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ARTICLE Nuclear and chloroplast DNA phylogeny reveals complex evolutionary history of Elymus pendulinus Genome Downloaded from www.nrcresearchpress.com by University of Nebraska Lincoln on 09/12/15 For personal use only.

Chi Yan, Qianni Hu, and Genlou Sun

Abstract: Evidence accumulated over the last decade has shown that allopolyploid genomes may undergo complex reticulate evolution. In this study, 13 accessions of tetraploid Elymus pendulinus were analyzed using two low-copy nuclear genes (RPB2 and PepC) and two regions of chloroplast genome (Rps16 and trnD-trnT). Previous studies suggested that Pseudoroegneria (St) and an unknown diploid (Y) were genome donors to E. pendulinus, and that Pseudoroegneria was the maternal donor. Our results revealed an extreme reticulate pattern, with at least four distinct gene lineages coexisting within this species that might be acquired through a possible combination of allotetraploidization and introgression from both within and outside the tribe Hordeeae. Chloroplast DNA data identified two potential maternal genome donors (Pseudoroegneria and an unknown species outside Hordeeae) to E. pendulinus. Nuclear gene data indicated that both Pseudoroegneria and an unknown Y diploid have contributed to the nuclear genome of E. pendulinus, in agreement with cytogenetic data. However, unexpected contributions from Hordeum and unknown aliens from within or outside Hordeeae to E. pendulinus without genome duplication were observed. Elymus pendulinus provides a remarkable instance of the previously unsuspected chimerical nature of some plant genomes and the resulting phylogenetic complexity produced by multiple historical reticulation events. Key words: Elymus pendulinus, allopolyploid, reticulate evolution, introgression, nuclear DNA, chloroplast DNA. Résumé : Les évidences obtenues au fil de la dernière décennie ont montré que les génomes allopolyploïdes peuvent connaître une évolution réticulée complexe. Dans ce travail, 13 accessions de l’Elymus pendulinus tétraploïde ont été étudiées en examinant deux gènes nucléaires a` faible nombre de copies (RPB2 et PepC) ainsi que deux régions du génome chloroplastique (Rps16 et trnD-trnT). Des études antérieures avaient suggéré que les espèces diploïdes sources des génomes chez l’E. pendulinus étaient Pseudoroegneria (génome St) et une autre espèce inconnue (génome Y), et que le Pseudoroegneria avait été le parent maternel. Les résultats de ce travail révèlent une réticulation extrême avec au moins quatre lignages génétiques distincts coexistant au sein de cette espèce, lesquels auraient pu survenir via une possible combinaison d’allotétraploïdisation et d’introgression provenant de l’intérieur et de l’extérieur des hordées. Les données chloroplastiques ont mis en évidence deux sources potentielles du génome maternel, soit le Pseudoroegneria et une espèce inconnue hors des hordées. Les données nucléaires ont indiqué que deux espèces, Pseudoroegneria et l’espèce diploïde source du génome Y, auraient contribué au génome nucléaire de l’E. pendulinus, conformément aux données cytogénétiques. Cependant, des contributions inattendues, sans duplication génomique, ont été notées en provenance du genre Hordeum et d’espèces additionnelles parmi les hordées et au-dela`. L’E. pendulinus fournit ainsi un cas remarquable de la nature chimérique, jusque la` insoupçonnée, de certains génomes de plantes et de la complexité phylogénétique qui résulte de plusieurs évènements de réticulation. Mots-clés : Elymus pendulinus, allopolyploïde, évolution réticulée, introgression, ADN nucléaire, ADN chloroplastique.

Introduction Hybridization and polyploidization have contributed greatly to speciation (Cui et al. 2006). Numerous studies have indicated that almost all angiosperm species are of polyploid origin or have been affected by recursive polyploidizations (Vision et al. 2000; Cui et al. 2006; Jiao et al. 2011). Studies involving the Hordeeae (syn. Triticeae) group (e.g., Elymus, Aegilops, and Triticum) emphasize the impact of hybridization and polyploidization on species evolution. Introgression through repeated hybridization followed by segmental gene duplication events are also reported to play critical roles in driving speciation (Kavanaugh et al. 2006; Antunes et al. 2007; Ragupathy et al. 2008). Ragupathy et al. (2008) demonstrated that a segmental duplication event mediated by an LTR retrotransposon occurred prior to the polyploidization, resulting in hexaploid wheat speciation. Introgression has been shown in some

instances to cause widespread genomic and epigenomic changes in a recipient species similar to those caused by the merger of divergent genomes during allopolyploid speciation (Liu and Wendel 2000; Liu et al. 2004; Shan et al. 2005; Wang et al. 2005). Mallet (2005) estimated that up to 25% of plant species produce viable offspring from interspecific mating. Numerous studies suggested that introgression has resulted in range expansion and niche shifts (Klier et al. 1991; Neuffer et al. 1999; Milne and Abbott 2000; Rieseberg et al. 2007). The occurrence of introgression events may also confound reconstruction of individual polyploidization events by creating complex reticulate patterns (Mason-Gamer et al. 2010). Previous studies indicated that introgression clearly has the potential for inducing significant evolutionary change in recipient species (Kavanaugh et al. 2006; Antunes et al. 2007; Ragupathy et al. 2008).

Received 3 January 2014. Accepted 12 February 2014. Corresponding Editor: C.L. McIntyre. C. Yan,* Q. Hu,* and G. Sun. Biology Department, Saint Mary’s University, 923 Robie Street, Halifax, NS B3H 3C3, Canada. Corresponding author: Genlou Sun (e-mail: [email protected]). *These authors contributed equally to this work. Genome 57: 97–109 (2014) dx.doi.org/10.1139/gen-2014-0002

Published at www.nrcresearchpress.com/gen on 27 February 2014.

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Elymus L., the largest genus in the tribe Hordeeae, is composed exclusively of allopolyploids and includes approximately 150 species as interpreted by Löve (1984). Cytological analyses have identified five basic genomes (St, H, Y, P, and W; genome symbols follow those of Wang et al. 1994) in the genus. The St genome found in all species of Elymus was supposedly donated by Pseudoroegneria (Nevski) Á Löve (Lu et al. 1991; Salomon and Lu 1994). The H, P, and W genomes are derived from Hordeum L., Agropyron Gaetn., and Australopyrum (Tzvelev) Á Löve, respectively; while the origin of the Y genome is still unclear and subject to debate (Dewey 1971; Torabinejad and Mueller 1993; Jensen and Salomon 1995; Liu et al. 2006; Sun and Salomon 2009). Elymus pendulinus (2n = 4x = 28) is a short-lived perennial, selfpollinating allotetraploid species. It inhabits central Asia and is characterized by drooping to strongly nodding spikes. Cytogenetic analyses revealed E. pendulinus as an allotetraploid with St and Y genomes (Dewey 1984; Jensen 1990). Later studies using molecular data confirmed this conclusion (Mason-Gamer et al. 2002, 2010; Sun and Salomon 2009). Molecular phylogenetic studies have demonstrated the complexity of the dynamic nature of polyploids, including relationships involving unexpected alien genetic materials (Cronn et al. 2003; Mason-Gamer 2004; Mahelka and Kopecký 2010), multiple origins of allopolyploid genome combinations (Wendel et al. 1991; Soltis et al. 1995; Cook et al. 1998; Doyle et al. 1999, 2002), together with introgression (or gene flow). These phenomena have been considered as the causes of shared polymorphism across ploidy level and (or) phylogenetic incongruence among loci (Weissmann et al. 2005; Hedrén et al. 2008; Brokaw and Hufford 2010). Keng and Chen (1963) examined the variations in spike length and lemma vestiture within E. pendulinus, but there has been little examination on its genetic variation, allopolyploid origin, and evolutionary history, all of which remain poorly explained (Jensen 1990; Lu et al. 1991; Zhou et al. 1999). In the present study, we analyzed 13 accessions of tetraploid E. pendulinus from the southern borders of Altai, Eastern Siberia, and the Far East of Russia using chloroplast and low-copy nuclear genes. To identify the maternal parent of E. pendulinus, we analyzed sequence data from two cpDNA loci, RPS16 and trnD-trnT, within a broad sample of diploid genera throughout the tribe. Next, we investigated the intra-specific evolution of E. pendulinus using two low-copy nuclear markers, RPB2 and PepC, analyzed making use of a database that included most of the diploid genera within Hordeeae and several StY species of Elymus. Finally, we used sequence analysis to explore variation among E. pendulinus accessions in terms of multiple origins and (or) introgression.

Materials and methods Plant materials and DNA extraction Thirteen accessions of E. pendulinus were used in this study (Table 1). DNA was extracted from fresh young leaf tissue using the method of Junghans and Metzlaff (1990). Herbarium specimens were made from mature plants and kept at the Biology Department, Saint Mary’s University. Two low-copy nuclear genes, the second largest subunit of RNA polymerase II (RPB2) and the phosphoenolpyruvate carboxylase (PepC), along with two chloroplast DNA sequences, ribosomal protein S16 (RPS16) and non-coding chloroplast DNA region trnD-trnT, from different accessions of E. pendulinus were amplified and sequenced. RPB2 and PepC sequences for some diploid species of Hordeeae, representing the St, H, I, Xu, W, P, E, Ns, Ta, A, S, Xp, F, O, Q, K, R, and D genomes, along with Bromus were obtained from published data (Sun et al. 2008; Helfgott and Mason-Gamer 2004), and included in the analyses. Plant materials with their accession numbers, genomic constitutions, geographical origins, and GenBank identification numbers are presented in Table 1.

Genome Vol. 57, 2014

DNA amplification and sequencing The low-copy nuclear genes RPB2 and PepC and cpDNA genes RPS16 and trnD-trnT were amplified by polymerase chain reaction (PCR) using the primers P6F/P6FR (Sun et al. 2007), PEPC-F/PEPC-R (Helfgott and Mason-Gamer 2004), RPS16F/RPS16R (Popp and Oxelman 2007), and TrnD/TrnT (Sun 2002), respectively. The amplification profile for the RPB2 gene was as follows: an initial denaturation at 95 °C for 4 min; 35–40 cycles of 95 °C for 40 sec, 52 °C for 40 sec, 72 °C for 90 sec; and a final cycle of 72 °C for 10 min. The PCR profile for amplifying the PepC gene was based on Helfgott and Mason-Gamer (2004). The PCR protocol for RPS16F/ RPS16R and TrnD/TrnT followed Popp and Oxelman (2007) and Sun (2002), respectively. The PCR products for the nuclear genes amplified from E. pendulinus were cloned into the pGEM-easy T vector (Promega Corporation, Madison, Wis., USA) according to the manufacturer’s instructions. The resulting plasmids were used to transform Escherichia coli DH5␣, and 10–20 colonies for each accession were randomly selected for screening. Each colony was transferred to 10 ␮L of LB broth with 0.1 mg/mL ampicillin. The solutions were incubated at 37 °C for 30 min before using 2 ␮L for PCR to check the presence of an insert using the same primers that were used for the original PCR amplification. For the solutions that were confirmed to contain the insert, the remaining 8 ␮L of solution was transferred to 5 mL LB broth and incubated at 37 °C overnight. Plasmid DNA was isolated using the Promega Wizard Plus Minipreps DNA Purification System (Promega Corporation, Madison, Wis., USA) according to manufacturer’s instructions. The PCR products amplified by cpDNA primers RPS16F/RPS16R and TrnD/TrnT were purified and then directly sequenced. Both the PCR products and plasmid DNAs were commercially sequenced by either MACROGEN (Seoul, Korea) or Taihe Biotechnology (Beijing, China). Both forward and reverse strands of PCR products or plasmid DNAs were sequenced independently to enhance the sequence quality. As Taq errors that cause substitutions are mainly random, it is unlikely that any two sequences would share identical Taq errors to create a false synapomorphy. Data analysis Automated sequence outputs were visually inspected with chromatographs. Multiple sequence alignments were made using ClustalX with default parameters and additional manual edits to minimize gaps (Thompson et al. 1997). Because of the high similarity of sequences in polyploids, PCR-mediated recombination could occur and yield chimeric products during PCR amplification (Saiki et al. 1988; Cronn et al. 2002). Recombinants can easily be identified as chimeras in the alignments. Alignments were inspected for chimeric sequences, and no recombinants were detected. Phylogenetic analysis using the maximum-parsimony (MP) method was performed with the computer program PAUP* ver. 4 beta 10 (Swofford 2003). All characters were specified as unweighted and unordered, and gap-only columns were excluded in the analyses. The most parsimonious trees were constructed by performing a heuristic search using the tree bisection-reconnection (TBR) branch-swapping method with the following parameters: MulTrees on and 10 replications of random addition sequences with the stepwise addition option. Multiple parsimonious trees were combined to form a strict consensus tree. Overall character congruence was estimated by the consistency (CI) and the retention index (RI). To infer the robustness of clades, bootstrap (BS) values with 1000 replications (Felsenstein 1985) were calculated by performing a heuristic search using the TBR option with Multree on. In addition to MP analysis, maximum-likelihood (ML) analyses were also performed. For ML analysis, eight nested models of sequence evolution were tested for each data set using PhyML 3.0 (Guindon and Gascuel 2003). For each data set, the general timereversible (GTR) (Lanave et al. 1984) substitution model led to the largest ML score compared with the other seven substitution Published by NRC Research Press

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Table 1. Taxa from the genera Bromus, Aegilops, Eremopyrum, Heteranthelium, Psathyrostachys, Secale, Taeniatherum, Agropyron, Australopyrum, Dasypyrum, Thinopyrum, Triticum, Pseudoroegneria, Hordeum, and Elymus used in this study, including their origin, accession number, and GenBank sequence number. Species

Accession No.*

Genome† Origin

Bromus catharticus Vahl Bromus sterilis L. Bromus tectorum L. Aegilops comosa Sibth. & Smith Aegilops comosa Sibth. & Smith Aegilops longissima Schweinf. & Muschl. Aegilops searsii Feldman & Kislev Aegilops sharonensis Eig Aegilops speltoides Tausch Aegilops tauschii Coss. Aegilops umbellulata Zhuk. Aegilops uniaristata Vis. Aegilops uniaristata Vis. Agropyron cristatum (L.) Gaertn. Agropyron fragile (Roth) P. Candargy Agropyron fragile (Roth) P. Candargy Agropyron mongolicum Keng. Australopyrum retrofractum (Vickery) Á. Löve

CN 32048 PI 229595 Kellogg s.n. G602 PI 551032 PI 542196 PI 599150 PI 542237 PI 499261 PI 486265 PI 276994 PI 276996 PI 554418 PI 383534 PI 598674 PI 598694 PI 598460 PI 531553

— — — M M SI SS SI S D U N N P P P P W

Australopyrum retrofractum (Vickery) Á. Löve

PI 533013

W

Australopyrum retrofractum (Vickery) Á. Löve

PI 533014

W

Australopyrum retrofractum (Vickery) Á. Löve

PI 547363

W

Eremopyrum bonaepartis (Spreng.) Nevski Eremopyrum bonaepartis (Spreng.) Nevski Eremopyrum distans (K. Koch) Nevski Eremopyrum orientale (L.) Jaub. & Spach Hordeum bogdanii Wilensky Hordeum bogdanii Wilensky Hordeum bogdanii Wilensky Hordeum bogdanii Wilensky

PI 203442 PI 219966 PI 193264 PI 203440 PI 531760 H 4014 H 7476 PI 499498

F F F F H H H H

Hordeum bogdanii Wilensky Hordeum bogdanii Wilensky Hordeum brachyantherum Nevski subsp. californicum (Covas & Stebbins) Bothmer, N. Jacobsen & Seberg Hordeum bulbosum L. Hordeum bulbosum L.

PI 499645 PI 531762 H 3317

RPB2‡

PepC‡

RPS16

trnD-trnN

HQ014410 HQ231839 − − − − − − − − − + + EU187438 − + + −

− − AY553239 AY553236 − − − − − − − − − − − − − −

− − − − + + + + + + + − + − + − + −

− + − − + + − + + − − − − − + − + +

−

−

−

+

EU187482

−

+

+

EU187470

−

+

+

+ + − − − + − EF596768

− − − AY553254 EU282293 − − −

+ − + + − − + −

+ − − + − − − −

H H H

N/A Iran N/A N/A Greece Turkey Israel Turkey China Turkey Turkey Istanbul, Turkey Former Soviet Union Kars, Turkey Kazakhstan Kazakhstan China Austr. Capital Terr., Australia New South Wales, Australia New South Wales, Australia New South Wales, Australia Ankara Girishk N/A Turkey China Pakistan China Inner Mongolia, China Xinjiang, China Tajikistan United States

EU18747 − +

− − −

− − −

− + −

H 3878 PI 440417

I I

Italy N/A

+ −

− −

− −

Hordeum chilense Roem. & Schult. Hordeum chilense Roem. & Schult. Hordeum comosum Presl. Hordeum cordobense Bothmer, N. Jacobsen & Nicora Hordeum flexuosum Steud. Hordeum marinum Huds. Hordeum marinum Huds. Hordeum marinum Huds. subsp. gussoneanum (Parl.) Thell. Hordeum marinum subsp. marinum Huds. Hordeum murinum Huds. Hordeum murinum L.

H 1816 H 1819 H 1181 H 6460

H H H H

Chile Chile Argentina Argentina

+ − + +

− EU282294, EU282295, EU282296 EU282297 − − −

− + − −

− − − −

H 2127 PI 304347 PI 304346 H 581

H Xa Xa Xa

Uruguay United Kingdom United States Greece

+ − − +

− EU282298 AY553258 −

− − − −

− − − −

H 121 CIho 15683 PI 247054

Xa Xu Xu

Greece United States United States

+ − −

− − −

− − −

Hordeum patagonicum (Haumann) Covas subsp. magellanicum (Parodi ex Nicora) Bothmer, Giles & N. Jacobsen Hordeum patagonicum (Haumann) Covas subsp. mustersii (Nicora) Bothmer, Giles & N. Jacobsen

H 1342

H

Argentina

+

− AY553259 EU282299, EU282300 −

−

−

H 1358

H

Argentina

+

−

−

−

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100

Genome Vol. 57, 2014

Table 1 (continued). Accession No.*

Genome† Origin

RPB2‡

PepC‡

RPS16

trnD-trnN

H 6052

H

Argentina

+

−

−

−

H 1353

H

Argentina

+

−

−

−

H 1352

H

Argentina

+

−

−

−

H 1236 CIho 15654 H 10070 H 7754 H 9152 H 1780 H 6439 PI 531791 RJMG 107 H 3140 A

H H H H H H H H I I

Argentina United States Russian China China Argentina Argentina Argentina N/A Cyprus

+ − − − + + + − − +

− EU282301 − − − − − EU282302 AY553260 −

− − − − − − − − − −

− − + + − − + − − −

H 7514 A PI 577112 PI 401351

I Q Q

China Turkey Iran

+ − +

− − −

− + −

− − −

PI 401354

Q

Iran

+

AY553255

+

−

H 10248 PI 632554 PI 228389 PI 228390 PI 228391 PI 282392 PI 330687 PI 330688 PI 401274 D 2844 PI 232128 PI 232134

St St St St St St St St St St St St

Tadzhikistan Uzbekistan Iran Iran Iran Iran Kandavan Pass, Iran Sirak-Sar, Iran Saqqez, Iran

− − − − EU282304 EU282305 − − − AY553264 − −

+ + − − − − − + − − − −

− − + + − − − + − − + −

Pseudoroegneria spicata (Pursh) Á. Löve Pseudoroegneria spicata (Pursh) Á. Löve

PI 232140 PI 236669

St St

− HQ231842

− −

− −

+ −

Pseudoroegneria spicata (Pursh) Á. Löve

PI 286198

St

HQ231843

−

−

−

Pseudoroegneria spicata (Pursh) Á. Löve

PI 506274

St

EF596746

−

+

−

Pseudoroegneria spicata (Pursh) Á. Löve Pseudoroegneria spicata (Pursh) Á. Löve Pseudoroegneria spicata (Pursh) Á. Löve

PI 516184 PI 537379 PI 537389

St St St

HQ231848 HQ231851 HQ231852

− − −

− − −

− − −

Pseudoroegneria spicata (Pursh) Á. Löve Pseudoroegneria spicata (Pursh) Á. Löve Pseudoroegneria spicata (Pursh) Á. Löve Pseudoroegneria spicata (Pursh) Á. Löve Pseudoroegneria spicata (Pursh) Á. Löve

PI 539873 PI 547154 PI 547162 PI 563869 PI 563872

St St St St St

HQ231853 HQ231854 HQ231855 HQ231856 HQ231857

− − − − −

− − − − −

− − − + +

Pseudoroegneria spicata (Pursh) Á. Löve Pseudoroegneria spicata (Pursh) Á. Löve

PI 598818 PI 598822

St St

− HQ231858

− −

− −

+ +

Pseudoroegneria spicata (Pursh) Á. Löve Pseudoroegneria spicata (Pursh) Á. Löve Pseudoroegneria stipifolia (Czern. ex Nevski) Á. Löve Pseudoroegneria stipifolia (Czern. ex Nevski) Á. Löve

PI 610986 PI 619445 PI 531751

St St St

Idaho, United States Whoming, United States United States British Columbia, Canada Washington, United States Washington, United States Oregon, United States Oregon, United States Washington, United States Idaho, United States Idaho, United States Oregon, United States Oregon, United States Montana, United States Oregon, United States Colorado, United States Utah, United States Nevada, United States Ukraine

− − HQ231837 HQ231838 − − EF596753 EF596751 EF596752 − HQ231840 HQ231841

EF596747 HQ231859 −

− − −

− + −

PI 313960

St

Russian Federation

AY553263 − EU282307, EU282308 EU282306

−

−

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Species Hordeum patagonicum subsp. patagonicum (Haumann) Covas Hordeum patagonicum (Haumann) Covas subsp. santacrucense (Parodi ex Nicora) Bothmer, Giles & N. Jacobsen Hordeum patagonicum (Haumann) Covas subsp. setifolium (Parodi ex Nicora) Bothmer, Giles & N. Jacobsen Hordeum pubiflorum Hook. f. Hordeum pusillum Nutt. Hordeum roshevitzii Bowden Hordeum roshevitzii Bowden Hordeum roshevitzii Bowden Hordeum stenostachys Godr. Hordeum stenostachys Godr. Hordeum stenostachys Godr. Hordeum vulgare L. Hordeum vulgare subsp. spontaneous (K. Koch) Thell Hordeum vulgare subsp. vulgare L. Henrardia persica (Boiss.) C.E. Hubb. Heteranthelium piliferum (Banks & Sol.) Hochst. Heteranthelium piliferum (Banks & Sol.) Hochst. Pseudoroegneria ferganensis Drobow Pseudoroegneria geniculata (Trin.) Á. Löve Pseudoroegneria libanotica (Hack.) D.R. Dewey Pseudoroegneria libanotica (Hack.) D.R. Dewey Pseudoroegneria libanotica (Hack.) D.R. Dewey Pseudoroegneria libanotica (Hack.) D.R. Dewey Pseudoroegneria libanotica (Hack.) D.R. Dewey Pseudoroegneria libanotica (Hack.) D.R. Dewey Pseudoroegneria libanotica (Hack.) D.R. Dewey Pseudoroegneria spicata (Pursh) Á. Löve Pseudoroegneria spicata (Pursh) Á. Löve Pseudoroegneria spicata (Pursh) Á. Löve

−

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101

Table 1 (continued). Accession No.*

Genome† Origin

RPB2‡

PepC‡

RPS16

trnD-trnN

Pseudoroegneria stipifolia (Czern. ex Nevski) Á. Löve Pseudoroegneria stipifolia (Czern. ex Nevski) Á. Löve Pseudoroegneria strigosa (M. Bieb.) Á. Löve Pseudoroegneria strigosa (M. Bieb.) Á. Löve Pseudoroegneria strigosa (M. Bieb.) Á. Löve

PI 325181

St

EF596748

−

+

+

PI 440095

St

Stavropol, Russian Federation Russian Federation

+

−

−

−

PI 531752 W6 14049 PI 499637

St St St

Estonia Russian Federation China

HQ231850 HQ231836 −

− − −

+ + −

Pseudoroegneria strigosa subsp. aegilopoides (Drobow) Á. Löve Pseudoroegneria strigosa subsp. aegilopoides (Drobow) Á. Löve Pseudoroegneria strigosa subsp. aegilopoides (Drobow) Á. Löve Pseudoroegneria strigosa subsp. aegilopoides (Drobow) Á. Löve Pseudoroegneria tauri (Boiss. & Balansa) Á. Löve Pseudoroegneria tauri (Boiss. & Balansa) Á. Löve Pseudoroegneria tauri (Boiss. & Balansa) Á. Löve Pseudoroegneria tauri (Boiss. & Balansa) Á. Löve Pseudoroegneria tauri (Boiss. & Balansa) Á. Löve Pseudoroegneria tauri (Boiss. & Balansa) Á. Löve Psathyrostachys juncea (Fischer) Nevski Psathyrostachys juncea (Fischer) Nevski Secale cereale L. Secale cereale L. Taeniatherum caput-medusae (L.) Nevski Taeniatherum caput-medusae subsp. asperum (L.) Nevski Taeniatherum caput-medusae subsp. caput-medusae (L.) Nevski Taeniatherum caput-medusae subsp. caput-medusae (L.) Nevski Taeniatherum caput-medusae subsp. caput-medusae (L.) Nevski Thinopyrum bessarabicum (Savul. & Rayss) Á. Löve Thinopyrum elongatum (Host) D.R. Dewey

PI 420842

St

Former Soviet Union

HQ231846

− − EU282309, EU282310 −

+

+

PI 440000

St

HQ231847

−

−

+

W6 13089

St

Stavro, Russian Federation Xinjiang, China

HQ231835

−

−

+

PI 531755

St

China

−

EU282311

−

−

PI 380644

St

Iran

−

−

−

PI 401319

St

Iran

−

EU282314, EU282315 EU282313

−

−

PI 401324

St

Iran

HQ231844

−

−

+

PI 401326

St

Iran

HQ231845

−

−

+

PI 401330

St

Toward Ahar, Iran

−

−

+

−

PI 380652

St

Iran

−

EU282312

−

−

PI 406469 PI 430871 Kellogg s.n. PI 573710 RJMG 189 PI 561091

Ns Ns R R Ta Ta

Former Soviet Union Former Soviet Union N/A Georgia N/A Siirt, Turkey

+ + − − − +

− − AY553266 − AY553268 −

+ − − + − +

+ − − + − −

PI 208075

Ta

Kars, Turkey

+

−

+

−

PI 220591

Ta

Afghanistan

+

−

−

−

PI 222048

Ta

Afghanistan

+

−

+

−

PI 531712

Eb

Estonia

EU187474

−

−

−

PI 142012

Ee

EU187439

−

+

+

Thinopyrum elongatum (Host) D.R. Dewey Triticum monococcum L. Triticum monococcum L. Elymus abolinii (Drobow) Tzvelev

RJMG 113 PI 191146 PI 531555

Ee AM AM StY

Odessa, Russian Federation N/A Spain N/A China

− − − −

− + − −

− − − −

Elymus abolinii (Drobow) Tzvelev

PI 531554

StY

Xinjiang, China

−

−

Elymus antiquus (Nevski) Tzvelev

PI 632564

StY

China

EU187443, EU18744 −

AY553269 − AJ007705 GQ844927, GQ844928 −

−

−

Elymus caucasicus (Koch) Tzvelev

PI 531573

StY

Estonia

−

−

Elymus ciliaris (Trin.) Tzvelev

PI 531575

StY

China

EU187454, EU187453 −

−

−

Elymus ciliaris (Trin.) Tzvelev

PI 564917

StY

−

PI 401277

StY

EF596749, EU187483 −

−

Elymus longearistatus (Boiss.) Tzvelev

Vladivostock, Soviet Far East Iran

−

−

Elymus longearistatus (Boiss.) Tzvelev

PI 401280

StY

North of Tehran, Iran

EU187447, EU187448

−

−

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Species

GQ844931, GQ844932 GQ844940, GQ844941 GQ844942, GQ844943 − GQ844950, GQ844951 −

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Table 1 (concluded). Species

Accession No.*

Genome† Origin

RPB2‡

PepC‡

RPS16

trnD-trnN

Elymus nevskii Tzvelev

PI 314620

StY

Former Soviet Union

−

−

−

Elymus semicostatus (Nees ex Steud.) Melderis

PI 271522

StY

India

−

−

−

Elymus semicostatus (Nees ex Steud.) Melderis

PI 207452

StY

Afghanistan

−

−

Elymus pendulinus (Nevski) Tzvelev

PI 499452

StY

China

EU187445, EU187446 −

GQ844952, GQ844953 GQ844956, GQ844957 −

−

−

Elymus pendulinus (Nevski) Tzvelev Elymus pendulinus (Nevski) Tzvelev Elymus pendulinus (Nevski) Tzvelev Elymus pendulinus (Nevski) Tzvelev Elymus pendulinus (Nevski) Tzvelev Elymus pendulinus (Nevski) Tzvelev Elymus pendulinus (Nevski) Tzvelev Elymus pendulinus (Nevski) Tzvelev Elymus pendulinus (Nevski) Tzvelev Elymus pendulinus (Nevski) Tzvelev Elymus pendulinus (Nevski) Tzvelev Elymus pendulinus (Nevski) Tzvelev Elymus pendulinus (Nevski) Tzvelev

BKA 0921 VOK 0728 VBG 0727 VOK 0724 VBG 0722 MES 0721 USS 0720 VLA 0719 VLA 0718 RUS 0716 ZAR 0715 ZAR 0714 AND 0713

StY StY StY StY StY StY StY StY StY StY StY StY StY

Primorski, Russia Primorski, Russia Primorski, Russia Primorski, Russia Primorski, Russia Primorski, Russia Primorski, Russia Primorski, Russia Primorski, Russia Primorski, Russia Primorski, Russia Primorski, Russia Primorski, Russia

Y, ? St, Y St, Y, ? St, Y St, Y, ? St, Y, ? St St, Y St St, Y St, Y St, ?, ? St, ?, ?

+ + + + + + + + + + + + +

+ + + + + + + + + + + + +

GQ844954, GQ844955 St1, St2, Y Y ?, ? St, Y St, Y St, Y St, Y St, Y, ? St, Y St, Y St, Y St, Y, ? St, Y, ?

Note: +, the sequence data has been recovered; −, the sequence data has not been recovered. *Number refers to the material used in this study. †Genome designations are according to Wang et al. (1994). ‡GenBank sequence number.

models: JC69 (Jukes and Cantor 1969), K80 (Kimura 1980), F81 (Felsenstein 1981), F84 (Felsenstein 1993), HKY85 (Hasegawa et al. 1985), TN93 (Tamura and Nei 1993), and custom (data not shown). The GTR model produced the highest log-likelihood value, so the GTR model was chosen for the ML analysis. For ML, the support of clades was assessed with the approximate-likelihood ratio test (aLRT) method, which is an alternative to the BS method for evaluating tree reliability (Anisimova and Gascuel 2006).

Results RPS16 analysis Thirteen accessions of E. pendulinus were analyzed together with an additional 34 RPS16 sequences from diploid species of Hordeeae. The data matrix contained 733 characters, of which 659 were constant, 43 were parsimony uninformative, and 31 were parsimony informative. MP analysis was conducted using Bromus catharticus as an outgroup. MP analysis produced 88 equally parsimonious trees (CI = 0.886, excluding uninformative characters; RI = 0.913). The ML heuristic search using the GTR model resulted in a single tree. The tree topologies generated by MP and ML analyses were similar to each other. An MP strict consensus tree with BS and ML aLRT values is shown in Fig. 1. Phylogenetic analyses based on RPS16 sequence data grouped all sequences from E. pendulinus into the Pseudoroegneria St genome clade, except for the sequence from the accession ZAR 0714 of E. pendulinus (Fig. 1). Within this clade, the sequences from three accessions of E. pendulinus (VOK 0724, VOK 0728, and VBG 0727) formed a subclade (BS = 83; aLRT = 0.864) and the accessions VLA 0719 and USS 0720 were put into a subclade (BS = 72; aLRT = 0.850). The accessions AND 0713, MES 0721, and BKA 0921 were grouped with two Pseudoroegneria accessions (Pseudoroegneria stipifolia PI 325181 and Pseudoroegneria gracillima PI 420842) and Thinopyrum elongatum (PI 142012) with weak support. Unexpectedly, the sequence from the E. pendulinus accession ZAR 0714 was not included in the St clade.

trnD-trnT analysis Thirteen trnD-trnT sequences from E. pendulinus and 38 sequences from GenBank were used in this analysis. The trnD-trnT data matrix of 51 sequences contained 900 characters, of which 73 were parsimony informative. MP analysis produced 295 equally parsimonious trees (CI = 0.797, excluding uninformative characters; RI = 0.764). The tree topologies generated by ML were similar to those generated by MP. An MP strict consensus tree with BS (1000 replicates) and ML aLRT values is shown in Fig. 2. Phylogenetic analyses based on trnD-trnT sequence data also grouped all sequences from E. pendulinus into the St genome clade, except accession ZAR0714 (BS = 55; aLRT = 0.786) (Fig. 2). Interestingly, the cpDNA trnD-trnT data tree also positioned the accession ZAR 0714 of E. pendulinus outside of the species of Hordeeae analyzed here. Within this clade, three accessions of E. pendulinus (VOK 0724, VOK 0728, and VBG 0727) formed an independent subclade (BS = 85; aLRT = 0.932). The accession AND 0713 of E. pendulinus grouped with one accession of P. stipifolia (PI 325181). RPB2 analysis As expected for allotetraploids, two different copies were recovered from six accessions (BKA 0921, VOK 0728, VOK 0724, VLA 0719, RUS 0716, and ZAR 0715); however, three distinct sequences were recovered from five accessions of E. pendulinus (VBG 0727, VBG 0722, MES 0721, ZAR 0714, and AND 0713), and one copy was recovered from two accessions (USS 0720 and VLA 0718). MP analysis using 100 RPB2 sequences together with two outgroup taxa, Bromus sterilis and B. catharticus, was conducted (332 parsimony informative characters, 1320 equally most parsimonious trees, CI = 0.573; RI = 0.859). The tree topology generated by ML analyses using the GTR model is similar to those generated by MP. An MP strict consensus tree with ML aLRT and MP BS (1000 replicates) values is shown in Fig. 3. The phylogenetic tree showed three different clades, representing the St, Y, and H genome groups. As expected, the sequences obtained from nine accessions of E. pendulinus (ZAR 0714, ZAR 0715, RUS 0716, VLA 0719, MES 0721, VBG 0722, VOK 0724, VBG 0727, and VOK 0728) that were amplified and sequenced were well separated into two Published by NRC Research Press

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Fig. 1. Strict consensus tree derived from RPS16 sequence data was conducted using a heuristic search with tree bisection-reconnection (TBR) branch swapping. Numbers above and below branches are bootstrap (BS) and maximum-likelihood approximate-likelihood ratio test (ML aLRT) values, respectively. Bromus catharticus was used as an outgroup. Consistency index (CI) = 0.886, retention index (RI) = 0.913.

100 0.998

70 0.950

70 0.895 72 0.850

St

53 0.792 83 0.864 0 864

66 0.877 88 0.911 93 1.000

52 0.788 88 0.870

different clades: one in the Pseudoroegneria-like (St genome) clade (BS = 83; aLRT = 0.98), another in the Y genome clade (BS = 96; aLRT = 0.97). Of the two accessions that had only one copy of the RPB2 sequences, USS 0720 had the St version and VLA 0718 the Y version. Within the Y genome clade, the E. pendulinus sequences were separated into two subclades (BS = 85 and 63). Unexpectedly, one of each of the sequences from five E. pendulinus accessions (AND 0713, MES 0721, VBG 0722, VBG 0727, and BKA 0921) was grouped with species of Hordeum (H, Xa, and I genomes) (BS = 100; aLRT = 1.00). These Hordeum-like sequences from E. pendulinus were put into two subclades. The first (BS = 91) is comprised of accessions AND 0713, MES 0721, VBG 0722, and VBG 0727 along with H. bogdanii; the second subclade contained BKA 0921 and AND 0713 Hordeum-like copy. PepC analysis Thirteen accessions of E. pendulinus were analyzed using the PepC gene sequence. Expectedly, two distinct PepC copies were

CN 32048 B. catharticus ZAR0714 E. pendulinus (StY) y y jjuncea ((Ns)) PI 406469 Psathyrostachys PI 547363 Aust. retrofractum (W) PI 533014 Aust. retrofractum (W) PI 208075 Taeniatherum caput-medusae (Ta) PI 222048 Taeniatherum caput-medusae (Ta) PI 561091 Taeniatherum caput-medusae (Ta) PI 191146 Triticum monococcum (AM) PI 499261 Aegilops speltoides (S) PI 599150 Aegilops g p searsii ((SS) PI 276994 Aegilops umbellulata (U) PI 486265 Aegilops tauschii (D) PI 542237 Aegilops sharonensis (SI) PI 542196 Aegilops longissima (SI) PI 554418 Aegilops uniaristata (N) PI 551032 Aegilops comosa (M) PI 573710 Secale cereale (R) PI 401354 Heteranthelium piliferum (Q) PI 142012 T. elongatum (Ee) AND0713 E. pendulinus (StY) MES0721 E. pendulinus (StY) BKA0921 E. pendulinus (StY) PI 325181 P. stipifolia (St) PI 420842 P. gracillima (St) VLA0719 E. pendulinus (StY) USS0720 E. pendulinus (StY) ZAR 0715 E. pendulinus (StY) PI 632554 P. geniculata (St) VLA 0718 E. pendulinus (StY) VBG0722 E. pendulinus (StY) H10248 P. ferganensis (St) PI 401330 P. tauri (St) VOK0724 E. pendulinus (StY) VOK0728 E. pendulinus (StY) VBG0727 E. E pendulinus d li (StY) RUS0716 E. pendulinus (StY) PI 330688 P. libanotica (St) PI 506274 P. spicata (St) PI 598674 Ag. fragile (P) PI 598460 Ag. mongolicum (P) PI 193264 Eremopyrum distans (F) PI 203440 Eremopyrum orientale (F) PI 203442 Eremopyrum bonaepartis (F) PI 577112 Henrardia persica (O) H1819 H. chilense (H) H7476 H.bogdanii (H)

recovered from nine accessions of E. pendulinus (PI 499452, ZAR 0715, RUS 0716, VLA 0718, USS 0720, MES 0721, VBG 0722, VOK 0724, and VBG 0727). Unexpectedly, three different copies of PepC sequences were detected from four accessions of E. pendulinus (AND 0713, ZAR 0714, VLA 0719, and BKA 0921), and only one copy was identified from accession VOK 0728. Phylogenetic analysis of the 80 sequences was performed using Bromus tectorum as an outgroup. The data matrix contained 1054 characters, of which 267 were constant, 395 were parsimony uninformative, and 392 were parsimony informative. Heuristic searches resulted in 1575 most parsimonious trees with a CI = 0.705, excluding uninformative characters, and RI = 0.838. The tree topologies generated by ML using the GTR model and MP analyses were similar to each other. An MP strict consensus tree with BS and aLRT values is shown in Fig. 4. The phylogentic analyses generated three large clades (Fig. 4), representing St genome sequences, Y genome sequences together with Hordeeae genome sequences (M, Ta, AM, R, and Ee), and a Published by NRC Research Press

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Fig. 2. Strict consensus tree derived from trnD-trnT sequence data was conducted using a heuristic search with tree bisection-reconnection (TBR) branch swapping. Numbers above and below branches are bootstrap (BS) and maximum-likelihood approximate-likelihood ratio test (ML aLRT) values, respectively. Bromus sterilis was used as an outgroup. Consistency index (CI) = 0.797, retention index (RI) = 0.764.

69 0.858

55 0.776

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96 0.980

67 0.896

57

St 55

85 0.932

0.786

67 0.881

51 0.772

0.756 56 0.880

100 0.957

87 0.950

100 1.000

Hordeum-like sequence. Thirteen E. pendulinus sequences were grouped with diploid species of Pseudoroegneria (St). The second clade was composed of the identified Y copies from tetraploid species of Elymus, E. pendulinus sequences, and the sequences from M, Ta, AM, R, and Ee genomes in Hordeeae. In this clade, nine accessions from E. pendulinus together with seven Y copies from additional tetraploid species of Elymus constructed a subclade, and the other three E. pendulinus sequences were grouped with R and Ee genomes sequences. Four sequences from E. pendulinus fell into the H clade together with diploid species of Hordeum, named Hordeum-like copies. Interestingly, a copy from E. pendulinus ZAR 0714 was also put outside of the species of Hordeeae analyzed here, which is consistent with the RPB2 nuclear gene and two

52 52 0.809 0.821

PI 203442 Eremopyrum bonaepartis (F) PI 533014 Aust. retrofractum (W) PI 531553 Aust. retrofractum (W) PI 533013 Aust. retrofractum (W) PI 203440 Eremopyrum orientale (F) PI 598460 Ag. mongolicum (P) PI 598674 Ag. fragile (P) PI 547363 Aust. retrofractum (W) PI 531752 P. strigosa (St) PI 401326 P. tauri (St) PI 420842 P. gracillima (St) PI563872 P. spicata (St) PI 142012 T. elongatum (Ee) W6 13089 P. strigosa subsp. aegilopoides (St) PI 401324 P. tauri (St) PI 228389 P. libanotica (St) W6 14049 P. strigosa subsp. aegilopoides (St) PI 440000 P. gracillima (St) PI 228390 P. libanotica (St) PI 330688 P. libanotica (St) PI 325181 P. stipifolia (St) pendulinus ((StY)) AND0713 E. p ZAR 0715 E. pendulinus (StY) MES0721 E. pendulinus (StY) USS0720 E. pendulinus (StY) VLA0719 E. pendulinus (StY) RUS0716 E. pendulinus (StY) VBG0727 E. pendulinus (StY) VOK0728 E. pendulinus (StY) VOK0724 E. pendulinus (StY) VLA 0718 E. E pendulinus (StY) VBG0722 E. pendulinus (StY) BKA0921 E. pendulinus (StY) PI 232140 P. spicata (St) PI232128 P. spicata (St) PI 563869 P. spicata (St) PI 598818 P. spicata (St) PI619445 P. spicata (St) PI598822 P. spicata p ((St)) PI 573710 Secale cereale (R) PI 542237 Aegilops sharonensis (SI) PI 551032 Aegilops comosa (M) PI 542196 Aegilops longissima (SI) PI 499261 Aegilops speltoides (S) H6439 H. stenostachys (H) PI 531762 H. bogdanii (H) H7754 H. roshevitzii (H) H10070 H. H roshevitzii h it ii (H) PI 406469 Psathyrostachys juncea (NS) ZAR0714 E. pendulinus (StY) PI 229595 B. sterilis

cpDNA data. Two distinct Hordeum-like copies were obtained from VBG 0727, and two distinct St copies from the accession BKA 0921 were also recovered. Within the St (Pseudoroegneria + Elymus) clade, all E. pendulinus accessions, except AND 0713, BKA 0921b, and VLA 0719, formed a well supported subclade (BS = 93; aLRT = 0.94). The sequences from AND 0713 and BKA 0921b were grouped into a separate subclade (BS = 99; aLRT = 0.99). The St copy from VLA 0719 was distinct from the other St copies. In the second clade, which contains the Y genome version of the sequence, AND 0713, BKA 0921b, and VLA 0719, diploid species of Hordeeae with the R, Ta, AM, Ee, and M genomes. The rest of the Y genome sequences of E. pendulinus were grouped with other tetraploid species of Elymus and were Published by NRC Research Press

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Fig. 3. Strict consensus tree derived from Rpb2 sequence data was conducted using a heuristic search with tree bisection-reconnection (TBR) branch swapping. Numbers above and below branches are bootstrap (BS) and maximum-likelihood approximate-likelihood ratio test (ML aLRT) values, respectively. Bromus catharticus was used as an outgroup. Consistency index (CI) = 0.573, retention index (RI) = 0.859.

91 0.945

55 79 0.956

95 1.000

77 0.814

100 1.000

99 0.974

82

100 0.999

0.998 100

100 0.998

1.000 98 0.991

100 1.000 62

100 1.000

67 0.927

100 0.995

92 0.963 57

71 0.946

0.911 63 0.782 70 0.957 57

83 0.978

0.853

99

65

1.000

91

81 0.803

0.926

0.905

71 00.994 994

55 0.994

0.690

63

67 0.932 94 0.998

100 0.978

86 100 0.831 0.993 64 94 0.747 0.914

71 0.831

63 85 0.905

96

63 0.754

0.973 100 1.000 100 0.997 100 0.995

64

0.781

Y+Triticeae

85 0.911

Pseudoroegneria+Elyymus

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91

Hordeum+Elymus

90 0.871

0.969

69

86 0.913

?

100 1.000

CN32048 B. catharticus PI229595 B. sterilis ZAR0714 E. pendulinus (StY) PI430871 Psathyrostachys juncea (Ns) PI406469 Psathyrostachys juncea (Ns) MES0721 E. pendulinus (StY) VBG0727 E. pendulinus (StY) AND0713 E. pendulinus (StY) VBG0722 E. pendulinus (StY) H499645 H. bogdanii (H) PI499498 H. bogdanii (H) H4014 H. bogdanii (H) H6052 H. H patagonicum t i (H) H9152 H. roshevitzii (H) H1181 H. comosum (H) H1780 H. stenostachys (H) H6439 H. stenostachys (H) H6460 H. cordobense (H) H1358 H. patagonicum (H) H1816 H. chilense (H) H3317 H. brachyantherum (H) H2127 H. flexuosum (H) H121 H. marinum (Xa) H581 H. marinum (Xa) H1236 H. pubiflorum (H) H1342 H. patagonicum (H) H1352 H. patagonicum (H) H1353 H. patagonicum (H) AND0713 E. pendulinus (StY) BKA0921 E. pendulinus (StY) H3140A 3140A H. vulgare l ssp. spontaneous (I) () H7514A H. vulgare ssp. vulgare (I) H3878 H. bulbosum (I) PI203442 Eremopyrum bonaepartis (F) PI219966 Eremopyrum bonaepartis (F) PI330687 P. libanotica (St) PI330688 P. libanotica (St) PI 531573 E. caucasicus (StY) PI401274 P. libanotica (St) PI228389 P. libanotica (St) VBG0722 E. pendulinus (StY) PI 564917 E. ciliaris (StY) MES0721 E. pendulinus (StY) AND0713 E. pendulinus (StY) VLA0719 E. pendulinus (StY) USS0720 E. pendulinus (StY) RUS0716 E. pendulinus (StY) VOK0724 E. pendulinus (StY) VBG0727 E. E pendulinus d li (S Y) (StY) VOK0728 E. pendulinus (StY) ZAR0714 E. pendulinus (StY) ZAR 0715 E. pendulinus (StY) PI 531554 E. abolinii (StY) PI 207452 E. semicostatus (StY) PI506274 P. spicata (St) PI610986 P. spicata (St) PI325181 P. stipifolia (St) PI440095 P. stipifolia (St) PI537389 P. spicata (St) W614049 P. strigosa (St) PI286198 P. spicata (St) W613089 P. strigosa subsp. aegilopoides (St) PI232134 P. spicata (St) PI236669 P. spicata (St) PI619445 P. spicata (St) PI547162 P. spicata p ((St)) PI563869 P. spicata (St) PI420842 P. gracillima (St) PI547154 P. spicata (St) PI440000 P. gracillima (St) PI537379 P. spicata (St) PI598822 P. spicata (St) PI232128 P. spicata (St) PI531752 P. strigosa (St) PI516184 P. spicata (St) PI228390 P. libanotica (St) PI401324 P. tauri (St) PI401326 P. tauri (St) PI539873 P. spicata (St) PI563872 P. spicata (St) PI 401280 E. longearistatus (StY) PI142012 Thinopyrum elongatum (Eb) PI531712 Thinopyrum bessarabicum (Eb) PI554418 Aegilops g p uniaristata ((N)) PI276996 Aegilops uniaristata (N) P383534 Ag. cristatum (P) PI598460 Agropyron mongolicum (P) PI598694 Agropyron fragile (P) ZAR0714 E. pendulinus (StY) ZAR 0715 E. pendulinus (StY) BKA0921 E. pendulinus (StY) VOK0724 E. pendulinus (StY) RUS0716 E. pendulinus (StY) VLA0719 E. pendulinus (StY) VLA 0718 E. pendulinus (StY) MES0721 E. pendulinus (StY) VBG0722 E. pendulinus (StY) VBG0727 E. pendulinus (StY) VOK0728 E. pendulinus (StY) PI 564917 E. ciliaris (StY) PI 531554 E. abolinii (StY) PI 207452 E. semicostatus ((StY)) PI 401280 E. longearistatus (StY) PI 531573 E. caucasicus (StY) PI401351 Heteranthelium piliferum (Q) PI401354 Heteranthelium piliferum (Q) PI533014 Aust. Retrofractum (W) PI547363 Aust. Retrofractum (W) PI208075 Ta. caput-medusae (Ta) PI222048 Ta. caput-medusae (Ta) PI220591 Ta. caput-medusae (Ta) PI561091 Ta. caput-medusae (Ta)

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Fig. 4. Strict consensus tree derived from PepC sequence data was conducted using a heuristic search with tree bisection-reconnection (TBR) branch swapping. Numbers above and below branches are bootstrap (BS) and maximum-likelihood approximate-likelihood ratio test (ML aLRT) values, respectively. Bromus tectorum was used as an outgroup. Consistency index (CI) = 0.705, retention index (RI) = 0.838.

56 97 0.977 100 1.000

98 0.991

99

100

100 1.000 100 0.996 89 0.973

93

0.788

57 0.769

58 0.791

95 0.956 60 0.873

53 93 0.930 55 65 100

88 0.856 99 0.998 62 93 0.950

0.994 96 70 0.860 80

0.940

62

78 0.885

100 65 0.996 0.909 99 0.946

0.988

67 0.818

100 1.000 100 1.000

65 0.749 64 0.756 82 100 0.998

0.871

Discussion Maternal donor of E. pendulinus The St genome is present in all species of Elymus and is a key component of this genus. It has been suggested that Pseudoroegeneria

?

well supported (BS = 100; aLRT = 1.00), and within this clade, nine accessions from Russia were placed into one subclade (BS = 62; aLRT = 0.87).

62 0.871

Y+Triticeae

0 732 0.732

62 0.773

Pseudoroegneeria+Elymus

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88 00.997 997

AND0713 E. pendulinus (StY) VBG0727 E. pendulinus (StY) H 1816 H. chilense (H) PI 531791 H. stenostachys(H) CIho 15684 H. pusillum (H) PI 531760 H. bogdanii (H) PI 304346 H. marinum (Xa) PI 304347 H. H marinum i (X ) (Xa) VLA0719 E. pendulinus (StY) VBG0727 E. pendulinus (StY) CIho 15683 H. murinum (Xu) PI 247054 H. murinum (Xu)-a PI 247054 H. murinum (Xu)-b PI 440417 H. bulbosum (I)-a PI 440417 H. bulbosum (I)-b PI 440417 H. bulbosum (I)-c RJMG 107 H. vulgare (I) RUS0716 E. pendulinus (StY) VBG0722 E. pendulinus (StY) USS0720 E. pendulinus (StY) BKA0921 E. E pendulinus (StY) VLA0718 E. pendulinus (StY) MES0721 E. pendulinus (StY) ZAR0714 E. pendulinus (StY) ZAR0715 E. pendulinus (StY) VOK0724 E. pendulinus (StY) PI499452 E. pendulinus (StY) PI531555 E. abolinii (StY) PI314620 E. nevskii (StY) PI 499637 P. strigosa (St)-a PI 499637 P. strigosa (St)-b PI 531755 P. strigosa subsp. aegilopoides(St) PI531575 E. ciliaris (StY) PI271522 E. semicostatus (StY) A 0 13 E. pendulinus AND0713 d l (S ) (StY) BKA0921 E. pendulinus (StY) PI 313960 P. stipifolia (St) PI 531751 P. stipifolia (St)-a PI 531751 P. stipifolia (St)-b PI 610986 P. spicata (St) D 2844 P. spicata (St) PI 282392 P. libanotica (St) PI 380652 P. tauri (St) PI 228391 P. libanotica (St) PI 401319 P. tauri (St) PI 380644 P. tauri (St)-a PI 380644 P. tauri (St)-b PI632564 E. E antiquus (StY) PI401277 E. longearistatus (StY) PI531573 E. caucasicus (StY) VLA0719 E. pendulinus (StY) PI 203440 Eremopyrum orientale (F) G602 Aegilops comosa (M) RJMG 189 Ta. caput-medusae (Ta) Triticum monococcum (AM) Kellogg s.n. Secale cereale (R) RJMG 113 Thinopyrum elongatum (Ee) AND0713 E. pendulinus (StY) BKA0921 E. pendulinus (StY) VLA0719 E. pendulinus (StY) PI531575 E. ciliaris (StY) PI314620 E. E nevskii (StY) PI531555 E. abolinii (StY) PI632564 E. antiquus (StY) PI271522 E. semicostatus (StY) PI499452 E. pendulinus (StY) ZAR0714 E. pendulinus (StY) RUS0716 E. pendulinus (St) VOK0728 E. pendulinus (StY) MES0721 E. pendulinus (StY) ZAR0715 E. pendulinus (StY) VOK0724 E. pendulinus (StY) USS0720 E. pendulinus (StY) VBG0722 E. pendulinus (StY) VLA0718 E. E pendulinus (StY) PI401277 E. longearistatus (StY) PI531573 E. caucasicus (StY) PI 401354 Heteranthelium piliferum (Q) ZAR0714 E. pendulinus (StY) Kellogg s.n. B. tectorum

Hordeu um+Elymus

62 0.607 84 90 0.899 0.840

100 0.928

is the maternal donor during polyploid speciation of the tetraploid species of Elymus (Dewey 1984). Chloroplast sequences revealed a very close relationship of Pseudoroegeneria to all species of Elymus (Redinbaugh et al. 2000; Mason-Gamer 2001; McMillan and Sun 2004; Liu et al. 2006). As expected, the cpDNA data indicated that most of the E. pendulinus accessions analyzed here have the St genome donor as maternal parent (Figs. 1 and 2), which is consistent with previous results. Published by NRC Research Press

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Yan et al.

The data for one accession, ZAR 0714, was exceptionally anomalous, having both cpDNA and nDNA that appears to be non-hordeaceous in origin. The accession ZAR 0714 has been morphologically and anatomically confirmed as E. pendulinus by A.V. Agafonov at the Central Siberian Botanical Garden of the Siberian Branch of the Russian Academy of Science, Novosibirsk (personal communication). The DNA was isolated from the seeds collected by A.V. Agafonov. RPS16 and trnD-trnT sequence trees revealed that the E. pendulinus accession ZAR 0714 is present outside of the Hordeeae clade, indicating two possible maternal donors to E. pendulinus. Both nuclear gene data also found a copy of sequence from ZAR 0714 present outside the Hordeeae in the phylogenetic trees (Figs. 3 and 4). It is not clear if an unknown donor from outside of the Hordeeae, if any, represented an entire genome from a third donor, or whether both cpDNA and nuclear gene are acquired through introgression by natural hybridization. The former hypothesis can be ruled out because our cytological observation found that this accession is tetraploid (data not shown). We suggest an alternative interpretation that introgression events occurred within the donor of ZAR 0714 for the chloroplast and nuclear genes in ancient time, as has been reported in several instances (Martinsen et al. 2001; Heuertz et al. 2006). A recent study by Koch and Matschinger (2007) suggested that chloroplast introgression has become less common in recent times. Elymus pendulinus has been shown to hybridize with other species (Lu et al. 1991; Salomon and Lu 1994; Zhou et al. 1999), demonstrating at least some potential to acquire genetic material through introgression. Moreover, both nuclear gene data of ZAR 0714 contained the St copy. The conclusion that Pseudoroegneria is the maternal donor of E. pendulinus is favored here. We proposed a hypothetical scenario that a successful hybridization event might lead to introgression, which have occurred repeatedly or continually on E. pendulinus ZAR 0714, leading to a morphological variation. Introgression shaped genome diversity within E. pendulinus Meiotic pairing data has indicated that the St genomes of both Elymus and Pseudoroegneria have a very high similarity (Dewey and Asay 1982; Jensen and Salomon 1995). Cytological and molecular data have also confirmed that Pseudoroegneria is the donor of the St genome in Elymus (Dewey 1984; Jensen 1990; Torabinejad and Mueller 1993; Jensen and Salomon 1995; Jones et al. 2000; Redinbaugh et al. 2000; Mason-Gamer et al. 2002; McMillan and Sun 2004; Xu and Ban 2004; Liu et al. 2006; Yan et al. 2011). Results from nuclear gene sequences are more complicated than those from the chloroplast genome. Our results indicate that E. pendulinus has experienced a very complex evolutionary history that has involved multiple hybridizations and polyploidization. The analyses of unlinked nuclear RPB2 and PepC gene sequences demonstrated intra-specific variation in natural E. pendulinus populations originating from Russia. The nuclear gene trees revealed more potential donors than were expected, including Hordeum (H) and an unknown donor, possibly from within or outside the tribe, in addition to the St and Y genomes. The presence of the St and Y genome within E. pendulinus is consistent with previous cytogenetic (Dewey 1984; Jensen 1990) and molecular data (Mason-Gamer et al. 2002; McMillan and Sun 2004). However, these two expected copies were not recovered from all accessions. In the RPB2 gene data, one of the expected copies (St and Y) was not recovered from BKA 0921, VLA 0718, and USS 0720. In addition, the PepC sequence data showed that St copy sequences were not recovered from accessions VOK 0728 and VBG 0727. While failure to obtain a particular gene copy from a polyploid species may not indicate the lack or loss of that gene copy, in this situation primers specific to the “missing” gene copy may clarify the case (Ge et al. 1999; Ferguson and Sang 2001; Doyle et al. 2002; Mahelka and Kopecký 2010). Furthermore, when we considered the two phylogenetic trees from nuclear genes, the St

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and Y copies either from RPB2 or PepC, or from both genes have been recovered in all accessions of E. pendulinus. The apparent involvement of Hordeum (genome H) as a third participant in the evolution of E. pendulinus comes as a surprise. Five out of 13 E. pendulinus accessions contained the Hordeumlike sequence in RPB2 data, and 3 out of 13 in PepC data. There is little cytogenetic evidence for the presence of an H genome in E. pendulinus. Hordeum-like PepC sequences from E. pendulinus have numerous insertions and deletions, some of which involve gain or loss of Strowaway-like transposable elements, which is consistent with a previous study in Elymus repens (StStH) (Mason-Gamer 2008). The RPB2 and PepC Hordeum-like copies were found in two accessions (AND 0713 and VBG 0727). A Hordeum-like RPB2 sequence was found in three accessions (MES 0721, VBG 0722, and VBG 0727) and a Hordeum-like PepC sequence in accession VLA 0719. The hypothesis of an entire H genome present in E. pendulinus can be ruled out because our cytological observation found that all accessions studied here are tetraploids without a third genome (data not shown). A direct contribution from Hordeum to E. pendulinus remains a possibility. Although StH and StY species are intersterile, there are many StHY allopolyploid species in the genus Elymus. The gene exchange may have occurred between the H and Y genome in StHY species. An alternative explanation might be that E. pendulinus acquired the sequences from the H genome through introgression. This phenomenon has been revealed for certain loci in E. repens (Mason-Gamer 2008) and in Elymus ciliaris (unpublished data). Another unexpected result is the apparent genetic contribution from species outside the Hordeeae to E. pendulinus. Multiple origins of E. pendulinus Our molecular results have demonstrated intra-specific variation in E. pendulinus in Russia. From the RPS16 cpDNA gene tree, three subclades composed of E. pendulinus accessions were examined in the St clade. The sequences from E. pendulinus AND 0713, MES 0721, and BKA 0921 fell into the clade with sequences from diploid P. stipifolia and P. gracillima, which indicated that they could be potential donors. The other two subclades in the RPS16 St clade are a monophyletic group with E. pendulinus accessions only. On the trnD-trnT tree, accession AND 0713 is grouped with P. stipifolia as well, and the same accessions (VBG 0727, VOK 0728, and VOK 0724) formed a subclade in St clade (BS = 85; aLRT = 0.93) as RPS16 (BS = 83; aLRT = 0.86). Based on our phylogenetic analyses, both the RPS16 and trnD-trnT cpDNA trees point to more than one potential maternal donor for E. pendulinus, suggesting that the Russian E. pendulinus used in this study may have originated from multiple sources of Pseudoroegneria. Within the RPB2 tree, four accessions of E. pendulinus (MES 0721, VBG 0727, AND 0713, and VBG 0722) are placed in a distinct monophyletic group (BS = 91; aLRT = 0.95) that is sister to the H. bogdanii; the other two Hordeum-like sequences (AND 0713 and BKA 0921) are grouped together into a distinguished subclade (BS = 100; aLRT = 1.00). This demonstrates that the RPB2 sequences in E. pendulinus might be introgressed from at least two different Hordeum diploids. In the RPB2 St clade, five accessions were placed into two subclades with weak support; as well as RPB2 Y clade, all E. pendulinus accessions are separated into two distinguished subclades. In the PepC tree, the Hordeum-like copies are also separated into two suclades (AND 0713 with VBG 0727; VLA 0719 with VBG 0727). In the St clade, nine accessions of E. pendulinus from Russia formed a subclade (BS = 93; aLRT = 0.94). The other three accessions (AND 0713, BKA 0921, and VLA 0719) do not have a close relationship with any of the diploid taxa sampled, and thus which St genome species was their progenitor remains unknown. A similar situation occurs in the Y clade as well, where the same three accessions (AND 0713, BKA 0921, and VLA 0719) in E. pendulinus are grouped with the Hordeeae genome (R, Ee, AM, Ta, and M), which are separated from the other Y copy of E. pendulinus. This phenomPublished by NRC Research Press

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enon has been reported for the EF-G gene (elongation factor) in tetraploid species of Elymus (Sun and Komatsuda 2010), where Y genome sequences were grouped with W and E sequences as well as sequences from many annual species (M, N, Ta, R, A, Q, etc.). All of the gene trees suggested multiple contributions from Pseudoroegneria, an unknown Y genome donor, and species of Hordeum, and reveal sequence divergence after polyploid formation.

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Acknowledgement This research was supported by discovery grants from the Natural Sciences and Engineering Research Council of Canada (238425), Canadian Foundation for Innovation, and a Senate Research Grant at Saint Mary’s University to G.S. Thanks go to A.V. Agafonov at the Central Siberian Botanical Garden of the Siberian Branch of the Russian Academy of Science, Novosibirsk, for providing the seeds of Elymus pendulinus; Lauren Davey for English editing of the manuscript; and two reviewers for their valuable comments.

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