﻿A phylogeny of the Triraphideae including Habrochloa and Nematopoa (Poaceae, Chloridoideae)

﻿Abstract To investigate the evolutionary relationships among species of the tribe Triraphideae (including two monotypic genera, Habrochloa and Nematopoa), we generated a phylogeny based on DNA sequences from nuclear ribosomal (ITS) and four plastid markers (rps16-trnK, rps16 intron, rpl32-trnL, and ndhA intron). Habrochloa and Nematopoa form a clade that is sister to Neyraudia and Triraphis. Member of the Triraphideae have paniculate inflorescences, 3-veined, marginally ciliate lemmas, usually with hairy lateral veins, that are apically bifid and awned from between a sinus. A description of the Triraphideae and key to the genera is provided, and the biogeography is discussed, likely originating in Africa.


Introduction
pointed out that Neyraudia R. Br. was perhaps an ally of Triraphis R. Br. since both genera possess slender microhairs and the two have keeled lemmas that are villous on the lateral veins . Based on DNA sequence studies Bouchenak-Khelladi et al. (2008) were first to show strong support for Neyraudia and Triraphis as being sister in the subfamily Chloridoideae Kunth ex Beilschm. Hilu and Alice (2001) and Bouchenak-Khelladi et al. (2008), using the same matK sequence marker placed these two genera in the subtribe Uniolinae Clayton, now a member of tribe Eragrostideae Stapf. Another DNA sequence study supported the placement of the Neyraudia-Triraphis clade as being sister to remaining species in the Chloridoideae and, subsequently, the tribe Triraphideae P.M. Peterson [based on subtribe Triraphidinae Stapf (1917)] was erected to include these two genera (Peterson et al. 2010). Using unpublished DNA sequence phylogenies (Peterson and Romaschenko, unpubl.), the monotypic Habrochloa C.E. Hubb., was added to the Triraphideae in the classification of all genera within the Poaceae (Soreng et al. 2015(Soreng et al. , 2017. Hubbard (1935Hubbard ( , 1957a transferred Triraphis longipes Stapf & C.E. Hubb. to Crinipes Hochst. (Arundinoideae) since it possessed a bearded callus, then later moved it to a new monotypic genus, Nematopoa C.E. Hubb. Nematopoa was included in the Arundinoideae by Clayton and Renvoize (1986). In more recent classifications (Soreng et al. 2015(Soreng et al. , 2017, Nematopoa longipes (Stapf & C.E. Hubb.) C.E. Hubb. was placed as a synonym of Triraphis as originally described. Based on unpublished DNA sequence phylogenies (Peterson and Romaschenko, unpubl.), Soreng et al. (2022) and Gallaher et al. (2022) placed Nematopoa in the Triraphideae. Therefore, the current concept of the Triraphideae consists of four genera, Habrochloa, Nematopoa, Neyraudia, and Triraphis.
In the present phylogenetic study, using DNA sequences from nuclear ribosomal (ITS) and four plastid markers (rps16-trnK, rps16 intron, rpl32-trnL, and ndhA intron), we include for the first time Habrochloa bullockii, Nematopoa longipes, and Neyraudia arundinacea (L.) Henrard with two other species of Neyraudia and five species of Triraphis. In addition, we include a description of the Triraphideae, key to the genera in the tribe, and hypothesize its biogeographical history.

Materials and methods
Detailed methods for DNA extraction, amplification, and sequencing are given in Romaschenko et al. (2012) and Peterson et al. (2010Peterson et al. ( , 2014aPeterson et al. ( , b, 2015aPeterson et al. ( , b, 2016. We used Geneious Prime 2020 (Kearse et al. 2012) for contig assembly of bidirectional sequences of ndhA intron, rpl32-trnL, rps16 intron, rps16-trnK and ITS regions, and implemented in Geneious Muscle algorithm (Edgar 2004) to align the sequences and adjust the final alignment. The maximum likelihood parameters for each region were estimated with GARLI 2.0 (Zwickl 2006) and were used as priors in Bayesian calculations to infer overall phylogeny. The Bayesian tree was constructed using MrBayes v3.2.7 (Huelsenbeck and Ronquist 2001;Ronquist et al. 2012). All compatible branches were saved. The Bayesian analysis was initiated with random starting trees sampling once per 100 generations and continued until the value of the standard deviation of split sequences dropped below 0.01 indicating convergence of the chains. The effective sample size (ESS) value for all the parameters was greater than 200 and the first 25% of the sampled values were discarded. Maximum likelihood bootstrap analyses (Felsenstein 1985) were performed using GARLI with 1000 repetitions. The resulted file containing 1000 trees from the bootstrap analysis was then read into PAUP* v.5.0 (Swofford 2000) to compute the majority rule consensus tree.
Our study was designed to test relationships among species residing in four genera (Habrochloa, Nematopoa, Neyraudia, and Triraphis) attributed to the Triraphideae. Representative species from all remaining tribes (Centropodieae P.M. Peterson, N.P. Barker & H.P. Linder, Cynodonteae Dumort., Eragrostideae Stapf, and Zoysieae Benth.) in the Chloridoideae have been included to test the monophyly of the tribe (Peterson et al. 2010). In addition, the phylogeny includes two species from the Danthonioideae, Danthonia compressa Austin and Merxmuellera drakensbergensis (Schweick.) Conert, and one species from the Panicoideae, Chasmanthium latifolium (Michx.) H.O. Yates, which was used as an outgroup.

Results and discussion
Thirty-five new sequences (16%) from five species (nine individuals) are newly reported in GenBank, along with all other sequences for 48 individuals and 41 species included in this study (Table 1). Total aligned characters, numbers of sequences, proportion of invariable sites, and other parameters are noted in Table 2. The resulting plastid and ITS topologies were inspected for conflicting nodes with ≥ 95% posterior probabilities. No supported conflict was found so plastid and ITS sequences were combined.
The Bayesian tree from the combined plastid and ITS regions is well resolved (Fig. 1). Most clades have posterior probabilities equal to 1.00 and additional bootstrap values of 90% or greater. There is strong support for Habrochloa bullockii + Nematopoa longipes sister to a monophyletic Neyraudia with three individuals of N. reynaudiana (Kunth) Keng ex Hitchc. sister to one individual of N. arundinacea (type of the genus) Table 1. Taxon voucher (collector, number, and where the specimen is housed), country of origin, and GenBank accession for DNA sequences of rps16-trnK, rps16 intron, rpl32-trnL, ndhA intron, and ITS regions; bold indicates new accession; a dash (-) indicates missing data, an asterisk (*) indicates sequences not generated in our lab.  Burbidge 1946 andPeterson et al. 2022) sister to T. schinzii Hack. and T. ramosissima Hack. sister to T. andropogonoides (Steud.) E. Phillips + T. purpurea Hack. Our molecular data clearly support independent recognition of Nematopoa since it is sister to Habrochloa and not a member of the Triraphis clade.
Habrochloa bullockii and Nematopoa longipes are clearly aligned within the Triraphideae, and together with Neyraudia and Triraphis, share the following salient morphological features: paniculate inflorescences, 3-veined, marginally ciliate lemmas, usually with hairy lateral veins, and lemmas that are apically bifid and awned from between the sinus Peterson et al. 2010;Clayton et al. 2016). Another probable synapomorphy for these four genera is possession of panicoid-type bicellular microhairs (long, narrow basal and terminal cells; Amarasinghe and Watson 1988).  verified the presence of panicoid bicellular microhairs for Habrochloa, Nematopoa, and Triraphis but indicate that they are absent in Neyraudia arundinacea. However, Clayton and Renvoize (1986) previously indicated that Neyraudia possesses slender microhairs similar to those in Triraphis.
Based on a sample containing Nematopoa, Neyraudia, and Triraphis, Gallaher et al. (2022) determined the crown age (10.62 Ma) and stem age (46.76 Ma) of the Triraphideae. Although at least three species of Neyraudia include tropical and temperate Asia in their distribution, Africa is the most likely area of origin for the Triraphideae since all four genera in the tribe include species distributed in Africa. In addition, the Triraphideae shares a common ancestor with Centropodieae, also from Africa and temperate Asia (Peterson et al. 2011). Because more than half of the genera of Chloridoideae reside in Africa and the larger tribes, i.e., the Eragrostideae Table 2. Characteristics of the five DNA regions (rps16-trnK, rps16 intron, rpl32-trnL, ndhA and ITS) and parameters used as priors in Bayesian analyses estimated with GARLI. 2.0.  Peterson et al. 2007Peterson et al. , 2010Peterson et al. , 2011Peterson et al. , 2014c.