Plectranthus edulis (Vatke) Agnew (Lamiaceae), locally known as Ethiopian potato syno. Ethiopian dinich, is one of the native Ethiopian edible tuber crops that has been significantly contributing to household food security for millions of subsistence farmers. However, its current production is declining to the extent of total extinction from several administrative regions where it used to be widely cultivated. It is one of the less researched crops regardless of being indigenous and its contribution to food security during time of scarcity. Therefore, we intended to assess the level of genetic diversity in 67 accessions, representing nine populations that were collected from diverse agro-ecologies in the country, using ISSR markers and hence, to generate a baseline information that assists marker assisted breeding, conservation and germplasm management efforts.In the present study, ten polymorphic ISSR markers were screened and optimized, that generated an average of 7.4 scorable bands per marker and revealed high overall percent polymorphism (95%), Nei’s gene diversity (h = 0.40) and Shannon index (I = 0.62) suggesting ISSR’s effectiveness in detecting high levels of genetic diversity. A considerably high overall populations gene diversity (Nei’s) (h = 0.32) and Shannon index (I = 0.47) were observed, revealing high potential of the populations for further breeding and conservation efforts particularly for population from Gurage administrative zone, which showed the highest values. Similarly, estimation of pairwise genetic distance revealed the importance of cross breeding population from Awi administrative zone to the rest populations. Analysis of hierarchical molecular variance (AMOVA) showed higher levels of genetic differentiation within populations (92%), and collection regions (94%) suggesting that either clonal mode of propagation in the crop or farmers selection pressure for important agronomic traits or both maintained the original heterozygosity in the crop. UPGMA phylogenetic analysis did not strictly group the populations based on their geographic region of origin, which could be attributed to the widely practiced tuber exchange and hence continuous human mediated exchange of genetic material and sharing of the same genetic base among the geographic regions.P. edulis populations. However, it is important to couple them with agro-morphological traits or codominant molecular markers to get more reliable information for use in breeding and conservation. Several of the potential administrative zones we covered are useful for P. edulis diversification and conservation. However, the crop is currently highly marginalized and this led to rapid decline in population size and loss of valuable agronomic traits. To address this challenge, there is an urgent need to take counteractive measures.The ISSR markers used in the present study were effective in revealing extent and patterns of genetic diversity in  Plectranthus edulis (Vatke) Agnew is locally known by several vernacular names among which, Ethiopian potato syno. Ethiopian dinich, is frequently used by the scientific community. It is an ancient tuber crop and native to Ethiopia. The crop belongs to family Lamiaceae, subfamily Nepetoideae, tribe Ocimeae, and genus Plectranthus [Plectranthus, such as P. esculentus (Livingstone potato), P. parviflorus (Sudan potato) and P. rotundifolius (Madagascar potato) [tranthus , 2. It htranthus , 4. The  potato) –7.P. edulis is primarily cultivated for food and as a source of income for millions of subsistence farmers, particularly in the country’s densely populated highlands and semi-highlands. The crop is also widely used as a folk medicine and a source of nectar for honeybee [In Ethiopia, honeybee , 9. Howehoneybee . Furtherhoneybee .Genetic diversity, the total genetic variation in a species, could be assessed using a particular method or a combination of methods such as agro-morphological traits, biochemical and/or DNA marker(s) methods. Because of their ability to generate more reliable information, DNA molecular markers have been used for more than 30 years in estimating genetic diversity values –14. ISSRP. edulis focusing only on agro-morphological diversity, ontogeny, micro-propagation, phytochemistry and nutritional analysis. As far as we know, very limited work is available using molecular DNA data such as EST-SSR [P. edulis populations collected from diverse agro-ecological regions in Ethiopia using ISSR markers. The generated information could be used as baseline in the future to support marker assisted breeding, conservation and germplasm management of this crop.So far, very limited research activities have been conducted on  EST-SSR  and no rP. edulis accessions, representing nine populations, were collected from the four main growing regions in the country  protocol , approxiTwenty-five ISSR primers (Source: Primer kit 900 (UBC 900) obtained from University of British Columbia, Vancouver, Canada) were used during the initial screening for variability and reproducibility. Ten  polymorphic primers capable of generating reproducible bands were selected and optimized for the study Table .Table 2L2O (15.2 μl), MgCl2 (25 mM) (3.0 μl), Taq buffer (10× reaction buffer S) (2.5 μl), dNTPs (1.25 mM) (1.0 μl), primer (20 pmol μl−1) (0.4 μl), Fire Pol DNA Polymerase (5 U μl−1) (0.4 μl),  and template DNA (10 ng µl−1) (2.5 µl). The amplification program was set at 94 °C for 4 min preheating and initial denaturation, followed by 40 cycles of denaturation at 94 °C for 15 s, primer annealing at specific annealing temperature for each primer , absent (0) and ambiguous (?). After successful scoring, the data were assembled into a binary data matrix with the samples in a row and the ISSR markers (loci) in column.The resulting data matrix was analyzed using appropriate software. POPGENE ver. 1.32  was usedThe ten screened and optimized ISSR primers produced 74 clear and scorable bands over the entire nine populations (an average of 7.4 per primer). The molecular weight of the fragments ranged between 200 and 2000 bp. UBC-834 produced the highest number of scorable bands (12), whereas UBC-835 and UBC-868 scored the lowest (5 bands each)  of polymorphic loci. The highest gene diversity (h = 0.48) and Shannon’s information index (I = 0.68) were recorded for UBC-812, followed by UBC-834, 844 and 854. UBC-835 showed the least gene diversity (h = 0.39) and UBC-866 showed the lowest Shannon’s information index (I = 0.58). Overall, there were 0.40 and 0.62, respectively, gene diversity and Shannon’s information index over the entire population , gene diversity h = 0.44) and Shannon’s information index (I = 0.62), followed by population from Central Highland . Populations from Metekel and Wolaita Sodo administrative zones showed the lowest values for PPL, h and I (Table 4 and Shap = 0.001) percentage of differentiation  due to variance within populations and regions than differentiation among populations and regions   value (Table m = 1.54)  Table . The lowue Table  and a hiThe populations pairwise genetic distance (D) ranged from 0.11 to 0.39. In this regard, in the magnitude order, individuals from the Awi administrative zone population were relatively distantly related to the populations of Wolaita Sodo, Metekel, and Illu Aba Bora administrative zones. Similarly, population from Jimma administrative zone was relatively distant from the populations of Wolaita Sodo and Metekel administrative zones. The lowest estimate of genetic distance was observed between individuals of the Central Highland and East Wollega administrative zone populations Table .Table 6NI followed by cluster II (24). Clusters III and IV included three accession each, while cluster V contained five accessions  and populations from Gamo Gofa, Metekel, Gurage and Illu Aba Bora administrative zones were grouped together (C1). Population from Wolaita Sodo administrative zone appeared as a monophyletic group (C2)  were grouped together under cluster 1 were grster II 2. ClusterAnalysis of principal components (PCoA) based on Nei’s  genetic ISSR marker system is one of the widely used molecular markers for assessing the extent and patterns of genetic diversity and for deducing phylogenetic relationships in a variety of crop species. Similarly, it has been used in a wide variety of plants worldwide to study genetic variation , 28.P. edulis populations. There have been similar reports in other endemic root and tuber crops such as sweet potato [Lens culinaris medikus) [Coffee arabica L.) [The ten reproducible ISSR markers selected and used in the present study showed an overall high level of polymorphism (95%) which implies their great usefulness in revealing and evaluating the level of genetic diversity among and within t potato , yam [29t potato  populatimedikus)  and coffbica L.)  from Ethbica L.) , and sesbica L.) .P. edulis populations could be attributed to the crop’s clonal propagation nature in which only few parts of the population can preserve the original genetic diversity in the basal population. Furthermore, the efforts of local farmers, especially the elders, to maintain the original genetic diversity by preserving tubers over generations could be another reason for the observed higher genetic diversity indices, although the population size is rapidly declining from vast areas where it used to be widely cultivated. Twenty EST-SSR DNA markers detected a larger number of alleles and revealed a similar increasing trend in genetic diversity in twelve P. edulis populations that were collected from diverse agro-ecologies in the country [The extent of genetic diversity in plant species is the result of one or more factors such as reproductive biology, life (evolutionary) history, geographic range of distribution, and various environmental factors that, in one way or another, affect mutation rates –37. In t country .P. edulis populations from Gurage, Central Highland, Awi, East Wollega and Jimma administrative zones, in order of magnitude, are important sources for improving the germplasm and taking immediate conservation actions.High level of genetic variability is desirable because it increases fitness and thus reduces the likelihood of local extinction . In thisP. edulis domestication and cultivation. In agreement with this, Rampersad et al. [On the other hand, the low values of percent polymorphism, gene diversity and Shannon’s information index observed in populations from Metekel and Wolaita Sodo administrative zones could be attributed to the smaller sample size used from these specific areas. In addition, the recent introduction and domestication of the crop to some areas, especially, the Metekel zone, which is a bit pocketed and relatively recently dominated by the Oromo and Awi ethnic groups, could be also the probable reason. Compared to other ethnic groups in the area, these ethnic groups are known to be more familiar with d et al.  suggestePlectranthus edulis populations revealed a lower extent of genetic differentiation among the populations and the regions that could result from historical or contemporary exchange of germplasms, especially tubers for immediate planting, between or among zones and regions included in this study. The higher values of the overall gene flow (Nm = 1.54) and lower overall Gst  we observed could also support this premise as suggested by Wright [m > 1), and hence migration, is a powerful force for decreasing differentiation among populations [y Wright  since thulations .Coccinia abyssinica) and yam, respectively. Similarly, Wodajo [Lepidium sativum, respectively.On the other hand, the significantly higher genetic differentiation within populations and regions could be attributed to the clonal propagation nature of the crop, an important aspect in maintaining heterozygosity in the basal population over generations regardless of the current population size fluctuation. This can be explained by the fact that the crop is historically sexually reproduced through seeds, which is still rarely practiced at research sites, and this has allowed the crop to accumulate heterozygosity in the basal population to some extent. Tadele et al.  and Nasc, Wodajo , and Sei, Wodajo  reportedP. edulis populations that could be a valuable source for selection breeding. For this purpose, individuals from the Awi administrative zone versus Wolaita Sodo, Metekel, Illu Aba Bora and Gurage administrative zones, showing considerable pairwise genetic distance, could be used as parental sources. On the other hand, Mantel test revealed that the genetic distance did not correlate with geographic distance (data not shown) which again confirms intensive seed tuber exchange between or among farmers or the recent divergence of the crop from a common genetic base. Moulin et al. [The wide range of pairwise genetic distance that we detected (the highest being more than 4× of the lowest) generally indicates the high genetic variability in n et al.  reportedP. edulis populations showed poor correlation between geographic origin and the patterns of clustering. Samples or populations from distant zones or regions have been found lumped together the same cluster and those from the same or nearby geographic areas have been placed under a different cluster and such grouping weakly supports the concept of “isolation by distance” [The genetic relationships between and among istance” . GeneralFrom the present study, we conclude that ISSR marker system is useful in estimating the extent of genetic diversity and generating valuable information for use in further breeding and conservation measures in indigenous crops. However, in order to generate a more reliable information, it is important to combine it with other marker systems such as agro-morphological traits-based genetic diversity assessment or co-dominant molecular marker systems, such as SNPs, which have a higher potential for estimating population genetic structure.P. edulis diversification and conservation. The higher gene flow among the different regions, coupled with the current decline in population size, may result in loss of valuable agronomic traits unless counteracting action is taken.On the basis of genetic diversity indices such as gene diversity, Shannon’s information index, and percent of polymorphic loci, Gurage, Central Highland, Awi, East Wollega and Jimma administrative zones are relatively better areas for Representative samples from all potential growing areas need to be collected exhaustively to provide a good estimate of the crop’s existing genetic diversity for use in improving it and reversing its current rapid genetic erosion.


Cytb to investigate genetic diversity and demographic history among 19 populations of P. flavescens in eastern the monsoon region of China. In a Bayesian clustering analysis of the microsatellite data, three distinct clades were present, and each population consisted of a mixture of individuals from the three clusters. An AMOVA of the data from both the microsatellite loci and Cytb revealed that genetic variation was mainly within each population. For the 543 individuals from the 19 regions, 77 unique haplotypes were obtained by DnaSP 4.0, and a median-joining network showed no obvious geographical pattern and displayed high gene flow and minimal population genetic structure among the 19 populations. According to a Mantel test, there was no significant association between genetic distance and geographic distribution and no isolation by distance. Mismatch distribution and neutrality tests showed no demographic expansion for the 19 populations. Microsatellite and mitochondrial DNA data suggested there was high gene flow and low differentiation among the populations. These results will help provide valuable information to study the migratory route of insects, especially important agricultural pests.To analyze genetic characters of migratory dragonflies, we used 10 microsatellite markers and a partial sequence of the mitochondrial gene  When it comes to long distance migrations, some animals, such as birds and whales, may be the most commonly thought of3. Yet, some dragonflies are as capable of long- distance travel. Insects in the order Odonata  are the most ancient invertebrates capable of flight and very diverse, with approximately 6000 species worldwide6. Mass migration of dragonflies has long been known and there are about 25–50 migratory species7. Pantala flavescens is well suited for phylogeographic studies due to its extensive migratory ranges, which spans mountain ranges, continents and oceans.Animal migration is one of the most remarkable phenomena in nature, in pursuit of improved safety, increased foraging opportunities, and higher reproductive outputPantala flavescens  may be the most common dragonfly in the world because it is very adaptable to different habitats and long-distance dispersal capability. It occurs in temperate and tropics regions, from lowland to montane, typically in coastal and open areas that span thousands of kilometers11. Isotopic evidence also suggests that its multigenerational journey may total over 18,000 km, with single individuals traveling over 6,000 km during the transoceanic trek from northern India to east Africa12.Anax junius (Drury 1773), the common green darner, is relatively high in the absence of any obvious phylogeographic pattern13. Libellula quadrimaculata , the four-spotted chaser or skimmer, showed high haplotype interconnection among samples collected within Asia, Europe and North America14.Such migratory behavior of odonate insects could homogenize genetic differentiation among populations by the exchange of individuals and genes among populations that are separated by large distances and thus impact the population structure of that species. For example, haplotype diversity in populations of P. flavescens using randomly amplified polymorphic DNA has also shown that genetic diversity is low and gene flow is high among five geographically isolated populations within India15. Based on sequence data using cytochrome oxidase 1 (CO1), high rates of gene flow are present among all studied geographic regions and genes are shared among individuals across the globe3. In addition, no significant genetic differentiation among Malaysian populations of P. flavescens was found, despite a high level of gene flow determined by mismatch distribution and neutrality tests, which also provided evidence of demographic expansion during the Pleistocene 11.Previous research on CO1 gene sequence analysis of the tiny dragonfly Nannophya pygmaea showed overall low genetic diversity among 68 N. pygmaea individuals collected over six habitats in Korea16. Although these geographic populations of N. pygmaea in Korea clustered into two groups, genetic isolation by distance was not detected16.A less-mobile species may be expected to show some evidence of haplotype clustering according to geographic region. A portion of mitochondrial b gene (Cytb) has often been used as a marker for studies of evolution and population structure and to resolve taxonomic conflicts in many animal groups17. In the present study, we used partial sequences of Cytb and microsatellite markers to analyze the population structure, genetic divergence and demographic history among individuals of P. flavescens collected during an intensive sampling in the eastern monsoon region of China. Such investigations can improve our knowledge of the migratory behavior of P. flavescens in China and may also contribute to investigations of the evolution of migration. Additionally, the sampling and analytical methods used here may provide a potential model to study the migration of other insects.Microsatellites are the most popular genetic marker owing to their high abundance, easily typed locus-specific codominance and Mendelian inheritance. The mitochondrial cytochrome P. flavescens, 542 sequences including 477 bases in the mtDNA Cytb genes were obtained. Of the total genetic characters, 430 bases were conserved, and 47 polymorphic sites were found in the alignment of 542 Cytb sequences, with 12 singleton polymorphic sites (20%) and 35 parsimony informative sites (80%). Genetic diversity and the distribution of haplotypes among different populations of P. flavescens based on the Cytb sequences are shown in Table From 583 individuals of Fst values ranged from −0.003 to 0.090, with the highest differentiation observed between CC and LF and the lowest between HEB and QF (Supplementary Table Fst < 0.15), and the other 105 populations with no differentiation (Fst < 0.05). The Fst matrix based on mitochondrial genes showed that 133 of the 171 population pairs had no significant differentiation  came from variation within the populations. Results of the AMOVA test based on mtDNA markers in different populations of D and and Fu’s FS statistic are statistically negative but not significant (P > 0.02) (Table D and Fu’s FS was negative (Table P > 0.02). The results showed that these populations were in a stable state, with no recent bottleneck or a rapid population expansion. However, for the other populations , which showed these populations had experienced a recent population expansion.When the 19 populations were regarded as a whole, Tajima’s 2) Table . For mosFst. The genetic parameters of these populations based on the 10 microsatellite markers among the 19 populations are summarized in Table Ar) ranged from 5.33 in population ZZ to 6.95 in CF (mean 5.94); 140 alleles were obtained, with 6 alleles (Na) across microsatellite loci in XX to 8.9 in HZ (mean 7.35). The mean effective number of alleles (Ne) across microsatellite markers was 3.06, ranging from 3.03 in HEB to 4.41 in GL. The mean value of Shannon’s index (I) across microsatellite markers was 1.46, ranged from 1.34 (GZ) to 1.66 in (HZ). The observed heterozygosity (Ho) ranged from 0.36 in QF to 0.55 in JX (mean 0.48) and expected heterozygosity (He) ranged from 0.64 in HEB to 0.75 in HZ (mean 0.68). The unbiased expected heterozygosity (uHE) ranged from 0.65 in HEB to 0.77 in CF (mean 0.69). Overall, there was a high level of genetic diversity for all microsatellite loci in the study regions.In a microsatellite analysis of 542 DNA samples from 19 populations using 10 microsatellite markers, Fisher’s test indicated 142 of 190 locus–population combinations deviated significantly from Hardy–Weinberg Equilibrium (HWE). According to a Micro-checker analysis, the null allele was very low, mostly less than 0.01 among 10 microsatellite markers; therefore, the presence of null alleles had almost no influence on the analysis of Fst matrix based on microsatellite data showed that 101 of the 171 population pairs had significant differentiation , and the other 106 population pairs had no differentiation (Fst < 0.05). The Bayesian clustering analysis revealed the presence of three distinct clusters, and each population was a mixture including individuals from three clusters  was genetic variation within populations.A high distribution proportion was obtained by the partial Bayesian method in Geneclass, and 95.7% (519/542) of the individuals were assigned to the 19 populations. The remaining 23 individuals might be migrants from other areas to the sampling regions. GENECLASS identified that all migratory individuals were offspring of migrants and that none were first-generation migrants.Z = 8299.1787, r = 0.0854, P = 0.804, and Cytb: Z = 2863.798, r = −0.1002, P = 0.147), which may be reflective of a lack of phylogenetic divergence among the individuals across the study areas. This result also suggested migratory dragonflies could colonize and exchange genes with local populations.Our observation showed no significant correlation between genetic distance and geographic distance of these populations based on the two types of markers  suggested that if Fst = 0, then the two populations lack differentiation; when Fst = 0.05–0.15, the populations are moderately differentiated, and when FST = 0.15–0.25, the populations are highly differentiated20. In our experiment, different populations had low to moderate genetic differentiation based on Fst values: from −0.003 to 0.09  of mtDNA were widely distributed in all populations, and the haplotypes in 19 populations formed three clusters, but had no obvious geographic divisions, which indicates that geographic barriers and climatic factors have little influence on migration of this dragonfly in different regions.The median-joining network revealed a close relationship among haplotypes, suggesting that Nm are often taken at face value as the approximate number of migrants moving among populations21. In our experiment, different populations had a high number of individuals dispersed (Nm = 4.326), thus avoiding the genetic differentiation that arises from genetic drift and explaining the low inter-subpopulation genetic variation. Our results showed a high rate of gene flow and lack of population differentiation among 19 studied populations. At the same time, genetic differentiation and geographic distance were not correlated, so isolation by distance does not appear to be a barrier for gene flow.Estimates of Nannophya pygmaea is very small and unlikely to migrate over large-scale regions, which likely contributes to its overall low diversity and genetic isolation by distance16. The endangered damselfly Coenagrion mercuriale  is a weak flier22, and significant genetic differentiation between sites can be prevented if sites are <2 km apart and not separated by a physical barrier. Nevertheless, dispersal by C. mercuriale is sufficiently restricted so that genetic structure can result from isolation by distance develops within 10 km22.A less-mobile species may be expected to harbor some evidence of haplotype clustering according to geographic region. P. flavescens could successfully colonize and adapt new habitats. They were able to disperse randomly and exchange genes with local populations which lead to high rates of gene flow among 19 geographic populations. However, the migration of P. flavescens on a global scale and the potential ecological impacts of their migratory behavior remain unknown and need further research.Our results suggested that P. flavescens were sampled from 19 geographic sites in 18 provinces in China, from June 2013 through October 2014 , and preserved at −20 °C in the refrigerator.Cytb were amplified using published primers23. Each PCR amplification was performed in 30 μL reactions with each reaction consisting of 15 μL of 2× Taq Master Mix solution , 1 μL of DNA template, 1 μL of each primer , and 12 μL of RNase-free water. The PCR reactions were performed in a Techne thermocycler (Germany) with the following program: 5 min initial denaturation step at 94 °C; 35 cycles of 45 s at 94 °C, 45 s at 55 °C, 1 min at 72 °C; and a final extension 5 min at 72 °C. Amplification products of Cytb were sequenced on an ABI 3730 DNA Sequencer . Mitochondrial DNA sequences were manually checked and aligned with ClustalX 1.8524, using the multiple alignment default parameters. Nucleotide composition, parsimony informative sites, variable sites and conserved sites were calculated with MEGA 625. Molecular diversity indices such as nucleotide diversity and haplotype diversity were analyzed in DnaSP 4.026. Genetic differentiation (fixation index Fst) between populations was calculated using mtDNA data and Arlequin 3.0 with 10000 permutations27. Analysis of molecular variation (AMOVA) based on mtDNA data, as implemented in Arlequin 3.027, was used to test for hierarchical genetic structure of the populations.Partial regions of the mitochondrial gene 28 software was used to construct a median-joining network. Demographic history changes were analyzed for P. flavescens using two neutrality tests, Tajima’s D (1989)29 and Fu’s Fs (1997)30, which explained a recent population bottleneck or population expansion. Mismatch distributions count the number of site differences between each pair of sequence in a sample and use the calculation result to build a histogram. According to the coalescent theory, a population usually shows a unimodal mismatch distribution following a population demographic expansion31.Network 2.0P. flavescens19. MICRO-CHECKER 2.2.3 was used to detect genotyping errors in microsatellite data due to null alleles, stuttering, or allele dropout using 1000 randomizations32. Deviations from Hardy–Weinberg equilibrium (HWE) in all the loci of each population and linkage disequilibrium between pairs of loci were assessed using Genepop 3.433. To study the genetic diversity of different geographic populations, we used GenAlEx 6.4134 to calculate mean number of alleles (Na), effective number of alleles (Ne), Shannon’s index (I); expected and observed heterozygosity (He and Ho) and unbiased expected heterozygosity (uHE). Allelic richness (Ar), fixation index (Fst), and inbreeding coefficient (Fis) among these sites were analyzed in FSTAT 2.9.335. Genetic differentiation between all pairs of populations was calculated in Arlequin 3.027.Total DNA was extracted from 583 individuals for PCR and genotyping for 10 microsatellite loci developed for 27, was used to test for hierarchical genetic structure of the populations. We used STRUCTURE 2.3.336 and its nonspatial algorithm to further assess the degree of population differentiation within and between the 19 populations based on microsatellite data. The allelic frequencies for different populations and the admixture model were used to class different individuals into corresponding population clusters. Simulation was run 7 times for each value of k for 106 iterations after a burn-in period of 30,000. To determine the optimal number of groups (K), we utilized both the log likelihood [ln Pr (X/K)] method as recommended by Pritchard et al.37 and the ΔK statistic of Evanno et al.36. Individuals of the first migrant generation for each population were detected using the L-home likelihood computation in GENECLASS 238.Analysis of molecular variation (AMOVA) based on microsatellite data, as implemented in Arlequin 3.0P. flavescens is influenced by distance. To determine whether movement patterns were limited by spatial scale, we ran isolation by distance analysis between pairwise linearized genetic and log-geographic distance data using a Mantel test in IBDWS 3.2339.Correlation tests were conducted between the geographic distance and the corresponding genetic differentiation matrix of these populations in the Mantel test. The geographical and genetic distances should be positively correlated if the dispersal of Supplement material


Mantella aurantiaca, Mantella crocea, and Mantella milotympanum. Although our restriction site‐associated DNA (RAD) sequencing approach identified clear genetic clusters, they do not align with current species designations, which has important conservation implications for these imperiled frogs. Moreover, our results suggest that levels of intraspecific color variation within this group have been overestimated, while species diversity has been underestimated. Within major genetic clusters, we observed distinct patterns of variation including: populations that are phenotypically similar yet genetically distinct, populations where phenotypic and genetic breaks coincide, and populations that are genetically similar but have high levels of within‐population phenotypic variation. We also detected admixture between two of the major genetic clusters. Our study suggests that several mechanisms—including hybridization, selection, and drift—are contributing to phenotypic diversity. Ultimately, our work underscores the need for a reevaluation of how polymorphic and polytypic populations and species are classified, especially in aposematic organisms.Intraspecific color variation has long fascinated evolutionary biologists. In species with bright warning coloration, phenotypic diversity is particularly compelling because many factors, including natural and sexual selection, contribute to intraspecific variation. To better understand the causes of dramatic phenotypic variation in Malagasy poison frogs, we quantified genetic structure and color and pattern variation across three closely related species,  This study—the first quantitative and objective exploration of color diversity in the Mantella genus—not only provides a foundation for future studies of color evolution in Malagasy poison frogs but also identifies several critical issues that should be more thoroughly considered in any investigation of aposematic organisms presumed to be polymorphic or polytypic.Given the lack of resolution in previous molecular studies, it is apparent that a high‐resolution genetic dataset is needed to both clarify relationships within this group and to determine whether observed color variants represent distinct species or morphs. In this study, we used a restriction site‐associated (RAD) sequencing approach, in combination with multiple matrix regression analysis, to compare variation in dorsal coloration and side and ventral patterning with genetic and geographic distance across the entire known range of three species of Malagasy poison frog. Specifically, our objectives were to (a) clarify genetic structure among populations of 22.1M. aurantiaca, M. crocea, and M. milotympanum, throughout their entire known range in central‐eastern Madagascar /(G + B) represented the y‐axis. From this color space, we calculated chroma and hue values following Krohn and Rosenblum . Preliminary analyses did not reveal an effect of sex on frog coloration or pattern, so sexes were lumped for the analyses presented here. However, our study design did not explicitly aim to quantify sexual dimorphism, and future studies can investigate this question with targeted sampling.2.3We extracted DNA from toe clips using Qiagen DNeasy extraction kits  generally following the manufacturer’s protocol with two modifications: 4 µl of 1 mg/ml RNase A was added to each sample after lysis, and DNA was eluted in 45 µl of 1× LTE buffer to maximize concentration. Prior to library preparation, we checked the quality of extracted DNA using agarose gels and quantified DNA using a Qubit 2.0 Fluorometer .We constructed a restriction site‐associated DNA (RAD) library following the protocol of Ali et al. , without2.4https://github.com/CGRL-QB3-UCBerkeley/RAD). We first demultiplexed raw fastq reads using internal barcode sequences and allowing for one mismatch in barcode sequence. We removed demultiplexed reads that did not include the expected restriction enzyme cut site at the beginning of the read, again allowing for one mismatch in cut site sequence, and also removed exact duplicates using Super Deduper (https://github.com/dstreett/Super-Deduper). To filter reads, we used Cutadapt . We only retained those reads that mapped uniquely to the reference. Using Picard (http://www.picard.sourceforge.net) and GATK  where at least 80% of individuals showed at least 3× coverage.We used genotype likelihoods instead of genotype calls whenever possible to account for uncertainty in our data. Because our data showed low to medium coverage , SNP and genotype calls based on allele counts could potentially cause bias or introduce noise in downstream analyses , with ten replicates per K value. We then used the Evanno method  of the covariance matrix of posterior genotype probabilities implemented in ngsTools . We ran NGSadmix with ten replicates for each K value and used the Evanno method to determine the most likely K value. We assigned admixed populations to the NGSadmix group from which more than 50% of its admixture was drawn. Finally, we calculated FST values for all possible pairwise population comparisons using the realSFS function of ANGSD.To characterize fine‐scale population structure, we performed an NGSadmix analysis within each major genetic cluster. We again estimated individual admixture proportions with the number of clusters ranging from one to one more than the total number of populations , other populations were geographically more proximate with varying degrees of genetic distance between them (as in Clusters B and C).We found that K = 2 based on Evanno et al., FST value between these two populations (0.43) was among the highest in our dataset . Additionally, the degree of phenotypic variability was much lower in this cluster than in the others, as side pattern was the only phenotypic trait that was significantly correlated with geographic distance when accounting for genetic distance . Phenotypically, populations within this cluster were relatively uniform, and there was little variation in either dorsal coloration or in side and ventral pattern . Variation in ventral patterning also remained significantly correlated with genetic distance after controlling for geographic distance . All three quantified phenotypic traits were significantly correlated with geography after controlling for genetic distance . Phenotypic discrepancies between the M. crocea and M. aurantiaca groups were most evident in dorsal coloration, particularly in dorsal chroma and hue . Despite this spatial segregation of phenotypes, we observed an exceptionally high degree of variation within populations in this group . Our NGSadmix results yielded a consistent signature of admixture in all M. cf. milotympanum populations that we sampled . Notably, candidate hybrids, or M. cf. milotympanum individuals, demonstrated higher within‐population variation in patterning characteristics, and also displayed novel pattern phenotypes not present in either “parental” species  and are in fact distinct from the other M. crocea populations included in this study  M. crocea populations form a distinct cluster with the two M. aurantiaca populations .Populations within this complex were highly structured at relatively small spatial scales, with distinct genetic groups emerging. Unexpectedly, genetic clusters identified in this study do not correspond to current species designations. Specifically, our results indicated that frog populations that have thus far been considered to be a green color variant of the  Figures  and 3. OM. milotympanum and M. crocea are conspecific, these intermediates were referred to as M. cf. milotympanum in the literature and assumed to be another phenotypic variant  that displayed intermediate genotypes and pattern phenotypes Figures  and 6. BM. aurantiaca and M. milotympanum are currently classified as critically endangered, while M. crocea is classified as vulnerable  and Cluster B . Due to the highly isolated and fragmented nature of Mantella populations, further intensive survey efforts will be important to identify any additional extant populations.The taxonomic resolution provided by our study has significant consequences for conservation efforts, given that s Figure . We recoM. crocea”) should be considered a distinct entity and prioritized given its extremely limited distribution . Due to the likely hybridization that we detected between Clusters B and C, and the fine‐scale population structure that we observed within each cluster, the status of Clusters B and C is less clear. Rather than revising taxonomy prematurely, we recommend additional studies on gene flow and migration, characteristics of frog calls, and mating behavior before species boundaries can be clarified with any certainty.Overall, while it is premature to delineate new species on the basis of our analyses—especially considering the significant conservation implications in this system—our study confirms that there are at least three genetically distinct groups that do not correspond to current species descriptions. At the least, it seems clear that Cluster A  that displayed intermediate genotypes and novel pattern phenotypes  and lizards (Zonosaurus sp. in M. aurantiaca and Zonosaurus madagascariensis in M. laevigata) . Describing novel phenotypic variants within a species should require—at the least—a high‐resolution genetic dataset to confirm intraspecific relationships. In addition, rather than characterizing human‐observed variants as morphs, we recommend that researchers specify the facet of phenotype measured, the sensory perspective used to quantify phenotype, and the level of biological organization where variation is observed. The way we define and delineate morphs is relevant in many systems, but carries particular significance in aposematic organisms, where novel color and pattern variants are regularly found. If we are overestimating intraspecific color variation and underestimating species diversity—as was found in this study—the classification of populations and species as polymorphic or polytypic may need to be reevaluated. Ultimately, it is time to give as much consideration to the conceptual and operational morph delineation as has been given to species.None declared.KK designed the research with input from EBR. KK performed the field work and lab work. KK and KB analyzed the data. KK and EBR wrote the paper with contributions from KB.


CYP405A2 between different populations of the common burnet moth Zygaena filipendulae from across the Western Palearctic region to illuminate the colonization history of this species. These data show two major clusters of Z. filipendulae populations possibly reflecting two different refugial populations during the last ice age. The two types of Z. filipendulae only co‐occur in Denmark, Sweden, and Scotland indicating that Northern Europe comprise the hybridization zone where individuals from two different refugia met after the last ice age. Bayesian phylogeographic and ecological clustering analyses show that one cluster probably derives from an Alpe Maritime refugium in Southern France with ancestral expansive tendencies to the British Isles in the west, touching Northern Europe up to Denmark and Sweden, and extending throughout Central Europe into the Balkans, the Peleponnes, and South East Europe. The second cluster encompasses East Anatolia as the source area, from where multiple independent dispersal events to Armenia, to the Alborz mountains in north‐western Iran, and to the Zagros mountains in western Iran are suggested. Consequently, the classical theory of refugia for European temperate species in the Iberian, Italian, and Balkan peninsulas does not fit with the data from Z. filipendulae populations, which instead support more Northerly, mountainous refugia.Northern and mountainous ice sheets have expanded and contracted many times due to ice ages. Consequently, temperate species have been confined to refugia during the glacial periods wherefrom they have recolonized warming northern habitats between ice ages. In this study, we compare the gene  Consequently, temperate species have been confined to refugia during the glacial periods wherefrom they have recolonized warming northern habitats between ice ages . In Europe, many temperate species have been shown to inhabit the Iberian, Italian, and Balkan peninsulas during glacials Hewitt, . FurtherPhylogeographic analyses can be used to unravel historical processes resulting in contemporary population distributions based on gene sequences  . When the preferred larval food plant Lotus corniculatus  are brightly colored day flying moths comprising 98 species distributed across most of Europe. e Figure  are verye Figure , p. 49 iZygaena filipendulae has been established as a model system to study the evolution and roles of its chemical defense compounds, the cyanogenic glucosides linamarin and lotaustralin  either preserved in ethanol or as dried specimens. Genomic DNA was isolated from moths or larvae using DNeasy Blood and Tissue kit (Qiagen 69504). CYP405A2 genes from the specimens were PCR amplified from genomic DNA diluted to a concentration of 10 ng/µl, using the following program: denaturation 94°C for 2 min followed by 30 rounds of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 1 min. Final extension was at 72°C for 7 min. Primers are shown in Supporting Information Table 2.2The fragments of CYP405A2 obtained from all the specimens were manually assembled into full‐length genomic sequences in MEGA7  sampler calculating posterior probabilities in a fully model‐based framework, thus controlling for uncertainty in haplotype relationships. A total of 43 concatenated exon sequences of CYP405A2  were available for the Bayesian phylogeographic clustering analysis. Haplotype reconstruction used 42 distinct haplotypes with 67 parsimony informative sites and further (178 ‐ 42) intermediate haplotypes for the most parsimonious haplotype network. The analysis was run with two Markov chain Monte Carlo initializations for 2,000,000 iterations each with 2,000 steps saved, taking the strict parsimony level option at minimum, ds = 0. The dimensionality of the data was p = 2 for geographical dimensions longitude and latitude. The posterior mass for the number of clusters strongly concentrates around 2, with the posterior probability of two clusters being >0.81. The two clusters are designated as the “Western‐Central‐Northern European” Z. filipendulae (European) and “Caucasian–Iranian” Z. filipendulae cluster (Caucasian), respectively. The root location is the same regardless if BPEC analyses are run with or without recombinant sites.Bayesian phylogeographic and ecological clustering was used to identify genetically distinct geographical population clusters and ancestral locations in 3Z. filipendulae, CYP405A2 sequences from 43 specimens were analyzed. Z. filipendulaeCYP405A2 sequences are between 92% and 99% identical to each other . The accuracy scores for the ancestral sequences are 0.998 and 0.997, respectively, implying that on average any base has a 99.8% or 99.7% chance of being correct , because Scotland was covered in ice before that timepoint. When using this calibration point, timings of other events in the phylogenetic tree appeared to have happened later than they logically could have, for example Z. lonicera should have split from Z. filipendulae only 22,000 years ago which seems highly unlikely. Consequently, the split between the European and the Caucasian cluster appear to be much earlier than the 15,000 years ago used to calibrate the tree and consequently, it is not possible to assign a reliable calibration time to the molecular clock tree  in central Europe Naumann, . None ofe Figure . As evidZ. filipendulae were assigned to two phylogeographic clusters with high posterior probabilities   Figure  by BPEC.) Figure , Table 1High posterior assignment probabilities to the European cluster were obtained for the following haplotypes (≥0.95): 1 Scotland West, 2 France West, 24 Scotland East II, 30 Amager I, 31 Citytwo I, 39 Iran Northeast, 40 Iran Northwest, 41 Armenia a, 43 Pyrenees. High posterior assignment probabilities to the “Caucasian–Iranian” cluster (Caucasian) were obtained for the following haplotypes (≥0.95): 4 England Early, 8 Poland West, 9 Poland Southeast, 11 Switzerland I, 12 Kvistgaard I. The remaining haplotypes share origins from both clusters in varying proportions, but usually with higher probabilities to the West‐Central‐North cluster (≥0.67), or in two cases assigned with equal probability to both clusters (0.50) (6 SDU I and 21 Poland South). Haplotypes 6 and 21 both had similarly low probabilities for two alternative clusters (posterior probabilities of 0.5), and so could not be unambiguously assigned to a single cluster. It is remarkable that in specimens tested from two neighboring populations in England, only differing in the timing of the end of diapause in spring (England Early and England Late), we found a 100% assignment to the Caucasian cluster for the early specimen, and a two‐third assignment to the European population for the late specimen. Another example is the haplotypes of both strands of the gene in the Armenian specimen, being assigned with high proportion to the predominant West‐Central‐North cluster though located in the Eastern region.Theoretical predictions of coalescent theory states that a root haplotype is expected to have a higher number of haplotype connections in the network, rather than being close to the tips. The fact that BPEC does provide a finite (probability) distribution of haplotype trees—as well as the existence of the underlying migration model—allows this method to incorporate the uncertainty in the haplotype rooting.4Z. filipendulae across the Western Palearctic region to infer their phylogeographic relationships. Only a fraction of the total genetic diversity within populations of a species can be seen in any study, relying on the number of specimens and populations analyzed. Most previous studies have relied on the mitochondrial COI gene and a sequence of 648 bp in length  and another in East Anatolia (Caucasian). The Alpe Maritime refugium has ancestral expansive tendencies to the British Isles in the west, touching Northern Europe up to Denmark and Sweden, and extending throughout Central Europe into the Balkans, the Peloponnese and South East Europe. The second cluster encompasses East Anatolia (Caucasian) as the source area, from where multiple independent dispersal events to Armenia, to the Alborz mountains in north‐western Iran, and to the Zagros mountains in western Iran are suggested  could indicate recent colonization events, as seen for the Carabid beetle Carabus arcensis on other Danish islands . Despite these differences, both populations are very closely related in the phylogenetic tree, although the BPEC analyses showed them to mostly belong to the two different clusters. The two Scottish populations are quite different from each other, one of them very similar to the English populations while the other is in the Caucasian cluster. This could signify that this population has been isolated since the initial colonization of Great Britain, while the other three British populations have been interbreeding to some extent.The two English L. corniculatus in this study. However, L. corniculatus is unfortunately routinely sown out along, for example, road banks all over Europe, and the seeds all originate from similar populations. Therefore, it is highly problematic to find enough samples of the “native” plant populations and to avoid dilution of the phylogenetic signal by sequences from the sown plants.Since insects cannot colonize new areas prior to the establishment of their food plant, it would have been ideal to include Z. filipendulae populations were found in this study which could reflect two different refugial populations during the last ice age, an Alpine/Maritime refugium in Southern France and another in East Anatolia. These two types of Z. filipendulae are only co‐occurring in Denmark, Sweden, and Scotland which could indicate that Northern Europe comprise the “hybridization zone” where individuals from the two different refugia met after the last ice age. BPEC analyses identified genetically distinct geographical population clusters and ancestral locations in Z. filipendulae and suggest an Alpine/Maritime refugium in Southern France and another in East Anatolia. Consequently, the classical theory of refugia for European temperate species in the Iberian, Italian, and Balkan peninsulas does not fit with the data from Z. filipendulae populations, which instead support more Northerly, mountainous refugia.Conclusively, two major groupings of The authors have no conflict of interest to declare.MZ conceived and designed the overall study. LD extracted DNA and sequenced genes from specimens. MZ assembled and aligned genes and produced phylogenetic trees. AH did Bayesian phylogeographic analyses. MZ and AH wrote the manuscript, and all authors revised the manuscript. Click here for additional data file.


Information on linkage disequilibrium (LD) and the extent of effective population size (Ne) has important implications for exploring the degree of biological diversity, for predicting underlying selection pressure, and for designing animal breeding programs. In this study, we assessed LD, Ne, and the distribution of minor allele frequency in six goat populations. Accordingly, the results of LD and Ne using a single nucleotide polymorphism (SNP) panel  are helpful for the sustainable conservation, proper management, and utilization of Chinese goat populations.2 and D’ values, respectively. The average r2 between all the populations varied little and ranged from 0.055 r2 for the Jining Grey to 0.128 r2 for the Guangfeng, with an overall mean of 0.083. Across the 29 autosomal chromosomes, minor allele frequency (MAF) was highest on chromosome 1 (0.321) and lowest on chromosome 25 (0.309), with an average MAF of 0.317, and showing the lowest (25.5% for Louping) and highest (28.8% for Qingeda) SNP proportions at MAF values > 0.3. The inbreeding coefficient ranged from 0.064 to 0.085, with a mean of 0.075 for all the autosomes. The Jining Grey and Qingeda populations showed higher Ne estimates, highlighting that these animals could have been influenced by artificial selection. Furthermore, a declining recent Ne was distinguished for the Arbas Cashmere and Guangfeng populations, and their estimated values were closer to 64 and 95, respectively, 13 generations ago, which indicates that these breeds were exposed to strong selection. This study provides an insight into valuable genetic information and will open up the opportunity for further genomic selection analysis of Chinese goat populations.Genome-wide linkage disequilibrium is a useful parameter to study quantitative trait locus (QTL) mapping and genetic selection. In many genomic methodologies, effective population size is an important genetic parameter because of its relationship to the loss of genetic variation, increases in inbreeding, the accumulation of mutations, and the effectiveness of selection. In this study, a total of 193 individuals were genotyped to assess the extent of LD and Ne in six Chinese goat populations using the SNP 50K BeadChip. Across the determined autosomal chromosomes, we found an average of 0.02 and 0.23 for r Capra hircus) are animals that are highly adaptable to diverse environmental conditions, are raised all over the world for meat, milk, skin, fibre, and manure production, and are the sole source of livelihood for many rural people and landless laborers 2 and D’ ribed by . The avesoftware . The LD Effective population size (Ne) was estimated based on the following equation: E(r2)−1) , where NNeP v1.1 . Time pos: T=12c . Ne was −5) and call rate (<0.95), respectively. About 7849 SNPs were removed using LD-based pruning when an r2 threshold of 0.2 was exceeded. After filtering, 39,552 SNPs (74.14%) were used for LD analysis. The SNPs spanned approximately 0.43 GB of the caprine autosomal genome. The total SNP numbers per each chromosome (CHI) are summarized in In the present study, 53,347 SNPs were used before quality control. About 5946 SNPs were removed, leaving 47,401 of the loci distributed over 29 autosomal chromosomes, which were used for downstream analysis. About 2663 SNPs were removed due to having a MAF of <0.05. About 1568 and 1715 SNPs were removed on the basis of their results for Hardy–Weinberg Equilibrium (<10A neighbor-joining (NJ) tree clustering method represents relationships based on Nei’s genetic distance. The NJ tree showed four major clades: the Louping and Arbas Cashmere goats clustered separately at distinct clades a. The QiIn the current study, the mean MAFs for all the autosomal SNPs were 0.276 (AC), 0.308 (QGD), 0.284 (NJ), 0.314 (JN), 0.255 (LP), and 0.258 (GF), as indicated in IS are shown in 2 and D′ values estimated between adjacent SNPs for the 29 autosomal chromosomes were 0.02 and 0.23, respectively. The average r2 results were lowest for the JN group (0.055) and highest for the GF group (0.128), with an overall mean of 0.083. The r2 measure is known to be a more robust parameter for bi-allelic marker analysis, and therefore requires a smaller sample size for accurate LD estimation than D’ [2) decreased with genetic distance for all groups . The mean LD (r2) showed the most rapid decline in the first five bins. Overall, the JN, QGD, and NJ goat breeds displayed the lowest LD values across all intervals as compared to AC, GF, and LP populations. For each of the 29 autosomal chromosomes, the inbreeding coefficient ranged from 0.064 to 0.085 with an average of 0.075.LD, rNe declined between 1000 and 100 generations ago across the six studied goat populations. In total, the estimated Ne had decreased in AC (from 2289 to 95), NJ (from 3908 to 109), (JN from 5695 to 164), LP (from 3469 to 121), QGD (from 5630 to 140), and GF (from 2308 to 64) at 13 generations ago, which reflected a downward trend in Ne for all breeds due to selection. A rapidly decreasing recent Ne was observed in the GF and AC, while the JN and QGD had a slow Ne decline trend  is a crucial population genetic parameter because of its relationship to the loss of genetic variation, increases in inbreeding, the accumulation of mutations, and the determination of the accuracy of genomic selection . The curIn this study, we found low to moderate LD among the six populations, which indicates that the Caprine SNP 50K BeadChip will likely be a feasible tool for the prediction of genomic breeding values in goat populations. From the result showing a decline in LD with genetic distance, we inferred the Ne values and showed a rapidly decreasing trend across the entire studied population. Moreover, this study revealed a decreasing trend of Ne with the estimated values closer to 64 for AC and 95 for QGD at 13 generations ago, implying that the decline in the Ne of these goat breeds should be avoided. Further confirmatory investigations for the current population are required on a larger population dataset.


Sarcoptes scabiei in host mammal communities have been reported based on microsatellite-level genetic studies in the last two decades. While one involves restrictions among different host taxa, the other is associated with predator–prey interactions between different host taxa. In contrast to these observations, the present study reports a possible irregular case of transmission of S. scabiei between herbivorous Japanese serow and omnivorous Caniformia mammals in Japan, though under very weak predator–prey relationships.Two transmission patterns of Sarcoptes mites isolated from omnivorous Caniformia , omnivorous Cetartiodactyla (wild boar) and herbivorous Cetartiodactyla (Japanese serow) in Japan were analyzed by amplifying nine microsatellite markers. Principal components analyses (PCA), Bayesian clustering analyses using STRUCTURE software, and phylogenetic analyses by constructing a NeighborNet network were applied to determine the genetic relationships among mites associated with host populations.DNA from 93 Sarcoptes mites from wild boars and Japanese serows was observed. Conversely, considerably close genetic relationships were detected between Caniformia-derived and Japanese serow-derived mites. Because the predator–prey interactions between the omnivorous Caniformia and herbivorous Japanese serow are quite limited and epidemiological history shows at least a 10-year lag between the emergence of sarcoptic mange in Japanese serow and that in Caniformia, the transmission of S. scabiei from Caniformia to Japanese serow is highly suspected.In all the analyses, the genetic differentiation of S. scabiei transmission patterns. This type of cryptic relationship of S. scabiei populations may exist in local mammalian communities worldwide and become a risk factor for the conservation of the remnant and fragmented populations of wild mammals.The close genetic relationships among mites beyond Host–taxon relationships and without obvious predator–prey interactions in Caniformia and Japanese serow deviate from previously reported The online version of this article (10.1186/s13071-019-3630-5) contains supplementary material, which is available to authorized users. Sarcoptes scabiei is a notorious pathogenic mite species (Acari: Astigmata: Sarcoptidae) that causes a highly contagious dermatitis known as sarcoptic mange in mammalian hosts  and wild boars (Sus scrofa leucomystax) , an omnivorous Cetartiodactyla (wild boar) and a herbivorous Cetartiodactyla (Japanese serow).In the present study, we aim to illuminate the neglected relationships of Sarcoptes mites derived from Japanese serows and those from omnivorous species in Caniformia.To our knowledge, the predator–prey relationships between these omnivorous Caniformia and Cetartiodactyla have not been previously reported, except for a few limited cases of predation of Japanese serow by feral dogs  and possFrom 1996 to 2016, we collected skin crusts from 93 mangy animals from 10 different prefectures in Japan: Tokyo, Kanagawa, Saitama, Gifu, Wakayama, Hyogo, Shimane and Yamaguchi on Honshu Island, and Oita and Saga on Kyushu Island , domestic/companion dogs (n = 11), raccoons (n = 1), Japanese martens (n = 1), Japanese serows (n = 7) and wild boars (n = 13)  isolation method for frozen skins proposed by Alasaad et al. [S. scabiei from the morphological criteria defined by Fain [The animals belonged to 17 populations from 6 species that consisted of raccoon dogs . Molecular analyses were conducted following the method of Alasaad et al. [Sarcoptes mites were amplified by multiplex polymerase chain reaction (PCR). Each 5 µl PCR mixture contained 1 µl of the isolated mite DNA together with the PCR mixture containing all primer pairs (each primer ranged from 0.04 to 0.1 µM), 200 µM dNTPs, 0.5 µl of 10× PCR buffer , 0.5 mM MgCl2 and 0.05 µl (0.5 U/reaction) Ex Taq . Fluorescent PCR amplicons were analyzed in an ABI PRISM 3130 Genetic Analyzer using GeneScan 500 Liz® Size Standard . Allele calling was performed using the GeneMapper® Software v.4.1 .After the crushing individual mites, the DNA of each d et al.  with slid et al. ) from SaO) and expected (HE) heterozygosity, linkage disequilibrium (LD) and Hardy–Weinberg equilibrium (HWE) tests were calculated using GENEPOP v.4.0. [P-values of LD tests were given by Chi-square tests and those of HWE were calculated by a Markov chain algorithm [Observed  of loci, to analyze the underlying population genetic model. First, to describe differentiation between mite populations, principal components analysis (PCA) was carried out using the package adegenet  of R v.3adegenet . Multivaadegenet . In thisenet [SA ) among menet [SA . These genet [SA .Sarcoptes mites and to support the former 2 analytical methods. We performed 100,000 MCMC (Marcov Chain Monte Carlo) simulations followed by 50,000 steps of burn-in for each K value (K = 1–17), and these calculations were repeated independently 10 times. Subsequently, the most likely clustering number of K was determined by the peak of ΔK calculated by the method of Evanno et al. [However, HWE in populations and LE between SSR loci are often violated in natural populations and are viewed as unrealistic . In sucho et al. . The webo et al.  was usedo et al. , and cluo et al. .O and HE ranged from 0.152 (Sarms 44) to 0.337 (Sarms 37) and from 0.501 (Sarms 38) to 0.881 (Sarms 33), respectively.One hundred and eight alleles from the 9 SSR loci of all 93 mites were detected Table ; they coSarcoptes mite populations associated with KN, GN, GCf, WN, HS, OCc and SmS , Sarms 34 and Sarms 40 , Sarms 34 and Sarms 45 , Sarms 40 and Sarms 41  and Sarms 40 and Sarms 45 , when tested in the whole population.A significant deviation from HWE was observed for some loci in The results of the PCA are shown in Fig. SA showed the 5 genetic clusters (Clusters A–E) in 93 mites  and pine marten (M. martes) were defined as “carnivores.” These animals are widely recognized as omnivores based on their food habits [Sarcoptes mite populations observed in their study did not conform to the foraging ecology of mammals, but to the taxonomy at the suborder level: (i) Caniformia in Carnivora as a substitute for “carnivore”; (ii) Suina in Cetartiodactyla as a substitute for “omnivore”; and (iii) Ruminantia in Cetartiodactyla as a substitute for “herbivore”.As previously described, Rasero et al.  compreheo et al.  lack acco et al.  did not d habits , 47. TheSarcoptes mite populations [Sarcoptes mites do not have free-living stages and individual hosts function as their ephemeral habitats in patchy environments [Sarcoptes mite populations in the European mammalian fauna and are consistent with the “Host–taxon phenomenon” formulated by Rasero et al. [As far as the present study is concerned, the taxonomy of our target species was as follows: domestic dog, raccoon dog, raccoon and Japanese marten belong to the suborder Caniformia in Carnivora; wild boar belongs to the suborder Suina in Cetartiodactyla; and Japanese serow belongs to the suborder Ruminantia in Cetartiodactyla. Deficiency of observed heterozygosity and deviation from HWE in some of the SSR loci in a few mite populations were also confirmed in the previous reports of other ulations , 18, 20.ronments . Bearingo et al. . MeanwhiSarcoptes mites from the herbivorous preys to their carnivorous predators. However, this one-directional concept is not supported in case of “very weak” prey-predator relationships, the epidemiological history and the ecology of Japanese serow. Rather, infection from Caniformia mammals to serows is highly suspected. Upon reviewing the recent epidemiological history of sarcoptic mange in Japan, it was seen that its outbreaks in Japanese serows started roughly ten years later than the epidemic in raccoon dogs [In the previous studies, the prey-predator pattern was described as “Curse of prey”  and tendoon dogs . From a oon dogs , 22. In oon dogs . During oon dogs , 25. It S. scabiei. Based on the data obtained from a questionnaire survey conducted by the Wildlife Management Office [From the view point of Japanese serow ecology, which is a territorial and monogamous species, usually living solitarily , the trat Office , most ofSarcoptes mites arises through both direct or indirect contact [Sarcoptes mites off the host is only a few days in the natural environment [Sarcoptes mite’s ecology and contact between host species, besides the transmission experiments, may furnish a clear answer for the observed gene flow between herbivore ruminants and Caniformia species.Given that the inter-species transmission of  contact , there a contact  and deer contact . There i contact . Althougironment , infectiironment , 53. FurSarcoptes mites from Caniformia species and Japanese serows do not belong to dominant genotypes in the Caniformia-serow derived communities in their locations  natural events of host migration and dispersion, or (ii) artificial translocation of Sarcoptes mites. Note that GCf4 and GCf7 from the domestic/companion dogs in Gifu belonged to the mite group from raccoon dogs and serows in Kyushu Island (OCc1-5 from Oita and SgN from Saga). Kyushu and Gifu are geographically distinct (> 550 km) and separated by sea. Thus, it is unlikely that this genetic relationship was formed by a natural event. Rather, this link could probably be owed to the artificial translocation of mangy animals, such as domestic/companion dogs. However, there are, of course, possible alternative hypotheses for the observed close genetic relationships between distant Sarcoptes mite populations, such as (i) an effect of a small sample size in mite populations between the two distant mite populations (i.e. limited observation and un-detection of similar genotypes), and (ii) a chance effect of mutation and convergence of genetic characteristics. Future research is needed for understanding of this type of genetic relationships and also the possible artificial impact for the expanding distribution of sarcoptic mange.Lastly, our data reveal that some Sarcoptes mite populations follow a “Host–taxon” regulation between wild boars and Caniformia with Japanese serow communities but deviate between the Caniformia and Japanese serow. To our knowledge, this result provides the first genetic evidence for a possible hidden transmission of sarcoptic mange between host species belonging to different taxa without unambiguous predator–prey systems. In the diverted transmission patterns of S. scabiei among multi-host systems worldwide, there may be cryptic relationships of S. scabiei populations in local mammalian communities. Although the effect of sarcoptic mange in wildlife species is still poorly understood [Capricornis species, sarcoptic mange infection from the Caniformia species, in addition to the possibility of artificial translocation of mangy animals (i.e. domestic/companion dogs), should be deliberated in wildlife management programs.The results of our study show that in Japan, derstood , the undderstood . For theAdditional file 1: Table S1. Results of Hardy–Weinberg equilibrium tests for each microsatellite locus for each Host-associated Sarcoptes mite population.Additional file 2: Figure S1. Population structure as inferred by STRUCTURE analysis of Sarcoptes mites in the present study. a The graph for K = 9 with the 93 mites. b The graph for K = 4 with 80 mites . Each mite genotype is represented by a single vertical bar plot. Each color represents one cluster, and the proportion of each color in each bar plot shows the likelihood of assignment in the inferred clusters. Mite populations associated with each host animals are separated by thick black lines. 1, TCf; 2, TN; 3, KN1-8; 4, GCf1-9; 5, GN1-23; 6, WCf; 7, WM; 8, WN1-26; 9, WP; 10, SgN1-2; 11, StCc; 12, WCc; 13, OCc1-5; 14, WS; 15, HS1-5; 16, SmS1-6; 17, YS. For host population abbreviations see Table


MPAs) designated for marine megafauna has been criticized due to the high mobility and dispersal potential of these taxa. However, dispersal within a network of small MPAs can be beneficial as connectivity can result in increased effective population size, maintain genetic diversity, and increase robustness to ecological and environmental changes making populations less susceptible to stochastic genetic and demographic effects . Here, we use both genetic and photo‐identification methods to quantify gene flow and demographic dispersal between MPAs of a highly mobile marine mammal, the bottlenose dolphin Tursiops truncatus. We identify three populations in the waters of western Ireland, two of which have largely nonoverlapping core coastal home ranges and are each strongly spatially associated with specific MPAs. We find high site fidelity of individuals within each of these two coastal populations to their respective MPA. We also find low levels of demographic dispersal between the populations, but it remains unclear whether any new gametes are exchanged between populations through these migrants . The population sampled in the Shannon Estuary has a low estimated effective population size and appears to be genetically isolated. The second coastal population, sampled outside of the Shannon, may be demographically and genetically connected to other coastal subpopulations around the coastal waters of the UK. We therefore recommend that the methods applied here should be used on a broader geographically sampled dataset to better assess this connectivity.The functioning of marine protected areas ( DemograLowe and Allendorf  distinguTursiops truncatus) exhibits hierarchical population structure, with the greatest divergence found between pelagic and coastal populations  as part of an overall European strategy (Natura 2000) to maintain or restore the species at “favourable conservation status.” Therefore, SACs (or Natura 2000 sites) have been designated in the coastal waters of several areas in EU Member States. Around the British Isles, such SACs are located in Moray Firth (Scotland), Cardigan Bay , and two areas on the west coast of Ireland, the Shannon Estuary and in western parts of Counties Galway and Mayo (West Connacht Coast) , in 2001–2005, 2007–2010, and 2013–2014 Figures  and here2.2The dataset comprising of altogether 97 unique samples included 85 samples already genotyped by Mirimin et al. . This se2.3n = 12).DNA was extracted from 12 new skin samples using the DNeasy Blood and Tissue kit from Qiagen. A total of 15 nuclear microsatellite loci , with K set to vary from 1 to 6, using 1,000,000 Markov Chain Monte Carlo (MCMC) iterations preceded by 1,000,000 burn‐in steps. Convergence of chains  was confirmed visually and the consistency of runs was checked by confirming that the variance in estimated ln Pr(X|K) was smaller within each K compared to the variance between the different Ks, and calculating the average posterior probability for each K. ∆K, which has been argued to be a better predictor of the number of populations, was also calculated following Evanno, Regnaut, and Goudet  that clusters individuals together based on genetic similarity to find the most likely number of populations. DAPC does not rely on any population genetic model  and is efficient at detecting hierarchical structure  values against different values of K and by examining individual assignment probability plots. Consistency of the runs was checked by examining the convergence of MCMC chains in TRACER 1.6.  were highly consistent in their inference of the most likely number of clusters and the individual assignment probabilities so the dataset was divided into three putative populations, l et al. , this poFST  estimated using the R package diveRsity and repeated 10 times. These pairwise values were compared to FST values calculated for two sets of ten individuals randomly drawn from within a single coastal population, Coastal Shannon or Coastal mobile. To supplement this analysis, the power to detect a significant moderate population differentiation, based on an FST value of ≥0.1 in a sample consisting of the allele frequencies from both coastal populations and using a sample size of ten individuals per “subpopulation” , was calculated by running 1,000 simulations in POWSIM 4.1  within the last two generations were estimated using BAYESASS  for the Coastal Shannon population was derived using LDNe, a method that uses linkage disequilibrium . Long‐term photo‐identification data are not available for the “pelagic” dolphins in this area. Social structure analyses were performed in SOCPROG 2.4 compiled version  was measured using two indices of the frequency of co‐occurrence: the half‐weight association index (HWI) and the simple ratio  was used to test if temporary or long‐lasting social bonds existed between individuals, and compared to the null association rate . The SLAR was fitted separately to the individuals encountered within and outside of the Shannon Estuary as the data showed that these groups did not associate with each other. Mathematical models representing simulated social structures, that is whether individuals had constant companionships or casual associates during the study  or across loci , suggesting that homozygosity was not due to allelic dropout. Therefore, the observed deviations from HWE across all populations and loci are most likely attributed to the structuring of the populations, i.e., Wahlund effect  within the Coastal mobile population and one locus (Dde61) within the Pelagic population were out of HWE  and using the ad hoc method by Evanno et al.  assignment probabilities instead of clustering together with the Coastal Shannon as was the case when all loci were included , o et al.  was threo et al. a. The mao et al. a. RemoviCoastal mobile cluster with 79% probability by STRUCTURE  were considered; the most likely number of coastal populations identified was two .These results were consistent with clustering probabilities calculated in TESS when only the biopsy samples of coastal dolphins  of dyads that were close relatives, with the Queller and Goodnight  among individuals assigned to the Coastal Shannon population, −0.04 (SD = 0.25) among the Coastal mobile, to −0.06 (SD = 0.13) among the Pelagic dolphins. The mean relatedness values within the Coastal Shannon  and the Coastal mobile (300 dyads) were also significantly higher compared to the relatedness of dyads when individuals were selected randomly, one from each of the two coastal populations . Allele richness (AR) and observed heterozygosity (HO) were lower in the two coastal populations compared to the pelagic population .No evidence of significant heterozygote deficiency was found across all loci in any of the populations (FST and Jost's D) between the pelagic and the two coastal populations and between the two coastal populations (defined with STRUCTURE), and this difference persisted after removing close relatives from the dataset  or Coastal mobile  individuals  indicating a true population differentiation that was not driven by the sampling of closely related individuals or uneven sampling. The simulations run in POWSIM 4.1  were second‐generation migrants from the Coastal Shannon population with 64% and 79% probability, respectively. One individual assigned to the Pelagic population by STRUCTURE (“bnd204”) had a 37% probability of being a first‐generation migrant and a 46% probability of being a second‐generation migrant from the Coastal mobile population. When the individual that was biopsied in the Shannon Estuary but genetically assigned to Coastal mobile population (“tt‐05‐03”) was grouped together with other Shannon individuals, it had a 19% probability of being a first‐generation migrant and a 70% probability of being a second‐generation migrant from the Coastal mobile population.When looking at individual posterior probabilities of migrant ancestry, two individuals from the 3.4p < 0.01, SD: p < 0.0001, and CV: p < 0.0001) indicating significant preferred short‐ (within sampling period) and long‐term (between sampling periods) companions. Moreover, the proportion of nonzero elements was larger in the random data compared to real data which suggests that some individuals may avoid others  model “casual acquaintances”  and within the Coastal mobile population it was 0.23 (SD = 0.21). The difference in the association indices between the two populations and especially the higher variation associated with the Coastal mobile may be linked to the lower number of encounters included in the social analysis .When testing for preferred and avoided companionships between and within the two coastal populations, the mean HWI in the real data was found to be significantly higher compared to the HWI of a permuted random dataset (mean: SE = 0.0128). Likewise, dolphins belonging to the Coastal mobile population were never photographed in the Shannon Estuary during the study period so their LIR in the Shannon Estuary was also zero. The LIR within the Shannon stayed fairly constant for approximately 100 days, followed by some fluctuations in the rate , hence their annual average lagged identification rate (LIR) was zero to any other study area, except to Brandon Bay where it was 0.0263  when the data from the two coastal populations were combined. However, this is likely attributed to the correlation of zero values in the combined dataset as no correlation was found between the two indices when testing for this separately for each population . Of fifteen dyads with significant associations , none had relatedness coefficient ≥0.45, but three dyads had coefficient values close to 0.25 indicating possible half‐siblings or cousins. No correlation was found between relatedness and spatial overlap within the Coastal Shannon  or the Coastal mobile population . Overall, these results indicate that close kinship may not strongly promote overall social associations in these two populations.When only the biopsied individuals with a sufficient number of photo‐identification encounters (≥3) were considered, a significant correlation was found between the relatedness coefficient  toward the end of the study period reflects a decrease in site fidelity that is likely explained by mortality and/or emigration. These results highlight that a high degree of site fidelity, especially evident in the Shannon Estuary SAC where data have been collected for over 12 years, is a key driver of fine‐scale population structure among coastal populations. A high degree of site fidelity among resident populations of bottlenose dolphins to certain local areas has been found in other parts of the world  in southern Australia, with greater relatedness found between males within schools than between schools  and partly because of the fact that the biopsy sampled animals did not necessarily have enough photo‐identification encounters for further social analyses.In this study, there is evidence of significant companionships within the two coastal populations, and it is possible that social bonds promote and maintain the observed social and genetic separation of these populations. The observed companionships did not seem to be linked to relatedness, but close associates were found both among kin and nonkin individuals, similar to a recent study by Louis et al. . In contthat is, <10% of the population growth is contributed by migrants from other populations regardless of whether they contribute to the gene flow or not. Recent migration rates between the different Irish bottlenose dolphin populations were nonsignificant  in all comparisons inferred using BAYESASS. However, one individual (“tt05‐03”) encountered over 9 years in the Shannon Estuary, was genetically assigned to the Coastal mobile population. It is interesting that this dolphin has never been photographed associating with the Coastal mobile population, but no close kin were found among the sampled individuals assigned to the Coastal Shannon population. Given that ~40% of the Coastal Shannon population have been biopsied (and genotyped) based on abundance estimates derived for this population varying between 114 and 140  represents <3% demographic dispersal between the coastal Irish populations, it seems unlikely that the contribution to the demographic processes are significant. However, this largely depends on the management targets set to the population in question and the power to detect changes in abundance, survival, or other demographic processes.Lowe and Allendorf  describe Berrow, , 2003, iCoastal Shannon and Coastal mobile populations , with more than double the amount of samples compared to females; thus these results should be treated with caution. Both Mirimin et al.  and LouiNykänen, , suggestCoastal mobile population were identified as likely second‐generation migrants originating from the Coastal Shannon population. However, whilst individual assignment methods, such as STRUCTURE, are believed to perform well at identifying migrant individuals (Putman & Carbone, Coastal mobile population. To date, only ~12% of this population occurring in Irish waters has been sampled, based on a median multisite abundance estimate of 189 dolphins derived for a wide area extending to the west and north‐west coast of Ireland (Nykänen, Two individuals strongly assigned to the Nykänen, . OverallCoastal North” metapopulation, Louis et al., Coastal Shannon population, which is, based on this study, both genetically and demographically isolated. Coupled with the relatively small effective population size, this makes Coastal Shannon especially vulnerable to any environmental or anthropogenic stressors. The Coastal mobile population occurring in Irish waters, on the other hand, may belong to this “Coastal North” metapopulation, and previous research has shown that at least some of these mobile coastal animals travel over distances at the scale of hundreds of kilometers (Cheney et al., Coastal North” metapopulation extending beyond Irish waters, transnational cooperation, monitoring and management may be needed. Six individuals from the west coast of Ireland have been matched on an ad hoc basis to photo‐identification catalogues comprised of animals ranging in the coastal waters of Scotland (Robinson et al., Nichols et al.  and LouiThe present study supports the delineation of the three populations occurring in Irish waters as separate management units based on the low genetic, social, and demographic dispersal between the populations, thus validating the current designation of separate SACs for the two coastal populations. The study also highlights the importance of distinguishing genetic and demographic connectivity so that gene flow can be differentiated from immigration that has no subsequent genetic contribution from the migrant to the local population. Even though the genetic connectivity between the different populations of bottlenose dolphins in this study was negligible and accompanied by moderate‐to‐strong genetic differentiation, quantification of migration rates and the degree of social connectivity have implications in the delineation of MUs, especially in cases where population structuring is less clear. With this information, the functioning of existing marine protected areas or networks can be better assessed and the need for designating new protected areas is evaluated.None declared.M.N., A.D.F., S.N.I., and E.R. conceptualized the work and the analyses. E.D. and M.N. performed laboratory work. M.N., L.M., and M.L analyzed the genetic data. M.N., M.O., A.E., and S.N.I. analyzed the photo‐identification data. M.N., S.N.I., E.R., A.D.F., M.O., and A.E. collected the genetic samples and photo‐identification data. M.N. wrote the manuscript. All authors approved the final manuscript.Analysis input files used for TESS, DAPC, STRUCTURE, diveRsity, and SOCPROG are deposited in Dryad (Supporting information Data  Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.