Organization, anatomy, and fungal endophytes of a Triassic conifer embryo†
This study was supported in part by funds from the National Science Foundation (ANT-0635477 and EAR-0542170). The authors thank the two anonymous reviewers whose comments greatly enhanced the manuscript.
• Premise of the study: Much is known about the Paleozoic conifers and the conifers assignable to extant families that appear in the Jurassic; however, relatively little is known about the transitional conifers of the early Mesozoic, especially the Voltziales. To better understand the evolution of this group, we aim to increase knowledge of voltzialean anatomy and morphology. A fossil embryo of the group is described in this study.
• Methods: Several permineralized seeds, one containing a well-preserved embryo, were collected from the early Middle Triassic Fremouw Peak locality in the Central Transantarctic Mountains. Samples were prepared using the standard acetate peel technique and studied in transmitted light.
• Key results: The embryo belongs to Parasciadopitys aequata, a member of the Voltziales. The embryo and megagametophyte tissues are exquisitely preserved. The embryo is colonized by two distinct fungi. Sporocarps of the fungi are found in the megagametophyte and in the space between the megagametophyte and nucellus. The additions of embryo and megagametophyte characters to the description of P. aequata have made it one of the most completely known fossil taxa reproductively.
• Conclusions: Preservation of fossil embryos, although rare, can expand the array of characters available in tracing the evolutionary history of plants. The embryo of P. aequata shares similarities with embryos of other extinct and extant conifer families. The association of the embryo with Combresomyces cornifer and Mycocarpon asterineum increases our understanding of the roles of microorganisms in Triassic ecosystems.
Structurally preserved remains have extensively contributed to our understanding of the internal organization of fossil plants. In some instances, preservation is so extraordinary that features such as starch grains (5), nuclei (4; 42), microgametophytes (56), sperm (47), apical cells (24), possible chromosomes (6), megagametophytes with archegonia (20, 21; 64), and embryos (e.g., 43; 37; 69; 64; 39) can be observed. Although the relative scarcity of fossil megagametophytes and embryos, compared to other preserved plant parts, render detailed comparisons difficult, they nevertheless depict a more complete picture of fossil plant anatomy and morphology while providing additional character states useful in determining ancestral traits in fossil plants. Here we report on an isolated seed containing both a cellularly preserved embryo and megagametophyte from the Triassic conifer seed cone Parasciadopitys aequata Yao, Taylor et Taylor (83).
Parasciadopitys aequata was originally described by 83 from permineralized peat collected on Fremouw Peak in the Central Transantarctic Mountains, Antarctica. 83 assigned the cone to the Taxodiaceae (= Cupressaceae s.l.) although it shares many features with seed cones assigned to the Voltziales, which are considered to be transitional or intermediate between the Carboniferous Cordaitales and modern conifer families (57; 80; 18). 18 have reported anatomically preserved compression fossils of Telemachus Anderson cones from the Triassic of Antarctica and indicate that Parasciadopitys is a permineralized equivalent of the compression taxon Telemachus. Cones of Telemachus are commonly placed in the Voltziales. A phylogenetic analysis of the compression and permineralized species demonstrated that Parasciadopitys is better grouped with the Voltziales, a heterogeneous grade group believed to include the ancestors of modern conifer families (57; 18). We agree with the conclusions of 18 that Parasciadopitys is better aligned with the Voltziales than with the Taxodiaceae.
Evidence of fungi and fungal-like organisms is frequently found in permineralized peat in the form of isolated vegetative hyphae, spores, and other propagules (see 78 and references therein). Several forms have been described, but we still lack information about the ecology of these organisms, their nutritional modes, and biological affinities. Mycocarpon asterineum Taylor et White (a putative endogonaceous zygomycete) and Combresomyces cornifer Dotzler, Krings, Agerer, Galtier et Taylor (Peronosporomycetes) are two fossil microorganisms from the Fremouw Peak locality, previously known only as dispersed reproductive structures. They are described here as occurring within the seed of P. aequata. In addition to describing embryo anatomy and organization in P. aequata, we also discuss the possible ecological relationship of the embryo and its endophytic microorganisms.
MATERIALS AND METHODS
Stratigraphy and specimen preparation
Parasciadopitys aequata has been described from the Fremouw Formation, Central Transantarctic Mountains. The Fremouw Formation is 620–750 m thick and was deposited by low-sinuosity, braided streams (3). Permineralized peat is found at a single locality, approximately 30 m below the top of the formation. Blocks of permineralized peat were likely rafted into their current position during a flood that caused them to be stranded on sand bars (79). The silica source for the permineralization was the dissolution of siliceous, volcanic detritus that was abundant in the Upper Fremouw Formation (79). Permineralized peat containing seeds of Parasciadopitys was collected from a saddle north of Fremouw Peak in the Queen Alexandra Range of the Transantarctic Mountains (84°17′41″S, 164°21′48″E; 2). The peat is dated as early Middle Triassic based on palynomorphs and nearby vertebrate fossils (19; 27). Peat blocks were sectioned and the polished surface etched with 49% hydrofluoric acid for 1–5 min. Cellulose acetate peels (22) were made from the prepared surface with some peels subsequently mounted on microscope slides using Eukitt (O. Kindler GmbH, Freiburg, Germany) as a mounting medium. Slides are housed in the Paleobotany Division of the Natural History Museum and Biodiversity Institute, University of Kansas, Lawrence, under accession numbers 23,991–24,008, 26,359–26,363, and 26,506–26,536. Peels and slides of the fossil seeds were made from blocks 10,028 Dbot; 10,147 F2; 10,160 D1-S2; 11,277 B2side bot; and 15,711 Ctop. A longitudinal section of an extant Pinus L. embryo (Triarch slide no. 10-6w, Ripon, Wisconsin, USA) from the teaching slides of the Paleobotanical Collections, KU Natural History Museum, was also examined to compare the contents of the fossil and extant embryo cells. All specimens were photographed using a Leica (Allendale, New Jersey, USA) DC500 digital camera attachment on a Leica DM 5000B compound microscope and a Leica MZ 16 dissecting microscope. Digital images were processed using Photoshop CS3 version 10.0 (1990–2007, Adobe Systems, San Jose, California, USA). High magnification (>640×) images were taken using immersion oil.
Cells of the megagametophyte vary in shape and size depending largely on their position relative to the embryo (Fig. 1A). Cells closest to and farthest from the embryo are typically smaller and more elongate than those located centrally between the nucellus and embryo (Fig. 1B). The smaller cells are approximately 38 by 46 µm, while the larger ones are 55 by 80 µm. Cells of the megagametophyte are loosely packed and intercellular spaces are common. Megagametophyte cells are filled with opaque matter of uncertain origin (Fig. 1C). Two types of unknown filamentous strands are also found within cells of the megagametophyte. Thinner strands are < 1 µm wide (Fig. 1C, arrow); more robust strands are ≥ 1.25 µm thick (Fig. 3A, B, arrows). In another specimen of P. aequata, the poorly preserved megagametophyte shows evidence of an archegonium (Fig. 2A, B). When viewed in cross section, the archegonium occurs in the center of the megagametophyte, and its remains are surrounded by degraded material. The archegonium is 312 µm wide at the thickest point and has several ovoid voids (Fig. 2B) that range from 82 to 137 µm long and 73 to 90 µm wide.
Description of the P. aequata embryo comes from a single well-preserved specimen that is present on several acetate peels. The embryo is oriented within the seed with its epicotyl directed toward the chalaza and the suspensor toward the micropylar end (Fig. 1A). A corrosion cavity is present between the cells of the megagametophyte and embryo and is most obvious at the base of the embryo (Fig. 1A, bottom). The embryo itself is 2.0 mm long and 530 µm wide. Cotyledons are 490 µm long and 195 µm wide; the hypocotyl is 1.1 mm long and 530 µm wide and the embryonic root cap (= calyptroperiblem), 419 µm long and 414 µm wide (Fig. 1A). The ratio of the hypocotyl-to-embryo lengths is 0.55 and of cotyledon-to-embryo lengths is 0.25. The ratios of embryonic-root-cap-to-embryo lengths and embryo-to-seed lengths are 0.21 and 0.66, respectively.
Cells of the embryo are nearly isodiametric and generally 18 by 24 µm, except in the provascular regions where they are more elongate and measure about 36 µm long. Intracellular contents are more abundant in the embryonic cells than in those of the megagametophyte. Most noticeable is the presence of relatively large (∼5 µm) spherical structures in the center of nearly all embryo cells (Fig. 3A). These subcellular structures are more elongated in the area of provascular tissue (Fig. 3B) where they measure ca. 3 by 18 µm. The cells of the embryo also contain smaller spherical bodies (Fig. 3A). Numerous strands fill the intracellular spaces (Fig. 3A). In the periphery of the embryo, elongate cells with degraded contents are occasionally present (Fig. 3G, arrowhead). Cells of the embryo are densely packed and have little intercellular space.
The embryo of P. aequata has two cotyledons (Figs. 1A, 3C). In some sections, provascular tissue can be seen in the cotyledons (Fig. 3C); provascular strands extend from the tip of the cotyledons to the shoot apical meristem (SAM) (Figs. 3B, C). The preservation of the SAM itself is poor, and the epicotyl does not appear to be well developed. The SAM of P. aequata is narrow and consists of a small number of cells (Fig. 3D). Surface initials are composed of a single layer of cells that undergo both anticlinal and periclinal divisions. Beneath this layer is the central mother cell zone that contains ∼4 layers of cells (Fig. 3D). The transitional zone that leads from the central mother cell zone into the rib meristem is only a few cells thick. The peripheral zone is also composed of only a few cells (Figs. 3B, D). Cells differentiating into procambium tissue can be seen in the hypocotyl (Fig. 3B), extending from the shoot to the root apical meristem.
The embryonic root cap of P. aequata consists of a meristematic area and developing root cap (Fig. 3E). All root tissues arise from a small group of initials that constitute the root apical meristem (Fig. 3E, arrow). The root apical meristem produces cells in an open organization that differentiate slightly into a column and pericolumn. The column is defined as the cylinder of meristematic cells that will become part of the embryonic stele of the root; the pericolumn is defined as the tissue that surrounds it and will become the cortex of the developed root. The cells of the pericolumn develop from the initials in the column (Fig. 3E). There is no junction zone present between the hypocotyl and embryonic root in P. aequata. The protoderm has given rise to a set of epidermal cells (Fig. 3F, arrow) that surround the entire embryo. The epidermal cells are generally smaller than the cells that make up the majority of the embryo. A layer of hypodermal cells is continuous from the apical portion of the embryo to the basal portion of the hypocotyl, where it becomes disrupted by the root cap. At the micropylar end of the seed, a group of degraded cell walls representing the fossilized remains of the suspensor are present (Fig. 3E, arrowhead). The cells of the suspensor are no longer preserved, but some evidence of its connection to the embryo can be seen at the root tip (Figs. 1A, 3E).
Fossilization of the P. aequata embryo occurred at a late stage of embryo development because the cotyledons appear to be well developed. No leaf primordia were formed in this specimen, indicating that either embryo development had not been completed or P. aequata did not form leaf primordia until it was closer to the time of germination.
Two different microorganisms occur within the P. aequata seed that contains the embryo. In a tangential-longitudinal section that only shows the megagametophyte, an oogonium of the peronosporomycete Combresomyces cornifer is present (Fig. 4A, B). The host plant tissues directly surrounding the oogonium are degraded more than elsewhere in the megagametophyte.
Immature individuals of the putative zygomycete Mycocarpon asterineum (Figs. 1A arrow, 4C, D) are also found within the seed between the megaspore membrane and the megagametophyte. The specimens are typically 200–450 µm in diameter and contain a single large spore (zygospore or azygospore) (Figs. 1A arrow, 4C, D). The wall (mantle) of the structure displays a distinct layer formed by interlaced hyphae. Hyphae can occasionally be found throughout the embryo (Figs. 3A, G) and are preserved as thin tubular strands extending for varying lengths within the tissues. Some occur intracellularly, while others appear to never penetrate cell walls. At this stage, the developing seed no longer has an open micropyle, and it is unclear how the microorganisms entered the seed or whether infection was pre- or postfertilization.
Although fossilized embryos can confer information about ancient plants, their rarity in the fossil record and range of preservation quality together with the lack of many stages in embryogenesis render it difficult to make meaningful comparisons with those of extant plants and other fossil embryos. In P. aequata, only a single late stage in embryo development has been preserved, which somewhat limits the comparisons that can be made across taxa.
Megagametophyte and embryo
Megagametophyte cell shapes are not consistent and may be a reflection of compression (Fig. 1B, C). In extant seeds, this compressed tissue is apparently caused by the growth and expansion of the embryo into the megagametophyte. For fossilized material, degradation of the delicate cell walls preceding permineralization or shrinkage may also be the cause. The distortion of cell shape makes it difficult to interpret a pattern of gametophyte development from this specimen. In the periphery of the embryo, elongate cells with degraded contents are occasionally found (Fig. 3G, arrowhead). These cells are similar in size and position to the secretory structures that are found in the embryos of some extant conifers (63).
The cellular contents of the megagametophyte and embryo tissues are interpreted as the permineralized remains of biological structures and not as preservational artifacts, such as crystal structures. Unfortunately, the fossil material in this study cannot be prepared for transmission electron microscopy (TEM), which is essential for unequivocally identifying organelles in fossil material (45). Because of the size of the small spherical objects in the megagametophyte and embryo tissues, the cell contents could represent the fossilized remains of starch grains and lipid or protein bodies, structures that are abundant in the cells of extant plant embryos (Fig. 5A, B). The visual similarities between the fossil material (Fig. 3A) and living specimens (Fig. 5A, B) are not enough evidence to reach a reliable conclusion; the natural variation in organelle sizes, starch grains, and lipid and protein bodies makes it impossible to positively identify these features (45). Given the position and ubiquitous occurrence of the large spherical objects (>5 µm) in the embryo, along with the elongation of these bodies in the provascular regions of the embryo (Fig. 3B), these bodies more probably represent fossilized nuclei. They appear in the center of each cell, and there is never more than one present (Figs. 3A, 5A). These structures become larger and more elongate in the area of the provascular tissue (Fig. 3B), which is consistent with their interpretation as nuclei because nuclei are enlarged and elongated in regions of cell division in extant plants (60; 36; Fig. 5C). We do not interpret these as the remains of coagulated cytoplasm because they are consistent in size and shape in all of the cells and other structures that are separate from the nuclei are also present, but the lack of TEM data makes it impossible to determine for certain (45). The thin strands present within cells (<1 µm wide; Fig. 1C) are interpreted as cytoplasmic strands and the more robust strands (≥1.25 µm thick and longer than the thin filaments; Figs. 3A, B, arrows) as fungal hyphae. These strands are the same size and shape as the hyphae that make up the sporocarp of M. asterineum. The ovoid voids within the archegonium could be the remains of vacuoles present in the archegonial egg or merely degraded material within the archegonium. Although these voids may appear to be individual archegonia in the illustrated cross section (Fig. 2A, B), it is clear when viewing successive cross sections that they are not archegonia. The voids change position from section to section and do not have the elongate shape characteristic of archegonia in gymnosperms (38). The brown material surrounding the archegonium (Fig. 2B) is interpreted as the remains of archegonial jacket cells.
Comparison with extant gymnosperm embryos
The cellular preservation of the P. aequata embryo makes it possible to compare the fossil embryo with similar features in extant and other fossil gymnosperm embryos. For example, the developing root in P. aequata exhibits the defined column and pericolumn that is present in most gymnosperms (63). Of the extant gymnosperms, only the embryos of the Podocarpaceae and Taxaceae lack a defined column and pericolumn (30; 67; 7; 63). The P. aequata embryo lacks a junction zone between the hypocotyl and root cap. Among extant conifers, embryos of the Pinaceae have junction zones, while those of the Podocarpaceae and Taxaceae do not (63). A continuous epidermis from the cotyledons to the root cap, similar to that seen in P. aequata, can be found in Taxus L. and Podocarpus L'Hér. ex Pers. (67; 63). The hypocotyl of P. aequata is more elongated than in members of the Cycadales (63). Developing secretory structures are found in the pith of P. aequata embryos and are known to occur throughout the conifers (63).
Cotyledon number varies greatly among the gymnosperms, even at the generic level. Embryos with two cotyledons can be found in species of the Cycadaceae, Ginkgoaceae, Araucariaceae, Sciadopityaceae, Cupressaceae, Cephalotaxaceae, Podocarpaceae, Taxaceae, and Ephedraceae (31). The Pinaceae are the only gymnosperms that rarely have embryos with only two cotyledons (31). Although the characters of the fossil embryo that can be compared to extant gymnosperms are few, it is notable that the features present in P. aequata are found in both ancestral and more derived conifer families (11). The Pinaceae, which are generally considered the most ancestral of all extant conifer families (11), appear to have the fewest embryo characters in common with the embryo of P. aequata.
Comparison to fossil embryos
Although rare, a number of fossil gymnosperm embryos have been described to date. These include members of the Araucariaceae (14; 13; 8; 82; 41; 62; 69, 6, 72, 73; 76, 77; 9), Pararaucariaceae (70; 74), Voltziales (43; 39; 29), Bennettitales (10; 65; 35; 61; 58; 75), and others of unknown affinities (37; 64). In some cases, the fossil embryos are only preserved with sufficient detail to determine that the specimens are in fact embryos. Only those embryos with preservation allowing for a meaningful comparison will be discussed here.
There are more fossil embryos described for the Araucariaceae than for any other group of plants; they have been found in Jurassic localities in Argentina (Patagonia), Japan, and India, as well as Cretaceous localities in New Zealand and England. One of the best-known fossil embryos is that of Araucaria mirabilis (Spegazzini) Windhausen (14; 13; 8; 69, 6). Embryos of A. mirabilis from the Cerro Cuadrado fossil forest measure up to 5 mm in length and 1 mm in diameter (14), and are much larger than those of P. aequata. Mature seeds of A. mirabilis measure 8–130 mm long and 2–6 mm wide (71), while the seed of P. aequata that contains the embryo is only 3 mm long and 1.6 mm wide. The embryo-to-seed ratio for A. mirabilis is 0.625, similar to the value for P. aequata (0.66), but the tissue proportions for the two embryos are very different. The cotyledons in P. aequata only extend approximately one fourth of the length of the embryo instead of half the length as seen in A. mirabilis (69). The hypocotyl in A. mirabilis extends for a quarter of the total embryo length in contrast to over half of the length in P. aequata. Both embryos have preserved nuclei that elongate in regions of provascular tissue and secretory cells in their cortex (69). In addition, the two share the same number of cotyledons (69). Like P. aequata, embryos of A. mirabilis have a column and pericolumn structure in their embryonic root cap; the embryonic root cap extends for about one fourth of the araucarian embryo (69), similar to P. aequata.
Araucarian embryos from the Upper Cretaceous of Japan are not as well preserved as those from Patagonia. Embryos of Araucaria nihongii Stockey, Nishida et Nishida possess two laminar cotyledons, similar to P. aequata (76). A series of resin canals occurs throughout the cotyledons and hypocotyl of A. nihongii. The embryonic root cap is differentiated into a column and pericolumn region (76). The preservation of Araucaria. nipponensis Stockey, Nishida et Nishida is not quite as good as the other araucarian embryos, but it is clear that these embryos had two cotyledons (77).
Jurassic rocks in southern England have also yielded preserved embryos. Embryos of A. sphaerocarpa Carruthers have two cotyledons that comprise nearly half of the embryo length. The embryo-to-seed ratio for this species is 0.44 (72). Araucaria brownii Stockey has been suggested as having four cotyledons, although the quality of preservation makes it impossible to say for certain (73).
Araucarites bindrabunensis 82 from Mesozoic strata in the Rajmahal Hills is another member of the Araucariaceae known to have fossil embryos. Vishnu-Mittre noted their presence in the fossil description, but did not elaborate on any of the features of the embryo. 62 later described an araucarian megastrobilus from the Rajmahal Hills with a dicotyledonous embryo, although the specimen was not assigned to any genus or species and was apparently distinct from A. bindrabunensis. A preserved araucarian embryo has also been described from Cretaceous rocks in New Zealand. Wairarapaia mildenhallii Cantrill et Raine, originally described as Araucarites sp. (41), has been demonstrated to have at least two cotyledons (9). The embryo-to-seed ratio for this specimen is about 0.50.
In summary, P. aequata and some of the fossil embryos assigned to the Araucariaceae share a common number of cotyledons, a column and pericolumn in the embryonic root cap, and the presence of secretory cells in the cortex of some embryos. They differ in the lack of a junction zone and in the relative proportions of the embryos. The embryo-to-seed ratio for some species is similar to P. aequata although the ratio appears to be quite variable in the fossil embryos.
Parasciadopitys aequata shares a mix of features with Pararaucaria patagonica Wieland (8; 70). Similar to A. mirabilis, P. patagonica has an embryonic root cap with a column and pericolumn, a similar embryo-to-seed ratio (0.67), and no junction layer (8; 70). Pararaucaria patagonica differs from Parasciadopitys aequata by lacking secretory cells and possessing 6–8 cotyledons (70). The ratios of the cotyledon-to-embryo length (0.42) and embryonic-root-cap-to-embryo length (0.10) also differ greatly from those of P. aequata, while the ratio of hypocotyl-to-embryo length of 0.50 is somewhat similar.
Fossil voltzialean embryos are known from the Pennsylvanian of Kansas (39; 29) and Permian of Texas (43, 44). Recent investigations suggest that P. aequata is a member of that group as well (18), extending the time range for voltzialean embryos from the Late Pennsylvanian to the early Middle Triassic. Seeds of Emporia cryptica Hernández-Castillo, Stockey, Rothwell et Mapes have embryos with six cotyledons, a well-defined corrosion cavity, and an embryo-to-seed ratio ranging from ca. 0.36–0.45 (39; 29). The Permian embryo of Moyliostrobus texanum Miller et Brown does not provide enough useful characters for any comparison. No cotyledons are distinguishable, and the presence of the embryo was demonstrated primarily by the occurrence of tracheids within tissues inside the nucellus (43, 44). The limited data on voltzialean embryos are indicative of very different embryo-to-seed ratios and number of cotyledons.
As mutualists, parasites, saprotrophs, or as organisms capable of spanning these categories, endophytic fungi are known to have significant effects on the diversity and structure of plant communities (15). Throughout the fossil record are several examples of fungi filling each of these roles. Interactions between fossil fungi and plants have also been described as occurring within seeds (55; 49; 68). Details of plant–fungal interactions in fossils, with a few exceptions (e.g., 33), are unfortunately often obscured by poor preservation and little evidence of host response.
Mycocarpon asterineum is a fungal fossil from the early Middle Triassic known to date only from dispersed specimens (81). The fossils are presumed to be zygomycetous and most probably represent mantled zygospores similar to those seen in some extant members of the Endogonales (e.g., 32), but the role of these fungi in the ecosystem cannot be evaluated due to the lack of demonstrable interactions with other organisms. The specimens of M. asterineum occur within the seed, outside of the megagametophyte and embryo tissues (Figs. 1A, 4D), and are interpreted as being immature due to a lack of the inner noncellular wall layer characteristic of fully developed individuals (81). No evidence of plant response to the fungus or indication of hyphal attachment has been found.
A single oogonium of the peronosporomycete Combresomyces cornifer is also found in the seed of P. aequata (Fig. 4A, B). Combresomyces cornifer was originally described from Visean (Middle Mississippian) cherts from central France (17) where the organism occurs exclusively in periderm cells of Lepidodendron rhodumnense Renault. The lycopsid shows no host reaction, and it remains uncertain whether C. cornifer was a symptomless endophyte, a parasite, or a saprotroph (17). Oogonia of C. cornifer were later discovered by 59 in silicified peat from the Middle Triassic of Antarctica. In contrast to the Visean fossils, the specimens from Antarctica occur dispersed and are generally much larger. Much of the nearby root, stem, ovule, and leaf tissue in the presence of the Triassic C. cornifer was moderately to heavily degraded. These authors suggested that C. cornifer functioned as a saprotroph in the Fremouw Peak ecosystem. The megagametophyte tissue surrounding the C. cornifer oogonia shows signs of degradation that are not seen elsewhere in the specimen, suggesting that the endophyte may have caused the damage. It is important to point out, however, that no hyphae have been found attached to the oogonium. It is most probable that the tissue breakdown occurred during the hyphal phase and was not done by the reproductive units. Given the size of C. cornifer oogonia and the absence of holes in the seed, it seems likely that the sporocarp was produced inside the host and was not incidentally washed into the seed.
Ecological role of the microbial endophytes
A major goal of studying fossil fungi is to discover their role in their ancient ecosystem. Below, we discuss the possibility of C. cornifer and M. asterineum being mutualists, saprotrophs, or parasites.
In a mutualistic relationship between a plant and a microbe, there is a two-way exchange of nutrients that benefits both organisms to some degree, although it can be variable over time (28). Such relationships can last for extended periods of time, i.e., weeks, months, or longer, depending on the organisms involved and the environment in which the association occurs (40). The exchange of nutrients in mutualistic relationships mostly occurs through haustoria produced by the fungus (26; 16). No evidence of haustoria or any other features of mutualistic fungi have been discovered in the fossil embryo or megagametophyte described in this paper.
Fungi in a saprotrophic phase, by definition, only destroy the host tissue of nonliving organisms. The preservation of P. aequata strongly suggests that the organism had not completely ceased metabolic activity before fossilization occurred. The nuclei present in megagametophyte and embryo cells indicate that at least some portions of the embryo tissue were physiologically active. Statistical studies of the cellular contents of Miocene leaves have shown that nuclei are among the rarest of organelles to appear in the fossil record, in contrast to the sequence of cell degradation observed in extant plants where the nuclei outlast other organelles (45; 46 and references therein). If our interpretation of the spherical bodies as nuclei is correct, a saprotrophic association of the embryo with fossil fungi is impossible.
In the case of C. cornifer, the fungus is associated with decay in the living plant tissue, suggesting an infection strategy consistent with a parasite. There is no evidence, however, of a host response to this infection other than a lack of tissue, which could have been caused by a host hypersensitive response (34; 12; 23). Although M. asterineum does not provide direct evidence of a parasitic interaction with P. aequata, its presence is consistent with some of the different infection strategies found in parasites, particularly a biotrophic infection strategy.
If they are parasites, there are several infection strategies that the microbes could employ. Microbial parasites employ three basic infection strategies: biotrophic, hemibiotrophic, and necrotrophic (40; 16). During necrotrophic infection, the fungus destroys the cells of the host as it proceeds through the tissues (51; 40). The interaction between C. cornifer and P. aequata exhibits this destruction, and thus, C. cornifer may have functioned as a necrotrophic parasite.
A biotrophic infection strategy depends upon the host to complete the microbe's life cycle (16), and the fungi derive their nutrients from living host cells using specialized haustoria (26; 50). Some of the features of this infection strategy can be seen in the interaction between M. asterineum and Parasciadopitys aequata, but much of this attribution to the type of infection is based on the absence of data, and so an infection strategy cannot reliably be attributed to the fungus.
The evidence from these fungi within Parasciadopitys seeds suggests ecosystem roles for C. cornifer not previously described. Combresomyces cornifer could be a necrotrophic parasite of P. aequata, while M. asterineum shows only the general features of a seed endophyte. The uncertainty in their exact ecological roles underscores the inherent difficulties in determining symbiotic relationships in the fossil record. In the case of P. aequata and its fungal endophytes, the relationship is not clear even though the embryo of P. aequata is exceptionally well preserved.
The plant remains that are known from the Fremouw Peak permineralized peat generally exhibit good tissue preservation. The embryo of P. aequata, however, displays a level of cellular preservation that is unique at this locality. For example, no nuclei or other subcellular contents have previously been described from the permineralized fossils, although this may be due to a bias in the kinds of plant tissues that are preserved. There is abundant permineralized leaf material at Fremouw Peak, but no known leaves are as well preserved as this P. aequata seed (see 52, 53; 1). This taphonomic bias may occur because the dominant trees were seasonally deciduous and thus the leaves were not physiologically active for a certain amount of time before fossilization. If this seed of P. aequata was in a dormant state at the time of fossilization, this fact may have contributed to its preservational detail.
A common criterion for excellent preservation of tissues is that microbial decomposition is (largely) excluded (66). Decay caused by microbes typically occurs at a faster rate in the presence of moisture, which is a condition that is essential for the formation of permineralized peats. Acidic water conditions, such as those in peat swamps, have also been suggested to hinder the degradation of plant material by microbes (54). It is clear that exceptional preservation of this P. aequata seed occurred even in the presence of microbes. There are several possible reasons why such detail was preserved, even while the seed was infected by organisms that could cause decay. These include rapid fossilization, possible protection of the tissues from more destructive microbes by fungi already in the seed, biocidal properties of secondary metabolites produced by the host plant, or a combination of two or more of these factors. There is some evidence that permineralization occurred near the time that the peat was first inundated with water. Studies have shown that submerged plant organs can lose soluble materials in a matter of hours and completely reach equilibrium with the water chemistry within days of being submerged (48; 66). The presence of subcellular structures in the embryo and megagametophyte tissues indicates that the seed did not reach this equilibrium point. While rapid permineralization likely contributed to the preservation of the embryo, it could not have been the only factor in its preservation. Since the microbes present in the seed of P. aequata had not yet destroyed the embryo, they might have in some way protected the seed from other microbes, which would have caused more immediate and rapid decay. Infection of plant tissues by fungi in a biotrophic phase of their lifecycle is known to protect the plant against more fungal infections (e.g., 25). A combination of rapid permineralization and protection from microbe degradation by a different fungus may have contributed to the exceptional preservation of this embryo at Fremouw Peak.
Preservation of fossil embryos, although rare, can expand the array of characters available in tracing the evolutionary history of plants. The embryo of P. aequata shares similarities with embryos of other extinct and extant conifer families. Such a position is not surprising given that P. aequata belongs to the Voltziales, a group considered to be transitional in conifer evolution. Unfortunately, only very few fossil conifers have been described to date with well-preserved embryos. For a meaningfully contribution to a phylogenetic analysis, more fossil embryos must be discovered and incorporated into a whole-plant reconstruction. Recent work by 18 indicates that the seed cone P. aequata was part of a plant for which the leaves and stem are known (Heidiphyllum elongatum leaves, Notophytum krauselii stems). The addition of an embryo to that reconstruction makes the resulting whole plant one of the best known structurally preserved early Mesozoic conifers.
This paper has also demonstrated how unusual preservation of plants can yield information on the ecology of other organisms in the fossil record. Specimens of M. asterineum and C. cornifer, fossil microbes from the Fremouw Peak peat in Antarctica, were previously only known from their dispersed reproductive structures. Further investigation into plant–fungal interactions at Fremouw Peak and in the fossil record in general will allow researchers to track changes in these interactions throughout geologic time.
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