Botanical Journal of the Linnean Society, 2011, 167, 19–46. With 2 figures Affinities in C3 Cyperus lineages (Cyperaceae) revealed using molecular phylogenetic data and carbon isotope analysis ISABEL LARRIDON1*, MARC REYNDERS1, WIM HUYGH1, KENNETH BAUTERS1, KOBEKE VAN DE PUTTE2, A. MUTHAMA MUASYA3, PASCAL BOECKX4, DAVID A. SIMPSON5, ALEXANDER VRIJDAGHS6 and PAUL GOETGHEBEUR1 1 Ghent University, Department of Biology, Research Group Spermatophytes, K. L. Ledeganckstraat 35, 9000 Gent, Belgium 2 Ghent University, Department of Biology, Research Group Mycology, K. L. Ledeganckstraat 35, 9000 Gent, Belgium 3 University of Cape Town, Botany Department, Rondebosch 7700, South Africa 4 Ghent University, Faculty of Bioscience Engineering, Laboratory of Applied Physical Chemistry – ISOFYS, Coupure 653, 9000 Gent, Belgium 5 Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3AB, UK 6 K.U.Leuven, Institute of Botany and Microbiology, Laboratory of Plant Systematics, Kasteelpark Arenberg 31, 3001 Leuven, Belgium Received 22 November 2010; revised 9 February 2011; accepted for publication 18 May 2011 Maximum likelihood and Bayesian analyses of nrDNA (ETS1f) and plastid DNA (rpl32-trnL, trnH-psbA) sequence data are presented for ‘C3 Cyperus’ (Cyperaceae). The term ‘C3 Cyperus’ indicates all species of Cyperus s.l. that use C3 photosynthesis linked with eucyperoid vegetative anatomy. Sampling comprises 77 specimens of 61 different taxa, representing nearly all previously recognized subdivisions of C3 Cyperus and the segregate genera Courtoisina, Kyllingiella and Oxycaryum. According to our results, the Cyperus clade is divided in six well-supported clades. The first of these clades (clade 1) forms three subclades largely corresponding to Cyperus sections Haspani, Incurvi and Diffusi. Clade 2 comprises the entirely New World C. section Luzuloidei sensu Denton (1978). Clade 3 is a highly diverse clade including two subclades: clade 3a, C. sections Pseudanosporum and Anosporum plus the segregate genera Courtoisina and Oxycaryum; and clade 3b, C. section Fusci. Clade 4 corresponds to C. section Alternifolii and clade 5 to C. section Leucocephali plus the segregate genus Kyllingiella. The sixth clade is a well-supported monophyletic clade encompassing all C4 Cyperus s.l. species (‘C4 Cyperus’). This study establishes a phylogenetic framework for future studies. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167, 19–46. ADDITIONAL KEYWORDS: Bayesian inference – Cypereae – Cyperoideae – molecular phylogeny – photosynthetic pathway – systematics. INTRODUCTION Cyperaceae have an almost cosmopolitan distribution (Govaerts et al., 2007). Sedges did not only evolve a large diversity of genera and species in the tropics and subtropics, but they are also often dominantly *Corresponding author. E-mail: isabel.larridon@ugent.be present in the vegetation of temperate and arctic regions (Carex L., c. 1800 spp.; Govaerts et al., 2011). Because of their ecological significance in wetlands and the important role of some Cyperaceae in the food chain of dry grasslands, knowledge of the biodiversity and evolution of this plant family is very valuable. Cyperaceae are grass-like plants, often with complex compound inflorescences, in which many adaptations © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167, 19–46 19 20 I. LARRIDON ET AL. such as reductions and contractions have occurred, complicating evolutionary reconstruction and classification. This has not only led to uncertain interpretations of the inflorescence and flowers (homology problems, e.g. Bruhl, 1991; Muasya et al., 2009b; Vrijdaghs et al., 2009, 2010), but also to conflicting classification systems (e.g. Clarke, 1908; Kükenthal, 1935–1936; Kern, 1974; Haines & Lye, 1983). Prior to the era of molecular phylogenetics, classifications based on cladistic analyses of matrices of morphological and anatomical character states were presented (Goetghebeur, 1986; Bruhl, 1995). Since then, molecular phylogenetic research has resulted in new insights into Cyperaceae. Molecular phylogenetic studies at family level have been largely based on plastid DNA: sequence data for rbcL (e.g. Muasya et al., 1998; Simpson et al., 2007) or rbcL and trnL-F (the trnL intron and the trnL–trnF intergenic spacer) (Muasya, Simpson & Chase, 2002; Muasya et al., 2009a). The latest molecular phylogenetic studies of Cyperaceae (Simpson et al., 2003, 2007; Muasya et al., 2009a) recognized only two subfamilies, Cyperoideae and Mapanioideae, which are easily distinguished by the structure of their reproductive units. Tribe Cypereae Nees (Cyperoideae) is defined as including all taxa sharing the Cyperus-type embryo and the similar Ficinia-type embryo (Van der Veken, 1965; Goetghebeur, 1998; Muasya et al., 2009a, b). Based on molecular data (Muasya, Simpson & Chase, 2002; Muasya et al., 2009a), two clades are recognized in Cypereae. The Ficinia Schrad. clade is predominantly characterized by spikelets with spirally arranged glumes and includes Scirpoides Ség., Dracoscirpoides Muasya (Muasya et al., 2009a, 2011), Hellmuthia Steud., Isolepis R.Br. and Ficinia (Muasya & de Lange, 2010). The Cyperus L. clade usually has spikelets with distichously arranged glumes. The generic limits in the Cyperus clade (c. 950 spp.) are notoriously controversial (Muasya et al., 2009b). When using the classification of Goetghebeur (1998), the Cyperus clade comprises a paraphyletic Cyperus s.s. as the core genus, in which 13 segregate genera are nested. These segregate genera (Alinula J.Raynal, Androtrichum (Brongn.) Brongn., Ascolepis Nees ex Steud., Courtoisina Soják, Kyllinga Rottb., Kyllingiella R.W.Haines & Lye, Lipocarpha R.Br., Oxycaryum Nees, Pycreus P.Beauv., Queenslandiella Domin, Remirea Aubl., Sphaerocyperus Lye and Volkiella Merxm. & Czech) were created because their species diverged significantly from typical Cyperus with respect to vegetative, floral and anatomical characters. Each of these segregates is circumscribed by a combination of morphological characters, including inflorescence and spikelet morphology, unit of dispersal, nutlet orientation and photosynthetic pathway (Bruhl & Wilson, 2007; Muasya et al., 2009b; Vrijdaghs et al., 2011). The presence of Kranz anatomy, linked with C4 photosynthesis, is an important character in classifying taxa within the Cyperus clade. This character has been used in the classification of Cyperus since Rikli (1895), long before the discovery of the C4 photosynthetic pathway. As already indicated by Raynal (1973) and Goetghebeur (1989), later authors such as Soros & Bruhl (2000), Muasya et al. (2002), Bruhl & Wilson (2007) and Besnard et al. (2009) confirmed that the C4 photosynthetic pathway arose only once in Cypereae, although it arose at least four separate times in Cyperaceae. In the Cyperus clade, C3 photosynthesis is characterized by the presence of the eucyperoid anatomy type (plesiomorphic), whereas C4 photosynthesis is linked with the chlorocyperoid anatomy type. Bruhl, Stone & Hattersley (1987) and Bruhl & Perry (1995) clarified the chlorocyperoid anatomy. According to Besnard et al. (2009), the first appearance of C4 photosynthesis in Cyperaceae probably occurred between 19.6 ± 4.9 and 10.1 ± 3.6 Ma in Bulbostylis DC., with the other C4 appearances occurring during the last 12 Ma, making C4 sedges generally younger than C4 grasses (Christin et al., 2008a, b). Although sedges generally occupy wetter habitats than grasses and commonly occur in wetlands and marshes, many C4 and a considerable number of C3 sedges occupy seasonally dry habitats. The C4 pathway, which raises the water-use efficiency compared with the C3 photosynthesis type (Sage, 2004), might have contributed to the colonization of drier habitats (Besnard et al., 2009). However, water limitation might not have been the most important factor for the success of C4 sedges. See Pyankov et al. (2010) for a discussion of C4 photosynthesis in European representatives of Cyperaceae and other families. Li, Wedin & Tieszen (1999) and Stock, Chuba & Verboom (2004) argued that fire resistance, optimized nitrogen uptake and resistance to chemical stress (salt and heavy metals) and higher levels of irradiance were probably more important. Figure 1 represents some of the morphological and habitat diversity of C3 Cyperus lineages. As discussed in more detail in Larridon et al. (2011), Cyperus is most commonly divided into two units, determined by the character states of an anatomical and of an inflorescence character set. As mentioned above, the vegetative anatomy in Cyperus is either eucyperoid or chlorocyperoid. Based on this character, Rikli (1895) divided Cyperus into two separate genera: Eucyperus and Chlorocyperus. The prefix Eu-, which suggests the inclusion of the type species of Cyperus in this group, should have led Britton (1907) to use one of Linnaeus’ (1753) original C3 Cyperus spp. as the type of Cyperus. However, he © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167, 19–46 AFFINITIES IN C3 CYPERUS LINEAGES REVEALED chose Cyperus esculentus L. instead (Huygh et al., 2010), a species with chlorocyperoid anatomy. The inflorescence in Cyperus is either composed of digitately clustered spikelets or is an anthela composed of spikes of spikelets. Clarke (1893) divided Cyperus into two subgenera based on these two inflorescence types: Pycnostachys and Choristachys. As demonstrated by Goetghebeur (1989), the two systems (based on anatomy type or inflorescence type) are not completely congruent. In the present study, molecular phylogenetic data and results of carbon isotope analysis of the Cyperus clade were analysed with the specific objectives of: (1) elucidating phylogenetic relationships focusing on the C3 Cyperus lineages in order to establish a phylogenetic framework for future studies of the Cyperus clade; (2) testing whether infrageneric taxa in Cyperus (e.g. Kükenthal, 1935–1936; Denton, 1978) are monophyletic; and (3) characterizing which photosynthetic system is used in previously unstudied taxa. For the accepted names of the taxa treated in this paper, we refer to the World Checklist of Cyperaceae (Govaerts et al., 2007, 2011). The classification used is that of Goetghebeur (1998). A paper documenting the necessary nomenclatural/taxonomical changes based on the results presented in this paper and further supported by morphological, embryological, ontogenetic and anatomical data will be published elsewhere (Larridon et al., in press). The final objective of the research on Cyperus carried out by the authors is to recircumscribe the genus so that it is monophyletic and to create a new infrageneric classification of the genus supported by both molecular and morphological data. MATERIAL AND METHODS SAMPLING Seventy-seven samples of 60 different taxa were used for this study. The samples with species names, voucher information, origin and GenBank accession numbers for the sequences are given in Table 1. Three sequences were used from a previous unpublished study (GenBank accession numbers GU135417, GU135444, GU135397; J. R. Abbott, K. M. Neubig, W. M. Whitten & N. H. Williams, unpubl. data). The other sequences were all newly generated for this study. Taxa within Cyperus were selected to represent a broad morphological and geographical range and to include a wide range of the traditionally recognized sections, subgenera and segregate genera (C3 Cyperus: Courtoisina, Kyllingiella and Oxycaryum; C4 Cyperus: Alinula, Ascolepis, Lipocarpha, Kyllinga, Pycreus, Queenslandiella, Remirea and Sphaerocyperus). As this study assesses relationships above the 21 rank of species, multiple species samples and infraspecific taxa were generally not used. The outgroup taxa were selected based on the family-wide analysis of plastid rbcL and trnL-F sequences by Muasya et al. (2009a). Taxonomic details for most taxa mentioned (such as author, place and date of publication, synonyms, distribution) follow Govaerts et al. (2007, 2011). More detailed information on the nomenclature of generic and subdivisional names of the Cyperus clade is given in Huygh et al. (2010), Larridon et al. (2011) and Reynders et al. (2011). DNA EXTRACTION Samples were either of wild origin, mostly collected during recent field expeditions (silica-dried), or sampled from plants cultivated at the Ghent University Botanical Garden. Additional dried leaf samples were selected from herbarium specimens (GENT). Total DNA was extracted from 100 mg fresh or 20 mg dried material using the GenElute™ Plant Genomic DNA Miniprep Kit (Sigma-Aldrich, USA) or the DNeasy Plant Mini Kit (Qiagen, Germantown, USA) following the manufacturers’ protocols. The material was first ground using a mortar and pestle with the addition of the extraction buffers and a knifepoint of sterilized sand. Additional DNA samples were provided by the DNA Bank at the Royal Botanic Gardens, Kew (UK). MARKERS Two non-coding plastid DNA markers were used in this study: the rpl32-trnL intergenic spacer of the small single-copy region of the plastid genome (Shaw et al., 2007) and the trnH-psbA intergenic spacer (e.g. Kress et al., 2005; Shaw et al., 2005, 2007; Dragon & Barrington, 2009). To compare the information held in the plastid and nuclear genomes, sequences of a nuclear region were also produced. Also, molecular phylogenetic studies solely based on plastid markers (e.g. Muasya et al., 2002, 2009a; Simpson et al., 2007) give insufficient resolution at lower taxonomic levels, indicating that a marker with a more rapid rate of evolution was needed to resolve the relationships in the Cyperus clade. Although Álvarez & Wendel (2003) rightly indicated the challenges of using non-singlecopy or low-copy nuclear markers, we chose to use a fragment of the external transcribed spacer 1 (ETS1f). We selected the ETS1f marker, not only because it displays a rapid rate of evolution compared with most plastid loci, but also because we found it can be readily amplified and sequenced even from poorly preserved plant material. The relative poor quality of DNA extracted from herbarium specimens © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167, 19–46 22 I. LARRIDON ET AL. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167, 19–46 AFFINITIES IN C3 CYPERUS LINEAGES REVEALED 23 Figure 1. Diversity of C3 Cyperus morphologies and habitats. A. Cyperus fuscus with anthelate inflorescence with clearly distichously glume arrangement growing in the Ghent University Botanical Garden. B. Cyperus pulchellus with whitish capitate inflorescence growing seasonally wet area near Mahajanga, Madagascar. C. Cyperus debilissimus with extremely elongated culms growing up and hanging down from the vegetation at edge of the forest in Andringitra National Park, Madagascar. D. Cyperus prolifer with rays arranged so inflorescence is spherical growing with a creeping rhizome in wet sand at Cirque Rouge near Mahajanga, Madagascar. E. Cyperus alternifolius with many, almost equally sized involucral bracts growing on a shaded riverbank near Mahajanga, Madagascar. F. Cyperus betafensis with anthelate inflorescence and well-developed involucral bracts growing at the edge of the forest in Andringitra National Park, Madagascar. Photographs (A) by M. Reynders and (B-F) by W. Huygh. 䉳 prevents effective use of single-copy nuclear genes. ETS1f, like the internal transcribed spacer (ITS) part of the nuclear ribosomal DNA (nrDNA), has already proved useful in resolving phylogenetic relationships in Cyperaceae, e.g. Uncinia Pers. (Starr, Harris & Simpson, 2003), Schoenoplectus (Rchb.) Palla (Yano & Hoshino, 2005), Fimbristylis Vahl (Yano & Hoshino, 2007) and Carex (Dragon & Barrington, 2009). PCR AMPLIFICATION, SEQUENCING AND ALIGNMENTS PCR amplification was performed using buffer solutions and Taq polymerase from Qiagen. Reactions were carried out using a Gradient Mastercycler (Eppendorf Inc., Hamburg, Germany). Amplification of rpl32-trnL was performed following the protocol and using the primers of Shaw et al. (2007) for rpl32-F: (5′-CAGTTCCAAAAAAACGTACTTC-3′); and trnL(UAG): (5′-CTGCTTCCTAAGAGCAGCGT-3′). Amplification of trnH-psbA was performed following the protocol of Shaw et al. (2005) and using adapted primers of Wanke (S. Wanke, unpubl. data) Pe-trnH: (5′-ATTCACAATCCACTGCCTTGAT-3′); and Pe-psbA: (5′-AATGCACACAACTTCCCTCTA-3′). Amplification of ETS1f was performed following the protocol and using the primers of Starr et al. (2003) for ETS1f: ETS-F (5′-CTGTGGCGTCGCATGAGTTG-3′) and 18S-R (5′-AGACAAGCATATGACTACTGGCAGG-3′). The PCR products were electrophoresed on 1% agarose gels in 1 ¥ Tris-acetate-EDTA (TAE) buffer (pH 8.0) and stained with ethidium bromide to confirm a single product. Sequencing was performed using the same primers used in the PCR reactions. Sequencing was run on an Applied Biosystems ABI 3130XL Genetic Analyser (Life Technologies, CA, USA). The software SequencherTM v4.8 (GeneCodes Corporation, Ann Arbor, MI, USA) was used to assemble forward and reverse sequences into contigs, inspect ABI chromatograms and edit nucleotides where needed. The sequences were aligned manually in PhyDE 0.995 (Müller, Müller & Quandt, 2008). To eliminate ambiguously aligned positions in the alignment as objectively as possible, the online program Gblocks v0.91b (Castresana, 2000) was used. The program was run with settings allowing for smaller blocks, gaps within these blocks and less strict flanking positions. Alignments are available from the first author by request. DATA ANALYSIS Phylogenetic hypotheses were produced using maximum likelihood (ML) and Bayesian inferences (BI). All analyses were first performed on the single marker data sets (ETS1f, rpl32-trnL, trnH-psbA). As no conflicting clades with a significant confidence value (i.e. with bootstrap / PP support) were revealed, a combined data set was constructed and analysed. The latter was subdivided into three partitions, corresponding to the single markers. The program RAxML v7.0.3 (Stamatakis, 2006) was used to execute the Rapid Bootstrapping algorithm for 500 replicates, combined with an ML search, using the GTRCAT model (Stamatakis, Hoover & Rougemont, 2008). Model parameters were optimized for each partition when analysing the combined data set. Following Van de Putte et al. (2010), two Bayesian phylogenetic (BI) analyses were carried out in MrBayes v3.1.2 (Ronquist & Huelsenbeck, 2003). For the first analysis, MrModeltest v2.3 (Nylander, 2004) was used to determine the model that best fits the data, applying Akaike’s information criterion. For the combined data set, a model was determined for each partition. This method is referred to as the BI-MrModeltest method. For the second analysis, a single general time-reversible model with rate variation across sites and a proportion of invariable sites was used. Rates and all model parameters were unlinked between all partitions of the combined data set. This method is referred to as the BI-GTR+I+G method. Two independent, parallel runs of one cold and three heated chains were run for 10 million generations each. Trees and parameter estimates were saved every 1000th generation. Convergence, associated likelihood values, effective sample size values and burn-in values of the different runs were verified with Tracer v1.4.1 (Rambaut & Drummond, 2007). Calculation of the consensus tree and the posterior © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167, 19–46 24 I. LARRIDON ET AL. Table 1. List of the samples used in the molecular study with species names, voucher information (* leaf sample courtesy of the collector/A. M. Muasya), origin and GenBank accession numbers for the sequences Taxon Voucher Origin ETS1f trnH-psbA rpl32-trnL Alinula paradoxa (Cherm.) Goetgh. & Vorster Ascolepis capensis (Kunth) Ridl. Courtoisina assimilis (Steud.) Maquet Courtoisina cyperoides (Roxb.) Soják Courtoisina cyperoides (Roxb.) Soják Cyperus ajax C.B.Clarke Cyperus albostriatus Schrad. Cyperus alternifolius L. Reid 1027 (GENT) South Africa HQ705964 – HQ705894 Hess 52/1760 (GENT) Angola HQ705957 – HQ705887 Faden et al. 96/119 (K*; Muasya et al., 2002) Faden et al. 96/456 (K*) Tanzania HQ705939 HQ705812 HQ705872 Tanzania HQ705940 HQ705813 – Madagascar HQ705941 HQ705814 HQ705873 DR Congo HQ705916 HQ705794 HQ705852 South Africa HQ705915 HQ705793 HQ705851 BG Ghent HQ705948 HQ705818 HQ705878 Madagascar HQ705917 HQ705795 HQ705853 Larridon et al. 2010–0326 (GENT) Larridon et al. 2010-0200 (GENT) Viane 1327 (GENT) Madagascar HQ705918 – – Madagascar HQ705919 HQ705796 HQ705854 Ivory Coast HQ705921 HQ705798 – Reynders et al. 090307/03 (GENT) Beentje 4774 (K – DNA Bank 29378) Cameroon, BG Ghent HQ705922 HQ705799 HQ705856 Madagascar HQ705930 – – Jongkind & Nieuwhuis 2847 (GENT) Larridon et al. 2010–0103 (GENT) Larridon et al. 2010–0154 (GENT) Larridon et al. 2010–0282 (GENT) Muasya & Knox 1021 (EA) Muasya & Knox 976 (EA; Muasya et al., 2002) Reynders & Sabulao 66 (GENT) Carter 9237 (GENT) Ghana HQ705954 HQ705823 HQ705884 Madagascar HQ705933 HQ705808 HQ705866 Madagascar HQ705932 HQ705807 HQ705865 Madagascar – HQ705806 HQ705864 Kenya HQ705926 HQ705802 HQ705859 Kenya HQ705944 – HQ705877 Philippines HQ705945 HQ705817 – USA HQ705907 HQ705788 HQ705844 Goetghebeur 5601 (GENT) Zardini 29789 (GENT) Cuba Paraguay HQ705959 HQ705908 HQ705827 HQ705789 HQ705889 HQ705845 Bryson 16965 (GENT) USA HQ705903 – HQ705840 Van der Veken 12823 (GENT) France HQ705904 HQ705785 HQ705841 Cyperus balfourii C.B.Clarke Cyperus betafensis Cherm. Cyperus betafensis Cherm. Cyperus buchholzii Boeck. Cyperus buchholzii Boeck. Cyperus chamaecephalus Cherm. Cyperus cuspidatus Kunth Cyperus debilissimus Baker Cyperus debilissimus Baker Cyperus debilissimus Baker Cyperus denudatus L.f. Cyperus dichrostachyus Hochst. ex A.Rich. Cyperus difformis L. Cyperus distinctus Steud. Cyperus elegans L. Cyperus entrerianus Boeck. Cyperus eragrostis Lam. Cyperus eragrostis Lam. Larridon et al. 2010-0261 (GENT) Malaisse & Goetghebeur 130 (GENT) Reid 726 (GENT) Goetghebeur 11516 (GENT) Dorr 2744 (GENT) © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167, 19–46 AFFINITIES IN C3 CYPERUS LINEAGES REVEALED 25 Table 1. Continued Taxon Voucher Origin ETS1f trnH-psbA rpl32-trnL Cyperus eragrostis Lam. Cyperus esculentus L. Goetghebeur 11494 (GENT) Goetghebeur 11303 (GENT) Larridon et al. 2010-0034 (GENT) Montes 1799 (K – DNA Bank 29366) Goetghebeur 5869 (GENT) BG Basel, BG Ghent HQ705905 HQ705786 HQ705842 BG Nantes, BG Ghent HQ705960 HQ705828 HQ705890 Madagascar HQ705925 HQ705801 HQ705858 Argentina – HQ705791 HQ705849 BG Ghent HQ705914 HQ705792 HQ705850 Øllgaard 74763 (GENT) Ecuador HQ705913 – – de Retz 67715 (GENT) Schessl 3316 (GENT) Muasya & Muthama 1269 (EA) Muasya 984 (EA, K; Muasya et al., 2002) Reid 902 (GENT) France Brazil Kenya HQ705946 HQ705943 HQ705927 – – HQ705803 – HQ705876 HQ705860 Kenya HQ705961 HQ705829 HQ705891 South Africa HQ705923 – – Goetghebeur 5868 (GENT) BG Ghent HQ705909 – – Van den Eynden 213 (GENT) Larridon et al. 2009-0076 (GENT) Larridon et al. 2010-0225 (GENT) Viane 681 (GENT) Ecuador HQ705910 – HQ705846 Kenya HQ705949 HQ705819 HQ705879 Madagascar HQ705931 – HQ705863 Venezuela HQ705911 – HQ705847 Goetghebeur 5866 (GENT) De Wolf 92-86 (GENT) BG Ghent Senegal HQ705962 HQ705935 HQ705830 – HQ705892 HQ705868 Larridon et al. 2010–0265 (GENT) Phillipson 1647 (GENT) Madagascar HQ705936 HQ705810 HQ705869 Madagascar HQ705950 – HQ705880 Larridon et al. 2010-0069 (GENT) Madagascar HQ705934 HQ705809 HQ705867 Chantaranothai et al. 814 (K – DNA Bank 29382) Larridon et al. 2010-0003 (GENT) Muasya & Knox 964 (EA) Thailand HQ705937 – HQ705870 Madagascar HQ705928 HQ705804 HQ705861 Tanzania HQ705929 HQ705805 HQ705862 Carter 6152 (GENT) USA HQ705912 HQ705790 HQ705848 Malaisse & Goetghebeur 1171 (GENT) Mwachala et al. 446 (EA) DR Congo HQ705938 HQ705811 HQ705871 Kenya HQ705924 HQ705800 HQ705857 Shaw 890 (K*) Hong Kong (China) HQ705963 HQ705831 HQ705893 Cyperus foliaceus C.B.Clarke Cyperus friburgensis Boeck. Cyperus friburgensis Boeck. Cyperus friburgensis Boeck. Cyperus fuscus L. Cyperus gardneri Nees Cyperus haspan L. Cyperus kerstenii Boeck. Cyperus leptocladus Kunth Cyperus luzulae (L.) Retz. Cyperus luzulae (L.) Retz. Cyperus marginatus Thunb. Cyperus molliglumis Cherm. Cyperus ochraceus Vahl Cyperus papyrus L. Cyperus pectinatus Vahl Cyperus pectinatus Vahl Cyperus phaeolepis Cherm. Cyperus plantaginifolius var. minor Cherm. Cyperus platystylis R.Br. Cyperus prolifer Lam. Cyperus purpureoviridis Lye Cyperus pseudovegetus Steud. Cyperus reduncus Hochst. ex Boeck. Cyperus renschii Boeck. Cyperus rotundus L. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167, 19–46 26 I. LARRIDON ET AL. Table 1. Continued Taxon Voucher Origin ETS1f trnH-psbA rpl32-trnL Cyperus schomburgkianus Nees Cyperus sp. (Diffusi) de Michel 2000 (GENT) Bolivia – HQ705821 HQ705882 Larridon et al. 2010-0215 (GENT) Laegaard 17222 (GENT) Madagascar HQ705920 HQ705797 HQ705855 Senegal HQ705947 – – Guyana HQ705906 HQ705787 HQ705843 BG Ghent HQ705951 HQ705820 HQ705881 Madagascar HQ705955 HQ705824 HQ705885 Madagascar HQ705956 HQ705825 HQ705886 South Africa Kenya HQ705902 HQ705901 HQ705784 HQ705783 HQ705839 HQ705838 Philippines, BG Ghent HQ705965 HQ705832 HQ705895 Muasya & Muthama 1262 (EA) Kenya HQ705952 – – Muasya & Muthama 1247 (EA) Reynders & Sabulao 26 (GENT) Mwachala 340 (EA) Kenya HQ705953 HQ705822 HQ705883 Philippines HQ705958 HQ705826 HQ705888 Kenya HQ705942 HQ705815 HQ705874 Zardini 18398 (GENT) Paraguay – HQ705816 HQ705875 Goetghebeur 11519 (GENT) Muasya 2490 (EA) South Africa, BG Ghent Kenya HQ705966 HQ705833 HQ705896 HQ705967 HQ705834 HQ705897 Faden et al. 96/48 (K*; Muasya et al., 2002) Goetghebeur 11520 (GENT) Tanzania HQ705968 HQ705835 HQ705898 BG Porto, BG Ghent HQ705900 HQ705782 HQ705837 Tanzania HQ705969 HQ705836 HQ705899 Cyperus submicrolepis Kük. Cyperus surinamensis Rottb. Cyperus textilis Thunb. Cyperus waterloti Cherm. Cyperus waterloti Cherm. Ficinia gracilis Schrad. Isolepis fluitans (L.) R.Br. Kyllinga nemoralis (J.R.Forst. & G.Forst.) Dandy ex Hutch. & Dalziel Kyllingiella microcephala (Steud.) R.W.Haines & Lye Kyllingiella polyphylla (A.Rich.) Lye Lipocarpha chinensis (Osbeck) J.Kern Oxycaryum cubense (Poepp. & Kunth) Palla Oxycaryum cubense (Poepp. & Kunth) Palla Pycreus polystachyos (Rottb.) P.Beauv. Queenslandiella hyalina (Vahl) Ballard Remirea maritima Aubl. Scirpoides holoschoenus (L.) Soják Sphaerocyperus erinaceus (Ridl.) Lye Jansen-Jacobs 521 (GENT) Goetghebeur 11517 (GENT) Larridon et al. 2010-0010 (GENT) Larridon et al. 2010-0043 (GENT) Muasya 2713 (BOL) Muasya & Knox 3195 (EA) Goetghebeur 11518 (GENT) Faden et al. 96/358 (K*; Muasya et al., 2002) probability (PP) of clades was based upon the trees sampled after the chains converged. Trees were drawn using FigTree v1.3.1 and Adobe Photoshop CS3. CARBON ISOTOPE ANALYSIS Carbon isotope analysis (d13C) was performed on 65 species to confirm their photosynthesis type. The © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167, 19–46 AFFINITIES IN C3 CYPERUS LINEAGES REVEALED measurements of 13C natural abundance in plant samples were performed using an elemental analyser (ANCA-SL, SerCon, UK) coupled to an isotope ratio mass spectrometer (20–20, SerCon, UK). The measured 13C/12C ratios are expressed as d13C values (‰) relative to the Vienna Pee Dee Belemnite (VPDB) standard: δ 13C = (R sample − R standard R standard ) × 1000 Rsample and Rstandard refer to the 13C/12C ratio in the sample and the standard, respectively. The working standard for the measurements was wheat flour with a d13C value of –27.01 ± 0.04‰ (certified by IsoAnalytical, UK). All analyses were performed in duplicate. RESULTS SEQUENCE ALIGNMENTS After alignment and application of Gblocks, the ETS1f alignment included 70 sequences of 400 bases, the rpl32-trnL alignment 63 sequences of 819 bases and the trnH-psbA alignment 58 sequences of 787 bases. The concatenated data set included 77 sequences and the Gblocks program retained 61% or 2006 characters of the original alignment. Most excluded regions came from the ETS1f region. PHYLOGENETIC ANALYSIS The three single-locus ML analyses revealed nearly identical topologies and bootstrap values. As expected, the clades supported by single-locus analyses received even greater support in the multi-locus ML analysis. In the various analyses, only minor conflicts concerning the position of the species in the C4 Cyperus clade were detected, but most nodes in this clade are not supported. Also, in the ETS1f single locus ML tree, Cyperus section Luzuloidei branches off as a separate clade, whereas in all other analyses, section Luzuloidei (clade 2; Fig. 2) forms a clade sister to a clade with C. sections Pseudanosporum and Anosporum, the segregate genera Courtoisina and Oxycaryum and C. section Fusci (clade 3a and clade 3b; Fig. 2). The three single-locus BI analyses did not differ significantly in tree topologies. The BI-MrModeltest and BI-GTR+I+G methods also revealed the same topologies, but branches received slightly higher support in the BI-GTR+I+G analysis. The multiplelocus BI topologies did not differ from the multiplelocus ML tree, except in C4 Cyperus, as mentioned above for the ML analyses. Evaluation of the multiple-locus BI analyses output shows that the 27 two runs of the BI-MrModeltest analysis converged on similar log likelihood (-17080) and parameter values. The burn-in value for both runs was determined at 1.5 million generations. The two runs of the BI-GTR+I+G analysis also converged on similar log likelihood (-16947 to -16948) and parameter values. The burn-in value for the BI-GTR+I+G method was determined at 2.5 million generations. The effective sample size (ESS) for the likelihood value of the combined runs was slightly higher for the BI-GTR+I+G analysis. The latter consisted of 1455.91 uncorrelated samples, whereas the BI-MrModeltest analysis comprised 1282.38 uncorrelated samples. Figure 2 shows the 50% majority consensus multiple-locus BI-GTR+I+G tree with the associated PP values and the bootstrap values of the multiplelocus ML tree. Only bootstrap values above 75% and posterior probabilities above 0.85 are shown. CARBON ISOTOPE ANALYSIS Table 2 lists all confirmed C3 Cyperus spp. and some of the C4 Cyperus spp. used in the molecular study. The data listed in this table were either obtained through carbon isotope analysis (d13C) performed on 65 species at Ghent University or taken from the literature (Bruhl & Wilson, 2007). For 15 species, the photosynthesis type was confirmed for the first time overall, and for an additional eight species [indicated in Table 2 by an asterisk (*)] this was confirmed for the first time using carbon isotope analysis. Out of a total of 98 species of the c. 187 C3 Cyperus species, the photosynthesis type is now confirmed (52%). Two sections as circumscribed by Kükenthal (1936) prove heterogeneous in this respect: ‘Cyperus section Glutinosi’ and ‘C. section Dichostylis’. DISCUSSION GENERIC CIRCUMSCRIPTION AND OUTGROUP RELATIONSHIPS The monograph of Cyperus by Kükenthal (1935– 1936) offers an abundant source of hypotheses concerning groups of related species (Table 3). Nevertheless, not all taxa of the genus were explicitly placed within specific sections, and the generic circumscription of Cyperus and its allies has been adapted since Kükenthal’s (1935–1936) revision. Also, it provides few clues as to how the taxa relate to each other or to possible outgroups. Previous molecular phylogenetic studies (Muasya et al., 1998, 2000, 2002, 2009a; Simpson et al., 2007), defined the closest related group within Cyperaceae as the Ficinia clade and place several segregate genera in the Cyperus clade. However, these studies do not © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167, 19–46 28 I. LARRIDON ET AL. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167, 19–46 AFFINITIES IN C3 CYPERUS LINEAGES REVEALED © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167, 19–46 29 Figure 2. 50% majority consensus multiple-locus BI-GTR+I+G tree with the associated PP values and the bootstrap values of the multiple-locus ML tree. Only bootstrap values above 75% and posterior probabilities above 85% are shown. 30 I. LARRIDON ET AL. Table 2. List of all confirmed C3 Cyperus species and some of the C4 Cyperus species used in the molecular study. The data listed in this table were either obtained through carbon isotope analysis (d13C) performed on 65 species at the Ghent University or taken from literature (Bruhl & Wilson, 2007). Of fifteen species, the photosynthesis type was confirmed for the first time overall, and, of an additional eight species (*), this was performed for the first time using carbon isotope analysis. The species included in Cyperus section Glutinosi by Kükenthal (1936) are in bold. Species C3/C4 References (d13C: value, voucher) Ficinia gracilis Schrad. Isolepis fluitans (L.) R.Br. Scirpoides holoschoenus (L.) Soják Androtrichum trigynum (Spreng.) H.Pfeiff. Courtoisina assimilis (Steud.) Maquet C3 C3 C3 C3 IL IL IL IL C3 (d13C: (d13C: (d13C: (d13C: -27.80, -30.44, -26.40, -27.44, Muasya 2365 GENT); Bruhl & Wilson, (2007) Leten s.n. GENT); Bruhl & Wilson, (2007) Goetghebeur 5246 GENT); Bruhl & Wilson, (2007) Goetghebeur 4764 GENT); Bruhl & Wilson, (2007) Courtoisina cyperoides (Roxb.) Soják Kyllingiella microcephala (Steud.) R.W.Haines & Lye Kyllingiella polyphylla (A.Rich.) Lye Kyllingiella simpsonii Muasya Oxycaryum cubense (Poepp. & Kunth) Palla Cyperus acuminatus Torr. & Hook. Cyperus ajax C.B.Clarke Cyperus albostriatus Schrad. Cyperus alternifolius L. Cyperus aquatilis R.Br. Cyperus balfourii C.B.Clarke Cyperus baronii C.B.Clarke Cyperus buchholzii Boeck. Cyperus burkartii Guaglianone Cyperus canus J.Presl & C.Presl Cyperus cephalotes Vahl Cyperus chalaranthus J.Presl & C.Presl Cyperus chamaecephalus Cherm. Cyperus colymbetes Kotschy & Peyr Cyperus concinnus R.Br. Cyperus constanzae Urb. Cyperus debilissimus Baker Cyperus deciduus Boeck. Cyperus dentatus Torr. Cyperus denudatus L.f. Cyperus dereilema Steud. Cyperus dichrostachyus Hochst. ex A.Rich. Cyperus difformis L. Cyperus diffusus Vahl Cyperus disjunctus C.B.Clarke Cyperus distinctus Steud. Cyperus drummondii Torr. & Hook. Cyperus entrerianus Boeck. Cyperus eragrostis Lam. Cyperus fertilis Boeck. C3 C3 IL (d13C: -28.14, Van der Veken 9037 GENT); Bruhl & Wilson, (2007) IL (d13C: -27.85, Coppejans 693 GENT); Bruhl & Wilson, (2007) Bruhl & Wilson, (2007) C3 C3 C3 IL (d13C: -31.38, Kornas 755 GENT); Bruhl & Wilson, (2007) IL (d13C: -25.40, Ole Sayalel 5320 EA) IL (d13C: -30.06, Zardini 18347 GENT); Bruhl & Wilson, (2007) C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 Bruhl & IL (d13C: IL (d13C: IL (d13C: Bruhl & IL (d13C: Bruhl & Bruhl & Bruhl & IL (d13C: IL (d13C: Bruhl & C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 IL (d13C: -35.34, Schatz 2789 GENT, d13C: -34.35, Beentje 4774 K) IL (d13C: -26.30, Denny 1283 GENT) Bruhl & Wilson, (2007) IL (d13C: -29.19, Ekman 6879 K) IL (d13C: -29.54, Messmer 895 K) Bruhl & Wilson, (2007) Bruhl & Wilson, (2007) IL (d13C: -27.26, Hess 51/53 GENT); Bruhl & Wilson, (2007) Bruhl & Wilson, (2007) IL (d13C: -26.80, Viane 2614 GENT); Bruhl & Wilson, (2007) C3 C3 C3 C3 C3 C3 C3 C3 Cyperus fischerianus A.Rich. Cyperus flaccidus R.Br. Cyperus foliaceus C.B.Clarke C3 C3 C3 IL (d13C: -29.44, Hess 52/158 GENT); Bruhl & Wilson, (2007) IL (d13C: -32.04, Viane 1327 GENT) Bruhl & Wilson, (2007) IL * (d13C: -28.82, Carter 9237 GENT); Bruhl & Wilson, (2007) Bruhl & Wilson, (2007) IL (d13C: -27.87, Zardini 29789 GENT); Bruhl & Wilson, (2007) IL (d13C: -29.59, Bryson 16965 GENT); Bruhl & Wilson, (2007) IL (d13C: -37.13, Van der Veken 8940 GENT); Bruhl & Wilson, (2007) Bruhl & Wilson, (2007) Bruhl & Wilson, (2007) Bruhl & Wilson, (2007) Wilson, -28.05, -30.20, -28.75, Wilson, -32.70, Wilson, Wilson, Wilson, -28.93, -29.56, Wilson, (2007) Hess 50/176 GENT); Bruhl & Wilson, (2007) Reid 726 GENT); Bruhl & Wilson, (2007) Harley 22939 GENT); Bruhl & Wilson, (2007) (2007) Dorr 2744 GENT) (2007) (2007) (2007) Lopez 29 GENT) Heckman 116 K); Bruhl & Wilson, (2007) (2007) © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167, 19–46 AFFINITIES IN C3 CYPERUS LINEAGES REVEALED 31 Table 2. Continued Species C3/C4 References (d13C: value, voucher) Cyperus friburgensis Boeck. Cyperus fuscus L. Cyperus gardneri Nees C3 C3 C3 Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus glaucophyllus Boeck. gracilis R.Br. gymnocaulos Steud. haspan L. hieronymi Boeck. humilis Kunth incomtus Kunth intricatus Schrad. ex Schult. kipasensis Cherm. laevis R.Br. laxus Lam. lecontei Torr. ex Steud. leptocladus Kunth leucocephalus Retz C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 Cyperus limosus Maxim. Cyperus luzulae (L.) Retz Cyperus mapanioides C.B.Clarke Cyperus marginatus Thunb. Cyperus megalanthus (Kük.) G.C.Tucker Cyperus michoacanensis Britton Cyperus microglumis D.A.Simpson Cyperus miliifolius Poepp. & Kunth Cyperus mirus C.B.Clarke Cyperus nayaritensis Tucker Cyperus ochraceus Vahl Cyperus palianparaiensis Govindarajalu Cyperus pectinatus Vahl Cyperus perennis (M.E.Jones) O’Neill Cyperus phaeolepis Cherm. Cyperus platycaulis Baker Cyperus platystylis R.Br. C3 C3 C3 C3 C3 IL (d13C: -34.30, Øllgaard 74763 GENT) IL (d13C: -28.52, De Retz 67715 GENT); Bruhl & Wilson, (2007) IL * (d13C: -31.33, Schessl 3316 GENT); Bruhl & Wilson, (2007) Bruhl & Wilson, (2007) IL (d13C: -30.33, Bruhl 5 GENT); Bruhl & Wilson, (2007) Bruhl & Wilson, (2007) IL (d13C: -25.66, Viane 908 GENT); Bruhl & Wilson, (2007) Bruhl & Wilson, (2007) Bruhl & Wilson, (2007) Bruhl & Wilson, (2007) Bruhl & Wilson, (2007) Bruhl & Wilson, (2007) Bruhl & Wilson, (2007) Bruhl & Wilson, (2007) Bruhl & Wilson, (2007) IL (d13C: -27.84, Reid 902 GENT); Bruhl & Wilson, (2007) IL * (d13C: -30.38, Chantaranothai 1630 K); Bruhl & Wilson, (2007) Bruhl & Wilson, (2007) IL (d13C: -28.78, Schessl 85/1–4 GENT); Bruhl & Wilson, (2007) Bruhl & Wilson, (2007) IL (d13C: -26.91, Hess 51/16 GENT); Bruhl & Wilson, (2007) Bruhl & Wilson, (2007) C3 C3 C3 C3 C3 C3 C3 Bruhl & Bruhl & Bruhl & IL (d13C: Bruhl & IL (d13C: Bruhl & C3 C3 C3 C3 C3 Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 IL (d13C: -26.76, De Wolf 92/86 GENT); Bruhl & Wilson, (2007) Bruhl & Wilson, (2007) IL (d13C: -29.80, Phillipson 1647 GENT) Bruhl & Wilson, (2007) IL (d13C: -27.35, Chantaranothai et al. 814 K); Bruhl & Wilson, (2007) Bruhl & Wilson, (2007) IL (d13C: -26.02, Reid 1816 GENT); Bruhl & Wilson, (2007) IL (d13C: -29.68, Carter 6152 GENT); Bruhl & Wilson, (2007) IL (d13C: -27.32, De Smet 77/23 GENT); Bruhl & Wilson, (2007) Bruhl & Wilson, (2007) IL * (d13C: -29.74, Audru 5375 P); Bruhl & Wilson, (2007) Bruhl & Wilson, (2007) IL (d13C: -26.54, Hess 52/779 GENT); Bruhl & Wilson, (2007) IL * (d13C: -24.03, Schessl 177/2 GENT); Bruhl & Wilson, (2007) IL (d13C: -33.91, Lam & Meeuse 5816 K) Bruhl & Wilson, (2007) IL (d13C: -26.24, Lambinon 82/85 GENT) Bruhl & Wilson, (2007) Bruhl & Wilson, (2007) IL * (d13C: -28.75, Viane 961 GENT); Bruhl & Wilson, (2007) prolifer Lam. pseudoleptocladus Kük. pseudovegetus Steud. pulchellus R.Br. pulcher Thunb. reduncus Hochst. ex Boeck. reflexus Vahl renschii Boeck. schomburgkianus Nees sciaphilus Cherm. seslerioides Kunth sexangularis Nees sphaerospermus Schrad. stradbrokensis Domin submicrolepis Kük. Wilson, Wilson, Wilson, -31.16, Wilson, -30.19, Wilson, (2007) (2007) (2007) Boorman 229 GENT) (2007) Viane 681 GENT) (2007) © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167, 19–46 32 I. LARRIDON ET AL. Table 2. Continued Species C3/C4 References (d13C: value, voucher) Cyperus surinamensis Rottb. C3 Cyperus sylvestris Ridl. Cyperus tenerrimus J.Presl & C.Presl Cyperus tenuispica Steud. Cyperus textilis Thunb. Cyperus trinervis R.Br. Cyperus uncinulatus Schrad. ex Nees Cyperus vaginatus R. Br. Cyperus virens Michx. var. minarum (Boeck.) Denton Cyperus virens Michx. var. montanus (Boeck.) Denton Cyperus virens Michx., as C. virens var. virens Cyperus xerophilus Cherm. Cyperus elegans L. C3 C3 C3 C3 C3 C3 C3 C3 IL (d13C: -28.68, Jansen-Jacobs 521 GENT); Bruhl & Wilson, (2007) Bruhl & Wilson, (2007) IL * (d13C: -31.13, Davidse 35095 GENT); Bruhl & Wilson, (2007) Bruhl & Wilson, (2007) IL * (d13C: -24.26, Bohnen 7744 GENT); Bruhl & Wilson, (2007) IL (d13C: -27.73, Wilson 8565 GENT) Bruhl & Wilson, (2007) IL (d13C: -28.64, Wilson 905 K); Bruhl & Wilson, (2007) Bruhl & Wilson, (2007) C3 Bruhl & Wilson, (2007) C3 Bruhl & Wilson, (2007) C3 C4 Cyperus esculentus L. Cyperus lacunosus Griseb. Cyperus oxylepis Nees ex Steud. Cyperus papyrus L. Cyperus trachysanthos Hook. & Arn. Ascolepis capensis (Kunth) Ridl. Lipocarpha chinensis (Osbeck) J.Kern C4 C4 C4 C4 C4 Bruhl & Wilson, (2007) IL (d13C: -11.46, Goetghebeur 5601 GENT); Bruhl & Wilson, (2007) IL (d13C: -12.83, Madsen 5310 GENT); Bruhl & Wilson, (2007) Bruhl & Wilson, (2007) IL (d13C: -11.63, Carter 9061 GENT); Bruhl & Wilson, (2007) IL (d13C: -11.12, Hess 50/88 GENT); Bruhl & Wilson, (2007) IL (d13C: -12.47, St. John 23599 K); Bruhl & Wilson, (2007) Pycreus polystachyos (Rottb.) P.Beauv. Queenslandiella hyalina (Vahl) Ballard Remirea maritima Aubl. C4 C4 IL (d13C: -10.77, IL (d13C: -10.35, Wilson, (2007) IL (d13C: -10.60, IL (d13C: -12.93, C4 IL (d13C: -11.56, MacDougal 3449 GENT); Bruhl & Wilson, (2007) C4 C4 answer questions concerning the affinities between the taxa within the Cyperus clade. In this study, the outgroup was chosen to represent various lineages in the Ficinia clade. As the C4 photosynthetic pathway arose only once in the Cyperus clade (e.g. Soros & Bruhl, 2000; Besnard et al., 2009), we can recognize at least two main infrageneric groups in Cyperus: (1) an eucyperoid subgenus, uniting plants without the Kranz syndrome and with an inflorescence generally composed of digitately clustered spikelets (or contracted to a head-like inflorescence); and (2) a chlorocyperoid subgenus, uniting plants with a chlorocyperoid anatomytype (Kranz syndrome) and an inflorescence composed of spikes of spikelets or condensed spikes. Using the data gathered in a previous study of the nomenclature of the Cyperus clade (Huygh et al., 2010; Larridon et al., 2011; Reynders et al., 2011), the two subgenera have been named respectively: Cyperus subgenus Anosporum (Nees) C.B.Clarke (1884) and Traore 33 GENT); Bruhl & Wilson, (2007) Malaisse & Goetghebeur 790 GENT); Bruhl & Reekmans 6441 GENT); Bruhl & Wilson, (2007) Mwachala 269 GENT); Bruhl & Wilson, (2007) subgenus Cyperus. Informally, we use the names C3 Cyperus and C4 Cyperus for these subgenera, respectively. RELATIONSHIPS IN C3 CYPERUS The Cyperus clade (Fig. 2) is sister to the Ficinia clade and consists of a paraphyletic C3 Cyperus, in which a well-supported monophyletic clade is nested, encompassing the species of the Cyperus clade that use C4 photosynthesis (C4 Cyperus). Table 3 shows the infrageneric classification of C3 Cyperus species according to Kükenthal (1935–1936), with the species listed using their currently accepted names (Govaerts et al., 2011). In contrast, Table 4 presents the preliminary subdivisional delimitations in C3 Cyperus based on the results of the molecular phylogenetic hypothesis obtained in this study. The clades (or their subclades) in C3 Cyperus (Fig. 2) largely concur with previously recognized taxa (Cyperus sections and sub- © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167, 19–46 AFFINITIES IN C3 CYPERUS LINEAGES REVEALED 33 Table 3. Infrageneric classification of C3 Cyperus species according to Kükenthal (1935–1936). The current synonymy for species names largely follows Govaerts et al. (2011) Cyperus subgenus Eucyperus pars Pycnostachys Section Species Glutinosi Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Luzuloidei Pseudanosporum Vaginati Diffusi Remarks constanzae Urb. elegans L. gardneri Nees lacunosus Griseb. oxylepis Nees trachysanthos Hook. & Arn. acuminatus Torr. & Hook. altsonii Kük. cellulosoreticulatus Boeck. columbiensis Palla distinctus Steud. drummondii Torr. & Hook. entrerianus Boeck. eragrostis Lam. hieronymi Boeck. incomtus Kunth intricatus Schrad. ex Schult. luzulae (L.) Retz. megalanthus (Kük.) G.C.Tucker ochraceus Vahl pseudovegetus Steud. reflexus Vahl sordidus J.Presl & C.Presl surinamensis Rottb. virens Michx. xanthostachyus Steud. boeckeleri Phil. elytropiptos Steud. platystylis R.Br. alternifolius L. canus J.Presl & C.Presl debilissimus Baker dioicus I.M.Johnst. gymnocaulos Steud. marginatus Thunb. phaeolepis Cherm. sexangularis Nees textilis Thunb. vaginatus R.Br. limiticola Larridon & Reynders ajax C.B.Clarke albopurpureus Cherm. albostriatus Schrad. balfourii C.B.Clarke baronii C.B.Clarke buchholzii Boeck. chalaranthus J.Presl & C.Presl chorisanthos C.B.Clarke derreilema Steud. diffusus Vahl felipponei Kük. fischerianus Schimp. ex A.Rich. friburgensis Boeck. C4 Cyperus C4 Cyperus C4 Cyperus C4 Cyperus Unplaced name Unplaced name Not seen by Kükenthal © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167, 19–46 34 I. LARRIDON ET AL. Table 3. Continued Cyperus subgenus Eucyperus pars Pycnostachys Section Incurvi Fusci Species Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Remarks glaucophyllus Boeck. helferi Boeck. kurzii C.B.Clarke laxus Lam. leptocladus Kunth longifolius Poir. multispicatus Boeck. nemoralis Cherm. pseudoleptocladus Kük. radians Nees et Meyen ex Kunth renschii Boeck. sylvestris Ridl. tabina Steud. ex Boeck. thorelii E.G.Camus trialatus (Boeck.) J.Kern xerophilus Cherm. duclouxii E.G.Camus anisitsii Kük. ankaizinensis Cherm. betafensis Cherm. chamaecephalus Cherm. consors C.B.Clarke dichromenaeformis Kunth disjunctus C.B.Clarke fertilis Boeck. filipes Benth. grandisimplex C.B.Clarke hoppiifolius Uittien hylophilus Cherm. inops C.B.Clarke longistylus Kük. mapanioides C.B.Clarke miliifolius Poepp. & Kunth molliglumis Cherm. neoguineensis Kük. pandanophyllum C.B.Clarke pearcei C.B.Clarke pedunculosus F.Muell. plantaginifolius Cherm. rufostriatus C.B.Clarke ex Cherm. sciaphilus Cherm. simplex Kunth subpapuanus Kük. tetraphyllus R.Br. dichrostachyus Hochst. ex A.Rich. difformis L. fuscus L. haematocephalus Boeck. ex C.B.Clarke pulcher Thunb. pulcherrimus Willd. ex Kunth reduncus Hochst ex Boeck. silletensis Nees submicrolepis Kük. trailii C.B.Clarke Not seen by Kükenthal (1936) © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167, 19–46 AFFINITIES IN C3 CYPERUS LINEAGES REVEALED 35 Table 3. Continued Cyperus subgenus Eucyperus pars Pycnostachys Section Haspani Leucocephali Anosporum Graciles Dichostylis Cyperus subgenus Juncellus Minuti Cyperus subgenus Mariscus Decidui Aristati Species Cyperus unicolor Boeck. Cyperus soongoricus Kar. & Kir. Cyperus commixtus Kük. Cyperus concinnus R.Br. Cyperus dentatus Torr. Cyperus denudatus L.f. Cyperus foliaceus C.B.Clarke Cyperus haspan L. Cyperus lecontei Torr. Cyperus pendulus Cherm. Cyperus pinetorum Britton Cyperus platycaulis Baker Cyperus prolifer Lam. Cyperus sphaerospermus Schrad. Cyperus subaequalis Baker Cyperus tenuispica Steud. Cyperus kipasensis Cherm. Cyperus leucocephalus Retz. Cyperus michoacanensis Britton ex C.B.Clarke Cyperus pulchellus R.Br. Cyperus schomburgkianus Nees Cyperus tenerrimus J.Presl & C.Presl. Cyperus cephalotes Vahl Cyperus colymbetes Kotschy et Peyr. Cyperus pectinatus Vahl Cyperus aquatilis R.Br. Cyperus breviculmis R.Br. Cyperus debilis R.Br. Cyperus flaccidus R.Br. Cyperus gracilis R.Br. Cyperus laevis R.Br. Cyperus mirus C.B.Clarke Cyperus trichodes Griseb. Cyperus trinervis R.Br. Isolepis levynsiana Muasya & D.A.Simpson Isolepis leucoloma (Nees) C.Archer Cyperus humilis Kunth Cyperus meeboldii Kük. Cyperus michelianus (L.) Link Cyperus seslerioides Kunth Cyperus tweediei C.B.Clarke Cyperus uncinulatus Schrad. ex Nees Cyperus hilairenus Steud. Juncellus minutus C.B.Clarke Cyperus limosus Maxim. Remarks Not seen by Kükenthal (1936) Unlikely distribution Isolepis Isolepis C4 Cyperus C4 Cyperus Uncertain (Kükenthal, 1936) Unplaced name Cyperus deciduus Boeck. Courtoisina assimilis (Steud.) Maquet Courtoisina cyperoides (Roxb.) Soják © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167, 19–46 36 I. LARRIDON ET AL. Table 4. Preliminary subdivisional delimitations in C3 Cyperus based on the presented results. The current synonymy for species names largely follows Govaerts et al. (2011). The distribution data were queried from Govaerts et al. (2011). Type species underlined Taxon Species Distribution Courtoisina Courtoisina assimilis (Steud.) Maquet Courtoisina cyperoides (Roxb.) Soják ex Fusci Cyperus reduncus Hochst. ex Boeck. Ethiopia to S Africa, Madagascar Chad to S Africa, Madagascar, Himalaya to Indo-China W Trop. Africa to Uganda Kyllingiella Kyllingiella Kyllingiella Kyllingiella Kyllingiella Tropical and S Africa, Indian Subcontinent Ethiopia to E Tropical. Africa Tanzania to Zambia E Tropical Africa Oxycaryum Oxycaryum cubense (Poepp. & Kunth) Palla ex Elegantes Cyperus gardneri Nees Cyperus section Alternifolii Cyperus alternifolius L. Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus section Anosporum Cyperus section Diffusi ex Incurvi ex Incurvi microcephala (Steud.) R.W.Haines & Lye polyphylla (A.Rich.) Lye simpsonii Muasya ugandensis R.W.Haines & Lye canus J.Presl & C.Presl dioicus I.M.Johnst. gymnocaulos Steud. marginatus Thunb. phaeolepis Cherm. sexangularis Nees textilis Thunb. vaginatus R.Br. Tropical and Subtropical Africa, tropical and Subtropical America Cuba, SE Mexico to NE Argentina Ethiopia to Mozambique, W Indian Ocean, Arabian Pen. Mexico to Colombia Mexico (Baja California Sur) Australia Kenya to S Africa Madagascar S Tropical and S Africa S Africa Australia Cyperus cephalotes Vahl Tropical Asia to NE Australia Cyperus colymbetes Kotschy & Peyr Cyperus pectinatus Vahl Sudan to Mozambique, Madagascar Tropical and S Africa, Madagascar Cyperus afromontanus Lye E Tropical Africa Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus W Central Tropical Africa to Malawi E Madagascar S Tropical and S Africa N Central Madagascar W Indian Ocean Tropical Africa, W Indian Ocean Central Madagascar Mexico (Chiapas) Tropical Africa. Argentina (Misiones) W South America to Paraguay Mexico to Central America Papua New Guinea Costa Rica to Panama Brazil (Bahia) Ethiopia to Malawi Tropical and Subtropical Asia to Queensland S Central China S Venezuela to NE Argentina Ethiopia to Malawi Kenya to Malawi Indo-China Andaman Island Mexico to Tropical America S Africa ajax C.B.Clarke albopurpureus Cherm. albostriatus Schrad. ankaizinensis Cherm. balfourii C.B.Clarke baronii C.B.Clarke betafensis Cherm. breedlovei G.C.Tucker buchholzii Boeck. burkartii Guagl. chalaranthus J. Presl & C. Presl chorisanthos C.B. Clarke cinereobrunneus Kük. costaricensis Gómez-Laur. davidsei G.C.Tucker derreilema Steud. diffusus Vahl duclouxii Camus felipponei Kük. fischerianus Schimp. ex A.Rich. glaucophyllus Boeck. helferi Boeck. kurzii C.B.Clarke laxus Lam. leptocladus Kunth © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167, 19–46 AFFINITIES IN C3 CYPERUS LINEAGES REVEALED Table 4. Continued Taxon Species Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus section Fusci Cyperus section Haspani longifolius Poir. matudae G.C.Tucker meistostylus S.T. Blake multispicatus Boeck. muniziae G.C.Tucker nemoralis Cherm. nyererei Lye pseudoleptocladus Kük. pseudopetiolatus G.C. Tucker renschii Boeck. rupicolus S.T.Blake sylvestris Ridl. tabina Steud. ex Boeck. thorelii Camus trialatus (Boeck.) J.Kern turrialbanus Gómez-Laur. xerophilus Cherm. W Indian Ocean SE Mexico (Chiapas) New Guinea Assam to W Jawa Brazil (São Paulo) Central Madagascar S Tanzania Tropical and S Africa Brazil (Bahia) Tropical Africa, Comoros Queensland to NE New South Wales Tropical Africa Central and S Tropical America N Vietnam S China to W Malesia Costa Rica to Panama Central Madagascar Cyperus dichrostachyus Hochst. ex A.Rich. Tropical and S Africa, Madagascar Cyperus difformis L. Cyperus fuscus L. Tropical and Subtropical Old World Macaronesia, Europe, Mediterranean to China S Africa Cape Province to KwaZulu-Natal Tropical Asia Assam to Vietnam E Kazakhstan Tropical Africa N South America to N Brazil S Venezuela to Argentina Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus section Graciles Distribution heamatocephalus Boeck. ex C.B.Clarke pulcher Thunb. pulcherrimus Willd. ex Kunth silletensis Nees soongoricus Kar. & Kir. submicrolepis Kük. traillii C.B.Clarke unicolor Boeck. Cyperus aquatilis R.Br. New Guinea to N and E Australia Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus N Australia N Australia Queensland, NE New South Wales N and E Australia New Caledonia, E Australia E Australia E Australia Queensland to NE New South Wales Queensland, NE New South Wales N and E Australia breviculmis R.Br. cristulatus S.T.Blake enervis R.Br. flaccidus R.Br. gracilis R.Br. laevis R.Br. mirus C.B.Clarke sculptus S.T.Blake stradbrokensis Domin trinervis R.Br. Cyperus afroalpinus Lye Zaïre to Kenya Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus N Somalia N and E Australia Zaïre to S Africa SE Canada to E USA Tropical and S Africa to N Australia Tropical Africa Tanzania (Mt Mnyera) Tropical and Subtropical Zambia Angola to W Tanzania SE USA commixtus Kük. concinnus R.Br. deciduus Boeck. dentatus Torr. denudatus L.f. foliaceus C.B.Clarke graciliculmis Lye haspan L. kasamensis Podlech kipasensis Cherm. lecontei Torr. ex Steud. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167, 19–46 37 38 I. LARRIDON ET AL. Table 4. Continued Taxon ex Diffusi Species Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Distribution microumbellatus Lye pendulus Cherm. pinetorum Britton platycaulis Baker prolifer Lam. purpureoviridis Lye sensilis Baijnath sphaerospermus Schrad. subaequalis Baker tenuispica Steud. Cyperus vandervekenii Reynders, Dhooghe & Goetgh. Cyperus section Incurvi ex Alternifolii Cyperus section Leucocephali Kenya (Shimba Hills) Madagascar Cuba (I. de la Juventud) Chad to KwaZulu-Natal, Madagascar Somalia to S Africa, W Indian Ocean Tanzania (Nguru Mts) KwaZulu-Natal Mozambique to S Africa Madagascar Tropical and Subtropical Old World to Central Asia Rwanda Cyperus almensis D.A.Simpson Brazil (Bahia: Pico das Almas) Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Paraguay E Madagascar S Tanzania to Zambia SE and S Brazil Central Madagascar SE Brazil E Australia W Tropical Africa to Angola New South Wales S Venezuela to Paraguay N South America, N Peru E Madagascar S Brazil Solomon Is. Mexico to Guatemala Tropical Africa Central and S Tropical America Central Madagascar Madagascar New Guinea E Madagascar Peru to Bolivia New Guinea to N. Australia Madagascar E Madagascar E Madagascar S Mexico to Tropical America Brazil (Bahia to Minas Gerais) Queensland Papua New Guinea E Australia anisitsii Kük. chamaecephalus Cherm. chinsalensis Podlech consors C.B.Clarke debilissimus Baker dichromenaeformis Kunth disjunctus C.B.Clarke fertilis Boeck. filipes Benth. grandisimplex C.B.Clarke hoppiifolius Uittien hylophilus Cherm. inops C.B.Clarke longistylus Kük. lundellii O’Neill mapanioides C.B.Clarke miliifolius Poepp. & Kunth molliglumis Cherm. multinervatus Bosser neoguinensis Kük. pandanophyllum C.B.Clarke pearcei C.B.Clarke pedunculosus F.Muell. plantaginifolius Cherm. rufostriatus C.B.Clarke ex Cherm. sciaphilus Cherm. simplex Kunth subcastaneus D.A.Simpson semifertilis S.T.Blake subpapuanus Kük. tetraphyllus R.Br. Cyperus androhibensis D.A.Simpson NW Madagascar Cyperus Cyperus Cyperus Cyperus Cyperus Brazil (Bahia: Pico das Almas) Indian Subcontinent, Indo-China SW Mexico (Sierra de Manantlán) Central Somalia SW Mexico brumadoi D.A.Simpson leucocephalus Retz. michoacanensis Britton ex C.B.Clarke microglumis D.A.Simpson nayaritensis G.C.Tucker © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167, 19–46 AFFINITIES IN C3 CYPERUS LINEAGES REVEALED Table 4. Continued Taxon Cyperus section Luzuloidei ex Diffusi Species Distribution Cyperus pulchellus R.Br. Cyperus schomburgkianus Nees Cyperus tenerrimus J.Presl & C.Presl Tropical Old World N South America to Brazil Mexico to Bolivia Cyperus acuminatus Torr. & Hook. USA to NE Mexico Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus altsonii Kük. cellulosoreticulatus Boeck. columbiensis Palla distinctus Steud. drummondii Torr. & Hook. entrerianus Boeck. eragrostis Lam. friburgensis Boeck. hieronymi Boeck. incomtus Kunth intricatus Schrad. ex Schult. luzulae (L.) Retz. megalanthus (Kük.) G.C.Tucker ochraceus Vahl pseudovegetus Steud. reflexus Vahl Cyperus Cyperus Cyperus Cyperus sordidus J.Presl & C.Presl surinamensis Rottb. virens Michx. xanthostachyus Steud. Guyana Venezuela, Bolivia, S Brazil Colombia SE USA, Bahamas SE USA to Tropical America Mexico to N Argentina, Caribbean Easter Island, America S Tropical America Paraguay to N Argentina Peru to N Argentina Costa Rica to N Argentina Mexico to Tropical America Mexico to Central America Tropical and Subtropical America Central and E USA Oklahoma to Mexico, Costa Rica to Argentina W Mexico Tropical and Subtropical America Tropical and Subtropical America S South America Cyperus section Cyperus platystylis R.Br. Pseudanosporum Cyperus section Radiantes Cyperus radians Nees & Meyen ex Kunth Affinity unknown (C3) ex ‘Dichostylis’ Cyperus ex ‘Dichostylis’ Cyperus ex ‘Dichostylis’ Cyperus ex Elegantes Cyperus ex Minuti Cyperus Cyperus Cyperus humilis Kunth seslerioides Kunth uncinulatus Schrad. ex Nees constanzae Urb. limosus Maxim. palianparaiensis Govind. perennis (M.E.Jones) O’Neill Affinity unknown (photosynthesis type not confirmed) ex Alternifolii Cyperus limiticola Larridon & Reynders ex Incurvi Cyperus marojejyensis Bosser ex Graciles Cyperus trichodes Griseb. ex ‘Dichostylis’ Cyperus tweediei C.B.Clarke (related to Cyperus arsenei O’Neill & Ben.Ayers C. uncinulatus?) (related to Cyperus hilairenus Steud. C. uncinulatus?) (related to Cyperus microbrunneus G.C.Tucker C. uncinulatus?) Unplaced name Cyperus boeckeleri Phil. Unplaced name Cyperus elytropiptos Steud. Tropical and Subtropical Asia, Australia SE China to W Malesia Mexico to Tropical America S USA to N Argentina S Mexico to Tropical America Caribbean Russian Far East, China to Vietnam India NW Mexico Central Madagascar Madagascar Jamaica Argentina (Tucumán) Mexico SE Brazil S Mexico to Central America Chili ? © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167, 19–46 39 40 I. LARRIDON ET AL. genera or segregate genera; e.g. Clarke, 1908; Kükenthal, 1935–1936; Haines & Lye, 1983). Androtrichum, which was indicated as the earliest branching taxon of the Cyperus clade (Muasya et al., 2009a), was not included in this study because of the lack of material suitable for DNA extraction. Clade 1 Clade 1 (Fig. 2) encompasses the three sections Diffusi, Haspani and Incurvi, of which Diffusi and Incurvi have pantropical distributions. Cyperus section Diffusi has important radiations of species in South America, Africa, Madagascar and Asia and C. section Incurvi has important radiations in South America, Madagascar and Oceania (Table 4). Species from both sections mostly occur in shaded forest habitats. Cyperus section Haspani has a mainly African distribution (Table 4), but also includes pantropical species (e.g. C. haspan L.). The three sections form clearly delineated subclades in clade 1. Species of section Haspani (23 spp.; Table 4) can be annuals or more often perennials, usually with poorly developed leaf blades, and an inflorescence of digitate clusters of three to seven spikelets. Kükenthal (1936) included 17 species in this section (of which three were unseen by him) corresponding to 15 currently accepted species (Table 3). Kükenthal (1936) placed C. deciduus Boeck. in C. section Decidui Kük. of his subgenus Mariscus because of its deciduous spikelets. However, he noted the similarity in habit with C. denudatus L.f and C. haspan. Since Kükenthal’s monograph of Cyperus (Kükenthal, 1936), several new species have been described in this section. In this study (Fig. 2), five species of section Haspani are included in the molecular study: C. denudatus, C. foliaceus C.B.Clarke, C. haspan, C. prolifer Lam. (Fig. 1D) and C. purpureoviridis Lye. This last species was described by Lye (1983) without clear indication of its affinities. However, in Haines & Lye (1983) C. purpureoviridis is listed among species belonging to section Diffusi. In the molecular phylogenetic hypothesis presented here (Fig. 2), the species clusters in section Haspani. The morphology of this species falls between sections Diffusi and Haspani, but a clear Haspani character is the well-developed creeping rhizome with relatively long internodes as in, for example, C. denudatus and C. prolifer. Cyperus section Incurvi (32 species; Table 4) includes a number of remarkable species. The section is characterized by having obtuse or shortly mucronate incurved glumes, which articulate at their saccate (pouched) and persistent base (Kükenthal, 1936). Kükenthal (1936) described this section and originally included 31 species (of which three were unseen by him). These correspond to 27 currently recognized species (Table 3). Since then new species have been described and several species have been placed in synonymy. Four Malagasy species (five accessions) of C. section Incurvi sensu Kükenthal (1936) were included in our molecular study (Fig. 2): C. betafensis Cherm. (Fig. 1F), C. chamaecephalus Cherm. (Gautier et al., 2010), C. molliglumis Cherm. and C. plantaginifolius Cherm. var. minor Cherm. Kükenthal (1936) included the strange Malagasy endemic C. debilissimus Baker (Fig. 1C) in his C. section Vaginati [ = C. section Alternifolii] based on the reduction of the leaves to leaf sheaths. Chermezon (1937) thought this species deserved its own section (C. section Debilissimi Cherm., nom. nud.; Larridon et al., 2011). However, our results place it with the other Malagasy species of Incurvi included in this study. Cyperus betafensis clusters with section Diffusi. Cyperus betafensis (Fig. 1F) differs in its habit from the other Malagasy Incurvi and Chermezon (1937) placed it in C. section Diffusi, together with C. ankaizinensis Cherm. At the same time, he placed the other endemic Malagasy Incurvi species in C. section Pandanophylli Cherm., nom. nud. (Larridon et al., 2011), based on their capitate inflorescences contrasting with the anthelate inflorescences of C. ankaizinensis and C. betafensis. The rainforest-dwelling, Malagasy endemics of C. section Incurvi (C. chamaecephalus, Cyperus hylophilus Cherm., C. molliglumis, C. multinervatus Bosser, C. pandanophyllum Cherm., C. plantaginifolius Cherm., C. rufostriatus C.B.Clarke ex Cherm. and C. sciaphilus Cherm.) are generally characterized by their broad leaves, purple leaf sheaths and long bracts much overtopping the capitate inflorescence. Their habit is rather unusual for the genus (Simpson, 1992; Gautier et al., 2010). Bosser (1955) also included C. marojejyensis Bosser in this group, but here we consider this relationship not well enough established. In our opinion, C. section Incurvi sensu Kükenthal (1936) is heterogeneous; the molecular phylogenetic hypothesis confirms it is polyphyletic. The absence from this study of the South American and Australasian species of the section, including the type (C. disjunctus C.B.Clarke; Larridon et al., 2011), makes it impossible to interpret the delimitations of the section correctly at this stage and to evaluate whether the Malagasy forest species should be considered as a separate group, as Chermezon (1937) indicated. Cyperus section Diffusi (43 species; Table 4) is generally characterized by a perennial habit, welldeveloped leaves often with purplish leaf sheaths, large unequal bracts, and frequently a large, rather diffuse inflorescence of digitate clusters of one to six spikelets. Kükenthal (1936) recognized 26 species in section Diffusi (of which two were unseen by him) © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167, 19–46 AFFINITIES IN C3 CYPERUS LINEAGES REVEALED agreeing with 30 currently accepted species (Table 3). Few of the species included by Kükenthal (1936) were South American. However, later studies revealed numerous overlooked species in the Neotropics (Gómez-Laurito, 1978; Gómez & Gómez-Laurito, 1982; Tucker, 1986, 2007; Guaglianone, 1990). Cyperus diffusus Vahl, interpreted by Kükenthal (1936) as a pantropical species, is now divided into several taxa: C. laxus Lam. (South America), C. buchholzii Boeck. (Africa) and C. diffusus s.s. (Asia). In this study (Fig. 2), seven species (10 accessions) of section Diffusi sensu Kükenthal (1936) from Africa (including Malagascar) are included (C. ajax C.B.Clarke, C. albostriatus Cherm., C. balfourii C.B.Clarke, C. buchholzii, C. leptocladus Kunth and C. renschii Boeck.) and three samples of the South American species C. friburgensis Boeck. However, the last species consistently appears in the clade corresponding to C. section Luzuloidei sensu Denton (1978), making section Diffusi sensu Kükenthal (1936) polyphyletic. Consequently, C. friburgensis needs to be excluded from section Diffusi. As mentioned above, the Malagasy C. betafensis (Fig. 1F) does not belong to section Incurvi (cf. Kükenthal, 1936) but to section Diffusi (cf. Chermezon, 1937). The two Malagasy species included (C. balfouri and C. betafensis) appear more closely related to each other than to species of section Diffusi occurring in mainland Africa. Clade 2 An entirely New World clade including C. section Luzuloidei sensu Denton (1978) (‘Luzulae group’) is sister to clade 3. This clade is a good example of a radiation of species in the New World. Of the 21 species in section Luzuloidei (Table 4), Denton (1978) included ten species in her ‘Luzulae group’, i.e. C. acuminatus Torr. & Hook., C. distinctus Steud., C. eragrostis Lam., C. intricatus Schrad. ex Schult., C. luzulae (L.) Retz., C. ochraceus Vahl, C. pseudovegetus Steud., C. reflexus Vahl, C. surinamensis Rottb. and C. virens Michx. She also included three more currently accepted species (Govaerts et al., 2011): C. entrerianus Boeck. in synonymy with C. luzulae, C. drummondii Torr. & Hook., as a variety of C. virens, and C. megalanthus (Kük.) G.C.Tucker, as a variety of C. pseudovegetus. Furthermore, Denton (1978) excluded six species previously included in section Luzuloidei by Kükenthal (1936; Table 3), i.e. C. altsonii Kük., C. cellulosoreticulatus Boeck., C. columbiensis Palla, C. hieronymi Boeck., C. incomtus Kunth and C. xanthostachyus Steud., and omitted one more species (C. sordidus J.Presl & C.Presl). In our molecular phylogenetic analysis, ten samples of the Luzulae group were included, encompassing seven of its 13 41 currently accepted species. For two of the species, more than one accession was included: (1) for C. eragrostis, a specimen was used collected in the USA where it occurs as a native plant, another specimen was collected in France where the species is naturalized, and a specimen from the Ghent University Botanical Garden was also used; (2) for C. luzulae, two specimens were used, one of which was collected in the wild, and the other cultivated in the Ghent University Botanical Garden. We can conclude here that Denton’s Luzulae group (Denton, 1978) forms a natural group of species. However, the relationships between the species in this group are not well resolved in the phylogenetic tree (Fig. 2). Because no species excluded by Denton (1978) were included in this study, we cannot comment on the monophyly of section Luzuloidei sensu Kükenthal (1936). The species C. friburgensis, previously included in section Diffusi (e.g. Kükenthal, 1936; Tucker, 2007) clusters in section Luzuloidei. We included several different specimens of C. friburgensis in the phylogenetic study to ensure its inclusion in section Luzuloidei was not erroneous. Clade 3 Clade 3 is highly diverse, as it not only includes several previously recognized sections, i.e. Anosporum, Pseudanosporum and Fusci, but also includes the segregate genera Courtoisina and Oxycaryum. This clade can be divided into two major subclades, clade 3a and clade 3b. Anosporum, Courtoisina and Oxycaryum have all been or are still recognized at generic level because of their highly unusual morphological characteristics. Anosporum and Oxycaryum are characterized by their (floating) aquatic lifeform and corky nutlets. Oxycaryum is also characterized by its spirally arranged glumes. Courtoisina is characterized by its yellowish green colour, flattened spikelets which disarticulate as a unit when mature, leaving the spikelet bract and prophyll behind, often conspicuously winged glumes, and linear–lanceolate to linear–oblong nutlets. A further paper will focus in more detail on the C3 Cyperus segregate genera and the necessary nomenclatural/ taxonomic changes to include these taxa into a monophyletic genus Cyperus (Larridon et al., in press). In this study (Fig. 2), an African and an American specimen of Oxycaryum cubense (Poepp. & Kunth) Palla are included in the phylogenetic analysis. Cyperus gardneri Nees, a Neotropical species, which Kükenthal (1936) placed in the mainly C4 Cyperus section Elegantes (as ‘C. section Glutinosi’), clusters together with Oxycaryum. Analysis of the photosynthesis type used by the species in C. section Elegantes shows that, of the six species included by Kükenthal (1936; Table 3), two use C3 photosynthesis (Table 2 in © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167, 19–46 42 I. LARRIDON ET AL. bold, see below). The limited material available of the other proven C3 species of C. section Elegantes, C. constanzae Urb., did not yield DNA fit for analysis. For the genus Courtoisina, we included Courtoisina assimilis (Steud.) Maquet and an African and a Malagasy sample of Courtoisina cyperoides (Roxb.) Soják. Kükenthal (1936) placed the two species of Courtoisina in Cyperus subgenus Mariscus based on its deciduous spikelets, but Mariscus has been shown to be polyphyletic (e.g. Lye, 1992). A Cyperus species previously classified in section Fusci (Cyperus reduncus Hochst. ex Boeck.; Kükenthal, 1936; Table 3) clusters with the two known Courtoisina spp. The other taxa clustering in this subclade belong to Cyperus section Anosporum (Cyperus pectinatus Vahl) and section Pseudanosporum (Cyperus platystylis R.Br). Clade 3b corresponds to C. section Fusci (11 spp.; Table 4). This relatively small section is characterized by its generally annual habit (all species except C. dichrostachyus and C. pulcher), often reduced number of anthers (one or two, rather than three), and frequently small glumes and nutlets. Kükenthal (1936) included 11 species in his key of the section and mentioned a twelfth species (C. soongoricus Kar. & Kir.) as unknown to him (Table 3). In this study (Fig. 2), four species are included in the phylogenetic analysis (C. dichrostachyus Hochst. ex A.Rich., C. difformis L., C. fuscus L. (Fig. 1A) and C. submicrolepis Kük.). We exclude C. reduncus from section Fusci as it clusters with Courtoisina (see above). Clade 4 The sampled species of C. section Alternifolii form a clade, which is sister to the clade including C. section Leucocephali and the genus Kyllingiella, and C4 Cyperus (Fig. 2). This section is typified by the umbrella sedge (C. alternifolius L.), a well-known ornamental (Fig. 1E). Section Alternifolii has a southern hemisphere distribution, occurring in Australia, South America (including dioecious species), Madagascar and southern Africa, where the diversity of the section is noticeably higher (Table 4). The section comprises nine species (Table 4) and is characterized by its leafless culms, and many, almost equally sized involucral bracts. Cyperus alternifolius ssp. flabelliformis Kük. is sometimes recognized as the separate species, C. involucratus Rottb., but here we treat C. alternifolius in the broad sense. Kükenthal (1936) included C. debilissimus in his key to this section (as ‘C. section Vaginati’), and mentioned C. paucispiculatus Cherm. [ = C. limiticola Larridon & Reynders] as unknown to him (Table 3). We included C. alternifolius, C. marginatus Thunb., C. phaeolepis Cherm., C. textilis Thunb. and C. debilissimus in the phylogenetic analysis. As mentioned above, C. debilissimus clusters with the Malagasy species of section Incurvi. The affinities of C. limiticola are as yet unknown (Larridon, Reynders & Goetghebeur, 2008). In our results, C. textilis clusters in between the three included C. alternifolius accessions (Fig. 2). As noted in Gordon-Gray (1995), two Southern African species are closely related to C. alternifolius, i.e. C. sexangularis Nees and C. textilis Thunb., and there is a need to carefully establish the differences, in particular between typical C. alternifolius and C. textilis. Clade 5 Clade 5 is formed by Cyperus section Leucocephali and the segregate genus Kyllingiella (Fig. 2). The close relationship of section Leucocephali and Kyllingiella is reflected in a marked resemblance in habit (small grass-like plants with a pale-coloured capitate inflorescence; see Fig. 1B) and in their preference for wet open grasslands. However, in Kyllingiella spp. the glumes are spirally arranged. In 1990, Simpson revised section Leucocephali, including seven species. Since then, two new species have been described. In this study, a specimen of C. schomburgkianus Nees from Bolivia is included. This specimen (Beck 25586) is a first record for the species from Bolivia. Simpson (1990) remarked on the disjunct distribution of this section across the tropics that might have implications for its monophyly. However, the recent discoveries of new species, and its intricate relationship with Kyllingiella, rather indicate a lack of knowledge of this group. A formal taxonomic revision with the inclusion of Kyllingiella in Cyperus will be published elsewhere (Larridon et al., in press). RELATIONSHIPS WITH C4 CYPERUS A well-supported monophyletic clade encompassing the C4 Cyperus s.l. species (C4 Cyperus; characterized by C4 photosynthesis linked with chlorocyperoid vegetative anatomy) forms the sixth clade of Cyperus. The C4 species C. cuspidatus Kunth (Bruhl & Wilson, 2007: chlorocyperoid anatomy, carbon isotope reading typical for C4) and its Malagasy sister species C. waterloti Cherm. (synonymized with C. cuspidatus in Govaerts et al., 2011), consistently form a clade outside the main C4 Cyperus clade (Fig. 2; Muasya et al., 2009a). Kükenthal (1936) placed C. cuspidatus in C. section Amabilis of his ‘subgenus Cyperus pars Pycnostachys’ (see above). The relationships in the main C4 Cyperus clade are not well resolved in this study. A molecular phylogenetic study focusing on C4 Cyperus is underway (W. Huygh, M. Reynders, I. Larridon, K. Bauters, A.M. Muasya, D.A. Simpson, P. Goetghebeur, unpubl. data). © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167, 19–46 AFFINITIES IN C3 CYPERUS LINEAGES REVEALED CARBON ISOTOPE ANALYSIS Carbon isotope analysis (d13C) was performed on 65 species to confirm their photosynthetic pathway (Table 2). In this study, emphasis was put on C. section Elegantes. This section was considered to belong in the group with C4 photosynthesis, but has inflorescences with digitate spikelet clusters (Kükenthal’s (1935–1936) pars Pycnostachys; see above). Cyperus section Elegantes (Clarke, 1883) is the correct name for Kükenthal’s (1936) section Glutinosi (Larridon et al., 2011). This section includes C. elegans L. (the type species), C. constanzae, C. gardneri, C. lacunosus Griseb., C. oxylepis Nees and C. trachysanthos Hook. & Arn. (Table 3). The carbon isotope analysis conducted during this study confirmed previous studies (e.g. Bruhl & Wilson, 2007), indicating that C. section Elegantes is heterogeneous. Cyperus constanzae and C. gardneri have carbon isotope readings typical for C3, whereas C. elegans, C. lacunosus, C. oxylepis and C. trachysanthos have carbon isotope readings typical for C4. Such heterogeneity can also be shown in other previously recognized sections using the data assembled by Bruhl & Wilson (2007), e.g. C. section Dichostylis sensu Kükenthal (1936). As well as two confirmed C4 species, i.e. C. meeboldii Kük. and C. michelianus (L.) Delile, Kükenthal (1936) also included several confirmed C3 species, namely C. uncinulatus Schrad. ex Nees, C. seslerioides Kunth and C. humilis Kunth. Cyperus andinus Palla ex Kük., another species included by Kükenthal (1936), is now considered to be a heterotypic synonym of C. seslerioides (Govaerts et al., 2011). The photosynthesis type of the last species included in C. section Dichostylis sensu Kükenthal (1936: 310), C. tweediei C.B. Clarke, has not yet been confirmed. However, in the protologue of this species, Clarke (1908: 4) clearly stated its affinity to C. uncinulatus and C. humilis, so we can tentatively identify it as a C3 Cyperus species. Since the publication of Kükenthal’s monograph of Cyperus (Kükenthal, 1935–1936), two more species have been described which are placed in the vicinity of C. uncinulatus, i.e. C. arsenei O’Neill & Ben.Ayers and C. microbrunneus G.C.Tucker. The phylogenetic position of this group of species in C3 Cyperus could not yet be verified. Cyperus section Graciles is another group that includes confirmed C3 Cyperus species. This section was not included in the molecular study because the specimens present in the GENT herbarium did not yield any useable DNA. Kükenthal (1936) included eight species in C. section Graciles, corresponding with 11 accepted species names (Table 3). Blake (1939) published a revision of C. section Graciles representing a more natural circumscription of this 43 section. After correspondence, Kükenthal (1943) accepted Blake’s opinions. Three species included by Kükenthal (1936) in section Graciles were no longer included by Blake (1939). Two of these, C. 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; Muasya, Simpson & Smets, 2006, 2007). A third species included in section Graciles by Kükenthal (1936), C. trichodes Griseb., was excluded most probably based on its highly unlikely distribution in Jamaica (Table 4); all other species of section Graciles are limited to Australia (Oceania). Furthermore, Blake (1939) had a quite different view of the synonymy and rank of some of the taxa included in section Graciles (see Table 4). For seven of the 11 species now included in section Graciles (Table 4), the photosynthesis type is confirmed as C3 (Table 2; Bruhl & Wilson, 2007). Cyperus radians Nees & Meyen ex Kunth was included by Kükenthal (1936) in section Diffusi, although Suringar (1898) placed it in its own section, Radiantes, an opinion which was shared by Kern (1974). Although generally considered as a eucyperoid species, no studies (carbon isotope analysis/molecular study) have as yet been performed to test this view. CONCLUSIONS From the maximum likelihood and Bayesian analyses of nrDNA (ETS1f) and plastid DNA (rpl32-trnL and trnH-psbA) sequence data presented here, we conclude that the Cyperus clade consists of a paraphyletic group with eucyperoid anatomy using C3 photosynthesis (C3 Cyperus; clades 1–5) and a wellsupported monophyletic clade with chlorocyperoid anatomy using C4 photosynthesis (C4 Cyperus). In C3 Cyperus, five major clades are recognizable. Clade 1 can be divided in three subclades largely corresponding to Cyperus sections Haspani, Incurvi and Diffusi. The other major clades respectively correspond to: clade 2, an entirely New World C. section Luzuloidei sensu Denton (1978); clade 3, a highly diverse clade including two subclades: clade 3a, sections Pseudanosporum and Anosporum and the segregate genera Courtoisina and Oxycaryum, and clade 3b, C. section Fusci; clade 4, C. section Alternifolii; and clade 5, C. section Leucocephali and the segregate genus Kyllingiella. This study establishes a phylogenetic framework for future studies in the diverse Cyperus clade. ACKNOWLEDGEMENTS We thank Pieter Asselman for his helpful suggestions with the lab work, Andy Vierstraete for performing the © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167, 19–46 44 I. LARRIDON ET AL. sequence reactions (Ghent University, Belgium) and Lazlo Csiba (Royal Botanic Gardens, Kew) for the extraction of DNA from the specimens in the Kew Herbarium (Kew DNA Bank). We thank the Department of Environment and Natural Resources (DENR Region 8), for providing a collecting permit for Cyperaceae in the Philippines, and the Ministry of Scientific Research and Innovation for providing a collecting permit for Cameroon (No. 082/MINRESI/B00/C00/ C10/C12), and the general secretariat of the AETFAT congress 2007 and the staff of the National Herbarium in Yaoundé and Limbe Botanic Gardens for their field assistance. 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 with organizing the expedition. The ANGAP Madagascar National Parks authority, the general secretariat of the AETFAT congress 2010 and the staff of the MBG office in Antananarivo are acknowledged for their support in securing collecting permits (No. 082/10/MEF/SG/DGF/DCB. SAP/SLRSE – I. Larridon) for Cyperaceae in Madagascar and their help with organizing the expedition. FUNDING 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 field expeditions were financed through 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 Álvarez I, Wendel JF. 2003. Ribosomal ITS sequences and plant phylogenetic inference. Molecular Phylogenetics and Evolution 29: 417–434. Archer C. 1998. A new combination in Isolepis. Bothalia 28: 41–42. Besnard G, Muasya AM, Russier F, Roalson EH, Salamin N, Christin P-A. 2009. Phylogenomics of C4 photosynthesis in sedges (Cyperaceae): multiple appearances and genetic convergence. Molecular Biology and Evolution 26: 1909–1919. Blake ST. 1939. (publ. 1940). Notes on Australian Cyperaceae 3. Proceedings of the Royal Society of Queensland 51: 32–50. Bosser J. 1955. Cyperacées nouvelles de Madagascar. Naturaliste Malgache 7: 119–121. Britton NL. 1907. The sedges of Jamaica (Cyperaceae). Bulletin of the Department of Agriculture Jamaica 5 (Suppl. 1): 1–19. Bruhl JJ. 1991. Comparative development of some taxonomically critical floral/inflorescence features in Cyperaceae. Australian Journal of Botany 39: 119–127. Bruhl JJ. 1995. Sedge genera of the world: relationships and a new classification of the Cyperaceae. Australian Systematic Botany 8: 125–305. Bruhl JJ, Perry S. 1995. Photosynthetic pathway-related ultrastructure of C3, C4 and C3-like C3-C4 intermediate sedges (Cyperaceae), with special reference to Eleocharis. Australian Journal of Plant Physiology 22: 1–10. Bruhl JJ, Stone NE, Hattersley PW. 1987. C4 acid decarboxylation enzymes and anatomy in sedges (Cyperaceae): first record of NAD-malic enzyme species. Australian Journal of Plant Physiology 14: 719–728. Bruhl JJ, Wilson KA. 2007. Towards a comprehensive survey of C3 and C4 photosynthetic pathways in Cyperaceae. In: Columbus JT, Friar EA, Porter JM, Prince LM, Simpson MG, eds. Monocots III/grasses IV. Aliso 23. Claremont, CA: Rancho Santa Ana Botanic Garden, 99–148. Castresana J. 2000. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Molecular Biology and Evolution 17: 540–552. Chermezon H. 1937. Cypéracées. In: Humbert H, ed. Flore de Madagascar. 29e Fam. Tananarive: Imprimerie Officielle, 1–335. Christin PA, Besnard G, Samaritani E, Duvall MR, Hodkinson TR, Savolainen V, Salamin N. 2008a. Oligocene CO2 decline promoted C4 photosynthesis in grasses. Current Biology 18: 37–43. Christin PA, Salamin N, Muasya AM, Roalson EH, Russier F, Besnard G. 2008b. Evolutionary switch and genetic convergence on rbcL following the evolution of C4 photosynthesis. Molecular Biology and Evolution 25: 2361– 2368. Clarke CB. 1883. Cyperaceae. In: Baker JG, ed. Contributions to the Flora of Madagascar 3. Journal of the Linnean Society, Botany 20: 279–299. Clarke CB. 1884. On the Indian species of Cyperus; with remarks on some others that specially well illustrate the subdivisions ot the genus. Journal of the Linnean Society, Botany 21: 1–202. Clarke CB. 1893. Cyperaceae. In: Hooker JD, ed. Flora of British India 6, 19. London: L. Reeve & Co., 585–672. Clarke CB. 1908. New genera and species of Cyperaceae. Kew Bulletin of Miscellaneous Information, Additional Series 8: 1–196. Denton MF. 1978. A taxonomic treatment of the Luzulae group of Cyperus. Contributions from the University of Michigan Herbarium 11: 197–271. Dragon JA, Barrington DS. 2009. Systematics of the Carex aquatilis and C. lenticularis lineages: geographically and ecologically divergent sister clades of Carex section Phacocystis (Cyperaceae). American Journal of Botany 96: 1896– 1906. Gautier L, Nusbaumer L, Larridon I, Callmander MW. 2010. Distribution of Cyperus chamaecephalus Cherm., a forest undergrowth species with inconspicuous inflorescences. Candollea 65: 364–367. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167, 19–46 AFFINITIES IN C3 CYPERUS LINEAGES REVEALED Goetghebeur P. 1986. Genera Cyperacearum. Een bijdrage tot de kennis van de morfologie, systematiek en fylogenese van de Cyperaceae-genera. PhD Thesis, Ghent University, Belgium. Goetghebeur P. 1989. Studies in Cyperaceae 9. Problems in the lectotypification and infrageneric taxonomy of Cyperus. Bulletin de la Société Royale de Botanique de Belgique 122: 103–114. Goetghebeur P. 1998. Cyperaceae. In: Kubitzki K, ed. The families and genera of vascular plants 4. Flowering plants – monocotyledons. Berlin: Springer-Verlag, 141–190. Gómez LD, Gómez-Laurito J. 1982. Plantae mesoamericanae novae 7. Phytologia 52: 227–229. Gómez-Laurito J. 1978. De Cyperacearum costaricensium notitate. Cyperus. Brenesia 14–15: 357–359. Gordon-Gray KD. 1995. Cyperaceae in Natal. In: Leistner OA, Momberg BA, eds. Strelitzia 2. Pretoria: National Botanical Institute, 1–218. Govaerts R, Simpson DA, Goetghebeur P, Wilson KL, Egorova T, Bruhl J. 2007. World checklist of cyperaceae. Sedges. Kew: Kew Publishing, Royal Botanic Gardens, xiii + 765 p. Govaerts R, Simpson DA, Goetghebeur P, Wilson KL, Egorova T, Bruhl J. 2011. World checklist of cyperaceae. Kew: The Board of Trustees of the Royal Botanic Gardens, Published on the Internet; available at http://www.kew.org/ wcsp/monocots/ accessed 25.03.2011. Guaglianone ER. 1990. Une especie nueva de Cyperus (Cyperaceae) de Misiones, Argentina. Darwiniana 30: 233– 236. Haines RW, Lye KA. 1983. The sedges and rushes of East Africa. Nairobi: East African National History Society. Huygh W, Larridon I, Reynders M, Muasya AM, Govaerts R, Simpson DA, Goetghebeur P. 2010. Nomenclature and typification of names of genera and subdivisions of genera in Cypereae (Cyperaceae): 1. Names of genera in the Cyperus clade. Taxon 59: 1883–1890. Kern JH. 1974. Cyperaceae. In: van Steenis CGGJ, ed. Flora malesiana, ser. 1. Den Haag: Junk, 107–187. Kress WJ, Wurdack KJ, Zimmer EA, Weigt LA, Janzen DH. 2005. Use of DNA barcodes to identify flowering plants. Proceedings of the National Academy of Sciences of the United States of America 102: 8369–8374. Kükenthal G. 1935–1936. Cyperaceae–Scirpoideae– Cypereae. In: Engler A, ed. Das pflanzenreich 4 (20) [Heft 101]. Berlin: Engelmann, 1–671. Kükenthal G. 1943. Neue oder nicht genügend bekannte Cyperaceen. Mitteilungen des Thüringischen Botanischen Vereins n.f., 50: 1–13. Larridon I, Huygh W, Reynders M, Muasya AM, Govaerts R, Simpson DA, Goetghebeur P. Accepted. Nomenclature and typification of names of genera and subdivisions of genera in Cypereae (Cyperaceae): 2. Names of subdivisions in Cyperus. Taxon 60: 868–884. Larridon I, Reynders M, Goetghebeur P. 2008. Cyperus limiticola, a new name for a Madagascan Cyperus (Cyperaceae). Novon 18: 187–188. Larridon I, Reynders M, Huygh W, Bauters K, Vrijdaghs 45 A, Leroux O, Muasya AM, Goetghebeur P. In press. Taxonomic changes in C3 Cyperus (Cyperaceae) supported by molecular phylogenetic data, morphology, embryology, ontogeny and anatomy. Plant Ecology and Evolution. Li M-R, Wedin DA, Tieszen LL. 1999. C3 and C4 photosynthesis in Cyperus (Cyperaceae) in temperate eastern North America. Canadian Journal of Botany 77: 18–209. Linnaeus C. 1753. Species plantarum. Stockholm. Lye KA. 1983. Studies in African Cyperaceae 25. New taxa and combinations in Cyperus L. Nordic Journal of Botany 3: 213–232. Lye KA. 1992. The history of the genus Mariscus. Lidia 3: 37–72. Muasya AM, Bruhl JJ, Simpson DA, Culham A, Chase MW. 2000. Suprageneric phylogeny of Cyperaceae: a combined analysis. In: Wilson KL, Morrison DA, eds. Monocots: systematics and evolution. Melbourne: CSIRO Publishing, 593–601. Muasya AM, de Lange PJ. 2010. Ficinia spiralis (Cyperaceae) a new genus and combination for Desmoschoenus spiralis. New Zealand Journal of Botany 48: 31–39. Muasya AM, Reynders M, Goetghebeur P, Simpson DA, Vrijdaghs A. 2011. Dracoscirpoides (Cyperaceae) – a new genus from Southern Africa, its taxonomy and floral ontogeny. South African Journal of Botany. doi:10.1016/ j.sajb.2011.05.011. Muasya AM, Simpson DA, Chase MW. 2002. Phylogenetic relationships in Cyperus s.l. (Cyperaceae) inferred from plastid DNA sequence data. Botanical Journal of the Linnean Society 138: 145–153. Muasya AM, Simpson DA, Chase MW, Culham A. 1998. An assessment of suprageneric phylogeny in Cyperaceae using rbcL DNA sequences. Plant Systematics and Evolution 211: 257–271. Muasya AM, Simpson DA, Smets E. 2006. Isolepis tenella, a new combination in Cyperaceae. Novon 16: 89–90. Muasya AM, Simpson DA, Smets E. 2007. Isolepis levynsiana, a new name for Cyperus tenellus (Cyperaceae). Novon 17: 59. Muasya AM, Simpson DA, Verboom GA, Goetghebeur P, Naczi RFC, Chase MW, Smets E. 2009a. Phylogeny of Cyperaceae based on DNA sequence data: current progress and future prospects. Botanical Review 75: 2–21. Muasya AM, Vrijdaghs A, Simpson DA, Chase MW, Goetghebeur P, Smets E. 2009b. What is a genus in Cypereae: phylogeny, character homology assessment and generic circumscription in Cypereae. Botanical Review 75: 52–66. Müller J, Müller K, Quandt D. 2008. PhyDE – phylogenetic data editor, version 0.995. Available at http://phyde.de/ Nylander JAA. 2004. Mrmodeltest v2. Program distributed by the author. Uppsala: Evolutionary Biology Centre, Uppsala University. Pyankov VI, Ziegler H, Akhani H, Deigele C, Lüttge U. 2010. European plants with C4 photosynthesis: geographical and taxonomic distribution and relations to climate parameters. Botanical Journal of the Linnean Society 163: 283– 304. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167, 19–46 46 I. LARRIDON ET AL. Rambaut A, Drummond AJ. 2007. Tracer v1.4. Available at http://beast.bio.ed.ac.uk/Tracer Raynal J. 1973. Notes cypérologiques 19. Contribution à la classification de la sous-famille des Cyperoideae. Adansonia sér. 2, 13: 145–171. Reynders M, Huygh W, Larridon I, Muasya AM, Govaerts R, Simpson DA, Goetghebeur P. Accepted. Nomenclature and typification of names of genera and subdivisions of genera in Cypereae (Cyperaceae): 3. Names of subdivisions in segregate genera of Cyperus. Taxon 60: 885–895. Rikli M. 1895. Beiträge zur vergleichenden Anatomie der Cyperaceen mit besonderer Berücksichtigung der inneren Parenchymscheide. Jahrbücher für Wissenschaftliche Botanik 27: 485–580. Ronquist F, Huelsenbeck JP. 2003. MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574. Sage RF. 2004. The evolution of C4 photosynthesis. New Phytologist 161: 341–370. Shaw J, Lickey EB, Beck JT, Farmer SB, Liu W, Miller J, Siripun KC, Winder CT, Schilling EE, Small RL. 2005. The tortoise and the hare II: Relative utility of 21 noncoding chloroplast DNA sequences for phylogenetic analysis. American Journal of Botany 92: 142–166. Shaw J, Lickey EB, Schilling EE, Small RL. 2007. Comparison of whole chloroplast genome sequences to choose noncoding regions for phylogenetic studies in angiosperms: the tortoise and the hare III. American Journal of Botany 94: 275–288. Simpson DA. 1990. A revision of Cyperus section Leucocephali. Kew Bulletin 45: 485–501. Simpson DA. 1992. A new species of Cyperus and a reassessment of Cyperus rufostriatus (Cyperaceae) from Madagascar. Notes on Madagascar Cyperaceae 1. Kew Bulletin 47: 745–751. Simpson DA, Furness CA, Hodkinson TR, Muasya AM, Chase MW. 2003. Phylogenetic relationships in Cyperaceae subfamily Mapanioideae inferred from pollen and plastid DNA sequence data. American Journal of Botany 90: 1071– 1086. Simpson DA, Muasya AM, Alves M, Bruhl JJ, Dhooge S, Chase MW, Furness CA Ghamkhar K, Goetghebeur P, Hodkinson TR, Marchant AD, Nieuborg R, Reznicek AA, Roalson EH, Smets E, Starr JR, Thomas WW, Wilson KL, Zhang X. 2007. Phylogeny of Cyperaceae based on DNA sequence data – a new rbcL analysis. In: Columbus JT, Friar EA, Porter JM, Prince LM, Simpson MG, eds. Monocots III/grasses IV. Aliso 23. Claremont, CA: Rancho Santa Ana Botanic Garden, 72–83. Soros CL, Bruhl JJ. 2000. Multiple evolutionary origins of C4 photosynthesis in the Cyperaceae. In: Wilson KL, Morrison DA, eds. Monocots: systematics and evolution. Melbourne: CSIRO, 629–636. Stamatakis A. 2006. RAxML-VI-HPC: maximum likelihoodbased phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22: 2688–2690. Stamatakis A, Hoover P, Rougemont J. 2008. A rapid bootstrap algorithm for the RAxML web servers. Systematic Biology 57: 758–771. Starr JR, Harris SA, Simpson DA. 2003. Potential of the 5′ and 3′ ends of the intergenic spacer (IGS) of rDNA in the cyperaceae: new sequences for lower-level phylogenies in sedges with an example from Uncinia Pers. International Journal of Plant Sciences 164: 213–227. Stock WD, Chuba DK, Verboom GA. 2004. Distribution of South African C3 and C4 species of Cyperaceae in relation to climate and phylogeny. Austral Ecology 29: 313–319. Suringar JV. 1898. Het geslacht Cyperus. Leeuwarden: Hugo Suringar. Tucker GC. 1986. New mesoamerican species of Cyperus (Cyperaceae). Rhodora 88: 503–513. Tucker GC. 2007. Systematics of Cyperus L. section Diffusi Kunth (Cyperaceae) in the neotropics. A Botânica no Brasil: Pesquisa, Ensino e Políticas Públicas Ambientais [58 Congresso Nacional de Botânica] 311–314. Van de Putte K, Nuytinck J, Stubbe D, Le HT, Verbeken A. 2010. Lactarius volemus sensu lato (Russulales) from northern Thailand: morphological and phylogenetic species concepts explored. Fungal Diversity 45: 99–130. Van der Veken P. 1965. Contribution à l’ embryographie systématique 1 des Cyperaceae-Cyperoideae. Bulletin du Jardin botanique de l’État à Bruxelles 35: 285–354. Vrijdaghs A, Muasya AM, Goetghebeur P, Caris P, Nagels A, Smets E. 2009. A floral ontogenetic approach to questions of homology within the Cyperoideae (Cyperaceae). Botanical Review 75: 30–51. Vrijdaghs A, Reynders M, Larridon I, Muasya AM, Smets E, Goetghebeur P. 2010. Spikelet structure and development in Cyperoideae (Cyperaceae): a monopodial general model based on ontogenetic evidence. Annals of Botany 105: 555–571. Vrijdaghs A, Reynders M, Muasya AM, Larridon I, Goetghebeur P, Smets E. 2011. Spikelet and floral morphology and development in Cyperus and Pycreus (Cyperaceae). Plant Ecology and Evolution 144: 44–63. Yano O, Hoshino T. 2005. Molecular phylogeny and chromosomal evolution of Japanese Schoenoplectus (Cyperaceae), based on ITS and ETS1f sequences. Acta Phytotaxonomica et Geobotanica 56: 183–195. Yano O, Hoshino T. 2007. Phylogenetic relationships and chromosomal evolution of Japanese Fimbristylis (Cyperaceae) using nrDNA ITS and ETS1f sequence data. Acta Phytotaxonomica et Geobotanica 57: 205–217. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167, 19–46