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High-temperature, acid-hydrolyzed remains of Polytrichum (Musci, Polytrichaceae) resemble enigmatic Silurian-Devonian tubular microfossils¹

Department of Botany, University of Wisconsin, Madison, Wisconsin 53706 USA

Received for publication January 3, 2000. Accepted for publication June 8, 2000.

	ABSTRACT

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Gametophytes and sporophyte components of two species of the evolutionarily early-divergent moss Polytrichum were separately subjected to high-temperature acid hydrolysis, and remains were examined by fluorescence microscopy and scanning electron microscopy. Remains included fragments of capsule, seta, leaves, stems, and calyptra. Cell walls of all remains were autofluorescent in violet and UV excitation, suggesting occurrence of resistant polyphenolic compounds. Calyptras of both species dissociated into smooth- walled, acutely branched filamentous associations of tubular cells with distinctively thickened cell junctions. In these aspects and measurements of wall dimensions made from SEMs, the hydrolysis-resistant Polytrichum calyptra remains were similar to several tubular microfossils described from Silurian and Lower Devonian deposits, whose provenance is unknown or ascribed to fungi. Our data suggest the possibility that at least some ancient tubular microfossils might have originated from Polytrichum-like early mosses. They add to increasing evidence that bryophytes left microfossil evidence for their presence millions of years earlier than is indicated by their macrofossil record.

Key Words: bryophytes • calyptra • microfossils • mosses • Polytrichum.

	INTRODUCTION

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The timing and the process of the origin of the first land plants have not been easy to determine because molecular data used to infer the phylogeny of embryophytes have been incongruent with the macrofossil record for the earliest land plants. Molecular sequence evidence and gene architecture (Lewis, Mishler, and Vilgalys, 1997 ; Qiu et al., 1998 ) as well as biochemical and ultrastructural data (Sztein et al., 1995, 1999 ; Brown and Lemmon, 1990, 1992, 1997 ) indicate that bryophytes diverged early from a monophyletic land plant (embryophyte) lineage. In contrast, the first recorded land plant macrofossils represent polysporangiate pretracheophytes and vascular plants (Kenrick and Crane, 1997 ), the earliest complete or partially complete specimens being Cooksonia from the mid-Silurian (Edwards, 1979 ; Edwards and Feehan, 1980 ). Unambiguous macrofossil remains of bryophytes are not found until much later, liverworts first appearing in the Devonian and mosses in the Carboniferous (Oostendorp, 1987 ). The macrofossil record has thus led some authorities to regard bryophytes as later appearing, morphologically reduced offshoots of vascular plants, a concept that has strongly influenced thinking about the direction of reproductive evolution in land plants.

An alternative explanation for the relatively late appearance of bryophyte macrofossils invokes the relatively low-preservation potential of intact bryophyte thalli. Bryophytes lack lignified tissues, and liverwort and hornwort gametophytes, in particular, are often of delicate construction. Consequently, early Paleozoic remains of undisputed bryophyte-like plants are rarely found as whole specimens, except under exceptional preservational conditions (Edwards, Duckett, and Richardson, 1995 ). However, the earliest bryophytes might have left more extensive microfossil remains, such as small fragments of decay-resistant tissues and spores. This idea was first suggested by Gray and Boucot (1977, 1978, 1980 ; Gray, Massa, and Boucot, 1982 ) who described Ordovician spores and cellular scraps that were distinctly different from vascular plants but were reminiscent of bryophytes. In particular, Ordovician spore tetrads resembled those of the modern liverwort Sphaerocarpos (Gray, 1985). Recently, comparative studies of extant bryophytes and ancient fossils have been used to test the hypothesis that bryophytes may also have left an early record of vegetative remains.

Kroken, Graham, and Cook (1996) subjected a variety of extant bryophytes to a high-temperature, acid hydrolysis procedure that more or less mimics the taphonomic changes experienced by ancient plants. In addition to spores, bryophyte sporangial epidermis and placenta, as well as leaves, stems, and rhizoids of mosses, were found to survive as microscopic remains, with their resistance attributed to the presence of autofluorescent polyphenolic compounds in the cell walls. Acid- hydrolyzed remains were interpreted as the parts of a bryophyte that have the potential to survive degradation long enough to become fossilized.

Further, morphometric comparisons of acid-hydrolyzed remains of extant early-divergent bryophytes and microfossils hypothesized to be the remains of early land plants suggested possible bryophyte-like origins of some enigmatic Ordovician, Silurian, and Devonian microfossils. For example, acid-hydrolyzed sporangial epidermis remains of the early-divergent moss Sphagnum (Kroken, Graham, and Cook, 1996 ) and the early-divergent liverwort Sphaerocarpos (Graham and Gray, in press ) closely resembled mid-late Ordovician scraps described by Gray, Massa, and Boucot (1982) . Sporangial epidermis of other liverworts yielded acid-hydrolysis-resistant remains that resembled Silurian-Devonian tubes and cell sheets (Kroken, Graham, and Cook, 1996 ).

These results, together with molecular data, strongly support the concept that: (1) earliest land plants resembled modern bryophytes, (2) bryophyte-like land plants predated the earliest polysporangiophytes and vascular plants, and (3) terrestrial embryophytes were present by the early-middle Ordovician (Strother, Al-Hajri, and Traverse, 1996 ). In this study, we extended the neontological-paleontological approach to another early-divergent moss, Polytrichum, comparing certain of its acid-hydrolysis resistant tissues to early Paleozoic microfossils whose source has previously been uncertain. Our proximate goal was to illuminate the origin of such remains and our ultimate goal to assess the relative importance of bryophytes in the early history of land plants.

	MATERIALS AND METHODS

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Polytrichum ohioense was collected from Oneida County, Wisconsin and identified by C. Trivett. Polytrichum commune was collected by L. Wilcox in Portage, Wisconsin and identified by L. E. Graham. Gametophores, setae, capsules, and calyptras from P. ohioense and calyptras from P. commune were isolated with a forceps and hydrolyzed separately. Several Polytrichum commune calyptras were immersed in a soil-water mixture in a Parafilm-sealed petri plate for 21 d in order to test their resistance to microbial decay.

Moss tissues were acid hydrolyzed in 15-mL Eppendorf tubes having three holes in the caps to allow for gas escape during boiling. Holes were made by heating a dissecting needle in a flame, then touching it to the cap of a closed tube. (In performing this procedure, care should be taken not to breathe volatilized plastic vapors.) Perforation of caps prevented opening of tubes (due to expanding gases) and entry of boiling water that would dilute the acid mixture.

Samples were dehydrated with glacial acetic acid for 5–10 min, then centrifuged. The glacial acetic acid was pipetted off, then a 9:1 v/v solution of acetic anhydride and concentrated sulfuric acid was added to samples. The volumetric proportion of plant material to acid mixture was at least 50:1 as recommended by Good and Chapman (1978) . Tubes were floated in a beaker of boiling water for at least 20 min, until the acid-plant mixture turned opaque and dark brown. Tubes were then centrifuged for 3–4 min at a moderate speed sufficient to sediment remains without packing them tightly at the bottom. The supernatant was removed, and the surviving material was washed with glacial acetic acid and centrifuged. The glacial acetic acid was then removed and the remains stored in distilled water at room temperature.

Microscopic observations were performed with a Zeiss Axioplan fluorescence microscope fitted with NEOFLUAR objectives, an HBO (50-W mercury) light source, and filter packs G365 FT395 LP 420 (ultraviolet: number 48 777 02) and 395–440 Ft 460 LP 470 (violet: number 444448 77 05) for excitation wavelengths of 365 nm and 395–440 nm. For scanning electron microscopy (SEM), surviving tissue scraps of Polytrichum ohioense were attached to an aluminum stub by double-sticky tape and gold coated. Remains of Polytrichum commune were transfered by pipette to a glass coverslip and dried on a hot plate for 5–10 min on low heat. The glass coverslips were attached to an aluminum stub by double sticky tape and then gold coated. Accelerating voltage was 15 kV. SEM micrographs of acid-hydrolyzed Polytrichum calyptras and original images of Late Silurian or Lower Devonian microfossils that had been published previously were digitally scanned and printed side by side for comparative analysis. Though replicate measurements of the dimensions of extant materials were made, and analogous measurements were performed on images of individual fossils, statistical comparison could not be performed because replicate fossils were not available.

	RESULTS

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Remains of acid-hydrolyzed Polytrichum ohioense included fragments of capsule, seta, leaves, stems, and calyptra. Only calyptras for P. commune were acid hydrolyzed, and remains were obtained. All remains were autofluorescent in violet and ultraviolet excitation. Capsule remains were unstructured clumps of organic material lacking defined cell outlines. Seta remains consisted of a single type of characteristic tissue—long, thin, tube-like structures that were arranged in parallel. Stem and leaf systems were recovered in nearly intact condition, and several kinds of tissues and cells were preserved. Capsule, seta, leaf, and stem remains are not further described here, as they could not be correlated with microfossils described in the literature, in contrast to calyptra remains.

Calyptra remains from P. ohioense and P. commune were very similar in that both primarily consisted of long, hollow, branched tubes. Cells at the exterior of an intact calyptra (Fig. 1) were segmented branched tubes. Tubular structure was maintained after hydrolysis, though overall integrity of the calyptra was lost (Fig. 2). Acid-hydrolyzed tubes tended to aggregate into tangled masses (Fig. 3). Individual tubes had smooth outer and inner walls (Figs. 5 and 7) that were thickened at joints between cells and at branch points (Fig. 5). Branching invariably occurred at an acute angle.

View larger version (107K): [in this window] [in a new window]   Figs. 1–2. Morphology of Polytrichum commune calyptra as viewed by SEM. 1. An entire, unhydrolyzed calyptra showing construction of loosely aggregated tubular cells. Scale bar = 0.5 mm. 2. Remains of an acid-hydrolyzed calyptra showing aggregations of branched, tubular cells. Scale bar = 42.9 mm.

View larger version (109K): [in this window] [in a new window]   Figs. 3–8. Comparison of acid-hydrolyzed remains of Polytrichum calyptras with previously published images of enigmatic Paleozoic microfossils. 3. Matted aggregate of P. ohioense calyptra cells viewed by SEM. Scale bar = 72.0 µm. This image is similar to an SEM of a matted aggregate of tubular microfossils reproduced as Fig. 4 (Edwards [1982 ]: fig. 84 reproduced by permission of Academic Press, Ltd., London, UK). Scale bar = 40 µm. 5. Portion of a tubular array of cells of P. commune in SEM view. Scale bar = 35.7 µm. Note the acute branching angle and thickened walls at intercellular junctions. 6. Similar branching and wall thickening pattern in enigmatic tubular microfossils viewed in light microscopy that were previously published (Wellman [1995 ]: fig. 2 in plate V reproduced by permission of Elsevier Science, Oxford, UK). Scale bar = 16.6 µm. 7. SEM view of a razor-cut end of a tubular cell of P. commune calyptra. Scale bar = 3.6 µm. Figure 7 is similar to Fig. 8 , an SEM of the cut end of a fossil previously published (Edwards [198 2]: fig. 76 reproduced by permission of Academic Press, Ltd., London, UK). Scale bar = 8.47 µm

Polytrichum calyptras exposed to soil microorganisms for 21 d remained intact, though they underwent a color change from very light tan to a golden brown or black color. A similar color change was observed in calyptra remains resulting from high-temperature acid hydrolysis. Microbe-exposed calyptras autofluoresced yellow-green in violet and blue-white in UV excitation, as did those exposed to high-temperature acid hydrolysis.

	DISCUSSION

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This study adds to the growing body of evidence supporting the concept that bryophytes appeared on land much earlier than their macrofossil record suggests, and most likely left early Paleozoic microfossils in the form of vegetative remains as well as spores. Our results also extend the variety of bryophyte tissues known to withstand high-temperature acid hydrolysis, our index of the taphonomic conditions experienced by ancient plants. Placental remains were recovered after acid hydrolysis of the hornwort Anthoceros (as were placentae and associated zygotes of the charophycean green alga Coleochaete) (Delwiche, Graham, and Thomson, 1989 ). Kroken, Graham, and Cook (1996) reported placental remains from several species of the moss Sphagnum after acid hydrolysis. They also reported sporangial (capsule epidermal) remains from Conocephalum conicum, Lophocolea heterophylla, and several species of Sphagnum. Sporangial epidermis was also recovered from the liverwort Sphaerocarpos (Graham and Gray, in press ). Sphagnum leaves were resistant to acid hydrolysis, and rhizoid remains could be recovered from the mosses Dicranum polysetum, Leucobryum glaucum, Mnium cuspidatum, and M. punctatum (Kroken, Graham, and Cook, 1996 ), as could nearly intact leaf and stem systems of Polytrichum ohioense and P. commune (this study).

High-temperature acid-hydrolysis-resistance of seta and calyptra, observed in this study of Polytrichum, has not been previously reported. The pseudosetae of Sphagnum and evanescent setae of Lophocolea were not resistant to acid hydrolysis (Kroken, Graham, and Cook, 1996 ). Of the Polytrichum remains observed in this study, only those of the calyptra bore significant resemblance to photographs of enigmatic microfossils previously published by Edwards (1979, 1982) , Edwards, Duckett, and Richardson (1995) , Gensel, Johnson, and Strother (1990) , and Wellman (1995) . Morphological and size similarities between the acid-hydrolyzed remains of extant Polytrichum and certain smooth-walled, branched tubular microfossils suggest that a calyptra-producing ancestor of modern Polytrichum could have been the source of the microfossils.

Molecular phylogenetic studies indicate that polytrichaceous mosses diverged relatively early within the monophyletic lineage represented by extant Musci, but later than Sphagnum and liverworts (Lewis, Mishler, and Vilgalys, 1997 ; Hyvönen et al., 1998 ). This may explain the appearance of smooth- walled, branched, tubular microfossils later in the fossil record (Silurian-Early Devonian) than the Ordovician microfossils that have been linked to liverwort and Sphagnum hydrolysis remains (Kroken, Graham, and Cook, 1996 ; Graham and Gray, in press).

The facts that (1) much of the Polytrichum thallus was recovered after high-temperature acetolysis, (2) the autofluorescence properties of moss remains resemble those of other resistant tissues, and (3) calyptras are resistant to microbial decay imply that cell walls of this moss are highly impregnated with polyphenolic compounds that decrease decay potential. Similarly autofluorescent walls of the desmid Staurastum (a member of the Charophyceae, the green algal lineage most closely related to land plants) contain polyphenolics that are both microbe-resistant (Gunnison and Alexander, 1975a, b ) and acid-hydrolysis resistant (Kroken, Graham, and Cook, 1996 ). The latter authors argued that bryophytes inherited the capacity for production of autofluorescent, wall-bound polyphenolic compounds from charophycean ancestors, expressing them in tissues where protection from microbial decay was likely to have adaptive value. Because the calyptra is believed to function as protection for the developing sporophyte, production of polyphenolic wall compounds in this structure may have had adaptive value for early mosses.

This study also suggests a new interpretation for the source of at least some of the Paleozoic smooth-walled, branched, tubular microfossils whose provenance has not been identified or have been previously regarded as possible fungal remains [see review of Paleozoic microfossils, including tubes, by Gensel, Johnson, and Strother (1990) ]. Edwards (1982) suggested that some of the Paleozoic tubes are remains of Nematothallus (Lang, 1937 ), a genus of Paleozoic fossil plant- like organisms (nematophytes) that lack modern representatives. Wellman (1995) noted that fossil tubes found in nonmarine deposits were likely terrestrial or freshwater in origin, and that they could have been derived from many sources including land plants, fungi, algae, or previously undocumented organisms.

Based on the evidence presented here, we suggest that at least some of the smooth-walled, branched, tubular microfossils known from the early Paleozoic represent calyptral remains of early mosses. This interpretation is supported by: (1) morphological similarity, (2) size similarity, and (3) resistant properties of both microfossils and extant Polytrichum calyptras. Our results suggest that correlation between other enigmatic microfossils and hydrolysis-resistant remains of extant bryophytes may be possible.

	FOOTNOTES

 
¹ The authors thank Heidi Barnhill of the UW Entomology SEM facility for technical assistance, Chris Trivett (Weber State University, Utah) for collections of Polytrichum ohioense, and Lee Wilcox, Department of Botany, for digital preparation of figures. Dianne Edwards (University College, Cardiff, Wales) and Charles Wellman (Sheffield University) critically read the manuscript prior to submission and provided helpful comments, as well as original images from their published work for use in this paper.

² Author for reprint requests (Tel: 608/262-2640; FAX: 608/262-7509; e-mail: lkgraham{at}facstaff.wisc.edu )

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