A Fossil Record of Spores before Sporophytes

Abstract

Because their resistant, sporopolleninous walls preserve a record of morphogenetic change during spore formation, fossil cryptospores provide a direct physical record of the evolution of sporogenesis during the algal–plant transition. That transition itself is a story of the evolution of development—it is not about phylogeny. Here, we review the fossil record of terrestrially derived spore/cryptospore assemblages and attempt to place these microfossils in their evolutionary context with respect to the origin of complex multicellularity in plants. Cambrian cryptospores show features related to karyokinesis seen in extant charophytes, but they also possess ultrastructure similar to that seen in liverworts today. Dyadospora, a cryptospore dyad recovered from sporangia of Devonian embryophytes, first occurs in the earliest Ordovician. Tetrahedraletes, a likely precursor to the trilete spore, first occurs in the Middle Ordovician. These fossils correspond to evolutionary novelties that were acquired during a period of genome assembly prior to the existence of upright, axial sporophytes. The cryptospore/spore fossil record provides a temporal scaffold for the acquisition of novel characters relating to the evolution of plant sporogenesis during the Cambrian–Silurian interval.

1. Introduction

Land plants are one of only four clades of eukaryotes that possess complex multicellular structure that derives from the development from an embryo. We use the terms embryophytes and land plants as equivalent, following Margulis and Schwartz [1], who recognized the Kingdom Plantae as systematically distinct from the largely unicellular Protista (protists). The embryophyte life cycle pairs a haploid (1n) phase possessing multicellular structures, called antheridia and archegonia, with a diploid (2n) phase growing from a multicellular embryo that produces a complex plant structure, the sporophyte. All land plants possess these basic features or some derivation thereof. None of the green (a + b chlorophyll-containing) algae that are ancestral to the land plants possess antheridia, archegonia or an embryo. The origin of the land plants, therefore, represents a major evolutionary transition from simple multicellular organization to complex multicellularity [2,3,4]. And unlike the case with animals, whose unicellular ancestors were fully aquatic, the evolution of complex multicellularity in land plants was driven by selection in terrestrial settings [5,6].

Historically, studies in comparative plant morphology [7,8], and to a small extent paleobotany [9,10], have addressed the evolution of the algal–land plant transition. In the latter 20th century, evolutionary biologists began to approach the study of embryophyte origins using phylogeny reconstruction based on phylogenetic systematics [11]. Phylogeny refers to the pattern of evolutionary relations among species. Phylogenetic studies based on morphological character traits have been followed by the rise in molecular phylogenetics, and now phylogenomics has continued to hone in on the phylogeny of the Streptophyta, the clade that contains the green algal ancestors plus the land plants. There are two aspects to modern phylogenetic and phylogenomic approaches that have appeared to have reached a consensus in recent years: the closest algal ancestor to the land plants, and the phylogenetic relation between the three bryophyte groups and the vascular plants at the base of the embryophyte tree. The third application of molecular phylogenetic approaches to the study of the origin of land plants is the construction of evolutionary trees, or time-trees, from molecular phylogenies, which use fossil calibrations to place dates on branch nodes within phylogenetic trees. All three of these molecular-based efforts to unravel the evolution of land plants from their algal ancestors have provided provisional answers to this problem; however, as pointed out recently [12,13], neither the algal sister nor the monophyly of the bryophytes has yet been resolved.

An apparent consensus that the Zygnematophyceae are sister to the Embryophyta [14,15,16,17,18] at the very least requires considerable gene loss [19], or “mosaic and reductive evolution” [20]. And, from a purely morphological perspective, species in the Coleochaetaceae clearly possess far more complex features that demonstrate adaptation to subaerial settings [21,22,23,24] than do species in the Zygnematophyceae, which are simply filamentous in terms of multicellular complexity.

When it comes to the timing of bifurcations of the green plant phylogeny, there is a considerable range of hypothesized “origins” to the land plants, ranging from deep into the Neoproterozoic (980 to 568 million years ago, or Ma) [25,26] to Cambro–Ordovician (515–474 Ma) [12,27] to lowermost Ordovician 486 Ma [28]. None of these molecular evolutionary trees shows land plant origins matching the fossil record of the first occurrence of the whole-plant fossil, Cooksonia, which is found in strata of Wenlock age (ca. 425 Ma) in Ireland [29] and in the Prague Basin [30]. This temporal gap of some 50 to 80 million years (Myr) continues to characterize a conceptual difference between molecular dating methods and the concreteness of the fossil record. While there is little doubt that the first appearance datum (FAD) of Cooksonia would have occurred after the true origin of the first land plant, it is highly unlikely that a time gap representing over 9% of the entire Phanerozoic rock record could be missing a plant fossil record. Paleobotanists have long argued that the sedimentary record was not conducive to fossil plant preservation during this interval [27,31,32,33,34], whether due to the ephemeral nature of projected primitive plant types or a lack of terrestrial deposition prior to the Devonian. That same interval, the Ordovician through lower Silurian, of course, is replete with a record of plant spores and cryptospores, many of which were washed into shallow marine habitats [35,36,37,38,39,40,41,42]. Tomescu et al. [43] have argued that there is no “missing” fossil record of bryophytes—they have a nominal potential for preservation that is comparable with that of vascular plants. If bryophytes are not found in any particular fossil assemblage, it could simply be that they were not present in the source community. In evolutionary time, the implication would be that we expect to find bryophyte fossils within a reasonable period of time after they had evolved.

The inherent nature of the evolution of the first land plants from their algal ancestors is slowly being unraveled by phylogenomic and transcriptomic studies that have demonstrated homologous components of ancestral (algal) and derived (plant) genomes. However, knowing scattered bits of the ancestral genetic toolkit [44,45,46] is insufficient at present to paint a convincing and comprehensive picture of a gradual origin of complex multicellular development in geologic time. Here, we rely on an early 20th century hypothesis based on classical plant developmental morphology, the antithetic, or interpolational hypothesis of F. O. Bower [7,47,48]. In studying morphogenetic change during bryophyte life cycles, Bower based his initial antithetic theory on the work of Čelakovsky [49] who studied the alternation of generations in cryptogams. For a comprehensive review of the history of Bower’s interpolational theory, see Haig [50].

Figure 1 presents a summary diagram of Bower’s interpolational hypothesis in the context of natural selection in aquatic vs. subaerial conditions. Bower proposed that in evolutionary time, the origin of the sporophyte generation began with a single diploid cell, the fertilized zygote, that remained attached to its amphibious gametophyte host. In the ancestral alga, that zygote would have undergone meiosis to produce (aquatic) biflagellate zoospores (Figure 1a). However, in ephemeral aquatic habitats with periodic subaerial exposure, there would have been selection pressure to encase those zoospores in resistant walls to allow for perennation (overwintering) (Figure 1b). Bower next predicted that the single zygote might delay meiosis and simply begin to divide mitotically to produce multiple diploid zygotes, each of which was still capable of undergoing meiosis to produce spores. This resulted in a hypothesized “zygote, or so-called, spore thallus” (Figure 1c). The next stage in his scenario was the “sterilization” of totipotent cells to form a vegetative tissue that was no longer capable of meiosis but continued to divide mitotically to form protective or nutritive tissues surrounding the zygote (or zygotes) (Figure 1d). Presumably, this first set of vegetative cells would eventually be associated with the future sporangium, but the essence of the hypothesis is that the further evolution of the vegetative cells (and tissues) of the sporophytic generation would become established as the developmental program of the plant embryo (Figure 1e). Of course, this is a vast oversimplification of what was required to evolve complex multicellularity in the land plants, but it presents an evolutionary developmental scaffold that serves as a starting point for comparison to both the fossil record and the serial acquisition of the genomics of plant development. The genius of Bower’s view was the recognition that the evolution of sporophyte development in geologic time occurred in reverse order to the manifestation of sporophyte development (growth) as seen in extant plants.

Here, we examine the nature of the early spore fossil record and tie that record to Bower’s basic scaffold—that of “spores before sporophytes.” We will first introduce the historical use of spores as land plant proxies and explain why that needs to be revised in terms of documenting the genomic assembly of the first embryophytes. This also entails revisiting a discussion about the definition of the term cryptospore, the spore-like microfossils that often occur in the fossil record as tetrads and dyads long before the advent of the trilete spore, the preferred propagule of the early vascular land plants. Next, we attempt to reframe the question of the origin of plants away from the traditional paleontological perspective, which has been focused on pattern of species relationships (phylogeny) rather than process of genomic assembly (evolution). Paleobotanical inquiry has been overly concerned with trying to pinpoint the timing of the origin of land plants. Far more interesting is the question of how embryonic complex multicellularity evolved in the lineage that became the land plants. That question of “how” involves combining theory with direct fossil evidence in the context of trying to piece together the long process of the evolution of plant embryophyty. There is no such thing as an embryophyte existing prior to the plant embryo—the multicellular structure that includes an inherent genomic program specifying the development of a complex, multicellular organism from a single fertilized egg cell. It is the serial assembly of that genomic package, through a combination of re-purposed prior algal genes and de novo genes unique to land plants, that constitutes the “origin” of land plants. For reasons cited below, it is the fossil record of cryptospores and trilete spores that yield the first clues to the serial origins of plant development. In doing so, the cryptospore/spore record provides a starting point for thinking about the subsequent genomic assembly of other aspects of plant development. Finally, we introduce the fossil record of Cambro–Ordovician sporomorphs and discuss their relation to the known record of land plant spores in the Silurian and the evolutionary origin of the land plant spore in the Ordovician. This leads to the final section on the relation between the evolution of the plant spore and the evolution of the overall plant sporophyte.

2. The Historical Use of Spores as Land Plant Proxies

The modern paleobotanical study of the oldest land plants was ushered in by Lang [51] who described the first clearly vascular plant, Cooksonia pertoni Lang from the Downtonian (Přídolí) of Wales [10]. This assemblage, which is very near the Siluro–Devonian boundary, marks the point in time after which land plant fossils become more and more common and well known through the remainder of the Devonian. There are a few outliers of vascular plant fossils subsequently recovered from Silurian rocks, including the well-established occurrence of Cooksonia in the Homerian (Silurian) of Ireland [29,52] and in the Middle Sheinwoodian (Silurian) in the Prague Basin, Bohemia [30], but any older land plant remains are truly problematic. Wellman et al. [35] recently reviewed claims of Ordovician land plants and found none to be credible. Bona fide trilete spores, e.g., Ambitisporites avitus Hoffmeister, Figure 2a, originally described from the lower Silurian of Libya [53], on the other hand, have stood the test of time, and there are now credible reports of trilete spores reported throughout the remainder of the Silurian [42,54].

This temporal lag between the arrival of the first trilete spores and the first vascular plants was well established by 1970 [55], and the recovery of trilete spores in the sedimentary rock record has subsequently served as a proxy for the contemporary presence of land plants. Trilete spores are shed by bryophyte species today; however, with the exception of Sphagnum, a clade that diversified during the Miocene [56], most spores of the true mosses (Musci) are rounded, alete, and without robust sporoderm [57]. Therefore, as a practical matter, the recovery of fossil trilete spores, beginning with Ambitisporites in the latest Ordovician/lowest Silurian, became a proxy record for vascular plants (tracheophytes), even though this is not strictly true—trilete spores found in the rock record are a proxy for embryophytes.

Another factor that favors the preservation of fossil spores over plant vegetative matter is sporopollenin—the highly chemically inert biopolymer that is found in the outer walls of spores and pollen grains. Sporopollenin protects the living cell from desiccation and photochemical damage, including UV-B. These deleterious environmental factors are directly due to subaerial exposure, and there is an underlying assumption from the paleobotanical perspective that land plants evolved sporopollenin in response to natural selection in subaerial environments. For example, recent research shows that plant spores and pollen grains vary their sporopollenin content in response to environmental factors associated with exposure to UV-B [58,59,60,61,62]. This implies that sporopollenin synthesis pathways seen in land plants today evolved under conditions of exposure to UV-B irradiance—the initial evolution of sporopollenin synthesis took place under subaerial conditions. Regardless of the evolutionary origin of sporopollenin synthesis, the resistant nature of the spore wall, in combination with the abundance of spore production as evidenced in early land plants, creates a reasonable expectation that the plant spore should precede the arrival of vegetative plant tissues in the fossil record [31,33,63].

Gray and Boucot [64] were the first to recognize that in the early Silurian, non-marine palynological assemblages were characterized by a previously unrecognized form of plant-like spores that are found as permanently bound spore tetrads. Their documentation of permanent spore tetrads from the Medina Group (New York state) was followed by the recovery of trilete spores (Figure 2a) and spore tetrads (Figure 2a) from the laterally equivalent Massanutten Sandstone in the Southern Appalachians of Virginia [65] and in the Tuscarora Formation (Figure 2c–e) in the Central Appalachians of Pennsylvania [66,67].

The initial claims of Jane Gray, that the spore tetrads were derived from land plants, were received with some degree of skepticism [68], but by 1984, the idea that early land plants were releasing their spores as permanent tetrads (and, dyads) rather than as trilete spores became formalized taxonomically by the creation of the term cryptospore [69]. Gray argued that the tetrad condition was consistent with spore release found in some extant bryophyte groups [34,70,71], which is true only for liverworts (Marcantiopsida), so Richardson’s cryptospore concept embraced the idea that spore tetrads may have been the evolutionary precursors to the trilete spore, paralleling the possibility that the bryophytes (liverworts) were the evolutionary precursors to the vascular plants. The general acceptance of a land plant as opposed to algal origin to cryptospore tetrads [70,71] was also reinforced by studies demonstrating heterochrony in extant bryophyte sporogenesis [72]. This, too, made it logical to think that the trilete spore was the evolutionary outcome of delaying sporangium maturation and tetrad release by adding a phase of spore separation to spore development prior to release. Such a process would have been facilitated by the tapetal production and release of callase late in spore development [73].

In any case, during the 1980s, the general acceptance of trilete spores as a proxy for land plants prior to their general occurrence in the fossil record was extended to the occurrence of spore tetrads, particularly those that were arranged in a tetrahedral configuration, a geometry that “predicts” future trilete spore formation. More accurately, tetrahedral spore tetrads, specifically Tetrahedraletes (Figure 2b–e), Cryptotetras (Figure 2h), and Rimosotetras (Figure 2l) became a proxy for the existence of embryophytes, not just vascular plants, since the closest living plants that release spores in tetrads are primarily liverworts. The initial recovery of Ordovician (Caradoc, now, Katian) tetrads from Libya [74] extended the recognition and acceptance of Late Ordovician spore tetrads as evidence of the presence of land plants in deposits that predate the first occurrence of actual plant mesofossils. That evidence now extends to Middle Ordovician deposits in Saudi Arabia [39,74,75], which contain exquisitely well-preserved dyads (Figure 2g) and tetrahedral tetrads (Figure 2h), and the Lower Ordovician (Tremadocian) in Australia [37], where the tetrahedral tetrad, Rimosotetras was recently recovered. By utilizing tetrahedrally arranged cryptospore tetrads as a proxy for land plants, the temporal gap with plant mesofossils has now increased to around 50 Myr (Tremadocian-Homerian). This is the present paradigm with regard to the fossil record of land plant origins: they originated during the lowermost Ordovician (c. 480 Ma), but for various reasons [31] are not preserved in the rock record until the Homerian (c. 430Ma). This still does not agree with molecular clock studies [25,26,27,28], all of which posit older origins to the embryophytes.

The term, cryptospore, itself, has two separate definitions [42,76,77,78], which are still in use today and can be cause for some confusion [78]. It is important to realize the distinction here because the varying definitions are closely tied to the use of cryptospores as earlier fossil proxies for land plants. Steemans initially proposed that cryptospores, whether in dyad, tetrad or monad form, were necessarily shed by land plants (embryophytes). They are, in palynological terminology, miospores [42]. This, in effect, guarantees that the fossil distribution of cryptospores corresponds to the fossil distribution of the Embryophyta. Because both cryptospore dyads and tetrahedral tetrads are known to occur in sporangia of Devonian plants [79,80,81,82,83,84,85] there is good evidence supporting the direct link between fossil embryophytes and cryptospores sensu Steemans. In addition, some liverworts shed spore tetrads normally today, and there is even one documented case of meiotic production of dyads in Haplomitrium gibbsiae, a basal liverwort [86].

In 2000, Strother and Beck [76] reported on a new discovery of spore-like microfossils from the Bright Angel Shale in the Grand Canyon. The term, “spore-like”, was required in this case because none of these Middle Cambrian palynomorphs possessed a trilete mark and none of them occurred in combinations of four, tetrahedrally. arranged spore bodies. However, the assemblages recovered from the estuarine facies of the Bright Angel Shale were very much unlike acritarch-dominated marine facies known globally from the Middle Cambrian. In fact, the non-marine provenance of the assemblage combined with the overall spore-like character of the palynomorphs led to a proposal to expand the use of the term, “cryptospore” to include all non-marine, spore-like palynomorphs [76,87,88,89]. This more liberal use of the term cryptospore was intended to accommodate spore-like microfossils that might be derived from “algae” or “proto-embryophytes” that had yet to achieve true land plant status—development via an embryo. Strother and Beck [76] argued that this broader, more inclusive term was needed to prevent their classification as acritarchs, a term which carries with it a connotation of marine algal phytoplankton. This also places cryptospores on a par with other palynomorph classes that constitute components of palynological assemblages. In pragmatic terms, it is relatively obvious when scanning a palynological strew slide containing lower Paleozoic residue to distinguish cryptospores from chitinozoans and acritarchs.

While it seems clear that some tetrads and cryptospore dyads have unambiguous connections to Siluro–Devonian land plants [most recently reviewed in [35]], others, particularly enveloped and sculpted enveloped tetrads and dyads, have not been recovered from fossil plant sporangia. Therefore, palynological assemblages containing cryptospores, which appear to be unambiguously related to embryophytes, invariably contain additional species whose botanical provenance is not known, although in many aspects they appear spore-like in overall character. This dilemma further emphasizes the need for a broader term to encompass spore-like palynomorphs whose systematic position cannot be proven to belong to the embryophytes. In addition, it would be prudent to divorce the class term, cryptospore, from its use as a proxy for the land plants. Cryptospores are really more of a class of palynomorphs, roughly on a par with chitinozoans, spores and acritarchs—rather than a sub-set of plant spores.

There are two more issues to discuss with respect to the use of the term cryptospore. One is the nature of spores and cryptospores as proxies for the presence of land plants in palynological assemblages. The second has to do with the more recent developments in molecular biology, and especially phylogenomics, over the last two decades, which have reawakened an interest in an evo-devo perspective in the origin [6,37,44,45,90,91] and early evolution [92,93,94] of land plants. Although the concept of “spores before sporophytes” was recognized over 100 years ago by Bower [7,47,50], it is only recently that the significance of this recognition of the evolution of plant development in the transition between the algae and the land plants has been applied to the study of the fossil record [6,45,94,95]. If spores evolved early on in the evolutionary transition from algae to land plants, then spores per se, including trilete spores, may not be a correct proxy for the existence of embryophytes—charophytic, subaerial algae may have been producing plant-like spores prior to the origin of sporophytic development from an embryo [94]. Thus, the cryptospore proxies that have been accepted as indicating the presence of land plants in strata deposited prior to the first macroscopic plant fossils, may be acting only as a proxy for the evolution of plant-like meiosis (sporogenesis) and not for the existence of embryonic development in land plants [37]. If the plant spore evolved in an evolutionary time before the sporophyte, paleobotanists can no longer use recovered spore tetrads as a proxy for the presence of land plants—only as a proxy for meiotic sporogenesis.

3. Phylogeny and the Evo-Devo Approach to the Evolution of Land Plants

When the pattern of evolutionary relationships is reduced to a bifurcating tree of higher taxa, even if those taxa are clades, we sometimes may feel as though the problem of the evolutionary history of the groups depicted has been solved as well. However, the bifurcations on a phylogeny are not equivalent in terms of the evolutionary processes, ultimately species originations, that took place in deep time to generate that evolutionary pattern. The split between the Embryophyta and the green algae is not equivalent, in an evolutionary sense, to lineage splits within the Charophyceae or another green algal group. Speciation events occurring within an algal group all occur within organisms whose morphological complexity is at the same level of simple multicellularity. Speciations within the Charophyceae do not require the de novo invention of an entirely new level of structural biological complexity. However, the bifurcation between the alga sister to the Embryophyta and the Embryophyta involved the evolution of complex multicellularity in the streptophyte lineage. That evolutionary step, the origin of the embryonic form of complex multicellularity sensu Knoll [2], has only occurred four times in the eukaryotic tree of life (eTOL): the brown and red algae, animals, and the land plants [96]. Both the red and brown algae are fundamentally marine in origin and distribution, and the animals, too, had an aqueous origin. The land plants, however, are fundamentally a subaerial group, although they derive from freshwater algal ancestors [5].

Just knowing the phylogeny of the land plants and their green algal outgroup does not fully explain the origin of the land plants: the pattern itself is not explanatory. Instead, we must look at the evolution of embryonic development for an understanding of how this phylogeny came to be. Embryonic development includes the entirety of a complex system of interacting tissues that derive from a single fertilized egg cell, the diploid zygote, as it undergoes a series of mitotic cell divisions followed by cell movements and differentiation into different cell types. This process of differentiation, itself, had to evolve over time, and that is what we need to investigate from a paleobotanical perspective if indeed we are to gain a proper understanding of plant origins in geologic time.

It is clear, therefore, that the “origin” of land plants does not constitute a singularity in geologic time. Rather, the “origin” took place over a period of time as the plant genome was assembled from a mix of reprogrammed prior algal genes combined with de novo genes [6,44,45]. We refer to this as a period of serial assembly of the plant regulatory genome. From a paleobotanical perspective, one might hope to see serial assembly manifest itself in a stratigraphically ordered sequence of plant-like fossils, but this is not the case. Instead, we are met with various fragments, but especially of cryptospores and spores, cuticle-like impressions of epidermal cells, and banded and smooth-walled tubes and fibrous elements. What these represent are characters that are composed of recalcitrant organic polymers. These features can be used to track serial assembly to the extent that they are the manifestations of regulatory pathways (Character Identity Networks, or ChINs sensu Wagner) [97] used in the developmental construction of morphology, or structural fingerprints sensu Rothwell and Tomescu [94,98].

These are essentially the requirements of an evo-devo approach to the study of plant origins as opposed to a phylogenetic approach. Neontologists are now engaged in discovering those prior algal genes that have been coöpted, or repurposed, for new functions in plants [44,45,46,90]. Paleobotanists can provide a geological timeline of character acquisition and show the temporal order of the serial acquisition of the underlying plant genome. The remainder of this review will pursue this program from this paleobotanical perspective, focusing on the cryptospore and spore fossil record, but with the goal of interpreting the evolution of sporogenesis during the algal–plant transition.

4. The Cambro-Ordovician Fossil Sporomorph Record

Leaving behind the controversial aspects of the use of the term cryptospore, we can begin to assemble a record of sporomorphs through the Cambrian–Silurian interval. The Early Paleozoic encompasses the time range during which some molecular “timetrees” posit the origin of land plants [12,27]. Figure 3 represents such a diagram, which demonstrates, in a semiquantitative manner, a general picture of early sporomorph evolution. The categories used in Figure 3 correspond to morphological categories used to group cryptospore taxa [78,99] and, with the addition of polyads, correspond roughly to the range chart (Figure 3) of Kenrick et al. [31]. The width (in mm) of each time bin corresponds to the number of species published in 28 systematic descriptions of cryptospore assemblages, so this provides only a first-order assessment of cryptospore stratigraphic distribution. In Figure 4, we have collapsed the various cryptospore categories into a single “cryptospore” taxon, corresponding to a liberal definition of cryptospores that is intended to include both miospores and ancestral “algal” spore types (see above discussion on the definition of cryptospore). There are no occurrences in the Floian, but at least two species are found earlier, so we assume they persist through to the Dapingian. The gray block and dotted line at the base of the trilete spore distribution corresponds to what we consider to be a lower Katian outlier based on Steemans et al. [100] that was systematically described by Wellman et al. [40]. This particular apparent outlier contains ornamented trilete spores that are not otherwise known until the Wenlock [101,102]. There are also trilete spores reported in the Sandbian-Hirnantian interval from Sweden, but we interpret these to be distorted leiospheres, where surface folds mimic trilete marks. In our view, the FAD of trilete spores are occurrences of Ambitisporites in Hirnantian sediments from Gondwana. This interpretation is reflected in Figure 3.

The polyads category is intended to show the various taxa that have been attributed to an evolving complex of charophyte algae that were adapting to subaerial conditions during the algal–plant transition [94]. There is some temporal overlap in the lowermost Ordovician between more typical Cambrian taxa and dyads and tetrads (Dyadospora and Rimosotetras) that share affinities with later cryptospore taxa considered to be miospores and hence belong to the embryophytes. These are discussed in Strother and Foster [37], who argued that their co-occurrence reinforces the probability that these older, topologically irregular forms are spores that had yet to evolve a plant-like meiosis. The occurrence of polyads in Darriwilian and younger strata reflects the distribution of Grododowon, which forms planar sheets of dyads (Figure 2j) in addition to irregular tetrad-derived topologies reported by Vavrdová [103].

The bulk of the cryptospore categories contain a mixture of spore types from different sources, and they are generally not considered to be phylogenetically distinct. This is especially so for any of the enclosed forms, whose enclosing envelopes can be interpreted to come from a number of different sources. Early studies of mainly Silurian and Ordovician cryptospores considered the likelihood that enveloped tetrads and dyads had retained either a resistant spore mother cell (SMC) wall or possibly the sporopollenous wall of a charophycean algal zygospore, relating to a developmental shift in the timing of sporopollenin from the diploid to the haploid phase, the so-called sporopollenin transfer hypothesis [21,94,104,105]. Because the normal condition in plant sporogenesis today is for the spore mother cell wall to disintegrate, the retention of a resistant-walled envelope weighs in as more of an algal trait [106]. The temporal distribution of enclosed dyads and tetrads, as expressed in the range chart (Figure 3), does not support the idea that enveloped forms pre-date naked cryptospore types. In all three cases, monads, dyads and tetrads, the naked forms are more abundant earlier than their enclosed counterparts. The presence of enclosing walls in the Cambrian cryptospore cohort is another issue altogether and is clearly not related to the retention of developmental stages within a sporangium. Overall, with the proviso that retention of a resistant wall around one or more spore-bodies is more reminiscent of a cyst, which implies an algal origin, the stratigraphic distribution of enclosed cryptospores types does not present a clear evolutionary signal with respect to the algal–plant transition.

In a study of cryptospores from the lower Silurian Tuscarora Fm, Johnson [67] observed that sometimes morphologically similar forms were preserved both with and without a thin outer envelope. This prompted her to suggest that, in some cases, the absence of an envelope might be due to a taphonomic loss. In the original study of the Darriwilian Hanadir Shale Member, Strother et al. [107] illustrated a single spore tetrad within a loose, thin, envelope, but in a systematic study of the same assemblage including a set of reprocessed samples, they were unable to recover any enveloped forms [39]. In spite of Johnson’s observations, the presence/absence of an enclosing membrane continues to be utilized as a generic (and supra-generic) character in cryptospore classifications [78]. In the end, in combination with possible taphonomic loss, the utility of enclosing walls as a clue to systematic affinity remains somewhat questionable. And, as noted by Steemans and Wellman, “The envelope absence/presence may be an unreliable character for taxonomic designation” [78].

Planar tetrads are another problem altogether, as this topological arrangement has no modern analog among cryptogamic land plants. Quadrisporites Hennelley is a case in point. This cruciate form retains asymmetric ornamentation in some species, reminiscent of peripheral spines seen in various chlorophycean freshwater algae [106]. Other species may contain what appears to be an excystment feature in the form of lateral pores. Although we have tabulated this taxon as a planar cryptospore tetrad (Figure 2), its relationship to the land plants has been questioned [78,108]. Alternatively, the spore-bodies in the Ordovician planar tetrad, Tetraplanarisporites, are very similar in form to other tetrads that are considered to be related to the embryophyte lineage. Therefore, in this case, the inclusion of a tetrad form that is not seen in Siluro–Devonian sporangia may be indicative of morphological variation in tetrad production in plant precursors.

Both regular cryptospore dyads and tetrahedral tetrads are well documented to occur in Devonian polysporangiates [81,82,83,84,85,109], so there is no doubt that these cryptospores are possibly of embryophyte origin. Their co-occurrence with Cambrian cryptospores in the Tremadocian of Australia [37] makes it likely that not all of these simple forms are guaranteed to be markers of embryophytic affinity, however. And, in an evo-devo approach to reconstructing the fossil evidence of the serial assembly of the plant sporophyte, both tetrads and dyads are now considered as evidence for the presence of plant-like (bryophytic) sporogenesis/meiosis [94,110], rather than as proxies for the existence of the plant sporophyte itself.

Figure 3. Stratigraphic distribution of cryptospore categories and early trilete spores. Here we have plotted the stratigraphic distribution of species grouped into traditionally defined categories of basic cryptospore types. Under embryophytic spores, “Imperf trilete” refers to Imperfectotriletes species, which are considered to be spores physically torn from isometric tetrads. The width of each distribution is proportional to the number of species reported for each stage. Greyed lines represent the inferred existence of taxa. The grayed segment in the trilete spores refers to an outlier occurrence [40,101] of trilete spores from a shallow well core in Saudi Arabia that is yet to be replicated. The species data were extracted from the following systematic studies: [37,38,39,66,67,75,88,101,102,103,111,112,113,114,115,116,117,118,119].

Figure 3. Stratigraphic distribution of cryptospore categories and early trilete spores. Here we have plotted the stratigraphic distribution of species grouped into traditionally defined categories of basic cryptospore types. Under embryophytic spores, “Imperf trilete” refers to Imperfectotriletes species, which are considered to be spores physically torn from isometric tetrads. The width of each distribution is proportional to the number of species reported for each stage. Greyed lines represent the inferred existence of taxa. The grayed segment in the trilete spores refers to an outlier occurrence [40,101] of trilete spores from a shallow well core in Saudi Arabia that is yet to be replicated. The species data were extracted from the following systematic studies: [37,38,39,66,67,75,88,101,102,103,111,112,113,114,115,116,117,118,119].

Figure 4. Stratigraphic distribution of cryptospores and trilete spores. In this stratigraphic distribution plot, we have combined all species occurrences of cryptospores s.l. into one distribution. The grayed segment in the trilete spores refers to an outlier occurrence [40,100] of trilete spores from a shallow well core in Saudi Arabia that is yet to be replicated. Data based on the same set of references as in Figure 2.

Figure 4. Stratigraphic distribution of cryptospores and trilete spores. In this stratigraphic distribution plot, we have combined all species occurrences of cryptospores s.l. into one distribution. The grayed segment in the trilete spores refers to an outlier occurrence [40,100] of trilete spores from a shallow well core in Saudi Arabia that is yet to be replicated. Data based on the same set of references as in Figure 2.

5. The Evolutionary Origin of the Plant Spore as Evidenced in the Fossil Record

The cryptospores represented as polyads in Figure 3 constitute a distinct palynological assemblage that has been documented in Laurentia [76,87,88,117,118,120,121,122] (see Figure 2m–q), and more recently, in China [116]. These spore-like microfossils are considerably different from the larger and more geometrically regular cryptospores found in Darriwilian and younger strata; they do not include forms with clear relationships with any extant spores of cryptogamic land plants. Wellman [33] initially concluded that these problematic Cambrian palynomorphs were either the resting cysts or “body cells” of some form of green algae. This interpretation, that the Cambrian cryptospore assemblages bear no relation to the origin of land plants, has continued to influence assessments of the early Paleozoic spore/cryptospore record [27,31,32,35,78], although Kenrick et al. [31] considered that the Cambrian forms may have come from green algae that inhabited freshwater or subaerial habitats.

It is important to note, however, that, parallel to the fact that Cambrian cryptospores are not found in the modern cryptogamic palynoflora, neither are they found in populations of extant green algae (Chlorophyceae plus Charophyceae). In other words, we have no modern analogs to Cambrian cryptospores found in either spore-bearing land plants or freshwater algae living today. A study designed specifically to look for such remains in extant subaerial algae, from a wide range of geographic habitats, showed that the laminae in vegetative charophyte cells subjected to desiccation [123] are not homologous to the laminated walls of Cambro–Ordovician cryptospores [110,118,120,121]. The extant terrestrial algal flora does not provide any modern analog to the Cambrian cryptospores; therefore, we must look elsewhere for a demonstration of the systematic affinities of these extinct, spore-like microfossils. Intriguingly, the complex topology of these spores provides distinct clues as to the developmental processes of sporogenesis—including peculiar aspects of cell division and the timing of wall formation. And, when placed in the context of the evo-devo argument of “spores before sporophytes”, (sensu, Bower 1908), it is possible to generate a working hypothesis of the evolution of sporomorph development, whose timing is based directly on the fossil record of cryptospores—such a model has been previously published [94] and will only be reviewed briefly here.

The synthesis presented by Strother and Taylor [94] is based on our own observations on the morphology and topology of fossil cryptospores [39,77,88,110,117,118,120,121] in combination with botanical studies on charophycean life cycles [124,125,126] and molecular and cellular studies on sporogenesis in bryophytes [72,127,128,129,130,131]. It begins with the hypothetical underpinnings of the interpolational hypothesis of Bower [47], who himself based his theory on the issue of the antithetic versus homologous origin of the alternation of generations in the plant life cycle as originally posited by Čelakovsky [49] in 1874. Bower [47] preferred the term interpolational, to antithetic, as it more accurately described his position on the de novo origin of the plant sporophyte and not with the origin of alternation per se [50]. The basic idea is encapsulated in the phrase, spores before sporophytes, which posits that in evolutionary time, the land plant spore evolved first, followed by the evolution of the vegetative plant body of the sporophyte generation. Thus, the evolutionary order, as would be seen in the fossil record, is opposite to the perceived developmental order observed in embryophyte life cycles today, where sporogenesis (and meiosis) occurs after the construction of the plant body. The idea that spores evolved first is reinforced by studies of sporogenesis in extant bryophytes. The details in the timing of pre-prophase band (PPB) formation and quadralobing in bryophyte sporocytes indicate that shifts from algal to bryophytic meiosis are likely due to heterochrony between the onset of cytokinesis and the application of sporopollenin to the developing (meio)spore wall [71]. Bryophytes show a range of microtubule organizing centers (MTOC’s) that span algal (centriolar) to embryophytic (diffuse) and include unique MTOC systems as well [131]. These developmental studies indicate that the fundamental aspects of cell division were actively undergoing natural selection during early bryophyte evolution: mitosis and meiosis were in play during the algal–plant transition.

Taking a step further back, it is apparent that in the charophycean algae today, meiosis is not at all like that seen in the bryophytes [124]. In Coleochaete, the fertilized zygote (zygospore), which would initially be considered diploid (2n) prior to germination, undergoes a series of up to five rounds of endoreduplications of the genetic material [132]. Here the zygote functions as a coenocyte, which then undergoes schizogony (cytokinesis) to produce up to 32 haploid, biflagellate zoospores. Thus, each initial fertilization event may result in anywhere from 2 to 32 zoospores. However, it is the decoupling between karyokinesis and cytokinesis that is significant here. Bear in mind that in the extant species, this fertilization result produces zoospores that function only in water. Any perennation function today is served by the resistant zygote.

In order to connect the extant life cycle of Coleochaete to the origin of the plant spore we need to assume the transfer of sporopolleninous wall formation from the zygote wall to the zoospore wall—the so-called “sporopollenin transfer hypothesis” [21,22,104,105,133]. Hemsley [104], Figure 1 made the logical assumption that the transfer would have involved four meiotic products, but the fossil record dictates otherwise. The application of sporopollenin as a molecular component of the spore wall occurred endogenously in cells that underwent a charophycean style of reduction division, not an embryophytic style of meiosis restricted to four meiospores per zygote. Independently of their relation to the embryophytes, both Adinosporus and Agamachates express sporopolleninous walls in spore–body combinations that indicate a decoupling between karyokinesis and cytokinesis in a manner that mimics reduction division in Coleochaete today. We refer to this as endogenous wall formation because in order to produce the combinations of cells (spores) within walls (envelopes) as seen in these species, the resistant component of cell walls must be generated centrifugally. It is simply not geometrically possible to generate combinations of enclosed tetrads and spore dyads within a single wall if the resistant wall was formed from the outside of the developing spore combination [95]. It is now possible to fit the topology of cryptospore walls, as seen in what appear to be irregular spore packets, into a general evolutionary model of spore wall formation (Taylor and Strother, in preparation), which we briefly summarize in the following section.

6. The Evolution of Spore Development

It has long been recognized that the fundamental unit of sporopollenin deposition is in the form of tri-partite lamellae (TPL) of unit membrane thickness (less than 50 nm) [134]. Developmentally, this consists of the excretion of sporopollenin precursors from inside the cell, through the cell membrane, and polymerization of the final resistant compound on the cell surface. Successive waves of deposition result in successive superposed lamellae, the youngest being closest to the cell surface. If these pulses of deposition occur over the entire surface of the cell, this results in a complete barrier, which we refer to here as a continuous layer. In the case of this type of wall in Cambrian cryptospores, TEM analyses reveal somewhat thicker individual continuous lamellae (termed laminae—greater than 50 nm), indicating the formation of substantially more robust walls, presumably in response to greater challenges presented by desiccation and/or UVB exposure in subaerial environments (Taylor and Strother in preparation). It is not until the advent of much thicker walls in the Ordovician that we begin to see a new type of sporopollenin deposition which we interpret to be the evolution of a chamber enclosed from the environment (sporangium), surrounded by a layer of nutritive cells depositing additional sporopollenin (tapetum) that is completely under the control of the parent organism. This organism may not have been an embryophyte, suggesting the existence of a phase of the genomic assembly that could be termed “sporangium before axial sporophyte”.

7. Conclusions

A modern history of phylogenetic thinking, combined with a heavy dose of paleontological uniformitarianism, has slowed the contribution of the fossil record to the more general understanding of the evolutionary origin of the land plants. A long-term focus on the phylogenetic relations among taxa has obfuscated the fact that the genomic assembly of the first embryophyte was a lengthy process. The strict requirement that cryptospores be defined as derived from embryophytes [42,78] has prevented us from recognizing which spore-like microfossils were the ancestral propagules of embryophyte ancestors. Paleontological uniformitarianism refers to the idea that we must base our assumptions of fossil affinities in comparison with modern forms—in this case, pre-existing plant spores. However, of course, the plant spore itself must have come from somewhere so there was never a guarantee that the precursor to the trilete spore would have been recognizable as a trilete spore. This is why we need a term like cryptospore as a label on microfossils that do not exist in today’s palynoflora.

We have begun the process of applying the biology of the freshwater and subaerial charophyte algae to the fossil spores and fragments that are recovered in the fossil record. This has been important in the interpretation of seemingly topological and geometric irregularity that runs rampant through the earliest Paleozoic cryptospore assemblages. However, with insight provided by TEM, we have been able to construct a consistent model of pre-embryophytic sporoderm ultrastructure that seems to be a logical first step toward the understanding of the evolution of the plant spore. These studies have indicated that the tetrahedral tetrads, like Cryptotetras and Tetrahedraletes, which are now accepted by paleobotanists as indicating the origin of land plants, are, rather, indicators of the earliest meiosis (sporogenesis), and there is no guarantee that this took place in a plant with a sporangium perched atop a sporophytic axis.

The fossil record, as we interpret it now, indicates the existence of a subaerial charophyte complex that was evolving in response to aeroterrestrial settings, and in doing so, was evolving toward a new phase of biological complexity. This is literally the evolution of development, and we are fortunate to have seen just a tiny piece of this evolutionary history in the form of sporopolleninous walls and other fragments of recalcitrant biopolymers that have survived fossilization. The next phase of this research program is to recognize which of these various fossil fragments and spore-like microfossils retain clues to their underlying, inherent developmental genomics, or ChINs, sensu Wagner [97]. The paleontological goal is to recognize the evolutionary process of the algal–plant transition and to place that transition into its correct place in geologic time.