Plant Syst Evol (2009) 277:117–142 DOI 10.1007/s00606-008-0111-2 ORIGINAL ARTICLE Palynological diversity and major evolutionary trends in Cyperaceae Anne Nagels Æ A. Muthama Muasya Æ Suzy Huysmans Æ Alex Vrijdaghs Æ Erik Smets Æ Stefan Vinckier Received: 4 April 2008 / Accepted: 23 September 2008 / Published online: 14 November 2008 Ó Springer-Verlag 2008 Abstract Pollen and orbicule morphology of 84 species, representing 52 genera from all tribes and subfamilies are investigated, in order to assess the systematic value of palynological data and to determine palynological evolutionary trends in Cyperaceae. A total of 90% of the species are examined for the first time with scanning electron microscopy. Pollen grains of Cyperaceae are oblate spheroidal to perprolate in shape, inaperturate to polyporate with opercula or pontopercula on pori or colpi. We distinguished seven different sexine ornamentation patterns. Orbicules occur in all species investigated. Pollen morphological variation within Cyperaceae is considerable and includes dispersal unit; number, location and degree of differentiation of apertural zones; and sexine ornamentation patterns. In subfamily Mapanioideae both tribes can be characterized by palynological synapomorphies. However, in subfamily Cyperoideae, the observed pattern of variation does not fit the most recent molecular phylogeny due to A. Nagels (&)
S. Huysmans
A. Vrijdaghs
E. Smets Laboratory of Plant Systematics, Institute of Botany and Microbiology, Kasteelpark Arenberg 31, P.O. Box 2437, 3001 Louvain, Belgium e-mail: annenagels@gmail.com A. M. Muasya Botany Department, University of Cape Town, Private Bag X3, Rondebosch 7701, South Africa E. Smets National Herbarium of the Netherlands, Leiden University Branch, P.O. Box 9514, 2300 RA Leiden, The Netherlands S. Vinckier Center for Transgene Technology and Gene Therapy, Flanders Institute for Biotechnology, K.U. Leuven, Campus Gasthuisberg, Herestraat 49, 3000 Louvain, Belgium high levels of homoplasy and polymorphism in major pollen characters. Keywords Cyperaceae
Cyperoideae
Mapanioideae
Orbicules
Palynology
Pollen
Pseudomonads
SEM Introduction Cyperaceae are the third largest family in the monocotyledons consisting of 109 genera and approximately 5,500 species (Govaerts et al. 2007). Recent phylogenetic studies based on molecular data have suggested to maintain only two subfamilies within Cyperaceae: Mapanioideae and Cyperoideae (Simpson et al. 2008; Muasya et al. 2008). In this new delimitation Mapanioideae comprise two tribes: Hypolytreae and Chrysitricheae, while the circumscription of Cyperoideae changed considerably to include taxa previously placed in Caricoideae and Sclerioideae (sensu Goetghebeur 1998) (Table 1). The palynology of Cyperaceae attracted quite some attention in the past, mainly because of the occurence of an unusual type of simultaneous microsporogenesis, which leads to the formation of pseudomonads (Selling 1947; Davis 1966) or kryptotetrads (Erdtman 1952). After meiosis of the microspore mother cell, one of the four nuclei enlarges and occupies the centre of the coenocytic cell, while the other three nuclei migrate to the narrow apex where they are separated by septa and subsequently degenerate (Shah 1962; Dunbar 1973; Strandhede 1973; Furness and Rudall 1999; Brown and Lemmon 2000; Simpson et al. 2003). This unusual pattern of microsporogenesis is only known in one other unrelated group: tribe Styphelieae in Ericaceae (Smith-White 1959). 123 118 A. Nagels et al. Table 1 Comparison of Cyperaceae classifications according to Goetghebeur (1998) and Simpson et al. (2008) and Muasya et al. (2008) Goetghebeur (1998) Simpson et al. (2008) Muasya et al. (2008) Subfamily Mapanioideae 1. Hypolytreae (?Exocarya, Capitularina) 1. Hypolytreae 2. Chrysitricheae (?Hellmuthia) 2. Chrysitricheae (?Exocarya, Capitularina) Subfamily Cyperoideae 1. Scirpeae 1.Scirpeae 2. Fuireneae 2. Fuireneae 3. Eleocharideae 3. Eleocharideae 4. Abildgaardieae 4. Abildgaardieae (?Arthrostylis, Actinoschoenus, Trachystylis) 5. Cypereae 5. Cypereae (?Hellmuthia) 6. Dulichieae 6. Dulichieae 7. Schoeneae 7. Schoeneae (?Arthrostylis, Actinoschoenus, Trachystylis, 8. Rhynchosporeae Rhynchospora) Subfamily Sclerioideae 1. Cryptangieae (-Exochogyne) 9. Cryptangieae (?Exochogyne) orbicules are reported in the Centrolepidaceae (Rowley and Dunbar 1996) and the Poaceae (e.g. El-Ghazaly and Jensen 1987; Vinckier and Smets 2001). In Cyperaceae, orbicules have been reported in Carex wallichiana Presc. (Shah 1962), Eleocharis palustris (L.) Roem. and Schult. (Carniel, 1971; Dunbar 1973), Hellmuthia Steud., Mapania Aubl., Capitularina Kern, Exocarya Benth. and Diplasia Rich. (Simpson et al. 2003; Vrijdaghs et al. 2006). Several authors have recognized different pollen types in Cyperaceae mainly based on pollen shape, pollen size, and number and type of apertures (see Table 2 for an overview). However, the number of species investigated with scanning electron microscope (SEM) is very limited and the recent availability of a Cyperaceae phylogeny offers great potential for evolutionary interpretations of the data. The major aim of the present study is to provide a palynological overview at family level in order to document with scanning electron microscopy, the pollen and orbicule morphology for a relevant sampling of all major clades. Our data are then used to assess the taxonomically useful characters, and to determine palynological evolutionary trends in Cyperaceae sensu Muasya et al. (2008). 2. Trilepideae 10. Trilepideae 3. Sclerieae 11. Sclerieae 4. Bisboeckelereae 12. Bisboeckelereae Materials and methods 13. Cariceae Sampling Subfamily Caricoideae 1. Cariceae Mapania-type pollen (sensu Simpson et al. 2003) differs in several aspects from the common wind pollinated, pyriform pseudomonads with one to six apertures (Furness and Rudall 1999; Van Wichelen et al. 1999). Mapania-type pollen, recorded in five genera of the mapanioid tribe Hypolytreae (Simpson et al. 2003), is spheroidal with only one distal aperture and the pollen grains appear to be sticky, suggesting animal pollination. Erdtman (1952) and Haines and Lye (1983) suggested that mapanioid pollen grains are not pseudomonads, and Koyama (1961) considered them as tetrads. Moreover, in contrast to the peripheral pollen arrangement in Cyperaceae, Mapaniatype pollen grains are centrally arranged in the locules (Kirpes et al. 1996; Simpson et al. 2003). In the anthers of seed plants, tiny (±1 lm) sporopollenin granules might occur on the radial and inner tangential walls of secretory tapetum cells. These granules are sometimes in close contact with the pollen grains and are called orbicules (Erdtman et al. 1961) or Ubisch bodies (Rowley 1962, probably in reminiscence and in honour to Kosmath 1927 and von Ubisch 1927). The distribution, morphology and function of orbicules in angiosperms have been reviewed by Huysmans et al. (1998, 2000). In Poales, 123 We sampled among all major clades that represent recently recognized taxonomic groups in Cyperaceae (Muasya et al. 2008). A total of 84 species representing 46 genera in Cyperoideae and six genera in Mapanioideae were investigated (see Appendix 1). We mainly used stamens from flowers fixed in FAA (70% ethanol, acetic acid, 40% formaldehyde, 90:5:5), which were collected in the field or in botanical gardens. We supplemented our sampling with herbarium material from the collections in BR and GENT, and living material. Preparation techniques were optimized for each category of sample: fixed, herbarium or fresh material, respectively. SEM observations Stamens of fixed flowers were dissected in 70% ethanol under a Wild M3 stereo microscope (Leica Microsystems AG, Wetzlar, Germany) and preserved in 70% ethanol. The dissected anthers were rinsed twice in 70% ethanol for 5 min and in a mixture (1:1) of 70% ethanol and DMM (dimethoxymethane) for 5 min. Subsequently the anthers were transferred to 100% DMM for 20 min, prior to critical-point drying (Balzers CPD 030). Palynological evolutionary trends in Cyperaceae 119 Table 2 Literature overview of previous palynological studies in Cyperaceae Reference Typology Diagnostic characters LM/SEM/ TEM Sampling Wodehouse (1935) Basic type Ovoid to pyriform, monoaperturate, granulate sexine LM 3 species, 2 genera Erdtman (1952, 1966, 1971) Carex-type Elongate, one distal porus ? three lateral pori/ colpi (majority of Cyperaceae) LM 65 species, 35 genera Type two Spheroidal, monoaperturate (Mapania and related genera) Two pollen types Pollen type identification key to native New Zealand Cyperaceae with two pollen types based on pollen shape (pyriform or paraboloid and spheroidal) LM 26 species, 13 genera Cranwell (1953) Shah (1962) Monocolpate, smooth exine LM 7 species, 5 genera Huang and Chung (1971) Identification key to 12 genera LM 102 species, 12 genera LM 31 species, 12 genera LM/SEM ? Padhye and Makde (1980) Cyperus-type Ellipsoid, one distal colpus, granulate/foveolate sexine (most frequent) Carex-type As defined by Erdtman (1966) Haines and Lye (1983) Mapania-type Spheroidal, monoporate (members of Hypolytreae) Carex-type One distal aperture ? three lateral apertures, psilate sexine Cyperus longus-type Pantoaperturate with one distal pore and 4–6 colpi LM/SEM Cyperus michelianustype Pantoaperturate with several pores Cladium mariscus-type Inaperturate Fernandez (1987) 19 species Schoenus nigricans-type Pantoaperturate with one distal pore and 4–6 colpi, but dimensions different from first type Bruhl (1995) Van Wichelen et al. (1999) Carex flacca-type Pantoaperturate with one distal pore and 4–5 colpi Carex hallerana-type Pantoaperturate with one distal pore and 4–5 equatorial pores Type one 1–6 apertures (most frequent) Type two [6 apertures (genera Baumea, Machaerina and Tricostularia) Type one Spheroidal/oblong ovoid, one distal ulcus (Mapanioideae) Type two Broadly obovoid, one distal ulcus ? three lateral pori (Sclerioideae and Caricoideae) LM 122 genera (according to Bruhl 1995) LM/SEM 30 species, 27 genera Different types Cyperoideae Moar and Wilmshurst (2003) 12 groups Pollen identification key to 15 genera of New Zealand Cyperaceae, mainly based on grain shape and aperture type LM 38 species, 15 genera Simpson et al. (2003) Mapania-type Pollen development, shape 9 species, 7 genera Pseudomonad-type Pollen development, shape LM/SEM/ TEM Herbarium material was treated with a modified enzyme-based method according to Schols et al. (2004a) slightly adjusted to the fragile nature of Cyperaceae pollen. The stamens were rehydrated in Na–cacodylate buffer (pH 7.3, 0.05 M) for ca. 36 h. Subsequently, the anthers were fixed in 2.5% glutaraldehyde in Na–cacodylate buffer for minimum 8 h and rinsed two times during 30 min in Na–cacodylate buffer. Next, the anthers were transferred to a solution of 8 ml Na–cacodylatebuffer, 0.03 g cellulase (Fluka Biochemica, cellulase of Trichoderma viride) and 0.1 g pectinase (Fluka Biochemica, pectinase of Aspergillus niger) for 24 h. The test tubes with the material were shaken with a shaking device (VEL GFL 3015). The material was then 123 120 dehydrated in a graded ethanol–DMM series and criticalpoint dried. Fresh material was immediately fixed in glutaraldehyde (2.5% in Na–cacodylate buffer) for minimum 8 h. Next, it was rinsed in buffer (2 9 30 min) and transferred to a graded buffer–ethanol series (30% ethanol, 50% ethanol, 70% ethanol). Subsequently, the anthers were transferred to a 1:1 mixture of ethanol and DMM for 5 min. Prior to critical-point drying, the material was moved to 100% DMM for 20 min. The dried anthers were mounted on stubs with double-sided adhesive carbon tape followed by the dissection of the anthers to make the pollen grains visible. The stubs were coated with gold with a SPIMODULETM Sputter Coater (SPI Supplies, West Chester, PA, USA). Images were obtained with a SEM (JEOL JSM6360). Comparative size measurements of orbicules and pollen were made on SEM-micrographs using Carnoy 2.0 (Schols et al. 2002). Only completely rehydrated pollen grains were measured. For each species investigated, 50 orbicules were measured and mean values and standard deviations were calculated. Terminology follows Punt et al. (2007) unless stated otherwise. Coding In total 11 characters were scored for three outgroups and 52 Cyperaceae genera (Appendix 2 and C). Three quantitative and continuous characters (polar axis, P/E ratio and orbicule size) were coded using Thiele’s gap weighting method as implemented by MorphoCode (Schols et al. 2004b), with n = 5, where n is the number of allowed character states in the Thiele formula (Thiele 1993). Noncontinuous characters were treated as unordered multistate characters (pollen shape, distinctness of apertures, presence or absence of distal aperture, lateral aperture number, lateral aperture shape, sexine ornamentation, orbicule shape and orbicule ornamentation) and were assigned the default weight of 1. A. Nagels et al. characters was negligible, so we decided not to show these results here and to use the molecular tree to optimize the pollen characters. Results The 84 species studied show considerable variation in both pollen and orbicule morphology. The palynological characters of the species studied are described below and summarized in Table 3 (pollen characteristics) and Table 4 (orbicule characteristics). Polarity and symmetry Pyriform, subprolate, prolate or perprolate pollen grains are heteropolar in shape and apertural system. These pollen grains have a broad distal pole and a narrower proximal one (e.g. Fig. 1a). At the distal pole an aperture is present whereas no aperture is observed at the proximal pole (e.g. Fig. 1B). Spheroidal pollen grains are isopolar in shape (Fig. 1c–g) but heteropolar in apertural system, with only a single aperture at one of the poles (e.g. Fig. 1c). The heteropolarity in pollen shape and apertural system of the pollen grains studied, is linked to the typical microsporogenesis of the pseudomonads and the arrangement of the pollen grains during development in the anther locules (Fig. 1v). Pollen size Pollen size varies significantly within many species investigated (Table 3). The equatorial diameter (E) ranges from 12.9 lm in Kyllinga sp. (Fig. 1d) to 30.72 lm in Cladium mariscus (Fig. 1a) and the polar axis (P) ranges from 12.89 lm in Lipocarpha nana (Fig. 1e) to 52.69 lm in Chrysitrix dodii (Fig. 1b). The majority of the species investigated have small to medium sized pollen with an equatorial diameter in the 15–30 lm range and a polar axis in the 20–40 lm range (Table 3). Character optimization Pollen shape Characters were optimized on a strict consensus tree obtained from a parsimony analysis of rbcL and trnL-F sequence data of all genera investigated (data from Muasya et al. 2008) using MacClade 4 (Maddison and Maddison 2001). This particular analysis was selected since it represents the most thoroughly sampled molecular analysis of the entire family available today. Adding the palynological characters (Appendix 3: Table 5) to the rbcL ? trnL-F data matrix produced a strict consensus tree with a slightly less resolved topology compared to the strict consensus tree based on molecular data only (Muasya et al. 2008). The impact on support values by adding the palynological 123 Pollen shape in equatorial view (P/E) ranges from suboblate (Capeobolus brevicaulis; Fig. 1f) to perprolate (Ficinia zeyherii; Fig. 1j) but is in general spheroidal (Fig. 1c–g) or (sub)prolate (Fig. 1h–i). Spheroidal pollen grains are thought to be dominant in, and restricted to, subfamily Mapanioideae. However, we also observed spheroidal pollen in 21 Cyperoideae species belonging to 16 genera (Table 3). On the other hand, (sub)prolate pollen grains are found in three mapanoid species (Figs. 1b, 2j–k). Pollen shape based on the P/E ratio is inadequate to describe the pear-shaped pollen grains in some genera (Becquerelia, Species P ± SD E ± SD (lm) (lm) Shape P/E Apertures Sexine # ? Shape Ornamentation Distinctness Ornamentation Figure Subfamily Cyperoideae Abildgaardieae Arthrostylis aphylla 21.46 ± 1.38 19.26 ± 2.10 1.12 ± 0.14 Spheroidal 1 distal ulcus, 6 lateral pori Granules, perf, me PO Microechinate, perf 1p–q, 2n Bulbostylis hispidula 31.14 ± 0.76 25.38 ± 0.62 1.27 ± 0.12 Subprolate 1 distal ulcus, 5 lateral colpi/pori Fossulate/neg. microreticulate PO Granulate, perf 1h Fimbristylis complanata 17.64 ± 3.10 15.15 ± 1.00 1.16 ± 0.12 Subprolate No apertures observed – – Neg. microreticulate, granulate 2a No apertures observed – – Neg. microreticulate, granulate 2b 3k Fimbristylis xyridis 25.95 19.74 1.32 Subprolate Palynological evolutionary trends in Cyperaceae Table 3 Summary of major pollen morphological characters for all species studied Bisboeckelereae 26.22 ± 0.21 18.70 ± 0.38 1.43 ± 0.01 Prolate 1 distal ulcus, 3 lateral pori Neg. microreticulate, mgr, perf PO Granulate, perf Carex capitata 28.94 ± 0.72 25.43 ± 1.09 1.15 ± 0.16 Subprolate 1 distal ulcus, 4 lateral pori Fossulate PO Granulate, perf Carex elata 35.93 ± 0.63 22.58 ± 1.09 1.59 ± 0.05 Prolate 1 distal ulcus, 4 lateral pori Neg. microreticulate PO 4 ap zones (pori) observed Neg. microreticulate, mgr, perf ? Granulate, perf – 1 distal ulcus, 4 lateral ap zones (pori) – Granules, mgr, perf PO Granulate, perf 3b – – Granulate, perf – 1 distal ulcus, 4 lateral ap zones (pori) Neg. microreticulate ? PO Neg.-microreticulaat/ fossulate, mgr, perf – mgr, perf Becqerelia cymosa* Cariceae – – – Fossulate 1i granulate, perf Carex monostachya* Collapsed Kobresia myosuroides 27.76 ± 1.53 Schoenoxiphium lehmannii Collapsed Schoenoxiphium sparteum – Uncinia rubra Collapsed – – – – Fossulate, PO Granulate, perf – Everardia montana* Collapsed – – – 1 distal ulcus, PO Granulate, perf – 4 lateral pori Neg. microreticulate, mgr, perf Exochogyne amazonica* 23.77 ± 0.96 Polyporate ? Fossulate PO Metareticulate ?, granulate, perf 3i Lagenocarpus rigidus subsp. rigidus* – 3 pori observed Granules, mgr, perf PO Granulate, perf – 27.01 ± 0.82 1.03 ± 0.05 Spheroidal – – – 20.51 – – Cryptangieae 23.62 ± 0.41 1.03 ± 0.06 Spheroidal – – 121 123 – 122 123 Table 3 continued Species P ± SD E ± SD (lm) (lm) Shape P/E Apertures Sexine Figure # ? Shape Ornamentation Distinctness Ornamentation 1 distal ulcus, 4 lateral pori/colpi ? Neg. microreticulate, perf, mgr PO Fossulate/granulate, perf – 20.03 ± 1.54 1.11 ± 0.00 Spheroidal 1 distal ulcus, 4 lateral pori Granules O Granulate, perf – Cypereae Courtoisina assimilis Collapsed Cyperus alternifolius** 22.17 ± 1.63 – – – Cyperus articulatus 22.71 ± 1.66 16.07 ± 1.55 1.42 ± 0.14 Prolate 1 distal ulcus, 4–5 lateral pori/colpi Granules, perf O Granulate, perf – Cyperus dubius 18.51 ± 1.30 18.68 ± 0.54 0.99 ± 0.09 Spheroidal 1 distal ulcus Granules, me O 2s, 3f Cyperus hemisphaericus* 22.57 ± 2.39 17.94 ± 1.74 1.27 ± 0.24 Prolate 4–6 lateral pori/colpi 1 distal ulcus, 5 lateral colpi Psilate, rugulate/ granulate Granules, me O Granulate/ microechinate, perf – Cyperus laevigatus 27.04 ± 0.61 20.77 ± 1.33 1.30 ± 0.05 Subprolate 1 distal ulcus Granules, me O Granulate, perf (f) – Cyperus rotundus 28.09 ± 1.76 21.49 ± 1.01 1.31 ± 0.09 Subprolate 1 distal ulcus Fossulate, mgr O Granulate, perf (f) 1s, 2u 4–5 lateral colpi 4–5 lateral pori/colpi 27.11 ± 3 17.35 ± 0.58 1.57 ± 0.21 Prolate 1 distal ulcus, 4 lateral pori Granules, perf O Granulate, perf 2r Ficinia bulbosa* 30.57 ± 2.85 21.89 ± 1.25 1.40 ± 0.10 Prolate 1 distal ulcus, 5 lateral pori Granules, perf O Granulate, perf – Ficinia capitellum Collapsed – – – 1 distal ulcus, 4–5 lateral colpi Doughnut –shaped granules, perf O Granulate, perf – Ficinia dunensis Collapsed – – – 1 distal ulcus, 4 lateral colpi Granules O Granulate, perf – Ficinia gracilis 26.41 20.68 1.28 Subprolate 1 distal ulcus, 3–4 lateral pori Granules PO Granulate, perf – Ficinia minutiflora 19.37 ± 0.28 18.40 ± 0.66 1.05 ± 0.03 Spheroidal 1 distal ulcus, 4 lateral pori Doughnut-shaped granules O Microreticulate, mgr 3g Ficinia polystachya 41.93 ± 0.48 22.66 ± 1.37 1.85 ± 0.10 Prolate 1 distal ulcus, 6 lateral colpi Doughnut-shaped granules O Granulate, perf – Ficinia radiata 23.19 ± 0.70 22.2 9 ± 1.81 1.04 ± 0.05 Spheroidal 1 distal ulcus, 5 lateral pori Granules, perf, mgr PO Granulate, perf – Ficinia tristachya Collapsed Ficinia zeyheri 33.81 ± 1.90 18.27 ± 1.13 Hellmuthia membranacea Isolepis antarctica 33.66 ± 1.54 28.08 ± 0.61 1.62 ± 0.11 Prolate 23.83 ± 0.85 17.91 ± 0.72 1.33 ± 0.08 (Sub)prolate – – – 2.3 ± 0.10 Perprolate – Granules, perf, mgr O Granulate, perf – 1 distal ulcus, 6 lateral colpi Doughnut-shaped granules PO Granulate, perf 1j 1 distal ulcus, 6 lateral pori 1 distal sulcus, 5–6 lateral pori/colpi Granules, me, perf O 2q Granules, mgr, perf O Fossulate/granulate, perf Granulate, perf 2t A. Nagels et al. Ficinia brevifolia Species P ± SD E ± SD (lm) (lm) Shape P/E Apertures Sexine # ? Shape Ornamentation Distinctness Ornamentation Figure Isolepis digitata 21.47 17.40 1.23 Subprolate 1 distal ulcus, 4 lateral pori Granules, perf O Granulate, perf 1v Isolepis prolifera 25.79 21.77 1.18 Subprolate 1 distal ulcus, 4–5 lateral pori Granules, mgr, perf O Granulate, perf – Isolepis setacea 27.81 22.64 1.23 Subprolate 1 distal ap zone, 5 Fossulate, mgr, perf lateral ap zones (pori) PO Fossulate/granulate, perf (f) 1l Kyllinga eximia 21.00 ± 1.99 20.44 ± 1.44 1.03 ± 0.06 Spheroidal 5 pori/colpi Granules, me O Microechinate, perf 1g Kyllinga flava 21.82 ± 0.82 16.39 ± 0.71 1.33 ± 0.04 (Sub)prolate 1 distal (s)ulcus, 4–5 lateral pori/colpi Granules, me O Microechinate, perf (f) 1m, r, 3a Kyllinga polyphylla** 22.44 ± 0.29 19.19 ± 0.07 1.17 ± 0.02 Subprolate 1 distal ulcus, 4 lateral colpi Granules, me O Microechinate – Kyllinga sp. 17.34 ± 1.28 12.93 ± 0.39 1.41 ± 0.15 Prolate 1 distal ulcus, 4 lateral colpi Granules, me O Microechinate, perf 1d Kyllingiella polyphylla 17.47 ± 1.51 15.96 ± 1.83 1.10 ± 0.06 Spheroidal 1 distal ulcus, 5–6–7 pori Granules, me, perf O Microechinate, perf – Lipocarpha nana 12.89 ± 0.47 13.24 ± 3.48 0.98 ± 0.13 Spheroidal 1 distal ulcus, 4 lateral pori Granules, me, perf O Microechinate, perf 1e, 2m Lipocarpha rehmannii 17.19 ± 1.18 25.06 ± .95 1 distal ulcus, 4–5 lateral pori Granules, me, perf O Microechinate, perf – Oxycaryum cubense 23.79 ± 2.51 20.73 ± 2.20 1.15 ± 0.10 Subprolate 1 distal ulcus, 5 lateral colpi Granules, mgr, perf PO Granulate, perf – Pycreus flavescens** 26.58 21.83 1.22 Subprolate 1 distal ulcus, 5 lateral colpi Granules, mgr (f) O Granulate, perf – Pycreus mundtii 18.27 16.79 1.09 Spheroidal 1 distal ulcus, 5 lateral colpi Granules, mgr O Fossulate/granulate, perf – 1 distal sulcus, 5 lateral Granules pori O Granulate, perf 4b 1 distal ulcus, min. 1 lateral porus PO Granulate, perf – Pycreus sanguinolentus 20.07 ± 1.83 Scirpoides holoschoenus** Collapsed 1.04 ± 0.14 Spheroidal 19.90 ± 1.68 1.02 ± 0.18 Spheroidal – – – Fossulate, mgr, perf Palynological evolutionary trends in Cyperaceae Table 3 continued Dulichieae Dulichium arundinaceum Eleocharideae Eleocharis acutangula 20.17 ± 0.46 1.29 ± 0.14 Subprolate 1 distal sulcus, 4 lateral Neg. microreticulate, ap zones ? perf, mgr PO Fossulate, granulate, perf (f) – 35.25 ± 3.26 23.62 ± 3.80 1.52 ± 0.27 Prolate No apertures observed – – Neg. microreticulate, granulate, perf 2p, 3d 37.54 ± 3.80 28.40 ± 0.59 1.32 ± 0.11 Subprolate 1 distal ulcus, 5 lateral colpi Fossulate, perf, mgr PO Granulate, perf 2o 123 123 Eleocharis palustris 26.15 ± 3.47 124 123 Table 3 continued Species P ± SD E ± SD (lm) (lm) Shape P/E Apertures Sexine Figure # ? Shape Ornamentation Distinctness Ornamentation 1 distal ulcus, 4 lateral pori Neg. microreticulate, mgr, perf PO Granulate, perf – 1 distal ulcus, 4 lateral pori Fossulate, mgr, perf PO Fossulate/granulate, perf 3c Fuireneae Fuirena abnormalis 30.52 ± 3.79 25.49 ± 3.07 1.21 ± 0.21 Subprolate Fuirena leptostachya 23.64 Pseudoschoenus inanis 30.85 ± 5.06 23.70 ± 3.87 1.33 ± 0.33 (Sub)prolate 1 distal ulcus, 5 lateral pori/colpi Neg. microreticulate, granules, mgr, perf PO Granulate, perf – Schoenoplectus senegalensis 29.22 ± 5.43 26.99 ± 3.75 1.08 ± 0.06 Spheroidal 1 distal ulcus; 4 lateral pori Fossulate, mgr, perf PO Granulate, perf 1n, o 22.52 ± 2.68 15.56 ± 0.68 1.45 ± 0.20 Prolate No apertures observed – – Neg. microreticulate 2c 21.50 ± 0.95 22.35 ± 0.88 0.96 ± 0.04 Spheroidal Polyporate Granules, perf, mgr PO Fossulate/granulate, perf 2d Suboblate/ spheroidal Polyporate Granules, perf, mgr PO Fossulate/granulate, perf 1f 20.96 1.13 Spheroidal Rhynchosporeae Rhynchospora sp. Schoeneae Baumea rubiginosa Capeobolus brevicaulis 20.75 23.69 0.88 Caustis flexuosa* Collapsed – – – – – – Granulate, perf – Caustis recurvata* Collapsed – – – 1 distal ulcus, 4 lateral ap zones observed Neg. microreticulate, mgr, perf PO Neg. microreticulate, mgr, perf – Cladium mariscus 42.00 ± 4.62 30.72 ± 2.15 1.38 ± 0.10 Prolate 0–1 distal (s)ulcus, 0–4 Fossulate, perf lateral pori/colpi PO Granulate, perf 1a, 2f, g Costularia humbertii* 33.11 ± 0.38 26.35 ± 1.14 1.26 ± 0.04 Subprolate 1 distal ulcus, min. 5 lateral colpi Fossulate, mgr, perf O Granulate, mgr – Gahnia lanigera* 26.83 ± 3.84 25.03 ± 1.42 1.08 ± 0.21 Spheroidal Polyaperturate Granules, mgr, perf PO Fossulate/granulate, perf – Machaerina flexuosa* – Schoenus nigricans* Collapsed Tetraria compar* 45.85 ± 5.96 Trianoptiles solitaria 21.6 23,29 – – 3 pori observed Granules, perf PO Granulate, perf – – – – 1 distal ulcus, 4 lateral colpi Neg. microreticulate, mgr, perf PO Granulate, perf 1t 25.90 ± 0.91 1.77 ± 0.21 Prolate 20.39 1.06 Spheroidal 1 distal ulcus, Fossulate, mgr, perf PO Granulate, perf – 1 distal ulcus, min. 3 lateral pori/colpi Fossulate, mgr,perf PO Granulate, perf – Scirpeae 34.89 ± 1.92 24.96 ± 2.01 1.40 ± 0.08 Prolate 1 distal ulcus, 4 lateral ap zones (pori) Fossulate, mgr, perf PO Granulate, perf – Eriophorum latifolium 28.05 ± 0.44 19.99 ± 1.97 1.41 ± 0.14 Prolate 1 distal ulcus, 4 lateral ap zones (colpi) Fossulate, mgr, perf PO Granulate, perf 2l A. Nagels et al. Amphiscirpus nevadensis* Species P ± SD E ± SD (lm) (lm) Phylloscirpus acaulis* Collapsed Scirpus sylvaticus* 24.80 ± 1.94 Trichophorum alpinum* Collapsed – Shape P/E – – 21.26 1.14 Sexine Figure # ? Shape Ornamentation Distinctness Ornamentation 1 distal ulcus, 4 lateral ap zones (pori) Fossulate, mgr, perf PO Neg.-microreticulate/ granulate, perf – 1 distal ulcus, 4 lateral ap zones (pori) Fossulate, mgr, perf PO Neg.-microreticulaat/ fossulate, mgr, perf – – 1 distal (s)ulcus, 4 pori Granules, mgr, perf PO Granulate, perf – Spheroidal/ Subprolate 1 distal ulcus, 4 poorly visible apertures Fossulate, mgr, perf PO Granulate, perf 2e 1 distal ulcus, 3–4 Neg. microreticulate, lateral ap zones (pori) perf (f) PO Granulate, perf – – – – Granulate, perf – 1 distal ulcus Granules, me, perf O Fossulate/rugulate, perf, 2h, 3e mgr PO Rugulate, perf, mgr, me – – 21.72 ± 1.74 1.15 ± 0.09 Subprolate – Apertures Sclerieae Scleria rugosa 24.32 Scleria terrestris** 28.35 ± 0.40 25.70 ± 1.44 1.13 ± 0.11 Spheroidal Palynological evolutionary trends in Cyperaceae Table 3 continued Trilepideae Afrotrilepis pilosa* Collapsed Coleochloa setifera 25.64 ± 1.63 – – – 21.24 ± 1.41 1.22 ± 0.15 Subprolate Subfamily Mapanioideae Chrysitricheae Chorizandra cymbaria* Few material Chorizandra enodis* 46.12 ± 5.46 – – – 28.79 ± 2.80 1.60 ± 0.14 Prolate 1 distal ulcus, no lateral Granules, me, perf apertures observed 1 distal ulcus, 4 lateral pori Fossulate/granules, mgr, PO perf Granulate, perf 2j Chrysitrix dodii 52.69 ± 1.05 29.08 ± 1.59 1.82 ± 0.13 Prolate 1 distal ulcus Granules, mgr O Granulate, perf 1b Lepironia articulata* 28.29 ± 2.86 23.18 ± 0.96 1.23 ± 0.17 Subprolate 1 distal ulcus, 0–4 lateral pori observed Granules PO Granulate, perf 2k Hypolytreae Diplasia karatifolia* Hypolytrum jenmanii subsp. jenmanii* 18.08 ± 1.19 18.01 ± 2.09 1.01 ± 0.14 Spheroidal 1 distal ulcus – – Microreticulate, mgr 1k 17.35 ± 1.92 15.85 ± 1.48 1.10 ± 0.14 Spheroidal 1 distal ulcus Granules – Microreticulate 3h Mapania cuatrecasasii* 18.56 ± 1.13 18.32 ± 0.69 1.01 ± 0.06 Spheroidal 1 distal ulcus Granules O Fossulate, perforate 2i 16.02 ± 1.35 15.26 ± 0.99 1.05 ± 0.11 Spheroidal 1 distal ulcus Granules, me – Microreticulate/ microechinate 1c, 3j Mapania linderi* Species are listed alphabetically; fixed material (without asterisk), herbarium material is indicated with an asterisk and living material with double asterisks. # aperture number, ap apertural, E equatorial axis, f few, mgr microgranules, me microechinae, neg. negative, O operculum, P polar axis, perf perforations, PO pontoperculum 125 123 126 A. Nagels et al. Table 4 Summary of major morphological orbicule characters for all species studied Species Diameter (lm) ± SD Shape Ornamentation Remark Figure 0.59 ± 0.11 Ang Sm – 4g Subfamily Cyperoideae Abildgaardieae Arthrostylis aphylla Bulbostylis hispidula 0.40 ± 0.07 Irr Mgr Fimbristylis complanata 0.29 ± 0.05 Irr Mgr – – – Fimbristylis xyridis 0.56 ± 0.07 Irr Me – – 0.43 ± 0.06 Sph Mgr – – Bisboeckelereae Becquerelia cymosa* Cariceae Carex capitata 0.45 ± 0.09 Irr Mgr – – Carex elata Carex monostachya* 0.56 ± 0.09 0.47 ± 0.10 Sph Sph Sm Mgr – – – – Kobresia myosuroides 0.41 ± 0.09 Ang Mgr – – Schoenoxiphium lehmannii 0.30 ± 0.06 Irr Mgr – – Schoenoxiphium sparteum 0.35 ± 0.07 Sph Mgr Em, aggr – Uncinia rubra 0.39 ± 0.10 Irr Mgr Aggr – Cryptangieae Exochogyne amazonica* 0.46 ± 0.07 Irr Mgr, me – – Lagenocarpus rigidus subsp. rigidus* 0.60 ± 0.13 Sph Me – – Cypereae Courtoisina assimilis 0.41 ± 0.08 Irr Me Aggr – Cyperus alternifolius** 0.52 ± 0.10 Sph Mgr (f) – – – Cyperus articulatus 0.44 ± 0.08 Sph Me, mgr Cyperus dubius 0.62 ± 0.12 Irr Mgr Cyperus haspan 0.86 ± 0.18 Sph Me – – Em – Cyperus hemispaericus* 0.46 ± 0.09 Ang Mgr, perf (f) – – Cyperus laevigatus Cyperus rotundus 0.72 ± 0.15 0.73 ± 0.16 Sph Irr Me Me – Aggr – 4a Ficinia brevifolia 0.58 ± 0.09 Sph – – – Ficinia bulbosa* 0.56 ± 0.10 Irr Mgr Thr – Ficinia capitellum 0.44 ± 0.07 Sph Sm – – Ficinia dunensis 0.59 ± 0.11 Sph Mgr – – Ficinia gracilis 0.69 ± 0.10 Sph Sm Aggr – Ficinia minutiflora 0.74 ± 0.14 Sph Mgr – – Ficinia polystachya 0.53 ± 0.07 Ang Mgr – Ficinia radiata 0.64 ± 0.14 Sph Sm, perf (f) Aggr 4c Ficinia tristachya 0.32 ± 0.06 Irr Mgr – – Ficinia zeyheri 0.66 ± 0.10 Sph Mgr (f) Aggr – Hellmuthia membranacea 0.53 ± 0.08 Irr Mgr – – Isolepis antarctica 0.42 ± 0.09 Irr Me – – Isolepis digitata 0.46 ± 0.08 Sph Mgr – – Isolepis prolifera Isolepis setaceae 0.47 ± 0.07 0.46 ± 0.09 Irr Irr Mgr Me – – – – Kyllinga eximia 1.28 ± 0.25 Irr Me Aggr – Kyllinga flava 0.65 ± 0.13 Irr Me Spheres 4l Kyllinga polyphylla** 0.69 ± 0.13 Irr Me Aggr – Kyllinga sp. 0.76 ± 0.15 Irr Me – 4i Kyllingiella polyphylla 0.75 ± 0.15 Irr Me – – 123 Palynological evolutionary trends in Cyperaceae 127 Table 4 continued Species Diameter (lm) ± SD Shape Ornamentation Remark Figure Lipocarpha nana 0.64 ± 0.12 Irr Me – – Lipocarpha rehmannii 0.58 ± 0.09 Irr Me – – Oxycarium cubense 0.37 ± 0.08 Irr Me – – Pycreus flavescens** 0.85 ± 0.15 Irr Me – – Pycreus mundtii 0.20 ± 0.09 Irr Mgr, me – 4f Pycreus sanguinolentus 0.53 ± 0.10 Irr Me – 4b Scirpoides holoschoenus** 0.45 ± 0.08 Irr Mgr Aggr – – Irr – Aggr – Dulichieae Dulichium arundinaceum Eleocharideae Eleocharis acutangula 0.36 ± 0.07 Irr – Ret aggr 4d, e Eleocharis palustris 0.24 ± 0.04 Irr Mgr, perf Aggr – Fuireneae Fuirena abnormalis 0.44 ± 0.05 Irr Mgr – – Fuirena leptostachya 0.33 ± 0.08 Irr Mgr Aggr – Pseudoschoenus inanis 0.49 ± 0.09 Irr Mgr – – Schoenoplectus senegalensis 0.46 ± 0.08 Ang Sm – – 0.37 ± 0.09 Irr Mgr – – Rhynchosporeae Rhynchospora sp. Schoeneae Baumea rubiginosa 0.97 ± 0.21 Sph Sm – – Capeobolus brevicaulis 0.62 ± 0.12 Irr Mgr Em – Caustis flexuosa* 0.57 ± 0.12 Irr Mgr Aggr – Caustis recurvata* 0.53 ± 0.10 Irr Me Thr – Cladium mariscus 0.58 ± 0.10 Irr Mgr – – Costularia humbertii* 0.47 ± 0.07 Irr Mgr Thr – Gahnia lanigera* 0.50 ± 0.07 Sph Mgr, me – – Machaerina flexuosa* Schoenus nigricans* 0.49 ± 0.08 0.47 ± 0.09 Sph Mgr (f) – – Irr Mgr – – Tetraria compar* 0.40 ± 0.06 Sph Sm – – Trianoptiles solitaria – Irr Sm Aggr – Scirpeae Amphiscirpus nevadensis* 0.48 ± 0.07 Irr Mgr – – Eriophorum latifolium 0.51 ± 0.07 Irr Mgr – – Phylloscirpus acaulis* 0.53 ± 0.11 Irr Mgr – – Scirpus sylvaticus* 0.43 ± 0.07 Irr Mgr Aggr – Trichophorum alpinum* 0.43 ± 0.09 Irr Mgr Aggr – Sclerieae Scleria rugosa 0.42 ± 0.06 Irr Mgr Em – Scleria terrestris** 0.51 ± 0.02 Irr Sm Aggr – Trilepideae Afrotrilepis pilosa* 0.51 ± 0.11 Irr Mgr Thr – Coleochloa setifera 0.41 ± 0.07 Irr Mgr, str – 4k Subfamily Mapanioideae Chrysitricheae Chorizandra cymbaria* 0.49 ± 0,09 Irr Mgr Thr – Chorizandra enodis* 0.50 ± 0.08 Sph Mgr (f), perf (f) Conn – Chrysitrix dodii 0.70 ± 0.12 Irr Mgr – – 123 128 A. Nagels et al. Table 4 continued Species Diameter (lm) ± SD Shape Ornamentation Remark Figure Lepironia articulata* 0.42 ± 0.07 Irr Mgr – – 0,51 ± 0,11 Sph Mgr – 4h Hypolytreae Diplasia karatifolia* Hypolytrum jenmanii subsp. jenmanii* 0.25 ± 0.10 Sph Sm Em – Mapania cuatrecasasii* 0.82 ± 0.19 Do Sm – 4j Mapania linderi* No orbicules observed – – – – Aggr Aggregates, ang angular, conn connected, do doughnut-shaped, em embedded, f few, irr irregular, mgr microgranules, me microechinae, perf perforations, sm smooth, sph spherical, str striae, thr threads Species are listed alphabetically; fixed material (without asterisk), herbarium material is indicated with an asterisk and living material with double asterisks Carex, Eleocharis, Fuirena, Hellmuthia, Isolepis, Lepironia and Kyllinga; Figs. 1i, r, 2k, o–q). We use the term pyriform to describe this characteristic pollen shape. The shape in polar view (amb), is circular (Fig. 1k), angular (Fig. 1l) or irregular (Fig. 1m). The peripheral arrangement of the pollen grains in the locules also has a major impact on the shape of mature pollen grains (Fig. 1v). Pollen is closely packed in a uniseriate layer which often results in more or less angular grains. Apertures The number of apertures ranges from zero (Eleocharis acutangula, Fimbristylis complanata, F. xyridis and Rhynchospora sp.; Fig. 2a–c, p) to more than eight (Baumea rubiginosa; Fig. 2d). In cases where the sexine ornamentation is not differentiated in the apertural region, the number of apertures is difficult to determine even on SEM observations (e.g. Scleria rugosa; Fig. 2e). Therefore, we judged it more appropriate to use the term ‘apertural zones’. Pentaaperturate pollen grains are most common in Cyperaceae (Table 3). These pollen grains usually have one distal aperture (often an ulcus) (e.g. Eriophorum latiolium; Fig. 2l) and four lateral apertures or apertural zones (e.g. Lipocarpha nana; Fig. 2m). In the species studied, the number of lateral apertures varies from three (e.g. Becquerelia cymosa; Table 3) to six (Arthrostylis aphylla; Fig. 2n), even within a genus, (e.g. Eleocharis; Fig. 2o–p). In Cladium mariscus, we observed some pollen grains lacking visible apertures and others with apertures within the same specimen (Fig. 2f–g). Monoaperturate pollen grains were found in only one cyperoid genus Coleochloa (Fig. 2h) and in four of the six mapanioid genera studied, Chrysitrix (Fig. 1b), Diplasia (Fig. 1k), Hypolytrum, and Mapania (Fig. 1c). We observed lateral apertures in the two other mapanioid genera Chorizandra (Fig. 2j) and Lepironia (Fig. 2k). In Cyperaceae apertures are pori (e.g. Baumea rubiginosa; Fig. 2d) or colpi (e.g. Kyllinga sp.; Fig. 1d) and the 123 shape of apertures can vary within the same species (e.g. Isolepis antarctica; Fig. 2t). In Cyperus rotundus we observed irregularly shaped apertures (Fig. 2u). In Hellmuthia membranacea (Fig. 2q) and Ficinia brevifolia (Fig. 2r), the pollen grains display depressions above and below the lateral apertures. Pollen grains of Carex elata (Fig. 1i) and Coleochloa setifera (Fig. 2h) possess an elevated thickened ring around the aperture (aspis). In a few species we observed papillae (e.g. Cyperus dubius; Fig. 2s). Our observations demonstrate that number and shape of apertures can vary within a genus and even within a single species (heteromorphic pollen). The plasticity in the development of apertural zones in Cyperaceae pollen considerably reduces the systematic value of characters such as the number and position of apertures. Sexine ornamentation We observed seven different sexine ornamentation patterns with occasionally intermediate forms, including microechinate (e.g. Kyllinga flava; Fig. 3a), granulate– perforate (e.g. Kobresia myosuroides; Fig. 3b), fossulate (e.g. Fuirena leptostachya; Fig. 3c), negative microreticulate (e.g. Eleocharis acutangula; Fig. 3d), fossulate– rugulate (Coleochloa setifera; Fig. 3e), psilate–rugulate (Cyperus dubius; Fig. 3f) and microreticulate (e.g. Ficinia minutiflora and Hypolytrum jenmanii subsp. jenmanii; Fig. 3g–h). However, the majority of species are granulate–perforate. Exochogyne amazonica shows a rather exceptional sexine with narrow, elevated ridges (Fig. 3i) that may suggest a metareticulum. Apertures are always covered with an operculum (e.g. Ficinia minutiflora and Kyllinga flava; Figs. 1m, 3a, g) or a pontoperculum (e.g. Bulbostylis hispidula and Becquerelia cymosa; Figs. 1h, 3k), except in Diplasia karatifolia (Fig. 1k). These opercula or pontopercula consist of several sporopollenin elements, which also show variation in ornamentation patterns: elements with microgranules and microechinae (Fig. 3a, b), elements with perforations Palynological evolutionary trends in Cyperaceae Fig. 1 Shape in equatorial or polar view (SEM). a Cladium mariscus. b Chrysitrix dodii. c Mapania linderi*, spheroidal pollen grain in equatorial view. d Kyllinga sp. e Lipocarpha nana. f Capeobolus brevicaulis, suboblate/spheroidal pollen grain in equatorial view. g Kyllinga eximia, spheroidal pollen grain. h Bulbostylis hispidula, subprolate pollen grain in equatorial view. i Carex elata, prolate pollen grain in equatorial view. j Ficinia zeyherii, perprolate pollen grain in equatorial view. k Diplasia karatifolia*, circular amb (polar view). l Isolepis setacea, angular amb (polar view). m Kyllinga flava, 129 irregular amb (polar view). n, o Schoenoplectus senegalensis. p, q Arthrostylis aphylla. n, p semi-collapsed pollen grain (equatorial view). o, q rehydrated pollen grain. r Kyllinga flava, pyriform pollen grain in equatorial view. s Cyperus rotundus, semi-rehydrated (left) and fully rehydrated (right) pollen grain. t Schoenus nigricans, collapsed pollen grain in equatorial view. v Isolepis digitata, overview of the pollen grain arrangement in the locule. Scale bar 10 lm. Species without asterisk fixed material, asterisk herbarium material, double asterisks living material 123 130 (Fig. 3b), fossulate (e.g. Fig. 2u), negative microreticulate (Fig. 3k), and doughnut-shaped granules (Fig. 3g). The ornamentation of these elements displays often similarities with the mesocolpial or mesoporial sexine ornamentation (e.g. Fig. 3a). 123 A. Nagels et al. Orbicules We observed orbicules in all species studied, except in Mapania linderi and Everardia montana. Size and shape of orbicules, and the ornamentation of the orbicular wall at Palynological evolutionary trends in Cyperaceae b Fig. 2 Apertural conditions (SEM). a–c Equatorial view of pollen grains without visible apertures. a Fimbristylis complanata. b Fimbristylis xyridis. c Rhynchospora sp. d Baumea rubiginosa, polyporate pollen grain (white arrows). e Scleria rugosa, poorly visible ulcus (black arrow) and lateral apertures (white arrows). f, g Cladium mariscus. f Pollen grain without visible apertures. g Equatorial view of pollen grain with ulcus (black arrow) and lateral apertures (white arrows). h Coleochloa setifera, monoaperturate pollen grain with aspis (white arrow). i Mapania cuatrecasasii*, polar view of collapsed pollen grain with a single ulcus (black arrow). j Chorizandra enodis*, equatorial view of pollen grain with two visible lateral apertures (white arrows). k Lepironia articulata*, lateral apertures (white arrows). l Eriophorum latifolium, pentaaperturate pollen grain, ulcus (black arrow) and two lateral apertural zones (white arrows) visible. m Lipocarpha nana, view at proximal pole, four lateral apertures (white arrows). n Arthrostylis aphylla, view at proximal pole, six lateral apertures (white arrows). o Eleocharis palustris, three lateral apertures visible (white arrows). p Eleocharis acutangula, equatorial view of pollen grain without visible apertures. q Hellmuthia membranacea, ring (white arrowhead) below lateral apertures (white arrows). r Ficinia brevifolia, depressions (white arrowheads) below and above lateral apertures (white arrows). s Cyperus dubius, pollen grain with papilla (white arrowhead). t Isolepis antarctica, equatorial view of pollen grain with distal sulcus (black arrow) and lateral pori (white arrows). u Cyperus rotundus, pollen grain with irregular shaped aperture (white arrowhead). Species without asterisk fixed material, asterisk herbarium material, double asterisks living material species level are summarized in Table 4. Orbicules often occur in high densities and are dispersed over the entire inner locule wall (Fig. 4a). Occasionally they are attached to the pollen grain wall (e.g. Pycreus sanguinoletus; Fig. 4b). In some species, aggregations of single orbicules occur (e.g. Ficinia radiata; Fig. 4c). The morphology of the locule wall reveals the position of underlying endothecial cells and thickenings (Fig. 4d). In Eleocharis acutangula (Fig. 4e), the orbicules form a reticulate pattern over the entire locule wall or the orbicules may be absent at rounded spots on the locule wall (Fig. 4d). Orbicules of Pycreus mundtii (Fig. 4f) are the smallest (ca. 0.20 lm), whereas the largest orbicules (ca. 1.28 lm) occur in Kyllinga eximia. Four shapes were distinguished: (1) angular as found in Arthrostylis aphylla (Fig. 4g); (2) more or less spherical as observed in Diplasia karatifolia (Fig. 4h); (3) doughnutshaped as present in Mapania cuatrecasasii (Fig. 4j) and (4) irregular as occurring in Kyllinga sp. (Fig. 4i). In some species, the orbicules are embedded in the locule wall (e.g. Hypolytrum jenmanii subsp. jenmanii). In Kyllinga flava we found spherical bodies, with a similar ornamentation as the orbicular wall, but much larger in size (±2.26 lm). These spherical bodies are scattered all over the locule wall but less dense than the orbicules (Fig. 4l). The majority of species have orbicules with a microgranulate (e.g. Kyllinga sp.; Fig. 4i) or a microechinate (e.g. Kyllinga flava; Fig. 4l) wall ornamentation. However, in some species the orbicular wall is smooth (Mapania cuatrecasasii; Fig. 4j). In Coleochloa setifera, striae were 131 observed (Fig. 4k) on the orbicular wall. Occasionally, perforations were present (Kyllinga sp.; Fig. 4i). We often observed similarities between the sexine ornamentation (or the ornamentation and shape of the sporopollenin granules on the opercula or pontopercula) and the ornamentation and shape of the orbicules (e.g. Kyllinga flava; Figs. 3a, 4l and Coleochloa setifera; Figs. 3e, 4k). Discussion Palynological data in a phylogenetic framework In order to determine possible synapomorphies and evolutionary trends in the Cyperaceae, we optimized the coded palynological characters on a phylogenetic hypothesis based on parsimony analysis of rbcL and trnLF sequence data of the genera investigated (Muasya et al. 2008, modified). Three pollen characters (number of lateral, apertures distinctness of apertures and sexine ornamentation) show some congruence with the molecular phylogeny and are illustrated (Fig. 5). The other eight palynological characters were also optimized but were incongruent with the phylogeny (not shown). There is a transition in the lateral aperture number from zero (outgroup and subfamily Mapanioideae) to polyporate (Baumea-clade) or most commonly to four (clade Cariceae–Dulichieae–Scirpeae and tribe Fuireneae) with in some cases an increase to five or even six lateral apertures (tribe Cypereae) (Fig. 5a). Evolution from a low to a higher aperture number has been suggested for several groups, for example, Alismatales (Chanda et al. 1988) and Dioscorea (Schols et al. 2005). An increased number of apertures may offer a potential selective advantage because it increases the number of prospective germination sites, thus facilitating contact between at least one aperture and the stigmatic surface (Furness and Rudall 2004). Occasionally a reversal from five to zero was identified, for instance in Fimbristylis. We observed also a shift from operculate pollen grains in the outgroup and the subfamily Mapanioideae to pontoperculate pollen in Cyperoideae with a reversal to operculate pollen in tribe Cypereae (Fig. 5b). It has been proposed that pollen aperture number and pattern is related to microsporogenesis (e.g. Blackmore and Crane 1998; Rudall and Bateman 2007) and that simultaneous cytokinesis allows variation in the interaction between nuclei, which is impossible in successive microsporogenesis, thus leading to heteromorphic pollen (Ressayre et al. 2002). Changes in aperture number are thus potentially easy to achieve (Furness and Rudall 2004). Granulate–perforate sexine occurs throughout the outgroup and the majority of Cyperaceae species sampled and can be considered as the plesiomorphic 123 132 Fig. 3 Sexine and aperture ornamentation (SEM). a Kyllinga flava, microechinate sexine with perforations (black arrow), operculate aperture with microechinate sporopollenin granules (white arrow). b Kobresia myosuroides, granulate (white arrow) sexine with perforations (black arrow), in the right angle is the apertural zone with pontoperculum visible. c Fuirena leptostachya, fossulate sexine with microgranules (white arrow) and perforations (black arrow). d Eleocharis acutangula, negative microreticulate sexine with perforations (black arrow) and microgranules (white arrow). e Coleochloa setifera, fossulate/rugulate sexine with perforations (black arrow) and microgranules (white arrow). Scale bar 1 lm. f Cyperus dubius, psilate/rugulate sexine with microgranules (white arrow). 123 A. Nagels et al. g Ficinia minutiflora, microreticulate sexine with thickenings on the crossings of the muri (black arrow), which resemble the orbicules; operculate aperture with doughnut-shaped sporopollenin granules (white arrow). Scale bar 2 lm. h Hypolytrum jenmanii subsp. jenmanii*, microreticulate sexine. i Exochogyne amazonica*, pollen grain with metareticulum, arrows indicate assumed apertures. j Mapania linderi*, microreticulate sexine with echinae (white arrow). k Becquerelia cymosa*, pontoperculate aperture with negative microreticulate ornamentation. gr Groove, ar areola, lu lumen, mu muri). Species without asterisk fixed material, asterisk herbarium material, double asterisks living material Palynological evolutionary trends in Cyperaceae 133 Fig. 4 Orbicules (SEM). a Cyperus rotundus, overview locule wall with orbicules. Scale bar 5 lm. b Pycreus sanguinoletus, orbicules attached to pollen grain wall (arrow). c Ficinia radiata, aggregations of orbicules (arrow). Scale bar 10 lm. d Eleocharis acutangula, overview locule wall with blank spots where pollen grains used to be; endothecial thickening (arrow). e Eleocharis acutangula, orbicules arranged in reticulate pattern. f Pycreus mundtii, smallest orbicules (0.20 lm). g Arthrostylis aphylla, angular orbicules. Scale bar 2 lm. h Diplasia karatifolia*, spherical orbicules. i Kyllinga sp., irregular orbicules (black arrow) with microgranules (white arrow). j Mapania cuatrecasasii*, doughnut-shaped orbicules (arrows). Scale bar 1 lm. k Coleochloa setifera, orbicules with striae (arrow). l Kyllinga flava, orbicules with microechinae (white arrow), spherical bodies with microechinae (black arrow). Species without asterisk fixed material, asterisk herbarium material, double asterisks living material condition in the group. Most other sexine ornamentation patterns have evolved more than once. Microechinate sexines are notably present in the Kyllinga-Oxycaryum clade (Fig. 5c) and could be a highly informative character to split certain genera that are most likely non-monophyletic, such as Cyperus (Muasya et al. 2002). Mapanioideae In Mapanioideae, several pollen characters have a distinct systematic value at tribal level: Hypolytreae is characterized by spheroidal, monad-like pollen grains (Figs. 1c, k, 2i) while Chrysitricheae have (sub)prolate pollen grains that are most likely pseudomonads (Simpson et al. 2003; Figs. 1b, 2j–k). Moreover, Chrystricheae display a peripheral pollen arrangement within the locule and the Hypolytreae a central pollen arrangement (Kirpes et al. 1996; Simpson et al. 2003). Members of Hypolytreae have monoaperturate pollen grains (Figs. 1c, k, 2i), in contrast to Chrysitricheae which possess besides monoaperturate pollen grains with a distal ulcus also pollen with several lateral apertures (Fig. 2j–k), a feature that is common in the Cyperoideae. Mapanioid pollen grains have always been reported as being monoaperturate (Haines and Lye 1983; 123 134 Fig. 5 Optimization of pollen characters on a plastid phylogeny of the Cyperaceae. a Number of lateral apertures. b Distinctness of apertures. c Sexine ornamentation. Strict consensus tree obtained from a parsimony analysis of rbcL and trnL-F sequence data (Muasya et al. 2008). Optimization was carried out using MacClade 4.04 123 A. Nagels et al. (Maddison and Maddison 2001). Ab Abildgaardieae, Bi Bisboeckelereae, Ca Cariceae, Chr Chrysitricheae, Cry Cryptangieae, Du Dulicheae, El Eleocharideae, Fu Fuireneae, Hy Hypolytreae, Ju Juncaceae, Rh Rhynchosporeae, Sc Scirpeae, Sch Schoeneae, Scl Scleriae, Tri Trilepideae, Tur Thurniaceae Palynological evolutionary trends in Cyperaceae Van Wichelen et al. 1999; Simpson et al. 2003) except for the observation of lateral apertures in Lepironia mucronata by Erdtman (1966, 1971). In literature, the sexine ornamentation of pollen grains in Mapanioideae has been described as (sub)reticulate (Simpson et al. 2003). This is only partially confirmed by our results (Fig. 3h, j), because we also observed granulate and rugulate pollen grains in Mapanioideae. Moreover, Hypolytreae and Chrysitricheae can be readily distinguished by their sexine pattern. Hypolytreae pollen grains are microreticulate (Fig. 3h, j), except those of Mapania cuatrecasasii (fossulate–perforate), while Chrysitricheae pollen grains are granulate or rugulate–granulate. We observed orbicules in all species studied, with exception of Mapania linderi. The systematic value of orbicules in Mapanioideae is rather low, rendering no support for the two tribes based on orbicular morphology. Based on nonmolecular data, Bruhl (1995) suggested that Mapanioideae comprise only one tribe (Hypolytreae), since Chrysitricheae were usually nested in other mapanioids. However, our data (Fig. 5) strongly supports the recognition of both tribes Hypolytreae and Chrysitricheae as inferred by Simpson et al. (2003, 2008) and Muasya et al. (2008). Tribe Trilepideae The position of this tribe is unresolved in the phylogenetic hypothesis in Fig. 5. In the recent molecular phylogenies of Simpson et al. (2008) and Muasya et al. (2008), however, this clade is sister to the rest of Cyperoideae although with weak support only. The monoporate pollen grains of Coleochloa setifera (Trilepideae) (Fig. 2h) offer support for Trilepideae as an early diverging lineage in Cyperoideae. This feature occurs in only one other cyperoid genus (Cladium) and in the closely related Mapanioideae. Pollen grains of Coleochloa setifera also have a rugulate sexine pattern (Fig. 3e) in common with Chorizandra cymbaria (Mapanioideae). The other species of Trilepideae studied have pollen with the plesiomorphic granulate–perforate sexine ornamentation. Further observations in Trilepideae are needed to search for useful systematic pollen characters, which can help to clarify the position of this tribe. Cyperoideae Pollen morphological variation within subfamily Cyperoideae is considerable. The different pollen shapes that occur in Cyperoideae have arisen several times in the phylogeny. Even within a single genus, for example Cyperus, we observed different pollen shapes (Table 3). This variability is probably caused by harmomegathic effects on the fragile Cyperaceae pollen grains. Furthermore, we conclude that the peripheral arrangement of the pollen grains in the 135 locules affects their shape at maturity. Species with pollen grains without visible apertures (Fig. 2a–c, f, p) are scattered over the Cyperoideae according to the most recent molecular phylogenies. Baumea rubiginosa (Fig. 2d), Gahnia lanigera and Capeobolus brevicaulis (Fig. 1f) have polyporate pollen grains similar to those of Machaerina sp. (Van Wichelen et al. 1999) (Fig. 5a). Baumea and Machaerina are sister genera in the tribe Schoeneae (Fig. 5), to which also the genus Gahnia belongs. A broader sampling of Lepidosperma and Neesenbeckia, the closest relatives of Baumea and Machaerina, is needed to determine if the polyporate condition is also present in other genera of this small clade. The number of apertures in the reminder of tribe Schoeneae varies between zero and six. The clade with tribes Scirpeae 1 and 2, Dulichieae and Cariceae, possess pollen grains with one distal aperture and four lateral apertures (Fig. 5a). This apertural system is also present in pollen grains of tribe Fuireneae (Fig. 5a). The rest of Cyperoideae has an aperture number varying between three and six, with most species having pentaaperturate pollen grains (Fig. 2m). Consequently, our observations oppose Shah (1962) and Padhye and Makde (1980), who stated that monocolpate pollen grains are the dominant type in Cyperaceae. Cypereae generally possess pollen grains with one distal aperture and five lateral apertures (Fig. 5a), but the number of apertures can vary within a genus and even in representatives of a single species, indicating a high level of plasticity in this clade. The presence of pontopercula is plesiomorphic in Cyperoideae and there is a reversal to opercula in tribe Cypereae (Fig. 5b). The sexine ornamentation in species of Cyperoideae is mostly described as granulate (Wodehouse 1935; Huang and Chung 1971; Padhye and Makde 1980), perforate (Moar and Wilmshurst 2003), psilate (Haines and Lye 1983) or foveolate (Padhye and Makde 1980). These sexine ornamentation descriptions should be treated with caution since most are based on LM observations only, in contrast with our more detailed SEM observations. Our results indicate that the granulate–perforate sexine ornamentation pattern (e.g. Fig. 3b) is most common in the Cyperaceae species examined and also in Juncaceae (Fig. 5c). We therefore conclude that the granulate–perforate sexine ornamentation is primitive in Cyperaceae with transitions to the other sexine ornamentations recognized. The six other sexine ornamentation patterns occur throughout the phylogeny. Microechinate pollen grains are present in tribe Cypereae (Cyperus hemisphaericus, Kyllinga, Lipocarpha, Kyllingiella) and in Arthrostylis aphylla (Abildgaardieae, sensu Muasya et al. 2008) and Mapania linderi (Hypolytreae, sensu Muasya et al. 2008) (Fig. 5c). Pollen grains of Ficinia minutiflora show a microreticulate sexine pattern similar to the pattern in some mapanioid genera (Fig. 3g). We conclude that at generic level sexine ornamentation 123 136 shows a high degree of plasticity which prevents the detection of a clear pattern. Although orbicule data have proven to be useful for evaluating systematic relationships in certain groups (Raj and El-Ghazaly 1987; Huysmans et al. 1997; Vinckier et al. 2000; Vinckier and Smets 2002), they only have minor taxonomic importance in Cyperoideae due to lack of variation. Most species possess spherical to irregularly-shaped orbicules with microgranules on the orbicule wall. We found some striking parallelisms between the shape and ornamentation of orbicules and sexine ornamentation or the ornamentation of the sporopollenin granules on the opercula and pontopercula (e.g. Hesse 1986; Huysmans et al. 1997; Vinckier et al. 2000). The systematic value of palynological data at tribal level in Cyperoideae is rather low as a consequence of high levels of homoplasy and polymorphism in most palynological characters. Sexine ornamentation patterns and pollination The majority of Cyperaceae are thought to be anemophilous. Entomophily in Cyperaceae is associated with taxa that are fragrant (e.g. Mapania) or that have coloured inflorescences (e.g. Ascolepis, Cyperus, Rhynchospora) (Goetghebeur 1998). It has been linked to forest habitats (Goetghebeur 1998), but the taxa with microechinate pollen occur in savannah or open woodland. Sexine projections are often associated with entomophily (e.g. Tanaka et al. 2004); taxa with microechinate pollen in our study (e.g. Kyllinga, Lipocarpha) may be entomophilous according to this hypothesis. However, some Betulaceae, Asteraceae and grasses with microechinate pollen grains are wind pollinated (Vinckier and Smets 2001). The presence of brightly coloured inflorescences (white, orange, brown) in Kyllinga could offer additional evidence for entomophily. However, the brightly coloured Ficinia radiata flowers produce granulate pollen grains, suggesting that alternative adaptations to promote entomophily may exist in different taxa or habitats. This shows that ecological studies are needed to conclude with certainty. The forest-dwelling genera of Hypolytreae with microreticulate pollen grains (e.g. Mapania and Diplasia), are thought to be adapted to invertebrate pollination by having sticky pollen due to the presence of lipids (Simpson et al. 2003). The differences in sexine ornamentation of the pollen grains observed in Hypolytreae and Chrysitricheae pollen do not reject the hypothesis that the groupings within subfamily Mapanioideae are based on the specialization of some taxa in terms of their pollination biology (Simpson et al. 2003). Pollen morphological similarities with other Poales In Juncaceae and Thurniaceae, the sister groups of the Cyperaceae (Muasya et al. 1998, 2000), the four meiotic 123 A. Nagels et al. products become viable, fertile pollen grains, which are dispersed as tetrads (e.g. Erdtman 1952, 1971; Furness and Rudall 1999). The pollen grains are ulcerate in both families (Dahlgren et al. 1985), which is also the case in basal and some other members of the Cyperaceae (Fig. 5a). Pollen tetrads in Juncaceae are operculate, and are described as granulate (Buchner and Weber 2000), a sexine ornamentation pattern which is also present in the Cyperaceae (Fig. 3b). Unlike in most Cyperaceae, pollen arrangement in the locule of the anther in Juncaceae is central (Kirpes et al. 1996). Sexine ornamentation, distinctness of apertures and pollen arrangement in Thurniaceae is not documented. Restionaceae have pollen grains dispersed as monads with a scrobiculate (perforate) sexine (Dahlgren et al. 1985). The latter is also reported in our study group (Table 3). Monoaperturate pollen grains occur in both families (Chanda 1966; Dahlgren et al. 1985). Like Juncaceae, the pollen grains of Restionaceae are centrally arranged in the locule in contrast to the peripheral pollen arrangement in most Cyperaceae. Some palynological similarities between Cyperaceae and Poaceae are observed. In both groups orbicules are present in huge numbers (Vinckier and Smets 2001). In several species of both families, orbicules are fused or connected with each other by sporopollineous threads forming a network of orbicules covering the locule wall (Vinckier and Smets 2001). Similar sexine ornamentations are observed in Cyperaceae and Poaceae species (e.g. negative microreticulate with microgranules/echinae and perforations) (Vinckier and Smets 2001; Datta and Chaturvedi 2004). Both groups possess species with spheroidal, monoaperturate pollen grains with an annulus (Skvarla et al. 2003; Datta and Chaturvedi 2004; Perveen 2006); and the peripheral arrangement of the pollen grains in the locule is dominant in Poaceae and Cyperaceae (Kirpes et al. 1996). Conclusions The pollen morphological variation encountered is far greater than was conceived so far. Our data support the delimitation of only two subfamilies. In Mapanioideae pollen polarity, pollen shape, aperture number and sexine ornamentation provide additional support for the recognition of two tribes (Hypolytreae and Chrysitricheae). Hypolytreae are characterized by monoaperturate, spheroidal pollen grains while Chrysitricheae have pear-shaped pseudomonads. In Cyperoideae we could identify an evolutionary trend concerning the number and localization of apertures. Early diversified groups lack lateral apertures, and polyporate pollen grains are an intermediate form towards the presence of four lateral apertures to five lateral apertures in the Palynological evolutionary trends in Cyperaceae derived Cypereae. A second evolutionary trend concerns the shift from the plesiomorphic granulate–perforate sexine to the other sexine patterns. No other distinct trends in character evolution could be discerned in subfamily Cyperoideae because of the high degree of homoplasy in some major pollen morphological features, thus restricting the taxonomic value of those palynological characters. Therefore, the use of pollen types in Cyperaceae is an oversimplification of the palynological variation present. In the literature there is controversy about the number of apertures in Cyperoideae pollen. This can be explained by (1) observation of collapsed or insufficient hydrated pollen grains, (2) apertural zones that are difficult to distinguish due to only slightly differentiated sexine ornamentation in the apertural regions, and (3) intraspecific variation in aperture number. Ultrastructural observations of living material are indispensable to provide evidence on the exact number of apertures and to investigate whether grains without externally visible apertures are inaperturate or omniaperturate. Further developmental studies on the pseudomonad or monad (Mapania-type pollen) nature of pollen grains in Cyperaceae are needed to answer phylogenetic questions such as, are there intermediate forms between monadcentral pollen arrangement and pseudomonad-peripheral pollen arrangement? Acknowledgments This research was financially supported by the research council of K. U. Leuven (OT/05/35) and the Fund for Scientific Research—Flanders (Belgium) (F.
W. O.—G.0268.04). S. Vinckier was a postdoctoral fellow of F.
W. O. in the course of this study. A. M. Muasya acknowledges the Norwegian Council for Higher Education Programme for Development Research & Education (NUFU project 53/03) for funding and Flora Research Permit from CapeNature (AAA005-00054-0028). We thank Prof. P. Goetghebeur from the University of Ghent and Prof. J. Rammeloo, Director of the National Botanic Garden of Belgium (BR) for the supply of living material and herbarium specimens. We are grateful to Anja Vandeperre for technical assitance. Dr. Ochoterena and Steven Janssens are acknowledged for their assistance with the character optimization. Appendix 1: Species studied are listed alphabetically; fixed material (without asterisk), herbarium specimens are indicated with an asterisks and living material with double asterisks Afrotrilepis pilosa (Ridl.) Gilly (*), Cameroon, P. Goetghebeur, 5182 (GENT). Amphiscirpus nevadensis S. Watson (*), USA, J. Bouharmont, 19926, BR-S
P. 917869 (BR). Arthrostylis aphylla R. Br., Australia, R.K. Harwood, RKH 1161. Baumea rubiginosa Boeck., Australia, J.J. Bruhl and Hodges, JH 792. 137 Becquerelia cymosa Brogniart (*), Brazil, M. Luceno, 33B (GENT). Bulbostylis hispidula (Vahl) R.W. Haines, Kenya, A.M. Muasya, AM 2466. Capeobolus brevicaulis (C.B. Clarke), J. Browning, South Africa, A.M. Muasya, AM 2203. Carex capitata L., Botanical Garden University Ghent, Belgium, P. Goetghebeur, PG 10466. Carex elata Lowe, cultivated in the Botanical Garden of the Institute of Botany and Microbiology (K.U.Leuven) Belgium, A. Vrijdaghs, AV11. Carex monostachya, A. Rich. (*), Senegal, C. Vanden Berghen, 8701 (BR) Caustis flexuosa, R. Br. (*), USA, E.F. Constable, 26663 (BR). Caustis recurvata Spreng. (*), Australia, P.K. Endress, 4381 (GENT). Chorizandra cymbaria R. Br. (*), Australia, K.L. Wilson and K. Frank, 8954 (GENT). Chorizandra enodis Nees, Australia, K.L. Wilson and K. Frank, 8922 (GENT). Chrysitrix dodii C.B. Clarke, South Africa, A.M. Muasya, AM 2797. Cladium mariscus (L.) Pohl, National Botanical Garden of Belgium, A. Vrijdaghs, AV06. Coleochloa setifera (Ridl.) Gilly, Kenya, A.M. Muasya, AM 2464. Costularia humbertii Bosser (*), Madagascar, J.S. Miller and P.P. Lowry, II4175 (GENT). Courtoisina assimilis (Steud.) P. Maquet, Kenya, A.M. Muasya, AM 2124. Cyperus alternifolius L. (**), Botanical Garden University Ghent Belgium, A. Vrijdaghs, 2001/1114. Cyperus articulatus L., Kenya, A.M. Muasya, AM 2168. Cyperus dubius Rottb., Kenya, A.M. Muasya, AM 2188. Cyperus haspan L., Kenya, A.M. Muasya, AM 2135 (EA). Cyperus hemisphaericus Boeckeler (*), Tanzania, E. Milne-Redhead and P. Taylor, 8053A (BR). Cyperus laevigatus L., Botanical Garden University Ghent, Belgium, P. Goetghebeur, PG 10202. Cyperus rotundus L., Kenya, A.M. Muasya, AM 2164. Diplasia karatifolia L.C. Rich (*), Bolivia, R. Rueda, 921 (GENT). Dulichium arundinaceum Britton, Botanical Garden University Ghent, Belgium, P. Goetghebeur, PG 9914. Eleocharis acutangula (Roxb.) Schult., Kenya, A.M. Muasya, AM 2437. Eleocharis palustris R. Br., Hortus Botanicus Lovaniensis Belgium, A. Vrijdaghs, AV07b. Eriophorum latifolium Hoppe, Hortus Botanicus Lovaniensis Belgium, A. Vrijdaghs, AV04. Everardia montana Ridley (*), Venezuela, P.E. Berry, O. Huber and J. Rosales, 4912 (GENT). 123 138 Exochogyne amazonica C.B. Clarke (*), Brasil, M. Aparecida da Silva, C. Proença, E. Cardoso and J.P. Paixão, 23.5.1994 (GENT). Ficinia brevifolia Nees, South Africa, A.M. Muasya, AM 2205 (BOL, EA, K). Ficinia capitellum Nees, South Africa, A.M. Muasya, AM 2206 (BOL, EA, K). Ficinia dunensis Levyns, South Africa, A.M. Muasya, AM 2242. Ficinia gracilis Schrad., Kenya, A.M. Muasya, AM 2571. Ficinia minutiflora C.B. Clarke, South Africa, A.M. Muasya, AM 2257 (BOL, EA, K). Ficinia polystachya Levyns, South Africa, A.M. Muasya, AM 2320. Ficinia radiata Kunth, South Africa, A.M. Muasya, AM 2262 (BOL, EA, K). Ficinia tristachya (Vahl) Nees, South Africa, A.M. Muasya, AM 2255. Ficinia zeyheri Boeckeler, South Africa, A.M. Muasya, AM 2209 (BOL, EA, K). Fimbristylis complanata (Retz.) Link, Kenya, A.M. Muasya, AM 2147. Fimbristylis xyridis R. Br., Australia, R.K. Harwood, RKH 1162. Fuirena abnormalis C.B. Clarke, Kenya, A.M. Muasya, AM 2192. Fuirena leptostachya Oliver, Kenya, A.M. Muasya, AM 2136. Gahnia lanigera (R. Br.) Benth. (*), Australia, B.J. Blaylock, 1227 (GENT). Hellmuthia membranacea (Thunb.) R.W. Haines and Lye, South Africa, A.M. Muasya, AM 2792 (KUL). Hypolytrum jenmanii C.B. Clarke subsp. jenmanii (*), Guyane, J.J. de Granville, F. Crozier, 13652 (GENT). Isolepis antarctica (Willd.) Roem. & Schult., South Africa, A.M. Muasya, AM 2247 (BOL, EA, K). Isolepis digitata Nees ex Schrad., South Africa, A.M. Muasya, AM 2258. Isolepis prolifera (Rottb.) R. Br., South Africa, A.M. Muasya, AM 2265. Isolepis setacea (L.) R. Br., Kenya, A.M. Muasya, AM 2547. Kobresia myosuroides Fiori & Paoletti, Botanical Garden University Ghent, Belgium, P. Goetghebeur, PG 10009. Kyllinga eximia C.B. Clarke, Kenya, A.M. Muasya, AM 2137. Kyllinga flava C.B. Clarke, Kenya, Musili, MM 009. Kyllinga polyphylla Thou. ex Link (**), Botanical Garden University Ghent, Belgium, A. Vrijdaghs, 2004/21768. Kyllinga sp. Rottb., Kenya, A.M. Muasya, AM 2658. Kyllingiella polyphylla, Kenya, A.M. Muasya, AM 2435. 123 A. Nagels et al. Lagenocarpus rigidus (Kunth) Nees subsp. rigidus (*), French Guiana, D. Torida-Marbot, 329 (GENT). Lepironia articulata (Retzius) Domin (*), Papua New Guinea, P. Goetghebeur and W. Vyverman, 6673 (GENT). Lipocarpha nana (A. Rich.) Cherm., Kenya, A.M. Muasya, AM 2194. Lipocarpha rehmannii (Ridl.) Goetgh., Kenya, A.M. Muasya, AM 3132. Machaerina flexuosa (Böckeler) Kern (*), Madagascar, J.S. Miller and A. Randrianasdo, 4382 (GENT). Mapania cutatrecasasii T. Koyama (*), Costa Rica, G. Herrera, 3282 (GENT). Mapania linderi Hutchinson (*), Ivory Coast, C.C.H. Jongkind, 4435 (GENT). Oxycarium cubense (Poepp. & Kunth) Palla, Kenya, Mwachala, M340. Phylloscirpus acaulis (Philippi) Goetghebeur and D.A. Simpson subsp. pachycaulis (*), Equador, S. Laegaard, S. Dhooge and E. Jones, 21519 (GENT). Pseudoschoenus inanis (Thunb.) Oteng- Yeboah, South Africa, A.M. Muasya, AM 3061. Pycreus flavescens Beauv. ex Rchb. (**), Botanical Garden University Ghent, Belgium, A. Vrijdags, 2005/ 0401. Pycreus mundtii Nees, Kenya, A.M. Muasya, AM 2156. Pycreus sanguinolentus (Vahl) Nees, Kenya, A.M. Muasya, AM 2157. Rhynchospora sp. Vahl, Australia, R.K. Harwood, RKH 1127. Schoenoplectus senegalensis (Hochst. ex Steud.) Palla, Kenya, Malombe 40. Schoenoxiphium lehmannii Kunth ex Steud., Kenya, Malombe, KG 96. Schoenoxiphium sparteum C.B. Clarke, Kenya, A.M. Muasya, AM 2566. Schoenus nigricans L., UK, K. De Wale, 1239 (GENT). Scirpoides holoschoenus (L.) Sojak (**), Botanical Garden University Ghent, Belgium, A. Vrijdaghs, 2003/1536. Scirpus sylvaticus L. (*), Botanical Garden University Ghent, Belgium, P. Goetghebeur, 5382 (GENT). Scleria rugosa R. Br., Australia, R.K. Harwood, RKH 1143. Scleria terrestris (L.) Fassett (**), Botanical Garden University Ghent, Belgium, A. Vrijdaghs, 21768. Tetraria compar H.C. Taylor (*), South africa, H.C. Taylor, 9996 (GENT). Trianoptiles solitaria (C.B. Clarke) Levyns, South Africa, A.M. Muasya, AM 3024 Trichophorum alpinum Pers. (*), USA, D. Collet, 670 (BR). Palynological evolutionary trends in Cyperaceae 139 Uncinia rubra Colenso ex Boott, Botanical Garden University Ghent, Belgium, P. Goetghebeur, PG 9727. Appendix 2: A detailed list of the characters and their states as defined for the optimization and the combined analysis. Characters 1, 2 and 9 are quantitative and continuous characters 1. Polar axis (0) (1) (2) (3) (4) 0 1 2 3 4 2. P/E (0) (1) (2) (3) (4) 0 1 2 3 4 3. (0) (1) (2) (3) (4) 4. (0) (1) (2) 5. 6. (0) (1) (2) 7. (0) (1) 8. (0) (1) (2) (3) (4) (5) (6) (\18) (\28) (\41) (\52) ([52) (\1) (\1.4) (\1.7) (\2.3) ([2.3) 9. Pollen shape suboblate (0.75–0.88) spheroidal (0.88–1.14) subprolate (1.14–1.33) prolate (1.33–2.00) perprolate ([2.00) Distal aperture absent 1 ulcus 1 sulcus Number lateral apertures (0) absent (1) 3 (2) 4 (3) 5 (4) 6 (5) [6 Shape lateral apertures colpi pori variable Distinctness of apertures pontoperculum operculum Sexine ornamentation microechinate granulate–perforate fossulate negative microreticulate fossulate–rugulate psilate–rugulate microreticulate Orbicule diameter (0) (1) (2) (3) (4) 0 1 2 3 4 (\0.3) (\0.6) (\0.9) (\1.2) ([1.2) 10. Orbicule shape (0) (1) (2) (3) angular irregular spherical doughnut-shaped 11. Orbicule ornamentation (0) (1) (2) (3) smooth microgranules microechinae striae Appendix 3 Table 5. Table 5 Data matrix with coded characters 1 2 3 4 Kyllinga 0,1 0,1 1,2,3 1,2 CyperusC4 1,2 0,1,2 1,2,3 1 Pycreus 1 0,1 1,2 1,2 Lipocarpha 0 0 1 CyperusC3 1 1 1 5 6 7 8 9 10 11 2,3 0 1 2 2,4 1 0 2,3,4 0,1,2 0,1,2 1,2 1,2 1 0,1,5 3 0,1 1 1,2 0,1,2 1 1,2 1 2,3 1 1 2 1,2 1 0 1 1 1 1,2 1,2 1,2 1 1 123 140 A. Nagels et al. Table 5 continued 1 2 3 4 5 6 7 8 9 Kyllingiella 0 1 1 1 3,4,5 1 Courtoisina ? ? ? 1 2 2 Oxycaryum 1 1 2 1 3 Ficinia 1,2,3 0,1,2,3,4 1,2,3,4 1 Isolepis 1 1 2,3 1,2 Hellmuthia 2 2 3 Scirpoides ? ? ? Fuirena 1,2 1 1,2 Schoenoplectus 2 1 Eleocharis 2 1,2 Bulbostylis 2 1 Fimbristylis 0,1 Arthrostylis Phylloscirpus 1 ? Amphiscirpus 2 Scirpus 1 Eriophorum 1 ? 2 1 0 1 2 1 0 1,2 0 1 2 ? 0 1 1,2,3,4 2 0,1,2 0,1 0,1,2 0,1 1,6 2,3,4 2 1,2 1,2 1 0,1 1,2 1 4 1 1 1 ? 1 1,2 ? ? ? 1 1 1 0 ? 1 2 1 1 1 0,1 0 1,2 1 1 2 1 0 0 1 0 1 2,3 0,1 0,3 0 1 1 1,2 0 1,3 2 1 3 2 1 1 1 0 1 1 2 0 0 ? 1 1,2 0,1 ? 3 1 ? 1 ? 1 1 4 2 1 1 0 1 0 1 1 1 0 0 0 1,3 1 3 1 2 1 1 1 1 0 1 1 2 1 2 1 1 1 1 0 2,3 2 1 3 1 2 0 1 1 1 0 1 Dulichium 1 1 2 2 2 ? 1 ? ? 0 2 Trichophorum ? ? ? 1,2 2 1 1 1 1 0 1 Schoenoxiphium ? ? ? 1 2 ? 1,2 1 0,1 0 1,2,3 Uncinia ? ? ? ? ? ? 1 1 1 0 1 Carex 2 1,2 2,3 1 2 1 1,2 0,1 1 0 1,2 Kobresia 1 1 1 1 2 1 0 1 1 0 1 Rhynchospora 1 2 3 0 0 ? 1 1 1 ? 3 Exochogyne 1 1 1 ? 5 1 1 1,2 1 0 1 Lagenocarpus ? ? ? 1 1 1 2 2 1 0 1 Cladium 3 1 3 0,1,2 0,1,2 2 1 1 1 0 1 Becquerelia Scleria 1 1,2 2 1 3 1,2 1 1 1 1,2 1 1 2 1 1 0,1 1 1 0 0 1 1 Costularia 2 1 2 1 3 0 1 1 1 1 1 Trianoptiles 1 1 1 1 1 2 1 0 ? 0 1 Baumea 1 1 1 1 5 1 2 0 3 0 1,2 Machaerina ? ? ? ? 1 1 2 1 1 0 1 Caustis ? ? ? 1 2 ? 1 1,2 1 0 1,3 Gahnia 1 1 1 2 5 2 2 1,2 1 0 1,2 Schoenus ? ? ? 1 2 0 1 1 1 0 1 Tetraria 3 3 3 1 3 0 2 0 1 0 1 Capeobolus 1 0 0,1 1 5 1 1 1 2 0 1,2 Coleochloa 1 1 2 1 0 ? 1 1,3 1 1 4 Chrysitrix 4 3 3 1 0 ? 1 1 2 1 1 Chorizandra 3 2 3 1 0,2 1 1,2 1 1 0 1,4 Lepironia 2 1 2 1 0,2 1 1 1 1 0 1 Mapania 0,1 0 1 1 0 ? 3 0 2 1 0,2,6 Hypolytrum Diplasia 0 1 1 0 1 1 1 1 0 0 ? ? 2 2 0 1 0 1 ? ? 6 6 Juncus ? ? ? 1 0 ? ? ? ? 1 1 Luzula ? ? ? 1 0 ? ? ? ? 1 1 Prionium ? ? ? 1 0 ? ? ? ? ? 1 123 10 11 Palynological evolutionary trends in Cyperaceae References Blackmore S, Crane PR (1998) The evolution of apertures in the spores and pollen grains of embryophytes. 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