University of Alberta

Paleobiology of Canadian amber: an exceptional record of Late Cretaceous , with contributions to additional taxa and the study of amber

by

Ryan Christopher McKellar

A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

Department of Earth and Atmospheric Sciences

©Ryan Christopher McKellar Fall 2011 Edmonton, Alberta

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••I Canada ABSTRACT

Canadian amber provides a glimpse of a Late Cretaceous (Campanian) ecosystem preserved in exquisite detail. Here we document one of the best- preserved assemblages of Hymenoptera (ants, bees, and wasps) in the Mesozoic. Contributions are also made to the study of a broader range of taxa and the development of amber research. Amber offers the best fossil record for diminutive terrestrial organisms with a low preservation potential, such as micro-hymenopterans. Canadian amber preserves a broad range of hymenopteran taxa, but is strongly biased towards smaller taxa, so members of 'Parasitica' (a grade of parasitoid hymenopterans) are most thoroughly represented. To date, 30 families, 38 genera and 70 of Hymenoptera have been identified in the assemblage: 36 of these species, six of the genera, and two of the families stem from the body of work summarized in this dissertation. Herein, Canadian amber specimens are contrasted against those recovered from other Cretaceous amber deposits, and large-scale palaeobiogeographic and stratigraphic range patterns are discussed. New taxa are documented within Neuroptera (lacewings) and (true bugs), as these specimens constitute important records for their respective families (Rhachiberothidae and Microphysidae). The most abundant and diverse assemblage of feathers and putative protofeathers yet to be recovered from Mesozoic amber is also described. Careful screening of Canadian amber collections for inclusions as small as micro-hymenopterans has yielded a wide range of inclusions of high scientific value. Contributions to the study of these specimens will promote additional research into a somewhat overlooked palaeontological resource. Finally, the versatility of stable isotope analyses in the study of amber is demonstrated. This technique is used to explore the source of Canadian amber, and the role of attacks in the formation of amber deposits. This new approach to the study of amber provides a more comprehensive account of conditions in the amber-producing forest, and allows palaeoecological hypotheses to be tested through modern analogues. ACKNOWLEDGEMENTS This work would not have been possible without the support, collaboration and mentorship of many people. I am greatly indebted to both of my supervisors, Brian Chatterton and Alex Wolfe, for their support, guidance and encouragement in all facets of my research. I would like to thank Allan Ashworth, Michael Caldwell, Karlis Muehlenbachs, and Felix Sperling for acting as my committee members and examiners - their time, expertise and guidance has been invaluable. My has been extremely supportive throughout all of my studies, and deserves a great deal of credit here. Collaborative research with Michael Engel (University of Kansas) and Jaime Ortega-Bianco (Universitat de Barcelona) in particular has expanded my understanding and experience with palaeoentomology. Work with Karlis Muehlenbachs and Ralf Tappert has greatly improved my grasp of stable isotope geochemistry and FTIR analyses, while research with Stacey Gibb has contributed to my understanding of ichnology and invertebrate palaeontology. Numerous other workers, particularly those that have acted as collaborators, provided invitations to contribute works, or edited and reviewed the papers encompassed by this dissertation have also provided opportunity for growth. As with most palaeontological projects, specimens have been essential in this study. I am extremely grateful to those that have donated specimens for my research, such as the Leuck family; or have provided access to museum collections, such as Andrew Bennett, Jim Troubridge, and Robert Skidmore (Canadian National Collection of and ); James Gardner and Brandon Strilisky (Royal Tyrrell Museum of Palaeontology); Philip Perkins (Harvard Museum of Comparative Zoology); Danny Shpeley and Felix Sperling (University of Alberta Strickland Entomological Museum); Andrew Locock (University of Alberta Palaeontology Museum); Nicola Howard (University of Alberta Laboratory for Vertebrate Palaeontology); Janet Waddington and Antonia Guidotti (Royal Ontario Museum); and Jocelyn Hudon (Royal Alberta Museum). Numerous friends and colleagues have also shared their experience, permitted the use of their equipment, or have assisted with fieldwork, including: Kevin Aulenback, Bruce Archibald, Amber Garrett, Will Hobbs, Shane and Vicki Leuck, Svetlana Kuzmina, Lindsey Leighton, Allan Lindoe, Sergei Matveev, Valerie McKellar, Darrin Molinaro, Heather Proctor, Thomas Stachel, Ralf and Michelle Tappert, and Graham Young. Funding was provided through NSERC (PGS D-3) and Alberta Ingenuity Fund (AIF Studentship) support, as well as NSERC Discovery Grants (to B.D.E. Chatterton and A.P. Wolfe). TABLE OF CONTENTS ABSTRACT i ACKNOWLEDGEMENTS iii LIST OF TABLES viii LIST OF FIGURES ix LIST OF SYMBOLS, NOMENCLATURE, OR ABBREVIATIONS xii

CHAPTER 1:AGENERAL INTRODUCTION TO CANADIAN AMBER 1

OBJECTIVES OF STUDY AND PROGRESSION OF PAPERS 1

INTRODUCTION 2

GEOLOGICAL SETTING 4

AMBER COLLECTION 5

PALAEOHABITAT 7

THE CANADIAN AMBER TREE 7

AGE OF CANADIAN AMBER 11

PHYSICAL AND CHEMICAL PROPERTIES 12

THE DIVERSITY OF CANADIAN AMBER INCLUSIONS 13

REFERENCES 19

CHAPTER 2: CORRELATION OF GRASSY LAKE AND CEDAR LAKE AMBERS USING

INFRARED SPECTROSCOPY, STABLE ISOTOPES, AND PALAEOENTOMOLOGY 26 INTRODUCTION 26 Historical background 26 Cedar Lake amber 26 Grassy Lake amber 27 Stratigraphic constraints 28 Objectives of study 29 MATERIALS AND METHODS 30 Materials 30 FTIR spectroscopy 30 Stable isotopes 31 Palaeoentomology 32 RESULTS 32 FTIR spectra 40 Stable isotopes 42 Amber morphology and physical properties 44 Palaeoentomology 46 DISCUSSION 47 Amber spectroscopy, weathering, and botanical source 47 Inferences from the stable isotopic composition of amber 49 Palaeoentomological and stratigraphic considerations 51 CONCLUSIONS 53 REFERENCES 55

CHAPTER 3: HYMENOPTERA IN CANADIAN CRETACEOUS AMBER (INSECTA) 63 INTRODUCTION 63 Canadian amber perspective on the Hymenoptera 65 Comparisons to other Cretaceous amber deposits 68 GEOLOGICAL SETTING 68 Occurrence 69 Collections 69 HYMENOPTERAN DIVERSITY IN THE ASSEMBLAGE 70 Evanioidea (? Aulacidae) 70 Ceraphronoidea (Ceraphronidae, ?Megaspilidae, Stigmaphronidae) 72 Trigonalyoidea (Maimetshidae) 76 Cynipoidea (Cynipidae, Figitidae, Liopteridae, Protimaspidae) 77 Diaprioidea (Diapriidae) 79 Platygastroidea (?Platygastridae, Scelionidae) 80 Serphitoidea (Serphitidae) 84 Mymarommatoidea (Mymarommatidae) 86 Chalcidoidea (?Eupelmidae, Mymaridae, Tetracampidae, ?Torymidae) 89 (Ichneumonidae, ) 92 Aculeata, the stinging wasps 94 Scolebythidae 94 Bethylidae 96 Chrysididae 97 97 Formicidae, the ants 98 Crabronidae, and other apoid wasps 99 Comments 100 DISCUSSION 100 Relative abundance of families 100 Diversity patterns and future expectations 102 The paucity of Aculeata, symphytans, and phytophages 103 CONCLUSIONS 104 REFERENCES 105

CHAPTER 4: CONTRIBUTIONS TO OTHER INSECT TAXA IN CANADIAN AMBER (HEMIPTERA,NEUROPTERA) 121 THE FIRST MESOZOIC MICROPHYSIDAE (HEMIPTERA) 121 Introduction 121 Materials and methods 122 Systematic Palaeontology 122 Key to the fossil genera and subgenera of Microphysidae 123 Popovophysa McKellar and Engel gen. nov. 123 Popovophysa entzmingeri McKellar and Engel sp. nov. 125 Discussion 128 A NEW THORNY LACEWING (NEUROPTERA: RHACHIBEROTHIDAE) 131 Introduction 131 Systematic Palaeontology 132 Albertoberotha McKellar and Engel, new 132 Albertoberotha leuckorum McKellar and Engel, new species 134 Discussion 136 REFERENCES 138

CHAPTER 5: CONTRIBUTION TO VERTEBRATE PALAEONTOLOGY IN CANADIAN AMBER 142 A DIVERSE ASSEMBLAGE OF LATE CRETACEOUS DINOSAUR AND BIRD FEATHERS FROM CANADIAN AMBER 142 Introduction 142 Results and discussion 144 Stage I morphotype, isolated filaments 144 Stage II morphotype, clustered filaments 144 Stage IV and V morphotypes, specialized barbules 145 Conclusions 148 Supplementary material 149 References 150

CHAPTER 6: GENERAL CONTRIBUTION TO THE STUDY OF AMBER 152 INSECT OUTBREAKS PRODUCE DISTINCTIVE CARBON ISOTOPE SIGNATURES IN DEFENSIVE RESINS AND FOSSILIFEROUS AMBERS 152 Introduction 152 Materials and methods 154 Samples 154 Methods 154 Results and discussion 155 MPB attack and resin carbon isotopes 155 Dominican amber andHymenaea resins 156 New Jersey amber 158 Conclusions 159 References 160 CHAPTER 7: GENERAL CONCLUSIONS 163 SUMMARY OF WORK 163 Advances in the palaeontology of Canadian amber 163 Advances in the understanding of Canadian amber 164 Advances in amber research 165 BROADER IMPLICATIONS FOR THE HYMENOPTERA 166 BROADER IMPLICATIONS FOR THE STUDY OF CANADIAN AMBER AND AMBER RESEARCH 167 FUTURE WORK 168 Palaeoentomology 168 Chemotaxonomy and stable isotope analyses 169 REFERENCES 171

APPENDIX 1: A MODIFIED TECHNIQUE FOR THE PREPARATION OF FRIABLE AMBER AND THE BULK-PROCESSING OF ITS INCLUSIONS 175

APPENDIX 2: STABLE ISOTOPE MEASUREMENTS FROM NORTH AMERICAN CRETACEOUS AMBERS 183

APPENDIX 3: SUPPLEMENTARY MATERIAL FOR CHAPTER 5 "CONTRIBUTION TO VERTEBRATE PALAEONTOLOGY IN CANADIAN AMBER" 184

APPENDIX 4: SUPPLEMENTARY TABLE OF 813C DATA FROM RESINS AND AMBERS 215 LIST OF TABLES

Table 1.1: families recorded from Canadian amber 17

Table 2.1: Palaeoentomological comparison between Grassy Lake and Cedar Lake amber 33 Table 3.1: Classification and list of extant families of Hymenoptera (adapted from Huber, 2009).. 64 Table 4.1: Checklist of described fossil Microphysidae 121 Table 4.2: Fossil Rhachiberothidae (thorny lacewings), updated from Engel and Grimaldi (2008).. 132 LIST OF FIGURES

Fig. 1.1 Subcrop diagram of Upper Cretaceous formations in the prairie provinces, with the Saskatchewan river system superimposed 3 Fig. 1.2 Stratigraphic diagram for the main Albertan amber-bearing units 4 Fig. 1.3 Surface collecting at the Grassy Lake tailings piles 4 Fig. 1.4 A surface-concentrated amber fragment at the Grassy Lake site 4 Fig. 1.5 Small rod-shaped amber pieces found within a sub-bituminous coal northwest of Drumheller 4 Fig. 1.6 Modern and Late Cretaceous cupressaceous foliage bearing on the identity of Canadian amber-producing trees 9 Fig. 1.7 FTIR spectra of Cretaceous Canadian ambers, select modern conifers, and additional fossil resins 10 Fig. 1.8 Species richness plot of arthropod inclusions in Canadian amber 14 Fig. 1.9 Diptera and Hymenoptera inclusions in Canadian amber 15 Fig. 1.10 Neuroptera, Coleoptera, and Hemiptera inclusions in Canadian amber. 16 Fig. 2.1 Subcrop diagram of Upper Cretaceous formations in the prairie provinces, with the Saskatchewan river system superimposed 27 Fig. 2.2 Stratigraphic diagram for Alberta through Santonian to Maastrichtian Stages 29 Fig. 2.3 FTIR spectra 41 Fig. 2.4 Stable isotope measurements for S13C and 8D 42 Fig. 2.5 Stable isotope comparisons and context: stable isotope compositions measured for Grassy Lake and Cedar Lake ambers, as compared to other North American Mesozoic ambers, and a wide range of published values 43 Fig. 2.6 Grassy Lake amber characteristics and hymenopteran inclusions 45 Fig. 2.7 Dendrogram of cluster analysis results obtained from the FTIR spectra of western Canadian Cretaceous ambers 47 Fig. 3.1 Apocritan phylogeny with Canadian amber inclusions indicated 66 Fig. 3.2 Photomicrographs of Ceraphronoidea, Trigonalyoidea, Cynipoidea, and Platygastroidea in Canadian amber 74 Fig. 3.3 Photomicrographs of Serphitoidea and Platygastroidea in Canadian amber 82 Fig. 3.4 Photomicrographs of Mymarommatoidea, Chalcidoidea, and Ichneumonoidea in Canadian amber 88 Fig. 3.5 Photomicrographs of Aculeata and fragmentary remains of larger taxa .95 Fig. 4.1 Popovophysa entzmingeri gen. et sp. nov. photomicrographs 126 Fig. 4.2 Popovophysa entzmingeri gen. et sp. nov. habitus drawings 127 Fig. 4.3 Albertoberotha leuckorum sp. nov. habitus diagram of holotype 135 Fig. 4.4 Albertoberotha leuckorum sp. nov. enlarged view of right foreleg 135 Fig. 4.5 Albertoberotha leuckorum sp. nov. forewing venation diagram 136 Fig. 4.6 Albertoberotha leuckorum sp. nov. habitus photograph of holotype ....136 Fig. 5.1 Feather evolutionary-development model (Prum, 1999), terminology (Lucas and Stettenheim, 1973), and Stage I and II specimens from Canadian amber 143 Fig. 5.2 Specialized barbules in Canadian amber 145 Fig. 5.3 Pigmentation in Canadian amber feathers 147 Fig. 6.1 Products of infestation in trees and resin 153 Fig. 6.2 813C values of modern resins and amber analogues 156 Fig. 6.3 Products of infestation in amber 157 Fig. Al.l Specimens prepared with new protocol 177 Fig. A3.1 Graph of specimen diameters for filamentous structures (Stage I and II) and barbules in Canadian amber, compared to other possible sources....200 Fig. A3.2 Photomicrographs of Stage I filaments in UALVP 52821 201 Fig. A3.3 Compound microscope images (b.f.) of Stage I filaments in UALVP 52821 202 Fig. A3.4 Dissecting and compound microscope images of Stage I filaments, fungi, and mammalian hair 203 Fig. A3.5 Compound microscope images (b.f.) of Stage II clusters in UALVP 52822 204 Fig. A3.6 Compound microscope images of coiled barbules (TMP 96.9.334) ..205 Fig. A3.7 Dissecting microscope images of coiled barbules (TMP 96.9.334)...206 Fig. A3.8 Compound microscope images of differentiated barbules with distinct pennulae in UALVP 52820, indicating preservation that is visually identical to Stage I and II morphotypes 207 Fig. A3.9 Compound microscope images of pennaceous barbs with reduced plumulaceous barbules 208 Fig. A3.10 SDCM and LSCM data for Stage I morphotype and TMP 96.9.997 - emission response microphotographs and emission spectra 209 Fig. A3.ll LSCM and additional photomicographs of UALVP 52821 210 Fig. A3.12 Photomicrographs of modern bird feathers for comparison of barbule structure and pigmentation patterns 211 LIST OF SYMBOLS, NOMENCLATURE, OR ABBREVIATIONS

Throughout this work, nomenclature and symbols are explained within the introductory portions of each chapter. Collection abbreviations that appear repeatedly within this work include: CNC-CAS, Canadian National Collection of Insects and Arthropods, Canadian Amber Series, Ottawa, Ontario, Canada CNCI, Canadian National Collection of Insects and Arthropods, Ottawa, Ontario, Canada MCZ, Harvard University Museum of Comparative Zoology, Cambridge, Massachusetts, USA RAM, Royal Alberta Museum, Edmonton, Alberta, Canada ROM, Royal Ontario Museum, Toronto, Ontario, Canada RTMP, Royal Tyrrell Museum of Palaeontology, Drumheller, Alberta, Canada TMP, specimens belonging to RTMP UALVP, University of Alberta Laboratory for Vertebrate Palaeontology, Edmonton, Alberta, Canada UASM, University of Alberta Strickland Entomology Museum, Edmonton, Alberta, Canada CHAPTER 1: A GENERAL INTRODUCTION TO CANADIAN AMBER1*

OBJECTIVES OF STUDY AND PROGRESSION OF PAPERS Canadian amber is approaching its 75th year of palaeoentomological study. Despite the length of time that this material has been known, collected, and studied, the largest advances in its palaeontological study have occurred predominantly within the last few decades, and existing collections still have much to offer. This dissertation represents the first step towards a comprehensive treatment of the inclusions in Canadian amber and placing these inclusions in a meaningful context. The main objective of this work is to advance the study of paleobiology in Canadian amber, with an emphasis on hymenopteran inclusions. Secondary objectives include contributing to the understanding of Canadian amber, and advancing amber research in general. Any advance in the study of Canadian amber faces a number of obstacles. First, there has been little in the way of synthetic work on the Canadian amber assemblage (e.g., McAlpine and Martin, 1969; Pike, 1995), so described taxa are scattered among the literature and reported taxa are often unverified. This renders comparisons between deposits difficult and limits palaeoecological inferences for any new taxa discovered. Second, it is unclear whether the two main sources of research material (Cedar Lake and Grassy Lake) represent a single amber deposit, justifying the treatment of their inclusions as a single collection. Chapter 2 addresses both of these longstanding issues, providing a synthesis of the systematic work conducted on Canadian amber inclusions, and utilizing multiple lines of evidence to demonstrate conclusively a single source for Canadian amber. Chapter 3 summarizes a number of alpha- papers on Hymenoptera in Canadian amber that have been completed during the course of this dissertation (McKellar and Engel, 2011a; McKellar and Engel, 2011b; Perrichot et al., in press), or are nearing completion (e.g., works on Scelionidae (McKellar and Engel, in prep.), Ichneumonidae (McKellar et al, in prep.) and Scolebythidae (Engel et al., in prep.)). As a result of the work encapsulated in Chapter 3, Hymenoptera are now the best understood of the insect orders in Canadian amber, and it is possible to make meaningful comparisons between this assemblage and other Cretaceous deposits.

*A version of this chapter has been published. McKellar, R.C., Wolfe, A.P. 2010. Canadian Amber, in: Penney, D. (Ed.), Biodiversity of fossils in amber from the major world deposits. Siri Scientific Press, Manchester, pp. 149—166.

1 Work on Hymenoptera within Canadian amber provided an opportunity to screen the entirety of the RTMP amber collection and view portions of the CNC- CAS collection, as well as to secure donations from private collectors and prepare new material from the Grassy Lake locality. During the course of this work, a secondary objective became apparent - describing inclusions of high scientific value beyond the Hymenoptera. Chapter 4 documents some of the new taxa described within Hemiptera (Microphysidae) and Neuroptera (Rhachiberothidae), as well as connotations for their higher-level taxa. Chapter 5 documents the largest and most diverse assemblage of feathers yet to be recovered from Mesozoic amber. These integumentary structures include morphotypes similar to the protofeathers found as compression fossils in association with dinosaur remains, as well as advanced morphologies indicative of specific behaviours. Together, these contributions help to raise the profile of Canadian amber, and broaden my experience in palaeoentomology. Creating the foundation for detailed work on the inclusions in Canadian amber (i.e., Chapter 2) also led to two advances for amber research in general. Analysis of the stable isotopic composition of numerous ambers demonstrated a pattern in the composition of carbon within certain deposits. Chapter 6 further examines this carbon-13 enrichment pattern in amber and its modern analogue, defensive resin. Ultimately, this demonstrates the role of insect attacks in the formation of some amber deposits, and the potential to obtain proxy data for palaeoclimate or palaeoecological events from the stable isotopic composition of amber. This new approach to the study of amber provides a better context for the biological inclusions within each deposit. Through a modification of existing preparation techniques (Appendix 1), it is also possible to extend the fossil record of Hymenoptera and other insect groups significantly within western Canada. This technique opens up the previously inaccessible record of biological inclusions in friable coal-associated ambers, and has the potential to fill in gaps in the Late Cretaceous and Paleocene fossil record of insects.

INTRODUCTION "Canadian amber" is a popular term for amber originating from the Campanian (Late Cretaceous) Grassy Lake locality in southern Alberta, which is also recovered as a secondary deposit along the shores of Cedar Lake in western Manitoba (McKellar et al., 2008, Chapter 2 herein). Although these two sites remain the most significant sources for arthropod inclusions, there are also a

2 number of additional Late Cretaceous and Paleogene strata in western Canada that bear appreciable quantities of amber, some of which is fossiliferous (Fig. 1.1).

^CANADAl

study area i^ff^ OKm 100 Km | | Paskapoo/Porcupine Hills/Scollard Undifferentiated Eastend/Frenchman and Ravenscrag ^H|| Horseshoe Canyon/St Mary River Undifferentiated Bearpaw Belly River/Oldman-Foremost Figure 1.1. A, Inset showing study area; B, Subcrop diagram of Upper Cretaceous formations in the prairie provinces, with the Saskatchewan River system superimposed. Fine lines and dark gray shading represent the course of river drainage and the position of lakes, respectively. Modified from fig. 24.1 of Dawson et al. (1994), and Energy, Mines and Resources Canada's (1985) Canada Drainage Basins map.

With increased exploration, it appears as though amber from western Canada will provide multiple windows into faunal and floral developments in the Campanian, Maastrichtian and Early Eocene (Archibald and Makarkin, 2004). This portion of the fossil record is of interest because it constitutes the last known diverse amber assemblages prior to the Cretaceous-Tertiary mass extinction event, as well as one of the few areas where both pre- and post-extinction assemblages can be compared. Here we focus upon the better understood Campanian Canadian amber, with some discussion of additional inclusion-bearing localities in early stages of investigation, namely other Cretaceous sites in Alberta and the Eocene deposit at Hat Creek, British Columbia.

3 GEOLOGICAL SETTING The most extensively studied Canadian amber deposit is that at Grassy Lake, Alberta, for which the in situ source is the Taber coal zone, near the top of the Foremost Formation (Fig. 1.2). This provides a range of geological constraints for both this amber and derived secondary occurrences downstream, of which the most notable occurs at Cedar Lake. Six coal seams make up the Taber coal zone, and two of these seams are exposed at Grassy Lake. Due to limited exposure, it

Figure 1.3. Surface collecting at the Grassy Lake tailings piles. 'JFi

Figure 1.4. A surface-concentrated amber fragment at the Grassy Lake site (1.4 cm).

DBuff sandstone with minor siltstone and mudstone; foreland or alluvial H Interbedded buff sandstone and olive green siltstone, mudstone and coal; coaly alluvial I Mauve shale, tuffaceous; paleosols and volcanic ash H Interbedded light grey sandstone, siltstone, dark grey shale and coal; paralic, variably coaly BDark grey shale with minor siltstone; fully marine ^Inclusion-bearing amber present Figure 1.2. Stratigraphic diagram for the main Albertan amber-bearing units (after Dawson et al., 1994; MacEachern and Hobbs, 2004; dates from Eberth & Hamblin, 1993; Eberth, 2002). Tertiary, Paleocene, Maastrichtian, abbreviated; Fm., Formation; Gp., Figure 1.5. Small rod-shaped amber pieces Group; * denotes diachronism. found within sub-bituminous coal Northwest of Drumheller. is unclear which of the six coal seams outcrop at Grassy Lake. Amber is found within the upper sub-bituminous coal, which is approximately 70 cm thick, as well as within the thin (30 cm) organic-rich shales overlying it (Pike, 1995). The distribution of amber is somewhat patchy within these sediments. Other ambers are found within or associated with a large number of coals or lignitic units throughout the strata of western Canada (McAlpine and Martin, 1969; Pike, 1993). A full account of western Canadian ambers is beyond the scope of this work, but it is worth noting that a small number of insect inclusions have been confirmed from a few of the reported Late Cretaceous deposits, including early Maastrichtian portions of the Horseshoe Canyon Formation in the vicinity of Drumheller, Alberta; late Campanian and earliest Maastrichtian portions of the Horseshoe Canyon Formation in the vicinity of Edmonton, Alberta (Appendix 1); and from amber in the late Campanian Pachyrhinosaurus bone bed within the Wapiti Formation near Grande Prairie, Alberta (Tanke, 1994). These strata and the Early Eocene Hat Creek Coal Formation near the village of Cache Creek, British Columbia, are predominantly interpreted as the product of broad marshes that deposited in marginal marine or lowland (Archibald and Makarkin, 2004) positions, respectively.

AMBER COLLECTION Canadian amber was first reported by Joseph Burr Tyrrell (1891) as a placer deposit along the shores of Cedar Lake, in western Manitoba. Cedar Lake amber or Chemawinite was initially collected in bulk and concentrated from among the woody debris lining the beach for the purpose of manufacturing varnish (McAlpine and Martin, 1969). It was not until the pioneering works of Carpenter et al. (1937) that insect inclusions were described from this deposit. Large collections of Cedar Lake amber were subsequently established at the Harvard University Museum of Comparative Zoology (Cambridge, Massachusetts), the Canadian National Collection of Insects and Arthropods (Ottawa, Ontario), and the Royal Ontario Museum (Toronto, Ontario). When the Grand Rapids Dam was completed at the outlet of Cedar Lake in 1965, the amber-rich beaches were submerged, rendering established collections the only source of material from the site. Recently, amber has begun to accumulate along the strandline once again (Poinar, 1992; Graham Young, pers. comm.), but its abundance has not yet approached the levels observed by early collectors. In 1963, P. Boston discovered amber with insect inclusions at a site 5 near Medicine Hat, in southern Alberta (McAlpine and Martin, 1966). Early work on the entombed fauna showed similarities to the assemblage known from Cedar Lake, prompting McAlpine and Martin (1969) to assign the latter deposit a Late Cretaceous age, and extend the term 'Canadian amber' to this new deposit. Strata bearing Campanian (Late Cretaceous) amber are exposed in outcrop at a number of sites in southern Alberta. The main locality for collecting Canadian amber is the Grassy Lake site; near the Village of Grassy Lake and southwest of Medicine Hat (full locality details are available from the Royal Tyrrell Museum of Palaeontology upon request). Here, abandoned pit mine operations have left behind large tailings piles (Fig. 1.3) comprised of portions of two of the uppermost coal seams within the Taber Coal Zone and some of the shale beds adjacent to these seams. Because amber nodules behave as resistant clasts within the loose tailings, and possess a specific gravity relatively close to that of water, they are concentrated on the surface each time it rains (Fig. 1.4). Larger pieces of amber are collected by hand or with forceps. These pieces can range in size from a few millimeters to 3.5 centimeters, but the majority of pieces are one centimeter or less in diameter (Pike, 1995). The amber itself is typically dark yellow in colour with dark brown drying lines, and is often coated with a weathering rind or carbonaceous matrix. These characteristics make the amber difficult to spot among the tailings. Bright sunlight elicits a mild green fluorescence from fractured surfaces on the amber nodules, aiding in their surface collection. Alternative methods for bulk collection employed at the site include the mechanical screening and specific gravity (floatation) separation techniques utilized in the study of collection biases by Pike (1993; 1995). Additional amber deposits with inclusions are associated with some of the Cretaceous and Tertiary coals in western Canada (localities are discussed above). Unfortunately, most of these deposits are composed of friable amber dominated by nodules 5 mm or smaller, that lack the robustness to survive prolonged exposure at the surface or even incautious handling. Trace amounts of amber are surface-collected from these sites as freshly weathered nodules, but often amber must be extracted from the coal face using a scalpel and forceps (Fig. 1.5). The study and preservation of inclusions within this amber requires specialized epoxy embedding techniques, such as those of Nascimbene and Silverstein (2000). While amber pieces from the main Canadian amber deposits are more robust, they too benefit from embedding preparation.

6 Amber deposits within Canada have not gained the notoriety of some other Cretaceous ambers, largely because this material has never been developed as a commercial resource for jewelry making or for amateur inclusion collectors. Most western Canadian amber is not a lucrative resource for development because it is typically too brittle, dark or diminutive to suit this purpose. Furthermore, amber is protected under the Historic Resources Act in Alberta, and similar legislation is in place within the other prairie provinces of Canada.

PALAEOHABITAT The preliminary nature of the study of inclusions within Canadian amber hampers their use in detailed analyses of palaeoenvironment. The best-studied taxa with environmental connotations are the Ceratopogonidae studied by Borkent (1995). Taxa within this dipteran family have distinctive larval substrates. The ceratopogonid species and sex ratios observed within Canadian amber are suggestive of a marginal marine setting with a mixture of moist sands, wetland pools, and salt marsh habitats in close proximity to one another (Borkent, 1995). Body fossils found in strata adjacent to the amber include dermal scutes from crocodilians (pers. obs.), and foraminiferal tests of Haplophragmoides spp., interpreted as an indicator of brackish water deposition (Ogunyomi and Hills, 1977). Aside from these indicative fossils, the main source for palaeohabitat information is the interpreted depositional environment, based predominantly on lithology and stratigraphy. Grassy Lake amber has been interpreted as occurring within a series of coal and shale beds related to cycles of lagoonal and salt marsh deposition in the area (Ogunyomi and Hills, 1977; Pike, 1995). This interpretation is supported by what is known of the entombed and associated taxa.

THE CANADIAN AMBER TREE Among the unresolved questions surrounding fossiliferous Late Cretaceous ambers from western Canada is the identity of their source tree. Several chemotaxonomic efforts alternately using infrared and nuclear magnetic resonance spectroscopy have suggested that Canadian amber was produced by conifers belonging to the family Araucariaceae (Lambert et al, 1996; Zobel, 1999; Kosmowska-Ceranowicz et al., 2001; Aber and Kosmowska-Ceranowicz, 2001). This inference follows earlier contentions that the source tree belonged within the genus Agathis (Langenheim and Beck, 1968; Langenheim, 1969). However, strictly chemotaxonomic assignments to Araucariaceae are problematic from

7 a biogeographical perspective, because no araucarian remains are known from Canadian amber (Borkent, 1995), nor does Agathis have any confirmed Mesozoic distribution in the northern hemisphere (Stockey, 1982). We have reexamined the botanical source of Grassy Lake amber in greater detail, benefiting from the integration of newly-discovered botanical remains and an extensive spectroscopic database garnered by Fourier-transform infrared (FTIR) microspectroscopy. Botanical remains are rare in Late Cretaceous ambers from western Canada, having previously been noted only by Borkent (1995), who reported "Glyptostrobus twigs" from Grassy Lake material. Vascular plants comprise less than one percent of total fossil inclusions, and our new observations are limited to isolated conifer needles and the tips of needle-bearing shoots (Fig. 1.6). Although cuticular details, such as stomata, are not preserved within these specimens, they appear to share a common botanical origin within the conifer family Cupressaceae. Needle phyllotaxy in each case is helically arranged, as in Parataxodium Arnold and Lowther, and never decussate as in the closely related genus Metasequoia Miki. The amber-hosted specimens compare favorably to exquisitely-preserved silicified Parataxodium foliage from the Horseshoe Canyon Formation of Alberta (Aulenback, 2009). Parataxodium is an extinct genus of Cretaceous cupressaceous tree, with close affinities to Bald cypress (Taxodium) and Chinese Swamp cypress (Glyptostrobus). It was once a common tree in floodplains, lowland forests, and swamps, spanning western North America northwards to Alaska (Arnold and Lowther, 1955; Mclver, 2002). Because these are the only botanical fossils documented to date from Cretaceous Canadian amber, we hypothesize that Parataxodium is the most probably source tree, and test this hypothesis using infrared spectroscopic data (Fig. 1.7). Canadian amber produces two distinct Fourier-Transform infrared (FTIR) spectral fingerprints, which were originally interpreted as indicating two distinct source trees (e.g., Christiansen and Pike, 2002). It has since been shown that this degree of spectroscopic variability is attributable to the amount of weathering and transport of the material ex situ, that is, beyond the primary sedimentary position in which it was originally hosted (McKellar et al., 2008, Chapter 2 herein). Thus, Cedar Lake, Horsethief Canyon, and to a variable extent Grassy Lake ambers all have somewhat muted FTIR spectra, characterized by a pronounced shift of the dominant C=0 stretching band to 1720 cm1 (Fig. 1.7). Fresher ambers retrieved directly from coals at localities in Edmonton, Drumheller, and Medicine Hat retain the 1695 cm1 position of the dominant C=0 absorbance band that

8 Figure 1.6. Modern and Late Cretaceous cupressaceous foliage bearing on the identity of Canadian amber- producing trees. A, Modern Metasequoia glyptostroboides foliage; B and F, Silicified remains of Parataxo- dium extracted from the Horsethief Canyon Formation (from Aulenback, 2009, reproduced with permission); C-E, H-G, conifer foliage recovered from Grassy Lake amber, including needle-bearing shoots (C-D, G), and close-up of an isolated needle demonstrating shrunken and coalified condition of remains (H). E is a line diagram illustrating the helically arranged phyllotaxy of needles in specimen D, which is evident in all the amber-hosted specimens. • Canadian Late Cretaceous ambers • Other ambers • Modern resins

Baltic amber

Modern Agathis austrahs

Cedar Lake, MB

Horsethief Canyon, AB

Grassy Lake, AB o

Italian Tnassic amber •s > "5 Edmonton, AB

Drumheller, AB

Medicine Hat, AB Vf^-A_ Giraffe Pipe, NT, Eocene Metasequoia sp. resinite

Modern Metasequoia glyptostroboides

Modern Thu/a standishn

Modern Chamaecyparis nootkatensis 4000 3500 3000 2500 2000 1500 1000 Wavenumber (cm"1) Figure 1.7. FTIR spectra of Cretaceous Canadian ambers, select modern conifers, and additional fossil resins Spectroscopic methods are described in detail by McKellar et al (2008) Grey lines indicate the regions of prominent spectral absorbance used in chemotaxonomic assignment, whereas the small black arrow indicates the shifting C=0 peak that we interpret as a weathering artifact The Italian Tnassic amber is attnbuted to the extinct family Cheirolepidiaceae (Roghi et al, 2006), while Baltic amber has affinities with Sciadopityaceae (Wolfe et al, 2009) 10 is characteristic of modern Cupressaceae. Furthermore, these unweathered specimens have far less muted fingerprint regions (1600-900 cm1), which again express considerable similarity to modern cupressaceous genera, while differing substantially from the spectrum of Agathis australis (Fig. 1.7). Taken together, the entombed botanical remains and the FTIR spectroscopic results suggest that cupressaceous amber-producing forests persisted in western Canada throughout much of the Late Cretaceous, and that Parataxodium was an important, if not dominant, element of this ecosystem. Much of the amber is stratigraphically associated with coal seams or adjacent shale units, and lignitic stringers within medium-grained sandstone, which is compatible with the envisaged ecology of Parataxodium as a swamp and floodplain inhabitant. Parataxodium was therefore quite likely involved in the genesis of some western Canadian Cretaceous coal measures, to an extent that remains to be determined.

AGE OF CANADIAN AMBER As an in situ exemplar of Canadian amber, material from Grassy Lake permits an absolute age assignment for the formation of the main insect-bearing amber deposit. The amber is approximately 78 to 79 million-years-old, based upon radiometric dates obtained from bentonites within the shales that encompass the Taber coal zone. Eberth and Deino (1992) provide a lower age bracket for the base of the Foremost Formation at 79.14±0.15 million-years-ago. The Taber coal zone occurs within the Foremost Formation, just beneath its contact with the overlying Oldman Formation. Bentonites from within and just above this interval have been dated at 78.2±0.2 million-years-ago (Goodwin and Deino, 1989; Penney, 2006), based upon argon-argon analyses of material from Kennedy Coulee, a nearby site within Montana, USA. Although it is not possible to determine which of the upper seams within the Taber coal zone outcrop at Grassy Lake, it is possible to constrain the amber deposit within a relatively short time span. Grande Prairie amber from the Pipestone Creek Pachyrhinosaurus bone bed occurs 27 meters beneath a bentonite that was potassium-argon dated at 73.27 ± 0.25 million-years-ago (Tanke, 1994). This establishes a limit for how young the amber could be, and places the time of deposition within an interval when most positions east and south of the area experienced a marine transgression of the western interior seaway. Ambers from the Horseshoe Canyon Formation in the vicinity of 11 Edmonton and Drumheller occur in association with laterally extensive coal seams. These seams have been assigned numbers throughout the much of southern Alberta, but correlation of the upper portions of this series between outcrop in the vicinity of Edmonton and the thoroughly investigated exposures near Drumheller is somewhat tentative (Campbell, 1993; Chen et al., 2005). At present, it is not possible to assign an absolute age to the Horseshoe Canyon Formation amber deposits. It appears as though Horsethief Canyon material from the vicinity of Drumheller is early Maastrichtian in age, while Edmonton material is late Campanian (Whitemud Creek site) and earliest Maastrichtian (Edmonton river valley site) in age.

PHYSICAL AND CHEMICAL PROPERTIES Unprepared collections of Grassy Lake amber at the Royal Tyrrell Museum of Palaeontology, in Drumheller, Alberta, and at the University of Alberta confirm Pike's (1993; 1995) observations that the average size of surface collected specimens is usually less than 1 cm, with an average mass of 0.26 g (surface collected) or 0.06 g (screened samples). In terms of volume, most Grassy Lake amber is representative of external resin flows (Schlauben) and is somewhat flattened in one dimension. Stalactite-like repetitive flows appear to contain elevated numbers of insect inclusions, and most of the incidences of multiple inclusions. Insect inclusions appear most abundant in positions adjacent to drying surfaces, and regularly have wings or appendages trapped within a drying surface. Nodules are often fragmented, resulting in blocky pieces with conchoidal fractures along their broken edges. Amber colours range from pale yellow to deep red-orange, but the bulk of the material is a rich orange-yellow. Amber morphologies other than Schlauben are represented by smaller numbers of nodules in Grassy Lake collections, including isolated drops that retain their original shape, as well as disc-shaped pieces often interpreted to represent resin pockets within the tree. Small quantities of milky or 'bone' amber, clouded by many tiny bubbles, are also present. Specimens that have been surface-exposed for prolonged periods of time develop a thin weathering patina (<1 mm thick), and lack the coating of carbonaceous material that is often attached to fresh specimens. Very small nodules (<5 mm in size) appear to develop a pervasive granular texture and become slightly crumbly as a result of extensive weathering. Canadian amber from both the Grassy Lake and Cedar Lake sites share the aforementioned physical characteristics, and also display a remarkably broad

12 range of C and H stable isotope compositions. 513C values range from -27.1%o to -20.0%o with a mean of -23.6%o, while 5D values range from -353.3%o to -252.5%o with a mean of -297.8%o (McKellar et al, 2008). Taken in the context of a shared source tree for the amber within the deposit, these ranges likely denote a wide variety of tree stress levels or pronounced canopy effects (513C values), and a variable water supply for the trees or pronounced weathering effects upon the amber (5D values) (Nissenbaum and Yakir, 1995; Murray et al., 1998). Work is currently underway to clarify the effects of some of these variables upon the isotopic composition of amber, and to characterize fully the additional amber deposits of Alberta.

THE DIVERSITY OF CANADIAN AMBER INCLUSIONS Research upon the inclusions within Canadian amber is largely within its preliminary stages. Although the Cedar Lake amber fauna was published upon as early as 1937 (Carpenter et al., 1937), the loss of the Cedar Lake collecting site and the uncertainty that surrounded its connection with Grassy Lake amber hampered further study. McAlpine and Martin (1969) provided a general overview of Canadian amber inclusions, but it was not until Pike (1995) completed his dissertation on Grassy Lake amber that a family level survey with a large specimen set was attempted. Subsequently, Skidmore (1999) provided a summary of specimen tag data for the Canadian National Collection of Insects and Arthropods amber collection, and McKellar et al. (2008, Chapter 2 herein) produced a literature-based summary of identified Canadian amber species and higher taxa. New taxa have since been introduced in the works of Liu et al. (2007), Engel and Grimaldi (2008; 2009), and McKellar and Engel (2009; 2011a; 201 lb). This list of known taxa has been further modified by subsequent work on Hymenoptera: the changes are summarized in Chapter 3. There are currently 134 valid species in 108 genera, 55 families, and 10 orders described from Canadian amber. In terms of identified material, these numbers are higher, with 170 species, 115 genera, 133 families and 23 orders represented. The work encompassed by this dissertation accounts for 38 species, 8 genera, and 4 of the families recognized within the deposit. Those insect orders that have the greatest recognized diversity are Hymenoptera (70 spp.), Diptera (50 spp.), and Hemiptera (23 spp.), largely reflecting the extent to which they have been studied. These taxa comprise 87% of the named species within the assemblage, and most species are based upon 13 single specimens. Assessing palaeodiversity in the Canadian amber assemblage is difficult at this time because of the strong bias created by differential research intensity among higher taxa. The species richness graph presented here (Fig. 1.8) should be used cautiously, as it is likely that continued research will strongly affect the relative diversity among arthropod orders. Groups such as (mites and ticks) appear abundant and diverse within the assemblage, yet this order is represented by only one described species within Bdellidae, based upon a single specimen (E wing, 1937).

2 2

.«** .eS® „«•.<* ^o^* .eS* ,e.<* c^ «<>.<* *&* kr/

Figure 1.8. Species richness plot of arthropod inclusions in Canadian amber (data updated from McKellar et al., 2008)

The best-studied insect order to date is Hymenoptera, with works on Megaspilidae, Braconidae, Serphitidae and Scelionidae (Fig. 1.9) (Brues, 1937); Megaspilidae, Stigmaphronidae, and Serphitidae (McKellar and Engel, 2011a; 2011b, Chapter 3); Mymaridae, Mymarommatidae, Tetracampidae and Trichogrammatidae (Yoshimoto, 1975); Chrysididae and Sphecidae (Evans, 1969); Dryinidae (Olmi, 1995); Protimaspidae (Kinsey, 1937); Liopteridae, Figitidae and Cynipidae (Liu et al, 2007); Maimetshidae (Perrichot et al., in press); and Formicidae (Wilson, 1985; Dlussky, 1999; Engel and Grimaldi, 2005). Dipteran species have been described from the families Atelestidae (Grimaldi and Cumming, 1999), Bibionidae (Peterson, 1975), Cecidomyiidae (Gagne, 1977), Ceratopogonidae and Chironomidae (Fig. 1.9) (Boesel, 1937; Borkent, 1995; Poinar et al., 1997), Culicidae (Poinar et al., 2000), Ironomyiidae (McAlpine, 1973), Lygistorrhinidae and (Blagoderov and Grimaldi, 2004), Phoridae and Sciadoceridae (McAlpine and Martin, 1966; Brown and Pike, 1990), 14 Stratiomyidae (Teskey, 1971) and Tipulidae (Krzeminski and Teskey, 1987; Brooks et al., 2007). Hemiptera are the only other insect order with a substantial number of named species, representing the families Jascopidae (Hamilton, 1971); Microphysidae (McKellar and Engel, in press); Canadaphidae, Drepanosiphidae and Palaeoaphidae (Essig, 1937; Richards, 1966; Heie, 1996; Kania and Wegierek, 2005; Heie 2006); Cretamyzidae and Mesozoicaphididae (Heie and Pike, 1992); Tajmyraphididae (Heie, 1996); andMargarodidaes./. (Beardsley, 1969). Neuroptera is represented by only two named species within Berothidae (Klimaszewski and Kevan, 1986) and Rhachiberothidae (Fig. 1.10) (McKellar and Engel, 2009), Coleoptera (Fig. 1.10) are represented by two species within Bruchidae (Poinar, 2005) and Scydmaenidae (O'Keefe et al., 1997), 15 Figure 1.10. A, Neuroptera: Rhachiberothidae: Albertoberotha leuckorum McKellar and Engel, 2009 (TMP 91.148.790); B, Coleoptera (UA specimen); C, Hemiptera: Aphidoidea (UA specimen), all specimens from Grassy Lake. is known from one species within Sphaeropsocidae (Grimaldi and Engel, 2006), as is Trichoptera within Electralbertidae (Botosaneanu and Wichard, 1983). There is good evidence for the presence of numerous additional families (Table 1.1) based upon the work of Skidmore (1999), which contains determining authorities for many specimens housed in the Canadian National Collection of Insects and Arthropods. The prevalence of small nodules within Canadian amber all but precludes the preservation of larger taxa, leaving many of the non-insect arthropods represented by fragmentary remains of limited taxonomic value. Arthropod specimens have been identified within orders such as Blattaria (roaches, Skidmore, 1999) Pseudoscorpionida (pseudoscorpions, Schawaller, 1991) and Isoptera (termites, Engel and Delclos, 2010), but few species have been described. It is not currently possible to distinguish between the actual rarity of other arthropod orders in the Canadian amber fauna, and the potential taphonomic constraints on their occurrence as inclusions. The diminutive Collembola 16 Table 1.1: Arthropod families recorded from Canadian amber

ARACHNIDA Canadaphidae Protimaspidae Acari Collembola Cercopidae Scelionidae Bdellidae BrachystomeUidae Cicadellidae Scolebythidae Camisiidae Hypogastruridae Cixiidae Serphitidae Cunaxidae Isotomidae Cretamyzidae Stigmaphronidae Erythraeidae Neanuridae Drepanosiphidae Tetracampidae Eupodidae Oncobryidae Fulgoridae Torymidae Gymnodamaeidae Poduridae Issidae Trichogrammatidae Hermanniidae Protentomobryidae Jascopidae Oribatidae Sminthuridae Margarodidaes./. Neuroptera Parasitidae Tomoceridae Membracidae Berothidae Mesozoicaphididae Chrysopidae Araneae Diptera Microphysidae Coniopterygidae Araneidae Anisopodidae Hemerobiidae Ctenidae Atelestidae Palaeoaphidae Raphididae Erigonidae Bibionidae Psyllidae Rhachiberothidae Huttoniidae Bombyliidae Reduviidae Sisyridae Lagonomegopidae Canthyloscelididae Tajmyraphididae Linyphiidae Cecidomyiidae Psocoptera Oonopidae Ceratopogonidae Hymenoptera Liposcelidae Theridiidae Chironomidae Aulacidae Sphaeropsocidae Culicidae Bethylidae Pseudoscorpiones Dolichopodidae Braconidae Rhaphidioptera Chernetidae Empididae Ceraphronidae Mesoraphidiidae Ironomyiidae Chrysididae HEXAPODA Lygistorrhinidae Crabronidae Thysanoptera Coleoptera Mycetophilidae Cynipidae Aeolothripidae Bruchidae Phoridae Diapriidae Lophioneuridae Carabidae Platypezidae Dryinidae Thripidae Chrysomelidae Psychodidae Eulophidae Cleridae Rhagionidae Eupelmidae Trichoptera Curculionidae Scatopsidae Figitidae Electralbertidae Dascillidae Sciadoceridae Formicidae Psychomyiidae Eucnemidae Sciaridae Ichneumonidae Helodidae Stratiomyidae Liopteridae Zygentoma Mordellidae Tipulidae Maimetshidae Lepismatidae Orthoperidae Megaspilidae Scydmaenidae Hemiptera Mymaridae Staphylinidae Adelgidae Mymarommatidae Trogositidae Anthocoridae Platygastridae Caliscelidae Proctotrupidae

(springtails) are best-represented with eight species described from the families BrachystomeUidae, Isotomidae, Neanuridae, Oncobryidae, Protentomobryidae, Sminthuridae and Tomoceridae (Folsom, 1937; Christiansen and Pike, 2002). Aranae (spiders) have also been described, with two species in the families Lagonomegopidae (Penney, 2004) and Oonopidae (Penney, 2006).

17 The only account of plant inclusions to date is provided above, within our discussion of the amber source tree. In addition to these specimens, there are numerous samples containing fungal hyphae, lichen fragments, and palynomorphs within the Royal Tyrrell Museum of Palaeontology collections, but these have not yet been described. In terms of abundance, Canadian amber is dominated by aphids (Fig. 1.10) and mites. Out of the 1155 individuals classified by Pike (1995), 338 aphids and 174 mites were catalogued. It is not uncommon to find multiple individuals within a single amber piece, and more than 30 aphids have been observed within one nodule. This contrasts with some other Cretaceous ambers, such as New Jersey amber, which contain coccids (scale insects) as the primary plant-feeders. The discrepancy between lower trophic tiers within insect assemblages is most likely attributable to differences in forest types or source plants for the amber within each deposit. In turn, inter-deposit differences in higher trophic tiers may be connected to the predominance of aphids or scale insects, and form a source for comparisons of predators and parasitoids between deposits. The relatively high diversity and abundance of the hymenopteran family Serphitidae within Canadian amber may suggest that this extinct family was associated with some of the entombed aphids or other potential hosts. Further work on the diversity and abundance of insect inclusions in Cretaceous amber has the potential to elucidate these palaeoecological relationships. It is hoped that by comparing the numerous ambers that occur within Late Cretaceous strata in western Canada to each other, it will be possible to provide an account of the amber producing forests and their constituents throughout much of this key time interval. The Albertan series of deposits is one of the few instances where a relatively consistent set of depositional environments have repeatedly preserved amber, and in many cases have preserved amber faunas. If these faunas are studied more extensively, they have the potential to provide a record often million years of insect without the uncertainty associated with comparisons across broad geographic areas or disparate environments. Furthermore, this time interval spans part of the adaptive radiation of insects alongside the rise of angiosperms. Comparison to amber faunas after the Cretaceous-Tertiary mass extinction also elucidates the effects of this event on insect biodiversity.

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Langenheim, J.H., 1969. Amber: a botanical inquiry. Science 163, 1157-1169. Langenheim, J.H., Beck, C.W., 1965. Infrared spectra as a means of determining botanical sources of amber. Science 149(3679), 52-55. Langenheim, J.H., Beck, C.W., 1968. Catalogue of infrared spectra of fossil resins (ambers): I North and South America. Botany Museum Leaflets, Harvard University 22(3), 65-120. Liu, Z., Engel, M.S., Grimaldi, D.A., 2007. Phylogeny and geological history of the cynipoid wasps (Hymenoptera: Cynipoidea). American Museum Novitates 3583, 1-48. MacEachern, J. A., Hobbs, T.W., 2004. The ichnological expression of marine and marginal marine conglomerates and conglomeritic intervals, Cretaceous Western Interior Seaway, Alberta and northeastern British Columbia. Bulletin of Canadian Petroleum Geology 52(1), 77-104. McAlpine, J.F., 1973. A fossil ironomyiid fly from Canadian amber (Diptera: Ironomyiidae). Ibid 105, 105-111. McAlpine, J.F., Martin, J.E.H., 1966. Systematics of Sciadoceridae and relatives

22 with descriptions of two new genera and species from Canadian amber and erection of family Ironomyiidae (Diptera: Phoroidea). Canadian Entomologist 98, 527-545. McAlpine, J.F., Martin, J.E.H., 1969. Canadian amber - a paleontological treasure-chest. Canadian Entomologist 101, 819-838. Mclver, E.E., 2002. The paleoenvironment of Tyrannosaurus rex from southwestern Saskatchewan, Canada. Canadian Journal of Earth Sciences 39,207-221. McKellar, R.C., Engel, M.S., 2009. A new thorny lacewing (Neuroptera: Rhachiberothidae) in Canadian Cretaceous amber. Journal of the Kansas Entomological Society 82(2), 114-121. McKellar, R.C., Engel, M.S., 2011a. The serphitid wasps (Hymenoptera: Proctotrupomorpha: Serphitoidea) of Canadian Cretaceous amber. Systematic Entomology 36(1), 192-208. McKellar, R.C., Engel, M.S., 2011b. New Stigmaphronidae and Megaspilidae (Hymenoptera: Ceraphronoidea) from Canadian Cretaceous amber. Cretaceous Research, doi:10.1016/j.cretres.2011.05.008. [In press, available online]. McKellar, R.C., Engel, M.S., (in press). First Mesozoic Microphysidae (Hemiptera): a new genus and species in Late Cretaceous amber from Canada. The Canadian Entomologist. McKellar, R.C., Wolfe, A.R, Tappert, R., Muehlenbachs, K., 2008. Correlation of Grassy Lake and Cedar Lake ambers using infrared spectroscopy, stable isotopes, and palaeoentomology. Canadian Journal of Earth Sciences 45(9), 1061-1082. Murray, A.R, Edwards, D., Hope, J.M., Boreham, C.J., Booth, W.E., Alexander, R.A., Summons, R.E., 1998. Carbon isotope biogeochemistry of plant resins and derived hydrocarbons. Organic Geochemistry 29(5-7), 1199— 1214. Nascimbene, P., Silverstein, H., 2000. The preparation of fragile Cretaceous ambers for conservation and study of organismal inclusions, in: Grimaldi, D.A. (Ed.), Studies on fossils in amber, with particular reference to the Cretaceous of New Jersey. Backhuys Publishers, Leiden, pp. 93-102. Nissenbaum, A., Yakir, D., 1995. Stable isotope composition of amber, in: Anderson K.B., Crelling J.C., (Eds.), Amber, resinite and fossil resins. American Chemical Society Symposium Series 617, 32^42.

23 Ogunyomi, O., Hills, L.V., 1977. Depositional environments, Foremost Formation (Late Cretaceous), Milk River area, southern Alberta. Bulletin of Canadian Petroleum Geology 25(5), 929-968. O'Keefe, S., Pike, T., Poinar, G., 1997. Palaeoleptochromus schaufussi (gen. nov., sp. nov.), a new antlike stone beetle (Coleoptera: Scydmaeniidae) from Canadian Cretaceous amber. Canadian Entomologist 129(3), 379-385. Olmi, M., 1995. Dryinids and embolemids in amber (Hymenoptera Dryinidae et Embolemidae). Redia 78(2), 253-271. Penney, D., 2004. Cretaceous Canadian amber spider and the palpimanoidean nature of lagonomegopids. Acta Palaeontologica Polonica 49(4), 579-584. Penney, D., 2006. Fossil oonopid spiders in Cretaceous ambers from Canada and Myanmar. Palaeontology 49(1), 229-235. Penney, D., Selden, P.A., 2006. First fossil Huttoniidae (Arthropoda: : Araneae) in late Cretaceous Canadian amber. Cretaceous Research 27, 442^46. Perrichot, V., Ortega-Bianco, J., McKellar, R.C., Delclos, X., Azar, D., Nel, A., Tafforeau, P., Engel, M.S., (in press). New and revised maimetshid wasps from Cretaceous ambers (Hymenoptera, Maimetshidae). ZooKeys. Peterson, B.V., 1975. A new Cretaceous bibionid from Canadian amber (Diptera: Bibionidae). Canadian Entomologist 107, 711-715. Pike, E.M., 1993. Amber taphonomy and collecting biases. Palaios 8, 411^419. Pike, E.M., 1995. Amber taphonomy and the Grassy Lake, Alberta, amber fauna. Ph.D. thesis. Department of Biological Sciences, University of Calgary, Calgary, Alberta. Poinar, G.O. Jr., 1992. Life in amber. Stanford University Press, Stanford, California. Poinar, G.O. Jr., 2005. A Cretaceous palm bruchid, Mesopachymerus antiqua, n. gen., n. sp. (Coleoptera: Bruchidae: Pachymerini) and biogeographical implications. Proceedings of the Entomological Society of Washington 107(2), 392-397. Poinar, G.O. Jr., Krantz, G.W., Boucout, A.J., Pike, T.M., 1997. A unique Mesozoic parasitic association. Naturwissenschaften 84, 321-322. Poinar, G.O. Jr., Zavortink, T.J., Pike, T., Johnston, P.A., 2000. Paleoculicis minutus (Diptera: Culicidae) n. gen., n. sp., from Cretaceous Canadian amber, with a summary of described fossil mosquitoes. Acta Geologica Hispanica 35(1-2), 119-128.

24 Roghi, G., Ragazzi, E., Gianolla, R, 2006. amber of the Southern Alps (Italy). Palaois 21, 143-154. Richards, W.R., 1966. Systematics of fossil aphids from Canadian amber (Homoptera: Aphididae). Canadian Entomologist 98, 746-760. Schawaller, W., 1991. The first Mesozoic pseudoscorpion, from Cretaceous Canadian amber. Palaeontology 34, 971-976. Skidmore, R.E., 1999. Checklist of Canadian amber inclusions in the Canadian National Collection of Insects. Research Branch Agriculture and Agri- Food Canada electronic publication. Available from www.biology.ualberta. ca/facilities/strickland/SKIDMORECNCCanadianAmberlnclusions.pdf [cited 22 December, 2009]. Stockey, R.A., 1982. The Araucariaceae: an evolutionary perspective. Review of Palaeobotany and Palynology 37, 133-154. Tanke, D.H., 2004. Mosquitoes and mud: the 2003 Royal Tyrrell Museum expedition to the Grande Prairie region (northwestern Alberta, Canada). Alberta Paleontological Society Bulletin 19(2), 3-31. Teskey, H.J., 1971. Anew soldier fly from Canadian amber (Diptera: Stratiomyidae). Canadian Entomologist 103, 1659-1661. Tyrrell, J.B., 1891. Fossil resin ("amber"). Canadian Geological Survey Annual Reports 5, 14-15. Wilson, E.O., 1985. Ants from the Cretaceous and Eocene amber of North America. Psyche 92, 205-216. Wolfe, A.P., Tappert, R., Muehlenbachs, K., Boudreau, M., McKellar, R.C., Basinger, J.F., Garrett, A., 2009. A new proposal concerning the botanical origin of Baltic amber. Proceedings of the Royal Society of London B: Biology 276, 3403-3412. Yoshimoto, CM., 1975. Cretaceous chalcidoid fossils from Canadian amber. Canadian Entomologist 107, 499-532. Zobel, A.M., 1999. Cedarite and other fossil resins in Canada, in: Investigations into amber: proceedings of the international interdisciplinary symposium Baltic amber and other fossil resins. Gdansk, Poland, pp. 241-245. CHAPTER 2: CORRELATION OF GRASSY LAKE AND CEDAR LAKE AMBERS USING INFRARED SPECTROSCOPY, STABLE ISOTOPES, AND PALAEOENTOMOLOGY1*

INTRODUCTION Historical background The Canadian amber deposits at Grassy Lake (southern Alberta) and Cedar Lake (western Manitoba) are renowned internationally because of the richness of their arthropod inclusions (Pike 1994). These ambers are particularly important because they were formed during a crucial time for understanding the adaptive radiations of modern insect groups and the co-evolutionary processes associated with the rise of angiosperms. Furthermore, these faunas closely predate the K/T extinction event, providing an important baseline with which to assess its consequences for terrestrial arthropods.

Cedar Lake amber The study of Cedar Lake amber can be traced as far back as 1890, and includes such famous personages as the geologist Joseph Tyrrell and the palaeoentomologist Frank Carpenter (Grimaldi 1996). Amber from this locality has been referred to as chemawinite or cedarite (Aber and Kosmowska- Ceranowicz 2001), and the former name has been incorrectly extended by some authors to encompass the bulk of Canadian amber. The most extensive reviews of this deposit are those of Carpenter et al. (1937) and subsequently McAlpine and Martin (1969). While both provide general overviews of the arthropod fauna, and the former includes detailed descriptions for some taxa, an exhaustive taxonomic survey of the Cedar Lake fauna has not been attempted. Amber along the beaches of Cedar Lake was once so plentiful that the Hudson's Bay company collected it for manufacturing varnish, and early collecting trips were able to collect > 100 kg of raw amber with relative ease. Later collections saw diminishing returns, but still found faunal inclusions in 2% of the raw amber collected (Mc Alpine and Martin 1969). Construction of the Grand Rapids dam, completed in 1965, inundated the amber-rich beach. This rendered the collections at the Harvard University Museum of Comparative Zoology (Boston), the Royal Ontario Museum (Toronto), and the Canadian National

*A version of this chapter has been published. McKellar, R.C., Wolfe, A.P., Tappert, R., Muehlen- bachs, K., 2008. Correlation of Grassy Lake and Cedar Lake ambers using infrared spectroscopy, stable isotopes, and palaeoentomology. Canadian Journal of Earth Sciences 45(9), 1061-1082. 26 Collection of Insects and Arthropods (Ottawa) the bases for most published work associated with the deposit. Given the importance of Cedar Lake amber, it has been characterized geochemically using both infrared spectroscopy (Langenheim and Beck 1968; Kosmowska-Ceranowicz et al. 2001; Aber and Kosmowska- Ceranowicz 2001), and stable isotopes (Nissenbaum and Yakir 1995). In recent syntheses on amber (e.g., Rice 1980; Poinar 1992; Grimaldi 1996), the provenance of Cedar Lake amber is given merely as 'near Medicine Hat', and the deposit has been assigned cautiously a Late Cretaceous age.

Grassy Lake amber Amber from Grassy Lake was first mentioned as a coal mine associated deposit "near Medicine Hat, Alberta" discovered by P. Boston in 1963 (McAlpine and Martin 1966, p.531). Aside from isolated references in the entomological literature, Grassy Lake amber was largely unexplored until Pike's (1995) dissertation on the locality. Pike (1995) provided a provisional taxonomy for many of the arthropod inclusions, as well as a detailed account of taphonomy and

Figure 2.1. (A) Inset showing study area. (B) Subcrop diagram of the Oldman and Foremost Formations in the prairie provinces, with the Saskatchewan River system superimposed. Light gray shading indicates the extent of the outcrop and subcrop of the Oldman-Foremost Formations and the Belly River Group. Fine lines and dark gray shading represent the course of river drainage, and the position of lakes, respectively. This diagram is modified from fig. 24.1 of Dawson et al. (1994), and Energy, Mines and Resources Canada's (1985) Canada Drainage Basins map. 27 collection biases. Subsequent collaborations described portions of the fauna in detail (e.g., Heie and Pike 1992, 1996; O'Keefe et al. 1997; Poinar et al. 1997), while others have reported on additional arthropod groups represented in the deposit (e.g., Yoshimoto 1975; Gagne 1977; Wilson 1985; Penney and Selden 2006). Thus, the palaeoentomology of Grassy Lake amber is somewhat better understood than that of the Cedar Lake deposit. Grassy Lake amber is associated with a thin (~70 cm thick) coal seam and two 30 cm thick overlying shale beds. Large portions of this coal seam (below the amber containing interval) have been strip-mined in the region south of the village of Grassy Lake, Alberta, and the resulting tailings piles are the main source of amber. The amber forms resistant nodule-shaped masses that weather out of the disturbed sediments and are easily surface-collected, but the distribution of amber within these sediments is variable. The majority of curated specimens from Grassy Lake are located at the Royal Tyrrell Museum in Drumheller, Alberta, Canada. Due to the relatively recent interest in Grassy Lake amber, basic analyses of physical properties, stable isotopic composition, and infrared spectra have been limited to date (e.g., Zobel 1999).

Stratigraphic constraints From these pioneering investigations it has become evident that the Cedar and Grassy Lake ambers might be related in at least a general sense. It was obvious to J.B. Tyrrell that Cedar Lake amber was a secondary deposit that must have originated somewhere within the Cretaceous of the prairies (Carpenter 1937). By following the course of the Saskatchewan River drainage upstream, and identifying potential source rocks that it incises, there are a wide range of Cretaceous strata that may have originally contained the amber (Fig. 2.1). The matter is complicated by the areal extent of lithologically-similar marginal to fully marine sediments, as well as flood plain deposits, that may host amber-rich coal measures. Fortunately, the arthropod assemblage contained in Cedar Lake amber suggests a Campanian age (McAlpine and Martin 1969), which greatly restricts the extent of potential source rocks (shaded area of Fig. 2.1). Even if the arthropods are assigned to a broader time interval (e.g., Santonian to Maastrichtian) the putative source formations are limited by the conditions required for amber accumulation. Large amber concentration deposits are generally found in association with coal and lignified plant tissues, which are

28 often associated with deltaic and coal-producing terrestrial and near-shore environments (Grimaldi 1996). This focuses attention on the Foremost Formation, and to a limited extent upon the younger Oldman, Dinosaur Park, and Horseshoe Canyon Formations, and the older Milk River Formation. Amber has a specific gravity lower than that of salt water, so a concentration deposit within distal, fully marine sediments is unlikely. This effectively excludes the marine Bearpaw Formation overlying the Foremost, and the marine Pakowki Formation that underlies it (Fig. 2.2). Within the Foremost Formation, there are six coal seams that are also referred to as the Taber coal zone. However, it is unclear which UJ Southern South- Eastern

Deadhorse Deadhorse that amber is not widespread Coulee Mbr Coulee Mbr within these units, and that Virgelle Virgelle z Mbr < Mbr its occurrence may be mildly z o Telegraph Telegraph diachronous. Creek Mbr Creek Mbr < Hanson Mbr Thistle COLORADO Objectives of study COLORADO Mbr GP Despite the longstanding Dowling GP Mbr suggestion that the Grassy Lake Legend Q = no data due to unconformity and Cedar Lake amber deposits are Q = fully-marine units coeval, detailed comparisons of Figure 2.2. Stratigraphic diagram for Alberta through Santonian to Maastrichtian Stages, modified from text- the amber from these two localities figure 1 of Braman (2001) and fig 24 3 of Dawson et al have not been attempted using (1994) 29 multiple lines of geochemical and palaeobiological evidence. The methodologies employed in this paper aim to establish new guidelines for comparisons between in situ and reworked amber deposits, and explore the potential effects of long­ distance transport on the characteristics of amber. The present study also contributes the largest stable isotopic data set generated from amber to date, obtained with sufficient stratigraphic control to address the source and fate of these important amber deposits.

MATERIALS AND METHODS Materials Specimens of Grassy Lake amber were compared to Cedar Lake amber, as well as other Cretaceous ambers from the Horseshoe Canyon Formation (early Maastrichtian) collected at localities near Drumheller and Edmonton, Alberta. Additional comparisons were made with samples with less stratigraphic constraint from the vicinity of Medicine Hat, Alberta (Campanian). Cretaceous amber used in stable isotope analyses was collected from the surface of two coal seams separated by 10 m stratigraphically, at the Motrin Bridge crossing north of Drumheller (base of section at 51°38'50.3"N, 112°54'30.8"W); from a single coal seam in a road cut near the Royal Tyrrell Museum of Palaeontology, west of Drumheller (51°28'41"N, 112°47'20"W); and from a single coal seam at the bottom of Edmonton's North Saskatchewan River valley (53°31'38"N 113°29'27"W). In all cases, the amber samples were collected from within coal- seams, or from shales directly adjacent to a coal. Additional samples within the FTIR spectral library at the University of Alberta came from deposits within Horsethief Canyon, Alberta (near 51°32'21.9"N, 112°52'06.8"W). Purchased specimens from the Raritan Formation (Turonian) at Sayerville, New Jersey, U.S.A. were also analyzed, but there is little control over their collection.

FTIR spectroscopy Fourier Transform Infrared Spectroscopy (FTIR) was used to determine the IR absorption characteristics of ambers from each deposit. Numerous thin flakes (~5-10 um thick) were taken from both freshly fractured and weathered surfaces of Grassy Lake amber pieces. Samples were taken from a wide range of amber morphological types (isolated drips, schlauben, runnels, and disks) and amber clarity grades (opaque/bone, clear, and clear with a variety of inclusions). These flakes were placed on a NaCl disk and their infrared absorption was determined in transmitted mode, at wavelengths ranging from 2.5 to 15 urn (wavenumbers from 650 to 4000 cm1), using a Thermo-Nicolet Nexus 470 FTIR spectrometer equipped with a Nicolet Continuum IR microscope. For each spectrum 200 individual interferograms were averaged. In order to minimize the continuum and to avoid the necessity of baseline corrections, only samples with a consistent thickness have been analyzed. In order to objectively compare the FTIR spectroscopic results obtained from western Canadian amber samples, hierarchical (agglomerative) cluster analysis was undertaken (e.g., Naumann et al. 1991). Fourteen FTIR spectra were included in this analysis, representing different weathering states of Grassy Lake amber («=8), Cedar Lake amber («=2), and single exemplars for each of Horsethief Canyon, Medicine Hat, Edmonton, and Drumheller ambers. The fingerprint regions of each spectrum (800-1100 cm1) were first normalized to unity, in order to offset the potential influence of variable sample thickness and angle of incidence. The clustering employs Ward's (1963) minimum variance algorithm, which was applied to a dissimilarity matrix of squared Euclidean distances to create the resulting dendrogram. Cluster analysis was performed with MVSP version 3.1 software (Kovach 1999).

Stable isotopes The carbon (513C) and hydrogen (8D) stable isotopic compositions were measured from portions of the same amber specimens used for FTIR spectroscopy, as well as from additional samples of similar morphology and grade. Specimens from Cedar Lake, Edmonton, Drumheller, Morrin Bridge, and the Raritan Formation were also analyzed isotopically, but these did not encompass as wide a range of amber grades and flow morphologies as were recognized at Grassy Lake. All specimens were fragments from freshly-broken surfaces that were cleaned with distilled water and air-dried, but were not chemically or thermally pre-treated. Amber samples of 2.7 mg were combusted at 800°C in vacuum-sealed quartz glass tubes, to which CuO (2 g), Cu (100 mg), and Ag (100 mg) had been added. 13 Values of 8 C and 5D were obtained from the C02 and H20 evolved during combustion using a Finnigan MAT-252 isotope-ratio mass spectrometer. The results are expressed in 8 notation relative to Vienna Standard Mean Ocean Water (VSMOW) for 8D, and the Vienna Pee Dee Belemnite (VPDB) for 813C. The precision for the lab procedures outlined above are ±3%o for 8D, and ±0.1 %o for 813C. 31 Palaeoentomology A faunal comparison was made between the arthropods from the Grassy and Cedar Lake ambers, using published records dedicated to these sites (e.g., Pike 1995; Skidmore 1999) as well as the compilation of all isolated taxonomic works that refer to material from either locality. Published images of taxa from these localities are somewhat limited, so in most cases the published identifications are accepted as definitive. Published identifications were augmented by first­ hand observations of specimens within the Royal Tyrrell Museum collection of Grassy Lake amber made using primary literature and keys (e.g., Goulet and Huber 1993). Select inclusions were photographed through a Wild M400 Photomakroskop with a Nikon D100 digital camera, and individual images were compiled using Auto-Montage software. The compiled taxonomic list comprising primary and secondary identifications is presented in Table 2.1. In all cases where the determining authority is unknown or the identification was uncertain, the taxa are presented with question marks. Comparison of the Grassy and Cedar Lake amber faunas are made at high levels of taxonomy (order and family), but emphasis is placed upon lower taxonomic ranks (genus and species).

32 Table 2.1: Palaeoentomological companson between Grassy Lake and Cedar Lake amber "x" markers indicate the specimen availability within studies on each taxon "x" indicates that authors only observed material from one locality, while "xx" indicates specimens from both localities were observed A bold "x" denotes a taxon that was confirmed by McKellar in a preliminary study of a portion of the Royal Tyrrell Museum's Grassy Lake amber collection Rare orders are marked with the number of known specimens, and all common orders were observed by McKellar The works of McAlpine and Martin, as well as many other determining authonties are cited in Skidmore (1999) The most recent taxonomic source available is listed preferentially here, for the sake of brevity

Taxon •s-S Source

ORDER HYMENOPTKRA Superfamily Ceraphronoidea Family Megaspihdae XX Skidmore 1999 Lygocerus dubitatus Brues, 1937 9 Brues 1937 Conostigmus cavannus McKellar & Engel, 201 lb XX McKellar & Engel, 2011b Family Stigmaphromdae XX McKellar & Engel, 2011b Tagsmiphron canadense Engel & Gnmaldi, 2009 X Engel & Gnmaldi, 2009 Tagsmiphron exitorum McKellar & Engel, 201 lb XX McKellar & Engel, 2011b Tagsmiphron leucki McKellar & Engel, 2011b XX McKellar & Engel, 201 lb Tagsmiphron spiculum McKellar & Engel, 2011b XX McKellar & Engel, 2011b Family Ceraphromdae XX McAlpme & Martin 1969

Superfamily Chalcidoidea Family Eulophidae XX McAlpine & Martin 1969 Family Eupelmidae X Pike 1995 Family Mymandae XX XX Skidmore 1999 Carpentenana tumida Yoshimoto, 1975 XX Yoshimoto 1975 Macalpima canadensis Yoshimoto, 1975 XX Yoshimoto 1975 Protooctonus masneri Yoshimoto, 1975 XX Yoshimoto 1975 Triadomerus bulbosus Yoshimoto, 1975 XX XX Yoshimoto 1975 Family Tetracampidae XX XX Skidmore 1999 Baeomorpha distincta Yoshimoto, 1975 XX XX Yoshimoto 1975 Baeomorpha dubitata Brues, 1937 XX XX Yoshimoto 1975 Baeomorpha elongata Yoshimoto, 1975 XX XX Yoshimoto 1975 Baeomorpha ovatata Yoshimoto, 1975 XX XX Yoshimoto 1975 Bouceklytus arcuodens Yoshimoto, 1975 XX Yoshimoto 1975 Distylopus bisegmentus Yoshvmolo, 1975 XX Yoshimoto 1975 Family Toryrmdae X Pike 1995 Family Tnchogrammatidae XX XX Skidmore 1999 Enneagmus pristmus Yoshimoto, 1975 XX Yoshimoto 1975

Superfamily Proctotrupoidea Family Diapnidae XX Skidmore 1999 Family Proctotrupidae (Serphidae) XX Skidmore 1999

Superfamily Evamoidea Family Gasteruptndae XX Skidmore 1999

Superfamily Ichneumonoidea Family Bracomdae XX XX Skidmore 1999 Diospilus allani Brues, 1937 X Brues 1937 Neoblacus facialis Brues, 1937 X Brues 1937 Pygostolus patriarchicus Brues, 1937 X Brues 1937 Family Ichneumomdae XX Skidmore 1999 Family Paxylommatidae XX Skidmore 1999

Superfamily Platygastroidea Family Platygastridae XX Skidmore 1999 Family Scehonidae XX XX Skidmore 1999 Baryconus fullen Brues, 1937 X Brues 1937 Proterosceho antennahs Brues, 1937 X Brues 1937

33 Superfamily Chrysidoidea Family Bethylidae XX Skidmore 1999 Family Chrysididae XX XX Skidmore 1999 Procleptes carpenteri Evans, 1969 X Evans 1969 Family Dryinidae XX Skidmore 1999 Dryinus canadensis Olmi, 1995 X Olmi1995

Superfamily Cynipoidea Family Cynipidae XX Liu et al., 2007 Tanaoknemus ecarinatus Liu & Engel, 2007 XX Liu et al., 2007 Family Figitidae XX Liu et al, 2007 Micropresbyteria caputipressa Liu & Engel, 2007 XX Liu et al., 2007 Anteucoila delicia Liu & Engel, 2007 XX Liu et al, 2007 Family Liopteridae XX Liu et al., 2007 Goerania petiolata Liu & Engel, 2007 XX Liu et al., 2007 Proliopteron redactus Liu & Engel, 2007 XX Liu et al., 2007 Family Protimaspidae XX XX Skidmore 1999 Protimaspis costalis Kinsey, 1937 X Kinsey 1937

Superfamily Mymarommatoidea Family Mymarommatidae XX XX Skidmore 1999 Archaeromma minutissima (Brues), 1975 XX XX Yoshimoto 1975 Archaeromma nearctica Yoshimoto, 1975 XX XX Yoshimoto 1975

Superfamily Serphitoidea Family Serphitidae Brues, 1937 XX XX McKellar & Engel, 201 la Jubaserphites ethani McKellar & Engel, 201 la XX McKellar & Engel, 201 la Serphites paradoxus Times, 1937 XX XX McKellar & Engel, 2011a Serphites bruesi McKellar & Engel, 201 la XX McKellar & Engel, 201 la Serphites hynemani McKellar & Engel, 2011a XX McKellar & Engel, 201 la Serphites kuzminae McKellar & Engel, 2011a XX McKellar & Engel, 2011a Serphites pygmaeus McKellar & Engel, 2011a XX McKellar & Engel, 2011a

Superfamily Apoidea Family Crabronidae X X Pike 1995; Evans 1969 Lisponema singularis Evans, 1969 X Evans 1969

Superfamily Vespoidea Family Formicidae XX Skidmore 1999 Cananeuretus occidentalis Engel & Grimaldi, 2005 X Engel & Grimaldi 2005 Canapone dentate Dlussky, 1999 X Dlussky 1999 Eotapinoma mcalpini Dlussky, 1999 X Dlussky 1999 Sphecomyrma canadensis Wilson, 1985 X Wilson 1985 ORDER DIPTERA "Nematocerans" Family Amsopodidae XX Skidmore 1999 (no det) Family Bibionidae XX McAlpine & Martin 1969 Plecia myersi Peterson, 1975 X Peterson 1975 Family Canthyloscehdidae (Synnenndae) X Pike 1995 Family Cecidomyndae XX XX Skidmore 1999 Cretocatocha mcalpinei Gagne, 1977 XX Gagne 1977 Cretocordylomyia quadnseries Gagne, 1977 XX Gagne 1977 Cretomiastor ferejunctus Gagne, 1977 XX XX Gagne1977 Cretowinnertzia angustala Gagne, 1977 XX Gagne 1977 Family Ceratopogomdae XX XX Skidmore 1999 Adelohelea glabra Borkent, 1995 XX XX Borkent 1995 Cuhcoides canadensis (Boesel), 1937 XX X Borkent 1995 Cuhcoides agamus Borkent, 1995 XX Borkent 1995 Cuhcoides annosus Borkent, 1995 XX XX Borkent 1995 Cuhcoides bullus Borkent, 1995 XX Borkent 1995 Cuhcoides fihpalpis Borkent, 1995 XX XX Borkent 1995 Cuhcoides obuncus Borkent, 1995 XX XX Borkent 1995 Cuhcoides tyrrelh (Boesel), 1937 XX X Borkent 1995 Dashyhelea sp XX Skidmore 1999 Heleageron arenatus Borkent, 1995 XX XX Borkent 1995 Atricuhcoides globosus (Boesel), 1937 XX X Borkent 1995 Atricuhcoides sp Borkent, 1995 XX XX Borkent 1995 Leptoconops primaevus Borkent, 1995 XX XX Borkent 1995 Minyohelea pumihs Borkent, 1995 XX Borkent 1995 Palaeobrachypogon aquilonms (Boesel), 1937 XX XX Borkent 1995 Palaeobrachypogon vetus Borkent, 1995 XX XX Borkent 1995 Palaeobrachypogon remmi Borkent, 1995 XX XX Borkent 1995 Peronehelea chrimikalydia Borkent, 1995 XX XX Borkent 1995 Protocuhcoides depressus Boesel, 1937 XX X Borkent 1995 Family Chironomidae XX XX Skidmore 1999 Metriocnemus sp Poinar et al, 1997 X Poinar et al 1997 Metriocnemus cretatus Boesel, 1937 X Boesel 1937 Spamotoma conservata Boesel, 1937 X Boesel 1937 Spaniotoma (Smittia) veta Boesel, 1937 X Boesel 1937 Family Culicidae X Poinar et al 2000 Paleocuhcus minutus Poinar et al, 2000 X Poinar etal 2000 Family Lygistorrhimdae XX Blagoderov & Gnmaldi 2004 Plesiognoriste carpenteri Blagoderov & Gnmaldi 2004 XX Blagoderov & Gnmaldi 2004 Family Mycetophihdae XX XX Blagoderov & Gnmaldi 2004 Syntemna fissurata Blagoderov & Gnmaldi, 2004 XX Blagoderov & Gnmaldi 2004 Saigusaia pikei Blagoderov & Gnmaldi, 2004 XX Blagoderov & Gnmaldi 2004 Synapha longistyla Blagoderov & Gnmaldi, 2004 XX Blagoderov & Gnmaldi 2004 Nedocosia canadensis Blagoderov & Gnmaldi, 2004 XX Blagoderov & Gnmaldi 2004 Zeluma occidentals Blagoderov & Gnmaldi, 2004 XX Blagoderov & Gnmaldi 2004 Lecadomleia parvistyla Blagoderov & Gnmaldi, 2004 XX Blagoderov & Gnmaldi 2004 Family Psychodidae XX Skidmore 1999 Sycorax sp XX Skidmore 1999 Family Scatopsidae XX XX Skidmore 1999 Family Sciandae XX XX Skidmore 1999 Family Tipuhdae (Limonndae) XX XX Skidmore 1999 Dicranomyia albertensis (Krzeminski & Teskey)1987 X Brooks etal 2007 Macalpina incomparabihs Krzeminski & Teskeyl987 X Krzeminski & Teskey 1987 Trirhonpura canadensis Kr?eminski & Teskey 1987 X Krzeminski & Teskev 1987

35 Brachycera Family Atelestidae XX XX Gnmaldi & Cumming 1999 Apalocnemis canadambns Gnmaldi & Cumming, 1999 X Gnmaldi & Cumming 1999 Archichrysotus mamtobus Gnmaldi & Cumming, 1999 X Gnmaldi & Cumming 1999 Cretodromia glaesa Gnmaldi & Cumming, 1999 X Gnmaldi & Cumming 1999 Cretoplatypalpus amencanus Gnmaldi & Cumming X Gnmaldi & Cumming 1999 Mesoplatypalpus carpenteri Gnmaldi & Cumming 1999 X Gnmaldi & Cumming 1999 Nemedromia campama Gnmaldi & Cumming, 1999 X Gnmaldi & Cumming 1999 Nemedromia telescopica Gnmaldi & Cumming, 1999 X Gnmaldi & Cumming 1999 Family Bombylndae XX Skidmore 1999 Family Dohchopodidae 9 Skidmore 1999 Family Empididae XX XX Skidmore 1999 Family Ironomyndae X McAlpme 1973 Cretonomyia pristina McAlpme, 1973 X McAlpine 1973 Family Platypezidae XX Skidmore 1999 Family Rhagiomdae 9 Skidmore 1999 Family Stratiomyidae XX McAlpine & Martin 1969 Cretaceogaster pygmaeus Teskey, 1971 X Teskey 1971

Cyclorrhapha Family Phondae XX XX Skidmore, 1999 Prionphora canadambra McAlpine & Martine, 1966 XX McAlpine & Martin 1966 Priophora intermedia Brown & Pike, 1990 XX XX Brown & Pike 1990 Pnophora longicostahs Brown & Pike, 1990 XX Brown & Pike 1990 Priophora setifemorahs Brown & Pike, 1990 XX Brown & Pike 1990 Family Sciadocendae XX XX Skidmore 1999 Sciadophora bostoni McAlpine & Martin, 1966 XX McAlpine & Martin 1966 ORDER. HEMIPTERA Suborder Auchenorrhyncha Superfamily Cicadoidea Family Cercopidae XX XX Skidmore 1999 Family Cicadellidae XX Skidmore 1999 Family Jascopidae XX XX Skidmore 1999 Jascopus notabihs Hamilton, 1971 X Hamilton 1971 Family Membracidae XX Skidmore 1999

Superfamily Fulgoroidea Family Cahscehdae XX Skidmore 1999 Family Cixndae XX Skidmore 1999 Family Fulgondae XX Skidmore 1999 Family Issidae 9 Skidmore 1999

Suborder Heteroptera Superfamily Cimicoidea Family Nabidae Skidmore 1999 Superfamily Coreoidea Family Anthocondae Skidmore 1999, McAlpine & Superfamily Reduvioidea Martin 1969 Family Reduvndae Skidmore 1999

Suborder Sternorrhyncha Superfamily Aphidoidea Family Adelgidae XX XX Skidmore 1999 Family Canadaphidae XX XX Skidmore 1999 Alloambna caudate Richards, 1966 XX Richards 1966 Alloambna infelicis Kama & Wegierek, 2005 X Kama & Wegierek 2005 Canadaphis carpenten Essig, 1937 X X Pike 1995, Essig 1937 Pseudambria longirostris Richards, 1966 XX Richards 1966 Family Cretamyzidae Heie & Pike, 1992 X Heie & Pike 1992 Cretamyzus pikei Heie & Pike, 1992 X Heie & Pike 1992 Family Drepanosiphidae XX Richards 1966 Amferella bostom Richards, 1966 XX Richards 1966 Family Palaeoaphidae XX XX Kama & Wegierek 2005 Ambraphis costalis Richards, 1966 XX Richards 1966 Ambraphis kotejai Kama & Wegierek, 2005 X Kama & Wegierek 2005 Longiradius foottitti Heie, 2006 X Heie 2006 Palaeoaphis archimedia Richards, 1966 XX Richards 1966 Palaeoaphidiella abdominahs Heie, 1996 X Heie 1996 Family Tajmyraphididae Grassyaphis pikei Heie, 1996 X Heie, 1996 Family indet Canaphis albertensis Heie, 2006 X Heie 2006 Superfamily Coccoidea Electrococcus canadensis Beardsley, 1969 X Beardsley 1969 Neutrococcus albertaensis Pike, 1995 X Pike 1995 (nom nudem) Superfamily Phylloxeroidea Family Mesozoicaphididae Heie & Pike, 1992 X Heie & Pike 1992 Albertaphis longirostris Heie & Pike, 1992 X Heie & Pike 1992 Calganaphis unguifera Heie & Pike, 1992 X Heie & Pike 1992 Campamaphis albertae Heie & Pike, 1992 X Heie & Pike 1992 Mesozoicaphus canadensis Heie & Pike, 1992 X Heie & Pike 1992 Mesozoicaphus electn Heie & Pike, 1992 X Heie & Pike 1992 Mesozoicaphus parva Heie & Pike, 1992 X Heie & Pike 1992 Mesozoicaphus tuberculata Heie & Pike, 1992 X Heie & Pike 1992 Superfamily Psylloidea Family PsylliHap XX .JUS SkiHmnre199Q

37 ORDER COLEOPTERA Family Bruchidae X Poinar 2005 Mesopachymerus antiqua Poinar, 2005 X Poinar 2005 Family Carabidae (larva) ? Skidmore 1999 Family Chrysomellidae XX Skidmore 1999 Family Cleridae XX Skidmore 1999 Family Curculionidae XX Skidmore 1999 Family Dascillidae XX Skidmore 1999 Family Eucmetidae X Pike 1995 Family Helodedae ? Skidmore 1999 Family Mordellidae XX Skidmore 1999 Family Orthoperidae ? Skidmore 1999 Family Scydmaeniidae XX xx Skidmore 1999 Palaeoleptochromus schaufussi O'Keefe, 1997 X O'Keefe etal. 1997 Family Staphylinidae XX xx Skidmore 1999 Family Trogositidae ? Skidmore 1999

ORDER STREPSIPTERA 1 Skidmore 1999 ORDER NEUROPTERA Family Berothidae ? Skidmore 1999 Family Rhachiberothidae X McKellar & Engel, 2009 Albertoberotha leuckomm McKellar & Engel, 2009 X McKellar & Engel, 2009 Family Coniopterygidae XX xx Skidmore 1999 Family Hemerobiidae X Klimaszewski & Kevan 1986 Plesiorobius canadensis Klimaszewski & Kevan, 1986 X Klimaszewski & Kevan 1986 Family Raphididae XX Skidmore 1999 Family Sisyridae XX ? Skidmore 1999

ORDER PSOCOPTERA Family Lepidoscelidae XX Skidmore 1999 Family Spaeropsocidae XX xx Skidmore 1999 Sphaeropsocites canadensis Grimaldi & Engel, 2006 X Grimaldi & Engel 2006 finhoeronw^v wL XX Skidmore 1999 ORDMR THYSANOPTERA Suborder Terebrantia Family Aeolothripidae XX Skidmore 1999 Family Thripidae XX Skidmore 1999 Suborder Tubilifera X Pike 1995 "Stem Group Thysanoptera" Family Lophioneuridae XX Skidmore 1999

ORDER TRICHOPTERA Family Electralbertidae X Pike 1995 Electralberta cretacica Botosaneanu & Witchard, 1983 X Botosaneanu & Wit. 1983 Family Psychomyidae ? Skidmore 1999

ORDER LEP1DOPTERA Superfamily Incurvaroidea 1 Skidmore 1999

ORDER PHASMIDA 4 Pike 1995 ORDER DERMAPTERA 1 Pike 1995 ORDER BLAITARIA 23 Skidmore 1999 ORDER ZORAPTERA 1 Skidmore 1999 ORDER 1SOPTERA 4 Skidmore 1999 ORDER PLECOPTERA 1 Skidmore 1999 ORDER COLLEMBOLA Family Brachystomelhdae XX Christiansen & Pike 2002 Belhngeria cornua Chnstiansen & Pike, 2002 XX Chnstiansen & Pike 2002 Family Hypogastrundae X Pike 1995 Family Isotomidae XX Chnstiansen & Pike 2002 Protoisotoma micromucra Chnstiansen & Pike XX Chnstiansen & Pike 2002 Family Neanundae X Pike 1995, (below) Campanunda electra Pike, 1995 X Pike 1995 (nom nudetri) Pseudoxenylla foveahs Chnstiansen & Pike, 2002 XX Christiansen & Pike 2002 Family Oncobryidae Christiansen & Pike, 2002 XX Chnstiansen & Pike 2002 Oncobrya decepta Chnstiansen & Pike, 2002 XX Chnstiansen & Pike 2002 Family Podundae XX Skidmore 1999 Family Protentomobryidae X x Pike 1995 Protentomobryapalhserifike, 1995 X Pike 1995 (nom nudem) Protentomobrya walkeri Folsom 1937 x Folsom 1937 Family Smmthundae XX Skidmore 1999 Brevimucronus anomalus Chnstiansen & Pike, 2002 XX Chnstiansen & Pike 2002 Keratopygos megalos Chnstiansen & Pike, 2002 XX Chnstiansen & Pike 2002 Family Tomocendae xx Skidmore 1999 Entomocerus minis Chnstiansen & Pike, 2002 xx Chnstiansen & Pike 2002 ORDERTHYSANURA &i4mor e -¥- more ~uORDH R AC AR1 Skidi Acanformes Suborder Orbatida Family Onbatidae XX xx Skidmore 1999 Superfamily Damaeoidea XX Skidmore 1999 Superfamily Gymnodamaeoidea Family Gymnodamaeidae XX Skidmore 1999 Superfamily Hermannielloidea XX Skidmore 1999 Superfamily Hermannioidea Family Hermanniidae XX Skidmore 1999 Superfamily Nothroidea Family Camisndae XX Skidmore 1999 Superfamily Oppioidea XX Skidmore 1999

Suborder Prostigmata Superfamily Family Bdelhdae XX XX Skidmore 1999 Bdella vetusta Ewing, 1937 X Ewing 1937 Family Cunaxidae XX Skidmore 1999 Superfamily Erythroidea XX Skidmore 1999 Family Erythraeidae XX XX Skidmore 1999 Leptus sp XX Skidmore 1999 Superfamily Family Eupodidae XX Skidmore 1999

Parasitiformes Suborder Mesostigmata Superfamily Parasitoidea Family Parasitidae Skidmore 1999 ORDER ARAN HAH Family Araneidae ? XX Skidmore 1999 Family Ctenidae XX Skidmore 1999 Family Erigonidae ? Skidmore 1999 Family Huttoniidae XX XX Skidmore 1999; Penney & Selden, 2006 Family Lagonomegopidae X Penney 2004 Grandoculus chemahawinensis Penney, 2004 X Penney 2004 Family Linyphiidae XX Skidmore 1999 Family Oonopidae X Penney 2006 Orchestina albertensis Penney, 2006 X Penney 2006 Family Theridiidae ? XX Skidmore 1999

ORDER PSEUDOSCORPIONiDA 4 Skidmore 1999

RESULTS FTIR spectra Comparison of the FTIR spectra of ambers from Cedar Lake and Grassy Lake indicate only minor differences (Fig. 2.3). There is a similar degree of variability within the spectra generated from different forms of Grassy Lake amber, as well as from weathered versus fresh surfaces. Nonetheless, the same absorption peaks can be identified throughout the 8-10 urn (1000-1250 cm1) range in all Grassy Lake and Cedar Lake spectra, although they are slightly more subdued in the latter. This window is considered to represent the primary fingerprinting region for the spectral characterization of ambers (Langenheim and Beck 1968). Compared to all of the FTIR spectra we have generated from Cretaceous amber localities in Alberta, Cedar Lake amber is the most similar to Grassy Lake amber (Fig. 2.3), while amber from Edmonton, Medicine Hat, Horsethief Canyon, and Drumheller, all differ to varying degrees. However, because the variability observed within Grassy Lake FTIR spectra is sufficient to encompass the spectra of certain other Late Cretaceous deposits as well as that of Cedar Lake amber, FTIR does not provide an exclusive match. For example, amber collected in Horsethief Canyon produces a fingerprint quite similar to that of Grassy Lake amber, but lacks the absorption peak near 1240 cm1 (8.06 urn) seen in all examples of Grassy Lake material. The FTIR spectra of Cedar Lake and Grassy Lake ambers presented here are generally comparable to previous reports (Langenheim and Beck 1968; Aber and Kosmowska-Ceranowicz 2001; Kosmowska-Ceranowicz et al. 2001). However, our results are the first to include comparisons with additional Cretaceous ambers from western Canada. Wavenumber (cm"1) 2000 1500 1000 900 800 700

Figure 2.3. FTIR spectra. (A) Inset depicting complete Grassy Lake 15 spectrum at wavelengths from 2.5 to 15 urn. Shading highlights the most informative region and fine lines highlight salient peaks. (B) Blow-ups of absorption be­ tween 5 and 15 urn, including the fingerprint region (brackets). From top to bottom: Horsethief Canyon - slightly oxidized Maastrichtian sample producing similar spectrum to that seen within Grassy Lake material; Grassy Lake 10,13,15, and 5 - a range of amber morphologies and grades, showing minor variations within their spectra, and many peaks lost or reduced in G.L. 5 due to weathering effects; Cedar Lake 1 -typical spectrum from this locality, showing extreme weathering effects; Medicine Hat, Edmonton, and Drumheller sam­ ples produced strikingly different spectra from that seen at G.L, whereas the latter two localities have nearly identical spectra and are represented here by a single line.

9 10 11 Wavelength (um) 41 Stable isotopes The stable isotopic ratios obtained from North American Cretaceous ambers are presented in Figure 2.4. The data have also been compiled as a co-isotopic plot (Fig. 2.5) that includes previous measurements from Nissenbaum and Yakir (1995). The new raw isotopic data are contained in Appendix 2. Both Grassy Lake and Cedar Lake ambers have SD values that exhibit a wide range of values. In the Grassy Lake samples, SD ranges from -351.9%o to -265.2%o, with a mean value of -301.8%o. The Cedar Lake material produced 5D values of -353.3%o to -252.5%o, with a mean value of -291.5 %o. All other North American ambers have much narrower ranges of 8D: Edmonton material ranged from -349.2%o to -338.6%o with a mean value of -343.4%o; amber collected in the Drumheller Valley ranged from -328.2%0 to -307.8%o with a mean of -320.7%o; amber from the 'upper coal seam' at the Morrin Bridge locality ranged from -311.8%o to -297.2%o with a mean of -303.8%o, while that from the 'lower coal seam' was distinct, ranging from -280.6%o to -270.0%o with a mean of-275.9%o. Finally, amber from New Jersey's Raritan Formation displayed values from -248.8%o to -227.6%o with a mean of-236.6%o. The new 5D data for Cedar Lake amber are consistent with the prior results of Nissenbaum and Yakir (1995), who published a single 8D value of -272%o, well within the range of new measurements presented here. However, their reported range of 5D values in amber (-270%o to -160%o) needs to be significantly extended, given the common occurrence of lower 8D values reported here (Fig. 2.4).

0 OOGBDflESDD QD Q Grassy Lake (n=28) 0 oo asxnncoBDO op i 301 8%o -23 8%o 0 0 (BOO 0 CO CD 0 Cedar Lake (n=13) (JSOOO (BD O 0 -291 5%o -23'3%o (£) dp New Jersey (n=5) oo o o oo -236'6%o -2l'8%o upper coal (n=3)Q 00 COO lower coal (n=3) Morrin Bridge (n=6) 9 CO O 0 303 8%o 275 9%o 24'8%o 0DOO 0 Drumheller Valley (n=6) o 0 0 0 0 -320 7%o 22 8%o coop Edmonton (n=6) 0 0 OOP 343 4%o 24 6%o

-375 -350 -325 -300 -275 -250 -225 -200 -30 -28 -26 -24 -22 -20 -18 5D (VSMOW) %o 6 C (VPDB) %o

Figure 2.4. Stable isotope measurements for 813C and 8D Value ranges, mean values, and sampling intensity within each locality

With respect to S13C, Grassy Lake amber ranged from -27.1%o to -20.9%o, with most samples falling between -24.6%o and -20.9%o. The mean S13C value for

42 Grassy Lake amber was -23.8%o. Cedar Lake material sampled here displayed a range of 513C values from -24.8%o to -20.0%o, with a mean value of-23.3%o. Other North American ambers displayed similar degrees of variability in 513C, but slightly different mean values (Fig. 2.4). 513C of amber from Edmonton ranged from -25.9%o to -23.6%o (mean: -24.6%o); samples from the Drumheller Valley ranged from -26.5%o to -20.7%o (mean: -22.8%o); Morrin Bridge upper and lower coal seams were not distinct and produced values from -27.6%o to -23.1%o (mean: -24.8%o); and New Jersey amber ranged from -23.2%o to -20.2%o (mean: -21.8%o). In the case of Cedar Lake amber, our measurements overlap the range of 813C values reported previously (Nissenbaum and Yakir 1995), and furthermore suggest that the -23.9%o to -21.4%o range in 813C values they reported should be extended to a range of -24.8%o to -20.0%o. The greater ranges of 513C values observed here (4.8%o for Cedar Lake material and 6.2%o for Grassy Lake amber) appear to reflect the greater number of analyses reported herein. This may compromise the utility of 513C values for fingerprinting the provenance of amber deposits, since the value ranges for individual deposits appear much greater than originally portrayed (Nissenbaum and Yakir 1995). The two deposits that form the focal point of this study have been circumscribed with ellipses (Fig. 2.5) to indicate the degrees with which their

LEGEND: North American Mesozoic Resinites • CL= Cedar Lake (U Cret) • GL= Grassy Lake (U Cret) = ERV= Edmonton(U Cret) o DV= Drumheller Valley (U Cret) A MUC= Morrin upper coal (U Cret) v MLC= Morrin lower coal (U Cret) O NJ= New Jersey Rantan (U Cret)

Other Mesozoic Resinites • IH= Israeli Hermon (Cret )* • BC= Burmese (Cret)* A P= Polish (Cret')* •« S= Spanish (')*

Cenozoic Resinites • PB= Polish Baltic (Eoc Oligo)* ©B= Baltic (Eoc-Oligo)* • 1= Sicilian (Mio)* yR= Romanian (Mio)* AM= Mexican (Oligo)*

Recent Resins iPR = P/nus sp* •A R = Araucana sp * • PC = Phillipine Copal*

I—| = Range of 813C values published with a single 5D value* -375 * Data from Nissenbaum and Yakir (1995) -28 -27 -26 -25 -24 -23 -22 -21 -20 -19 -18

6nX (VPDB) %. Figure 2.5. Stable isotope comparison and context Stable isotope compositions measured for Grassy Lake and Cedar Lake ambers, as compared to other North American Mesozoic ambers and a wide range of published values Ellipses outline the range of values seen at the two focal localities 43 isotopic values overlap, and their relation to the isotopic composition of ambers from other localities. The first observation is that the 5D and 513C values of amber are in general uncorrelated with each other. With respect to the results from Cedar Lake and Grassy Lake ambers, these two populations have by far the greatest dispersion in both 513C and 8D values. Yet these broad isotopic ranges overlap almost completely, with the exception of two extremely negative 513C values from Grassy Lake amber, and that the 8D values of this deposit are on average about 10%o lower, relative to Cedar Lake amber. All other Mesozoic ambers produced comparatively narrow ranges of 8D values, and more highly variable ranges of 813C. This does not create an unequivocal match, but strongly suggests that Grassy Lake material is the only single source capable of producing the range of values seen in Cedar Lake amber. Other localities (e.g., Drumheller Valley material) may develop broader ranges in values with future analyses, but the differences in mean values seen here suggests that the overlap in values will not be as extensive as it is between Grassy Lake and Cedar Lake amber.

Amber morphology and physical properties Amber from Grassy Lake and Cedar Lake possess a distinctive set of shared physical characteristics. The size of amber masses from Grassy Lake is usually less than 1 cm, with rare specimens reaching up to 3.5 cm in length (see Fig. 2.6a, and compare to fig. 15 of McAlpine and Martin 1969), while pieces recovered from Cedar Lake are "for the most part smaller than a pea [while some] were found as large as a robin's egg" (Tyrrell 1891, p. 14). The majority of amber from both deposits represent portions of massive flows (schlauben) and bizarre disc­ shaped morphologies (often interpreted as resin pockets within the tree, but seen to include palynomorphs and fungal hyphae within our material). Isolated droplets and runnels make up the remainder of the samples (see Fig. 2.6b). In terms of colour, amber from each locality ranges from pale yellow to deep orange-red, with the most samples a rich orange-yellow. Clarities or grades within each deposit range from very clear to rare examples of milky or 'bone' amber, clouded by minute bubbles. Grassy Lake amber often has a thin rind of carbonized material, which is replaced by a thin weathering rind (< 1mm thick) in surface exposed specimens. Prolonged exposure creates a granular texture and slightly crumbly amber masses - this becomes pervasive in small specimens (< 5mm). Cedar Lake amber consistently lacks a carbonized rind, but often displays a surface patina or granular texture. Very dark flow lines are evident within specimens from both localities, and the incidence of arthropod inclusions is approximately one in every Figure 2.6. (A) A sample of Grassy Lake amber surface collected (note colour and nodule size predomi­ nance). Scale is in cm. (B) Range of amber morphologies and clarity grades: lower half of circle arrangement composed of runnels containing bulk of insect inclusions (polished specimen at 3 o'clock position), as well as stalactites (9 o'clock position) and schlauben (6 o'clock position); disk-shaped nodules in upper half of circle, with partially removed carbonaceous rinds (10-11 o'clock), milky weathering rind (1 o'clock), and results of tumble-polishing (2 o'clock); inner circlet displays range of colours and shapes seen within isolated droplets; centerpiece runnel displays near opacity due to darkly-coloured drying surfaces. Scale is in cm. (C) Archaeromma mmutissimum (Brues), (Hymenoptera: Mymarommatidae) female specimen 0.5 mm long, RTMP 96.9.178: this species is found at both Grassy Lake and Cedar Lake. (D) A nearcticum Yoshimoto, 1975, male specimen 0.32 mm long, from Grassy Lake, RTMP 96.9.176: this species is known from both G.L. and C.L. localities. (E) ?Paxylommatine hymenopteran, 1.3 mm long, RTMP 96.9.192: this subfamily found only at Grassy Lake to date. (F) Serphitid hymenopterans Serphites n. sp. A (R. McKellar, in prep.) each approximately 1.6 mm long, from Grassy Lake, RTMP 96.9.183: this genus is restricted to the Cretaceous and is known from both G.L. and C.L. localities.

45 50 sampled amber pieces (McAlpine and Martin 1969; Pike 1995). Amber collected from other Albertan sources, such as the vicinity of Drumheller and Edmonton (early Maastrichtian, Horseshoe Canyon Fm. in both cases), and the vicinity of Medicine Hat (Campanian, Belly River Group), is physically different from the material found at Grassy Lake and Cedar Lake. In all of these additional localities, the amber collected was typically very small (1-5 mm), drop-shaped or cylindrical, and medium-to-dark in colour (ranging from yellow to a deep, rich red, with the bulk of material dark orange or red in colour). Material from all three of these localities is usually very brittle - so much so that it is difficult to collect it in any great quantity. These amber samples do not appear to represent massive flows, but instead isolated drips and perhaps the cylindrical fillings of internal resin ducts: samples 1 cm or larger are rare. Pike (1993) indicated that some of the Maastrichtian amber collected near Edmonton and Grande Prairie had been observed to contain insect inclusions, but amber collected from Edmonton differs substantially in morphology and inclusion abundance, and material collected near Grande Prairie falls within a different drainage system than Cedar Lake.

Palaeoentomology Taxonomic studies have suggested that the Cedar Lake and Grassy Lake amber are similar, usually portraying this relatedness as a result of comparable environmental conditions during their formation (e.g., Penney and Selden 2006). Unfortunately, the majority of the low-level taxonomic work has not yet been completed for both localities, and few studies make direct comparisons between the taxa found within the different deposits. Of the available published data, family-level taxonomy is usually the lowest level achieved. The family-level taxonomy of the Grassy Lake amber and Cedar Lake amber faunas are contrasted in Table 2.1, and supplemented with any lower-level data available. At the order-level, 10 out of 23 taxa are shared between the two deposits (the 13 that are present only in Grassy Lake material are exceedingly rare taxa within this deposit and were likely found due to sampling intensity). At the level of families or superfamilies, 41 taxa are in common, while 72 are unique to one deposit. Comparisons made above the species level are not very informative because most Cretaceous amber faunas are very similar, thus species are emphasized here. Of the species described from these localities, 25 can be found in both localities, while 111 are only present within one deposit or the other (see exemplars in Figs. DISCUSSION A synthesis of the spectroscopic, stable isotopic and entomological data generated from western Canadian Late Cretaceous ambers is necessary to assess their potential relationships, and most importantly the provenance of secondary deposits such as the Cedar Lake amber. We emphasize that any single fingerprinting methodology is unlikely to provide unequivocal correlations, necessitating the use of multiple approaches.

Amber spectroscopy, weathering, and botanical source Although compelling similarities exist between the Grassy Lake and Cedar Lake FTIR spectra (Fig. 2.3), these are insufficient for unambiguous correlation. In a general sense, this is because the FTIR spectra of most Cretaceous ambers have pronounced over-arching similarities. This sentiment echoes those of Langenheim (1993, p. 155), who suggested that: "the older the amber, the more similar the spectra usually become", and furthermore that: "different spectra may well indicate different plant sources ... but very similar spectra do not necessarily mean the same source". Cluster analysis of FTIR spectra from western Canadian ambers («=14) confirms some of these difficulties, while reducing the uncertainty inherent in purely visual assessments of FTIR spectrosopic results. The resulting

- Drumheiier(Dv i) "dendrogram (Fig. 2.7) Edmonton (ERV1) indicates that two distinct Medicine Hat (MH 1) populations exist among f Grassy Lake (GL 15a) Grassy Lake (GL 5a) the analyzed samples: Grassy Lake (GL 5b) one containing Horsethief Grassy Lake (GL 10a) Grassy Lake (GL 13b) Canyon, Cedar Lake, Grassy Lake (GL 13a) and variably-weathered Grassy Lake (GL 15a) Horsethief(HTI) specimens of Grassy Lake

r Grassy Lake (GL 10) amber (n=\\), the other Cedar Lake (CL1) containing ambers from Cedar Lake (CL 2) r- —I— 40 30 20 10 Drumheller, Medicine Hat, Squared Euclidean distance and Edmonton (M=3). The Figure 2.7. Dendrogram of cluster analysis results obtained from the FTIR spectra of western Canadian dissimilarities expressed Cretaceous ambers. Two chemically-distinct populations within the smaller cluster are evident: one containing Grassy Lake, Cedar Lake, and Horsethief Canyon ambers, the other represented by are far greater than those samples from Edmonton, Drumheller, and Medicine Hat. between representatives of the larger 'Grassy Lake' cluster. These results have several implications. First, spectral differences associated with different weathering states of Grassy Lake amber (Fig. 2.3) exert only minor effects, given that all specimens, as well as those obtained from Cedar Lake amber, cluster together closely. Second, the two major clusters defined above (Fig. 2.7) are entirely compatible with the following assignment to discrete source rocks: the Horseshoe Canyon Formation for ambers from Drumheller, Medicine Hat, and Edmonton, and the slightly-older Foremost Formation for the larger 'Grassy Lake' cluster (Fig. 2.2). Third, the FTIR results verify that the primary spectral impact of subaerial weathering is the progressive muting of absorption peaks, as first proposed for reworked Baltic amber (Beck 1986). Cedar Lake amber, which is presumed to have been transported fluvially at least 500 km from its source, produces FTIR spectra that are comparable to weathered specimens of Grassy Lake amber, both of which have subdued peaks relative to fresher specimens (Fig. 2.3). From this assessment, it appears that the variability of FTIR spectra previously reported for Cedar Lake amber (Beck 1986; Aber and Kosmowska-Ceranowicz 2001; Kosmowska-Ceranowicz et al. 2001) merely reflects differences in weathering states and, potentially, development in FTIR spectroscopic technology, rather than multiple sources as suggested by some authors (e.g., Christiansen and Pike 2002). Conventional FTIR analyses involved samples >100 mg, typically represented by a composite of small fragments embedded in KBr pellets, and resulting in loss of control with respect to the weathering states of the materials being considered. The use of micro-FTIR spectroscopy here allows discrete samples <1 mg to be routinely measured, typically from freshly fractured surfaces, and without need to embed the amber in KBr prior to analysis. Accordingly, we consider the progressive diminution of absorption peaks within the 'Grassy Lake' amber series (Fig. 2.3) and corresponding cluster analysis results (Fig. 2.7) to capture differential weathering among ambers of similar, and possibly identical, original provenance. The inferences drawn above are corroborated by the physical properties of the amber in question. Samples obtained from Drumheller, Medicine Hat, and Edmonton are representative of the majority of coal-associated Cretaceous amber within Alberta: they are highly brittle and typically darker in color; whole specimens >1 cm3 are rare. Grassy Lake amber from the Foremost Formation has both greater strength and a lighter color relative to other Alberta ambers, which is likely attributable to a lower degree of thermal maturation and oxidation 48 (Poinar and Mastalerz 2000). Much of the Grassy Lake amber is hosted in shale and not coal, which may diminish the extent to which the amber is subjected to compaction, heating, and diagenetic alteration in situ. The implication of these arguments is that weathering of this amber occurred primarily during secondary transport following exhumation. The quality of entomological fossil records from both Grassy Lake and Cedar Lake ambers is potentially an important consequence of these collective attributes, all the more since this palaeontological resource is unmatched elsewhere in the Cretaceous of western Canada. Unfortunately, the chemical changes that occur during amber diagenesis are not completely understood, particularly for ambers of this age (Grimaldi et al. 2000). This renders difficult any meaningful use of the FTIR data in an effort to suggest a botanical source for these ambers through comparisons to modern conifer resins. Although a source tree within the Araucariaceae has been tentatively advanced for the Grassy Lake and Cedar Lake ambers (Kosmowska- Ceranowicz et al. 2001), none of the botanical inclusions or palynomorphs observed to date confirm an araucarian source tree. More comprehensive accounts of botanical inclusions and spectroscopic data may shed light on the composition of source forests, but no firm conclusions can be drawn at present.

Inferences from the stable isotopic composition of amber The analysis of amber stable isotopic signatures was initiated by Nissenbaum and Yakir (1995). These authors surmised that the modest range of 813C values determined from a range of different ambers makes this characteristic a relatively useful tool for accurate correlation. Our results from western Canadian Cretaceous ambers produced 513C ranges of 6.2%o and 4.8%o from Grassy Lake and Cedar Lake, respectively, proving that variability within a single deposit may be considerably larger than previously envisaged from a limited number of analyses. Although these results might be viewed as diminishing the utility of S13C as a fingerprinting tool, the opposite conclusion may prove equally tenable. This is because the ranges of 813C values from Grassy Lake and Cedar Lake ambers produce overlap and produce nearly identical mean values (Fig. 2.4), despite the large amplitude of values. The exceptional range of amber 513C values can be reconciled by variable plant palaeo-physiological factors, including growth rate and carbon partitioning between different components of the source tree (Murray et al. 1998). Attendant factors, such as forest canopy structure, the presence of pests and pathogens, and palaeoclimate, may also be implicated in modulating

49 amber 813C values. In the case of Grassy Lake amber, variability in 813C values appears to correlate with the morphology of the amber (Appendix 2). Resin flow morphologies relating to limb breakage and bark wounding have been identified using direct analogy to modern conifers (Pinaeceae, Cupressaceae, and Araucariaceae); these morphologies correspond to the most highly-negative 813C values from the range observed. These samples appear representative of relatively small volumes of released resin. On the other hand, the more massive flow morphologies that comprise the majority of recovered amber samples can be ascribed to traumatic responses, or induced resinosis (Grimaldi 1996). These samples are associated with relatively high 513C values. Although more analyses are required to firmly establish these relationships, they remain consistent with the notion that trees experiencing the traumatic stresses that engender rapid and copious resin production have a reduced ability to metabolically fractionate against 13C (Nguyen Tu et al. 1999). Accordingly, the ranges of 513C values reported here may be attributed to variable forest conditions and tree health. As with 813C, the 8D values from Cedar Lake and Grassy Lake ambers are highly variable, with substantial but not exclusive overlap (Fig. 2.5). In contrast to the 813C results, however, discrepancies arise between maximum and mean 8D values obtained from the two ambers. The ~10%o increase of mean 8D values seen in Cedar Lake amber, relative to Grassy Lake specimens, is of the magnitude and direction predicted from weathering effects. A relative 8D increase of ~19%o has been attributed to weathering effects in previous studies (Nissenbaum and Yakir 1995). If Cedar Lake 8D values are corrected for this effect, the resulting values fall directly within the range of measured Grassy Lake amber 8D values. However, such effects are relatively minor in the context of either the ranges of 8D values obtained from Grassy Lake and Cedar Lake specimens (almost 70%o), or occasional extremely negative 8D value (e.g., -353.3%o; Fig. 2.4). As with 813C values, a potential explanation for the broad range of 8D values may reside in distinct flow characteristics among the analyzed samples. Isolated drops and bubble-rich (bone) amber consistently produced the lowest 8D values, while specimens from massive and multilayered flows (schlauben and runnels) anchor the isotopically-enriched portion of the 8D spectrum. The former group is likely to represent constitutive resins that solidified rapidly at the site of discrete injuries, while the latter population may be attributable to more extensively induced resinosis. As defensive resins contain a greater proportion of volatile 50 (mono- and sequi-) terpenoids relative to constitutive counterparts (Trapp and Croteau 2001), the possibility exists that differential degrees of volatilization exert a first-order control on amber 8D, with greater volatile losses resulting in increased 5D values. Finally, the highly-negative 5D values reported here from western Canadian Cretaceous ambers are noteworthy from a palaeoclimate perspective. These samples are unlikely to be over-printed by contamination, because most post-depositional isotopic artifacts lead to an increase in 5D values. Fractionation of SD between modern conifer resins and their local meteoric waters is in the order of -170%o to -200%o (Nissenbaum and Yakir 1995), which is directly comparable to the 5D fractionation reported from many plant lipids (Sauer et al. 2001). This similarity is unlikely to be fortuitous, given that considerable biochemical commonalities exist among pathways of isoprenoid synthesis (Bouvier et al. 2005), whether the end-product is a plant lipid or, in the case of amber, a diverse suite of terpenoid moieties. On these grounds, our results suggest that the water source that supported the Grassy Lake amber-producing forest had on occasion 5D values as low as -150%o to -180%o, with a more central tendency in the -60%o to -120%o range. This broad range is difficult to explain, as it is roughly equivalent to modern precipitation 5D values spanning southern Ontario (-65%o) to the central Northwest Territories (165%o) (Birks et al. 2004). Thus, we suggest that local water sources may have been highly variable during intervals of amber production in the palaeo-forest. This variability could potentially involve dramatic changes in water sources, relative humidity, ambient temperatures, or some combination of these factors, but our data do not provide any clear indication of cause.

Palaeoentomological and stratigraphic considerations Because Grassy Lake amber is represented in published works by as many as ten times the number of studied specimens documented from Cedar Lake material, there is an inherent sampling bias in any palaeoentomological correlation between these deposits. Very few deliberate comparative studies of these faunas exist (Table 1), and the present study is the first to summarize exhaustively the information available to date. Although a number of taxa have been reinvestigated, it remains impossible to ascertain to what extent further examinations of the fauna from each locality will influence the overall picture of their assemblages. With this in mind, the absence of several rare orders from

51 Cedar Lake amber that occur sporadically in material from Grassy Lake can not be viewed as a criterion for differentiation: in all likelihood sampling intensity can be invoked. Similarly, just over one third of the superfamilies and families from the two ambers have shared representation. However, taxonomic works at these levels have typically been restricted one deposit or the other. If only studies considering both deposits are included, 40% of superfamilies and families occur in both deposits. These differences provide a good reflection of the amount of work dedicated to either deposit: of the 75 taxa at this rank that have been recorded, only seven are documented from Cedar Lake amber alone (Table 1). Similar one-sidedness is evident from studies at the species level: the shared proportion of species is 19% when studies restricted to one or the other deposit are considered, but rises to 36% in investigations addressing both Grassy Lake and Cedar Lake material simultaneously. The two most comprehensive taxonomic inventories to date address portions of the orders Hymenoptera (Yoshimoto 1975) and Diptera (Borkent 1995), providing some indication of the trend that accompanies increasingly detailed taxonomic scrutiny. These studies portray the Grassy Lake and Cedar Lake amber faunas as remarkably similar, sharing 7 of 13 hymenopteran species, and 15 of 18 dipteran species. These similarities prompted Borkent (1995) to postulate that both amber deposits formed in similar ecological situations relative to surrounding habitats, given nearly identical species and sex ratios observed among representatives of the biting midges (Diptera: Ceratopogonidae). Indeed, the dipterans in general, and the diminutive ceratopogonids in particular, provide a strong basis for the palaeoentomological correlation of the Grassy Lake and Cedar Lake ambers. Borkent's (1995) results imply that both ambers formed in immediate proximity to the mixture of aquatic habitats required by specific taxa of larval ceratopogonids (e.g., moist sands, wetland pools, salt-marshes). Although this group of insects is known from other Cretaceous ambers (e.g., Grogan and Szadziewski 1988; Penalver et al. 2007), the abundance of shared species in Grassy Lake and Cedar Lake ambers is noteworthy. This is because traps capable of capturing a nearly identical suite of ceratopogonid taxa, in similar sex ratios, are unlikely to have been widespread. The documentation of at least 25 shared arthropod species in the Grassy Lake and Cedar Lake deposits can be placed in a stratigraphic context to further constrain the possible age of amber formation, as well as the distribution of suitable palaeoenvironments. It is difficult to envisage significant production of

52 arthropod-rich amber outside of the Taber coal zone at the top of the Foremost Formation, as this is one of few lithologies associated with marginal marine deposition in the Campanian. Bentonites within the shale that contains the Taber coal zone produce radiometric ages between 78.2 and 79.0 Ma (Goodwin and Deino 1989; Pike 1995), constraining the formation of Grassy Lake amber to this interval. The Milk River Formation is the nearest underlying non-marine unit, which is overlain uncomformably by marine shales of the Pawkowki Formation that separate it from the lower Foremost Formation (Stelck et al. 1976). The Milk River Formation has been dated at -83.5 Ma (Braman 2001). Above the Foremost Formation, the non-marine Oldman and Dinosaur Park formations appear in close succession (Dodson 1971), but only in the uppermost portion of the Dinosaur Park Formation is another coal-rich interval encountered: the Lethbridge coal zone, dated -74.9 Ma (Eberth and Hamblin 1993). Bearpaw Formation marine sediments overlie the Lethbridge coal zone (Fig. 2.2). Within this stratigraphic framework, the closest non-marine units to the known source of Grassy Lake amber in the Taber coal zone are -3 Ma younger (Lethbridge coal zone) or -5 Ma older (Milk River Formation). Given a maximum estimated duration for insect species in the order of 3-10 Ma (Grimaldi and Engel 2005), it becomes highly improbable that 25 species would simultaneously bridge either of these temporal gaps. Furthermore, the Lethbridge coal zone is not known to produce appreciable quantities of amber at the type section of the Dinosaur Park Formation (Eberth and Hamblin 1993), and the Milk River Formation contains neither substantial coals nor concentrations of plant remains. The probability that the Cedar Lake fauna originated from anywhere other than the six coal measures of the upper Foremost Formation is significantly reduced by the outlined stratigraphic constraints placed on the palaeoentomological record.

CONCLUSIONS Although Cedar Lake amber cannot be assigned to the Grassy Lake deposit with absolute certainty without characterizing each and every potential in situ source, the balance of evidence pointing to a common source is overwhelming. At the very least these two deposits should be treated as equivalent, if not identical. Faunal inclusions show that the two ambers were formed at a similar time and in similar palaeoenvironments. Grassy Lake amber is one of few Alberta ambers of sufficient strength to withstand fluvial transport in excess of 500 km while

53 retaining exquisitely-preserved arthropod inclusions. The physical properties of Cedar Lake amber, augmented by FTIR spectroscopic and stable isotopic fingerprints, support this correlation. From a purely stratigraphic perspective, coal seams and associated shales from the upper Foremost Formation become the most viable source for the Cedar Lake amber deposit and its associated fauna. In turn, the Grassy Lake outcrop is the only known upstream seam from the Foremost that produces a faunal record of this quality. The Grassy Lake and Cedar Lake amber deposits also serve to evaluate the effects of long-distance transport on the physical and chemical properties of amber. In a general sense, it appears that peaks within FTIR spectra become more subdued as weathering progresses, while 8D values become increasingly greater, and amber specimen size is reduced by comminution during transport. Beyond the minor differences between these deposits, their internal variability is likely an excellent source of information concerning the conditions that triggered the formation of amber within Late Cretaceous forests. If the relationships between amber morphology and stable isotope composition proposed here are verified by studies of additional amber deposits (as has been done in Chapter 6), the potential exists to specify the underlying environmental cues that lead to massive resin releases in ancient forests.

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Amber taphonomy and the Grassy Lake, Alberta, amber fauna. PhD. Thesis. Department of Biological Sciences, University of Calgary, Calgary, Alberta. Poinar, G.O. Jr., 1992. Life in amber. Stanford University Press, Stanford. Poinar, G.O. Jr., 2005. A Cretaceous palm bruchid, Mesopachymerus antiqua, n. gen., n. sp. (Coleoptera: Bruchidae: Pachymerini) and biogeographical implications. Proceedings of the Entomological Society of Washington 107(2), 392-397. Poinar, G.O. Jr., Mastalerz, M., 2000. Taphonomy of fossilized resins: determining the biostratinomy of amber. Acta Geologica Hispanica 35(1- 2), 171-182.

60 Poinar, G.O. Jr., Krantz, G.W., Boucout, A.J., Pike, T.M., 1997. A unique Mesozoic parasitic association. Naturwissenschaften 84, 321-322. Poinar, G.O. Jr., Zavortink, T.J., Pike, T., Johnston, P.A., 2000. Paleoculicis minutus (Diptera: Culicidae) n. gen., n. sp., from Cretaceous Canadian amber, with a summary of described fossil mosquitoes. Acta Geologica Hispanica 35(1-2), 119-128. Rice, P.C., 1980. Amber: the golden gem of the ages. Van Nostrand Reinhold Company. New York. Richards, W.R., 1966. Systematics of fossil aphids from Canadian amber (Homoptera: Aphididae). The Canadian Entomologist 98, 746-760. Sauer, P.E., Eglinton, T.I., Hayes, J.M., Schimmelmann, A., Sessions, A.L., 2001. Compound-specific D/H ratios of lipid biomarkers from sediments as a proxy for environmental and climatic conditions: Geochimica et Cosmochimica Acta 65, 213-222. Skidmore, R.E., 1999. Checklist of Canadian amber inclusions in the Canadian National Collection of Insects. Research Branch Agriculture and Agri- Food Canada electronic publication. Available from www.biology.ualberta. ca/facilities/strickland/SKIDMORECNCCanadianAmberlnclusions.pdf [cited 22 December, 2009]. Stelck, C.R., Wall, J.H., Sutherland, G., 1976. Field trip A-5 Guidebook: Mesozoic stratigraphy in central Alberta foothills near Drumheller, May 16-18, 1976. Geological Association of Canada Mineralogical Association of Canada Joint Annual Meeting 1976, Edmonton, Alberta. Teskey, H.J., 1971. A new soldier fly from Canadian amber (Diptera: Stratiomyidae). The Canadian Entomologist 103, 1659-1661. Trapp, S., Croteau, R., 2001. Defensive resinosis biosynthesis in conifers. Annual Review of Plant Physiology and Plant Physiology 52, 689-724. Tyrrell, J.B., 1891. Fossil resin ("amber"). Canada Geological Survey, Annual Report 5, 14-15. Ward, J.H., 1963. Hierarchical grouping to optimize an objective function. Journal of the American Statistical Association 58, 236-244. Wilson, E.O., 1985. Ants from the Cretaceous and Eocene amber of North America. Psyche 92, 205-216. Yoshimoto, CM., 1975. Cretaceous chalcidoid fossils from Canadian amber. The Canadian Entomologist 107, 499-532.

61 Zobel, A.M., 1999. Cedarite and other fossil resins in Canada, in: Investigations into amber: proceedings of the international interdisciplinary symposium Baltic amber and other fossil resins, Gdansk, Poland, pp. 241-245.

62 CHAPTER 3: HYMENOPTERA IN CANADIAN CRETACEOUS AMBER (INSECTA)1*

INTRODUCTION The Hymenoptera are a hyperdiverse insect order consisting of approximately 145,000 described species, with an estimated 0.5 to 1.2 million modern species (Gaston, 1991; Grimaldi and Engel, 2005; Huber, 2009), and perhaps rivaling Coleoptera as the most diverse insect order (Grissell, 1999; Sharkey 2007; Huber, 2009). The order is commonly known for its social members, such as some wasps or bees, the latter of which are important pollinators (Michener, 2007); or ants, which constitute a sizeable part of terrestrial biomass (Holldobler and Wilson, 1990). However, these taxa constitute only a fraction of total hymenopteran diversity. The vast majority of hymenopterans are actually parasitoids - internal or external parasites that kill their hosts (Quicke, 1997; Grimaldi and Engel, 2005; Huber, 2009). Parasitoids are key members of many ecosystems and often limit populations of other insects groups (LaSalle and Gauld, 1993). Because of their host selectivity, parasitoids are employed extensively as biological control agents for pest management. Although stinging hymenoptera (Aculeata) and agricultural pests, such as sawflies (symphytans), are conspicuous members of modern ecosystems and are familiar to most people, the parasitic Hymenoptera, not surprisingly, comprise the majority of Cretaceous fossils for the order, and are the main focus of this review. The relationships between Hymenoptera and other holometabolous insect orders, as well as the arrangement and composition of many of the taxa within the Hymenoptera are somewhat unresolved. Sharkey (2007) and Heraty et al. (2011) have recently provided reviews of hymenopteran phylogeny, so little additional detail remain to be presented here. Although there is strong support for the of the order (e.g., Vilhelmsen, 1997, 2001; Grimaldi and Engel, 2005; Sharkey, 2007), many internal relationships are disputed, so we have adhered to a relatively conservative taxonomy (Table 3.1). Historically, the order has been divided into the grades 'Symphyta' (sawflies, wood wasps, and orussids), and 'Parasitica' (the numerous parasitoid lineages within the Apocrita), leading to the Aculeata (ants, bees, and wasps).

* A version of this chapter has been submitted as a solicited review article. McKellar, R.C., Engel, M.S., (in review) Hymenoptera in Canadian Cretaceous amber (Insecta). Cretaceous Research.

63 Table 3.1: Classification and list of extant families of Hymenoptera (adapted from Huber, 2009).

ARCHIHYMENOPTERA Rhopalosomatidae Infraorder Xyelomorpha Bradynobaemdae Superfamily Xyeloidea Formicidae Xyehdae Scolndae NEOHYMENOPTERA Vespidae Infraorder Tenthredinomorpha Superfamily Apoidea Superfamily Pamphiloidea Heterogynaidae Megalodontesidae Ampuhcidae Pamphilndae Sphecidae Superfamily Tenthredinoidea Crabronidae Argidae Colletidae Blasticotomidae Hahctidae Cimbicidae Andrenidae Dipnomdae Mehttidae Pergidae Megachilidae Tenthredimdae Apidae Infraorder Cephomorpha Infraorder Proctotrupomorpha Superfamily Cephoidea Superfamily Proctotrupoidea Cephidae Proctotrupidae Infraorder Siricomorpha Vanhomndae Superfamily Siricoidea Ropronndae Sincxdae Austronndae Anaxyehdae Peradenndae Infraorder Xiphydriomorpha Helondae Superfamily Xiphydrioidea Pelecimdae Xiphydnidae Proctorenyxidae Euhymenoptera Superfamily Diaprioidea Infraorder Orussomorpha Monomachidae Superfamily Orussoidea Diapnidae Orussidae Maamingidae Suborder Apocrita Superfamily Cynipoidea Infraorder Stephanomorpha Austrocyrupidae Superfamily Stephanoidea Ibalndae Stephamdae Lioptendae Infraorder Evaniomorpha Figitidae Superfamily Trigonalyoidea Cynipidae Tngonalyidae Superfamily Platygastroidea Superfamily Megalyroidea Nixomidae Megalyridae Sparasionidae Superfamily Ceraphronoidea Scehomdae Megaspihdae Platygastridae Ceraphromdae Superfamily Mymarommatoidea Superfamily Evanioidea Mymarommatidae Aulacidae Superfamily Chalcidoidea Gasteruptndae Mymaridae Evanndae Tnchogrammatidae Infraorder Vespomorpha Aphehnidae Superfamily Ichneumonoidea Sigmphondae Bracomdae Eulophidae Ichneumomdae Tanaostigmatidae Parvorder Aculeata Eupelmidae Superfamily Chrysidoidea Rotoitidae Plumarndae Encyrtidae Scolebythidae Euchantidae Sclerogibbidae Agaomdae Dryinidae Penlampidae Embolemidae Ormyndae Bethyhdae Torymidae Chrysididae Tetracampidae Superfamily Vespoidea Eurytomidae Sierolomorphidae Pteromahdae Tiphndae Leucospidae Pompihdae Chalcididae Mutilhdae Sapygidae Symphytans are almost unknown from Mesozoic amber, with the exception of a single member of Anaxyelidae from Early Cretaceous Spanish amber (Ortega-Bianco et al., 2008) and isolated orussid specimens from Late Cretaceous Siberian and New Jersey amber (Vilhelmsen, 2004). Therefore, the portions of the hymenopteran phylogeny most pertinent to our discussion are within the suborder Apocrita, and to a limited extent, the parvorder Aculeata. The composition of the Canadian amber hymenopteran assemblage is presented in this context, with a strong focus on the non-aculeate Apocrita, and with superfamilies forming the major units for most of the discussion. Relationships among the apocritan superfamilies are highly unstable, with the most recent reviews and syntheses (e.g., Sharkey, 2007; Vilhelmsen et al, 2010a; Heraty et al., 2011) still suggesting a largely unresolved or weakly supported topology. We have adopted a relatively conservative outline of proposed superfamily relationships due to this uncertainty (Fig. 3.1). Finer details regarding the placement and monophyly of each of the families or superfamilies discussed are presented within their respective sections.

Canadian amber perspective on the Hymenoptera The high diversity and wide range of biology that characterizes modern Hymenoptera appears to have been largely established by the Cretaceous, when the insect-bearing amber deposits first became abundant, and the fossil record of insects was greatly improved (Rasnitsyn and Quicke, 2002; Grimaldi and Engel, 2005). Within Canadian amber, the hymenopteran assemblage is composed almost exclusively of parasitoids. These taxa offer insights into the antiquity of parasitoid associations, through comparison to modern relatives with known biology. Additionally, the obligate parasitoid-host relationships of many of these taxa also provide evidence for the presence of other groups within the Cretaceous amber-producing forest, even though these host groups may not be preserved, or have yet to be recovered as inclusions themselves. The reliance of parasitoids on specific hosts may also make them viable as ecological indicators, but this aspect of their ecology remains largely inaccessible in Mesozoic ambers due to partial representation and the limited study of each assemblage. Canadian amber offers a unique window on the evolution of the Hymenoptera. This deposit constitutes the last known diverse insect assemblage in the Mesozoic (Grimaldi and Engel, 2005). Its proximity to the end-Cretaceous extinction event makes the deposit an important point for comparisons across MYA

Protimaspidae, Cynipidae /Figitidae*, joptendae

Figure 3 1. Apocntan phylogeny with Canadian amber inclusions indicated. Thickened hori­ zontal lines indicate the extent of the fossil record for each taxon; "X" indicates taxon pres­ ence in Canadian amber, with families listed along the right margin; the degree of stippling within the illustrated exemplar corresponds to the extent of our study within the RTMP and CNC-CAS collections, and those families illustrated are indicated with an asterisk. (Modified from Gnmaldi and Engel, 2005, fig. 11-9; McKellar and Engel, 2011a, fig. 5b; McKellar and Engel 2011b, figs. 2a, 4e; Pernchot et al., in press, fig. 14; McKellar and Engel in prep.).

66 the event, as well as to previous assemblages within the Mesozoic. It helps to refine our understanding of the impacts Late Cretaceous and early Tertiary events had on insects, while at the same time establishing the cast of taxa for later developments. Capturing Late Cretaceous diversity is particularly important for Hymenoptera because the group appears to have undergone two major adaptive radiations, one in the Mesozoic (e.g., Rasnitsyn, 2002; Grimaldi and Engel, 2005), followed by the rise of its social members in the Tertiary (e.g., Grimaldi and Agosti, 2000; Engel, 2001). Canadian amber provides a glimpse of the order during the latter part of its Mesozoic radiation (alongside the rise of angiosperms), and indicates which groups were still present near the end of the era. Interestingly, Mesozoic representatives of'parasitica' display some family-level extinctions across the end-Cretaceous boundary. Although the end- Cretaceous extinction is considered to have had little effect on insects (e.g., Labandeira and Sepkoski, 1993; Rasnitsyn, 2002; Grimaldi and Engel, 2005), the loss of some hymenopteran families coupled with a reduction in the diversity of others, suggests that there may be perceptible ecological effects around this time. An improved understanding of the biology of the affected groups (through their modern relatives and amber syninclusions) may provide insights into terrestrial changes associated with this time interval. There are a number of caveats for the study of Canadian amber that should be considered throughout this work. It should be noted that the record provided from amber is generally biased towards small taxa and those that live within the amber-producing forest or utilize the resin, and may be affected by such factors as temperature fluctuations or other controls on the production and viscosity of resin (e.g., Pike, 1995; Zherikhin, 2002; Martinez-Delclos et al. 2004). In the case of Canadian amber, the record is strongly biased toward smaller insects, with collected amber pieces typically displaying a length of less than 1 cm and rarely reaching 3.5 cm in length (Pike, 1995; McKellar et al., 2008). Furthermore, Canadian amber does not appear to have been exposed to long distance transport prior to burial, so it may sample a somewhat narrow range of marginal-marine habitats. The comprehensive account of Ceratopogonidae (Diptera) in the deposit suggested a range of larval habitats that is consistent with these restrictions (Borkent, 1995), as does our knowledge of the regional geology and the stable isotopic composition of the amber itself (McKellar et al., 2008). In addition to these considerations, the total number of inclusions documented for Canadian amber is deceptively high, and should not be viewed as a true measure of research 67 extent. This figure is attributable to the sheer number of pieces that contain either mites (Acari) or aphids (Aphidoidea), which constitute 29% and 15% of the total assemblage observed by Pike (1995), respectively. The deposit is still within the early stages of study, and even within the largest collection (RTMP), orders such as Hymenoptera are present within only 297 of the more than 2812 inclusion- bearing catalogued amber pieces. Finally, end-Cretaceous extinctions within Hymenoptera should be inferred with caution: the fossil record for the group is largely non-existent during the Paleocene, and the Canadian amber fauna pre­ dates the end of the Cretaceous by approximately 13 Ma.

Comparisons to other Cretaceous amber deposits Due to the taphonomic constraints of working on amber inclusions (Pike, 1995; Zherikhin, 2002; Martinez-Delclos et al., 2004), some of the most meaningful comparisons are made between amber assemblages. Throughout this work we compare the Canadian amber assemblage to a number of other Cretaceous deposits. The details of each repeatedly mentioned deposit's age are provided here to limit redundancy. Amber deposits with a substantial hymenopteran component include: Siberian (Taymyr) amber (Santonian, Late Cretaceous in age); New Jersey amber (Turonian, Late Cretaceous in age); Charentese (French) amber (Albian-Cenomanian, mid-Cretaceous in age); Burmese (Myanmar) amber (Late Albian, Early Cretaceous in age); Spanish amber (Albian, Early Cretaceous in age); and Lebanese amber (Barremian-Aptian, Early Cretaceous in age). Most of these deposits and their biodiversity have been reviewed within the edited volume of Penney (2010). Comprehensive lists of global insect fossil deposits, including compression fossil sites, are available within the work of Rasnitsyn and Quicke (2002), Martinez-Delclos et al. (2004), and Grimaldi and Engel (2005).

GEOLOGICAL SETTING "Canadian amber" is a general term for amber that has been collected from either Late Cretaceous (Campanian) strata at the Grassy Lake locality in southern Alberta, or a secondary deposit of this material along the shores of Cedar Lake in western Manitoba (McAlpine and Martin, 1969; McKellar et al., 2008). There are also a number of unnamed coal-associated Cretaceous amber deposits in western Canada, that have produced little in the way of insect inclusions (McAlpine and Martin, 1969; Pike, 1993), and the Eocene Hat Creek deposit, that is under preliminary investigation in British Columbia (Archibald and Makarkin, 2004,

68 Lawrence et al., 2008). Here we focus on the Grassy Lake locality because it is the in situ source for most of the Cretaceous inclusions studied to date. The known biodiversity and geological setting of Canadian amber have recently been reviewed by Pike (1995), McKellar et al. (2008, i.e., Chapter 2), and McKellar and Wolfe (2010, i.e., Chapter 1). These works have drawn together data from regional geology, the palaeoentomological and palaeobotanical records, Fourier-transform infrared spectroscopy of amber, and the stable isotope composition of H and C in amber. The resulting interpretation is that Grassy Lake amber was formed by cupressaceous trees (likely belonging to the genus Parataxodium Arnold and Lowther), and that the amber was deposited within a lagoon or salt marsh setting with little pre-burial transport.

Occurrence The Grassy Lake site is an abandoned pit mine near the Village of Grassy Lake, southwest of Medicine Hat, in southern Alberta. At this site, the uppermost coal seams within the Taber Coal Zone (Foremost Formation) have been mined, and the resulting tailings piles are slowly eroding, concentrating amber upon their surfaces. Although it is unclear exactly which of the six Taber Coal Zone seams outcrop and were mined at the site, the age of the amber is relatively well-constrained. The amber is approximately 78-79 million-years-old, based on radiometric dates obtained from bentonites within the shales that encompass the Taber coal zone (Goodwin and Deino, 1989; Eberth and Deino, 1992). Full locality details are available from the Royal Tyrrell Museum of Palaeontology.

Collections Major holdings of Canadian amber inclusions are found within the collections of the Royal Tyrrell Museum of Palaeontology (RTMP, TMP specimen numbers), the Canadian National Collection of Insects and Arthropods (CNC, CNC-CAS specimen numbers), the Harvard Museum of Comparative Zoology (MCZ), and the Royal Ontario Museum (ROM, which incidentally contains many of the types originally listed as MCZ specimens). Minor collections are also held by the University of Alberta Strickland Entomology Museum (UASM), University of Alberta Laboratory of Vertebrate Palaeontology (UALVP), and the Manitoba Museum. Our study has focused primarily on material within the CNC, RTMP, and UASM collections, which together contain approximately 4500 inclusions.

69 HYMENOPTERAN DIVERSITY IN THE ASSEMBLAGE Among the insect orders represented in Canadian amber, Hymenoptera have recently surpassed Diptera in terms of the number of recognized species (58 spp. compared to 50 spp.) to become the most speciose order in the deposit (updated from McKellar and Wolfe, 2010). The number of species recognized largely reflects the extent to which the individual orders have been studied. Hymenoptera were first reported from Canadian amber in the collaborative work of Carpenter et al. (1937). In this work Brues (1937) described ceraphronoids, ichneumonoids, mymarommatoids, platygastroids and serphitoids, and Kinsey (1937) described cynipoids. Subsequently, Evans (1969) has described Chrysididae and Sphecidae, Yoshimoto (1975) has described chalcidoids and mymarommatoids, Ponomarenko (1981) has described Dryinidae, and Formicidae have been described in a number of publications (e.g., Wilson, 1985; Dlussky, 1999). With the exception of Yoshimoto's (1975) work, most of these contributions have described single species. In recent years, there has been a resurgence in work on the deposit: Engel and Grimaldi (2005) have revisited formicids, Liu et al. (2007) have described cynipoids, Perrichot et al. (2011) have described a trigonyaloid, and McKellar and Engel have provided comprehensive accounts of the serphitids (2011a), ceraphronoids (2011b), and platygastroids (in prep.), from the deposit. Here we provide a summary of the current knowledge of Hymenoptera within the deposit, including comments on some taxa recorded from the deposit (e.g., Pike, 1995; Skidmore, 1999; McKellar et al., 2008) that have not been confirmed through subsequent work.

Evanioidea (?Aulacidae) The superfamily is currently considered to consist of three families, Aulacidae, Evaniidae, and Gasteruptiidae (Grimaldi and Engel, 2005; Engel, 2006; Penalver et al., 2010), although some workers treat Aulacidae as a subfamily within Gasteruptiidae (e.g., Zhang and Rasnitsyn, 2007). At one point, up to three fossil families were recognized as well, including Andreneliidae, Cretevaniidae, and Praeaulacidae (Penalver et al., 2010). Based on inconclusive placement within phylogenetic analyses (Basibuyuk et al., 2002) Andreneliidae has been variably recognized as a family (e.g., Zhang and Rasnitsyn, 2007) or placed within Evaniidae (e.g., Engel, 2006). Cretevaniidae has also had its contents transferred into Evaniidae (Basibuyuk et al., 2002; Zhang et al., 2007; Zhang and Rasnitsyn, 2007). At a larger scale, there has been some doubt regarding the monophyly of

70 the Evanioidea (e.g., Dowton and Austin, 2001), but the most recent cladistic analyses (Deans and Whitfield, 2003; Deans et al., 2006; Vilhelmsen et al., 2010a; Heraty et al., 2011) have suggested that the group is likely monophyletic. The Evanioidea display moderate modern diversity, with approximately 1000 species in 39 genera (Mason, 1993). In terms of biology, modern Evaniidae are predators within roach oothecae (e.g., Brown, 1973), while Gasteruptiidae are predators specific to wasp and solitary bee larvae (e.g., Engel, 1995), and Aulacidae are less specialized parasitoids of buprestid and cerambycid wood- boring beetles and xiphydriid wood wasps (Carlson, 1979; Grimaldi and Engel, 2005). The feeding strategies of the superfamily have been examined in a phylogenetic context by Basibuyuk et al. (2002), and a more complete account of their fossil history has been provided by Nel et al. (2004).

Current status of study Evanioids have been reported only once from Grassy Lake amber, by Skidmore (1999). This record is based on specimen tag data for CNC-CAS 155, which was determined as Gasteruptiidae sensu lato by A.P. Rasnitsyn in 1988. Although we have not yet observed this specimen, the inclusion should probably be referred to the family Aulacidae, as this has been the case with other specimens attributed to Gasteruptiidae s.l. by Rasnitsyn in (Grimaldi et al., 2002), due to a slight difference in family concepts (e.g., Dowton et al, 1997; Rasnitsyn, 2002). It appears as though evanioids are rare within the deposit, as all specimens observed within the RTMP collection that were tentatively identified as members of the superfamily (i.e., work notes and determinations on the specimen slides) have subsequently been shown to belong to Serphitidae (McKellar and Engel, 2011a). No members of Evaniidae have been recovered to date.

Comments Evanioids are somewhat uncommon as fossils, but their near-absence in Canadian amber is puzzling. This superfamily appears better-represented in other Cretaceous ambers. Evaniids are known from three species in Cenomanian and Santonian (Late Cretaceous) Russian ambers (Rasnitsyn, 1975). Two genera and three species of Evaniidae have been described from New Jersey amber, based on four specimens (Basibuyuk et al., 2000; Engel, 2006; Grimaldi and Nascimbene, 2010). The family has been identified in amber from Charentes, France (Perrichot et al., 2010). Burmese amber has provided two specimens that have been described as distinct species and genera within Evaniidae (Basibuyuk et al., 2000; Engel, 2006; Ross et al., 2010), as well as a single species of Aulacidae, and two monospecific genera of Gasteruptiidae s.l. (Cockerell, 1917a,b; Ross et al., 2010). Spanish amber has provided five species in two evaniid genera (Penalver and Delclos, 2010; Penalver et al., 2010). Lebanese amber also contains three evaniid species within three different genera (Basibuyuk et al., 2002; Deans et al., 2004; Azar et al., 2010). The lack of Canadian amber evaniids is particularly strange because they are fairly well-represented in slightly older North American New Jersey amber. Furthermore, their roach (Blattaria) hosts are present in the Canadian assemblage. It is expected that further collecting will provide additional representatives of the superfamily with preservation sufficient for formal description.

Ceraphronoidea (?Ceraphronidae, Megaspilidae, Stigmaphronidae) Ceraphronoidea consists of two extant families, Ceraphronidae and Megaspilidae. In the Cretaceous, two additional families existed alongside megaspilids - Stigmaphronidae and Radiophronidae. Recent cladistic analyses including fossil material have suggested that within Ceraphronoidea, Radiophronidae are a distinct , likely basal to (Stigmaphronidae + (Megaspilidae + Ceraphronidae)) (Ortega-Bianco et al., 2010, 2011a), while Stigmaphronidae are a monophyletic group basal to Megaspilidae + Ceraphronidae (Engel and Grimaldi, 2009, Davis et al., 2010; Ortega-Bianco et al., 2010), although ceraphronids perhaps render megaspilids paraphyletic, a possibility highlighted by certain extant taxa intermingling the traditional distinctions of the family (Miko and Deans, 2009). Although the Ceraphronoidea have been consistently supported as a monophyletic group, their placement among the Apocrita is debated. Some works support their placement within the Evaniomorpha (e.g., Rasnitsyn, 1975, 2002; Gibson, 1985; Dowton et al., 1997; Sharkey, 2007; Davis et al., 2010; Vilhelmsen et al., 2010a), while others have placed them in Proctotrupomorpha, near Platygastroidea (e.g., Ronquist et al., 1999; Sharkey and Roy, 2002). These competing hypotheses have recently been reviewed in detail (Engel and Grimaldi, 2009; Ortega-Bianco et al., 2010), and most of the fossil material for the superfamily has received coverage in these works and that of Penalver and Engel (2006). The Ceraphronoidea consist of an estimated 2000 modern species, and are known to act as parasitoids of Coccoidea (Hemiptera), Cecidomyiidae (Diptera), dipteran puparia, Lepidoptera, Mecoptera, Neuroptera, and Thysanoptera; and hyperparasitoids of Aphididae (Hemiptera) and Braconidae (Masner, 1993). The two extant families are cosmopolitan in distribution (Johnson and Musetti, 2004). Based on the high relative abundance and diversity of the superfamily in some amber deposits, such as New Jersey amber (Engel and Grimaldi, 2009) and Spanish amber (Ortega-Bianco et al., 2011a), it appears as though the superfamily may have been more successful in the Cretaceous than it is today.

Current status of study Ceraphronoids are uncommon inclusions within Canadian amber (Figs. 3.2a-c). To date, only seven specimens have been documented from the CNC, UASM and RTMP collections. Lygocerus (-Dendrocerus) dubitatus Brues was the first ceraphronoid described, and was based on a single ROM specimen with poor preservation (Brues, 1937). Although L. dubitatus was tentatively attributed to Megaspilidae, subsequent work has not clarified the details or placement of this taxon, and it is commonly treated as incertae sedis. Megaspilids are exceptionally rare within Mesozoic amber. Only two specimens of Conostigmus cavannus McKellar and Engel are known from Canadian amber. These, along with single specimens of both Conostigmus dolicharthrus Alekseev and Rasnitsyn and Prolagynodes penniger Alekseev and Rasnitsyn from Siberian amber, constitute the only Mesozoic representatives of the family (Alekseev and Rasnitsyn, 1981; McKellar and Engel, 2011). The first member of Stigmaphronidae described from the deposit was Tagsmiphron canadense Engel and Grimaldi (Engel and Grimaldi, 2009). Subsequent work has provided an additional three species within this genus: T. exitorum McKellar and Engel, T. leucki McKellar and Engel, and T. spiculum McKellar and Engel (McKellar and Engel, 2011). With the exception of C. cavannus, all species described from the deposit have been based on single specimens. Despite reports of Ceraphronidae within Canadian amber (McAlpine and Martin, 1969; McKellar et al., 2008), the family appears to be absent. McAlpine and Martin (1969) listed seven specimens within this family, but the specimen tag data of Skidmore (1999) do not support this list, and the only specimen reported as potentially belonging to Ceraphronidae (CNC-CAS 538) was apparently determined as a chrysidid by A.R Rasnitsyn in 1988.

Comments Megaspilids have a very limited fossil record, consisting mainly of Eocene Baltic amber representatives, with two additional species known from Siberian

73 Figure 3.2. Photomicrographs of Ceraphronoidea, Tngonalyoidea, Cynipoidea, and Platygastroi- dea in Canadian amber (A) Conostigmus cavannus (Megaspilidae) lateral view with metasoma partially missing, (B) habitus diagram of Conostigmus cavannus, (C) Tagsmiphron spiculum (Stigmaphronidae) lateral view, (D) Ahstemiam cellula (Maimetshidae) lateroventral view, wmg apices and most of metatarsi missing, (E) undescnbed Figitidae in lateral view, (F) Scelionidae (McKellar et al, in prep) lateral view, wmg apices strongly deflected by drying line in amber, (G) partial sceliomd in lateral view, with spider syninclusion adjacent to scale bar and partly ob­ scured by drying line in amber, (H) Scelionidae (McKellar et al, in prep) dorsal view Scale bars 1 mm (A-H) (Modified from McKellar and Engel, 201 lb, figs la, 3d,e, Pemchot et al, in press, fig 13a)

74 amber, and two species known from compression fossils in the Miocene Rubielos de Mora Basin of Spain (Penalver and Engel, 2006). They have also been reported from Burmese amber, but not described (Ross et al., 2010). The presence of Conostigmus as rare inclusions in Canadian amber makes sense given that a specimen of the genus has also been recovered from the slightly older (Santonian), Laurasian amber of . The rarity of these inclusions in both assemblages implies that the family was a relatively minor component of the Late Cretaceous hymenopteran fauna. The Stigmaphronidae have recently been reviewed by Engel and Grimaldi (2009), and additional taxa have been described by McKellar and Engel (2011) and Ortega-Bianco et al. (2011a). Taxa described in these works indicate that stigmaphronids played a much larger role in the Cretaceous fauna than the other ceraphronoid families. In many Cretaceous amber deposits, the family is one of the largest components of the hymenopteran assemblage, yet in Canadian amber only four specimens have been recovered, all of which belong within Tagsmiphron. This low diversity and abundance contrasts strongly with the New Jersey and Spanish amber assemblages, but a wide range exists between the various Cretaceous ambers. Siberian amber contains three individuals within Allocotidus, Hippocoon, and Stigmaphron (Kozlov, 1975; Engel and Grimaldi, 2009); New Jersey amber contains 11 specimens within Elasmophron and Tagsmiphron (Engel and Grimaldi, 2009); Burmese amber has provided two specimens within Burmaphron (Engel and Grimaldi, 2009); Spanish amber has yielded a staggering 54 specimens representing the genera Burmaphron, Elasmophron, Hippocoon, Libanophron, and Tagsmiphron (Ortega-Bianco et al., 201 la); and Alaskan (Cenomanian, Late Cretaceous) and Lebanese amber have each provided a single specimen of Allocotidus and Lebanophron, respectively (Muesebeck, 1963; Engel and Grimaldi, 2009). The reduced Alaskan and Lebanese assemblages are likely a result of limited hymenopteran study within these deposits, but in Burmese amber, there is the possibility that the low diversity and abundance reflects the actual faunal composition. Stigmaphronids are conspicuously absent from Charentese amber, perhaps as a result of the habitat sampled by this deposit, or sampling bias induced by the conditions of amber production (Perrichot et al., 2007b). Canadian amber contains the last record of the family prior to its disappearance at the end of the Cretaceous. Its assemblage suggests that Tagsmiphron may have been the last remaining genus of the family, if taphonomic or palaeobiogeographic factors were not responsible for the 75 observed composition. Even though few ceraphronoids have been collected from Canadian amber, Ceraphronidae and Radiophronidae are conspicuously absent from the assemblage. The lack of ceraphronids within the last diverse Cretaceous assemblage could be viewed as additional support for the group's post-Cretaceous origin within Megaspilidae (Engel and Grimaldi, 2009), as could the lack of Baltic amber exemplars. The absence of radiophronids is less informative, as they are currently known from nine specimens in Spanish amber (Ortega-Bianco et al., 2010), and it is unclear whether their range is restricted to the Early Cretaceous. Both of these ranges bear further examination, which is only possible once more data have been obtained from other amber deposits.

Trigonalyoidea (Maimetshidae) Maimetshidae are an enigmatic family found only in the Cretaceous. Members of the family have been described mostly from amber inclusions (e.g., Rasnitsyn, 1975; Perrichot et al., 2004; Perrichot et al., in press), but are also known from compression fossils (e.g., Rasnitsyn, 1990; Rasnitsyn et al., 1998; Rasnitsyn and Brothers, 2009). Nothing is known regarding the biology of the group, but specimens have been recovered as syninclusions alongside Dolichopodidae (Diptera), Sphecidae, and indeterminate Hymenoptera (Perrichot et al., 2004). Other taxa within Trigonalyoidea are known to utilize parasitoids within symphytan and lepidopteran larvae (Mason, 1993). The presence of maimetshids in Siberian, Canadian, French, Spanish, and Lebanese amber deposits, as well as compression fossils in Orapa, Botswana (Rasnitsyn and Brothers, 2009; Perrichot et al., in press) indicates that the family likely had a cosmopolitan distribution in the Cretaceous. The systematic placement of Maimetshidae is becoming more and more resolved (Perrichot et al., in press). Some authors have recognized the family within an expanded concept of Ceraphronoidea sensu Rasnitsyn (2002) or Stephanoidea sensu Ortega-Bianco et al. (2010) (e.g., Rasnitsyn, 1975; Ronquist et al., 1999; Rasnitsyn and Brothers, 2009; Perrichot, 2009; Vilhelmshen et al., 2010). Others have considered the group as belonging within Megalyridae (e.g., Shaw, 1988; 1990). The cladistic analysis of Vilhelmsen et al. (2010b) recognized Maimetshidae as the sister group to Trigonalyidae, a classificatory position followed herein and by Perrichot et al. (in press). Other relationships within Stephanoidea sensu Ortega-Bianco et al. (2010) are not as well resolved, and the 76 placement of Ceraphronoidea (as used here) among either the Evaniomorpha or Proctotrupomorpha is debated (see further discussion under "Ceraphronoidea").

Current status of study and comments A single maimetshid species has been described from Canadian amber, Ahstemiam cellula McKellar and Engel (Perrichot et al., in press). This new genus and species was based upon a single male specimen, the only individual recovered to date (Fig. 3.2d). It is somewhat odd that Trigonalyoidea are not better represented within the deposit, as numerous maimetshid specimens have been documented (Perrichot et al., in press) from amber in France (10 specimens belonging to Guyomaimetsha enigmatica Perrichot et al), Spain (8 specimens belonging to Iberomaimetsha rasnitsyni Ortega-Bianco et al., and /. nihtmara Ortega-Bianco et al.), and Lebanon (1 specimen belonging to Ahiromaimetsha najlae). Trigonalyidae are also known from both of these deposits (Nel et al., 2003; Perrichot et al., 2010; Pefialver and Delclos, 2010). Two specimens of Maimetsha arctica Rasnitsyn have also been described from Siberian amber, but have subsequently deteriorated so new material is needed (Perrichot et al., in press). The lack of exemplars in thoroughly-studied New Jersey amber (Grimaldi and Nascimbene, 2010), and their low abundance in Canadian and Siberian amber suggests that the superfamily may not have been as well represented in the habitats sampled by northern amber during the Cretaceous.

Cynipoidea (Cynipidae, Figitidae, Liopteridae, Protimaspidae) Cynipoidea includes approximately 3000 described extant species (Ronquist, 1999), with an estimated 17000 species yet to be described (Norlander, 1984; Liu et al., 2007). Although the superfamily contains the Cynipidae, a secondarily phytophagous family commonly known as gall wasps, the majority of its members are parasitoids. This includes basal taxa that are parasitoids of wood-boring insect larvae (e.g., Liu and Norlander, 1994), and more derived groups that are either parasitoids of various larvae within the Holometabola, or hyperparasitoids of aphids or psyllids through braconid or chalcidoid larvae (Buffington et al., 2005; Liu et al., 2007). The phylogenetic position of Cynipoidea has been unstable, with some analyses placing it as the sister group to Diapriidae (Rasnitsyn, 2002), or Platygastroidea (Castro and Dowton, 2006); while others have suggested a close relationship to both Proctotrupoidea and Platygastroidea + Chalcidoidea

77 (Dowton and Austin, 1994), or Platygastroidea and Proctotrupoidea (Vilhelmsen et al., 2010a). The analyses of Heraty et al. (2011) have suggested either a sister group relationship between Cynipoidea and Platygastroidea, or Cynipoidae and a clade composed of Proctotrupoidea, Diaprioidea, Mymarommatoidea, and Chalcidoidea. Additional details on relationships within the superfamily have been provided in the work of Liu et al. (2007) and references therein.

Current status of study Protimaspis costalis Kinsey (Protimaspidae) was the only cynipoid recognized from Canadian amber until the work of Liu et al. (2007). This work described Proliopteron redactus Liu and Engel (Liopteridae: Proliopterinae), Goerania petiolata Liu and Engel (Liopteridae: Goeraniinae), Micropresbyteria caputipressa Liu and Engel (Figitidae: subfamily incertae sedis), Anteucoila delicia Liu and Engel (Figitidae: Eucoilinae), and Tanaoknemus ecarinatus Liu and Engel (Cynipidae: subfamily incertae sedis). Subsequent to these descriptions, few additional cynipoids have been recovered from the deposit or discovered within the RTMP collections. Those specimens that have been discovered have yet to be described, but appear to belong within the family Figitidae (Fig. 3.2e).

Comments The fossil record of Cynipoidea has recently been reviewed by Liu et al. (2007) and Liu and Engel (2010). The research effort surrounding specimens in Cretaceous amber has largely been directed at inclusions in Canadian, New Jersey, and Siberian amber. This renders comparisons between a number of deposits difficult, but permits some well-supported comparisons between the two main North American deposits. While Canadian amber contains at least six individuals belonging to species within the families Protimaspidae, Liopteridae, Figitidae, and Cynipidae; New Jersey amber contains at least three specimens belonging to species in Stolamissidae and eucoiline Figitidae; and Siberian amber contains five species distributed among subfamilies of Figitidae (Liu et al., 2007). New Jersey and Canadian amber each contain their own unique extinct family, Stolamissidae and Protimaspidae respectively, and they share members of eucoiline Figitidae. Canadian amber appears unique among Cretaceous deposits in that it contains Liopteridae and a putative member of Cynipidae. Modern liopterids are thought to act as parasitoids on wood-boring insects such as buprestid beetles or siricid

78 wasps, and modern eucoilines are parasitoids of calyptrate fly larvae and those of cyclorrhaphans (Ritchie, 1993; Ronquist, 1999). Most of these host groups would be feasible in a Cretaceous ecosystem, with the exception of the Calyptrata, which appear to be largely a Tertiary group (Grimaldi and Engel, 2005). The cynipoid taxa documented from Canadian amber are of interest because they include some of the earliest records for what may be secondarily phytophagous hymenopterans. Liu et al. (2007) tentatively suggested that Tanaoknemus may belong within Cynipidae, and constitute an earlier record than definitive cynipids, such as Kinseycynips Liu and Engel known from Baltic amber.

Diaprioidea (Diapriidae) Diaprioidea are a moderately diverse hymenopteran superfamily, and Diapriidae consist of approximately 2300 described species in 150 genera, with an estimated 2200 undescribed species globally (Johnson, 1992; Masner, 1993). Diapriids are generally endoparasitoids of dipteran larvae and pupae, or those of ants (Formicidae) and rove beetles (Coleoptera: Staphylinidae) (Loiacono, 1987; Masner, 1993; Lak and Nel, 2009). Diaprioidea, includes the families Diapriidae, Maamingidae and Monomachidae, which were recently removed from Proctotrupoidea (Sharkey, 2007), a paraphyletic group (Dowton et al., 1997; Dowton and Austin, 2001). A fourth, extinct family is also known from Cretaceous amber of New Jersey and Spain (Engel, unpublished data). Analyses of metasomal characters have not supported Diaprioidea, and have brought the monophyly of Diapriidae into question (Vilhelmsen et al., 2010a). Diapriidae have variably been placed as sister to Cynipoidea (Rasnitsyn, 1988; Sharkey and Roy, 2002), or to Chalcidoidea + Platygastroidea (Dowton et al., 1997; Castro and Dowton, 2006), but the monophyly and placement of the family remains largely unresolved (Sharkey, 2007; Vilhelmsen et al., 2010a). The most recent analyses (Heraty et al., 2011) have suggested that Diaprioidea may either be the monophyletic sister group to Chalcidoidea, or a grade leading up to Chalcidoidea.

Current status of study Mc Alpine and Martin (1969) were the first to mention Diapriidae within Canadian amber, listing two specimens within the CNC-CAS collection. Schluter (1978) presumably referred to one of these two specimens, identifying it as indeterminate within Diapriidae (Perrichot and Nel, 2008). In 1989, A.P. Rasnitsyn determined

79 one of the specimens (CNC-CAS 661a) as Diapriidae cf. Ismarinae (Skidmore, 1999). This latter identification is almost certainly correct, as Ismarinae display a rather distinctive morphology within the family (Masner, 1993). We have found no additional exemplars of diapriidae within the RTMP or UASM collections, suggesting that this family is exceedingly rare within the assemblage.

Comments In general, Diapriidae are rare as inclusions in Mesozoic amber. Perrichot and Nel (2008) provided a summary of the family's fossil record, and described the first belytine from Late Albian amber of south-western France. Lak and Nel (2009) added another diapriid from these deposits but left the subfamilial assignment as uncertain. This work indicated that a single ismarine species has also been described from Aptian amber of Japan (Fujiyama, 1994). Otherwise, an indeterminate diapriinae has been reported from Siberian amber (Zherikhin and Sukatsheva, 1973), an indeterminate ismarine has been reported from the Cenomanian amber of north-western France (Schluter, 1978), and indeterminate diapriids have been reported from the Turonian Timmerdyakh amber of Russia (Rasnitsyn, 1980), Burmese amber (Grimaldi et al., 2002), and Early Albian amber from Spain (Engel, unpublished data). If one of the diapriids observed within the Canadian amber assemblage indeed belongs within Ismarinae, it would constitute the third record of this subfamily in the Mesozoic.

Platygastroidea (?Platygastridae, Scelionidae) Platygastroidea is composed of four families, Nixoniidae, Sparasionidae, Scelionidae, and Platygastridae (Table 3.1). Although Scelionidae was synonymized with Platygastridae by Sharkey (2007) based on the of Scelionidae (Austin and Field, 1997; Dowton and Austin, 2001; Murphy et al., 2007; Johnson et al., 2008). The family has been recognized with weak support in subsequent morphological analyses (Vilhelmsen et al., 2010a) and it would appear that removal of the basal lineages Nixoniini and Sparasionini to familial rank is a more suitable solution than a retrograde classification pooling everything into Platygastridae s.l., a system in which much hierarchical as well as distinctive morphological and biological information is obscured. The superfamily is considered to be monophyletic (e.g., Austin et al., 2005; Vilhelmsen et al., 2010a), but its placement within Apocrita is far from resolved. Morphological analyses have suggested that Platygastroidea may be the sister group to Ceraphronoidea

80 (Ronquist et al., 1999), or its families may be placed in an unresolved trichotomy with Ceraphronoidea (Sharkey and Roy, 2002). Conversely, molecular analyses have suggested a potential sister group relationship between Platygastroidea and Chalcidoidea (Dowton and Austin, 2001). This relationship has found some support within the work of Heraty et al. (2011), but the Platygastroidea have also been recovered as the sister group to the remainder of Proctotrupomorpha within this work. Platygastroidea is a relatively large group of parasitoids, with approximately 4460 described modern species, and a further 6000 species estimated (Masner, 1993; Austin et al., 2005). Both Scelionidae and Platygastridae display a nearly cosmopolitan distribution, with scelionids acting as idiobiont parasitoids within eggs produced by a wide range of insects and spiders, and platygastrids acting as koinobiont parasitoids within immature hemipterans and cecidomyiid dipterans (Masner, 1993; Austin et al., 2005). Johnson et al. (2008) have provided a summary of all described fossils within the superfamily.

Current status of study Platygastroids are present with such high abundance and diversity in Cretaceous amber deposits, that some have suggested the group reached its pinnacle prior to the Tertiary (Grimaldi et al., 2002; Grimaldi and Engel, 2005). Despite their common occurrence as amber inclusions, remarkably few platygastroid taxa have been described from Cretaceous amber (Brues, 1937; Schluter, 1978; Nel and Azar, 2005; Johnson et al., 2008). Only two scelionid species have been described from Canadian material, Baryconus fulleri Brues and Proteroscelio antennalis Brues, based on two individuals (Brues, 1937). The number of recognized taxa will increase significantly in the near future, as a full account of the 65 identifiable platygastroids in the RTMP and UASM collections (McKellar et al., in prep., Figs. 3.2f-h, 3.3a) suggests an additional 16 species, as well as what appears to be a number of new genera, all within Scelionidae. Interestingly, only two poorly- preserved individuals of P. antennalis are among these specimens, and no further B. fulleri material has been recovered (although there is one new species that appears to belong within this genus). Comparable taxonomic work is underway for the Platygastroidea in Spanish amber (Ortega-Bianco et al., unpublished data), which will soon permit comparisons between Early and Late Cretaceous amber representatives. Based on our observations of the RTMP and UASM collections, we 81 Figure 3.3. Photomicrographs of Platygastroidea and Serphitoidea m Canadian amber. (A) five conspecific male Sceliomdae in various orientations and states of preservation (McKellar et al., in prep.), (B) Serphites hynemam (Serphitidae) in lateral view, (C) three conspecific male S. para­ doxus syninclusions, (D) female (left) and male with everted genitalia (right) belonging to 5. hyne- mani. Scale bars 1 mm (A-D). (Modified from McKellar et al., 2008, fig. 6f).

82 suggest that records of Platygastridae in Canadian amber (Skidmore, 1999; McKellar et al., 2008) need to be critically re-examined. Although the RTMP and UASM specimens display a wide range of flagellar article numbers and vary significantly in the relative lengths of their metasomal terga, all specimens appear to be better accommodated within Scelionidae than Platygastridae. This contrasts with determinations in the CNC-CAS collection, where five platygastrids have been listed along with 44 scelionids (Skidmore, 1999).

Comments At this point, it is difficult to draw comparisons between our material and that of other amber deposits, because they are in the early stages of study. As with most Cretaceous amber deposits, Scelionidae are the dominant hymenopteran taxon in terms of both abundance and diversity. Preliminary results from the study of Albian (Early Cretaceous) Spanish amber suggest that there are fewer species present in the Spanish assemblage, that they comprise the majority of hymenopteran inclusions, and that they too belong exclusively to Scelionidae (J. Ortega-Bianco, pers. comm.). Currently, only a single scelionid species has been described from the Cenomanian amber of France (Cenomanoscelio pulcher Schluter, with three additional species in open nomenclature), and two scelionid species have been described from Lebanese amber (Cretaxenomerus jankotejai Nel and Azar and Proteroscelio gravatus Johnson et al. (Schluter, 1978; Nel and Azar, 2005; Johnson et al., 2008). Canadian amber also contains members of Proteroscelio. Additional family-level reports include: platygastrids from Charentese amber (Perrichot et al., 2010), scelionids from Burmese amber (Ross et al., 2010), and platygastrids from Spanish amber (Penalver and Delclos, 2010), but it is unclear whether changes in the status of Scelionidae have affected which family has been reported from each deposit. Comparisons to other Cretaceous deposits are restricted almost exclusively to comments on relative abundance. Pike (1995) demonstrated that Platygastroidea account for approximately 29% of hymenopteran inclusions in Canadian amber: both Skidmore (1999) and our own experience with the RTMP and UASM collections have produced similar results (near 32%) on a larger scale, under less controlled collecting conditions. A preliminary overview of inclusions in Siberian amber (Zherikhin and Sukatsheva, 1973) has shown that platygastroids account for approximately 37% of all hymenopteran inclusions (Austin et al., 2005). This may suggest a similar trapping frequency or habitat, but until more is known about the systematics of

83 platygastroids in other deposits, this is merely speculation. The presence of numerous platygastroids within Canadian amber would be expected based on their modern biology. Potential host taxa, such as spiders and hemipterans make up a large portion of the assemblage (Pike, 1995), implying abundant prey. In the deposit scelionids are often found as syninclusions with these taxa (Fig. 3.2g). Other interesting features of the Canadian amber assemblage include single-sex congregations of up to five conspecific scelionid syninclusions (Fig. 3.3a), possibly suggesting sexual behaviour.

Serphitoidea (Serphitidae) Serphitids are another family that are known exclusively from Cretaceous amber, and that have proven difficult to place in a systematic framework. The family was originally placed within the Proctotrupoidea (=Serphoidea at the time) on the basis of its wing venation, but Braes (1937) also noted a two-segmented petiole, as in Formicidae, and a head similar to that of Chalcidoidea. It was not until the work of Kozlov and Rasnitsyn (1979) that an affinity to members of the Mymarommatoidea was noted, based upon shared petiolar morphology. When Kozlov and Rasnitsyn (1979) described the Siberian amber serphitids and mymarommatids, they considered the latter a subfamily of the Serphitidae, but Mymarommatoidea had previously been treated as distinct superfamily, and subsequent work has also recognized it at the this level (e.g., Noyes and Valentine, 1989; Gibson, 1993; Gibson et al., 1999; 2007) with the Serphitoidea distinct (Grimaldi and Engel, 2005). Recent classification has suggested the combination of Serphitoidea and Mymarommatoidea within the nanorder Bipetiolarida and the combination of these superfamilies with Chalcidoidea to form the parvorder Chalcidones (Engel, 2005). The larger-scale placement of these taxa is largely unresolved (for further discussion see "Chalcidoidea" section). As Serphitidae are extinct, and their relationships are contentious, almost nothing is known about their biology. Their occurrence in numerous amber deposits suggests a Laurasian or cosmopolitan distribution.

Current status of study Serphites paradoxus Braes was among the first wasps described from Canadian amber, and the two males Braes described within the genus were also the basis for the family Serphitidae (Brues, 1937). More than 70 years later, McKellar and Engel (2011) revisited the family within the CNC-CAS, RTMP and UASM

84 collections. Based on 60 additional specimens, this work described a further five species and one new genus, including: Serphites bruesi McKellar and Engel, S. hynemani McKellar and Engel, S. kuzminae McKellar and Engel, S. pygmaeus McKellar and Engel, and Jubaserphites ethani McKellar and Engel (Figs. 3.3b—d). The family was found to be second only to Scelionidae in terms of both abundance and diversity within the deposit.

Comments Serphitidae are much more abundant within Canadian amber than in most other Cretaceous deposits, yet display comparatively low genus-level diversity. In Siberian amber, 13 specimens have been reported and are distributed among the species Microserphites parvulus Kozlov and Rasnitsyn, Serphites dux Kozlov and Rasnitsyn, S. gigas Kozlov and Rasnitsyn, and Aposerphites solox Kozlov and Rasnitsyn (Kozlov and Rasnitsyn, 1979). In New Jersey amber, four specimens have been recovered, representing two Serphites species (Engel et al. in press). Neither Charentese amber (Perrichot et al., 2007b; 2010), nor Lebanese amber (Azar et al., 2010) have provided any specimens. Burmese amber has yielded approximately 24 specimens, and these are presently being described (Ross et al, 2010; M.S. Engel, unpublished data). Only four specimens have been recovered from the Spanish amber deposits, representing Aposerphites angustus Ortega-Bianco et al., Microserphites soplaensis Ortega-Bianco et al., Serphites lamiak Ortega-Bianco et al., and S. silban Ortega-Bianco et al. (Ortega-Bianco et al., 2011c). In the Canadian assemblage, all but one specimen belong within Serphites. This, combined with the wide range of body sizes and morphologies observed in Canadian Serphites, suggests that they may have filled niches occupied by other serphitid genera elsewhere. Much of the genus-level diversity within Serphitidae may have been lost prior to their end-Cretaceous disappearance, but this will remain pure speculation until the validity of the genera has been tested, and their distribution, ranges, potential hosts and habitats are better understood. Serphitids within Canadian amber have provided a unique opportunity to view fine anatomical details for this extinct family. Many male specimens have fully-everted genitalia, making distinctions between otherwise similar species possible (McKellar and Engel, 2011). In some cases males are found in single-sex accumulations (Fig. 3.3c) of up to six syninclusions, or together with a conspecific female (Fig. 3.3d), hinting at sexual behaviour or confirming allotypes. 85 Difficult to discern features, such as the presence of a possible netrion and metasomal laterotergites, have also been observed in specimens from the deposit (Gibson et al. 2007, McKellar and Engel, 2011), and some of these features are phylogenetically informative. Work is currently under way to complete a phylogenetic analysis (Engel et al., in prep.) that will encompass Serphitoidea and Mymarommatoidea from Spanish amber (Ortega-Bianco et al., 2011c; 20lid), Burmese (Myanmar) amber (Engel and Grimaldi, 2007a), New Jersey amber (Engel and Grimaldi, 2007a; Engel et al., in press), Siberian amber (Kozlov and Rasnitsyn, 1979), and the newly described taxa from Canadian amber (McKellar and Engel, 2011).

Mymarommatoidea (Mymarommatidae) Mymarommatoidea is considered to be a monophyletic group (Gibson 1986; Vilhelmsen and Krogmann, 2006; Gibson et al., 2007; Heraty et al., 2011), and has been suggested to either form the sister group to Serphitoidea (Grimaldi and Engel, 2005; Ortega-Bianco et al., 20 lid), form a component of Serphitoidea (Kozlov and Rasnitsyn, 1979; Rasnitsyn et al., 2004), or form the sister group to Chalcidoidea with Serphitoidea unresolved (Gibson, 1986; Ronquist et al., 1999). Within Mymarommatoidea, the Mymarommatidae were recently supplemented by two additional families, Alavarommatidae, and Gallorommatidae. The former family was based on a single species in Spanish amber (Ortega-Bianco et al., 201 Id); while the latter family was based on isolated Cretaceous species from Siberian (Kozlov and Rasnitsyn, 1979) and French Cenomanian amber (Schliiter, 1978), and now contains additional species from Burmese (Engel and Grimaldi, 2007) and Spanish amber (Ortega-Bianco et al., 201 Id). Mymarommatoids are instantly recognizable due to their two-segmented petiole, reduced venation and minute size. Beyond their unique morphology, mymarommatoids are noteworthy in that there are currently more fossil species described (16 spp.) than there are extant species described (12 spp.) - although it has been suggested that approximately 13 additional extant species awaiting description (Gibson et al., 2007; Ortega-Bianco et al., 2011d). If the preservational constraints for such minute insects are taken into account, this superfamily appears to have been much more diverse in Cretaceous ecosystems than it is today (Engel and Grimaldi, 2007). Little is known about the biology of the superfamily, but eggs have been suggested as a potential host life-stage based upon mymarommatoid morphology (Yoshimoto, 1984). Specimens have

86 been collected from a range of moist forest habitats, such as bracket fungi, leaf litter, and soils, and despite their rarity in collections, they appear to have a nearly global distribution (Gibson, 1993; Gibson et al., 2007 and references therein).

Current status of study Although mymarommatids are relatively common inclusions in Canadian amber, few taxa have been described from this family. Yoshimoto (1975) viewed what is now Mymarommatidae as a subfamily within Mymaridae (Chalcidoidea) when he documented Archaeromma minutissimum (Brues) and A. nearcticum Yoshimoto. Gibson et al. (2007) later revised the work of Yoshimoto. Through a more comprehensive examination of the superfamily, these authors found that Protooctonus Yoshimoto was actually a mymarommatid, not a mymarine, as originally described. Thus, the genus was synonymized with Archaeromma, producing a third mymarommatid species within the deposit, A. masneri (Yoshimoto). These three species are represented by 23 individuals divided among the ROM, CNC-CAS, and MCZ collections. We have recently begun work on 24 new specimens from the RTMP and UASM collections, and have recovered at least two additional morphospecies (Figs. 3.4a, b). To date, no representatives of Gallorommatidae have been recovered from Canadian amber, leaving Archaeromma as the sole mymarommatoid genus in the deposit, with a moderately high abundance and species-level diversity.

Comments The high clarity and low suspended particulate load of Canadian amber make it one of the few Mesozoic deposits in which specimens as small as mymarommatids are easily observed for detailed study. With this said, numerous dark drying lines in the amber make it difficult to spot the specimens within the early stages of amber nodule screening and preparation. It appears as though this has had some impact on the apparent abundance of mymarommatids within various collections. In the 1281 CNCI amber specimens detailed by Skidmore (1999), 20 individuals of Mymarommatidae were listed (approximately 1.5% of inclusions). Meanwhile, in the RTMP collection of more than 2965 specimens, 28 mymarommatids appear in museum catalogues (of which we can verify at least 12, or between 0.4% and 0.9% of all inclusions), and in the subset of RTMP collections analyzed by Pike (1995) 12 mymarommatids were listed out of 1155 inclusions (approximately 1% of all findings). In approximately four kilograms Figure 3.4. Photomicrographs of Mymarommatoidea, Chalcidoidea, and Ichneumonoidea in Canadian amber (A) Archaeromma nearcttcum (Mymarommatidae) in oblique dorsal view (B) undescnbed Archaer- omma (McKellar et al, m prep ) in lateral view (C) undescnbed, new species within Chalcidoidea, likely within Torymidae (McKellar et al, in prep ), in dorsal view, with wings contorted due to interaction with drying lines in amber, and with large piece of frass posteriorly (upper right of image) (D, E) undescnbed, new ichneumonids in oblique dorsal view (D), and lateral view (E), (Kopylov et al, in prep ) (F) taphonomi- cally damaged braconid m lateral view, with antenor portion of head displaced to the antenor, and thin-cuti- cled areas, such as the metasoma and antennae deformed Scale bars 0 5 mm (A,B), 1 mm (C-F) (Modified from McKellar et al, 2008, figs 6d,e) of bulk amber prepared from Grassy Lake, we have encountered 11 additional specimens. This last figure is significantly higher than would be expected based on the other collections observed, and the sampling analyses of Pike (1995). It is hoped that the new specimens will permit a broader and more detailed examination of Campanian mymarommatoids, as the amber has been collected and prepared with an eye toward such small inclusions. Samples have been slide mounted and polished into thin sections, permitting the extensive use of compound microscopy.

88 Unless the forest that produced Canadian amber sampled a specific habitat where mymarommatoids were particularly common, their relatively high incidence as inclusions provides strong support for the group having been better- represented in the Cretaceous than it is today. It is unclear whether the lack of Gallorommatidae and Alavarommatidae in both Canadian and New Jersey amber indicates their absence in North America at this point, but Archaeromma is the only genus known from either deposit. Archaeromma appears to have reached its greatest diversity within the fauna sampled by Canadian amber. Three species have been described therein, as opposed to the two species known from Siberian amber, or the single species known from each of the Japanese, New Jersey, Spanish and Burmese deposits (Ortega-Bianco et al., 201 Id). All of the additional material viewed within the RTMP collection clearly belongs to Mymarommatidae, and appears to belong to Archaeromma as well, but more detailed study will be necessary for a confident attribution.

Chalcidoidea (?Eupelmidae, Mymaridae, Tetracampidae, ?Torymidae) Remarkably, the Chalcidoidea are one of the most extensively documented of hymenopteran lineages in Canadian amber. The systematics of this superfamily is notoriously difficult and although chalcidoids are recorded from various other Cretaceous ambers including Alaskan amber (Hurd et al., 1958; Usinger and Smith, 1957; Langenheim et al., 1960), Siberian amber (Zherikhin and Sukatcheva, 1973), Charentese amber (Perrichot et al., 2010), and Burmese amber (Ross et al., 2010), few have been studied critically and much work remains to be completed on the Mesozoic record of Chalcidoidea. Chalcidoidea have been variably proposed as the sister group to Mymarommatoidea (e.g., Rasnitsyn, 2002; Gibson et al, 2007; Heraty and Darling, 2009), Platygastroidea (Dowton and Austin, 2001), Diapriidae (Dowton et al., 1997), Diaprioidea (Castro and Dowton, 2006), or to Mymarommatidae + Maamingidae (Vilhelmsen et al., 2010a). It is noteworthy that mymarommatids were not included in any of the molecular studies (e.g., Dowton et al., 1997; Dowton and Austin, 2001; Castro and Dowton, 2006) until the work of Heraty et al. (2011). The latter work suggested that Mymarommatoidea is most likely the sister group to Diaprioidea + Chalcidoidea. Despite the variability in placement, the superfamily is strongly supported as monophyletic (Vilhelmsen et al., 2010a; Heraty et al, 2011). The Chalcidoidea are almost exclusively parasitoids, although some notable exceptions do exist, such as the famous fig wasps which are secondarily 89 phytophagous. None of these phytophages are known in Cretaceous amber, their earliest records being from Eocene-Oligocene compression fossils of the Florissant Formation (Brues, 1910) and Early Miocene inclusions in Dominican amber (Pefialver et al. 2006). Nonetheless, most chalcidoids are important parasitoids heavily used in biological control programs. Their hosts range across life stages within 13 orders of insects, and include some arachnids and nematodes as well (Gibson et al., 1999). The superfamily is considered one of the most diverse within Hymenoptera. Estimates for total diversity within Chalcidoidea are as high as 375,000 to 500,000 modern species (Heraty and Gates, 2003; Noyes, 2003), with approximately 21,250 species currently described (Noyes, 1998; Gibson et al., 1999). The superfamily is thought to have undergone explosive radiation after the Mesozoic, but the Paleocene gap in their fossil record has partially obscured the nature of this radiation, and not all of the component families diversified at the same time (Heraty and Darling, 2009).

Current status of study The chalcidoids were the first group of Hymenoptera to receive focused attention during the early days of Canadian amber research. Yoshimoto (1975) made the most sizeable contribution to their study in Canadian amber. He described six species in Tetracampidae: Baeomorpha distincta Yoshimoto; B. dubitata Brues; B. elongata Yoshimoto; B. ovata Yoshimoto; B. acuodens Yoshimoto; Distylopus bisegmentus Yoshimoto, and one species in Trichogrammatidae, Enneagmus pristinus Yoshimoto. The trichogrammatid has subsequently been transferred to Mymaridae (Huber, 2005; Heraty and Darling, 2009). He also described four species in Mymaridae: Carpenteriana tumida Yoshimoto; Macalpinia canadensis Yoshimoto; and Triadomerus bulbosus Yoshimoto (the fourth species, Protooctonus masneri Yoshimoto, was later transferred to Mymarommatidae by Gibson et al. (2007)). The family-level placement of both Baeomorpha and Distylopus has been questioned, because both genera possess a long curved calcar that does not match well with that observed in modern representatives of Tetracampidae (Gumovsky and Perkovsky, 2005), but this has not yet resulted in a revised systematic placement. In addition to the work of Yoshimoto (1975), Torymidae and Eupelmidae have subsequently been noted by Pike (1995). Eulophidae was noted by McAlpine and Martin (1969) and also as a determination by Yoshimoto (for CNC-CAS 82) within the work of Skidmore (1999). The record of Eulophidae appears to be

90 erroneous, as Yoshimoto (1975) listed specimen "CAS 82" as the male allotype for the tetracampid Baeomorpha distincta. Furthermore, the eulophid record of McAlpine and Martin (1969) is actually based on the work of Langenheim et al. (1960) which provided tentative identifications of inclusions within Alaskan amber. No specimens have been described from these three families, and we have yet to encounter any specimens that are readily attributable to these families within the RTMP or UASM collections. This said, a number of undescribed specimens exist, some of which are assuredly new taxa. One specimen that we have observed (Fig. 3.4c) is approximately 2.5 times the length of any chalcidoid reported from the deposit, and preliminary key work (Gibson, 1993) suggests referral to Torymidae, supporting the work of Pike (1995). Further work on these specimens will provide more confident attribution.

Comments With the limited amount of work conducted on other Mesozoic fossil chalcidoids, there is little to compare the Canadian assemblage to, aside from the much later, Eocene Baltic amber. Not surprisingly, chalcidoids are much better represented in Baltic amber, with numerous species described across the families Encyrtidae, Eucharitidae, Eupelmidae, Mymaridae, Perilampidae, Tetracampidae and Torymidae (Heraty and Darling, 2009 and references therein). This is the result of much more extensive collection and study of Baltic amber. Further work on Mesozoic deposits will provide a source for more appropriate comparisons. At this point, a putative record of Eupelmidae exists from Charentese amber (Perrichot et al., 2010), Eulophidae has been reported from Alaskan amber (Langenheim et al., 1960), and Chalcididae and Mymaridae have been reported from Burmese amber (Ross et al., 2010), but none of this material has been described or critically examined. If some of the new material from Canadian amber does indeed represent Torymidae, the group will join Mymaridae and Tetracampidae as one of the only chalcidoid families currently recognized in the Mesozoic (Heraty and Darling, 2009). As one of the first groups to be thoroughly documented, Chalcidoidea played a key role in demonstrating that Cedar Lake amber shares many species with Grassy Lake amber, supporting a common source for the material. Yoshimoto demonstrated a 54% overlap in the chalcidoid species he studied from the two localities, and this pattern was later confirmed (83% species overlap) by Borkent (1995) in his work on Ceratopogonidae (Diptera) from these sites.

91 Ichneumonoidea (Ichneumonidae, Braconidae) Ichneumonoidea has been subdivided into a varying number of families, but most recent treatments have settled upon just two families, the Ichneumonidae and Braconidae (Gauld and Bolton, 1988; Grimaldi and Engel, 2005; Perrichot et al., 2009). The superfamily is typically considered to be monophyletic (e.g., Gauld and Bolton, 1988; Vilhelmsen et al., 2010a) and closely related to Aculeata (Dowton and Austin, 1994) if not the sister group to Aculeata (Rasnitsyn, 2002; Dowton et al., 1997; Sharkey, 2007). This relationship has not always been recovered in molecular analyses (e.g., Dowton and Austin, 2001; Castro and Dowton, 2006; Heraty et al., 2011) and is still somewhat uncertain (Sharkey, 2007). The Ichneumonoidea are among the most diverse hymenopteran superfamilies, with an estimated 40,000 modern species in Braconidae and a further 60,000 in Ichneumonidae, although relatively few actually are described at around 23,300 and 17,600, respectively(Gauld and Bolton, 1988; Wahl and Sharkey, 1993; Huber, 2009). The superfamily displays a wide range of biology, including endo- and ectoparasitoids with idiobiont or koinobiont strategies, and utilizes a very broad range of host taxa, sometimes acting as hyperparasitoids (Wahl and Sharkey, 1993).

Current status of study To date, three species have been described within Braconidae from Canadian amber. These include Diospilus allani Brues, Neoblacus facialis Brues, and Pygostolus patriarchicus Brues (Brues, 1937). Ichneumonids have also been reported (Skidmore, 1999) based on CNC-CAS specimen tag data (one poorly preserved specimen, and one specimen determined as Paxylommatinae by A.P. Rasnitsyn in 1988). Although we have not had opportunity to observe these last two specimens, we can confirm that the specimens reported so far are fairly representative for the proportions of these families in the deposit. The RTMP and UASM collections have furnished 13 identifiable specimens, of which five belong to Ichneumonidae, and eight to Braconidae. Preliminary work suggests that there are at least two species of ichneumonids and at least two species of braconids beyond those already recognized from the deposit (Figs. 3.4d-f). One of the ichneumonids displays many similarities with Eubaeus Townes (D.S. Kopylov, pers. comm.) known from Siberian amber (Townes, 1973). Given the range of

92 biology known for modern ichneumonoids and our current uncertainty regarding determinations, little can be deduced from the Canadian amber specimens. Townes (1973) suggested that Eubaeus was likely a parasitoid of symphytans, and its presence in both Siberian and Canadian amber would suggest additional faunal overlap between these regions in the Late Cretaceous. The material available suggests that there are strong taphonomic constraints on the ichneumonoids preserved within the deposit. The largest specimen we have observed is 3 mm long, and nearly all specimens display relatively poor preservation, with strong deformation in thin-cuticled areas such as the metasoma and antennae (Fig. 3.4f). Exposure prior to full encapsulation is likely a limiting factor in the preservation of larger specimens, and those preserved are often distorted by remobilization of the resin prior to hardening. Comparatively large body sizes in Ichneumonoidea also suggest that some members may have been able to free themselves from the resin.

Comments Ichneumonoidea are much better represented by compression assemblages than in contemporaneous amber deposits. This is apparently a result of the much larger number of specimens sampled by compression collections, as even Ichneumonidae are minor constituents of some compression assemblages (e.g., Kopylov, 2010). Despite their comparatively large size, or perhaps partly as a result of it, few identifiable ichneumonoid remains are found in most Mesozoic amber assemblages. Canadian amber is similar to most other deposits in this regard. It contains a relatively sparse record for ichneumonoids, particularly Ichneumonidae. Elsewhere, the braconid Protorhyssalus goldmani Basibuyuk and Quicke has been described from New Jersey amber (Basibuyuk and Quicke, 1999; Grimaldi and Nascimbene, 2010). Siberian amber has yielded three ichneumonid species in distinct genera (Townes, 1973), and a further four species, plus two additional new genera have been described recently from the region (Kopylov, in prep.). Braconids have also been documented from the region's deposits (Zherikhin and Sukatsheva, 1973), but no specimens have been described. Both Charentes amber (Perrichot et al., 2010) and Spanish amber (Penalver and Delclos, 2010; Ortega-Bianco et al., 2009, 2011) have records of Braconidae, while Burmese amber (Ross et al., 2010) has been reported to contain braconids as well as ichneumonids. At present, it is not possible to draw any firm conclusions about the very

93 low number of ichneumonid specimens recovered from Canadian amber, and reported from some other Mesozoic deposits, but some speculation is possible. If their biology contributes to the likelihood of becoming an amber inclusion, the proportionally high number of braconid inclusions may be related to their use of hemimetabolous insects such as aphidoids, coccoids, and psocopterans as hosts, while ichneumonids largely utilize holometabolous insects (Wahl and Sharkey, 1993). Groups such as Aphidoidea and Psocoptera make up a sizeable portion of the Canadian amber assemblage (Pike, 1995) and apparently the Siberian assemblage as well (Zherikhin and Sukatsheva, 1973), while Coccoidea are abundant in New Jersey (Grimaldi et al., 2000) and Spanish amber (Alonso et al., 2000). A better understanding of the ichneumonoids and potential hosts present in all Cretaceous ambers may make speculations about host relationships testable, as well as refine our understanding of their role as principal parasitoids in Late Mesozoic ecosystems.

Aculeata, the stinging wasps In general, aculeates appear to be under-represented in Cretaceous ambers (Evans, 1969), although this situation has changed much in the intervening decades (e.g., Grimaldi and Engel, 2005; Engel and Grimaldi, 2006; Engel, 2008). With this said, Canadian amber entombs a unique cross-section of small aculeates, including the families Scolebythidae, Bethylidae, Chrysididae, Dryinidae, Formicidae, and Crabronidae. There are a limited number of specimens for each of the aculeate families, and we have not yet had opportunity to work on much of this material extensively, so most of our discussion is arranged around the families present and is general in nature.

Scolebythidae Scolebythidae were only recently recognized in Canadian amber (Engel, Ortega- Bianco, and McKellar, in prep.). Two species have been described based on three inclusions (Figs. 3.5a, b), and it appears as though these species belong to two different and new genera. Elsewhere, scolebythids are known from a single species in Spanish amber (Engel, Ortega-Bianco, and McKellar, in prep.). Three monotypic genera have also been described from Lebanese amber: Libanobythus milkii Prentice and Poinar; Uliobythus terpsichore Engel and Grimaldi; and Zapensia libanica Engel and Grimaldi (Azar et al., 2010). A single female of Boreobythus turonius Engel and Grimaldi has also been described from New

94 Figure 3.5. Photomicrographs of Aculeata and fragmentary of larger taxa. (A, B) one of the two new Scole- bythid species described from the deposit (A), and a habitus diagram (B) in dorsal view (Engel et al., in prep). (C) undescribed Bethylidae (McKellar and Engel, in prep.) in oblique dorsal view (D) undescribed Formicidae (McKellar and Engel, in prep ) in oblique anterior view, with gaster curled beneath trunk and with most legs swept together against a flow line (to lower left of image). (E) Fragmentary remains of larger specimen, apparently Bracomdae in lateral view, with three prominent filaments representing the ovipositor and ovipositor sheaths, and to the right of these are faint impressions of the wings (upper right of image), metasoma (central right), and hmd legs (lower right of image) in a drying line Scale bars 1mm. (Modified from Engel et al., in prep.) Jersey amber (Engel and (inmaldi, 2U07). Although the tossil taxa described to date appear to be rather finely subdivided with numerous monotypic genera, these divisions have withstood cladistic analyses (Engel and Grimaldi, 2007; Engel, Ortega-Bianco, and McKellar, in prep.). In all deposits where scolebythids have been encountered, they constitute a rare component of the assemblage, but their morphological disparity suggests that they were once relatively diverse. Scolebythids are rarely encountered in modern collections, and appear to have been much more diverse in the past, with six modern species and 10 fossil species described (Azevedo et al., 2011; Engel, Ortega-Bianco, and McKellar, in prep.). The only available biological data for the family suggests that they are gregarious ectoparasitoids of cerambycid and anobiid beetles (Brothers, 1981; Melo, 2000). Neither of these coleopteran families is known from Canadian 95 amber, but this does not necessarily offer much insight: beetles within the assemblage are in the very early stages of study, with only two species described to date (O'Keefe et al., 1997; Poinar, 2005; McKellar and Wolfe, 2010; McKellar and Engel, in prep.), and it is likely scolebythids simply favor wood-boring beetles of suitable size and biology rather than these two families specifically.

Bethylidae Bethylidae have been reported from Canadian amber, with two specimens determined by L. Masner in 1974 (Skidmore, 1999). We can confirm their presence based on RTMP material (Fig. 3.5c), but this material has yet to be described. Our work on the RTMP collection has shown that representatives of this family are rare, but not exceedingly so. The identifiable RTMP specimen appears to belong within the subfamily Epyrinae based on preliminary key work (Finnamore and Brothers, 1993). Evans (1973) made passing mention of a Celonophamia Evans species within Canadian amber, but this material was never described. It is possible that the RTMP specimen belongs to this genus. Bethylids are relatively well-known in other Cretaceous ambers, and their fossil history has recently been reviewed by Perrichot and Nel (2008). The family has been reported but not described from French Cretaceous amber (Perrichot and Nel, 2008), New Jersey amber (Grimaldi et al, 2002; Grimaldi and Nascimbene, 2010; Engel, unpublished data), and Spanish amber (Penalver and Delclos, 2010; Ortega-Bianco and Engel, unpublished data). Four species have been described from Burmese amber: Apenesia electriphila Cockerell, Bethylitella cylindrella Cockerell, Epyris atavellus Cockerell, and Sclerodermas quadridentatum Cockerell (Ross et al., 2010). Six bethylid inclusions have also been reported from Siberian amber (Zherikhin and Sukatsheva, 1973), with two species described, Archaepyris minutus Evans and Celonophamia taimyria Evans (Evans, 1973). If the RTMP specimen belongs to Celonophamia, it would indicate additional faunal overlap between Siberian and Canadian amber. Bethylids are diverse within modern ecosystems with 2325 nominal species, a cosmopolitan distribution, and greater abundance in the tropics (Finnamore and Brothers, 1993; Huber, 2009). Known hosts for modern representatives include Lepidoptera and Coleoptera larvae (Nagy, 1969; Doutt, 1973; Finnamore and Brothers, 1993); both of these orders are little-known in the Canadian amber assemblage (Pike, 1995; McKellar et al., 2008).

96 Chrysididae This family is currently represented by a single described species within Canadian amber, Procleptes carpenteri Evans (Evans, 1969). Skidmore (1999) also provided a single record of Chrysididae in the CNCI collection (Chrysididae: Cleptinae, specimen CNCI-CAS 538, determined by A.P. Rasnitsyn in 1988). The RTMP and UASM collections have yet to yield any additional individuals. Compared to other Cretaceous ambers, the rarity of chrysidids in the deposit is not surprising. The group is presently unknown from Burmese and Lebanese amber, and has been reported but not described from Spanish amber (Penalver and Delclos, 2010). In addition, undescribed species are represented in New Jersey amber (Engel, unpublished data). Two species, Hypocleptes rasnitsyni Evans and Protamisega khatanga Evans were described from Siberian amber based on 11 specimens (Evans, 1973). The rarity of chrysidids in amber may be related to host taxa availability, or the habitats sampled by the various amber-producing forests. Chrysidids are represented by approximately 3000 described modern species, and there are an estimated 1000 species awaiting description (Kimsey and Bohart, 1990). Their modern distribution is cosmopolitan, with temperate deserts as their region of greatest known diversity (Finnamore and Brothers, 1993). Chrysididae are known to act as parasitoids or cleptoparasites of a wide range of insects, including lepidopterans, phasmatids, symphytans, vespids, and apoids (Kimsey and Bohart, 1990). Evans (1969) viewed Procleptes as very similar in morphology to extant Cleptes Latreille and suggested that this was likely indicative of either a symphytan host or utilization of phasmatid eggs. Four decades after the formation of this hypothesis, symphytan inclusions are still unknown from the deposit, while putative phasmatid remains have been encountered only rarely (Pike, 1995; McKellar et al., 2008). This might suggest phasmatids as a more likely host group for Procleptes, but other taxa cannot be ruled out given the extent of study within the deposit, and the apparent under- representation of symphytans in amber.

Dryinidae Dryinus canadensis (Ponomarenko) is the only dryinid currently recognized from the deposit, and is known from a single specimen (Olmi, 1995). Pike (1995) reported a second specimen within this family, but the specimen was subsequently described and included as a paratype of Serphites pygmaeus

97 McKellar and Engel (Serphitidae) (McKellar and Engel, 2011). Dryinids known from other Mesozoic amber deposits (Olmi et al., 2010) include: Aphelopus palaeophoenicius Olmi described from Lebanese amber; Cretodryinus zherichini Ponomarenko and Dryinus antiquus (Ponomarenko) described from Siberian amber; and Burmanteon olmii Engel and Hybristodryinus resinicolus Engel described from Burmese amber (Engel, 2005b; Olmi et al., 2010). The family has not yet been collected from New Jersey amber (Grimaldi and Nascimbene, 2010) or Charentese amber (Perrichot et al., 2010). Modern dryinids are moderately diverse, with approximately 1600 species occupying a cosmopolitan distribution (Coelho et al, 2011, and references therein). They are parasitoids of both immature and adult plant hoppers (Auchenorrhyncha), and display specialized chelate protarsi in most females, in order to handle their prey (Finnamore and Brothers, 1993). Two of their most commonly preyed-upon auchenorrhynchan families, Cicadellidae and Membracidae, have been reported from Canadian amber (Skidmore, 1999). There also exists the possibility that the extinct family Jascopidae (Hamilton, 1971) may have been attacked. Further work on both the hymenopteran and hemipteran components of the assemblage will hopefully elucidate some of these biological relationships.

Formicidae, the ants Ants within Canadian amber have been few, but have figured prominently as they were originally reported as part of the early work on Mesozoic formicids. Sphecomyrma canadensis Wilson followed upon the description of ants from New Jersey (Wilson et al., 1967) and Siberian amber (Dlussky, 1975; 1983). The two specimens of Sphecomyrma in the CNC-CAS collection were later joined by Canapone dentata Dlussky and Eotapinoma macalpini Dlussky, described based upon two additional CNC-CAS specimens (Dlussky, 1999). Cananeuretus occidentalis Engel and Grimaldi is the most recently-described formicid from the deposit, based upon two TMP specimens (Engel and Grimaldi, 2005). We are currently in the process of describing a fifth formicid species from Canadian amber, based on material donated by the Leuck family. The single specimen (Fig. 3.5d) appears to show many similarities to C. occidentalis, but the degree to which it is contorted within the amber has rendered specimen preparation and description difficult. At this point, it is obvious that the specimen has more numerous mandibular teeth and a more prominent nodus than C. occidentalis but

98 the fine details of its morphology await study. Fossil ants have recently been reviewed in a number of works (e.g., Engel and Grimaldi, 2005; Ward, 2007; Perrichot et al., 2007a). As classified by Engel and Grimaldi (2005) following the work of Bolton (2003), the Canadian amber formicids represent the subfamilies Sphecomyrminae, Dolichoderinae, and perhaps Aneuretinae and Ponerinae. This places Canadian amber behind the New Jersey, and Siberian assemblages in terms of recognized species-level diversity, but apparently at the forefront of subfamily-level diversity. The contrast is strongest when compared to Santonian Siberian amber, which contains five species solely in Sphecomyrminae (Engel and Grimaldi, 2005). These diversity differences suggest that the habitat associated with each amber deposit played an important role in their formicid assemblages. Similarities, such as the presence of sphecomyrmines and dolichoderines in numerous Laurasian amber deposits, have also been viewed as support for a Laurasian origin and subsequent dispersal of these groups (Perrichot et al., 2007a).

Crabronidae, and other apoid wasps Lisponema singularis Evans, 1969 is the only apoid wasp known from Canadian amber, and was described based on a single male in the MCZ collection. Evans (1969) commented on the marked similarity between the amber inclusion and modern Spilomena Shuckard (Crabronidae), which he viewed as a clear indication of the fossil exemplar having nested in pre-existing hollows. Perhaps more interesting is that Spilomena is a solitary wasp that preys on thrips (Evans, 1969). If the morphological similarity implies a similar biology, it may suggest that L. singularis preyed on the thysanopterans that are present in low numbers within the amber assemblage. Modern apoid wasps display a range of parasitoid to primitive social lifestyles, typically preying upon lepidopteran larvae, orthoperoids or spiders (Finnamore, 1993). The family is quite large among the apoid wasps, with approximately 8745 described species (Pulawski, 2011). Apoid wasps are relatively rare inclusions in other Cretaceous ambers. Described crabronid species include: Psolimena electra Antropov from New Jersey amber (Grimaldi and Nascimbene, 2010); Taimyrisphex pristinus Evans (tentatively placed within the family), as well as Cretoecus spinicoxa Budrys and Pittoecus pauper Evans from Siberian amber (Evans, 1973); Cretospilomena familiaris Antropov and Prolemistus apiformis Antropov from Burmese amber. Additional taxa have been reported from Burmese amber (Ross et al., 2010),

99 although all of the species originally described within the family by Antropov (2000), under the retrograde classification as 'Sphecidae', have subsequently been transferred to appropriate places among the apoid wasp families (Bennett and Engel, 2006; Ross et al., 2010). Apoid wasps have also been reported from Charentese amber (Perrichot et al., 2010), Spanish amber (Penalver and Delclos, 2010), and Lebanese amber (Prentice, 1994; Ohl, 2004). Bennett and Engel (2006) have reviewed the fossil record of apoid wasps, including their Tertiary representatives.

Comments Canadian amber captures a mixture of fossorial and flying aculeates, but has not yet provided any large aculeates. The largest aculeate described was Procleptes carpenteri, at slightly more than 3.2 mm in total body length. Based on the limited number of formicids, it appears as though the entombed assemblage was not biased toward ground-level and lower trunk sampling, although this deficiency may be habitat-related (Perrichot et al., 2007b). Most Cretaceous ambers show relatively low numbers of ants (Engel and Grimaldi, 2005), so their low abundance here should not be taken as an absolute indicator. The lack of large aculeates suggests that either resin viscosity was insufficient to trap these hymenopterans, or that relatively small resin flows were insufficient to fully entomb and preserved them. Fragmentary remains of larger taxa are occasionally found within the deposit, such as disassociated setae, wing fragments, or ovipositors (Fig. 3.5e). Thus, it is suggested that the scarcity of large resin flows (e.g., Pike 1995) is likely the controlling factor in the preservation of large aculeates and other hymenopteran groups, such as ichneumonoids, within the deposit.

DISCUSSION Relative abundance of families The differences in Hymenoptera representation between deposits may reflect underlying differences between the types of amber-producing forests, conditions within the forests, or taphonomic constraints. New Jersey amber and Canadian amber would be expected to bear similar wasp assemblages because the deposits themselves show numerous similarities. Both appear to be the product of cupressaceous trees, both occur on the same continent - even if it was periodically divided by the Western Interior Seaway (Kauffman, 1984), and the two deposits are reasonably close in terms of age (Grimaldi et al., 2000, McKellar et al., 2008). Despite these similarities, recent work on families such as Serphitidae, Stigmaphronidae, and Mymarommatoidea, shows that there are pronounced differences in the relative abundance of the hymenopteran families and that comparatively few genera are shared between these deposits. This suggests that potential host groups for the wasps may have played a large role in the composition of each assemblage. In turn, palaeolatitude and climate conditions (temperate vs. subtropical settings) may have dictated which host groups were available (Grimaldi et al., 2000). The palaeolatitude hypothesis is supported by some of the family-level work conducted on Siberian amber. Although Canadian and Siberian amber were formed on different continents, they are separated by only about 5 Ma (Grimaldi and Engel, 2005) and little more than 6° of palaeolatitude (Martinez-Delclos et al., 2004). Zherikhin and Sukatsheva (1973) have demonstrated similarities to the Canadian assemblage in terms of family-level diversity and relative abundance for Hymenoptera. Within both Siberian and Canadian amber, Serphitidae is the second most abundant group after Scelionidae, and groups such as Mymarommatoidea and Cynipoidea are relatively well-represented. Subsequent taxonomic work has shown that genera shared between the two deposits include relatively common and widespread taxa, such as Serphites (Serphitidae), as well as a compliment of otherwise uncommon taxa, such as Conostigmus (Megaspilidae) and possibly Celonophamia (Bethylidae) and Eubaeus (Ichneumonidae). The full extent of faunal overlap will only be addressed through more detailed study of the deposits, but current knowledge suggests that this line of research may provide insights into Cretaceous palaeobiogeography and ecology. The faunal contents of amber deposits may also reflect the conditions under which they were formed. If forest fires, infestations, or physical perturbations were responsible for the release of resin within some amber- producing forests, the entombed assemblages may be related to these events. The assemblages may be biased toward representing groups common during a specific season, or those that utilize resources such as injured trees. Some deposits are known to occur in association with fusainized remains (e.g., Grimaldi et al., 2000; Brasier et al., 2009; Najarro et al., 2010), which would suggest fire may have played a role in amber genesis. Alternatively, the stable isotopic composition of the amber itself may provide some insight into the events that led to amber production (McKellar et al., 2011). Although most insect assemblages have not been studied thoroughly enough to test these potential influences, they bear consideration in future work.

Diversity patterns and future expectations One of the more interesting trends emerging within the Canadian amber assemblage, when compared to earlier Cretaceous assemblages, is what appears to be a reduction in higher-level taxa (genera or families) coincident with an increase in the number of species within the remaining groups. This pattern appears in a number of well-represented groups. Within Mymarommatoidea, Archaeromma is the only genus present, but the genus displays its highest species-level diversity in the Cretaceous (more than four species). In Ceraphronoidea, particularly within Stigmaphronidae, only Tagsmiphron is present, but it is represented by at least three species. Within Serphitoidea, Serphites is the only large serphitid present, but the genus is represented by five species. This pattern is not universal: in groups such as the formicids, there are a wide range of supraspecific taxa with few representatives. The groups that demonstrate a reduction in higher-level taxa are in many cases those that appear to have experienced extinctions near the end of the Cretaceous (Stigmaphronidae and Serphitidae) or appear to be less diverse in modern ecosystems than they were in the Cretaceous (Mymarommatoidea). While it is tempting to interpret the reduction in higher-level taxa as an indicator of the decline of certain families leading up to the end of the Cretaceous, it should be noted that most of these comparisons are based on very small sample sizes. Until a better understanding of all Cretaceous amber assemblages is reached, and the effects of taxonomic decisions, palaeoecology, or palaeobiogeographic distributions on the Canadian records can be ruled out, most interpretation must be tentative. If patterns persist after the Hymenoptera have been studied to the same extent in a number of other deposits, and the taxonomic units have been assessed, an explanation will be necessary, and hopefully apparent. Based on what is known of contemporary ambers, a natural question is which additional families would be expected based on findings in other Cretaceous amber deposits? Given the similarities between the Canadian and Siberian assemblages, it seems likely that additional chrysidids and bethylids will be recovered (Evans, 1973), as well as at least some members of Gallorommatidae. More representatives of Evanioidea would be expected, as would specimens of Evaniidae. Both of these taxa are widespread within Cretaceous ambers and Canadian amber contains inclusions of their host groups.

The paucity of Aculeata, symphytans, and phytophages The near-absence of Aculeates reported in the assemblage is largely a product of their scarcity as inclusions, but is also partly related to the extent of their study. We have encountered additional exemplars of Bethylidae, Scolebythidae and Formicidae. These taxa are represented by only six specimens in the more than three-thousand inclusions we have examined in detail (RTMP and UASM collections). The exceptionally low numbers of Aculeata and the absence of symphytans bear further explanation. Symphytans are nearly unknown from Mesozoic ambers, with few examples from Siberian, New Jersey and Spanish amber (Grimaldi et al., 2002). The lack of symphytans in Canadian amber may be partly related to their reduced diversity relative to apocritans (Sharkey, 2007), particularly outside of cool temperate regions (i.e., proportional representation). Regardless of their diversity in the region, symphytans are intimately tied to plants, and their presence would be expected within amber producing forests. Their larvae predominantly feed within cones, on leaf tissues, or on fungi within wood (Grimaldi and Engel, 2005). Symphytan biology, coupled with their excellent compression fossil record (Rasnitsyn, 2002), suggests that their interaction with resin must exert a strong control on the amber record for the group. Perhaps the simplest explanation is a taphonomic one. The majority of inclusions in Canadian amber are small, and the hymenopterans are no exception. Microhymenopterans are well-represented, while the opposite is true of groups that typically have larger members. In groups with a range of sizes, only the smallest taxa are represented. This is particularly evident in aculeates, and has been noted previously (Evans, 1969; 1973). It is likely that both aculeates and symphytans are under-represented because they are not readily encapsulated in a single, small resin flow. Furthermore, large taxa are either strong enough to escape small flows or strong enough fliers to reduce their chances of being blown into a resin flow. The near-absence of phytophagous Hymenoptera is much easier to explain. While primary phytophages, such as symphytans, were likely excluded from the amber record because of their size, it appears as though secondarily phytophagous groups, such as Cynipidae and Agaonidae, simply were not present. These families did not exist at the time, or were at least in the very early stages of their evolutionary development. The putative record of Cynipidae provided by Liu et al. (2007) from Canadian amber is the earliest recorded occurrence of the family, and fossil Agaonidae are not known until the Eocene (Penalver et al, 2006).

CONCLUSIONS It is difficult to make generalizations about the hymenopteran assemblage in Canadian amber, because of potential taphonomic influences within the deposit as well as the infancy of modern work on the fauna. This is further complicated by the uncertainty associated with comparing this deposit to others that may have been produced under dramatically different conditions, in geographically disparate locations and separated by millions of years. Despite these complications, some distribution and range patterns do appear to be taking shape as additional taxa are added to the assemblage, and more groups are reported. In particular, our observations provide additional support for the idea that some hymenopteran groups were widespread in the Cretaceous, even though they currently have austral distributions or have their highest diversity within this region. This pattern has been observed in Scolebythidae, which are now known from Canadian amber, as well as Lebanese, Spanish, and New Jersey amber, but appear to be a relict in the modern austral region (Engel and Grimaldi, 2007). The suggestion that some groups were more diverse in the Cretaceous than they are now is also supported by some of the inclusions in Canadian amber. Groups such as the Scelionidae, Mymarommatidae, and Ceraphronoidea are represented by many more inclusions than would be expected on the basis of their modern representatives. Part of this increase in representation may be a result of the warm temperate conditions at the time, but Scelionidae in particular display a wide range of morphologies (and likely diversity) compared to modern collections. As one of the best-studied groups, hymenopterans provide a clear indication that we are far from reaching the plateau of the species discovery curve (e.g., Bebber et al., 2007; Bernard et al., 2010) within the deposit. 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THE FIRST MESOZOIC MICROPHYSIDAE (HEMIPTERA) Introduction Microphysids are a small group of diminutive, predatory plant bugs found predominantly on tree bark, but also in decaying wood, or in wet plant litter. Members of this family are poorly known, because of their small size (1.5 to 3 mm), cryptic habitats, and limited distribution (Schuh and Slater 1995). Recent species appear to have a Holarctic distribution with some putative South African exceptions (Schuh and Slater 1995, Schuh and Stys 1991). The Recent fauna is composed of approximately 30 species in either five (e.g., Schuh and Slater 1995) or four genera (Popov et al. 2008, followed here), and until now, the fossil record for the family has consisted often species described from Eocene Baltic and Rovno ambers, and all documented within the last 10 years (Table 4.1).

Table 4.1. Checklist of described fossil Microphysidae. [Note that the species Loricula {Loricula) ablusa Popov 2006 and Loricula {Myrmedobia) kerneggerorum Popov 2006 have been transferred to the Anthocoridae (Popov and Herczek 2009), and that species are listed from youngest to oldest here].

Taxon Age Deposit

Loricula {Myrmericuld) perkovskyi Putshkov and Popov 2003 Late Eocene Rovno amber Loricula {Eocenophysa) damzeni Popov 2004 Middle Eocene Baltic amber Loricula {Loricula) ceranowiczae Popov 2004 Middle Eocene Baltic amber Loricula {Loricula) finitima Popov 2006 Middle Eocene Baltic amber Loricula {Loricula) polonica Popov and Herczek 2008 Middle Eocene Baltic amber Loricula {Myrmericuld) heissi Popov 2006 Middle Eocene Baltic amber Loricula {Myrmericuld) ocellata Popov 2006 Middle Eocene Baltic amber Loricula {Myrmedobia) pericarti Popov 2004 Middle Eocene Baltic amber Loricula {Myrmericuld) samlandi Popov 2006 Middle Eocene Baltic amber Tytthophysa sylwiae Popov and Herczek 2009 Middle Eocene Baltic amber Popovophysa entzmingeri gen. et sp. nov. Campanian Canadian amber

* A version of this chapter has been published as two separate papers. McKellar, R.C., Engel, M.S., 2009. A new thorny lacewing (Neuroptera: Rhachiberothidae)from Canadian Cretaceous amber. Journal of the Kansas Entomological Society 82(2), 141-121. McKellar, R.C., Engel, M.S., (in press). First Mesozoic Microphysidae (Hemiptera): a new genus and species in Late Creta­ ceous amber from Canada. The Canadian Entomologist. Herein we provide documentation of the first Mesozoic Microphysidae based on the remains of two males preserved in Late Cretaceous amber from the uppermost Foremost Formation in the vicinity of Grassy Lake, southern Alberta. The known biodiversity of this amber deposit and its geological setting have recently been reviewed by Pike (1995), McKellar et al. (2008, Chapter 2), and McKellar and Wolfe (2010, Chapter 1). These works suggest that Grassy Lake amber was formed approximately 78-79 m.y.a., by cupressaceous plants (likely belonging to the genus Parataxodium Arnold and Lowther), and that the amber was deposited within a lagoon or salt marsh setting without significant pre-burial transport.

Materials and methods Amber specimens were embedded in epoxy, slide-mounted, and polished into thin sections for optimal viewing and long-term preservation. This technique largely follows that described by Nascimbene and Silverstein (2000) for preparing fragile amber. The thickness of the mounted specimens limited the use of compound microscopy in this study. Descriptive terminology generally follows that of Schuh and Slater (1995) and Stys (1962). Anatomical abbreviations include 'tr.' for transverse, and 'long.' for longitudinal. Measurements in the description are those of the holotype, with those of the paratype in parentheses. All measurements were made using an ocular micrometer on an Olympus SZ60 stereomicroscope, and supplemental observations were made with an Olympus BX51 compound microscope. Photomicrographs were prepared using a Nikon D1X camera attached to an Infinity K-2 long-distance microscope lit with Microptics fiber optic flashes. A Zeiss Axio Imager. A1 compound microscope was used for higher magnification photomicrographs ('b.f.' denotes bright field photographs, 'd.f denotes dark field photographs).

Systematic Palaeontology Order Hemiptera Linnaeus Suborder Heteroptera Latreille Infraorder Cimicomorpha Leston et al. Family Microphysidae Dohrn Key to the fossil genera and subgenera of Microphysidae [Modified from Popov and Herczek (2009): these authors also keyed all known fossil species.]

1. Oval body outline; hemelytra with convex anterior margin and no clear distinction between corium and membrane; cuneus indistinct, exocorium broad and reaching apex of cuneus Tytthophysa Popov and Herczek — Oblong body outline; hemelytra with nearly straight anterior margin and distinct corium, cuneus, and membrane; exocorium narrow and reaching costal fracture 2

2(1). Hemelytra with single, large, closed cell in membrane, bordered by thick veins and with short, clavate processus corial; endocorium with single subhyaline cell; metacoxae widely separated; labial segment IV shortest Popovophysa gen. nov. — Hemelytra with variable closed cells in membrane, typically with elongate processus corial, and often with additional longitudinal veins; endocorium without subhyaline cells; metacoxae close to each other; labial segment I typically shortest (Loricula Curtis) 3

3(2). Labium thin and reaching middle of mesosternum; apex of labial segment II near or reaching base of head Loricula (Loricula) Curtis — Labium thickness variable and only reaching procoxa; apex of labial segment II not reaching base of head 4

4(3). Labium thick and reaching base of procoxa; apex of labial segment II positioned at midlength of compound eye Loricula (Myrmedobia) Barensprung — Labium thin and reaching midlength or apex of procoxa; apex of labial segment II not reaching base of head Loricula (Myrmericuld) Popov

Popovophysa McKellar and Engel gen. nov. Type species. Popovophysa entzmingeri McKellar and Engel sp. nov.

Etymology. The new genus-group name is a combination of Popov (in honour of Yuri Popov, of the Paleontological Institute, Russian Academy of Sciences - recognizing his contributions to the family), and physa (Greek, 'bellows or bubble'), a component of the family name. The name is feminine.

Diagnosis. Male. Compound eyes prominent, hemispherical, positioned posterolaterally on head; ocelli present, dorsally protuberant; antennae slender, 4-segmented, antennomere I shortest, antennomere IV longer than antennomere II. Labium inserted anteriorly on head, 4-segmented, reaching apices of procoxae, gently arched; segment I greatly reduced, segment IV (apicalmost) shortest, II and Ill subequal in length. Pronotum trapezoidal, with distinct anterior collar, collar longer than postocular length of head and approximately 6 times wider than long; callosities not prominent; transverse groove distinct, well impressed. Metacoxae separated; tarsi dimerous; pretarsal claws simple, arolium absent. Endocorium apically with single posterior cell bordered by thick veins; membrane with one thick-veined cell present, with strong processus corial (pronounced veinal stub). Hind wing with simple distal abscissa of R+M. In most aspects, similar to Ciorulla Pericart, but differentiated by presence of short labial segment IV and single subhyaline cell in endocorium. Differentiated from all Microphysinae by widely separated metacoxae, and hind wing with unbranched distal abscissa of R+M.

Description. Macropterous; body form not rounded or oval as in coleopteriform species. Head prognathous, elongate; clypeus horizontal. Hemelytra long, entirely covering abdomen, subtransparent except clavus somewhat more heavily sclerotized, membrane transparent and faintly infumate, corium, clavus, and cuneus weakly coriaceous; corium not densely covered with microtrichia, such microtrichia sparsely scattered on corium and absent from membrane; costal margin in corium narrow (= narrow exocorium); costal fracture well developed, demarcating distinct cuneus. Hind wing lightly infumate; R, M, and Cu distinct; R+M and Cu forming distal abscissae extending to, but not reaching, wing apex. Abdomen elongate ovoid, lateral borders convex, segments transverse; pygophore (sternum IX) elongate, bluntly rounded at apex; parameres not visible.

Discussion. The new genus shares many characteristics with Ciorulla, the sole genus of Ciorullinae, including a similar hemelytral form, and widely separated metacoxae. Popovophysa is readily distinguished from Ciorulla by the single vein-bound cell within its endocorium (two such cells, or spots, are present in Ciorulla). The distal abscissa of R+M is simple in Popovophysa, not forked as in most modern Microphysinae (but this feature is unknown for Ciorullinae in which the holotype, and only known specimen, has not been dissected or prepped further: Pericart 1974, Popov 2004). As in all New World taxa of Microphysinae, labial segment I is greatly reduced. Popovophysa entzmingeri McKellar and Engel sp. nov. Figs. 4.1,4.2

Material examined. Holotype: Male, UASM 22323; deposited in the University of Alberta Strickland Entomology Museum, Edmonton, Alberta, Canada; Grassy Lake amber, Campanian, Late Cretaceous. Small segment of spider's web present as syninclusion. Paratype: Male, TMP 96.9.334; deposited in the Royal Tyrrell Museum of Palaeontology, Drumheller, Alberta, Canada; Grassy Lake amber, Campanian, Late Cretaceous. Syninclusions are fragments of two feathers attributable to aquatic birds.

Etymology. The specific epithet is a patronym for Arthur (Art) Entzminger, the late father of Vicki Leuck, the type specimen's collector.

Diagnosis. As for the genus by monotypy {vide supra).

Description. Male. Measurements. Total body length (from apices of hemelytra to apex of clypeus) 1.63 mm (1.70 mm), maximum width near 0.45 mm; head length 0.25 mm, width across compound eyes (near 0.24 mm); labial segment I length 0.09 mm, segment II length 0.13 mm, segment III length 0.19 mm, segment IV length 0.08 mm; compound eye length near 0.13 mm; preocular head length near 0.14 mm; interocular distance near 0.06 mm; antennomere I length 0.10 mm; antennomere II length 0.20 mm; antennomere III length 0.18 mm; antennomere IV length (0.23 mm); pronotal length (medial) (near 0.16 mm), anterior width (approximately 0.18 mm), posterior width (near 0.38 mm); mesoscutum width (near 0.28 mm), medial length (near 0.10 mm); scutellum anterior width (near 0.14 mm), medial length (0.18 mm); hemelytra length 1.13 mm (1.2 mm), maximum width 0.44 mm; exocorium length 0.56 mm; cuneus length 0.24 mm; abdomen length near 0.60 mm, maximum width (near 0.40 mm); pygophore length 0.18 mm. Head. Length greater than width, preocular portion of head elongate, nearly equal to combined ocular and postocular lengths (Figs. 4.1 A, H, 4.2A); head rapidly narrows anterior to antennal insertion; antennal insertion almost contacting compound eye; ocellocular distance nearly two ocellar diameters due to dorsal prominence of ocelli, weak carinae posterior to ocelli converge posteriorly; compound eyes separated from pronotum by less than one-half compound eye length; vertex, frons, clypeus, and labial segment I all with few, coarse, erect, elongate setae (Fig. 4.1C); setae very sparse, erect, and much finer on labial segments II-IV; labium robust and extending to apex of procoxa;

Figure 4.1. Popovophysa entzmingeri gen. et sp. nov. photomicrographs: (A-F) hoiotype, UASM 22323: (A), ventrolateral habitus, apices of antennae are missing; (B), dorsolateral habitus, arrow indicates callosity; (C), head, ventrolateral view, d.f., arrow indicates protuberant lateral ocellus; (D), protibial apex, d.f., arrow indicates tenet setae; (E), mesotibial apex, b.f.; (F), metatibial apex, b f; (G, H), paratype, TMP 96.9.334: (G), dorsal habitus; (H), ventral habitus. Scale bar 0.5 mm in A, B, G, H; 0.2 mm in C; 0.05 mm in D-F. antennomeres II-IV with dense cover of inclined, elongate setae, with lengths comparable to that of antennomere widths, antennomere I apparently glabrous. Thorax. Pronotum markedly narrowed anteriorly (Figs. 4.1G, 4.2B), posterior border approximately two times longer than anterior border; lateral margins of posterior lobe strongly carinate (Fig. 4. IB) [= plate-like edging of Popov (2004)]; posterior border gently concave; carinae on posterior lobe continue around lateral margins of anterior lobe, with lateral borders sinuate; anterior lobe with calli low and broadly-domed, yet distinct in oblique view; surface texture of callosities unclear in type specimen; sulcus posterior to collar interrupted on ventral surface by at least four fine longitudinal carinae (Figs. 4.1 A, 4.2A). Mesoscutum comparatively long, subequal to length of scutellum, with pronounced dorsal convexity (Figs. 4.1G, 4.2B). Scutellum somewhat kite-shaped

Figure 4.2. Popovophysa entzmingeri gen. et sp. nov. habitus drawings: (A), holotype, UASM 22323, ventrolateral habitus, apices of antennae are missing; (B), paratype, TMP 96.9.334, dorsal habitus, distal portions of legs and much of the hind wings are omitted due to limited visibility, the head is inclined to the right. Scale bars 0.5 mm in A and B. in dorsal view, with anterior corner sunken and deeply incised into posterior margin of mesoscutum, posterior corner and central body of scutellum with strong dorsal convexity, forming longitudinal ridge. Pronotum, mesoscutum, scutellum and clavus all bearing fine, inclined setae in moderate density and lengths - with no glabrous patches apparent, but with anterodorsal surface of mesoscutum bearing reduced pilosity. Hemelytra with anterior (outer) margins of corium nearly straight, roughly parallel (Figs. 4.1G, 4.2A, 4.2B); cuneus apex subacute. Legs predominantly thin, with femora approximately twice as thick as tibiae and subequal in length, except metatibiae, with length 1.2 times metafemoral length; setae relatively dense on most leg surfaces and elongate (with lengths similar to or greater than widths of their respective leg subcomponents); setae suberect on ventral surfaces of most leg components, and inclined on dorsal surfaces; apex of protibia with increased density and length of setae; protibial apex expanded with distinct tibial comb terminating dorsally in spine as long as basal tarsomere, and apparently with ventral fossula spongiosa (Fig. 4. ID) composed of fine, elongate tenet setae; mesotibial apex with at least three diminutive, erect spines (Fig. 4.IE); metatibial apex with circlet of approximately ten diminutive, suberect spines (Fig. 4. IF); basal tarsomere on all tarsi with two subapical setae, each at least twice as long as tarsomere. Abdomen. Anterior to pygophore, abdomen with ovoid outline (Figs. 4.1H, 2A); pygophore significantly narrower than rest of abdomen; sternites with high ventral convexity medially (following contours of pygophore), and recurved laterally, forming flattened 'wings' in lateral one-quarter of sternite width. Thick and moderately elongate setae dense and inclined on sternites, setae longer in posterior and lateral positions; pygophore with increased density and lengths of setae, especially on posterior and lateral surfaces, longest setae similar in length to tarsi. Colouration. Specimen cuticle pulled away from amber surface during diagenesis (Figs. 4.1 A, 4.IB, 4.1G, 4.1H), producing many surfaces on both specimens with mottled or metallic colouration - these are artifacts of preservation. Original colouration likely consisting of dark brown or chestnut- coloured head (Fig. 4.1C), antennae, thorax, and lateral extensions of abdomen, with paler brown labium, legs, tarsi, and perhaps ventromedial abdomen.

Discussion To date, there has been no cladistic analysis of relationships within the Microphysidae. Such an analysis is beyond the scope of the present work, but Popovophysa displays a suite of characters that clearly indicates it belongs within the family, yet cannot be accommodated within any of the existing genera. Characters such as the absence of visible metathoracic scent gland grooves, presence of a single processus corial, presence of distal branching in the hind wing R+M, presence of fusion between the dorsal laterotergites and mediotergites, presence of fusion between ventral laterotergite 8 and gonocoxite I, and the absence of spermatheca have been suggested as autapomorphies for Microphysidae in a large-scale morphological analysis of Cimicomorpha (Schuh and Stys 1991). All visible characters in Popovophysa match these states, with the exception of an unbranched R+M apex in the hind wing. Based upon morphological characters, Schuh and Stys (1991) placed Microphysidae as the basal sister group to the remainder of the Miriformes (Joppeicidae + ((Thaumastocorinae + Xylastodorinae) + (Miridae + (Vianaidinae + Tingidae s. s.)))), which is the sister group to Cimiciformes ((Medocostidae + (Nabinae + Prostemmatinae)) + (Lasiochilidae + (Plokiophilidae + (Lytocoridae + (Anthocoridae + (Cimicidae + Polyctenidae)))))). Subsequent molecular analyses generally supported the monophyly of Cimiciformes, but did not agree on the position of Microphysidae, placing Joppeicidae and Microphysidae as basal sister groups to the Cimiciformes lineage (Tian et al. 2008). A subsequent preliminary treatment, largely focused on the aberrant family Curaliidae, recovered a joppeicoid clade (Joppeicidae + (Velocipedidae + Curaliidae)) as basal, with Microphysidae as sister to the remaining families of the Cimiciformes (Schuh et al. 2008). A larger combined molecular and morphological approach (Schuh et al. 2009) confirmed the position of Microphysidae as basalmost within the Cimiciformes. The Schuh et al. (2009) study also recovered a reduced number of morphological unambiguous synapomorphies for the Microphysidae (as represented by Loricula elegantula), including the presence of distal branching in the hind wing R+M, and the presence of fusion between the dorsal laterotergites and mediotergites. Additional characters, including the presence of sensory structures on the membrane across the entire length of the cell-forming veins, and the fusion of ventral laterotergite 8 with the first gonocoxite in females, were not recovered when combined with molecular data (Schuh et al. 2009). Within Microphysidae, Popovophysa entzmingeri shares many features with the extant, monotypic, Central Asian genus Ciorulla Pericart. Both genera have proportionally large and strongly convex compound eyes; widely separated metacoxae on either side of a posteriorly truncated metasternum; dorsally prominent ocelli; antennal insertion adjacent to the compound eye; and subhyaline cells within the endocorium. The inclusion of P. entzmingeri within Ciorulla is not possible because the Cretaceous species lacks numerous diagnostic characters for Ciorulla, and by extension, the subfamily Ciorullinae erected by Popov (2004). In particular, the new species lacks the short opisthognathous head, elongate labial segment IV, short labial segment II, and second subhyaline cell within the endocorium that are diagnostic for Ciorulla. Although P. entzmingeri is most similar to Ciorulla, it shares numerous characteristics with members of Microphysinae, particularly Loricula (Myrmericula) Popov. Diagnostic microphysine characteristics within the new species include the shape and size of the pronotum and labium, proportions of the labial segments (with segments II and III longest), and the presence of a medial longitudinal groove on the mesosternum. Within Microphysinae, the new species meets all of the additional diagnostic criteria for Loricula (sensu Popov 2004), including: a head with an elongate preocular area, obvious ocelli, and slender antennae with an elongate antennomere II; a pronotum that is broad (tr.) and short (long.), narrowing anteriorly to one-half of its posterior width, with a collar situated distinctly ahead of the anterior pronotal lobe, an anterior lobe with distinct calli and a well-impressed transverse groove, and a posterior lobe with a convex posterior margin. Popovophysa entzmingeri also displays a fossula spongiosa composed of fine, elongate tenet setae similar to structures documented by Weirauch (2007, fig. 6c) in Loricula. Within Loricula, P. entzmingeri is most similar to Loricula (Myrmericula) perkovskyi Putshkov and Popov, a fossil specimen described from Late Eocene Ukrainian (Rovno) amber. These species share similar labial lengths and proportions (segments II and III of comparable length), similar antennomere proportions and setation, and nearly identical pronotal and mesothoracic configurations (see Popov 2004, fig. 4), but differ notably in the presence of tibial setae and a short labial segment IV in P. entzmingeri. Despite the numerous similarities outlined above, P. entzmingeri differs from all Microphysinae in the form of its wing venation, and metacoxae, which closely match Ciorullinae. The intermingling of microphysine and ciorulline traits within the new species suggests that these subfamilies may not be distinct, but this remains untested by phylogenetic analysis. Characteristics observed in combination within the Canadian amber species support Popov's (2004) treatment of Myrmericula and Myrmedobia Barensprung as subgenera of Loricula. Popov (2004) noted that Eocene specimens of Loricula {Myrmericula) perkovskyi from Ukrainian amber, and Loricula {Myrmedobia) pericarti Popov from Baltic amber, display a distinct collar anterior to the pronotum (characteristic of many modern Loricula spp.) in combination with carinate lateral borders on the pronotum (characteristic of many modern Myrmedobia spp.). The presence of both characters in fossil species reduces the number of diagnostic characters supporting these taxa at the generic level. Popovophysa entzmingeri, predates other fossils by approximately 30 million years, and also has a distinct collar and prominent lateral carinae. In terms of palaeobiogeography and palaeoecology, the new material from Canadian amber is only moderately informative. Aside from being the first Mesozoic record of the family, the two specimens described herein are the first New World fossils for the Microphysidae. These specimens already display a proportionally short labial segment I, something that is characteristic of modern Nearctic microphysids. This is not necessarily indicative of any large-scale pattern, as the fossil record for the family is sparse, and only the monotypic genera Mallochiola Bergoth and Chinaola Blatchley are currently recognized in the Nearctic (Schwartz 1989; Wheeler 1992; Popov 2004). Ecological relationships of the Canadian amber specimens may have been similar to those of modern microphysids, but there are no direct relationships indicated by syninclusions within the amber. Modern members of the family are known to prey upon small arthropods such as mites, aphids and psocids (Popov et al. 2008), all of which are common inclusions in Grassy Lake amber.

A NEW THORNY LACEWING (NEUROPTERA: PVHACHIBEROTHIDAE) FROM CANADIAN

CRETACEOUS AMBER

Introduction The thorny lacewings comprise a family of enigmatic, relict species related to the mantispid lacewings (Mantispidae) and beaded lacewings (Berothidae). Species most closely resemble berothids, the family in which they were originally placed (Tjeder, 1959), but have distinctly raptorial forelegs, resembling in this respect the condition in Mantispidae. While some have argued that rhachiberothids are a basal branch of Mantispidae (Willmann, 1990, 1994), the prevailing evidence supports a relationship closer to Berothidae (Aspock and Mansell, 1994; Aspock et al., 2001). Today the family is restricted to sub-Saharan Africa with only 13 described species - five in the genus Rhachiberotha Tjeder, seven in the genus Mucroberotha Tjeder, and one in the genus Hoelzeliella Aspock and Aspock (Aspock and Mansell, 1994; Aspock and Aspock, 1997). However, during the geological past the thorny lacewings were effectively global in distribution and with nearly as many species recorded as are alive today and with a more impressive array of morphologies, reflective of a significant past diversity. Indeed, the family has dwindled in species and abundance and would appear that the family experienced its greatest diversity during the Cretaceous. Fossils are exclusively found in amber from both the Paleogene, in Baltic and Parisian deposits, and the Cretaceous, with species known from New Jersey (Grimaldi, 2000), France (Nel et al, 2005), Myanmar (Engel, 2004), Spain (Engel, pers. obs.), Lebanon (Nel et al., 2005), and now western Canada (Table 4.2).

Table 4.2. Fossil Rhachiberothidae (thorny lacewings), updated from Engel and Grimaldi (2008).

; This amber is likely of Baltic origin (Jarzembowski, 1999)

Herein we report the discovery of a new genus and species of thorny lacewing in Late Cretaceous (Campanian) amber from Canada, representing the first rhachiberothid and the third neuropteran family from these deposits. Previous studies have documented two berothids and an immature chrysopid in Canadian amber (Klimaszewski and Kevan, 1986; Engel and Grimaldi, 2008). Increased study of Canadian material will likely add to this diversity and has the potential to provide additional immature fossil specimens for comparison with modern rhachiberothids (e.g., Minter, 1990).

Systematic Palaeontology Family Rhachiberothidae Tjeder, 1959 Albertoberotha McKellar and Engel, new genus Type species. Albertoberotha leuckorum McKellar and Engel, new species.

Diagnosis. Body with moderately dense, long setae; extensive fringe of setae along margin of wing; legs densely setose with reduced setae on protibia. Flagellum relatively short, with 27 articles; antennal scape elongate (length 8 times width); pedicel one-half length of scape; both with dense, fine setae more prominent apically (Fig. 4.3). Postocular lobes not reduced, bearing setae. Pronotum elongate and narrower than head. Profemur with basal protuberance, width gradually tapering to apex, with no mid-length expansion in breadth, longer than procoxa; profemoral spines in two rows arranged as in Figure 4.4; protibial spines in single row, uniform in size and orientation; short first tarsomere on protarsus, additional recurved spines on first and second tarsomeres, long third tarsomere. Forewing with fuscous areas (Fig. 4.5); trichosors present; costal space constricted near Sc-R fusion; all c-sc crossveins unbranched; single sc-r crossvein proximal to Rs origin, separated from origin by two vein-widths; Sc termination in apical third of wing; two r-rs crossveins, lr-rs nearly equidistant from Rs origin and 2r-rs crossvein, 2r-rs proximal to Sc-R fusion by one length of crossvein; Rs with four branches, posteriormost branch subdivided much more proximally than other branches; no crossveins between Rs branches; two rs-ma crossveins present; one ma-mp crossvein present; one mp-cua crossvein present, both mp-cua and 1 rs-ma slightly distal to MP origin; lcup-a crossvein near origin of CuP; two main branches of anal vein diverge near wing base, with single crossvein between. Hind wing with one r-rs crossvein distal to tangent of origin of fourth Rs branch; single rs-ma crossvein distally; details of wing apex and base unclear due to wing curvature and overlap.

Etymology. The new genus-group name is a combination of Alberta (the province of origin) and the generic name Berotha. The name is feminine.

Remarks. Albertoberotha is most similar to Chimerhachiberotha Nel et al., 2005 from Lebanese amber of Neocomian age. Although these monospecific genera share a basal protuberance of the profemur and some aspects of wing venation, significant differences separate them. There are many more c-sc crossveins in Albertoberotha (~33 veins, compared to -18 veins in Chimerhachiberotha), and there are fewer branches within Rs (four in Albertoberotha, compared to five in Chimerhachiberotha). Albertoberotha lacks a median expansion of the profemur and apical expansion of the protibia. It also has proportionally longer spines on the profemur and a single row of more pronounced and uniform protibial spines. The pedicel of Albertoberotha is much more cylindrical and elongate (not globulous). Additionally, Albertoberotha appears to be characterized by generally dense and long setation, compared to Chimerhachiberotha.

Albertoberotha leuckorum McKellar andEngel, new species (Figs. 4.3^1.6) Diagnosis. As for the genus {vide supra).

Description. Male. Total body length 1.95 mm (abdomen slightly curled ventrally); forewing length 3.25 mm, width ~1.0 mm; hind wing length ~2 mm, width -0.6 mm. Body preserved in dark brown color, legs pale brown with darker joints and tarsi (Fig. 4.6); integument imbricate and impunctate. Head round in dorsal view, with minor swelling on vertex, and with numerous thin setae as long as scape. Compound eye large and ovoid. Flagellar articles uniform in size, shape and setation; articles slightly elongate, with whorl of setae same length as article positioned apically, and whorl of half-length setae basally. Mouthparts not clearly visible except for maxillary palps. Pronotum two-thirds width of head and nearly square in dorsal view; setation similar to that of head. Meso- and metanotum sparsely setose medially, lateral setation similar to pronotum. Meso- and metathoracic legs long and thin, densely covered with apically directed, short, thin setae; sporadic, robust setae on apical regions of tibia. Metafemur and metatibia mildly sinuous near joint. Meso- and metatarsus cylindrical, with first tarsomere slightly longer than all others combined; second to fifth tarsomeres gradually diminishing in length. Prothoracic leg robust, highly spinose across inner surfaces of profemur and protibia (Fig. 4.3); procoxa elongate and compressed, shorter than profemur; profemur expanded towards base, with basal protuberance bearing circlet of splayed, thickened spines; erect spines of variable length and distribution forming two rows down remainder of profemur; protibia with single row of apically inclined spines regularly spaced and more robust apically; short, apically directed setae dense on outer surface and sides of profemur, reduced and more sparse on protibia; protarsus comparatively short and stout; first tarsomere markedly short, strongly curved outward (basally), with hooked, recumbent apical spine twice tarsomere length, and few minute, suberect spines on inner surface; second tarsomere shorter than first, with similar spine array; third tarsomere slightly longer than first, with apical taper. Forewing elongate sub-oval in shape; marginal portions of veins and trichosors with tufts of long, fine setae forming dense fringe around entire wing; shorter, fine setae sparse on dorsal and ventral surfaces of longitudinal veins, absent from crossveins.

Figures 4.3-4.4. Albertoberotha leuckorum n. sp. 4.3, habitus diagram of holotype, ventral (oblique) aspect, all but right forewing omitted for clarity, bubbles obscuring detail indicated in grey. 4.4, enlarged view of right foreleg, detailing spines and setae on profemur and protibia. Scale bars = 1 mm. Figures 4.5-4.6. Albertoberotha leuckorum n. sp. 4.5, forewing venation diagram, setae omitted, anterior edge of wing curled ventrally, dashed line indicates reconstruction across amber fracture, and shading denotes fuscous areas. 4.6, habitus photograph of holotype, ventral (oblique) aspect. Scale bars = 1 mm.

Hind wing setation and outline similar to forewing; Sc vein heavily sclerotized. Abdomen curled ventrally and crumpled; froth of fine bubbles obscuring posterior tip, except for protrusion of two talon-shaped paramere-mediunci; short, fine setae scattered across abdomen, with longer setae on posterior margins of terga.

Material examined. Holotype: Royal Tyrrell Museum of Palaeontology, Drumheller, Alberta, Canada, TMP 91.148.790; Campanian amber; Canada, Alberta, Grassy Lake locality (site specifics available from RTMP).

Etymology. The specific epithet is a patronym honoring the Leuck family for their assistance in collecting Grassy Lake amber.

Discussion The new record reported here is significant because it extends the North American temporal range of the family later into the Late Cretaceous, and it expands its distribution to include higher latitude areas on the west side of the western interior seaway (Table 4.2). This adds support to the hypothesis that the Rhachiberothidae were nearly global in their distribution until perhaps the Eocene. The great morphological disparity between^, leuckorum and existing taxa also contributes to the concept of greater family diversity in the Cretaceous period. Albertoberotha also intermingles traits previously used to segregate the thorny lacewings in to two subfamilies, possessing postocular, setose tubercles while simultaneously having the inner row of protibial spines and no sc-r crossveins distal to origin of Rs. This combination of traits further supports the notion that many of the Cretaceous rhachiberothids form a grade leading to the modern genera and inclusion of these into a subfamily renders the basal group paraphyletic (Engel and Grimaldi, 2008). Given the remarkable morphological diversity of characters seen among Cretaceous rhachiberothids relative to the modern fauna, it is likely that a far greater diversity has yet to be recovered. Indeed, even in single deposits dramatically different genera have been found (e.g., Nel et al., 2005) and so it is to be greatly hoped that further exploration in other amber deposits will reveal additional taxa to help fill out our picture of Mesozoic and Paleogene lacewing diversity. REFERENCES Aspftck, U., Aspock, H., 1997. Studies on new and poorly-known Rhachiberothidae (Insecta: Neuroptera) from subsaharan Africa. Annalen des Naturhistorischen Museums in Wien, Serie B, Botanik und Zoologie 99, 1-20. Aspock, U., Mansell, M.W., 1994. A revision of the family Rhachiberothidae Tjeder, 1959, stat. n. (Neuroptera). Systematic Entomology 19(3), 181- 206. Aspock, U., Plant, J.D., Nemeschkal, H.L., 2001. Cladistic analysis of Neuroptera and their systematic position within Neuropterida (Insecta: Holometabola: Neuropterida: Neuroptera). Systematic Entomology 26(1), 73-86. Engel, M.S., 2004. Thorny lacewings (Neuroptera: Rhachiberothidae) in Cretaceous amber from Myanmar. Journal of Systematic Palaeontology 2(2), 137-140. Engel, M.S., Grimaldi, D.A., 2008. Diverse Neuropterida in Cretaceous amber, with particular reference to the paleofauna of Myanmar (Insecta). Nova Supplementa Entomologica 20, 1-86. Grimaldi, D., 2000. A diverse fauna of Neuropteroidea in amber from the Cretaceous of New Jersey, in: Grimaldi, D., (Ed.), Studies on Fossils in Amber, with Particular Reference to the Cretaceous of New Jersey. Backhuys Publishers; Leiden, pp. 259-303. Jarzembowski, E.A., 1999. British amber: A little-known resource. Estudios del Museo de Ciencias Naturales de Alava (Numero Especial 2) 14, 133-140. Klimaszewski, J., Kevan, D.K.M., 1986. Anew lacewing-fly (Neuroptera: Planipennia) from Canadian Cretaceous amber, with an analysis of its fore wing characters. Entomological News 97(3), 124-132. McKellar, R.C., Wolfe, A.P., 2010. Canadian amber, in: Penney, D., (Ed.), Biodiversity of fossils in amber from the major world deposits. Siri Scientific Press, Manchester, pp. 149-166. McKellar, R.C., Wolfe, A.P., Tappert, R., Muehlenbachs, K., 2008. Correlation of Grassy Lake and Cedar Lake ambers using infrared spectroscopy, stable isotopes, and palaeoentomology. Canadian Journal of Earth Sciences 45(9), 1061-1082. Minter, L.R., 1990. A comparison of the eggs and first-instar larvae of Mucroberotha vesicaria Tjeder with those of other species in the families Berothidae and Mantispidae (Insecta: Neuroptera), in: Mansell, M.W., Aspock, H., (Eds.), Advances in Neuropterology, Proceedings of the Third International Symposium on Neuropterology. Berg en Dal, Kruger National Park, 1988. Pretoria, RSA, pp. 115-129. Nascimbene, P., Silverstein, H., 2000. The preparation of fragile Cretaceous ambers for conservation and study of organismal inclusions, in: Grimaldi, D., (Ed.), Studies of fossils in amber, with particular reference to the Cretaceous of New Jersey. Backhuys Publishers, Leiden, pp. 93-102. Nel, A., Perrichot, V., Azar, D., Neraudeau, D., 2005. New Rhachiberothidae (Insecta: Neuroptera) in Early Cretaceous and Early Eocene ambers from France and Lebanon. Neues Jahrbuch fur Geologie und Palaontologie, Abhandlungen 235(1), 51-85. Pericart, J., 1974. Une espece nouvelle de Microphysidae representant un genre nouveau, et une espece nouvelle d'Anthocoridae (Rhynchota, Hemiptera). Bulletin de la Societe Entomologique de France 79(9-10), 253-257. Pike, E.M., 1995. Amber taphonomy and the Grassy Lake, Alberta, amber fauna. Ph.D. thesis. Department of Biological Sciences, University of Calgary, Calgary, Alberta. Popov, Y.A., 2004. New microphysids (Heteroptera: Cimicomorpha, Microphysidae) from Baltic amber and taxonomy of this family. Prace Muzeum Ziemi 47, 97-107. Popov, Y.A., 2006. New microphysids (Heteroptera, Cimicomorpha, Microphysidae) of the Baltic Eocene amber from the collection of Ernst Heiss. Denisia 19, 571-579. Popov, Y.A., Herczek, A., 2009. A new peculiar minute bug (Hemiptera, Heteroptera, Cimicomorpha, Microphysidae) from the Eocene Baltic amber. Denisia 26, 151-154. Popov, Y.A., Herczek, A., Kanja, I., 2008. One more microphysid from the Eocene Baltic amber (Heteroptera: Cimicomorpha, Microphysidae). Genus 19(4), 611-617. Putshkov, P. V., Popov, Y.A., 2003. The first find of Microphysidae from Ukrainian (Rovno) amber (Heteroptera, Cimicomorpha). Annals of the Upper Silesian Museum in Bytom, Entomology 12, 81-85. Schlilter, T., 1978. Zur Systematik und Palokologie harzkonservierter Arthropoda einer Taphozonose aus dem Cenomanium von NW-Frankrecih. Berliner Geowissenschaftliche Abhandlugen, Reihe A, Geologie und Palaontologie 9, 1-150. Schuh, R.T., Slater, J. A., 1995. True Bugs of the World (Hemiptera: Heteroptera): Classification and Natural History. Cornell University Press; Ithaca, New York. Schuh, R.T., Stys, P., 1991. Phylogenetic analysis of cimicomorphan family relationships (Heteroptera). Journal of the New York Entomological Society 99(3), 298-350. Schuh, R.T., Weirauch, C, Henry, T.J., Halbert, S.E., 2008. Curaliidae, a new family of Heteroptera (Insecta: Hemiptera) from the eastern United States. Annals of the Entomological Society of America 101(1), 20-29. Schuh, R.T., Weirauch, C, Wheeler, W.C., 2009. Phylogenetic relationships within the Cimicomorpha (Hemiptera: Heteroptera): A total-evidence analysis. Systematic Entomology 34(1), 15^48. Schwartz, M.D., 1989. New Records of Palearctic Heteroptera in New York State: Microphysidae and Miridae. Journal of the New York Entomological Society 97(1), 111-114. Stys, P., 1962. Venation of metathoracic wings and notes on the relationships of Microphysidae (Heteroptera). Acta Societatis Entomologicae Czechosloveniae 59(3), 234-239. Tian, Y, Zhu, W, Li, M., Xie, Q., Bu, W, 2008. Influence of data conflict and molecular phylogeny of major in cimicomorphan true bugs (Insecta: Hemiptera: Heteroptera). Molecular Phylogenetics and Evolution 47(2), 581-597. Tjeder, B., 1959. Neuroptera-Planipennia: The lace-wings of southern Africa. 2. Family Berothidae, in: Hanstrom, B., Brinck, P., Rudebeck, G., (Eds.), South African Life [Volume 6]. Swedish Natural Science Research Council; Stockholm, pp. 256-314. Weirauch, C, 2007. Hairy attachment structures in Reduviidae (Cimicomorpha, Heteroptera), with observations on the fossula spongiosa in some other Cimicomorpha. Zoologischer Anzeiger 246, 155-175. Whalley, P.E.S., 1980. Neuroptera (Insecta) in amber from the Lower Cretaceous of Lebanon. Bulletin of the British Museum of Natural History, Geology Series 33, 157-164. Whalley, P.E.S., 1983. Fera venatrix gen. and sp. n. (Neuroptera, Mantispidae) from amber in Britain. Neuroptera International 2(4), 229-233. Wheeler, A.G. Jr., 1992. Chinaola quercicola rediscovered in several specialized plant communities in the southeastern United States (Heteroptera: Microphysidae). Proceedings of the Entomological Society of Washington 94(2), 249-252. Willmann, R., 1990. The phylogenetic position of the Rhachiberothinae and the basal sister-group relationships within the Mantispidae (Neuroptera). Systematic Entomology 15(2), 253-265. Willmann, R., 1994. Die phylogenetische Position ursprunghcher Mantispidae (Insecta, Planipennia) aus dem Mesozoikum und Alt-Tertiar. Verhandlungen des Naturwissenschaftlichen Vereins in Hamburg 34, 177-203. CHAPTER 5: CONTRIBUTION TO VERTEBRATE PALAEONTOLOGY IN CANADIAN AMBER1*

A DIVERSE ASSEMBLAGE OF LATE CRETACEOUS DINOSAUR AND BIRD FEATHERS FROM CANADIAN AMBER

Introduction Although amber offers unparalleled preservation of feathers (Schlee and Glockner, 1978; Davis and Briggs, 1995; Alonso et al, 2000; Perrichot et al., 2008), only isolated specimens of uncertain affinity have been reported from the Late Cretaceous (Grimaldi and Case, 1995). This contrasts with the rich Early Cretaceous compression assemblage from northeastern China (Ji et al., 1998; Norell and Xu, 2005; Xu and Guo, 2009), leaving a substantial temporal gap in our understanding of feather evolution. Late Cretaceous amber from Grassy Lake, Alberta (late Campanian) is derived from lowland cupressaceous conifer forests that occupied the margin of the Western Interior Seaway, and is best known for its diverse insect inclusions (McKellar and Wolfe, 2008). Eleven feather or protofeather specimens were recovered from over four-thousand Grassy Lake amber inclusions screened at the Royal Tyrrell Museum of Palaeontology (TMP) and University of Alberta (UALVP). These structures have disparate morphologies that span four evolutionary stages for feathers (Prum, 1999; Prum and Brush, 2002). Specimens include filamentous structures similar to the protofeathers of non-avian dinosaurs that are unknown from modern birds (Brush, 2000; Currie and Chen, 2001; Xu et al., 2010), as well as advanced bird feathers displaying pigmentation and adaptations for flight and diving. The currently-accepted (Prum, 1999; Prum and Brush, 2002) evolutionary- developmental model for feathers (Fig. 5.1 A) consists of a Stage I morphology characterized by a single filament: this unfurls into a tuft of filaments (barbs) in Stage II. In Stage III, either some tufted barbs coalesce to form a rachis (central shaft) (Ilia), or barbules (segmented secondary branches) stem from the barbs (Illb), then these features combine to produce tertiary branching (Illa+b). Barbules later differentiate along the length of each barb, producing distal barbules with hooklets at each node to interlock adjacent barbs and form a closed pennaceous (vaned) feather (Stage IV). Stage V encompasses a wide range of

* A version of this chapter is currently in press. McKellar, R.C., Chatterton, B.D.E., Wolfe, A.P., Currie, P. J., (in press). A diverse assemblage of Late Cretaceous dinosaur and bird feathers from Canadian amber. Science. Figure 5.1. Feather evolutionary-developmental model (Prum, 1999), terminology (Lucas and Stettenheim, 1973), and Stage I and II specimens from Canadian amber. (A) Feather stages out­ lined within text. Green denotes calamus or equivalent; blue, barbs; purple, rachis; red, barbule internodes; d.b., distal barbules; r., ramus; p.b., proximal barbules. (B) Field of filaments cut obliquely (Stage I), UALVP 52821. (C) Filament clusters variably oriented (Stage II), UALVP 52822. (D) Close-up of C, showing filaments that comprise clusters -dark coloration due to pig­ mentation and filament outer walls that appear thicker than in isolated filaments. Scale bars 1 mm (B,C), 0.1 mm (D). [See Appendix 3 for additional figures and details]. additional vane and subcomponent specializations. Most modern birds possess Stage IV or V feathers, or secondary reductions from these stages (Stettenheim, 1973; Prum, 1999). Modern feathers exhibit a range of morphologies that are tied to their variable functions and are observable in some of their finest subunits, the barbules (Lucas and Stettenheim, 1972). This is particularly important in the study of amber-entombed feathers, as preservation is biased toward feather subcomponents, which provide the basis for our morphological comparisons. Our morphological comparisons are supplemented heavily in the supporting material for this chapter (Appendix 3). Most paragraphs within the "Results and discussion" section have a corresponding section within this appendix, or are greatly expanded upon in the appendix. Figures with an "A3" prefix are also found within Appendix 3. Results and discussion Stage I morphotype, isolated filaments Stage I is represented by UALVP 52821, which contains a dense forest of regularly-spaced, flexible filaments with a mean diameter of 16.4 ±4.2 urn (Figs. 5.IB, A3.1-A3.4). Filaments are hollow with the internal cavity comprising -60% of total diameter, have no obvious internal pith, and taper apically. Where surface texture is observable, filaments bear a faint cross-hatching pattern but lack surface topography. The filaments are not plant or fungal remains because they lack cell walls and are relatively large. Comparatively small diameters and a lack of cuticular scales imply they are not mammalian hairs, as does direct comparison to a hair from this amber deposit. Their closest morphological match is the filamentous covering found of non-avian dinosaurs such as the compsognathid Sinosauropteryxprima (Chen et al., 1998). The amber-entombed specimens are slightly finer than those of Sinosauropteryx, which may have been distorted by compression and permineralization. The amber filaments display a wide range of pigmentation, ranging from nearly transparent to dark (Fig. A3.2). No larger- scale color patterns are apparent. (Additional specimen details are provided in supporting material, Appendix 3.)

Stage II morphotype, clustered filaments The Stage II morphotype (Figs. 5.1C, D, A3.5) consists of tightly adpressed clusters approximately 0.2 mm in width and composed of filaments that are otherwise similar to the Stage I filaments. Five clusters are preserved together in UALVP 52822. As in Stage II primitive feathers (Prum, 1999), filaments in each cluster appear to diverge from a common basal region without branching, but no rachis is visible where the clusters exit the amber. These filaments bear some resemblance to fibrils that compose pycnofibers (tufted filaments) in pterosaur compression fossils (Kellner et al., 2009), except the amber specimens lack the secondary organization observed in pycnofiber bundles. The most morphologically-comparable compression fossils are protofeathers associated with the dromaeosaurid Sinornithosaurus millenii (Xu et al., 2001). These clusters exhibit generally comparable sizes and shapes to the amber specimens, and even have the more loosely-bundled appearance distally where individual filaments have more variable lengths. (Additional specimen details are provided in supporting material, Appendix 3.) Stage IV and V morphotypes, specialized barbules In contrast to Stages I and II, additional specimens from Canadian amber have barbules specialized for discrete functions. In TMP 96.9.334 (Figs. 5.2A-C, A3.6, A3.7), a thickened rachis is surrounded by numerous barbules with tightly coiled bases. The barbules undergo three or more complete whorls, and are composed of semi-flattened internodes (~120 um long, 9 urn wide) separated by weakly expanded nodes (~12 um wide). This coiling cannot be attributed to interaction

Figure 5.2. Specialized barbules in Canadian amber. (A) Coiled barbules surrounding thickened rachis (arrow), cut obliquely, TMP 96.9.334. (B) Close-up of coils in isolated barbule. (C) Semi- flattened internodes and weakly expanded node of A - diffuse, variable barbule pigmentation pro­ duces pale overall color. (D) Isolated barb with differentiated barbules and thickened ramus, in spider's web, UALVP 52820. (E) Barbules near distal tip of D, with clearly defined distal and proximal barbule series (left and right sides of ramus, respectively). (F) Close-up of distal barbule in E, showing nodal prongs and ventral tooth on basal plate (arrow) adjacent to abrupt transition into pennulum - banded pattern of dark pigmentation within basal plate, and diffuse dark pigmen­ tation within pennulum, suggest a gray or black feather (Chandler, 1916). Scale bars 0.4 mm (A), 0.2 mm (B,D,E), 0.05 mm (C,F). [See Appendix 3 for additional figures and details]. between barbules and resin during amber polymerization as it only occurs at the base of each barbule. Modern seedsnipes and sandgrouse (Cade and Maclean, 1967; Maclean, 1983) possess belly feathers with similar basal barbule coiling, which allows water to be retained for transport to the nest for distribution to nestlings or for cooling incubating eggs. Grebes also have coiled barbules that absorb water into plumage, facilitating diving by modifying buoyancy, reducing hydrodynamic turbulence, and improving insulation (Fjeldsa, 2004). In all of these instances, the keratin of coiled barbules interacts with water to uncoil and absorb water through capillary action (Maclean, 1983). The high number of coils in TMP 96.9.334 is most similar to that reported from grebes (Fjeldsa, 2004, Chandler, 1916), implying that the Cretaceous barbules are related to diving behavior. Barbules displaying all characteristics necessary for forming vaned feathers are also present in Canadian amber (Fig. 5.2D-F, A3.8). These were likely borne by an animal capable of flight. Within UALVP 52820, barbules of unequal lengths arise from either side of the barb, producing a differentiated series of longer proximal (-0.42 mm) and shorter distal (-0.24 mm) elements, all having spinose nodal projections. Barbules are widely spaced along a thick ramus (barb shaft) adapted for rigidity, and are strongly differentiated to interlock with adjacent barbs to form a vane. Based on the presence of a rachis in TMP 96.9.334 and differentiated barbules in UALVP 52820, these specimens can be assigned conservatively to Stages IV and V, and are attributed to Late Cretaceous birds. The remaining six feathers are fragmentary downy and contour feathers (Fig. 5.3). Although they offer limited insight concerning the identity or behavior of their bearer, their structure and pigmentation bear directly on feather evolutionary stages. Four of the six feather fragments in TMP 96.9.997 (Figs. 5.3A-D, A3.9) are aligned. Superficially, these exhibit an intermediate morphology (Stage lib) proposed for an Early Cretaceous (late Albian) French amber specimen (Perrichot et al., 2008). In the Canadian specimens, as in the French material, the main axis preserved is short (3.7 mm) and weakly denned, dorsoventrally flattened, and composed of fused secondary branches in an opposite arrangement. However, in the Canadian specimens, intense pigmentation in each internode produces a beaded appearance, highlighting segmentation that is otherwise difficult to discern based on barbule diameter variation (Fig. 5.3C). Segmentation identifies the finest branches as barbules attached to narrow rami, and not barb equivalents attached to a rachis. Figure 5.3. Pigmentation in Canadian amber feathers. (A-D) Semi-pennaceous feathers, TMP 96.9.997: (A) six barbs; (B) close-up of box in A, arrow indicates unpigmented ramus; (C) detail of ramus and barbule bases; (D) dark-field microphotograph of C, showing brown coloration with ramus and basal internodes minimally-pigmented. Density and distribution of pigments (Chandler, 1916; Dove, 2000) consistent with medium- to dark-brown modern feathers. (E) Unpigmented downy bar- bules, TMP 79.16.12. (F-K) Poorly differentiated, flattened barbules: (F) partial overview of 16 pen- naceous barbs, TMP 96.9.553; (G) close-up of F, showing variable, diffuse pigmentation within bar­ bule bases (ventral plates translucent, dorsal flanges pigmented); (H) unpigmented, isolated barb with juvenile mite, TMP 96.9.546; (I) central portion of isolated barb, TMP 94.666.15; (J) dark-field micro­ photograph of I, showing overall color; (K) banded pigmentation within basal plate of proximal bar­ bule in I, indicating 5-6 component internodes. (L) Reduced pennaceous barbs from non-interlocking region of dark-brown and white mottled chicken contour feather for comparison. Scale bars 0.5 mm (A), 0.2 mm (B,E,F,H-nJ,L), 0.04 mm (C,D,G,K). [See Appendix 3 for additional figures and details].

This interpretation identifies these small specimens as subcomponents of a larger feather, such as basal barbs on a contour feather (Lucas and Stettenheim, 1972), and not a distinct stage in feather evolution lacking barbules (Perrichot et al., 2008). This interpretation likely extends to the French material as well. Pigmentation is preserved with fidelity in all additional specimens. While downy feathers are consistently transparent, and would have been white in life, pennaceous feathers are more variable with diffuse, transparent, and mottled patterns of pigmentation (Fig. 5.3E-L) that match those observed in modem birds (Chandler, 1916; Dove, 2000).

Conclusions Although neither avian nor dinosaurian skeletal material has been found in direct association with amber at the Grassy Lake locality, fossils of both groups are present in adjacent stratigraphic units. Hadrosaur footprints are found in close association with the amber, and younger (late Campanian and Maastrichtian) strata of western Canada contain diverse non-avian dinosaur (Currie, 2005) and avian (Longrich, 2009; Buffetaut, 2010) remains. There is currently no way to refer the feathers in amber with certainty to either birds or the rare small theropods from the area (Currie, 2005). However, the discovery of end-members of the evolutionary-developmental spectrum in this time interval, and the overlap with structures found only in non-avian dinosaur compression fossils, strongly suggests that the protofeathers described here are from dinosaurs and not birds. Given that Stage I filaments were present in densities relevant for thermoregulation and protection, and that comparable structures are preserved as coronae surrounding compression fossils, it becomes apparent that protofeathers had important non-omamental functions. Specialized barbule morphologies, including basal coiling, suggest that Campanian feather-bearers had already evolved highly specialized structures similar to those of modem grebes to enhance diving efficiency. Canadian amber provides examples of Stages I through V of Pram's (1999) evolutionary-developmental model for feathers. None of the additional morphotypes observed in compression fossils of non-avian dinosaurs (Xu and Guo, 2009; Xu et al., 2010) or amber (Perrichot et al., 2008) were found here, suggesting that some morphotypes may not represent distinct evolutionary stages, or may not have persisted into the Late Cretaceous. The snapshot of Campanian feather diversity from Canadian amber is biased towards smaller feathers, subcomponents of feathers, feathers that are molted frequently, and feathers in body positions that increase their likelihood of contacting resin on tree trunks. Despite these limitations, the assemblage demonstrates that numerous evolutionary stages were present in the Late Cretaceous, and, perhaps more importantly, that plumage already served a range of functions in both dinosaurs and birds. Supplementary material There are 30 pages of supplementary material available in Appendix 3. This appendix includes additional details for materials and methods in this study, and supporting data for the conclusions drawn in this chapter. The supplementary material contains additional details on Stage I and II morphotype identifications, including comparisons to: modern mammalian hair, fossil mammalian hair, fungal and plant remains, degraded or taphonomically-altered feather remains, pterosaur pycnofibers, Sinosauropteryx prima integumentary structures, and Sinornithosaurus millenii integumentary structures. The use of additional chemical and morphological comparison techniques is discussed for Stage I and II specimens, and chemical results based primarily on laser scanning confocal microscopy are presented. Further details are provided on the morphology, pigmentation, and taphonomy of each of the specimens encompassed in this chapter, and all specimens are illustrated in a series of 11 full-page figures. References Alonso, J. Arillo, A., Barron, E., Corral, J.C., Grimalt, J., Lopez, R., Martinez- Delclos, X., Ortuno, V., Penalver, E., Trincao, P.R., 2000. Anew fossil resin with biological inclusions in Lower Cretaceous deposits from Alava (Northern Spain, Basque-Cantabrian Basin). Journal of Paleontology 74(1), 158-178. Brush, A.H., 2000. Evolving a protofeather and feather diversity. American Zoologist 40, 631-639. Buffetaut, E., 2010. A basal bird from the Campanian (Late Cretaceous) of Dinosaur Provincial Park (Alberta, Canada). Geological Magazine 147, 469^72. Cade, T.J., Maclean, G.L., 1967. Transport of Water by Adult Sandgrouse to Their Young. Condor 69, 323-343. Chandler, A.C., 1916. A study of the structure of feathers, with reference to their taxonomic significance. University of Californian Publications on Zoology 13, 243^146. Chen, P.-J., Dong, Z.-M., Zhen, S.-N., 1998. An exceptionally well-preserved theropod dinosaur from the Yixian Formation of China. Nature 391,147- 152. Currie, P.J., 2005. Theropods, including birds, in: Currie, P.J., Koppelhus, E.B., (Eds.), Dinosaur Provincial Park: A Spectacular Ancient Ecosystem Revealed. Indiana University Press, Bloomington, pp. 367-397. Currie, P.J., Chen, P.-J., 2001. Anatomy of Sinosauropteryxprima from Liaoning, northeastern China. Canadian Journal of Earth Sciences 38, 1705-1727. Davis, P.G., Briggs, D.E.G., 1995. Fossilization of feathers. Geology 23, 783-786. Dove, C.J., 2000. A descriptive and phylogenetic analysis of plumulaceous feather characteristics in Charadriiformes. Ornithological Monographs 51, 1-163. Fjeldsa, J., 2004. The Grebes: Podicipedidae. Oxford University Press, New York. Grimaldi, D., Case, G.R., 1995. A feather in amber from the Upper Cretaceous of New Jersey. American Museum Novitates 3126, 1-6. Ji, Q., Currie, P.J., Norell, M.A., Ji, S.-A., 1998. Two feathered dinosaurs from northeastern China. Nature 393, 39^12. Kellner, A.W.A., Wang, X., Tischlinger, H., de Almeidos Campos, D., Hone, D.W., Meng, X., 2009. The soft tissue of Jeholopterus (Pterosauria, Anurognathidae, Batrachognathinae) and the structure of the pterosaur wing membrane. Proceedings of the Royal Society of London B, Biological Sciences 277, 321-329. Longrich, N., 2009. An ornithurine-dominated avifauna from the Belly River Group (Campanian, Upper Cretaceous) of Alberta, Canada. Cretaceous Research, 30, 161-177. Lucas, A.M., Stettenheim, P.R., 1972. Avian Anatomy: Integument. US Department of Agriculture, pp. 235-279. Maclean, G.L., 1983. Water transport by sandgrouse. BioScience 33, 365-369. McKellar R.C., Wolfe, A.P., 2010. Canadian amber, in: Penney, D. (Ed.), Biodiversity of fossils in amber from the major world deposits. Siri Scientific Press, Manchester, pp. 149-166. Norell, M., Xu, X., 2005. Feathered dinosaurs. Annual Review of Earth and Planetary Sciences 33, 277-299. Perrichot, V., Marion, L., Neraudeau, D., Vullo, R., Tafforeau, P., 2008. The early evolution of feathers: fossil evidence from Cretaceous amber of France. Proceedings of the Royal Society of London B, Biological Sciences 275, 1197-1202. Prum, R.O., 1999. Development and evolutionary origin of feathers. Journal of Experimental Zoology 285, 291-306. Prum, R.O., Brush, A.H., 2002. The evolutionary origin of and diversification of feathers. Quarterly Review of Biology 77, 261-295. Schlee, D., Glockner, W., 1978. Bernstein: Bernsteine und Bernstein-Fossilien. Stuttgarter Beitrage ziir Naturkunde (C) 8, 1-72. Stettenheim, P., 1973. The bristles of birds. Living Bird 12, 201-234. Xu, X., Guo, Y., 2009. The origin and early evolution of feathers: insights from recent paleontological and neontological data. Vertebrata PalAsiatica 47, 311-329. Xu, X., Zheng, X., You, H., 2010. Exceptional dinosaur fossils show ontogenetic development of early feathers. Nature 464, 1338-1341. Xu, X., Zhou, Z.-H., Prum, R.O., 2001. Branched integumental structures in Sinornithosaurus and the origin of feathers. Nature 410, 200-204. CHAPTER 6: GENERAL CONTRIBUTIONS TO THE STUDY OF AMBER1*

INSECT OUTBREAKS PRODUCE DISTINCTIVE CARBON ISOTOPE SIGNATURES IN DEFENSIVE RESINS AND FOSSILIFEROUS AMBERS

Introduction The stable isotopic composition of plant carbon has the capacity to record physiological stress, such as that induced by insect attack, because 13C discrimination is reduced under impaired conditions (Farquhar et al., 1989). Although carbon stable isotopes have been considered from a range of plant tissues (e.g., Francey et al., 1982; Loadner et al., 1997; Grocke et al., 1999; Jahren and Sternberg, 2003), relatively little attention has been paid to exuded secondary metabolites (Murray et al., 1989; Nissenbaum and Yakir, 1995; Nissenbaum et al., 2005; Stern et al., 2008; Dal Corso et al., 2011), despite their excellent potential for preservation in the fossil record as amber (Grimaldi, 1996a; Langenheim, 2003). Resin retains its original stable isotopic composition throughout maturation with a high degree of fidelity (Nissenbaum and Yakir, 1995; Dal Corso et al., 2011), making analogies between modern resin and fossil amber tenable. Within a framework of actualistic palaeontology, we examined the role of wood-boring insects as a potential cause for amber production by investigating the isotopic response of modern resins generated in direct response to a major insect outbreak event. Mountain pine beetle (Dendroctonus ponderosae, Coleoptera: Curculionidae: Scolytinae; hereafter MPB) is currently devastating western North American forests with pronounced economic, ecological and biogeochemical consequences (Kurz et al., 2009). Climate change, specifically warmer winter temperatures, has allowed MPB to infest extensive tracts of lodgepole pine {Pinus contorta var. latifolia, Fig. 6.1) already weakened by multiple years of drought. MPB larvae over-winter in galleries excavated in the host tree's cambium, where they feed on blue stain fungus {Grosmannia clavigera, Ascomycota). Adults emerge in late summer, disperse, and form mass attacks guided by combinations of pheromones and volatile resin constituents released during pioneer attacks (Safranyik and Wilson, 2006). This strategy frequently overcomes the constitutive

* A version of this chapter has been published. McKellar, R.C., Wolfe, A.P., Muehlenbachs, K., Tappert, R., Engel, M.S., Cheng, T., Sanchez-Azofeifa, G.A., 2011. Insect outbreaks produce dis­ tinctive carbon isotope signatures in defensive resins andfossiliferous ambers. Proceedings of the Royal Society of London B: Biological Sciences, doi: 10.1098/rspb. 2011.0276. \ ~r$',\ ?>& * M J'"- '

Figure 6.1. Products of infestation in trees and resin, (a) Foliage colour changes due to MPB attack in west-central Alberta, photo courtesy J. Cooke, University of Alberta, (b) Pitch-out tubes at base of MPB infested lodgepole pine trunk, inset: detail of single tube. Scale bar 1 cm (b).

(pre-formed) oleoresin defense system of the host tree (known as pitch-out), while MPB boring introduces fungi that act as a food source as galleries develop. Once galleries and the spread of fungi have girdled the tree, there is further impairment of both induced (secondary) resin production, and the mechanics of water circulation in the phloem (Francheschi et al., 2005). Foliage of infested trees first dulls then yellows and reddens over one to two years following a successful MPB attack (Kurz et al., 2009). By the time foliage of infested trees becomes visibly impacted on a large scale, MPB has often dispersed to new hosts. The example of MPB provides a powerful analogy to explore 813C patterns in geologically-relevant volumes of plant resins and test hypotheses concerning the origins of amber deposits. In the course of developing a database of resin and amber stable isotopic compositions for the major world deposits, sufficient material was acquired and analyzed to examine in detail two major deposits that are putatively associated with insect damage, Dominican and New Jersey amber. Abundant deposits of fossiliferous Miocene (Iturralde-Vinent and MacPhee, 1996) amber from the Santiago region of the Dominican Republic have been attributed with confidence to the leguminous tree genus Hymenaea (Langenheim, 1969), for which modern taxa produce large volumes of resin in response to injury (Langenheim, 1969; Langenheim, 2003). Dominican amber is considered to have formed as a result of catastrophic events in an established moist tropical forest (Poinar and Poinar, 1999). Hurricane damage has been invoked as the most likely factor leading to the massive production of resin within this forest (Grimaldi, 1996b; Penney, 2008), ultimately producing the amber deposit. The large number of bark beetles preserved as inclusions within the amber (Poinar and Poinar, 1999; Penney, 2010) suggest an alternate cause, and provide an unique opportunity for comparison to modern resins produced as a direct result of infestation by scolytine beetles such as MPB. Late Cretaceous (Turonian) amber from New Jersey has been associated with both forest fires and the activities of wood-boring beetles with associated pathogenic fungi (Grimaldi et al., 2000). Although amber from this deposit is chemically homogenous and potentially originates from a single source conifer taxon, three distinct visual categories emerge from the census of thousands of specimens examined (Grimaldi et al., 2000). About ~70% of specimens are turbid, and contain the majority of insect inclusions as well as copious wood particulate material directly comparable to that produced by insect boring (Grimaldi et al., 2000). Most remaining specimens comprise small droplets that are optically transparent, but a third category includes rare specimens of opaque amber having a frothed appearance from microscopic gas inclusions associated with burning (Grimaldi et al., 2000). Only the opaque amber can be related directly to fire events given that, beyond its distinctive bubble-rich appearance, inclusions of fusainized wood are also found within. The presence of fusainized plant remains within the surrounding clay has led to the suggestion that the entire amber deposit resulted from forest fires (Grimaldi et al., 2000), but it remains unclear whether this sedimentological association is the result of taphonomic processes (Brasier et al, 2009; Najarro et al., 2010).

Materials and methods Samples We sampled lodgepole pine resins in an experimental forest plot near Grande Prairie, Alberta, Canada (55°05'13"N, 118°12'54"W), in both 2008 and 2009. Lodgepole pine resins were collected from infested and uninfested trees in close proximity in order to rule out the effects of microhabitat variability. Resin was collected from fresh flows on the lower trunk or from freshly trimmed branches. For New Jersey and Dominican amber, isotopic measurements were made from marginal cuttings of barren and insect-bearing specimens, avoiding bubbles and all particulate inclusions.

Methods Established methods were employed for carbon stable isotopic measurements and amber palaeoentomological investigations (Nissenbaum et al., 2005; McKellar et al., 2008). All isotopic data are reported in delta notation (813C) relative to VPDB with a precision of ±0.1 %o. Modern resins were first heated (50°C) for 12 hours under vacuum to remove the most volatile resin components and environmental water. Supplementary experiments with modern and subfossil Hymenaea resin showed no measurable fractionation associated with heating. All statistical differences between populations of resin and amber 813C values were assessed using Welch two-sided Mests with 95% confidence intervals. Mean values for each sample population are reported ±1 standard deviation. All results and sample descriptions are available in Appendix 4.

Results and discussion MPB attack and resin carbon isotopes During the 2008 MPB outbreak expansion, we observed 813C enrichment in resins from trees in the earliest stages of infestation (known as green attack), compared to adjacent healthy control trees (Fig. 6.2). The difference between 2008 newly infested green attack trees (mean 513C =-27.0 ±0.9l%o) and uninfested control trees (mean 513C =-28.2 ±0.68%o) is highly significant (p=0.018), with resins from infested trees consistently enriched relative to neighbouring healthy trees subjected to identical microclimatic conditions. In one tree where superimposed resin flows could be dissected sequentially to capture conditions prior to and 13 during MPB attack, resin 5 C values shifted from -28.2%0 (pre-2008) to -26.7%o (2008). Freshly produced resins from uninfested lodgepole pine trees having been subjected to water stress for four months since excavation and bagging of the root mass yielded mean 513C =-26.9 ±0.81%o. A similar isotopic enrichment to that induced by MPB attack can thus be induced rapidly by water stress alone, supporting the notion that phloem interruption is the most likely mechanism mediating isotopic enrichment in both cases. Additional lodgepole pine resins from the Grande Prairie test plot were collected in 2009, following a relatively harsh winter and late spring that slowed MPB expansion. Surviving infested trees produced new defensive resins that registered a return to pre-infestation values (mean 813C =-28.2 ±0.80%o), indistinguishable from either 2008 or 2009 healthy control trees (Fig. 6.2). Both surviving populations are in stark contrast with trees developing visible yellow foliage (mean 813C =-26.4 ±0.22%o). By far the most enriched sample was from a tree entering red attack (813C =-23.2%o), which provides an important end- member because few red-

5l3C(VPDB)%o attack specimens retain the -30 -29 -28 -27 -26 -25 -24 -23 -22 -21 -20 capacity to exude new resin. I I I I I I I I I I I Modern Pinus contorta resins These analyses demonstrate MPB 2008 control trees that the trajectory of MPB -28 2 ±0 68%o outbreak in lodgepole ¥ ™," W MPB 2008 green attack -27 0 ±0 91%o pine resin is isotopically MPB 2009 control, surviving trees discernible in real time: MPB -28 0 ±0 65%o 13 (•_>_5_J MPB 2009 infested, surviving trees attack induced positive 8 C -28 2 ±0 80%o excursions averaging 1.1 %o MPB 2009 CTE J MPB 2009 that intensify if the tree yellowing -26 4 ±0 22%o 23 2%o red attack P •••! Root-bound nursery trees eventually succumbs, but are -26 9±0 81%o rapidly reversed in cases of 1—1—1—1—1—1—r T T rebound. Hymenaea resins, copal and amber Modern Hymenaea courbai il resin -28 3±0 71%o Dominican amber and Sub-recent Colombian copal Hymenaea resins -27 5 ±0 55%o 1 -T Barren Dominican amber -26 0±1 01% Modern Hymenaea courbaril DP D CD PCD Dominican amber, 13 insect inclusions resin (mean 6 C =-28.3 -24 9 ±1 54%o Dominican amber, ±0.71%o)andsubfossil bormg-beetle inclusions -23 6±125%o Hymenaea copal (mean 813C 1 1 1 1 1 r~ T T T =-27.5 ±0.55%o) constrain New Jersey Cretaceous amber the isotopic composition of Barren TL rxupq • QD g -22 3 ±1 19°/oo resins derived from wounding Turbid of otherwise healthy trees. -21 9±0 98%o Values from Dominican Opaque *?* -20 6 ±0 37%o amber are enriched relative I I 1 1 1 1 1 1 1 1 1 -30 -29 -28 -27 -26 -25 -24 -23 -22 -21 -20 to these baseline values 513C(VPDB)%o (Fig. 6.2), and progress from Figure 6.2. 813C values of modern resins and amber specimens barren of insects analogues. The mean and standard deviation of each ,3 sample population are shown beneath the data. Colours (mean 8 C =-26.0 ±1.01%o), correspond to inferred levels of tree ecophysiological to those with inclusions of stress, based on consensus of the extent of observed insect damage and attendant mean exudate 813C value within generalist insects unrelated each population, ranging from healthy (dark green) to to infestation (mean 513C =- near-death (red). 24.9 ±1.54%o), and finally to amber containing obligate tree-boring beetles of the subfamilies Scolytinae and Platypodinae (Curculionidae, Fig. 6.3b) (mean 813C =-23.6 ±1.25%o). The latter population's isotopic composition is significantly different (p=0.011) from that of other Dominican amber specimens and Hymenaea resins.

Figure 6.3. Products of infestation in amber (a) Ophrynopus peritus Engel, an orussid wasp in Dominican amber which attacks wood-boring beetles, (b) Cenocephalus quasiexquisitus Davis & Engel, a platypodine boring-beetle in Dominican amber with wood particulates, (c) New Jersey amber physical classes, left to right: clear amber droplet, turbid amber with multiple flows, opaque amber with weathering rind, (d) Boreobythus turonius Engel & Grimaldi, a scolybethid wasp that attacks wood-boring beetles, shrouded by turbid NJ amber. Scale bars 1 mm (a-d).

When compared to the MPB data set, the distribution of 813C values observed in Dominican amber strongly suggests that many of the pieces containing boring beetles were produced at times of increased tree stress, likely as a direct result of insect attacks. In a survey of 2924 Dominican amber arthropod inclusions, 115 platypodine beetles were observed (Poinar and Poinar, 1999), while others have noted that scolytines comprise approximately 7% of insect inclusions within the deposit (Cognato and Grimaldi, 2009). Wood-boring beetles are thus a prominent group within the entombed fauna, and their activities may have contributed meaningfully to amber production. For example, remaining barren Dominican amber specimens present intermediate 813C values relative to Hymenaea resins produced as a result of mechanical damage and those containing fossil insects (Fig. 6.2). This suggests that mechanical injury alone was insufficient to produce the isotopic compositions observed in the latter population. New Jersey amber All three visual classes of New Jersey amber (Fig. 6.3c) were analyzed isotopically, revealing a progression of 813C enrichment between fragments of transparent (mean 813C =-22.3 ±1.19%o), turbid (mean 613C =-21.9 ±0.98%o), and opaque amber (mean S13C =-20.6 ±0.37%o). This distribution indicates that many amber specimens containing insect inclusions and plant debris were formed from resins produced under conditions of pronounced ecophysiological stress, thus supporting the association between insect-mediated damage and defensive resin production. The further enrichment observed in opaque New Jersey amber is consistent with the loss of isotopically-depleted volatile moieties during fire events, as implied independently by the record of bubble and fusain inclusions. Although the direction and amplitude of these isotopic shifts are consistent with the MPB results, we note that the baseline 813C of New Jersey amber is consistently enriched (by ~5%o) relative to modern lodgepole pine resins. We suggest that combinations of source tree metabolism, fundamental differences in the Cretaceous carbon cycle (Jarvis et al., 2006), and slight degrees of long-term resin diagenesis explain these differences, but that the original fingerprint of tree physiological stress is nonetheless recorded by amber isotopic compositions. Despite these potential influences, the distribution of 813C values retains a clear pattern within the deposit, which is directly comparable to the pattern produced by MPB infestation. One limitation of invoking wood-boring beetles in the genesis of New Jersey amber is their relative scarcity as inclusions within the amber. In a survey of 1032 New Jersey amber arthropod inclusions (Grimaldi et al., 2000), 37 identifiable beetles were recovered, two of which belonged to live wood- boring groups (Cupedoidea). Scolytinae and Platypodinae are known from Early Cretaceous Burmese amber, and Scolytinae are also present in Early Cretaceous Lebanese amber (Cognato and Grimaldi, 2009), but both groups have yet to be recovered from New Jersey amber. While the presence of obligate beetle parasitoids, such as wasps of the families Megalyridae, Orussidae, and Scolebythidae (Grimaldi et al., 2000; Engel and Grimaldi, 2007), provides indirect evidence for wood-boring beetles, at present, the abundance of wood particulates within turbid amber provides the strongest independent evidence for wood-boring (Fig. 6.3d). These indirect inferences are now augmented convincingly by the results of our isotopic analyses. Conclusions The direction and magnitude of resin 813C values from Dominican and New Jersey amber mirrors that expressed in MPB-infested pine forests (Fig. 6.2). In each instance, a strong case can be made for the association of insects to physiological stress in host trees, which in turn has the potential to become recorded in the 513C of resins synthesized shortly after attack. As a corollary, not all ambers preserving inclusions of wood-boring insects have strongly enriched isotopic values, as expected given opportunities for recovery that may equally be registered by 813C. For example, although a 1.3%o increase in mean 813C values was observed in Dominican amber samples containing boring beetles, there is substantial overlap with specimens lacking direct evidence of insect attack. We envisage that much of the isotopic variability documented has origins in water stress mediated by insects and their fungal symbionts, which affects both the quantity and quality of defensive resins. Water stress results in the progressive enrichment of the carbon isotopic composition of photosynthetic products by reducing stomatal conductance and hence the ability to discriminate against 13C (Farquhar et al., 1989). We have shown here that this isotopic effect is propagated to secondary metabolites. Boring insect attacks induce the same general pattern of isotopic enrichment produced by drought alone because the attacks interfere with water transport in host trees. As a corollary, pre-conditioning by drought clearly has been a factor in the magnitude and severity of current MPB outbreaks (Kurz et al, 2009). In modern and ancient ecosystems, 813C values from tree resins constitute a novel early indicator of insect outbreak events. This tool can be applied to ambers as old as the Mesozoic, and is expressed in both conifer and angiosperm resins. More intensive work is required to disambiguate the isotopic effects specific to insects and plant water stress, but our work addressing MPB suggests that resin composition responds faster than foliage colour, and hence can diagnose the severity of an outbreak more rapidly than satellite remote sensing, with implications for management. The analogies with both Cretaceous and Miocene ambers provide new lines of evidence supporting the active participation of insects in the original production of resins now preserved as amber, and correlate nicely with the diversity of wood-boring insects and their associates recovered from these deposits. Carbon stable isotopic ratios in fossil resins, together with detailed palaeoentomological consideration of associated inclusions, can be exploited to better understand this important dimension of plant-insect co- evolution. References Brasier, M., Cotton, L., Yenney, I., 2009. First report of amber with spider webs and microbial inclusions from the earliest Cretaceous (c. 140 Ma) of Hastings, Sussex. Journal of the Geological Society of London 166, 989- 997. Cognato, A.I., Grimaldi, D., 2009. 100 million years of morphological conservation in bark beetles (Coleoptera: Curculionidae: Scolytinae). Systematic Entomology 34, 93-100. Dal Corso, J., Preto, N., Kustatscher, E., Mietto, P., Roghi, G., Jenkyns, H.C., 2011. Carbon-isotope variability of Triassic amber, as compared with wood and leaves (Southern Alps, Italy). Palaeogeography, Palaeoclimatology, Palaeoecology 302(3-4), 187-193. Engel, M.S., Grimaldi, D.A., 2007. Cretaceous Scolebythidae and phylogeny of the family (Hymenoptera: Chrysidoidea). American Museum Novitates 3568, 1-16. Farquhar, G.D., Ehleringer, J.R., Hubick, K.T., 1989. Carbon isotope discrimination and photosynthesis. Annual Review of Plant Physiology and Plant Molecular Biology 40, 503-537. Francey, R.J., Farquhar, G.D., 1982 An explanation of 13C/12C variations in tree rings. Nature 297, 28-31. Franceschi, V.R., Krokene, P., Christiansen, E., Krekling, T., 2005 Anatomical and chemical defenses of conifer bark against bark beetles and other pests. New Phytologist 167, 353-375. Grimaldi, D.A., 1996a. Amber: window to the past. American Museum of Natural History Press, New York. Grimaldi, D.A., 1996b. Captured in amber. Scientific American 274(4), 84-91. Grimaldi, D., Shedrinsky, A., Wampler, T.P. 2000. A remarkable deposit of fossiliferous amber from the Upper Cretaceous (Turonian) of New Jersey, in: Grimaldi, D., (Ed.), Studies on Fossils in Amber, with Particular Reference to the Cretaceous of New Jersey. Backhuys Publishers, Leiden, pp 1-76. Grocke, D.R., Hesselbo, S.P., Jenkyns, H.C., 1999. Carbon-isotope composition of Lower Cretaceous fossil wood: ocean-atmosphere chemistry and relation to sea-level change. Geology 27, 155-158. Iturralde-Vinent, M.A., MacPhee, R.D.E., 1996. Age and paleogeographical origin of Dominican amber. Science 273, 1850-1852. Jahren, A.H., Sternberg, L.S.L., 2003. Humidity estimate for the middle-Eocene Arctic rainforest. Geology 31, 463^66. Jarvis, I., Gale, A.S., Jenkyns, H.C., Pearce, M.A., 2006. Secular variation in Late Cretaceous carbon isotopes: a new 813C carbonate reference curve for the Cenomanian-Campanian (99.6-70.6 Ma). Geological Magazine 143(5), 561-608. Kurz, W.A., Dymond, C.C., Stinson, G., Rampley, G.J., Neilson, E.T., Carroll, A.L., Ebata, T., Safranyik, L., 2009. Mountain pine beetle and forest carbon feedback to climate change. Nature 452, 987-990. Langenheim, J.H., 1969 Amber: a botanical inquiry. Science 163, 1157-1169. Langenheim, J.H. 2003. Plant Resins: Chemistry, Evolution, Ecology, and Ethnobotany. Timber Press, Portland. Loader, N.J., Robertson, I., Barker, A.C., Switsur, V.R., Waterhouse, J.S., 1997. An improved technique for the batch processing of small wholewood samples to a-cellulose. Chemical Geology 136, 313-317. McKellar, R.C., Wolfe, A.R, Tappert, R., Muehlenbachs, K., 2008 Correlation of Grassy Lake and Cedar Lake ambers using infrared spectroscopy, stable isotopes, and palaeoentomology. Canadian Journal of Earth Sciences 45, 1061-1082. Murray, A.R, Edwards, D., Hope, J.M., Boreham, C.J., Booth, W.E., Alexander, R.A., Summons, R.E., 1989 Carbon isotope biogeochemistry of plant resins and derived hydrocarbons. Organic Geochemistry 29, 1199-1214. Najarro, M., Penalver, E., Perez-De La Fuente, R., Ortega-Bianco, J., Menor- Salvan, C, Barron, E., Soriano, C, Rosales, I., Lopez Del Valle, R., Velasco, R, Tornos, R, Daviero-Gomez, V., Gomez, B., Delclos, X., 2010. Review of the El Soplao amber outcrop, Early Cretaceous of Cantabria, Spain. Acta Geologica Sinica, English Edition 84(4), 959-976. Nissenbaum, A., Yakir, D., 1995. Stable isotope composition of amber, in: Anderson K.B., Crelling J.C., (Eds.), Amber, resinite and fossil resins. American Chemical Society Symposium Series 617, 32-42. Nissenbaum, A., Yakir, D., Langenheim, J.H., 2005. Bulk carbon, oxygen, and hydrogen stable isotope composition of recent resins from amber- producing Hymenaea. Naturwissenschaft 92, 26-29. Penney, D., 2008. Dominican amber spiders: a comparative palaeontological- neontological approach to identification, faunistics, ecology and biogeography. Siri Scientific Press, Manchester. Penney, D., 2010. Dominican amber, in: Penney, D., (Ed.), Biodiversity of fossils in amber from the major world deposits. Siri Scientific Press, Manchester, pp. 22-41. Poinar, G.O. Jr., Poinar, R., 1999. The amber forest: a reconstruction of a vanished world. Princeton University Press, Princeton. Safranyik, L., Wilson, B., 2006. The Mountain Pine Beetle: A Synthesis of Biology, Management, and Impacts in Lodgepole Pine. Natural Resources Canada, Canadian Forest Service, Victoria. Stern, B., Lampert Moore, CD., Heron, C, Pollard, A.M., 2008. Bulk stable light isotopic ratios in recent and archaeological resins: towards detecting the transport of resins in antiquity? Archaeometry 50, 351-370. CHAPTER 7: GENERAL CONCLUSIONS

SUMMARY OF WORK To reiterate from Chapter 1: the main objective of this work is to advance the palaeontological study of Canadian amber, with an emphasis on hymenopteran inclusions. Secondary objectives include contributing to the understanding of Canadian amber, and advancing amber research in general. Here, the advances associated with this dissertation are outlined, and large-scale implications are discussed.

Advances in the palaeontology of Canadian amber At this point, we finally have a complete picture of the hymenopteran representatives in the Royal Tyrrell Museum of Palaeontology collection (as well as numerous supplementary specimens from the Canadian National Collection and University of Alberta collections). Although some taxa remain to be formally described, almost all identifiable specimens have received coverage herein. This places Canadian amber in the midst of an international effort to document the Hymenoptera in New Jersey amber (Turonian, Late Cretaceous), French Charentese amber (Albian-Cenomanian, mid-Cretaceous), Burmese (Myanmar) amber (Late Albian, Early Cretaceous), and Spanish amber (Albian, Early Cretaceous). As a result, it will soon be possible to conduct even more comprehensive assemblage comparisons, as well as cladistic analyses that encompass a broad range of Cretaceous taxa. Canadian amber holds a special place in such work, as it is the last diverse Cretaceous assemblage. Furthermore, Canadian amber offers some of the best preservation among known Cretaceous deposits, making taxonomic descriptions and comparisons possible in unmatched detail. Many of the large-scale biostratigraphic, palaeobiogeographic, and palaeoecological implications for the taxa encompassed by this dissertation have been discussed within the context of the hymenopteran inclusions in Canadian amber (Chapter 3). Other taxa examined, particularly thorny lacewings (Neuroptera: Rhachiberothidae), support the same general patterns observed in Hymenoptera. Modern rhachiberothids are known only from 13 described species in sub-Saharan Africa (Aspock and Aspock, 1997), yet the family appears to have been cosmopolitan and more diverse in the Cretaceous. Rhachiberothids have been recovered from Canadian, New Jersey, French, Burmese, Spanish, and Lebanese amber (McKellar and Engel, 2009, and references therein), and display a wider range of morphology than is known in the modern fauna, suggesting greater Cretaceous diversity. Scolebythids (Aculeata: Chrysidoidea) show many similarities to this pattern. They appear to have a predominantly austral relict distribution (Engel and Grimaldi, 2007, Azevedo et al., 2011), and are represented by six modern species and ten fossil species within Canadian, New Jersey, Spanish, and Lebanese amber (Chapter 3). Other hymenopteran groups that appear to have enjoyed greater Cretaceous diversity include Ceraphronidae, Mymarommatidae, and Scelionidae (Chapter 3). Furthermore, families such as Serphitidae and Stigmaphronidae, are known exclusively from the Cretaceous, yet appear to have been relatively diverse and widespread at the time. Taken together, these observations support the hypothesis that there have been significant changes in the distribution and diversity of many insect lineages after the Cretaceous, possibly as a result of climate change (Ander, 1942; Eskov, 1992; Grimaldi, 1992; Grimaldi and Engel, 2005). The composition of the Canadian amber assemblage also suggests a more immediate cause for extinction of serphitids and stigmaphronids (see below, under "Broader implications for the Hymenoptera").

Advances in the understanding of Canadian amber Two of the most persistent problems associated with the study of Canadian amber have been largely resolved within this body of work. These problems relate to the source of Cedar Lake amber, and the botanical source of both Cedar Lake and Grassy Lake amber. Although early studies (e.g., Carpenter, 1937) suggested that Cedar Lake amber must have originated somewhere within Alberta or Saskatchewan, and palaeoentomological work indicated that the two deposits were of similar age (McAlpine and Martin, 1969) and formed in similar environments (Borkent, 1995), there was still uncertainty regarding the source of Cedar Lake amber. This uncertainty was well-founded: it appeared as though Canadian amber produced two distinct FTIR spectra (e.g., Christiansen and Pike, 2002). Re- analysis of a large sample set of amber pieces from both Cedar Lake and Grassy Lake resolved this issue, indicating that the differences in FTIR spectra were simply a result of weathering effects (Chapter 2). Coupled with the extent of faunal overlap in the amber assemblages, and the similarity of stable isotopic compositions and physical properties, there is now strong support for treating the two localities as a single collection. This has a large impact on the study of Canadian amber inclusions because it is now possible to place Cedar Lake amber in a stratigraphic context. This also has an impact on sample sizes for future work - the F.M. Carpenter collection of Cedar Lake amber at the Harvard Museum of Comparative Zoology reportedly consists of 180 Kg of unprocessed material (McAlpine and Martin, 1969). To put this into context, most of the specimens examined in this dissertation stem from 30 Kg or less of Grassy Lake amber. The second longstanding issue with Canadian amber also has its origins in infrared spectroscopic analyses and chemotaxonomic assignments. Early IR spectroscopic work on the deposit suggested a source tree within Araucariaceae (Langenheim and Beck, 1968; Langenheim, 1969), and this was supported by subsequent analyses (e.g., Lambert et al., 1996; Zobel, 1999; Kosmowska- Ceranowicz et al., 2001; Aber and Kosmowska-Ceranowicz, 2001), yet the known distribution of this tree family contradicts this assignment (Stockey, 1982; Grimaldi et al., 2000). Through a broader FTIR spectroscopic comparison of Cretaceous ambers from western Canada and modern resins, we were able to demonstrate a cupressaceous source tree (Chapter 2). The only identifiable botanical remains we observed support this conclusion, and also suggest that the tree may belong to the genus Parataxodium (Chapter 1). This finding provides a firmer understanding of the type of forest that produced Canadian amber, and suggests that the numerous coal-associated ambers of western Canada may be directly comparable to the main deposit of Canadian amber.

Advances in amber research The application of stable isotope geochemistry to amber deposits represents a relatively new development for amber research. Early work demonstrated that amber retains the isotopic composition of the original tree resin with a high degree of fidelity (Nissenbaum and Yakir, 1995). This indicated that the composition of amber would be useful in identifying deposits (as used in Chapter 2), but its application to larger problems, such as constructing terrestrial 813C curves, is still in its infancy (Dal Corso et al., 2010). Within this body of work, we have demonstrated that intra-deposit variability in 813C values provides insights into palaeoecological events (Chapter 6). This work also provides further support for the fidelity and ecological sensitivity of stable isotopes in Mesozoic amber: such support is key if larger palaeoclimate questions are to be addressed with these data. The technique for processing friable amber that was developed during the course of study (Appendix 1) may seem trivial, but it opens up an entirely new avenue for amber research. The exceedingly fragile ambers that have been reported in association with coals in western Canada have been known for quite some time (e.g., McAlpine and Martin, 1969; Pike, 1993), but have remained inaccessible from a palaeoentomological perspective. The modified embedding technique allows these ambers to be processed in bulk, and has already generated a significant number of inclusions with respect to the amount of time invested in collecting and processing material. This technique offers a high-resolution record of well-preserved terrestrial arthropods that spans approximately 10 Ma. The most promising aspect of this new approach is an improvement in Paleocene representation. When combined with FTIR spectroscopy and stable isotope analyses of the entombing amber, this represents a major advance for the amber- based fossil record.

BROADER IMPLICATIONS FOR THE HYMENOPTERA At least two major implications for hymenopteran systematics are inferred from the Canadian amber assemblage. For the Ceraphronoidea, there are still no fossil representatives for Ceraphronidae, while both Megaspilidae and Stigmaphronidae are represented within Canadian amber. Although this is negative evidence, it provides additional support to the hypothesis that Ceraphronidae indeed may be a paraphyletic group, derived from within Megaspilidae after the Eocene (Engel and Grimaldi, 2009, Chapter 3). Much stronger evidence exists that Scelionidae (Platygastroidea) is paraphyletic, with Platygastridae originating from within Scelionidae after the Cretaceous. In both Canadian amber and Spanish amber, Scelionidae is the only one of these two families to be conclusively documented. In Canadian amber, Scelionidae are present with a broad range of characters, encompassing many of those used to diagnose modern Platygastridae (Masner, 1993), or suggested as ground plan characters for the family (Masner and Huggert, 1989). This is a clear indication that the two families were not distinct before the end of the Mesozoic, contrary to previous suggestions based on Canadian amber platygastroids (Masner and Huggert, 1989). In the context of other Cretaceous ambers, Canadian amber stands out in that it seems to have reduced genus-level diversity in many of the hymenopteran families that either disappear after the Cretaceous, or appear to have reached their pinnacle during the Cretaceous (Chapter 3). Previous studies have suggested that diversity differences between Canadian amber and slightly older New Jersey amber are likely the result of palaeolatitude (Grimaldi et al., 2000), and that the range contractions or loss of diversity in some groups may be the result of Tertiary climate change (Grimaldi and Engel, 2005). The similarity between Canadian amber and Siberian (Taymyr) amber (Santonian, Late Cretaceous in age) supports the palaeolatitude hypothesis, but also provides a possible immediate cause for the extinction of Serphitidae and Stigmaphronidae. In both of these deposits aphids (Aphidoidea) are the most abundant plant-feeders, and Serphitidae is second only to Scelionidae in terms of abundance and diversity (Zherikhin and Sukatsheva, 1973; Kozlov and Rasnitsyn, 1979). In direct contrast, New Jersey amber (Grimaldi et al., 2000) and Spanish amber (Alonso et al., 2000), contain scale insects (Coccoidea) as the primary plant-feeders, and have Ceraphronoidea as the second most abundant and diverse wasp group. This suggests that Serphitidae and Stigmaphronidae may have been parasitoids on aphids and scale insects, respectively. Therefore, the end-Cretaceous disappearance of aphid families specialized for feeding on conifers (Heie, 1996; Grimaldi and Engel, 2005), such as Canadaphidae, Cretamyzidae, Mesozoicaphididae, Palaeoaphididae, and Tajmyraphididae, is a probable cause for the extinction of serphitids (McKellar and Engel, 2011). Similarly, the extinction of stigmaphronids may be attributable to the loss of Electrococcidae, the only coccoid family known from both Canadian and New Jersey amber (Koteja, 2000). Climate change likely played a key role in the demise of these families and the range contractions of other hymenopteran taxa. Forests with Metasequoia as a major component show dramatic distribution changes as a result of climate change (Liu et al., 2007), and either this genus or a close relative appears to have been the source of Canadian amber.

BROADER IMPLICATIONS FOR THE STUDY OF CANADIAN AMBER AND AMBER RESEARCH The multidisciplinary approach to the study of Canadian amber adopted within this study, particularly the incorporation of stable isotope data, demonstrates that there are new lines of evidence that can be brought to bear on palaeoecological problems. Most previous work has relied primarily upon inclusions within amber (e.g., Borkent, 1995; Witchard et al., 2009), or the geological setting and remains of other taxa associated with an amber deposit (e.g., Grimaldi et al., 2000; Brasier et al., 2009), in order to examine palaeoecology or address events that may have led to amber production. We have demonstrated that proxy data for palaeoecological events (Chapter 6), and perhaps terrestrial climate conditions, can be derived from the stable isotopic composition of amber itself. When this is coupled with palaeoentomological data, palaeobotanical data obtained from inclusions, and chemotaxonomic data obtained from FTIR spectroscopy of the amber, a comprehensive picture of conditions in the amber-producing forest results. Because many of the western Canadian amber deposits are found in direct association with dinosaur remains (Tanke, 2004; Chapter 1), this approach provides new insights into habitat conditions for a much broader range of terrestrial taxa. These amber deposits may also provide a broader perspective on terrestrial conditions leading up to the Cretaceous-Tertiary extinction event. From a palaeoentomological standpoint, one of the most exciting implications of this work is that there is the possibility to examine well-preserved insect assemblages leading up to and across the Cretaceous-Tertiary boundary. Although insects are generally considered to have been little-affected by the end- Cretaceous extinction (e.g., Labandeira and Sepkoski, 1993; Grimaldi and Engel, 2005) there is almost no body fossil record of insects within this time interval. Examining in situ amber assemblages may finally reveal when insect families, such as Stigmaphronidae and Serphitidae disappear, and clarify the conditions surrounding their extinction.

FUTURE WORK Palaeoentomology Although all of the Hymenoptera inclusions in the RTMP collection have received coverage in this dissertation, much valuable work remains to be completed on this material. Ultimately, I aim to complete all of the alpha taxonomy on the Hymenoptera within the RTMP collection, and revisit the type material and any unexamined specimens within the CNC-CAS and ROM collections. Once this work is complete, taxonomy of the inclusions will be fully supported, and the vast amount of unprepared material within Harvard's MCZ collection and private collections will be approachable. In terms of phylogenetic work for the taxa encompassed in this dissertation, an international collaboration is currently underway, led by Michael Engel (University of Kansas). Alpha taxonomy for groups such as Serphitoidea, Mymarommatoidea, Ceraphronoidea, and Platygastroidea are nearing a degree of completion where cladistic analyses are feasible for representatives in Canadian, New Jersey, Spanish, and Burmese amber. Parasitic hymenoptera are considered to be excellent indicators of ecological disturbance and prone to extinction, due to their high trophic tier and specialized host requirements (LaSalle and Gauld, 1993). As a more complete picture of other Cretaceous amber assemblages develops, it will be of particular interest to observe variations in the group's representation, and how they fare across the Cretaceous-Tertiary extinction. Western Canada offers a unique opportunity to pursue a high-resolution record of insects in amber, with numerous coal-associated ambers. Now that it is possible to examine these friable ambers for inclusions (Appendix 1), the region offers one of the most complete amber records for the Late Cretaceous, as well as a valuable extension into the 'Paleocene gap' in the fossil record of insects. Material examined during the course of this dissertation has also yielded a number of insect inclusions that await description in additional orders. Highlights include: new representatives of Coleoptera (Mordellidae, Scirtidae, and Scydmaenidae), with Mordellidae in direct association with floral remains; new Trichoptera; and a range of immature insects, including a number of individuals preserved within a cocoon. In addition to these specimens, the RTMP and CNC- CAS collections contain a vast number of undescribed species or unverified records. One only has to refer to Table 2.1 (Chapter 2) to gain a full appreciation for the number of higher taxa that have been reported with no described material. Much work remains to be done on these specimens, and they will undoubtedly provide many interesting new records and insights into the amber-producing forest.

Chemotaxonomy and stable isotope analyses During the course of this dissertation contributions were made to a large database of amber and resin stable isotope data. This database now spans most of the world's major amber deposits, extending from the Triassic to subfossil copals and modern resins, and includes source tree data generated through FTIR analyses. Aside from providing an alternative terrestrial 513C record for much of the Mesozoic and Cenozoic, these data offer the opportunity to interrogate additional palaeoecological questions. Work is currently underway to assess whether arthropod inclusions in Eocene Baltic amber are actually encased in amber with an isotopic composition reflective of their modern habitats. This addresses Eocene climate conditions as well as the taphonomy of Baltic amber. Work has also begun on modern resins from the kauri trees (Agathis australis, Araucariaceae) of New Zealand. By comparing resins generated in various habitats it is hoped that we can further examine the ecological sensitivity of stable isotopes in resins, and by extension, amber. From an Albertan perspective, the coupled FTIR and stable isotope data promise to elucidate changes and conditions in palaeoforests throughout the Late Cretaceous and Early Paleocene. REFERENCES Aber, S.W., Kosmowska-Ceranowicz, B., 2001. Bursztyn i inne zywice kopalne swiata. Kredowe zywice kopalne Ameryki Polnocnej: cedaryt (czemawinit), jelinit. Polski Jubiler 2(13), 22-24. [In Polish]. Alonso, J., Arillo, A., Barron, E., Corral, J.C., Grimalt, J., Lopez, R., Martinez- Delclos, X., Ortufio, V., Pefialver, E., Trincao, PR., 2000. A new fossil resin with biological inclusions in Lower Cretaceous deposits from Alava (Northern Spain, Basque-Cantabrian Basin). Journal of Paleontology 74(1), 158-178. Ander, K., 1942. Die Insektenfauna des baltischen Bernsteins nebst damit verknupften zoogeographischen Problemen. Lunds Universitets Arsskrift, 2 Afdeling, Medicin samt Matematiska och Naturvetenskapliga Amnen 38, 1-82. Aspock, U., and Aspock, H., 1997. Studies on new and poorly-known Rhachiberothidae (Insecta: Neuroptera) from subsaharan Africa. Annalen des Naturhistorischen Museums in Wien, Serie B, Botanik und Zoologie 99, 1-20. Azevedo, CO., Xu, Z., Beaver, R.A., 2011. Anew species of Pristapenesia Brues (Hymenoptera, Scolebythidae) from Asia. Zootaxa 2750, 60-64. Borkent, A., 1995. Biting midges in the Cretaceous amber of North America (Diptera: Ceratopogonidae). Backhuys Publishers, Leiden. Brasier, M., Cotton, L., Yenney, I., 2009. First report of amber with spider webs and microbial inclusions from the earliest Cretaceous (c. 140 Ma) of Hastings, Sussex. Journal of the Geological Society of London 166, 989- 997. Carpenter, F.M., 1937. Introduction, in: Insects and arachnids from Canadian amber. University of Toronto Studies, Geological Series, 40, 7-13. Christiansen, K., and Pike, E., 2002. Cretaceous Collembola (Arthropoda, Hexapoda) from the Upper Cretaceous of Canada. Cretaceous Research 23, 165-188. Dal Corso, J., Preto, N., Kustatscher, E., Mietto, P., Roghi, G., & Jenkyns, H.C., 2011. Carbon-isotope variability of Triassic amber, as compared with wood and leaves (Southern Alps, Italy). Palaeogeography, Palaeoclimatology, Palaeoecology 302(3-4), 187-193. Engel, M.S., Grimaldi, D.A., 2007. Cretaceous Scolebythidae and phylogeny of the family (Hymenoptera: Chrysidoidea). American Museum Novitates 3568, 1-16. Engel M.S., Grimaldi, D.A., 2009. Diversity and phylogeny of the Mesozoic wasp family Stigmaphronidae (Hymenoptera: Ceraphronoidea). Denisia 26, 53-68. Eskov, K.Y., 1992. Archaeid spiders from Eocene Baltic amber (Chelicerata: Araneida: Archaeidae) with remarks on the so-called 'Gondwanan' ranges of Recent taxa. Neues Jahrbuch fur Geologie und Palaontologie, Abhandlungen 185, 311-328. Grimaldi, D. A., 1992. Vicariance biogeography, geographic extinctions, and North American Oligocene tsetse flies, in: Novacek, M.J., Wheeler, Q.D. (Eds.), Phylogeny and Extinction. Columbia University Press, New York, pp. 178-204. Grimaldi, D., Engel, M.S., 2005. Evolution of the Insects. Cambridge University Press, Cambridge. Grimaldi, D., Shedrinsky, A., Wampler, T.P., 2000. A remarkable deposit of fossiliferous amber from the Upper Cretaceous (Turonian) of New Jersey, in: Grimaldi, D. (Ed.), Studies on Fossils in Amber, with Particular Reference to the Cretaceous of New Jersey, Backhuys Publishers, Leiden, pp 1-76. Heie, O.E., 1996. Palaeoaphididae and Tajmyraphididae in Cretaceous amber from Alberta, Canada (Hemiptera: Aphidinea). Annals of the Upper Silesian Museum - Entomology 6-7, 97-103. Kosmowska-Ceranowicz, B., Giertych, M., Miller, H., 2001. Cedarite from Wyoming: infrared and radiocarbon data. Prace Muzeum Ziemi 46, 77-80. Koteja, J., 2000. Scale insects (Homoptera, Coccinea) from Upper Cretaceous New Jersey amber, in: Grimaldi, D. (Ed.), Studies on Fossils in Amber, with Particular Reference to the Cretaceous of New Jersey, Backhuys Publishers, Leiden, pp 147-229. Kozlov, M.A., Rasnitsyn, A.P., 1979. On the limits of the family Serphitidae (Hymenoptera, Proctotrupoidea). Entomologicheskoe Obozrenie 58(2), 402^16. [In Russian]. Labandeira, C.C., Sepkoski, J.J. Jr., 1993. Insect diversity in the fossil record. Science 261(5119), 310-315. Lambert, J.B., Johnson, S.C, Poinar, G.O. Jr., 1996. Nuclear magnetic resonance characterization of Cretaceous amber. Archaeometry 38(2), 325-335. Langenheim, J.H., 1969. Amber: a botanical inquiry. Science 163, 1157-1169. Langenheim, J.H., Beck, C.W., 1968. Catalogue of infrared spectra of fossil resins (ambers): I North and South America. Botanical Museum Leaflets, Harvard University, 22(3), 65-120. LaSalle, J., Gauld, I.D., 1993. Hymenoptera: their diversity, and their impact on the diversity of other organisms, in: LaSalle, J., Gauld, I.D. (Eds.), Hymenoptera and Biodiversity. CAB International Wallingford, UK, pp. 1-26. Liu, Y-J., Arens, N.C., Li, C-S., 2007. Range change in Metasequoia: relationship to palaeoclimate. Botanical Journal of the Linnean Society 154, 115-127 Masner, L., 1993. Superfamily Platygastroidea, in: Goulet, H., Huber, J.T. (Eds.), Hymenoptera of the world: an identification guide to families. Research Branch Agriculture Canada Publication, Ottawa, pp. 558-565. Masner, L., Huggert, L., 1989. World review and keys to genera of the subfamily Inostemmatinae with reassignment of the taxa to the Platygastrinae and Sceliotrachelinae (Hymenoptera: Platygastridae). Memoirs of the Entomological Society of Canada 147, 1-214. McAlpine, J.F., Martin, J.E.H., 1969. Canadian amber - a paleontological treasure-chest. The Canadian Entomologist 101, 819-838. McKellar, R.C., Engel, M.S., 2009. A new thorny lacewing (Neuroptera: Rhachiberothidae) in Canadian Cretaceous amber. Journal of the Kansas Entomological Society 82(2), 114-121. McKellar, R.C., Engel, M.S., 2011. The serphitid wasps (Hymenoptera: Proctotrupomorpha: Serphitoidea) of Canadian Cretaceous amber. Systematic Entomology 36(1), 192-208. Nissenbaum, A., Yakir, D., 1995. Stable isotope composition of amber, in: Anderson K.B., Crelling, J.C., (Eds.), Amber, resinite and fossil resins. American Chemical Society Symposium Series 617, 32^12. Pike, E.M., 1993. Amber taphonomy and collecting biases. Palaios 8, 411-419. Stockey, R.A., 1982. The Araucariaceae: an evolutionary perspective. Review of Palaeobotany and Palynology 37, 133-154. Tanke, D.H., 2004. Mosquitoes and mud: the 2003 Royal Tyrrell Museum expedition to the Grande Prairie region (northwestern Alberta, Canada). Alberta Paleontological Society Bulletin 19(2), 3-31. Witchard, W., Grohn, C, Seredszus, F., 2009. Aquatic insects in Baltic amber. Verlag Kessel, Remagen-Oberwinter. Zherikhin, V.V., Sukatsheva, I.D., 1973. On the Cretaceous insect-bearing „ambers" (retinites) from North Siberia, in: Narchuk, E.P., (Ed.), Problems in Insect Palaeontology: XXIV annual lectures in memory of N. A. Kholodovsky (1-2 April 1971), Nauka Press, Leningrad, pp. 3-48. [In Russian]. Zobel, A.M., 1999. Cedarite and other fossil resins in Canada, in: Investigations into amber: proceedings of the international interdisciplinary symposium Baltic amber and other fossil resins. Gdansk, Poland, pp. 241-245.

174 APPENDIX 1: A MODIFIED TECHNIQUE FOR THE PREPARATION OF FRIABLE AMBER AND THE BULK-PROCESSING OF ITS INCLUSIONS1*

INTRODUCTION Specialized techniques for the preparation of brittle amber have been widely documented (e.g., Grimaldi, 1993; Pike, 1995, Corral et al., 1999; Nascimbene and Silverstein, 2000). These techniques have proven effective in the stabilization and long-term preservation of otherwise fragile or unstable amber pieces, and represent an important advance for the study of Mesozoic amber inclusions in particular. Recent reviews of these techniques include those of Nascimbene and Silverstein (1999), and Penney and Green (2010). Most of the reported techniques for stabilizing and polishing brittle ambers share several features in common. They depend primarily on embedding the amber specimen in epoxy resin or bioplastic. Embedding stabilizes the amber to facilitate polishing and has the added benefit of filling in fractures to reduce internal refraction and reflection. This is typically done after rinsing the amber of debris, examining the pieces for surface impressions, and then polishing planar 'windows' or tumbling the specimens to peer beneath any external weathering rind. Variations on this general protocol include the use of immersion baths of glycerine, sugar water, water, Canada balsam, cedarwood oil, or mineral oils to approximate the refractive index of the specimen (Grimaldi, 1993) and improve visibility of inclusions (for screening or temporary mounting). Also, some workers advocate the use of a vacuum chamber while embedding (e.g., Nascimbene and Silverstein, 1999), as this drives embedding medium into cracks within the specimen, further clarifying and reinforcing the amber. Although these myriad techniques have greatly improved the study and preservation of brittle amber, they have proven too costly (in terms of both time and materials) for the processing of very small and exceedingly fragile amber specimens. These friable ambers are more brittle than most of the material processed for inclusions, apparently as a result of more extensive thermal maturation or oxidation prior to collection (Grimaldi, 1996). Friable amber often crumbles under even cautious handling, which precludes the earliest steps in most existing protocols. The specimens typically cannot be scrubbed while rinsing, and cannot be subjected to preliminary polishing to peer beneath the exterior

* A version of this appendix is in preparation for publication. At present, authors consist of: McK- ellar, R.C., and Tappert, R., but additional workers will likely be involved in the final contribution, as we wish to describe part of an amber assemblage utilizing this technique. weathering rind. Often, these limitations can be overcome by examining the specimens in an immersion bath to check for inclusions (the authors have had success with glycerine in particular). However, many of these specimens have a deeply crazed or fractured outer surface that cannot be properly rinsed of the immersion liquid or will spall within the immersion bath. These restrictions have precluded detailed study of inclusions in numerous deposits with trace amounts of amber. In western Canada, many of the low grade (sub-bituminous) coals have amber associated with their upper surfaces, or preserved within the coal itself (McKellar and Wolfe, 2010). These amber pieces are typically very small, with an average size of approximately 5 mm, and larger pieces reaching 1 cm in their greatest dimension. Indurate pieces are exceedingly rare, and most pieces must be handled cautiously with forceps. These occurrences of amber have been noted in previous publications (McAlpine and Martin, 1969; Pike, 1993), but they have not been developed as a palaeontological resource because of the difficulties associated with their collection and study. Here we provide a modified technique for the collection and preparation of friable amber and illustrate some of the inclusions recovered with the technique.

THE PROTOCOL Collecting Most of the material that we have examined has been collected directly from shallow excavations into coal seams (10 cm or less in depth) within the Cretaceous strata of Alberta, Canada. Alternatively, amber pieces have often been found in moderate concentrations within the shales or fine, lignitic sandstones that commonly occur directly above or below these seams in western Canada. Amber pieces have also been surface-collected from slumped or eroded coal faces along rivers and streams, but these are typically more resilient than the pieces extracted directly from coal. Due to the small size and fragility of the amber pieces, most were gathered with fine forceps into vials with cotton or tissue packing in order to prevent damage. Coals and shales were flaked apart to search for small pieces in situ, and these pieces were extracted with the pointed tip of fine forceps. This technique yielded many small droplets or rod-shaped pieces (Fig. ALIA). Multi- layered amber pieces representative of repetitive flows were seldom encountered, but were typically the largest pieces collected from any given horizon. When amber pieces fragmented during extraction or collection, the pieces were retained, and usually transferred to a separate container for further processing as a single specimen.

Figure Al.l. Specimens prepared with new protocol. (A) Raw Paleocene amber pieces from the vicinity of Wabamun Lake, Alberta. (B) Embedded specimens from the same locality as A and generally similar to the raw material prior to embedding, demonstrating clearing achieved through embedding, and also surface clarity on surface contacting adhesive film. The area to left and below the lines marked with arrows is the smooth surface left once the adhesive tape used in embed­ ding is removed, outside of these lines is the frosted surface created by the mould. (C) Overview of a polished Maastrichtian friable amber piece from the Edmonton region, Albera. Upper arrow indicates hemipteran inclusion, lower arrow Parataxodium-type foliage. (D) Detail of hemipteran inclusion, demonstrating quality of preservation in friable amber.

Specimen embedding Due to the fragility of the amber pieces, most were soaked and gently rinsed with water in order to remove loose sediment, but few were strong enough to permit scrubbing. Specimens were removed to a separate vial to air dry. Individual amber pieces were generally grouped by size, then affixed in rows to the bottom of a disposable embedding mould using fine forceps or a moistened brush to manipulate amber pieces. We chose to use 22 mm by 22 mm square moulds with tear-down sides (e.g., Polysciences "Peel-Away moulds") in order to facilitate aligning specimens and batch processing. These moulds are supplied in sheets that can be trimmed to provide the appropriate number of mounts, are easily labeled and stacked (in the vacuum chamber), and utilize manageable batch sizes for embedding epoxy. Most importantly, disposable moulds do not require any significant force or flexion to release the embedded specimens from the mould - thereby preventing the amber pieces from cracking during handling of the mount. Specimens were affixed to the bottom of the moulds using a transparent double- sided tape. We used "X-Film DX" double sided adhesive film available through the German company Modulor, but this is not the only alternative available. This particular film was chosen because it demonstrated good adhesion (specimens were easily mounted), it did not interact with the epoxy resin (the tape was easily peeled from the bottom of the embedded specimens without leaving a residue), and the tape's backing film is both flexible and strong (the tape did not leave fragments in the embedding resin). Depending on the size of the amber pieces or fragments, they were mounted in groups of 2-25 specimens. Specimens were aligned in rows with sufficient space between individuals to allow single specimens to be cut out of the grouping with a rock saw equipped with a kerf blade: this typically requires little more than a 1 mm gap. Cutting out individual pieces permits viewing or polishing specimens in a different orientation if it becomes necessary. The trays of aligned amber pieces were filled with a low-viscosity epoxy resin as an embedding medium. We utilized Buehler "Epo-THIN" epoxy because of its long cure and working times, good penetration, and high clarity and hardness. This product is widely used in the preparation of petrographic thin-sections, but there are many alternatives available. Epoxy was poured into a vacant corner of each mould, and allowed to slowly rise around the amber pieces, in order to reduce the number of bubbles produced on the surface of each amber piece. The epoxy was poured until the tallest specimen in each mould was 2-3 mm below the surface of the epoxy, and the moulds were transferred to a vacuum chamber. The filled mould sheets were exposed to vacuum (-27 inHg or 91 kPa) for approximately five minutes, then the vacuum was released (driving resin into the low-pressure areas created). This process was repeated three or more times, until the frothing produced in the resin at the beginning of each vacuum cycle had diminished. Pouring epoxy to a greater depth than the tallest specimen concentrated most of the bubbles produced during the vacuum process upon the surface of the epoxy, and not on the amber specimens themselves. In cases where bubbles clung to the amber pieces, they were delicately swept away with a pin or probe. Mould trays were then placed in a fume hood and the epoxy was allowed to cure for 12 hours or more.

Polishing and slide-mounting embedded specimens After the epoxy mounts were peeled from their disposable moulds, the upper surface of each block provided good specimen visibility. With the exception of small bubbles concentrated around the margins of the upper surface, most regions were vitreous and clear. The sides of the epoxy mount that were in contact with the mould had reduced visibility, because the minor surface texture of the mould was reproduced in the epoxy. Peeling the adhesive film from the bottom of each epoxy block provided a clear window directly beneath each amber specimen, with visibility nearly as good as that produced by the upper surface of the epoxy block (Fig. A LIB). After a preliminary examination for inclusions, the bottom surface of each epoxy block was repeatedly ground using wet/dry sandpaper submerged in a shallow water bath. Bulk material was removed by hand-grinding with 220-grit sandpaper in intervals of approximately 30 seconds, but lapidary wheels are an excellent alternative for all steps of grinding and polishing. Depending on the size of the amber pieces within the epoxy block, and their internal visibility, the ground surface was periodically made smooth with a series of 800- and 1200- grit papers followed by polishing in an aluminum oxide ("Alumina G" polishing compound) slurry. This created a viewing surface with good optical properties, and allowed us to scan through amber pieces for inclusions that otherwise may have been hidden by the amber's poor internal visibility. In cases where the amber pieces were completely obscured by matrix and it was not possible to remove this material prior to embedding, the upper surface of the epoxy block was polished as well, permitting a clear view through the amber pieces. When an inclusion was identified, we removed as much material as possible from the polished face, then either cut the inclusion-bearing amber piece from its surrounding mount, or affixed the entire mount to a petrographic slide. We used low-viscosity epoxy to mount the embedding block on a slide, or if it was necessary, to embed the cut­ out inclusion a second time. (Re-embedding specimens produced a larger piece of resin that was easier to manipulate.) Once specimens were attached to slides, material was removed from the upper surface by grinding and polishing it using the same technique as the initial 'search'. A cover slip was applied to the finished surface using epoxy. If it was apparent that a different viewing orientation was necessary to study the inclusion properly, either the specimen block was ground at an angle to achieve this orientation, or the block was mounted at the edge of the slide, and a cover slip was applied to that margin, providing an additional viewing surface.

RESULTS AND DISCUSSION The major differences between our protocol and that of Nascimbene and Silverstein (2000) are: 1) screening amber pieces for inclusions after they have been embedded; 2) the use of adhesive tape to hold specimens down during embedding; and 3) the use of a single, minimal allotment of epoxy resin for vacuum-embedding. As with most existing protocols, Nascimbene and Silverstein (2000) relied upon polishing or tumbling amber specimens in their initial search for inclusions. They also relied on removing vacuum-impregnated amber pieces from an initial epoxy bath, and then placing them in partially-set epoxy, in order to keep them from floating in the final epoxy mount. These differences between protocols may seem minimal, but they have a pronounced effect on the amount each specimen must be handled or processed in an unsupported state, and how much time and supplies must be devoted to each amber piece. Our modified technique permits the bulk-processing of friable and exceptionally fragile amber, by reducing the damage associated with handling the amber, and providing an efficient means of screening such material for inclusions. Handling of unsupported specimens is reduced primarily through eliminating all but the most essential handling prior to embedding. Although we typically clean our specimens prior to embedding, even this is not strictly necessary. The only handling that is absolutely necessary is placing specimens on an adhesive film. Even preliminary cleaning is not essential to the search for inclusions once both the upper and lower surface of the mould have been ground down to form windows in the outer rind of the amber. Other steps, such as preliminary polishing or transferring amber pieces between epoxy batches can be eliminated completely. Additional advantages of the new protocol include the ability to position broken pieces with inclusions back together prior to embedding. The adhesive on the double-sided tape used for mounting is sufficiently thick to orient specimens properly, and a small amount of cyanoacrylate glue delivered with a pin is sufficient to hold the pieces together throughout the embedding process. Batch processing of amber for inclusions has the added benefit of saving time and materials. Preliminary tumbling to screen material in bulk can take many weeks, and risks damage to near-surface inclusions and particularly fragile amber pieces (Grimaldi, 1993). Polishing individual pieces by hand as part of the screening process is less damaging with fragile ambers, but is difficult with specimens as small as those we are dealing with, and would destroy the majority of friable specimens. Early embedding permits the polishing of windows into as many as 25 pieces of amber simultaneously, with the option of extracting and preserving specific amber pieces at any stage of the grinding and polishing process. Batch processing reduces the amount of expendables, such as epoxy resin and slides that are necessary to process a given amount of amber. Furthermore, the use of double-sided tape eliminates the strict timing and wasted epoxy that has been necessary in previous protocols. The new protocol has shown great promise in processing the friable ambers associated with coals in western Canada. These previously unworked deposits now yield inclusions with the level of efficiency that is required for assemblage studies (Fig. A1.1C, D). When prepared properly, friable amber offers a valuable extension to the fossil record derived from the major amber deposits of the world. REFERENCES Corral, J.C, Lopez Del Valle, R., Alonso, J., 1999. El ambar cretacico de Alava (Cuenca Vasco-Canabrica, norte de Espafia). Su colecta y preparation. Estudios del Museo de Ciencias Naturales de Alava 14(special publication 2), 7-21. Grimaldi, D., 1993. The care and study of fossiliferous amber. Curator 36(1), 31-49. Grimaldi, D.A., 1996. Amber: window to the past. Harry N. Abrams Inc. in association with The American Museum of Natural History, New York. McAlpine, J.F., Martin, J.E.H., 1969. Canadian amber - a paleontological treasure-chest. The Canadian Entomologist 101, 819-838. McKellar R.C., Wolfe, A.P., 2010. Canadian amber, in: Penney, D. (Ed.), Biodiversity of fossils in amber from the major world deposits. Siri Scientific Press, Manchester, pp. 149-166. Nascimbene, P., Silverstein, H., 2000. The preparation of fragile Cretaceous ambers for conservation and study of organismal inclusions, in: Grimaldi, D.A. (Ed.), Studies on fossils in amber, with particular reference to the Cretaceous of New Jersey. Backhuys Publishers, Leiden, pp. 93-102. Penney, D., Green, D.L, 2010. Introduction, preparation, study & conservation of amber inclusions, in: Penney, D. (Ed.), Biodiversity of fossils in amber from the major world deposits. Siri Scientific Press, Manchester, pp. 5-21. Pike, E.M., 1993. Amber taphonomy and collecting biases. Palaios 8, 411^419. Pike, E.M., 1995. Amber taphonomy and the Grassy Lake, Alberta, amber fauna. Ph.D. thesis. Department of Biological Sciences, University of Calgary, Calgary, Alberta. APPENDIX 2: STABLE ISOTOPE MEASUREMENTS FROM NORTH AMERICAN CRETACEOUS AMBERS Sample Location Age Amber Observations (Colour/Grade; Morphology; Notes)

eriar I ake amber f Tom Harvard's Museum of Comparative Zoology, s less thaini 3mm i_n size ec ar -a ce, Manito ?a eavy weathering ec ar -a ce, Manito )a :d, isolated drip, imino. r• weatherinweathering ec ar La ce, Manito la i orange, dnpID,'mino, mi r patina , ,, edar Lake, Manitoba edium orange milky, flow fragment, highly eathered 14 -alee, Manitoba patina _ecar ..a ce, Manito ?a patina „a ce, Manito >a , , „_„ -.aernent,"partly weathered -a ce, Manito )a e, orange, lsolatecrdnp, minor patina -ake, Manitoba — Trange, clear, flow fragment, blocky and unweathered -ake, Manitoba ^ampanian Medium orange, drip fragment, minor patina Kecar .a ce, Mamto ?a Kam )aman Yellow, clear, tiny isolated dridnp -a ce, Mamto >a Kam )aman range, milky, -ake, Manitoba -am )aman issenbaum am cw fragment,' pervasively weathered

rassy T ake amber LI Grassy Lake, Alberta Campaman -?ITT Deep orange, flattened disk, 'internal', thm oxidative

grassy Lake, Alberta .le orange, isolated drop jrassy Lake, Alberta P&ediu m orange, clear, massive flow, milky strn ;ers jrassy Lake, Alberta one grade, massive flow, dark orange rind of c ar

Grassy Lake, Alberta olden yellow„ clear, massivmassivie flow,flow, botanicabptar l inclusion Grassy Lake, Alberta >eep orange, clear, massive flow,-•" carbo-irbon-ncn h Grassy Lake, Alberta ale yellow, clear, massive flow, palynolynomorpn h

Grassy Lake, Alberta Campaman Dark yellow, massive flow, stringer stains, oxidation

Grassy Lake, Alberta Campaman 'eatnered surface of amber mass (medium orange, attened disk\ grassy Lake, A )erta a ej " jrassy -ace, A )erta La e? jrassy ..ace,A jerta :JIo jrassy -ace, A jerta ; yellow, isolated drip, ..... , jrassy -ace,A jerta le grade, subsphencal mass, dark oxidative crust jrassy Lake, A berta : oi bone grade and dark orange, subsphencal,

cylindrical dnp, weathered to granular ISO atec c np, weathered to granular m lso atec c n), so id, unweat lered 5 lso atec c n ), so id, unweat lered :, isolated dn), so id. unweathered ulti-1 ow runnel, c ark flow lines multi-flow runnel, dark flow lines edium orange, flattened 'internal disk, ottled bone/medium orange, nodular, heavily weathered,, soft, GL34 Grassy Lake, Alberta Campaman Mottled bone/medium orange. nodular, heavily GL35 Grassy Lake, Alberta Campaman Dark orange, multi-flow, inci aphids,scehonid,chiro

rassy Lake, Alberta Campaman Dark orange, multi-flow, inci aphids, indet dipteran m Srassy Lake, Alberta Campaman Medium orange, flattened internal disk rmtnn (river valley^ amber •n monton « Maastnchtian -25 9 -349 Medium orange, cloudy, weathered to granular, Edmonton, A jerta /Jaastnchtian -339 Pale yellow, isolated drop, solid Ec monton, A jerta |aastri' c: ltjan -344 Mec fum orange, cylindrical fragment, crumbling m Ec monton, A >erta /laastric: ltian -343 Met lum orange, drip end, crumbling Ec monton, A /laastric ltian -HI Mec lum orange, sheet flow fragment, solid mi Edmonton, A •laastnchtian -339 Medium orange, spherical drop, granular i Bndgi upper and lower coal seams >ert:rfa Maastnc ltial n tec c is i-orange, drop et in e, crum >erta Maastn, c lltia n tec c is i-orange, c ro )e t in e, crum )erta 4aastnc ltian tec c is i-orange, c ro )eti n e, crum), )erta ..,/laastri_ c ltian tec c is i-orange, c ro) et in e, crum jerta Maastnchtian teqdish-orange; dropjetjn jgnitic sha e; crumb berta Maastnchtian Jark orange, oroplef m ligmtic shale, pervasivel; weathered

"heller V, /laastric ltian lie,orange, cylindrical flowJragme:ien t 4aastnc ltian edium orange, cylindrical flow fra] /laastric ltian A— «.„Te( :rejd OT.eranulaanularr wwui h carbon rind Maastnc ltian 'cylil ow fragment /laastric ltian turn orangeS, spherical mass, solid, /taastnchtian orange, isolated drop, weathered granular

New Jersey Rantart Frn amber (purchased^ NJ1 Sayerville, New Jersey Turoniau n -?m— Butterscotch, multiple flows, inci erythraeid, eratopogomd, wppd debns , , NJ2 Sayerville, New Jersey Turoman sample glutterscatch, multiple flows, inci indet Hymenopte: leaked bubble-clouding, wood cjebns, , e, New Jersey uronian h, ctoudedwitn bubbles and wood debi e, New Jersey uronian —• -. ear, large flow fragment, unweat e, New Jersey uronian : ear, large flow fragment, patina 1 e, New Jersey uronian ear, flow tip fragment, subsphencal APPENDIX 3: SUPPLEMENTARY MATERIAL FOR CHAPTER 5 "CONTRIBUTION TO VERTEBRATE PALAEONTOLOGY IN CANADIAN AMBER"1*

MATERIALS AND METHODS Established methods were employed for the collection and preparation (Nascimbene and Silverstein, 2000) of amber inclusions. Epoxy-embedded amber nodules were slide-mounted and polished, and cover slips were applied to optimize views and ensure long-term preservation of the inclusions. Total slide thickness ranged from 1.8 mm to 8.5 mm, with the thickest mounts at times challenging the resolving power of compound microscopy. A suite of modern bird feathers and hair samples were directly compared to the amber- entombed specimens, as were morphological atlases on the microscopic structure of mammalian hairs (Hausman, 1920; Brunner and Coman, 1974) and feathers (Chandler, 1916; Dove, 2000). Modern comparative specimens were either epoxy- embedded or examined unaltered, depending on the degree of magnification required. All specimens were photographed using a Canon PowerShot A640 camera attached to a Zeiss Stereo Discovery. V8 microscope, or Zeiss Axio Imager.Al compound microscope ('b.f.' denotes bright field photographs, 'd.f.' denotes dark field photographs). Images usually encompass multiple focal planes and were compiled using Axiomat or Helicon Focus software. All measurements were taken either digitally using Axiomat, or on a Wild M5 dissecting microscope equipped with an ocular micrometer. The inherent limitations of working with amber governed our approach to the Canadian amber specimens, and consequently we focused our work on morphological comparisons and morphometric analyses. The nature and rarity of these specimens precludes destructive sampling until additional specimens are recovered. Potentially contentious specimens, such as the Stage I and II morphotypes, were subjected to additional non-destructive sampling. Spinning disk confocal microscopy (SDCM) and laser scanning confocal microscopy (LSCM) were utilized. SDCM data were obtained using a Hamamatsu Orca R2 camera on an inverted Olympus 1X81 microscope with a Yokogawa CSU-10 spinning disc confocal head (examining excitation at 491 nm and 561 nm). LSCM data were obtained with a Leica SP5 microscope using a 20x 0.5 Na objective

* A version of this appendix is currently in press as supplementary online material. McKellar, R.C., Chatterton, B.D.E., Wolfe, A.P., Currie, P. J., (in press). A diverse assemblage of Late Creta­ ceous dinosaur and bird feathers from Canadian amber. Science. and acousto-optical tunable filters (examining excitation at 405 nm). Results of these analyses, as well as additional morphological details on all specimens are presented here. Institutional abbreviations: TMP, Royal Tyrrell Museum of Palaeontology, Drumheller, Alberta, Canada; RAM, Royal Alberta Museum, Edmonton, Alberta; RM, Redpath Museum, McGill University, Montreal, Quebec, Canada, modern bird collection; UALVP, University of Alberta Laboratory of Vertebrate Palaeontology, Edmonton, Alberta. SUPPLEMENTARY TEXT Additional details of Stage I and II morphotype identifications A major concern regarding the specimens identified as Stage I or II morphotypes is whether other possible interpretations are tenable. Within the main text we briefly summarize these possibilities and the bases for their rejection. Here we provide full details of the work underpinning our conclusions.

Comparison to modern mammalian hairs In general, the Stage I and II morphotypes reported are of a smaller diameter than most mammalian hairs and do not appear to possess cuticular scales. More specifically, the filaments measured from UALVP 52821 have a mean diameter of 16.4±4.2 um («=80), with minimum and maximum diameters of 6.2 um and 27.1 um, respectively. In UALVP 52822, filaments have a mean diameter of 17.9±5.0 um («=28), and range between 10.7 um and 31.0 um. The UALVP 52822 filaments are loosely bundled into five distinct clusters. The three clusters that have definite edges and appear to represent a complete cross-section of the bundle measure 213 um, 233 um and 325 um in diameter at their narrowest. The diameters observed for Stage I and II filaments therefore fall just within the lowest range of values known for modern mammal hair. Mammal hair has been studied extensively, and the two main types that have been documented across a wide range of taxa, with attention to both overall diameter and cuticular scale patterns, are underhairs (understory fur) and guard hairs. Given that the Stage I and II filaments overlap with only the finest known mammal hairs, and furthermore given differences between modern and Cretaceous mammalian faunas, we conducted detailed comparisons to pelages that represent both the smallest known underhair diameters (Hausman, 1920) and contain the widest taxonomic range of organisms, including numerous marsupials (Brunner and Coman, 1974). Because the latter work was based mainly upon guard hairs (typically of slightly larger diameter than underhairs (Brunner and Coman, 1974), but more likely to enter in contact with tree resin), measurements were taken from the narrowest part of each exemplar. Underhair diameters listed for 162 species of mammals (Hausman, 1920) yielded a mean value of 59.5±82.3 um, ranging from 6.8 um to 680 um. Measurements of guard hair diameters for 75 species of Australian mammals (Brunner and Coman, 1974) yielded a mean value of 48.2±37.8 um, a minimum of 9.4 um, and a maximum of 168 um. Although these samples clearly display a wide range of diameters, all modern specimens within the low end of the spectrum were united by two morphological features. In almost all cases of diameters below 25 um, the medulla (hollow core) of the hair was discontinuous, being subdivided along its length into either a uniserial ladder or aeriform lattice arrangement (Hausman, 1920; Brunner and Coman, 1974). This was typically observed in conjunction with coarse, diamond-shaped cuticular scales arranged with a maximum of two to three scales fitting within one hair- width and resulting in a jagged margin on the hair when viewed in longitudinal section (Hausman, 1920; Brunner and Coman, 1974). Stage I and II filaments differ markedly from this arrangement. The filaments are hollow, with an outer wall that comprises approximately 40% of the total diameter, and is further reduced within apical portions of the filaments. The hollow nature of the filaments is best illustrated in UALVP 52821, where patchy translucency and broken edges demonstrate that the filaments have a circular cross-section, and that their cores are hollow (Figs. A3.3, A3.4A, A3.11; see also appendix section regarding specimen LSCM analyses and taphonomic considerations). Within areas where the filaments are preserved as nearly opaque masses due to darker pigmentation, they nonetheless preserve faint cross-hatching of very fine light and dark spots (Figs. A3.4B-D). Within areas where the filaments are translucent, the outer wall clearly does not possess a jagged margin, which would be clearly observed if cuticular scales were present.

Comparison to fossil mammalian hair In Canadian amber, there is currently one hair fragment known (TMP 96.9.998). This specimen (Figs. A3.4E, F) is in the process of being studied and described, but our preliminary analysis already indicates a number of distinctions between it and the Stage I and II morphotypes described herein. The hair fragment is significantly wider than any of the filaments preserved (approximately 56 um in diameter) and reveals faint indications of fine, closely-spaced cuticular scales when viewed with dark-field microscopy. Additional observations suggest that the specimen lacks a broad medullary cavity and that the medulla is likely discontinuous in either an aeriform lattice or multiserial ladder pattern, once again in contrast to any of the Stage I and II filaments reported. Additional Mesozoic fossil hair specimens from the Early Cretaceous of France include two fragments preserved in three dimensions within amber (Vullo et al., 2010). These specimens have observed diameters that range from 32^18 um and from 49-78 um, respectively, and possess cuticular scales that are smoothly undulate with an intermediate spacing (Vullo et al., 2010). Preservational characteristics of the hair fragments described by these authors are similar to those observed for both hair and the Stage I morphotype filaments from Canadian amber.

Comparison to fungal and plant remains In general, the Stage I and II morphotypes reported can be differentiated from plant and fungal remains based upon their comparatively large size, lack of septae, and preservational characteristics. Most fungal hyphae branch and exhibit a diameter range from 1-15 urn, but the known range extends from 0.5 urn to 1 mm (Hudson, 1986). Cell walls in hyphae are generally thin (often 0.2 urn or less), with chitin as the main structural component (Burnett, 1986). In amber, this combination of features typically results in filamentous fungi that are easily observed as mycelia (larger mats of hyphae). These appear vitreous or white when examined under reflected light (Fig. A3.4G). Conceivably, groups such as the Zygomycetes (bread moulds) could produce coenocytic hyphae (those lacking internal septae) of similar overall morphology to UALVP 52821. However, a subparallel, non-branching, centimeter-scale series of such hyphae lacking any adventitious septae or terminal sporangia seems highly improbable (Talbot, 1971). Furthermore, UALVP 52821 displays pigmentation and an outer wall thickness that do not match the preservational characteristics of fungi within this amber deposit. Many of the characteristics that separate Stage I and II morphotypes from fungal remains also distinguish them from plant remains. The Stage I and II morphotypes exhibit no evidence of longitudinal subdivision within their hollow cores, and their diameters are roughly twice to thrice those of xylem cells found in the deposit. Furthermore, xylem cells are typically polygonal in cross-section and when encountered in Canadian amber, are typically present as adjoined series of cells that form blocky fragments of tissue that have been carbonized or perhaps fusainized. Sclerenchyma fibers (commonly referred to as bast or plant fibers) are the most likely component of woody plants to exhibit the general shape, size, lack of pitting, thickened outer walls, and undivided elongate forms (Catling and Grayson, 1982) observed in the amber filaments. Although some sclerenchyma fibers used in textiles have comparable mean diameters to those observed in Stage I and II filaments, these are never heavily pigmented, are nearly pentagonal or hexagonal in cross-section with uniformly thick walls, taper at both apices, and exhibit a wide array of apicular morphologies (Von Bergen and Krauss, 1942; Catling and Grayson, 1982). Furthermore, the plant remains we have recognized in Cretaceous ambers from western Canada (McKellar and Wolfe, 2010), particularly those that breach the surface of their encapsulating amber nodule, are generally preserved as carbonized remains that preserve little surface detail at the cellular level.

Comparison to degraded or taphonomically-altered feather remains An alternate interpretation of the Stage II morphotype we describe is that it represents a series of degraded feather rachi that have decayed to the point of exposing their internal filamentous structure. The morphology of such structures has recently been explored (Lingham-Soliar et al., 2010) through biodegradation, using keratin consuming fungi. This has revealed the underlying structure of the rachis, indicating that filaments that once comprised rachi bear distinct nodes directly comparable to those of barbules, quite unlike the filaments recovered from amber. The inferred Stage II clusters could also be construed as a result of poorly-preened feathers, in which the barbs have clumped together. Although this alternative is more difficult to discount, we note that, unlike typical barbs, the filaments that comprise the Stage II clusters we describe possess circular cross- sections, in absence of any indication of a rachis from which they could have originated.

Comparison to pterosaur pycnofibers Pycnofibers are bushy fibers found in association with pterosaur remains: these have an average diameter between 0.2 and 0.5 mm, and are apparently composed of finer fibrils of unknown original structure or composition (Kellner et al., 2009). Compared to UALVP 52821, there is an overlap in the known diameters of the clusters, and they both appear to have sub-centimeter lengths. In the case of pycnofibers, the component fibrils appear to be much more tightly bound, particularly near the apex of the pycnofiber, which makes their distinction much more difficult than the loosely-bound Stage II filaments observed in amber.

Sinosauropteryx prima comparison In terms of compression fossils, the Stage I morphotype filaments observed in Canadian amber are most comparable to protofeathers from Sinosauropteryx prima. The integumentary structures of S. prima display a range of lengths, from ~4 mm to at least 4.0 cm, depending on the specimen and their body position (Chen et al., 1998; Currie and Chen, 2001). These independent filaments range in thickness from easily observed 0.2 mm filaments to those that are considerably smaller than 0.1 mm (Currie and Chen, 2001). The filaments are hollow and round in cross-section (Ji and Ji, 1997) and may have been secondary branches of larger structures (Currie and Chen, 2001) or isolated filaments (Prum and Brush, 2002). Although the UALVP 52821 specimen does not display filaments with diameters as large as the maximum reported from S. prima, they are consistent with the finer filaments found in this specimen, and fall within the range of observed lengths. As with the filaments from S. prima, UALVP 52821 filaments are hollow with circular cross-sections. It must be noted that compression, permineralization, and lack of definition may all have contributed to some degree of distortion of the original dimensions of filaments associated with S. prima. Compression potentially flattens otherwise cylindrical filaments, whereas permineralization may increase the apparent thickness of the outer wall. The lack of definition between individual filaments in S. prima may also yield overestimates of original filament thicknesses.

Sinornithosaurus millenii comparison The UALVP 52822 clustered filaments described as a Stage II morphotype are most similar to compression fossils surrounding Sinornithosaurus millenii. In S. millenii, although there is no direct evidence of a rachis (as with the amber specimens), barbules are clearly clustered into independent tufts with compressed widths of 1-3 mm and lengths of up to 4.5 cm (Xu et al, 2001; Prum and Brush, 2002). These clustered filaments appear to have been attached basally, or in one example, inferred to have arisen from a central rachis (Xu et al, 2001; Prum and Brush, 2002). Although no direct measurements of the filaments that comprise each cluster have been presented by Xu et al (2001) they appear to be of sub-millimeter diameter similar to the filaments observed in amber. As in the Sinosauropteryx prima protofeathers, the clusters found with Sinornithosaurus millenii are likely to have expanded diameters as a result of filament splaying during compression. Their displacement from the body suggests that the clusters associated with S. millenii were not immediately buried (Xu et al, 2001), so the main limitations on the degree of filament splaying would have been the length of time the clusters were allowed to decay, and the rigidity with which the filaments were fixed in the clusters. Additional morphological observation techniques Due to the current rarity of specimens, destructive sampling is not possible with the Canadian amber material (including crack-out studies utilizing scanning electron microscopy). Synchrotron x-ray microtomography has recently demonstrated great promise for studying small-scale inclusions within amber. This imaging technique has demonstrated unmatched resolution of fine structures (Soriano et al., 2010), yet has been unsuccessful in the analysis of amber- entombed hair specimens (Vullo et al., 2010) comparable to the Stage I and II filaments described here, likely as a result of low density contrast. This leaves, beyond light microscopy, confocal microscopy as the primary source of additional data on the Canadian amber specimens (described below).

Chemical comparison to mammalian hairs As mammalian hairs constitute the most similar structures in terms of both overall morphology and preservational characteristics, we sought additional analyses to compare the chemical composition of the Stage I and II morphotypes to hair. The identification of a-keratin or p-keratin in the putative protofeathers would provide strong support for our structural inferences, because these proteins are specific to the integumentary structures of mammals and reptiles, respectively. The presence of P-keratin has been demonstrated in filaments associated with the non-avian theropod Shuvuiia deserti through the use of immunohistochemical responses, measured utilizing P-keratin specific antibodies that were tagged with fluorescent markers and subjected to LSCM (Schweitzer et al, 1999). Such testing is, at present, impossible for specimens such as UALVP 52821 and UALVP 52822 because they do not provide enough volume for analysis, and furthermore cannot be dissociated from the entombing amber matrix. Moreover, the gymnospermous resin has permeated filaments during amber polymerization, which is problematic for such analyses because it is autofluorescent, impermeable, highly insoluble, and contains trace quantities of various amino acids of botanical origin (Poinar, 1992; Langenheim, 2003). Finally, Canadian amber is not readily sectioned as it fractures conchoidally. Taken together, these characteristics temper our expectations for successful immunohistochemical analyses of amber-borne filaments at present, should additional specimens be located to allow destructive sampling. Fortunately, we can interrogate this issue non-destructively with confocal microscopic approaches. Analysis by LSCM and SDCM Given these caveats, we turned to LSCM and SDCM to assess the composition of the filaments. Keratin is known to autofluoresce with a predictable emission profile (Wu and Qu, 2006). This makes possible a comparison of fluorescence patterns amongst UALVP 52821, UALVP 52822, and unambiguous feather fragments within the deposit. Ideally, differences between the excitation and emission profiles of the specimens would permit comparison between these specimens, as well as a wider range of inclusions within the deposit, in order to rule out conclusively the alternative origins for the filaments discussed above. UALVP 52821 was compared to TMP 96.9.997 with both SDCM and LSCM. TMP 96.9.997 is both strongly-pigmented and has completely transparent barbule sections in close proximity to the slide's cover slip. It has a total slide thickness of approximately 2.5 mm, and as one of the thinnest specimens in the feather series is the most likely to produce a clear excitation response from keratin alone. These specimens were exposed to a wide range of excitation wavelengths (405 nm, 488 nm, and 561 nm - UV was not possible due to the pronounced autofluorescence of amber at these wavelengths). The responses of the pigmented keratin, clear keratin, and surrounding amber were contrasted in TMP 96.9.997 and compared to areas of similar visible response in UALVP 52821. Analysis of TMP 96.9.997 illustrated the limitations of this approach, as autofluorescence from the amber was strong at all observed excitation wavelengths. Focusing on keratin within TMP 96.9.997 did not provide an emission profile that was distinguishable from that of the amber in terms of peak values (Figs. A3.10A-C), but the intensity produced by keratin provided additional visibility of anatomical details. When UALVP 52821 was analyzed, an identical pattern emerged (Figs. A3.10D-F), but the background interference from the surrounding amber was much greater (because the total slide thickness in the area sampled was approximately 5 mm). Although these data do not demonstrate conclusively the presence of keratin within either specimen, LSCM imaging confirmed the hollow structure of the filaments in UALVP 52821 (Fig. A3.11). Furthermore, three-dimensional viewing indicated that the pigmented portion of each filament is surrounded by a thin layer that emits with slightly greater intensity than the surrounding amber. This layer may represent either a reflective surface where the specimen has pulled away from the amber, or a region of different composition. The latter appears more likely given the apparent thickness of this feature (3-5 um where measurable). Similar elevated emission intensities were observed from both keratin in TMP 96.9.997 and some narrow fractures within the amber of UALVP 52821; thus the observations are inconclusive. The pigmented layer within UALVP 52821 is readily visible and appears to be thin and nearly circular in cross-section. The internal area bounded by the pigmented layer lacks any visible structures, and appears to have been hollow in life. These observations are strongly supported by instances where the filaments are cross­ cut by either the polished surface of the amber (Fig. A3.1 IB), or the edge of the amber piece itself. Where the filaments are cut cleanly, the hollow core appears as an oblong shape free of structure. This would be expected if the filaments possess a nearly circular cross-section, as their orientation within the amber specimen typically produces oblique sections. Where the filaments breach the surface of the amber nodule, their outlines are rounded and appear circular. If and when additional representatives of the Stage I and II morphotypes are recovered from Canadian amber, we plan to pursue chemical analyses to a much greater extent, particularly once a sufficient archive exists to allow destructive sampling. In the interim, we are open to suggestions for additional techniques from the community.

Detailed descriptions of individual specimens and consideration of taphonomy and preservation UALVP 52821 (Stage I morphotype): UALVP 52821 exhibits complex taphonomy: resin remobilization prior to hardening has sheared off the basal portions of the filaments, and has introduced a series of offsets or micro-faults running through many of them. It also appears as though minor decay and the escape of trapped gasses have resulted in fragmentation of the outer wall in many filaments. This has produced a number of perforations in some of the filaments: these are visible as semicircular incisions of filament margins that correspond to fragments of the outer wall found floating in the amber (Figs. A3.2, A3.4A). The complete margin of some of these holes is also visible in some places (Fig. A3.4A inset), providing a clear indication of the thickness of the outer wall, and confirming the hollow interior of the filaments. Additionally, the filaments appear to have been arranged in rows at the time of inclusion within the amber mass, which may reflect either their original arrangement or clumping within the resin (Fig. A3.2). Their form of preservation, particularly their patchy translucency, is similar to that of both feather remains and a hair fragment recovered from the deposit (Figs. A3.4E, F). The alternation of fine light and dark spots that appears to form a cross-hatch pattern on the surface of some filaments may have a taponomic origin. This pattern is similar to that observed in insect cuticles that have pulled away from the encapsulating amber within the deposit, and does not necessarily indicate genuine primary topography.

UALVP 52822 (Stage II morphotype): The clusters of filaments that run parallel to the longest axis of UALVP 52822 (Fig. 5.1C) interact with a dark drying line, partly obscuring the separation between individual filaments at the apex of the cluster. Also, all clusters breach the exterior surface of the amber nodule, limiting their observed lengths and any potential to observe basal attachments. The filaments within each cluster converge basally, regardless of orientation or their preserved lengths. Within the same amber nodule are a single aphidoid hemipteran, potential insect frass pieces, and a few partial strands of a spider's web.

TMP 96.9.997 and TMP 96.9.1036 (superficially Stage lib morphotype): TMP 96.9.997 is close to, but does not extensively contact a drying line within the amber. This has produced a few small areas where dark staining appears to spread outward from individual barbules. In TMP 96.9.1036, the contact with a drying line is greater, as is the areal extent of the darkened surroundings. Exposure and weathering of the latter specimen explains at least some of the lack of pigmentation in barbules near the apex of the barb (Fig. A3.9).

TMP 96.9.553, TMP 94.666.15 and TMP 96.9.546 (pennaceous barbs): In TMP 96.9.553, resin flow and interaction with a drying line has caused barbules on at least three of the barbs to draw inward toward the ramus. In TMP 94.666.15, barbules near the apex and base of the barb are similarly swept inward due to resin flow (Figs. 5.31, J). In this specimen, interaction with the drying line is fairly extensive, and may have caused the darker color. In TMP 96.9.546, interaction with a drying line has created dark margins surrounding basal barbules (Fig. 5.3H), but has had little other effect. A mite that appears to be a juvenile oribatid (H. Proctor det.) is found in association with TMP 96.9.546, but appears to be within a different flow region in the amber, and not directly associated with the feather fragment. TMP 79.16.12 (down feather): The tuft of downy barbules within TMP 79.16.12 converges basally (Fig. 5.3E), but each is truncated at the edge of the amber nodule. Within the amber nodule are six specimens of the dipteran Adelohelea glabra Borkent, at least 4 partial aphidoid hemipteran specimens, and a few isolated strands of spider web. TMP 96.9.334 (coiled barbules): Although the feather portion preserved within the amber nodule does not appear to encompass any barbs, the presence of specialized barbules and a broad rachis suggest an advanced Stage IV morphotype for TMP 96.9.334 (Figs. A3.6, A3.7). A prominent drying line within the nodule suggests resin flowed toward the apex of the rachis, sweeping many of the barbules inward toward the rachis, and causing some of the barbules to tear free and rotate (their nodes show that they face the opposite direction). Most of these barbules were probably attached to an unpreserved barb ramus that was basal to the preserved section of feather (as the barb ramus is not preserved). There is a fragment of what may be barb ramus preserved on the surface of the amber nodule (Fig. A3.7B), but preservation is too poor for identification. Those barbules that do not terminate on this questionable fragment exit the edge of the amber piece basally with no indication of attaching to the rachis segment (Fig. A3.7). The amber slice that entombs the feather is slightly less than 2.25 mm thick, so it is possible that the window of preservation occurred between barb rami, but this would require a relatively wide spacing of barb rami. Posterior to the microphysid hemipteran in the amber nodule, and well removed from rachis, a second set of barbules splays outward in the opposite direction (Fig. A3.6B). Unless the rachis has completely folded back on itself outside the window of preservation (and against the direction of resin flow), this second set of barbules is difficult to explain as anything other than the remains of a second feather. UALVP 52820 (Stage V, vanedfeather fragment): UALVP 52820 is caught within a large mass of tangled spider's web (Fig. A3.8). To preserve the web, the amber nodule was not polished to a thin wafer. As a result, there are numerous drying lines to contend with. Aside from the feather, only a few potential insect frass pellets are found within the amber nodule.

Additional notes on pigmentation and structure of Canadian amber feather specimens One of the most interesting aspects of the Canadian amber assemblage is the preservation of pigments within the specimens. Pigmentation has recently been described from a number of non-avian theropods (Li et al., 2010; Zhang et al., 2010) and fossil birds (Vinther et al., 2008; 2010; Clarke et al., 2010; Zhang et al., 2010). This work has hinged upon the identification of melanosomes (pigment bodies within organelles) with distinctive shapes and arrangements, through the use of scanning electron microscopy (Vinther et al., 2010). Although preservation is exceptional within amber, examination of the insect assemblage has demonstrated that diagenetic alteration has had a profound effect on the coloration of the insect remains, and techniques such as melanosome observation are likely the only way to precisely identify the original colors of the feather specimens. Unfortunately, it is not possible to subject the Canadian amber to the destructive sampling required to access the melanosomes for SEM examination. This limits the discussion to pigment intensity and distribution, and comparison with works that have mapped these patterns in modern feathers (Chandler, 1916; Dove, 2000).

UALVP 52821 (Stage I morphotype): The filaments of UALVP 52821 exhibit a wide range of diffuse (non-localized) pigmentations, ranging from near-transparency to heavily-pigmented, nearly opaque (Fig. A3.2). Pigmentation along the length of each filament appears to be relatively consistent, but taphonomic influences complicate this observation, and limit any inferences of the original colors. These specimens appear to have ranged in color from near-white (unpigmented) to near-black (heavily pigmented). No large-scale pigmentation patterns, such as banding created by a series of neighboring filaments with similar pigmentation can be inferred, although this may be an effect of the small sample size.

UALVP 52822 (Stage II morphotype): Much of the dark coloration in stage II morphotype specimens (UALVP 52822) is attributable to preserved pigments; however, it is not possible to observe the distribution of pigments within these structures as they are nearly opaque (Fig. A3.5). This, combined with a lack of modern analogues, limits our interpretation to suggesting tentatively a dark brown or black overall color for the filament clusters.

TMP 96.9.997 and TMP 96.9.1036 (superficially Stage lib morphotype): Dark-field microphotography (Fig. A3.9) and comparison between the Canadian amber specimens (TMP 96.9.997 and TMP 96.9.1036) and epoxy-embedded modern feathers shows that the density and distribution of pigments (Chandler, 1916; Dove, 2000) preserved in the fossil material is consistent with a medium- to dark-brown plumage (Fig. A3.12). The ramus and proximal three to four barbule nodes lack or have reduced pigmentation, as do the basal sections of distal barbule nodes (Fig. A3.9B). Within distal barbule nodes, pigment is concentrated in oblong masses, leaving clear nodes in addition to clear bases within the internodes (Fig. A3.9E). Barbules are approximately 6 um in diameter and gradually taper away from their nodes, which appear to bear three elongate (3 um) prongs (Dove, 2000). This barbule type conforms to the reduced plumulaceous barbules described within the basal regions of some contour feathers (Lucas and Stettenheim, 1972), but the barbs and their barbules are present in a sparse and strictly aligned pennaceous pattern that does not match well with observed modern exemplars.

TMP 96.9.553, TMP 94.666.15 and TMP 96.9.546 (pennaceous barbs): In these specimens, the barbules on both sides of each barb are of pennaceous morphology (Lucas and Stettenheim, 1973), with ventral plates (blade-like bases) that gradually narrow and become more cylindrical toward their apices (Figs. 5.3F-K). Barbules range in length from 0.15 mm to 0.35 mm and appear to be composed of 10 to 12 distinct nodes. Barbules on these partial feathers appear to lack differentiation into the smooth proximal and hooked distal series on either side of the barb. The barbules do not correspond well to modern reduced pennaceous morphologies, because barbules on either side of the barb are of relatively even lengths, comparable shapes, and lack hooklets at their nodes. Within one amber piece containing 16 examples of this morphotype (TMP 96.9.553, Fig. 5.3F), the individual barbs appear to converge upon a shared base. These specimens might be identified as a variation on the open pennaceous (non-interlocking) terminal regions of barbs within contour feathers (Lucas and Stettenheim, 1972), but this interpretation would require that the bases of individual barbs were drawn together taphonomically, due to torsion within viscous resin. Pigmentation is present within two of the three specimens with flattened barbs. In both of these specimens, the dorsal flange (cylindrical portion) of the basal internode is darkly pigmented, while the ventral plate bears reduced pigmentation within its ventral margin (Figs. 5.3I-K). Interrupted pigmentation is apparent within many of the ventral plates, reflecting segmentation within the base of each barbule (Fig. 5.3K), as pigmentation is only present within the apical portions of the subsequent internodes. In each of the two specimens that possess pigmentation, its intensity and distribution are comparable to dark brown modern feathers (Chandler, 1916); however, amber thickness and interactions with drying lines within the amber preclude more detailed analysis.

TMP 79.16.12 (down feather): TMP 79.16.12 possesses tufted barbules that lack pigmentation, with thin, flattened internodes (approximately 8 um in width, 18 um in breadth, and 170 um in length) ending in moderately inflated nodes (25 um in diameter) with three weak nodal points (Fig. 5.3E). Individual barbules appear to converge on a short rachis, although none is apparent within the amber itself. Taken together, these features suggest an understory position within the plumage, and the overall appearance of the specimens is similar to that of natal or juvenile down (Lucas and Stettenheim, 1972). These barbules appear transparent, and would have been white in life.

TMP 96.9.334 (coiledbarbules): Pigmentation is diffuse and variable within the barbules of TMP 96.9.334 (Fig. A3.6): the overall color would likely have been pale or white. Interestingly, the basal internodes within each barbule appear to be consistently of a slightly darker color than their apical equivalents. Structurally, these barbules exhibit a form of basal coiling that is analogous to that found in some modern birds, such as sandgrouse, seedsnipes, and grebes. In these modern examples, the coils are used to sequester water within the plumage. This coiling differs significantly from the curled barbule bases observed in many taxa (e.g., Fig. A3.12D), in that the barbules undergo full rotations, and when exposed to water, they straighten, drawing water in by capillary action (Cade and Maclean, 1967). In the modern taxa that exhibit basal coiling, this structure is either used for transport of water to the nest (in sandgrouse, possibly in seedsnipes) or as a means of altering the hydrodynamic properties of the bird, in order to facilitate diving (in grebes) (Cade and Maclean, 1967; Maclean, 1983; Fjeldsa, 2004). Although neither of these groups exhibit as many basal coils as those observed in TMP 96.6.334, grebes appear to exhibit slightly more coils than the other taxa. Grebes possess 2-3 basal coils (Chandler, 1916; Fjeldsa, 2004; Figs. A3.12F-G), while sandgrouse possess 1.5-2 full coils (Maclean, 1983), and seedsnipes have less developed basal coiling (Cade and Maclean, 1967). The basal coils in TMP 96.9.334 may have served either of the functions known in the modern avifauna, but the high number of coils suggests that they are more likely to have been employed in diving behavior, as they would have sequestered a comparatively large volume of water.

UALVP 52820 (Stage V, varied feather fragment): The preserved feather section of UALVP 52820 is entombed within a thick piece of amber and crosses multiple drying lines, making color observations difficult. Transmitted light microphotographs (Fig. A3.8) reveal a banded pattern of dark pigmentation within the basal plate and diffuse dark pigmentation within the pennulum, suggesting perhaps a grey or black feather (Chandler, 1916). Although this is only a partial feather, the ramus (barb shaft) is expanded dorsoventrally, with a distinct dorsal ridge bordered by ledges, a characteristic of rami adapted to form strong vanes for flight (Lucas and Stettenheim, 1972). Furthermore, distal series barbules each display a distinct, narrow pennulum, and a moderately elongate, narrow ventral tooth on the apex of a broad basal plate. These are adaptations for interlocking with adjacent barbs to form a vane (Lucas and Stettenheim, 1972). Fig. A3.1 Graph of specimen diameters for filamentous structures (Stage I and II) and barbules in Canadian amber, compared to other possible sources. Circles indicate mean value, vertical lines 1 SD, boxes show observed ranges or reported ranges for majority of specimens (Hudson, 1986), and arrows indicate ranges beyond graph area accompanied by maximum value.

UALVP 52821 filaments (n=70)

UALVP 52822 filaments (n=28) +o- Guard hair survey (n=75) -o- Underhair survey (n=161) -o ->• 680

Fungal hyphae range -*-1000 TMP 96.9.997 barbule range • TMP 79.16.12 barbule range

I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—J- 0 50 100 150 200 Diameter (urn) Fig. A3.2 Photomicrographs of Stage I filaments in UALVP 52821. (A) Field of individual filaments cut obliquely, illustrating distribution of filaments; (B) close-up of boxed area within A, showing apparent grouping of filaments (arrow) and color variation between filaments when illuminated from above.

201 Fig. A3.3 Compound microscope images (b.f.) of Stage I filaments in UALVP 52821. (A) Area where filaments are truncated by outer surface of amber nodule (pebbled amber surface in upper-right of figure), arrow indicates one of the faults running through the filaments; (B) hollow central region of a filament (arrow); see figures A3.4 and A3.11 also. Fig.A3.4 Dissecting and compound microscope images of Stage I filaments, fungi, and mammalian hair. (A) Degraded portion of Stage I filament apex in UALVP 52821; vertical arrows indicate regions where there are holes in the outer wall, angled arrows indicate pieces of the outer wall floating within the amber; inset shows holes with complete outlines at double the magnification of A (d.f.); (B-D) apparent surface texture of Stage I filament in UALVP 52821, (B) filament adjacent to arrow displays faint cross-hatching pattern of light and dark areas, (C) filament adjacent to arrow displays clearer cross-hatching, perhaps as a result of a nearby bend in the filament (d.f.), (D) multiple filaments display faint texture where they have pulled away from the surrounding amber (d.f.); (E) TMP 96.9.998, mammalian hair from Canadian amber, with thick cortex and discontinuous medulla, most likely displaying a multiserial ladder or aeriform lattice pattern adjacent to arrow (b.f.); (F) TMP 96.9.998, showing faint traces of cuticular scales adjacent to arrows (d.f); (G) mat of fungal hyphae (white filaments near bottom of image) contrasted against pair of Stage I filaments (larger, dark filaments near top of image) in UALVP 52821. Fig. A3.5 Compound microscope images (b.f.) of Stage II clusters in UALVP 52822. (A) Distal tip of cluster in Fig. IC, showing tapered apices of filaments and loose bundling within a cluster, also with apparent dark, diffuse pigmentation; (B) proximal truncation of cluster in Fig. IC, showing tightly adpressed filaments at point where cluster is cross-cut by the edge of the amber nodule (arrow); (C) loose bundling apparent within other clusters in the same piece of amber, these clusters are more obliquely oriented within the nodule, and may show variable pigmentation within filaments (toward upper-left of figure). Fig.A3.6 Compound microscope images of coiled barbules (TMP 96.9.334). (A) Specimen overview showing coiled barbule bases (predominantly within the lower left of figure) surrounding thick, flattened rachis (arrow); reddish-brown areas are the result of a prominent drying line within the amber (b.f); (B) oblique section through cluster of coiled barbules surrounding a microphysid hemipteran, with portions of second feather posterior to microphysid (TMP 96.9.334, microphotograph); (C) straight apical barbule sections exhibiting variable diffuse pigmentation (b.f); (D, E) close-ups of straight barbule nodes and intemodes, showing flattened intemodes that twist slightly along their length and exhibit a linear pattern as a result of either ultrastructure or pigment granule distribution (b.f).

205 Fig. A3.7 Dissecting microscope images of coiled barbules (TMP 96.9.334). (A) Specimen overview (opposite to Fig. A3.6A), showing coiled barbule bases (predominantly within lower, central part of figure) surrounding thick, flattened rachis (vertical arrows); base of rachis (lower arrow) recessed with respect to surface of amber piece as a result of weathering; reddish-brown areas are the result of a prominent drying line within the amber; (B) close-up of rachis base, vertical arrow indicates fragment of possible barb ramus that is too poorly preserved to permit confident identification, inclined arrows indicate a few of the many individual barbules that exit the edge of the amber piece without making contact with the rachis (this lack of attachment appears to be characteristic of most of the barbules, although they are crowded basally).

0.5 mm

206 Fig. A3.8 Compound microscope images of differentiated barbules with distinct pennulae in UALVP 52820, indicating preservation that is visually identical to Stage I and II morphotypes. (A) Isolated barb with differentiated barbules and thickened barb shaft ensnared in spider web (microphotograph) (B) overview of barbules near base of barb, and surrounding spider web, (b.f); (C) overview of barbules near distal tip of barb, with clearly defined distal and proximal barbule series (left and right sides of ramus, respectively), distinguished by the sharp transition between the base and pennulum within the distal series barbules (arrow), (b.f); (D) close-up of proximal barbule, showing distribution of pigmentation, and nodal prongs, (b.f); (E) close-up of distal barbule, showing distribution of pigmentation, nodal prongs, and ventral tooth upon basal plate (arrow) adjacent to abrupt transition into pennulum (b.f). Fig.A3.9 Compound microscope images of pennaceous barbs with reduced plumulaceous barbules. (A) Overview of pigmented barb, TMP 96.9.997 (b.f); (B) close-up of boxed area in A, showing weak ramus and unpigmented basal barbules, as well as distribution of pigment within subsequent barbules (b.f); (C) dark-field image of same feather region, showing apparent feather color created by pigmentation, as well as distribution of pigmentation within barb components; (D) overview of variably pigmented barb with elongate ramus tip, TMP 96.9.1036, (b.f., micro-panorama compiled using Helicon Focus); (E) close-up of pigment distribution within basal barbules of D. Fig.A3.10 SDCM and LSCM data for Stage I morphotype and TMP 96.9.997 - emission response microphotographs and emission spectra. (A) Autofluorescence of TMP 96.9.997 when exposed to laser based excitation at 491 nm (green, filter for emission wavelength et525/50) and 561 nm (red, filter for emission wavelength et620/60), showing marginally brighter spots where only keratin is preserved; (B) normalized emission spectrum for sampling points on TMP 96.9.997 when excited at 405 nm, emission from amber (ROI 1) peaks between 480—490 nm, similar, but progressively more muted peaks for unpigmented keratin (ROI 3) and pigmented regions of barbule (ROI 2); (C) map of sampling points and emission intensity between 540 and 550 nm TMP 96.9.997; (D) autofluorescence of UALVP 52821 when exposed to laser based excitation at 491 nm (green) and 561 nm (red); (E) emission spectrum for sampling points on UALVP 52821 when excited at 405 nm, emission from amber (ROI 1) peaks broadly near 540 nm; similar, but progressively more muted peaks for unpigmented outer wall of filament when cut obliquely (ROI 2); unpigmented outer wall of filament when cut longitudinally (ROI 4); and pigmented layer (ROI 3); (F) map of sampling points and emission intensity between 540 and 550 nm in UALVP 52821, arrow indicates rounded outline produced where filament breaches surface of amber piece (the pebbled surface at the lower right of the image, also visible in Fig. A3.10D).

jnmj 11=775 «2 = 775 d* = 0 |m«I B £ Fig. A3.ll LSCM and additional photomicographs of UALVP 52821. (A) Three-dimensional scan of UALVP 52821 at 405 nm excitation (mapping emission intensity between 411 run and 766 nm); fine white lines correspond to vertical section planes presented in panels to the left of and below the main figure. Arrow indicates circular cross-section of one filament apex, directly comparable to B. Brackets delimit filaments cut obliquely, demonstrating outer wall (bright) surrounding thin pigmented layer (dark) and hollow core (comparable to the surrounding amber), this pattern is also found within longitudinal sections of the filaments within this piece of amber. (B) Dissecting microscope photomicrograph of filaments in the same region of the amber specimen as A, illustrating appearance of filaments when sectioned obliquely along polished surface, arrows indicate oblong internal voids exposed at the section plane.

0.1 mm Fig.A3.12 Photomicrographs of modern bird feathers for comparison of barbule structure and pigmentation patterns. (A) Plumulaceous barbules from the afterfeather of a pheasant (b.f); (B) dark-field image of A, showing pigment distribution and resulting dull-brown coloration; (C) close-up of barbules in A, showing pigment concentration near nodes - although somewhat more diffuse, this is comparable to pigmentation in Fig. A3.8 (b.f.); (D) partially curled barbule bases in the plumulaceous basal barbs within a body contour feather of a kiwi {Apteryx owenii, RM 5440), for comparison to coiled barbule bases in Figs. A3.6 and A3.7, and also an example of diffuse pigmentation (b.f.); (E) single barb from white belly feather of a grebe (Aechmophorus occidentalis, RAM Z279.78.2), illustrating coiled barbule bases, predominantly with two basal coils (dissecting microscope); (F) combined focal-plane image of different barbule from same feather as E, providing overview of coiling (d.f.); (G) single image of the barbule bases that are partly obscured in G, due to the orientation of the barbules (d.f).

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Langenheim, J.H., 2003. Plant Resins: Chemistry, Evolution, Ecology, and Ethnobotany. Timber Press, Portland, pp. 23-50. Li, Q., Gao, K.-Q., Vinther, J., Shawkey, M.D., Clarke, J.A., D'Alba, J., Meng, Q., Briggs, D.E.G., Prum, R., 2010. Plumage color patterns of an extinct dinosaur. Science 327, 1369-1372. Lingham-Soliar, T., Bonser, R.H.C., Wesley-Smith, J., 2010. Selective biodegradation of keratin matrix in feather rachis reveals classic bioengineering, Proceedings of the Royal Society of London B: Biological Sciences 277, 1161-1168. Lucas, A.M., Stettenheim, P.R., 1972. Avian Anatomy: Integument. US Department of Agriculture, pp. 235-279. Maclean, G.L., 1983. Water transport by sandgrouse. BioScience 33, 365-369. McKellar R.C., Wolfe, A.P., 2010. Canadian amber, in: Penney, D. (Ed.), Biodiversity of fossils in amber from the major world deposits. Siri Scientific Press, Manchester, pp. 149-166. Nascimbene, P., Silverstein, H., 2000. 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Zhang, F., Kearns, S.L., Orr, P.J., Benton, M.J., Zhou, Z., Johnson, D., Xu, X., Wang, X., 2010. Fossilized melanosomes and the colour of Cretaceous dinosaurs and birds. Nature 463,1075-1078. APPENDIX 4: Supplementary Table of 8I3C data from resins and ambers Tree or Sample Resin description Notes S"C%» amber ID (VPDB1 piece ID

_2fIPl S MPR ™M™{ sanylp Onlfected -Inlv 1T> yel Whole sample used (small sample)

BC-2 6096 Pale yellow; slightly granular Collected adjacent to gall on branch -28.1

BC-6 6098 Pale yellow; minor particulates; somewhat tacky -27.4 BI-2 old 6100 Dark orange, clear; crystalline; prominent weathering Sample obtained beneath weathering surface -28.2 resin surface Wood fragment removed -27.6 BC-5 6101 Pale yellow; tacky; minor-moderate particulates Combined resin from two flows to obtain necessary sample -28.4 BC-1 6106 Pale yellow; granular; minor particulates; partly tacky weight Small sample -28.3 BC-44 6108 Pale yellow; minor particulates; granular but slightly tacky

_2ft BinPR infesteSa d sampleP fs (collected July 1fT>: fF W ^le lerale iryellowt ; some particulates; faint drying surface May be splash-down from neighbouring tree, (reanalysis ?2%A confirmed) BI-2 6097 Pale yellow; granular ext; some minor particulates -26.7

BI-6 6099 Milky yellow; clean; bubble clouded -27.3

BI-5 6104 Pale yellow; mostly granular; minor-moderate Wood fragment removed, whole sample used -27.4 particulates BI-4 6105 Pale yellow; granular; minor particulates; partly tacky Dark red previous flow trimmed off of underside -25.6

BI-1 6107 Pale yellow; granular; minor particulates; slightly tacky Older resin flow from trunk, rerun with fresh material below -27.6 (lb) BI-lb 6214 Medium yellow; slightly granular; minor particulates; Small resin sample, directly from trimmed branch surface, -26.3 very tacky minor surface patina removed 2009 MPB'control' samples (collected August 16^: "3SX DC-1 6145 Transparent; tresh, ran heavily in vial Small diameter tree -28.7 DC-2 6147 Pale yellow; fresh flow Droplet string on trunk surface -27.4 DC-4 6149 Medium yellow; partially set clear flow Tree trunk with long longitudinal gash, droplet string on trunk -28.6 DC-5 6151 Medium yellow; partially set flow surface From a pitch out tube -27.3 DC-6 6153 Transparent; fresh flow; very runny From trunk surface -27.2 BC-16 6155 Transparent; fresh flow; very runny -28.6 BC-8 6159 Pale yellow, clear; fresh flow From fresh wound, stringer of droplets, slightly oxidized but -27.4 BC-10 6163 Medium yellow, clear still tacky Foliage bordering upon yellowing, from a fresh wound -27.8 BC-11 6165 Pale yellow, clear; runny (BC-13 is dead, replacement neighbour) crown restricted to very -28.3 CC-13 6167 Dark yellow, clear; tacky but very viscous; droplet high up, otherwise healthy YELLOWING tree, droplet stringer from longitudinal gash -26.6 BC-7 6157 Medium yellow; fresh flow YELLOWING tree, runnel of drips from trunk -26.2 BC-9 6161 Pale/medium yellow, clouded; slightly crystalline and surface-oxidized 20009 MPB 'infested' ssample s (collected August 16^: DI-5F1 6146 Mediuleaium yellow; clear Fresh from pitch out tube, "38"T

DI-2 6148 Medium yellow; clear From trunk wound, may be slightly aged -28.7

DI-4 6150 Partially cloudy trunk and pitch flows pale, crystalline Large diameter tree, limited infestation, -28.6 with low viscosity component and no particulates DI-5 6152 Pale yellow; clear; droplets (near B-10) -28.8 BI-16 6156 Clear; very runny Fresh flow from pitch-out tube -28.0

BI-8 6160 Medium yellow; slightly milky; very runny Fresh flow from pitch-out tube -27.5

BI-9 6162 Pale yellow; very clear Fresh flow from pitch-out tube on trunk -26.8

BI-11 6166 Pale yellow; clear; runny YELLOWING tree; from fresh wound -26.2

BI-13 6168 Medium yellow; clear droplet, slightly thickened but still YELLOWING tree; from fresh trunk wound -26.4 tacky DI-6 6154 Honey-coloured; cloudy but moderately fluid RED tree; fresh flow from pitch-out tube; (tree 20 m from B-7 -23.2 end of transect)

Edmonton samples, root-hailed nursery trees, originally from Niton Junction, Algeria: PC-1 6206 Pale yellow; clear; moderately tacky Frorombasm b e of trunk TTT PC-2 6207 Transparent; clean; runny From base of trunk -26.0

PC-3 6208 Milky; granular, very tacky From base of trunk; trimmed of organic particulates and -26.0 weathering crust PC-4 6209 Pale yellow; clear; moderately tacky From base of trunk -27.1

PC-5 6210 Pale yellow; milky/granular; slightly pliable, not very From base of trunk -26.7 tacky PC-6 6211 Medium yellow; granular/crystalline; minor particulates From base of trunk; mostly solidified older resin flow -26.4 PC-7 6212 Medium orange; crystalline; clear From base of trunk; surface patina removed -27.9

New Jersey Amher samples tTiironian, T,ate Cretaceous^:

Clear DropletsiTjRckgrpund production under a wide range of conditions): Barren -23.2

NJ-5 5926 Golden yellow; flow fragment Barren, minor surface patina removed -21.3 NJ-6 5927 Golden yellow; droplet tip, spherical Barren -20.5

NJ-7 6112 Pale yellow, clear; thin droplet shaft Barren -22.7

NJ-10 6115 Medium yellow, clear; drip tip Barren -22.6

NJ-12 6117 Medium yellow; droplet tip Barren, small sample -22.1

NJ-13 6118 Light red; slightly granular; multi-flow end Barren -24.8

NJ-15 6120 Deep red, clear; massive flow fragment Barren, weathering rind removed -22.7

NJ-16 6121 Medium red, clear; massive flow Barren, weathering rind removed -21.2

NJ-17 6122 Medium red, clear; massive flow Barren, weathering rind removed -21.3

NJ-20 6125 Medium yellow, clear; slab flow Botanical surface impression, sampled to avoid impression -23.5

NJ-21 6126 Medium yellow, clear; slab flow? Wood fragments, otherwise barren; sampled to avoid inclusions -23.8

StNJ-6b 6073 Pale yellow, clear; slab flow? Barren -21.3

StNJ-7 5985 Pale to medium yellow; runnel or drip fragment Barren -23.1

StNJ-8 5986 Medium yellow; drip fragment Barren -21.0

StNJ-10 5988 Medium yellow; runnel fragment Barren -22.0

Turbid Amber (contains almost all inclusions, makes up almost 70% of deposit, putatively .attack-relatedJi: NJ-1 5922 Butterscotch colour; turbid; numerous larger wood chips Mite (Acan: Erythraeidae?), midge (Diptera: -21.9 Ceratopogonidae?), sampled to avoid inclusii NJ-18 6123 Medium orange; massive flow; slightly granular Botanical inclusion (indeterminate) -21.4

StNJ-9 5987 Deep orange; runnel fragment Botanical inclusion (indeterminate) -22.8 StNJ-lb 6068 Rich orange; multi-flow runnel Midge (Diptera: Chironomidae?) -22.9

StNJ-2b 6069 Caramel; multi-flow runnel; some larger particulates Obscured hemipteran or thysanuran -21.6

StNJ-3b 6070 Deep yellow; slab flow; numerous particulates Hemipterans (3) -22.9

StNJ-5b 6072 Deep reddish-brown Indeterminate partial appendage, bubble inclusions, specimen -22.6 semi-polished NJ-19 6124 Medium yellow; droplet tip, multi-flow fragment?; minor Barren -21.0 particulates StNJ-4b 6071 Deep orange; multi-flow runnel; particulate-rich Barren -20.1

Op#°.ue Amber (bubble clouded, bone-grade amber.putatiyely fire-related or affected):, ,„,,.,,,,, ... , , , NJ-ll 6116 Medium yellow, clear; droplet tip, multi-flow fragment? Surface densely frothed with bubbles, material beneath analyzed -20.6

NJ-3 5928 Butterscotch Heavily clouded with bubbles and wood fragments -20.2

NJ-14 6119 Medium orange; massive flow piece? Heavy external rind of bubbles, material beneath analyzed -21.0

Dominican amber samples (Miocene):

Barren 'scrap' amber (may potentially be,trimmings from pieces with inclusions): Dom-7 6136 Medium orange, multi-flow Barren? -26.5

Dom-9 6138 Rich orange; heavy surface patina; massive flow fragment Barren?, trimmed of patina -24.9

Dom-15 6144 Yellow-orange; multi-flow fragment; minimal particulates Barren except for bark fragment and frass -26.8

Dom-4 5935 Pale yellow Hymenoptera: Apidae -26.8

Dom-5 5956 Honey yellow Isolated insect appendages -23.3 Dom-6 5957 Honey yellow Hymenoptera: Formicidae?, indeterminate appendages -24.4

Dom-10 6139 Medium yellow, multi-flow fragment Hymenoptera: Formicidae, Coleoptera, Diptera, indeterminate -23.1 larvae (2) Dom-11 6140 Rich orange; multi-flow fragment; deep oxidation on one side Hymenoptera: Ichneumonoidea and fungal hyphae -28.0 Dom-13 6142 Rich orange; multi-flow fragment Disarticulated Trichoptera?, frass -23.6

Dom-14 6143 Medium orange; multi-flow fragment; minor particulates Posterior half of indeterminate insect, frass -25.3

Dom-8 6137 Yellowish-orange, multi-flow fragment Clastic matrix around specimen avoided -24.4

DomIso-2 6170 Pale orange Thysanura and Coleoptera: Curculionidae: Platypodinae -26.2

DomIso-6 6174 Medium yellow Thysanura -24.5

DomIso-7 6175 Deep orange Botanical inclusion (Fabaceae: Acacia?) -24.3

Wood-boring beetle inclusions: Coleoptera: Curculionidae: Platypodinae, mite (Acari) -23.4 Dom-1 5952 Honey yellow Coleoptera: Curculionidae: Platypodinae, mite, wood debris -24.8 Dom-2 5953 Deep yellow, multi-flow fragment Coleoptera: Curculionidae: Platypodinae, larger wood fragments -25.5 Dom-3 5954 Deep yellow; two flows Coleoptera: Curculionidae: Scolytinae -22.9 DomIso-3 6171 Pale yellow Coleoptera: Curculionidae: Scolytinae -22.8 DomIso-4 6172 Pale yellow Coleoptera: Curculionidae: Scolytinae -25.2 DomIso-5 6173 Deep orange Coleoptera: Curculionidae: Platypodinae -24.5 DomIso-8 6176 Medium orange Coleoptera: Curculionidae: Platypodinae -22.6 DomIso-9 6177 Medium yellow Domlso-10 6178 Pale yellow Coleoptera: Curculionidae: Platypodinae (2 syninclusions) -22.1

Domlso-ll 6179 Deep yellow Coleoptera: Curculionidae: Platypodinae (8 syninclusions) -22.5

Hymenaea resin and copal:

Modern Hymenaea courbaril resin, commercial samples from Brazil: Minor oxidation rind removed; few bubble inclusions -27.5 Hym-1 6195 Honey-yellow; clear Minor oxidation rind removed; few bubble and frass inclusions -28.1 Hym-2 6126 Honey-yellow; clear Minor oxidation rind removed -28.4 Hym-3 6199 Honey-yellow; clear Minor oxidation rind removed; minor particulates present -29.2 Hym-4 6201 Honey-yellow; clear Minor oxidation rind removed -26.9 Hym-5 6203 Honey-yellow; clear Minor oxidation rind removed; minor particulates present -28.8 Hym-6 6205 Honey-yellow; clear

Sub-recent Colpmbian copal (estimated <250.years old) from Hymenaea sp.: Sampled beneath polished surface; branch segment impression, -27.9 bubble inclusions Cop-1 6173 Pale yellow, clear; massive flow Sampled beneath polished surface; many Isoptera, bubble and -27.1 Cop-2 6174 Pale yellow, clear; massive flow frass inclusions Sampled beneath polished surface; Isoptera (3) -27.4 Cop-3 6175 Pale yellow, clear; multiple flows Sampled beneath polished surface; partial Coleoptera and -28.4 Cop-4 6176 Pale yellow, clear; laminar flow Aranaea, numerous wood fragments

Cop-5 6177 Pale yellow, clear; massive flow Sampled beneath opaque rind; few plant fragments and bubbles -27.0

Cop-6 6178 Pale yellow, clear Sampled beneath polished surface; many bubble inclusions, -27.2 partial isopteran? FJFfects. nfheaiinpflynwnapa raurharil resins until no further (measurable) vnla^es are shed: fym-C-1 6r95 Honey-yellow Listed as sample Hym-1 above, control. -27.5

Hym-H-1 6196 Honey-yellow clear Other half of Hym-C-1, heated beyond melting under vacuum, -27.6 until no further volatiles were released Hym-C-2 6126 Honey-yellow clear Listed as sample Hym-2 above, control. -28.1

Hym-H-2 6127 Honey-yellow clear Other half of Hym-C-2, heated beyond melting under vacuum, -28.4 until no further volatiles were released Hym-C-3 6199 Honey-yellow clear Listed as sample Hym-3 above, control. -28.4

Hym-H-3 6200 Honey-yellow clear Other half of Hym-C-3, heated beyond melting under vacuum, -28.3 until no further volatiles were released Hym-C-4 6201 Honey-yellow clear Listed as sample Hym-4 above, control. -29.2

Hym-H-4 6202 Honey-yellow clear Other half of Hym-C-4, heated beyond melting under vacuum, -29.1 until no further volatiles were released Hym-C-5 6203 Honey-yellow clear Listed as sample Hym-5 above, control. -26.9

Hym-H-5 6204 Honey-yellow clear Other half of Hym-C-5, heated beyond melting under vacuum, -27.3 until no further volatiles were released Hym-C-6 6205 Honey-yellow clear Listed as sample Hym-6 above, control. -28.8

Hym-H-6 6206 Honey-yellow clear Other half of Hym-C-6, heated beyond melting under vacuum, -28.8 until no further volatiles were released

Notes: Sample abbreviations are as follows: for trees within the 2008 MPB transect, "BC" and "BI" are prefixes that respectively denote resin from control and infested trees within numbered pairs tagged by Alberta Sustainable Resource Development; within the 2009 MPB transect, "BC" and "BI" remain the same, but are supplemented by "CC" (a replacement neighbour chosen for a dead control tree), and "DC" and "DI" (additional control and infested trees along the transect tagged by Alberta Sustainable Resource Development or identified by R.C.M. in the field); within the New Jersey amber samples, "NJ" samples are commercial samples held by the lab of A.P.W. while "StNJ" samples are those of the University of Alberta Strickland Entomology Museum; within the Dominican amber samples, "Dom" identifies commercial samples from the La Toca mine (Santiago area), while the "Domlso" series are commercial specimens from the vicinity of Santiago.