Ambystoma barbouri) are in decline as their distribution lies mostly within rapidly developing areas in the Nashville Basin. Information regarding the partitioning of genetic variation among populations of A. barbouri and the taxonomic status of these populations relative to northern populations and their congener, the Small-mouthed Salamander (A. texanum), have important implications for management and conservation of this species. Here we combined mitochondrial sequencing and genome-wide single nucleotide polymorphism (SNP) data generated using Genotyping-by-Sequencing (GBS) to investigate patterns of genetic variation within Tennessee populations of A. barbouri, to assess their relationship to populations in Kentucky, and to examine their phylogenetic relationship to the closely related A. texanum. Results from phylogenetic reconstructions reveal a complex history of Tennessee A. barbouri populations with regards to northern populations, unisexual A. barbouri, and A. texanum. Patterns of mitochondrial sequence variation suggest that A. barbouri may have originated within Tennessee and expanded north multiple times into Kentucky, Ohio, Indiana, and West Virginia. Phylogenetic reconstructions based on genome-wide SNP data contradict results based on mitochondrial DNA and correspond to geographic and taxonomic boundaries. Variation in allele frequencies at SNP genotypes, as identified by multivariate analyses and Bayesian assignment tests, identified three evolutionary significant units (ESUs) for A. barbouri within Tennessee. Collectively, these results emphasize the need for prioritizing conservation needs for Tennessee populations of A. barbouri to ensure the long-term persistence of this species.In Tennessee, populations of the state endangered Streamside Salamander ( Ambystoma barbouri, A. barbouri, but can be mitigated by management actions aimed at maintaining genetic diversity. Information regarding A. barbouri’s taxonomy, population structure and patterns of genetic variation are currently needed to prioritize conservation needs and to efficiently allocate management resources. Genomic tools are increasingly being used to improve recovery and management planning in at-risk species, including salamanders, by informing taxonomic relationships, demographic histories, and biologically meaningful units of conservation that will preserve genetic diversity and the long-term adaptive potential of species [Genetic variation, population structure, and demographic history are increasingly recognized as important factors for the design of effective conservation strategies . Streams species –7.A. barbouri and its sister-species A. texanum  are a concern for precisely identifying targets of conservation. Adults of these two species are nearly indistinguishable based on external morphological features alone and were previously considered to be conspecific. They can be differentiated using scanning electron microscopy on the number and shape of maxillary and premaxillary teeth, larval life-history traits, and choice of breeding environment [A. texanum and A. barbouri [A. texanum and A. barbouri are not reciprocally monophyletic; A. texanum is recovered as a clade nested within A. barbouri from Tennessee, suggesting that pond-breeding A. texanum were more recently derived from a stream-breeding A. barbouri ancestor from Central Tennessee. However, reconstructions based on two nuclear gene sequences resulted in reciprocally monophyletic clades for the two species, consistent with a history where A. barbouri and A. texanum are older independent lineages derived from a shared common ancestor. Additional sequence data are needed to resolve unanswered phylogenetic questions as these earlier reconstructions were based on a limited number of molecular markers  and representative outgroups.Taxonomic uncertainties surrounding the evolutionary relationship between ironment , 9. In abarbouri . ReconstA. barbouri. Mitochondrial haplotypes are most closely related to mitochondrial haplotypes found south of the Ohio River, and southwest of the Kentucky River [A. barbouri to unisexual ambystomatids has not been explored.Mitochondrial evidence also informs on the origins of unisexual ambystomatid populations (family Ambystomatidae) that are common in the Great Lakes region of North America. Unisexual ambystomatids exhibit a unique reproductive mode whereby male sperm activates egg development, but contributes variable amounts of nuclear genetic material depending on compatibility with unrelated cytoplasmic DNA (termed kleptogenesis) . While tky River ; howeverA. barbouri in Tennessee and discontinuous populations in Kentucky, Indiana, Ohio, and West Virginia. Phylogenetic reconstructions of A. barbouri populations based on mitochondrial sequence data (913 bp) from the D-loop and an adjacent intergenic spacer suggest that Tennessee A. barbouri populations are both ecologically and genetically distinct from more northern populations [A. barbouri from Tennessee were smaller and less developed than individuals from Kentucky. Also, laboratory behavioral assays show that Tennessee A. barbouri were similar to western Kentucky individuals, but less active than individuals from northern populations [A. barbouri. However, conclusions from this study were limited as these reconstructions are based on a single mitochondrial gene from only three individuals from a few populations within a kilometer of each other in Rutherford County.At a finer scale, uncertainties also exist regarding the relationship among isolated populations of ce data 9 bp from ulations . These dce data 9 bp from A. barbouri populations in Tennessee are in decline, and the accompanying loss of genetic variation in small populations further threatens the long-term persistence of this species [A. barbouri are limited to a handful of mitochondrial and nuclear sequences used for phylogenetic studies [Observations from field surveys suggest that  species . Mainten studies , 10. Gen studies .A. barbouri as well as the taxonomic relationship between A. barbouri and A. texanum in order to prioritize conservation needs and inform management practices aimed at maximizing long-term persistence of this species. Mitochondrial sequence data and genome wide SNP genotypes were used to (1) further examine. the phylogenetic relationship between A. barbouri and A. texanum, (2) review the taxonomic relationship of disjunct populations of A. barbouri in Tennessee, relative to northern populations, (3) evaluate patterns of genetic differentiation between geographically isolated populations of A. barbouri within the state of Tennessee and (4) estimate within-population genetic variation and examine demographic history of Tennessee A. barbouri. Taxonomic relationships, units of conservation, and geographic partitioning of genetic variation are discussed in the context of establishing conservation priorities and designing effective management strategies for this species.Here we investigate patterns of genetic variation within and among populations of Tennessee A. barbouri in the form of adult tail clips, eggs, and whole larvae were obtained from field surveys or from collaborators , Davidson County (D3), Rutherford County , Sumner County , Wilson County , and Williamson County (Wil2). A total of five A. texanum individuals were sampled; these included four individuals from  and one from Craighead Co., Arkansas (AKT1). Outgroups included two A. mabeei (North Carolina), two A. maculatum (AAFB), and one A. talpoideum (AAFB).Tissue samples of borators , Table 1Ambystoma [DNA was extracted from tail clips, eggs, and larvae using the EZNA Tissue DNA Mini kit (OMEGA BIO-TEK) following the manufacturer’s protocol except that DNA was eluted in water. Approximately 1200 bp of the mitochondrial D-loop was targeted for PCR amplification using primers developed by Shaffer and McKnight  Table 2Table 2. mbystoma –18. Polymbystoma   and Bayesian optimality criteria were used for phylogenetic analyses. Maximum-likelihood analyses were performed using the software RAxML [Phylogenetic reconstructions were estimated for all unique mitochondrial D-loop haplotypes and an additional 53 re RAxML  on the Cre RAxML  under thre RAxML  also on re RAxML , implemere RAxML  using Bare RAxML , 27. A mA. barbouri in Tennessee, 17 individuals from five populations of A. barbouri in Kentucky, five individuals from two populations of A. texanum, and two sampled outgroups A. talpoideum (N = 1) and A. mabeei  greater than 0.5 were removed to filter out possible paralogs. The final SNP dataset was then filtered to remove loci deviating from Hardy-Weinberg equilibrium (p < 0.05), loci genotyped in less than 60% of individuals, and SNPs with a minor allele frequency (MAF) less than 0.01. Only one SNP was retained per sequenced tag.The equences . We usedequences  to generA. barbouri populations were estimated using the R-package DiveRsity v. 1.9.9 [P), allelic richness (AR), and observed and expected heterozygosity (Ho and He). Estimates of P, AR, Ho, and He were estimated for the entire SNP dataset and for the subset of SNPs that were polymorphic within each population.Estimates of within population genetic variation for 12 Tennessee v. 1.9.9 . SummaryE) was estimated for Tennessee A. barbouri populations using both the linkage disequilibrium (LD) method and the heterozygote-excess method [EEstimator v2 [E estimation [Effective population size  as implemented in Arlequin 3.5 [Population pairwise FT values  and G”STlues [ST  were estiveRsity . Hierarcquin 3.5 , 38. ResR (v. 2.1.4) [find.clusters function was first used to identify the optimal value of K based on a Bayesian Information Criterion (BIC) process. The optimal K was then used to perform a DAPC analysis to describe the relationship between the genetic clusters. Individual membership probabilities were assessed using the function compoplot. DAPC analysis was performed on three hierarchical datasets; these included 1) 12 populations of A. barbouri in Tennessee, 2) all sampled Tennessee and Kentucky A. barbouri populations, and 3) all A. barbouri populations and four A. texanum individuals from AAFB.The optimal number of genetic clusters (K) based on genomic SNPs was estimated using both a multivariate approach and a Bayesian-based assignment method. Discriminant Analysis of Principal Component (DAPC) was performed using the ‘adegenet’ package in . 2.1.4) , 40. TheA. barbouri populations using the program STRUCTURE 2.3.4 [A. barbouri populations were not included in assignment analyses due to small sample sizes at these sites. Values of K (number of populations) ranged from 1 to 12 populations with 20 replicate runs per value of K; MCMC simulations were performed for a burn-in of 250,000 iterations and an additional 1,000,000 iterations were retained for the final analysis. Results were summarized using the software package CLUMPAK [K) was determined using the Delta-K method [Bayesian assignment tests were performed on Tennessee RE 2.3.4 . Kentuck CLUMPAK . The optK method  as impleK method . The Dist = years before present) as implemented in the software BPP v. 4.4 [A. barbouri and A. texanum populations. Loci represented by fewer than 100 individuals were removed from the analysis. Joint species-delimitation and species-tree reconstructions were performed using the A11 model, where individuals were assigned to sampled populations. Run parameters were as follows: uniform rooted trees were used as the species model prior, the theta prior (θ = 4NEμ) was assigned a gamma distribution with α = 3 and β = 0.04 and the prior for tau (τ = tμ) was gamma distributed with α = 3 and β = 0.2. Three independent MCMC runs were implemented, each with a burn-in of 5,000, a sample frequency of 10 and a total of 50,000 iterations. Species delimitation and species-tree topologies with the highest posterior probabilities from the A11 runs were used as input for the A00 analyses. The A00 model estimates divergence time parameters and long-term NE, assuming fixed species assignments and a fixed species tree. The priors for A00 were the same as priors used for the A11 model. Three independent runs were also performed for the A00 analysis, each with a burn-in of 5,000, a sample frequency of 10 and a total of 50,000 iterations. MCMC chains were pooled and absolute estimates of τ were converted to years before present (YBP) in the BPPR using the genome wide mutation rate estimate for the vertebrate nuclear genome where μ = 1.21 х10−9 [A multispecies coalescent (MSC) model was used to examine taxonomic boundaries, estimate species trees, and estimate population divergence times  and 100% bootstrap support (bs) for Bayesian and ML analysis, respectively. In Clade II, A. barbouri from Wilson Co.  and Davidson Co. (population D3) formed a monophyletic clade that also includes Kentucky A. barbouri populations from southwest of the Kentucky River . Clade I and Clade II were recovered as sister clades in both analyses with strong support . Clade III included all A. texanum individuals  with the exception of a single A. texanum haplotype obtained from Genbank ; this sequence is recovered as basal to Clade III in both ML and Bayesian analyses. Clade IV groups with Clade III and included Tennessee A. barbouri individuals from Rutherford Co. , Bedford Co. (B6), and Williamson Co.; pp = 100, bs = 71%). Clade V included all Kentucky individuals from populations northeast of the Kentucky River, Ohio, and Indiana . Finally, Clade VI is sister to Clade V and included all Tennessee A. barbouri from Sumner Co. , a single haplotype from Wilson population W3, and three haplotypes sequenced from Rutherford population R7 . On a larger scale, all A. barbouri and A. texanum are part of the same well-supported monophyletic group in both analyses , such that all A. texanum haplotypes are nested within A. barbouri.Both Bayesian and ML phylogenetic reconstructions of mitochondrial D-loop haplotypes identified six major clades across plotypes . The Cla6 ± 5.9 х 106; 220 individuals were retained after removal of low coverage individuals . A total of 1.56 х 106 loci were recovered from the de novo assembly in ipyrad and 440,629 loci were retained after filtering. Additional filtering for SNPs  recovered 1,169 SNPs for the dataset that included only Tennessee A. barbouri, 516 SNPs in the dataset that included all sampled A. barbouri populations in Tennessee and Kentucky, and 500 SNPs in the dataset that included all A. barbouri and A. texanum populations.The average number of retained sequence reads per individual generated from sequencing of GBS libraries was 5.86 х 10o and He; however, this population had a low proportion of polymorphic SNPs overall. Rutherford County populations (R1 and R7) had the lowest estimates of Ho and He, when considering only polymorphic loci and for the entire SNP dataset.Much of the genetic variation within our SNP dataset was in the form of fixed differences between populations . The proNE) were generated for five populations with adequate sampling (N≥20), including S7, S8, R1, R7, and D3 (NE = 15 (R1) to NE = 108 (108) (S8); the upper confidence interval for S8 was ∞, indicating low signal for this dataset. Results from the heterozygote excess method were inconclusive (∞) for two of the five analyzed populations. Estimates from the remaining three populations ranged from 24 (D3) to 406 (S7); however, CIs included ∞ for two of these estimates (S7 and S8).Estimates of effective population size  to highly differentiated (FST = 0.703 for S5/R1). In general, populations north of the Cumberland River  showed evidence of long-term isolation from populations south of the Cumberland River. Pairwise FST estimates comparing populations north and south of the Cumberland River averaged 0.606 ± 0.047, while FST estimates among Tennessee populations north of the Cumberland River averaged only 0.078 ± 0.041. Populations south of the Cumberland were further structured; populations in Wilson and Davidson Counties  were diverged from populations further south in Williamson, Rutherford, and Bedford counties. Estimates of pairwise FST comparing populations within Wilson/Davidson averaged 0.104 ± 0.035 and within Williamson, Rutherford and Bedford Counties averaged 0.060 ± 0.060, while between group pairwise FST values were much higher, averaging 0.284 ± 0.060. Pairwise G”ST were very similar to FST estimates and demonstrated the same hierarchical pattern.Pairwise F fashion . OverallA. barbouri recovered from mitochondrial phylogenetic reconstructions were further examined using genome-wide SNP genotypes in an AMOVA framework (The three monophyletic clades of Tennessee ramework . Most vaK = 3) best represented genetic variation among populations sampled within Tennessee  between A. texanum and A. barbouri was 1.6 million YBP. All Sumner A. barbouri formed a monophyletic group with Kentucky populations as a monophyletic sister group. Divergence time estimates indicated that Sumner populations and Kentucky populations shared a common ancestor < 500,000 YBP. All A. barbouri populations south of the Cumberland River were monophyletic  and were further split into two groups that corresponded to clades recovered in the mitochondrial gene tree. Davidson and Wilson populations were recovered together in the first group  and Rutherford, Bedford, and Williamson populations  formed the second group.The A11 species tree reconstruction with the highest posterior identified the same Tennessee clades recovered in our mitochondrial gene trees with the exception of the placement of A. barbouri. Despite the documented decline of A. barbouri populations in the Nashville Basin, results from GBS-derived SNP genotyping indicate that estimates of genetic variation in extant populations of Tennessee A. barbouri are similar to SNP-based datasets examined from other ambystomatids [A. barbouri with more northern populations and with its sister species A. texanum. Despite marked differences in phylogenetic reconstructions based on mtDNA sequencing and nuclear genotypes, collectively these results indicate that Tennessee is genetically unique from northern A. barbouri and A. texanum.Patterns of genomic variation and taxonomic relationships identified here have important implications for developing management strategies aimed at the long-term conservation of tomatids , 49. ParA. barbouri populations in the Nashville Basin, maintenance of adaptive genetic variation is particularly critical as these populations are faced with habitat alteration by urbanization in addition to climate change [O ranged from 0.165 to 0.269 for populations of the Mole Salamander  and were slightly higher for populations of the Marbled Salamander  [Eurycea bislineata). Fusco et al. [O from urban, suburban and rural salamander populations  and concluded that genetic variation had been maintained, despite habitat disturbance. However, estimates of heterozygosity may not be directly comparable across independent studies as filtering parameters in bioinformatic pipelines can influence population genetic summary statistics. Stringent filtering criteria may preferentially retain loci in conserved regions of the genome and downwardly bias estimates of genetic variation [A. texanum and A. barbouri populations based on microsatellite markers. Micheletti and Storfer [A. barbouri distributed throughout Kentucky, Ohio, and Indiana; the average He from their study ranged from 0.67–0.81, respectively. Few conclusions can be made by comparing results from these prior microsatellites studies to our results based on SNP genotypes; microsatellites are known to have a much higher rate of mutation than nucleotide substitutions, increasing the expected amount of allelic diversity for a given population size [Loss of genetic variation resulting from rapid population decline threatens the long-term success of conservation efforts due to loss of adaptive potential and fixation of deleterious alleles . For A. e change . Evaluat7–0.298) . Anothero et al.  reportedariation . There h Storfer  estimateion size .E) may also signal a decline in the genetic health of at-risk populations. Our estimates of NE were low (NE from the LD method averaged 58.0 across five populations), but were not unusual for LD-derived estimates of NE in salamanders. Published estimates of NE reported for amphibians have typically been under 100 [A. californiense; NE values of 11–64) [A. leorae; NE values of 17–45) [A. macrodactylum; NE = 23–2070 [A. macrodactylum, Savage et al. [E for 47 breeding populations and more than half of these estimates were less than 50. Life history factors may provide some explanation for low NE in this group as salamanders often exhibit high variance in reproductive success and larval survival within populations. Variance in reproductive success will increase relatedness among individuals in a population, inflating linkage disequilibrium across loci and reducing NE.A reduction in estimates of contemporary effective population size   and for  23–2070 . In A. me et al.  estimateE and other demographic parameters in at-risk species can be challenging due to the sample sizes required for LD detection. Many threatened taxa are inherently rare and prohibitively difficult to sample in large numbers. For robust estimation of NE, it is recommended that sample sizes be greater than 30 for most systems [E estimates can be downwardly biased and confidence intervals can be large (or infinite) due to inadequate signal in the dataset [E analysis to populations with sample sizes > 20 individuals, which left only five populations for NE estimation. The infinite upper confidence interval for our NE estimate in population Sumner 8 was likely due to an inadequate sample size for this population (N = 21).Obtaining robust estimates of N systems . When th dataset . We limiA. barbouri in Tennessee that involves populations of unisexual ambystomatids, A. texanum, and populations of A. barbouri from the northern core region . Our results support findings by Bogart et al. [A. barbouri. The phylogenetic reconstruction shown here further details this relationship whereby unisexual ambystomatids likely shared a maternal common ancestor with A. barbouri populations in Kentucky and populations from Middle Tennessee. This pattern, coupled with phylogenies based on nuclear genes, points to a history of repeated hybridization events initiated by an A. barbouri maternal ancestor in the southern portion of its range.Mitochondrial gene-tree reconstructions suggest a complex biogeographic history for t et al.  demonstrA. texanum is nested within present-day A. barbouri, rendering A. barbouri paraphyletic. This phylogenetic pattern contradicts the scenario proposed by Kraus and Petranka [A. barbouri descended from the more widespread pond-breeding A. texanum. Species-tree reconstructions based on genome-wide SNP data are incongruent with relationships recovered from the mitochondrial trees. This species-tree topology adheres to conventional taxonomic and geographical boundaries  and may facilitate rapid diversification [With regards to their mitochondrial lineage, Petranka , that stundaries , where A species . Incongr species , 66. Detfication .A. barbouri. Mitochondrial-based phylogenetic reconstructions and partitioning of genetic variation at SNP loci  identify the same three genetically distinct groups in Tennessee; these include a northern cluster, a central cluster, and a southern cluster. The Cumberland River appears to have served as a major barrier to gene flow between the northern and the central/southern clusters. The central and southern clusters both occur south of the Cumberland River and are divided by smaller regional drainage patterns. Individuals in the northern and central clusters occupy the Old Hickory Lake and Lower Stones River watersheds, respectively. The central cluster is bordered by the Cumberland River to the North and by the East Fork of the Stones River to the east. The southern cluster includes populations from three watersheds including the West Fork Stones River Watershed, Upper Duck River Watershed, and Mill Creek Watershed; the southern cluster (with the exception of the Wil2 population) is bordered by the Stones River to the north and by the Duck River to the south.The hydrogeography of the Nashville Basin, together with cyclic glacial movements during the Pleistocene, may have shaped contemporary patterns of genetic variation in Tennessee populations of A. barbouri populations are not monophyletic with respect to northern populations, which may reflect a history of repeated range expansions from Tennessee into the northern end of A. barbouri’s present day distribution. The estimated timing of divergence between the three Tennessee clusters falls within the mid-late Pleistocene. Glacial advances during the Pleistocene did not extend into Kentucky and Tennessee and this region likely served as a refuge for both terrestrial and aquatic fauna [A. barbouri northward from Tennessee may explain the polyphyletic relationships of contemporary Tennessee A. barbouri with respect to populations in the north. Specifically, mitochondrial A. barbouri populations in northeastern Kentucky, Ohio, and Indiana (Clade V) are nested within Tennessee A. barbouri as are mitochondrial haplotypes from ambystomatid unisexuals and A. texanum. Genetic evidence of repeated northward expansions has been reported for the Eastern Woodrat (Neotoma floridana) [Mitochondrial-based phylogenetic reconstructions revealed that Tennessee ic fauna . As glacoridana)  and otheoridana) , 71).A. barbouri in Tennessee. First, patterns of genetic variation in mitochondrial and nuclear genomes support the assignment of three genetically distinct units for management that warrant the designation of evolutionary significant units (ESUs), where ESUs are defined as groups of populations that show phylogeographic differentiation for mtDNA haplotypes and divergence in allele frequencies in nuclear markers [Results from our genomic survey have specific implications for the design of management and conservation strategies that may improve the long-term persistence of  markers , 73. TheA. barbouri, Tennessee populations are geographically disjunct from the majority of A. barbouri breeding populations [A. barbouri should be prioritized for conservation planning as these populations are both genetically diverse and evolutionarily distinct from populations in the northern part of their distribution. The distinctiveness of these populations is further evidenced by observed differences in reproductive life-history traits, including mean diameter of early stage ovum size and number of eggs per clutch [A. barbouri breeding sites in Tennessee may be instrumental to ensuring the long-term viability of this species as a whole.Evolutionarily and phylogenetically distinctive populations contribute disproportionately to genetic diversity and should be ranked highly in regards to conservation priority. Geographically peripheral populations are frequently observed to be genetically less-redundant than more central populations and are more likely to possess potentially adaptive genetic variation . For A. ulations  and occur clutch . Prioritr clutch . Howeverr clutch . ProtectS1 Table(DOCX)Click here for additional data file.S1 FigA. barbouri in Tennessee B.) sampled populations of A. barbouri in Tennessee and Kentucky, and C.) all sampled populations of A. barbouri and two representative populations of A. texanum.Assignment probabilities from DAPC analysis for A.) sampled populations of (DOCX)Click here for additional data file.


Carassius fish, or Hibuna, which is mainly found in Lake Harutori on Hokkaido, Japan. Although Hibuna in Lake Harutori has been protected as a symbol of the Natural Monument of Japan, it has recently suffered population decline. To establish an effective conservation strategy for Hibuna, we investigated its origin, reproductive mode, and genetic diversity, with reference to the surrounding wild populations, using nuclear microsatellites and mitochondrial gene sequences. Our genetic analyses revealed that the main ploidy of Hibuna was triploid or tetraploid, and it reproduces gynogenetically. However, no co-existing sexual diploid Carassius was detected among our samples, suggesting that the sexual diploids and the gynogenetic population including Hibuna would be at risk of co-extirpation. In addition, Hibuna showed high genetic/clonal diversity and most Hibuna had nonindigenous mitochondrial haplotypes that are mostly identical to those reported from goldfish. These results indicate that Hibuna most probably originated from hybridization between indigenous gynogenetic triploids and goldfish introduced about 100 years ago, involving rare sexual reproduction. This spontaneous long-term field experiment exemplifies the recently documented diversification process of gynogenetic Carassius via complex interploidy gene flow. Although the priority to be placed on the conservation of Hibuna is controversial, the maintenance of gynogenetic Carassius, including Hibuna, requires strategic conservation of sexual populations.Conservation of sperm-dependent asexual (gynogenetic) species is challenging due to their complicated ecological dynamics, which requires the stable coexistence with their sperm-providing sexual relatives, who often share similar niches. A symbolic but vulnerable gynogenetic animal is the scarlet  The reproductive mode  of protected species is one of the key determinants of genetic diversity that affects the effective population size (Ne) and selection dynamics , 2. GynoCarassius fish is interesting subject for studying evolution and conservation of asexual vertebrates. There has been taxonomical confusion in this genus, but currently at least three species are recognized: the crucian carp (Carassius carassius), the Japanese white crucian carp (C. cuvieri), and the wild goldfish, C. auratus-complex , whidangered , 16. Howdangered , 18. Theelopment .C. auratus-complex, the populations attracting particular attention are those including scarlet crucian carp, locally called “Hibuna”, which shows unique scarlet body color variants, instead of the normal brownish silver color, at maturity and is found in several distant river systems on Hokkaido, the northernmost island of Japan [Carassius individuals showing a scarlet body color as “Hibuna” regardless of their ploidy. Particularly famous is the Carassius population including Hibuna in Lake Harutori, a coastal lagoon (surface area of 0.36 km2 and average water depth of 2.3 m) in Kushiro, Hokkaido [Among the of Japan . Since aof Japan , we hereHokkaido . This haHokkaido . Since tHokkaido , 22. HowHokkaido . FortunaCarassius [de novo mutations. Then, at least triploid Hibuna has been inferred to be derived from de novo mutations in the pigmentation-related genes of the triploid Carassius that result in the unique scarlet body color of Hibuna [C. auratus-complex [de novo mutations or goldfish, and to investigate its genetic composition with reference to goldfish and other indigenous ordinary triploids.Despite its symbolic status, the possible artificial origin of the Hibuna in Lake Harutori has been disputed by the recorded introduction of about 3,000 goldfish into the lake in 1916, before the first report of Hibuna in 1922 , 22. Altarassius . The genf Hibuna , 23. How-complex . This in-complex , 24, 25.In this study, the ploidy and genetic characteristics of the scarlet crucian carp, Hibuna, collected from Lake Harutori and the Abashiri River, which flows into the Sea of Okhotsk in Hokkaido, as one of the other known habitats of Hibuna , were inLake Harutori, the habitat of Hibuna, has been listed as a Natural Monument of Japan since 1937, where fish sampling is prohibited. Thus, we acquired permission for sampling from Kushiro City and Hokkaido Prefectural governments. For conservation reasons, we performed non-destructive sampling, with fish being released back into the lake after collecting a small amount of fin tissue, following a careful procedure. All procedures were conducted in accordance with the Guidelines for Proper Conduct of Animal Experiments , ARRIVE C. auratus-complex populations in Hokkaido are believed to be derived from Honshu Island [Hokkaido Island is the northernmost Japanese island, separated from Honshu Island by Tsugaru Strait (130–140 m in depth) and from Sakhalin Island by Soya Strait (50–60 m in depth) . The freu Island , it is su Island .C. auratus-complex lineages referred to as clades C5 and C6, which are widespread in mainland China [To test the role of goldfish in the origin of Hibuna, we referenced previously reported genetic characteristics of goldfish. Wang et al. (2013) reported mitochondrial gene genealogy of goldfish, covering the range of goldfish strains in China and Japan . Their rnd China , 37.C. auratus-complex specimens [Using this population background history to test the “goldfish origin hypothesis” for Hibuna, we first compared the mitochondrial haplotype of Hibuna with the mitochondrial sequences of goldfish deposited in a DNA database . In addipecimens  collecteC. auratus-complex and 42 Hibuna specimens, were collected from Lake Harutori  and the Abashiri River (38 specimens) on Hokkaido using a hand net and a cast net  were sequenced for all of the newly collected specimens. A primer pair, IF-f_L14724 and IF-r_H15915 [™ Terminator Cycle Sequencing FS Ready Reaction Kit v3.1, Applied Biosystems) using the primer IF-f [b, LC699673–LC699680). Previously reported sequence data from wild Japanese C. auratus-complex populations collected from Hokkaido and Tohoku [C. auratus-complex and two haplotypes of sister species, C. cuvieri, as an outgroup using RAxML [Partial gene regions of mitochondrial cytr_H15915 , and GoTmer IF-f . All mitd Tohoku  and thosd Tohoku  , especially in the eastern Hokkaido populations, where no diploid individuals were found  group of Hibuna specimens from Lake Harutori, which was higher than the observed average nucleotide diversity of 0.0061 of 11 analyzed wild non-Hibuna populations from Hokkaido (Five haplotypes were detected from Hibuna using 885-bp cytHokkaido .Carassius collected from the Abashiri River. Some Hibuna specimens (8 from Lake Harutori) showed regional mitochondrial haplotypes, which were also found in other wild Hokkaido populations and included in Japanese clades B1 and B3  showed mitochondrial haplotypes that were identical or very similar to previously reported goldfish haplotypes  from goldfish (or their descendants) to the triploid lineage and maintenance of the alleles are likely to have occurred via the following scenarios : (i) HybCarassius via rare sexual reproduction. The observed high genetic diversity among Hibuna strongly suggests that genetic diversity of sexual relatives would have contributed to the genetic diversity of gynogenetic triploid Carassius, as recently shown for Japanese populations [Carassius. However, considering the negative aspect of genetic disturbance regarding the origin of Hibuna, when considering their priority for conservation, the preservation of other wild surrounding populations in eastern Hokkaido needs to be taken into account. Additionally, it is necessary to prevent the further spread of Hibuna or the introduction of goldfish into natural waters. Fortunately, in the case of Lake Harutori, it is expected that the dispersal of Hibuna is limited and can be managed because of their protection by law and the small river basin size .Hibuna is a symbolic fish and its habitat (Lake Harutori) was designated as a Natural Monument of Japan in 1937. This study revealed that Hibuna was derived from hybridization between introduced goldfish (or their descendants) and native gynogenetic triploid ulations . Thus, HCarassius in 24 localities across Hokkaido in 1977–1981 reported that triploids were dominant in most localities (18 out of 24 locations) [Carassius was at a very low frequency (not observed). Some of these diploid and triploid Carassius populations might already be at risk of extirpation. Considering the need for sexual diploids to maintain triploids, the decline of diploids has probably occurred recently. Although the causes of such a very low frequency of diploids are unknown, their fitness may have been decreased by anthropogenic impacts such as deterioration of the quality of their spawning grounds and habitat [Carassius, there is a need to identify the actual cause of the population decline and clarify the factors contributing to their stable coexistence. With respect to the latter, the leading explanations are lower parasite resistance [Carassius  . Thirty  habitat , which msistance , 50 and sistance , 52. In lineages ), some elineages , 53.Poecilia fish [Ambystoma salamander [Cobitis loach [Carassius, several studies reported the existence of triploid males [Carassius [Carassius has shown the ability to use divergent lineages in the diploid C. auratus-complex as a sperm donor [Carassius by Hokkaido triploid populations, where sexual diploid Carassius is rare or absent. This possibility should be confirmed by ecological investigation on the reproduction of the triploids.Another possibility for explaining the maintenance of triploids despite the very low frequency or absence of sexual diploids is that triploids might avoid the risk of extirpation by acquiring parthenogenesis or by changing the species of the sexual sperm donor. However, evolution to sperm-independent parthenogenesis has not yet been observed in any animals . The swilia fish , 55, Amblamander , and Cobis loach , 57. In id males , 59, altid males , 60–63. arassius –66, suchrm donor , 37, andrm donor –69. ThusCarassius fish via interploidy gene flow involving rare sexual reproduction. Thus, Hibuna’s unique scarlet body color symbolizes the complexity of reproduction among vertebrates. However, the priority to be placed on the conservation of Hibuna relative to that of the surrounding populations should be reconsidered because the observed dominance of triploids in most Hokkaido Carassius populations implies a hidden risk of their co-extirpation. There is thus a need for studies on ecological and physiological differences between the diploid and triploid fish in order to establish appropriate conservation policies for the Carassius populations, especially the diploids, on Hokkaido. The maintenance of sexual diploid populations will also stabilize triploid populations by providing sperm donors and facilitating the genetic diversity of triploids via interploidy gene flow.In summary, our study revealed that the artificial origin of Hibuna involved hybridization with goldfish. The unintended 100-year field experiment after the introduction of goldfish into the lake exemplified various reproductive modes and complex diversification processes occurring in S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 TableAccessions with identical sequences to the one from other accessions are shown in parentheses.(XLSX)Click here for additional data file.S4 TableThe statistics of Hibuna populations are shown in bold.(XLSX)Click here for additional data file.


Gevuina avellana Mol., Proteaceae) is a native tree of Chile and Argentina of edible fruit-type nut. We applied two approaches to contribute to the development of strategies for mitigation of the effects of climate change and anthropic activities in G. avellana. It corresponds to the first report where both tools are integrated, the MaxEnt model to predict the current and future potential distribution coupled with High-Resolution Melting Analysis (HRM) to assess its genetic diversity and understand how the species would respond to these changes. Two global climate models: CNRM-CM6-1 and MIROC-ES2L for four Shared Socioeconomic Pathways: 126, 245, 370, and 585  were evaluated. The annual mean temperature (43.7%) and water steam (23.4%) were the key factors for the distribution current of G. avellana (AUC = 0.953). The future prediction model shows to the year 2040 those habitat range decreases at 50% (AUC = 0.918). The genetic structure was investigated in seven natural populations using eight EST-SSR markers, showing a percentage of polymorphic loci between 18.69 and 55.14% and low genetic differentiation between populations . According to the discriminant analysis of principal components (DAPC) we identified 10 genetic populations. We conclude that high-priority areas for protection correspond to Los Avellanos and Punta de Águila populations due to their greater genetic diversity and allelic richness.The Chilean hazelnut ( Gevuina avellana Mol. is a native tree of Chile and Argentina, monotypic of the genus Gevuina (Proteaceae) . The. TheGevue family . In Chilcuttings ,14,15,16cuttings . At the cuttings . A phylo contain ,22,23. Gsymptoms . Varioussymptoms ,27,28,29symptoms ,32,33. Tsymptoms ,35.Native plants are one of our most powerful tools for protecting and conserving healthy and resilient ecosystems. Successful native plant restoration facilitates the conservation of innumerable species interactions, such as between plants, their consumers, and their pollinators, that provide the foundation of ecosystem services upon which humans and wildlife depend . When thThe maximum entropy modeling (MaxEnt) is a multivariate approach that estimates the distribution of a species by finding the probability distribution of maximum entropy, subject to constraints representing our incomplete information about the distribution . The maxMonitoring global biodiversity from space through remote sensing geospatial patterns has a high potential to add to our knowledge acquired by field observation. Nevertheless, some essential biodiversity variables (EBVs) are not directly measurable by remote sensing from space, specifically the EBV class genetic composition . To asseG. avellana Mol. present in Chile. It corresponds to the first known report of the genetic structure and spatial distribution described for the species in the Region, which was developed mainly in natural populations that are not part of the National System of Protected Wilderness Areas of the State (SNASPE) administered by the National Forest Corporation (CONAF). At the same time, another of the objectives of this research is to propose an alternative methodology that may successfully replace more expensive molecular analyses or difficult interpretation, which could benefit conservation genetic studies using the analysis of HRM, which once optimized; is a fast, low-cost technique, especially when massive plant genotyping is required. Finally, the study aims at professionals interested in the conservation or ecological restoration of plants  who wish to incorporate in their research alternative methodologies to their areas of specialization. When species of which little or nothing is known, it is advisable to use MaxEnt Model and HRM Analysis as a strategy for gathering rapid and integral information.The present work seeks to contribute to the knowledge of the northernmost known natural populations of G. avellana distribution at the regional level. Therefore, it was relevant and transcendental for the successful development of the research to rely on the empirical and ancestral knowledge that we were able to record from the local peasant communities. These communities base part of their subsistence economy on the collection of fruits that they later market locally, either as dried or processed fruits (mainly as flour). As a result of this collaborative work and information provided by the locals and transmitted generationally, we were able to categorize geographically, phenologically, and phenotypically several centuries-old, natural populations of G. avellana not previously described that, for the most part, were part of peasants’ properties or other private sectors surrounding them. With this, we were able to select, evaluate and rescue trees using tissue culture techniques (unpublished data) with contrasting characteristics of interest and adapted to the edaphoclimatic zones of the region  where the presence of in Range . A totalin Range . G. avellana in Central Chile (Maule Region) and project the possible variations that this habitat could suffer under different environmental scenarios, twenty-three different environmental variables were analyzed using the MaxEnt tool. Validation of our model allowed us to arrive at AUC values of 0.953 for the potential current habitat of G. avellana, indicating that the prediction results are very accurate, which can be used for subsequent analysis , and their ideal habitat would move and concentrate in the highest sectors of the Andes mountains in height ranges that go from 520 to 1200 meters above sea level or higher.avellana , Table 2G. avellana. G. avellana from each population, using the EST-SSR marker Ga38. The shape of the melting curves revealed the difference between the genotypes and showed that all genotypes could be easily distinguished based on their melting curves.The genotypes tested with eight EST-SSR markers and selected as HRM Standard or Reference Profile  generated unique melting curves over different temperature ranges . The molecular markers evaluated subsequently in each population allowed the detection between three and five melting curves . Figure G. avellana. The optimal combination of the eight EST-SSR markers allowed the discrimination of all the individuals analyzed. On the other hand, the effective number of alleles per locus (Ne) for the total of the local populations was 1.40 ranging from 1.18 (Canelillo) to 1.55 (Punta de Águila). The coefficient of genetic differentiation (GST = 0.0431) indicates that 4.31% of the genetic diversity is found between populations and 95.69% within populations. The percentage of polymorphism in the analyzed populations remained in a range of 18.69%–55.14%, with the populations of Punta de Águila and Los Avellanos showing the highest percentages of polymorphism . The situation that is maintained for these populations when analyzing Private alleles (Pa) whose values range between 59 for the Punta de Águila population and 53 for Los Avellanos.G. avellana analyzed indicate that the highest percentage of variation is within the populations with 95%. followed by the genetic diversity present among the populations, with a total of 5% of the observed variance. Genetic differentiation between populations was moderate  (The results of molecular variance (AMOVA) for the seven populations of < 0.001) .The value of the Bayesian information criterion (BIC) based on the binary matrix (0 and 1) with the combined data of the eight EST-SSR markers showed that clusters useful to describe the data are K = 10 .ST) in all populations fluctuated between 0.0339 and 0.0368  Group 10 is made up of genotypes from Las Lomas, Roblería, Los Avellanos, Armerillo, and Punta de Águila; and (c) Group 8 is represented by genotypes from all populations.G. avellana described in the present work was developed by guiding us in the historical structure of ecological niche modeling reported in other publications ..G. avellThe advantage of HRM analysis is that Real-Time PCR system software provides a table of dissociation temperature data automatically (including clustering graphs), and for the construction of the binary matrix, only must be analyzed the forms of melting curves, which makes the process something fast, precise, easy to interpret and analyze, and above all, an independent process, since high-cost external services are not required to obtain results. Even more, when it is required to analyze small fragments , it alloG. avellana plants propagated by in vitro culture , it allows us to know genetic clusters that can be biologically meaningful structures and reflect interesting biological processes. For this, we must define the number of clusters that are useful to describe our data . DAPC ca country .G. avellana populations present in the Maule Region, information that will allow the local community and government entities to make conscientious, better informed and objective decisions in a timely manner when implementing strategies aimed at guaranteeing effective plans for the conservation, recovery, make visible, and potential use of the species.Based on the Model MaxEnt results and HRM Analysis, we could make some decisions in terms of conservation. For example, if we aim to protect populations containing significant genetic variability  in areasG. avellana, due to its ethnobotanical characteristics, is a species with a high potential for domestication [G. avellana is restricted to the collection of its fruits in their natural state; however, less than thirty percent of the total inventory is considered suitable for collection, due to the difficult access to the populations and their high dispersion space [Native plants are often promoted to solve various environmental sustainability issues . Promotitication . The exptication , though tication . Severaltication . Currenton space . Thus, ion space .2 and associated shifts in temperature and precipitation are all expected to impact plant ecophysiology, distribution, and interactions with other organisms [Currently, climate change is becoming a serious challenge around the world, with significant threats to food security, ecosystems, economic stability, and water resources . The imprganisms . Accordirganisms . The charganisms . To mitirganisms . A recomrganisms .G. avellana, generating the greatest amount of information that could be quickly socialized and transferable in such a way as to encourage and invite the scientific community, farmers, local community, and government agencies to develop and accelerate conservation strategies and ecologically sustainable use of the species. Our biotechnological approach allowed us to contribute significantly to one of the key and fundamental questions in this type of study related to what and where to restore. The criteria that we address to develop an effective plan for conversation and/or sustainable use in G. avellana not only considered a survey of genetic and ecological information of the species in the region but also included the development of a fast and efficient system of in vitro propagation of individuals of high genetic and phytosanitary quality selected based on their genetic, phenotypic, phenological plasticity, and agroforestry characteristics of productive interest (unpublished data). This point is really important since Chile has committed to restoring 600.000 ha of native forest by the year 2035; however, the quantity and quality of native plants that are required to be produced were not considered; When evaluating whether the nurseries currently meet the minimum seedling quality standards based on their morphophysiological attributes, it was concluded that the restoration commitments desired by 2035 would not be achieved until 2181 [G. avellana, due to its agroforestry and productive characteristics, should be seriously considered as an alternative for the development of a crop of regional interest. If we consider, for example, that the commercial plantations in the Maule Region of its homonymous European Hazelnut (Corylus avellana L.) have increased their representation significantly, going from 30 ha in 1999 to 24.455 ha in 2021, with a representation of this species at the country level by over 80%, becoming, in addition, the fourth most important fruit crop at the country level [G. avellana as an ecological crop, more environmentally friendly, and with regional identity is a completely viable alternative to implement. However, at this point, we consider that the social, cultural, ecological, and environmental focus should not be lost; therefore, we propose not to think of G. avellana for the development of a massive monoculture but rather the development of a “domestication limited to small-scale” fully regulated (through Technology Transfer), which allows both the conservation of genetic diversity and the sustainable use of the species and specifically contributes to improving the local communities’ quality of life. These communities are made up mostly of peasants whose families own small extensions of land that they have inherited generationally, where there are remnants of native forests—that is, fragmented forests of native species, including G. avellana. Lands that, due to the edaphoclimatic and topographical conditions of the area, are generally not suitable for the establishment of other types of crops that allow the development of agricultural activities. These lands have soils with low water availability, often eroded, with streams that are difficult to access, where, in addition, in the case of G. avellana, although there are natural populations within these lands, the production of its fruits is uneven and insufficient and makes it unfeasible to base the family economy on a permanent and profitable activity. Under this social and cultural reality, and as there are no incentives from the state towards these communities to protect and maintain these native resources, the peasants and locals who own these lands dedicate themselves to working in other economic activities, and with regard to the management of the use of their land, they often make decisions to sell part of it (invoking the change in land use regulations) or they simply choose to cut down the remnants of native forest to replace them with forest plantations and more economically lucrative fruit trees such as Pine (Pinus radiata), Eucalyptus  and, in recent years, European hazelnut (Corylus avellana). According to the above, and with the information generated in this study, it is feasible to develop an adequate platform for multipurpose technology transfer in G. avellana and transform it into an adequate alternative for reforestation, since its benefits and scope in different areas  have not been really valued or dimensioned.Based on the above, our focus of attention was focused on making visible a phytogenetic resource that is almost unknown at the regional level and with great potential for domestication and exploitation, such as til 2181 . With rery level , thinkinGevuina avellana Mol. located in two Andean sectors of the Maule Region  , located between 34°41′ and 36° 33′ south latitude A. Young untains) . These pG. avellana in Chile was obtained from Global Biodiversity Information Facility   A. The cuG. avellana based on its current presence dataset. In particular, 22 bioclimatic layers and one topographic variable (elevation) were obtained from the WorldClim database . Variables were retrieved at a spatial resolution of 30 arc-seconds (~1 km) for the current distribution and 2.5 arc-minutes (~21 Km) for future distributions. Projections of future climate change to predict the potential distribution of G. avellana were evaluated using the so-called Scenario Model Intercomparison Project (ScenarioMIP) under Phase 6 of the Coupled Model Intercomparison Project (CMIP6). Two global climate models (GCMs), CNRM-CM6-1 and MIROC-ES2L for the four shared socioeconomic pathways (SSPs), 126, 245, 370, and 585  were evaluated.Twenty-three variables were retrieved as predictors to model the potential environmental niche of The georeferenced longitude and latitude variables were analyzed with QGIS version 3.20 software and duplicate data were eliminated. Geographic location map was made through the Geographical Information System (GIS) using tools ArcGIS (ArcMap 10.03 software). The coordinates were exported to CVS format and the climatic variables in ASCII format, both integrated into MaxEnt software version 3.4.4 for further analysis . First, www.retsch.com (accessed on 20 September 2022)). To carry out the exploratory analysis of the genetic variability of the populations, genomic DNA was extracted from 60 trees from different natural populations of G. avellana. They were selected based on characteristics of interest (G. avellana populations.The genomic DNA was extracted from 500 mg of lyophilized leaves of Chilean hazelnut according to the protocol CTAB described by . The leaTM (Thermo Fisher) and displayed in 1% agarose gel electrophoresis. The final concentration was adjusted to 10 ng µL−1.The nucleic acids purity and concentration were estimated on a Multiskan Sky HighG. avellana [G. Avellana, because their characteristics were adequate for analysis HRM [For the exploratory analysis of the genetic variability of individuals of interest, the so-called Nonanchored Inter Simple Sequence Repeat (ISSR) markers were used . Differeavellana ,81,82. Wysis HRM .G. avellana is summarized in nine steps described in Figure 13. This consisted of selecting adult trees from the seven populations under study with contrasting phenological and phenotypic characteristics of different kinds  and introduce explants of each one of them through tissue culture techniques (in vitro culture) that would allow having a core collection representative of the diversity found. That is, DNA sample variants were isolated and conventional PCR conditions were standardized on these genotypes with expressed sequence tag-simple sequence repeat (EST-SSR) markers described for the species [The strategy designed to apply the HRM analysis in  species . Of a toG. avellana genotypes that presented variations in their nucleotide sequences were selected and evaluated by HRM. To assess the reproducibility of the HRM method, PCR reactions were performed in triplicate for each genotype for the eight EST-SSR primers  . For the primers , Step 8.ulations , Step 9.G. avellana Mol. were genotyped with eight expressed sequence tag-simple sequence repeat (EST-SSR) markers, based on a cDNA library of the species: Ga94, Ga38, Ga88, Ga90, Ga36, Ga7d, Ga92, and Ga49 [−1 (1 µL of each) were used directly in 10 µL reaction volumes containing 1X Kapa HRM Fast Master Kit , 2.5 mM MgCl2, and 200 nM of each primer. PCR amplification, DNA melting, and end point fluorescence level acquiring PCR amplifications were performed on AriaMx Real-Time PCR System . PCR protocol was established for all SSRs, under the following conditions: 40 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 30 s, and extension at 72 °C for 20 s. High-resolution melting analysis was performed after PCR amplification at the temperature ramping and fluorescence acquisition setting recommended by the manufacturer, which is, the temperature ramping up to 95 °C and rising by 0.2 °C/10 s.The populations of and Ga49 . NucleicG. avellana from seven natural populations of the Maule Region were analyzed. HRM profiles of each individual were compared to representative standard curves for each marker (HRM Standard) . Then, we established a directory with the data using the following parameters: (1) dat <- read.table (binary data matrix in TXT format), (2) obj <- genind to define how our data is composed , (3) parameter “grp<-find.clusters (max.n.clust = 40)” to define and form the population groups, and (4) parameter “dapc1<-dapc ” were used to choose the PCs, number of clusters, and the number of eigenvalues that explained the highest percentage of the accumulated variance”, which finally allowed obtaining the basic scatterplots using function (5) “scatter (dapc1)”.Two hundred and eighty trees of tandard) . One Bintandard)  GenAlex tandard)  and Adegtandard) . DAPC is Jombart . Using gG. avellana with HRM analysis can be a fast and cost-effective tool, suitable to replace other genotyping technologies such as DNA sequencing by capillary electrophoresis. We conclude that the HRM methodology described in this work can be used as a reference and applied to the study of other native species of interest.It was shown that EST-SSR genotyping in G. avellana Mol. They are located in the Andean sectors of the Coastal Range and Los Andes range, areas that, by the year 2040, will decrease by 50%, moving mainly to the Andes range above 1200 meters above sea level. Along with this, according to analysis HRM and the discriminant analysis of principal components (DAPC) we conclude that high-priority areas for protection correspond to Los Avellanos and Punta de Águila populations due to their greater genetic diversity and allelic richness.Based on the ecological niche modeling developed by MaxEnt, we determined that currently the ideal areas in the Maule Region to establish G. avellana, and the interest shown in wanting to be part of a solution to a problem that affects us all as a society related to negative anthropogenic impacts and climate change on our plant genetic resources, we conclude that the key for a successful implementation of a conservation program in G. avellana, is to actively include the participation of the peasant community to maintain this synergy between them and the scientific world.According to the valuable interaction that allowed us to establish a synergy between the real needs of the peasant community dedicated to the activity of collection of fruits in


Elaeis guineensis, in Malaysia and Indonesia is Elaeidobius kamerunicus. The weevil species is not native to these countries, but it was introduced from Cameroon, West Africa, in 1981 to improve pollination efficiency, thus improving bunch formation and yield. Forty years after the weevil introduction, recent reductions in bunch yield reported in Malaysia caused by poor bunch formation could be associated with a decrease in pollination efficiency due to the founder effect. Several genetic diversity studies of weevil populations based on morphological and mitochondrial markers have been carried out; however, the studies did not provide sufficient evidence for explaining the genetic variation, particularly at the intra-species level. This study aims to develop a set of robust E. kamerunicus-specific nuclear DNA markers to directly assess the genetic diversity of weevil populations. The marker development lays the foundation for future applications by extending the survey into larger areas where the oil palm is cultivated to obtain a more comprehensive understanding of the genetic diversity and inbreeding occurrence status of E. kamerunicus in these introduced regions. This could facilitate sustainable genetic monitoring and conservation planning of E. kamerunicus, especially in non-native oil palm-growing countries.The main pollinator for the African oil palm, Elaeidobius kamerunicus Faust) was introduced from Cameroon, West Africa, to Malaysia in 1981, and subsequently, to other oil palm-growing countries as well. This study aims to develop a set of robust E. kamerunicus-specific nuclear DNA markers to directly assess the genetic diversity of the weevil populations. A total of 19,148 SNP and 223,200 SSR were discovered from 48 weevils representing three origins  using RAD tag sequencing. Subsequent filtering steps further reduced these to 1000 SNP and 120 SSR. The selected 220 SNP exhibited a polymorphism information content (PIC) of 0.2387 (±0.1280), and 8 SSR had the PIC of 0.5084 (±0.1928). These markers were found to show sufficient polymorphism, making it possible to assign 180 weevils into three major clusters from Ghana, Cameroon, and Southeast Asia . These DNA markers successfully confirmed the Cameroon origin of the Southeast Asian cluster. However, the presence of null alleles in the SSR markers, due to limited flexibility of the probe design on the short RAD tags, led to an underestimation of heterozygosity within the populations. Hence, the developed SNP markers turned out to be more efficient than the SSR markers in the genetic diversity assessment of the E. kamerunicus populations. The genetic information provides useful insight into developing guidelines for the genetic monitoring and conservation planning of E. kamerunicus. The oil palm-pollinating weevil ( Elaeis guineensis Jacq., originated from West Africa. The earliest record of its introduction to Southeast Asia is found regarding the planting of four dura palms in Bogor Botanical Garden, Indonesia, in 1848 [Elaeidobius in West Africa [Thrips hawaiiensis was identified in Malaysia as an alternative pollinator to the native weevil, but the species was far less efficient and usually absent from young plantings [Oil palm,  in 1848 , supplyi in 1848 . The oilt Africa . In the lantings . The oillantings .Elaeidobius kamerunicus Faust, 1898 (Coleoptera: Curculionidae) was chosen after the species was studied to be the most predominant oil palm pollinating weevil in Cameroon. The species is also well adapted to both wet and hot seasons, and has a high pollen carrying capacity among the other Elaeidobius species [Elaeidobius kamerunicus is holometabolous, completing its development on post anthesis oil palm male inflorescence [The species  species . In addirescence . Adult frescence . The weerescence .E. kamerunicus species in the form of pupae was imported from Plantations Pamol du Cameroon, Lobe Estate, Cameroon, to Malaysia [In 1981, the Malaysia . A totalMalaysia ,12,13. HMalaysia . The relMalaysia .E. kamerunicus probably suffered a bottleneck effect that weakened their adaptability to local climate change, making them vulnerable to parasitic nematode infection [E. kamerunicus populations were conducted to determine the presence of a bottleneck effect, but multivariate analysis based on 15 morphological characteristics of E. kamerunicus mainly explained the variation between male and female insects, rather than between geographical regions [A declining trend of FFB, however, has occasionally been reported in Malaysia since the 1990s. Low rainfall and over-pruning, causing a reduction in the formation of female inflorescences, could be contributing factors to this trend. In addition, the descendant population of introduced nfection ,16,17,18 regions ; therefoCOI, COII and a nuclear gene fragment of arginine kinase (AK) were used in estimating the phylogeography of the E. kamerunicus populations [Hence, geneticists started adopting molecular markers to directly measure the genetic diversity of this important weevil population. Mitochondrial marker ulations . The Souulations . Howeverulations . Nuclearulations . Simple ulations . Neverthulations .E. kamerunicus-specific nuclear marker systems, i.e., single nucleotide polymorphisms (SNP) and simple sequence repeats (SSR), using RAD (restriction site associated DNA) tag sequencing, and then to assess the effectiveness and applicability of the two marker sets to capture the expected genetic patterns in the population.We hypothesize that the designed novel DNA markers will be effective to measure the inbreeding effect among the Malaysia populations, as well as to estimate the genetic diversity of the other introduced region (Indonesia), and to contrast with this effect with those of the origin countries (Cameroon and Ghana). Hence, the aim of this study was to develop two robust TM Tick DNA kit , according to the manufacturer’s protocol. This was to ensure good quality DNA for the RAD tag sequencing and to minimize the risk of cross-contamination from other organisms, particularly nematodes, which are usually present in the abdominal segment of weevils. Subsequently, the evaluation of marker applicability for genetic diversity studies was performed, based on a larger set of 180 weevils representing two native populations (from Cameroon and Ghana) and 10 introduced populations in Malaysia and Indonesia  and Indonesia  were collected for marker discovery and development. Tissues from the head and membranous part of the hind wings of each weevil were sampled for DNA extraction using Analytik Jenandonesia ; each poMsll restriction enzyme. Digested DNA fragments were subjected to 150 bases paired-end sequencing using Illumina NextSeq500 V2 platform . Standard pre-processing steps were taken after the de-multiplexing of the library groups using Illumina bcl2fastq 2.17.1.14 software . Raw sequences per sample were obtained by the de-multiplexing of library groups into samples, according to their inline barcodes (with a maximum of 2 mismatches or Ns) and verification of the restriction site. All reads were processed by an adaptor clipper, and reads with a final length shorter than 20 bases were discarded as well. The next step was quality trimming by removing reads containing Ns and at the 3′-end to get the average Phred quality score (Q > 20) within a window size of 10 bases. Processed sequence reads were pooled and clustered using the CD-HIT-EST program at 95% similarity cut-off [The genomic DNA of each individual weevil was digested using the  cut-off . The ali cut-off . Variant cut-off  by folloftp://ftp.ncbi.nlm.nih.gov/genomes/refseq/bacteria/assembly_summary.txt (accessed on 26 January 2018). This step was performed to remove possible contaminants from weevil-specific sequences. In addition, as E. kamerunicus belongs to the order Coleoptera, those identified markers that were mapped to genomes of beetle species  in the NR database were selected for this study. Additionally, those unmapped markers with a >90% call rate calculated in the PLINK program [TM Genotyping by Sequencing  ranging from 54 °C to 60 °C; the maximum Tm difference between the forward and the reverse primer was set at 0.5 °C. A total of 120 primer pairs with an M13-tailed forward primer for subsequent amplicon dye-labeling [FullSSR suite v1.0  was adoplabeling  were synA), expected heterozygosity (He), observed heterozygosity (Ho), polymorphism information content (PIC) [is) [is across the populations using the PowerMarker v3.25 program [Only SNP and SSR markers with a call rate >95% were included in the genetic diversity analyses . At the nt (PIC) , and inbIC) [is) , were geIC) [is)  for SSR IC) [is)  to constIC) [is) . In addiIC) [is) , HiefstaIC) [is)  and PegaIC) [is) , followiIC) [is) . The freIC) [is)  using thIC) [is) . Allelic program . o per SNP was 0.2917 (±0.2221), which was close to the mean He per SNP = 0.2982 (±0.1742). In this study, NA = 435 were detected, with the average of 1.9773 alleles per SNP  alleles, ranging from 4 to 13 alleles . All eight SSR markers indicated a significant deviation from the Hardy–Weinberg equilibrium (HWE) at p = 0.05 threshold (2 < 0.5)  . NAF < 0.05), moderate (0.05 ≤ NAF < 0.20), or large  ranging from 0.0341 to 0.3361 and the mean of 0.1903 (±0.1243) . There aF ≥ 0.20 . Thus, tA mean (CAM = 1.6500 and GHN = 1.3318), Ho (CAM = 0.2417 and GHN = 0.0992), and PIC (CAM = 0.1810 and GHN = 0.0833) in both native populations, compared to the mean values across populations . Interestingly, these four diversity parameters were comparable among the introduced populations and higher than the mean values across populations. The genetic diversity of 12 assayed weevil populations was analyzed using 220 SNP and 8 SSR, respectively, to compare the applicability of both marker systems. For SNP, MAF of the CAM population (0.8222) and Ghana (GHN) population (0.9199) were higher than 0.7974 (±0.0409), which is the MAF mean . This alis, ranging from −0.1146  to −0.0244 (CAM), except for the GHN population, with 0.0330. The SSR markers, however, indicated the opposite trend of Fis , in which all 12 populations showed positive values in the range between 0.2931  to 0.5857 . As this was observed due to the presence of null alleles in some markers, allelic frequencies were then adjusted for re-estimation of SSR-based Fis. After the adjustment, the SSR-based Fis were reduced, ranging from −0.0246 (CAM) to 0.1173 (SS). Particularly, the SBH (−0.0024), CWK (−0.0030), and CAM (−0.0246) populations recorded negative Fis values (A ≥ 3.0000) than did the SNP, leading to a lower MAF mean (0.6370 ± 0.0665)  . HoweverIn general, the mean pairwise Nei’s genetic distance (D) among the 12 assayed populations calculated using SNP and SSR was 0.0329 and 0.1330, respectively. The lowest genetic relatedness between the GHN population and the CAM population was revealed by SNP (D = 0.1452) and SSR (D = 0.3841). However, the closest related population pair detected in both marker systems varied: NPM-CPM pair (D = 0.011) in SNP and CPM-SR pair (D = 0.0367) in SSR. The genetic relatedness among populations was subsequently visualized in a UPGMA phenogram, based on D estimates . Both SNRAD tag sequencing is a cost-effective and rapid method for discovering DNA markers, especially for non-model species with no prior genome references ,49,50,51Taxus florinii, were suitable for primer design [E. kamerunicus reference genome, the identification of weevil-specific SNPs and SSRs, based on cross-mapping with reference genomes of the order Coleoptera, was found to be promising. A similar SNP marker discovery using RAD tag sequencing for a coconut leaf beetle without a reference genome was reported in [Although discovery of SSR is more feasible, even with a single reference genome, only 0.28% of the identified SSR in this study were suitable for primer design. This was due to short generated RAD tag sequences, which usually spanned between 150 bp and 300 bp , therebyr design . In thatorted in . As an eorted in .A of SNP = 435 was observed to be 8.7 times higher than the NA of SSR at 50.Despite the reduced number of DNA markers, 220 SNP and 8 SSR were found to be informative. Markers with PIC values ≥0.5 are usually considered as highly informative, 0.25–0.5 as moderate, and <0.25 as low . For bi-A of multi-allelic SSR, the marker system had more advantages in explaining within-cluster variation than inter-clusters  GHN, (ii) CAM, and a cluster of (iii) introduced populations in Malaysia and Indonesia. With a higher mean Nclusters C,D. PrevE. kamerunicus between Central and West Africa [From the UPGMA phenogram, the cluster of the introduced populations in Malaysia and Indonesia was more closely related to CAM than GHN, which corresponded to the introduction event of the weevil from Cameroon to Southeast Asia. Furthermore, separate genetic clustering between Ghana, Central Africa, and Cameroon, West Africa, is in agreement with that obtained in a previous study reporting the difference in mitochondrial genetic clustering of t Africa . is < 0), suggesting an absence of inbreeding in these populations. These findings might further support those of previous studies. Morphometric measurements of E. kamerunicus in Malaysia and Indonesia were not significantly different from those of the native Liberia weevils [E. kamerunicus has also been reported to be stable [o in native GHN and CAM populations compared to the introduced populations, leading to a homozygosity deficit (Fis > 0) in the CAM population. One possible reason for this was non-random sampling, where these samples were collected from a limited number of palms, thereby not representing the heterozygosity of the real populations. Hence, random sampling from a wider range of the native population is recommended in future studies.Interestingly, 11 weevil populations indicated excessive SNP-based heterozygosity  to obtain consistent and high call rates for more reliable results in future studies. In addition, PCR-based SNP markers can be cost-effective when genotyping a large number of samples. The Seq-SNP genotyping takes 3 days and costs USD 7 for each DNA sample genotyped with 163 SNPs in 261 cucumber varieties [These 220 informative SNP markers can be deployed as a PCR-based genotyping method, such as Seq-SNP , Kompetitive Allele Specific PCR (KASP)   and TaqMarieties . This feE. kamerunicus using RAD tag sequencing was promising. The applicability of informative SNP in the genetic diversity assessment of weevil populations was observed to be superior to SSR. The main limitation of SSR is the null allele, which is common in RAD tag sequences. The presence of null allele in SSR markers from this study has led to an upward bias in inbreeding coefficient estimates. The inbreeding coefficient that was estimated using SNP-based data has recorded negative values in introduced E. kamerunicus populations indicating inbreeding is not being observed for now. For further confirmation, we propose expanding the survey to more populations across Malaysia, Indonesia, and Papua New Guinea using the developed specific nuclear DNA markers. Collaborative studies can be performed among industry players in oil palm-growing countries. This would obtain a more comprehensive genetic diversity concerning weevil populations in these regions. The information will be used to create guidelines regarding conservation planning and sustainability monitoring of this important pollinator species, from the estate to the national level. The discovery of nuclear DNA markers for


Sassafras tzumu (Hemsl.) Hemsl., as an Arctic Tertiary relict woody species, is an ecologically and economically important deciduous tree endemic to southern China. Nonetheless, the genetic resources and backgrounds of S. tzumu are still lacking and remain largely unclear. Here, we predicted 16,215 candidate polymorphic nuclear microsatellite (nSSR) loci from the assembled nucleus databases of six geographic-distant individuals of S. tzumu via CandiSSR. Among these nSSRs, the di- (75.53%) and tri-nucleotide (19.75%) repeats were the most abundant, and 27 new polymorphic SSRs were developed and characterized in 136 individuals from six natural populations of S. tzumu. The majority of the above 27 SSRs  presented moderate polymorphism (mean PIC = 0.356), and the transferability of these markers in other Sassafras species was high (85.19%). A moderately low level of genetic diversity and a high variation (FST = 0.286) of six wild populations of S. tzumu were illuminated by 16 selected polymorphic nSSRs, with the average expected heterozygosity (HE) of 0.430 at the species level and HE ranging from 0.195 to 0.387 at the population level. Meanwhile, a bottleneck effect was shown in two populations. Consistent with the results of the principal coordinate analysis (PCoA) and phylogenetic trees, structure analysis optimally divided these six S. tzumu populations into two clusters, and the further strong population subdivision appeared from K = 2 to K = 5, which corresponded to two evolutionarily significant units (ESUs). Moreover, the significant correlation between genetic and geographic distance was tested by the Mantel test , clarifying the effect about isolation by distance (IBD), which could be possibly explained by the low gene flow (Nm = 0.625), a relatively high degree of inbreeding (FIS = 0.166), a relatively large distribution, and mountainous barriers. Above all, our research not only enlarged the useful genetic resources for future studies of population genetics, molecular breeding, and germplasm management of S. tzumu and its siblings but also contributed to proposing scientific conservation strategies and schemes for the better preservation of S. tzumu and other Sassafras (Lauraceae) species. Sassafras J. Presl (Lauraceae) contains three extant species with a disjunct distribution between East Asia and North America: S. tzumu (Hemsl.) Hemsl. is located in the Yangtze River basin and its southern areas in continental China; S. randaiense (Hayata) Rehder is merely distributed in Central Taiwan; whereas S. albidum (Nutt.) Nees is found in the east of North America  with 100,000 MCMC (Markov Chain Monte Carlo) repetitions and 100,000 burn-in periods. We assumed that the studied populations conformed to the admixture model and independent allele frequencies. The obtained results from STRUCTURE were further interpreted by STRUCTURE HARVESTER [K method [K) that corresponded to the change of lnp(D) between consecutive K values for the calculation of the optimal K. The most likely number of cluster (K) was identified as the one that maximized lnp(D) and/or ΔK.Bayesian methods were utilized to reveal the optimal number of genetic clusters  and trinucleotide (19.75%) repeats were the most abundant. A total of 27 novel nSSRs with polymorphism were utilized to illuminate the genetic diversity of 136 individuals in six populations of S. tzumu, resulting most markers (88.89%) showing high or moderate polymorphism (PIC = 0.356). The majority of nSSRs (85.19%) could be successfully transferred to the other two Sassafras species, and some of them showed polymorphism. The population genetic results revealed a moderately low genetic diversity of S. tzumu with HE ranging from 0.195 (LL) to 0.387 (BYS) at the population level, and the average HE was 0.430 at the species level. The bottleneck effect appeared in some populations (YX and JGS). The population differentiation was high (FST = 0.286), and the majority of variations (71.60%) were within the populations. Consistent with the results of the principal coordinate analysis (PCoA) and phylogenetic trees, structure analysis optimally divided these six S. tzumu populations into two clusters, and the further strong population subdivision appeared from K = 2 to K = 5, which corresponded to two evolutionarily significant units (ESUs). Moreover, the significant correlation between genetic and geographic distance was tested by the Mantel test , clarifying the effect about isolation by distance (IBD), which could be possibly explained by the low gene flow (Nm = 0.625), a relatively high degree of inbreeding (FIS = 0.166), a relatively large distribution, and mountainous barriers. Taken together, our research gives a deeper understanding of the population genetic diversity and structure of S. tzumu in subtropical China and will contribute to the conservation and utilization of S. tzumu and the other Sassafras (Lauraceae) species.In this study, we first identified 16,215 potential polymorphism nSSR loci based on the assembled nuclear genome database of six individuals of


Fucus vesiculosus, with a particular focus on two forms: attached and free‐living. Here we demonstrate that sympatric populations representing the two forms display marked differences in characteristics of reproduction and genetic diversity. Asexual reproduction was ubiquitous in the free‐living form despite being almost entirely absent in the attached form, while signals of polyploidy were common in both forms despite the distinct reproductive modes. Gene flow within and between the forms differed, with barriers to gene flow occurring between forms at various spatial scales due to the reproductive modes employed by individuals of each form. The divergent genetic characteristics of F. vesiculosus demonstrate that intraspecific differences can influence the properties of populations with consequential effects on the whole ecosystem. The differing genetic patterns and habitat requirements of the two forms define separate but closely associated ecological entities that will likely display divergent responses to future changes in environmental conditions.Genetic characteristics of populations can have substantial impacts on the adaptive potential of a species. Species are heterogeneous, often defined by variability at a range of scales including at the genetic, individual and population level. Using microsatellite genotyping, we characterize patterns underlying the genetic heterogeneity in marine macroalga  Fucus vesiculosus. Sympatric populations display marked differences in characteristics of reproduction, genetic diversity and population structure.Using microsatellite genotyping we characterise patterns underlying the genetic heterogeneity in two forms of marine macroalga  Although clonality within the attached Baltic Sea F. vesiculosus form is more commonly documented, unattached forms are also observed to display clonal growth. An embedded form of F. vesiculosus in the western Baltic Sea near Glücksburg (Germany) reproduces entirely by clonal growth  in the study populations and assessed the spatial genetic structure across the study area. We hypothesized that the attached form reproduces primarily sexually and the free‐living form primarily asexually. We also hypothesized spatial structuring among all populations, although connectivity between sympatric populations was predicted to be relativity high. Finally, based on previous studies, we expected that polyploidy would be absent in the Baltic Sea F. vesiculosus, yet we also found a substantial proportion of potential polyploid samples in our study populations. This allowed us to consider the association between genome size and form.In this work, we investigate the reproductive mode and spatial genetic structure in the Baltic Sea 22.1Fucus vesiculosus thalli were sampled during 2017–2018 from 20 locations within the Baltic Sea along the Swedish coast of the Northern Baltic proper, the Finnish coasts of the Archipelago Sea and the Gulf of Finland, the Estonian coast of the Gulf of Riga, and the German coast of the Arkona basin  following the standard kit protocol and PL1 buffer for cell lysis. Eight polymorphic microsatellite loci – L20, L38, L58, L85, L94  and checked by eye. A sizable proportion of individuals displayed more than two alleles in at least one of the loci studied. Thorough validity checks were performed including repeat DNA extractions, PCR reactions, and ABI plate setups with new reagents to determine the validity of the third and/or fourth allele. The trend of ≥3 observed alleles in the electropherograms was consistent and repeatable for the apparent polyploid specimens. Allele peaks for apparent polyploid specimens were often of near to equal amplitudes and thus, as accurate allele determination is essential for population genetic analysis, all called alleles were kept for determining the genotype. This is problematic, as the allele dosage of polyploid samples cannot be assessed from the genotype of the sample. For instance, an individual sample appearing as genotype AB may indeed be AB if it is diploid, but AAB or ABB if it is triploid, and AAAB, AABB, or ABBB if tetraploid. As a result, we could not use standard inference of diploid genotypic data, but used Meirmans  softwareHs; Nei, shc; both corrected for sample size ). These clonal MLGs represented 2%–10% of the total samples within each population and only 1% of the total attached sample. Tests of clonal diversity confirm that clonal MLGs at AS6 and TZ8 are likely per chance identical genotypes as a result of random mating while clonal MLGs at AS3 likely represent true clonal lineages (Appendix The proportion of MLGs detected was significantly larger in the attached form Appendix . Rarefac3.3Hs) did not differ significantly (range: 0.58–0.64), but Hs differed significantly between the two forms  varied widely, with a mean RhoST of 0.20 including a single ramet per clonal lineage per population (range: 0.01–0.52) and 0.24 including all ramets  and in the whole data (p .007), but not in the free‐living form  was an order of magnitude larger within compared to between forms  when compared to previous studies in the Baltic Sea  related species , or it can be a result of multiplication of the whole genome of a single parent species (autopolyploidy). In the Baltic Sea,  Colman,  and spect Isæus, . This meF. vesiculosus, F. evanescens  and the Gulf of Riga  are the only known areas with a potential for multiple species occurring sympatrically. Apart from  HELCOM,  have bee HELCOM, . Thus, iF. vesiculosus and F. radicans occur both sympatrically and allopatrically  results in an even greater reduction of fitness , alongside the frequent occurrence of adventitious branches , forms were significantly genetically differentiated from each other. However, this hierarchical level explained only a minor part (1%) of the total variance in the data and the among‐populations level explained more than an order of magnitude more  or sexual reproduction and subsequent dispersal of propagules to a new location. Both modes of reproduction could take place in either attached or free‐living populations and moreover, these alternatives are not mutually exclusive. Our study does not provide direct answers to the origin of the free‐living populations, but clonal diversity and distribution, and the spatial structure of the population allow us to discuss these scenarios. Free‐living forms of Fucus spp. populations have classically been assumed to derive from attached populations through clonal growth whereby pieces of thalli  would detach from attached individuals, float freely and eventually aggregate in still locations and persist over a longer time . In one potential scenario, propagules with different genotypes gather in sheltered locations and free‐living population arise when emerging individuals fragment further at their own pace, resulting in a mix of clonal and unique MLGs. In an alternative scenario, detached pieces of thalli from one or more populations immigrate to a free‐living population. This would result in unique MLGs in the free‐living population, but shared clonal MLGs would then also be expected. While we confirmed that the nearest attached populations were not sources of the free‐living populations due to the lack of shared clonal MLGs, this scenario is feasible, if the immigrants are derived from a larger area and founder effect boosts genetic differentiation among populations. Detached thalli have a large dispersal capability ; data curation (lead); formal analysis (lead); funding acquisition (lead); investigation (lead); methodology (lead); project administration (lead); resources ; supervision ; visualization (lead); writing – original draft (lead). Jaanika Blomster: Conceptualization ; supervision ; writing – review and editing . Ellen Schagerström: Conceptualization ; resources ; writing – review and editing . Perttu Seppä: Formal analysis (supporting); writing – review and editing .Funding for this project was provided through grants from the Walter and Andrée de Nottbeck Foundation and the Onni Talas Foundation.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.https://doi.org/10.6084/m9.figshare.19361759.This article has earned an Open Data badge for making publicly available the digitally‐shareable data necessary to reproduce the reported results. The data is available at Appendix S1Click here for additional data file.