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American Journal of Botany 100(12): 2450–2457, 2013 ; http://www.amjbot.org/ © 2013 Botanical Society of America

American Journal of Botany 100(12): 2450–2457. 2013.

One of the most striking features of humid tropical forest ecosystems is the abundance of bryophytes growing on the leaves of other plants ( Pócs, 1982 ; Sonnleitner et al., 2009 ). These epiphylls grow on a variety of plant families ( Pócs, 1982 ), but all host species have evergreen, leathery, or papyra-ceous leaves and live in warm climates with high rainfall ( Sonnleitner et al., 2009 ). When found outside the tropics, the host plants are restricted to humid, typically maritime climates ( Vitt et al., 1973 ; Pypker et al., 2006 ; Risk et al., 2011 ). Bryo-

phyte gametophytes generally lack a true cuticle ( Proctor, 1979 ), one possible reason for their scarcity as fossils, but they should be more common given that bryophyte epiphylls adhere tightly to plant leaves and their host plants live near depositional settings with high preservation potential. Indeed, plant leaves are by far the most common type of macrofossil in terrestrial sediments, and bryophytes in the humid tropics may cover over 50% of the host’s leaf surface ( Sonnleitner et al., 2009 ). Given the abundance of bryophyte epiphylls in modern tropical and maritime ecosystems the almost total ab-sence of epiphyllous bryophytes in the geological record is diffi cult to explain.

The fossil record for epiphyllous mosses is particularly scanty. This is greatly at odds with the relatively more exten-sive fossil record of nonepiphyllous mosses, which originate in the Carboniferous Period almost 330 million years ago ( Hübers and Kerp, 2012 ). Macrofossils of Muscites poly-trichaceus Renault and Zeiller were reported as a true moss from the Upper Carboniferous sediments of the Commentry Basin of France ( Renault and Zeiller, 1888 ; Lignier, 1914 ; Stewart and Rothwell, 2010 ). Subsequently, an older moss specimen was described, Muscites plumatus B.A. Thomas (Bryales; Thomas, 1972 ), collected from the Lower Carbonif-erous Drybrook Sandstone of the Forest of Dean in Glouces-tershire (UK). However, these two previously described “oldest” nonepiphyllous moss macrofossils have been ques-tioned by Hübers and Kerp (2012) based upon a lack of diag-nostic features attributable to mosses. Hübers and Kerp (2012) question the affi nity of Muscites polytrichaceus Renault and Zeiller (1888) and M. bertrandi Lignier (1914) as mosses

1 Manuscript received 14 June 2013; revision accepted 10 October 2013. This study was made possible through major fi nancial support from

National Science Foundation Grant (EAR-0643290) to BBS and JCM and a Marie Curie Excellence Grant (MEXT-CT-2006-042531) to JCM. Student grants to RSB for fi eld work came from the Colorado Scientifi c Society, the Evolving Earth Foundation, The Field Dreams Program at The Field Museum, the Geological Society of America, the Paleontological Society, and the Western Interior Paleontology Society. The AFM equipment used for this work was funded by Science Foundation Ireland (SFI: 07/IN1/B031). Colin Carney helped with fi eld work that produced the fossil material. Ken MY P’ng in the Nanovision Center at Queen Mary University of London was integral during the SEM process. Ian Glasspool is thanked for accessioning and maintaining the Dakota Formation collection at The Field Museum. JCM acknowledges current funding from SFI (11/PI/1103) and the European Research Council (2011-StG279962). RSB acknowledges current funding from the Peter Buck Fellowship program at the National Museum of Natural History, Smithsonian Institution, Washington, D.C., USA.

6 Author for correspondence (e-mail: barclay.rich@gmail.com)

doi:10.3732/ajb.1300209

NEW METHODS REVEAL OLDEST KNOWN FOSSIL EPIPHYLLOUS MOSS: BRYIIDITES UTAHENSIS GEN. ET SP. NOV. (BRYIDAE) 1

RICHARD S. BARCLAY 2,6 , JENNIFER C. MCELWAIN 3 , JEFFREY G. DUCKETT 4 , MAARTEN H. VAN ES 5 , ANIKA S. MOSTAERT 3,5 , SILVIA PRESSEL 4 , AND BRADLEY B. SAGEMAN 2

2 Northwestern University, Department of Earth & Planetary Sciences, Technological Institute, 2145 Sheridan Road, Evanston, Illinois 60208-3130 USA; 3 University College Dublin, School of Biology and Environmental Science, Belfi eld, Dublin 4,

Ireland; 4 Life Sciences, Plants Division, Natural History Museum, Cromwell Road, London, SW7 5BD, UK; and 5 University College Dublin, Conway Institute of Biomolecular and Biomedical Research, Belfi eld, Dublin 4, Ireland

• Premise of the study: Epiphyllous bryophytes are a highly characteristic feature of many humid tropical forest ecosystems. In contrast to the extensive fossil record for the leaves of their host plants, the record is virtually nonexistent for the epiphylls themselves, despite a fossil record for mosses that begins in the Middle Carboniferous Period, 330 million years ago.

• Methods: Epifl uorescence optical microscopy, scanning electron microscopy, and atomic force microscopy were employed to investigate an intimate association between a newly discovered epiphyllous moss and a Lauraceae plant host from the middle Cretaceous.

• Key results: We describe the oldest fossil specimen of an epiphyllous moss, Bryiidites utahensis gen. et sp. nov., identifi ed from an individual specimen only 450 µm long, situated on an approximately one millimeter square fossil leaf fragment. The moss epiphyll is exquisitely preserved as germinating spores and short-celled protonemata with transverse and oblique cross-walls closely matching those of extant epiphyllous mosses on the surface of the plant-leaf hosts.

• Conclusions: The extension of the epiphyll record back to the middle Cretaceous provides fossil evidence for the appearance of epiphyllous mosses during the diversifi cation of fl owering plants, at least 95 million years ago. It also provides substantive evidence for a tropical maritime climate in central North America during the middle Cretaceous.

Key words: atomic force microscopy; Bryophyta; Cretaceous; Dakota Formation; epiphyll; Lauraceae; paleobotany; protonema.

2451December 2013] BARCLAY ET AL.—OLDEST KNOWN FOSSIL EPIPHYLLOUS MOSS

the two fossils both comprise anchoring haptera branch systems unique to the extant genus Ephemeropsis ( Pressel and Duckett, 2009 ).

Here we describe from a single specimen, a new species of epiphyllous moss, Bryiidites utahensis gen. et sp. nov. ( Fig. 1 ). This specimen represents the only fossil occurrence to date of an epiphyllous moss germinating from spores on a leaf surface. In addition, the specimen was preserved in sediments that are temporally well-constrained as Late Cenomanian (early Late Cretaceous, 95 Ma; Elder et al., 1994 ; Sageman et al., 2006 ; Laurin and Sageman, 2007 ; Barclay et al., 2010 ; Meyers et al., 2012 ), which extends the fossil record of epiphyllous mosses by at least 40 million years.

MATERIALS AND METHODS

Dispersed fossil plant cuticle was extracted from a rock sample using a three-step maceration process. The rock was treated with concentrated hydro-chloric acid (HCl, 37%) to release carbonates, a saturated solution of pyrophos-phate (Na 4 P 2 O 7, 0.1M) to disperse clays, and hydrofl uoric acid (HF, 48%) to dissolve the remaining silicate minerals. Each chemical treatment was followed by sieving (125 µm) and dilution steps. The sample containing the epiphyllous moss was extracted from rock sample RSB0730, collected from a measured stratigraphic section in Wahweap Creek (RSB0726) on Bureau of Land Management

because the compression and impression fossils do not show enough typical features. They also dismiss M. plumatus Thomas (1972) as the oldest described moss because it lacks a clear cell pattern. Replacing these two previously “oldest” macrofossils of moss are several morphotypes attributable to the Sphagnales, dated consistently using pollen and a sin-gle-zircon Pb/Pb date from a volcanic tuff at the base of the Ortelsdorf Formation in Germany. Regardless of which for-mation holds the oldest verifi able nonepiphyllous moss, they both date to the Middle Carboniferous Stage (late Visean), almost 330 million years ago ( Thomas, 1972 ; Hübers and Kerp, 2012 ).

For epiphyllous mosses, two fossil specimens need to be con-sidered. Both have previously been ascribed to the extant genus Ephemeropsis , one from the Eocene of Germany ( Mägdefrau, 1968 ) and another from the Miocene of New South Wales in Australia ( Selkirk, 1974 ). The taxonomic assignment of these fossil specimens might appear to be problematic at fi rst sight, i.e., they lack the common key characters that are diagnostic of moss protonemata and rhizoids, viz. tip-growing multicellular uniaxial fi laments comprising short or elongate cells with trans-verse or oblique cross-walls and with lateral side branches pro-ducing either similar fi laments or gametophore buds or asexual propagules (gemmae or tubers; Duckett et al., 1998 ). However,

Fig. 1. Epifl uorescence image of Bryiidites utahensis type specimen (The Field Museum, Chicago, Illinois, USA: PP54677). Inset shows oblique cell division to form new side branch, with adjacent spore.

2452 AMERICAN JOURNAL OF BOTANY [Vol. 100

property in southwestern Utah, USA. The type specimen of the fossil epiphyll (PP54677) is permanently stored on an SEM stub in the Paleobotany Collec-tions at The Field Museum in Chicago, Illinois, USA.

The stratigraphic position of the sample containing the fossil epiphyl-lous moss is plotted against a stratigraphic section of the Middle Member of the late Cenomanian Dakota Formation, situated 30.6 m below the contact with the Tropic Shale (Appendix S1; see Supplemental Data with the online version of this article). The contact between the overlying Tropic Shale and the Middle Dakota Member represents a major marine fl ooding surface identifi able in outcrop and in the subsurface throughout the region ( Elder et al., 1994 ; Laurin and Sageman, 2001 , 2007 ). This transgressive surface is related to the peak global sea-level high stand that occurred soon after, in the early Turonian Stage ( Haq et al., 1987 ). The individual stratigraphic section containing the fossil epiphyll was correlated to a previously devel-oped regional stratigraphic framework, developed using detailed biostratig-raphy and sequence stratigraphy and extending from Cedar City to the western edge of Lake Powell in southwest Utah ( Uličný, 1999 ; Laurin and Sageman, 2001 , 2007 ).

Stable carbon isotope values have helped to further constrain the age of the fossil epiphyll to about one million years prior to the Cenomanian-Turonian boundary ( Barclay et al., 2010 ). Sample RSB0730 was collected below a major carbon cycle perturbation at the end of the Cenomanian Stage, known as Ocean Anoxic Event 2 ( Schlanger and Jenkyns, 1976 ). This event was characterized by a positive 4‰ shift in δ 13 C from organic carbon ( Sageman et al., 2006 ), a large carbon isotope shift identifi ed in the fi eld area from terrestrial plant material (including data from section RSB0726; Barclay et al., 2010 ). The geologic units that marked the onset of Ocean Anoxic Event 2 were traced (at the scale of individual beds; Elder et al., 1994 ), to the type locality for the Cenomanian-Turonian boundary in Pueblo Colorado ( Sageman et al., 2006 ; Meyers et al., 2012 ) using litho-, bio-, and tephro-stratigraphy ( Elder, 1988 ; Kowallis et al., 1989 ; Elder et al., 1994 ). As a result of the robust temporal and sequence stratigraphic framework developed for the region, and for the section that contains the fossil epiphyll in particular, the fossil epiphyll can be reliably placed within the Late Cenomanian Stage at 95 million years ago ( Obradovich, 1993 ; Sageman et al., 2006 ; Meyers et al., 2012 ).

The use of three complementary microscope techniques was required to interpret fully the relationship between the fossil epiphyll and the host plant leaf. The epiphyll specimen was fi rst imaged under fl uorescent light with a Leica DFC300FX camera attached to a Leica DM 2500 microscope using a band pass excitation fi lter (I3) in the range of 450-490 nm ( Fig. 1 ). The z-stacking software package Automontage (Syncroscopy, Cambridge, UK) was used to produce a ‘stacked image’ from ~15 raw images of the fossil specimen to ensure all details of relief represented within the fi nal image were focused. Two additional microscopy techniques were employed to in-vestigate the specimen at higher resolution. Scanning electron microscopy (SEM) was implemented because it provided the micron-scale resolution required to investigate many of the small features of the fossil surface. The specimen was mounted onto a standard SEM stub, sputter-coated in gold, and investigated at high vacuum in a FEI Quanta 3D FEG dual beam scan-ning electron microscope (FEI, Hillsboro, Oregon, USA). The SEM only pro-vided horizontal measurements, but we were investigating a three-dimensional fossil. Therefore, we used atomic force microscopy (AFM) for the fi rst time on any macrofossil plant material to obtain quantitative measurements at the nanometer scale in three dimensions, for both the epiphyll and the host leaf surface.

The leaf and epiphyll surfaces were scanned directly using AFM (JPK NanoWizard II AFM from JPK Instruments, Germany). AFM constructed a 3D topographical image of the surface by scanning a nanometer-scale tip over the surface ( Morris et al., 2010 ). High-resolution images were made directly on the unmodifi ed sample using Olympus AC240TS silicon probes with typical tip radius of 9 nm, spring constants of approximately 2 N/m and resonance frequency of 70 kHz. Scans were made in intermittent-contact mode in air. Due to the relatively rough topography of both the fossil leaf host and fossil epiphyll, most images were restricted to a 10 µm scan size. Standard JPK control software was used to analyze and process the AFM data, and Hugin Panorama Photo Stitcher (HUGIN 2010, version 4.0, free panorama stitcher, http://hugin.sourceforge.net/download ) was used to cre-ate the mosaic in Figs. 2 and 3 .

An inverted light microscope integrated with the AFM was used to identify features on the fossil leaf as “landmarks” (resin glands and cracks were visible using both epifl uorescence and transmitted light) to identify the approximate location of the cantilever scan area. By conducting overlapping sequential

Figs. 2–4. Three-dimensional mosaic of Bryiidites utahensis using atomic force microscopy (AFM), relocated using epifl uorescence image as a blue-print. 2. Unaligned AFM height map of Bryiidites utahensis from multiple scans. (Colored height scale starts at black for lower-most part of scan, passing through shades of orange, ending at the top of scan with white). 3. Aligned mosaic of AFM scans. Colored height scale in fi nished mosaic ( Fig. 3 ) is 3 µm. 4. Epifl uorescence image of Bryiidites utahensis on host leaf surface. White arrows indicate same cluster of spores relocated during AFM imaging.

scans, a 3D-mosaic of the spores and leaf surface was produced ( Fig. 3 ). The process was repeated until the scanned area was reliably matched to portions of the epifl uorescence image ( Fig. 4 ). SEM images were related back to both the epifl uorescence image and the AFM scans using the same landmark features identifi ed while creating the AFM mosaic.

2453December 2013] BARCLAY ET AL.—OLDEST KNOWN FOSSIL EPIPHYLLOUS MOSS

the spheres could have been coalifi ed side-branches. A 3D-mosaic ( Figs. 2, 3 ), assembled from overlapping AFM images, revealed elongated shallow grooves ( Figs. 5, 6 ) in the position of the fi lament cells visible with epifl uorescence.

Filament trough width (4-7 µm), fi lament trough depth (1-3 µm), sphere diameter (10.5-12 µm), and sphere height (2-3 µm) were measured using AFM ( Figs. 2, 3, 5, 7, 8 ). Horizontal measure-ments of all structures made with AFM were confi rmed by SEM ( Figs. 6, 9 ). Filament width (8-15 µm) was measured us-ing epifl uorescence, defi ned by the outer edges of the nonfl uo-rescing cell walls. Both SEM and AFM images show that the spheres have been fl attened and some are split open ( Figs. 2, 3, 5–9 ).

The fossil leaf host (morphotype DSU01) was ascribed to the Lauraceae based upon seven epidermal features preserved on the cuticle of the host leaf ( Barclay et al., 2010 ). These include a paracytic stomatal complex, a randomly dispersed stomatal distribution, and a stomatal pore position that is level with the epidermal cells, as well as the presence of trichomes, papillae, striations, and especially glandular resin cells embedded in the upper and lower epidermis. These traits are commonly associ-ated with the Lauraceae ( Coiffard et al., 2009 ) and are typically used for the identifi cation and classifi cation of angiosperms based solely upon epidermal features of the leaves ( Bandulska, 1926 ; Metcalfe and Chalk, 1950 ; Baranova, 1987 ; Barclay et al., 2007 ). Stem fragments were present in these deposits, but the cell walls on stem-derived cuticles are elongated rectangles, whereas the cell shapes on the leaf host were polygonal/rounded, which is a typical shape for cells on the upper leaf surface ( Barclay et al., 2007 ). In addition to rounded/polygonal cell shape, the absence of stomata on the host leaf surface provides stronger support for the epiphyll growing on an upper surface of a leaf, as almost all angiosperms have stomata only on a single leaf surface, invariably the lower surface ( Barclay et al., 2012 ). On the leaf host (morphotype DSU01), stomata are al-ways abundant on the lower surface (295 stomata per mm 2 for this sample—RSB0730; Barclay et al., 2010 ), whereas they are absent on the upper surface, which is true for the specimen car-rying the fossil epiphyll. Both cell shape and lack of stomata on the host leaf surface suggest that the fossil epiphyll was grow-ing on an upper side of the leaf host.

DISCUSSION

A survey of fi lamentous structures across mosses, hornworts, liverworts, algae, cyanobacteria, and fungi, both extant and fos-sil, reveals matching counterparts only in the mosses ( Webster and Weber, 1980 ; Goffi net and Shaw, 2008 ; Graham et al., 2009 ; Taylor et al., 2009 ; Stewart and Rothwell, 2010 ; Moore et al., 2011 ). The absolute signature of moss protonemata is side branch initials marked by oblique septa. Typical protonemata, as pro-duced by most bryalean mosses and exemplifi ed by the ‘funari-alean’ type, consist of multicellular, uniaxial, regularly branching fi laments with new cells being added either by division of the apical cell or by the formation of side branch initials, usually im-mediately behind the cross walls along the primary axis. Main axes comprise elongate cylindrical cells with oblique cross walls (caulonemata); side branches have shorter cells separated by transverse walls (chloronemata—similar to those produced by germinating spores) that subsequently differentiate into caulone-mata. In a range of moss families, including those with numerous epiphytic and epiphyllous taxa, protonemata are distinct from

SYSTEMATIC PALEOBOTANY

Class — Bryophyta.

Subclass — Bryidae Engl.

Order — Incertae sedis.

Family — Incertae sedis.

Type Genus — Bryiidites Barclay, Duckett, McElwain, van Es, Mostaert, Pressel, et Sageman, gen. nov.

Generic and specifi c diagnosis — Germinating dark-walled spores 10.5-12 μ m in diameter producing uniaxial sparsely branched fi laments 8-15 μ m wide, comprising short cells with transverse or oblique cross walls.

Type species — Bryiidites utahensis Barclay, Duckett, McEl-wain, van Es, Mostaert, Pressel, et Sageman, sp. nov.

Etymology — Referring to the assignment to the subclass of moss, and “utahensis” for the discovery of the fossil in the state of Utah, USA.

Holotype hic designatus — PP54677 ( Figs. 1–9 ) (designated here; deposited in the Paleobotany Collections at The Field Mu-seum, Chicago, Illinois, USA).

Locality — Holotype extracted from rock sample RSB0730, collected within stratigraphic section RSB0726 of the Middle Dakota Formation. Sampled 10.56 meters above the contact with the Lower Dakota Formation, readily identifi able in out-crop as a pebble conglomerate. The stratigraphic section is lo-cated on public land, managed by the Utah Bureau of Land Management. Global positioning system (GPS) coordinates for the base of the continuous vertical section are: N 37.16806 ° , W 111.71047 ° , using the NAD27 datum on a handheld Garmin Navigator GPS.

Stratigraphic position and age — Middle Dakota Formation, Late Cenomanian Stage.

RESULTS

Epifl uorescence microscopy provides the best overall view of the structure of the epiphyll specimen ( Fig. 1 ), a result that became apparent only after conducting AFM and SEM micros-copy. Imaging the whole specimen using AFM is impractical due to time constraints, and certain features of the epiphyll that were obvious under epifl uorescence were “invisible” using conventional SEM techniques. Under epifl uorescence, cross walls of the cells on the fossil epiphyll are visible as intercon-nected, nonfl uorescing dark rectangles on the brightly emitting leaf surface (inset in Fig. 1 ). Epifl uorescence shows that the fossil epiphyll comprises a series of sparsely branched uniaxial fi laments associated with 25 darkened spheres, some of which were investigated under SEM ( Figs. 5–9 ). The fi laments are composed of shortly rectangular cells with transverse cross walls, with side branch sites marked by oblique septa. No at-tempt was made to investigate all of the darkened spheres iden-tifi ed under epifl uorescence, leaving the possibility that some of

2454 AMERICAN JOURNAL OF BOTANY [Vol. 100

fi laments of Cephaleuros are highly irregular in outline, very thick-walled, and have highly variable widths. Fungi can also be excluded as hyphal systems generally have narrower widths and lack closely spaced transverse septa.

The fossil might at fi rst sight resemble a myxomycete capil-litium; however: (1) the presence of oblique and transverse septa; (2) the fact that the presumed spores are connected to the fi lamentous system; and (3) the lack of any ornamentation on the spores in the fossil again excludes this group. Beside moss protonemata the only other possible candidates are fi lamentous cyanobacteria. Both have fi laments comprising short cells, but in cyanobacteria heterocysts are clearly distinct along these, cells are frequently swollen, main axes are often, although not always, multiseriate, and branching involves very different cell division patterns ( Gugger and Hoffman, 2004 ) from the single oblique septum seen in the fossil. Thus we conclude that the fossil represents an epiphyllous moss protonema. It is also note-worthy that the fossil epiphyll was found on the upper leaf sur-face where extant epiphylls almost invariably occur.

The 3D-mosaic created by assembling overlapping detailed AFM scans ( Figs. 2, 3 ), illustrated that only elongated, shallow grooves remained in the position where fi lament cells were vis-ible with epifl uorescence ( Figs. 5, 6 ). These should have been raised features under AFM and SEM, because in life the fi la-ments were originally rectangular. That only shallow depres-sions remain in the surface of the host helps to explain why the fossil epiphyll was “invisible” with traditional microscopy and also may help to explain why fossil epiphylls have not been commonly identifi ed. The depressions replacing the fi laments indicate possible enzymatic digestion of the leaf host by the epiphyll. Although the production of adhesive substances by attaching protonemal and rhizoidal fi laments in bryophytes is documented ( Pocock and Duckett, 1985 ; Pressel and Duckett, 2009 ), whether the same secretions also contain enzymes that digest the leaf surface of the host requires further research.

We interpret many of the darkened spheres initially recog-nized under epifl uorescence to be spores. This conclusion is based upon the observation that under epifl uorescence ( Fig. 1 ) the spheres are directly attached to the branching fi laments, and when the spheres were relocated and examined in detail, the spheres are hollow ( Figs. 3, 7–9 ) and have no internal nor external ornamentation when imaged using AFM and SEM techniques ( Figs. 2, 3, 5–9 ), a common trait of spores. Due to time con-straints—especially for AFM, but also for SEM—not every dark-ened sphere visible under epifl uorescence was relocated, and therefore not all of the darkened spheres were investigated in de-tail. As a result, we cannot confi rm that each of the darkened spheres visible under epifl uorescence was a spore. The possibility remains that some of these darkened spheres (under epifl uores-cence) could have been in the very early phase of side branch ini-tiation because the range of fi lament widths (8-15 µm) overlaps with the diameter of the darkened spheres (10-12.5 µm). However, for the spores that were examined in detail, we were able to make precise measurements of their horizontal dimensions using AFM and SEM, as well as nanometer-scale measurements of their height using AFM.

The spores were probably originally spherical and subse-quently fl attened during postdepositional compaction of the sedi-ments ( Figs. 5–9 ). This process also appears to have split open the spores, revealing the smooth internal wall ( Fig. 9 ). Flattening probably caused a slight increase in width, but caused a more sub-stantial vertical reduction by 4-5 times (based upon reduced spore height). The small diameter of the fossil spores (10.5-12.0 µm

this typical funarialean type in being essentially monomorphic with upright and horizontal fi laments comprising short cells with transverse septa and with side branches produced at right angles to the main axis from the center of the mother cell. Here too, however, formation of a side branch initial always involves depo-sition of a new oblique wall. This latter protonemal type closely matches that of the fossil ( Nehira, 1984 ; Goode et al., 1993 ; Duckett et al., 1998 ; Duckett et al., 2004 ). Of the other two bryo-phyte lineages, hornworts do not produce fi lamentous protone-mata and the same is true for the vast majority of liverworts. The leafy liverwort Cephalozia has a transient uniaxial fi lamentous juvenile stage, but these fi laments have only transverse cross walls and are unbranched ( Schuster, 1966 ).

A wide range of other organisms that might occur on leaf surfaces also produce a fi lamentous stage. Among these one can easily exclude epiphyllous/parasitic green algae as they never produce regular branching and oblique cross walls. Typi-cally, epiphyllic members of the Trentopohliales have fi laments of thick-walled bulging cells with irregular outlines and the parasitic green alga Cephaleuros forms a fl at thallus and lacks the spore profi les seen in the fossil. Furthermore, subcuticular

Figs. 5–6. Detailed images of leaf surface and fossil spores (indicated by white arrows). 5. Three-dimensional tilted image of a fossil spore cre-ated using AFM. 6. SEM image of a fossil spore. Trough to right of spores in ( Fig. 5 ) and left in ( Fig. 6 ) are former locations of protonemata where surface has been etched.

2455December 2013] BARCLAY ET AL.—OLDEST KNOWN FOSSIL EPIPHYLLOUS MOSS

branching sites (Duckett & Pressel, personal observations). Though the identity of these has never been investigated they most likely belong within a range of families with short-celled protonemata and which contain numerous epiphytic taxa e.g., Leskeaceae, Pterigynandraceae, Cryphaeaceae, Neckeraceae, and Orthotrichaceae ( Pócs, 1982 ; Goode et al., 1993 ) and ex-emplified by Orthotrichum lyellii W.J. Hooker & Taylor ( Fig. 10 ). This moss together with two other epiphytic Orthotri-chum species, O. diaphanum Schrader ex Bridel and O. affi ne Schrader ex Bridel produce extensive protonemal colonies as epiphytes and occasionally on evergreen leaves. In vitro culti-vation of epiphytes and epiphylls ( Duckett et al., 2004 ) demon-strates that sporelings of Ulota crispa Bridel (Orthotrichaceae; Fig. 11 ) and mature protonemal systems of Cryphaea hetero-malla (Hedw.) D.Mohr (Cryphaeaceae; Fig. 12 ) exhibit the same morphology of shortly cylindrical cells with transverse end walls found in Bryiidites utahensis . Entosthodon obtusus Lindberg (Funariaceae) and Ulota crispa illustrate typical spore germination in mosses with the emergence of fi laments from spores (indicated by black arrows in Figs. 13 and 11 , respec-tively) similar to the dark rounded structures visible in epifl uo-rescence images of Bryiidites utahensis ( Figs. 1, 4 ). In Fig. 13 , Entosthodon illustrates the elongate cells typical of funarialean protonemata. A further frequent feature of wild moss protone-matal colonies is the presence of numerous spores, again mir-roring their occurrence in the fossil.

The foregoing assumes that the fossil was growing on a leaf attached to the parent plant. An alternative possibility is that the fossil-bearing leaf had fallen from a tree and become colonized by spores from terricolous mosses falling on it. This would seem less likely because of the density of the spores on the fos-sil and the fact that terricolous mosses almost always have pro-tonemata with elongated cells (i.e., funarialean type).

Thus, with this suite of features ruling out alternatives we identify the fossil as an epiphyllous moss and name it appropri-ately as Bryiidites utahensis gen. et sp. nov. on the basis of its unique resemblance to the germinating spores and protonemata of a range of mosses (see Systematic Paleobotany, above).

Modern epiphyllous mosses are typically found in humid cli-mates, with increased abundance and diversity in regions with a warm and equable climate ( Pócs, 1982 ; Sonnleitner et al., 2009 ). The fossil epiphyll provides another line of evidence, from a botanical perspective, that paleoclimate conditions in Southwestern Utah were both warm and humid during the mid-dle Cretaceous ( Barclay et al., 2010 ), despite a paleolatitude of 36 ° N ( Scotese, 1991 ; Sageman and Arthur, 1994 ). Previous evidence for warm temperatures at these latitudes came from coeval paleotemperature estimates based upon δ 18 O estimates from benthic foraminifera ( Huber et al., 2002 ). Bottom water temperatures at Blake Nose (30 ° N paleolatitude) were 19 ° C in the late Cenomanian, far warmer than modern temperatures of 2 ° C ( Huber et al., 2002 ). Latitudinal gradients in sea surface temperature from the equator toward the poles were also sig-nifi cantly lower during Cenomanian–Turonian time, with a mean 0.10 ° C per degree latitude gradient, only a quarter as strong as for modern day ( Huber et al., 2002 ). Air temperatures in the equatorial Atlantic during the late Cenomanian may have reached as high as 33 ° C, based upon the Tex 86 geochemical proxy and δ 18 O from planktonic foraminifera ( Forster et al., 2007 ), and the poles were similarly very warm with evidence for low-temperature-sensitive crocodilians on Axel Heiberg Is-land (paleolatitude of 72 ± 4 ° N; Tarduno et al., 1998 ). The paleodepositional setting for the Dakota Formation was the

after compaction) places them at the low end of the moss range ( Clarke, 1979 ), and rules out the majority of extinct and extant pollen grains, most of which have diameters larger than 12 µm. Typical wind-pollinated plants have pollen grains that range from 20 to 40 µm in diameter, and wind-dispersed pollen grains also tend to be smaller than pollen dispersed by animals ( Whitehead, 1983 ). The majority of fungal spores with simple, round, unornamented, and nonfl agellate morphology can also be ruled out, as they typically measure considerably less than 10 µm in diameter ( Webster and Weber, 1980 ).

Typical epiphyllous bryophytes in tropical forests include hundreds of species of leafy liverworts in the Lejeuneaceae, a few species in the hornwort genus Dendroceros , and a variety of pleurocarpous mosses, including the hookerioid genera Ephemeropsis , Crossomitrium ( Daltonia ), and Fabronia , plus various members of the Cryphaeaceae and Erpodiaceae ( Pócs, 1982 ; Goode et al., 1993 ). Apart from Ephemeropsis , these mosses are identifi ed from their leafy shoots and sporophytes but also frequent as epiphylls are moss protonemata comprising short cells with transverse septa and oblique septa at side

Figs. 7–9. AFM and SEM images of the same Bryiidites utahensi s spore (same location of spore indicated by white arrows). 7. AFM height map. 8. 3D tilted AFM image. Height scale (black to white) is 3 µm for both AFM images. 9. SEM image exposing hollow interior of spore.

2456 AMERICAN JOURNAL OF BOTANY [Vol. 100

North America with the Lauraceae. The precise temporal dating of the fossil within the numerically constrained Dakota Forma-tion of Southwestern Utah fi nally provides agreement between the fossil record and molecular-clock dating techniques that currently place the diversifi cation of pleurocarpous mosses alongside the evolution of fl owering plants in the Cretaceous ( Ignatov and Shcherhakov, 2007 ; Newton et al., 2007 ).

LITERATURE CITED

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shoreline of the western margin of the Western Interior Seaway in North America ( Roberts and Kirschbaum, 1995 ; Laurin and Sageman, 2007 ). The soils that constitute the Dakota Formation in southwestern Utah are dominated by gleysols and coal hori-zons, with pedogenic features that suggest everwet conditions prevailed during deposition ( Laurin and Sageman, 2001 ; Barclay et al., 2010 ). Together, these paleoclimatic features support the botanical evidence from the epiphyllous moss for a warm and humid climate in the late Cenomanian of southwest-ern Utah.

The fossil host leaf morphotype (DSU01) shares seven char-acters in common with Hypodaphnis zenkeri (Engl.) Stapf ( Barclay et al., 2010 ), the only species in the basal genus of the angiosperm family Lauraceae ( Rohwer, 2000 ). The Lauraceae evolved in the early Late Cretaceous, and have been identifi ed as fossils from abundant charcoalifi ed reproductive structures (fl owers and fruits) and macrofossil leaves from multiple sedi-mentary deposits almost 100 million years old ( Crepet et al., 2004 ). Today, Lauraceae are pan-tropical in distribution, with limited presence in warm temperate climates ( Rohwer, 2000 ). This specimen of Bryiidites utahensis suggests that the associa-tion of epiphyllous mosses with their particular plant hosts dates to at least 95 million years ago, with the fi rst record in

Figs. 10–13. Transmitted light images of extant moss protonemata. 10. Orthotrichum lyellii (Orthotrichaceae) with side branch sites marked by oblique septa, which produces a characteristic y-shaped pattern. 11. Ulota crispa (Orthotrichaceae) germinating from spores. 12. Cryphaea heteromalla (Cryphaeaceae) exhibiting protonemal morphology typical of epiphylls. 13. Entosthodon obtusus (Funariaceae) germinating from spores.

2457December 2013] BARCLAY ET AL.—OLDEST KNOWN FOSSIL EPIPHYLLOUS MOSS

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