The Operculum in Pollen of Monocotyledons

Within monocotyledons, monosulcate pollen is the predominant type and probably represents the plesiomorphic condition, but considerable variation occurs in sulcus morphology. An operculum is an exine thickening that covers most of an aperture. Monocot opercula are usually associated with sulci, although they can occur in ulcerate apertures, as in Poaceae. There are several other aperture types closely related to the monosulcate-operculate type, and confusion occurs in the palynological literature between monosulcate-operculate, pontoperculate, disulculate, disulcate, and zona-aperturate (zonasulculate or zonasulcate) pollen. Transmission electron microscopy (TEM) was used to determine the distribution of the thick apertural intine and to accurately identify these aperture types. Operculate pollen most frequently was present in Asparagales (particularly Agavaceae, Doryanthaceae, lridaceae, and Tecophilaeaceae), Liliales (particularly Liliaceae, Melanthiaceae, and Uvulariaceae), and relatively infrequently among commelinid monocots, except for some Arecaceae, Dasypogonaceae, and Poales. Thus, we conclude that opercula have probably evolved several times independently within monocots, particularly in taxa from dry or seasonally dry habitats, and that this adaptation may be related to their function in protecting the aperture. Two transformation series of related aperture types are proposed, one of which involves monosulcate-operculate pollen, although further testing will be required.


INTRODUCTION
The monosulcate condition is the most common aperture type found in monocotyledons and is probably the plesiomorphic state (Furness and Rudall 1999).However, there is considerable diversity among monosulcate apertures.Sulci may be diffuse, extended, insulate (with scattered islands of exine), operculate, or with banded opercula (Halbritter and Hesse 1993).As Halbritter and Hesse (1993) pointed out, it is necessary to examine unacetolysed pollen in a turgid state to accurately observe details of the sulcus, since acetolysis causes the collapse of thin-walled monocot pollen and the removal of structures associated with the sulcus, including opercula.
Here we review the distribution of operculate pollen in monocots in a systematic context.In the palynological literature, the term "operculum" refers to an exinous thickening which covers a large part of an aperture (e.g., Punt et al. 1994).The operculum may be fused to the surrounding exine at both ends; apertures with this type of structure are termed "pontoperculate."Since the pontoperculum effectively divides the sulcus into two, when viewed using scanning electron microscopy (SEM) such an aperture may be indistinguishable from the disulculate aperture type.Similarly, confusion may arise between monosulcate-operculate and zona-aperturate (zonasulcate or zonasulculate) apertures.The orientation of monocot pollen apertures cannot be determined without reference to the tetrad stage of pollen development, and examination of sections of fresh pollen using TEM is also necessary in order to determine the position of the thickened apertural intine and thus the type of aperture.There is therefore much confusion in the pollen literature about the application of the terms monosulcate-operculate, pontoperculate, disulculate, disulcate, zonasulculate, and zonasulcate (or meridionosulcate ).
In the higher asparagoid clade, opercula are rare or absent, with the exception that several genera of Agavaceae have pontoperculate pollen and some have a sulcus that appears to be intermediate between operculate and pontoperculate (Alvarez and Kohler 1987;Ambwani and Kumar 1993).Some Laxmanniaceae have zonasulculate apertures (Chanda and Ghosh 1976), although aperture orientation requires confirmation by examination of tetrads, and sections showing the position of the thickened intine have not been examined to date.Disulculate apertures are a synapomorphy for tribe Amaryllideae (Amaryllidaceae: Schulze 1984;Snijman and Linder 1996).

DISCUSSION
Monosulcate-operculate pollen is primarily a monocot character, since opercula are rare in other basal angiosperms with monosulcate pollen.However, opercula are not restricted to monocots, as they occur on the colpate or colpatederived apertures of some eudicots (though they are sometimes removed by acetolysis), including some Caryophyllaceae, some Euphorbiaceae, and some Rosaceae (Erdtman 1952;Eide 1981;Lobreau-Callen eta!. 2000).Within monocots, opercula are particularly characteristic of the orders Asparagales (especially lower asparagoids), Liliales, and Poales, and have thus probably evolved several times, perhaps as a result of similar selection pressures (Fig. 13).The function of the operculum is probably to shield the cytoplasm from desiccation via the pollen aperture, although it may also provide protection against entry of pathogens such as bacteria and fungi via the aperture.Protection from dehydration could account for the presence of opercula in many species from relatively dry (or seasonally dry) habitats, such as many Iridaceae and Tecophilaeaceae.Opercula are noticeably absent from Alismatales and Zingiberales (Fig. 13), which occur in moist or wet habitats, and often have inaperturate (omniaperturate) pollen (Furness and Rudalll999).Blackmore and Barnes (1986) demonstrated that in Crocosmia Xcrocosmiiflora (Lemoine ex Anonymous) N. E. Br. (Iridaceae) the operculum assists in sealing the large sulcus, which folds inwards during dehydration.Opercula may occur in either insect-pollinated or wind-pollinated species.In Poaceae, which are wind pollinated, there is a reduced aperture that is almost completely covered by the operculum.
The operculum is a specialized region of exine that is -"'-'-'-'-'-'-'-'-'--'-'-'-'-'-'-'-'-""  usually thinner and has a simpler structure than the exine elsewhere on the pollen grain.For example, in Zigadenus Michx.(Melanthiaceae-Liliales) the operculum lacks the thick foot layer of the remaining exine (Furness and Rudall 2003;Fig. 3, 4).The operculum rests on top of a thick, often channelled, apertural intine and in this way a pontoperculate grain can be distinguished from a disulculate one.Disulculate pollen has two distinct apertures separated by thin nonapertural intine (e.g., Pauridia, Hypoxidaceae-Asparagales: Simpson 1983).In zona-aperturate pollen the intine is thick and often channelled beneath the ring-like aperture and thin beneath the exine of the rest of the grain (e.g., Aristea ecklonii Baker, Iris reticulata, Iridaceae-Asparagales: Furness and Rudall 2003).The operculum in Poaceae is associated with a structure known as the Zwischenkorper, a lenticular body composed of pectic polysaccharides located between the operculum and the intine, which hydrates at germination and presses away the operculum before the pollen tube tip emerges (Heslop-Harrison and Heslop-Harrison 1980).
We propose two transformation series of monocot aperture types: (1) monosulcate, monosulcate-operculate, pontoperculate, disulculate, zonasulculate (Fig. 14; see Series A) and (2) monosulcate, extended sulcate, disulcate, zonasulcate (meridionosulcate) (Fig. 14; see Series B).In the first series (Fig. 14, Series A), if the thickened intine beneath the operculum of a pontoperculate grain is lost, and the exine of the operculum becomes thicker, disulculate apertures will result.These changes could equally work in the opposite direction.Thus, monosulcate-operculate pollen may give rise to pontoperculate, disulculate, and zonasulculate or vice versa, and these aperture types can occur within single species, as in Agave americana L. and A. wightii L. (Ambwani and Kumar 1993).Relatively simple changes in the proportions of exine and intine would enable these transformations to take place and the developmental controls affecting exine and intine production in pollen require further investigation.There may also be a separate transformation series between monosulcate (non-operculate) and both disulcate and zonasulcate types (Fig. 14, Series B).Disulcate apertures are relatively rare in monocots but occur in Tofieldia (Tofieldiaceae-Alismatales), in which Huynh (1976) demonstrated the presence of sulci at both the distal and proximal poles.Extension of either a single sulcus (with extended sulcate as an intermediate condition) or two sulci around the grain would produce the zonasulcate (meridionosulcate) condition.
The developmental controls giving rise to an operculum are unknown, but the position of the operculum may relate to that of the microtubule organizing centers (MTOCs) during meiosis.Sheldon andDickinson ( 1983, 1986) disrupted meiosis in microsporocytes of Lilium L. (Liliaceae), which has monosulcate pollen, using centrifugation or colchicine treatment, then cultured them to maturity.Some microsporocytes underwent only the first meiotic division to produce dyads rather than tetrads; these developed a sulcus with an "island" of exine in the center, similar to a monosulcateoperculate aperture.Sheldon and Dickinson demonstrated that in "normal" Lilium pollen the location of the MTOC at the second meiotic division corresponds with the sulcus position, but in pollen with only one meiotic division the MTOC corresponds with the patterned exine in the center of the sulcus.The relationship between the MTOCs and the operculum of monosulcate-operculate Liliaceae such as Erythronium L. would be an interesting topic for future investigation.

Fig. 13 .
Fig. 13.-0perculate pollen mapped on a diagram of monocot relationships, with orders as in the Chase et al. (2000) classification.
Fig. 14.-Two proposed transformation series of pollen apertures, Series A and B, all in distal polar view.Series A includes monosulcate-operculate pollen.