Author Manuscript Paul Peterson ORCID iD: 0000-0001-9405-5528 Research Article Phylogeny, classification, and biogeography of Afrotrichloris, Apochiton, Coelachyrum, Dinebra, Eleusine, Leptochloa, Schoenefeldia, and a new genus, Schoenefeldiella (Poaceae: Chloridoideae: Cynodonteae: Eleusininae) Running title: Peterson et al.: Phylogeny,classification, and biogeography of Afrotrichloris, Coelachyrum, Dinebra, Eleusine, Leptochloa, Schoenefeldia, and Schoenefeldiella Paul M Peterson1*, Konstantin Romaschenko1,2, Yolanda Herrera Arrieta3, and Maria S. Vorontsova4 1 Department of Botany, National Museum of Natural History, Smithsonian Institution, Washington, DC 20013-7012, USA 2 M.G. Kholodny Institute of Botany, National Academy of Sciences, Kiev 01601, Ukraine 3 Instituto Politécnico Nacional, CIIDIR Unidad-Durango-COFAA, Durango, C.P. 34220, Mexico 4 Comparative Plant and Fungal Biology, Royal Botanic Gardens, Kew, Richmond Surrey, TW9 3AE, UK *Author for correspondence. E-mail: peterson@si.edu. Tel.: 1-202-633-0975. Fax: 1-202786-2653. This is the author manuscript accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/jse.12803. This article is protected by copyright. All rights reserved. Author Manuscript ORCID (http://orcid.org): PMP, 0000-0001-9405-5528; KR, 0000-0002-7248-4193. Received XX April 2021; Accepted XX XXX 2021; Article first published online xx Month 20XX Abstract―To investigate the evolutionary relationships among species of Afrotrichloris, Apochiton, Coelachyrum, Dinebra, Eleusine, Leptochloa, and Schoenefeldia of subtribe Eleusininae a phylogeny based on DNA sequences from nine gene regions (ITS, rps16trnK, rps3, rps16, rpoC2, rpl32-trnL, ndhF, ndhA, ccsA) is presented. Previous molecular phylogenies indicated that Coelachyrum was polyphyletic and Schoenefeldia was paraphyletic with Afrotrichloris embedded within it. Apochiton burttii was embedded in the Coelachyrum clade paired with C. longiglume, C. poiflorum was placed outside of Coelachyrum and sister to Eleusine, and Schoenefeldia is paraphyletic with its two species forming a grade sister to Afrotrichoris. Our molecular phylogeny supports recognition of a new genus, Schoenefeldiella, and a new combination, Schoenfeldiella transiens. In addition, we provide generic emendations for Coelachyrum which now includes five species including a new combination proposed here, Coelachyrum burttii, and Eleusine which now includes 11 species. Key words biogeography, Coelachyrum, grasses, ITS, molecular systematics, plastid DNA sequences, Poaceae, Schoenefeldiella, taxonomy 1. Introduction The tribe Cynodonteae currently includes 850 species in 94 genera and 21 subtribes, and it has received high support as a monophyletic lineage in recent molecular analyses (Peterson et al., 2014b, 2015a, 2015b, 2016; Soreng et al., 2015, 2017). Within the This article is protected by copyright. All rights reserved. Author Manuscript subtribe Eleusininae (236 species in 27 genera) Peterson et al. (2015b) found Coelachyrum Hochst. & Nees to be polyphyletic because C. lagopoides (Burm.f.) Senaratna, Apochiton burttii C.E. Hubb., and C. poiflorum Chiov. form a grade sister to Eleusine Gaertn., and Schoenefeldia Kunth to be paraphyletic with respect to two samples of Afrotrichloris martinii Chiov. Coelachyrum is a heterogeneous group of five annual or perennial semi-desert grasses that have been grouped together based on having paniculate inflorescences composed of loose to dense racemes, these often spaced (racemose) or clustered (digitate) on a central axis; 3-veined, broadly elliptic to ovate, rounded-on-the-back lemmas; and dorso-ventrally flattened caryopses with free pericarps that are highly ornamented and often rugulose (Phillips, 1974, 1995; Clayton et al., 2006; Cope, 2007). The genus was described by Hochstetter & Nees von Esenbeck (1842) based on Coelachyrum brevifolium Hochst. & Nees, the type species. Twelve years later, Steudel (1854) transferred this species into Eleusine along with 15 other species. Recently, Coelachyrum yemenica (Schweinf.) S.M. Phillips was transferred to Disakisperma Steud. based on shared morphology and sequence similarity of five DNA markers (Snow et. al., 2013). The most recent treatment of Eleusine includes 10 species, and eight of these were found in a strongly supported clade sister to Coelachyrum poiflorum in a molecular DNA sequence study (Liu et al., 2011; Peterson et al. 2015b; Soreng et al., 2017). Apochiton C.E. Hubb. is a monotypic genus known only from Tanzania and is characterized in having an annual habit, panicle branches often reduced to racemes; 3veined, short-awned lemmas; awned palea keels; and trigonous caryopses with free This article is protected by copyright. All rights reserved. Author Manuscript pericarps (Hubbard, 1936, 1974a). The species has never been attributed to any other genus and Hubbard (1936) mentions in his description that the unusual grain (free pericarp) is only known in a few species of Eragrostis Wolf, Sporoboleae [=Sporobolinae], Sclerodactylon Stapf, Dinebra Jacq., Eleusine, Dactyloctenium Willd., and Coleachyrum. However, Afrotrichloris, Austrochloris Lazarides, Daknopholis Clayton, Tetrapogon Desf., and the Unioliinae Clayton also have grains with a free pericarp (Clayton & Renvoize, 1986). For most of the 20th century Dinebra was represented by three species (Clayton & Renvoize, 1986; Watson & Dallwitz, 1992). Based on results of molecular DNA sequence studies, many species formerly placed in Leptochloa s.l. were transferred to an expanded Dinebra now with ±23 species, while Leptochloa s.s. was reduced to eight species with recognition of Diplachne P. Beauv. (2 spp.), Disakisperma (4 spp.), and Trigonochloa P.M. Peterson & N. Snow (2 spp.) [Peterson et al., 2012, 2014a, 2015a; Snow & Peterson, 2012; Snow et al., 2013, 2018, in prep.; Soreng et al., 2017]. Schoenefeldia includes two species, S. gracilis Kunth (the type) and S. transiens (Pilg.) Chiov., both characterized in having a solitary, paired or digitate racemes and spikelets with long-awned lemmas with the awns braided along the culm (Hubbard, 1974b; Cope, 2007). Schoenefeldia transiens can be separated from S. gracilis in having 2-flowered spikelets with the upper floret sterile and awned, the perennial habit, and flat leaf blades 5−35 cm long. Afrotrichloris Chiov. also includes two species, A. martinii and A. hyaloptera Clayton. Afrotrichloris hyaloptera can be separated from A. martinii by possessing This article is protected by copyright. All rights reserved. Author Manuscript shorter lateral lemmas lobes with a longer central awn and longer inflorescence spikes (Clayton, 1967). Clayton (1967) indicated that Afrotrichloris bears some similarity to Trichloris E. Fourn. ex Benth., although the two species traditionally placed in Trichloris now reside within Leptochloa P. Beauv. s.s. (Peterson et al. 2012; 2015b). Clayton also mentioned that A. hyaloptera is not unlike Enteropogon Nees and that Afrotrichloris probably represents different lines of divergence from a common Chloris Sw. stock. Using plastid (rps16-trnK, rps3, rps16, rpoC2, rpl32-trnL, ndhF, ndhA, ccsA) and nuclear ITS DNA sequence markers, we present a new phylogenetic analysis of 85 species in 24 genera of Eleusininae, emphasizing those species surrounding Coelachyrum, Apochiton, Schoenefeldia, and Afrotrichloris, along with Dinebra, Leptochloa, and Eleusine. We expand the number of species sampled to include all five accepted species of Coelachyrum and both species of Afrotrichloris (Clayton et al., 2006; Snow et al., 2013). In addition, we discuss morphological and anatomical characters supporting relationships, discuss biogeography, and propose changes to the classification of studied taxa. 2. Material and Methods 2.1. Taxon sampling Our sampling consists of 141 samples, representing 85 species of grasses, of which 84 are included in the Cynodonteae, and one is included in the Zoysieae. In addition, we include all six species that have been attributed to Coelachyrum and two species of Afrotrichloris (Clayton et al., 2006). Outside of the Cynodonteae, Sporobolus indicus (L.) R. Br. was chosen as the out group. A complete list of taxa, voucher information, and This article is protected by copyright. All rights reserved. Author Manuscript GenBank numbers can be found in Appendix S1. All collections gathered by PMP after 1998 were collected in silica but the majority of samples used in this study were taken from herbarium specimens. 2.2. DNA extraction, amplification, and sequencing All procedures were performed in the Laboratory of Analytical Biology (LAB) at the Smithsonian Institution. DNA isolation, amplification, and sequencing of rpl32-trnL spacer and ndhA intron (small single copy region); rps16-trnK spacer and rps16 intron (large single copy region); ndhF, ccsA, rpoC2, and rps3 (encoding region); and ITS were accomplished following procedures outlined in Peterson et al. (2010a, 2010b; 2012; 2014a). We specifically targeted plastid regions that proved to be most informative in our previous studies on chloridoid grasses (Peterson et al., 2010a, 2010b, 2011, 2012, 2014a, 2014b, 2014c; 2015a, 2015b, 2016; Liu et al., 2011). 2.3. Phylogenetic analyses We used Geneious 5.3.4 (Drummond et al., 2011) for contig assembly of bidirectional sequences of rpl32-trnL, ndhA, ndhF, rps3, rps16, rps16-trnK, rpoC2, ccsA, and ITS regions, and Muscle (Edgar, 2004) to align consensus sequences and adjust the final alignment (see Elect. Suppl. for sequence alignments). 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 the 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 This article is protected by copyright. All rights reserved. Author Manuscript 1). The ML analysis was conducted with GARLI 0.951 (Zwickl, 2006). The ML bootstrap analysis was performed with 1000 replicates, with 10 random addition sequences per replicate. The output file containing trees of ML found for each bootstrap dataset was then read into PAUP where the majority rule consensus tree was constructed. Bootstrap (BS) values of 90−100% were interpreted as strong support, 70−89% as moderate, and 50−69% as weak. Bayesian posterior probabilities (PP) were estimated using a parallel version of MrBayes v3.1.2 (Huelsenbeck & Ronquist, 2001; Ronquist & 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. Posterior probabilities (PP) of 0.95−1.00 were considered to be strong support. In the phylogram (Fig. 1) the native distribution of each species is indicated by color as follows: North America (red), South America (tan), Africa (dark green), Australia + Pacific (dark purple), and Eurasia (light purple). 2.4. Assessment of incongruence and data combining strategy 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. If no supported conflict was found, plastid and ITS sequences were combined. Where This article is protected by copyright. All rights reserved. Author Manuscript conflicting topologies were found, the datasets for inconsistently placed taxa were duplicated in the matrix. One set of the taxon was represented by the corresponding plastid sequences only, the other taxon set by only ITS sequences. The remaining positions for the truncated datasets were then coded as missing data. We use this ‘taxon duplication’ approach (Pirie et al., 2008; Pelser et al., 2010; Peterson et al., 2015a, 2016) to resolve our phylogenetic tree minimizing the diffusing effects of taxa with strongly supported incongruence between the plastid and ITS data, and to represent their alternative placements in relation to the remaining phylogenetic groups among which relationships are congruent (see Fig. 1). The combination of data is advantageous because it can provide better backbone support for nodes through the use of plastid data, and improve resolution for terminal nodes within the main phylogenetic groups by using ITS sequences. 3. Results 3.1. Phylogenetic analyses Twenty percent (167/823) of the sequences used in our study are newly reported here (Appendix S1). Characteristics of the nine regions are given in Table 1. Forty-seven sequences of rps3 (61%), 43 for rpoC2 (56%), and 30 for ndhF (41%), 13 for ccsA (19%), 15 for ITS (11%), 10 for rpl32-trnL (8%), 5 for rps16-trnK (5%), 3 for ndhA (4%), and 3 for rps16 (3%) are newly reported. Sequence alignment length for ndhA is 1150 bp, rps16-trnK is 1079 bp, ccsA is 954 bp, rps16 is 898, rpl32-trnK is 884 bp, rpoC2 is 808 bp, ndhF is 765 bp, ITS is 746 bp, and rps3 is 612 bp. Four hundred fortysix sequences or 35% (446/1269) were missing. This article is protected by copyright. All rights reserved. Author Manuscript 3.2. Incongruence between ITS and combined plastid phylograms The ITS signal places the Dinebra clade as sister to Aeluropus lagopoides−Odyssea paucinervis (PP = 1, BS = 80 in the Aeluropodinae) outside the Eleusininae, whereas the combined plastid signal places the Dinebra clade within Eleusininae (PP = 1, BS = 68). The topology among the species of Dinebra in the ITS- and plastid-derived trees is also different (Fig. 1A & B). In the ITS-derived tree Dinebra chinensis (L.) P.M. Peterson & N. Snow is sister to all remaining species in the genus which form two clades (labeled A and B on Fig. 1A). Clade A (PP = 1, BS = 100) contains two varieties of D. panicea (Retz.) P.M. Peterson & N. Snow (PP = 1, BS =96) that are sister to D. squarrosa (Pilg.) P.M. Peterson & N. Snow−D. retroflexa (Vahl.) Panz. (PP= 0.95, BS = 64), and these are sister to D. marquisensis (F. Br.) P.M. Peterson & N. Snow−D. xerophila (P.M. Peterson & Judz.) P.M. Peterson & N. Snow. Clade B contains an Australian clade with a monophyletic D. decipiens (R. Br.) P.M. Peterson & N. Snow with three varieties sister to D. divaricatissima (S.T. Blake) P.M. Peterson & N. Snow. Sister to D. decipiens−D. divaricatissima is D. ligulata (Lazarides) P.M. Peterson & N. Snow−D. southwoodii (N. Snow & B.K. Simon) P.M. Peterson & N. Snow (PP = 1, BS = 100). The basal member of the Australian clade is D. neesii (PP = 1, BS = 87), and sister to the Australian clade is D. haareri (Stapf & C.E. Hubb.) P.M. Peterson & N. Snow−D. somalensis (Stapf) P.M. Peterson & N. Snow (PP = 1, BS = 98). Sister to D. haareri−D. somalensis + the Australian clade is D. nealleyi (Vasey) P.M. Peterson & N. Snow−D. scabra (Nees) P.M. Peterson & N. Snow (PP = 1, BS = 97). Sister to D. nealleyi−D. scabra + D. haareri−D. This article is protected by copyright. All rights reserved. Author Manuscript somalensis + the Australian clade is D. aquatica (Scribn. & Merr.) P.M. Peterson & N. Snow −D. coerulescens (Steud.) P.M. Peterson & N. Snow + D. caudata (K. Schum.) P.M. Peterson & N. Snow −D. viscida (Scribn.) P.M. Peterson & N. Snow (PP = 1, BS = 98). In the plastid-derived tree there also are two clades (labeled A’ and B’ in Fig. 1B). In clade A’, the two varieties of D. panicea (PP = 1, BS = 98) are sister to D. marquisensis−D. xerophila (PP = 1, BS = 98). Sister to D. panicea + D. marquisensis−D. xerophila is D. retroflexa−D. squarrosa (PP = 1, BS = 97), and all of these are sister to D. chinensis (PP = 1, BS = 79). In clade B’ the monophyletic Australian clade includes D. divaricatissima embedded with the three varieties of D. decipiens and this is sister to D. nealleyi−D. scabra (PP = 1, BS = 84). Dinebra haareri−D. somalensis (PP = 1, BS = 100) is sister (PP = 1, BS = 98) to D. nealleyi−D. scabra + the Australian clade, and all of these form a clade (PP = 0.99, BS = 66) with D. panicoides (J. Presl) P.M. Peterson & N. Snow−D. viscida + D. aquatica−D. caudata sister to D. coerulescens (PP = 1, BS = 84). 3.3. Phylogenetic tree of Eleusininae The ML tree from the combined plastid and ITS regions is well resolved with strong support (PP = 1, BS ≥ 98) for the Eleusininae and the following genera: Afrotrichloris, Astrebla F. Muell., Ceolachyrum s.s., Diplachne, Disakisperma, and Eleusine (Fig. 1A & B). Diplachne is sister to all remaining genera in the Eleusininae (PP = 1, BS = 97). Coelachyrum is polyphyletic because C. poiflorum. is sister to all species of Eleusine (PP This article is protected by copyright. All rights reserved. Author Manuscript = 1, BS = 98). Apochiton burttii C.E. Hubb. is embedded within Coelachyrum and sister to C. longiglume Napper, and this pair is sister to C. brevifolium Hochst. & Nees + C. piercei (Benth.) Bor (PP = 1, BS = 100). Coelachyrum lagopoides (type) is sister to the rest of the Coelachyrum s.s. clade. Eleusine is monophyletic with E. jaegeri Pilg.+ E. multiflora Hochst. ex A. Rich. sister to remaining species in the genus. Eleusine floccifolia (Forssk.) Spreng. is sister (PP = 0.96, BS = 88) to E. africana Kenn.-O'Byrne + E. indica (L.) Gaertn. + E. tristachya (Lam.) Lam. + E coracana (L.) Gaertn. + E. kigeziensis S.M. Phillips. Seven accessions of E. africana form a clade (PP = 0.95, BS = 56) and these are sister to a polytoma that includes E. indica (polyphyletic), E. tristachya (PP = 1, BS 71), and E. coracana + E. kegeziensis (PP = 87, BS = 68). The Leptochloa clade is moderately supported (PP = 1, BS = 84) with L. crinita (Lag.) P.M. Peterson & N. Snow and L. pluriflora (E. Fourn.) P.M. Peterson & N. Snow forming a clade showing little variation sister to a clade containing L. chloridiformis Parodi–L. virgata (L.) P. Beauv. (PP = 0.56), where a single individual of L. virgata (Rimachi 8359) is sister to two accessions of L. chloridiformis, rendering L. virgata polyphyletic. Leptochloa longa Griseb. is sister to the L. crinita−L. pluriflora + L. chloridiformis−L. virgata clade. Leptochloa digitata (R. Br.) Domin is the first split followed by two accessions of L. anisopoda (Scribn. ex B.L. Rob.) P.M. Peterson, and L. exilis (Renvoize) P.M. Peterson. Austrochloris Lazarides + Astreba F. Muell. (PP = 1, BS = 97) are sister to remaining Eleusininae genera (PP = 0.98, BS = 65; Fig. 1B). The next split includes the Disakisperma clade (PP = 1, BS =100) sister to remaining Eleusininae. Schoenefeldia This article is protected by copyright. All rights reserved. Author Manuscript appears polyphyletic with its two species in a grade sister to a monophyletic Afrotrichloris (PP = 1, BS = 100). Schoenefeldia gracilis is basal and sister to S. transiens + Afrotrichloris. 4. Discussion 4.1. Phylogeny We found strong support for the following lineages recognized as genera: Afrotrichloris, Astrebla, Coelachyrum, Dinebra, Diplachne, Disakisperma, Eleusine, and Leptochloa. However, our Coelachyrum clade is paraphyletic because it includes Apochiton burttii in a subclade with C. longiglume. These two species share morphological synapomorphies of an annual habit, a paniculate inflorescence, and pedicelled spikelets, whereas the other three species of Coelachyrum are perennial and have a raceme inflorescence (the branches inserted digitally or along the main axis), and nearly sessile spikelets. Therefore, we make the new combination for Apochiton below and emend Coelachyrum to include short-awned lemmas and paleas, and trigonous caryopses. Chiovenda (1915) was first to recognize the affinities of Coelachyrum poiflorum by placing it in Eleusine. However, he mentions two other species, Eleusine somalensis Hack. and E. yemensis (Schweinf) Chiov. that, based on our earlier molecular studies, are placed in Dinebra and Disakisperma, respectively (Peterson et al., 2012; Snow et al., 2013). Coelachyrum poiflorum [≡ Eleusine poiflora (Chiov.) Chiov.] has hairy florets, villous lemmas villous with spreading hairs on the midvein and flanks below, and villous palea keels (Phillips, 1995). The only distinguishing character separating C. poiflorum This article is protected by copyright. All rights reserved. Author Manuscript from other species of Eleusine is hairy florets. Therefore, it seems best to align C. poiflorum within Eleusine, splitting at the same hierarchical level as Coelachyrum in our tree (Fig. 1A), rather than erect a new genus. Our new phylogeny of Eleusine is topologically similar to that found in Liu et al. (2011) in their investigation of low copy nuclear genes. However, we found a moderately supported E. jaegeri + E. multiflora clade (PP = 1, BS = 89), whereas Liu et al. (2011) found a strongly supported E. jaegeri + E. floccifolia clade (BS = 90−100, PP = 1) for both nuclear trees derived from EF-1a and Pepc4 markers. We did not include E. intermedia (Chiov.) S.M. Phillips in our current study, and this may have affected our topology. We verify for the first time using molecular data that Leptochloa longa aligns within Leptochloa and the number of species within the genus increases to eight. However, exact placement of L. longa within the Leptochloa clade is tentative because we were only able to sequence the rps3 marker for this species, although morphologically L. longa fits easily into the genus. Overall topology among the species of Leptochloa in our phylogeny is similar to that found in Peterson et al. (2015a). There are still three species that have historically been placed in Leptochloa: L. malayana (C.E. Hubb.) Jansen ex Veldkamp, L. monticola Chase, and L. tectoneticola (Backer) Jansen ex Veldkamp, that have not been included in a molecular DNA phylogeny and need further study. The unresolved placement of the Dinebra clade (inside or outside of the Eleusininae) has been documented previously in Peterson et al. (2012, 2015a, 2016). Incongruence between the plastid and nuclear DNA signal is common, especially within the Poaceae, This article is protected by copyright. All rights reserved. Author Manuscript and more study of Dinebra is necessary to untangle its evolutionary history, although it appears to be another case of hybridization and subsequent genomic introgression (Peterson et al., 2016). In addition, there are four species currently placed in Dinebra: D. perrieri (A. Camus) Bosser., D. polycarpha S.M. Phillips, D. simoniana (N. Snow) P.M. Peterson & N. Snow, and D. srilankensis (N. Snow) P.M. Peterson & N. Snow, that have not been included in a molecular DNA phylogeny and require further study. The inclusion of Afrotrichloris hyaloptera in our current study as a strongly supported sister to A. martinii provides evidence for retaining Afrotrichloris as a separate genus characterized in having a racemose inflorescence, 3−5-veined upper glumes shorter than the lemma, and deeply cleft lemma apices that can be awned (Watson & Dallwitz, 1992; Phillips, 1995; Clayton et al., 2006). Afrotrichloris shares many morphological features with Schoenefeldia, including, racemose inflorescences, long-awned lemmas, and caryopses with free pericarps. However, in our phylogeny of Schoenefeldia is paraphyletic, with the two species of Schoenefeldia in a grade sister to Afrotrichoris. We are left with two options to interpret the taxonomy of these four species: A) recognize all four species in Schoenefeldia or B) erect a new genus for S. transiens becasue it does not pair with the type, S. gracilis. We chose B and describe a new genus, Schoenefeldiella below. 4.2. Biogeography The Eleusininae (crown age of 25.85 Ma), a diverse assemblage now consisting of 28 genera with addition of Schoenefeldiella, that may have originated in Africa because it shares a common ancestor with subtribe Dactylocteniinae P.M. Peterson, Romasch. & Y. This article is protected by copyright. All rights reserved. Author Manuscript Herrera, also of African origins (Peterson et al., 2015a, 2016, in press). Both species of Afrotrichloris (African), five species of Coelachyrum (primarily African spreading to Asia), 10 of the 11 species of Eleusine Gaertn. (primarily African, Asian, and Australasian; only E. tristachya radiated to South America), and a single species each of Schoenefeldia and Schoenefeldiella occur in Africa (Liu et al., 2011; Clayton et al., 2016; Peterson et al., in press). Dinebra (±23 species, African, Australasian, and North American) also seems to have African origins as portrayed in our analyses. Dinebra chinensis (native to east Africa, Asia, Malaysia, Papua New Guinea, Phillipines, and Japan) is depicted as the basal split in our ITS-derived clade and in the plastid-derived A’ clade; whereas in the plastid-derived B’ clade the first bifurcation D. coerulescens (African) sister to remaining species and the second bifurcation D. haareri + D. somalensis (African) is sister to remaining species in the genus. The origins of Leptochloa is more difficult to speculate about because the basal species, L. digitata is endemic to Australia and shares a common ancestor with the remaining species of Leptochloa, many of which are widespread in the western hemisphere (4 spp.) or restricted to South America and/or the Caribbean (2 spp.), and Panama and Trinidad (1 spp.) [Snow et al., in prep]. As mentioned previously, there are still three species only tentatively placed in Leptochloa; two of these, L. malayana and L. tectoneticola from tropical Asia, Indo-China, and Malaysia were initially placed in Diplachne (Veldkamp, 1971; Clayton et al., 2016). 5. Taxonomy This article is protected by copyright. All rights reserved. Author Manuscript 5.1 Coelachyrum Hochst. & Nees, Linnaea 16(2): 221. 1842. gen. emend. Type: Coelachyrum brevifolium Hochst. & Nees. Description: Annuals or perennials, tufted, sometimes stoloniferous. Culms 7−90 cm long, geniculately ascending or decumbent, usually rooting at the lower nodes. Leaf sheaths glabrous; ligules membranous, sometimes with a ciliate fringe, apex usually truncate; blades linear or lanceolate, occasionally rounded or cordate near base, flat. Inflorescence a panicle with pedicelled spikelets (C. burttii & C. longiglume) or composed of racemes, these either digitately inserted or racemosely inserted along a main axis with nearly sessile spikelets. Spikelets 3−14-flowered, ovate, elliptic, or oblong, laterally compressed; disarticulation below the glumes, the spikelet falling as a unit; glumes shorter than the spikelet, 1−3 (−7)-veined, membranous, persistent, apex obtuse to acute, sometimes mucronate, and occasionally short-awned (C. burttii); lemmas 3veined, membranous, glabrous, asperulous, or hairy, apex truncate, obtuse or acute to mucronate, occasionally short-awned (C. burttii); paleas shorter than the lemma, 2keeled, the keels ciliate or glabrous, occasionally the keels extending into awns (C. burttii); stamens 3. Caryopses ellipsoid, ovoid, oblong or reniform, concavo-convex flattened or trigonous (C. burttii), the surface granular or rugose, pericarp free. Distribution: Five species comprise Coelachyrum, which are found in west and east tropical Africa and tropical Asia in Arabia, Pakistan, and India (Phillips, 1995; Cope, 2007). 5.2 Coelachyrum burttii (C.E. Hubb.) P.M. Peterson, comb. nov. ≡ Apochiton burttii C.E. Hubb., Hooker's Icon. Pl. 34: t. 3319. 1936. Type: Tanzania, Tanganyika Territory, This article is protected by copyright. All rights reserved. Author Manuscript Kondoa Irangi District near Sambala, 19 May 1929, B.D. Burtt 2602 (holotype: K000366648 [image!]; isotype: US-1646885!). 5.3 Eleusine Gaertn., Fruct. Sem. Pl. 1:7. 1788. gen. emend. Type: Eleusine coracana (L.) Gaertn. (≡ Cynosurus coracanus L.). Description: Tufted annuals or perennials, sometimes rhizomatous or stoloniferous. Culms 10−200 cm tall, erect, geniculately ascending or decumbent; internodes flattened and elliptical in cross section. Leaf sheaths usually shorter than the internodes, keeled; ligules membranous, apex truncate, ciliate; blades smooth, occasionally with scattered papillose-based hairs, mostly flat. Inflorescence with digitate, subdigitate, or with a main axis of racemes, the spikelets biseriate on one side of the triangular rachis, sessile. Spikelets 6–18-flowered, ovate, laterally compressed; disarticulation above the glumes and between the florets; glumes shorter than the spikelets, usually 1-veined but occasionally 2−7-veined, ovate, membranous, apex acute to obtuse, occasionally mucronate; lemmas 3-veined, membranous, the veins usually glabrous or occasionally with shaggy hairs below (E. poiflora), margins glabrous or sometimes hairy, apex acute, obtuse or truncate, occasionally mucronate; paleas shorter than the lemmas, 2-veined, the veins glabrous or occasionally with hairs (E. poiflora); stamens 3. Caryopses mostly trigonous but occasionally dorsally or laterally compressed or concavo-convex, the surface granular, striate or rugose, hilum punctiform, and pericarp free. Distribution: With the inclusion of Eleusine poiflora (≡ Coelachyrum poiflorum Chiov. = Coelachyrum stoloniferum C.E. Hubb.), there are now 11 species of Eleusine of which nine are found in Africa; of these nine E. indica is a pantropical weed and E. This article is protected by copyright. All rights reserved. Author Manuscript poiflora extends into Somalia, Djbouti, Saudi Arabia, Yemen, and Oman; E. tristachya is native to South America; and E. coracana (finger millet) is known only from cultivation (Phillips, 1972, 1995; Peterson et al., 2001, 2015; Cope, 2007). 5.4 Schoenefeldiella P.M. Peterson, gen. nov. – Type: Schoenefeldiella transiens (Pilg.) P.M. Peterson [≡ Schoenefeldia transiens (Pilg.) Chiov.]. Diagnosis: Schoenefeldiella differs from Schoenefeldia in having 2-flowed spikelets with the upper floret sterile and awned, the perennial habit, and flat leaf blades 5−35 cm long. Description: Plants perennial, tufted. Culms 70−120 cm tall, erect or geniculately ascending. Leaf blades 5−35 cm long, 1−5 mm wide, flat, scaberulous or smooth below, glabrous or loosely pilose. Inflorescence with 2−4 racemes digitately inserted; racemes 10−20 cm long, straight or flexuous, 1-sided and 2-rowed with sessile spikelets, rachis 0.5−0.8 mm wide; disarticulation below the spikelets. Spikelets 3.5−5 mm long, 2flowered, laterally compressed, the lower floret perfect the upper reduced (vestigial) and sterile; glumes 2−5 mm long, 1-veined; lower glume 1.5−3 mm long, awned, the awn 1−2.5 mm long; upper glumes 3.5−5 mm long, unawned; perfect lemmas 3−4 mm long, 3-veined, membranous, awned, margins ciliate, the awns 25−45 mm long usually twisting and closely appressed to the raceme, apically inserted between a minutely bifid apex; sterile lemmas 0.4−1 mm long, awned, the awn 10−25 mm long; callus pilose, obtuse; paleas hyaline, 2-veined; stamens 3, anthers 0.5−1 mm long. Caryopses ellipsoid, laterally compressed, hilum punctiform, pericarp free. This article is protected by copyright. All rights reserved. Author Manuscript Distribution: Schoenefeldiella transiens is found in is Eastern Africa from Ethiopia, South Sudan, Somalia, Uganda, Kenya, Tanzania, Zimbabwe, Mozambique, and South Africa (Hubbard, 1974b; Phillips, 1974, 1995; Gibbs Russell et al., 1990; Cope, 1999; Mashau & Götze, 2014). Schoenefeldiella transiens (Pilg.) P.M. Peterson, comb. nov. ≡ Chloris transiens Pilg., Bot. Jahrb. Syst. 51: 418. 1914 ≡ Schoenefeldia transiens (Pilg.) Chiov., Res. Sci. Somal. Ital. 1: 186. 1916 – Lectotype (designated by C.E. Hubbard in Fl. Trop. E. Afr., Gramineae 2: 309. 1974): TANZANIA. Pare District, Kwa Sengiwa-Maji ya juu, steppe below Kilimanjaro, 900 m, 14 Dec 1901, C. Uhlig 882 (B10 0002186 [image!]; isolectotype: EA00000056 [image!]). Etymology: The name is derived from Schoenefeldia because both genera share many morphological features. Acknowledgements We thank the National Geographic Society Committee for Research and Exploration (Grant No. 8848–10, 8087–06) for field and laboratory support, the Smithsonian Institution’s Restricted Endowments Fund, the Scholarly Studies Program, Research Opportunities, Atherton Seidell Foundation, Biodiversity Surveys and Inventories Program, Small Grants Program, the Laboratory of Analytical Biology, and the United States Department of Agriculture, all for financial support. We would also like to acknowledge Robert J. Soreng for many extended discussions pertinent to the manuscript and Jeffery M. Saarela and Neil Snow for providing helpful comments on the manuscript. References This article is protected by copyright. All rights reserved. Author Manuscript Chiovenda E. 1915. Planta novae vel minus notae e regione aethiopica. Annali di Botanica (Rome) 10: 371−415. Clayton WD. 1967. Studies in the Gramineae: XIII. Kew Bulletin 20: 99–110. Clayton WD, Renvoize SA. 1986. 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All rights reserved. Author Manuscript branches are posterior probabilities; numbers below branches are bootstrap values; color indicates where the sample was collected, usually indicative of native status; vertical bars indicate our classification; scale bar = 0.9% substitutions/site. Supplementary Material The following supplementary material is available online for this article at http://onlinelibrary.wiley.com/doi/XXXXX/suppinfo: Appendix S1. Taxon voucher (collector, number, and where the specimen is housed), country of origin, and GenBank accession for DNA sequences of rps3, rps16-trnK, rps16 intron, rpoC2, rpl32-trnL, ndhF, ndhA intron, ccsA, and ITS regions (bold indicates new accession); a dash (–) indicates missing data; an asterisk (*) indicates sequences not generated in our lab. Table 1. Characteristics of the nine regions, rps3, rps16-trnK, rps16 intron, rpoC2, rpL32-trnL, ndhF, ndhA intron, ccsA, and ITS, and parameters used in Bayesian analyses indicated by Akaike Information Criterion (AIC). Total aligned characters Number of sequences Likelihood score (lnL) ITS Ove rall 1150 954 6949 746 769 5 73 80 70 690 135 825 30 3 13 (41%) (4%) (19%) rps3 ndhF ndhA intron 612 107 9 898 808 884 765 77 101 92 77 120 5 3 43 (3%) (56%) (8% ) 1804. 4012 3463. 51 .8 46 3041. 68 456 3779.9 4493. 3048.4 2.75 0 11 5 47 Number of new sequences ccsA Comb ined plasti d data rps1 rpL3 rps16 6rpoC2 2intron trnK trnL (61% (5%) ) 10 This article is protected by copyright. All rights reserved. 154 15 167 (22% (20 (11%) ) %) 11973 .22 Author Manuscript Number of substitution types 6 Model for amongsites rate variation gam ma 6 6 6 6 6 6 6 gam gamm gamm gam gamm gamm gamm ma a a ma a a a - 6 - - gamm a - 1.673 1.37 64 867 1.176 20 3.507 34 0.97 2.1864 1.382 1.4828 820 0 02 2 1.269 70 3.415 2.83 39 812 1.138 39 5.160 54 1.93 3.0386 3.410 3.9574 638 7 70 7 3.244 44 0.486 0.47 63 934 0.256 02 0.303 43 0.36 0.5267 0.705 0.5945 141 5 11 5 1.925 07 0.768 1.70 92 272 1.007 98 2.045 51 1.03 1.6527 2.762 1.2725 125 2 00 5 0.954 72 4.822 2.86 23 646 1.926 45 10.40 906 1.52 3.9414 3.614 3.9789 720 3 63 1 4.871 50 1.000 1.00 00 000 1.000 00 1.000 00 1.00 1.0000 1.000 1.0000 000 0 00 0 1.000 00 0.432 0.30 86 958 0.392 82 0.397 45 0.37 0.3268 0.361 0.3003 470 0 19 2 0.233 98 0.151 0.14 27 992 0.107 61 0.145 83 0.13 0.1286 0.136 0.1588 243 9 61 2 0.208 19 0.168 0.14 85 703 0.166 87 0.234 01 0.12 0.1703 0.147 0.1622 790 4 84 7 0.250 67 0.247 0.39 01 348 0.332 71 0.222 72 0.36 0.3741 0.354 0.3786 497 7 36 0 0.307 16 Proportion of invariable sites 0.687 0.10 99 426 0.333 35 0.000 00 0.18 0.2954 0.345 0.5469 741 4 23 9 Substitution model TVM +G TV M+ G GTR +I+G TIM3 GT TPM3 +I+G R+G uf+G Gamma shape parameter (α) 0.792 1.05 707 499 0.970 74 0.570 86 Substitution rates Character state frequencies - - - - - 0.239 03 - TPM3 uf+G - GTR +I+G - 0.99 0.7835 0.959 0.9667 039 5 69 2 - 0.970 93 - This article is protected by copyright. All rights reserved. TVM +I+G