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Scientific Reports Volume 11, Article Number: 21803 (2021) Cite this article

Understanding today’s genetic diversity, population structure, and tree species evolutionary history can provide information for resource management and conservation activities, including response to the pressures of climate change. Cercis canadensis (Eastern Bauhinia) is an economically valuable understory tree species native to the United States (USA), and it is also important for forest ecosystems and wildlife health. Here, we record and explain the population genetics and evolutionary history of this deciduous tree species in its distribution range. In this study, we used 12 microsatellite markers to survey 691 wild-type trees sampled at 74 collection points in 23 states in the eastern United States. The high genetic diversity and limited gene flow are revealed in the wild natural forest stands of C. canadensis, and its population is explained by two main genetic clusters. These findings indicate that the ancient population bottleneck occurred at the same time as the last glacial peak (LGM) in North America. The structure of the current population may have originated from an ancient population in the eastern United States, which survived the LGM and then split into two contemporary clusters. The data shows that since the last glacial event, the population has expanded from one to several refuges behind the glacier that now occupy the current geographic range of the species. Our in-depth understanding of the genetic variation preserved in this species can guide future efforts to protect and utilize the most adaptable populations with the greatest genetic and structural diversity.

The genetic structure and demographics of many North American plant species are greatly affected by climate fluctuations that occurred during the Pleistocene period1,2. During the last glacial peak (LGM) 3, 4, and 5 that occurred approximately 18,000-21,000 years ago, the Laurentide Ice Sheet extended from the northernmost tip of North America to 39°N3. These events reduced the distribution of many temperate tree species and forced them to enter glacial refuges, including unglaciated southern regions and suitable microenvironments that exist in northern glacial regions1,6. In the eastern United States (US), many refuges that have historically been adjacent or closely occurring have been identified, but the location and number of refuges are still under debate6,7. These refuges are poorly represented in the fossil record, but the spatial genetic structure and evolutionary history of many species have been used as evidence of historical refuges6,8,9,10.

According to the hypothesis of "range shift after the last glacial maximum", many temperate species re-settled and spread to their current distribution areas after LGM11,12. The results of this recolonization process can be inferred from the genetic structure of the existing plant populations, manifested as a decrease in genetic diversity along the colonization route, as well as different spatial genetic clusters within the newly expanded species range2,13,14. In European temperate plant species, patterns of reduced genetic diversity within a certain range are common15. This trend is not so obvious in North American tree species, such as Carya cordiformis [Wagenh.] K. Koch (Bitterwood Pecan) and C. ovata [Mill.] K. Koch (shagbark hickory), which are distributed in their 15 species. The genetic homogeneity of North American plant species can be explained by the slow expansion into new areas after the glacier, the emergence of many refuges nearby, and the high gene flow over time15,16. However, phylogenetic studies provide evidence that the range dynamics of post-glacial species populations contribute more to the genetic diversity of current temperate tree species than any other ecological force (for example, Central Margin Theory17, especially for the northern part). In terms of population) the edge of species distribution 11.

The distribution of tree species, their genetic diversity and population structure are affected by many factors, including climate fluctuations, population events, ecological and environmental variables, and their unique biology11, 18, and 19 However, the role of glaciers in the distribution, range, genetic variation, and spatial genetic structure of outcrossing species across a wide range of geographic areas is unclear, especially in the eastern United States. Based on the current species distribution and population structure of temperate tree species in the eastern United States, we assessed the spatial population structure of the widely distributed understory Cercis canadensis L. (C. canadensis var. canadensis L.; Fabaceae; Eastern redbud). Cercis canadensis is a good system for studying the role of LGM in tree species in the eastern United States because of its wide and continuous geographic distribution in the eastern United States without any major geographic barriers.

Cercis canadensis is a self-incompatible deciduous tree 20 native to the Midwest and East of the United States and northeastern Mexico 21,22. This species grows well in partial shade and can adapt well to various climatic conditions and altitudes. It can be found in the cold hardiness zone of the United States Department of Agriculture 4 to 923,24. This relatively small ornamental tree is characterized by its wide, colorful, umbrella-shaped canopy 25, and has become a popular landscape tree 23 for its heart-shaped leaves, compact shape, and early spring flowers.

When microsatellite sites were used to examine the fine, small, and scattered populations of C. canadensis, wild trees maintained high genetic diversity, gene flow, and moderate to high genetic differentiation27. Although C. canadensis is ecologically important, the understanding of contemporary genetic diversity, temporal and spatial genetic structure, gene flow, and past evolutionary history of this species in the continental United States is limited. In order to solve this knowledge gap, we used the microsatellite loci to complete the following tasks: (1) Describe the genetic diversity of the wild populations of C. canadensis in the United States; (2) Infer the pattern of the spatial genetic structure of C. canadensis; ( 3) Reveal the evolutionary demographic data in its native range. We hypothesize that the wild population of C. canadensis will have genetic diversity, and the genotype will be spatially clustered within its native range. We also hypothesize that the genetic structure will be consistent with the expansion that occurred in one of several southern glacier refuges. More specifically, we aim to explore the following questions: (1) Do genetic diversity and population structure patterns reflect the evidence from the Northern Glacier Reserve? We expect to detect high genetic diversity with a clear population structure in the northern range, otherwise the trend of genetic diversity from high to low from south to north indicates that the Canadian C. canadensis relocates from one or more southern glacier refuges to the north. (2) Is there any evidence of miniature refuges within the current distribution of C. canadensis species? If there are multiple mini-refuges nearby, especially in the southern region, we hope to be able to detect the genetic homogeneity of the studied population.

C. canadensis leaf samples were collected by authors, collaborators, and citizen scientists (see Acknowledgements), who collected specimens from the native range of the species in 23 states in the Midwest and Eastern United States (Table 1). The use of trees in this study complies with international, national and/or institutional guidelines. Identify the plants according to the collection guidelines provided to our collectors and confirmed by the co-authors (certified specimens stored in the Vascular Herbarium of the University of Tennessee, catalog number TENN-V-0246136). For each collection point, at least 10 uncultivated C. canadensis trees within a radius of one mile were selected and their geographic coordinates were recorded. Collect 5 to 7 disease-free young leaves from each tree, sandwich them between sheets of absorbent paper, and store them in paper envelopes at ambient room temperature until processing. Leaf samples from 1,193 individual trees were collected at 117 collection points. In order to avoid over-representation of trees in a geographic area, we randomly selected a subset of collection locations from a geographic area with more than one sampling location. A total of 790 trees were used in this study, representing 79 collection sites, which span across Canada. C.

From each tree, 60 to 100 mg of dry leaf tissue is used to isolate DNA. The samples were homogenized four times with a Beadmill 24 homogenizer (Fisher Scientific, Pittsburgh, Pennsylvania, USA) for 30 seconds each, 30 seconds each, and kept in liquid nitrogen for 5 minutes between each crushing step. Qiagen DNeasy Plant Mini Kit (Qiagen, Valencia, California, US) is used to isolate genomic DNA (gDNA) from crushed samples and make the following minor modifications to the protocol provided by the manufacturer. Specifically, 2% w/v polyvinylpyrrolidone (PVP) was mixed into the lysis buffer (AP1). Then add 8 μl RNase to each sample tube and incubate in a 65°C water bath for 45 minutes. Gently invert each sample tube every two minutes to thoroughly mix the sample. Finally, the sample is incubated at -20 °C for at least one hour. If there are any visible residual debris and elution buffer, wash the spin column with ethanol. Heat the elution buffer to 65 °C, then add 50 µl to the spin column twice. The concentration of gDNA was quantified using an ND1000 UV-Vis spectrophotometer (NanoDrop Technologies, Wilmington, Delaware, US), and the gDNA was stored at -20 °C until further use.

Initially, gDNA was isolated from five Canadian C. canadensis individuals in the gardens of the University of Tennessee (Knoxville, Tennessee, USA) and used to evaluate 68 candidate microsatellite loci. Based on the successful amplification and the consistency of PCR product size with published data, primers for 12 polymorphic microsatellite loci (Table 2) were selected for this study. The polymerase chain reaction (PCR) was used to amplify microsatellite sites in a 10 µl reaction mixture, which contained the following: 1 µl gDNA, 1 µl 10 µM forward and reverse primers, 0.5 µl dimethyl sulfoxide, 4 µl GoTaq G2 Hot Start Master Mix (Promega Corp, Madison, Wisconsin US) and 2.5 µl sterile molecular grade water. To ensure the validity of the data, a negative control (a reaction mixture with water instead of any DNA sample) and a positive control (a DNA sample from the initial screening, amplified on all microsatellite primers) were added to each primer pair tested ). Use Eppendorf Thermocycler (Eppendorf AG, Hamburg, Germany) to complete the DNA amplification with 12 microsatellite sites in all samples in a 96-well plate. The thermal curve is as follows: initial denaturation at 94°C for 3 minutes, then 35 One denaturation cycle was at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 30 seconds, and finally at 72°C for 4 minutes.

Use QIAxcel capillary electrophoresis system (Qiagen) to observe the amplified PCR products, and use 15/600 bp internal alignment markers and 25 to 500 bp DNA ladders for analysis. The above procedure was used to amplify and visualize all C. canadensis gDNA samples for each of the 12 microsatellite loci. Reactions that did not produce any amplification products were rerun before being considered as missing data. Discard samples with ≥ 40% missing data. In addition, collection sites with more than four samples with ≥ 40% missing data were excluded from the data set.

Using the Excel macro FLEXBIN version 28, the original allele size is converted to allele classes. In this program, alleles are classified into base pair (bp) size categories by statistical similarity. This binning genetic data set is used for all the following statistical analyses, which were done using R version 3.5.329. The data was cloned and corrected using R package POPPR version 2.8.230,31 to identify the existence of cloned individuals at the collection site level. For each collection site, only the multilocus genotype (MLG) was used to obtain an unbiased estimate of the allele frequency from the data set 32.

The R package POPPR is used to calculate the total number of alleles at each locus, the observed heterozygosity (H2O; the number of heterozygotes present at the locus divided by the sample size), and the expected heterozygosity (HE; calculated as each locus Expected heterozygosity 33), and linkage disequilibrium (rbard); non-random association of alleles between loci). In addition, the Shannon-Weiner diversity index (H) was calculated for each collection site using POPPR. H Consider the allele richness and uniformity of allele distribution. In the POPPR package, the number of unique private alleles in the collection site and different loci is estimated. Allele richness (Ar) is a measure of the rare allele count of each locus, estimated using the package HIERFSTAT version 0.04-2235. Allelic richness is used as an estimate of the long-term evolutionary potential for adaptation and persistence in a given population36,37. Use the HIERFSTAT package to calculate genetic fixation index (FST), inbreeding coefficient (FIS) and allelic differentiation (F'ST)38,39. GenAlEx 6.5 software (Peakall & Smouse, 2006; Peakall & Smouse, 2012) was used to estimate gene flow (Nm). In the program, Nm is estimated as the effective migration number for each site based on F statistics.

The STRUCTURE version 2.3.440 program with the mixed model was used to analyze the population structure in the native range of wild C. canadensis trees. This Bayesian clustering method using Monte Carlo Markov Chain (MCMC) method is used with the following parameters: 500,000 aging cycles and 1,000,000 MCMC repetitions for 30 K values ​​between 1 and 18 Independent chain. The result output is visualized through STRUCTURE HARVESTER web version 6.9441. Use the Evanno method to calculate the optimal K value, which is the index of population clusters existing in the data set. The estimate of the ΔK standard obtained from STRUCTURE HARVESTER was visualized using POPHELPER 2.2.643 which merged 30 independent chains. R package MAPS version 3.3.044 and PLOTRIX version 3.8-145 are used to generate a pie chart of the mixing ratio when K = 2.

Several model-free methods were used to study the population structure of C. canadensis samples. Use Nei genetic distance in POPPR46,47 to construct Neighbor Connection (NJ) dendrogram. Principal component discriminant analysis (DAPC) was implemented using ADEGENET version 2.1.148 to visualize the potential genetic structure of the species in its wide geographic range. This is a two-step multivariate analysis used to investigate genetic variation within populations between sampling collection sites. First, perform principal component analysis (PCA), and then select the number of PCA vectors (to minimize DAPC overfitting to account for most of the variance). Then, use a selected number of PCAs to reveal differences between groups while minimizing intra-group variation, and use discriminant analysis to sort collection sites into different groups49,50. In addition, the missing value is calculated as the average allele frequency, and cross-validation analysis is performed to select the appropriate number of PCs.

Distance isolation (IBD) is estimated using Mantel test 51,52, using Euclidean distance, with 10,000 permutations in the VEGAN version 2.5-653 package. IBD examines the correlation between genetic distance and geographic distance between individuals in a data set. The Mantel test was implemented at 74 collection points while treating the entire data set as a group.

POPPR with 10,000 permutations was used to perform analysis of molecular variance (AMOVA)54 by classifying individuals into stratified groups to assess the degree of molecular variance within, between, and between collection points. The population level includes: (1) 74 collection points as a level group; (2) two groups based on STRUCTURE analysis; (3) divided by hot continent (mountain province), hot continent, warm continent, subtropical, grassland The five major ecological regions are divided into four major groups (see supporting data Figure S2) in the Midwest and East of the United States (Bailey, 1994). C. canadensis warm land sub-collection points and hot land sub-collection points are grouped together. Since C. canadensis is distributed in a wide range of climates and altitudes, we tested the influence of regional climate models on the population structure of C. canadensis in the AMOVA analysis.

In order to investigate and explain the evolutionary history of C. canadensis, we used the DIYABC program version 2.155,56, which utilizes the approximate Bayesian calculation (ABC) statistical method. For this analysis, based on the STRUCTURE results, the collected individuals were combined into two main groups. In order to clarify the evolutionary history of C. canadensis, we analyzed the competition scenarios in the two ABC steps. In the first step, we tested five demographic scenarios using 200,000 simulated pseudo-observation data sets (POD), among which: (1) the first two scenarios show that the current two main groups are gradually diverging from the ancient population, (2) The third scenario shows a single, two-way split between the contemporary group and the ancient unsampled group, and (3) the last two scenarios are based on the divergence assumptions between the current group and two independent ancient unsampled groups. Once the analysis of these scenarios is completed, the two scenarios that produced higher logistic regression support in the first step are selected as the basis for the second step of evaluating ABC. In the second step, seven scenarios were constructed to solve the possibility of bottlenecks in the evolutionary history of species. More than 1,000,000 pseudo-observation data sets (POD) are simulated under the hypothetical prior parameter range of each scene. Estimate the posterior probability of the comparison schemes to select the most supported scheme55.

Twelve microsatellite sites were amplified from 790 Canadian trees sampled in this study. Due to missing data (missing ≥ 40% SSR), 5 out of 79 collection points were excluded, and 49 individuals from the remaining 74 collection points were discarded, resulting in 691 individuals out of 74 collection points. In addition, after deleting two cloned individuals, 689 unique multilocus genotypes from 74 collection points were retained for further data analysis (Table 1). The overall average null allele or missing data in the entire data set was 2.89% (see Supporting Information Figure S1). Nei has a genetic diversity index (HE) value of 0.67 in the 74 collection points studied, ranging from 0.32 (Anderson County, TN10) to 0.68 (Sabi County, NE1) (Table 1). In addition, a weak (rbard = 0.05, P value = 0.01) but significant linkage disequilibrium value was detected in the data set. 19 private alleles were detected in 74 collection points (Table 1). In addition, 9 of the 12 microsatellite loci produced private alleles, and the largest number of private alleles were recovered from locus 168a (Pa = 4, Table 2). The number of alleles per locus ranges from 6 to 13, with an average of 10 alleles per locus (Table 2). The overall allele richness (Ar) ranges from 1.23 at locus 995a to 1.79 at locus 780b, with an average of 1.55, which means that there is a high allele richness in wild C. canadensis individuals. The observed heterozygosity (H2O) of all loci was 0.32, ranging from 0.01 (locus 995a and 680a) to 0.99 (locus 127spa). The overall expected heterozygosity (HE) for all 12 microsatellite loci is high (HE = 0.69), ranging from 0.52 (locus 995a) to 0.86 (locus 780b).

The overall Shannon-Weiner diversity index (H) for the 12 loci is 1.42, ranging from 0.84 (locus 995a) to 2.19 (locus 780b; Table 2). In addition, high population fixation (FST = 0.19; range from 0.05 to 0.53; Table 2) and population differentiation (F'ST = 0.19; range from 0.05 to 0.54) were identified in the C. canadensis population. We estimate the inbreeding coefficient (FIS) for all loci to be 0.43, indicating that there are too many homozygotes in the Canadian C. canadensis population studied (Table 2). The average estimated gene flow is 0.75, which indicates that a limited amount of gene flow has occurred in the study population (Table 2).

Using Nei's genetic distance, we estimated the paired FST values ​​between 74 collection points, which ranged from 0.02 to 0.33 (see Supporting Information Table S1). The structural results showed that the best ΔK = 2, which means that in its wide native range, the Canadian C. canadensis collection sites are divided into two main clusters. The collection points in the northernmost collection area of ​​the United States (Ohio to Nebraska) and south-central to north-central (from Texas to Nebraska) are the first clusters (designated as the northern genetic cluster) Part (Figure 1). The remaining collection points along the Atlantic coastline from the northeast (New York) to south-central (Mississippi) belong to the second cluster (designated as the southern genetic cluster). The constructed NJ dendrogram shows that there are two main populations (except KS and TX collection sites, which are not grouped with any main population), which supports the discovery of STRUCTURE with two genetic clusters (Figure 2). In addition, the distribution of collection points in the two main groups (NJ dendrogram) is similar to the distribution of collection points in the two STRUCTURE-based clusters. The DAPC double plot further confirmed the existence of genetic structure, mainly two overlapping clusters along the x-axis (Figure 3). Therefore, based on other analyses used in this study, the grouping of individuals of C. canadensis in Canada is best explained by two genetic clusters (Figure 1-3). These analyses also show that most collection sites (except for two from Georgia) are grouped according to their geographic origin.

(A, B, C). The structural bar chart represents the genetic clusters (ΔK = 2-3) between 74 collection points of Canadian Bauhinia (A and B). Each vertical bar represents an individual sample, and the color of the bar represents the distribution probability that the individual belongs to one of the identified clusters. The mixing coefficient pie chart inferred by STRUCTURE (ΔK = 2), drawn in the eastern United States (C) and Canada C. in the 74 collection locations used in this study.

An adjacency tree of 74 Canadian Bauhinia collection points based on Nei genetic distance. The numbers indicate the percentage of bootstrapping support using 1,000 replications (threshold is set to 70%).

Principal component discriminant analysis (DAPC) of Cercis canadensis individuals in 23 states in the United States. The first 47 principal components explained 94% of the individual variation of C. canadensis in the data set. Here, allele 154 at locus 199a explains 12% of the variance on the first axis, and allele 102 at locus 220a explains 13% of the variance on the first axis (threshold = 0.06). The data is constructed using 1,000 permutations. Discriminant analysis feature values ​​are also provided.

In the analysis of molecular variation (AMOVA), the first data arrangement showed that most of the genetic variation existed in 74 collection points (74.2%, P <0.001) (Table 3). There were also significant differences between collection locations (25.8%, P <0.001). When the data set was divided into two genetic clusters based on the STRUCTURE results, 69.2% (P <0.001) of genetic variation was attributed to the location of the collection site (Table 3). There were also significant differences between two different genetic clusters (13.9%, P <0.001) and collection points within the cluster (17%, P <0.001) (Table 3). When the data set is divided according to the major ecological zone groups distributed by C. in Canada, only 7.9% (P <0.001) of the variability can be attributed to between ecological zone groups, and 18.6% (P <0.001)) can be attributed to Variability between collection points within the group (Table 3). Most genetic variation is explained between individuals within the collection site, rather than between population or group levels in all three test scenarios (Table 3). Nevertheless, the degree of variation observed within the collection point and between clusters reveals the existence of genetic structure. Therefore, the AMOVA results are consistent with the stratified fixed index and indicate the existence of a demographic structure. However, when the data is divided by major ecological regions, the amount of variation within the group is the lowest. This finding suggests that the division of trees within the ecoregion cannot be expected to explain the genetic differentiation and population structure observed in the wild population of C. canadensis in Canada. The results of distance isolation analysis show that in the Canadian C. canadensis population, the geographic distance effect is weak, but it is linearly related to genetic distance (r = 0.08, P <0.001) (see Supporting Information Figure S2).

However, the DIYABC program using the ABC method supports the existence of population structure and found evidence of an ancient bottleneck event that occurred in the wild population of C. canadensis in Canada. Starting from the first step of the analysis, two possible scenarios were selected based on the posterior relative support (Scenario 2, posterior probability (P) = 0.39 and scenario 3, posterior probability (P) = 0.37; Figure 4A). In these analyses, scenario 2 provides evidence that the contemporary Canadian C. canadensis population originated from an ancient population in the southeastern United States, and later the northern population (the first genetic cluster) and the southern population (the second genetic cluster) separated . Alternatively, scenario 3 indicates that the current two C. canadensis groups (northern and south) have been separated from an ancient, unsampled group (Figure 4A). In the second step of ABC analysis (Figure 4B), principal component analysis and relative posterior probability test show that scenario 2a (posterior probability (P) = 0.74, Figure 4B) is the most supported and therefore has the greatest possibility Accurately described the obvious evolutionary process in the native stand of C. canadensis. Therefore, we infer from scenario 2a, from an ancient population, a group of Canadian C. in the south).

(A, B). The possible DIYABC evolution scenario of the evolutionary history of Bauhinia in Canada. Here, the current C. canadensis populations are distributed in the north (N1) and south (N2). In addition, N1b and N2b represent the populations of N1 and N2 before the bottleneck event. On the right side of each scene, the time scale represents the timeline of each event (t = 0 is the current time, t1-db = the occurrence of a bottleneck, t1/t2 = the split between the population and the original population). We analyzed our data using two ABC steps, resulting in five competitive scenarios in the first step (4A), and seven scenarios where bottleneck events may occur in the second step of analysis (4B). Scenario 2a (B) from step 2 received the most support in the DIYABC analysis, and the timeline of the bottleneck (t1-db) and divergence (t1) events of scenario 2a is given in units of generations. For each case, the value of the relative posterior probability (P) is reported.

The estimated posterior parameters of Scenario 2a indicate that the population bottleneck occurred about 4,950 generations ago (722-9,650 generations in the simulation data set), about 25,000 years ago, considering that the average time for the Canadian C. canadensis tree to reach reproductive maturity is six to Seven years 57 (Figure 4B). Therefore, the bottleneck event is most likely to occur during the last ice age, which ended approximately 21,000 years ago3,5. Later, the northern population diverged from the southern population (102 to 1,490 generations in the simulated data set) before about 493 generations (Figure 4B). The post-mortem analysis provides goodness of fit for this situation. The original data set is well embedded in the previous POD population and nested in the rear POD population (for details on this analysis, please refer to the support information table) S2).

The Canadian C. wild population sampled in the native range of the United States reveals a high level of genetic diversity and population differentiation, the existence of population structure, limited gene flow, and the ancient bottleneck that coincides with the last ice age in the northern United States in time. We detected the presence of geographic clusters longitudinally in the southern region (along the US coastal plain) and the northern region. Evolutionary history analysis revealed an ancient bottleneck event that occurred in the southern C. canadensis population, and then the northern population and the southern population of C. canadensis diverged.

When comparing populations in the ecoregions in which they were collected, the ecoregion name has nothing to do with the population structure and genetic diversity of the wild population of C. canadensis in Canada. The low genetic variation of C. canadensis across ecological regions is not surprising, because the tree species can adapt well to various soil types, environmental conditions and habitats without any major geographic barriers, and in the eastern United States, however, we found Evidence of relatively high genetic diversity among the northernmost collection points (IA, IN, MI, NE, and OH, and the mid-latitudes of North America), which are located on the periphery of the contemporary northern range of the region. Species. A reasonable explanation for this difference between the northernmost samples compared to the southern collection site is the possibility of a major refuge or mixture among small and medium-sized but genetically rich populations in the refuge contact area 58,59,60 . This effect is most obvious in species that reproduce through long-distance gene flow and local adaptation between sensitive individuals at the margins of the distributed population. However, unlike European temperate species, eastern North American species maintain a high degree of genetic diversity in northern populations61,62. This high level of genetic diversity in the northern population of C. canadensis can be maintained through long-distance seed dissemination events during the expansion of the northern refuge from behind the glacier62,63.

The ability of C. canadensis to maintain high genetic diversity may be affected by many factors, including wide and continuous geographic distribution, outcrossing reproductive systems, and large effective population sizes59,64,65,66. Many other temperate tree species can maintain a high degree of genetic diversity over a wide geographic range even in the presence of environmental pressures 11, 14, 58, pressure from insects and plant pathogens 67, 68, and human interference 67, 69, 70 Sex and allele richness. A study using microsatellite sites revealed high genetic variation among five Asian Bauhinia species, with an average of 5.7 alleles per site71. A recent study of a small and dispersed population of C. canadensis also determined that the trees of this species maintain high genetic diversity and allelic richness throughout their native range27, which are comparable to Asian Bauhinia species and several other species. Hardwood species are consistent 66, 72, 73, 74, 75, 76.

In addition to high genetic variability, the C. canadensis population also shows extensive morphological variability under different environmental conditions20,26,77,78,79. For example, it was found that the shape, size, surface pilose and other structural characteristics of Bauhinia leaves are closely related to environmental factors such as temperature and moisture content79,80,81. In Bauhinia, these characteristics may be derived from local adaptation to different climate pressures26,82,83. The morphological variation of C. canadensis led to efforts to distinguish the species into the following three varieties: C. canadensis var. canadensis L., distributed in the mesophyte habitat of the eastern United States, and C. canadensis var. mexicana (Rose) M. Hopkins (Mexican redbud) and C. canadensis var. texensis (S. Watson) M. Hopkins (Texas redbud), commonly found in the semi-arid areas of central Mexico and southwestern Texas 26,84,85,86. However, due to the highly continuous morphological variation pattern of the Canadian C. canadensis population within its range, the validity of this subspecific classification has been questioned. In addition, current phylogenetic studies cannot provide sufficient support to verify these divisions84,87. Because C. canadensis var. Mexico and C. canadensis var. texensis is not represented in our research, and our data will not help solve this problem.

Widely distributed large populations of tree species usually have low genetic differentiation, and the population structure within their geographic range is limited15, 60, 65, 66, 68, 88. The populations of Viburnum rufidulum Raf.89 and Cornus florida L.68 are temperate tree species, widely distributed in the southeastern United States, with low genetic differentiation and weak population structure. In the populations of V. rufidulum and C. florida, high levels of gene flow through pollen and seed transmission may reduce genetic variability75,89. In contrast to these studies, the high degree of genetic differentiation observed in the widely distributed C. canadensis population may be due to the limited gene flow and the population history of this species.

Similar to many other self-incompatible forest tree species20, the gene flow of C. canadensis depends on various pollen and seed dispersal mechanisms. The flying distance of insect pollinators varies from one mile to several miles 90,91, which will limit the long-distance gene flow through the spread of pollen between trees. The seed pods and seeds of C. canadensis are relatively heavy and usually fall near the parent tree. In the next few years, the surviving offspring will grow as non-reproductive seedlings 57,92 and produce half-sibling "neighborhoods" 89,93,94,95,96 on local spatial scales. Several mammals that feed on seeds, such as Eastern Wood Rats (Neotoma floridana Ord) 97 and birds including Quail 98, contribute to the dissemination of C. canadensis seeds to a certain extent. Small rodents and deer may eat repeatedly from the same tree, and therefore carry closely related half-sib propagules (if eaten when seeds are mature), the distance is limited to the retention time of feces 57,97,99,100,101. In order to fully understand gene flow patterns and predict changes in the distribution pattern of C. canadensis in Canada, it may be helpful to understand the seed and pollen dispersal methods of animals related to this species and the efficacy of seed transportation. However, seed transport efficiency may be limited to the relatively short distance these animals move during foraging. The consumption rate of fruits by animals is also dependent on Canadian fruits as emergency food in late autumn or winter. This behavior will reduce the efficiency of functional seed dissemination 57,97,99,100,101. These events may restrict gene flow to short distances, create spatial genetic structures, and increase the possibility of inbreeding at the local level, as revealed by the fine-scale level assessment of Canadian C. 27,69,102. We also collected samples of C. canadensis from New York (United States), which represent individuals that occurred farther north than the reported geographic range of the species. These individuals may also be caused by the escape of open pollination after the introduction of C. canadensis into the management landscape.

The structural analysis of the C. canadensis data set revealed that there are two geographically distinct clusters, designated as the northern cluster and the southern cluster, which are separated in the longitudinal northwest and southeast directions along the Kentucky-Tennessee-Mississippi transition zone. From this evidence, there is no barrier in the southern Appalachian Mountains, because populations belonging to the northern structural group are found on both sides of the Appalachian Mountains. The existence of only two genetic clusters is consistent with the simple postglacial lineage theory proposed for eastern North American tree species59,103. The most recent glacial event ended approximately 21,000 years ago. The conclusion reached is that northern and temperate tree species have moved to the mid-latitudes of the eastern United States, where many species have survived bottleneck refuge populations104. According to the scenario supported by our DIYABC, the refuge in the southeast may also be the main post-ice refuge for C. in Canada. This situation is further supported by several phylogenetic studies, which indicate that the population of the southeastern United States is an important large postglacial refuge for many temperate species 14,103,104. Modern temperate species, including Fagus grandifolia Ehrh. (American beech), Acer rubrum L. (red maple) and C. florida (flowering dogwood) probably originated from this southeast refuge 4,14,105. Cercis canadensis also has the same geographic distribution as these temperate tree species, and modern wild populations of C. canadensis are ubiquitous in the area.

Our analysis also reveals support for several possible mini-refuges in the eastern United States, which is evident in genetic differences between populations and substructures that lack different centers. Several studies of different tree species have shown that refuges exist in the eastern United States, such as the southern Appalachian Mountains, the Southeast Coastal Plain, and the Lower Mississippi Valley (McLachlan et al., 2005; Potter et al., 2011). The postglacial C. canadensis population from this geographic range may have spread north to establish the current species distribution. The post-glacial populations of other tree species in this range adapted to semi-arid to arid environments9,14,103 and showed the same obvious adaptability characteristics in the midwestern C. canadensis population. In addition, the high genetic diversity and allelic richness of the modern Canadian C. canadensis population can also support the existence of many refuges or fragmented refuges.

Phylogenetic studies of other tree species and animals indicate that they have survived as hidden mini-refuges in the north 104,106,107,108. Due to insufficient Late Pleistocene fossil data in the northern region, it is difficult to conclude that the most supported DIYABC evolutionary scenario of Canadian C. shows that the Canadian C. canadensis population continued to exist in the southern population during the last ice age. Therefore, we also found very little evidence that this species may have a hidden refuge in the north. In contrast, pre-glacial Canadian C. canadensis was distributed in populations in the Midwest and Southeastern United States, and later survived one or more post-glacial refuges in the Midwest and Southeastern United States. Due to the long-term population isolation in the refuge zone, the refuge population behind the glacier in the Midwest of the United States may have diverged from the large refuge population in the southeast, resulting in a genetically differentiated northern spatial cluster 70,95. In addition, the northern population after this glacier may later migrate from the Midwestern United States to its current distribution.

The ancestors of the North American Bauhinia species are believed to have originated in moderate climatic conditions and may have spread to North America through the North Atlantic land bridge 81,84,109. According to multiple studies, the ancestral Bauhinia population adapted to a drier environment, and then spread to the northern hemisphere in the middle of the Miocene 84,87. Then, as suggested by DIYABC Scene 2a, this ancestral, unsampled, Miocene Bauhinia population may have produced the southern C. canadensis population.

This economically and ecologically important deciduous tree species has many ideal morphological changes and ornamental characteristics, including leaf color and texture, flower color changes, drought tolerance, pathogen resistance, and various architectural forms 20, 26, 57. The fruits and seeds of C. canadensis are eaten by several birds and small mammals57,97,99,101, and many pollinators rely on this tree as an early food source110. In the United States, there are more than three dozen cultivars available for commercial use, and the sales of seedlings of this species contribute more than $27 million 71,111 in the United States each year. Combined with the recently introduced new horticultural varieties with highly desirable characteristics, the value of the adaptability characteristics that may exist in different geographic regions supports the importance of protecting the local diversity of Canadian C. These populations are genetic pools of potential variability that can provide breeding programs with the resources needed to improve selected traits (for example, limit the productivity of seed pods in landscape specimens), and provide additional resources for the development of high-value cultivars for commercial trade chance. Future work should also focus on identifying important adaptive characteristics in wild populations that can be used to help ensure Canadian C.

After the manuscript is accepted, the data will be made public and stored in the tree demon depository.

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This work was partly supported by the United States Department of Agriculture (USDA; Grant 58-6062-6), the National Institute of Food and Agriculture (NIFA; Hatch Project 1009630: TEN00495) and the University of Tennessee Open Publication Support Fund. We are very grateful to more than 52 individual contributors, friends and family members for their enthusiastic sampling assistance. They helped us collect leaf tissue samples and coordinate collection of wild redbud trees from all over the United States. Without your every support, this work would not happen. In addition, we sincerely thank Adrienne Gorny (Cornell University), David Held (Auburn University), Caterina Villari and Megan Buland (University of Georgia), Chris Wyman (University of Tennessee), Christine Nalepa and John Banask (University of North Carolina) Agriculture And Consumer Services), Cory Tanner (Clemson University), Donn Johnson and Lizabeth Herrera (University of Arkansas), Erfan Vafaie (Texas A&M University), Eric Day (Virginia Tech), Eric Rebek (Okla Homer State University), Erin Pfar (Rutgers University), Frank Hale (University of Tennessee), Gary Bachman (Mississippi State University), Grace Pietsch (University of Tennessee), Jackie Lee (University of Arkansas), Jason Griffin (Kansas State University) ), Juang-Horng Chong (Clemson University), John Olive (Auburn University), Katherine Kilbourne (Tennessee Department of Agriculture), Matt Ginzel and Geoffrey Williams (Purdue University), Michelle Clayson (Michigan, USA) , Natalie Diesel and Robbie Doerhoff (from Missouri), Nathan Schiff (United States Department of Agriculture, Mississippi), Philip Marshall (Wallonia Nursery, Indiana), Raymond Moore (Tennessee Valley Management Bureau, Alabama), Rob Pival (University of Tennessee), Ron Winston (Florida), Sandra Wilson (University of Florida), Sarah White (Clemson University), Scott Goldman (Tennessee) Department of Agriculture), Scott Ludwig (Amvac Chemical Corp., Texas), Shimat Joseph (University of Georgia), Stephen Clarke, Mr. Horne, Ms. Standard and Mr. Gras (US Department of Agriculture and Forestry Service, Texas), Steve Meyers (Mississippi State University), Sydney Everhart and Eldon Everhart (University of Nebraska-Lincoln), and Will Hudson (University of Georgia).

Department of Entomology and Plant Pathology, University of Tennessee, Knoxville, Tennessee, USA

Meher Ony, Robert N. Trigiano, Marcin Nowicki, Sarah L. Boggess, and Denita Hadziabdic

Department of Plant Science, University of Tennessee, Knoxville, Tennessee, USA

Department of Forest Resources, University of Minnesota, St. Paul, Minnesota, USA

Department of Entomology, Purdue University, West Lafayette, Indiana, USA

Department of Plant Pathology, University of Nebraska, Lincoln, Nebraska, USA

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DH, WK and RT conceived and designed experiments, including main concepts and proof outlines. All authors assisted in sample collection and preparation. MO conducted experiments and SB contributed to these processes. MO, MN, SB and DH troubleshoot the technical details of the experiment. MO, MN, JZ and DH contributed to data analysis. SE provides technical advice on data preparation and analysis. All authors contributed to the interpretation of the results, manuscript writing and editing. All authors provided important feedback to shape the experiment, analyze, and finally produce the manuscript.

The author declares no competing interests.

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Ony, M., Klingeman, WE, Zobel, J. etc. The genetic diversity of Bauhinia in North America reveals an ancient population bottleneck that originated after the last glacier peak. Scientific Report 11, 21803 (2021). https://doi.org/10.1038/s41598-021-01020-z

DOI: https://doi.org/10.1038/s41598-021-01020-z

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