships of commonly used celery accessions and explore the efficiency of the primers used for cultivars identification. Analysis of molecular variance (AMOVA) of Apium graveolens L. var. dulce showed that approximately 43% of genetic diversity was within accessions, 45% among accessions, and 22% among horticultural types. The neighbor-joining tree generated by unweighted pair group method with arithmetic mean (UPGMA), and population structure analysis, as well as principal components analysis (PCA), separated the cultivars into clusters corresponding to the geographical areas where they originated. Genetic distance analysis suggested that genetic variation within Apium graveolens was quite limited. Genotypic diversity showed any combinations of 55 genic SSRs were able to distinguish the genotypes of all 30 accessions.Celery ( Apium graveolens L.) is a biennial species from the family of Apiaceae with 2n = 2x = 22 chromosomes. It originated from the Mediterranean basin and several cultivated types are grown worldwide for consumption. Besides the wild (Apium chilanse) species and celeriac (Apium graveolens L. var. rapaceum) species, both coming from Western countries, common celery (Apium graveolens L. var. dulce) cultivars are generally classification based on their origin as celery (cultivars introduced from Western countries), local celery (Chinese celery) and the middle type .Celery  test marker polymorphism on a set of celery cultivars; (2) assess the genetic variation existing in the materials used; (3) detect the genetic diversity and population structure of these materials and (4) explore the efficiency of the primers used for cultivar identification.A list of the samples investigated in this study is given in In the previous work  we minedWhen the 140 developed SSR markers were used for genotyping the set of 30 accessions, 23 markers had no clear bands or were amplified only in very few accessions. These markers were excluded from further analyses, reducing the number of good quality markers to 117 (83.57%). Among those good quality markers, 54 were monomorphic and 63 (53.85%) were polymorphic on the 30 accessions. The successful amplification rate and polymorphism rate were similar to those of our previous work  , though The number of loci per SSR ranged from two to five, with a mean value of 2.71, which was similar to that of previous studies on celery using EST-SSRs (2.68) , but lowThe number of loci per SSR and the PIC values in our study were low. Generally, it was believed that these indexes for genomic-derived SSRs were significantly higher than EST-SSRs, as indicated by the reports on flax , wheat , levant Ho) and expected heterozygosity (He). The former varied from 0 to 0.73 (mean 0.13), while the latter varied from 0.07 to 0.68 (mean 0.33). The mean Ho and He values were similar to the previous results of 0.14 and 0.36, respectively [He values showed that 85.48% of the markers were in the range from 0 to 0.3, which was a very low heterozygosity  for all pairs of horticultural types with at least two accessions per type  from var. dulce (cultivated celery). Compared to the wild species, var. rapaceum was closer to var. dulce, which may be due to the fact that the studied var. rapaceum belonged to cultivars. The observed distances between wild species, celeriac species and var. dulce accessions corresponded to the sexual compatibility of the two species with var. dulce accessions, which can be further proved by the fact that marker transfer rate was 100% in var. rapaceum, but only 54.84% in wild type. What’s more, both celeriac and wild species were the closest to the cluster of celery accessions, but the most distant from the cluster of local celery accessions. This result was supported by the differences in their origins.PCA analysis unambiguously separated the wild and the var. dulce and var. rapaceum was 0.34, with a range from 0.26 (C123) to 0.72 (C1). The largest genetic distance (0.72) was between C1 and C83, while the least genetic distance of 0.02 was found between C29 and C97. The average genetic distance of wild species was 2.42 with a range from 2.06 to 3.22, which was much larger than cultivated accessions (less than 0.7). Overall, seventy percent of the genetic distance between any two cultivars was no more than 0.4 and only thirty percent were larger than 0.4  and 1 μL of template (aprox. 20 ng/μL). PCR was performed as follows: denaturation at 94 °C for 5 min, followed by 38 cycles of 30 s at 94 °C, 30 s at Tm , 1 min at 72 °C and a final step at 72 °C for 10 min. PCR products were firstly detected by agarose gel electrophoresis and the products possessing single band or only a few bands were subjected to 7% polyacrylamide gel to separate alleles. With regard to those had no bands or multiple bands, we optimized the PCR condition to get better products for separation of alleles. PCR products were mixed with a volume of loading buffer and then denatured at 95 °C for 10min before being loaded on the polyacrylamide gel.The SSR markers were developed through celery transcripotme sequencing . PrimersHo) and expected heterozygosity (He) were calculated using the Popgene software version 1.31 [Ho represents the estimated proportion of observed heterozygotes at a given locus for co-dominant markers. He, estimated using the Levene algorithm [SSR alleles were scored manually starting from the smallest to the largest-sized bands. The presence or absence of each single fragment was coded as 1 or 0, respectively, and scored for a binary data matrix. Scored data from polymorphic loci were used to calculate the polymorphism information content (PIC) according to Equation (1):ch locus . Observeion 1.31 . Ho reprlgorithm , represeAnalysis of molecular variance (AMOVA)  between A genetic similarity matrix was constructed and Nei’s genetic distance  was calcIn order to see whether scoring more loci is likely to increase the genotypic diversity, or whether one has reached a plateau, we used the software MultiLocus ver. 1.3b  to estimThis was the first attempts at celery genetic and genotypic diversity analysis using SSR markers developed from transcriptome sequencing. The AMOVA analysis indicated that the largest part of genetic diversity was within populations, while genetic diversity found among populations was low. The geneetic distance of wild species was much larger than that of cultivated accessions, suggesting the wider genetic diversity of the wild species, while the diversity within cultivars was quite limited. PCA analysis revealed that accessions of the same horticultural types were well clustered together. The UPGMA dendrogram and population structure clearly separated wild species from cultivars, and further divided the cultivars into two clusters, corresponding to the geographical areas from where they originated. Genotypic diversity analysis suggested that 29 markers were needed to identify 99% of genotypes and any combinations of 55 SSR markers were able to distinguish genotypes of all 30 accessions. Given that the genetic similarity of commonly used accessions was high, we need to develop more and higher polymorphic markers to efficiently distinguish closely related varieties. This study would provide a common ground for celery accessions identification, breeding and protection of breeders’ rights.


Aedes koreicus was recorded out of its native range for the first time in Belgium in 2008. Since then, several other European populations or single individuals have been observed throughout Europe with reports from Italy, Switzerland, European Russia, Slovenia, Germany and Hungary. The Italian population seems to be the only one that is expanding rapidly, so the Swiss population very likely derives from it.The East Asian mosquito species Ae. koreicus was found in a cemetery vase in 2016 in the city of Wiesbaden, Germany. In the following year the finding was confirmed and an established population could be proven over an area of about 50 km2. The morphological identification of the first larva was confirmed by sequencing of a region within the nad4 sequence. A study of adult females showed that the morphological characteristics of this population are not identical to the populations from Belgium and Italy. The eggs and larvae were found together with Aedes j. japonicus in the same breeding sites and ovitraps, as well as with other indigenous mosquito species such as Culex pipiens/Culex torrentium, Aedes geniculatus and Anopheles plumbeus.In a surveillance program for invasive mosquito species, a single larva of Since the newly discovered population in Germany shows different morphological characteristics to the populations in Belgium and Italy, it seems to originate from an independent introduction. It remains unknown how the introduction took place. A further spread similar to the one in northern Italy can be assumed for the future due to similar climatic conditions.The online version of this article (10.1186/s13071-018-3199-4) contains supplementary material, which is available to authorized users. Aedes (Hulecoeteomyia) koreicus (Edwards) is native to Korea, north-eastern China and eastern Russia [Aedes (Hulecoeteomyia) japonicus japonicus , which has spread over North America and central Europe in recent years [Aedes koreicus is supposed to have a similar potential for becoming introduced to remote countries [Ae. koreicus was shown to be present over an area of about 3000 km2 [Ae. koreicus is spreading quite dramatically in northern Italy, comparable to the expansion of Ae. j. japonicus in Germany [Ae. koreicus in Europe come from Russia [The container-breeding mosquito species n Russia . It appen Russia  only front years . Aedes kountries . Indeed,ountries . It is dountries . In 20133000 km2 . The pat3000 km2 , 9. Ther3000 km2 . This sh Germany , 12  and Anopheles plumbeus (Stephens) in the townships. In 2013, a surveillance program was initiated to check water containers in the cemeteries of the KABS member municipalities for the presence of Ae. j. japonicus.aponicus  and showaponicus . The firtzerland , 22. Sintzerland , 23 and tzerland , 24. TheAe. koreicus was found in 2016 in Wiesbaden, a city in the northern part of the area. In the following year, a survey was started to validate this finding and to evaluate the establishment and distribution of the population. Additionally, a detailed morphological study was performed for the discrimination of the species to Ae. j. japonicus and to identify the morphological characteristics and therefore the presumptive origin of the population.Within this program, a single larva of Ae. j. japonicus. Within this framework, cemeteries of the KABS member municipalities were investigated once a year and all larvae found in cemetery vases and water basins were collected and stored in glass vessels containing 50 ml of 70% ethanol. The collections usually took place in late August or early September, depending on rainfall and temperature which influence the hatching and speed of development. The larvae were morphologically identified in the laboratory using a stereomicroscope with a magnification up to 100×, following the keys of Becker et al. [In 2013, the KABS started a surveillance program for the early detection of invasive mosquito species, primarily r et al. , Mohrig r et al.  and TanaAe. koreicus using the descriptions by Tanaka [During this surveillance program, a single larva collected from a cemetery in the city of Wiesbaden in September 2016 was morphologically identified as y Tanaka . The larAfter this detection, a survey was started in Wiesbaden the following year in order to investigate whether the larva was part of an established population and to evaluate the actual distribution of the species. The city is situated in the Rhine-Main area, about 20 km west of Frankfurt/Main, next to the river Rhine and bound by the low mountain range High Taunus in the north-west and the mouth of the River Main in the south-east. It has a temperate climate with an annual mean temperature of 10.4 °C and an average annual precipitation of 653 mm  , 28. TheAe. koreicus was collected in the Sonnenberg District, located north-east of the city center; therefore this point was chosen as the center of the investigation area. Nine sampling sites were determined within a radius of 2 km, including two cemeteries, three locations in the forest, three private gardens and one industrial area to find the range of the infested area in the surroundings of the first detection. The elevation of these sites ranges between 180–260 m.The first larva of Between one and five ovitraps were placed at each site, depending on the size and environmental conditions. Locations near bushes and hedges hidden from passers-by and also allowing rainwater to refill the traps were preferred. The traps were assembled from black plastic trays (25 × 18 × 14 cm)  with brown clay flowerpots (17 × 14 cm diameter) as oviposition substrate which were placed upside down inside the trays. Initially the traps were filled with 2 l of tap water and protected from overflowing by a hole 4 cm below the rim at each short side. During the investigation period the water levels were maintained by both rainwater and manual refilling when sampling the traps.Ovitraps were collected three times between July and October 2017. Eggs were removed from the flower pots with a soft brush and put into 500 ml of tap water for hatching. Hatched larvae as well as the larvae directly collected from the ovitraps were reared to the fourth-instar, killed and stored in 70% ethanol until identification and counting of the larvae. About 100 individuals from five different sampling sites were reared to adult stage for morphological investigation of the females. Cemetery vases and water basins at the Sonnenberg cemetery were monitored twice, on July 4 and August 14.Additionally, the routine surveillance program was continued in 8 other cemeteries in Wiesbaden. The cemeteries were sampled once at the beginning of September. Their elevation ranged between 88–230 m.via the sequencing of a fragment of the mitochondrial nad4 gene in addition to morphological identification. DNA extraction  was performed according to the manufacturer’s protocol and the extracted DNA was kept frozen at -20 °C until further use.The larva collected in 2016 was identified nad4 sequence was amplified by PCR [A region within the d by PCR  using thAn initial denaturation (30 s at 94 °C) was followed by 35 cycles of denaturation at 94 °C for 15 s, annealing at 53 °C for 45 s and elongation at 68 °C for 60 s. The cycling ended with a final elongation step at 68 °C for 5 min.via agarose gel electrophoresis  the amplicon was prepared for sequencing using the Monarch PCR & DNA cleanup-kit (New England BioLabs) following the manufacturer’s protocol. Sequencing was carried out by Eurofins Genomics .After checking the size of the PCR product Females were studied to determine the morphological characteristics of the introduced specimens. In Belgium and Italy the morphological variant from the Korean island Jeju-do was reported , 6, whicAnother examined difference was the characteristic pattern of lines on the scutum, which had a silver-white color in the Belgian population, whereas all Korean individuals showed yellowish-brown or golden-yellow scales. Although the pattern of pale scales on the abdominal terga revealed a high degree of variation, the Jeju-do and Belgian specimens sometimes missed the basolateral patches, whereas the basomedian patches were usually very thin .Ae. koreicus and Ae. japonicus, additional characters were examined comparatively using individuals of Ae. j. japonicus collected in Wiesbaden and other locations in the range of the south-west German population (see next section). These are the scutal pattern [Ae. j. japonicus, the presence of pale scales on the costa and the coloration of the hindfemur [Since there seem to be some uncertainties in the discrimination of the two closely related invasive species  pattern , the scaindfemur   was present at all nine sites. Vases and water basins in the Sonnenberg cemetery were positive for larvae of Ae. koreicus, Ae. j. japonicus, Cx. pipiens/Cx. torrentium and An. plumbeus. In the ovitraps, numbers of larvae of Ae. koreicus ranged between 3–131 with an average number of 50 larvae per positive trap . Altogether, 16 of the 27 ovitrap samples were positive for Ae. koreicus. In these traps, the species was associated with Ae. j. japonicus twice, with Cx. pipiens/Cx. torrentium nine times and with both species four times. On both sampling dates in August, 7 ovitraps were positive for Ae. koreicus whereas in October only 2 ovitraps were positive, both of which had been found positive before. The ovitraps in the Sonnenberg cemetery were positive on all three sampling dates  geniculatus (Oliver) (3/8) and An. plumbeus (6/8) were also found. In 2 cemetery sampling sites, all 5 species were present. In the city of Mainz south of the Rhine, Ae. j. japonicus was found in 5 of the 12 cemeteries screened in 2017. All 12 cemeteries were negative for invasive mosquito species (IMS) in the year before.Ongoing sampling in the framework of the KABS surveillance program in Wiesbaden outside of the 2 km radius showed 3 more cemeteries to be positive for ies Fig. . Culex pnad4 region resulted in a sequence of 381 bp (GenBank: MK069483). The output of the BLAST search showed 99% identity with sample sequences of Ae. koreicus from Germany (KT962063) and Belgium (JF430392), 100% similarity with a shorter sequence (333 bp) from Belgium (KJ623735) and 99% identity with shorter sequences from Belgium  and from Korea . The closest similarity to Ae. j. japonicus was 92% similarity to a sample sequence from USA (DQ470164).The sequencing of the amplified Ae. koreicus and showed a dark subbasal band in all examined females of Ae. j. japonicus  albopictus (Skuse) and Ae. j. japonicus, the introduction could be due to the transport of plants or used tires [Ae. koreicus and most of them negative for Ae. j. japonicus. Furthermore, Kampen et al. [Ae. koreicus further westwards when sampling cemetery vases and other breeding sites, but they did detect Ae. j. japonicus in the area of Wiesbaden.Together with the three newly discovered sites in the city of Wiesbaden in the routine surveillance program of the KABS in 2017, the area colonized by ed tires , 33. In ed tires . Betweenn et al.  did not Ae. koreicus in Italy shows that the species not only spreads actively, but also seems to be transported passively by trade of goods and road vehicles to more remote areas. It is assumed that the expansion to the south and west is mainly due to dense road connections in these directions [Ae. koreicus is apparently also distributed by vehicle transport. In 2015, the species was detected on highway E35 in ovitraps for Ae. albopictus surveillance north of the Alps [Ae. albopictus [Ae. koreicus has been made in the surveillance program along the German highways coming from Switzerland [The distribution of rections . In Switbopictus . So far,tzerland   and from Slovenia [Ae. koreicus was found as larvae only in 2013 among samples of Ae. j. japonicus. In 2014 and 2015, only Ae. j. japonicus could be found. It seems to be unlikely that Ae. j. japonicus displaced Ae. koreicus because both species are reported to occur sympatrically in Korea, colonizing the same breeding sites, with Ae. koreicus having a better adaptation to urban areas [Ae. koreicus and Ae. albopictus was observed [Ae. koreicus and Ae. j. japonicus in Germany.The coexistence of Slovenia , but no an areas . In Italobserved , 39, whiAedes koreicus could not only be found in suburban areas with a high number of gardens and rural areas, but also frequently in and near forests. Similar results were observed by Baldacchino et al. [Ae. koreicus in forested than in urban areas with gravid traps which should reveal the preferred breeding site habitats, as with ovitraps. This shows that the species is not only restricted to the townships but can also colonize natural habitats.o et al. , who cauAe. koreicus in Belgium was described as the morphological variant from the Korean island Jeju-do [The population of  Jeju-do . The aut Jeju-do  and Tana Jeju-do . In WiesIn the individuals examined from the new population in Wiesbaden, the pedicel scales were mainly pale, while in Belgium they showed a characteristic pattern of dorsal and lateral pale spots. The erect fork scales on the vertex were usually completely dark, which is also described from the Korean mainland. The scales on the antepronotum were all broad and pale, while in Belgian and Jeju-do individuals crescent-shaped scales were also present. The main difference between the two forms is found in the basal pale bands of the hind tarsus . The indAe. koreicus in Germany was not introduced from the Belgian or Italian/Swiss populations but originates from an independent introduction, most likely from the Korean peninsula and not from Jeju-do island. In this case, this is the first evidence for multiple introductions of this species to Europe. It is also possible that the Italian, Russian, Slovenian, German and Hungarian introductions were independent of the Belgian population, while the Swiss population very likely descends from the Italian one [Ae. j. japonicus to North America [Ae. koreicus. By characterizing the German individuals as a different morphological variant, derivation from the Belgian or Italian/Swiss populations can be excluded. This is also the first report of the morphological variant of Ae. koreicus from the Korean mainland outside of its native range. In European Russia and Hungary, the collected specimens were not characterized morphologically [This leads us to the conclusion that the newly emerged population of lian one . There i America  and Euro America , 41, so ogically , 18 and ogically .Aedes koreicus could clearly be identified and distinguished from Ae. j. japonicus morphologically, which is widely distributed and abundant in south-western Germany. Even though there is a high intraspecific variability in some of the examined characteristics, the morphological comparison revealed less variable and only slightly overlapping characteristics that allow an unambiguous identification. The main diagnostic characters for the distinction of the two species were the base of the hind femur, the pale basal scales on hind tarsomere IV and the subspiracular patch.Ae. koreicus, while all individuals of Ae. j. japonicus showed a dark subbasal band Additional file 2:Table S2. Results of the morphological study. Examined features with numbers of observed characteristics of Ae. koreicus and Ae. j. japonicus females. (PDF 276 kb)Additional file 3:Figure S1. Dorsal view of the scutum (a) and abdomen (b) of Ae. koreicus. The arrow shows the anterior dorsocentral line. (TIF 4283 kb)Additional file 4:Figure S2. Larval characteristics of Ae. koreicus (left) and Ae. j. japonicus (right). a Pecten. b Apical spines on saddle. (TIF 2898 kb)


Carcharhinus sorrah and the Blacktip shark Carcharhinus limbatus are among the most exploited species. In this study, we investigated the population genetic structure of C. sorrah (n = 327) along the coasts of the Arabian Peninsula and of C. limbatus (n = 525) along the Arabian coasts, Pakistan, and KwaZulu‐Natal, South Africa, using microsatellite markers . Our findings support weak population structure in both species. Carcharhinus sorrah exhibited a fine structure, subdividing the area into three groups. The first group comprises all samples from Bahrain, the second from the UAE and Yemen, and the third from Oman. Similarly, C. limbatus exhibited population subdivision into three groups. The first group, comprising samples from Bahrain and Kuwait, was highly differentiated from the second and third groups, comprising samples from Oman, Pakistan, the UAE, and Yemen; and South Africa and the Saudi Arabian Red Sea, respectively. Population divisions were supported by pairwise FST values and discriminant analysis of principal components (DAPC), but not by STRUCTURE. We suggest that the mostly low but significant pairwise FST values in our study are suggestive of fine population structure, which is possibly attributable to behavioral traits such as residency in C. sorrah and site fidelity and philopatry in C. limbatus. However, for all samples obtained from the northern parts of the Gulf (Bahrain and/or Kuwait) in both species, the higher but significant pairwise FST values could possibly be a result of founder effects during the Tethys Sea closure. Based on DAPC and FST results, we suggest each population to be treated as independent management unit, as conservation concerns emerge.The increase in demand for shark meat and fins has placed shark populations worldwide under high fishing pressure. In the Arabian region, the spot‐tail shark  During this event, approximately 23–15 million years ago, small isolated water pools formed along The Gulf's seafloor, which are thought to have had an important effect on the origin, dispersal, and speciation of several elasmobranch groups , South Africa, the UAE, and Yemen . Samples from South Africa originated from sharks caught in mesh nets as part of a bather protection program . A breakdown of sex and size composition for all samples is available in Supporting Information Table C. sorrah females with a total of 78 pups were collected from Deira fish market, Dubai, the UAE. These samples were not included in the population structure analysis but were instead used to detect null alleles by checking for genotype mismatches  between pups and their known mothers , characterized by a deep‐V hull design, portable fuel gallons, and an engine >400 horse power. Tissue sampling was randomized by collecting no more than ten samples of each species on the same day. The only exception was Pakistan where landings of Total genomic DNA from Red Sea samples was extracted following the protocol described in Spaet et al. . DNA fro2.2C. sorrah, DNA amplification was performed using 15 species‐specific polymorphic loci . All samples were scored blindly, without knowledge of the sampling location to avoid any unintentional bias. To account for genotyping errors, we used standard likelihood‐based methods as implemented in the program GENEPOP  and a test for linkage disequilibrium were performed using ARLEQUIN  adjustment in R  for each species. Five runs were generated per K value tested, with K ranging from 1 to 10. Simulations were run with a burn‐in period of 100,000 steps, followed by a Markov chain Monte Carlo (MCMC) iteration of 100,000 steps. The length of the burn‐in period was verified by ensuring that the Ln P(D) and the likelihood of the runs had stabilized. A correlated allele frequency model was used with sampling site as location prior and admixture were assumed, as recommended when population structure is likely to be subtle , which is a model choice criterion to select for the true value of K  was performed using the R package adegenet. DAPC has the advantage of using predefined clusters identified using a clustering algorithm. It optimizes variance among these clusters, while minimizing the variance within them to illustrate differences between them .Isolation by distance (IBD) was tested using a Mantel test implemented in the R package adegenet. The geographic distance between locations was measured in kilometers using Google Maps . Measures of geographic distance were taken as straight lines drawn along the coast and then plotted against genetic distances (corrected AIc) , under three possible mutation models: the infinite allele model (IAM), the single‐step mutation model (SMM), and the two‐phase model (TPM).33.13.1.1Ho ± SE) ranging from 0.64 ± 0.16 in Bahrain to 0.69 ± 0.15 in Yemen. Allelic richness ranged from 3.5 ± 0.4 (Bahrain) to 3.9 ± 0.4 (the UAE and Yemen). The value of FIS, an inbreeding coefficient measure that calculates the proportion of the variance in the subpopulation contained in an individual  and pups (n = 78) than the rest of loci   . All pairwise comparisons involving Bahrain showed high genetic distance, irrespective of geographic distance (data not shown).The DAPC scatterplot supported weak fine‐scale genetic differentiation into three groups. The first group comprises all samples from Bahrain, the second from the UAE and Yemen, and the third from Oman Figure a. A neig3.1.2HO = 0.73 ± 0.04) and lowest for Bahrain (HO = 0.62 ± 0.05). Allelic richness did not greatly differ between sampling locations, ranging from 3.6 ± 0.3 (Yemen) to 4.3 ± 0.5 (Bahrain)   Table . The proFST = 0.01, p < 0.001) . Yet, all pairwise measures involving Bahrain and Kuwait showed high genetic distance, irrespective of geographic distance (data not shown).The DAPC scatterplot also supported population subdivision between three groups. The first group comprises all samples from Bahrain and Kuwait, the second from Oman, Pakistan, the UAE and Yemen, and the third from South Africa and the Saudi Arabian Red Sea )   Figure a, yet a AIc for C. limbatus was similar among sexes ; W = 18,951, p = 0.008, Wilcoxon's rank‐sum test)  in both species  a variation in sample size among sampling locations (n = 51–96)   . Furthermore, the seasonal variation in water temperature in The Gulf is largest for Bahrain and Kuwait , indicating that temperatures are less stable at Bahrain and Kuwait ,  Castro, . This suC. sorrah and C. limbatus, respectively , which could promote population closure on finer geographic scales. The distinctiveness of all samples from Bahrain and Kuwait from all other sampling locations could be the result of founder effects during the Tethys Sea closure. Overall, our study suggests that conservationists and resource managers should treat each of the three mentioned groups as separate conservation units.Findings of this study have contributed to our understanding of the population structure of None declared.C. limbatus and C. sorrah from Kuwait, Bahrain, UAE, Oman, Yemen, and Pakistan, analyzed and interpreted the data, and wrote the manuscript. Julia Spaet provided tissue samples for C. limbatus from Saudi Arabia and critically reviewed and edited the manuscript. Geremy Cliff provided tissue samples for C. limbatus from South Africa. All authors approved the final version of the manuscript.Dareen Almojil designed the study, collected samples for https://doi.org/10.5061/dryad.811nr62.The entire dataset used in this study has been deposited in the Dryad Repository,  Click here for additional data file.