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

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

Haiqing Yu1,2,XingFan 1,ChunZhang1, Chunbang Ding3, Xiaoli Wang3 & Yonghong Zhou1,2*

1Triticeae Research Institute, Sichuan Agricultural University, Dujiangyan 611830, Sichuan, People’s Republic of China; e-mail: [email protected] 2 Key Laboratory of Crop Genetic Resources and Improvement, Ministry of Education, Sichuan Agricultural University, Yaan 625014, Sichuan, People’s Republic of China 3 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 ribosomal internal transcribed spacer (ITS) sequences were analyzed for eighteen Pseudoroegneria (St), two (EeSt), two Douglasdeweya (StP), three Lophopyrum (Ee andEb), three (P), two (H), two (W) and two (Ns) accessions. The main results were: (i) Pseudoroegneria gracillima, P. stipifolia, P. cognata and P. strigosa (2x) were in one clade, while P. libanotica, P. tauri and P. spicata (2x) were in the other clade, indicating there are the differentiations of St genome among diploid Pseudoroegneria species; (ii) P. geniculata ssp. scythica, P. geniculata ssp. 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 unreasonable to treat P. geniculata ssp. scythica and P. geniculata ssp. pruinifera as the subspecies of P. geniculata,andtheyshouldbe transferred to a new Trichopyrum, which consists of species with EeSt genomes. It is also suggested that one of the diploid donor of D. wangii and D. deweyi is derived from Agropyron species, and it is reasonable to treat tetraploid species with 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 exceptionally drought tolerant and have excellent for- age quality (Dewey 1984). Pseudoroegneria is a genus in Triticeae (Poaceae) L¨ove (1984) and Dewey (1984) suggested that the with Pseudoroegneria strigosa (M. Bieb.) Á. L¨ove as taxonomic treatment for Triticeae species should be the type species (L¨ove 1980). Morphologically, the based on genomic constitutions and this view has been species in this genus are caespitose, long-anthered and widely accepted (Lu 1994; Wang et al. 1994; Zhou et al. cross-pollinating perennials. Pseudoroegneria was built 1999; Yen et al. 2005b). The cytogenetic studies indi- around one genome designated St,whichisoneof cated that there are St, StSt, StP and EeSt genomic the most important basic genomes (St, H, P, W, constitutions in Pseudoroegneria and all of them are Ns and E) in perennial Triticeae. The St genome is diploids or tetraploids (Dewey 1984; Wang et al. 1986; the donor genome of the species in the polyploid gen- Jensen et al. 1992; Liu & Wang 1993). Yen et al. (2005a) era Roegneria (StY), Douglasdeweya (StP), established a new genus Douglasdeweya C. Yen, J.L. (StH, StYH and StYW), (StYP), Elyt- Yang & B.R. Baum, and the species with StP genomes rigia (EeSt and EbEeSt)andPascopyrum (StHN- in Pseudoroegneria were transferred to Douglasdeweya. sXm) (Dewey 1984; L¨ove 1984; Yen & Yang 1990; Yen It has also been reported that three tetraploid species in et al. 2005a,b). The species in Pseudoroegneria are dis- Roegneria C. Koch: Roegneria alashanica Keng, Roeg- tributed in the Northern Hemisphere, and occurred on neria elytrigioides C.YenetJ.L.YangandRoegne- open rocky hillsides from the Middle East and Tran- ria magnicaespes (D.F.Cui)L.B.Cai,containStSt scaucasia across Central Asia and Northern China to genomes and should be treated as the species of Pseu- Western North America. Pseudoroegneria grasses are 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. L¨ove

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

2 P. cognata (Hack.) Á. L¨ove Y0756 St Kuqa, , 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. L¨ove PI 565009 StSt Russian Federation EF014227, EF014228

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

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

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

8 P. gracillima (Nevski) Á. L¨ove PI 420842 St Russian Federation EF014234

9 P. kosaninii (Nabelek) Á. L¨ove PI 237636 — EF014235, EF014236, EF014237

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

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

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

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

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

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

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

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

18 P. tauri (Boiss. & Balansa) Á. L¨ove 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 EF014250 C. 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. L¨ove

23 Lo. bessarabicum (Savul & Rayss) PI 531712 Eb Estonia,Russian Federation L36506a C. Yen, J.L. Yang & Y. Yen

24 Lo. elongatum (Host) Á. L¨ove PI 531719 Ee France EF014249

25 Lo. elongatum (Host) Á. L¨ove 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. L¨ove

29 Au. pectinatum (Labill.) Á. L¨ove ssp. pectinatum D3438 W L36483a

30 Au. pectinatum (Labill.) Á. L¨ove ssp. retrofractum PI 531553 W Australia L36484a (J.W. Vickery) Á. L¨ove

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. L¨ove

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 tetraploid Material and methods species, Elytrigia caespitosa (C. Koch) Nevski, Elytri- materials gia caespitosa ssp. nodosa (Nevski) Tzvelev and Pseu- A total of 34 Triticeae accessions, including 18 Pseu- doroegneria geniculata ssp. scythica (Nevski) Á. L¨ove, dorogeneria accessions with different genomic constitutions have been treated in two different genera based on (i.e. the St, StSt,andEeSt genomes together with P. morphological classification, they were suggested to be kosaninii and P. geniculata ssp. pruinifera), two species of Elytrigia (EeSt genomes), three accessions of Lophopyrum treated in a new genus Trichopyrum Á. L¨ove because of e b their similar genome formula EeSt (Liu & Wang 1993; (E and E genome), two species of Douglasdeweya (StP genomes), three accessions of Agropyron (P genome), two Yen et al. 2005b). In addition, the genomic constitu- accessions of Australopyrum (W genome), two species of tions of octoploid Pseudoroegneria kosaninii (Nabelek) Hordeum (H genome) and two species of Psathyrostachys Á. L¨ove and hexaploid Pseudoroegneria geniculata ssp. (Ns genome), were used in this study. Bromus cathar- pruinifera (Nevski) Á. L¨ove are obscure. Therefore, the ticus Vahl was used as outgroup. All the seed mate- phylogenetic relationships and taxonomic treatment of rials were kindly provided by American National Plant several species related to Pseudoroegneria are still in Germplasm System (Pullman, WA, USA) and Triticeae Re- dispute. search Institute, Sichuan Agricultural University (Sichuan, To understand the phylogenetic relationships People’s Republic of China). These seeds were germi- among the species of Pseudoroegneria, Elytrigia, Dou- nated and grown in the perennial nursery. The mature were carefully identified by Profs. Chi Yen, Jun- glasdeweya and Lophopyrum, in the present study we liang Yang and Yonghong Zhou. The taxa, accession num- sequenced and analyzed the nuclear ribosomal inter- bers, genomic constitutions, geographic origins and Gen- nal transcribed spacer (ITS) fragments of Pseudoroeg- Bank accession numbers (Benson et al. 2007) are listed neria and related species. The aims were as follows: in Table 1. The nomenclature and genome symbols of the (i) to investigate the differentiation of St genome in species used in this study follow the opinions of L¨ove diploid Pseudoroegneria species; (ii) to evaluate the (1984), Dewey (1984), Wang et al. (1994) and Yen et al. phylogenetic relationships of Pseudoroegneria species; (2005b). and (iii) to demonstrate the intergeneric relationships DNA extraction and purification among Pseudoroegneria, Elytrigia, Douglasdeweya and The leaf samples for each material were collected from Lophopyrum. mature plants in the perennial nursery of Triticeae Re- Phylogenetic relationships in Pseudoroegneria 501 search Institute, and ground in liquid nitrogen in a Results 1.5 mL microfuge tube. DNA was extracted and puri- fied with a slight modification of the cetyltrimethylam- Sequence analysis of ITS regions monium bromide procedure outlined in Doyle & Doyle The length of sequences ranged from 212 to 221 bp in (1990). the ITS1 region and from 215 to 217 bp in the ITS2 re- gion. The 5.8S subunit was the most conserved region ITS amplification, cloning and sequencing and was 164 bp in length for all the sequences except The amplification of ITS regions was done using primers ITS-4 (5’-TCCTCCGCTTATTGATATGC-3’) and ITS-L for one clone from Pseudoroegneria geniculata (Trin.) (5’-TCGTAACAAGGTTTCCGTAGGTG-3’) (Baldwin Á. L¨ove, which has a 5-bp insertion (CATCG) at posi- 1992; Hsiao et al. 1995). The PCR reaction was carried out tion 261 of the alignment. The average of G+C content in a total volume of 25 µL containing 1× reaction buffer, was 62%. The aligned sequences yielded 608 characters. 1.5 mM MgCl2 ,0.5µM of each primer, 200 µMofeach Most of the sequence variations occurred in the spacer dNTP (TakaRa Biotechnology Co., Ltd, Dalian, China), regions. In the ITS1 and ITS2 regions, 71 and 77 vari- 0.5 units of ExTaq Polymerase (TakaRa), with an addi- able characters were detected respectively. There was a tion of 10% dimethyl sulfoxide (Buckler & Holtsford 1996; 4-bp deletion (GTGG) in all accessions of H, Ns, P and Buckler et al. 1997) and sterile water to the final volume. W genomes and a 6-bp indel (TTTTCA) from position The thermocycling profile consisted of an initial denatu- ration at 94 ◦C for 3 min, followed by 35 cycles of 1 min 55 to 60 in Et. caespitosa, Et. caespitosa ssp. nodosa, at 94 ◦C, 1 min at 52 ◦C, 1 min at 72 ◦C and final exten- P. geniculata ssp. scythica and P. geniculata ssp. pru- sion of 8 min at 72 ◦C. PCR reactions of each accession inifera (Fig. 1). Multiple base pairs insertion, deletion were carried out in four independent reactions in an ABI or substitution was not found in the ITS2 region. 9700 thermal cycler (Applied Biosystems, CA, USA). Am- plification products were mixed and purified using the Gel Phylogenetic analysis of Pseudoroegneria and the re- Extraction Kit (50) (Omega, GA, USA) and linked into a lated genera pMD18–T Easy Vector Systems according to the manufac- The topologies of the two analyses (ML and BI) were turer’s instruction (TakaRa). Three to five positive clones nearly consistent. The ML tree is shown in Figure 2. for each species were randomly selected and sequenced by Sunbiotech Co., Ltd (Beijing, China). The sequences used The percent of bootstrap values and posterior prob- in this study have been deposited with GenBank (Ben- abilities are indicated above and below the branches, son et al. 2007) under the accession numbers EF014226– respectively. Three major clades (Clade A, H and Ns) EF014251). were formed. The Ns clade (Psathyrostachys)wasin- cluded together with the H clade (Hordeum)ina Sequence alignment and phylogenetic analysis basal polytomy. The clade A consisted of all the Pseu- The boundaries of the ITS regions were determined by doroegneria species together with species in Elytri- comparison with the ITS sequence ofPseudoroegneria liban- gia, Lophopyrum, Douglasdeweya, Agropyron and Aus- otica (Hackel) D. R. Dewey (GenBank accession number tralopyrum. Five groups were detected within the clade L36501) (Hsiao et al. 1995). The ITS sequence alignment A. The Group I consisted of Et. caespitosa, Et. caespi- was executed with Clustal X program (Thompson et al. 1997). Gaps were coded as binary characters by their pres- tosa ssp. nodosa, P. geniculata ssp. scythica, P. genic- ence/absence, and were used for the phylogenetic analy- ulata ssp. pruinifera, P. geniculata, P. alashanica, P. ses. elytrigioides, P. strigosa (2x, 4x), Pseudoroegneria spi- PAUP* 4.0b10 (Swofford 2002) was used to find the cata (Pursh) Á. L¨ove (4x), P. kosaninii, Lophopyrum best maximum likelihood (ML) tree by performing a heuris- elongatum (Host) Á. L¨ove, Lophopyrum bessarabicum tic search with tree-bisection-reconnection branch swapping (Á. L¨ove) C. Yen, J. L. Yang et Y. Yen, Pseudoroegne- and 100 random addition replicates. Topological robustness ria gracillima (Nevski) Á. L¨ove, Pseudoroegneria cog- was assessed by bootstrap analysis with 100 replications us- nata (Hack.) Á. L¨ove and Pseudoroegneria stipifolia ing as-is sequence addition. The SYM+G model of evolu- (Czern. ex Nevski) Á. L¨ove. The clones with 8–bp dele- tion was used in the ML analysis based on the result of MrModeltest 2.2 (Nylander 2004). The fit of 24 ML mod- tion of P. alashanica and P. elytrigioides formed the els to the data set was tested, and base change frequen- Group II. The Group III comprised Agropyron crista- cies, proportion of variable characters and shape of the tum (L.) Gaertner, Agropyron puberulum (Boiss. ex gamma distribution were estimated in the Modeltest anal- Steud.) Grossh., Douglasdeweya wangii C. Yen, J. L. ysis. The best model to the data was obtained by the Hi- Yang&B.R.Baum,Douglasdeweya deweyi (K. B. erarchical Likelihood Ratio Tests (Posada & Crandall 1998; Jensen,S.L.Hatch&J.K.Wipff)C.Yen,J.L.Yang Roalson & Friar 2004). Bayesian inference (BI) for phy- &B.R.Baum,Australopyrum pectinatum (Labill.) Á. logenetic analysis was executed with MrBayes 3.1.2 (Ron- L¨ove ssp. pectinatum and Australopyrum pectinatum quist & Huelsenbeck 2003). The same model and numbers (Labill.) Á. L¨ove ssp. retrofractum (J.W.Vickery)Á. of base change frequencies set in the ML search were used in BI analysis with starting from a random tree. Markov L¨ove. The Group IV contained one clone of P. kosaninii. chain Monte Carlo chain length for analyses was 8×106 The Group V included P. kosaninii, P. strigosa (4x), generations with trees sampled every 100 generations and Pseudoroegneria strigosa ssp. aegilopoides (Drobow) Á. the first 2×104 trees were discarded as burn-in. Additional L¨ove, P. spicata (2x), P. libanotica and Pseudoroegne- runs with the same conditions produced the same topology ria tauri (Boiss. & Balansa) Á. L¨ove. Six monophyletic with insignificant differences in posterior probability of any clades were found in the clade A, which correspond to node. the five genomic types (EeSt, Ee/Eb, StSt, P and W). 502 H. Yu et al.

P. geniculata ssp. scythica-6 P. geniculata ssp. pruinifera-5 Et. caespitosa ssp. nodosa-22 Et. caespitosa-21 A. cristatum-26 A. cristatum-27 Au. pectinatum ssp. pectinatum-29 Au. pectinatum ssp. retrofractum-30 A. puberulum-28 D. deweyi-19 D. wangii-20 H. bogdanii-31 H. brevisubulatum-32 Lo. bessarabicum-23 Lo. elongatum-24 Lo. elongatum-25 P. alashanica-1-1 P. alashanica-1-2 P. cognata -2 P. elytrigioides-3-1 P. elytrigioides-3-2 P. gracillima-7 P. gracillima-8 P. geniculata-4-1 P. geniculata-4-2 Ps. huashanica-33 Ps. juncea-34 P. kosaninii-9-1 P. kosaninii-9-2 P. kosaninii-9-3 P. libanotica-10 P. libanotica-11 P. strigosa ssp. aegilopoides-17 P. stipifolia-14 P. s pi cata-12 P. s pi cata-13 P. strigosa-15 P. strigosa-16-1 P. strigosa-16-2 P. taur i -18 Consensus

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, P and W genomes and a 6-bp indel of the species of EeSt genomes. The first number after species name refers to the accession numbers shown in Table 1. The second number after species name refers to the different clones.

Et. caespitosa, Et. caespitosa ssp. nodosa, P. genicu- phologically, P. strigosa have long awns and equal lata ssp. scythica and P. geniculata ssp. pruinifera were glumes, P. spicata have slender awns with unequal clearly included in the EeSt clade. The Ee/Eb clade glumes, whereas P. tauri and P. libanotica have no comprised Lo. elongatum and Lo. bessarabicum. Dou- awns with unequal glumes. P. stipifolia have rough glasdeweya wangii, D. deweyi, A. cristatum and A. pu- rachis densely covered by pricklets (Yen et al. 2007). berulum were clustered into the P clade. The endemic The molecular evidence based on nuclear RNA poly- Australian grasses Au. pectinatum ssp. pectinatum and merase II (RPB2) reported a 39 bp MITE stowaway Au. pectinatum ssp. retrofractum were grouped into the element insertion in the region of RPB2 gene for P. spi- W clade. Two different clones of P. geniculata were in cata and P. stipifolia, while P. tauri and P. libanotica one StSt clade, and the clones with 8–bp deletion of P. did not have this insertion (Sun et al. 2007). The Pseu- alashanica and P. elytrigioides formed the other StSt doroegneria diploid species also have wide distribution. clade. They are distributed from Ciscaucasica (P. stipifolia) to Middle East (P. libanotica and P. tauri) and North- Discussion ern China (P. strigosa ssp. aegilopoides), then reach to Western North America (P. spicata). All the evidences Differentiation of St genome in diploid Pseudoroegne- suggested that St genome in different diploid species ria species has been modified to a large extent and has differenti- Hybrids between the diploid species with St genome ated from each other. The results on ML and BI trees have almost complete chromosome pairing, but with in the present study indicated that diploid Pseudoroeg- high or complete sterility, indicating different versions neria species were clustered into two distinct groups, of St genome (i.e. the St1, St2, etc.) existed in diploid which suggested differentiations of St genome among Pseudoroegneria species (Stebbins & Pun 1953). Mor- the diploid Pseudoroegneria species. Phylogenetic relationships in Pseudoroegneria 503

P. geniculata ssp. scythica-6(EeSt) e Et. caespitosa-21(E St) e 100 Et. caespitosa ssp. nodosa-22(EeSt) E St 100 P. geniculata ssp. pruinifera-5(---) P. kosaninii-9-2(----) 71 Lo. elongatum -25(Ee) 0.01 e 100 Lo. elongatum-24(E ) Ee/Eb Lo. bessarabicum-23(Eb) 96 P. geniculata-4-2(StSt) 100 P. geniculata-4-1(StSt) StSt Group I P. strigosa-16-2(StSt) 87 P. s pi c ata-13(StSt) P. gracillima-7(St) P. gracillima-8(St) P. cognata-2(St) P. strigosa-15(St) P. alashanica-1-1(StSt) P. elytrigioides-3-1(StSt) P. stipifolia-14(St) Clade A 59 P. elytrigioides-3-2(StSt) Group II 97 P. alashanica-1-2(StSt) StSt A. cristatum-26(P) A. puberulum-28(P) 56 92 A. cristatum-27(P) P 100 D. wangii-20(StP) Group III D. deweyi-19(StP) 100 Au. pectinatum ssp. pectinatum-29(W) 56 100 Au. pectinatum ssp. retrofractum-30(W) W 91 P. kosaninii-9-1(----) Group IV P. kosaninii-9-3(----) P. strigosa-16-1(StSt) P. strigosa ssp. aegilopoides-17(St) P. s pi c at a -12(St) Group V 85 P. libanotica-10(St) P. libanotica-11(St) P. tauri-18(St) 100 H. bogdanii-31(H) H 100 H. brevisubulatum-32(H) 72 Ps. huashanica-33(Ns) Ns 96 Ps. juncea-34(Ns) B. catharticus-35

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. The first number after species name refers to the accession numbers shown in Table 1. The second number after species name refers to the different 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 to Group V) included.

Phylogenetic relationships of polyploid Pseudoroegneria Group V was clustered with the diploid Pseudoroegne- species ria species, which indicated that P. kosaninii seemed to In this study, one type of ITS sequences in P. alashan- possess one St genome. The ITS sequence in Group I ica and P. elytrigioides were clustered with tetraploid was grouped with the species of EeSt genomes with no speciesP. geniculata, P. spicata (4x), and P. strigosa bootstrap value support. The genomes in P. kosaninii (4x), which indicated that they were actually distantly were thus unclear. related with each other. The tetraploid species P. alashanica and P. elyt- Phylogenetic relationships between Pseudoroegneria rigioides were closely related based on morphological and the related genera and cytological evidence (Lu 1994; Cai 1997). Morpho- Cytologically, P. geniculata contains StSt genomes, logically, P. alashanica and P. elytrigioides were first while P. geniculata ssp. scythica possesses similar EeSt recognized as the species of Roegneria (Keng 1959; Yen genomes in Et. caespitosa and Et. caespitosa ssp. no- & Yang 1984). Lu (1994) reported the genomic con- dosa, and the genome constitution of P. geniculata ssp. stitutions of R. elytrigioides as StSt and combined it pruinifera is still unknown (Dewey 1984; Liu & Wang to Pseudoroegneria. Zhang et al. (2002) indicated that 1989, 1993). In this study, P. geniculata was not clus- R. alashanica contains one St genome and the other tered together with P. geniculata ssp. scythica and P. genome is still unknown. Liu et al. (2006) used the StSt geniculata ssp. pruinifera, indicating the distinctness as the genomic constitutions of R. alashanica for phy- between P. geniculata and its two subspecies. P. genicu- logenetic analysis of Elymus based on ITS data. In the lata ssp. scythica, P. geniculata ssp. pruinifera, Et. cae- present study, P. alashanica and P. elytrigioides were spitosa and Et. caespitosa ssp. nodosa were distinctly grouped together. formed the EeSt clade with 6–bp indel in ITS1 regions. P. kosaninii is an octoploid species and the genome The same 6-bp substitution was also found in the same constitutions are still unknown. Three different ITS site of ITS sequence from Elytrigia intermedia (Host) sequences in P. kosaninii were detected. One type in Nevski (EeEbSt genomes) (Li et al. 2004). The result 504 H. Yu et al.

Diploid Tetraploid Polyploid

P. strigosa P. geniculata P. gracillima P. alashanica P. cognata P. elytrigioides (StSt) P. stipifolia P. s pi cata Group I P. strigosa P. kosaninii (----)

Pseudoroegneria (St) P. spicata P. libanotica P. geniculata ssp. scythica Group V P. taur i Et. caespitosa (EeSt) P. strigosa ssp. aegilopoides Et. caespitosa ssp. nodosa

Lo. elongatum Lophopyrum (Ee, Eb) P. geniculata ssp. pruinifera (---) Lo. bessarabicum D. wangii (StP) D. deweyi

A. cristatum Agropyron (P) A. puberulum

Fig. 3. Schematic representation of phylogenetic relationships of species in Pseudoroegneria and the related genera. The donor genera are drawn directly on the branches of the phylogeny, diploid, tetraploid and polyploid taxa are mapped to the right. Rectangles indicate the closely related species with the same 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- Douglasdeweya. lata ssp. pruinifera is probably EeEbSt.Itisthusun- In order to reveal the phylogenetic relationships reasonable to treat P. geniculata ssp. scythica and P. of Pseudoroegneria and the related genera clearly, a geniculata ssp. pruinifera as the subspecies of P. genicu- schematic picture (Fig. 3) was drawn based on the evi- lata. According to L¨ove’s principles, Et. caespitosa, Et. dences from the present study combined with the previ- caespitosa ssp. nodosa, Et. intermedia, P. geniculata ous cytological and morphological evidences. According ssp. scythica and P. geniculata ssp. pruinifera should to the result of ML tree, Group I and Group V repre- be combined to a new genus designated as Trichopy- sent different St genome among diploid Pseudoroegne- rum (Yen et al. 2005b). ria species. Mason-Gamer et al. (2002) reported that diploid Pseudoroegneria species, Lo. elongatum and Lo. bessa- Acknowledgements rabicum were in one clade based on the molecular evi- dences from rpoA, tRNA spacers, restriction sites and The authors are thankful to the Program for Changjiang their combined data. The morphological tree also in- Scholars and Innovative Research Team in University (PC- dicated that diploid Pseudoroegneria species and Lo. SIRT), China (No. IRT 0453), the National Natural Science elongatum formed a clade (Seberg & Frederiksen 2001). Foundation of China (No. 30670150, 30470135); the Science In the present study, Lo. elongatum and Lo. bessara- and Technology Bureau of Sichuan Province, Education Bu- bicum were clustered with P. gracillima, P. stipifolia, reau of Sichuan Province, China, for the financial support. P. cognata and P. strigosa, which suggested E and St We particularly thank American National Plant Germplasm genomes are closely related. The result is congruous System for providing seeds. with the previous morphological and molecular anal- ysis. Wang & Hsiao (1989) suggested that E genome References of Lo. elongatum and J genome of Lo. bessarabicum are the same genomes. Wang et al. (1994) designed Baldwin B.G. 1992. Phylogenetic utility of the internal tran- the genome of Lo. bessarabicum as Eb and the genome scribed spacers of nuclear ribosomal DNA in plants: an ex- 1 of Lo. elongatum as Ee. However, Jauhar (1990) pro- ample from the Compositae. Mol. Phylogenet. Evol. : 3–16. b e Benson D.A., Karsch-Mizrachi I., Lipman D.J., Ostell J. & posed that E and E genomes are not homologous but Wheeler D.L. 2007. GenBank. Nucleic Acids Res. 35 (Data- homoeologous. The close affinity between Lo. bessara- base Issue): D21–D25. bicum and Lo. elongatum was also implied in this study. Buckler E.S. IV & Holtsford T.P. 1996. Zea systematics: riboso- D. wangii and D. deweyi were clustered together mal ITS evidence. Mol. Biol. Evol. 13: 612–622. Buckler E.S. IV, Ippolito A. & Holtsford T.P. 1997. The evolution and were distinctly related to Agropyron species. This for ribosomal DNA: divergent paralogues and phylogenetic provides strong evidence that one of the diploid donor implications. Genetics 145: 821–832. of D. wangii and D. deweyi is derived from Agropyron Cai L.B. 1997. A taxonomical study on the genus Roegneria C. species. The similar result was obtained from cytolog- Koch from China. Acta Phytotaxonomica Sinica 35: 148–177. ical studies by Wang et al. (1986) and Jensen et al. Dewey D.R. 1984. The genomic system of classification as a guide to intergeneric hybridization with the perennial Triticeae, (1992). Therefore, it is reasonable to transfer tetraploid pp. 209–280. In: Gustafson J.P. (ed.), Gene Manipulation in species with StP genomes from Pseudoroegneria to Plant Improvement, Plenum Press, New York. Phylogenetic relationships in Pseudoroegneria 505

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