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Genetic drift, historic migration, and limited gene flow contributing to the subpopulation divergence in wild sea beet (Beta vulgaris ssp. maritima (L.) Arcang)
Evolution of sea beet
Tehseen Muhammad Massub Formal analysis Methodology Writing – original draft 1
Wyatt Nathan A. Formal analysis Writing – review & editing 2
https://orcid.org/0000-0003-1197-2078
Bolton Melvin D. Project administration Resources Supervision Writing – review & editing 2
Fugate Karen K. Formal analysis Methodology Writing – review & editing 2
Preister Lisa S. Formal analysis Methodology Writing – review & editing 2
https://orcid.org/0000-0002-4558-8448
Yang Shengming Formal analysis Methodology Writing – review & editing 3
Ramachandran Vanitharani Formal analysis Methodology Writing – review & editing 2
Li Xuehui Formal analysis Methodology Writing – review & editing 1
https://orcid.org/0000-0001-6189-5985
Chu Chenggen Conceptualization Data curation Formal analysis Investigation Methodology Writing – original draft Writing – review & editing 2 *
1 Department of Plant Sciences, North Dakota State University, Fargo, ND, United States of America
2 USDA-ARS, Edward T. Schafer Agricultural Research Center, Sugarbeet and Potato Research Unit, Fargo, ND, United States of America
3 USDA-ARS, Edward T. Schafer Agricultural Research Center, Cereal Research Unit, Fargo, ND, United States of America
Al-Judaibi Awatif Abid Editor
University of Jeddah, SAUDI ARABIA
Competing Interests: The authors have declared that no competing interests exist.

* E-mail: chenggen.chu@usda.gov
6 9 2024
2024
19 9 e030862622 4 2024
26 7 2024
https://creativecommons.org/publicdomain/zero/1.0/ This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Cultivated beet (Beta vulgaris L. ssp. vulgaris) originated from sea beet (B. vulgaris ssp. maritima (L.) Arcang), a wild beet species widely distributed along the coasts of the Mediterranean Sea and Atlantic Ocean, as well as northern Africa. Understanding the evolution of sea beet will facilitate its efficient use in sugarbeet improvement. We used SNPs (single nucleotide polymorphisms) covering the whole genome to analyze 599 sea beet accessions collected from the north Atlantic Ocean and Mediterranean Sea coasts. All B. maritima accessions can be grouped into eight clusters with each corresponding to a specific geographic region. Clusters 2, 3 and 4 with accessions mainly collected from Mediterranean coasts are genetically close to each other as well as to Cluster 6 that contained mainly cultivated beet. Other clusters were relatively distinct from cultivated beets with Clusters 1 and 5 containing accessions from north Atlantic Ocean coasts, Clusters 7 and Cluster 8 mainly have accessions from northern Egypt and southern Europe, and northwest Morocco, respectively. Distribution of B. maritima subpopulations aligns well with the direction of marine currents that was considered a main dynamic force in spreading B. maritima during evolution. Estimation of genetic diversity indices supported the formation of B. maritima subpopulations due to local genetic drift, historic migration, and limited gene flow. Our results indicated that B. maritima originated from southern Europe and then spread to other regions through marine currents to form subpopulations. This research provides vital information for conserving, collecting, and utilizing wild sea beet to sustain sugarbeet improvement.

http://dx.doi.org/10.13039/100007917 Agricultural Research Service 3060-21000-045-000D https://orcid.org/0000-0001-6189-5985
Chu Chenggen http://dx.doi.org/10.13039/100018478 Beet Sugar Development Foundation 634 https://orcid.org/0000-0001-6189-5985
Chu Chenggen Sugarbeet Research and Education Board of Minnesota and North Dakota 633 https://orcid.org/0000-0001-6189-5985
Chu Chenggen This research is funded by USDA-ARS CRIS project No. 3060-21000-045-000D, the Beet Sugar Development Foundation (BSDF), and the Sugarbeet Research and Education Board of Minnesota and North Dakota (SBREB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data AvailabilityAll relevant data are within the manuscript and its Supporting Information files. Raw data are publicly available online through USDA-BeetBase (https://beetbase.scinet.usda.gov/).
Data Availability

All relevant data are within the manuscript and its Supporting Information files. Raw data are publicly available online through USDA-BeetBase (https://beetbase.scinet.usda.gov/).
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pmcIntroduction

Domestication and continuous artificial selection in breeding efforts often leads to a reduction in genetic diversity in modern crop cultivars [1–3]. The loss of genetic diversity has significantly narrowed the genetic base of modern crop varieties [4], thereby reducing adaptability of crops to cope with unfavourable conditions. To enhance the buffering capacity of crops against environmental changes, it is crucial to broaden genetic diversity, thus facilitating the introduction of beneficial genetic variations. Sugarbeet (Beta vulgaris L. ssp. vulgaris) is an important crop that provides a significant source of sucrose [5] but is facing a myriad of disease and pest issues, and additionally is not well-suited for growth in diverse environments [6]. Low genetic diversity in sugarbeet cultivars has become a critical issue that decreases sustainability of the sugarbeet industry [7–9]. It is very important to mine new gene pools to bring in new genetic variations for developing cultivars with more resilience to stresses.

Wild relatives of sugarbeet are likely important genetic sources for adaptive variations crucial for the development of new cultivars [10,11]. Sea beet (Beta vulgaris ssp. maritima (L.) Arcang., according to classification adopted by the US National Plant Germplasm System (NPGS), referred to as B. maritima hereafter), the wild progenitor of cultivated beet, has emerged as a primary reservoir of genetic diversity for the enhancement of sugarbeet [11–16]. To fully exploit the potential of B. maritima for sugarbeet improvement, a more profound understanding of its ecological dynamics, geographical spread and ability to acclimate to the environment is needed [17,18].

Wild B. maritima populations extend along the Mediterranean basin (Algeria, Egypt, France, Greece, Israel, Italy, Lebanon, Morocco, Spain, Tunisia, Turkey, etc.) and the Atlantic coast (Belgium, Denmark, France, Germany, Netherlands, Spain, United Kingdom, etc.), and introduced to many countries in Asia, Central Europe, north and south America (https://powo.science.kew.org/). The observed clinal pattern of B. maritima is likely a result of post-glacial biogeographic processes along the Atlantic coast, with the Mediterranean Basin serving as a population refuge during the last ice age [19]. Previous studies have reported a gradient of variation during the evolution of B. maritima, including the shift from an annual to a biennial growth habit [20–23]. In recent years, research has focused on identifying B. maritima accessions to improve sugarbeet resistance to diseases such as Cercospora leaf spot caused by Cercospora beticola Sacc. [24], rhizomania caused by beet necrotic yellow vein virus (BNYVV) [25], sugar beet cyst nematode (Heterodera schachtii Schmidt) [26], powdery mildew caused by the fungus Erysiphe polygoni (formerly Erysiphe betae) [27], Aphanomyces root rot caused by Aphanomyces cochlioides [28], and yellow wilt caused by Fusarium oxysporum f. sp. betae [29], the development of a smooth root phenotype [30], and salt and drought tolerance [31], but utilization of the genetic diversity in B. maritima for sugarbeet improvement remains limited.

Using SNPs (single nucleotide polymorphisms) from microarrays, Andrello et al. [32] analyzed 1,512 individuals from 1080 accessions of Beta germplasm which grouped all accessions into nine clusters, revealing genetic structure in the germplasm partially corresponding to geographical patterns. However, due to confounding differences between sea beet and cultivated beets, a single geographic origin for cultivated beet domestication could not be definitively determined. High-throughput next-generation sequencing technologies offer opportunities for the efficient discovery of SNPs that cover the entire genome to precisely calculate genetic distance within wild germplasm collections or between wild germplasm and cultivated crops. Tehseen et al. [16] used SNPs generated from a GBS (genotype-by-sequencing) platform to analyze 1,928 accessions of B. vulgaris germplasm lines that included 607 B. maritima accessions and found a subset of 329 B. maritima accessions that was more genetically diverse from sugarbeet, indicating their potential for introducing novel genetic variations from B. maritima to enhance sugarbeet improvement. Sandell et al. [33] sequenced the genome of 606 Beta germplasm lines that included 239 B. maritima accessions and 285 sugarbeet lines and identified two genetically distinct groups of sea beet with one from the Atlantic coast and the other from the Mediterranean Sea area. Notably, accessions collected from Greece exhibited the closest genetic proximity to sugarbeet in that study. Therefore, genetic diversity studies provide an efficient way of identifying B. maritima accessions with greater potential for broadening the genetic base of sugarbeet.

Besides improving the efficient utilization of B. maritima accessions in sugarbeet breeding, genetic diversity analysis is expected to also reveal population structure and gene flow during evolution of sea beet, thereby providing information that is useful for conserving and maintaining genetic diversity in B. maritima. Preservation of B. maritima becomes urgent since the habitats of sea beet along seashores have been altered by the erection of barriers to preserve the seacoast and the recreational use of beaches and estuaries [34]. Since genetic diversity in wild germplasm changes over time due to mutation, selection, genetic drift, and gene flow [35], analysis of genetic resources regarding population distribution, population structure, allelic frequency change, genetic distance, origin of domestication, etc., all provide important information for conserving and maintaining diversity. Therefore, it is essential to use markers that covering the entire genome to accurately investigate diversity of all B. maritima accessions.

Using SNPs generated through a GBS platform, we evaluated all publicly available B. maritima accessions that were collected along the coasts of the Atlantic Ocean, extending latitudinal from the English Channel to the Moroccan coast next to the strait of Gibraltar and longitudinally across the Mediterranean basin from the Balearic to Levantine seas. The objectives in this study were: 1) to evaluate population structure of the worldwide B. maritima collection, 2) elucidate clinal variation and admixture patterns of B. maritima populations, 3) estimate dynamic gene flow during B. maritima evolution and sugarbeet domestication, and 4) generate knowledge that will assist in maintaining the genetic diversity of B. maritima to sustain sugarbeet improvement.

Material and methods

Plant materials

A total of 599 B. maritima accessions from the U.S. National Plant Germplasm System (NPGS) and USDA-ARS sugarbeet genetics program at Fargo, ND were used in the current research. Accessions were collected or introduced from 25 countries and were divided into seven regions of the world including Africa (58 accessions, native collected), Asia (six accessions, two accessions that were from China and Uzbekistan may be introduced), northern Europe (181 accessions, native collected), southern Europe (179 accessions, native collected), western Europe (153 accessions, native collected), and North America (22 accessions, may be introduced) (Tables 1 and S1). In addition, a set of 30 cultivated beet lines including three fodder beet, three table beet, and 24 sugarbeet that came from different origins (S1 Table) were used as a reference to compare genetic distance among B. maritima accessions. These 30 lines were selected according to phylogenetic analysis in Tehseen et al. [16] to represent cultivated beet.

10.1371/journal.pone.0308626.t001 Table 1 List and origin of 599 B. maritima accessions used in the current study with their putative geographic regions.

Region	Countries (number of lines)	Total	
Africa	Egypt (26), Morocco (31), Tunisia (1)	58	
Asia	China (1), Georgia (1), Uzbekistan (1), India (2), Israel (1)	6	
Northern Europe	Denmark (21), Ireland (49), Jersey Island (2), UK (109)	181	
Southern Europe	Croatia (1), Cyprus (1), Greece (56), Italy (100), Portugal (6), Spain (8), Turkey (7)	179	
Western Europe	Belgium (3), France (145), Germany (2), Netherlands, (2), Guernsey Island (1)	153	
North America	United States	22	

SNP genotyping through a genotype by sequencing (GBS) platform

All materials were grown in a greenhouse room at 18–27 ˚C under a 16-h light/8-h dark regime. For GBS analysis, 0.1 g of fresh leaves were collected after 4-leaf stage from 7 to 10 plants of each accession and immediately lyophilized in a Virtis Freezemobile 35EL (SP Scientific, Inc., Warminster, PA, USA) for two days. The dried tissue was ground to a fine powder using a 1600 MiniG SPEX homogenizer (SPEX, Inc., Metuchen, NJ, USA). Genomic DNA was extracted from dried tissue using a KingFisher Flex DNA purification system (KingFisher, Inc., Falls Church, VA, USA). DNA samples were then fragmented using NsiI and BfaI restriction enzymes, and barcoded adapters were ligated to the DNA fragments to differentiate between each accession. GBS sequencing libraries were created using the method of Hilario et al. [36] by amplifying barcoded DNA using a 96-plex plate, then purifying and quantifying the PCR product before sequencing with an Illumina HiSeq 2000 system (Illumina, Inc., San Diego, CA, USA). The sequenced fragments were compared to the sugarbeet genome sequence assembly EL10.2 of sugarbeet line EL10 [37]. Raw SNP data was filtered to remove SNPs with > 20% missing data and genotype imputation was done using Beagle v5.0 [38], thereby reducing the missing data rate to 0% and retaining only bi-allelic SNPs.

Preparing SNP data for population structure analysis

Management of high-throughput SNP data was carried out using the wrapper function of R package SambaR [39]. Briefly, raw data was imported as a.vcf file using the R package vcfR and then converted to a genlight object using the package adegenet v2.1.3 [40–42]. SNPs were filtered through function filterdata in R package SambaR to remove SNPs with a minor allele frequency less than 0.05. Linkage disequilibrium (LD) decay was calculated using PLINK v1.9 [43], and SNPs on each chromosome that were apart over the LD decay distance were selected through PLINK v1.9 for population structure analysis.

Phylogenetic analysis of B. maritima accessions and cultivated beet lines

To create an overview of the genetic distance among B. maritima accessions and between wild sea beet and cultivated beet, Tassel v5.0 was used to generate a phylogenetic tree of all genotypes via UPGMA (unweighted pair group method with arithmetic mean) hierarchical clustering [44]. Genetic distances were calculated using the Interactive Tree Of Life (iTOL v5) with phylogenetic clade tree data [45].

Population structure analysis

Population structure of 599 B. maritima accessions and 30 cultivated beet lines was determined using both the Bayesian-model based clustering method [46] and the non-model-based discriminant analysis of principal components (DAPC) [40]. The Bayesian-model based clustering was conducted using STRUCTURE v.2.3.4, with ten independent replicates run for each putative subpopulation with the number of subpopulations (K) set from 2 to 10 under the admixture model. Burn-in period was set at 10,000 and MCMC (Markov chain Monte Carlo) replications were set at 50,000. Delta K (ΔK) was used to estimate the optimal number of subpopulations based on the change in log probability of data between successive structure iterations using Structure Harvester [47].

DAPC was determined using R package adegenet v2.1.3 [41], with the k-means function used to find the maximum number of subpopulations with maximizing variations and the optimum number of clusters (subpopulations) corresponding to the lowest BIC (Bayesian information criterion) value. The optimal number of clusters was used in the DAPC analysis via the dapc function, which transformed data using PCA (principal component analysis) and performed a discriminant analysis on the retained principal components to form linear combinations of SNP markers with the largest between-group variance and the smallest within-group variance, to describe the clusters, i.e. subpopulations. In both k-means and dapc analyses, 200 principal components were retained for sufficiently explaining 85% of the variance.

Genetic diversity analyses among subpopulations

Genetic differences between subpopulations were estimated using the stamppFst function in R package StAMPP v.1.6.1 [48]. Genome-wide Fst was calculated according to Weir and Cockerham [49] to estimate the proportion of genetic variance in a subpopulation relative to the total genetic variance in the whole collection and to compare similarities among subpopulations. The function stamppNeisD in the same R package was utilized to measure Nei’s genetic distance [50] among subpopulations, which is used to estimate effects of mutation and genetic drift accounting for allelic change during evolution.

To compare genomic variations among subpopulations, the average major allele frequency calculated within every 1-Mb region on each chromosome was compared among subpopulations. The variances of the average allele frequency within the same genomic region across subpopulations were used as indicators of the variation level among subpopulations. Average major allele frequency and their variance across subpopulations were calculated using Microsoft Excel. Genomic regions that have a variance of major allele frequency above the 75th percentile were considered as regions significantly varied across subpopulations during B. maritima divergence.

Genetic diversity analyses within subpopulations

Within each subpopulation, nucleotide diversity (π) and Watterson estimator (allelic richness) were calculated using the R package SambaR [39] to measure genetic variation among individuals in the subpopulation and minor allele frequency. Tajima’s D (balance between rare and common alleles) was calculated using the same tool to measure gene frequency deviation from neutral evolution, i,e. any selective pressure existing during B. maritima evolution. Since all sub-populations consisted of varying numbers of genotypes, scaled values of diversity indices were utilized to provide a more comprehensive explanation of the data.

Results

Genotypic markers distribution

GBS platform generated approximately 520,000 SNPs using EL10.2 as the reference genomic assembly. From these, a group of 148,317 SNP markers was selected by removing SNPs with minor allele frequency and greater than >20% missing data rate. Chromosomes 5 and 6 had the most markers with 19,115 (12.89%) and 19,140 (12.90%) markers, respectively, while chromosome 9 had the least with 14,277 (9.63%) SNPs. Across the entire genome, the marker density was, on average, 3.81 markers per kilobase (kb). Chromosome 5 had the highest marker density (3.54 markers/kb) while chromosome 1 had the lowest density (4.07 markers/kb) (Fig 1A). Analysis using the set of 148,317 SNPs in 599 B. maritima accessions and 30 cultivated beet lines found linkage disequilibrium (LD) decayed on average within 28 kb (R2 = 0.2) (Fig 1B). A subset of 77,180 SNPs was obtained after LD pruning using threshold of R2 = 0.2 and was then used for population structure analysis.

10.1371/journal.pone.0308626.g001 Fig 1 Distribution and density of single nucleotide polymorphisms (SNPs) across nine chromosomes (A) and average linkage disequilibrium (LD) decay (B) in the collection of 599 Beta maritima accessions and 30 cultivated beet lines.

Phylogenetic analysis of B. maritima accessions with cultivated beet lines

Phylogenetic analysis of 599 B. maritima accessions and 30 cultivated beet lines showed that B. maritima accessions were grouped into two distinct clusters, with one cluster distinct from cultivated lines and the other related to cultivated lines (Fig 2). The cluster related to cultivated lines contained 276 accessions with 60.9% of accessions collected from southern Europe, whereas the cultivated-distinct cluster included 323 accessions with 51.1% collected from northern Europe and 35.0% collected from western Europe (Table 2). This agreed with reports from Sandell et al. [33] and Tehseen et al. [16] that B. maritima accessions from northern and western Europe are more genetically diverse to cultivated beet.

10.1371/journal.pone.0308626.g002 Fig 2 Phylogenetic tree developed from 599 Beta maritima accessions and 30 cultivated beet lines using SNPs covering the whole genome.

The red lines in the cluster clades indicate the cultivated beet lines.

10.1371/journal.pone.0308626.t002 Table 2 Distribution of Beta maritima accessions in two distinct phylogenetic clusters defined by phylogenetic analysis using B. maritima collection and 30 cultivated beet lines.

Cluster	Region	Number of lines	Percentage within cluster (%)	
cultivated-related (276 accessions)	
	Africa	28	10.1	
	Asia	6	2.2	
	North America	18	6.5	
	Northern Europe	16	5.8	
	Southern Europe	168	60.9	
	Western Europe	40	14.5	
cultivated-distinct (323 accessions)	
	Africa	30	9.3	
	North America	4	1.2	
	Northern Europe	165	51.1	
	Southern Europe	11	3.4	
 	Western Europe	113	35.0	

Population structure in B. maritima population

Population structure analysis of B. maritima accessions via STRUCTURE indicated that 3, 5, 7, or 8 subpopulations may occur in the collection although 5 or 8 subpopulations was most likely (Figs 3 and S1). Though no clear population structure could be concluded from STRUCTURE analysis, the grouping of subpopulations by this program agreed with the geographic distribution of the accessions. For example, if the collection was divided into three subpopulations, accessions can be grouped into Atlantic, north African and Mediterranean regions. If five subpopulations were used to describe the collection, subpopulations would group accessions into English Channel, French Atlantic, Moroccan coast near Gibraltar, the Mediterranean Sea, and Levantine and Aegean Sea coasts.

10.1371/journal.pone.0308626.g003 Fig 3 The plot of K versus ΔK for determination of the optimum number of subpopulations within 599 B. maritima accessions.

Optimal subpopulations are equal to K values with maximal ΔK values. Data was generated using the computer program STRUCTURE v2.3.4.

The DAPC method [41] determined that eight clusters was the optimum number of subpopulations in the collection (Figs 4 and S2, S1 Table), which agreed with the cluster grouping in STRUCTURE when K = 8. In detail, Cluster 1 contained 173 accessions with 125 from northern Europe and 46 from western Europe. Cluster 2 had 110 accessions with 59 from southern Europe and 21 from western Europe, but it also contained 22 accessions from north America, 4 accessions from Asia, and 7 accessions from Africa. Cluster 3 only contained 24 accessions with 15 from Morocco. Cluster 4 had 104 accessions with 88 from southern Europe. Cluster 5 carried 114 accessions with 42 from northern Europe (mainly admixture from United Kingdom) and 68 from western Europe (mainly the French Atlantic coast). Cluster 6 included 25 accessions from different regions in addition to the 30 cultivated beets, and thus is corresponding to cultivated beet. Cluster 7 contained 34 accessions with 19 from Egypt and 15 from south of the Aegean Sea in southern Europe, and Cluster 8 had 15 accessions that were all collected from the Atlantic coast of Morrocco (Tables 3 and S1; Fig 4). Overall, accessions in Clusters 2, 3 and 4 are closest to the cultivated cluster (Cluster 6), those in Cluster 7 are the most distinct from cultivated beet lines. Clusters 1, 5 and 7 are all distinct to the cultivated cluster but accessions in Clusters 1 and 5 showed closer relationship which agrees that two clusters were physically close along the north Atlantic Ocean seashores. Interestingly, the geographic distribution of clusters agreed well with the marine current direction in Mediterranean Sea [51] and the north Atlantic Ocean [52] (S3 Fig), suggesting genetic divergence in B. maritima likely caused by the marine currents, facilitating the dispersion B. maritima to different regions and subsequent isolation.

10.1371/journal.pone.0308626.g004 Fig 4 Genetic structure of the 599 B. maritima accessions and 30 cultivated beet lines analyzed using DAPC (discriminant analysis of principal components) methodology.

A. Genetic relationships amongst clusters. The length of bars for each cluster indicates variations within each cluster. Bars in different color correspond to different clusters. B. Geographic distribution of clusters defined by DAPC method. The geographic map is based on NASA world map (https://data.nasa.gov/). Detailed list of accessions in each cluster is available in Tables 3 and S1. Location of Cluster 6 is not shown since it is mainly corresponding to worldwide collected cultivated beet lines.

10.1371/journal.pone.0308626.t003 Table 3 Distribution of Beta maritima accessions in eight clusters as defined by DAPC (discriminant analysis of principal components) methodology across all collection regionsa.

Cluster	Cultivated beet	Africa (Egypt)	Africa (Morocco)	Asia	North America	North Europe	South Europe	West Europe	Total	
1	0 	0	0	0	1	125	1	46	173	
2	0	7	0	4	16	3	59	21	110	
3	0	0 	15	0	2	0	7	0	24	
4	0	0	1*	0	1	0	88	14	104	
5	0	0	0	0	2	42	2	68	114	
6	30	0	1	2	0	11	7	4	55	
7	0	19	0	0	0	0	15	0	34	
8	0	0	15	0	0	0	0	0	15	
Total	30	26	32	6	22	181	179	153	629	
a The numbers in bold font style indicate majority of accessions in the corresponding cluster.

* This is the only accession from Tunisia.

Genetic differentiation among B. maritima subpopulations

Pairwise comparison of the fixation index Fst among all subpopulations revealed moderate to high population differentiation (Table 4), which agreed with results from the DAPC analysis above. Low comparison Fst values (high genetic similarity) were found between Clusters 2 and 4, and between Clusters 1 and 5, whereas high Fst values (low genetic similarity) were observed when comparing Clusters 7 or 8 with any other clusters, which agreed with the geographic origins of Clusters 7 and 8 that are more isolated. Clusters 1 and 5 were also distinct from the others as their Fst values, when comparing with other clusters, were relatively high (Table 4), fitting with their locations at north Atlantic Ocean seashores that are far away from other locations. In contrast, Fst values of population comparisons among Clusters 2, 3 and 4 from the seashores of the Mediterranean Sea including the Balearic and Alboran seas were relatively low, which agrees with their close geographic distances or the circular marine current prevailing in those waters [51]. Nei’s genetic distance estimated from comparison among clusters agreed well with the corresponding Fst values observed (Table 4).

10.1371/journal.pone.0308626.t004 Table 4 Genetic diversity among subpopulations represented by fixation index (Fst) and Nei’s genetic distance estimated using a worldwide collection of 599 Beta maritima accessions with 30 cultivated beet lines a.

Cluster	1	2	3	4	5	6	7	8	
1	-	0.31	0.20	0.31	0.05	0.47	0.53	0.33	
2	0.77	-	0.19	0.06	0.16	0.17	0.37	0.34	
3	0.32	0.41	-	0.16	0.11	0.46	0.57	0.27	
4	0.69	0.10	0.30	-	0.17	0.28	0.44	0.34	
5	0.06	0.38	0.22	0.38	-	0.33	0.44	0.26	
6	0.82	0.19	0.80	0.39	0.64	-	0.65	0.62	
7	0.80	0.56	0.82	0.70	0.92	0.83	-	0.75	
8	0.49	0.73	0.30	0.61	0.45	0.77	0.95	-	
a Table entries above the diagonal show the calculated Weir and Cockerham’s Fst; entries below the diagonal show estimates of Nei’s genetic distance.

Comparison of major allele frequencies along chromosomes across all eight clusters found all chromosomes carried regions that had higher levels of variation for allele distribution (Fig 5 and S2 Table), of which, chromosomes 1, 7 and 9 had relatively larger regions with higher allelic variation, and chromosomes 3, 5 and 6 had relatively lower variation. Regions with higher allelic variation may carry genes that help sea beet adapt to different conditions and result in divergence of B. maritima during evolution. In addition, major allele frequency in cultivated beet cluster (cluster 6) remained higher than the frequencies in other clusters (Fig 5), consistent with the fact that cultivated beet has undergone higher selection pressure.

10.1371/journal.pone.0308626.g005 Fig 5 Major allele frequency distribution on each chromosome across all clusters defined by DAPC (discriminant analysis of principal components) methodology.

Red rectangular boxes with dashed lines indicate genomic regions with a higher level of variation among clusters, and the regions were determined by the above 75th percentile of the major allele frequency variance.

Genetic diversity within B. maritima subpopulations

The scaled nucleotide diversity (π), Watterson estimator (ø) and Tajima’s D value indicate genetic diversity in subpopulations with less interference due to subpopulation size. Estimation of these scaled genetic diversity indices in eight subpopulation of B. maritima defined by the DAPC method indicated that Clusters 1 through 5 had similar diversity with no obvious selection pressure (Table 5). However, Cluster 5, that contains accessions mainly from the French Atlantic coast and seashore of the United Kingdom, exhibited slightly higher but non-significant level of diversity. Clusters 2, 3 and 4 showed a little higher allelic richness when combining the estimations of nucleotide diversity and Watterson estimator, in agreement with the circular marine current in the western Mediterranean Sea and the Tyrrhenian Sea that likely increased allele exchange among these three clusters (Table 5 and S3 Fig).

10.1371/journal.pone.0308626.t005 Table 5 Population genetic indices estimated within each cluster (subpopulation) as defined by DAPC (discriminant analysis of principal components) methodology in 599 Beta maritima accessions and 30 cultivated beet lines.

Cluster	Scaled (π)	Watterson scaled	Tajima’s D scaled	
1	0.24	0.15	0.09	
2	0.29	0.17	0.13	
3	0.24	0.20	0.04	
4	0.26	0.17	0.10	
5	0.30	0.17	0.14	
6	0.15	0.16	-0.01	
7	0.07	0.10	-0.03	
8	0.12	0.13	-0.01	

Clusters 6, 7 and 8 showed a relatively low level of genetic diversity with the estimated Tajima’s D being negative (Table 5), indicating selection pressure had been applied to those clusters. Cluster 6 contains mainly cultivated beet lines and agrees with beet cultivars have been under through high selection pressure. The non-natural gene frequency change in Clusters 7 (accessions from north Egypt and south Greece along the Levantine and Aegean Seas) and Cluster 8 (accessions from Morrocco) may be largely due to the smaller population size within clusters, in addition to the possible selection pressure exerted by the unique environmental conditions in these two isolated regions.

Discussion

The genetic structure of plant populations is influenced by a combination of environment and demographic processes, encompassing historical and contemporary gene flow. Typically, this pattern is assessed indirectly on a broad scale, primarily at the population level [32]. Exploring the mechanisms driving range shifts and factors influencing population formation are crucial questions in evolutionary biology and ecological genetics, particularly within the framework of climate change [19]. The observed patterns in the current study of B. maritima populations offer insight into significance of geographic features, contributing to our understanding of how these factors jointly influence the distribution of genetic diversity and population structure at the broader levels of whole population, as well as at individual genetic levels amongst the accessions in the subpopulations.

Sandell et al. [33] found the B. maritima accessions they investigated could be divided into two genetically distinct groups. In this study, our phylogenetic analysis of 599 B. maritima accessions and 30 cultivated beet lines also revealed two major groups with one being closely related and the other distinct to cultivated beet lines. The cultivated-related group had accessions from the Mediterranean Sea coast whereas the cultivated-distinct group contained accessions predominantly from the seashores of the north Atlantic Ocean. Distinctions between Atlantic and Mediterranean populations of B. maritima were also documented in other reports [32,53], and this phylogeographic distribution pattern has also been indicated by variations in chloroplast [13] and patterns of allozyme [54].

By using DAPC analysis, eight distinct clusters of B. maritima were identified and each clearly aligned to a defined geographic region. The DAPC analysis in this research largely agrees with findings of Andrello et al. [32] that grouped 1080 accessions of Beta germplasm lines into nine clusters. Our analysis focused mainly on B. maritima accessions and enabled us to assign each cluster to a specific geographical region. All clusters from DAPC analysis represent segments of a continuous genetic gradient extending from south-east to north-west, which indicates that the genetic structure is influenced by a combination of local genetic drift and limited gene flow.

In B. maritima, gene flow can occur through pollen dispersal and the spread of seed. Pollen flow is constrained by factors such as wind and plant density and occurs primarily over short distances [55]. Seed dispersal, on the other hand, is facilitated by seawater movements, leading to seeds being washed away from the shores [13,31). Combining our B. maritima clusters defined by DAPC with marine current direction data in the Mediterranean Sea [51] and Atlantic Ocean [52], the observed patterns of population structure align well with the marine circulation, as indicated in previous reports [19,56]. Our findings suggest a comprehensive understanding of the genetic architecture in B. maritima, where both spatial and genetic factors play integral roles.

The Mediterranean region has been recognized as the diversity center of B. maritima [10,13,53,56]. Of the eight clusters identified in this research, Cluster 2 contained the most accessions from the coast of western and southern Europe (France, Italy and Greece) along the Mediterranean Sea, and it also contains accessions collected from almost all regions including Asia, Africa, North America, and other regions of Europe (S1 Table), which indicated that region of Cluster 2 is likely to be the center of origin of B. maritima. Cluster 4, with a majority of accessions from southern Europe (Italy and Greece), exhibited the closest genetic distance to Cluster 2 (Table 4) and cultivated beet, i.e., Cluster 6 (Fig 4), suggesting it is likely the center of origin for cultivated beet domestication. Sandell et al. [33] further suggested that Greece might have served as the site of domestication for the ancestral sugarbeet, as B. maritima accessions in that region were found to be the closest wild relatives to sugarbeet.

Clusters 1, 3, 5, 7 and 8 are likely the result of colonization of seeds spread from the Mediterranean region. Cluster 7 contains accessions from Egypt and southern Greece, and a few from Turkey and southern Italy. Cluster 7 likely originated from sea beet drift from southern Europe and followed by colonization and spread along the shores of the Levantine Sea through the circular marine current of that region (S3 Fig). Cluster 3 could be accessions that originated from Cluster 2 and colonized the southwest seashore of the western Mediterranean (S3 Fig). Due to the circular marine currents within the western Mediterranean and Tyrrhenian Seas, accessions in Cluster 3 were most closely related to accessions in Clusters 2 and 4 (Fig 3). Accessions in Cluster 8 may be originated from Cluster 3 and colonized the Atlantic seashore of Morrocco since accessions in Cluster 8 were closely related to Cluster 3 (Table 4). Clusters 1 and 5 may also have originated from Cluster 3 but were dispersed via the marine currents of the Atlantic Ocean towards the north. This observation is supported by the low comparison values of Fst and Nei’s distance between Cluster 3 and either Cluster 1 or 5, which suggest a relatively close relationship among these clusters (Table 4). Comparison of DNA sequences of genomic regions exhibiting higher allele frequency variation (Fig 5) may facilitate the identification of genes to help elucidate the evolution of B. maritima.

Genetic diversity within each subpopulation provided additional insight supporting the potential divergence process of B. maritima. The higher allelic richness observed in clusters 2, 3, and 4 aligns with increased chances for allele exchange amongst them due to circular marine currents across the western Mediterranean and Tyrrhenian Sea (S3 Fig). Accessions within these clusters would constitute valuable genetic resources for identifying novel alleles. The low level of genetic diversity within accessions in Clusters 7 and 8 is consistent with the geographic isolation of these accessions due to marine current direction, which limited their allele exchange with accessions in other clusters, consequently reducing allelic richness (Fig 5). The narrow genetic basis due to limited allele richness in two clusters further increased their vulnerability to environmental conditions further reducing allelic richness by environmental selection pressure (Table 5). Our finding agreed with Veloso et al. [57] for role of ocean currents in species dispersal. Overall, accessions in clusters 1, 5, 7 and 8 that are more distinct from clusters 2, 3, 4, and 6 are likely to provide an important source of novel variations to broaden the genetic base and increase the sustainability of sugarbeet, and they need to be considered for prior conservation to maintain genetic diversity.

In summary, an analysis of 599 B. maritima accessions and 30 cultivated beet lines using DAPC methodology grouped the publicly available accessions of B. maritima into eight subpopulations, with each subpopulation localizing to a distinct geographic region. Population structure analysis elucidated the clinal variation and admixture patterns during evolution in the B. maritima collection. Further, the genetic diversity analysis among subpopulations confirmed southern Europe’s role as the center of diversity for B. maritima, identified relationships among subpopulations, and revealed patterns of dynamic gene flow contributing to B. maritima divergence. The knowledge obtained from this study offers new insights that will be valuable for the preservation of genetic diversity within B. maritima and the effective utilization of novel alleles and variations to enhance sugar beet improvement.

Supporting information

S1 Fig Population structure of 599 B. maritima accessions as a function of the number of subpopulations (K) between 2 to 10.

Different colors in each row represent different subpopulations. Each bar represents the estimated membership of subpopulations for a single genotype.

(TIF)

S2 Fig Average Bayesian Information Criterion (BIC) plotted against 1–20 putative clusters to provide the optimum number of subpopulations, equivalent to the number at the lowest position, in the world-wide collection of 599 B. maritima accessions.

(TIF)

S3 Fig Geographical distribution of B. maritima clusters along with the marine current direction in the Atlantic Ocean (A) and the Mediterranean Sea (B). The geographic map is based on NASA world map (https://data.nasa.gov/). Black arrows indicate marine current direction of the Atlantic Ocean according to Offshore Engineering [52] and that of the Mediterranean Sea according to Pascual et al. [51]. Red circles with numbers indicate the clusters defined by DAPC (discriminant analysis of principal components) methodology with Cluster 1 contained accessions from northern and western Europe, Cluster 2 had accessions mainly from southern and western Europe, and few accessions from north America, Asia and Africa, Cluster 3 contained accessions from Morocco, Cluster 4 had accessions from southern Europe, Cluster 5 carried accessions from northern Europe (mainly from United Kingdom) and western Europe (mainly from Atlantic coast of French), Cluster 6 is corresponding to 30 cultivated beets used as the reference (it’s not shown in the picture), Cluster 7 contained accessions from Egypt and south of the Aegean Sea in southern Europe, and Cluster 8 had accessions collected from the Atlantic coast of Morrocco.

(TIF)

S1 Table List of 599 B. maritima accessions and 30 cultivated beet lines in eight clusters as defined by DAPC (discriminant analysis of principal components) methodology with collection locations for each accession.

(DOCX)

S2 Table Average major allele frequencies on each chromosome of B. maritima across the eight clusters as defined by DAPC (discriminant analysis of principal components) methodology.

(DOCX)

This research is supported by the USDA-ARS CRIS project No. 3060-21000-045-000D, the Beet Sugar Development Foundation (BSDF), and the Sugarbeet Research and Education Board of Minnesota and North Dakota (SBREB). Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. The U.S. Department of Agriculture is an equal opportunity provider and employer.

10.1371/journal.pone.0308626.r001
Decision Letter 0
Al-Judaibi Awatif Abid Academic Editor
© 2024 Awatif Abid Al-Judaibi
2024
Awatif Abid Al-Judaibi
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version0
4 Jun 2024

PONE-D-24-16245Genetic drift, historic migration, and limited gene flow contributing to the subpopulation divergence in wild sea beet (Beta vulgaris ssp. maritima)PLOS ONE

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Reviewer #1: First, I extend my gratitude to the authors for their outstanding work and well-constructed text. In this study, authors investigated 599 sea beet (Beta maritima) accessions collected from the north Atlantic Ocean and Mediterranean Sea coasts. It is a comprehensive study about the distribution and origin of sea beet and cultivated beet. The title explains the research properly, and the results are described well in details. I think the manuscript contains a good research question and explains the migration route of wild beet along the north Atlantic Ocean and Mediterranean Sea coasts. In the future, the findings will be useful to identify novel genomic regions related to resistance against abiotic/biotic stress factors in Beta vulgaris. However, I have some comments as follows. I recommend minor revisions.

Here are my comments:

-In Materials and Methods, authors did not describe the growth conditions of beet accessions. Authors should mention how many accessions they used from each of the following: Africa, Asia, northern Europe, southern Europe, western Europe, eastern Europe, and North America.

-In Figures 4 and S3, why did not authors show the location of Cluster 6? That map does not show the locations correctly unless I have missed something. For example, I do not see Greece and Italy in Cluster 2 (please see discussion, line 306). Why do you think the Cluster 2 is the center of origin for sea beet and cultivated beet? You may highlight the experimental results for clearer explanation. In addition, each cluster (1-8) must be briefly described in the legend of figure S3.

Reviewer #2: Correct name: Beta vulgaris subsp. maritima (L.) Arcang.

Adding to the title and in the first citation of the text the authorship of the species -> Verify: www.tropicos.org

I suggest adding the botanical family name to the title or in the keywords.

I suggest adding the scientific name of the causal agents of the diseases mentioned in paragraph 3 of the Introduction.

I congratulate the authors on this present work, of fundamental importance for the establishment or better management of genetic improvement programs, as well as for the conservation of wild species.

Reviewer #3: The origin of cultivated plants and the identification of the most closely related native populations, as well as their crop wild relatives, is a highly topical subject, given its potential for the genetic improvement of crops. Following on from other work that has been carried out on sugar beet, the submitted manuscript analyses more than six hundred samples of Beta maritima from various regions of the world. The results presented are interesting and improve the knowledge of the phylogenetic relationships of the populations, particularly those occurring in the Mediterranean region.

In general, the manuscript is well-written, well-structured, and easy to understand.

Apart from a few minor suggestions/comments (listed below), there is one aspect that I would like to highlight because I think it should be corrected: some of the samples used are from areas where Beta maritima is not native, but naturalised (this is the case with the samples from North America). I think this should be mentioned/analysed throughout the text (material and methods, results and discussion, tables, etc.). This aspect can even add value to the discussion, as hypotheses can be put forward for the origin of the populations in the regions where Beta maritima is naturalised.

Other comments:

Ln. 54 – ‘Sea beet (Beta vulgaris L. ssp. maritima Arcang., referred to as B. maritima hereafter)’ – Currently, some of the world's most important taxonomic databases (e.g. Plants of the World Online - POWO; and World Flora Online - WFO) only accept Beta vulgaris subsp. vulgaris and consider Beta vulgaris subsp. maritima as a synonym. This does not invalidate the work developed, but it can be mentioned in the text that the work carried out used the classification adopted by the US National Plant Germplasm System (NPGS).

ln. 59 – ‘Wild B. maritima populations extend along the Mediterranean basin and the Atlantic coast.’ – This description should be more detailed (see POWO) and the regions where the species is introduced should be indicated.

Ln. 76 – ‘Tehseen et al’ - Tehseen et al. The full stop is missing.

Ln. 108 to 111 – Detail the distribution (native or non-native) in the mentioned regions. In Table 1, mark (e.g. with a *) the accessions collected in regions where the taxa is not native (e.g. North America). Check the values in this table: the total is 601 and not 599 as it should be.

Check the legend of Fig. 1 (ln 524) - references to A and B are missing.

In Results section, I think Fig. 3 (Ln. 208) could be made supplementary and Fig. S3 (ln. 231) be included in the main text. The latter is quite enlightening. However, its graphic presentation could be improved.

ln. 273 – ‘… is influenced by a combination of landscape characteristics.’ - I suggest replacing 'landscape' (which has aesthetic connotations) with environment.

Ln. 300 – In the discussion, this manuscript could also consider the results of the paper Veloso et al. 2021 (Genetic Diversity and Population Structure of Wild Beets (Beta spp.) from the Western Iberian Peninsula and the Azores and Madeira Islands) which also highlights the importance of ocean currents in species dispersal.

Finally, I suggest complementing the discussion by including some conservation proposals, namely the prioritisation of certain populations / regions taking into account their phylogenetic affinities and genetic diversity.

**********

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Reviewer #3: No

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10.1371/journal.pone.0308626.r002
Author response to Decision Letter 0
Submission Version1
7 Jun 2024

Response to Editor:

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Response: the figures were reproduced using NASA world map (https://data.nasa.gov/), and ocean current direction was manually added according to the reference listed.

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Response: added.

7. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Response: all references were checked.

Responses to Reviewers' comments:

Reviewer #1:

First, I extend my gratitude to the authors for their outstanding work and well-constructed text. In this study, authors investigated 599 sea beet (Beta maritima) accessions collected from the north Atlantic Ocean and Mediterranean Sea coasts. It is a comprehensive study about the distribution and origin of sea beet and cultivated beet. The title explains the research properly, and the results are described well in details. I think the manuscript contains a good research question and explains the migration route of wild beet along the north Atlantic Ocean and Mediterranean Sea coasts. In the future, the findings will be useful to identify novel genomic regions related to resistance against abiotic/biotic stress factors in Beta vulgaris. However, I have some comments as follows. I recommend minor revisions.

Here are my comments:

- In Materials and Methods, authors did not describe the growth conditions of beet accessions.

Response: information was added.

- Authors should mention how many accessions they used from each of the following: Africa, Asia, northern Europe, southern Europe, western Europe, eastern Europe, and North America.

Response: number of accessions were added for each region. The region ‘eastern Europe’ is deleted since the two lines have not good marker data and were not included in the analysis.

-In Figures 4 and S3, why did not authors show the location of Cluster 6?

Response: it has two reasons: 1) the cluster 6 is corresponding to cultivated beet that selected worldwide, not a specific location is assigned; and 2) B. maritima accessions in the cluster 6 are spread similarly in several other clusters with no dominant amount from a specific region, it is impossible to assign a location for those accessions.

- That map does not show the locations correctly unless I have missed something. For example, I do not see Greece and Italy in Cluster 2 (please see discussion, line 306).

Response: Thank you so much for pointing this out. When making the figure, we only thought to find regions and try to make them distinct, but clusters 2 and 4 are very close and their regions are largely overlapped. We thus enlarged the region of the cluster 2 to let it covers Italy and Greece, and it is more consistent with our results. Great thanks!

- Why do you think the Cluster 2 is the center of origin for sea beet and cultivated beet? You may highlight the experimental results for clearer explanation.

Response: Cluster 2 is the center of origin for sea beet simply because it contains accessions collected from almost all regions, the location of regions for Cluster 2 is determined based on the location that majority of the accessions were collected. Accessions in Cluster 2 but collected from the other regions are indication of they were migrated from the region we defined, indicating regions assigned to major accessions in Cluster 2 at Mediterranean Sea coast likely to be the center of sea beet. However, the sea beet accessions close to Cluster 6 that equivalent to cultivated beet were largely collected in Greece, indicating Greece might be the center of cultivated beet domesticated from sea beet.

- In addition, each cluster (1-8) must be briefly described in the legend of figure S3.

Response: Great thanks for this suggestion, cluster descriptions were added accordingly. We appreciate your comments to improve the manuscript.

Reviewer #2:

- Correct name: Beta vulgaris subsp. maritima (L.) Arcang. Adding to the title and in the first citation of the text the authorship of the species -> Verify: www.tropicos.org I suggest adding the botanical family name to the title or in the keywords.

Response: Great thanks, the scientific name was corrected in the title and in the text.

- I suggest adding the scientific name of the causal agents of the diseases mentioned in paragraph 3 of the Introduction.

Response: the causal agents of the diseases were added.

- I congratulate the authors on this present work, of fundamental importance for the establishment or better management of genetic improvement programs, as well as for the conservation of wild species.

Response: great thanks and appreciate your comments for improving the manuscript.

Reviewer #3:

The origin of cultivated plants and the identification of the most closely related native populations, as well as their crop wild relatives, is a highly topical subject, given its potential for the genetic improvement of crops. Following on from other work that has been carried out on sugar beet, the submitted manuscript analyses more than six hundred samples of Beta maritima from various regions of the world. The results presented are interesting and improve the knowledge of the phylogenetic relationships of the populations, particularly those occurring in the Mediterranean region.

In general, the manuscript is well-written, well-structured, and easy to understand.

Apart from a few minor suggestions/comments (listed below), there is one aspect that I would like to highlight because I think it should be corrected: some of the samples used are from areas where Beta maritima is not native, but naturalised (this is the case with the samples from North America). I think this should be mentioned/analysed throughout the text (material and methods, results and discussion, tables, etc.). This aspect can even add value to the discussion, as hypotheses can be put forward for the origin of the populations in the regions where Beta maritima is naturalised.

Response: We agree the location of some accessions not the location they were collected, as you indicated that accessions from North America may be donated by some institution oversea. Since the number of such accessions are small and they didn’t affect the results much, we didn’t mention about them in the discussion. Actually, we just started a new project using conserved gene sequence to investigate the evolution of B. maritima accessions as well as cultivated beets, which will be better to differentiate B. maritima accessions and then narrow down region of the origin.

Other comments:

Ln. 54 – ‘Sea beet (Beta vulgaris L. ssp. maritima Arcang., referred to as B. maritima hereafter)’ – Currently, some of the world's most important taxonomic databases (e.g. Plants of the World Online - POWO; and World Flora Online - WFO) only accept Beta vulgaris subsp. vulgaris and consider Beta vulgaris subsp. maritima as a synonym. This does not invalidate the work developed, but it can be mentioned in the text that the work carried out used the classification adopted by the US National Plant Germplasm System (NPGS).

Response: Thanks. We made the change accordingly.

ln. 59 – ‘Wild B. maritima populations extend along the Mediterranean basin and the Atlantic coast.’ – This description should be more detailed (see POWO) and the regions where the species is introduced should be indicated.

Response: More details were added according to POWO, but we are not sure if it’s correct for mentioning accessions were introduced. I heard that few B. maritima accessions from California, USA might be natively collected, but no further information to indicate if it’s true.

Ln. 76 – ‘Tehseen et al’ - Tehseen et al. The full stop is missing.

Response: Corrected. Thanks!

Ln. 108 to 111 – Detail the distribution (native or non-native) in the mentioned regions. In Table 1, mark (e.g. with a *) the accessions collected in regions where the taxa is not native (e.g. North America). Check the values in this table: the total is 601 and not 599 as it should be.

Response: Detailed information was added, accordingly. It should be 599 accessions. The two accessions from eastern Europe have not good marker data and were removed from analysis later, but we forget to change the table, accordingly.

Check the legend of Fig. 1 (ln 524) - references to A and B are missing.

Response: Thanks for finding this. We changed the figure but forget to change the figure legends. It’s now corrected.

In Results section, I think Fig. 3 (Ln. 208) could be made supplementary and Fig. S3 (ln. 231) be included in the main text. The latter is quite enlightening. However, its graphic presentation could be improved.

Response: Thanks for this comment. We originally put Figure S3 in the main text, but considered the figure was published, we then move it as supplemental file. This did flag out by academic editor and the journal. We have to redo the figure but still think it’s better to put it as the supplementary.

ln. 273 – ‘… is influenced by a combination of landscape characteristics.’ - I suggest replacing 'landscape' (which has aesthetic connotations) with environment.

Response: Thanks. We changed it as suggested.

Ln. 300 – In the discussion, this manuscript could also consider the results of the paper Veloso et al. 2021 (Genetic Diversity and Population Structure of Wild Beets (Beta spp.) from the Western Iberian Peninsula and the Azores and Madeira Islands) which also highlights the importance of ocean currents in species dispersal.

Response: Thanks. The reference is cited.

Finally, I suggest complementing the discussion by including some conservation proposals, namely the prioritisation of certain populat

Attachment Submitted filename: response to reviewers.docx

10.1371/journal.pone.0308626.r003
Decision Letter 1
Al-Judaibi Awatif Abid Academic Editor
© 2024 Awatif Abid Al-Judaibi
2024
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Submission Version1
29 Jul 2024

Genetic drift, historic migration, and limited gene flow contributing to the subpopulation divergence in wild sea beet (Beta vulgaris ssp. maritima (L.) Arcang)

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10.1371/journal.pone.0308626.r004
Acceptance letter
Al-Judaibi Awatif Abid Academic Editor
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2024
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https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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