Phylogenetics of Miscanthus, Saccharum and Related Genera

Home , Eriochrysis, Eulalia (plant), Eulaliopsis, Hemarthria, Imperata, Lophopogon

J Plant Res (2002) 115:381–392 © The Botanical Society of Japan and Springer-Verlag Tokyo 2002 Digital Object Identiﬁer (DOI) 10.1007/s10265-002-0049-3

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 Introduction and related genera in the Saccharum complex. Three DNA regions were sequenced, including the trnL intron and the Tribe Andropogoneae (Poaceae) includes many species trnL-F intergenic spacer of the plastid genome and the ITS with high economic value, including the C4 grasses Saccha- region of nuclear ribosomal DNA (nrDNA). Because it was rum officinarum L. (sugarcane), Sorghum bicolor (L.) more variable, the ITS region proved most suitable for phy- Moench (sorghum) and Zea mays L. (maize). Subtribe Sac- logenetic reconstruction at this level, and the results indi- charinae Griseb. includes Saccharum L. and Miscanthus cate that Miscanthus s.l. and Saccharum s.l. are polyphyletic. Anderss., the latter having considerable potential as a bio- A set of species from Saccharum section Ripidium (clade a) mass crop for renewable energy production and raw mate- do not group closely with any members of Saccharum s.l.. A rial for the cellulose and paper industries (Bullard et al. number of Miscanthus species from eastern or south- 1995; Clifton-Brown and Lewandowski 2000). Saccharinae eastern Asia represent a monophyletic group with a basic according to Clayton and Renvoize (1986), also include chromosome number of 19 (clade b), but the other species Eriochrysis P. Beauv., Eulalia Kunth, Eulaliopsis Honda, from Africa and the Himalayas are clearly excluded. There Homozeugus Stapf., Imperata Cyr., Lophopogon Hack., is support for a monophyletic Saccharum s.s. clade including Microstegium Nees, Pogonatherum P. Beauv., Polytrias S. officinarum and S. spontaneum that is sister to Miscanthus Hack. and Spodiopogon Trin. The subtribe is morphologi- s.s. (clade c). There is no evidence to support the division cally defined by their terminal inflorescence (except Eulali- of some Saccharum s.l. into the genera currently known as opsis and Pogonatherum) of solitary or digitate racemes and Erianthus and Narenga. Saccharum contortum (= Erianthus paired similar spikelets. The paired spikelets are often plu- contortus), S. narenga (= Narenga porphyrocoma) and mose; the callus is rounded or truncate. The lower glume is Erianthus rockii, group more closely with Miscanthus fus- mostly thin, and the lower floret is usually reduced to a cus, a species from the Himalayas and also with the African sterile lemma. The upper lemma is entire or bilobed and Miscanthus s.l. species (= Miscanthidium, clade d). can have a glabrous awn. Despite these characteristics the only known morphological synapomorphy for Saccharinae Key words Erianthus • Miscanthus • Molecular • would be their bisexual paired spikelets. Other Andropogo- Saccharum • Sugarcane • Systematics neae 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 Schizach- rium (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 • • • T.R. Hodkinson M.W. Chase M.D. Lledó S.A. Renvoize sugarcane and in which the taxonomy is particularly con- Royal Botanic Gardens, Kew, Richmond, Surrey, UK fused (Daniels and Roach 1987). There is a need to charac- T.R. Hodkinson (*) • N. Salamin terise this complex more comprehensively. Department of Botany, Trinity College, University of Dublin, Dublin 2, The monophyletic status of many genera within Saccha- Ireland Tel. +353-16081128; Fax +353-16081147 rinae is also in doubt. The most widely debated is Saccharum e-mail: [email protected] itself. Saccharum s.l. (Clayton and Renvoize 1986) has been Springer-VerlagTokyoJournal of Plant ResearchJ Plant Res102650918-94401618-0860s10265-002-0049-30049Bot Soc Jpn and Springer-VerlagOriginal Article

382 divided into a number of other genera including Erianthus However, they also recognised that Eriochrysis, Eulalia, Michaux, Narenga Bor and Ripidium Trin., but Clayton and Imperata, Miscanthus, Saccharum and Spodiopogon form a Renvoize (1986) chose to combine all of these genera under closely knit group in which the phylogenetic relationships Saccharum because the characters used to define them were are unclear. Saccharum is considered by many as the closest thought to be more suited to infra-generic categorisation. relative of Miscanthus, and these two genera frequently They argued that the division of awned (Erianthus) and hybridise (Sobral et al. 1994). awnless species is artificial and the separation of Narenga, The species of Miscanthus can be distinguished from with its coriaceous glumes, is trivial because this is no more those of Saccharum by their tough inflorescence rachis and than an extreme expression of a trend found elsewhere in both spikelets of a pair being pedicellate, although the the genus. Saccharum s.l. has approximately 40 species pedicels are of different lengths. Most members of Miscant- (Clayton and Renvoize 1986), Erianthus approximately 20 hus s.l. are native to eastern or south-eastern Asia (China, and Narenga only two (Adati and Shiotani 1962). Saccha- Japan and neighbouring regions); two species are known rum s.s. (Price 1963; Daniels and Roach 1987) is distributed from the Himalayas and four from southern Africa (Fig. 1). throughout the tropics and subtropics due to cultivation, but In the past, the African species have been placed in a sep- the species are native to south-eastern Asia. Erianthus is cos- arate genus, Miscanthidium Stapf, mainly on the basis of mopolitan and, according to Celarier (1956), can be divided their elongate inflorescence axis and short racemes, but into two subgenera, one with two anthers and an American these differences were not considered sufficient to warrant distribution; the other with three anthers and an Old World separation from Miscanthus by Clayton and Renvoize distribution. The Old World distribution of Saccharum s.l. is (1986) because M. floridulus (Labill.) Warb. Ex. K. Schum. given in Fig. 1. The New World species are predominantly & Lauterb. and M. fuscus (Roxb.) Benth. [= Sclerostachya found in North America. fusca (Roxb.) A. Camus] in Asia also have elongate inflo- The taxonomic status of Miscanthus is also in a state of rescence axes. flux, and little is known about the identity and inter- Groups of species at sectional rank within Miscanthus relationships of its species. According to Clayton and have been recognised, and a key to Miscanthus species was Renvoize (1986), Miscanthus s.l. comprises approximately given in Hodkinson et al. (1997). The most comprehensive 20 species and appears well-defined morphologically. 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 Using DNA sequence data of nuclear and plastid DNA broadly agreed with the treatments of Honda (1930) and regions, this study aimed to assess the monophyly of Mis- Adati and Shiotani (1962). There are four African Miscant- canthus and Saccharum and their phylogenetic relationships hus species not included in the genus by Lee (1964b, c, d) to other Saccharinae. DNA sequencing is particularly well namely: M. ecklonii (Nees) Mabb., M. junceus (Stapf) suited for phylogenetic studies and has been used exten- Pilger, M. sorghum (Nees) Pilger and M. violaceus (K. sively for such purposes at many different taxonomic levels Schum.) Pilger. Himalayan M. fuscus was also excluded (Chase et al. 1993; Hsiao et al. 1994; Soltis et al. 1999; from Miscanthus by Lee (and recognised as Sclerostachya Salamin et al. 2002). The internal transcribed spacer (ITS) fusca), and has been included by Clayton and Renvoize regions of nuclear ribosomal DNA (for a review see Baldwin (1986). Miscanthus brevipilus Hand.-Mazz., M. changii, Y.N. et al. 1995), and the trnL intron and trnL-F intergenic spacer Lee, and M. eulaliodes Keng ex. Hand.-Mazz., which were of plastid DNA (hereafter trnL-F; Taberlet et al. 1991; Gielly listed by Lee (1964b, c, d), have not been evaluated here and Taberlet 1994; Hopper et al. 1999; Molvray et al. 1999; because they were not available for study. Miscanthus trans- Chase et al. 2000; Lledó et al. 2000) were used to construct morrisonensis appears to intergrade with M. sinensis on a phylogenetic hypotheses with parsimony methods. The ITS morphological level. Another taxon known as M. conden- region has proven useful for phylogenetic studies at this tax- satus Hackel has been given species rank by previous onomic level in various plant groups, including grasses authors, but falls within the normal range of morphological (Baldwin et al. 1995; Hsiao et al. 1995a, b, 1999; Hodkinson variation found in M. sinensis (Koyama 1987). Lee (1964a) et al. 2000; Grass Phylogeny Working Group 2001; recognised M. condensatus on the basis of leaf anatomy Hodkinson et al. 2002a) and so has the trnL-F region (Briggs because it differs in having unclosed bundles in the midrib. and Johnson 2000; Briggs et al. 2000). A number of grass In addition, M. condensatus only grows at low elevation in ITS sequences have been published and/or deposited in the coastal zone of Japan. We have treated M. condensatus GenBank. We have utilised these sequences to provide a here as M. sinensis subsp. condensatus (Hackel) T. Koyama more comprehensive sample of Saccharinae (Table 1). We (following Koyama 1987). 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. AY116299, AY116263, 75 Hodkinson 15. 1969-19004 Cymbopogon citratus L. AY116258, 129 Hodkinson 129 Cymbopogon citratus L. AF019823, (a) Hsiao et al. 1999 Anthristiriinae Themeda triandra Forsk. AY116261, MWC9286, (a) Salamin s.n. Themeda triandra Forsk. AF019820 Hsiao et al. 1999 Chionachninae Chionachne cyathopoda F.Muell. ex AF019819 Hsiao et al. 1999 Benth. Rottboellinae Hemarthria uncinata R.Br. AF019821 Hsiao et al. 1999 Saccharinae Erianthus rockii Keng AF345216, (a) Chen et al. unpublished E. rockii Keng AF345217, (b) Chen et al. unpublished Eulalia irritans (R.Br.) Kuntze AY116298, AY116242, 137 Adams 1756 (= Poganatherum irritans) E. quadrinervis (Hack.) Kuntze AY116303, AY116251, 134 Polunin et al. 3294 E. trispicata (Schult.) Henrard AY116291, 138 Clarkson 10062 E. villosa (Thunb.) Nees AY116302, 132 Devenish 1282 Imperata cylindrica (L.) P.Beauv. AY116297, AY116262, 122 Marsden 3 Raeuschel I. cylindirca (L.) P.Beauv. Raeuschel AF345653, (a) Chen et al. unpublished I. cylindirca (L.) P.Beauv. Raeuschel AF092512, (b) Tsai and Chou unpublished Miscanthus sp. Anderss. AY116283, AY116243, 155 Phillips 155 M. ecklonii (Nees) Mabb. AY116290, AY116264, 86 du Toit 2347 M. floridulus (Labill.) Warb. ex. K. Schum. AY116278, AY116248, 72 Hodkinson 30. 1978-1387 & Lauterb. M. floridulus (Labill.) Warb. ex. K. Schum. AY116280, TRH1 (a) Scally s.n. & Lauterb. M. floridulus (Labill.) Warb. ex. K. Schum. AY116281, TRH2 (b) Scally s.n. & Lauterb. M. fuscus (Roxb.) Benth. AY116286, AY116265, 83 Mangelsdorf 1955 US56-5-5 M. junceus (Stapf) Pilger AY116288, AY116254, 88 Tinley 1060 M. junceus (Stapf) Pilger AY116289, AY116255, 89 Simon 2309 M. nepalensis (Trin.) Hack. AY116292, AY116252, 25 Hodkinson 1 M. oligostachyus Stapf AY116277, AY116249, 16 Hodkinson 13 M. oligostachyus Stapf AY116279, AY116245, 161 Hodkinson 161 384

Table 1. Continued

Tribe Subtribe Genus/species GenBank, ID number* Voucher or reference

M. sacchariflorus (Maxim.) Benth. & AJ426564, AY116247, 61 Hodkinson s.n. 1987-2727 Hook. “Purpurascens” M. sacchariflorus (Maxim.) Benth. & AY116282, AY116246, 5791 Renvoize 5791 Hook. M. sinensis Anderss. var. variegatus Beal. AY116276, 1 Hodkinson 33 M. sinensis Anderss. AJ426565, AJ426571, 5 Hodkinson 40. 1978-1389 M. sinensis Anderss. subsp. condensatus AY116270, AJ426573, 7 Renvoize s.n. 1969-19091 (Hackel) T. Koyama M. sinensis Anderss. “Gracillimus” AY116274, 28 Hodkinson s.n. MB94/05 M. sinensis Anderss. “Roland” AY116272, 29 Hodkinson s.n. MB94/06 M. sinensis Anderss. AJ426566, AJ426572, 30 ADAS MB94/07 M. sinensis Anderss. “Grosse Fontane” AY116273, 31 Hodkinson PN95/01 M. sinensis Anderss. “Yakushimanum” AY116275, 63 Hodkinson 21. 1987-1148 M. transmorrisonensis Hayata AY116271, AY116250, 65 Hodkinson 20. 1990-2748 Saccharum arundinaceum Retz. AY116295, 118 Shiu Ying Hu 11199 S. arundinaceum Retz. AF345201, (a) Chen et al. unpublished S. arundinaceum Retz. AF345202, (b) Chen et al. unpublished S. barberi Jeswiet AF331657, (a) Chen et al. unpublished S. barberi Jeswiet AF345199, (b) Chen et al. unpublished S. contortum (Ell.) Nutt. (= Erianthus AY116287, MWC9284, (a) Salamin s.n. contortus Baldw. Ex Ell.) S. contortum (Ell.) Nutt. (= Erianthus AY116256, 121 Renvoize 3797 contortus Baldw. Ex Ell.) S. fallax Balansa (= Narenga fallax AF345213 Chen et al. unpublished (Balansa) Bor) S. fulvus R.Br. (= Erianthus fulvus (R.Br.) AF345218, (a) Chen et al. unpublished Kunth) S. fulvus R.Br. (= Erianthus fulvus (R.Br.) AF345219, (b) Chen et al. unpublished Kunth) S. narenga Wall. (= Narenga AF345233, (a) Chen et al. unpublished porphyrocoma (Hance) Bor) S. narenga Wall. (= Narenga AF345234, (b) Chen et al. unpublished porphyrocoma (Hance) Bor) S. officinarum L. (sugarcane cv.) AY116284, AY116253, 104 Kew 1973-12242 S. ravennae Murr. (= Erianthus ravennae AF019824, (a) Hsiao et al. 1999 Beauv.) S. ravennae Murr. (= Erianthus ravennae AY116296, MWC9285 (b) Salamin s.n. Beauv.) S. robustum Brandes & Jesw. ex Grassl AF345237, (a) Chen et al. unpublished S. robustum Brandes & Jesw. ex Grassl AF345238, (b) Chen et al. unpublished S. sinense Roxb. AF345242, (a) Chen et al. unpublished S. sinense Roxb. AF345243, (b) Chen et al. unpublished S. spontaneum L. AY116285, AY116259, 119 Stewart 26672 Spodiopogon sibiricus Trin. AY116300, AY116257, 128 Lancaster 210 Sorghinae Sorghum australiense Garber & Synder SAUO4788 Sun et al. 1994 S. bicolor (L.) Moench SBU04789 Sun et al. 1994 S. halepense (L.) Pers. AY116293, AY116244, 6 Hodkinson 10. 1966-54209 S. laxiflorum F.M. Bailey SLU04791 Sun et al. 1994 S. macrospermum E.D. Garber SMU04798 Sun et al. 1994 S. versicolor Anderss. SVU04795 Sun et al. 1994 Tripsacinae Tripsacum australe Cutler & E.Anders. TAU46653, (a) Buckler and Holtsford 1996 T. australe Cutler & E.Anders. TAU46654, (b) Buckler and Holtsford 1996 Zea perennis (Hitchc.) Reeves & AF019818 Hsiao et al. 1999 Mangelsd. Z. diploperennis Iltis, Doebley & Guzman AY116294, AY116260, 164 Hodkinson 164 Z. luxurians (Durieu) R.M.Bird ZLU46594 Buckler and Holtsford 1996 Z. mays L. AF019811 Hsiao et al. 1999 Arundinelleae Arundinella nepalensis Trin. AF019816 Hsiao et al. 1999 Erianchneae Eriachne triseta Nees ex. Steud. AF019811 Hsiao et al. 1999 Paniceae Cenchrinae Cenchrus ciliaris L. AF019832 Hsiao et al. 1999 C. incertus M.A. Curt. AY116301, MWC9279 Salamin s.n. Pennisetum purpureum Schum. AF345232 Chen et al. unpublished P. macrourum Trin. AY116266, 117 Hodkinson 117 P. setaceum (Forsk.) Chiov. AF019833 Hsiao et al. 1999 Digitariinae Digitaria sanguinalis (L.) Scop. AF019826 Hsiao et al. 1999 D. sanguinalis (L.) Scop. AY116268, 110 Hodkinson 110 385

Table 1. Continued

Tribe Subtribe Genus/species GenBank, ID number* Voucher or reference

Setariinae Echinochloa colona Link AJ133708 Wu unpublished E. crus-galli (L.) Beauv. AJ133707, (a) Wu unpublished E. crus-galli (L.) Beauv. AY116269, 125 Hodkinson 125 Panicum bisulcatum Thunb. AF019829, (a) Hsiao et al. 1999 P. virgatum L. AY116267, 120 Hodkinson 120 Setaria parviﬂora (Poir.) M. Kerguelen AF019831 Hsiao et al. 1999 Stenotaphrum micranthum (Desv.) C.E. AF019830 Hsaio et al. 1999 Hubbard

did not apply the incongruence length difference (ILD) test prised 30 cycles, each with 1 min denaturation at 97∞C, 1 min (or similar congruence tests) as they have been shown to be annealing at 51∞C, and an extension of 3 min at 72∞C. A final ineffective in identifying combinability of data and in some extension of 7 min at 72∞C was also included. Amplified, cases have been proven to be misleading (Yoder et al. 2001). double-stranded DNA fragments were purified using We chose to base our decision to combine on the pattern of Promega Wizard PCR minicolumns and sequenced using major clades and their respective bootstrap percentages Taq Dye-Deoxy Terminator Cycle Sequencing Kits of following Reeves et al. (2001). Applied Biosystems on an Applied Biosystems 373 or 377 automated DNA sequencer, all according to the manufac- turer’s protocols and with the same primers as the initial amplification. Sequence editing and assembly of the com- Materials and methods plementary strands used Sequence Navigator and AutoAs- sembler programs (Applied Biosystems). Each position was Specimens individually inspected to be sure that both strands agreed. Specimens were collected from the living collections at the Royal Botanic Gardens, Kew, Surrey, UK and ADAS, Data analysis Arthur Rickwood Research Station, Cambridge, UK (a consultancy and research organisation for agriculture, food, DNA sequences were aligned by eye or with CLUSTAL W rural development and environment in the UK and over- (Thompson et al. 1995) with subsequent manual correction seas). Voucher specimens of each accession are listed in following the guidelines of Kelchner (2000). Gaps were Table 1. Genus names follow the treatment of Clayton and coded as missing data. The resulting matrices were analysed Renvoize (1986). The provenance of most Miscanthus using heuristic search options of PAUP*4.0b8 (Swofford sinensis specimens is unknown due to their long use in 1998). Searches included 1,000 replicates of random addi- horticulture. tion 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 DNA extraction MULPARS (keeping multiple equally most parsimonious trees) on. Internal support was assessed using 1,000 boot- DNA was extracted from 0.5–1.0 g of young fresh leaf strap replicates (Felsenstein 1985) with MULPARS on, but material using a modified 2X CTAB procedure of Doyle saving no more than 20 trees per replicate to reduce time and Doyle (1987), precipitated using 100% ethanol or iso- spent swapping on large numbers of trees, and SPR swap- propanol for at least 48 h at –20∞C, pelleted and washed ping. A combined analysis of trnL-F and ITS data was also with 70% ethanol and purified via caesium chloride/ethid- performed using the same parameters as above. A number ium bromide (1.55 g/ml) gradient centrifugation with subse- of species from the tribes Paniceae, Arundinelleae and quent dialysis to remove salts. Ethidium bromide was Eriachneae were chosen as outgroups for the analyses extracted with H2O-saturated butanol. DNA was then because these have been shown to belong to the same sub- stored in TE buffer (10 mM Tris–HCl; 1 mM EDTA; pH 8.0) family as the Andropogoneae, but are clearly separate from at –80∞C until use. 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 Results primer, ITSF, described by White et al. (1990) and the reverse primer 26SE of Sun et al. (1994). The plastid trnL Analysis of ITS intron and trnL-F intergenic spacer were amplified as one piece using the c and f primers described by Taberlet et al. The aligned ITS matrix was 874 base pairs (bp) long; 279 (1991). The thermal cycling (Applied Biosystems 480) com- sites were variable and 198 of these were potentially infor- 386 mative. Figure 2 shows one of 1,520 equally most parsimo- equal to (clade b and c), or greater than (clade d) the same nious trees for ITS sequence data with groups consistent in clades in the individual ITS analysis. Therefore, we based all shortest trees marked as solid lines. It has 999 steps, with our decision to combine on a close examination of the a consistency index (CI) of 0.46 and a retention index (RI) clades present in the trees and the support for each of these. of 0.71. The combined trnL-F and ITS matrix was 2,147 bp long and There is moderate support (80% bootstrap percentage; contained 329 variable sites of which 149 were potentially BP) for the monophyly of a group including Andropogo- informative. Analysis produced 32,039 trees of length 614, neae and Arundinella (Arundinelleae), but no support CI of 0.69 and RI of 0.68 (Fig. 4). Neither Miscanthus s.l. or greater than 50 BP for Saccharinae Griseb. as defined by Saccharum s.l. are monophyletic. A monophyletic Miscant- Clayton and Renvoize (1986). Many species of Saccharinae hus s.s. group (group b) can be identified (100 BP). Miscant- group more closely with species of Sorghinae, Chionachin- hus floridulus, M. sinensis and M. transmorrisonensis group inae or Tripsacinae. Themeda triandra of the Anthistiriinae together with 95 BP. Saccharum officinarum/S. spontaneum is sister to the rest of Andropogoneae. (group c; 95 BP) are sister to Miscanthus s.s. in all equally Miscanthus s.l. is polyphyletic in all shortest trees. most parsimonious trees, but this is not supported by BP. However, a core group of Miscanthus species including M. The African Miscanthus species, M. fuscus and Saccharum floridulus, M. sacchariflorus, M. sinensis, M. sinensis subsp. contortum form a clade with 86 BP (group d). Miscanthus condensatus, M. oligostachyus and M. transmorrisonensis nepalensis falls in an isolated position. forms a clade (group b) supported with 99 BP. Five Saccha- rum species (group c), S. barberi, S. officinarum (a culti- vated sugarcane accession), S. robustum, S. sinense and S. spontaneum are sister to the core Miscanthus clade in all Discussion equally most parsimonious trees. Another group of Saccha- rum s.l. species (group a; Fig. 2), including S. arundinaceum Phylogenetics of Miscanthus, Saccharum and related genera and S. ravennae (= Ripidium or Erianthus section Ripid- ium), forms a monophyletic group in all shortest trees sister On the basis of DNA data, both Miscanthus s.l. and Saccha- to Imperata, which is distinct from other Saccharum species rum s.l. are polyphyletic. There is support for monophyletic but with <50 BP. groups within the Saccharum complex, but these do not cor- There is weak support (67 BP) for a clade (group d) con- respond to current taxonomic groupings. Among these are a taining the African Miscanthus species, M. junceus and M. well-supported core group of Miscanthus species (group b; ecklonii, the Himalayan M. fuscus and a number of Saccha- Figs. 2 and 4), including M. floridulus, M. oligostachyus, M. rum species including S. contortum (= Erianthus contortus), sacchariflorus, M. sinensis, M. sinensis subsp. condensatus S. narenga (= Narenga porphyrocoma) and Erianthus rockii, and M. transmorrisonensis and a core group of Saccharum but no support for the inclusion of these with the core group species (group c) corresponding to Saccharum s.s. (Price of Miscanthus species (no combination of E. rockii has 1963; Daniels and Roach 1987; Irvine 1999). It has a basic been made with Saccharum and, therefore, we treat it as chromosome number of 19 that is unique in Saccharinae for Erianthus despite believing it should be combined with which the basic number of 10 predominates. Miscanthidium or Saccharum; we are currently investigating Price (1963) and Daniels and Roach (1987) included six its nomenclature further). There is 88 BP support for the species in Saccharum s.s. (S. barberi, S. edule, S. officinarum, grouping of M. fuscus, S. narenga and E. rockii. These data S. robustum, S. sinense and S. spontaneum). However, S. indicate that the closest relatives of the African Miscanthus robustum is often synonymised with S. spontaneum; S. bar- species and M. fuscus are a group of Saccharum s.l. species beri or S. sinense are also often included with S. officinarum. and not other Miscanthus s.s. species. Miscanthus nepalensis We included five of these species in our analyses and found is also clearly separate from the core monophyletic south- them to form a sister group to Miscanthus s.s. in all equally eastern Asian Miscanthus group and associates more closely most parsimonious trees for ITS sequence data and for the with members of Eulalia and Sorghum, but this does not combined ITS and trnL-F analysis. These findings would receive >50 BP. support the conclusion of Clayton and Renvoize (1986), who considered Saccharum the closest relative of Miscant- hus s.s. Therefore, our results indicate that Saccharum s.s. is Analysis of trnL-F and combined ITS and trnL-F more closely related to Miscanthus s.s. than other members of Saccharum or the “Saccharum complex”. The other The aligned trnL-F matrix was 1,042 bp long; 132 sites were Saccharum species (Saccharum complex) group more variable and 26 of these were potentially informative. Anal- closely with other Miscanthus species such as the African ysis of the trnL-F matrix produced 17,920 equally most par- Miscanthus s.l. (= Miscanthidium) and the Himalayan (M. simonious trees (125 steps, CI = 0.93, RI = 0.89) with poor nepalensis and M. fuscus). Saccharum is separated from internal support (Fig. 3). The trnL-F data were joined with Miscanthus primarily on the basis of its fragile rachis (Mis- the ITS data in a combined analysis. No conflict between canthus has a tough rachis, not breaking up at maturity) and major clades of the trnL-F and the ITS analyses was iden- one of its paired spikelets being sessile (in Miscanthus both tified. Furthermore, the bootstrap support values of the spikelets of the pair are pedicellate, although they are not of major clades in the combined ITS and trnL-F analysis were equal length). 387

Fig.2 2. Parsimony tree of ITS sequence data for subtribe Saccharinae Numbers below branches are bootstrap percentages above 50%. and related genera. One of 1,520 equally most parsimonious trees of Groups found in all shortest trees are indicated by solid lines (groups length = 999; CI = 0.46, RI = 0.71. Values above branches are steps. not found in all shortest trees by a dotted line) 388

3 Fig. 3. Parsimony tree for Mis- canthus, 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 The results of our analysis with ITS and combined ITS types, one grouping with Miscanthus s.s. and the other with and trnL-F data indicate that Saccharum s.l. is not mono- Saccharum s.s. in our analysis. The ITS region is represented phyletic. The taxonomy of Saccharum is complex, and inter- by numerous tandem repeats in plant genomes (Baldwin et specific and intergeneric hybrids further confuse the al. 1995), and in most species the repeat units are homoge- situation (Daniels et al. 1975; Al-Janabi et al. 1994). Basic nised (via concerted evolution; Cronn et al. 1996) so that chromosome number varies (D’Hont et al. 1995, 1996), and one sequence predominates. It is likely that there has been polyploidy adds further complexity to systematic studies. considerable hybridisation and introgression between Mis- Modern day sugarcane has 2n = 80 with a basic chromosome canthus and Saccharum (Al-Janabi et al. 1994), and in some number of 10; its wild relative, S. spontaneum, has 2n = 40– Saccharum species such as S. robustum a mixture of both 128 (D’Hont et al. 1995). In Andropogoneae x = 10 predom- Saccharum and Miscanthus ITS repeat types exist, which inates (Burner 1991). Most varieties of sugarcane are provides evidence of intergeneric hybridisation. Miscanthus derived from crosses between S. officinarum (noble cane) has also been implicated by others in the evolution of S. offi- and S. spontaneum, but introgression between these and cinarum. Al-Janabi et al. (1994) showed that one Miscant- other species of the Saccharum complex (such as Erianthus) hus species from New Guinea, with a high chromosome has also been shown (Besse et al. 1996; Janoo et al. 1999a, number of 192, had the same plastid restriction sites as the b). majority of the Saccharum genus, suggesting that hybridis- A number of groups can be identified within Saccharum ation had occurred. s.l. from the ITS sequence data (Fig. 2), but these do not 389

4 Fig. 4. Parsimony tree for Mis- canthus, 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 According to Grassl (1972) New World Erianthus species Narenga. The genus Erianthus was separated from Saccha- are distinct from the Old World species in many morpho- rum by Michaux in 1803 and based on the New World logical attributes. The New World species have only two species E. saccharoides Michaux. The division was based anthers, whereas the Old World species have three. New primarily on the possession of an awn in Erianthus. There is World species also have floral parts with strong awns and little evidence from ITS data to support such a division. Eri- large seeds (primarily for animal dispersal) whereas Old anthus has also been split into New and Old World (section World species are adapted for wind dispersal (Grassl 1972). Ripidium) groups (Grassl 1972; Besse et al. 1996). A restric- These Old World Erianthus species were treated as Ripid- tion enzyme study of plastid DNA from various members of ium by Trinius in 1820 and Grassl (1972). Grassl (1972) Saccharinae by Sobral et al. (1994) showed that Miscanthus, agreed with Trinius that Ripidium ravennae (L.) Trin. was Narenga, Saccharum and Sclerostachya form a monophyl- separate from both Erianthus and Saccharum and created a etic group that was different from the Old World Erianthus number of new combinations within Ripidium including R. species (including E. arundinaceus and E. ravennae), which arundinaceum (Retz.) Grassl, R. bengalense (Retz.) Grassl, were more closely related to Sorghum bicolor. Two of the R. elephantinum (Hook f.) Grassl (treated by some as a syn- five Old World Erianthus species (E. ravennae and E. arun- onym of E. ravennae), R. kanashiroi (Ohwi) Grassl and R. dinaceus) studied by Sobral et al. (1994) were included in procerum (Roxb.) Grassl. Our analysis, in accordance with our study and were also found to be distinct from other Grassl (1972) indicates that Ripidium is a distinct group Saccharum s.l. species. because these species grouped together (group a; Fig. 2), 390 but separate from other Saccharum s.l. in all equally most from this analysis which genus they belong to or what their parsimonious trees. closest relatives are. They are included by some in the genus There is support (67 BP in the ITS tree; 86 BP in the Diandranthus (Trin.) L. Liu. Two anthers are also found in a combined tree) for a clade (group d; Figs. 2 and 4) contain- number of New World Saccharum s.l. species, but there is no ing the African Miscanthus species, M. fuscus (Himalayan) evidence to link these with M. nepalensis in our analyses. and Saccharum narenga (Narenga porphyrocoma; Hima- The ITS and the combined trees indicate that the African layan), Erianthus rockii (China/Burma/Indo-China) and Miscanthus taxa and M. fuscus (Himalayan) may not form a Saccharum contortum (Erianthus contortus; New World). monophyletic group with Miscanthus s.s. Saccharum contor- There is, however, no support for the monophyly of tum groups with these taxa, and it is highly probable that Narenga as its two species do not group together. Grassl these species are more closely allied to members of Saccha- came to this conclusion in 1972 and chose to combine rum s.l. than to Miscanthus. The African species of Miscant- Narenga with Sclerostachya because both have a chromo- hus (= Miscanthidium) have a basic chromosome number some count of 2n = 30 and behave similarly in crosses with (x) of 15 compared with 19 in Miscanthus s.s. They possess Saccharum (Grassl 1972). Saccharum fallax (= Narenga fal- an elongated inflorescence axis compared with Miscanthus lax) would not appear to be part of this group on the basis s.s., which are all subdigitate (except M. floridulus) and have of the ITS sequence data. Therefore, there is support for a leathery/papery (coriaceous), two-keeled glumes compared group that would include Sclerostachya and species assigned with papery/membranous, one-five nerved, glumes in to Erianthus (= Saccharum), Miscanthus s.l. (M. fuscus), and Miscanthus s.s. Miscanthus fuscus has an elongated inflo- Narenga. The closest allies of this group would, on the basis rescence axis like M. floridulus, but its spikelets have of sequence data, appear to be the Miscanthidium segre- coriaceous glumes like the African Miscanthus species. gates of Miscanthus s.l., but also Saccharum contortum, a There is, therefore, good molecular and morphological north American species. evidence to separate M. fuscus (Sclerostachya fusca) and Sorghum is polyphyletic in the ITS analysis and some the African Miscanthus species (= Miscanthidium) from species are embedded within the group of related genera of Miscanthus s.s. the “Saccharum complex” in our analysis (Fig. 2). It is not Miscanthus section Triarrhena (M. sacchariflorus) is usually included in this complex, but is one of the six genera monotypic and part of a polytomy with M. oligostachyus that has been successfully hybridised with Saccharum (section Karyiasua) and the species of Miscanthus section (Narenga, Miscanthidium, Miscanthus, Narenga, Scle- Miscanthus in the ITS analysis. Data from the DNA finger- rostachya and Sorghum). Furthermore, genetic maps of Sac- printing technique AFLP (Hodkinson et al. 2002b) indicate charum and Sorghum based on single dose DNA markers that M. oligostachyus is sister to a monophyletic group con- (Guimaraes et al. 1997) and RFLPs (Dufour et al. 1997) taining species of M. sections Miscanthus and Triarrhena have demonstrated high colinearity between Saccharum (M. floridulus, M. sacchariflorus, M. sinensis and M. trans- and Sorghum genomes. morrisonensis). Miscanthus oligostachyus and the other members of M. section Kariyasua (M. tinctorius and M. changii, not sequenced) have few racemes in comparison to Infrageneric relationships of Miscanthus the other sections and can be separated on morphological grounds from M. section Triarrhena by their possession of The subclades of Miscanthus in the ITS and combined trees awns and short spikelet callus hairs. They are restricted in do not fully agree with the sections defined by Lee (1964b, distribution to Japan. Miscanthus section Kariyasua is sister c). The core group of Miscanthus represents the south- to this group. Our data, therefore, would support the inclu- eastern Asian species of Miscanthus sections Kariyasua, sion of three of the sections in Miscanthus s.s. (Sinensis, Miscanthus and Triarrhena of Lee (1964b, c). Data from Triarrhena and Kariyasua) instead of the four defined by chromosome numbers support the monophyly of some of Lee (1964b, c, d) because Lee also chose to recognise the south-eastern Asian Miscanthus. The sections Kariya- Miscanthus section Diandra. It is not possible, from our sua, Miscanthus and Triarrhena all have a basic chromo- results, to evaluate the monophyly of these sections except some number (x) of 19, which is not found in any related for section Miscanthus, which is monophyletic in our anal- taxon (x = 10 predominates in the Andropogoneae) and ysis (95 BP in the combined analysis). 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. Conclusions Amplified fragment length polymorphism (AFLP) DNA fingerprinting (Vos et al. 1995; Reeves et al. 1998; Carolan On the basis of the DNA sequence data Miscanthus s.l. and et al. 2002) data showed that M. nepalensis was clearly sep- Saccharum s.l. are polyphyletic. Saccharum section Ripid- arate from other Miscanthus species (Hodkinson et al. ium is separate from all other Saccharum species in our 2002b). Species of section Diandra resemble Miscanthus s.s. analysis (group a, Figs. 2 and 4) and may be best treated as on morphological grounds, but possess two anthers, instead the genus Ripidium Trin. following Grassl (1972). of the usual three, and a basic chromosome number of five Six Miscanthus species form a well-supported clade or ten instead of 19 (Mehra and Sharma 1975). It is not clear (group b; Fig. 2), and these have a unique basic chromo- 391 some number of 19. Miscanthus ¥ giganteus Greef & Deuter Baldwin BG, Sanderson MJ, Porter JM, Wojciechowski MF, Cambell ex Hodkinson & Renvoize (Hodkinson and Renvoize CS, Donoghue MJ (1995) The ITS region of nuclear ribosomal DNA: a valuable source of evidence on angiosperm phylogeny. Ann Mo 2001), an allopolyploid hybrid of M. sinensis and M. saccha- Bot Gard 82:247–277 riflorus, would also form part of this group. The Himalayan Besse P, McIntyre CL, Berding N (1996) Ribosomal DNA variations in species M. fuscus groups more closely with the African Mis- Erianthus, a wild sugarcane relative (Andropogoneae – Sacchari- canthidium and Saccharum contortum than with the south- nae). Theor Appl Genet 92:733–743 Briggs BG, Johnson LAS (2000) Hopkinsiaceae and Lyginiaceae, two eastern Asian Miscanthus s.s. Miscanthus section Diandra of new families of Poales in Western Australia with revisions of Lee (1964d), also found in the Himalayan region, and rep- Hopkinsia and Lyginia. Telopea 8:477–502 resented by M. nepalensis, does not group with any member Briggs BG, Marchant AD, Gilmore S, Porter CL (2000) A molecular phylogeny of Restionaceae and allies. In: Wilson KL, Morrison DA of Miscanthus s.l.. Recently, its species have been recognised (eds) Monocots – systematics and evolution. Proc 2nd Int Conf Com- as a separate genus, Diandranthus (Trin) L. Liu, and our parative Biol Monocots, Sydney 1998. CSIRO, Melbourne, pp 661– molecular analysis would add weight to this separation. 667 Species corresponding to Saccharum s.s. form a sister Buckler ES 4th, Holtsford TP (1996) Zea systematics: ribosomal ITS evidence. Mol Biol Evol 13:612–622 group to Miscanthus s.s. (group c; Figs. 2 and 4). Two differ- Bullard MJ, Heath MC, Nixon PM (1995) Shoot growth, radiation ent ITS sequences from S. robustum are individually shown interception and dry matter production and partitioning during the to be related to Miscanthus and Saccharum, respectively, establishment phase of Miscanthus sinensis “Giganteus” grown at two densities in the UK. Ann Appl Biol 126:94–102 and the former provides evidence of hybridisation between Burner DM (1991) Cytogenetic analyses of sugarcane relatives these closely allied genera. A further clade (group d; Figs. 2 (Andropogoneae: Saccharinae). Euphytica 54:125–133 and 4) contains species of Miscanthus s.l. and Saccharum s.l. Carolan JC, Hook ILI, Walsh JJ, Hodkinson TR (2002) Using AFLP (including some of their segregate genera Narenga, Miscant- markers for species differentiation and assessment of genetic vari- ability of in vitro-cultured Papaver bracteatum (Section Oxytona). In hidium, and Sclerostachya). 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