| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Systematics |
2Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3AB, UK; 3Department of Botany, University of Dublin, Trinity College, Dublin 2, Ireland; 4East African Herbarium, National Museums of Kenya, P.O. Box 45166-00100, Nairobi, Kenya
Received for publication November 15, 2002. Accepted for publication February 13, 2003.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Key Words: Chrysitricheae • Cyperaceae • Hypolytreae • Mapanioideae • phylogenetics, pollen • pseudomonad • rps16 • trnL-F
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
). The family has considerable economic importance; many members are serious agricultural weeds, whereas others provide food, fuel, and medicines together with construction, weaving, and perfumery materials (Simpson and Inglis, 2001
). They also have importance in conservation as dominant components of many wetland ecosystems and are reliable indicators of habitat deterioration in such systems.
Cyperaceae subfamily Mapanioideae (sensu Goetghebeur, 1998
) comprise two tribes, Hypolytreae and Chrysitricheae. Tribe Hypolytreae (sensu Simpson, 1992
; Goetghebeur, 1998
) comprise nine genera, Capitularina Kern, Diplasia Rich., Exocarya Benth., Hypolytrum Rich., Mapania Aubl., Mapaniopsis C.B. Clarke, Paramapania Uittien, Principina Uittien, and Scirpodendron Zipp. ex Kurz, although Simpson (1996)
reduced Mapaniopsis to sectional level within Mapania. Tribe Hypolytreae are pantropical, with a center of diversity in the Asia-Pacific region. Principina is only known from one collection on the island of Principe off the west coast of Africa. Many species are large, robust plants of the herb layer in tropical wet forests and their margins. Tribe Chrysitricheae (sensu Goetghebeur, 1998
) comprise four genera: Chrysitrix L., Chorizandra R.Br., Hellmuthia Steud., and Lepironia Rich. This group has a predominantly Southern Hemisphere distribution, and species occur in a variety of habitats ranging from tropical wet forest to open, swampy places. In contrast to the above, Bruhl (1995)
treated both tribes as a single unit, i.e., tribe Hypolytreae in subfamily Caricoideae. Thus there is disagreement between authors over subfamily and tribal delimitation.
Understanding the relationship of Mapanioideae to the rest of Cyperaceae has been problematic, because morphological characters in Cyperaceae are often difficult to interpret in terms of structural homology. In Mapanioideae this is exemplified by the inflorescence, a key source of character data generally in Cyperaceae. There is a particular specialization of inflorescence structure into units termed spicoids (cf. Kukkonen, 1984
; Simpson, 1992
), which comprise 2–13 scale-like floral bracts. The two lowest bracts are opposite, keeled, and often enclose the upper bracts (when the latter are present). Sometimes they are more or less united. The lower bracts subtend a single stamen, but the upper bracts are usually empty. There is a single gynoecium, which is not subtended by a floral bract. The whole structure is subtended and partially to fully hidden by a larger glume-like bract (spicoid bract). These are further aggregated into spikes. The homology of these units is still unclear. Some workers regard them as a derived type of spikelet (the basic inflorescence unit found in most other Cyperaceae) (Dahlgren et al., 1985
). Others view the spicoid as a flower in which the regular trimerous structure of the cyperaceous flower has been disturbed (Goetghebeur, 1986
, 1998
).
Studies of the tribes have mainly comprised regional floristic or monographic treatments of the genera (e.g., Koyama, 1970
; Kern, 1974
; Simpson and Koyama, 1998
; Alves and Thomas, 2002
) or work carried out as part of broader classifications of Cyperaceae (Bruhl, 1995
; Goetghebeur, 1998
). There is also a worldwide revision of the genus Mapania (Simpson, 1992
).
DNA sequencing studies are helping to build a better understanding of within- and between-tribe relationships in Cyperaceae. Muasya et al. (1998)
provided the first family-wide DNA-based phylogenetic trees which used the rbcL gene and included five species from tribe Hypolytreae [Hypolytrum bullatum C.B. Clarke, Hypolytrum nemorum (Vahl) Spreng., Mapania cuspidata (Miq.) Uittien sensu lato, M. meditensis D.A. Simpson, and Scirpodendron bogneri S.S. Hooper] and three in tribe Chrysitricheae [Chrysitrix capensis L., Hellmuthia membranacea (Thunb.) Haines and Lye and Lepironia articulata (Retz.) Domin]. Muasya et al. (2000)
expanded on this work to include morphological data. Both studies showed Hypolytreae and Chrysitricheae as sister to the rest of Cyperaceae. Tribal nomenclature followed Bruhl (1995)
, with all the taxa placed under Hypolytreae. However, within this group the taxa formed two subclades that corresponded to Hypolytreae and Chrysitricheae sensu Goetghebeur (1998)
. The one exception was Hellmuthia membranacea, which was placed well away from the Hypolytreae/Chrystricheae clade in Scirpeae (sensu Bruhl, 1995
).
Pollen development in most Cyperaceae is highly unusual, as three of the four products of meiosis degenerate (e.g., Juel, 1900
; Tanaka, 1939
; Khanna, 1963
; Dunbar, 1973
; Strandhede, 1973
; Makde, 1982
; Kirpes et al., 1996
; Furness and Rudall, 1999
; Brown and Lemmon, 2000
), forming pollen described as "pseudomonads" (Selling, 1947
). Before meiosis the microsporocytes become elongated. Microsporogenesis is simultaneous (reviewed in Furness and Rudall, 1999
), and after meiosis one of the four nuclei becomes larger and occupies the center of the wedge- or pear-shaped coenocytic cell, while the other three migrate to the narrower apex. These three smaller nuclei are separated off by septa at the narrower apex. Then they degenerate, and their remnants are enclosed by a thick intine. A thin exine develops around the whole pseudomonad, often with one indistinct aperture at the broader base, although aperture number varies (e.g., Furness and Rudall, 1999
; van Wichelen et al., 1999
). This pattern of pollen development, in which three of the four products of meiosis abort, is known in only one other unrelated group of angiosperms: tribe Styphelieae in Ericaceae (Smith-White, 1959
). Erdtman (1944
, 1952
) observed that the pollen of Mapania and allies differs morphologically from that of other Cyperaceae, being spheroidal and thick-walled with a distinct ulcerate aperture, and suggested that further cytological investigation was required. He described Chrysitrix pollen as corresponding to this Mapania-type, although his illustration (Erdtman, 1952
; p. 142, Fig. 78C) clearly shows a wedge-shaped grain. Whether pollen of Mapania and allies is a pseudomonad or not has remained an open question since detailed ultrastructural and ontogenetic investigations have not been carried out previous to this study. Koyama (1969
; pp. 219–220) described morphological differences in the pollen of mapanioid genera "[which] confirm Erdtman's remarks" and stated that the pattern of exine sculpture in mapanioid pollen differs from the usual granule type found in normal cyperaceous pollen.
The aim of this study was to investigate the relationships of Hypolytreae and Chrysitricheae by reconstructing their phylogeny using pollen data and plastid DNA sequence (trnL-F intron, trnL-F intergenic spacer, and rps16 intron) data. These DNA regions have proven to be appropriate for other systematic studies including Oxelmann et al. (1997)
, Reeves et al. (2001)
, Hodkinson et al. (2002a
, b
), and Muasya and Simpson (2002)
. We wanted to shed light on questions of subfamilial and tribal delimitation. We also wanted to examine the important question of whether the pollen grains are pseudomonads or not by studying the pollen of Mapania and related taxa using light microscopy and scanning and transmission electron microscopy.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Light microscopy (LM) and transmission electron microscopy (TEM)
It was necessary to examine different stages of pollen development. Inflorescences or anthers of different sizes were therefore collected for each taxon if possible. Material was placed in glutaraldehyde fixative (2.5% glutaraldehyde in 0.1 mol/L cacodylate buffer, pH 7.2) or Karnovsky's fixative (2% paraformaldehyde and 2.5% glutaraldehyde in 0.05 mol/L phosphate buffer, pH 7.2; Glauert, 1975
) in the field. Material was stored in fixative until it was brought back to the laboratory where this was replaced with fresh fixative, de-aerated under vacuum, and fixed overnight at 4°C. Some material was stored for longer in fixative at 4°C prior to embedding. Samples were then washed in cacodylate or phosphate buffer, treated with 1% osmium tetroxide for 3 h at room temperature, washed again, and dehydrated through a graded ethanol series. Material was embedded in medium-grade LR white resin (London Resin Company, Reading, UK) in gelatin capsules.
Material fixed in the field in FPA was transferred to 70% ethanol with glycerine and stored. Prior to embedding it was washed in 70% ethanol to remove the glycerine, then treated with osmium tetroxide, dehydrated, and embedded as above. Preservation of the protoplasm was not optimal in the material fixed with FPA, and the cell contents appeared degraded.
Sectioning was carried out using a Reichert Ultracut (Leica, Milton Keynes, UK). Semi-thin (ca. 1 µm) sections were cut using a dry glass knife, stained with toluidine blue, mounted in DPX (Aldrich Chemical Company, Gillingham, UK) and examined to determine the stage of pollen development. Suitable stages were photographed using a Nikon Labophot light microscope (Nikon, Kingston, UK) with a Nikon AFX-II camera attachment and normal brightfield optics. Ultra-thin (gold) sections were cut using a diamond knife, stained with uranyl acetate and lead citrate (Reynolds, 1963
) in an LKB Ultrostainer (Leica), before examination using a JEOL JEM-1210 transmission electron microscope (JEOL, Welwyn, Garden City, UK) at 80 kV.
Scanning electron microscopy (SEM)
Material of Mapania tenuiscapa was prepared by dissecting pollen from anthers fixed in glutaraldehyde. This was dehydrated through an ethanol series and air-dried onto a specimen stub. The stub was sputter-coated with platinum using a Balzers SPD 030 sputter-coater (Balzers Ltd., Balzers, Liechtenstein) and examined using a Hitachi S-2400 scanning electron microscope (Hitachi, Wokingham, UK) at 18 kV.
DNA sequencing
Sequence data were obtained from 35 taxa (see Supplementary Data accompanying the online version of this paper) including 14 from Mapanioideae. Total DNA was extracted using the modified cetyltrimethylammonium bromide (CTAB) method of Doyle and Doyle (1987)
from material collected in silica gel or herbarium specimens and precipitated in isopropanol for 2–4 wk. A few extractions were purified using cesium chloride/ethidium bromide gradients; the remainder were cleaned with Concert Rapid Polymerase Chain Reaction (PCR) Purification System (GibcoBRL, Gaithersburg, Maryland, USA)) spin columns. The trnL intron, trnL-F intergenic spacer (both hereafter trnL-F), and rps16 intron were each amplified as one complete piece using the following forward and reverse primers: trnL-F (cf., Taberlet et al., 1991
) and rps16 (F, R2; Oxelman et al., 1997
).
Standard polymerase chain reaction (PCR) protocols were followed. After some experimentation optimum results were achieved using ca. 100 ng/µL of total DNA, 1x Taq buffer (Promega, Madison, Wisconsin, USA), 2 mmol/L of magnesium chloride, 0.2 mmol/L of dNTPs, 100 ng of each primer, and 2.5 units of Taq polymerase (Promega) per 100-µL reaction. The thermal cycling (Applied Biosystems 480, Foster City, California, USA [ABI]) comprised 30 cycles, each with 1 min denaturation at 97°C, 1 min annealing at 51°C, and an extension of 3 min at 72°C. A final extension of 7 min at 72°C was also included. The PCR products were cleaned using the Concert Rapid PCR Purification System (Gibco BRL). Sequencing of the PCR products was carried out using cycle sequencing with ABI Big Dye terminators run on either an ABI 377A automatic sequencer at the Royal Botanic Gardens, Kew, or an ABI 310 Genetic Analyzer at the University of Dublin (following the manufacturer's protocol). Sequences were assembled and edited using Sequencher (Gene Codes Corporation, Ann Arbor, Michigan, USA).
Phylogenetic analyses
Heuristic parsimony analyses were carried out using PAUP* 4.0b10 software (Swofford, 1998
) on a Macintosh G4. The trnL intron and trnL-F intergenic spacer were treated as one region in the analyses (referred to as trnL-F). Searches were conducted with equally weighted characters (Fitch, 1971
), 1000 replicates of random addition sequence, and tree-bisection-reconnection (TBR) branch-swapping, with no more than 10 trees saved per replicate to save swapping on suboptimal islands and the MulTrees and steepest descent options in effect. Internal support was estimated using 1000 replicates of bootstrap (Felsenstein, 1985
). Bootstrap trees were built using simple taxon addition with TBR swapping, retaining groups with frequencies >50% in the bootstrap consensus tree. The following categories were used to describe levels of bootstrap support: weak = 50–74%; moderate = 75–84%; and strong = 85–100%.
The decision to combine molecular data sets was based on the pattern of major clades and their respective bootstrap percentages following Reeves et al. (2001)
. We did not apply the incongruence length difference (ILD) or similar congruence tests as they have been shown to be ineffective in identifying combinability of data and in some cases have been proven to be misleading (Yoder et al., 2001
). In addition to the molecular data we included pollen type as an unordered character in the combined analysis with the following states: (0) permanent tetrahedral tetrads, (1) pseudomonads, or (2) Mapania-type. Pollen types, based on original observations and records from the literature, are summarized in Table 1.
|
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
(Table 1; Figs. 1A–D, 2A–F, and 3A–F), except for Principina. The taxon examined with SEM, Mapania tenuiscapa, has spheroidal pollen with a finely reticulate surface and a single ulcus (Fig. 1A–D). The large, prominent ulcus has an annulus and a finely reticulate aperture membrane (Fig. 1D). Material adhering to the pollen surface is probably lipid (Fig. 1A and C). Sections through the anther locules of M. tenuiscapa and Diplasia karataefolia show that they are filled with spheroidal pollen (the central arrangement of Kirpes et al., 1996
) and the grains at the edge of the locule are often orientated with the single aperture towards the secretory tapetum (Figs. 2A–C and 3A–C). Orbicules (or Ubisch bodies), which are sporopollenin bodies produced by the secretory tapetum, are found close to the aperture in Mapania and along the edge of the tapetum, together with lipid, in Diplasia (Figs. 2D and 3C, D). Beneath the ulcus is an area of thickened intine (Figs. 2B–D and 3C, D), forming an oncus (Hyde, 1955
) where germination proteins are stored; this appears larger and more degenerate in Mapania. As the material was fixed in the field, it could not be de-aerated under vacuum and so preservation of the intine and cytoplasm is in some cases not as good as it would be in laboratory-fixed material (e.g., Fig. 2C). This thickened intine is continuous with the thinner intine surrounding the rest of the grain (Figs. 2D and 3C, D). The exine is lamellated at the edges of the ulcus (Figs. 2C, D and 3C, D); some of this is possibly endexine, although developmental studies would be needed to confirm this. There is also exineous material inside the ulcus and granules on the surface; these coalesce to form a membrane in Diplasia (Figs. 2C, D and 3C, D). The ulcus forms a cavity that is filled with material, probably derived from the tapetum; the pollen coat (or pollenkitt) comprises lipid and proteins (Figs. 2D and 3D). This material also extends between the columellae of the non-apertural wall, which has a foot layer; the tectum is discontinuous in Mapania and appears more continuous and granular in Diplasia (Figs. 2F and 3E). Lipid and orbicules are found adjacent to the exine in Diplasia (Fig. 3E). In Mapania the larger vegetative nucleus is positioned close to the wall away from the aperture, and the smaller generative nucleus lies between it and the apertural intine (Fig. 2E). The generative nucleus forms the twisted or spindle-shaped sperm cells, of which there are probably two, as in Carex (trinucleate pollen: Furness and Rudall, 1999
); one is shown in Diplasia (Fig. 3F).
|
|
|
, all members of Chrysitricheae sensu Goetghebeur (1998)
and in other tribes of Cyperaceae (Table 2; Figs. 4, 5, 6, 7, and 8). The presence of pseudomonads is confirmed in Capitularina, Chorizandra, Exocarya, and Hellmuthia for the first time.
|
|
|
|
|
|
; Furness and Rudall, 1999
). At the apex, the three nonfunctional nuclei may be cut off by septa, as in Hellmuthia (Fig. 8A–B) and form tetrahedral tetrads with one cell much larger than the other three. Tetrads in Hellmuthia are in two parallel rows in longitudinal sections through the anther locule with the larger cell of each tetrad towards the secretory tapetum (Fig. 8A). A thin exine develops around the outside of the whole tetrad or pseudomonad (Fig. 8B). The septa and the smaller, nonfunctional nuclei then degenerate and the nuclear remnants (which appear electron-dense using TEM) are generally surrounded by thick intine which fills the apex (Fig. 7B–C). The timing and extent of intine development may vary between genera; in Hellmuthia the intine is thick while the septa are visible (tetrad stage, Fig. 8B), but in Capitularina there is only a little intine at the apex and beneath the aperture by the time the functional nucleus has undergone mitosis (Fig. 4B–E).
The arrangement of the mature pseudomonads in the anther locule mostly corresponds to the peripheral arrangement of Kirpes et al. (1996)
, so that two rows are seen in longitudinal sections and the pseudomonads are in a ring in transverse sections, although there are some central pseudomonads (Figs. 4A, 6C, 7A, and 8C–D). There are orbicules along the inner edge of the secretory tapetum in Capitularina, Exocarya, and Hellmuthia (Figs. 4E, 7D, and 8E). The peripheral pseudomonads are oriented so that the basal pore is adjacent to the secretory tapetum (Figs. 4E, 6C, 7D, and 8D–E). The pore is indistinct and covered with a granular exineous membrane (Figs. 4E, 6D, 7D–E, and 8E). There is some thickened intine beneath the pore (Figs. 4E, 6D, 7D–E, and 8E) and usually much thicker intine at the apex where the electron-dense remnants of the nonfunctional nuclei are sealed in by this intine (Figs. 6E, 7B–C, and 8B and D). The exine is thin, with a foot layer, short columellae, and a granular semi-tectate surface (Figs. 4D, 6F, and 7F). There may be some endexine at the aperture (e.g., in Capitularina: Fig. 4E), but the nonapertural exine appears entirely ectexineous, as in most monocots. However, there is some variation in the exine between genera; for example, the foot layer in Chrysitrix is granular (Fig. 6F).
The functional nucleus divides to form a vegetative and generative nucleus (Fig. 4B). The generative nucleus divides again to form two spindle-shaped sperm cells in many Cyperaceae (Furness and Rudall, 1999
), one of which is visible in Capitularina close to the pseudomonad wall (Fig. 4F). In Hellmuthia the vegetative nucleus is located close to the wall between the apex and the ulcus (Fig. 8F).
Permanent tetrahedral tetrads
In Juncaceae and Thurniaceae, the sister groups to the Cyperaceae (Muasya et al., 1998
, 2000
), all four meiotic products reach maturity and pollen is dispersed as permanent tetrahedral tetrads (Table 1).
Analysis of trnL-F and rps16
For trnL-F 941 sites were included in the analysis, of which 464 were variable and 272 were parsimony-informative. The analysis resulted in two equally most parsimonious trees, each of 930 steps with a consistency index (CI) of 0.67 and a retention index (RI) of 0.80. One of these trees is illustrated in Fig. 9. The two trees differ only in their placement of Rhynchospora alba; in the tree not illustrated, this species is placed between the Carex/Dulchium/Eriphorum clade and the rest of Cyperaceae. For rps16, 883 characters were included, of which 383 were variable and 249 were potentially parsimony-informative. This analysis yielded 20 equally most parsimonious trees, each of 740 steps with a CI of 0.72 and an RI of 0.82. Three nodes, indicated by arrows in the single tree illustrated (Fig. 10), were not recovered in the strict consensus tree. Amplification of Diplasia karataefolia, Hellmuthia membranacea, and Lepironia articulata proved unsuccessful for rps16, and no sequence data were available for Dulichium arundinaceum, thereby excluding the taxa from this analysis.
|
|
Combined analysis
For this analysis 1825 sites were included, of which 848 were variable and 522 were potentially parsimony-informative. The analysis yielded five equally most parsimonious trees each with 1729 steps, with a CI of 0.69 and an RI of 0.81. One node, indicated by the arrow in the single tree illustrated (Fig. 11), was not recovered by the strict consensus tree. Bootstrap percentages were generally stronger and the overall topology was similar to the trnL-F analysis, except that Juncaceae formed a monophyletic group, as in the rps16 analysis. Hypolytreae/Chrysitricheae again formed a strongly supported group (100%) and were resolved into two subgroups with low bootstrap support (67%/68%). Hypolytreae were paraphyletic.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Apart from the difference in pollen shape, the structure of the aperture, a large ulcus with a lamellated exine layer and an oncus of thick intine, is very different from the aperture of the pseudomonads in the other taxa. In addition, the pollen of Mapania and Diplasia appears to be sticky (lipid adhering to the surface) and, together with floral morphology and habit, indicates that Mapania and related genera are animal pollinated (Lorougnon, 1973
; Simpson, 1992
). This may have been a factor leading to the loss of pseudomonads in some members of Mapanioideae, while they have been retained in others. It would indicate that the shift in pollination vector ocurred late in evolutionary terms and that the subfamily was already canallized into pseudomonad production. The evolutionary significance of pseudomonads in Cyperaceae is not yet understood. Possibly the wedge or pear shape, with a pointed apex, is a streamlined shape adapted to wind pollination.
Evolutionary-developmental studies are needed to investigate the genetic controls leading to monads or pseudomonads. Mutants such as QUARTET in Arabidopsis give rise to pollen in permanent tetrads (analogous to the situation in Juncaceae-Thurniaceae) in a taxon that normally produces monads (Preuss et al., 1994
). The polarity exhibited by Cyperaceae pseudomonads, in which the functional and nonfunctional nuclei occupy separate domains, is an interesting example of cell polarity worthy of further investigation (Brown and Lemmon, 2000
). Twell et al. (1998)
suggested that early gametophytic gene expression controlled equal quadripartite division by simultaneous microsporogenesis and influenced microspore polarity during later stages of pollen development in angiosperms. The breakdown of some of these controls may possibly lead to the abortion of three of the four nuclei in most Cyperaceae. Ranganath and Rao Nagashree (2000)
pointed out that meiotic cell division in microsporocytes of most Cyperaceae is asymmetric, unlike the condition in most angiosperms. This has important implications for the fate of the daughter cells and parallels the process of megasporogenesis in some angiosperms. Investigation of early gametophytic gene expression in Cyperaceae would be necessary to further understand the causes of this asymmetic division.
Tetrahedral tetrads are indicative of simultaneous microsporogenesis, which also occurs in Cyperaceae (see above), and this is a synapomorphy for the Cyperaceae-Juncaceae-Thurniaceae clade (Furness and Rudall, 1999
, 2000
). Many monocotyledons have successive microsporogenesis, although the simultaneous type characterizes some groups (Furness and Rudall, 1999
, 2000
). Muller in Kern (1974)
pointed out the similarity in pollen development among Juncaceae and Cyperaceae. In Juncaceae the exine forms around all four microspores of the tetrad (known as a calymmate tetrad), whereas the inner walls are thinner. This is similar to most Cyperaceae except that in this family three of the potential microspores degenerate.
Subfamily and tribal delimitation
The overall tree topology resolved by our analyses shows features congruent with previous family studies based on morphological (Bruhl, 1995
; Simpson, 1995
), molecular (Muasya et al., 1998
), and combined data (Muasya et al., 2000
). These include members of Juncaceae as sister to Cyperaceae and (with the exception of Simpson, 1995
) Hypolytreae/Chrysitricheae as sister to the rest of Cyperaceae. Although Bruhl (1995)
chose not to recognize it, our analysis suggests that Mapanioideae are a distinct unit (with moderate to high bootstrap support [83–100%]). This conclusion is supported by the high percentages of bootstrap support in all other studies of the group and also by the morphological characteristics of the group, notably the presence of the spicoid. Whatever its homology, the spicoid appears to represent a synapomorphy for the group.
Within Mapanioideae, Goetghebeur (1998)
characterized Hypolytreae as "large-leaved species, usually (with) many spikelets per inflorescence and (very) poorly differentiated embryos" (p. 151), whereas Chrystricheae are described as having "a reduced vegetative apparatus and inflorescence and probably a highly differentiated embryo" (p. 151). Yet these tribes are somewhat ill-defined. "Reduced vegetative apparatus" really only applies to Lepironia and Chorizandra, both of which have leaves reduced to bladeless sheaths. Some authors, such as Kern (1974)
, have suggested they are congeneric. Moreover, a reduced inflorescence is seen in members of both tribes, such as Mapania meditensis and Lepironia articulata. With regard to embryotype, Goetghebeur (1986)
noted that the embryo of Exocarya sclerioides is weakly differentiated, placing it within Hypolytreae, whereas that of Capitularina involucrata is unknown. Although embryotype is undoubtedly an important character, it has caused problems with other groups, for example in Bulbostylis, which some authors, such as Haines and Lye (1983)
, have combined with Abildgaardia, although in overall morphology they are distinct. We undertook a combined phylogenetic analysis (data not presented) using embryotype as an additional character but this had no influence on the topologies resolved. Thus embryotype may not be appropriate for distinguishing the two tribes, and it appears difficult to justify separation of the tribes on morphological grounds alone.
Nevertheless, our analyses show some consistent features, in particular the placement of Exocarya sclerioides and Capitularina involucrata (both members of tribe Hypolytreae) as sister to the group that otherwise comprises tribe Chrystricheae. The most consistent resolution is found in the combined analysis, supporting the argument of Reeves et al. (2001)
that combining data sets can aid systematic inference. A significant feature of this analysis is that the two groups reflect pollen type, one with Mapania-type pollen, the other with pseudomonads. Overall, our analyses make Hypolytreae polyphyletic, which indicates that current tribal circumscriptions need to be modified.
The placement of Diplasia is variable between analyses. In the trnL-F analysis it is sister to the rest of Mapanioideae while in the combined analysis it is grouped with Mapania, Scirpodendron, and Hypolytrum (68% bootstrap), although in the consensus tree it forms a polytomy within this group. Diplasia is a monospecific genus from the South American tropics. Morphologically it is well placed within the group that has Mapania-type pollen, as suggested by the combined analysis. We were unable to obtain a rps16 sequence for this taxon, which may account for its unstable position, and further investigation is necessary. The inclusion of taxa that had missing data in the combined analysis had little effect on the overall outcome of the analysis. We conducted a combined analysis with these taxa excluded (data not presented) but the result was similar. The effects of including taxa with missing character data in phylogenetic analyses have been widely discussed (e.g., Wilkinson, 1995
; Wiens, 1998
; Anderson, 2001
). Such work suggests that the inclusion of missing taxa from one gene in a combined analysis does not reduce phylogenetic accuracy. Thus we included these taxa to maintain a reasonable level of sampling.
Apparent biogeographical discontinuities are also highlighted by these groupings if the distributions of the taxa are considered. Genera in the larger group comprising mostly tribe Hypolytreae are all equatorial in distribution, whereas those in the group comprising mostly tribe Chrysitricheae occur only in the Southern Hemisphere. Goetghebeur (1998)
noted that the distribution of tribe Chrysitricheae indicates a Gondwanan origin. However, both Capitularina and Exocarya are also Southern Hemisphere taxa, Capitularina occurring in northern New Guinea and the Solomon Islands, Exocarya in southeastern New Guinea and northeastern Australia. Thus their placement with Chrysitricheae again seems more appropriate than with Hypolytreae.
The assertion by several authors (e.g., Holttum, 1948
; Kern, 1974
; Goetghebeur, 1986
) that Scirpodendron is the most "archaic" sedge has been disputed by others (e.g., Bruhl, 1995
; Muasya et al., 1998
), who have resolved Scirpodendron nested well within Hypolytreae (sensu Bruhl, 1995
). We have confirmed the latter and have additionally shown that Scirpodendron is in the group with specialized Mapania-type pollen that we believe evolved late in the history of Mapanioideae. However, there appears to have been an early division in Cyperaceae, with one lineage becoming the Mapanioideae in which the spicoid evolved as a specialized trait within the group and the other lineage giving rise to the rest of Cyperaceae.
Chrysitrix was described by Goetghebeur (1998)
as "somewhat aberrant" in tribe Chrystricheae on account of its unusual floral structure. This comprises an additional lateral spicoid, without male flowers, next to a spicoid of typical construction. However, our work indicates that, despite this specialization, Chrysitrix is well placed with other members of the tribe. The placement of Hellmuthia away from Mapanioideae is a feature commented on in other studies that have included this genus (e.g., Bruhl, 1995
; Muasya et al., 1998
, 2000
). With additional pollen data there now seems little doubt that Hellmuthia is better placed elsewhere and that its inflorescence structure is not, as suggested by Haines and Lye (1976)
, homologous to the spicoid-type inflorescence. Floral development studies are needed to elucidate inflorescence structure in both these genera.
A feature of both groups within Mapanioideae is that taxa assigned to the same genera do not necessarily group together, and these separations are often supported by high bootstrap percentages. For example, in the combined analysis Mapania tenuiscapa is sister to Hypolytrum testui, and Chorizandara sphaerocephala is sister to Chrysitrix capensis rather than C. cymbaria. Either we are dealing with more taxa than are currently recognized or conversely fewer, larger genera. Morphologically Hypolytrum and Mapania are close; they overlap in a number of characters and are reliably separated only by the number of floral bracts within in each spicoid (2–3 in Hypolytrum, 4–7 in Mapania). This suggests a larger grouping. However, the pattern is more complicated. All the taxa sampled in Mapania have a number of features in common (e.g., the same number of structures in the spicoid and a similar fruit exocarp structure). Morphologically they form a natural group which, along with other Asian-Pacific species, are assigned to Mapania sect. Pandanophyllum. Thus an alliance between M. tenuiscapa and Hypolytrum testui seems unlikely, even if Hypolytrum and Mapania were congeneric. Likewise, a generic relationship between Chrysitrix capenesis and Chorizandra cymbaria seems unlikely; both genera are morphologically distinct, particularly in the structure of inflorescence (Chrysitrix capensis has a lateral spike, which is characteristic for this genus; in Chorizandra cymbaria there is a single pseudolateral spike, again characteristic for the genus). Further sampling is needed to clarify the patterns emerging here.
Our work has supported the recognition of Mapanioideae and has revealed some new patterns of relationship. These suggest that grouping within the subfamily is based on the specialization of some taxa in terms of their pollination biology, a feature reflected in the molecular data. Mapania-type pollen is a synapomorphy for this specialized group and indicates that a reversal from pseudomonads to monads, with associated changes in pollen structure, has occurred. If the current tribal names Hypolytreae and Chrysitricheae are to be applied to these groups, the tribes will have to be recircumscribed. We propose continued recognition of both tribes, with Hypolytreae comprising taxa with Mapania-type pollen and Chrysitricheae for the remaining taxa with pseudomonad pollen (Table 2). Of the two taxa not included in our analysis, we have placed Principina in Hypolytreae on the basis of its morphological similarity to both Hypolytrum and Mapania. Paramapania has Mapania-type pollen and is morphologically similar to Mapania; it is also placed in Hypolytreae.
|
FOOTNOTES |
|---|
5 Author for reprint requests (d.simpson{at}rbgkew.org.uk
)
|
LITERATURE CITED |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Anderson J. S. 2001 The phylogenetic trunk: maximal inclusion of taxa with missing data in an analysis of the Lepospondyli (Vertebrata, Tetrapoda). Systematic Biology 50: 170-193[CrossRef][ISI][Medline]
Brown R. C. B. E. Lemmon 2000 The cytoskeleton and polarization during pollen development in Carex blanda (Cyperaceae). American Journal of Botany 87: 1-11
Bruhl J. J. 1995 Sedge genera of the world: relationships and a new classification of the Cyperaceae. Austalian Systematic Botany 8: 125-305[CrossRef]
Carniel K. 1962 Beiträge zur Entwicklungsgeschichte des sporogonen Gewebes der Gramineen und Cyperaceen. II. Cyperaceae. Österreichisches Botanisches Zeitschrift 109: 81-95[CrossRef]
Cranwell L. M. 1953 New Zealand pollen studies. The monocotyledons. Bulletin. Auckland Institute and Museum 3: 1-91
Dahlgren R. M. T. H. T. Clifford P. F. Yeo 1985 The families of monocotyledons. Springer-Verlag, Berlin, Germany
Doyle J. J. J. L. Doyle 1987 A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin 19: 11-15
Dunbar A. 1973 Pollen development in the Eleocharis palustris group (Cyperaceae). I. Ultrastructure and ontogeny. Botaniska Notiser 126: 197-254[ISI]
Erdtman G. 1944 Pollen morphology and plant taxonomy. II. Notes on some monocotyledonous pollen types. Svensk Botanisk Tidskrift 38: 163-168
Erdtman G. 1952 Pollen morphology and plant taxonomy. Angiosperms. Almqvist and Wicksell, Stockholm, Sweden
Felsenstein J. 1985 Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783-783[CrossRef][ISI]
Fitch W. M. 1971 Toward defining the course of evolution: minimum change for a specific tree topology. Systematic Zoology 20: 406.[CrossRef][ISI]
Furness C. A. P. J. Rudall 1999 Microsporogenesis in monocotyledons. Annals of Botany 84: 475-499
Furness C. A. P. J. Rudall 2000 The systematic significance of simultaneous cytokinesis during microsporogenesis in monocotyledons. In K. L. Wilson and D. A. Morrison [eds.], Monocots: systematics and evolution, 189–193. CSIRO, Melbourne, Victoria, Australia
Glauert A. M. 1975 Fixation, dehydration and embedding of biological specimens. Practical methods in electron microscopy, vol. 3, part 1. North-Holland, Amsterdam, Netherlands
Goetghebeur P. 1986 Genera Cyperacearum. Ph.D. dissertation. University of Ghent, Belgium
Goetghebeur P. 1998 Cyperaceae. In K. Kubitzki [ed.], The families and genera of vascular plants, vol. 4, 141–190. Springer-Verlag, Berlin, Germany
Haines R. W. K. A. Lye 1976 Studies in African Cyperaceae 14. The genus Hellmuthia Steud. Botaniska Notiser 129: 61-67[ISI]
Haines R. W. K. A. Lye 1983 The sedges and rushes of East Africa. East African Natural History Society, Nairobi, Kenya
Hodkinson T. R. M. W. Chase D. Lledo N. Salamin S. A. Renvoize 2002a Molecular phylogeny of Miscanthus s.l., Saccharum and related genera (Saccharinae, Andropogoneae, Poaceae) using DNA sequences from the ITS nuclear ribosomal DNA and the plastid trnL-F regions. Journal of Plant Research 115: 381-392[CrossRef][ISI][Medline]
Hodkinson T. R. M. W. Chase C. Takahashi I. J. Leitch M. D. Bennett S. A. Renvoize 2002b The use of DNA sequencing (ITS and trnL-F), AFLP and fluorescent in-situ hybridization to study allopolyploid Miscanthus (Poaceae). American Journal of Botany 89: 279-286
Holttum R. 1948 The spikelet in Cyperaceae. Botanical Review 14: 525-541[ISI]
Hyde H. A. 1955 Oncus, a new term in pollen morphology. New Phytologist 54: 255-256[CrossRef]
Juel H. O. 1900 Beiträge zur Kenntniss der Tetradentheilung. Jahrbuch für Wissenschaftliche Botanik 35: 626-659
Kern J. H. 1974 Cyperaceae. In C. G. G. J. van Steenis [ed.], Flora Malesiana series 1,. 7: 435-753
Khanna P. 1963 Male gemetophyte and obturator in Cyperus rotundus L., and C. triceps Endl. (= Kyllinga triceps Rottb). Current Science 32: 133-134
Kirpes C. C. L. G. Clark N. R. Lersten 1996 Systematic significance of pollen arrangement in microsporangia of Poaceae and Cyperaceae: review and observations on representative taxa. American Journal of Botany 83: 1609-1622[CrossRef][ISI]
Koyama T. 1969 Delimitation and classification of the Cyperaceae-Mapanioideae. In J. E. Gunkel [ed.], Current topics in plant science, 201–228. Academic Press, New York, New York, USA
Koyama T. 1970 The American species of the genus Hypolytrum (Cyperaceae). Darwiniana 16: 49-92
Kukkonen I. 1984 On the inflorescence structure in the family Cyperaceae. Annales Botanici Fennici 21: 257-264[ISI]
Lambert A. M. 1970 Formation et différenciation des grains de pollen chex les luzules (structures et ultrastructures). Bulletin de L'Académie et de la Société Lorraine des Sciences 9: 147-149
Lorougnon G. 1973 Le vecteur pollinique chez les Mapania et les Hypolytrum Cypéracées de sous-bois des forêts tropicales ombrophiles. Bulletin Jardin Botanique National de Belgique 43: 33-36[CrossRef]
Makde K. H. 1982 Pollen development in the Cyperaceae. Journal of the Indian Botanical Society 61: 242-249
Meyer N. A. S. Yaroshevskaya 1976 The phylogenetic significance of the development of pollen grain walls in Liliaceae, Juncaceae and Cyperaceae. In I. K. Ferguson and J. Muller [eds.], The evolutionary significance of the exine, 91–100. Academic Press, London, UK
Muasya A. M. J. J. Bruhl D. A. Simpson A. Culham M. W. Chase 2000 Suprageneric phylogeny of Cyperaceae: a combined analysis. In K. L. Wilson and D. A. Morrison [eds.], Monocots: systematics and evolution, 593–601. CSIRO, Melbourne, Victoria, Australia
Muasya A. M. D. A. Simpson 2002 A monograph of the genus Isolepis. Kew Bulletin 56: 257-362
Muasya A. M. D. A. Simpson M. W. Chase A. Culham 1998 An assessment of suprageneric phylogeny in Cyperaceae using rbcL DNA sequences. Plant Systematics and Evolution 211: 257-271[CrossRef][ISI]
Oxelmann B. M. Lidén D. Berglund 1997 Chloroplast rps16 intron phylogeny of the tribe Sileneae (Caryophyllaceae). Plant Systematics and Evolution 206: 393-410[CrossRef][ISI]
Preuss D. S. Y. Rhee R. W. Davis 1994 Tetrad analysis is possible in Arabidopsis with mutation of the QUARTET (QRT) genes. Science 264: 1458-1460
Ranganath R. M. N. Rao Nagashree 2000 Selective cell elimination during microsporogenesis in sedges. Sexual Plant Reproduction 13: 53-60[CrossRef][ISI]
Reeves G. M. W. Chase P. Goldblatt P. Rudall M. F. Fay A. V. Cox B. Lejeune T. Sousa-Chies 2001 Molecular systematics of Iridaceae: evidence from four plastid DNA regions. American Journal of Botany 88: 2074-2087
Reynolds E. S. 1963 The use of lead citrate at high pH as an electron opaque stain in electron microscopy. Journal of Cell Biology 17: 208-212
Selling O. H. 1947 Studies in Hawaiian pollen statistics. II. The pollens of Hawaiian phanerogams. Bishop Museum special publication 38. Bishop Museum, Honolulu, Hawaii, USA
Sharma M. 1967 Pollen morphology of Indian monocotyledons. Journal of Palynology Special Volume. Palynological Society of India, Lucknow, India
Simpson D. A. 1992 A revision of the genus Mapania. Royal Botanic Gardens, Kew, Surrey, UK
Simpson D. A. 1995 Relationships within Cyperales. In P. Rudall, P. J. Cribb, D. F. Cutler, and C. J. Humphries [ eds.], Monocotyledons: systematics and evolution, vol. 2, 497–509. Royal Botanic Gardens, Kew, Surrey, UK
Simpson D. A. 1996 New taxa and combinations in Mapania (Cyperaceae) from South America. Kew Bulletin 51: 729-740[CrossRef]
Simpson D. A. C. A. Inglis 2001 Cyperaceae of economic, ethnobotanical and horticultural importance: a checklist. Kew Bulletin 56: 257-360
Simpson D. A. T. Koyama 1998 Cyperaceae. In T. Santisuk and K. Larsen [eds.], Flora of Thailand, vol. 6, 247–485. Royal Forest Department, Bangkok, Thailand
Smith-White S. 1959 Pollen development patterns in Epacridaceae: a problem in cytoplasm-nucleus interaction. Proceedings of the Linnean Society of New South Wales 84: 8-35
Strandhede S. O. 1973 Pollen development in the Eleocharis palustris group (Cyperaceae). II. Cytokinesis and microspore degeneration. Botaniska Notiser 126: 255-265[ISI]
Swofford D. L. 1998 PAUP*: phylogenetic analysis using parsimony (*and other methods), version 4. Sinauer Associates, Sunderland, Massachusetts, USA
Taberlet P. L. Gielly G. Pautou J. Bouvet 1991 Universal primers for the amplification of three non-coding regions of the chloroplast DNA. Plant Molecular Biology 17: 1105-1109[CrossRef][ISI][Medline]
Tanaka N. 1939 Chromosome studies in Cyperaceae. V. Pollen development of Carex grallatoria Maxim. var. Heteroclita Kük. ex Matsum. Japanese Journal of Genetics 15: 153-157[CrossRef]
Tanaka N. 1941 Chromosome studies in Cyperaceae. XII. Pollen development in five genera with special reference to Rhynchospora. Botanical Magazine, Tokyo 55: 55-65
Twell D. S. K. Park E. Lalanne 1998 Asymmetric division and cell-fate determination in dev
