Author for correspondence: Roy C. Brown Tel: +1 337 482 6757 Email: firstname.lastname@example.org
•Fossil spores from mid-Ordovician deposits (475 million yr old) are the first indication of plants on land and predate megafossils of plants by 30–50 million yr. Sporopollenin-walled spores distinguish land plants from algae, which typically have heavy-walled zygotes that germinate via meiosis into motile or protonemal cells.
•All land plants are embryophytes with spores produced by the sporophyte generation. It is generally assumed that retention of the zygote and delay in meiosis led to matrotrophic embryo development and intercalation of the diploid sporophyte before spore production.
•However, new data on the cell biology of sporogenesis in extant bryophytes suggest that spores were produced directly from zygotes in protoembryophytes. The mechanism of wall transfer from zygote to meiospores was a three-phase heterochrony involving precocious initiation of cytokinesis, acceleration of meiosis, and concomitant delay in wall deposition. In bryophyte sporogenesis, cytokinesis is typically initiated in advance of meiosis, and quadrilobing of the cytoplasm is followed by development of a bizarre quadripolar spindle that assures coordination of nuclear distribution with predetermined spore domains.
•This concept of the innovation of sporogenesis at the onset of terrestrialization provides a new perspective for interpreting fossil evidence and understanding the evolution of land plants.
The evolution of land plants from algal ancestors is marked by the innovation of sporogenesis, a unique developmental process by which the haploid cells resulting from meiosis (meiospores) are covered with a complex desiccation-resistant, sporopollenin-impregnated wall known as sporoderm. Using two lines of evidence, the fossil record and the cell biology of extant bryophytes, we have developed a concept of the origin of sporogenesis independent of the origin of alternation of generations. In this scenario, the sporopollenin wall typically covering the algal zygote was transferred directly to meiospores without an intervening embryo and sporophyte generation. Evolution of this developmental process was the result of heterochronies; precocious initiation of cytokinesis, acceleration of meiosis and delay in wall deposition, resulting in sporoderm deposition on meiospores rather than zygote (Fig. 1). Accordingly, moist sites on the Paleozoic landscape would have been populated by simple and fragile thalloid or even filamentous gametophytes that left no fossil record. Fertilization of eggs by motile male gametes resulted in zygotes, each of which immediately underwent meiosis, resulting in a tetrad of spores. On land, the production of four spores from each zygote, capable of both survival and dispersal, would be immeasurably more advantageous than any number of meiospores designed for dispersal in water. Obviously the eventual intercalation of mitosis before meiosis increased the number of cells entering the meiotic pathway, but the critical transition from aquatic to terrestrial habitat was made possible by the expedient transfer of wall from zygote directly to meiospores. The earliest progenitors of land plants would be equipped with the reproductive advantage of producing desiccation-tolerant spores.
Materials and Methods
Immunofluorescence was carried out according to methods previously published (Brown & Lemmon, 1995) as modified specifically for bryophytes (Brown & Lemmon, 2006). In summary, capsules were dissected and a small sample of sporocytes were stained in aceto-orcein to determine the stage of development and the remainder were fixed overnight at 4°C in 4% formaldehyde freshly prepared from paraformaldehyde in microtubule-stabilizing buffer (Brown & Lemmon, 1995). Sporocytes were spread onto coverslips coated with Mayer’s egg albumen histological adhesive and covered by a thin agarose-gelatin film. Cells were permeabilized with a combined enzyme/detergent mixture (Brown & Lemmon, 2006). Following a thorough buffer wash, cells were incubated with a 1 : 160 000 dilution of mouse monoclonal antibody (G9) followed by a 1 : 100 dilution of rhodamine red anti-mouse immunoglobulin G (IgG) to label γ-tubulin, and a 1 : 100 dilution of rat monoclonal anti-∂tubulin followed by a 1 : 100 dilution of fluorescein anti-rat IgG to label microtubules. The G9 antibody was raised in mouse against bacterially expressed S. pombeγ-tubulin; its specificity in bryophytes has been thoroughly characterized (Shimamura et al., 2004). Following several washes in water, nucleic acids were stained with 4′,6-diamidino-2-phenylindole (DAPI) in d-H2O, before mounting in Prolong antifade reagent (Molecular Probes, Eugene, OR, USA). Fluorescence was examined with a Leica SR5 confocal laser scanning microscope (CLSM). Series of images were sequentially collected in the Z-axis and ImageJ software (http://rsbweb.nih.gov/ij/) used to generate projections for illustrations.
Results and Discussion
The fossil record
The fossil record provides evidence that spores were produced by the pioneering land plants and that attributes of sporogenesis continued to evolve over a period of 40–50 million yr. The first confirmation of plant life on land is an abundance of sporopollenin-walled spores without any evidence of the plants that produced them. This early period, recognized by Gray (1993) as the Eoembryophytic, lasted some 50 million yr, from c. 476 to 432 million yr ago. The oldest palynomorphs, which have been designated cryptospores, constitute an assemblage of monads, dyads and tetrads, all of which remain attached and enclosed in a tight-fitting envelope or synoecosporal wall (Wellman & Gray, 2000; Taylor & Strother, 2008). After this initial period, monads, dyads and tetrads surrounded by sporopollenin-containing walls but without envelopes appeared and were followed by free hilate monads and trilete spores (Wellman & Gray, 2000).
Although it is generally assumed that the early plants were at the bryophyte grade of organization with monosporangiate sporophytes, no fossil evidence of the plants/plant progenitors that produced this diversity of spores has been found (Wellman & Gray, 2000; Wellman et al., 2003). The first megafossils of plants occur 65 million yr after the earliest fossil spores and they are usually interpreted as primitive vascular plants (protracheophytes) (Wellman & Gray, 2000). The first bryophyte megafossils are liverwort-like from the Devonian and contemporaneous with tracheophytes (Edwards et al., 1995).
Cell biology of sporogenesis in extant bryophytes
Extant bryophytes exhibit an unexpected diversity in sporogenesis, providing ontogenetic clues to decipher the great enigma of millions of years of fossil spores without fossils of the plants that could have produced them.
Bryophyte meiosis is characterized by a curious reversal of the usual sequence of nuclear division followed by cytokinesis (Fig. 2). Cytokinesis is typically initiated before meiosis and sporocytes become quadrilobed into the four future spore domains, leaving the undivided diploid nucleus in the central cytoplasm (Brown & Lemmon, 1988a). We interpret this precocious initiation of cytokinesis before meiosis as a clue to the mechanism for transfer of wall deposition from zygote to meiospores. If occurring in algal–plant transitional forms, this could in itself account for the oldest fossil spores, the cryptospores, which seem to be imperfectly divided tetrads enclosed by a synoecosporal wall. Precocious quadrilobing of sporocyte cytoplasm occurs in all three major taxa of bryophytes: mosses, hornworts and liverworts. It is most extreme in two of the three classes of liverworts, the Treubiopsida and Jungermanniopsida, and less so in hornworts and mosses (Brown & Lemmon, 1988a).
Quadrilobing, which accelerates meiosis by pre-establishment of division planes and definition of spore domains, can involve both the cytoskeleton and cytoplasmic components. Brown & Lemmon (2006, 2009) have recently discovered that quadrilobing in certain liverworts is preceded by premeiotic bands of microtubules marking the future cytokinetic planes before the nucleus enters prophase I (Fig. 3). The bands may control quadrilobing by allowing cytoplasmic growth only between the sites marked by the bands, thus creating furrows and/or preventing sporopollenin deposition in the furrows. Whatever the mechanisms responsible, quadrilobing contributes to wall deposition on spores, or at least spore domains, as opposed to surrounding the entire premeiotic cell.
In mosses, hornworts and some liverworts, precocious quadrilobing is associated with monoplastidic meiosis, a pleisiomorphy of land plants that is probably a legacy from algal ancestors (Graham, 1993; Renzaglia et al., 1994; Brown & Lemmon, 1997). Some charophycean algae, the group most closely related to land plants, appear to have monoplastidic meiosis, but nothing is known of the cytoskeleton because of fixation difficulties resulting from the heavy zygote wall (Graham, 1993). Monoplastidic meiosis is found in all major clades of plants with the exception of seed plants (reviewed by Brown & Lemmon, 1997). In this distinctive type of meiosis, quadripolarity is established in early prophase by two divisions of a single plastid and migration of the four plastids to tetrahedral positions in the future spore domains where they establish second division poles. The discovery of both monoplastidic meiosis (Renzaglia et al., 1994) and premeiotic bands (Brown et al., 2010) in the relictual liverwort Blasia pusilla provides further evidence of the importance of such developmental mechanisms in the acceleration of meiosis and the production of free spores.
Deeply lobed sporocytes of certain liverworts produce pre-patterned exine precursors before chromosomal division (Fig. 3), providing evidence that wall deposition itself is initiated in the diploid cell during meiotic prophase (Brown et al., 1986). Other liverworts (e.g. some species of Sphaerocarpos and Riccia) produce a permanently adhered tetrad of spores (Proskauer, 1954; Schuster, 1992). Spore wall ornamentation develops over the entire circumference of the spore tetrad and is continuous from one spore surface to the next. Unlike free tetrads of other bryophytes, germination is from the unspecialized distal surface.
Spindle ontogeny in quadrilobed sporocytes
We suggest that once cytokinesis was initiated before meiosis, a quadripolar spindle was required for coordination with the predetermined spore domains. An amazing variety of microtubule organizing centres (MTOCs) have been discovered in meiosis of bryophytes (Brown & Lemmon, 2007; Brown et al., 2010). MTOCs are recognized by concentrations of γ-tubulin (Fig. 4), a molecule universally associated with nucleation of microtubules in eukaryotic cells (Ovenchkina & Oakley, 2001; Schmit, 2002; Wiese & Zheng, 2006). In land plants, γ-tubulin has been released from a tight association with centrioles that is typical of animal and algal cells (Shimamura et al., 2004; Brown & Lemmon, 2007). A diffuse MTOC residing at the nuclear envelope organizes anastral spindles in euphyllophytes (Stoppin et al., 1994; Schmit, 2002). Studies of bryophytes suggest that plant γ-tubulin is a pleiomorphic entity that moves in a cell cycle-specific manner to different locations where, in association with other molecules, it nucleates the microtubule systems that drive cell division and morphogenesis (Brown & Lemmon, 2007). In bryophytes, it can be concentrated into discrete forms of MTOCs: polar organizers (POs), plastid-MTOCs, as well as nuclear envelope MTOCs. All of these MTOCs are known to organize the quadripolar spindles in bryophyte meiosis (Brown & Lemmon, 2006, 2009; Brown et al., 2010).
In mosses and hornworts, quadripolar spindles are organized by plastid-MTOCs (Brown & Lemmon, 1997, 2007). In liverworts, now considered to be the earliest divergent land plants and therefore sister to all other land plants (Qiu, 2008), all forms of MTOCs occur (Brown & Lemmon, 2006, 2007, 2009; Brown et al., 2010). This amazing diversity in spindle ontogeny, which is in stark contrast to the uniformity of anastral spindle origin in euphyllophytes and centrosomal spindle origin in algal and animal cells, is strong evidence that there was an early explosive radiation in developmental mechanisms associated with the evolution of sporogenesis.
Although unorthodox, the quadripolar spindle functions to distribute chromosomes normally during the two divisions of meiosis (Brown & Lemmon, 1988a, 1997). Four poles of the first division spindle serve to properly orient spindle axes with respect to the predetermined spore domains. The initially quadripolar spindle forms from four cones of microtubules emanating from MTOCs located in the four spore domains (Fig. 5). The plus end interaction of these opposing systems forms a quadripolar microtubule system (QMS) that encloses and shapes the nucleus in prophase. The nuclear envelope breaks down and as homologues attach to microtubules of the QMS, the poles converge in pairs toward a single spindle axis that terminates at opposite cleavage furrows. Thus, the mature metaphase I spindle is functionally bipolar with a pair of poles straddling opposite cleavage furrows.
Quadripolar spindles regularly result in telophase I nuclei located at, often arching over, opposite cleavage furrows. Although phragmoplasts typically form between the nuclei, they generally disperse without directing wall deposition and second division, with rare exceptions, occurs in the undivided sporocyte. The two spindles of meiosis II, which are formed with poles in pairs of spore domains, are oriented perpendicular to the axis of first division (Brown & Lemmon, 1988a, 1997). Thus, the quadripolar sporocyte achieves proper placement of tetrad nuclei, one in each of the predetermined spore domains. Phragmoplasts form among the four haploid nuclei and the tetrad of spores is simultaneously cleaved by wall deposition along the predetermined cytokinetic planes.
Of the > 20 000 species of bryophytes (mosses, liverworts and hornworts), we know of only one exception to the precocious determination of future cleavage planes of the tetrad before meiosis. Sporocytes of the complex thalloid liverwort Conocephalum conicum are apolar, the spindles variously oriented, and spore domains are determined after meiosis via radial microtubule systems emanating from the tetrad nuclei (Brown & Lemmon, 1988b; Brown et al., 2010) in a manner more typical of meiosis in euphyllophytes.
The complex sporoderm is deposited layer by layer and it is reasonable to suppose that in evolution of sporogenesis, the first layer may have been deposited around the entire zygote before lobing of the cytoplasm occurred with subsequent layers walling the spore domains. This would account for the monads and variety of enclosed dyads and tetrads found in the fossil record before appearance of free tetrads. We suggest that accelerated meiosis, and especially precocious quadrilobing, was an important mechanism employed by protoembryophytes. Accelerated meiosis coupled with a delay in wall deposition made it possible for every zygote to produce a tetrad of walled spores, each of which was adapted for survival and dispersal in the terrestrial habitat, producing new gametophyte plants with novel genotypes. In this scenario, the Eoembryophytic epoch was a period of cytological innovations that solved the problem of producing walled meiospores suitable for colonizing the land surface.
All land plants today are embryophytes; the zygote and young diploid embryo are retained on the parent plant, which provides nourishment and shelter for the developing sporophyte. Neither the fossil record nor ontogeny of any modern land plant provides clues to evolution of the diploid multicellular sporophyte. Obviously, the zygote had to remain without sporoderm to develop into a multicellular sporophyte. A delay in entry of the zygote into the meiotic pathway could then allow for the intercalation of mitosis. These mitotic divisions may have first served simply to increase the number of cells entering meiosis but later gave rise to embryo/sporophyte development.
The extant bryophytes are living fossils that recapitulate the evolutionary history of sporogenesis and retain vestiges of an impressive diversity of developmental strategies employed in its early evolution. The hypothesis presented here points to a central role for sporogenesis in early colonization of land by providing spores that could serve as dispersal units of sexual reproduction. This concept explains the enigma of an abundance of fossil palynomorphs/spores before plant megafossils. In this case, Occam’s razor seems apropos; there are no fossils of associated sporophytes because there were no sporophytes when spores were first produced. Blackmore & Barnes (1987) hypothesized that two factors contributed to evolution of the embryophyte spore wall; transfer of the sporopollenin wall from zygotes to meiospores, and the fusion of single lamina into multilaminate walls. Early fossil spores provided sufficient evidence for Taylor & Strother (2008) to detect steps in the evolution from single lamina typical of algal walls to the multilaminate spore walls of embryophytes. The fossil record, however, cannot provide evidence that the wall derived directly from zygote walls. The authors therefore concluded that the synoecosporal wall of early cryptospores represented the remains of spore mother cell walls shed from a multicellular sporophyte. The cell biology of extant bryophytes, however, does provide evidence for an evolutionary transfer of the zygote wall directly to meiospores. Both lines of evidence support the astute hypothesis proposed by Blackmore & Barnes (1987) for the origin of sporogenesis, a singular and underappreciated event of unparalleled importance in the history of life on Earth.
Doctors Steve Blackmore, Thomas Pesacreta, Karen Renzaglia, and Charles Wellman read and commented on an earlier draft of the manuscript. We thank Dr Tetsuya Horio for the gift of the G9 antibody.