A phylogeny of species near Agrostis supporting the recognition of two new genera, Agrostula and Alpagrostis (Poaceae, Pooideae, Agrostidinae) from Europe

Abstract Based on a molecular DNA phylogeny of three plastid (rpl32-trnK, rps16 intron, and rps16-trnK) and nuclear ITS regions investigating 32 species of Agrostidinae, we describe two new genera, Agrostulagen. nov. with a single species and Alpagrostisgen. nov. with four species; provide support for five species in a monophyletic Podagrostis; and include a small sample of 12 species of a monophyletic Agrostis s.s. (including the type and most species of Neoschischkinia), that separates into two clades corresponding to A.subg.Agrostis and A.subg.Vilfa. Agrostula differs from Agrostis in having leaf blades with pillars of sclerenchyma which are continuous between the adaxial and abaxial surface of the blades, dorsally rounded glumes with blunt to truncate and erose to denticulate apices, florets ½ the length of the glumes, lemmas equally wide as long, widest at (or near) apex, apices broadly truncate, irregularly 5 to 7 denticulate to erose, awnless, anthers longer than the lemmas, and rugose-papillose caryopses. Alpagrostis differs from Agrostis in having geniculate basally inserted awns and truncate lemma apices with lateral veins prolonged from the apex in (2)4 setae. The following eight new combinations are made: Agrostulatruncatula, Agrostulatruncatulasubsp.durieui, Alpagrostisalpina, Alpagrostisalpinavar.flavescens, Alpagrostisbarceloi, Alpagrostissetacea, Alpagrostissetaceavar.flava, and Alpagrostisschleicheri. In addition, we provide a key separating Agrostula and Alpagrostis from Agrostis s.s. and other genera previously considered as synonyms of Agrostis; lectotypify Agrostisalpina Scop., A.schleicheri Jord. & Verl., A.truncatula Parl., and A.truncatulavar.durieui Henriq.; and neotypify A.setacea Curtis.


Introduction
The genus Agrostis L. includes ca. 224 species worldwide and is placed in subtribe Agrostidinae Fr., supersubtribe Agrostidodinae Soreng, tribe Poeae R.Br., and supertribe Poodae L. Liu in subfamily Pooideae Benth. . The length of the palea was recognized by Trinius (1820, 1824) as an important character in separating species of Agrostis into two groups, those with short paleas in A. sect. Trichodium (Michx.) Trin. and those with longer paleas in A. sect. Vilfa (Adans.) Roem. & Schult. The term "Trichodium net", based on observations of the Swedish scientist T. Vestergren, to describe the lemma epidermis of Agrostis which bear a fine-meshed network when observed under high magnification, is found almost exclusively in those species with short paleas (Björkman 1960).
Podagrostis (Griseb.) Scribn. & Merr. was initially described as a section of Agrostis (Grisebach 1852) and was recently updated and revised by Sylvester et al. (2019aSylvester et al. ( , b, 2020 to include ten species native to the western hemisphere. Five additional species of Agrostis were transferred into Podagrostis in Sylvester et al. (2020) of which P. bacillata (Hack.) Sylvester & Soreng and P. rosei (Scribn. & Merr.) Sylvester & Soreng are newly included in our molecular analysis using nuclear internal transcribed spacer (ITS) and three plastid DNA (rpl32-trnK, rps16 intron, and rps16-trnK) markers. Previously, P. thurberiana (Hitchc.) Hultén was included in a phylogenetic study based on morphology and three plastid regions, and the taxon was weakly supported as the sister group of a strongly supported Agrostis + Polypogon Desf. clade (Soreng et al. 2007). A limitation of that analysis was that only a single species was included for each of the three genera. No molecular study has included more than two species to test the monophyly of this putative lineage (Saarela et al. 2017). The salient characters separating Podagrostis from Agrostis are: a) floret usually equaling or subequaling the glumes, sometimes slightly shorter but reaching past ¾ the length of the glumes, b) palea well-developed, usually reaching from (2/3) ¾ to almost the apex of the lemma, c) presence of a glabrous or distally hairy rachilla extension emerging from under the base of the palea as a slender short stub up to 1.4 mm long (rudimentary in most florets of P. rosei), and d) lemmas unawned or with a short straight mucro 0.2-0.6 mm long, inserted medially or in the upper half of the lemma, not surpassing the glumes (awn 1.6-2 mm long, inserted in lower 1/3 of lemma, straight or geniculate and usually not surpassing glumes in P. rosei) [Sylvester et al. 2020]. Sáez and Rosselló (2000) described Agrostis barceloi L. Sáez & Rosselló from the northern mountains of Mallorca (Balearic Islands) placing it in the Agrostis alpina Scop. complex along with A. curtisii Kerguélen and A. schleicheri Jord. & Verl. These four species share the following synapomorphies: geniculate basally inserted awns and truncate lemma apices that bare lateral setae (extension of the lateral veins) [Romero García et al. 1988a, b;Sáez and Rosselló 2000] [Romero García et al. 1988a, b].
Neoschischkinia Tzvelev (1968) (Tzvelev 1968). Valdés and Scholz (2006)  The main goals of this study were to estimate the phylogenetic relationships of species near or sister to Agrostis based on ITS and three plastid DNA regions (rpl32-trnK, rps16 intron, and rps16-trnK) for species of Agrostidinae and provide names for two clades that align near but not within Agrostis s.s. In addition, we provide a key separating Agrostula, gen. nov., and Alpagrostis, gen. nov., from Agrostis s.s. and other genera considered as synonyms of Agrostis. We propose lectotypes for Agrostis alpina, A. schleicheri, A. truncatula and A. truncatula var. durieui Henriq., and a neotype for A. setacea.

Phylogenetic analyses
Detailed methods for DNA extraction, amplification, and sequencing are given in Romaschenko et al. (2012) and Peterson et al. (2014Peterson et al. ( , 2015aPeterson et al. ( , b, 2016. We used Geneious Prime 2020 (Kearse et al. 2012) for contig assembly of bidirectional sequences of rpl32-trnL, rps16 intron, rps16-trnK, and ITS regions, and Muscle (Edgar 2004) to align consensus sequences and adjust the final alignment. We identified models of molecular evolution for the cpDNA and nrDNA regions using jModeltest (Posada 2008) and applied maximum-likelihood (ML) and Bayesian searches to infer overall phylogeny. The combined data sets were partitioned in accordance with the number of markers used. Nucleotide substitution models selected by Akaike's Information Criterion, as implemented in jModelTest v.0.1.1, were specified for each partition ( Table 1). The ML analysis was conducted with GARLI 0.951 (Zwickl 2006). The ML bootstrap analysis used 1000 replicates with 10 random addition sequences per replicate. The tree file from the ML result was read into PAUP where the majorityrule consensus tree was constructed. Bayesian posterior probabilities (PP) were estimated using a parallel version of the MrBayes v3.2.7 (Huelsenbeck and Ronquist 2001;Ronquist and Huelsenbeck 2003) where the run of eight Markov chain Monte Carlo iterations was split between an equal number of processors. Bayesian analysis was initiated with random starting trees and was initially run for four million generations, sampling once per 100 generations. The analysis was run until the value of the standard deviation of split sequences dropped below 0.01 and the potential scale reduction factor was close to or equal to 1.0. The fraction of the sampled values discarded as burn in was set at 0.25.
It is critically important to include the type species of genera and other higher taxa when doing molecular studies to know you are using the name correctly as intended by the original author. The following species are the types of their respective genera and are included in our analyses: Agrostis canina L. (type conserved), Calamagrostis canescens (Weber) Roth, Chascolytrum subaristatum (Lam.) Desv., Gastridium ventricosum (Gouan) Schinz & Thell., Neoschischkinia elegans (= Agrostis tenerrima), Podagrostis aequivalvis Trin., and Triplachne nitens (Guss.) Link.
Our study was designed to test relationships of three of the four species (A. alpina, A. curtisii, and A. schleicheri) of the Agrostis alpina group, all five species that have been attributed to Neoschischkinia (N. elegans, N. nebulosa, N. pourretii, N. reuteri, and N. truncatula), Podagrostis, Gastridium P. Beauv., Triplachne Link, and representative samples of Agrostis, Calamagrostis Adans., and Chascolytrum Desv. All of these genera have been found in a clade in previous molecular analyses and in our unpublished trees investigating a large number of species in Agrostis, Calamagrostis, Cinnagrostis Griseb., and Koeleria Pers. (Saarela et al. 2017;Barberá et al. 2019a, b;Peterson et al. 2019). Previous analyses of Polypogon found members of the genus nested in a grade within Agrostis and there was incongruence between the plastid and nuclear signals (Saarela et al. 2017;Romaschenko et al. unpubl.). We do not address this question here (i.e., Polypogon is not included in our sampling) since we lack a large sample of species within Agrostis and it is beyond the scope of our study. Echinopogon caespitosus C.E. Hubb. in subtribe Echinopogoninae Soreng was chosen as the outgroup since it lies outside of the Agrostidinae, but inside supersubtribe Agrostidodinae Tkach et al. 2020).

Taxonomy
Herbarium acronyms follow Index Herbariorum (Thiers, continuously updated). In this treatment glabrous means without pubescence (in the sense of slender, relatively soft hairs). Smooth indicates no prickle-hairs with broad bases and/or hooked or pointed apices (i.e., pubescence can occur on a smooth surface, and a rough or scabrous surface can be glabrous). Specimens in the United States National Herbarium (US) and the Real Jardín Botánico Herbarium (MA) were reviewed for this study, in addition to Romero Zarco (1987), Romero García et al. (1988a, Sáez and Rosselló (2000), Clayton et al. (2006), Cope andGray (2009), andPortal (2009) were consulted during preparation of the descriptions. Beyond types (some only seen in images), only material from herbaria where specimens have been checked and verified by the authors are cited. Parts of the generic key were adapted from Sylvester et al. (2020).

Phylogeny
A total of 176 new sequences from 33 species (48 individuals) are reported in Gen-Bank (Table 1). Total aligned characters for individual regions and other parameters are noted in Table 2. The resulting plastid and ITS topologies were inspected for conflicting nodes (see Fig. 1) with ≥ 80% bootstrap support (BS) and/or posterior probabilities (PP) ≥ 0.95. No supported conflict was found so plastid and ITS sequences were combined.
The ML tree from the combined plastid and ITS regions ( Fig. 1) is well resolved (posterior probabilities identified in the Bayesian analysis are included on the ML tree, and most clades include a PP = 1), with strong support (BS ≥ 96-100) for the following clades: two species of Gastridium, four accessions of Triplachne nitens, an Agrostis s.s. clade that includes two subclades A and B, three accessions of Agrostis truncatula, and the Agrostis alpina-A. curtisii-A. schleicheri clade; moderate support (BS = 84-86%) for seven species of Calamagrostis and two species of Chascolytrum; and weak support (BS = 57%) for five species of Podagrostis. Chascolytrum is basal followed by, in order of divergence, a clade with Agrostis truncatula sister to Calamagrostis, a clade with Gastridium sister to Triplachne which is sister to the remaining species in the Agrostis s.l. clade (PP = 1, BS = 66). In Agrostis s.l., Podagrostis is sister to the Agrostis alpina-A. curtisii-A. schleicheri clade and the Agrostis s.s. clade.

Discussion
Our molecular sampling of five species of Podagrostis is the largest to date. In an earlier Romaschenko et al. (unpubl.) study of the three species then in the genus, P. humilis (Vasey) Björkman exhibited incongruence with the nuclear ITS signal aligning within the Podagrostis clade and the plastid signal aligning as sister to Agrostis s.s. in a grade with the Agrostis alpina-A. curtisii-A. schleicheri clade at the base. The addition of P. bacillata and P. rosei in our analysis eliminated this anomaly. In an earlier study primarily using different DNA markers with only P. aequivalvis and P. rosei (as Agrostis rosei Scribn. & Merr.), Saarela et al. (2017) found P. rosei to be part of a well-supported clade with four Chinese species of Deyeuxia Clarion ex P. Beauv. and Calamagrostis bolanderi Thurb. + P. aequivalvis. Although C. bolanderi's placement in a strongly supported lineage with P. aequivalvis provides support for its transfer to Podagrostis, we hesitate to include it here because it may represent a separate hybrid between Podagrostis and Calamagrostis (Sylvester et al. 2020 Affinities of Agrostis truncatula are unclear, given the lack of support for its position (PP = 0.52) in the phylogeny sharing a common ancestor with Calamagrostis rather than aligning within Agrostis s.l. Agrostis truncatula has many unique morphological characteristics and differs from other species of Agrostis in having the combination of perennial habit, leaf blades with pillars of sclerenchyma that are continuous between the adaxial and abaxial surface of the blades, dorsally rounded glumes with blunt to truncate and erose to denticulate apices, open and diffuse panicles, florets ½ the length of the glumes, lemmas equally wide as long, widest at (or near) apex, apices broadly truncate, irregularly 5 to 7 denticulate to erose, awnless, anthers longer than the lemmas, and rugose-papillose caryopses. We, thus, describe Agrostula gen. nov. below based on the single species, A. truncatula, with two subspecies. We find no support for recognizing Neoschischkinia (Tzvelev 1968;Valdés and Scholz 2006), since four of the five species attributed to the genus align in the Agrostis s.s. clade: A. nebulosa, A. reuteri, and A. tenerrima (type of Neoschischkinia) in Agrostis subg. Vilfa (clade B), and A. pourretii in A. subg. Agrostis (clade A); whereas A. truncatula is phylogenetically isolated from Agrostis (Agrostula). All these species exhibit unusual characteristics not commonly found within Agrostis, i.e., diffuse, open panicles with divaricate and capillary branches, trapezoid lemmas with truncate apices, and caryopses with transverse furrows. However, without molecular DNA evidence, earlier systematists could not predict the complicated phylogenetic history of Agrostula truncatula.
Our rationale for recognizing the Agrostis alpina complex in a new genus, Alpagrostis gen. nov., is straightforward. Much like Podagrostis, there are salient morphological  features, i.e., geniculate basally inserted awns and truncate lemma apices with setaceous lateral veins, and there is strong clade support as sister to Agrostis s.s. The branch length of the Alpagrostis clade is moderately long indicating genetic differentiation shared among its members separating it from other clades. Sáez and Rosselló (2000) suggested that Agrostis barceloi, a tetraploid (2n = 28), is closely related to A. schleicheri, a hexaploid (2n = 42), and might have originated by the splitting of the shared ancestral lineage. The morphological features shared by A. barceloi and A. schleicheri suggest they may be derived from the diploids, A. alpina (2n = 14) or A. curtisii (2n = 14) since the former two species are geographically and genetically isolated (Sáez and Rosselló 2000). Massó et al. (2016) surveyed 40 of the 100 known individuals of the extremely narrow endemic, A. barceloi, for allozyme diversity, showing all loci to be monomorphic or with fixed heterozygosity consistent with allopolyploid origin (interspecific hybridization process and subsequent chromosome duplication) [Stebbins 1947;Crawford 1989;Soltis and Soltis 2000]. The Agrostis s.s. clade is divided into two strongly supported A and B clades that correspond to species that align in the Agrostis subg. Agrostis (clade A) or Agrostis subg. Vilfa (Adans.) Rouy (clade B) [≡ A. sect. Vilfa (Adans.) Roem. & Schult.]. As mentioned in the introduction, palea length is an important character used to separate these two subgenera and all species in clade A have paleas ≤1/3 the length of the lemma as expected, sometimes rudimentary or absent as in e.g. A. mertensii Trin., A. subpatens Hitchc. However, not all species in clade B have paleas ½-2/3 the length of the lemma since A. tenerrima has paleas 1/6 the length of the lemma and only about 0.1 mm long. This is not terribly surprising since hybrids among species of Agrostis are often fertile, and inter-subgeneric hybrids include A. canina × A. stolonifera L., a cross between the type of each subgenus of Agrostis (Widén 1971;Belanger et al. 2003;Watrud et al. 2004). In the future we intend to publish a large phylogeny of Agrostis with a Type. Agrostis truncatula Parl. Diagnosis. The one species of Agrostula differs from all other species of Agrostis by its glumes being dorsally rounded, not keeled, smooth throughout, and with apices blunt to truncate and erose to denticulate. Further differentiation can be made by the combination of perennial habit, leaf blades with pillars of scleren chyma that are continuous between the adaxial and abaxial surface of the blades, panicles open and diffuse, florets ½ the length of the glumes, lemmas equally wide as long, widest at (or near) apex, apices broadly truncate, irregularly 5 to 7 denticulate to erose, awnless, paleas c. ½ the length of the lemma, anthers longer than the lemma, caryopsis surface rugose-papillose, and its ecology, being found growing in very shallow soils.
Distribution and ecology. Iberian Peninsula and northern Africa, distributed in France, Spain, Portugal, and Morocco. Found in Mediterranean, Iberian-Atlantic and cold temperate, often high-elevation, environments of the Pyrenees. Forms part of pioneer grassland species assemblages which grow on very shallow and sandy 'skeleton' soils, apparently reliant on climatic humidity in addition to precipitation for its water supply. Usually flowering from June to July.
Notes. Agrostula truncatula also differs in its leaf blade anatomy from most other species of Agrostis in having pillars of sclerenchyma which are continuous between the adaxial and abaxial surface of the blades. These continuous pillars of sclerenchyma are exceptionally thick and found only on the margins and central vein in subsp. truncatula, while subsp. durieui has thinner continuous sclerenchyma packets in the margins, central and primary veins (Romero García and Blanca López 1988: fig. 4C-F). Costal cells and intercostal long cells of the abaxial blade surface are also distinct, with A. truncatula differing from species of Agrostis in the Iberian Peninsula in having paired s 0 -z cells in the costal zone, and l 3 type long cells in the intercostal zone (Romero García and Blanca López 1988). Stomata are also apparently absent on the abaxial blade surface, a character found in only a few other species in the Iberian Peninsula, i.e., Agrostis reuteri and Alpagrostis setacea (Romero García & Blanca López, 1988

Notes.
Romero García et al. (1988a) provide a key to differentiate the two subspecies. The typical subspecies has ligules as long or longer than wide with acute apices, conduplicate leaf blades that recurve at anthesis, and shorter lemmas 0.5-0.8 mm long whereas Agrostula truncatula subsp. durieui has ligules wider than long with truncate apices, flat, rarely conduplicate leaf blades that do not recurve at anthesis, and longer lemmas (0.7-)0.9-1(-1.2) mm long. Portal (2009)  Diagnosis. The species of Alpagrostis differ from Agrostis by a combination of characters in having plants densely tufted with only intravaginal innovations, leaves mainly basal, basal leaf blades involute and setaceous or filiform, conduplicate and acute, 0.1-1.2 mm in diameter as folded or rolled, ligules longer than they are wide, spikelets generally > 3 mm long, lemma apices truncate with lateral veins prolonged from the apex in 2 (A. setacea) or 4 setae 0.1-0.5 mm long, and, crucially, and lemmas with a well-developed awn, 3-7.4 mm long, inserted basally c. 0.1-0.4 mm from the base of the lemma, conspicuously twisted and geniculate.
Distribution and ecology. Europe and Mediterranean. Found in cold temperate, often high-elevation environments, often found growing on nutrient poor soils. Usually flowering from June to August.
Notes. All caryopses examined from herbarium specimens had a liquid lipid endosperm or were shriveled with a deep sulcus, implying that fresher specimens likely had a liquid endosperm. Agrostis sect. Bromidium (Nees & Meyen) E. Desv. shares many characteristics with Alpagrostis, such as lemma apices terminating in scabrous setae, well-developed, thickened, twisted and geniculate awns inserted in the lower 1/3 of the lemma, palea < 1/3 the length of the lemma, caryopses with liquid to semiliquid endosperm. Based on molecular DNA studies, Romaschenko et al. (unpubl.) and Tkach et al. (2020) found Bromidium to align within Agrostis s.s.
Alpagrostis barceloi differs somewhat from the other species in the genus, in terms of the panicle branches and pedicels being smooth, spikelets sometimes being shorter, 1-veined upper glumes, and awn sometimes inserted slightly higher up the lemma.  Notes. This species is included in Alpagrostis based on its similar morphology, although this needs to be confirmed in molecular analyses. Certain characteristics sometimes differ from the other species in the genus, i.e., spikelets and lemmas sometimes shorter, insertion of the awn sometimes higher on the lemma, panicle branches and pedicels smooth or scaberulous. Alpagrostis barceloi shares with other member of the genus, conduplicate leaf blades, truncate lemma apices with setaceous extensions of the lateral veins, and ecologically is a strict orophyte, much like A. alpina and A. schleicheri (Sáez and Rosselló 2000). There is one specimen in the British Museum Herbarium, originally from "Curtis's garden" (BM-001144085), which may be taken as representative of the species." Philipson was possibly referring to this specimen. On the neotype there are three different collections on the same sheet. The specimen on the upper left of the sheet is BM-001144085 ( Fig. 2A).   Key to differentiate taxa of Agrostula and Alpagrostis from Agrostis and other genera previously considered as synonyms of Agrostis by Watson and Dallwitz (1992) and Clayton and Renvoize (1986)  Floret equaling or subequaling the glumes, sometimes slightly shorter but reaching past ¾ the length of the glumes, usually with a short rachilla prolongation emerging behind the palea (sometimes absent in many florets of P. rosei and P. humilis so check many spikelets); paleas well-developed, usually reaching from (2/3) ¾ to almost the apex of the lemma; lemmas muticous or with a short straight awn 0.2-0.6 mm long, inserted medially or in the upper half of the lemma, not surpassing the glumes (awn well-developed, 1.6-2 mm long, inserted in lower 1/3 of lemma, straight or geniculate and usually not surpassing glumes in P. rosei Floret notably shorter than the glumes, usually 1/3-3/4 the length of the glumes, rarely longer, without a trace of a rachilla prolongation; paleas well-developed, poorly-developed, or absent, when well-developed reaching from ½-¾ the length of the lemma; lemmas muticous, with a short straight awn 0. Glumes keeled, usually scabrous (at least in part), rarely upper glume smooth throughout, apices obtuse to acute-acuminate, rarely blunt to truncate, rounded to muticous; palea absent or rudimentary to ¾ the length of the lemma; panicles open and diffuse to condensed and spikelike; lemmas usually longer than wide (rarely equally wide as long), usually narrowed towards the apex, apices variable, ranging from somewhat broadly to usually narrowly truncate, usually with 2 to 5 dents (sometimes aristulate), to blunt and entire, awnless or with an awn 0.2-6+ mm long; anthers sometimes longer to usually shorter than the lemma; caryopsis surface usually smooth; perennials or annuals; usually growing from well-developed soils, less often from shallow soils, and generally reliant on soil moisture for their water supply; cosmopolitan ..