A M ERIC AN MUSEUM NOVITATES Number 3908, 37 pp. September 28, 2018 Biological Inclusions in Amber from the Paleogene Chickaloon Formation of Alaska DAVID A. GRIMALDI,1 DAVID SUNDERLIN,2 GEORGENE A. AAROE,2 MICHELLE R. DEMPSKY,2 NANCY E. PARKER,2 GEORGE Q. TILLERY,2 JACLYN G. WHITE,2 PHILLIP BARDEN,3 PAUL C. NASCIMBENE,1 AND CHRISTOPHER J. WILLIAMS4 ABSTRACT The Chickaloon Formation in south-central Alaska contains rich coal deposits dated very close to the Paleocene-Eocene boundary, immediately beneath which occur dispersed nodules of amber along with abundant remains of Metasequoia, dicots, and monocots. The nodules are small (less than 10 mm in length), nearly 10,000 of which were screened, yielding several inclu- sions of fungi and plant fragments, but mostly terrestrial arthropods: 29 specimens in 10 orders and 13 families. The fungi include resinicolous hyphae and a dark, multiseptate hyphomycete. Plants include wood/bark fragments and fibers, and the microphylls of a bryophyte (probably a moss, Musci). Among the arthropods are arachnids: mites (Acari: Oribatida), Pseudoscorpi- onida, and the bodies and a silken cocoon of spiders (Araneae). Insecta include Blattodea, Thysanoptera, Hemiptera (Heteroptera and Aphidoidea), Coleoptera (Dermestidae: Megatomi- nae), Trichoptera, Diptera (Chironomidae: Tanypodinae), and Hymenoptera (Formicidae: For- micinae). Nymphal aphids predominate (65% of the arthropod individuals), which were probably feeding on the source tree, likely Metasequoia. There is a bias in preservation toward small arthropods (mean body length 0.75 mm) that are surface-dwelling (nonwinged) stages and taxa. Chickaloon amber contains the most northerly fossil records of pseudoscorpions, thrips, Dermestidae, and Cenozoic ants and mites, so the deposit is contributing unique data on high-latitude paleodiversity of the Paleogene hothouse earth. 1 Division of Invertebrate Zoology, American Museum of Natural History, New York. 2 Department of Geology & Environmental Geosciences, Lafayette College, Easton, Pennsylvania. 3 Department of Biology, New Jersey Institute of Technology, Newark. 4 Department of Earth & Environment, Franklin & Marshall College, Lancaster, Pennsylvania. Copyright © American Museum of Natural History 2018 ISSN 0003-0082 2 AMERICAN MUSEUM NOVITATES NO. 3908 INTRODUCTION The late Paleocene through early Eocene is biotically one of the most significant periods of the Cenozoic. The earth was in a hothouse climate phase with ice-free poles, having an equitable global climate with a shallow latitudinal temperature gradient evidenced by the mean annual temperature (MAT) of ~5° C. Earth’s temperature gradually rose through the Paleocene, then spiked at the Paleocene-Eocene Thermal Maximum (PETM) (Bains et al., 1999; Katz et al., 1999), an event lasting some 200,000 years around 55 Ma, with global tem- peratures rising by some 5°−8° C. Approximately half the species of benthic foraminiferans became extinct during this event, and there was profound turnover of Paleocene mammal faunas into the radiation of modern orders in the Early Eocene (Gingerich, 2003; McInerney and Wing, 2011). This brief episode is correlated with a massive infusion of isotopically negative carbon into the biosphere, called the Carbon Isotope Event (CIE). Its cause is debated: volcanism (e.g., North Atlantic Volcanic Province); release of huge stores of meth- ane from slumping of continental shelves (Bains et al., 1999); and possibly even cometary impact. After the PETM subsided, temperatures gradually rose to another Cenozoic peak at the Early Eocene Climatic Optimum. One of the most compelling examples of the global hothouse climate derives from the Early to middle Eocene Eureka Sound Group of Ellesmere and Axel Heiberg Islands in the Canadian arctic (Eberle and Greenwood, 2012). At approximately 75° N paleolatitude (just a few degrees south of the current latitude), there existed lush forests of ferns, conifers, birches, oaks, and ginkgo, inhabited by lizards, turtles, alligators, early tapirs, and plagiome- nid mammals similar to flying lemurs, among many others. Seasonal ranges are estimated from 2°−3° C to 20° C and an annual precipitation of 120 cm/yr (Eberle and Greenwood, 2012). Similar conditions, including mangrove swamps, were circumpolar at approximately the same paleolatitudes in present-day Siberia (Suan et al., 2017). Not only did the polar warmth facilitate dispersal and spread of Laurasian biotas, but, given the direct and well- established relationship between area and species diversity (MacArthur and Wilson, 1963; Lomolino, 2000), the expansion of earth’s habitable landmass must have vastly increased the diversification of all life, not just mammals. Another biotic event of substantial biological significance around the Paleocene-Eocene transition, and well into the Eocene, was the deposition of massive quantities of tree resin, which fossilized into amber. Perhaps stimulated by the warm overall paleoclimate, the Eocene is arguably the most prolific geological period for amber, resulting in huge deposits, for exam- ple, on the southern Baltic (Larsson, 1978; Weitschat and Wichard, 2010), in India (Rust et al., 2010), and elsewhere. The effects on ecosystems were not inconsequential (large quantities of unrecycled carbon are locked in these amber deposits), but the really important consequence of all this amber is paleontological, providing an archive of delicate, highly diverse ancient life preserved with unmatched microscopic fidelity. Here we report biological inclusions in the most northerly deposit of fossiliferous amber from the Cenozoic, a finding with implications for biogeography and paleoclimatology. 2018 GRIMALDI ET AL.: PALEOGENE BIOLOGICAL INCLUSIONS IN AMBER 3 149˚ 03.75’W 149˚ 02.5’ 149˚ 00’ 148˚ 57.5’ 148˚ 55’ 148˚ 52.5’ W 61˚ 45’N 700 600 N COLLECTION SITE 400 500 400 Coyote Lake 400 Evan Jones Mine M4o0C0orseee k Wishbone 500 672 WISHBONE HIL60L0 Slipper3 00 Eska Creek KnCoreb ek 300 BuffCraleoe k 50L0ake531 400 500 Lake200 200 d. 300ALASKA 300 e R 200 Area enlarged 396 Seventeen 200 nesvill Mile Lake 200 Jo SUTTON To 200 Palmer Glenn Highway 61˚ 42.5’N FIG. 1. Chickaloon amber collection site in the reclaimed Evan Jones Mine. Contour intervals in meters. Geologic and Paleoenvironmental Setting The field collections of dispersed amber in this study were recovered from an exposure of the upper Chickaloon Formation in the reclaimed Evan Jones Mine on the north slope of Wishbone Hill near Sutton, Alaska (61°44.5′N/148°56.4′W) (fig. 1). The Chickaloon Formation is exposed there and elsewhere throughout the east-west trending Matanuska Valley in south- central Alaska and overall comprises a ~1500 m thick sequence predominantly made up of sandstones, mudrocks, and coals of Late Paleocene/Early Eocene age (Flores and Stricker, 1993; Trop et al., 2003; Neff et al., 2011). Zircon fission track and K/Ar dates on ash partings in the upper Chickaloon Formation at the collection site provide age constraint, placing the Paleo- cene-Eocene boundary within the Chickaloon’s Premier Coal Zone (Triplehorn et al., 1984; Flores and Stricker, 1993) (fig. 2). The overlying Early Eocene Jonesville coal zone in the Chick- aloon is in conformable contact with Eocene-age Wishbone Formation conglomerates exposed at the top of Wishbone Hill (Trop et al., 2003; Neff et al., 2011). The ammonoid-bearing, Late Cretaceous Matanuska Formation unconformably underlies the Chickaloon Formation and represents marine conditions before regional regression and basin fill. Trop et al. (2003) provided a depositional model of the Matanuska Valley/Talkeetna Moun- tains forearc basin, indicating progressive basin fill during which Chickaloon sediments were deposited in floodplain fluvio-lacustrine conditions that gave way to estuarine depositional settings along the basin axis to the southwest. Based on geological and paleomagnetic data, the Chickaloon was likely deposited near its present latitude (~62° N) (see Sunderlin et al., 2011). Site-specific studies at the Evan Jones Mine by Flores and Stricker (1993) and Neff et al. (2011) described facies associations interpreted as meander-channel, crevasse-splay, and floodplain- mire depositional environments. The dispersed amber described in this study was recovered 4 AMERICAN MUSEUM NOVITATES NO. 3908 Contact with overlying Wishbone Formation KEY Covered transitional Conglomerate interval of interbedded sand and conglomerate X-bedded sand Siltstone Coals in the Shale Jonesville Coal Zone Carbonaceous sand Coal/coaly shale Channel sands Black Zone Grey Zone Base of current terraced exposure Covered interval – mining reclamation – most of Premier Coal Zone m 5 Dispersed amber in coaly shales 2 Base of exposed Chickaloon Fm. section FIG. 2. Generalized stratigraphy of the upper Chickaloon Formation at the reclaimed Evan Jones Mine (modified from Neff et al., 2011). Amber collections were made from coaly shales near the base of the accessible succession. from a single horizon (fig. 2) of coaly mudrock lithofacies in close proximity—but not attached to—permineralized cupressaceous (Metasequoia) wood (Williams et al., 2010). Other paleofloral remains in carbonate-cemented mudrocks and fine sandstones of the upper Chickaloon Formation include abundant Metasequoia shoots and cones, leaves of dip- terid ferns and the monocot Haemanthophyllum, Equisetites axes, rare fragments of putative palm fronds, numerous types of reproductive bodies with affinities to those of the modern Platanus and Acer (among others), and a diverse magnoliid and eudicot foliar assemblage preserved as compressions/impressions (Wolfe et al., 1966; Sunderlin et al., 2011). Faunal remains in the formation include a freshwater gastropod fauna (Viviparidae) (Walker et al., 2009) and a chelydrid turtle carapace (Hutchison and Pasch, 2004) Leaf physiognomy-based methods for estimating paleoclimatic parameters suggest that the Chickaloon flood basin was temperate, with MAT (11−14.6° C) and mean annual precipitation (MAP; 120−180 cm/yr) estimates much higher than the present-day climate of southern Alaska (Sunderlin et al., 2011). Dicot-leaf herbivory in the forms of margin feeding, hole-feeding, and skeletonization was documented by Sunderlin et al. (2011) and Brannick et al. (2012) and is believed to have been caused by insect cohabitants. Foliar damage frequency (proportion of leaves damaged) and intensity (area damaged on each leaf) is low in comparison with other floras of similar age, paleoenvironment, and paleoclimate (Sunderlin et al., 2011, 2014). 2018 GRIMALDI ET AL.: PALEOGENE BIOLOGICAL INCLUSIONS IN AMBER 5 MATERIALS AND METHODS A field collection of 9677 dispersed amber pieces was hand picked (“picking” method) from the surface of the study horizon and examined in the lab under stereomicroscopy (7×−45× mag- nification) for included faunal and floral remains. When necessary, pieces were ground on a lapi- dary wheel with discs of P800−P2500 grit to provide clear views into the pieces. Amber pieces with inclusions were embedded in EpoTek 301-2 synthetic resin, for stabili- zation of the brittle amber during preparation. Embedded pieces were trimmed with a water- fed diamond-edge trim saw, and carefully ground and polished using 600, 800, 1200, and 2400 grit emery papers (Buehler, Inc.) on a water-fed lapidary wheel. Final preparations commonly were 1−2 mm thick, to optimize observation of inclusion details at high magnifications. Inclu- sions were observed by applying the amber piece to a glass microscope slide using a drop of glycerin or water, and covering with a glass cover slip. General observations up to 150× were made using several stereoscopes (Wild, Nikon, Leitz), as well as a Wild compound scope at 100× and 200×; the latter was also used for measurements and illustrations (with an attached drawing tube). For observation at high magnification (400×), a Nikon Eclipse E600 compound scope was used with a Nikon Plan Fluor ELWD (extended-length working distance) 40×/0.60 objective. The Nikon Eclipse and a Nikon SMZ1500 stereoscope were used for photomicrog- raphy, along with NIS Elements software for z-stacking and scales. To further evaluate preservation of an ant inclusion, specimen AMNH WH-1 was examined using X-rays, examined under multiple parameters with a Bruker (Kontich, Belgium) Skyscan 1275 Micro-CT system at the New Jersey Institute of Technology. The specimen was assessed under live imaging with X-ray energies ranging from 20−70 kV, as well as detector settings rang- ing from ~100–750 msec. While the amber itself exhibited differential X-ray attenuation and penetration relative to the surrounding air, under no permutation of X-ray energies or detec- tor exposure settings did the inclusion itself resolve, revealing no detectable density differen- tial between the cuticle/body cavity of the ant inclusion and the amber matrix. RESULTS AND DISCUSSION Amber Collections Each examined piece of amber was categorized by morphology and size (table 1). Amber nodules that appear to originally have been formed internally in wood or under bark (“blisters” in Pike, 1993) comprise ~58% of the collection, while pieces exhibiting drip or “flow” (Pike, 1993) morphologies (i.e., runnels) and those that could not be categorized are less represented (~28% and ~14%, respectively). Large pieces (>10 mm in long axis, ~>0.25 g) comprise less than 1% of the collection, while medium (5–10 mm) and small (<5 mm, ~<0.03 g) pieces make up most of the material (~12% and 87%, respectively). Pike (1993), in his study of Late Cretaceous amber from southwestern Canada, emphasized the need to examine small amber pieces for inclusions, noting this size fraction’s disproportion- ate likelihood (by weight) to contain inclusions. Even though a sampling strategy of “picking” 6 AMERICAN MUSEUM NOVITATES NO. 3908 Table 1. Chickaloon Formation amber collection by morphological and size categories. Blister Flow Uncategorized Total Small (<5 mm) 4632 2500 1259 8391 Medium (5-10 mm) 936 250 66 1252 Large (>10 mm) 30 2 2 34 TOTAL 5598 2752 1327 9677 may lead to underrepresentation of small amber pieces (as compared to “floating”) (Pike, 1993), we made a pointed effort to collect across the size spectrum in the Chickaloon study horizon, including small nodules <0.2 g. Thus, we feel that our amber collection is minimally biased against sampling inclusions in the ways against which Pike (1993) cautioned. Amber Inclusions Fungi Figures 3, 10A AMNH LC-D4: A small piece containing very fine, filamentous hyphae with a very uni- form thickness ca. 2−3 μm (fig. 3A−C). Most hyphal strands are sinuous and straight, some squiggly. The hyphae are very webby and possibly interconnecting (their density makes it dif- ficult to tell), but they definitely branch. The hyphae grew on a dark mass of substrate that is partially preserved near the surface of the amber piece, with a dark reddish “halo” over the surface of the substrate and most hyphae (indicating some pyritization and/or oxidation). This substrate may be bark, since there are fragments of discernable bark fibers nearby (fig. 4A−C). Interestingly, the hyphae appear to have been growing into the fresher, lighter-yellow resin, since the fine, delicate filaments are perfectly arranged and undisturbed by any flow. The filaments do not appear to be sheathed bacteria, since their cores do not appear to have cell chains. Sheathed bacteria are reported to occur in amber (Schmidt and Schäfer, 2005; Girard et al., 2009), although various lines of other data led Speranza et al. (2015) to conclude that these reports are instead of fungal hyphae. Saint Martin and Saint Martin (2017) disputed the interpretation of hyphae, even though Speranza et al. (2015) used confocal laser microscopy to detect the diagnostic presence of chitin polysaccharides, which occur in fungi but not bac- teria. The filaments in Chickaloon amber are very similar to the webby hyphae of certain resinicolous ascomycetes that thrive on conifers, Mycocaliciales, which are known from the present through the Cenozoic (Tuovila et al., 2013). The genus Chaeonthecopsis, for example, FIG. 3. Fungal inclusions. A−C (AMNH LC-D4): Hyphae of apparent resinicolous fungus (?Mycocaliciales), penetrating into core of amber. A. Most of hyphal mass. B, C. Different focal planes of same area, at higher magnification. D−G. Clumps of a hyphomycete with multiseptate phragmaconidia growing on substrate (AMNH GC-A8), possibly of a sooty mold (Capnodiales), or closely related to the enigmatic Eocene hypho- mycete Casparytorula. D. Entire mass. E. Portion of fungal mass at higher (200×) magnification. F. Detail, 400×. G. Dispersed phragmaconidia adjacent to the sessile masses. 2018 GRIMALDI ET AL.: PALEOGENE BIOLOGICAL INCLUSIONS IN AMBER 7 8 AMERICAN MUSEUM NOVITATES NO. 3908 FIG. 4. Plant remains. A−C. Bark or wood fibers, AMNH WH-13. D−F, Microphylls of a moss (AMNH WH-2). D. Entire specimen. E. Larger microphyll at higher magnification. F. Tip of smaller microphyll, 200×. 2018 GRIMALDI ET AL.: PALEOGENE BIOLOGICAL INCLUSIONS IN AMBER 9 FIG. 5. Arachnids. A−E. Two fragments of the same piece containing portion of a spider egg sac, shown in different views and magnifications (AMNH LC-B3a, b). D, E. Highest magnification, showing how silk strands are cabled and woven. F. Spider (unidentified), ventral view (AMNH LC-D7). G. Pseudoscorpion, family indet. AMNH GC-A8b. H. Mite, Acari: Oribatida (family indet.), ventral view (AMNH LC-D1). See figure 7D for another spider inclusion. 10 AMERICAN MUSEUM NOVITATES NO. 3908 FIG. 6. Aphid apterae/nymphs (Hemiptera: Aphidoidea). A. One complete and one partial aphid. B. Complete aphid, dorsal view (AMNH LC-A2). C. Complete aphid, dorsal view (AMNH LC-D3). D. Complete aphid, lateral view (AMNH WH-9). E. AMNH WH-5, showing remains of four aphids.