Biologia 63/4: 498—505, 2008Section Cellular and Molecular BiologyDOI: 10.2478/s11756-008-0091-2

Phylogenetic relationships of species in Pseudoroegneria (Poaceae:Triticeae) and related genera inferred from nuclear rDNA ITS(internal transcribed spacer) sequences

Haiqing Yu1,2, Xing Fan1, Chun Zhang1, Chunbang Ding3, XiaoliWang3

& Yonghong Zhou1,2*1Triticeae Research Institute, Sichuan Agricultural University, Dujiangyan 611830, Sichuan, People’s Republic of China;e-mail: zhouyh@sicau.edu.cn2 Key Laboratory of Crop Genetic Resources and Improvement, Ministry of Education, Sichuan Agricultural University,Yaan 625014, Sichuan, People’s Republic of China3 College of Biology and Science, Sichuan Agricultural University, Yaan 625014, Sichuan, People’s Republic of China

Abstract: To evaluate the phylogenetic relationships of species in Pseudoroegneria and related genera, the nuclear ribosomalinternal transcribed spacer (ITS) sequences were analyzed for eighteen Pseudoroegneria (St), two Elytrigia (EeSt), twoDouglasdeweya (StP), three Lophopyrum (Ee andEb), three Agropyron (P), two Hordeum (H), two Australopyrum (W)and two Psathyrostachys (Ns) accessions. The main results were: (i) Pseudoroegneria gracillima, P. stipifolia, P. cognata andP. strigosa (2x) were in one clade, while P. libanotica, P. tauri and P. spicata (2x) were in the other clade, indicating thereare the differentiations of St genome among diploid Pseudoroegneria species; (ii) P. geniculata ssp. scythica, P. geniculatassp. pruinifera, Elytriga caespitosa and Et. caespitosa ssp. nodosa formed the EeSt clade with 6–bp indel in ITS1 regions;and (iii) Douglasdeweya wangii, D. deweyi, Agropyron cristatum and A. puberulum comprised the P clade. It is unreasonableto treat P. geniculata ssp. scythica and P. geniculata ssp. pruinifera as the subspecies of P. geniculata, and they should betransferred to a new genus Trichopyrum, which consists of species with EeSt genomes. It is also suggested that one of thediploid donor of D. wangii and D. deweyi is derived from Agropyron species, and it is reasonable to treat tetraploid specieswith StP genomes into Douglasdeweya.

Key words: Pseudoroegneria; Douglasdeweya; Trichopyrum; internal transcribed spacer; phylogeny; genome.

Abbreviations: BI, Bayesian inference; bp, base pairs; ITS, internal transcribed spacer; ML, maximum likelihood.

Introduction

Pseudoroegneria is a genus in Triticeae (Poaceae)with Pseudoroegneria strigosa (M. Bieb.) Á. Love asthe type species (Love 1980). Morphologically, thespecies in this genus are caespitose, long-anthered andcross-pollinating perennials. Pseudoroegneria was builtaround one genome designated St, which is one ofthe most important basic genomes (St, H, P, W,Ns and E) in perennial Triticeae. The St genome isthe donor genome of the species in the polyploid gen-era Roegneria (StY), Douglasdeweya (StP), Elymus(StH, StYH and StYW), Kengyilia (StYP), Elyt-rigia (EeSt and EbEeSt) and Pascopyrum (StHN-sXm) (Dewey 1984; Love 1984; Yen & Yang 1990; Yenet al. 2005a,b). The species in Pseudoroegneria are dis-tributed in the Northern Hemisphere, and occurred onopen rocky hillsides from the Middle East and Tran-scaucasia across Central Asia and Northern China toWestern North America. Pseudoroegneria grasses are

exceptionally drought tolerant and have excellent for-age quality (Dewey 1984).Love (1984) and Dewey (1984) suggested that the

taxonomic treatment for Triticeae species should bebased on genomic constitutions and this view has beenwidely accepted (Lu 1994; Wang et al. 1994; Zhou et al.1999; Yen et al. 2005b). The cytogenetic studies indi-cated that there are St, StSt, StP and EeSt genomicconstitutions in Pseudoroegneria and all of them arediploids or tetraploids (Dewey 1984; Wang et al. 1986;Jensen et al. 1992; Liu &Wang 1993). Yen et al. (2005a)established a new genus Douglasdeweya C. Yen, J.L.Yang & B.R. Baum, and the species with StP genomesin Pseudoroegneria were transferred to Douglasdeweya.It has also been reported that three tetraploid species inRoegneria C. Koch: Roegneria alashanica Keng, Roeg-neria elytrigioides C. Yen et J. L. Yang and Roegne-ria magnicaespes (D. F. Cui) L. B. Cai, contain StStgenomes and should be treated as the species of Pseu-doroegneria (Lu 1994; Zhou et al. 1999; Zhang et al.

* Corresponding author

c©2008 Institute of Molecular Biology, Slovak Academy of Sciences

Phylogenetic relationships in Pseudoroegneria 499

Table 1. Species and accessions used in this study.

No. Species Accession No. Genome Geographic origin GenBank

Pseudoroegneria (Nevski) Á. Love

1 P. alashanica (Keng) B.R. Lu Z2006 StSt Yinchuan, Ningxia, China AY740796a,AY740797a

2 P. cognata (Hack.) Á. Love Y0756 St Kuqa, Xinjiang, China EF014226

3 P. elytrigioides (C. Yen et J.L. Yang) B.R. Lu Z2005 StSt Changdu, Tibet, China AY740798a,AY740799a

4 P. geniculata (Trin.) Á. Love PI 565009 StSt Russian Federation EF014227,EF014228

5 P. geniculata ssp. pruinifera (Nevski) Á. Love PI 547374 — Ural, Russian Federation EF014229

6 P. geniculata ssp. scythica (Nevski) Á. Love PI 502271 EeSt Russian Federation EF014231

7 P. gracillima (Nevski) Á. Love PI 440000 St Stavropol, Russian Federation EF014233

8 P. gracillima (Nevski) Á. Love PI 420842 St Russian Federation EF014234

9 P. kosaninii (Nabelek) Á. Love PI 237636 — Turkey EF014235,EF014236,EF014237

10 P. libanotica (Hackel) D.R. Dewey PI 228389 St Iran AY740794a

11 P. libanotica (Hackel) D.R. Dewey PI 343188 St Iran EF014238

12 P. spicata (Pursh) Á. Love PI 547161 St Oregon, United States AY740793a

13 P. spicata (Pursh) Á. Love PI 232124 StSt Washington, United States EF014239

14 P. stipifolia (Czern. ex Nevski) Á. Love PI 325181 St Stavropol, Russian Federation EF014240

15 P. strigosa (M. Bieb.) Á. Love PI 499637 St Urumqi, Xinjiang, China AY740795a

16 P. strigosa (M. Bieb.) Á. Love PI 531752 StSt Estonia, Russian Federation EF014241,EF014242

17 P. strigosa ssp. aegilopoides (Drobow) Á. Love PI 595164 St Xinjiang, China EF014243

18 P. tauri (Boiss. & Balansa) Á. Love PI 401323 St Iran EF014244

Douglasdeweya C. Yen, J.L. Yang & B.R. Baum

19 D. deweyi (K.B. Jensen, S.L. Hatch & J.K. Wipff) PI 531756 StP Caucasus, Russian Federation EF014250C. Yen, J.L. Yang & B. R. Baum

20 D. wangii C. Yen, J.L. Yang & B. R. Baum PI 401330 StP Tabriz, Iran EF014251

Elytrigia Desvaux

21 Et. caespitosa (C. Koch) Nevski PI 547311 EeSt Russian Federation EF014245

22 Et. caespitosa ssp. nodosa (Nevski) Tzvelev PI 547345 EeSt Ukraine EF014247

Lophopyrum Á. Love

23 Lo. bessarabicum (Savul & Rayss) PI 531712 Eb Estonia,Russian Federation L36506a

C. Yen, J.L. Yang & Y. Yen

24 Lo. elongatum (Host) Á. Love PI 531719 Ee France EF014249

25 Lo. elongatum (Host) Á. Love PI 547326 Ee France L36495a

500 H. Yu et al.

Table 1. (continued)

No. Species Accession No. Genome Geographic origin GenBank

Agropyron Gaertner

26 A. cristatum (L.) Gaertner H10154 P Altai, Xinjiang, China AY740890a

27 A. cristatum (L.) Gaertner H10066 P Altai, Xinjiang, China AY740891a

28 A. puberulum (Boiss. Ex Steud.) Grossh. PI 229573 P Iran L36482a

Australopyrum (Tzvelev) Á. Love

29 Au. pectinatum (Labill.) Á. Love ssp. pectinatum D3438 W Australia L36483a

30 Au. pectinatum (Labill.) Á. Love ssp. retrofractum PI 531553 W Australia L36484a

(J.W. Vickery) Á. Love

Hordeum L.

31 H. bogdanii Wilensky PI 531761 H China AY740876a

32 H. brevisubulatum (Trin.) Link Y1604 H Fuyun, Xinjiang, China AY740877a

Psathyrostachys (Nevski) Á. Love

33 Ps. huashanica Keng ex P. C. Kuo PI 531823 Ns Shanxi, China L36499a

34 Ps. juncea (Fisch.) Nevski PI 314521 Ns Russian Federation L36500a

Bromus L.

35 B. catharticus Vahl S20004 — Kunming, Yunnan, China AF521898a

a These GenBank accession No. were published previously in GenBank (Benson et al. 2007).

2002; Ding et al. 2005). Although the three tetraploidspecies, Elytrigia caespitosa (C. Koch) Nevski, Elytri-gia caespitosa ssp. nodosa (Nevski) Tzvelev and Pseu-doroegneria geniculata ssp. scythica (Nevski) Á. Love,have been treated in two different genera based onmorphological classification, they were suggested to betreated in a new genus Trichopyrum Á. Love because oftheir similar genome formula EeSt (Liu & Wang 1993;Yen et al. 2005b). In addition, the genomic constitu-tions of octoploid Pseudoroegneria kosaninii (Nabelek)Á. Love and hexaploid Pseudoroegneria geniculata ssp.pruinifera (Nevski) Á. Love are obscure. Therefore, thephylogenetic relationships and taxonomic treatment ofseveral species related to Pseudoroegneria are still indispute.To understand the phylogenetic relationships

among the species of Pseudoroegneria, Elytrigia, Dou-glasdeweya and Lophopyrum, in the present study wesequenced and analyzed the nuclear ribosomal inter-nal transcribed spacer (ITS) fragments of Pseudoroeg-neria and related species. The aims were as follows:(i) to investigate the differentiation of St genome indiploid Pseudoroegneria species; (ii) to evaluate thephylogenetic relationships of Pseudoroegneria species;and (iii) to demonstrate the intergeneric relationshipsamong Pseudoroegneria, Elytrigia, Douglasdeweya andLophopyrum.

Material and methods

Plant materialsA total of 34 Triticeae accessions, including 18 Pseu-dorogeneria accessions with different genomic constitutions(i.e. the St, StSt, and EeSt genomes together with P.kosaninii and P. geniculata ssp. pruinifera), two species ofElytrigia (EeSt genomes), three accessions of Lophopyrum(Ee and Eb genome), two species of Douglasdeweya (StPgenomes), three accessions of Agropyron (P genome), twoaccessions of Australopyrum (W genome), two species ofHordeum (H genome) and two species of Psathyrostachys(Ns genome), were used in this study. Bromus cathar-ticus Vahl was used as outgroup. All the seed mate-rials were kindly provided by American National PlantGermplasm System (Pullman, WA, USA) and Triticeae Re-search Institute, Sichuan Agricultural University (Sichuan,People’s Republic of China). These seeds were germi-nated and grown in the perennial nursery. The matureplants were carefully identified by Profs. Chi Yen, Jun-liang Yang and Yonghong Zhou. The taxa, accession num-bers, genomic constitutions, geographic origins and Gen-Bank accession numbers (Benson et al. 2007) are listedin Table 1. The nomenclature and genome symbols of thespecies used in this study follow the opinions of Love(1984), Dewey (1984), Wang et al. (1994) and Yen et al.(2005b).

DNA extraction and purificationThe leaf samples for each material were collected frommature plants in the perennial nursery of Triticeae Re-

Phylogenetic relationships in Pseudoroegneria 501

search Institute, and ground in liquid nitrogen in a1.5 mL microfuge tube. DNA was extracted and puri-fied with a slight modification of the cetyltrimethylam-monium bromide procedure outlined in Doyle & Doyle(1990).

ITS amplification, cloning and sequencingThe amplification of ITS regions was done using primersITS-4 (5’-TCCTCCGCTTATTGATATGC-3’) and ITS-L(5’-TCGTAACAAGGTTTCCGTAGGTG-3’) (Baldwin1992; Hsiao et al. 1995). The PCR reaction was carried outin a total volume of 25 µL containing 1× reaction buffer,1.5 mM MgCl2 , 0.5 µM of each primer, 200 µM of eachdNTP (TakaRa Biotechnology Co., Ltd, Dalian, China),0.5 units of ExTaq Polymerase (TakaRa), with an addi-tion of 10% dimethyl sulfoxide (Buckler & Holtsford 1996;Buckler et al. 1997) and sterile water to the final volume.The thermocycling profile consisted of an initial denatu-ration at 94◦C for 3 min, followed by 35 cycles of 1 minat 94◦C, 1 min at 52◦C, 1 min at 72◦C and final exten-sion of 8 min at 72◦C. PCR reactions of each accessionwere carried out in four independent reactions in an ABI9700 thermal cycler (Applied Biosystems, CA, USA). Am-plification products were mixed and purified using the GelExtraction Kit (50) (Omega, GA, USA) and linked into apMD18–T Easy Vector Systems according to the manufac-turer’s instruction (TakaRa). Three to five positive clonesfor each species were randomly selected and sequenced bySunbiotech Co., Ltd (Beijing, China). The sequences usedin this study have been deposited with GenBank (Ben-son et al. 2007) under the accession numbers EF014226–EF014251).

Sequence alignment and phylogenetic analysisThe boundaries of the ITS regions were determined bycomparison with the ITS sequence ofPseudoroegneria liban-otica (Hackel) D. R. Dewey (GenBank accession numberL36501) (Hsiao et al. 1995). The ITS sequence alignmentwas executed with Clustal X program (Thompson et al.1997). Gaps were coded as binary characters by their pres-ence/absence, and were used for the phylogenetic analy-ses.

PAUP* 4.0b10 (Swofford 2002) was used to find thebest maximum likelihood (ML) tree by performing a heuris-tic search with tree-bisection-reconnection branch swappingand 100 random addition replicates. Topological robustnesswas assessed by bootstrap analysis with 100 replications us-ing as-is sequence addition. The SYM+G model of evolu-tion was used in the ML analysis based on the result ofMrModeltest 2.2 (Nylander 2004). The fit of 24 ML mod-els to the data set was tested, and base change frequen-cies, proportion of variable characters and shape of thegamma distribution were estimated in the Modeltest anal-ysis. The best model to the data was obtained by the Hi-erarchical Likelihood Ratio Tests (Posada & Crandall 1998;Roalson & Friar 2004). Bayesian inference (BI) for phy-logenetic analysis was executed with MrBayes 3.1.2 (Ron-quist & Huelsenbeck 2003). The same model and numbersof base change frequencies set in the ML search were usedin BI analysis with starting from a random tree. Markovchain Monte Carlo chain length for analyses was 8×106generations with trees sampled every 100 generations andthe first 2×104 trees were discarded as burn-in. Additionalruns with the same conditions produced the same topologywith insignificant differences in posterior probability of anynode.

Results

Sequence analysis of ITS regionsThe length of sequences ranged from 212 to 221 bp inthe ITS1 region and from 215 to 217 bp in the ITS2 re-gion. The 5.8S subunit was the most conserved regionand was 164 bp in length for all the sequences exceptfor one clone from Pseudoroegneria geniculata (Trin.)Á. Love, which has a 5-bp insertion (CATCG) at posi-tion 261 of the alignment. The average of G+C contentwas 62%. The aligned sequences yielded 608 characters.Most of the sequence variations occurred in the spacerregions. In the ITS1 and ITS2 regions, 71 and 77 vari-able characters were detected respectively. There was a4-bp deletion (GTGG) in all accessions ofH,Ns,P andW genomes and a 6-bp indel (TTTTCA) from position55 to 60 in Et. caespitosa, Et. caespitosa ssp. nodosa,P. geniculata ssp. scythica and P. geniculata ssp. pru-inifera (Fig. 1). Multiple base pairs insertion, deletionor substitution was not found in the ITS2 region.

Phylogenetic analysis of Pseudoroegneria and the re-lated generaThe topologies of the two analyses (ML and BI) werenearly consistent. The ML tree is shown in Figure 2.The percent of bootstrap values and posterior prob-abilities are indicated above and below the branches,respectively. Three major clades (Clade A, H and Ns)were formed. The Ns clade (Psathyrostachys) was in-cluded together with the H clade (Hordeum) in abasal polytomy. The clade A consisted of all the Pseu-doroegneria species together with species in Elytri-gia, Lophopyrum, Douglasdeweya, Agropyron and Aus-tralopyrum. Five groups were detected within the cladeA. The Group I consisted of Et. caespitosa, Et. caespi-tosa ssp. nodosa, P. geniculata ssp. scythica, P. genic-ulata ssp. pruinifera, P. geniculata, P. alashanica, P.elytrigioides, P. strigosa (2x, 4x), Pseudoroegneria spi-cata (Pursh) Á. Love (4x), P. kosaninii, Lophopyrumelongatum (Host) Á. Love, Lophopyrum bessarabicum(Á. Love) C. Yen, J. L. Yang et Y. Yen, Pseudoroegne-ria gracillima (Nevski) Á. Love, Pseudoroegneria cog-nata (Hack.) Á. Love and Pseudoroegneria stipifolia(Czern. ex Nevski) Á. Love. The clones with 8–bp dele-tion of P. alashanica and P. elytrigioides formed theGroup II. The Group III comprised Agropyron crista-tum (L.) Gaertner, Agropyron puberulum (Boiss. exSteud.) Grossh., Douglasdeweya wangii C. Yen, J. L.Yang & B. R. Baum, Douglasdeweya deweyi (K. B.Jensen, S. L. Hatch & J. K. Wipff) C. Yen, J. L. Yang& B. R. Baum, Australopyrum pectinatum (Labill.) Á.Love ssp. pectinatum and Australopyrum pectinatum(Labill.) Á. Love ssp. retrofractum (J. W. Vickery) Á.Love. The Group IV contained one clone of P. kosaninii.The Group V included P. kosaninii, P. strigosa (4x),Pseudoroegneria strigosa ssp. aegilopoides (Drobow) Á.Love, P. spicata (2x), P. libanotica and Pseudoroegne-ria tauri (Boiss. & Balansa) Á. Love. Six monophyleticclades were found in the clade A, which correspond tothe five genomic types (EeSt, Ee/Eb, StSt, P andW).

502 H. Yu et al.

Consensus P. tauri-18 P. strigosa-16-2 P. strigosa-16-1 P. strigosa-15 P. spicata-13 P. spicata-12 P. stipifolia-14

P. strigosa ssp. aegilopoides-17 P. libanotica-11 P. libanotica-10 P. kosaninii-9-3 P. kosaninii-9-2 P. kosaninii-9-1 Ps. juncea-34 Ps. huashanica-33 P. geniculata-4-2 P. geniculata-4-1 P. gracillima-8 P. gracillima-7 P. elytrigioides-3-2 P. elytrigioides-3-1 P. cognata -2 P. alashanica-1-2 P. alashanica-1-1 Lo. elongatum-25 Lo. elongatum-24 Lo. bessarabicum-23 H. brevisubulatum-32 H. bogdanii-31

D. wangii-20 D. deweyi-19 A. puberulum-28 Au. pectinatum ssp. retrofractum-30 Au. pectinatum ssp. pectinatum-29 A. cristatum-27 A. cristatum-26 Et. caespitosa-21 Et. caespitosa ssp. nodosa-22 P. geniculata ssp. pruinifera-5 P. geniculata ssp. scythica-6

Fig. 1. The comparison of partial sequences in the ITS1 region. The boxed regions show a 4-bp deletion in all accessions of H, Ns, PandW genomes and a 6-bp indel of the species of EeSt genomes. The first number after species name refers to the accession numbersshown in Table 1. The second number after species name refers to the different clones.

Et. caespitosa, Et. caespitosa ssp. nodosa, P. genicu-lata ssp. scythica and P. geniculata ssp. pruinifera wereclearly included in the EeSt clade. The Ee/Eb cladecomprised Lo. elongatum and Lo. bessarabicum. Dou-glasdeweya wangii, D. deweyi, A. cristatum and A. pu-berulum were clustered into the P clade. The endemicAustralian grasses Au. pectinatum ssp. pectinatum andAu. pectinatum ssp. retrofractum were grouped into theW clade. Two different clones of P. geniculata were inone StSt clade, and the clones with 8–bp deletion of P.alashanica and P. elytrigioides formed the other StStclade.

Discussion

Differentiation of St genome in diploid Pseudoroegne-ria speciesHybrids between the diploid species with St genomehave almost complete chromosome pairing, but withhigh or complete sterility, indicating different versionsof St genome (i.e. the St1, St2, etc.) existed in diploidPseudoroegneria species (Stebbins & Pun 1953). Mor-

phologically, P. strigosa have long awns and equalglumes, P. spicata have slender awns with unequalglumes, whereas P. tauri and P. libanotica have noawns with unequal glumes. P. stipifolia have roughrachis densely covered by pricklets (Yen et al. 2007).The molecular evidence based on nuclear RNA poly-merase II (RPB2) reported a 39 bp MITE stowawayelement insertion in the region of RPB2 gene for P. spi-cata and P. stipifolia, while P. tauri and P. libanoticadid not have this insertion (Sun et al. 2007). The Pseu-doroegneria diploid species also have wide distribution.They are distributed from Ciscaucasica (P. stipifolia)to Middle East (P. libanotica and P. tauri) and North-ern China (P. strigosa ssp. aegilopoides), then reach toWestern North America (P. spicata). All the evidencessuggested that St genome in different diploid specieshas been modified to a large extent and has differenti-ated from each other. The results on ML and BI treesin the present study indicated that diploid Pseudoroeg-neria species were clustered into two distinct groups,which suggested differentiations of St genome amongthe diploid Pseudoroegneria species.

Phylogenetic relationships in Pseudoroegneria 503

Ps. juncea-34(Ns) Ps. huashanica-33(Ns)

H. bogdanii-31(H) H. brevisubulatum-32(H)

B. catharticus-35

P. libanotica-11(St)

P. strigosa ssp. aegilopoides-17(St)

P. tauri-18(St)

P. kosaninii-9-3(----) P. strigosa-16-1(StSt)

P. libanotica-10(St) P. spicata-12(St)

P. kosaninii-9-1(----)

A. cristatum-27(P)

D. deweyi-19(StP) D. wangii-20(StP)

Au. pectinatum ssp. retrofractum-30(W) Au. pectinatum ssp. pectinatum-29(W)

A. puberulum-28(P)A. cristatum-26(P)

P. stipifolia-14(St)

P. alashanica-1-2(StSt) P. elytrigioides-3-2(StSt)

P. alashanica-1-1(StSt) P. elytrigioides-3-1(StSt)

P. strigosa-15(St)

P. gracillima-7(St) P. gracillima-8(St) P. cognata-2(St)

P. strigosa-16-2(StSt) P. spicata-13(StSt)

P. geniculata-4-2(StSt) P. geniculata-4-1(StSt)

Lo. elongatum-24(Ee) Lo. elongatum -25(Ee)

Lo. bessarabicum-23(Eb)

P. kosaninii-9-2(----)

P. geniculata ssp. scythica-6(EeSt)

Et. caespitosa ssp. nodosa-22(EeSt)P. geniculata ssp. pruinifera-5(---)

Et. caespitosa-21(EeSt)

92

100

96

59

71

72

100

56

100

0.01

100

100

P

Ns

W

H

EeSt

Ee/Eb

100 StSt

StSt

Clade A

Group I

Group III

Group IV

Group V

Group II

100

100

96

97

91

85

100

56

87

Fig. 2. Phylogenetic tree inferred using maximum likelihood. Branch lengths are proportional to the inferred number of substitutions.Numbers above and below clades indicate the percent of bootstrap support and the Bayesian posterior probability, respectively. Thefirst number after species name refers to the accession numbers shown in Table 1. The second number after species name refers to thedifferent clones. Capital letters in parentheses indicate the genome type of the species. The genome type (EeSt, Ee/Eb, StSt, P,W,H and Ns) of a monophyletic group is given to the right. The clade A indicates the polyphyletic group with five groups (Group I toGroup V) included.

Phylogenetic relationships of polyploid PseudoroegneriaspeciesIn this study, one type of ITS sequences in P. alashan-ica and P. elytrigioides were clustered with tetraploidspeciesP. geniculata, P. spicata (4x), and P. strigosa(4x), which indicated that they were actually distantlyrelated with each other.The tetraploid species P. alashanica and P. elyt-

rigioides were closely related based on morphologicaland cytological evidence (Lu 1994; Cai 1997). Morpho-logically, P. alashanica and P. elytrigioides were firstrecognized as the species of Roegneria (Keng 1959; Yen& Yang 1984). Lu (1994) reported the genomic con-stitutions of R. elytrigioides as StSt and combined itto Pseudoroegneria. Zhang et al. (2002) indicated thatR. alashanica contains one St genome and the othergenome is still unknown. Liu et al. (2006) used the StStas the genomic constitutions of R. alashanica for phy-logenetic analysis of Elymus based on ITS data. In thepresent study, P. alashanica and P. elytrigioides weregrouped together.P. kosaninii is an octoploid species and the genome

constitutions are still unknown. Three different ITSsequences in P. kosaninii were detected. One type in

Group V was clustered with the diploid Pseudoroegne-ria species, which indicated that P. kosaninii seemed topossess one St genome. The ITS sequence in Group Iwas grouped with the species of EeSt genomes with nobootstrap value support. The genomes in P. kosaniniiwere thus unclear.

Phylogenetic relationships between Pseudoroegneriaand the related generaCytologically, P. geniculata contains StSt genomes,while P. geniculata ssp. scythica possesses similar EeStgenomes in Et. caespitosa and Et. caespitosa ssp. no-dosa, and the genome constitution of P. geniculata ssp.pruinifera is still unknown (Dewey 1984; Liu & Wang1989, 1993). In this study, P. geniculata was not clus-tered together with P. geniculata ssp. scythica and P.geniculata ssp. pruinifera, indicating the distinctnessbetween P. geniculata and its two subspecies. P. genicu-lata ssp. scythica, P. geniculata ssp. pruinifera, Et. cae-spitosa and Et. caespitosa ssp. nodosa were distinctlyformed the EeSt clade with 6–bp indel in ITS1 regions.The same 6-bp substitution was also found in the samesite of ITS sequence from Elytrigia intermedia (Host)Nevski (EeEbSt genomes) (Li et al. 2004). The result

504 H. Yu et al.

Diploid Tetraploid

Group V

Group I

P. strigosa

P. gracillima

P. cognata

P. stipifolia

P. geniculata

P. alashanica

P. elytrigioides (StSt)

P. spicata

P. strigosa

P. spicata

P. libanotica

P. tauri

P. strigosa ssp. aegilopoides

P. geniculata ssp. scythica

Et. caespitosa (EeSt)

Et. caespitosa ssp. nodosa

Lo. elongatum

Lo. bessarabicum

A. cristatum

A. puberulum

D. wangii

D. deweyi (StP)

P. geniculata ssp. pruinifera (---)

P. kosaninii (----)

Lophopyrum (Ee, Eb)

Pseudoroegneria (St)

Agropyron (P)

Polyploid

Fig. 3. Schematic representation of phylogenetic relationships of species in Pseudoroegneria and the related genera. The donor generaare drawn directly on the branches of the phylogeny, diploid, tetraploid and polyploid taxa are mapped to the right. Rectangles indicatethe closely related species with the same ploidy and genomes. Lines connect the taxon names with their respective parental species.Dashed lines represent ambiguous parental affiliation. Genomic constitutions are given in boldface.

suggested that the genomic constitution of P. genicu-lata ssp. pruinifera is probably EeEbSt. It is thus un-reasonable to treat P. geniculata ssp. scythica and P.geniculata ssp. pruinifera as the subspecies of P. genicu-lata. According to Love’s principles, Et. caespitosa, Et.caespitosa ssp. nodosa, Et. intermedia, P. geniculatassp. scythica and P. geniculata ssp. pruinifera shouldbe combined to a new genus designated as Trichopy-rum (Yen et al. 2005b).Mason-Gamer et al. (2002) reported that diploid

Pseudoroegneria species, Lo. elongatum and Lo. bessa-rabicum were in one clade based on the molecular evi-dences from rpoA, tRNA spacers, restriction sites andtheir combined data. The morphological tree also in-dicated that diploid Pseudoroegneria species and Lo.elongatum formed a clade (Seberg & Frederiksen 2001).In the present study, Lo. elongatum and Lo. bessara-bicum were clustered with P. gracillima, P. stipifolia,P. cognata and P. strigosa, which suggested E and Stgenomes are closely related. The result is congruouswith the previous morphological and molecular anal-ysis. Wang & Hsiao (1989) suggested that E genomeof Lo. elongatum and J genome of Lo. bessarabicumare the same genomes. Wang et al. (1994) designedthe genome of Lo. bessarabicum as Eb and the genomeof Lo. elongatum as Ee. However, Jauhar (1990) pro-posed that Eb and Ee genomes are not homologous buthomoeologous. The close affinity between Lo. bessara-bicum and Lo. elongatum was also implied in this study.D. wangii and D. deweyi were clustered together

and were distinctly related to Agropyron species. Thisprovides strong evidence that one of the diploid donorof D. wangii and D. deweyi is derived from Agropyronspecies. The similar result was obtained from cytolog-ical studies by Wang et al. (1986) and Jensen et al.(1992). Therefore, it is reasonable to transfer tetraploidspecies with StP genomes from Pseudoroegneria to

Douglasdeweya.In order to reveal the phylogenetic relationships

of Pseudoroegneria and the related genera clearly, aschematic picture (Fig. 3) was drawn based on the evi-dences from the present study combined with the previ-ous cytological and morphological evidences. Accordingto the result of ML tree, Group I and Group V repre-sent different St genome among diploid Pseudoroegne-ria species.

Acknowledgements

The authors are thankful to the Program for ChangjiangScholars and Innovative Research Team in University (PC-SIRT), China (No. IRT 0453), the National Natural ScienceFoundation of China (No. 30670150, 30470135); the Scienceand Technology Bureau of Sichuan Province, Education Bu-reau of Sichuan Province, China, for the financial support.We particularly thank American National Plant GermplasmSystem for providing seeds.

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Received October 20, 2007Accepted April 12, 2008