Spodoptera frugiperda) is important as it can help mitigate crop damage, and highlight areas at risk of outbreaks or evolving insecticide resistance. Determining population structure in invasive FAW has been a challenge due to genetic mutations affecting the markers traditionally used for strain and haplotype identification; mitochondrial cytochrome oxidase I (COIB) and the Z-chromosome-linked Triosephosphate isomerase (Tpi). Here, we compare the results from COIB and Tpi markers with highly variable repeat regions (microsatellites) to improve our understanding of FAW population structure in Africa. There was very limited genetic diversity using the COIB marker, whereas using the TpiI4 marker there was greater diversity that showed very little evidence of genetic structuring between FAW populations across Africa. There was greater genetic diversity identified using microsatellites, and this revealed a largely panmictic population of FAW alongside some evidence of genetic structuring between countries. It is hypothesised here that FAW are using long-distance flight and prevailing winds to frequently move throughout Africa leading to population mixing. These approaches combined provide important evidence that genetic mixing between invasive FAW populations may be more common than previously reported.Understanding the population structure and movements of the invasive fall armyworm (FAW,  Spodoptera frugiperda) is a highly invasive crop pest in Africa, Asia and Australasia1. It is native to North America where it is largely migratory, surviving winters in southern Florida and Texas before migrating north as the temperature warms, though there is some evidence that parts of Central and South America, such as Puerto Rico, have more resident populations that rarely interact with FAW from elsewhere in America4. The migratory nature of FAW means that it has a strong flight ability, and some individuals can disperse as far as 300 miles before oviposition5. Wherever it disperses to, the effects are devastating, causing millions of tonnes of crops to be lost, resulting in huge economic losses as well as food shortages6.The fall armyworm  and the Z-chromosome-linked Triosephosphate isomerase (Tpi) haplotypes12. There are two Tpi markers used for FAW, TpiE4 that is based on variation in exon 4 which can differentiate between the corn and rice strains, and TpiI4 that is based on intronic variation and has six recorded haplotypes  that can differentiate between strains and populations12. However, there is some disagreement between COIB and TpiE4 haplotypes in FAW in Africa for strain identification, with evidence suggesting that the COIB haplotypes are less reliable in distinguishing between invasive populations across Africa and Asia13. This disagreement means that most individuals are identified as the rice strain with the COIB marker, and the same individuals are then identified as the corn strain with the TpiE4 marker. Given that the majority of samples have been collected on maize plants it has been suggested that the most accurate marker is likely to be the TpiE4 marker13. However, this confusion with the COIB and TpiE4 markers is most likely due to the hybridization of the corn and rice strains which has occurred in the invasive populations since FAW left its native range14. Furthermore, the COIB and Tpi markers show very little variation in the invasive FAW populations, for example, only two COIB haplotypes  and four TpiI4 haplotypes  were previously reported in South Africa and India13.There is currently a lot known about FAW population structure and movements in its native range , enabling farmers to deal with outbreaks and minimise crop losses13. Understanding the source of the FAW population via population genetics approaches is, therefore, an important area of research in curbing further spread, and this study addresses this by using microsatellites to determine population structure and genetic diversity across Africa.There is also strong evidence that the invasive populations in Asia are originally from Africa, with FAW from both continents showing similar haplotype frequencies and the same mutation affecting the COIB strain identification markerEpisyrphus balteatus and Sphaerophoria scripta) microsatellites revealed high levels of genetic mixing, suggesting frequent migratory movements across Europe over a very large geographical scale, predominately occurring along the North–South axis15. Additionally, microsatellites can also be used to detect genetic differentiation at much smaller scales, such as between reduviid bugs (Triatoma dimidiate) in neighbouring villages in Guatemala16.Microsatellites are highly variable, repeat regions of DNA that are useful when studying genetic mixing within insect populations at continental scales. For example, in two hoverfly species  between 2017 and 2019. We used traditional strain and haplotyping methods for FAW , as well as eight highly variable FAW microsatellite loci to determine genetic structure and mixing across countries.19, and the TpiE4/TpiI4 product was sequenced using Sanger sequencing. The expected strain discordance between the COIB and TpiE4 markers was observed in all countries, with the markers only reporting the same strain in 19% of samples . However, when using the TpiE4 marker, the proportion reported as the corn strain was similar across all five countries . Those larvae identified as corn strain by the COIB marker in Ghana (N = 45), Rwanda (N = 16), Sudan (N = 6) and Zambia (N = 8) were sequenced using Sanger sequencing to determine the haplotype. All larvae were identified as CSh4 suggesting very little genetic differentiation based on COIB haplotypes.The COIB marker was analysed using an enzyme based PCR assayIn all countries, the intronic TpiI4 marker identified both corn and rice strain FAW, with the corn strain (82–99%) being more common compared to the rice strain 1–18%)  when all individuals were considered together (Table index of association (rbarD) metric measures how likely individuals are to be the same at one particular locus in relation to other loci, and how this compares to other individuals, and can give a good indication of linkage between loci20. This metric was calculated and suggested a possible high chance of linkage between three pairs of loci   and each suggested that there was very little evidence of population differentiation across Africa24  suggesting high levels of population mixing , but low for others (e.g. Spf1592 and Spf789) , or between sampling years . The amova suggested, however, that FAW from each country were genetically different to FAW from other countries .Population differentiation was further analysed using an amova to determine if the genetic distance between individuals varies by country, location within country or sampling year  and LnPr(K) methods25. Based on Delta K there were three genetically distinct clusters in FAW , the most likely number of clusters was five  begins to plateau after K = 3 we propose that population structure of FAW in Africa is best described by three genetic clusters. To identify potential substructure in FAW from the four countries exhibiting evidence of genetic similarity , a separate analysis was performed in STRUCTURE. This identified 3 genetic clusters as the most likely scenario, based on both DeltaK and LnPr(K), and further confirmed high levels of mixing between the countries, with no strong evidence of substructure identified  and then using Discriminant Analysis Principal Components (DAPC). This approach determines the number of possible clusters by running successive K-means clustering, and selecting the most suitable cluster based on Bayesian Information Criterion and the number of PCs to keep was calculated to be 7 using the a-score26. This method identified three clusters as the best model based on BIC (BIC = 120.67) in FAW , whereas in previous population genetics studies using microsatellites in FAW from Paraguay and Brazil, no loci were found to be out of HWE18. Based on this range of alleles, previous work found genetic differentiation between northern and southern FAW populations across Brazil and Paraguay, as well as gene flow across all populations sampled8. This indicates that despite a recent bottleneck there is still sufficient genetic diversity in microsatellite regions to enable population genetic studies of FAW in Africa.Although FAW in Africa are likely to have undergone a population bottleneck at the time of invasion, the range of alleles for each locus identified in this study (3 to 13) was similar to that previously reported from Paraguay and Brazil (3 to 15)GST, Hedrick’s GST and Jost’s D). This indicates that these populations mix frequently, and no strong genetic structure is evident. This was supported by the amova which showed that most of the genetic variance was occurring between individuals.Populations from the six countries  did not show strong signs of population differentiation when using traditional measures 32, it is important to consider the implications of frequent, long-distance flights that seem to be occurring in FAW.This lack of population differentiation between countries provides evidence consistent with FAW undergoing long distance migratory flights in Africa creating a panmixia of populations. Additionally, there was no evidence of genetic differentiation between samples from different sampling locations within the same country, confirming that populations are mixing within countries. This has significant consequences for the evolution and spread of insecticide resistance, as resistance alleles can spread rapidly throughout each country and across Africa. This is an important finding as insecticide resistance (organophosphate and pyrethroid resistance) has already been reported in FAW in China, so is highly likely to be present in Africa28. Further investigation with microsatellites using clustering approaches show that whilst the countries included in this study are similar genetically , others are more differentiated (e.g. Sudan). We conclude from this that genetic mixing of FAW populations is occurring widely across Africa, however, there are some FAW possibly forming resident and partially segregated populations, as seen in parts of South America and the Caribbean7. Alternatively, the possibility that FAW have not been in Africa long enough to evolve population differentiation should also be considered.The evidence of panmixia contrasts with previous results based on the COIB and Tpi markers, which analysed FAW samples from across Africa and found evidence of genetic differentiation between geographically widespread countries9, or no clear pattern of division between populations28. Our study using microsatellites found that the two most genetically distinct populations are the most northerly and most southerly populations. African countries located further south  showed more similarities to each other compared with countries further north  . This pattern of genetic separation coincides with the known migratory routes of the congeneric African armyworm (Spodoptera exempta) in eastern Africa, which follow the movement of the dominant winds each season, typically moving moths towards the north-west from Kenya and northern Tanzania, and a more south-westerly movement across southern Africa from Malawi34. This is also aligned with the movement of the inter-tropical convergence zone (ITCZ), with the wind direction  being broadly south-easterly north of the equator and north-easterly south of the equator34. Based on the high levels of mixing between FAW populations alongside this evidence of some genetic structuring between northern and southern populations, it is hypothesised that FAW may also follow the movement of the dominant winds if they are migratory in Africa as, like many other insects, they rely on wind to support high-altitude long-distance flights36.Previous reports based on COIB and Tpi suggested a possible east–west divide between FAW populationsThis study highlights the benefits of using multiple approaches to study genetic diversity, with evidence presented for both widespread genetic mixing between populations alongside some segregation between countries. This is most likely due to a proportion of FAW adults undergoing long-distance migratory flights whilst the remaining FAW form more sedentary, resident populations. These results provide important evidence that genetic mixing between FAW populations throughout Africa may be more common than previously reported. This has important consequences for FAW management when considering factors such as the spread of insecticide resistance and crop infestations across borders.FAW larvae for sequencing with COIB:TpiI4:TpiE4 were collected from Ghana , Malawi , Rwanda , Sudan  and Zambia  and stored in ethanol. FAW larvae for microsatellite analysis were collected from Ghana , Kenya , Malawi , Rwanda , Sudan , and Zambia  and stored in ethanol. Full collection details are provided in Supplementary Table DNA was extracted from samples following the standard protocol for tissue in the Qiagen DNeasy Blood and Tissue kit. DNA was stored in buffer AE at − 20 °C. The protocol was altered slightly for extracting DNA from larvae collected in Sudan, these modifications were 200 μl ATL and an additional 200 μl 1 × SSC before incubation and the DNeasy Spin Column was centrifuged at 13,000 RPM.27) and TpiI4/TpiE4 . Cycling parameters for both COIB and TpiI4/TpiE4 were 94 °C 10 min, 33 cycles of 94 °C 1 min, 55 °C 1 min, 72 °C 1 min and then a final extension of 72 °C for 5 min. Following COIB amplification, the product was incubated at 37 °C for 2 h with 1 µl EcoRV restriction enzyme and 2 µl NEBuffer to determine FAW strain. EcoRV cuts the amplicon at position 1182 bp if the sample is from the rice strain resulting in two visible bands, and does not cut for the corn strain resulting in one larger band when the product is run on a gel electrophoresis (Table 1164A1287), corn h2 (A1164G1287), corn h3 (G1164A1287), corn h4 (G1164G1287) and rice (T1164A1287)27 and unidirectional Sanger sequencing was used to verify the COIB corn haplotypes. Sequencing reactions contained 0.75 µL BigDye® Reaction Mix, 1.70 µL 5 × BigDye® Sequencing Buffer, 0.32 µL 10 µM Forward primer, 5–20 ng template DNA and H2O to supplement the reaction to 10 µL. The sequencing reaction was preincubated for 1 min at 96° C followed by 25 cycles of: 10 s at 96° C; 5 s at 50° C; 4 min at 60° C. Excess incorporated dye-terminators were removed using EDTA/Ethanol precipitation before resuspending in 13 µL Hi-Di® formamide and capillary gel electrophoresis on an ABI 3500 Genetic Analyzer. Strain identification was carried out using the Tpi marker by Sangar sequencing following the same protocol as for COIB based on nucleotide variation in exon-4 (TpiE4), where the corn strain has base C183, the R strain has base T183 and hybrids  have C/T18327. Tpi Intron 4 (TpiI4) was used to determine TpiI4 haplotypes based on 18 previously recorded highly variable positions37. For sequencing analysis, raw sequences were assembled and aligned using ClustalW in BioEdit39. Statistical analysis on strain and haplotype distributions for TpiE4 were carried out in R using a Poisson GLM followed by a Chi2 test using the amova function. Based on the TpiI4 haplotypes, an amova was carried out using the POPPR package as this gave details of the variance explained within and between samples and populations , from which a P value was calculated using a randomization test with 999 permutations. The genetic distance computed for the amova was also used for Principal Components Analysis (PCA) using the prcomp function in R.DNA was amplified for strain identification using COIB  or 0.5 µl PCR product . Genotyping results were viewed on Thermo Fisher Connect™. The threshold for successful amplification was > 100RFU, and for heterozygotes the minor peak was > 50% of the major peak. Alleles were called based on size measurements and peaks determined to be the same allele if size measurements were within 0.5 nucleotides of each other .40. Hardy–Weinberg equilibrium was tested using the PEGAS R package41. The frequency of null alleles was determined using the Chakraborty et al. (1994) formula through the POPGENREPORT R package42. Heterozygosity and F-statistics were calculated using the HIERFSTAT package43. Genetic differentiation was measured using the MMOD package (Gst and Jost’s D)44. Linkage disequilibrium was calculated using an association index using the POPPR package45 and by composite linkage disequilbrium using GenePop (v 4.7)46. An Analysis of Molecular Variance (AMOVA) to determine population differentiation based on genetic distance was carried out using the adonis2 function from the VEGAN package in R for all variables47 and country was looked at using an amova with the POPPR package as this gave details of the variance explained within and between samples and populations45, from which a P value was calculated using a randomization test with 999 permutations. The genetic distance computed for the amova was also used for Principal Components Analysis (PCA) using the prcomp function in R. To identify population clusters, a Discriminant Analysis of Principle Components (DAPC) was carried out after clusters were identified de novo  using the find.clusters function in the ADEGENET package in R26. Optimum number of K was selected based on BIC 40. HardySupplementary Information.


Ancient genomes uncover multiple migratory pulses to Japan with the spread of wet rice farming and the rise of the state. Prehistoric Japan underwent rapid transformations in the past 3000 years, first from foraging to wet rice farming and then to state formation. A long-standing hypothesis posits that mainland Japanese populations derive dual ancestry from indigenous Jomon hunter-gatherer-fishers and succeeding Yayoi farmers. However, the genomic impact of agricultural migration and subsequent sociocultural changes remains unclear. We report 12 ancient Japanese genomes from pre- and postfarming periods. Our analysis finds that the Jomon maintained a small effective population size of ~1000 over several millennia, with a deep divergence from continental populations dated to 20,000 to 15,000 years ago, a period that saw the insularization of Japan through rising sea levels. Rice cultivation was introduced by people with Northeast Asian ancestry. Unexpectedly, we identify a later influx of East Asian ancestry during the imperial Kofun period. These three ancestral components continue to characterize present-day populations, supporting a tripartite model of Japanese genomic origins. The Japanese archipelago has been occupied by humans for at least 38,000 years. However, its most radical cultural transformations have only occurred within the past 3000 years, during which time its inhabitants quickly transitioned from foraging to widespread rice farming to a technologically advanced imperial state . High levels of endogenous human DNA were preserved in 12 of these samples , which wf3  Japanese populations using the statistic ; Mbuti) . Our resWe further investigated the genome-wide autosomal affinities of our ancient Japanese individuals to continental populations using principal components analysis (PCA). We projected ancient individuals onto the genetic variation of present-day populations in the SGDP dataset from South and Central Asia, Southeast and East Asia, and Siberia  . These show that all East Asian individuals in our dataset since the beginning of the Jomon period have a higher affinity to Jomon than the earlier diverged Hoabinhian but have a lower affinity in comparison to DevilsCave_N; this supports the inferred phylogeny of three distinct hunter-gatherer lineages in East Eurasia, rather than a previously proposed model in which Jomon is a mixture of Hoabinhian and East Asian-related lineages  (The separation of the Jomon from other populations  supportsable S5] . These rWe apply population genetic modeling to estimate the timing of the appearance of the Jomon lineage. Our approach makes use of genome-wide patterns of runs of homozygosity (ROHs) to identify the demographic scenario that best fits the ROH spectrum observed in our oldest and highest coverage sample, JpKa6904 (see note S2). The distribution of ROH tracts reflects the effective population size and the time to the most recent common ancestor between two copies of haplotypes within an individual  (33). Thf4 . Among the Upper Paleolithic individuals tested, only Yana_UP is significantly closer to Jomon than Han, Dai, or Japanese, respectively (Z > 3.366). This affinity is still detectable even if we replace these reference populations with the other Southeast and East Asians (table S6), supporting gene flow between the ancestors of Jomon and Ancient North Siberians, a population widespread in North Eurasia before the LGM , where i and j are any pairs of the three Jomon groups (fig. S13). These Jomon individuals similarly do not display any diversity in genetic affinity to continental populations when grouped by geography  (fig. S14). The only observable difference within the Jomon individuals is a slightly higher affinity between sites located on Honshu, implying an insular effect with restricted gene flow between Honshu and other islands (fig. S15). Overall, these results show limited spatial and temporal genetic variation within the Jomon population, supporting the idea of near-complete isolation from the rest of Asia for thousands of years.We lastly investigated potential temporal and spatial variation within the Jomon population. Three temporal groups, defined by the Initial, Early, and Middle-Late-Final stages of the Jomon period, show similar levels of shared genetic drift with ancient and present-day continental populations, implying little or no genetic impact from outside of the archipelago over these subperiods (P = 0.00003), as opposed to a morphological assessment that groups these two individuals as a part of the Jomon lineage  (Z > 3.0): those from the West Liao River basin in Northeast China (WLR_BA_o and HMMH_MN), Baikal , and Northeast Siberia (Ekven_IA). This affinity is not observable in two more individuals  who came from the same archaeological site as the other Bronze Age West Liao River individual (WLR_BA_o)  werP > 0.05) for three of these: Baikal hunter-gatherers and the West Liao Middle Neolithic or Bronze Age individuals with a high level of Amur River ancestry . The admixture model was confidently supported  that the level of Jomon ancestry is comparable between these two Yayoi individuals (Z = 1.309). These results imply an approximately equal ratio of indigenous hunter-gatherer and migrant contribution to the Yayoi communities associated with this northwestern Kyushu site. This parity is particularly notable when compared to agricultural migrations in western Eurasia, where minimal hunter-gatherer contribution is observed in many regions, including the archipelago of Britain and Ireland  into the Liaodong Peninsula (northwestern part of the Korean Peninsula) and then reaches the archipelago via the Korean Peninsula . Thus, the Kofun are genetically distinct from the Yayoi in terms of their ancestral components, as supported from our outgroup f3, PCA, and ADMIXTURE clusters  . This admixture model was confidently supported with P > 0.05 for only 5 of 59 populations tested. We then applied qpAdm to quantify the genetic contribution from Yayoi and each of these sources in turn (table S9). The two-way admixture model was subsequently rejected in an additional two populations due to a lack of support from different reference sets. The remaining three populations  show 20 to 30% contributions to the Kofun (fig. S19). These three all share strong genetic drift (highlighted by the green square in fig. S17), characterized with a major component of broadly East Asian ancestry in their ADMIXTURE profiles (fig. S6). To further screen the source of additional ancestry in the Kofun individuals, we tested a three-way admixture by replacing the Yayoi ancestry with Jomon and Northeast Asian ancestry (table S10). Only Han were successfully modeled as a source of ancestry in the model . Despite not showing statistically significant gene flow with Yayoi to the exclusion of Jomon (Z = 2.487) . Only one candidate was found to better fit a two-way mixture for Kofun, a population of the Late Bronze Age and Iron Age individuals from the Yellow River basin (YR_LBIA)  and the Kofun (1:2.5) . Second,f4 (fig. S21). Although some of the ancient populations exhibit higher affinity to Kofun than Japanese, none of them is supported by qpAdm as an additional source of ancestry present in the Kofun (see note S3 and table S15). Unexpectedly, no ancient or modern population shows additional gene flow with the present-day Japanese to the exclusion of the Kofun. Our admixture modeling further confirms that the present-day Japanese population is sufficiently explained by Kofun ancestry without increasing Jomon or Yayoi ancestry or without introducing an additional ancestor represented by present-day Southeast or East Asians or Siberians (table S16). We also find that the modern Japanese population has the same set of ancestral components as the three-way admixture in the Kofun individuals (table S17), with a slightly increased level of East Asian ancestry in the modern Japanese compared with the Kofun individuals sampled (fig. S22). This suggests a certain level of genetic continuity but not absolute. A strict model of continuity between the Kofun and the modern Japanese population  is rejected (table S18) (P = 0.769). These results suggest that the genetic profile of three major ancestral components established by the state formation period has become a foundation for present-day Japanese populations, as also supported from dental and nonmetrical cranial traits   and 5. TOur data provide evidence of a tri-ancestry structure for present-day Japanese populations , refininThe lineage ancestral to Jomon is proposed to have originated in Southeast Asia with a deep divergence from other ancient and present-day East Asians . The JapThe spread of agriculture is often marked by population replacement, as documented in the Neolithic transition throughout most of Europe, with only minimal contributions from hunter-gatherer populations observed in many regions . This imThe continental component inherited by the Yayoi is best represented in our dataset by the Middle Neolithic and Bronze Age individuals from the West Liao River basin with a high level of Amur River ancestry   from six archaeological sites across the west and central parts of the Japanese archipelago were sampled. Detailed information of each site is provided in note S1, including archaeological contexts and radiocarbon dates that are taken from OxCal4.4 , before all libraries were merged into one bam per individual. Two final processing steps were carried out on the merged files: Indels were realigned using Genome Analysis Toolkit (GATK) version 3.7-0  . The kinship estimation for all ancient individuals was done using “Relationship Estimation from Ancient DNA” (READ) . We genotyped all ancient individuals for the biallelic SNP sites present on two different reference panels in which they were merged to the following: (i) the Human Origin Array (HOA) consisting of 594,896 SNP sites for 1963 modern, ancient, and reference genomes (n = 112), filtered for transversions and global minor allele frequencies of 1%, with the options of “killr2: YES,” “r2thresh: 0.2,” “numoutlieriter: 0,” “lsqproject: YES,” and “autoshrink: YES.” With the exception of “Yayoi_1,” we included ancient individuals with a coverage of at least 100,000 SNPs.PCA was conducted using the smartpca (v16000) from the EIGENSOFT package (v7.2.0) (n = 189) and present-day populations from the HOA . SNPs were pruned for sites in linkage disequilibrium using PLINK v1.90b4.4 (r2 threshold of 0.2 (--indep-pairwise 50 5 0.2), leaving 186,856 SNPs for analysis. We ran 10 replicates with random seeds for the number of clusters (K) from 2 to 12 and chose the run with the minimum cross-validation error for plotting.We used ADMIXTURE v.1.3.0 (Maximum likelihood trees were inferred under various admixture models using TreeMix (v1.13) (f statistics using qp3Pop (v300) and qpDstat (v662) with the f4 mode in the AdmixTools v6.0 package  and split time (T) of the Jomon lineage, we applied coalescent simulations of the Out-of-Africa model . Fitting of the model was measured as an approximate Bayes factor (aBF) , Kusunda (n = 2), Papuan (n = 14), Dai (n = 4), Ami (n = 2), Naxi (n = 3) (n = 1) (n = 1) (n = 1) (To model admixture events in the prehistory of Japan, we applied qpWave v600 and qpAdm v1000 in the AdmixTools v6.0 package to the merged datasets of ancient East Eurasians and the SGDP populations (We used DATES v753 (


Dioon holmgrenii De Luca, Sabato et Vázq.Torres is an endangered species; it is endemic and its distribution is restricted to the biogeographic province of the Mexican Pacific Coast. The aim of this work was to determine the diversity and genetic structure of nine populations. The genetic diversity parameters and Wright’s F statistics were determined with six microsatellite loci. The genetic structure was determined by using the Structure software and by a discriminant analysis. The genetic diversity of the populations was high. The proportion of polymorphic loci was 0.89, the observed heterogeneity was higher (Ho = 0.62 to 0.98) than expected (He = 0.48 to 0.78), and the fixation index was negative (IF = −0.091 to −0.601). Heterozygous deficiency (FIT = 0.071) was found at the species level and heterozygotes excess (FIS = −0.287) at the population level. The genetic differentiation between populations was high (FST = 0.287), with the number of migrants less than one. Three groups of populations were differentiated, and the variation within populations, between populations, and between groups was: 65.5, 26.3, and 8.2%, respectively. Multiple factors explain the high genetic diversity, while the genetic structure is due to geographic barriers. Community reserves are urgent in at least one most diverse population of each group. Cycads are gymnosperms that originated possibly in the Carboniferous and Permian periods of the Paleozoic era, between 345 to 280 million years ago ,2,3, whiCurrently, there are two families—Cycadaceae and Zamiaceae—, 10 genera, and 356 species in the world, distributed mainly in subtropical areas and, to a lesser extent in equatorial habitats with low temperature and humidity ,13. MexiDioon is a neotropical genus with 16 species, with 15 of them endemic to Mexico and one to Honduras; except for the non-evaluated species, all of them are threatened . As. AsST vae (0.04) , Cycas g (0.054) , Zamia a (0.897) . The posulations .D. holmgrenii consists of depressions and elevations with significant unevenness , as well as their proximity to the distribution area of D. caputoi and D. planifolium that mixed with D. holmgrenii in the late Pleistocene era [On the other hand, clustering between ulations . In thiscene era , supportD. holmgrenii populations can be related to life history and biogeographic history [D. holmgrenii is restricted to the Xolapa tectonostratigraphic terranes. The Xolapa terrain originated between the Late Cretaceous and Paleocene (99 to 54 Million years), but joined the Mixtec and Zapotec tectonic terrains between the Eocene and Middle Miocene (54 to 16 million years) [Dioon genus originated in the late Paleocene and early Eocene (~56 million years) in the north of the American continent and migrated to the south [Dioon expanded throughout Mexico, where the change from humid to xeric habitat caused the greatest speciation during the Oligocene and late Miocene [D. holmgrenii belongs, expanded towards the Pacific coast and it diversified due to the aridification process that occurred during the Miocene (23 to 5 million years) [D. holmgrenii has as ancestors to D. planifolium Salas-Mor., Chemnick & T.J. Greg., D. argenteum T.J. Greg., Chemnick, Salas-Mor. & Vovides, D. purpusii, D. caputoi, and D. califanoi De Luca & Sabato [Dioon first populated the Mixtec and Zapotec lands and later the union of the Xolapa terrain with these lands; the Dioon genus dispersed in the Xolapa terrain, where the speciation of D. holmgrenii possibly occurred, to the limit of the Mixtec land. All the above indicates that colonization of the Dioon genus occurred from west to east, and this fact reinforces the hypothesis of precursor (west of Río Verde) and derived (east of Río Verde) populations of D. holmgrenii.The distribution of diversity among  history ,43. In a history . In thisn years) . Althougn years) ,83,84, the south . Through Miocene . The sou Miocene ,17, to wPossibly, the recent colonization of the eastern Río Verde populations and a slight associated founder effect , could eDioon holmgrenii has evolutionary potential to adapt to changing climate conditions due to its high genetic diversity, which allows its sustainable management [nagement . The resnagement . Likewisnagement  or meteonagement .D. holmgrenii populations [This implies to include all or most of the populations in the conservation plans. The distribution of the genetic diversity into three groups allows to guide conservation efforts and at least one population from each group should be integrated into an in situ conservation program. Sites with the presence of cycads must be declared or included in the Natural Protected Areas (NPA) lists ,87,88. Nulations ,88,90.D. holmgrenii populations it is necessary to implement UMAs aimed at reproducing, marketing, and planting plants in deforested sites. Through the UMAs, the owners or possessors can obtain benefits to avoid livestock and agriculture in the lands with the presence of this species. The foregoing is of vital importance in populations of the group three, where habitat fragmentation due to land use change is greater . However, the process has been delayed for several years, under the argument that the species is listed as “in danger of extinction” in the NOM-059-SEMARNAT-2010  and in tD. holmgrenii  were collected in nine populations of lmgrenii  located lmgrenii . The min® gDNA Plant DNA extraction kit from Invitrogen , and using a DNA extraction robot . The quality (absorbance at 260/280 nm) and the DNA concentration were measured with an ultra-low volume spectrometer .DNA extraction was performed on 100 mg of tissue with the commercial ChargeSwitchD. edule [Zamia integrifolia L.f. [−1 with HPLC water. PCR (polymerase chain reaction) was performed in a thermal cycler . All PCR products were verified on 2% agarose gels. Denaturation, alignment, and extension temperatures were adjusted according to the results observed in the agarose gels.Six nuclear microsatellite primers developed for D. edule  and 10 dlia L.f.  were tes2, 0.5 μL of 10 mM DNTP, 5 μL of 10 pM of each forward primer pair (2.5 μL each), 5 μL of 10 pM of each reverse primer pair (2.5 μL of each), 0.25 μL of 5 units of Taq DNA polymerase  and 2.0 μL of template DNA (20 ng μL−1) and 6.65 μL of HPLC water. The multiplex PCR program consisted of: 5 min of initial denaturation at 94 °C; followed by 35 cycles of 30 s of denaturation at 94 °C; one minute of alignment at 58.1 (group 1), 57.3 (group 2) or 58.9 °C (group 3); 2 min extension at 72 °C, with a 15 min final extension at 72 °C.The microsatellite amplification of all the samples was carried out by multiplex PCR, where the primers with similar alignment temperatures and with different size (bp) were grouped into three groups . The pri®, Hercules, CA, USA) and then frozen at −20 °C for three minutes before starting electrophoresis. The electrophoresis of the PCR products was carried out on a DNA sequencer . Each electrophoresis reaction consisted of 2 μL of PCR product, 0.25 μL of Size Standard LIZ®500, and 7.75 μL of formamide. The electropherograms were analyzed with the GeneMapper® software version 4.0  [The multiplex PCR products were denatured at 96 °C for three minutes in a thermal cycler  , to consGenetic diversity parameters such as the percentage of polymorphic loci (PLP), average number of alleles (NA), effective number of alleles (EA), expected heterozygosity (He), observed heterozygosity (Ho), and fixation index (F) for each population were determined with the InfoGene software version 2016 . A variaThe numbers of total (NTA) common (NCA), rare (NRA), and exclusive (NAE) alleles were identified for each population. Alleles were called rare when their frequency was less than 0.05, and common when their frequency was equal to or higher than 0.05 . The excIS), between populations (FST) and considering all populations as one (FIT). The number of migrants per generation (Nm) was calculated with Nm = (1 − FST)/(4 FST) [ST values between pairs of populations were calculated. The relationship between Nei’s genetic distances and geographic distances was determined with the Mantel test [The F statistics  were est/(4 FST) . Additio/(4 FST) , geograptel test  using thtel test .In order to evaluate the genetic structure between populations, the Structure V.2.4 software was used . This coDioon holmgrenii has a high genetic diversity. The high diversity of D. holmgrenii is the result of the combined effect of its interbreeding system, longevity, variety, and relative extensive geographic distribution, as well as the evolutionary history that it shares with other species. The genetic structure is defined by its evolutionary history and biogeographic history; likewise, the Río Verde and the mountains act as a geographical barrier for the differentiation of population groups. Knowledge of diversity and genetic structure allows creating the in situ conservation strategies for D. holmgrenii.


Metaphire vulgaris (a member of the Clitellata class) is widely distributed across China, and has important ecological functions and medicinal value. However, investigations into its genetic diversity and differentiation are scarce. Consequently, we evaluated the genetic diversity of five populations of M. vulgaris  in Yancheng, China via the mitochondrial COI gene and the novel microsatellites developed there. A total of nine haplotypes were obtained by sequencing the mitochondrial COI gene, among which NYYZ and QDDY populations had the greatest number of haplotypes (nh = 5). Further, the nucleotide diversity ranged from 0.00437 to 0.1243. The neighbor-joining trees and the TCS network of haplotypes indicated that earthworm populations within close geographical range were not genetically isolated at these small scale distances. Results of the identification of microsatellite molecular markers revealed that the allele number in 12 microsatellite loci ranged from 4 to 13. The observed heterozygosity ranged from 0.151 to 0.644, whereas the expected heterozygosity ranged from 0.213 to 0.847. The polymorphism data content of most sites was >0.5, which indicated that the designed sites had high polymorphism. Structural analysis results indicated that GM, HD, and NYYZ had similar genetic structures across the five populations. The Nei’s genetic distance between HD and NYYZ populations was the smallest (Ds = 0.0624), whereas that between HD and QDY populations was the largest (Ds = 0.2364). The UPGMA tree showed that HD were initially grouped with NYYZ, followed by GM, and then with QDDY. Furthermore, cross-species amplification tests were conducted for Metaphire guillelmi, which indicated that the presented markers were usable for this species. This study comprised a preliminary study on the genetic diversity of M. vulgaris, which provides basic data for future investigations into this species.The earthworm species  Earthworms have key roles in myriad soil processes, including soil turnover, aeration, and drainage, and the breakdown and incorporation of organic matter . StudiesAmynthas corticis)  and microsatellites are extensively employed for species identification, population genetic diversity, and genetic differentiation . mtDNA iorticis) . Furtherorticis) .Metaphire vulgaris using RAD sequencing technologies. M. vulgaris belongs to the genus Metaphire of the family Megascolecidae, which can be found in many provinces across China, including Jiangsu, Shanghai, Zhejiang, and Guizhou  in the laboratory. The extracted DNA was stored at −20°C for an extended duration.All earthworm samples were selected from five sites in Yancheng City, Jiangsu Province, China and grouped according to their geographic origin . A totalhe lumen . Once thM. vulgaris (KJ137279.1), Metaphire guillelmi (KT429017.1), and Metaphire californica (KP688581.1) from NCBI . Amplifications were performed in 25 μl reaction volumes containing 12.5 μl of 2 × Taq Master Mix (Dye Plus), 1 μl of genomic DNA, 0.5 μl of each primer, and 10.5 μl of deionized water. The PCR procedure was as follows: an initial denaturation step at 95°C for 5 min, followed by 32 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, with a final extension at 72°C for 5 min. After being detected by agarose gel electrophoresis, the PCR products were sent to the TSINGKE Biotech Company  for sequencing. Species identification was performed via DNA barcoding by sequencing a fragment of the COI gene. Ultimately, we obtained 78 M. vulgaris and 22 M. guillelmi earthworms.The primers used for gene amplification were designed with reference to the entire mtDNA sequences of M. vulgaris specimen was used for RAD-seq, where RAD library construction and Illumina sequencing were conducted by Novogene Bioinformatics Technology Co. Ltd.  following the standard protocol. Approximately 1.287 G bases of raw reads were obtained from the RAD library, with average Q30 and GC contents of 92.41 and 43.51%, respectively. Subsequent to the filtering and assembly of the raw reads, we obtained 33,069 contigs, with average contig lengths of 346 bp. A total of 1975 microsatellites were obtained that were suitable for the design of primers. The primers were designed using the primer 3.0 subprogram of the SR search software . A total of 20 pairs of primers were randomly designed and employed to amplify the DNA templates of 3 M. vulgaris individuals, of which 12 pairs of primers produced clean products. These primers were labeled with fluorescent dye 5′ 6-FAM, 5′ HEX, or 5′ TAMRA for randomly testing the amplification in 24 M. vulgaris individuals. Finally, 12 microsatellite loci with high polymorphisms and 12 corresponding primers were successfully screened  were sampled for cross-amplification analysis. All PCR products were sent to the SINGKE Biotech Company  for genotyping using an ANI 3730 Genetic Analyzer (Applied Biosystem).Except for different primers, the reaction system and reaction conditions were as above. The PCR products were also checked using a 1% agarose gel electrophoresis method. To validate the developed microsatellites in other M. guillelmi as an outgroup. The confidence levels at nodes after 1000 repetitions employed the Bootstrap method , observed heterozygosity (HO), expected heterozygosity (HE), polymorphism information content (PIC) values, and Hardy–Weinberg equilibrium test for each locus. Arlequin 3.0 software  per locus. Through an AMOVA analysis using Arlequin 3.0 software, the distribution patterns of genetic diversity were compared. Popgene 3.2 software was employed to calculate the genetic distance (DS), and construct a phylogenetic tree via UPGMA. An analysis of the population genetic structure was performed with Structure 2.3.4 software . The average frequencies of T, C, A, and G were 32.6, 22.3, 27.9, and 17.2%, respectively. The A + T contents (60.5%) were higher than the C + G contents (39.5%). The nucleotide diversity (π), nh, and Hd are presented in M. guillelmi haplotype. There were nine haplotypes within the five populations of M. vulgaris, among two haplotypes (Hap 1 and Hap 2) were found in four populations, two haplotypes (Hap 3 and Hap 9) were found in two populations, and the remaining five haplotypes were designated as “private haplotypes”  were obtained, of which 78 were lotypes” .M. guillelmi outgroup was obviously different from the M. vulgaris group as one branch, and five populations of M. vulgaris were divided into two large branches. A total of nine haplotypes were distributed between the two branches, which was similar to the aggregation of the overall TCS network , a genetic diversity assessment of 73 individuals in 5 populations of M. vulgaris, and cross-species amplification of polymorphic microsatellite markers from M. guillelmi, showed that most microsatellite loci could be successfully amplified , and from 0.213 to 0.847 , respectively. The average PIC was 0.571, while the highest was 0.823 for the Mv07 locus, and the lowest was 0.200 for the Mv08 locus.An innovative design of the 12 microsatellite loci and their corresponding 12 pairs of primers  to 5.250 (QDDY), where the average allele number was 4.42 (E ranged from between 0.504 (QDY) and 0.633 (HD). The average of the observed and expected heterozygosity was 0.426 and 0.581, respectively. The PIC was from between 0.446 and 0.546, as the lowest in the QDY population, and the highest in the HD population, with an average of 0.504. The best K (K = 3) values were obtained from the Structure Harvester (see text footnote 1). The genetic structure of the population was analyzed using Structure 2.3.4 software, setting K = 3, that is, the five populations could be divided into three genetic groups  (The Nwas 4.42 . The rand green) . All fivF-statistics (IS), genetic differentiation (FST), and inbreeding coefficient in the overall populations (FIT). The FIS ranged from −0.107 (Mv11) to 0.466 (Mv09), with an average value of 0.255. The FST ranged from 0.011 (Mv07) to 0.164 (Mv03), with an average value of 0.085. The FIT ranged from −0.018 (Mv11) to 0.522 (Mv12), with an average of 0.318. From these three indices, it was observed that there was a certain inbreeding phenomenon; however, the genetic differentiation coefficient was small, which indicated that the degree of genetic differentiation of the population was not high.atistics  were estDs) of Nei was calculated by Popgene v3.2 software. The results indicated that the genetic distance between the HD and NYYZ populations was the smallest (Ds = 0.0624) and the genetic distance between the HD and QDY populations was the largest (Ds = 0.2364)  . The gen 0.2364)  revealedith QDDY .Lumbricus rubellus, 2006; Aporrectodea longa (Ude), 2012; Lumbricus terrestris, 2016)  . At presymorphic . Cross-sumbricus .M. vulgaris in Yancheng City. In the present study, the QDY population had the lowest genetic diversity and the HD population had the highest. The COI gene fragment species produced 9 haplotypes in 78 samples of the M. vulgaris population with a nucleotide diversity of π = 0.01088 ± 0.00633. This was lower than Amynthas triastriatus in China by Dong Yan (π = 0.0309)  studied by Dima Souleman, the population of M. vulgaris showed a moderate genetic diversity. The results based on mitochondrial COI gene and microsatellites showed that the genetic diversity of QDY and GM populations was low, whereas that of the HD population was the highest. However, the evaluation of genetic diversity with different molecular markers may give different results  , which wectively . The NA,rrestris  (HO: fro results . The conis study .DS) was calculated to evaluate the level of differentiation between populations. The DS values between QDY and any other populations was greater than 0.16, which implied that they possessed medium genetic differentiation. The FST = 0.09349 (P < 0.01) based on microsatellite markers also indicated that genetic differentiation had occurred between the five populations, forming different genetic clusters. According to Bayesian analysis in Structure 2.3.4 software, the five populations of M. vulgaris were divided into three genetic clusters. The AMOVA results revealed that the source of genetic differences emerged primarily from within the populations. However, the phylogenetic NJ tree and network based on the species haplotypes of the mitochondrial gene showed no obvious lineage structure. The results of population differentiation based on mitochondrial COI gene and microsatellite molecular markers were inconsistent, which may have been because microsatellites are nuclear genes, while COI are cytoplasmic genes, and the two have different inheritance patterns  can be found below: NCBI GenBank, COI1 accession numbers: Ethical review and approval was not required for the animal study because Earthworms are common soil animals and are not listed in IUCN Red List of Threatened Species.HL: conception and design. NX: development of methodology. ZQ: acquisition of data. YF, NX, ZQ, and JC: analysis and interpretation of data. YF, HR, and HL: writing, review, and/or revision of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The global increase in the cultivation of native wild plants has raised concerns regarding potential risks associated with translocating genetic lineages beyond their natural range. This study aimed to investigate whether agricultural cultivation of neo-crops (a) accounts for the levels of genetic diversity present in wild populations, and whether (b) cultivated populations are genetically divergent from wild populations and thus pose a potential threat to wild genetic diversity.The Cape Floristic Region (CFR), located along the southern Cape of South Africa.Cyclopia Vent. (Fabaceae), a CFR endemic neo-crop cultivated for the production of a herbal infusion referred to as Honeybush tea. Wild and cultivated populations for three of three widely cultivated Honeybush species  were screened. Genetic diversity and differentiation were measured and compared between wild and cultivated groups.High Resolution Melt analysis (HRM) coupled with Sanger sequencing was used to screen three non-coding chloroplast DNA loci in Across all asseccions, a total of 17 haplotypes were detected, four of which were shared between wild and cultivated populations, while the remaining 13 were only detected in wild populations. Genetic diversity and differentiation was significantly higher in wild populations than in cultivated populations.If no guidelines exist to facilitate the introduction of native wild plant taxa to a cultivated setting, wild genetic diversity patterns are likely to be compromised by cultivated populations. In the case presented here, cultivation represents a genetic bottleneck, failing to account for rare haplotypes, and may have disrupted species boundaries by initiating interspecific hybridization. More empirical work is required to evaluate the extent to which neo-crop cultivation poses a risk to wild genetic resources in the CFR and globally. Commercial trade of wild crop and medicinal plants relies predominantly on material sourced from wild populations. However, consumer demand for ’natural products’ and products manufactured from renewable resources has promoted an increase in the domestication and cultivation of wild crop species , referreCarex caryophyllea Latourr (Cyperaceae), Phragmites australis (Cav.) Steud, Gene flow from cultivated to wild populations is particularly common in traditional crop systems  and can Catha edulis [Vahl] Forssk. ex Endl., Celastraceae), genotypes sourced from wild populations in Ethiopia were used to establish cultivated populations in Yemen and Kenya, yet both regions support genetically distinct natural qat populations that could have formed the basis for local cultivation  contained a combination of haplotypes sourced from multiple, geographically separate, wild populations, lacking the phylogeographic structuring present in wild populations   R.Dahlgren, Fabaceae) (Cyclopia Vent (Fabaceae)—have been raised   and possabaceae) . As a ren raised .Cyclopia species and involved the selection of individuals with commercially favorable traits , and cross pollination experiments . This study explores the prediction that cultivated Cyclopia populations will fail to reflect the spatial distribution and diversity of chloroplast lineages present in the wild. Thus, the current state of honeybush cultivation, and likely neo-crop cultivation in general, may represent a genetic risk to the integrity of wild populations occurring in close proximity to cultivated populations.This study is the first to describe the levels of genetic diversity among wild and cultivated populations of an endemic crop plant originating from the CFR, focusing specifically on three widely cultivated sis HRM,  coupled C. intermedia E.Mey, C. subternata Vogel., and C. longifolia Vogel.) represent the most widely cultivated Honeybush taxa, cultivated in the Western Cape and Eastern Cape provinces of South Africa (C. intermedia), a widespread coastal lowland obligate seeder (C. subternata ) and a critically endangered Eastern Cape endemic, riparian specialist with a mixed post fire response of facultative seeding and sprouting (C. longifolia). Since life history traits and range size impact a species’ demographic history  were subject of recent phylogeographic studies  were amplified using Cyclopia specific primers and subsequently screened for nucleotide variation via HRM curve analysis. Samples were run in duplicates and HRM clustering was conducted on a single population basis following the recommendations of atpI-atpH intergenic spacer and 36 individuals for the ndhA intron were PCR amplified using the reverse primers and following PCR protocols of . Each base-call was assigned a quality score using the PHRED base-calling program (https://doi.org/10.6084/m9.figshare.12624620.v1) was then used to assign each sample its respective haplotype identity based on HRM clustering.Sequences were assembled using  program . Sequenc program  and visu program , and werR (V 3.5.1) (TCS (v 1.2.1)  between wild and cultivated populations were tested via Mantel tests (Hs.test function implemented in the adegenet (v 2.1.1) library, . This distance measure treats alignment gaps as evolutionary events and all gaps were reduced to a single base pair prior to analysis. Support for population clustering was assessed via a bootstrap analysis with 9999 replicates, implemented using the aboot function implemented in poppr.All analyses were performed in V 3.5.1) . The genV 3.5.1)  construcv 1.2.1) . As TCS el tests ; using Hlibrary, . Geneticlibrary,  using th library . Three a library  and G””s library , and a m library , using t library . Populat library , calculaatpI-atpH intergenic spacer and 339 bp from the ndhA intron) with an overall GC content of 28.1%. The alignment contained 22 polymorphic sites including nine transitions, ten transversions, and three indels (two of 7 bp and one of 71 bp). Haplotype frequency within populations and nucleotide variation among haplotypes are summarized in Of the 504 samples screened for haplotype variation, seven (six wild and one cultivated) failed to PCR amplify despite repeated efforts and the final dataset consisted of 497 samples. High Resolution Melt analysis with haplotype confirmation by sequencing revealed 17 cpDNA haplotypes with 100% specificity for all three loci . The final concatenated dataset consisted of 794 bp  and cultivated C. subternata populations, but were not present in any wild C. subternata populations screened.The SP network  revealedCyclopia populations, based on pairwise population genetic distance resulted in weak grouping of species. Cultivated populations, however, exhibited little differentiation and generally clustered together based on species . The remaining wild C. subternata populations were more divergent and did not cluster with other C. subternata populations. The C. intermedia and C. subternata populations sampled from Garcia’s pass (GAR) exhibited no genetic differentiation (both fixed for haplotype F). In general, wild C. intermedia populations tended to be genetically distinct, supporting unique haplotypes and formed no clear clusters in the Neighbor Joining population tree .Clustering of  species . Similarle group . Wild C.ion tree . CultivaC. subternata and C. intermedia, genetic structuring was detected in wild populations, with 60.1% and 83.3% of genetic variation detected among populations for the two species, respectively . In contrast, no structuring was detected in cultivated populations of these species, with 4.3% and 1.7% of variation structured within cultivated populations for C. intermedia and C. subternata, respectively . No genetic structuring was found for wild or cultivated C. longifolia populations, as all populations shared the same common haplotype and only two rare haplotypes , but no differences in gene diversity was detected between wild and cultivated C. longifolia populations. Mean genetic differentiation  was higher in all wild populations than in cultivated populations. All population differentiation and diversity measures are summarized in In the cases of N and M,  were detThis study set out to explore haplotype diversity patterns in wild and cultivated populations of Honeybush, an endemic neo-crop from the Cape Floristic Region (CFR) of South Africa. Cultivated populations appear to have originated from a small number of founding populations and/or individuals and represent a genetic bottleneck. Thus, cultivated populations tend to lack the genetic diversity and phylogeographic structuring present in wild populations and may represent a genetic threat to wild populations if gene-flow occurs.Cyclopia breeding material originating from multiple wild populations ; C. subternata populations from the Tsitsikamma and/or Outeniqua mountains (these wild populations share the same common haplotype found in cultivated populations); while C. longifolia originates from its only known wild source, the Van Stadens River system. These findings are consistent with those from recent microsatellite analysis of C. subternata  was confirmed to have been established from commercial seed and did not differ in dominant haplotypes from other cultivated populations. Furthermore, Harkerville shared a rare haplotype with cultivated material from Uniondale (U)—where putative hybrids were detected . This rare haplotype was only detected in wild C. intermedia populations located in the Swartberg Mountains (SWB) and may be evidence of possible chloroplast capture . Secondly, C. intermedia and C. subternata have been found to successfully produce hybrid offspring with other members of the genus under experimental conditions, and C. intermedia material from the Swartberg was included in the initial Honeybush cultivation trials . However,  capture  resultin capture  or under capture  and it in trials , Fig. 1.Cyclopia populations than cultivated populations, with multiple cases of near complete haplotype turnover among wild populations  in addition to rare cases of cultivated plants intentionally established in natural vegetation . The chloroplast genome is maternally inherited with no recombination in the majority of angiosperms  haplotypes from the United States to native Chinese populations resulted in a hybrid swarm that disrupted local cpDNA genome frequency and ecological processes due to vigorous growth displacing native plant species , C. genistoides (near threatened), C. maculata (near threatened), C. plicata (endangered), and C. sessiliflora (near threatened). However, it is unlikely that all neo-crops will exhibit the levels of phylogeographic structuring, genetic diversity and, conservation requirements of Cyclopia, and more work is required to define the extent to which wild genetic resources are at risk during neo-crop cultivation practices, particularly in species rich landscapes such as the Cape of South Africa.Until the genetic risks are better understood, formal guidelines should be developed to facilitate sustainable cultivation of neo-crops. In the case of Honeybush, an ecology-centric approach, as the one outlined by 10.7717/peerj.11462/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.11462/supp-2Supplemental Information 2Working example and R script used to assign haplotype identities to assecions based on HRM clustering results.Click here for additional data file.10.7717/peerj.11462/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.11462/supp-4Supplemental Information 4Click here for additional data file.


Bombina bombina) have rapidly declined during recent decades. Extensive agricultural land use has fragmented the landscape, leading to habitat disruption and loss, as well as eutrophication of ponds. In Northern Germany (Schleswig‐Holstein) and Southern Sweden (Skåne), this population decline resulted in decreased gene flow from surrounding populations, low genetic diversity, and a putative reduction in adaptive potential, leaving populations vulnerable to future environmental and climatic changes. Previous studies using mitochondrial control region and nuclear transcriptome‐wide SNP data detected introgressive hybridization in multiple northern B. bombina populations after unreported release of toads from Austria. Here, we determine the impact of this introgression by comparing the body conditions (proxy for fitness) of introgressed and nonintrogressed populations and the genetic consequences in two candidate genes for putative local adaptation (the MHC II gene as part of the adaptive immune system and the stress response gene HSP70 kDa). We detected regional differences in body condition and observed significantly elevated levels of within individual MHC allele counts in introgressed Swedish populations, associated with a tendency toward higher body weight, relative to regional nonintrogressed populations. These differences were not observed among introgressed and nonintrogressed German populations. Genetic diversity in both MHC and HSP was generally lower in northern than Austrian populations. Our study sheds light on the potential benefits of translocations of more distantly related conspecifics as a means to increase adaptive genetic variability and fitness of genetically depauperate range margin populations without distortion of local adaptation.Northern range margin populations of the European fire‐bellied toad  of the fire‐bellied toad were introgressed by Southern specimens from Austria. Introgressed Swedish populations were elevated in body weight and allelic diversity of an adaptive immune gene (MHCII), while diversity at a heat shock protein gene stayed unaffected. This points towards a benefit of edge populations from long‐distance translocation. HSPs are molecular chaperons which have been repeatedly discussed as important players in the adaptation to various perturbations of the environment, most notably heat stress  is a highly threatened amphibian species, especially in Northern Europe. After the last glacial period, B. bombina recolonized the European continent out of putative refugia near the Black Sea in two distinct expansions , Northern Germany (Schleswig‐Holstein), and Eastern Austria in 2016  as described by Peig and Green . This wiWe compared body length, body mass, and the SMI among five groups: Northern (German and Swedish) populations, which we found to have been genetically introgressed by Austrian toads, German and Swedish populations putatively free of genetic introgression  and associated pairwise comparisons of mean values among groups.2.3B. bombina  and two African clawed frog (Xenopus sp.) sequences provided from Xenbase  for the identification of the exon/intron margins  and the other used a B. bombina HSP70 kDa reference sequence retrieved via Sanger sequencing during primer development. We then parsed the resultant mapping files using SAMtools v1.3.1   using Geneious v9.1.6 , no site‐specific selection test was performed.To identify putative amino acid and expressed protein variation of the HSP70 kDa gene, we translated the exons of 33.1SE], respectively; Figure SE], p = .002). In comparison, the German populations were significantly heavier and had a higher SMI than both the Austrian and the Swedish populations. There was no significant difference in body mass or SMI among introgressed and nonintrogressed populations in Germany.Southern lineage toads (Austria) were—on average—significantly smaller than Northern lineage toads Figure . There w3.2Among the analyzed 297 specimens from 17 populations, we found 3 northern lineage (AA*) and 4 southern lineage (DEG*) haplotypes . In the site‐specific analysis, however, the single‐likelihood ancestor counting (SLAC) method identified two sites under positive/diversifying selection and one under negative/purifying selection. The fixed effects likelihood (FEL) method identified the same three sites under selection plus additional three sites under positive, and additional two sites under negative selection. Almost all sites under positive selection, except for one, are located in putative antigen‐binding sites (ABS) known from human leukocyte antigen genes (HLA), which are part of the MHC gene complex , we plotted the proportion of shared alleles within and between regions using a Circos plot Figure . In AustWe counted the total number of MHC II exon 2 alleles in Austrian, Swedish, German, and combined northern (German and Swedish) populations for each individual and per group Table . The aveB. bombina populations, all located northwest of lake Neusiedl and in Vienna.We found ubiquitous alleles  at high frequency in all German and the majority of the Austrian populations. In contrast, in Sweden, these ubiquitous alleles were only present in two out of four populations, and at lower frequencies Figure . In Swed3.4The different exon fragments of the HSP70 kDa gene differed substantially in their number of polymorphic sites. We found one polymorphic site in exon 1, none in exons 2 and 3, seven in exon 4, and 17 in exon 5. In order to detect differences between alleles from nonintrogressed and introgressed populations, an allele network including all the 25 polymorphic sites was constructed. Nine different alleles were identified  carrying the same expressed polymorphism  by phenylalanine (F) within a leucine‐rich region (LRR)). Each of these alleles occurred only in one out of the 57 analyzed individuals. The genewise dN/dS ratio was 0.038. As we only found a single nonsynonymous substitution, this estimate is not precise. Still, it deviates highly significantly from the neutral expectation  and is hence indicative of negative (purifying) selection on HSP70 kDA in the geographical regions analyzed here.Using the known reading frame of the Asian fire‐bellied toad , we translated each 4B. bombina originating from two northern countries, both independently introgressed by toads from the southern lineage  and genetic variation at expressed genes in B. bombina populations may lead to an increased fitness . The introgression may have provided adaptive alleles at many loci  and the yellow‐bellied toad (B. variegata)—which occur where the species’ distribution ranges overlap—appear to be selected against  population score significantly lower in average morphometric trait values  when compared to their Northern German counterparts. These Swedish populations mark the northern margin of the species distribution and may hence struggle with suboptimal habitats and adverse climatic conditions. Our finding supports the hypothesis that introgression of southern genotypes into these northernmost B. bombina populations to be generally genetically less diverse at these loci, when compared to conspecific southern populations in Austria. These lower levels of genetic diversity could reflect a combination of local adaptation, colonization history, edge effects, and declines in population size during the last century. A reduced genetic variability of these northern populations has also been observed at neutral molecular markers . We find northern Introgression from a geographically distant lineage into a genetically isolated population instantly increases the diversity in a population's gene pool  and an excess of nonsynonymous substitutions is commonly observed, as typical for protein domains with antigen‐binding function  selection  consistently occurred at high frequencies across all populations. The ratio among these alleles was shifted toward allele 1 in Austria and the northern introgressed populations. This and the occurrence of a specific HSP allele ; Data curation (lead); Formal analysis ; Investigation ; Visualization ; Writing‐original draft (lead); Writing‐review & editing . Katrin Kiemel: Data curation (supporting); Formal analysis ; Investigation ; Visualization ; Writing‐original draft (supporting); Writing‐review & editing . Michael Westbury: Formal analysis (supporting); Investigation (supporting); Supervision (supporting); Writing‐original draft (supporting); Writing‐review & editing . Maike Lauritsen: Data curation (supporting); Formal analysis (supporting); Investigation (supporting); Visualization (supporting); Writing‐review & editing (supporting). Marijke Autenrieth: Formal analysis (supporting); Visualization (supporting); Writing‐review & editing (supporting). Günter Gollmann: Resources (supporting); Writing‐review & editing (supporting). Silke Schweiger: Resources (supporting); Writing‐review & editing (supporting). Marika Stenberg: Resources (supporting); Writing‐review & editing (supporting). Per Nyström: Resources (supporting); Writing‐review & editing (supporting). Hauke Drews: Resources (supporting); Writing‐review & editing (supporting). Ralph Tiedemann: Conceptualization (lead); Data curation (supporting); Formal analysis (supporting); Funding acquisition (lead); Investigation (supporting); Project administration (lead); Resources (lead); Supervision (lead); Writing‐original draft (supporting); Writing‐review & editing .Appendix S1Click here for additional data file.


Acer caudatifolium using chloroplast DNA to explore the relationships of terrain and local climate heterogeneity with range shifts and demography in Taiwan. Our results showed that the extant populations have shifted upward and northward to the mountains since the Last Glacial Maximum. The distributional upshift of A. caudatifolium is in contrast to the downward expansion of its closest relative in Taiwan, A. morrisonense. The northern populations of A. caudatifolium have acquired multiple-source chlorotypes and harbor high genetic diversity. However, effective gene flow between the north and south is interrupted by topography, geographic distance, north-south differences in October rainfall, and other climate heterogeneities, blocking southward genetic rescue. In addition, winter monsoon-driven rainfall may cause regional differences in the phenological schedule, resulting in adaptive effects on the timing of range shift and the genetic draft of chlorotype distribution. Terrain, distance, and local climate also differentiate the northernmost populations from the others, supporting the previous taxonomic treatment of Acer kawakamii var. taitonmontanum as an independent variety.Under climate fluctuation, species dispersal may be disturbed by terrain and local climate, resulting in uneven spatial-genetic structure. In addition, organisms at different latitudes may be differentially susceptible to climate change. Here, we tracked the seed dispersal of  Both environmental conditions and dispersibility may be limiting factors for changes in the distribution ranges of species. The measurement of ecological niche is one way to link the suitable environmental conditions for species in which their fitness can be maintained or increased. These theoretical niche ranges are the fundamental (Grinnellian) niches  and coulGene flow and demographic dynamics may be subject to spatial and environmental influences ,6. GivenAcer caudatifolium Koidzumi, was selected as the research object. Acer caudatifolium is widely distributed at low to high elevations, is endemic to the island of Taiwan . Th. ThFST/(1 − FST) , respectD and the demographic or spatial expansion time (τ) were selected as the responses. The latitude, longitude, and altitude were taken as the geographic predictors, and the five climatic factors  were used as the environmental predictors. The optimal model was chosen using backward selection by the stepAIC function in the R package MASS [Population genetic patterns can also be influenced by demographic dynamics, which can be spatially and environmentally related. We, therefore, tested whether the demographic change was associated with geographic and environmental factors. The generalized linear model (GLM) was conducted in the R package stats , and Tajage MASS .p = 0.001 and 0.012, respectively, τ = 0.643–8.350 and 0.135–7.924 in the demographic and spatial expansion models, respectively). These estimates do not include monomorphic populations, which does not imply constant demography of these populations but rather an inability to perform mismatch analysis.The cpDNA estimation revealed that northern Taiwan is a hotspot of genetic diversity, whereas the southern populations are almost monomorphic in chlorotype (Hap6). The northern population comprises Hap4, Hap6, and their derived chlorotypes . MismatcInference of population genetic barriers by the Monmonier algorithm suggested that most of the geographic barriers are in northern Taiwan C–E. The p = 0.213, Model selection in MLPE indicated that IBD had the lowest AIC and BIC, followed by IBD + IBE, which had a very similar AIC value  and spatial expansion time comprised both spatial and environmental factors, although certain factors were excluded under backward selection , whereas only prec10 significantly predicted spatial expansion time (p = 0.010) (Model selection in GLM suggested that the null (empty) model could not be rejected by any alternative model in demographic expansion time. However, the optimal models for predicting the extent of population dynamics (Tajima’s election . Under t= 0.010) . Togethep < 0.001) indicates the predicted models of Acer caudatifolium are reliable. The greatest contributing factor, Tmax2, was highly correlated with temperature-related Bioclims , Tavg, Tmax, Tmin, vapr, AET, and PET  in Pearson’s correlation  A,B. Bothrelation . Accordirelation C,D. ThesA. caudatifolium into higher mountain ranges from the narrow habitats in the southwestern area of Taiwan. The ENM speculates that the south was more suitable for A. caudatifolium growth than north-central Taiwan, although the northeast may also have potentially suitable habitats . Among ure 5 of ,13,14. Iure 5 of . Howeverure 5 of  or the cure 5 of . A. morrisonense, the chlorotypes of A. caudatifolium are differentiated from north to south . Howeveure 5 of ). DuringA. caudatifolium have wings that aid spreading, maple seed dispersal is unexpectedly distance-limited and aggregated [The distinct chlorotype composition suggests the northernmost populations as a unique evolutionary significant unit from the southern populations. The formation of the northeast-southwest geographic barrier may be attributable to environmentally based dispersal bias. The disruption of gene flow by terrain according to the mountain-barrier effect is more significant in the northern populations, particularly for blocking gene flow between the southern and northernmost populations on and around the Datun volcano group . Environmentally determined migratory characters, e.g., seed dispersibility, may constrain the extent of range shift. Although the seeds of gregated . The wargregated , resultiA. caudatifolium typically turn yellow in October, and differences in late autumn rainfall between northeastern and southwestern Taiwan may result in differences in leaf duration and next-year seed yield . The significant correlation between prec10 and the timing of range change implies that autumn rainfall was one of the key factors affecting adaptive migration under paleoclimate change. In addition, since selection may eliminate maladaptive migrants, such a phenological difference may affect the success of colonization, thereby interrupting north-south gene flow. The greatest contributing factor of ENM, Tmax2, are highly correlated with other temperature-related factors  between the rainy northeast and arid southwest in Taiwan has affected forest phenology ,38. XyleD, indicating that autumn rainfall, summer sunshine, and evapotranspiration jointly determine the extent of population renewal and demographic structure. Precipitation, solar radiation, and evapotranspiration may affect photosynthesis, nutrient accumulation, growth, and yield simultaneously. These environmental factors related to survival and reproduction are undoubtedly crucial to population genetic structure via local adaptation and affect the chlorotype distribution via the genetic draft, particularly for populations in central to northern Taiwan. Our results show that local climate heterogeneity combined with the influence of geography  reduces the ability of the northernmost chlorotypes to enter the south, resulting in genetic differentiation from the south. Prec10, srad6, srad7, and AET were related to Tajima’s Seasonality is more pronounced in northern Taiwan than in southern Taiwan. The higher genetic diversity of the northern populations promotes their adaptability and enables local adaptation to long-term seasonal and climate changes. By contrast, the southern margin may be more susceptible to climate change , as refl


Helleborus niger (Ranunculaceae). We applied a set of phylogeographic analyses based on restriction-site associated DNA sequencing (RADseq) data and plastid DNA sequences to a range-wide sampling of populations. These analyses were supplemented with species distribution models generated for the present and the Last Glacial Maximum (LGM). We used exploratory analyses to delimit genomically coherent groups and then employed demographic modeling to reconstruct the history of these groups. We uncovered a deep split between two major genetic groups with western and eastern distribution within the Southern Limestone Alps, likely reflecting divergent evolution since the mid-Pleistocene in two glacial refugia situated along the unglaciated southern margin of the Alps. Long-term presence in the Southern Limestone Alps is also supported by high numbers of private alleles, elevated levels of nucleotide diversity and the species’ modeled distribution at the LGM. The deep genetic divergence, however, is not reflected in leaf shape variation, suggesting that the morphological discrimination of genetically divergent entities within H. niger is questionable. At a shallower level, populations from the Northern Limestone Alps are differentiated from those in the Southern Limestone Alps in both RADseq and plastid DNA data sets, reflecting the North-South disjunction within the Eastern Alps. The underlying split was dated to ca. 0.1 mya, which is well before the LGM. In the same line, explicit tests of demographic models consistently rejected the hypothesis that the partial distribution area in the Northern Limestone Alps is the result of postglacial colonization. Taken together, our results strongly support that forest understory species such as H. niger have survived the LGM in refugia situated along the southern, but also along the northern or northeastern periphery of the Alps. Being a slow migrator, the species has likely survived repeated glacial-interglacial circles in distributional stasis while the composition of the tree canopy changed in the meanwhile.Glacial refugia of alpine and subnival biota have been intensively studied in the European Alps but the fate of forests and their understory species in that area remains largely unclear. In order to fill this gap, we aimed at disentangling the spatiotemporal diversification of disjunctly distributed black hellebore  The climatic changes during the Quaternary have had a major impact on the distribution of many plant and animal species by inducing shifts, expansions, contractions, and fragmentations of ranges as well as population extirpations . In exteThe Eastern Alps have a clearly tripartite tectonic structure, with the siliceous Central Eastern Alps flanked by the calcareous Northern and Southern Limestone Alps . During Rosa pendulina based on somewhat ambiguous Amplified Fragment Length Polymorphism evidence. In contrast, a refugium in the southeastern Alps and the adjacent Balkan Peninsula was strongly supported, for instance, for Cyclamen purpurascens , larch (Larix decidua), and dwarf mountain pine (Pinus mugo) formed a vegetation belt between alpine and lowland grasslands; in addition, spruce (Picea abies) and Scots pine (Pinus sylvestris) occurred in the alluvial plains. Mesophytic broad-leaved forest trees, such as beech (Fagus sylvatica), were absent from the Alpine fringe, but survived in refugia further south, in particular in the northwestern Balkan Peninsula . It is a herbaceous perennial with a robust rhizome and the only representative of H. section Helleborus  were reported, among others, from Italy  vegetation belt  with a strong preference for carbonate bedrock  forests or krummholz (H. niger subsp. macranthus (Freyn) Schiffn.  The Northern Limestone Alps were colonized postglacially from a single cold stage refugium in the weakly glaciated Southern Limestone Alps and/or adjacent areas in the northwestern Balkan Peninsula  individuals per population for the DNA extraction . In 2019g for 5 min. The clear supernatant was transferred onto a prefilter and centrifuged at 10,000 g for 1 min. Cleaning of the extract by binding the DNA to magnetic beads, repeated ethanol precipitation and recovery of DNA was carried out by an InnuPure C16 robot . RNA was digested by adding 4 μl RNase A solution (100 mg/ml), followed by incubation for 5 min at room temperature. The quality of DNA was checked on an agarose gel. The DNA concentration was estimated using a Qubit 4 fluorometer .DNA was extracted from silica gel dried leaf material using the innuPREP Plant DNA Kit II . Dry leaf material (100 mg) was transferred together with two 4 mm steel balls into a 2 ml reaction tube and frozen for 10 min in liquid nitrogen. The material was immediately ground using a Retsch  swing mill for 4 min. Subsequently, 500 μl lysis solution SLS and 20 μl Proteinase K were added and mixed vigorously by pulsed vortexing for 5 s. During lysis, tubes were incubated at 65°C for 30 min. Lysis was stopped by addition of 100 μl precipitation buffer P and samples were vortexed again for 5 s, followed by incubation at room temperature for 5 min and centrifugation at 10,000 H. niger , and three individuals from two populations of H. foetidus as outgroup, using the restriction enzyme PstI (New England Biolabs) and a protocol adapted from Single-digest RADseq libraries were prepared from three individuals from each of 40 populations of https://github.com/wtsinpg/illumina2bam) and the program process_radtags.pl implemented in Stacks v. 1.35  for demographic modeling, the 10 individuals with the lowest proportion of missing data were selected from each STRUCTURE group (see below). For those individuals, SNPs were exported in vcf format with the program populations and further filtered using VCFtools 0.1.15  and finally the --write_single_snp flag was used to select only a single (first) SNP per fragment, to minimize the chance of selecting linked loci. For this dataset, all individuals sampled from the same locality were defined as a population. For the second dataset, later used for phylogenetic tree reconstruction, three individuals of s 0.1.15  for a maH. niger samples , the optimal grouping of populations was determined using Bayesian clustering in STRUCTURE 2.3.4, using the admixture model with uncorrelated allele frequencies (K (number of groups) ranging from 1 to 10 were carried out using a burn-in of 10,000 MCMC iterations followed by 100,000 additional iterations. The optimal K was identified as the K, where the increase in likelihood started to flatten out, the results of replicate runs were similar, and the clusters were non-empty. Additionally, the deltaK criterion was employed, reflecting an abrupt change in likelihood of runs at different K (For the quencies . Ten repferent K . To estiferent K .H. niger and H. foetidus), we computed a maximum likelihood (ML) phylogeny using RAxML v. 8.2.8 (https://www.biostars.org/p/55555/) and Felsenstein’s ascertainment bias correction was further used to account for missing invariant sites  founder event  Western Group vs. Eastern Group and (ii) South-Eastern Subgroup vs. North-Eastern Subgroup. For both comparisons, we translated three scenarios  into thrs that defines the fraction of the ancestral population (nuA) founding each daughter population, where nuA*s represents the island population and nuA*(1−s) represents the mainland population. We enforced an upper limit of 0.5 for s.To differentiate the scenarios of a pre-glacial vs. a post-glacial split of the populations, we dated the splits between groups using the Bayesian program BPP (see below). For both pairwise comparisons, we tested eight island demographic models . These eight models represent the three main hypotheses and modifications with ancestral or recent migration included to explain additional features of the 2D-JSFS . While ti; In the optimization of the models, we initially optimized 3-fold perturbed random starting parameters followed by two rounds of 2-fold and one round of 1-fold perturbed parameters to estimate the log-likelihood of the SFS given the model. The number of replicates in each round was 10, 20, 30, and 60, respectively. Across all analyses, we used the optimized parameter sets of each replicate to simulate the 2D-JSFS, and a multinomial approach to estimate the log-likelihood of the 2D-JSFS given the model. The models were compared using the Akaike information criterion (AIC), and the replicate with the highest likelihood for each model was used to calculate AIC scores, ∆AIC scores, and Akaike weights ωi; .via model selection  to absolute time, the function msc2time.r implemented in the R package bppr was used (μ (substitution rate per site per generation) and generation time from the “mcmc file” returned by BPP , we chose generation times of 5, 10, and 20 years to represent a range of genealogical scenarios for the species. We emphasize that the demographic generation time is not the time until the first reproduction but the average time between two successive generations within a population  containing 11.6 μl of RedTaq ReadyMix (Sigma-Aldrich), 1.5 μl of BSA , 0.8 μl of each primer (10 μM) rpoB and psbMR  following the manufacturer’s instructions. Sequencing was then carried out at Eurofins Genomics  using the primers rpoB, trnCGCAF, and ycf6F . Scans were saved as grayscale in bitmap format. The program tpsDig264 version 2.25 (Morphometry was performed on rosette leaves of ion 2.25  was usedshapiro.test). Non-normally distributed variables were transformed to normality using the BoxCox function of the library hdrcde (cor function in order to detect undesirable high correlations (Pearson’s correlation coefficient r ≥ 0.90). We ran a principle component analysis (PCA) using the eigen function. A discriminant analysis (DA) was carried out to explore morphological separability of the two main genetic groups  using the lda function from the MASS library  to avoid effects of different value ranges. In order to remove size effects potentially exerted by the environment or the developmental stage of the leaves, we normalized the metric characters 1–13 by division through the sum of their values calculated for every individual. Therefore, subsequent analyses are solely based on leaf shape. The transformed variables were tested for normal distribution using the Shapiro-Wilk test  and the paleoclimate (ending 1983), the delta-method was applied in a two-step approach including climate data having a temporal overlap of some decades between both data sets. We used the CRU TS4.01 data set (r > |0.7|).Because of the rough terrain of mountain regions within the study area, the coarse spatial resolution of available historic climate data sets does not allow reconstructing the migration history of species since the LGM. We, hence, applied a statistical downscaling procedure previously used in several studies on climate change effects . The “dealeoView . CurrentaleoView ,b availadata set  with a sdata set , and recH. niger at the end of the LGM and under current conditions. These models relate species occurrences to the five environmental variables described above at a resolution of 1 km. SDMs were based on generalized linear models (GLMs) starting with a niche determined using the climate of the current distribution of H. niger. However, this entailed a clearly too narrow species’ niche as the model predicted an extinction of the species, i.e., no cell within the study region was predicted to be suitable at 12,100 BP  to project the environmental suitability of the study area for H. niger  followinSD = 0.16) million. The denovo_map.pl pipeline (ustacks) identified a mean coverage of 10.68× (±1.01) over all samples in the catalog . Relationships within the two clades were not supported (BS < 50%). However, the North-Eastern Subgroup was resolved as monophyletic sister-group to two northern populations of the South-Eastern Subgroup (264 and 267).A RAxML phylogeny based on 4,914 SNPs  that wasDetailed results of our demographic modeling are given in s; i = 0.8; 21 = 5.1, m12 = 1.7). The second-best model  of the ancestral population.In all comparisons, models of vicariance fitted better to our data than founder event models . We perfi = 0.51; 12 = 6.7, m21 = 0.2; i = 0.2) included an old founder event with exponential population growth of the North-Eastern Subgroup and continuous asymmetric migration from the North-Eastern to the South-Eastern Subgroup followed by a period with stable population sizes.Similarly, in the pairwise comparison between the South-Eastern and the North-Eastern Subgroup, the best fitting model  DC .The ranges of the Western and the Eastern Groups in the Southern Limestone Alps are likely parapatric; according to our knowledge, they are not separated by a distribution gap , 3A. PreH. niger subsp. macranthus and the typical subspecies  had the third-highest loading on the second PCA axis we suggest that –based on leaf shape variation – the traditional discrimination of two subspecies within H. niger is highly questionable as it does not reflect the main genetic break.The deep genetic divergence between the Western and the Eastern Group is not reflected in leaf shape variation; other sources of morphological variation such as flowers could not be explored due to the phenological state of the herbarium vouchers. Leaf shape was deemed an important source of characters discriminating between bspecies . Howeverbspecies , only thbspecies . Even ifWithin the Eastern Group, populations from the Northern Limestone Alps are differentiated from those in the Southern Limestone Alps in both RADseq and plastid data sets  gene flow from north to south. Specifically, in the pairwise comparison of the South-Eastern and the North-Eastern Subgroups, the best-fitting model  was a viH. niger in the Northern Limestone Alps, seems to be autochthonous due to its occurrence in a natural habitat, i.e., undisturbed forest vegetation in a deep gorge with difficult access, and the presence of the “correct” plastid haplotype. While an anthropogenic introduction of population 29 from eastern parts of the Northern Limestone Alps cannot be fully rejected given this evidence, a long-distance dispersal event explaining the haplotype of population 30 seems highly unlikely.A remarkable exception from the overall good correlation between genetic relatedness and geographic proximity is presented by population 30 from the western distribution limit in the Northern Limestone Alps. This population, which grows in the vicinity of a chapel, was hypothesized to be a garden escape already during fieldwork. Its likely non-indigenous status is underlined by the presence of a plastid haplotype from the Western Plastid Lineage otherwise present in geographically distant, western-most populations in the Southern Limestone Alps  can be found at: PS, CD, EZ, KH, and WW conceived the study. CD and many other colleagues (listed in the acknowledgments) collected the samples. JW, WW, AG, and KH compiled the distribution and climatic data and produced the species distribution models. EZ and DL carried out the molecular lab work. EZ and PK analyzed the genomic data. BF analyzed the genetic data. CD produced and analyzed the morphometric data. EZ and PS wrote major parts of the manuscript, with exception of the parts about SDMs written by KH. All authors read and edited the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.