1Romaschenko & al. ? Systematics and evolution of needle grassesTAXON ? Article version: 9 Dec 2011: 27 pp. INTRODUCTION Peripheral Stipeae, clarification of what is in and what is outside the tribe. ? Stipeae Martinov are cool-season or tem- perate C3 grasses placed in subfamily Pooideae Benth. (GPWG, 2001). We estimate that the Stipeae s.str. include between 572 and 670 species with the number varying depending on how finely the Asian taxa are divided. The broadest molecular studies in Pooideae (GPWG, 2001; Davis & Soreng, 2007; Soreng & al., 2007; Bouchenak-Khelladi & al., 2008; Schneider & al., 2009) place the origin of Stipeae after the separation of Brachy elytreae Ohwi, Lygeeae J. Presl, and Nardeae W.D.J. Koch, among the remaining tribes Phaenospermateae Renvoize & Clayton s.l. (including Duthieinae Pilg. ex Potztal), Meliceae Link ex Endl. plus Brylkinieae Tateoka, Diarrheneae C.S. Campb., and core Pooideae (Brachypodieae Harz, Bromeae Martinov plus Hor- deeae Martinov (= Triticeae Dumort.), Poeae R. Br. including Aveneae Dumort.). Although Diarrheneae are usually resolved as sister to core Pooideae, the exact phylogenetic relationships of Stipeae within this set has varied among studies. A few gen- era placed in Stipeae s.l. clearly do not belong there: Milium L. (Clayton & Renvoize, 1986), Cyathopus Stapf, and Dichelachne Endl. (Tzvelev, 1989) have b een resolved within Poeae based on molecular data (D?ring, 2009; Schneider & al., 2009; Soreng & al., 2007), and Streptachne R. Br. (Tzvelev, 1989) has been accepted by other agrostologists as a synonym of Aristida L. in Aristidoideae Caro. Subtribe Duthieinae is sometimes placed in Aveneae, Sti- peae, or Phaenospermateae. In its broadest current sense Duthie- inae includes: Anisopogon R. Br., Danthoniastrum (J. Holub) 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, peterson@si.edu 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 micromor- phological 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 (mono- typic), 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). 2Romaschenko & al. ? Systematics and evolution of needle grasses TAXON ? Article version: 9 Dec 2011: 27 pp. Holub, Metcalfia Connert, Pseudodanthonia Bor & C.E. Hubb., Sinochasea Keng, Duthiea Hack., and Stephanachne Keng (Baum, 1973; Clayton & Renvoize, 1986; Holub, 1998; Wu & Phillips, 2006; Soreng & al., 2011). Based on molecular data, and morphological extension of that, Megalachne Steud. (D?ring, 2009) and Podophorus Phil. should be placed in Poeae (Clayton & Renvoize, 1986; Soreng & al., 2011), rather than in Duthieinae (Soreng & al., 2003). Uncertainty concerning the relationships of the American and Chinese elements of subtribe Duthieinae (sensu Clayton & Renvoize, 1986), led Soreng & al. (2003) and Wu & Phillips (2006) to place the genera of this subtribe within Stipeae, and accept Phaenospermateae as a monotypic tribe. Although accumulating molecular DNA data support placing Duthieinae in Phaenospermateae, the relation- ship of Duthieinae and Phaenospermateae to Stipeae remains poorly resolved and controversial (GPWG, 2001; Soreng & al., 2003, 2011; Davis & Soreng, 2007, 2010; Romaschenko & al., 2008, 2010; Schneider & al., 2009). Anisopogon was added to Duthieinae (Soreng & al., 2011) based on molecular data (GPWG, 2001; Davis & Soreng, 2007, 2010). Morphological inferences. ? Stipeae s.str. (excluding the above-mentioned elements and ignoring Ampelodesmos Link) are mostly tussock-forming grasses characterized by having single-flowered spikelets without rachilla extensions that dis- articulate above the glumes; florets with a distinct, sometimes sharp, often bearded callus, lemmas that are rounded on the back, (3?)5?9-nerved, and often concealing the palea (if the palea is exposed when the floret is closed, then the palea is coriaceous), terminally awned (or from between short lobes) where the awn is the result of fusion between the central and two lateral vascular traces, the awn usually geniculate and twisted in the proximal segment and sometimes caducous, plumose or scabrous; flow- ers with three, sometimes two, linear lodicules that are slightly indurate at maturity and glabrous (with venation weaker than in other subfamilies but often more distinct than in other Pooideae), often penicillate anthers tips, glabrous ovaries, caryopses with a long-linear hilum, small embryos with compound starch and no lipid; and small-sized chromosomes with a base number x = 7, 8, 9, 10, 11, or 12, for which the ancestral base number is uncertain. Unlike most Pooideae genera, the surfaces of the leaves occa- sionally have unicellular microhairs (Barkworth, 2007; Clayton & Renvoize, 1986; Soreng & Davis, 1998). The number of genera accepted in the Stipeae varies widely in modern treatments. Tzvelev (1976, 1989) accepted four gen- era in the former Soviet Union and 18 in the World, Clayton & Renvoize (1986) accepted nine in the World, Barkworth (2007) accepted 13 in the U.S.A. and Canada, and Soreng & al. (2003) list 18 accepted genera of Stipeae for the New World (see Table 1). These genera (excluding Ampelodesmos, which when 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 spe- cies, 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). Genus (or group isolated from a genus in which it has been included) Tzvelev (1976) Clayton & Renvoize (1986) Tzvelev (1989) Soreng & al. (2003) Barkworth & al. (2007) Our opinion, here Achnatherum P. Beauv. y (20) Stipa y (20) y (36) y (27 FNA na- tive, 7 Mexico, 1 New Zea- land, 21 Old World; ca. 56 in total) y, p.p. (21) Aciachne Benth. n/a y (1) y (1) y (3) n/a y (3) Amelichloa Arriaga & Barkworth n/a presumably in Stipa presumably in Stipa Nassella and Achnatherum y (5) y, tentatively, needs further study (5) Ampelodesmos Link n/a y, tribe Poeae (1) y, tribe Ampelo- desmeae (1) y, Stipeae subtribe (1) y, Stipeae (1) y, tentatively in subtribe Ampelo- desminae (1) Anatherostipa (Hack. ex Kuntze) Pe?ailillo n/a presumably in Stipa presumably in Stipa y (11) n/a not mono- phyletic, p.p. typic (8) Anemanthele Veldkamp n/a presumably in Stipa presumably in Stipa n/a n/a y (1) Austrostipa S.W.L. Jacobs & J. Everett n/a presumably in Stipa presumably in Stipa y y (63) y (63) 3Romaschenko & al. ? Systematics and evolution of needle grassesTAXON ? Article version: 9 Dec 2011: 27 pp. Celtica F.M. V?zquez & Barkworth n/a presumably in Stipa Stipa Macrochloa y (1) y (1) ?Eriocoma Nutt. group? n/a Oryzopsis y (3) Achnatherum Achnatherum y (29) Hesperostipa (M.K. Elias) Barkworth n/a presumably in Stipa presumably in Stipa y (5) y (5) y (5) Jarava Ruiz & Pav. n/a Stipa Stipa y (59) y (50) y, p.p. mono- phyletic (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 sect. Neotrinia Stipa Achnatherum Achnatherum Achnatherum y (1) 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., P.M. Peterson & Soreng n/a presumably in Stipa presumably in Stipa Jarava Jarava y (31) Patis Ohwi n/a Stipa y (1) (1 in FNA in Piptatherum) (1 in FNA in Piptatherum) y (3) Piptatheropsis Romasch., P.M. Peterson & Soreng n/a Oryzopsis presumably in Stipa Piptatherum Piptatherum y (5) 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- merly of Achnatherum and Jarava) n/a presumably in Stipa presumably in Stipa Achnatherum, Jarava Achnatherum, Jarava y, 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 Stipa) n/a presumably in Stipa unknown Achnatherum Achnatherum y (1) Stipa L. y (300) y (300) y (300) y y (200) y (110 min.) ?Stipella-Inaequiglumes group? (former Stipa sects.) Stipa sect. Stipella and Inaequiglumes Stipa Stipa a/a n/a y (2) ?Timouria Roshev. group? (includes some Achnatherum spp.) Achnatherum sect. Timouria Stipa Achnatherum n/a n/a y (5) 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, Podo phorus?Poeae s.l. Table ?. Continued. Genus (or group isolated from a genus in which it has been included) Tzvelev (1976) Clayton & Renvoize (1986) Tzvelev (1989) Soreng & al. (2003) Barkworth & al. (2007) Our opinion, here 4Romaschenko & al. ? Systematics and evolution of needle grasses TAXON ? Article version: 9 Dec 2011: 27 pp. represent Stipeae s.str. or the ?core Stipeae?. Generic boundar- ies among the genera in the Stipeae are problematic, especially within Achnatherum P. Beauv., Jarava Ruiz & Pav., Stipa L., Oryzopsis Michx., and Piptatherum P. Beauv. Difficult and controversial delimitations among these led agrostologists to adopt a broad concept of the genus Stipa to encompass all of the currently accepted genera except Oryzopsis, Aciachne Benth., and Piptochaetium J. Presl in the New World (Spegazzini, 1901; Hitchcock, 1935, 1951) and to split Piptatherum in the Old World from New World Oryzopsis s.l. (Freitag, 1975, 1985). Various studies were performed that described new genera and emend generic limits (Parodi, 1947, 1960; Barkworth, 1983, 1990, 1993; Jacobs & Everett, 1996; Pe?ailillo, 1996, 2002, 2003, 2005; Rojas, 1997; Torres, 1997a, b, c; Barkworth & Tor- res, 2001; Cialdella & Giussani, 2002; V?zquez & Barkworth, 2004; Arriaga & Barkworth, 2006; Cialdella & al., 2007; Ro- maschenko & al., 2008, 2010; Barber & al., 2009). Phylogenetic inferences for Stipeae based on traditional morphological characters are few. Based on morphological features the most comprehensive review was made by Tzvelev (1977) where phylogenetic weight was assigned to such charac- ters as shape of the lemma and callus, and development of awn indumentum. Tzvelev?s general phylogenetic system suggested there were two major lineages: (1) Stipa s.str. (including long lanceolate lemmas, strongly developed awn indumentums, and sharp callus) and; (2) Piptatherum (including short, hairless lem- mas with a caducous awn, and a blunt callus). Piptatherum was thought to have originated from more primitive Achnatherum- like species, whereas Ptilagrostis Griseb. and Achnatherum chinense (Hitchc.) Tzvelev were considered to be intermediate taxa between Achnatherum and Stipa s.str., and Achnatherum and Piptatherum, respectively. Piptochaetium and Nassella (Trin.) E. Desv. were considered to be close relatives, and Erio- coma Nutt. was thought to be a vicariant branch of Piptatherum in the New World. Over the years most of the American species of Stipa s.l. were placed in endemic New World genera, such as Anatherostipa (Hack. ex Kuntze) Pe?ailillo (Pe?ailillo, 1996) and Jarava, or into two genera (Achnatherum and Piptatherum) thought to be shared with Asia. Thomasson (1978, 1980, 1981, 1982, 1985) was first to document the phylogenetic importance of the lemma epider- mal pattern in Stipeae. Barkworth & Everett (1987) used this information to delineate hypothetical relationships among genera, pointing out that Stipa and Piptatherum have elon- gated lemma epidermal cells with sinuous lateral walls (pattern also revealed in Miocene-dated spikelets of the fossil genus Berriochloa M.K. Elias; Thomasson, 1978, 1982, 1985), and that Achnather um and Austrostipa S.W.L. Jacobs & J. Everett have short lemma epidermal cells with slightly sinuous to strait lateral walls. However, Barkworth & Everett (1987) followed Tzvelev (1977) in emphasizing the shape of the lemma and callus and development of awn indumentum rather than lemma epidermal pattern, and therefore postulated a similar phyloge- netic history. Hesperostipa (M.K. Elias) Barkworth and the ?Obtusa group? of Parodi (1946; included in Anatherostipa), and Nassella and Piptochaetium sensu Tzvelev were thought to be two pairs of closely related genera. Molecular inferences. ? Evolutionary relationships within Stipeae based on studies of molecular characters have not been clearly or fully elucidated (Jacobs & al., 2000, 2007; Barkworth & al., 2008; Romaschenko & al., 2008, 2010), and these as well as those based on morphology remain controver- sial (Barkworth & Everett, 1987; Barkworth, 1993; Chiapella, 2008). Prior to the study of Romaschenko & al. (2010), only a small fraction of the Stipeae s.str. genera and generic diversity were sampled, and outgroup sampling remained poor. Ampelodesmos, which is very different from Stipeae s.str. in gross morphology of the spikelet, but similar in anatomy and cytology to members of this tribe (Decker, 1964), appears from molecular data to be nested in Stipeae (Davis & Soreng, 2007, 2010; Hsiao & al., 1999; Romaschenko & al., 2008, 2010; Soreng & Davis, 1998). The genus has recently been included in Stipeae (Barkworth, 2007), transferred into the genus Stipa (Columbus & Smith, 2010), and is sometimes placed in the monotypic subtribe Ampelodesminae Conert (Soreng & al., 2003, 2011). In a series of morphological studies (Barkworth, 1990, 1993; Barkworth & Torres, 2001; Cialdella & Giussani, 2002) and in a phylogenetic study using molecular characters (Jacobs & al., 2000), Nassella and Piptochaetium were found to be sister genera. In more recent molecular phylogenetic analyses based on nrDNA ITS sequences, Jacobs & al. (2007) found that the Piptatherum-Oryzopsis complex along with Stipa s.str., Ampelodesmos, Anisopogon, Hesperostipa, and Piptochaetium were among early diverging lineages; Austrostipa was depicted as a derived clade with Anemanthele Veldkamp embedded; and Nassella was more closely related to Jarava than to Piptochae- tium (Cialdella & al., 2007). However, there was little statistical support for this structure and much polyphyly of genera was evident in these trees. In a phylogenetic analysis of 14 genera of Stipeae using four plastid loci, Cialdella & al. (2010) found only three mono- phyletic genera, Austrostipa, Hesperostipa, and Piptochaetium. Their primary focus was to elucidate relationships of Aciachne and Amelichloa Arriaga & Barkworth, both portrayed as para- or polyphyletic. In a combined analysis of ITS and four plastid loci (Romaschenko & al., 2008), Stipa s.str. and Piptatherum, along with Ampelodesmos, Piptochaetium, Anatherostipa, Hesperostipa, and Ptilagrostis were found to lie among the poorly resolved sets of basal lineages of Stipeae that share the lemma epidermal pattern of elongate cells with sinuous lateral walls. We identified this lemma epidermal pattern as ?saw-like? because the sidewalls of the lateral cells have a serrate appearance (Romaschenko & al., 2008, 2010). This contrasted sharply with the phylogenetic scheme proposed by Tzvelev (1977). The remainder of the Stipeae genera sampled were resolved in a well-supported achnatheroid clade consist- ing of: (1) a ?major American clade? containing New World Achnatherum, Jarava s.str. (excluding species of the former Stipa subg. Pappostipa Speg., all of which were placed within Jarava by Pe?ailillo, 2003, and elevated to generic status by Romaschenko & al., 2008, as Pappostipa (Speg.) Romasch., P.M. Peterson & Soreng), and Nassella with Amelichloa nested 5Romaschenko & al. ? Systematics and evolution of needle grassesTAXON ? Article version: 9 Dec 2011: 27 pp. within it; and (2) a core achnatheroid clade containing Aus- trostipa and Asian species of Achnatherum embedded with Eurasian species of Piptatherum (Romaschenko & al., 2010). We identified this pattern as ?maize-like? because it resembles the surface of an ear of corn by having short fundamental cells with straight sidewalls and square to rounded, closely placed silica bodies (Romaschenko & al., 2008, 2010). Based on five plastid and nuclear ITS sequences, Romaschenko & al. (2010) conducted a molecular phylogenetic study of all 21 genera of Stipeae. They presented a stepwise 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 Macro chloa Kunth to be sister to all other Stipeae, Achnatherum and Pip- tatherum to be polyphyletic, and provided support for recog- nizing the following monophyletic genera: Achnatherum s.str., Aciachne, Amelichloa, Austrostipa, Hesperostipa, Jarava s.str., Ortachne Nees ex Steud., Pappostipa, Piptatherum s.str., Pipto- chaetium, Ptilagrostis s.str., Stipa s.str., and Trikeraia Bor. Using four plastid regions, Romaschenko & al. (2011) conducted a phylogenetic analysis of the short-spikeleted spe- cies of the Stipeae. They recognized a Eurasian Piptatherum clade, a new North American genus, Piptatheropsis Romasch., P.M. Peterson & Soreng, and resurrected Patis Ohwi to include three species, two from Eurasia and one from North America. The main objectives of the present paper are to provide a better resolved and more highly supported phylogenetic hypoth- esis for the currently accepted genera and infrageneric groups within Stipeae compared to our previous study (Romaschenko & al., 2010). Particular effort is made to investigate the close relationships among the American genera Hesperostipa, Pipto- chaetium, and Anatherostipa that was suggested by Thomasson (1978, 1982, 1985) based on a study of lemma epidermal fea- tures. We add four plastid gene regions (rps3, rpl32-trnL, rps16- trnK, rps16 intron) to those we used previously (trnK-matK, matK, trnH-psbA, trnL-F, ndhF). We significantly expand our earlier survey of Stipeae by sampling an additional 65 species including some of uncertain taxonomic position. We test the monophyly of the achnatheroid clade and its correlation with specialized lemma epidermal anatomy, and test the monophyly of the remaining Stipeae lineages (excluding Macrochloa) that lack the achnatheroid lemma epidermal anatomy. We compare phylogenetic trees based on plastid and ITS datasets, discuss previous molecular and morphological studies where appro- priate, correlate lemma micromorphological characters and chromosome base numbers with our hypotheses based on our phylograms, interpret biogeographical relationships, and evalu- ate the phylogenetic signal of plastid inversions and indels. MATERIALS AND METHODS Taxon sampling. ? The Stipeae sample (voucher infor- mation and GenBank numbers are given in Appendix 1 in the Electronic Supplement) consists of all 23 to 26 accepted genera (Soreng & al., 2003, 2011), has enhanced coverage within ma- jor infrageneric lineages of polyphyletic genera detected in our previous studies (Romaschenko & al., 2008, 2010), and has im- proved focus on the taxonomic and geographical diversity within the tribe. At least two exemplars from each non-monotypic genus and internal group previously resolved have been selected to facilitate a more accurate interpretation of the generic concepts in the tribe. The total data set of 156 accessions representing 139 species comprises 7279 aligned nucleotide positions, 6639 bp from the plastid data, and 640 bp nuclear ribosomal ITS data (Table 2). Nine hundred and fifty sequences are newly reported to GenBank (Appendix 1). We included the type species of all infrageneric groups and genera sampled. In order to determine the phylogenetic limits of Stipeae and its relationships with other tribes we included six of seven peripheral genera currently placed in Phaenospermateae (Schneider & al., 2009; Soreng & al., 2011), some of which have been classified within Stipeae (Avdulov, 1931; Tzvelev, 1977; Wu & Phillips, 2006), and Bryl- kinia F. Schmidt (Brylkinieae-Meliceae), Triniochloa Hitchc. (Meliceae), Diarrhena P. Beauv. (Diarrheneae), and Dielsiochloa Pilg. (Poeae). Brachyelytrum erectum (Schreb.) P. Beauv., Nardus Table ?. Summary of nine plastid regions and nrDNA ITS used in this study. trnK- matK matK trnH- psbA trnL-F rps3 ndhF rpl32- trnL rps16- trnK rps16 intron Plastid 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 6Romaschenko & al. ? Systematics and evolution of needle grasses TAXON ? Article version: 9 Dec 2011: 27 pp. stricta L., and Lygeum spartum L. were included as outgroups based on their well-documented early diverging positions in sub- family Pooideae (Hilu & al., 1999; GPWG, 2001; Soreng & al., 2003, 2007, 2011; Davis & Soreng, 2007; Bouchenak-Khelladi & al., 2008; Schneider & al., 2009). DNA extraction, amplification, and sequencing. ? The plant tissue was disrupted using Qiagen TissueLyser, and DNA was isolated using a BioSprint 96 DNA Plant Kit (Qiagen, Va- lencia, California, U.S.A.). For amplification, the genomic DNA was combined with 1? reaction buffer (200 mM Tris-HCl, 500 mM NH4 ; Bioline Biolase Taunton, Madison, Wisconsin, U.S.A.) without Mg++, 2 mM MgCl2, 200 mM dNTPs, 1.5 ?l of Taq polymerase (Bioline Biolase Taunton), and 40 pmol/?l each of forward and reverse primers. We targeted nine chloroplast DNA regions from the large single copy (LSC) and the small single copy (SSC) regions of the genome: trnK-matK (intron, LSC), matK (coding region, LSC), trnH?GUG-(rps19)-psbA (coding region, spacer, LSC), trnL-trnF (intron, spacer, LSC), rps3 (coding region, LSC), ndhF (coding region, SSC), rpl32-trnLUAG (spacer, SSC), rps16- trnK (spacer, LSC), and rps16 (intron, LSC). The trnK-5?matK intergenic spacer (IGS) was amplified and sequenced using the primers trnK3914F (Johnson & Soltis, 1995) and trnK660SR (Romaschenko & al., 2008). The primers trnK660SF and matK1412SR (Romaschenko & al., 2008) were used to amplify the 5?-end portion (555 bp) of matK. The trnH?GUG-psbA region was amplified with primers trnHf??GUG (Tate & Simpson, 2003) and psbA3f (Sang & al., 1997). In Stipeae, as in most monocots (Wang & al., 2008), this region encompasses the entire copy of the rps19 gene embedded between the trnH?GUG and psbA genes. Within Stipeae the lengths and variability (%) of the components of this region are: trnH?GUG-rps19 IGS (inverted repeat, IRA)?195 bp (5.1%), rps19 (IRA)?216 bp (4.1%), and rps19-psbA IGS (LSC)?165 bp (22%). The trnH?GUG-psbA re- gion has been used for phylogenetic inferences (Shaw & al., 2005, 2007) and barcoding purposes (Kress & al., 2005, 2009; Kress & Erickson, 2009). The trnL-trnF region which includes the trnL intron, the 3?trnL exon, and the trnL-trnF intergenic spacer, was ampli- fied using primers 5?trnLUAA(f) and trnFGAA(c) (Taberlet & al., 1991). The rps3 gene was amplified and sequenced using primers rps3C29F and rps3C697R (Peterson & al., 2010a, b). Variability rate and ease of amplification make it suitable for phylogenetic study, especially when working with older herbar- ium specimens. For the ndhF gene we amplified and sequenced the variable 3?-end (783 bp) with the primers ndhF1311F and ndhF2091R (Romaschenko & al., 2010). The region rpl32-trnLUAG was amplified and sequenced with primers trnLUAG and rpl32-F (Shaw & al., 2007). The sequences contain the entire rpl32-trnLUAG IGS and a small portion of the trnLUAG gene. The rps16-trnK IGS was amplified and sequenced with rpS16-900F and 3914PR primers (Peterson & al., 2010a, 2010b). Since the rpS16-900F primer is placed at the 3?-end of rps16 intron the amplified region contains the entire 3?rps16 exon and rps16-trnK IGS. For amplification of the rps16 intron the primers rpS16R and rpS16F were used (Peterson & al., 2010a, b). The amplification parameters that we found to be effective across a wide range of the taxa for the plastid regions were: initial denaturation phase of 4 min at 94?C; followed by 35 cycles of denaturation at 94?C for 40 s, annealing phase at 50?C?56?C for 40 s, extension phase at 72?C for 1 min 30 s, and final extension at 72?C for 10 min. We used 50?C?51?C of primer annealing temperature for all coding plastid regions. The entire nuclear ribosomal ITS region was amplified using primers ITS4 (White & al., 1990) and ITS5A (Stanford & al., 2000) with the following thermocycler settings: 4 min at 94?C; followed by 35 cycles of 94?C for 30 s, 55?C for 30 s, 72?C for 1 min 20 s, and a final extension at 72?C for 10 min. All PCR products were cleaned with ExoSAP-IT (USB, Cleveland, Ohio, U.S.A.). DNA sequencing was performed with BigDye Terminator Cycle Sequencing v.3.1 (PE Applied Biosystems, Foster City, California, U.S.A.) according to the following parameters: 80?C, 5 min; 25 or 30 cycles of 95?C for 10 s, 50?C for 5 s and 60?C for 4 min. Sequenced prod- ucts were analyzed on an ABI PRISM 3730 DNA Analyzer 7900HT (ABI). All regions except rpl32-trnL, rps16 intron, and 3?rps16-5?trnK were sequenced in one direction. Relatively short regions (500?750 bp) covered by our primers were easily interpreted allowing us to accumulate sequences from different parts of the genome for phylogenetic inference (Shaw & al., 2005, 2007). The rpl32-trnL, rps16 intron, and 3?rps16- 5?trnK were sequenced in both directions and the program Sequencher v.4.8 (Gene Codes Corp., Ann Arbor, Michigan, U.S.A., 1991? 2007) was employed to produce the contig sequence for the entire region. Phylogenetic analyses. ? Sequences alignment was done manually using BioEdit v.7.0.5.3 (Hall, 1999). The in- dels, minute inversions, and other regions for which alignment was considered ambiguous, were excluded from the analyses. OLIGO v.7.33 (Rychlik, 2009) was implemented to investi- gate the nature of putative minute inversions detected in the rpl32-trnL IGS (between 671?676 bp of aligned sequence) and rps19-psbA IGS of the trnH-psbA region. The objectives were to define these minute inversions and determine if they were reversed point mutations or constitute stable loops of the single-stranded hairpin formations (Downie & Palmer, 1992; Kelchner & Wendel, 1996; Kim & Lee, 2005; Bain & Jansen, 2006; Catalano & al., 2009; Lehtonen & al., 2009). The results of this analysis were used as additional characters or support for the putative phylogenetic groups (see Appendix 2 in the Electronic Supplement). These mutations were plotted on the plastid phylogram (Fig. 1). The amount of excluded data for each region is presented in Table 2. No data was excluded from matK, rps3, and ndhF. All gaps were treated as missing data. We conducted maximum likelihood and Bayesian analy- ses to infer phylogenies. The maximum likelihood analysis was conducted with the program GARLI v.0.951 (Zwickl, 2006). Bayesian and maximum likelihood analyses yielded trees with visually similar topology, i.e., the trees are visually the same, but some branch lengths could differ minutely. A test run of Bayesian analysis for the combined plastid dataset under the single GTR?+?G model yielded the same topology and posterior probability (PP) values as the Bayesian analysis for 7Romaschenko & al. ? Systematics and evolution of needle grassesTAXON ? Article version: 9 Dec 2011: 27 pp. a partitioned dataset performed under models suggested by MrModeltest v.1.1b (Nylander, 2002) for separate regions. The Akaike information criterion models are indicated in Table 2 (Kimura, 1981; Tavar?, 1986; Posada & Crandall, 1998). Bootstrap analyses were performed under maximum likeli- hood algorithm using GARLI (Zwickl, 2006) and were set for 1000 bootstrap replicates. The majority-rule trees were then constructed in PAUP* v.4.0b10 (Swofford, 2000). Bootstrap (BS) values of 90%?100% were interpreted as strong support, 70%?89% as moderate, 50%?69% as weak, and those under 50% were not reported. We identify some clades as ?group? in regular script when there is no formal genus name for them (e.g., ?Miliaceae group?), or with the genus in italics when there was at least one species included with others that have not been formally transferred (e.g., ?Eriocoma group?). Bayesian posterior probabilities were estimated using the program MrBayes v.3.01 (Huelsenbeck & Ronquist, 2001; Ronquist & al., 2005) with DNA substitution models selected using the program MrModeltest v.1.1b (Nylander, 2002). The plastid dataset was then partitioned into four subsets (1: trnk- matK?+?rps3?+?rps16-trnK?+?rpl32-trnL; 2: matK?+?trnL-F?+?rps16 intron; 3: trnH-psbA; 4: ndhF) and were processed implement- ing different parameters suggested by Akaike information cri- terion (Table 2). The ITS data were calculated separately. Each Bayesian analysis was initiated with random starting trees and initially run for two million generations, sampling once per 100 generations. The analysis was continued until the value of 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 was set at 0.25. The test of alternative phylogenetic hypotheses was ac- complished using parametric bootstrapping (Huelsenbeck & al., 1995; Swofford & al., 1996; Goldman & al., 2000) as imple- mented in Mesquite v.2.6 (Maddison & Maddison, 2009). The best-scoring maximum likelihood tree (the optimal topology for unconstrained dataset) and simulation model parameters were obtained using GARLI (Zwickl, 2006) for the single GTR?+?G model of sequence evolution in maximum likelihood searches. The same procedure was repeated for the maximum likelihood searches with monophyly constraints consistent with the research or alternative hypothesis (other than the null hy- pothesis). The constraint topology and model parameters were used to simulate 1000 data matrices equal in size to the original matrix using Mesquite v.2.6. These parameters were then used in PAUP* to find the most parsimonious trees constructed un- der topological constraints and the most parsimonious uncon- strained trees. Differences in tree length for constrained and unconstrained searches for each of the 1000 simulated matri- ces were calculated and plotted as histograms using Mesquite v.2.6. The distribution of tree length differences between two potential topologies was estimated. If the difference between constrained and unconstrained topologies fell outside the 95% confidence interval of this distribution (P < 0.05), the alterna- tive hypothesis was rejected. Scanning electron microscopy. ? Lemma ultrastructure was studied using dry mature seeds sampled from herbarium specimens from the majority of the Stipeae species used in the phylogenetic analysis. To remove epicuticular wax the lemmas were cleaned in xylene for four hours. Samples were mounted and then covered with gold from a vacuum spray gun (JII-4X, Japan). The ultrastructure of the lemma was studied at varying magnifications using a Jeol (JSM35C, Japan) scanning electron microscope. We illustrate the lemma epidermal pattern (LEP) for 44 species (Figs. 3?4); 32 of these have never been published; eight of these (Achnatherum hymenoides (Roem. & Schult.) Bark- worth, Achnatherum stillmanii (Bol.) Barkworth, Oryzopsis asperi folia Michx., Piptatheropsis canadensis (Poir.) Romasch. & al., Piptatheropsis micrantha (Trin. & Rupr.) Romasch. & al., Piptatherum miliaceum (L.) Coss., Ptilagrostis kingii (Bol.) Barkworth, Ptilagrostis mongolica (Turcz. ex Trin.) Griseb.) are of much higher quality and/or complement ink drawings; three of these (Celtica gigantea (Link) F.M. V?zquez, Macro chloa tenacissima (Loefl. ex L.) Kunth, Nassella neesiana (Trin. & Rupr.) Barkworth) confirm previous work (Thomasson, 1978, 1980; Barkworth, 1983; Barkworth & Everett, 1987; V?squez & Barkworth, 2004); and one replaces an erroneously published pattern for Stipa pennata L. (V?squez & Barkworth, 2004). Cytogenetics. ? We surveyed the original literature and floristic treatments for chromosome numbers reported for spe- cies of Stipeae, and discuss the possible evolution of base chro- mosome numbers in the tribe (see Appendix 3 in the Electronic Supplement for reported chromosome numbers). RESULTS Analysis of plastid sequences. ? Since very little differ- ence and no contradictions in generic composition or arrange- ment were observed among maximum likelihood trees in the individual plastid analyses, the data were combined. The combined plastid data provides a rather well-resolved tree, mostly with high backbone support (Fig. 1). The first split on the phylogenetic tree strongly indicates (BS = 99, PP = 1.00) the Phaenospermateae (BS = 89, PP = 1.00) to be sister to the remainder of the Pooideae represented by the tribes Meliceae, Poeae, Diarrheneae, and Stipeae (BS = 99, PP = 1.00). There is strong support for clades of Meliceae-Brylkinieae (BS = 99, PP = 1.00), Diarrhenaeae-Poeae (BS = 100, PP = 1.00), and Stipeae (BS = 100, PP = 1.00). However, the relationships among these clades are weakly supported (BS = 66, PP = 0.86). Within Stipeae, the monotypic genus Macrochloa is re- solved as sister to a clade of all remaining taxa with strong support (BS = 99, PP = 1.00). Above this split Stipeae comprise three well-supported clades, each with a saw-like LEP, and we refer to these clades as SL1, SL2, and SL3. A fourth lineage that is the sister of SL3 we designate as the ?achnatheroid clade? (AC). The first saw-like lineage (SL1; BS = 99, PP = 1.00) con- sists of two geographically distinct clades: one is primarily distributed in Eurasia, which we call the ?Eurasian saw-like lin- eage (ESL),? the other is distributed in the Americas, which we call the ?American saw-like lineage (ASL)?. The ESL (BS = 86, 8Romaschenko & 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 = 100, PP = 1.00), which we refer to as the ESL1 that includes the type of Stipa (S. pennata). This clade is split into two strongly supported clades: (1) a clade that includes S. purpurea Griseb., S. subsessiliflora (Rupr.) Roshev., S. regeliana Hack., S. bun- geana Trin., and S. capillacea Keng (BS = 96, PP = 1.00) and (2) a clade that includes S. pennata, S. brauneri (Pacz.) Klokov, S. barbata Desf., S. caucasica Schmalh., S. capillata L., and S. breviflora Griseb. (BS = 100; PP = 1.00). The other is a weakly supported, taxonomically complex clade (BS = 68, PP = 0.82), which we refer to as the ESL2 clade that includes Ptila- grostis s.str. as well as several monotypic or ditypic genera such as Ampelodesmos, Psammochloa Hitchc., Achnatherum splen- dens (Trin.) Nevski (?Neotrinia?, Romaschenko & al., 2010), Oryzopsis asperifolia (the only American representative of this clade), Trikeraia, and Orthoraphium Nees. The ESL2 clade includes a strongly supported Ampelodesmos-Psammochloa- ?Neotrinia? clade (BS = 99, PP = 1.00) in which Ampelodesmos is sister to a clade of Psammochloa and ?Neotrinia? (BS = 100, PP = 1.00). The other, weakly supported clade (BS = 66, PP = 0.86) within ESL2 includes Oryzopsis asperifolia as sister to a strongly supported Trikeraia-Orthoraphium-Ptilagrostis clade (BS = 100, PP = 1.00). A strongly supported clade (BS = 96, PP = 1.00) of Trikeraia species is part of a clade that includes Orthoraphium roylei Nees and five species of Ptilagrostis; their relationships are not well-resolved. The monophyly of Ptilagrostis s.str. (excluding P. kingii and P. pelliotii (Danguy) Grubov) is weakly supported (BS = 64, PP = 0.83). However, two clades within Ptilagrostis are strongly supported (BS = 100, PP = 1.00): one includes Ptilagrostis mongolica (the type) and P. malyshevii Tzvelev; and the other includes P. luquensis P.M. Peterson & al., P. junatovii Grubov, and P. dichotoma Keng ex Tzvelev. The ASL clade is weakly supported (BS = 58, PP = 0.93), and comprises two clades: ASL1 and the ASL2. The ASL1 clade has no support and splits into two major clades: (1) a clade (BS = 69, PP = 0.75) that encompasses Ptilagrostis kingii as sister to the strongly supported Piptatheropsis clade (BS = 100, PP = 1.00) that includes five former members of North American Piptatherum (called the ?Piptatheropsis group? by Romaschenko & al., 2010; and 2011?as a new genus); and (2) a South American clade (BS = 95, PP = 1.00) that includes a monophyletic Ortachne and Pappostipa, each with strongly supported (BS = 100 and PP = 1.00) crown nodes. The ASL2 clade is strongly supported (BS = 94, PP = 1.00) and includes a monophyletic Hesperostipa (BS = 100 and PP = 1.00) from North America as sister to a strongly supported clade (BS = 90, PP = 1.00) of the remaining species. Among these remaining species there is a single clade of the pan-American genus Pipto- chaetium (BS = 100, PP = 1.00) as sister to a clade containing the South American genera Aciachne, Anatherostipa and Loren- zochloa Reeder & C. Reeder. Anatherostipa s.l. is polyphyletic, since two species, A. hans-meyeri (Pilg.) Pe?ailillo and A. rosea (Hitchc.) Pe?ailillo, are united in a clade with Lorenzochloa (BS = 100, PP = 1.00) that is sister to a strongly supported (BS = 100, PP = 1.00) clade that includes Aciachne plus the remaining Anatherostipa s.str. species (BS = 97, PP = 1.00). The Aciachne clade, composed of two species, is strongly supported (BS = 100, PP = 1.00), and this is sister to the Anatherostipa s.str. clade (BS = 100, PP = 1.00) composed of four species. The sister to the SL1 clade, which includes all remaining species (clades SL2, SL3, and AC) is moderately supported (BS = 77, PP = 0.95). The SL2 clade (BS = 100, PP = 1.00) contains the far East-Asian Patis which in this tree includes P. obtusa (Stapf) Romasch. & al. (? Piptatherum kuoi S.M. Phillips & Z.L. Wu) and P. coreana (Honda) Ohwi (? Achnatherum corea- num (Honda) Ohwi) and is sister to a strongly supported clade (BS = 100, PP = 1.00) including SL3 and the AC. The SL3 clade (BS = 66, PP = 0.71) encompasses the morphologically isolated North American species Achnatherum stillmanii (?Stillmania?) as sister to a strongly supported clade of Eurasian Piptatherum s.str. (BS = 100, PP = 1.00), including P. coerulescens (Desf.) P. Beauv., the type of this genus. The strongly supported AC (BS = 100, PP = 1.00) contains three major clades. The ?core achnatheroid clade? (CAC, BS = 85, PP = 0.96) is sister to a moderately supported clade (BS = 84, PP = 1.00) that is divided into two highly supported sister clades: the Asian ?Timouria Roshev. group? (A2, PP = 100, PP = 1.00); and the ?major American clade? (MAC, BS = 100, PP = 1.00). CAC includes the monotypic western Mediterranean genus Celtica F.M. V?zquez & Barkworth as sister to a mod- erately supported clade (BS = 81, PP = 1.00) that consists of an Australian clade (BS = 52, PP = 0.95) and a Eurasian clade of Achnatherum (A1, PP = 0.91). The Australian clade includes Anemanthele, a monotypic genus from New Zealand, as sis- ter to a monophyletic Austrostipa (BS = 86, PP = 1.00). The A1 clade encompasses 10 species of Achnatherum, including A. calamagrostis (L.) P. Beauv., the type of the genus, and two species of Piptatherum sect. Virescentia Roshev. ex Freitag (P. virescens (Trin.) Boiss. and P. paradoxum (L.) P. Beauv.). The two representatives of Piptatherum sect. Miliacea Roshev. ex Freitag (P. miliaceum (L.) Coss. and P. thomasii (Duby) Kunth) form a strongly supported clade (?Miliacea group,? BS = 100, PP = 1.00) that is sister to the A1 clade. However, this sister relationship is poorly supported (PP = 0.67). Thus, the Eurasian Piptatherum species are resolved in three distinct clades: Patis in SL2, Piptatherum s.str. in SL3, the ?Miliacea group? and P. sect. Virescentia in CAC. North American spe- cies formerly treated in Piptatherum are contained in SL1 in the genus Piptatheropsis. Stipa capensis Thunb. and S. parvi- flora Desf. (of the monotypic Stipa sects. Stipella Tzvelev and Inaequeglumes (Bor) F.M. Vazquez & Devesa) of arid Medi- terranean origin are united without any support and appear as 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. 9Romaschenko & al. ? Systematics and evolution of needle grassesTAXON ? Article version: 9 Dec 2011: 27 pp. Brachyelytrum erectu Nardus stricta Phaenosperma globosa Danthoniastrum compactum Anisopogon avenaceus Duthiea brachypodium Sinochasea trigyna Diarrhena fauriei Diarrhena japonica Macrochloa tenacissima Stipa brauneri Stipa pennata Ampelodesmos mauritanicus Psammochloa villosa Achnatherum splendens Oryzopsis asperifolia Trikeraia pappiformis Trikeraia hookeri Orthoraphium roylei Ptilagrostis mongolica Ptilagrostis dichotoma Ptilagrostis junatovii Ptilagrostis luquensis Ptilagrostis kingii Piptatheropsis micrantha Piptatheropsis shoshoneana Piptatheropsis pungens Ortachne rariflora Ortachne breviseta Pappostipa major Pappostipa chrysophylla Pappostipa vaginata Pappostipa hieronimusii Pappostipa speciosa Hesperostipa spartea Hesperostipa comata Hesperostipa neomexicana Piptochaetium avenaceum Piptochaetium featherstonei Piptochaetium brachyspermum Piptochaetium panicoides Anatherostipa hans-meyeri Anatherostipa rosea Aciachne acicularis Aciachne flagellifera Anatherostipa venusta Anatherostipa mucronata Lorenzochloa erectifolia Anatherostipa rigidiseta Piptatherum holciforme Piptatherum ferganense Piptatherum angustifolium Piptatherum hilariae Celtica gigantea Austrostipa scabra Austrostipa tenuifolia Austrostipa campylachne Austrostipa semibarbata Stipa capensis Stipa parviflora Achnatherum calamagrostis Piptatherum miliaceum Achnatherum sibiricum Achnatherum inebrians Achnatherum bromoides Piptatherum virescens Piptatherum paradoxum Achnatherum/Timouria saposhnikovii Achnatherum chinense Achnatherum caragana Achnatherum robustum Achnatherum parishii Achnatherum latiglume Achnatherum occidentale subsp. californicum Achnatherum occidentale Achnatherum nelsonii Jarava media Jarava plumosula Jarava pseudoichu Jarava ichu Jarava castellanosii Jarava scabrifolia Amelichloa caudata Amelichloa clandestina Nassella clarazii Nassella pfisteri Nassella neesiana Nassella trichotoma Nassella filiculmis 100 99 100 100 100 99 100 96 100 83 100 94 90 100 95 99 100 98 100 97 100 92 100 100 82 95 78 95 86 100 85 99 100 100 89 100 100 97 70 1.00 99 1.00 1.00 1.00 1.00 0.97 1.00 0.83 1.00 0.86 1.00 1.00 1.00 0.75 0.99 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.62 1.00 1.00 1.00 1.00 1.00 1.00 0.96 0.99 MAC 1.00 0.79 1.00 1.00 1.00 1.00 1.00 86 0.82 68 L a d d e r M a i z e - li ke L EP CAC AC Diarrhena obovata Dielsiochloa floribunda Triniochloa stipoides Brylkinia caudata Stephanachne nigrescens Stephanachne pappophorea Lygeum spartum Ptilagrostis malyshevii Stipa caucasica Stipa capillata Stipa breviflora Stipa capillacea Stipa bungeana Stipa regeliana Stipa subsessiliflora Stipa purpurea Stipa barbata Piptatheropsis canadensis Piptatheropsis exigua Piptochaetium montevidense Anatherostipa obtusa Patis obtusa Patis coreana 100 1.00 Achnatherum stillmanii Piptatherum coerulescens Piptatherum munroi Piptatherum aequiglume Piptatherum songaricum Piptatherum sogdianum Piptatherum fedtschenkoi 1.00 Anemanthele lessoniana Austrostipa nitida Austrostipa nodosa Austrostipa pycnostachya Austrostipa flavescens Austrostipa trichophylla Austrostipa juncifolia Austrostipa hemipogon1.00 Piptatherum thomasii Achnatherum turcomanicum Achnatherum jacquemontii Achnatherum pekinense Achnatherum brandisii Achnatherum confusum Achnatherum pubicalyx Ptilagrostis pelliotii Jarava annua Achnatherum wallowaense Achnatherum hendersonii Achnatherum curvifolium Achnatherum scribneri Achnatherum perplexum Achnatherum pinetorum Achnatherum lettermanii Achnatherum richardsonii Achnatherum aridum Achnatherum lobatum Achnatherum lemmonii Achnatherum hymenoides Achnatherum diegonense Achnatherum constrictum Achnatherum eminens Achnatherum multinode Achnatherum acutum 100 89 100 84 99 99 54 95 100 100 96 69 99 64 100 99 99 98 96 77 100 66 100 100 100 85 81 100 52 98 85 90 85 99 72 94 87 95 84 98 100 76 62 87 73 81 55 56 75 99 77 91 100 A1 A2 A3 A4 Patis Stillmania Miliacea gr. Neotrinia Piptatherum Achnatherum Eriocoma gr. Timouria gr. Pseudoeriocoma gr. Piptatheropsis 1.00 100 1.00 99 1.00 1.00 Metcalfia mexicana1.00 0.86 0.63 1.00 1.00 1.00 0.86 66 100 1.00 1.00 1.00 1.00 0.94 1.00 1.00 1.00 66 0.74 1.00 1.00 58 0.93 69 1.00 1.00 1.00 1.00 1.00 0.94 1.00 100 1.00 77 0.95 100 1.00 0.71 1.00 1.00 1.00 100 0.95 1.00 1.00 1.00 Austrostipa macalpinei Austrostipa compressa79 1.00 65 1.00 1.00 0.67 0.91 1.00 1.00 0.98 1.00 1.00 1.00 1.00 88 1.00 1.00 1.00 1.00 1.00 1.00 0.98 65 0.97 1.00 1.00 0.98 1.00 57 66 1.00 60 0.95 0.94 57 U no rde red S a w - l ike L EP U no rde red S a w - li ke L EP O rde red S a w - li ke L EP M a i z e - li ke L EP North America South America Mediterranean Eurasia Australia and New Zealand CTGAAA TTTCAG TATCAG CTGGAA ATGGAA ATGAAA CTAAAA CCGAAA CCCAAA 1 1 1ii i 2 3 4 5 6 7 1i 1i 1i 1ii 1i 1i 1i ? ?? rpl32-trnL - 8 bp deletion (104) - 5 bp deletion (214) rps16-trnK ? - 16 bp insertion (214) ? - 5 bp insertion (830) ? ? trnL-F ? - 10 bp deletion (89) - 7 bp insertion (110) - 7 bp insertion (777) - 5 bp insertion (115) ? - 21 bp repeat insertion (343) ? - 5 bp insertion (777) ? - 1 bp insertion (396) ? - 20 bp insertion (571) - 21 bp insertion (170) (except Metcalfia) trnH-psbA rps16 intron 0 TTGAAA ASL ESL SL1 SL2 SL3 ESL1 ESL2 ASL1 ASL2 7 7 7 7 - li ke LE P Stipella 1 Xi Yi GGTCAG TTTTAG Stipeae Brachyelytreae Nardeae Lygeeae Phaenospermateae Meliceae Poaeae Diarrheneae 3 0 0 0 0 0 2 ? ? ? Xi Yi 1 4 6 5 No rps19-psbA loop sequence? ? 61 0.64 10 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 of the central Asian species Timouria saposhnikovii Roshev., A. chinense, A. caragana (Trin.) Nevski, and Ptilagrostis pelliotii. The MAC is strongly supported (BS = 100, PP = 1.00) and is split into two separate clades: the ?Eriocoma group? (A3, BS = 98, PP = 1.00), and a complex clade (BS = 100, PP = 1.00) that includes the ?Pseudoeriocoma group? (A4, BS = 70, PP = 1.00) and representatives of Jarava, Nassella, and Amelichloa. The ?Eriocoma group? and ?Pseudoeriocoma group? include North American species currently placed in Achnatherum but they are resolved in separate, strongly supported clades. Spe- cies of Achnatherum within AC have maize-like LEPs and are scattered in four clades: two Eurasian (A1, A2) and two of North American origin (A3, A4). Achnatherum splendens (?Neotrinia?) of the SL1/ESL2 clade, and A. stillmanii (?Still- mania?) of the SL3 clade, have saw-like LEPs. Jarava annua (Mez) Pe?ailillo is located in an unsupported grade between the ?Eriocoma group? A3, and the other mem- bers of MAC. These other members of MAC form a strongly supported clade (BS = 100, PP = 1.00) that includes a grade of J. media (Speg.) Pe?ailillo and J. plumosula (Nees ex Steud.) F. Rojas (members of former Stipa subg. Ptilostipa Speg.? Spegazzini, 1901; Roig, 1964), the ?Pseudoeriocoma group? A4), Jarava s.str., Amelichloa, and Nassella. Jarava plumosula is sister to a clade (BS = 89, PP = 1.00) that includes an unre- solved trichotomy of the ?Pseudoeriocoma group? (A4, BS = 70, PP = 1.00), Jarava s.str. (BS = 100, PP = 1.00, including the type, J. ichu Ruiz & Pav.), and the Amelichloa-Nassella clade (BS = 97, PP = 1.00). The ?Pseudoeriocoma group? (A4) is composed of five south-western North American species, primarily distributed in Mexico. Plastid DNA minute inversions and indels. ? Minute inversions were detected in rpl32-trnL and trnH-psbA. How- ever, only in data from the trnH-psbA region were we able to find cases of clear phylogenetic utility. We identified a stem- loop hairpin formation involving a small region flanking the inverted repeat (IRA-LSC). All taxa in our dataset (except Dielsio chloa, which lacks the loop) exhibited a hairpin for- mation characterized by relatively long and conserved stem sequences (averaging 19 bp in length) linked by a short loop (6 bp). The poly-A end of IRA is sometimes partially involved in the stem formation. Polymorphic sequences in the trnH-psbA hairpin loop and inverted loop were mapped on our plastid tree. This loop proved to be inverted in some sequences (Fig. 1: Xi, Yi, 1i and 1ii). The sequences from the loop also exhibited base mutations that accumulate in a phylogenetically informative order in some cases (Fig. 1: sequence 0 gives rise to 1 and frequently reverses except in Stipeae above Macrochloa; 1 independently gives rise to 2, and (within Stipeae) to 3, 4, 5, and 6; and 6 gives rise to 7). The change from state 1 to state 6 marks the MAC clade, and this is not reversed, but does give rise to state 7 in some Achnatherum A3 elements. In this loop sequence CTGGAA (type ?0? in Fig. 1) is common among members of early diverging lineages of Pooideae (Brachy- elytrum P. Beauv., Danthoniastrum, Duthiea, Phaenosperma) but is absent in Stipeae. This sequence is also characteristic of Diarrhena. Nardus L. and Lygeum Loefl. ex L. exhibit specific inversions Xi and Yi of unknown sequence origin. For a more detailed discussion of the stem-loop hairpins see Appendix 2. Thirteen indels were detected in four of our sequenced plastid regions: six in rpl32-trnL, three in rps16-trnK, one in rps16 intron, and three in trnL-F. These are identified by their starting position within the aligned sequences and are mapped on the tree (Fig. 1). In rpl32-trnL a 21 bp insertion (170) was found in all representatives of the Phaenospermateae except Metcalfia. The 7 bp insertion (110) indicates a possible alter- native relationship between plastids of three species of Stipa (S. purpurea, S. subsessiliflora, S. regeliana). Another 7 bp insertion (777) confirms the monophyly of four species in the ESL1 clade (this taxon set is also supported by minute inver- sion 1i?). The 8 bp deletion (104) supports the monophyly of the entire AC clade. Within AC a 5 bp insertion (830) adds support for the entire Australia?New Zealand clade, and a 5 bp deletion (214) supports a central and east Asian subclade of six species within Achnatherum A1: A. inebrians (Hance) Keng ex Tzvelev, A. pekinense (Hance) Ohwi, A. brandisii (Mez) Z.L. Wu, A. confusum (Litv.) Tzvelev, A. sibiricum (L.) Keng ex Tzvelev, and A. pubicalyx (Ohwi) Keng. The rps16-trnK region exhibited two independent inser- tions in the ASL2 clade of different lengths. A 1 bp insertion (396) marks Aciachne and Anatherostipa s.str. from the Anath- erostipa hans-meyeri?A. rosea?Lorenzochloa clade. This is followed by a 20 bp insertion (571) marking the separation of the Anatherostipa s.str. from the Aciachne clade. The Asian Achnatherum A1 clade of six species, mentioned above, is ad- ditionally supported by a 16 bp insertion (214). In the rps16 intron a 5 bp insertion (777) is unique to the Patis clade. A trnL-F 5 bp insertion (115) supports the monophyly of S. purpurea, S. subsessiliflora, S. regeliana, S. bungeana, and S. capillacea. A 10 bp deletion (89) marks the crown node for the Piptatherum s.str. clade. A 21 bp repeat insertion (343) is confined to ESL2 and appears three times in that clade. It was detected in Psammochloa?Achnatherum splendens (?Neo- trinia?) clade, Trikaraia clade, and the Ptilagrostis s.str. clade. Analysis of ITS sequences. ? The ITS phylogenetic tree is poorly resolved with little backbone support (Fig. 2). The bifurcation between Brachyelytrum, Nardus-Lygeum, and the rest of the Pooideae has weak support (BS = 60, PP = 0.94). The Phaenospermateae tribe is not monophyletic as its ele- ments are distributed among the first three branches after the Brachyelytrum and Nardus-Lygeum split. The only supported clade within Phaenospermateae (BS = 100, PP = 1.00) con- tains two species of Stephanachne and Sinochasea. Triniochloa (Meliceae) and Dielsiochloa (Poeae) are united with minimal 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. 11 Romaschenko & al. ? Systematics and evolution of needle grassesTAXON ? Article version: 9 Dec 2011: 27 pp. 1.00 0.99 0.64 1.00 100 68 AC Brachyelytrum erectum Nardus stricta Danthoniastrum compactum Anisopogon avenaceus Duthiea brachypodium Sinochasea trigyna Diarrhena fauriei Diarrhena japonica Stipa brauneri Stipa pennata Ampelodesmos mauritanicus Psammochloa villosa Achnatherum splendens Oryzopsis asperifolia Trikeraia pappiformis Trikeraia hookeri Ptilagrostis malyshevii Ptilagrostis dichotoma Ptilagrostis junatovii Ptilagrostis luquensis Ptilagrostis kingii Piptatheropsis micrantha Piptatheropsis shoshoneana Piptatheropsis pungens Ortachne rariflora Ortachne breviseta Pappostipa major Pappostipa chrysophylla Pappostipa vaginata Pappostipa hieronimusii Pappostipa speciosa Hesperostipa spartea Hesperostipa comata Hesperostipa neomexicana Piptochaetium avenaceum Piptochaetium featherstonei Piptochaetium brachyspermum Piptochaetium panicoides Anatherostipa hans-meyeri Anatherostipa rosea Aciachne acicularis Aciachne flagellifera Anatherostipa venusta Anatherostipa mucronata Anatherostipa rigidiseta Piptatherum holciforme Piptatherum fedtschenkoi Piptatherum angustifolium Piptatherum hilariae Austrostipa scabra Austrostipa tenuifolia Austrostipa campylachne Austrostipa semibarbata Stipa capensis Achnatherum calamagrostis Piptatherum miliaceum Achnatherum sibiricum Achnatherum inebrians Achnatherum bromoides Ptilagrostis pelliotii Achnatherum chinense Achnatherum caragana Achnatherum robustum Achnatherum perplexum Achnatherum occidentale subsp. californicum Achnatherum occidentale Achnatherum nelsonii Jarava media Jarava plumosula Amelichloa caudata Amelichloa clandestina Nassella clarazii Nassella pfisteri Nassella neesiana Nassella trichotoma Nassella filiculmis L a d d e r - lik e M a i z e - l ike LE P Diarrhena obovata Dielsiochloa floribunda Metcalfia mexicana Stephanachne nigrescens Stephanachne pappophorea Ptilagrostis mongolica Stipa caucasica Stipa capillata Stipa breviflora Stipa capillacea Stipa bungeana Stipa regeliana Stipa subsessiliflora Stipa purpurea Stipa barbata Piptatheropsis canadensis Piptatheropsis exigua Piptochaetium montavidense Anatherostipa obtusa Patis obtusa Achnatherum stillmanii Piptatherum coerulescens Piptatherum munroi Piptatherum songaricum Piptatherum sogdianum Piptatherum ferganense Anemanthele lessoniana Austrostipa nitida Austrostipa nodosa Austrostipa pycnostachya Austrostipa flavescens Austrostipa juncifolia Austrostipa hemipogon Piptatherum thomasii Achnatherum turcomanicum Achnatherum jacquemontii Achnatherum pekinense Achnatherum brandisii Achnatherum confusum Achnatherum pubicalyx Achnatherum/Timouria saposhnikovii Jarava annua Achnatherum wallowaense Achnatherum hendersonii Achnatherum scribneri Achnatherum parishii Achnatherum pinetorum Achnatherum lettermanii Achnatherum richardsonii Achnatherum aridum Achnatherum lobatum Achnatherum lemmonii Achnatherum hymenoides Achnatherum diegonense Achnatherum constrictum Achnatherum eminens Achnatherum multinode Achnatherum acutum A1 A2 A3 A4 Stillmania Miliacea gr. Neotrinia Piptatherum Achnatherum Eriocoma gr. Timouria gr. Pseudoeriocoma Piptatheropsis Piptatherum virescens Piptatherum paradoxum Stipa parviflora Celtica gigantea Orthoraphium roylei Piptatherum aequiglume Brylkinia caudata Phaenosperma globosa Lygeum spartum 60 83 100 78 52 81 83 99 98 68 69 69 77 81 84 83 90 72 81 83 74 62 81 99 91 94 54 91 94 87 100 95 92 98 70 66 97 95 100 77 100 89 100 97 97 82 69 81 99 75 99 86 99 100 0.94 Macrochloa tenacissima MAC gr. 57 96 68 66 60 63 Austrostipa compressa Austrostipa macalpinei 99 52 54 60 Jarava pseudoichu Jarava ichu Jarava castellanosii Jarava scabrifolia 91 99 60 65 68 1.00 1.00 0.70 0.55 0.72 0.73 0.61 0.98 0.76 0.84 0.83 1.00 1.00 0.67 1.00 1.00 0.67 1.00 0.63 1.00 0.93 1.00 1.00 1.00 52 0.99 0.95 0.57 1.00 0.61 0.56 1.00 0.96 1.00 0.54 0.91 0.83 1.00 1.00 0.67 1.00 1.00 0.82 0.86 1.00 1.00 0.96 0.99 1.00 1.00 0.99 0.99 0.94 0.90 1.00 1.00 1.00 1.00 1.00 1.00 0.99 0.77 0.81 0.62 1.00 1.00 1.00 1.00 0.93 1.00 0.97 0.87 1.00 0.98 0.57 0.98 0.81 1.00 1.00 0.86 0.98 1.00 0.67 0.64 1.00 0.53 0.99 1.00 1.00 U no rde red S a w - li ke L EP O rde red S a w - li ke LE P North America South America Mediterranean Eurasia Australia and New Zealand SL2 SL3 ASL2 Stipella 0.54 Triniochloa stipoides 1.00 0.79 0.74 0.54 0.99 0.89 1.00 0.50 1.00 0.99 U no rde red S a w - li ke L EP 1.00 0.53 0.51 98 54 87 60Stipeae +Pappostipa Brachyelytreae Nardeae Lygeeae Phaenospermateae Poaeae Meliceae Diarrheneae LE P 12 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 = 0.76). Most major clades identified in the plastid tree (Fig. 1) are not detected in the ITS tree (Fig. 2). Only SL3 with poor support (PP = 0.67), SL2 (analysis includes only Patis obtusa), AC (including Pappostipa s.str.) with strong support (BS = 94, PP = 1.00), and MAC with poor support (PP = 0.62) are found in the ITS tree. Species found in the plastid ESL2 clade are placed into two remote clades in the ITS tree, one with a strongly supported Ptilagrostis s.str. (BS = 96, PP = 100), and one with a moderately supported set of the other ESL2 mem- bers (BS = 81, PP = 100) (excluding Othoraphium which aligns near Patis). Ampelodesmos is sister to a moderately supported Psammochloa-?Neotrinia?-Oryzopsis-Trikeraia clade (BS = 83, PP = 1.00). The phylogenetic relationships of Psammochloa and ?Neotrinia? are strongly supported (BS = 98, PP = 1.00). This clade is sister to the Oryzopsis-Trikeraia clade (BS = 57, PP = 0.67) wherein Oryzopsis is sister to the monophyletic Trikeraia (BS = 99, PP = 1.00). In the ITS analysis, in contrast to the plastid analysis, Hesperostipa (BS = 99, PP = 1.00) is sister to the AC-Pappostipa clade. Piptochaetium (BS = 83, PP = 1.00) and Ortachne s.str. (BS = 90, PP = 1.00) are again well-resolved. The union of Piptatheropsis with Ptilagrostis kingii as sister appears as in the plastid tree, but with poor support (PP = 0.61). A clade of Aciachne and Anatherostipa s.l. is weakly supported (BS = 68, PP = 1.00) here and not in the plastid tree. However, we lack ITS data for Lorenzochloa. The AC clade is strongly supported (BS = 94, PP = 100). There is poor support for Celtica as sister to all the other mem- bers of AC (BS = 54, PP = 0.99). However, the plastid CAC clade is not resolved in the ITS tree, it collapses into a series of unsupported subclades and a large polytomy. Five notable relationships well-supported by ITS data and also resolved in the plastid CAC clade are: (1) a clade of two species of Old World Piptatherum of sect. Virescentia (BS = 95, PP = 0.99); (2) a clade of Piptatherum sect. Miliacea species (BS = 100, PP = 1.00); (3) an Austrostipa-Anemanthele clade (PP = 0.94); (4) a strongly supported clade of some Eurasian Achnatherum A1 members (BS = 92, PP = 1.00) (however, other A1 elements, A. calamagrostis, A. turcomanicum Tzvelev, and A. jacque- montii (Jaub. & Spach) P.C. Kuo & S.L. Lu, are scattered in our trees); and (5) a strongly supported Pappostipa clade (BS = 91, PP = 1.00). The MAC clade appears intact in the ITS tree (except for the ejection of Jarava annua), but has poor support (PP = 0.62). MAC includes four principal clades placed in a polytomy, and these are similar to the plastid clades: (1) ?Eriocoma group? (A3, PP = 0.81); (2) a strongly supported Jarava s.str. clade (BS = 91, PP = 1.00); (3) a portion of the ?Pseudoeriocoma group? is strongly supported and this includes Achnatherum acutum (Swallen) Valdes-Reyna & Barkworth, A. eminens (Cav.) Barkworth, and A. multinode (Scribn. ex Beal) Valdes- Reyna & Barkworth (BS = 99, PP = 1.00), but this does not include A. constrictum (Hitchc.) Valdes-Reyna & Barkworth or A. diegoense (Swallen) Barkworth; and (4) a weakly sup- ported Amelichloa?Achnatherum diegoense?Nassella clade (PP = 0.64). This latter clade includes a weakly supported clade of five species of Nassella (BS = 68, PP = 0.98) as sister to the weakly supported clade of two species of Amelichloa (strongly supported, BS = 99, PP = 1.00) with Achnatherum diegonense (BS = 65, PP = 0.87). Testing alternative phylogenetic hypotheses. ? There are four clades that have markedly different relationships based on the plastid (p, Fig. 1), and nuclear (n, Fig. 2) datasets: (1) Hesperostipa, (2) Pappostipa, (3) Ptilagrostis s.str., and (4) Piptatherum s.str.?Achnatherum stillmanii. We examined the robustness of each of these clades under the constrained alter- native topologies. (1) In the plastid tree (p) Hesperostipa is placed within ASL2 as sister to the Piptochaetium-Anatherostipa-Lorenzo- chloa-Aciachne clade (hypothesis A); based on ITS (n), Hes- perostipa is sister to the Pappostipa-AC clade (hypothesis B). The heuristic search using the plastid dataset under the con- straints (to be part of, or sister to, the AC clade as in ITS) indi- cates that 21 extra steps (constituting 0.83% of the entire tree length) were needed for Hesperostipa to be resolved as sister to AC. The analysis of distribution of tree length differences in Mesquite v.2.6 revealed that only eight extra steps would be needed if the hypothesis of a sister-group relationship between Hesperostipa and AC were true. Therefore, hypothesis B was rejected at P < 0.05, and it was concluded that Hesperostipa and Pappostipa?AC could not form a clade based on the plas- tid data. Hypothesis A was tested in the nuclear dataset and the difference between unconstrained and constrained most parsimonious trees resulted in 10 extra steps for the nuclear dataset (0.74%). The analysis of the distribution of tree length differences yielded an equal to higher number of extra steps allowed at P < 0.05 as likely outcome if hypothesis A were true. Therefore, hypothesis A, namely that Hesperostipa belongs to ASL (Fig. 1), is accepted. (2) Pappostipa was resolved as member of ASL (p) and was placed as nested within AC (n). The two hypotheses re- 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 & W?ndisch (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. 13 Romaschenko & al. ? Systematics and evolution of needle grassesTAXON ? Article version: 9 Dec 2011: 27 pp. 14 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 observed more than 0.05% of the time. Thus both hypotheses were rejected. (3) Ptilagrostis s.str. was resolved as member of ESL2 (p) and was placed as sister to a clade of Patis, Ortachne, Pip- tatheropsis, Piptochaetium, Anatherostipa, and Aciachne (n). The two hypotheses required 13 (p; 0.51%) and 22 (n; 1.47%) extra steps. The analysis of the distribution of tree length dif- ferences was expected to be observed more than 0.05% of the time. Thus, both hypotheses were rejected. (4) Piptatherum s.str.?Achnatherum stillmanii was re- solved as sister to AC (p) and was placed as sister to the re- maining Stipeae excluding Macrochloa (n). The two hypoth- eses required nine (p; 0.36%) and 23 (n; 1.5%) extra steps. The analysis of the distribution of tree length differences was expected to be observed more than 0.05% of the time. Thus, both hypotheses were rejected for all datasets. Lemma micromorphology. ? Lemma epidermal pattern (LEP) is diagnosed by several characters: length and shape of the walls of the fundamental cells (FC), length and shape of silica cells (SC), presence or absence of silica bodies (SB), presence, absence, or frequency of cork cells (CC), presence or absence of prickles (hooks), shape of the prickle base, etc. (Thomasson, 1978; Ellis, 1979; Romaschenko & al., 2008, 2010). ? Saw-like LEP (SLP) pattern is common in Stipeae and widespread among grasses outside of this tribe (Fig. 3A?Y; Finot & al., 2006; Liu & al., 2010; Peterson, 1989; Romaschenko & al., 2008; Thomasson, 1986; Valdes-Reyna & Hatch, 1991). Features of this type are the presence of elongate (usually very long, i.e., more than 2? longer than wide) FC with sinuate to lobate sidewalls (Romaschenko & al., 2008). The FC alternate with SC containing SB. Cork cells are usually paired with SC and situated adjacent to the proximal end-wall of the SC/SB pairs of cells. Within Stipeae we distinguished six major sub- types of SLP. 1. Macrochloa SLP (Fig. 3B, E?J, V, Y) as a non-CC vari- ant has FC of variable length that are 3?7 times longer than SC that contain SB, and often alternate with SB?CC pairs. The silica bodies in these species are round or slightly elongated and the CC are square to crescent-shaped. Prickles (with rounded prickle base) and unicellular microhairs are usually present and the sidewalls are thick, dentate (Macrochloa, Oryzopsis, Patis obtusa, Piptatherum: Fig. 3B, H, V, Y) or lobate (Ampelodes- mos, Psammochloa, ?Neotrinia?, Trikeraia: Fig. 3E?G, I, J). In Trikeraia the lemma surface appears striate at less than 10? magnification. This striate pattern is homogeneous within the Ampelodesmos-Psammochloa-?Neotrinia?-Trikeraia group with the exception of Trikeraia hookeri (Stapf) Bor (Fig. 3J) which has slightly elongated SB (not rounded as in others) and often has a shallow contraction near the middle. Cork cells were not observed in Patis obtusa (Fig. 3V). 2. Stipa SLP (Fig. 3C, D) is characteristic of the ESL1 clade which encompasses the members of Stipa s.str. The FC are square to twice as long as wide, of nearly uniform shape and alternate with SC in a regular (ordered) pattern. All other SLP subtypes do not have consistent pairing of SC with FC (un- ordered). The SC are ornamented with square-based prickles and sometimes have adjacent dorsally compressed CC. The sidewalls of the FC are thick and deeply sinuous. 3. Ptilagrostis SLP (Fig. 3K?N, U, W as non-CC variant) has FC that are of variable length and are 2?7 times longer than SC, and often alternate with SC-CC pairs. Silica bodies vary in length and are rectangular with straight walls or with 2?5 shallow contractions. The CC are square. The sidewalls of the FC are sinuous, not thickened. Prickles and unicellular microhairs are often present. These characteristics are found in Ptilagrostis s.str., Ptilagrostis kingii, and Piptatheropsis (Fig. 3L?N). The CC are less abundant within ASL1 than in Ptila- grostis s.str. Within the Ptilagrostis SLP we recognize three variations: Orthoraphium roylei (Fig. 3K) with extremely long and irregularly placed SB with multiple (up to five) shallow contractions, ?Stillmania? LEP that resembles Orthoraphium with the sidewalls of the SB almost straight and without con- strictions (CC not observed), and Patis coreana (Fig. 3U) with elongated silica bodies often with single deep constriction at the mid-point (CC not observed). 4. Hesperostipa SLP (Fig. 3T) has FC that are long, with deeply sinuous and thick sidewalls. Prickles and unicellular microhairs are present, and CC and SB were not observed. 5. Aciachne SLP (Fig. 3O, Q?S; including Ortachne, Pe- ?ailillo, 2005) has FC that are long with lobate, thick sidewalls and rounded silica bodies sometimes with shallow contractions that are 2?6 times shorter than the FC, and CC that are right- angled to crescent-shaped. Small prickles are sparse. Apart from Aciachne (Fig. 3S) we also recognize variations of this subtype in Anatherostipa mucronata (Griseb.) F. Rojas (Fig. 3R), A. rosea (Fig. 3Q), and Lorenzochloa (Fig. 3O). The LEP of A. mucronata differs in having SB of irregular shape, slightly elongate with 1?2 shallow to deep contractions, and CC were not observed in A. mucronata. 6. Anatherostipa SLP (Fig. 3P) is found in the remaining species of Anatherostipa s.str. It is distinguished by lemma surfaces densely covered with prickles on the SC. It includes elongated FC with lobate, thick walls, producing a striate ap- pearance on the surface that is also densely covered with prick- les above the SC. The SB are irregularly shaped and rare, and CC were not observed. ? Maize-like LEP (MLP) is confined to Stipeae. Within Sti- peae this LEP is specific to achnatheroid grasses and, with the exception of Pappostipa, is restricted to the AC clade. Species with this pattern have thin-walled FC that are approximately equal in length and width to significantly shorter than wide (with few exceptions) with mostly straight sidewalls. The FC are oval, round, square-round, or longitudinally compressed, and all have often square-cornered or sometimes round SB that regularly alternate with FC. In the typical syndrome, where SB are densely packed and regularly alternate with compressed FC, the lemma surface resembles the surface of a fruiting in- florescence (?ear?) of corn (maize). SC-CC pairs are scarce to absent. We distinguished four MLP subtypes, two of which occur in a single species. 1. Celtica MLP (Fig. 4A) is one of the two exceptions within the MLP that have very long FC (5?10 times longer than SC) with thick sidewalls. However, the sidewalls of the 15 Romaschenko & al. ? Systematics and evolution of needle grassesTAXON ? Article version: 9 Dec 2011: 27 pp. 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)]. 16 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 are round and CC were not observed. This pattern is somewhat transitional between SLP and MLP. 2. Miliacea MLP (Fig. 4D) is a thin-walled version of the Celtica MLP. The FC are 3?5 times longer than SB, and have straight sidewalls. The SB are round, very rarely associated with CC, and the CC are crescent-shaped. 3. Stipella MLP (Fig. 4B) is found only in ?Stipella? (Stipa capensis). This pattern has large CC that are wider than SB in the adjacent SC. Additionally, the sidewalls of FC are thick and slightly sinuous. The CC are usually regularly associated with SB, which is unusual for achnatheroids (see also S. parviflora note below in MLP subtype 4). 4. Achnatherum MLP (Fig. 4C, E?S) is widespread among achnatheroids, and is found in Pappostipa (Romaschenko & al., 2008). Species in Achnatherum s.str., Austrostipa, Ane- manthele, ?Timouria group?, ?Eriocoma group?, ?Pseudoerio- coma group?, Jarava, and Amelichloa have this pattern where the CC are scarce but are prevalent in the ?Eriocoma group?. The length of the SB varies within each group. A pattern of equally narrow (longitudinally compressed) FC and SB was found in the ?Pseudo eriocoma group?. Jarava annua (Fig. 4N) and Amelichloa (Fig. 4S) share an unusual version of this MLP subtype by having irregularly shaped SB and variable length FC. Stipa parvi flora (S. sect. Inaequiglumes (Bor) F.M. Vazquez & Devesa) shares a typical MLP and lacks prominent CC and thick FC sidewalls but is distinct from the Stipella MLP (described above for Stipa capensis). ? Ladder-like LEP (Fig. 4T) is thought to be unique to Sti- peae and restricted to Nassella. This pattern includes longitu- dinally compressed FC and SC that are frequently of similar size. The SB do not fill the lumen of the SC, thus the FC and SC are sometimes indistinguishable. Both types of cells are thin-walled and aligned in regular alternate rows. Cork cells were not observed. DISCUSSION Phylogenetic relationships of early diverging lineages in Pooideae. ? The position of tribe Brachyelytreae as sister to Nardeae plus Lygeeae and the rest of the subfamily Pooideae is confirmed by numerous phylogenetic analyses (Davis & Soreng, 1993, 2007, 2010; Clark & al., 1995; Catal?n & al., 1997; Barker & al., 1999; Hilu & al., 1999; Hsiao & al., 1999; Soreng & Davis, 2000; GPWG, 2001; Mathews & al., 2002; D?ring & al., 2007; Soreng & al., 2007; Bouchenak-Khelladi & al., 2008; Schneider & al., 2009). Our analysis based on the nine plastid regions confirms the position of Phaenospermateae after the separation of Nardeae-Lygeeae, as sister to the re- maining Pooideae. These are followed by Meliceae as sister to Diarrheneae?core Pooideae?Stipeae. This scenario does not contradict the maximum parsimony strict consensus topology of Davis & Soreng (2010). All these tribes, and nodes support- ing tribe arrangement, are strongly supported, except for the order of Meliceae as sister to Stipeae plus Diarrheneae-Poeae. The latter pair has low BS support in our trees. In our analyses Phaenospermateae s.l. have moderate sup- port, and Phaenosperma is nested among Duthieinae genera. Monophyly of Phaenospermateae is also supported by the presence of a unique 21 bp insertion in rpl32-trnL, which is absent only in Metcalfia. Since Phaenospermateae are excluded from the Meliceae?core Pooideae?Diarrheneae?Stipeae clade with strong support, placement of Phaenospermateae and/or Duthieinae within a broadly defined Stipeae is rejected. Lineages and reticulation within Stipeae. ? Relation- ships within Stipeae have been debated and analyzed over the last 50 years (Freitag, 1975, 1985; Tzvelev, 1976; Clayton & Renvoize, 1986; Barkworth & Everett, 1987; Arriaga & Bark- worth, 2006; Barkworth, 2007; Jacobs & al., 2007; Barkworth & al., 2008; Romaschenko & al., 2008, 2010, 2011). Although the general consensus, prior to Romaschenko & al. (2008, 2010), was that achnatheroid-type morphology, with medium- sized spikelets with blunt calluses and unspecialized awns, gave rise to all other more specialized types of spikelet mor- phology, our DNA data strongly refute this. Taxa with achnath- eroid-type spikelet morphology do not form a monophyletic group, but those with achnatheroid LEP do, and genera with achnatheroid spikelet morphology and SLP are mostly derived from genera with a different set of features such as those found in Macrochloa. Macrochloa is strongly supported as sister to the remain- ing Stipeae. It has many peculiar features, such as long, bifid prophylls; unique leaf anatomy; short, truncate, velutinous lig- ules; protruding lemma lobes (V?zquez & Barkworth, 2004), along with an unordered SLP (common among Phaenosper- mateae elements and in our SL clades). We have found it useful to divide Stipeae into three groups: (1) Macrochloa with multiple autapomorphic features; (2) clades with SLP, which we refer to as SL lineages; and (3) those with MLP or ladder-like LEP, which we call achnatheroid grasses. The lemma epidermal patterns in the Macrochloa and SL lineages are mutually exclusive to the pattern found in ach- natheroid grasses. Even though the achnatheroid grasses are monophyletic, it is clear that they are derived at some level from an ancestor with SLP. The SL lineages, as they appear in the plastid tree (Fig. 1), do not form a monophyletic group. They resolve in three clades that diverge in a stepwise order; SL1, SL2, and SL3, with SL3 sister to the AC. Even though SL3 is weakly supported, an iden- tical monophyletic group of SL3 taxa is found in both plastid and ITS trees, with identical internal branching order at the base. Although SL1 is strongly supported as the sister group to the set of SL2, SL3, and AC, there is only moderate support for the sister-group relationship of SL2 to SL3/AC. This significant weakness in the phylogenetic structure among SL lineages is reiterated by the different, although unsupported, relationships detected in the ITS analysis (Fig. 2). In the ITS tree SL2 and SL3 are resolved within two successive diverging clades of a non-monophyletic SL1, while Hesperostipa and Pappostipa are removed from the SL1/ASL1 clade in the plastid tree and placed as sister clades within AC in the ITS tree. The achnatheroid clade, which includes only taxa with maize-like or ladder-like LEP, is monophyletic (strongly 17 Romaschenko & al. ? Systematics and evolution of needle grassesTAXON ? Article version: 9 Dec 2011: 27 pp. supported, see Fig. 1), and is comprised of three subclades: (1) an Austral-Eurasian CAC with some isolated African elements, (2) an east Asian ?Timouria group?, and (3) an exclusively New World MAC. The stepwise order of SL2 and SL3, as sister to AC, and CAC as sister to ?Timouria group? plus MAC, are moderately to strongly supported and geographically consistent with a Eurasian origin of AC in the plastid tree. This geographi- cal arrangement is contradicted by the ITS tree, which suggests an American origin of AC from the ancestors of Hesperostipa and Pappostipa. However, comparison of the distribution of tree length differences between the plastid and ITS trees (see results) lead us to favor the plastid hypothesis of Hesperostipa belonging within SL1 ASL clade. The SLP clade including an ESL and an ASL lineage was detected in our previous study based on five plastid regions (see clades 1 and 2 in Romaschenko & al., 2010). With addi- tion of four more plastid regions the SL1 clade is now strongly supported (Fig. 1). The combination of the Eurasian and American SLs in one clade is not consistent with the topology obtained from ITS. Despite alternative hypotheses generated from different datasets, only inferences based on the plastid analysis had strong bootstrap support. We conclude that the incongruence between the ITS and plastid tree is perhaps due to stochastic processes and/or independent reticulation events in the ITS evolution during the origin of Stipa, Ptilagrostis, Orthoraphium, and Pappostipa, although we have not tested for this (Humphreys & al., 2010). Within ESL Stipa s.str. is strongly supported, but in a narrower sense than previously understood by taxonomists. We detected two strongly sup- ported clades, which provide the first insight into infrageneric structure of Stipa based on molecular data. One clade includes S. capillacea, S. bungeana (both of S. sect. Leiostipa Dumort.), and three taxa of disputed placement. These latter three, Stipa purpurea (S. sect. Barbatae Junge), S. subsessiliflora (S. sect. Pseudoptilagrostis Tzvelev), and S. regeliana (S. sect. Rege- lia Tzvelev), are sometimes referred to different sections of Stipa, or included in Ptilagrostis (Roshevits, 1934; Tzvelev, 1974, 1977; Freitag, 1985). The monophyly of these three taxa is supported by a 7 bp insertion in rpl32-trnL. The strongly supported clade of the above five species (Fig. 1) is marked by a 5 bp insertion in the trnL-F region (unknown for S. capil- lacea and S. regeliana due to lack of trnL-F sequences). Stipa purpurea, S. subsessiliflora, and S. regeliana typically pos- sess purplish glumes, relatively short awns, and short glumes with entire apices. Based on these characters Roshevits (1934) placed S. purpurea and S. subsessiliflora in Ptilagrostis. Based on morphological characters that resemble Ptilagrostis, Tzvelev (1976, 1977) and Freitag (1985) placed Stipa subsessiliflora in S. sect. Pseudoptilagrostis Tzvelev. In a detailed study of anatomical and gross morphological features of Ptilagrostis, S. subsessiliflora was thought to be closer to Ptilagrostis than to Stipa s.str. (Barkworth, 1983). Additionally, a numerical analysis (Barkworth, 1983) placed S. subsessiliflora between representatives of Ptilagrostis and Achnatherum (A. cala- magrostis, A. caragana). The seemingly transitional nature of morphological features between Stipa and Ptilagrostis for S. purpurea, S. regeliana, and S. subsessiliflora, and some additional Achnatherum-like features for S. subsessiliflora, suggests reticulate or homoplasious origins. Widely divergent sequences of ITS (i.e., clones) have been found and these are the subject of a forthcoming paper (Romaschenko & al., in prep.). The LEP of S. subsessiliflora is clearly of the saw-like type (Fig. 3C), in agreement with the placement of this species in Stipa. Even though the above three species of Stipa are resolved in a grade, their sequences differ very little. Since they share the same 7 bp insertion in rpl32-trnL (Fig. 1), evidence of common origin is supported. A second Stipa s.str. clade, supported by plastid and ITS analyses, comprises S. breviflora as sister to the strongly supported plastid subclade of five very closely related species that share a 7 bp insertion in the rpl32-trnL region and an inversion type ?1? in the trnH-psbA region (Fig. 1). These five species have been placed in four sections of Stipa accord- ing to Tzvelev (1976): sects. Leiostipa (S. capillata), Smirnovia Tzvelev (S. caucasica), Barbatae (S. barbata, S. brauneri), and Stipa (S. pennata). Within Stipa s.str. it appears that S. bun- geana shares ITS characteristics with these five species and shares a plastid type with S. capillacea, indicating possible additional rounds of reticulation within the genus. The Ampelodesmos-Psammochloa-?Neotrinia?-Oryzopsis- Trikeraia set (a grade in ESL2, see Fig. 1) all have SLPs, and, with one exception, are characterized by stout Eurasian species, usually more than one meter tall that occur in a diverse array of open habitats (Mediterranean scrub, Mongolian sand-dunes, arid steppe, and open mountain slopes of the Tibet-Qinghai pla- teau). The single species of Oryzopsis s.str. is exceptional as it is of moderate stature (Barkworth, 2007) and endemic to North American shady cool temperate forests. Except for Trikeraia with three species, the other genera are monotypic. All the genera have unusual characteristics. Ampelodesmos, Psammo- chloa, and Trikeraia are rhizomatous (an uncommon charac- ter in Stipeae) while ?Neotrinia? (Achnatherum splendens) forms massive tussocks. All members of this group, along with Macrochloa and Celtica, have well-developed lemma lobes flanking the central awn (lobes setiform in Ampelodesmos and Trikeraia), and such lobes are consistently small or absent elsewhere in the tribe. Achnatherum splendens was placed in Achnatherum sect. Neotrinia Tzvelev (Tzvelev, 1976). How- ever, molecular data (Romaschenko & al., 2010; Figs. 1?2 of this paper), and SLP (Fig. 3G) strongly advocate its position among Eurasian SLP lineages. Achnatherum splendens seems most closely related to Psammochloa villosa (Trin.) Bor based on molecular data. Ampelodesmos has the most unusual spikelet features in Stipeae with multiple florets per spikelet and a prolonged rachilla above the uppermost floret (Stipeae otherwise have one floret and no rachilla extension, not even a rudiment of one), slender terminal untwisted undifferentiated awns (Stipeae awns are often twisted at the base), hairy ovaries (otherwise known only in Orthoraphium and Patis), and three lanceolate lodicules (2 or 3 in Stipeae) that are dorsally and marginally ciliate (Stipeae lodicules are glabrous except in Psammo- chloa) and strongly vascularized (Stipeae lodicules are usu- ally faintly vascularized, except in Psammochloa). However, unlike other phylogenetically isolated Mediterranean taxa (e.g., 18 Romaschenko & al. ? Systematics and evolution of needle grasses TAXON ? Article version: 9 Dec 2011: 27 pp. Macrochloa, Celtica, and Stipa capensis), Ampelodesmos has a strongly supported sister relationship with the Asian pair of Psammochloa-?Neotrinia?. Additionally, these three species share an inverted loop sequence in the trnH-psbA region of the chloroplast genome. It is most parsimonious to assume that the plastid sequence of Psammochloa-?Neotrinia? originated by T?A mutation in the already inverted ?1i? sequence found in the Ampelodesmos plastid, rather than by a separate inversion and two mutations, or by five base changes from uninverted sequences. Soreng & Davis (2000) suggested Ampelodesmos could be an ancient hybrid between Stipeae and an unknown ancestor, but that remains unproven. The shared geography of the ASL(Fig. 1) lends additional support to the possible common origin of all the American taxa with SLPs, except Oryzopsis. However, the crown node is weakly supported and it splits into three clades: (1) Pip- tatheropsis plus Ptilagrostis kingii, (2) Ortachne-Pappostipa, and (3) Hesperostipa-Piptochaetium-Anatherostipa-Aciachne- Lorenzochloa. There is no support for the joining of clades 1 and 2 (above) as sisters in ASL1 (Fig. 1) but ASL2 has strong support. The following is a detailed discussion of each of these clades. (1) The North American genus Piptatheropsis, as pre- viously depicted in Romaschenko & al. (2010, 2011), is only distantly related to the Asian Piptatherum (Fig. 1). The sister to Piptatheropsis is Ptilagrostis kingii (weakly supported in the plastid tree), a North American species with transitional features between Piptatheropsis and Ptilagrostis (Barkworth, 1983), but we have no evidence that it is directly related to Asian Ptilagrostis s.str. The only other North American species of Ptilagrostis, P. porteri (Rydb.) W.A. Weber (not included in the present study) has a more complicated history and will be the subject of a forthcoming paper. Piptatheropsis?Ptilagrostis kingii are characterized by having the longest stem in the hair- pin formation in the trnH-psbA region among Stipeae. This pro- vides some evidence for independent evolution of this group. (2) The Ortachne-Pappostipa sister relationship was strongly supported in the plastid analysis (Fig. 1). However, this relationship is difficult to reconcile since these species have little in common in their habits, chromosome numbers, and LEPs. Pappostipa species have MLP (Romaschenko & al., 2008) while Ortachne has an unordered SLP (Pe?ailillo, 2005) similar to that of Piptatheropsis (Fig. 3N), Ptilagrostis kingii (Fig. 3M), Aciachne (Fig. 3S) and some species of Anath- erostipa (Fig. 3Q?R). The Pappostipa clade was nested within, or sister to the AC in the ITS tree (Fig. 2). The probabilities of alternative placement of Pappostipa within ASL in the ITS tree was statistically insignificant. The LEP type being incompat- ible with that of other ASL genera and alternative phylogenetic placements in plastid and ITS trees indicates that Pappostipa arose from an ancient allopolyploid hybridization event. (3) Some shared features of the Hesperostipa-Pipto- chaetium-Anatherostipa-Aciachne-Lorenzochloa clade have been reported in Hesperostipa, Anatherostipa, Piptochae- tium, and the Miocene fossil genus Berriochloa (Thomasson, 1978; Barkworth & Everett, 1987; Pe?ailillo, 2005). These include paleas with projecting keel apices and similarities in their unordered SLP. The Hesperostipa-Piptochaetium-Anath- erostipa-Aciachne-Lorenzochloa clade was previously detected in phylogenetic studies (Cialdella & al., 2010; Romaschenko & al., 2010), but had not received strong support until now (Fig. 1). Results of our ITS tree place Hesperostipa outside ASL at the base of AC. However, the test of statistical significance of the differences in length between constrained and unconstrained trees showed a high probability that Hesperostipa should be included in ASL. The existing incongruence possibly reflects the high level of homoplasy in our ITS data. As a result, we postulate the correct phylogenetic position for Hesperostipa is (seen in Fig. 1) within ASL2 as sister to Piptochaetium, Anatherostipa, Aciachne, and Lorenzochloa. The lemma morphology of Hesperostipa corresponds well to what was found in Berriochloa. The LEPs of H. comata (Trin. & Rupr.) Barkworth, H. neomexicana (Thurb.) Barkworth (Thomasson, 1978), and H. spartea (Trin.) Barkworth (Fig. 3T) look similar to those found in Berriochloa minuta Elias and B. maxima Elias (Thomasson, 1978). Thomasson (1978) described the lineage of Berriochloa-Hesperostipa-Piptochaetium as having LEPs with fundamental cells (FC) with deeply sinuous sides and end-walls, lacking silica bodies (SB) and cork cells (CC), and he estimated the time of separation of Hesperostipa and Pip- tochaetium to be late Pliocene. According to our data the SB/ CC pairs are common in SLP lineages (Fig. 3B, E?J, L, Y), outside of Stipeae (Fig. 3A), and are normally present in the Piptatheropsis?Ptilagrostis kingii clade (Fig. 3M?N). This suggests that the Berriochloa-Hesperostipa-Piptochaetium lin- eage (where short cells are absent) is a derivative of an ancestral extinct LEP where both types of short cells (silica and cork) evolved, and from which Piptatheropsis, Ptilagrostis kingii, and Ortachne are scarcely modified. Another piece of evi- dence is that the sister to Piptochaetium is the Anatherostipa- Aciachne-Lorenzochloa clade, where scarce SB were found in Anatherostipa mucronata and SB/CC pairs are present in Aciachne (Fig. 3R?S), representing stages of this loss. A ?pappose? subset of Anatherostipa species (A. rosea, A. hans-meyeri) is characterized by the presence of long hairs at the apex of the lemma, abundant SBs (Fig. 3Q), and a LEP that resembles Lorenzochloa and Ortachne. Our plastid tree clearly separates the A. rosea?A. hans-meyeri?Lorenzochloa clade from the remaining Anatherostipa s.str., which aligned with Aciachne in a strongly supported subclade (Fig. 1). The latter subclade is marked by single bp insertion in rps16-trnK, while the internal clade of remaining Anatherostipa s.str. is marked by a 20 bp insertion in the same region (Fig. 1). Such evidence reveals that Anatherostipa, as currently understood (Pe?ailillo, 1996), is polyphyletic. All plastid analyses, even when we excluded Pappostipa as putative conflicting taxon (results not shown), resolve Ortachne s.str. as an isolated lin- eage with no support for sister relationships between it and other ASL clades (other than Pappostipa). Ptilagrostis and Patis are placed as potential sisters to the main body of ASL in the ITS analyses. A character that Ptilagrostis shares with ASL is a chromosome base number of x = 11, but this is common among American SLP species (Barkworth, 1983). 19 Romaschenko & al. ? Systematics and evolution of needle grassesTAXON ? Article version: 9 Dec 2011: 27 pp. The SLP lineage 2 (SL2), i.e., Patis, is a newly detected strongly supported clade that includes two far East Asian, stout (to 1 m tall), broad- and flat-leaved species, Patis coreana and P. obtusa. The distribution of Patis coreana and P. obtusa overlap in the mesic temperate forests of eastern China (area of Hubei, Shaanxi, and Zhejiang provinces; Ohwi, 1941, 1942; Wu & Philips, 2006), and occurs outside the ecological ranges of all but one other Asian Stipeae species, Achnatherum pe- kinense (including A. extremorientale (H. Hara) Keng). Patis coreana and P. obtusa share a 5 bp insertion in the rps16 intron (Fig. 1) and these two species were recognized as being anoma- lous within their former respective genera (Wu & Phillips, 2006). Patis coreana was originally described in Stipa and has been placed in Achnatherum and Orthoraphium by Ohwi (1941, 1953). These two species of Patis apparently represent a distinct and isolated lineage since they do not align in our phylogenetic analysis with Stipa, Orthoraphium, Piptatherum, or Achnatherum (Romaschenko & al., 2011). The LEP of Pa- tis coreana is clearly of the saw-like type (Fig. 3U), whereas Patis obtusa, differs in having rounded SB resembling those of Piptatherum s.str. (Fig. 3Y) except that the SB/CC pairs are scarce, and side-walls of the FC are thicker. A third species, Patis racemosa (Sm.) Romasch. & al. from North America, has been transferred into the genus (Romaschenko & al., 2011). Patis coreana and P. obtusa have prominent transverse veinlets between the longitudinal veins of the glumes (these are visible but faint in P. racemosa), and all three species have wide, long flag-leaf blades and underdeveloped or absent basal leaf-blades (i.e., they have cataphylls). The SLP lineage 3 (SL3) or Piptatherum-?Stillmania? is weakly supported in the plastid tree (Fig. 1) and the ITS tree (Fig. 2). In each tree ?Stillmania? is sister to a strongly sup- ported Piptatherum (2). The phylogenetic position of SL3 is contradictory among the three analyses. In the plastid analysis it is rendered as sister to the AC and in the ITS tree it is placed as sister to Ampelodesmos-Psammochloa-Oryzopsis-Trikeraia. The LEP of ?Stillmania? (Fig. 3W) is of the saw-like type and resembles that of Ptilagrostis (Fig. 3L; Barkworth, 1983) and Piptatherum (Fig. 3Y), but differs by having SBs of irregular length and alternation, and the lack of associated cork cells (CC). The provisional name ?Stillmania? is used to repre- sent Achnatherum stillmanii from the western part of North America. It was once placed in Ptilagrostis by Elias (1942), although Barkworth (1983) provided morphological and nu- merical evidence for placing it in Achnatherum. It shares some morphological features with Achnatherum, but differs by not having a MLP, possessing notched paleas with extended keels, and prominent lemma lobes. The Piptatherum s.str. clade received strong support in all phylogenetic analyses (Figs. 1?2) (Romaschenko & al., 2010). Members of this clade share a 10 bp deletion in the trnL-F region. Morphological characters supporting the Piptatherum s.str. clade are dorsally compressed spikelets, linear disarticu- lation scars, and basally unfused and well-separated lemma margins (Romaschenko & al., 2010). This combination of char- acteristics is consistent throughout this clade and do not appear in Piptatheropsis, Patis, or the ?Miliacea group?. Historically, other morphological features used to broadly circumscribe Pip- tatherum (Roshevitz, 1951; Freitag, 1975; Tzvelev, 1976; Dorn, 2001; Soreng, & al., 2003; Wu & Phillips, 2006; Barkworth, & al., 2008), such as small anthoecia, dark, often glossy and coriaceous lemmas, and well-exposed paleas, are found in Pa- tis, Piptatheropsis, and various Eurasian taxa including species of Piptatherum sect. Miliacea (P. miliaceum, P. thomasii) and sect. Virescentia (P. virescens, P. paradoxum). These features all appear to result from convergence (Romaschenko & al., 2010, 2011). The AC is strongly supported in the phylogenetic analyses (Figs. 1?2) and is marked by a unique 8 bp deletion in the rpl32- trnL region. This terminal clade differs from Macrochloa and the SLP clades by having a distinct MLP with shortened FC with straight to slightly sinuate side-walls. The CAC was detected in previous studies (Romaschenko & al., 2008, 2010) but without statistical support. In the cur- rent analysis it received moderate bootstrap support in the plastid tree (Fig. 1). Within CAC the monotypic Mediterra- nean genus Celtica (V?zquez & Barkworth, 2004) is sister to the remainder, which includes clades of: Stipa capensis and S. parviflora, Anemanthele and Austrostipa, Eurasian ?Mili- acea group? (Piptatherum miliaceum, P. thomasii), and Eur- asian Achnatherum (A1). The LEP of Celtica is unusual among achnatheroids because it has long FC (Fig. 4A). However, the side-walls of the FC are straight which is in agreement with the LEP concept described for achnatheroid grasses, but it may represent the transitional form between saw-like and maize- like LEPs. Assuming x = 12 is the base chromosome number, Celtica is an octoploid (2n = 96), which is the highest known ploidy level in Stipeae (also found in Ampelodesmos). Two other Mediterranean taxa, Stipa capensis and S. parvi- flora, were placed at the base of Achnatherum (A1) plus the ?Miliacea group?, but without support in the plastid analysis (Fig. 1). Tzvelev (1976) placed S. capensis in a monotypic Stipa sect. Stipella and Freitag (1985) added S. parviflora to the sec- tion based on these two taxa having an unusually short palea. V?zquez & Devesa (1996) accepted the placement of S. capen- sis, but placed S. parviflora in another monotypic Stipa sect. Inaequiglumes. Our phylogenetic and LEP evidence indicates that both taxa should be removed from Stipa and treated as members of the AC clade. The LEPs of S. capensis and S. parvi- flora are different; S. parviflora is typical maize-like (Fig. 4C) and S. capensis is unique among achnatheroids because it has enlarged cork cells often associated with silica cells/bodies (Fig. 4B). The ITS tree did not resolve these taxa as a pair and there was only low support for their phylogenetic isolation. Thus, the phylogenetic placements of S. capensis (2n = 18, 26, 34, 36) and S. parviflora (2n = 28) remain somewhat ambiguous. The ?Miliacea group? consists of two closely related species (Piptatherum miliaceum, P. thomasii) that were placed in Pip- tatherum (Roshevits, 1951; Freitag, 1975; Tzvelev, 1976) after the application of the name Oryzopsis to non-North American taxa was discontinued (Romaschenko & al., 2011). This group shares a chromosome number of 2n = 24 with the Piptatherum s.str. clade and Achnatherum (A1). However, the set of morpho- logical characters outlined for Piptatherum s.str. in this study is 20 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 P. miliaceum and P. thomasii are not coriaceous, the disarticula- tion scars are circular (transversally elliptic in Piptatheropsis), and the lemma borders are basally fused (pers. obs.). The LEP of the?Miliacea group? represents a third unique type in the achnatheroid clade (Fig. 4D), which together with phylogenetic evidence and other morphological differences support the isola- tion of this group from Piptatherum and Achnatherum. Achnatherum as broadly circumscribed (our A1 to A4, ?Neotrinia?, and ?Stillmania?) includes 35 North American spe- cies, about 20 species from Eurasia, and one from New Zealand. Following Tzvelev (1976), the species currently accepted by the Flora of China in Achnatherum (Wu & Philips, 2006) are attrib- utable to five general groups with the following characteristics: (1) callus short, often obtuse; awn straight or flexuous, articu- lated; lemma membranous with apical lobes (Achnatherum sect. Achnatherum; Stipa sect. Lasiagrostis Steud., type: A. cala- magrostis?Freitag, 1985; V?zquez & Devesa, 1996); (2) callus short, often obtuse; awn straight; lemma at maturity becoming dark and coriaceous, with apical lobes, only marginally cover- ing the palea (Achnatherum sect. Aristella (Trin.) Tzvelev, type: A. bromoides (L.) P. Beauv.?Tzvelev, 1976; Stipa [unranked] Aristella Trin.; Stipa sect. Aristella (Trin.) Steud.?Freitag, 1985; V?zquez & Devesa, 1996); (3) callus conspicuous, of- ten acute; awn once or twice geniculate; lemma at maturity becoming dark and coriaceous, with absent or inconspicuous lobes, and completely covering the palea (Achnatherum sect. Achnatheropsis (Tzvelev) Prob., type: A. sibiricum?Freitag, 1985; Stipa sect. Achnatheropsis Tzvelev?Freitag, 1985; Tzvelev, 1976; Achnatherum sect. Protostipa Tzvel.; Stipa ser. Sibiricae Roshev.?Bor, 1970); (4) callus short, obtuse; awn straight, caducous; lemma pilose, membranous, covering the palea by 2/3, with apical lobes; palea minutely protruding above the lemma apex (Achnatherum sect. Neotrinia, type: A. splen- dens?Tzvelev, 1976); and (5) callus short, obtuse; awn straight, caducous; lemma pilose, membranous, covering most of the palea, with apical lobes; palea shorter than lemma (Timouria, type: T. saposhnikowii?Roshevitz, 1916]. Our molecular study provides strong evidence for Ach- natherum sect. Achnatheropsis being a natural group. It in- cludes Achnatherum sibiricum, A. confusum, A. pekinense, and A. brandisii (Roshevits, 1934; Bor, 1970; Tzvelev, 1976, 1977; Freitag, 1985), along with A. pubicalyx and A. inebrians that have somewhat transitional features between this section and A. sect. Aristella. The Achnatherum-A1 clade (including the type of Achnatherum) received strong support in the ITS tree and little support in the plastid tree (PP = 0.91) (Figs. 1?2). The crown node within this group is marked by two plastid indels, a 16 bp insertion in rps16-trnK and a 5 bp deletion in rpl32-trnL (Fig. 1). The other members of Achnatherum s.l. resolved as groups outside of CAC, within A2, A3, and A4 (?Eriocoma?, ?Pseudoeriocoma?, and ?Timouria groups?) or as isolated species in SL1 and SL3 (?Neotrinia? and ?Stillmania? lineages). More sampling in Achnatherum is needed to deter- mine whether species with membranous lemmas (Achnatherum sect. Achnatherum) and species with coriaceous lemmas (sect. Aristella) represent natural groups. Our current analysis confirms the position of Piptatherum virescens and P. paradoxum (Piptatherum sect. Virescentia) in CAC (Romaschenko & al., 2010). These taxa share the same LEP as the majority of the other members of AC (Fig. 4I) and align with A. bromoides (sect. Aristella) in the plastid analysis with moderate support or without support in the ITS tree. The sister relationship of A. bromoides and P. sect. Virescentia is more plausible since the taxa share dark and coriaceous lem- mas with a blunt callus, persistent awns, and similar habitats. Achnatherum bromoides, P. paradoxum, and P. virescens have a somewhat specialized lemma form that resembles that found in Piptatherum s.str., Piptatheropsis, and the ?Miliacea group?. The evident polyphyly of Achnatherum s.l. and Piptatherum s.l. rein- forces the suggestion of Thomasson (1980: 235) that ?similarities among taxa in the shape of the anthoecia are not in themselves sufficient to interpret phylogenetic relationships in Stipeae?. The monophyly of the Austrostipa-Anemanthele group is weakly supported in the plastid and ITS trees (Figs. 1?2). The crown node is marked by a 5 bp insertion in the rpL32-trnL re- gion. In the ITS tree Anemanthele is embedded within Austro- stipa, consistent with results of Jacobs & al. (2000). However, in our plastid tree Anemanthele is sister to Austrostipa with weak support. The subgeneric structure within Austrostipa is poorly represented in the study. Of the several recovered clades only two represent separate subgenera: Austrostipa subg. Longiaristatae S.W.L. Jacobs & J. Everett (1996; A. compressa (R. Br.) S.W.L. Jacobs & J. Everett and A. mac alpinei (Reader) S.W.L. Jacobs & J. Everett), and Austrostipa subg. Austrostipa (A. campilachne (Nees) S.W.L. Jacobs & J. Everett, A. semi- barbata (R. Br.) S.W.L. Jacobs & J. Everett, and A. hemipogon (Benth.) S.W.L. Jacobs & J. Everett). The most densely sam- pled subgenus, Austrostipa subg. Falcatae S.W.L. Jacobs & J. Everett (1996; A. scabra (Lindl.) S.W.L. Jacobs & J. Everett, A. nitida (Summerh. & C.E. Hubb.) S.W.L. Jacobs & J. Everett, A. nodosa (S.T. Blake) S.W.L. Jacobs & J. Everett, A. tenui- folia (Steud.) S.W.L. Jacobs & J. Everett, and A. trichophylla (Benth.) S.W.L. Jacobs & J. Everett), was polyphyletic in all our analyses. Based on our phylogenetic trees and the peculiar morphological types found in Austrostipa-Anemanthele, this strictly Australasian group presumably shares a common an- cestor with Asian achnatheroids, both sharing the MLP (Fig. 4E?I) and general growth habit. The ?Timouria group? (A2) received strong support in our plastid tree (Romaschenko & al., 2010). It encompasses Timouria, which was first described as a monotypic genus (Ro- shevits, 1916) and then included in a monotypic section of Ach- natherum (as A. saposhnikovii; Tzvelev, 1976). In our plastid tree the ?Timouria group? included Achnatherum caragana and A. chinense, which were aligned by Tzvelev (1976) and Frei- tag (1985), respectively, in Achnatherum sect. Neotrinia and Stipa sect. Lasiagrostis, and also Ptilagrostis pelliotii. Despite differences in gross morphology among its members they all share rather small (3?4 mm long), elliptic, 2-toothed, pubescent lemmas with caducous awns, 3-veined glumes, and setaceous leaf blades. The LEP in the ?Timouria group? is maize-like, often with extremely short fundamental cells (Fig. 4G?M). Molecular evidence and LEP place Ptilagrostis pelliotii within 21 Romaschenko & al. ? Systematics and evolution of needle grassesTAXON ? Article version: 9 Dec 2011: 27 pp. AC, far removed from the SL1 Ptilagrostis clade. The well- developed awn indumentum in P. pelliotii, approaching that of ?true? Ptilagrostis, could be a result of convergent evolution. Despite morphological similarities to members of Ach- natherum (A1), the ?Timouria group? represents an independent branch of Asian achnatheroid grasses with caducous awns and compressed, elliptic florets, whereas other Asian achnatheroids have persistent awns and terete, fusiform florets. The unusual characteristics of the ?Timouria group? led Roshevits (1951) to describe subtribe Timouriinae to join smallest-spikeleted Asian and American genera, including Eriocoma (based on E. hymen- oides (Roem. & Schult.) Rydb.), Piptatherum, Oryzopsis, as well as Piptochaetium and Nassella in a narrow sense (Parodi, 1944, 1947). According to our plastid tree the ?Timouria group? shares a common ancestor with MAC, which includes some of the largest genera in American Stipeae, such as the ?Eriocoma group? (Achnatherum-A3), Jarava, and Nassella, with 29, 37, and 115 species, respectively. The MAC was detected in previous phylogenetic analyses (Romaschenko & al., 2008, 2010, 2011), and received strong support in our plastid tree. The monophyly of MAC is also sup- ported by base mutation ?6? in the loop sequence in trnH-psbA region. The first split within MAC separates: (1) ?Eriocoma group? (Achnatherum-A3) which includes the majority of North American Achnatherum species, from (2) an assemblage of Jarava, ?Pseudoeriocoma group? (Achnatherum-A4), Nassella, and Amelichloa. Ignoring Jarava annua for the moment, these can be considered sister groups and are strongly supported in our plastid tree. In the ?Eriocoma group? (A3) A. wallowaense J. Maze & K.A. Robson, A. hendersonii (Vasey) Barkworth, and A. hy- menoides have short, elliptic, slightly laterally compressed florets, and lemmas with caducous awns (a Timouria-like char- acter according to Roshevits, 1951). These three taxa form a grade leading to other ?Eriocoma group? taxa that have terete and fusiform florets and lemmas with persistent awns (Fig. 1). The overall morphological pattern of fusiform florets in the ?Eriocoma group? is similar to that in some species in the Asian Achnatherum (A1) clade, especially to those taxa in A. sect. Achnatheropsis that have a crown of hairs at the distal end of the lemma surrounding the base of the awn. This similarity lead Barkworth (1993) to transfer American species formerly placed in Stipa (Hitchcock, 1925a, b) to Achnatherum. Another group of five species within MAC (Achnatherum diegonense, A. constrictum, A. eminens, A. multinode, A. acu- tum) appears to have a separate evolutionary history, and is called the ?Pseudoeriocoma group? (A4). In both trees the ?Pseudoerio- coma group? aligns in a subclade with Jarava and Nassella rather than with the ?Eriocoma group? (Figs. 1?2). This group- ing was detected by Barkworth & al. (2008) based on a dataset which included ITS and plastid data. In our study this group is moderately supported in our plastid tree (Fig. 1), but in our ITS tree (Fig. 2) A. constrictum is aligned in a grade with Jarava, and A. diegonense is sister to Amelichloa caudata?A. clandestina. These results could be attributed to homoplasy within the ITS tree, or to past hybridization events. Since not all of the species of American Achnatherum were employed in our current study, the number of species allied to the ?Pseudoeriocoma group? is uncertain. In the ITS tree (Fig. 2) a smaller strongly supported ?Pseudoeriocoma group? of A. acutum, A. eminens, and A. mul- tinode was sister to Jarava plumosula?J. media?A. constrictum; these two Jarava species were formerly placed in Stipa subg. Ptilostipa (Spegazzini, 1901; Roig, 1964). Jarava as broadly applied is obviously polyphyletic in our analyses, but separation of plumose and long-awned sect. Ptilostipa from other Jarava s.str. is consistent with our previous analyses (Romaschenko & al., 2008, 2010) where larger sets of Jarava and Nassella taxa were included. The separation of Jarava plumosula and J. me- dia (Stipa sect. Ptilostipa) from the ?Pseudoeriocoma group? is consistent with the taxonomic distribution of relatively long (not less than half the length of the lemma) and hairy paleas, with two well-developed veins, a characteristic that these groups share with the ?Eriocoma group?. In contrast, in Jarava s.str. and Nassella the paleas are relatively short, glabrous or scarcely hairy, and the veins are faint or absent. The origin of the ladder- like LEP in Nassella is apparently derived from MLP ancestors which is found in species of Jarava, the ?Timouria group? (A2), the ?Eriocoma group? (A3), and the ?Pseudoeriocoma group? (A4) (Romaschenko & al., 2010). According to the current dataset the sister-group relation- ships between Jarava s.str. and Nassella remain unsupported. In previous phylogenetic analyses the recently described Ameli- chloa (Arriaga & Barkworth, 2006) was found to be embedded within Nassella (Barkworth & al., 2008; Romaschenko & al., 2008, 2010), or sister to Nassella as in our present analysis using a smaller dataset (Figs. 1?2). However, these relationships are not consistent with the LEP pattern where Nassella has funda- mental and silica cells that are compressed and undifferentiated forming a ladder-like LEP (Fig. 4T) and Amelichloa has MLP with well developed silica bodies and associated cork cells (Fig. 4S). Species of Amelichloa have relatively long and hairy paleas with well-developed veins, features shared with members of the ?Eriocoma? and ?Pseudoeriocoma groups?, and Stipa sect. Ptilostipa. Along with Pappostipa, the phylogenetic affiliation of Amelichloa represents one of the remaining puzzles in the evolutionary history of Stipeae, where the molecular evidence is at variance with established morphological patterns. Lemma epidermal pattern polarized by the molecular data. ? The utility of lemma epidermal patterns for phylo- genetic studies of Stipeae as postulated by Tzvelev (1977), Thomasson (1978, 1980, 1982, 1985), and Barkworth & Everett (1987), has never been questioned. In contrast, many gross mor- phological floret characters traditionally used in determining taxonomic or phylogenetic inferences in this tribe (i.e., length and indumentum of the awn; size and shape of the lemma; and sharpness of the callus; Freitag, 1975, 1985; Tzvelev, 1975), evi- dently have evolved independently many times within Stipeae. Our results reinforce these suggestions since these adaptive traits are not consistent within any recovered clades but are found within many lineages. One test of our phylogenetic hypotheses is to compare the distribution of phylogenetically conservative morphological and anatomical characteristics. In Poaceae many characteristics involved in adaptations to dispersal and seedling establishment 22 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- versal. For example, awns and points of insertion of awns, pubescence patterns, number of florets per spikelet, size of flo- rets, callus shape, elongated rhizomes and stolons, and annual life-cycle have all evolved repeatedly and, in many instances, reversed (Barkworth & Everett, 1987; Davidse, 1987; Soreng & al., 2007). However, we have found evolutionarily conservative anatomical characteristics that are consistent with molecular phylogenetic hypotheses and these support our interpretations. The phylogenetic trees presented in this paper effectively polar- ize the distribution of lemma epidermal characteristics (Figs. 1?2). We provide evidence that the SLP with long, often deeply sinuate fundamental cells is the ancestral state in Stipeae. In its general form this same LEP is common outside of Stipeae (Thomasson, 1986; Peterson, 1989; Valdes-Reyna & Hatch, 1991; Columbus, 1996; Snow, 1996; Finot & al., 2006; Liu & al., 2010) while MLP and ladder-like LEP are not otherwise known within Pooideae. Within Stipeae (ignoring Pappostipa for the moment) the SLP is present in Macrochloa, SL1, SL2, and SL3 (Fig. 1) up to the point of divergence of the AC where it is replaced by the MLP with often short fundamental cells and abundant silica bodies. Within AC there are no known reversals to the ancestral condition. No dataset examined so far has provided evidence for a single, initial bifurcation between Stipeae with SLP grasses (SL1, SL2, SL3; Fig. 3A?Y) and the AC, which possess MLP (Fig. 4A?T). More likely, three to six lineages with SLP di- verged independently before the origin of the AC clade. Stipa s.l., Piptatherum s.l., and Achnatherum s.l. were revealed in the current study as polyphyletic based on molecular data and their LEP. Only a few species in these three genera are evidently mis- classified as evidenced by LEP types, and we have tentatively provided new names or group names on the trees to identify these. Stipa pennata (type) and Achnatherum calamagrostis (type) reside firmly in the SLP and the MLP, respectively. Pri- mary stepwise divergence of several SLP lineages leading to the divergence of the AC is marked by newly acquired features of the lemma and this polarizes the LEP distribution in Stipeae in such a way that the achnatheroid grasses are clearly derived from among the SLP lineages. Within the AC a few achnatheroid taxa vary in their MLP, and these can be interpreted as transitional from unordered SLP to the typical and more common MLP. For instance, in Celtica the fundamental cells are long, and have thick walls but they are considered to represent MLP since the fundamental cells? walls are straight as in other species in AC. The ?Mili- acea group? (Piptatherum miliaceum, P. thomasii) is similar to Celtica but has thin walls. In Stipa capensis (?Stipella?) the fundamental cells are somewhat sinuous-walled and thick but are short as in the MLP. Within AC a highly modified ladder-like LEP is found only in species of Nassella wherein the silica cells are lacking silica bodies and are almost indistinguishable from the longi- tudinally highly compressed fundamental cells. Since the clade of Nassella is deeply nested within AC and all other branches have MLP it is apparent that the Nasselloid ladder-like LEP is derived from the MLP. Biogeography. ? Despite the deep history of unambigu- ous Stipeae fossils in the Miocene, and possible Stipeae fossils from the mid-Oligocene of North America (Elias, 1942; Gal- breath, 1974; Thomasson, 1978, 1980, 1987, 2005), the biogeo- graphical history within Stipeae remains difficult to interpret. However, the plastid tree (Fig. 1) provides some clues. We are reasonably confident that Macrochloa is sister to the rest of the extant elements of Stipeae. Today Macrochloa consists of a single Mediterranean species, but the nearest relatives of Sti- peae come from all over the world so extinction in other regions may have left us only this one Mediterranean element. Several clades within Stipeae are evidently geographically pure. The ASL1 and MAC clades are strictly New World. CAC is strictly Old World with a probable expansion into Australasia and Africa. The ?Timouria group? is strictly South-East Asian. As for the SL1/ESL clade, the North American monotypic genus Oryzopsis belies the otherwise strictly Eurasian nature of the clade. As for the SL3 clade, Achnatherum stillmanii of North America is sister to a strictly Eurasian group of Piptatherum s.str. At a minimum, if we interpret Fig. 1 at face value, starting with Macrochloa in the Mediterranean, there are four evident dispersal events into the New World, and one to Australasia. For the reverse direction, of Stipeae dispersal to the Old World from the New World, we must postulate at least six dispersal events. However, along the backbone of the tree (Fig. 1) there are few unambiguous directions of dispersal or patterns of vicariance, particularly as there is only weak support at several pivotal nodes. Chromosome numbers. ? There is much uncertainty about the pleisomorphic base chromosome number in Pooideae. Outside of Stipeae, Brachyelytrum has a base chromosome number of x = 11 (2n = 22), Nardus of x = 13 (2n = 26), and Lygeum of x = 10 (2n = 40; see Appendix 3 for list of chromo- some numbers). Within Phaenospermateae (sensu Schneider & al., 2009), counts of x = 7 (2n = 14) have been reported for Danthoniastrum and Duthiea (Kozuharov & Petrova, 1991; Watson & Dallwitz, 1992) and x = 12 (2n = 24) is known for Phaenosperma and Stephanachne. Meliceae have base chromo- some numbers of x = 9, 10, and perhaps 12. The base number is ambiguous for Diarrheneae where 2n numbers are [36?], 38, and 60, and x might be 5, 6, [9?] 10, or 19. Only in the tribes Bromeae, Triticeae, and Poeae s.l. (the core Pooideae) the base chromosome number is obviously x = 7. Chromosome numbers have been reported for more than 120 species of Stipeae, 86 of which we included in our study (see Appendix 3). Of these 120 about 26 appear to be diploid with possible base numbers of x = 7, 8, 9, 10, 11, or 12. De- spite the high frequency of polyploid series in Stipeae three hypotheses have been suggested for their base chromosome number: x = 7 (Tzvelev, 1977), x = 12 (Avdulov, 1931), and x = 11 (Clayton & Renvoize, 1986; Hilu, 2004). The possibility that one of these numbers might be basal for the entire Poaceae has also been thoroughly discussed (Avdulov, 1931; De Wet, 1987; Hilu, 2004; Hubbard, 1948; Hunziker & Stebbins, 1987; Raven, 1975; Stebbins, 1982, 1985). The evidence for x = 7 being the original base chromosome number in Stipeae is weak, and is contradicted by the presence 23 Romaschenko & al. ? Systematics and evolution of needle grassesTAXON ? Article version: 9 Dec 2011: 27 pp. of this possible base number in only three highly derived lin- eages of our plastid tree. Eleven counts possibly based on x = 7 are present in AC (CAC and Nassella) and in Piptochaetium; all of these are polyploid, and five are 2n = 28 (tetraploid). Chromosome numbers in most other Stipeae represent different polyploid series based on x = 12 or x = 11. A base number of x = 12 is the most common one among diploids in Stipeae, occurring in AC and in all SL lineages. In our dataset 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- pretable as being based on x = 12. Several taxa with 2n = 48 are most likely tetraploid, but most of the other taxa are more comfortably interpreted as tetraploid based on x = 9 (at 2n = 36), rather than as triploids based on x = 12. A base of x = 11 also occurs widely among diploids, oc- curring in Aciachne, Piptatheropsis, and Ptilagrostis of the SL lineages. Several studies (Johnson, 1945; Probatova & Sokolovskaya, 1980) confirmed the presence of a stable 2n = 22 karyotype in Stipeae that spans almost the entire SL1 clade including its Eurasian (ESL) and American (ASL) subclades. In Stipa s.str. 45 species have counts of 2n = 44 (tetraploid). Fifteen other diploid and polyploid taxa of SL and AC lineages are easily interpreted as tetraploids or hexaploids based on x = 11, including several New World species of Achnatherum and Hesperostipa, and one species each of the following genera: Anemanthele, Jarava s.l. (J. plumulosa), Macrochloa, Nassella, 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 species of Piptochaetium with 2n = 28); or as initially x = 11, and having lost two chromosomes. None of the possible phylogenetic scenarios represented in our analyses (Figs. 1?2) supports a single event of descending or ascending disploidy in Stipeae between x = 11 and 12. In the plastid phylogeny the chromosome number patterns in Stipeae begin with Macrochloa which is known to have 2n = 24, 36, 40, 64, and 66, as well as 72. These could be based on x = 8, 10, 11 or 12, or even 16 for this species. The first split between x = 11 and x = 12 in Stipeae is more confidently associated with the crown node of SL1. Examination of the SL1 clade reveals an x = 11 series that spans the entire ASL clade, occurring in diploid Piptatheropsis, one polyploid Piptochaetium (x = 7 is also pos- sible for the genus), tetraploid Hesperostipa, and hexaploid and octoploid Pappostipa. A second split between x = 11 and x = 12 in SL1 could be associated with the crown node for the ESL clade. In this case, an x = 11 series is represented by tetraploid Stipa and diploid Ptilagrostis. An x = 12 series is represented by a group of stout, rhizomatous to tussock-forming SLP taxa with putative tetraploid (2n = 48) karyotypes. The latter group includes Ampelodesmos and ?Neotrinia? (2n = 42, 48). The second SLP lineage (SL2) encompasses putative tetraploids? Patis coreana (2n = 46; Tateoka, 1986) and P. racemosa (2n = 46; Johnson, 1945). The third SLP lineage, SL3, is represented by the diploid Old World genus Piptatherum s.str. with 2n = 24 (x = 12 in all five species with counts). Thus, there is no clear case here for x = 11 over x = 12. Recent studies have provided new insight into the evolu- tion of the grass genome (Paterson & al., 2004; Salse & al., 2004, 2008, 2009; Wei & al., 2007), including the facility of certain chromosomes to fuse and possibly split. These studies illustrate that earlier karyological studies can often be usefully interpreted in light of a molecular phylogeny (Levin, 2002). Given the data we have, x = 12 seems more likely to be the base chromosome number for Stipeae, but x = 11 cannot be ruled out. CONCLUSION Based on analyses presented in this paper and on unpub- lished data 33 groups that we envision to represent genera within Stipeae are listed in Table 1. Eleven of these groups currently are monotypic: Ampelodesmos, Anemanthele, Celtica, ?Inaequi- glumes?, Macrochloa, ?Neotrinia?, Oryzopsis, Orthoraphium, Psammochloa, ?Stillmania?, and ?Stipella?. Thirteen of these generic groups are distributed in North America, ten are found in Asia, nine are in South America, and seven are in Europe. Stipa still might be the largest genus with between 110 and 232 species (depending on how finely the species are divided) distributed in Eurasia; Nassella has approximately 117 species distributed in the Americas; and Austrostipa has 62 species endemic to Australia and New Zealand (Klokov & Ossyczn- juk, 1976; Martinovsk?, 1980; Moraldo, 1986; Edgar & Connor, 2000; Barkworth, 2007; Everett & al., 2009; Soreng & al., 2009). ACKNOWLEDGMENTS We thank the following organizations and people: the Smithso- nian Institution?s Restricted Endowment Fund, the Scholarly Stud- ies Program, Research Opportunities, Atherton Seidell Foundation, Biodiversity Surveys and Inventories Program, National Museum of Natural History-Small Grants, and Laboratory of Analytical Biology (LAB) all for financial support; Lee Weigt, Gabriel Johnson, Jeffrey Hunt, David Erickson, Kenneth Wurdack, and Andrea Ormon for sup- port and consultation while working at LAB; the National Geographic Society Committee for Research and Exploration (grant number 8087- 06) for field and laboratory support; the Fulbright Scholar Program to KR for a research visit to the Smithsonian Institution; the Komarov Botanical Institute, Russian Academy of Sciences for the opportunity to work with herbarium collections, and Nikolai Czvelev and Dmitry Geltman for consultation and permitting us to sample Stipeae speci- mens; Asuncion Cano Echevarr?a, Oscar Tovar Serpa?, Dorita Su- sanibar Cruz, Mar?a Isabel La Torre Acuy, Jenny Vanessa Rojas Fox, Isidoro S?nchez Vega, Socorro Gonz?lez Elizondo, Nancy Refulio- Rodr?guez, Diego Leonel Salariato, Adela Mar?a Panizza, Fernando O. 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February 2012: Electronic Supplement, 1 pp. 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 S1 Romaschenko & al. ? Systematics and evolution of needle grassesTAXON ? Article version: 9 Dec 2011: Electr. Suppl., 5 pp. 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: Brachyelytr u m erect u m (Schreb.) P. Beauv., Soreng 744 0 (US), U.S.A., Maryland (from New York), EU489398, EU489184, EU489251, EU489326, JF698121, GU254790, JF6979 71, JF698 425, JF698272, EU489105; BRYLKINIEAE: Br ylkinia caudata (Munro) F. Schmidt, Ping s.n. (US), China, Jilin or Sichuan, GU254914, GU254725, GU254835, GU254957, JF698122, GU254780, JF6979 72, JF698 426, JF698273, GU254647; DIARRHENEAE: Diarrhena fauriei (Hack.) Ohwi, Koba s.n. (US), Japan, Nagono, ?, ?, JF698 5 49, JF697770, JF698125, JF6978 79, JF6979 75, JF698 429, JF698276, JF697717; Diarrhena japonica Franch. & Sav., Koba s.n. (US), Japan, Kangawa, ?, ?, JF698 5 5 0, JF697771, JF698126, JF6978 8 0, JF6979 76, JF698 430, JF698277, JF697718; Diarrhena obovata (Gleason) Brandenburg, Soreng 7439 (US), U.S.A., Maryland (from Michagan), GU254922, GU254730, GU254834, GU254956, JF698127, GU254783, JF6979 77, JF698 431, JF698278, GU254669; LYGEEAE: Lygeum spartu m L., Soreng 369 8 & Soreng (US), Spain, Analucia, JF698 614, JF697823, JF698 5 53, ?, JF698141, JF697883, JF6979 9 0, JF698 4 4 5, JF698292, JF697721; MELICEAE: Triniochloa stipoides (Kunth) Hitchc., Peterson 20581, Soreng & Romasc henko (US), Peru, Cuzco, JF698 6 4 0, ?, JF698 5 8 0, ?, JF698209, JF697911, JF698 0 5 8, JF698 515, JF698363, JF69774 8 ; NARDEAE: Nardus stricta L., Soreng 7497, Soreng & Gillespie (US), Greece, Thessaly, EU489432, EU489217, EU489285, EU489360, JF698143, GU254791, JF6979 93, JF698 4 4 8, JF698295, EU489143; PHAENOSPERMATIDEAE: Anisopogon avenaceu s R. Br., Soreng 59 05, Peterson & Jacobs (US), Australia, New South Wales, GU254933, GU254682, GU254823, GU254946, JF69810 8, GU254769, JF6979 5 9, JF69 8 413, JF69825 8, GU254657; Danthoniastru m compact u m (Boiss. & Heldr.) Holub, Soreng 7520-1, Soreng & Gillespie (US), Greece, Pelopponisos, GU254907, GU254720, GU254836, GU254958, JF698124, GU254779, JF6979 74, JF698 428, JF698275, GU254646; D u t hiea brach y podium (P. Candargi) Keng & Keng f., Soreng 5358 , Peterson & Sun (US), China, Sichuan, GU254934, GU254683, GU254819, GU254947, JF698129, GU254793, JF6979 78, JF698 433, JF69828 0, GU254656; Metcalfia mexicana (Scribn.) Conert, Peterson 188 0 8 & Valdes-Reyna (US), Mexico, Coahuila, ?, ?, ?, ?, ?, JF697884, JF6979 92, JF698 4 47, JF698294, JF697722; Phaenosperma globosa Munro ex Benth., Soreng 5325, Peterson & Sun (US), China, Sichuan, GU254935, GU254684, JF698 5 5 4, GU254948, JF698156, GU254792, JF698 0 0 8, JF698 4 62, JF69 8310, GU254655; Sinochasea trig yna Keng, Soreng 5644 , Peterson & Sun (US), China, Xizang, GU254932, GU254681, GU254824, ?, JF69819 0, GU254778, JF698 039, JF698 49 6, JF698344, GU254645; Stephanachne nigrescens Keng, Miehe 94-547-9, Miehe & W?ndisc h (Marberg U., Fac. Geograph.), China, Sichuan, JF698 628, JF697836, JF698 5 6 8, ?, JF698191, JF6978 9 9, JF698 0 4 0, JF698 49 7, JF698345, JF697736; Stephanachne pappophorea (Hack) Keng, Yunatov 138 (LE), China, Xinjiang, JF698 629, JF697837, JF698 5 69, ?, JF698192, JF6979 0 0, JF698 0 41, JF698 49 8, JF698346, JF697737; POEAE: Dielsiochloa f loribunda (Pilg.) Pilg., Peterson 20445, Soreng, Romasc henko & Susanibar Cruz (US), Peru, Huancavelica, JF698 612, JF697821, JF698 5 51, JF697772, JF698128, JF697881, ?, JF698 432, JF698279, JF697719; STIPEAE: Ac hnatherum acu t u m (Swallen) Vald?s-Reyna & Barkworth, Valdes-Reyna 1444 (US), Mexico, Coahuila, JF698 5 81, JF697791, JF698 516, JF69774 9, JF698 0 5 9, JF6978 4 6, JF697912, JF69836 4, JF698210, JF6976 8 8 ; Ac hnatherum aridum (M.E. Jones) Barkworth, Tiehm 13518 & Nachlinger (US), U.S.A., Nevada, EU489382, EU489170, EU489237, EU489311, JF698 0 6 0, JF69 78 47, JF697913, JF698365, JF698211, EU489089; Ac hnatherum brandisii (Mez) Z.L. Wu, Soreng 560 0 , Peterson & Sun (US), China, Xizang, JF698 5 82, JF697792, JF698 517, ?, JF698 0 61, JF6978 4 8, JF697914, JF698366, JF698212, JF6976 8 9; Ac hnatherum bromoides (L.) P. Beauv., Romasc henko 439 & Didukh (KW), Ukraine, Crimea, GU254905, GU254708, GU254845, ?, JF698 0 62, GU254734, JF697915, JF698367, JF698213, GU254624; Ac hnatherum calamagrostis (L.) P. Beauv., P yke 164 (US), Spain, Huesca, GU254899, GU254698, GU254851, GU254940, JF698 0 63, GU254743, JF697916, JF69 8368, JF69 8214, GU254638; Ac hnatherum caragana (Trin.) Nevski, Goloskokov s.n. (US), Kazakhstan, Dzhungar Alatau, GU254915, GU254690, GU254852, GU254973, JF698 0 6 4, GU254732, JF697917, JF698369, JF698215, GU254631; Ac hnatherum c hinense (Hitchc.) Tzvelev, Petrov s.n. (LE), China, Gansu, GU254900, GU254691, GU254860, ?, JF698 0 6 5, GU254735, JF697918, JF698370, JF698216, GU254630; Ac hnatherum confu s u m (Litv.) Tzvelev, Kot ukhov s.n. (LE), Kazakhstan, South Altai, JF698 5 83, JF697793, JF698 518, ?, JF698 0 6 6, ?, JF697919, JF698371, JF698217, JF69769 0 ; Ac hnatherum constrict u m (Hitchc.) Vald?s-Reyna & Barkworth, Sohns 1362 (US), Mexico, San Luis Potosi, JF698 5 8 4, JF69 779 4, JF69 8 519, JF69 775 0, JF69 8 0 67, JF69 78 4 9, JF69 7920, JF698372, JF698218, JF697691; Ac hnatherum c ur vifoliu m (Swallen) Barkworth, Wilkens 166 0 (US), U.S.A., New Mexico, ?, ?, JF698 521, ?, JF698 0 69, JF6978 51, ?, JF698374, JF698220, ?; Ac hnatherum diegoense (Swallen) Barkworth, Fiander 5778 (US), U.S.A., California, JF698 5 8 6, JF69779 6, JF698 522, JF697751, JF698 0 70, JF6978 52, JF697922, JF698375, JF698221, JF697692; Ac hnatherum eminens (Cav.) Barkworth, Peterson 10952 & Annable (US), Mexico, Zacatecas, JF698 5 8 7, JF69779 7, JF698 523, JF697752, JF698 0 71, JF6978 53, JF697923, JF698376, JF698222, JF697693; Ac hnatherum hender- sonii (Vasey) Barkworth, Maze 10 0 0 & Robson (US), U.S.A., Washington, JF698 5 8 8, JF69779 8, JF698 524, JF697753, JF698 0 72, JF6978 5 4, JF697924, JF698377, JF698223, JF69769 4; Ac hnatherum h y menoides (Roem. & Schult.) Barkworth, Saarela 205 (UBC), U.S.A., California, GU254894, GU254710, GU254844, GU254972, JF698 0 73, GU254733, JF697925, JF698378, JF698224, GU254632; Ac hnatherum inebrians (Hance) Keng ex Tzvelev, Soreng 5393, Peterson & Sun (US), China, Gansu, GU254903, GU254695, GU254847, GU254990, ?, GU254750, JF697926, JF698379, JF698225, GU254626; Ac hnatherum jacq uemontii (Jaub. & Spach) P.C. Kuo & S.L. Lu, Neubauer s.n. (US), Afganistan, Sare Bulbul, JF698 5 8 9, JF69779 9, JF698 525, ?, JF698 0 74, JF6978 5 5, JF69 7927, JF69 838 0, JF69 8226, JF69 769 5; Ac hnatherum latiglu me (Swallen) Barkworth, Le Roy s.n. (US), U.S.A., California, JF698 5 9 0, JF69 78 0 0, JF698 526, ?, JF698 0 75, JF6978 5 6, JF697928, JF698381, JF698227, ?; Ac hnatherum lemmonii (Vasey) Barkworth, Halse 616 4 (BRY), U.S.A., Oregon, JF698 5 91, JF6978 01, JF698 527, JF69775 4, JF698 0 76, JF6978 57, JF697929, JF698382, JF698228, JF69769 6 ; Ac hnatherum lettermanii (Vasey) Barkworth, Hart man 72830 (GREE), U.S.A., New Mexico, JF698 5 92, JF6978 02, JF698 528, JF69775 5, JF698 0 77, JF6978 5 8, JF697930, JF698383, JF698229, JF69769 7; Ac hnatherum lobatu m (Swallen) Barkworth, Peterson 10 66 0 & Annable (US), Mexico, Coahuila, JF698 5 93, JF6978 03, JF698 529, JF69775 6, JF698 0 78, JF69 78 5 9, JF69 7931, JF69838 4, JF698230, JF69769 8 ; Ac hnatherum m ultinode (Scribn. ex Beal) Vald?s-Reyna & Barkworth, Hoge 264 (US), Mexico, Coahuila, JF698 5 9 4, JF6978 0 4, JF698 530, JF697757, JF698 0 79, JF6978 6 0, JF697932, JF698385, JF698231, JF69769 9; Ac hnatherum nelsonii (Scribn.) Barkworth, Saarela 593 (UBC), Canada, British Columbia, GU254895, GU254713, GU254854, GU254971, JF698 0 8 0, GU254761, JF697933, JF698386, JF698232, GU254633; Ac hnatherum occidentale (Thurb. ex S. Watson) Barkworth, Saarela 594 , Sears & Maze (UBC), Canada, British Columbia, GU254896, GU254714, GU254855, GU254970, JF698 0 81, GU254754, JF697934, JF698387, JF698233, GU254634; Ac hnatherum occidentale subsp. californicu m (Merr. & Burtt Davy) Barkworth, Howell 36354 (US), U.S.A., California, EU489383, EU489171, EU489238, EU489312, JF698 0 82, GU254740, JF697935, JF69838 8, JF698234, EU489090; Ac hnatherum parishii (Vasey) Barkworth, Roos 4895 & Roos (US), U.S.A., California, EU489385, GU254716, EU489240, EU489314, JF698 0 83, GU254756, JF697936, JF698389, ?, EU489092; Ac hnatherum pekinense (Hance) Ohwi, Nefedova s.n. & Pasc henko (LE), Russia, Far East (Ne- zhina), JF698 5 9 5, JF6978 0 5, JF698 531, ?, JF698 0 8 4, JF6978 61, JF697937, JF69839 0, JF698235, JF69770 0 ; Ac hnatherum perplexu m Hoge & Barkworth, Page 2494 (US), U.S.A., New Mexico, JF698 5 9 6, JF6978 0 6, JF698 532, JF69775 8, JF698 0 8 5, JF6978 62, JF697938, JF698391, JF698236, JF697701; Ac hnatherum pinetorum (M.E. Jones) Barkworth, Bostick 4875 (US), U.S.A., Nevada, JF698 5 9 7, JF6978 0 7, JF698 533, JF69775 9, JF698 0 8 6, JF6978 63, JF697939, JF698392, JF698237, JF697702; Ac hnatherum p u bicaly x (Ohwi) Keng, Kozlov 124 (LE), China, Hebei, JF698 5 9 8, JF69 78 0 8, JF69 8 534, ?, JF698 0 8 7, JF6978 6 4, JF6979 4 0, JF698393, JF698238, JF697703; Ac hnatherum richardsonii (Link) Barkworth, Peterson 1839 9, Saarela & Smith (US), Canada, Alberta, EU489386, EU489173, JF698 535, EU489315, JF698 0 8 8, JF6978 6 5, JF6979 41, JF698394, JF698239, EU489093; Ac hnatherum robu s t u m (Vasey) Barkworth, Peterson 10583 & Annable (US), Mexico, Coahuila, GU254906, GU254715, GU254856, GU254969, JF698 0 8 9, GU254755, JF6979 42, JF698395, JF698240, GU254635; Ac hnatherum scribneri (Vasey) Barkworth, Hazlett 120 9 0 (GREE), U.S.A., New Mexico, JF698 5 9 9, JF6978 0 9, JF698 536, JF69776 0, JF698 0 9 0, JF6978 6 6, JF6979 43, JF698396, JF698241, JF69770 4; Ac hnatherum sibiric u m (L.) Keng ex Tzvelev, Soreng 5104 , Peterson, Wang & Zh u (US), China, Hebei, GU254904, GU254696, GU254846, ?, JF698 0 91, GU254741, JF6979 4 4, JF698397, JF698242, GU254610; Ac hnatherum splendens (Trin.) Nevski, Soreng 5121, Peterson, Wang & Z h u (US), China, Inner Mongolia, GU254913, GU254687, GU254818, GU254951, JF698 0 92, GU254787, JF6979 4 5, JF698398, JF698243, GU254668; Ac hnatherum stillmanii (Bol.) Barkworth, Hoover 4614 (US), U.S.A., California, JF698 6 0 0, JF697810, JF698 537, JF69 7761, JF698 0 93, JF6978 67, JF69 79 4 6, JF69 8399, JF69 824 4, JF69 770 5; Ac hnatherum t urcomanicu m Tzvelev, Goncharov 162 & Grigoriev (LE), Tajikistan, Novabad, JF698 6 01, JF697811, JF698 538, ?, JF698 0 9 4, ?, ?, ?, JF698245, JF69770 6 ; Ac hnatherum wallowaense J. Maze & K. A. Robson, Maze S2 Romaschenko & al. ? Systematics and evolution of needle grassesTAXON ? Article version: 9 Dec 2011: Electr. Suppl., 5 pp. 10 0 6 , Robson & Henn (US), U.S.A., Oregon, JF698 6 02, JF697812, JF698 539, ?, JF698 0 9 5, JF6978 6 8, JF6979 47, JF698 4 0 0, JF698246, JF69770 7; Aciachne acic ularis Laegaard, Peterson 13931 & Ref ulio Rodrig uez (US), Peru, Ancash, GU254930, GU254673, GU254865, GU254988, JF698 0 9 6, GU254806, JF6979 4 8, JF698 4 01, JF698247, GU254625; Aciachne f lagellifera L?gaard, Laegaard 19436 (AAU), Ecuador, Tungurahua, GU254893, GU254672, GU254877, GU254987, JF698 0 9 7, GU254805, JF6979 4 9, JF698 4 02, JF698248, GU254654; A melichloa caudata (Trin.) Arriaga & Barkworth, Peterson 1139 8 & Annable (US), Argentina, Mendoza, EU489388, EU489175, EU489241, EU489317, JF698 0 9 8, GU254764, ?, JF698 4 03, ?, EU489095; A melichloa clandestina (Hack.) Arriaga & Barkworth, Barkworth 5103 (US), U.S.A., Texas, GU254898, GU254717, GU254853, GU254968, JF698 0 9 9, GU254765, JF6979 5 0, JF698 4 0 4, JF698249, GU254636; A m pelodesmos mauritanic u s (Poir.) T. Durand & Schinz, P yke 702 (BC), Spain, Tarragona, GU254911, GU254722, GU254832, GU254955, JF69810 0, GU254797, JF6979 51, JF698 4 0 5, JF69825 0, GU254667; Anatherostipa hans-meyeri (Pilg.) Pe?ailillo, Peterson 20 6 45, Soreng, Romasc henko & Gonzalez-Elizondo (US), Bolivia, La Paz, EU489391, EU489177, EU489244, EU489319, JF698101, GU254804, JF6979 52, JF698 4 0 6, JF698251, EU489098; Anatherostipa mu cronata (Griseb.) F. Rojas, Peterson 19551, Soreng, Salariato & Panizza (US), Argentina, Jujuy, GU254887, GU254697, GU254861, GU254985, JF698102, GU254803, JF6979 53, JF698 4 0 7, JF698252, GU254611; Anatherostipa obt u sa (Nees & Meyen) Pe?ailillo, Peterson 13811 & Ref ulio Rodrig uez (US), Peru, Ancash, JF698 6 03, JF697813, JF698 5 4 0, JF697762, JF698103, JF6978 69, JF6979 5 4, JF698 4 0 8, JF698253, JF69770 8 ; Anatherostipa rigidi- seta (Pilg.) Pe?ailillo, Beck s.n. (LPB), Bolivia, La Paz, GU254890, GU254699, GU254869, GU254984, JF698104, GU254809, JF6979 5 5, JF698 4 0 9, JF698254, GU254612; Anatherostipa rosea (Hitchc.) Pe?ailillo, Laegaard 10 86 4 (AAU), Ecuador, Azuay, GU254889, GU254671, GU254867, GU254986, JF698105, GU254795, JF6979 5 6, JF698 410, JF698255, GU254652; Anatherostipa venusta (Phil.) Pe?ailillo, P f ister 9394 (US), Chile, Region II, GU254885, GU254680, GU254870, GU254983, JF69810 6, GU254801, JF6979 57, JF698 411, JF698256, GU254613; Anemanthele lessoniana (Steud.) Veldkamp, Travers s.n. in hb. Mez I.3236 (US), New Zealand, South Isl. (Marlborough), ( JF698 6 0 4, JF69 7814, JF698 5 41, ?, JF698107, JF69 78 70, JF69 79 5 8, JF69 8 412, JF698257, JF69770 9; A u s trostipa camp ylachne (Nees) S.W.L. Jacobs & J. Everett, Peterson 14267, Soreng, Rosenberg & MacFarlane (US), Australia, Western Austra- lia, GU254902, GU254694, GU254848, GU254975, JF698109, GU254737, JF6979 6 0, JF698 414, JF698259, GU254627; A u s trostipa compressa (R. Br.) S.W.L. Jacobs & J. Everett, Peterson 14514 , Soreng & Rosenberg (US), Australia, Western Australia, EU489393, EU489179, EU489246, EU489321, JF698110, ?, ?, ?, JF698260, EU489100; A u s trostipa f lavescens (Labill.) S.W.L. Jacobs & J. Everett, Spju t 7182 (US), Australia, Western Australia, JF698 6 0 5, JF697815, JF698 5 42, JF697763, JF698111, JF6978 71, JF6979 61, JF698 415, JF698261, JF697710; A u s trostipa hemipogon (Benth.) S.W.L. Jacobs & J. Everett, Simon 13449 (US), Australia, South Australia, JF698 6 0 6, JF697816, JF698 5 43, JF69776 4, JF698112, JF6978 72, JF6979 62, JF698 416, JF698262, JF697711; A u s trostipa juncifolia (Hughes) S.W.L. Jacobs & J. Everett, Peterson 14502 , Soreng, Rosenberg & MacFarlane (US), Australia, Western Australia, JF698 6 0 7, JF69 7817, JF69 8 5 4 4, JF69 776 5, JF69 8113, JF6978 73, JF69 79 63, JF69 8 417, JF69 8263, JF69 7712; A u s trostipa macalpinei (Reader) S.W.L. Jacobs & J. Everett, Peterson 14292 , Soreng, Rosenberg & MacFarlane (US), Australia, Western Australia, JF698 6 0 8, ?, JF698 5 4 5, JF69776 6, ?, JF6978 74, JF6979 6 4, ?, JF69826 4, JF697713; A u s trostipa nitida (Summerh. & C.E. Hubb.) S.W.L. Jacobs & J. Everett, Cant y 216 4 (US), Australia, South Australia, JF698 6 0 9, JF69 7818, JF69 8 5 4 6, JF69 7767, JF69 8114, JF69 78 75, JF69 79 6 5, JF69 8 418, JF69 8265, JF69 7714; A u s trostipa nodosa (S.T. Blake) S.W.L. Jacobs & J. Everett, Ward 203 (KW), Australia, New South Wales, JF698 610, JF697819, JF698 5 47, JF69776 8, JF698115, JF6978 76, JF6979 6 6, JF698 419, JF698266, JF697715; A u s trostipa py cnostach ya (Benth.) S.W.L. Jacobs & J. Everett, Peterson 14318 , Soreng & Rosenberg (US), Australia, Western Australia, EU489394, EU489180, EU489247, EU489322, JF698116, JF6978 7 7, ?, JF698 420, JF698267, EU489101; A u s trostipa scabra (Lindl.) S.W.L. Jacobs & J. Everett, Peterson 14442 , Soreng & Rosenberg (US), Australia, Western Australia, EU489395, EU489181, EU489248, EU489323, JF698117, GU254738, JF6979 67, JF698 421, JF698268, EU489102; A u s trostipa semibarbata (R. Br.) S.W.L. Jacobs & J. Everett, Simon 13439 (US), Australia, South Australia., GU254901, GU254693, GU254849, GU254974, JF698118, GU254736, JF6979 6 8, JF698 422, JF698269, GU254628; A u s trostipa tenuifolia (Steud.) S.W.L. Jacobs & J. Everett, Peterson 14248 , Soreng & Macfarlane (US), Australia, Western Australia, EU489397, EU489183, EU489250, EU489325, JF698119, GU254739, JF6979 69, JF698 423, JF698270, EU489104; A u s trostipa trichoph ylla (Benth.) S.W.L. Jacobs & J. Everett, Peterson 14324 , Soreng & Rosenberg (US), Australia, Western Australia, JF698 611, JF697820, JF698 5 4 8, JF697769, JF698120, JF6978 78, JF6979 70, JF698 424, JF698271, JF697716; Celtica gigantea (Link) F. M. V?zquez & Barkworth, P yke 705 (BC), Spain, Albacete, GU254919, GU254726, GU254843, GU254961, JF698123, GU254775, JF6979 73, JF698 427, JF698274, GU254642; Hesperostipa comata (Trin. & Rupr.) Barkworth, Saarela 595, Sears & Maze (UBC), Canada, British Columbia, EU489399, EU489185, EU489252, EU489327, JF698130, GU254812, JF6979 79, JF698 434, JF698281, EU489106; Hesperostipa neomexicana (Thurb.) Barkworth, Peterson 18934 & Valdes-Reyna (US), Mexico, Nuevo Leon, EU489400, EU489186, GU254840, EU489328, JF698131, GU254808, JF6979 8 0, JF69 8 435, JF69 8282, EU489107; Hesperostipa sparte a (Trin.) Barkworth, Holmes 214 (US), U.S.A., (Missouri?), EU489401, EU489187, EU489253, EU489329, JF698132, GU254745, JF6979 81, JF698 436, JF698283, EU489108; Jarava annua (Mez) Pe?ailillo, Peterson 15614 & Soreng (US), Chile, Region I, JF698 613, JF697822, JF698 5 52, JF697773, JF698133, JF69 78 82, JF69 79 82, JF69 8 437, JF69 828 4, JF69 7720; Jarava castellanosii (F.A. Roig) Pe?ailillo, Peterson 10336 & Annable (US), Argentina, Jujuy, EU489405, EU489191, EU489256, EU489333, JF698134, GU254770, JF6979 83, JF698 438, JF698285, EU489112; Jarava ich u Ruiz & Pav., Peterson 20745, Soreng, Romasc henko & Gonzalez Elizondo (US), Peru, Puno, EU489415, EU489202, EU489267, EU489344, JF698135, GU254763, JF6979 8 4, JF698 439, JF69 8286, EU489124; Jarava media (Speg.) Pe?ailillo, Peterson 19337, Soreng, Salariato & Panizza (US), Argentina, La Rioja, EU489419, EU489205, EU489272, EU489347, JF698136, GU254758, JF6979 8 5, JF698 4 4 0, JF698287, EU489129; Jarava plu mosula (Nees ex Steud.) F. Rojas, Peterson 20471, Soreng, Romasc henko & Susanibar Cruz (US), Peru, Ayacucho, EU489422, EU489207, EU489275, EU489350, JF698137, GU254757, JF6979 8 6, JF698 4 41, JF69828 8, EU489133; Jarava pseudoich u (Caro) F. Rojas, Peterson 20736 , Soreng, Romasc henko & Gonzalez Elizondo (US), Peru, Puno, EU489424, EU489209, EU489277, EU489352, JF698138, GU254762, JF6979 8 7, JF698 4 42, JF698289, EU489135; Jarava scabrifolia (Torres) Pe?ailillo, Peterson 11712 & Annable (US), Argentina, Salta, EU489425, EU489210, EU489278, EU489353, JF698139, GU254760, JF6979 8 8, JF69 8 4 43, JF69829 0, EU489136; Lorenzochloa erectifolia (Swallen) Reeder & C. Reeder, Peterson 14074 & Ref ugio Rodrig uez (USM), Peru, Junin, JN882350 , ?, JN882351 , ?, ?, ?, ?, ?, ?, ?; Macrochloa tenacissima (Loefl. ex L.) Kunth, P yke 701 (BC), Spain, Almer?a, GU254912, GU254723, GU254833, GU254978, JF698142, GU254782, JF6979 91, JF698 4 4 6, JF698293, GU254648; Nassella clarazii (Ball) Barkworth, Peterson 11651 & Annable (US), Argentina, Catamarca, EU489436, EU489221, EU489289, EU489364, JF698144, GU254766, JF6979 9 4, ?, JF698296, EU489147; Nassella filic ulmis (Delile) Barkworth, Soreng 70 0 9 & Soreng (US), Chile, Region VIII, EU489439, EU489224, EU489292, EU489367, JF698145, GU254768, JF6979 9 5, JF698 4 4 9, JF698297, EU489150; Nassella neesiana (Trin. & Rupr.) Barkworth, Peter- son 10258 & Annable (US), Argentina, Jujuy, EU489444, EU489228, EU489297, EU489371, ?, GU254767, JF6979 9 6, JF69 8 4 5 0, JF69 8298, EU489155; Nassella pfisteri (Matthei) Barkworth, Soreng 7017a & Soreng (US), Chile, Region VIII, EU489446, EU489229, EU489299, EU489372, JF698146, JF697885, JF6979 9 7, JF698 4 51, JF698299, EU489157; Nassella trichotoma (Nees) Hack. ex Arechav., Peterson 1150 6 & Annable (US), Argentina, San Juan, EU489451, EU489232, EU489305, EU489376, JF698147, GU254742, JF6979 9 8, JF698 4 52, JF69830 0, EU489164; Ortachne breviseta Hitch., Laegaard 128 02 (AAU), Argentina, Neuqu?n, GU254924, GU254674, GU254868, GU254989, JF698148, GU254810, JF6979 9 9, JF698 4 53, JF698301, GU254666; Ortachne rarif lora (Hook. f.) Hughes, Mariz 924 (CONC), Chile, Region XI, GU254925, GU254675, GU254864, GU254952, JF698149, GU254771, JF698 0 0 0, JF69 8 4 5 4, JF698302, GU254665; Orthoraphiu m roylei Nees, Soreng 5261, Peterson & Sun (US), China, Yunnan, GU254881, GU254703, GU254858, GU254979, JF698150, GU254753, JF698 0 01, JF69 8 4 5 5, JF69 8303, GU254617; Oryzopsis asperifolia Michx., Saarela 384 (UBC), Canada, Alberta, GU254908, GU254686, GU254821, GU254964, JF698151, GU254788, JF698 0 02, JF698 4 5 6, JF698304, GU254653; Pappostipa chr ysoph ylla (E. Desv.) Romasch., Peterson 19220 , Soreng, Salariato & Panizza (US), Argentina, Mendoza, EU489407, EU489193, EU489258, EU489335, JF698152, GU254781, JF698 0 03, JF698 4 57, JF698305, EU489114; Pappostipa hieronym u sii (Pilg.) Romasch., Peterson 1148 8 & Annable (US), Argentina, San Juan, EU489410, EU489197, EU489262, EU489339, JF698153, JF697886, JF698 0 0 4, JF69 8 4 5 8, JF69 830 6, EU489118; Pappostipa major (Speg.) Romasch., Soreng 7222 & Soreng (US), Chile, Region IX, EU489427, EU489212, EU489280, EU489355, JF69815 4, GU254798, JF698 0 0 5, JF69 8 4 5 9, JF69 8307, EU489138; Pappostipa speciosa (Trin. & Rupr.) Romasch., Peterson 11549 & Annable (US), Argentina, San Juan, EU489426, EU489211, EU489279, EU489354, ?, GU254772, JF698 0 0 6, JF698 4 6 0, JF69830 8, EU489137; Pappostipa vaginata (Phil.) Romasch., Peterson 11744 & Annable (US), Argentina, Salta, EU489431, EU489216, EU489284, EU489359, JF698155, Appendix ?. Continued. S3 Romaschenko & al. ? Systematics and evolution of needle grassesTAXON ? Article version: 9 Dec 2011: Electr. Suppl., 5 pp. GU254816, JF698 0 0 7, JF698 4 61, JF698309, EU489142; Patis coreana (Honda) Ohwi, Liou 10 85 (US), China, Hebei, JF698 5 8 5, JF69779 5, JF698 520, ?, JF698 0 6 8, JF6978 5 0, JF697921, JF698373, JF698219, ?; Patis obt u sa (Stapf) Romasch., P.M. Peterson & Soreng, Soreng 4531 & Kelly (US), China, Sichuan, JF698 619, JF697828, JF698 5 5 9, JF697778, JF698166, JF6978 91, JF698 018, JF698 472, JF698320, JF697727; Pip tatheropsis canadensis (Poir.) Romasch., P.M. Peterson & Soreng, Raymond & Ku c yniak s.n. (US), Canada, Quebec, JF698 616, JF697825, JF698 5 5 6, JF697775, JF698159, JF6978 8 8, JF698 011, JF69 8 4 6 5, JF69 8313, JF697724; Pip tatheropsis exig ua (Thurb.) Romasch., P.M. Peterson & Soreng, Reveal 1073 (KW), U.S.A., Colorado, GU254927, GU254677, GU254859, GU254976, JF698161, GU254752, JF698 013, JF698 4 67, JF698315, GU254663; Pip tatheropsis micrantha (Trin. & Rupr.) Romasch., P.M. Peterson & Soreng, Peterson 18437, Saarela & Smith (US), Canada, Alberta, GU254926, GU254676, GU254866, GU254950, JF698167, GU254815, JF698 019, JF698 473, JF698321, GU254664; Pip tatheropsis p ungens (Torr.) Romasch., P.M. Peterson & Soreng, Hermann 134 07 (US), Canada, Alberta, JF698 622, JF697831, JF698 5 62, JF69 7781, JF69 8172, JF6978 9 4, JF69 8 024, JF69 8 478, JF69 8326, JF69 7730; Pip tatheropsis s hoshoneana (Curto & Douglass M. Hend.) Romasch., P.M. Peterson & Soreng, Eno 13 (US), U.S.A., Idaho, GU254928, GU254688, GU254841, GU254941, JF698173, GU254814, JF698 025, JF698 479, JF698327, GU254662; Pip tatherum aequiglu me (Duthie ex J.D. Hooker) Roshev., Koelz 1389 (US), India, Himachal Pradesh, JF698 615, JF697824, JF698 5 5 5, JF697774, JF698157, JF6978 8 7, JF698 0 0 9, JF698 4 63, JF698311, JF697723; Pip tatherum ang u s tifoliu m (Munro ex Regel) Roshev., Nepli 144 (LE), Tajikistan, West Pamir (Khorog), GU254923, GU254707, GU254825, GU254944, JF69815 8, GU254811, JF698 010, JF698 4 6 4, JF698312, GU254659; Pip tatherum coerulescens (Desf.) P. Beauv., Soreng 3765 (US), Greece, Attica, GU254936, GU254685, GU254822, GU254949, JF698160, GU254789, JF698 012, JF698 4 6 6, JF698314, GU254639; Pip tatherum fedts c henkoi Roshev., Botc hantzev 1559 (LE), Tajikistan, Pamir, JF698 617, JF697826, JF698 5 57, JF697776, JF698162, JF6978 8 9, JF698 014, JF698 4 6 8, JF698316, JF697725; Pip tatherum ferganense (Litw.) Roshev., Kamelin 10 0 (LE), Tajikistan, Guis- sar (Varzob), JF698 618, JF697827, JF698 5 5 8, JF697777, JF698163, JF6978 9 0, JF698 015, JF698 4 69, JF698317, JF697726; Pip tatherum hilariae Pazij, Potaliev 343 (LE), Tajikistan, Pamir, GU254929, GU254679, GU254828, GU254943, JF69816 4, GU254807, JF698 016, JF698 470, JF698318, GU254660; Pip tatherum holciforme (M. Bieb.) Roemer & Schultes, Didukh 1203 (KW), Ukraine, Yalta, GU254931, GU254670, GU254829, GU254945, JF698165, GU254817, JF698 017, JF69 8 471, JF69 8319, GU254658; Pip tatherum miliaceu m (L.) Coss., Gillet t 16 0 94 (US), Jordan, Azraq, GU254918, GU254729, GU254838, GU254960, JF698168, GU254776, JF698 020, JF698 474, JF698322, GU254643; Pip tatherum m unroi (Stapf) Mez, Soreng 5686 , Peterson & Sun (US), China, Sichuan, JF698 621, JF697830, JF698 5 61, JF69778 0, JF698170, JF6978 93, JF698 022, JF698 476, JF698324, JF697729; Pip tatherum para- doxu m (L.) P. Beauv., P yke 831 (BC), Spain, Catalu?a, GU254916, GU254727, GU254862, GU254977, JF698171, GU254744, JF698 023, JF698 477, JF698325, GU254622; Pip tatherum sogdianum (Grig.) Roshev., Konkov 1637 (LE), Tajikistan, Sughd, JF698 623, ?, JF698 5 63, ?, JF698174, JF6978 9 5, ?, JF698 4 8 0, JF698328, JF697731; Pip tatherum songaricu m (Trin. & Rupr.) Roshev., Goloskokov s.n. (LE), Kazakhstan, Ili-Pribalhashje, JF698 624, JF697832, JF698 5 6 4, JF697782, JF698175, JF6978 9 6, JF698 026, JF698 4 81, JF698329, JF697732; Pip tatherum thomasii (Duby) Kunth, P yke 833 (BC), Spain, Catalu?a, JF698 620, JF697829, JF698 5 6 0, JF697779, JF698169, JF6978 92, JF698 021, JF698 475, JF698323, JF697728; Pip tatherum virescens (Trin.) Boiss., Romasc henko 445 & Didukh (KW), Ukraine, Yalta, GU254917, GU254728, GU254837, GU254959, JF698176, GU254777, JF698 027, JF698 4 82, JF698330, GU254644; Pip to- chaetiu m avenaceum (L.) Parodi, Soreng 7739 & Romasc henko (US), U.S.A., Maryland, GU254883, GU254705, GU254872, GU254981, JF698177, GU254799, JF698 028, JF698 4 83, JF698331, GU254615; Pip tochaetiu m brach y s permu m (Speg.) Parodi, Peterson 11252 & Annable (US), Argentina, La Pampa, EU489452, EU489233, EU489306, EU489377, JF698178, GU254802, JF698 029, JF698 4 8 4, JF698332, EU489165; Pip tochaetiu m featherstonei (Hitchc.) Tovar, Peterson 10314 & Annable (US), Argentina, Jujuy, GU254882, GU254704, GU254873, GU254980, JF698179, GU254796, JF698 030, JF698 4 8 5, JF698333, GU254616; Pip tochaetiu m montevidense (Spreng.) Parodi, Peterson 20486 , Soreng, Romasc henko & Su sanibar Cruz (US), Peru, Ayacucho, JF698 625, JF69 7833, JF698 5 6 5, JF697783, JF69818 0, JF6978 9 7, ?, JF698 4 8 6, JF698334, JF697733; Pip tochaetiu m panicoides (Lam.) E. Desv., Soreng 7011 & Soreng (US), Chile, Region VIII, EU489453, EU489234, EU489307, EU489378, JF698181, GU254794, JF698 031, JF698 4 8 7, JF698335, EU489166; Psammochloa villosa (Trin.) Bor, Safronova 952 (LE), Mongolia, Suhe-Batorsk, GU254892, GU254678, GU254820, GU254939, JF698182, GU254786, JF698 032, JF698 4 8 8, JF69 8336, GU254651; P tilagrostis dichotoma Keng ex Tzvelev, Soreng 5647, Peterson & Sun (US), China, Zizang, GU254880, GU254702, GU254874, GU254965, JF698183, GU254749, JF698 033, JF69 8 4 8 9, JF69 8337, GU254618; P tilagrostis junatovii Grubov, Kot ukhov s.n. (LE), Russia, West Altai, GU254879, GU254701, GU254842, ?, JF69818 4, GU254748, JF698 034, JF698 49 0, JF698338, GU254619; P tilagrostis kingii (Bol.) Barkworth, Peirson 10 819 (US), U.S.A., California, GU254937, GU254712, GU254827, GU254942, JF698185, GU254813, JF698 035, JF698 491, JF698339, GU254661; P tilagrostis lu q uensis P.M. Peterson, Soreng & Z.L. Wu, Soreng 5383, Peterson & Sun (US), China, Gansu, GU254878, GU254700, GU254875, ?, JF698186, GU254747, JF698 036, JF698 492, JF698340, GU254620; P tilagrostis malysc hevii Tzvelev, Ikonnikov 21570 (LE), Krygyzstan or Tajikistan, ?, JF698 626, JF697834, JF698 5 6 6, ?, JF698187, ?, ?, JF698 493, JF698341, JF697734; P tilagrostis mongholica (Turcz. ex Trin.) Griseb., Koloskov s.n. (LE), Kyrgyzstan, ?, GU254886, GU254689, GU254876, GU254938, JF69818 8, GU254746, JF698 037, JF698 49 4, JF698342, GU254621; P tilagrostis pelliotii (Danguy) Grubov, Gr u bov 1815 (LE), Mongolia, Trans-Altai Gobi, JF698 627, JF697835, JF698 5 67, ?, JF698189, JF6978 9 8, JF698 038, JF698 49 5, JF698343, JF697735; Stipa barbata Desf., P yke 704 (BC), Spain, Granada, JF698 630, JF697838, JF698 570, JF69778 4, JF698193, JF6979 01, JF698 0 42, JF698 49 9, JF698347, JF697738; Stipa brauneri (Pacz.) Klokov, Romasc henko 418 (BC), Ukraine, Crimea, JF698 631, JF697839, JF698 571, JF69778 5, JF698194, JF6979 02, JF698 0 43, JF698 5 0 0, JF698348, JF697739; Stipa brevif lora Griseb., Soreng 5449 & Peterson (US), China, Qinghai, JF698 632, ?, JF698 572, ?, JF698195, JF6979 03, JF698 0 4 4, JF69 8 5 01, JF698349, JF69774 0 ; Stipa bungeana Trin., Soreng 5397, Peterson & Sun (US), China, Gansu, JF698 633, JF6978 4 0, JF69 8 573, JF69778 6, JF698196, JF6979 0 4, JF698 0 4 5, JF698 5 02, JF698350, JF697741; Stipa capensis Thunb., P yke 703 (BC), Spain, Tarragona, GU254921, GU254724, GU254826, GU254963, JF698197, GU254773, JF698 0 4 6, JF698 5 03, JF698351, GU254640; Stipa capillacea Keng, Soreng 5382 , Peterson & Sun (US), China, Sichuan, JF698 634, ?, JF698 574, ?, JF698198, JF6979 0 5, JF698 0 47, JF698 5 0 4, JF698352, JF697742; Stipa capillata L., Romasc henko 60 6 (KW), Kazakhstan, Jambyl, JF698 635, JF6978 41, JF698 575, JF69778 7, JF698199, JF6979 0 6, JF698 0 4 8, JF698 5 0 5, JF698353, JF697743; Stipa caucasica Schmalh., Ro- masc henko 635, Su sanna & Kudratov (BC), Tajikistan, Zeravshan, JF698 636, JF6978 42, JF698 576, JF69778 8, JF69820 0, JF6979 0 7, JF698 0 4 9, JF698 5 0 6, JF69835 4, JF69774 4; Stipa parvif lora Desf., Romasc henko 74 & Romo (US), Spain, Almer?a, GU254920, GU254719, GU254839, GU254962, JF698201, GU254774, JF698 0 5 0, JF698 5 0 7, JF698355, GU254641; Stipa pennata L., Romasc henko 466 (BC), Ukraine, Lugansk, GU254891, GU254718, GU254857, GU254967, JF698202, GU254759, JF698 0 51, JF698 5 0 8, JF698356, GU254637; Stipa p urp urea Griseb., Soreng 550 9, Peterson & Sun (US), China, Xizang, JF698 637, JF6978 43, JF698 57 7, JF69778 9, JF698203, JF6979 0 8, JF698 0 52, JF698 5 0 9, JF698357, JF69774 5; Stipa regeliana Hack., Tz velev 694 (LE), Tajikistan, ?, JF698 638, JF6978 4 4, JF698 578, ?, JF698204, JF6979 0 9, JF698 0 53, JF698 510, JF69835 8, JF69774 6 ; Stipa su b sessilif lora (Rupr.) Roshev., I vanov s.n. (LE), Kyrgyzstan, ?, JF698 639, JF6978 4 5, JF698 579, JF69779 0, JF698205, JF697910, JF698 0 5 4, JF698 511, JF698359, JF697747; Timouria saposhnikovii Roshev., Soreng 5475, Peterson & Sun (US), China, Gansu, GU254897, GU254692, GU254850, ?, JF69820 6, GU254731, JF698 0 5 5, JF698 512, JF698360, GU254629; Trikeraia hookeri (Stapf) Bor, Koelz 2328 (US), India, Jammu and Kashmir, GU254909, GU254711, GU254830, GU254953, JF698207, GU254785, JF698 0 5 6, JF698 513, JF698361, GU254650; Trikeraia pappiformis (Keng) P.C. Kuo & S.L. Lu, Soreng 5653, Peterson & Sun (US), China, Xizang, GU254910, GU254721, GU254831, GU254954, JF69820 8, GU254784, JF698 0 57, JF698 514, JF698362, GU254649. Appendix ?. Continued. S4 Romaschenko & al. ? Systematics and evolution of needle grassesTAXON ? Article version: 9 Dec 2011: Electr. Suppl., 5 pp. 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 . Br ylkinia 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 P tilagrostis lu q uensis , 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 Ac hnatherum latiglu me , A. perplexu m , A. pinetorum , and A . s cribneri (?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 Pip tochaetiu m brach y s permu m . Its presence in two serially diverging lineages in the Stillmania /Pip tatherum (P2) clade could be mapped as having been gained twice, or gained once and lost once. In our study A m pelodesmos 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, A m pelodesmos 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 Pip tatherum and Pip tatheropsis (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 P tilagrostis 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 an- giosperms. Cladistic s 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. C urr. 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. Ph y totaxa 1: 3?20. Appendix ?. Chromosome numbers of taxa included in the phylogenetic analyses. A c h n a t h e r u m bromoides (L.) P. Beauv. ? 2n = 18, 28 (Tzvelev, 1976; Freitag, 1985; V?zquez & Devesa, 1996); A . calamagrostis (L.) P. Beauv. ? 2n = 24 (Martinovsk?, 1980; V?zquez & Devesa, 1996); A . caragana (Trin.) Nevski ? 2n = 24 (Freitag, 1985); A . confu s u m (Litv.) Tzvelev ? 2n = 24 (Tzvelev, 1976); A . c ur vifoliu m (Swallen) Barkworth ? 2n = 44 (Barkworth, 2007); A . eminens (Cav.) Barkworth ? 2n = 44, 46 (Brown, 1951; Barkworth, 2007); A . hen- dersonii (Vasey) Barkworth ? 2n = 34 (Barkworth, 2007); A . h y menoides (Roem. & Schult.) Barkworth ? 2n = 46, 48 (Johnson, 1945; Barkworth, 2007); A . jacq uemontii (Jaub. & Spach) P.C. Kuo & S.L. Lu ? 2n = 24 (Freitag, 1985); A . latiglu me (Swallen) Barkworth ? 2n = 70 (Barkworth, 2007); A . lemmonii (Vasey) Barkworth ? 2n = 34 (Barkworth, 2007); A . let termanii (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. californicu m (Merr. & Burtt Davy) Barkworth ? 2n = 36 (Johnson, 1962); A . pekinense subsp. eff u s u m (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 . robu s t u m (Vasey) Barkworth ? 2n = 64 (Barkworth, 2007); A . s cribneri (Vasey) Barkworth ? 2n = 40 (Barkworth, 2007); A . sibiric u m (L.) Keng ex Tzvelev ? 2n = 22, 24 (Avdulov, 1931); A . s plendens (Trin.) Nevski ? 2n = 48, 42 (Tzvelev, 1976; Freitag, 1985); A . t urcomanicu m Tzvelev ? 2n = 24 (Freitag, 1985); A cia c h n e acic ularis Laegaard ? 2n = 22 (Reeder & Reeder, 1968; Macfarlane & Watson, 1980; Davidse & Pohl, 1994); A . f lagellifera L?gaard ? 2n = 22 (Reeder & Reeder, 1968; Macfarlane & Watson, 1980); A m p e l o d e s m o s mauritanicu s (Poir.) T. Durand & Schinz ? x = 12; 2n = 48, 96 (Decker, 1964; Macfarlane & Watson, 1980; Martinovsk?, 1980); A n a t h e r o s ti p a hans-meyeri (Pilg.) Pe?ailillo ? 2n = 22 (Davidse & Pohl, 1994); A n e m a n t h e l e les- soniana (Steud.) Veldkamp ? 2n = 40?44 (Murray & al., 2005); B r a c h y e l y t r u m erect u m (Schreb.) P. Beauv. ? x = 11; 2 x = 22 (Macfarlane & Watson, 1980; Wu & Phillips, 2006); B r y l kinia caudata (Munro) F. Schmidt ? 2n = 40 (Macfarlane & Watson, 1980; Wu & Phillips, 2006); C e l tic a gigantea (Link) F.M. V?zquez & Barkworth ? 2n = 96 (V?zquez & Devesa, 1996; V?zquez & Barkworth, 2004); D a n t h o nia s t r u m compact u m (Boiss. & Heldr.) Holub ? 2n = 14 (Kozuharov & Petrova, 1991); Dia r r h e n a 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); D u t hie a brach y podium (P. Candargi) Keng & Keng f. ? x = 7 (Watson & Dallwitz, 1992); He s p e r o s ti p a comata (Trin. & Rupr.) Barkworth ? 2n = 36, 44, 46 (Barkworth, 2007); H . neomexicana (Thurb.) Barkworth ? 2n = 44 (Barkworth, 2007); H . s partea (Trin.) Barkworth ? 2n = 44, 46 (Barkworth, 2007); J a r a v a ich u Ruiz & Pav. ? 2n = 40 (Davidse & Pohl, 1994); J. plu mosula (Nees ex Steud.) F. Rojas ? 2n = 44 (Avdulov, 1931; Covas & Bocklet, 1945; Matthei, 1965); L o r e n z o c h l o a erectifolia (Swallen) Reeder & C. Reeder ? 2n = 22 (Reeder & Reeder, 1968); Ly g e u m spartu m L. ? 2n = 16, 40 (Myers, 1947; Djabeur & al., 2008); M a c r o c h l o a tenacissima (Loefl. ex L.) Kunth ? n = 12; 2n = 24, 40, 64, 66, 72 (V?zquez & Devesa, 1996; V?zquez & Barkworth, 2004); N a r d u s stricta L. ? 2n = 26 (Avdulov, 1931; Barkworth, 2007); N a s s e l l a neesiana (Trin. & Rupr.) Barkworth ? 2n = 28 (Matthei, 1965; Barkworth, 2007); N . trichotoma (Nees) Hack. ex Arechav. ? 2n = 36 (Barkworth, 2007); O r y z o p si s asperifolia Michx. ? 2n = 46, 48 (Johnson, 1945); P a p p o s ti p a major (Speg.) Romasch. ? 2n = 60 (Roig, 1964); P. s peciosa (Trin. & Rupr.) Romasch. ? 2n = 66, 68, 74 (Stebbins & Love, 1941; Roig, 1964; Matthei, 1965; Barkworth, 2007); P a ti s coreana (Honda ex Nakai) Ohwi ? 2n = 46 (Tateoka, 1986); P h a e n o s p e r m a globosa Munro ex Benth. ? x = 12; 2n = 24 (Avdulov, 1931; Macfarlane & Watson, 1980; Wu & Phillips, 2006); Pi p t a t h e r o p si s canadensis (Poir.) 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The grass genera of the world: Descriptions, illustrations, identification, and information retrieval; including synonyms, morphology, anatomy, physiology, phytochemistry, cytology, classification, pathogens, world and local distribution, and references. Version: 23 Apr. 2010. http://delta-intkey.com/grass/index.htm. Wu, Z. & Phillips, S.M. 2006. Stipeae. Pp. 188?212 in: Wu, Z., Raven, P.H. & Hong, D.Y. (eds.), Flora of China, vol. 22, Poaceae . Beijing: Science Press; St. Louis: Missouri Botanical Garden Press. (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); Pi p t a t h e r u m coerulescens (Desf.) P. Beauv. ? 2n = 24 (Kergu?len, 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. m unroi (Stapf) Mez ? 2n = 24 (Moinuddin & al., 1994); P. paradoxu m (L.) P. Beauv. ? 2n = 24 (Martinovsk?, 1980); P. songaric u m (Trin. & Rupr.) Roshev. ex Nikitina ? 2n = 24 (Tzvelev, 1976); P. virescens (Trin.) Boiss. ? 2n = 24 (Avdulov, 1931; Freitag, 1975; Tzvelev, 1976); Pi p t o c h a e tiu m avenaceum (L.) Parodi ? 2n = 22, 28 (Cialdella & Giussani, 2002; Barkworth, 2007); P ti l a g r o s ti s 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); S t e p h a n a c h n e nigrescens Keng ? 2n = 24 (Watson & Dallwitz, 1992); S. pappophorea (Hack) Keng ? 2n = 24 (Watson & Dallwitz, 1992); S ti p a barbata Desf. ? 2n = 44 (V?zquez & 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; V?zquez & Devesa, 1996); S. capillata L. ? 2n = 44 (Avdulov, 1931; Tzvelev, 1976; Freitag, 1985; V?zquez & Devesa, 1996); S. caucasica Schmalh. ? 2n = 44 (Tzvelev, 1976; Freitag, 1985); S. parvif lora Desf. ? 2n = 28 (Freitag, 1985; V?zquez & Devesa, 1996); S. pennata L. ? 2n = 44 (Avdulov, 1931; Tzvelev, 1976; Freitag, 1985); Trinio c h l o a stipoides (Kunth) Hitchc. ? 2n = 32 (Brown, 1951; Reeder, 1971). Appendix ?. Continued.