Siraitia grosvenorii, an economically important plant species with high medicinal value, is endemic to subtropical China. To determine the population structure and origin of cultivated S. grosvenorii, we examined the variation in three chloroplast DNA regions  and two orthologous nuclear genes (CHS and EDL2) of S. grosvenorii in 130 wild individuals  and 21 cultivated individuals using a phylogeographic approach. The results showed three distinct chloroplast lineages, which were restricted to different mountain ranges, and strong plastid phylogeographic structure. Our findings suggest that S. grosvenorii likely experienced ancient range expansion and survived in multiple refuges in subtropical China during glacial periods, resulting in population fragmentation in different mountainous areas. Our results also demonstrated that wild populations in Guilin  share the same gene pool as cultivated S. grosvenorii, suggesting that current cultivars were collected directly from local wild resources, consistent with the principles of “nearby domestication.” The results of this study provide insights into improving the efficiency of S. grosvenorii breeding using a genetic approach and outline measures for the conservation of its genetic resources. Siraitia grosvenorii, an economic plant species distributed in subtropical China. Our results clarify not only the population genetic structure of S. grosvenorii, but also determin the geographic origins of cultivated S. grosvenorii. The current study provide important information for the utilization and conservation of the valuable genetic resources of S. grosvenorii. The manuscript presents the first phylogeographic analysis of  S. grosvenorii was introduced from Guangxi into other provinces in southern China. In addition to its use as Chinese traditional medicine , Dayaoshan, Yunkai, Jiulianshan, and Wugongshan Mountains at an altitude of 400–1400 meters. Global climatic fluctuations, particularly Quaternary climate oscillations, might have profoundly shaped the distribution range and genetic structure of plants  , inter‐simple sequence repeat (ISSR), and amplified fragment length polymorphism (AFLP) markers, which revealed a high level of genetic differentiation among the wild populations  fragments and two orthologous nuclear genes. We aimed to (1) investigate the genetic structure of S. grosvenorii, and infer its main potential refugial locations and demographical history, and (2) determine the geographical origin and ancestral populations of cultivated S. grosvenorii. Information on the population structure and demographical history of S. grosvenorii is important for the genetic improvement of its cultivars and the conservation of its wild germplasm.In this study, we report the results of the phylogeographical analysis of 22.1S. grosvenorii were collected in this study, including 130 individuals selected from 13 wild populations , 4 cultivated male individuals, and 17 female individuals from 7 cultivated accessions . Details of the sampling are shown in Table S. grosvenorii exhibits clonal reproduction and has a small population size, the individual plants were sampled from sites located >10 m apart  and EID1‐like F‐box protein 2 (EDL2), were selected. CHS has been used in phylogeographic studies  was obtained from the National Center for Biotechnology Information , and its orthologs were identified using the OrthoMCL database (http://orthomcl.org/orthomcl/). The nuclear genes were PCR amplified with sequence‐specific primers designed using Primer3Web (http://primer3.wi.mit.edu/). Primer details are given in Table 2+ plus), 4 μL of dNTP mix (2.5 μM), 0.5 μL of each primer (50 mM), and 2.5 U of ExTaq DNA polymerase . The amplification program was as follows: predenaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 55 or 58°C (Table Genomic DNA was extracted from the dried leaves using the modified cetyltrimethylammonium bromide (CTAB) method  were amplified from 151 individuals and aligned individually, then they were concatenated using the concatenate sequence plugin in PhyloSuite 1.2.2 , and nucleotide diversity (π) were calculated using DNAsp 5.0  and nuclear genes (CHS and EDL2) were constructed using Network 5.0  was selected as the outgroup. The F81 + F model was selected as the most appropriate substitution model based on Bayesian information criterion (BIC) implemented in IQ‐TREE ModelFinder  method and implemented with the IQ‐TREE 1.6.12 program  and within‐population genetic diversity (HS) were calculated using PERMUT 2.0  were performed using Arlequin 3.11  to 3.0 × 10−9 (maximum) substitutions per site per year was used as a range of average mutation rates, as reported for synonymous sites in the chloroplast genes of angiosperm species .Divergence times for the three chlorotype‐based lineages, identified based on cpDNA data, were estimated using BEAST 1.6.1  using 1000 parametric bootstrap replicates in Arlequin 3.11. All tests performed in Arlequin 3.11 and DNAsp 5.0 used default settings.The historical population dynamics of  Tajima,  and Fu aFu & Li,  using DN, Fs Fu,  using Ar, Fs Fu,  in DNAsp33.1h and π values of wild populations were 0.822 and 0.00978, respectively  was higher than the average within‐population genetic diversity (HS = 0.036), resulting in high population differentiation . A permutation test revealed a significant phylogeographic structure in S. grosvenorii . The results of overall AMOVA  were identified, among which twenty‐five were unique to a single population. Haplotype H12 was shared by seven populations , making it the most common and the most widely distributed haplotype  and 0.00406 (range: 0–0.00422), respectively (Table h (0.932) and π (0.00422) were found in the JLS and SG populations, respectively  and 0.00264 (range: 0–0.00315), respectively (Table h (0.774) and π (0.00315) were found in population JLS and JX, respectively  was higher than the HS of these genes . Although the NST value of CHS (0.464) was higher than the corresponding GST value (0.420), the NST value of EDL2 (0.344) was lower than the corresponding GST value (0.349), the difference was not significant (p > .05), suggesting that the phylogeographic structure of S. grosvenorii based on the nuclear gene data was also not significant. The results of AMOVA (Table CHS sequence among populations (50.95%) was only slightly higher than that within populations (49.05%). In contrast to the results of cpDNA and CHS data analyses, the among‐population variation in EDL2 (37.86%) was lower than the within‐population variation (62.14%) , but the among‐lineage variation in both CHS and EDL2 was much lower  , as well as the nonsignificant SSD and HRag statistics (p > .05) of CHS. However, the nonsignificant Tajima's D and Fu and Li's D values of the two nuclear genes and the significant SSD and HRag statistics of EDL2 also rejected the hypothesis of rapid expansion ; the Fu's Fs values of MD lineage in CHS data were significant and negative ; and the remaining Tajima's D, Fu and Li's D, and Fu's Fs values were nonsignificant (Table HRag statistics of the three lineages in CHS data and those of MD and YK lineages in EDL2 data were nonsignificant (p > .05)  and early Pleistocene (2.58 to 0.80 Mya)  Figure , implyinFeliner,  but alsoS. grosvenorii experienced an ancient expansion event that brought ancestral haplotypes H12 and E3 to different parts of the range, which resulted in the origin of numerous derived haplotypes in these isolated locations; for example, 25 CHS haplotypes and 6 EDL2 haplotypes, which were private to a single population. Nevertheless, the nuclear haplotypes H3, E1, and E8 occurred in MD (HZ population only) and YD lineages, and E6 was shared between the MD and YK lineages. These results suggest limited pollen dispersal among lineages within S. grosvenorii.Haplotypes shared among the three lineages  represent the internal nodes of the network, could be considered as ancestral, and would predate the divergence of populations. We did not find a firm recent population expansion signal in our nuclear dataset. The distribution of these haplotypes across all regions could probably be attributed more to the persistence of ancestral polymorphisms than to recent gene flow and dispersal. Thus, the most likely scenario is that 4.2S. grosvenorii is documented to have been cultivated in Longsheng, Yongfu, and Lingui counties of Guilin for at least 140 years  and several nuclear haplotypes with cultivars, indicating that the main cultivars currently grown in Yongfu, Lingui, and Longsheng originated from wild populations distributed in Guilin, Guangxi, consistent with the principals of “nearby domestication.” Indeed, the initial cultivation of S. grosvenorii followed traditional practices, whereby plants were directly collected from the local wild resources and then cultivated in the field , it is obvious that gene‐rich pools of wild relatives have not been exploited for the improvement of cultivars. The three genetic lineages identified in this study contained a large number of unique haplotypes  is found in the Yunkai Mountains and therefore deserves particular attention as a prime ex situ conservation target. Additionally, we propose that populations from the MD lineage and YD lineage  should be utilized as the plant material or core gene pool for breeding and crop improvement, for which both the in situ and ex situ methods should be adopted.The populations of MD lineage showed higher genetic diversity, and the populations of YD and YK lineages also exhibited a certain level of genetic diversity. Although wild populations of  Figures , Table 75S. grosvenorii distributed in subtropical China. Our cpDNA results showed that three main lineages of the present populations of S. grosvenorii occupy different mountain ranges and have been isolated from each other for a long period of time. S. grosvenorii likely experienced an ancient range expansion and survived in multiple refuges during the glacial periods, resulting in fragmentation in different mountainous areas. S. grosvenorii cultivars originated from wild populations MES, BL, and JT in Guilin, Guangxi, consistent with the principle of nearby domestication. This phylogeographical study also provides a genetic basis for the utilization and conservation of S. grosvenorii germplasm.The molecular data collected in the current study permit the first phylogeographic analysis of Bingbin Xie: Conceptualization ; data curation (lead); formal analysis (lead); investigation ; methodology (lead); software (lead). Bowen Lai: Conceptualization ; data curation (supporting); formal analysis (supporting); writing – original draft (lead); writing – review and editing (supporting). Liping Chen: Data curation (supporting); formal analysis (supporting); investigation ; methodology (supporting); software (supporting). Sujuan Wei: Conceptualization ; methodology (supporting); supervision (supporting); validation (supporting); writing – review and editing . Shaoqing Tang: Conceptualization ; funding acquisition (lead); project administration (lead); supervision (lead); validation (lead); visualization (lead); writing – review and editing .The author declares that they have no conflict of interest.


Paramacrobiotus fairbanksi was described from Alaska (USA) based on integrative taxonomy and later reported from various geographical locations making it a true cosmopolitan species. The ‘Everything is Everywhere’ (EiE) hypothesis assumes that microscopic organisms have unique features that help them to inhabit many different environments, meaning they can be considered cosmopolitan. In the present work we report four new populations of Pam. fairbanksi from the Northern Hemisphere which suggests that the ‘EiE’ hypothesis is true, at least for some tardigrade species. We also compared all known populations of Pam. fairbanksi at the genetic and morphological levels. The p-distances between COI haplotypes of all sequenced Pam. fairbanksi populations from Albania, Antarctica, Canada, Italy, Madeira, Mongolia, Spain, USA and Poland ranged from 0.002% to 0.005%. In total, twelve haplotypes (H1-H12) of COI gene fragments were identified. We also report statistically significant morphometrical differences of species even though they were cultured and bred in the same laboratory conditions, and propose epigenetic factor as a main cause rather than temperature, predation risk and food availability. Furthermore, we also discuss differences in the potential distribution of two Paramacrobiotus species. Currently there are 33 families, 159 genera, 1464 species and 21 additional subspecies within this phylum1. Paramacrobiotus fairbanksi Schill, Förster, Dandekar & Wolf, 20103 was described from Alaska (USA) and reported from the Antarctic, Italy, Poland and Spain4 9. It is a large-size (up to 800 μm) parthenogenetic Paramacrobiotus found mostly in mosses and can be shortly characterized by white or transparent cuticle without pores, three bands of teeth in the oral cavity, three macroplacoids and microplacoid in pharynx (richtersi group), smooth lunules under all claws, granulation on all legs, and eggs with reticulated conical processes without caps or spines. Pam. fairbanksi is a triploid species8 inhabiting various locations throughout the globe. The species is an omnivore, i.e., it feeds on algae, cyanobacteria, fungi, nematodes and rotifer10. However, dietary preferences have been observed to differ between juveniles and adults .The Phylum Tardigrada currently consists of th century12 suggests that microorganisms and small invertebrates should have a cosmopolitan distribution. Microscopic organisms are often considered cosmopolitan species, as, the presence of specific adaptations allows them to inhabit most environments. These adaptations include a) the possibility of easy passive dispersion , b) the presence of very resistant spore stages  that help to survive extreme conditions, and c) the presence of asexual or parthenogenetic reproduction, allowing for rapid increase in the number of individuals16. Cosmopolitism was strongly suggested for many tardigrade species in the past, however, the suggestion was later undermined (e.g. ref19). At present, we have strong and compelling evidence of a wide distribution of some tardigrade taxa, which means that we return to the concept of cosmopolitism of at least some species of tardigrades (e.g. ref22) which can support the hypothesis ‘Everything is Everywhere’ (EiE) for tardigrades. According to Gąsiorek et al21, “a species may be termed as cosmopolitan if it was recorded in more than one zoogeographic realm”. There are four tardigrade species known from more than one zoogeographic realm, i.e., Echiniscus testudo23 , Milnesium inceptum24 Morek, Suzuki, Schill, Georgiev, Yankova, Marley & Michalczyk, 2019, Pam. gadabouti22 Kayastha, Stec, Mioduchowska and Kaczmarek. 2023 and Pam. fairbanksi. Furthermore, two parthenogenetic species from the genus Paramacrobiotus, i.e., Pam. fairbanksi and Pam. gadabouti, are contenders as they show a wide distribution that supports the hypothesis EiE.The ‘Everything is Everywhere’ hypothesis, which was proposed at the beginning of the 20Pam. fairbanksi from all known localities of this species in Albania, the Antarctic, Canada, Italy, Mongolia, Poland, Portugal (Madeira) and the USA. We also discuss genetic and morphological differences between them and consider the general distribution of Pam. fairbanksi.In the present paper we compare different populations of 25. The moss samples  were dried post extractions and were deposited at the Department of Animal Taxonomy and Ecology, Institute of Environmental Biology, Adam Mickiewicz University in Poznań, Uniwersytetu Poznańskiego 6, 61–614 Poznań, Poland.Four moss samples from trees and rocks were collected in 2018 (Mongolia) and 2019   with the rotifers and nematodes added as food ad libitum. All cultures were kept in the environmental chamber at a temperature of 18°C and in darkness.Specimens of the populations from Albania, Canada, Madeira and Mongolia were cultured in the Department of Animal Taxonomy and Ecology  according to the protocol described by Roszkowska et al.28. Slides were then placed in an incubator and dried for two days at ca. 60°C. Dried slides were sealed with transparent nail polish and examined under an Olympus BX41.Specimens were extracted from cultures and prepared for light microscopy analysis. They were mounted on microscope slides in a small drop of Hoyer’s medium and secured with a cover slip29. Macroplacoid length sequence is given according to Kaczmarek et al.30. The pt ratio is the ratio of the length of a given structure to the length of the buccal tube, expressed as a percentage31. The pt values always provided in italics. Morphometric data were handled using the “Parachela” ver. 1.8 template available from the Tardigrada Register32. Tardigrade taxonomy follows Bertolani et al.33. Genus abbreviations follow Perry et al.34.All measurements are given in micrometers [μm]. Structures were measured only if their orientation was suitable. Body length was measured from the anterior extremity to the end of the body, excluding the hind legs. Buccal tubes, claws and eggs were measured according to Kaczmarek & MichalczykPam. fairbanksi was identified in vivo using light microscopy (LM). To obtain voucher specimens, DNA extractions were made from individuals using a Chelex® 100 resin (Bio-Rad) extraction method provided by Casquet et al.35 with modifications described in Stec et al.25. We sequenced three molecular markers, which differ in effective mutation rates: two nuclear fragments (18S rRNA and 28S rRNA) and one mitochondrial fragment (COI). All DNA fragments were amplified according to the protocols described in Kaczmarek et al.9, with primers listed in Before genomic DNA extraction, each specimen of 39. Comparison of obtained molecular markers with those deposited in GenBank and homology search were performed using BLAST application . The COI haplotypes were generated using the DnaSP v5.10.01 program41 and were translated into amino acid sequences using the EMBOSS-TRANSEQ application42 to check for internal stop codons and indels. Then all sequences obtained in our study, and the sequences downloaded from the GenBank database as originating from Pam. fairbanksi, were aligned with CLUSTALW using default settings. Alignment sequences were trimmed to 689, 572 and 574 bp for 28S rRNA, 18S rRNA and COI barcodes, respectively. The calculation for the uncorrected pairwise distances (p-distances) was performed for COI sequences using the MEGA X43.The amplified nuclear and mitochondrial barcode sequences were edited using the BIOEDIT softwarePam. fairbanksi were deposited at the Department of Animal Taxonomy and Ecology, Institute of Environmental Biology, Adam Mickiewicz University in Poznań, Uniwersytetu Poznańskiego 6, 61–614 Poznań, Poland.All obtained sequences have been deposited in GenBank (for the accession numbers please see Network 4.6.1.3 software (www.fluxuxengineering.com). The median-joining algorithm (MJ)44 and substitution rates with the weight of 3 for transitions and 1 for transversions (transition: transversion ratio (ti:tv)) were applied. The star contraction pre-processing was generated to delete all superfluous median vectors and links. Additionally, the maximum parsimony post-processing was calculated. In turn, signatures of population expansion, equilibrium or decline in Pam. fairbanksi were inferred from the neutrality tests calculation  computed in the DnaSP v5.10.01 program and Arlequin v.3.5. software47. Analyses were performed with 1 000 replicates.Reconstruction of genetic relationships among COI haplotypes and genealogical connections was carried out using the Pam. fairbanksi. Measurements of the body and buccal tube length  were used as the dependent and the populations as grouping variables. Normal distribution in residuals was checked using the Shapiro test. Other morphometric traits, i.e., stylet support insertion points (SSIP), external width of buccal tube (BTEW) and placoids  were also analysed. All the analyses were performed in R 4.1.349. The level of statistical significance was considered at p < 0.05. In the case of post hoc tests, only statistically significant results were presented. Principal Component Analysis (PCA) was performed using the R script from Stec et al.48. The analysis was performed for data from eggs and animals. For animals, both absolute values (raw measurements in μm)  and relative pt values  were used. For eggs, absolute values (raw measurements in μm) were used. All analyses were carried out using the R software program49. The “imputePCA” function of the R package “missMDA ver. 1.17” was used to impute missing data in the animal data set using the PCA imputation technique50. Cross-validation (function “estim_ncpPCA”) was used to determine the number of components utilized to impute the missing data. The PCA function of the software “FactoMineR ver. 2.3”51 was used to perform PCAs on the scaled data. The software “ggplot2 ver. 3.3.2”, “plyr ver. 1.8.6”, and “gridExtra ver. 2.3” were used to depict PCAs53. The presence of a structure in the PCA data was tested using a randomization approach on the eigenvalues and statistics according to Björklund54 and an in-house R script developed by MV in Stec et al.48. PERMANOVA was done on the PCs with the R packages “vegan ver. 2.5.6” and “pairwiseAdonis ver. 0.3”55, with the species hypothesis generated by phylogenetic techniques as the independent variable. Using the Benjamini-Hochberg correction, the -level for multiple post hoc comparisons was adjusted independently for adult and egg56. In total, 106 tardigrade specimens  were measured and later used in the analyses for animals. Furthermore, differences in egg morphology between populations were studied and tested using ANOVA. Egg bare diameter (EBD), full diameter (EFD) and processes height (PH) were characters for the populations used as the dependent variable to determine compared groups and Bonferroni corrections. In total, 100 tardigrade eggs  were measured and used in the analyses. All the analyses were performed in R 4.1.0. The level of statistical significance was considered at p < 0.05. Only statistically significant results were presented for post hoc tests.We used the Analysis of Variance (ANOVA) test with post hoc comparison of pairs of measurements, applying Bonferroni correction to statistically analyze the differences in morphometrics between different populations of Pam. fairbanksi populations was assembled in Corel Photo-Paint 2021.A map of the known distribution of Pam. fairbanksi and Pam. gadabouti. The ENM was performed with the use of the Maxent algorithm, ver. 3.4.4.48. MaxEnt performs the model with the fewest possible occurrence data and takes presence-only (PO) data. The model generates models of habitat appropriateness by handling continuous and categorical variables using regularization parameters58. The raster package in R was used to extract climatic raster values, and for ENM evaluation, version 0.3.1 of ENMevaluate in R was used. The bioclimatic variables available in MERRAclim Dataset 19 were used as environmental variables for Maxent modelling. We used MERRAclim Dataset because it provides a global set of satellite-based bioclimatic variables that includes Antarctica, which is one of the locations for Pam. fairbanksi. The 19 global bioclimatic datasets from the 2000s at 5 arcminutes resolution 59 consist of temperature layers (BIO1-BIO11) and humidity layers (BIO12-BIO19). The temperature layers are in degrees Celsius multiplied by 10 and the humidity layers are in kg of water/kg of air multiplied by 10000059. The receiver operating characteristic (ROC) plot’s area under curve (AUC) was used to assess the model’s accuracy57. AUC describes the relationship between the proportion of correctly-anticipated presences and the proportion of absences of mistakenly-projected species in the model60. The AUC gauges the effectiveness of the model with a value between 0 and 1. Furthermore, AUC values > 0.9 indicate excellent accuracy, 0.7 to 0.9 indicate good accuracy, and values below 0.7 indicate low accuracy62. The jackknife test was used to estimate the model’s variable relevance. The localities for Pam. fairbanksi are from Pam. gadabouti from Kayastha et al.22. The coordinate list is provided in SM.01 and the R script for ENM in SM.02.An ecological niche modelling (ENM) approach was used to predict the current potential distribution of 3No significant differences were shown by the ANOVA test performed on BL between the studied populations  and the Polish population (p=0.004), while pt values of specimens from the Madeiran population were higher than the Polish population (p=0.003) and pt values of specimens from the Alaskan population were higher than the Madeiran population (p=0.018) and the Mongolian population (p=0.0001)  . The ANOThe ANOVA performed on EFD measurements of eggs  showed significant differences between all the populations . Eggs inpt: ψ=13.14 p<0.001, ϕ=0.43 p<0.001; eggs: ψ=17.62 p<0.001, ϕ=0.50 p<0.001). The first two components of the PCA of animals’ absolute measured value (pt indices (The randomization test in PCA demonstrated that only the first two PCs explained greater variation than was anticipated by the null model (no data structure) for both animal and egg datasets (SM.11). As a result, only the initial two PCs were maintained and used for additional investigation and interpretation. Furthermore, the ψ and ϕ statistics of the PCA were significantly distinct from what they anticipated under the null assumption   that differed from it by one mutational step. One haplotype (haplotype H2 from Spain) differed from central haplotype H4 by two mutational steps. In several geographical regions, i.e., the USA, Albania, Italy, Poland and Spain there were regional endemic haplotypes. Surprisingly, the second haplotype that occurred in different localities was haplotype H1 and this haplotype was common for three populations, from Mongolia, Poland and Antarctica.The COI sequences of  0.003%) . In totaPam. fairbanksi from Albania, Canada, Madeira and Mongolia were 917–1547 bp-long , they showed only one nucleotide substitution. A comparison was performed with the sequences from the following geographical localities: Antarctica (GenBank: MN9603029), Poland (GenBank: MH664941-4263), USA (GenBank: EU03807864) and Italy (GenBank: MK041027-298). The 28S rRNA molecular marker was very conservative, and was 694–805 bp-long. No nucleotide substitution was found for all obtained sequences even after comparing (and trimmed to 689 bp) with GenBank sequences from Antarctica (GenBank: MN960306 – MN9603079) and Poland (GenBank: MH66495063). Nevertheless, one unidentified base was found in the sequence originating from the Polish population.In turn, the 18S rRNA sequences of  bp-long  and no nDemographic expansion was preliminarily tested based on the value of neutrality tests that confirmed a neutral model of observed polymorphism. Negative significant values for Tajima’s D were found, indicating a high number of low-frequency polymorphisms in the COI sequences dataset and potential population size expansion . In turnPam. fairbanksi and Pam gadabouti. The study is limited to bioclimatic variables. The stimulated model predicted good accuracy for the overall model with an AUC for Pam. fairbanksi of 0.826 and excellent accuracy for the overall model with an AUC for Pam gadabouti of 0.924. The suitability for Pam. fairbanksi seems moderate  is unlikely to be true. Similarly, the suggestions that harsh conditions in Antarctica may favor laying larger eggs while in cultures the eggs are smaller because of the lack of such selective pressure9 seems untrue as the egg size of specimens from Antarctica overlaps with egg sizes of specimens from Albania, Canada and Mongolia, which were sampled from cultured populations in the present study.Kaczmarek et al.66. Various studies combining COI variation and phenotypic plasticity were conducted throughout different invertebrates’ phyla, including tardigrades69, proving the marker’s accuracy in this group of organisms. The result showed high genetic homogeneity between organisms with wide geographical distribution together with clearly visible morphological differences known as phenotypic plasticity67.Cytochrome oxidase subunit I gene (COI) sequences is one of the most reliable barcodes to investigate genetic variation with phenotypic plasticity since COI is a genetic marker with a high genetic variation compared to multiple other DNA barcodes70 or corals71, suggesting that occasionally COI may fail as a barcode marker due to hybridization events. Many studies have already shown that Wolbachia  can increase the speciation rate and can affect COI haplotypes73. However, the nuclear markers tested for Pam. fairbanksi have been consistent for the studied populations. No molecular markers that correspond with morphological features in tardigrades have been suggested so far. Future studies with higher-resolution markers designed for intrapopulation variation should be performed to determine if any pattern of genetic diversity concordant with morphological variation can be observed.Furthermore, several studies uncovered data incongruence between mitochondrial and nuclear markers, e.g., for earthwormsPam. fairbanksi can be explained by phenotypic plasticity. This explanation is supported by the fact that populations from Poland and Antarctica share the same haplotype, H1, but vary in EFD measurements of eggs, which is the most variable trait in the analysis. Several distant geographic populations share the same COI haplotype, H1 or H4, yet it does not make them morphologically consistent within the haplotypes. These populations have, as shown in the study, a low evolutionary rate, and the inter-population variation develops under variable conditions experienced in different locations. The exact causes and mechanisms of the phenotypic plasticity in the morphology of adults and eggs of Pam. fairbanksi remains unknown, although, it has unsurprisingly been shown, that some physical traits differ in chosen cultured tardigrades depending on the temperature and food abundance7. If the morphological variation in Pam. fairbanksi is an effect of phenotypic plasticity, it is unclear which factors could cause various morphotype expressions. The specimens from Mongolia, Albania, Canada and Madeira that were measured in our study come from populations cultured in similar laboratory conditions but were started with different counts of founders of various ages, kept in variable densities and with different numbers of generations that had passed culture, so no answer can be proposed at this moment.Based on morphometric and genetic analyses, it is possible that the subtle morphological variation observed in geographically remote populations of 74), which tardigrades’ habitats clearly are. Furthermore, phenotypic plasticity has been widely observed in other invertebrates like corals (e.g. Pseudopterogorgia bipinnata Verrill 186475), scallops , marine invertebrates, gastropods , rotifers 77) and many more. No concordant genetic variation was observed, but a large and discrete differentiation of morphotypes was present and was always associated with external environmental factors such as temperature, predation risk and food availability82.Phenotypic plasticity, in morphology and other aspects of phenotype, such as life history traits, is seen as an advantage for thriving in heterogeneous environments 85 and the scorpion species Liocheles australasiae (Fabricus 1775) are parthenogenetic for multiple generations in captivity87 and are widely distributed89. Similar cases are found in many animals and plants (ref93)). However, Baker et al.84 also suggested that parthenogenesis indicates morphological variation as a result of epigenetic mechanisms. Furthermore, Mioduchowska et al.72 provided molecular evidence of the presence of the bacterial endosymbiont Wolbachia based on next generation sequencing in tardigrades. Wolbachia have an effect on the evolution as well as the ecology of their hosts, and have been found to cause effects including cytoplasmic incompatibility, feminization, male killing, and induced parthenogenesis94 It has been noted that at the intraspecific level, even individuals from the same population can undergo morphological changes in their characters to diversify within niches available to the species95. Similarly, Kihm et al.88 proposed epigenetic factors as a main cause for variability in tardigrade Dactylobiotus ovimutans egg morphology, although the population was cultured under controlled laboratory conditions. Despite being rare, it is known that intraspecific variation is caused by external environmental conditions, epigenetics and seasonality96. Therefore, it is also likely that the morphological differences that we observed in the present study might be due to epigenetic factors, as the studied populations were cultured under controlled laboratory conditions.The phenomenon where parthenogenetic  lineages occupy a wider geographical range, but sexual populations are restricted to a limited area, is termed ‘geographical parthenogenesis’97. It effectively predicts habitat suitability for rare and poorly studied taxa99. Pam. fairbanksi presence is linked to the presence of suitable microhabitats, like moss patches, and their life strategy can make them less likely to be affected by general climatic conditions. However, bioclimatic variables used in the study may be a good predictor of the possibility of the occurrence of suitable microhabitats. We investigated the possible distribution of Pam. fairbanksi and compared it with other widely distributed species of the genus Paramacrobiotus, i.e., Pam. gadabouti. Paramacrobiotus fairbanksi already reported from various continents exhibit a cosmopolitan distribution covering different types of environments, whereas Pam. gadabouti, although also potentially cosmopolitan, has a clear affinity to areas with a Mediterranean climate. Its distribution is poorly known due to lack of sampling in many habitats. Such differences clearly show us that even when we consider some of the species to be cosmopolitan, specific patterns of distribution can be completely different. However, we must also stress that the number of known localities for both species is relatively low and, in the future, when the number of records of these species will be higher, a distribution pattern may look different.Ecological niche modelling is an important and useful tool that has been used to address issues in many fields of basic and applied ecologyParamacrobiotus fairbanksi described originally from Alaska, USA, is now known from almost all zoogeographic realms. The identification of this species is possible based on morphometric characters alone because of the overlap in measurements of all measured structures. Moreover, the analysis shows low genetic variability among Pam. fairbanksi populations from various geographical locations, which may in general suggest that interspecies genetic variability in tardigrades is very low too or could be’ the effects of Wolbachia infection. The species fits the ‘Everything is Everywhere’ hypothesis and is an example of a parthenogenetic species with wide distribution. Despite very low genetic variation, some indiscrete morphological variations were observed. Since all the studied populations were cultured and bred in the same laboratory conditions, such variation may have been caused by epigenetic effects, and were not the result of different temperatures, food sources and seasonality.


Arctocephalus australis is a marine apex predator of Pacific and Atlantic waters, with a disjunct set of populations in Peru and Northern Chile [Peruvian fur seal (Pfs)] with controversial taxonomic status. We demonstrate, using complete genome and reduced representation sequencing, that the Pfs is a genetically distinct species with an admixed genome that originated from hybridization between the SAfs and the Galapagos fur seal  ~400,000 years ago. Our results strongly support the origin of Pfs by homoploid hybrid speciation over alternative introgression scenarios. This study highlights the role of hybridization in promoting species-level biodiversity in large vertebrates.Hybridization is widespread and constitutes an important source of genetic variability and evolution. In animals, its role in generating novel and independent lineages (hybrid speciation) has been strongly debated, with only a few cases supported by genomic data. The South American fur seal (SAfs)  Genomic analyses reveal the Peruvian fur seal originated from hybrid speciation between the Galapagos and South American fur seals. Homoploid hybrid speciation (HHS) refers to the stable establishment of hybrid populations attaining evolutionary independence from their parental donor species without change in ploidy  is endemic to the Galapagos Archipelago  is distributed in New Zealand and Southern Australia  has two disjunct distribution ranges: Most rookeries occur continuously from the southern Atlantic coast of southern Brazil to the southern Pacific coast of Chile. The second range begins 2000 km northward, runs from the north-central Chilean coast to the central Peruvian coast, and is concentrated in relatively few reproductive colonies, the largest in Punta San Juan (PSJ), central-southern Peru . Thustralia  (20). Thern Peru  (21). SeArctocephalus gazella) used as an outgroup, was 24.6× (±6.2) (table S1). The filtered whole-genome panel consisted of ~13.3 million single-nucleotide polymorphisms (SNPs) distributed in 3892 scaffolds. We also generated a double digest Restriction-site Associated DNA sequencing (ddRAD-seq) SNP dataset resulting in a filtered dataset of 47 individuals from all species and populations with a total of 3198 segregating SNPs distributed in 6168 scaffolds  . Our PerBy definition, an essential requirement of the HHS model is that the new hybrid lineage originated without change in chromosome number; specifically, it should not be polyploid  with both the whole-genome  and ddRAD-seq datasets. In both cases, species were well separated, and individuals of the same species clustered together, including the SAfs of different populations  .FST (ddRAD-seq data) was concordant with the above results, presenting a low to moderate population differentiation (0.03 to 0.09) between the four South American populations (table S3). FST between these localities and the Pfs was higher, ranging from 0.13 (Pfs-SAfs Chile) to 0.18 . FST between recognized species ranged from 0.20 (SAfs Chile-NZfs) to 0.40 (Gfs-NZfs) (table S3).Genetic differentiation between populations and species estimated with K = 3 had the lowest cross-validation error; fig. S5), with individuals of each species appearing as a genetically homogeneous group  grouped with the Galapagos individuals in the PCA . They alWe next estimated the phylogenetic relationships between the individual genomes using a multispecies coalescent species tree approach with ASTRAL-III . The genGiven the discordant phylogenetic position of the Pfs in different analyses, we next quantified the genomic frequency of the three different topologies considering Pfs, Gfs, and one of the SAfs genomes . We splif3 and f4-statistics to investigate signals of admixture between populations by splitting up the genome into 1331 blocks of 10,000 SNPs each. The f3-statistic explicitly tests whether a taxon of interest C results from admixture between two others (A and B): A significantly negative f3-statistic supports the admixture hypothesis, while a positive value is not informative ; signals were similar regardless of the choice of SAfs localities . Although substantially weaker, we also detected an introgression signal between the NZfs and Gfs. The f4-statistics use four-taxa topologies in the form , and when A is an outgroup, it allows testing past introgression between B and either C or D. A significantly positive f4 implies gene flow between B and D, and a significantly negative f4 implies gene flow between B and C. Here, A is either NZfs or Afs, B is SAfs or Gfs, and we fixed Pfs as D to ease visualization (this only affects the sign). Because the phylogenetic position of the Pfs as a sister to the Gfs or the SAfs was unclear, we tested both topologies. We found highly significant signals of gene flow between Pfs and both Gfs and SAfs (f4 < 0.003). These results strongly supported that the Pfs was involved in past gene flow with both the Gfs and the SAfs, although these statistics cannot discern the direction of the introgressions nor distinguish between a scenario of continuous gene flow and HHS.We applied the f-branch metric to summarize the f4 ratio values (an estimation of the ancestry proportions in an admixed population). We used the three SAfs genomes from different localities and the Pfs and Gfs as sister species. We found strong evidence of introgression (P < 0.01) between the Pfs and SAfs regardless of population choice for the SAfs  analysis . In thisf4 values as estimated above. The Pfs grouped with the SAfs, closest to the population from Chile and then to Argentina and Falklands, although the latter two groups had minimal branch lengths (fig. S11). However, about 49% of the Peruvian genome originated from an introgression event from the Gfs. Given the lack of differentiation between Argentina and Falklands samples of the SAfs, we ran a second analysis combining them as an Atlantic population (f3-statistics) showed similar results . In all four networks, the Pfs was found as a hybrid between Gfs and SAfs, with the genomic contribution of the Gfs being higher (~70%). A smaller (~6%) introgressive event from the SAfs to the NZfs was detected.Tg and Ts; Tg and Ts to 0 shows their posterior distribution tending to 0, which, in this limit, would render all scenarios equivalent to the HHS scenario (Tg and Ts to 300 generations increased the posterior probability of the HHS scenario to >0.99 (fig. S16).Because our results highly supported a hybrid origin for the Pfs, we next used an approximate Bayesian computation approach to test whether the species originated by HHS or by alternative hybridization scenarios  and is sscenario . ConversTo estimate when the hybridization resulting in the Pfs lineage would have occurred, we used a multispecies coalescent model (MSC) in the presence of introgression implemented in BP&P program. As input, we used the topology derived from the species network with the two hybridization events obtained with the previous methods  and 5000We have used a suite of phylogenetic and population genetic methods to explore the origin of the Pfs lineage. Despite different assumptions and temporal sensitivity, all methods concurred and suggested that the Pfs is an evolutionarily independent lineage that originated from an ancient hybridization, not a recent introgression event, between the Gfs and the SAfs. Furthermore, our ABC test strongly supported the hypothesis that the Pfs originated from instantaneous HHS . HHS shoFurthermore, data from the past demographic monitoring of Pfs populations suggest that Pfs individuals are fully viable and that PSJ and other rookeries are long-term reproductive colonies. For example, in a recent census during four reproductive seasons in Peru (2016–2019), there were individuals from all age classes  in all years (Some form of RI (extrinsic or intrinsic) between a new hybrid population and its putative parental species is necessary to initiate and maintain evolutionary independence , ~5000 km south of the Galapagos Islands  of any of its parental species. Besides, the Pfs originated >0.4 Ma, a time interval of comparable magnitude to the time of speciation for other recognized pinniped species (3) were obtained with piglet notch pliers from pups or recently dead animals found ashore, stored in 70% ethanol, and cryopreserved at −20°C. We used selective and nonlethal sampling without the application of immobilizing drugs. The appropriate catching and sampling permits were obtained from the appropriate authorities for the respective locations. Genomic DNA extractions were carried out with DNeasy Blood & Tissue Kit (QIAGEN) following the manufacturer’s protocol. DNA yield was quantified using a spectrophotometer (NanoDrop Lite), and quality was assessed on a 2% agarose gel.In addition to the previously sequenced genomes of the NZfs, Gfs, and SAfs (from Argentina)  were prepared in-house following Kess For each sample, 20 ng of genomic DNA was digested with restriction enzymes. P1 and P2 barcode combinations were ligated to the generated fragments for individual identification before pooling samples. DNA fragments between 355 and 555 bps were selected using 1.5% agarose gel electrophoresis. These fragments were purified with the Invitrogen PureLink Quick Gel Extraction Kit and magnetic bead purification to remove fragments below 250 bps. Genomic libraries were sequenced on an Illumina HiSeq 2500 platform using a single-end 2× 100-bp reads module.www.bioinformatics.babraham.ac.uk/projects/fastqc, accessed 1 March 2018). The closest reference genome available to the focal clade, the Afs A. gazella , and miscalling indels were locally realigned by Genome Analysis Toolkit v3.8  v0.929 .To estimate the ploidy level of our focal species , we used ploidyNGS, a python program that allows the identification of ploidy level using only the sequencing reads of the WGS data    following Lopes et al.  of our WGS samples were assembled by mapping raw reads with PALEOMIX v1.2.13.2 against the complete mitochondrial genome of We also sequenced an mtDNA control region fragment of the ddRAD-seq samples of the fur seals from the Peruvian coast (PSJ and Isla Foca) as described previously (https://github.com/simonhmartin/twisst (accessed 10 March 2022) and http://evomics.org/learning/population-and-speciation-genomics/2018-population-and-speciation-genomics/topology-weighting/.For Twisst analysis (topology weighting by an iterative sampling of subtrees) (f3 and f4-statistics (f3-statistic explicitly tests whether a taxon of interest C is the result of admixture between two others (A and B), considering the product of allelic differentials between (c − a)(c − b): Negative values suggest that allelic frequencies c are intermediate at many populations, which is consistent with admixture. In contrast, positive results are not informative about the admixture. f4-statistics uses unrooted four-population phylogenies to visualize shared genetic drift among taxa  topology, without invoking admixture, the allele frequency difference between A and B (a − b) and between C and D (c − d) should be unrelated and hence results in f4 = [(a − b)(c − d)] = 0. Significantly positive f4 implies gene flow between A and C or B and D. Significantly negative f4 implies gene flow between A and D or B and C. As used here, when A is an outgroup, it allows testing past introgression between B and either C or D. A significantly positive f4 implies gene flow between B and D, and a significantly negative f4 implies gene flow between B and C. Significant f4 values may also be interpreted as rejecting the given topology  and significantly negative (z < −3) values reject the null hypothesis  and the f4-ratio statistics  and derived (B) sites in a four-taxa asymmetric phylogeny {, O} with P1 to P3 being in-groups and O being the outgroup. Under the null hypothesis that P1 and P2 descend from an ancestor that diverged earlier from the ancestral P3, derived alleles B should be found at the same proportion in P1 and P2. Gene flow between P2 and P3 will lead to an excess of ABBA patterns and a positive D-statistic; gene flow between P1 and P3 will lead to a surplus of BABA patterns and a negative D-statistics C metric uses f4 ratio to assess gene flow evidence in branches and internal descendants of a hypothesized phylogeny. The (bf)C statistic reflects the excess sharing of alleles between population or taxon C and the descendants of the branch labeled b  to transform the VCF file to the PHYLIP format as needed. We first considered all three SAfs genomes  together (run_hyde.py) and next tested each SAfs individual .The HyDe program performs hypothesis tests on triples of taxa to detect hybridization events, including hybrid speciation, using phylogenetic invariants estimated from the genomic data  to estimate admixture graphs. The SNP dataset used above for the Dsuite analyses was thinned to one SNP each 25 kb, resulting in 96,026 SNPs with no missing data. The best-fit graph was estimated using five independent runs of command find_graphs. Confidence intervals were estimated using qpgraph_resample_snps with 100 bootstraps. The above methods used statistics  estimated from individual SNPs.Given the evidence of admixture between the taxa from the initial analyses, we next estimated the evolutionary history of the group while accounting for introgression. First, we used TreeMix, a method that estimates the patterns of population splits and historical events of admixture by using a set of SNPs of extant populations. Next, we used admixtools2 in R  and a maximum of three reticulations to reduce runtime. On the basis of the previous analysis , we considered SAfs from Argentina, Falklands, and Chile as a unique taxon. The hybridization network tree was visualized with Dendroscope v3.7.4.We also inferred phylogenetic networks (that account for ILS and introgression) with PhyloNet v3.8.2  as implemented in BP&P v4.1.4 (We used the approximate Bayesian computation method implemented on DIYABC v2.1.0 (Tg and Ts). We consider this a reasonable minimum time for an offshoot population to be considered a genetically different population from its parent and for the method to be able to statistically differentiate these scenarios from the HHS “instantaneous” origin . However, to test the effect of this limitation on our results, we ran two additional analyses: one analysis with Tg and Ts set to a minimum of 300 generations and the other setting these parameters to a minimum of 0. Note that as Tg and Ts approached 0, this essentially renders the alternative scenarios 2 to 4 equivalent to the HHS scenario.The distribution of the priors for the parameters is presented in fig. S14. The population size priors for the three species were set to a large interval but those for the ancestral populations that ultimately originated the Pfs (in scenarios 2 to 4) were narrower . As priors, for the time of the divergence of Gfs and Pfs and the hybridization, we used the estimates from the BP&P analyses. For the non-HHS  scenarios, we first set a minimum of 100 generations between the origin of the population(s) and the introgression or hybridization that originated the Pfs  was computed with the logistic regression based on the 40,000 simulated data closest to the observed data and the four intermediate values. In addition, pre-evaluation scenario-prior combinations and model checking for the best scenario were performed with PCA with the default options. Model checking, as described in DIYABC manual, “is a PCA in the space of summary statistics using datasets simulated with the prior distributions of parameters and the observed data as well as datasets from the posterior predictive distribution that are represented on each plane of the PCA. If the model fits the data well, then one should see on each PCA plane a wide cloud of datasets simulated from the prior, with the observed dataset in the middle of a small cluster of datasets from the posterior predictive distribution.” The settings for this analysis were logit transformation of parameters on 10,000 simulated data closest to the observed data and 1000 datasets simulated from the posterior. We used DIYABC analyses only to test the evolutionary scenarios, not to estimate the time parameters; as in DIYABC with ddRAD-seq SNP dataset, absolute times are not informative . We additionally estimated the posterior distribution of the


Gymnema sylvestre (Retz.) R. Br. ex Schult. is a well-known medicinal plant against diabetes in India. There is as such no organized cultivation in India, and the plant is still being collected from the wild for their therapeutic uses. It is, therefore, important to estimate the genetic diversity and population genetic structure of G. sylvestre to ascertain the genetically diverse germplasm. The present study, therefore, was undertaken to analyze the genetic variability in 118 accessions belonging to 11 wild populations of G. sylvestre using directed amplification of minisatellite-region DNA (DAMD) and inter simple sequence repeats (ISSR).H = 0.26, I = 0.40, PPL = 80.89%) at a species level, while the average genetic diversity at the population level was low. Among the 11 populations studied, PCH and UTK populations showed maximum genetic diversity, followed by KNR and AMB, while TEL population revealed the lowest genetic diversity. AMOVA and Gst values (0.18) revealed that most of the genetic variations are found within populations and very less among populations, and higher gene flow (Nm = 2.29) was found to be responsible for the genetic homogenization of the populations. The clustering pattern resulting from the UPGMA dendrogram was in congruence with STRUCTURE and PCoA, segregating all the 11 populations into two main genetic clusters: cluster I  and cluster II (populations of South India). The clustering patterns obtained from all three statistical methods indicate that the genetic structure in G. sylvestre populations corresponds to the geographical diversity of the populations and represents a strong genetic structure.The present genetic analyses of 11 populations with 25 markers (8 DAMD and 17 ISSR) revealed significant genetic diversity (The genetically diverse populations identified during the present study could be a potential genetic resource for further prospecting and conserving this important plant resource.The online version contains supplementary material available at 10.1186/s43141-023-00497-7. Gymnema R. Br. (Apocynaceae) comprises approximately of 37 species worldwide [Gymnema sylvestre (Retz.) R. Br. ex Schult., commonly known as “Gurmar” or “Madhunashini,” is an important medicinal plant because of its reported anti-diabetic properties [G. sylvestre is a perennial, slow-growing, woody climber, with very small pale yellow-colored flowers in lateral umbel cyme that reaches up to 8–10 m height or more on the host plant. The presence of various triterpenoids such as gymnemic acid, gymnemasaponins, gymnemosides, gymnemanol, gymnemagenin, and gymnemasin along with several other glycosides such as gymsyloside F and gymsyloside G makes it a valuable plant in pharmaceuticals [G. sylvestre also has hypolipidemic, hepatoprotective [G. sylvestre has been used as an important ingredient in the preparations of many poly-herbal formulations for diabetes such as D-400 or Diabecon [The genus orldwide , and aboorldwide . Among toperties . It has operties . In Indioperties , 6, coveeuticals . Apart fotective , neuroprotective , immunomotective , cardiopotective , anti-inotective , anti-caotective , antibacotective , and antotective . G. sylvDiabecon , IME-9 [Diabecon , and BGRDiabecon .G. sylvestre is one of the 242 highly traded medicinal plants in India. The increased commercial demand for this highly valuable plant species has resulted in its overexploitation by local traders, and a big threat is operating on the species in the wild [G. sylvestre has been declared near threatened (NT) in Maharashtra, vulnerable (VU) in West Bengal, Madhya Pradesh, Chhattisgarh, Andhra Pradesh, and endangered (EN) in Rajasthan [the wild . In addithe wild . Accordiajasthan . It is tEphedra foliata [Curcuma [Bergenia ciliata [Illicium griffithii [Leptadenia pyrotechnica [Rauwolfia tetraphylla [Decalepis salicifolia [There are many molecular markers available these days to analyze the genetic diversity and population genetic structure in plants. The most frequently used molecular markers are directed amplification of minisatellite-region DNA (DAMD)  and inte foliata  Curcuma [Curcuma , Bergeni ciliata , and Illiffithii . ISSR matechnica , Rauwolfraphylla , and Decicifolia  of the fG. sylvestre using molecular markers [G. sylvestre using a combination of DAMD and ISSR markers seems to be a maiden attempt.There are a few reports available on the genetic diversity of  markers –40. TherG. sylvestre. A closely related species Tylophora indica (Burm. f.) Merr. was used as the out-group . In the present analysis, we have considered a total of 118 accessions of 11 populations of Total genomic DNA from silica-dried leaf tissues was isolated using the CTAB method . The quaThe 20 DAMD markers available in the public domain  were cusG. sylvestre, of which eight markers resulted in distinct and reproducible fragments that were subsequently used for further amplification of the entire set of 11 populations. The amplification with DAMD marker was carried out in a 20 μl reaction mixture containing 4 μl of template DNA (80 ng), 2 μl of 10 mM dNTP mix (2.5 mM each dNTP), 1.2 μl of 25 mM MgCl2, 2.5 μl of suitable 10 × assay buffer (Taq Buffer A) supplied along with the enzyme, 1.5 μl of 10 μM primer, and 0.33 μl of 3.0 U Taq DNA polymerase  using a Proflex PCR System . After initial denaturation at 94 °C for 4 min, each cycle consisted of 1 min of denaturation at 94 °C, 2 min of annealing at (50 °C–55 °C), and 2.5 min extension at 72 °C, along with 7 min final extension at the end of 40 cycles. The amplification pattern of 118 G. sylvestre individuals is shown by the representative gel profile of marker FvIIex8C  was carried out in a 20 μl reaction volume, containing 1 μl of 10 mM dNTP mix (2.5 mM each dNTP), 1 μl of 25 mM MgCl2, 2.5 μl of suitable 10 × assay buffer (Taq Buffer A) supplied along with the enzyme, 1.5 μl of 10 μM primer, and 0.33 μl of 3.0 U Taq DNA polymerase  using a Proflex PCR System . After initial denaturation at 94 °C for 4 min, each cycle consisted of 1 min of denaturation at 94 °C, 1 min of annealing at (50 °C–54 °C), and 2 min extension at 72 °C along with 7 min final extension at the end of 35 cycles. The amplification pattern of 118 G. sylvestre individuals is shown by the representative gel profile of marker UBC-809  were finally selected and profiles of the complete set of 118 individuals of Z-statistics to test the correlation (r) between the three pairs of datasets  and to ascertain goodness of fit between these markers computed using MXCOMP module of NTSYS-pc software version 2.02e [G. sylvestre individuals with the out-group species which was visualized in MEGA11 software [Data analysis with the two markers (8 DAMD and 17 ISSR) was carried out for all 25 reproducible profiles showing consistent banding patterns. A binary data matrix was generated indicating the presence (1) or absence (0) of a band for each marker in the assay. The data matrices  were analyzed statistically to evaluate the relative informativeness of the two markers independently and cumulatively, by calculating the percentage of polymorphic loci (PPL), polymorphic information content (PIC), resolving power (Rp), diversity index (DI), effective multiplex ratio (EMR), and marker index (MI) for each marker , 44. Manon 2.02e . Sequenton 2.02e  using thsoftware . Geneticsoftware  were calsoftware  both by PPL, observed number of alleles (Na), effective number of alleles (Ne), Nei’s gene diversity (H) [I) [Ht), genetic diversity within populations (Hs), Nei’s genetic differentiation coefficient among populations (Gst), and gene flow among populations (Nm) were calculated in POPGENE program version 1.32 [The 11 populations considered in the present study were assumed to be in Hardy–Weinberg equilibrium for estimating the parameters of genetic diversity at population and species levels. The various genetic diversity parameters viz., sity (H) , Shannon (H) [I) , total gion 1.32  using cuion 1.32  using thion 1.32 .G. sylvestre were done in GenAlEx program version 6.5 [r) between pair-wise geographic and genetic distances of 11 populations and to evaluate the isolation by distance (IBD) model using cumulative marker data.Hierarchical partitioning of genetic variability at three strata, i.e., within populations, among populations, and among regions were examined by analysis of molecular variance (AMOVA) and clustering of populations in principal coordinate analysis (PCoA) for spatial representation of the relative genetic distances among all the individuals of sion 6.5  using cusion 6.5  was alsoK with prior population information was inferred using the admixture model with correlated allele frequencies. The program was run 20 times for each “K,” ranging from 1 to 12. Each run consisted of a burn-in time of 50,000 and a MCMC replication of 150,000. The number of K was obtained in a web-based python program STRUCTURE HARVESTER version 0.6.94 [K” groups that best fit the data.A cumulative dataset consisting of a set of individuals genotyped using multilocus markers was evaluated for clustering of populations using the Bayesian approach (Markov chain Monte Carlo (MCMC)) implanted in STRUCTURE software version 2.3 , 58. Thin 0.6.94  by implen 0.6.94 . STRUCTUA total of 155 loci were amplified with 8 DAMD markers in 118 individuals of 11 populations, with an average of 19.38 loci per marker. Out of these 155 loci, 128 were polymorphic corresponding to 82.58% polymorphism, with an average of 16 polymorphic loci per marker. The size of amplified products varied approximately from 170 to 1600 bp. Among the 8 DAMD markers, 33.6 showed maximum polymorphism (90.48%), whereas the FvIIex8 showed minimum (71.43%). The marker URP38F came up with the maximum Rp value (37.86), whereas HBV with minimum (21.44). The PIC value was found to be maximum (0.30) for the marker FvIIex8c, whereas minimum (0.20) for 14C2. Among the 8 DAMD markers, two DAMD markers were found to be reasonably informative with PIC values > 0.25 and < 0.50 , whereas the remaining six markers were slightly informative with their PIC values < 0.25  whereas the UBC-866 showed minimum (50.00%). The marker UBC-835 came up with the maximum Rp value (30.83), whereas UBC-840 with minimum (13.05). The PIC value was found to be maximum (0.33) for the marker UBC-842 whereas minimum (0.12) for UBC-808. Out of 17 ISSR markers, seven markers were found to be reasonably informative with PIC values > 0.25 and < 0.50 , whereas rest of the ten markers were slightly informative with their PIC values < 0.25  revealed 80.89% polymorphism with PIC, Rp, DI, EMR, and MI values of 0.24, 24.41, 0.30, 11.76, and 3.51, respectively (Table r = 1) for each pair of data sets. The Mantel test indicates the effectiveness and suitability of both marker systems in estimating the genetic diversity and population genetic structure in natural populations of G. sylvestre  were found almost equal for both the marker systems. However, the Rp, EMR, and MI values of the DAMD were found to be higher  than the ISSR  indicating the higher informativeness of DAMD over ISSR markers in estimating the genetic diversity of Na, Ne, H, I, and PPL; the PCH  and UTK population  were found to be the most diverse, closely followed by KNR  and AMB population , whereas TEL population was found to be least diverse  among 11 populations of G. sylvestre , but the average genetic diversity of a population  was found much lower than the genetic diversity at the species level. This could be due to the differences in genetic diversity of different populations, from PCH and UTK (PPL = 59%) to TEL (PPL = 29.37%) with an average of 45.12% polymorphism per population. In the total genetic diversity of G. sylvestre (Ht = 0.26), most of the part was found to be contributed by genetic diversity within populations  closely followed by Central-Indian region with four populations , while the North-Indian region with two populations showed the lowest genetic diversity , which indicates that of the total genetic variations of species, there are only 18% genetic variations among populations, and most of the genetic variations, i.e., 82%, are within populations. This means that these 11 populations are genetically less differentiated from each other (Table Nm = 2.29) among 11 populations which is responsible for lowering the genetic differentiation among populations , most of the part  was found to be occupied by genetic diversity within populations , followed by among regions (22%), and low among populations (16%) Table . This reer Table . This wans Table . The popns Table .Table 4AG. sylvestre populations revealed a very strong positive correlation  between geographic distance and genetic distance, indicating that the current genetic structure of the G. sylvestre populations is according to the geographical distances and isolation between populations  to 0.9627 (BDK v/s LAL) and the inter-population genetic distances ranged from 0.0380 (BDK and LAL) to 0.2142 (BDK v/s KNR). From these values, the genetic identities and genetic distances are found to be congruent with the geographical distances between populations Table . Mantel G. sylvestre into two major clusters: cluster I contained all six populations of the North and Central Indian region while cluster II contained all five populations of the South Indian region. In cluster, I, the BDK and LAL populations of the North Indian region are clearly separated out as an independent sub-cluster Ia from the sub-cluster Ib of four Central Indian populations . In the sub-cluster Ib, the Chhattisgarh population (DHM) clearly separated out as an independent sub-cluster Ib2 from sub-cluster Ib1 of the rest of the three populations of Madhya Pradesh . In cluster II, MBL and TEL populations were segregated out as two different independent sub-clusters  from the rest of the three populations  comprising sub-cluster IIb2. Furthermore, in this sub-cluster IIb2, the KNR population got segregated as an independent sub-cluster IIb2b from the sub-cluster IIb2a of AMB and UTK populations . Cluster I comprised all 60 individuals of six populations of North and Central India, while cluster II contained rest of the 58 individuals belonging to five populations of South India. The three axes showed 48.62%, 64.59%, and 76.64% cumulative variations, respectively  of G. sylvestre from 11 assumed populations. The cluster I contained all 60 individuals from six natural populations of North and Central India , which showed an average ancestry membership participation coefficient of 95.5%  to the inferred cluster I. Cluster II contained all 58 individuals from five natural populations of South India  which showed an average ancestry membership participation of 93.9%  to the inferred cluster II. Therefore, the Bayesian assignment of 118 G. sylvestre individuals of the 11 assumed geographic populations emerged as the existence of two genetic clusters (Table Fst) which was obtained from STRUCTURE analysis for the two genetic clusters was 0.37 and 0.26, respectively, which indicates that there is adequate genetic differentiation within each genetic cluster.The UPGMA-based population dendrogram clustered all 11 assumed populations of ely Fig. . BayesiaG. sylvestre are dwindling due to its exploitation for therapeutic uses in traditional and modern medical systems, thorough information on the species genetic diversity and population genetic structure is of utmost priority for its sustainable use, conservation, and management plans. Therefore, the present study was undertaken to analyze the genetic diversity and population structure in 11 representative populations of G. sylvestre sampled from the North, Central, and Southern parts of India. The genetic diversity of a species is the product of billions of years of evolution, and it should be studied and protected for a healthy ecosystem and its functions. As the genetic variations do not recover as quickly as population size, it must be preserved appropriately. The varied gene pool of a species allows it to adapt to the changing climatic circumstances and anthropogenic stress so that it can grow and persist in nature, and it is also significant in its environment because it supports the diversity of other related species [As the wild populations of  species . Genetic species –64.Trichosanthes dioica and Mangifera indica, SPAR (single primer amplification reaction) techniques involving more than one DNA marker system produce more realistic genetic variation [G. sylvestre were either limited to a narrower geographical area or had fewer individuals or both [G. sylvestre was used in the analyses of genetic diversity in 11 natural populations of G. sylvestre which revealed high levels of polymorphism by both the markers independently (DAMD = 82.58% and ISSR = 79.93%) as well as cumulatively (80.89%), indicating that G. sylvestre has a broad genetic base. Each marker system was found to be capable of detecting considerable genetic diversity in G. sylvestre demonstrating their discriminative efficacy and application in the current investigation. ISSR polymorphism (79.93%) obtained from the present study corroborates with earlier studies carried out in G. sylvestre [G. sylvestre [G. sylvestre populations with minisatellite marker system (DAMD marker) showing considerable genetic polymorphisms (82.58%) which is reinforced by the studies conducted in other medicinally important plant species from tropical or sub-tropical climate of India such as Jatropha curcas (DAMD = 92.02%) [Musa acuminata (DAMD = 88.46%) [Curcuma longa (DAMD = 84.40%) [Ephedra foliata (DAMD = 79.70%) [G. sylvestre.Genetics has a predominant role in natural population conservation. The increasing importance of genetics in conservation may be attributed to the availability of various kinds of molecular markers, and they also have an impact on our capacity to answer important conservation concerns . Previouariation , 68. All or both –40. Usinylvestre , 34, 36.ylvestre . Present 92.02%) , Musa ac 88.46%) , Curcuma 84.40%) , and Eph 79.70%) . In the G. sylvestre revealed that PCH and UTK populations have maximum genetic diversity closely followed by KNR and AMB populations while TEL showed the least genetic diversity. Among the four highly diverse populations, three  are from the South-Indian region and one (PCH) is from the Central Indian region. The high genetic diversity of these three South-Indian populations  corroborates with the high diversity found at lower latitudes as compared to higher latitudes. These populations are having lowest three latitudes among all the 11 studied populations. Many previous studies have also come up with the idea that high genetic diversity is found in plants and vertebrates at lower latitudes [G. sylvestre populations revealed a considerably high level of genetic diversity at the species level  which is comparable to high genetic diversity indices value of other outcrossing plant species like Justicia adhatoda  [Trifolium repens  [Asarum mandshuricum  [H = 0.26) for G. sylvestre was found to be higher than the Nei’s gene diversity calculated for dicotyledons (H = 0.17), long-lived perennials (H = 0.18), and widespread plants (H = 0.18) [G. sylvestre (H = 0.26) is within the range of “0.17–0.33” which has been estimated earlier for outcrossing plants [H is also comparable to the previously estimated value of H  in G. sylvestre [G. sylvestre has prominent pollinia and entomophily for cross-pollination, and the most frequent insects for cross-pollination are from the order; Hymenoptera (bees and wasps), Lepidoptera (butterflies and moths), and Diptera (flies) [G. sylvestre could be predominantly explained by its life form , breeding system (outbreeding due to conspicuous entomophily), seed (comose seed) wind dispersal mechanism, and geographic range (widely distributed in the entire peninsular Indian region). Generally, long-lived perennials with an outbreeding system, wind dispersal mechanism, and wide geographic spread have a high genetic diversity [G. sylvestre (H = 0.26) was found to be remarkably high, the average genetic diversity per population of G. sylvestre (H = 0.16) was found significantly lower than this total genetic diversity for the species because the average genetic diversity per population gets reduced by differences in genetic diversity by populations. As different populations have to face different climatic conditions, as well as their respective population size also differ from one another so, each population prevails in a different amount of genetic diversity. However, when we compare our estimated genetic diversity of G. sylvestre (H = 0.26) with other species of its group Apocynaceae; Asclepiadoideae  [G. sylvestre which are responsible for its high genetic diversity, the increasing anthropogenic pressure like habitat destruction and fragmentation and global climate change might be reducing its genetic diversity and fitness. Another important feature of G. sylvestre which was observed in the wild is that it also produces new progenies by clonal propagation and, in nature, its seed germination rate is also very poor [The intra-population genetic diversity analysis for 11 natural populations of atitudes . Also, tatitudes . As the  = 0.39) , Trifoli = 0.44) , and Asa = 0.42) . In gene = 0.42) , 77. Nei = 0.18) . The Neig plants . The valylvestre . Like ma (flies) . Besidesiversity . Outcros = 0.31) , it seemery poor . These tGst ranges from zero to one, and the value of Nm ˃ one and ˂ four is considered moderate [Gst = 0.18) obtained from the present analyses showed low genetic differentiation among 11 different populations of G. sylvestre which was created by a moderate rate of migration of genetic materials (Nm = 2.29) among these 11 populations. Being an outcrossing species, the significant gene flow (in the form of pollen and comose seeds) happens to be responsible for lowering the genetic differentiation among populations of species, although it is not proven yet that either entomophily or comose seeds are contributing more towards the gene flow. AMOVA also revealed only 16% of genetic variations present among the populations and most of the genetic variations are present within populations (62%). Results obtained from Gst, AMOVA, Ht, and Hs showed that of the total genetic variations of the species, most of them are occurring within the populations. In a former study, it was found that the outcrossing plant species with wind dispersal mechanism and long-life spans like G. sylvestre has maximum genetic variations within populations while the selfing plant species have maximum genetic variations among populations [Genetic drift, natural selection, recombination, and mutation are the major evolutionary forces responsible for making each population of a species genetically distinct from another population of the same species; therefore, population diversification occurs and over millions of years of time evolution of new species takes place, as these evolutionary forces are helping in maintaining the required genetic differentiation between different populations of a species and also provide the basis for local adaptation to different climatic conditions for each population which is crucial for the evolutionary potential of species. On the other hand, gene flow among populations is the unifying force that binds geographically separated populations into a single evolutionary unit-the species. Without gene flow between populations, populations will become genetically distinct from one another, and if extinction happened to one population, that would result in a significant loss of a particular genetic variation . The valmoderate . The valulations .G. sylvestre were in congruence with geographical diversity. The IBD model also suggested that the current genetic structure of 11 different populations is in accordance with the geographical distances. Population genetic structure analyses using STRUCTURE resulted into two major genetic clusters (K = 2) from the 11 distant populations of G. sylvestre suggesting that the moderate amount of gene flow (Nm = 2.29) which has homogenized the nearby populations thus highlighting two major genetic clusters prominently, with an average 5.3% admixing of individuals in which the cluster I showed 4.5% admixing to cluster II and cluster II showed 6.1% admixing to cluster I. Most of the admixing were shown by the peripheral populations like DHM = 9.3%  and TEL = 22.9% (from South Indian region), respectively. The population genetic structure of G. sylvestre with two genetic clusters or two genetic groups (K = 2) obtained from the present analyses reveals an intermediate type of species sub-division which is strictly in congruence to the geography and can also be called two geographical groups: the first group of North and Central Indian individuals and the second group of South Indian Individuals. These two genetic clusters show local adaptation to two different geographical areas, respectively, which is good for the evolutionary potential of a species. Within each genetic cluster, there is significant genetic divergence among populations which means all the populations are also locally adapted. The UPGMA and PCoA analyses have supported the result of STRUCTURE as they also came up with two genetic clusters in G. sylvestre.The genetic identities and genetic distances between 11 populations of G. sylvestre using cumulative marker data (ISSR + DAMD) with a larger dataset covering a large niche of species were thought to be more informative than previous studies in the species. Considering the medicinal importance of G. sylvestre, overexploitation, and depleting populations, different conservation strategies are required for the protection of diverse populations to maintain the species gene pool. Populations like PCH, UTK, KNR, and AMB with maximum genetic diversity should be prioritized for conservation, whereas populations with low genetic diversity, such as MBL and TEL, require adequate management to maximize the diversity. Besides, there is an urgent need to develop promising tissue culture protocols to increase the regeneration process to safeguard existing natural populations and to introduce maximum individuals in impoverished areas to maintain diversity. Results obtained from the present study may be helpful in the identification, collection, and prioritization of genetically varied G. sylvestre individuals for its improvement and conservation. The establishment of commercial agro-techniques and standardized harvesting protocols may reduce the pressure on populations of medicinal plants growing in the wild.Genetic polymorphisms from the cumulative data were found to be more appropriate as single type of molecular marker does not provide the best estimate of genome-wide variability in an organism. Comprehensive analyses of genetic diversity and population genetic structure in Additional file 1:Fig. S1a. A representative profile of G. sylvestre DNA (Gs001 to Gs118) amplified by DAMD primer FVIIex8c. ‘M’ represents a low range DNA ruler (100bp-3kb). Outgroup species DNA is shown at the last (OG). Fig. S1b. A representative profile of G. sylvestre DNA (Gs001 to Gs118) amplified by ISSR primer UBC-809. ‘M’ represents a low range DNA ruler (100bp-3kb). Outgroup species DNA is shown at the last (OG).Additional file 2:Fig. S2. Correlation between geographical and genetic distances of populations of G. sylvestre in India, showing geographical distance (in Km) in X axis and Nei’s genetic distance (cumulative marker data) in Y axis.Additional file 3:Fig. S3. Bayesian analysis of 118 individuals of G. sylvestre in STRUCTURE software showing, a. Bar plot representing individuals arranged according to its most likely ancestry b. Evanno table highlighting that value of K which describes the genetic groups of species c. Graph showing the peak corresponding to that value of K which tells genetic groups of species estimated by Evanno method.Additional file 4:Table S1. Mantel Z-statistics carried out for three pairs of data matrices  in G. sylvestre.Additional file 5:Table S2. Region-wise diversity statistics of G. sylvestre calculated for the three major regions using POPGENE software.