Systematics and Evolution of the Needle Grasses (Poaceae: Pooideae: Stipeae) Based on Analysis of Multiple Chloroplast Loci, ITS, and Lemma Micromorphology

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TAXON — Article version: 9 Dec 2011: 27 pp. Romaschenko & al. • Systematics and evolution of needle grasses

Systematics and evolution of the needle grasses (Poaceae: Pooideae: Stipeae) based on analysis of multiple chloroplast loci, ITS, and lemma micromorphology

Konstantin Romaschenko,1,2 Paul M. Peterson,2 Robert J. Soreng,2 Nuria Garcia-Jacas,1 Oksana Futorna3 & Alfonso Susanna1

1 Laboratory of Molecular Systematics, Botanic Institute of Barcelona (CSIC-ICUB), Passeig del Migdia s.n., 08038, Barcelona, Spain 2 Department of Botany, National Museum of Natural History, Smithsonian Institution, Washington, D.C., 20013, U.S.A. 3 M.G. Kholodny Institute of Botany, National Academy of Sciences of Ukraine, 01601 Kiev, Ukraine Author for correspondence: Paul M. Peterson, [email protected]

Abstract We conducted a molecular phylogenetic study of the tribe Stipeae using nine plastid DNA sequences (trnK-matK, matK, trnH-psbA, trnL-F, rps3, ndhF, rpl32-trnL, rps16-trnK, rps16 intron), the nuclear ITS DNA regions, and micromorphological characters from the lemma surface. Our large original dataset includes 156 accessions representing 139 species of Stipeae representing all genera currently placed in the tribe. The maximum likelihood and Bayesian analyses of DNA sequences provide strong support for the monophyly of Stipeae; including, in phylogenetic order, Macrochloa as remote sister lineage to all other Stipeae, then a primary stepwise divergence of three deep lineages with a saw-like (SL) lemma epidermal pattern (a plesiomorphic state). The next split is between a lineage (SL1) which bifurcates into separate Eurasian and American clades, and a lineage of three parts; a small Patis (SL2) clade, as sister to Piptatherum s.str. (SL3), and the achnatheroid clade (AC). The AC exhibits a maize-like lemma epidermal pattern throughout. AC consists of a core clade of Austral-Eurasian distribution and a “major American clade” of North and South American distribution. The base chromosome number for Stipeae is somewhat ambiguous but based on our survey it seems most likely to be x = 11 or 12. Our phylogenetic hypothesis supports the recognition of the following genera and groups (listed by region): Eurasia—Achnatherum, “Miliacea group”, “Neotrinia” (monotypic), Orthoraphium (monotypic), Patis (also 1 from North America), Piptatherum s.str., Psam- mochloa (monotypic), Ptilagrostis, Stipa, “Timouria group”, and Trikeraia; Mediterranean—Ampelodesmos (monotypic), Celtica (monotypic), Macrochloa (monotypic), and “Stipella-Inaequiglumes group”; Australasia —Anemanthele (monotypic), and Austrostipa; North America (NA)—“Eriocoma group”, Hesperostipa, Oryzopsis (monotypic), Piptatheropsis, “Pseudoeriocoma group”, and “Stillmania” (monotypic); South America—Aciachne, Amelichloa (also NA), Anatherostipa (s.str.), Jarava (polyphyletic), Lorenzochloa, Nassella (also NA), Ortachne, Pappostipa (also NA), and Piptochaetium (also NA). Monophyly of Phaenospermateae including Duthieinae is demonstrated, and its inclusion within or treatment as sister to Stipeae is rejected.

Keywords biogeography; evolution; grasses; lemma micromorphology; molecular systematics; phylogeny; plastid DNA sequences; Poaceae; Stipeae

Supplementary Material Appendices 1 to 3 (in the Electronic Supplement) and the alignments are available in the Supplementary Data section of the online version of this article (http://ingentaconnect.com/content/iapt/tax).

INTRODUCTION Pooideae (Brachypodieae Harz, Bromeae Martinov plus Hor- deeae Martinov (= Triticeae Dumort.), Poeae R. Br. including Peripheral Stipeae, clarification of what is in and what is Aveneae Dumort.). Although Diarrheneae are usually resolved outside the tribe. — Stipeae Martinov are cool-season or tem- as sister to core Pooideae, the exact phylogenetic relationships perate C3 grasses placed in subfamily Pooideae Benth. (GPWG, of Stipeae within this set has varied among studies. A few gen- 2001). We estimate that the Stipeae s.str. include between 572 era placed in Stipeae s.l. clearly do not belong there: Milium L. and 670 species with the number varying depending on how (Clayton & Renvoize, 1986), Cyathopus Stapf, and Dichelachne finely the Asian taxa are divided. The broadest molecular studies Endl. (Tzvelev, 1989) have b een resolved within Poeae based on in Pooideae (GPWG, 2001; Davis & Soreng, 2007; Soreng & al., molecular data (Döring, 2009; Schneider & al., 2009; Soreng 2007; Bouchenak-Khelladi & al., 2008; Schneider & al., 2009) & al., 2007), and Streptachne R. Br. (Tzvelev, 1989) has been place the origin of Stipeae after the separation of Brachy elytreae accepted by other agrostologists as a synonym of Aristida L. in Ohwi, Lygeeae J. Presl, and Nardeae W.D.J. Koch, among the Aristidoideae Caro. remaining tribes Phaenospermateae Renvoize & Clayton s.l. Subtribe Duthieinae is sometimes placed in Aveneae, Sti- (including Duthieinae Pilg. ex Potztal), Meliceae Link ex Endl. peae, or Phaenospermateae. In its broadest current sense Duthie- plus Brylkinieae Tateoka, Diarrheneae C.S. Campb., and core inae includes: Anisopogon R. Br., Danthoniastrum (J. Holub)

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Holub, Metcalfia Connert, Pseudodanthonia Bor & C.E. Hubb., sharp, often bearded callus, lemmas that are rounded on the back, Sinochasea Keng, Duthiea Hack., and Stephanachne Keng (3–)5–9-nerved, and often concealing the palea (if the palea is (Baum, 1973; Clayton & Renvoize, 1986; Holub, 1998; Wu exposed when the floret is closed, then the palea is coriaceous), & Phillips, 2006; Soreng & al., 2011). Based on molecular terminally awned (or from between short lobes) where the awn is data, and morphological extension of that, Megalachne Steud. the result of fusion between the central and two lateral vascular (Döring, 2009) and Podophorus Phil. should be placed in Poeae traces, the awn usually geniculate and twisted in the proximal (Clayton & Renvoize, 1986; Soreng & al., 2011), rather than in segment and sometimes caducous, plumose or scabrous; flow- Duthieinae (Soreng & al., 2003). Uncertainty concerning the ers with three, sometimes two, linear lodicules that are slightly relationships of the American and Chinese elements of subtribe indurate at maturity and glabrous (with venation weaker than in Duthieinae (sensu Clayton & Renvoize, 1986), led Soreng & other subfamilies but often more distinct than in other Pooideae), al. (2003) and Wu & Phillips (2006) to place the genera of this often penicillate anthers tips, glabrous ovaries, caryopses with a subtribe within Stipeae, and accept Phaenospermateae as a long-linear hilum, small embryos with compound starch and no monotypic tribe. Although accumulating molecular DNA data lipid; and small-sized chromosomes with a base number x = 7, 8, support placing Duthieinae in Phaenospermateae, the relation- 9, 10, 11, or 12, for which the ancestral base number is uncertain. ship of Duthieinae and Phaenospermateae to Stipeae remains Unlike most Pooideae genera, the surfaces of the leaves occa- poorly resolved and controversial (GPWG, 2001; Soreng & al., sionally have unicellular microhairs (Barkworth, 2007; Clayton 2003, 2011; Davis & Soreng, 2007, 2010; Romaschenko & al., & Renvoize, 1986; Soreng & Davis, 1998). 2008, 2010; Schneider & al., 2009). Anisopogon was added The number of genera accepted in the Stipeae varies widely to Duthieinae (Soreng & al., 2011) based on molecular data in modern treatments. Tzvelev (1976, 1989) accepted four gen- (GPWG, 2001; Davis & Soreng, 2007, 2010). era in the former Soviet Union and 18 in the World, Clayton Morphological inferences. — Stipeae s.str. (excluding the & Renvoize (1986) accepted nine in the World, Barkworth above-mentioned elements and ignoring Ampelodesmos Link) (2007) accepted 13 in the U.S.A. and Canada, and Soreng & al. are mostly tussock-forming grasses characterized by having (2003) list 18 accepted genera of Stipeae for the New World (see single-flowered spikelets without rachilla extensions that dis- Table 1). These genera (excluding Ampelodesmos, which when articulate above the glumes; florets with a distinct, sometimes included in Stipeae is placed in subtribe Ampelodesminae),

Table . Comparison of recent classifications of genera and phylogenetically isolated lineages in the tribes Stipeae and Ampelodesmeae with our proposed arrangement.a “y” = accepted, otherwise the genus in which the taxon is placed is specified (qualified by “presumably” where a genus was published later and the taxonomy was not explicit as to the species’ placement in the source); parentheses enclose estimated numbers of species, if given in the source, or determinable from that; “p.p.” = pro parte; a non-italicized “group” name implies the name is informal here and an italicized “group” name implies the published genus has not been formally emended to include some elements included here; “n/a” indicates the genus or group was neither within geographic purview of, nor otherwise mentioned in, the source; FNA = Barkworth & al. (2007). Clayton Genus (or group isolated from a genus in Tzvelev & Renvoize Tzvelev Soreng & al. Barkworth Our opinion, which it has been included) (1976) (1986) (1989) (2003) & al. (2007) here Achnatherum P. Beauv. y (20) Stipa y (20) y (36) y (27 FNA na- y, p.p. (21) tive, 7 Mexico, 1 New Zea- land, 21 Old World; ca. 56 in total) Aciachne Benth. n/a y (1) y (1) y (3) n/a y (3) Amelichloa Arriaga & Barkworth n/a presumably presumably Nassella and y (5) y, tentatively, in Stipa in Stipa Achnatherum needs further study (5) Ampelodesmos Link n/a y, tribe Poeae y, tribe y, Stipeae y, Stipeae (1) y, tentatively (1) Ampelo- subtribe (1) in subtribe desmeae (1) Ampelo- desminae (1) Anatherostipa (Hack. ex Kuntze) Peñailillo n/a presumably presumably y (11) n/a not mono- in Stipa in Stipa phyletic, p.p. typic (8) Anemanthele Veldkamp n/a presumably presumably n/a n/a y (1) in Stipa in Stipa Austrostipa S.W.L. Jacobs & J. Everett n/a presumably presumably y y (63) y (63) in Stipa in Stipa

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Table . Continued. Clayton Genus (or group isolated from a genus in Tzvelev & Renvoize Tzvelev Soreng Barkworth Our opinion, which it has been included) (1976) (1986) (1989) & al. (2003) & al. (2007) here Celtica F.M. Vázquez & Barkworth n/a presumably Stipa Macrochloa y (1) y (1) in Stipa “Eriocoma Nutt. group” n/a Oryzopsis y (3) Achnatherum Achnatherum y (29) Hesperostipa (M.K. Elias) Barkworth n/a presumably presumably y (5) y (5) y (5) in Stipa in Stipa Jarava Ruiz & Pav. n/a Stipa Stipa y (59) y (50) y, p.p. monophyletic (30) Lorenzochloa Reeder & C. Reeder n/a Ortachne y (1) Ortachne n/a y (1) nested within Anatherostipa p.p. non-typic, needs further study Macrochloa Kunth n/a Stipa Stipa y (2) y (1) y (1) “Miliacea group” (former Piptatherum sect.) n/a Stipa Piptatherum Piptatherum Piptatherum y (2) Nassella (Trin.) E. Desv. n/a y (15) y (10) y (115) y (116) y (117) “Neotrinia” (former Achnatherum sect.) Achnatherum Stipa Achnatherum Achnatherum Achnatherum y (1) sect. Neotrinia Ortachne Nees ex Steud. n/a y (3) y (2) y (3) n/a y (2) Orthoraphium Nees n/a Stipa y (2) n/a n/a y (1) Oryzopsis Michx. n/a y (35) y (1) y (1) y (1) y (1) Pappostipa (Speg.) Romasch., n/a presumably presumably Jarava Jarava y (31) P.M. Peterson & Soreng in Stipa in Stipa Patis Ohwi n/a Stipa y (1) (1 in FNA in (1 in FNA in y (3) Piptatherum) Piptatherum) Piptatheropsis Romasch., P.M. Peterson & n/a Oryzopsis presumably Piptatherum Piptatherum y (5) Soreng in Stipa Piptatherum P. Beauv. y (50) Oryzopsis y (50) y (7) y (30) y, p.p. (32) Piptochaetium J. Presl n/a y (30) y (20) y (35) y (27) y (35) Psammochloa Hitchc. n/a y (1) y (1) n/a n/a y (1) Pseudoericoma group (clambering spp. for- n/a presumably presumably Achnatherum, Achnatherum, y, Jarava merly of Achnatherum and Jarava) in Stipa in Stipa Jarava Jarava p.p. & Achnatherum p.p. (7 min.) Ptilagrostis Griseb. y (9) Stipa y (9) y (2) y (9) y (8) “Stillmania” (formerly in Achnatherum or n/a presumably unknown Achnatherum Achnatherum y (1) Stipa) in Stipa Stipa L. y (300) y (300) y (300) y y (200) y (110 min.) “Stipella-Inaequiglumes group” Stipa sect. Stipa Stipa a/a n/a y (2) (former Stipa sects.) Stipella and Inaequiglumes “Timouria Roshev. group” (includes some Achnatherum Stipa Achnatherum n/a n/a y (5) Achnatherum spp.) sect. Timouria Trikeraia Bor n/a y (2) y (2) n/a n/a y (3) a Genera placed in Stipeae by only one of the compared classifications are exempted from the table since they are now understood to belong to other tribes: Streptachne (= Aristida)—Aristideae; Metcalfia—Phaenospermateae; Cyathopus, Dichelachne, Megalachne, Milium, Podophorus —Poeae s.l.

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represent Stipeae s.str. or the “core Stipeae”. Generic boundar- Molecular inferences. — Evolutionary relationships ies among the genera in the Stipeae are problematic, especially within Stipeae based on studies of molecular characters have within Achnatherum P. Beauv., Jarava Ruiz & Pav., Stipa L., not been clearly or fully elucidated (Jacobs & al., 2000, 2007; Oryzopsis Michx., and Piptatherum P. Beauv. Diff icult and Barkworth & al., 2008; Romaschenko & al., 2008, 2010), and controversial delimitations among these led agrostologists to these as well as those based on morphology remain controver- adopt a broad concept of the genus Stipa to encompass all of the sial (Barkworth & Everett, 1987; Barkworth, 1993; Chiapella, currently accepted genera except Oryzopsis, Aciachne Benth., 2008). Prior to the study of Romaschenko & al. (2010), only a and Piptochaetium J. Presl in the New World (Spegazzini, 1901; small fraction of the Stipeae s.str. genera and generic diversity Hitchcock, 1935, 1951) and to split Piptatherum in the Old were sampled, and outgroup sampling remained poor. World from New World Oryzopsis s.l. (Freitag, 1975, 1985). Ampelodesmos, which is very different from Stipeae s.str. Various studies were performed that described new genera and in gross morphology of the spikelet, but similar in anatomy emend generic limits (Parodi, 1947, 1960; Barkworth, 1983, and cytology to members of this tribe (Decker, 1964), appears 1990, 1993; Jacobs & Everett, 1996; Peñailillo, 1996, 2002, from molecular data to be nested in Stipeae (Davis & Soreng, 2003, 2005; Rojas, 1997; Torres, 1997a, b, c; Barkworth & Tor- 2007, 2010; Hsiao & al., 1999; Romaschenko & al., 2008, 2010; res, 2001; Cialdella & Giussani, 2002; Vázquez & Barkworth, Soreng & Davis, 1998). The genus has recently been included 2004; Arriaga & Barkworth, 2006; Cialdella & al., 2007; Ro- in Stipeae (Barkworth, 2007), transferred into the genus Stipa maschenko & al., 2008, 2010; Barber & al., 2009). (Columbus & Smith, 2010), and is sometimes placed in the Phylogenetic inferences for Stipeae based on traditional monotypic subtribe Ampelodesminae Conert (Soreng & al., morphological characters are few. Based on morphological 2003, 2011). features the most comprehensive review was made by Tzvelev In a series of morphological studies (Barkworth, 1990, (1977) where phylogenetic weight was assigned to such charac- 1993; Barkworth & Torres, 2001; Cialdella & Giussani, 2002) ters as shape of the lemma and callus, and development of awn and in a phylogenetic study using molecular characters (Jacobs indumentum. Tzvelev’s general phylogenetic system suggested & al., 2000), Nassella and Piptochaetium were found to be there were two major lineages: (1) Stipa s.str. (including long sister genera. In more recent molecular phylogenetic analyses lanceolate lemmas, strongly developed awn indumentums, and based on nrDNA ITS sequences, Jacobs & al. (2007) found that sharp callus) and; (2) Piptatherum (including short, hairless lem- the Piptatherum-Oryzopsis complex along with Stipa s.str., mas with a caducous awn, and a blunt callus). Piptatherum was Ampelodesmos, Anisopogon, Hesperostipa, and Piptochaetium thought to have originated from more primitive Achnatherum- were among early diverging lineages; Austrostipa was depicted like species, whereas Ptilagrostis Griseb. and Achnatherum as a derived clade with Anemanthele Veldkamp embedded; and chinense (Hitchc.) Tzvelev were considered to be intermediate Nassella was more closely related to Jarava than to Piptochae- taxa between Achnatherum and Stipa s.str., and Achnatherum tium (Cialdella & al., 2007). However, there was little statistical and Piptatherum, respectively. Piptochaetium and Nassella support for this structure and much polyphyly of genera was (Trin.) E. Desv. were considered to be close relatives, and Erio- evident in these trees. coma Nutt. was thought to be a vicariant branch of Piptatherum In a phylogenetic analysis of 14 genera of Stipeae using in the New World. Over the years most of the American species four plastid loci, Cialdella & al. (2010) found only three mono- of Stipa s.l. were placed in endemic New World genera, such as phyletic genera, Austrostipa, Hesperostipa, and Piptochaetium. Anatherostipa (Hack. ex Kuntze) Peñailillo (Peñailillo, 1996) Their primary focus was to elucidate relationships of Aciachne and Jarava, or into two genera (Achnatherum and Piptatherum) and Amelichloa Arriaga & Barkworth, both portrayed as para- thought to be shared with Asia. or polyphyletic. Thomasson (1978, 1980, 1981, 1982, 1985) was first to In a combined analysis of ITS and four plastid loci document the phylogenetic importance of the lemma epider- (Romaschenko & al., 2008), Stipa s.str. and Piptatherum, mal pattern in Stipeae. Barkworth & Everett (1987) used this along with Ampelodesmos, Piptochaetium, Anatherostipa, information to delineate hypothetical relationships among Hesperostipa, and Ptilagrostis were found to lie among the genera, pointing out that Stipa and Piptatherum have elon- poorly resolved sets of basal lineages of Stipeae that share gated lemma epidermal cells with sinuous lateral walls (pattern the lemma epidermal pattern of elongate cells with sinuous also revealed in Miocene-dated spikelets of the fossil genus lateral walls. We identified this lemma epidermal pattern as Berriochloa M.K. Elias; Thomasson, 1978, 1982, 1985), and “saw-like” because the sidewalls of the lateral cells have a that Achnatherum and Austrostipa S.W.L. Jacobs & J. Everett serrate appearance (Romaschenko & al., 2008, 2010). This have short lemma epidermal cells with slightly sinuous to strait contrasted sharply with the phylogenetic scheme proposed by lateral walls. However, Barkworth & Everett (1987) followed Tzvelev (1977). The remainder of the Stipeae genera sampled Tzvelev (1977) in emphasizing the shape of the lemma and were resolved in a well-supported achnatheroid clade consist- callus and development of awn indumentum rather than lemma ing of: (1) a “major American clade” containing New World epidermal pattern, and therefore postulated a similar phyloge- Achnatherum, Jarava s.str. (excluding species of the former netic history. Hesperostipa (M.K. Elias) Barkworth and the Stipa subg. Pappostipa Speg., all of which were placed within “Obtusa group” of Parodi (1946; included in Anatherostipa), Jarava by Peñailillo, 2003, and elevated to generic status by and Nassella and Piptochaetium sensu Tzvelev were thought Romaschenko & al., 2008, as Pappostipa (Speg.) Romasch., to be two pairs of closely related genera. P.M. Peterson & Soreng), and Nassella with Amelichloa nested

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within it; and (2) a core achnatheroid clade containing Aus- monophyly of the achnatheroid clade and its correlation with trostipa and Asian species of Achnatherum embedded with specialized lemma epidermal anatomy, and test the monophyly Eurasian species of Piptatherum (Romaschenko & al., 2010). of the remaining Stipeae lineages (excluding Macrochloa) that We identified this pattern as “maize-like” because it resembles lack the achnatheroid lemma epidermal anatomy. We compare the surface of an ear of corn by having short fundamental cells phylogenetic trees based on plastid and ITS datasets, discuss with straight sidewalls and square to rounded, closely placed previous molecular and morphological studies where appro- silica bodies (Romaschenko & al., 2008, 2010). priate, correlate lemma micromorphological characters and Based on five plastid and nuclear ITS sequences, chromosome base numbers with our hypotheses based on our Romaschenko & al. (2010) conducted a molecular phylogenetic phylograms, interpret biogeographical relationships, and evalu- study of all 21 genera of Stipeae. They presented a stepwise ate the phylogenetic signal of plastid inversions and indels. model for the evolution of Stipeae comprising two initial deep bifurcations or splits followed by two further bifurcations, all highly correlated with geography. They found Macrochloa MATERIALS AND METHODS Kunth to be sister to all other Stipeae, Achnatherum and Pip- tatherum to be polyphyletic, and provided support for recog- Taxon sampling. — The Stipeae sample (voucher infor- nizing the following monophyletic genera: Achnatherum s.str., mation and GenBank numbers are given in Appendix 1 in the Aciachne, Amelichloa, Austrostipa, Hesperostipa, Jarava s.str., Electronic Supplement) consists of all 23 to 26 accepted genera Ortachne Nees ex Steud., Pappostipa, Piptatherum s.str., Pipto- (Soreng & al., 2003, 2011), has enhanced coverage within ma- chaetium, Ptilagrostis s.str., Stipa s.str., and Trikeraia Bor. jor infrageneric lineages of polyphyletic genera detected in our Using four plastid regions, Romaschenko & al. (2011) previous studies (Romaschenko & al., 2008, 2010), and has im- conducted a phylogenetic analysis of the short-spikeleted spe- proved focus on the taxonomic and geographical diversity within cies of the Stipeae. They recognized a Eurasian Piptatherum the tribe. At least two exemplars from each non-monotypic genus clade, a new North American genus, Piptatheropsis Romasch., and internal group previously resolved have been selected to P.M. Peterson & Soreng, and resurrected Patis Ohwi to include facilitate a more accurate interpretation of the generic concepts three species, two from Eurasia and one from North America. in the tribe. The total dataset of 156 accessions representing 139 The main objectives of the present paper are to provide a species comprises 7279 aligned nucleotide positions, 6639 bp better resolved and more highly supported phylogenetic hypoth- from the plastid data, and 640 bp nuclear ribosomal ITS data esis for the currently accepted genera and infrageneric groups (Table 2). Nine hundred and fifty sequences are newly reported within Stipeae compared to our previous study (Romaschenko to GenBank (Appendix 1). We included the type species of all & al., 2010). Particular effort is made to investigate the close infrageneric groups and genera sampled. In order to determine relationships among the American genera Hesperostipa, Pipto- the phylogenetic limits of Stipeae and its relationships with chaetium, and Anatherostipa that was suggested by Thomasson other tribes we included six of seven peripheral genera currently (1978, 1982, 1985) based on a study of lemma epidermal fea- placed in Phaenospermateae (Schneider & al., 2009; Soreng & tures. We add four plastid gene regions (rps3, rpl32-trnL, rps16- al., 2011), some of which have been classified within Stipeae trnK, rps16 intron) to those we used previously (trnK-matK, (Avdulov, 1931; Tzvelev, 1977; Wu & Phillips, 2006), and Bryl- matK, trnH-psbA, trnL-F, ndhF). We significantly expand our kinia F. Schmidt (Brylkinieae-Meliceae), Triniochloa Hitchc. earlier survey of Stipeae by sampling an additional 65 species (Meliceae), Diarrhena P. Beauv. (Diarrheneae), and Dielsiochloa including some of uncertain taxonomic position. We test the Pilg. (Poeae). Brachyelytrum erectum (Schreb.) P. Beauv., Nardus

Table . Summary of nine plastid regions and nrDNA ITS used in this study. trnK- trnH- rpl32- rps16- rps16 Plastid matK matK psbA trnL-F rps3 ndhF trnL trnK intron dataset ITS Number of taxa 152 146 155 128 151 151 146 151 153 156 151 Average sequence length (SL) 564 555 573 649 688 783 724 716 743 5995 578 Aligned sequence length (SLal) 589 555 663 754 688 783 954 814 839 6639 640 Number of excluded characters 51 0 68 78 0 0 192 9 47 447 66 Proportion of excluded characters [%] 8.7 – 10.3 10.3 – – 20.1 1.1 5.6 6.7 10.3 Number of variable characters (VC) 175 147 139 249 142 321 410 321 218 2122 378 VC/SL (% variability) 31.0 26.5 24.3 38.4 20.6 41.0 56.6 44.8 29.3 35.4 65.4 VC/SLal (% variability) 29.7 26.5 20.0 33.0 20.6 41.0 43.0 39.4 26.0 32.0 59.1 Tree length 152 115 136 153 183 505 528 348 241 2514 1475 Best-fit model of nucleotide substitution according to Akaike information criterion TVM TVMef SYM TVMef TVM K81uf+G TVM TVM TVMef GTR+G GTR+G

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stricta L., and Lygeum spartum L. were included as outgroups The amplification parameters that we found to be effective based on their well-documented early diverging positions in sub- across a wide range of the taxa for the plastid regions were: family Pooideae (Hilu & al., 1999; GPWG, 2001; Soreng & al., initial denaturation phase of 4 min at 94°C; followed by 35 2003, 2007, 2011; Davis & Soreng, 2007; Bouchenak-Khelladi cycles of denaturation at 94°C for 40 s, annealing phase at & al., 2008; Schneider & al., 2009). 50°C–56°C for 40 s, extension phase at 72°C for 1 min 30 s, DNA extraction, amplification, and sequencing. — The and final extension at 72°C for 10 min. We used 50°C–51°C of plant tissue was disrupted using Qiagen TissueLyser, and DNA primer annealing temperature for all coding plastid regions. was isolated using a BioSprint 96 DNA Plant Kit (Qiagen, Va- The entire nuclear ribosomal ITS region was amplified using lencia, California, U.S.A.). For amplification, the genomic primers ITS4 (White & al., 1990) and ITS5A (Stanford & al., DNA was combined with 1× reaction buffer (200 mM Tris-HCl, 2000) with the following thermocycler settings: 4 min at 94°C; 500 mM NH4 ; Bioline Biolase Taunton, Madison, Wisconsin, followed by 35 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for U.S.A.) without Mg++, 2 mM MgCl2, 200 mM dNTPs, 1.5 μl 1 min 20 s, and a final extension at 72°C for 10 min. of Taq polymerase (Bioline Biolase Taunton), and 40 pmol/μl All PCR products were cleaned with ExoSAP-IT (USB, each of forward and reverse primers. Cleveland, Ohio, U.S.A.). DNA sequencing was performed We targeted nine chloroplast DNA regions from the large with BigDye Terminator Cycle Sequencing v.3.1 (PE Applied single copy (LSC) and the small single copy (SSC) regions of Biosystems, Foster City, California, U.S.A.) according to the the genome: trnK-matK (intron, LSC), matK (coding region, following parameters: 80°C, 5 min; 25 or 30 cycles of 95°C LSC), trnH GUG-(rps19)-psbA (coding region, spacer, LSC), for 10 s, 50°C for 5 s and 60°C for 4 min. Sequenced prod- trnL-trnF (intron, spacer, LSC), rps3 (coding region, LSC), ucts were analyzed on an ABI PRISM 3730 DNA Analyzer ndhF (coding region, SSC), rpl32-trnLUAG (spacer, SSC), rps16- 7900HT (ABI). All regions except rpl32-trnL, rps16 intron, trnK (spacer, LSC), and rps16 (intron, LSC). The trnK-5′matK and 3′rps16-5′trnK were sequenced in one direction. Relatively intergenic spacer (IGS) was amplified and sequenced using the short regions (500–750 bp) covered by our primers were easily primers trnK3914F (Johnson & Soltis, 1995) and trnK660SR interpreted allowing us to accumulate sequences from different (Romaschenko & al., 2008). The primers trnK660SF and parts of the genome for phylogenetic inference (Shaw & al., matK1412SR (Romaschenko & al., 2008) were used to amplify 2005, 2007). The rpl32-trnL, rps16 intron, and 3′rps16- 5′trnK the 5′-end portion (555 bp) of matK. The trnH GUG-psbA region were sequenced in both directions and the program Sequencher was amplified with primers trnHf GUG (Tate & Simpson, 2003) v.4.8 (Gene Codes Corp., Ann Arbor, Michigan, U.S.A., 1991– and psbA3f (Sang & al., 1997). In Stipeae, as in most monocots 2007) was employed to produce the contig sequence for the (Wang & al., 2008), this region encompasses the entire copy entire region. of the rps19 gene embedded between the trnH GUG and psbA Phylogenetic analyses. — Sequences alignment was genes. Within Stipeae the lengths and variability (%) of the done manually using BioEdit v.7.0.5.3 (Hall, 1999). The in- components of this region are: trnH GUG-rps19 IGS (inverted dels, minute inversions, and other regions for which alignment repeat, IRA)—195 bp (5.1%), rps19 (IRA)—216 bp (4.1%), and was considered ambiguous, were excluded from the analyses. rps19-psbA IGS (LSC)—165 bp (22%). The trnH GUG-psbA re- OLIGO v.7.33 (Rychlik, 2009) was implemented to investi- gion has been used for phylogenetic inferences (Shaw & al., gate the nature of putative minute inversions detected in the 2005, 2007) and barcoding purposes (Kress & al., 2005, 2009; rpl32-trnL IGS (between 671–676 bp of aligned sequence) Kress & Erickson, 2009). and rps19-psbA IGS of the trnH-psbA region. The objectives The trnL-trnF region which includes the trnL intron, the were to define these minute inversions and determine if they 3′trnL exon, and the trnL-trnF intergenic spacer, was ampli- were reversed point mutations or constitute stable loops of the fied using primers 5′trnLUAA(f) and trnFGAA(c) (Taberlet & single-stranded hairpin formations (Downie & Palmer, 1992; al., 1991). The rps3 gene was amplified and sequenced using Kelchner & Wendel, 1996; Kim & Lee, 2005; Bain & Jansen, primers rps3C29F and rps3C697R (Peterson & al., 2010a, b). 2006; Catalano & al., 2009; Lehtonen & al., 2009). The results Variability rate and ease of amplification make it suitable for of this analysis were used as additional characters or support phylogenetic study, especially when working with older herbar- for the putative phylogenetic groups (see Appendix 2 in the ium specimens. For the ndhF gene we amplified and sequenced Electronic Supplement). These mutations were plotted on the the variable 3′-end (783 bp) with the primers ndhF1311F and plastid phylogram (Fig. 1). The amount of excluded data for ndhF2091R (Romaschenko & al., 2010). each region is presented in Table 2. No data was excluded from The region rpl32-trnLUAG was amplified and sequenced matK, rps3, and ndhF. All gaps were treated as missing data. with primers trnLUAG and rpl32-F (Shaw & al., 2007). The We conducted maximum likelihood and Bayesian analy- sequences contain the entire rpl32-trnLUAG IGS and a small ses to infer phylogenies. The maximum likelihood analysis portion of the trnLUAG gene. The rps16-trnK IGS was amplified was conducted with the program GARLI v.0.951 (Zwickl, and sequenced with rpS16-900F and 3914PR primers (Peterson 2006). Bayesian and maximum likelihood analyses yielded & al., 2010a, 2010b). Since the rpS16-900F primer is placed trees with visually similar topology, i.e., the trees are visually at the 3′-end of rps16 intron the amplified region contains the the same, but some branch lengths could differ minutely. A entire 3′rps16 exon and rps16-trnK IGS. For amplification of test run of Bayesian analysis for the combined plastid dataset the rps16 intron the primers rpS16R and rpS16F were used under the single GTR + G model yielded the same topology and (Peterson & al., 2010a, b). posterior probability (PP) values as the Bayesian analysis for

6 TAXON — Article version: 9 Dec 2011: 27 pp. Romaschenko & al. • Systematics and evolution of needle grasses

a partitioned dataset performed under models suggested by specimens from the majority of the Stipeae species used in the MrModeltest v.1.1b (Nylander, 2002) for separate regions. The phylogenetic analysis. To remove epicuticular wax the lemmas Akaike information criterion models are indicated in Table 2 were cleaned in xylene for four hours. Samples were mounted (Kimura, 1981; Tavaré, 1986; Posada & Crandall, 1998). and then covered with gold from a vacuum spray gun (JII-4X, Bootstrap analyses were performed under maximum likeli- Japan). The ultrastructure of the lemma was studied at varying hood algorithm using GARLI (Zwickl, 2006) and were set for magnifications using a Jeol (JSM35C, Japan) scanning electron 1000 bootstrap replicates. The majority-rule trees were then microscope. constructed in PAUP* v.4.0b10 (Swofford, 2000). Bootstrap We illustrate the lemma epidermal pattern (LEP) for 44 (BS) values of 90%–100% were interpreted as strong support, species (Figs. 3–4); 32 of these have never been published; eight 70%–89% as moderate, 50%–69% as weak, and those under of these (Achnatherum hymenoides (Roem. & Schult.) Bark- 50% were not reported. We identify some clades as “group” worth, Achnatherum stillmanii (Bol.) Barkworth, Oryzopsis in regular script when there is no formal genus name for them asperifolia Michx., Piptatheropsis canadensis (Poir.) Romasch. (e.g., “Miliaceae group”), or with the genus in italics when there & al., Piptatheropsis micrantha (Trin. & Rupr.) Romasch. & was at least one species included with others that have not been al., Piptatherum miliaceum (L.) Coss., Ptilagrostis kingii (Bol.) formally transferred (e.g., “Eriocoma group”). Barkworth, Ptilagrostis mongolica (Turcz. ex Trin.) Griseb.) are Bayesian posterior probabilities were estimated using of much higher quality and/or complement ink drawings; three the program MrBayes v.3.01 (Huelsenbeck & Ronquist, 2001; of these (Celtica gigantea (Link) F.M. Vázquez, Macro chloa Ronquist & al., 2005) with DNA substitution models selected tenacissima (Loefl. ex L.) Kunth, Nassella neesiana (Trin. & using the program MrModeltest v.1.1b (Nylander, 2002). The Rupr.) Barkworth) confirm previous work (Thomasson, 1978, plastid dataset was then partitioned into four subsets (1: trnk- 1980; Barkworth, 1983; Barkworth & Everett, 1987; Vásquez & matK + rps3 + rps16-trnK + rpl32-trnL; 2: matK + trnL-F + rps16 Barkworth, 2004); and one replaces an erroneously published intron; 3: trnH-psbA; 4: ndhF) and were processed implement- pattern for Stipa pennata L. (Vásquez & Barkworth, 2004). ing different parameters suggested by Akaike information cri- Cytogenetics. — We surveyed the original literature and terion (Table 2). The ITS data were calculated separately. Each floristic treatments for chromosome numbers reported for spe- Bayesian analysis was initiated with random starting trees and cies of Stipeae, and discuss the possible evolution of base chro- initially run for two million generations, sampling once per mosome numbers in the tribe (see Appendix 3 in the Electronic 100 generations. The analysis was continued until the value of Supplement for reported chromosome numbers). standard deviation of split sequences dropped below 0.01 as the convergence diagnostic value (Huelsenbeck & Ronquist, 2001). The fraction of the sampled values discarded as burn-in RESULTS was set at 0.25. The test of alternative phylogenetic hypotheses was ac- Analysis of plastid sequences. — Since very little differ- complished using parametric bootstrapping (Huelsenbeck & ence and no contradictions in generic composition or arrange- al., 1995; Swofford & al., 1996; Goldman & al., 2000) as implement were observed among maximum likelihood trees in the mented in Mesquite v.2.6 (Maddison & Maddison, 2009). The individual plastid analyses, the data were combined. best-scoring maximum likelihood tree (the optimal topology The combined plastid data provides a rather well-resolved for unconstrained dataset) and simulation model parameters tree, mostly with high backbone support (Fig. 1). The first split were obtained using GARLI (Zwickl, 2006) for the single on the phylogenetic tree strongly indicates (BS = 99, PP = 1.00) GTR + G model of sequence evolution in maximum likelihood the Phaenospermateae (BS = 89, PP = 1.00) to be sister to the searches. The same procedure was repeated for the maximum remainder of the Pooideae represented by the tribes Meliceae, likelihood searches with monophyly constraints consistent with Poeae, Diarrheneae, and Stipeae (BS = 99, PP = 1.00). There is the research or alternative hypothesis (other than the null hy- strong support for clades of Meliceae-Brylkinieae (BS = 99, PP pothesis). The constraint topology and model parameters were = 1.00), Diarrhenaeae-Poeae (BS = 100, PP = 1.00), and Stipeae used to simulate 1000 data matrices equal in size to the original (BS = 100, PP = 1.00). However, the relationships among these matrix using Mesquite v.2.6. These parameters were then used clades are weakly supported (BS = 66, PP = 0.86). in PAUP* to find the most parsimonious trees constructed un- Within Stipeae, the monotypic genus Macrochloa is re- der topological constraints and the most parsimonious uncon- solved as sister to a clade of all remaining taxa with strong strained trees. Differences in tree length for constrained and support (BS = 99, PP = 1.00). Above this split Stipeae comprise unconstrained searches for each of the 1000 simulated matri- three well-supported clades, each with a saw-like LEP, and we ces were calculated and plotted as histograms using Mesquite refer to these clades as SL1, SL2, and SL3. A fourth lineage v.2.6. The distribution of tree length differences between two that is the sister of SL3 we designate as the “achnatheroid potential topologies was estimated. If the difference between clade” (AC). constrained and unconstrained topologies fell outside the 95% The first saw-like lineage (SL1; BS = 99, PP = 1.00) con- confidence interval of this distribution (P < 0.05), the alterna- sists of two geographically distinct clades: one is primarily tive hypothesis was rejected. distributed in Eurasia, which we call the “Eurasian saw-like lin- Scanning electron microscopy. — Lemma ultrastructure eage (ESL),” the other is distributed in the Americas, which we was studied using dry mature seeds sampled from herbarium call the “American saw-like lineage (ASL)”. The ESL (BS = 86,

7 Romaschenko & al. • Systematics and evolution of needle grasses TAXON — Article version: 9 Dec 2011: 27 pp.

PP = 1.00) consists of two clades: the Stipa s.str. clade (BS = since two species, A. hans-meyeri (Pilg.) Peñailillo and A. rosea 100, PP = 1.00), which we refer to as the ESL1 that includes the (Hitchc.) Peñailillo, are united in a clade with Lorenzochloa (BS type of Stipa (S. pennata). This clade is split into two strongly = 100, PP = 1.00) that is sister to a strongly supported (BS = supported clades: (1) a clade that includes S. purpurea Griseb., 100, PP = 1.00) clade that includes Aciachne plus the remaining S. subsessiliflora (Rupr.) Roshev., S. regeliana Hack., S. bun- Anatherostipa s.str. species (BS = 97, PP = 1.00). The Aciachne geana Trin., and S. capillacea Keng (BS = 96, PP = 1.00) and clade, composed of two species, is strongly supported (BS = (2) a clade that includes S. pennata, S. brauneri (Pacz.) Klokov, 100, PP = 1.00), and this is sister to the Anatherostipa s.str. clade S. barbata Desf., S. caucasica Schmalh., S. capillata L., and (BS = 100, PP = 1.00) composed of four species. S. breviflora Griseb. (BS = 100; PP = 1.00). The other is a The sister to the SL1 clade, which includes all remaining weakly supported, taxonomically complex clade (BS = 68, PP species (clades SL2, SL3, and AC) is moderately supported (BS = 0.82), which we refer to as the ESL2 clade that includes Ptila- = 77, PP = 0.95). The SL2 clade (BS = 100, PP = 1.00) contains grostis s.str. as well as several monotypic or ditypic genera such the far East-Asian Patis which in this tree includes P. obtusa as Ampelodesmos, Psammochloa Hitchc., Achnatherum splen- (Stapf) Romasch. & al. (≡ Piptatherum kuoi S.M. Phillips & dens (Trin.) Nevski (“Neotrinia”, Romaschenko & al., 2010), Z.L. Wu) and P. coreana (Honda) Ohwi (≡ Achnatherum corea- Oryzopsis asperifolia (the only American representative of this num (Honda) Ohwi) and is sister to a strongly supported clade clade), Trikeraia, and Orthoraphium Nees. The ESL2 clade (BS = 100, PP = 1.00) including SL3 and the AC. The SL3 clade includes a strongly supported Ampelodesmos-Psammochloa- (BS = 66, PP = 0.71) encompasses the morphologically isolated “Neotrinia” clade (BS = 99, PP = 1.00) in which Ampelodesmos North American species Achnatherum stillmanii (“Stillmania”) is sister to a clade of Psammochloa and “Neotrinia” (BS = 100, as sister to a strongly supported clade of Eurasian Piptatherum PP = 1.00). The other, weakly supported clade (BS = 66, PP = s.str. (BS = 100, PP = 1.00), including P. coerulescens (Desf.) 0.86) within ESL2 includes Oryzopsis asperifolia as sister to a P. Beauv., the type of this genus. strongly supported Trikeraia-Orthoraphium-Ptilagrostis clade The strongly supported AC (BS = 100, PP = 1.00) contains (BS = 100, PP = 1.00). A strongly supported clade (BS = 96, three major clades. The “core achnatheroid clade” (CAC, BS = PP = 1.00) of Trikeraia species is part of a clade that includes 85, PP = 0.96) is sister to a moderately supported clade (BS = Orthoraphium roylei Nees and five species of Ptilagrostis; 84, PP = 1.00) that is divided into two highly supported sister their relationships are not well-resolved. The monophyly of clades: the Asian “Timouria Roshev. group” (A2, PP = 100, PP Ptilagrostis s.str. (excluding P. kingii and P. pelliotii (Danguy) = 1.00); and the “major American clade” (MAC, BS = 100, PP Grubov) is weakly supported (BS = 64, PP = 0.83). However, = 1.00). CAC includes the monotypic western Mediterranean two clades within Ptilagrostis are strongly supported (BS = genus Celtica F.M. Vázquez & Barkworth as sister to a mod- 100, PP = 1.00): one includes Ptilagrostis mongolica (the type) erately supported clade (BS = 81, PP = 1.00) that consists of an and P. malyshevii Tzvelev; and the other includes P. luquensis Australian clade (BS = 52, PP = 0.95) and a Eurasian clade of P.M. Peterson & al., P. junatovii Grubov, and P. dichotoma Achnatherum (A1, PP = 0.91). The Australian clade includes Keng ex Tzvelev. Anemanthele, a monotypic genus from New Zealand, as sis- The ASL clade is weakly supported (BS = 58, PP = 0.93), ter to a monophyletic Austrostipa (BS = 86, PP = 1.00). The and comprises two clades: ASL1 and the ASL2. The ASL1 A1 clade encompasses 10 species of Achnatherum, including clade has no support and splits into two major clades: (1) a A. calamagrostis (L.) P. Beauv., the type of the genus, and two clade (BS = 69, PP = 0.75) that encompasses Ptilagrostis kingii species of Piptatherum sect. Virescentia Roshev. ex Freitag as sister to the strongly supported Piptatheropsis clade (BS = (P. virescens (Trin.) Boiss. and P. paradoxum (L.) P. Beauv.). 100, PP = 1.00) that includes five former members of North The two representatives of Piptatherum sect. Miliacea Roshev. American Piptatherum (called the “Piptatheropsis group” by ex Freitag (P. miliaceum (L.) Coss. and P. thomasii (Duby) Romaschenko & al., 2010; and 2011—as a new genus); and (2) Kunth) form a strongly supported clade (“Miliacea group,” a South American clade (BS = 95, PP = 1.00) that includes a BS = 100, PP = 1.00) that is sister to the A1 clade. However, monophyletic Ortachne and Pappostipa, each with strongly this sister relationship is poorly supported (PP = 0.67). Thus, supported (BS = 100 and PP = 1.00) crown nodes. The ASL2 the Eurasian Piptatherum species are resolved in three distinct clade is strongly supported (BS = 94, PP = 1.00) and includes clades: Patis in SL2, Piptatherum s.str. in SL3, the “Miliacea a monophyletic Hesperostipa (BS = 100 and PP = 1.00) from group” and P. sect. Virescentia in CAC. North American spe- North America as sister to a strongly supported clade (BS = 90, cies formerly treated in Piptatherum are contained in SL1 in PP = 1.00) of the remaining species. Among these remaining the genus Piptatheropsis. Stipa capensis Thunb. and S. parvi- species there is a single clade of the pan-American genus Pipto- flora Desf. (of the monotypic Stipa sects. Stipella Tzvelev and chaetium (BS = 100, PP = 1.00) as sister to a clade containing Inaequeglumes (Bor) F.M. Vazquez & Devesa) of arid Medi- the South American genera Aciachne, Anatherostipa and Loren- terranean origin are united without any support and appear as zochloa Reeder & C. Reeder. Anatherostipa s.l. is polyphyletic, sister to the “Miliacea group” plus A1 clade.

Fig. . Phylogram of maximum likelihood tree from analysis of plastid data. Numbers above branches are bootstrap values; numbers below branches are posterior probability values; taxon colour indicates native distribution; 13 indels, one stem loop hairpin formation, and lemma epidermal patterns (LEP) are mapped on the tree.

8 TAXON — Article version: 9 Dec 2011: 27 pp. 0 Romaschenko &Brachyelytrum al. • Systematics erectum Brachyelytreae and evolution of needle grasses 100 X Nardus stricta Nardeae i Lygeeae 1.00 Yi Lygeum spartum 0 Danthoniastrum compactum 66 Anisopogon avenaceus 89 0.86 57 0 Duthiea brachypodium 100 1.00 Metcalfia mexicana Phaenosperma globosa Phaenospermateae 1.00 0 (except Metcalfia) 100 Stephanachne pappophorea 0.63 84 1.00 1i Stephanachne nigrescens 1.00 Sinochasea trigyna 99 2 Brylkinia caudata 99 Meliceae 1 No rps19-psbA loop sequence 1.00 Triniochloa stipoides 1.00 100ĺ Dielsiochloa floribunda Poaeae 1.00 54 Diarrhena obovata 0 1.00 95 Diarrhena fauriei Diarrheneae 1.00 Diarrhena japonica 3 Macrochloa tenacissima 99 96 Stipa purpurea Stipa subsessiliflora 1.00 Ÿ1.00 69 Stipa regeliana LEP 99 100 0.94 Stipa bungeana ESL1 1.00 Stipa capillacea 1.00 Stipa breviflora 100 Stipa capillata Stipa caucasica 66 1.00 100 86 Stipa barbata

1 Ordered 0.86 ESL i 1.00 1.00 Stipa brauneri Stipa pennata 99 1 1i Ampelodesmos mauritanicus 100 Psammochloa villosa like - w a S 1.00 1ii ESL2 68 Ÿ 1.00 Achnatherum splendens Neotrinia 0.82 Oryzopsis asperifolia LEP 66 96 Trikeraia pappiformis 0.86 100 Ÿ 1.00 Trikeraia hookeri 1.00 Orthoraphium roylei 100 Ptilagrostis mongolica 0.74 64 1.00 Ptilagrostis malyshevii 100 Ÿ0.83 Ptilagrostis luquensis 99 100 1.00 4 83 Ptilagrostis junatovii SL1 1.00 1.00 0.97 Ptilagrostis dichotoma Ptilagrostis kingii 69 99 Piptatheropsis exigua 0.75 100 1.00 Piptatheropsis shoshoneana 1.00 99 Piptatheropsis micrantha

Piptatheropsis LEP 98 Piptatheropsis canadensis 1.00 Stipeae ASL1 1.00 Piptatheropsis pungens 100 Ortachne rariflora 95 1.00 Ortachne breviseta Pappostipa major like - w a S Unordered 1.00 100 96 Pappostipa chrysophylla 58 1.00 82 Pappostipa vaginata ASL 1.00 0.93 0.99 Pappostipa hieronimusii Pappostipa speciosa 100 Hesperostipa spartea 1.00 95 Hesperostipa comata 1.00 Hesperostipa neomexicana like - e z i a M 94 100 Piptochaetium avenaceum Piptochaetium featherstonei ASL2 1.00 1.00 99 99 77 1i Piptochaetium brachyspermum 1.00 98 Piptochaetium panicoides 1.00 90 0.94 1.00 Piptochaetium montevidense LEP 1.00 100 Anatherostipa hans-meyeri 1.00 61 Anatherostipa rosea 0.64 Lorenzochloa erectifolia 100 Aciachne acicularis 97 1.00 Aciachne flagellifera 1.00 100 Anatherostipa venusta 100 Anatherostipa mucronata 1.00 92 1.00 Anatherostipa rigidiseta 1.00 Anatherostipa obtusa 100 Patis obtusa SL2 1.00 Patis coreana Patis 1i Achnatherum stillmanii Stillmania 66 1i Piptatherum coerulescens SL3 0.71 100 78 Piptatherum aequiglume Ÿ 1.00 Piptatherum holciforme 1.00 95 100 0.62 Piptatherum ferganense 1.00 Piptatherum songaricum 1.00 Piptatherum munroi Piptatherum like - w a S Unordered 77 100 Piptatherum sogdianum 1.00 Piptatherum angustifolium 0.95 100 Piptatherum hilariae trnH-psbA 1.00 Piptatherum fedtschenkoi Celtica gigantea Anemanthele lessoniana Xi GGTCAG 100 Austrostipa scabra 60 52 1.00 Austrostipa nitida Yi 79 TTTTAG 0.95 0.95 Austrostipa compressa 0 100 85 1.00 Austrostipa macalpinei CTGGAA CAC 86 85 Austrostipa nodosa 1.00 0.96 1.00 1.00 65 Austrostipa tenuifolia 1 CTGAAA 1.00 Austrostipa pycnostachya 98 Austrostipa flavescens 1i 81 Austrostipa trichophylla TTTCAG 1.00 1.00 85 Austrostipa juncifolia 1ii Austrostipa campylachne TATCAG 1.00 90 Austrostipa semibarbata 2 TTGAAA 1.00 Austrostipa hemipogon 5 Stipa capensis Stipella 3 ATGGAA Stipa parviflora 100 Piptatherum miliaceum Miliacea gr. 4 ATGAAA 1.00 Piptatherum thomasii Achnatherum calamagrostis 0.67 Achnatherum turcomanicum 5 CTAAAA 100 AC Achnatherum jacquemontii 6 CCGAAA 1.00 0.91 85 Achnatherum bromoides 0.99 99 Piptatherum virescens 1.00 7 CCCAAA Piptatherum paradoxum A1 LEP Achnatherum inebrians Achnatherum 87 Achnatherum pekinense 1.00 94 Achnatherum brandisii rpl32-trnL 1.00 72 Achnatherum confusum 99 Achnatherum sibiricum 0.98 - 21 bp insertion (170) 1.00 Achnatherum pubicalyx 100 Achnatherum chinense - 8 bp deletion (104) Achnatherum caragana Timouria gr. 1.00 95 Achnatherum/Timouria saposhnikovii 1.00 Ptilagrostis pelliotii A2

- 5 bp deletion (214) 100 Achnatherum wallowaense M a i z e - like like - e z i a M 1.00 Achnatherum hendersonii - 5 bp insertion (830) 98 Achnatherum hymenoides 84 1.00 81 7 Achnatherum latiglume - 7 bp insertion (110) 76 62 1.00 Achnatherum curvifolium 1.00 Achnatherum scribneri 1.00 1.00 73 7 - 7 bp insertion (777) 7 Achnatherum perplexum 0.94 87 7 Achnatherum pinetorum A3 1.00 Achnatherum lettermanii rps16-trnK Achnatherum richardsonii Eriocoma gr. 55 100 Achnatherum aridum - 1 bp insertion (396) 6 MAC 0.98 65 57 Achnatherum parishii 1.00 0.97 56 Achnatherum robustum - 20 bp insertion (571) Achnatherum lobatum 99 Achnatherum occidentale subsp. californicum Achnatherum lemmonii 75 - 16 bp insertion (214) 1.00 Achnatherum nelsonii 0.79 Achnatherum occidentale Jarava annua Jarava media rps16 intron Jarava plumosula 100 70 Achnatherum diegonense - 5 bp insertion (777) 1.00 89 1.00 Achnatherum constrictum 91 Achnatherum eminens A4 1.00 1.00 77 Achnatherum multinode trnL-F 88 0.98 Achnatherum acutum Pseudoeriocoma gr. Jarava pseudoichu Ÿ - 5 bp insertion (115) 1.00 100 Jarava ichu - 10 bp deletion (89) 1.00 100 Jarava castellanosii Ÿ 1.00 Jarava scabrifolia - 21 bp repeat insertion (343) Amelichloa caudata Ÿ Amelichloa clandestina 97 Nassella clarazii 1.00 Nassella pfisteri Nassella neesiana LEP Nassella trichotoma

North America Mediterranean Nassella filiculmis

L a d d e r r e d d a L - like like - Australia and New Zealand South America Eurasia 9 Romaschenko & al. • Systematics and evolution of needle grasses TAXON — Article version: 9 Dec 2011: 27 pp.

The “Timouria group” (A2, BS = 100, PP = 1.00) consists sequence CTGGAA (type “0” in Fig. 1) is common among of the central Asian species Timouria saposhnikovii Roshev., members of early diverging lineages of Pooideae (Brachy- A. chinense, A. caragana (Trin.) Nevski, and Ptilagrostis elytrum P. Beauv., Danthoniastrum, Duthiea, Phaenosperma) pelliotii. but is absent in Stipeae. This sequence is also characteristic of The MAC is strongly supported (BS = 100, PP = 1.00) and Diarrhena. Nardus L. and Lygeum Loefl. ex L. exhibit specific is split into two separate clades: the “Eriocoma group” (A3, BS inversions Xi and Yi of unknown sequence origin. For a more = 98, PP = 1.00), and a complex clade (BS = 100, PP = 1.00) detailed discussion of the stem-loop hairpins see Appendix 2. that includes the “Pseudoeriocoma group” (A4, BS = 70, PP = Thirteen indels were detected in four of our sequenced 1.00) and representatives of Jarava, Nassella, and Amelichloa. plastid regions: six in rpl32-trnL, three in rps16-trnK, one in The “Eriocoma group” and “Pseudoeriocoma group” include rps16 intron, and three in trnL-F. These are identified by their North American species currently placed in Achnatherum but starting position within the aligned sequences and are mapped they are resolved in separate, strongly supported clades. Spe- on the tree (Fig. 1). In rpl32-trnL a 21 bp insertion (170) was cies of Achnatherum within AC have maize-like LEPs and found in all representatives of the Phaenospermateae except are scattered in four clades: two Eurasian (A1, A2) and two Metcalfia. The 7 bp insertion (110) indicates a possible alter- of North American origin (A3, A4). Achnatherum splendens native relationship between plastids of three species of Stipa (“Neotrinia”) of the SL1/ESL2 clade, and A. stillmanii (“Still- (S. purpurea, S. subsessiliflora, S. regeliana). Another 7 bp mania”) of the SL3 clade, have saw-like LEPs. insertion (777) confirms the monophyly of four species in the Jarava annua (Mez) Peñailillo is located in an unsupported ESL1 clade (this taxon set is also supported by minute inver- grade between the “Eriocoma group” A3, and the other mem- sion 1i ). The 8 bp deletion (104) supports the monophyly of bers of MAC. These other members of MAC form a strongly the entire AC clade. Within AC a 5 bp insertion (830) adds supported clade (BS = 100, PP = 1.00) that includes a grade of support for the entire Australia–New Zealand clade, and a 5 bp J. media (Speg.) Peñailillo and J. plumosula (Nees ex Steud.) deletion (214) supports a central and east Asian subclade of six F. Rojas (members of former Stipa subg. Ptilostipa Speg.— species within Achnatherum A1: A. inebrians (Hance) Keng Spegazzini, 1901; Roig, 1964), the “Pseudoeriocoma group” ex Tzvelev, A. pekinense (Hance) Ohwi, A. brandisii (Mez) A4), Jarava s.str., Amelichloa, and Nassella. Jarava plumosula Z.L. Wu, A. confusum (Litv.) Tzvelev, A. sibiricum (L.) Keng is sister to a clade (BS = 89, PP = 1.00) that includes an unre- ex Tzvelev, and A. pubicalyx (Ohwi) Keng. solved trichotomy of the “Pseudoeriocoma group“ (A4, BS = The rps16-trnK region exhibited two independent inser- 70, PP = 1.00), Jarava s.str. (BS = 100, PP = 1.00, including tions in the ASL2 clade of different lengths. A 1 bp insertion the type, J. ichu Ruiz & Pav.), and the Amelichloa-Nassella (396) marks Aciachne and Anatherostipa s.str. from the Anath- clade (BS = 97, PP = 1.00). The “Pseudoeriocoma group” (A4) erostipa hans-meyeri–A. rosea–Lorenzochloa clade. This is is composed of five south-western North American species, followed by a 20 bp insertion (571) marking the separation of primarily distributed in Mexico. the Anatherostipa s.str. from the Aciachne clade. The Asian Plastid DNA minute inversions and indels. — Minute Achnatherum A1 clade of six species, mentioned above, is ad- inversions were detected in rpl32-trnL and trnH-psbA. How- ditionally supported by a 16 bp insertion (214). In the rps16 ever, only in data from the trnH-psbA region were we able to intron a 5 bp insertion (777) is unique to the Patis clade. find cases of clear phylogenetic utility. We identified a stem- A trnL-F 5 bp insertion (115) supports the monophyly of loop hairpin formation involving a small region flanking the S. purpurea, S. subsessiliflora, S. regeliana, S. bungeana, and inverted repeat (IRA-LSC). All taxa in our dataset (except S. capillacea. A 10 bp deletion (89) marks the crown node for Dielsiochloa , which lacks the loop) exhibited a hairpin for- the Piptatherum s.str. clade. A 21 bp repeat insertion (343) mation characterized by relatively long and conserved stem is confined to ESL2 and appears three times in that clade. It sequences (averaging 19 bp in length) linked by a short loop was detected in Psammochloa–Achnatherum splendens (“Neo- (6 bp). The poly-A end of IRA is sometimes partially involved trinia”) clade, Trikaraia clade, and the Ptilagrostis s.str. clade. in the stem formation. Polymorphic sequences in the trnH-psbA Analysis of ITS sequences. — The ITS phylogenetic tree hairpin loop and inverted loop were mapped on our plastid tree. is poorly resolved with little backbone support (Fig. 2). The This loop proved to be inverted in some sequences (Fig. 1: Xi, bifurcation between Brachyelytrum, Nardus-Lygeum, and the Yi, 1i and 1ii). The sequences from the loop also exhibited base rest of the Pooideae has weak support (BS = 60, PP = 0.94). mutations that accumulate in a phylogenetically informative The Phaenospermateae tribe is not monophyletic as its ele- order in some cases (Fig. 1: sequence 0 gives rise to 1 and ments are distributed among the first three branches after the frequently reverses except in Stipeae above Macrochloa; 1 Brachyelytrum and Nardus-Lygeum split. The only supported independently gives rise to 2, and (within Stipeae) to 3, 4, 5, clade within Phaenospermateae (BS = 100, PP = 1.00) con- and 6; and 6 gives rise to 7). The change from state 1 to state 6 tains two species of Stephanachne and Sinochasea. Triniochloa marks the MAC clade, and this is not reversed, but does give (Meliceae) and Dielsiochloa (Poeae) are united with minimal rise to state 7 in some Achnatherum A3 elements. In this loop support.

Fig. . Phylogram of maximum likelihood tree from analysis of nuclear ITS data. Numbers above branches are bootstrap values; numbers below branches are posterior probability values; taxon colour indicates native distribution; lemma epidermal patterns (LEP) are mapped on the tree.

10 TAXON — Article version: 9 Dec 2011: 27 pp. RomaschenkoBrachyelytrum & al. •erectum SystematicsBrachyelytreae and evolution of needle grasses 83 Nardus stricta Nardeae Lygeeae 1.00 Lygeum spartum 100 Stephanachne nigrescens 1.00 78 Sinochasea trigyna 0.70 Stephanachne pappophorea Phaenosperma globosa Phaenospermateae 60 Danthoniastrum compactum 0.94 Duthiea brachypodium 0.54 Metcalfia mexicana 0.72 52 Anisopogon avenaceus 0.73 Dielsiochloa floribunda Poaeae 0.50 Brylkinia caudata Meliceae 0.74 Triniochloa stipoides 0.61 Diarrhena obovata 0.79 98 Diarrhena fauriei Diarrheneae 1.00 0.55 Diarrhena japonica Macrochloa tenacissima Ampelodesmos mauritanicus LEP 81 98 Psammochloa villosa 0.84 1.00 83 1.00 Achnatherum splendens Neotrinia 1.00 57 Oryzopsis asperifolia 99 0.67 Trikeraia pappiformis 0.83 1.00 Trikeraia hookeri Achnatherum stillmanii Stillmania SL3 Piptatherum coerulescens 0.67 100 68 Piptatherum munroi 1.00 Piptatherum aequiglume 1.00 69 Piptatherum hilariae 1.00 Piptatherum angustifolium Piptatherum Piptatherum fedtschenkoi 0.99 Piptatherum holciforme 77 Piptatherum songaricum 0.63 69 1.00 Piptatherum sogdianum 0.93 Piptatherum ferganense 0.76 Stipa capillacea like - w a S Unordered 87 Stipa regeliana 1.00 Stipa purpurea Stipa subsessiliflora LEP 0.89 91 Stipa bungeana 0.99 Stipa breviflora 60 Stipa capillata 0.98 Stipa barbata Stipeae Stipa caucasica 63 Stipa brauneri Ordered 0.54 Stipa pennata Ptilagrostis malyshevii 96 like - w a S Ptilagrostis mongolica 1.00 Ptilagrostis luquensis Ptilagrostis junatovii 52 Ptilagrostis dichotoma 0.99 0.99

Orthoraphium roylei LEP SL2 Patis obtusa 83 Piptochaetium avenaceum Piptochaetium featherstonei 1.00 74 62 Piptochaetium brachyspermum 0.95 81 Piptochaetium panicoides 0.57 1.00 Piptochaetium montavidense Ptilagrostis kingii 81 Piptatheropsis exigua 0.61 1.00 Piptatheropsis shoshoneana 0.56 84 Piptatheropsis canadensis Piptatheropsis 83 1.00 Piptatheropsis pungens 0.96 Piptatheropsis micrantha 90 Ortachne rariflora 1.00 Ortachne breviseta Anatherostipa rosea 72 0.54 Aciachne acicularis 0.91 Aciachne flagellifera 68 like - w a S Unordered Anatherostipa hans-meyeri 1.00 54 Anatherostipa mucronata Anatherostipa venusta 0.83 81 1.00 Anatherostipa rigidiseta Anatherostipa obtusa ASL2 99 Hesperostipa neomexicana 1.00 66 Hesperostipa comata 0.67 Hesperostipa spartea 60 Celtica gigantea Jarava annua 1.00 Stipa parviflora 0.82 Pappostipa major 0.86 91 AC 94 Pappostipa chrysophylla 1.00 1.00 94 Pappostipa hieronimusii +Pappostipa 87 Pappostipa vaginata 1.00 0.96 Pappostipa speciosa Achnatherum jacquemontii 0.53 95 Piptatherum paradoxum 0.99 Piptatherum virescens 54 66 Anemanthele lessoniana 0.99 Austrostipa nodosa 0.90 97 95 Austrostipa tenuifolia 1.00 1.00 Austrostipa pycnostachya 100 Austrostipa scabra 0.94 1.00 Austrostipa nitida 99 Austrostipa compressa 1.00 Austrostipa macalpinei Austrostipa juncifolia 0.77 77 Austrostipa campylachne 89 Austrostipa flavescens 1.00 100 0.99 Austrostipa semibarbata 1.00 Austrostipa hemipogon Achnatherum calamagrostis

Achnatherum turcomanicum LEP Achnatherum inebrians 92 Achnatherum pubicalyx A1 1.00 Achnatherum brandisii 98 Achnatherum confusum Achnatherum 1.00 Achnatherum sibiricum 70 Achnatherum pekinense 0.99 Achnatherum bromoides Achnatherum/Timouria saposhnikovii Ptilagrostis pelliotii A2

Achnatherum caragana like - e z i a M Achnatherum chinense Timouria gr. 100 Piptatherum miliaceum 1.00 Piptatherum thomasii Miliacea gr. Stipa capensis Stipella 100 Achnatherum wallowaense 54 1.00 Achnatherum hendersonii 0.86 97 Achnatherum pinetorum 1.00 Achnatherum lettermanii 0.51 60 Achnatherum scribneri 99 Achnatherum occidentale subsp. californicum 0.98 Achnatherum nelsonii 1.00 0.81 81 Achnatherum occidentale A3 0.81 0.67 0.64 Achnatherum lemmonii Eriocoma gr. Achnatherum aridum 52 Achnatherum hymenoides North America 1.00 0.53 Achnatherum richardsonii 69 Achnatherum perplexum 82 Achnatherum parishii 0.99 97 Achnatherum lobatum South America 1.00 1.00 Achnatherum robustum 91 Jarava ichu Jarava pseudoichu MAC 1.00 99 0.62 1.00 Jarava castellanosii Mediterranean Jarava scabrifolia Jarava media 86 Jarava plumosula 60 0.93 Achnatherum constrictum 1.00 99 Achnatherum acutum Eurasia 1.00 75 Achnatherum eminens A4 0.97 Achnatherum multinode Pseudoeriocoma 65 Achnatherum diegonense gr. 0.87 99 Amelichloa caudata 1.00 Amelichloa clandestina Australia and New Zealand Nassella clarazii 0.64 68 68 0.98 Nassella pfisteri 0.57 Nassella neesiana 0.98 100

Nassella trichotoma LEP

1.00 Nassella filiculmis 11 L a d d e r - like like - r e d d a L Romaschenko & al. • Systematics and evolution of needle grasses TAXON — Article version: 9 Dec 2011: 27 pp.

The monophyly of Stipeae is poorly supported (only PP = (A3, PP = 0.81); (2) a strongly supported Jarava s.str. clade 0.76). Most major clades identified in the plastid tree (Fig. 1) (BS = 91, PP = 1.00); (3) a portion of the “Pseudoeriocoma are not detected in the ITS tree (Fig. 2). Only SL3 with poor group” is strongly supported and this includes Achnatherum support (PP = 0.67), SL2 (analysis includes only Patis obtusa), acutum (Swallen) Valdes-Reyna & Barkworth, A. eminens AC (including Pappostipa s.str.) with strong support (BS = (Cav.) Barkworth, and A. multinode (Scribn. ex Beal) Valdes- 94, PP = 1.00), and MAC with poor support (PP = 0.62) are Reyna & Barkworth (BS = 99, PP = 1.00), but this does not found in the ITS tree. Species found in the plastid ESL2 clade include A. constrictum (Hitchc.) Valdes-Reyna & Barkworth are placed into two remote clades in the ITS tree, one with a or A. diegoense (Swallen) Barkworth; and (4) a weakly sup- strongly supported Ptilagrostis s.str. (BS = 96, PP = 100), and ported Amelichloa–Achnatherum diegoense–Nassella clade one with a moderately supported set of the other ESL2 mem- (PP = 0.64). This latter clade includes a weakly supported clade bers (BS = 81, PP = 100) (excluding Othoraphium which aligns of five species of Nassella (BS = 68, PP = 0.98) as sister to the near Patis). Ampelodesmos is sister to a moderately supported weakly supported clade of two species of Amelichloa (strongly Psammochloa-“Neotrinia”-Oryzopsis-Trikeraia clade (BS = supported, BS = 99, PP = 1.00) with Achnatherum diegonense 83, PP = 1.00). The phylogenetic relationships of Psammochloa (BS = 65, PP = 0.87). and “Neotrinia” are strongly supported (BS = 98, PP = 1.00). Testing alternative phylogenetic hypotheses. — There This clade is sister to the Oryzopsis-Trikeraia clade (BS = 57, are four clades that have markedly different relationships based PP = 0.67) wherein Oryzopsis is sister to the monophyletic on the plastid (p, Fig. 1), and nuclear (n, Fig. 2) datasets: (1) Trikeraia (BS = 99, PP = 1.00). In the ITS analysis, in contrast Hesperostipa, (2) Pappostipa, (3) Ptilagrostis s.str., and (4) to the plastid analysis, Hesperostipa (BS = 99, PP = 1.00) is Piptatherum s.str.–Achnatherum stillmanii. We examined the sister to the AC-Pappostipa clade. Piptochaetium (BS = 83, robustness of each of these clades under the constrained alter- PP = 1.00) and Ortachne s.str. (BS = 90, PP = 1.00) are again native topologies. well-resolved. The union of Piptatheropsis with Ptilagrostis (1) In the plastid tree (p) Hesperostipa is placed within kingii as sister appears as in the plastid tree, but with poor ASL2 as sister to the Piptochaetium-Anatherostipa-Lorenzo- support (PP = 0.61). A clade of Aciachne and Anatherostipa chloa-Aciachne clade (hypothesis A); based on ITS (n), Hes- s.l. is weakly supported (BS = 68, PP = 1.00) here and not in perostipa is sister to the Pappostipa-AC clade (hypothesis B). the plastid tree. However, we lack ITS data for Lorenzochloa. The heuristic search using the plastid dataset under the con- The AC clade is strongly supported (BS = 94, PP = 100). straints (to be part of, or sister to, the AC clade as in ITS) indi- There is poor support for Celtica as sister to all the other mem- cates that 21 extra steps (constituting 0.83% of the entire tree bers of AC (BS = 54, PP = 0.99). However, the plastid CAC length) were needed for Hesperostipa to be resolved as sister clade is not resolved in the ITS tree, it collapses into a series to AC. The analysis of distribution of tree length differences of unsupported subclades and a large polytomy. Five notable in Mesquite v.2.6 revealed that only eight extra steps would be relationships well-supported by ITS data and also resolved in needed if the hypothesis of a sister-group relationship between the plastid CAC clade are: (1) a clade of two species of Old Hesperostipa and AC were true. Therefore, hypothesis B was World Piptatherum of sect. Virescentia (BS = 95, PP = 0.99); rejected at P < 0.05, and it was concluded that Hesperostipa (2) a clade of Piptatherum sect. Miliacea species (BS = 100, PP and Pappostipa–AC could not form a clade based on the plas- = 1.00); (3) an Austrostipa-Anemanthele clade (PP = 0.94); (4) tid data. Hypothesis A was tested in the nuclear dataset and a strongly supported clade of some Eurasian Achnatherum A1 the difference between unconstrained and constrained most members (BS = 92, PP = 1.00) (however, other A1 elements, parsimonious trees resulted in 10 extra steps for the nuclear A. calamagrostis, A. turcomanicum Tzvelev, and A. jacque- dataset (0.74%). The analysis of the distribution of tree length montii (Jaub. & Spach) P.C. Kuo & S.L. Lu, are scattered in differences yielded an equal to higher number of extra steps our trees); and (5) a strongly supported Pappostipa clade (BS allowed at P < 0.05 as likely outcome if hypothesis A were true. = 91, PP = 1.00). Therefore, hypothesis A, namely that Hesperostipa belongs to The MAC clade appears intact in the ITS tree (except for ASL (Fig. 1), is accepted. the ejection of Jarava annua), but has poor support (PP = 0.62). (2) Pappostipa was resolved as member of ASL (p) and MAC includes four principal clades placed in a polytomy, and was placed as nested within AC (n). The two hypotheses re- these are similar to the plastid clades: (1) “Eriocoma group” quired 16 (p; 0.63%) and 31 (n; 2.05%) extra steps. The analysis

Fig. . Saw-like lemma epidermal pattern (LEP) found in all lineages of Stipeae with exception of Achnatheroid clade and Pappostipa. A, Stephan achne nigrescens [Miehe 94-547-9, Miehe & Wündisch (Marburg University)]. B, Macrochloa tenacissima [Pyke 701 (BC)]. C, Stipa subsessiliflora [Ivanov s.n. (LE)]. D, Stipa pennata [Romaschenko 466 (BC)]. E, Ampelodesmos mauritanicus [Pyke 702 (BC)]. F, Psammochloa villosa [Safronova 952 (LE)]. G, Achnatherum splendens [Soreng 5121, Peterson, Wang & Zhu (US)]. H, Oryzopsis asperifolia [Saarela 384 (UBC)]. I, Trikeraia pappiformis [Soreng 5653, Peterson & Sun (US)]. J, Trikeraia hookeri [Koelz 2328 (US)]. K, Orthoraphium roylei [Soreng 5261, Peterson & Sun (US)]. L, Ptilagrostis mongholica [Koloskov s.n. (LE)]. M, Ptilagrostis kingii [Peirson 10819 (US)]. N, Piptatheropsis micrantha [Peterson 18437, Saarela & Smith (US)]. O, Lorenzochloa erectifolia [Peterson 14074 & Tovar (US)]. P, Anatherostipa rigidiseta [Beck s.n. (LPB)]. Q, Anatherostipa rosea [Laegaard 10864 (AAU)]. R, Anatherostipa mucronata [Peterson 19551, Soreng, Salariato & Panizza (US)]. S, Aciachne acicularis [Peterson 13931 & Refulio Rodriguez (US)]. T, Hesperostipa spartea [Holmes 214 (US)]. U, Patis coreana [Liou 1085 (US)]. V, Patis obtusa [Soreng 4531 & Kelley (US)]. W, Achnatherum stillmanii [Hoover 4614 (US)]. Y, Piptatherum ferganense [Kamelin 100 (LE)]. cc = cork cell; fc = fundamental cell; mh = macrohair; sb = silica body.

12 TAXON — Article version: 9 Dec 2011: 27 pp. Romaschenko & al. • Systematics and evolution of needle grasses

13 Romaschenko & al. • Systematics and evolution of needle grasses TAXON — Article version: 9 Dec 2011: 27 pp.

of the distribution of tree length differences was expected to be and sometimes have adjacent dorsally compressed CC. The observed more than 0.05% of the time. Thus both hypotheses sidewalls of the FC are thick and deeply sinuous. were rejected. 3. Ptilagrostis SLP (Fig. 3K–N, U, W as non-CC variant) (3) Ptilagrostis s.str. was resolved as member of ESL2 has FC that are of variable length and are 2–7 times longer (p) and was placed as sister to a clade of Patis, Ortachne, Pip- than SC, and often alternate with SC-CC pairs. Silica bodies tatheropsis, Piptochaetium, Anatherostipa, and Aciachne (n). vary in length and are rectangular with straight walls or with The two hypotheses required 13 (p; 0.51%) and 22 (n; 1.47%) 2–5 shallow contractions. The CC are square. The sidewalls extra steps. The analysis of the distribution of tree length dif- of the FC are sinuous, not thickened. Prickles and unicellular ferences was expected to be observed more than 0.05% of the microhairs are often present. These characteristics are found in time. Thus, both hypotheses were rejected. Ptilagrostis s.str., Ptilagrostis kingii, and Piptatheropsis (Fig. (4) Piptatherum s.str.–Achnatherum stillmanii was re- 3L–N). The CC are less abundant within ASL1 than in Ptila- solved as sister to AC (p) and was placed as sister to the re- grostis s.str. Within the Ptilagrostis SLP we recognize three maining Stipeae excluding Macrochloa (n). The two hypoth- variations: Orthoraphium roylei (Fig. 3K) with extremely long eses required nine (p; 0.36%) and 23 (n; 1.5%) extra steps. and irregularly placed SB with multiple (up to five) shallow The analysis of the distribution of tree length differences was contractions, “Stillmania” LEP that resembles Orthoraphium expected to be observed more than 0.05% of the time. Thus, with the sidewalls of the SB almost straight and without con- both hypotheses were rejected for all datasets. strictions (CC not observed), and Patis coreana (Fig. 3U) with Lemma micromorphology. — Lemma epidermal pattern elongated silica bodies often with single deep constriction at (LEP) is diagnosed by several characters: length and shape the mid-point (CC not observed). of the walls of the fundamental cells (FC), length and shape 4. Hesperostipa SLP (Fig. 3T) has FC that are long, with of silica cells (SC), presence or absence of silica bodies (SB), deeply sinuous and thick sidewalls. Prickles and unicellular presence, absence, or frequency of cork cells (CC), presence microhairs are present, and CC and SB were not observed. or absence of prickles (hooks), shape of the prickle base, etc. 5. Aciachne SLP (Fig. 3O, Q–S; including Ortachne, Pe- (Thomasson, 1978; Ellis, 1979; Romaschenko & al., 2008, 2010). ñailillo, 2005) has FC that are long with lobate, thick sidewalls • Saw-like LEP (SLP) pattern is common in Stipeae and and rounded silica bodies sometimes with shallow contractions widespread among grasses outside of this tribe (Fig. 3A–Y; that are 2–6 times shorter than the FC, and CC that are right- Finot & al., 2006; Liu & al., 2010; Peterson, 1989; Romaschenko angled to crescent-shaped. Small prickles are sparse. Apart & al., 2008; Thomasson, 1986; Valdes-Reyna & Hatch, 1991). from Aciachne (Fig. 3S) we also recognize variations of this Features of this type are the presence of elongate (usually very subtype in Anatherostipa mucronata (Griseb.) F. Rojas (Fig. long, i.e., more than 2× longer than wide) FC with sinuate to 3R), A. rosea (Fig. 3Q), and Lorenzochloa (Fig. 3O). The LEP lobate sidewalls (Romaschenko & al., 2008). The FC alternate of A. mucronata differs in having SB of irregular shape, slightly with SC containing SB. Cork cells are usually paired with SC elongate with 1–2 shallow to deep contractions, and CC were and situated adjacent to the proximal end-wall of the SC/SB not observed in A. mucronata. pairs of cells. Within Stipeae we distinguished six major sub- 6. Anatherostipa SLP (Fig. 3P) is found in the remaining types of SLP. species of Anatherostipa s.str. It is distinguished by lemma 1. Macrochloa SLP (Fig. 3B, E–J, V, Y) as a non-CC vari- surfaces densely covered with prickles on the SC. It includes ant has FC of variable length that are 3–7 times longer than SC elongated FC with lobate, thick walls, producing a striate ap- that contain SB, and often alternate with SB–CC pairs. The pearance on the surface that is also densely covered with prick- silica bodies in these species are round or slightly elongated and les above the SC. The SB are irregularly shaped and rare, and the CC are square to crescent-shaped. Prickles (with rounded CC were not observed. prickle base) and unicellular microhairs are usually present and • Maize-like LEP (MLP) is confined to Stipeae. Within Sti- the sidewalls are thick, dentate (Macrochloa, Oryzopsis, Patis peae this LEP is specific to achnatheroid grasses and, with the obtusa, Piptatherum: Fig. 3B, H, V, Y) or lobate (Ampelodes- exception of Pappostipa, is restricted to the AC clade. Species mos, Psammochloa, “Neotrinia”, Trikeraia: Fig. 3E–G, I, J). with this pattern have thin-walled FC that are approximately In Trikeraia the lemma surface appears striate at less than equal in length and width to significantly shorter than wide 10× magnification. This striate pattern is homogeneous within (with few exceptions) with mostly straight sidewalls. The FC the Ampelodesmos-Psammochloa-“Neotrinia”-Trikeraia group are oval, round, square-round, or longitudinally compressed, with the exception of Trikeraia hookeri (Stapf) Bor (Fig. 3J) and all have often square-cornered or sometimes round SB that which has slightly elongated SB (not rounded as in others) and regularly alternate with FC. In the typical syndrome, where SB often has a shallow contraction near the middle. Cork cells were are densely packed and regularly alternate with compressed not observed in Patis obtusa (Fig. 3V). FC, the lemma surface resembles the surface of a fruiting in- 2. Stipa SLP (Fig. 3C, D) is characteristic of the ESL1 florescence (“ear”) of corn (maize). SC-CC pairs are scarce to clade which encompasses the members of Stipa s.str. The FC absent. We distinguished four MLP subtypes, two of which are square to twice as long as wide, of nearly uniform shape occur in a single species. and alternate with SC in a regular (ordered) pattern. All other 1. Celtica MLP (Fig. 4A) is one of the two exceptions SLP subtypes do not have consistent pairing of SC with FC (un- within the MLP that have very long FC (5–10 times longer ordered). The SC are ornamented with square-based prickles than SC) with thick sidewalls. However, the sidewalls of the

14 TAXON — Article version: 9 Dec 2011: 27 pp. Romaschenko & al. • Systematics and evolution of needle grasses

Fig. . Maize-like and ladder-like lemma epidermal patterns (LEP) characteristic of Achnatheroid grasses. A, Celtica gigantea [Pyke 705 (BC)]. B, Stipa capensis [Pyke 703 (US)]. C, Stipa parviflora [Romaschenko 74 & Romo (US)]. D, Piptatherum miliaceum [Gillet 16094 (US)]. E, Ane- manthele lessoniana [Mez 13236 (US)]. F, Austrostipa scabra [Peterson 14442, Soreng & Rosenberg (US)]. G, Achnatherum pubicalyx [Kozlov 124 (LE)]. H, Achnatherum turcomanicum [Goncharov 162 & Grigoriev (LE)]. I, Piptatherum virescens [Romaschenko 445 & Didukh (KW)]. J, Timouria saposhnikovii [Soreng 5475, Peterson & Sun (US)]. K, Achnatherum caragana [Goloskokov s.n. (US)]. L, Achnatherum chinense [Petrov s.n. (LE)]. M, Ptilagrostis pelliotii [Grubov 1815 (LE)]. N, Jarava annua [Peterson 15614 & Soreng (US)]. O, Achnatherum hymenoides [Saarela 205 (UBC)]. P, Achnatherum occidentale [Saarela 594, Sears & Maze (UBC)]. Q, Achnatherum eminens [Peterson 10952 & Annable (US)]. R, Jarava ichu [Peterson 20745, Soreng & Romaschenko (US)]. S, Amelichloa clandestina [Barkworth 5103 (US)]. T. Nassella neesiana [Peterson 10258 & Annable (US)].

15 Romaschenko & al. • Systematics and evolution of needle grasses TAXON — Article version: 9 Dec 2011: 27 pp.

FC are straight as is the common condition in the MLP. The SB In our analyses Phaenospermateae s.l. have moderate sup- are round and CC were not observed. This pattern is somewhat port, and Phaenosperma is nested among Duthieinae genera. transitional between SLP and MLP. Monophyly of Phaenospermateae is also supported by the 2. Miliacea MLP (Fig. 4D) is a thin-walled version of the presence of a unique 21 bp insertion in rpl32-trnL, which is Celtica MLP. The FC are 3–5 times longer than SB, and have absent only in Metcalfia. Since Phaenospermateae are excluded straight sidewalls. The SB are round, very rarely associated from the Meliceae–core Pooideae–Diarrheneae–Stipeae clade with CC, and the CC are crescent-shaped. with strong support, placement of Phaenospermateae and/or 3. Stipella MLP (Fig. 4B) is found only in “Stipella” (Stipa Duthieinae within a broadly defined Stipeae is rejected. capensis). This pattern has large CC that are wider than SB in Lineages and reticulation within Stipeae. — Relation- the adjacent SC. Additionally, the sidewalls of FC are thick and ships within Stipeae have been debated and analyzed over the slightly sinuous. The CC are usually regularly associated with last 50 years (Freitag, 1975, 1985; Tzvelev, 1976; Clayton & SB, which is unusual for achnatheroids (see also S. parviflora Renvoize, 1986; Barkworth & Everett, 1987; Arriaga & Bark- note below in MLP subtype 4). worth, 2006; Barkworth, 2007; Jacobs & al., 2007; Barkworth 4. Achnatherum MLP (Fig. 4C, E–S) is widespread among & al., 2008; Romaschenko & al., 2008, 2010, 2011). Although achnatheroids, and is found in Pappostipa (Romaschenko & the general consensus, prior to Romaschenko & al. (2008, al., 2008). Species in Achnatherum s.str., Austrostipa, Ane- 2010), was that achnatheroid-type morphology, with medium- manthele, “Timouria group”, “Eriocoma group”, “Pseudoerio- sized spikelets with blunt calluses and unspecialized awns, coma group”, Jarava, and Amelichloa have this pattern where gave rise to all other more specialized types of spikelet mor- the CC are scarce but are prevalent in the “Eriocoma group”. phology, our DNA data strongly refute this. Taxa with achnath- The length of the SB varies within each group. A pattern of eroid-type spikelet morphology do not form a monophyletic equally narrow (longitudinally compressed) FC and SB was group, but those with achnatheroid LEP do, and genera with found in the “Pseudo eriocoma group”. Jarava annua (Fig. achnatheroid spikelet morphology and SLP are mostly derived 4N) and Amelichloa (Fig. 4S) share an unusual version of this from genera with a different set of features such as those found MLP subtype by having irregularly shaped SB and variable in Macrochloa. length FC. Stipa parviflora (S. sect. Inaequiglumes (Bor) F.M. Macrochloa is strongly supported as sister to the remain- Vazquez & Devesa) shares a typical MLP and lacks prominent ing Stipeae. It has many peculiar features, such as long, bifid CC and thick FC sidewalls but is distinct from the Stipella MLP prophylls; unique leaf anatomy; short, truncate, velutinous lig- (described above for Stipa capensis). ules; protruding lemma lobes (Vázquez & Barkworth, 2004), • Ladder-like LEP (Fig. 4T) is thought to be unique to Sti- along with an unordered SLP (common among Phaenosper- peae and restricted to Nassella. This pattern includes longitu- mateae elements and in our SL clades). dinally compressed FC and SC that are frequently of similar We have found it useful to divide Stipeae into three groups: size. The SB do not fill the lumen of the SC, thus the FC and (1) Macrochloa with multiple autapomorphic features; (2) SC are sometimes indistinguishable. Both types of cells are clades with SLP, which we refer to as SL lineages; and (3) thin-walled and aligned in regular alternate rows. Cork cells those with MLP or ladder-like LEP, which we call achnatheroid were not observed. grasses. The lemma epidermal patterns in the Macrochloa and SL lineages are mutually exclusive to the pattern found in achnatheroid grasses. Even though the achnatheroid grasses are DISCUSSION monophyletic, it is clear that they are derived at some level from an ancestor with SLP. Phylogenetic relationships of early diverging lineages in The SL lineages, as they appear in the plastid tree (Fig. 1), Pooideae. — The position of tribe Brachyelytreae as sister to do not form a monophyletic group. They resolve in three clades Nardeae plus Lygeeae and the rest of the subfamily Pooideae that diverge in a stepwise order; SL1, SL2, and SL3, with SL3 is confirmed by numerous phylogenetic analyses (Davis & sister to the AC. Even though SL3 is weakly supported, an iden- Soreng, 1993, 2007, 2010; Clark & al., 1995; Catalán & al., tical monophyletic group of SL3 taxa is found in both plastid 1997; Barker & al., 1999; Hilu & al., 1999; Hsiao & al., 1999; and ITS trees, with identical internal branching order at the Soreng & Davis, 2000; GPWG, 2001; Mathews & al., 2002; base. Although SL1 is strongly supported as the sister group to Döring & al., 2007; Soreng & al., 2007; Bouchenak-Khelladi the set of SL2, SL3, and AC, there is only moderate support for & al., 2008; Schneider & al., 2009). Our analysis based on the the sister-group relationship of SL2 to SL3/AC. This significant nine plastid regions confirms the position of Phaenospermateae weakness in the phylogenetic structure among SL lineages is after the separation of Nardeae-Lygeeae, as sister to the re- reiterated by the different, although unsupported, relationships maining Pooideae. These are followed by Meliceae as sister to detected in the ITS analysis (Fig. 2). In the ITS tree SL2 and Diarrheneae–core Pooideae–Stipeae. This scenario does not SL3 are resolved within two successive diverging clades of a contradict the maximum parsimony strict consensus topology non-monophyletic SL1, while Hesperostipa and Pappostipa of Davis & Soreng (2010). All these tribes, and nodes support- are removed from the SL1/ASL1 clade in the plastid tree and ing tribe arrangement, are strongly supported, except for the placed as sister clades within AC in the ITS tree. order of Meliceae as sister to Stipeae plus Diarrheneae-Poeae. The achnatheroid clade, which includes only taxa with The latter pair has low BS support in our trees. maize-like or ladder-like LEP, is monophyletic (strongly

16 TAXON — Article version: 9 Dec 2011: 27 pp. Romaschenko & al. • Systematics and evolution of needle grasses

supported, see Fig. 1), and is comprised of three subclades: (1) additional Achnatherum-like features for S. subsessiliflora, an Austral-Eurasian CAC with some isolated African elements, suggests reticulate or homoplasious origins. Widely divergent (2) an east Asian “Timouria group”, and (3) an exclusively New sequences of ITS (i.e., clones) have been found and these are the World MAC. The stepwise order of SL2 and SL3, as sister to subject of a forthcoming paper (Romaschenko & al., in prep.). AC, and CAC as sister to “Timouria group” plus MAC, are The LEP of S. subsessiliflora is clearly of the saw-like type moderately to strongly supported and geographically consistent (Fig. 3C), in agreement with the placement of this species in with a Eurasian origin of AC in the plastid tree. This geographi- Stipa. Even though the above three species of Stipa are resolved cal arrangement is contradicted by the ITS tree, which suggests in a grade, their sequences differ very little. Since they share the an American origin of AC from the ancestors of Hesperostipa same 7 bp insertion in rpl32-trnL (Fig. 1), evidence of common and Pappostipa. However, comparison of the distribution of origin is supported. A second Stipa s.str. clade, supported by tree length differences between the plastid and ITS trees (see plastid and ITS analyses, comprises S. breviflora as sister to the results) lead us to favor the plastid hypothesis of Hesperostipa strongly supported plastid subclade of five very closely related belonging within SL1 ASL clade. species that share a 7 bp insertion in the rpl32-trnL region and The SLP clade including an ESL and an ASL lineage was an inversion type “1” in the trnH-psbA region (Fig. 1). These detected in our previous study based on five plastid regions five species have been placed in four sections of Stipa accord- (see clades 1 and 2 in Romaschenko & al., 2010). With addi- ing to Tzvelev (1976): sects. Leiostipa (S. capillata), Smirnovia tion of four more plastid regions the SL1 clade is now strongly Tzvelev (S. caucasica), Barbatae (S. barbata, S. brauneri), and supported (Fig. 1). The combination of the Eurasian and Stipa (S. pennata). Within Stipa s.str. it appears that S. bun- American SLs in one clade is not consistent with the topology geana shares ITS characteristics with these five species and obtained from ITS. Despite alternative hypotheses generated shares a plastid type with S. capillacea, indicating possible from different datasets, only inferences based on the plastid additional rounds of reticulation within the genus. analysis had strong bootstrap support. We conclude that the The Ampelodesmos-Psammochloa-“Neotrinia”-Oryzopsis- incongruence between the ITS and plastid tree is perhaps due Trikeraia set (a grade in ESL2, see Fig. 1) all have SLPs, and, to stochastic processes and/or independent reticulation events with one exception, are characterized by stout Eurasian species, in the ITS evolution during the origin of Stipa, Ptilagrostis, usually more than one meter tall that occur in a diverse array Orthoraphium, and Pappostipa, although we have not tested of open habitats (Mediterranean scrub, Mongolian sand-dunes, for this (Humphreys & al., 2010). Within ESL Stipa s.str. is arid steppe, and open mountain slopes of the Tibet-Qinghai pla- strongly supported, but in a narrower sense than previously teau). The single species of Oryzopsis s.str. is exceptional as it understood by taxonomists. We detected two strongly sup- is of moderate stature (Barkworth, 2007) and endemic to North ported clades, which provide the first insight into infrageneric American shady cool temperate forests. Except for Trikeraia structure of Stipa based on molecular data. One clade includes with three species, the other genera are monotypic. All the S. capillacea, S. bungeana (both of S. sect. Leiostipa Dumort.), genera have unusual characteristics. Ampelodesmos, Psammo- and three taxa of disputed placement. These latter three, Stipa chloa, and Trikeraia are rhizomatous (an uncommon charac- purpurea (S. sect. Barbatae Junge), S. subsessiliflora (S. sect. ter in Stipeae) while “Neotrinia” (Achnatherum splendens) Pseudoptilagrostis Tzvelev), and S. regeliana (S. sect. Rege- forms massive tussocks. All members of this group, along with lia Tzvelev), are sometimes referred to different sections of Macrochloa and Celtica, have well-developed lemma lobes Stipa, or included in Ptilagrostis (Roshevits, 1934; Tzvelev, flanking the central awn (lobes setiform in Ampelodesmos 1974, 1977; Freitag, 1985). The monophyly of these three taxa and Trikeraia), and such lobes are consistently small or absent is supported by a 7 bp insertion in rpl32-trnL. The strongly elsewhere in the tribe. Achnatherum splendens was placed in supported clade of the above five species (Fig. 1) is marked by Achnatherum sect. Neotrinia Tzvelev (Tzvelev, 1976). How- a 5 bp insertion in the trnL-F region (unknown for S. capil- ever, molecular data (Romaschenko & al., 2010; Figs. 1–2 of lacea and S. regeliana due to lack of trnL-F sequences). Stipa this paper), and SLP (Fig. 3G) strongly advocate its position purpurea, S. subsessiliflora, and S. regeliana typically pos- among Eurasian SLP lineages. Achnatherum splendens seems sess purplish glumes, relatively short awns, and short glumes most closely related to Psammochloa villosa (Trin.) Bor based with entire apices. Based on these characters Roshevits (1934) on molecular data. placed S. purpurea and S. subsessiliflora in Ptilagrostis. Based Ampelodesmos has the most unusual spikelet features on morphological characters that resemble Ptilagrostis, Tzvelev in Stipeae with multiple florets per spikelet and a prolonged (1976, 1977) and Freitag (1985) placed Stipa subsessiliflora rachilla above the uppermost floret (Stipeae otherwise have in S. sect. Pseudoptilagrostis Tzvelev. In a detailed study of one floret and no rachilla extension, not even a rudiment of anatomical and gross morphological features of Ptilagrostis, one), slender terminal untwisted undifferentiated awns (Stipeae S. subsessiliflora was thought to be closer to Ptilagrostis than awns are often twisted at the base), hairy ovaries (otherwise to Stipa s.str. (Barkworth, 1983). Additionally, a numerical known only in Orthoraphium and Patis), and three lanceolate analysis (Barkworth, 1983) placed S. subsessiliflora between lodicules (2 or 3 in Stipeae) that are dorsally and marginally representatives of Ptilagrostis and Achnatherum (A. cala- ciliate (Stipeae lodicules are glabrous except in Psammo- magrostis, A. caragana). The seemingly transitional nature chloa) and strongly vascularized (Stipeae lodicules are usu- of morphological features between Stipa and Ptilagrostis for ally faintly vascularized, except in Psammochloa). However, S. purpurea, S. regeliana, and S. subsessiliflora, and some unlike other phylogenetically isolated Mediterranean taxa (e.g.,

17 Romaschenko & al. • Systematics and evolution of needle grasses TAXON — Article version: 9 Dec 2011: 27 pp.

Macrochloa, Celtica, and Stipa capensis), Ampelodesmos has their unordered SLP. The Hesperostipa-Piptochaetium-Anath- a strongly supported sister relationship with the Asian pair of erostipa-Aciachne-Lorenzochloa clade was previously detected Psammochloa-“Neotrinia”. Additionally, these three species in phylogenetic studies (Cialdella & al., 2010; Romaschenko & share an inverted loop sequence in the trnH-psbA region of the al., 2010), but had not received strong support until now (Fig. 1). chloroplast genome. It is most parsimonious to assume that the Results of our ITS tree place Hesperostipa outside ASL at the plastid sequence of Psammochloa-“Neotrinia” originated by base of AC. However, the test of statistical significance of the T→A mutation in the already inverted “1i” sequence found in differences in length between constrained and unconstrained the Ampelodesmos plastid, rather than by a separate inversion trees showed a high probability that Hesperostipa should be and two mutations, or by five base changes from uninverted included in ASL. The existing incongruence possibly reflects sequences. Soreng & Davis (2000) suggested Ampelodesmos the high level of homoplasy in our ITS data. As a result, we could be an ancient hybrid between Stipeae and an unknown postulate the correct phylogenetic position for Hesperostipa ancestor, but that remains unproven. is (seen in Fig. 1) within ASL2 as sister to Piptochaetium, The shared geography of the ASL(Fig. 1) lends additional Anatherostipa, Aciachne, and Lorenzochloa. The lemma support to the possible common origin of all the American morphology of Hesperostipa corresponds well to what was taxa with SLPs, except Oryzopsis. However, the crown node found in Berriochloa. The LEPs of H. comata (Trin. & Rupr.) is weakly supported and it splits into three clades: (1) Pip- Barkworth, H. neomexicana (Thurb.) Barkworth (Thomasson, tatheropsis plus Ptilagrostis kingii, (2) Ortachne-Pappostipa, 1978), and H. spartea (Trin.) Barkworth (Fig. 3T) look similar and (3) Hesperostipa-Piptochaetium-Anatherostipa-Aciachne- to those found in Berriochloa minuta Elias and B. maxima Elias Lorenzochloa. There is no support for the joining of clades 1 (Thomasson, 1978). Thomasson (1978) described the lineage and 2 (above) as sisters in ASL1 (Fig. 1) but ASL2 has strong of Berriochloa-Hesperostipa-Piptochaetium as having LEPs support. The following is a detailed discussion of each of these with fundamental cells (FC) with deeply sinuous sides and clades. end-walls, lacking silica bodies (SB) and cork cells (CC), and (1) The North American genus Piptatheropsis, as pre- he estimated the time of separation of Hesperostipa and Pip- viously depicted in Romaschenko & al. (2010, 2011), is only tochaetium to be late Pliocene. According to our data the SB/ distantly related to the Asian Piptatherum (Fig. 1). The sister CC pairs are common in SLP lineages (Fig. 3B, E–J, L, Y), to Piptatheropsis is Ptilagrostis kingii (weakly supported in outside of Stipeae (Fig. 3A), and are normally present in the the plastid tree), a North American species with transitional Piptatheropsis–Ptilagrostis kingii clade (Fig. 3M–N). This features between Piptatheropsis and Ptilagrostis (Barkworth, suggests that the Berriochloa-Hesperostipa-Piptochaetium lin- 1983), but we have no evidence that it is directly related to Asian eage (where short cells are absent) is a derivative of an ancestral Ptilagrostis s.str. The only other North American species of extinct LEP where both types of short cells (silica and cork) Ptilagrostis, P. porteri (Rydb.) W.A. Weber (not included in evolved, and from which Piptatheropsis, Ptilagrostis kingii, the present study) has a more complicated history and will be and Ortachne are scarcely modified. Another piece of evi- the subject of a forthcoming paper. Piptatheropsis–Ptilagrostis dence is that the sister to Piptochaetium is the Anatherostipa- kingii are characterized by having the longest stem in the hair- Aciachne-Lorenzochloa clade, where scarce SB were found pin formation in the trnH-psbA region among Stipeae. This pro- in Anatherostipa mucronata and SB/CC pairs are present in vides some evidence for independent evolution of this group. Aciachne (Fig. 3R–S), representing stages of this loss. (2) The Ortachne-Pappostipa sister relationship was A “pappose” subset of Anatherostipa species (A. rosea, strongly supported in the plastid analysis (Fig. 1). However, A. hans-meyeri) is characterized by the presence of long hairs this relationship is difficult to reconcile since these species at the apex of the lemma, abundant SBs (Fig. 3Q), and a LEP have little in common in their habits, chromosome numbers, that resembles Lorenzochloa and Ortachne. Our plastid tree and LEPs. Pappostipa species have MLP (Romaschenko & clearly separates the A. rosea–A. hans-meyeri–Lorenzochloa al., 2008) while Ortachne has an unordered SLP (Peñailillo, clade from the remaining Anatherostipa s.str., which aligned 2005) similar to that of Piptatheropsis (Fig. 3N), Ptilagrostis with Aciachne in a strongly supported subclade (Fig. 1). The kingii (Fig. 3M), Aciachne (Fig. 3S) and some species of Anath- latter subclade is marked by single bp insertion in rps16-trnK, erostipa (Fig. 3Q–R). The Pappostipa clade was nested within, while the internal clade of remaining Anatherostipa s.str. is or sister to the AC in the ITS tree (Fig. 2). The probabilities of marked by a 20 bp insertion in the same region (Fig. 1). Such alternative placement of Pappostipa within ASL in the ITS tree evidence reveals that Anatherostipa, as currently understood was statistically insignificant. The LEP type being incompat- (Peñailillo, 1996), is polyphyletic. All plastid analyses, even ible with that of other ASL genera and alternative phylogenetic when we excluded Pappostipa as putative conflicting taxon placements in plastid and ITS trees indicates that Pappostipa (results not shown), resolve Ortachne s.str. as an isolated lin- arose from an ancient allopolyploid hybridization event. eage with no support for sister relationships between it and (3) Some shared features of the Hesperostipa-Pipto- other ASL clades (other than Pappostipa). chaetium-Anatherostipa-Aciachne-Lorenzochloa clade have Ptilagrostis and Patis are placed as potential sisters to been reported in Hesperostipa, Anatherostipa, Piptochae- the main body of ASL in the ITS analyses. A character that tium, and the Miocene fossil genus Berriochloa (Thomasson, Ptilagrostis shares with ASL is a chromosome base number 1978; Barkworth & Everett, 1987; Peñailillo, 2005). These of x = 11, but this is common among American SLP species include paleas with projecting keel apices and similarities in (Barkworth, 1983).

18 TAXON — Article version: 9 Dec 2011: 27 pp. Romaschenko & al. • Systematics and evolution of needle grasses

The SLP lineage 2 (SL2), i.e., Patis, is a newly detected other morphological features used to broadly circumscribe Pip- strongly supported clade that includes two far East Asian, stout tatherum (Roshevitz, 1951; Freitag, 1975; Tzvelev, 1976; Dorn, (to 1 m tall), broad- and flat-leaved species, Patis coreana and 2001; Soreng, & al., 2003; Wu & Phillips, 2006; Barkworth, P. obtusa. The distribution of Patis coreana and P. obtusa & al., 2008), such as small anthoecia, dark, often glossy and overlap in the mesic temperate forests of eastern China (area coriaceous lemmas, and well-exposed paleas, are found in Pa- of Hubei, Shaanxi, and Zhejiang provinces; Ohwi, 1941, 1942; tis, Piptatheropsis, and various Eurasian taxa including species Wu & Philips, 2006), and occurs outside the ecological ranges of Piptatherum sect. Miliacea (P. miliaceum, P. thomasii) and of all but one other Asian Stipeae species, Achnatherum pe- sect. Virescentia (P. virescens, P. paradoxum). These features kinense (including A. extremorientale (H. Hara) Keng). Patis all appear to result from convergence (Romaschenko & al., coreana and P. obtusa share a 5 bp insertion in the rps16 intron 2010, 2011). (Fig. 1) and these two species were recognized as being anoma- The AC is strongly supported in the phylogenetic analyses lous within their former respective genera (Wu & Phillips, (Figs. 1–2) and is marked by a unique 8 bp deletion in the rpl32- 2006). Patis coreana was originally described in Stipa and trnL region. This terminal clade differs from Macrochloa and has been placed in Achnatherum and Orthoraphium by Ohwi the SLP clades by having a distinct MLP with shortened FC (1941, 1953). These two species of Patis apparently represent with straight to slightly sinuate side-walls. a distinct and isolated lineage since they do not align in our The CAC was detected in previous studies (Romaschenko phylogenetic analysis with Stipa, Orthoraphium, Piptatherum, & al., 2008, 2010) but without statistical support. In the cur- or Achnatherum (Romaschenko & al., 2011). The LEP of Pa- rent analysis it received moderate bootstrap support in the tis coreana is clearly of the saw-like type (Fig. 3U), whereas plastid tree (Fig. 1). Within CAC the monotypic Mediterra- Patis obtusa, differs in having rounded SB resembling those nean genus Celtica (Vázquez & Barkworth, 2004) is sister to of Piptatherum s.str. (Fig. 3Y) except that the SB/CC pairs are the remainder, which includes clades of: Stipa capensis and scarce, and side-walls of the FC are thicker. A third species, S. parviflora, Anemanthele and Austrostipa, Eurasian “Mili- Patis racemosa (Sm.) Romasch. & al. from North America, acea group” (Piptatherum miliaceum, P. thomasii), and Eur- has been transferred into the genus (Romaschenko & al., 2011). asian Achnatherum (A1). The LEP of Celtica is unusual among Patis coreana and P. obtusa have prominent transverse veinlets achnatheroids because it has long FC (Fig. 4A). However, the between the longitudinal veins of the glumes (these are visible side-walls of the FC are straight which is in agreement with but faint in P. racemosa), and all three species have wide, long the LEP concept described for achnatheroid grasses, but it may flag-leaf blades and underdeveloped or absent basal leaf-blades represent the transitional form between saw-like and maize- (i.e., they have cataphylls). like LEPs. Assuming x = 12 is the base chromosome number, The SLP lineage 3 (SL3) or Piptatherum-“Stillmania” is Celtica is an octoploid (2n = 96), which is the highest known weakly supported in the plastid tree (Fig. 1) and the ITS tree ploidy level in Stipeae (also found in Ampelodesmos). (Fig. 2). In each tree “Stillmania” is sister to a strongly sup- Two other Mediterranean taxa, Stipa capensis and S. parvi- ported Piptatherum (2). The phylogenetic position of SL3 is flora, were placed at the base of Achnatherum (A1) plus the contradictory among the three analyses. In the plastid analysis “Miliacea group”, but without support in the plastid analysis it is rendered as sister to the AC and in the ITS tree it is placed (Fig. 1). Tzvelev (1976) placed S. capensis in a monotypic Stipa as sister to Ampelodesmos-Psammochloa-Oryzopsis-Trikeraia. sect. Stipella and Freitag (1985) added S. parviflora to the sec- The LEP of “Stillmania” (Fig. 3W) is of the saw-like type and tion based on these two taxa having an unusually short palea. resembles that of Ptilagrostis (Fig. 3L; Barkworth, 1983) and Vázquez & Devesa (1996) accepted the placement of S. capen- Piptatherum (Fig. 3Y), but differs by having SBs of irregular sis, but placed S. parviflora in another monotypic Stipa sect. length and alternation, and the lack of associated cork cells Inaequiglumes. Our phylogenetic and LEP evidence indicates (CC). The provisional name “Stillmania” is used to repre- that both taxa should be removed from Stipa and treated as sent Achnatherum stillmanii from the western part of North members of the AC clade. The LEPs of S. capensis and S. parvi- America. It was once placed in Ptilagrostis by Elias (1942), flora are different; S. parviflora is typical maize-like (Fig. 4C) although Barkworth (1983) provided morphological and nu- and S. capensis is unique among achnatheroids because it has merical evidence for placing it in Achnatherum. It shares some enlarged cork cells often associated with silica cells/bodies (Fig. morphological features with Achnatherum, but differs by not 4B). The ITS tree did not resolve these taxa as a pair and there having a MLP, possessing notched paleas with extended keels, was only low support for their phylogenetic isolation. Thus, the and prominent lemma lobes. phylogenetic placements of S. capensis (2n = 18, 26, 34, 36) and The Piptatherum s.str. clade received strong support in all S. parviflora (2n = 28) remain somewhat ambiguous. phylogenetic analyses (Figs. 1–2) (Romaschenko & al., 2010). The “Miliacea group” consists of two closely related species Members of this clade share a 10 bp deletion in the trnL-F (Piptatherum miliaceum, P. thomasii) that were placed in Pip- region. Morphological characters supporting the Piptatherum tatherum (Roshevits, 1951; Freitag, 1975; Tzvelev, 1976) after s.str. clade are dorsally compressed spikelets, linear disarticu- the application of the name Oryzopsis to non-North American lation scars, and basally unfused and well-separated lemma taxa was discontinued (Romaschenko & al., 2011). This group margins (Romaschenko & al., 2010). This combination of char- shares a chromosome number of 2n = 24 with the Piptatherum acteristics is consistent throughout this clade and do not appear s.str. clade and Achnatherum (A1). However, the set of morpho- in Piptatheropsis, Patis, or the “Miliacea group”. Historically, logical characters outlined for Piptatherum s.str. in this study is

19 Romaschenko & al. • Systematics and evolution of needle grasses TAXON — Article version: 9 Dec 2011: 27 pp.

not consistent with that of the “Miliacea group”. The lemmas of Our current analysis confirms the position of Piptatherum P. miliaceum and P. thomasii are not coriaceous, the disarticula- virescens and P. paradoxum (Piptatherum sect. Virescentia) in tion scars are circular (transversally elliptic in Piptatheropsis), CAC (Romaschenko & al., 2010). These taxa share the same and the lemma borders are basally fused (pers. obs.). The LEP LEP as the majority of the other members of AC (Fig. 4I) and of the“Miliacea group” represents a third unique type in the align with A. bromoides (sect. Aristella) in the plastid analysis achnatheroid clade (Fig. 4D), which together with phylogenetic with moderate support or without support in the ITS tree. The evidence and other morphological differences support the isola- sister relationship of A. bromoides and P. sect. Virescentia is tion of this group from Piptatherum and Achnatherum. more plausible since the taxa share dark and coriaceous lem- Achnatherum as broadly circumscribed (our A1 to A4, mas with a blunt callus, persistent awns, and similar habitats. “Neotrinia”, and “Stillmania”) includes 35 North American spe- Achnatherum bromoides, P. paradoxum, and P. virescens have a cies, about 20 species from Eurasia, and one from New Zealand. somewhat specialized lemma form that resembles that found in Following Tzvelev (1976), the species currently accepted by the Piptatherum s.str., Piptatheropsis, and the “Miliacea group”. The Flora of China in Achnatherum (Wu & Philips, 2006) are attrib- evident polyphyly of Achnatherum s.l. and Piptatherum s.l. rein- utable to five general groups with the following characteristics: forces the suggestion of Thomasson (1980: 235) that “similarities (1) callus short, often obtuse; awn straight or flexuous, articu- among taxa in the shape of the anthoecia are not in themselves lated; lemma membranous with apical lobes (Achnatherum sect. sufficient to interpret phylogenetic relationships in Stipeae”. Achnatherum; Stipa sect. Lasiagrostis Steud., type: A. cala- The monophyly of the Austrostipa-Anemanthele group is magrostis—Freitag, 1985; Vázquez & Devesa, 1996); (2) callus weakly supported in the plastid and ITS trees (Figs. 1–2). The short, often obtuse; awn straight; lemma at maturity becoming crown node is marked by a 5 bp insertion in the rpL32-trnL re- dark and coriaceous, with apical lobes, only marginally cover- gion. In the ITS tree Anemanthele is embedded within Austro- ing the palea (Achnatherum sect. Aristella (Trin.) Tzvelev, type: stipa, consistent with results of Jacobs & al. (2000). However, A. bromoides (L.) P. Beauv.—Tzvelev, 1976; Stipa [unranked] in our plastid tree Anemanthele is sister to Austrostipa with Aristella Trin.; Stipa sect. Aristella (Trin.) Steud.—Freitag, weak support. The subgeneric structure within Austrostipa 1985; Vázquez & Devesa, 1996); (3) callus conspicuous, of- is poorly represented in the study. Of the several recovered ten acute; awn once or twice geniculate; lemma at maturity clades only two represent separate subgenera: Austrostipa subg. becoming dark and coriaceous, with absent or inconspicuous Longiaristatae S.W.L. Jacobs & J. Everett (1996; A. compressa lobes, and completely covering the palea (Achnatherum sect. (R. Br.) S.W.L. Jacobs & J. Everett and A. macalpinei (Reader) Achnatheropsis (Tzvelev) Prob., type: A. sibiricum—Freitag, S.W.L. Jacobs & J. Everett), and Austrostipa subg. Austrostipa 1985; Stipa sect. Achnatheropsis Tzvelev—Freitag, 1985; (A. campilachne (Nees) S.W.L. Jacobs & J. Everett, A. semi- Tzvelev, 1976; Achnatherum sect. Protostipa Tzvel.; Stipa ser. barbata (R. Br.) S.W.L. Jacobs & J. Everett, and A. hemipogon Sibiricae Roshev.—Bor, 1970); (4) callus short, obtuse; awn (Benth.) S.W.L. Jacobs & J. Everett). The most densely sam- straight, caducous; lemma pilose, membranous, covering the pled subgenus, Austrostipa subg. Falcatae S.W.L. Jacobs & palea by 2/3, with apical lobes; palea minutely protruding above J. Everett (1996; A. scabra (Lindl.) S.W.L. Jacobs & J. Everett, the lemma apex (Achnatherum sect. Neotrinia, type: A. splen- A. nitida (Summerh. & C.E. Hubb.) S.W.L. Jacobs & J. Everett, dens—Tzvelev, 1976); and (5) callus short, obtuse; awn straight, A. nodosa (S.T. Blake) S.W.L. Jacobs & J. Everett, A. tenui- caducous; lemma pilose, membranous, covering most of the folia (Steud.) S.W.L. Jacobs & J. Everett, and A. trichophylla palea, with apical lobes; palea shorter than lemma (Timouria, (Benth.) S.W.L. Jacobs & J. Everett), was polyphyletic in all type: T. saposhnikowii—Roshevitz, 1916]. our analyses. Based on our phylogenetic trees and the peculiar Our molecular study provides strong evidence for Ach- morphological types found in Austrostipa-Anemanthele, this natherum sect. Achnatheropsis being a natural group. It in- strictly Australasian group presumably shares a common an- cludes Achnatherum sibiricum, A. confusum, A. pekinense, and cestor with Asian achnatheroids, both sharing the MLP (Fig. A. brandisii (Roshevits, 1934; Bor, 1970; Tzvelev, 1976, 1977; 4E–I) and general growth habit. Freitag, 1985), along with A. pubicalyx and A. inebrians that The “Timouria group” (A2) received strong support in have somewhat transitional features between this section and our plastid tree (Romaschenko & al., 2010). It encompasses A. sect. Aristella. The Achnatherum-A1 clade (including the Timouria, which was first described as a monotypic genus (Ro- type of Achnatherum) received strong support in the ITS tree shevits, 1916) and then included in a monotypic section of Ach- and little support in the plastid tree (PP = 0.91) (Figs. 1–2). natherum (as A. saposhnikovii; Tzvelev, 1976). In our plastid The crown node within this group is marked by two plastid tree the “Timouria group” included Achnatherum caragana and indels, a 16 bp insertion in rps16-trnK and a 5 bp deletion in A. chinense, which were aligned by Tzvelev (1976) and Frei- rpl32-trnL (Fig. 1). The other members of Achnatherum s.l. tag (1985), respectively, in Achnatherum sect. Neotrinia and resolved as groups outside of CAC, within A2, A3, and A4 Stipa sect. Lasiagrostis, and also Ptilagrostis pelliotii. Despite (“Eriocoma”, “Pseudoeriocoma”, and “Timouria groups”) or as differences in gross morphology among its members they all isolated species in SL1 and SL3 (“Neotrinia” and “Stillmania” share rather small (3–4 mm long), elliptic, 2-toothed, pubescent lineages). More sampling in Achnatherum is needed to deter- lemmas with caducous awns, 3-veined glumes, and setaceous mine whether species with membranous lemmas (Achnatherum leaf blades. The LEP in the “Timouria group” is maize-like, sect. Achnatherum) and species with coriaceous lemmas (sect. often with extremely short fundamental cells (Fig. 4G–M). Aristella) represent natural groups. Molecular evidence and LEP place Ptilagrostis pelliotii within

20 TAXON — Article version: 9 Dec 2011: 27 pp. Romaschenko & al. • Systematics and evolution of needle grasses

AC, far removed from the SL1 Ptilagrostis clade. The well- the number of species allied to the “Pseudoeriocoma group” is developed awn indumentum in P. pelliotii, approaching that of uncertain. In the ITS tree (Fig. 2) a smaller strongly supported “true” Ptilagrostis, could be a result of convergent evolution. “Pseudoeriocoma group” of A. acutum, A. eminens, and A. mul- Despite morphological similarities to members of Ach- tinode was sister to Jarava plumosula–J. media–A. constrictum; natherum (A1), the “Timouria group” represents an independent these two Jarava species were formerly placed in Stipa subg. branch of Asian achnatheroid grasses with caducous awns and Ptilostipa (Spegazzini, 1901; Roig, 1964). Jarava as broadly compressed, elliptic florets, whereas other Asian achnatheroids applied is obviously polyphyletic in our analyses, but separation have persistent awns and terete, fusiform florets. The unusual of plumose and long-awned sect. Ptilostipa from other Jarava characteristics of the “Timouria group” led Roshevits (1951) to s.str. is consistent with our previous analyses (Romaschenko & describe subtribe Timouriinae to join smallest-spikeleted Asian al., 2008, 2010) where larger sets of Jarava and Nassella taxa and American genera, including Eriocoma (based on E. hymen- were included. The separation of Jarava plumosula and J. me- oides (Roem. & Schult.) Rydb.), Piptatherum, Oryzopsis, as dia (Stipa sect. Ptilostipa) from the “Pseudoeriocoma group” well as Piptochaetium and Nassella in a narrow sense (Parodi, is consistent with the taxonomic distribution of relatively long 1944, 1947). According to our plastid tree the “Timouria group” (not less than half the length of the lemma) and hairy paleas, shares a common ancestor with MAC, which includes some of with two well-developed veins, a characteristic that these groups the largest genera in American Stipeae, such as the “Eriocoma share with the “Eriocoma group”. In contrast, in Jarava s.str. group” (Achnatherum-A3), Jarava, and Nassella, with 29, 37, and Nassella the paleas are relatively short, glabrous or scarcely and 115 species, respectively. hairy, and the veins are faint or absent. The origin of the ladder- The MAC was detected in previous phylogenetic analyses like LEP in Nassella is apparently derived from MLP ancestors (Romaschenko & al., 2008, 2010, 2011), and received strong which is found in species of Jarava, the “Timouria group” (A2), support in our plastid tree. The monophyly of MAC is also sup- the “Eriocoma group” (A3), and the “Pseudoeriocoma group” ported by base mutation “6” in the loop sequence in trnH-psbA (A4) (Romaschenko & al., 2010). region. The first split within MAC separates: (1) “Eriocoma According to the current dataset the sister-group relation- group” (Achnatherum-A3) which includes the majority of North ships between Jarava s.str. and Nassella remain unsupported. American Achnatherum species, from (2) an assemblage of In previous phylogenetic analyses the recently described Ameli- Jarava, “Pseudoeriocoma group” (Achnatherum-A4), Nassella, chloa (Arriaga & Barkworth, 2006) was found to be embedded and Amelichloa. Ignoring Jarava annua for the moment, these within Nassella (Barkworth & al., 2008; Romaschenko & al., can be considered sister groups and are strongly supported in 2008, 2010), or sister to Nassella as in our present analysis using our plastid tree. a smaller dataset (Figs. 1–2). However, these relationships are In the “Eriocoma group” (A3) A. wallowaense J. Maze & not consistent with the LEP pattern where Nassella has funda- K.A. Robson, A. hendersonii (Vasey) Barkworth, and A. hy- mental and silica cells that are compressed and undifferentiated menoides have short, elliptic, slightly laterally compressed forming a ladder-like LEP (Fig. 4T) and Amelichloa has MLP florets, and lemmas with caducous awns (a Timouria-like char- with well developed silica bodies and associated cork cells (Fig. acter according to Roshevits, 1951). These three taxa form a 4S). Species of Amelichloa have relatively long and hairy paleas grade leading to other “Eriocoma group” taxa that have terete with well-developed veins, features shared with members of and fusiform florets and lemmas with persistent awns (Fig. 1). the “Eriocoma” and “Pseudoeriocoma groups”, and Stipa sect. The overall morphological pattern of fusiform florets in the Ptilostipa. Along with Pappostipa, the phylogenetic affiliation “Eriocoma group” is similar to that in some species in the Asian of Amelichloa represents one of the remaining puzzles in the Achnatherum (A1) clade, especially to those taxa in A. sect. evolutionary history of Stipeae, where the molecular evidence Achnatheropsis that have a crown of hairs at the distal end of is at variance with established morphological patterns. the lemma surrounding the base of the awn. This similarity Lemma epidermal pattern polarized by the molecular lead Barkworth (1993) to transfer American species formerly data. — The utility of lemma epidermal patterns for phylo- placed in Stipa (Hitchcock, 1925a, b) to Achnatherum. genetic studies of Stipeae as postulated by Tzvelev (1977), Another group of five species within MAC (Achnatherum Thomasson (1978, 1980, 1982, 1985), and Barkworth & Everett diegonense, A. constrictum, A. eminens, A. multinode, A. acu- (1987), has never been questioned. In contrast, many gross mor- tum) appears to have a separate evolutionary history, and is called phological floret characters traditionally used in determining the “Pseudoeriocoma group” (A4). In both trees the “Pseudoerio- taxonomic or phylogenetic inferences in this tribe (i.e., length coma group” aligns in a subclade with Jarava and Nassella and indumentum of the awn; size and shape of the lemma; and rather than with the “Eriocoma group” (Figs. 1–2). This group- sharpness of the callus; Freitag, 1975, 1985; Tzvelev, 1975), evi- ing was detected by Barkworth & al. (2008) based on a dataset dently have evolved independently many times within Stipeae. which included ITS and plastid data. In our study this group is Our results reinforce these suggestions since these adaptive moderately supported in our plastid tree (Fig. 1), but in our ITS traits are not consistent within any recovered clades but are tree (Fig. 2) A. constrictum is aligned in a grade with Jarava, and found within many lineages. A. diegonense is sister to Amelichloa caudata–A. clandestina. One test of our phylogenetic hypotheses is to compare the These results could be attributed to homoplasy within the ITS distribution of phylogenetically conservative morphological tree, or to past hybridization events. Since not all of the species and anatomical characteristics. In Poaceae many characteristics of American Achnatherum were employed in our current study, involved in adaptations to dispersal and seedling establishment

21 Romaschenko & al. • Systematics and evolution of needle grasses TAXON — Article version: 9 Dec 2011: 27 pp.

are relatively prone to parallelism, convergence, and even re- Biogeography. — Despite the deep history of unambigu- versal. For example, awns and points of insertion of awns, ous Stipeae fossils in the Miocene, and possible Stipeae fossils pubescence patterns, number of florets per spikelet, size of flo- from the mid-Oligocene of North America (Elias, 1942; Gal- rets, callus shape, elongated rhizomes and stolons, and annual breath, 1974; Thomasson, 1978, 1980, 1987, 2005), the biogeo- life-cycle have all evolved repeatedly and, in many instances, graphical history within Stipeae remains difficult to interpret. reversed (Barkworth & Everett, 1987; Davidse, 1987; Soreng & However, the plastid tree (Fig. 1) provides some clues. We are al., 2007). However, we have found evolutionarily conservative reasonably confident that Macrochloa is sister to the rest of anatomical characteristics that are consistent with molecular the extant elements of Stipeae. Today Macrochloa consists of phylogenetic hypotheses and these support our interpretations. a single Mediterranean species, but the nearest relatives of Sti- The phylogenetic trees presented in this paper effectively polar- peae come from all over the world so extinction in other regions ize the distribution of lemma epidermal characteristics (Figs. may have left us only this one Mediterranean element. Several 1–2). We provide evidence that the SLP with long, often deeply clades within Stipeae are evidently geographically pure. The sinuate fundamental cells is the ancestral state in Stipeae. In ASL1 and MAC clades are strictly New World. CAC is strictly its general form this same LEP is common outside of Stipeae Old World with a probable expansion into Australasia and (Thomasson, 1986; Peterson, 1989; Valdes-Reyna & Hatch, Africa. The “Timouria group” is strictly South-East Asian. As 1991; Columbus, 1996; Snow, 1996; Finot & al., 2006; Liu & for the SL1/ESL clade, the North American monotypic genus al., 2010) while MLP and ladder-like LEP are not otherwise Oryzopsis belies the otherwise strictly Eurasian nature of the known within Pooideae. Within Stipeae (ignoring Pappostipa clade. As for the SL3 clade, Achnatherum stillmanii of North for the moment) the SLP is present in Macrochloa, SL1, SL2, America is sister to a strictly Eurasian group of Piptatherum and SL3 (Fig. 1) up to the point of divergence of the AC where s.str. At a minimum, if we interpret Fig. 1 at face value, starting it is replaced by the MLP with often short fundamental cells with Macrochloa in the Mediterranean, there are four evident and abundant silica bodies. Within AC there are no known dispersal events into the New World, and one to Australasia. reversals to the ancestral condition. For the reverse direction, of Stipeae dispersal to the Old World No dataset examined so far has provided evidence for a from the New World, we must postulate at least six dispersal single, initial bifurcation between Stipeae with SLP grasses events. However, along the backbone of the tree (Fig. 1) there (SL1, SL2, SL3; Fig. 3A–Y) and the AC, which possess MLP are few unambiguous directions of dispersal or patterns of (Fig. 4A–T). More likely, three to six lineages with SLP di- vicariance, particularly as there is only weak support at several verged independently before the origin of the AC clade. Stipa pivotal nodes. s.l., Piptatherum s.l., and Achnatherum s.l. were revealed in the Chromosome numbers. — There is much uncertainty current study as polyphyletic based on molecular data and their about the pleisomorphic base chromosome number in Pooideae. LEP. Only a few species in these three genera are evidently mis- Outside of Stipeae, Brachyelytrum has a base chromosome classified as evidenced by LEP types, and we have tentatively number of x = 11 (2n = 22), Nardus of x = 13 (2n = 26), and provided new names or group names on the trees to identify Lygeum of x = 10 (2n = 40; see Appendix 3 for list of chromo- these. Stipa pennata (type) and Achnatherum calamagrostis some numbers). Within Phaenospermateae (sensu Schneider (type) reside firmly in the SLP and the MLP, respectively. Pri- & al., 2009), counts of x = 7 (2n = 14) have been reported for mary stepwise divergence of several SLP lineages leading to Danthoniastrum and Duthiea (Kozuharov & Petrova, 1991; the divergence of the AC is marked by newly acquired features Watson & Dallwitz, 1992) and x = 12 (2n = 24) is known for of the lemma and this polarizes the LEP distribution in Stipeae Phaenosperma and Stephanachne. Meliceae have base chromo- in such a way that the achnatheroid grasses are clearly derived some numbers of x = 9, 10, and perhaps 12. The base number from among the SLP lineages. is ambiguous for Diarrheneae where 2n numbers are [36?], 38, Within the AC a few achnatheroid taxa vary in their MLP, and 60, and x might be 5, 6, [9?] 10, or 19. Only in the tribes and these can be interpreted as transitional from unordered Bromeae, Triticeae, and Poeae s.l. (the core Pooideae) the base SLP to the typical and more common MLP. For instance, in chromosome number is obviously x = 7. Celtica the fundamental cells are long, and have thick walls but Chromosome numbers have been reported for more than they are considered to represent MLP since the fundamental 120 species of Stipeae, 86 of which we included in our study cells’ walls are straight as in other species in AC. The “Mili- (see Appendix 3). Of these 120 about 26 appear to be diploid acea group” (Piptatherum miliaceum, P. thomasii) is similar with possible base numbers of x = 7, 8, 9, 10, 11, or 12. De- to Celtica but has thin walls. In Stipa capensis (“Stipella”) the spite the high frequency of polyploid series in Stipeae three fundamental cells are somewhat sinuous-walled and thick but hypotheses have been suggested for their base chromosome are short as in the MLP. number: x = 7 (Tzvelev, 1977), x = 12 (Avdulov, 1931), and x Within AC a highly modified ladder-like LEP is found = 11 (Clayton & Renvoize, 1986; Hilu, 2004). The possibility only in species of Nassella wherein the silica cells are lacking that one of these numbers might be basal for the entire Poaceae silica bodies and are almost indistinguishable from the longi- has also been thoroughly discussed (Avdulov, 1931; De Wet, tudinally highly compressed fundamental cells. Since the clade 1987; Hilu, 2004; Hubbard, 1948; Hunziker & Stebbins, 1987; of Nassella is deeply nested within AC and all other branches Raven, 1975; Stebbins, 1982, 1985). have MLP it is apparent that the Nasselloid ladder-like LEP is The evidence for x = 7 being the original base chromosome derived from the MLP. number in Stipeae is weak, and is contradicted by the presence

22 TAXON — Article version: 9 Dec 2011: 27 pp. Romaschenko & al. • Systematics and evolution of needle grasses

of this possible base number in only three highly derived lin- Recent studies have provided new insight into the evolu- eages of our plastid tree. Eleven counts possibly based on x = tion of the grass genome (Paterson & al., 2004; Salse & al., 7 are present in AC (CAC and Nassella) and in Piptochaetium; 2004, 2008, 2009; Wei & al., 2007), including the facility of all of these are polyploid, and five are 2n = 28 (tetraploid). certain chromosomes to fuse and possibly split. These studies Chromosome numbers in most other Stipeae represent illustrate that earlier karyological studies can often be usefully different polyploid series based on x = 12 or x = 11. A base interpreted in light of a molecular phylogeny (Levin, 2002). number of x = 12 is the most common one among diploids in Given the data we have, x = 12 seems more likely to be the base Stipeae, occurring in AC and in all SL lineages. In our dataset chromosome number for Stipeae, but x = 11 cannot be ruled out. 2n = 24 is recorded for Old World Achnatherum (CAC; A1) and Piptatheropsis, Piptatherum s.str., “Miliacea” and “Virescens groups”. Despite this, there are few polyploids that are inter- CONCLUSION pretable as being based on x = 12. Several taxa with 2n = 48 are most likely tetraploid, but most of the other taxa are more Based on analyses presented in this paper and on unpub- comfortably interpreted as tetraploid based on x = 9 (at 2n = lished data 33 groups that we envision to represent genera within 36), rather than as triploids based on x = 12. Stipeae are listed in Table 1. Eleven of these groups currently A base of x = 11 also occurs widely among diploids, oc- are monotypic: Ampelodesmos, Anemanthele, Celtica, “Inaequi- curring in Aciachne, Piptatheropsis, and Ptilagrostis of the glumes”, Macrochloa, “Neotrinia”, Oryzopsis, Orthoraphium, SL lineages. Several studies (Johnson, 1945; Probatova & Psammochloa, “Stillmania”, and “Stipella”. Thirteen of these Sokolovskaya, 1980) confirmed the presence of a stable 2n = generic groups are distributed in North America, ten are found 22 karyotype in Stipeae that spans almost the entire SL1 clade in Asia, nine are in South America, and seven are in Europe. including its Eurasian (ESL) and American (ASL) subclades. Stipa still might be the largest genus with between 110 and In Stipa s.str. 45 species have counts of 2n = 44 (tetraploid). 232 species (depending on how finely the species are divided) Fifteen other diploid and polyploid taxa of SL and AC lineages distributed in Eurasia; Nassella has approximately 117 species are easily interpreted as tetraploids or hexaploids based on x = distributed in the Americas; and Austrostipa has 62 species 11, including several New World species of Achnatherum and endemic to Australia and New Zealand (Klokov & Ossyczn- Hesperostipa, and one species each of the following genera: juk, 1976; Martinovský, 1980; Moraldo, 1986; Edgar & Connor, Anemanthele, Jarava s.l. (J. plumulosa), Macrochloa, Nassella, 2000; Barkworth, 2007; Everett & al., 2009; Soreng & al., 2009). and Pappostipa. Old World Achnatherum sibiricum is 2n = 22 and 24 (x = 11 and 12). Piptochaetium fimbriatum has counts of 2n = 42 and 44, and could be interpreted as x = 7 (as are other ACKNOWLEDGMENTS species of Piptochaetium with 2n = 28); or as initially x = 11, and having lost two chromosomes. We thank the following organizations and people: the Smithso- None of the possible phylogenetic scenarios represented in nian Institution’s Restricted Endowment Fund, the Scholarly Stud- our analyses (Figs. 1–2) supports a single event of descending ies Program, Research Opportunities, Atherton Seidell Foundation, or ascending disploidy in Stipeae between x = 11 and 12. In the Biodiversity Surveys and Inventories Program, National Museum of plastid phylogeny the chromosome number patterns in Stipeae Natural History-Small Grants, and Laboratory of Analytical Biology begin with Macrochloa which is known to have 2n = 24, 36, 40, (LAB) all for financial support; Lee Weigt, Gabriel Johnson, Jeffrey 64, and 66, as well as 72. These could be based on x = 8, 10, 11 Hunt, David Erickson, Kenneth Wurdack, and Andrea Ormon for sup- or 12, or even 16 for this species. The first split between x = 11 port and consultation while working at LAB; the National Geographic and x = 12 in Stipeae is more confidently associated with the Society Committee for Research and Exploration (grant number 8087- crown node of SL1. Examination of the SL1 clade reveals an x 06) for field and laboratory support; the Fulbright Scholar Program to = 11 series that spans the entire ASL clade, occurring in diploid KR for a research visit to the Smithsonian Institution; the Komarov Piptatheropsis, one polyploid Piptochaetium (x = 7 is also pos- Botanical Institute, Russian Academy of Sciences for the opportunity sible for the genus), tetraploid Hesperostipa, and hexaploid and to work with herbarium collections, and Nikolai Czvelev and Dmitry octoploid Pappostipa. A second split between x = 11 and x = Geltman for consultation and permitting us to sample Stipeae speci- 12 in SL1 could be associated with the crown node for the ESL mens; Asuncion Cano Echevarría, Oscar Tovar Serpa†, Dorita Su- clade. In this case, an x = 11 series is represented by tetraploid sanibar Cruz, María Isabel La Torre Acuy, Jenny Vanessa Rojas Fox, Stipa and diploid Ptilagrostis. An x = 12 series is represented Isidoro Sánchez Vega, Socorro González Elizondo, Nancy Refulio- by a group of stout, rhizomatous to tussock-forming SLP taxa Rodríguez, Diego Leonel Salariato, Adela María Panizza, Fernando with putative tetraploid (2n = 48) karyotypes. The latter group O. Zuloaga, and Stephan Beck for help with our field expeditions to includes Ampelodesmos and “Neotrinia” (2n = 42, 48). The Peru, Bolivia, and Argentina; Sun Hang for help doing fieldwork in second SLP lineage (SL2) encompasses putative tetraploids— China; Sabine Miehe for providing samples from China, Hidehisa Patis coreana (2n = 46; Tateoka, 1986) and P. racemosa (2n = Koba for providing samples from Japan, Samuel Pyke for provid- 46; Johnson, 1945). The third SLP lineage, SL3, is represented ing samples from North Africa, and Jeffery M. Saarela for providing by the diploid Old World genus Piptatherum s.str. with 2n = 24 samples from North America; Michael Pirie, Ana Maria Cialdella, (x = 12 in all five species with counts). Thus, there is no clear Osvaldo Morrone†, and three anonymous reviewers for suggesting case here for x = 11 over x = 12. improvements to the manuscript.

23 Romaschenko & al. • Systematics and evolution of needle grasses TAXON — Article version: 9 Dec 2011: 27 pp.

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Thomasson, J.R. 1980. Paleoeriocoma (Gramineae, Stipeae) from the Valdes-Reyna, J. & Hatch, S.L. 1991. Lemma micromorphology in Miocene of Nebraska: Taxonomic and phylogenetic significance. the Eragrostideae (Poaceae). Sida 14: 531–549. Syst. Bot. 5: 233–240. Vázquez, F.M. & Barkworth, M.E. 2004. Resurrection and emenda- Thomasson, J.R. 1981. Micromorphology of the lemma in Stipa robusta tion of Macrochloa (Gramineae: Stipeae). Bot. J. Linn. Soc. 144: and Stipa viridula (Gramineae: Stipeae): Taxonomic significance. 483–495. S. W. Naturalist 26: 211–214. Vázquez, F.M & Devesa, J.A. 1996. Revisión del género Stipa L. y Thomasson, J.R. 1982. Fossil grass anthoecia and other plant fossils Nassella Desv. (Poaceae) en la Península Ibérica e islas Baleares. from arthropod burrows in the Miocene of Western Nebraska. Acta Bot. Malac. 21: 125–189. J. Paleontol. 56: 1011–1017. Wang, R., Cheng, C., Chang, C., Wu, C., Su, T. & Chaw, S.M. 2008. Thomasson, J.R.1985. Miocene fossil grasses: Possible adaptation in Dynamics and evolution of the inverted repeat-large single copy reproductive bracts (lemma and palea). Ann. Missouri Bot. Gard. junctions in the chloroplast genome of monocots. B. M. C. Evol. 72: 843–851. Biol. 8: 36. DOI: 10.1186/1471-2148-8-36. Thomasson, J.R. 1986. Lemma epidermal features in the North Ameri- Watson, L. & Dallwitz, M.J. 1992 onwards. The grass genera of the can species of Melica and selected species of Briza, Catabrosa, world: Descriptions, illustrations, identification, and information Glyceria, Neostapfia, Pleuropogon, and Schizachne (Gramineae). retrieval; including synonyms, morphology, anatomy, physiology, Syst. Bot. 11: 253–262. phytochemistry, cytology, classification, pathogens, world and Thomasson, J.R. 1987. Fossil grasses: 1820–1986 and beyond. Pp. local distribution, and references. Version: 23 Apr. 2010. http:// 159–167 in: Soderstrom, T.R., Hilu, K.W., Campbell, C.S. & Bark- delta-intkey.com/grass/index.htm. worth, M.E. (eds.), Grass systematics and evolution. Washington, Wei, F., Coe, E., Nelson, W., Bharti, A.K., Engler, F., Butler, E., D.C.: Smithsonian Institution Press. Kim, H., Goicoechea, J.L., Chen, M., Lee, S., Fuks, G., San- Thomasson, J.R. 2005. Berriochloa gabeli and Berriochloa huletti chez-Villeda, H., Schroeder, S., Fang, Z., McMullen, M., Davis, (Gramineae, Stipeae), two new grass species from the late Miocene G., Bowers, J.E., Paterson, A.H., Schaeffer, M., Gardiner, J., Ash Hollow Formation of Nebraska and Kansas. J. Paleontol. 79: Cone, K., Messing, J., Soderlund, C. & Wing, R.A. 2007. Physi- 185–199. cal and genetic structure of the maize genome reflects its complex Torres, A.M. 1997a. Nassella (Gramineae) del noroeste de la Argentina. evolutionary history. PLoS Genet. 3(7): e123. DOI: 10.1371/journal Comis. Invest. Ci. (Buenos Aires) 13: 5–45. .pgen.0030123. Torres, A.M. 1997b. Stipa (Gramineae) del noroeste de la Argentina. White, T.J., Bruns, T., Lee, S. & Taylor, J. 1990. Amplifications and Comis. Invest. Ci. (Buenos Aires) 13: 46–67. direct sequencing of fungal ribosomal RNA genes for phylogenet- Torres, A.M. 1997c. Nicoraella (Gramineae) un nuevo genero para ics. Pp. 315–322 in: Innis, M., Gelfand, D., Sninsky, J. & White, T. America del Sur. Comis. Invest. Ci. (Buenos Aires) 13: 69–77. (eds), PCR protocols: A guide to methods and applications. San Tzvelev, N.N. 1974. Zametki o tribe Stipeae Dum. semeistva zlakov Diego: Academic Press. (Poaceae) V SSSR. (Notes of the tribe Stipeae Dum., Fam. Poaceae) Wu, Z. & Phillips, S.M. 2006. Stipeae. Pp. 188–212 in: Wu, Z., Raven, in URSS). Novosti Sist. Vyssh. Rast. 11: 4–21. P.H. & Hong, D.Y. (eds.), Flora of China, vol. 22, Poaceae. Beijing: Tzvelev, N.N. 1976. Zlaki SSSR. Leningrad: Nauka Publishers. Science Press; St. Louis: Missouri Botanical Garden Press. Tzvelev, N.N. 1977. [On the origin and evolution of Feathergrasses Zwickl, D.J. 2006. Genetic algorithm approaches for the phylogenetic (Stipa L.)]. Pp. 139–150 in: Lebedev, D.V. & Karamysheva, Z.V. analysis of large biological sequence datasets under the maxi- (eds.), Problemy ekologii, geobotaniki, botanicheskoi geografii i mum likelihood criterion. Ph.D. dissertation, University of Texas, floristiki. Leningrad: Academiya Nauk SSSR. Austin, U.S.A. Tzvelev, N.N. 1989. The system of grasses (Poaceae) and their evolution. Bot. Rev. (Lancaster) 55: 141–203.

27 TAXON 61 (1) • February 2012: Electronic Supplement, 1 pp. Romaschenko & al. • Systematics and evolution of needle grasses

International Journal of Taxonomy, Phylogeny and Evolution

Electronic Supplement to

Systematics and evolution of the needle grasses (Poaceae: Pooideae: Stipeae) based on analysis of multiple chloroplast loci, ITS, and lemma micromorphology

Konstantin Romaschenko, Paul M. Peterson, Robert J. Soreng, Nuria Garcia-Jacas, Oksana Futorna & Alfonso Susanna

1 TAXON — Article version: 9 Dec 2011: Electr. Suppl., 5 pp. Romaschenko & al. • Systematics and evolution of needle grasses

Appendix . Species and vouchers sampled for DNA, arranged by tribe, species, voucher (lead collector and number and co-collector[s]), country, lower po- litical unit, GenBank accession numbers (those in bold are new) by DNA region: trnK-matK, matK, trnH-psbA, trnL-F, rps3, ndhF, rpl32-trnL, rps16-trnK, and rps16 intron, ITS. BRACHYELYTREAE: Brachyelytrum erectum (Schreb.) P. Beauv., Soreng 7440 (US), U.S.A., Maryland (from New York), EU489398, EU489184, EU489251, EU489326, JF698121, GU254790, JF697971, JF698425, JF698272, EU489105; BRYLKINIEAE: Brylkinia caudata (Munro) F. Schmidt, Ping s.n. (US), China, Jilin or Sichuan, GU254914, GU254725, GU254835, GU254957, JF698122, GU254780, JF697972, JF698426, JF698273, GU254647; DIARRHENEAE: Diarrhena fauriei (Hack.) Ohwi, Koba s.n. (US), Japan, Nagono, –, –, JF698549, JF697770, JF698125, JF697879, JF697975, JF698429, JF698276, JF697717; Diarrhena japonica Franch. & Sav., Koba s.n. (US), Japan, Kangawa, –, –, JF698550, JF697771, JF698126, JF697880, JF697976, JF698430, JF698277, JF697718; Diarrhena obovata (Gleason) Brandenburg, Soreng 7439 (US), U.S.A., Maryland (from Michagan), GU254922, GU254730, GU254834, GU254956, JF698127, GU254783, JF697977, JF698431, JF698278, GU254669; LYGEEAE: Lygeum spartum L., Soreng 3698 & Soreng (US), Spain, Analucia, JF698614, JF697823, JF698553, –, JF698141, JF697883, JF697990, JF698445, JF698292, JF697721; MELICEAE: Triniochloa stipoides (Kunth) Hitchc., Peterson 20581, Soreng & Romaschenko (US), Peru, Cuzco, JF698640, –, JF698580, –, JF698209, JF697911, JF698058, JF698515, JF698363, JF697748; NARDEAE: Nardus stricta L., Soreng 7497, Soreng & Gillespie (US), Greece, Thessaly, EU489432, EU489217, EU489285, EU489360, JF698143, GU254791, JF697993, JF698448, JF698295, EU489143; PHAENOSPERMATIDEAE: Anisopogon avenaceus R. Br., Soreng 5905, Peterson & Jacobs (US), Australia, New South Wales, GU254933, GU254682, GU254823, GU254946, JF698108, GU254769, JF697959, JF698413, JF698258, GU254657; Danthoniastrum compactum (Boiss. & Heldr.) Holub, Soreng 7520-1, Soreng & Gillespie (US), Greece, Pelopponisos, GU254907, GU254720, GU254836, GU254958, JF698124, GU254779, JF697974, JF698428, JF698275, GU254646; Duthiea brachypodium (P. Candargi) Keng & Keng f., Soreng 5358, Peterson & Sun (US), China, Sichuan, GU254934, GU254683, GU254819, GU254947, JF698129, GU254793, JF697978, JF698433, JF698280, GU254656; Metcalfia mexicana (Scribn.) Conert, Peterson 18808 & Valdes-Reyna (US), Mexico, Coahuila, –, –, –, –, –, JF697884, JF697992, JF698447, JF698294, JF697722; Phaenosperma globosa Munro ex Benth., Soreng 5325, Peterson & Sun (US), China, Sichuan, GU254935, GU254684, JF698554, GU254948, JF698156, GU254792, JF698008, JF698462, JF698310, GU254655; Sinochasea trigyna Keng, Soreng 5644, Peterson & Sun (US), China, Xizang, GU254932, GU254681, GU254824, –, JF698190, GU254778, JF698039, JF698496, JF698344, GU254645; Stephanachne nigrescens Keng, Miehe 94-547-9, Miehe & Wündisch (Marberg U., Fac. Geograph.), China, Sichuan, JF698628, JF697836, JF698568, –, JF698191, JF697899, JF698040, JF698497, JF698345, JF697736; Stephanachne pappophorea (Hack) Keng, Yunatov 138 (LE), China, Xinjiang, JF698629, JF697837, JF698569, –, JF698192, JF697900, JF698041, JF698498, JF698346, JF697737; POEAE: Dielsiochloa floribunda (Pilg.) Pilg., Peterson 20445, Soreng, Romaschenko & Susanibar Cruz (US), Peru, Huancavelica, JF698612, JF697821, JF698551, JF697772, JF698128, JF697881, –, JF698432, JF698279, JF697719; STIPEAE: Achnatherum acutum (Swallen) Valdés-Reyna & Barkworth, Valdes-Reyna 1444 (US), Mexico, Coahuila, JF698581, JF697791, JF698516, JF697749, JF698059, JF697846, JF697912, JF698364, JF698210, JF697688; Achnatherum aridum (M.E. Jones) Barkworth, Tiehm 13518 & Nachlinger (US), U.S.A., Nevada, EU489382, EU489170, EU489237, EU489311, JF698060, JF697847, JF697913, JF698365, JF698211, EU489089; Achnatherum brandisii (Mez) Z.L. Wu, Soreng 5600, Peterson & Sun (US), China, Xizang, JF698582, JF697792, JF698517, –, JF698061, JF697848, JF697914, JF698366, JF698212, JF697689; Achnatherum bromoides (L.) P. Beauv., Romaschenko 439 & Didukh (KW), Ukraine, Crimea, GU254905, GU254708, GU254845, –, JF698062, GU254734, JF697915, JF698367, JF698213, GU254624; Achnatherum calamagrostis (L.) P. Beauv., Pyke 164 (US), Spain, Huesca, GU254899, GU254698, GU254851, GU254940, JF698063, GU254743, JF697916, JF698368, JF698214, GU254638; Achnatherum caragana (Trin.) Nevski, Goloskokov s.n. (US), Kazakhstan, Dzhungar Alatau, GU254915, GU254690, GU254852, GU254973, JF698064, GU254732, JF697917, JF698369, JF698215, GU254631; Achnatherum chinense (Hitchc.) Tzvelev, Petrov s.n. (LE), China, Gansu, GU254900, GU254691, GU254860, –, JF698065, GU254735, JF697918, JF698370, JF698216, GU254630; Achnatherum confusum (Litv.) Tzvelev, Kotukhov s.n. (LE), Kazakhstan, South Altai, JF698583, JF697793, JF698518, –, JF698066, –, JF697919, JF698371, JF698217, JF697690; Achnatherum constrictum (Hitchc.) Valdés-Reyna & Barkworth, Sohns 1362 (US), Mexico, San Luis Potosi, JF698584, JF697794, JF698519, JF697750, JF698067, JF697849, JF697920, JF698372, JF698218, JF697691; Achnatherum curvifolium (Swallen) Barkworth, Wilkens 1660 (US), U.S.A., New Mexico, –, –, JF698521, –, JF698069, JF697851, –, JF698374, JF698220, –; Achnatherum diegoense (Swallen) Barkworth, Fiander 5778 (US), U.S.A., California, JF698586, JF697796, JF698522, JF697751, JF698070, JF697852, JF697922, JF698375, JF698221, JF697692; Achnatherum eminens (Cav.) Barkworth, Peterson 10952 & Annable (US), Mexico, Zacatecas, JF698587, JF697797, JF698523, JF697752, JF698071, JF697853, JF697923, JF698376, JF698222, JF697693; Achnatherum hendersonii (Vasey) Barkworth, Maze 1000 & Robson (US), U.S.A., Washington, JF698588, JF697798, JF698524, JF697753, JF698072, JF697854, JF697924, JF698377, JF698223, JF697694; Achnatherum hymenoides (Roem. & Schult.) Barkworth, Saarela 205 (UBC), U.S.A., California, GU254894, GU254710, GU254844, GU254972, JF698073, GU254733, JF697925, JF698378, JF698224, GU254632; Achnatherum inebrians (Hance) Keng ex Tzvelev, Soreng 5393, Peterson & Sun (US), China, Gansu, GU254903, GU254695, GU254847, GU254990, –, GU254750, JF697926, JF698379, JF698225, GU254626; Achnatherum jacquemontii (Jaub. & Spach) P.C. Kuo & S.L. Lu, Neubauer s.n. (US), Afganistan, Sare Bulbul, JF698589, JF697799, JF698525, –, JF698074, JF697855, JF697927, JF698380, JF698226, JF697695; Achnatherum latiglume (Swallen) Barkworth, Le Roy s.n. (US), U.S.A., California, JF698590, JF697800, JF698526, –, JF698075, JF697856, JF697928, JF698381, JF698227, –; Achnatherum lemmonii (Vasey) Barkworth, Halse 6164 (BRY), U.S.A., Oregon, JF698591, JF697801, JF698527, JF697754, JF698076, JF697857, JF697929, JF698382, JF698228, JF697696; Achnatherum lettermanii (Vasey) Barkworth, Hartman 72830 (GREE), U.S.A., New Mexico, JF698592, JF697802, JF698528, JF697755, JF698077, JF697858, JF697930, JF698383, JF698229, JF697697; Achnatherum lobatum (Swallen) Barkworth, Peterson 10660 & Annable (US), Mexico, Coahuila, JF698593, JF697803, JF698529, JF697756, JF698078, JF697859, JF697931, JF698384, JF698230, JF697698; Achnatherum multinode (Scribn. ex Beal) Valdés-Reyna & Barkworth, Hoge 264 (US), Mexico, Coahuila, JF698594, JF697804, JF698530, JF697757, JF698079, JF697860, JF697932, JF698385, JF698231, JF697699; Achnatherum nelsonii (Scribn.) Barkworth, Saarela 593 (UBC), Canada, British Columbia, GU254895, GU254713, GU254854, GU254971, JF698080, GU254761, JF697933, JF698386, JF698232, GU254633; Achnatherum occidentale (Thurb. ex S. Watson) Barkworth, Saarela 594, Sears & Maze (UBC), Canada, British Columbia, GU254896, GU254714, GU254855, GU254970, JF698081, GU254754, JF697934, JF698387, JF698233, GU254634; Achnatherum occidentale subsp. californicum (Merr. & Burtt Davy) Barkworth, Howell 36354 (US), U.S.A., California, EU489383, EU489171, EU489238, EU489312, JF698082, GU254740, JF697935, JF698388, JF698234, EU489090; Achnatherum parishii (Vasey) Barkworth, Roos 4895 & Roos (US), U.S.A., California, EU489385, GU254716, EU489240, EU489314, JF698083, GU254756, JF697936, JF698389, –, EU489092; Achnatherum pekinense (Hance) Ohwi, Nefedova s.n. & Paschenko (LE), Russia, Far East (Ne- zhina), JF698595, JF697805, JF698531, –, JF698084, JF697861, JF697937, JF698390, JF698235, JF697700; Achnatherum perplexum Hoge & Barkworth, Page 2494 (US), U.S.A., New Mexico, JF698596, JF697806, JF698532, JF697758, JF698085, JF697862, JF697938, JF698391, JF698236, JF697701; Achnatherum pinetorum (M.E. Jones) Barkworth, Bostick 4875 (US), U.S.A., Nevada, JF698597, JF697807, JF698533, JF697759, JF698086, JF697863, JF697939, JF698392, JF698237, JF697702; Achnatherum pubicalyx (Ohwi) Keng, Kozlov 124 (LE), China, Hebei, JF698598, JF697808, JF698534, –, JF698087, JF697864, JF697940, JF698393, JF698238, JF697703; Achnatherum richardsonii (Link) Barkworth, Peterson 18399, Saarela & Smith (US), Canada, Alberta, EU489386, EU489173, JF698535, EU489315, JF698088, JF697865, JF697941, JF698394, JF698239, EU489093; Achnatherum robustum (Vasey) Barkworth, Peterson 10583 & Annable (US), Mexico, Coahuila, GU254906, GU254715, GU254856, GU254969, JF698089, GU254755, JF697942, JF698395, JF698240, GU254635; Achnatherum scribneri (Vasey) Barkworth, Hazlett 12090 (GREE), U.S.A., New Mexico, JF698599, JF697809, JF698536, JF697760, JF698090, JF697866, JF697943, JF698396, JF698241, JF697704; Achnatherum sibiricum (L.) Keng ex Tzvelev, Soreng 5104, Peterson, Wang & Zhu (US), China, Hebei, GU254904, GU254696, GU254846, –, JF698091, GU254741, JF697944, JF698397, JF698242, GU254610; Achnatherum splendens (Trin.) Nevski, Soreng 5121, Peterson, Wang & Zhu (US), China, Inner Mongolia, GU254913, GU254687, GU254818, GU254951, JF698092, GU254787, JF697945, JF698398, JF698243, GU254668; Achnatherum stillmanii (Bol.) Barkworth, Hoover 4614 (US), U.S.A., California, JF698600, JF697810, JF698537, JF697761, JF698093, JF697867, JF697946, JF698399, JF698244, JF697705; Achnatherum turcomanicum Tzvelev, Goncharov 162 & Grigoriev (LE), Tajikistan, Novabad, JF698601, JF697811, JF698538, –, JF698094, –, –, –, JF698245, JF697706; Achnatherum wallowaense J. Maze & K. A. Robson, Maze

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Appendix . Continued. 1006, Robson & Henn (US), U.S.A., Oregon, JF698602, JF697812, JF698539, –, JF698095, JF697868, JF697947, JF698400, JF698246, JF697707; Aciachne acicularis Laegaard, Peterson 13931 & Refulio Rodriguez (US), Peru, Ancash, GU254930, GU254673, GU254865, GU254988, JF698096, GU254806, JF697948, JF698401, JF698247, GU254625; Aciachne flagellifera Lægaard, Laegaard 19436 (AAU), Ecuador, Tungurahua, GU254893, GU254672, GU254877, GU254987, JF698097, GU254805, JF697949, JF698402, JF698248, GU254654; Amelichloa caudata (Trin.) Arriaga & Barkworth, Peterson 11398 & Annable (US), Argentina, Mendoza, EU489388, EU489175, EU489241, EU489317, JF698098, GU254764, –, JF698403, –, EU489095; Amelichloa clandestina (Hack.) Arriaga & Barkworth, Barkworth 5103 (US), U.S.A., Texas, GU254898, GU254717, GU254853, GU254968, JF698099, GU254765, JF697950, JF698404, JF698249, GU254636; Ampelodesmos mauritanicus (Poir.) T. Durand & Schinz, Pyke 702 (BC), Spain, Tarragona, GU254911, GU254722, GU254832, GU254955, JF698100, GU254797, JF697951, JF698405, JF698250, GU254667; Anatherostipa hans-meyeri (Pilg.) Peñailillo, Peterson 20645, Soreng, Romaschenko & Gonzalez-Elizondo (US), Bolivia, La Paz, EU489391, EU489177, EU489244, EU489319, JF698101, GU254804, JF697952, JF698406, JF698251, EU489098; Anatherostipa mucronata (Griseb.) F. Rojas, Peterson 19551, Soreng, Salariato & Panizza (US), Argentina, Jujuy, GU254887, GU254697, GU254861, GU254985, JF698102, GU254803, JF697953, JF698407, JF698252, GU254611; Anatherostipa obtusa (Nees & Meyen) Peñailillo, Peterson 13811 & Refulio Rodriguez (US), Peru, Ancash, JF698603, JF697813, JF698540, JF697762, JF698103, JF697869, JF697954, JF698408, JF698253, JF697708; Anatherostipa rigidiseta (Pilg.) Peñailillo, Beck s.n. (LPB), Bolivia, La Paz, GU254890, GU254699, GU254869, GU254984, JF698104, GU254809, JF697955, JF698409, JF698254, GU254612; Anatherostipa rosea (Hitchc.) Peñailillo, Laegaard 10864 (AAU), Ecuador, Azuay, GU254889, GU254671, GU254867, GU254986, JF698105, GU254795, JF697956, JF698410, JF698255, GU254652; Anatherostipa venusta (Phil.) Peñailillo, Pfister 9394 (US), Chile, Region II, GU254885, GU254680, GU254870, GU254983, JF698106, GU254801, JF697957, JF698411, JF698256, GU254613; Anemanthele lessoniana (Steud.) Veldkamp, Travers s.n. in hb. Mez I.3236 (US), New Zealand, South Isl. (Marlborough), ( JF698604, JF697814, JF698541, –, JF698107, JF697870, JF697958, JF698412, JF698257, JF697709; Austrostipa campylachne (Nees) S.W.L. Jacobs & J. Everett, Peterson 14267, Soreng, Rosenberg & MacFarlane (US), Australia, Western Austra- lia, GU254902, GU254694, GU254848, GU254975, JF698109, GU254737, JF697960, JF698414, JF698259, GU254627; Austrostipa compressa (R. Br.) S.W.L. Jacobs & J. Everett, Peterson 14514, Soreng & Rosenberg (US), Australia, Western Australia, EU489393, EU489179, EU489246, EU489321, JF698110, –, –, –, JF698260, EU489100; Austrostipa flavescens (Labill.) S.W.L. Jacobs & J. Everett, Spjut 7182 (US), Australia, Western Australia, JF698605, JF697815, JF698542, JF697763, JF698111, JF697871, JF697961, JF698415, JF698261, JF697710; Austrostipa hemipogon (Benth.) S.W.L. Jacobs & J. Everett, Simon 13449 (US), Australia, South Australia, JF698606, JF697816, JF698543, JF697764, JF698112, JF697872, JF697962, JF698416, JF698262, JF697711; Austrostipa juncifolia (Hughes) S.W.L. Jacobs & J. Everett, Peterson 14502, Soreng, Rosenberg & MacFarlane (US), Australia, Western Australia, JF698607, JF697817, JF698544, JF697765, JF698113, JF697873, JF697963, JF698417, JF698263, JF697712; Austrostipa macalpinei (Reader) S.W.L. Jacobs & J. Everett, Peterson 14292, Soreng, Rosenberg & MacFarlane (US), Australia, Western Australia, JF698608, –, JF698545, JF697766, –, JF697874, JF697964, –, JF698264, JF697713; Austrostipa nitida (Summerh. & C.E. Hubb.) S.W.L. Jacobs & J. Everett, Canty 2164 (US), Australia, South Australia, JF698609, JF697818, JF698546, JF697767, JF698114, JF697875, JF697965, JF698418, JF698265, JF697714; Austrostipa nodosa (S.T. Blake) S.W.L. Jacobs & J. Everett, Ward 203 (KW), Australia, New South Wales, JF698610, JF697819, JF698547, JF697768, JF698115, JF697876, JF697966, JF698419, JF698266, JF697715; Austrostipa pycnostachya (Benth.) S.W.L. Jacobs & J. Everett, Peterson 14318, Soreng & Rosenberg (US), Australia, Western Australia, EU489394, EU489180, EU489247, EU489322, JF698116, JF697877, –, JF698420, JF698267, EU489101; Austrostipa scabra (Lindl.) S.W.L. Jacobs & J. Everett, Peterson 14442, Soreng & Rosenberg (US), Australia, Western Australia, EU489395, EU489181, EU489248, EU489323, JF698117, GU254738, JF697967, JF698421, JF698268, EU489102; Austrostipa semibarbata (R. Br.) S.W.L. Jacobs & J. Everett, Simon 13439 (US), Australia, South Australia., GU254901, GU254693, GU254849, GU254974, JF698118, GU254736, JF697968, JF698422, JF698269, GU254628; Austrostipa tenuifolia (Steud.) S.W.L. Jacobs & J. Everett, Peterson 14248, Soreng & Macfarlane (US), Australia, Western Australia, EU489397, EU489183, EU489250, EU489325, JF698119, GU254739, JF697969, JF698423, JF698270, EU489104; Austrostipa trichophylla (Benth.) S.W.L. Jacobs & J. Everett, Peterson 14324, Soreng & Rosenberg (US), Australia, Western Australia, JF698611, JF697820, JF698548, JF697769, JF698120, JF697878, JF697970, JF698424, JF698271, JF697716; Celtica gigantea (Link) F. M. Vázquez & Barkworth, Pyke 705 (BC), Spain, Albacete, GU254919, GU254726, GU254843, GU254961, JF698123, GU254775, JF697973, JF698427, JF698274, GU254642; Hesperostipa comata (Trin. & Rupr.) Barkworth, Saarela 595, Sears & Maze (UBC), Canada, British Columbia, EU489399, EU489185, EU489252, EU489327, JF698130, GU254812, JF697979, JF698434, JF698281, EU489106; Hesperostipa neomexicana (Thurb.) Barkworth, Peterson 18934 & Valdes-Reyna (US), Mexico, Nuevo Leon, EU489400, EU489186, GU254840, EU489328, JF698131, GU254808, JF697980, JF698435, JF698282, EU489107; Hesperostipa spartea (Trin.) Barkworth, Holmes 214 (US), U.S.A., (Missouri?), EU489401, EU489187, EU489253, EU489329, JF698132, GU254745, JF697981, JF698436, JF698283, EU489108; Jarava annua (Mez) Peñailillo, Peterson 15614 & Soreng (US), Chile, Region I, JF698613, JF697822, JF698552, JF697773, JF698133, JF697882, JF697982, JF698437, JF698284, JF697720; Jarava castellanosii (F.A. Roig) Peñailillo, Peterson 10336 & Annable (US), Argentina, Jujuy, EU489405, EU489191, EU489256, EU489333, JF698134, GU254770, JF697983, JF698438, JF698285, EU489112; Jarava ichu Ruiz & Pav., Peterson 20745, Soreng, Romaschenko & Gonzalez Elizondo (US), Peru, Puno, EU489415, EU489202, EU489267, EU489344, JF698135, GU254763, JF697984, JF698439, JF698286, EU489124; Jarava media (Speg.) Peñailillo, Peterson 19337, Soreng, Salariato & Panizza (US), Argentina, La Rioja, EU489419, EU489205, EU489272, EU489347, JF698136, GU254758, JF697985, JF698440, JF698287, EU489129; Jarava plumosula (Nees ex Steud.) F. Rojas, Peterson 20471, Soreng, Romaschenko & Susanibar Cruz (US), Peru, Ayacucho, EU489422, EU489207, EU489275, EU489350, JF698137, GU254757, JF697986, JF698441, JF698288, EU489133; Jarava pseudoichu (Caro) F. Rojas, Peterson 20736, Soreng, Romaschenko & Gonzalez Elizondo (US), Peru, Puno, EU489424, EU489209, EU489277, EU489352, JF698138, GU254762, JF697987, JF698442, JF698289, EU489135; Jarava scabrifolia (Torres) Peñailillo, Peterson 11712 & Annable (US), Argentina, Salta, EU489425, EU489210, EU489278, EU489353, JF698139, GU254760, JF697988, JF698443, JF698290, EU489136; Lorenzochloa erectifolia (Swallen) Reeder & C. Reeder, Peterson 14074 & Refugio Rodriguez (USM), Peru, Junin, JN882350, –, JN882351, –, –, –, –, –, –, –; Macrochloa tenacissima (Loefl. ex L.) Kunth, Pyke 701 (BC), Spain, Almería, GU254912, GU254723, GU254833, GU254978, JF698142, GU254782, JF697991, JF698446, JF698293, GU254648; Nassella clarazii (Ball) Barkworth, Peterson 11651 & Annable (US), Argentina, Catamarca, EU489436, EU489221, EU489289, EU489364, JF698144, GU254766, JF697994, –, JF698296, EU489147; Nassella filiculmis (Delile) Barkworth, Soreng 7009 & Soreng (US), Chile, Region VIII, EU489439, EU489224, EU489292, EU489367, JF698145, GU254768, JF697995, JF698449, JF698297, EU489150; Nassella neesiana (Trin. & Rupr.) Barkworth, Peter- son 10258 & Annable (US), Argentina, Jujuy, EU489444, EU489228, EU489297, EU489371, –, GU254767, JF697996, JF698450, JF698298, EU489155; Nassella pfisteri (Matthei) Barkworth, Soreng 7017a & Soreng (US), Chile, Region VIII, EU489446, EU489229, EU489299, EU489372, JF698146, JF697885, JF697997, JF698451, JF698299, EU489157; Nassella trichotoma (Nees) Hack. ex Arechav., Peterson 11506 & Annable (US), Argentina, San Juan, EU489451, EU489232, EU489305, EU489376, JF698147, GU254742, JF697998, JF698452, JF698300, EU489164; Ortachne breviseta Hitch., Laegaard 12802 (AAU), Argentina, Neuquén, GU254924, GU254674, GU254868, GU254989, JF698148, GU254810, JF697999, JF698453, JF698301, GU254666; Ortachne rariflora (Hook. f.) Hughes, Mariz 924 (CONC), Chile, Region XI, GU254925, GU254675, GU254864, GU254952, JF698149, GU254771, JF698000, JF698454, JF698302, GU254665; Orthoraphium roylei Nees, Soreng 5261, Peterson & Sun (US), China, Yunnan, GU254881, GU254703, GU254858, GU254979, JF698150, GU254753, JF698001, JF698455, JF698303, GU254617; Oryzopsis asperifolia Michx., Saarela 384 (UBC), Canada, Alberta, GU254908, GU254686, GU254821, GU254964, JF698151, GU254788, JF698002, JF698456, JF698304, GU254653; Pappostipa chrysophylla (E. Desv.) Romasch., Peterson 19220, Soreng, Salariato & Panizza (US), Argentina, Mendoza, EU489407, EU489193, EU489258, EU489335, JF698152, GU254781, JF698003, JF698457, JF698305, EU489114; Pappostipa hieronymusii (Pilg.) Romasch., Peterson 11488 & Annable (US), Argentina, San Juan, EU489410, EU489197, EU489262, EU489339, JF698153, JF697886, JF698004, JF698458, JF698306, EU489118; Pappostipa major (Speg.) Romasch., Soreng 7222 & Soreng (US), Chile, Region IX, EU489427, EU489212, EU489280, EU489355, JF698154, GU254798, JF698005, JF698459, JF698307, EU489138; Pappostipa speciosa (Trin. & Rupr.) Romasch., Peterson 11549 & Annable (US), Argentina, San Juan, EU489426, EU489211, EU489279, EU489354, –, GU254772, JF698006, JF698460, JF698308, EU489137; Pappostipa vaginata (Phil.) Romasch., Peterson 11744 & Annable (US), Argentina, Salta, EU489431, EU489216, EU489284, EU489359, JF698155,

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Appendix . Continued. GU254816, JF698007, JF698461, JF698309, EU489142; Patis coreana (Honda) Ohwi, Liou 1085 (US), China, Hebei, JF698585, JF697795, JF698520, –, JF698068, JF697850, JF697921, JF698373, JF698219, –; Patis obtusa (Stapf) Romasch., P.M. Peterson & Soreng, Soreng 4531 & Kelly (US), China, Sichuan, JF698619, JF697828, JF698559, JF697778, JF698166, JF697891, JF698018, JF698472, JF698320, JF697727; Piptatheropsis canadensis (Poir.) Romasch., P.M. Peterson & Soreng, Raymond & Kucyniak s.n. (US), Canada, Quebec, JF698616, JF697825, JF698556, JF697775, JF698159, JF697888, JF698011, JF698465, JF698313, JF697724; Piptatheropsis exigua (Thurb.) Romasch., P.M. Peterson & Soreng, Reveal 1073 (KW), U.S.A., Colorado, GU254927, GU254677, GU254859, GU254976, JF698161, GU254752, JF698013, JF698467, JF698315, GU254663; Piptatheropsis micrantha (Trin. & Rupr.) Romasch., P.M. Peterson & Soreng, Peterson 18437, Saarela & Smith (US), Canada, Alberta, GU254926, GU254676, GU254866, GU254950, JF698167, GU254815, JF698019, JF698473, JF698321, GU254664; Piptatheropsis pungens (Torr.) Romasch., P.M. Peterson & Soreng, Hermann 13407 (US), Canada, Alberta, JF698622, JF697831, JF698562, JF697781, JF698172, JF697894, JF698024, JF698478, JF698326, JF697730; Piptatheropsis shoshoneana (Curto & Douglass M. Hend.) Romasch., P.M. Peterson & Soreng, Eno 13 (US), U.S.A., Idaho, GU254928, GU254688, GU254841, GU254941, JF698173, GU254814, JF698025, JF698479, JF698327, GU254662; Piptatherum aequiglume (Duthie ex J.D. Hooker) Roshev., Koelz 1389 (US), India, Himachal Pradesh, JF698615, JF697824, JF698555, JF697774, JF698157, JF697887, JF698009, JF698463, JF698311, JF697723; Piptatherum angustifolium (Munro ex Regel) Roshev., Nepli 144 (LE), Tajikistan, West Pamir (Khorog), GU254923, GU254707, GU254825, GU254944, JF698158, GU254811, JF698010, JF698464, JF698312, GU254659; Piptatherum coerulescens (Desf.) P. Beauv., Soreng 3765 (US), Greece, Attica, GU254936, GU254685, GU254822, GU254949, JF698160, GU254789, JF698012, JF698466, JF698314, GU254639; Piptatherum fedtschenkoi Roshev., Botchantzev 1559 (LE), Tajikistan, Pamir, JF698617, JF697826, JF698557, JF697776, JF698162, JF697889, JF698014, JF698468, JF698316, JF697725; Piptatherum ferganense (Litw.) Roshev., Kamelin 100 (LE), Tajikistan, Guis- sar (Varzob), JF698618, JF697827, JF698558, JF697777, JF698163, JF697890, JF698015, JF698469, JF698317, JF697726; Piptatherum hilariae Pazij, Potaliev 343 (LE), Tajikistan, Pamir, GU254929, GU254679, GU254828, GU254943, JF698164, GU254807, JF698016, JF698470, JF698318, GU254660; Piptatherum holciforme (M. Bieb.) Roemer & Schultes, Didukh 1203 (KW), Ukraine, Yalta, GU254931, GU254670, GU254829, GU254945, JF698165, GU254817, JF698017, JF698471, JF698319, GU254658; Piptatherum miliaceum (L.) Coss., Gillett 16094 (US), Jordan, Azraq, GU254918, GU254729, GU254838, GU254960, JF698168, GU254776, JF698020, JF698474, JF698322, GU254643; Piptatherum munroi (Stapf) Mez, Soreng 5686, Peterson & Sun (US), China, Sichuan, JF698621, JF697830, JF698561, JF697780, JF698170, JF697893, JF698022, JF698476, JF698324, JF697729; Piptatherum paradoxum (L.) P. Beauv., Pyke 831 (BC), Spain, Cataluña, GU254916, GU254727, GU254862, GU254977, JF698171, GU254744, JF698023, JF698477, JF698325, GU254622; Piptatherum sogdianum (Grig.) Roshev., Konkov 1637 (LE), Tajikistan, Sughd, JF698623, –, JF698563, –, JF698174, JF697895, –, JF698480, JF698328, JF697731; Piptatherum songaricum (Trin. & Rupr.) Roshev., Goloskokov s.n. (LE), Kazakhstan, Ili-Pribalhashje, JF698624, JF697832, JF698564, JF697782, JF698175, JF697896, JF698026, JF698481, JF698329, JF697732; Piptatherum thomasii (Duby) Kunth, Pyke 833 (BC), Spain, Cataluña, JF698620, JF697829, JF698560, JF697779, JF698169, JF697892, JF698021, JF698475, JF698323, JF697728;Piptatherum virescens (Trin.) Boiss., Romaschenko 445 & Didukh (KW), Ukraine, Yalta, GU254917, GU254728, GU254837, GU254959, JF698176, GU254777, JF698027, JF698482, JF698330, GU254644; Pipto- chaetium avenaceum (L.) Parodi, Soreng 7739 & Romaschenko (US), U.S.A., Maryland, GU254883, GU254705, GU254872, GU254981, JF698177, GU254799, JF698028, JF698483, JF698331, GU254615; Piptochaetium brachyspermum (Speg.) Parodi, Peterson 11252 & Annable (US), Argentina, La Pampa, EU489452, EU489233, EU489306, EU489377, JF698178, GU254802, JF698029, JF698484, JF698332, EU489165; Piptochaetium featherstonei (Hitchc.) Tovar, Peterson 10314 & Annable (US), Argentina, Jujuy, GU254882, GU254704, GU254873, GU254980, JF698179, GU254796, JF698030, JF698485, JF698333, GU254616; Piptochaetium montevidense (Spreng.) Parodi, Peterson 20486, Soreng, Romaschenko & Susanibar Cruz (US), Peru, Ayacucho, JF698625, JF697833, JF698565, JF697783, JF698180, JF697897, –, JF698486, JF698334, JF697733; Piptochaetium panicoides (Lam.) E. Desv., Soreng 7011 & Soreng (US), Chile, Region VIII, EU489453, EU489234, EU489307, EU489378, JF698181, GU254794, JF698031, JF698487, JF698335, EU489166; Psammochloa villosa (Trin.) Bor, Safronova 952 (LE), Mongolia, Suhe-Batorsk, GU254892, GU254678, GU254820, GU254939, JF698182, GU254786, JF698032, JF698488, JF698336, GU254651; Ptilagrostis dichotoma Keng ex Tzvelev, Soreng 5647, Peterson & Sun (US), China, Zizang, GU254880, GU254702, GU254874, GU254965, JF698183, GU254749, JF698033, JF698489, JF698337, GU254618; Ptilagrostis junatovii Grubov, Kotukhov s.n. (LE), Russia, West Altai, GU254879, GU254701, GU254842, –, JF698184, GU254748, JF698034, JF698490, JF698338, GU254619; Ptilagrostis kingii (Bol.) Barkworth, Peirson 10819 (US), U.S.A., California, GU254937, GU254712, GU254827, GU254942, JF698185, GU254813, JF698035, JF698491, JF698339, GU254661; Ptilagrostis luquensis P.M. Peterson, Soreng & Z.L. Wu, Soreng 5383, Peterson & Sun (US), China, Gansu, GU254878, GU254700, GU254875, –, JF698186, GU254747, JF698036, JF698492, JF698340, GU254620; Ptilagrostis malyschevii Tzvelev, Ikonnikov 21570 (LE), Krygyzstan or Tajikistan, –, JF698626, JF697834, JF698566, –, JF698187, –, –, JF698493, JF698341, JF697734; Ptilagrostis mongholica (Turcz. ex Trin.) Griseb., Koloskov s.n. (LE), Kyrgyzstan, –, GU254886, GU254689, GU254876, GU254938, JF698188, GU254746, JF698037, JF698494, JF698342, GU254621; Ptilagrostis pelliotii (Danguy) Grubov, Grubov 1815 (LE), Mongolia, Trans-Altai Gobi, JF698627, JF697835, JF698567, –, JF698189, JF697898, JF698038, JF698495, JF698343, JF697735; Stipa barbata Desf., Pyke 704 (BC), Spain, Granada, JF698630, JF697838, JF698570, JF697784, JF698193, JF697901, JF698042, JF698499, JF698347, JF697738; Stipa brauneri (Pacz.) Klokov, Romaschenko 418 (BC), Ukraine, Crimea, JF698631, JF697839, JF698571, JF697785, JF698194, JF697902, JF698043, JF698500, JF698348, JF697739; Stipa breviflora Griseb., Soreng 5449 & Peterson (US), China, Qinghai, JF698632, –, JF698572, –, JF698195, JF697903, JF698044, JF698501, JF698349, JF697740; Stipa bungeana Trin., Soreng 5397, Peterson & Sun (US), China, Gansu, JF698633, JF697840, JF698573, JF697786, JF698196, JF697904, JF698045, JF698502, JF698350, JF697741; Stipa capensis Thunb., Pyke 703 (BC), Spain, Tarragona, GU254921, GU254724, GU254826, GU254963, JF698197, GU254773, JF698046, JF698503, JF698351, GU254640; Stipa capillacea Keng, Soreng 5382, Peterson & Sun (US), China, Sichuan, JF698634, –, JF698574, –, JF698198, JF697905, JF698047, JF698504, JF698352, JF697742; Stipa capillata L., Romaschenko 606 (KW), Kazakhstan, Jambyl, JF698635, JF697841, JF698575, JF697787, JF698199, JF697906, JF698048, JF698505, JF698353, JF697743; Stipa caucasica Schmalh., Ro- maschenko 635, Susanna & Kudratov (BC), Tajikistan, Zeravshan, JF698636, JF697842, JF698576, JF697788, JF698200, JF697907, JF698049, JF698506, JF698354, JF697744; Stipa parviflora Desf., Romaschenko 74 & Romo (US), Spain, Almería, GU254920, GU254719, GU254839, GU254962, JF698201, GU254774, JF698050, JF698507, JF698355, GU254641; Stipa pennata L., Romaschenko 466 (BC), Ukraine, Lugansk, GU254891, GU254718, GU254857, GU254967, JF698202, GU254759, JF698051, JF698508, JF698356, GU254637; Stipa purpurea Griseb., Soreng 5509, Peterson & Sun (US), China, Xizang, JF698637, JF697843, JF698577, JF697789, JF698203, JF697908, JF698052, JF698509, JF698357, JF697745; Stipa regeliana Hack., Tzvelev 694 (LE), Tajikistan, –, JF698638, JF697844, JF698578, –, JF698204, JF697909, JF698053, JF698510, JF698358, JF697746; Stipa subsessiliflora (Rupr.) Roshev., Ivanov s.n. (LE), Kyrgyzstan, –, JF698639, JF697845, JF698579, JF697790, JF698205, JF697910, JF698054, JF698511, JF698359, JF697747; Timouria saposhnikovii Roshev., Soreng 5475, Peterson & Sun (US), China, Gansu, GU254897, GU254692, GU254850, –, JF698206, GU254731, JF698055, JF698512, JF698360, GU254629; Trikeraia hookeri (Stapf) Bor, Koelz 2328 (US), India, Jammu and Kashmir, GU254909, GU254711, GU254830, GU254953, JF698207, GU254785, JF698056, JF698513, JF698361, GU254650; Trikeraia pappiformis (Keng) P.C. Kuo & S.L. Lu, Soreng 5653, Peterson & Sun (US), China, Xizang, GU254910, GU254721, GU254831, GU254954, JF698208, GU254784, JF698057, JF698514, JF698362, GU254649.

S3 TAXON — Article version: 9 Dec 2011: Electr. Suppl., 5 pp. Romaschenko & al. • Systematics and evolution of needle grasses

Appendix . A discussion of the stem-loop hairpin formation in trnH-psbA for the Stipeae. The type “1” loop sequence is probably a result of G→A substitution in the type “0”. It is characteristic in the majority of Stipeae and is also found in Aniso- pogon, Metcalfia, Stephanachne pappophorea, Sinochasea, and Triniochloa. Brylkinia has a type “2” loop sequence, probably a result of C→T substitution in the type “1”. Dielsiochloa lacks the loop sequence but preserves stem sequences of 17 bp length. Type “3” probably originated independently from type “1”, as a result of C→A substitution in type “0” (as mapped it would involve a two-step change from type 1 by initial reversal to type “0”). Type “3” is unique to Macrochloa. The most parsimonious interpretation of types 4, 5, and 6, is that they originated directly from type “1” by a single base substitution: C→A in type “4”; G→A in type “5”; and T→C in type “6”. Type “7” appears to have arisen from a G→C substitution in type “6”. Type “4” supports the monophyly of Ptilagrostis luquensis, P. junatovii, and P. dichotoma. Type “5” is found in Stipa capensis within the CAC clade and is present in all three geographically isolated samples of this species (Romaschenko, unpub.). Type “6” is specific to MAC, adding support to the monophyly of American achnatheroids. Type “7” is found in Achnatherum latiglume, A. perplexum, A. pinetorum, and A. scribneri (“Eriocoma group”).

Only the type “1” loop sequences produced inversions in Stipeae. Within the Pooideae inversion “1i” also appears in Stephanachne nigrescens (Phaeno- spermateae clade in Fig. 1). It also supports monophyly of the union of all sampled representatives of Stipa sections Leiostipa, Barbatae, Stipa, and Smirnovia, and is found independently in Piptochaetium brachyspermum. Its presence in two serially diverging lineages in the Stillmania/Piptatherum (P2) clade could be mapped as having been gained twice, or gained once and lost once. In our study Ampelodesmos contains type “1i” loop sequence which is in agreement with the sequence reported by Barkworth & al. (2008) [GenBank accession number EU204684]. It provides interpretation for the origin of the 1ii inversion found in the Psammochloa-“Neotrinia” clade. Since we did not find the original sequence for the 1ii inversion we assumed that it resulted not from the direct inversion of one of the polymorphic types but rather from mutation (T→A substitution) in the already inverted sequence. Presence of the 1i inversion in a sister, Ampelodesmos supports this assumption. Outside of Stipeae the stem of the single-stranded hairpin formation varies from 14 to 19 bp with correspondent ∆G from −4.7 to −18.6 kcal/mol, a value that indicates low to high likelihood of spontaneous formation (Downie & Palmer, 1992; Kelchner & Wendel, 1996; Kim & Lee, 2005; Lehtonen & al., 2009; Bain & Jansen, 2006; Catalano & al., 2009). Within the Stipeae the hairpin stem is generally 19 bp with higher likelihood of spontaneous formation derived from correspondent value of ∆G = −21.3 to −22.6 kcal/mol. Higher numbers of nucleotide pairs involved in hairpin stem formation result in decreasing the magnitude of ∆G, thus enhance the stability of the stem sequences. The long stem exhibits very few changes throughout the entire dataset. Longer hairpin formations (22 bp; ∆G = −21.3 to −21.8 kcal/mol) were found in Stipeae among the members of the Piptatherum and Piptatheropsis (25 bp; ∆G = −21.8 to −22.0 kcal/mol). The most prominent hairpin stem formation of 25 bp (without gaps) with the lowest free energy value of ∆G = −31.7 kcal/mol was detected for Ptilagrostis kingii. Bain, J.F. & Jansen, R.K. 2006. A chloroplast DNA hairpin structure provides useful phylogenetic data within tribe Senecioneae (Asteraceae). Canad. J. Bot. 84: 862–868. Catalano, S.A., Saidman, B.O. & Vilardi, J.C. 2009. Evolution of small inversions in chloroplast genome: A case study from a recurrent inversion in angiosperms. Cladistics 25: 93–104. Downie, S.R. & Palmer, J.D. 1992. Restriction site mapping of the chloroplast DNA inverted repeat: A molecular phylogeny of the Asteridae. Ann. Missouri Bot. Gard. 79: 266–283. Kelchner, S.A. & Wendel, J.F. 1996. Hairpins create minute inversions in noncoding regions of chloroplast DNA. Curr. Genet. 30: 259–262. Kim, K.J. & Lee, H.L. 2005. Widespread occurrence of small inversions in the chloroplast genomes of land plants. Molec. Cells 19: 104–113. Lehtonen, S., Myllys, L. & Huttunen, S. 2009. Phylogenetic analysis of non-coding plastid DNA in the presence of short inversions. Phytotaxa 1: 3–20.

Appendix . Chromosome numbers of taxa included in the phylogenetic analyses. Achnatherum bromoides (L.) P. Beauv. — 2n = 18, 28 (Tzvelev, 1976; Freitag, 1985; Vázquez & Devesa, 1996); A. calamagrostis (L.) P. Beauv. — 2n = 24 (Martinovský, 1980; Vázquez & Devesa, 1996); A. caragana (Trin.) Nevski — 2n = 24 (Freitag, 1985); A. confusum (Litv.) Tzvelev — 2n = 24 (Tzvelev, 1976); A. curvifolium (Swallen) Barkworth — 2n = 44 (Barkworth, 2007); A. eminens (Cav.) Barkworth — 2n = 44, 46 (Brown, 1951; Barkworth, 2007); A. hendersonii (Vasey) Barkworth — 2n = 34 (Barkworth, 2007); A. hymenoides (Roem. & Schult.) Barkworth — 2n = 46, 48 (Johnson, 1945; Barkworth, 2007); A. jacquemontii (Jaub. & Spach) P.C. Kuo & S.L. Lu — 2n = 24 (Freitag, 1985); A. latiglume (Swallen) Barkworth — 2n = 70 (Barkworth, 2007); A. lemmonii (Vasey) Barkworth — 2n = 34 (Barkworth, 2007); A. lettermanii (Vasey) Barkworth — 2n = 32, 66 (Johnson, 1962; Barkworth, 2007); A. nelsonii (Scribn.) Barkworth — 2n = 36, 44 (Johnson, 1962; Barkworth, 2007); A. occidentale (Thurb. ex S. Watson) Barkworth — 2n = 36 (Johnson, 1962; Barkworth, 2007); A. occidentale subsp. californicum (Merr. & Burtt Davy) Barkworth — 2n = 36 (Johnson, 1962); A. pekinense subsp. effusum (Maxim.) T. Koyama — 2n = 24 (Tzvelev, 1976; Tateoka, 1986); A. pinetorum (M.E. Jones) Barkworth — 2n = 32 (Barkworth, 2007); A. richardsonii (Link) Barkworth — 2n = 44 (Barkworth, 2007); A. robustum (Vasey) Barkworth — 2n = 64 (Barkworth, 2007); A. scribneri (Vasey) Barkworth — 2n = 40 (Barkworth, 2007); A. sibiricum (L.) Keng ex Tzvelev — 2n = 22, 24 (Avdulov, 1931); A. splendens (Trin.) Nevski — 2n = 48, 42 (Tzvelev, 1976; Freitag, 1985); A. turcomanicum Tzvelev — 2n = 24 (Freitag, 1985); Aciachne acicularis Laegaard — 2n = 22 (Reeder & Reeder, 1968; Macfarlane & Watson, 1980; Davidse & Pohl, 1994); A. flagellifera Lægaard — 2n = 22 (Reeder & Reeder, 1968; Macfarlane & Watson, 1980); Ampelodesmos mauritanicus (Poir.) T. Durand & Schinz — x = 12; 2n = 48, 96 (Decker, 1964; Macfarlane & Watson, 1980; Martinovský, 1980); Anatherostipa hans-meyeri (Pilg.) Peñailillo — 2n = 22 (Davidse & Pohl, 1994); Anemanthele lessoniana (Steud.) Veldkamp — 2n = 40–44 (Murray & al., 2005); Brachyelytrum erectum (Schreb.) P. Beauv. — x = 11; 2x = 22 (Macfarlane & Watson, 1980; Wu & Phillips, 2006); Brylkinia caudata (Munro) F. Schmidt — 2n = 40 (Macfarlane & Watson, 1980; Wu & Phillips, 2006); Celtica gigantea (Link) F.M. Vázquez & Barkworth — 2n = 96 (Vázquez & Devesa, 1996; Vázquez & Barkworth, 2004); Danthoniastrum compactum (Boiss. & Heldr.) Holub — 2n = 14 (Kozuharov & Petrova, 1991); Diarrhena fauriei (Hack.) Ohwi — 2n = 38 (Macfarlane & Watson, 1980); D. japonica Franch. & Sav. — 2n = 38 (Lee, 1967; Macfarlane & Watson, 1980); D. obovata (Gleason) Brandenburg — 2n = 60 (Macfarlane & Watson, 1980); Duthiea brachypodium (P. Candargi) Keng & Keng f. — x = 7 (Watson & Dallwitz, 1992); Hesperostipa comata (Trin. & Rupr.) Barkworth — 2n = 36, 44, 46 (Barkworth, 2007); H. neomexicana (Thurb.) Barkworth — 2n = 44 (Barkworth, 2007); H. spartea (Trin.) Barkworth — 2n = 44, 46 (Barkworth, 2007); Jarava ichu Ruiz & Pav. — 2n = 40 (Davidse & Pohl, 1994); J. plumosula (Nees ex Steud.) F. Rojas — 2n = 44 (Avdulov, 1931; Covas & Bocklet, 1945; Matthei, 1965); Lorenzochloa erectifolia (Swallen) Reeder & C. Reeder — 2n = 22 (Reeder & Reeder, 1968); Lygeum spartum L. — 2n = 16, 40 (Myers, 1947; Djabeur & al., 2008); Macrochloa tenacissima (Loefl. ex L.) Kunth — n = 12; 2n = 24, 40, 64, 66, 72 (Vázquez & Devesa, 1996; Vázquez & Barkworth, 2004); Nardus stricta L. — 2n = 26 (Avdulov, 1931; Barkworth, 2007); Nassella neesiana (Trin. & Rupr.) Barkworth — 2n = 28 (Matthei, 1965; Barkworth, 2007); N. trichotoma (Nees) Hack. ex Arechav. — 2n = 36 (Barkworth, 2007); Oryzopsis asperifolia Michx. — 2n = 46, 48 (Johnson, 1945); Pappostipa major (Speg.) Romasch. — 2n = 60 (Roig, 1964); P. speciosa (Trin. & Rupr.) Romasch. — 2n = 66, 68, 74 (Stebbins & Love, 1941; Roig, 1964; Matthei, 1965; Barkworth, 2007); Patis coreana (Honda ex Nakai) Ohwi — 2n = 46 (Tateoka, 1986); Phaenosperma globosa Munro ex Benth. — x = 12; 2n = 24 (Avdulov, 1931; Macfarlane & Watson, 1980; Wu & Phillips, 2006); Piptatheropsis canadensis (Poir.) Romasch., P.M. Peterson & Soreng ― 2n = 22 (Barkworth, 2007); P. exigua (Thurb.) Romasch., P.M. Peterson & Soreng — 2n = 22 (Barkworth, 2007); P. micrantha (Trin. & Rupr.) Romasch., P.M. Peterson & Soreng — 2n = 22, 24 (Johnson, 1945; Barkworth, 2007); P. pungens

S4 TAXON — Article version: 9 Dec 2011: Electr. Suppl., 5 pp. Romaschenko & al. • Systematics and evolution of needle grasses

Appendix . Continued. (Torr.) Romasch., P.M. Peterson & Soreng — 2n = 22, 24 (Johnson, 1962; Barkworth, 2007); P. shoshoneana (Curto & Douglass M. Hend.) Romasch., P.M. Peterson & Soreng — 2n = 20 (Curto & Henderson, 1998; Barkworth, 2007); Piptatherum coerulescens (Desf.) P. Beauv. — 2n = 24 (Kerguélen, 1975); P. holciforme (M. Bieb.) Roemer & Schultes — 2n = 24 (Johnson, 1945; Freitag, 1975; Tzvelev, 1976); P. miliaceum (L.) Coss. — 2n = 24 (Avdulov, 1931; Freitag, 1975); P. munroi (Stapf) Mez — 2n = 24 (Moinuddin & al., 1994); P. paradoxum (L.) P. Beauv. — 2n = 24 (Martinovský, 1980); P. songaricum (Trin. & Rupr.) Roshev. ex Nikitina — 2n = 24 (Tzvelev, 1976); P. virescens (Trin.) Boiss. — 2n = 24 (Avdulov, 1931; Freitag, 1975; Tzvelev, 1976); Piptochaetium avenaceum (L.) Parodi — 2n = 22, 28 (Cialdella & Giussani, 2002; Barkworth, 2007); Ptilagrostis junatovii Grubov — 2n = 22 (Tzvelev, 1976); P. kingii (Bol.) Barkworth — 2n = 22 (Johnson, 1945); P. mongholica (Turcz. ex Trin.) Griseb. — 2n = 22 (Tzvelev, 1976; Probatova & Sokolovskaya, 1980; Freitag, 1985; Tateoka, 1986); Stephanachne nigrescens Keng — 2n = 24 (Watson & Dallwitz, 1992); S. pappophorea (Hack) Keng — 2n = 24 (Watson & Dallwitz, 1992); Stipa barbata Desf. — 2n = 44 (Vázquez & Devesa, 1996); S. brauneri (Pacz.) Klokov — 2n = 44 (Avdulov, 1931; Tzvelev, 1976); S. capensis Thunb. — 2n = 18, 26, 34, 36 (Avdulov, 1931; Tzvelev, 1976; Freitag, 1985; Vázquez & Devesa, 1996); S. capillata L. — 2n = 44 (Avdulov, 1931; Tzvelev, 1976; Freitag, 1985; Vázquez & Devesa, 1996); S. caucasica Schmalh. — 2n = 44 (Tzvelev, 1976; Freitag, 1985); S. parviflora Desf. — 2n = 28 (Freitag, 1985; Vázquez & Devesa, 1996); S. pennata L. — 2n = 44 (Avdulov, 1931; Tzvelev, 1976; Freitag, 1985); Triniochloa stipoides (Kunth) Hitchc. — 2n = 32 (Brown, 1951; Reeder, 1971). Avdulov, N.P. 1931. Karyo-systematische Untersuchung der Familie Gramineen. Trudy Prikl. Bot. Suppl. 43: 1–352. Barkworth, M.E. 2007. Stipeae. 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