Tricostate Mosses and Associated Bryophilous Fungi from The

TRICOSTATE MOSSES AND ASSOCIATED BRYOPHILOUS FUNGI FROM THE

EARLY CRETACEOUS OF VANCOUVER ISLAND (CANADA)

Glenn W.K. Shelton

A Thesis Presented to

The Faculty of Humboldt State University

In Partial Fulfillment of the Requirements for the Degree

Master of Science in Biology

Committee Membership

Dr. Alexandru M.F. Tomescu, Committee Chair

Dr. Ruth A. Stockey, Committee Member

Dr. Stephen C. Sillett, Committee Member

Dr. Michael R. Mesler, Graduate Coordinator

May 2015

ABSTRACT

TRICOSTATE MOSSES AND ASSOCIATED BRYOPHILOUS FUNGI FROM THE

EARLY CRETACEOUS OF VANCOUVER ISLAND (CANADA)

Glenn W.K. Shelton

Two novel fossil mosses (Tricosta plicata gen. et sp. nov. and

Krassiloviella limbelloides gen. et sp. nov.) and several fungal morphotypes associated with one of these mosses are described here based on anatomically preserved material from the Early Cretaceous (Valanginian-136 Ma) Apple Bay locality of Vancouver Island (Canada). The mosses provide additions to the extremely short list of anatomically preserved bryophytes currently described from the pre-Cenozoic and have tricostate leaves (i.e., bearing three leaf veins or costae per leaf), a trait only known in a few other Mesozoic fossil mosses. Although the sporophytes of Tricosta and Krassiloviella are unknown, the high level of anatomical detail preserved in these fossils allows for whole-plant reconstructions of the gametophytes and the recognition of a new family of pleurocarpous mosses,

Tricostaceae fam. nov. The bryophyte-inhabiting (bryophilous) fungi, known exclusively in association with the gametophytes of T. plicata, are represented by

ii several distinct morphologies including: epiphyllous hyphae and appressoria, intracellular haustoria and cell aggregates, and endophytic perithecioid fruiting bodies embedded within shoot tips. This is the first occurrence of bryophilous fungi documented in the fossil record. Both the moss gametophytes and the bryophilous fungi are common throughout the Apple Bay material, yet inconspicuous. Their characterization holds promise for future studies of anatomically preserved material in terms of documenting past bryofloras and mycofloras, both of which are fascinating but little-studied components of paleo- ecosystems.

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ACKNOWLEDGEMENTS

I would like to thank my committee for their perseverance in making it through this tome the with still a healthy mind for comments and discussion. A special thanks goes to my advisor, Dr. Alexandru “Mihai” Tomescu and all of the HSU Biology

Department staff and faculty who have helped shape me academically throughout the past five years as an undergraduate and Master’s Candidate. It should also be said that, the inspirational environment and solid work ethic found in the “Lanphere-Reiss-Tomescu

Lab” (in part, my office), were great motivators for me during my years there (2010-

2015). This work is supported in part by: the Natural Sciences and Engineering Research

Council of Canada grant A-6908 to R. A. Stockey; the HSU Biology Department’s

Master’s Student Grant; and the Gregory Mark Jennings Award (HSU Biology).

TABLE OF CONTENTS

ABSTRACT ...... ii

ACKNOWLEDGEMENTS ...... iv

LIST OF TABLES ...... viii

LIST OF FIGURES ...... ix

INTRODUCTION ...... 1

TRICOSTA PLICATA GEN. ET SP. NOV...... 4

Fossil Record of Pre-Cenozoic Mosses ...... 4

Materials and Methods ...... 7

Systematics ...... 9

Description ...... 12

Habit, branching, shoot architecture, and stem anatomy ...... 12

Leaf morphology and anatomy ...... 20

Specialized branches ...... 29

Discussion ...... 35

The tricostate condition ...... 35

Tricostate analogues in extant mosses ...... 36

Tricostate mosses in the fossil record ...... 38

Taxonomic placement of Tricosta plicata gen. et sp. nov...... 40

Pleurocarpous mosses in the pre-Cenozoic fossil record ...... 48

Gametangia in the fossil record ...... 51

Outstanding questions ...... 53

Conclusions ...... 56

KRASSILOVIellA LIMBELLOIDES GEN. ET SP. NOV...... 58

Introduction ...... 58

Materials and Methods ...... 59

Systematics ...... 61

Description ...... 64

Habit, branching, shoot architecture, and stem anatomy ...... 64

Leaf morphology and anatomy ...... 72

Specialized branch ...... 80

Discussion ...... 84

The tricostate condition ...... 84

Taxonomic placement of Krassiloviella limbelloides gen. et sp. nov...... 84

Moss-animal interactions ...... 88

Conclusions ...... 88

IMPLICATIONS OF TRICOSTA AND KRASSILOVIA ...... 90

BRYOSYMBIOTIC FUNGI ...... 92

Introduction ...... 92

Materials and Methods ...... 93

Description of Fungal Morphotypes ...... 95

Tubular septate hyphae ...... 95

Moniliform hyphae ...... 105

Intracellular cell aggregations ...... 109

Phragmospores and dictyospores ...... 114

Aperturate amerospores ...... 118

Perithecioid fruiting bodies ...... 118

Interfoliar stromata ...... 121

Haustoria and other fungal types ...... 124

Discussion ...... 127

Ascomycete specialists ...... 127

Biotrophs or decomposers? ...... 128

Life history of the Tricosta plicata bryophilous fungi ...... 129

Systematics ...... 131

Conclusions ...... 135

REFERENCES ...... 137

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LIST OF TABLES

Table 1. Summary of Tricosta plicata gen. et sp. nov., Krassiloviella limbelloides gen. et sp. nov., and a comparison with species of Tricostium...... 41

Table 2. Comparison1 of Tricosta plicata gen. et sp. nov. to some monopodially branched pleurocarpous mosses2,3...... 46

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LIST OF FIGURES

Figure 1. Habit of Tricosta plicata gen. et sp. nov. Tuft of gametophytes in various planes of section; stems are traced on image at right; note single perithecioid fruiting body in transverse section (e.g., arrowheads); scale bar = 2 mm; P13957 Btop #16...... 13

Figure 2. Habit, branching, shoot architecture, and stem anatomy of Tricosta plicata gen. et sp. nov. 2A. Shoot in longitudinal section; narrow arrowheads show antheridia (upper arrowhead = sac; lower = stalk); all other arrowheads indicate perigonial branches; scale bar = 200 µm; P15425 C bot #38a. 2B. Stems in transverse sections showing radially arranged cortical cells; scale bar = 100 µm; P13957 A #2. 2C. Detail of 2B; note few, scattered, narrow cells near stem center; scale bar = 50 µm; P13957 A #2. 2D. Composite image of much-branched shoot in longitudinal section; arrowhead at far left shows vegetative branch; all other arrowheads represent positions of perigonial branches; scale bar = 500 µm; P15425 Cbot #56a. 2E. Stem in longitudinal section; scale bar = 100 µm; P13957 A #2. 2F. Detail of 2E showing fusiform cortical cells; scale bar = 50 µm; P13957 A #2...... 15

Figure 3. Branching architecture of Tricosta plicata gen. et sp. nov. reconstructed from serial sections (P15425 Cbot #1a-#92a); red dots represent positions of perigonia; broken lines at apex of main stem indicate uncertain branch arrangement; other broken lines indicate saw cuts; scale bar = 1 mm...... 16

Figure 4. Shoot architecture and stem anatomy of Tricosta plicata gen. et sp. nov. 4A. Vegetative shoot tip in longitudinal section; scale bar = 200 µm; P15425 Cbot #55a. 4B. Detail of 4A showing a group of faintly colored apical cells; scale bar = 50 µm; P15425 Cbot #55a. 4C. Darkened shoot tip showing scale-like leaf primordial (arrowheads); scale bar = 50 µm. P16435 Ctop #15. 4D. Branch primordium in longitudinal section; subtending leaf distanced one or two cells from primordium; thick arrowhead indicates base of scale-like structure surrounding primordium; note dark hypha within costa (thin arrowhead); scale bar = 50 µm; P13957 Btop #25. 4E. Branch primordium in longitudinal section directly subtended by leaf; thick arrowhead indicates scale-like structure; subtending leaf base indicated by thin arrowhead; scale bar = 50 µm; P16435 Ctop #14. 4F. Shoot transverse section showing leaf central costa (thick arrowhead) and one lateral costa (thin arrowhead) attached below point of leaf divergence; scale bar = 50 µm; P13131 Dtop #12c. 4G. Serial section of Figure 4F just above point of leaf divergence (with central and lateral costa, arrowheads); scale bar = 50 µm; P13131 Dtop #13c. 4H. Transverse section of shoot just above branching point showing unistratose and strongly plicate leaves with three costae per leaf (abaxial surface of one leaf underlined; C = costa); note paradermal section of part of leaf base (alar cells, arrowhead); scale bar = 100 µm; P13131 Dtop #3c...... 19

Figure 5. Rhizoids of Tricosta plicata gen. et sp. nov. 5A. Base of gametophyte tuft showing several stems in transverse section bearing rhizoids; scale bar = 200 µm; P13256 Cbot #19. 5B. Stem in transverse section (at left) surrounded by smooth-walled rhizoids; arrowheads indicate oblique end-walls within rhizoids; scale bar = 100 µm; P13256 Cbot #36. 5C. Smooth-walled rhizoids in transverse (thick arrowheads) and longitudinal sections (at left); thin arrowhead indicates oblique end-wall; scale bar = 50 µm; P13256 Cbot #34...... 21

Figure 6. Tricosta plicata gen. et sp. nov. leaf model. 6A. Cell morphology; note thickened cell walls at mid-leaf (right side), representing cells whose walls bear superficial and intercellular fungal hyphae; tracings are from 9 different leaves (specimen numbers listed here from base to apex, and left to right): P13131 Dtop #4c; P13957 Btop #52; P15425 Cbot #47a; P15425 Cbot #49a; P15425 Cbot #49a; P15425 Cbot #47a; P15425 Cbot #14a; P15425 Cbot #54a; P3957 Btop #157. 6B. Series of leaf tracings (right) in transverse section from base to apex demonstrate strong plication throughout; photos (below, with leaves highlighted) correspond to serial tracings; leaf sections from 7 different leaves; specimen numbers correspond to serial tracings from base to apex: P13029 D #21; P 13029 D #21; P15425 Cbot #26a; P15425 Cbot 22a; P13131 Dtop #3c; P15425 Cbot #16a; P15425 Cbot #11a; scale bar = 200 µm (A and B)...... 23

Figure 7. Leaf anatomy of Tricosta plicata gen. et sp. nov. 7A. Shoot transverse section showing tricostate leaves with strong plications (two leaves highlighted); arrowhead indicates cells of alar region; scale bar = 50 µm; P15422 A #1. 7B. Perigonial shoot in transverse section (center indicated by asterisk); innermost leaves (ca. 4) perigonial, other leaves vegetative; note protruding bistratose abaxial costa-layer in distal leaf sections (thin arrowheads); thick arrowhead indicates bundle of epiphyllous fungal hyphae; scale bar = 100 µm; P15425 Cbot #16a. 7C. Leaf transverse section showing three-layered median costa (ab-, adaxial surfaces traced; arrowheads = costal layers); scale bar = 30 µm; P15422 A #1. 7D. Leaf transverse section showing three-layered central costa (below) and bi-layered lateral costa (above); scale bar = 50 µm; P15422 A #1. 7E. Leaf transverse section showing three-layered median costa (below); note alar region (above) and adjacent lateral costa (arrowhead); scale bar = 50 µm; P15422 A #1. 7F. Costa in longitudinal section showing linear cells with tapered (arrowhead) or transverse end- walls; scale bar = 50 µm; P16435 Ctop #13. 7G. Costa in paradermal section showing linear cells with tapered or transverse end-walls, and juxtacostal cells (arrowheads); scale bar = 100 µm; P15425 Cbot #54a. 7H. Two leaf bases in longitudinal section showing few cells (arrowheads) of two different alar regions; scale bar = 50 µm; P13957 Btop #52. 7I. Alar region in transverse section; arrowhead indicates adjacent lateral costa; note stem center occupied by hyphae (section is just below perithecioid fruiting body); scale bar = 50 µm; P13957 Btop #25...... 26

Figure 8. Leaf anatomy of Tricosta plicata gen. et sp. nov. 8A. Shoot in transverse section showing inflated alar cells of (shaded) clasping leaf base (arrowhead = central costa); scale bar = 100 µm; P13131 Dtop #6c. 8B. Densely foliated shoot in longitudinal

section (leaf bases at left, stem at right); arrowhead indicates inflated alar cell; scale bar = 50 µm; P16435 Ctop #10. 8C. Cells of leaf base in paradermal section (center) and few alar cells in section (thick arrowhead); thin arrowhead = lateral costa; scale bar = 50 µm; P13957 Btop #52. 8D. Alar region in paradermal section; scale bar = 50 µm; P15425 C bot #44a. 8E. Leaf cells in paradermal section showing thin walls indicative of the absence of fungal hyphae or taphonomic alterations; scale bar = 30 µm; P15425 Cbot #49a. 8F. Leaves in paradermal section showing laminal cell shapes near leaf base (lower left) and in lower half of leaf (right); scale bar = 100 µm; P15425 Cbot #47a. 8G. Laminal cell shapes in distal half of leaf; scale bar = 50 µm; P15425 Cbot #49a. 8H. Leaves in paradermal sections showing cell shapes of mid-leaf; scale bar = 100 µm; P15425 Cbot #49a. 8I. Leaf paradermal section showing cell shapes at mid-leaf; note fungi (arrowheads); scale bar = 100 µm; P15425 Cbot #48a. 8J. Paradermal section showing leaf cell shapes at mid-leaf; scale bar = 100 µm; P15425 Cbot #48a. 8K. Leaf apex in paradermal section; note leaf margins not shown; scale bar = 50 µm; P13957 Btop #157. 8L. Detail of 8K showing laminal cell shapes and median costa (upper arrowhead shows linear cells of adaxial costal layer; lower arrowhead shows short cells of abaxial costal layer); scale bar = 50 µm; P13957Btop #157...... 28

Figure 9. Perigonia of Tricosta plicata gen. et sp. nov. 9A. Several perigonia in transverse sections (e.g., asterisks), some of these showing antheridia (arrowheads); scale bar = 200 µm; P15425 Cbot #13a. 9B. Perigonial shoot in transverse section showing antheridium at center and innermost perigonial leaves (e.g., arrowheads) with weak costae and plication; scale bar = 100 µm; P15425 Cbot #25a. 9C. Perigonium in longitudinal section showing well-preserved swollen axis (thick arrowhead) bearing several leaf bases and incompletely preserved antheridial stalk (thin arrowhead; note base of sac attached to stalk); scale bar = 100 µm; P15425 Cbot #47a. 9D. Perigonium in longitudinal section showing incompletely preserved swollen axis (thick arrowhead) and well-preserved antheridial stalk (thin arrowhead; note base of sac attached to stalk); scale bar = 100 µm; P15425 Cbot #38a. 9E. Perigonium in longitudinal section showing incompletely preserved axis (thick arrowhead), antheridial stalk (thin arroewhead), and oblong antheridial sac attached to stalk; scale bar = 100 µm; P15425 Cbot #36a. 9F. Antheridial stalk in longitudinal section showing triseriate and tiered organization; arrowhead indicates base of antheridial sac; scale bar = 50 µm; P15425 Cbot #38a. 9G. Antheridial stalk in transverse section showing triseriate organization; scale bar = 50 µm; P15425 Cbot #28a. 9H. Perigonium in transverse section showing convoluted antheridial jacket and innermost perigonial leaf; scale bar = 50 µm; P15425 Cbot #38. 9I. Antheridial jacket in longitudinal section showing irregular and narrow cell shapes (e.g., inset; scale bar = 10 µm); scale bar = 50 µm; P15425 Cbot #38...... 32

Figure 10. Probable perichaetia of Tricosta plicata gen. et sp. nov. 10A. Stem (dashed line) and specialized lateral branch (?perichaetium; arrowhead) in longitudinal section; scale bar = 300 µm; P13957 Btop #129. 10B. Stem and specialized lateral branch (arrowhead) in longitudinal section; scale bar = 300 µm; P13957 Btop #131. 10C. Stem and specialized lateral branch (arrowhead) in longitudinal section; scale bar = 300 µm; xi

P13957 Btop #132. 10D. Detail of 10C showing incompletely preserved perichaetium axis (bottom), erect leaves and elongate leaf cells; arrowhead indicates putative archegonium; scale bar = 100 µm; P13957 Btop #132. 10E. Detail of 10B showing swollen axis (thick arrowhead) and erect leaves with elongate cells; apex bears delicate elongate structure (archegonium; thin arrowhead); scale bar = 100 µm; P13957 Btop #131. 10F. Detail of 10E showing archegonium in longitudinal section; note elongate laminal cells at right; scale bar = 50 µm; P13957 Btop #131. 10G. Detail of 10D showing archegonial venter and incompletely preserved base (arrowhead) in longitudinal section; note absence of tiered, triseriate stalk; scale bar = 50 µm; P13957 Btop #132. 10H. Oblique section of archegonium above level of shoot tip (not shown) showing narrow neck canal at center (arrowheads), single layer of neck cells (n) and few layers of delicate venter cells (v); scale bar = 20 µm; P13957 Btop #121. 10I. Longitudinal section of specialized shoot showing delicate tissue attached directly to apex; scale bar = 50 µm; P13957 Btop #89...... 34

Figure 11. Branching architecture of Krassiloviella limbelloides gen. et sp. nov. 11A. Reconstructed branching architecture showing a main stem (ms) with missing tip and a long, reiterative stem with tip missing (top); note branching along main stem restricted to distal portion; dashed lines represent portions of stem missing in between slabs; scale bar = 2 mm; P17596 Cbot #1-#68 and P17596 D #1-#45. 11B. Reconstructed branching architecture with main stem bearing putative gametangial branch (arrowhead) directly subtended by short, narrow branch; dashed lines represent portions of stem removed by saw cut; scale bar = 2 mm; P25 Bbot #1b-#73b...... 66

Figure 12. Stem anatomy of Krassiloviella limbelloides gen. et sp. nov. 12A. Oblique section of stem showing arrangement of cortical cells and absence of conducting strand; note epidermis not preserved; scale bar = 100 µm; P25 Bbot #8b. 12B. Cortical cells in longitudinal section showing thin walls and tapered end walls; scale bar = 30 µm; P25 Bbot #13b. 12C. Longitudinal section of stem showing well-defined epidermis of dark cells (thin arrowhead) and minute lateral branch (asterisk); note most cortex in stem and branch not preserved; scale bar = 200 µm; P17596 Cbot #51...... 68

Figure 13. Shoot architecture of Krassiloviella limbelloides gen. et sp. nov. 13A-13C. Shoots in longitudinal section showing branches (arrowheads) and numerous leaf bases; scale bars = 1 mm; 13A-13C: P25 Bbot #27b, P25 Bbot #30b, P25 Bbot #41b. 13D. Detail of Fig. 12C showing epidermis (arrowhead) composed of rectangular cells; most cortex not preserved (bottom of image); scale bar = 50 µm; P17596 Cbot #51. 13E. Shoot in longitudinal section showing entire branch tip (arrowhead); scale bar = 300 µm; P25 Bbot #27b...... 69

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Shoot in longitudinal section showing erect, densely arranged leaves; note slightly wider divergence angle of leaf subtending branch (arrowhead); scale bar = 1 mm; P17596 Cbot #45. 14D. Detail of 14A showing concave shape to remaining tip (suggesting herbivory); note darkened outer cell layers of stem tip and adjacent leaves; scale bar = 150 µm; P17596 D #37. 14E. Shoot cross section showing helically arranged, overlapping leaves and characteristic incomplete preservation of laminae; scale bar = 300 µm; P15388 A #23. 14F. Branch with tip removed by taphonomic processes; note crystalline structures at remaining branch tip (arrowheads); scale bar = 200 µm; P25 Bbot #41b. 14G. Branch with entire tip and short, delicate leaves; note at least one contorted leaf base (arrowhead); scale bar = 150 µm; P17596 Cbot #53...... 71

Figure 15. Rhizoids of Krassiloviella limbelloides gen. et sp. nov. 15A. Transverse section of gametophyte base showing dense rhizoids; note three costa bases of single leaf (arrowheads); scale bar = 200 µm; P17345 Ctop #18a. 15B. Transverse section of gametophyte base showing dense rhizoids attached to stem; few rhizoids attached to abaxial surface of central costa (arrowhead); scale bar = 200 µm; P17345 Ctop #14a. 15C. Detail of 15A showing smooth-walled rhizoids with oblique end-walls (arrowheads); scale bar = 50 µm; P17345 Ctop #18a. 15D. Leaf axil in longitudinal section showing rhizoid base (arrowhead) attached to adaxial surface of costa; scale bar = 50 µm; P25 Bbot #12b. 15E. Paradermal section of incompletely preserved leaf showing few rhizoids (R = rhizoids and the area magnified in 15F) attached to leaf surface; scale bar = 300 µm; P17596 Cbot #51. 15F. Detail of 15E showing rhizoid bases (thick arrowheads) and oblique end-walls (thin arrowheads; also shown in inset, scale bar = 10 µm); scale bar = 30 µm; P17596 Cbot #51...... 73

Figure 16. Leaf anatomy and morphology of Krassiloviella limbelloides gen. et sp. nov. 16A. Paradermal section showing entire leaf length; thick arrowheads indicate central costa, thin arrowheads indicate left lateral costa (right lateral costa not shown); at right, leaf model superimposed over same image; scale bar = 1 mm; P17596 Cbot #66. 16B- 16F. Series of leaf transverse sections from near base (16B) to the apex (16F); leaves traced adaxially; scale bars B-D = 100 µm; E-G = 50 µm; C and D: P13131 Dbot #17c; E: P13131 Dbot #10c; F: P13131 Dbot #63c; G: P13131 Dbot #49c...... 74

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rectangular cells of epidermis (e.g., arrowhead); scale bar = 50 µm; P17596 Cbot #44. 17F. Paradermal section of leaf showing central costa (right), lateral costa (left), and laminal cells forming longitudinal files (in between arrowheads); larger juxtacostal cells adjacent to costae; scale bar = 200 µm; P17596 Cbot #66. 17G. Apiculate leaf tip (apiculus) in paradermal section; note minute dentition (arrowhead); scale bar = 50 µm; P25 Bbot #1b. 17H. Oblique section of stem showing leaf base (traced abaxially) just above point of divergence of lateral costae (left and right arrowheads); central costa (arrowhead at bottom) incompletely preserved and attached to stem; scale bar = 200 µm; P25 Bbot #2b. 17I. Oblique section of shoot showing leaf base below point of divergence; arrowheads indicate positions of costae; scale bar = 200 µm; P25 Bbot #2b...... 76

Figure 18. Leaf anatomy of Krassiloviella limbelloides gen. et sp. nov. 18A. Longitudinal section through central costa; scale bar = 100 µm; P17596 Cbot #66. 18B. Leaf transverse section below midleaf showing slightly incurved leaf margin; note darkened epidermis; scale bar = 50 µm; P13131 Dbot #22c. 18C. Transverse section of planar leaf margin and lateral costa near midleaf; note ab- and adaxial costal epidermis; scale bar = 50 µm; P13131 Dbot #22c. 18D. Leaf base in paradermal section showing central costa (right), laminal cell morphology, and lateral costa (left); scale bar = 50 µm; P17596 Cbot #51. 18E. Leaf base in paradermal section showing central costa (right), laminal cell morphology, and lateral costa (left); scale bar = 50 µm; P25 Bbot #34b. 18F. Paradermal section of incompletely preserved leaf base (stem at left) showing cells near the alar region just above point of leaf attachment; scale bar = 50 µm; P17596 D #30. 18G. Composite image of leaf transverse section above midleaf showing three homogeneous costae and incompletely preserved lamina; scale bar = 50 µm; P13131 Dbot #10c. 18H. Paradermal section near leaf apex showing laminal cells (bottom) and isodiametric cells of epidermis (arrowhead); scale bar = 50 µm; P13131 Dbot# 19c. 18I. Paradermal section of leaf apex showing few linear costal cells (thin arrowhead), laminal cells (center), epidermal cells (thick arrowhead); scale bar = 50 µm; P13131 Dbot #23c. 18J. Leaf paradermal section near mid-leaf showing central costa (right), median costa (left) and small isodiametric laminal cells; note larger juxtacostal cells; scale bar = 50 µm; P17596 Cbot #66. 18K. Leaf paradermal section above mid-leaf showing central costa (right), median costa (left) and small isodiametric laminal cells; note larger juxtacostal cells; scale bar = 50 µm; P17596 Cbot #66...... 79

incompletely preserved delicate tissue (above shoot tip) of probable gametangia and paraphyses; scale bar = 50 µm; P25 Bbot #9b. 19J. Magnification of 19E showing darkened bases (arrowheads) attached to shoot tip and incompletely preserved delicate tissue (above shoot tip) of probable gametangia and paraphyses; note missing material due to taphonomic processes (asterisks); scale bar = 50 µm; P25 Bbot #12b. 19K. Magnification of 19D showing probable uniseriate paraphysis (thin arrowhead) and gametangial bases (thick arrowheads); scale bar = 50 µm; P25 Bbot #11b...... 82

Figure 20. Probable archegonial branch tip of Krassiloviella limbelloides gen. et sp. nov. 20A. Longitudinal section at the periphery of specialized branch tip showing two dark bases (bottom); above base at left is dark several-celled structure (?embryo; arrowhead); the delicate pale tissue (center) is the probably remnants of several “archegonia”; scale bar = 50 µm; P25 Bbot #7b. 20B. Serial section adjacent 20A showing two dark bases (bottom); base at right directly attached (thin arrowhead) to column of pale tissue (archegonium) with putative several-celled embryo (thick arrowhead); scale bar = 50 µm; P25 Bbot #8b...... 83

Figure 21. Gametophytes of Tricosta plicata gen. et sp. nov. harboring bryophilous fungi. 21A. Longitudinal section of densely foliated shoot; note stem (= s) anatomy not preserved; scale bar = 500 µm; P15425 Cbot #65a. 21B. Shoot in oblique section hosting several fungal morphotypes; note dense fungal stromata (arrowheads) away from leaf axils; scale bar = 300 µm; P13616 Etop #21b. 21C. Longitudinal section of shoot tip showing terminal position of fruiting body (fb); note subtending branch at left; scale bar = 100 µm; P13957 A #7...... 96

Figure 22. Tubular septate hyphae and haustoria on and in the leaves of Tricosta plicata gen. et sp. nov. 22A. Leaves in section showing regularly branched, tubular, septate hypha running along the leaf surface; scale bar = 20 µm; P15425 Cbot #56a. 22B. Large-diameter hypha with dark and ornamented walls; one leaf cell in transverse section is occupied by delicate fungal haustorium (arrowhead) suspended by small-diameter filament; scale bar = 20 µm; P15425 Cbot #54a. 22C. Magnification of 22B showing delicate haustorium suspended in center of host cell by small-diameter filament (arrowhead); scale bar = 10 µm; P15425 Cbot #54a. 22D. Leaf paradermal section showing hyphae along leaf cell wall outlines, endophytic cell aggregates (arrowheads at right), and fungal material which may represent developing endophytic aggregate (left arrowhead); note highly degraded costa at left in longitudinal view; scale bar = 50 µm; P15425 Cbot #46a. 22E. Magnification of 22D showing hyphae (e.g., arrowheads) along the leaf cell wall outlines (inter-and extracellularly); dark intracellular fungi (near center) may represent developing endophytic aggregate; scale bar = 20 µm; P15425 Cbot #46a. 22F. Delicate haustorium (arrowhead) at center of leaf cell; scale bar = 20 µm; P15425 Cbot #46a...... 98

Figure 23. Fungal hyphae, appressoria, and haustoria on and in leaves of Tricosta plicata gen. et sp. nov. 23A. Leaf paradermal section showing hyphae along host cell wall

xv outlines (= darkened thickenings of cell wall outlines); note hypha running through degraded costa (arrowhead at right); and epiphytic hypha bearing round cells (arrowhead at bottom); scale bar = 50 µm; P15425 Cbot #48a. 23B. Magnification of 23D, showing knob-like appressorium and evidence of penetration peg (pp); note top half of appresorium-bearing filament missing; scale bar = 5 µm; P15425 Cbot #52a. 23C. Magnification of 23A showing epiphytic hypha bearing 3 or 4 round cells alternately; scale bar = 10 µm; P15425 Cbot #48a. 23D. Hypha running superficially (extracellularly) over leaf cell wall outlines; laterally-produced appressorium (ap) shown positioned over anticlinal wall of juxtacostal cell; scale bar = 20 µm; P15425 Cbot #52a. 23E. Magnification of 23F showing haustorium suspended intracellularly by small- diameter hyphae (arrowheads); scale bar = 10 µm; P15425 Cbot #49a. 23F. Paradermal section showing hyphae along leaf cell wall outlines (= darkened thickenings of cell wall outlines); note intracellular aggregations (wide arrowheads), hyphal fragments radiating from juxtacostal cells (thin arrowheads at right; costa not shown), and delicate haustorium (thin arrowhead at left); scale bar = 50 µm. P15425 Cbot #49a. 23G. Magnification of 23F showing hyphae along leaf cell wall outlines (arrowheads) and intracellular aggregation (at right); scale bar = 20 µm; P15425 Cbot #49a...... 100

Figure 24. Fungi associated with leaves of Tricosta plicata gen. et sp. nov. 24A. SEM of leaf cell in section showing points of penetration by hyphae (thin arrowheads) and traces of intercellular hyphae (thick arrowhead); scale bar = 5 µm; P15425 Cbot #38a. 24B. SEM of 24C showing juxtacostal leaf cells in paradermal section (costa at right); evidence of penetration peg (thick arrowhead at right), traces of intercellular hyphae (thin arrowheads), and hypha connected to intracellular aggregate (thick arrowhead at left); scale bar = 20 µm; P15425 Cbot #38a. 24C. Light micrograph of 24B showing juxtacostal leaf cells in paradermal section (costa at right); arrowhead indicates hypha connected to intracellular aggregate; scale bar = 20 µm; P15425 Cbot #38a. 24D. Thick bundle of hyphae (center) running along leaf surface; scale bar = 50 µm; P15425 Cbot #43a. 24E and 24F. Serial transverse sections of leaf margin enveloped by bundle of hyphae; bundle (thick arrowheads) bridges adjacent leaf; thin arrowheads indicate an intracellular aggregate; asterisk in 24E indicates large crystal where lamina incompletely preserved; scale bars = 20 µm; 24E: P15425 Cbot #47a; 24F: P15425 Cbot #48a. 24G. Leaf in transverse section showing bundle of organized hyphae; hyphal bases at left perpendicular to leaf surface; arrowheads indicate hyphae within costa; scale bar = 30 µm; P15425 Cbot #47a...... 103

Figure 25. Fungal appressoria on leaves of Tricosta plicata gen. et sp. nov. 25A. Paradermal section of leaf base showing appressoria (arrowheads) born on epiphyllous hyphae; note hyphae following host cell wall outlines at far left; scale bar = 30 µm; P15425 Cbot #42a. 25B. Magnification of Figure 25A showing appressorium and indication of penetration hypha (arrowhead); scale bar = 5 µm; P15425 Cbot #42a. 25C. SEM of epiphyllous hyphae with perforations (arrowheads) indicative of penetration peg bases; scale bar = 10 µm; P15425 Cbot #43a...... 104

xvi

Figure 26. Epiphyllous moniliform hyphae on Tricosta plicata gen. et sp. nov. 26A. Section near leaf axil showing numerous moniliform chains, some in connection (e.g., arrowheads) with tubular hyphae; scale bar = 30 µm; P15425 Cbot #68a. 26B. Moniliform hypha running across leaf surface in transverse section; scale bar = 20 µm; P15425 Cbot #54a. 26C. Section near leaf axil showing moniliform hyphae; one chain (top) shown running across surface of leaf; other chains and tubular hyphae occur in dense mass (below); scale bar = 20 µm; P15425 Cbot #64a. 26D. Branched, epiphyllous moniliform hypha; scale bar = 10 µm; P15425 Cbot #68a. 26E. Moniliform hypha (four cells shown at top) connected to tubular hypha; scale bar = 20 µm; P15425 Cbot #51a. 26F. Leaf in section with adhered cluster of smooth-walled fungal cells probably representing condensed moniliform hyphae; scale bar = 20 µm; P15425 Cbot #42a. 26G. SEM of moniliform hyphae showing minute holes (arrowheads) which correspond to bases of penetration hyphae; scale bar = 5 µm; P15425 Cbot #43a. 26H. Dense cluster of smooth-walled fungal cells probably representing cluster of moniliform hyphae; scale bar = 20 µm; P15425 Cbot #38a...... 107

Figure 27. Epiphyllous ornamented moniliform hyphae on Tricosta plicata gen. et sp. nov. 27A. Leaves in transverse section showing large-diameter moniliform chains with ornamented walls; one chain attached to tubular hypha (right arrowhead), another attached directly to leaf costa (top arrowhead); thin arrowhead at left indicates intracellular aggregate connected to tubular hypha; note degraded leaf costae (asterisks); scale bar = 50 µm; P15425 Cbot #49a. 27B. Large-diameter, moniliform chain (attached to leaf at right) bearing hyaline lateral cell connected to tubular hypha (arrowhead); inset (scale bar = 5 µm) shows micro-echinate ornamentation; scale bar = 20 µm; P15425 Cbot #52a. 27C. Branched and ornamented moniliform hypha attached to leaf surfaces; scale bar = 20 µm; P15425 Cbot #50a. 27D. Large-diameter moniliform hypha (with short branch) showing smooth to scarcely ornamented walls; scale bar = 20 µm; P15425 Cbot #43a. 27E. SEM of large-diameter moniliform chain attached to incompletely preserved leaf surface (below); arrowhead indicates micro-echinate process; scale bar = 5 µm; P15425 Cbot #38a...... 108

Figure 28. Intracellular aggregates within leaves of Tricosta plicata gen. et sp. nov. 28A. Leaves in section harboring intracellular fungal aggregates showing connection with tubular hyphae (arrowheads); scale bar = 30 µm; P15425 Cbot #46a. 28B. Few leaves in section showing several fungal aggregates and moniliform chains (arrowheads); aggregates at left show evenly-spaced distribution (occurring in every other cell); scale bar = 30 µm; P15425 Cbot #49a. 28C. Leaf in longitudinal section showing intracellular moniliform aggregate and penetration hypha (arrowhead) within host anticlinal wall; large structure probably a plant spore; inset (scale bar = 5 µm) shows thickened leaf cell wall (arrowhead) surrounding penetration hypha; scale bar = 20 µm; P15425 Cbot #68a. 28D. Coiled moniliform aggregate surrounded by incomplete (ruptured) leaf cell wall (arrowheads); scale bar = 10 µm; P15425 Cbot #49a. 28E. Minute moniliform hypha in connection with (arrowhead) intracellular aggregate; scale bar = 10 µm; P15425 Cbot #46a. 28F. SEM of few leaf cells in paradermal section showing intracellular aggregate xvii in connection with intercellular hypha (arrowhead) through anticlinal wall; scale bar = 10 µm; P15425 Cbot #43a. 28G. Leaf in longitudinal section showing two intracellular aggregates, upper aggregate in connection with small-diameter moniliform chain; scale bar = 20 µm; P15425 Cbot #49a...... 111

Figure 29. Leaf paradermal section (costa at left) showing several dark intracellular aggregates spaced in regular pattern; scale bar = 50 µm; P15425 Cbot #49a...... 112

Figure 30. Leaf in section showing ruptured periclinal walls (r) along one leaf surface; part of moniliform chain (underneath pyrite aggregates at left) shown attached to leaf by hypha (arrowhead); hyphal shown on opposite leaf surface; scale bar = 20 µm; P15425 Cbot #49a...... 113

Figure 31. Phragmospores and dictyospores within gametophyte shoots of Tricosta plicata gen. et sp. nov. 31A. Triseriate dictyospore attached to leaf surface; degenerated cells at center of propagule indicate germination or previous attachment to hyphae; scale bar = 20 µm; P13616 Etop #19a. 31B. Triseriate dictyospore attached to leaf surface by at least one hypha (thin arrowhead); thick arrowhead indicates biseriate dictyospore in transverse section; large amerospore shown at bottom left; scale bar = 20 µm; P15425 Cbot #49a. 31C. Pair of light-colored triseriate dictyospores attached to hyphae (thin arrowheads) and adaxial surface of leaf costa in transverse section; dictyospore at right connected (thick arrowhead) to leaf at top; scale bar = 20 µm; P15425 Cbot #32a. 31D. Triseriate dictyospore in contact with leaf surface (at bottom); scale bar = 20 µm; P13616 Etop #18b. 31E. Biseriate dictyospore in between leaves; scale bar = 10 µm; P15425 Cbot #51a. 31F. Biseriate dictyospore showing physical connection (arrowhead) with branched tubular hypha; scale bar = 20 µm; P15425 Cbot #52a. 31G. Biseriate dictyospore showing incomplete material at one end (arrowhead) indicating previous hyphal attachment to leaf surface at left; "pp” = base of penetration peg on lateral knob- like appressorium; scale bar = 20 µm; P15425 Cbot #51a. 31H. Four-celled phragmospore attached at left to abaxial surface of incompletely preserved leaf; arrowhead indicates aperture in cross-wall of propagule; scale bar = 20 µm; P13957 Btop #126. 31I. Four-celled phragmospore with both ends incomplete; dark amerospore at left in transverse section shows slit (arrowhead); scale bar = 10 µm; P15425 Cbot #64a. 31J. Four-celled phragmospore (top, center) with both ends incomplete; incompletely preserved leaves in section at bottom bear branched hyphae (below, left) and dark bundle of hyphae (right); scale bar = 20 µm; P15425 Cbot #52a. 31K. Four-celled phragmospore with ends entire and attached below to highly degraded leaf surface; scale bar = 10 µm; P15425 Cbot #52a...... 116

xviii

phragmospore at least three cells long (at lower right) scattered in between leaf bases; fragments at upper left may represent cells of same propagule type; inset (scale bar = 10 µm; P15425 Cbot #41’a): magnification of 32A at attachment to leaf surface showing laterally-produced infection hypha (arrowheads indicate base and tip of infection hypha) penetrating leaf anticlinal wall; scale bar = 30 µm; P15425 Cbot #47a...... 117

Figure 33. Aperturate amerospores scattered within shoots of Tricosta plicata gen. et sp. nov. 33A. Loose aggregate of amerospores (single-celled propagules) in between leaves (hyphae at upper right following cell wall surfaces of leaf in section); arrowheads indicate slit-like apertures; scale bar = 20 µm; P15425 Cbot #48a. 33B. Magnification of 33A showing amerospore in transverse section; arrowheads indicate thickened margins of slit; scale bar = 5 µm; P15425 Cbot #48a. 33C. Magnification of 33A showing oval amerospore with hilum (arrowhead) near middle; adjacent spore (top left) shows slit (s) and pale thickenings of the slit margin; scale bar = 5 µm; P15425 Cbot #48a. 33D. Amerospore showing thick wall and slit (arrowhead); scale bar = 10 µm; P15425 Cbot #64a. 33E. Thick-walled amerospore in transverse section showing slit (arrowhead) and shape altered by crystal (asterisk); scale bar = 5 µm; P15425 Cbot #38a. 33F. Leaf in paradermal section bearing two dark amerospores, moniliform chains and intracellular aggregate; arrowheads indicate amerospore connection with hyphae; scale bar = 30 µm; P15425 Cbot #43a. 33G. Light-colored amerospore (hilum indicated by arrowhead) in contact with (but not attached to) a scattered aggregate of small fungal cells; scale bar = 10 µm; P15425 Cbot #38a. 33H. Amerospore showing slit (arrowhead); spore open along slit; scale bar = 10 µm.; P15425 Cbot #54a...... 120

Figure 34. Perithecioid fruiting bodies embedded in shoot tips of Tricosta plicata gen. et sp. nov. 34A. Two gametophyte shoots bearing fruiting bodies (arrowheads) embedded within their tips; top in transverse section; bottom in oblique section; scale bar = 300 µm; P13957 Btop #9. 34B. Shoot in oblique section bearing subspherical fruiting body (shown in midlongitudinal section); arrowhead indicates ostiole surrounded by funnel- shaped leaf base (traced abaxially); inset (scale bar = 50 µm; P13957 Btop #127): fruiting body in near-midlongitudinal section showing funnel-shaped leaf base around ostiolar neck; scale bar = 100 µm. P13957 A #11. 34C. Fruiting body in midtransverse section; note thin, single-layered fruiting body wall (arrowhead) and disorganized fungal contents; scale bar = 100 µm; P13957 Btop #9. 34D. Ostiolar region of fruiting body in near-midlongitudinal section; thin arrowheads indicate short ostiolar neck; funnel-shaped leaf base surrounding ostiolar neck; fruiting body wall not preserved (thick arrowheads indicate previous position of wall); note contents loosely aggregated, incompletely preserved; scale bar = 50 µm; P13957 Btop #126. 34E. Fruiting body and shoot in oblique section showing few layers of stem cortex around fruiting body; arrowhead indicates fruiting body wall; note extremely pale fungal tissue within host cortical cells; scale bar = 100 µm; P13957 Btop #11. 34F. Fruiting body contents in 34C showing thick hypha (arrowhead) bearing bases of reproductive hyphae; scale bar = 20 µm; P13957 Btop #9. 34G. Bases of reproductive hypae; arrowheads indicate septae; scale bar = 10 µm; P13957 Btop #9. 34H. Ostiolar neck in transverse section showing xix darkened cells (arrowheads) of leaf base surrounding ostiolar neck; scale bar = 50 µm; P13957 Btop #140. 34I. Fruiting body in midtransverse section showing single-layered wall, disorganized fungal contents, and few intracellular hyphae (arrowheads) external to fruiting body wall; scale bar = 100 µm; P13957 Btop #138. 34J. Fruiting body wall in section composed of single layer of hyphae (arrowhead); scale bar = 20 µm; P13957 Btop #138. 34K. Transverse section near base of fruiting body showing bases of reproductive structures (arrowheads); scale bar = 20 µm; P13957 Btop #9...... 123

Figure 35. Interfoliar stromata on Tricosta plicata gen. et sp. nov. 35A. Transverse section showing interfoliar stroma with at least five cavities (asterisks); arrowheads indicate previous stroma attachment to leaf surface at right; scale bar = 50 µm; P13616 Etop #15b. 35B. Fungal stroma bearing three cavities (arrowhead indicates smallest cavity); stromatic tissue at top left (thin arrowhead) envelopes darkened leaf margin (thick arrowhead at left); scale bar = 50 µm; P13616 Etop #15b. 35C. Few incompletely preserved cavities at center (in oblique sections) within stroma attached to surfaces of leaves (arrowheads); scale bar = 50 µm; P13616 Etop #19b. 35D. Stroma attached to leaf with single cavity; arrowheads indicate fungal contents; scale bar = 50 µm; P13616 Etop #19b. 35E. Stromatic cavity showing bases of reproductive structures (thin arrowheads); W = cavity wall; scale bar = 30 µm; P13616 Etop #15b...... 126

xx 1

INTRODUCTION

The aim of this thesis is two-fold: 1) to provide detailed descriptions and

taxonomic assessments of two novel Early Cretaceous mosses, and 2) to characterize

in detail fossil fungi associated with one of the novel mosses described here. While

the mosses are represented by numerous anatomically and three-dimensionally

preserved gametophyte specimens, the fungi are represented by several distinct

types, these found in either highly specific positions (e.g., shoot tips or within

individual leaf cells) or scattered throughout the gametophyte shoots. The fossil

record of mosses in the pre-Cenozoic (>66 Ma) is represented by less than 100

described species and among these, anatomical preservation is extremely rare (e.g.,

Oostendorp, 1987; Ignatov, 1990; Taylor et al., 2009; see “Fossil Record of Pre-

Cenozoic Mosses”) making the mosses described here a welcome addition the fossil record. And to the best of my knowledge, this is the first account of bryophyte- inhabiting fungi (bryophilous fungi) in the fossil record.

The vegetative moss gametophyte is composed of an axis (referred to as a

“stem”), multicellular rhizoids, simple and sessile leaves with a blade-like lamina, and occasionally additional structures along the stem (paraphyllia) or around branch primordia (pseudoparaphyllia) (Goffinet et al., 2009). Leaves of extant mosses are typically unistratose and bear a single leaf vein (a.k.a. costa or nerve), a divided leaf vein, or no nerve at all (= ecostate; the term ecostate may also be used when the leaf vein is insignificant in length; Goffinet et al., 2009) (see “The tricostate condition”).

2 Two new fossil mosses are described here in which the leaves bear three well-

developed costae, the costae arising separately at the leaf base (= the tricostate

condition), a trait unknown in extant mosses. However, three other mosses

described as having tricostate leaves are known from the Mesozoic fossil record, and

one of these is coeval with the mosses described here (Krassilov, 1973; Ignatov and

Shcherbakov, 2011a, b) (see “Tricostate mosses in the fossil record”).

Specimens used in this study come from the Early Cretaceous

(Valanginian—136 Ma) Apple Bay flora of Vancouver Island (British Columbia,

Canada) (e.g., Stockey and Rothwell, 2009). The fossils are found in fist-sized carbonate concretions and preserved by cellular permineralization (e.g., Stockey and

Rothwell, 2009; also see “Materials and Methods”) wherein cell walls are encased in dissolved minerals (e.g., calcium carbonate), and cell wall organic matter is rearranged at the sub-cellular level preserving anatomy of the plant tissues (e.g.,

Taylor et al., 2009), potentially throughout the entire plant. Material from this locality is largely fragmentary (with numerous plant groups represented; e.g.,

Stockey and Rothwell, 2009; also see “Materials and Methods”), but the small gametophyte plants of bryophytes such as mosses are the perfect size to allow for whole or nearly whole plants to be preserved within the concretions. Other tricostate mosses previously described from the fossil record are known as leaves or shoot fragments from Eastern Asia and preserved as compressions (Krassilov, 1973;

Ignatov and Shcherbakov, 2011a, b), a type of preservation lending much less anatomical detail for taxonomic comparisons (see “Taxonomic placement of

3 Tricosta plicata gen. et sp. nov.” and “Taxonomic placement of Krassiloviella

limbelloides gen. et sp. nov.”). However, gross morphology such as branching

patterns and leaf shapes may be more readily apparent in compressions than in

permineralized material (Taylor et al., 2009).

The gametophytes of T. plicata and K. limbelloides have been reconstructed as

part of this thesis. These reconstructions along with detailed descriptions of their

anatomy and morphology (based on numerous specimens) are used to make taxonomic

comparisons with extinct and extant mosses. Comparisons reveal that the two mosses are

species new to science, each representing a new genus and species (viz, Tricosta plicata gen. et sp. nov. and Krassiloviella limbelloides gen. et sp. nov.), and both belong to a new family (Tricostaceae fam. nov.) within the pleurocarpous mosses (or pleurocarps), a very large and diverse group of mosses well represented in extant bryofloras (ca. 5300-6600 species; O’Brien, 2007). The names of newly described taxa—Tricostaceae, Tricosta plicata, and Krassiloviella limbelloides—will be formalized in future publications.

The bryophilous fungi described here (associated exclusively with the gametophytes of Tricosta plicata; see “Bryosymbiotic Fungi”) are represented by sterile

(e.g., hyphae and infection structures) and fertile structures (viz, perithecioid fruiting bodies). In addition to detailed descriptions of their morphologies, broad taxonomic comparisons are made with extant bryophilous fungi; these comparisons along with a greater understanding of their infection and reproductive structures may allow more meaningful taxonomic assessments in future studies.

4 TRICOSTA PLICATA GEN. ET SP. NOV.

Fossil Record of Pre-Cenozoic Mosses

Compared to an estimated 13,000 extant moss species (Goffinet et al., 2009), the pre-Cenozoic moss fossil record with only ca. 70 described species (e.g.,

Oostendorp, 1987; Ignatov, 1990; Taylor et al., 2009) represents only a small fraction of known moss diversity. In contrast, Cenozoic fossil mosses are more numerous, many representing modern orders, families, and genera with few referable to extant species (e.g., Miller, 1984; Taylor et al., 2009). Although the placement of pre-Cenozoic fossil mosses into modern groups is less common and more tentative, some of these fossils resemble modern groups, demonstrating exquisite preservation and their potential utility in moss systematics.

The oldest unequivocal moss fossils are from the Carboniferous (Middle

Mississippian, late Visean) of eastern Germany (Hübers and Kerp, 2012). These fossils consist of leaf fragments with cuticular preservation, some resembling the extinct Protosphagnales (Oostendorp 1987; Ignatov, 1990) and other fragments with unresolved affinities. From the Permian of Antarctica, Merceria augustica Smoot et

Taylor (1986) most closely resembles extant Bryidae. Several mosses preserved as compressions have been reported from the Permian of Russia (Oostendorp, 1987;

Ignatov, 1990). Some of these have leaves with dimorphic cells similar to those of

Sphagnum L., but they also display costae, a character not seen in extant Sphagnales.

5 The order Protosphagnales Neuburg was erected to accommodate these extinct

Sphagnum-like mosses. Many other mosses from the same collections are best

attributed to the Bryales sensu lato (Oostendorp, 1987). De Souza et al. (2012)

described two mosses preserved as compressions in the Permian (Guadalupian) of

Brazil; one of these, Capimirinus riopretensis De Souza, Branco, et Vargas shows sparse branching and a single putative sporophyte attached to a short lateral shoot.

Several compressed early Mesozoic fossil mosses described by Krassilov

(1973) and Ignatov and Shcherbakov (2011a, b) display well-preserved leaf areolation and some show branching patterns. Among them, the genus Tricostium

Krassilov is represented by three species known from the Lower Triassic to Middle-

Upper Jurassic of Russia and Mongolia. These fossils have leaves with three darkened striations interpreted as costae which extend from the base well into the leaf tip. Reissinger (1950) described fossils resembling extant species of Sphagnum from the Early Jurassic (Oostendorp, 1987). Two mosses with highly branched gametophytes and lateral bud-like structures, which have been interpreted as gametangial shoots, are described as Baigulia Ignatov, Karasev, et Sinitsa and

Bryokhutuliinia ingodensis Ignatov from the Upper Jurassic of Russia (Ignatov et al.

2011). The combination of highly branched gametophytes and lateral bud-like structures suggest those fossils may be pleurocarpous mosses.

Moss diversisty from the Cretaceous consists of at least seven genera:

Muscites Brongniart, Yorekiella Krassilov, Bryokhutuliinia Ignatov, Tricostium,

Vetiplanaxis N.E. Bell, Campylopodium Bescherelle, and Eopolytrichum Konopka,

6 Herendeen, Merrill, et Crane. Throughout the Cretaceous several species preserved

as compressions from Russia (Ignatov and Shcherbakov, 2011a), Mongolia

(Krassilov, 1982), Germany (Ettingshausen and Debey, 1859), and Tennessee, USA

(Berry, 1928) have been described as species of the morphogenus Muscites. More

complete preservation is seen in Lower Cretaceous compression fossils from Russia:

Yorekiella pusilla Krassilov (Aptian-Barremian) (Krassilov, 1973), Bryokhutuliinia obtusifolia Ignatov et Shcherbakov and Tricostium longifolium Ignatov et

Shcherbakov (Ignatov and Shcherbakov, 2011a). Species of Vetiplanaxis, a Middle

Cretaceous (late Albian) genus comprising four species preserved in Burmese

amber, are most comparable to the Hypnodendrales (Hedenäs et al., 2014).

Exceptional charcoalified moss gametophytes and sporophytes have been described

by Konopka et al. (1997; 1998) from the Late Cretaceous (late Santonian) of

Georgia, USA; these fossils, described as Campylopodium allonense Konopka,

Herendeen et Crane and Eopolytrichum antiquum Konopka, Herendeen, Merrill, et

Crane, are assigned to the families Dicranaceae and Polytrichaceae, respectively.

Most of the moss fossil record is represented by compression fossils. In pre-

Cenozoic moss fossils, anatomical preservation is very rare. Prior to this study,

anatomical preservation includes cuticular preservation of the Mississippian moss

leaves reported by Hübers and Kerp (2012), charcoalification of the Late Cretaceous

material described by Konopka et al. (1997; 1998), permineralization in the Permian

moss, Merceria Smoot et Taylor (Smoot and Taylor, 1986), and the preservation of

Vetiplanaxis in Burmese amber from the Middle Cretaceous (Hedenäs et al., 2014).

7 Exquisitely preserved plant remains are present in carbonate marine concretions from Jurassic, Cretaceous, Paleogene and Neogene sediments worldwide (e.g., Stockey

and Rothwell, 2006), many of which contain remains of anatomically preserved

bryophytes (e.g., Steenbock et al., 2011; Tomescu et al., 2012). Here I describe an

anatomically preserved Early Cretaceous moss based on abundant permineralized

specimens from the Apple Bay locality (Vancouver Island, British Columbia, Canada).

This moss is described as a new genus and species characterized by highly branched

gametophytes with numerous perigonia on short lateral branches and tricostate leaves, a

trait not recognized in extant mosses and documented only in a few Mesozoic fossils. It

is one of the most complete fossil mosses yet described, represents the earliest

unequivocal record for pleurocarpy, and defines a new family within Superorder

Hypnanae. Along with other tricostate mosses (fossil genus Tricostium), this moss brings

to light a long extinct aspect of moss morphological diversity.

Materials and Methods

Numerous moss gametophyte shoots are preserved by cellular

permineralization in at least 23 carbonate concretions from the Apple Bay locality.

The mosses are part of an allochthonous fossil assemblage deposited in nearshore

marine sediments (e.g., Stockey and Rothwell, 2009). The concretions are collected

from sandstone (greywacke) beds exposed on the northern shore of Apple Bay,

Quatsino Sound, on the west side of Vancouver Island, British Columbia, Canada

(50°36’21” N, 127°39’25” W; UTM 9U WG 951068) (e.g., Stockey and Rothwell,

8 2009). The concretion-containing layers are regarded as Longarm Formation

equivalents and have been dated by oxygen isotope analyses to the Valanginian

(Early Cretaceous, ca. 136 Ma; D. Gröcke pers. comm., 2013).

This Early Cretaceous flora includes lycophytes, equisetophytes, at least 10

families of ferns (Smith et al., 2003; Hernandez-Castillo et al., 2006; Little et al.,

2006a, b; Rothwell and Stockey, 2006; Stockey et al., 2006; Vavrek et al., 2006;

Rothwell et al., 2014) and numerous gymnosperms (Stockey and Wiebe, 2008;

Stockey and Rothwell, 2009; Klymiuk and Stockey, 2012; Klymiuk et al., 2015;

Rothwell and Stockey, 2013; Rothwell et al., 2014; Ray et al., 2014), fungi (Smith et

al., 2004; Bronson et al., 2013) and a lichen with heteromerous thallus organization

(Matsunaga et al., 2013). This flora is emerging as the most diverse assemblage of fossil bryophytes known worldwide with leafy and thalloid liverworts and more than

20 distinct moss morphotypes currently recognized (Tomescu et al., 2012). The mosses represent pleurocarpous, polytrichaceous, and leucobryaceous types, as well as several morphotypes of unresolved affinities including at least three distinct tricostate types.

Fossil-containing concretions are sliced into slabs and sectioned using the cellulose acetate peel technique (Joy et al., 1956). Slides are prepared using Eukitt, xylene-soluble mounting medium (O. Kindler GmbH, Freiburg, Germany).

Micrographs are taken using a Nikon Coolpix E8800 digital camera on a Nikon

Eclipse E400 compound microscope. Images are processed using Photoshop

(Adobe, San Jose, California, USA).

9 The moss described here is found in numerous slabs: P13029 D; P13032 F;

P13131 D; P13171 E; P13172 G; P13174 C; P13175 E; P13218 F; P13256 C; P13311 I;

P13308 J; P13483 C; P13616 E; P13957 A, B,C; P14560 B; P15393 B; P15422 A , B;

P15425 C; P15800 C; P16435 C; P17515 B. Specimens used for this study come from:

P13029 D; P13131 Dtop; P13171 Etop; P13174 Cbot; P13256 Cbot; P13308 Jtop;

P13616 Etop; P13957 A, B, C; P15422 A; P15425 Cbot; P16435 Ctop. All specimens

and preparations are housed in the University of Alberta Paleobotanical Collections

(UAPC-ALTA), Edmonton, Alberta, Canada.

Systematics

Class—Bryopsida Rothm.

Subclass—Bryidae Engl.

Superorder—Hypnanae W.R. Buck, Goffinet and A.J. Shaw

Order—Incertae sedis

Family—Tricostaceae fam. nov.

Familial diagnosis—Gametophyte plants pleurocarpous. Stems regularly to irregularly pinnately branched, central conducting strand absent. Cortical cells thin- walled, hyalodermis or thick-walled outer cortex lacking. Paraphyllia absent.

Leaves helically arranged with three costae and conspicuous alar regions. Laminal cells isodiametric to elongate, not linear. One to few gametangia borne on lateral specialized shoots (perigonia, perichaetia).

Type genus—Tricosta gen. nov.

10 Generic diagnosis—Gametophytes much-branched. Leaves isophyllous, imbricate and densely covering stems. Branch primordia arising one or very few cells above subtending leaf. Multicellular rhizoids smooth. Leaves tricostate with costae symmetrically arranged, arising separately in leaf base and homogeneous in transverse section. Alar regions small. Laminal cells elongate, becoming isodiametric distally; laminal cells smooth, thin-walled, with rhombic, oval, repand or isodiametric shapes. Perigonia sessile on lateral branches, with single antheridia.

Perigonial leaves like vegetative leaves but smaller. Perichaetia sessile, lateral along main stems, with few archegonia. Perichaetial leaves different from vegetative leaves.

Etymology—Tricosta for the tricostate leaves.

Type species—Tricosta plicata sp. nov.

Specific Diagnosis—Gametophytes in spreading tufts at least 20 mm high, main stems once-pinnate, roughly complanate. Branches inserted at 40-70º angles and

0.1-1.1 mm intervals. Stem diameter 0.2 mm basally to 0.12 mm apically, 10-14 cells across, epidermal cells narrower than cortical cells; branch diameter smaller than stem diameter. Leaves dense, 10-20 leaves mm-1 along stem; 3/8 phyllotaxis.

Rhizoids at stem base only, ca. 24 µm diameter. Leaves straight, with 40-55º

divergence angle. Leaves ca. 2.0 mm long, 0.5 mm wide at base, up to 0.9 mm mid-

leaf. Leaves ovate, with entire margin and acute apex, strongly plicate throughout;

plications form adaxially concave longitudinal folds, associated with costae. Leaf

lamina ca. 18 cells wide between median and lateral costae, ca. 15 cells between

11 lateral costae and leaf margin. Costae strong (ca. 0.9 of leaf length), central costa

percurrent, up to 6-8 cells wide (each cell 6-9 µm diameter), composed of three

layers (1-2 layers distally). Abaxial cells of costa short (to isodiametric), becoming larger in diameter toward leaf apex; comparable to those of lamina in the leaf apex.

Median costae up to 55 µm wide, 30-40 µm thick; lateral costae 35 µm wide, 25-40

µm thick. Alar regions up to 9 cells wide, 5 cells tall; cells prominently inflated in

transverse sections (diameter up to 34 µm), globose to elongate (up to 54 µm) in

longitudinal sections. Lamina ca. 13-19 µm thick; laminal cells forming oblique

(and longitudinal) files in base and mid-leaf; distally laminal cells form longitudinal

files. Lamina cells at leaf base up to 5:1 (length:width ratio) and rectangular to

rhombic; mid-leaf cells 2-3:1 and up to 35 µm, rhombic, repand or oval; distally,

cells isodiametric, up to 23 µm diameter. Perigonia ca. 1 mm long overall.

Perigonial axes up to 200 µm long, 115 µm thick; axes bear ca. 4 erect or spreading leaves ca. 0.9 mm long, similar to vegetative leaves but with plications weak or absent on innermost leaves. Antheridia oblong, up to 350 µm long, 150 µm wide, borne on triseriate stalks. Antheridial jacket with narrow irregularly shaped cells.

Perichaetial axes with few straight, erect leaves inserted at steep angle. Perigonial leaf cells narrow (ca. 4.5:1 and 40 µm long), rectangular and rhombic in lower part of leaf. Archegonia at least 200 µm long.

Etymology—specific epithet plicata for the marked, characteristic plication of the leaves.

Holotype hic designatus—To be determined; UAPC-ALTA.

12 Paratypes—To be determined.

Locality— Apple Bay locality, Quatsino Sound, northern Vancouver Island, British

Columbia (lat. 50º36’21” N, long. 127º39’25” W; UTM 9U WG 951068).

Stratigraphic position and age—Longarm Formation equivalent; Valanginian, ca. 136

Ma (Early Cretaceous).

Description

Habit, branching, shoot architecture, and stem anatomy

Tricosta plicata is represented by more than 100 distinct gametophyte shoots.

The gametophytes are diminutive, solitary or tufted (one tuft measures ca. 22 mm in

height; Fig. 1). The most completely preserved individual shoot, whose detailed

branching architecture was reconstructed based on serial sections, is a 9 mm long

fertile fragment (Fig. 2D). The base of this shoot is characterized by more widely

spaced leaves and thicker stem, while the apical region bears more densely spaced

leaves on a narrower stem; the tip is incompletely preserved but flanked by perigonia

(Fig. 3). Branching is frequent, irregularly to regularly pinnate, and roughly

complanate (Fig. 3). Branches are inserted at intervals of 0.1-1.1 mm, and at angles

of 40-70º (Figs. 2A, 2D, 3). The basal 3.8 mm of the shoot bears no branches. Most

branches along the main stem are relatively short (up to 0.85 mm) and unbranched.

However, one lateral from the main stem generates a complex branching system originating perpendicular to the main stem with up to four orders of branching and

surpassing the main stem in length (Fig. 3).

15 Figure 2. Habit, branching, shoot architecture, and stem anatomy of Tricosta plicata gen. et sp. nov. 2A. Shoot in longitudinal section; narrow arrowheads show antheridia (upper arrowhead = sac; lower = stalk); all other arrowheads indicate perigonial branches; scale bar = 200 µm; P15425 C bot #38a. 2B. Stems in transverse sections showing radially arranged cortical cells; scale bar = 100 µm; P13957 A #2. 2C. Detail of 2B; note few, scattered, narrow cells near stem center; scale bar = 50 µm; P13957 A #2. 2D. Composite image of much-branched shoot in longitudinal section; arrowhead at far left shows vegetative branch; all other arrowheads represent positions of perigonial branches; scale bar = 500 µm; P15425 Cbot #56a. 2E. Stem in longitudinal section; scale bar = 100 µm; P13957 A #2. 2F. Detail of 2E showing fusiform cortical cells; scale bar = 50 µm; P13957 A #2.

17 Stem diameters range from 0.2 mm basally to 0.12 mm apically (with branch

diameters consistently smaller than stem diameters) and stems are ca. 10-14 cells

across (Figs. 2B, 2C). Transverse sections show an epidermis of 16-23 µm diameter

cells and a cortex composed of slightly larger (16-35 µm diameter) circular to

polygonal cells with evenly thickened walls (Figs. 2B, 2C). The stem (and branch)

center is occasionally occupied by one to a few narrow cells (5-12 µm in diameter)

but shows no clear organization into a central conducting strand (Fig. 2C).

Longitudinal sections show fusiform cortical cells 57-75 µm long and up to 18-23

µm wide (Figs. 2E, 2F). Epidermal cells in longitudinal sections are 35-60 µm long.

Vegetative shoot tips are incompletely preserved and show a wide range of variation in preservation. The tips are composed of either several large cells faint in color (Figs. 4A, 4B), or several small cells darker in color (Fig. 4C)—the different colors probably indicate different states of decomposition or whether the tips were actively dividing. Some of the shoots show leaf primordia (Fig. 4C) and branch primordia in longitudinal sections (Figs. 4D, 4E). Branch primordia occur in leaf axils, separated by at least one cell from their subtending leaf, and slightly sunken in the stem tissue. They are dome-shaped, up to 60 µm wide and 40 µm tall, and consist of several small cells. Each branch primordium is covered by at least one over-arching scale-like structure (Figs. 4D, 4E) but preservation precludes resolving the origin of these structures. Whether they are derived from the delicate

19 Figure 4. Shoot architecture and stem anatomy of Tricosta plicata gen. et sp. nov. 4A. Vegetative shoot tip in longitudinal section; scale bar = 200 µm; P15425 Cbot #55a. 4B. Detail of 4A showing a group of faintly colored apical cells; scale bar = 50 µm; P15425 Cbot #55a. 4C. Darkened shoot tip showing scale-like leaf primordial (arrowheads); scale bar = 50 µm. P16435 Ctop #15. 4D. Branch primordium in longitudinal section; subtending leaf distanced one or two cells from primordium; thick arrowhead indicates base of scale-like structure surrounding primordium; note dark hypha within costa (thin arrowhead); scale bar = 50 µm; P13957 Btop #25. 4E. Branch primordium in longitudinal section directly subtended by leaf; thick arrowhead indicates scale-like structure; subtending leaf base indicated by thin arrowhead; scale bar = 50 µm; P16435 Ctop #14. 4F. Shoot transverse section showing leaf central costa (thick arrowhead) and one lateral costa (thin arrowhead) attached below point of leaf divergence; scale bar = 50 µm; P13131 Dtop #12c. 4G. Serial section of Figure 4F just above point of leaf divergence (with central and lateral costa, arrowheads); scale bar = 50 µm; P13131 Dtop #13c. 4H. Transverse section of shoot just above branching point showing unistratose and strongly plicate leaves with three costae per leaf (abaxial surface of one leaf underlined; C = costa); note paradermal section of part of leaf base (alar cells, arrowhead); scale bar = 100 µm; P13131 Dtop #3c.

20 primordium tissue or the epidermis of the surrounding stem (i.e., either a “scale leaf”

or pseudoparaphyllium origin, respectively; sensu Newton and De Luna, 1999)

cannot be determined. The branch primordia, incompletely preserved in all

specimens, are bordered by a palisade of radially arranged cells with circular to

wedge-shaped cells (up to 10 x 24 µm) in longitudinal sections (Figs. 4D, 4E).

One specimen represents the base of a small tuft (i.e., several shoots

originating from few parent stems) that is covered in rhizoids (Fig. 5A). The rhizoids are dense with 17-30 µm diameters, and reach up to 700 µm from the stems.

Rhizoid bases have been observed in stem transverse sections. The rhizoids are multicellular with characteristic oblique end-walls (Figs. 5B, 5C) and branching was not observed.

Tricosta plicata is isophyllous and leaves are imbricate and densely covering the stems with ca. 9 leaves mm-1 in proximal regions of the shoots and up to 23

leaves mm-1 distally (e.g., Fig. 2A). Phyllotaxis is helical, following a 3/8

phyllotactic ratio. Longitudinal sections show erect leaves with divergence angles of

40-55º and wider angles where leaves subtend branches (Fig. 2A). Paraphyllia were not observed.

Leaf morphology and anatomy

In terms of overall shape, the leaves are symmetrical, ovate, have entire

margins, and are broadly attached to the stems (Figs. 6, 4F, 4G). The leaves are ca.

0.5 mm wide at the base, reaching a maximum width of 0.9 mm and length of 2.1

Figure 5. Rhizoids of Tricosta plicata gen. et sp. nov. 5A. Base of gametophyte tuft showing several stems in transverse section bearing rhizoids; scale bar = 200 µm; P13256 Cbot #19. 5B. Stem in transverse section (at left) surrounded by smooth- walled rhizoids; arrowheads indicate oblique end-walls within rhizoids; scale bar = 100 µm; P13256 Cbot #36. 5C. Smooth-walled rhizoids in transverse (thick arrowheads) and longitudinal sections (at left); thin arrowhead indicates oblique end-wall; scale bar = 50 µm; P13256 Cbot #34.

23 Figure 6. Tricosta plicata gen. et sp. nov. leaf model. 6A. Cell morphology; note thickened cell walls at mid-leaf (right side), representing cells whose walls bear superficial and intercellular fungal hyphae; tracings are from 9 different leaves (specimen numbers listed here from base to apex, and left to right): P13131 Dtop #4c; P13957 Btop #52; P15425 Cbot #47a; P15425 Cbot #49a; P15425 Cbot #49a; P15425 Cbot #47a; P15425 Cbot #14a; P15425 Cbot #54a; P3957 Btop #157. 6B. Series of leaf tracings (right) in transverse section from base to apex demonstrate strong plication throughout; photos (below, with leaves highlighted) correspond to serial tracings; leaf sections from 7 different leaves; specimen numbers correspond to serial tracings from base to apex: P13029 D #21; P 13029 D #21; P15425 Cbot #26a; P15425 Cbot 22a; P13131 Dtop #3c; P15425 Cbot #16a; P15425 Cbot #11a; scale bar = 200 µm (A and B).

24 mm. Incomplete preservation of leaf tips permits only close approximation of total

leaf length. Leaf apices are acute (Figs. 8K, 8L).

Leaves are unistratose, strongly plicate, and tricostate (Figs. 4H, 4F, 4G, 6,

7A, 7B). The leaf margins are unistratose and gently recurved (curved abaxially)

throughout (Fig. 6). Plication forms three adaxially concave longitudinal folds, each

associated with a costa. The median fold and costa extend from the leaf base into

the apex (i.e., percurrent), whereas the two lateral folds and costae are shorter,

extending from the leaf base to somewhat below the apex (i.e., “failing”; Figs. 8K,

8L). At the widest point, the lamina is ca. 18 cells wide between the median and

lateral costae and ca. 15 cells between the lateral costae and leaf margin (Fig. 6).

Median costae end apically within 4-5 cells from the leaf margin while lateral costae end 3-4 cells from the margin (Fig. 6). The three costae of a leaf originate separately and slightly below the level of leaf divergence (Figs. 4F, 4G).

Median costae are ca. 55 µm wide and 27-42 µm thick in the basal half of the leaf, while lateral costae are ca. 35 µm wide and 25-42 µm thick (Figs. 4H, 6, 7A,

7B). Costae are tristratose at the base, becoming bistratose in the upper half of the leaf and, in transverse sections, they typically protrude more abaxially than adaxially, especially in distal sections. Costae consist of cylindrical elongate cells which form three layers: adaxial, median and abaxial (Figs. 7C-7E). In paradermal and longitudinal sections, costal cells are 40-138 µm long with one or both ends tapered (Figs. 7F, 7G). Adaxial and median costa layers are up to six cells wide

26 Figure 7. Leaf anatomy of Tricosta plicata gen. et sp. nov. 7A. Shoot transverse section showing tricostate leaves with strong plications (two leaves highlighted); arrowhead indicates cells of alar region; scale bar = 50 µm; P15422 A #1. 7B. Perigonial shoot in transverse section (center indicated by asterisk); innermost leaves (ca. 4) perigonial, other leaves vegetative; note protruding bistratose abaxial costa-layer in distal leaf sections (thin arrowheads); thick arrowhead indicates bundle of epiphyllous fungal hyphae; scale bar = 100 µm; P15425 Cbot #16a. 7C. Leaf transverse section showing three-layered median costa (ab-, adaxial surfaces traced; arrowheads = costal layers); scale bar = 30 µm; P15422 A #1. 7D. Leaf transverse section showing three-layered central costa (below) and bi-layered lateral costa (above); scale bar = 50 µm; P15422 A #1. 7E. Leaf transverse section showing three-layered median costa (below); note alar region (above) and adjacent lateral costa (arrowhead); scale bar = 50 µm; P15422 A #1. 7F. Costa in longitudinal section showing linear cells with tapered (arrowhead) or transverse end-walls; scale bar = 50 µm; P16435 Ctop #13. 7G. Costa in paradermal section showing linear cells with tapered or transverse end-walls, and juxtacostal cells (arrowheads); scale bar = 100 µm; P15425 Cbot #54a. 7H. Two leaf bases in longitudinal section showing few cells (arrowheads) of two different alar regions; scale bar = 50 µm; P13957 Btop #52. 7I. Alar region in transverse section; arrowhead indicates adjacent lateral costa; note stem center occupied by hyphae (section is just below perithecioid fruiting body); scale bar = 50 µm; P13957 Btop #25.

28 Figure 8. Leaf anatomy of Tricosta plicata gen. et sp. nov. 8A. Shoot in transverse section showing inflated alar cells of (shaded) clasping leaf base (arrowhead = central costa); scale bar = 100 µm; P13131 Dtop #6c. 8B. Densely foliated shoot in longitudinal section (leaf bases at left, stem at right); arrowhead indicates inflated alar cell; scale bar = 50 µm; P16435 Ctop #10. 8C. Cells of leaf base in paradermal section (center) and few alar cells in section (thick arrowhead); thin arrowhead = lateral costa; scale bar = 50 µm; P13957 Btop #52. 8D. Alar region in paradermal section; scale bar = 50 µm; P15425 C bot #44a. 8E. Leaf cells in paradermal section showing thin walls indicative of the absence of fungal hyphae or taphonomic alterations; scale bar = 30 µm; P15425 Cbot #49a. 8F. Leaves in paradermal section showing laminal cell shapes near leaf base (lower left) and in lower half of leaf (right); scale bar = 100 µm; P15425 Cbot #47a. 8G. Laminal cell shapes in distal half of leaf; scale bar = 50 µm; P15425 Cbot #49a. 8H. Leaves in paradermal sections showing cell shapes of mid-leaf; scale bar = 100 µm; P15425 Cbot #49a. 8I. Leaf paradermal section showing cell shapes at mid-leaf; note fungi (arrowheads); scale bar = 100 µm; P15425 Cbot #48a. 8J. Paradermal section showing leaf cell shapes at mid-leaf; scale bar = 100 µm; P15425 Cbot #48a. 8K. Leaf apex in paradermal section; note leaf margins not shown; scale bar = 50 µm; P13957 Btop #157. 8L. Detail of 8K showing laminal cell shapes and median costa (upper arrowhead shows linear cells of adaxial costal layer; lower arrowhead shows short cells of abaxial costal layer); scale bar = 50 µm; P13957Btop #157.

29 basally, becoming one to two cells wide apically with cells 8-16 µm in diameter.

The abaxial layer is up to eight cells wide basally (cells 6-9 µm in diameter) and just

one or two cells wide distally (cells up to 23 µm in diameter; Fig. 7C).

Distinct but relatively small alar regions are present at the leaf base corners

(Figs. 4H, 6, 7A, 7H, 7I, 8A-8D). They are up to 9 cells wide and five cells tall with

cells prominently inflated in transverse sections (widths ca. 15-34 µm; e.g., Figs. 7I,

8A) and globose to elongate (lengths up to 54 µm) in paradermal and longitudinal

sections (e.g., Figs. 8B, 8D). Laminal cells (Figs. 6, 8E-8L) are 13-19 µm thick throughout and in paradermal sections towards the leaf base, they are up to 5:1 and

62 µm long with elongate and rectangular to rhombic shapes. In the mid-leaf, cells are ca. 2-3:1 and ca. 25 µm long (up to 35 µm) with mostly rhombic or oval shapes.

In the distal half of the leaf, cells become isodiametric with 10-23 µm diameters.

Laminal cells adjacent to the costae are comparable in size to neighboring laminal cells and have various, typically elongate shapes: rhombic, repand, rectangular, and isodiametric (Figs. 6, 7G). Throughout the basal half of the leaf, laminal cells typically form oblique files; whereas, longitudinal files (of isodiametric cells) are typical in the distal half. Walls of laminal cells are smooth and thin (ca. 1.0 µm thick; Fig. 8E).

Specialized branches

At least two specimens preserve perigonia. One of these is an extensively

branched gametophyte with diminutive perigonial shoots borne apically or laterally

on nearly all branches (Figs. 3, 2A, 9A). Perigonial axes are 115-200 µm long, 95-

30 115 µm thick, and bear ca. 4 leaves (Figs. 9B-9E). The perigonial leaves are erect or

spreading and anatomically similar to cauline leaves except for smaller size (e.g.,

lengths ca. 0.9 mm), weaker plications, and weaker costae of the innermost

perigonial leaves (Figs. 9B, 9H). Furthermore, perigonial leaf apices are acute with

rounded tips. All perigonial axes bear one antheridium at their tip. The antheridia

are oblong (up to 350 µm long and 150 µm wide; Fig. 9E), and borne on triseriate

stalks (145-150 µm long and 44-50 µm thick; Figs. 9D-9G). The stalks consist of short squat cells forming ca. 10-14 tiers. Stalk cells are 16-23 µm wide and 7-23 µm long in longitudinal sections, generally smaller at the stalk base and becoming larger distally. Antheridial jackets in paradermal sections consist of narrow (7-8 µm) irregularly shaped cells (Fig. 9I). Paraphyses and sperm cells were not observed.

At least three gametophytes bear putative perichaetia (Figs. 10A-10I). These specialized branches are borne laterally along main stems which occur near the periphery of an extensively branched tuft of gametophytes. The numerous other shoot tips of the tuft are vegetative, incompletely preserved, or occupied by perithecioid fungal fruiting bodies (see “Perithecioid fruiting bodies”). The perichaetia are composed of a bud-like axis with few densely arranged, straight, and erect leaves that are crowded from the leaf bases to near the apices (apices not preserved; Figs. 10A-10E). The leaves are composed of narrow cells (up to 4.5:1 and 40 µm long ca. mid-leaf) with rectangular, rhombic, or linear shapes throughout the lower half of the leaf (Figs. 10D-10F, 10H). The branch tips are conic (Fig. 10D) or narrowly dome-shaped (Fig. 10I) and bear a single narrow

32 Figure 9. Perigonia of Tricosta plicata gen. et sp. nov. 9A. Several perigonia in transverse sections (e.g., asterisks), some of these showing antheridia (arrowheads); scale bar = 200 µm; P15425 Cbot #13a. 9B. Perigonial shoot in transverse section showing antheridium at center and innermost perigonial leaves (e.g., arrowheads) with weak costae and plication; scale bar = 100 µm; P15425 Cbot #25a. 9C. Perigonium in longitudinal section showing well-preserved swollen axis (thick arrowhead) bearing several leaf bases and incompletely preserved antheridial stalk (thin arrowhead; note base of sac attached to stalk); scale bar = 100 µm; P15425 Cbot #47a. 9D. Perigonium in longitudinal section showing incompletely preserved swollen axis (thick arrowhead) and well-preserved antheridial stalk (thin arrowhead; note base of sac attached to stalk); scale bar = 100 µm; P15425 Cbot #38a. 9E. Perigonium in longitudinal section showing incompletely preserved axis (thick arrowhead), antheridial stalk (thin arroewhead), and oblong antheridial sac attached to stalk; scale bar = 100 µm; P15425 Cbot #36a. 9F. Antheridial stalk in longitudinal section showing triseriate and tiered organization; arrowhead indicates base of antheridial sac; scale bar = 50 µm; P15425 Cbot #38a. 9G. Antheridial stalk in transverse section showing triseriate organization; scale bar = 50 µm; P15425 Cbot #28a. 9H. Perigonium in transverse section showing convoluted antheridial jacket and innermost perigonial leaf; scale bar = 50 µm; P15425 Cbot #38. 9I. Antheridial jacket in longitudinal section showing irregular and narrow cell shapes (e.g., inset; scale bar = 10 µm); scale bar = 50 µm; P15425 Cbot #38.

34 Figure 10. Probable perichaetia of Tricosta plicata gen. et sp. nov. 10A. Stem (dashed line) and specialized lateral branch (?perichaetium; arrowhead) in longitudinal section; scale bar = 300 µm; P13957 Btop #129. 10B. Stem and specialized lateral branch (arrowhead) in longitudinal section; scale bar = 300 µm; P13957 Btop #131. 10C. Stem and specialized lateral branch (arrowhead) in longitudinal section; scale bar = 300 µm; P13957 Btop #132. 10D. Detail of 10C showing incompletely preserved perichaetium axis (bottom), erect leaves and elongate leaf cells; arrowhead indicates putative archegonium; scale bar = 100 µm; P13957 Btop #132. 10E. Detail of 10B showing swollen axis (thick arrowhead) and erect leaves with elongate cells; apex bears delicate elongate structure (archegonium; thin arrowhead); scale bar = 100 µm; P13957 Btop #131. 10F. Detail of 10E showing archegonium in longitudinal section; note elongate laminal cells at right; scale bar = 50 µm; P13957 Btop #131. 10G. Detail of 10D showing archegonial venter and incompletely preserved base (arrowhead) in longitudinal section; note absence of tiered, triseriate stalk; scale bar = 50 µm; P13957 Btop #132. 10H. Oblique section of archegonium above level of shoot tip (not shown) showing narrow neck canal at center (arrowheads), single layer of neck cells (n) and few layers of delicate venter cells (v); scale bar = 20 µm; P13957 Btop #121. 10I. Longitudinal section of specialized shoot showing delicate tissue attached directly to apex; scale bar = 50 µm; P13957 Btop #89.

35 putative archegonium (at least 200 µm long) composed of a delicate pale-colored tissue.

The body of these delicate structures is probably multistratose, as the delicate tissue is at least a few cells across in serial sections (Figs. 10F, 10G). The archegonia lack a distinct stalk (e.g., Fig. 10G) and in one instance, the delicate tissue attaches directly to the shoot tip (Fig. 10I). Another specimen shows an archegonium in oblique-longitudinal section

(approximately 100-200 µm above the shoot tip). The archegonium is in section at the junction between venter and neck (i.e., showing a well-defined canal at center surrounded by a single layer of neck cells and few layers of extremely delicate venter cells; Fig.

10H).

Discussion

The tricostate condition

The costa (also termed midrib or nerve) is a multistratose region of the moss

leaf forming a longitudinal band anatomically different from the rest of the lamina

and playing a role in the conduction of water and photosynthates. Most moss leaves

bear a single costa that varies greatly in anatomy and length among taxa (Goffinet et

al., 2009). The condition in which a costa is divided at the base or along its length

(e.g., Goffinet et al., 2009) is treated as a single “forked” costa, which makes sense

from a developmental standpoint. Whereas, mosses that lack costae (or have costae

of an insignificant length; ecostate) are found among diverse lineages (e.g.,

Sphagnum L., Buxbaumia Hedw., Erpodium Brid., Pleurophascum Lindb., and

Hedwigia Beauv.). Mosses bearing multiple costae per leaf (pluricostate or

36 multicostate) are found typically among pleurocarpous taxa (e.g., Thamniopsis M.

Fleisch., Antitrichia Brid., and Neckera Hedw.; e.g., Goffinet et al., 2009). Extant

pluricostate mosses typically bear two short costae per leaf while instances of two

strong costae (e.g., some Hookerales) or more than three costae are rare (e.g.,

Antitrichia, which features a median costa and a variable number of shorter accessory costae; e.g., Lawton, 1971). None of these pluricostate conditions

conforms to the tricostate condition of Tricosta plicata. In this context, the tricostate

condition (in which three strong costae originate independently at the leaf base and

extend well beyond the midleaf) present in both T. plicata and the Mesozoic genus

Tricostium, clearly sets these species apart from all other known living and extinct

mosses.

Tricostate analogues in extant mosses

Although no mosses with three strong costae are recognized in modern

bryofloras, a few extant mosses exhibit multilayered bands of cells additional to the

central costa that are comparable to the tricostate condition in Tricosta plicata. Two

types of features present in some mosses can be morphologically similar to lateral

costae: (1) multistratose longitudinal thickenings (or multistratose “streaks”)

composed of cells more or less similar to those of the lamina, and (2) multistratose

intramarginal limbidia (intramarginal borders or teniolae) which are bands of cells

running parallel and few cells internal to the leaf margin.

Examples of multistratose thickenings similar to costae are seen in

Coscinodon arctolimnius Steere and C. cribrosus Spruce (Grimmiaceae). Leaves of

37 Coscinodon Spreng. bear a central costa and two lateral multistratose thickenings

that run along leaf plications (Hastings and Deguchi, 1997). These multistratose

thickenings consist of cells anatomically similar to the those of the costa (in the basal half of the leaf of C. cribrosus and throughout the leaf in C. arctolimnius ssp. higuchii Hastings et Deguchi). While the multistratose thickenings of Coscinodon are comparable to costae in featuring elongated cells, costae and multistratose thickenings are probably developmentally different as suggested by: (1) cells in the streaks shorter than those of the central costa; (2) irregular width, thickness, and position of the streaks on the leaf; and (3) an absence of cell differentiation in the streaks that is seen in the costa (viz, stereids are present in the costae and not in the streaks).

Other structures comparable to lateral costae are multistratose intramarginal limbidia. Examples are seen in Calymperes Sw., Teniolophora W.D. Reese, and

Limbella Müll. Hal (e.g., Gradstein et al., 2001). In Calymperes and Teniolophora, the cross-sectional anatomy of their limbidia is more simple than that of their costae, suggesting a laminal rather than a costal origin. In Limbella tricostata Bartr.

(=Sciaromium tricostatum Mitt.) the intramarginal limbidia have cross-sectional anatomy similar to that of the costa (e.g., Lawton, 1971). Although the intramarginal limbidia of Limbella are positioned only one to two cells away from the leaf margin (e.g., Lawton, 1971), among extant mosses, these structures are most similar to the lateral costae of Tricosta.

38 Studies of leaf development in mosses are rare (e.g., Frey, 1970), and none

has addressed the homology of multistratose structures of the lamina. Ttherefore, I

can only base my comparisons on anatomy. Overall, multilayered structures of the

lamina known in extant mosses that approach the tricostate condition are

anatomically different from, and probably not homologous to costae, as suggested

above. While the intramarginal limbidia of Limbella are most similar to the lateral

costae of Tricosta, the differences between these two genera are numerous (e.g.,

stem branching, laminal cell areolation, costal anatomy). Furthermore, while it is

obvious that Tricosta has a leaf margin 10-15 cells wide, it is not clear whether the

cells external to the limbidium of Limbella represent a very narrow leaf margin (1-2

cells wide) or are part of the limbidium (e.g., Lawton, 1971). Together, these show

that extant moss diversity does not include any structures equivalent to the lateral

costae of Tricosta.

Tricostate mosses in the fossil record

Moss gametophytes with tricostate leaves have been previously reported only

from Mesozoic (Triassic to Early Cretaceous) rocks in Russia and Mongolia (and

potentially extending from the Permian; Ignatov and Shcherbakov, 2011b), where

they are preserved as compressions (Krassilov, 1973; Ignatov and Shcherbakov,

2011a, b). Such mosses have been assigned to the genus Tricostium Krassilov with

three species: Tricostium triassicum Ignatov et Shcherbakov, T. papillosum

Krassilov and T. longifolium Ignatov et Shcherbakov. The genus Tricostium is diagnosed as having flat, imbricate, and unistratose leaves with three costae

39 (Krassilov, 1973). The diagnosis is based on the type species, T. papillosum

collected from Middle-Upper Jurassic (Callovian-Oxfordian) rocks, near the mouth

of the Umalta River, in eastern Russia. The collection consists of a few shoot

fragments up to three millimeters long and several detached leaves and leaf

fragments (Krassilov, 1973). Details of branching architecture are unknown, except

for a single specimen bearing a branch that diverges at a 45º angle. Leaves are ovate

with clasping bases, ca. 1.8 mm long, 0.8-1.0 mm wide, and have acute to obtuse

tips. Laminal cells are isodiametric (15-18 µm) to slightly elongate and with

granulose faces. The cell faces have been interpreted as papillate with 8-10 papillae

per cell. Median costae are ca. 50 µm wide and percurrent; lateral costae are

narrower, extend ca. 0.67 to 0.9 of the leaf length, and arise independently from the

median costa (Krassilov, 1973).

Tricostium triassicum (Upper Permian?—Lower Triassic of Mongolia) is

represented by a single leaf, nearly complete except for the apex and basal-most region (Ignatov and Shcherbakov, 2011b). The leaf shows a conspicuous darkened midrib (ca. 80 µm wide) which extends the entire leaf length (ca. 4.5 mm). Narrow, dark lateral striations are seen running nearly the entire length, more or less parallel to the margin. It is unclear whether these lateral striations are costae, plications or taphonomic artifacts, especially given that only one leaf of this type is known. For instance, there are multiple lateral striations per-side of the leaf, lateral striations are asymmetric (i.e., occurring at different distances from the leaf margin), and similar and discontinuous striations are seen in the base of the specimen oriented obliquely

40 to the leaf margin and midrib (Ignatov and Shcherbakov, 2011b). These traits are not consistent with the lateral costae of a tricostate moss.

Tricostium longifolium (Early Cretaceous of southeastern Russia) is represented by several leafy shoots and a few isolated leaves (Ignatov and

Shcherbakov, 2011a). Stems are 0.3 mm wide, sparsely foliated (ca. 1.6 leaves per mm) and sparsely branched (two branching points, ca. 5 mm interval). Leaves are 4-

6 mm long and up to 1.5 mm wide, diverging from the stem at 15-45˚ angles, and have a recurved distal half. Leaves seen in face-view bear a central costa thicker than the lateral costae. All costae reach at least 0.9 the leaf length. Laminal cells are isodiametric and13-17 µm in diameter (Ignatov and Shcherbakov, 2011a).

Taxonomic placement of Tricosta plicata gen. et sp. nov.

Justification for a new genus.

The unique nature of three strong costae per leaf suggests a close relationship among all tricostate mosses. However, aside from the tricostate leaves, Tricosta plicata is similar to Tricostium only in terms of leaf divergence angles (ca. 40 – 45˚), leaf width (ca. 1.0 mm), and in having strong costae with short laminal cells (Table

1). Of the three species of Tricostium, T. papillosum is most similar to Tricosta plicata, comparing favorably in leaf shape and length as well as width of the central costa (Table 1). However, Tricosta plicata differs from T. papillosum in many respects, including branching angle, leaf density, leaf profile, leaf apex, laminal cell arrangement, laminal cell shape, laminal cell dimensions, and leaf cell wall texture

(Table 1).

41 Table 1. Summary of Tricosta plicata gen. et sp. nov., Krassiloviella limbelloides gen. et sp. nov., and a comparison with species of Tricostium.

Character Tricosta Krassiloviella Tricostium Tricostium Tricostium plicata limbelloides longifolium papillosum triassicum Stem length 22 mm 20 mm 10 mm 3.9 mm ? (min.)

Branch lengths 500 µm 0.3-1 mm 5.5-7.5 mm 1.3 mm ? (min.)

Distance 480 µm 0.8-4.0 mm 5.0 mm ? ? between branches (min.)

Stem width 0.2 mm 370-430 µm ca. 0.3 mm ? ?

Branching (41)-55-(75º) 25-45º ca. 25-35(60º) ca. 43º ? angle

Density of dense, (11)-18- 4.5-6 leaves sparse, ca. 1.6 dense, ca. 3-5 ? foliation (23) leaves mm- mm-1 leaves mm-1 leaves mm-1 1

Leaf erect-spreading ±erect (5-20º) 15-45º at base; patent (25-40º) ? divergence (38-55º) distal half of leaf recurved)

Leaf straight straight recurved straight ? orientation

Leaf shape ovate narrowly lanceolate ovate (to narrowly (outline) lanceolate narrowly lanceolate

ovate) (or oblong?)

Leaf concavity plicate concave some keeled flat (slightly flat (to undulate?) concave?)

Leaf margin entire mostly entire, ? serrate distally entire minute dentition in apex

Leaf length ca. 2.0 mm 4-5 mm 4-6 mm 1.2-1.8 mm 4-5 mm

Leaf width 0.8-1.0 mm up to 0.9 mm up to 1.5 mm ca. 0.5-1 mm) 0.9 mm

42 Character Tricosta Krassiloviella Tricostium Tricostium Tricostium plicata limbelloides longifolium papillosum triassicum Leaf apex acute (to acute (tip acute obtuse to acute acute? acuminate?) apiculate)

Leaf base clasping clasping truncate? clasping (or truncate? auriculate?)

Median costa at least 0.95 at least 0.95, at least 0.9 0.9-0.95 at least 0.8 length (failing to percurrent (failing to percurrent) percurrent)

Median costa ca. 54 µm 70-125 µm 60-80 µm ca. 50 µm 80 µm width Lateral costae at least 0.9 at least 0.95, at least 0.9 0.7-0.9 >at least 0.8 lengths percurrent, merging in apex Lateral costae ca. 35 µm 50-80 µm ca. 25 µm ca. 20-30 µm 30-40 µm widths Alar region conspicuous; inconspicuous; ? ? ? cells inflated, cells oval, globose to irregular, or cylindric, isodiametric, diameters 15-32 12-24 (up to 40) µm (up to 54 µm long, 12 (up µm long) to 16) µm wide, 2.5:1 Laminal cell oblique files longitudinal oblique files? longitudinal longitudinal arrangement near mid-leaf; files files files longitudinal files in distal half Laminal cell rhombic, oval, isodiametric polygonal, quadrate to shapes repand, oval to isodiametric, or (rounded or isodiametric short isodiametric quadrate polygonal?) rectangular Laminal cell up to 5:1 (40 up to 4:1 13-17 µm 15-18 µm ca. 13-16 dimensions µm) basally; 2- basally (40 µm wide 3:1 at mid-leaf µm); 1.33:1 and (ca. 25 µm and 8-12 µm up to 35 µm); lengths (up to isodiametric and 18 µm) above up to 23 µm the base distally Leaf cell wall absent absent ? thickened ? thickenings corners? Laminal cell smooth smooth ? pluripapillate ? surface texture (8-10 papillae per cell)

43 Second, the difference in modes of preservation leads to a strong disparity

between the type and number of taxonomically informative characters as well as the

degree of morphological and anatomical detail available. Fossils belonging to

Tricostium provide information on few characters, including leaf shape, size, angle of divergence, and leaf density along the stems, as well as branching pattern (if present) and leaf areolation (Table 1). Tricosta plicata preserves information on

several additional characters including branching architecture, phyllotaxis, stem

diameters, stem anatomy, detailed leaf anatomy from various planes of section,

costal anatomy, and fertile structures (perigonial and potentially perichaetial shoots).

Consequently, Tricosta plicata is characterized in much more detail than Tricostium.

Third, Tricostium is defined chiefly on leaf characters, as the fossils lack

detail on other characters. Thus none of the species is reconstructed as a whole plant

and Tricostium is best regarded as a morphogenus (i.e., a taxon defined based only

on a subset of characters of the whole plant; Bell and York, 2007) erected for moss

leaves displaying a tricostate condition. In contrast, Tricosta plicata is a natural

taxon based on a whole-plant concept for the gametophyte. Taken together, all these

considerations warrant placement of the Apple Bay material in the new genus,

Tricosta.

Tricosta plicata as a hypnanaean pleurocarp.

Despite the lack of information on sporophytes, several gametophytic

features of Tricosta plicata place it among the superorder Hypnanae (=

pleurocarpous mosses). In the strict sense, pleurocarpy refers to the production of

44 sporophytes (thus, archegonia) on typically bud-like lateral shoots, although there

are exceptions to this criterion both within the Hypnanae and in a few lineages basal

to this clade (e.g., La Farge-England, 1996; Newton, 2007). Because sporophytes or

unequivocal perichaetia are not known in Tricosta plicata, direct evidence for

pleurocarpy is not available for this species. And, the taxonomic literature on extant

mosses fails to provide a cohesive set of criteria for recognizing pleurocarpous

mosses in the absence of sporophytes. This is not surprising, given that bryologists

interested in using morphological and anatomical characters of living mosses usually

have access to entire populations, both phases of the life cycle, and developmental

traits for taxonomic decisions. Gametophyte characters can, nevertheless, be used to

demonstrate the pleurocarpous condition.

An in-depth survey of the bryological literature relating to pleurocarpy

reveals that, among extant mosses, the co-occurrence of a set of prominent

gametophyte features indicates a pleurocarpous condition: (1) an abundance of short

lateral branches bearing bud-like perigonia (antheridium-bearing branches), a feature

that indicates a similar perichaetial branching pattern (N.E. Bell, pers. comm., 2014;

L. Hedenäs, pers. comm., 2014); (2) monopodial and much-branched (±pinnate)

primary stems; (3) pluricostate, (4) homocostate, (5) and strongly plicate leaves; (6)

leaf cells elongate (in Tricosta, ca. 2:1 to 3:1) and rhombic at mid leaf, with (7) thin

walls, and (8) arranged in oblique files; (9) the presence of well-differentiated alar regions (alar cells inflated); and (10) the absence of a central conducting strand in the stems (e.g., Lawton, 1971; Vitt, 1982; 1984; Hedenäs, 1994; La Farge-England,

45 1996; Newton and De Luna, 1999; Ignatov and Shcherbakov, 2007; Newton, 2007;

Goffinet et al., 2009). None of these characters considered individually is

exclusively diagnostic of pleurocarpous mosses, yet they each occur only

sporadically in non-pleurocarpous mosses, and are not known to occur in

combination in any extant non-pleurocarpous moss. Consequently, this combination

of traits is indicative of the Hypnanae and places Tricosta in this clade.

Justification for a new hypnanaean family.

Within the moss superorder Hypnanae (= pleurocarpous mosses), Tricosta

compares most favorably with the Pilotrichaceae (Hookeriales) and the Hypnales

(Table 2). The vast majority of pleurocarp diversity belongs to the Hypnales which

comprises more than 40 families and 400 genera (Goffinet et al., 2009). There are

numerous families within this group that have several conspicuous traits in common

with Tricosta plicata e.g., monopodial and pinnate branching, an absence of

paraphyllia, lack of central conducting strand in the stems, leaves helically arranged,

conspicuous alar regions, and laminal cell morphology (Lawton, 1971; Vitt, 1982;

Chiang, 1995; Gradstein et al., 2001; Goffinet et al., 2009; Eckel, 2011; Ramsay,

2012a; Ramsay, 2012b). Families with a combination of these traits (including

Pilotrichaceae of the Hookeriales) are included in Table 2. Of these families,

Pilotrichaceae, Amblystegiaceae, Hypnaceae, Pylaisiadelphaceae, and

Sematophyllaceae show the greatest similarity to Tricosta (Table 2). However, in

addition to the tricostate and plicate leaves with strong costae which make Tricosta different from all living and extinct mosses, these families exhibit sets of differences

46 Table 2. Comparison1 of Tricosta plicata gen. et sp. nov. to some monopodially branched pleurocarpous mosses2,3.

Character Pilotrichaceae Amblystegiaceae Regmatodontaceae Hypnaceae Rhytidiaceae Pylaisiadelphaceae Sematophyllaceae Tricosta plicata

Branching irregular to irregular to irregular to pinnate pinnate pinnate irregular to pinnate irregular to pinnate subpinnate subpinnate pinnate

Stem absent usually present weak present narrow usually absent absent absent conducting strand

Paraphyllia absent rare absent usually absent absent absent? absent absent

Leaf straight straight to straight often falcate often ±secund straight, few falcate occasionally straight orientation falcate-secund or falcate- secund, rarely secund falcate-secund

Leaf some concave rarely plicate, some concave often concave plicate, rugose some concave concave strongly plicate surface some concave (or plicate) topography

Costa(ae) strong, double mostly single, single short and single, strong short and double or short and double or three, strong often variable double or none none absent

Laminal various short to linear short to elongate mostly linear linear mostly linear mostly linear short to cell shape elongate

Laminal smooth or smooth or rarely smooth smooth or strongly smooth, sometimes smooth or papillose smooth cell surface papillose, prorulose papillose porose, papillose porose or not prorulose

Alar cells undifferentiated not to strongly not or barely usually well- well- few, quadrate, well-differentiated few, well- differentiated differentiated differentiated, differentiated usually not inflated differentiated, quadrate to inflated (rarely inflated quadrate)

Table 2. Comparison1 of Tricosta plicata gen. et sp. nov. to some monopodially branched pleurocarpous mosses2,3 (continued).

1differences from Tricosta plicata are highlighted 2classification follows Goffinet et al. (2009); Pilotrichaceae belongs to Hookeriales; all other families belong to Hypnales 3sources of character states: Lawton (1971), Vitt (1982), Chiang (1995), Gradstein et al. (2001), Goffinet et al. (2009), Eckel (2011), Ramsay (2012a, b)

48 worthy of erecting a new family to accommodate Tricosta (Table 2). Additional differences not listed in Table 2 include: stem anatomy (in Pilotrichaceae: few outer layers of the cortex with narrow, thick-walled cells and typically, a hyalodermis), perigonia containing only a single antheridium (rare among mosses; viz, Sphagnum and Buxbaumia), and to the best of my knowledge, isodiametric distal leaf cells

(present in Tricosta). Together, the differences suggest that none of these families is a good placement for Tricosta. These differences, along with the unique tricostate condition, warrant erection of a new family, Tricostaceae.

Pleurocarpous mosses in the pre-Cenozoic fossil record

Few pre-Cenozoic mosses have been discussed in terms of putative pleurocarpy. However, in such discussions, similarities have been inferred based on characters that are not exclusively diagnostic of pleurocarpy when considered independently (e.g., much-branched gametophytes) or else on equivocal reproductive structures. Uskatia Neuburg described from the Late Permian of

Russia has been compared to pleurocarps by Oostendorp (1987) based on abundantly branched pinnate stems with small leaves. However, Ignatov and Shcherbakov

(2007) have suggested that, despite the pinnate branching, Uskatia probably belongs to a different group due to the presence of leaves attached to the stem only by their costa, a character unknown in mosses. Numerous shoots of the moss Capimirinus riopretensis are known from Permian rocks (Guadalupian) of Paraná, Brazil (De

Sousa et al., 2012). These compressions and impressions show sparse dichotomous branching, leaves ca. 1.4 x 0.5 mm, and a single putative sporophyte in organic

connection with a short lateral shoot. However the sporophyte nature of this

structure is equivocal. Aside from the lack of anatomical information, the sporophyte’s dimensions are unusually small—the seta-like structure is only 0.9 mm long while the sporangium-like structure is 0.4 x 0.2 mm. Because of the uncertain nature of this structure and the lack of other informative characters, Capimirinus De

Souza, Branco, et Vargas is best considered incertae sedis within the mosses.

Several well-preserved shoots of the moss Palaeodichelyma sinitzae Ignatov et Shcherbakov have been described from the Upper Jurassic (Lower Cretaceous?) of

Transbaikalia in southern Russia (Ignatov and Shcherbakov, 2007). Some characters of Palaeodichelyma Ignatov et Shcherbakov suggest a pleurocarpous origin such as the lateral bud-like structures and other traits seen in the pleurocarpous

Fontinalaceae e.g., strong costae, some leaves keeled, tristichous leaf arrangement, elongate laminal cells with transverse end-walls (Ignatov and Shcherbakov, 2007).

However, pleurocarpy of Palaeodichelyma is conjectural, mostly because the nature of its lateral bud-like structures is not understood, and laminal cells with transverse end-walls are rare among the pleurocarpous mosses.

Another genus, Bryokhutuliinia, represented by three species preserved as compressions in the Upper Jurassic (Lower Cretaceous?) of southern Russia and

Mongolia (Ignatov and Shcherbakov, 2007; Ignatov et al., 2011; Ignatov and

Shcherbakov, 2011a) shows pinnately branched shoots and bud-like structures (or terminal rosette-like structures in one species) interpreted as gametangial branches.

Leaves are ecostate, arise in a complanate fashion, and laminal cells that are

rectangular and elongate (variable, but ca. 5:1). Although pinnate branching is

indicative of pleurocarpy and some of the leaf traits suggest Hookerialean affinities

(e.g., ecostate and complanate leaves; Ignatov and Shcherbakov, 2007), further

evidence is necessary to argue unequivocally for pleurocarpous affinities of this

moss.

Vetiplanaxis, represented by four exquisitely well-preserved species from

Middle Cretaceous (late Albian) Burmese amber, is most comparable to the

pleurocarpous Hypnodendrales but this assignment is tentative (Hedenäs et al.,

2014). One species shows just a few branches produced in a pinnate fashion near the

shoot tip which, in addition to the traits comparable to some extant members of the

Hypnodendrales (long, relatively thin-walled laminal cells at mid-leaf; narrow costae; teeth on the abaxial surface of the costa; and marginal leaf cells slightly differentiated), suggests pleurocarpous affinities.

Overall, among the pre-Cenozoic mosses, Palaeodichelyma, Bryokhutuliinia,

and Vetiplanaxis compare most favorably to extant pleurocarps (e.g., Hedenäs et al.,

2014). However, in these taxa, proposed or putative pleurocarpous affinities are based on one or only a few criteria (e.g., general appearance, pinnate branching) encountered in pleurocarpous mosses and suggestive of various extant lineages, rather than on a well-defined, extensive set of diagnostic criteria. In this context, the suite of traits listed above in support of pleurocarpous affinities for Tricosta plicata, provides the strongest evidence to date for pleurocarpy in the pre-Cenozoic.

As the oldest well-supported pleurocarp, Tricosta provides a “hard”

minimum age for the clade—Valanginian, 136 Ma. This age is broadly consistent

with divergence times estimated on molecular phylogenies for the majority of the

pleurocarps, including the Hypnanae (Newton et al., 2007), but falls close to the

upper limit of the predicted interval (165-131 Ma) and is younger than the predicted

origin of the pleurocarpous condition (194-161 Ma). A more relaxed set of

morphological and anatomical criteria for recognizing pleurocarpy would include

Bryokhutuliinia and Palaeodichelyma among the pleurocarps, pushing the minimum

age of the clade to the Jurassic-Cretaceous boundary, ca. 145 Ma. Only including

the equivocal Uskatia and Capimirinus among the pleurocarps would bring the

minimum age of the clade, based on fossils, into the Late or Middle Permian, more

than 250 Ma.

Gametangia in the fossil record

The fossil record of antheridia borne on free-living gametophytes is sparse.

A few early vascular plant gametophytes from the Rhynie chert (Remyophyton delicatum Kerp, Trewin et Hass, Kidstonophyton discoides Remy et Hass,

Lyonophyton rhyniensis Remy et Remy) show well preserved antheridia and

antheridiophores (Taylor et al., 2009). To the best of my knowledge, Eopolytrichum

antiquum (Konopka et al., 1997) is the only instance of preservation of antheridia in

the moss fossil record. Aside from that, a very small number of equivocal splash

cups or perigonia are known (Townrow, 1959; Ignatov and Shcherbakov, 2007; De

Souza et al., 2012), which makes the antheridia preserved in Tricosta plicata a

welcome addition to this sparse fossil record. Each perigonium of Tricosta plicata bears a single antheridium, a condition that is apparently rare among mosses (seen in

Sphagnum—single antheridia in leaf axils—and Buxbaumia—perigonia with single antheridia; Schofield and Hébant, 1984; Goffinet and Buck, 2013) and, to the best of my knowledge, unique among the pleurocarpous mosses, which typically have few to several antheridia per perigonium (personal observation). Unfortunately, the taxonomic and evolutionary significance of this character is difficult to assess, as the literature on extant mosses does not include an extensive survey of moss perigonia

(A.E. Newton, pers. comm., 2014).

At least three shoots of Tricosta plicata (preserved in a single concretion) display perichaetium-like structures (Fig. 10A-10I). The features of these specialized later branches (except the bud-like architecture and lateral position) are different from those seen in the perigonial shoots of Tricosta, which is consistent with a perichaetial nature (e.g., Schofield and Hébant, 1984; Goffinet et al., 2009;

Goffinet and Buck, 2013). Furthermore, the delicate structures (presumably archegonia) seen in sections at branch apices differ from the antheridia of Tricosta in several respects: the “archegonia” have a much paler color; more incomplete preservation (i.e., more delicate); the absence of a well-defined stalk that delimits the body from the base (as in moss antheridia); the absence of a jacket or sac; and perhaps most importantly, an apparently multistratose body (Fig. 10F, 10G). To the best of my knowledge, the only fossil bryophyte in which archegonia have been

53 preserved is the leafy liverwort, Naiadita Brodie from the Upper Triassic Rhaetic

Flora of England (Harris, 1938).

Lastly, the presence of only one sex per gametophyte on fertile Tricosta specimens suggests dioicy. One extensively branched gametophyte (Fig. 3) bears numerous perigonia, whereas another tuft of gametophytes with hundreds of branches (Fig. 1) bears only a few “perichaetia.” The other shoot tips in the latter specimen are vegetative, incompletely preserved, or occupied by single terminal perithecioid fruiting bodies. The shoot tips occupied by fungal fruiting bodies were probably archegonial (see “Perithecioid fruiting bodies”). Although I cannot exclude the possibility that Tricosta gametophytes are monoicous and bear gametangia of both types, the fact that the most extensive specimens are unisexual is consistent with dioicy.

Outstanding questions

The absence of extant mosses morphologically or anatomically equivalent to tricostate mosses (a relatively extensive group with at least eight distinct types known; Tomescu et al., 2012; personal observation), emphasizes the uniqueness of this group and evokes questions about moss evolution. While such questions remain unanswered for now, they provide ideas for discussion and detailed studies in bryophyte development and morphology.

Why is the tricostate condition absent in extant mosses? The answer to this question may revolve around the function of costae, which could be (1) to conduct water for faster diffusion throughout the leaf lamina, or (2) to provide structural

integrity, i.e., maintain leaf shape during desiccation. When grown in an aquatic medium, costate mosses tend to develop ecostate leaves (Zastrow, 1934). This could

be explained by the lack of pressures which favor development of a costa in drier

conditions—i.e., no need for improved leaf conduction and the absence of desiccation. However, not all ecostate mosses are aquatic and some costate mosses are aquatic (e.g., Ochyra and Vanderpoorten, 1999). Given the diminutive size of the leaves of Tricosta and many other costate mosses, their water relations would probably not benefit significantly from specialized structures enhancing leaf conduction. These suggest that a role of costae in desiccation-related leaf structural integrity has more traction in explaining the presence of multiple costae.

Do trends in the presence of costae provide any insight to their evolutionary history? In the broadest sense, extant mosses illustrate a trend from ecostate leaves in the most basal lineages (Takakiopsida, Sphagnopsida, Andreaeopsida) to costate leaves in more derived lineages (Oedipodiopsida, Polytrichopsida, Bryopsida).

Independent instances of further reduction or elaboration of the costa are found in some lineages within these groups (Goffinet et al., 2009; Olsson et al., 2009).

However, the oldest mosses known in the fossil record have costate leaves (Hubers and Kerp, 2012). A denser stratigraphic and taxonomic sampling of the moss fossil record will be needed to test for the presence of evolutionary trends in leaf morphology.

Do all costae share the same developmental pathway and are there anatomical signatures in fossil mosses for the mode of development of the costa?

Some costae form early in leaf development by periclinal divisions in specific

regions of the unistratose leaf primordia (Frey, 1970). In contrast, multistratose

streaks in the leaf lamina (or leaf margin) are formed later in development (Goffinet et al., 2009). Developmental information of this type is not available for Tricosta, but even if it was, the very few studies of leaf development in extant mosses do not allow for conclusive inferences on developmental differences diagnostic of costae vs. multistratose streaks.

Finally, with regards to the pleurocarpous mosses, how well do gametophytic characters reflect phylogeny? More broadly, this question relates to the fundamental conundrum raised in the taxonomic placement of many fossil mosses by the severe disparity between characters available to paleobotanists versus bryologists studying extant mosses. In the particular case of Tricosta, the absence of sporophytic characters, widely used in family-level taxonomy of extant mosses, precludes traditional comparisons and unequivocal placement within the pleurocarpous mosses. The alternative to this is comparison based on gametophytic characters. However, although incorporating gametophytic characters into phylogenetic studies is not uncommon (e.g.,

Hedenäs, 1994; Newton and De Luna, 1999), such comparisons are rendered difficult by the lack of extensive and detailed surveys of gametophytic characters relevant to family- level taxonomy for the pleurocarps. A survey of this type would be a monumental undertaking given that many morphological and anatomical traits are, to date, not evenly covered across all taxa (if at all) and therefore not available in the literature. However, such work could significantly strengthen the morpho-anatomical framework of moss

taxonomy and would certainly improve the precision of taxonomic placement of fossil

mosses, many of which are known only from the gametophyte phase.

Conclusions

Throughout the Mesozoic and late Paleozoic, anatomical preservation among fossil mosses is rare (Smoot and Taylor, 1986; Konopka et al., 1997; 1998; Hübers and Kerp, 2012; Hedenäs et al., 2014). The anatomical and morphological detail preserved in Tricosta plicata allows for the most complete reconstruction of a fossil moss gametophyte to date. Additionally, Tricosta plicata represents a newly

recognized family, genus, and species and the first bryophyte component described

from the Early Cretaceous Apple Bay flora of Vancouver Island.

A combination of gametophytic traits indicates that Tricosta is a hypnanaean

(pleurocarpous) moss. Few other pre-Cenozoic fossil mosses have been reported as

putative pleurocarps (e.g., Uskatia, Capimirinus, Palaeodichelyma, Bryokhutuliinia,

and Vetiplanaxis), but Tricosta plicata provides the strongest and oldest evidence for

pleurocarpy in the pre-Cenozoic. The antheridia of Tricosta plicata add to a very

sparse fossil record of gametangia. Among mosses, only Eopolytrichum antiquum

(Konopka et al., 1997), preserves unequivocal gametangia (antheridia). Tricosta

plicata also exhibits probable perichaetia with incompletely preserved archegonia.

The only known instance of archegonia in the bryophyte fossil record is in Naiadita,

a Triassic leafy liverwort (Harris, 1938).

Finally, studies of anatomically preserved fossil bryophytes emphasize the need,

also stressed elsewhere (Câmara and Kellogg, 2010), for thorough and taxonomically broad anatomical surveys of extant bryophytes. Such studies would both enhance the precision of taxonomic placement of fossils and increase resolution of overall moss systematics and phylogeny.

KRASSILOVIELLA LIMBELLOIDES GEN. ET SP. NOV.

Introduction

Bryophytes originated in the early Paleozoic (e.g., Hubers and Kerp, 2012) and, among them, mosses subsequently gave rise to several easily-recognized major lineages

(e.g., sphagnalean, polytrichalean, dicranalean, and pleurocarpous mosses). Today, there are more than 13,000 described moss species (Goffinet et al., 2009) with several fossils from the Cenozoic representing extant lineages (especially those known from amber;

Taylor et al., 2009). Only a small number of mosses have been reported from the pre-

Cenozoic (ca. 70 described species; e.g., Oostendorp, 1987; Ignatov, 1990; Taylor et al.,

2009). Fossil mosses described from pre-Cenozoic rocks (>66 Mya) and which have been placed into a modern taxonomic framework are almost exclusively acrocarpous mosses.

The oldest fossil moss remains consist of leaf fragments with cuticular preservation known from the Carboniferous (Middle Mississippian, late Visean) of

Germany (Hübers and Kerp, 2012). Some of these fragments resemble the extinct

Protosphagnales (Hübers and Kerp, 2012) while others are too incompletely preserved for taxonomic placement. A few Permian mosses have been convincingly compared to extant groups such as the Bryidae (Merceria augustica Smoot et Taylor; Smoot and

Taylor, 1986) and the Sphagnales or Protosphagnales (Oostendorp, 1987; Ignatov, 1990).

Throughout the Mesozoic, few fossils have been compared rather tentatively to

59 pleurocarpous mosses (e.g., Palaeodichelyma Ignatov et Shcherbakov, Bryokhutuliinia

Ignatov, Vetiplanaxis N.E. Bell, Tricosta gen. nov., and Uskatia Neuburg; Oostendorp,

1987; Ignatov and Shcherbakov, 2007; Ignatov et al., 2011; Ignatov and Shcherbakov,

2011a; Hedenäs et al., 2014).

Most fossil mosses are preserved as compressions, while anatomical preservation

(especially from the pre-Cenozoic deposits) is rare (e.g., Taylor et al., 2009). Aside from the moss described here, anatomical preservation among mosses includes cuticular preservation of Mississippian leaf fragments (Germany; Hübers and Kerp, 2012), permineralization in the Permian moss, Merceria (Antarctica; Smoot and Taylor, 1986) and the Early Cretaceous Tricosta (Canada; see “Tricosta plicata gen. et sp. nov.”), preservation of the Middle Cretaceous Vetiplanaxis in Burmese amber (Hedenäs et al.,

2014), and the charcoalified Late Cretaceous Eopolytrichum Konopka and

Campylopodium Bescherelle (USA; Konopka et al., 1997; 1998).

The Early Cretaceous moss described here adds to the sparse record of pre-

Cenozoic anatomically preserved mosses. Comparisons with extinct and extant mosses indicate that this plant represents a new genus and species, Krassiloviella limbelloides, within the extinct pleurocarpous family Tricostaceae. This moss provides another example of the unique tricostate condition once widespread in the Northern Hemisphere during the Mesozoic.

Materials and Methods

At least 20 moss gametophytes of a common morphology are preserved by cellular

60 permineralization in more than 15 calcium carbonate concretions as part of an allochthonous fossil assemblage deposited in nearshore marine sediments (e.g., Stockey and Rothwell, 2009). The concretions are included in greywacke sandstone beds exposed on the northern shore of Apple Bay, Quatsino Sound, on the west side of Vancouver

Island, British Columbia, Canada (50°36’21” N, 127°39’25” W; UTM 9U WG 951068)

(e.g., Stockey and Rothwell, 2009). Layers containing the concretions are regarded as

Longarm Formation equivalents and have been dated by oxygen isotope analyses to the

Valanginian (Early Cretaceous, ca. 136 Ma; D. Gröcke, pers. comm., 2013).

This Early Cretaceous flora includes lycophytes, equisetophytes, at least 10 families of ferns (Smith et al., 2003; Hernandez-Castillo et al., 2006; Little et al., 2006a, b;

Rothwell and Stockey, 2006; Stockey et al., 2006; Vavrek et al., 2006; Rothwell et al.,

2014) and numerous gymnosperms (Stockey and Wiebe, 2008; Stockey and Rothwell,

2009; Klymiuk and Stockey, 2012; Klymiuk et al., 2015; Rothwell and Stockey, 2013;

Rothwell et al., 2014; Ray et al., 2014), fungi (Smith et al., 2004; Bronson et al., 2013), and a thalloid lichen with heteromerous organization (Matsunaga et al., 2013).

Moreover, this flora is probably the most diverse assemblage of fossil bryophytes known worldwide, with leafy and thalloid liverworts and more than 20 distinct moss morphotypes currently recognized (Tomescu et al., 2012). Pleurocarpous, polytrichaceous, and leucobryaceous mosses are known, as well as several morphotypes of unknown affinities including at least two more distinct tricostate types.

Fossil-containing concretions were sliced into slabs and sectioned using the cellulose acetate peel technique (Joy et al., 1956). Slides were prepared using Eukitt, a

xylene-soluble mounting medium (O. Kindler GmbH, Freiburg, Germany). Micrographs

were taken using a Nikon Coolpix E8800 digital camera on a Nikon Eclipse E400

compound microscope. Images were processed using Photoshop (Adobe, San Jose,

California, USA).

Krassiloviella limbelloides has been identified in the following rock slabs: 16 C;

P25 B; P13131 D; P13174 C; P13734 B; P13997 C; P14403 C; P15053 C; P15157 B, C;

P15388 A; P15800 C; P16366 B; P16459 C; P17345 C; P17596 B, C, D. Specimens

used for this study come from: P25 B-bottom, P13131 D-bottom; P13997 C-bottom;

P14403 C-bottom; P15388 A; P15800 C-bottom; P16366 B-bottom; P17345 C-top;

P17596 B-bottom, C-bottom, D. All specimens and preparations are housed in the

University of Alberta Paleobotanical Collections (UAPC-ALTA), Edmonton, Alberta,

Canada.

Systematics

Class—Bryopsida Rothm.

Subclass—Bryidae Engl.

Superorder—Hypnanae W.R. Buck, Goffinet and A.J. Shaw

Order—incertae sedis

Family—Tricostaceae gen. nov.

Emended diagnosis—Gametophyte plants pleurocarpous. Stems regularly to irregularly pinnately branched, central conducting strand absent. Cortical cells thin-walled, hyalodermis or thick-walled outer cortex lacking. Paraphyllia absent. Leaves helically

arranged with three costae. Laminal cells isodiametric to elongate, not linear; alar

regions well differentiated or not. One to few gametangia borne on lateral specialized

shoots (perigonia, perichaetia).

Type genus—Krassiloviella gen. nov.

Generic diagnosis—Gametophytes solitary or in tufts. Stems terete, with cortical cells

equal in diameter across stem, with evenly thickened walls. Rhizoids present in basal portions of shoots. Shoots isophyllous. Leaves imbricate, erect, straight, symmetrical, tricostate. Costae very strong, arising separately; lateral costae thinner than central costa.

Distinct epidermis on both ab- and adaxial surface of costae, consisting of squat

(transversely elongate) or isodiametric cells; costae otherwise homogeneous. Alar

regions minute, weakly differentiated. Laminal cells smooth, thin-walled; in leaf base slightly elongate, inflated with oblong, oval, irregular, or isodiametric shapes; above leaf base laminal cells quadrate to isodiametric. (Sporophytes unknown).

Etymology—Krassiloviella, after the late Dr. Valentin A. Krassilov for his contributions to Mesozoic floras (especially in Eastern Asia), which include treatments of numerous bryophytes. It was Dr. Krassilov’s keen eye that first detected the tricostate condition in fossil mosses from Eastern Russia.

Type species—Krassiloviella limbelloides sp. nov.

Specific Diagnosis—Gametophyte branching irregular to pinnate, complanate, concentrated distally along main stems. Branches at regular intervals (1-4 mm) and 25-

45º angles. Stems 350-450 µm in diameter, 25 cells across; stem epidermis distinct, consisting of smaller cells. Rhizoids smooth-walled, 12-14 µm diameter; some rhizoids

63 present on leaves. Leaves 4.5-6 mm-1, with 3/8 helical phyllotaxis, diverge from stem at

5-20º angles. Leaves lanceolate, concave throughout, up to 5 mm long and 0.9 mm wide.

Leaf margin planar or slightly incurved (curved adaxially), entire or minutely dentate.

Leaf apex acute to apiculate; lamina up to three cells thick between costae in leaf apex.

Leaf margin widest (6-10 cells) in basal half of leaf, tapering to one cell wide in apex.

Costae parallel leaf margin, percurrent, converging in apex; composed of thin-walled cells. Costae 50-80 µm thick. Median costae 12-16 cells wide (125-90 µm wide from leaf base to apex), ca. 7 cells thick. Lateral costae ca. 8 cells wide (80-70 µm wide from leaf base to apex), 5-7 cells thick. Costal cells 30-100 µm long, 5-10 µm diameter; epidermal cells of costae 10-12 µm. Alar regions poorly differentiated, ca. 25-50 cells.

Lamina cells at leaf base up to 4:1, 12-24 µm (up to 40 µm) long, 12-16 µm wide; above base, lamina cells up to 1.33:1, 8-10 µm, forming longitudinal files. Juxtacostal cells larger than other lamina cells.

Etymology—specific epithet limbelloides for the overall similarity in leaf morphology with species of the genus Limbella Müll. Hal., (Amblystegiaceae).

Holotype hic designatus—To be determined; UAPC-ALTA.

Paratypes—To be determined.

Locality— Apple Bay locality, Quatsino Sound, northern Vancouver Island, British

Columbia (lat. 50º36’21” N, long. 127º39’25” W; UTM 9U WG 951068).

Stratigraphic position and age—Longarm Formation equivalent; Valanginian, ca. 136

Ma (Early Cretaceous).

Description

Habit, branching, shoot architecture, and stem anatomy

Krassiloviella limbelloides is represented by more than 20 gametophyte shoots.

Sporophytes are unknown. The gametophytes are solitary or occur in tufts and at least 2

cm long. Reconstructions of two extensive specimens based on serial sections (Figs.

11A, 11B) show irregularly to pinnately branched gametophyte stems with more-or-less

complanate branching. One of these specimens is at least 10.5 mm long (Figs. 11A, 14A-

14C), unbranched in the proximal 6 mm, and with branches arising in the distal 4.5 mm.

A well-developed reiterative stem (ca. 12 mm long) diverges from the main stem ca. 4 mm from the distal end (the tip of the main stem is not preserved; Fig. 11A). The reiterative stem is unbranched, except for two minute (≤ 100 µm long) branches, and its tip is missing (Figs. 11A, 14A, 14D). The other extensive specimen (Figs. 11B, 13A-

13C, 19A), at least 11 mm long, is branched throughout with at least one lateral demonstrating second-order branching near the remaining tip of the main stem. The main stem continues for less than 0.5 mm past the dichotomy that generated the specialized branch, but its tip is missing (Fig. 11B). Branching of K. limbelloides occurs at regular intervals (the length of which varies between specimens; 0.8-4.0 mm) and at 25-45º angles (Figs. 13A-13C).

Stems are terete and lack a conducting strand (Fig. 12A). They are 370-430 µm

in diameter and ca. 25 cells across. Cortical cells are round to polygonal in transverse

sections, 60-140 µm long, and equal in diameter (11.5-18.5 µm) across the stem. Their

67 walls are evenly thin (<1 µm) and end-walls are oblique or tapered (Fig. 12B). The well- defined epidermis consists of darker cells with slightly thicker walls, rectangular shapes, and dimensions 9-14 µm thick, 11.5-16 µm wide, and 9-17 µm tall (Figs. 12C, 13D).

Branches are 0.3-1.0 mm long, 150-235 µm wide, < 15 cells across (Figs. 13A-

13C, 13E) and composed of mostly of short, inflated cells. Most branch tips are incompletely preserved due to either taphonomy (e.g., stream abrasion and tips replaced by crystals; Fig. 14F) or perhaps truncation by herbivores (Figs. 14A, 14D; see

Discussion below). Truncated tips have a characteristically concave outline with the outermost one to few layers of cells darkened. Leaves adjacent to the truncated tips conform to the same concave shape and bear dark cells at the remaining leaf tips; this is consistent with an herbivore removing the entire shoot tip (Figs. 14A, 14D).

Leaves are isophyllous, follow a 3/8 phyllotaxis with imbricate arrangement, and densely cover the stems (4.5-6 leaves mm-1) (e.g., Figs. 13A-13C, 14A, 14E). Leaves diverge from the stems at 5-20º angles (with wider angles where leaves subtend branches) and are straight (Fig. 14C) or rarely recurved (Fig. 14B). In one longitudinal section, a short branch (< 1 mm long) bears leaves < 3 mm long that are erect or delicate and contorted (Fig. 14G). However, leaves on most branches are similar to stem leaves, albeit slightly smaller. Paraphyllia were not observed.

One gametophyte base bears dense multicellular rhizoids (Figs. 15A-15C) which are 12-14 µm in diameter, smooth-walled, and have oblique end-walls (Fig. 15C). In

Figure 14. Shoot architecture of Krassiloviella limbelloides gen. et sp. nov. 14A. Shoot in longitudinal section showing several leaf bases and incomplete tip; scale bar = 1 mm; P17596 D #37. 14B. Shoot longitudinal section showing several leaf bases and leaf divergence angles; arrowhead = recurved leaf; scale bar = 1 mm; P17596 D #30. 14C. Shoot in longitudinal section showing erect, densely arranged leaves; note slightly wider divergence angle of leaf subtending branch (arrowhead); scale bar = 1 mm; P17596 Cbot #45. 14D. Detail of 14A showing concave shape to remaining tip (suggesting herbivory); note darkened outer cell layers of stem tip and adjacent leaves; scale bar = 150 µm; P17596 D #37. 14E. Shoot cross section showing helically arranged, overlapping leaves and characteristic incomplete preservation of laminae; scale bar = 300 µm; P15388 A #23. 14F. Branch with tip removed by taphonomic processes; note crystalline structures at remaining branch tip (arrowheads); scale bar = 200 µm; P25 Bbot #41b. 14G. Branch with entire tip and short, delicate leaves; note at least one contorted leaf base (arrowhead); scale bar = 150 µm; P17596 Cbot #53.

addition to stems, rhizoids are found attached to leaves near the leaf base either abaxially

(Fig. 15B) or adaxially (Fig. 15D). In one paradermal section, rhizoids are attached to

leaf tissue away from the leaf base (Figs. 15E, 15F), but it is unclear whether the rhizoids

are attached to the lamina or costae due to incomplete preservation.

Leaf morphology and anatomy

The leaves of Krassiloviella limbelloides are symmetrical, lanceolate, concave

throughout, and broadly attached to the stems (Figs. 16, 17C, 17I). They are up to 5 mm

long and 0.9 mm wide (ca. 500 µm wide at the base) (Fig. 16A). Margins are planar

(rarely slightly incurved; Figs. 16, 17C, 18B) and entire (or minute dentation near the leaf

tip; Figs. 17G). Leaf apices are acute and apiculate (Fig. 17G).

Leaves are tricostate with a unistratose lamina throughout most of the leaf length

(e.g., Fig. 16). Laminae are delicate, as evidenced by their typically incomplete preservation (e.g., Figs. 14E, 16, 17C). Where unistratose, the lamina is 11.5-14 µm

thick. However, the lamina is bistratose or tristratose in areas where costae converge at

the leaf apex (Figs. 17A, 17B). The three costae arise separately (Figs. 17H, 17I). The

lateral costae run parallel to the leaf margin and converge well into the apex (thus all

three costae are percurrent; Fig. 16). The leaf margin is 6-10 cells in the basal half of the

leaf, 4-6 cells at mid-leaf, and one cell in the apex. At the widest point of the leaf, the

lamina is 15 cells between the median and lateral costae (Fig. 16).

Figure 16. Leaf anatomy and morphology of Krassiloviella limbelloides gen. et sp. nov. 16A. Paradermal section showing entire leaf length; thick arrowheads indicate central costa, thin arrowheads indicate left lateral costa (right lateral costa not shown); at right, leaf model superimposed over same image; scale bar = 1 mm; P17596 Cbot #66. 16B-16F. Series of leaf transverse sections from near base (16B) to the apex (16F); leaves traced adaxially; scale bars B-D = 100 µm; E-G = 50 µm; C and D: P13131 Dbot #17c; E: P13131 Dbot #10c; F: P13131 Dbot #63c; G: P13131 Dbot #49c.

Figure 17. Leaf anatomy of Krassiloviella limbelloides gen. et sp. nov. 17A. Leaf tip transverse section below level at which one lateral costa (left) merges with central costa; arrowheads indicate three costae; lamina bistratose in between central and right lateral costae; scale bar = 50 µm; P13131 Dbot #63c. 17B. Leaf tip transverse section above level at which one lateral costa (at left in 17A) merged with central costa (arrowhead); lamina is bi- or tristratose in between central and right lateral costae; scale bar = 50 µm; P13131 Dbot #49c. 17C. Transverse section of shoot showing tricostate, concave leaves with planar margins (two leaves traced adaxially); asterisk represents center of incompletely preserved stem; scale bar = 200 µm; P13131 Dbot #17c. 17D. Leaf costa in oblique section showing more or less face-view of epidermis (arrowhead); scale bar = 50 µm; P17596 Cbot #47. 17E. Leaf costa in longitudinal section showing small rectangular cells of epidermis (e.g., arrowhead); scale bar = 50 µm; P17596 Cbot #44. 17F. Paradermal section of leaf showing central costa (right), lateral costa (left), and laminal cells forming longitudinal files (in between arrowheads); larger juxtacostal cells adjacent to costae; scale bar = 200 µm; P17596 Cbot #66. 17G. Apiculate leaf tip (apiculus) in paradermal section; note minute dentition (arrowhead); scale bar = 50 µm; P25 Bbot #1b. 17H. Oblique section of stem showing leaf base (traced abaxially) just above point of divergence of lateral costae (left and right arrowheads); central costa (arrowhead at bottom) incompletely preserved and attached to stem; scale bar = 200 µm; P25 Bbot #2b. 17I. Oblique section of shoot showing leaf base below point of divergence; arrowheads indicate positions of costae; scale bar = 200 µm; P25 Bbot #2b.

Median costae are 95-125 µm wide in the lower half of the leaf and becoming 70-

90 µm wide in the apex; lateral costae are 50-80 µm wide, becoming 35-70 µm wide in the apex (e.g., Figs. 16B-16F, 17A-17C, 17F). Median costae are 12-16 cells wide and 7 cells thick, whereas lateral costae are 8 cells wide and 5-7) cells thick. Costae are ca. 50-

80 µm thick and have a thin (6-10 µm) epidermis on both ab- and adaxial surfaces (Figs.

17D, 17E, 18H, 18I). The cells of this epidermis are isodiametric or rectangular and small (5-12 µm across). The middle layers of the costae are homogeneous, composed of thin-walled cells (walls ca. 1 µm thick) (Figs. 18B, 18C, 18G). In longitudinal sections, costal cells are 70-100 µm long proximally and 30-50 µm long distally, mostly 5-10 µm in diameter, and tapered at one or both ends (Fig. 18A).

Laminal cells are smooth and thin-walled (walls ca. 1 µm thick) (Figs. 18D, 18E,

18F, 18H-18K). In the leaf base, laminal cells are 2-2.5:1 (up to 4:1 in leaves of specialized branches) with mostly oval shapes and dimensions 12-40 µm long and 12-16

µm wide (Figs. 18D, 18E). Alar regions are small, weakly differentiated, and incompletely preserved (Fig. 18F); they occupy roughly 25-50 cells with morphology comparable to other cells in the leaf base. Above the base, laminal cells form longitudinal files (Figs. 17F, 18H-18K) of mostly isodiametric shapes (or oval; up to

1.33:1); they are small, typically 8-10 µm diameters and up to 18 µm long. One or two rows of larger cells 14-35 µm long and 25 µm wide occupy juxtacostal positions (Figs.

17F, 18J, 18K).

Specialized branch

One specimen bears a specialized (gametangial?), bud-like lateral branch near the

remaining stem tip (Figs. 11B, 19) directly subtended by a narrow vegetative lateral

branch 300 µm long, 150 µm diameter (Figs. 11B, 19A). Several other short lateral

branches are borne on this specimen, but none is bud-like. The bud-like branch is a thick

axis ca. 250 x 250 µm with densely arranged, erect leaves (innermost leaves < 2 mm

long) with cells in the leaf base up to 4:1 (Fig. 19H). The shoot tip is expanded (flat to

shallowly convex) and bears at least 5 incompletely darkened bases at its center ca. 50

µm wide, 8 cells across, and 5 cells high (e.g., Figs. 19I, 19J), as well as delicate leaf

bases around the edges (Figs. 19B-19G, 19I-19K). A delicate, incompletely preserved

tissue (gametangia?) occupies the area above the bases (e.g., Figs. 19I, 19J). At the periphery of the expanded shoot tip, one column of the gametangial tissue is attached directly to a darkened base (viz, without an antheridial stalk) suggesting an archegonial nature (Figs. 20A, 20B). Also at the shoot tip periphery is an incompletely preserved,

dark cellular body ca. 55 µm long and 42 µm thick (6 cells long and 4 cells wide; Fig.

20A) that may represent a developing embryo (Figs. 20A, 20B). At least one uniseriate

paraphysis is preserved composed of cells ca. 36 µm long and 15 µm in diameter (Fig.

19K).

Figure 19. Probable perichaetium of Krassiloviella limbelloides gen. et sp. nov. 19A. Longitudinal section of shoot bearing a probable gametangial branch (thick arrowhead), subtended by short branch (thin arrowhead); tip of main stem not shown; scale bar = 500 µm; P25 Bbot #11b. 19B-19G. Serial longitudinal sections of gametangial branch shown in 19A showing flat-topped axis bearing densely-arranged erect leaves and bases of probable gametangia (arrowheads) attached to tip; scale bar in G applies to B-G = 150 µm; B-G: P25 Bbot #9b-#14b. 19H. Paradermal section of a “gametangial” branch-leaf showing elongate laminal cells (center); scale bar = 50 µm; P25 Bbot #6b. 19I. Magnification of 19B showing darkened bases (arrowheads) attached to shoot tip and incompletely preserved delicate tissue (above shoot tip) of probable gametangia and paraphyses; scale bar = 50 µm; P25 Bbot #9b. 19J. Magnification of 19E showing darkened bases (arrowheads) attached to shoot tip and incompletely preserved delicate tissue (above shoot tip) of probable gametangia and paraphyses; note missing material due to taphonomic processes (asterisks); scale bar = 50 µm; P25 Bbot #12b. 19K. Magnification of 19D showing probable uniseriate paraphysis (thin arrowhead) and gametangial bases (thick arrowheads); scale bar = 50 µm; P25 Bbot #11b.

Discussion

The tricostate condition

The tricostate condition in which three costae originate separately at the leaf base is only known in pre-Cenozoic fossil mosses (i.e., Tricostium Krassilov, Tricosta gen. nov., and Krassiloviella gen. nov.) and clearly sets these plants apart from all other living and extinct mosses (see “Tricosta plicata gen. et sp. nov.”; Krassilov, 1973; Ignatov and

Shcherbakov, 2011a, b). Few extant mosses possess leaf features similar to the lateral costae of a tricostate moss. These include multistratose thickenings of the lamina in some species of Coscinodon Spreng. (Grimmiaceae; Hastings and Deguchi, 1997) and intramarginal limbidia in Limbella tricostata Bartr. (Amblystegiaceae; Lawton, 1971) and some species of Calymperes Sw. (Calyperaceae) and Teniolophora W.D. Reese

(Pottiaceae) (Gradstein et al., 2001). Only in Limbella Müll. Hal. is the anatomy of these lateral accessory structures (limbidia) closely comparable to that of the costa (e.g.,

Lawton, 1971). While these structures of Limbella are the best modern analogues to the lateral costae of extinct tricostate mosses, their marginal position suggests a different developmental pathway from costae (Goffinet et al., 2009). Differences in the central conducting strand, leaf length, and costal anatomy distinguish Limbella from K. limbelloides. Thus, overall, extant moss diversity does not include any structures equivalent to the lateral costae of tricostate mosses.

Taxonomic placement of Krassiloviella limbelloides gen. et sp. nov.

Generic level placement.

Mosses that share the tricostate condition include the genus Tricostium reported

from Mesozoic (Triassic or Permian(?) to Early Cretaceous) rocks in Russia and

Mongolia (Krassilov, 1973; Ignatov and Shcherbakov, 2011a, b) and Tricosta plicata gen. et sp. nov. from the Apple Bay locality of Vancouver Island (see “Tricosta plicata gen. et sp. nov.”). Although both these fossils possess tricostate leaves, the disparity in preservation of their characters leads to differences in taxonomic status. Namely, while

Tricostium is a morphogenus (see “Tricostate mosses in the fossil record”) based on leaf compressions that preserve little anatomical detail (e.g., laminal cell areolation), T. plicata is represented by numerous permineralized shoots with detailed preservation of numerous characters (e.g., branching architecture, leaf and stem anatomy). As a result, comparisons between K. limbelloides and Tricosta are more extensive and informative.

Overall, such comparisons indicate that despite the shared tricostate condition, significant

differences between K. limbelloides and the other tricostate mosses warrant erection of a

new genus.

Krassiloviella limbelloides is similar in some respects to the entire range of

morphology seen in the three species of Tricostium in having straight, lanceolate leaves with margins entire or toothed distally; leaves up to 5 mm long with an acute apex and

strong costae (Table 1). However, several traits of K. limbelloides distinguish it from

from Tricostium: leaves erect with narrow divergence angles, a narrow leaf margin, wide

costae merging distally, and smooth laminal cells (Table 1). Of the different species of

Tricostium, K. limbelloides is most similar to T. papillosum (Middle-Upper Jurassic,

Russia; Krassilov, 1973) but has longer leaves with a narrowly lanceolate shape (versus

ovate in T. papillosum), narrower leaf divergence angles, narrowly acute leaf apices, much wider costae (that converge in the leaf apex), and smooth (versus pluripapillose) leaf cells (Table 1).

Comparisons with Tricosta plicata reveal several similarities consistent with

placement in the same family, Tricostaceae (see below). Similarities include:

monopodial main stems, branching intervals shorter distally along main stems, branches

short, stems without a central conducting strand, cortex of ±equal diameter (composed of

thin-walled cells), 3/8 helical phyllotaxis, leaves narrow, straight, with ±entire margins

and a narrow apex, costae very strong and homogeneous, costae ending in or near the

apex, laminal cells smooth, short (especially in the apex) and with more elongate laminal

cell shapes in the leaf base (Table 1). Krassiloviella limbelloides is different from

Tricosta in having more delicate leaves that are erect and borne at very narrow

divergence angles; leaves lanceolate (up to 5 mm), concave, with narrow, plane margins;

an inconspicuous alar region; costae wider, thicker, and converging in the apex; and

laminal cells mostly isodiametric and forming longitudinal files (Table 1).

Pleurocarpy and family-level placement.

Taken alone, the pinnate branching, homocostate and pluricostate leaves of K.

limbelloides provide a strong case for pleurocarpy (see “Tricosta plicata as a hypnanaean

pleurocarp”)—the production of gametangia on short, bud-like lateral branches (e.g., La

Farge-England, 1996; Newton, 2007). Furthermore, the co-occurrence of several

additional features suggests pleurocarpous affinities for this species: main stems

monopodial, presence of second-order branching, stems without a conducting strand, and

87 thin laminal cell walls (see “Tricosta plicata as a hypnanaean pleurocarp”). The combination of these features has been used as a proxy for pleurocarpy in the absence of sporophytes based on an extensive survey of the bryological literature (see “Tricosta plicata as a hypnanaean pleurocarp”).

Additional evidence in support of pleurocarpy in K. limbelloides comes from the specialized bud-like lateral branch preserved on one shoot of K. limbelloides. This incompletely preserved branch is probably a perichaetium bearing the remnants of archegonia. If that is true, the lateral position of the branch (Fig. 19) indicates pleurocarpy, as the production of archegonia on short, bud-like lateral branches is the defining feature of pleurocarpous mosses.

The family Tricostaceae was erected to accommodate some much-branched, pleurocarpous, tricostate mosses (see “Taxonomic placement of Tricosta plicata gen. et sp. nov.”). Other than pleurocarpy, many features diagnostic of Tricostaceae are also seen in K. limbelloides: stems regularly to irregularly pinnately branched; central conducting strand absent; cortical cells thin-walled; absence of hyalodermis or thick- walled outer cortex; paraphyllia absent; leaves helically arranged with three costae; some laminal cells isodiametric, never linear; and, probably, few gametangia per specialized gametangial branch. The only notable difference is that whereas K. limbelloides has inconspicuous alar regions, Tricostaceae has conspicuous alar regions. However, this single difference is far outweighed by the similarities which justify the inclusion of K. limbelloides in the Tricostaceae. Consequently, I have emended the familial diagnosis to include mosses with or without conspicuous alar regions.

Moss-animal interactions

One shoot tip shows evidence of herbivory, likely by a micro-invertebrate, in the form of a concave truncation of a shoot apex in which the outermost layers of remaining cells are darkened (Fig. 14A, 14D). The uniform shape of missing tissue across both stem and adjacent leaves strongly suggests that the shoot tip was removed by an herbivore and not by fungi. For example, the presence of a stromatic structure or fruiting body at the shoot tip would almost certainly leave behind remnants of hyphae in and on host tissues and no such hyphae were found. The shoot tip was probably not altered by taphonomic processes either because characterstic signs of a taphonomic breakage a the tip—e.g., crystal-shaped vacancies—are absent. If the cell darkenings represent wound- response by the plant, this would indicate the interactions preceded taphonomic processes. This is probably one of very few moss-animal interactions documented in the fossil record, especially for the pre-Cenozoic.

Conclusions

Krassiloviella limbelloides is distinct from all other living and extinct mosses. It shares the tricostate condition with few other Mesozoic fossils, from which it is otherwise different in several respects. Krassiloviella limbelloides adds a second well-characterized bryophyte to the Early Cretaceous Apple Bay flora of Vancouver Island and another genus to the extinct, pleurocarpous Tricostaceae.

The level of detail preserved in fossils from the Early Cretaceous Apple Bay locality allows for detailed reconstructions and thorough discussions of the evolution of

89 anatomical features otherwise lost to harsher taphonomic processes. The preservation of extremely delicate structures such as antheridia (see “Specialized branches”) and lichen thalli (Matsunaga et al., 2013) is a testament to this, as is the microscopic evidence of herbivory and perichaetia in K. limbelloides. This level of detail is promising in terms of a continued study and thorough evaluation of the taxonomy and life history of Early

Cretaceous bryophytes and cryptogams from Apple Bay.

IMPLICATIONS OF TRICOSTA AND KRASSILOVIA

The newly described species Tricosta plicata and Krassiloviella limbelloides

and the family Tricostaceae provide new insights for the fields of bryology and

paleobotany. Possessing the rare morphological trait of having only three costae per

leaf, seen exclusively in the fossil record, is a testament to this. And the discussion

of costal analogues above (see “Tricostate analogues in extant mosses”), which hints

at a potential homology between such basic gametophyte structures as costae and

limbidia, demonstrates the degree to which bryophyte morphology, anatomy, and

development still have to be explored. A tantalizing scenario can be extrapolated

from this potential homology: if costae and some costa-like structures such as limbidia were demonstrated to be homologous, there may be cases of “living fossils”—that is, where extant counterparts are discovered after fossils have been

described, in this case, extant mosses with tricostate leaves—in today’s bryofloras.

The level of anatomical detail preserved in fossils from Apple Bay is rare, especially among mosses (see “Fossil Record of Pre-Cenozoic Mosses”). In assessing the taxonomic affinities of Tricosta plicata and Krassiloviella limbelloides, I encountered

the conundrum of having several traits preserved in the fossils (viz, detailed gametophyte

anatomy) without comprehensive detail on those traits in the literature on extant mosses.

The paucity of studies comparing anatomy among pleurocarpous moss gametophytes is due in part to an overwhelming diversity of pleurocarpous mosses (ca. 5300-6600 species; O’Brien, 2007). Also, moss sporophytes are emphasized in morphometric

studies as the sporophytes are an incredibly rich source of characters (e.g., Goffinet et al.,

1999) but sporophytes are not known for the newly described species here. It does not

follow that a classification of pleurocarpous mosses based solely on gametophyte

characters would be worthless or uninformative. Rather, the number of gametophytic

characters used in comparisons of extant mosses is too low for the paleobryologist to

make fine-level taxonomic assessments of their fossils even if numerous gametophyte

characters are preserved. Many developmental characters, which provide another rich

source for studies of extant bryophyte gametophytes (e.g., Mishler and De Luna, 1991),

have not yet been explored in the Apple Bay mosses. However the combination of

numerous specimens and anatomical preservation of delicate structures in the fossils

described here (e.g., gametangia, branch primordia, and entire shoot tips) is a promising

area for future research.

Of course, assigning taxonomic affinities is only “half-the-battle” and perhaps less than half of the significance in describing new fossil taxa. The addition of morphological diversity for instance—such as the striking tricostate condition among mosses, first described by V.A. Krassilov (1973)—is testament to the need for continued exploration in the field of paleobotany. In the same light, characterizing fossil taxa provides hard evidence of extinct morphologies and clues to the life histories of extinct plants, a dynamic element of plant evolution not attainable from living plants.

BRYOSYMBIOTIC FUNGI

Introduction

Bryophilous or bryosymbiotic fungi are fungi that are consistently, but not necessarily exclusively, associated with bryophytes. Although they are more common than one would expect (e.g., members of the Polytrichaceae are nearly always infected by fungi; Döbbeler, 1997), bryophilous fungi are not intensely studied (e.g., Döbbeler, 1997;

Davey and Currah, 2006). Most of the major fungal lineages (ascomycetes, zygomycetes, basidiomycetes) include bryophilous representatives, but the vast majority of bryophilous fungi are ascomycetes (more than 300 described species; Racovitza, 1959;

Felix 1988; Döbbeler, 1997; Davey and Currah, 2006). These fungi, the overwhelming majority of which are obligate species, have been documented in association with several major lineages of mosses and liverworts (especially leafy liverworts); they occur on a variety of gametophyte and sporophyte substrates, and exhibit a variety of of nutritional modes (Racovitza, 1959; Felix 1988; Döbbeler, 1997; 2002; Davey and Currah, 2006).

While fungi have a rich fossil record (Taylor et al., 2015), the record of bryophyte fossils is much less extensive (e.g., Oostendorp, 1987, Taylor et al., 2009); and, to date, there is no record of fossil bryophilous fungi. This is due in part to the rarity of anatomically preserved bryophytes and beacuase bryophytes tend to show few macroscopic symptoms of fungal infection, collections and descriptions of bryophytes

(fossil and extant) are biased towards exquisite specimens, and there is a general lack of

93 collaboration between bryologists and mycologists (e.g., Döbbeler, 1997; Davey and

Currah, 2006; Taylor et al., 2009).

Here is documented a diverse assemblage of fungi on the gametophytes of an extinct moss species, Tricosta plicata from the Apple Bay locality in the Lower

Cretaceous of Vancouver Island. Anatomical preservation of the moss and fungi allows for recognition of several morphotypes, some of which occupy specialized niches

(bryophilous fungi s.s.) within the moss shoots. This description is the third addition to the fossil record of fungi at Apple Bay and, to the best of my knowledge, the first record of fossil bryophilous fungi worldwide.

Materials and Methods

At least seven distinct fungal morphotypes with a few subtypes are preserved by cellular permineralization in 16 carbonate concretions as part of an allochthonous fossil assemblage deposited in nearshore marine sediments (e.g., Stockey and Rothwell, 2009).

Some of the morphotypes occur with each other, and are either epiphytic, endophytic, or dispersed among the gametophyte shoots of the extinct moss species, Tricosta plicata.

Concretions containing these fossils are included in greywacke sandstone beds exposed on the northern shore of Apple Bay, Quatsino Sound, on the west side of Vancouver

Island, British Columbia, Canada (50°36’21” N, 127°39’25” W; UTM 9U WG 951068)

(e.g., Stockey and Rothwell, 2009). Layers containing the concretions are regarded as

Longarm Formation equivalents and have been dated by oxygen isotope analyses to the

Valanginian (Early Cretaceous, ca. 136 Ma) (D. Gröcke pers. comm., 2013).

This Early Cretaceous flora includes lycophytes, equisetophytes, at least 10 families

of ferns (Smith et al., 2003; Hernandez-Castillo et al., 2006; Little et al., 2006a, b;

Rothwell and Stockey, 2006; Stockey et al., 2006; Vavrek et al., 2006; Rothwell et al.,

2014) and numerous gymnosperms (Stockey and Wiebe, 2008; Stockey and Rothwell,

2009; Klymiuk and Stockey, 2012; Klymiuk et al., 2015; Rothwell and Stockey, 2013;

Rothwell et al., 2014; Ray et al., 2014), fungi (Smith et al., 2004; Bronson et al., 2013), a thalloid lichen (Matsunaga et al., 2013), and mosses (Tomescu et al., 2012). This flora is probably the most diverse assemblage of fossil bryophytes known worldwide including leafy and thalloid liverworts and more than 20 distinct moss morphotypes currently recognized (Tomescu et al., 2012), representing pleurocarpous, polytrichaceous, and leucobryaceous mosses as well as several morphotypes with unresolved affinities.

Fossil-containing concretions were sliced into slabs and sectioned using the cellulose acetate peel technique (Joy et al., 1956). While thin-sections (rather than acetate peels) are ideal for studying complex three-dimensional fossil fungi (Taylor et al.,

2015), the premise for study of these rock slabs was initially bryological and required serial acetate peels for high-resolution morphological reconstructions (e.g., Sections 1.2 and 1.3). Slides were prepared using Eukitt, a xylene-soluble mounting medium (O.

Kindler GmbH, Freiburg, Germany). Micrographs were taken using a Nikon Coolpix

E8800 digital camera on a Nikon Eclipse E400 compound microscope. Images were processed using Photoshop (Adobe, San Jose, California, USA).

The fungal morphotypes have been identified in the following rock slabs: P13172

G; P13174 C; P13175 E; P13311 I; P13616 E; P13957 A, B, C; P15422 A; P15425 C;

P15800 C; P16435 C. Specimens used for this study come from: P13616 Etop; P13957

A, B, C; P15422 A. All specimens and preparations are housed in the University of

Alberta Paleobotanical Collections (UAPC-ALTA), Edmonton, Alberta, Canada.

Description of Fungal Morphotypes

Seven distinct fungal morphotypes are associated with the gametophytes of

Tricosta plicata that occur in several different locations on these gametophytes: (1) basal

portions of shoots in leaf axils (Fig. 21A), (2) away from leaf axils and near leaf costae

(Fig. 21B), (3) in between adjacent leaves and towards leaf apices (Fig. 21B), and (4) embedded within shoot tips (Fig. 21C). Whereas some of these fungal morphotypes are found in more than one location, others appear to be associated preferentially with only one location.

Tubular septate hyphae

Tubular septate hyphae are known from gametophytes in all rock slabs used in this study. The hyphae are mostly 2-5 µm in diameter and ubiquitous on the moss leaves

(epiphyllous). Branching, if present, occurs at regular intervals (ca. 30 µm; Fig. 22A).

Individual cells of the hyphae are 7-9 µm long, thin-walled, and light to dark in color

(Fig. 22B).

In paradermal sections of the moss leaves, hyphae of this type run along the leaf

surface (superficially), often following the leaf cell outlines (e.g., Figs. 22D, 22E, 23A,

Figure 22. Tubular septate hyphae and haustoria on and in the leaves of Tricosta plicata gen. et sp. nov. 22A. Leaves in section showing regularly branched, tubular, septate hypha running along the leaf surface; scale bar = 20 µm; P15425 Cbot #56a. 22B. Large- diameter hypha with dark and ornamented walls; one leaf cell in transverse section is occupied by delicate fungal haustorium (arrowhead) suspended by small-diameter filament; scale bar = 20 µm; P15425 Cbot #54a. 22C. Magnification of 22B showing delicate haustorium suspended in center of host cell by small-diameter filament (arrowhead); scale bar = 10 µm; P15425 Cbot #54a. 22D. Leaf paradermal section showing hyphae along leaf cell wall outlines, endophytic cell aggregates (arrowheads at right), and fungal material which may represent developing endophytic aggregate (left arrowhead); note highly degraded costa at left in longitudinal view; scale bar = 50 µm; P15425 Cbot #46a. 22E. Magnification of 22D showing hyphae (e.g., arrowheads) along the leaf cell wall outlines (inter-and extracellularly); dark intracellular fungi (near center) may represent developing endophytic aggregate; scale bar = 20 µm; P15425 Cbot #46a. 22F. Delicate haustorium (arrowhead) at center of leaf cell; scale bar = 20 µm; P15425 Cbot #46a.

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Figure 23. Fungal hyphae, appressoria, and haustoria on and in leaves of Tricosta plicata gen. et sp. nov. 23A. Leaf paradermal section showing hyphae along host cell wall outlines (= darkened thickenings of cell wall outlines); note hypha running through degraded costa (arrowhead at right); and epiphytic hypha bearing round cells (arrowhead at bottom); scale bar = 50 µm; P15425 Cbot #48a. 23B. Magnification of 23D, showing knob-like appressorium and evidence of penetration peg (pp); note top half of appresorium-bearing filament missing; scale bar = 5 µm; P15425 Cbot #52a. 23C. Magnification of 23A showing epiphytic hypha bearing 3 or 4 round cells alternately; scale bar = 10 µm; P15425 Cbot #48a. 23D. Hypha running superficially (extracellularly) over leaf cell wall outlines; laterally-produced appressorium (ap) shown positioned over anticlinal wall of juxtacostal cell; scale bar = 20 µm; P15425 Cbot #52a. 23E. Magnification of 23F showing haustorium suspended intracellularly by small-diameter hyphae (arrowheads); scale bar = 10 µm; P15425 Cbot #49a. 23F. Paradermal section showing hyphae along leaf cell wall outlines (= darkened thickenings of cell wall outlines); note intracellular aggregations (wide arrowheads), hyphal fragments radiating from juxtacostal cells (thin arrowheads at right; costa not shown), and delicate haustorium (thin arrowhead at left); scale bar = 50 µm. P15425 Cbot #49a. 23G. Magnification of 23F showing hyphae along leaf cell wall outlines (arrowheads) and intracellular aggregation (at right); scale bar = 20 µm; P15425 Cbot #49a.

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23F, 23G, 25A). Traces of hyphae are common within host cell walls (intercellular; Figs.

24A, 24B), while intracellular tubular hyphae are only found occasionally, typically

occurring in the cells of leaf costae (Figs. 4D, 23A, 24G). Laminal cells of Tricosta

plicata in the absence of hyphae are thin-walled (Fig. 8E). Some paradermal and

transverse leaf sections show hyphae grouped into bundles and adhering to the leaf

surface (Fig. 24D, 24G). Serial transverse sections of one pair of leaves show a bundle of

hyphae wrapped around the margin of a leaf and bridging over to the adjacent leaf via

hyphae perpendicular to the leaf surface (Figs. 24E, 24F). There are four appressoria (or

appressorium-like structures) derived from these hyphae which are preserved in leaf

paradermal sections (Figs. 23B, 25A, 25B, 31G). Two appressoria occur near leaf axils

(Figs. 25A, 25B) where each appressorium represents a single cell borne laterally along

hyphae (e.g., Fig. 25B) or intercalary between filaments (Fig. 25A); these appressoria are

irregular in shape and up to ca. 25 µm across. The other two appressoria occur away

from leaf axils and the represent lateral, knob-like extensions from tubular filaments

(Figs. 23B, 23D, 31G); these appressoria are 3-5 µm across. The hyphae which produce

the appressoria follow the host cell wall outlines. The bases of penetration pegs which correspond to small lateral hyphae (Fig. 25C) can be seen on the appressoria (Figs. 23B,

25B, 31G) and on tubular hyphae (Fig. 25C). In some sections, anticlinal walls of moss leaf cells display traces of penetration pegs, sometimes numerous traces per leaf cell (Fig.

24A).

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These tubular septate hyphae are found connected with several other distinct

fungal morphotypes described here: moniliform chains (Figs. 26A, 26E, 27B),

endophytic cell aggregations (e.g., Figs. 24B, 24C, 28A, 28B), multicellular elongate

propagules (Figs. 31B, 31C, 31F).

Moniliform hyphae

Moniliform hyphae are known from gametophytes in all rock slabs used in this

study. They occur as chains of spherical, subspherical or slightly elongate cells with 3.5-

14 µm diameters. Cells are light to dark in color and are smooth or ornamented with

micro-echinate processes (Figs. 27A-27E). These hyphae (or chains) can be branched

with up to two orders of branching (Figs. 26D). Moniliform hyphae have been found

connected with both tubular hyphae (Figs. 26A, 26E, 27B) and endophytic cell

aggregations (Figs. 28G, 28F). There are two subtypes:

1) Small diameter (2.5-5 µm): light colored with smooth-walled cells, ubiquitous

deep in leaf axils, especially in basal portions of moss shoots (Figs. 26A-26C, 26E). The

cells of this chain subtype can bear one or few small holes (ca. 315-375 nm diameter) on

the cell surface; the holes correspond to the bases of minute penetration pegs (Fig. 6G).

Globular aggregates up to 30 µm across, are found adhering to leaf laminae away from

leaf axils (Fig. 26F) or scattered (Fig. 26H). The aggregates are composed of many

tightly-packed cells 3.5-5 µm diameters and likely represent folded and aggregated small- diameter moniliform chains.

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2) Larger diameter (6-10 µm): dark colored with thicker-walled cells, smooth or

ornamented with micro-echinate processes < 1 µm (Figs. 27A-27E), and adhering to leaf surfaces throughout the shoots.

Intracellular cell aggregations

Intracellular (or endophytic) fungal cell aggregates occur as groups of up to 12

thin-walled cells (light in color) hosted within moss leaf cells (e.g., Figs. 28A-28G).

Individual fungal cells are spherical (or molded to the shape of adjacent structures) and 4-

(5-7)-10 µm in diameter. The spherical cells of the aggregations have been found` connected to minute penetration pegs (Fig. 28C) or larger diameter hyphae (e.g., Figs.

28A, 28B) that cross the anticlinal walls of plant host cells. Thickening of the host cell wall surrounding penetration pegs (Fig. 28C) may represent evidence of host response.

In many instances, the aggregates clearly represent un-branched moniliform chains coiled within host leaf cells (e.g., Figs. 28C, 28D). Other aggregates appear as extremely densely-packed cells (e.g., Fig. 29). Many aggregations occur in physical connection with moniliform hyphae (e.g., Figs. 28B, 28E, 28G).

Endophytic cell aggregates occur throughout the gametophyte shoots (abundant in one specimen; Fig. 21A). In some leaf paradermal sections, the endophytic fungi occupy every other cell creating a checkerboard pattern of infected cells (Figs. 28B, 29). Some leaf cells show a well-defined rupture on one periclinal wall as evidence of having been occupied by the fungal aggregates (e.g. Fig. 30); the same host cells show tubular hyphae organized into a bundle on the leaf surface opposite the rupture. These hyphae produce

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Figure 28. Intracellular aggregates within leaves of Tricosta plicata gen. et sp. nov. 28A. Leaves in section harboring intracellular fungal aggregates showing connection with tubular hyphae (arrowheads); scale bar = 30 µm; P15425 Cbot #46a. 28B. Few leaves in section showing several fungal aggregates and moniliform chains (arrowheads); aggregates at left show evenly-spaced distribution (occurring in every other cell); scale bar = 30 µm; P15425 Cbot #49a. 28C. Leaf in longitudinal section showing intracellular moniliform aggregate and penetration hypha (arrowhead) within host anticlinal wall; large structure probably a plant spore; inset (scale bar = 5 µm) shows thickened leaf cell wall (arrowhead) surrounding penetration hypha; scale bar = 20 µm; P15425 Cbot #68a. 28D. Coiled moniliform aggregate surrounded by incomplete (ruptured) leaf cell wall (arrowheads); scale bar = 10 µm; P15425 Cbot #49a. 28E. Minute moniliform hypha in connection with (arrowhead) intracellular aggregate; scale bar = 10 µm; P15425 Cbot #46a. 28F. SEM of few leaf cells in paradermal section showing intracellular aggregate in connection with intercellular hypha (arrowhead) through anticlinal wall; scale bar = 10 µm; P15425 Cbot #43a. 28G. Leaf in longitudinal section showing two intracellular aggregates, upper aggregate in connection with small-diameter moniliform chain; scale bar = 20 µm; P15425 Cbot #49a.

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Figure 29. Leaf paradermal section (costa at left) showing several dark intracellular aggregates spaced in regular pattern; scale bar = 50 µm; P15425 Cbot #49a.

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114 branches that penetrate the host cells and are connected to the endophytic aggregates

(e.g., Figs. 24B, 24C, 28A, 28B).

Phragmospores and dictyospores

More than ten multicellular propagules have been found on gametophytes of

Tricosta plicata. They are oblong to spindle-shaped or club-shaped (Fig. 32A), smooth- walled, and attached to leaf surfaces (Figs. 31A-31D, 31F, 31H, 31K) or scattered in between leaves (Figs. 31E, 31I). They are composed of cells ca. 5 µm across, with dark or light-colored walls. One exceptional propagule type (subtype 2) is composed of much larger cells up to 15 µm across. The propagules fall into four subtypes:

1) Uniseriate four-celled phragmospores ca. 15 µm long and 5 µm wide (Figs.

31H-31K). One of these bears an aperture in the center of a cross wall (Fig. 31H).

2) Phragmospores at least 7 cells long with dark, thick walls. The cells decrease in size from large and globose (ca. 15 µm diameter) to cells ca. 10 µm across, lending to an overall tapered or club-shaped propagule (Figs. 32A, 32B). One of these propagules, attached to a leaf surface, bears a small infection hypha penetrating the periclinal wall of an adjacent leaf cell (Fig. 32B inset); the same propagule shows a scar at the tapered end

(Fig. 32A), probably where the propagule was attached to a conidiogenous hypha.

3) Biseriate dictyospores with four tiers, 16 µm long, and 9 µm wide (Figs. 31B,

31E-31G).

4) Triseriate dictyospores 8-10 tiers long, up to 20 µm wide, and 60 µm long

(Figs. 31A-31D).

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Figure 32. Several-celled phragmospore with intracellular penetration peg on Tricosta plicata gen. et sp. nov. 32A. Club-shaped phragmospore (attached to leaf surface above) at least seven cells long with scar at tapered end (thin arrowhead); below, hypha on adjacent leaf probably bears lateral conidiogenous branch (thick arrowhead) covered by pyrite aggregates; scale bar = 20 µm; P15425 Cbot #41’a. 32B. Club-shaped phragmospore at least three cells long (at lower right) scattered in between leaf bases; fragments at upper left may represent cells of same propagule type; inset (scale bar = 10 µm; P15425 Cbot #41’a): magnification of 32A at attachment to leaf surface showing laterally-produced infection hypha (arrowheads indicate base and tip of infection hypha) penetrating leaf anticlinal wall; scale bar = 30 µm; P15425 Cbot #47a.

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Few of the propagules in types 1), 3), and 4) have one or both ends complete

(Figs. 31D, 31K) while all other propagules either show evidence of germination or of

having been connected to hyphae (Figs. 31A-31C, 31F-31J, 32A). Some of these

propagules are in physical connection with tubular hyphae, either at their ends or sides

(Figs. 31B, 31C, 31F). Propagules occur singly except in one instance where two light-

colored triseriate dictyospores occur side-by-side and attached to the adaxial surface of a leaf costa (Fig. 31C).

Aperturate amerospores

Unicellular propagules lacking internal divisions (amerospores; Taylor et al.,

2015) are known from a single rock slab (P15425 C). They are abundant and scattered

throughout the moss shoots (Figs. 31B, 31I, 33A-33H). One loose cluster of more than

ten propagules occurs next to a leaf surface but is not attached to the leaf (Fig. 33A).

Cells are round to reniform in side-view, ca. 18 µm long and 12 µm wide. Walls are

thick and dark in color (with one exception; Fig. 33G). Each propagule bears a single

slit-like longitudinal aperture with thickened margins (e.g., Figs. 31I, 33A-33D, 33H) and a hilum occurs half way along the length of the propagule (Fig. 13C, 12G). In one instance, an amerospore is shown attached to hyphae (Fig. 33F).

Perithecioid fruiting bodies

Ascomycetous perithecioid fruiting bodies are known from a single rock

concretion (P13957). At least 45 spherical or subspherical fruiting bodies with 140-175

µm diameters occur embedded in tissues of shoot tips (Figs 21C, 34A). The infected

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gametophytes are part of a much branched 20 mm high tuft. Each infected shoot tip is

subtended by a branch that diverges less than 0.5 mm from the tip (Figs. 21C, 34A).

Fruiting bodies occur singly and are embedded centrally in the shoot tip in up to 5 layers

of stem tissue (e.g., Figs. 34A, 34C, 34E, 34I). Diameters of infected stems are

hypertrophied with infected stem diameters ranging from 225-250 µm compared to ca.

200 µm diameter uninfected stems. Fruiting body walls are up to 10 µm thick and

composed of one layer (rarely more than one) of dark, septate hyphae (Figs. 34C, 34I,

34J). The cells of moss cortex adjacent to the fruiting body bear pale contents (e.g., Fig.

34E) of possible fungal origin. Fruiting bodies have a single apical ostiole 20-40 µm wide (Figs. 34B, 34D, 34H) and a very short ostiolar neck 20-50 µm long (Figs. 34B,

34D). The ostiolar neck is surrounded by the bases of apical leaves of the gametophyte shoot (Figs. 34B, 34D). The leaf bases are darkened and diverge at a wide angle (ca. 45º) forming a funnel-shaped structure (Figs. 34B, 34D, 34H).

Several fruiting bodies contain material that is difficult to interpret (Figs. 34I,

34J). Lining the interior of other fruiting bodies are the bases of reproductive or sterile structures (viz, asci, conidia, paraphyses, or pseudoparaphyses; Figs. 34F, 34G, 34K) arising from a hymenial layer (Fig. 34F).

Interfoliar stromata

Interfoliar stromatic structures are known from a single rock slab (P13616 E).

More than five stromatic structures occur in between leaves and positioned at least 0.5

mm from the leaf axils (Fig. 21B). The stromata are broadly attached to the surfaces of

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Figure 34. Perithecioid fruiting bodies embedded in shoot tips of Tricosta plicata gen. et sp. nov. 34A. Two gametophyte shoots bearing fruiting bodies (arrowheads) embedded within their tips; top in transverse section; bottom in oblique section; scale bar = 300 µm; P13957 Btop #9. 34B. Shoot in oblique section bearing subspherical fruiting body (shown in midlongitudinal section); arrowhead indicates ostiole surrounded by funnel- shaped leaf base (traced abaxially); inset (scale bar = 50 µm; P13957 Btop #127): fruiting body in near-midlongitudinal section showing funnel-shaped leaf base around ostiolar neck; scale bar = 100 µm. P13957 A #11. 34C. Fruiting body in midtransverse section; note thin, single-layered fruiting body wall (arrowhead) and disorganized fungal contents; scale bar = 100 µm; P13957 Btop #9. 34D. Ostiolar region of fruiting body in near-midlongitudinal section; thin arrowheads indicate short ostiolar neck; funnel-shaped leaf base surrounding ostiolar neck; fruiting body wall not preserved (thick arrowheads indicate previous position of wall); note contents loosely aggregated, incompletely preserved; scale bar = 50 µm; P13957 Btop #126. 34E. Fruiting body and shoot in oblique section showing few layers of stem cortex around fruiting body; arrowhead indicates fruiting body wall; note extremely pale fungal tissue within host cortical cells; scale bar = 100 µm; P13957 Btop #11. 34F. Fruiting body contents in 34C showing thick hypha (arrowhead) bearing bases of reproductive hyphae; scale bar = 20 µm; P13957 Btop #9. 34G. Bases of reproductive hypae; arrowheads indicate septae; scale bar = 10 µm; P13957 Btop #9. 34H. Ostiolar neck in transverse section showing darkened cells (arrowheads) of leaf base surrounding ostiolar neck; scale bar = 50 µm; P13957 Btop #140. 34I. Fruiting body in midtransverse section showing single-layered wall, disorganized fungal contents, and few intracellular hyphae (arrowheads) external to fruiting body wall; scale bar = 100 µm; P13957 Btop #138. 34J. Fruiting body wall in section composed of single layer of hyphae (arrowhead); scale bar = 20 µm; P13957 Btop #138. 34K. Transverse section near base of fruiting body showing bases of reproductive structures (arrowheads); scale bar = 20 µm; P13957 Btop #9.

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both adjacent leaves (Figs. 35A-35C) or, less commonly, to only one of them (Fig. 35D).

The stromata, which are incompletely preserved, conform to the leaf plications and have irregular shapes and sizes, up to 200 µm across and 50 µm thick (Figs. 35A-35D). The stromatic tissue is composed of densely-packed septate hyphae with ca. 3-5 µm diameters. The stromata contain closely-spaced cavities separated by single layers of hyphae (e.g., Figs. 35A, 35B, 35E). The cavities have rounded shapes in section, are up to 70 µm across and lined with a single pseudoparenchymatous layer (Fig. 35E) from which bases of hyphae protrude (e.g., Figs. 35B, 35C, 35E).

Haustoria and other fungal types

Aside from the seven fungal morphotypes described above, a few other fungal

structures are found associated with the gametophytes of Tricosta plicata, some too

poorly understood or incompletely preserved to be included here. Several delicate

haustorial structures are common throughout the moss shoots (e.g., Figs. 22B, 22C, 22F)

and are suspended within host cells by extremely narrow-diameter hyphae (Figs. 22C,

23E). These haustoria are probably conspecific with the intracellular aggregates and represent a phase of infection prior to the formation of the several-celled aggregates (see

“Life history of the Tricosta plicata bryophilous fungi”). Several sections show another,

rather uncommon fungal arrangement consisting of round fungal cells (3-5 µm diameter) borne laterally on tubular hyphae (Figs. 23A, 23C), the round cells being densely arranged (up to 6 µm intervals along hyphae) in an alternate fashion.

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Discussion

The bryophilous syndrome is best defined as a specialized interaction between a

fungus and its bryophyte host. Although there is much variation in the form of the

interaction (e.g., parasitic, pathogenic, obligate, generalist, etc.), the tendency of

bryophilous fungi is to have highly specialized life cycles and structural adaptations

(Racovitza, 1959; Felix 1988; Döbbeler, 1997; 2002; Davey and Currah, 2006). Not

surprisingly, this specialization is most often attributed to the co-evolutionary history of

the associates (e.g., Döbbeler, 1997; 2002; Davey and Currah, 2006). For example, the

cell wall chemistry of mosses is complex, containing distinct lignin-like compounds that are highly resistant to decay (Davey and Currah, 2006). Complex wall chemistry is consistent with the observed trend in which bryophilous fungi show specialized life cycles and structural adaptations.

Ascomycete specialists

The fungal associates of Tricosta plicata described here display growth patterns

that are best explained as a bryophilous syndrome. The fungi follow growth habits of

extant bryophilous fungi including the sorting of morphotypes based on position within the moss gametophyte (i.e., niche specialization, as detailed in the descriptions of individual fungal morphotypes), and hyphae following the host cell outlines (Racovitza,

1959; Felix 1988; Döbbeler, 1997; 2002; Davey and Currah, 2006). Furthermore, in studies of extant bryophytes, fungal associates occur most frequently (in many cases exclusively) on one or few host species (Döbbeler, 1997; 2002; Davey and Currah, 2006).

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Consistent with these observations, gametophytes of T. plicata provide the only known substratum for the fungi described here.

The vast majority of bryophilous fungi (Racovitza, 1959; Felix 1988; Döbbeler,

1997; Davey and Currah, 2006) and the majority of endophytic fungi (Taylor et al., 2015) belong to the phylum Ascomycota. With the exception of the aperturate unicellular propagules (amerospores), all of the fungal morphotypes associated with T. plicata compare favorably with the ascomycetes. In addition to the clear pattern of ascomycetes as the dominant players among bryophilous fungi, a few lines of evidence directly support ascomycete affinities for most of the Apple Bay morphotypes: fruiting bodies with perithecioid morphology, and narrow, tubular, and regularly-septate hyphae without clamp connections. Although moniliform hyphae and multicellular propagules are known in other groups of fungi (e.g., Webster and Weber, 2007), the aspect of these morphotypes at Apple Bay is strikingly similar to comparable structures in the

Ascomycota (see Systematics below), and they are physically connected to ascomycetous hyphae.

Biotrophs or decomposers?

Generally speaking, it is thought that whereas biotrophic fungi elicit cell- or tissue-level responses in their plant hosts, decomposers feeding on dead plant material will leave no such traces. The fossil fungi show growth patterns and host plant responses consistent with a biotrophic life history. For example, most of the tubular hyphae of the

Apple Bay bryophilous fungi are found in superficial or intercellular positions, like those of extant biotrophic bryophilous fungi (e.g., Felix, 1988; Döbbeler, 1997). In stems

129 occupied by perithecioid fruiting bodies, host response is demonstrated in at least three distinct ways. 1) Host stem cortex is expanded to accommodate the girth of the fruiting body (Fig. 34A, 34C, 34E). 2) The apical leaves of infected shoots form a funnel-shaped structure from leaf bases (Figs. 34B, 34D, 34H). 3) Leaf cell wall thickenings occur surrounding penetration hyphae (Fig. 28C). Additionally, the fact that one of the Tricosta plicata gametophytes hosting fungi bears structures as delicate as antheridia (Fig. 9), and that gametophytes occupied by the fruiting bodies are exquisitely preserved and show no signs of necrosis (e.g., Figs. 2C-2F, 7I, 35) indicates that the gametophytes were most likely alive when colonized by the fungi.

Despite their apparently bryophilous nature, there are indications that some of the fungi had more marked effects on their hosts. The moss shoot that bears the delicate antheridia exhibits decomposition of stem and leaf tissues most likely due to fungal activity (e.g., Figs. 2A, 2D, 21A). Some leaf costae host intracellular fungal hyphae

(Figs. 23A, 24G) more consistent with a necrotrophic habit (e.g., Felix, 1988; Döbbeler,

1997) and, in some cases, leaves bearing intracellular fungal aggregates in cells adjacent the costae show incompletely preserved costae with darkened tissue, indicating partial decomposition (e.g., Fig. 29).

Life history of the Tricosta plicata bryophilous fungi

Some of the fungal morphotypes described on Tricosta plicata are clearly conspecific, as demonstrated by the instances of physical connection documented in this material. These include the tubular septate hyphae, endophytic cell aggregations, moniliform chains, and multicellular propagules (Figs. 24B, 24C, 26A, 26E, 27B, 28A,

130

28B, 28G, 28F, 31B, 31C, 31F 33F). The patterns of connection of these different

morphotypes suggest a sequence of infection of the moss leaves by these fungi.

Dispersal.

It is unknown whether the multicellular propagules are conidia or ascospores.

Nevertheless, some of the propagules are scattered throughout the moss shoots and some are attached to the leaves via tubular septate hyphae, suggesting a role in dispersal. Thus, these multicellular propagules give rise to the hyphae which in turn generate the different infection structures observed on the moss leaes (e.g., Figs. 31F, 32A, 32B).

Exploratory hyphae and production of infection structures.

Tubular septate hyphae are ubiquitous on the moss leaves, occurring superficially

and running preferentially along the cell wall outlines. From these hyphae, appressoria

(e.g., Figs. 23G, 25B, 31G) and moniliform hyphae (see below) form small diameter infection or penetration pegs which develop a delicate haustorium within individual leaf cells adjacent to the costae (Figs. 22C, 22F, 23E, 28C, 28F). The haustoria are eventually replaced inside the cells by tightly packed and folded moniliform chains (intracellular aggregates). Stages intermediate between haustoria and intracellular aggregates show dark, irregularly shaped fungal material suspended within a host cell by hyphae (Figs.

22D, 22E) in a similar way to that observed in the haustoria.

Dissemination from host leaf cells.

Dissemination of endophytic aggregations (or folded moniliform chains) from

individual leaf cells involves proliferation of the moniliform chain outside of the host

131

cell. This occurs through a rupture of the cell wall (e.g., Figs. 28E, 28G) and eventual exit

of the entire aggregate from the cell (e.g., Figs. 28D, 30).

Cyclic re-infection.

Both tubular septate hyphae and epiphytic moniliform hyphae show evidence of

small diameter infection pegs (Figs. 25C, 26G). This suggests that moniliform chains,

like the tubular hyphae, have the potential to (re-)infect moss leaf cells. Moniliform chains are formed in large numbers not only through the process of single-host-cell infections (i.e., those formed endophytically) but also from tubular septate hyphae external to the moss cells (those formed epiphytically; Fig. 26A). The large numbers of moniliform chains and the presence of infection pegs on both tubular septate hyphae and moniliform hyphae suggest a role in cyclic re-infection.

Systematics

The fungal fruiting structures (perithecioid fruiting bodies and stromata) and the

infection structures of the Apple Bay fungi are not completely understood, which renders

detailed comparisons with extant taxa tenuous at this time. In particular, the absence of

reproductive content from the fruiting structures (e.g., asci, ascospores, or pycnidia)

limits the level at which comparisons can be made with extant bryophilous fungi. Also,

detailed reconstructions of the infection structures (appressoria, penetration hyphae, and

intracellular haustoria) will be needed to make meaningful taxonomic comparisons.

Nonetheless, many extant bryophilous fungi (associated with both leafy liverworts and

mosses) share traits with some of the morphotypes described in this study. Extensive

descriptions and reviews of bryophilous mycofloras (Racovitza, 1959; Döbbeler, 1978;

132

1997; Davey and Currah, 2006; Döbbeler, 2002; 2010) highlight similarities between extant fungi and the Apple Bay bryophilous fungi. These comparisons may inform future studies of fossil bryophilous fungi.

Hyphae.

Numerous extant bryophilous species produce hyphae that occur intercellularly or extend superficially, following exactly the host cell wall outlines (e.g., Racovitza, 1959), in the same way as the hyphae of the T. plicata fungi. However, among these species there is considerable variation in hyphal morphology (e.g., wall thickness, degree of branching, cell lengths) and morphology of the host-cell-association. A more meaningful taxonomic comparison of the Apple Bay hyphae would require a more detailed characterization of the geometry and positioning of infection structures with respect to the host cells. Moniliform hyphae comparable to those of the Apple Bay fungi are not represented in the literature on bryophilous fungi.

Globose cell aggregates.

The aggregations of many small cells scattered in between or attached to leaves

(Figs. 26F, 26H) probably represent aggregated moniliform hyphae (see “2. Moniliform

Hyphae” above). If the aggregates are instead discrete structures, they may be comparable to asexual fructifications as seen in Sporodesmium syntrichiae Racov.

(1959).

Appressoria and haustoria.

The two irregularly-spaced and irregularly-shaped Apple Bay appressoria (Figs.

25A, 25B) are comparable to those of species such as Nectria hylocomii Döbbeler

133

(Hypocreales; 1997). All four of the Apple Bay appressoria form at cell junctions (Figs.

23B, 23D, 25A, 25B, 31G), a trait seen in many species including Nectria egans Corner

(Döbbeler, 1997) and Calonectria frullaniae Racovitza (Hypocreales; 1959). The knob- like laterally-produced appressoria in the fossil fungi (Figs. 23B, 23D, 31G) are comparable to few species including the dothideomycetes Leptomeliola scapaniae

Racovitza and L. hypnorum Racovitza (1959). Delicate haustoria suspended within

individual leaf cells by small-diameter hyphae (Figs. 22B, 22C, 22F, 23E) are seen in few

species including Pseudonectria crozalsiana Grelet (Hypocreales; Racovitza, 1959).

Intracellular aggregates.

Intracellular aggregates or moniliform chains filling individual bryophyte leaf cells are seen in Nectria muscivora Burk. et Br. and Leptomeliola scapaniae. However, these aggregates are few-celled and either have been produced from an intracellular mycelium (N. muscivora) or are composed of irregularly shaped cells (Leptomeliola scapaniae) (Döbbeler, 1978).

Multicellular propagules.

The four-celled Apple Bay phragmospores (Figs. 31H-31K) compare favorably in shape and wall thickness to ascospores of some Leptomeliola Höhn. species (Racovitza,

1959) and conidia of the sordariomycete Pleosphaeria lophoziae Racovitza (1959). The biseriate conidia of Macrosporium commune Rabenhorst (incertae sedis; Racovitza,

1959) are similar in morphology to those described above (Figs. 31E-31G), and the

triseriate ascospores of few species including Teichospora jungermannicola Mass.

134

(Pleosporales; Racovitza, 1959) are comparable to the Apple Bay material (Figs. 31A-

31D).

Perithecioid fruiting bodies.

Several extant bryophilous fungi produce perithecioid fruiting bodies at the tips

(embedded or not) of bryophyte gametophytes (e.g., Racovitza, 1959; Döbbeler 2002;

Döbbeler, 2010; Davey and Currah, 2006). Some of these fruiting bodies are formed within stromata (e.g., Bryostroma Döbbeler, Dothideales; 1978) while others are non-

stromatic perithecia as in Bertia axillaris Racovitza (?incertae sedis; 1959), Nectria

gymnophila Döbbeler and Ticonectria testudinea Döbbeler (Hypocreales; 2002).

However, none of these non-stromatic species are known to form fruiting bodies with a single-layered wall, like those observed at the tips of the T. plicata gametophytes (Figs.

34C, 34I, 34J).

Several lines of evidence indicate that the shoot tips of T. plicata infected by perithecioid fruiting bodies were perichaetia. The tips of uninfected branches in the same gametophyte tuft show direct and circumstantial evidence of a perichaetial nature (see

“Specialized branches” under “Tricosta plicata gen. et sp. nov.”; Fig. 10). The presence of such reproductive shoot tips suggests that many other branch tips in the tuft were also reproductive, as production of numerous reproductive shoots per fertile gametophyte is typical of pleurocarpous mosses. The high occurrence of infected shoot tips could be another indication that many of these tips were reproductive, given that reproductive tips are known sinks for photosynthates—perichaetia are “one of the most distinct and nutrient-rich microniches in bryophytes” (Döbbeler, 2010, p. 404). Furthermore, another

135 gametophyte bears no shoot-tip-inhabiting, perithecioid fruiting bodies despite being heavily infected by other types of fungal structures and despite producing numerous perigonia (see “Specialized branches” under “Tricosta plicata gen. et sp. nov.”; Fig. 3).

This is consistent with preferential selection of perichaetial shoots by the Apple Bay perithecioid fruiting bodies, a trait documented in some extant bryophilous fungi

(Döbbeler, 2002; 2010). Lastly, the modification of apical perichaetial leaves into trumpet-shaped structures seen in infections caused by extant Nectria gymnophila and

Ticonectria testudinea (Döbbeler, 2010) is similar, yet not equivalent, to the funnel- shaped leaves of shoot tips infected by the Apple Bay perithecioid fruiting bodies (Figs.

34B, 34D).

Conclusions

The fungi described here provide the first evidence for the occurrence of bryophilous fungi in the fossil record. The Tricosta bryophilous fungi and add a third component to the Early Cretaceous fungal diversity at Apple Bay, previously represented by Quatsinoporites cranhamii Smith, Currah et Stockey (2004) and Spataporthe taylori

Bronson, Klymiuk, Stockey et Tomescu (2013). Compared to the total number of extant fungi, the known richness of bryophilous fungi represents only a small fraction, although new species are being described (e.g., Döbbeler, 1997; Davey and Currah, 2006). The

Apple Bay fungi described here may represent new taxa, but more in-depth comparisons with the known extant diversity are needed to corroborate this idea. Both the tissue-level interactions and the consistent co-occurrence of these fungi with the gametophytes of

136

Tricosta plicata demonstrate that those interactions as well as a high level of host specificity had evolved in moss-inhabiting fungi prior to the Early Cretaceous. Finally, these small and relatively inconspicuous bryophilous fungi from Apple Bay represent a cautionary tale that emphasizes the need to expand our search image and maintain a keen eye for fossil fungi.

137

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