Scolitantides orion), whose caterpillars feed only on a few species of sedum and are guarded by ants. In Poland, this butterfly has survived only in one region and it is critically endangered. It is important to examine the genetic condition and demography of its extant populations, to help to create an effective conservation plan for the species. We studied the demography and genetic structure of the two largest populations inhabiting opposite banks of the Vistula River. Captured individuals were marked and released to estimate population sizes. Both populations were small, and they fluctuated in numbers, but adults were twice as numerous on the western riverbank. Analyses of various genetic markers have shown that the genetic variation is low in each population. Likewise, the genetic diversity between the two populations is low, which indicates that the gene flow exists, despite the river acting as a geographical barrier. The occasional migration occurs more frequently from the west to the east, which is consistent with the dominant regional wind direction.The disappearance of many butterfly species is currently observed in Europe, as most of them display strict habitat preferences and/or food specializations. A good example of such a species is the chequered blue butterfly , which is an example of the dramatic decline in the population number. We studied the two largest remaining populations inhabiting opposite banks of the river. Mark-release-recapture studies showed that both populations were small, and they fluctuated in numbers, but adult individuals were twice as numerous on the western site. Genetic analyses were carried out using a mitochondrial  and nuclear markers . We found out that genetic variation was low at both sites but higher in the smaller eastern population. This pattern is likely to be better explained by past distribution, when the butterfly, as a continental species used to be much more widespread in the east. However, the genetic differentiation between populations was low. This could suggest that the existing gene flow is facilitated by dominant regional wind direction, which may also contribute to a better genetic condition of the western population. Finally, a comparison of the obtained COI sequences with others available enabled us to reveal the phylogeographic pattern of the S. orion from different localities within its range. Butterflies are considered to be sensitive to environmental changes . The maiScolitantides orion  distributed irregularly across Palaearctic from Spain to Japan [S. orion to a viable level [It is suggested that as many as a quarter of European butterflies are threatened . Moreoveto Japan . Its Eurto Japan . The speto Japan , but in to Japan , and itsle level .S. orion from different localities within its range.Here, we investigated for the first time a genetic structure and demography of the chequered blue butterfly. Our studies were conducted in a highly isolated part of its distribution range in Poland. The two chosen local populations were the largest of all known in the country and coincidentally the only populations that were large enough to carry out thorough demographic and genetic research. Mark-release-recapture sampling (MRR) enabled us to estimate seasonal population size and the mean lifespan of adults. Then, using both mitochondrial and nuclear markers, we assessed genetic structure of the populations and examined the differentiation between them to detect possible gene flow. In this text, we also discuss implications of our findings for the regional viability assessment of the butterfly. In addition, based on a comparison of obtained sequences of the cytochrome oxidase subunit (COI) and others available in the GenBank database, we reveal the phylogeographic pattern of the Scolitantides orion  is one of the smallest European butterflies, with the wingspan of about 25–28 mm. Sexual dimorphism is moderately conspicuous. The upper side of females is very dark greyish-brown, with blue flush restricted to basal part or absent. In males, the blue markings often cover most of the wings, but their intensity can vary considerably. Six subspecies are recognized throughout its range, of which three occur in Europe, i.e., S. o. orion Pallas, 1771  , and S. o. ultraornata Verity, 1937 (southern Fennoscandia) [The chequered blue butterfly Siberia) , S. o. pscandia) .Sedum species . Local populations are usually small, but may form a metapopulation system [The butterfly inhabits warm, often rocky slopes scarcely covered by vegetation, with significant presence of larval food plants i.e., some n system . There an system . Eggs arn system .S. orion was always a very local species, recorded in four areas. It became extinct in Lower Silesia, Pieniny Mountains and Bieszczady Mts., and has probably survived only in the valley of the Vistula River on a few sites in the area of Kazimierz Dolny [In Poland rz Dolny . In the rz Dolny , but it rz Dolny .During intensive exploration carried out between 2014 and 2016 in the area of Kazimierz Dolny only three local populations were found there. In the case of one of them, only few individuals were observed. Hence, this study was restricted to two sites about 9 km apart and separated by unfavourable biotopes, including: farmlands, village buildings, and the Vistula River about 300 m wide. They were Parchatka (P1) and Janowiec (P2).Solidago canadensis). A smaller patch of the habitat  was located at a distance of about 200 m. It covered the southern slope of the Vistula River valley. However, due to very low numbers of observed individuals, it was excluded from the analyses. In the first half of the 20th century, xerothermic grasslands and scrubs were much more prevalent in the area and there were likely a lot of interconnected patches of suitable habitats.The site of Parchatka (P1) is located on the eastern bank of the Vistula River in vicinity of Parchatka village and encompassed sunny edges of the ravine covered with patches of xerothermic vegetation, with a total area of about 0.13 ha . The ravPinus sylvestris (The site of Janowiec (P2) is situated at the edge of the Vistula River valley, on the west bank of the river, and encompassed about 2.5 ha of open sandy grasslands surrounded by the Rhamno-Prunetea scrub and low trees, mainly Scots pines lvestris . The areBoth populations were sampled in 2015 and 2016 with the mark-release-recapture method (MRR). The phenology of the butterfly at both sites was similar and the main difference was the appearance of a few individuals of the second generation in the P2 population. The sampling covered the entire flight period of the focal species in the first brood. The P1 site was visited between 5 May and 11 June in 2015 and between 4 May and 1 June in 2016, 15 and 13 times respectively. In P2, the sampling was carried out between 30 April and 17 June in 2015 and between 29 April and 7 June in 2016. There were 15 visits in both seasons. One person spent about 2–3 h on the site during each sampling day and studies were performed on days with favourable weather, between 10 am and 5 pm. Butterflies were captured with an entomological net, marked on the underside of their hind-wings with unique identity codes using a fine-tipped waterproof pen, and then immediately released at the place of capture. Due to a small size and complicated wing pattern it was not possible to write a number as it is widely practiced in studies of other lycaenids, thus a combination of colour dots and dashes was applied . This mac) [ê = (1 − ϕ)−1 − 0.5 [The data collected were analysed with the Cormack–Jolly–Seber and constrained Jolly–Seber models (POPAN), using MARK 8.0 software ,20. The c) , an appr−1 − 0.5  and temp−1 − 0.5 .S. orion was sampled in 2018 for genetic studies. Tissue samples were taken using a nonlethal method from 34 adults of S. orion from P1 population and 31 butterflies from P2 population. Small fragments of hind wings (~2 mm2) were torn off using tweezers for genetic testing, and then individually stored in 95% ethanol. All procedures were approved by the Regional Director for Environmental Protection in Lublin, Poland (permit no.: WPN.6401.57.2018.MPR). DNA was extracted from small wing clippings using the Genomic Mini Kit (A&A Biotechnology), following the manufacturer’s protocol, and eluted in 100 µL of Tris buffer.A total number of 65 individuals of Fnull). Markers that showed high values of Fnull  and monomorphic loci (Macu9 and Macu15) were excluded from further analysis  and observed (HO) heterozygosities, and the mean polymorphic information content (PIC). Linkage disequilibrium between loci was tested with Genepop v4.0 [FIS in each population were calculated with Fstat 2.9.3 [S. orion were estimated for six microsatellite loci by calculating the FST values, which quantify the variance of allele frequencies, in Fstat 2.9.3, as well as RST values using Genepop v4.0. The significance of FST value was ascertained with 1000 permutations and interpreted using Wright’s scale [Ne) was estimated using the linkage disequilibrium method employed in LDNe [The remaining six loci were then analysed using the Cervus 3.0.3 program for determining the number of alleles per locus (A) and their size ranges, as well as to calculate the expected  and two nuclear genes (EF-1α and Wgl). Universal primers for barcoding gene COI, LepF1 and LepR1  were useFST and ΦST values. All values were statistically tested in Arlequin.Haplotype reconstruction of the nuclear genes (EF-1α and Wgl) from genotype data was conducted using the algorithms provided in PHASE as implemented in DnaSP v.5.10 . For allBEAST 1.75 [Cupido minimus, JQ996391) and the scarce large blue  downloaded from GenBank. A consensus tree accessing the posteriori probability values of each clade was generated using TreeAnnotator 1.6.1 [Due to small numbers of haplotypes identified in both Polish populations of the chequered blue butterfly and very few ND5, EF-1α and Wgl gene sequences available in the GenBank database, we conducted phylogenetic analysis only for the mtCOI gene. Phylogenetic analyses were performed on 93 sequences of the mtCOI gene fragment (579 bp), of which 64 were from Poland , and the other 29 sequences were from the GenBank database . PhylogeAST 1.75 . The subAST 1.75 . A totalAST 1.75 . Trees wor 1.6.1 . Haplotyor 1.6.1 .The number of marked individuals varied between 19 in 2015 (P1) and 74 in 2016 P2, . Most ofThe population size was significantly higher in 2016, both in P1 and P2 population. In three datasets, males were more numerous than females by about 30% and only for the P1 population predominance of females was detected in 2015. However, it is worth noting that the total number of marked individuals was in this case relatively low compared to the other datasets .The maximum duration between captures of an individual reached 17 and 11 days for males and females, respectively. The maximum number of recaptures on different days for an individual was 5 for males and 6 for females. The mean number of days between the first and last capture varied between 2.17 and 3.86 for males and 1.89 and 2.43 for females.φ), while for the two other φ was sex-dependent and hence it was possible to calculate survival rates for both sexes separately. However, capture probabilities (p) were sex-dependent only for P1 from 2015 and P2 from 2016.For two datasets (P1 from 2015 and P2 from 2016) the model assumed a constant  survival rate  and six microsatellite loci (A) was low and very similar in both examined populations, at 5.83 (P1) and 5.67 (P2), respectively. The parameters of genetic variation, i.e., expected heterozygosity (HE), observed heterozygosity (HO), and the mean polymorphic information content (PIC) estimated on the basis of microsatellite loci, were similarly low in both populations, although slightly higher in the P1 population . There was no significant linkage disequilibrium among any pair of the loci under study, indicating that the studied loci were most probably not linked. Deviations from Hardy–Weinberg equilibrium were significant after Bonferroni correction for two loci , but only in the P1 population  ranged from 4 (Macu44) to 12 alleles , on average Nite loci . The meapulation . The genpulation .Ne of 29.4, with 95% parametric confidence intervals (CI): 16.0–70.8, whereas for the P2 population, the Ne was 41.6 with a large 95% parametric CI: 19.5–209.8.GeneClass2 identified eight individuals (23.5%) from the P1 population as being possible first-generation migrants, and there was a significant probability that six individuals (19.4%) did not originate from the P2 population in which they were sampled. For the P1 population, LDNe revealed an We discovered only two distinct haplotypes for mtCOI gene, of which the one appearing in P1 population was new and not previously reported . The hapThe parameters of genetic variation in all sequenced genes in both populations, are summarized in S. orion populations in Poland were low but significantly different from zero . In agreement with these results, the individual microsatellite genotypes of S. orion from both Polish populations showed overlapping distributions in the PCoA plot and did not show any obvious subgroups ; and moderate for the EF-1α gene . Low values of FST = 0.013 (p > 0.05) and ΦST = 0.051 (p < 0.05) were also obtained for the concatenated nuclear gene sequences, EF-1α_Wgl. Low and not significantly different from zero values of FST and ΦST = 0.054 (p > 0.05) were observed also in mtCOI gene. Only in the mitochondrial ND5 gene genetic differentiation values were very great (FST and ΦST = 0.584) and significantly different from zero (p < 0.05), which translated into equally high values of these parameters obtained for the concatenated COI and ND5 gene sequences . However, it should be emphasized that the high genetic diversity values derived from the ND5 gene sequence were the result of just one variable site in this gene fragment that differentiated the two populations.According to Wright’s scale , pairwisubgroups . SimilarS. orion possessing the H1 haplotype  than to Parchatka (ca 12 km) [S. orion population in Poland still reflects historic distribution, when the butterfly used to be much more widespread, to a much greater extent than the present situation, which was strongly influenced by habitat fragmentation. This kind of relationship was also found in other endangered lycaenids, namely Phengaris arion [Pseudophilotes bavius [Melitaea cinxia [Another possible explanation of the low genetic differentiation between the studied populations is as follows. At the moment, except for the Parchatka site, only single individuals are observed in scattered localities in the east. In the 1990s, a 12 km) . Therefois arion  and Pseus bavius , as wella cinxia .Ne) is often important for determining the conservation status of threatened populations and species [Ne values for the chequered blue were rather low and similar in both populations. The values seem to be not much lower than the estimated census sizes, but the information provided by the two numbers does not correspond temporally: demographic data were collected in 2015 and 2016, and the samples for genetic analyses in 2018. However, it can be assumed that in 2018, as in the previous years, the P2 population was 2–3 times more numerous than the P1 population. Thus, Ne/N ratio in both populations should be similar, indicating genetic similarity of the populations and the presence of gene flow between them. Restored or created patches of biotopes need to exist in close proximity to enable gene flow between populations and the spontaneous colonization of new patches of habitat. Finally, even if we assume sufficient gene flow between the populations, we must remember that the total regional population size is very small and very sensitive to genetic deterioration and even extinction, especially taking into consideration seasonal fluctuations in numbers of the Polish populations detected in our studies. Hence, actions dedicated to the protection, improvement, and enlargement of existing habitats are vital to the survival of S. orion in the region. Actions undertaken in Finland are a good example [This study could contribute to the preparation of an appropriate and effective plan for the species protection in the future. Estimation of an effective population size . This could be an example of clinal variation as in the case of other lycenids [S. o. parvula seems to be a clearly distinct lineage and its status is certain.Our results have some implications for the species taxonomy, namely subspecific division. The dominant COI haplotype in Polish populations is the same as that identified in Finland, and it differs only slightly from those found in Norway, Germany, Switzerland, and Romania. Therefore, it may be concluded that there are no phylogenetic grounds for the separation of Nordic subspecies lycenids ,72. HoweThe steppe species do not show a uniform phylogeographic pattern in eastern Central Europe, but they share a unique genetic signature and some distinct units were formed in now isolated steppic habitats . We founMelitaea cinxia) populations, which formed some not totally genetically isolated units when compared in north-south and east-south directions. Polish M. cinxia population had mixed ancestry as mtCOI haplotypes from Central and Eastern  lineages were detected [S. orion. In Europe, intensive sampling in the southern part of the range is especially important, since this area may be a refugium for genetic diversity of some species, including butterflies [S. orion, are expected to be generally less diverse [The chequered blue butterfly displays similar but less complex phylogeographic diversity to the Glanville fritillary (detected . Other sdetected ,75,76, bdetected  and mammdetected  show a sterflies . However diverse .S. orion suggests that most of the individuals from the Polish populations are similar to Finish ones, while there are no phylogenetic grounds for the separation of the Nordic subspecies S. o. subornata from S. o. orion, and the Iberian subspecies S. o. parvula is certain. Molecular data suggest an interesting phylogeographic pattern in S. orion. However, to gain a complete insight into this pattern, it is important to fill in the gaps in the geographic range of the species with genetic data. Our results can help to create an effective conservation plan for the species in the future.Mark-release-recapture studies revealed that both studied populations were small, and they fluctuated in numbers, but on the western site, adults were twice as numerous. We found out that genetic variation was largely shared between both sites, but higher for the smaller eastern population. This pattern could be better explained by past distribution, when the butterfly used to be much more widespread on the eastern bank of the river. However, the genetic differentiation between two localities was low. This could suggest an existing gene flow, despite the Vistula River acting as a geographic barrier. We hypothesize that the occasional migration of individuals occurs more frequently from the west to the east, which is consistent with the dominant regional wind direction. It may also contribute to a better genetic condition of the western population. The analysis of available COI sequences of


Amorphophallus albus P. Y. Liu & J. F. Chen (Araceae) is a plant species with extremely small populations (PSESP) and an important economic crop endemic to dry-hot valleys along the Jinsha River. In order to gain information for sustaining the development and conservation of A. albus, we studied the genetic diversity and population structure of this species using microsatellite markers (SSR). In this study, we analysed 364 individuals belonging to 24 populations, including four wild populations and three ex-situ cultivated populations, collected in the provinces Yunnan, Sichuan and Hubei.A. albus possesses moderate genetic diversity with the percentage of polymorphic loci (PPL) from 69.23 to 100%, an expected heterozygosity (He) of 0.504 and an average Shannon’s Information Index (I) 0.912. Analysis of molecular variance (AMOVA) indicated that most of the variance (71%) resided within populations and the estimated gene flow (Nm) was 0.61. The results of UPGMA cluster tree, STRUCTURE analyses together with the Mantel test  indicated that geographically closely located populations are clustered together with some exceptions.The population genetic analyses indicated that A. albus still possesses moderate genetic variation in most of the studied populations, and for now, most cultivated populations were naturally distributed but still some reintroduction exists. For sustaining the present genetic variation, some protections measures are necessary for the wild populations and also for the cultivated ones with high genetic diversity.Our results showed that  Amorphophallus albus P. Y. Liu & J. F. Chen (Araceae) is a herbaceous perennial plant species occurring along the Jinsha River in southern Sichuan and northern Yunnan. It is growing in open forests between 800 to 1000 m altitude on arid locations [A. albus makes this species the second most cultivated Amophophallus species after A. konjac K. Koch in China [A. albus is one of the pillars in agriculture of counties along the Jinsha River. For example, in Jinyang, the cultivation area is more than 3333 ha with commodity production more than 30,000 kg and a production value of about 120 million Yuan every year [A. albus are important factors for the breeding of drought-resistant varieties [A. albus was listed as a potential targeted PSESP  for the China National Key Program of Survey and Germplasm Conservation of Plant Species with Extremely Small Populations in Southwest China [ocations , 3. The in China . At presery year . Moreovearieties . Since ist China .Abies ziyuanensis L.K. Fu & S.L. Mo (He = 0.337) [Elaeagnus mollis Diels  [He) of endemic plant species analyzed by microsatellite is 0.42, whilst for species with narrow distribution is 0.56 and 0.62 for widespread species, respectively. At present, studies focusing on genetic diversity of Araceae species were valued mostly by the first generation of molecular markers including RFLP [Amorphophallus paeoniifolius (Dennst.) Nicolson and Xanthosoma sagittifolium (L.) Schott were analysed by microsatellites [Genetic diversity is the variation of the genetic material of organisms and the basis for adaptation of species to the natural environment . Charact= 0.337) , Elaeagn 0.3815) . Accordi 0.3815) , the aveing RFLP , RAPD [1ing RFLP , 20, AFLing RFLP –23, onlytellites , 25, andtellites . Among ttellites .A. albus from 24 populations including four wild populations and 17 in-situ cultivated populations as well as three ex-situ populations for following purposes: 1) to explore the trends of natural formation and evolution; 2) to provide a theoretical basis for conservation; 3) to reveal the net of introduction into the present cultivation area and 4) to determine the origin of this species.In the present study, we used 13 pairs of microsatellite loci to analyse the genetic diversity and population structure of A. albus (Table PIC) range from 0.439 to 0.869 with an average of 0.683, which indicate a high polymorphism and their suitability for genetic analysis, the null allele frequency range from 0.013 to 0.523 with an average of 0.195, meanwhile, only three loci  were detected with null alleles through Micro-checker software [Na) was 3.619, with a range from 1.846 (SDC/TWC) to 4.615 (HLX). The average effective allele number (Ne) was 2.372, with a range from 1.541 (SLC) to 3.404 (LIZ). The average Shannon’s Information Index (I), observed heterozygosity (Ho), expected heterozygosity (He) are 0.912, 0.528, 0.504 on average, respectively. The percentage of polymorphic loci (PPL) of each population ranged from 69.23 to 100%. Based on the results, population LIZ  and HLX  showed the highest genetic diversity, while population SLC  and SDC  showed the lowest genetic diversity.In this study, we finally collected 364 individuals from 24 populations including four wild populations and 17 in-situ populations together with three ex-situ populations, each populations’ information are listed in Table software . In totaFst) and estimated gene flow (Nm) was 0.29 and 0.61, respectively. The results of F-statistics in each locus are shown in Table Fis) of most loci were less than zero with an average of − 0.04. The estimated population differentiation coefficient (Fst) of each locus ranged from 0.221 to 0.419, with an average of 0.321, the average gene flow (Nm) of all the loci was 0.560, almost identical to the results calculated by AMOVA.According to the results of AMOVA analysis, about 29.23% of the total genetic variation occurred among populations, whereas the remaining 70.77% of the variation occurred within populations Table . The estNei’s genetic distance  and genetic distances  of population pairs were listed in Table S1. Among all the populations, the farthest genetic distance and lowest genetic identity existed in SJX and SDC, while MYZ and HB had the nearest genetic distance and highest genetic identity. The dendrogram based on nce Fig.  by BayesA. albus from 24 populations in Sichuan, Yunnan, Hubei Province and they all expressed high polymorphism with an average PPL of 95.19%. According to the results, we observed a moderate genetic diversity of this species . In comparison, the genetic diversity observed was lower than in other studied Amorphophallus species using microsatellite markers, e.g., in A. paeoniifolius  [He = 0.42) summarized by Nybom [A. albus, which is a perennial herb with limited distribution showing self-incompatible mating system and endozoochory, it is supposed to have relatively higher genetic diversity. However, as an important economic crop, A. albus was inevitably disturbed by human activities such as habitat destruction and over excavation in recent years similar to A. konjac [A. albus can hardly be found in nature. Moreover, most farmers, who cultivated this species for commercial purposes, tend to use asexual reproduction to get more corms and shorter life cycles [A. albus, and the gene flow from Jingyang to Pingshan showed a trend of expanding towards east along the river. This pattern could also be observed from other species native to the dry and hot valleys along the Jinsha River [In this study, 13 microsatellite loci were analyzed to reveal the genetic diversity and population structure of = 1.172) , but higby Nybom , 34. Genby Nybom . For A. . konjac . Conseque cycles . This fiha River , 38.A. albus indicated a high level of differentiation (Fst = 0.29) and low gene flow (Nm = 0.61) among populations. According to Wright [Fst > 0.25/Nm < 1. High genetic differentiation may result from heterogeneous environments [A. albus is pollinated by rove beetles . This small insect pollination mating system and the complex geography may have limited gene flow among populations and therefore promoted genetic differentiation of this species [A. albus possess traits for seed dispersal by birds, but this could not be observed.The genetic analysis of o Wright , populatronments . Though ronments . Additioronments , 43, and species . MoreoveFis) in most loci were less than zero . PCR amplification was performed under the following conditions: 95 °C for 3 min, 32 cycles of 95 °C for 30 s, annealing at 56–60 °C for 30 s, and elongation at 72 °C for 30 s, and a final extension step at 72 °C for 5 min. The PCR products were separated and visualized using the QIAxcel capillary gel electrophoresis system .The genomic DNA was extracted from approximately 5 g of dried leaves of each collected sample using the modified CTAB method . DNA conN), average number of alleles (Na), effective number of alleles (Ne), Shannon’s information index (I), expected heterozygosity (He) and observed heterozygosity (Ho), fixation index (F) and percentage of polymorphic loci (PPL) were detected using GeneAlex version 6.0. F-statistics  were estimated for each locus across all populations using Fstat version 2.9.3.2. Genetic distances and genetic identity between each pair of accessions were measured from shared allele frequencies using PopGene 32. A dendrogram was constructed based on Nei’s genetic distance matrix using the MEGA version 4 software using the unweighted pair group method and the arithmetic averages (UPGMA) algorithm [Fst / (1-Fst) with 1000 random permutations was conducted using NTSYSpc software version 2.10e [Data from QIAxcel capillary gel electrophoresis were analyzed by GeneMarker V. 2.2.0 to get allele fragment data. Micro-checker software was used to detect whether null alleles were present  and CERVlgorithm . An anallgorithm . A Mantelgorithm  to compaon 2.10e . The geoon 2.10e . Admixtuon 2.10e .Additional file 1: Table S1. Paired Nei’s genetic distance  and genetic identity  of 24 populations of Amorphophallus albus


Psylliodes attenuata (Coleoptera: Chrysomelidae: Psylliodes) is a common pest of Cannabis sativa, including cultivars of both industrial hemp and medicinal marijuana. Both the larval and adult stages of this beetle can cause significant damages to C. sativa, resulting in substantial crop losses. At present, little is known about the populations of this pest, including its genetic diversity. In this study, we obtained 281 P. attenuata samples from nine field sites representing broad industrial hemp productions in China and analyzed their DNA sequences at the mitochondrial COI gene, the insect DNA barcode. Our analyses revealed a total of 48 haplotypes, with 28 being found only in one specimen each while the remaining 20 were shared by two or more specimens each. Of the 20 shared haplotypes, eight were shared among local populations often from far away locations, consistent with recent long-distance dispersals. However, the observed putative long-distance dispersals have not obscured the significant genetic differentiations among the regional populations from northeastern, eastern, central and southwestern China. Interestingly, haplotype network analyses suggest evidence for potential mitochondrial recombination in natural populations of this species. We briefly discuss the implications of our results on its evolution, center of diversity, route of spread, and pest management strategies in hemp fields.The hemp flea beetle  Cannabis sativa L.) is an ancient crop of Asian origin that has been traditionally cultivated in many regions of Europe and Asia, mainly as a source of textile fibers [C. sativa plants have also been developed into many other uses, including paper, biocomposites, functional foods, construction materials, cosmetics, biofuel and personal care [C. sativa is of significant medical and recreational interests due to its productions of the psychoactive compound D-9-tetrahydrocannabinol (THC) and/or other cannabinoids such as cannabidiol (CBD). CBD and THC have shown pharmacological properties in the treatment of mood disorders, pain, cancer, diabetes, and inflammatory and neurodegenerative diseases [C. sativa that can be grown. For example, according to EU legislation (No 809/2014 of 17 July 2014), production of hemp is permitted if the content of THC is less than 0.2% [C. sativa is only allowed for the purpose of producing hemp fiber in jurisdictions with valid license and appropriate security measures.Industrial hemp  1803 (Coleoptera: Chrysomelidae: Psylliodes), commonly known as hemp flea beetle or hop flea beetle, is geographically broadly distributed. It has been found throughout Eurasia, from the United Kingdom and France to the Middle East, eastern Siberia, Japan and China [P. attenuata) has been found on several plants such as hops and nettles, especially in the spring [Cannabis growth facilities [Like all agricultural crops, hemp is susceptible to infections and damages by a variety of pathogens and pests. In p losses . Psyllioe spring ,10, it mcilities ,12,13. Icilities .Sirex noctilio populations in China [Spissistilus festinus in the United States [Despite its broad distribution and significant economic impact on the hemp industry, we know very little about the genetic diversity and phylogeography of hemp flea beetle. Molecular markers, such as those based on nuclear and organelle DNA sequences, have become increasingly popular for investigating the diversity and genetic relationships among populations of insects . Specifiin China  and to id States .P. attenuata populations from six provinces in China and obtained their DNA sequences at the mitochondrial COI gene. The sequence data were used to infer the diversity and relationships among geographic populations of P. attenuata in China. The relevance of our results to the control of this pest in hemp fields will be discussed.In this study, we collected samples of P. attenuata. The remaining five, including two each in Hunan and Yunnan provinces and one in Heilongjiang province, did not have P. attenuata at the time of our sampling. The sampling site information, including hemp cultivar and the number of hemp flea beetles used for subsequent analyses, are presented in For this study, we broadly sampled hemp flea beetles in four hemp growth regions in China: northeastern China (including Heilongjiang and Jilin provinces), central-east China (Shandong and Anhui provinces), south-central China (Hunan province), and southwestern China (Yunnan province). Within four provinces Heilongjiang, Anhui, Hunan, and Yunnan, we sampled three sites from each province. For the remaining two provinces Shandong and Jilin, only one site was sampled in each. Among the 14 sampled sites, nine were found to be infested by ® Insect DNA kits (Omega Bio-Tek) according to the manufacturer’s instructions. The extracted DNA samples were stored in an −20 °C freezer until use. The quality and quantity of each extracted DNA sample was assessed using Nanodrop (Thermo Scientific Nanodrop 2000). To obtain the COI barcoding fragment, the general invertebrate COI amplification primers (the forward primer LCO1490 (5′-GGTCAACAAATCATA AAGATATTGG-3′) and the reverse primer HC02198, (5′-TAAACTTCAGGGTGACCAAAAAAT CA-3′)) were used to amplify the target sequence of P. attenuata in PCR reactions [2O. The thermocycler procedure was as follows: initial denaturation for 1 min at 94 °C, followed by 20 cycles of increasing the annealing temperature by 0.5 °C every cycle at 94 °C for 1 min, annealing at 45 °C for 90 s, and extension at 72 °C for 1 min, and 20 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 90 s, and extension at 72 °C for 1 min, with a final extension at 72 °C for 5 min [w/v) agarose gel (1 × TAE), alongside a DNA marker . After electrophoresis at 150 V for 40 min, the PCR products were visualized using ethidium bromide and ultraviolet light. Finally, products clearly visible after electrophoresis were sent to a commercial company  for sequencing in both directions.A total of 281 adult hemp flea beetles were analyzed. For each beetle, its genomic DNA was isolated using the E.Z.N.A.or 5 min . PCR proAll COI sequences were checked manually in BioEdit v7.0.9.0 to assess their quality . The alin = 1000 replications.Common parameters of genetic diversity, including the number of haplotypes, number of private haplotypes, and the haplotype and nucleotide diversities, were calculated using DNAsp v6 . The hapP. attenuata samples was estimated based on the analysis of molecular variance (AMOVA). Genetic differences among pairs of geographic populations were assessed based on the traditional STF values. In addition, the relationship between genetic distance and geographic distance was assessed using the non-parametric Mantel test, based on data on the two distance measures between all pairs of specimens . In the Mantel test, the pairwise genetic distance between specimens was calculated as the number of nucleotide differences between their COI sequences, while the pairwise geographic distance was calculated as direct physical distance based on their longitudinal and latitudinal coordinates that was transformed logarithmically. The AMOVA, pairwise population STF, and Mantel test were conducted using the GenAlEx V 6.5 software, with statistical significance for both the AMOVA and STF values derived based on 1000 permutations [We investigated the geographic distributions of mitochondrial COI single nucleotide polymorphisms and haplotypes within and among geographic populations, using the program GenAlEx 6.5 . The conutations . P. attenuata specimens were analyzed for their mitochondrial COI sequence polymorphisms. These 281 specimens were captured in nine sites in six provinces. Our BLAST search comparisons for each of the 281 COI sequences revealed that our sequences shared >99% of nucleotide identity to that of a known COI DNA sequence from P. attenuatus . Thus, the sequencing results confirmed that the morphological identifications were correct and that all our samples belonged to the same species. The obtained COI DNA sequences were aligned, with the total alignment contained 645 bp. Among the 645 aligned nucleotide sites for the 281 specimens, 600 were conserved, 45 were variable, and there was no insertion or deletion. Of the 45 variable sites, 16 were singleton sites and 29 were parsimony-informative, with the less frequent nucleotide shared by at least two specimens at each of the 29 sites. The average base compositions of A, G, C, T in the COI gene fragments were 31.0%, 15.40%, 17.40%, and 36.10%, respectively, with the average content of A + T about twice of that of C + G.In this study, a total of 281 Based on nucleotide polymorphisms, the 281 COI sequences were grouped into 48 haplotypes. The detailed distributions of the 48 haplotypes are shown in Among the 48 haplotypes, two, H1 and H33, were especially abundant, representing 91 and 61 specimens, respectively . As mentP. attenuata. These eight haplotypes accounted for >70% (198/281) of our total specimens  had positive D values, consistent with balancing selection in these three local populations. The remaining six local populations had negative D values, consistent with recent selective sweep, or population expansion after a recent bottleneck. However, only three of the nine D values were statistically significantly different from 0 . The three local populations included one each from Anhui, Jilin, and Heilongjiang (Daqin) , with alThe relationships among mitochondrial COI haplotypes are illustrated in The haplotype network analyses revealed a similar pattern but with additional information and complexity . SpecifiP. attenuata. The results suggest potential genetic differentiation among the geographic populations as well as evidence for recent migrations and/or incomplete lineage sorting. To further quantify the contributions of geographic separation to the observed nucleotide polymorphisms, we estimated the pairwise population genetic relationships using the standard Wright’s STF using the program DnaSP 6.0. Our results showed that geographic separation contributed significantly to the observed sequence variations, with an STF of 0.59635 (p < 0.001). However, between individual pairs of local populations, the STF values ranged widely, from −0.01205 to 0.88233   were consistent with those shown above based on the overall and pairwise variance . Further < 0.05) , a resulP. attenuata, a common hemp pest, from nine geographic locations in six provinces representing the broad range of commercial hemp production in China. These hemp flea beetles were analyzed for their patterns of COI DNA sequence variation among specimens from within and between the local populations. Our results identified that 45 of the sequenced 645 bp mitochondrial COI fragments as being variable within the total population sample. These polymorphic sites separated the 281 specimens into 48 COI haplotypes. These haplotypes were present in variable frequencies, from 1/281 to 91/281. Eight of the 48 haplotypes were found in at least two of the nine local populations, and these eight haplotypes accounted for >70% of all specimens. Each of the remaining 40 haplotypes was only found in one of the nine local populations. Most haplotypes showed geographic-specific distribution patterns. The differential haplotype distributions resulted in significant population differentiation among most local populations in China. Our results provide the first survey of genetic variation on this important pest of hemp. Below we discuss our obtained results, including the implications for the management of hemp flea beetles. In this study, we collected samples of http://v3.boldsystems.org/index.php/databases). As a result, our data should facilitate future comparative studies with other flea beetles in the genus Psylliodes and other insects.Similar to previous studies on other insects ,28,29,30The 48 COI haplotypes showed a range of relationships with each other. Based on their sequence similarities, these haplotypes were clustered into three large groups , with the B group being the intermediates of A and C groups through multiple mutational steps. Interestingly, there are several cases of haplotypes forming loops/reticulate networks. While such loops represent uncertainties in the evolutionary relationships among the observed haplotypes, the observed loop, involving haplotypes H1, H3, H6 and H16 with one mutational step between adjacent members , is consWe identified two haplotypes, H1 and H33, in very high frequencies. H1 showed a broad geographic distribution, found in central, eastern, and northern China. However, it was most frequently found in Anhui province. Its phylogenetic distribution in both the NJ tree and the network indicates that H1 was located at the root/center of many other haplotypes and likely represented a relatively ancient haplotype in the Chinese population of the hemp flea beetle. Seventeen other haplotypes differed from H1 by only one or a few mutational steps, with many of these associated haplotypes found in central and eastern China . The resP. attenuata was likely an ancient species within China and that geographic separation has played a dominant role in the species history. The sampled regions in China also differ in their climate and other geographic conditions. Thus, it is possible that these factors have also played a role in the distribution of these haplotypes. In six of the nine local populations, as well as in the total sample, the Tajima’s D values were negative, consistent with a recent population expansion, likely after population bottlenecks. Interestingly, these COI sequence diversity results are also consistent with the recent cultivation history of hemp in China. Because of the associations with psychoactive compounds such as THC and the negative stigma about psychoactive compounds in China since the end of the opium war, hemp cultivation has been very limited and highly regulated since at least the mid-20th century, despite its long history of cultivation in China. Consequently, due to its narrow host range in favor of hemp plants, P. attenuata has likely experienced a population bottleneck in China since the mid-20th century. However, due to recent surging interests for both hemp fibers and its medicinal properties, hemp cultivation has been increasing rapidly. Such a rapid expansion of host hemp plants could have resulted in the rapid expansion of hemp flea beetles and the generation of many rare (singleton) haplotypes, but which are closely related to the major haplotypes such as H1 and H33. At present, the adaptive significance of the observed rare haplotypes in P. attenuata is not known.The presence of divergent haplotypes in different geographic regions and the observed significant genetic differentiation suggest that P. attenuata. However, we found that certain haplotypes or closely related haplotypes were broadly distributed, consistent with long-distance dispersal or incomplete lineage sorting of this pest. Hemp flea beetles breed once a year, with their mating, feeding and living all closely associated with hemp plants. Thus, long-distance dispersal by their own flight, wind, and other natural means would be highly unlikely to account for haplotype sharing between northeastern, eastern, and central China for several of the haplotypes such as H1, H6, H7, and H24. Instead, the observed haplotype sharing among fields located thousands of kilometers apart was likely due to incomplete lineage sorting and/or recent anthropogenic activities such as human travel and trade. Indeed, anthropogenic activities have been found to impact the distributions of a variety of plant pathogens and pests [P. attenuata, where no haplotype was found shared with those from other geographic areas, the Yunnan haplotypes were found closely related to those in other regions separated by one or a few mutations, consistent with their recent shared histories. Indeed, more extensive sampling may reveal additional sharing of COI haplotypes among geographic populations.Our statistical analyses of the observed COI sequence variations revealed that geographic separation played an important role in the population structure of nd pests ,34,35,36P. attenuata diversity in China. However, additional samples of natural hemp populations and uses of DNA sequence information from other genes, including those from the nuclear genomes, are needed in order to identify the true center(s) of origins for P. attenuata. In China, based on our observations, the method for controlling P. attenuata mainly relies on chemical pesticides, including cypermethrin, abamectin and acetamiprid. In practice, farmers typically spray pesticides onto Cannabis seedlings to prevent P. attenuata from killing the young seedlings. At present, there is no measure to control this pest at later stages of Cannabis growth. However, as shown in our sampling, hemp flea beetles are commonly found during the late stages of Cannabis growth which can result in serious crop losses. Given the oligophagous and monovoltine nature of this pest, one control and prevention measure would be to use crop rotation to eliminate the food source of P. attenuata for one to several years, to significantly reduce or entirely eliminate the reproduction of this pest. Similarly, given the observed evidence for potential gene flow between distinct geographic populations of P. attenuata due to anthropogenic factors, the second measure would be to ensure that the commercial hemp seeds are free of hemp flea beetles  before they are shipped from seed centers to distinct locations for commercial planting and hemp production.Among the nine local populations, those from Shandong, Jilin and Hunan contained a high number of haplotypes, high nucleotide and haplotype diversities, and broad distributions of their haplotypes across the phylogenetic tree. Together, these results suggested that these three regions, especially Jilin, were likely the centers of P. attenuata populations in China. Our analyses of 281 specimens identified a total of 48 haplotypes in various frequencies and distribution patterns. We found that local populations located close to each other were genetically very similar. However, regional populations from different parts of China were significantly differentiated. On the other hand, the sharing of COI haplotypes among diverse geographic regions is consistent with incomplete lineage sorting and/or recent long-distance dispersals, likely due to anthropogenic activities such as human travel and trade. The adaptive significance of the observed COI sequence variations awaits further investigation.In this study, sequences of a 645 bp fragment of the mitochondrial barcode gene COI were used to analyze the genetic diversity and structure of


Pax Mongolica. The genetic history beyond the WEu haplogroups, notably detectable on both sides of Mongolia, was more difficult to explain. For this reason, we moved to the analysis of entire mitogenomes (N = 147). Although it was not completely possible to identify specific lineages that evolved in situ, two major changes in the effective  population size were reconstructed. The more recent one, which began during the late Pleistocene glacial period and became steeper in the early Holocene, was probably the outcome of demographic events connected to western Eurasia. The Neolithic growth could be easily explained by the diffusion of dairy pastoralism, as already proposed, while the late glacial increase indicates, for the first time, a genetic connection with western Eurasian refuges, as supported by the unusual high frequency and internal sub-structure in Mongolia of haplogroup H1, a well-known post-glacial marker in Europe. Bronze Age events, without a significant demographic impact, might explain the age of some mtDNA haplogroups. Finally, a diachronic comparison with available ancient mtDNAs made it possible to link six mitochondrial lineages of present-day Mongolians to the timeframe and geographic path of the Silk Route.Mongolia is located in a strategic position at the eastern edge of the Eurasian Steppe. Nomadic populations moved across this wide area for millennia before developing more sedentary communities, extended empires, and complex trading networks, which connected western Eurasia and eastern Asia until the late Medieval period. We provided a fine-grained portrait of the mitochondrial DNA (mtDNA) variation observed in present-day Mongolians and capable of revealing gene flows and other demographic processes that took place in Inner Asia, as well as in western Eurasia. The analyses of a novel dataset  of mtDNAs highlighted a clear matrilineal differentiation within the country due to a mixture of haplotypes with eastern Asian (EAs) and western Eurasian (WEu) origins, which were differentially lost and preserved. In a wider genetic context, the prevalent EAs contribution, larger in eastern and central Mongolian regions, revealed continuous connections with neighboring Asian populations until recent times, as attested by the geographically restricted haplotype-sharing likely facilitated by the Genghis Khan’s so-called  MongoliSilk Road (founded by the Chinese Han dynasty in 130 BCE) that played a major role in the economic, demographic and cultural processes shaping the history of several Eurasian populations . At its peak (1206–1368 CE), the empire stretched from present-day Poland in the west to Korea in the east, and from Siberia in the north to the Gulf of Oman and Vietnam in the south, covering approximately 22% of Earth’s total land and with a population of over 100 million people. At the beginning, Genghis Khan used to destroy most infrastructures along the Silk Route, but eventually he decided to adopt a politics of supporting and facilitating commercial and cultural exchanges between regions under his dominion (Pax Mongolica (1280–1360 CE), indicating a pacific and flourishing period characterized by commercial, cultural, religious, and scientific exchanges between western and eastern populations, including trades between nomadic groups and urban centers  provided several pieces of information. Evidence deriving from the mtDNA haplogroups shared between Afanasievo and Yamnaya people supports an eastward migration from the Pontic-Caspian steppes . The pren = 4), Bayankhongor (n = 2), Bayan-Ölgii (n = 216), Bulgan (n = 5), Darkhan-Uul (n = 1), Dornod (n = 370), Dornogovi (n = 26), Dundgovi (n = 1), Govi-Altai (n = 8), Khentii (n = 132), Khovd (n = 429), Khövsgöl (n = 307), Ömnögovi (n = 2), Övörkhangai (n = 8), Selenge (n = 4), Sükhbaatar (n = 246), Töv (n = 10), Ulaanbaatar (n = 2), Uvs (n = 132), Zavkhan (n = 167); the remaining samples (n = 348) belonged to individuals who did not provide province information and therefore were associated to an “unspecified” group. Provinces with less than 30 individuals were grouped into three geographic macro-areas by considering their geographic position, biome, and orography: “Gobi Desert” , “Khangai Mountains” (Arkhangai and Övörkhangai), “Near Ulaanbaatar”   Mongolian provinces: Arkhangai (nbaatar) .DNA extraction and mtDNA control-region sequencing were performed as in https://empop.online/) under accession number EMP00853. A total of 2,133 haplotypes encompassed the entire mitochondrial control region , while 2,335 haplotypes encompassed at least the HVS1 segment (nps 16024–16365). The sequences were aligned to the revised Cambridge Reference Sequence    using Se12920.1) . Conside12920.1) .Several mtDNA sequence variation parameters were estimated by using DnaSP 5.1 software . NucleotThe Mongolian maternal gene pool was further compared with a Eurasian dataset encompassing 546 bps of the control region (nps 16024–16569) obtained from 30,400 individuals from 69 countries/geographic areas . Four poThe entire mitogenome sequences of 147 individuals, representative of different macro-haplogroups, were obtained  by usinghttp://www.phylotree.org/) (The IGV (Integrative Genomics Viewer) software  was usedee.org/)  using boee.org/)  and SAM2https://empop.online/) under accession number EMP00853.All complete mitogenomes (N = 147) are available in GenBank under accession numbers OL619795-OL619941 and in the EMPOP mtDNA population database  tree, built with an updated version of mtPhyl v.5.003 and checked with MEGAX software. One published L3 sequence (accession number DQ341081) was used as outgroup to reconstruct time estimates and demographic trends in BEAST v2.6.6 , as prevThe concomitant analysis of 2,335 HVS1 sequences from modern Mongolians identified 160 polymorphic sites (excluding gaps and ambiguous sites) with a Nei’s nucleotide diversity (π or Pi) of 0.00809, and a very high haplotype diversity (Hd = 0.986). This genetic diversity is heterogeneously distributed within Mongolia . The “NeThe overall 2,420 Mongolian mtDNAs were classified into different 413 lineages and sub-lineages, ultimately grouped into 18 macro-haplogroups  and 121 belonging to WEu haplogroups  . ThroughIn order to provide a timeframe to the mtDNA inputs from the west, we focused on the WEu mitogenomes providing Bayesian coalescence ages of internal nodes. Most of these lineages, with a western Eurasian origin but now also identified in Mongolia, coalesced during and soon after the last glacial maximum  . The BSPMongolia is one of the most sparsely populated countries in the world, but complex population interactions occurred across this Eastern Steppe region over the past several millennia . Here weThe majority of mtDNAs belong to haplogroups typical of eastern Asian populations whose frequency decreases from eastern to western regions. An opposite pattern could be observed for those lineages characteristic of western Eurasia. Overall, both EAs and WEu haplogroups contributed to create the mtDNA differentiation currently detectable in Mongolia, as highlighted by the PCA . The WEuin situ. A few and more recent events have been also reconstructed through the analysis of modern and ancient mitogenomes, some during the Bronze Age, others in the last three thousand years. As if haplogroup H1 (and its sub-clades) might suggest a direct link between Europe and Mongolia, six sub-lineages identified in ancient mitogenomes perfectly match the timeframe and path of the Silk Route and can be still identified in present-day Mongolians. Finally, rather than finding long-distance traces of the Mongol Empire expansion to the west, we identified continuous and recent  connections with neighboring Eastern Asian populations. The geographically restricted sharing of haplotypes from typical EAs mtDNA lineages might represent an outcome of Genghis Khan’s so-called Pax Mongolica still detectable in present-day Mongolians.The gene pool of present-day Mongolians reflects gene flows and demographic processes that occurred over the past several millennia across the Eurasian Steppe, thus representing an important key to reconstruct the genetic history of Inner Asia as well as western Eurasia. The analyses of a large set  of mtDNAs allowed us to identify peculiarities of the mitochondrial gene pools of different Mongolian regions. A clear matrilineal differentiation was identified across the country due to the differential contribution of mitochondrial lineages of eastern Asian and western Eurasian origins. The EAs contribution was probably linked to continuous interactions with neighboring regions until present days, presumably including those related to the Mongol Empire expansions (1206–1368 CE). The inputs from the west were more difficult to pinpoint. Therefore, we moved to the analysis of entire mitogenomes (N = 147), which allowed us to date the WEu lineages and to reconstruct demographic trends across time. After the first migration of Paleolithic hunter-gatherers, the major increases of the population size could be linked to post-glacial late Pleistocene expansions and to changes towards a more sedentary lifestyle during the Holocene. However, the lack of Mongolia-specific lineages did not allow to directly study mitogenomes that evolved


Ammotragus lervia Pallas 1777) is an ungulate species, native to the mountain ranges of North Africa. In the second half of the twentieth century, it was successfully introduced in some European countries, mainly for hunting purposes, i.e. in Croatia, the Czech Republic, Italy, and Spain. We used neutral genetic markers, the mitochondrial DNA control region sequence and microsatellite loci, to characterize and compare genetic diversity and spatial pattern of genetic structure on different timeframes among all European aoudad populations. Four distinct control region haplotypes found in European aoudad populations indicate that the aoudad has been introduced in Europe from multiple genetic sources, with the population in the Sierra Espuña as the only population in which more than one haplotype was detected. The number of detected microsatellite alleles within all populations (< 3.61) and mean proportion of shared alleles within all analysed populations (< 0.55) indicates relatively low genetic variability, as expected for new populations funded by a small number of individuals. In STRUCTURE results with K = 2–4, Croatian and Czech populations cluster in the same genetic cluster, indicating joined origin. Among three populations from Spain, Almeria population shows as genetically distinct from others in results, while other Spanish populations diverge at K = 4. Maintenance of genetic diversity should be included in the management of populations to sustain their viability, specially for small Czech population with high proportion of shared alleles (0.85) and Croatian population that had the smallest estimated effective population size (Ne = 5.4).The aoudad ( Newly created populations are often established with relatively few individuals of unknown genetic background and therefore may be particularly susceptible to loss of genetic variation due to inbreeding and genetic drift4. Within such recently established populations the genetic diversity might decrease, whereas population differentiation might increase over time5. Reduced gene flow and the low level of genetic diversity can also enhance the rate of inbreeding, which is considered to reduce a populations’ adaptability to a changing environment. Homozygous recombination of deleterious recessive alleles become more likely, which may lead to inbreeding depression6.During the nineteenth and twentieth century, large number of non-native animal species have been widely introduced in Europe. One of the most important drivers for such introductions was hunting, with introductions aiming to create or improve hunting opportunities, especially when native game species had become scarce7. Consequently, both distribution and genetics of ungulate populations across Europe have been profoundly influenced by such introductions8. Among ungulates introduced into Europe, the aoudad  is one of the species that was successfully established in the wild beyond its natural range8, with established populations in Croatia, Czech Republic, Italy, and Spain9.Among all introduced groups of mammal species in Europe, ungulates stand out from the others with 73.5%, since this is one of the most important game groups in all European countries10. As a result of mostly poaching and habitat loss, population numbers of aoudad in its native range are declining12, classifying this species as Vulnerable in the IUCN Red List13. The aoudad is a generalist herbivore and extremely plastic in its utilization of available food resources14. It is a polygynous species with high reproductive rate. Large and strong individuals have high fitness and reproductive success15, increasing their potential to colonise different localities whenever conditions are appropriate11.The aoudad, or Barbary sheep, is native to the mountain ranges of North Africa. Small groups scattered irregularly on large territories from Mauritania in the west to the Red Sea in the east show a typical pattern of the distribution of the species in the wild16. The population in the Czech Republic was established following the escape of several individuals from the Plzeň Zoo in 1976, and it consisted of a few dozen animals17, while today this population is believed to be extinct . In Italy, one small population of about 20 individuals is known to be present at the Beigua Regional Park in the province of Savona (Liguria) since 200715. Previously, another population was present in the province of Varese (Lombardy), where a group of six individuals  escaped from a private enclosure in 1993 and established a small breeding population that reached the maximum size of about 20 individuals. This population has been eradicated by the personnel of the Province of Varese accordingly to a plan approved by the Italian National Institute for Environmental Protection and Research (ISPRA) since 2005 . In Sierra Espuña, Spain, 36 aoudads, from zoos in Germany and Morocco, were intentionally introduced by the regional administrations in 1970. This founding population reproduced with great success and naturally dispersed very rapidly from their release area to nearby montane enclaves. Its current population, estimated at around 2000 individuals, is still expanding19. In 1972 sixteen animals from Sierra Espuña were released into La Palma island, Canary Islands, and established successfully since then18.In Croatia, during 2002 a number of aoudads of unknown origin were illegally released in the southern Dinaric region (Mosor Mountain). Current data obtained by GPS tracking of aoudad movements showed that the population is limited to the Mosor Mountain range and it is highly unlikely that it will expand its range beyond that area20. There is no information on environmental impacts for the other non-native aoudad populations15.So far, there is no evidence of the negative impact of the aoudad on host ecosystems in mainland Spain, except for La Palma island, where they have critically affected the survival and diversity of native, endemic flora and caused high levels of soil erosion5, as adaptation in such species occurs mainly through selection on pre-existing genetic variation21. Since scientific literature about the genetic structure of European aoudad populations is scarce, it is thus important to study genetic diversity and structure of those populations to assess their sustainability. Unfortunately, no comprehensive genetic analyzes have yet been carried out on original aoudad populations from their native range, except for a few studies of specific populations . In addition, insight into the genetic structure and origin of European aoudad populations might contribute to the ex-situ conservation of the species that is threatened in its native range.When the introduction of species into new areas is human-mediated, the new populations are often founded by only a few individuals that are completely isolated from the source populations. In the case of the European aoudad populations, sudden and substantial reduction in effective population size during first introductions and lack of gene flow into the established populations are assumed to have led to the loss of genetic variation through genetic drift. Severe reductions in genetic diversity may limit viability and adaptive potential of introduced populations under environmental change23. We aimed to understand how current levels of genetic diversity and structuring vary among European aoudad populations that differ with regard to the time and source of introduction.Here we report the first attempt to characterize the genetic diversity and population genetic structure of all non-native European aoudad populations. We used neutral genetic markers, mitochondrial DNA (mtDNA) control region sequence and microsatellite loci, to characterize and compare genetic diversity and spatial pattern of genetic structure on different timeframes among study populations. While variability of maternally inherited and more conserved mtDNA control region reflect maternal lineages present in the founder individuals, microsatellites, due to their codominant nature and high variability can reflect more recent events that shaped current genetic structureBased on samples collected from all known locations in Europe, the objectives of this study were to characterize the patterns of neutral genetic structure of recently established non native populations of aoudad and gain insights into the number of maternal lineages of these populations.24. In the full sample, four mtDNA haplotypes . None of the samples collected in Italy were successfully sequenced, due to the low quality of DNA. Low quality of the DNA isolated from faecal samples is known to dramatically reduce the genotyping success of such samplesHaplotype Amle01 was the most frequent (70%) and the only one found in Croatian, Czech and La Palma samples. Samples from Almeria were monomorphic for Amle02 haplotype, therefore haplotype and nucleotide diversity was zero in these populations. Samples from Sierra Espuña comprised of three haplotypes  with haplotype diversity of 0.69 and nucleotide diversity of 0.06. Observed four haplotypes were defined by 59 variable sites of which 11 were parsimony informative. The least number of mutation (12) was present between haplotypes Amle02 and Amle03, while the haplotype Amle04 stood out as remarkably divergent from all others, with the maximum of 55 variable sites separating Amle04 and Amle02  to 9 (INRA040), with a mean number of alleles per locus of 5.07 (Supplementary Table f estimator ranged between − 0.029 (Almeria) to 0.070 (Sierra Espuña) with a mean positive value of 0.302 ± 0.057.Linkage disequilibrium was observed for: 14 pairs of loci in Croatia, four pairs in Sierra Espuña, and for one pair of loci in La Palma and Almeria. After applying FDR corrections, linkage disequilibrium was significant only for MM12/SR-CSRP12 and TGLA073/SR-CSRP12 in Croatia. The multilocus value of the Estimated effective population size was the smallest in Croatian population reflecting the small number of founding individuals, and the largest in Sierra Espuña.Mean proportion of shared alleles between all individuals within each population was: 0.56 in Sierra Espuña, 0.57 in La Palma, 0.66 in Croatia, 0.75 in Almeria and 0.85 in the Czech population.ST value was 0.296 (95% CI = 0.249–0.347). The lowest pairwise FST value was observed between populations from Czech and Sierra Espuña (FST = 0.109), while the highest value was found between Croatian and Almeria populations (FST = 0.459) .Global FAmong STRUCTURE runs, the highest ΔK value was observed for K = 3, followed by that for K = 4  between individuals from this population and the largest estimated effective population size of 36.25. There is no available data on subspecies/lineages of aoudads used in introductions to Sierra Espuña (or in preceding introductions to Frankfurt and Ain Sebad Zoos), nor published mtDNA sequences from original populations that would allow for identification of subspecies/lineages present in this population, but variability of detected haplotypes might reflect introductions from multiple distinct lineages. This is especially valid for haplotype Amle04, which differs by at least 46 mutations from other haplotypes detected in this population, indicating its origin from evolutionary distinct units. Reported numbers of mutations between haplotypes from same subspecies of ungulates are usually lower. For example, in the study of chamois (Rupicapra sp.)26, native European mountain ungulate, D-loop haplotypes from the same subspecies were up to 15 mutations apart. In its native range in North Africa, six aoudad subspecies have been described, based on their distribution and morphological differences in coat color and horns27. Derouiche et al.22 aimed to further evaluate the validity of the subspecies and determine the geographical limits of the valid ones using mitochondrial cytochrome b gene. Based on the haplotypes obtained, their results indicated a deep Mediterranean-Saharan genetic break in the species, suggesting the presence of two highly distinct evolutionary lineages.Sierra Espuña population is known to have originated from individuals from the Frankfurt Zoo in Germany and the Ain Sebad Zoo in Casablanca, Moroccoar was less than 3.61 in all analysed populations) and mean proportions of shared alleles within all populations (> 0.55) which confirm their poor genetic diversity. The theoretical expectations for the loss of allelic variation after bottlenecks are described by Nei et al.28 who showed that critical factors determining this loss are the size of founding population and the growth rate of a newly established population. They indicated that the average number of alleles per locus will be more sensitive to founding population size than heterozygosity. This is illustrated in most of sampled aoudad populations, in which allelic richness is substantially low , where it was attributed to isolation and poor management practices. Largest number of detected alleles and the highest observed heterozygosity in Sierra Espuña population is another indication of the highest genetic variability in this population, supporting the theory of multiple origins.All analyzed aoudad populations in Europe were only recently introduced, with relatively small number of founding individuals, so it is expected that their genetic diversity is low in comparison to the viable free mating populations of native ungulates. This is confirmed by the number of detected alleles within all populations  back in 1975; so that it presumably belongs to the Saharan aoudad subspecies, while the other ones analyzed in our study might be of admixed origin, based on the fact that it is a population formed by individuals coming from the Casablanca Zoo in Morocco , and from the Frankfurt Zoo, of unknown origin. In addition, Almeria population had the second highest mean proportion of shared alleles (0.75), lower only than the small Czech population.30. Implications of detected low genetic diversity on management of these populations can be discussed only after management goals are clearly defined. Commonly accepted practice is to increase genetic variability of introduced species by introducing more individuals with different genetic background. The effects of such practices were reviewed by Dlugosch and Parker31 who quantitatively summarized the genetic diversity data available at the time for 80 introduced or invasive species of animals, plants and fungi. Their review shows that multiple introductions had only small positive effects on diversity on average, disapproving the argument that multiple introductions have been critical in providing genetic rescue from severe and deleterious founder effects in most cases. According to this review, increases in genetic diversity caused by multiple introductions and/or gene flow occur after many decades, during which time the range expansion of the founder population is successful. Later introductions of genetically distinct individuals can increase genetic diversity through admixture, and the strength of this effect will depend on the reproductive rate of the individuals from different “waves” of introductions.Management of European aoudad populations varies between countries and the views change with new insights of their coexistence with native species and ecosystemsA. lervia individuals from Croatia , Italy  and Spain . These populations represent the overall distribution range of free-ranging aoudad populations in Europe. Also, we obtained samples from a captive population from the Czech Republic  and those of the Saharan aoudad, A. l. sahariensis, in Almeria, Spain; 36°49′48″N 2°24′29″W.From 2016 to 2018, we collected 92 samples of 17. Since the aoudad population in Italy is small and we were not able to collect any tissue samples, we collected seven fresh faeces samples. In Spain 47 tissue samples were collected from three locations: Sierra Espuña n = 17, La Palma n = 15, during regular hunting, and from captivity in Almeria n = 15. Geographic locations of the sampled populations are presented in Fig. In Croatia, 34 tissue samples were collected during regular hunting. In the Czech Republic, four blood samples were collected from live aoudad because they are protected and not huntedSampling was done according to the Ethical and Welfare Standards presented in the , Regulation on the Protection of Animals Used for Scientific Purposes , with the approval of the Bioethical Committee for the Protection and Welfare of Animals of the University of Zagreb Faculty of Agriculture (UR.BR. 251-71-29-02/19-21-1). All the research was done in accordance with arrive guidelines.32. For the fluorescent labeling of PCR fragments, we used the M13-tailed primer method33. Singleplex PCRs were performed in a final 8 µL reaction volumes consisting of 1 × GoTaq G2 Hot Start Colorless Master Mix (Promega), 0.2 µM of each reverse and M13 primer, 0.05 µM of forward primer and 1 µL of template DNA. Information about PCR hybridization temperatures of all loci and thermocycler programme is given in Beja-Pereira et al.32. PCR products were analyzed on an ABI 3730 Genetic Analyser with GeneScan 350 ROX internal size standard (Applied Biosystems) by Macrogen (South Korea). GeneMapper v3.2. (Applied Biosystems) was used for scoring alleles.Total genomic DNA from tissue samples was extracted using the Promega Wizard Genomic Purification Kit following the manufacturer’s instructions. From faeces samples, DNA extraction was done using a liquid handling robot (Hamilton Starlet) and optimization by Applied Biosystems MagMAX DNA extraction Kit. All individuals were genotyped using fifteen polymorphic microsatellite loci: BM143, BM302, BM415, BM1443, BM1818, ETH225, ILSTS030Q, INRA005, INRA040, MAF70, MB25, MM12, SR-CSRP12, SR-CSRP24, TGLA07334, following the PCR protocol given in Mereu et al.34. The PCR products were sequenced in forward direction using an ABI 3730 automated DNA sequencer (Applied Biosystems). Electropherograms were visually inspected using Applied Biosystems SEQSCAPE software and the sequences were trimmed to 767 bp which could be unequivocally called.Mitochondrial control region was amplified using tRNAPro and tRNAPhe primers35. The presence of tandem repeats in the 5′ part of the control region led to ambiguity in the position of the gaps and for this reason we excluded the ambiguous region from final sequences used in further analyses. A final alignment was obtained by merging the two trimmed fragments at positions 15,448–15,568 and 15,730–16,214 with respect to the GenBank sequence (accession number FJ207522) and consisted of 606 nucleotides. DnaSP v.6 was used to estimate number of haplotypes, haplotype (Hd) and nucleotide (π) diversity36. The evolutionary relationships between the haplotypes were analyzed by a median-joining (MJ) network37 which was constructed using PopART38. The parameter ε was set to zero (default) to obtain a sparse spanning network.The resulting sequences were aligned with two previously published sequences of the aoudad complete mitogenome (GenBank accession numbers FJ207522 and EF466060) using the ClustalW algorithm implemented in MEGA X39 was used to check amplified microsatellite genotypes for large allele dropout, scoring errors due to stuttering and the presence of null alleles. Null allele frequencies were then estimated for each locus and population using the expectation maximization algorithm40 implemented in FreeNA41. Polymorphism Information Content (PIC) was calculated for each microsatellite marker per population using CERVUS v.3.0.742. GENETIX v.4.0543 was used to estimate the mean number of alleles, the observed heterozygosity (Ho), expected hererozygosity (He) and f estimator of FIS44 per locus and population. Linkage disequilibrium was tested among all pairs of loci in each population using a permutation procedure (n = 1000) using GENETIX v.4.05. Departures from Hardy–Weinberg equilibrium (HWE) were tested for each population using the score tests for heterozygote deficiency implemented in GENEPOP v.4.7.245. Allelic richness [Nar(g)] and private allelic richness [Npar(g)] were estimated using a rarefaction procedure implemented in HP-RARE46, where g represents the minimum number of alleles observed at a locus in one of the populations . The minimum number of genes in analysed populations was 28 , so this was used as a basis for rarefraction.For all population based analyses we used only genotypes from populations with more than 10 samples . MICRO-CHECKER 2.2.347, as implemented in NEESTIMATOR 2.0148. As suggested by Waples and Do49, we excluded alleles with frequencies below 0.02 in order to avoid bias caused by rare alleles.We estimated effective population size (Ne) for each population using the linkage disequilibrium methodhttps://www.microsoft.com/en-ca/microsoft-365/excel).Finally, to estimate the genetic similarity of individuals within populations, we estimated the mean proportion of shared alleles between all individuals within each population using custom script in Microsoft Excel 2016 , with K ranging from 1 to 7, were carried out with 106 iterations after a burn-in period of 105 iterations. We considered no admixture model and uncorrelated allele frequencies. The most likely number of clusters (K) was estimated by estimation of the ΔK statistic that reflects the rate of change of log-likelihood values between sets of runs with successive K values, using STRUCTURE HARVESTER51. To account for label switching between results of different runs with same K, results from 10 runs with selected K were combined using CLUMPP v.1.1.252. Results were displayed graphically using DISTRUCT v.1.153 and modified with Microsoft PowerPoint 2016 .We estimated overall and pairwise genetic differentiation with the Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.


Tilia cordata Mill. is a valuable tree species enriching the ecological values of the coniferous‐dominated boreal forests in Europe. Following the historical decline, spreading of Tilia sp. is challenged by the elevated inbreeding and habitat fragmentation. We studied the geographical distribution of genetic diversity of Tilia cordata populations in Lithuania. We used 14 genomic microsatellite markers to genotype 543 individuals from 23 wild‐growing populations. We found that Tilia cordata retained high levels of genetic diversity . AMOVA, Bayesian clustering, and Monmonier's barrier detection indicate weak but significant differentiation among the populations (FST = 0.037***) into geographically interpretable clusters of (a) western Lithuania with high genetic heterogeneity but low genetic diversity, bottleneck effects, (b) relatively higher genetic diversity of Tilia cordata on rich and most soils of midland lowland, and (c) the most differentiated populations on poor soils of the coolest northeastern highland possessing the highest rare allele frequency but elevated inbreeding and bottleneck effects. Weak genetic differentiation among the Tilia cordata populations in Lithuania implies common ancestry, absence of strong adaptive gradients, and effective genetic exchange possible mediated via the riparian networks. A hypothesis on riparian networks as gene flow mediators in Tilia cordata was raised based on results of this study. Tilia cordata in Lithuania contains the autochthonous populations of high genetic diversity representing the pre‐historical gene pools, that can be promoted, and the escapes from urban sources of low diversity, that must be contained.Gene pool of  Mixed Norway spruce (Picea abies) Scots pine forests are common in the northeastern and western highlands. Midland lowland contains relatively richer soils and is dominated by broadleaved tree species. According to the forest inventory from 2010, the forest sites with over 50% dominance of Tilia cordata comprise 6,500 ha in Lithuania, which is 0.32% of the total area of forest land in Lithuanian. Tilia cordata is more frequent on rich soils in the forests of midland lowland, especially its southwestern and northern parts , aspen (Populus tremula), birch (Betula pendula), Norway spruce, hornbeam (Carpinus betulus), black and gray alders , Norway maple  . However, the northern PAPI population contained features less common elsewhere: pale bark with less expressed farrows, pronounced multiple forking of stems. The Tilia cordata sites were carefully chosen for natural origin and location by avoiding proximity to the urban areas, usually within forest tracts of various size. We used an electric drill to sample the sawdust from 20 to 25 adult overstory Tilia cordata trees per population. We selected the trees spaced at least 20 meters away from each other within a zigzag sampling path covering ca. 2–5 ha depending on the density of Tilia cordata at a site. In most of the stands we sampled, there were sites with more than 50% dominance of Tilia cordata.A total of 543 a Figure , Table 12.2The DNA was extracted from silica gel‐dried wood sawdust collected by drilling with electric bore ca. 1 cm deep into the trunk  was calculated for each population and region based on maximum‐likelihood method  and coalescent theory using MIGRATE_N software  observed in the data  is higher than He expected under mutation‐drift equilibrium  than SMM (stepwise mutation model) . The mode shift tests for deviations from so‐called L shape distribution of allele frequencies usually indicate recent bottlenecks. This test assumes that the bottleneck‐free populations possess high number of low‐frequency alleles which follow an L shape appearance of the allele frequency histogram  and Dest  the frequency‐based differentiation indexes Gst Jost,  with GENst Jost, .Tilia cordata in Lithuania. We set the burn‐in period length for posterior distribution to 105 and the number of MCMC iterations to 105, the K range from 1 to 10, each replicated 10 times. We used the correlated allele frequency model, no admixture, and the LOCPRIOR option for the three regions indicated in Table K values from 2 to 6. We did not consider deploying higher than K = 6 groups because the individual Bayesian assignment started to “decay” so that for a significant number of individuals the membership proportions become similar between the K groups tested. Within each run of K from to 10 clusters, the most appropriate replication out of 10 was identified by (a) calculating for each individual the maximum likelihood for belonging to a single cluster out of N clusters and (b) averaging these maximum likelihoods over all individuals for each single run and (c) selecting a single run out of 10 replicated runs with the highest mean likelihood. In this way, we obtained the structure, which assigned individuals to a single cluster with the highest likelihoods.We used the Bayesian clustering approach implemented in STRUCTURE soft. ver. 2.2.3  but returned 8 alleles in T. platyphyllos and the locus Tc927 few alleles in T. cordata and 15 alleles in T. platyphyllos.We also screened for possible natural hybridization events between 33.1Tilia cordata in our material. All the loci consistently contained two alleles per individual, indicating no polyploidy in Tilia cordata. The set of 14 loci generated a total of 178 different alleles . For most loci, the expected heterozygosity (He) values varied at about 0.8 indicating highly polymorphic material , and therefore were excluded from further analysis of genetic structure and diversity  in the western region 1 was significantly lower than in the remaining regions  and inbreeding coefficient (FIS)  Table . Howeveref followed that of He with the lowest values in western highland and highest—in eastern highland  and 3 , while for the remaining regions, the p‐values were well above the significance threshold and ranged from 0.2 to 0.4 . The set of loci that returned the strongest Dest‐based differentiation was Tc920 (Dest = 0.24), Tc4 (0.15), Tc915 (0.13), Tc963 (0.25), Tc7 (0.14). These five loci also were the most polymorphic with highest ranking Na and He values , strong differentiation among populations within region , and the remaining 96.26% between individuals within populations , indicating some isolation by distance and substructuring among Tilia cordata populations in Lithuania.The Mantel test showed weak but significant linear correlation between the genetic and geographical distances . Here, we need to note the morphotype difference between SVEK1 and SVEK2 (not scored), which was the reason for sampling these two geographically adjacent stands. In SVEK2, straight and single‐stemmed trees dominated, while SVEK1 contained large number of curvy trees with multiple forking. Both the stands had no signs of coppicing. At K = 4 to 5, the PAPI population in the northeast emerged with no associates at all and the homogeneity of the midland lowland populations starts to be more pronounced . Therefore, we consider the K = 6 structure interpretable as optimum in our material.Evanno's deltaK method suggested 0 Figure . However0 Figure  and seek0 Figure . At K = s Figure . At K = K = 6 clusters revealed the following structure  hige Figure ; (b) a re Figure .The additional STRUCTURE runs to screen for a finer structure by analyzing a subset of populations revealed (a) a subdivision of the western region 1 into northern 1A and southern 1B, in this way separating the former Eastern Prussian territory from the rest (b) slight differentiation of the two northern populations of PAPI and ZAGA from the rest in the midland highland Figure .The Monmonier's barriers along marked shifts in allele frequencies were not as helpful in finding geographical consistent genetic structures Figure . The barThe surface plot of the interpolated Nei et al.  genetic T. cordata and T. platyphyllos, we examined the putative T. platyphyllos marker allele frequencies for the loci Tc8 and Tc927. For the locus Tc8 with the most common 140 and 146 bp alleles at 99% frequency  exceeded those found by Logan et al. (Tilia cordata in Poland (He = 0.53 – 0.58), Russia (He = 0.56–0.59), and Austria (He = 0.61) as well as average He values in the UK . Asexual reproduction in Tilia is expected to increase when moving northwards due to harsher climate for sexual reproduction . T. platyphyllos does not occur naturally in the forests and is present in urban parks of Lithuania. JUOD population is in the coastal spit, presently a national park, formerly a famous coastal resort of Eastern Prussia, where we personally found small, planted groups of T. platyphyllos some 2–3 km away from the sampling site. The single case tracked by us indicates that spontaneous hybridization or escapes are possible and likely to intensify in the future. To confirm that, Semerikova et al.  of park trees into nearby forests. Therefore, artificial establishments may constitute a significant spreading source of Tilia cordata back into the forests and leave a notable signature into the ancient autochthonous gene pools. This observation is confirmed by relatively higher genetic heterogeneity of Tilia cordata in region 1  fragmentation drift in natural gene pools and (b) aftereffects of spreading from urban sources of low diversity  that could be caused can be strong fragmentation due to reduction of the effective population size under higher selective pressure in this coolest region in the country (as indicated by the significant bottleneck effect). There also is a relatively higher urbanization pressure in eastern region 3 , the situation with genetic diversity was different—high allelic diversity but elevated inbreeding and bottleneck effects detected  create favorable environment for 4.3Tilia cordata in Lithuania contrast with Erichsen et al.  than elsewhere. In our material, there also is a tendency for a stronger northward than southward directed gene flow  high genetic heterogeneity of the populations in western Lithuania, (2) genetically homogeneous population composition in the midland lowland, and (3) unique genetic group present in high frequency in the northeastern highland. We interpret the high genetic heterogeneity of the western Lithuanian Tilia cordata populations as (a) possible effects of provenance transfer, especially in the former Eastern Prussia, where different management regime and seed collection sources are expected , coastal lowland (1B) mid‐northern (2A), mid‐southern (2B), northeastern (3), and southeastern (3) Lithuania is 7, 3, 2, 4, and 3, respectively. In general, presence of environmental heterogeneity and microhabitat diversity helps forming gene pool diversity and so strengthening species for challenges of climate change  were most differentiated in Lithuania. There could be several reasons for that (a) adaptation to cooler climate, drier and poorer sites in this region, (b) gene flow from the east as discussed above on high rare allele frequency in this region, (c) stronger genetic drift effects due to relatively stronger fragmentation caused by to higher urban pressure and harsher environment for Tilia in this region.The Tilia cordata populations. Such diversity reduction is especially undesirable at the northern edge of species advancement, where fragmentation of Tilia cordata gene pool is still profound and the environment for sexual reproduction is less favorable . Conservation efforts could consider capturing the genetic diversity of Tilia cordata within each of the western, midland, and eastern genetic clusters in Lithuania by identifying autochthonous stands. Asexual reproduction, presence of old‐growth trees, and variable age structures could be considered as the main criteria for the autochthonous populations. The commercial value of Tilia cordata trees can be one but not a decisive factor for selecting the conservation stands. Natural population management should favor preservation of old‐growth trees of Tilia cordata in mixed forest stands ; Formal analysis ; Funding acquisition ; Methodology ; Software ; Supervision ; Writing‐original draft . Ruta Kembryte: Investigation . Jurata Buchovska: Investigation . Virgilijus Baliuckas: Conceptualization ; Funding acquisition ; Methodology ; Resources . Darius Kavaliauskas: Investigation .Supplementary MaterialClick here for additional data file.