Molecular Phylogenetics and Evolution 77 (2014) 296–306

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Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev

Evolutionary pattern of rDNA following polyploidy in Leymus (Triticeae: Poaceae) Xing Fan a,b,1, Jing Liu a,1, Li-Na Sha a,1, Gen-Lou Sun c, Zhi-Qin Hu a, Jian Zeng d, Hou-Yang Kang a, Hai-Qin Zhang a, Yi Wang a, Xiao-Li Wang e, Li Zhang e, Chun-Bang Ding e, Rui-Wu Yang e, You-Liang Zheng a,b, Yong-Hong Zhou a,b,⇑ a

Triticeae Research Institute, Sichuan Agricultural University, Wenjiang 611130, Sichuan, China Key Laboratory of Crop Genetic Resources and Improvement, Ministry of Education, Sichuan Agricultural University, Yaan 625014, Sichuan, China Biology Department, Saint Mary’s University, Halifax NS B3H 3C3, Canada d College of Resources and Environment, Sichuan Agricultural University, Wenjiang 611130, Sichuan, China e College of Life Sciences, Sichuan Agricultural University, Yaan 625014, Sichuan, China b c

a r t i c l e

i n f o

Article history: Received 15 March 2013 Revised 20 March 2014 Accepted 16 April 2014 Available online 26 April 2014 Keywords: Leymus rDNA Polyploidy Evolutionary pattern Geographic origin

a b s t r a c t Ribosomal ITS polymorphism and its ancestral genome origin of polyploid Leymus were examined to infer the evolutionary outcome of rDNA gene following allopolyploid speciation and to elucidate the geographic pattern of ITS variation. The results demonstrated that different polyploids have experienced varying fates, including maintenance or homogenization of divergent arrays, occurrence of chimeric repeats and potential pseudogenes. Our data suggested that (1) the Ns, P/F, and St genomic types in Leymus were originated from Psathyrostachys, Agropyron/Eremopyrum, and Pseudoroegneria, respectively; (2) the occurrence of a higher proportion of Leymus species with predominant uniparental rDNA type might associate with the segmental allopolyploid origin, nucleolar dominance of alloploids, and rapid radiation of Leymus; (3) maintenance of multiple parental ITS types in allopolyploid might result from long generation times associated to vegetative multiplication, number and chromosomal location of ribosomal loci and/or recurrent hybridization; (4) the rDNA genealogical structure of Leymus species might associate with the geographic origins; and (5) ITS sequence clade shared by Leymus species from Central Asia, North America, and Nordic might be an outcome of ancestral ITS homogenization. Our results shed new light on understanding evolutionary outcomes of rDNA following allopolyploid speciation and geographic isolation. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction Ribosomal RNA is the central component of ribosomes. In eukaryote genomes, the 18S, 5.8S, and 26S rRNA genes (rDNA), which are separated by internal transcribed spacers ITS1 and ITS2, are organized into a shared transcription unit, and repeated transcription units form multigene families in tandem arrays on one or several non-homologous chromosomes (Eickbush and Eickbush, 2007). The genes are highly conserved in both linear sequences and secondary structures even between eukaryotes and prokaryotes. ITS sequences have a nucleotide substitution rate high enough to generate intra- and inter-specific variability, ⇑ Corresponding author at: Triticeae Research Institute, Sichuan Agricultural University, Wenjiang 611130, Sichuan, China. E-mail address: [email protected] (Y.-H. Zhou). 1 These authors contributed equally. http://dx.doi.org/10.1016/j.ympev.2014.04.016 1055-7903/Ó 2014 Elsevier Inc. All rights reserved.

making them most commonly used for phylogenetic reconstruction (Álvarez and Wendel, 2003). The accumulation of such variability and hence the observed divergence among nrDNA paralogs are often affected by the process of concerted evolution. Although the mechanisms of concerted evolution are not entirely clear, unequal crossing over and biased gene conversion were the principal explanation of this process (Eickbush and Eickbush, 2007). Concerted evolution rapidly homogenizes the sequences of ribosomal DNA tandem repeats, such that sequences within a lineage are usually identical (Arnheim et al., 1980). It has been suggested that purifying selection was also likely responsible for high conservation of the coding regions (Nei and Rooney, 2005). However, recent studies have shown that presence of multiple paralogs, recombinant mosaic sequences or a mixture of both within a genome are phenomena of ITS evolution (Álvarez and Wendel, 2003; Bao et al., 2010; Xiao et al., 2010). Nei and Rooney (2005) pointed out that ribosomal genes or spacers have undergone at

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the most partial conversion or been subjected to a mixed process of concerted and birth-and-death evolution. Analysis of rDNA sequences in Ensis has also demonstrated that birth-and-death processes might be responsible for evolution of the ribosomal genes and ITS regions (Vierna et al., 2010). Under a birth-and-death model, gene duplications generate new genes that can persist in the genome for long time, degenerate into pseudogenes, or even being deleted. Moreover, the evolution of rDNA in hybrids or allopolyploids may be complex and several evolutionary outcomes were observed which differ in the way the parental rDNA loci interact. In some species, such as Arabidopsis (O’Kane et al., 1996) and Triticum (Zhang et al., 2002), two divergent rDNA copies may be retained and evolve independently without interaction. In other cases, such as in allopolyploids of cotton (Wendel et al., 1995), Nicotiana (Volkov et al., 1999), and Tragopogon (Lim et al., 2008), one rDNA type would be lost either through loss of an entire duplicated array or via interlocus homogenization, the latter even comprising directional concerted evolution. The third evolutionary pathway is a mosaic of two different rDNA types, potentially homogenizing to a new rDNA type unlike that of either parent donor. Obviously, if rDNA have undergone incomplete concerted evolution or been subjected to a mixed process of concerted and birth-and-death evolution, different types of paralogous and orthologous rDNA sequences may be present in the genome, often including silenced and non-functional rDNA sequences referred to as pseudogenes. The increasing evidences involving intraspecific rDNA polymorphisms highlight the need to determine the effect these have on the level of homogenization and the subsequent evolution of these repeated sequences within a species. Kadmon and Allouche (2007) emphasized the role of area and geographical isolation as the basic determinants of species diversity and diversification. Heterogeneous ecological habitats following geographical isolation are important in determining genetic differentiation among species and even promoting speciation (Kadmon and Allouche, 2007; Wen, 1999). Integrating phylogenetic information and evolutionary pattern of gene sequences with geographical distributions and ecological habitats of closely related species can be used to reveal the relative evolutionary importance of ecological divergence and geographical isolation (Graham et al., 2004). Despite the concerted and birth-and-death evolution, accumulated evidences showed that ITS polymorphism within individual should not be considered as an exceptional occurrence. Apart from the fact that ITS sequences have been used as an efficient tool for phylogenetic studies and have also been proven successful in detecting hybridization events (Sang et al., 1995; Liu et al., 2006; Sha et al., 2008), it has been reported that significant intraspecific divergence in the ITS region was response to geographical isolation (Aguilar et al., 1999; Feliner et al., 2004; Pramual et al., 2012). Moreover, Aguilar and Feliner (2003) showed that the ITS sequences of Armeriamaderensis species have synapomorphic changes and proposed that geographically isolated organisms should allow more substitutions in the ITS region to be preserved against gene flow from other congeners. The genus Leymus in the wheat tribe (Poaceae: Triticeae) includes about 30 species that distribute from the arctic to the subtropics and tropic alpine regions, from the seacoast to the Qinghai-Tibetan Plateau. All the Leymus species are considered to be allopolyploids (tetraploid, hexaploid, octoploid, decaploid, and dodecaploid) with two basic genomes, Ns and Xm. The Ns was originated from Psathyrostachys, while Xm represented a genome of unknown origin (Wang et al., 1994). To date, the St, Ns, Eb/Ee, P, and F genomes have been presumed as the potential donor of the Xm genome (Shiotani, 1968; Dewey, 1984; Löve, 1984; Zhang and Dvorak, 1991; Sun et al., 1995; Fan et al., 2009; Sha et al., 2010). Morphologically, Leymus species exhibit large variation with

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absent to strong rhizomes, single to multiple spikelets per node, erectly involute to loosely flat leaf, and subulate to lanceolate to absent glumes. The natural habitats of Leymus species, accompanied by geographic dispersal, include saline or alkaline lands, dry or semidry areas, as well as shady and moist forests. As heterogeneous ecological habitats following widely geographic distribution exhibited in Leymus, an unavoidable question concerns whether the pattern of rDNA variation is associated with geographic origin. In this study, we used cloning and sequencing of multiple repeats per individual to examine ITS polymorphism within and among Leymus allopolyploids and assess nucleotide diversity among homologous ITS repeats and their ancestral genome origins. Our objectives were to infer the evolutionary outcome of reuniting two divergent ITS repeat types in Leymus allopolyploids and elucidate the geographic pattern of ITS genetic variation within Leymus. 2. Materials and methods 2.1. Plant materials ITS region of 25 species and one variety of Leymus (including a total of 28 Leymus accessions) and 11 diploid taxa representing the St, Ns, E, P and F genomes were sequenced. ITS sequences for 24 accessions representing other 19 diploid Triticeae genomes along with Bromus inermis were obtained from published data (Hsiao et al., 1995). In addition, the sequences from four published ITS sequence (L. angustus, EF601999 and EF602001; L. chinensis, EF601991; L. tianschanicus, EF602025) (Sha et al., 2008) were also included in analysis. Plant materials with accession numbers, genomic constitutions, geographical origins, and GenBank identification numbers are presented in Table S1. The seed materials of Leymus with PI and W6 numbers were kindly provided by American National Plant Germplasm System (Pullman, Washington, USA), and Leymus duthiei var. longearistatus was kindly provided by Dr. S. Sakamoto (Kyoto University, Japan). The seed materials of Leymus with ZY and Y numbers were collected from the field by the authors of this paper. The plants and voucher specimens of the Leymus species are deposited at the Herbarium of Triticeae Research Institute, Sichuan Agricultural University, China (SAUTI). 2.2. DNA extraction, ITS amplification and sequencing DNA extraction followed a standard CTAB protocol (Doyle and Doyle, 1987) from one accession per species. The ITS region of nuclear ribosomal DNA was amplified by PCR using the primers of ITS4 and ITS5 (Hsiao et al., 1995). To decrease the chance of PCR drift and PCR selection, a 75 ll reaction mixture was separated into five reactions, and PCR products were pooled together after amplification. The amplification was carried out in a 15 ll reaction mixture containing 0.3 U of high-fidelity Ex Taq DNA polymerase (TaKaRa Biotechnology Co. Ltd., Dalian, China), 1 reaction buffer, 1.5 mM MgCl2, 1.2 mM of each dNTP, 0.5 lM of each primer, with an addition of 8% dimethylsulfoxide (DMSO) and water to the final volume. Sequence artifacts may arise due to Taq DNA polymerase error in PCR-based rDNA analysis (Qiu et al., 2001). If the putative rDNA chimerical sequence was obtained using above high-fidelity Ex Taq DNA polymerase, Pfu DNA polymerase (Promega) was used to exclude the potential risk of high-fidelity Ex Taq DNA polymerase error. PCR products were cloned into the pMD18-T vector (TaKaRa) following the manufacture’s instruction. The cloned PCR products were sequenced in both directions by Sunbiotech Company (Beijing, China). To avoid the influence of higher GC content during ITS sequencing, 8% DMSO was added to the sequencing

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reactions. The sequences for each accession of prior presumed donor were determined based on at least 5 independent clones. For each Leymus taxa, 11–15 randomly selected clones were sequenced to recover all the possible ITS sequences from the donor species.

2.3. Data analysis Multiple sequences were aligned using ClustalX (Thompson et al., 1999) with default parameters and additional manual edits to minimize gaps. To reduce the possible impact of PCR artifacts, unique substitutions in single clone was removed from the analysis. The basic sequence statistics, including nucleotide frequencies, GC content, transition/transversion (ns:nv) ratio and variability in different regions of the sequences were calculated by MEGA 4 (Tamura et al., 2007). Ribosomal pseudogenes have been identified based on mutations in conserved regions or nucleotide diversity in a nucleotide sequence (Thornhill et al., 2007). Nucleotide diversity was estimated by Tajima’s p in DnaSP 4.10.9 (Rozas et al., 2005). Highly conserved 5.8S motifs (M1: CGATGAAGAACGyAGC; M2: GAATTGCAGAAwyC; M3: TTTGAAyGCA, where y = C/T and w = A/ T) in sequence alignments were checked with MEME package (http://www.sdsc.edu/MEME/meme/website/html). Sequences not matching these motifs at all positions were considered to be pseudogenes. Recombination was examined by the maximum chi-square method in the RecombiTEST (Piganeau et al., 2004), the pairwise homoplasy index (PHI) in SplitsTree (Huson and Bryant, 2006), and GARD (genetic algorithm recombination detection) method in HYPHY (Pond and Frost, 2005). To estimate the number of haplotype, clone genealogy was conducted by coalescent simulations using the Median-Joining model as implemented in the Network v4.0 software (Bandelt et al., 1999). Demographic history in the sampled Leymus lineages was estimated using mismatch distributions and Fu’s Fs statistics in the program DnaSP 4.10.9 (Rozas et al., 2005). Significance of Fs-values was calculated with the simulated distribution of random samples (1000 steps). Three data matrixes (Matrix I, II, and III) were separately used to carry out phylogenetic analyses, and putative pseudogenes were excluded. Matrix I, including all the ITS sequences of Leymus and the sequences from Psathyrostachys (Ns), Pseudoroegneria (St), Thinopyrum bessarabicum (Eb), Lophopyrum elongatum (Ee), Agropyron (P), and Eremopyrum (F), was used to generate a phylogenetic network using SplitsTree 4 (Huson and Bryant, 2006) with the NeighborNet algorithm (Bryant and Moulton, 2004). Phylogenetic networks account for the evolutionary role of reticulate events, presumably arising from recombination, by demonstrating conflicting signal as loops or reticulations (Hudson, 1998). Matrix II, including all the Ns genomic sequences of Leymus and Psathyrostachys, was used to create a maximum likelihood (ML) tree. To further estimate the possible origin of Xm genomic ITS sequences in Leymus, a third matrix (Matrix III), including all the non-Ns genomic sequences of Leymus and those sequences from 33 diploid taxa representing 23 basic genomes in Triticeae, were used to generate a ML tree. Bromus inermis was used as outgroup. ML analysis was performed using PAUP*4.0b10 (Swofford DL, Sinauer Associates, http://www.sinauer.com). The evolutionary model was determined using ModelTest v3.0 with Akaike information criterion (AIC) (Posada and Crandall, 1998). The optimal models identified for both Matrix II and Matrix III were GTR + G. ML heuristic searches were performed with 100 random addition sequence replications and TBR branch swapping algorithm. The robustness of the trees was estimated by bootstrap support (BS) (Felsenstein, 1985). BS-value less than 50% was not included in figures.

3. Results and discussion 3.1. ITS sequence analysis Twenty-five species and one variety of Leymus (including a total of 28 Leymus accessions) were sampled and were first analyzed with its presumed diploid donor from Psathyrostachys, Pseudoroegneria, Thinopyrum, Lophopyrum, Agropyron, and Eremopyrum. Eleven to fifteen clones for each accession of Leymus and five clones for each presumed donor species were sequenced. Consequently, a total of 441 ITS sequences were obtained. Analysis of the three conserved motifs within the 5.8S region identified 47 potential pseudogenes from 386 ITS sequences in Leymus, 3 potential pseudogenes from 55 ITS sequences in diploid donor species. It was suggested that potential pseudogenes showed increased sequence diversity and might reflect relaxed selection due to little functional constraint (Wernegreen and Moran, 2000; Xiao et al., 2010). Consistent with this suggestion, the present result confirmed that the sequence diversity estimated by nucleotide diversity (p) across the ITS region of all putative pseudogenes (p = 0.0270) was remarkably higher than that for presumed functional sequences (p = 0.0173), and the Tajima’s D value in putative pseudogenes ( 2.2424) (P = 0.05) is less negative than that in presumed functional sequences ( 2.5832) (P < 0.001). These putative pseudogenic ITS types were excluded in the further analyses because the presence of pseudogenic ITS sequence may lead to altered phylogenetic inference. Comparison of the remaining 391 sequence showed that the DNA sequence ranged in length from 212 bp to 225 bp in the ITS1 region, from 215 to 218 bp in the ITS2 region, and 164 bp long in the 5.8S rDNA. The GC content ranged from 59.3% (clone LMU593) to 64.4% (clone LAN303) for the ITS1 region, and from 53.6% (LSH193) to 61.0% (LTR142, LTI544, and LTI551) for the 5.8S region, and from 60.3% (LPE267) to 66.6% (LLE204) for the ITS2 region.

3.2. Ancestral genome origin of ITS sequence When putative pseudogenic ITS sequences were excluded, network analysis of 396 sequences (including five published ITS sequences: L. angustus, EF601999 and EF602001; L. chinensis, EF601991; L. tianschanicus, EF602025; Psathyrostachys fragilis, L36498) showed that the ITS sequences of Leymus species were split into the Ns and non-Ns genomic types (Fig. 1A). As expected, the Ns genomic types of Leymus were grouped with the sequences from Psathyrostachys, indicating that Ns genomic types of Leymus were originated from Psathyrostachys species, because Psathyrostachys as the Ns genome donor to Leymus has been confirmed (Fan et al., 2009; Wang and Jensen, 1994; Zhang et al., 2006). The non-Ns genomic types were scattered into different network clade. To further estimate the origin of non-Ns genomic types in Leymus, all the non-Ns genomic sequences were included in ML analysis under the GTR + G substitution model, together with one representative of ITS sequence from 32 diploid taxa representing 23 basic genomes in Triticeae. ML analysis yielded a log-likelihood score of 2911.588 for the best tree (gamma shape parameter = 0.511; the assumed nucleotide frequencies: A = 0.2263, C = 0.2808, G = 0.2936, T = 0.1993). Two distinct non-Ns genomic ITS types, which were separately grouped with the sequences from diploid species, were recognized in ML analysis (Clade I and II) (Fig. 2). In Clade I, eleven non-Ns genomic haplotypes, including nine from L. tianschanicus (LTI541, LTI544, LTI545, LTI547, LTI548, LTI551, LTI553, LTI554, and EF602025) and two from L. angustus (EF602001 and EF601999), were grouped with those from Agropyron (P) and Eremopyrum (F) in 72% bootstrap support. Previous single-copy nuclear Acc1 gene data suggested that the

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Fig. 1. Phylogenetic network inferred from a total of 396 ITS sequences from Leymus and its putative diploid donor species (A). The sequences fall into two major clades, with the Ns genomic ITS types to the left of the dotted line and the non-Ns genomic ITS types to the right. The numbers after species names refer to the clone number. The sequences from Psathyrostachys species are highlighted with red box. The sequences labeled by the number of asterisk indicate three type of potentially chimeric ITS repeats. Abbreviations of species names are listed in the bottom right corner of the figure. Different color labeled the geographic information of Leymus species. Boxed subset provides mismatch distribution and Fs statistic for the Ns genomic ITS types of Leymus species (B). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

P/F genome was closely related to the Xm genome (Fan et al., 2009), which also be favored by chroloplast DNA sequence (Sha et al., 2010; Culumber et al., 2011). Taking all into consideration, it can be suggested that the P/F genome might contribute to the origin of the Xm genome of Leymus species, and the eleven nonNs genomic ITS types in Leymus might originate from Agropyron/ Eremopyrum. The ML analysis also showed that ten non-Ns genomic sequences including eight from L. angustus (LAN302, LAN303, LAN308, LAN309, LAN311, LAN313, LAN319, LAN320) and two from L. chinensis (LCH260 and EF601991) were clustered with Pseudoroegneria strigosa (PST561) (Fig. 2, the Clade II). The non-Ns genomic sequences had a 4-bp TGGG insertion, while this insertion was absent in the Ns and P/F-like genomic type of Leymus. Single-copy nuclear DMC1 gene data showed that the St-copy sequences of L. angustus and L. chinensis were grouped with Pseudoroegneria with moderate statistic support (73% bootstrap support and 90% posterior probability), indicating that the Pseudoroegneria genome might contribute to Leymus species by recurrent hybridization (Sha et al., unpublished data). Therefore, the presence of St genomic ITS types in Leymus represent contribution of the Pseudoroegneria genome. Since Leymus has originated through polyploidization speciation, chimeric ITS repeat may be produced by intergenomic recombination following polyploidization. Phylogenetic network analysis also showed three reticulate clusters formed by ten sequences (labeled by asterisk), which is an outcome of potential recombination events (LQI222 for recombination I; LTI549, LTI550, and LTI552 for recombination II; LAN301, LAN307, LAN317, LQI228, LTI555, and LRAM382 for recombination III) (Fig. 1A). These ten sequences were scattered into distinct clades and did not group with any ITS sequence from diploid species in ML phylogenetic tree

(Fig. 2). The PHI test found significantly recombination (p = 0.00086) for these ten sequences, while when these ten sequences were excluded from the phylogenetic network, the p value for the PHI test is not significant (p = 0.5567). Recombination was also found using the maximum Chi-Square test (P = 0.039). The presence of recombinant ITS type could reflect true biological recombination events or artifacts of PCR (Kovarik et al., 2005). The chance of artifacts of PCR has been significantly minimized in experiments since PCR reaction mixture with DMSO and different DNA polymerases were used for amplification and cloning. A lower proportion of recombinant molecules (10 of 443 sequenced clones, i.e., 2.26%) were recovered from sampled plants, indicating that this problem was indeed limited. Therefore, we prefer the explanation of recombinant genomic rDNA molecules. One potential breakpoint at position 196 (near the binding site between ITS1 and 5.8S region) in the putative recombinant sequences was found using HyPhy GARD analyses. This finding further supports the presence of recombination identified by PHI and maximum Chi-Square test, indicating that recombinant may occurred between ITS1 and ITS2 region. However, it is difficult to estimate the ancestral/parental origins of recombinant genomic rDNA repeat with extant diploids because a precise knowledge of the ancestral conditions involving polyploidy speciation was absent. 3.3. Predominant Ns genomic rDNA type in Leymus In 24 of 25 Leymus polyploid species examined (all except LTI), ITS types of the Ns genome are far more than that of the Xm genome (the majority of species had only Ns genomic type of ITS sequences), indicating that Leymus have a higher proportion of species with predominant Ns genomic rDNA type. There are three

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Fig. 2. Maximum-likelihood tree derived from ITS sequences from non-Ns genomic haplotypes of Leymus and its related diploid taxa representing 23 basic genomes in Triticeae. Numbers above nodes are bootstrap values P50%. The capital letters in bracket indicate the genome type of the diploid Triticeae plants.

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possible explanations for this phenomenon. The first one is that origin of the Xm genome might also associate with that of the Ns genome. DNA hybridization analysis of L. arenarius (Bödvarsdóttir and Anamthawat-Jónsson, 2003) and several other taxa (Zhang and Dvorak, 1991) suggested that Leymus may be a segmental allopolyploid derived from two distinct Psathyrostachys species. It is thus possible that predominant Ns genomic rDNA type is present in a segmental allopolyploid with majority of Ns-genome material. Given that predominant Ns genomic rDNA type might result from a segmental allopolyploid origin of Leymus species, the presence of the P/F and St genomic rDNA types in several Leymus might result from recurrent hybridization or incomplete lineage sorting. Recurrent hybridization hypothesis is likely because sympatric distribution among Agropyron/Eremopyrum, Pseudoroegneria, and Leymus species have provide an opportunity in physical proximity for hybridization events. The hypothesis of incomplete lineage sorting may also explain the occurrence of the P/F and St genomic rDNA types in Leymus because a history of incomplete lineage sorting has been reported as a not surprising phenomena not only for diploid lineages (Mason-Gamer and Kellogg, 1996; Escobar et al., 2011) but also for polyploid species (Fan et al., 2009, 2013; Mahelka and Kopecky´, 2010) within the tribe Triticeae. The second explanation is that the predominance of Ns genomic rDNA type may be an epigenetic outcome accompanied by polyploidization. Epigenetic expression patterns of ribosomal loci may influence subsequent evolutionary patterns of rDNA homogenization and retention (Kovarik et al., 2008; Bao et al., 2010). Kovarik et al. (2008) suggested that epigenetic silencing of rDNA loci makes them less vulnerable to homogenization and more likely to be lost, perhaps thousands or millions of years later. It has been suggested that Leymus originated about 11–12 million year ago (MYA) (Fan et al., 2009). If epigenetic bias of the rDNA loci due to nucleolar dominance occurred in the Xm genome in Leymus, epigenetic silencing of rDNA loci on the Xm genome should be more subject to ultimate mutational obliteration. Nucleolar dominance in the Xm genome under a relatively long-time evolutionary history of Leymus might account for the predominance of Ns genomic rDNA type. Thirdly, it has been reported that due to biased concerted evolution after allopolyploidization, one of the parental rDNA units can be eliminated from the genome of initial hybrids, suggesting the predominance of another parental rDNA type (Volkov et al., 1999; Matyasek et al., 2007). Considering the possibility of the elimination and/or replacement of rDNA units following biased concerted evolution (Volkov et al., 1999), the predominance of Ns genomic rDNA type in Leymus may be an outcome of the elimination and/or replacement of Xm genomic rDNA units. However, the question is whether a higher proportion of species (In 24 of 25 polyploid species) with predominant Ns genomic rDNA type was attributed to the possibility that all sampled Leymus species have undergone the elimination and/or replacement of Xm genomic rDNA units following biased concerted evolution after allopolyploidization. Because sequence elimination and/or replacement (e.g., Xm genomic rDNA) is likely to be an irreversible process, we can envision a scenario that a monophyletic allopolyploid group following rapid radiation events is enriched for lineages with unidirectional homogenized rDNA repeats (e.g., Ns genomic rDNA). Previous studies based on single-copy nuclear Acc1 data has suggested that most extant Leymus species are considered to be derived from the rapid diversification of an ancestor lineage during the Miocene (Fan et al., 2009). Rapid radiation is often characterized by star-like topology, little sequence variation, rapid speciation, and diverse morphology and ecological habitats (Seehausen, 2004). In this study, all the Ns genomic ITS haplotypes in phylogenetic network formed one star-like radiation and had generally relatively low sequence divergence (ranging from 0.2% in many cases to 4.3%). Unimodal mismatch distributions and significantly nega-

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tive values of Fs statistics (Fs = 2.3261, P < 0.05) within star-like topology suggested rapid lineage expansions (Fig. 1B). Consistent with the Acc1 gene data, the present results also indicated the occurrence of rapid diversification in Leymus. Given rapid radiation, an initial allopolyploid lineage with one of parental ITS repeats (e.g., Ns genomic ITS repeats) due to biased concerted evolution was rapidly diversified into ecologically different Leymus species, and each maintained the same predominant genomic rDNA type as ancestral lineage. Rapid radiation thus might promote the rapid diversification of an ancestor lineage with predominant Ns genomic rDNA type, and subsequent attribute to a higher proportion of species with predominant Ns genomic rDNA type in Leymus. 3.4. Geographical structure of ITS variation in Leymus Under the GTR + G substitution model, ML analysis of all the Ns genomic ITS sequences (putative pseudogenic ITS sequences were excluded) generated a log-likelihood score of 2273.459 for the best tree (gamma shape parameter = 2.249; the assumed nucleotide frequencies: A = 0.2264, C = 0.3005, G = 0.2690, T = 0.2041). In the ML tree, ITS sequences from Leymus species were split into twelve groups (Fig. 3). Group I, II, VII, VIII, and IX are comprised of the sequences from the Qinghai-Tibetan Plateau. Group III included all the sequences from Central Asia and northwest China, and seven sequences from North American L. innovatus (LIN61, LIN62, LIN64, LIN65, LIN66, LIN69, and LIN70), as well as four sequences from Nordic. Group IV consisted of the sequences of L. arenarius from Nordic. Group V included the sequences of L. coreanus from the Far East of Russia. Group VI contained all the sequences from North America. Group X is comprised of the sequences of L. chinensis from north China. Group XI included the sequences of L. duthiei from the Hengduan Mountainous regions of China and those of L. duthiei var. longearistatus from Japan. Group XII contained the sequences of L. komarovii from northeast China. These results indicated that the ITS genealogical structure is congruent with the geographic ranges of Leymus speices. Heterogeneous environment, gene-flow, unidirectional concerted evolution, and rapid gene turnover may associate with the geographic structure of ITS variation in Leymus. Wen (1999) pointed out that similar natural habitats may have exerted similar selection pressures and led to similar morphological adaptations among species, while the different habitats could have contributed to the morphological distinctions. Morphologically, the Leymus species from the QinghaiTibetan Plateau usually have leaf sheath glabrous, rachis and rachilla pubescent, and glume shorter than spikelet. Most Leymus species from Central Asia have strong rhizomes and tall plant figures. Whereas the Leymus species distributed in warm and moist climate conditions (e.g., L. duthiei and L. duthiei var. longearistatus) are distinguished from the other Leymus species by its highly reduced glumes and loosely flat leaf. Similar natural selection not only promotes similar morphological characteristics in Leymus but also triggers genetic homogeneity in rDNA variations. Divergent natural selection due to heterogeneous environment drives genetic divergence of Leymus lineages and has also attributed to the evolutionary divergence of rDNA gene. Gene-flow via recurrent hybridization has been demonstrated to be frequent in the genus (Sha et al., 2008; Fan et al., 2009), and thus can cause different ITS copies to meet within the same genome. Gene-flow and concomitant contacts between different ribotypes from sympatric Leymus species might share sympatric rDNA allele and subsequent strengthen allopatric genetic differentiation. The present ML analysis showed that the Ns genomic ITS sequences of all Leymus species from the Qinghai-Tibetan Plateau, Central Asia, and North American did not form monophyly but form between-species gene clustering pattern (Fig. 3, Group I, II, III and VI). The Median-Joining

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Fig. 3. Maximum-likelihood tree inferred from Ns genomic ITS sequence of Leymus and Psathyrostachys species. Numbers with bold above nodes are bootstrap values P50%. Different color labeled the geographic information of Leymus species. Abbreviations of species names are listed in the bottom right corner of the figure. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

(MJ) network further confirmed that the ITS sequences from different species with sympatric distribution shared the same haplotype, and in many case the ITS sequences of a species were scattered in

different clusters (Figs. 4 and 5). Moreover, some sequences such as LFL171 (Fig. 4A), LMU597 (Fig. 4A), LSA123 (Fig. 5A), LSA125 (Fig. 5A), LTI543 (Fig. 5B) and LTI546 (Fig. 5B) were differentiated

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Fig. 4. Median-Joining networks (MJ) based on the Ns genomic ITS sequence of Leymus species from the Qinghai-Tibetan Plateau. MJ networks of Ns genomic ITS sequence in the Group I of ML tree (A). MJ networks of Ns genomic ITS sequence in the Group III of ML tree (B). Haplotypes in network are represented by circles. Numbers along network branches indicate the position of mutation between nodes. Abbreviations of species names are listed in the bottom right corner of the figure.

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Fig. 5. Median-Joining networks (MJ) based on the Ns genomic ITS sequence of Leymus species from North American and Central Asia. MJ networks of Ns genomic ITS sequence of Leymus species from North America (A). MJ networks of Ns genomic ITS sequence of Leymus species from Central Asia (B). Haplotypes in network are represented by circles. Numbers along network branches indicate the position of mutation between nodes. Abbreviations of species names are listed in the figure.

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from other sequences by more than 10 mutational steps, which is indicative of an excess of rare variants created by recurrent hybridization between sympatric Leymus species. Gene-flow by recurrent hybridization among sympatric Leymus species can inhibit rDNA evolution by preventing natural selection from establishing local genetic differences, while limited gene-flow due to significant geographical barriers (e.g., the Qinghai-Tibetan Plateau and the Bering Strait) appeared to be associated with strong genetic differentiation among the different geographical groups. Unidirectional concerted evolution could be related to geographical differentiation among Leymus species, since the monophyly of Ns genomic ITS sequences within Leymus species (e. g. L. komarovii, L. coreanus, L. duthiei and L. duthiei var. longearistatus) was identified in phylogenetic tree. Because rapid gene turnover following birth-and-death evolution can lead to creation of species-specific gene clusters, we cannot rule out the possibility that the monophyly of ITS sequences within Leymus species may be resulted from rapid rDNA gene duplication and loss. The clear separation of ITS sequences according to their geographic areas may be a consequence of rapid gene turnover. However, the intragenomic polymorphisms of L. innovatus and L. arenarius do not readily fit the geographical structure of ITS variation (Fig. 3, Group III). The ITS genealogical analysis showed that five sequences from North American L. innovatus (LIN61, LIN64, LIN65, LIN69, and LIN70) and four sequences from Nordic L. arenarius (LAR10, LAR13, LAR20, and LAR21) were grouped with those sequences from Central Asian Leymus species (Figs. 3 and 5B), and LAR22 was sister to those sequences from L. komarovii (Fig. 3). Two possible hypotheses for these cases that are not fully concordant with the geographical structure can be envisaged. One hypothesis is that retardation in concerted evolution might cause old gene-flow events to remain tractable. Data from morphology (Dewey, 1984), geographic distribution (Zhi and Teng, 2005), and molecular evidence (Fan et al., 2009) have suggested that Leymus originated in Central Asia. Ancestral polyploidization events occurred in Central Asia could lead to the presence of retardation in concerted evolution. It is thus possible those ITS sequences (LIN61, LIN64, LIN65, LIN69, LIN70, LAR10, LAR13, LAR20, and LAR21) scattered in Central Asia sequences may be representative of homogenization of ancestral ITS variation. Secondly, considering the fact that the habitats preferred by most Central Asia Leymus species are cold and dry alpine meadow, it cannot be ruled out that ITS sequence clade shared by Leymus growing in cold climates (North American alpine, Nordic, and Central Asian alpine) might have been acquired independently by convergence.

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constitutions? Several factors may be responsible for changing the tempo of intergenomic homogenization. Vegetative reproduction may be one reason for maintenance of parental ITS sequences (Sang et al., 1995), because frequent reproduction via strong rhizomes in Leymus may prolong generation time significantly. L. angustus, L. tianschanicus, and L. chinensis have stronger rhizomes than many other species which exhibited single type of ITS sequences. Difference in the number and chromosomal localities of rDNA arrays in Leymus may be another factor that affects the tempo of intergenomic homogenization. For example, in situ hybridization experiments have shown that L. angustus has a total of twelve 45S rDNA sites, which appeared at both ends of some chromosomes (Ørgaard and Heslop-Harrison, 1994). Yang et al. (2007) reported that in L. duthiei and L. duthiei var. longearistatus, four chromosomes carried 45S rDNA loci, which appeared at the termini of the short arms. It can also not be ruled out that recurrent hybridization with different populations of P genome lineage and introgression of St-genome during polyploidization might prolong generation time to promote the maintenance of parental sequences, because polyploids often originated many times from independent populations of their progenitor, especially when polyploids and their parental species coexist on a large geographic scale. In fact, L. angustus, L. tianschanicus, L. chinensis and the P and St genome lineages have a sympatric distribution in Central Asia, which provides the physical proximity for hybridization. The other factors that may affect the tempo of concerted evolution, however, cannot be assessed here because the epigenetic modifications, locus loss, locus jumping, copy amplification, sequence transposition, gene conversion by chromatid exchange, and genetic interaction of rDNA arrays in Leymus are unknown (Sang et al., 1995; Kovarik et al., 2008). Acknowledgments This study was funded by the National Natural Science Foundation of China (Nos. 30900087, 31200252, 31270243, 31101151, 31301349), the research fund for Large-scale Scientific Facilities of the Chinese Academy of Sciences (Grant No. 2009LSF-GBOWS-01), Special Fund for Agro-Scientific Research in the Public Interest of China (No. 201003021), and the Science and Technology Bureau (No. 2060503) and Education Bureau of Sichuan Province. We are very grateful to American National Plant Germplasm System (Pullman, Washington, USA) providing the part seed materials. We also thank three anonymous reviewers for their very useful comments on the manuscript.

3.5. Maintenance of parental types of ITS repeats in Leymus Appendix A. Supplementary material Concerted evolution, via unequal crossing-over or gene conversion, can homogenize different parental genomes in an allopolyploid so that only one parental genome type may be seen in the hybrid (Sang et al., 1995). In this study, only the Ns type of ITS repeat was found in twenty-one species, which is in agreement with the suggestion that concerted evolution is the main mechanism maintaining uniformity within members of the Leymus rDNA multigene family. The monophyly of Ns genomic ITS sequences within L. komarovii, L. coreanus, L. duthiei and L. duthiei var. longearistatus are also indicative of unidirectional concerted evolution scenario. However, maintenance of two or more parental types of ITS repeats was observed in only four allopolyploids, L. angustus (Ns-, P-, and St-type), L. tianschanicus (Ns- and P-type), and L. chinensis (Ns- and St-type), indicating that concerted evolution has not homogenized the parental ITS sequences in these allopolyploid. Why do L. angustus, L. tianschanicus and L. chinensis fail to show intergenomic homogenization between parental loci whereas the remaining Leymus species do, despite having the same genomic

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