Lobaria pulmonaria and two Macaronesian endemics, L. immixta and L. macaronesica, based on microsatellites. We utilize population genetic approaches to explore population subdivision and evolutionary history of these taxa on the Canary Islands, Madeira, Azores, and the western Iberian Peninsula. A common feature in all species was the deep divergence between populations on the Azores, a pattern expected by the large geographic distance among islands. For both endemic species, there was a major split between archipelagos. In contrast, in the widespread L. pulmonaria, divergent individuals were distributed across multiple archipelagos, suggesting a complex evolutionary history involving repeated migration between islands and mainland.Macaronesia is characterized by a high degree of endemism and represents a noteworthy system to study the evolutionary history of populations and species. Here, we compare the population-genetic structure in three lichen-forming fungi, the widespread  Many of the world’s volcanic island systems are characterized by radiations in specific genera6. In volcanic islands, one main driver of environmental change is the erosion stage of the island, which creates substantial environmental change on a time scale of millions of years and can lead to massive altitudinal differences and hence, to differences in plant communities7.The study of large-scale geographic genetic patterns can provide valuable insight regarding the evolutionary history of taxa. Archipelagos of volcanic origins are of a particular interest because one can safely assume the absence of habitat connectivity between islands. Exceptions exist in areas where sea levels have receded, leading to connectivity between islands surrounded by shallow sea. Volcanic islands are fascinating systems to study owing to their isolation from large mainland populations, and their high degree of endemism4. Island groups within Macaronesia have been the cradle of recent radiations in several plant and fungal genera10. Thus, due to its complex, but well-known volcanic history, its geographic isolation from mainland sites, its high diversity and high degree of endemism, Macaronesia has for a long time been a major playground for evolutionary biologists and biodiversity researchers.One of the most distinctive places in the world to study the evolutionary history of taxa is Macaronesia, a group of volcanic archipelagos in the Atlantic Ocean, consisting of the Azores, Madeira and nearby islands, the Canary Islands, the Selvagem Islands and the Cape Verde Islands. Macaronesia harbors a highly diverse biota, comprising many endemics12 investigated the genus Roccella in North America and Europe including Macaronesia and found that North American and Macaronesian species formed sister clades. Only a single species in the genus, R. elisabethae, was found to be endemic to Macaronesia. Sérusiaux et al.4 found that multiple lichen fungi belonging to the genus Nephroma were neoendemics which originated in Macaronesia and subsequently spread to the European continent.However, few prior studies have investigated the genetic relationships among lichenized fungi on the Macaronesian islands. Tehler et al.Lobaria pulmonaria and two closely related taxa, the Macaronesian endemics L. immixta and L. macaronesica, which are sister taxa14. Lobaria pulmonaria is a lichen associated with old forest habitats in Europe, Asia, Africa and North America. All three species are epiphytes and share the same primary photobiont, the green-alga Symbiochloris reticulata, which is mainly vertically transmitted via soredia in L. pulmonaria, although photobiont switches do occur occasionally16.We used six fungal nuclear microsatellite loci to investigate the geographic genetic patterns in three lichen fungi, the wide-spread Lobaria lichens, i.e. with the woody plant species the lichen is growing on: laurel trees and heather.Using population genetic approaches on a large dataset comprising microsatellite repeats, we compare the genetic patterns across species. Second, we explore the genetic relationships among geographic regions in each species. Finally, we compare our results with the phylogeographic patterns of phorophyte species of the L. immixta, 21.7% in L. macaronesica, and 24.6% in L. pulmonaria. These results illustrate that a considerable percentage of thalli in each species were morphologically poorly developed so that reliable morphology-based species identifications were not possible, and they caution against uncritically including poorly developed specimens into population genetic studies of lichen-forming fungi.To identify the lichen species, we confirmed our field identifications with quantitative RealTime PCR. We found 16.5% of mismatches between field species identifications and molecular identifications in L. immixta, population trees indicated that populations on the Canary Islands and Madeira were related to one another, but also isolated, as indicated by them being occupied by separate clades , with minimal gene flow between these groups and the Azores cluster. The Iberian genotypes grouped with those from the Azores  geographic sampling. The site in Newfoundland, Canada showed some similarity to sites on the Iberian Peninsula and the single site investigated in Ireland showed similarity to the Azores , indicating substantial population subdivision. The amount of genetic differentiation between populations differed between species, with L. immixta showing lowest and L. pulmonaria showing the highest differentiation among Madeiran populations.There was high genetic differentiation among archipelagos for all species, as indicated by highly significant pairwise FST values and geographic distance in linear models. The relationships were strongest and explained the most variance in Lobaria immixta (R2= 0.48), followed by L. macaronesica (R2 = 0.38). Lobaria pulmonaria had a statistically significant, but somewhat weaker signal of isolation by distance, where the geographic distance explained only 7% of the variance in pairwise FST data.All species showed significant isolation by distance Fig. , as indiLobaria pulmonaria and its two endemic relatives, L. immixta and L. macaronesica, was that all three species showed high divergence between archipelagos and significant isolation by distance. This pattern was expected considering the large geographic distance between archipelagos and the lack of stepping-stone habitats characteristic of oceanic island biota. Overall, the population models in DAPC provided evidence for a hierarchical island model. This is consistent with the fact that the studied archipelagos were far apart, supporting divergence, but some islands in closer proximity had the opportunity for occasional gene flow. Previous studies comparing Swiss and Canadian populations of L. pulmonaria have found substantial differentiation, as expected in populations situated on different continents17. So, not surprisingly, the occurrence and frequency of gene flow appears to decrease with geographic distance in L. pulmonaria, which is also evident in our data on isolation by distance. Significant differentiation over large geographic scales has been shown in other lichen fungi as well24.A unifying motive in the microsatellite repeat data of L. pulmonaria has a complex population history in Macaronesia, the coexistence of divergent groups of genetic clusters on the same archipelago suggesting recurrent immigrations from sites on the European continent, but less from the North American continent. By comparison, the population histories of the two endemic species were more straightforward. Homogeneity of groups of genetic clusters within archipelagos rejects the hypothesis of repeated recent migration among archipelagos in the endemics. For both endemic species, each archipelago contained numerous private alleles. Thus, the populations on archipelagos evolved by genetic drift and/or by accumulation of new mutations, causing divergence.Taken together, our data indicate that Nephroma inferred the colonization of mainland sites from sites on the Macaronesian islands4. Second, an investigation of a liverwort showed that (1) sites in Macaronesia hosted a hidden diversity in the liverwort comparable to the radiation in higher plants at the genus level, and (2) low-diversity sites in western Europe were recolonized from Macaronesian refugial populations25. This diversity pattern was not evident in L. pulmonaria (the only species with more occurrences outside of than within Macaronesia)—sites on the mainland were highly diverse - see also26 - and were differentiated from island sites. If mainland sites received L. pulmonaria from the islands, this migration must have occurred a long time ago because the observed substantial differentiation of the large island and mainland populations would require that many generations have passed.Recent studies of lichen fungi and bryophytes emphasize the importance of Macaronesia for the colonization of sites in continental Europe. First, a study of lichen fungi in the genus Parmelina carporrhizans, unidirectional gene flow to the Macaronesian Islands was inferred27. Our data for Lobaria pulmonaria show a contrasting pattern with repeated gene flow between mainland and Macaronesian Islands and an overall higher differentiation among populations.In contrast, for the lichen L. pulmonaria to 6.9–11.9 Myr BP when it diverged from its Asian relative Lobaria tuberculata, and the divergence of the two Macaronesian endemic species from L. pulmonaria to 5.5–9.9 Myr BP28. Thus, the endemic species are likely to represent neo-endemics that may have evolved on the Macaronesian Islands after diverging from the older and widespread species, L. pulmonaria. This is similar to what has been found for Macaronesian Nephroma species4.A previous study dated the origin of L.pulmonaria: most island populations were genetically distinct from populations on the mainland. This reflects a history of low migration to the islands and genetic drift and diversification of island populations, which seem to have evolved from the continental populations and accumulated private alleles, which might have resulted from mutation or from genetic drift leading to loss of variation. Populations of L. pulmonaria from the Canary Islands were related to the Iberian Peninsula and those from the Azores to the Irish population. Divergence between geographic regions was also confirmed by our analysis of private and shared alleles. All regions contained a comparatively high number of private alleles in L. pulmonaria, with few alleles shared between regions. Hence, gene flow between archipelagos or between continental populations and archipelagos is rather low.In all species, we found substantial differentiation among archipelagos, indicating long-term isolation. This was also true for the widespread species, L. pulmonaria have found that differentiation between regional populations is mainly dependent on the geographic scale of sampling30. On a small spatial scale, individuals of this species showed spatial autocorrelation in genotypes and alleles due to local deposition of diaspores33, but no or little differentiation was found among forested areas within a pasture woodland29 or even among sites tens of km apart within a region17. However, significant differentiation among geographic regions has been found when different refugial regions were included in the analysis34, or when populations growing in different habitats such as floodplain forests and mountain ridges were compared within one region35.Previous studies of L. pulmonaria could be due to the overarching role of stepping-stone habitats leading to effective gene flow. In volcanic archipelagos situated off the continental shelf and surrounded by ocean, the islands themselves represent the few stepping stones, but there are no additional populations in between. Thus, we expected a substantial amount of genetic differentiation between sites on different archipelagos, which was indeed found.The lack of genetic differentiation between subsets of a landscape or a forested region in L.pulmonaria. Interestingly, sites on the Iberian Peninsula unexpectedly grouped with the site investigated on Newfoundland . Moreover, the site in Ireland was more similar to Azorean populations, rather than to the geographically closer sites on the Iberian Peninsula.However, genetic distance was not simply a function of geographic distance for some of the studied populations of 28. In L. macaronesica, the single known small population of this species on the Iberian Peninsula is genetically distinct from the Macaronesian island populations and could be the remains of what might have been a much larger population during phases of past, moister climate.A particularly interesting facet of our data set was the relationship between island and mainland sites in the endemic species. Both endemic species are likely to have evolved on the Macaronesian islands, given their divergence timeL. immixta is known from the Iberian Peninsula grouped within the Azorean populations  had a similar result40.For an oceanic archipelago, the Azores host relatively few endemic plant taxa, a pattern which has been ascribed to a less variable paleoclimate because climatic variability can drive radiationsLobaria are dependent on a woody plant community. Two phorophyte taxa of the lichens have also been investigated in a phylogeographic context in Macaronesia. In the heather Erica scoparia, a western European and Macaronesian shrub which is a frequent phorophyte of Lobaria sp. on the Macaronesian Islands, Azorean populations were far more diverse than those in western continental Europe40. The authors explained this pattern by recurrent migration to the Azores and extinctions on the mainland. In L. pulmonaria, populations on the Azores were diverse and consisted of divergent genetic clusters, but in contrast with the pattern of E. scoparia, populations located on the Iberian Peninsula, Newfoundland and Ireland showed high diversity as well. A study targeting the phylogeography of L. pulmonaria on the European continent using microsatellite repeat data on thousands of samples showed no tendency for continental populations to have low allelic richness26. This could be the case if this lichen spread efficiently into sites affected by Pleistocene glaciations.Epiphytic lichens such as the investigated species of Laurus nobilis L., Laurus azorica (Seub.) Franco). Laurus azorica is a common phorophyte of Lobaria lichens in Macaronesia, whereas the drought resistant Laurus nobilis on the mainland is only rarely hosting the species. In the Laurus species complex, chloroplast haplotypes of Macaronesian L. azorica were closely related to those of L. nobilis from the western Mediterranean and from Northwestern Africa. The ‘Macaronesian’ haplotype occupied northwestern Africa, the Canary Islands and Madeira. A second, closely related haplotype occupied the Azores, and two others the western Mediterranean. The authors inferred the existence of multiple refugia in Laurus, and strong range dynamics in the western Mediterranean part of the range41. Climatic reconstructions showed that large, suitable areas for Laurus existed in the Pliocene, but during the last glacial maximum, these were reduced to the Mediterranean Basin and the Macaronesian islands42. These areas could also have enabled the survival of Lobaria spp., given that the climate was moist enough. With some exceptions , current sites where Laurus is found in northern Africa do not harbor Lobaria spp. as the climate is too dry, indicating that the ecological niches of the lichens and their phorophyte do only partially overlap. The large differentiation between Azorean and Canarian/Madeiran populations in all of our study species appears to somewhat resemble the phylogeographic pattern of Laurus. We would not have expected a close similarity of genetic patterns due to the differences in life history including dispersal syndrome in trees vs. lichens and as we used different loci.The second phorophyte of our study species investigated in a phylogeographic context was laurel tree , which were connected by a rather recent (1 Ma) event8. The Selvagem archipelago (27 Ma) is situated between the Canary Islands and Madeira and is characterized by a dry climate and of low elevation (≤ 153 m) vegetation lacking forest45. Due to lack of suitable forest habitat, Selvagem could not have served as a steppingstone between the other archipelagos for our study species in recent time. Madeira consists of two main islands: Madeira (< 5.6 Ma) and the older Porto Santo (14 Ma)47.The age of the three archipelagos is well documented. Island age decreases from East to West on the Canary Island archipelago: Fuerteventura, 20.6 Ma; Lanzarote, 15.5 Ma; Gran Canaria, 14.5 Ma; La Gomera, 12.0 Ma; Tenerife, ~ 7.5 Ma; La Palma, 2.0 Ma; El Hierro, 1.12 Ma49. The studied islands are Santa Maria (8.12 Ma), Sao Miguel (4.01 Ma)50, Terceira (3.52 Ma), Faial (0.73 Ma), Pico (0.25 Ma)51, and Flores (2.16 Ma)52. The occurrence of a land bridge connecting Pico and Faial at the maximum of the last glaciation  has been reported54. It seems unlikely that this land bridge would have led to increased connectivity between the high-altitude forest types characteristically inhabited by Lobaria lichens, unless the habitat suitable for Lobaria spp. occurred at considerably lower elevations in the past.The Azores were formed along spreading midoceanic ridges at the joint of the African, American and European platesLobaria  to nine (Madeira) populations, depending on island size and availability of populations of ria Fig. ; Table 1ria Fig. . Within 55.Samples were collected along a transect through the population in large populations. If the local population size was small, we deviated from this sampling scheme and sampled lichens from all trees situated within approximately 1 hectare of forest. We attempted to include three specimens per species from each sampled tree. Our species identifications in the field were sometimes difficult as many specimens were poorly developed. Hence, we had to rely on molecular species identifications by RealTime PCRLobaria pulmonaria, L. immixta and L. macaronesica. Lobaria pulmonaria is a widespread epiphytic lichen in large parts of the northern hemisphere, with a few occurrences in the southern hemisphere56. The other two species are endemic to Macaronesia but are both known from one spot on the mainland in Portugal (Sintra). From this site, L. immixta had been reported by Christa and Josef Poelt in 196157. This is the second report of this species from this site. Moreover, L. macaronesica was discovered in Sintra in 2007 by C. Scheidegger13. For the endemic species, all samples from Sintra were found inside of the Royal Gardens, while L. pulmonaria also occurred in natural forests located in the vicinity.Our study species were 13, we utilized RealTime PCR for species identifications. The RealTime PCR used a small (10 bp), species-specific stretch of the Internal Transcribed Spacer (ITS) region. For protocols, see Werth et al.55.DNA was extracted following the manufacturer’s protocol using the DNeasy 96 plant kit . Each thallus was inspected for the presence of apothecia and parasites, and only thallus parts free of these structures were utilized for DNA extractions. Field identifications of the lichen material was done by SW and CS, lab identifications by SW. As many specimens were not well developed and lacked the diagnostic propagules58. Only six out of eight loci worked for all species and these were used for the analyses to ensure comparability among data sets; these markers were fungus-specific according to Widmer et al.59. We recently developed additional microsatellite loci for L. pulmonaria60, but the variability in the present set of six markers was highly suitable for a regional-scale study58.The genotyping of microsatellites followed Werth et al.59, 200 nmol LPu28F, and 200 nmol LPu28R-VIC. Primer sequences not shown above are given in Walser et al.61. Fluorescent labels VIC, PET, NED, and 6FAM were used, supplied by Life Technologies  or Sigma-Aldrich . Additionally, each reaction contained 2.5 μL Qiagen multiplex PCR mix , 0.5 μL diluted DNA (c. 0.5–10 ng) and H2O to 5 μL. Cycling conditions were 95 °C for 15 min; 35 cycles of 95 °C for 15 s, 52 °C for 30 s, 72 °C for 1 min; followed by a final elongation step of 60 °C for 30 min. Fragment analyses were run on an automated capillary sequencer . Alleles were genotyped with an internal size standard (LIZ500) using GeneMapper version 3.7 .All markers were run in a single multiplex PCR. For two loci, we used primers optimized to work in all species. Each PCR reaction contained 200 nmol LPu09F, 200 nmol LPu09R-PET, 350 nmol of LPu15F, 350 nmol LPu15R-PET, 200 nmol LPu23F-6FAM, 200 nmol LPu23R, 200 nmol LPu24F2-VIC , 200 nmol LPu24R, 300 nmol LPu25F3-NED , 300 nmol LPu25R 5′-CAT GAA ACG GTT TTG GTT GA-3′ In order to compare regional genetic structures among endemic and widespread species, we utilized extensive microsatellite datasets of each species to quantify the overall genetic relationships between sites, to infer the grouping of populations, and to analyze the partitioning of genetic variability upon sites and geographic regions.62. The genetic relationships between all studied sites were evaluated for each species using the chord distance DC63, as implemented in the ‘gendist’ module of PHYLIP. Neighbor-joining trees were constructed with ‘neighbor’, and a majority-rule consensus tree was computed using the ‘consense’ module of PHYLIP. The consensus trees were midpoint rooted with FigTree version 1.2.164 and visualized on a map with the geo-spatial information system GenGIS version 2.4.165.For each species, 1000 bootstrapped microsatellite allele frequency datasets were generated using the ‘seqboot’ module of PHYLIP version 3.6967. After an initial run for values of K up to 39 (number of sites), an additional 10 runs were performed for the optimum partition in each species. Admixture analysis (data not shown) was run and none of 1402 individuals (L. immixta), three of 1499 (L. macaronesica), and nine of 1239 samples (L. pulmonaria) were found to be admixed when a Bonferroni correction for multiple testing had been applied to the data. Neighbor-joining trees depicting the relationships between clusters were generated in BAPS using Nei’s genetic distance, averaged over loci69. The major groupings were mapped in ArcGIS version 10 (ESRI). To quantify genetic differentiation between geographic regions, population pairwise FST values were computed in Arlequin version 3.570 for each of the three fungal species.In order to analyze the main grouping in the data of each species and to infer the number of genetic groups (K) in each species, sites were clustered in a mixture analysis using BAPS version 5.471. Thus, it is possible to distinguish e.g. island models of gene flow from hierarchical island models or stepping-stone models73. DAPC was implemented in the R-package ‘adegenet’ v. 2.1.374 in R v. 3.6.175.Discriminant Analysis of Principal Components (DAPC) can provide valuable information about the most likely population model of a set of sampling sites, as different geneflow patterns lead to different genetic clusteringST values were calculated in R using the ‘hierfstat’ package v. 0.04-22 and geographic distances between sites were calculated with the package ‘geosphere’ v. 1.5-10 using the ‘distm’ and ‘distGeo’ functions. Significance of isolation by distance was assessed with linear regression models, where FST was the response variable and geographic distance the predictor.To assess whether there was isolation by distance, pairwise F76.The current data set consists of six fungus-specific microsatellite data and all allele frequency are accessible in the Dryad repositorySupplementary Table S1.


Oryza meridionalis  and an Oryza rufipogon like taxa that have been shown to have a chloroplast genome sequence that is closer to that of O. meridionalis than to O. rufipogon from Asia. Rare plants of intermediate morphology have been observed in the wild despite a reported reproductive barrier between these two species. We now report the resequencing of plants from 26 populations including both taxa and putative hybrids. A comparison of chloroplast and nuclear genome sequences indicated re-combinations that demonstrated hybridisation in both directions. Individuals with intermediate morphology had high nuclear genome heterozygosity consistent with a hybrid origin. An examination of specific genes  revealed the presence of heterozygotes with alleles from both parents suggesting that some wild plants were early generation hybrids. These plants may have low cross-fertility preserving the continuation of the two distinct species. Repeated backcrossing of these rare hybrids to one parent would explain the plants exhibiting chloroplast capture. These observations suggest that reticulate evolution is continuing in wild Oryza populations and may have been a key process in rice evolution and domestication.The wild rice gene pool, i.e., AA-genome, in Australia is geographically and genetically distinct from that in Asia. Two distinct taxa are found growing together in northern Australia,  Pisum (Opuntia (Elymus)  ; baobabs(Elymus) ; and Adeenanthos . Much efenanthos . Morpholenanthos . Geneticenanthos , polymorenanthos , and incenanthos . Next-geenanthos .Oryza) belongs to the tribe Oryzeae—a member of the grass subfamily Ehrhartoideae. The genus Oryza comprises 11 genome groups of which the AA-genome group (with eight diploid species) is the most diverged group, distributed throughout the world, except for, Antarctica (Oryza species), BBCC (three Oryza species), CC (three Oryza species), CCDD (three Oryza species), EE (one Oryza species), FF (one Oryza species), GG (three Oryza species), HHJJ (two Oryza species), HHKK (one Oryza species), and KKLL (one Oryza species)  . Vicariaspecies) . The estears ago . Four Orustralia . Of thesr length . More real forms . O. rufiustralia . Australrom Asia . This inrom Asia .Oryza species, especially in crosses between O. sativa and its wild relatives , resulting in hybrid sterility  has been observed in these wild rice populations, which were assumed to be due to hybridisation between the ype taxa . These wWe have now reexamined the origins of these hybrids by re-sequencing and analysing the nuclear genomes of these wild rice populations including wild plants of hybrid appearance.Australian wild rice is widely distributed from the south of Townsville to the northern parts of Cape York Peninsula, Queensland, Australia. Fresh leaves of wild rice from 26 different geographical sites were collected in the two consecutive years 2015 and 2016 by 1 to identify any sequencing error before performing further analysis. Sequences were trimmed at both 0.05 and 0.01 quality scores to truncate low-quality reads. Based on the percentage of loss of reads and bases, 0.01 trimmed reads were considered for further analysis. The total Illumina raw reads for the 26 samples ranged from 56,349,078 to 104,325,654 bp. The total raw nucleotides ranged between 8,416,673,405 and 15,628,960,467 bp. After trimming, the total 0.01 trimmed reads ranged from 54,631,067 to 98,924,768 and nucleotides from 7,925,007,230 to 14,260,593,039 bp . Samples were kept in liquid nitrogen to prevent thawing throughout the pulverisation process. DNA was extracted from pulverised leaf tissue of wild rice samples following the CTAB method . The qua3,039 bp .O. rufipogon type taxa (= Taxon A) and O. meridionalis (= Taxon B) was 384.8 and 354.9 Mb, respectively . Sequence data for the Australian O. rufipogon like taxon is reported in Prior to final mapping, the best mapping parameters were evaluated with the three different alignment quality thresholds: 1.0 length fraction (LF) and 0.95 similarity fraction (SF), 1.0 LF and 0.90 SF and 1.0 LF and 0.85 SF. Illumina paired-end reads were then mapped to the reference genome Os-Nipponbare-Reference-IRGSP-1, which was downloaded at Rice Annotation Project Database  with theectively . Other dO. meridionalis, and the reference chloroplast genome of O. rufipogon type taxa obtained from O. meridionalis and O. rufipogon type taxa  = 1.0604; R(b) = 2.5331, R(c) = 0.5698; R(d) = 0.9914; R(e) = 2.5331; R(f) = 1; gamma = 0.587. Phylogenetic reconstruction was performed using PAUP* version 4 software  were investigated to test for nuclear introgression from the putative parental populations. Among these, WR44, WR52, and WR153 were reported by ype taxa , and Ory ex Watt  were ali ex Watt  with defdelTest2  on the Esoftware  with maxseed shattering 4 (sh4) on chromosome 4, four starch synthesis related genes on chromosome 6  and granule bound starch synthase II (GBSSII) on chromosome 7. Plausible hybridisation generations were determined based on the level of heterozygosity across the whole genome of these hybrids.Single nucleotide polymorphism (SNP) genotypes of six genes located on chromosomes 4, 6, and 7 from hybrids and putative parental taxa were aligned, with respect to Os-Nipponbare-Reference-IRGSP-1 with CLC-GWB to screen for contributing donor parents in genotypes of hybrid populations. These genes include GBSSI, SSI, SBEI, and ALK) from two putative parental populations (WR24 and WR81), one hybrid and outgroup species were first aligned with MAFFT version 7.407 (O. sativa spp. japonica cv. Nipponbare was considered an outgroup species (A). We simulated a total of 5 four-taxon tests to determine biparental nuclear geneflow between hybrid and two putative parental taxa. We estimated Patterson’s D-statistic based on the random distribution of ABBA-BABA polymorphisms in a four-taxon test .Four taxon (ABBA-BABA) test as a measure of geneflow was performed with Hybrid Check version 1 software . Four geon 7.407  with defxon test . The D-sr2 = 0.2 (which recursively eliminates SNPs within the sliding window if r2 is greater than 0.2). To investigate the relationship of population structure with geographic distance, a subset of genome-wide SNP data  from 26 wild rice genotypes  were used to perform isolation by distance (IBD) analysis using Mantel test  to assess the genetic variation in wild rice populations using plink version 1.9 . To ensutel test  with thetel test . Nei  contained an O. rufipogon type chloroplast genome and an O. meridionalis type nuclear genome.The analysis of phylogenetic relationships based on the whole chloroplast and nuclear genome had suggested that chloroplast capture was present in Australian wild rice populations . The wholis type  while thype taxa . While Wt genome . This reO. sativa spp. japonica cv. Nipponbare reference genome. Of the six genes, one gene (sh4) on chromosome 4 is associated with seed shattering while the other five genes are starch synthesis-related genes located on chromosome 6  and chromosome 7 (GBSSII). From SNP data, it was clearly shown that the six genes of WR62 received alleles from both O. rufipogon type taxa and O. meridionalis parents. SBEI and GBSSII had heterozygous SNP genotypes with both O. rufipogon type taxa and O. meridionalis alleles in all of five hybrids  also had heterozygous loci for ALK and SSI, respectively. Sequence alignment images of SNP genotypes of sh4 gene , ALK gene , and GBSSII   was found in WR44, followed by 5,917,274 in WR62, 5,291,708 in WR52, 5,295,508 in WR161 and 4,764,266 in WR153 (O. rufipogon type taxa was applied to the above-mentioned formula (2) for WR62, which exhibited O. rufipogon type nuclear genome while the genome size of O. meridionalis was used for the other hybrids. A high level of genetic variation was observed in both WR44 (0.69%) and WR62 (0.6%) across the whole genome followed by WR161 (0.34%), WR52 (0.32%), and WR153 (0.31%) in relation to O. sativa spp. japonica cv. Nipponbare .SNP were analysed for six genes on chromosomes 4, 6, and 7 were analysed to assess the genetic contribution of the putative parents to the natural hybrid populations in relation to  hybrids . However,202 bp)  showed h,202 bp)  while WR,202 bp) . In all in WR153 . Similarpponbare . The levD-statistic) was performed to test for biallelic mutation patterns (ABBA-BABA) to determine geneflow between the hybrids and two putative perennial parental populations in a topology {, O. sativa spp. japonica cv. Nipponbare}. The length of a MAFFT aligned sequence was 4,473 bp with a total of four blocks: ; ; ; and . The D-statistic value based on the distribution of AABB and BABA on each block showed significant deviation for all four-taxon trees  and hybrid while the positive value suggests geneflow between O. meridionalis (WR81) and hybrid. In all cases, O. sativa spp. japonica cv. Nipponbare was an outgroup. The Z-score for all four-taxon tests was significant (p < 0.001).A four-taxon test . The number of allelic positions where the variants were located was determined. The total number of variants ranged from 1,918,262 in WR24 to 7,310,928 in WR81 , and the least heterozygosity was found in O. rufipogon type taxa (0.16%). The SNPs that were uniquely present in individuals were determined to define the genetic relationships between individuals. The rare or unique heterozygous SNP position (%) ranged from 4 to 48%. The highest number of unique heterozygous SNP positions was in O. rufipogon type taxa and the lowest number in O. meridionalis, indicating O. rufipogon type taxa might be subject to more genetic drift compared to O. meridionalis populations due to their smaller population size.To estimate intra- and interspecific variations in the wild rice gene pool, variants comprised of five different types: SNV, insertions, deletions, MNV, and replacement were assessed across the whole genome of twenty-one putative parental taxa  formed another group  of 26 wild rice genotypes were first processed to remove linkage for PCA analysis. A total of 490,655 SNP sites were retained that fell below the linkage threshold. PCA was performed on linkage-pruned sites to determine the genetic groupings of wild rice populations. PCA analysis showed two clear clusters: (1) one group formed with hybrids  and one putative parental taxon (WR24) and (2) all er group . The firR2 = −0.008, p-value = 0.42) between Nei’s pairwise genetic distance and geographic distances (R2) was 0.008 with a p-value of 0.42. This indicated genetic differentiation was not significantly increased with geographic distance, indicating genetic connectivity in wild rice populations across a wide range.Isolation by distance analysis based on a Monte-Carlo test with 999 permutations clearly showed that there was no significant relationship  differed from each other by 34 variants  and O. sativa spp. Nipponbare cv. japonica (125 variants). In the Australian O. rufipogon, ancient chloroplast capture has occurred from O. meridionalis  due to recent bidirectional chloroplast capture from these parents. The recent introgressive hybridisation demonstrated here may be an explanation of both the apparent chloroplast capture in the Australia O. rufipogon populations and the wild hybrids.This study confirms two types of the chloroplast genome in hybrid populations introgressed from one of their parental taxa. A total of five hybrids were studied of which only one hybrid (WR62) exhibited an  origins . Chlorop origins . Unidire origins  or nucle origins  from one origins . Unlike  origins . In this origins . Brozynstic tree . Despitevariants . The O. dionalis , howeverO. meridionalis. These observations clearly demonstrate an ongoing hybridisation and introgression persisting in the wild rice populations evidenced by the presence of viable hybrids between O. rufipogon type taxa and O. meridionalis being found at very low frequency. Earlier findings postulated that interspecific crossability between an O. meridionalis and a perennial Asian O rufipogon was modest but asymmetric  and O. rufipogon produce low fertility pollen (<10%) as found with putative annual strains (<20–40%)  and panicle fertility (66.9%), however, geographical distance seems to be the reason for the development of reproductive barriers  . Intra-sbarriers . They obocations .D-statistic values from the four-taxon test indicate a nuclear geneflow from both putative parents into hybrids. Early generation hybrids exhibited higher heterozygosity and polymorphic variations than that of the later generations of hybrids. Repeated backcrossing between F1/F2 offspring and O. meridionalis may lead to comparatively low heterozygous SNP variation in the later-generation of hybrids. PCA analysis also reveal that early generation hybrids were clustered with O. rufipogon type taxa and later generation hybrids (except for WR161) form a group with O. meridionalis. Reproductive barriers underpin a termination of hybridisation and thereby, introduce introgression (SEED DEVELOPMENT 1) on chromosome 6 is detected in O. meridionalis and O. sativa (SEED DEVELOPMENT 1 gene (sdv1-m) in wild rice strain from the Jpn2 site  appears to evoke segregation distortion (1:2:0) in a BC4F2 progeny. Therefore, offspring carrying homozygous recessive alleles (sdv1-m) cause seed abortion at the seed development stage. O. sativa and O. meridionalis, then backcrossed with O. sativa to obtain backcrossing progeny. Despite homozygous recessive alleles, offspring bear seed if they are self-fertilised. O. rufipogon from Asia or Australia does not have this abortive gene. Backcrossing with O. meridionalis along with selfing may reduce seed abortion in later-generation hybrids.The positive and negative significant gression . An inte. sativa . A recesO. rufipogon type taxa are distinguished with open panicles and short awns from O meridionalis, which exhibits long awns with closed panicles. However, awn lengths sometimes overlapped  were searched to discover extremely rare apparent hybrids based upon their intermediate morphology. This study has confirmed the hybrid identity of these extremely rare plants. The Asian O. rufipogon samples that have been sequenced all show the presence of an O. rufipogon chloroplast that is distinct from that of O. meridionalis and the Australian O. rufipogon like taxa  remain as distinct populations despite the extremely rare hybridisation events between these two taxa confirmed in this study. The reproductive barrier between them resulting in low pollen fertility requires the backcrossing of the hybrids in subsequent generations.ike taxa . The twoO. sativa ssp. japonica cv. Nipponbare. This finding is, in agreement with the result reported by O. meridionalis and 2,564,013 SNPs in O. rufipogon, compared to Asian O. rufipogon  and cultivated O. sativa ssp. indica , with respect to O. sativa ssp. japonica cv. Nipponbare. A molecular study using restriction fragment length polymorphism (RFLP) markers revealed that cultivated rice retained 40% of the wild alleles during rice domestication  had a more heterozygous and repetitive genome than O. rufipogon (Taxon A), resulting in a higher number of scaffolds in the egnoem of this taxon. Mating systems play an important role in acquiring genetic variations. Perennials exhibit outcrossing mating system, which results in a higher degree of genetic variation than annuals while selfing is a feature of annuals  in dispersing Oryza species over large distances is quite evident  can be found below: RH conceived the project. AF and RH collected field samples. SH, AF, and RH analysed the data. SH wrote the first draft 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Coreiusguichenoti of dam constructions in the upper reaches of the Yangtze River, we analyzed the genetic diversity and population structure of 12 populations collected in 2009 and 2019 using mitochondrial DNA (mtDNA) control regions. There was no significant difference in genetic diversity between 2009 and 2019 (P > 0.05), but the population structure tended to become stronger. Genetic differentiation (FST) among five populations  collected in 2009 was not significant (P > 0.05). However, some populations collected in 2019 were significantly differentiated (P < 0.05), indicating that the population structure has undergone change. A correlation analysis showed that the genetic diversity of the seven populations collected in 2019 was significantly negatively correlated with geographical height , indicating that the populations at high elevations were more vulnerable than those at low elevations. In order to prevent the further decrease of genetic diversity and population resources, some conservation and restoration suggestions, such as fish passage and artificial breeding, are put forward.To investigate the genetic effects on the population of  Coreiusguichenoti   is one of the endemic fishes and an important economic fish in the upper reaches of Yangtze River in China  has not been reported. In this study, we used mitochondrial DNA (mtDNA) control region to analyze the genetic diversity and structure of 377 individuals from 12 populations of C.guichenoti, which were collected from the upper reaches of the Yangtze River in 2009 and 2019. The population diversity and structure were compared between 2009 and 2019. Different groups were compared within the same sampling time, and among different populations in the same group. Understanding the genetic diversity and structure of historical and current populations will help to make the right management decisions and identify feasible conservation measures that contribute to the conservation and restoration of wild resources , the Xufu section of the Jinsha River (SF), the Jiangan section of the Yangtze River (JA), the Luxian section of the Yangtze River (LX) and the Beibei section of the Jialing River (BB) in 2009. A total of seven populations were collected in the Xiqu section of Jinsha River (XQ), Hongmenchang section of Jinsha River (HMC), Longchi section of Minjiang River (LC), Caiba section of Yangtze River (CB), Xuzhou section of Yangtze River (XZ), Jiangyang section of Yangtze River (JY) and Liemian section of Jialing River (LM) in 2019 .To reduce the influence of different ages on the experimental error, 308 samples aged around 3 years were selected from 377 individuals, according to the relationship between age and body length ; Table 1mtDNA control regions in all populations. The PCR conditions, electrophoresis and sequencing were as described in Primers DL1  and DH2   were setmtDNA control regions  using Coreiusheterodon (Genbank no. FJ376082.1) and Abbottinarivularis (GenBank no. AP011257.1) as outgroups. The median connection network (MJN) method (FST) among populations were estimated using Genepop v. 2.4 (Haplotype number (H), Haplotype diversity (h), and nucleotide diversity (π) were used to estimate genetic diversity in  regions . Genetic regions . Based o) method  was usedp v. 2.4 .mtDNA control region. First, Tajima’s D (BSP) was implemented in BEAST v. 1.5.4  model in IMA2 program (mtDNA control region. Running IMa2 required two steps (M mode and L mode). Firstly, in M mode, the model parameter function is estimated based on 6 ×108 MCMC steps, according to the aging cycle of 200,000 steps. Secondly, the model parameter function estimated by M mode is used to estimate the offset rate in L mode.Using the isolation with migration  between log-transformed genetic distance and log-transformed geographic distance was determined by using 2, 000 permutations of Distance Isolation (IBD) Web Service v. 3.1.6  and JA (1). All differences of genetic variation (h and π) of 12 populations was not significant by paired t-test (P > 0.05).Genetic diversity analysis of 12 populations was performed based on YB, XQ and HMC), middle group , and lower group (BB and LM). AMOVA analysis of five populations  collected in 2009 showed no significant molecular differences in mtDNA among populations, between populations and within populations  from 2019 showed that the intrapopulation variance (97.05%) was significant  collected in 2009 were not significant (P > 0.05). The genetic differentiation between XQ and JA, HMC, and BB was not significant. Two neighborhood link trees were constructed based on the FST values in five populations (2009) and seven populations (2019). BB and LX populations clustered together, and SF, JA, and YB populations clustered in adjacency trees , Hap3 (n = 32), Hap4 (n = 20), and Hap11 (n = 11) was different at different sites. Hap3 and Hap4 were not observed in the down group (BB and LM). The analysis of the aggregation method based on the IM model showed that the level of gene flow between populations was very limited .Mantel test showed that geographical distance was significantly associated with genetic differentiation in five populations , suggesting that the upstream region may be the breeding and distribution center of the Yangtze River and its tributaries (t-test (P > 0.05), indicating that there was no significant decrease in genetic diversity from 2009 to 2019, through the mean value of genetic diversity in 2009 was higher than that in 2019. Based on this result, it appears that the breeding of C.guichenoti has not been completely disrupted by dams. Some individuals may not need to go upstream to spawn and may be able to breed in scattered habitats, even if they are severely affected by dams.In this study, we assessed genetic variation in twelve populations of ulations . Adults butaries . There wP > 0.05), and most of the total genetic variation occurred among individuals within groups, which was insignificant neither (P > 0.05). The FST values showed that the population structure of each sampling site was not significant. Moreover, these five groups do not form independent groups in terms of population structure, and each individual actually has an equal probability of belonging to any of these groups in any analysis. There was no obvious systematic geographic structure among the haplotypes of the samples from different geographical locations. The sequence differences within and between groups were small, indicating high genetic similarity among different groups and relatively shallow population history. The genetic differentiation of the five populations was significantly correlated with geographical distance. After spawning, this species usually flows downstream with floods, and when rivers are in flood, juvenile and adult fish subsequently swim upstream  in the Yangtze River, and this was also attributed to spawning of drifting eggs and larvae . Significant differences among all populations have not yet developed, but differences among individuals within populations are emerging. To explain this, the dam restricted the genetic links between the upstream and downstream populations of C.guichenoti, splitting them into small, isolated populations. A strong, though occasionally permeable, barrier to transmission may exist to limit the flow of genes between sites. In this case, the species’ habitat becomes fragmented, which not only affects the spawning and development environment of C.guichenoti, but also impedes upstream and downstream migration and gene exchange. Thus, weak correlation between the observed genetic differentiations and geographical distances was found in seven populations  while it is significant negative correlation in five populations . As the hydrological environment changes, the individuals within the population become different. Then there may be a strong demographic structure in the future. Furthermore, compared with large populations, small populations are more prone to inbreeding and loss of genetic diversity due to genetic drift and bottlenecks  can lead to gene rescue or recovery . IncreasFurthermore, in order to maintain heterozygosity and genetic diversity, mature adults should be collected for artificial propagation from different locations across the distribution, especially considering the high genetic diversity among groups . Aquacul


Toxicodryas. We used dated phylogeographic inferences, population clustering analyses, demographic model selection, and paleo‐distribution modeling to conduct a phylogenomic and historical demographic analysis of this genus. Our results revealed significant population genetic structure within both Toxicodryas species, corresponding geographically to river barriers and divergence times from the mid‐Miocene to Pliocene. Our demographic analyses supported the interpretation that rivers are indications of strong barriers to gene flow among populations since their divergence. Additionally, we found no support for a major contraction of suitable habitat during the last glacial maximum, allowing us to reject both the refuge and river‐refuge hypotheses in favor of the river‐barrier hypothesis. Based on conservative interpretations of our species delimitation analyses with the Sanger and ddRAD data sets, two new cryptic species are identified from east‐central Africa. This study highlights the complexity of diversification dynamics in the African tropics and the advantages of integrative approaches to studying speciation in tropical regions.The relative roles of rivers versus refugia in shaping the high levels of species diversity in tropical rainforests have been widely debated for decades. Only recently has it become possible to take an integrative approach to test predictions derived from these hypotheses using genomic sequencing and paleo‐species distribution modeling. Herein, we tested the predictions of the classic river, refuge, and river‐refuge hypotheses on diversification in the arboreal sub‐Saharan African snake genus  Toxicodryas. We used dated phylogeographic inferences, population clustering analyses, demographic model selection, and paleo‐distribution modeling to conduct a phylogenomic and historical demographic analysis of this genus. Our demographic analyses supported the interpretation that rivers have represented strong barriers to gene flow among populations since their divergence, and we found no support for a major contraction of suitable habitat during the last glacial maximum, allowing us to reject both the refuge and river‐refuge hypotheses in favor of the river barrier hypothesis.We tested the predictions of the classic river, refuge, and river‐refuge hypotheses of tropical rainforest evolution on diversification in the arboreal Sub‐Saharan African snake genus  The “river hypothesis” in which species and populations diverged across riverine barriers  river hypothesis: boundaries between population distributions should correspond to riverine barriers and the ages of populations should be relatively old, corresponding to the ages of the rivers; (b) refuge hypothesis: population distributions should be concordant with locations of hypothesized rainforest refugia during cold, dry periods, and populations are predicted to be relatively young, corresponding to the Pleistocene glaciation cycles; (c) river‐refugia hypothesis: population distributions should be correlated with the locations of rainforest refugia and bounded by rivers barriers, or will have been confined to refugial locations and additionally subdivided by rivers. Under this scenario, the timing of population splits should correspond to ages of rivers but would be expected to show patterns of range expansion and contraction for niche model projections to the Pleistocene.Toxicodryas as a model system to test multiple predictions derived from these hypotheses. The genus Toxicodryas consists of two species of large‐bodied, rear‐fanged, venomous sub‐Saharan African snakes, T. blandingii, and T. pulverulenta  through fieldwork and from various museums (see Table We obtained 20 specimens of 2.2b (cyt b) were PCR‐amplified for each individual using standard primers . Electropherograms were edited manually in Geneious v5.6.7 , and genomic DNA (gDNA) was extracted using the Maxwell RSC system (Promega). The nuclear gene c‐mos and the mitochondrial gene cytochrome SbfI (restriction site 5′‐CCTGCAGG‐3′) and MspI (restriction site 5′‐CCGG‐3′). The resulting double digestion products were then bead‐cleaned with AmpureXP beads (Agencourt) and individually barcoded using custom oligonucleotide adapters. Pooled samples were size‐selected to a mean insert length of 541 base pairs (bp) (487–595 bp range) with internal standards with a Pippin Prep™ . Resulting postligation products were amplified for eight cycles with a high‐fidelity polymerase . An Agilent TapeStation was used to determine the final fragment size distribution and concentration of each pool. Library pools were combined in equimolar amounts for sequencing on one Illumina HiSeqX lane (with a 10% PhiX spike‐in and 150 bp paired‐end reads).We also sequenced genome‐wide anonymous nuclear markers for each individual following a modified version of the ddRADseq protocol of Peterson et al., . For eacIllumina reads from the ddRAD libraries were processed using STACKS v. 2.4  and n  until we identified the parameters at which the maximum number of polymorphic loci were available across 80% (r = 0.8) of the population. For our data, this was found to be M = 5 and n = 15. Further parameters were tested in POPULATIONS separately for each species and for the genus as a whole in order to balance missing data and number of polymorphic loci. Within T. blandingii and T. pulverulenta, the percent missing data were low  and no further processing was needed, and r = 0.8 was used. Because of dissimilarity between the two species causing high levels of missing data in the combined data set, further restrictions were implemented. For the genus‐wide data set, we set r = 0.5 and p = 4 [p is the minimum number of populations in which a locus must be present (here 4/5)]. This approach increased the number of informative loci, but also the amount of missing data. For each of our three separate data sets, we generated a data set comprising only a single random SNP per locus , and another data set comprising full‐length sequences for all loci (for use in phylogenetic reconstruction).We aimed to produce three separate ddRAD data sets, including one for 2.3b) and implemented bModelTest in Beast v. 2.6.2 to assess all possible substitution models for each gene using a Bayesian approach , and one at the Naja + Hemachatus node (minimum age: 17.0 Mya). Fossil ages and placement were based on Head et al. . A discriminant analysis of principal components (DAPC) was run using Adegenet v. 2.1.1 . This program uses a binned multidimensional folded site frequency spectrum , a wrapper for ∂a∂i , divergence without gene flow (Model 2), divergence with secondary contact (Model 3), and divergence with gene flow (Model 4). Priors for both models were drawn from uniform distributions for population size: 10,000–1,000,000 haploid individuals , divergence time: 20,000–2,000,000 generations, migration rate: 0.000005–0.005 corresponding to 0.05–5 migrants per generation. We then coarsened our empirical site frequency spectra to 10 bins each. Our out‐of‐bag error rates were calculated, and 500 random forest classifiers were simulated using 100,000 pseudo‐observed data sets for each model. A confusion matrix was calculated to determine how often the correct model was selected and posterior probability for the “best” model was estimated for each species.We created folded multidimensional site frequency spectrums for the two T. blandingii clades and the two Central African T. pulverulenta clades. The West African T. pulverulenta clade was excluded because of its small sample size. This program implements a founder effect algorithm using a steppingstone model, assuming that each colonizing event is associated with a founder event, to determine whether a population shows signatures of expansion or equilibrium isolation‐by‐distance  was used to conduct a species delimitation analysis on our ddRAD data set. As described above for the demographic analysis, this program uses a binned multidimensional folded site frequency spectrum  that could be visually identified by the authors. Duplicate records were removed, and points were thinned within a distance of 10 kilometers using the spThin package , and research‐grade locality records from iNaturalist  index was used to test for model extrapolation  consisted of 1,237 bp, including indels. Both genes were represented in all samples with the exception of c‐mos for the outgroup Contia longicaudae. Information on samples used in the Sanger analysis, including museum catalog number and GenBank accession number, can be found in the T. blandingii data set consisted of 7,231 loci with 5.0% missing data, and an effective mean per‐sample depth of coverage of 83.6x ± 12.0x. Our T. pulverulenta data set consisted of 4,471 loci with 7.3% missing data, and an effective per‐sample mean depth of coverage of 77.9x ± 14.6x. The concatenated ddRAD data set used for phylogenetic analyses had a length of 450,512 bp and 3,024 SNPs.Our concatenated c‐mos and cyt 3.2T. blandingii, in West and Central Africa, respectively, and three divergent lineages of T. pulverulenta, one from West Africa and two in Central Africa, north and south of the Congo River . Diversification within each species is estimated to have taken place primarily in the Pliocene, with the two clades in T. blandingii diverging around 4.3 Mya, the West African clade of T. pulverulenta diverging around 3.3 Mya, and the two Central African clades diverging around 1.9 Mya . While it is possible that small sample sizes for several of our populations may have negatively impacted the power of our demographic analyses, our values for posterior probability and out‐of‐bag error rate are similar to those obtained by Smith and Carstens . Accordingly, the strength of the founder effect (q) for each population was generally small, and the founder distance (d) was large .Population size analyses for demographic signatures of range expansion versus equilibrium isolation‐by‐distance strongly rejected the expansion model for each of the four populations tested . Our analysis was run twice with random seeds to check for consistency, and both runs gave highly similar results. Similarly, DelimitR identified divergence without gene flow as the best model for T. blandingii and divergence with gene flow for T. pulverulenta. For both species, models suggesting lack of divergence or present‐day gene flow had very low probabilities  and two are distributed in Central Africa, separated by the western Congo River. The boundary between the two clades within T. blandingii is delimited somewhere between the Sanaga River in Cameroon and the Congo River in the DRC, and, while the species distribution reaches this area, no genetic sampling is available. Both rivers have frequently been interpreted as population barriers in other terrestrial vertebrates  and T. pulverulenta (east of the Niger Delta) represent cryptic new species that are in the process of being formally described (Greenbaum et al. T. pulverulenta, separated by the Congo River will be conservatively described as one species based on the results of our STRUCTURE v. 2.3.4 analysis (Falush et al., In light of the Miocene/Pliocene divergence times and lack of gene flow between these five clades, it is likely that they represent distinct evolutionary lineages. Based on these molecular results, significant differences in scale counts, and even venom toxicity, the Central African populations of 4.5The nature of the intervening habitat surrounding rainforest refugia during the Pleistocene has been widely debated (reviewed in Couvreur et al., Toxicodryas species are generally characterized as arboreal across rainforest and woodland habitats and the two species exhibit widely overlapping distributions in West and Central Africa (Chippaux & Jackson, Toxicodryas may be a result of the relatively reduced dependence of arboreal snakes on moist habitats, as reflected by their distribution in both woodland and rainforest. The stability of Toxicodryas habitat through the Pleistocene supports the hypothesis that rainforest composition shifted to dryer woodlands surrounding rainforest refugia, instead of a more dramatic shift to strict savanna habitat. Southward shifts in species suitability may correspond with predicted forest distribution shifts of White (Similar paleo‐distribution studies on frogs have suggested substantial habitat contraction in Central Africa during the Pleistocene (Leaché et al., of White  and Alleof White , suggest5The complexity of geographic barriers in West and Central Africa, and the association of refugia with areas of high surface relief or riparian zones (Hofer et al., Toxicodryas from throughout their range would be ideal for fully understanding the demographic history of these species.It is important to note, however, that as a result of the difficulty in finding and obtaining tissues from arboreal, venomous, Central African snakes, the small sample sizes used in this analysis may have influenced the inference of our population demographic parameters (Hale et al., These results highlight the importance of using an integrative, multidisciplinary approach to statistically distinguish among competing hypotheses to explain high levels of geographically concentrated species biodiversity. Moving beyond pure pattern‐based inference, a deeper and more nuanced understanding of the production, partitioning, and maintenance of diversity in complex landscapes may lead to inference of environmental and evolutionary processes that accumulate terrestrial biodiversity in tropical areas, which coincide in many cases with Global Biodiversity Conservation Hotspots (Hrdina & Romportl, None declared.K.E.A., A.T.P., and R.M.B. designed the study. E.G., C.K., M.‐O. R, and J.P. provided tissues. K.E.A., P.M.H. and V.V.S. performed the laboratory work. K.E.A., P.M.H and W.P.T.N analyzed the data. K.E.A wrote the manuscript. E.G., P.M.H., J.P., M.‐O. R and R.M.B. edited the manuscript.Supplementary MaterialClick here for additional data file.


Hipparchia semele), a butterfly endemic to Europe and of growing conservation concern in North-West Europe, and report on its population genetics, in a fragmented, anthropogenic landscape in Belgium. Our study included samples from 23 different locations nested in two regions and additional historical samples from two locations. We assessed contemporary, long-distance dispersal based on genetic assignment tests and investigated the effect of habitat loss and fragmentation on the population genetic structure and genetic variation using data of nine microsatellite loci.To set up successful conservation measures, detailed knowledge on the dispersal and colonization capacities of the focal species and connectivity between populations is of high relevance. We developed species-specific nuclear microsatellite molecular markers for the grayling (Ne) .Detected dispersal events covered remarkably long distances, which were up to ten times larger than previously reported colonisation distances, with the longest movement recorded in this study even exceeding 100 km. However, observed frequencies of long-distance dispersal were low. Our results point to the consequences of the strong population decline of the last decades, with evidence of inbreeding for several of the recently sampled populations and low estimates of effective population sizes  populations in highly fragmented, anthropogenic landscapes.The online version contains supplementary material available at 10.1186/s12862-021-01953-z. In recent years, terrestrial insect declines have been reported worldwide, with habitat loss and fragmentation as main drivers of population decline in anthropogenic landscapes . HabitatHipparchia semele) is a butterfly species endemic to Europe that is highly affected by habitat loss and fragmentation, in particular in NW Europe. It is listed as ‘Threatened’ in Flanders (northern Belgium) [The grayling (Belgium)  and as ‘Belgium)  and the Belgium) . In mostBelgium) , 10 embeBelgium) . SimilarBelgium) . HeathlaBelgium) , 13. EurBelgium) . The graBelgium) .For the conservation of species in fragmented biotopes that need additional measures relative to standard vegetation management, species action plans should address the coordination of conservation and management actions on a landscape-scale, encompassing a network of suitable habitats and metapopulations . ConservIn demographic studies, dispersal is often estimated with direct methods such as mark-release-recapture (MRR) studies, the dynamics of patch colonization and patch extinctions or data on range expansions. Other mobility indices of insect species, such as mobility assessment (e.g. very sedentary versus highly mobile) and relative flight speed, are usually based on expert judgements . These mWhile previous studies on the grayling focussed on habitat use and mobility , 23, 24,Hipparchia semele  has a widespread distribution, but it is absent in the northernmost parts and the highest European mountains [Within Europe, the grayling ountains . See Addountains . Due to ountains , 55% in ountains . The spehttp://waarnemingen.be). For our genetic study, we sampled the locations in northern Belgium where a minimum of ten individuals were observed in one year in the time period 2015–2019. Sampling occurred during the summer of 2020. We assumed that each sampling location contained a single deme; a local population consisting of closely related individuals that form a distinct gene pool, and where mating is random. We sampled 23 different locations nested in two different regions: the Belgian coast in north-west Belgium (five locations) and the inland Campine region located in the north-east of Belgium (18 locations)  of 30 grayling butterflies, except for the location Maatheide (MAH) and Teutelberg (TEB), where only 2 and 25 individuals were detected, respectively. This resulted in a total of 657 non-destructive wing-clip samples. Wing-clip sampling does not reduce flight ability nor does it affect survival rate , althouate  . Details on microsatellites and PCR conditions are given in Additional file Before extraction of genomic DNA  and population-specific genetic diversity , with and without each of these loci. Six loci were discarded during these steps and the two samples of location Maatheide (MAH) were discarded due to missing data at more than 3 loci. The following data analyses were performed based on nine microsatellite loci and 641 unique samples from 24 sampling locations . A total of 63 alleles, with an average of 3.6 alleles per locus, were observed and the dataset had an overall proportion of missing data of 3.7%. For additional information, see Additional file In total, 701 butterfly wing clips were genotyped. Five microsatellite loci were discarded from the analyses due to stutter peaks and subsequent difficulty in allele scoring. Hence, we worked with 15 microsatellite loci. Next, we examined the assumptions of Hardy–Weinberg equilibrium and of no linkage disequilibrium (LD). We used GENEPOP v4.3  to test Genetic assignment methods of dispersal inference make use of individual genotypes and population allele frequencies to estimate where individuals were born or not . GENECLAN), average number of observed alleles per locus (A), effective number of alleles (Ae), average observed heterozygosity (Ho), Nei’s overall expected heterozygosity (He) and the mean inbreeding coefficient (FIS) using GenAlEx v6.501 [IS corrected for the potential presence of null alleles with the program INEst 2.2 [Ar) and number of private alleles (Ap) adjusted for sample size, based on a minimum sample size of 13 individuals. To detect significant differences in genetic diversity indices, ANOVA analysis were conducted in R [He from the values expected at mutation–drift equilibrium (He > He eq)  for each sampling location by using the linkage disequilibrium method (LDNe) implemented in the program NeEstimator v2 [Ne, FIS, Ar and He were tested with univariate linear regressions in R [For each sampled location, we calculated the average number of individuals genotyped per locus  when there is no differentiation between populations and one at complete differentiation. Differences in the distribution of genotypes between all population pairs were assessed through calculation of 95% confidence intervals (C.I.) of the G’ST values with a bias-corrected bootstrapping method (1000 bootstraps). Using the ISOLDE program in GENEPOP v4.3, we tested for an overall and regional pattern of genetic heterogeneity over geographic distances with the isolation-by-distance-test (IBD) [ST-estimates (FST/(1–FST)) for pairs of populations and the Euclidian geographic distance in kilometres between sampling locations. We set the number of permutations for Mantel test to 100 000 and the rest of the parameters on default parameter settings. Next, we used GenAlEx v6.501 to estimate levels of hierarchical structuring within populations, among populations and among regions by analysis of molecular variance (AMOVA) with populations nested within two regions  (999 permutations). Bayesian analyses of population structure was performed with the programs STRUCTURE v2.3.4 [We used the R package DiveRsity to calculate global, regional and pairwise genetic differentiation between populations. We calculated G’ST  and addiST [EST  based onst (IBD) . We usedE v2.3.4  and BAPSE v2.3.4  (AdditioThe population assignment test using the Bayesian method identified seven putative first-generation long-distance dispersers p ≤ 0.01) Table . Putativ Table . Ar: 3.26 and 2.83 resp., ANOVA test p < 0.05). Genetic diversity ranged from 2.8 to 4.3 alleles/locus and values of expected heterozygosity (He) ranged from 0.35 to 0.46. Corrected allelic richness values (Ar) ranged from 2.6 to 3.7 and the corrected number of private alleles (Ap) ranged from 0.0 to 0.1  and in twelve of the seventeen (70%) recent inland populations   coastal populations  and in five of the seventeen (30%) recent inland populations   and 54 (ZBN) individuals using the Random Mating model  and a negative trend between Ar and the distance to the nearest population . The other tested correlations were not significant.We found higher levels of genetic diversity  in the inland populations than in the coastal populations (ST = 0.059 (95% C.I.: 0.046–0.073) and DEST = 0.014 (95% C.I.: 0.009–0.020)). Regionally, genetic differentiation was even lower between the inland populations  and DEST = 0.007 (95% C.I.: 0.002–0.013)) and slightly higher between the coastal populations  and DEST = 0.017 (95% C.I.: 0.008–0.029)). Both global and regional differentiation were significant . Overall, pairwise population G’ST-values ranged from − 0.012 to 0.160  between 97 of the 276 population pairs. IBD-analysis showed that genetic variability was only globally structured as indicated by the significant linear relationship . Within the inland region, we found no significant isolation by distance . The IBD-analyses of the coastal region showed a positive trend . So there is weak evidence for isolation-by-distance in this region  studies observed covered distances of 1.2 km , 1.5 km 2). In contrast, previous MRR studies in the region [Phengaris (Maculinea) alcon, Vanden Broeck et al. [P. alcon from MRR studies was 0.5 km and the maximum distance recorded from spontaneous colonization data so far was 1.7 km [The dispersal distances observed in this study are substantially longer than the ones reported in previous MRR studies. In this study, we covered the entire distribution area of the grayling in northern Belgium . For exk et al.  found evs 1.7 km . To get Although we provide evidence for dispersal ability of the grayling over unexpected long distances, the observed frequencies of long-distance dispersal events were low 1.2%). This observed low frequency of long-distance dispersal can, at least partly, be explained by the low genetic differentiation between populations, which limits the power of the assignment tests to detect dispersal events . Further.2%. ThisAtrytonopsis sp. on the highly urbanized coast of North Carolina (USA). Based on Amplified Fragment Length Polymorphic (AFLP) molecular markers, Leidner and Haddad [However, the majority of the detected long-distance dispersal occurred between inland populations, while no dispersal between the coastal populations was detected. Even with less strict detection methods, the vast majority of detected dispersal events occurred between the inland populations, with sparse dispersal events within the coastal region and between regions Table : S8. In d Haddad  found thd Haddad  and on Id Haddad . Furtherd Haddad , 63. Dued Haddad . These aIn contrast to the coastal region, the grayling inland populations are principally surrounded by a matrix of woodland, heathland and meadows. Woodlands are often considered to be dispersal barriers for butterflies of open habitats , but Nowicki et al.  showed tWhen habitat fragmentation hampers gene flow, populations can suffer from loss of genetic diversity reflected by low effective population sizes, resulting in inbreeding and extinction risks. We did find evidence of inbreeding in several populations and low estimates for effective population sizes . Inbreeding increases levels of homozygosity and exposes deleterious recessive alleles, which can result in inbreeding depression and reduced population fitness , 67. TheFor further investigating the potential effects of fragmentation on genetic diversity, we included samples collected two decades ago (dated 2001) from two inland populations (MEH and TEB) and compared the genetic diversity with recent samples of the corresponding locations (dated 2020). Levels of genetic diversity, in terms of effective population size and allelic richness, were similar between the recent and historical samples for both locations. However, for the location MEH, there was no evidence of inbreeding in the historical sample, while we found weak evidence of inbreeding in the current population. This may indicate that the levels of diversity did not change over the past two decades, but that a consistent low average population size (we estimated low effective population sizes and the census population sizes have been decreasing over decades ) caused Phengaris alcon [Plebejus argus (unpublished data) occured in the same inland Campine region of this study. The results of these studies and our study on H. semele were obtained using the same methods. P. alcon is listed as ‘Critically endangered’, while P. argus is listed as ‘Endangered’ in Flanders [P. argus populations. The study on P. argus also found a low number of dispersers within the inland region and no significant Isolation-by-distance.Previous studies on the two heathland butterfly species is alcon  and PlebFlanders . In termx population combinations. The presence of null alleles causes an increase in homozygosity, which means that levels of inbreeding could be inflated . Additied (e.g. ), and aned , 7130, 7Based on the habitat characteristics and the genetic differentiation and mobility, we can assign grayling populations in northern Belgium to two different Functional Conservation Units (FCUs) (cf. ); the coFor the coastal conservation unit (FCU 1), as current inter-population connection is likely extremely low, we suggest that the emphasis should be on creating new habitat between existing populations in order to improve population network connectedness. Furthermore, translocation actions such as reinforcements should be considered, as spontaneous exchange of individuals between populations is likely hindered by anthropogenic barriers to gene flow along the Belgian coast and because of the observed inbreeding. The inland conservation unit (FCU 2) consists of a larger metapopulation, in terms of the number of demes and occupied territory, and connectivity between sub-populations is likely higher and less hindered by anthropogenic barriers compared to the coastal populations. However, also for FCU 2, restored and/or new heathland habitat have likely a low chance to be colonized spontaneously within a short time frame. Over the past years, there has been a strong decline of the grayling in Northern Belgium , which iTranslocations should be conducted according to IUCN guidelines . To avoiThis genetic study on the grayling showed that dispersal distances covered by grayling butterflies were much larger than previously assumed. Long-distance movements are however scarce. Estimated effective population sizes were low and several populations showed evidence for inbreeding, which reflect the consequences of strong population declines during the last decades. Although genetic differentiation was found to be low, both within as between regions, there was a significant differentiation between the coastal and inland region. Therefore, we suggest to treat the two regions as as two separate conservation units. Urgent measures  are needed for the sustainable conservation of the species in northern Belgium.Additional file 1.S1. Distribution of Hipparchia semele in North-West Europe. S2. Samples and sampling sites of Hipparchia semele. S3. DNA extraction. S4. Microsatellite development. S5. Microsatellite loci and PCR-conditions. S6. Estimation of the frequency of null alleles with the Dempster method. S7. Hardy-Weinberg equilibrium and Linkage Disequilibrium testing. S8. Population assignment test: Frequency based method. S9. Genetic bottlenecks. S10. Estimation of effective population size (Ne). S11. STRUCTURE and BAPS analysis. S12. Genetic diversity statistics. S13. The effect of null alleles on the FIS-values. S14. Pairwise G’ST values. S15. Isolation-by-distance analyses. S16. Levels of hierarchical structuring within populations, among populations and among regions estimated by analyses of molecular variance (AMOVA). S17. Plots of the Principal Component Analysis (PCoA). S18. Detailed maps of the land-use in northern Belgium.


Stratiotes aloides which has become rare in several areas. The preservation of genetic diversity within a species is a prerequisite for survival under changing environmental conditions. To evaluate the level of genetic diversity within and among populations of Stratiotes aloides, we investigated samples from waterbodies across Europe using AFLP. Low genetic diversity among samples from the same population was found, proving that stands consist of few clones which propagate clonally. Nevertheless, most populations showed differences compared to other populations indicating that there is genetic diversity within the species. The analyzed samples formed two groups in STRUCTURE analyses. The two groups can be further subdivided and mainly follow the major river systems. For conserving the genetic diversity of Stratiotes aloides, it would thus be preferable to focus on conserving individuals from many different populations rather than conserving selected populations with a higher number of individuals per population. For reintroductions, samples from the same river system could serve as founder individuals.Intense land use and river regulations have led to the destruction of wetland habitats in the past 150 years. One plant that is affected by the reduction in appropriate habitats is the macrophyte  Stratiotes includes the sole living species S. aloides L. (water soldier) and is a member of the Hydrocharitaceae which belong to the order Alismatales within monocots. During the Tertiary and Quaternary periods, there were up to twenty different species of the genus Stratiotes in Europe and Asia [S. aloides, stands in one waterbody are often formed by clonal individuals of the same sex [Stratiotes aloides is distributed from northern Middle Europe in the West to Siberia in the East.The monotypic genus and Asia  . Althouratiotes ,2,18. Esilometer . The mainditions ,20. Sinc species . Knowled species .Stratiotes aloides populations across Europe to obtain insights into the circumference of the genepool of the species. These results could be helpful to find answers to conservation issues such as status of a population in a particular locality or possible source populations for recolonizations in habitats where Stratiotes aloides has already become extinct.Here, in this study, we aim to investigate the genetic diversity of After excluding 102 replicates, the final matrix used for analyses contained 345 individuals and 1320 fragments.Results based on uncorrected p-distances and Hamming distances gave the same clustering patterns in neighbor-joining (NJ) dendrograms and principal coordinate analysis (PCoA). The same was found for the pair of Dice distances and Jaccard distances. Therefore, we used only results based on uncorrected p-distances and Dice distances for further analyses.NJ dendrograms based on uncorrected p and Dice distances both showed a star-like shape with a backbone of relative short branches lacking bootstrap support greater than 75% . They diSTRUCTURE analysis gave the highest value of ΔK for K = 3 plus a few other suboptimal K values  in the aFST distances from hierarchical AMOVAs, those based on groupings according to the STRUCTURE results gave the highest values, and also gave the highest values over all PCoA.Principal coordinate analyses based on the two distance methods (uncorrected p and Dice) gave very similar clustering patterns, with uncorrected p-distances showing a higher total sum of coordinates . The firFST value of 0.97  was estimated with uHe = 0.213 in the overall analysis of all samples together. Analysis of separate populations gave the highest Nei’s H-value of H = 0.031 for the commercial samples from Austria (Stauden Hameter population 8) and the lowest value (H = 0.000) for populations from the UK . Shannon’s index was estimated to be I = 0.344 in the overall analysis of all samples together. In the analysis of separate populations, the highest and lowest values were found in the same populations for Nei’s H  among the tested pairs of matrices  between the water systems, genetic diversity between populations is expected to be high.Here, in this study, we examined the genetic diversity of FST value (0.97) when analyzing the populations separately which shows a high level of genetic differences between the populations and a very low level of genetic differences within the populations, indicating that the populations consist mainly of clones of few genetically different individuals. Considering that the populations propagate vegetatively, and that today, there is no gene flow between the populations via transfer of individuals from one population to another, the relations between the populations could show historical connections between populations. This explains why no, or if only a medium, correlation between geographic distance and genetic distance of the populations is found. The observed correlation between geographic distances and genetic distances based on AMOVA grouped by water bodies has to be viewed with some precaution, as the grouping based on water bodies is, of course, a geography-related grouping and will, therefore, already have a slight bias towards a stronger correlation. Nevertheless, we can still see that there is some correlation between geographic distance and genetic distance when we look at it at the level of waterbodies. The populations from waterbodies of the central European highlands and plains in particular have a genepool which is different from the genepool shared by populations from other regions in Europe. However, it looks like that geneflow between populations has occurred. The fact that water soldier populations from waterbodies along the Rhine seem to be a mixture of genepools from British and Danube genepools might be explained by the historic watercourse of the river Rhine with pervious headwaters of the Danube being directed to the Rhine and a common delta of the Rhine and Thames [Stratiotes and Cook and Urmi-König [Stratiotes aloides fruits exo- and endo-zoochorically and if they are migrating over longer distances, seeds and thus genetic information can be transferred between localities. A further point that has to be kept in mind, when investigating relationships among European water soldier populations, is the fact that Stratiotes aloides has a long history as an ornamental plant [Stratiotes aloides date back around 45 million years [Stratiotes aloides across Europe to form two main groups which can be further subdivided. Roughly, the two groups can be referred to as a central northern Europe-group (CE) and a western–southern–eastern Europe group.Indeed, we did find a high d Thames . A second Thames ). The cod Thames . There ad Thames  and Orsed Thames  give an mi-König  as well mi-König  mention al plant ,2. Unexpon years , the obson years . SummingStratiotes aloides across its distributional range showed a much higher level of genetic diversity within the examined populations [Ottelia acuminata where high levels of genetic differences between the investigated populations were found, but little diversity within the populations [Previous studies of ulations . As the ulations  are not ulations .Stratiotes aloides found in Europe are remnants of much larger and connected populations. For example, in the Danube flood plains around Vienna, Stratiotes aloides was still very common by the mid-19th century, around 100 years later, this species was already mentioned to be rare [All still present-day populations of  be rare  . Short (approx. 5–7 cm) pieces of leaves were collected and immediately dried in silica gel. From several populations, herbarium specimens were collected and deposited in the herbarium of the University of Natural Resources and Life Sciences, Vienna (WHB). Herbarium accession numbers are indicated in the table of accessions  using the DNeasy Plant Mini Kit (Qiagen), mainly according to the manufacturer’s protocol. Exceptions were elution of DNA from the columns, which was performed with two steps of 50 µL of elution buffer each. RNA was digested after DNA extraction using 1 µg RNAse A and incubated at 37 °C for 30 min.DNA was extracted from 20 mg silica gel dried leaf material per individual. The material was ground into a fine powder in 2 mL tubes together with three glass beads in a Tissue-Lyser  with 20 sQuality control of the DNA extracts was performed photometrically using a NanoDrop 2000 spectrometer. To check RNA digestion, samples were loaded on a 0.8% agarose gel.All DNA extracts that met the quality criteria were adjusted to 100 ng/µL and used for AFLP fingerprinting. Preparation of AFLP samples mainly followed the original protocol  with sliEcoR I and Mse I) and ligation of double-stranded adaptors to the resulting restricted fragments were performed in one step in a thermal cycler (37 °C for 2 h followed by a hold at 10 °C). Reactions comprised 1.1 μL 10× T4 DNA ligase buffer , 1.1 μL 0.5 M NaCl, 0.55 μL BSA , 50 μM Mse I adaptors , 5 μM EcoR I adaptors (genXpress), 1 U Mse I restriction endonuclease , 5 U EcoR I restriction endonuclease (New England BioLabs), 67 U T4 DNA ligase (Promega), and 5.5 μL DNA (100 ng/µL) and were made up to a total volume of 11 μL with sterile water. Ligated DNA fragments were diluted 10-fold with TE buffer (0.1%). Preselective amplification reactions contained 1 μL 10× polymerase buffer , 0.2 U DreamTaq DNA polymerase , 0.1 μL dNTPs , 0.55 μL preselective primer pairs , 2 μL diluted restriction ligation product, and were brought to a total volume of 10 μL with sterile water. Amplification was carried out with the following profile: 2 min at 72 °C, 20 cycles of 20 s denaturing at 94 °C, 30 s annealing at 56 °C, 2 min extension at 72 °C, and a final extension step for 30 min at 60 °C. The preselective PCR products were diluted 10-fold with sterile water. Reactions for selective amplification contained 1 μL 10× Polymerase buffer (ThermoFisher Scientific), 0.1 U DreamTaq DNA polymerase (ThermoFisher Scientific), 0.1 μL dNTPs , 0.55 μL Mse I-primer , 0.55 μL EcoR I-primer , and 2 μL diluted preselective amplification product and were brought to a total volume of 10 μL with sterile water. They were carried out in with the following profile: 2 min at 94 °C, 9 cycles of 10 s at 94 °C, 30 s at 65–57 °C (reducing the temperature at 1 °C per cycle), 2 min at 72 °C, 25 cycles of 10 s at 94 °C, 30 s at 56 °C, 2 min at 72 °C and a final extension for 30 min at 60 °C. All PCR steps and incubations were carried out in an Eppendorf Mastercycler Gradient. The selective PCR products were purified using Sephadex G-50 Superfine  applied to a MultiScreen-HV 96-Well Plate (Millipore) in three steps of 200 μL each (5× g sephadex in 60 mL 1× TE-buffer) and settled at 750× g . The same speed was used for centrifugation of the samples , again for 5 min. One microliter of the eluate was combined with 15 μL HiDi and 0.25 μL LIZ 600  and denatured for 3 min at 95 °C before running them on a capillary sequencer .Restriction of genomic DNA with two restriction enzymes  were chosen after testing seven different primer combinations in a preliminary test. The selected primer combinations generated clear and not too many bands, thus decreasing the risk of fragments co-migrating by chance, but still with sufficient variability to distinguish the samples.The selective primer pairs . After pre-analysis using default settings, sizing profiles of all samples were checked and where necessary, manually corrected. Most of these corrections concerned the 20 bp peak of the size standard. These peaks were often not correctly recognized by the GeneMarker program. High-quality sizing profiles (score > 90) were obtained for all samples. A panel of scorable fragments was established for each primer combination, and fragments between 30 and 600 bp were scored. The relative fluorescent unit (RFU) threshold was set at 40. Automatic scoring was conducted using Local Southern peak call, peak saturation, baseline subtraction, spike removal, pull up correction, and a stutter peak filter of 5% . The resAll three primer combinations were combined in a single matrix and analyzed together. Different distance measures were tested for their power to resolve relationships with our dataset. Distance matrixes were calculated in PAUP* v4.0a167   using the R packages “ecodist”  and “scav3.5.2.2 .To investigate further significant groupings of the included individuals, we used the programs STRUCTURE v.2.3.4 ,40,41,42FST values from AMOVAs, binary distances calculated with GenAlEx and geographic distances  and calculations of population statistics were conducted using Arlequin v3.5.2.2 . The Excv 1.2.3; ). Calculv 1.2.3; , Shannonv 1.2.3;  and perc