J Plant Res (2002) 115:381–392 Digital Object Identifier (DOI) 10.1007/s10265-002-0049-3 © The Botanical Society of Japan and Springer-Verlag Tokyo 2002 ORIGINAL ARTICLE Trevor R. Hodkinson • Mark W. Chase • M. Dolores Lledó • Nicolas Salamin • Stephen A. Renvoize Phylogenetics of Miscanthus, Saccharum and related genera (Saccharinae, Andropogoneae, Poaceae) based on DNA sequences from ITS nuclear ribosomal DNA and plastid trnL intron and trnL-F intergenic spacers Received: February 4, 2002 / Accepted: June 19, 2002 / Published online: August 28, 2002 Abstract DNA sequences were used to assess the monophyly and inter-relationships of Miscanthus, Saccharum and related genera in the Saccharum complex. Three DNA regions were sequenced, including the trnL intron and the trnL-F intergenic spacer of the plastid genome and the ITS region of nuclear ribosomal DNA (nrDNA). Because it was more variable, the ITS region proved most suitable for phylogenetic reconstruction at this level, and the results indicate that Miscanthus s.l. and Saccharum s.l. are polyphyletic. A set of species from Saccharum section Ripidium (clade a) do not group closely with any members of Saccharum s.l.. A number of Miscanthus species from eastern or southeastern Asia represent a monophyletic group with a basic chromosome number of 19 (clade b), but the other species from Africa and the Himalayas are clearly excluded. There is support for a monophyletic Saccharum s.s. clade including S. officinarum and S. spontaneum that is sister to Miscanthus s.s. (clade c). There is no evidence to support the division of some Saccharum s.l. into the genera currently known as Erianthus and Narenga. Saccharum contortum (= Erianthus contortus), S. narenga (= Narenga porphyrocoma) and Erianthus rockii, group more closely with Miscanthus fuscus, a species from the Himalayas and also with the African Miscanthus s.l. species (= Miscanthidium, clade d). Key words Erianthus Saccharum • Sugarcane • • Miscanthus Systematics • Molecular T.R. Hodkinson • M.W. Chase • M.D. Lledó • S.A. Renvoize Royal Botanic Gardens, Kew, Richmond, Surrey, UK T.R. Hodkinson (*) • N. Salamin Department of Botany, Trinity College, University of Dublin, Dublin 2, Ireland Tel. +353-16081128; Fax +353-16081147 e-mail: trevor.hodkinson@tcd.ie Springer-VerlagTokyoJournal of Plant ResearchJ Plant Res102650918-94401618-0860s10265-002-0049-30049Bot Soc Jpn and Springer-VerlagOriginal Article • Introduction Tribe Andropogoneae (Poaceae) includes many species with high economic value, including the C4 grasses Saccharum officinarum L. (sugarcane), Sorghum bicolor (L.) Moench (sorghum) and Zea mays L. (maize). Subtribe Saccharinae Griseb. includes Saccharum L. and Miscanthus Anderss., the latter having considerable potential as a biomass crop for renewable energy production and raw material for the cellulose and paper industries (Bullard et al. 1995; Clifton-Brown and Lewandowski 2000). Saccharinae according to Clayton and Renvoize (1986), also include Eriochrysis P. Beauv., Eulalia Kunth, Eulaliopsis Honda, Homozeugus Stapf., Imperata Cyr., Lophopogon Hack., Microstegium Nees, Pogonatherum P. Beauv., Polytrias Hack. and Spodiopogon Trin. The subtribe is morphologically defined by their terminal inflorescence (except Eulaliopsis and Pogonatherum) of solitary or digitate racemes and paired similar spikelets. The paired spikelets are often plumose; the callus is rounded or truncate. The lower glume is mostly thin, and the lower floret is usually reduced to a sterile lemma. The upper lemma is entire or bilobed and can have a glabrous awn. Despite these characteristics the only known morphological synapomorphy for Saccharinae would be their bisexual paired spikelets. Other Andropogoneae have paired spikelets, but one of these is usually either male or sterile. However, many exceptions exist, such as some species of Ischaemum (Ischaemninae) and Schizachrium (Andropogoninae), which have bisexual paired spikelets. Saccharinae, therefore, are poorly defined, and their monophyly remains insufficiently evaluated. Systematists have used the term “Saccharum complex” to describe a subset of the Saccharinae (Erianthus, Miscanthus, Narenga, Saccharum and Sclerostachya) implicated in the origin of sugarcane and in which the taxonomy is particularly confused (Daniels and Roach 1987). There is a need to characterise this complex more comprehensively. The monophyletic status of many genera within Saccharinae is also in doubt. The most widely debated is Saccharum itself. Saccharum s.l. (Clayton and Renvoize 1986) has been 382 divided into a number of other genera including Erianthus Michaux, Narenga Bor and Ripidium Trin., but Clayton and Renvoize (1986) chose to combine all of these genera under Saccharum because the characters used to define them were thought to be more suited to infra-generic categorisation. They argued that the division of awned (Erianthus) and awnless species is artificial and the separation of Narenga, with its coriaceous glumes, is trivial because this is no more than an extreme expression of a trend found elsewhere in the genus. Saccharum s.l. has approximately 40 species (Clayton and Renvoize 1986), Erianthus approximately 20 and Narenga only two (Adati and Shiotani 1962). Saccharum s.s. (Price 1963; Daniels and Roach 1987) is distributed throughout the tropics and subtropics due to cultivation, but the species are native to south-eastern Asia. Erianthus is cosmopolitan and, according to Celarier (1956), can be divided into two subgenera, one with two anthers and an American distribution; the other with three anthers and an Old World distribution. The Old World distribution of Saccharum s.l. is given in Fig. 1. The New World species are predominantly found in North America. The taxonomic status of Miscanthus is also in a state of flux, and little is known about the identity and interrelationships of its species. According to Clayton and Renvoize (1986), Miscanthus s.l. comprises approximately 20 species and appears well-defined morphologically. However, they also recognised that Eriochrysis, Eulalia, Imperata, Miscanthus, Saccharum and Spodiopogon form a closely knit group in which the phylogenetic relationships are unclear. Saccharum is considered by many as the closest relative of Miscanthus, and these two genera frequently hybridise (Sobral et al. 1994). The species of Miscanthus can be distinguished from those of Saccharum by their tough inflorescence rachis and both spikelets of a pair being pedicellate, although the pedicels are of different lengths. Most members of Miscanthus s.l. are native to eastern or south-eastern Asia (China, Japan and neighbouring regions); two species are known from the Himalayas and four from southern Africa (Fig. 1). In the past, the African species have been placed in a separate genus, Miscanthidium Stapf, mainly on the basis of their elongate inflorescence axis and short racemes, but these differences were not considered sufficient to warrant separation from Miscanthus by Clayton and Renvoize (1986) because M. floridulus (Labill.) Warb. Ex. K. Schum. & Lauterb. and M. fuscus (Roxb.) Benth. [= Sclerostachya fusca (Roxb.) A. Camus] in Asia also have elongate inflorescence axes. Groups of species at sectional rank within Miscanthus have been recognised, and a key to Miscanthus species was given in Hodkinson et al. (1997). The most comprehensive effort to subdivide the genus was made by Lee (1964b, c, d), 1 Fig. 1. Distribution of Miscanthus and Saccharum sensu lato species in the Old World. The major areas of distribution are shown by rings, but exclude occasional records from elsewhere. Saccharum officinarum (sugarcane) is not included because it has a widespread distribution due to cultivation 383 who separated the Asian species into four sections that broadly agreed with the treatments of Honda (1930) and Adati and Shiotani (1962). There are four African Miscanthus species not included in the genus by Lee (1964b, c, d) namely: M. ecklonii (Nees) Mabb., M. junceus (Stapf) Pilger, M. sorghum (Nees) Pilger and M. violaceus (K. Schum.) Pilger. Himalayan M. fuscus was also excluded from Miscanthus by Lee (and recognised as Sclerostachya fusca), and has been included by Clayton and Renvoize (1986). Miscanthus brevipilus Hand.-Mazz., M. changii, Y.N. Lee, and M. eulaliodes Keng ex. Hand.-Mazz., which were listed by Lee (1964b, c, d), have not been evaluated here because they were not available for study. Miscanthus transmorrisonensis appears to intergrade with M. sinensis on a morphological level. Another taxon known as M. condensatus Hackel has been given species rank by previous authors, but falls within the normal range of morphological variation found in M. sinensis (Koyama 1987). Lee (1964a) recognised M. condensatus on the basis of leaf anatomy because it differs in having unclosed bundles in the midrib. In addition, M. condensatus only grows at low elevation in the coastal zone of Japan. We have treated M. condensatus here as M. sinensis subsp. condensatus (Hackel) T. Koyama (following Koyama 1987). Using DNA sequence data of nuclear and plastid DNA regions, this study aimed to assess the monophyly of Miscanthus and Saccharum and their phylogenetic relationships to other Saccharinae. DNA sequencing is particularly well suited for phylogenetic studies and has been used extensively for such purposes at many different taxonomic levels (Chase et al. 1993; Hsiao et al. 1994; Soltis et al. 1999; Salamin et al. 2002). The internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (for a review see Baldwin et al. 1995), and the trnL intron and trnL-F intergenic spacer of plastid DNA (hereafter trnL-F; Taberlet et al. 1991; Gielly and Taberlet 1994; Hopper et al. 1999; Molvray et al. 1999; Chase et al. 2000; Lledó et al. 2000) were used to construct phylogenetic hypotheses with parsimony methods. The ITS region has proven useful for phylogenetic studies at this taxonomic level in various plant groups, including grasses (Baldwin et al. 1995; Hsiao et al. 1995a, b, 1999; Hodkinson et al. 2000; Grass Phylogeny Working Group 2001; Hodkinson et al. 2002a) and so has the trnL-F region (Briggs and Johnson 2000; Briggs et al. 2000). A number of grass ITS sequences have been published and/or deposited in GenBank. We have utilised these sequences to provide a more comprehensive sample of Saccharinae (Table 1). We also present a combined analysis of ITS and trnL-F data. We Table 1. Grass species and associated voucher specimens used in the study. Vouchers are deposited at Royal Botanic Gardens, Kew (K). *ID number represents identification number used in this study to differentiate taxa with the same name Tribe Subtribe Genus/species GenBank, ID number* Voucher or reference Andropogoneae Andropogoninae Andropogon gerardii Vit. Cymbopogon citratus L. Cymbopogon citratus L. Themeda triandra Forsk. Themeda triandra Forsk. Chionachne cyathopoda F.Muell. ex Benth. Hemarthria uncinata R.Br. Erianthus rockii Keng E. rockii Keng Eulalia irritans (R.Br.) Kuntze (= Poganatherum irritans) E. quadrinervis (Hack.) Kuntze E. trispicata (Schult.) Henrard E. villosa (Thunb.) Nees Imperata cylindrica (L.) P.Beauv. Raeuschel I. cylindirca (L.) P.Beauv. Raeuschel I. cylindirca (L.) P.Beauv. Raeuschel Miscanthus sp. Anderss. M. ecklonii (Nees) Mabb. M. floridulus (Labill.) Warb. ex. K. Schum. & Lauterb. M. floridulus (Labill.) Warb. ex. K. Schum. & Lauterb. M. floridulus (Labill.) Warb. ex. K. Schum. & Lauterb. M. fuscus (Roxb.) Benth. M. junceus (Stapf) Pilger M. junceus (Stapf) Pilger M. nepalensis (Trin.) Hack. M. oligostachyus Stapf M. oligostachyus Stapf AY116299, AY116263, 75 AY116258, 129 AF019823, (a) AY116261, MWC9286, (a) AF019820 AF019819 Hodkinson 15. 1969-19004 Hodkinson 129 Hsiao et al. 1999 Salamin s.n. Hsiao et al. 1999 Hsiao et al. 1999 AF019821 AF345216, (a) AF345217, (b) AY116298, AY116242, 137 Hsiao et al. 1999 Chen et al. unpublished Chen et al. unpublished Adams 1756 AY116303, AY116251, 134 AY116291, 138 AY116302, 132 AY116297, AY116262, 122 Polunin et al. 3294 Clarkson 10062 Devenish 1282 Marsden 3 AF345653, (a) AF092512, (b) AY116283, AY116243, 155 AY116290, AY116264, 86 AY116278, AY116248, 72 Chen et al. unpublished Tsai and Chou unpublished Phillips 155 du Toit 2347 Hodkinson 30. 1978-1387 AY116280, TRH1 (a) Scally s.n. AY116281, TRH2 (b) Scally s.n. AY116286, AY116265, 83 AY116288, AY116254, 88 AY116289, AY116255, 89 AY116292, AY116252, 25 AY116277, AY116249, 16 AY116279, AY116245, 161 Mangelsdorf 1955 US56-5-5 Tinley 1060 Simon 2309 Hodkinson 1 Hodkinson 13 Hodkinson 161 Anthristiriinae Chionachninae Rottboellinae Saccharinae 384 Table 1. Continued Tribe Subtribe Sorghinae Tripsacinae Arundinelleae Erianchneae Paniceae Cenchrinae Digitariinae Genus/species GenBank, ID number* Voucher or reference M. sacchariflorus (Maxim.) Benth. & Hook. “Purpurascens” M. sacchariflorus (Maxim.) Benth. & Hook. M. sinensis Anderss. var. variegatus Beal. M. sinensis Anderss. M. sinensis Anderss. subsp. condensatus (Hackel) T. Koyama M. sinensis Anderss. “Gracillimus” M. sinensis Anderss. “Roland” M. sinensis Anderss. M. sinensis Anderss. “Grosse Fontane” M. sinensis Anderss. “Yakushimanum” M. transmorrisonensis Hayata Saccharum arundinaceum Retz. S. arundinaceum Retz. S. arundinaceum Retz. S. barberi Jeswiet S. barberi Jeswiet S. contortum (Ell.) Nutt. (= Erianthus contortus Baldw. Ex Ell.) S. contortum (Ell.) Nutt. (= Erianthus contortus Baldw. Ex Ell.) S. fallax Balansa (= Narenga fallax (Balansa) Bor) S. fulvus R.Br. (= Erianthus fulvus (R.Br.) Kunth) S. fulvus R.Br. (= Erianthus fulvus (R.Br.) Kunth) S. narenga Wall. (= Narenga porphyrocoma (Hance) Bor) S. narenga Wall. (= Narenga porphyrocoma (Hance) Bor) S. officinarum L. (sugarcane cv.) S. ravennae Murr. (= Erianthus ravennae Beauv.) S. ravennae Murr. (= Erianthus ravennae Beauv.) S. robustum Brandes & Jesw. ex Grassl S. robustum Brandes & Jesw. ex Grassl S. sinense Roxb. S. sinense Roxb. S. spontaneum L. Spodiopogon sibiricus Trin. Sorghum australiense Garber & Synder S. bicolor (L.) Moench S. halepense (L.) Pers. S. laxiflorum F.M. Bailey S. macrospermum E.D. Garber S. versicolor Anderss. Tripsacum australe Cutler & E.Anders. T. australe Cutler & E.Anders. Zea perennis (Hitchc.) Reeves & Mangelsd. Z. diploperennis Iltis, Doebley & Guzman Z. luxurians (Durieu) R.M.Bird Z. mays L. Arundinella nepalensis Trin. Eriachne triseta Nees ex. Steud. Cenchrus ciliaris L. C. incertus M.A. Curt. Pennisetum purpureum Schum. P. macrourum Trin. P. setaceum (Forsk.) Chiov. Digitaria sanguinalis (L.) Scop. D. sanguinalis (L.) Scop. AJ426564, AY116247, 61 Hodkinson s.n. 1987-2727 AY116282, AY116246, 5791 Renvoize 5791 AY116276, 1 AJ426565, AJ426571, 5 AY116270, AJ426573, 7 Hodkinson 33 Hodkinson 40. 1978-1389 Renvoize s.n. 1969-19091 AY116274, 28 AY116272, 29 AJ426566, AJ426572, 30 AY116273, 31 AY116275, 63 AY116271, AY116250, 65 AY116295, 118 AF345201, (a) AF345202, (b) AF331657, (a) AF345199, (b) AY116287, MWC9284, (a) Hodkinson s.n. MB94/05 Hodkinson s.n. MB94/06 ADAS MB94/07 Hodkinson PN95/01 Hodkinson 21. 1987-1148 Hodkinson 20. 1990-2748 Shiu Ying Hu 11199 Chen et al. unpublished Chen et al. unpublished Chen et al. unpublished Chen et al. unpublished Salamin s.n. AY116256, 121 Renvoize 3797 AF345213 Chen et al. unpublished AF345218, (a) Chen et al. unpublished AF345219, (b) Chen et al. unpublished AF345233, (a) Chen et al. unpublished AF345234, (b) Chen et al. unpublished AY116284, AY116253, 104 AF019824, (a) Kew 1973-12242 Hsiao et al. 1999 AY116296, MWC9285 (b) Salamin s.n. AF345237, (a) AF345238, (b) AF345242, (a) AF345243, (b) AY116285, AY116259, 119 AY116300, AY116257, 128 SAUO4788 SBU04789 AY116293, AY116244, 6 SLU04791 SMU04798 SVU04795 TAU46653, (a) TAU46654, (b) AF019818 Chen et al. unpublished Chen et al. unpublished Chen et al. unpublished Chen et al. unpublished Stewart 26672 Lancaster 210 Sun et al. 1994 Sun et al. 1994 Hodkinson 10. 1966-54209 Sun et al. 1994 Sun et al. 1994 Sun et al. 1994 Buckler and Holtsford 1996 Buckler and Holtsford 1996 Hsiao et al. 1999 AY116294, AY116260, 164 ZLU46594 AF019811 AF019816 AF019811 AF019832 AY116301, MWC9279 AF345232 AY116266, 117 AF019833 AF019826 AY116268, 110 Hodkinson 164 Buckler and Holtsford 1996 Hsiao et al. 1999 Hsiao et al. 1999 Hsiao et al. 1999 Hsiao et al. 1999 Salamin s.n. Chen et al. unpublished Hodkinson 117 Hsiao et al. 1999 Hsiao et al. 1999 Hodkinson 110 385 Table 1. Continued Tribe Subtribe Genus/species GenBank, ID number* Voucher or reference Setariinae Echinochloa colona Link E. crus-galli (L.) Beauv. E. crus-galli (L.) Beauv. Panicum bisulcatum Thunb. P. virgatum L. Setaria parviflora (Poir.) M. Kerguelen Stenotaphrum micranthum (Desv.) C.E. Hubbard AJ133708 AJ133707, (a) AY116269, 125 AF019829, (a) AY116267, 120 AF019831 AF019830 Wu unpublished Wu unpublished Hodkinson 125 Hsiao et al. 1999 Hodkinson 120 Hsiao et al. 1999 Hsaio et al. 1999 did not apply the incongruence length difference (ILD) test (or similar congruence tests) as they have been shown to be ineffective in identifying combinability of data and in some cases have been proven to be misleading (Yoder et al. 2001). We chose to base our decision to combine on the pattern of major clades and their respective bootstrap percentages following Reeves et al. (2001). Materials and methods Specimens Specimens were collected from the living collections at the Royal Botanic Gardens, Kew, Surrey, UK and ADAS, Arthur Rickwood Research Station, Cambridge, UK (a consultancy and research organisation for agriculture, food, rural development and environment in the UK and overseas). Voucher specimens of each accession are listed in Table 1. Genus names follow the treatment of Clayton and Renvoize (1986). The provenance of most Miscanthus sinensis specimens is unknown due to their long use in horticulture. DNA extraction DNA was extracted from 0.5–1.0 g of young fresh leaf material using a modified 2X CTAB procedure of Doyle and Doyle (1987), precipitated using 100% ethanol or isopropanol for at least 48 h at –20∞C, pelleted and washed with 70% ethanol and purified via caesium chloride/ethidium bromide (1.55 g/ml) gradient centrifugation with subsequent dialysis to remove salts. Ethidium bromide was extracted with H2O-saturated butanol. DNA was then stored in TE buffer (10 mM Tris–HCl; 1 mM EDTA; pH 8.0) at –80∞C until use. prised 30 cycles, each with 1 min denaturation at 97∞C, 1 min annealing at 51∞C, and an extension of 3 min at 72∞C. A final extension of 7 min at 72∞C was also included. Amplified, double-stranded DNA fragments were purified using Promega Wizard PCR minicolumns and sequenced using Taq Dye-Deoxy Terminator Cycle Sequencing Kits of Applied Biosystems on an Applied Biosystems 373 or 377 automated DNA sequencer, all according to the manufacturer’s protocols and with the same primers as the initial amplification. Sequence editing and assembly of the complementary strands used Sequence Navigator and AutoAssembler programs (Applied Biosystems). Each position was individually inspected to be sure that both strands agreed. Data analysis DNA sequences were aligned by eye or with CLUSTAL W (Thompson et al. 1995) with subsequent manual correction following the guidelines of Kelchner (2000). Gaps were coded as missing data. The resulting matrices were analysed using heuristic search options of PAUP*4.0b8 (Swofford 1998). Searches included 1,000 replicates of random addition sequence (saving no more than 100 trees per replicate to reduce time spent swapping large islands of trees) with subtree pruning regrafting (SPR) branch swapping with MULPARS (keeping multiple equally most parsimonious trees) on. Internal support was assessed using 1,000 bootstrap replicates (Felsenstein 1985) with MULPARS on, but saving no more than 20 trees per replicate to reduce time spent swapping on large numbers of trees, and SPR swapping. A combined analysis of trnL-F and ITS data was also performed using the same parameters as above. A number of species from the tribes Paniceae, Arundinelleae and Eriachneae were chosen as outgroups for the analyses because these have been shown to belong to the same subfamily as the Andropogoneae, but are clearly separate from it (Hsaio et al. 1999; Giussani et al. 2001; Grass Phylogeny Working Group 2001). DNA sequencing The ITS region was amplified by PCR using the forward primer, ITSF, described by White et al. (1990) and the reverse primer 26SE of Sun et al. (1994). The plastid trnL intron and trnL-F intergenic spacer were amplified as one piece using the c and f primers described by Taberlet et al. (1991). The thermal cycling (Applied Biosystems 480) com- Results Analysis of ITS The aligned ITS matrix was 874 base pairs (bp) long; 279 sites were variable and 198 of these were potentially infor- 386 mative. Figure 2 shows one of 1,520 equally most parsimonious trees for ITS sequence data with groups consistent in all shortest trees marked as solid lines. It has 999 steps, with a consistency index (CI) of 0.46 and a retention index (RI) of 0.71. There is moderate support (80% bootstrap percentage; BP) for the monophyly of a group including Andropogoneae and Arundinella (Arundinelleae), but no support greater than 50 BP for Saccharinae Griseb. as defined by Clayton and Renvoize (1986). Many species of Saccharinae group more closely with species of Sorghinae, Chionachininae or Tripsacinae. Themeda triandra of the Anthistiriinae is sister to the rest of Andropogoneae. Miscanthus s.l. is polyphyletic in all shortest trees. However, a core group of Miscanthus species including M. floridulus, M. sacchariflorus, M. sinensis, M. sinensis subsp. condensatus, M. oligostachyus and M. transmorrisonensis forms a clade (group b) supported with 99 BP. Five Saccharum species (group c), S. barberi, S. officinarum (a cultivated sugarcane accession), S. robustum, S. sinense and S. spontaneum are sister to the core Miscanthus clade in all equally most parsimonious trees. Another group of Saccharum s.l. species (group a; Fig. 2), including S. arundinaceum and S. ravennae (= Ripidium or Erianthus section Ripidium), forms a monophyletic group in all shortest trees sister to Imperata, which is distinct from other Saccharum species but with <50 BP. There is weak support (67 BP) for a clade (group d) containing the African Miscanthus species, M. junceus and M. ecklonii, the Himalayan M. fuscus and a number of Saccharum species including S. contortum (= Erianthus contortus), S. narenga (= Narenga porphyrocoma) and Erianthus rockii, but no support for the inclusion of these with the core group of Miscanthus species (no combination of E. rockii has been made with Saccharum and, therefore, we treat it as Erianthus despite believing it should be combined with Miscanthidium or Saccharum; we are currently investigating its nomenclature further). There is 88 BP support for the grouping of M. fuscus, S. narenga and E. rockii. These data indicate that the closest relatives of the African Miscanthus species and M. fuscus are a group of Saccharum s.l. species and not other Miscanthus s.s. species. Miscanthus nepalensis is also clearly separate from the core monophyletic southeastern Asian Miscanthus group and associates more closely with members of Eulalia and Sorghum, but this does not receive >50 BP. Analysis of trnL-F and combined ITS and trnL-F The aligned trnL-F matrix was 1,042 bp long; 132 sites were variable and 26 of these were potentially informative. Analysis of the trnL-F matrix produced 17,920 equally most parsimonious trees (125 steps, CI = 0.93, RI = 0.89) with poor internal support (Fig. 3). The trnL-F data were joined with the ITS data in a combined analysis. No conflict between major clades of the trnL-F and the ITS analyses was identified. Furthermore, the bootstrap support values of the major clades in the combined ITS and trnL-F analysis were equal to (clade b and c), or greater than (clade d) the same clades in the individual ITS analysis. Therefore, we based our decision to combine on a close examination of the clades present in the trees and the support for each of these. The combined trnL-F and ITS matrix was 2,147 bp long and contained 329 variable sites of which 149 were potentially informative. Analysis produced 32,039 trees of length 614, CI of 0.69 and RI of 0.68 (Fig. 4). Neither Miscanthus s.l. or Saccharum s.l. are monophyletic. A monophyletic Miscanthus s.s. group (group b) can be identified (100 BP). Miscanthus floridulus, M. sinensis and M. transmorrisonensis group together with 95 BP. Saccharum officinarum/S. spontaneum (group c; 95 BP) are sister to Miscanthus s.s. in all equally most parsimonious trees, but this is not supported by BP. The African Miscanthus species, M. fuscus and Saccharum contortum form a clade with 86 BP (group d). Miscanthus nepalensis falls in an isolated position. Discussion Phylogenetics of Miscanthus, Saccharum and related genera On the basis of DNA data, both Miscanthus s.l. and Saccharum s.l. are polyphyletic. There is support for monophyletic groups within the Saccharum complex, but these do not correspond to current taxonomic groupings. Among these are a well-supported core group of Miscanthus species (group b; Figs. 2 and 4), including M. floridulus, M. oligostachyus, M. sacchariflorus, M. sinensis, M. sinensis subsp. condensatus and M. transmorrisonensis and a core group of Saccharum species (group c) corresponding to Saccharum s.s. (Price 1963; Daniels and Roach 1987; Irvine 1999). It has a basic chromosome number of 19 that is unique in Saccharinae for which the basic number of 10 predominates. Price (1963) and Daniels and Roach (1987) included six species in Saccharum s.s. (S. barberi, S. edule, S. officinarum, S. robustum, S. sinense and S. spontaneum). However, S. robustum is often synonymised with S. spontaneum; S. barberi or S. sinense are also often included with S. officinarum. We included five of these species in our analyses and found them to form a sister group to Miscanthus s.s. in all equally most parsimonious trees for ITS sequence data and for the combined ITS and trnL-F analysis. These findings would support the conclusion of Clayton and Renvoize (1986), who considered Saccharum the closest relative of Miscanthus s.s. Therefore, our results indicate that Saccharum s.s. is more closely related to Miscanthus s.s. than other members of Saccharum or the “Saccharum complex”. The other Saccharum species (Saccharum complex) group more closely with other Miscanthus species such as the African Miscanthus s.l. (= Miscanthidium) and the Himalayan (M. nepalensis and M. fuscus). Saccharum is separated from Miscanthus primarily on the basis of its fragile rachis (Miscanthus has a tough rachis, not breaking up at maturity) and one of its paired spikelets being sessile (in Miscanthus both spikelets of the pair are pedicellate, although they are not of equal length). 387 2 Fig. 2. Parsimony tree of ITS sequence data for subtribe Saccharinae and related genera. One of 1,520 equally most parsimonious trees of length = 999; CI = 0.46, RI = 0.71. Values above branches are steps. Numbers below branches are bootstrap percentages above 50%. Groups found in all shortest trees are indicated by solid lines (groups not found in all shortest trees by a dotted line) 388 3 Fig. 3. Parsimony tree for Miscanthus, Saccharum and related genera for the trnL-F intron and spacer regions of plastid DNA. One of 17,920 equally most parsimonious trees. Length = 125, CI = 0.93, RI = 0.89. Values above branches are steps. Numbers below branches are bootstrap percentages above 50% Saccharum robustum had two different ITS sequence types, one grouping with Miscanthus s.s. and the other with Saccharum s.s. in our analysis. The ITS region is represented by numerous tandem repeats in plant genomes (Baldwin et al. 1995), and in most species the repeat units are homogenised (via concerted evolution; Cronn et al. 1996) so that one sequence predominates. It is likely that there has been considerable hybridisation and introgression between Miscanthus and Saccharum (Al-Janabi et al. 1994), and in some Saccharum species such as S. robustum a mixture of both Saccharum and Miscanthus ITS repeat types exist, which provides evidence of intergeneric hybridisation. Miscanthus has also been implicated by others in the evolution of S. officinarum. Al-Janabi et al. (1994) showed that one Miscanthus species from New Guinea, with a high chromosome number of 192, had the same plastid restriction sites as the majority of the Saccharum genus, suggesting that hybridisation had occurred. The results of our analysis with ITS and combined ITS and trnL-F data indicate that Saccharum s.l. is not monophyletic. The taxonomy of Saccharum is complex, and interspecific and intergeneric hybrids further confuse the situation (Daniels et al. 1975; Al-Janabi et al. 1994). Basic chromosome number varies (D’Hont et al. 1995, 1996), and polyploidy adds further complexity to systematic studies. Modern day sugarcane has 2n = 80 with a basic chromosome number of 10; its wild relative, S. spontaneum, has 2n = 40– 128 (D’Hont et al. 1995). In Andropogoneae x = 10 predominates (Burner 1991). Most varieties of sugarcane are derived from crosses between S. officinarum (noble cane) and S. spontaneum, but introgression between these and other species of the Saccharum complex (such as Erianthus) has also been shown (Besse et al. 1996; Janoo et al. 1999a, b). A number of groups can be identified within Saccharum s.l. from the ITS sequence data (Fig. 2), but these do not 389 4 Fig. 4. Parsimony tree for Miscanthus, Saccharum and related genera for the combined data matrix of ITS and the trnL-F intron and spacer regions of plastid DNA. One of 32,039 equally most parsimonious trees. Length = 614, CI = 0.69, RI = 0.68. Values above branches are steps. Numbers below branches are bootstrap percentages above 50%. Groups found in all shortest trees are indicated by solid lines (groups not found in all shortest trees by a dotted line) correspond to previous groupings such as Erianthus or Narenga. The genus Erianthus was separated from Saccharum by Michaux in 1803 and based on the New World species E. saccharoides Michaux. The division was based primarily on the possession of an awn in Erianthus. There is little evidence from ITS data to support such a division. Erianthus has also been split into New and Old World (section Ripidium) groups (Grassl 1972; Besse et al. 1996). A restriction enzyme study of plastid DNA from various members of Saccharinae by Sobral et al. (1994) showed that Miscanthus, Narenga, Saccharum and Sclerostachya form a monophyletic group that was different from the Old World Erianthus species (including E. arundinaceus and E. ravennae), which were more closely related to Sorghum bicolor. Two of the five Old World Erianthus species (E. ravennae and E. arundinaceus) studied by Sobral et al. (1994) were included in our study and were also found to be distinct from other Saccharum s.l. species. According to Grassl (1972) New World Erianthus species are distinct from the Old World species in many morphological attributes. The New World species have only two anthers, whereas the Old World species have three. New World species also have floral parts with strong awns and large seeds (primarily for animal dispersal) whereas Old World species are adapted for wind dispersal (Grassl 1972). These Old World Erianthus species were treated as Ripidium by Trinius in 1820 and Grassl (1972). Grassl (1972) agreed with Trinius that Ripidium ravennae (L.) Trin. was separate from both Erianthus and Saccharum and created a number of new combinations within Ripidium including R. arundinaceum (Retz.) Grassl, R. bengalense (Retz.) Grassl, R. elephantinum (Hook f.) Grassl (treated by some as a synonym of E. ravennae), R. kanashiroi (Ohwi) Grassl and R. procerum (Roxb.) Grassl. Our analysis, in accordance with Grassl (1972) indicates that Ripidium is a distinct group because these species grouped together (group a; Fig. 2), 390 but separate from other Saccharum s.l. in all equally most parsimonious trees. There is support (67 BP in the ITS tree; 86 BP in the combined tree) for a clade (group d; Figs. 2 and 4) containing the African Miscanthus species, M. fuscus (Himalayan) and Saccharum narenga (Narenga porphyrocoma; Himalayan), Erianthus rockii (China/Burma/Indo-China) and Saccharum contortum (Erianthus contortus; New World). There is, however, no support for the monophyly of Narenga as its two species do not group together. Grassl came to this conclusion in 1972 and chose to combine Narenga with Sclerostachya because both have a chromosome count of 2n = 30 and behave similarly in crosses with Saccharum (Grassl 1972). Saccharum fallax (= Narenga fallax) would not appear to be part of this group on the basis of the ITS sequence data. Therefore, there is support for a group that would include Sclerostachya and species assigned to Erianthus (= Saccharum), Miscanthus s.l. (M. fuscus), and Narenga. The closest allies of this group would, on the basis of sequence data, appear to be the Miscanthidium segregates of Miscanthus s.l., but also Saccharum contortum, a north American species. Sorghum is polyphyletic in the ITS analysis and some species are embedded within the group of related genera of the “Saccharum complex” in our analysis (Fig. 2). It is not usually included in this complex, but is one of the six genera that has been successfully hybridised with Saccharum (Narenga, Miscanthidium, Miscanthus, Narenga, Sclerostachya and Sorghum). Furthermore, genetic maps of Saccharum and Sorghum based on single dose DNA markers (Guimaraes et al. 1997) and RFLPs (Dufour et al. 1997) have demonstrated high colinearity between Saccharum and Sorghum genomes. Infrageneric relationships of Miscanthus The subclades of Miscanthus in the ITS and combined trees do not fully agree with the sections defined by Lee (1964b, c). The core group of Miscanthus represents the southeastern Asian species of Miscanthus sections Kariyasua, Miscanthus and Triarrhena of Lee (1964b, c). Data from chromosome numbers support the monophyly of some of the south-eastern Asian Miscanthus. The sections Kariyasua, Miscanthus and Triarrhena all have a basic chromosome number (x) of 19, which is not found in any related taxon (x = 10 predominates in the Andropogoneae) and may represent a synapomorphy for this clade. Miscanthus nepalensis, section Diandra, is separate from this core Miscanthus clade in all equally most parsimonious trees, but there is no support >50 BP for this separation. Amplified fragment length polymorphism (AFLP) DNA fingerprinting (Vos et al. 1995; Reeves et al. 1998; Carolan et al. 2002) data showed that M. nepalensis was clearly separate from other Miscanthus species (Hodkinson et al. 2002b). Species of section Diandra resemble Miscanthus s.s. on morphological grounds, but possess two anthers, instead of the usual three, and a basic chromosome number of five or ten instead of 19 (Mehra and Sharma 1975). It is not clear from this analysis which genus they belong to or what their closest relatives are. They are included by some in the genus Diandranthus (Trin.) L. Liu. Two anthers are also found in a number of New World Saccharum s.l. species, but there is no evidence to link these with M. nepalensis in our analyses. The ITS and the combined trees indicate that the African Miscanthus taxa and M. fuscus (Himalayan) may not form a monophyletic group with Miscanthus s.s. Saccharum contortum groups with these taxa, and it is highly probable that these species are more closely allied to members of Saccharum s.l. than to Miscanthus. The African species of Miscanthus (= Miscanthidium) have a basic chromosome number (x) of 15 compared with 19 in Miscanthus s.s. They possess an elongated inflorescence axis compared with Miscanthus s.s., which are all subdigitate (except M. floridulus) and have leathery/papery (coriaceous), two-keeled glumes compared with papery/membranous, one-five nerved, glumes in Miscanthus s.s. Miscanthus fuscus has an elongated inflorescence axis like M. floridulus, but its spikelets have coriaceous glumes like the African Miscanthus species. There is, therefore, good molecular and morphological evidence to separate M. fuscus (Sclerostachya fusca) and the African Miscanthus species (= Miscanthidium) from Miscanthus s.s. Miscanthus section Triarrhena (M. sacchariflorus) is monotypic and part of a polytomy with M. oligostachyus (section Karyiasua) and the species of Miscanthus section Miscanthus in the ITS analysis. Data from the DNA fingerprinting technique AFLP (Hodkinson et al. 2002b) indicate that M. oligostachyus is sister to a monophyletic group containing species of M. sections Miscanthus and Triarrhena (M. floridulus, M. sacchariflorus, M. sinensis and M. transmorrisonensis). Miscanthus oligostachyus and the other members of M. section Kariyasua (M. tinctorius and M. changii, not sequenced) have few racemes in comparison to the other sections and can be separated on morphological grounds from M. section Triarrhena by their possession of awns and short spikelet callus hairs. They are restricted in distribution to Japan. Miscanthus section Kariyasua is sister to this group. Our data, therefore, would support the inclusion of three of the sections in Miscanthus s.s. (Sinensis, Triarrhena and Kariyasua) instead of the four defined by Lee (1964b, c, d) because Lee also chose to recognise Miscanthus section Diandra. It is not possible, from our results, to evaluate the monophyly of these sections except for section Miscanthus, which is monophyletic in our analysis (95 BP in the combined analysis). Conclusions On the basis of the DNA sequence data Miscanthus s.l. and Saccharum s.l. are polyphyletic. Saccharum section Ripidium is separate from all other Saccharum species in our analysis (group a, Figs. 2 and 4) and may be best treated as the genus Ripidium Trin. following Grassl (1972). Six Miscanthus species form a well-supported clade (group b; Fig. 2), and these have a unique basic chromo- 391 some number of 19. Miscanthus ¥ giganteus Greef & Deuter ex Hodkinson & Renvoize (Hodkinson and Renvoize 2001), an allopolyploid hybrid of M. sinensis and M. sacchariflorus, would also form part of this group. The Himalayan species M. fuscus groups more closely with the African Miscanthidium and Saccharum contortum than with the southeastern Asian Miscanthus s.s. Miscanthus section Diandra of Lee (1964d), also found in the Himalayan region, and represented by M. nepalensis, does not group with any member of Miscanthus s.l.. Recently, its species have been recognised as a separate genus, Diandranthus (Trin) L. Liu, and our molecular analysis would add weight to this separation. Species corresponding to Saccharum s.s. form a sister group to Miscanthus s.s. (group c; Figs. 2 and 4). Two different ITS sequences from S. robustum are individually shown to be related to Miscanthus and Saccharum, respectively, and the former provides evidence of hybridisation between these closely allied genera. A further clade (group d; Figs. 2 and 4) contains species of Miscanthus s.l. and Saccharum s.l. (including some of their segregate genera Narenga, Miscanthidium, and Sclerostachya). On the basis of our evidence, the genus Miscanthidium should be recognised and a number of new taxonomic combinations made. Miscanthus fuscus, Saccharum contortum, S. narenga and Erianthus rockii should be combined with Miscanthidium. Miscanthidium also includes M. capense (Nees) Stapf. (= Miscanthus ecklonii), M. sorghum (Nees) Stapf. (= Miscanthus sorghum), M. teretifolium (Nees) Stapf. (= Miscanthus junceus) and M. violaceum (K. Schum) Robyns (= Miscanthus violaceus). Therefore, we can recognise the distinction between Saccharum s.s., Miscanthus s.s., Ripidium (= Saccharum section Ripidium) and a newly defined Miscanthidium group, and there is evidence to support the monophyly of each of these. The other members of the “Saccharum complex” are more difficult to position. More studies are needed to find a better way of subdividing the remaining members of the Saccharinae (including the Saccharum complex) into genera or infrageneric taxa. Acknowledgments This work was supported by the Ministry of Agriculture, Fisheries and Food, UK (MAFF project code QA3580) the Irish Higher Education Authority, and the Enterprise Ireland International Collaboration Scheme (IC/2001/029). We would like to thank Mike Bullard and Peter Nixon at ADAS, UK, for the collection and maintenance of a large living collection of Miscanthus. We thank Louise Scally and Steve Waldren at Trinity College Dublin for the provision of two Miscanthus floridulus accessions and Mary Thorpe of the Living Collections Department, Royal Botanic Gardens, Kew, UK for her assistance throughout this project. Thanks also to Mike Fay for his assistance with the molecular analysis and Derek Clayton for his assistance with the World Grasses Database and general interpretation of results. References Adati S, Shiotani I (1962) The cytotaxonomy of the genus Miscanthus and its phylogenetic status. 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