Aliso: A Journal of Systematic and Floristic Botany Aliso: A Journal of Systematic and Floristic Botany Aperture Pattern and Microsporogenesis in Asparagales Aperture Pattern and Microsporogenesis in Asparagales

The aperture pattern of pollen grains is a character defined as the number, shape, and position of apertures. Although this character is highly variable in angiosperms, two states are particularly wide spread. Pollen grains with one polar aperture occur frequently in basal angiosperms and monocots while tricolpate pollen is a synapomorphy of the eudicots. Many morphological characters are the result of a compromise between selective forces (acting on morphology) and developmental constraints (limiting the range of possible morphologies). To investigate what are the respective roles of devel opment and selection in the determination of aperture pattern in angiosperms, we have chosen to study the characteristics of cell division during male meiosis, since it has been shown that aperture pattern is determined during microsporogenesis. The present study focuses on Asparagales. From a selection of species belonging to the major families of Asparagales, we described the type of cytokinesis, the way callose is deposited, the shape of the tetrad, as well as the shape and position of apertures within the tetrad. We show that although pollen morphology is quite uniform in Asparagales (most species produce monosulcate pollen), the characteristics of cell division during male meiosis vary among families. A highly conserved developmental sequence is observed in higher Asparagales whereas lower Asparagales, and particularly Iridaceae, display different ways of achieving cell division. This paper presents the first large-scale investigation of intersporal wall formation in a group of plants-Asparaga les-conducted using simple methods of staining and obser vation. Our main objective was to explore the developmental pathway of microsporogenesis from a cellular point of view, in a range of species representing the taxonomic diversity of Asparagales, with a focus on higher Asparagales, in order to understand the conservativeness of aperture pattern.


INTRODUCTION
Pollen morphology is extremely diversified in angiosperms. Many studies have focused on the structure of pollen grains and particularly on the ornamentation of the pollen wall. However, a high diversity is also found in the apertures, and particularly in the aperture pattern (Erdtman 1952;Huynh 1976;Thanikaimoni 1986;Harley and Zavada 2000). Variation in aperture pattern is expressed in the shape and number of apertures, as well as the position of apertures within the tetrad, visible when microsporogenesis is completed. The pollen grains in monocots are characterized by a relatively conserved aperture pattern since many species produce monosulcate pollen. However, the monosulcate type is not a strict rule in monocots (Harley and Zavada 2000) and variation occurs in several families such as Alismataceae (Furness and Rudall 1999), Araceae (Grayum 1992), Liliaceae and Tofieldiaceae (Huynh 1976;Rudall et al. 2000), Arecaceae (Harley and Baker 2001), Dioscoreaceae (Caddick et al. 1998), and Hemerocallidaceae (Huynh 1971). The Hemerocallidaceae family belongs to the order Asparagales, which has been subject to detailed phylogenetic analyses (Chase et al. 1995;Rudall et al. 1997;Fay et al. 2000). Rudall et al. ( 1997) surveyed the evolution of cytokinesis and pollen aperture type in Asparagales and concluded that simultaneous cytokinesis is apomorphic for the order, with a reversal to the successive type in higher Asparagales. As with the rest of monocots, most asparagalean species produce monosulcate pollen grains. Other aperture types have been recorded in families such as Hemerocallidaceae, characterized by the predominance of trichotomosulcate pollen (Roth et al. 1987), or Iridaceae, in which among others, zonasulcate, disulcate, trichotomosulcate, or inaperturate types are encountered (Goldblatt andLe Thomas 1992, 1993). It is noteworthy that variation is mostly found in families belonging to the lower asparagalean grade, whereas taxa from the higher Asparagales clade consistently produce monosulcate pollen. Moreover, monosulcate pollen grains can be produced by either successive or simultaneous cell division, whereas trichotomosulcate pollen occurs only when cytokinesis is of the simultaneous type (Rudall et al. 1997).
The type of cytokinesis is one of the different parameters that have been shown to play a role in the determination of aperture pattern (Res sayre 2001;Ressayre et al. 2002). Other parameters involved are the characteristics of intersporal wall formation during male cytokinesis (centrifugal or centripetal callose deposition), the shape of the tetrad (tetragonal, tetrahedral, or rhomboidal), and the position of apertures within the tetrad (polar or grouped at the last point of contact between the microspores).
Little information is available concerning intersporal wall formation in Asparagales. The process has been described in detail for a handful of species, such as Convallaria majalis L. (Waterkeyn 1962), Hemerocallis fulva L., and Sanseviera trifasciata Prain. (Longly and Waterkeyn 1979). Cytokinesis is described as being successive for all three species, and cell division involves the formation of three centrifugal cell plates (one formed after the first meiotic division, two formed after the second meiotic division) meeting with callose ingrowths at the junction with the callose wall surrounding the tetrad. Cell plates are covered by additional callose  (Longly and Waterkeyn 1979;Ressayre 2001).
This paper presents the first large-scale investigation of intersporal wall formation in a group of plants-Asparagales-conducted using simple methods of staining and observation. Our main objective was to explore the developmental pathway of microsporogenesis from a cellular point of view, in a range of species representing the taxonomic diversity of Asparagales, with a focus on higher Asparagales, in order to understand the conservativeness of aperture pattern.

MATERIALS AND METHODS
For this study, 14 species were selected, representing most major families of the higher Asparagales, as well as a few families of the lower Asparagales. Fresh floral buds of different sizes, and therefore at different stages of microsporogenesis, were collected from plants grown in botanical gardens or found on the campus of the Universite Paris-Sud (Table 1). Floral buds were immediately dissected to extract the anthers. One anther per bud was squashed and mounted in aceto-carmine in order to identify the stage of the bud (Fig. I, 2). When meiosis was in progress, the remaining anthers were squashed in aniline blue (modified from Arens 1949), which revealed callose wall formation by epifluorescence (Fig. 3, 4). When the tetrad stage was observed, half of the remaining anthers were squashed in congo red (Stainier eta!. 1967), in order to visualize the position of apertures within the tetrad (Fig. 5, 6), and the remaining half were mounted in aniline blue, to visualize callose walls just before the release of pollen grains. Intersporal wall formation of the first and second meiotic divisions is represented in Fig. 7-18 and 19-30, respectively. Mature pollen was also mounted in congo red. Aceto-carmine and congo red preparations were observed in transmission light with a Zeiss Axiophot 1980-773 Kew microscope. The epifluorescence Zeiss Axiophot microscope was used with filter set 01 (excitation 345, emission 425 nm long pass) for aniline blue staining.

DISCUSSION
The microsporogenesis pathway leading to monosulcate po llen, a feature of monocots a nd basal angiospe rms, was known to invo lve either successive or simultaneous cytokinesis. The results obtained in thi s study show that in hi gher Asparagales, a clade characterized by successive cytokinesis (Rudall et a l. 1997), the whole process of microsporogenesis is highly conserved . All species examined di splayed identical features concerning the formation of the callosic cell walls and the distribution of sulci within the tetrad. Intersporal walls are formed by centrifugally ex pa nding cell pl ates, with sli ght ingrowths of callose at th e junction between th e cell plates and the callose wall surrounding the tetrad (indicated by an arrow on Fig. 22). What had been described for two spec ies (Long ly and Waterkeyn 1979) can therefore be considered as a general feature of the hi g her Asparagales.
The clade consisting of Asphodelaceae, He merocallidaceae, and Xanthorrhoeaceae is sister to hi gher Asparaga les . Asphodel aceae, which produce monosulcate pollen through simultaneous cytokinesis (Rudall et al. 1997), achieve the cell wall formation by means of centrifugal cell plates a in hi gher Asparagales. Thi s is also the case for Heme rocallidaceae (data not shown) in which , interesting ly, Hemerocallis di splays a type of cytokinesis inte rmedi ate between successive and simultaneous cytokinesis (Cave 1955). Xanthorrh.oea is rep011ed as be ing successive (Rudall et a l. 1997). Our results show th at in lower A paragales, the microsporogenes is pathway assoc iated with a successive type of cytokinesis is achieved by way of centrifugally g rowing cell plates, exactly like in hi gher Asparagales . A s imultaneous cytokinesis is, however, the most common conditi on in lower Asparaga les. In thi s case, cell wall formation can be achieved by centrifugal cell pl ates, a situation encountered in Asphodelaceae and re lated families , Nadot, Penet, Dreyer, Forchioni , and Ressay re ALISO   or by callose growing centripetally (data not shown), such as in Iridaceae or Tecophilaeaceae. In spite of this variation observed in microsporogenesis, the pollen produced is monosulcate in most Asparagales. This raises the question of the role played by cytokinesis and cell wall formation during microsporogenesis in the determination of aperture pattern (Ressayre et al. 2002). A more thorough investigation of microsporogenesis in lower Asparagales is currently being conducted and will allow us to further explore this point.