Sinocalycanthus chinensis Cheng et S. Y. Chang , which has a unique systematic status, is listed as a national second‐class protected plant of China. In this study, the genetic diversity, performance, and fitness of F1 progeny from crosses between the Damingshan (DMS) population of S. chinensis and pollen parents from the Daleishan (DLS) and Longxushan (LXS) populations were examined. The DLS population has a relatively small population size, low genetic diversity, and considerable geographical and genetic distances from the DMS population relative to the LXS population. Compared with naturally occurring seeds, DLS‐sired seeds had the highest thousand‐seed weight, starch content, fat content, germination rate, germination index, and emergence rate, but the lowest protein content. Naturally occurring, open‐pollinated seeds had the lowest thousand‐seed weight, starch content, and fat content, but the highest protein content. Compared with natural F1 progeny, DMS × DLS seedlings had the highest genetic diversity, photosynthetic parameters, and growth characteristics, except for leaf mass ratio and stem mass ratio. Under strong light, DMS × DLS seedlings exhibited a Fv/Fm value of 0.75, while the other two seedling types exhibited Fv/Fm values of 0.65. DLS‐sired seeds had the most vigorous growth characteristics except for leaf mass ratio and stem mass ratio. These results suggest that genetic rescue by transplanting seedlings from the DLS population or hand pollination with pollen from the DLS population would be effective methods to reduce inbreeding depression and obtain strong offspring with high genetic diversity and fitness in the DMS population. Genetic rescue by transplanting seedlings from the DLS population or hand pollination with pollen from the DLS population would be effective methods to reduce inbreeding depression and obtain strong offspring with high genetic diversity and fitness in the DMS population. It can be concluded that the geographical distance between source and recipient populations and the population size and genetic diversity of the source populations are the most important considerations for genetic rescue , commonly known as Chinese sweet shrub  and Longxushan (LXS) populations were examined. The size and genetic diversity of the DLS and LXS populations and the geographical distances and genetic distances between the DLS and DMS populations and the LXS and DMS populations were compared. We aimed to assess the efficacy of genetic rescue in S. chinensis. Specially, we aimed to determine whether the pollen parent population used for genetic rescue had differential effects on genetic diversity, performance and fitness of resulting F1 progeny. These results will enhance the theoretical basis for the genetic rescue of the endangered S. chinensis, thus playing a key role in the conservation of this endangered species.Both the genetic diversity and the performance of the progeny have been used separately to evaluate the effect of habitat fragmentation on the fitness of populations in different plant species and regions  deciduous shrub with glossy green leaves and camellia‐like flowers measuring up to 4″ in diameter , Linan City, Zhejiang Province, China. The DMS population, briefly described in Table 2.3S. chinensis populations located at two sites with different population sizes, levels of genetic diversity, and relative geographical and genetic distances from the DMS seed parent were used as pollen parents. One is located at Longxushan Mountain (LXS) in Anhui Province, China; this population is small with relatively high genetic diversity and with short geographical and low genetic distances from the DMS population  in Tiantai County, Zhejiang Province, China, and it is a medium‐sized population located a long geographical distance from the DMS population  seeds , DMS × LXS (pollen) seeds  and control seeds were germinated in an illuminated incubator  with 30/15°C and 12‐hr/12‐hr light/dark cycles at 80% humidity. Seeds were immersed in HSeeds were considered to be germinated when their radicle length exceeded 2 mm , stomatal conductance (Gs), transpiration rate (Tr), and intercellular CO2 concentration (Ci) between 08:30 and 11:30. Three leaves per plant were chosen, and six consecutive measurements were performed . The photosynthetically active radiation (PAR) was maintained at 800 μmol m2 concentration of 400 ppm, and a relative humidity of 70%. We used a red‐blue LED light source attached to the system to produce steady photosynthetically active radiation (PAR). Prior to the measurements, we allowed the mature leaf to acclimate under a PAR of 2,000 μmol m−2 s−1 for 30 min to avoid photo‐inhibition. As soon as the value stabilized, we exposed the leaves to a series of PAR values for 20 min or so in the following order: 2,000, 1,500, 1,200, 1,000, 800, 600, 400, 200, 100, 50, 20, and 0 μmol m−2 s−1. The temporal interval between each concentration was 3 min. We fitted the entire photosynthetic light response curve in Origin 8.0 (OriginLab) as a binary linear equation by calculating the maximum of the net photosynthetic rate (Pn) as Pmax. We utilized the following definitions. The light intensity at the maximum Pn value (Pmax) was defined as the light saturation point (LSP). The light intensity at a zero Pn value was defined as the light compensation point (LCP). The Pn at a maximum PAR of zero was defined as the dark respiration point (Rd).To construct light response curves, we obtained all photosynthesis measurements between 09:30 and 11:00 (Beijing time) based on a mature leaf from each plant at a leaf temperature of 25°C, a CO−2 s−1) were measured between 12:00 and 14:00 with a portable chlorophyll fluorometer . Measurements were performed on the third undamaged adult leaf from the top of each plant after 30 min of dark adaptation using light exclusion clips. For each measurement, three leaves per plant from three randomly selected plants per measurement were sampled. The data from three measurements of three leaves were averaged and used as the mean for each plant. The variable‐to‐maximum fluorescence ratio (Fv/Fm), which has been used to express the maximum photosystem II (PSII) photochemical efficiency, was calculated.Chlorophyll fluorescence parameters under strong light  to minimize overlap, scanned , and analyzed using the WinRhizo software package  to obtain the root volume (RV), total root length (RL), root surface area (RSA), and number of root tips (RT).2.8SD values. One‐way ANOVA was used to test the significance of the differences among treatments. All the analyses, including principal component analysis (PCA), were conducted using SPSS 19.0 software (IBM Corp.). All graphs were produced using SigmaPlot 13.0 software (Systat Software Inc.).The data are presented as mean ± 2.92, 1 U of Taq DNA polymerase (Promega Inc.), 20 ng of template DNA, 20 mg/ml bovine serum albumin (BSA), 20 pmol of forward primer, 7.5 pmol of reverse primer, and 0.5 mM concentrations of each dNTP. The amplification reaction was performed with a PTC 220 Thermal Cycler . The touchdown program included an initial 5‐min denaturation at 94°C, followed by 10 cycles of 1 min at 94°C, 1 min at 45°C (touchdown for 1°C every cycle), and 1.5 min at 72°C, followed by 25 cycles of 1 min at 94°C, 1 min at 48.8°C, and 1.5 min at 72°C, with an 8‐min final extension at 72°C.When the seedlings were 20 days old, 30 healthy seedlings were randomly selected from among the F1 DLSH and LXSH progeny as well as control progeny for genetic diversity analysis using the sequence‐related amplified polymorphism (SRAP) method. One fresh tender leaf from one seedling was crushed into powder in liquid nitrogen, and the genomic DNA was extracted for analysis. Total DNA concentration was determined according to the method described by Li and Jin . Univers2O in the PCR. For every sample, amplification was conducted in triplicate.The PCR amplification products were loaded onto a 1.6% agarose gel (containing 3.5 mg/ml ethidium bromide) with 0.5 × TBE buffer. Electrophoresis was conducted for 1.5 hr at 100 V. After electrophoresis, the gels were visualized with a GIS‐2008 gel imaging and analysis system . The Gel Doc XR system was used for imaging (Bio‐Rad Laboratories). For a negative control, template DNA was replaced with ddHP), Shannon's information index , and Nei's  or absence (0). The number of polymorphic loci, the percentage of polymorphic loci , DLSH seedlings exhibited a Fv/Fm value of 0.75, while the other two seedlings exhibited a Fv/Fm value of 0.65  were polymorphic. Table h and I values, with averages of 0.2734 and 0.4085, respectively. The genetic diversity of F1 progenies, as estimated by h and I, placed DLSH as the most diverse, followed by LXSH and, lastly, the control progeny  that produced clear and reproducible DNA fragments were selected and used for genotyping. A total of 50 dominant loci were amplified with an average of 6.25 loci per primer. Among these, 39 loci  seems to be a good predictor of plant fitness because even minor changes in plant physiological status can have direct and significant consequences on plant reproduction. The value of Fv/Fm is almost constant for different plant species measured under nonstressful conditions (0.8 < Fv/Fm < 0.86) , while LXS pollen, typified by relatively lower genetic distance from the DMS population . Zhu and Li ; funding acquisition (supporting); methodology (supporting); project administration (supporting); resources (supporting); supervision (supporting); validation , visualization , writing – original draft (lead), writing – review and editing (lead). Caihong Qi: Data curation , formal analysis , investigation , methodology , writing – review and editing (supporting). Jingjing Gu: Data curation , formal analysis , investigation , methodology , writing – review and editing (supporting). Zexin Jin: Conceptualization (lead), funding acquisition (lead), project administration (lead), resources (lead), supervision (lead), validation , visualization , writing – review and editing (supporting).Figure S1Click here for additional data file.Figure S1_captionClick here for additional data file.


Mya arenaria is one of the most ancient invaders of European coasts and is present in many coastal ecosystems, yet little is known about its genetic structure in Europe. We collected 266 samples spanning a latitudinal cline from the Mediterranean to the North Sea and genotyped them at 12 microsatellite loci. In parallel, geometric morphometric analysis of shell outlines was used to test for associations between shell shape, latitude and genotype, and for a selection of shells we measured the thickness and organic content of the granular prismatic (PR), the crossed-lamellar (CL) and the complex crossed-lamellar (CCL) layers. Strong population structure was detected, with Bayesian cluster analysis identifying four groups located in the Mediterranean, Celtic Sea, along the continental coast of the North Sea and in Scotland. Multivariate analysis of shell shape uncovered a significant effect of collection site but no associations with any other variables. Shell thickness did not vary significantly with either latitude or genotype, although PR thickness and calcification were positively associated with latitude, while CCL thickness showed a negative association. Our study provides new insights into the population structure of this species and sheds light on factors influencing shell shape, thickness and microstructure.The soft-shell clam  Mya arenaria Linnaeus, 1758, is a marine bivalve that occurs in numerous intertidal infaunal communities across Europe and North America. While this species has a relatively high dispersal potential during the planktonic larval stage and as juveniles [M. arenaria appears to have been largely influenced by human-mediated translocations. Although deliberate introductions have been documented in the literature [The soft-shell clam terature , the majterature  or via sterature ,5.M. arenaria was subsequently reintroduced into Europe by the Vikings during the 13th to 15th centuries [Present in Europe during the Pliocene , the sofenturies ,9, makinenturies . Followienturies ,12, on tenturies , around enturies  and in tenturies ,18.M. arenaria being an important species in many European coastal ecosystems, little is known about its population genetic structure, especially when compared to other European shellfish species [M. arenaria from North America, both of which included a single European population [Despite  species ,21,22,23pulation ,25, onlypulation ,27. In aM. arenaria populations, although these were locally restricted to the Southern coast of Ireland, North Wales and the Netherlands. This study found clear evidence for genetic differentiation at microsatellites but not mtDNA [More recently, microsatellite markers have been developed for the soft-shell clam, which, in contrast to the studies described above, have uncovered relatively strong population genetic structure and higher levels of genetic diversity in North America ,27,28. OCharacterizing population genetic structure not only provides information on patterns of gene flow and genetic drift, but it may also shed light on the extent to which morphological or other phenotypic traits are plastic versus under genetic control ,30. The M. arenaria. While Swan [One obvious shell morphological trait where great variation among individuals can be observed is shape. Shell shape variation has been studied in a variety of molluscan species and it appears to be significantly influenced by both environmental and genetic effects, as well as their interaction ,38,39. Aile Swan  and Emerile Swan  have proM. arenaria is composed of four main layers. These are the outermost periostracum, the granular prismatic (PR), the crossed-lamellar (CL) and the complex crossed-lamellar (CCL) layers [3) crystals and inter-crystalline biomineral organic matrix. Differences in the energetic cost of producing and maintaining different shell structures and components [M. arenaria is widespread and locally abundant, combined with the availability of recently developed microsatellites [The shell of ) layers   and 30 s at 72 °C; and final elongation step of 15 min at 60 °C (see a). Fluorescently labeled PCR products were resolved by electrophoresis on an ABI 3730xl capillary sequencer and allele sizes were scored by three independent observers using GeneMarker v. 2.6.2 (Softgenetics®). Samples that failed to genotype at four or more loci were excluded from subsequent analyses.All of the samples were genotyped at 12 previously characterized microsatellite loci ,28. Thes0 °C see  for Ta. p-values were corrected for the table-wide false discovery rate (FDR) according to the procedure described by Benjamini & Hochberg [Na), allelic richness (Ar), observed heterozygosity (Ho) and expected heterozygosity (He). Finally, we calculated standardized multilocus heterozygosity (sMLH) for each individual using the R package inbreedR v. 0.3.2 [The R package Pegas v. 0.12  was usedHochberg . Next, tHochberg  was usedv. 0.3.2 .Fst values using Arlequin v. 3.5.2.2 [K groups by dividing the dataset in such a way that maximizes Hardy-Weinberg and linkage equilibria within the resulting groups. Each individual is then attributed a group membership value (Q) that varies from 0 to 1, with the latter indicating full group membership. We ran five simulations for each value of K between one and 10. We set the burn-in period and Markov chain Monte Carlo repetitions to 105 and 106, respectively. The most likely number of genetic groups was evaluated using the maximal average value of Ln P (D), a model choice criterion that estimates the posterior probability of the data, and the ΔK procedure described by Evanno et al. [To test for the presence of population structure, we implemented a number of different approaches. First, we calculated pairwise  3.5.2.2 , where s 3.5.2.2 ,60. Thir 3.5.2.2  to carryo et al. . A geometric morphometric approach  based onn = 262; Outlines of orthogonal lateral and anterior views of the left shell valves , capturing 90.1% of the total shape variance, were used as new shape variables and analyzed using multivariate analysis of variance (MANOVA) to test for significant effects of location of origin and shell length (size) on shape variances. To visualize shell outline differences at the extremes of the morphospace, we generated deformation grids and iso-deformation lines through mathematical formalization of thin plate spline (TPS) analysis . Shell mijk is the kth thickness observation from PCj and site i , Siteij is the random intercept and εijk is the error, which are assumed to be normally distributed with an expectation of zero and variances p-values in mixed models [Generalized linear mixed models (GLMMs) were then used to explain variation in shell shape with respect to a number of predictors. Specifically, the scores of the first three shape PCs were modelled as a function of standardized latitude, the first two genetic PCs, sMLH, shape  and their two-way interactions. Shell length was also included in the model to control for possible effects of within-population size variation, and collection site was fitted as random effect. Scores from the first three shape PCs were used as response variables within the same model to account for the interdependence of multiple shape variables that simultaneously describe variation in shell outlines as a whole . This moariables . Due to d models , we consA total of 167 shells were characterized in terms of both total thickness and the thickness of the three individual layers . The left shell valves were set in polyester resin  blocks and sliced longitudinally along their axis of maximum growth c using aThicknessijk is the kth thickness observation from layer j  and site i , Siteij is the random intercept for layer j, which is assumed to be normally distributed with an expectation of zero and variance εijk is the normally distributed error with a mean of zero and different variances per layer j changing exponentially with the variance covariate gPC2ik. This variance structure was chosen over others because it minimized the AICc value  and collection site, which was included as random effect. In the second model, the thicknesses of the three shell layers were combined into a single response variable and the following predictor variables were analyzed: standardized latitude, the first two genetic PCs, sMLH, shell layer  and their two-way interactions. Shell length and collection site were also included, as in the model of whole shell thickness. The thickness of the three layers was analyzed within the same GLMM to simultaneously model common and divergent effects on each layer as well as to reduce the probability of type I error. Non-significant interaction terms were then rejected and only factors that minimized the AICc value  were retCc value . Mean efCc value .M. arenaria specimens were selected from two populations Lisbon (LIS) and Saint Andrews (SAN) to test for differences in shell organic content between low and high latitudes. We removed the periostracum by sanding before isolating pieces of PR and CCL. Shell pieces were cleaned, air-dried and then finely ground. We tested 10 milligrams of this powdered shell with a thermogravimetric analyzer . The samples were subjected to constant heating from ~25 °C to 700 °C at a linear rate of 10 °C min−1 under a dynamic nitrogen atmosphere and weight changes were recorded. The proportion of organic matrix (wt%) was then estimated as the proportion of weight loss during the thermal treatment between 150 °C and 550 °C (n = 20) was then modeled as a function of collection site and shell layer to test for latitudinal differences in shell organic content. Pairwise contrasts with a standard Bonferroni correction were then used to test for differences in wt% between the layers both within and between sampling locations.We performed thermogravimetric analyses (TGA) to estimate the weight proportion (wt%) of organic matrix within the two dominant shell layers, PR and CCL. A random subsample of five d 550 °C . The wt%https://figshare.com) (DOI: 10.6084/m9.figshare.11949546).The microsatellite data used to generate this study are publibly available from the Figshare repository . Significance was lost after excluding ITA  but the overall relationship became significant again after both ITA and LIS were excluded . Results of the PCA confirmed the greater magnitude of differentiation of the Italian population by clearly resolving individuals from ITA as a separate cluster  within our dataset and to derive group membership coefficients (Q) for each individual. The number of groups is often identified using the maximal value of Ln P(D), a model-choice criterion that estimates the posterior probability of the data. However, Ln P(D) often plateaus or continues to increase slightly after the true value of K has been reached [P(D) rising steeply until around K = 4 and then increasing gradually towards a peak at K = 9  but there was no effect of shell size .PCA performed on harmonic coefficients revealed marked variation of shell outlines among individuals in both lateral and anterior views. The first three PCs accounted for 90.1% of the shape variability and a scatterplot of PC1 versus PC2 revealed appreciable variation among the nine populations across the morphospace . MultivaSubsequently, we attempted to identify specific shell features making the greatest contributions towards shell shape variation through comparisons of outlines at the extremes of the morphospace . We furtp < 0.01, effect size = 0.27, 95% CI = 0.07 to 0.45). Consequently, shell shape appears not to be influenced by any of the predictor variables with the possible exception of a weak effect of age.Finally, we constructed a generalized linear mixed model (GLMM) to explore the effects of latitude, body size and genotype  on shell shape variation, as described in the Materials and methods. Shell shape was not significantly associated with latitude or genotype , but a sIn order to investigate how shell thickness may be related to latitude, shell length and genotype, we constructed two GLMMs, the first of whole shell thickness and the second of the thickness of the individual layers . Variation in whole shell thickness was not associated with any of the predictor variables, with the exception of shell length , indicatz = 3.69, p < 0.01), indicative of increasingly calcified prismatic layers at high latitudes . In addition, significant differences in the organic content of the two layers were detected in the low-latitude population , but not in the high-latitude population .To investigate differential patterns of organic content deposition in the two main shell layers at different latitudes, we quantified the proportion of organic matrix in the PR and CCL layers for each of five individual shells taken from the two extremes of the latitudinal range (LIS and SAN). The PR layer was characterized by a significantly higher wt% of organic content in shells from warm temperate regions in comparison to cold temperate regions  of the Mediterranean M. arenaria populations. Genomic data would also be desirable as these might allow divergence times to be estimated and the strength of support for alternative recolonization pathways to be evaluated sensu [Although our sampling design was far from exhaustive, the broad geographical coverage of our study allowed us to capture a number of interesting patterns. First and most obviously, samples from the Adriatic Sea (ITA) were strongly differentiated from the Atlantic populations. Deep genetic divergence between the Mediterranean and the Atlantic is a common pattern found in native European marine invertebrates ,72, whicerranean ,12. Addierranean  suggesteed sensu . M. arenaria populations may superficially resemble that observed in other marine species native to Europe, where Atlantic populations are divided into a northern and a southern genetic cluster [M. arenaria was at least partially anthropogenic, following its introduction to Denmark between the 13th and 15th centuries [The pattern of genetic structure we uncovered among the Atlantic  cluster ,73,74. Henturies ,9. This M. arenaria samples from Portugal showed high genetic affinity with populations from along the continental coast of the North Sea and the entrance of the Baltic Sea (Le Crotoy (LCT), Balgzand (TEX), Sylt (SYL) and Kiel (KIE)). This finding is again consistent with the notion that man-mediated translocation played an important role in shaping the genetic structure of M. arenaria across Europe. In this particular case, the most parsimonious explanation for the observed pattern would be that soft-shell clams from the North Sea coast were introduced into Portugal, either deliberately or inadvertently due to the fact that M. arenaria can be easily transported with ballast water [Finally, we could show that st water ,5. In list water  have alrM. arenaria. One possible explanation could be that our predictor variables were too crude to capture meaningful variation in key abiotic or biotic variables. In particular, although our broad geographic coverage maximized variation in a suite of climatic and other variables, this may have hindered the detection of relatively subtle effects. This issue could potentially be circumvented by sampling populations over a much finer geographic scale, as this would effectively control for large-scale sources of variability while facilitating more quantitative investigation of specific factors such as substrate typology and sediment size.An EFA of shell outlines uncovered differences in shell shape among the collection sites, but none of the predictors fitted in our GLMM could explain a significant proportion of the total variation. Although it is not necessarily surprising that the genetic variables were unrelated to variation in shell shape, studies of other bivalve species have reported strong effects of local environmental conditions, substrate type and predation pressure ,41,44,753 saturation and reduced metabolic rates [M. arenaria, predation acts most heavily upon young individuals that are present only in the upper layer of the substrate, while predation risk for older individuals buried deeper in the substrate is negligible [Geographic patterns of molluscan skeletal production have typically been explained by two paradigms: poleward reduction of predation pressure ,77 and iic rates ,77,78,79gligible ,81. Give3 deposition [Surprisingly, our results are also not consistent with poleward skeletal reductions due to increased calcification costs at high latitudes, as we did not observe any change in total shell thickness along a latitudinal cline. However, shell organic content was reduced in samples from the more northerly, colder environment, which is in line with suggestions that shell organics have higher production costs than CaCOposition ,47. Simiposition , where tposition . LikewisPrevious studies of a variety of marine and freshwater invertebrates have provided support for a plastic nature of shell morphology. This conclusion has been reached either due to the absence of a link between population genetic structure and morphological variation ,34,36 orM. arenaria across Europe and to relate this to latitudinal variation in shell shape, thickness, microstructure and organic content. We uncovered strong population structure, consistent with the known involvement of humans in reintroducing this species to Europe as well as a long history of both deliberate and unintentional long-distance translocations. Additionally, we were unable to find any genetic effects on individual variability in shell shape and thickness, consistent with previous studies of other shellfish species reaching the conclusion that shell morphology is largely plastic [M. arenaria was latitude, which is associated with variation in numerous variables, both biotic and abiotic.We used microsatellites to characterize the population genetic structure of  plastic ,35,36. S


Kidderia subquadrata, a small mollusk that inhabits intertidal rocky island ecosystems throughout the WAP. Using a phylogeographical approach, we examined the spatial patterns of genetic diversity in this brooder species to test the hypothesis of strong genetic structure in incubating organisms and the hypothesis of glacial refugia in organisms with limited dispersion. We found evidence of strong genetic structure among populations of the WAP and a recent expansion in the South Shetland Islands. Our findings are concordant with the predictions that incubating organisms, abundant in Antarctica, present a strong genetic structure among their populations and also support the hypothesis of glacial refugia in organisms with limited dispersion. The effect of the coastal current pattern in the WAP is suggested as a driver to the local spatial dynamics of the genetic diversity distribution. Although genetic information about this microbivalve is still scarce, the knowledge reported here has increased our understanding of the evolutionary patterns of this organism that is endemic to the Southern Ocean.It is well established that Antarctic biodiversity has been strongly influenced by rapid climatic fluctuations during the Quaternary. Marine invertebrates from Antarctica constitute an interesting lens through which to study the impacts of the last glacial periods as glaciation impacted the distribution and intraspecific genetic variation of these animals. However, the impact on the spatial genetic distribution and historical demography of local processes in areas adjacent to the West Antarctic Peninsula (WAP) is less clear. Here we present new genetic information on the bivalve  For example, the poor dispersers, which include many species, present seemingly high levels of endemism in the SO3. The high prevalence of brooders can be explained partially by glacial cycles4. This explanation is based on the glacial refugium hypothesis5. In this scenario, the hypothetical glacial refuges in the Antarctic would have allowed the Antarctic species to be protected during the interglacial periods6, and processes of expansion and contraction could have taken place7. Currently, numerous evidence of an increase in population size during the early stages of the last glaciation seems to support the glacial contractions and interglacial expansions were a recurrent pattern that eventually led to species adapting to cold conditions during the Late Quaternary10. Species colonizing different environments have managed to survive through the Pliocene and Pleistocene glaciation cycles by shifting between habitable areas of polynyas or ice fracture zones under former or extant Antarctic ice shelves13. Some hypotheses that have emerged to explain the survival of species in the last glacial periods are derived from the Expansion‐Contraction Model14. One of these hypotheses is in situ persistence in Antarctic glacial refuges16. The in situ persistence scenario suggests the presence of one or several isolated refugia on the shelf; these refugia are associated with strong population bottlenecks16. A second hypothesis is that of island refuges, which proposes that shallow marine species survived out of the Antarctic continental shelf in adjacent Antarctic islands such as the South Shetland Islands (SSIs)17. This area has been defined as an in situ refuge associated to volcanoes or areas of geothermal activity18. These refuges have been particularly informative in reconstructing the periglacial and postglacial history of marine organisms20. Finding a pronounced geographical structure may provide evidence of putative refugial areas23. Refugial populations are usually composed of subsets of the genetic diversity and long-term isolation of populations within geographically separate refugia will lead to genetic differentiation24.Studies based on Southern Ocean (SO) biodiversity have been focused on eurybathy, circumpolarity and the high prevalence of brooders25 lists 57 species and 41 genera, of which only 12 genera are still represented in the SO; most of the remaining genera occur at present in the seas north of the Polar Front26 and a few still live around Antarctica (12.5%)25. Moreover, the analysis of species richness in bivalve families revealed that the distribution has high richness north of the Antarctic peninsula and low richness to the south in, for example in families like Pectinidae, Nuculidae, Mytilidae, Gaimardiidae35. Antarctic bivalves have a varied range of reproductive strategies. In some the sexes are separate (gonochoric organisms) while others are simultaneous hermaphrodites37, some display indirect development and external fertilization (planktonic larvae)38 while others exhibit direct development in which the embryos are retained by the female, who provides parental care to the offspring (brooder species)45.Despite the fact that several genera of bivalves have survived the glacial periods, a reduced number of these persist in Antarctic benthic systems and their biogeography patterns have yet to be exhaustively studied. The review of Seymour Island Paleocene molluscsK. subquadrata Pelseneer (1903)47 is a brooder species with a 4.5 mm average shell length47. They incubate their offspring until the juvenile stage and have been reported inhabiting the rocky intertidal of islands adjacent to the West Antarctic Peninsula (WAP)48. No genetic information on this species has been reported prior to this work. In this paper, we aim to increase the available information about the Antarctic biodiversity and microbivalve species. To that end, we have developed a spatial analysis of the genetic diversity in populations of K. subquadrata from the WAP to advance the understanding of the evolutionary history of these microbivalves that inhabit Antarctica.The microbivalve (CoxI) and a suite of phylogeographic analyses to estimate the spatial genetic diversity and the historical demography of this species.Based on the absence of the larval phase, we hypothesized that due to a low dispersal potential in this species we would find a high spatial genetic structure among populations with evidence of in situ refugia in the SSIs. To test this hypothesis, we used the mitochondrial molecular marker, Cytochrome oxidase I ST = 0.87, p-value = 0.0001). The highest values of genetic differentiation measured with SAMOVA analysis were detected when the populations were separated into 4 groups  with values of FCT = 0.92   throughout the geographical range studied51. Similar results were reported in other works on this Antarctic limpet; these studies, whose sampling sites covered a large geographical range, suggest the existence of a single panmictic unit52. More recently, for sea star brooders, researchers have concluded that broadcasters are less spatially structured than brooders53.Our analysis supports the genetic differentiation among sampled populations of K. subquadrata may have been enhanced by the Bransfield Strait current system. The surface circulation in Bransfield Strait is influenced by the Antarctic Coastal Current (ACC) and the Antarctic Slope Front54. Seaward of the South Shetland Islands (SSIs), the southwestward slope currents persist over the long term, they are originated by the Antarctic Slope Current that flows around the Antarctic Peninsula55. The water masses of the ACC transport nutrients west of the SSIs and enter the area through the Bransfield Strait flowing strongly to the east-northeast along the southern margin of the SSIs56. The Weddell Sea water enters through the Bransfield Strait mainly from the north-east and flows in a south-west direction along the Antarctic Peninsula. A similar process has been proposed to explain a genetic barrier identified in the Antarctic annelids of the family Phyllodocidae family in the strait north of Deception Island57, which coincides with the oceanic front that is generated by the intrusion of seawater masses from the Weddel Sea in the Bransfield Strait55. Therefore, the structure in K. subquadrata populations could be explained by the system of currents in the Bransfield Strait that forms a cyclonic circulation59. In addition, Livingston Island showed the presence of unique haplotypes that are separated by more than 10 mutational steps from the ancestral polymorphic haplotype but lie geographically lying within the SSIs. This undoubtedly requires a more detailed explanation and further investigation. According to a modified map from Barlett (2018), Livingston Island has low bathymetry (100–200 m)60. This pattern could result in water retention processes that increase the retention of marine invertebrate in the island, thus acting as a sink that results in a strong spatial genetic differentiation of its K. subquadrata population. However, the number of individuals analyzed in this study is small and an increased sample size would provide a more complete picture of the spatial process in this locality and could reveal the actual phylogeographic pattern.The local oceanographic dinamyc may have also played a relevant role in the spatial interaction among populations in the interglacial periods in the SO. The spatial genetic structure pattern of 61. It now appears that the continental shelf was not ice-covered equally across the Antarctic coastline, which allowed some ice-free refuges for fauna during the glacial maxima62. According to the literature, the last glacial maximum occurred 20,000 to 17,000 years ago and West Antarctica and the adjacent islands were apparently covered in ice63. The deglaciation of Signy Island and the SSIs began 14,000—11,000 years ago and spread during the Holocene until 8,000—6,000 years ago67. Strong signals of recent demographic expansion as well as founder effect associated with recolonization can be inferred from our results in the haplotype network and DIYABC analysis. Therefore, a potential historical demographic hypothesis could be that strong genetic subdivision is a consequence of a pattern of multiple glacial refugia with a Pleistocene post-glacial expansion21.On the other hand, glacial activity in Antarctica has been suggested as a driver of phylogeographic patterns for species by causing isolation and the presence of in situ glacial refugesK. subquadrata that were collected as part of our samples lived as epibionts on the macroalgae. Macroalgal rafting has been suggested to explain the low genetic differentiation of marine communities across the Subantarctic region68, and recent investigations have revealed the arrival of invasive species that reached the Antarctic continent alive on kelp rafts70. Thus, a potential explanation for this unusual spatial pattern of the genetic distribution in K. subquadrata populations could be that they have used macroalgae as a transport vector. This hypothesis implies some degree of successful colonization and posterior isolation of this unique haplotypes. In the case of Doumer Island, the southernmost locality, we identified exclusively private haplotypes, they displayed low genetic diversity and the highest FST values, suggesting a process known as leptokurtic dispersion73. Since that time it has probably served as its own refuge and undergone divergence in isolation19. Similar patterns of genetic structure have been reported in the marine bivalves Arctica islandica74 in the Northern Hemisphere.In particular, the most widely distributed and frequents haplotypes were shared by five islands: Signy, Penguin, King George, Greenwich and Deception. The Signy and Doumer Islands are the most geographically distance localities within the study’s sampling range. The DIYABC reveals that Signy and Doumer are the ancestral populations and probably after the original split, Signy island constituted a source population (refuge) for the population of SSIs. Interestingly, the haplotype network showed that Signy also has a unique haplotype that is different from the ancestral polymorphic haplotype and more closely related to the unique Doumer haplotypes. Some individuals of 75. It is worth noting that cryptic species are commonly found in brooding species in the Antarctic Peninsula in, for example, echinoderms such as ophiuroids, echinoids and brooding pygnogonids78. The study of the bivalve Lissarca notorcadensis was one of the first to show indications of cryptic speciation in Antarcic bivalves27. New evidence of two cryptic lineages in the Antarctic Peninsula was recently reported in the bivalve Aequiyoldia eightsii, with an estimation of genetic distance of 5.79% from CoxI35. Considering K. subquadrata’s intrinsically low dispersal and the evidence of the probable occurrence of in situ refuges revealed in this study, a cryptic speciation process could potentially explain the genetic diversity in the population of Doumer Island. The samples obtained from Doumer Island were collected in a protected bay surrounded by glaciers, semi-isolated from the marine currents since there is only one entrance to the bay; these geographical features indicate that gene flow out of bay could be difficult. Here, based on CoxI we report a genetic distance of 2% among the central haplotype (H1) and the two unique haplotypes from Doumer Island; this value is within the limit established in the existing literature79 for inferring a speciation process. Intraspecific divergence with CoxI is rarely greater than 2% in the phylogeographic analyses; even so it can serve as an effective tool in recognizing putative new lineages81. Moreover, there are reports of distinct glacial refugia in the SSIs and Antarctic Peninsula harboring cryptic species that have diverged recently in micro-allopatry84.Non‐pelagic development has been described in the Antarctic fauna, where brood protection appears dominant42; its greatest utility is in non-model species where there are no previous genetic data, since its use enables comparisons with previous studies. However, recent studies have reported significant dissimilarities between genetic diversity and structure encountered in marine species depending on the genetic markers used85. Differences in patterns of genetic structure have been linked to the fact that organelle DNA is more sensitive to introgression and/or rapid sweeps (due to selection or strong genetic drift) than is nuclear DNA86. Therefore, it is imperative to develop new nuclear markers  in Antarctic marine invertebrates. Further studies could increase the number of populations sampled in the Southern Ocean to test for the existence of local adaptation using recently developed genomics and transcriptomics tools.In conclusion, we report new evidence of a strong spatial genetic structure in a brooder microbivalve species unique to the WAP. We also suggest possible presence of in situ glacial refuges and we infer a cryptic speciation in progress. Mitochondrial DNA has been widely used in numerous studies of phylogeographyK. subquadrata were collected from the following islands  gene two pair primers were designed. Two pairs of primers were used: the first corresponds to kg1F (5′-TTG GGC TGG GTT AAT AGG TACA-3′) and kg7R (5′-GAA AAC CAG CAA ACA TAG CA-3′) flanking a fragment with a total length of 861 bp; the second pair corresponds to kg7F: (5′- TGC TAT GTT TGC TGG TTT TC -3′) and kg8R (5′- CCC AAA AAG ACA TTT GAC CC -3′), which were used to recover a fragment of 279 bp. The CoxI gene amplified a total length of 1140 bp, coding 380 amino acids.The total DNA of each individual was isolated with Quick—DNA plus (Zymo Research) commercial kit following the procedures described by the manufacturer. Given the absence of genetic information for the genus 2, 0.2 mM each dNTP, 0.25 μM each primer, 0.2X BSA, 1 μL DNA concentrate (10—50 ng), 0.6 units GoTaq DNA polymerase (Promega) and H2O to reach the final volume. The CoxI gene was amplified using the following thermocycling profile: an initial denaturation step (95 °C for 5 min); 35 cycles of amplification ; and a final extension step (72 °C for 8 min). The PCR products were purified using E.Z.N.A. Cycle Pure PCR Purification kits (Omega Bio-tek) and sequenced in both directions by the sequencing service of Macrogen Inc. Company (www.macrogen.com). Sequences were aligned in Geneious R10.2.4 software87 and manually edited to resolve unclear base calls. CoxI consensus sequences were translated into amino acids using the invertebrate mitochondrial genetic code to check for stop codons. Alignments were performed using the default settings of the ClustalW alignment algorithm implemented in Geneious .All PCRs were performed in a final volume of 25 μL containing 1X PCR buffer, 3.5 mM MgClK. subquadrata we used standard diversity indexes: number of haplotypes (k), number of segregating sites (S), haplotype diversity (H) and the average number of paired differences between sequences (π) for each region using DnaSP v588. Pairwise genetic differentiation (pairwise FST) was estimated to determine the genetic structure among populations using the ARLEQUIN program v.3.5189. To test the hypothesis of isolation by distance (IBD) we used the relationship between genetic (FST⁄1-FST)90 and the geographic distances among Signy, King George, Penguin, Deception, Greenwich, Livingston and Doumer Islands to perform a Mantel test91 executed in the R environment with the Vegan (2.5–2 version) package92.To estimate the levels of genetic polymorphism in populations of 89. Next, we developed a set of analyses using the geographic coordinates of each sampled island. Here, we analyzed the population structure without an a priori cluster hypothesis using a spatial analysis of molecular variance (SAMOVA)93. In this approach, sample sites are clustered based on a simulated procedure that aims to maximize the proportion of total genetic variance caused by differences among populations groups.To test for spatial genetic differentiation, we calculated a global FST by performing a global AMOVA analysis with the ARLEQUINK. subquadrata we estimated the number and composition of panmictic groups and the spatial boundaries among them using a clustering method based on the Bayesian model computed with the GENELAND package, version 4.0.094 in the R environment version 2.4.192. This software implements a Markov Chain Monte Carlo (MCMC) procedure to determine the best clustering of samples with regard to genetic and geographic information. Geographic information is considered as the Bayesian prior level, so clusters corresponding to spatially structured groups are considered to be more probable than clusters that are randomly distributed in space. Five × 106 MCMC iterations were sampled every 1000 steps with a 500-step burn-in period, and a maximum number of clusters K = 8 was run to estimate the model parameters and posterior probabilities of group membership.To infer the spatial pattern of genetic diversity in CoxI haplotypes for the whole dataset were characterized using median joining networks in HapView (http://www.cibiv.at/~greg/haploviewer)95.Finally, the genealogical relationships among 96 and the Fu Fs statistic97 were performed. The Tajima D test is based on the fact that in a neutral model, estimates of the number of sites segregating and the average number of nucleotide differences are correlated. Fu’s test is based on the model of infinite sites without recombination; it gives the probability of observing a random sample with the number of alleles equal to or less than the value observed, given the observed level of diversity and the assumption that all alleles are selectively neutral. Both statistics were calculated in the ARLEQUIN. In addition, we estimated past population dynamics over time using the Bayesian skyline plot method implemented in BEAST 298. To develop this analysis, we selected the five sample sites  that shared the most frequently observed haplotype; as described earlier, these sample sites showed a star-like topology in the haplotype network. We conducted two independent Bayesian MCMC runs using the generalized time-reversible (GTR) model with a gamma distribution (G), previously estimated with JModelTest299 and mutation rates calibrated for CoxI sequences of bivalves101 1.0% Myr-1. Substitution rates were modified to a tenfold evolutionary rate (10% per million), considering the correction for time dependence of molecular rates at the population level103. The two independent MCMC calculations were run for 1.5 × 107 generations (sampled every 1000 iterations), and the first 10% of the trees were discarded as burn-in. The convergence of runs was confirmed with Tracer v1.6104, ensuring a minimum of 600 effective samples for each statistic (ESSs). The median and corresponding credibility intervals of the Bayesian skyline plot were depicted with Tracer v1. 6.To examine the historical population demography, the Tajima D test105 to tested for different population demographic scenarios. This software evaluates population histories using Approximate Bayesian Computation (ABC) with genetic data, by testing scenarios that are built through a combination of population divergence, admixture and population size changes. Following the recommendations of Cabrera and Palsbøll106 to improve DIYABC’s ability to reveal the true demographic model, we focused on simple contrasting models and reduced the number of candidate scenarios after a set of hierarchical analysis beginig with 5 preliminary scenarios (Fig. –9 and máximum 1 × 10–4 and the mutation model used was kimura 2 parameters. For the historical models, priors were set by default and in accordance with the recommendations of the authors of the software, we performed 2,000,000 simulations.In order to better understand the population history of the species we used the program DIYABC v2.1ios Fig. . SubsequSupplementary information.


Odontomachus chelifer (Latreille) (Ponerinae) is a ground-dwelling, predominantly carnivorous ant whose colonies may contain multiple egg-laying queens and are potentially susceptible to border effects in the Brazilian savanna known as Cerrado. The ecology and natural history of O. chelifer is well studied, but very little is known about the genetic diversity of O. chelifer colonies. In this study, we developed microsatellite markers for the study of genetic variation in O. chelifer. We created a microsatellite-enriched library that resulted in the development and characterization of 22 markers, of which 18 were found to be polymorphic in the population studied. The mean expected heterozygosity was 0.59, whereas the mean rarified allelic richness was determined as 4.27 alleles per locus. The polymorphism level detected was similar to genetic diversity estimates found in other poneromorph ant species. The microsatellites developed here are likely to be useful for the investigation of colony structure, functional polygyny, breeding system, and population genetics in O. chelifer. Moreover, the description of O. chelifer’s genetic diversity is crucial for its conservation and maintenance of its ecological role in the Cerrado savanna. Odontomachus chelifer (Latreille) (Formicidae: Ponerinae)  and each queen’s reproductive activity is mediated by queen–queen dominance interactions, even in mature colonies, with highly ranked queens laying more eggs and foraging less frequently outside the nest   is a gronerinae) . The spenerinae) . Additionerinae) . Odontomthe nest . In the the nest , which sthe nest . Even th but see .O. chelifer, which will enable future studies on genetic variation and maintenance of O. chelifer populations.Microsatellites—or SSR (simple sequence repeats)—are frequently used as a molecular tool to study genetic diversity in ant colonies. These molecular markers are repetitive sequences (one to six nucleotide repeats), distributed in tandem along the DNA and frequently and randomly distributed across the genome in most eukaryote species . MicrosaO. chelifer were sampled in Fazenda Campininha, a Cerrado savanna reserve located in the city of Mogi-Guaçu (22°18′S e 47°11′W), in the state of São Paulo, southeastern Brazil. Via active searching, we collected 15–20 workers from 18 O. chelifer nests. Ant voucher specimens are deposited at the Museu de Zoologia da Universidade Estadual de Campinas .Colonies of DNA extraction was performed using methods described by O. chelifer were used to build a microsatellite-enriched library, based on protocol previously described in 8 and (GT)8 biotin-linked probes, followed by recovery with streptavidin magnetic-coated beads . The selected fragments were cloned into the pGEM-T vector (Promega) and transformed into competent Escherichia coli (XL1-Blue strain). Recombinant colonies were identified by colorimetric white-blue detection using X-gal. Plasmid DNA was then extracted using an alkaline lysis method and inserts were sequenced on a 3500 Genetic Analyzer sequencer . We sequenced 103 clones and the electropherograms were edited using CLC Genomics Workbench v. 4.9 software . Vector, adapter, and restriction site sequences were removed using Seqman . Additionally, sequences were compared with entries of the public National Center for Biotechnology Information database using Blastn (m between 45 and 65°C (maximum of 3°C difference between both primers in the pair), and 4) presence of A or T bases at the 3′ end (to reduce probability of self-pairing). We also avoided designing primers that allow the formation of homo or heterodimers. For automating the genotyping on the 3500 Genetic Analyzer sequencer (Applied Biosystems), an M13 sequence tail (5′-CACGACGTTGTAAAACGAC-3′) was added to the 5′ end of each forward primer  (mean ± SE) was 0.59 ± 0.05, with the highest values found in the Och6 (0.89), Och3 (0.86), and Och8 (0.80) loci. Very low HE values were identified in Och88 (0.07) and Och15 (0.08)  . Rarifie5 (0.08) . The mea5 (0.08) . We foun5 (0.08) . FinallyO. chelifer, resulting in 22 markers, 18 of which were found to be polymorphic in the population studied. Even though some markers displayed low polymorphic content, most proved to be highly informative (PIC > 0.5) to access genetic diversity in O. chelifer (Pachycondyla inversa (5–12 alleles; Hypoponera opacior (9–21 alleles; Pachycondyla luteipes (2–8 alleles; A microsatellite-enriched library was developed for chelifer . DiversiO. chelifer genetic variation.Despite the availability of microsatellite markers for other Ponerinae or for other ant species , interchO. chelifer.Eleven microsatellite loci exhibited deviations from HWE, indicating violation of one or more assumptions of the Hardy–Weinberg model, namely presence of selection, migration and/or mutation, finite population size, and non-random mating . AdditioO. chelifer have already been studied in Brazilian forests and savannas (Although the behavior and ecology of


Chamaecrista fasciculata, their Bradyrhizobium symbionts and rhizosphere free-living bacteria at a small spatial scale to evaluate the extent to which symbiotic partners will have similar SGS and to understand how plant hosts choose among nodulating symbionts. We found SGS across all sampled plants for both the host plants and nodulating rhizobia, suggesting that both organisms are influenced by similar mechanisms structuring genetic diversity or shared habitat preferences by both plants and microbes. We also found that plant genetic identity and geographic distance might serve as predictors of nodulating rhizobia genetic identity. Bradyrhizobium elkanii was the only type of rhizobia found in nodules, which suggests some level of selection by the host plant.In natural plant populations, a fine-scale spatial genetic structure (SGS) can result from limited gene flow, selection pressures or spatial autocorrelation. However, limited gene flow is considered the predominant determinant in the establishment of SGS. With limited dispersal ability of bacterial cells in soil and host influence on their variety and abundance, spatial autocorrelation of bacterial communities associated with plants is expected. For this study, we collected genetic data from legume host plants,  Several studies have recognized plants as one of the most important factors shaping soil microbial community structure ,2,3,4. IHordeum vulgare L. rhizosphere microbiome composition. Therefore, with dispersal-limited bacterial and fungal communities and host influence on microbial variety and abundance, spatial autocorrelation of rhizosphere communities is expected [While most studies have focused on differences in soil microbial communities associated with different plant species, a few studies have addressed these differences within a single species and found that microbial community structure and composition can also vary according to intraspecific genotypic differences of host plants ,13,14,15expected ,17. HoweNodulating rhizobia, which begin as free-living soil microbes, also experience variable selection as a result of changes in host genetic identity, resource availability, temperature, moisture and the soil biotic community. Both interspecific ,19 and iChamaecrista fasciculata (Michx.) Greene (Partridge Pea), an annual native legume (Fabaceae), is widely distributed in the eastern U.S. The species exhibits intraspecific morphological variation and wide ecological tolerance of different soil types and habitats [Bradyrhizobium is the only genus of rhizobia that is symbiotic with C. fasciculata, but many different genetic variants of Bradyrhizobium are able to nodulate in C. fasciculata [habitats . As a sphabitats  and to mhabitats . Given ihabitats . Bradyrhciculata ,34,35,36Bradyrhizobium are phylogenetically diverse within a site but do not have a strong genetic structure across large geographic distances. This led them to suggest that the scale at which successful symbioses are established may be very fine and determined by plant x rhizobia genotypic interactions, rather than soil traits. In this study, we tested for the presence of similar patterns of fine-scale genetic structure between C. fasciculata host plants and their nodulating Bradyrhizobium symbionts. We expected that plants in close physical proximity would be more genetically similar than those more distant from one another due to having gravity dispersed seeds. We also expected that nodulating rhizobia would exhibit a spatial scale of genetic structure that is concordant with their host plants due to genotype x genotype interactions, limited dispersal of bacteria in the soil, and enhancement of a suitable symbiont pool from continual host plant presence. Lastly, we also characterized members of order Rhizobiales, which includes Bradyrhizobium, from plant rhizospheres to test the hypothesis that the bacterial strains in the nodules are a subset of available bacteria in the soil. Previous studies have surveyed genetic diversity and distributions of rhizobia communities in the soil and nodules of different plant hosts [Bradyrhizobium symbionts and provides understanding of the degree to which host plants choose certain microbial genotypes from the soil. Additionally, examination of fine-scale genetic structure of multiple interacting species has rarely been conducted. The proposed study enhances our understanding of the role of bacterial symbionts in local adaptation of plants and the degree to which plant hosts act as a predictor to determine genetic structure of soil microbes.The findings of ref.  indicatent hosts ,37,38,39IS) were consistently positive, indicating heterozygote deficiency across all plots  for plant kinship coefficient over distance classes (p = 0.001 for bF = −0.0138) (ij) between individuals. Significant positive values of Fij were found at short distances (<43 m), indicating that neighboring individuals had a higher genetic relatedness than random pairs of individuals. Negative values  of Fij occurred at larger distances (F), F(1) and Sp statistics across all plots were −0.0121, 0.045 and 0.0127, respectively  . Since t−0.0138)  and prin−0.0138) , we comb−0.0138) . The aveistances . Mean vaectively . The p-vtruA sequences from nodulating rhizobia. The aligned length of the truA sequence set was 497 nucleotides (~67% coverage of truA gene in Bradyrhizobium). All sequences most closely resembled Bradyrhizobium elkanii truA sequences in BLAST-n searches in GenBank (NCBI 1988). Additionally, the phylogeny of truA sequences indicates that all recovered sequences from C. fasciculata cluster exclusively with B. elkanii and with 1.0 posterior probability support (truA haplotypes were found. Only plot 1 demonstrated a significant regression slope (bF) for rhizobia kinship coefficient over distance classes    . When pl= 0.046) , but we = 0.041) .p = 0.001, p = 0.99, p = 0.26, p < 0.0001 for both, A significant relationship between plant genetic and geographic distances was found , B. canariense (22%), and unidentified Bradyrhizobium strains (9%)  . RhizobiC. fasciculata, including heaviness of the seeds and bee pollination, suggest that plants should exhibit an isolation by distance pattern. The SGS pattern detected for host plants in the studied plots was consistent with IBD, showing an increase in genetic distance between pairs of plants as they increased in physical distance from one another. Additionally, the kinship coefficients gradually decrease from very positive to very negative over the distance of sampling. Discovering statistically significant fine scale genetic structure at most of our studied distances is concordant with the existence of very restricted seed dispersal events among plants and suggests that plants are not randomly distributed even at this fine scale. Values for the Sp statistic in this study, ranging 0.0048–0.0139, are comparable to another study on this species with reported Sp statistic of 0.00746 [C. fasciculata.In many plant species, seed dispersal has a peak distribution at or close to the maternal plant and progressively fewer seeds are dispersed at greater distances from the maternal plant ,41,42. T 0.00746 . The mea 0.00746 , which aC. fasciculata as well. Additionally, limited dispersal ability of bacterial cells in the soil due to passive means is expected to maintain structure if there is not extensive soil disturbance. Although we did not find a significant kinship coefficient for pairs of rhizobia at most geographic distances, the mean sp statistic in rhizobia followed the same pattern observed for plant hosts. That is, we found a significant bF for rhizobia and plant hosts in plot 1 and the combined plots. This finding suggests a common mechanism that influences genetic structure of plants and rhizobia. It may be that host plants directly influence their rhizobia symbionts by selecting for certain strains or that plant hosts and their nodulating rhizobia are influenced by similar environmental factors that structure genetic diversity. In our study, we focused on only one housekeeping gene, truA, to identify the nodulating rhizobia and estimate genetic diversity of this community. Although this does not capture genome-wide diversity, it is a suitable genetic marker that was considered highly informative of phylogenetic relationships in Bradyrhizobium by refs. [truA, which indicates an ability to discriminate among most of the rhizobia symbionts we recovered. In another study, the nucleotide diversity of Rhizobium leguminosarum biovar viciae isolates from Vicia cracca L. acquired using two housekeeping genes, glnII and recA, and one nodulation gene, nodC, revealed relatively close values of nucleotide diversity for all of the three studied genes  [Given the presence of fine-scale structure in host plants and widely documented genotype x genotype interactions between legumes and their symbionts, we expected to find significant genetic structure in nodulating rhizobia of by refs. ,48. Our ctively) . This stC. fasciculata is similar to studies of other legumes. In ref. [truA to study genetic diversity of nodulating rhizobia in C. fasciculata across Mississippi, 9–25 haplotypes and 0.831–0.954 for haplotype diversity was reported at a regional scale. Our finding of six haplotypes and substantial haplotype diversity in a single site indicates that populations can harbor diverse symbionts even at the smallest spatial scales. In a fine scale study (<2 m) of diversity patterns in microbial communities associated with roots of Bistorta vivipara L., a perennial herb, it was revealed that similarities between the structure of bacterial and fungal communities were stronger in soils than in roots [While a literature review found no fine-scale studies of genetic structure in nodulating rhizobia or plant endophytes for comparison to our results, we have found that the number of haplotypes recovered for  In ref. , which ain roots . The rooBradyrhizobium nodulating C. fasciculata [C. fasciculata studied here belonged to Bradyrhizobium elkanii  K. Larsen, the plant genotype explained only 4.91% of the variation in nodulating rhizobia [Selection of only one species by the host plants may reduce the SGS pattern of kinship in nodulating rhizobia. A non-significant Mantel test also supports this idea by showing that genetic distance of isolates is not associated with geographic distance between pairs of nodules , following [TruA, a housekeeping gene involved in translation and ribosomal biogenesis [Bradyrhizobium strains [truAB-F/R primers specific for Bradyrhizobium [truA. Successfully amplified products (one sequence per sample) were cleaned by adding 0.2x Antarctic Phosphatase buffer, 5 units of Exonuclease I, and 1.25 units of Antarctic Phosphatase , to 7 μL of PCR product. This mixture was heated to 37 °C for 15 min followed by 80 °C for 15 min. Once the samples were cleaned, cycle sequencing was conducted in 10 μL reactions using either the forward or reverse primer and Big Dye version 3.1 . Forward and reverse primer sequences were generated for all individuals using the PCR primers. Sequenced samples were dried and sent to the Arizona State University DNA Lab for capillary electrophoresis. Rhizobia forward and reverse sequences were edited and assembled into a consensus sequence for each sample using Sequencher version 4.7 . Sequences were aligned using the alignment algorithm in Geneious v.10.2.5 .One to two nodules per plant were surface sterilized with 1% hypochlorite for 5 min then washed in sterile water. Later, they were placed in 70% ethanol for 5 min and then were washed three times with sterile distilled water each for 1 min. After surface sterilization, nodules were ground to release rhizobia, and this mixture was plated on solid agar MAG medium  in petriollowing ,69, was ollowing ,72). Priogenesis  and that strains ,48,74 wan = 70 plants) using 0.5 g of soil and the FastDNA™ SPIN Kit for soil isolation . We were unsuccessful in getting truA to amplify consistently in these samples. Thus, we used the 16S rRNA gene to characterize rhizosphere rhizobia communities because this gene has been broadly used in prokaryote taxonomy and phylogenetic reconstructions and sequences are widely available across prokaryotic lineages [For bacterial community sequencing of rhizosphere samples, whole-community DNA was extracted from each rhizosphere sample  using QIIME2 . This soO), expected heterozygosity (HE), and inbreeding coefficient (FIS) using GenAlEx version 6.503 [p-value of 0.001, which is adjusted using the sequential Bonferroni correction for multiple comparisons [F) of pairwise statistics (Fij) on ln (distance) using 9999 randomizations of the individual spatial positions and obtaining 95% confidence intervals (CIs) after bootstrapping (1000 iterations) using SPAGeDi 1.5 [F/(1 − F(1)), where F(1) is the mean F(ij) between individuals belonging to the first distance interval and bF is the regression slope of F(ij) on rij  [(1) can be considered a good estimate of the kinship between pairs of neighbors, on the condition that the first distance interval contains enough pairs of individuals to obtain reasonably precise F(1) values [The plant dataset was tested for presence of unique multilocus genotypes using the Poppr package  in RStudon 6.503 . For eacparisons . A Princparisons  to exploparisons . The plaparisons ), which parisons . SeventeGeDi 1.5 . Spatiali and j) . F(1) ca) values . StandartruA from each nodule was checked against GenBank sequences via BLAST-n searches [truA sequences were aligned in Geneious v.10.2.5  using the internal alignment algorithm and compared against five reference sequences of taxonomically valid Bradyrhizobium retrieved from GenBank [B. canariense BTA1 (Accession: JX064276), B. elkanii USDA 76 (Accession: JX064277), B. japonicum USDA 6 (Accession: JX064272), B. liaoningense USDA 3622 (Accession: JX064278), and B. yuanmingense CCBAU 10,071 (Accession: JX064271). These five strains are informative for identifying Bradyrhzobium species which associate with plants from Fabaceae [The sequence of searches  to ident GenBank  in a phyFabaceae . We usedFabaceae ,91 to seFabaceae  as the bFabaceae  was usedFabaceae . We condFabaceae .truA haplotype sequences were converted to SPAGeDi 1.5 input using haplotype codes generated by SPADS 1.0 [F) of pairwise statistics (Fij) on ln (distance) using 9999 randomizations of the individual spatial positions and obtaining 95% confidence intervals (CIs) after bootstrapping (1000 iterations) [Analysis of spatial genetic structure (SGS) of nodulating rhizobia was conducted for each plot separately and all plots combined using SPAGeDi 1.5 . RhizobiPADS 1.0 . All setrations) , and sevtruA sequences using Mega v.6 [Isolation-by-distance for the plant hosts and nodulating rhizobia was investigated separately by comparing pairwise population genetic and geographic distances in a Mantel test . PairwisMega v.6 . GeograpMega v.6  based onMega v.6 . The ManMega v.6 , and 100Mega v.6  was alsoC. fasciculata in the rhizospheric soil.Sequences of Rhizobiales 16S rRNA that were generated from rhizosphere samples were compared against sequences in GenBank  through Chamaecrista fasciculata and at the fine scale studied here. Here we showed that there is fine-scale genetic structure in the host plant and its nodulating rhizobia, and that plant genotype along with geographic distance contribute minimally to explaining genetic distance among nodulating rhizobia. The data indicate that co-located host plant individuals are likely to be more genetically similar than those more distant probably because large seeds are maintained close to the maternal plant. The low dispersal ability of bacteria in soil and specificity between legume and rhizobia species may explain the common pattern of structure between rhizobia and their host plants. Nevertheless, the small amount of variation in nodulating rhizobia genetic distances explained by host genetic distance and geographic distance suggests that there may be other environmental factors influencing these interactions. We also found that the only rhizobia strain retrieved from sampled host plant nodules belonged to Bradyrhizobium elkanii, which was also the most common form in the rhizosphere. This finding reveals a relatively high degree of plant selection among available rhizobia in soil. To understand to what degree this strain-specific legume rhizobia symbioses can develop, further studies in particular habitats are needed.No study to date has been conducted on genetic diversity and structure of legume rhizobia system within


Bombus spp.) should be particularly vulnerable to genetic bottlenecks, since homozygosity at the sex-determining locus leads to costly diploid male production (DMP). The Tree Bumblebee (Bombus hypnorum) has rapidly colonized the UK since 2001 and has been highlighted as exemplifying the genetic paradox of invasion. Using microsatellite genotyping, combined with the first genetic estimates of DMP in UK B. hypnorum, we tested two alternative genetic hypotheses (‘bottleneck’ and ‘gene flow’ hypotheses) for B. hypnorum's colonization of the UK. We found that the UK population has not undergone a recent severe genetic bottleneck and exhibits levels of genetic diversity falling between those of widespread and range-restricted Bombus species. Diploid males occurred in 15.4% of reared colonies, leading to an estimate of 21.5 alleles at the sex-determining locus. Overall, the findings show that this population is not bottlenecked, instead suggesting that it is experiencing continued gene flow from the continental European source population with only moderate loss of genetic diversity, and does not exemplify the genetic paradox of invasion.Genetic bottlenecks can limit the success of populations colonizing new ranges. However, successful colonizations can occur despite bottlenecks, a phenomenon known as the genetic paradox of invasion. Eusocial Hymenoptera such as bumblebees ( This reduction in population size creates a sampling effect on alleles, reducing genetic diversity . Such a sl-CSD). In sl-CSD, allelic diversity at a single locus, combined with haplodiploidy, determines an individual's sex [Genetic bottlenecks are potentially even more harmful in the Hymenoptera  due to single-locus complementary sex determination  [Vespa velutina) has successfully colonized much of southern Europe following a founding event inferred to involve a single multiply-mated queen [Diploid male production (DMP) is costly since diploid males are inviable or sterile ,12 or prus spp.) –18. In aus spp.) ,20. Despus spp.) ,22, withus spp.) –27. For ed queen .Apis mellifera, represent some of the most important insect pollinators of food crops and wild plants [sl-CSD, in range-expanding eusocial bee species [Eusocial Hymenoptera perform essential services across natural and farmed ecosystems . In partd plants , a role d plants –31). Howd plants ,32–35. G species ,26.B. hypnorum) represents a highly successful range-expanding bumblebee. Having been initially recorded in southern England in 2001 [B. hypnorum extends across continental Europe and Asia [B. hypnorum arrived in the UK from the closest neighbouring area of its pre-2001 range, northern France [The Tree Bumblebee  being observed in three of 13 colonies reared from field-collected queens [B. hypnorum population has four alleles and that the founding population consisted of one or two multiply mated queens [B. hypnorum in the UK despite an apparently severe genetic bottleneck has been cited as a prime example of the genetic paradox of invasion [A previous study suggested that d queens . These dd queens . Consequinvasion .B. hypnorum [B. hypnorum population, in which queen mating frequency was estimated at 1.7 mates per queen [B. hypnorum has expanded its range westwards within continental Europe across Germany and Belgium from the middle of the twentieth century [B. hypnorum truly exemplifies the genetic paradox of invasion is uncertain. This suggestion is also consistent with the findings of a recent RAD-seq study that showed similar levels of genetic diversity and no evidence of structuring between six UK B. hypnorum populations and one in northern France [However, the previous work  did not hypnorum ,40–43, phypnorum . Recentler queen , suggest century ,45,46, sn France .B. hypnorum. Under the ‘bottleneck hypothesis', a small number of individuals founded the entire UK population in a single, chance event [B. hypnorum population will show low genetic diversity, evidence of a recent severe genetic bottleneck (i.e. at the time of colonization) and high levels of DMP. By contrast, under the ‘gene flow hypothesis’, colonization of the UK by B. hypnorum represented part of an ongoing, large-scale westward range expansion, with a larger founding population and subsequent continued immigration from continental European populations [B. hypnorum population will show high genetic diversity, no recent severe genetic bottleneck and low levels of DMP.Therefore, we defined two contrasting genetic hypotheses to characterize the mode of colonization of the UK by ce event . This hyulations . This hyB. hypnorum's colonization of the UK truly represents a genetic paradox of invasion. To this end, within a representative UK population of B. hypnorum, we pursued two aims. First, we quantified genetic diversity in workers  and mal2.(a)(i)B. hypnorum in Norwich, Norfolk, UK, were used to test for a recent reduction in effective population size (bottleneck), i.e. one occurring at the time of colonization [B. hypnorum workers were sampled non-lethally [2 area across two consecutive summers . Of these, 645 workers  were genotyped at up to 14 microsatellite loci (median = 11 loci), following methods described in detail in Crowther et al. [et al. [Workers from a population of nization . A totallethally  from a 2(ii)et al. [AR), and mean observed (Ho) and expected heterozygosity (He).All 645 worker genotypes from Crowther et al.  were useB. hypnorum study population. The first was a sign test implemented in the programme BOTTLENECK 1.2.02 [B. hypnorum study population. The M-ratio defines the ratio between allelic diversity and allele size range at a locus, with bottlenecks leading to the stochastic loss of rare alleles and a subsequent reduction of the M-ratio [Using single workers randomly selected from each of 89 independent clusters among the worker genotypic data identified with COLONY v. 2  (i)B. hypnorum, males were sampled from two sources. First, 380 male pupae were sampled from 20 mature B. hypnorum colonies collected in the field in Norfolk and Suffolk, UK, over two consecutive years . These 20 colonies each provided 7–24 randomly sampled male pupae for genotyping .To estimate levels of DMP in B. hypnorum colonies reared from 107 queens collected from field sites in Surrey, Greater London, and Norfolk (Norwich), UK, during spring 2018 . In total, 37 of the 107 field-collected queens reared at least one adult offspring , with nine colonies producing only workers, six colonies producing only males and 22 colonies producing both workers and males. Of the 28 colonies that produced males, 12 were assigned as ‘first-brood male’ producers, which were defined as colonies in which either any males eclosed within one week of first worker eclosion (ncolonies = 6) or only males and no workers eclosed (ncolonies = 6) ; the remaining 16 were assigned as ‘late male’ producers, which were defined as colonies in which all males eclosed later than one week after first worker eclosion . From the 28 male producing colonies, a total of 232 adult males were sampled for genotyping , comprising 25 first-brood males, which were defined as males that either eclosed within one week of first worker eclosion or were produced by colonies producing no workers, and 207 late males, which were defined as males that eclosed later than one week after first worker eclosion.Second, to allow for survivorship biases in field-collected nests , adult males were sampled from n = 612, i.e. 380 pupal males from field-collected nests plus 232 adult males from colonies reared from field-collected queens) were genotyped at the same microsatellite loci as were used for the 2014/2015 worker samples described above. However, because in males one locus (BTMS0132) proved monomorphic, data analysis in males was based on a median (range) of 13 (3–13) polymorphic loci.All sampled males ((ii)Diploid males were assigned as those phenotypic males that were heterozygous at two or more microsatellite loci across two independent rounds of genotyping . All males accepted as diploid  were heterozygous at a mean (range) of 5.5 (2–9) loci.The diploid male data were then used to produce three estimates of the frequency of colonies exhibiting DMP: (i) from the field-collected colonies; (ii) from the colonies reared from field-collected queens; and (iii) from the latter colonies after correcting for sampling error arising when males were sampled for genotyping .(iii)et al. [B. hypnorum population [By applying the formula of Adams et al.  and an apulation  were useAll data analyses were carried out using R, unless stated otherwise.3.(a)Across the 645 workers and 14 microsatellite loci, the median number (range) of alleles per locus was 5.0 (3–11), mean allelic richness was 5.9, and mean observed and expected heterozygosity were, respectively, 0.48 and 0.51.p = 0.49).The sign test found no evidence of a recent bottleneck, as the number of loci that had an excess of heterozygosity (7.0 of 14 loci) was not significantly different from that expected . Unexpectedly, all Bombus species exhibited M-ratios under 0.7 (B. hypnorum population relative to UK populations of other Bombus species.The M-ratio (mean ± standard error) was 0.38 ± 0.05. This fell within the range of the mean M-ratios for the five reference  species , and ovender 0.7 , indicat(b)(i)Across the 612 males and 13 microsatellite loci, the median (range) number of alleles per locus was 5.0 (3–8), and mean allelic richness was 5.3 , broadly matching the results from the worker genotypes.a).All 20 field-collected colonies produced diploid offspring  and diploid male pupae were found in one of them (5%), accounting for 4.5% of all genotyped male pupae a.Figureb,c). At the level of individual males, four of 25 (16.0%) first-brood males and 29 of 207 (14.0%) late males were found to be diploid , with diploid males accounting for 14.2% of all genotyped males.Genotyping of adult males from the 37 colonies reared from field-collected queens showed that, of the 32 colonies that produced diploid offspring , five (15.6%) produced diploid males . These five DMP colonies comprised four of 12 (33.3%) first-brood male producing colonies and one of 16 (6.3%) late male producing colonies b,c. At tOf the 26 colonies reared from field-collected queens, producing diploids and retained after correcting for sampling error, four (15.4%) produced diploid males .Overall, therefore, 15.4–15.6% of colonies reared from field-collected queens produced diploid males. Moreover, given that male diploidy was genetically confirmed in only 33.3% of first-brood male producing colonies and 16.0% of first-brood males, first-brood male production was a poor indicator of DMP, as previously it has been assumed that 100% of first-brood male producing colonies would be diploid male producing colonies and 100% of first-brood males would be diploid males.(ii)D) for field-collected colonies, colonies reared from field-collected queens and colonies reared from field-collected queens after correcting for sampling error were 1/20, 5/32 and 4/26, respectively, or 0.05, 0.156 and 0.154, respectively (as above). Combined with published data on the relative frequencies of singly, doubly and triply mated queens in the main study population [p) of 0.029, 0.094 and 0.093, respectively, and hence estimates of the number of alleles at the sex-determining locus (N) of 69.0, 21.3 and 21.5, respectively . Taking the last estimate to be the most accurate, and adding the calculated boundary values, led to an estimated number  of alleles at the sex-determining locus in the UK B. hypnorum population of 21.5 (13.0–30.8) alleles.Estimates of the proportion of colonies exhibiting DMP  to discriminate between two hypotheses for colonization of the UK by this range-expanding species. The bottleneck hypothesis  predicted low genetic diversity, a recent severe genetic bottleneck and high levels of DMP. The gene flow hypothesis  predicted high genetic diversity, no recent severe genetic bottleneck and low levels of DMP. Our results showed relatively high allele numbers at microsatellite loci and (as estimated from DMP levels) at the sex-determining locus, no evidence of a recent severe genetic bottleneck and relatively low levels of DMP. Therefore, they do not support the bottleneck hypothesis and instead support the gene flow hypothesis. Consequently, although B. hypnorum has rapidly and successfully expanded its range, it does not represent an example of the genetic paradox of invasion.We analysed genotypic data from a UK population of the Tree Bumblebee ((a)B. hypnorum population numbered as few as one or two multiply-mated queens [et al. [B. hypnorum population having been founded in a single event by as few as two multiply-mated queens.Previous evidence suggested that the founding UK d queens . Howeverd queens  would yi [et al.  and maleB. hypnorum population  were both higher than values reported in Belgian and Estonian B. hypnorum populations for which comparable data exist  [et al. [B. hypnorum populations and a French population. In addition, expected heterozygosity in the study B. hypnorum population was, on average, intermediate between values found by previous studies in common, established, and widespread European Bombus species and scarce, range-restricted, and/or declining species . Specifically, it was lower than values for 7/8 populations of common species, and higher than those for 7/8 populations of scarce declining species . Combined, these genetic diversity comparisons again support the lack of a severe bottleneck in the colonization of the UK by B. hypnorum.Expected heterozygosity and allelic richness at microsatellite loci in the study  = 1.94) , while H [et al.  found siB. hypnorum is rapidly expanding its range across the UK, and possibly recently did so across north-western Europe [B. hypnorum queens in the UK [B. hypnorum study population shows a level of expected heterozygosity lower than values found in common European Bombus species provisionally supports this idea . However, the idea requires full testing by sampling an extensive series of B. hypnorum populations across the UK and continental Europe.Given that n Europe ,45,46, on Europe . This isn Europe , where sn the UK . The facBombus species are annual insects that typically undergo one generation per year. Some, including B. hypnorum, have been suggested to exhibit facultative bivoltinism, i.e. two colony cycles (and hence two generations) per year [B. hypnorum's colonization of the UK in or shortly before 2001 and the worker sampling in the current study. Based on previous power analyses [N to 2.5 N generations after the initial bottleneck, where N equals the founding population size  immediately after a putative bottleneck [B. hypnorum, one would expect the number of immigrating queens in the founding year and in each subsequent year to have been relatively high.per year , but theanalyses , our sigttleneck . TherefoB. hypnorum greater than the levels found in five other UK Bombus species B. hypnorum, frequencies of DMP colonies were 5% for mature, field-collected colonies and 15.4–15.6% for colonies reared from field-collected queens. Given observed levels of facultative polyandry in the main study population [B. hypnorum from continental Europe found no diploid males. However, in these studies, colony sample sizes were lower, i.e. 13 colonies [p = 0.169). Therefore, at most, DMP frequency is only moderately higher in the UK than in continental European B. hypnorum populations. This is again consistent with the gene flow hypothesis, qualified by some loss of genetic diversity having occurred at the dispersal front represented by the UK B. hypnorum population.In the study population of pulation , these vcolonies  or 10 cocolonies , such thB. hypnorum population [b). If this was the case in the first-brood male producing colonies in the previous study [Previously, a frequency of DMP colonies of 23.1% (3 of 13 colonies) was reported in a UK pulation , but thius study , and 6.3us study  can be eV. velutina population was founded by a single polyandrous queen [B. terrestris population was founded by two monandrous queens [B. hypnorum population do not support the occurrence of a severe genetic bottleneck.High DMP frequencies are characteristic of populations of eusocial Hymenoptera known from other evidence to have suffered severe bottlenecks during the colonization of new ranges. For example, the French us queen , and DMPus queen . Similars queens , and DMPs queens . CorrespB. hypnorum population  was 21.5 (13.0–30.8) alleles. This estimate is consistent with the conclusion from the microsatellite data that the UK B. hypnorum population has not undergone a severe bottleneck, with even the lower bound exceeding the previous estimate of this number [B. florilegus, a species that has undergone a severe range contraction across its Japanese range [B. hypnorum population is again consistent with the lack of an extreme reduction in genetic diversity in the latter population.The estimated number of alleles at the sex-determining locus in the UK s number  by over se range . The conB. hypnorum colonies reared from field-collected queens suggests that DMP decreased colony productivity (DMP colonies (n = 5): mean nworkers = 18, mean ngynes = 0, mean nmales= 15; non-DMP colonies (n = 32): mean nworkers = 34, mean ngynes = 6, mean nmales= 43). This suggestion is in line with previous findings in ants and bees [Combining the genetic and productivity data in the B. hypnorum population showed relatively high genetic diversity, no evidence of a recent severe genetic bottleneck and low levels of DMP, matching predictions from the gene flow hypothesis. Hence, colonization of the UK by B. hypnorum does not represent an example of the genetic paradox of invasion [B. hypnorum undergoing a recent westward range expansion within Europe [Argiope bruennichi) [Coenagrion scitulum) [. In such cases, the central genetic phenomena are progressive loss of genetic diversity across an invasion front and its consequent impact on adaptability [B. hypnorum's distinctive pollinating role in the UK [In conclusion, genetic data from the UK invasion ,38 or aninvasion ,20. Alonn Europe ,45,46, oennichi)  and the citulum) . In suchtability ,62, alonn the UK  remains