Pericarp diversity in Cyperaceae tribe Scirpeae

Accepted Manuscript Title: Pericarp diversity in Cyperaceae tribe Scirpeae Author: K˚are Arnstein Lye PII: DOI: Reference:

S0367-2530(16)30088-3 http://dx.doi.org/doi:10.1016/j.flora.2016.05.011 FLORA 50981

To appear in: Received date: Revised date: Accepted date:

15-1-2016 23-5-2016 28-5-2016

Please cite this article as: Lye, K˚are Arnstein, Pericarp diversity in Cyperaceae tribe Scirpeae.Flora http://dx.doi.org/10.1016/j.flora.2016.05.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Flora 211: 001-008, 2016

Pericarp diversity in Cyperaceae tribe Scirpeae Kåre Arnstein Lye K. A. Lye ([email protected]), Dept. of Ecology and Natural Resource Management, Norwegian University of Life Sciences, Høgskolevegen 12, PO Box 5003, NO-1432 Ås, Norway

Highlights  X-ray analysis is used to describe fruit wall diversity in Cyperaceae.  Variation in fruit wall structure corresponds to DNA diversity in five genera.  Silica is present in all genera and in 26 of 29 investigated species.  Plants from regions with an intense UV radiation regime have a thick epidermis wall.  A calcium plate in the epidermis develops only in one species.

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ABSTRACT Transverse sections of the pericarp or fruit wall of 28 species and 30 taxa from all genera of family Cyperaceae tribe Scirpeae sensu Goetghebeur (1998) were investigated using SEM and x-ray analysis to study cell types and presence of different elements. Two genera described after my analyses were completed (Calliscirpus and Rhodoscirpus) are excluded from this investigation; also the genus Cypringlea described in 2003. Particular attention was paid to variation in cell types and presence of silicon and other elements in the different parts of the pericarp. This is a novel systematic method as previous investigations of elements in the pericarp have focused on the morphology of silicon phytoliths and not the actual occurrence of Si and other elements in the various cells. Four of seven investigated and accepted genera have silicon phytoliths, but Si is present in all genera and in 25 of 28 investigated species or in 27 of 30 taxa. Other elements were present in minor amounts. However, the pericarp of Trichophorum uniflorum (Trautv.) Malyschev & Lukitsch. from arctic Russia had no silicon, but a conspicuous Ca-plate in the epidermis cells. Plant species with a pericarp with a thick silicon layer are better adapted to endozoochorous seed dispersal by protecting the seed from digestive acids and the abrasive action of grinding in birds’ gizzards; also the mineral layer and/or a thick outer epidermis wall will protect against an intense UV radiation regime, such as the high Andes and the arctic. The new information discovered through x-ray analysis of the pericarp has the possibility to increase the reliability in analyses of fossil fruits of Cyperaceae. Fruit wall anatomy provides good characters for generic delimitations in some instances, and provides significant infrageneric variation in other genera. Of the seven investigated genera only Trichophorum and Phylloscirpus were heterogeneous as regards fruit characters. Keywords: silicon, paleobotany, X-ray analysis, Eriophorum, Phylloscirpus, Trichophorum

Introduction The fruit in the family Cyperaceae is a small to fairly large nut with copious endosperm and a small basal embryo (Dahlgren and Clifford, 1982); it is often named nutlet or achene. The seed has a thin testa, free from the pericarp (Figs. 1-3); raphe and chalaza are usually conspicuous (Goetghebeur, 1998). The presence and morphology of silica bodies, often named phytoliths, in the pericarp (Prychid et al., 2003) are well known from many genera and species of Cyperaceae (Schuyler, 1971; Haines and Lye, 1983; Goetghebeur and Van den Borre, 1989; Tucker and Miller, 1990; Lye, 2000), although the phytoliths have mainly been studied from surface view and the identification of silica and other elements in the pericarp is rarely undertaken (Menapace and Wujek, 1985). Phytolith is a Greek word meaning “plant stone”. I have used the word mainly about various silicon rich outgrowth like conical papillae and anvil like projections, while Si-rich flat structures are named silicon plates or silicon platforms. In other tribes silicon is sometimes present in more amorphous forms or as

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granules, and are then named Si-rich areas. Marek (1958) presented good drawings of transverse sections of the pericarp of most European species of tribe Scirpeae, and OtengYeboah (1972) included a photograph of a transverse section of the pericarp of Phylloscirpus acaulis (Phil.) Goetgh. & D. A. Simpson as well as photographs of pericarps from some other tribes of the family. Earlier anatomical studies of Cyperaceae have been concerned with mainly vegetative anatomy (Mehra and Sharma, 1965; Metcalfe, 1971), floral ontogeny (Vrijdaghs et al., 2005), inflorescence development (Reutemann et al., 2012 and 2015), embryo features (van der Veken, 1965; Vanhecke, 1974; Verbelen, 1970) and surface structure of diaspores (Schuyler, 1971; Haines and Lye, 1983; Goetghebeur and Van den Borre, 1989; Lye, 2000), and have therefore never discovered the diversity in silica deposition. The structure of the pericarp is of major importance for seed dispersal. In Cyperaceae many ecological dispersal types are established on the basis of the dispersing agents (van der Pijl, 1972), viz. wind (anemochory), water (hydrochory) and animals (zoochory); in the latter case the diaspores are either attached outside the animals (epizoochory) or they are eaten and passed through the alimentary system (endozoochory). Plants dispersed by wind have light fruits with hairy flat or smooth attachments such as Eriophorum and Trichophorum alpinum (L.) Pers., or flat achenes like Fimbristylis pterygosperma R.Br. and F. caloptera Latz (Lye, 2000). Water is the most important vector for dispersal in the family Cyperaceae (Leck and Schütz, 2005), and also anemochorous species are often secondarily dispersed by water after landing on a wet surface. Plants with diapores adapted to hydrochory either have so small and light fruits that they do not penetrate the surface water film (e.g. Scirpus sylvaticus L.) or they have diaspores which float because they have corky tissues or large air-filled cells in the pericarp like Bolboschoenus maritimus (L.) Palla (Lye, 2000). Animals are the second most important vector for dispersal, most importantly birds (Brochet et al., 2010), less commonly mammals (Kjellsson, 1985; Carter, 1993) or insects, particularly ants, and then named myrmecochory (Sernander, 1906; Gaddy, 1986). Diapores with hooks (in e.g. Uncinia) or setae with recurved barbs (in e.g. Eleocharis, Rhynchospora and Scirpus) are adapted to become attached to the fur, feathers or feet of animals, especially birds, but when landing on water they are dispersed by hydrochory (Ridley, 1930; Outred, 2002; Leck and Schütz, 2005). Waterfowl (ducks, geese and swans) eat large amounts of fruits of species of Cyperaceae; in teal (Anas crecca) 54% of the fruits of Schoenoplectiella mucronata (L.) Jung & Choi ingested were evacuated in a viable condition, and germinability was increased by gut passage (Brochet et al, 2010). In another experiment Bolboschoenus maritimus was the only species of ten which had diaspores germinating better after having passed through the digestive tract of the mallard (Anas platyrhynchos), and 51% of the diaspores were recovered in the feces (Wongsriphuek et al., 2008). It has been shown that germination in Bolboschoenus maritimus, Schoenoplectus lacustris (L.) Palla and S. acutus (Bigelow) Á. Löve and D. Löve is dependent on improved permeability of the pericarp (Clevering, 1995; Lacroix and Mosher, 1995). Bell in Leck and Schütz (2005) reported an increase in germination from 7 to 69 % after avian gut scarification for Eleocharis dietrichiana (Boeck.) C.B.Cl. Thus the pericarp thickness and structure is of paramount importance for water and gas permeability and elimination of dormancy. Neither gibberellic acid nor available

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nutrients improved germination of Carex diaspores (Bekker et al., 1998). However, biochemical changes such as transforming lipid storage materials to starch are important for cessation of dormancy in the Carex flava group (Schmid, 1980). Most species of Cyperaceae need light for germination, for some low temperature stratification is needed, for others specified oxygen levels alternating with flooding and drying are important (Leck and Schütz, 2005), and Schoenoplectiella hallii (A. Gray) Lye needs ethylene produced by decaying organic materal for germination (Baskin et al., 2003). The genus Scirpus L. as described by Linnaeus (1753 & 1754) consists of a very heterogeneous group of species at present considered to belong to many different genera and placed in six different tribes in subfamily Cyperoideae sensu Goetghebeur (1998). The 24 species described by Linnaeus in 1753 are now placed in the following genera and tribes (Govaerts et al., 2007): Scirpus with the type of the genus S. sylvaticus L. in tribe Scirpeae, Eleocharis R. Br. in tribe Eleocharideae, Bolboschoenus Palla, Schoenoplectus Palla and Schoenoplectiella Lye in tribe Fuireneae, Bulbostylis Kunth and Fimbristylis Vahl in Abildgaardieae, Isolepis R. Br., Cyperus L. and Scirpoides Séguier in Cypereae and Scleria Berg. in Sclerieae. Also Schoenus compressus L. has for many years been placed in the genus Scirpus, but is now recognized as Blysmus compressus (L.) Link in tribe Dulichieae. On the other hand Eriophorum cyperinum L. is now placed in the genus Scirpus and Eriophorum alpinum L. is now the type species of the genus Trichophorum. According to Goetghebeur (1998) Cyperaceae tribe Scirpeae Kunth ex Dumort (1827) consists of six genera, viz. Scirpus L. (20 species), Eriophorum L. (20 species), Phylloscirpus C.B. Clarke (4 species), Oreobolopsis T. Koyama & Guaglian. (3 species), Amphiscirpus Oteng-Yeb. (1 species) and Trichophorum Pers. (8-10 species) including Eriophorella Holub. Dhooge et al. (2003) described a new genus Zameioscirpus Dhooge & Goetgh. (3 species) in this tribe; it is most similar to Phylloscirpus. Also three other genera belonging to tribe Scirpoideae are described since Goetghebeur’s (1998) revision, viz. Cypringlea (Strong, 2003), Calliscirpus (Gilmour et al., 2013) and Rhodoscirpus (Léveillé-Bourret et al., 2015), but these genera are not investigated in this paper. In addition to these seven genera an eighth genus Erioscirpus Palla (2-3 species) has been separated from Eriophorum. Goetghebeur (1998) writes about this genus: "Erioscirpus, with its remarkable species in the Himalayas, might well deserve generic status, as suggested by its rather deviating embryo shape". Erioscirpus differs from Eriophorum as its stem is lacking central air tissue (Sharma, 1973), the flower has different bristles (Palla, 1896) and the plant has a very different habit, being confined to vertical rock- or soil-walls drying completely out for months. However, Erioscirpus has only recently been studied by molecular methods and is now transferred to tribe Cypereae (Yano et al., 2012). Palla (1896) also described another new genus, viz. Eriophoropsis Palla based on the single species Eriophorum virginicum L. with slightly different anatomical characteristics. Eriophoropsis has not been accepted by other authors (Tucker and Miller, 1990; Ball and Wujek, 2002). Eriophorella Holub has the same type species as Trichophorum, and is therefore not described here. Khokhrjakov (1985) described a new genus Maximowiczia based on the single species Eriophorum japonicum Maxim. (Scirpus maximoviczii C.B.Cl.). This species has the typical

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habit of Eriophorum, but differs from the other species in this genus in having six long bristles with outgrowths (Koyama, 1958). However, Khokhrjakov (1989) had to change this name to Maximowicziella due to homonymy, non Maximowiczia Rupr. (1856). Tzvelev (1999) described the genus Kreczetoviczia with the type species K. cespitosa (L.) Tzvelev, and also including K. pumila (Vahl) Tzvelev and K. uniflora (Trautv.) Tzvelev, all species by most authors today placed in the genus Trichophorum. The classification of tribe Scirpeae in Goetghebeur (1998) is only partly supported by recent phylogenetic analyses. When using combined rbcL and trnL-F data in both maximum parsimony and Bayesian analyses tribe Scirpeae sensu Goetghebeur (1998) comes out as a group separate from Dulichieae and Cariceae (Dhooge et al., 2003; Dhooge and Goetghebeur, 2004; Dhooge, 2005). However when using data from ETS-1f or combined ETS-1f and trnL-F data at least Amphiscirpus nevadensis (S. Watson) Oteng Yeboah comes outside the Scirpeae cluster (Dhooge, 2005). However, in Léveillé-Bourret et al. (2014) Amphiscirpus clusters with Phylloscirpus and Zameioscirpus. According to this paper Trichophorum (including Oreobolopsis and Cypringlea) seems to be the genus sister to Carex and tribe Cariceae. If this is true the genus Trichophorum has to be excluded from Scirpeae, but in future phylogenetic studies it may cluster differently. Both Dhooge and Goetghebeur (2004) and Muasya et al. (2009) show that Eriophorum L. is not well separated from Scirpus L. s.str., and that Oreobolopsis T. Koyama & Guaglian. is not well separated from Trichophorum Pers. Both phylogenetic analyses show Phylloscirpus separated from but closely related to Zameioscirpus, and that the complex TrichophorumOreobolopsis is the group most prominently separated from the other genera of tribe Scirpeae. Muasya et al. (2009) investigated more species than Dhooge and Goetghebeur (2004) and also throw light on the relationship between Trichophorum Pers. s.str. and the segregated genus Kreczetoviczia Tzvelev. They found T. alpinum (L.) Pers. to cluster with T. pumilum (Vahl) Schinz & Thell. [= Kreczetoviczia pumila], while T. caespitosum (L.) Hartm. [K. caespitosa, type of genus Kreczetoviczia] cluster with Oreobolopsis and various American and Asian species of the genus Trichophorum, including T. rigidum (Steud.) Goetgh., which is investigated in this paper. Similar results are presented by Léveillé-Bourret et al. ( 2014); they also show that the recently described genus Cypringlea cluster with Trichophorum. The aim of this paper was to evaluate both the genera accepted by Goetghebeur (1998) and those not accepted [Eriophoropsis, Erioscirpus, Kreczetoviczia and Maximowicziella] using new characters, with particularly emphasis on the genus Erioscirpus by Goetghebeur included in Eriophorum, but which I had studied in Nepal and found very different. However, since Yano et al. (2012) have found this genus to belong to tribe Cypereae, this part of the work is given less emphasis. Material and methods Sections of achenes of twenty eight species (or thirty taxa) in seven genera of Cyperaceae tribe Scirpeae sensu Goetghebeur (1998) were investigated using SEM and x-ray analysis to study cell types and presence of different minerals (Table 1). Plants from the north temperate genera Scirpus, Eriophorum (including Eriophoropsis), Trichophorum (including

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Kreczetoviczia) were collected in Scandinavia or Canada. Achenes from these collections were supplemented by achenes from other collections in the Oslo herbarium, Norway (O), while achenes from the genera Amphiscirpus, Erioscirpus, Oreobolopsis, Phylloscirpus and Zameioscirpus were sampled from the herbaria at Århus (AAU), Denmark or Kew (K), England. Fruits of Scirpus maximoviczii (Maximovicziella japonica) were provided by Takuji Hoshino from Okayama, Japan. Care was taken to ensure that all fruits investigated were fully mature. Vouchers from own collections are stored in the herbaria at the University of Oslo (O) or the Norwegian University of Life Sciences (NLH); see Table 1. All European species were investigated as well as about 1/3 of the total number of species from the tribe, including all species which are the types of the seven accepted genera investigated as well as the three investigated genera not accepted. All measurements (Table 2) are given in µm or atomic percent. Usually 3-4 fruits from each collection were studied. Since the mineral content in the various structures to a large extent depends on its age and development, statistical calculations are not undertaken; only presence or absence and location of silica and other minerals in different parts of the pericarp has systematic value. This work was started in 2008 with collection of material, and most of the x-ray and SEM work was carried out in 2009-2011. Thus two genera described later, viz. Calliscirpus (Gilmour et al., 2013) and Rhodoscirpus (Léveillé-Bourret et al., 2015) were not included; the same applies for the genus, Cypringlea, described in 2003 (Strong, 2003). The cross-sections were done by hand-made cuts with the exception of that of Trichophorum pumilum, where the pericarp was so hard that a microtome had to be used. The SEM photography and energy dispersive x-ray spectroscopy analysis (EDXA) was performed with a Zeiss EVO – 50 – EP scanning electron microscope combined with an INCA Energy 350 energy dispersive x-ray instrument from Oxford Instruments Analytical. This microanalysis is a technique used for elemental analysis of a sample. A high energy beam of x-rays is focused into the sample being studied. The beam may excite an electron from an inner shell of an element, ejecting it from the shell and creating an electron hole, which is immediately filled by an electron from an outer higher-energy shell; the difference in energy between the higher-energy shell and the lower-energy shell is released in the form of an xray. The number and energy of the x-rays emitted from a specimen is then measured by an energy-dispersive spectrometer. The method relies on the fundamental principle that each element has a unique atomic structure allowing unique sets of peaks on its x-ray emission spectrum. Results The pericarp in species of Cyperaceae tribe Scirpeae consists of three layers: (a) an epidermis (epicarp) with cells wider than tall with few exceptions, and usually with prominent amounts of silica (absent in Phylloscirpus boliviensis, Trichophorum uniflorum (Trautv.) Malyschev & Lukitsch. and Zameioscirpus muticus Dhooge & Goetgh. only), (b) a middle layer (mesocarp) 2-12 cells thick with little or no silica, and (c) an inner fibrous layer (endocarp) sometimes containing vessels but never containing silicon. The epicarp is stable and changes little from one collection to the other, but the thickness of the mesocarp

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sometimes vary considerably from collection to collection (see data on Eriophorum vaginatum in Table 2). The SEM and x-ray investigation of the pericarp show that silica is present in small to moderate amounts in all investigated species except the three species mentioned above. However, the pericarp mainly consists of carbon (50-80%), oxygen (1540%) and 0.1-1% other elements (silicon, potassium, calcium, aluminium, magnesium and chlorine); only the silica phytoliths and various special silica layers contained 3-14% silicon (Table 2, I). Potassium was present in amounts up to 0.6% in cell walls, other elements constitute 0.1-0.2% only. Only Trichophorum uniflorum has accumulated a thin layer of calcium in the outermost part of the pericarp. Four of seven investigated genera have pericarps in which silica is accumulated as phytoliths. The pericarp similarities between Scirpus s.str. and Eriophorum (including Eriophoropsis and Maximovicziella) and between Oreobolopsis and part of Trichophorum (including Kreczetoviczia) were congruent to molecular phylogenetic results. Variation in pericarp characters Thickness of pericarp in µm and number of cells in section (Table 2 columns A-B) The pericarp in species of Cyperaceae tribe Scirpeae is extremely variable in thickness, from very thin and fragile in Eriophorum (Fig. 1) and slightly thicker in Trichophorum cespitosum (Fig. 2) to very thick and hard in Trichophorum pumilum (Fig. 3), T. uniflorum and Amphiscirpus (Fig. 4). Scirpus cyperinus from Canada, Trichophorum alpinum from Norway and Erioscirpus comosus (in tribe Cypereae) from Himalaya all have a pericarp less than 15 µm thick, while in Amphiscirpus nevadensis from New Mexico the pericarp is 100150 µm thick. However, individual variation can be considerable, i.e. one collection of Eriophorum vaginatum had a pericarp 15-20 µm thick (from Fauske, North Norway), another 45-50 µm thick (from Geiranger, South Norway). The number of cells across the pericarp vary from 2(3) to 12; most genera have a 3-6 cell thick pericarp. Only Amphiscirpus, Trichophorum pumilum and T. uniflorum are atypical with an 8-10 (-12) cell thick pericarp. Epidermis cells wider than long versus isodiametric or taller than wide (Table 2 column C) In 23 species in 10 genera the epidermis cells of the pericarp are wider than long. However, Phylloscirpus acaulis subsp. pachycaulis, P. boliviensis (Barros) Dhooge & Goetgh., Trichophorum pumilum and Zameioscirpus have epidermis cells taller than wide. Presence of silica phytoliths (Table 2 column D) Silica phytoliths were present in epidermis cells of the pericarp in 18 out of 28 species. Three genera, Amphiscirpus, Phylloscirpus and Zameioscirpus are entirely lacking such phytoliths, while in Trichophorum they are present in two species and absent in three. Thickness of outer epidermis cell wall in µm (Table 2 column E) The outer cell wall of the epidermis of the pericarp is very thin, usually 0.5-1 µm in all species except in species from the Andes (Trichophorum rigidum, Phylloscirpus and Zameioscirpus) as well as the two subarctic species Trichophorum pumilum and T. uniflorum. In Zameioscirpus muticus this cell wall is 6-10 µm thick.

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Silica and calcium sheet in inner cell wall of epidermis (Table 2 columns F-G-H) A silica sheet is present in the epidermis cells of the pericarp in 25 of 28 species; this sheet is lacking in the three species Phylloscirpus boliviensis, Trichophorum uniflorum and Zameioscirpus muticus. The sheet is usually 0.5-3 µm thick, but particularly in the genus Eriophorum the thickness of the sheet is very variable even within the same species, e.g. in E. latifolium from an alpine locality the sheet is 10-15 µm thick, while in a lowland locality it was 2-3 µm thick. The sheet is continuous in 18 species and discontinuous (broken) at the tangential cell walls in 8 species. However, the distinction between a continuous and a discontinuous silica sheet is sometimes obscure, such as in Scirpus and Trichophorum. A calcium sheet was found in a single species, Trichophorum uniflorum from arctic Russia. Silicon in atomic percent in phytoliths and in cell walls of cells below epidermis (Table 2 columns I-J) Excepting the three species completely lacking silicon in their pericarp, all pericarps from the other species have conspicuous amounts of silicon varying from 0.6% in the South American species Phylloscirpus pachycaulis and Zameioscirpus muticus to 10.6% in Trichophorum pumilum from North Norway. Silicon is either absent or present in minor amounts (0.1-0.8%) in cells below epidermis in the pericarp; only a few species in two closely related genera, Scirpus and Eriophorum, had higher amounts (1.6-3.0%) of silicon in cell walls below the epidermis. Notes on individual genera As treated here Cyperaceae tribe Scirpeae Kunth ex Dumort (1827) contains ten genera and about 75 species, but only seven were investigated in this paper. Genera investigated but not accepted as relevant genera in tribe Scirpeae are not numbered and placed in square brackets. 1. Amphiscirpus Oteng-Yeb. in Notes Roy. Bot. Gard. Edinburgh 33 (2): 308 (1974).; type and only species: A. nevadensis (S.Watson) Oteng-Yeb. Fig. 4. Originally separated from Scirpus mainly on the basis of the culm anatomy (OtengYeboah, 1974), but it also differs from the slightly similar genus Schoenoplectus in its prominently ciliate ligules (Smith, 2002). Bruhl (1995) associated this plant with the high Andean genus Phylloscirpus, from which Amphiscirpus differs prominently in its much thicker pericarp consisting of many more cells, in being more encrusted with silica and in having the silica layer in the epidermis cells discontinuous (see Table 2). This genus differs from all other genera of the tribe due to its very thick (100-130 µm) pericarp. The fruit is more similar to species in tribe Fuireneae than to other species in tribe Scirpeae. 2. Calliscirpus C.N. Gilmour, J.R. Starr & Naczi in Kew Bull. 68: 98 (2013); type species: C. criniger (A. Gray) C.N. Gilmour, J.R. Starr & Naczi

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A genus clearly distinct from all other genera in tribe Scirpeae (Gilmour et al., 2013; (Léveillé-Bourret et al., 2014), but it was described after my SEM and x-ray investigation had been finished and is thus not included in this paper. 3. Cypringlea M.T. Strong in Novon 13: 123 (2003).; type species: C. analecta (Beetle) M.T. Strong This genus is considered similar to Trichophorum, but differs in “habitat, leaf blade development and morphology, inflorescence morphology, and the possession of rudimentary perianth bristles” (Strong, 2003; Reznicek and Elizondo, 2008). According to Gilmour et al. (2013) and Léveillé-Bourret et al. (2014) it clusters with species of Trichophorum and Oreobolopsis, and may eventually prove to be part of a Trichophorum s. lat. It was not included in my SEM and x-ray investigation. [Eriophoropsis Palla in Bot. Zeitung, 2. Abt., 54 (1): 148, in obs., 151, in clavi (1896); type species: Eriophoropsis virginica (L.) Palla This genus has been traditionally almost always placed in the genus Eriophorum. In fruit characters it is indistinguisable from the genus Eriophorum (see Tucker and Miller, 1990 and table 2). However, E. virginica is easily recognized with its late flowering season and conspicuous reddish brown perianth segments. Other species with reddish inflorescences have unlike E. virginica solitary spikelets, but it is not a significant generic level character.] 4. Eriophorum L. in Spec. Pl. 1: 52 (1753); type species: E. vaginatum L. See Jarvis (2007). Fig. 5. This genus is homogeneous and easily recognized based on its numerous (10-25) long smooth filiform perianth segments. The North American species E. crinigerum (A.Gray) Beetle, which has six barbed perianth bristles 2-3 times as long as the fruit (Ball and Wujek, 2002) is now placed in a new genus Calliscirpus (Gilmour et al., 2013). The East Asian species E. japonicum Maxim. is now included in the genus Scirpus because of its fewer setae (Koyama, 1958) and molecular relationship (Léveillé-Bourret et al., 2014). The pericarp in all species of Eriophorum is very similar to that of the genus Scirpus, which is in accordance with the close phylogenetic similarity between these two genera (Muasya et al., 2009; Hinchliff and Roalson, 2013; Léveillé-Bourret et al. 2014). However, the thickness of the silica sheet is very variable from 0.5-15 µm. Koyama (1958) included the traditional species of Eriophorum as section Vaginati of the genus Scirpus, a solution supported by both molecular data and fruit structure. [Erioscirpus Palla in Bot. Zeitung, 2. Abt., 54 (1): 148, in obs. (1896); type species: E. comosus (Wallr.) Palla This genus is traditionally placed in the genus Eriophorum, but is a xerophilous plant, which differs in culm anatomy and bristle morphology (Palla, 1896; Mehra and Sharma, 1965; Sharma, 1973). Molecular data places Erioscirpus in tribe Cypereae (Yano et al., 2012). The pericarp in Erioscirpus has the silica sheet of the lower part of the epidermis cells discontinuous, while in Eriophorum it is continuous. While species of Eriophorum always

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grow in wet habitats, species of Erioscirpus colonize dry ridges and hill tops (Yano et al., 2012).] [Kreczetoviczia Tzvelev in Bot. Zhurn. (Moscow and Leningrad) 84 (7): 112 (1999); type species: K. caespitosa (L.) Tzvelev Fig. 6. This genus was described recently to cover north temperate species of Trichophorum without long flattened perianth segments. Trichophorum is a very heterogeneous genus both morphologically and regarding fruit anatomy. However, the pericarp of the type species of Kreczetoviczia, viz. K. cespitosa, (figs. 2 and 7) is very similar to that of the type species of the genus Trichophorum, viz. T. alpinum (fig. 11), while the fruit structure of two other species, viz. K. pumila (fig. 12) and K. uniflora, is very different (compare figs. 11 and 12). The genus Kreczetoviczia is accepted as a good genus by some Russian botanists and in some databases.] [Maximovicziella A. P. Khokhr. in Analiz Fl. Kolymskogo Nagorya 15 (1989); type species: M. japonica (Maxim.) A. P. Khokhr. Syn. Scirpus maximowiczii C.B. Cl. In habit this species is similar to the genus Eriophorum, but like Scirpus it has six perianth segments only, and the perianth bristles are unique in being scabrid (sometimes antrorsely barbed) and at least three times as long as the nutlet. The plant was originally described as Eriophorum japonicum Maxim., but is now placed in the genus Scirpus because of its fewer setae (Koyama, 1958) and molecular relationship (Léveillé-Bourret et al., 2014). The pericarp is identical to that of Eriophorum and Scirpus. In the Panarctic Flora (Elven, 2016) as well as in Russia it is accepted as a good genus.] 5. Oreobolopsis Koyama & Guaglione in Darwiniana 28: 79 (1988); type species: O. tepalifera Koyama & Guagl. Fig. 7. This genus is characterized by a medium thick pericarp and presence of silica in both the inner and outer cell wall of the epidermis cells (table 2). The genus is closely related to the heterogeneous genus Trichophorum, and genetically clusters with T. cespitosum (Muasya et al., 2009), the type species of the genus Kreczetoviczia. However, the silica sheet in these species is different (see table 2 and compare fig. 8 with fig. 7), continuous in Trichophorum cespitosum, discontinuous in both species of Oreobolopsis. In Léveillé-Bourret et al. (2014) Oreobolopsis clusters with T. rigidum. Since the genus Oreobolopsis was described before Kreczetoviczia, Trichophorum cespitosum is likely to be included in Oreobolopsis rather than in Kreczetoviczia, if future research show T. cespitosum to be generically separate from T. alpinum. 6. Phylloscirpus C.B. Clarke. in Bull. Misc. Inform., Addit. Ser. 8: 45 (1908); type species: P. acaulis (Phil.) Goetgh. & D.A. Simpson. Fig. 8. This genus is most similar to Zameioscirpus, but differs in its eligulate leaves (Dhooge et al., 2003; Dhooge and Goetghebeur, 2004; 2007). In pericarp structure this genus is heterogeneous since P. boliviensis and P. acaulis subsp. pachycaulis have isodiametric epidermis cells, while P. acaulis subsp. acaulis and P. deserticola have broad and short epidermis cells. Otherwise P. boliviensis is very distinct in lacking silica in the pericarp.

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7. Rhodoscirpus Léveillé-Bourret, Donadio & J.R. Starr in Taxon 64: 940 (2015); type species: R. asper (J. & C. Presl) Léveillé-Bourret, Donadio & J.R. Starr A genus clearly distinct from all other genera in tribe Scirpeae (Léveillé-Bourret et al., 2015), but it was described after my SEM and x-ray investigation had been finished and is thus not included in this paper. 8. Scirpus L. in Sp. Pl. 1: 51 (1753); type species: S. sylvaticus L. See Jarvis (2007). Fig. 9. This genus is homogeneous when 23 of the 24 species described by Linnaeus (1753) are excluded. Bristles vary from smooth to barbellate, rarely absent. Pericarp structure is similar to that of Eriophorum. The European species of Scirpus have much smaller fruits than those of Eriophorum. However, some American species of Scirpus have somewhat larger fruits (Schuyler, 1964; 1967; Whitmore and Schuyler, 2002), but Scirpus fruits are generally smaller than Eriophorum fruits. The fruits of Nordic species of Scirpus weigh 0.06-0.15 mg, while those of Eriophorum weigh 0.26-1.11 mg (Lye, 1993). The close similarity between Scirpus and Eriophorum is also documented through molecular works (Dhooge and Goetghebeur, 2004; Simpson et al., 2008; Muasya et al., 2009; Léveillé-Bourret, 2014). 9. Trichophorum Persoon in Syn. Pl. 1: 69 (1805); type species: T. alpinum (L.) Pers. Figs. 10-11. This genus is the most heterogeneous and is the least understood of all genera in tribe Scirpeae. It is sometimes reported as the sister genus to Carex (Léveillé-Bourret, 2014). T. alpinum differs from all species in having a very thin pericarp and flattened bristles somewhat similar to those of the genus Eriophorum. A Californian species, Scirpus clementis M.E. Jones, was transferred to the genus Trichophorum by Smith (1995), but later transferred to the genus Oreobolopsis by Dhooge and Goetghebeur (2002). Trichophorum pumilum and T. uniflorum are atypical species in the genus due to their very thick pericarps only shared by Amphiscirpus nevadensis in tribe Scirpeae. The pericarp of these two species are most divergent from the type species T. alpinum. It is therefore surprising that in Muasya et al. (2009) T. alpinum cluster with T. pumilum and not with T. cespitosum, which has a pericarp similar to that of T. alpinum. T. pumilum differs from T. alpinum both in its thick pericarp and different isodiametric epidermis cells without phytoliths and a discontinuous silica sheet (Table 2). The pericarp of T. cespitosum differs from that of T. alpinum only in its somewhat thicker pericarp. T. uniflorum is very atypical in lacking silica in the pericarp; instead the outermost part of the fruit consists of a thin layer of calcium (not observed in any other species investigated). 10. Zameioscirpus Dhooge & Goetgh. in Plant Syst. Evol. 243: 75 (2003); type species: Z. atacamensis (Philippi) Dhooge & Goetgh. Fig. 12. This genus is well documented by Dhooge et al. (2003) both as regards classical morphology and anatomy and phylogenetic analysis. The pericarp differs from that of all other investigated genera in having distinct square to round air-filled cells in the outer cell layer being 3 times longer and wider than the cells in the two inner layers. The outer cell wall is 3-10 µm thick, and silica is present in small amounts as 1 µm thick Si-sheets mainly in the lower cell wall of the large epidermis cells. Z. muticus has the thickest outer cell wall

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of the fruit of all investigated species (6-10 µm thick). According to pericarp structure Zameioscirpus can only be compared with the genus Phylloscirpus. This fits well with the results obtained in molecular phylogentic analyses (Dhooge et al., 2003; Léveillé-Bourret et al., 2014). Dhooge et al. (2003) separate Zameioscirpus from the similar Phylloscirpus on its ligulate leaves (eligulate in Phylloscirpus). However, Phylloscirpus has the silica sheet of the epidermis continuous if present, while in Zameioscirpus this is broken at the tangential cell walls. Z. muticus Dhooge & Goetgh. is aberrant in completely lacking silica in the cell walls. Key to the investigated genera of Cyperaceae tribe Scirpeae [including Erioscirpus now in tribe Cypereae] based on diaspore and particular periderm structure Three genera Calliscirpus, Cypringlea and Rhodoscirpus are not included. Eriophorum, Scirpus (including Maximowicziella) and Trichophorum p.p. cannot be separated on pericarp structure alone. Erioscirpus was placed in Eriophorum until recently. For an alternative key to all genera including vegetative characters see Léveillé-Bourret et al. (2015). 1. Pericarp thick (75-130 µm) with 9-12 cell layers; phytoliths absent; silica sheet in inner epidermis cell wall discontinuous or absent 2 1. Pericarp thin (10-60 µm) with 2-8 cell layers; phytoliths present or absent; silica sheet in inner epidermis cell wall continuous, discontinuous or absent 3 2. Pericarp 100-130 µm thick; silica sheet 3-4 µm thick Amphiscirpus 2. Pericarp 75-90 µm thick; silica sheet to 1 µm thick Trichophorum p.p. (T. pumilum; T. uniflorum) 3. Epidermis cells isodiametric or taller than wide 4 3. Epidermis cells wider than tall 7 4. Silica sheet in inner cell wall of epidermis absent 5 4. Silica sheet in inner cell wall of epidermis present 6 5. Outer epidermis cell wall 6-10 µm thick Zameioscirpus muticus 5. Outer epidermis cell wall 3-4 µm thick Phylloscirpus boliviensis 6. Silica sheet in inner cell wall of epidermis discontinuous Zameioscirpus p.p. 6. Silica sheet in inner cell wall of epidermis continuous Phylloscirpus p.p. 7. Silica phytoliths absent Trichophorum p.p. (T. rigidum) 7. Silica phytoliths present 8 8. Silica sheet in inner cell wall of epidermis discontinuous 9 8. Silica sheet in inner cell wall of epidermis continuous 10 9. Outer epidermis cell wall 0.5-1 µm thick; fruit surrounded by filiform perianth segments Erioscirpus (now in tribe Cypereae) 9. Outer epidermis cell wall 1-2 µm thick; fruit surrounded by scale-like perianth segments Oreobolopsis 10. Fruit surrounded by 10-25 long filiform smooth perianth segments Eriophorum 10. Fruit surrounded by up to 6 filiform smooth or scabrid bristle like perianth segments 11 11. Fruit surrounded by 6 smooth filiform or compressed perianth segments many times longer than the fruit 12

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11. Fruit surrounded by up to 6 filiform or scabrid bristle like perianth segments up to twice as long as the fruit, or if prominently scabrid sometimes longer 13 12. Inflorescence a single small terminal spikelet; perianth segments white, flattened Trichophorum p.p. (T. alpinum) 12. Inflorescence with many spikelets; perianth segments more greyish and terete Scirpus p.p. 13. Inflorescence a single small terminal spikelet Trichophorum p.p. (including T. cespitosum) 13. Inflorescence lax or capitate, consisting of few to numerous spikelets Scirpus p.p. Discussion This study has added a number of novel anatomical characters useful in discriminating taxa in Cyperaceae tribe Scirpeae. Since all genera and most of the species have been subject to molecular phylogenetic analysis (Dhooge et al., 2003; Dhooge, 2005; Simpson et al., 2008; Muasya et al., 2009; Yano et al., 2012; Léveillé-Bourret et al., 2014, 2015), it is possible to evaluate which characters are most useful to discriminate the various taxa on a generic and specific level. Some pericarp characters correlate very well with molecular classification, others less so. Variation and functionality of the pericarp

One of the most interesting discoveries was the total absence of silicon in three species in three different genera (Table 2, column F). Although this loss is probably genetically based, the evolution may have been triggered by an alkaline environment, where Si is present in minor amounts. While presence or absence of silicon cannot be used as a taxonomic character to discriminate genera, it is useful in separating species, and the characteristics of the silica sheet is important. Since silica tubercles or phytoliths are developed in all species of three genera and lacking in all species of three other genera (Table 2, column D), they are of considerable taxonomic value. The presence and absence of Si phytoliths in different species in the heterogeneous genus Trichophorum may indicate that this genus needs rearrangement. Almost the same variation is seen in the presence of a continuous versus a discontinuous (broken) silica sheet (Table 2, column G). The outer cell wall of the epidermis is often only 0.5-1 µm thick in species growing in temperate regions, while in species from the high Andes at 3300-4840 m and in arctic Siberia this cell wall is 2-10 µm thick (Table 2, column E). A thick outer epidermis cell wall is particularly pronounced in the South American genera Phylloscirpus and Zameioscirpus. This may be an adaptation to survive in an overexposed UV radiation regime, as a thick cell wall will filter much of the damaging radiation; this is particularly important for the species lacking a silicon layer, viz. Phylloscirpus boliviensis, Trichophyllum uniflorum and Zameioscirpus muticus. Species lacking a silica sheet all have a fruits with a 3-10 µm thick outer epidermis cell wall. The shape and size of epidermis cells are other differential characters (table 2, column C). Most genera and species have fairly small epidermal cells, which are wider than tall and about 3-5 µm tall and about 10 µm wide. However, Zameioscirpus has epidermis cells 10-15 µm tall and usually taller than wide. Also Trichophorum pumilum has about 15 µm tall epidermis cells, which are taller than wide, but other species of this genus have the cells

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wider than tall. The same applies to the genus Phylloscirpus, where two species have wide cells and two other species 15-25 µm tall cells; P. boliviensis has the largest epidermis cells of all investigated species in Cyperaceae tribe Scirpeae. Achenes with such large air-filled cells usually float well on the water and are hydrochorous (Lye, 2000). Thickness of pericarps in µm (Table 2, column A) and number of cells in transverse section (Table 2, column B) are not useful discriminating characters with the exception that the genus Amphiscirpus is well separated from all other genera in tribe Scirpeae on the thickness of the pericarp in both µm and number of cells in transverse section. Since Amphiscirpus has a thick-walled achene very similar to that of the genus Schoenoplectus, it is probably better adapted to the abrasive action in birds’ gizzards and the digestive acids in stomachs than other species in tribe Scirpeae, and thus germination may be increased after passing the birds’ guts due to increased permeability of the pericarp like in several species of Bolboschoenus and Schoenoplectus (Brochet et al., 2010). Mueller and van der Valk (2002) used lignin content of seeds to measure seed coat durability in Schoenoplectus, but for species with thinner pericarp the thickness of the silicon platform may be more useful. Wongsriphuek et al (2008) found that size of diaspores was unrelated to resistance to digestion by birds, while Soons et al. (2008) and Figuerola et al. (2010) concluded that small diaspores survived gut passage better. Despite the great variation in silicon content in phytoliths and silica sheets, from 0.6 to 10.6 atomic percent, these values (Table 2, column I) seem to be of little discriminating value between genera and species. Perhaps the values are more dependent on age of fruit or available silicon in the soil. Cell walls below the epidermis (Table 2, column J) contain so little silicon in all species that this character is of no value for discriminating between species. The structure of the pericarp is adapted for different types of seed dispersal. Winddispersed plants like species of Eriophorum and Trichophorum alpinum have a thin pericarp usually 10-20 µm thick with hairy flat or smooth attachments. However, these species are also dispersed by water, and in E. vaginatum an interesting divergent evolution occurs: (1) among populations with pericarp 15-25 µm thick evolution is probably towards ligher diaspores facilitating better wind transport, while (2) among population with 40-50 µm thick pericarp evolution is possibly towards heavier and thus more nutrient rich diaspores with better adaptation to germination in a nutrient poor environment. Many of the characters outlined above will be useful in discriminating species not only in present day classifications, but also in paleobotany identifying fossil material (JiménezMejias and Martinetto, 2013; Martinetto et al., 2014). Systematic implications

The fact that I found no real differences in the pericarp between species of Eriophorum and Scirpus is interesting in relation to molecular research, as Léveillé-Bourret et al. (2014) noted the paraphyly of Eriophorum within Scirpus. However, Koyama (1958) is the only botanist, who has included the genus Eriophorum in Scirpus, but he also included many genera in Scirpus now known to belong to other tribes, e.g. Fuirena, Isolepis and Schoenoplectus. However, more species should be investigated and more coding markers should be developed before taxonomic changes are considered (Léveillé-Bourret et al., 2014).

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Of the genera in tribe Scirpeae investigated here, two genera, Eriophoropsis and Maximovicziella, have fruit structures identical to those in the genus Eriophorum and Scirpus; they only differ from Eriophorum in their different perianth structures and/or number or colour of bristles; Maximovicziella differs from its nearest relative Scirpus in its different setae (Koyama, 1958; Léveillé-Bourret et al., 2014). However, these two genera are not worth retaining as different bristle-types (or presence/absence of bristles) are frequently found within the same genus; for such variations in particularly Eleocharis, Rhynchospora and Fuirena see Haines and Lye (1983). The genus Kreczetoviczia proposed by Tzvelov (1999) is also split off from another genus (Trichophorum) mainly because of different perianth segments. However, while the genera Eriophorum, Eriophoropsis, Maximovicziella and Scirpus form a group of closely related species both morhologically, genetically and as regards fruit structure, the genus Trichophorum is very heterogeneous as regards fruit structure, type (or presence/absence) of perianth segments, general morphology as well as genetically. Therefore, Trichophorum may have to be split into two or more genera when more genetical and morphological data become available. As regards perianth structure the genus Kreczetoviczia is strikingly different from the type species of Trichophorum, but regarding pericarp structure the type species of Kreczetoviczia is more similar to the type species of Trichophorum than it is to other species of Kreczetoviczia. According to molecular data (Dhooge and Goetghebeur, 2004; Muasya et al., 2009; Léveillé-Bourret et al., 2014, 2015) a monophyletic genus Trichophorum can perhaps only be obtained if the genera Kreczetoviczia and Oreobolopsis are united with Trichophorum. Consequently the genus Trichophorum should not be split unless future more detailed and decisive molecular data becomes available, preferably after a full revision of the genus. I thus accept seven of the eleven investigated genera as good genera in tribe Scirpeae, viz. Amphiscirpus, Eriophorum, Oreobolopsis, Phylloscirpus, Scirpus, Trichophorum and Zameioscirpus. This means that I found Goethebeur’s (1998) classification correct for all genera except for Erioscirpus, which is a good genus in tribe Cypereae as shown by Yano et al. (2012). However, with more available molecular data Oreobolopsis may have to be united with Trichophorum and possibly Eriophorum with Scirpus. The present position of the genus Amphiscirpus within tribe Scirpeae is not fully documented. Acknowledgements The SEM photography and energy dispersive x-ray analysis (EDXA) was made possible through Elin Ørmen at the Department of Plant and Environmental Sciences, Microscopy Division, Norwegian University of Life Sciences. I also thank the curators at the herbaria AAU, K and O

for permission to sample fruits from their collections. Fruits of Scirpus maximowiczii (Maximovicziella) were kindly provided by Takuji Hoshino from Okayama, Japan. I also thank two very knowledgable anonymous reviewers, who have greatly improved this paper.

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Goetghebeur, P., Simpson, D.A. 1991. Critical notes on Actinoscirpus, Bolboschoenus, Isolepis, Phylloscirpus and Amphiscirpus (Cyperaceae). Kew Bull. 46: 169–178. Goetghebeur, P., Van den Borre, A. 1989. Studies in Cyperaceae 8. A revision of Lipocarpha, including Hemicarpha and Rikliella. Wageningen Agr. Univ. Pap. 89-1: 1–87. Govaerts, R., Simpson, D.A., Bruhl, J., Egorova, T., Goetghebeur, P., Wilson, K. 2007. World checklist of Cyperaceae. Kew Publishing. Online: http://apps.kew.org/wcsp/home.do Haines, R.W., Lye, K.A. 1983. The sedges and rushes of East Africa. East African Natural History Society, Nairobi. Hinchliff, C.E., Roalson, E.H. 2013. Using Supermatrices for Phylogenetic Inquiry: An Example Using the Sedges. Syst. Biol. 62: 205-219. Jarvis, C. 2007. Order out of Chaos. Linnaean Plant Names and their Types. The Linnaean Society of London. Jiménez-Mejías, P., Martinetto, E. 2013. Toward an accurate taxonomic interpretation of Carex fossil fruits (Cyperaceae): A case study in section Phaeocystis in the Western Palearctic. Amer. J. Bot. 100: 1580–1603. Kjellsson, G. 1985. Seed fate in a population of Carex pilulifera L. II. Seed predation and its consequences for dispersal and seed bank. Oecologia 67: 424–429. Koyama, T. 1958. Taxonomic study of the genus Scirpus Linné. J. Fac. Sci. Univ. Tokyo, sect. 3, Bot. 7: 271–366. Koyama, T., Guaglione, E.R. 1987. Oreobolopsis, a new genus of Cyperaceae (Scirpeae) from Bolivia, South America. Darwiniana 28: 79–85. Khokhrjakov, A. P. 1985. Flora Magadanskoi Oblasti. Nauka, Moscow. Khokhrjakov, A. P. 1989. Analiz Flora Kolymskogo Nagorya. Nauka, Moscow. Lacroix, C., Mosher, C. 1995. Early development and viability testing of embryos of Scirpus acutus Muhl. Aquatic Bot. 50: 117–125. Leck, M.A. & Schütz, W. 2005. Regeneration of Cyperaceae, with particular reference to seed ecology and seed banks. Perspect. Plant Ecol. Evol. Syst. 7: 95-133. Léveillé-Bourret, D., Gilmour, C. N., Starr, J. R., Naczi, R.F.C., Spalink, D., Sytsma, J. 2014. Searching for the sister to sedges (Carex): resolving relationships in the Cariceae-DulichieaeScirpoeae clade (Cyperaceae). Bot. J. Linn. Soc. 176: 1–21. Léveillé-Bourret, E., Donaldio, S., Gilmour, C. N., Starr, J. R. 2015. Rhodoscirpus (Cyperaceae: Scirpeae), a new South American sedge genus supported by molecular, morphological, anatomical and embryological data. Taxon 64 (5): 931–944. Linnaeus, C. 1753. Species Plantarum, ed. 1, vol. 1. Laurentii Salvii, Holmiae. Linnaeus, C. 1754. Genera Plantarum, ed. 5. Laurentii Salvii, Holmiae. Lye, K.A. 1993. Diaspore production in Norwegian Cyperaceae. Lidia 3: 81–108. Lye, K.A. 2000. Achene structure and function of structure in Cyperaceae. pp. 615–628 in K.L. Wilson and D.A. Morrison (eds.) Monocots: Systematics and Evolution. CSIRO, Melburne. Marek, S. 1958. A study of the anatomy of fruits of European genera in the subfamilies Scirpoideae Pax, Rhynchosporoideae Aschers. et Graebner and some genera of Caricoideae Pax. Monographiae Botanicae 6: 151–177. Martinetto, E.,Bouvet, D., Vassio, E., Magni, P., Jiménez-Mejías, P. 2014. A new protocol for the collection and cataloguing of reference material for the study of fossil Cyperaceae fruits: The Modern Xarpoloical Collection. Review Paleobot. Palynology 201: 56–74. Mehra, P. N., Sharma, O.P. 1965. Epidermal silica cells in the Cyperaceae. Botan. Gaz. 126: 53–58.

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Menapace, F.J., Wujek, D.E. 1985. Scanning electron microscopy as an aid to the sectional placement of taxa within the genus Carex (Cyperaceae): sections Lupulinae and Vesicariae. Micron and Microscopica Acta 16: 213–214. Metcalfe, C.R. 1971. Anatomy of the Monocotyledons vol. 5. Cyperaceae. Oxford University Press, Oxford. Muasya, A.M., Simpson, D.A., Verboom, G.A., Goetghebeur, P., Naczi, R..F.C., Chase, M.W., Smets, E. 2009. Phylogeny of Cyperaceae based on DNA sequence data: Current progress and future prospects. Bot. Review 75: 2–21. Mueller, M.H., van der Valk, A.G. 2002. The potential role of ducks in wetland seed dispersal. Wetlands 22: 170–178. Oteng-Yeboah, A.A. 1972. Taxonomic studies in Cyperaceae. Ph.D. thesis, University of Edinburgh. 392 pp. Oteng-Yeboah, A.A. 1974. Four new genera in Cyperaceae-Cyperoideae. Notes Roy. Bot. Gard. Edinburgh 33: 307–310. Outred, H.A. 2002. Traveling seeds? Examples from New Zealand. Biologist 49: 173–178. Palla, E. 1896. Zur Systematik der Gattung Eriophorum. Botanische Zeitung 1896, Heft VIII: 141– 155. Prychid, C.J., Rudall, P.J., Gregory, M. 2003. Systematics and biology of silica bodies in monocotyledons. The Botanical Review 69 (4): 377–440. Reutemann, A.G., Lucero, L.E., Guarise, N.J., Vegetti, A. 2012. Structure of the Cyperaceae inflorescence. Bot. Rev. 78: 184–204. Reutemann, A.G., Vegetti, A., Pozner, R. 2015. Inflorescence development in Abildgaardieae (Cyperaceae, Cyperoideae). Flora 210: 3–12. Reznicek, A., Elizondo, M.S.G. 2008. Cypringlea (Cyperaceae) revisited, a new combination and status. Acta Botanica Mexicana 83: 13–23. Ridley, R.N. 1930. The Dispersal of Plants Throughout the World. Ashford, Kent. Sernander, R. 1906. Entwurf einer Monographie der europäischen Myrmekochoren. Kgl. Svenska Vetenskapsakad. Handl. 41: 1-410. Schmid, B. 1980. Carex flava L. s.l. im Lichte der r-Selektion. Ph.D. Dissertation, University of Zürich, Zürich. Schuyler, A.E. 1964. A biosystematic study of the Scirpus cyperinus complex. Proc. Acad. Nat. Sci. Philadelph. 115: 283–311. Schuyler, A.E. 1967. A taxonomic revision of North American leafy species of Scirpus. Proc. Acad. Nat. Sci. Philadelph. 119: 295–323. Schuyler, A.E. 1971. Scanning Electron Microscopy of Achene Epidermis in Species of Scirpus (Cyperaceae) and Related Genera. Proc. Acad. Nat. Sci. Philadelph. 123: 29–52. Sharma, O.P. 1973. Anatomy of Eriophorum comosum. Phytomorphology 23: 17–24. Simpson, D.A. et al. 2008. Phylogeny of Cyperaceae based on DNA sequence data – a new rbcL analysis. Aliso 23: 72–83. Smith, S.G. 1995. New combinations in North American Schoenoplectus, Bolboschoenus, Isolepis, and Trichophorum (Cyperaceae). Novon 5: 97–102. Smith, S.G., 2002. Amphiscirpus. In: Flora of North America Editorial Committee (Eds.), Flora of North America. Oxford University Press, New York, Oxford, pp. 27–28. Soons, M.B., van der Vlugt, C., van Lith, B., Heil, G.W., Klaassen, M. 2008. Small seed size increases the potential for dispersal of wetland plants by ducks. Journ. Ecol. 96: 619–627. Strong, M.T. 2003. Cypringlea, a new genus of Cyperaceae from Mexico. Novon 13: 123–132.

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Tucker, G.C., Miller, N.G. 1990. Achene microstructure in Eriophorum (Cyperaceae): Taxonomic implications and paleobotanical applications. Bull. Torrey Bot. Club 117: 266–283. Tzelev, N.N. 1999. Ob obeme i nomenklature nekotorykh rodov sosudistykh rastenii evropeiskoi Rossii. Bot. Zhurn. (Moscow & Leningrad) 84: 109–118. van der Pijl, L. 1972. Principles of Dispersal in Higher Plants, 2nd ed. Springer-Verlag, Berlin, Heidelberg, New York. van der Veken, P. 1965. Contribution a l'embryographie systématique des Cyperaceae – Cyperoideae. Bull. Jard. Bot. État. Bruxelles 35: 285–352. Vanhecke, L. 1974. Embryography of some genera of the Cladiinae and the Gahniinae (Cyperaceae) with additional notes on their fruit anatomy. Bull. Jarb. Bot. Nat. Belg. 44: 367–400. Verbelen, J.P. 1970. Systematische embryografie van de Cyperaceae – Rhynchosporineae. Biol. Jaarb. Dodonaea 38: 151–166. Vrijdaghs, A., Caris, P., Goetghebeur, P., Smets, E. 2005. Floral ontogeny in Scirpus, Eriophorum and Dulichium (Cyperaceae) with special reference to the perianth. Ann. Bot. 95: 1199–1209. Whittemore, A.T., Schuyler, A.E. 2002. Scirpus. In: Flora of North America Editorial Committee (Eds.), Flora of North America. Oxford University Press, New York, Oxford, pp. 8–21. Wongsriphuek, C., Dugger, B.D., Bartuszevige, A.M. 2008. Dispersal of wetland plant seeds by mallards: influence of gut passage on recovery, retention, and germination. Wetlands 28: 290– 299. Yano, O., Ikeda, H., Watson, M. F., Rajbhandari, K. R., Jin, X.-F., Hoshino, T. Muasya, A. M., Ohba, H. 2012. Phylogenetic position of the Himalayan genus Erioscirpus (Cyperaceae) inferred from DNA sequence data. Bot. J. Linn. Soc. 170: 1–11.

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TEXT TO FIGURES Fig. 1. Transverse section of achene of Eriophorum scheuchzeri with very thin pericarp separate from the seed. Scale 0.1 mm. From Lye 30061 (Labrador, Canada). Fig. 2. Transverse section of Trichophorum cespitosum [Kreczetoviczia cespitosa] achene showing the pericarp and the seed with the testa separate from the pericarp. Scale: 0.1 mm (100 µm). From Lye 17259 (Flora, Norway). Fig. 3. Transverse section of Trichophorum pumilum achene showing the ery thick pericarp and the seed with the testa separate from the pericarp. Scale: 0.1 mm (100 µm). From Lye 18699 (Porsanger, Norway). Fig. 4. Amphiscirpus nevadensis pericarp and part of seed showing the epidermis cell layer with large amounts of silica embedded in the lower cell wall (A), the 6-10 cell thick middle layer of the pericarp (B), the 2-3 cell thick tube-like inner layer of the pericarp (C), the seed wall or testa (D) and the layer of phosphorus or phytin globoids (E). Photographed uncoated using variable pressure. Scale: 10 µm. From Crowell 2 (New Mexico, U.S.A.). Fig. 5. Transverse section of pericarp of Eriophorum vaginatum showing the outer cell layer with mamillate silica tubercles (phytoliths) from a thin basal silica plate and the 3 cell thick layer without silica below. Note that the silica layer is continuous through the walls of the neighbouring cells. Photographed uncoated using variable pressure. Scale: 10 µm. From Lye 20343 (Nordreisa, Norway). Fig. 6. Transverse section of the pericarp of Trichophorum cespitosum [Kreczetoviczia cespitosa] showing outermost cells of pericarp with continuous silica deposition, middle pericarp of 3-4 cells and the fibrous inner pericarp. Photographed uncoated using variable pressure. Scale: 10 µm. From Lye 17259 (Ringebu, Norway). Fig. 7. Transverse section of the pericarp of Oreobolopsis tepalifera showing outermost cells of pericarp with strong silica deposition on inner cell wall, the 5-6 cell thick middle pericarp and one cell thick fibrous inner pericarp. Note that the silica layer is not continuous, but broken at the cell walls of the neighbouring cells. Photographed uncoated using variable pressure. Scale: 10 µm. From Caballos 458 (Caxatapa, Bolivia).

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Fig. 8. Transverse section of Phylloscirpus deserticola pericarp showing the epidermis cell layer with silica embedded mostly in the upper cell wall, the 2-3 cell thick middle layer, and the 1-2 cell thick tube-like inner layer of the pericarp. Scale: 10 µm. From Solomon 15819 (Bolivia). Fig. 9. Transverse section of Scirpus sylvaticus pericarp showing the epidermis cell layer with silica embedded mostly in the lower cell wall, the 2-3 cell thick middle layer, and the fibrous inner layer of the pericarp. Scale: 10 µm. From Lye 13901 (Stord, Norway). Fig. 10. Transverse section of Trichophorum alpinum pericarp showing the epidermis cell layer with a continuous layer of silica embedded in the lower cell wall, the 2-3 cell thick middle layer, and the inner fibrous layer. Scale: 10 µm. From Lye 18715 (Porsanger, Norway). Fig. 11. Transverse section of Trichophorum pumilum [Kreczetoviczia pumilum] pericarp showing the epidermis cell layer with slight amounts of silica embedded in the lower cell wall (A), the 5-7 cell thick middle layer (B), the inner layer with vessels (C), and the fruit wall oe testa (D). Scale: 20 µm. The small white irregular globoids loosely scattered in the epidermis layer (s) are made of up to 6.1% sulphorus and 5.3% calcium. From Lye 18699 (Porsanger, Norway). Fig. 12. Section of the pericarp of Zameioscirpus atacamensis showing silica mainly embedded as a very thin white curved line in the innermost wall of the epidermis cells. Photographed uncoated using variable pressure. Scale: 10 µm. From Cabrera 3557A (Banos del Toro, Chile).

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

22

Table 1: List of plants used for SEM photography and x-ray analysis of achenes in transverse section [all from Cyperaceae tribe Scirpeae except Erioscirpus, previously Eriophorum, now a genus in tribe Cypereae]. Vouchers are in the herbaria indicated. Species Amphiscirpus nevadensis Eriophorum angustifolium Eriophorum angustifolium Eriophorum brachyantherum Eriophorum callitrix Eriophorum gracile Eriophorum latifolium Eriophorum latifolium Eriophorum russeolum Eriophorum scheuchzeri Eriophorum scheuchzeri Eriophorum vaginatum Eriophorum vaginatum Eriophorum vaginatum Eriophorum virginicum Erioscirpus comosus Erioscirpus microstachyus Oreobolopsis inversa Oreobolopsis inversa Oreobolopsis tepalifera Oreobolopsis tepalifera Phylloscirpus acaulis subsp. acaulis Phylloscirpus acaulis subsp. pachycaulis Phylloscirpus boliviensis Phylloscirpus boliviensis Phylloscirpus deserticola Phylloscirpus deserticola Scirpus cyperinus Scirpus maximowiczii Scirpus radicans

Country U.S.A. Norway Norway Norway Greenland Norway Norway Norway Norway Canada Norway Norway Norway Norway Canada NW India India Peru Peru Bolivia Bolivia Argentina Ecuador Ecuador Ecuador Bolivia Ecuador Canada Japan Norway

Region New Mexico M. & R., Stranda Troms, Kvænangen Hedmark, Alvdal Moskusoksefjorden Hedmark Oppland, Ringebu Akershus, Asker Finnmark Newfoundland Troms, Kvænangen M. & R., Stranda Nordland, Fauske Troms New Brunswick Jannsar

Locality Sierra, North Salem Geiranger, near Dalsnibba Alteidet, Brødskift Tunstad in Sølndalen Ankerplassen Eidskog, Langemyra Fåvang, Hågøygarden Oppsjøen, eastern side Kautokeino, Goaskinruoppi Labrador, Red Bay Kvænangsfjellet Geiranger, near Dalsnibba Svartvasshaugen Nordreisa, Sappen Kent, near Pointe Sapin Kalsi

Coordinates 107°13'W, 32°43'N 07°13'E, 62°03'N 22°09'48''E, 70°02'11''N 10°31'20''E, 62°06'05''N ca. 22°30'E, 73°36'N 12°15'35''E, 60°05'42''N 10°16'40''E, 61°24'24''N 10°23'18''E, 59°48'56''N 22°52'56''E, 69°02'59''N 56°25'21''W, 51°44'42''N 21°33'32''E, 69°53'54''N 07°13'E, 62°03'N 15°22'29''E, 67°18'10''N 21°17'14''E, 69°33'01''N 64°50'21''W, 46°59'18''N 77°50'E, 30°31'N

Ancash

Huascarán Nat. Park

77°46'W, 8°53'S

La Paz region La Paz, Nor Yungas

Loa aza, Caxatapa Yungas (Unduavi)

ca. 67° W & 16° S ca. 67°40' W & 16°10' S

Cotopaxi prov. Chimborazo prov. Pichincha prov. La Paz region Tungurahua prov. Prince Edward Isl. Hokkaido Telemark

Parque nc. de Cotopaxi Chimborazo, Refúgio Volcán Cayambe páremo Murillo, Lago Zongo road Ambato – Guaranda Malpeque Bay Mt.Daisetsu, Hakuundake Heddal, Notodden airport

78°28'W, 00°38'S 78°50'W, 01°28'S 78°01'W, 00°00'S 68°08'W, 16°18'S 78°51'W, 01°24'S 63°44'W, 46°25'N 142°54'27''E, 43°39'43''N 09°12'25''E, 59°33'54''N

Altitude 920 m 40 m 610 m 240 m 790 m 209 m 370 m 30 m 390 m 920 m 340 m 110 m 5m

±4200 m 4280 m 3870 m ±3950 m 4840 m 4450 m 4670 m ±4050 m 3m 2180 m 18 m

Collector Crowell Lye Lye Lye Vaage Wischmann Lye Lye Lye Lye Lye Lye Lye Lye Lye Gamble Gamble Smith Lægaard Caballos Paterson Eyerdam Lægaard Lægaard Øllgaard Solomon Lægaard Lye Hoshino Lye

Number 2 17223 15924 19434 s.n. s.n. 17165 16969 20481 30061 18707 17222 18800 20343 30156 [1891] 23471 9987 22246 458 1381 24303 101436 102805 1208 15819 54782 30160 22117 21305

Herbarium K NLH NLH NLH-879 O O-98113 NLH NLH NLH O O-75411 NLH O-89916 NLH O K K AAU AAU K AAU K AAU AAU AAU K AAU O O-85894

23

Scirpus sylvaticus Scirpus sylvaticus Trichophorum alpinum Trichophorum alpinum Trichophorum cespitosum Trichophorum cespitosum Trichophorum pumilum Trichophorum rigidum subsp. rigidum Trichophorum rigidum subsp. ecuadoriense Trichophorum uniflorum Zameioscirpus atacamensis Zameioscirpus atacamensis Zameioscirpus muticus

Norway Norway Norway Norway Norway Norway Norway Peru Ecuador Russia Chile Argentina Peru

Hordaland Østfold, Moss Oppland, V. Toten Troms Sogn og Fjordane Oppland, Ringebu Finnmark Ancash Azuay prov. Sakha Rep., Yakutia Coquimbo Rioja prov. Ancash

Stord, Haga Solbakken west of E6 Eina, near Tune school Nordreisa, Øvre Sappen Flora, Krokane Rønningen - Svinslåa Porsanger, Storbukta Cordillera Negra, Huaraz W of Paas Lena River, Chekurovka Banos del Toro Sarmiento, Leoncito Cordillera Negra, Huaraz

05°31'50''E, 59°48'14''N 10°41'56''E, 59°27'06''N 10°37'31''E, 60°37'06''N 21°16'48''E, 69°32'43''N 05°04'30''E, 61°35'37''N 10°05'31''E, 61°27'08''N 25°08'40''E, 70°17'31''N 79°15'W, 02°46'S ca. 127°30' E & 71°03' N 71°17'W, 29°58'S

5m 45 m 410 m 140 m 8m 725 2m 4200 m 3700 m < 20 m 3300 m 3400 m 4200 m

Lye Lye Lye Lye Lye Lye Lye Renvoize Lægaard Solstad et al. Cabrera Biurrun Renvoize

13901 23941 18204 18715 17259 27742 18699 5163 55049 05/0660 3557A 5150 5144

NLH O-252184 O-89943 O-75405 NLH O-284639 O-75419 AAU AAU O K AAU AAU

24

Table 2: Characteristics of the fruit wall of 9 genera and 29 species (30 taxa) of Cyperaceae tribe Scirpeae detected and measured through SEM and x-ray analysis [including Erioscirpus, previously Eriophorum, now a genus in tribe Cypereae]. A = Thickness of fruit wall in µm. B = number of cells in transverse section. C = epidermis cells wider than long (W) versus isodiametric or taller than wide (I). D = presence (1) or absence (0) of silica phytoliths (tubercles). E = thickness of outer cell wall in µm. F = thickness of silica sheet in inner cell wall of epidermis in µm (excluding silica phytoliths). G = silica sheet in inner epidermis cell wall. H = presence of Ca in epidermis cells. I = silicon in atomic percent in phytoliths or silica plates (mean of 5 & maximum value). J = silicon in atomic percent in cell walls of cells below epidermis. Species Amphiscirpus nevadensis Eriophorum angustifolium Eriophorum brachyantherum Eriophorum callitrix Eriophorum gracile Eriophorum latifolium Eriophorum latifolium Eriophorum russeolum Eriophorum scheuchzeri Eriophorum vaginatum Eriophorum vaginatum Eriophorum vaginatum Eriophorum virginicum Erioscirpus comosus Erioscirpus microstachyus Oreobolopsis inversa Oreobolopsis inversa Oreobolopsis tepalifera Oreobolopsis tepalifera Phylloscirpus acaulis subsp. acaulis Phylloscirpus acaulis subsp. pachycaulis Phylloscirpus boliviensis Phylloscirpus deserticola Phylloscirpus deserticola Scirpus cyperinus

Country U.S.A. Norway Norway Greenland Norway Norway Norway Norway Norway Norway Norway Norway Canada NW India India Peru Peru Bolivia Bolivia Argentina Ecuador Ecuador Bolivia Ecuador Canada

Collector Crowell Lye Lye Vaage Wischmann Lye Lye Lye Lye Lye Lye Lye Lye Gamble Gamble Smith Lægaard Caballos Paterson Eyerdam Lægaard Lægaard Solomon Lægaard Lye

Number 2 15924 19434 [1929] O-98113 17165 16969 20481 18707 17222 18800 20343 30156 [1891] 23471 9987 22246 458 1381 24303 101436 102805 15819 54782 30160

A 100-130 12-18 18-20 15-20 20-25 15-20 20-25 15-20 15-18 45-50 15-20 15-25 20-25 10-15 15-20 40-45 40-50 30-35 35-40 20-30 40-50 35-40 25-30 25-30 7-10

B 8-12 3-4 3-4 3-4 3-4 3-4 4-5 3-4 3-4 5-6 3-4 3-4 3-4 3-4 5 5-6 5-6 5-6 5-6 4-5 4-5 2-3 3-4 3-4 2-3

C W W W W W W W W W W W W W W W W W W W W I I W W W

D 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 1

E 1-2 < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 0.5-1.0 0.5 1-2 1-3 1-2 1-2 2-3 1-2 3-4 2-3 2-3 < 0.5

F 3-4 1 0.5 0.5 filled 10-15 2-3 1-2 1-2 1-2 0.5 0.5 2 2 0.5 2-3 obscure 2-3 2-3 0.5 0.5 absent 0.5 1 0.5-1

G broken continuous continuous continuous continuous continuous continuous continuous continuous continuous continuous continuous continuous broken broken broken broken broken broken continuous continuous absent continuous continuous continuous

H -

I 7.8 & 9.1 3.3 & 4.6 2.3 & 3.6 3.0 & 3.5 3.9 & 5.0 3.9 & 4.3 3.4 & 4.8 3.0 & 3.4 1.0 & 2.0 2.8 & 3.3 3.7 & 4.6 3.7 & 5.6 3.6 & 4.7 4.6 & 5.2 2.0 & 2,8 5.3 & 6.8 3.7 & 4.3 4.7 & 5.0 4.3 & 5.2 2.4 & 2.7 0.6 & 1.8 0&0 1.5 & 2.0 2.7 & 3.1 4.9 & 6.6

J 0.1 0.9-1.9 0.1-0.4 0-0.2 0.2-0.3 0.4-0.8 0.6-3.0 0-0.2 0.1 0.3-1.6 0-0.1 0.2-0.3 0.2-0.6 0.5-0.8 0.1-0.2 0.2 -0.3 0.1-0.2 0.1-0.5 0.1-0.3 0.1-0.3 0-0.1 0 0.1-0.3 0.1-0.4 0.6-1.7

25

Scirpus maximowiczii Scirpus radicans Scirpus sylvaticus Scirpus sylvaticus Trichophorum alpinum Trichophorum alpinum Trichophorum cespitosum Trichophorum cespitosum Trichophorum pumilum Trichophorum rigidum subsp. rigidum Trichophorum rigidum subsp. equadoriense Trichophorum uniflorum Zameioscirpus atacamensis Zameioscirpus atacamensis Zameioscirpus muticus

Japan Norway Norway Norway Norway Norway Norway Norway Norway Peru Ecuador Russia Chile Argentina Peru

Hoshino Lye Lye Lye Lye Lye Lye Lye Lye Renvoize Lægaard Solstad Cabrera Biurrun Renvoize

22117 21305 13901 23941 18204 18715 17259 27742 18699 5163 55049 05/0660 3557A 5150 5144

20-25 20-25 20-22 20-25 10-15 10-12 35-45 30-40 75-90 45-50 30-50 80-90 40-50 50-60 20-30

4-5 3 3-4 3-4 3 2-3 4-5 5-6 8-10 5-6 5-8 6-10 3 3-4 3-4

W W W W W W W W I W W W I I I

1 1 1 1 1 1 1 1 0 0 0 0 0 0 0

< 0.5 < 0.5 < 0.5 < 0.5 < 0.5 0.5-1 0.5 0.5 1-2 4-5 2-3 3-4 5-7 3-8 6-10

0.5 0.5-1 0.5-1 0.5-1 0.5 1-2 1-2 0.5 0.5-1 2-3 1-2 absent 0.5 1 absent

continuous continuous continuous continuous continuous continuous continuous continuous broken broken broken absent broken broken absent

++ -

6.5 & 7.7 4.4 & 6.3 2.3 & 3.1 6.3 & 7.1 2.0 & 2.8 3.5 & 5.5 4.6 & 5.7 3.2 & 4.1 10.6 & 14.3 4.2 & 5.4 4.3 & 4.7 0&0 1.7 & 2.4 0.6 & 1.1 0&0

0.1-0.2 0.1-0.8 0.1-0.3 0.1 0.1-0.5 none 0.2-0.4 0.1-0.4 0.2-0.5 0.1-0.2 0.1-0.3 0 0.1-0.3 0.05 0