==== Front PLoS One PLoS ONE plos plosone PLoS ONE 1932-6203 Public Library of Science San Francisco, CA USA 10.1371/journal.pone.0235475 PONE-D-20-12057 Research Article Biology and Life Sciences Genetics Departures from Diploidy Polyploidy Tetraploidy Biology and Life Sciences Evolutionary Biology Evolutionary Systematics Phylogenetics Phylogenetic Analysis Biology and Life Sciences Taxonomy Evolutionary Systematics Phylogenetics Phylogenetic Analysis Computer and Information Sciences Data Management Taxonomy Evolutionary Systematics Phylogenetics Phylogenetic Analysis Research and Analysis Methods Database and Informatics Methods Bioinformatics Sequence Analysis Biology and Life Sciences Evolutionary Biology Population Genetics Biology and Life Sciences Genetics Population Genetics Biology and Life Sciences Population Biology Population Genetics Biology and Life Sciences Genetics Departures from Diploidy Polyploidy Biology and Life Sciences Ecology Ecological Metrics Species Diversity Ecology and Environmental Sciences Ecology Ecological Metrics Species Diversity Biology and Life Sciences Organisms Eukaryota Plants Grasses Barley Research and analysis methods Database and informatics methods Bioinformatics Sequence analysis DNA sequence analysis Phylogenetic analysis of two single-copy nuclear genes revealed origin of tetraploid barley Hordeum marinum Origin of tetraploid barley Hordeum marinumYin Bo Data curationMethodologySoftwareValidationWriting – original draft1 Sun Genlou ConceptualizationProject administrationResourcesWriting – review & editing2 Sun Daokun ResourcesSupervisionWriting – review & editing1 http://orcid.org/0000-0002-1730-9421Ren Xifeng ConceptualizationData curationFormal analysisFunding acquisitionInvestigationWriting – original draft1* 1 College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, China 2 Biology Department, Saint Mary's University, Halifax, NS, Canada Ma Wujun Editor Murdoch University, AUSTRALIA Competing Interests: The authors have declared that no competing interests exist. * E-mail: robert04@example.com 30 6 2020 2020 15 6 e023547526 4 2020 16 6 2020 © 2020 Yin et al2020Yin et alThis 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.Sea barley Hordeum marinum is an important germplasm resource. However, the origin of this tetraploid H. marinum subsp. gussoneanum is still unclear, which has caused great perplexity to the exploration and utilization of germplasm resources. We used two single-copy nuclear genes, thioredoxin-like gene (TRX) and waxy1 gene encoding granule-bound starch synthase (WAXY1) to analyze 41 accessions of Hordeum marinum. The phylogenies of different genes told different story of evolution of tetraploids of H. marinum subsp. gussoneanum. The phylogenetic trees showed that two distinct copies of sequences from both genes were detected for some accessions of the tetraploids of H. marinum subsp. gussoneanum, and diploid marinum might also contribute to the origin and evolution of the tetraploid gussoneanum. Our findings suggested that tetraploid more likely originated from the diploids of H. marinum subsp. gussoneanum and another ancestor that might be an extinct unknown diploid species. Homogenization of gene in tetraploids also occurred after polyploidization as both TRX and WAXY1 sequences in some accessions of tetraploid H. marinum subsp. gussoneanum cannot be distinguished, indicating the complicated evolution of this tetraploid. Research supported by the Earmarked Fund for China Agriculture Research System (CARS-5). The Natural Sciences and Engineering Research Council of Canada (RGPIN-2018-05433) to GS. Data AvailabilityAll relevant data are within the manuscript and its Supporting Information files.Data Availability All relevant data are within the manuscript and its Supporting Information files. ==== Body Introduction The genus Hordeum in Triticeae consists of cultivated and wild barleys, including about 30 species with diploid (2n = 2x = 14), tetraploid (2n = 4x = 28) and hexaploid (2n = 6x = 42) taxa [1]. Hordeum species were proposed having four basic genome groups: I, H, Xa, and Xu [2]. Sea barley is a species in the Xa genome group [3], contains two subspecies: H. marinum subsp. marinum (2n = 2x = 14) and gussoneanum (2n = 2x = 14 and 2n = 4x = 28). The marinum and gussoneanum diploid forms can be distinguished by their morphology, but coexist throughout the Mediterranean region [3, 4]. The tetraploid gussoneanum is only found in the farthest eastern Mediterranean, from there to the east to Asia [4, 5]. Tetraploid subspecies was suggested as a suitable material for the improvement of barley in genetics and genomics, and investigation of polyploid evolution [6]. Over the last 50 years, many efforts have been made to clarify the relationships between tetraploid and diploid sea barley, while no phylogenic consensus exists and the origin of the tetraploid sea barley of Xa-genome remains controversy [2, 5, 7–10]. The definition of genomes between Triticeae and many other crops often through cytogenetic characterization of chromosomes and the analysis of their chromosome pairing behavior between interspecific and intergeneric crosses [11]. Fluorescence in situ hybridization (FISH)-based karyotypes were used to clarify the relationships and the origin between their polyploids and diploids in Xa-genome species [3, 5, 6]. The autopolyploid origin of tetraploid gussoneanum was investigated by a series of cytogenetic methods, including C-banding karyotypes analysis and the meiotic behaviour in hybrid process [12], as well as molecular phylogenetic analysis from the chloroplast loci examination, geographical information and the ecological data [4]. Recent molecular phylogenetic analyses with single copy nuclear markers suggested allopolyploid origin of tetraploid gussoneanum, with Xa genome from diploid of gussoneanum and another genome from unclear diploid progenitor [3, 5, 13]. The marinum was also suggested as the second progenitor [2, 14], while other results indicated the second progenitor was an unclear extant or a probable extinct diploid of the H. marinum [13, 15]. TRX gene encodes a thioredoxin-like protein, which is a small-molecular-weight thermostable protein and plays an important role in plant growth and development. Orthologous genes have been isolated from many Hordeum species and the coding region is highly conserved among the Hordeum species [16]. The TRX gene was confirmed to be a single-copy nuclear gene and showed a high level of interspecific sequence polymorphisms. It has been used to explore the phylogeny of Hordeum species [14, 17, 18, 19]. The granule-bound starch synthase (GBSSI or WAXY1) gene is involved in the synthesis of starch granules and can produce the most abundant proteins related to starch granulocytes. It has been cloned from many plants including rice, maize, barley and wheat [20]. The WAXY1 gene exists in a single copy in almost all the species studied and has been used to explore relationships among genera within tribes or among species within genera [21, 22, 23]. Here we used two single-copy nuclear genes: thioredoxin-like gene (TRX) and waxy1 gene encoding granule-bound starch synthase (WAXY1) to explore the origin of tetraploid species of Hordeum marinum subsp. gussoneanum. Materials and methods Plant materials Six accessions of polyploid H. marinum subsp. gussoneanum, and 33 diploid species of H. marinum subsp. marinum and H. marinum subsp. gussoneanum were sequenced in our study, which are mainly distributed in the west coast of the Mediterranean Sea and the Middle East, including 4 accessions from Portugal, 11 from Spain, 12 from Greece and 3 from Bulgaria. While the tetraploid species are mainly distributed in the Middle East, such as Turkey, Iran and Afghanistan. Classification of Hordeum species/subspecies follows von Bothmer et al. [1]. The seeds were friendly provided by the Nordic Genetic Resource Center (NordGen). The detailed information of collector and donor for each species of Hordeum can be found from the Nordic Genetic Resource Center's Web site (www.nordgen.org/index.php/en/content/view/full/344). Seeds of each accession were germinated, transplanted and grown in a greenhouse. DNA was extracted from young leaf tissue collected from 5 to 10 plants of each accession using the method of Stein et al. [24]. Plant materials with accession number, genome constitution, country of origin, and GenBank identification numbers were given in Table 1. The sequences of other Triticeae species were downloaded from the NCBI database and included in the phylogenetic analysis, including Aegilops, Psathyrostachys, Secale, Australopyrum, Taeniatherum, Triticum, Pseudoroegneria and Hordeum (S1 Table). 10.1371/journal.pone.0235475.t001Table 1 The species, accession number, genome and origin of 39 sea barley used in this study. Species Accession no. Genome Origin TRX WAXY1 H.marinum subsp. marinum Hudson H41 Xa Turkey + + H56 Xa Germany + + H87 Xa Jordan + + H90 Xa Greece + + H109 Xa Greece + + H126 Xa Greece + + H155 Xa Greece + + H588 Xa Greece + + H607 Xa Greece + + H624 Xa Greece + + H631 Xa Greece + + H759 Xa Greece + + H158 Xa Portugal + + H508 Xa Spain + + H512 Xa Spain + + H515 Xa Spain + + H518 Xa Spain + + H524 Xa Spain + + H546 Xa Spain + + H559 Xa Spain + + H560 Xa Spain + + H568 Xa Spain + + H. marinum subsp. gussoneanum (Parlatore) Thellung H161 Xa Portugal + + H162 Xa Portugal + + H163 Xa Portugal + + H299 Xa Bulgaria + + H534 Xa Spain + + H563 Xa Spain ++ + H581 Xa Greece + + H598 Xa Greece + + H608 Xa Greece + + H823 Xa Bulgaria + + H837 Xa Bulgaria + + H. marinum subsp. gussoneanum (Parlatore) Thellung H64 XaXa Soviet Union + + H81 XaXa Afghanistan + + H208 XaXa Turkey + + H800 XaXa Iran + + H824 XaXa Iran + + H825 XaXa Turkey + + DNA amplification and sequencing The single copy nuclear gene TRX and WAXY1 sequences were amplified by polymerase chain reaction (PCR) using the primers TRXF/R [17] and WAXY1F/R [21], respectively. The amplification profile for the TRX gene was as follows: an initial denaturation at 95°C for 4 min; 14 cycles of 95°C for 40 s denaturing and 72°C for 90 s extension. The amplification profile for the WAXY1 gene was as follows: an initial denaturation at 94°C for 3 min; 35 cycles of 94°C for 30 s, 65°C for 40 s, 72°C for 1 min; and a final cycle of 72°C for 20 min. PCR was carried out in a 20 μL reaction mixture, containing 30 ng template DNA, 0.2μM of each primer, 1.5 mM MgCl2, 0.2 mM of each deoxynucleotide (dATP, dCTP, dGTP, dTTP), 1 U of high fidelity Taq DNA polymerase (Biolabs, New England), and distilled deionized water to the final volume. To enhance the sequence quality, each PCR product from the two nuclear genes was independently amplified twice, and cloned into pGEMeasy T vector (Promega Corporation, Madison, Wis., USA) following the manufacturer's instruction. At least 10 clones from each species were randomly selected. In order to detect the presence of an insert, each colony was firstly transferred to 10 μL of LB broth with 0.1 mg/mL Ampicillin antibiotics. These solutions were incubated at 37°C for 30 min, then 2 μL was used as template to check the presence of an insert by PCR using the initial primers. For the colonies that were detected to contain the insert, the remaining 8 μL of solution was transferred to another 5 mL of LB broth (with Ampicillin antibiotics) and incubated at 37°C overnight for plasmid DNA isolation. Sequencing was done commercially by the Taihe Biotechnology (Beijing, China). Data analysis Automated sequence outputs were visually inspected using chromatographs. Multiple sequence alignments were made using ClustalX using default parameters [25]. We checked the existence of recombinants in our research by closer inspection of sequence alignment before phylogenetic analysis, and no such recombinants were found. A phylogenetic tree of sequences was constructed using the maximum likelihood (ML) algorithm in MEGA7, with 1000 bootstrap replicates, and a 60% cut off was used in the analysis. All characters were specified as unweighted and unordered, and all gap-only columns were excluded in the analyses. The most parsimonious trees were obtained by performing a heuristic search using the Tree Bisection-Reconnection (TBR) with the following parameters: Mul-Trees on and 10 replications of random addition sequences with the stepwise addition option. A strict consensus tree was usually generated from multiple parsimonious trees. Overall character congruence was estimated by the consistency index (CI) and the retention index (RI). Bootstrap (BS) values with 1000 replications were calculated by performing a heuristic search using the TBR option with Multree on, and were used to infer the robustness of clades [26]. In addition, Bayesian analyses were performed using MrBayes 3.1 [27]. Each data was tested to find the best-fitting model of sequence evolution by jModelTest 2.1.10 [28], using default parameters. Each data was estimated by the Akaike information criterion (AIC) [29], Bayesian information criterion (BIC) [30], and a decision theoretic performance-based approach (DT) [31]. The BIC was used to select model because of its high accuracy [28]. The HKY+G and TrN+G+I substitution models led to the best BIC scores for TRX and WAXY1, respectively (S2 Table). Therefore, the HKY+G (for TRX) and TrN+G+I (for WAXY1) models were used in the Bayesian analysis using MrBayes 3.1 [27]. The program was run with the standard setting of two analyses in parallel, each with four chains, and estimates of convergence of results by calculating standard deviation of split frequencies between analyses. In total, 3040 000 generations for TRX and 835000 generations for WAXY1 were run to make the standard deviation of split frequencies below 0.01. Samples were taken every 1000 generations. For all analyses, the first 25% of samples from each run were discarded as burn-in to ensure the stationary of the chains. A majority rule consensus tree was generated from the remaining sampled trees. Bayesian posterior probability (PP) values were obtained, and were used to test the robustness of clades. Results Genetic diversity analysis and neutrality test To reveal the genetic diversity of TRX between the diploid H. marinum subsp. gussoneanum and the diploid H. marinum subsp. marinum, genetic analysis and neutrality test in three populations were performed (Table 2). The number of haplotypes (H = 4), the haplotype diversity (Hd = 0.671), the number of polymorphic sites (S = 5), the per-site nucleotide diversity (θ = 0.00075 ± 0.00063) and the nucleotide diversity (π = 0.00246) were observed in the diploid H. marinum subsp. marinum. While just one haplotype was observed in the diploid H. marinum subsp. gussoneanum population. 10.1371/journal.pone.0235475.t002Table 2 Estimates of nucleotide diversity per base pair and test statistics for selection at TRX gene in different barley populations. The three populations are T-g-4x (H. marinum subsp. gussoneanum4x), T-m-2x (H. marinum subsp. marinum 2x) and T-g-2x (H. marinum subsp. gussoneanum 2x) respectively. Note: the gaps/missing/data were excluded; *robert04@example.com level. Population No. of accessions No. of Haplotypes (H) Haplotyp diversity (Hd) Number of polymorphic sites (S) Theta (per site) from S (θ) Nucleotid diversity (π) Tajima's D test Fu and Li's D* test Fu and Li's F* test T-g-4x 6 14 0.971 120 0.01466±0.00372 0.05071 0.90768 0.84171 0.99820 T-m-2x 22 4 0.671 5 0.00075±0.00063 0.00246 2.21537* 1.17564 1.69999* T-g-2x 11 1 0.000 0 0.00000±0.00000 0.00000 0.00000 0.00000 0.00000 All 39 18 0.881 125 0.03215±0.00922 0.03089 -0.24721 0.68653 0.40200 In addition to these, Tajima and Fu and Li's tests also were measured in three populations. Values obtained from three populations all are positive. However, only diploid species of H. marinum subsp. marinum population showed significant Tajima and Fu and Li's F values (2.21537 and 1.69999, respectively). As shown in Table 3 for WAXY1, the number of haplotypes (H = 5), the haplotype diversity (Hd = 0.727), the number of polymorphic sites (S = 15), the per-site nucleotide diversity (θ = 0.00222 ± 0.00140) and the nucleotide diversity (π = 0.00955) were observed in the diploid species of H. marinum subsp. marinum population. The number of haplotypes (H = 3), the haplotype diversity (Hd = 0.709), the number of polymorphic sites (S = 2), the per-site nucleotide diversity (θ = 0.00089 ± 0.00068) and the nucleotide diversity (π = 0.00114) were observed in the diploid species of H. marinum subsp. gussoneanum population. Compared with the diploid H. marinum subsp. marinum population, 1.8% haplotype diversity (Hd) and 0.841% nucleotide diversity (π) reduction were found in the diploid H. marinum subsp. gussoneanum population. 10.1371/journal.pone.0235475.t003Table 3 Estimates of nucleotide diversity per base pair and test statistics for selection at WAXY1 gene in different barley populations. The three populations are W-g-4x (H. marinum subsp. Gussoneanum 4x), W-m-2x (H. marinum subsp. marinum 2x) and W-g-2x (H. marinum subsp. gussoneanum 2x), respectively. Note: the gaps/missing/data were excluded; *robert04@example.com level. Population No. of accessions No. of Haplotypes (H) Haplotyp diversity (Hd) Number of polymorphic sites (S) Theta (per site) from S (θ) Nucleotid diversity (π) Tajima's D test Fu and Li's D* test Fu and Li's F* test W-g-4x 6 17 0.993 58 0.00813±0.00292 0.02781 0.70277 -0.35238 -0.05040 W-m-2x 22 5 0.727 15 0.00222±0.00140 0.00955 2.73675** 1.52352** 2.19348** W-g-2x 11 3 0.709 2 0.00089±0.00068 0.00114 0.85048 0.99697 1.07842 All 39 16 0.901 65 0.01961±0.00596 0.02371 0.49478 0.26653 0.41601 Both Tajima's D, and Fu and Li's tests were positive for the diploid H. marinum subsp. marinum population and the diploid H. marinum subsp. gussoneanum population, while negative for the tetraploid species of H. marinum subsp. gussoneanum population in Fu and Li's tests. Tajima's D and Fu and Li's values were observed significantly for the diploid H. marinum subsp. marinum population. However, Tajima's D and Fu and Li's tests did not show significantly for other two. Phylogenetic analysis of TRX sequence The DNA were amplified and cloned from 6 accessions of polyploid species and 33 diploid species. A total of 69 sequences (45 sequences generated in this study, and 24 downloaded from GenBank) were used for phylogenetic analysis. Two distinct copies of sequences were recovered from each tetraploid gussoneanum accession. The 19 TRX sequences from H. marinum subsp. gussoneanum and H. murinum subsp. murinum and 49 TRX sequences from other diploid Hordeum species together with one TRX sequence from the specie of Psathyrostachys juncea were phylogenetically analyzed. Psathyrostachys juncea was used as the outgroup. The data matrix contained 1157 characters, of which 622 were constant, 204 were parsimony uninformative, and 331 were parsimony informative. Maximum parsimony analysis produced 645 equally parsimonious trees with CI = 0.775 and RI = 0.894 (excluding uninformative characters). The separated Bayesian analyses using the HKY+G model led to the mean log-likelihood values of identical trees is –5772.39 and –5707.94. The minor difference between the tree topologies in Bayesian trees and those generated by MP was observed. The consensus tree with PP value produced by Bayesian analysis was shown in Fig 1. 10.1371/journal.pone.0235475.g001Fig 1 Strict consensus trees derived from TRX sequence data was conducted using the HKY+G model by Bayesian analysis. The main topologies generated by Maximum parsimony analysis are similar. Numbers above and below branches are Bayesian posterior probability (PP) values and bootstrap values, respectively. Psathyrostachys juncea was used as the outgroup. Consistency index (CI) = 0.775, retention index (RI) = 0.894. Geographic distribution of species: western Mediterranean (green), central Mediterranean (purple), eastern Mediterranean (blue). Phylogenetic analyses based on TRX sequence data grouped most sequences from the Xa-genome species of H. marinum subsp. gussoneanum and H. marinum subsp. marinum into a clade with 0.73 PP support (BS = 100%), All sequences from diploid species of H. marinum subsp. marinum formed a clade with 0.85 PP support (BS = 97%), and there are two subclades with PP = 0.96 (BS = 98%) and PP = 0.86 (BS = 96%) internally. All diploid species of H. marinum subsp. gussoneanum formed a clade in 0.76 PP support (BS = 100%). One each copy from 6 tetraploid species of H. marinum subsp. gussoneanum was grouped closely with diploid species of H. marinum subsp. gussoneanum and H. marinum subsp. marinum, while sequences from another copy of each 6 tetraploid H. marinum subsp. gussoneanum were grouped into an independent clade with PP = 0.98 (BS = 100%) (Fig 1). Phylogenetic analysis of WAXY1 sequence A total of 76 WAXY1 sequences from 39 accessions, including 6 polyploid H. marinum and 33 diploid species of H. marinum and other diploid species in Triticeae downloaded from GenBank, were obtained. Psathyrostachys juncea was used as the outgroup in phylogenetic analysis. The data matrix contained 1299 characters, of which 871 were constant, 159 were parsimony uninformative, and 269 were parsimony informative. Maximum parsimony analysis produced 173 equally parsimonious trees with CI = 0.624 and RI = 0.912 (excluding uninformative characters). The separated Bayesian analyses using the TrN+G+I model led to the mean log-likelihood values of identical trees was –6036.17 and –5979.37. The minor difference between the tree topologies in Bayesian trees and those generated by MP was observed. The consensus tree with PP value produced by Bayesian analysis was shown in Fig 2. 10.1371/journal.pone.0235475.g002Fig 2 Strict consensus trees derived from WAXY1 sequence data was conducted using the TrN+G+I model by Bayesian analysis. The main topologies generated by Maximum parsimony analysis are similar. Numbers above and below branches are Bayesian posterior probability (PP) values and bootstrap values, respectively. Psathyrostachys juncea was used as the outgroup. Consistency index (CI) = 0.624, retention index (RI) = 0.912. Geographic distribution of species: western Mediterranean (green), central Mediterranean (purple), eastern Mediterranean (blue). Phylogenetic analyses based on WAXY1 sequence data grouped all sequences from the Xa-genome species with 0.86 PP support (BS = 63%) (Fig 2). All the diploid H. marinum subsp. gussoneanum and H. marinum subsp. marinum were grouped into a clade with 1.00 PP support (BS = 100%), and sequences from diploid H. marinum subsp. marinum formed two subclades with 1.00 PP support (BS = 99%) and 0.56 PP support (BS = 91%). One each copy from 6 tetraploid H. marinum subsp. gussoneanum formed a clade with 1.00 PP support (BS = 93%). The rest sequences from another copy of 6 tetraploid H. marinum subsp. gussoneanum and all sequences from diploid H. marinum subsp. gussoneanum formed a subclade with 1.00 PP support (BS = 84%), while five of the sequences from diploid H. marinum subsp. gussoneanum were grouped in one clade with 0.55 PP support (BS = 74%), included in this clade was the sequence H64-1 from tetraploid H. marinum subsp. gussoneanum (Fig 2). Discussion Origin of polyploids in tetaploid H. marinum The relationships within the Xa genome have received considerable attention, partly on account of the importance of germplasm resources in barley, and partly because many previous phylogenetic research data sets have failed to reach a consensus view of the relationships among the Xa genome [32]. Phylogenetic relationships within Xa genome species are still unclear. Polyploid species present numerous opportunities and challenges to molecular phylogenetic studies. The maternally-inherited chloroplast genome data of polyploid is generally easy to obtain, and they provide more potentially informative data but many are incomplete. While data from single-copy or low-copy markers is especially informative [4, 33]. Phylogenies Hordeum species including Xa genome species have been previously reported using TRX and RPB2 sequences [14, 34]. While the origin and evolution of Xa-genome tetraploids has been controversy. Although homologous origin hypothesis [4, 12] or heterologous origin hypothesis [2, 13, 14, 15] have been proposed in previous studies, no enough evidence and data confirm that H. marinum subsp. gussoneanum is autopolyploids or allopolyploids. Therefore, we used two single-copy nuclear genes TRX and WAXY1 to infer phylogenetic relationships between diploid species and tetraploid species of H. marinum species. The phylogenetic tree of TRX showed that the accessions of tetraploid H. marinum subsp. gussoneanum such as H81, H800, H819 and H2303 are more likely autopolyploid. Two copies of sequences in each these accessions were placed into closely related the sister sub-clade, indicating that the two genomes in each these accessions might be less differentiated, which makes we speculate that they have a homologous origin. While the two copies of sequences from the accessions of tetraploid H. marinum subsp. gussoneanum such as H64, H208, H824 and H825 were placed into distinct clades, showing that their two genomes are quite different. We speculated that the genomes in each these accessions might come from different sources. The phylogenetic tree of TRX showed that H64, H208, H824 and H825 are heterologous origins, H819, H2303, H81, and H800 are likely homologous origins. While the phylogenetic tree of WAXY1 showed that H64, H81, H208, H800, H824, and H825 are all heterogeneous origins. The reason for this phenomenon might be due to different genes having different evolutionary history, the high degree of differentiation of the TRX gene and the large variability in different species of the Triticeae have occurred [14, 19]. One each copy of sequences from H800, H819 and H2303 is close to the diploid H. marinum subsp. gussoneanum and another each copy is close to the diploid H. marinum subsp. marinum in the phylogenetic tree of TRX, while only diploid H. marinum subsp. gussoneanum and tetraploid H. marinum subsp.gussoneanum were grouped in the same subclade and the diploid H. marinum subsp. marinum was grouped in a subclade alone in the phylogenetic tree of WAXY1. Results suggested that diploid H. marinum subsp. gussoneanum and tetraploids are closely related and share partially similar gene sequences, which suggests the diploid H. marinum subsp. gussoneanum might be one of the ancestors of the tetraploid H. marinum subsp. gussoneanum. This was consistent with the putative involvement of diploids H. marinum subsp. gussoneanum as a diploid ancestor of tetraploids of H. marinum subsp. gussoneanum [3]. Similarly, genomic in situ hybridization (GISH) revealed the diploid forms of gussoneanum would be a maternal diploid ancestor of H. marinum tetraploids [3], as provided by molecular phylogenies based on chloroplast DNA [4, 35]. While some tetraploids of H. marinum subsp. gussoneanum and diploids of H. marinum subsp. marinum were grouped in a sister-subclade, indicating diploid marinum might also contribute to the origin and evolution of the tetraploid gussoneanum [3]. Our results also suggested that the second genome donor to tetraploid gussoneanum remains unknown, and it might be an extinct species. Based on the two nuclear gene sequence data, we still could not determine the second genome donor to tetraploid gussoneanum since only tetraploid accession H824 was grouped into one cluster with diploid for both genes, other tetraploid accessions changed when they were analyzed with two distinct genes. These results suggested that different genes might experience different evolutionary history. In order to detangle the origin of tetraploid gussoneanum, the more genes used in the study, the better results will be obtained. Many polyploid species have likely originated repeatedly, including genetically different parent species. This supports the multiple origin hypothesis of the parents [11]. The heterogeneous origin can also be seen from the geographical distribution of diploids and tetraploids. Our findings indicated that the tetraploid gussoneanum might be originated in two or more geographic origin center [3]. Some studies indicated that tetraploids are mainly distributed in the Middle East and Eurasia, and some appeared in North America. While diploids are widely distributed along the Mediterranean coast as well as in Portugal and Spain. Considering the important impact of climate changing on survival and evolution of species, we speculate that the extinction of another ancestor might be related to climate change. Previous studies also mentioned that the climate or living environment is not suitable for the ancestors of the tetraploid H. marinum subsp. gussoneanum growth as the glaciers melting [36, 37], and it became extinct after the formation of tetraploids of H. marinum subsp. gussoneanum, resulting in a genetic bottleneck and the loss of some inherited parental information [4, 38]. In summary, Our findings favor heterogeneous origin of the tetraploid H. marinum subsp. gussoneanum. Homogenization of gene in tetraploids also occurred after polyploidization as both TRX and WAXY1 sequences in some accessions of tetraploid H. marinum subsp. gussoneanum cannot be distinguished. Supporting information S1 Table Taxa from Aegilops, Psathyrostachys, Secale, Taeniatherum, Australopyrum, Triticum, Pseudoroegneria and Hordeum used in this study. (DOC) Click here for additional data file. S2 Table Maximum likelihood fits of nucleotide substitution models. (DOC) Click here for additional data file. 10.1371/journal.pone.0235475.r001 Decision Letter 0 Ma Wujun Academic Editor © 2020 Wujun Ma2020Wujun MaThis 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 28 May 2020 PONE-D-20-12057 Phylogenetic analysis of two single-copy nuclear genes revealed origin of tetraploid barley Hordeum marinum PLOS ONE Dear Dr. Ren, Thank you for submitting your manuscript to PLOS ONE. 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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ********** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The study used two single-copy nuclear gene markers to classify a collection of 33 Hordeum marinum species, in order to draw a phylogenetic consensus map and reveal the origin of the tetraploid subspecies in Hordeum marinum. #### If my synopsis of the article is wrong, please discard my comments and find an alternative reviewer.###### It is a potentially article because of the novelty of the work and new findings revealed by one of the gene marker derived from the TRX gene. Compared to previous work [refer to reference number 3], the study has found similar results that the gussoneanum (4x) was clustered into two clades by the TRX gene marker, one of the clade was grouped with the Xa genome, but the other clade was not grouped into any genome. In comparison, by using the WAXY1 gene marker, all of the gussoneanum (4x) were grouped together, but overlapped with some accessions from the gussoneanum (2x) group. My question is: is that possible the unclustered clade may carry a new genome that was never reported before, or the overlapped group the gussoneanum (4x) and gussoneanum (2x) accessions is the transition stage from the gussoneanum (2x) type evolving to the gussoneanum (4x) type? By reading the manuscript, the answer seems unclear. Comments on other parts of the manuscript: 1. It is unclear why nuclear TRX and WAXY1 gene markers were selected out of numerous other genes. Authors must have very sound reasons to do so. As a result, the reasons should be elaborated in the introduction part. Why they are selected? If they have been used for classification before, what are the main findings? If they were not used before, what's the advantages of using them and expected outcomes? 2. There are a few places where the font of the words are different from the rest. I suggest using line numbers on pages to identify sentences when resubmit. 3. Reference 25: format 4. Fig.1 and Fig.2 sentence "Geographic distribution of ...." sentence is incomplete, the denotation for the "red line" is missing. 5. The last paragraph or "conclusion" paragraph needs to rewrite. This part should only have results from the study and concluding remarks from the authors, so the citation is redundant. Again, the word "angiosperms" is inappropriate in presenting here. Reviewer #2: This paper was written very well. Sea barley Hordeum marinum is an important germplasm resource. This paper suggested that tetraploid more likely originated from the diploids of H. marinum subsp. gussoneanum and the other ancestor that might be an extinct unknown diploid species. Homogenization of gene in tetraploids also occurred after polyploidization as both TRX and WAXY1 sequences in some accessions of tetraploid H. marinum subsp. gussoneanum cannot be distinguished, indicating the complicated evolution of this tetraploid. Line H824 was an interesting line, the gene was grouped into one cluster with 2X for both genes. But other 4X lines changed when they were analysed with two distinct genes. It means that the more genes used in the study, the better results will be obtained. Discuss this in the paper. There are some other minor issues which need to be addressed. Page 8: Abstract: "H. marinum subsp. gussoneanum, diploid marinum" from two sentences, insert "and". P13: Rewrite this sentence "To reveal the genetic differentiation between the diploid H. marinum subsp. Gussoneanum and the diploid H. marinum subsp. marinum used TRX, genetic analysis and neutrality test in three populations were performed." P13: change 1 from "While just 1 haplotype" to one. 1 to 9 should be written out. Reference: Italic Hordeum marinum for the reference 4. Double check other references. ********** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose "no", your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at robert04@example.com. Please note that Supporting Information files do not need this step. 10.1371/journal.pone.0235475.r002 Author response to Decision Letter 0 Submission Version1 15 Jun 2020 June 15, 2020 Dear Wujun Ma: Thanks for providing us with an opportunity to revise our manuscript. We have taken all comments from reviewers into consideration, and made a revised version of this manuscript. We invited Professor Genlou Sun (Saint Mary University of Canada) and Professor Hong Luo (Clemson University) to revise this manuscript. I am sending you our revised manuscript along with a point-by-point description of the changes made for your reviewing. I hope that our manuscript will meet your standards for publication in Plos one. Sincerely yours, Xifeng Ren Description of the changes Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf Response: Thanks for your suggestion. Changes were made according to the suggestions. 2. We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service. Whilst you may use any professional scientific editing service of your choice, PLOS has partnered with both American Journal Experts (AJE) and Editage to provide discounted services to PLOS authors. Both organizations have experience helping authors meet PLOS guidelines and can provide language editing, translation, manuscript formatting, and figure formatting to ensure your manuscript meets our submission guidelines. To take advantage of our partnership with AJE, visit the AJE website (http://learn.aje.com/plos/) for a 15% discount off AJE services. To take advantage of our partnership with Editage, visit the Editage website (www.editage.com) and enter referral code PLOSEDIT for a 15% discount off Editage services. If the PLOS editorial team finds any language issues in text that either AJE or Editage has edited, the service provider will re-edit the text for free. Upon resubmission, please provide the following: a) The name of the colleague or the details of the professional service that edited your manuscript b) A copy of your manuscript showing your changes by either highlighting them or using track changes (uploaded as a *supporting information* file) c) A clean copy of the edited manuscript (uploaded as the new *manuscript* file) Response: Thanks for your suggestion. Changes were made according to the suggestions. We have carefully edited this manuscript. Reviewer #1 Reviewer #1: The study used two single-copy nuclear gene markers to classify a collection of 33 Hordeum marinum species, in order to draw a phylogenetic consensus map and reveal the origin of the tetraploid subspecies in Hordeum marinum. If my synopsis of the article is wrong, please discard my comments and find an alternative reviewer. It is a potentially article because of the novelty of the work and new findings revealed by one of the gene marker derived from the TRX gene. Compared to previous work [refer to reference number 3], the study has found similar results that the gussoneanum (4x) was clustered into two clades by the TRX gene marker, one of the clade was grouped with the Xa genome, but the other clade was not grouped into any genome. In comparison, by using the WAXY1 gene marker, all of the gussoneanum (4x) were grouped together, but overlapped with some accessions from the gussoneanum (2x) group. My question is: is that possible the unclustered clade may carry a new genome that was never reported before, or the overlapped group the gussoneanum (4x) and gussoneanum (2x) accessions is the transition stage from the gussoneanum (2x) type evolving to the gussoneanum (4x) type? By reading the manuscript, the answer seems unclear. Response: Thanks for your suggestion. Because the limited number of genes used in the experiment, the origin of tetraploids of H. marinum subsp. gussoneanum has not been fully explored. Yes, the unclustered clade may carry a new genome that was never reported before. It may come from an extinct species. Similar results have appeared in previous literature reports. Comments on other parts of the manuscript: 1. It is unclear why nuclear TRX and WAXY1 gene markers were selected out of numerous other genes. Authors must have very sound reasons to do so. As a result, the reasons should be elaborated in the introduction part. Why they are selected? If they have been used for classification before, what are the main findings? If they were not used before, what's the advantages of using them and expected outcomes? Response: Thanks for your comments. We have added this information in the introduction. The nuclear TRX and WAXY1 genes were selected as markers because they are relatively conservative during the evolution of Triticeae, and they have also been used in phylogenetic studies. 2. There are a few places where the font of the words are different from the rest. I suggest using line numbers on pages to identify sentences when resubmit. Response: Thanks for your suggestion. Changes were made according to the suggestions. 3. Reference 25: format Response: Thanks for your suggestion. We have corrected the format error of the reference. 4. Fig.1 and Fig.2 sentence "Geographic distribution of ...." sentence is incomplete, the denotation for the "red line" is missing. Response: Red is only labeled as tetraploids of H. marinum subsp. gussoneanum, and its geographical distribution is shown in Table 1. For readability, we have changed the red label to black, with pictures and notes. 5. The last paragraph or "conclusion" paragraph needs to rewrite. This part should only have results from the study and concluding remarks from the authors, so the citation is redundant. Again, the word "angiosperms" is inappropriate in presenting here. Response: Thanks for your suggestion. We have revised the conclusion. Reviewer #2 Reviewer #2: This paper was written very well. Sea barley Hordeum marinum is an important germplasm resource. This paper suggested that tetraploid more likely originated from the diploids of H. marinum subsp. gussoneanum and the other ancestor that might be an extinct unknown diploid species. Homogenization of gene in tetraploids also occurred after polyploidization as both TRX and WAXY1 sequences in some accessions of tetraploid H. marinum subsp. gussoneanum cannot be distinguished, indicating the complicated evolution of this tetraploid. Line H824 was an interesting line, the gene was grouped into one cluster with 2X for both genes. But other 4X lines changed when they were analysed with two distinct genes. It means that the more genes used in the study, the better results will be obtained. Discuss this in the paper. Response: Thanks for your suggestion. We added relevant discussion on this. There are some other minor issues which need to be addressed. Page 8: Abstract: "H. marinum subsp. gussoneanum, diploid marinum" from two sentences, insert "and". Response: Thanks for your suggestion. Changes were made according to the suggestions. P13: Rewrite this sentence "To reveal the genetic differentiation between the diploid H. marinum subsp. Gussoneanum and the diploid H. marinum subsp. marinum used TRX, genetic analysis and neutrality test in three populations were performed." Response: Thanks for your suggestion. Changes were made according to the suggestions. P13: change 1 from "While just 1 haplotype" to one. 1 to 9 should be written out. Response: Thanks for your suggestion. Changes were made according to the suggestions. Reference: Italic Hordeum marinum for the reference 4. Double check other references. Response: Thanks for your suggestion. We have corrected the format error of all reference. Attachment Submitted filename: Response to reviewers.doc Click here for additional data file. 10.1371/journal.pone.0235475.r003 Decision Letter 1 Ma Wujun Academic Editor © 2020 Wujun Ma2020Wujun MaThis 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 Version1 17 Jun 2020 Phylogenetic analysis of two single-copy nuclear genes revealed origin of tetraploid barley Hordeum marinum PONE-D-20-12057R1 Dear Dr. Ren, We're pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. 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Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact robert04@example.com. Kind regards, Wujun Ma Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: 10.1371/journal.pone.0235475.r004 Acceptance letter Ma Wujun Academic Editor © 2020 Wujun Ma2020Wujun MaThis 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. 19 Jun 2020 PONE-D-20-12057R1 Phylogenetic analysis of two single-copy nuclear genes revealed origin of tetraploid barley Hordeum marinum Dear Dr. Ren: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact robert04@example.com. If we can help with anything else, please email us at robert04@example.com. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Wujun Ma Academic Editor PLOS ONE ==== Refs References 1 von Bothmer R , Salomon B , Linde-laursen I . Variation for crossability in a reciprocal, interspecific cross involving Hordeum vulgare and H. lechleri . Euphytica . 1995 ; 84 (3 ):183 –187 . 2 Blattner FR . Progress in phylogenetic analysis and a new infrageneric classification of the barley genus Hordeum (Poaceae: Triticeae) . Breed Sci . 2009 ; 59 (5 ):471 –480 . 3 Carmona A , Friero E , De Bustos A , Jouve N , Cuadrado A . The evolutionary history of sea barley (Hordeum marinum) revealed by comparative physical mapping of repetitive DNA .
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