Towards a new classification of the giant paraphyletic genus Cyperus (Cyperaceae): phylogenetic relationships and generic delimitation in C 4 Cyperus

Kenneth Bauters

In the last decade, efforts to reconstruct suprageneric phylogeny of the Cyperaceae have intensified. We present an analysis of 262 taxa representing 93 genera in 15 tribes, sequenced for the plastid rbcL and trnL-F (intron and intergenic spacer). Cyperaceae are monophyletic and resolved into two clades, here recognised as Mapanioideae and Cyperoideae, and the overall topology is similar to results from previous studies. Within Cyperoideae, Trilepideae are sister to rest of taxa whereas Cryptangieae, Bisboeckelerieae and Sclerieae are resolved within Schoeneae. Cladium and Rhynchospora (and Pleurostachys) are resolved into clades sister to the rest of Schoeneae, lending support to the recognition of these taxa in separate tribes. However, we retain these taxa in Schoeneae pending broader sampling of the group. The phylogenetic position of 40 species in 21 genera is presented in this study for the first time, elucidating their position in Abildgaardieae (Trachystylis), Cryptangieae (Didymiandrum, Exochogyne), Cypereae (Androtrichum, Volkiella), Eleocharideae (Chillania), and Schoeneae (Calyptrocarya, Morelotia). More sampling effort (more taxa and the use of more rapidly evolving markers) is needed to resolve relationships in Fuireneae and Schoeneae.

Cypereae form one of the largest and most complex tribes of the sedge family (Cyperaceae). Recently, two clades have been revealed within the tribe, the largest of which includes the giant genus Cyperus and its closest allies. However, thirteen genera of the generally accepted classification of Goetghebeur (1998) appear to be nested within Cyperus. The taxonomic status of many of these taxa has been under discussion since they are based on different combinations of a limited set of derived characters. Pycreus, the largest of these segregate genera, is characterised by laterally compressed dimerous pistils of which the derivation from the general trimerous situation was not yet understood. It shares this pistil with Kyllinga and Queenslandiella that both are, as is Pycreus, embedded in the Cyperus clade which uses C4 photosynthesis. The recent insights from molecular phylogenetics make a reevaluation possible of the taxonomic status of the thirteen different segregate genera of Cyper...

The sedge genera Alinula, Ascolepis, Kyllinga, Lipocarpha, Pycreus, Queenslandiella, Remirea, Sphaerocyperus and Volkiella (Cyperaceae) were recognised at generic level because they possess specialised inflorescence and/or flower characters. However, recent molecular phylogenetic analyses show that these genera are all nested in a paraphyletic Cyperus s.s. and therefore should be viewed as part of a broadly circumscribed genus Cyperus. For all species of Alinula and for the single species of Queenslandiella, Remirea and Sphaerocyperus, Cyperus names were already published by other authors. For the species of Lipocarpha and Volkiella, Cyperus names and a new sectional classification are published in a separate paper including a detailed molecular phylogenetic hypothesis for these taxa. Based on a study of herbarium specimens and literature, in this paper, twenty species of Ascolepis, seventeen species of Kyllinga, and six species of Pycreus, which do not yet have a validly published ...

bs_bs_banner Botanical Journal of the Linnean Society, 2013, 172, 106–126. With 3 figures Towards a new classification of the giant paraphyletic genus Cyperus (Cyperaceae): phylogenetic relationships and generic delimitation in C4 Cyperus ISABEL LARRIDON1*, KENNETH BAUTERS1, MARC REYNDERS1, WIM HUYGH1, A. MUTHAMA MUASYA2, DAVID A. SIMPSON3 and PAUL GOETGHEBEUR1 1 Research Group Spermatophytes, Department of Biology, Ghent University, K. L. Ledeganckstraat 35, 9000 Gent, Belgium 2 Botany Department, University of Cape Town, Rondebosch 7700, South Africa 3 Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3AB, UK Received 2 April 2012; revised 14 December 2012; accepted for publication 14 December 2012 Maximum likelihood and Bayesian inference analyses of nuclear ribosomal DNA (ETS1f) and plastid DNA (rpl32-trnL, trnH-psbA) sequence data are presented for ‘C4 Cyperus’ (Cyperaceae). The term ‘C4 Cyperus’ encompasses all species of Cyperus s.l. that use C4 photosynthesis linked with chlorocyperoid vegetative anatomy. Sampling comprises 107 specimens of 104 different taxa, including many of the subdivisions of C4 Cyperus s.s. and all C4 segregate genera (Alinula, Ascolepis, Kyllinga, Lipocarpha, Pycreus, Queenslandiella, Remirea, Sphaerocyperus and Volkiella). According to our results, C4 Cyperus is a well-supported monophyletic clade nested in C3 Cyperus. Despite the lack of resolution along the backbone of the C4 Cyperus clade and for some internal branches, several well-supported clades can be distinguished. The first clade in C4 Cyperus is formed by Cyperus cuspidatus and C. waterloti. Other recognizable and well-supported clades correspond to segregate genera, i.e. Ascolepis, Lipocarpha including Volkiella, and Kyllinga. Species of C4 Cyperus s.s. form a core grade in which the C4 segregate genera are embedded. Pycreus, the largest segregate genus composed of c. 120 species, is not monophyletic as it includes several C4 species of Cyperus s.s. This study establishes a phylogenetic framework for revising the classification and character evolution in Cyperus s.l. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172, 106–126. ADDITIONAL KEYWORDS: Cypereae – Cyperoideae – molecular phylogeny – paraphyly – species radiation – systematics. INTRODUCTION Cyperaceae (the sedge family) has an almost cosmopolitan distribution and plays a dominant role in wetland vegetation. The many reductions and convergences in the inflorescences of Cyperaceae have impeded evolutionary reconstruction (homology questions, e.g. Bruhl, 1991; Vrijdaghs et al., 2009, 2010; Muasya et al., 2009b) and classification (e.g. Clarke, 1908; Kükenthal, 1935–36; Kern, 1974; Haines & Lye, 1983; Bruhl, 1995; Goetghebeur, 1998). Based on recent molecular phylogenetic studies, Cyperaceae *Corresponding author. E-mail: isabel.larridon@ugent.be 106 consists of two main clades, corresponding to subfamilies Cyperoideae and Mapanioideae (Simpson et al., 2003, 2007; Muasya et al., 2009a). In Cyperoideae, two clades stand out because of their extraordinary species diversity: (1) the clade corresponding to the predominantly temperate tribe Cariceae (c. 1950 spp.); and (2) the clade corresponding to the mainly tropical tribe Cypereae (c. 1120 spp.). Together, they cover nearly three-fifths of the species diversity in Cyperaceae (Govaerts et al., 2012). Recent molecular phylogenetic studies of Cyperaceae (Simpson et al., 2003, 2007; Muasya et al., 2009a) have shown Cypereae sensu Goetghebeur (1998) to be monophyletic, but the generic delimita- © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172, 106–126 C4 CYPERUS PHYLOGENY (CYPERACEAE) 107 Table 1. The genera in Cypereae currently accepted by Govaerts et al. (2012), plus the recently published genus Dracoscirpoides (Muasya et al., 2012) and the recent phylogenetic novelty Erioscirpus (Yano et al., 2012). The segregate genera using the C4 photosynthetic pathway are underlined. The taxa indicated by an asterisk were recently included in Cyperus (Larridon et al., 2011b) Cypereae Ficinia clade ? Cyperus clade Dracoscirpoides Muasya (3 spp.) Erioscirpus Palla (2 spp.) Hellmuthia Steud. (1 sp.) Ficinia Schrad. (75 spp.) Isolepis R.Br. (76 spp.) Scirpoides Ség. (4 spp.) Androtrichum (Brongn.) Brongn. Alinula J.Raynal (4 spp.) Ascolepis Nees ex Steud., (22 spp.) Courtoisina Soják (2 spp.)* Kyllinga Rottb. (74 spp.) Kyllingiella R.W.Haines & Lye (4 spp.)* Lipocarpha R.Br. (36 spp.) Oxycaryum Nees (1 sp.)* Pycreus P.Beauv. (114 spp.) Queenslandiella Domin (1 sp.) Remirea Aubl. (1 sp.) Sphaerocyperus Lye (1 sp.) Volkiella Merxm. & Czech (1 sp.) tions in Cypereae remain controversial (Muasya et al., 2009b). In the past, members of Cypereae were circumscribed as having spikelets with distichous glumes and reduced, perianthless flowers (e.g. Kükenthal, 1935–36). However, neither the distichy of the glumes nor the absence of a perianth can be regarded as phylogenetically informative characters (e.g. Vrijdaghs et al., 2006; Muasya et al., 2009a, b, in press). Currently, members of Cypereae are circumscribed by the presence of a Cyperus-type embryo or the similar Ficinia-type embryo (Van der Veken, 1965; Goetghebeur, 1998; Muasya et al., 2009a, b). The presence of various combinations of characters (e.g. reduced flowers, reduced and/or contracted inflorescences) and convergent morphologies has led to the misinterpretation of the relationships of many lineages of Cypereae. A number of taxa (belonging especially to Erioscirpus Palla, Ficinia Schrad, Hellmuthia Steud., Isolepis R.Br., Kyllingiella R.W.Haines & Lye, Oxycaryum Nees, Scirpoides Séq.) have been allocated to various tribes in Cyperaceae, including Scirpeae, Rhynchosporeae, Hypolytreae and Schoeneae (e.g. Kunth, 1837; Nees von Esenbeck, 1842; Steudel, 1854–55; Clarke, 1908). However, extensive anatomical (Kranz anatomy), embryographical and molecular phylogenetic studies (e.g. Van der Veken, 1965; Goetghebeur, 1986, 1998; Bruhl, 1995; Muasya et al., 2001a, 2009a, b; Muasya, Simpson & Chase, 2002; Simpson et al., 2003, 2007; Larridon et al., 2011a, b; Yano et al., 2012) have revealed that these genera are closely related to Cyperus L. Consequently, the reinterpretation of the morphological characters of these genera in the context of Cypereae is required. On the basis of molecular phylogenetic studies (e.g. Simpson et al., 2007; Muasya et al., 2009a), two clades are recognized in Cypereae: (1) the Ficinia clade; and (2) the Cyperus clade. The first, smaller clade (c. 160 spp.) consists of several genera with a mainly southern African distribution, a ficinoid habit (hemicryptophytes, culm scapose, inflorescence capitate and appearing pseudolateral with main involucral bract being stem-like) and mostly spiral glumes. The basalmost branches include species with perianth parts (Dracoscirpoides Muasya, Erioscirpus, Hellmuthia; Vrijdaghs et al., 2006; Muasya et al., 2012; Yano et al., 2012). Prior to the embryographical study of Van der Veken (1965), most of these genera had been classified in or near Scirpus L. The second, larger, pantropical clade (c. 950 spp.), with mostly distichous glumes, comprises a paraphyletic Cyperus s.s. as the core genus (c. 700 spp.), in which at least 12 segregate genera are nested (Goetghebeur, 1998; Govaerts et al., 2012; see Table 1). The branch leading to Androtrichum (Brongn.) Brongn. (two species) appears to be at the base of the Cyperus clade (Muasya et al., 2002, in press), but this needs further confirmation. Although molecular phylogenetic studies have revealed that all of these genera are nested in Cyperus (e.g. Muasya et al., 2002; Larridon et al., 2011a), there has been considerable discussion about whether to include these taxa in Cyperus. Contemporary treatments either recognize the segregate genera as separate from Cyperus (e.g. Bruhl, 1995; Goetghebeur, 1998; Govaerts et al., 2007, 2012) or merge them into © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172, 106–126 108 I. LARRIDON ET AL. Cyperus at an infrageneric rank (e.g. Kükenthal, 1935–36; Haines & Lye, 1983; Lye, 1997). The Cyperus clade includes a grade of branches characterized by C3 photosynthesis (C3 Cyperus, c. 190 spp.), which were well resolved in a combined analysis of ETS1f, trnH-psbA and rpl32-trnL (Larridon et al., 2011a). In C3 Cyperus, most sections of the classification according to Kükenthal (1935–36) were confirmed. Larridon et al. (2011b) included the C3 segregates Courtoisina Soják, Oxycaryum and Kyllingiella in Cyperus, supported by molecular data, combined with morphology, embryography, ontogeny and anatomy. Nested in C3 Cyperus is a highly diverse clade (C4 Cyperus, c. 760 spp.) with the C4 photosynthetic pathway as a synapomorphy (e.g. Muasya, Simpson & Chase, 2001b; Muasya et al., 2002, 2009a, in press; Besnard et al., 2009; Larridon et al., 2011a). The nine C4 segregate genera represent c. 30% of diversity in the C4 Cyperus clade. Figure 1 shows some of the morphological diversity of C4 Cyperus lineages. They are generally considered to be well-delimited entities (e.g. Goetghebeur, 1998) and are circumscribed by a combination of morphological characters, including inflorescence and spikelet morphology, unit of dispersal and nutlet orientation (e.g. Muasya et al., 2009b; Vrijdaghs et al., 2011; Reynders et al., 2012; Figure 2). However, the mutual relationships of the taxa in C4 Cyperus still need to be determined. PARAPHYLY AND MODERN CLASSIFICATION STRATEGIES With the advancement of molecular phylogenetic research, species relationships and evolutionary patterns in giant genera provide new and valuable opportunities to study evolutionary processes. Often, these giant genera appear to contain derived lineages that have, up to now, been considered as separate genera (e.g. Acacia Mill., Miller & Bayer, 2001; Carex L., Starr & Ford, 2009; Croton L., Berry et al., 2005; Euphorbia L., Steinmann & Porter, 2002; Salvia L., Walker et al., 2004). The development of new classifications, encompassing the concept of monophyly for these large paraphyletic entities and their segregate genera, has been highly challenging. Three main strategies can be implemented: (1) splitting; (2) accepting paraphyletic taxa; and (3) lumping. Splitting paraphyletic taxa into a large number of small genera has been proposed for a number of large genera (e.g. Acacia; Maslin, Miller & Seigler, 2003). The decision on where to split needs to be based on a well-resolved phylogenetic hypothesis, and there are challenges to identifying diagnostic characters for the segregate entities and controversies about name application (Acacia; e.g. Moore et al., 2010, 2011; Smith & Figueiredo, 2011; Thiele et al., 2011). A second, less popular, strategy is a classification in which various segregate genera are upheld which are themselves monophyletic, but remain part of a paraphyletically circumscribed giant genus. The use of paraphyletic genera has been defended by some authors (e.g. Brummitt, 1996; Brummitt & Sosef, 1998), but has been strongly opposed by others (e.g. Nelson, Murphy & Ladiges, 2003). The third and most popular strategy when dealing with paraphyletic giant genera is the lumping of all the segregates into a broader circumscribed genus (e.g. in Euphorbia; Steinmann & Porter, 2002). A negative consequence of lumping is that it can become difficult to describe clearly the giant genus as a whole. OBJECTIVES In the present study, molecular phylogenetic data of the Cyperus clade were analysed: (1) to determine the mutual relationships of the taxa (i.e. genera, sections, species) included in C4 Cyperus; (2) to test whether the segregate genera and infrageneric taxa in C4 Cyperus (Kükenthal, 1935–36; Govaerts et al., 2012) are monophyletic; and (3) to examine the most suitable classification strategy for C4 Cyperus. Papers documenting the necessary nomenclatural/ taxonomical changes based on the results presented in this article and more detailed studies of several of the larger C4 segregates will be published elsewhere. This study is part of a larger research project aimed at recircumscribing Cyperus as a monophyletic unit and at creating a new infrageneric classification of the genus supported by both molecular and morphological data. MATERIAL AND METHODS One hundred and seven samples from 104 different taxa were used for this study. Sixty-seven sequences from 23 species were used from a previous study (Larridon et al., 2011a). The other 213 sequences from 81 different taxa were newly generated for this study. The samples with species names, voucher information, origin and GenBank accession numbers for the sequences are given in Table 2. 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. As this study assesses relationships above the rank of species, multiple species samples and infraspecific taxa were generally not used. The outgroup taxa were selected on the basis of the results of previous molecular phylogenetic analyses of Cypereae by Muasya et al. (2002, 2009a) and Larridon et al. (2011a). Taxonomic information for © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172, 106–126 C4 CYPERUS PHYLOGENY (CYPERACEAE) Figure 1. See caption on next page. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172, 106–126 109 110 I. LARRIDON ET AL. Figure 1. Morphological diversity in C4 Cyperus. A, Cyperus cyperoides (L.) Kuntze with strongly contracted spikes of spikelets in an anthelate inflorescence. B, Pycreus polystachyos (Rottb.) P.Beauv. with spikes of spikelets in an anthelate inflorescence. C, Cyperus laevigatus L. with a reduced inflorescence consisting of only a few sessile spikelets in a pseudolateral inflorescence. D, Lipocarpha chinensis (Osbeck) J.Kern with three sessile pseudospikelets. E, Cyperus capitatus with a capitate inflorescence. F, Kyllinga polyphylla Willd. ex Kunth with a capitate inflorescence of reduced, deciduous spikelets. G, Cyperus ustulatus A.Rich. with contracted spikes of spikelets in an anthelate inflorescence. H, Cyperus waterloti Cherm. with an inflorescence of digitately clustered spikelets. Photographs A–G taken by M. Reynders in the Ghent University Botanical Garden, H taken by W. Huygh at Cirque Rouge near Mahajanga, Madagascar. Figure 2. Three-dimensional reconstruction of the spikelet evolution in the Cyperus clade. The illustrations were drawn in Rhinoceros 3D® (Mc Neel, Seattle, WA, USA) by M. Reynders. The basic Cyperus spikelet with distichous glumes developed several times independently into lineages with spiral glumes. In addition, deciduous spikelets originated several times and, from there, different reduction lineages can be identified resulting in single-flowered spikelets. In the extreme situation, the bracts subtending the spikelets behave like glumes bearing the strongly reduced spikelets. Difficult interpretation of the latter resulted in the classification of these taxa among various Cyperaceae tribes before their affinity with Cyperus had been resolved. most taxa mentioned (such as author, place and date of publication, synonyms, distribution) follows Govaerts et al. (2007, 2012). The molecular phylogenetic hypothesis obtained was compared with the classification of Kükenthal (1935–36). Detailed information on the nomenclature of generic and subdivisional names of the Cyperus clade (including the synonymy of the names used by Kükenthal) is given in Huygh et al. (2010), Larridon et al. (2011c) and Reynders et al. (2011). 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, BR). The DNA extraction protocol, markers (ETS1f, rpl32-trnL and trnH-psbA) and material and methods for polymerase chain reaction (PCR) amplification and sequencing and obtaining alignments used in this study follow Larridon et al. (2011a). © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172, 106–126 C4 CYPERUS PHYLOGENY (CYPERACEAE) 111 Table 2. 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 (herbarium) Origin ETS1f trnH-psbA rpl32-trnL Alinula paradoxa (Cherm.) Goetgh. & Vorster Ascolepis brasiliensis (Kunth) Benth. ex C.B.Clarke Ascolepis eriocauloides (Steud.) Nees ex Steud. Ascolepis hemisphaerica Peter ex Goethg. Ascolepis protea Welw. Reid 1027 (GENT) South Africa HQ705964 – HQ705894 Larridon et al. 2010-0304 (GENT) Madagascar HE993954 HE993894 HE993685 De Wilde s.n. (BR) Congo HE993955 HE993895 – Reekmans 6729 (GENT) Burundi HE993956 – – Malaisse & Kisimba 695 (GENT) Malaisse & Goetghebeur 846 (GENT) Hess 52/1581 (GENT) Congo HE993957 HE993896 HE993686 Congo HE993958 HE993897 – Angola HE993959 HE993898 HE993687 Goetghebeur 11516 (GENT) Muasya & Ramdhani 2722 (BOL) Laegaard et al. 17024 (GENT) Goetghebeur 10744 (GENT) Reynders & Sabulao 15 (GENT) Goetghebeur 11988 (GENT) Rostad s.n. GENT Jongkind & Nieuwhuis 2847 (GENT) Muasya et al. 2529 (EA) Muasya & Muthama 1251 (EA) Goetghebeur 5601 (GENT) Muasya & Knox 954 (EA) Goetghebeur 11303 (GENT) Carter 4355 (GENT) Goetghebeur 4329 (GENT) Muasya & Muthama 1269 (EA) Dhondt 9 (GENT) BG Ghent South Africa HQ705948 HE993960 HQ705818 HE993899 HQ705878 HE993688 Senegal HE993961 HE993900 HE993689 BG Ghent Philippines HE993962 HE993963 HE993901 HE993902 HE993690 HE993691 BG Ghent USA Ghana HE993964 HE993965 HQ705954 HE993903 HE993904 HQ705823 HE993692 – HQ705884 Kenya Kenya HE993966 HE993967 HE993905 – HE993693 HE993694 Cuba Tanzania BG Nantes, BG Ghent Florida South Africa Kenya HQ705959 HE993968 HQ705960 HE993969 HE993970 HQ705927 HQ705827 – HQ705828 HE993906 HE993907 HQ705803 HQ705889 – HQ705890 HE993695 HE993696 HQ705803 Madagascar HE993971 – HE993697 Burkina Faso Philippines HE993972 HE993973 HE993908 – HE993698 HE993699 Kenya HQ705961 HQ705829 HQ705891 Morocco, BG Ghent Kenya HE993975 HE993974 HE993910 HE993909 – HE993700 Morocco, BG Ghent Netherlands Ecuador HE993976 HE993977 HQ705910 HE993911 HE993912 – HE993701 HE993702 HQ705846 Kenya HQ705949 HQ705819 HQ705879 Somalia HE993978 HE993913 HE993703 Hawaii HE993979 HE993914 HE993704 Ascolepis pusilla Ridl. Cyperus alopecuroides Rottb. Cyperus alternifolius L. Cyperus aterrimus Hochst. ex Steud. Cyperus bulbosus Vahl Cyperus capitatus Vand. Cyperus compressus L. Cyperus congestus Vahl Cyperus croceus Vahl Cyperus cuspidatus Kunth Cyperus dives Delile Cyperus dubius Rottb. Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus elegans L. endlichii Kük. esculentus L. filiculmis Vahl fulgens C.B.Clarke haspan L. Cyperus fallax Cyperus Cyperus impubes Steud. var. (Cherm.) Kük. iria L. javanicus Houtt. Cyperus kerstenii Boeck. Cyperus laevigatus L. 053 Cyperus laevigatus L. 138 Cyperus laevigatus L. 142 Cyperus longus L. Cyperus luzulae (L.) Retz. Cyperus marginatus Thunb. Cyperus meeboldii Kük. Cyperus meyenianus Kunth Desmet 77/13 (GENT) Reynders & Sabulao 60 (GENT) Muasya 984 (EA, K; Muasya et al., 2002) Goetghebeur 10201 (GENT) Larridon et al. 2009-0033 (GENT) Goetghebeur 10202 (GENT) Farjon 217 (GENT) Van den Eynden 213 (GENT) Larridon et al. 2009-0076 (GENT) Kilian & Lobin 6848 (GENT) Fosberg 47227 (GENT) © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172, 106–126 112 I. LARRIDON ET AL. Table 2. Continued Taxon Voucher (herbarium) Origin ETS1f trnH-psbA rpl32-trnL Cyperus papyrus L. Cyperus pectinatus Vahl Goetghebeur 5866 (GENT) Larridon et al. 2010-0265 (GENT) Porembski 624 (GENT) Samain 2005-001 (GENT) Shaw 890 (K*) Unknown s.n. (GENT) Laegaard 15909 (GENT) Goetghebeur 5965 (GENT) Goetghebeur 4908 (GENT) Muasya & Muthama 1247 (EA) BG 20051035G (GENT) BG Ghent Madagascar HQ705962 HQ705936 HQ705830 HQ705810 HQ705892 HQ705869 Ivory Coast Kenya Hong Kong (China) Ecuador Zimbabwe BG Ghent Cameroon Kenya HE993980 HE993981 HQ705963 HE993982 HE993983 HE993985 HE993984 HQ705953 HE993915 HE993916 HQ705831 HE993917 HE993918 HE993920 HE993919 HQ705822 HE993705 HE993706 HQ705893 HE993707 HE993708 HE993710 HE993709 HQ705883 BG Poznan, BG Ghent Madagascar HE993986 HE993921 HE993711 HQ705955 HQ705824 HQ705885 Madagascar HQ705956 HQ705825 HQ705886 South Africa Kenya South Africa Philippines, BG Ghent BG Ghent Kenya HQ705902 HQ705901 HE993987 HE993988 HQ705784 HQ705783 – HE993922 HQ705839 HQ705838 HE993712 HE993713 HE993989 HE993990 – HE993923 HE993714 HE993715 Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus Cyperus pustulatus Vahl rigidifolius Steud. rotundus L. rubiginosus Hook.f. rupestris Kunth sp. sphacelatus Rottb. spiralis Larridon Cyperus strigosus L. Cyperus waterloti Cherm. Cyperus waterloti Cherm. Ficinia gracilis Schrad. Isolepis fluitans (L.) R.Br. Kyllinga alata Nees Kyllinga brevifolia Rottb. Kyllinga bulbosa P.Beauv. Kyllinga chlorotropis Steud. Kyllinga nemoralis (J.R.Forst. & G.Forst.) Dandy ex Hutch. & Dalziel Kyllinga odorata Vahl Kyllinga polyphylla Willd. ex Kunth Kyllinga pulchella Kunth Lipocarpha albiceps Ridl. Lipocarpha chinensis (Osbeck) J.Kern Lipocarpha comosa J.Raynal Lipocarpha filiformis (Vahl) Kunth Lipocarpha kernii (Raymond) Goetgh. Lipocarpha micrantha (Vahl) G.C.Tucker Lipocarpha nana (A.Rich.) Cherm. Lipocarpha rehmannii (Ridl.) Goetgh. Lipcarpha salzmaniana Steud. Pycreus africanus (S.S.Hooper) Reynders Pycreus alleizettei Cherm. Pycreus bipartitus (Torr.) C.B.Clarke Larridon et al. 2010-0010 (GENT) Larridon et al. 2010-0043 (GENT) Muasya 2713 (BOL) Muasya & Knox 3195 (EA) Acocks 22902 (BR) Reynders and Sabulao 68 (GENT) Goetghebeur 11989 (GENT) Muasya & Gerhke 2606 (EA) Goetghebeur 11518 (GENT) Philippines, BG Ghent HQ705965 HQ705832 HQ705895 Strong 3485 (GENT) Beeckman Z35 (GENT) USA Congo HE993991 HE993992 HE993924 HE993925 HE993716 HE993717 Muasya & Knox 991 (EA) Hess 52/195 (GENT) Reynders & Sabulao 26A (GENT) Mincier 1027 (GENT) Kenya Angola Philippines – HE994025 HE994029 HE993926 HE993944 HE993948 HE993718 HE993748 HE993752 Zambia HE994028 HE993947 HE993751 Vanden Berghen 7913a (GENT) Laegaard 21195 (GENT) Senegal HE994030 HE993949 HE993753 Burkina Faso HE994026 HE993945 HE993749 Luceño 186 (GENT) Brazil HE994032 HE993951 – Larridon et al. 2010-0041A (GENT) Larridon et al. 2010-0320 (GENT) Luceño 28 (GENT) Madagascar HE994031 HE993950 HE993754 Madagascar HE994027 HE993946 HE993750 Brazil HE994033 HE993952 – Leeuwenberg 8527 (GENT) Congo HE993994 HE993927 – Larridon et al. 2010-0299 (GENT) Goetghebeur 11990 (GENT) Madagascar HE993993 – HE993719 BG Ghent HE993995 HE993928 HE993720 © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172, 106–126 C4 CYPERUS PHYLOGENY (CYPERACEAE) 113 Table 2. Continued Taxon Voucher (herbarium) Origin ETS1f trnH-psbA rpl32-trnL Pycreus capillifolius (A.Rich.) C.B.Clarke Pycreus cataractarum C.B.Clarke Pycreus elegantulus (Steud.) C.B.Clarke Pycreus fibrillosus (Kük.) Cherm. Pycreus flavescens (L.) P.Beauv. ex Rchb. Pycreus flavescens (L.) P.Beauv. ex Rchb. subsp. microglumis Lye Pycreus flavidus (Retz.) T.Koyama (Py021) Pycreus gracillimus Chiov. Pycreus intactus (Vahl) J.Raynal Cyperus ‘Pycreus’ juncelliformis Peter & Kük. Pycreus longistolon (Peter & Kük.) Napper Pycreus macranthus (Boeck.) C.B.Clarke Pycreus macrostachyos (Lam.) J.Raynal Muasya & Knox 999 (EA) Kenya HE993996 – HE993721 De Wilde 1452 (GENT) Cameroon HE993997 – HE993722 Unknown 348 (GENT) Kenya HE993998 HE993929 HE993723 Schmitz 7479 (GENT) Congo HE994005 – HE993729 Goetghebeur 10224 (GENT) BG Ghent HE993999 HE993930 HE993724 Malaisse & Goetghebeur 390 (GENT) Congo HE994000 HE993931 – Reynders & Sabulao 45 (GENT) Lewalle 2112 (GENT) Reid 609 (GENT) Philippines HE994001 HE993932 HE993726 Burundi South Africa HE994002 HE994003 HE993933 – – HE993727 Malaisse & Goetghebeur 409 (GENT) Congo HE994004 – HE993728 Muasya & Knox 1027 (EA) Kenya HE994006 HE993934 HE993730 Edwards 1038 (GENT) South Africa HE994007 – HE993731 Muasya with Kirika, Obunyali & Musili 2471 (EA) Goetghebeur 4826 (GENT) Kenya HE994008 HE993935 HE993732 Argentina HE994009 – HE993733 Richards 8409 (GENT) Robinson 3478 (GENT) Congo Zambia HE994010 HE994011 HE993936 – – HE993734 Robinson 2310 (GENT) Muasya & Knox 1018 (EA) Unknown 368 (GENT) Zambia Kenya Kenya HE994012 HE994013 HE994014 – HE993937 – HE993735 HE993736 HE993737 Muasya & Knox 940 (EA) Tanzania HE994015 HE993938 HE993738 Milne-Redhead & Taylor 9184 (GENT) Muasya & Muthama 1263 (EA) Goetghebeur 11519 (GENT) Tanzania HE994016 – HE993739 Kenya HE994017 HE993939 HE993740 South Africa, BG Ghent BG Ghent HQ705966 HQ705833 HQ705896 – – HE993741 Kenya HE994018 – HE993742 Congo HE994020 HE993940 HE993744 Madagascar HE994019 – HE993743 Kenya – HE993941 HE993725 Pycreus megapotamicus (A.Dietr.) Nees Pycreus melanacme Nelmes Pycreus melas (Ridl.) C.B.Clarke Pycreus micromelas Lye Pycreus mundtii Nees Pycreus nigricans (Steud.) C.B.Clarke Pycreus nuerensis (Boeck.) S.S.Hooper Pycreus pauper (Hochst. ex A.Rich.) C.B.Clarke Pycreus pelophilus (Ridl.) C.B.Clarke Pycreus polystachyos (Rottb.) P.Beauv. Pycreus polystachyos (Rottb.) P.Beauv. subsp. holocericeus (Link) T.Koyama Pycreus pumilus (L.) Nees Pycreus reductus Cherm. 017 Pycreus reductus Cherm. 046 Pycreus rehmannianus C.B.Clarke Reynders and Sabulao 64 (GENT) Muasya & Muthama 1264 (EA) Dhondt 11 (GENT) Larridon et al. 2010-0161 (GENT) Muasya & Knox 1022 (EA) © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172, 106–126 114 I. LARRIDON ET AL. Table 2. Continued Taxon Voucher (herbarium) Origin ETS1f trnH-psbA rpl32-trnL Pycreus rhizomatosus C.B.Clarke Pycreus sanguinolentus (Vahl) Nees Pycreus smithianus (Ridl.) C.B.Clarke Pycreus xantholepis Nelmes Queenslandiella hyalina (Vahl) Ballard Remirea maritima Aubl. Gereau & Dumetz 3259 (GENT) Kwika & Mundi 21 (GENT) Reekmans 7531 (GENT) Madagascar HE994021 HE993942 – Kenya HE994022 – HE993745 Burundi HE994023 HE993943 HE993746 Reekmans 9809 (GENT) Muasya 2490 (EA) Burundi Kenya HE994024 HQ705967 – HQ705834 HE993747 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 Namibia HE994034 HE993953 HE993755 Scirpoides holoschoenus (L.) Soják Sphaerocyperus erinaceus (Ridl.) Lye Volkiella disticha Merxm. & Czech Faden et al. 96/358 (K*; Muasya et al., 2002) Müller & Giess 493 (GENT) Alignments are available from the first author on request. Phylogenetic hypotheses were produced using maximum likelihood (ML) and Bayesian inference (BI) analyses. All analyses were first performed on the single-marker datasets (ETS1f, rpl32-trnL, trnHpsbA). As no conflicting clades with a significant confidence value were revealed, a combined dataset was constructed and analysed. The latter was subdivided into three partitions, corresponding to the single markers. The program RAxML v7.2.8 (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 dataset. Bayesian phylogenetic (BI) analyses were carried out in MrBayes v3.1.2 (Ronquist & Huelsenbeck, 2003). For the analysis, MrModeltest v2.3 (Nylander, 2004) was used to determine the model that best fitted the data, applying the Akaike Information Criterion. For the combined dataset, a model was determined for each partition. This method is referred to as the BI method. Four independent, parallel runs of one cold and three heated chains were run for 30 million generations each. Trees and parameter estimates were saved every 1000 generations. The analyses were run on a high-performance computer at Ghent University (Stevin Supercomputer Infrastructure, ICT Department). Convergence, associated likelihood values, effective sample size (ESS) values and burn-in values of the different runs were verified with Tracer v1.5 (Rambaut & Drummond, 2007). Calcula- tion of the consensus tree and the posterior probability (PP) of clades was based on the trees sampled after the chains converged. Trees were drawn using FigTree v1.3.1 and Adobe Photoshop CS3. RESULTS SEQUENCE ALIGNMENTS After alignment and application of Gblocks v0.91b (Castresana, 2000), the ETS1f alignment included 105 sequences of 953 bases, the rpl32-trnL alignment 94 sequences of 1334 bases and the trnH-psbA alignment 81 sequences of 1364 bases. The concatenated dataset included 108 sequences and the Gblocks program retained 57%, or 2101 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 greater support in the multi-locus ML analysis. In the various analyses, only minor conflicts concerning the position of some C4 Cyperus spp. in the backbone of the C4 Cyperus clade were detected. Most nodes in the backbone of this clade had little or no support. The three single-locus BI analyses did not differ significantly in tree topologies. The multi-locus BI topologies did not differ from the multi-locus ML tree, except for some of the C4 Cyperus spp. in the main polytomy, as mentioned above for the ML analyses. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172, 106–126 C4 CYPERUS PHYLOGENY (CYPERACEAE) Evaluation of the multi-locus BI analysis output showed that the four runs converged on similar log likelihood (–23 908) and parameter values. The burn-in value for all runs was determined at three million generations. ESS for the likelihood value of the combined runs consisted of 1397.38 uncorrelated samples. Figure 3 shows the 50% majority consensus multilocus BI tree with the associated PP values and the bootstrap values of the multi-locus ML tree. Only bootstrap values above 75% and PPs above 0.85 are shown. DISCUSSION WITH C3 CYPERUS AFFINITIES In Cypereae, the Cyperus clade is sister to the Ficinia clade, here used as outgroup represented by species of Scirpoides, Isolepis and Ficinia (Fig. 3). The Cyperus clade is strongly supported as monophyletic, but includes several lineages which are currently recognized at the generic level. As in Larridon et al. (2011a), C3 Cyperus spp. form a grade at the base of Cyperus (Fig. 3; Table 3). The clade sister to the C4 Cyperus clade is formed by Cyperus section Leucocephali Cherm. ex Kük. sensu Larridon et al. (2011b) (Fig. 3). Although the species of this section use C3 photosynthesis (e.g. Bruhl & Wilson, 2007; Larridon et al., 2011a), they occur in open grassland habitats which are generally dominated by species using C4 photosynthesis. This suggests that the species of Cyperus section Leucocephali have characters (e.g. geophytic hemicryptophtes, resprouting immediately at the start of the wet season and dying back on onset of the dry season, photosynthesis at high temperatures and irradiation) which make them fitter to survive in these habitats than most other C3 Cyperus spp. C4 CYPERUS RADIATION Our molecular phylogenetic hypothesis shows very short branch lengths for most of the C4 Cyperus clade when compared with the C3 Cyperus grade and the deepest nodes of the C4 Cyperus clade, suggesting a rapid diversification of the clade. Endress (2011: 370) wrote: ‘Many structural innovations originated in several clades [of angiosperms] and in special cases could become key innovations, which likely were hotspots of diversification’. The evolution of C4 photosynthesis in Cypereae can be considered as a key innovation, being the cause of a burst of speciation as a result of: (1) increased fitness in drier habitats (Besnard et al., 2009); (2) optimized nitrogen uptake; and (3) improved resistance to higher irradiance, fire and chemical stress caused by salt and heavy metals 115 (Li, Wedin & Tieszen, 1999; Stock, Chuba & Verboom, 2004). Based on our results and on literature and herbarium data on the distribution of species, we hypothesize that the evolution of the C4 photosynthetic pathway in Cypereae occurred in East Africa. This region, particularly present-day Tanzania, is the centre of diversity for C4 Cyperus spp. In addition, all segregate lineages and most sections are represented in the East African flora. Outside Africa, the Cyperus clade is either represented by widespread species or by taxa which evolved locally as a result of smaller radiations originating from dispersal events. AFFINITIES IN C4 CYPERUS The basal nodes Several early branches of the C4 Cyperus clade are strongly supported (Fig. 3). The first subclade, also retrieved in previous studies (e.g. Muasya et al., 2002, in press; Larridon et al., 2011a), is represented by Cyperus cuspidatus Kunth (and its Malagasy relative C. waterlotii Cherm.). Kükenthal (1935–36) placed the species of this clade in Cyperus section Amabiles C.B.Clarke. Although homogeneous, this section is only held together by characters which probably represent the plesiomorphic condition in C4 Cyperus, such as spikelets arranged in digitate clusters (as in many C3 Cyperus spp. vs. generally spikes of spikelets in C4 Cyperus) and multi-nerved glumes with an excurrent mucro. Species of Cyperus sections Amabiles, Aristati Nees and Rupestres C.B.Clarke show similar characters. After the C. cuspidatus clade, the next branches of our molecular phylogenetic hypothesis include species of the segregates Alinula J.Raynal, Ascolepis Nees, Lipocarpha R.Br., Queenslandiella Domin and Volkiella Merxm. & Czech, and of Cyperus section Rupestres (i.e. C. rupestris Kunth and C. meeboldii Kük.). The relationship between the two species of Cyperus section Rupestres is strongly supported in our analysis. Taxonomically, this section is well circumscribed by several synapomorphies, such as swollen stem bases and a tendency to reduced flowers, each with a single stigma branch and a single stamen. The exact position of its corresponding clade remains to be confirmed, but its position among the early branches of the C4 Cyperus clade seems acceptable. Queenslandiella The monotypic Queenslandiella is currently recognized as a separate genus, based on its laterally flattened, dimerous gynoecia and its deciduous spikelets. Queenslandiella has multi-nerved glumes with an excurrent mucro, suggesting that it is an early branching lineage of C4 Cyperus (Fig. 3). When dried, it has a strong curry odour, a character it shares with © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172, 106–126 116 I. LARRIDON ET AL. Figure 3. Phylogenetic hypothesis for the Cyperus clade: 50% majority consensus multi-locus Bayesian inference (BI) tree with the associated posterior probability (PP) values and the bootstrap values of the multi-locus maximum likelihood (ML) tree. Only bootstrap values > 75% and posterior probabilities > 85% are shown. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172, 106–126 C4 CYPERUS PHYLOGENY (CYPERACEAE) 117 Alinula According to the current circumscription, Alinula includes four species (Goetghebeur, 1998; Govaerts et al., 2012). Only one species, Alinula paradoxa (Cherm.) Goetgh. & Vorster, is included in this study (Fig. 3). From a morphological point of view, this species differs significantly from the other three. In our opinion, the current circumscription of Alinula does not represent a natural group. Haines & Lye (1983), who treated Alinula as a subgenus in Cyperus, included A. paradoxa in Cyperus subgenus Fimbricyperus Lye separate from the other Alinula spp. In our molecular phylogenetic hypothesis, A. paradoxa clusters among the early branches of the C4 Cyperus clade. More research is needed to reveal its exact relationships. Alinula lipocarphioides (Kük.) J.Raynal has been shown to be closer to Lipocarpha (Muasya et al., 2009a, in press). University, Gent, unpubl. data). Lipocarpha appears to be paraphyletic, including Ascolepis and Volkiella (Fig. 3). The first diverging branch is formed by Lipocarpha kernii (Raymond) Goetgh. and L. rehmannii (Ridl.) Goetgh. (Fig. 3), formerly placed in a separate genus Rikliella J.Raynal. Although these species strongly resemble Lipocarpha, prophyll and glumes have not been observed around the flower. Therefore, Goetghebeur & Van den Borre (1989) interpreted Rikliella as a highly evolved lineage of Lipocarpha. However, on the basis of our phylogenetic trees, it is unclear whether the partial inflorescences should be interpreted as pseudospikelets or as true spikelets with spiral glumes (which occur in at least three other lineages of the Cyperus clade; Muasya et al., in press). Sister to this clade is a clade comprising Ascolepis and Lipocarpha s.s. (Fig. 3). Ascolepis spikelets are characterized by a single large glume subtending a flower and the loss of the spikelet prophyll. In Lipocarpha s.s., the first branching clade is formed by L. micrantha (Vahl) G.C.Tucker (Fig. 3), which is characterized by a reduction of the glume. This clade is followed by the rest of Lipocarpha s.s., which also includes the monotypic Volkiella (Fig. 3). Volkiella possesses both a spikelet prophyll and a glume, and is included in a subclade with L. albiceps Ridl. and L. comosa J. Raynal (Fig. 3). These two Lipocarpha spp. are characterized by a well-developed, firm and often darkcoloured prophyll which falls off the rachis separately from the flower and its glume. In other Lipocarpha spp., the prophyll is hyaline and falls off together with the nutlet and glume. Volkiella shares the more rigid prophyll with the two abovementioned species. Volkiella disticha Merxm. & Czech is, in many aspects, a special, highly derived species differing from Lipocarpha by the distichous arrangement of the spikelets on the rachis. A more elaborate study of Lipocarpha, integrating molecular phylogeny and morphology, will be presented in another paper (K. Bauters et al., Ghent University, Gent, unpubl. data). Ascolepis–Lipocarpha clade A well-supported clade in our molecular phylogenetic hypothesis includes the genera Ascolepis, Lipocarpha and Volkiella (Fig. 3), which are all characterized by strongly reduced deciduous spikelets grouped into pseudospikelets (spikes of spikelets). Our results confirm that Ascolepis and Lipocarpha are closely related, as already observed by Muasya et al. (2002). Their relatively early branching position in C4 Cyperus is corroborated by the presence of a small, weakly differentiated Cyperus-type embryo, which is also common in C3 Cyperus and in the early branches of the Ficinia clade (M. Reynders, Ghent The hard polytomy The vast majority of C4 Cyperus spp. are included in an unresolved polytomy (Fig. 3), which can also be found in all previous molecular phylogenetic studies (e.g. Muasya et al., 2002, 2009a, b). As it has not been possible to resolve this polytomy, even when using fast mutating plastid and nuclear markers, additional markers need to be tested, as well as other techniques based on next-generation sequencing (e.g. Harrison & Kidner, 2011). However, in our molecular phylogenetic study, several subclades and the relationships between some taxa are strongly supported (Fig. 3). These taxa are discussed below. Table 3. C3 Cyperus species included in the phylogeny and the sections they represent Species Section Cyperus haspan Cyperus section Haspani (Kunth) C.B. Clarke Cyperus section Luzuloidei (Kunth) C.B. Clarke Cyperus section Anosporum (Nees) Pax Cyperus section Alternifolii (Kunth) C.B. Clarke Cyperus section Leucocephali Cherm. ex Kük. Cyperus luzulae Cyperus pectinatus Cyperus alternifolius and C. marginatus Cyperus spiralis C. squarrosus L., another species showing many of the presumed plesiomorphic characters of the clade. Cyperus squarrosus falls among the basal nodes in an internal transcribed spacer (ITS) analysis of Cyperus (C.S. Reid, Louisiana Department of Wildlife and Fisheries, Baton Rouge, unpubl. data). © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172, 106–126 118 I. LARRIDON ET AL. Table 4. Comparison between the noncore Pycreus species and the core Pycreus clade (with the exception of Cyperus laevigatus) Character Noncore species Core Pycreus clade Glumes Mucro Anthela Multi-nerved Usually present, excurrent Well-developed with long and narrow spikelets, often with second-order branches Isodiametric Mostly opportunistic and lowland concentrated Widespread and common on roadsides and rice fields Midrib with only three nerves Not present or rarely shortly excurrent Often condensed or reduced, especially in therophytic species Isodiametric to strongly elongate Often very specialized Narrow distribution, occurring in high-altitude bogs, salt marshes, floating on open water, etc. Nutlet epidermal cells Ecology Distribution and habitat C4 Cyperus s.s. One subclade of C4 Cyperus s.s. which is strongly supported in our molecular phylogenetic hypothesis (Fig. 3) contains species belonging to Cyperus sections Papyrus (Willd.) Thouars (C. papyrus L., C. dives Delile, C. alopecuroides Rottb.) and Rotundi C.B.Clarke (C. rotundus L., C. longus L., C. endlichii Kük., C. rigidifolius Steud.). These species are all characterized by a narrowly to broadly winged rachilla with deciduous or persistent wings. Several other sections which are not represented in the current analysis, i.e. Cyperus sections Brevifoliati C.B.Clarke, Exaltati (Kunth) C.B.Clarke and Fastigiati Kük., share these characters. Cyperus compressus L. (Cyperus section Compressi Nees) also clusters in this clade (Fig. 3). Kyllinga Kyllinga Rottb. forms a strongly supported monophyletic clade (Fig. 3). There is weak support for the Kyllinga clade as sister to a clade including C. iria L., C. croceus Vahl and C. fulgens C.B.Clarke. Kyllinga is delimited by the combination of a head-like inflorescence, deciduous spikelets and laterally flattened gynoecia. Three subclades can be recognized in the current molecular phylogenetic hypothesis (Fig. 3). A detailed molecular phylogenetic study of Kyllinga, including amplified fragment length polymorphism (AFLP) data, is being prepared (W. Huygh et al., Ghent University, Gent unpubl. data). Remirea and Sphaerocyperus The monotypic genera Remirea Aubl. and Sphaerocyperus Lye remain unresolved in C4 Cyperus (Fig. 3). Both taxa are characterized by a series of empty scales below the flower-bearing glume. For this reason, affinities with Schoeneae or Rhynchosporeae have been suggested (Fenzl, 1836: 144; Bentham, 1883: 1038; Ridley, 1884: 165; Pax, 1888: 116; Baillon, 1894: 377; Clarke, 1901–02: 267; Kükenthal, 1944: 200–209). In addition, Remirea has corky rachilla internodes. Pycreus Pycreus P.Beauv. is here retrieved as a paraphyletic entity including several Cyperus spp. (Fig. 3). In Pycreus, relationships are poorly resolved, although good resolution is obtained for some smaller clades of related species. Furthermore, one large clade is well supported and contains the majority of the sections and species in addition to C. laevigatus L. (Fig. 3). This clade is referred to as the ‘core Pycreus clade’. The Pycreus species which are not included in the core Pycreus clade all belong to four of Kükenthal’s (1935–36) sections, namely Cyperus section Albomarginati Kük., Cyperus section Lancei Kük., nom. superfl., Cyperus section Polystachyi (C.B.Clarke) Kük., nom. illeg., Cyperus section Pumili Kük. and Cyperus section Rhizomatosi Kük. Their mutual relationships remain unresolved, but their position outside the core Pycreus clade can be justified as the species in these sections possess plesiomorphic characters in contrast with the species in the core Pycreus clade (Table 4). Among the early branching lineages, two smaller clades are well supported (Fig. 3). Pycreus longistolon (Peter & Kük.) Napper and P. macrostachyos (Lam.) J.Raynal are strongly supported together. Kükenthal (1935–36) classified P. longistolon in Cyperus section Lancei, nom. superfl., a section which appears to be artificial as the species only share rather large and dark glumes. Pycreus macrostachyos was included in Cyperus section Albomarginati [as C. albomarginatus (Mart. & Schrad. ex Nees) Steud.]. The inclusion of P. longistolon in Cyperus section Albomarginati seems to be appropriate in view of the overall habit of the plants (except for the stolons), the large dimensions of th e spikelets, glumes and nutlets, and the wide, hyaline glume margins. However, the last character is less conspicuous than in P. macrostachyos. Another well-resolved subclade corresponds to Cyperus section Polystachyi, nom. illeg., and is characterized by typically elongated nutlets and a winged rachilla. Pycreus pelophilus C.B.Clarke is an excep- © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172, 106–126 C4 CYPERUS PHYLOGENY (CYPERACEAE) tion in having broad nutlets. Nevertheless, it was placed in this section by Kükenthal (1935–36) and this relationship is confirmed here (Fig. 3). The strongly supported inclusion of C. aterrimus (Fig. 3) in the early branching lineages of Pycreus is noteworthy as this species has triangular nutlets, a different inflorescence and overall larger dimensions of the glumes and nutlets compared with Pycreus. Cyperus aterrimus Hochst. ex Steud. is strongly supported as sister to P. nuerensis (Boeckeler) S.S.Hooper, which it resembles in its growth form, dark-coloured inflorescence and Afromontane distribution. Cyperus kerstenii Boeckeler and C. congestus Vahl also appear to be associated with the early branching Pycreus lineages, although without support. We found no morphological characteristics to support this relationship, especially as both species have deciduous glumes, a character which does not occur in Pycreus. The presence of species with triangular nutlets in Pycreus suggests a reversion of the dimerization of the gynoecium. Recently, Vrijdaghs (2006) and Reynders et al. (2012) showed that gynoecia in Cyperoideae originate from an annular primordium on which stigma primordia originate. This offers more flexibility for the positioning of stigma branches with respect to the restrictions previously assumed based on the anatomical studies by Blaser (1941a, b). The core Pycreus clade only includes Pycreus spp., except for C. juncelliformis Peter & Kük. and C. laevigatus. Cyperus juncelliformis is a true Pycreus, but its name has never been combined into Pycreus. Therefore, its name is mentioned as ‘Pycreus’ juncelliformis in Figure 3. The association of C. laevigatus with the core Pycreus clade seems to be strong. It was verified by including three separate samples of C. laevigatus, and this relationship also occurred in the analyses of the three markers separately (M. Reynders, Ghent University, Gent, unpubl. data). Whereas Pycreus is characterized by laterally flattened dimerous gynoecia, C. laevigatus has dorsiventrally flattened dimerous gynoecia. This might represent either an intermediate state between a trimerous Cyperus ancestor and Pycreus or a derived state from a Pycreus ancestor. Moreover, the vascularization pattern in the rachilla of C. laevigatus differs from the pattern in rachillas of several Pycreus spp. studied by Vrijdaghs et al. (2011). Shared characters of C. laevigatus and Pycreus are the rather glossy glumes and their ecology. ETS1f sequences of the species in the core Pycreus clade (except C. laevigatus) show a large duplication of 140 bp, which is a strong additional argument that this represents a natural group. In the core Pycreus clade, several species clusters are resolved 119 (Fig. 3). Pycreus flavidus (Retz.) T.Koyama clusters with ‘Pycreus’ juncelliformis, corresponding to Kükenthal’s (1935–36) Cyperus section Globosi (C.B.Clarke) Kük. The inclusion of P. niger (Ruiz & Pav.) Cufod. is morphologically supported by the similar nutlets and the shape of the glumes. In contrast, the inclusion of P. flavescens (L.) P.Beauv. ex Rchb. ssp. microglumis Lye is remarkable and needs further investigation. Morphologically, the species cluster of P. capillifolius (A.Rich.) C.B.Clarke and P. reductus Cherm. shows resemblances to Cyperus section Globosi, but this relationship remains unresolved in the current study. In addition, species of Cyperus section Sulcati Kük., nom. illeg., are distributed between two clades, although the species of this section all share peculiar glumes with a furrow on both sides. Pycreus sanguinolentus (Vahl) Nees and P. bipartitus (Torr.) C.B.Clarke are smaller representatives of this section, whereas P. mundtii Nees and P. megapotamicus (A.Dietr.) Nees are taller plants with long culms with spaced leaves that form floating mats on open water. The clustering of P. melanacme Nelmes with this section needs further investigation, as this is, in many ways, a rather distinct therophytic species. Pycreus africanus (S.S.Hooper) Reynders, P. smithianus (Ridl.) C.B.Clarke, P. cataractarum C.B.Clarke, P. fibrillosus (Kük.) Cherm. and P. gracillimus Chiov. form a well-resolved clade. Pycreus africanus belongs to Pycreus section Tuberculati Cherm. (Reynders & Goetghebeur, 2010). Pycreus smithianus and P. cataractarum share many characters, such as a contracted inflorescence, straight rachilla, bright white glumes and a Guineo-Congolean distribution, with a preference for habitats by running water. Kükenthal (1935–36) included both species in Cyperus section Propinqui (C.B.Clarke) Kük. Pycreus fibrillosus and P. gracillimus both have a plant base covered with fibrous remains of old leaf sheaths, an inflorescence reduced to only a few spikelets, a flexuous rachilla and a Zambesian distribution in Afromontane habitats. These species were placed in Cyperus section Propinqui and Cyperus section Latespicati Kük., respectively, by Kükenthal (1935–36) based on their pale vs. dark glumes. As this character seems to depend on altitude (many species of Cyperus s.l. growing above 2000 m have dark-coloured glumes), it is not considered as reliable for sectional delimitation. Therefore, these two sections are likely to be polyphyletic. A final strongly supported clade contains species belonging to Cyperus section Latespicati (P. alleizettei Cherm.) and Cyperus section Flavescentes Kük. (P. flavescens, P. rehmanianus C.B.Clarke) sensu Kükenthal (1935–36). Pycreus xantholepis Nelmes, a tall therophyte, shares its yellow glume colour and © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172, 106–126 120 I. LARRIDON ET AL. nutlet shape with P. alleizettei and P. flavescens. The inclusion of P. melas (Ridl.) C.B.Clarke [Cyperus section Globosi] needs further investigation, as this species is morphologically distinct. RE-EVALUATION OF THE GENERIC STATUS OF THE SEGREGATE LINEAGES In this section of the paper, we re-evaluate the generic status of the segregate lineages based on the currently available knowledge about these taxa. This is a combination of morphological, anatomical, ontogenetic and embryographical data, and the results of previous and current molecular phylogenetic studies. Alinula Goetghebeur & Vorster (1988) included four species in this genus. A species from eastern Africa was originally described as Ficinia lipocarphioides Kük. based on the presence of a hypogynous disc around the base of the fruit. However, after studying its inflorescence morphology and chlorocyperoid anatomy, Raynal (1973) hypothesized that the species was intermediate between Ascolepis and Mariscus Vahl, and eventually placed it in a new genus Alinula (Raynal, 1977). Three more species were added to Alinula, after a complex taxonomical trajectory (e.g. Goetghebeur, 1977; Goetghebeur & Vorster, 1988; Haines & Lye, 1983). In our opinion, the current circumscription of Alinula does not represent a natural group, although A. lipocarphioides, A. malawica (J.Raynal) Goetgh. & Vorster and A. peteri (Kük.) Goetgh. & Vorster show clear morphological affinities, such as the presence of pseudospikelets. However, as pseudospikelets also occur in other, more distantly related taxa of Cypereae (e.g. Ascolepis and Lipocarpha), their presence is, in our opinion, insufficient for generic delimitation. Moreover, A. lipocarphioides has been shown to be nested in the Lipocarpha clade (Muasya et al., in press). Ascolepis The head-like inflorescence of Ascolepis consists of clusters of single-flowered spikelets, sometimes with a rudimentary second glume. Typically, the spikelet prophyll does not develop, but the only glume subtending the single flower is always well developed and larger than the bract which subtends the spikelet. In other species, the glume encloses the flower completely and wings are often developed, possibly for wind dispersal. In other species, the glume is strongly elongated and/or brightly coloured, which gives the inflorescence heads an Asteraceae-like appearance (e.g. as in Ascolepis protea Welw.), suggesting insect pollination. Raynal (1973) postulated the origin of Ascolepis from a mariscoid ancestor. However, Goetghebeur (1980) argued that, although glume and nutlet are shed together in Ascolepis, the rachilla remains fixed on the rachis in contrast with Mariscus. Mariscus was an artificial genus grouping together members of Cypereae with deciduous spikelets. Our results concur with Muasya et al. (2002) in resolving Ascolepis and Lipocarpha as sister taxa. Morphological differentiation in these two taxa shows that similar functional inflorescences originated in both groups using different organs (e.g. As. protea vs. L. comosa). In Lipocarpha, the spikelet bract is strongly developed, whereas the glume subtending the flower is reduced. In Ascolepis, the spikelet bract is rudimentary, whereas the glume subtending the flower is strongly developed. Because of the morphological diversity of the inflorescence, rachilla and glumes among the different subgroups in Ascolepis, Goetghebeur (1986) considered the possibility that Ascolepis is a complex of convergent lineages which developed a similar inflorescence Bauplan. A more thorough molecular investigation of Ascolepis is needed to test the monophyly of this taxon. Kyllinga Kyllinga is characterized by the combination of laterally flattened gynoecia, deciduous spikelets with a reduced number of flowers and capitate inflorescences. The close relationship of Kyllinga with Cyperus has always been acknowledged, and various authors have treated Kyllinga at the subgeneric level in Cyperus (e.g. Kükenthal, 1935–36; Haines & Lye, 1983). However, Kyllinga has always been considered as a homogeneous, natural entity, as illustrated by the fact that several authors have maintained Kyllinga as a separate genus whilst lumping Mariscus, Pycreus, Torulinium Desv. ex Ham. and Juncellus C.B.Clarke in Cyperus (Lye, 1972, 1982; Tucker, 1983). The monophyly of Kyllinga is confirmed by our results, where it is retrieved as a strongly supported clade (Fig. 3). As (1) Kyllinga is nested in C4 Cyperus, (2) capitate inflorescences with reduced, deciduous spikelets (i.e. pseudospikelets) are encountered in various lineages in C4 Cyperus, such as Cyperus section Bulbocaules (C.B.Clarke) Kük., Ascolepis, Lipocarpha and Remirea, and (3) laterally flattened gynoecia also occur in Pycreus and Queenslandiella, which are not immediately related, there are, in our opinion, no sufficient arguments to warrant generic status for Kyllinga. Lipocarpha Lipocarpha spp. generally have a highly specialized inflorescence consisting of a spike of highly reduced spikelets, with each spikelet, subtended by a bract, containing an abaxial prophyll and an adaxial glume subtending the flower. A few Lipocarpha spp. have © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172, 106–126 C4 CYPERUS PHYLOGENY (CYPERACEAE) lost the glume subtending the flower, although some rudiments of it remain visible (Goetghebeur & Van den Borre, 1989). These species were originally classified in a separate genus Hemicarpha, based on the reduction of the glume and the presence of a pseudolateral inflorescence (Nees von Esenbeck, 1834). As the type species of Hemicarpha Nees, H. isolepis Nees [accepted name: L. hemisphaerica (Roth) Goetgh.], does not show this reduction, Hemicarpha was synonymized with Lipocarpha (Goetghebeur & Van den Borre, 1989). Lipocarpha micrantha, which belongs to this group, is sister to all other Lipocarpha spp. studied, including Volkiella. Haines & Lye (1971, 1983) and Goetghebeur & Van den Borre (1989) considered Rikliella to represent a final reduction step of a Lipocarpha spikelet, in which the spikelet prophyll and glume subtending the flower are lost, resulting in a perfect pseudospikelet with flowers in the axil of the spikelet bracts. Hemicarpha was indicated as the transitional stage between Lipocarpha and Rikliella. Hemicarpha and Rikliella are no longer recognized at the generic level (Goetghebeur & Van den Borre, 1989; Govaerts et al., 2012). Our results place the two species of Rikliella (L. rehmannii and L. kernii) on a separate, strongly supported branch, and not as a specialized lineage of Lipocarpha. This questions previous interpretations of its inflorescence Bauplan. As in Alinula, Ascolepis and Kyllinga, we do not consider the presence of pseudospikelets sufficient to warrant generic status for Lipocarpha. Furthermore, in this study, Lipocarpha is found to be paraphyletic, containing Ascolepis and Volkiella. A more detailed study of Lipocarpha and Rikliella will be published elsewhere (K. Bauters et al., Ghent University, Gent, unpubl. data). Pycreus Pycreus is the largest segregate genus in C4 Cyperus. Furthermore, it is morphologically and ecologically diverse. The close relationship between Cyperus and Pycreus has never been doubted, as Pycreus only differs from Cyperus s.s. in its laterally flattened gynoecia. These gynoecia also occur in Kyllinga and Queenslandiella, which, in contrast with Pycreus, also have deciduous spikelets. The generic status of these taxa has always been controversial, and their status strongly correlated with the taxonomic value granted to laterally flattened gynoecia. From our results, it is evident that taxa with laterally flatttened gynoecia are not sister groups, and Kyllinga is strongly supported as a separate entity. Therefore, we can conclude that there have been multiple independent origins of lateral gynoecia in Cypereae. Our current molecular phylogenetic study includes species representing all 13 sections of Kükenthal 121 (1935–36). Although relationships between the different sections remain poorly resolved, several patterns require further attention. Pycreus is not monophyletic, as species that Kükenthal (1935–36) included in Cyperus sections Albomarginati, Polystachyi, nom. illeg., Pumili and Rhizomatosi are found in the main C4 Cyperus polytomy (Fig. 3). Many species of these sections share several plesiomorphic characters which also occur in C4 Cyperus, whereas species in the core Pycreus clade show more evolved character states (see Table 4). As in Kyllinga, we do not consider laterally flattened gynoecia sufficient to maintain Pycreus at the generic level, especially as it was resolved as polyphyletic in the present study. Queenslandiella Queenslandiella is a third taxon nested in the C4 Cyperus polytomy, which is characterized by laterally flattened gynoecia. It shares the open inflorescence with Pycreus (which is the plesiomorphic condition in C4 Cyperus). However, it has most often been considered to be related to Kyllinga, with which it shares deciduous spikelets, and keeled and multi-nerved glumes (Chermezon, 1919; Ballard, 1932, 1933; Koyama, 1976). The species has always been placed in or near Cyperus. However, even when included in Cyperus, it was most often retained in its own section or subgenus (Kern, 1974; Govindarajalu, 1975; Haines & Lye, 1983). As with the other specialized, short-lived and monotypic segregate lineages, Queenslandiella has also accumulated many peculiar characters which isolate it from the other C4 Cyperus taxa. These characters include the large proportions of glumes and nutlets compared with most other Cyperus spp., vegetative anatomy (Govindarajalu, 1975) and embryo type (Van der Veken, 1965). Several Cyperus spp. have been considered to be closely related to Queenslandiella, including C. soyauxii Boeckeler, which has similar deciduous spikelets with similar glumes and a similar embryo (Kükenthal, 1936; Van der Veken, 1965), but trimerous pistils (Goetghebeur, 1986). Lye (1983) described C. micromariscus Lye, which is only known from its type collection in Tanzania. This plant also has an open inflorescence with deciduous spikelets and laterally flattened pistils comparable with Queenslandiella, but differs in the small glumes and nutlets and different habit. Therefore, Lye (1983) assumed a different origin of this species and placed it in its own Cyperus subgenus Micromariscus Lye (Haines & Lye, 1983). The relationship of Queenslandiella to both C. soyauxii and C. micromariscus needs further confirmation. As for the segregates above, we do not consider the specialized characters of Queenslandiella sufficient to warrant recognition at the generic level. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172, 106–126 122 I. LARRIDON ET AL. Remirea Remirea is another monotypic entity with special adaptations to its coastal habitat. It is characterized by a capitate inflorescence with deciduous spikelets. Each spikelet contains a few empty glumes at the base and a corky rachilla which envelops the fruit. For these reasons, it had been classified among Rhynchosporeae (Fenzl, 1836; Bentham, 1883; Pax, 1888; Baillon, 1894; Clarke, 1901–02; Kükenthal, 1944; Haines & Lye, 1983). However, Nees von Esenbeck (1834) had already placed Remirea correctly in Cypereae. After Kunth (1837) gave a correct interpretation of the spikelet, this opinion was followed by Chermezon (1922), Kern (1958, 1974), Oteng-Yeboah (1975), Hooper (1983) and Goetghebeur (1986, 1998). Remirea is nested in the main C4 Cyperus polytomy, similar to Sphaerocyperus (Fig. 3), which also has empty glumes in the lower part of the spikelets. The relationship between these two taxa remains unclear. However, we do not believe empty glumes at the base of the spikelets to be sufficient as a generic character considering that other links with C4 Cyperus are clear. The corky rachilla is also observed in C. odoratus L. (formerly in the genus Torulinium), a species with multiple flowers in which the rachilla breaks up into individual segments. The affinity between Remirea and C. odoratus needs further investigation. Sphaerocyperus The deciduous spikelets of the monotypic Sphaerocyperus have six or seven distichously arranged glumes, only one of which bears a maturing nutlet. The sole species has variously been placed in Actinoschoenus Benth., Cyperus, Schoenus L. and Rhynchospora Vahl before it was described as a separate genus Sphaerocyperus (Lye, 1972). Like Remirea, we consider the empty glumes as insufficient to retain this taxon as a separate genus nested in a paraphyletic Cyperus with which it shares clear morphological affinities. Volkiella Volkiella is a rare monotypic taxon from southwestern Africa (mainly Namibia), and can be seen as an extremely specialized lineage adapted to psammophytic habitats. When described, Volkiella was considered to be intermediate between Cyperus and Lipocarpha (Merxmüller & Czech, 1953). The relationship with Lipocarpha was explained by the similar presence of the two ‘floral scales’ (‘hypogynen Skalen’), for which the correct interpretation is not yet clear, but the relationship with Cyperus was assumed on the basis of the distichous placement of the ‘Glumae’, which are, in fact, the spikelet bracts and thus not homologues of the glumes in Cyperus and other sedges. This initial interpretation was followed by Van der Veken (1965) and Raynal (1973), but was later correctly interpreted by Goetghebeur (1986, 1998). As in several other lineages, such as Ascolepis, Lipocarpha and Alinula, Volkiella shows highly derived pseudospikelets with a Bauplan comparable with that of Lipocarpha, possessing a spikelet bract, a spikelet prophyll, a proximal glume subtending the single flower and a spikelet bract larger than the glume. Peculiarly, in Volkiella, the spikelets are distichously arranged on the spike axis, whereas this position is spiral in all other C4 Cyperus spp. Although Volkiella shows an abundance of autapomorphic, derived characters which isolate it from all other C4 Cyperus spp., it is nested in Lipocarpha and should thus be sunk into Cyperus together with Lipocarpha. BASIS FOR A MODERN CLASSIFICATION OF CYPERUS From the current and previous molecular phylogenetic analyses, it is evident that the classification of Goetghebeur (1998) in Cypereae can no longer be upheld without accepting paraphyletic genera. Although most of the segregate genera are morphologically well circumscribed, the rapid diversification of the Cyperus clade has resulted in several nested paraphyletic entities (e.g. the genus Volkiella is nested in the genus Lipocarpha, which is nested in the group of C4 Cyperus spp. formerly known as Mariscus, and C4 Cyperus is, in turn, nested in C3 Cyperus). Moreover, most morphological characteristics used for the delimitation of the different genera related to Cyperus appear to have a high level of homoplasy in the Cyperus clade (e.g. spiral glumes, dorsiventrally flattened dimerous pistils, deciduous spikelets, pseudospikelets; Fig. 2). Subsequently, different combinations of the same sets of these morphological characters have been used to circumscribe most taxa. Larridon et al. (2011a, b) placed the C3 segregate genera in C3 Cyperus based on a well-resolved phylogenetic hypothesis combined with morphological, embryographical, ontogenetic and anatomical data. In that study, a classification for the Cyperus clade was suggested in which two subgenera were recognized. Although Cyperus subgenus Anosporum (Nees) C.B.Clarke (C3 Cyperus) is currently circumscribed as a paraphyletic entity (Larridon et al., 2011a, b), the single origin of the C4 photosynthetic pathway, a clear apomorphy for the C4 Cyperus clade, forms a sufficiently strong argument for the use of an evolutionary approach restricted to the subgeneric level in Cyperus. For the lower level classification, a cladistic approach was followed in circumscribing only monophyletic sections and, subsequently, the segregate genera will be included in existing or new sections in Cyperus. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172, 106–126 C4 CYPERUS PHYLOGENY (CYPERACEAE) This classification can be extended to include the different taxa of the C4 Cyperus clade (Cyperus subgenus Cyperus). However, as most segregate genera are nested in a hard polytomy with many species from different sections of Cyperus s.s. and as the lower level relationships in several segregate genera are poorly resolved, it is currently premature to build a new sectional classification for the largest part of Cyperus subgenus Cyperus. A joint international effort will be necessary to expand the current phylogenetic studies with more DNA markers and taxa. This will then serve as a basis for the growing modern classification of the giant genus Cyperus. CONCLUSIONS From the data presented here, we conclude that the Cyperus clade consists of a paraphyletic C3 Cyperus and a well-supported monophyletic C4 Cyperus clade. Nine segregate genera are nested in C4 Cyperus, i.e. Alinula, Ascolepis, Lipocarpha, Kyllinga, Pycreus, Queenslandiella, Remirea, Sphaerocyperus and Volkiella, most of which are monophyletic. Because they are nested in the Cyperus clade, and as a consequence of the multiple origins of the characters used to circumscribe them, we suggest that all nine C4 Cyperus segregate genera should be included in a more broadly circumscribed Cyperus. This study establishes a phylogenetic framework for future studies of the different C4 Cyperus sections and segregates, and for the taxonomic inclusion of the C4 segregate genera into Cyperus s.l. ACKNOWLEDGEMENTS We thank Pieter Asselman (Ghent University) for his helpful suggestions with the laboratory work and Andy Vierstraete (Ghent University) for performing the sequence reactions. We thank the Department of Environment and Natural Resources (DENR Region 8) for providing a collecting permit for Cyperaceae in the Philippines. We are grateful for the invitation of the East African Herbarium (National Museums of Kenya, Nairobi) and the Kenya Wildlife Service for the permission access to collect sedges in protected areas in Kenya and their help in 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 help in securing collecting permits (N°082/10/MEF/SG/DGF/DCB.SAP/SLRSE – Isabel Larridon) for Cyperaceae in Madagascar and for their help in organizing the expedition. This work was supported by research grants of the Special Research Fund (BO5622, BO7418, BOF, Ghent University, Belgium) and the Department of Biology, Ghent Uni- 123 versity, Belgium. The field expeditions were financed by travel grants of the Research Foundation – Flanders (FWO) and the Leopold III-Fund and with support of the Department of Biology, Ghent University, Belgium. 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