Phylogeny of New World Stipeae (Poaceae): an evaluationof the monophyly of Aciachne and Amelichloa

Ana M. Cialdella*, Diego L. Salariato, Lone Aagesen, Liliana M. Giussani,Fernando O. Zuloaga and Osvaldo Morrone

Instituto de Botanica Darwinion, Labarden 200, San Isidro, B1642HYD Buenos Aires, Argentina

Accepted 8 February 2010

Abstract

The tribe Stipeae, with nearly 550 species, includes 28 core genera, of which 13 occur in America: Achnatherum, Aciachne,Amelichloa, Anatherostipa, Hesperostipa, Jarava, Nassella, Ortachne, Oryzopsis, Pappostipa, Piptatherum, Piptochaetium, and

Ptilagrostis. Based on 37 species representing 14 Stipeae genera, and using four chloroplast markers and morphological characters,we provide a phylogenetic hypothesis of the New World Stipeae, with our focus on Amelichloa and Aciachne. Parsimony analyses

included two approaches: (i) a multiple-sequence alignment where gaps were treated as missing or coded, (ii) using direct sequences

by direct optimization as implemented by the program POY v.4.0.2870. Analyses under direct optimization were conducted usingthe molecular data sets independently and combined, and with morphological data. Different cost regimes were explored and the

one producing the highest congruence between partitions was chosen. Among the genera considered, only Piptochaetium,Austrostipa, and Hesperostipa were resolved as monophyletic, while Achnatherum, Amelichloa s.l., Anatherostipa, Jarava, and

Nassella were polyphyletic, and Aciachne was polyphyletic or paraphyletic. As a result, Amelichloa can be restricted to amonophyletic group if including A. brachychaeta, A. ambigua, A. clandestina, and A. caudata, or it should be considered within

Nassella. The phylogenetic position of species of Aciachne suggests inbreeding and outbreeding events with species of Anatherostipa,Ortachne, and Hesperostipa.

� The Willi Hennig Society 2010.

The Stipeae comprises nearly 550 species found inboth hemispheres, mainly in temperate and warmtemperate grasslands of Africa, Australia, Eurasia, andAmerica (Barkworth, 1993; Barkworth et al., 2008). Thetribe was established by Kunth (1815), and its membersare mostly recognized by the presence of one-floweredspikelets with the rachilla articulated above the glumesand without extension, and lemmas with a single,terminal awn or a sharp point.

Although the delimitation of the Stipeae underwentseveral changes through the removal of unrelated genera(Barkworth and Everett, 1987), the remainder is con-sidered a natural and well defined monophyletic group(Hsiao et al., 1999; Jacobs et al., 2000, 2007). At

present, the core Stipeae (subtribe: Stipeae s.s.) isconsidered well delimited, although subtribes Ampel-odesminae and Duthieinae are sometimes included asisolated Stipeae s.l. elements (Soreng et al., 2003; Wuand Phillips, 2006), while the relationship of thesesubtribes to tribe Phaenospermateae is ambiguous.

Within the tribe, generic circumscription has changeddramatically, especially in recent years, based on mor-phological and molecular characters. Bentham (1882)recognized only two genera in the tribe, Stipa L. andOryzopsis Michx. In America, Hitchcock (1935, 1951)accepted Piptochaetium J. Presl and Nassella E. Desv., aconcept followed in most North American floras. InSouth America, Spegazzini (1901) accepted AciachneBenth., Oryzopsis, and Stipa. The genus Stipa, asoriginally conceived, was viewed as the core of thetribe: it was reviewed by Spegazzini (1901, 1925) who

C L A 3 1 0 B Dispatch: 24.3.10 Journal: CLA CE: Valarmathi

Journal Name Manuscript No. Author Received: No. of pages: 17 PE: Shyamala

*Corresponding author:

E-mail address: anacialdella@darwin.edu.ar

� The Willi Hennig Society 2010

Cladistics

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also established 12 subgenera based mainly on spikeletcharacters. This broad concept was followed in manydifferent treatments during the past century (Hitchcock,1935, 1951; Clayton and Renvoize, 1986; Nicora andRugolo de Agrasar, 1987; Curto, 1998; Renvoize, 1998).Recently, several subgenera of Stipa were raised to thegeneric rank on the basis of anatomical and morpho-logical characters, and new genera were also established(Barkworth and Everett, 1987; Barkworth, 1990; Jacobsand Everett, 1996; Penailillo, 1996, 2002, 2003; Torres,1997; Barkworth and Torres, 2001; Wheeler et al., 2002;Arriaga and Barkworth, 2006; Romaschenko et al.,2008). As currently understood, the Stipeae includes 28genera, of which 13 occur in America (Soreng et al.,2003; Arriaga and Barkworth, 2006; Romaschenkoet al., 2008): four of them inhabit both North andSouth America (Amelichloa, Arriaga & Barkworth,Piptochaetium, Nassella, and Piptatherum P. Beauv.);four are restricted to North America [PtilagrostisGriseb., Hesperostipa (M. K. Elias) Barkworth, Oryz-opsis, and Achnatherum P. Beauv.]; and five genera arepresent only in South America [Aciachne, Anatherostipa(Hack. ex Kuntze) Penailillo, Ortachne Nees ex Steud.,Jarava Ruiz & Pav. (the latter with one species reachingMexico), and Pappostipa (Speg.) Romasch., P.M. Pet-erson & Soreng (with one species reaching Mexico andthe USA)]. Four genera, endemic to Europe, Africa,Asia, or Australia, are also cultivated or introduced inAmerica (Soreng et al., 2003): Austrostipa S. W. L.Jacobs & J. Everett, Macrochloa Kunth, the recentsegregate genus Celtica F.M. Vazquez & Barkworth,and Stipa.

Although there is no doubt about the monophyly ofthe core Stipeae, serious discrepancies have arisenregarding the taxonomic and phylogenetic delimitationof its genera. Using four chloroplast markers (rpl16,rpoA, trnL-F, and trnT-L) and morphological charac-ters, we test the monophyly of the fully sampled generaAmelichloa and Aciachne, and provide a new phyloge-netic hypothesis of the New World members of theStipeae. The relationships among the 14 genera of thetribe occurring in America are presented and discussed.

An update to Genera of Stipeae occurring in America

For clarification on the generic limits, we present anupdate of the taxonomy and the criteria followed in thisstudy.

Achnatherum. Generic boundaries of Achnatherumhave been controversial for many years: Hitchcock(1925, 1935, 1951) included the short-awned species inOryzopsis and the long-awned ones in Stipa, Tzvelev(1976) restricted Achnatherum to nearly 20 speciesdistributed in subtropical regions of Eurasia, NorthAfrica, and North America, while Clayton and Renvo-ize (1986) included Achnatherum in Stipa, and Rojas

(1998) considered it as a synonym of Jarava. Ascurrently circumscribed (Jacobs and Everett, 1997;Penailillo, 2003; Arriaga and Barkworth, 2006; Roma-schenko et al., 2008), Achnatherum is a worldwide genuswith 36 species cited for America (Soreng et al., 2003).

Aciachne. This is a New World genus with threespecies ranging from Costa Rica to Argentina and Chile(Lægaard, 1987) 1. The genus is diagnosed by severalcharacters, including small pulvinate habit, acicularinvolute blades, and indurate glumes, lemma, and palea.

Amelichloa –This genus, recently established by Arri-aga and Barkworth (2006), was based on five speciespreviously classified in Achnatherum, Jarava, or Nassel-la. Four species out of five are found in South America,while A. clandestina is restricted to southern USA,northern Mexico and Colombia. Arriaga and Bark-worth (2006) described Amelichloa as having primarilybasal leaves rigid and with a sharp tip, caryopses withthree longitudinal ribs and persistent stylar bases, andcleistogamous axillary panicles in basal leaf sheaths.

Anatherostipa. Originally named as a section of Stipa(Kuntze, 1898), and then called ‘‘group Obtusae’’ orsect. Obtusae by Parodi (1946, 1950, respectively), it wasrecently raised to the generic rank (Penailillo, 1996). Thegenus comprises 11 New World species defined byhaving cylindrical florets with glumes membranaceous,longer or equal to the florets, lemma margins somewhatopen, palea flat, subequal to the lemma, blunt callus,and awn straight or curved, sometimes reduced to asharp point.

Austrostipa. This genus was established by Jacobs andEverett (1996), including approximately 60 Australasianspecies, three of them introduced in America. Austro-stipa is recognized by its florets, dark at maturity, toughin texture, often with brownish hairs, the lemma marginsoverlapping, and the callus long and pungent.

Hesperostipa. Formerly described as a section of Stipa(Elias, 1942), it was subsequently raised to the genericrank, including five North American species (Bark-worth, 1993). Based on morphological and anatomicalcharacters, Barkworth and Everett (1987) and Bark-worth (1990) suggested that Hesperostipa was related toPiptochaetium and Nassella, while Cialdella and Gius-sani (2002) resolved the genus as closely related toPiptochaetium.

Jarava and Pappostipa. Ruiz and Pavon (1794)included in their new genus Jarava species with theupper portion of the lemma bearing long hairs, thesehairs forming an apical pappus. Later, the genus wastreated as a section (Trinius and Ruprecht, 1842), or as asubgenus of Stipa (Spegazzini, 1901; Caro and Sanchez,1973). Recently, several authors (Jacobs and Everett,1997; Matthei et al., 1998; Rojas 1998; Penailillo, 2002,2003) reinstated Jarava at the generic level, expandingits morphological concept. As a result, Soreng et al.(2003) recognized 59 South American species in Jarava.

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Romaschenko et al. (2008) raised a group of 23 speciesof Stipa subg. Pappostipa to the generic rank on thebasis of having macrohairs on the proximal portion ofthe awn (column), while restricting other species toJarava s.s.

Nassella. The genus Nassella was traditionally appliedto species with short florets (Parodi, 1947), and thenexpanded to include species of Stipa with long florets aswell, all with an apical crown. As currently circum-scribed (Barkworth, 1990; Jacobs et al., 2000; Bark-worth and Torres, 2001; Soreng et al., 2003) it includesnearly 115 species distributed from Canada to Uruguayand Argentina. Based on a study of 22 species, Cialdellaet al. (2007) found Nassella to be monophyletic, withseveral synapomorphies: lemma margins strongly over-lapping, palea nerveless, apex of the lemma modifiedinto a crown, and the lemma body indurate.

Ortachne. This genus includes three species, distrib-uted from Costa Rica to Chile and Argentina. It hasbeen considered similar to Stipa s.l., differing in havingspikelets with glumes shorter than the floret, a mem-branaceous lemma, awns without a conspicuous artic-ulation, a blunt and hairy callus, and palea equal to thelemma in length (Parodi, 1953). The genus was alsoaccepted by Roig (1978), Clayton and Renvoize (1986),Nicora and Rugolo de Agrasar (1987), and Penailillo(2005).

Oryzopsis. This genus has been broadly interpreted asincluding Eurasian and North American species, or asincluding a single species, the North American O.asperifolia Michx. (Jacobs et al., 2000; Soreng et al.,2003). Barkworth (2007) described this monotypicOryzopsis as having spikelets with membranaceousglumes, shorter than or equal to the floret, hairy lemmawith overlapping slightly involute margins, and decid-uous awn.

Piptatherum. This genus has been considered as asection of Urachne (Trinius and Ruprecht, 1842) (nom.superfl. = Piptatherum), under Oryzopsis s.l. (Benthamand Hooker, 1883), restricted to Eurasian species(Johnson, 1945; Freitag, 1975), or expanded to includeNew World species (Barkworth, 1993; Soreng et al.,2003)2 . Piptatherum is fairly easy to recognize, mainly bya combination of morphological characters includingdorsally compressed florets, with indurate lemma andpalea, and lemma margins not overlapping, leaving thepalea partially exposed.

Piptochaetium. First circumscribed to include specieswith short, obovoid florets, blunt callus, and deciduousawn (Hitchcock, 1935, 1951), this genus was thenexpanded to comprise New World species with longcallus, terete florets, and persistent awn (Parodi, 1944).Piptochaetium was resolved as monophyletic by molec-ular data and morphological characters (Cialdella andGiussani, 2002; Cialdella et al., 2007; Barber et al.,2009), and comprises 36 species including involute

lemma margins, bi-keeled palea, palea longer than thelemma, and lemma apex with a conspicuous crown.

Ptilagrostis. This genus comprises 11 species, presentin alpine or subalpine habitats, most of them occurringin Asia, with two species in western USA. The genusincludes perennial, densely tufted plants with filiformleaves, florets with a short callus, lemma papery withmargins generally not overlapping and apex usually2-toothed (Peterson et al., 2005; Wu and Phillips, 2006).

Materials and methods

Taxon sampling

Thirty-seven species representing 14 genera of thetribe Stipeae were included in the molecular andmorphological data matrix (see Appendices 1 and 2):five species of Amelichloa (all species of the genus), sevenspecies of Achnatherum (seven American spp. of about66 worldwide, of which three are introduced in Americaand 33 are indigenous ⁄endemic to America), threespecies of Aciachne (3 ⁄3), two species of Anatherostipa(2 ⁄11), two species of Austrostipa (2 ⁄60), two species ofHesperostipa (2 ⁄5), three species of Jarava (3 ⁄32), fourspecies of Nassella (4 ⁄115), one species of Ortachne(1 ⁄3), Oryzopsis asperifolia (1 ⁄1), one species of Pappo-stipa (1 ⁄23), one species of Piptatherum (one Old Worldspecies of 30 worldwide and six native species to NorthAmerica), four species of Piptochaethium (4 ⁄36), andone species of Ptilagrostis (1 ⁄11). For Pappostipavaginata and Ortachne erectifolia (Swallen) Clayton,two specimens per species were used. Sampled speciesrepresent most of the morphological and geographicalvariability of the New World genera of Stipeae. Poaholciformis J. Presl. was used as outgroup.

Morphological characters

A total of 14 morphological characters were includedin the matrix; 13 were selected from Cialdella et al.(2007), and one more character, lemma apex, was added(Appendices 3 and 4). These characters were chosen torepresent diagnostic features for the genera and toelucidate phylogenetic groupings.

DNA isolation, amplification, and sequencing

Total DNA was extracted from fresh or silica-gel-dried leaves, using one of the modified CTABprotocols (Murray and Thompson, 1980; Saghai-Maroof et al., 1984; Doyle and Doyle, 1987). To extractDNA from a few herbarium specimens, the DNeasyPlant Mini Kit (Qiagen, Hilden, Germany) was used.Following extraction, DNA was amplified using thepolymerase chain reaction (PCR) for four chloroplast

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regions: (i) the rpl16 intron (rpl16) of the gene encodingribosomal protein L16, (ii) the trnL intron and trnL-trnFintergenic spacer (trnL-F), (iii) the gene of the alphasubunit of the RNA-polymerase (rpoA), and (iv) thetrnT-trnL intergenic spacer (trnT-L). Amplification ofthe rpl16 intron was accomplished in one fragment usingprimers F71 of Jordan et al. (1996) and R1661 ofKelchner and Clark (1997), or in two fragments usingthe additional primers F584 and R584 (Cialdella et al.,2007). For some sequencing reactions, two additionalprimers were also employed: R270 (Zhang, 2000), andF80 (Cialdella et al., 2007).The trnL-F region wasamplified in one or two fragments using primers C, D,E, and F as of Taberlet et al. (1991). When these primersfailed, the primers Cii, Fdw (Cialdella et al., 2007), E760(5¢-GGAGTGCGAMGAGAACTCAATG-3¢) and D760(5¢-CATTGAGTTCTCKTCGCACTCC-3¢) were used.For the amplification of the rpoA gene, RPOA1,RPOA2, RPOA5, and RPOA6 primers were used(Petersen and Seberg, 1997). The intergenic spacertrnT-L was amplified in one fragment using primersa ⁄b of Taberlet et al. (1991) or in two fragments usingthe additional primers F340 (5¢-TATGGCTCGGACG-AATAATCT-3¢) and R600 (5¢-GATTCTATGTTCT-CCTTAG-3¢). The 25 lL PCR reaction contained20 ng mL)1 DNA template, and a final concentrationof 1 · PCR buffer minus Mg, 5 mm MgCl2, 0.025 mm

dNTP each, 0.2 mm each primer, and 1.25–3 units TaqPolymerase recombinant from Invitrogen Life Technol-ogies. PCR amplifications were carried out under thefollowing conditions for most species: (i) trnT-L andtrnL-F: 1 cycle of 94 �C for 5 min, 34 cycles of 94 �C for30 s, 48 �C for 1 min, 72 �C for 1 min 30 s, and a finalextension cycle of 72 �C for 7 min; (ii) rpl16 and rpoA: 1cycle of 94 �C for 4 min, 34 cycles of 94 �C for 1 min,52 �C for 1 min, 72 �C for 2 min 30 s, and a finalextension cycle of 72 �C for 7 min. In addition, PCRadditives (bovine serum albumin and dimethyl sulfox-ide) were used to increase the yield, specificity, andconsistency of PCR reactions. Macrogen, Inc. (Seoul,Korea) performed cleaning of PCR products using theMontage PCR purification kit from Millipore, followingthe manufacturer�s protocol. Sequencing reactions werealso performed by Macrogen Inc. using an MJ ResearchPTC-225 Peltier Thermal Cycler and the ABI PRISMBigDyeTM Terminator Cycle Sequencing Kits withAmpliTaq DNA polymerase (Applied Biosystems, Fos-ter City, CA, USA), following the protocols supplied bythe manufacturer. Single-pass sequencing was per-formed on each template using selected primers sentby us (see above). The fluorescent-labelled fragmentswere purified from the unincorporated terminators withan ethanol precipitation protocol. Samples were resus-pended in distilled water and subjected to electrophore-sis in an ABI PRISM 3730XL sequencer (96 capillarytype, Applied Biosystems). To edit and assemble the

sequences, we used the program Chromas Pro v.1.41(Technelysium Pty, Ltd, Tewantin, Australia) and Bio-edit v.7.0.9.0 (Hall, 1999). A total of 38 taxa wereincluded in the trnL-F (with 24 new sequences), trnT-L,and rpoA data sets (with 40 new sequences), whereas 37taxa were used in the rpl16 data set because Anathero-

stipa orurensis could not be sequenced, including a totalof 23 new sequences. The species that could not becompletely sequenced are indicated in Appendix 1. Allsequences were submitted to GenBank (http://www.ncbi.nlm.nih.gov 3); voucher information and acces-sion numbers are provided in Appendix 1.

Data analyses

Parsimony analyses were conducted using the molec-ular data sets independently and in combination. Inaddition, the molecular data were analysed in combina-tion with the morphological data matrix.

A dynamic homology approach

DNA sequence data were analysed using direct opti-mization as implemented in the program POY v.4.0.2870(Varon et al., 2008). POY constructs phylogenetichypotheses directly without the intervening step ofmultiple sequence alignment (Wheeler, 1996, 2001).During the direct optimization procedure, POY incor-porates insertion and deletion events in addition to basesubstitutions. Consequently, base changes and indels areminimized during tree search, and POY selects the tree ⁄sthat requires the fewest changes to fit the input sequences.When analysing with POY (or any algorithm implement-ing sequence alignment), the cost of opening a gap andthe cost of a base changemust be specified a priori. Directoptimization has often been used in combination withsensitivity analysis (sensu Wheeler, 1995; Wheeler andHayashi, 1998; Giribet, 2003), where several cost regimesare explored and subsequently evaluated by congruencemeasures, as for example the incongruence length differ-ence 4(ILD) index (Mickevich and Farris, 1981; Farriset al., 1995; Aagesen et al., 2005) to select the cost set thatyields the highest congruence between the data parti-tions. For this purpose, the molecular data set wasanalysed using five different cost regimes: 2:1:1, 2:2:1,2:3:1, 2:5:1, 2:7:1 (substitution cost: opening gap cost:affine gap cost). Because congruence is improved whenindels are treated as single events using affine gap cost(Aagesen et al., 2005; Pons and Vogler, 2006), all the costregimes studied included affine gap cost where the gapopening value was higher than that of the gap extension.

Congruence among the partitions was measured usingthe ILD index (Mickevich and Farris, 1981; Aagesenet al., 2005) as proposed by Wheeler (1995).

In direct optimization, the computational timedecreases when the sequences are divided in small

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fragments (Giribet, 2001). Considering this, sequenceswere split in highly conserved regions (primer conservedregions): rpl16 (five fragments), trnL-F (four fragments),rpoA (four fragments), and trnT-L (seven fragments).

For the phylogenetic searches, the strategy imple-mented in the ‘‘search’’ command of POY v.4.0.2870was used. This command includes Wagner trees, treebisection–reconnection (TBR) swapping, ratchet, and afinal round of tree fusing. The analyses were run with amaximum execution time of 24–120 h depending on thedata, and the optimal length was hit independently atleast three times. When the morphological data werecombined with the molecular data, all morphologicalcharacters were treated as unordered and with the samecost as the nucleotide substitutions (cost = 2).

Bremer support values (BS), (Bremer, 1994) werecalculated in POY 4 using the command ‘‘calculate_sup-ports:bremer’’, performing independent constrainedsearches for each node, using 20 initial Wagner treesper node, and swapping by TBR. In the context ofdynamic homology, sequence fragments rather thanindividual nucleotides constitute the characters. There-fore during resampling methods such as jackknife orbootstrap, entire sequence fragments—not individualnucleotides—are resampled. In the present study we useonly 20 fragments; hence, for calculating the jackknifesupport values, the dynamic alignments were trans-formed into static alignments conserving their cost, andthe static matrices were exported using the command‘‘phastwinclad’’ (Smith and Wheeler, 2004; Arango andWheeler, 2007). When character transformations havedifferent prior weights ⁄cost (as in this case), supportvalues obtained by bootstrapping (Felsenstein, 1985) orjackknifing (Farris et al., 1996) can be distorted, pro-ducing either under- or overestimation of the frequen-cies (Goloboff et al., 2003). In order to avoid distortionof the frequencies, we used symmetric resampling (SR)support (Goloboff et al., 2003). The matrix exportedfrom POY was loaded with TNT v.1.1 and thesymmetric resampling support was calculated using50 000 replicates, change probability of 0.33, four initialWagner trees, and holding three trees per replicate.

Implied alignments (Wheeler, 2003) were obtainedfrom each tree as yielded by POY to analyse and codephylogenetically informative indels common to all trees.Indels and morphological characters were optimizedusing TNT v.1.1. All matrices and implied alignmentsfrom POY are available upon request from the authors.

A multiple-sequence alignment approach

To compare the results from the dynamic homologyanalysis with a conventional two-step analysis, wecarried out a multiple sequence alignment followed byparsimony analysis of the static homology alignments.Multiple alignments of the sequences (rpl16, trnL-F,

trnT-L, and rpoA) were generated with Muscle v.3.6(Edgar, 2004) with default settings and improved byvisual inspection using the program Bioedit v.7.0.9.0(Hall, 1999). Two matrices were produced: a matrix withgaps generated by the alignment process but consideredas missing data, and a matrix with those indels coded aspresence or absence as implemented in the programFastGap v.1.1 (Borchsenius, 2009), following the ‘‘sim-ple indel coding’’ method of Simmons and Ochoterena(2000). Both aligned matrices were analysed usingmaximum parsimony in TNT v.1.1 (Goloboff et al.,2008), treating all characters as equally weighted andexcluding non-informative data. Heuristic searchesinvolved 1000 replicates using random addition se-quences and TBR branch swapping, and holding 10trees per replicate; trees found were then submitted to anew round of TBR branch swapping. Nodal supportwas assessed with 1000 jackknife replicates (Farris et al.,1996; J.K.) of 10 random addition sequences, holdingfour trees per replicate and using the default removalprobability (P = 0.36). The average support (AS) wascalculated as the sum of individual support per branchdivided by the number of total nodes (number oftaxa—2). Data matrices were deposited in TreeBASE(http://www.treebase.org/treebase), submission name‘‘Ana Cialdella’’ and reviewer�s PIN code 3115.

Results

Molecular data

Sequence variation. Within the ingroup taxa, thelength of the unaligned sequences of the rpl16 variedfrom 1148 bp in Oryzopsis asperifolia to 1218 bp inHesperostipa comata; the trnL-F sequences varied from961 bp in Achnatherum multinode to 988 bp in Ameli-chloa brevipes; while in rpoA, sequences varied from1341 bp in Jarava media (Speg.) Penailillo to 1384 bp inAchnatherum bromoides. Finally, the shortest sequencefor the trnT-L was found in Achnatherum psilantherumKeng ex Tzvelev, with 833 bp; the longest was found inHesperostipa comata (Trin. & Rupr.) Barkworth, with966 bp.

Dynamic homology analyses

Individual Molecular Analyses. In 5the sensitivity anal-yses, the congruence measure (ILD index) identified inall partitions the parameter set 2-1-1 (substitutioncost = 2, opening gap cost = 1, extension gapcost = 1) as the ‘‘most congruent parameter set’’(Table 1). Under this cost regimen, the monophyly ofAmelichloa was never obtained by any of the molecularregions. Amelichloa brevipes was included in a clade withPappostipa vaginata in all analyses, while other species

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of Amelichloa were resolved as monophyletic only withthe trnT-L partition. The monophyly of the genusPiptochaetium was recovered with all the partitionsexcept trnT-L; the genus Hesperostipa was resolved asmonophyletic only in the analyses with trnL-F and rpoA,while the two species of the genus Austrostipa wereincluded in a clade only with the rpl16 and trnT-L

sequences. All other genera represented here by morethan one species were not monophyletic.

Combined Molecular Analyses. All trees obtainedusing the combined data set under the five differentcost sets yielded a clade comprising Amelichloa brachy-chaeta–A. caudata–A. clandestina and A. ambigua(Fig. 1). Similar results were obtained with the clades

Table 1

Tree lengths for the different partitions analysed (rpl16, trnL-F, rpoA, trnT-L, TOT: four regions combined) and congruence value (incongruence

length difference, ILD 15) at different parameter sets (left column, substitution cost : gap extension cost : gap opening cost)

rpl16 trnL-F rpoA trnT-L TOT ILD

2:1:1 784 547 483 616 2488 0.023

2:1:2 852 618 550 689 2781 0.026

2:1:3 909 685 607 754 3043 0.029

2:1:5 1019 813 716 878 3558 0.037

2:1:7 1125 937 815 996 4048 0.043

Fig. 1. Strict consensus of nine most parsimonious trees (L = 2488) using rpl16, trnL-F, rpoA, and trnT-L combined data set, under the parameter

set 2-1-1 (substitution cost = 2, opening gap cost = 1, extension gap cost = 1). Support values are indicated above branches as Bremer

support ⁄ symmetric resampling support. Bars represent taxonomic groups as discussed in the text.

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of Amelichloa brevipes–Pappostipa vaginata, Piptochae-tium, Austrostipa, Hesperostipa, and clades A and B.

When the four chloroplast regions were combinedunder the optimal cost regimen, the nine shortest trees of2488 steps were obtained (Table 2). The consensus treeof these topologies (Fig. 1) showed Achnatherum chingii

(Hitchc.) Keng and Oryzopsis asperifolia as the mostbasal taxa, while the rest of the species were grouped intwo well supported clades: clade A (12 BS, 97% SR) andclade B (5 BS, 96% SR).

Clade A comprises species of Amelichloa (exceptA. brevipes),Nassella, Jarava,Achnatherum,Austrostipa,Piptatherum, and Ptilagrostis. The first three genera,together with Achnatherum eminens, A. multinode, andA. diegoense, were united in a strongly supportedsubclade (10 BS, 99% SR) of New World species(‘‘New World subclade’’), while Old World taxa[Austrostipa, Piptatherum, Achnatherum inebrians(Hance) Keng, and A. bromoides] were resolved in aweakly supported sister subclade (6 BS, 52% SR).Achnatherum psilantherum and Ptilagrostis porteri(Rydb.) W.A. Weber were located basally in clade A.

Within clade A, the species of Amelichloa (except forA. brevipes) were resolved as monophyletic in a moder-ately supported subclade (10 BS, 67% SR). ThisAmelichloa set was resolved in a highly supported sistergroup relationship with the subclade of Nasella pampa-grandensis and N. melanosperma (17 BS, 99% SR).

Regarding the Old World taxa included in clade A,Austrostipa was recovered in a strongly supportedsubclade (21 BS, 100% SR) as sister to Piptatherummiliaceum (9 BS, 68% SR). This latter clade is weaklysupported as sister to the clade of Achnatherum inebriansand A. bromoides.

Clade B (5 BS, 96% SR) comprises species ofAciachne,Anatherostipa, Piptochaethium, Hesperostipa, andOrtachne erectifolia, as well as Pappostipa vaginata andAmelichloa brevipes. The genus Piptochaethium wasrecovered in a strongly supported clade (31 BS, 100%SR) that is sister to the clade includingOrtachne,Aciachneacicularis, A. pulvinata Benth., and Anatherostipa brevis

(Torres) Penailillo (11 BS, 99% SR). The genus Hesp-erostipa was resolved as monophyletic (12 BS, 100%SR) and related to Anatherostipa orurensis F. Rojas andAciachne flagellifera Laegaard (3 BS, 86% SR). Theplacement of Pappostipa vaginata samples confirmsthe phylogenetic position detected previously by Ciald-ella et al. (2007), and supports its recent segregationfrom Jarava (Romaschenko et al., 2008). Amelichloabrevipes was highly supported as the sister species ofPappostipa.

Several indels, as recovered from the implied sequencealignments, support some groups: (i), the rpl16 data setincluded two indels shared by the species of Austrostipa(1 bp each); (ii), in the trnL-F data set the species ofPiptochaetium had one indel of 10 bp; two indels of 1 bpwere shared by the species of Austrostipa; an indel of13 bp was present in the species of Hesperostipa; theclade of Amelichloa (except A. brevipes) together withNassella pampagrandensis and N. melanosperma sharedan indel of 5 bp 6, and; an indel of 1 bp was present inJarava media and J. plumosula; (iii) in the rpoAsequences the species of Piptochaethium shared twoindels of 1 and 2 bp; an indel of 5 bp supported theclade A; two indels of 1 bp support the sister clade toPtilagrostis porteri in clade A; two indels of 1 and 6 bpwere shared by Jarava media and J. plumosula (Nees exSteud.) F. Rojas; a gap of 16 bp was shared by Nassellaarcuata (R. E. Fr.) Torres and N. novari Torres; (iv) thetrnT-L alignment included an indel of 10 bp shared bythe species of Piptochaethium, and an indel of 2 bp waspresent in a major subclade, within clade B, comprisingspecies of Aciachne, Anatherostipa, Hesperostipa,Ortachne, and Piptochaetium.

Combined molecular and morphological data

The analyses with the molecular and morphologicaldata sets combined resulted in 88 optimal trees of 2664steps (Table 2). The consensus tree obtained (Fig. 2) isless resolved that the consensus obtained with just themolecular data set; nevertheless, both consensus treeswere very similar. The position of Amelichloa resolvedby molecular characters was recovered again with theaddition of the morphology, as were clade A, clade B,and the monophyletic genera Piptochaetium,Austrostipa,andHesperostipa. A few new relationships were detectedwhen the data were combined. Within clade A, Jaravamedia and J. plumosula joined Nassella arcuata andN. novari (3 BS, 99% SR). Within clade B, the species ofAciachne are united in a clade as a paraphyletic grade toAnatherostipa brevis and Ortachne (3 BS, 96% SR).

Multiple-sequence alignment analyses

The alignment of the total matrix with the fourmarkers combined (rpl16, rpoA, trnL-F and trnT-L)

Table 2

Number of most parsimonious trees and their length for each data set

[rpl16, trnL-F, rpoA, trnT-L, MOL (DNA): the four DNA regions

combined, TOT(DNA + MORPHOLOGY): four DNA regions and

morphology combined], when using the cost set substitution: 2, gap

opening: 1, gap extension: 1

Partition

No. optimal

trees

No.

steps

rpl16 1 784

trnL-F 34 547

rpoA 12 483

trnT-L 4 616

MOL (DNA) 9 2488

TOT (DNA + MORPHOLOGY) 88 2664

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resulted in 4815 characters, of which 201 (4.17%) wereparsimony-informative. Additionally, 169 gaps werecoded, of which 52 (30.8%) were parsimony-informa-tive, representing a final total of 5.07% informativecharacters. A total of 18.5% of missing data wereintroduced in the multiple alignment, of which 8.16%were generated by the addition of indels.

Parsimony analyses, with gaps treated as missingcharacters, generated 180 shortest trees of length = 333(Ci = 0.61; Ri = 0.87). The consensus tree retained 21nodes with jackknife value (JK) ‡ 50% and AS = 49%.When parsimony analyses were conducted coding thegaps as present ⁄absent, a total of 36 most parsimonioustrees (L = 437, Ci = 0.81, Ri = 0.85) were recovered;24 nodes of the consensus tree were supported with JK> 50% and the AS was higher (55%).

Results obtained from the analyses with gaps asmissing data or coded present ⁄absent were largelyidentical, and 82% of the clades were common to both

consensus trees. Both resolution and support valuesimproved when gaps were included in the analyses,revealing a notable phylogenetic signal among indelcharacters. This phylogenetic signal uncovers discrep-ancies in the position of Ptilagrostis porteri, in themonophyly of Amelichloa p.p., and in the position ofAciachne flagelifera. However, when including sequencelength information, the final consensus tree differed onlyin minor aspects from the results obtained with dynamichomology. Therefore the following discussion is basedon the results obtained with the dynamic homologyapproach.

Discussion

Both DNA data alone, and molecular data combinedwith morphology, supported two major clades in theStipeae (clades A and B, Figs 1 and 2), withAchnatherum

Fig. 2. Strict consensus of 88 most parsimonious trees (L = 2664) using a morphological and molecular (rpl16, trnL-F, rpoA, and trnT-L) combined

data set, under the parameter set 2-1-1 (substitution cost = 2, opening gap cost = 1, extension gap cost = 1 morphological change = 2). Support

values are indicated above branches as Bremer support ⁄ symmetric resampling support. Bars represent taxonomic groups as discussed in the text.

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chingii from China and Oryzopsis asperifolia both placedoutside the union of these major clades. This result doesnot agree with the hypothesis postulated by Roma-schenko et al. (2008), who resolved Piptochaetium,Anatherostipa, and Ptilagrostis as ‘‘basal lineages’’ ofthe tribe. In our analyses, Piptochaetium and Anathero-

stipa are embedded within clade B, while Ptilagrostisporteri appears in a sister position to the remainder ofclade A (Figs 1 and 2), similar to the results presentedby Barkworth et al. (2008), who resolved this species ina similar isolated relationship in their chloroplastphylogeny. Anatherostipa resolved as polyphyletic (orpotentially paraphyletic in multiple alignment analyses),a condition that contradicts the results published usinglarger sets of overlapping or different species publishedby Barkworth et al. (2008) and Romaschenko et al.(2008). Our results agree, at least in part, with theoverall phylogenetic structure detected by Jacobs et al.(2007), who postulated that members of the Piptathe-rum ⁄Oryzopsis complex, including species of Achnathe-rum, are among the earliest divergent lineages in thetribe.

Our two major clades agree with those resolved byCialdella et al. (2007) and Barber et al. (2009), mainlycorrelated with the basic chromosome number.Although data from chromosome counts are still poorlydocumented, clade A includes taxa with aneuploidseries of chromosome counts and a few representativeswith basic chromosome number x = 11 (Bowden andSenn, 1962; Probatova and Sokolovskaya, 1980; Ree-der, 1984; Strid and Andersson, 1985; Watson andDallwitz, 1992 onwards), while clade B comprisesgenera with basic chromosome number x = 11 (Bow-den, 1960; Love and Love, 1981;7 Watson and Dallwitz,1992 onwards).

Clade A

Clade A comprises species of Achnatherum, Austro-stipa, Jarava, Nassella, Piptatherum, and Amelichloa(except for A. brevipes), with Ptilagrostis porteri as asister taxon. Molecular data such as substitutions andan indel of 5 bp (from implied alignments of the rpoAtrees) supported this clade; no morphological characterssustained it. A strongly supported New World subclade,including Jarava, Nassella, Amelichloa, and New Worldspecies of Achnatherum, is defined within clade A. Thisset of genera corresponds to the ‘‘Major AmericanClade’’ detected by Romaschenko et al. (2008). Ptila-grostis porteri and the Old World species of Austrostipa,Piptatherum, and Achnatherum appeared, in a poorlysupported clade, on branches preceding this MajorAmerican Clade. When adding morphology (Fig. 2), theOld World species are in an unresolved polytomy, withPtilagrostis porteri remaining as sister to the rest of theclade. Achnatherum is clearly polyphyletic, in concur-

rence with previous studies (Jacobs et al., 2007; Bark-worth et al., 2008).

The genus Jarava was represented in our treatment byfour species: Jarava vaginata [now treated as Pappostipavaginata (see discussion) in clade B], and J. media,J. plumosula, and J. leptostachya (Griseb.) F. Rojas inclade A. Our results indicate that Jarava, as broadlycircumscribed by Penailillo (2002, 2003), is indeed apolyphyletic genus in concurrence with previous studies(Cialdella et al., 2007; Jacobs et al., 2007; Romaschenkoet al., 2008; Barber et al., 2009). Jarava media andJ. plumosula are sisters in rpl16, trnT-L, combinedDNA, and total evidence analyses. Both species wereincluded by Spegazzini (1901) in subg. Ptilostipa ofStipa, based on the presence of macrohairs on the distalportion of the awn or all over it. Jarava leptostachya,included by Spegazzini (1901) in subg. Jarava based onthe presence of a glabrous awn, and macrohairs at theapex of the lemma forming a pappus, resolved in a sisterposition to the Nassella p.p.–Amelichloa subclade inboth our molecular and combined analyses. This resultdiffers from that summarized by Romaschenko et al.(2008), who placed J. leptostachya in a well supportedJarava s.s. core clade, together with J. ichu and otherspecies (none of which we sampled here), but, as in thatstudy, J. plumosula and J. media were not united with,or not significantly supported as monophyletic with, thecore Jarava.

This study, together with previous published accounts(Barkworth et al., 2008; Romaschenko et al., 2008),reinforces the common belief that the broad genericconcept of Jarava proposed by Penailillo (2002, 2003) isclearly polyphyletic; a detailed morphological andmolecular study, including more species, is needed toredefine generic boundaries in Jarava and related taxa.However, it was apparently correct of Penailillo toremove all remaining indigenous South American spe-cies from the genus Stipa.

Four species of Amelichloa (A. brachychaeta, A. amb-

igua, A. clandestina, and A. caudata, but not A. brevipes)are united in a weakly supported clade, with no internalstructure resolved. When analysing rpl16, trnT-L, andthe combined sequence data set, and when addingmorphology, these four Amelichloa species appearedtogether with Nassella melanosperma (Stipa subg. Step-hanostipa Speg.) and N. pampagrandensis (Stipa subg.Nassella Speg.). This group of Amelichloa and Nassellaspecies was supported by an indel of 5 bp (from theimplied alignment of the trnL-F trees), and agrees withRomaschenko et al.�s (2008) findings that resolved aclade of A. caudata and A. clandestina nested withinNassella, as sister to a subclade of species of Nassellasubg. Stephanostipa.

The remote position of the South American Ameli-chloa brevipes, and its placement as sister to Pappostipavaginata within clade B, indicate that Amelichloa is

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polyphyletic and in need of reinterpretation. Amelichloa,with A. brevipes removed, could be restricted to amonophyletic group that includes A. brachychaeta,A. ambigua, A. clandestina, and A. caudata, or it couldbe included within Nassella, as suggested by Barkworthet al. (2008).

The genus Nassella, here represented by fourspecies, was polyphyletic in all analyses, contradictingprevious results (Jacobs et al., 2000, 2007; Cialdellaet al., 2007; Romaschenko et al., 2008; Barber et al.,2009). While Nassella pampagrandensis and N. mela-nosperma are related to species of Amelichloa, aspreviously stated, N. arcuata and N. novari are in adistant position and strongly related one to the otherspecies. Since Nassella is one of the largest genera ofthe Stipeae, with variable morphological characters, amore inclusive study is needed to confirm the poly-phyly of the genus and its relationship to Jarava(excluding Pappostipa).

The genus Austrostipa is united with Piptatherummiliaceum among the Old World genera of clade A.Monophyly of Austrostipa, previously resolved byJacobs et al. (2000, 2007) and Romaschenko et al.(2008), was also found in this analysis; it wasrecovered in rpl16 and trnT-L (by nucleotide substi-tutions and two indels in both markers) in our DNAand combined DNA and morphological analyses.Lemma margins strongly overlapping (char. 0), anda persistent awn (char. 4) support the monophyly ofthe genus.

Piptatherum miliaceum resolved as the sister taxon ofAustrostipa when analysing the trnT data set and in thecombined analyses. The Austrostipa–Piptatherum clade,however, is weakly supported, and marked only byindurated paleas and lemmas (chars 10 and 11).

The genus Achnatherum is polyphyletic, our resultsconcurring with previous studies using different markers(Jacobs et al., 2000, 2007; Barkworth et al., 2008).Romaschenko et al. (2008) detected a monophyleticgroup of New World species, but did not analyse OldWorld species. Achnatherum chingii is, as previouslymentioned, resolved outside the union of clades A and Bin all our analyses. The other Old World species sampledhere, A. inebrians, A. bromoides, and A. psilantherum,are resolved within clade A. However, relationshipsamong these species are poorly resolved, and they areoutside the Major American Clade as outlined byRomaschenko et al. (2008) or as detected here, whichincluded the New World Achnatherum8 taxa. Finally, theremaining studied species of the genus are within theNew World subclade of clade A. Achnatherum diegoense

is sister to the rest of this subclade, while A. eminens andA. multinode are, in the molecular analysis (but not thecombined morphological and molecular analysis),strongly supported as a sister clade to some species ofJarava (Stipa subg. Ptilostipa).

Clade B

Clade B is exclusively of the New World and containsspecies of Aciachne, Anatherostipa, Hesperostipa, Pipto-chaetium, and Ortachne erectifolia, with Amelichloabrevipes and Pappostipa vaginata (= Jarava vaginata)included in a clade as sister groups. This clade is definedmorphologically by its indurate lemmas (char. 11),although this character reverses in Anatherostipa andOrtachne.

Monophyly of Piptochaetium is clearly supported inboth molecular and total evidence analyses, in agree-ment with Cialdella et al. (2007), Jacobs et al. (2007),and Barber et al. (2009). We found nucleotide substitu-tions and indels in trnL-F, trnT-L, and rpoA thatsupported this clade.

Hesperostipa neomexicana and H. comata are united,based on DNA and combined DNA–morphology, asreported by Jacobs et al. (2000), who used both com-bined morphological ⁄anatomical and molecular (ITS)data, and Romaschenko et al. (2008) using plastid andITS characters separately. Monophyly ofHesperostipa issupported by a number of molecular changes (molecularsubstitutions and one indel), and defined morphologi-cally by its acute callus (char. 3) and a conspicuouscrown (char. 7).

Hesperostipa is resolved in a polytomy with Anath-erostipa, Aciachne, Ortachne 9, and Piptochaetium in thetotal evidence analysis, while the molecular consensustree shows the genus in a well supported clade withone species of Anatherostipa and one species ofAciachne.

Anatherostipa orurensis is resolved as sister toAciachne flagellifera and the genus Hesperostipa in themolecular consensus tree (Fig. 1), but this relationshipis not supported when morphology is added (Fig. 2).On the other hand, Anatherostipa brevis is, in both totalevidence and molecular consensus trees, embedded in astrongly supported clade with Ortachne and two speciesof Aciachne (except A. flagellifera). The latter resultagrees with previous hypotheses by Parodi (1946), whoplaced Anatherostipa species under Stipa group Obtu-sae, with Ortachne, and by Barkworth and Everett(1987), who considered Lorenzochloa (= Ortachne),Aciachne, and species of Anatherostipa (under Stipa) inthe informal Obtusae group. We can conclude thatAnatherostipa is, as currently circumscribed, a poly-phyletic genus in need of a complete phylogenetic studyof all its species, along with a complete sampling inOrtachne 10.

When using molecular data, Aciachne was resolvedas polyphyletic as its species are present in twodifferent clades: Aciachne acicularis and A. pulvinata,together with Anatherostipa brevis and Ortachne erecti-folia, are resolved in a well supported clade; whereasAciachne flagellifera, Anatherostipa orurensis, and both

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species of Hesperostipa are united in a separate non-sister clade. The relationship between Aciachne andAnatherostipa in both clades suggests a possiblehybridization event with Ortachne and Hesperostipaas probable parental lineages. However, when addingmorphology to the molecular data, Aciachne is para-phyletic, as A. flagellifera is in a basal position of theclade, which includes Ortachne, Anatherostipa brevis,and the remaining two species of Aciachne. Morpho-logically, this clade combines four characters: indu-rated glumes (char. 8); glumes shorter than the floret(char. 12); and a non-geniculate awn (char. 6) withoutdisarticulation with the lemma (char. 9). Aciachne,Anatherostipa, Hesperostipa, and Ortachne have speciesgrowing at high elevations of the Andes or in season-ally dry grasslands; these genera differ by severalmorphological features, the habit and texture of bractsof the spikelet.

Pappostipa vaginata is related, in all analyses, toAmelichloa brevipes in a strongly supported cladedefined by several molecular characters (substitutionsand one indel). The position of P. vaginata in clade Bagrees with previous results published by Cialdella et al.(2007) and Romaschenko et al. (2008).

General considerations

New World species of Stipeae are clearly dividedinto two different clades. Some morphological, cyto-logical, and geographical distributions are correlatedwith one or the other lineage. Among the generaconsidered, only Piptochaetium, Austrostipa, and Hesp-erostipa were resolved as monophyletic; Achnatherum,Amelichloa s.l., Anatherostipa, Jarava, and Nassellawere polyphyletic; while Aciachne, depending on theanalysis, was either poly- or paraphyletic. In particular,Amelichloa can be restricted to a monophyletic group ifincluding A. brachychaeta, A. ambigua, A. clandestina,and A. caudata, or it should be considered withinNassella. The phylogenetic position of species ofAciachne suggests inbreeding and outbreeding eventswith species of Anatherostipa, Ortachne, and Hespero-stipa. Finally, additional chromosome counts areneeded, particularly in those genera where no cytolog-ical data are available.

Acknowledgements

This research was supported by CONICET (grant5453) and ANPCyT (grants 13374, 32664, and 01286).Field collections were carried out by funds awarded bythe National Geographic Society (grants 7792-05 and8365-07) and by Myndel Botanical Foundation. We alsothank the staff of the Darwinion, who kindly helped usthroughout.

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Appendix 1

Species of Stipeae sequenced for molecular phyloge-netic analysis, taxa, voucher specimens from whichDNA was extracted for sequencing, and GenBankaccession numbers: rpl16, trnL-F, rpoA, and trnT-L.

*, Sequence partially incomplete; –, sequence notobtained.

Achnatherum bromoides (L.) P. Beauv., Ivaniva s.n.(MO), GU191945*, GU192008, GU191984,GU192048*. Achnatherum chingii (Hitchc.) Keng, Bouf-ford et al., 28773 (MO), GU191941*, GU192004,

GU191980, GU192044*. Achnatherum diegoense

(Swallen) Barkworth, Barkworth 2003 ⁄1 (COAH),GU191954*, GU192017, GU191993, GU192057. Achn-atherum eminens (Cav.) Barkworth, Zuloaga 9639 (SI),GU191958, GU192022, GU191998, GU192062. Achn-atherum inebrians (Hance) Keng, Ho et al., 230 (MO),GU191940*, GU192003*, GU191979, GU192043*.Achnatherum multinode (Scribn. ex Beal) Valdes-Reyna& Barkworth, Zuloaga 9646 (SI), GU191957,GU192021, GU191997, GU192061. Achnatherum psi-

lantherum Keng ex Tzvelev, Ho et al., 2898 (MO),GU191942, GU192005, GU191981, GU192045.

Aciachne acicularis Laegaard, Sulekic 2782 (SI),DQ887376, DQ887426, GU191974, GU192038. Aci-

achne flagellifera Laegaard, Laegaard 54503 (MO),GU191939*, GU192002, GU191978, GU192042. Aci-

achne pulvinata Benth., Laegaard 55350 (MO),GU191938*, GU192001, GU191977, GU192041.

Amelichloa ambigua (Speg.) Arriaga & Barkworth,Morrone 5762 (SI), GU191959*, GU192023*,GU191999*, GU192063. Amelichloa brachychaeta

(Godr.) Arriaga & Barkworth, Morrone 5509 (SI),GU191948, GU192011, GU191987, GU192051. Ameli-

chloa brevipes (E. Desv.) Arriaga & Barkworth, Humano3 (SI), GU191950, GU192013, GU191989, GU192053.Amelichloa caudata (Trin.) Arriaga & Barkworth, Mor-rone 5461a (SI), GU191949, GU192012, GU191988*,GU192052*. Amelichloa clandestina (Hack.) Arriaga &Barkworth, Zuloaga 9637 (SI), GU191955, GU192018,GU191994, GU192058.

Anatherostipa brevis (Torres) Penailillo, Sulekic 2554(SI), DQ887377, DQ887427, GU191975, GU192039.Anatherostipa orurensis F. Rojas, Peterson et al., 12987(MO), –, GU192019, GU191995, GU192059.

Austrostipa elegantisima (Labill.) S.W.L. Jacobs & J.Everett, Peterson 14289 (MO), GU191943, GU192006,GU191982, GU192046. Austrostipa nodosa (S.T. Blake)S.W.L. Jacobs & J. Everett, Hill 5099 (MO), GU191952,GU192015, GU191991, GU192055.

Hesperostipa comata (Trin. & Rupr.) Barkworth, sincolector 7431, GU191937, GU192000, GU191976,GU192040*. Hesperostipa neomexicana (Thurb.) Bark-worth, Zuloaga 9662 (SI), GU191956, GU192020*,GU191996, GU192060.

Jarava leptostachya (Griseb.) F. Rojas, Cialdella 417(SI), DQ887381, DQ887431, GU191970, GU192034.Jarava media (Speg.) Penailillo, Cialdella 242 (SI),DQ887382, DQ887432, GU191971*, GU192035. Jaravaplumosula (Nees ex Steud.) F. Rojas, Cialdella 258 (SI),DQ887384, DQ887434, GU191972, GU192036.

Nassella arcuata (R.E. Fr.) Torres, Cialdella 183 (SI),DQ887388, DQ887440, GU191968, GU192032.Nassella

melanosperma (J. Presl) Barkworth, Morrone 5139 (SI),DQ887396, DQ887447, GU191967, GU192031.Nassella

novari Torres, Cialdella 308 (SI), DQ887399, DQ887450,GU191969, GU192033. Nassella pampagrandensis

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(Speg.) Barkworth, Cialdella 163 (SI), DQ887451,DQ887400, GU191964, GU192028.

Oryzopsis asperifolia Michx., sin colector, GU191944,GU192007, GU191983, GU192047*.

Ortachne erectifolia (Swallen) Clayton, (a) Morrone4724 (SI), GU191947, GU192010, GU191986,GU192050; (b) Wood 5263 (MO), GU191953*,GU192016, GU191992, GU192056.

Pappostipa vaginata (Phil.) Romasch., (a) Cialdella264 (SI), DQ887386, DQ887436, GU191963*,GU192027; (b) Peterson 19222 (US), GU191946,GU192009, GU191985, GU192049.

Piptatherum miliaceum (L.) Coss., Rugolo 2135 (SI),DQ887408, DQ887459, GU191973, GU192037. Pipto-chaetium lasianthum Griseb., Giussani 318 (SI),DQ887413, DQ887465, GU191962, GU192026. Pipto-chaetium medium (Speg.) Torres, Giussani 317 (SI),DQ887415, DQ887467, GU191961, GU192025. Pipto-chaetium montevidensis (Spreng.) Parodi, Cialdella 16(SI), DQ887416, DQ887468, GU191965, GU192029.Piptochaetium napostaense (Speg.) Hack., Cialdella 55(SI), DQ887417, DQ887469, GU191966, GU192030.

Poa holciformis J. Presl, Soreng 7100 (US),DQ887425, DQ887481, GU191960, GU192024.

Ptilagrostis porteri (Rydb.) W.A. Weber, Barkworth99117 (COAH), GU191951*, GU192014, GU191990,GU192054.

Appendix 2

List of specimens considered for morphological anal-yses. Those marked (*) were used for DNA sequencing.

Aciachne acicularis Laegaard. ARGENTINA. Salta:Dpto. Oran, Piedra Azul, 3650 m, 13 Apr 1999, Sulekicet al., 2782*(SI). Aciachne flagellifera Laegaard. ECUA-DOR. Cotopaxi: 4000–4100 m, 00�56¢S 078�25¢W, 14May 1985, S. Lægaard 54154 (AAU, QCA). ECUA-DOR. Imbabura: 4150 m, 00�25¢N 78�20¢W, 06 Jun1985, S. Lægaard 54503* (AAU). Aciachne pulvinata

Benth. ECUADOR. Bolıvar: 4300 m, 01�24¢S078�55¢W, 02 Oct 1985, S. Lægaard 55350 (AAU).BOLIVIA. Cochabamba: 4350 m, 27 Jan 1996, NurRitter & John Wood 2828* (MO). Achnatherum bromo-

ides (L.) P. Beauv. Without data, Ivanina s ⁄n (MO).Achnatherum chingii (Hitchc.) Keng. Without data,Boufford et al.28773* (MO). CHINA. Qinghai: 3550–3650 m, 32�06¢N 097�16¢E, 24 Aug 1996, T.N. Ho, B.Bartholomew, M. Watson & M. Gilbert 2310 (MO).Achnatherum eminens (Cav.) Barkworth. MEXICO.Coahuila: Bosques de la montana, cerca de casa J.Valdes, camino de Saltillo a Los Lirios, 25� 23¢16¢� N100� 42¢34¢� W, 2115 m, 7 Oct 2007, Zuloaga et al.,9639* (SI). Achnatherum diegoense (Swallen) Bark-worth. USA. California: San Diego, Proctos Valleyroad, about 3 mi. From crossroads in Jamul, 5 Apr

2003, Barkworth 2003 ⁄1* (COAH). Achnatherum ineb-

rians (Hance) Keng. Without data, Ho et al.230* (MO).Achnatherum multinode (Scribn. ex Beal) Valdes-Reyna& Barkworth. MEXICO. Coahuila: Bosques de lamontana, cerca de casa J. Valdes, camino de Saltillo aLos Lirios, 25� 23¢16¢� N 100� 42¢34¢� W, 2115 m, 7 Oct2007, Zuloaga et al., 9646* (SI). Achnatherum psilanthe-

rum Keng ex Tzvelev. Without data, Ho et al., 2898*(MO). Amelichloa ambigua (Speg.) Arriaga & Bark-worth. ARGENTINA. Rıo Negro: Gral. Roca, ruta nac.22, km 1065, a 111 km de Gral. Roca camino a ChoeleChoel, 39�05¢19¢� S 66� 21¢ 36¢� W, 1 Dec 2006, Morroneet al., 5762* (SI). Amelichloa brachychaeta (Godr.)Arriaga & Barkworth. ARGENTINA. Buenos Aires:Reserva Otamendi, Nov 2006,Morrone 5508, 5509* (SI).Rio Negro: Gral. Roca, ruta nac 22, km 1065, a 111 kmde Gral. Roca, camino a Choele Choel, 39� 05¢ 19¢�S66�21¢ 36¢� W, 1 Dec 2006, Morrone et al., 5764 (SI);Avellaneda, ruta nac. 22, a 101 km de Choele Choelcamino a Rıo Colorado, 39� 04¢ 23�� S 64� 31� 49 ��W,Morrone et al., 5774 (SI). Amelichloa brevipes (E. Desv.)Arriaga & Barkworth. ARGENTINA. Santa Cruz:Perito Moreno, Humano 3* (SI). Amelichloa caudata

(Trin.) Arriaga & Barkworth. ARGENTINA. BuenosAires: Gral. Pueyrredon, Sierra de los Padres, 1 Ene2007, Giussani & Morrone 356 (SI); Tornquist, ParqueProvincial Sierra de la Ventana, base del cerro BahıaBlanca, 29 Oct 2006, Morrone et al., 5461a* (SI);Magdalena, Atalaya, alrededores de la Ea. R. Favaloro,15 Dic 2007, Morrone & Giussani 5812 (SI). Amelichloa

clandestina (Hack.) Arriaga & Barkworth. MEXICO.Coahuila: Parque Recreativo El Chorro, 25� 23� 11�� N100� 47� 11�� W, 7 Oct 2007, Zuloaga et al., 9637* (SI).Anatherostipa brevis (Torres) Penailillo. ARGENTINA.Salta: Dpto. Santa Victoria, entre Cie¢naga Abra yNazareno,

6 Mar 1999, Sulekic & Cano 2554* (SI). Anatherostipaorurensis F. Rojas. BOLIVIA, Potosi: Sud Lipez,4140 m, 18 March 1993, P.M. Peterson, R.J. Soreng &

S. Laegaard 12987* (MO). Austrostipa nodosa (S.T.Blake) S.W.L. Jacobs & J. Everett, Hill 5099* (MO).Austrostipa elegantissima (Labill.) S.W.L. Jacobs & J.Everett. AUSTRALIA. 16 km NE of Bindoon on Hwy95 (Great Northern), 13 Oct 1998, Peterson et al.,14276*(MO), 1428 (MO). Hesperostipa comata (Trin. &Rupr.) Barkworth. MEXICO. Veracruz: Carretera deCatemaco a Coyame, 3 km de Coyame, 1 Oct 2001,Zuloaga et al., 7431* (SI). Hesperostipa neomexicana

(Thurb.) Barkworth. MEXICO. Coahuila: Rancho LosAngeles, propiedad de la UAAN, 25� 06� 30�� N 100� 59�22�� W, Zuloaga et al., 9662* (SI). Jarava leptostachya

(Griseb.) F. Rojas. ARGENTINA. Jujuy: Dpto. Dr.Manuel Belgrano, entre Leon y Nevado de Chani, Pie dela Cuesta, 2600 m, 6 Mar 1963, Fabris et al., 3965(BAA). Dpto. Cochinoca, Abra Pampa, Cerro Huancar,3500 m, 17 Feb 1963, Cabrera et al., 15272 (BAA).

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Dpto. Humahuaca, ruta 9, de Tres Cruces a Humahu-aca, Ayo. Puente del Diablo, a pocos km de Tres Cruces,22� 55� S65� 33�W, 3690 m, 20 Feb 2002, Cialdella et al.,486 (SI). Dpto. Susques: ruta 70, cruce con ruta 16, entreSalina de Olaroz y Coranzulı, 4300 m, 23� 07� S 66� 31�W, 16 Mar 1994, Mulguraet al., 1301 (SI). Dpto.Tumbaya, camino de Purmamarca al Abra de Lipan,3700 m, 14 Feb 1985, Kiesling et al., 5205 (SI). Dpto.Yavi, Abra de Yavi, 3400 m, 20 Feb 1963, Cabreraet al., 15321 (BAA). Salta. Dpto. Los Andes, ruta 51, deSan Antonio de los Cobres a Viaducto La Polvorilla,4170 m, 18 Feb 2002, Cialdella et al., 417* (SI).BOLIVIA. La Paz: Prov. Omasuyos, en la ruta LaPaz-Huarina, a la altura de Laja, 3900 m, 28 Feb 1950,Krapovickas et al., 7063 (SI). Jarava media (Speg.)Penail. ARGENTINA. Jujuy: Dpto. Humahuaca,Humahuaca, falda de los cerros, 3050 m, 16 Feb 1931,Parodi 9695 (BAA). Huacalera, 2700 m, 23 Feb 1955,Cabrera 11983 (BAA). Dpto. Tumbaya, CienagaGrande, 11 km al S de El Moreno, 3500 m, 19 Feb1987, Nicora et al., 8893 (SI); ruta 52, 40 km antes dePurmamarca, 23� 41� S 65� 42� W, 3870 m, 19 Feb 2002,Cialdella et al., 468 (SI). Salta:Dpto. La Poma: Palermooeste, camino hacia la toma de agua, 2820 m, 13 Feb2002, Cialdella et al., 242* (SI). Tucuman: Dpto. Tafı,ruta 307, camino de El Infiernillo a Amaicha del Valle,2840 m, 11 Feb 2002, Cialdella et al., 179 (SI). Jaravaplumosula (Nees ex Steud.) F. Rojas. PERU. Lima:Prov. Canta, Road to Canta, km 74, near Yasao tooutside of Canta, 18 May 1981, Sullivan et al., 967 (SI).ARGENTINA. Jujuy: Dpto. Humahuaca, Mina Agu-ilar, Espinazo del Diablo, 3800 m, 21 Mar 1973,Ruthsatz s.n. (BAA 14563). Dpto. Tilcara, Tilcara,2600 m, 28 Jan 1943, Cabrera 7713 (BAA); Laguna deVolcan, 23� 55� S 65� 28� W, 2100 m, 21 Feb 2002,Cialdella et al., 534 (SI). Dpto.Yavi, Abra de Yavi,3400 m, 20 Feb 1963, Cabrera et al., 15333 (BAA).Salta. Dpto. Cachi, ruta 42, Parque Nacional LosCardones, camino hacia Seclanta¢s, 2850 m, 14 Feb2002, Cialdella et al., 258* (SI). Nassella arcuata (R. E.Fr.) Torres. ARGENTINA. Jujuy: Dpto. Cochinoca,Abra Pampa, Cerro Huancar, 3500 m, 17 Feb 1963,Cabrera et al., 15274 (BAA);entre Rı¢o Doncellas yCasabindo, 3540 m, 8 Feb 1995, Deginani et al., 512(SI). Dpto. Humahuaca, Ruta 9, de Tres Cruces aHumahuaca, Ayo. Puente del Diablo, 3690 m, 20 Feb2002, Cialdella et al., 490 (SI). Dpto. Tilcara, Huacalera,Pampa Corral, 4000 m, 2 Mar 1955, Cabrera 12133(BAA). Dpto. Yavi, Yavi Chico, 3420 m, 6 Mar 1940,Meyer s.n. LIL 14931 (BAA, SI). Salta: Dpto. Rosariode Lerma, desvı¢o de Ruta 51, entre Santa Rosa deTastil y Alfarcito, 3380 m, 21 Feb 1987, Nicora et al.,9002 (SI). Tucuman: Dpto. Tafı, ruta 307, camino de ElInfiernillo a Amaicha del Valle, 2840 m, 11 Feb 2002,Cialdella et al.183* (SI). Nassella melanosperma

(J. Presl) Barkworth. ARGENTINA. Buenos Aires:

Pdo. Tandil, Sierra de las Animas, La Cascada, 281 m,15 Nov 2004, Morrone et al., 5139* (SI). BRAZIL. RioGrande do Sul: Pelotas, Beira de estrada, 10 Nov 1954,da Costa Sacco 217 (SI). Nassella novari Torres.ARGENTINA. Jujuy: Dr. Manuel Belgrano, entreLeon y Nevado de Chani, Las Cuevas, 3000 m, III-1963, Fabris et al., 4043 (LP). Tumbaya, subida dePurmamarca a Abra de Pives, 3000 m, 24 Apr 1975,Cabrera et al., 26350 (LP, SI); al N del volcan, 2150 m,18 Mar 1973, Ruthsatz 14629 (BAA); Purmamarca,Tascal, 3400 m, 15 Feb 1963, Cabrera et al., 15155 (LP).Salta: Dpto. Cachi, 46 km de Cachi, camino a Salta,3060 m, 19 Mar 1972, Krapovickas et al., 21976 (SI).Dpto. Chicoana, ruta 33, de Cachi a Ciudad de Salta,pasando desvıo a ruta 42, 3210 m, 15 Feb 2002,Cialdella et al., 308* (SI). Nassella pampagrandensis

(Speg.) Barkworth. ARGENTINA. Jujuy: Santa Bar-bara, Cachipunco, 13 Feb 1964, Fabris et al., 5222 (LP).Tilcara, Yala de Monte Carmelo, 2900 m, 19 ⁄21 Jan1966, Fabris et al., 6482 (LP). Tumbaya, Chilcayo,2300 m, 5 Jan 1966, Fabris et al., 6102 (LP); Volcan,cerros, 10 Mar 1958, Cabrera and Marchionni 12935(BAA, LP); Chilcayo, Finca del Dr. Gronda, 2800 m,Deginani et al., 363 (SI). Tucuman: Dpto. Tafı, ruta 307,camino de Tafı del Valle a Amaicha del Valle, 1 kmantes de El Infiernillo, 2790 m, 10 Feb 2002, Cialdellaet al., 163* (SI). Ortachne erectifolia (Swallen) Clayton.PERU. Huanuco: 21 Jul 1982, Renvoize 4366 (MO).COLOMBIA. Arauca: 4200 m, 31 Dec 1985, J.R.I.Wood 5263* (MO). ECUADOR. Loja: 3300 m, 4�37�S79�2�2, 26 Nov 1998, Laegaard 19274 (MO).Oryzopsis

asperifolia Michx.5989*, Peterson 18407 Pappostipa

vaginata (Phil.) Romasch. ARGENTINA. Jujuy: Dpto.Cochinoca, Abra Pampa, Cerro Huancar, 3500 m, 22Feb 1963, Cabrera et al., 15406 (BAA). Dpto. Humahu-aca, Huacalera, 2700 m, 23 Feb 1955, Cabrera 11987(BAA). Dpto. Susques, subida a Alto Los Chorrillos,4100 m, 16 Feb 1980, Cabrera et al., 31759 (SI). Dpto.Tilcara, Posta de Hornillos, 2400 m, 10 Feb 1959,Cabrera 13180 (BAA). Dpto. Tumbaya, subida dePurmamarca a Abra de Pives, 3700 m, 24 Apr 1975,Cabrera et al., 26354 (SI); ruta 52, 40 km antes dePurmamarca,23�41�S 65�42� W, 3870 m, 19 Feb 2002,Cialdella et al., 464 (SI); Tumbaya, 2100 m, 15 Mar1973, Ruthsatz s.n. (BAA 14638); ruta 52, 40 km antesde Purmamarca, 3870 m, 19 Feb 2002, Cialdella et al.,464 (SI). Dpto. Yavi, Abra de Yavi, 3400 m, 20 Feb1963, Cabrera et al., 15335 (BAA). Salta. Dpto. Cachi,ruta 42, Parque Nacional Los Cardones, camino haciaSeclanta¢s, 2730 m, 14 Feb 2002, Cialdella et al., 264*(SI). Piptatherum miliaceum (L.) Coss. ARGENTINA.Buenos Aires: Distrito Federal, Quinta de Lezica,Caballito, 24 Dec 1910, Hicken s.n., SI 13458 (SI); LaPampa: Santa Rosa, naturalizada en la calle ArgentinoValle 555, 18 Ene 2003, Rugolo 2135* (SI). Piptochae-tium lasianthum Griseb. ARGENTINA. Buenos Aires:

15Ana M. Cialdella et al. / Cladistics (2010) 1–17

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Tandil, Rta prov. 74, Sierra del Tigre, campos junto a laReserva del Tigre, 31 ⁄12 ⁄2002, Giussani 318* (SI).Piptochaetium lejopodum (Speg.) Henrard. ARGEN-TINA. Buenos Aires: Tornquist, Villa Arcadia, Cerrodel Amor, 1 km de Sierra de la Ventana pasando por V.Arcadia,460 m, 12 ⁄11 ⁄2004, Morrone 5111* (SI). Pipto-chaetium medium (Speg.) Torres. ARGENTINA. Bue-nos Aires: Tandil, Rta prov. 74, Sierra del Tigre, camposjunto a la Reserva del Tigre, 31 ⁄12 ⁄2002, Giussani 317*(SI). Piptochaetium montevidense (Spreng.) Parodi.ARGENTINA. Entre Rıos: Gualeguaychu, ruta 16,km 58, de Gualeguaychu a Gualeguay, 5 Nov 2001,Cialdella 16* (SI). Piptochaetium napostaense (Speg.)Hack. ARGENTINA. Tucuman: Tafı, camino desdeTafı del Valle hacia Las Carreras, a 2 km de Tafı delValle, Cerro El Pelado, 2130 m, 9 Feb 2002, Cialdella55* (SI). Poa holciformis J. Presl. CHILE. RegionMetropolitana. Santiago: M. Rio Yeso, above Embalsedel Yeso and up to and around Termas del Plomo, 13Jan 2002, Soreng 7157* (US). Ptilagrostis porteri

(Rydb.) W.A. Weber. USA. Colorado: Park, south sideof Guanella Pass on W side of Forest Road 118, 8 Aug1999, Barkworth 99117* (COAH).

Appendix 3

List of characters and character states. A descriptionof the characters and character states and their range of

variation are presented as considered in the cladisticanalysis.

0. Lemma margins: flat, strongly overlapping = 0;flat, not or only slightly overlapping = 1; slightlyinvolute = 2; conspicuously involute = 3. 1. Palea

shape: flat on the abaxial surface = 0; bikeeled on theabaxial surface = 1. 2. Palea length: half the length ofthe lemma to equal the length of the lemma = 0; longerthan the lemma = 1; shorter than half the length of thelemma = 2. 3. Callus shape: acute or subacute = 0;blunt or truncate = 1. 4. Awn: persistent = 0; decidu-ous = 1. 5. Compression of the floret : absent or slightlycompressed = 0; laterally and conspicuously com-pressed = 1; dorsally and conspicuously com-pressed = 2. 6. Awn or awn-like tip: straight orcurved, not geniculate = 0; geniculate or bigenicu-late = 1. 7. Conspicuous crown: absent = 0; pres-ent = 1. 8. Glumes texture: membranous = 0;indurated = 1. 9. Disarticulation of the awn or awnlike

tip and the lemma: absent = 0; present = 1. 10. Paleatexture: membranous = 0; indurated = 1. 11. Lemma

texture: membranous = 0; indurated = 1. 12. Glumes

length: longer than the floret = 0; equal to the flo-ret = 1; shorter than the floret = 2. 13. Lemma apex:

minutely 2-toothed or without any tooth = 0; with onetooth = 1; with 2 conspicuous teeth = 2.

16 Ana M. Cialdella et al. / Cladistics (2010) 1–17

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Appendix 4

Morphological matrix used in the analyses. Numbers in the first row represent the characters described in Appendix3, together with their codification. Numbers within brackets show a polymorphism for the particular character of thespecies involved. Dashes indicate that a character is inapplicable for a taxon.

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Poa holciformis 1 1 0 1 - 1 - 0 0 - 0 0 [12] 0

Achnatherum bromoides 1 0 0 1 0 0 0 0 0 1 0 0 0 2

Achnatherum chingii 1 0 0 0 0 0 1 0 0 1 0 0 0 2

Achnatherum diegoense 1 0 0 0 1 0 1 0 0 1 0 0 0 2

Achnatherum eminens 1 0 0 0 1 0 0 0 0 1 0 0 0 0

Achnatherum inebrians 1 0 0 0 1 0 1 0 0 1 0 0 0 0

Achnatherum multinode 1 0 0 0 0 0 0 0 0 1 0 0 0 2

Achnatherum psilantherum 1 0 0 0 1 0 1 0 0 1 0 0 0 0

Aciachne acicularis [12] 0 0 1 0 0 0 0 1 0 1 1 2 0

Aciachne flagellifera [12] 0 0 1 0 0 0 0 1 0 1 1 2 0

Aciachne pulvinata [12] 0 0 1 0 0 0 0 1 0 1 1 2 0

Amelichloa ambigua 1 0 0 1 0 0 1 0 0 1 0 1 0 0

Amelichloa brachychaeta 1 0 0 1 0 0 1 0 0 1 0 1 0 0

Amelichloa brevipes 1 0 0 1 0 0 1 0 0 1 0 1 0 2

Amelichloa caudata 1 0 0 1 0 0 1 0 0 1 0 1 0 0

Amelichloa clandestina 1 0 0 1 0 0 1 0 0 1 0 1 0 2

Anatherostipa brevis 1 0 0 1 0 0 0 0 0 1 0 0 1 0

Anatherostipa orurensis 1 0 0 1 1 0 0 0 0 1 0 0 0 0

Austrostipa elegantissima 0 0 [02] 0 0 0 1 0 [01] 1 1 1 0 1

Austrostipa nodosa 0 0 0 0 0 0 1 0 [01] 1 1 1 0 [02]

Hesperostipa comata 1 0 0 0 0 0 1 1 0 1 1 1 0 0

Hesperostipa neomexicana 1 0 0 0 1 0 1 1 0 1 1 1 0 0

Jarava leptostachya 1 0 [02] 0 1 0 0 0 0 1 0 1 0 0

Jarava media 1 0 [02] 0 0 0 0 0 0 1 0 1 0 0

Jarava plumosula 1 0 2 0 1 0 0 0 0 1 0 1 0 0

Pappostipa vaginata(a) 1 0 [02] 0 0 0 1 0 0 1 0 1 0 0

Pappostipa vaginata(b) 1 0 [02] 0 0 0 1 0 0 1 0 1 0 0

Nassella arcuata 0 0 2 0 [01] 0 1 1 0 1 0 1 0 0

Nassella melanosperma 0 0 2 0 0 0 1 1 0 1 0 1 0 0

Nassella novari 0 0 2 0 1 0 1 1 0 1 0 1 0 0

Nassella pampagrandensis 0 0 0 1 1 0 1 1 0 1 0 1 0 0

Ortachne erectifolia(a) 1 0 0 1 0 0 0 0 0 0 0 0 2 0

Ortachne erectifolia(b) 1 0 0 1 0 0 0 0 0 0 0 0 2 0

Oryzopsis asperifolia 0 0 0 1 1 0 0 0 0 1 1 1 [12] 0

Piptatherum miliaceum 1 0 0 1 1 2 0 0 0 1 1 1 0 0

Piptochaetium lasianthum 3 1 1 1 1 0 1 1 0 1 1 1 0 0

Piptochaetium medium 3 1 1 0 0 0 1 1 0 1 1 1 0 0

Piptochaetium montevidense 3 1 1 1 1 1 [01] 1 0 1 1 1 0 0

Piptochaetium napostaense 3 1 1 0 0 0 1 1 0 1 1 1 0 0

Ptilagrostis porteri 1 0 0 1 0 0 1 0 [01] 1 [01] [01] [01] 2

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1 AUTHOR: Laegaard, 1987 has been changed to Lægaard, 1987 so that this

citation matches the Reference List. Please confirm that this is correct.

2 AUTHOR: Soreng, 2003 has been changed to Soreng et al., 2003 so that

this citation matches the Reference List. Please confirm that this is correct.

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(Please note that it is the responsibility of the author(s) to ensure that all

URLs given in this article are correct and useable.)

4 AUTHOR:ILD has been spelt out as incongruence length difference, is that

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5 AUTHOR: ‘‘In the sensitivity analyses, the congruence measure (ILD index)

identified in all partitions the parameter set 2-1-1 (substitution cost = 2,

opening gap cost = 1, extension gap cost = 1) as the ‘‘most congruent

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6 AUTHOR: ‘‘the clade of Amelichloa (except A. brevipes) together with

Nassella pampagrandensis and N. melanosperma shared an indel of 5

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8 AUTHOR: Achnatheum changed to Achnatherum.

9 AUTHOR: Orthachne changed to Ortachne.

10 AUTHOR: Orthachne changed to Ortachne.

11 AUTHOR: Author: Jacobs, S. W. L., Barkworth, M. E., Hsiao C. 2000. -

publisher added (CSIRO Publishing), is that correct?

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where it should be cited; or delete from the Reference List.

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