Untangling nucleotide diversity and evolution of the H genome in polyploid Hordeum and Elymus species based on the single copy of nuclear gene DMC1.

Numerous hybrid and polypoid species are found within the Triticeae. It has been suggested that the H subgenome of allopolyploid Elymus (wheatgrass) species originated from diploid Hordeum (barley) species, but the role of hybridization between polyploid Elymus and Hordeum has not been studied. It is not clear whether gene flow across polyploid Hordeum and Elymus species has occurred following polyploid speciation. Answering these questions will provide new insights into the formation of these polyploid species, and the potential role of gene flow among polyploid species during polyploid evolution. In order to address these questions, disrupted meiotic cDNA1 (DMC1) data from the allopolyploid StH Elymus are analyzed together with diploid and polyploid Hordeum species. Phylogenetic analysis revealed that the H copies of DMC1 sequence in some Elymus are very close to the H copies of DMC1 sequence in some polyploid Hordeum species, indicating either that the H genome in theses Elymus and polyploid Hordeum species originated from same diploid donor or that gene flow has occurred among them. Our analysis also suggested that the H genomes in Elymus species originated from limited gene pool, while H genomes in Hordeum polyploids have originated from broad gene pools. Nucleotide diversity (π) of the DMC1 sequences on H genome from polyploid species (π = 0.02083 in Elymus, π = 0.01680 in polyploid Hordeum) is higher than that in diploid Hordeum (π = 0.01488). The estimates of Tajima's D were significantly departure from the equilibrium neutral model at this locus in diploid Hordeum species (P<0.05), suggesting an excess of rare variants in diploid species which may not contribute to the origination of polyploids. Nucleotide diversity (π) of the DMC1 sequences in Elymus polyploid species (π = 0.02083) is higher than that in polyploid Hordeum (π = 0.01680), suggesting that the degree of relationships between two parents of a polyploid might be a factor affecting nucleotide diversity in allopolyploids.

Introduction

Hybridization and polyploidization have played a central role in the history of plant evolution, and contributed greatly to plant diversification and speciation [1], [2]. Much attention has been drawn to studying the evolutionary consequences of polyploid species in both genome size and contents, with the advances in molecular methods over the last two decades [3], [4]. Polyploid genome origins and evolution have also been the focus of plant evolutionists [1], [5]. Increasing evidences have demonstrated the complexity of the dynamic nature of polyploids. Many polyploids are proved to involve multiple origins in space and time [1], [5], together with introgression (or gene flow) [6]–[8]. Both multiple origins [9], [10] and gene flow [6]–[8] have been considered as the causes of shared polymorphism across ploidy level and/or phylogenetic incongruence among loci. However, whether gene flow among independent formations is regular occurrence following polyploid species have rarely been tested in ployploid taxa [11].

The tribe Triticeae contains several important cereal crops such as wheat, barley and rye, as well as forage crops. The tribe combines a wide variety of biological mechanisms and genetic systems which makes it an excellent group for research in evolution, genetic diversity, taxonomy, and speciation in plants [12]. According to Löve [13] and Dewey's [14] classification, genus Hordeum and Elymus are two relative large genera in the tribe Triticeae.

The genus Hordeum comprises 31 species (including cultivated barley, H. vulgare ssp. vulgare) and exists at the diploid, tetraploid, and hexaploid levels with a basic chromosome number x = 7. Based on cytogenetic analyses, the diploid species in Hordeum were classified into four monogenomic groups: H, I, Xa, and Xu genome group [15], [16], which were supported by isoenzyme analysis [17] and molecular data [18]–[22]. The H genome group is not only the largest genome group in Hordeum (including 14 diploid species, 7 tetraploid species, 4 hexaploid species, and 2 species existing at three ploidy levels (2×, 4×, 6×), and distributed widely from central Asia to the Americas), but also widely present in polyploid species in Elymus, Stenostachys and Pascopyrum.

Within the genus Elymus are approximately 50 allotetraploid species that combined both H and St genomes, and distributed throughout the world in non-tropical areas, from northern Greenland in the north to Tierra del Fuego in southernmost South America [23]. The St haplome originated from the genus Pseudoroegneria [14]. It has been confirmed that the H haplomes in Elymus were contributed by different Hordeum diploids [24]–[30]. Phylogenetic analyses based on phosphoenolpyruvate carboxylase, β-amylase, granule-bound starch synthase I [29] and disrupted meiotic cDNA (DMC) [30], suggested few potential Hordeum diploid species as H-genome donors to Elymus species. The tetraploid H. jubatum might have involved in the origin of StH Elymus [29]. However, the role of polyploid Hordeum species in the origin of StH Elymus remains to be studied. It is not clear whether gene flow across polyploid Hordeum and Elymus species has occurred following polyploid speciation. Recent studies led to the conclusion that the polyploid probably originated multiple times [1], [5], which are often considered as a potential source of increased genetic variation in polyploids. However, how much genetic variation is contributed by the diploid progenitors and the degree of gene flow among the independent origins are the two major factors determining the genetic diversity in polyploids. Yet, the extent and role of gene flow among polyploids in evolution remains enigmatic.

In present study, DMC1 data from the allopolyploid StH Elymus are analyzed together with diploid and polyploid Hordeum species. The objectives of this analysis are: (1) to explore the possible role of polyploid Hordeum species in the origin of StH Elymus; (2) to determine whether gene flow has occurred between polyploid Hordeum and Elymus; and (3) to examine the level of nucleotide polymorphism in the H genomes from Elymus, Hordeum diploids and polyploids. Answering these questions will provide new insights into the formation of these polyploid species, and the potential role of gene flow among polyploid species during polyploid evolution.

Materials and Methods

Samples

The present study includes 18 tetraploid (22 accessions) StH Elymus, 9 polyploid Hordeum species. All diploid Hordeum species and other diploid Triticeae species representing the St, W, P, and E genomes were included for analyses (Table 1). Bromus arvensis and B. sterilis were used as outgroups. The single copy nuclear gene disrupted meiotic cDNA (DMC1) has been applied to phylogenetic analyses in Triticeae species. The DMC1 sequences used in this study were collected from previously published sources [21], [30]–[35].

Table 1

Taxa used in this study.

Species	Ploidy	Accession no.	Genome	Origin*	GenBank accession no.	Authors
Agropyron cristatum Gaertn.	2×	H4349	P	Turkey	AF277241	Petersen & Seberg, 2000
Australopyrum retrofractum (Vickery) Á. Löve	2×	H6723	W	Australia	AF277251	Petersen & Seberg, 2000
Bromus arvensis L.		C618		NA	DQ247821	Petersen et al., 2006
Bromus sterilis L.		OSA420		Denmark	AF277234	Petersen & Seberg, 2000
Elymus canadensis L.	4×	PI 531567	StH	Alberta, Canada	EU366405, EU366406	Sha et al. 2009
Elymus caninus (L.)L	4×	H3169	StH	Västmanland, Sweden	H3169L, H3169k	Sun & Zhang, 2011
		PI314621	StH	NA	EU366407, EU366408	Sha et al., 2009
Elymus confusus E. Desy	4×	PI 598463	StH	Russian Federation	598463k	Sun & Zhang, 2011
		W6 21505	StH	NA	GQ855188	Wang et al., unpublished
Elymus cordilleranus Davidse &Pohl	4×	H6486	StH	Cajamarca, Peru	H6486k, H6486Y	Sun & Zhang, 2011
Elymus dentatus (Hook. F.) Tzvelev	4×	PI 628702	StH	Altay, Russian	PI 628702	Sun & Zhang, 2011
Elymus fibrosus (Schrenk) Tzcelev	4×	H10339	StH	Pelkosniemi, Finland	H10339K	Sun & Zhang, 2011
Elymus gayanus Desv.	4×	W6-13828	StH	Santa Cruz, Argentia	W6-13828L,W6-13828K	Sun & Zhang, 2011
Elymus hystrix L.	4×	H5495	StH	Canada	H5495R, H5495K	Sun & Zhang, 2011
	4×	PI531616	StH	NA	EU366415, EU366416	Sha et al. 2009
Elymus lanceolatus (Scribn. & Smith) Gould	4×	PI 236663	StH	Maryland, United States	PI 236663K	Sun & Zhang, 2011
Elymus multisetus (JG Sm.) Burtt Davy	4×	W6-20963	StH	California, United States	W6-20963Y, W6-20963R	Sun & Zhang, 2011
Elymus sibiricus L.	4×	PI 619579	StH	Xinjiang, China	GQ855198, EU366409	Sha et al. 2009
Elymus tranchycaulus (Link) Gould ex Shinn	4×	PI 537323	StH	Utah, United States	PI537323L	Sun & Zhang, 2011
		PI372500	StH	NA	GQ855191	Wang et al., unpublished
Elymus transhyrcanus	4×	PI383579	StH	NA	GQ855193, GQ855194	Wang et al., unpublished
Elymus violaceus (Hormem.) Feilberg	4×	H10588	StH	Julianehåb, Greenland	H10588Y	Sun & Zhang, 2011
Elymus virescens (Piper) Gould	4×	H10584	StH	Julianehåb, Greenland	H10584Y, H10584K	Sun & Zhang, 2011
Elymus virginicus	4×	PI490361	StH	NA	GQ855195, GQ855196	Wang et al., unpbulished
Elymus wawawaiensis J. Carlson ex Barkworth	4×	PI 610984	StH	Washington, United States	GQ855197, EU366410	Sha et al. 2009
Elymus wiegandii Femald	4×	PI 531708	StH	Aylwin, Quebec, Canada	PI531708K	Sun & Zhang, 2011
Hordeum arizonicum Covas	6×	H2144	HHH	Mexico	GU734674, GU734675, GU734676	Wang & Sun 2011
H. bogdanii Wilensky	2×	H4014	H	Pakistan	AY137412	Petersen & Seberg, 2003
H. brachyantherum Nevski subsp. brachyantherum	4×	H2348	HH	U.S.A.	GU734677, GU734678	Wang &Sun 2011
H. brachyantherum Nevski subsp. californicum (Covas & Stebbins) Bothmer, N. Jacobsen& Seberg	2×	H1942	H	U.S.A.	AF277260	Petersen & Seberg, 2003
H. brevisubulatum (Trin.) Link subsp. violaceum Boiss. & Hohen.	2×	H315	H	Iran	AY137396	Petersen & Seberg, 2003
H. bulbosum L.	2×	H3878	I	Italy	AY137411	Petersen & Seberg, 2003
H. chilense Roem. & Schult.	2×	H1819	H	Chile	AY137408	Petersen & Seberg, 2003
H. comosum C. Presl	2×	H1181	H	Argentina	AY137400	Petersen & Seberg, 2003
H. cordobense Bothmer, N. Jacobsen & Nicora	2×	H6429	H	Argentina	AY137415	Petersen & Seberg, 2003
H. depressum (Scribn. & J. G. Sm.) Rydb.	4×	H2008	HH	U.S.A.	GU734670, GU734671	Wang &Sun 2011
H. erectifilium Bothmer, N. Jacobsen& R.B. JØrg.	2×	H1150	H	Argentina	AF277259	Petersen & Seberg, 2003
H. euclaston Steud.	2×	H1263	H	Argentina	AY137401	Petersen & Seberg, 2003
H. flexuosum Nees ex Steud.	2×	H1133	H	Argentina	AY137399	Petersen & Seberg, 2003
H. fuegianum Bothmer, Jacobsen & Jørgensen	4×	H1418	HH	Argentina	GU734665, GU734666	Wang & Sun 2011
H. intercedens Nevski	2×	H1940	H	U.S.A	AY137409	Petersen & Seberg, 2003
H. jubatum L.	4×	H2013	HH	Mexico	GU734672, GU734673	Wang &Sun 2011
H. lechleri (Steud.) Schenck	6×	H1451	HHH	Chile	GU734667	Wang & Sun 2011
H. marinum Huds. subsp. marinum	2×	H546	Xa	Spain	AY137397	Petersen & Seberg, 2003
H. marinum Huds. subsp. gussoneanum (Parl.) Thell.	2×	H299	Xa	Bulgaria	AF277257	Petersen & Seberg, 2003
H. murinum L. subsp. glaucum (Steud.) Tzvelev	2×	H801	Xu	Iran	AF277258	Petersen & Seberg, 2003
H. muticum J. Presl	2×	H958	H	Bolivia	AY137398	Petersen & Seberg, 2003
H. parodii Covas.	6×	H1458	HHH	Argentina	GU734668, GU734669	Wang & Sun 2011
H. patagonicum (Hauman) Covas subsp. magellanicum (Parodi & Nicora) Bothmer, Giles & N. Jacobsen	2×	H6209	H	Argentina	AY137414	Petersen & Seberg, 2003
H. patagonicum (Hauman) Covas subsp. mustersii (Nicora) Bothmer, Giles & N. Jacobsen	2×	H1358	H	Argentina	AY137405	Petersen and Seberg, 2003
H. patagonicum (Hauman) Covas subsp. patagonicum	2×	H1319	H	Argentina	AY137403	Petersen & Seberg, 2003
H. patagonicum (Hauman) Covas subsp. santacrucense (Parodi & Nicora) Bothmer, Giles & N. Jacobsen	2×	H1493	H	Argentina	AY137406	Petersen and Seberg, 2003
H. patagonicum (Hauman) Covas subsp. setifolium (Parodi & Nicora) Bothmer, Giles & N. Jacobsen	2×	H1357	H	Argentina	AY137404	Petersen & Seberg, 2003
H. procerum Nevski	6×	H1166	HHH	Argentina	GU734661, GU734662, GU734663, GU734664	Wang & Sun 2011
H. pubiflorum Hook. f.	2×	H1296	H	Argentina	AY137402	Petersen & Seberg, 2003
H. pusillum Nutt.	2×	H2038	H	New Mexico	AY137410	Petersen & Seberg, 2003
H. roshevitzii Bowden	2×	H7202	H	China	AY137416	Petersen & Seberg, 2003
H. stenostachys Godr.	2×	H1783	H	Argentina	AY137407	Petersen & Seberg, 2003
H. tetraploidum Covas.	4×	H6198	HH	Argentina	GU734679, GU734680	Wang & Sun 2011
Lophopyrum elongatum (Host) Á. Löve	2×	H6692	Ee	Israel	AF277246	Petersen & Seberg, 2000
Psathyrostachys fragilis (Boiss.) Nevski subsp. fragilis	2×	H917	Ns	Iran	AF277261	Petersen & Seberg, 2000
Psa. stoloniformis Baden	2×	H9182	Ns	China	AF277264	Petersen & Seberg, 2000
Pseudoroegneria spicata (Pursh) Á. Löve	2×	H9082	St	U.S.A.	AF277245	Petersen & Seberg, 2000
Taeniatherum caput-medusae (L.) Nevski	2×	H10254	Ta	Russia	AF277249	Petersen & Seberg, 2000
Thinopyrum bessarabicum (Savul. & Rayss) Á. Löve	2×	H6725	Eb	Russia	AF277254	Petersen & Seberg, 2000

NA: Information not available from previous publication.

Data Analysis

Multiple sequence alignments are made using Clustal X with default parameters [36] with manual adjustment. Phylogenetic analysis using the maximum-parsimony (MP) method is performed with the computer program PAUP* ver. 4 beta 10 [37]. All characters are specified as unweighted and unordered, and gaps are excluded in the analyses. Most-parsimonious trees are obtained by performing a heuristic search using the Tree Bisection-Reconnection (TBR) option with MulTrees on, and ten replications of random addition sequences with the stepwise addition option. Multiple parsimonious trees are combined to form a strict consensus tree. Overall character congruence is estimated by the consistency index (CI), and the retention index (RI). In order to infer the robustness of clades, bootstrap values with 1000 replications [38] are calculated by performing a heuristic search using the TBR option with MulTrees on.

In addition to maximum parsimony analysis, maximum-likelihood (ML) analysis is performed. For ML analysis, 8 nested models of sequence evolution were tested for the data set using PhyML 3.0 [39]. The general time-reversible (GTR) [40] substitution model led to a largest ML score compared to the other 7 substitution models: JC69, K80, F81, F84, HKY85, TN93 and custom (data not shown). As the result, the GTR model was used for the ML analysis. The ML analysis was performed using the Mac OS X UNIX version of GARLI v. 0.95 [41]. The runs were set for an unlimited number of generations, and automatic termination following 10,000 generations without a significant (lnL increase of 0.01) topology change. Thirty analyses were run with random starting tree topologies, and the tree with best score was used to represent gene tree. Branch support (BS) was estimated based on 100 ML bootstrap replicates in GARLI.

Nucleotide diversity was estimated by Tajima's π [42] and Watterson's θ [43] statistics. The former measure quantifies the mean percentage of nucleotide differences among all pairwise comparisons for a set of sequences, whereas the latter is simply an index of the number of segregating (polymorphic) sites. Tests of neutral evolution were performed as described by Tajima [42], and Fu and Li [44]. The above calculations were conducted by the software program DnaSP v5 [45].

Results

Total of 87 sequences from 18 tetraploid (22 accessions) StH Elymus, 9 polyploid Hordeum species, 24 diploid Hordeum species/subspecies and 8 other diploid Triticeae species were analyzed. Sequence comparisons revealed five large insertions/deletions (indels). Compared to other sequences aligned here, one copy sequence (H1166s4) from hexaploid H. procerum and the sequence from diploid H. cordobense (AY134715) showed a 24 bp insertion at position 206. One sequences from E. confusus (PI 598463k) showed a 23 bp deletion at position 352. An 82 bp insertion was found in the sequences from polyploid species H. fuegianum, H. jubatum, and H. tetraploidum, and diploid Australopyrum velutinum and Taeniatherum caput-medusae as reported by Wang and Sun [35], and Petersen and Seberg [31], respectively. The sequence from E. trachycaulus (PI 537323L) showed a 30 bp insertion at position 1054. A 15 bp deletion was detected in the sequences from E. canadensis (EU366405), E. cordilleranus (H6486k), E. hystrix (EU366415), H. arizonicum (H2144s3), H. brachyantherum subsp. californicum (AF277260), H. brachyantherum subsp. brachyantherum (H2348s2), H. depressum (H2008s1) and H. procerum (H1166s3).

The 89 (including two outgroups) aligned 1221 bp DMC sequences showed 794 constant, 221 variable and parsimony-uninformative, and 206 parsimony-informative sites. Parsimony analysis using Bromus arvensis and B. sterilis as outgroup produced 740 equally parsimonious trees with a consistency index (CI) of 0.693, and a retention index (RI) of 0.848. Maximum likelihood analysis across 30 GARLI runs generated likelihood score ranging from –lnL6349.08703 to –lnL6355.83219. ML tree with BS is shown in Figure 1.

Figure 1

The best scoring ML tree was selected from 30 GARLI analyses under a GTR model.

Numbers above branches are ML bootstrap support values, and the numbers below branches are bootstrap support values.

Two copies of sequences each from E. caninus, E. cordilleranus, E. hystrix, E. sibiricus, E. virginicus and E. wawawaiensis were well separated into two distinct groups, one grouped with the sequences from H genome, and another with St genome from Pseudoroegneria (Fig. 1). As unexpected, the sequence (GQ855194) from E. transhyrcanus formed clade with Lophopyrum elongatum and Thinopyrum bessarabicum with 90% BS in ML and 78% BS support in MP. The second copy of the sequence from E. transhyrcanus (GQ855193) was sister to the sequences from E. canadensis, E. sibiricus and E. multisetus. The sequences from polyploid Hordeum species fall into the clade with sequences from diploid Hordeum except one each from polyploid H. arizonicum, H. fuegianum, H. jubatum, and H. tetraploidum (Fig. 1). Also included in this clade are the sequences from Elymus species which were from the H genome. One sequence from H. arizonicum was clustered with the St genome sequences from E. cordilleranus, E. hystrix, E. canadensis, and E. wiegandii. An Asian diploid H. brevisubulatum subsp. violaceum is sister to polyploid H. lechleri, H. parodii, H. procerum, H. tetraploidum, E. lanceolatus, E. confusus, E. virescens, E. wawawaiensis, E. caninus. Within this clade, the sequence from E. lanceolatus is sister to the sequence from H. lechleri, H. parodii, H. procerum, H. tetraploidum with highly support (87% BS in ML, 85% in MP). One copy each from polyploid H. arizonicum, H. brachyantherum subsp. brachyantherum, H. depressum, H. procerum, E. cordilleranus, E. hystrix and American diploid species H. brachyantherum subsp. californicum formed a moderately supported clade (83% BS in ML, and 62% BS in MP) (Fig. 1).

Based on grouping of the sequences in phylogenetic analysis, we further separately analyzed nucleotide variation of DMC1 gene in the H genome from Hordeum polyploids and diploids, and Elymus. Some of the putative H copies of sequences from Elymus and Hordeum polyploids were not clearly put into the H clade. These sequences (PI 537323L, GQ855194 and W6-13828K from Elymus, H6198s2, H2013s2, H1418s2 and H2144s2 from Hordeum) were excluded for nucleotide diversity analysis. Estimates of nucleotide polymorphism, π and θw, were shown separately for the H genome of Elymus, Hordeum polyploid and diploid species (Table 2). The number of polymorphic sites (56) in the H genome of polyploid Hordeum is much lower than that (90) in its diploid donor species. The estimates of nucleotide diversity in the H genome of diploid Hordeum studied were θw = 0.02693, π = 0.01488. The estimates of nucleotide diversity in the H genome of polyploid Hordeum studied were θw = 0.0168, π = 0.01734. The number of polymorphic sites, the estimates of nucleotide diversity θw, π for the H genome in Elymus species was 80, 0.02774 and 0.02083, respectively. The Tajima [42], and Fu & Li's [44] tests were conducted on each data set. The Tajima, and Fu and Li's values of the diploid Hordeum H genome were −1.95371 (P<0.05) and −2.65118 (P<0.05), respectively, which showed significant departure from neutrality, while the Tajima, and Fu and Li's values of the polyploid Hordeum H genome were −0.34466 and −0.80485, respectively. The Tajima's D was −1.19959 and the Fu and Li's D was −1.29141 for Elymus H genome.

Table 2

Estimates of nucleotide diversity and test statistics for selection at DMC1 in polyploidy and diploid H genome.

	N	h	n	s	π	θw	Fu & Li's D	Tajima's D
Hordeum genome
H (diploid)	24	21	1025	90	0.01488±0.00298	0.02693±0.00905	−2.65118*	−1.95371*
H (polyploid)	16	16	1023	56	0.01680±0.00159	0.01734±0.00652	−0.80485	−0.34466
Elymus
H	12	12	1002	80	0.02083±0.00318	0.02774±0.01098	−1.29141	−1.19959

The N is the number of sequences analyzed, h is the number of haplotypes, n is the number of the sites (excluding sites with gaps/missing data), s is the number of segregating sites, π is the average pairwise diversity, θw is the diversity based on the number of segregating sites.

: Significant at α = 0.05.

Discussion

Origin of H genome in StH-genome Elymus species based on single copy nuclear gene DMC1 has previously been discussed [30]. DMC1 sequence data also showed a reticulate relationship of American polyploid species and diploid Hordeum [35]. However, the relationship of the H genome in polyploid Hordeum and Elymus was not previously explored.

The maximum parsimonious analysis grouped 24 sequences from Hordeum diploid and polyploid species together with 94% bootstrap supported value, maximum likelihood analysis also grouped these sequences together with highly supported value of 97% (Fig. 1). Only 3 Hordeum diploid H genome species, H. brevisubulatum subsp. violaceum, H. brachyantherum subsp. californicum, and H. bogdanii were grouped together with the sequences from Elymus H genome, indicating that the H genomes in Elymus originated from limited Hordeum diploid species, whereas the H genomes in polyploid Hordeum species were contributed by relative large Hordeum diploids. One concern is that the number of sequences from Elymus H genome is less than the number of sequences from Hordeum polyploids analyzed here, which may bias the comparison. However, phylogeny of Elymus StStHH allotetraploids based on three nuclear genes including relative large sample of Elymus species suggested that the one diploid Hordeum species, H. brachyantherum subsp. californicum (Syn: H. californicum Covas & Stebbins), is the possible H- genome donor to Elymus species [29], which also indicated that H genome in Elymus species originated from limited Hordeum diploid. However, the published data indicated that many Hordeum diploid species have contributed to the origin of polyploids in this genus, more than 10 diploid species were suggested to be the potential donors to polyploids in Hordeum [22], [35], [46]. Taken these together, it can be concluded that the H genomes in Elymus species have originated from limited gene pool, while H genomes in Hordeum polyploids have originated from broad gene pools.

Polyploid formation is a prominent mode of speciation in the flowering plant. Recent molecular data indicated that polyploid speciation is often more complex than initially thought [47], which is also the case in the tribe Triticeae [8], [22], [28], [29], [48], [49]. Molecular studies suggested polyploid species in many genera have multiple origins rather than single origin [1], [5], [47], [50]. It was suggested that the fates of polyploid populations of independent origins varied depending on the amount of genetic variation initially contributed by the diploid progenitors [50]. Studies have demonstrated that genetic diversity in polyploids is often similar to or higher than their diploid progenitors [47], [51], [52]. It is worth comparing the nucleotide diversities among the H genomes from Elymus, polyploid and diploid Hordeum species. This may offer an opportunity to address the potential evolutionary outcomes of polyploidization. Nucleotide diversity (π) of the DMC1 sequences from polyploid species (π = 0.02083 in Elymus, π = 0.01680 in polyploid Hordeum) is higher than that in diploid Hordeum (π = 0.01488). The estimates of D were significantly departure from the equilibrium neutral model at this locus in diploid Hordeum species (P<0.05), suggesting an excess of rare variants in diploid species. These rare variants may not contribute to the origination of polyploids. Phylogenetic analyses indeed indicated that not all diploids have contributed to the origination of polyploids in Hordeum and Elymus. Why is the genetic variation in polyploids higher than in diploid even the polyploids originated from limited number of diploid detected here? It has demonstrated that polyploidization resulted in the genome wide gene duplication which not only enables allopolyploids to tolerate more genomic variation than their progenitors, but also provides new opportunity to create functional diversification between homoeologous genes over time [52]–[54]. After gene duplicated, one of the copies may undergo mutations, if mutations are not deleterious, the mutations will not be removed by natural selection. Nucleotide diversity of this copy of gene in polyploids will be higher than that in their progenitors. Recent studies on the evolutionary rates of duplicated genes in polyploids compared to their diploid relatives showed that the evolutionary rates appear to be different among different homoeologous locus pairs [55]–[58]. Barrier et al. [59] found that the floral regulatory genes APETALA1 (ASAP1) and APETALA3 (APETALA3/TM6) are evolving much faster in the polyploid species than in the diploids. Analysis of nucleotide sequence diversity (π) of RPB2 revealed that nucleotide diversity (π) of RPB2 on the St genome in tetraploid Elymus was higher than that in the diploid Pseudoroegneria St genome [60].

The degree of relationships between two parents of a polyploid was suggested as a general factor affecting the amount of genomic sequence variation in allopolyploids [54]. In a study on interspecifc crosses of Brassica found that the overall amount of genomic change in AC (or CA) tetraploids was much lower than that in the AB (or BA) tetraploids. This was because the genetic distance between the A (B. rapa) and C (B. oleracea) was much closer than that between the A and B (B. nigra) [61]. A study on the timing and rate of genome variation in triticale following allopolyploidization also suggested the degree of the relationship between the parental genomes was the key factor in determining the rate of genomic sequences variation occurring during intergeneric allopolyploidization [54]. It was well demonstrated that genus Hordeum is monophyletic genus [21], [22], and polyploids originated from the diploid species in this genus. While the Elymus StH genomic species originated from the St genome donor Pseudoroegneria and H genome donor Hordeum species. The genetic distance of parental genomes in polyploid Hordeum is much closer than that between St and H genomes. The degree of relationships between two parents of a polyploid might be factor affecting nucleotide diversity in allopolyploids. This might explain that nucleotide diversity (π) of the DMC1 sequences in Elymus polyploid species (π = 0.02083) is higher than that in polyploid Hordeum (π = 0.01680). This speculation needs to be further studied.

One of objectives of this study is to explore the possible role of polyploid Hordeum species in the origin of StH Elymus and whether the gene flow has occurred between polyploid Hordeum and Elymus species. The Hordeum H genome diploids are the H genome donor to both Elymus StH species and polyploid species in Hordeum. Phylogenetic analysis revealed that the H copy of DMC1 sequence in E. lanceolatus is very close to the H copy of DMC1 sequence in polyploid H. procerum, H. lechleri, H. parodii, and H. tetraploidum, indicating that the H genome in E. lanceolatus and those four polyploid Hordeum species originated from the same diploid donor. Alternative explanation is that gene flow has occurred among E. lanceolatus and H. procerum, H. lechleri, H. parodii, and H. tetraploidum. Elymus cordilleranus and E. hystrix formed a group with ployploid H. arizonicum, H. brachyantherum subsp. brachyantherum, H. depressum, H. procerum, and diploid H. brachyantherum subsp. californicum. This grouping is explained either by common origin of H genome in these Elymus and Hordeum polyploids or by gene flow among these polyploids. One copy of the DMC1 sequences from North American hexaploid H. arizonicum was grouped with St-copy sequences from American E. canadensis, E. hystrix, E. wiegandii and E. cordilleranus (Fig. 1). The diploid H. pusillum and tetraploid H. jubatum was suggested as the parental parents for H. arizonicum [22], [46], [62]. cpDNA analysis suggested that H. pusillum could be the maternal parent of H. arizonicum [63]. Previous analysis of DMC1 data suggested that H. brachyantherum subsp. californicum might be one diploid genome donor of H. arizonicum, and the second genome donor of H. arizonicum might be the common ancestor of H. brachyantherum subsp. brachyantherum, and showed that one copy of DMC1 sequences of H. arizonicum fall outside the Hordeum clade of the tree [35]. DMC1 data here suggested if the St genome was not the donor species to one copy of genome in H. arizonicum, gene flow has occurred between H. arizonicum and some Elymus StH genome species during evolutionary process.

Analysis of β-amylase data revealed that one of the H. jubatum genome was placed together with Elymus species [29]. The role of H. jubatum in the Elymus evolutionary history has been suggested, a tetraploid similar to H. jubatum might have been involved in the history of Elymus, either through introgression between the Elymus and H. jubatum, or through a direct contribution from H. jubatum like species to Elyums [29]. Our DMC1 data showed that one of the H. jubatum genome with H. tetraploidum, H. fuegianum, T. caput-medusae and Aust. velutinum grouped together, Elymus virescens as sister to this group. Our result not only did not contradict to the suggestion that H. jubatum was involved at some stage in the history of StStHH Elymus [29], but also further expanded to that several ployploid Hordeum species might have involved in the evolution of StStHH Elymus through gene flow among them.

The study on the polyploid formation in Tragopogon (Asteraceae) indicated a lack of gene flow among polyploid plants of independent origin, even when they co-occur, suggesting potential reproductive barriers among separate lineages in polyploid species [50]. Sequence analysis of 12 nuclear loci representing 6 genes on tetraploid Capsella bursa-pastoris and its close diploid relative C. rubella showed that polyploid speciation need not result in immediate and complete reproductive isolation, and the postpolyploidization hybridization and introgression can contribute significantly to genetic variation in newly formed polyploid [64]. Molecular characterization of a diagnostic DNA marker for domesticated tetraploid wheat indicated gene flow from wild tetraploid wheat to hexaploid wheat [65]. Our results suggested that gene flow among different polyploids in Triticeae species might play an important role in polyploid speciation and evolution.