Received: 24 Jun 2013 | returned for revision: 30 Sep 2013 | revision received: 23 Jan 2014 | accepted: 27 Jan 2014 | not published online ahead of inclusion in print and online issues || © International Association for Plant Taxonomy (IAPT) 2014
INTRODUCTION
The species in subfamily Chloridoideae Kunth ex Beil
schm. and tribe Cynodonteae Dumort. share two structural traits: all exhibit Kranz or C4 leaf anatomy (except two spe
cies of Ellisochloa P.M.Peterson & N.P.Barker from southern Africa; Peterson & al., 2011) and most have chloridoid bicel
lular microhairs (broad, short terminal cell the same thickness as the basal cell) present on leaf surfaces (Peterson & al., 2007, 2010a). Based on leaf anatomical structure, two main subtypes of C4 photosynthesis—classical NADME and PCK—have been observed (Hattersley & Watson, 1992). The Cynodonteae currently consists of at least 78 genera and there appear to be no definitive morphological features that differentiate Cyno
donteae from Chloridoideae since most of the structural varia
tion in the entire subfamily is exhibited in the tribe. However, Cynodonteae receive moderate to high support as a mono
phyletic lineage in molecular analyses (Hilu & Alice, 2001;
Columbus & al., 2007; Peterson & al., 2010a).
Reveal (2004) pointed out that Martinov (1820) used Chlor
ideae as a tribal name. However, Martinov’s “Chlorideae” was
not validly published as it corresponds to the fifth order in Kunth’s (1815) system, as one of 10 sections, an order or sec
tion within a family being an example of a misplaced rank
denoting term (Art. 5.1 & 37.6 of the ICN, McNeill & al., 2012).
Therefore, we revert back to next oldest name, Cynodonteae Dumort.
Recent molecular DNA studies within the subfamily Chloridoideae, specifically in the tribe Cynodonteae, have not been exhaustive, and they lack an adequate sample of species to assess phylogenetic relationships and determine monophyly of the genera (Hilu & Alice, 2001; Columbus
& al., 2007; BouchenakKhelladi & al., 2008; Peterson & al., 2010a). The most comprehensive estimates of the phylogeny of the Chloridoideae to date have been presented in Peterson
& al. (2010a) where 246 species included in 95 genera were sampled for seven markers (ndhA intron, ndhF, rps16-trnK, rps16 intron, rps3, rpl32-trnL, ITS). The resulting classifica
tion from this work recognized 13 subtribes within the Cyno
donteae. Gouinia E.Fourn. ex Benth. & Hook.f., Schencko- chloa J.J.Ortiz (included in Gouinia), Triplasis P.Beauv., and Vaseyochloa Hitchc. were placed in subtribe Tridentinae
A molecular phylogeny and classification of the Cteniinae, Farragininae, Gouiniinae, Gymnopogoninae, Perotidinae, and Trichoneurinae (Poaceae: Chloridoideae: Cynodonteae)
Paul M. Peterson,1 Konstantin Romaschenko1,2 & Yolanda Herrera Arrieta3
1 Smithsonian Institution, Department of Botany, National Museum of Natural History, Washington D.C. 20013-7012, U.S.A.
2 M.G. Kholodny Institute of Botany, National Academy of Sciences, Kiev 01601, Ukraine 3 Instituto Politécnico Nacional, CIIDIR Unidad Durango-COFAA, Durango, Mexico Author for correspondence: Paul M. Peterson, [email protected]
DOI http://dx.doi.org/10.12705/632.35
Abstract The classification of twenty Cynodonteae genera (Bewsia, Craspedorhachis, Ctenium, Dignathia, Farrago, Gouinia, Gymnopogon, Latipes, Leptocarydion, Leptothrium, Lophacme, Lopholepis, Mosdenia, Perotis, Schenckochloa, Toliara, Trichoneura, Trigonochloa, Triplasis, Vaseyochloa) has been poorly understood. The goals of this study were to reconstruct the evolutionary history of the tribe Cynodonteae, emphasizing these twenty genera using molecular data with increased species sampling. A phylogenetic analysis was conducted on 159 samples, of which 94 species (140 individuals) were in the Cynodonteae, using four plastid (rpl32-trnL spacer, ndhA intron, rps16-trnK spacer, rps16 intron) and nuclear ITS 1&2 (ribo
somal internal transcribed spacer regions) sequences to infer evolutionary relationships and revise the classification. Strong support was found for six lineages. Craspedorhachis, Ctenium, Dignathia, Gymnopogon, Trichoneura, and Triplasis appear monophyletic; Leptothrium is paraphyletic with Latipes inermis embedded within; Perotis is paraphyletic with Lopholepis and Toliara embedded within; and Gouinia is monophyletic with the resurrection of Schenckochloa. The molecular results sup
port the recognition of six subtribes; five (Cteniinae, Farragininae, Gymnopogoninae, Perotidinae, Trichoneurinae) are newly described. A new genus, Tridentopsis, is described; five new combinations, Leptothrium inerme, Perotis arenacea, Perotis ornithocephala, Tridentopsis eragrostoides, and Tridentopsis mutica are made; and Holboellia ornithocephala is lectotypified.
Keywords classification; ITS; phylogeny; plastid DNA sequences; Tridentopsis
Supplementary Material The Electronic Supplement (with Appendix S1) and the alignments are available in the Supplementary Data section of the online version of this article at http://ingentaconnect.com/content/iapt/tax
Keng & Keng f. In that same classification, Farrago Clay
ton, Latipes Kunth (included in Leptothrium Kunth), Lepto- carydion Hochst. ex Stapf, Leptothrium, and Lophacme Stapf (sometimes erroneously spelled Lophachme) were placed in Chloridoideae–incertae sedis (not yet placed in a tribe); and Bewsia Goossens, Craspedorhachis Benth., Ctenium Panz., Dignathia Stapf, Gymnopogon P.Beauv., Lopholepis Decne., Mosdenia Stent, Perotis Aiton, and Trichoneura Andersson were placed in the Cynodonteae–incertae sedis (not yet placed in a subtribe). Toliara Judz. and Trigonochloa P.M.Peterson
& N.Snow are newly described genera and have been shown to have affinities to Perotis (Judziewicz, 2009; Peterson & al., 2012; Snow & Peterson, 2012).
The aim of our paper is to discern the evolutionary re
lationships and present a new classification of 20 target gen
era. These 20 genera of uncertain affinities include: Bewsia (monotypic, Africa), Craspedorhachis (3 species, Africa), Ctenium (20 species, Africa, South America, North America, Asia), Dignathia (5 species, Africa, Asia), Farrago (monotypic, endemic to Tanzania), Gouinia (10 species, South and North America; including Schenckochloa), Gymnopogon (14 spe
cies, South and North America, Asia), Leptocarydion (mono
typic, Africa and Asia), Leptothrium (3 species, Asia, Africa, and South America; including Latipes), Lophacme (2 species, Africa), Lopholepis (monotypic, Asia), Mosdenia (monotypic, Africa), Perotis (14 species, Africa, Asia, Australasia), Toliara (monotypic, endemic to Madagascar), Trichoneura (8 species, Africa, South and North America, Asia), Trigonochloa (2 spe
cies, Africa and Asia), Triplasis (2 species, North and South America), and Vaseyochloa (monotypic, endemic to Texas, U.S.A.) (Smith, 1971; Clayton & al., 1974, 2006; Gibbs Russell
& al., 1991; Phillips, 1995; Cope, 1999; Raimondo, 2001). In addition to these 20 target genera, we sample 37 species that represent 12 subtribes known to be included in the Cynodon
teae (Peterson & al., 2010a, 2011, 2012).
Using plastid (rpl32-trnL, ndhA, rps16-trnK, rps16) and nuclear ITS DNA sequence markers, we present a new phyloge
netic analysis of Cynodonteae that includes these 20 genera that were previously assigned to either the Cynodonteae or Chlorid
oideae. The number of species sampled within many of these 20 genera is expanded and for the first time we are including species of Craspedorhachis, Farrago, Latipes, Leptocarydion, Leptothrium, and Schenckochloa (Peterson & al., 2010a, 2012).
In addition, we discuss morphological and anatomical charac
ters supporting relationships and propose changes to the clas
sification.
MATERIALS AND METHODS
Taxon sampling. — The taxon sampling consists of 159 samples, representing 113 species of grasses, of which 110 species are included in subfamily Chloridoideae; these are partitioned to represent the following five tribes and sub
tribes: Centropodieae with a single species, Triraphideae with two species, Eragrostideae with eight species, Zoysieae with five species, Cynodonteae: Tripogoninae with two species,
Pappophorinae with three species, Traginae with two species, Aeluropodinae with three species, Hilariinae with two species, Muhlenbergiinae with two species, Scleropogoninae with a single species, Boutelouinae with a single species, Monantho
chloinae with a single species, Triodiinae with a single spe
cies, Eleusininae with thirteen species, and Orcuttiinae with five species (see Electr. Suppl.: Appendix S1). We removed the following genera from our study because they did not align within or among clades containing the 20 target genera:
Allo lepis Soderst. & H.F.Decker, Brachychloa S.M.Phillips, Cleistogenes Keng, Dactyloctenium Willd., Jouvea E.Fourn., Lepturidium Hitchc. & Ekman, Neobouteloua Gould, Orinus Hitchc., Pogononeura Napper, and Sohnsia Airy Shaw; and we lack leaf material for two species placed in the Cynodon
teae–incertae sedis: Hubbardochloa Auquier and Kampo- chloa Clayton, and Oxychloris Lazarides which lies within the Eleusininae (Peterson & al., in prep.; Soreng & al., 2013, see suprageneric classification). Our sampling was primar
ily focused on 20 genera (Bewsia, Craspedorhachis, Ctenium, Dignathia, Farrago, Gouinia, Gymnopogon, Latipes, Lepto- carydion, Leptothrium, Lophacme, Lopholepis, Mosdenia, Perotis, Schenckochloa, Toliara, Trichoneura, Trigonochloa, Triplasis, Vaseyochloa) that are morphologically similar, and most of these genera have been previously aligned near one another (Peterson & al., 2010a). The dataset for these 20 gen
era includes sixtyfive percent (58/89) of the species currently placed in these genera by Clayton & al. (2006). A complete list of taxa, voucher information, and GenBank numbers can be found in Appendix S1 (Electr. Suppl.). Outside of the Chlorid
oideae, one species of Danthonioideae (Danthonia compressa Austin), one species from Aristidoideae (Aristida gypsophila Beetle), and one species of Panicoideae (Chasmanthium lati- folium (Michx.) H.O.Yates, phylogenetic root) were chosen as the outgroup. All collections gathered by PMP after 1998 were collected in silica but the majority of samples used in this study were selected from herbarium specimens.
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 singlecopy region), rps16-trnK spacer and rps16 intron (large singlecopy region), and ITS was accomplished following procedures outlined in Peterson & al. (2010a, b). We specifically targeted four of the plastid regions which proved to be most informative in our previous studies on chloridoid grasses (Peterson & al., 2010a, b, 2011, 2012).
Phylogenetic analyses. — We used Geneious v.5.3.4 (Drummond & al., 2011) for contig assembly of bidirectional sequences of rpl32-trnL, ndhA intron, rps16 intron, rps16-trnK, and ITS regions, and we used Muscle (Edgar, 2004) to align consensus sequences and adjust the final alignment (Supple
mentary Data includes sequence alignments). We identified models of molecular evolution for the cpDNA and nrDNA regions using jModelTest v.0.1.1 (Posada, 2008). We applied maximumlikelihood (ML) and Bayesian searches to infer overall phylogeny. The combined datasets were partitioned in accordance with the number of the markers used. Nucleotide
substitution models selected by Akaike’s information criterion, as implemented in jModelTest, were specified for each partition (Table 1). The ML analysis was conducted with GARLI v.0.951 (Zwickl, 2006). We used the standard option in PAUP* v.4.0b10 (Swofford, 2000) to generate the number of the parsimony in
formative characters for the five DNA regions (Table 1). 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 majorityrule consensus tree was constructed. Bootstrap (BS) values of 90%–100%
were interpreted as strong support, 70%–89% as moderate, and 50%–69% as weak. We also calculated the uncorrected pvalue in PAUP to demonstrate the sequence divergence among mem
bers of Leptothrium and Latipes.
Bayesian posterior probabilities (PP) were estimated using parallel version of the MrBayes v.3.1.2 (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 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 burnin was set at 0.25.
Posterior probabilities (PP) of 0.95–1.00 were considered as strong support.
In the phylograms the native distribution of each species is indicated as follows: North America (red), South America (tan), Central Africa (Tanzania, Kenya; dark green), Southern Africa (light green), and Southeast Asia (purple).
RESULTS
Phylogenetic analyses. — Fortyeight percent (320/660) of the sequences used in our study are newly reported here and in GenBank, and only 16.7% (132/792) are missing (Electr.
Suppl.: Appendix S1). Total aligned characters for individual regions are noted in Table 1. Plastid rpl32-trnL had the highest sequencing success of 94.3% of taxa recovered across the entire dataset. Recovery of other regions ranged from 66% to 90%.
Analysis of combined plastid and ITS sequences. — There was hard incongruence within the Gymnopogoninae clade be
tween the plastid (combined) and ITS tree. The plastid tree depicts a monophyletic Gymnopogon (BS = 100, PP = 1.00), whereas the ITS tree renders Gymnopogon paraphyletic and in
cludes Bewsia and Lophacme embedded within Gymnopogon.
Therefore, in our combined plastid–ITS analysis we removed
Table 1. Summary of the four plastid regions and nrDNA ITS used in maximumlikelihood and Bayesian searches indicated by Akaike’s information criterion.
Characteristic rpl32-trnL ndhA
intron rps16
intron rps16-trnK
Combined plastid
data ITS
Overall combined dataset
Total aligned characters 991 1124 989 954 4158 822 4980
Sequencing success (%) 94.3 66 80 85 82 90 83
Number of new sequences 78 (52%) 49 (47%) 65 (52%) 68 (51%) 260 (51%) 78 (55%) 338 (52%)
Likelihood score (−lnL) 7224.00 7181.83 5282.39 6408.54 17040.45
Number of substitution types 6 6 6 6 – 6
Model for amongsite rate variation gamma gamma gamma gamma – gamma
Substitution rates 1.2412
2.0479 0.4612 1.6880 1.7126 1.0000
1.2742 2.6666 0.5249 1.6465 2.525 1.0000
1.0553 1.3476 0.2844 1.2456 1.7866 1.0000
1.0989 2.7853 0.4664 1.4675 2.3991 1.0000
– 1.2515
2.9776 1.7603 0.8442 5.0446 1.0000 Character state frequencies 0.3599
0.1355 0.1315 0.3730
0.3665 0.1373 0.1484 0.3476
0.3914 0.1097 0.1646 0.3341
0.3006 0.1383 0.1421 0.4188
– 0.2450
0.2053 0.2457 0.3039
Proportion of invariable sites 0.1858 0.2778 0.2883 0.0378 – 0.2547
Substitution model GTR + I + G TVM + G TIM3 + I + G TIM3 + G – GTR + I + G
Gamma shape parameter (α) 1.2015 1.3218 1.6015 1.2233 – 1.0682
Number of parsimonyinformative charactersa 251 259 184 259 953 328 1281
a Supplementary data from parsimony analysis.
0.03
Perotis leptopus Ctenium newtonii Baldwin 13423
Trichoneura mollis Napper 550 Trichoneura grandiglumis Reihmer 6040 Schenckochloa barbata
Craspedorhachis rhodesiana
Latipes inermis = Leptothrium inerme Trichoneura grandiglumis Smook 7068
Ctenium brevispicatum Filgueiras 3251 Ctenium aromaticum
Gymnopogon chapmanianus
Mosdenia leptostachys Smook 1145 Tridens flavus
Gouinia virgata var. virgata (as G. longiramea) Swallen 2818
Leptocarydion vulpiastrum Peterson 24238
Gymnopogon ambiguus Thomas 168039 Leptocarydion vulpiastrum Peterson 23863
Trichoneura mollis Bogdan 4173
Lopholepis ornithocephala Trichoneura lindleyana Howell 9435
Gymnopogon grandiflorus Gymnopogon foliosus Gouinia gracilis
Leptothrium senegalense Belsky 336 Trichoneura elegans Silveus 812
Leptocarydion vulpiastrum Mboya 565
Gymnopogon floridanus
Farrago racemosa
Gymnopogon spicatus Quarin 4144
Craspedorhachis africana Litt 125 Trichoneura eleusinoides subsp. limpopoensis Pienaar 394
Leptocarydion vulpiastrum Bidgood 50038 Trichoneura eleusinoides Seydel 448
Perotis vaginata Gouinia paraguayensis
Dignathia hirtella Maggi 353
Ctenium newtonii Laegaard 18327 Gouinia virgata var. robusta
Trichoneura elegans Silveus 7312 Gouinia virgata var. virgata Villareal 8105
Gymnopogon ambiguus Becker 1488 Gouinia latifolia var. guatemalensis Martinez 29764
Dignathia gracilis
Leptothrium senegalense Peterson 24239
Gymnopogon burchellii Swallen 8524 Lophacme digitata Gouinia virgata var. virgata Rzedowski 14670
Mosdenia leptostachys Scheickerdt 1542
Perotis patens Klaassen 1305 Ctenium newtonii var. productum
Dignathia hirtella Barkhadle 353 Tridens muticus Gould 9978
Trichoneura weberbaueri Tridens flavus var. chapmanii
Leptothrium senegalense Laegaard17937
Gymnopogon brevifolius Peterson 24437 Perotis indica Peterson 23880
Craspedorhachis africana Laegaard 16193
Perotis somalensis
Gymnopogon aristiglumis
Bewsia biflora Davidse 6471
Craspedorhachis rhodesiana
Dignathia villosa
Bewsia biflora Peterson 23905 Trichoneura elegans Lundell 15030
Leptothrium senegalense Peterson 24196 Dignathia hirtella McCallum Webster 5251 Gouinia latifolia var. guatemalensis Beetle 918
Perotis patens Godfrey 1661 Ctenium somalense Gouinia brasiliensis
Vaseyochloa multinervosa
Gymnopogon spicatus Renvoize 3677
Craspedorhachis africana Fisher 504
Gymnopogon legrandii Triplasis americana
Trichoneura lindleyana Bentley 319
Trichoneura eleusinoides subsp. limpopoensis Davidse 5859 Tridens muticus Peterson 21997
Ctenium cirrhosum
Perotis hildebrandtii Peterson 23814
Gymnopogon brevisetus
Perotis patens Moss 11760
Craspedorhachis africana Schweickerdt 484 Craspedorhachis rhodesiana
Ctenium elegans
Perotis hildebrandtii Renvoize 1784
Craspedorhachis africana Fisher 459
Toliara arenacea
Perotis indica Peterson 23872 Perotis rara
Leptothrium rigidum Triplasis purpurea Peterson 14238
Ctenium brevispicatum Filgueiras s.n.
Trichoneura grandiglumis Smook 4896 Neesiochloa barbata
Triplasis purpurea Peterson 24420
Perotis hordeiformis
Gymnopogon brevifolius Hill 15844 Trichoneura eleusinoides Oliver 6696
Trigonochloa uniflora Snow 6978
Gymnopogon ambiguus Nelson 1392 Trigonochloa uniflora Greenway14075
Perotis patens Kruger 21750 Pappophorum pappiferum
Gymnopogon swallenii
Craspedorhachis rhodesiana
= Perotis ornithocephala
= Perotis arenacea
OUTGROUP
Cynodonteae
TRIODIINAE
TRIPOGONINAE
ORCUTTIINAE
HILARIINAE
SCLEROPOGONINAE MUHLENBERGIINAE
TRAGINAE
Triraphideae Eragrostideae
Zoysieae
PAPPOPHORINAE
BOUTELOUINAE
82 1.00
100 1.00
100 1.00
0.61 84 1.00 93
1.00 89 1.000.73
MONANTHOCHLOINAE
0.75 1.00 100
1.00
0.99
0.97
1.00 1.00
1.00 1.00
1.00
0.94 1.00
0.98
0.92
1.00 1.001.00
1.00 1.00 1.001.00
1.00
1.00 1.00
1.00
1.00 0.99
1.00
1.00 1.00
1.00
1.00
1.00
0.98
1.00 1.00
1.00
1.00
1.00
1.00 1.00
0.69 1.00
0.91
ELEUSININAE
GOUINIINAE
1.00
1.00 1.00
0.54 1.00 1.00
1.00 1.00 0.94
1.000.94 1.00
1.00
0.99 0.70
0.99 1.00
1.00 0.99 1.001.00
0.99 0.98
1.00
1.00
1.00 0.84
1.00 1.00
1.00
0.990.79 0.56
0.74
0.99 1.00
1.00
1.00
0.99
1.00 1.000.54
0.75
0.980.86
0.66 1.00
0.60
1.001.00
0.59 1.00
56 98
100 100
62 100
75
68
69
95 95
97 97
68 100
55 97
64 79
95 5776
86 57 100
58
99 98 68
100
100
100 99
89 81
86 67
60 90
60 65 68 89
6868 59
100 97
73 100 63 100
65
100 95
98
100
84 86
98 100
8167
67
100 100
9468 68
100 92
100
100 86 73
67 8383
81
100
100
100
100 54
8384
98 94 100
CTENIINAE
TRICHONEURINAE
FARRAGININAE
PEROTIDINAE
GYMNOPOGONINAE Centropodieae
100 1.00
1.00 0.9790
100
= Tridentopsis mutica
North America
Central Africa Southern Africa Southeast Asia South America
AELUROPODINAE
0.96
Fig. 1. Phylogram of maximumlikelihood tree from analysis of combined plastid and ITS sequences. Numbers above branches are bootstrap values; numbers below branches are posterior probabili
ties; clades outlined in dashed lines are discussed in text; scale bar = 3% sequence divergence: color indicates native distri
bution (see legend).
ITS sequences of Bewsia and Lophacme. There were no other examples of conflict between the two datasets.
The ML tree from the combined plastid (rpl32-trnL, ndhA, rps16-trnK, rps16) and ITS regions (Fig. 1) is well resolved with strong support for the six major clades labeled as: Cteniinae, Farragininae, Gymnopogoninae, Perotidinae, and Trichoneur
inae (all at BS = 100, PP =1.00) and Gouiniinae (BS = 95, PP
= 1.00). The FarragininaePerotidinae clade (BS = 65, PP = 0.84) is sister to the Gymnopogoninae clade (BS = 100, PP = 1.00); these are sister (BS = 68, PP = 0.98) to the Cteniinae
Trichoneur inae clade (BS = 58, PP = 0.98); and all five sub
tribes are sister with strong support (BS = 100, PP = 1.00) to the Gouiniinae. The Orcuttiinae is sister to these six major clades that form a monophyletic lineage (BS = 0.95, PP = 1.00).
The Gouiniinae clade consists of a monophyletic Gouinia (BS = 97, PP = 1.00) sister to Vaseyochloa multinervosa; these are sister (BS = 55, PP =0.54) to Tridens muticus (BS = 100, PP = 1.00). Tridens flavus L., the type for the genus, is sis
ter to Pappophorum pappiferum (Lam.) Kuntze and is clearly aligned with the Pappophorinae. Schenckochloa barbata is sister to the Gouinia-Vaseyochloa-Tridens clade (BS = 100, PP = 1.00), and all are sister (BS = 0.68, PP = 0.94) to a mono
phyletic Triplasis (BS = 97, PP = 1.00). Within Gouinia: the G. brasiliensis–G. paraguayensis (BS = 79, PP = 1.00) pair is sister to G. latifolia–G. virgata (BS = 57, PP = 0.94).
The Cteniinae clade consists of a monophyletic Ctenium with two distinct subclades: C. newtonii–C. elegans–C. soma - lense (BS = 100, PP = 1.00) sister to C. aromaticum–C. cirrho- sum–C. brevispicatum (BS = 99, PP =1.00).
The Trichoneurinae clade consists of a monophyletic Trichoneura with three distinct subclades with four accessions of T. eleusinoides (BS = 86, PP = 0.99) sister to the T. gran- diglumis–T. mollis (BS = 90, PP = 1.00) and T. weberbaueri–
T. lindleyana–T. elegans (BS = 60, PP = 1.00) clade.
The Farragininae clade includes Farrago racemosa Clay
ton sister to a monophyletic Craspedorhachis (BS = 97, PP = 1.00). To test the repeatability of our work we included four samples of the Craspedorhachis rhodesiana Rendle that were collected all by Strohbach 5699 but placed on different her
barium sheets. All sequences were identical.
The Perotidinae clade includes a paraphyletic Perotis (BS = 84, PP = 1.00) sister to Mosdenia leptostachys (Ficalho
& Hiern) Clayton. Embedded within Perotis are Lopholepis ornithocephala (Hook.) Steud. and Toliara arenacea Judz.
Within the Perotis clade four different accessions of Perotis patens Gand. (BS = 81, PP = 0.99) are sister to Toliara; and all of these are sister to P. leptopus–P. vaginata (BS = 98, PP
= 1.00). Perotis rara R. Br. forms an unsupported clade with Lopholepis ornithocephala; and this is sister to the P. somalen- sis–P. hildebrandtii clade (BS = 94, PP = 0.99).
Within the Gymnopogoninae clade a monophyletic Dig- nathia (BS = 100, PP = 1.00) with three species is sister to a paraphyletic Leptothrium (BS = 100, PP = 1.00) that includes Latipes inermis. Latipes inermis forms a clade with Lepto- thrium rigidum (BS = 67, PP = 1.00). Gymnopogon appears monophyletic with strong support (BS = 100, PP = 1.00) and is sister to Lophacme-Bewsia (BS = 90, PP = 0.97). Four
accessions of Leptocarydion vulpiastrum (De Not.) Stapf are sister to the Bewsia-Lophacme-Gymnopogon clade (BS = 100, PP = 1.00); and all of these (BS = 100, PP = 1.00) are sister to the Dignathia-Leptothrium clade (BS = 0.92, PP = 1.00).
Analysis of ITS sequences. — The ML tree based on data from the nrITS region is remarkably well resolved and very similar to the combined plastid–ITS tree (Fig. 2). There is strong support (BS = 100, PP = 1.00) for four of the major clades (Cteniinae, Farragininae, Perotidinae, Trichoneurinae) and moderate support for the Gouiniinae (BS = 87, PP = 1.00) and Gymnopogoninae (BS = 78, PP = 1.00). All six subtribes form a monophyletic clade (BS = 73, PP = 0.94). Within the Gouiniinae clade, Tridens muticus is sister to Gouinia; and within the Gymnopogoninae clade, Lophacme and Bewsia bi- flora render Gymnopogon paraphyletic.
Analysis of plastid sequences. — The ML tree based on combined plastid sequences (Fig. 3) depicts the Cteniinae, Far
ragininae, Perotidinae, and Trichoneurinae clades with strong support (BS = 98–100, PP = 1.00), the Gymnopogoninae with moderate support (BS = 84, PP = 1.00), and the Gouiniinae with weak support (BS = 61, PP = 0.64). All six subtribes form a monophyletic clade (BS = 88, PP = 1.00). Resolution and support for clades within the Perotidinae are lacking and within Perotis there is only a single strongly supported clade with two accessions of P. indica whereas within the combined plastid–ITS tree there are five strongly supported clades within Perotis and six highly supported clades within the ITS tree.
The plastid tree resolves a strongly supported (BS = 100, PP = 1.00), monophyletic Gymnopogon.
DISCUSSION
Within these 20 genera of Cynodonteae we found strong support for six lineages we treat as subtribes: the Cteniinae, Farragininae, Gouiniinae, Gymnopogoninae, Perotidinae, and Trichoneurinae. Craspedorhachis, Ctenium, Dignathia, Gym- nopogon, Trichoneura, and Triplasis all appear monophyletic;
Leptothrium is paraphyletic with Latipes inermis Chiov. em
bedded within; Perotis is paraphyletic with Lopholepis and Toliara embedded within; and Gouinia is monophyletic with the resurrection of Schenckochloa.
Gouiniinae. — With a limited sample of species per genus, Gouinia, Tridens, Triplasis, and Vaseyochloa were included in a clade (Columbus & al., 2007) and later formally treated in the subtribe Tridentinae (Peterson & al., 2010a). However, the type for Tridens (T. flavus (L.) Hitchc.) was not included. In our study Tridens flavus is clearly sister to Pappophorum (Fig. 1), a member of the Pappophorinae. Tridens muticus is separated from T. flavus in ValdesReyna’s (2003) key by containing nar
row panicles with appressed, ascending branches. The palea nerves of T. flavus are glabrous and not long hairy as in T. muti- cus and both species of Triplasis, also a member of the Gouinia clade. Based on a trigonous caryopsis, glumes shorter than the florets, a triangular rachilla, and anatomical differences, Ortíz (1991) erected Schenckochloa to include a single species native to western Brazil, S. barbata (Hack.) J.J.Ortíz. Therefore, we
0.06
Ctenium newtonii var. productum Gouinia virgata var. robusta Reeder 4714
Leptocarydion vulpiastrum Bidgood 50038 Trichoneura lindleyana Bentley 319
Mosdenia leptostahys Schweickerdt 1542
Latipes inermis
Farrago racemosa Trichoneura lindleyana Howel 9435
Trichoneura eleusinoides subsp. limpopoensis Pienaar 394 Trichoneura eleusinoides subsp. limpopoensis Davidse 5859 Triplasis americana
Gouinia brasiliensis
Gymnopogon grandiflorus Triplasis purpurea Peterson 14238
Perotis patens Godfrey 1661 Pappophorum pappiferum
Perotis patens Kruger 21750
Dignathia hirtella Maggi 353 Trichoneura weberbaueri Gouinia latifolia var. guatemalensis Martinez 29764
Craspedorhachis rhodesiana Tridens flavus var. chapmanii
Gouinia paraguayens
Leptothrium senegalense Peterson 24196
Gymnopogon brevisetus Leptothrium senegalense Belsky 336
Trichoneura grandiglumis Reihmer 6040
Craspedorhachis africana Litt 125
Perotis hildebrandtii Trichoneura mollis Bogdan 4173
Ctenium elegans Gouinia virgata var. virgata Villareal 8105
Gymnopogon swallenii Schenckochloa barbata
Trichoneura eleusinoides Seydel 448
Lophacme digitata
Gymnopogon ambiguus Nelson 1392 Leptothrium senegalense Peterson 24239 Triplasis purpurea Peterson 24420
Ctenium cirrhosum Gouinia gracilis
Perotis leptopus Craspedorhachis rhodesiana Trichoneura elegans Silveus 812
Dignathia hirtella Barkhadle 353
Bewsia biflora Peterson 23905 Perotis patens Klaassen 1305 Craspedorhachis africana Fisher 504 Craspedorhachis rhodesiana Neesiochloa barbata
Vaseyochloa multinervosa
Ctenium aromaticum Gouinia virgata (as G. longiramea) Swallen 2818
Perotis indica Peterson 23880 Trichoneura elegans Silveus 7312
Gymnopogon aristiglumis
Gymnopogon legrandii Gouinia latifolia var. guatemalensis Beetle 918
Perotis hordeiformis Trichoneura mollis Napper 550
Tridens muticus Gould 9978
Perotis somalensis
Ctenium brevispicatum Filgueiras s.n.
Perotis indica Peterson 23872
Bewsia biflora Davidse 6471 Gouinia virgata var. virgata Rzedowski 14670
Trichoneura eleusinoides Oliver 6696 Ctenium somalense
Ctenium newtonii Baldwin 13423
Trigonochloa uniflora Snow 6978
Gymnopogon ambiguus Becker 1488 Perotis rara
Gymnopogon spicatus Renvoize 3677 Gymnopogon spicatuss Quarin 4144
Gymnopogon foliosus Tridens muticus Peterson 21997
Tridens flavus
Trichoneura grandiglumis Smook 4896
Toliara arenacea
Gymnopogon brevifolius Peterson 24437 Leptocarydion vulpiastrum Peterson 24238 Dignathia hirtella McCallum 5251
Trichoneura elegans Lundell 15030
Leptocarydion vulpiastrum Mboya 565
Gymnopogon ambiguus Thomas 168039
Ctenium newtonii Laegaard 18327
Perotis vaginata
Gymnopogon brevifolius Hill 15844 Trichoneura grandiglumis Smook 7068
Craspedorhachis africana Fisher 459 Craspedorhachis africana Laegaard 16192
Ctenium brevispicatum Filgueiras 3251
Leptothrium rigidum
Leptocarydion vulpiastrum Peterson 23863 Craspedorhachis rhodesiana
OUTGROUP
1.00
1.00
1.00
1.00 0.99
0.67 0.990.57
1.00 1.00 1.00
0.64
0.94
0.94
0.77 1.00
1.00 1.00
1.00
0.99 1.00 0.97
0.99 0.890.99
1.00
1.00
1.00
1.00 1.00
1.00 0.73
1.00
1.00 0.99
0.94
0.740.70 0.770.99
1.00 0.97
1.00
0.76 1.000.97
0.96
0.99 0.910.99
1.00
0.91 1.00
1.00
1.00
1.00 1.00
1.001.00 0.94
1.00 1.001.00
1.00
1.00 0.80
0.950.99
1.00
0.99
1.00
0.99 0.99
1.00
1.00
1.00 1.00
1.00 0.86 1.00 99
96
79
79
61
73 7261
93 100
100
87 100 100
90 63
100
69
1.00 7899 5962
100
100 85 65
51 6982 55
100
61
53 99
8272
78 5580 53
71 98
73 58
100 100
100
100
100
100
100 100 100 1.00
52 83
92 100 90
67 94
78 70
100 98
51 53
90
76
100
100
74
89 82
73
100
92 98
86 64
99 96
PAPPOPHORINAE
GOUINIINAE
TRIPOGONINAE TRAGINAE
SCLEROPOGONINAE
MONANTHOCHLOINAE BOUTELOUINAE
Eragrostideae Zoysieae Centropodieae
Triraphideae
HILARIINAE MUHLENBERGIINAE
ELEUSININAE
ORCUTTIINAE
0.88
CTENIINAE
TRICHONEURINAE
FARRAGININAE
PEROTIDINAE
GYMNOPOGONINAE Cynodonteae
= Tridentopsis mutica
= Perotis arenacea
= Leptothrium inerme North America
Central Africa Southern Africa Southeast Asia South America
TRIODIINAEAELUROPODINAE
0.99
Fig. 2. Phylogram of maximumlikelihood tree from analysis of ITS sequences. Numbers above branches are bootstrap values; numbers below branches are posterior probabilities; clades outlined in dashed lines are discussed in text; scale bar = 6% sequence divergence; color indicates native distribution (see legend).
0.01
Craspedorhachis rhodesiana Triplasis purpurea Peterson 14238
Craspedorhachis africana Laegaard 16192 Neesiochloa barbata
Dignathia hirtella Maggi 353
Lophacme digitata Tridens flavus var. chapmanii
Gymnopogon foliosus
Leptocarydion vulpiastrum Peterson 24238 Gouinia paraguayensis
Perotis patens Godfrey 1661
Dignathia gracilis Greenway 10449
Ctenium newtonii Baldwin 13423 Gouinia gracilis
Triplasis americana
Ctenium brevispicatum Filgueiras 3251
Trichoneura grandiglumis Smook 7068
Toliara arenacea
Leptocarydion vulpiastrum Mboya 565 Gouinia virgata var. virgata Rzedowski 14670
Craspedorhachis rhodesiana
Mosdenia leptostachys Smook 1145 Craspedorhachis rhodesiana
Perotis leptopus
Leptocarydion vulpiastrum Bidgood 50038
Gymnopogon brevifolius Peterson 24437 Trigonochloa uniflora Snow 6978
Trichoneura mollis Napper 550 Gouinia latifolia var. guatemalensis Martinez 29764
Gymnopogon ambiguus Thomas 168
Ctenium elegans
Trichoneura elegans Lundell 15030 Gouinia virgata var. robusta
Ctenium cirrhosum
Craspedorhachis africana Schweickerdt 484
Perotis rara
Leptocarydion vulpiastrum Peterson 23863 Tridens muticus Gould 9978
Dignathia hirtella Barkhadle 353
Trichoneura eleusinoides subsp. limpopoensis Davidse 5859 Ctenium brevispicatum Filgueiras s.n.
Trigonochloa uniflora Greenway 14075
Latipes inermis Pappophorum pappiferum
Craspedorhachis africana Litt 125
Perotis indica Peterson 23880
Perotis hildebrandtii Renvoize 1784 Perotis vaginata
Ctenium newtonii Laegaard 18327 Ctenium aromaticum
Ctenium newtonii var. productum
Trichoneura lindleyana Bentley 319
Mosdenia leptostachys Schweickerdt 1542 Vaseyochloa multinervosa
Perotis patens Moss 11760
Gymnopogon spicatus Quarin 4144Gymnopogon aristiglumis Triplasis purpurea Peterson 24420
Gymnopogon spicatus Renvoize 3677
Craspedorhachis rhodesiana Trichoneura grandiglumis Smook 4896 Trichoneura eleusinoides Seydel 448
Gymnopogon chapmanianus Leptothrium rigidum
Leptothrium senegalense Peterson 24196
Trichoneura grandiglumis Reihmer 6040
Leptothrium senegalense Belsky 336
Trichoneura weberbaueri
Gymnopogon ambiguus Nelson 1392 Dignathia villosa
Gouinia virgata var. virgata Villareal 8105
Perotis patens Klaassen 1305 Ctenium somalense Tridens flavus
Perotis somalensis Gouinia latifolia var. guatemalensis Beetle 9181
Perotis patens Kruger 21750
Trichoneura eleusinoides subsp. limpopoensis Pienaar 394
Craspedorhachis africana Fisher 504
Bewsia biflora Davidse 6471 Perotis hildebrandtii Peterson 23814
Farrago racemosa
Dignathia hirtella McCallum 5251
Leptothrium senegalense Laegaard17937
Gymnopogon ambiguus Becker 1488 Gymnopogon brevifolius Hill 15844 Tridens muticus Peterson 21997
Lopholepis ornithocephala Perotis indica Peterson 23872
Leptothrium senegalense Peterson 24239 Perotis hordeiformis
Gymnopogon burchellii Gouinia brasiliensis
Craspedorhachis africana Fisher 459
Bewsia biflora Peterson 23905
Trichoneura elegans Silveus 812
Gymnopogon legrandii 1.00
1.00
1.00
1.00
1.00
1.00 1.00
1.00
1.00 1.00
1.00
0.92 0.67
0.79
0.83 0.64
0.88 1.00
1.00
0.98 0.99
0.97 0.97
1.00 1.00
0.98 0.98
0.98 0.88
1.00
1.00
1.00
1.00
1.00 1.00
1.00 1.00
1.00 0.68
1.00 0.74
0.89
1.00
1.00 1.00
1.00
1.00 1.00
0.94
0.90 0.980.98
0.94 0.79 1.00
1.00 1.00
1.00
1.00 0.97
0.91 1.00
0.75
0.80 0.96
1.00 0.63
0.61
1.00 1.00
0.98
1.00 1.00 0.84
1.000.98 0.99
0.96 1.00
1.00
1.00 0.510.99 0.70
0.691.00 0.99 1.00
Cynodonteae
96
100
100 99
98
91 100
100
79
70 99
74 61
98 70
71 99
100
79 76
68 98
79 79
74
88
59
100 59
90 1.0097
100 99
100 98
90
100
88 65
61
71 97
100
98 98
75 100
83
98 90
1.00100
91 100
85 84
78
84 55
97 99
100 85
89 74
78
10069 76
90 100
100
100 85 88
86
98
OUTGROUP
TRIPOGONINAE
TRAGINAE
SCLEROPOGONINAE
MONANTHOCHLOINAE BOUTELOUINAE
Eragrostideae
Zoysieae Centropodieae Triraphideae
HILARIINAE
MUHLENBERGIINAE
ELEUSININAE ORCUTTIINAE
PAPPOPHORINAE
North America
Central Africa Southern Africa Southeast Asia South America
GOUINIINAE
Schenckochloa barbata
Gouinia virgata (as G. longiramea) Swallen 2818
CTENIINAE
TRICHONEURINAE
FARRAGININAE
PEROTIDINAE
GYMNOPOGONINAE
Gymnopogon brevisetus
Gymnopogon floridanus Gymnopogon swallenii
Gymnopogon grandiflorus 52
52
98
55
= Tridentopsis mutica
= Perotis ornithocephala
= Perotis arenacea
= Leptothrium inerme TRIODIINAE
AELUROPODINAE
Fig. 3. Phylogram of maximumlikelihood tree from analysis of combined plastid sequences. Numbers above branches are bootstrap values; numbers below branches are posterior probabilities; clades outlined in dashed lines are discussed in text; scale bar = 1% sequence divergence; color indi
cates native distribution (see legend).
resurrect subtribe Gouiniinae to now include Gouinia, Tridens muticus, Triplasis, Schenckochloa, and Vaseyochloa, all from the New World (Peterson & al., 2007). Since Vaseyochloa is sister to Gounia s.str. (BS = 97, PP = 1.00, Fig. 1) and is mor
phologically distinct with caryopses that have two distinct hornlike style bases, we are inclined to be conservative and retain its use. The subtribal circumscription in Peterson & al.
(2007) is still tenable since it was prepared to include Gouinia (including Schenckochloa) and Vaseyochloa. However, after reviewing the morphologies of the species in the Gouinia clade the following trends seem worth mentioning: palea keels are strongly winged to somewhat winged, or at least expanded near the base, and spikelets terminate in a rachilla extension (Swallen, 1935; OrtízDiaz, 1993). Based on having paleas that are not widened or bowedout below and caryopses that are dorsally flattened, deeply concave to folded on the dorsal or hilar surface, and thickened towards the margins below, we place Tridens muticus in a new genus, Tridentopsis. We have preliminary data for 12 of the 17 species currently placed in Tridens (Peterson & Romaschenko, in prep.) and have included in the generic description of Tridentopsis below the morpho
logical features of Tridens muticus and Tridens eragrostoides (Vasey & Scribn.) Nash, the only other species we are aware of that aligns within the new genus.
Cteniinae. — Within the Cynodonteae, Ctenium is mor
phologically and genetically distinct. The genus is character
ized by having inflorescences of a single or sometimes 2–6 curved pectinate racemes; spikelets with two basal sterile florets, one fertile floret, and 1–3 rudimentary florets above the fertile; upper glumes that are 2 or 3veined with a dorsal, subapical awn. The Ctenium clade contains two strongly sup
ported, geographically separated subclades (New World and Africa), each with three species. However, this result might be erroneous since we are lacking data for 14 Ctenium species.
It is also possible that Kampochloa brachyphylla Clayton lies within the Cteniinae, since it is readily mistaken for Ctenium but the spikelets lack two lower sterile florets (Clayton, 1967a).
In the future we hope to obtain material of Kampochloa Clay
ton, known only from Angola.
Trichoneurinae. — The Trichoneura clade, like Ctenium, is genetically distinct and has a geographical signal. A cen
tral African clade (T. grandiglumis–T. mollis) is sister to a New World clade (T. weberbaueri–T. lindleyana–T. elegans) and these together are sister to a southern African clade (T. eleusinoides; see Fig. 1). We have sampled seven of the eight known species of Trichoneura, so this seems plausible.
Napper (1963) indicated that Pogononeura biflora Napper was probably related to Trichoneura or Leptochloa. However, Pogononeura has recently been reanalyzed and apparently aligns near the Pappophorinae or Tragiinae and not in the Sporobolinae as previously reported (Peterson & al., 2010a;
Peterson & al., in prep.). Unique morphological characteristics for the Trichoneur inae include: inflorescences with numerous unilateral racemes borne along a central axis; cuneate, and laterally compressed spikelets with glumes longer than the florets; and dorsally compressed caryopses that are plano or concavoconvex.
Farragininae. — Farrago racemosa, as described by Clayton (1967b), is an interesting Tanzanian endemic that was thought to be a close relative of Dignathia, both placed in the Zoysiinae by Clayton & Renvoize (1986). The senior authors (PMP & KR) traveled to Namatumuzi, Tanzania (type locality) in 2012 to gather material of this enigmatic grass characterized in having dorsally compressed fertile spikelets with longawned somewhat coriaceous glumes and delicate, hyaline lemmas that are dorsally hairy. Our DNA analyses clearly place Farrago as sister to Craspedorhachis. The latter African genus also has dorsally compressed spikelets with delicate, hyaline lemmas less firm than the glumes but differs from Farrago in having a perennial habit and conspicuously winged lower glumes and 3veined lemmas (unveined in Farrago) (Watson & Dallwitz, 1992). Craspedorhachis was thought to have affinities with Willkommia Hack. and Microchloa R.Br. (Clayton & Renvoize, 1986), both recently realigned in the Tragiinae and Eleusin
inae, respectively (Peterson & al., 2010a). Based on only rps3, Peterson & al. (2010a) reported that Craspedorhachis africana was embedded within Perotis but they attributed this result to a lack of variation within the marker.
Perotidinae. — Peterson & al. (2010a) reported that Perotis was polyphyletic by containing Lopholepis ornithocephala, and these were sister to Mosdenia. Even though the laterally com
pressed spikelet of Lopholepis has undergone profound mor
phological change and resembles a bird’s head in outline, other characters are consistent with those found in Perotis since it has leaf blades that are broad and clasping at the base with antrorse
ciliate margins, a single narrow raceme, and glumes exceeding the length of the florets. Clayton (1972) mentions Lopholepis ornithocephala as being the “third member” of an alliance with Leptothrium senegalense and L. rigidum Kunth (see discussion under Gymnopogoninae). Toliara arenacea was also found to be sister to Perotis in Peterson & al. (2012), and when initially described by Judziewicz (2009) was thought to be most closely related and morphologically similar to Perotis. However, when compared to Perotis, Toliara lacks awned glumes, has only two stamens, and has a pubescent ovary (Judziewicz, 2009). Perotis is clearly paraphyletic and we make the necessary combinations by transferring these two species to Perotis below. Mosdenia and Trigonochloa both align as strongly supported successive sisters to Perotis in our analyses (Peterson & al., 2010a, 2012;
Snow & Peterson, 2012). Mosdenia leptostachys differs from species of Perotis in having stolons and unawned glumes and Trigonochloa differs from Perotis in having trigonous caryop
ses that have a narrow but deep sulcus on the hilar side (Gibbs Russell & al., 1991; Snow & Peterson, 2012). Character trends within the African and southeast Asian Perotidinae include:
lanceolate to lanceolateovate leaf blades that are broad and clasping at the base with antrorseciliate margins; racemose inflorescence of one to several branches, if one, then usually narrow; and glumes that exceed the single fertile floret.
Gymnopogoninae. — Relationships among members of the Gymnopogoninae clade were previously elucidated, in part, when the Bewsia-Gymnopogon clade was depicted as sister to Dignathia (BS = 83, PP = 1.00; see fig. 3 in Peterson & al., 2010a). We expand this here to include Dignathia-Leptothrium
