Taxonomic changes in C3 Cyperus (Cyperaceae) supported by molecular data, morphology, embryography, ontogeny and anatomy

Plant Ecology and Evolution 144 (3): 327–356, 2011 doi:10.5091/plecevo.2011.653 REGULAR PAPER Taxonomic changes in C3 Cyperus (Cyperaceae) supported by molecular data, morphology, embryography, ontogeny and anatomy Isabel Larridon1,*, Marc Reynders1, Wim Huygh1, Kenneth Bauters1, Alexander Vrijdaghs2, Olivier Leroux3, A. Muthama Muasya4, David A. Simpson5 & Paul Goetghebeur1 Ghent University, Department of Biology, Research Group Spermatophytes, K.L. Ledeganckstraat 35, BE-9000 Gent, Belgium K.U.Leuven, Institute of Botany and Microbiology, Laboratory of Plant Systematics, Kasteelpark Arenberg 31, BE-3001 Leuven, Belgium 3 Ghent University, Department of Biology, Research Group Pteridology, K.L. Ledeganckstraat 35, BE-9000 Gent, Belgium 4 University of Cape Town, Botany Department, Rondebosch 7700, South Africa 5 Royal Botanic Gardens, Kew, Richmond, Surrey, UK-TW9 3AB, United Kingdom *Author for correspondence: isabel.larridon@ugent.be 1 2 Background and aims – Recent molecular studies validate a broad deinition of Cyperus (Cyperaceae) uniting genera previously scattered in Cyperoideae. First indication of their afinity with Cyperus was obtained through embryography. Cyperus consists of a paraphyletic C3 Cyperus and monophyletic C4 Cyperus. In this study, we aim to check and clarify the putative positions of the segregate genera in C3 Cyperus. Additional information is given and remarks are made on the position of some as yet unplaced species or sections in the C3 Cyperus phylogeny. Methods – Embryos of Cyperus constanzae and C. gardneri were cleared and drawn. Inlorescences of selected C3 Cyperus species were investigated using scanning electron and light microscopy. Histochemical tests were performed to assess the presence of suberin in the ‘corky’ tissue of the nutlets of Cyperus pectinatus. Key results – Embryography not only supports tribal classiication in Cyperoideae, it is also phylogenetically informative in C3 Cyperus. Morphology and ontogeny support molecular phylogenetic results suggesting the inclusion of the segregate genera in C3 Cyperus as new sections or in established sections, and conirm the need to broaden the circumscription of some of these sections. Conclusion – Although less diverse than C4 Cyperus, C3 Cyperus includes clades which evolved an exceptional morphological diversity compared to its limited species numbers. The segregate genera Courtoisina (deciduous spikelets), Kyllingiella (spirally-arranged glumes) and Oxycaryum (spirallyarranged glumes and dorsiventrally lattened dimerous gynoecia), and the taxon Anosporum (recognised at sectional, subgeneric or generic level) are here included in C3 Cyperus (= Cyperus subg. Anosporum) as sections or included in an existing section (Kyllingiella is included in Cyperus sect. Leucocephali). A formal taxonomic revision is presented with relevant new names and combinations, synonyms, diagnoses and identiication keys. Key words – Anatomy, Cyperus, Cyperaceae, embryography, molecular phylogeny, morphology, ontogeny, taxonomy. INTRODUCTION In the tropics and subtropics, Cyperus is the largest genus in the family Cyperaceae. Cyperus s. str. includes c. 700 species (Govaerts et al. 2011). Our recent molecular phylogenetic studies validate a broad deinition of Cyperus uniting genera previously scattered in Cyperoideae (Simpson et al. 2007, Muasya et al. 2009a, Larridon et al. 2011b). The seminal embryographical study by Van der Veken (1965) gave the irst indication of a close relationship between these taxa. After studying the embryos of 342 Cyperoideae species, Van der Veken (1965) not only concluded that the uniformity of the embryos of Cyperus species supports the wide concept of the genus, but he also revealed the presence of embryos of the Cyperus-type in many taxa previously placed near Scirpus (e.g. Ascolepis, Ficinia, Isolepis, Lipocarpha, Kyllingiella and Oxycaryum). Van der Veken (1965) studied the embryos of thirty C3 Cyperus species. Using maximum likelihood and All rights reserved. © 2011 National Botanic Garden of Belgium and Royal Botanical Society of Belgium – ISSN 2032-3921 Pl. Ecol. Evol. 144 (3), 2011 Bayesian analyses of nrDNA (ETS1f) and cpDNA (rpl32trnL and trnH-psbA) sequence data, Larridon et al. (2011b) concluded that the Cyperus clade consists of a paraphyletic C3 Cyperus (the Cyperus clade species using C3 photosynthesis linked with eucyperoid vegetative anatomy) in which a monophyletic C4 Cyperus is nested (uniting the Cyperus clade species using C4 photosynthesis linked with chlorocyperoid vegetative anatomy). In C3 Cyperus, ive major clades are recognisable (ig. 1) (Larridon et al. 2011b). Clade 1 can be divided in three subclades largely corresponding to Cyperus sect. Haspani, C. sect. Incurvi and C. sect. Diffusi. The other major clades respectively correspond to: (clade 2) an entirely New World C. sect. Luzuloidei sensu Denton (1978), (clade 3) a highly diverse clade including C. sect. Fusci, C. sect. Pseudanosporum and C. sect. Anosporum, and the segregate genera Courtoisina and Oxycaryum, (clade 4) C. sect. Alternifolii, and (clade 5) C. sect. Leucocephali and the segregate genus Kyllingiella. The morphological diversity of Cyperus translates into a large number of published names. Compared to C4 Cyperus, a smaller number of names of genera and of subdivisions of genera were described in C3 Cyperus (Huygh et al. 2010, Larridon et al. 2011a, Reynders et al. 2011). Of the names of genera and of subdivisions of genera described for C3 Cyperus, a surprisingly large number relate to a single, relatively small clade (ig. 1, clade 3). The number of names published for taxa belonging to clade 3, relects morphological diversity of this clade. Based on molecular data (Muasya et al. 2002, 2009a), two clades are recognised in Cypereae. Traditionally, Cypereae were described as having distichously organised spikelets and trimerous lowers without a perianth (e.g. Kükenthal 1936). However, Muasya et al. (2002, 2006, 2007, 2009a, 2009b) and Vrijdaghs et al. (2005, 2006, 2009) demonstrated that spirally organised spikelets and lowers with at least remnants of a perianth occur in the Ficinia clade in Cypereae. In the Cyperus clade, given that segregate genera such as Oxycaryum and Kylingiella are nested within it (Muasya et al. 2002, 2009a, Larridon et al. 2011b), spirally organised spikelets also occur. “Perianthless lowers” still hold, although in this ontogenetic study, we investigated: (1) the lexibility of the spikelet structure in C3 Cyperus s. str. and its segregate genera Courtoisina, Oxycaryum and Kyllingiella to establish the range of variation on the spikelet model as proposed by Vrijdaghs et al. (2010), (2) the variation in spikelet and loral structure present in the segregate genera, and (3) the variation in spikelet and loral structure from the developmental standpoint. The nature of the ‘corky’ tissue surrounding the nutlets of Cyperus pectinatus Vahl was studied using histochemical techniques. The molecular phylogenetic hypothesis of Larridon et al. (2011b) demonstrated the need to adapt the current infrageneric classiication of Cyperus as a whole, and more speciically of C3 Cyperus, to accommodate several segregate genera (i.e. Courtoisina, Oxycaryum and Kyllingiella). In addition, several species need to be moved between different sections. This paper provides the necessary formal nomenclatural and taxonomic changes and adds further morphological, embryographical and ontogenetic support for these taxonomic changes. 328 Taxonomic history As mentioned above, a surprisingly large number of taxa relate to clade 3 of the molecular phylogenetic hypothesis of Larridon et al. (2011b) (ig. 1), i.e. Cyperus sect. Anosporum (Pax) Nees, Cyperus sect. Pseudanosporum C.B.Clarke, Courtoisina Soják, and Oxycaryum Nees. Although the species of this clade have been placed in a number of different segegrate genera and / or subdivisions of Cyperus (or even Scirpus in the case of Oxycaryum cubense (Poepp. & Kunth) Palla) to relect the morphological diversity of this clade, they have some characters in common. For example, species of C. sect. Anosporum, C. sect. Pseudanosporum and Oxycaryum have nutlets with corky thickenings and Courtoisina and Oxycaryum have a tendency towards globose spikeletclusters (ig. 2D & E). The other taxa introduced here are C. sect. Leucocephali Cherm. ex Kük., Kyllingiella R.W.Haines & Lye, C. sect. Dichostylis sensu Kükenthal (1936), and C. sect. Graciles (Benth.) Kük. Cyperus sect. Anosporum and C. sect. Pseudanosporum – Nees (1834a) established Anosporum Nees as a monotypic genus based on the species Cyperus monocephalus Roxb. This species differs from Cyperus s. str. by its unusual habit and its nutlets which are surrounded by a corky tissue. Various authors, unknowingly, described other monotypic genera based on the same conspicuous species (Hydroschoenus Zoll. & Moritzi, Trentepohlia Boeck.). A second species, Cyperus pectinatus Vahl, served as type for the genus Atomostylis. The two species described in this genus are now both seen as synonyms of C. pectinatus. Boeckeler (1869, 1870) included several more species in Anosporum based on a similarity of the nutlets, including Anosporum pallidum Boeck. [= Cyperus platystylis R.Br.] and Anosporum cubense (Poepp. & Kunth) Boeck. [≡ Oxycaryum cubense]. Most authors at that time included Oxycaryum in Scirpus because of its spirally-arranged glumes. Both Nees (1834a) and Boeckeler (1869) considered the corky thickenings of the nutlet to be a perigynium. This inluenced Nees (1834a) to include Anosporum in the tribe Hypolytreae. However, Boeckeler (1869) placed Anosporum close to Cyperus. Clarke (1884) showed the perigynium theory to be incorrect. As Clarke (1884) is a survey of the Indian Cyperus species, he treated Cyperus cephalotes Vahl as the only species of Cyperus subg. Anosporum. In addition, Clarke (1884) described a new monotypic Cyperus section Pseudanosporum for the only other Asian species included in Anosporum by Boeckeler (1869), i.e. Cyperus platystylis. Clarke (1884) agreed with Steudel (1854) in placing C. platystylis somewhere near Kunth’s Alternifolii, of which it has the inlorescence, digitate spikelets and subexalate rachilla. However, in the same paper he remarked that even assuming that the corkiness of the nutlets is adaptive, there is much to connect C. platystylis with Anosporum. Clarke (1884) stated that in his opinion Anosporum should not be maintained as separate from Cyperus if C. platystylis is included in it as it does not have any of the traits which characterise Anosporum (the pedicel, the permanent style or the entire stigma), the only clearly shared character being the corkiness of the nutlet. In recent years most botanists shared the view of Kükenthal (1936), who regarded Cyperus cephalotes, C. co- Larridon et al., Taxonomic changes in C3 Cyperus (Cyperaceae) Figure 1 – Simpliied 50% majority consensus multiple-locus BI-GTR+I+Γ tree with the associated posterior probabilities (PP) values based on ig. 2 of Larridon et al. (2011b). Only PP values above 85% are shown. lymbetes Kotschy & Peyr. and C. pectinatus as belonging to a section Anosporum in Cyperus. However, Lye (1981) stated that Anosporum is suficiently different from Cyperus to warrant generic separation, at least when such genera as Alinula, Mariscus, Kyllinga, Pycreus, Remirea and Torulinium are accepted. Later, Haines & Lye (1983) treated Anosporum at subgeneric level in Cyperus. Lye (1981), like Boeckeler (1869) and Chermezon (1924) before him, considered the possibility of a relationship of Anosporum with Oxycaryum based on the similarity of their nutlets. Goetghebeur (1986) considered glume arrangement to be a more important character than corkiness of the nutlets. Courtoisina – The genus Courtoisina was irst established under the name Courtoisia by Nees (1834a) to accommodate the Indian species previously known as Kyllinga cyperoides Roxb. Clarke (1894) combined the African species Cyperus assimilis Steud. in Courtoisia. However, the name Courtoisia Nees (Nees 1834a) is a younger homonym of Cour329 Pl. Ecol. Evol. 144 (3), 2011 toisia Marchand (Lichenes) (Marchand 1830). Soják (1979), Raizada & Bennet (1981) and Rauschert (1982) published new names and combinations for Courtoisia Nees, most of which proved superluous. Wilson (1983) protested with good reason against the publication of superluous names. When renaming the genus, Soják (1979) combined Courtoisina cyperoides (Roxb.) Soják, but failed to combine Courtoisia assimilis (Steud.) C.B.Clarke, nom. illeg. Maquet (1988) later published the correct combination for this species, Courtoisina assimilis (Steud.) Maquet. Rauschert (1982) published the superluous and illegitimate name Pseudomariscus and made the equally illegitimate combination P. cyperoides (Roxb.) Rauschert. As with Soják (1979), the other known species was left unnamed. However, Rauschert (1982) did make the combination P. olivaceus (Boeck.) Rauschert for Oxycaryum cubense. Raizada & Bennet (1981) published superluous new names for both the genus and its two species. The proposal by Vorster (1986) to conserve Courtoisia Nees against Courtoisia Marchand was not accepted at the nomenclatural sessions during the 14th International Botanical Congress in Berlin (1987). The name Courtoisina Soják should be used (Brummitt 1989). Most recent authors consider Courtoisina to be a distinct genus (e.g. Goetghebeur 1986, 1998, Vorster 1996, Govaerts et al. 2007, 2011), although some consider it to be a part of Cyperus s. lat. (Haines & Lye 1983, Lye 1983, 1992). The most obvious characters used to support Courtoisina at generic rank are the strongly lattened spikelets which disarticulate as a unit when mature, leaving the spikelet bract and prophyll behind and the conspicuously winged glumes. Lye (1983) made the combination Cyperus subg. Courtoisia (Nees) Lye, and later (Lye 1992) he made the illegitimate combination Cyperus subg. Courtoisina (Soják) Lye for the same taxon. Publishing the name Cyperus subg. Courtoisia (Nees) Lye was unfortunate, because although the generic name Courtoisia Nees is an illegitimate later hononym, the subgeneric name Cyperus subg. Courtoisia is legitimate with priority from its date of publication (1983) (Huygh et al. 2010). Consequently, it is the correct name for the taxon at subgeneric rank in Cyperus, although Courtoisina Soják is the correct name at generic rank. Oxycaryum – The genus Oxycaryum, either considered as monotypic, or sometimes divided into several closely related taxa (Palla 1908), is widely distributed. A great number of synonyms have been published in other genera as Anosporum, “Crepidocarpus”, Cyperus, Isolepis, Kyllinga, Mariscus and Scirpus. Such inconsistent interpretations show that this plant unites characters which are more or less typical for one of these genera. Oxycaryum cubense was irst described in the tribe Scirpeae s. lat. as Scirpus cubensis Poepp. & Kunth because of its spirally-arranged glumes. However, the genus Scirpus, as interpreted by Linnaeus (1753), and accepted with some modiications by later authors (e.g. Boeckeler 1868–1877, Clarke 1908, Chermezon 1937), proved to be a very heterogeneous assemblage of species. Since the embryographical study of Van der Veken (1965) and the division of Scirpus, Oxycaryum is included in the Cypereae. As mentioned above, Boeckeler (1869), Chermezon (1924) and later Lye (1981) suggested a possible relationship 330 of Anosporum with Oxycaryum based on the corky nutlets. Van der Veken (1965) mentioned a certain similarity of the embryos of both taxa. Goetghebeur (1986) considered Oxycaryum to be related to Cyperus itself, of which he believed it to be an early evolutionary lineage based on the spirally arranged glumes. However, Goetghebeur (1986) does not support uniting Oxycaryum with Anosporum because of their differing morphology (nutlet morphology excepted). Cyperus sect. Leucocephali – Steudel (1854) described Cyperus pulchellus R.Br. as a new species Sorostachys kyllingioides Steud. (based on a different type specimen) in a separate genus Sorostachys. Only Lye (1981) accepted this genus, including only two species, Cyperus leucocephalus Retz. and Cyperus pulchellus. Lye (1981) placed Sorostachys close to C3 Cyperus and possibly even to Kyllingiella (Haines & Lye 1978). Later, Lye (1983) reduced Sorostachys to a subgenus in Cyperus. Clarke (1884) incorrectly placed C. leucocephalus in sect. Platystachyi, based on the presence of C. sphaerocephalus var. leucocephalus Kunth [= C. niveus var. leucocephalus (Kunth) Fosberg] in Kunth’s Platystachyi (Kunth 1837). As a consequence, Clarke (1884) also placed Sorostachys in synonymy of sect. Platystachyi. Based on Clarke’s (1884) mistake, Kern (1974) lectotypiied Cyperus sect. Platystachyi with C. leucocephalus Retz. However, Larridon et al. (2011a) superseded Kern’s (1974) choice, by giving preference to C. niveus. This is because Cyperus niveus was included in the original circumscription of C. sect. Platystachyi given by Kunth (1837) and its characters it the description of this group, in contrast to C. leucocephalus. Simpson (1990) clearly explained the differences between sections Leucocephali and Platystachyi. Furthermore, C. sect. Leucocephali and C. sect. Platystachyi, as originally circumscribed, both form well-deined natural groups in Cyperus and following the subgeneric classiication of Goetghebeur (1998) they respectively belong in C. subg. Anosporum (C3 photosynthesis – eucyperoid anatomy) and C. subg. Cyperus (C4 photosynthesis – chlorocyperoid anatomy) and are in so not closely related. Kükenthal (1936) validated Chermezon’s (1931) C. sect. Leucocephali. However, at the same time, Kükenthal (1936) reduced the name Sorostachys to the synonymy of sect. Platystachyi, but placed its only species (S. kyllingioides) in synonymy of C. leucocephalus in sect. Leucocephali. This confusion is probably also the result of Clarke’s (1884) error. Simpson (1990) includes seven species in his revision of C. sect. Leucocephali. He considered C. pulchellus and C. leucocephalus as separate species, and includes three of the others also included by Kükenthal (1936), i.e. C. schomburgkianus Nees, C. tenerrimus J.Presl & C.Presl, and C. michoacanensis Britton ex C.B.Clarke. C. zanzibarensis C.B.Clarke (accepted species name, Govaerts et al. 2007, 2011) is placed in the synonymy of C. pulchellus, while C. coronarius (Vahl) Kunth (accepted species name, Govaerts et al. 2007, 2011) is placed in the synonymy of C. leucocephalus and the recently described C. microglumis D.A.Simpson (Simpson 1990) and C. nayaritensis G.C.Tucker (Tucker 1983) are also included. Since Simpson’s (1990) publication, two additional species of this section were described by Simpson, i.e. Cyperus androhibensis D.A.Simpson (Simpson 1992) from Madagascar Larridon et al., Taxonomic changes in C3 Cyperus (Cyperaceae) and Cyperus brumadoi D.A.Simpson (Simpson 1993) from Brazil. Kyllingiella – Steudel (1842) described Kyllinga microcephala Steud. from Ethiopia. This species resembles Kyllinga in several respects (white capitate inlorescence and general habit). However, it differs in having spirally-arranged glumes. Richard (1850) renamed the species Isolepis kyllingioides A.Rich. Boeckeler (1870) in his turn transferred this plant to Scirpus as Scirpus kyllingioides (A.Rich.) Boeck. Most authors regarded this plant as a Scirpus. Clarke (1893) associated this species in Scirpus with another Cyperus species which often has spirally-arranged glumes, i.e. Scirpus michelianus L. [≡ Cyperus michelianus (L.) Delile]. Based on the results of e.g. Van der Veken’s (1965) embryographical study (Cyperus-type embryo) and Druyts-Voets (1970) anatomical study (eucyperoid stem and leaf anatomy), Haines & Lye (1978) established the genus Kyllingiella based on the species Kyllingiella microcephala (Steud.) R.W.Haines & Lye. As accepted by Govaerts et al. (2007, 2011), Kyllingiella includes four species. Cyperus sect. Dichostylis sensu Kükenthal (1936) – Huygh et al. (2010) explained that the name Dichostylis P.Beauv. ex T.Lestib. (Lestiboudois 1819: 39) is illegitimate since in its protologue another name (Echinolytrum Desv. (Desvaux 1808) [= Fimbristylis Vahl]) was cited in synonymy. As circumscribed by Kükenthal (1936) this is a very heterogeneous group of species, and included two conirmed C4 Cyperus species Cyperus meeboldii Kük. and C. michelianus (L.) Link (Bruhl & Wilson 2007). However, Kükenthal (1936) also placed four C3 Cyperus species in this section (C. humilis Kunth, C. seslerioides Kunth, C. tweediei C.B.Clarke and C. uncinulatus Schrad. ex Nees). A last species, C. hilairenus Steud., was mentioned by Kükenthal as uncertain with possible afinity to C. uncinulatus. Since then, two more species have been described with possible afinity to C. uncinulatus: C. arsenei O’Neill & Ben.Ayers and C. microbrunneus G.C.Tucker. Cyperus sect. Graciles – Bentham (1878: 254) published his Graciles as a group of unspeciied rank under the not validly published name “Cyperus sect. Eucyperus”. He diagnosed “Cyperus sect. Eucyperus” as follows: “Spikelets lat, the [rachis] not winged or rarely with an exceedingly narrow border. Style 3-cleft. Nut equally triquetrous.” and “Cyperus [unranked] Graciles” as: “Spikelets spreading, pale-coloured, in a single sessile cluster or solitary. Glumes obtuse or very shortly pointed. Nuts short.” Bentham (1878) included ive species, i.e. Cyperus tenellus L.f., C. gracilis R.Br., C. enervis R.Br., C. debilis R.Br. and C. laevis R.Br. Kükenthal (1936: 292) formally published this taxon at sectional rank and included eight species in Cyperus sect. Graciles. Blake (1939) published a thorough revision of C. sect. Graciles, in which he used a more natural circumscription for this section. After correspondence, Kükenthal (1943) accepted Blake’s opinions. Three species included by Kükenthal (1936) in C. sect. Graciles were no longer included by Blake (1939). Two of these, i.e. Cyperus tenellus L.f. and C. leucoloma Nees, have since been moved to the genus Isolepis, as I. levynsiana Muasya & D.A.Simpson and I. leucoloma (Nees) C.Archer respectively (Archer 1998; Muasya et al. 2002, 2006, 2007). A third species included in C. sect. Graciles by Kükenthal (1936), Cyperus trichodes Griseb., was excluded most likely based on its deviant distribution in Jamaica, while all other C. sect. Graciles species are limited to north and east Australia, and some of its surrounding islands. Furthermore, Blake (1939) had a quite different view on the synonymy and rank of some of the taxa included in C. sect. Graciles. MATERIAL AND METHODS Plant material and morphology We examined a large number of herbarium specimens (from the herbaria B, BM, BR, GENT, EA, K, MO, P, TAN, U, UPS, WAG mainly; abbreviations according to Holmgren et al. 1990), supplemented with own observations in the ield, and from collections in the Ghent University Botanical Garden. Additional information on species and (type) specimens was obtained from literature (incl. protologues) and the databases http://plants.jstor.org/, http://www.tropicos.org/ and Govaerts et al. (2011). Images of spikelets and nutlets were taken with a Nikon SMZ800 stereoscopic microscope, equipped with a Nikon digital camera DXM1200 (Nikon, Tokyo, Japan). The images were edited with Adobe Photoshop CS3 (Adobe Systems Inc., San Jose, USA). The macroscopic photos were taken during expeditions in the ield. Embryography The embryos of two species were studied and drawn, based on the methods described in Van der Veken (1965). For Cyperus gardneri Nees, embryos were studied and drawn from the specimen Schessl 3316 (GENT), and for Cyperus contanzae Urb. from the specimen Ekman 6879 (K). These embryos were compared with the embryos of C3 Cyperus species studied by Van der Veken (1965). Table 1 lists the species of which the embryographs are displayed in ig. 3. The embryos of clade 3 species (ig. 1) (Larridon et al. 2011b) were assembled and overlaid onto the Bayesian inference tree to trace their morphological evolutionary transformations (ig. 4). Ontogeny Inlorescences of the species studied were collected in the ield and at the Ghent University Botanical Garden (table 2) and subsequently ixed in FAA (70% ethanol, acetic acid, 40% formaldehyde, 90/5/5). Spikelets and loral buds were dissected in 70% ethanol under a Wild M3 (Leica Microsystems AG, Wetzlar, Germany) stereo microscope equipped with a cold-light source (Schott KL1500; Schott-Fostec LLC, Auburn, NY, USA). The prepared material was washed twice with 70% ethanol for 5 min and then placed in a mixture (1/1) of 70% ethanol and DMM (dimethoxymethane) for 5 min. Subsequently, the material was transferred to 100% DMM for 20 min, before it was CO2 critical point dried using a CPD 030 critical point dryer (BAL-TEC AG, Balzers, Liechtenstein). The dried samples were mounted on aluminium stubs using Leit-C and coated with gold with a SPI-ModuleTM Sputter Coater (SPI Supplies, West-Chester, PA, USA). Images were 331 Pl. Ecol. Evol. 144 (3), 2011 A B C D E F G H 332 Larridon et al., Taxonomic changes in C3 Cyperus (Cyperaceae) Table 1 – List of the species of which the embryographs are displayed in ig. 3. The classiication used for the sections is that of Kükenthal (1936), but the correct names are used for his sections (Larridon et al. 2011a). For the segregate genera we refer to Goetghebeur (1998) and Govaerts et al. (2007, 2011). Three relevant C4 Cyperus species included are indicated. Supraspeciic taxon Species C. sect. Alternifolii C. alternifolius L. C. sect. Anosporum C. pectinatus Vahl C. sect. Cyperus C. esculentus L. (C4) C. sect. Dichostylis sensu Kükenthal (1936) C. humilis Kunth, C. seslerioides Kunth, C. uncinulatus Schrad. ex Nees, C. michelianus (L.) Link (C4) C. sect. Diffusi C. ajax C.B.Clarke, C. diffusus Vahl C. sect. Elegantes C. constanzae Urb., C. elegans L. (C4), C. gardneri Nees C. sect. Fusci C. dichrostachyus Hochst. & A.Rich., C. difformis L., C. haematocephalus Boeck. ex C.B.Clarke, C. fuscus L., C. reduncus Hochst. ex Boeck., C. submicrolepis Kük., C. unicolor Boeck. C. sect. Graciles C. gracilis R.Br., C. tetraphyllus R.Br. C. sect. Haspani C. deciduus Boeck., C. haspan L. C. sect. Incurvi C. fertilis Boeck., C. mapanioides C.B.Clarke, C. simplex Kunth C. sect. Leucocephali C. tenerrimus J.Presl & C.Presl C. sect. Luzuloidei C. eragrostis Lam., C. incomtus Kunth C. sect. Pseudanosporum C. platystylis R.Br. Courtoisina Courtoisina assimilis (Steud.) Maquet, Courtoisina cyperoides (Roxb.) Soják Kyllingiella Kyllingiella microcephala (Steud.) R.W.Haines & Lye Oxycaryum Oxycaryum cubense (Poepp. & Kunth) Palla obtained on a Jeol JSM-6360 (Jeol, Tokyo) at the Laboratory of Plant Systematics (K.U. Leuven). Since in Cyperus s. lat. most spikelets have many lowers, and consequently in order to avoid the use of abstract numbers, (lower subtending) glumes are numbered from young (1) to old (x). based anti-fade solution (Citiluor AF1, Citiluor Ltd., UK). Immunoluorescence was observed with an epiluorescence microscope equipped with UV-illumination (Olympus BX51). Untreated sections were observed as control for intrinsic autoluorescence. Anatomy For detailed anatomical observation of mature nutlets, FAAixed material was dehydrated in a graded ethanol series, embedded in Technovit 7100 resin (Heraeus Kulzer, Wehrheim, Germany), sectioned, stained and mounted following Leroux et al. (2007). Phloroglucinol/HCl staining was performed on hand-cut sections using 2% (w/v) phloroglucinol in 95% (v/v) ethanol for 5 min, and subsequently mounting in 33% (v/v) hydrochloric acid. Sections were observed with a Nikon Eclipse E600 microscope and images were recorded using a Nikon digital camera DXM1200. For determination of suberin presence we applied a berberine/aniline blue luorescent staining procedure as described by Brundett et al. (1988). Sections were irst stained in 0.1% (w/v) berberine hemi-sulphate (Sigma; C.I. 75160) for 1 hour. After thorough washing, sections were stained with 0.5% (w/v) aniline blue (Sigma, C.I. 42755) for 30 min, washed with distilled water, and mounted in a glycerol- RESULTS Habit and habitat The taxa treated in this paper and the habitats in which they occur are illustrated in ig. 2. Cyperus pectinatus, Courtoisina cyperoides and Oxycaryum cubense and their related species occur in wetlands. Courtoisina cyperoides grows rooted in mud, e.g. in swamps (ig. 2C) and in rice ields (ig. 2D). Cyperus pectinatus (ig. 2B) and Oxycaryum cubense (ig. 2F) grow in loating mats in open water. Cyperus pulchellus (Cyperus sect. Leucocephali) and its related species have a preference for seasonally dry grasslands (ig. 2G) which is atypical for C3 Cyperus. Species of C. sect. Leucocephali and the segregate genus Kyllingiella are characterised by the presence of whitish capitate inlorescences (ig. 2H) and show adaptations to their dryer environment (often thickened base and/or remaining old leaf sheaths). ◄ Figure 2 – A, inlorescence of Cyperus pectinatus (picture taken by M. Reynders in Madagascar); B, habitat of C. pectinatus (picture taken by W. Huygh in Madagascar); C, habitat and D, inlorescence of Courtoisina cyperoides (pictures taken by A.M. Muasya in Madagascar); E, inlorescence and F, habitat of Oxycaryum cubense (pictures taken by R. Carter in Lowndes County, Georgia, U.S.A.); G, habitat and H, inlorescence of Cyperus pulchellus (pictures taken by W. Huygh in Madagascar). 333 Pl. Ecol. Evol. 144 (3), 2011 Figure 3 – Embryographs of C. constanzae (Ekman 6879, K) and C. gardneri (Schessl 3316, GENT) (own data) and relevant species studied by Van der Veken (1965). Species of which the embryos are in grey were included in the molecular phylogenetic study of Larridon et al. (2011b). Embryography The embryos of 31 C3 Cyperus species and of three relevant C4 Cyperus species (C. elegans, C. esculentus L. and C. michelianus) are displayed in ig. 3 and listed in table 1. The embryographs of C. constanzae and C. gardneri were newly produced for this study, the other 32 were published in Van der Veken’ study (1965). The embryographs are shown according to their known (grey = included in the molecular study of Larridon et al. 2011b) or inferred relationships. The embryos of C. sect. Haspani species are small and inconspicuous in shape. In C. sect. Diffusi, C. sect. Incurvi and C. sect. Luzuloidei, the embryos are noticeably larger and have a slightly asymmetrical development of the coleoptile. The embryo of C. constanzae (placed in a section with C. elegans (C4) by Kükenthal 1936) shows the most resemblance to the embryos of species of C. sect. Diffusi and C. sect. Incurvi. The embryograph of C. elegans is also shown; its shape and size are typical for most C4 Cyperus species (illustrated here by the embryograph of the C4 Cyperus species C. esculentus, lectotype of the name Cyperus L.). The embryos of C. sect. Pseudanosporum, C. sect. Anosporum, C. reduncus Hochst. ex Boeck., Courtoisina, C. gardneri, Oxycaryum and C. sect. Fusci are overlaid onto clade 3 of the Bayesian inference tree of Larridon et al. (2011b) (igs 1 & 4). The embryo of Cyperus reduncus is very similar to that of two Courtoisina species (characterised by the strongly asymmetrical development of the coleoptile). The embryos of Courtoisina and Cyperus reduncus share further similarities with those of C. sect. Anosporum and C. sect. Pseudanosporum sensu Kükenthal (1936) and with those 334 Figure 4 – Embryographs overlaid onto clade 3 (Anosporum– Courtoisina–Oxycaryum–Cyperus sect. Fusci clade) of the Bayesian inference tree of Larridon et al. (2011b). Larridon et al., Taxonomic changes in C3 Cyperus (Cyperaceae) of Oxycaryum cubense and C. gardneri. The embryos of the species of C. sect. Fusci are also very similar; a small embryo with a tendency towards an asymmetrical development of the coleoptile. The embryos of C. gracilis and C. tetraphyllus (C. sect. Graciles) show resemblance both to the embryo of C. alternifolius (shape) and to some of the embryos of C. sect. Diffusi and C. sect. Incurvi (size). Cyperus tenerrimus and Kyllingiella microcephala have embryos which are very similar in shape and size. The embryos of C. humilis, C. seslerioides and C. uncinulatus are rather small and show resemblance both to the embryos of C. tenerrimus and K. microcephala and to the smaller embryos of C. sect. Fusci species (C. haematocephalus, C. difformis and C. dichrostachyus). The embryo of C. humilis is conspicuously shaped. The embryograph of C. michelianus is also displayed in ig. 6 to illustrate its difference to the embryos of the C3 Cyperus species of C. sect. Dichostylis sensu Kükenthal (1936). Table 2 – Voucher data and origin of the C3 Cyperus species used in the ontogenetic study. Species Courtoisina assimilis (Steud.) Maquet Courtoisina cyperoides (Roxb.) Soják Cyperus colymbetes Kotschy & Peyr. Cyperus pectinatus Vahl Cyperus pulchellus R.Br. Kyllingiella polyphylla (A.Rich.) Lye Oxycaryum cubense (Poepp. & Kunth) Palla Voucher Larridon et al. 2009-0001 (GENT) Larridon et al. 2010-0261 (GENT) Origin Mwachala 341 (EA) Kenya Larridon et al. 2010-0265 (GENT) Muasya 2131 (EA) Kenya Madagascar Madagascar Kenya Muasya 2435 (EA) Kenya Mwachala 340 (EA) Kenya C A P B Gp D W W ss W gy gy s G Gp P ss W P B B iB Ra E B Figure 5 – SE micrographs and macroscopic images of spikelets in Cyperus pectinatus. A, spikelet subtended by a bract, with proximally the spikelet prophyll, followed by numerous glumes; B, part of partial inlorescence with a rachis and involucral bract, and some spikelets (the prophyll of one of the spikelets is coloured red, the bract subtending the spikelet yellow); C–E, proximal part of a spikelet, with in yellow, the bract subtending the spikelet, in red the spikelet prophyll [proximally, several glumes are empty (D, arrowed); alternating with the prophyll, only a scar of the proximal glume can be observed (E, encircled); the next glume is coloured in blue; the wings of this glume envelop the alternate, higher glume (coloured green); in the axil of the green coloured glume, the scars of loral parts are visible (stamens and gynoecium, coloured respectively yellow and purple)]. Abbreviations: B, bract; G, glume; Gp, proximal glume; gy, gynoecium; iB, involucral bract; P, prophyll; Ra, rachis; s, stamen; W, wing. 335 Pl. Ecol. Evol. 144 (3), 2011 F G G F B G o G W o s o sg sg s ov s ov s W s G G s s C sg s s sg o sg s s sg s D A G sg A st a a a st sg sg a ov G Rl a st a ov st a W a B a ov ff C ov f f nu a a Cu a ov W F 336 E D f G Larridon et al., Taxonomic changes in C3 Cyperus (Cyperaceae) ◄ Figure 6 – SE micrographs of the earliest loral ontogenetic stages in Cyperus pectinatus. A, distal part of a developing spikelet with glumes subtending a fower at different developmental stages; the spikelet apex is open, and immediately below it, successively and alternately, new glume primordia appear, so that the oldest glumes and lowers are situated proximally [at both sides of each lower, the wings of the alternate, higher glume are visible (arrowed)]; B, detail of new glume primordium (coloured green), with in its axil a yet undifferentiated lower primordium (coloured blue); C, later stage of loral development, with already visible three stamen primordia (coloured yellow), and a primordial gynoecium (coloured purple) consisting of an annular ovary primorium surrounding a central ovule primordium; D, idem as in “C”, later developmental stage; the ovary wall is growing up and envelopping the central ovule; on the top of the ovary wall, two adaxially situated and one baxial stigma primordia appear. Abbreviations: F, lower primordium; G, glume (primordium); o, ovule primordium; ov, ovary wall primordium; s, stamen (primordium); sg, stigma primordium; W, wing; *, rachilla apex. ◄ Figure 7 – SE micrographs of the loral ontogeny in Cyperus pectinatus. A, adaxial view of a developing lower; style and stigma branches are growing (purple), while the formation of anther and ilament in the two stamens (yellow) is completed; B, apical view of a a section through the middle part of a developing spikelet [two alternate lowers are visible, and the wing of a higher glume (arrowed)]; C, semi-mature pistil and stamen [along the ribs of the pistil, formation of cork is starting (arrowed)]; D, lateral-abaxial view of semi-mature lower with three stamens [on the top of each anther, a small apiculus is formed (encircled); below the anthers, the connective continues in the upper part of the ilament (arrowed); at this stage, ilaments are fused at their bases]; E, nutlet with three persistent withered stamens (notice that the stamens in these samples remain small with respect to the gynoecium); F, section through a hollow culm, with large peripheral cavities. Abbreviations: a, anther; Cu, culm; f, ilament; G, glume; nu, nutlet; ov, ovary wall primordium; Rl, rachilla; sg, stigma primordium; st, style; W, wing. Morphology and spikelet and loral ontogeny Cyperus sect. Anosporum and C. sect. Pseudanosporum – Spikelets of Cyperus pectinatus and C. colymbetes are distichously organised (igs 5 & 8). Lateral spikelets are subtended by a bract (ig. 5A & B), have a spikelet prophyll (ig. 5A–C), and the irst glumes are empty (ig. 5D). In C. pectinatus, the proximal glume can be dehiscent, with only a scar of it remaining in the spikelet (ig. 5E). The spikelet of C. pectinatus and C. colymbetes has an indeterminate rachilla (ig. 6A, ig. 8A & B). Immediately below the rachilla apex, new glumes originate alternately (ig. 6A, ig. 8A & B). Soon, in the axil of a newly originated glume, a lower primordium appears (igs 6B & 8B). The lower primordium differentiates into two adaxially situated and one abaxially situated stamen primordia, the latter being slightly retarded (igs 6C & 8C). At this stage, a gynoecium is originating from an annular ovary wall primordium surrounding a central ovule primordium (igs 6C & 8C). Next, on the top of the raising ovary wall, opposite the stamen primordia, three stigma primordia appear (igs 6D & 8D). Subsequently, the stamen primordia differentiate into ilament and anther, while the ovary wall envelops the central ovule, forming a style. The stigma branches grow up, protruding highly above the stamens (ig. 7A & B, ig. 8D & E). At this stage, the formation of a corky tissue along the ribs of the ovary begins (ig. 7C). On the top of the anthers, a small, unconspicuous apiculus is formed (ig. 7D & E). In the specimens studied, the stamens remain small compared to the gynoecium (ig. 7E). The culm is hollow, and large peripheral cavities are formed (ig. 7F). Figure 9 shows the nutlets of the three species included in Cyperus sect. Anosporum by Kükenthal (1936), C. cephalotes (ig. 9A), C. colymbetes (ig. 9B) and C. pectinatus (ig. 9C), and of the single species of C. sect. Pseudanosporum, C. platystylis (ig. 9D). Corky thickenings are obvious on the nutlets of all four species. In C. cephalotes, C. colymbetes and C. pectinatus the thickenings are concentrated at the base of the nutlets (ig. 9A–C), in C. platystylis the corky tissue is more evenly present along the three ridges of the trigonous nutlet (ig. 9D). A transverse section through a mature nutlet of C. pectinatus shows the embryo surrounded by a scleriied pericarp, as well as two lateral ridges consisting of parenchymatous cells (ig. 9E). To conirm the ‘corky’ nature of these lateral ridges we performed a berberine/aniline blue luorescent staining procedure, which has widely been used to stain suberised and ligniied cell walls (Brundrett et al. 1988). As suberin is the main constituent of cork this dye was used to check the presence of suberin in the lateral ridges. Berberin/ aniline blue stained cell walls in the ridges yellowish green (ig. 9F), whereas unstained control sections only displayed weak autoluorescence (ig. 9G), suggesting that cell walls in the ridges are suberised and/or ligniied. The negative phloroglucinol/HCl test (data not shown) and the absence of blueautoluorescence in the parenchymatous cells of the ridges (ig. 9H) further suggested that these are non-ligniied. In conclusion, these experiments suggest that the ridges of the nutlets of C. pectinatus are suberised and that the nutlets can indeed be called ‘corky’. Courtoisina and Cyperus reduncus – In Courtoisina cyperoides, spikelets are grouped in clusters, each subtended by a bract (ig. 10A). A spikelet cluster results from prophyll branching, i.e. in the spikelet prophyll, a secondary, tertiary etc. axis originates (ig. 10B–D). In both species, glumes have pronounced wings and a conspicuous mucro (e.g. in ig. 10E). The glumes are distichous upon an indeterminate rachilla (e.g. in ig. 11A) and subtend each a lower. The lower consists of a trimerous ovary and three stamens, which, at the early ontogenetic stages, grow faster than the gynoecium (ig. 11B), but later the developing stigma branches protrude above the stamens (ig. 11C). The ovary develops into a long trimerous nutlet (ig. 11D). The spikelets and nutlets of Cyperus reduncus (ig. 12A & B), Courtoisina assimilis (ig. 12C & D) and Courtoisina cyperoides (ig. 12E & F) are shown in ig. 12. The fusiform shape of the nutlets of these species is unusual in Cypereae. The glumes of all three species are mucronate. The spikelets in all three species are deciduous as a unit at maturity. However, in Cyperus reduncus the spikelets also break up easily in between the glumes. 337 Pl. Ecol. Evol. 144 (3), 2011 5 Figure 8 – SE micrographs of the loral ontogeny in Cyperus colymbetes. A, apical view of a distichously organised spikelet with indeterminate rachilla apex, with glumes and the lowers they subtend at different developmental stages (“1” is the most recent glume primordium); B, detail of the distal part of a spikelet, with the open rachilla apex and two glumes (green) with each in the axil an undifferentiated lower primordium (blue); C, detail of a developing lower at early ontogenetic stage; D, adaxial view of a developing lower; at this stage, the rising ovary wall envelopes the central ovule, and two adaxial and one abaxial stigma primordia are present on the top of it; the stamen primordia are differentiating into ilament and anther; E, lateral view of a developing lower (stamens are coloured yellow, the gynoecium purple; a wing of a higher, alternate glume is visible). Abbreviations: a, anther; f, ilament; F, lower primordium; G, glume (primordium); o, ovule primordium; ov, ovary wall primordium; s, stamen; sg, stigma primordium; st, style; W, wing; *, rachilla apex. B 3 1 F 2 4 A G 6 F G s o s ov sg s C G st sg a f a sg sg a st D sg ov a a f E G f f Oxycaryum and Cyperus gardneri – The spikelets of Oxycaryum cubense are spirally organised, with numerous glumes, each subtending a lower (ig. 13A & B). Along the rims of the glumes, hairs with a length of more than 0.5 mm grow (ig. 13C). The rachilla is indeterminate (ig. 13B). New glumes originate immediately below the rachilla apex in a tristichous arrangement, and in the axil of a new glume, soon a lower primordium appears (ig. 13D & E). The lower primordium differentiates into three stamen primordia, two adaxial and an abaxial one, and an annular ovary wall primordium surrounding a central ovule primordium (ig. 13F). On the top of the ovary wall, two laterally situated stigma primordia appear (ig. 14A). The stigma primordia grow out into stigma branches, soon protruding above the developing stamens (ig. 14B–E). Simultaneously, the stamen primordia differentiate into ilament and anther. On the top of the anthers, a conspicuous apiculus is formed (ig. 14D). At this stage, the glumes are well developed, with a large mucro (ig. 14E). Subsequently, the style and stigma branches elongate further, being forced to fold within the available space within the glume subtending the lower, with the stamens remaining relatively small (ig. 14F & G). Figure 15 shows a spikelet and nutlets of Cyperus gardneri (ig. 15A & B) and a spikeletcluster and nutlet of Oxycaryum cubense (ig. 15C & D). In C. gardneri, the glumes are distichously arranged and the nutlets are trigonous (trimerous gynoecium). In O. cubense, the glumes are spirally-arranged and the nutlets appear awkwardly lattened (dorsiventrally 338 W lattened dimerous gynoecium). Both species show corky thickenings on the nutlets (ig. 15B & D). The branching pattern and general appearance of the inlorescences of these species also shows similarities. Cyperus sect. Leucocephali and Kyllingiella – In Cyperus pulchellus, the spikelets are organised in clusters, each cluster being subtended by a bract (ig. 16A). A cluster originates by prophyll branching, where in the axil of the prophyll of the main spikelet a secondary spikelet originates, which at its turn has a tertiary spikelet in the axil of its prophyll, and so on (ig. 16B & C). The glumes are distichously placed, with each lower surrounded by the wings of the alternate, higher glume (ig. 16D & E). In Kyllingiella polyphylla, spikelets have spirally-arranged, elongate glumes which envelop the whole spikelet (ig. 17A). The rachilla is indeterminate, and immediately below its apex, new glumes originate in a spiral sequence (ig. 17B & C). Soon, in the axil of a new glume, a lower primordium originates, which differentiates into a stamen primordium and a loral apex (ig. 17C–E). The stamen primordium enlarges, becoming as large as the developing gynoecium, followed by the formation an anther and ilament (ig. 18A & B). Meanwhile, the ovary wall envelops the central ovule, and on its top, two lateral, or three (two adaxially and one abaxially situated) stigma primordia are visible (ig. 18B–E). When there are three stigma branches, this can also be the result of the splitting of one of the two originally formed stigma branches (ig. 18E). Flowers can also have two stamens (ig. Larridon et al., Taxonomic changes in C3 Cyperus (Cyperaceae) A B C D E F G H Figure 9 – A, nutlet with undivided style and two remaining stamens of C. cephalotes (Heckman 166, K); B, nutlet with part of style and two stamens remaining of C. colymbetes (Denny 1283, GENT); C, nutlet of C. pectinatus (De Wolf 92-86, GENT); D, nutlet of C. platystylis (Goetghebeur 6684, K); E, transverse section through a mature nutlet of C. pectinatus (Larridon et al. 2010-0265, GENT); F, berberineaniline blue stained section showing yellowish green stained cell walls; G, unstained control sections showing weak autoluorescence; H, combined image showing blue autoluorescence of ligniied sclerenchyma (top panel) and bright-ield image of the same section (bottom panel). 339 Pl. Ecol. Evol. 144 (3), 2011 P G A sA B B B P sA G Rl D E tA sA G W P C A B F G a G a F sg sg sg G f ov Rl D G nu C 340 Larridon et al., Taxonomic changes in C3 Cyperus (Cyperaceae) ◄ Figure 10 – SE micrographs of the spikelet structure and development in Courtoisina cyperoides. A, two bracts (green), each subtending a spikelet-cluster; B–C, spikelet prophyll (red), subtending a secondary spikelet (sA) of which the proximal part is visible [the prophyll of the secondary axis (not coloured) at its turn subtends a tertiary spikelet (tA, blue)]; D, detail of prophyll branching, the prophyll and rachilla of the main spikelet are coloured in red; E, glume with conspicuous mucro. Abbreviations: B, bract; G, glume; P, prophyll; Rl, rachilla; sA, secondary axis (or spikelet); tA, tertiary axis; W, wing. ◄ Figure 11 – SE micrographs of the spikelet structure and loral development in Courtoisina assimilis. A, distal part of a developing spikelet with the apical zone of the spikelet (red), several distichously placed glumes (green), and the lower primordia each glume subtends (blue); the lowest lower has already three stamen primordia (yellow) and a primordial gynoecium (purple); B, adaxial view of a developing lower, with on the top of the ovary wall three stigma primordia; at this stage, ilaments and anthers are formed; C, part of a spikelet; D, nutlet with persistent style and withered stigma branches (the arrow indicates where the glume is cut). Abbreviations: a, anther; f, ilament; F, lower primordium; G, glume; nu, nutlet; ov, ovary wall primordium; Rl, rachilla; sg, stigma primordium. A B C D E F Figure 12 – A, spikelet and B, nutlet of Cyperus reduncus (Madsen 6136, GENT); C, spikelet and D, nutlet of Courtoisina assimilis (Hooper & Townsend 1588, GENT); E, spikelet and F, nutlet of Courtoisina cyperoides (Malaisse & Goetghebeur 161, GENT). 341 Pl. Ecol. Evol. 144 (3), 2011 A D s s gy 5 G s 3 s 2 B s 6 G4 G7 s F E C 1 F gy G F o ov s s s G G sg s s sg o ov A sg s G sg sg sg sg s a s s sg D a a a st f f B st sg s a G sg ov s s C 342 G f ov f F G a E G f Larridon et al., Taxonomic changes in C3 Cyperus (Cyperaceae) ◄ Figure 13 – SE micrographs of the spikelet structure and loral development in Oxycaryum cubense. A–B, spikelets [encircled is a developing lower of which the stamens (yellow) are visible after removing a glume]; C, rim of a glume, with long, curled hairs; D, apical view of a developing spikelet [six (primordia of) intact glumes (green) and the scar of a seventh (G7) are visible, each subtending a lower (primordium); the most recently formed glume immediately under the rachilla apex is coloured blue, the irst lower primordium (in the axil of the third glume, arrowed) is coloured red]; E, detail of glume (green) subtending a lower primordium (red); F, apical view of a developing lower, with three stamen primordia (yellow) and an annular overy wall primordium surrounding a central ovule primordium (purple). Abbreviations: F, lower primordium; G, glume; gy, primordial gynoecium; o, ovule primordium; ov, ovary wall primordium; s, stamen; sg, stigma primordium; *, rachilla apex. ◄ Figure 14 – SE micrographs of the loral development in Oxycaryum cubense. A, apical view of a developing lower (two stigma primordia originate laterally on the top of the annular ovary wall primordium); B–D, lateral-abaxial view of a developing lower [at these successive stages, the stamen primordia (yellow) start differentiating into anther and ilament; the stigma primordia are growing up, forming stigma branches (purple); on the top of each anther, a conspicuous apiculus is formed (encircled)]; E, adaxial view of a semi-mature lower and the glume that subtends it [the glume has a large mucro (encircled); at this stage, the gynoecium (purple), with a long style, is larger than the stamens]; F, idem, at a later stage [the style (encircled) has become so long that style and stigma branches are folded within the available space]; G, detail of the ovary and style base. Abbreviations:a, anther; f, ilament; G, glume; gy, primordial gynoecium; o, ovule primordium; ov, ovary wall primordium; s, stamen; sg, stigma primordium; st, style. A B C D Figure 15 – A, spikelet and B, nutlet of Cyperus gardneri (Schessl 3316, GENT); C, partial inlorescence of Oxycaryum cubense (Guillen et al. 2257, GENT); D, nutlet of O. cubense (Kalliola et al. 2257, GENT). 18C) or two stamen primordia of which only one develops (ig. 18F). The nutlet is obovate (ig. 18G). DISCUSSION Spikelet structure All spikelets studied in C3 Cyperus concur with the spikelet model as proposed by Vrijdaghs et al. (2010). Compared with our previous study in the C4 species of Pycreus and C. laevigatus L. (Vrijdaghs et al. 2011), in the distichous species studied, concaulescence and epicaulescence are less conspicuously present. On the other hand, the lexibility of primordia in the axil of a glume to develop into either a lower or into a secondary spikelet is present C3 Cyperus as well. In Courtoisina cyperoides and Cyperus pulchellus, prophyll branching occurs, which indicates that prophylls, even in Cypereae, still have the potential to form a primordium in their 343 Pl. Ecol. Evol. 144 (3), 2011 sg sg sg a st W B P B P G ov f D A B B P’ B P C E Figure 16 – SE micrographs of spikelet and loral development in Cyperus pulchellus. A, two bracts with spikelet clusters resulting from prophyll branching; B, detail of spikelet cluster, with main axis (yellow) in bract (green) [in the prophyll of the main axis (yellow, P), a secondary axis is present (red); in the prophyll of the secondary axis (red, P’), a tertiary axis (blue) can be seen]; C, detail of a spikelet with a secondary spikelet (blue) in the axil of the spikelet prophyll (removed, scar arrowed); D, abaxial view of a developing lower [a single stamen (yellow), a gynoecium with three stigma branches (purple), and a wing of the higher, alternate lower is visible]; E, abaxial view of the gynoecium (purple) of a lower of which the stamen is removed [higher, and mostly hidden, is a younger lower (glume removed) and distally a glume hiding the apical part of the spikelet; the wings of the alternate, higher glumes are visible (arrowed)]. Abbreviations:a, anther; B, bract; f, ilament; G, glume; ov, ovary wall primordium; P, spikelet prophyll; P’, secondary spikelet prophyll; s, stamen; sg, stigma primordium; st, style; W, wing. axil, and that this primordium can be developed into a secondary axis (spikelet). Prophyll branching in C3 Cyperus was already described by Guarise & Vegetti (2008) for Cyperus sect. Luzuloidei. In most Cyperoideae, the spikelet prophyll is empty, with exception of Dulichieae and Caricae, where the spikelet prophyll subtends a lower. Whether primordia subtended by a glume (and we consider the spikelet prophyll as a irst glume) develop into lower or axis depends on phytohormonal regulation (Smith 1967). One might expect that: (1) presence or absence of a primordium in the axil of a glume/prophyll, (2) development of a primordium subtended by a glume/prophyll into lower or secondary spikelet, in all Cyperoideae are regulated by the same underlying genetic and developmental programmes. In Cypereae, the branching lexibility at spikelet level is high compared with the other 344 cyperoid subtaxa, by prophyll branching or by dedoublement of the spikelet primordium itself, or by the formation of secondary spikelets in the axil of glumes observed in Pycreus pumilus (L.) Nees (Vrijdaghs et al. 2011). This slightly blurs the cyperoid spikelet concept as “ultimate inlorescence branch” (Vrijdaghs et al. 2010). In Cyperus pectinatus, the prophyll as well as the proximal glumes are empty. Moreover, the irst glume alternating with the prophyll may be dehiscent. In such spikelets, an apparently unusual dispostion of the prophyll and irst glumes can be observed (ig. 5C & E). Floral ontogeny All lowers in C3 Cyperus concur with the general cyperoid loral ontogenetic model as proposed by Vrijdaghs et al. Larridon et al., Taxonomic changes in C3 Cyperus (Cyperaceae) (2009). The perianth is totally absent. Observed conspicuous reduction tendencies in androecium and gynoecium are the reduction of number of stigma branches and of stamens. In Oxycaryum cubense and Kyllingiella polyphylla, the stigma branches can be reduced from three to two (here, the word “reduction” is pehaps misleading, since in our opinion, it is rather a reorganisation of the gynoecium due to the development of the ovary wall from an annular primordium and to the ontogeny of the vascular bundles which link from the loral organ primordia to the stele; Reynders et al. accepted). In the latter species, the number of stigma branches can be two or three, and if three, this can be either by development from three stigma primordia, or by development from two stigma primordia of which one undergoes splitting during its development (ig. 18B, D & E). In K. polyphylla, the number of stamens varies between one and two. Here, a literal meaning can be given to the term “reduction” as shown in igure 19F, where two stamen primordia are formed, but only one develops into a stamen. In Cyperus pulchellus, the number of stamens is apparently ixed to one. In the other species studied, like in many other cyperoid species, the development of the abaxial stamen, especially at early ontogenetic stages, may be retarded a little bit with respect to the development of the two adaxial stamens (ig. 16D, ig. 17D & E). Cyperus sect. Anosporum and C. sect. Pseudanosporum Based on molecular phylogenetic data (ig. 1) (Larridon et al. 2011b), Cyperus sect. Anosporum and C. sect. Pseudanosporum are very closely related. Cyperus platystylis is the sister species of C. pectinatus. Possibly, C. platystylis is an intermediate on the evolutionary lineage leading from a more typical Cyperus morphology to the more specialised morphology of the three species included in C. sect. Anosporum by Kükenthal (1936), i.e. C. cephalotes, C. colymbetes and C. pectinatus. Morphological resemblance between all four species (e.g. tightly imbricate rather glossy and thick glumes), their shared habitat preference (wetlands, ig. 2B) and their adaptations to this habitat (corky nutlets, ig. 9; air cavities, ig. 7F), and embryology (igs 3 & 4) all support the inclusion of C. sect. Pseudanosporum in a broader circumscribed C. sect. Anosporum (see Taxonomic treatment). These corky thickenings allow the nutlets to loat. The corky nutlets of these species are often distributed inside their glumes and with the short stamens still attached to the base of the nutlet; this might give the nutlets even more buoyancy (air bubble?). Courtoisina Although the habit of the two Courtoisina species corresponds with that of Cyperus s. str., authors as Goetghebeur (1986, 1998), Vorster (1996), Govaerts et al. (2007, 2011) recognised Courtoisina as a distinct genus based on the combination of several differentiating characters. The characters identifying Courtoisina include spikelets disarticulating as a unit when mature leaving the spikelet bract and prophyll behind, winged glumes, and an unusually differentiated Cyperus-type embryo. However, one other C3 Cyperus species, i.e. Cyperus reduncus (included in Cyperus sect. Fusci by Kükenthal 1936), closely resembles the two Courtoisina species. It shares the therophytic habit, typical yellowish green colour, the long laccid leaves and leaf-like primary involucral bracts, and the spikelets disarticulating as a unit when mature, leaving the spikelet bract and prophyll behind. Additionally, in Cyperus reduncus, the rachilla of the spikelet can easily be broken at any point between glumes. In this species the nutlets are still distributed separately (nutlet = unit of dispersal). In Courtoisina assimilis and Courtoisina cyperoides the glumes closely envelop the few or single maturing nutlets. Furthermore, the glumes of the two Courtoisina species are clearly winged (ig. 12C & E), helping wind-distribution of the spikelet as a unit (spikelet = unit of dispersal). Though the glumes of Cyperus reduncus lack the conspicuous wings (ig. 12A), they are otherwise very similar to those of Courtoisina, but there are more glumes per spikelet. Also, Cyperus reduncus shares the oddly elongated nutlets (ig. 12B, D & F) and the unusually differentiated Cyperus-type embryo (strongly asymmetrical development of the coleoptile, igs 3 & 4) with Courtoisina. The molecular phylogenetic hypothesis of Larridon et al. (2011b) (igs 1 & 4), conirms the very close relationship of Courtoisina and Cyperus reduncus, and veriies its phylogenetic position in C3 Cyperus. Consequently, in the formal taxonomic treatment (see below) the two previously recognised Courtoisina species are included in Cyperus and put in their own section together with Cyperus reduncus. The characters previously used to separate Courtoisina from Cyperus have recently been shown to be of little taxonomic value (Muasya et al. 2009a, 2009b). These characters are homoplasies; they have arisen multiple times in different Cyperus lineages. For example, deciduous spikelets occur not only in the two Courtoisina species and Cyperus reduncus, but also in another, not closely related C3 Cyperus species, Cyperus deciduus Boeck., and in many C4 Cyperus species. Winged glumes, another “Courtoisina character”, also occur in different, unrelated lineages of the Cyperus clade like Ascolepis and Kyllinga. Unlike Clarke (1893), Kükenthal (1936), Podlech (1960), and Gordon-Gray (1995), we consider the infraspeciic distinction between the African and Asian specimens of Courtoisina cyperoides unjustiied. These authors defended a distinction at infraspeciic rank (subspecies or variety) by the presence of an excurving mucro of c. 0.5 mm present in the African specimens, but absent in the Asian specimens of Courtoisina cyperoides. However, in the type specimen of Courtoisina cyperoides (Wallich 3537, from India) the debated mucros are clearly present. Based on biogeography and morphology we place the origin of the section in Africa, where Cyperus reduncus most closely resembles its related C3 Cyperus species. Courtoisina cyperoides and Courtoisina assimilis represent further evolutionary steps away from the typical Cyperus characters (reduction of the number of glumes, conspicuously winged midrib). As mentioned above, the glumes of Courtoisina cyperoides from some Asiatic specimens illustrate the loss of the generally present mucros. Oxycaryum and Cyperus gardneri As mentioned above, Oxycaryum cubense was included in different genera based on its aberrant morphology (spirallyarranged glumes and dimerous dorsiventrally lattened gyn345 Pl. Ecol. Evol. 144 (3), 2011 F G F G G G G s fa G A B G D fa s G F G C E sg sg sg sg a s a st ov ov a f f ov B C A nu ov ff D 346 E F G Larridon et al., Taxonomic changes in C3 Cyperus (Cyperaceae) ◄ Figure 17 – SE micrographs of spikelet and loral development in Kyllingiella polyphylla. A, apical view of a spikelet, with spirally placed glumes, each subtending a lower (encircled); B, detail of the distal part of a spikelet, with the spikelet apex, and several glumes, each subtending a lower, at different developmental stages; C, detail of spikelet apex; D, detail of two glumes, each with a lower primordium, at successive stages; E, detail of a glume (green), with a lower primordium differentiating into a loral apex (purple) and a lateral stamen primordium (yellow). Abbreviations: F, lower primordium; fa, loral apex; G, glume; s, stamen; *, rachilla apex. ◄ Figure 18 – SE micrographs of spikelet and loral development in Kyllingiella polyphylla. A–C, detail of a developing lower at three successive stages [the stamen primordium (yellow) starts differentiating into ilament and anther; the ovary wall (purple) is covering the central ovule, and on its top two laterally positioned stigma primordia grow out]; D–E, gynoecia with two and three stigma branches (encircled), and splitting of one of the stigma branches (arrowed); F, lower with a not further developing stamen primordium (arrowed); G, nutlet, with persistent withered ilament. Abbreviations: a, anther; f, ilament; nu, nutlet; ov, ovary wall; s, stamen; sg, stigma branch (primordium); st, style. oecia / nutlets, igs 2E, 13, 14 & 15). However, the two key characteristics used to recognise Oxycaryum have originated multiple times in Cyperus (e.g. Muasya et al. 2009a, 2009b, Reynders et al. accepted, Vrijdaghs et al. 2011). A reversal to spiral glume arrangement (as found in the Ficinia clade) has occurred several times in the Cyperus clade (usually distichous glume arrangement), i.e. in Oxycaryum cubense and Kyllingiella (C3 Cyperus) and in Cyperus michelianus (C4 Cyperus). Dimerous dorsiventrally lattened nutlets also originated multiple times independently in the Cyperus clade, i.e. in Oxycaryum cubense and in various C4 Cyperus lineages. Consequently, there is clear justiication to include Oxycaryum in C3 Cyperus. The use of the C3 photosynthetic pathway (linked with eucyperoid anatomy), the presence of a Cyperus-type embryo (igs 3 & 4), and its phylogenetic position in C3 Cyperus (ig. 1) (Larridon et al. 2011b) further substantiate this inclusion. Molecular phylogenetical data (ig. 1) (Larridon et al. 2011b) also indicate a close relationship with Cyperus gardneri, a species morphologically resembling Oxycaryum cubense to some extent: somewhat contracted inlorescence, corky nutlets (ig. 15B), and a similar embryo (igs 3 & 4). In the taxonomic treatment below, the genus Oxycaryum is combined into Cyperus at sectional rank including Oxycaryum cubense and C. gardneri. Cyperus sect. Leucocephali and Kyllingiella The species of Cyperus sect. Leucocephali and those of the small genus Kyllingiella show a marked resemblance in habit (small to medium-sized grasslike plants with a thickened base and a pale-coloured capitate inlorescence, ig. 2H & G). Also, they share a preference for seasonally dry open grasslands; this is rather unusual for C3 Cyperus species which generally prefer forests and marshes. In this context, the phylogenetic position of C. sect. Leucocephali and Kyllingiella as sister clade to the C4 Cyperus clade (ig. 1) (Larridon et al. 2011b) might indicate a transitional stage towards C4 physiology which is relected by their enhanced drought resistance. The only character to uphold Kyllingiella as a distinct genus is the spiral arrangement of its glumes. As mentioned above, recent studies (Muasya et al. 2002, 2006, 2007, 2009a, 2009b, Simpson et al. 2007, Larridon et al. 2011b) showed that the presence of the spirally-arranged glumes is not a phylogenetically informative character, as this glume arrangement arose many time in Cypereae. Also in Cyperus pulchellus, the glume arranged is not entirely distichous (ig. 16A, B & E). Therefore, Kyllingiella is included here in C3 Cyperus, and more speciically into C. sect. Leucocephali. In 1990, Simpson published a revision of C. sect. Leucocephali including seven species. Since then, he described two additional species (Simpson 1992, 1993) in this section. In 1992, when Simpson described C. androhibensis, it was the irst recorded specimen of C. sect. Leucocephali in Madagascar. However, due to several recent inds of C. pulchellus in Madagascar, we now feel C. androhibensis should not be upheld as a separate species. In our opinion, the type (and only) specimen of C. androhibensis is an aberrant / not very well developed specimen of C. pulchellus. Consequently, C. androhibensis is here placed in synonymy of the widely distributed C. pulchellus. Cyperus sect. Dichostylis sensu Kükenthal (1936) p.p. Although possessing a small, congested, globose inlorescence with numerous spikelets as Cyperus sect. Leucocephali, Kükenthal (1936) nor Simpson (1990) considered Cyperus seslerioides to belong in C. sect. Leucocephali. Kükenthal (1936) placed C. seslerioides in his ‘C. sect. Dichostylis’. Simpson (1990) did not include C. seslerioides in C. sect. Leucocephali because of its ovate-lanceolate, excurrent glumes and trigonous achenes. However, some similarity can be seen between the embryos of Cyperus seslerioides and the embryos of C. tenerrimus and Kyllingiella microcephala (ig. 3). Several other C3 Cyperus species were included in C. sect. Dichostylis sensu Kükenthal (1936) by Kükenthal or have since been described: C. humilis, C. tweediei, C. uncinulatus, C. arsenei, C. microbrunneus and possibly C. hilairenus. It should be mentioned that the C3 photosynthetic pathway has only been conirmed in C. humilis, C. seslerioides and C. uncinulatus (Bruhl & Wilson 2007). Tucker (1983) placed his new species C. microbrunneus in C. sect. Dichostylis sensu Kükenthal (1936) based on its small size, narrow leaves, densely capitate, rayless inlorescence, oblong-lanceolate spikelets, one stamen per lower and small stipitate achenes. Although the species included in C. sect. Dichostylis sensu Kükenthal (1936) deinitely share some characters, this group of species is also obviously heterogeneous / polyphyletic as it includes both C3 and C4 Cyperus species. Molecular phylogenetic study is required to determine the relationships between the species in this group and their phylogenetic position in Cyperus. 347 Pl. Ecol. Evol. 144 (3), 2011 Table 3 – A preliminary classiication of C3 Cyperus. For more details on the nomenclature and typiication of the sections see Larridon et al. (2011a). Clade 1 Cyperus sect. Haspani Cyperus sect. Incurvi (probably polyphyletic) Clade 2 Cyperus sect. Diffusi Cyperus sect. Luzuloidei Clade 3 Cyperus sect. Courtoisina Cyperus sect. Oxycaryum Clade 4 Cyperus sect. Fusci Cyperus sect. Alternifolii Clade 5 C3 Cyperus Cyperus subg. Anosporum Cyperus Cyperus sect. Anosporum Cyperus sect. Leucocephali Unplaced C3 Cyperus Cyperus sect. Graciles Cyperus sect. Dichostylis sensu Kük. p.p. / Cyperus sect. Humiles Cyperus sect. Radiantes Clade 6 C4 Cyperus Cyperus subg. Cyperus Selected species (see table 4 of Larridon et al. 2011b). Unresolved, includes more than 500 Cyperus s.str. species and nine segregate genera (Huygh & Reynders et al. unpubl. data). Cyperus constanzae Urb. Kükenthal (1936) included Cyperus constanzae in C. sect. Glutinosi Kük., nom. illegit. As explained by Larridon et al. (2011b), the correct name for this section is C. sect. Elegantes C.B.Clarke (Clarke 1883: 288). Kükenthal (1936) included six species in this section, four species use the C4 348 photosynthetic pathway and two species (C. constanzae and C. gardneri) use C3 photosynthesis (Bruhl & Wilson 2007, Larridon et al. 2011b). Molecular phylogenetic study revealed a close relationship of C. gardneri with Oxycaryum cubense (ig. 1) (Larridon et al. 2011b). However, the position of C. constanzae in C3 Cyperus remains unknown. The embryo of C. constanzae shows most resemblance to the embryos of species of C. sect. Diffusi and C. sect. Incurvi (ig. 3). Based on its general morphology such a relationship is possible, but molecular phylogenetic conirmation is necessary to place this taxon in its correct section. Cyperus sect. Graciles This section was not included in the molecular study of Larridon et al. (2011b). For seven of the 11 species the photosynthesis type was conirmed as C3 (Bruhl & Wilson 2007, Larridon et al. 2011b). Although Blake (1939) suggested a relationship of Cyperus sect. Graciles with C. sect. Haspani, this is unlikely since the embryos of the C. sect. Graciles species included in this study do not at all resemble those of C. sect. Haspani (ig. 3). The embryos of C. gracilis and C. tetraphyllus show much more resemblance both to the embryo of C. alternifolius (shape) and to some of the embryos of C. sect. Diffusi and C. sect. Incurvi (size) (ig. 3). Based on embryographical data alone, it is impossible to clearly indicate the phylogenetic position of C. sect. Graciles. Furthermore, the morphology of the species of C. sect. Graciles does not show obvious similarities with just one of the Cyperus sections mentioned above. Molecular phylogenetic conirmation is needed here. TAXONOMIC TREATMENT Cyperus sect. Anosporum (Nees) Pax (Pax 1887: 107) – Anosporum Nees (Nees 1834a: 287) – Cyperus subg. Anosporum (Nees) C.B.Clarke (Clarke 1884: 34) – Type: Cyperus monocephalus Roxb. [= Cyperus cephalotes Vahl]. Hydroschoenus Zoll. & Moritzi (Moritzi 1846: 95). Trentepohlia Boeck., nom. rej. (Boeckeler 1858: 249). Cyperus sect. Pseudanosporum C.B.Clarke (Clarke 1884: 117) – Cyperus sect. Natantes C.B.Clarke, nom. illegit. (Clarke 1893: 597). Cyperus sect. Cephalotes J.V.Suringar, nom. illegit. (Suringar 1898: 76). Cyperus sect. Nudicaules Cherm., nom. invalid. (Chermezon 1922: 3). Diagnosis – Perennials, adapted to an aquatic (often loating) lifestyle. Glumes tightly imbricate, rather glossy and thick. Style unbranched, shortly branched or more deeply branched. Nutlets dark surrounded by paler corky tissue (at least on angles and at apex). Habitat – Growing in swamps or pools, either loating in deep water or emergent with roots in mud. Distribution – Species 4, tropical Africa, Asia and Australia. Species 1. Cyperus cephalotes Vahl (Vahl 1805: 311) – Anosporum cephalotes (Vahl) Kurz (Kurz 1876: 159) – Type: India, Nico- Larridon et al., Taxonomic changes in C3 Cyperus (Cyperaceae) Key to the species of Cyperus sect. Anosporum 1. 1’. 2. 2’. 3. 3’. Leaves reduced to their leaf-sheaths; involucral bracts 1–2 short, rigid and rather sharp........................2 Leaves not reduced................................................................................................................................3 Culms 3–5 mm thick, sharply triangular to winged; involucral bract 1.............................C. colymbetes Culm 0.5–2 mm thick, rounded-angular; involucral bracts 1–2..........................................C. pectinatus Bracts 3–5 leaf-like, 5–30 cm long; inlorescence capitate.................................................C. cephalotes Bracts 4–12 leaf-like, 30–80 cm long; inlorescence anthelate...........................................C. platystylis bar Islands, Vahl s.n. (holo-: C). Cyperus monocephalus Roxb. (Roxburgh 1820: 193) – Anosporum monocephalum (Roxb.) Nees (Nees 1834a: 287). Cyperus monocephaloides Roxb. ex Nees, nom. invalid. (Nees 1834b: 92). Hydroschoenus kyllingioides Zoll. & Moritzi (Moritzi 1846: 95). Trentepohlia bifoliata Boeck. (Boeckeler 1858: 249). Cyperus hookerianus Thwaites (Thwaites 1864: 342). Cyperus monogynus Boeck. (Boeckeler 1868: 565). Cyperus natans Buch.-Ham. ex C.B.Clarke, nom. invalid. (Clarke 1884: 34). Ungeria monocephala (Roxb.) Nees ex C.B.Clarke, nom. invalid. (Clarke 1884: 34). Ficinia foliaceobracteata H.Pfeiff. (Pfeiffer 1921: 35). Cyperus cephalotes var. grandiceps Kük. (Kükenthal 1943: 4). Distribution – Tropical Asia to Northeastern Australia. Description – Lye (1981: 187). 2. Cyperus colymbetes Kotschy & Peyr. (Kotschy & Peyritsch 1867: 49) – Anosporum colymbetes (Kotschy & Peyr.) Boeck. (Boeckeler 1869: 26) – Original type: Sudan, Bahrel-Ghasal, Tinne s.n., (holo-: W, destroyed during the war, Kotschy & Peyritsch 1867: t. XXIV). Distribution – Sudan to Mozambique, Madagascar. Description – Lye (1981: 188). 3. Cyperus pectinatus Vahl (Vahl 1805: 298) – Anosporum pectinatum (Vahl) Lye (Lye 1981: 188) – Type: Guinea, Dahomey, Ouidah, Isert s.n. (holo-: C). Cyperus nudicaulis Poir. (Poiret 1806: 240) – Anosporum nudicaule (Poir.) Boeck. (Boeckeler 1869: 26). Atomostylis cyperiformis Steud. (Steudel 1855: 315). Atomostylis lavescens Steud. (Steudel 1855: 315). Distribution – Tropical and Southern Africa, Madagascar. Description – Lye (1981: 188). 4. Cyperus platystylis R.Br. (Brown 1810: 214) – Type: Australia, New South Wales, Hawkesbury, Brown 5907 (holo-: BM). Cyperus pallidus Nees, nom. illegit. (Nees 1834b: 79) – Anosporum pallidum Boeck. (Boeckeler 1870: 412). Cyperus luitans Buch.-Ham. ex C.B.Clarke, nom. invalid. (Clarke 1884: 118). Distribution – Tropical and subtropical Asia, Australia. Description – Kern (1974: 618). Cyperus sect. Courtoisina (Soják) Larridon, comb. nov. – Courtoisina Soják, Časopis Národního muzea, řada přírodovědecká 148: 193. 1979 (Soják 1979) – Courtoisia Nees, nom. illegit., non Marchand (1830) (Nees 1834a: 286) – Indocourtoisia Bennet & Raizada, nom. illegit. (Raizada & Bennet 1981: 432) – Pseudomariscus Rauschert, nom. illegit. (Rauschert 1982: 559) – Cyperus subg. Courtoisia (Nees) Lye (Lye 1983: 230) – Cyperus subg. Courtoisina (Soják) Lye, nom. illegit. (Lye 1992: 52) – Type: Cyperus pseudokyllingioides Kük. as nomen novum of Courtoisia cyperoides (Roxb.) Nees (Kyllinga cyperoides Roxb.). Diagnosis – Medium-sized therophytes yellowish green with long laccid leaves and leaf-like primary involucral bracts, strongly lattened spikelets which disarticulate as a unit when mature leaving the spikelet bract and prophyll behind, glumes often conspicuously winged (except in C. reduncus). Some authors (Haines & Lye, 1983; Goetghebeur, 1998) report a strong odour (curry scent). Habitat – Often growing on temporarily wet sandy soils. Distribution – Species 3, widely distributed in tropical Central, East and South Africa, one also in Madagascar, India and Southeast Asia. Species 1. Cyperus assimilis Steud. (Steudel 1842: 584) – Courtoisia assimilis (Steud.) C.B.Clarke (Clarke 1894: 596) – Mariscus assimilis (Steud.) Podlech (Podlech 1960: 523) – Indocourtoisia assimilis (Steud.) Bennet & Raizada (Raizada & Bennet 1981: 432) – Courtoisina assimilis (Steud.) Maquet (Maquet 1988: 265). – Type (designated here): Ethiopia, Schimper 1252 (lecto-: B; isolecto-: BR, G, GOET, HEID, K, L, M, P, S, STU, WAG). Distribution – Ethiopia to South Africa, Madagascar. Description – Haines & Lye (1983: 174). 2. Cyperus pseudokyllingioides Kük. (Kükenthal 1936: 501) – Kyllinga cyperoides Roxb. (Roxburgh 1820: 182) – Mariscus cyperoides (Roxb.) A.Dietr. (Dietrich 1832: 348) – Courtoisia cyperoides (Roxb.) Nees (Nees 1834a: 286) – Cyperus pseudokyllingioides Kük. var. pseudokyllingioides (Kükenthal 1936: 501) – Courtoisina cyperoides (Roxb.) Soják (Soják 1979: 193) – Indocourtoisia cyperoides (Roxb.) Bennet & Raizada (Raizada & Bennet 1981: 432) – Pseudomariscus cyperoides (Roxb.) Rauschert (Rauschert 1982: 559) – Type (lectotype designated here): India, Wallich 3537 (holo-: ?; isolecto-: P). Cyperus kleinianus Hochst. ex Steud., nom. invalid. (Steudel 1854: 71). 349 Pl. Ecol. Evol. 144 (3), 2011 Key to the species of Cyperus sect. Courtoisina 1. 1’. 2. 2’. Glumes winged, 2–4(–12) per spikelet...................................................................................................2 Glumes not winged, 5–25 per spikelet..................................................................................C. reduncus Glumes 2(–3) per spikelet....................................................................................C. pseudokyllingioides Glumes 4(–12 per spikelet.....................................................................................................C. assimilis Key to the species of Cyperus sect. Oxycaryum 1. Glumes distichously-arranged, style-branches 3, nutlets trigonous (tropical and subtropical America)..........................................................................................................................................C. gardneri 1’. Glumes spirally-arranged, style-branches 2, nutlets dorsiventrally plano-convex (tropical and subtropical America and Africa).......................................................................................C. blepharoleptos Courtoisia cyperoides var. africana C.B.Clarke, nom. invalid. (Clarke 1893: 596). Cyperus pseudokyllingioides var. africanus C.B.Clarke ex Kük. (Kükenthal 1936: 501). Distribution – Himalaya to Indo-China, Chad to South Africa, Madagascar. Description – Haines & Lye (1983: 175). 3. Cyperus reduncus Hochst. ex Boeck. (Boeckeler 1868: 580) – Type: Ethiopia, Schimper s.n. (holo-: B, destroyed during the war?; iso-: M). Cyperus aristatus Hook.f. & Thomson ex C.B.Clarke, nom. invalid. (Clarke 1884: 90). Distribution – Chad to South Africa, Madagascar. Description – Haines & Lye (1983: 160). Cyperus sect. Oxycaryum (Nees) Larridon, comb. nov. – Oxycaryum Nees, in Martius, Flora Brasiliensis 2(1): 90. 1842 (Nees 1842) – Scirpus sect. Oxycaryum (Nees) Beetle (Beetle 1944: 263) – Type: Oxycaryum schomburgkianum Nees [= Cyperus blepharoleptos Steud.]. “Crepidocarpus Klotzsch ex Boeck.”, nom. invalid. (Boeckeler 1870: 414). “Scirpus sect. Cubenses Cherm.”, nom. invalid. (Chermezon 1937: 156). Diagnosis – Aquatic, often loating plants. Inlorescence anthelate (with partial inlorescences capitate) to capitate. Spikelets with several distichously or spirally-arranged glumes. Stamens 3. Style 3-id or 2-id. Nutlet trigonous or slightly dorsiventrally lattened, conspicuously corky. Habitat – Floating in water or growing in wet soil. Distribution – Species 2, tropical and subtropical America and Africa. Species 1. Cyperus blepharoleptos Steud. (Steudel 1854: 28) – Type: Senegal, Leprieur s.n. (holo-: P00462624; iso-: P00462625, P00462626). Scirpus cubensis Poepp. & Kunth (Kunth 1837: 172) – Anosporum cubense (Poepp. & Kunth) Boeck. (Boeckeler 1869: 350 26) – Oxycaryum cubense (Poepp. & Kunth) Palla (Palla 1908: 169) – Type: Cuba, Poeppig s.n. (holo-: ?; iso-: P). Oxycaryum schomburgkianum Nees (Nees 1842: 90) – Type: Guyana, Schomburgk 371 (holo-: W; iso-: BM, K, P). Mariscus foliosissimus Steud. (Steudel 1854: 65). Courtoisia olivacea Boeck. (Boeckeler 1861: 331) – Pseudomariscus olivaceus (Boeck.) Rauschert (Rauschert 1982: 559). Scirpus ablepharus Griseb. (Grisebach 1866: 240) – Anosporum ablepharum (Griseb.) Maury (Maury 1890: 125). “Crepidocarpus cubensis (Poepp. & Kunth) Klotzsch ex Boeck.”, nom. invalid. (Boeckeler 1870: 414). Anosporum cubense var. gracile Boeck. (Boeckeler 1870: 414) – Scirpus cubensis var. gracilis (Boeck.) Beetle (Beetle 1944: 146). Isolepis echinocephala Oliv. (Oliver 1875: 167). Anosporum schinzii Boeck. (Boeckeler 1888: 46) – Oxycaryum schinzii (Boeck.) Palla (Palla 1908: 169). Crepidocarpus schinzii Klotzsch ex Boeck., nom. invalid. (Boeckeler 1888: 46). Anosporum paraguayense Maury (Maury 1890: 124) – Oxycaryum paraguayense (Maury) Palla (Palla 1908: 169) – Scirpus cubensis var. paraguayensis (Maury) Kük. ex Barros (Barros 1935: 150) – Scirpus paraguayensis (Maury) Herter (Herter 1943: 161) – Oxycaryum cubense f. paraguayense (Maury) Pedersen (Pedersen 1995: 138). Anosporum piliferum Maury (Maury 1890: 124) – Oxycaryum piliferum (Maury) Palla (Palla 1908: 169) – Scirpus piliferus (Maury) Pickel (Pickel 1937: 124). Oxycaryum guianense Palla (Palla 1908: 169). “Kyllinga scirpina Rchb. ex C.B.Clarke”, nom. invalid. (Clarke 1894: 620). Distribution – Tropical and subtropical Africa, America. Description – Lye (1971: 282–284). 2. Cyperus gardneri Nees (Nees 1842: 34) – Type: Brazil, Gardner 1213 (holo-: BM; iso-: G, K, NY, P, TCD, US). Distribution – Cuba, Southeastern Mexico to Northeastern Argentina. Description – Diego-Pérez et al. (2001: 18, in Spanish). Larridon et al., Taxonomic changes in C3 Cyperus (Cyperaceae) Key to the species of Cyperus sect. Leucocephali 1. Glumes distichously-arranged................................................................................................................2 1’. Glumes spirally-arranged.......................................................................................................................9 2. Inlorescence ± dense (sub-)globose cluster of spikelets; stamen 1 (tropical Old World and Australia).....................................................................................................................................................3 2’. Inlorescence ± dense (sub-)globose cluster of spikelets or more loose half-globose cluster of spikelets; stamens 1, 2 or 3 (Neotropics)........................................................................................................5 3. Leaf-blades 0.4–0.6 mm wide; spikelets 2–5.5 × ± 1 mm; glumes 0.8–1 × 0.4–0.5 mm; achene widely obovoid or subglobose, 0.3–0.4 × 0.25–0.4 mm (Somalia).............................................C. microglumis 3’. Leaf-blades usually > 0.6 mm wide; spikelets > 1 mm wide; glumes > 1.2 mm long; achenes > 0.5 mm long........................................................................................................................................................4 4. Spikelets 4–8 × 1–2 mm wide; glumes 1.2–1.5 × 0.3–0.4 mm; achenes 0.5–0.8 × 0.2–0.3 mm (tropical Old World and Australia)....................................................................................................C. pulchellus 4’. Spikelets 2.5–6.5 × 2–2.5 mm; glumes 1.5–2.5 × ± 0.6 mm; achenes 1.2–1.6 × 0.3–0.4 mm (Indian Subcontinent, Indo-China)...........................................................................................C. leucocephalus 5. Inlorescence with up to 12 spikelets; achene ellipsoid, distinctly trigonous, 0.9–1.3 × 0.5–0.6 mm (Mexico)....................................................................................................................C. michoacanensis 5’. Inlorescence with more than 12 spikelets; achene narrowly cylindrical, cylindrical, obovoid or subglobose, obscurely trigonous, 0.2–0.4 mm wide....................................................................................6 6. Inlorescence bracts usually 5–8; inlorescence usually a loose half-globose cluster of spikelets; glumes 1.45–2 × 0.8–1 mm, prominently nerved; stamen 1 (Central America to Colombia).. ...........................................................................................................................................C. tenerrimus 6’. Inlorescence bracts 3–4(–5); inlorescence a dense, congested cluster of spikelets; glumes indistinctly nerved or nerveless; stamens 2 or 3........................................................................................................7 7. Stamens 3; glumes 2.3–3.6 × 0.8–1.2 mm; nutlet 1.2–1.7 × ± 0.3 mm, dark brown to black; plants often lowering male and female separately (protandry) (South America)...............C. schomburgkianus 7’. Stamens 2...............................................................................................................................................8 8. Glumes 2.1–2.6 × 0.6–1.2 mm; nutlet 1–1.3 × 0.3–0.4 mm, dark brown; mature anthers and gynoecia present at the same time (Mexico)...................................................................................C. nayaritensis 8’. Glumes 1.7–2 × 0.6–0.8 mm; nutlet 0.5 × 0.2 mm, pale brown to dark grey brown; plants sometimes lowering male and female separately (protandry) (Brazil)..................................................C. brumadoi 9. Inlorescence greenish; glumes 2–2.5 mm long including a 0.5 mm long recurved mucro....... .................................................................................................................................................C. spiralis 9’. Inlorescence whitish; glumes not mucronate......................................................................................10 10. Inlorescence head (2–)3(–4) mm in diam.; spikelets ± 2 mm long..................................C. acholiensis 10’. Inlorescence head larger; spikelets longer...........................................................................................11 11. Culms 5–40 cm × 0.2–0.5 mm; inlorescence head 3–8 × 3–5 mm; nutlets 0.5–0.8 mm long................ ...........................................................................................................................................C. kyllingiella 11’. Culms 30–62 cm × 0.7–1.5 mm; inlorescence head 3–7 × 5–9 mm; nutlets 1.3–1.7 mm long.............. .............................................................................................................................................C. simpsonii Cyperus sect. Leucocephali [Chermezon 1931: 17, nom. nud.] Cherm. ex Kük. (Kükenthal 1936: 276) – Cyperus [unranked] Leptolepides Boeck. (Boeckeler 1868: 588) – Cyperus ([unranked] Leptolepides) [unranked] Capitati Boeck. (Boeckeler 1868: 588) – Type: Cyperus leucocephalus Retz. Sorostachys Steud. (Steudel 1854: 71) – Cyperus subg. Sorostachys (Steud.) Lye (Lye 1983: 230). Kyllingiella R.W.Haines & Lye (Haines & Lye 1978: 176). Diagnosis – Small to medium-sized grass-like plants with a pale-coloured head-like inlorescence of numerous small spikelets and small, narrow, membranous glumes. Habitat – Open, seasonally dry habitats, especially grasslands. Distribution – 12 species, wide distribution throughout the tropics. Species 1. Cyperus acholiensis Larridon, nom. nov. – Kyllingiella ugandensis R.W.Haines & Lye (Haines & Lye 1978: 177), non Cyperus ugandensis Chiov. – Type: Uganda, Kertland 111 (holo-: MHU). Distribution – Kenya, Tanzania, Uganda. Description – Haines & Lye (1978: 177). 2. Cyperus brumadoi D.A.Simpson (Simpson 1993: 701) – Type: Brazil, Bahia, Carvalho, Brito & Santos 2617 (holo-: CEPEC; iso-: K). Distribution – Brazil. Description – Simpson (1993: 701). 3. Cyperus kyllingiella Larridon, nom. nov. – Kyllinga microcephala Steud. (Steudel 1842: 597) – Isolepis kyllingi351 Pl. Ecol. Evol. 144 (3), 2011 oides A.Rich., nom. illegit. (Richard 1850: 502) – Scirpus microcephalus (Steud.) Dandy (Dandy 1956: 366) – Scirpus kyllingioides (A.Rich.) Boeck., nom. illegit. (Boeckeler 1870: 733) – Isolepis microcephala (Steud.) Lye (Haines & Lye 1971: 480) – Kyllingiella microcephala (Steud.) R.W.Haines & Lye (Haines & Lye 1978: 176), non Cyperus microcephalus R.Br. – Type: Ethiopia, Schimper 650 (holo-: P; iso-: BR, G, K, MO, P, S, WAG). Distribution – Tropical and southern Africa, Indian subcontinent. Description – Haines & Lye (1983: 142). Distribution – South America: Bolivia (Beck 25586; LPB, GENT), Brazil, Guyana, Venezuela. Description – Simpson (1990: 495). 4. Cyperus leucocephalus Retz. (Retzius 1788: 11) – Sorostachys leucocephalus (Retz.) Lye (Lye 1981: 190) – Type: India, König s.n. (holo-: LD). Scirpus coronarius Vahl (Vahl 1805: 261) – Isolepis coronaria (Vahl) Roem. & Schult. (Roemer & Schultes 1817: 113) – Cyperus coronarius (Vahl) Kunth (Kunth 1837: 44). Kyllinga pierreana E.G.Camus (Camus 1910: 290). Distribution – Northeastern India, Bangladesh, Myanmar, Thailand, Vietnam. Description – Simpson (1990: 494). 11. Cyperus spiralis Larridon, nom. nov. – Isolepis polyphylla A.Rich. (Richard 1850: 503) – Kyllingiella polyphylla (A.Rich.) Lye (Haines & Lye 1983: 143), non Cyperus polyphyllus Vahl – Type: Ethiopia, Quartin Dillon s.n. (holo-: P; iso-: P). Distribution – Ethiopia to east tropical Africa. Description – Haines & Lye (1983: 143). 5. Cyperus michoacanensis Britton ex C.B.Clarke (Clarke 1908: 5) – Type: Mexico, Pringle 3427 (holo-: VT; iso-: K, NY). Cyperus patzcuarensis C.B.Clarke ex Kük., nom. invalid. (Kükenthal 1936: 277). Distribution – Mexico. Description – Simpson (1990: 500). 6. Cyperus microglumis D.A.Simpson (Simpson 1990: 492) – Type: Somalia, Beckett 217 (holo-: K; iso-: EA). Distribution – Somalia. Description – Simpson (1990: 492). 7. Cyperus nayaritensis G.C.Tucker (Tucker 1983: 161) – Type: Mexico, Nayarit, Feddema 418 (holo-: DUKE; iso-: ENCB, MICH). Distribution – Mexico. Description – Simpson (1990: 499). 8. Cyperus pulchellus R.Br. (Brown 1810: 213) – Sorostachys pulchellus (R.Br.) Lye (Lye 1981: 189) – Type: Australia, Brown 5917 (holo-: K; iso-: L). Sorostachys kyllingioides Steud. (Steudel 1854: 71) – Cyperus sorostachys Boeck., nom. superl. (Boeckeler 1868: 588). Cyperus zanzibarensis C.B.Clarke (Clarke 1901: 323). Cyperus androhibensis D.A.Simpson (Simpson 1992: 745). Distribution – Tropical Africa, Madagascar, India, Philippines, northern Australia. Description – Simpson (1990: 490). 9. Cyperus schomburgkianus Nees (Nees 1840: 393) – Type: Guyana, Schomburgk 810 (holo-: B, destroyed during the war; iso-: BM, G, K, TCD). Cyperus leucanthus Schrad. ex Nees (Nees 1842: 18) – Cyperus schomburgkianus var. leucanthus (Schrad. ex Nees) Kük. (Kükenthal 1936: 277). Cyperus schomburgkianus var. trilobatus Kük. (Kükenthal 1936: 277). 352 10. Cyperus simpsonii (Muasya) Larridon, comb. nov. – Kyllingiella simpsonii Muasya, Kew Bulletin 57: 997. 2002 (Muasya 2002) – Type: Tanzania, Ole Sayalel 5320 (holo-: EA; iso-: K). Distribution – Democratic Republic of Congo, Tanzania, Zambia. Description – Muasya (2002: 997). 12. Cyperus tenerrimus J.Presl & C.Presl (Presl & Presl 1828: 166) – Type: Mexico, Haenke s.n. (holo-: PR). Cyperus cymbiformis Liebm. (Liebmann 1850: 208). Cyperus wawrai Boeck. (Boeckeler 1874: 363). Distribution – Central America: Mexico, Guatemala, Nicaragua, El Salvador, Costa Rica, Panama; South America: Colombia. Description – Simpson (1990: 497–498). CONCLUSIONS The segregate genera Courtoisina, Oxycaryum and Kyllingiella are here included in Cyperus. Courtoisina and Oxycaryum are combined in Cyperus as sections, whereas Kyllingiella is included in an expanded Cyperus sect. Leucocephali. Cyperus sect. Pseudanosporum is placed in synonymy of C. sect. Anosporum. The inclusion of these segregates in C3 Cyperus (Cyperus subg. Anosporum) is based on the phylogenetic hypothesis presented by Larridon et al. (2011b) (ig. 1), and is here corroborated using morphology, embryology, spikelet and loral ontogeny, and anatomy. Table 3 presents a preliminary classiication of C3 Cyperus. ACKNOWLEDGEMENTS We thank the curators of B, BM, BR, GENT, EA, K, MO, P, TAN, U, UPS and WAG for the loan of material, and the numerous collectors of the specimens studied. Many thanks to Richard Carter (Valdosta State University Herbarium (VSC), Valdosta, GA, U.S.A.) for the pictures of Oxycaryum cubense. We are grateful for the invitation of the East African Herbarium (National Museums of Kenya, Nairobi) and the Kenya Wildlife Service for the permission to access and to collect sedges in the protected areas of Kenya and for their help organising the expedition. The ANGAP Madagascar National Parks authority, the general secretariat of the AETFAT congress 2010 and the staff of the MBG ofice in Antananarivo are acknowledged for their support in securing collecting permits (N°082/10/MEF/SG/DGF/DCB.SAP/SLRSE - Isabel Larridon) for Cyperaceae in Madagascar and their Larridon et al., Taxonomic changes in C3 Cyperus (Cyperaceae) help organising the expedition. This work was supported by research grants of the Special Research Fund (BO5622, BO7418, BOF, Ghent University, Belgium) and the Department of Biology, Ghent University, Belgium. The ield expeditions were inanced trough travel grants of the Research Foundation - Flanders (FWO) and the Leopold III-Fund, and with support of the Department of Biology, Ghent University, Belgium. REFERENCES Archer C. (1998) A new combination in Isolepis. Bothalia 28: 41– 42. Barros M. (1935) Ciperáceas argentinas 2. Géneros Kyllingia Rottb., Scirpus L., y Carex L. 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