Tests for isolation by distance (IBD) are the most commonly used method of assessing spatial genetic structure. Many studies have exclusively used mitochondrial DNA (mtDNA) sequences to test for IBD, but this marker is often in conflict with multilocus markers. Here, we report a review of the literature on IBD, with the aims of determining (a) whether significant IBD is primarily a result of lumping spatially discrete populations, and (b) whether microsatellite datasets are more likely to detect IBD when mtDNA does not. We also provide empirical data from four species in which mtDNA failed to detect IBD by comparing these with microsatellite and SNP data. Our results confirm that IBD is mostly found when distinct regional populations are pooled, and this trend disappears when each is analysed separately. Discrepancies between markers were found in almost half of the studies reviewed, and microsatellites were more likely to detect IBD when mtDNA did not. Our empirical data rejected the lack of IBD in the four species studied, and support for IBD was particularly strong for the SNP data. We conclude that mtDNA sequence data are often not suitable to test for IBD, and can be misleading about species’ true dispersal potential. The observed failure of mtDNA to reliably detect IBD, in addition to being a single-locus marker, is likely a result of a selection-driven reduction in genetic diversity obscuring spatial genetic differentiation. When individual dispersal distances are smaller than a species’ range, genetic drift acting on neutral genetic markers will over time result in a positive correlation between genetic differentiation between locations and the geographic distance separating them, a pattern known as “isolation by distance” (IBD)5. IBD is currently the most frequently tested hypothesis in studies of spatial genetic structure, and is considered to provide a suitable means of indirectly estimating a species’ dispersal ability7. The method is also considered important in the management of exploited species. For example, in high-dispersal marine species in which the existence of clear stock boundaries is unlikely, tests for IBD can help to define regional geographic units that respond to exploitation relatively independently8.Species with wide distributions are rarely completely panmictic9. It has been suggested that the power of other approaches  is far greater than that of Mantel tests in detecting a relationship when one is present10. Another problem, and one that affects IBD per se rather than merely the way in which it is identified, concerns its use under inappropriate conditions. Most prevalently, IBD can be strongly affected by the existence of distinct regional genetic clusters11. For example, secondary contact between populations that have been isolated from each other in glacial refugia12 or the existence of sister lineages in adjacent biogeographical regions13, will give the impression of limited dispersal ability even when each population is genetically homogeneous within its range. The fact that some regional genetic clusters display physiological adaptations to the region they occupy, and some are morphologically distinguishable from each other15 suggests that significant IBD may often be the result of inappropriately lumping distinct cryptic species. When these regional units are analysed individually20 or when stratified Mantel tests are applied and locations of populations are permuted within clusters21, the pattern of IBD is often no longer found, and the species’ true dispersal potential is revealed. Nonetheless, due to their simplicity and the fact that geographic distance can serve as a proxy for numerous other factors that limit gene flow between locations, tests for IBD will likely continue to be used as a standard method of assessing a species’ dispersal ability. As such, they represent a simple baseline method against which to test more complex landscape genetic approaches22.Despite their popularity, tests for IBD are often criticised. One recent debate concerns the use of Mantel tests in assessing relationships between genetic and spatial data24, but the implications of this on tests for IBD remain poorly explored. With the increased application of next-generation sequencing approaches in species-level studies, the information content of the markers that can be used for IBD has increased by several orders of magnitude. It is thus timely to determine how traditional markers such as single-locus mtDNA sequence data or a limited number of microsatellite loci perform relative to the much larger SNP data sets.One aspect of IBD that has received little prior attention is the question which genetic markers are most suitable to detect it, and under which conditions. Numerous studies have dealt with the fact that the power of different genetic markers to detect genetic structure can differ considerably, and may result in conflicting findingsad-hoc Google Scholar search using the search terms DNA barcoding, isolation by distance and mitochondria returned 17 800 hits. Although not all these studies would have conducted tests for IBD using this single locus, the result suggests that the use of mtDNA in tests for IBD is very common, and, despite the existence of more powerful alternatives, remains a standard method to assess species’ dispersal potential. We then compared microsatellite and selectively neutral single nucleotide polymorphism (SNP) datasets generated using double-digest restriction site associated DNA sequencing  with mtDNA data in four cases where no IBD was found with the latter marker. The data sets used in this study originate from widespread marine species with high dispersal ability  along continuous coastlines. The finding that the alternative markers consistently found strong evidence for IBD when none was found with mtDNA when accounting for the existence of spatially distinct population clusters suggests that the exclusive use of the latter can result in questionable conclusions about species’ dispersal ability.In the present paper, we searched the literature to get an overview of the performance of mtDNA compared to other markers. An 26), and along the coast of temperate southern Australia. The aim was to determine whether significant IBD is mostly the result of inappropriately pooling distinct regional genetic clusters . We only included species with high dispersal potential  because in low-dispersal species (i.e. whose offspring remains in the parent habitat) genetic structure is often not clearly linked with contemporary biogeography27. We also searched for (b) studies that compared IBD between mtDNA and microsatellites. For (b), various combinations of the following search terms were used in Google Scholar to identify suitable papers: isolation by distance (or isolation by geographic distance), IBD, mitochondria (or mtDNA), discrepancy, contrasting, conflicting, microsatellite. A table was then constructed for each, and general trends discussed.To gain an overview about the performance of mtDNA in identifying IBD, we searched for (a) studies that used this marker to test for IBD along the South African coastline , and two South African teleosts (the goby Psammogobius knysnaensis and the sardine Sardinops sagax). These species were selected because (a) comprehensive range-wide genetic data sets were available and (b) none exhibits mtDNA-based genetic structure, even though co-distributed species with similar dispersal potential may comprise multiple regional evolutionary lineages28. The mtDNA data were obtained from the following studies: N. atramentosa and S. diemenensis26; P. knysnaensis29; S. sagax: B. Chiazzari, unpubl. data. Data from other markers included microsatellites for the Australian gastropods , and SNP data for the South African teleosts. The latter were generated for the present study. Briefly, DNA was extracted from fin clip or muscle tissue using the cetyltrimethylammonium bromide (CTAB) method32. Double-digest restriction site associated DNA sequencing (ddRADseq) was performed at the Molecular Ecology Laboratory at Flinders University following the protocol of ddRADseq.25 with restriction enzymes SbfI and MseI and modifications as described in Brauer et al.33. Libraries were sequenced on an Illumina HiSeq 2000 platform at the McGill University and Genome Québec Innovation Centre. BayeScan v2.034 was then used to identify and exclude loci potentially under selection on the basis of having significantly elevated allele frequency differences between populations. We specified default settings and a false discovery rate of 5%. In order to create data sets comprising selectively neutral loci, we excluded 203 outlier SNPs identified with BayeScan from the total sardine data set  and 572 outlier SNPs from the goby data set , and used only the remaining loci to test for IBD. Sample sites for all four species are shown in Fig. We compared IBD in four marine species that occur along continuous marine regions in temperate southern Australia and South Africa. These include two Australian gastropods  simple linear regression. The Mantel tests were performed with the R package adegenet 1.4–042. This program tests for significant IBD by comparing the observed correlation with a histogram of simulated correlation categories and their frequency under the assumption of no IBD. We selected Spearman rank correlations43 and 999 matrix permutations. The program also creates scatterplots of geographic vs. genetic distance, that depict the density between points. Densities between points in a scatterplot can be used to determine whether the data originated from a single genetic cline or from two or more distinct regional clusters for each of which the test for IBD should be performed separately11. To confirm that the neutral SNP data did not comprise multiple genetic clusters, the k-means clustering algorithm in adegenet was used to identify the number of clusters that maximise between-cluster variation. Simple linear regression to corroborate the results of the Mantel tests was performed in SigmaStat 1.0 .Relationships between genetic and geographic distance matrices were calculated using (a) a Mantel testThe datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.Parechinus angulosus, based on mtDNA data), but this result was considered unreliable because of a small sample size20.A synthesis of studies on the South African and southern Australian coastlines indicated that significant IBD was found much more frequently when these tests were performed on data sets that originated from multiple (M) biogeographical regions than when tests were performed within single regions (S) Fig. . This trAcanthochiton garnoti, Cyclograpsus puncatus, Oxystele tigrina, O. variegata20 and Tetraclita serrata44 in South Africa, as well as Donax deltoides45 and Haliotis rubra46 in Australia.Particularly clear examples of significant IBD that was no longer found when data from individual biogeographical regions were analysed separately include A comparison of IBD between mtDNA and microsatellites in studies that used both markers revealed that marker-specific discrepancies are common, and were found in almost half of the studies reviewed Fig. . MicrosaF-statistics between sites identified no IBD for any of the mtDNA data sets .The Mantel test results were confirmed by linear regression analysis, with the exception of the microsatellite data of 22, we reanalysed the sardine SNP data set , and the power of the latter was only slightly below that of the complete data set (0.97).As IBD analyses can be affected by the number of sites49. It has been used rather uncritically ever since, and it is still not fully understood why it is often in conflict with nuclear markers23, although the smaller effective population size50, which results in a higher rate of lineage sorting51, is often cited. It is likely that the increasing popularity of DNA barcoding will result in an increase in the number of studies that will exclusively use mtDNA sequences to test for IBD, as it is now possible to readily apply this approach on entire communities (e.g.54). For that reason, a critical assessment of the power of mtDNA in detecting IBD is timely.Tests for isolation by distance that employed mtDNA sequence data became popular in the 1990s, when this became the marker of choice because of its faster mutation rate and smaller genome size compared to allozymes47, and non-significant results questionable. It is noteworthy that several additional studies we reviewed reported IBD for only one of these markers even though both were used in other analyses58, perhaps because the other was in conflict. Intuitively, one would expect a comprehensive data set of hypervariable microsatellites to be more suitable to detect IBD, not only because of their high mutation rate60 but also because different combinations of alleles arise in each generation and are sorted over geographical space61, making microsatellites useful to study spatial patterns at geographic scales as small as tens of meters63. As such, it is possible that some of the studies in which significant IBD was found with mtDNA data, but not microsatellites, may be a result of the latter employing microsatellite loci with comparatively little variation and/or a small number of loci, in addition to potential technical artefacts such as large allele dropout and null alleles. Any general conclusions about the usefulness of microsatellites to detect IBD is thus hampered by the fact that, unlike mtDNA sequences, the microsatellite data sets used in different studies are not directly comparable. However, a detailed assessment of the factors responsible for this discrepancy is beyond the scope of the present meta-analysis. Our results for the Australian gastropods, which employed highly informative microsatellite data sets suitable to detect self-recruitment31, and for which the same sites and individuals were used as for the corresponding mtDNA data, represent an important contribution towards facilitating more direct comparisons between the two marker types. In the light of these findings, it is clear that reliable results at a minimum require congruence between multiple marker types.Our review of studies that used mtDNA together with microsatellites indicated that the two markers were often in conflict, with microsatellites more likely to identify IBD when mtDNA did not. We are aware of no studies that employed power analyses to test for the probability of type II error, but linear regression analyses of our empirical data  hypothesis, which assumes a saturation plateau beyond which genetic diversity no longer increases75 may describe mtDNA evolution more adequately. At the population level, the low level of mtDNA diversity is a result of strong variation-reducing selection that may include strong purifying selection76 or selective sweeps driven by physiological constraints within environmentally homogeneous regions77. Interestingly, this selection not only affects non-synonymous sites but also third character codon positions where mutations do not result in amino acid changes80. Although there is no consensus, explanations such as linkage of sites affected by synonymous mutations to regions under intense selection84 or codon biases during translation86 have been invoked. The consequence of such processes , is that mutations and reversions in mitochondrial genes at the population level are typically limited to relatively few sites, and homoplasies are common87. In the context of IBD, it is possible that the same mutations arising independently across the physiologically constrained range of a population may obscure already limited neutral genetic differentiation driven by geographic distance. It has been suggested that the combination of variation-reducing selection at the population or species level, and adaptive selection at the between-species level, makes mtDNA particularly powerful for the molecular differentiation of species because it increases the ‘barcoding gap’87. However, the very departures from the expectations of selective neutrality that are so useful for DNA barcoding may be partly responsible for making this marker unsuitable to detect IBD.Mitochondrial DNA sequence data originate from a single locus and, unlike in microsatellite data, there is thus no differentiation in the frequency of alleles from different loci from one generation to the next that can generate spatial genetic structure. Nonetheless, the high mutation rate of mtDNA compared to nuclear DNA88. This suggests that for selectively neutral loci, marine populations should also have very high genetic diversity, and this has been confirmed using nuclear DNA92. However, as explained in the previous paragraph, the assumption of selective neutrality does not apply to mtDNA. A meta-analysis of >1600 animal species91 showed that within-species (or population level) mtDNA diversity is very similar across animal phyla, irrespective whether they have large or small census population sizes, or whether they are aquatic or terrestrial. An explanation for the low genetic diversity of large populations is provided by Gillespie’s77 ‘genetic draft’ hypothesis, which states that beneficial mutations are more likely to arise in large populations, and these result in recurrent selective sweeps that reduce genetic diversity at the mitogenome level. Low variation in animal mtDNA, irrespective of census population size, range or habitat type has been confirmed by numerous phylogeographic and DNA barcoding studies97. These considerations indicate that there is little reason to assume that the failure of mtDNA to reliably detect IBD is limited to marine species, and this is confirmed by the fact that of the taxa used for Fig. 98 that may be under divergent selection, the artefact of identifying IBD when distinct populations are pooled shown in Fig. Many marine species have very large census population sizes22. Together, these factors indicate that a sampling design hampered by insufficient financial commitment, capacity and sampling effort may result in spurious results that can be misleading about a species’ realized dispersal. Future research needs to focus on the amount of data required to reliably identify IBD when it is present. Our novel SNP data indicate that high throughput sequencing approaches may be suitable to resolve the problem of discrepancies between mtDNA and nuclear markers evident in numerous previous studies. The presence of significant positive relationships between genetic and geographic distance in species where no such relationship was evident for mtDNA data was particularly clear for the neutral SNP data, where the power to correctly detect IBD was maximal and the probability of a type II error was zero. However, given the costs that are presently involved in generating and analysing genome-wide SNP data, this type of marker could be seen as a standard against which less informative but more affordable genetic markers could be measured. We conclude that mtDNA data can be problematic when testing for IBD, as they often do not provide reliable information on a species’ true dispersal potential.In addition to the issues in identifying IBD that are related to marker type and evolutionary history, the power to detect IBD is also related to the number of populations included in the analysisSupplementary Information


Meoma ventricosa and its parasitic pea crab Dissodactylus primitivus) exhibits comparable population genetic structures in the Caribbean Sea and how the observed patterns match connectivity regions from predictive models and other taxa. Highly contrasting patterns were found: the host showed genetic homogeneity across the whole studied area, whereas the parasite displayed significant differentiation at regional and local scales. The genetic diversity of the parasitic crabs (both in microsatellites and COI) was distributed in two main groups, Panama–Jamaica–St Croix on the one hand, and the South‐Eastern Caribbean on the other. At a smaller geographical scale, Panamanian and Jamaican parasite populations were genetically more similar, while more genetic differentiation was found within the Lesser Antilles. Both species showed a signature of population expansion during the Quaternary. Some results match predictive models or data from previous studies  while others do not . The sharp dissimilarity of genetic structure of these codistributed species outlines the importance of population expansion events and/or contrasted patterns of gene flow. This might be linked to differences in several life history traits such as fecundity (higher for the host), swimming capacity of larval stages (higher for the parasite), and habitat availability (higher for the host).Evolution and population genetic structure of marine species across the Caribbean Sea are shaped by two complex factors: the geological history and the present pattern of marine currents. Characterizing and comparing the genetic structures of codistributed species, such as host–parasite associations, allow discriminating the relative importance of environmental factors and life history traits that influenced gene flow and demographic events. Using microsatellite and Cytochrome Oxidase I markers, we investigated if a host–parasite pair (the heart urchin  Sea level fluctuations, related to the Quaternary climatic oscillations since 2.5 million years ago (Ma), changed the geography and ecology of the region. Eight climatic cycles have been recorded since 800,000 years ago  and microsatellites, we addressed the following questions: (1) Do host and parasite exhibit comparable genetic structures? (2) Do these structures correspond to the connectivity regions from predictive models in the Caribbean area? (3) What are the respective contributions of Quaternary sea levels fluctuations   and HCO2198 (5′‐TAAACTTCAGGGTGACCAAAAAATCA‐3′) , the effective number of haplotypes (Ne), the haplotype diversity (h), the mean pairwise differences among haplotypes (MPD), and the nucleotide diversity (π). Haplotype networks (Minimum Spanning Networks) were constructed using MINSPNET  in order to find the grouping that maximizes the genetic variance among groups. Analysis of molecular variance  a Spatial Analysis of MOlecular VAriance , using the software Mantel 1.19 (life.bio.sunysb.edu/morph/soft‐mult.html). The geographical distance corresponded to the shortest distance avoiding islands/strips of land and was calculated using the path tool in Google Earth. IBD was performed for the whole dataset and inside the “Lesser Antilles” connectivity region (excluding St Croix) defined by Kool et al. (We tested isolation by distance (IBD) with a Mantel test  The sum of squared deviation (SSD) between the observed distribution of the number of nucleotide differences and the unimodal mismatch distribution expected from population expansion , using a pairwise divergence rate of 1.52% per million years  for the present dataset. The retained parameter values were selected according to the instructions of POWSIM manual .For FIS) using FSTAT. The significance of FIS was evaluated for each species using permutation tests: one testing for heterozygote excess and the other testing for heterozygote deficiency.Using Genepop 4.2.2, the frequency of assumed null alleles was calculated, and linkage disequilibrium was tested for each locus pair within each species Rousset, . We usedFST  between different populations using SPAGeDi 1.4 . For M. ventricosa, we also estimated FST values adjusted for null alleles with the software FreeNA . We evaluated the possibility of isolation by distance (IBD) by a Mantel test (FST vs. km) using the software Mantel 1.19. IBD was performed for the whole dataset and inside the “Lesser Antilles” connectivity region (excluding St Croix) defined by Kool et al. , alpha inferred, allele frequencies correlated among populations, and possibility of null alleles. For D. primitivus, we used STRUCTURE with values of K between 1 and 14 and 10 independent simulations, using the following parameters: running chain lengths of 100,000, admixture model , alpha inferred, and allele frequencies correlated among populations. We determined the most likely value of K using the original method  and the method of Evanno, Regnaut, and Goudet , we used STRUCTURE 2.3.4  separated by 10 substitutions . Group 1 included Panama, Jamaica, St Croix, Saint Barthélemy, Antigua, and Guadeloupe, and group 2 was composed of Martinique, Bequia, Canouan, and Barbados. ΦCT values for other possible patterns of population structure were close . K = 3 is associated with the segregation of Barbados and K = 4 with the segregation of Antigua. ΦCT values from AMOVA (among regions) were equal to 0.666 (p < .001) for D. primitivus and 0 for M. ventricosa (p = .53) (Appendix Φte Table . None waus Table . All ΦST 1 Table . Jost's Appendix . For D. Appendix .ST vs. km) showed a correlation between genetic and geographic distances, indicative of isolation by distance for the whole dataset  and when only the Lesser Antilles were considered  .The Mantel test for crabs (ΦAppendix . All theD. primitivus (seven groups), Fu's FS was negative and significant for two groups , and mismatch analyzes did not reject the null hypothesis of pure demographic expansion nor a spatial expansion for all groups . Mismatch analyzes rejected the null hypothesis of pure demographic expansion  but did not reject the spatial expansion null hypothesis . Assuming a divergence rate of 1.52% per million years for COI   for each locus for the large majority (96%) of sampling sites. Conversely, two loci in M. ventricosa  had null allele frequencies higher than 0.10 in most of the sites. In D. primitivus, there was no heterozygote deficiency in any population, whereas five populations showed heterozygote excess  in M. ventricosa. In D. primitivus, the mean Allelic Richness (AR) was 7.2 (±0.7) and did not significantly differ among sites . In M. ventricosa, the mean AR was 6.3 (±0.2) and did not significantly differ among sites .In both ) Narum, . For D. ss Table . In M. vKB Table . The aveFST results of the microsatellites were different between the two species . FCT values from AMOVA (among regions) were equal to 0.026 (p < .0001) for D. primitivus and 0.00001 for M. ventricosa (p = .42) (Appendix es Table . The larot Table . Only thot Table . In D. p 0 Table . The higAppendix .FST vs. km), both for the whole dataset  and within the Lesser Antilles . No isolation by distance was detected for sea urchin populations for the whole dataset  or within the Lesser Antilles .Isolation by distance of microsatellites in crab populations was detected by Mantel tests  of genetic clusters was one. In D. primitivus, the most probable K was four for the original method and two for the Evanno method. The bar plot for K = 2  were identified  across the entire investigated geographic area.The two interacting species exhibit highly contrasting genetic structures within the Caribbean Sea. The genetic diversity of the parasitic crab M. ventricosa is unknown while the one of D. primitivus is approximately 2 weeks .Both species exhibited signs of population expansion but a more recent expansion or a larger population size for the sea urchin host might explain the sharp contrast between the two species. It is likely that there are different magnitudes of gene flow taking into account the dissimilar dispersal abilities of the two species. Whereas both adult crabs and sea urchins are able to move, such movements are very local (tens of meters). Therefore, dispersal capacity is related to pelagic larval stages. It is conventional to consider pelagic larval duration (PLD) as the main contributor to dispersal distance, although the relationship of PLD with genetic structure varies between species Dawson, . The PLDM. ventricosa across the Caribbean Sea suggests such a lack of local adaptation potential. There are no data available for comparing parasitic success or infection consequences among populations of the pair M. ventricosa–D. primitivus. It would be interesting to acquire such data and compare them with those already obtained in Jamaica . This cannot be explained by geographical distance, because the distance between Martinique and the Grenadines  is comparable to the distance between Martinique and Guadeloupe. A potential explanation is linked to the different speeds of currents flowing between Guadeloupe and Martinique vs. Martinique and the Grenadines  drifting in currents with speeds of several tens of centimeters per second, as it was demonstrated in other decapod larvae  despite its geographical proximity to other islands. Moreover, some cases of gene flow calculated between Barbados and its neighboring islands were asymmetric. This corroborates the study of Roberts , who est4.4M. ventricosa is a coral‐associated organism that could have thrived with the coral reef expansion during these interglacial times  (Miller et al., BD, CDR, HAL, QJ, RAW, TR conceived and designed the experiments; BD, CDR, HAL, QJ, SM, TR performed the sampling; MB and QJ performed the laboratory work; BD, CDR, HAL, QJ, RAW analyzed the data; BD, CDR, HAL, RAW, TR contributed reagents/materials/analysis tools; BD, CDR, HAL, MB, QJ, RAW, SM, TR wrote the manuscript.None declared. Click here for additional data file.


Montenegrina is the second most speciose door snail genus in Europe. It is an obligate rock-dwelling land snail and has, compared to its complex systematics, a rather small distribution range in the western parts of the Balkan Peninsula. The different taxa themselves show a very narrow and patchy distribution range. As Montenegrina is comprehensively sampled over the whole distribution range, it is a perfect study system for general questions on speciation and morphological differentiation in land snails. To study the amount of gene flow between geographically close or co-occurring populations, highly polymorphic markers are needed.Clausiliidae (door snails) are gastropods with a very high diversity concerning shell morphology, especially of their complex closing apparatus, which provides the most important diagnostic traits for classification of taxa. Due to the high variability, a high number of taxa has been described, though their systematics and taxonomy is partially controversially discussed. Montenegrina populations . The number of alleles per locus ranged from 2 to 27. No significant linkage disequilibria between the same two loci were found in all three tested populations. The deviation from Hardy–Weinberg equilibrium reveal only for two loci a significant deviation from HWE in more than one population (Mont_5483 and Mont_4477).Thirteen microsatellite loci with a tetranucleotid repeat were isolated and tested in three geographically close Montenegrina and might reveal new results about speciation processes in co-occurring taxa. Furthermore, these microsatellite loci could also be tested in other clausiliid species.The 13 newly established genetic markers will help to gain better insights to the population genetic structure of  The gastropod family Clausiliidae (door snails) is one of the most speciose among land snails and is distributed in four continents with diversity hot-spots in southern Europe, southeastern Asia and the northwestern part of South America . Their eMontenegrina is an obligate rock-dwelling door-snail genus comprising 106 taxa [Montenegrina a perfect study system for general questions on speciation and morphological diversification of rock-dwelling gastropods. Due to the high morphological variability species delimitation is sometimes challenging in Montenegrina, especially in the case of co-occurring morphotypes. Therefore, estimating the amount of gene flow is crucial.Montenegrina on the basis of next generation sequencing data and tested them in two species, which occur very close together (about 25 km apart). (1) Montenegrina dofleini prespaensis Nordsieck, 1988 occurs along the western and southern shores of the Prespa Lake . For this study we selected specimen from two populations that live in close vicinity: MprP from Psarades (15 specimens) and MprPE from Panagia Eleousa Cave (20 specimens). (2) Montenegrina stankovici  lives along the eastern shore of Lake Ohrid (Macedonia). The studied population MstSV was collected in Sveti Naum (20 specimens).To test for possible gene flow between populations or morphotypes and to get insights into population structure in general, highly polymorphic nuclear genetic markers are needed, which can be applied to a high number of individuals at moderate cost. Therefore, we established 13 microsatellite loci for For DNA extraction we used the Qiagen spin column kit  and followed the manufacturer’s protocol. Microsatellite markers were established by ecogenics GmbH  based on seven individuals. Size-selected fragments from genomic DNA were enriched for SSR content by using magnetic streptavidin beads and biotin-labeled GTAT, GATA, AAAC and AAAG repeat oligonucleotides. The SSR-enriched library was analysed on an Illumina MiSeq platform using the Nano 2 × 250 v2 format. After assembly, 13,656 contigs or singlets contained a microsatellite insert with a tetra- or a trinucleotide of at least six repeat units or a dinucleotide of at least ten repeat units. Suitable primer design was possible in 2905 microsatellite candidates, of which 36 were positively tested for functionality and polymorphism. This was done with fluorescent labelled M13 tails that were added to the forward primers . The priM. d. prespaensis and 20 of M. stankovici—were genotyped. For each locus the observed (Ho) and the expected (He) heterozygosity as well as linkage disequilibria and the deviation from Hardy–Weinberg-equilibrium (HWE) was calculated per population in Genepop 4.2 [Montenegrina species, further alleles are found at this locus. Consequently, the 13 microsatellite markers are suitable for population genetic studies in the rock-dwelling land snail genus Montenegrina.In total 55 individuals—35 of epop 4.2  (Table 2epop 4.2 . Thus, tepop 4.2 . The resMontenegrina. We expect to test gene flow between syntopically occurring congeners, as well as among parapatric and allopatric populations of conspecific taxa. We hope that this will promote the better understanding of speciation patterns and speciation processes in obligate rock-dwelling gastropods. Furthermore, they could also be tested in other clausiliid species.The newly established genetic markers will be useful in many further investigations within


Pachyrhizus Rich. ex DC.) are leguminous crops native to South and Central America that are grown for their tuberous roots but are seed‐propagated. Using a landscape genetic approach, we examined correlations between environmental factors and phylogeographic patterns of genetic diversity in Pachyrhizus landrace populations. Molecular analyses based on chloroplast DNA sequencing and a new set of nuclear microsatellite markers revealed two distinct lineages, with strong genetic differentiation between Andean landraces (lineage A) and Amazonian landraces (lineage B). The comparison of different evolutionary scenarios for the diversification history of yam beans in the Andes using approximate Bayesian computation suggests that Pachyrhizus ahipa and Pachyrhizus tuberosus share a progenitor‐derivative relationship, with environmental factors playing an important role in driving selection for divergent ecotypes. The new molecular data call for a revision of the taxonomy of Pachyrhizus but are congruent with paleoclimatic and archeological evidence, and suggest that selection for determinate growth was part of ecophysiological adaptations associated with the diversification of the P. tuberosus–P. ahipa complex during the Mid‐Holocene.Understanding the distribution of crop genetic diversity in relation to environmental factors can give insights into the eco‐evolutionary processes involved in plant domestication. Yam beans ( In legume crops, the change from indeterminate to determinate growth habit between wild and cultivated forms is one of the traits frequently associated with this “domestication syndrome”  are a good model for studying the need for manipulating whole‐plant resource allocation and its role in driving the evolution of plant architectural traits during plant domestication. Unlike most legume crops, yam beans are grown not for their seeds, which contain high levels of toxic polyphenols, but for their tuberous roots, which have high starch, vitamin, and protein contents as well as high levels of micro‐ and macronutrients  P. tuberosus (Lam.) Spreng., which is known both in the wild and as a cultivated species; and (iii) P. ahipa (Wedd.) Parodi, which is endemic to Bolivia and known exclusively as a cultivated species. The most widespread species, P. tuberosus, forms a complex of four cultivar groups: (i) “Ashipa,” (ii) “Chuin,” (iii) “Yushpe,” and (iv) “Jíquima.” Cultivars differ in leaf morphology, ecology, and traditional cultivation methods . In contrast, Chuins and Yushpe thrive on rich alluvial soils and are grown on floodplains (várzea) that are inundated for 4–6 months a year , in particular, is unknown, as no wild parent has yet been identified. This study represents the most comprehensive molecular study to date on the genus Pachyrhizus and aims at shedding light on the domestication history of yam beans in South America. We present new molecular evidence based on chloroplast DNA sequencing and a set of new nuclear microsatellite markers. Using a landscape genetic approach and approximate Bayesian computation (ABC), we examine correlations between environmental factors and phylogeographic patterns of genetic diversity within Pachyrhizus landrace populations, and evaluate different scenarios for the diversification of yam beans in the Peruvian Andes. Results are discussed through the lens of historical ecology.Little is known about the origins and history of South American yam beans. The origin of the Bolivian “Ajipa”  from field and herbarium specimens spanning the whole South American distribution range of the genus Table . Total gUUG‐5′rps16  of DNA template, 1× CoralLoad PCR Buffer, 1.5 mM MgCl2, 5% DMSO, 0.1 μg/μl BSA, 0.16 μM each of both forward and reverse primers , dNTPs at 66 μM final concentration each, and 0.5 units of Taq DNA Polymerase . For both regions, PCR cycling conditions were 7 min at 94°C, followed by 35 cycles of 1 min at 94°C, 1 min at 55°C, and 2 min at 72°C. Amplification ended with 10 min at 72°C. PCR products were purified and sequenced in both directions at the Eurofins sequencing facility. Sequences were aligned and manually edited using CodonCode Aligner 6.0.2 . Sequences were deposited in GenBank under the accession numbers KT923495–KT923560 and KT924295–KT924350  (Table A), allelic richness corrected for sample size (AR), unbiased expected (HE) heterozygosity , which estimates genetic differentiation based on shared alleles rather than based on mean heterozygosity  identified at each sampling site (6) Table . Mean nuity Nei, , and F‐sty Jost, , was usety Jost, .K clusters, assuming Hardy–Weinberg equilibrium and linkage equilibrium within each cluster, to reconstruct hypothetical ancestral populations. A narrow dataset consisting of one copy of each unique MLG (n = 39) was used to avoid autocorrelation due to repeated MLGs. K was set to vary between 1 and 10. For each value of K, ten independent runs based on 100,000 Markov chain Monte Carlo iterations with a burn‐in period length of 10,000 generations were performed, assuming admixture and uncorrelated allele frequencies. No group priors were defined. The optimal number of clusters was determined using the ΔK method proposed by Evanno, Regnaut, and Goudet . We retained all principal components (PCs) during the preliminary variable transformation to conserve all the variation in the original data. The number of PCs retained for the discriminant analysis was determined based on α‐scores  were performed in R  . Three sets of competing scenarios were compared . Confidence in scenario choice was assessed by evaluating type I and type II error rates over 100 pseudo‐observed datasets (PODs) simulated using parameter values drawn from prior distributions and LDA‐transformed summary statistics , reflecting the fact that most landraces consisted of few geographically discrete genotypes that were fixed for one allele at most loci and differed from each other by one or two alleles only (Table P. tuberosus) showed the most variability, with the highest levels of genetic diversity recorded in Ecuador and Peru. In Peru, Ashipas cultivated by Tupi‐speaking groups (Cocama) in Dept. Loreto were distinct from Ashipas grown by Pano‐speaking groups (Shipibo) in Dept. Ucayali . Similarly, we found two genotypes of Chuins, one of which was predominantly found in Cocama villages (MLG 36), and the other in Shipibo villages (MLG 37). The two genotypes differed by one locus only  and were both also detected among Ashipas.Matrices of pairwise genetic differentiation tests are given in Table ly Table . The AshP. ahipa) showed similarly low levels of genetic variability. Northern twining and southern bushy morphotypes were associated with distinct genotypes but differed by only one locus . Whereas northern accessions were fixed for AIP3091, southern accessions were fixed for allele AIP3084 , albeit to a genotype different from those cultivated in the Peruvian and Bolivian Amazon. This “Yungas type” was characterized by the fixation of two alleles, AIP1750 and AIP3171, which were otherwise detected among only four Peruvian Ashipas  and in the Brazilian “Jacatupé”  was found to be genetically uniform, with only one genotype detected across the cultivar's entire distribution area. Although sharing unusual morphological characters, notably a determinate growth habit, Jíquima and Ajipa were genetically distinct . They were also both characterized by unique alleles at locus AIP30  and a shared allele at locus AIP23 (AIP2369) that was otherwise detected among wild accessions only. Genetic outlier analyses strongly suggested that this locus may be under disruptive selection  = −2.3, p > .99; Fig. 32) and AIP31 (AIP3166), while Jíquima was fixed for a private allele at AIP36 (AIP3670).The Ecuadorian Jíquima  clustered apart from cultivated accessions (TC and AC). At K = 4, STRUCTURE separated P. ahipa (AC) from P. tuberosus (TC) and further distinguished two wild subpopulations  available, PW055 (from Panama) and PW056 (from Ecuador), clustered both with TW, although PW056 showed signs of admixture with TC.Bayesian structuring analyses suggested that data were best represented by two populations. At s Figure . The onlK = 5  from P. tuberosus . As with STRUCTURE, DAPC separated wild P. tuberosus accessions  into two distinct geographic subpopulations (W1 and W2), but additionally separated P. tuberosus cultivars in two subgroups (B1 and B2)  Figure . Five suPhylogenetic relationships among genetic clusters were further explored using Neighbor–Net algorithm. The topology of the phylogenetic network was in agreement with patterns identified using Bayesian and nonparametric clustering methods, with a primary split separating wild accessions (lineage W) from cultivated accessions, and a second split separating lineage A from lineage B Figure .Pachyrhizus species, except P. ahipa for which no specific haplotype was found. Seven chloroplast haplotypes were resolved from the concatenated sequences of trnH‐psbA and trnQ‐rps16  diversity were generally concordant with nuclear data and confirmed the taxonomic status of 6 Figure a. Eight 6 Figure .P. tuberosus cultivars, the reverse complement (R) was predominantly found among highland Andean accessions  and TCA (carrying the R configuration). Most Peruvian accessions (TC) carried the TCW haplotype, while TCA was detected only in TC364. In contrast, all Bolivian accessions (AC and TC) carried the TCA haplotype. TCA also predominated among highland cultivars from Ecuador, while the lowland Jíquima group carried the TCW haplotype.P. tuberosus or of P. ahipa, Brazilian Jacatupé accessions carried the P. tuberosus haplotype for the trnH‐psbA region (L configuration), but the P. ahipa haplotype for the trnQ‐rps16 region, resulting in a distinct TC1 haplotype. TC1 was also detected in two Peruvian Ashipas collected near Tarapoto, TC359 and TC534. The only other accessions for which there was discordance between trnH‐psbA and trnQ‐rps16 were two Peruvian accessions, a Yushpe from Pucallpa (TC595) and an Ashipa collected near Cusco (TC537), both of which carried a unique combination of cpDNA haplotypes (TC2). Together with TC364, these were the only Peruvian accessions which carried the R form of the cpDNA inversion  and genetic differentiation between landrace populations, but no significant influence of elevation (ALT) or geographic distance  on the structure of genetic diversity (Table P = .875), with a posterior probability of .129. Landrace populations segregated into “xeric” and “mesic” ecotypes, with a clear separation between populations belonging to either lineage A or lineage B for bioclimatic variables related to rainfall  and Jíquima (JI) diverged from P. tuberosus (TC)  Fig. . DivergeTC) Fig. . ConfideTC) Fig. , with a 44.1P. ahipa is only known in cultivation in the subtropical east Andean valleys of Bolivia and northern Argentina   with P. tuberosus, which further supports the hypothesis that P. ahipa and P. tuberosus share a progenitor–derivative relationship.Although PDS has mostly been applied to wild species, it can be extended to scenarios for the domestication and diversification of crop plants. PDS offers a plausible explanation for the drastic reduction in genetic diversity we found in 4.2P. ahipa (lineage A) and P. tuberosus (lineage B) and highlighted the importance of precipitation seasonality as the main factor affecting the structure of genetic diversity among Pachyrhizus landrace populations. Self‐pollination may have played an important role in facilitating the adaptation of Pachyrhizus cultivars to different environments by accelerating the fixation of beneficial alleles , they never occur together in farmers' fields  are grown predominantly in the Yungas, a transitional zone between the Andean highlands and Amazon lowland rainforests characterized by warm tropical climate with no dry season, while Ajipa (P. ahipa) is only cultivated in the semiarid inter‐Andean valleys. Genetic analyses confirmed that all P. tuberosus accessions from the Yungas belonged to the Ashipa type. Like most Ashipas from Ecuador but unlike those from Peru, all Bolivian Ashipas carried the R form of the cpDNA inversion. Tapia and Sørensen  and the derivate species (P. ahipa).In northern Bolivia, Sørensen  showed t4.3P. ahipa, the Ecuadorian Jíquima was genetically distinct and carried a different form of the 9‐pb cpDNA inversion. Ajipa and Jíquima shared only one allele at locus AIP23 and clustered together in STRUCTURE analyses when this locus was included, but no longer when the locus was removed. Neutrality tests showed AIP23 to be likely under positive selection, suggesting that morphological similarities between the two cultivars result from convergent evolution under farmers' selection for adaptations to similar environmental constraints rather than from a shared origin.Despite sharing many phenotypical characters with P. ahipa in Bolivia, the current distribution range of the Jíquima cultivar is very limited and restricted to the Manabí and Guayas provinces of coastal Ecuador. The 460 mm annual precipitation rate in the region is lower than in any of the other habitats where P. tuberosus is found, but comparable to the 400–700 mm annual precipitation rate in Bolivian inter‐Andean valleys. While leaf shape differs between the two cultivars, Ajipa and Jíquima share important agromorphological characters, notably determinate growth and short racemes, but also photothermal neutrality  content, known as “Pitichuin,” and low DM varieties, known as “Cocotichuin.” Purple Pitichuins are the most common, while yellow and purple Cocotichuins are only known from Shipibo communities. Chuins are only found along the Ucayali River, where they are grown by Tupi‐speaking groups (Cocama) and Pano‐speaking groups (Shipibo), each of which grew distinct genotypes of Chuins. The diversity of unrelated vernacular names for Ashipa landraces in Peru and Ecuador suggests that Ashipas represent the ancestral landrace population from which all other cultivars derived  up until the Late Intermediate Period (AD 1000–AD 1500) , whereas Ugent et al. (P. ahipa).Drought‐tolerant ecotypes were probably also brought down to the valleys on the western slopes on the Andes, where archeological evidence indicates that “Ajipa‐like” cultivars were cultivated on the Peruvian coast as early as 4000 BP Figure b. Remain) Towle, . Remains) Towle,  and AD 1) Towle, , but the) Towle,  and Yaco) Towle,  identifit et al. , 1986 idPachyrhizus cultivation was briefly resumed on the Peruvian coast during the Late Intermediate Period as climatic conditions improved and Chimú‐Inca resettled coastal lowlands for the last time , and the Apurímac, Ene, and Mantaro river valleys in central eastern Peru, where Debouck et al. , should also be given priority for collecting germplasm which could, if it (still) exists, help resolve the history of P. ahipa in Peru.The persisting ambiguity about the taxonomic status of these plant remains is mainly due to the absence of cultivars with determinate growth in Peru nowadays (Sørensen et al., k et al.  suggesteP. tuberosus and P. ahipa landrace populations, it is urgent to document and safeguard genetic resources in Pachyrhizus wild relatives. Pachyrhizus panamensis, in particular, represents an important taxonomic gap which needs to be addressed. Like Ajipa and Jíquima, P. panamensis distribution is restricted to SDTFs. Pollen grains of P. ahipa are morphologically distinct from pollen grains of P. tuberosus and support a close relationship between P. ahipa and P. panamensis (Sørensen, P. panamensis, P. tuberosus, and P. ahipa cannot be fully resolved, however, because of the rarity of P. panamensis herbarium specimens.Given the limited genetic variation in P. tuberosus and P. ahipa and offers insights into the evolutionary history of yam beans in the Andes. Molecular data suggest the two species share a progenitor–derivative relationship, with environmental factors playing an important role in driving selection for divergent ecotypes adapted to contrasted environments. Future collection missions in Peru and Ecuador might bring new elements that will help refine the scenario we propose.In conclusion, while gray areas remain, this study sheds a new light on phylogenetic relationships between The authors declare no conflict of interest.https://doi.org/10.5061/dryad.7gp80. Chloroplast DNA sequences were deposited in GenBank under accession numbers KT923495–KT923560 and KT924295–KT924350.Data for this study are available from the Dryad Digital Repository:  Click here for additional data file. Click here for additional data file.


As bats have recently been described to harbor many different viruses, several studies have investigated the genetic co-variation between viruses and different bat species. However, little is known about the genetic co-variation of viruses and different populations of the same bat species, although such information is needed for an understanding of virus transmission dynamics within a given host species. We hypothesized that if virus transmission between host populations depends on events linked to gene flow in the bats, genetic co-variation should exist between host populations and astroviruses.Myotis nattereri) within and among populations at different geographical scales in Germany. Further, we correlated the observed bat population structure to that of partial astrovirus sequences (323–394 nt fragments of the RNA-dependent RNA polymerase gene) obtained from the same bat populations. Our analyses revealed that the studied bat colonies can be grouped into three distinct genetic clusters, corresponding to the three geographic regions sampled. Furthermore, we observed an overall isolation-by-distance pattern, while no significant pattern was observed within a geographic region. Moreover, we found no correlation between the genetic distances among the bat populations and the astrovirus sequences they harbored. Even though high genetic similarity of some of the astrovirus haplotypes found in several different regions was detected, identical astrovirus haplotypes were not shared between different sampled regions.We used 19 nuclear and one mitochondrial microsatellite loci to analyze the genetic population structure of the Natterer’s bat  contains supplementary material, which is available to authorized users. With their particular social, ecological, physiological and immunological traits, bats provide unique hosts for many viruses to co-evolve with e.g. , 2). Ind. Ind2]).Myotis nattereri Kuhl 1817 sensu lato ).d 100 km . Our resd 100 km  as we alM. nattereri do not overlap between geographic regions  and mutation rate between bats and viruses. In contrast to higher Eukaryotes such as the bat host, RNA viruses are subject to higher selective pressures and combined with a high mutation rate allow continuous and rapid adaptation to changing environmental conditions [No association was found when correlating the genetic distances of the different astrovirus sequences with their bat host genetic distances. We had originally expected that if the transmission route of astrovirus sequences resembles the pattern of host gene flow, genetic co-variation between astroviruses and host populations should be detected, especially across regions. The reason why no such correlation was found for nditions , 76, 77.nditions , 75. Thenditions . MoreoveM. nattereri within Germany, swarming sites act as stepping-stones for gene flow, as indicated by an overall isolation-by-distance pattern and the absence of such a significant pattern on a local scale. The observed population genetic structure indicates that no apparent strong barriers to gene flow exist within our study area for the bat host. While putative viral species were mostly shared between geographic regions, no haplotypes were shared for any putative viral species. Despite the observed genetic differentiation between the three geographic regions in the bat host and to a certain extent also in the harbored astroviruses, we did not detect a correlation between host and virus genetic distances. This could potentially be due to differences in genetic drift, selective pressure, population size and mutation rate between bats and viruses. Further studies with a higher astrovirus sample size and with specific simultaneous sampling during autumn mating at swarming sites are required to shed further light on the host-virus relationship between bats and their astroviruses.In summary, our findings suggest that for Additional file 1:Figure S1. Summary of the log-likelihood values from the 20 independent runs conducted with Structure for the number of genetic clusters (K) set to a minimum of 1 and a maximum of 10. The left graph shows the log-likelihood results of the runs for each K, whereas the right graph shows Delta K plotted against K. The most likely number of genetic clusters is three using both methods. Figure S2. Comparisons of the STRUCTURE runs for K = 3 with (top) or without (bottom) the LOCPRIOR option. Table S1. Table of sampling times and associated screening for astroviruses. AstV = astrovirus, BY = Bavaria, MV = Mecklenburg Western Pomerania, NRW = North Rhine Westphalia. AstV positive samples with a length of less than 279 nt could not be assigned to a specific haplotype, but are nevertheless included in the number of AstV positive samples presented here. Database S1. R-script used for the permutation test. With this script we tested how likely it is to have no overlap in virus haplotypes across regions and whether virus haplotypes are more different between regions than expected by chance. (DOCX 318 kb)Supplementary material. Additional file 2:Myotis nattereri. Table of alleles per locus for each individual bat used for the population genetic analyses on the Natterer’s bat (Myotis nattereri) determined using Genemapper v 5.0 (Applied Biosystems). (XLSX 75 kb)Genotype table


Maurolicus muelleri, based on the restriction-site associated DNA sequencing method, and preformed population connectivity and genetic diversity analyses in a subset of samples collected from the Bay of Biscay. Our study proves the method valid for obtaining genome-wide markers in this species and provides the first insights into the population genomics of M. muelleri. Importantly, the genomic resources developed here are made available for future studies and set the basics for additional endeavors on this issue.Mesopelagic fish are largely abundant poorly studied fish that are still intact, but which, due to their potentially great added value, will be imminently exploited by humans. Therefore, studies that provide information to anticipate the anthropogenic impact on this important resource are urgently needed. In particular, knowledge about their connectivity, potential adaptation and resilience are needed. This information can be obtained through the analysis of genome-wide markers which are now relatively easily and cost-efficiently discovered thanks to high-throughput sequencing technologies. Here, we have generated thousands of SNP markers in  The lack of physical barriers in the ocean and large populations sizes generally results in low genetic differentiation in marine fishes , which rAmong the poorly studied marine organisms are mesopelagic fish, i.e., those inhabiting the deep scattering layer, that represent the largest fish biomass in the ocean  and playMaurolicus muelleri.Several studies have focused on deciphering species boundaries and population connectivity within mesopelagic fishes using genetic variants, but they are all based on allozymes , single M. muelleri in the Northeast Atlantic  through pelagic trawling on board the R/V Emma Bardán and Ramón Margalef in September 2016. Catches were collected between 106 and 212 m and during daytime. Fish where frozen at -20°C until DNA extraction. Genomic DNA was extracted from about 20 mg of tissue using the Wizard® Genomic DNA Purification kit  following manufacturer’s instructions for “Isolating Genomic DNA from Tissue Culture Cells and Animal Tissue.” Extracted DNA was suspended in Milli-Q water and concentration was determined with the Quant-iT dsDNA HS assay kit using a Qubit® 2.0 Fluorometer (Life Technologies). DNA integrity was assessed by electrophoresis, migrating about 100 ng of GelRedTM-stained DNA on an agarose 1.0% gel.Specimens of SbfI restriction enzyme and ligated to modified Illumina P1 adapters containing 5 bp unique barcodes. Pools of 30 or 32 individuals were sheared using the Covaris® M220 Focused-ultrasonicatorTM Instrument (Life Technologies) and size selected to 300–500 pb by cutting agarose migrated DNA. After Illumina P2 adaptor (including 5 pb index) ligation, each library was amplified using 14 PCR cycles. The three pools, each with one unique Illumina index, were combined and paired-end sequenced (100 pb) on an Illumina HiSeq2000. Generated RAD-tags were analyzed using Stacks version 1.44 (process_radtags with default parameters and removing the last 10 bases of the end of the reads. Putative orthologous tags (stacks) per individual were assembled using ustacks with a minimum depth of coverage required to create a stack (m) of 3 and a maximum nucleotide mismatches (M) allowed between stacks of 2, 4, or 6. Catalogs of RAD loci were assembled using cstacks with number of mismatches allowed between sample tags when generating the catalog (n) of 3, 6, and 9 for M-values of 2, 4, and 6, respectively. Matches of individual RAD loci to the catalog were searched using sstacks. RAD loci found in at least 75% of the individuals were selected using populations and used to calculate . Using PLINK version 1.07  were prepared following the methods of ion 1.44 . Qualityion 1.07 , SNPs wi 2.0.8.3 .FST values per group pairs were calculated following the Genepop 4.3  hypothetical ancestral was calculated with the Bayesian clustering approach implemented in STRUCTURE . Average coverage per individual ranges from 6 to 20× (with an average of 11×). Number of RAD-tags per individual increases with number of reads used for stack formation and does not reach a maximum value even when more than 100K reads are used . This places the number of SbfI cut sites in M. muelleri above 32 000, which is larger than in other teleost species . The number of tags present in more than 75% of the individuals increases with increasing M because more common loci can be found when these are composed by more alleles. Consequently, larger M-values results in more alleles and thus more variable positions , inbreeding coefficient (FIS), and expected (He) and observed heterozygosity (Ho) values are congruent across the three parameter sets used. Expectedly, using only variable sites or selecting those with at least 0.05 MAF increases nucleotide diversity and both, expected and observed heterozygosity (Table 2). For all set of parameters and position subsets used, observed heterozygosity is lower than expected both overall and when calculated per catch. Per catch, values of π and He are similar, whereas values of Ho and FIS differ . In general, catches with low observed heterozygosity are also those with highest inbreeding coefficient  and vice versa , although this does not hold for catches 9012 and 9029 with present large Ho, but medium FIs values with respect to the others. Similar to other studies , all individuals display admixed representation of each of the hypothetical ancestral population, suggesting genetic connectivity within the area of study. Interestingly, catch 9018 shows an ancestry pattern that differs from the rest of the catches, yet, this difference is not visible in the PCA plots , where no differentiation among groups can be observed. In accordance with the hypothesis of high connectivity within the area of study, FST values between pairs of populations are low (Table 3), although, consistent with the pattern observed in the structure analyses, pairs including catch 9018 are those with highest FST values.In the Bayesian population structure analyses  population differentiation within the area of study, referred to as the Wahlund effect , (ii) prFigure 1), supports previous observations that individuals are grouped according to their age . This data will contribute to shed light on phylogeographic patterns within the species (Overall, we have validated the RAD-seq based SNP discovery method using the  species , detect  species , and to  species .NR-E designed the study, performed the analyses, interpreted the data, and wrote the manuscript. PÁ collected the samples, contributed to the interpretation of the data and revised the manuscript. XI contributed to the interpretation of the data and revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Persea americama Mill.), harbors a wide genetic diversity of this species, whose identification may provide the grounds to not only understand its unique population structure and domestication history, but also inform the efforts aimed at its conservation. Although molecular characterization of cultivated avocado germplasm has been studied by several research groups, this had not been the case in Mexico. In order to elucidate the genetic structure of avocado in Mexico and the sustainable use of its genetic resources, 318 avocado accessions conserved in the germplasm collection in the National Avocado Genebank were analyzed using 28 markers [9 expressed sequence tag-Simple Sequence Repeats (SSRs) and 19 genomic SSRs]. Deviation from Hardy Weinberg Equilibrium and high inter-locus linkage disequilibrium were observed especially in drymifolia, and guatemalensis. Total averages of the observed and expected heterozygosity were 0.59 and 0.75, respectively. Although clear genetic differentiation was not observed among 3 botanical races: americana, drymifolia, and guatemalensis, the analyzed Mexican population can be classified into two groups that correspond to two different ecological regions. We developed a core-collection by K-means clustering method. The selected 36 individuals as core-collection successfully represented more than 80% of total alleles and showed heterozygosity values equal to or higher than those of the original collection, despite its constituting slightly more than 10% of the latter. Accessions selected as members of the core collection have now become candidates to be introduced in cryopreservation implying a minimum loss of genetic diversity and a back-up for existing field collections of such important genetic resources.Mexico, as the center of origin of avocado ( Persea americana Mill.) has been insufficiently studied in its own center of origin despite the wide genetic diversity the region hosts. Understanding its unique population structure and domestication history, as well as exploring strategies for its conservation, has been a long-neglected priority.Mexican avocado  P. americana var. drymifolia (Schlecht. & Cham.) Blake (Mexican race), (2) P. americana var. guatemalensis L. Wms. , and (3) P. americana var. americana Mill. (West Indian race)  Kopp  Table  and theie) Table . These be) Table . In addims) Kopp  and var.b et al. —are alsoAccording to archeological evidence, avocado consumption began more than 7,000 years ago Smith,  and it hP. americana var. drymifolia) collected from different localities across Mexico—aims to conserve, as backup, these accessions in different institutions.Genetic diversity plays an important role not only in preservation of biodiversity, but also of ecological and cultural aspects, such as diet and economy , var. guatemalensis (15), var. americana (12), var. costareicensis (3), and hybrids (31)] was available for 165 accessions, while information about geographical origin was available for 299 accessions. Three other Persea species  are also included in the collection. Fresh leaves collected from individual trees in the field were transferred to the laboratory and freeze-dried on the same day. Total DNA was extracted by a modified tropical-plant-specific protocol , observed heterozygosity (Ho), unbiased expected heterozygosity (uHe), and fixation index (F) were calculated to determine the genetic diversity and the past inbreeding. Allele frequencies of each botanical race and deviation from Hardy-Weinberg equilibrium (HWE) of loci were calculated for the subset of genotyping data on 165 accessions with botanical race information. All these values were calculated using GenAlEx 6.5  and private allelic richness (PAR) for each botanical race were measured via rarefaction by HP-Rare v.1.0 . Linkage disequilibrium (LD) between pairs of markers was tested by Genpop on the Web .A total of 319 accessions of the BNGA collection were genotyped. One accession was not included due to low yield of amplicon. The botanical race information on the 165 accessions had been previously determined by a genebank curator regarding the fruit-skin characteristics and anise-like odor of the leaf. Number of total alleles scored (K) for the whole set of accessions showing both admixture and non-mixture by correlated allele frequency models.The membership compositions of each cluster, inferred by STRUCTURE analysis  from 1 to 8; each K was simulated 5 times with a burn-in of 10,000 iterations before collecting data, and 10,000 iterations of the Markov-Chain Monte Carlo (MCMC) method. Secondly, to obtain accurate results, parameter estimates were increased to a burn-in of 100,000 iterations and 100,000 iterations of MCMC with K from 1 to 5 with 5 iterations. The optimum K value was determined from log probability of data at each step of the MCMC, Pr(X|K), and ad hoc statistic ΔK of Evanno et al. , in what we consider to be a manageable sub-collection and in agreement with previous reports that establish that CC should contain ≥10% elements from the original collection  LD between pairs of markers in 344 (91.0%) combinations in all 318 accessions. The botanical race-wise values for significant LD were 205 (54.2%), 125 (33.1%), and 5 (1.3%) combinations for drymifolia, guatemalensis, and americana, respectively. For costaricensis, only six marker combinations were valid to calculate LD due to the small number of samples, none of which was significant.The 28 markers that were analyzed (9 EST-SSR and 19 SSRs) detected a total of 547 alleles ranging from 4 to 34 alleles with an average of 19.1 per locus, out of which, 393 (71.7%) were rare alleles present in frequency less than 0.05. Except for LMAV20, all the markers tested were not under HWE (Table R) and private allele richness (PAR) in the population were: 5.95 and 0.63 for drymifolia, 6.13 and 0.89 for guatemalensis, 6.22 and 0.69 for americana, and 3.82 and 0.90 for costaricensis, respectively ] and the Evanno's ΔK. Estimate of population divergence of allele frequencies in these two inferred populations from ancestral frequencies was 0.14 (Cluster A) and 0.05 (Cluster B), respectively.Genetic assignment methods implemented in STRUCTURE  and 0.71 (Cluster B). Accessions were assigned to one of the clusters when more than 80% of the genetic background belonged to such cluster. The number of accessions assigned to each cluster was 71 (22.3%) for Cluster A and 188 (59.1%) for Cluster B. Fifty-nine (18.6%) individuals were considered to be of admixed origin (80> A and B >20)  occur about five times more often than those in Cluster A (15) for drymifolia. Geographical distribution of Clusters A and B was represented on a map , and being 36 the smaller CC to meet this criterion, the core reference set of 36 individuals was selected from the 298 individual accessions. Scores obtained from the evaluation of CC36 were compared with those of other CCs as well as with the original collection Table . In addiA smaller ANE  means the diversity in the original collection is homogeneously represented by the CC. On the other hand, ENE (average distance between each CC sample and nearest CC sample) and E (average distance between CC samples) indicate the dispersion of data for the CC, where higher values stand for a better representation of extreme values. Values of ANE decrease as size of the CC increases; this is because members of a larger CC fill the gap of the smaller CC decreasing the average distance between the selected elements of the CC and those of the original collection.E-value increases as the number of CC increases; this indicates that extreme individuals are being included as the extreme values begin to form a single cluster.ENE's largest value in CC36 indicates that this CC set is the one with the largest dispersion. The Allele coverage (CA) reaches over 80% when CC size exceeds 36 and will continue to increase as CC becomes larger. This is explained by the large proportion of rare alleles in the total number of alleles detected ; the larger the CC, the higher the number of rare alleles included in it. The frequency of rare alleles in CC36 was 197/352 (56.0%). Members of CC36 were evenly distributed on the dendrogram drawn by neighbor-joining method using the same dataset for CC selection. Compared with the distribution of a smaller number of the CC, CC36 members filled the non–represented branches by those CC12 and CC24 selected members  and 3 (8.3%) for guatemalensis; 12 (3.8%) and 1 (2.8%) for americana; 3 (0.9%) and 2 (5.6%) for costaricensis; 31 (9.7%) and 3 (8.3%) for hybrids; as well as 148 (46.4%) and 14 (39%) for unknown. The ratio of genetic groups determined by STRUCTURE analysis of the original collection and CC36 were almost the same. A list of the 36 accessions is found in Supplementary Table The distribution patterns of the 298-original collection and CC36 were compared with geographical origin, botanical race distribution, and ratio of genetic group determined by STRUCTURE analysis. The original collection consists of accessions collected from 17 States in Mexico and 7 other countries. Elements selected in CC36 were collected from 9 Mexican States and one other country (Costa Rica), encompassing the most representative area of distribution of the geographic origin of accessions in the original collection. Components of botanical races and species in the original collection and CC36 were 104 (32.6%) and 11 (30.6%) for drymifolia. Cuiris-Pérez et al. (drymifolia). Galindo-Tovar et al.  values of the BNGA collection (He = 0.75) were smaller than those of 42 accessions of avocado varieties preserved in several institutes in Spain (He = 0.831), which consist of a mixture of drymifolia, guatemalensis, and americana collected from a broad geographical range . This high genetic diversity of the BNGA collection has derived mainly from the presence of rare alleles, which account for 71.7% (393/548) of total alleles. These rare alleles give significant values to Mexican avocado and emphasize the importance of conservation.In this work, a considerable number of accessions (318 accessions) from the BNGA collection were used to illustrate the genetic diversity and background of avocado present in Mexico. As the BNGA collection in CEBAJ-INIFAP contains accessions from a wide geographical range in the country, it served as an optimal data source to infer genetic diversity and background of avocado present in Mexico. The result of this study demonstrated that this collection has achieved as much genetic diversity as other international avocado collections. Expected genetic diversity  were analyzed together, but not when the analysis was performed within each botanical race. They concluded that the LD arose from the genetic structure of the different botanical races used in their study.Human imposed selection, intentional, or otherwise, modifies genetic diversity as it limits the number of lineages that are maintained for propagation. Although avocado is a subtropical tree species which predominantly outcrosses, we observed a deviation from HWE as well as a significant level of LD between pairs of markers in the materials used in this study. This could be explained by the extensive number of alleles being analyzed, yet it may suggest a domestication bottleneck process experienced differently by each avocado botanical race. Use of unlinked or weakly-linked genetic regions is recommended for STRUCTURE analysis to make meaningful inferences , our initial analysis included a small number of individuals of guatemalensis (15), americana (12), and costarricensis (3), as well as some non-P. americana species. We contrasted this analysis with one that only included drymifolia and no significant difference was found in this comparison (data not shown). The same approach was carried out with CC36 and, although the LD decreases at several loci, the overall high LD tendency prevailed. Microsatellite markers used in this study consisted in est-SSR and genomic SSR markers. Both markers suggested interlocus LD, and showed no evidence of selection at the est-SSR loci corresponding to the markers analyzed in this study. Currently, genome sequencing of Mexican avocado is in process , and our Cluster B with their group II (humid semi-warm to hot semi-dry). This congruence between genetic clustering and climate distribution clustering might imply that avocado in Mexico may have evolved separately in two climatic regions, which could have also led to differences in both est- and genomic microsatellite markers.Previous studies succeeded in grouping based on the botanical races using genetic classification by STRUCTURE analysis (Gross-German and Viruel, Other than the geographical/environmental factors, this grouping may have some relation with possible multiple domestication of avocado in Mexico. Clegg et al.  suggest Since genetic flow persists both among cultivated botanical races and non-cultivated wild avocadoes, correlation between race-identification based on morphological characteristics Table  and genoMolecular studies in clustering analysis have provided general agreement between botanical-race classification and the employed molecular markers with some exceptions (Ashworth and Clegg, Long-term conservation of commercially important tree species is one of the most urgent and challenging strategies aimed to assure the availability of such resources for future generations. This becomes particularly difficult with recalcitrant species. In the present work, we developed a core collection from 298 genotypes conserved in the BNGA in CEBAJ-INIFAP by PCA-K means clustering method with 28 microsatellite markers.The objective of the study was to identify a core-collection that contained genetic diversity representative of the original collection. Agro-morphological traits were excluded as a selection criterion since their inclusion would have impacted the genotypic selection process, possibly affecting the desired optimal allelic representation. Despite this exclusion, the selected CC ended up successfully representing the original collection in terms of geographical distribution, representation of botanical races, and genetic group assignment determined by STRUCTURE analysis. However, specifically designed CCs may be selected for other purposes based on different selection criteria.in vitro and cryopreservation techniques have proven effective in safeguarding recalcitrant species with a biotechnological approach (González-Arnao et al., Currently, the BNGA collection is maintained as field trees. This conservation method presents possible threats by natural disaster, as pest and diseases are inevitable. Although a backup conservation approach has been already implemented among field collections in different localities, such collections are still at potential risk. As an alternative, recent advances in The analysis of the BNGA avocado collection by molecular markers did not establish a clear difference among avocado botanical races. Our results suggest that the two genetic groups inferred were admixed and have contributed to the development of the current genetic structure of such populations, which might present evidence for putative environmental and/or domestication history of the species. This information, combined with agro-morphological characteristics, could prove useful when used for association studies, important in the acceleration of breeding procedures.The selected core collection efficiently represents the diversity of the original collection and is a suitable candidate set for long-term cryopreservation conservation. This strategy may serve as a model for the conservation of other important recalcitrant species.RM conceived and designed the experiments; EH, ME, MC, and EB provided reagents/materials/analysis tools; LG and RM performed the laboratory procedures; RM, LG, EB, and ME analyzed the data; LG and RM wrote the paper; EH, ME, MC, and EB edited and provided critical review of the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Glossina fuscipes fuscipes, which is an important species in Tanzania and Kenya. The main objective of the study was to provide baseline data to determine the genetic variability and divergence of G. f. fuscipes in the Lake Victoria basin of Tanzania and Kenya in order to guide future vector control efforts in the region.Tsetse flies (Diptera: Glossinidae) are sole vectors for trypanosomiasis, which affect human health and livestock productivity in Africa. Little is known about the genetic diversity of G. f. fuscipes from 8 sites along the shores of Lake Victoria were screened for genetic polymorphisms at 19 microsatellite loci. Samples were collected from two sites in Kenya and six sites in Tanzania. Four of the Tanzanian sites were located in the Rorya district, on the eastern shores of Lake Victoria, while the other two sites were from Ukerewe and Bukoba districts from the southern and western Lake Victoria shores, respectively. Four genetically distinct allopatric clusters were revealed by microsatellite analysis, which sorted the sampling sites according to geography, with sites separated by as little as ~65 km belonging to distinct genetic clusters, while samples located within ~35 km from each other group in the same cluster.Two hundred and seventy five G. f. fuscipes analyzed from the Lake Victoria Uganda shores. From a control perspective these results suggest that for sites within the same genetic cluster, control efforts should be carried out in a coordinated fashion in order to avoid re-invasions. Future work should focus on better quantifying the extent and spatial patterns of the observed genetic discontinuities of the G. f. fuscipes populations along the Tanzanian shores. This will aid in their control by providing guidelines on the geographical extent of the area to be treated at the same time.Our results suggest that there is ongoing genetic admixture within sampling sites located ~35 km from each other, while sites located ~65 km apart are genetically isolated from each other. Similar patterns emerged from a parallel study on The online version of this article (doi:10.1186/s13071-017-2201-x) contains supplementary material, which is available to authorized users. Glossina) remain to be insects of economic and medical importance in sub-Saharan Africa. They transmit pathogenic trypanosomes that cause sleeping sickness to humans and nagana to livestock [Tsetse flies  under Pan African Tsetse and Trypanosomiasis Eradication Campaign (PATTEC) initiative [G. f. fuscipes sampled in Tanzania and Kenya and relates them to the recommendations for guiding future vector control efforts in the region.Little is known on the population structure and gene flow of itiative . Lake ViGlossina fuscipes fuscipes were sampled at 8 localities around Lake Victoria, with 2 sites in Kenya (KIS and MAN) and 6 sites in Tanzania. These sites include 4 locations on the border of Tanzania and Kenya in the Rorya district , one in the Bukoba district at the border between Uganda and Tanzania (BUK), and one approximately in between these two areas in the Ukerewe district  according to the manufacturer’s instructions. PCR and genotyping were conducted for microsatellite markers at 19 previously identified loci that are distributed across the genome . PCR conST values between all sampling sites were calculated in Arlequin v 3.5 [. The optimal number of clusters in the data was calculated using the ad hoc statistic ΔK in STRUCUTURE HARVESTER [distruct package was used to plot the results. We also used discriminant analysis of principal components (DAPC) [adegenet package [The program Genepop 4.2  was usedin v 3.5 . Signifiin v 3.5 . The optARVESTER . The STRARVESTER , and diss (DAPC)  conducte package  to deter package .e) for the clustered populations were estimated under the linkage disequilibrium model with random mating included in the program NeEstimator v2 [To investigate patterns of migration and identify any probable migrants within our dataset, we used GeneClass2 . GeneClamator v2 . All allR) ranged from 2.89 to 4.94 across the sites. No significant differences were observed between observed and expected heterozygosities . These values are similar to the ones reported for G. f. fuscipes populations around Lake Victoria shores in Uganda [G. f. fuscipes flies in this region harbor a wealth of genetic diversity and thus have the long term potential to adapt to ecological changes in the area.In the present study, a total of 275 tsetse flies were genotyped at 19 microsatellite loci. According to inbreeding coefficient values for each population, no loci deviated significantly from Hardy-Weinberg equilibrium  recorded in this study is consistent with earlier reports on the same species in other areas of East Africa and is likely a consequence of genetic drift. In the riverine and lacustrine tsetse species, such as G. f. fuscipes, wet season dispersion from dry season refugia is thought to establish demes in which genetic drift leads to differentiation [G. tachinoides in Ghana [G. p. gambiensis in Niayes-Senegal [G. p. palpalis in Cote d’Ivore [G. p. palpalis) in Cameroon indicated the species formed a large panmixia population suggesting suppression to be the best option for the vector control [G. swynnertoni, a savannah species from northern Tanzania, have been reported to have the high level of gene flow [Fntiation , 18. Simin Ghana , 36, G. -Senegal , 37 and  d’Ivore . Howeverene flow . Geograpene flow .Fig. 3BaThe presence of admixed genotypes within clusters 2 and 3 Fig. . HoweverST = 0.341; FST = 0.308 and FST = 0.286) recovered at some sampling sites  were not uniform among sampling sites, and given the confidence intervals for clusters 3 and 4 (Fig. is study . These dFrom a control perspective these results suggest that, given the genetic homogeneity among sampling sites from the Rorya district, these sites should be controlled and monitored together to guard against reinvasion from neighboring untreated areas. A similar strategy should be adopted for the region including the two Kenyan sampling sites. However, given that the microsatellite data show that Kenya and the Rorya district samples are genetically distinct even though they are only ~65–80 km apart, control and monitoring effort may not necessarily need to include large geographical areas. Future studies should include a more uniform geographical representation of tsetse infested areas than in the current study to quantify the extent and spatial pattern of the genetic discontinuities found in this study among sampling sites located at varying geographical distances.