A polar dinosaur feather assemblage from Australia

Journal Pre-proof A polar dinosaur feather assemblage from Australia Martin Kundrát, Thomas H. Rich, Johan Lindgren, Peter Sjövall, Patricia Vickers-Rich, Luis M. Chiappe, Benjamin P. Kear PII:

S1342-937X(19)30285-0

DOI:

https://doi.org/10.1016/j.gr.2019.10.004

Reference:

GR 2233

To appear in:

Gondwana Research

Received Date: 4 July 2019 Revised Date:

26 August 2019

Accepted Date: 1 October 2019

Please cite this article as: Kundrát, M., Rich, T.H., Lindgren, J., Sjövall, P., Vickers-Rich, P., Chiappe, L.M., Kear, B.P., A polar dinosaur feather assemblage from Australia, Gondwana Research, https:// doi.org/10.1016/j.gr.2019.10.004. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 International Association for Gondwana Research. Published by Elsevier B.V. All rights reserved.

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A polar dinosaur feather assemblage from Australia

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Martin Kundráta,*, Thomas H. Richb, Johan Lindgrenc, Peter Sjövalld, Patricia

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Vickers-Riche, Luis M. Chiappef and Benjamin P. Kearg,**

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a

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Košice 04154, Slovakia

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b

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c

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d

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e

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3122, Australia, and School of Earth, Atmosphere and Environment, Monash University, Monash, Victoria

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3800, Australia

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f

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Angeles CA 90007, USA

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g

Center for Interdisciplinary Biosciences, Technology and Innovation Park, University of Pavol Jozef Šafárik,

Melbourne Museum, Museums Victoria, 11 Nicholson Street, Carlton, Victoria 3053, Australia

Department of Geology, Lund University, Sölvegatan 12, 223 62 Lund, Sweden RISE Research Institutes of Sweden, Chemistry and Materials, PO Box 857, 501 15 Borås, Sweden

Faculty of Science and Technology, Swinburne University of Technology, John Street, Hawthorn, Victoria

The Dinosaur Institute, Natural History Museum of Los Angeles County, 900 Exposition Boulevard, Los

Museum of Evolution, Uppsala University, Norbyvägen 16, 752 36 Uppsala, Sweden

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* Corresponding author at: Center for Interdisciplinary Biosciences, Technology and Innovation Park, University of Pavol Jozef Šafárik, Košice 04154, Slovakia.

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** Corresponding author.

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E-mail addresses: [email protected] (M. Kundrát), [email protected] (B.P. Kear)

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Keywords:

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Mesozoic birds

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Aves

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Paraves

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melanosomes

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Early Cretaceous

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ABSTRACT

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Exceptionally preserved Mesozoic feathered dinosaur fossils (including birds) are famous,

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but recognized from only very few localities worldwide, and are especially rare in the

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Southern Hemisphere. Here we report an assemblage of non-avian and avian dinosaur

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feathers from an Early Cretaceous polar (around 70°S) environment in what is now

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southeastern Australia. The recovered remains incorporate small (10–30 mm long) basal

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paravian-like tufted body feathers, open-vaned contour feathers, and asymmetrical bird-like

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wing feathers that possess high-angled barbs with possible remnants of barbicels — amongst

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the geologically oldest observed to date. Such morphological diversity augments scant

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skeletal evidence for a range of insulated non-avian theropods and birds inhabiting extreme

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southern high-latitude settings during the Mesozoic. Although some of these fossil feathers

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exhibit what may be residual patterning, most are uniformly toned and preserve rod-shaped

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microbodies, as well as densely-packed microbody imprints on the barbules that are

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structurally consistent with eumelanosomes. Geochemical analysis detected no identifiable

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residual biomolecules, which we suspect were lost via hydrolysis and oxidization during

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diagenesis and weathering. Nevertheless, an originally dark pigmentation can be reasonably

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inferred from these melanic traces, which like the coloured feathers of modern birds, might

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have facilitated crypsis, visual communication and/or thermoregulation in a cold polar

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habitat.

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1. Introduction

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Mesozoic non-avian dinosaur and bird remains with preserved feathers are iconic, and

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have been documented scientifically for almost 160 years (Kaye et al., 2019). However, this

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long history of research is disproportionately biased towards discoveries from the Northern

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Hemisphere (Brocklehurst et al., 2012). In contrast, feathered dinosaur fossils from the

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Southern Hemisphere are extremely rare, and thus far limited to a single enantiornithine bird

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skeleton with body and tail plumage remnants (de Souza Caravalho et al., 2015), together

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with a handful of isolated feather traces (most recently summarised by Prado et al., 2016a),

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all from the Lower Cretaceous (upper Aptian) Crato Formation of Brazil. The only other

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direct evidence of Mesozoic dinosaur plumage hitherto reported from the Austral Gondwanan

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landmasses are three enigmatic ‘bird’ feathers found in Lower Cretaceous (middle–upper

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Aptian) deposits of the Koonwarra Fossil Bed in Victoria, southeastern Australia (Talent et

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al., 1966; Waldman, 1970). Although additional feather specimens have since been

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mentioned in the literature (e.g., Vickers-Rich, 1991; Chiappe, 1996; Kellner, 2002; Poropat,

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2018; Poropat et al., 2018), these have never been fully described. Moreover, subsequent

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excavations have uncovered a total of ten feathers that reveal not only substantial

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morphological and taxonomic diversity, but also microstructural details potentially evincing

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colour, patterning and adaptive implications of what constitute the first demonstrable

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dinosaur feather assemblage recorded from beyond the Mesozoic southern polar circle.

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Here, we document both new and existing feather occurrences from the Koonwarra

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Fossil Bed, and undertake microscopic and spectroscopic analyses to determine their

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ultrastructural and biomolecular preservation. Our results contribute to the growing picture of

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rich terrestrial ecosystems that once occupied an unusual Early Cretaceous (Hauterivian–

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early Albian) cold-temperate polar environment in what is now southeastern Australia (see

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Poropat et al., 2018 for an overview). Dinosaurs were a conspicuous component of these

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biotas, and included a range of euornithopods (Rich and Rich, 1989; Rich and Vickers-Rich,

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1999; Herne et al., 2018; Herne et al., 2019), ankylosaurians (Barrett et al., 2010), possible

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ceratopsians (Rich et al., 2014), non-avian theropods (Benson et al., 2010; Barrett et al.,

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2011; Benson et al., 2012; Fitzgerald et al., 2012), and stem birds (Close et al., 2009). The

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bird remains comprise only an incomplete enantiornithine furcula (Close et al., 2009);

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however, a few other fragmentary enantiornithine bones have been identified from mid-

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Cretaceous (upper Albian–mid-Cenomanian) strata of the Toolebuc and Griman Creek

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formations in northeastern Australia (Molnar, 1986; Kurochkin and Molnar 1997; Molnar,

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1999; Kear et al., 2003; Bell et al., 2019).

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1.1. Institutional abbreviations

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GSV, Geological Survey of Victoria (Melbourne, Australia). MON, Monash University

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(Melbourne, Australia). NMV, Melbourne Museum, Museums Victoria (Melbourne,

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Australia). QUT, Queensland University of Technology (Brisbane, Australia). RISE,

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Research Institutes of Sweden (Borås, Sweden). UOM, University of Melbourne

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(Melbourne, Australia). UU, Uppsala University (Uppsala, Sweden).

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1.2. Collection history and setting of the Koonwarra Fossil Bed

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The Koonwarra Fossil Bed was discovered in 1961 during road works on the South

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Gippsland Highway ~2 km outside the town of Koonwarra, and 145 km southeast of

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Melbourne (see detailed locality map in Bean, 2017, p. 11, fig. 3). An initial systematic

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excavation conducted in January 1962 by a joint team from UOM, NMV and GSA examined

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the north side of the road cutting, and recovered the first fossil feathers (NMV P186979,

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NMV P250595) subsequently described by Talent et al. (1966). In 1966, a group of staff and

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students from MON collected samples from the opposing southern side of the highway and

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found a third feather (NMV P26059) later described by Waldman (1970). This same site was

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visited again in 1981 by a field party from UOM and NMV, who uncovered three additional

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feathers (NMV P162963, NMV P165474, NMV P160550), two of which have been briefly

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discussed (Vickers-Rich, 1991; Chiappe, 1996) and figured (Kellner, 2002) in thematic

108

reviews. Intermittent collecting has also brought to light four more as yet undocumented

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feather specimens found in 1973 (NMV P32192), 1985 (NMV P231783), and acquired by the

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NMV in 2014 (NMV P250594, NMV P250624) through a bequest from the estate of Dr Peter

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Duncan (Melbourne), an avid private collector of fossils from the Koonwarra Fossil Bed (see

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Poropat, 2018 for a popular account).

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The NMV additionally conducted a series of test excavations in 2013, and again with the

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logistic assistance of VicRoads (https://www.vicroads.vic.gov.au) in 2018, to re-explore the

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historically fossiliferous exposures of the Koonwarra Fossil Bed, and examine new localities

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up to 50 meters to the East, and 100 meters to the West of the main road cutting. This entire

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area is now designated as the Koonwarra Fish Beds Geological Reserve and has been heritage

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listed for site protection (Cayley and Cairns, 2017). The NMV surveys compiled a

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stratigraphic log and identified the most productive fossil-bearing layers (Fig. 1).

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Lithologically, the Koonwarra Fossil Bed comprises around eight metres of laminated

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siltstones and claystones interpolated between massive subarkose sandstone beds (Waldman,

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1971; Drinnan and Chambers, 1986; Bean 2017). An approximately 1.8 metres thick package

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of densely laminated strata in the middle and lower half of the sequence has produced most of

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the fossils. Dettmann (1986) and Seegets-Villiers and Wagstaff (2016) both correlated this

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section with the Cyclosporites hughesii spore-pollen Zone, which is possibly Barremian to

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predominantly Aptian in age. However, Gleadow and Duddy (1980) and Lindsay (1982)

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reported fission-track dates based on volcanogenic apatites extracted from around seven

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metres higher in the succession, which constrain the age to 115±6 Myr, with a lower bound of

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118±5 Myr estimated from samples taken immediately below the main fossiliferous horizons;

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this equates to the middle–late Aptian (Poropat et al., 2018).

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Waldman (1971) initially reconstructed the depositional setting of the Koonwarra Fossil

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Bed as an inland lacustrine habitat subject to anoxia, possibly caused by annual winter

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freezing (evidenced from conspicuous mass mortality accumulations of fish remains).

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However, Douglas and Williams (1982), Jell and Duncan (1986) and Drinnan and Chambers

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(1986) revised this interpretation, instead envisaging a restricted low-energy waterbody

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affected by flooding from episodic high rainfall, and intermittently connected to a larger

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adjacent lake system. Drinnan and Chambers (1986) and Dettmann (1986) further inferred a

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surrounding montane forest vegetation and markedly seasonal climate, which concurs with a

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recent analysis of the sediment geochemistry (Tuite et al., 2016), identifying substantial

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terrigenous organic input and combustion-related polycyclic aromatic hydrocarbons (PAHs)

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consistent with high atmospheric pO2 and periodic local forest fires (see also Tossolini et al.,

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2018).

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Regionally, the Koonwarra Fossil Bed forms part of an extensive volcanogenic

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sedimentary succession that accumulated in a high-latitude Australian-Antarctic rift basin

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during the Cretaceous (see Poropat et al., 2018 for summary). Precise palaeolatitude

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estimates (sensu Torsvik et al., 2012; van Hinsbergen et al., 2015) situate the Koonwarra

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Fossil Bed localities at 75.36±3º–78.32±3º S, based on the 110–120 Myr interval computed

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using Paleolatitude.org (http://www.paleolatitude.org/). This concurs with other global plate

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reconstructions, which indicate palaeolatitudes substantially >60º S (Matthews et al., 2016),

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but slightly exceeds a previous site specific estimate of 68.32º S at 118±5 Myr (Bean, 2017),

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derived using data from Seton et al. (2012), together with GPlates (https://www.gplates.org/)

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developed by Müller et al. (2018). Irrespectively, all of these calculations contextually place

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the Koonwarra Fossil Bed feather assemblage well within the Mesozoic southern polar circle.

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Accumulated fossil (Douglas and Williams, 1982; Douglas, 1986; Rich et al., 1988; Rich and

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Vickers-Rich, 1997), sedimentological (Constantine et al., 1998) and isotopic data (Gröcke,

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1997; Ferguson et al., 1999; Price et al., 2012) further infer seasonally cool-temperate to cold

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conditions (potentially with winter freezing) throughout the Aptian, with prolonged periods

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of polar darkness (Poropat et al., 2018) that must have impacted on the evolution of

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ecosystems in southeastern Australia at that time.

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2. Material and methods

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2.1. Specimen curation

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All of the Koonwarra Fossil Bed feather remains have been deposited in the Victorian

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state government palaeontology collection at NMV (Table 1). Following NMV institutional

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registration protocols, each feather specimen has been assigned a unique catalogue accession

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number with the suffix ‘A’ and ‘B’ denoting associated part (‘A’), versus counterpart (‘B’)

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sections where relevant.

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2.2. Microscopy

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We inspected the Koonwarra Fossil Bed feather specimens using a Leica DM RXE stereo microscope equipped with a Leica DFC 550 camera and LAS v.4.2 software supplied by the

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manufacturer. All measurements were taken using high-resolution digital micrographs

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imported into ImageJ (Rasband, 1997–2009; Abramoff et al., 2004).

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Field emission gun scanning electron microscopy (FEG-SEM) was conducted at UU, and

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examined uncoated samples on a Zeiss Supra 35-VP (Carl Zeiss SMT) incorporating a low

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vacuum VPSE detector, Robinson BSD backscattered electron-imaging attachment, and

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coupled EDX Apex 4 (Ametekh) EDS-detector for dispersive X-ray microanalysis.

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2.3. Spectroscopy

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We selected an isolated sample of the barbs and barbules, together with surrounding

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sediment, from the darkly colored counterpart section NMV P165474B. As a historical

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museum specimen, collection and storage under sterile conditions was impossible to verify;

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however, we freshly exposed a small surface area that was seemingly not treated with either

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preservatives or consolidants. This was then subjected to a detailed spectroscopic analysis

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using time-of-flight secondary ion mass spectrometry (ToF-SIMS) at RISE. ToF-SIMS

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enables precise in situ detection of residual biomolecules (see discussions in Colleary et al.,

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2015; Lindgren et al., 2015a; Lindgren et al., 2018; Li et al., 2018), but is minimally

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destructive, and thus provides an optimal technique for examining the exceptionally rare

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Koonwarra Fossil Bed feathers. We utilized a TOFSIMS IV instrument (IONTOF GmbH)

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with 25 keV Bi3+ primary ions, and low-energy electron flooding for charge compensation.

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Positive and negative ion data were acquired under static conditions with optimization for

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high mass resolution at m/∆m ≈5.000 (lateral resolution at 3–5 µm).

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3. Results and discussion

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3.1. Description of the fossil feathers

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The Koonwarra Fossil Bed feathers include tufted plumes up to 14 mm long. NMV

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P160550 consists of a dark calamus (often not preserved in ‘carbonized’ feather traces: Foth,

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2012) and short rachis supporting a dense bundle of parallel barbs; these are non-branching

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and were seemingly flexible in life (Fig. 2A). NMV P160550 thus differs from the

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pycnofibres of pterosaurs (Yang et al., 2019), as well as the integumentary coverings of some

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ornithischian dinosaurs (Zheng et al., 2009; Godefroit et al., 2014) and non-avian theropods,

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such as Sinosauropteryx (Currie and Chen, 2001) and Beipiaosaurus (Xu et al., 2009), which

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tend to comprise either solitary filaments or clumps of curving fibres that were potentially

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organized into branching units (Kundrát, 2004), or incorporated basal sheath-like structures

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(Saitta et al., 2018). NMV P160550 otherwise more closely resembles the compound

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‘protofeathers’ found on the body and limbs of various paravians, including Sinornithosaurus

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(Xu et al., 2001; McKellar et al., 2011), Yi (Xu et al., 2015), Aurornis (Godefroit et al.,

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2013a), Eosinopteryx (Godefroit et al., 2013b), Serikornis (Lefèvre et al., 2017), Caihong

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(Hu et al., 2018) and Anchiornis (Saitta et al., 2018); notably though, there is no evidence of

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distal bifurcation as posited from the detached contour feathers of Anchiornis (Saitta et al.,

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2018).

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NMV P250594 and NMV P250595 represent partially decomposed body feathers with

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barbs preserved as tangled mats that fray distally. NMV P250594 incorporates a mass of

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elongate filaments (Fig. 2B) similar to the compound plumage of Sinornithosaurus (Xu et al.,

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2001). On the other hand, NMV P250595 clearly preserves a tapering rachis with dark edges

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and a light core that could represent a diagenetically compacted lumen comparable to those

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reported in filamentous dinosaur plumage (Schweitzer et al., 1999; Mayr et al., 2002; Foth,

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2012). At least 12 barbs are visible along the incomplete vane of NMV P250595 (Fig. 2C).

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These are all low-angled (up to about 12º) and alternately branching. They lack obvious

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barbules, which might be a taphonomic artefact (Xu and Gou, 2009; Ji et al., 1998; Lefèvre et

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al., 2017; Saitta et al., 2018), but is also similar to some basal paravian feathers (Grimaldi and

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Case, 1995; Perrichot et al., 2008), such as those of Serikornis (Lefèvre et al., 2017, although

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see Saitta et al., 2018 for a conflicting interpretation).

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NMV P231783 is a very small (under 5 mm long) radially branching feather with a short

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calamus that resembles an avian neoptile feather (Foth, 2011). The vane is damaged and has

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irregular gaps but was clearly bilaterally symmetrical. The rachis integrates alternately

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branching barb rami that retain visible barbules extending to the apex (Fig. 2D). This is

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strikingly similar to the open-vaned tail plumage described in some non-avian coelurosaurs

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(Xing et al., 2016a).

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Possible avian feathers include NMV P162963, which has a thin rachis and

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symmetrically branching barbs angled at about 45º (although these are distorted proximally,

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perhaps by sediment mobilization during burial: e.g., Prado et al., 2016b). Interlocking

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barbules contribute to an open contour-like vane (Fig. 3A), which is reminiscent of the body

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plumage in Archaeopteryx (Foth et al., 2014), as well as the limb feathers of

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Protoarchaeopteryx and Caudipteryx (Ji et al. 1998), Eosinopteryx (Godefroit et al., 2013b),

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Serikornis and Anchiornis (Hu et al., 2009; Lefèvre et al., 2017; Saitta et al., 2018), and

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Caihong (Hu et al., 2018). Noticeably, however, the calamus is robust with a curving

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centreline that accords with the primary covert of Archaeopteryx (Kaye et al., 2019).

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NMV P250624 comprises the incomplete distal section of an open vane that had a thin

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rachis and closed pennaceous tip; no barbules are evident (Fig. 3B). In contrast, NMV

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P186979 and NMV P165474 are virtually intact contour feathers (Fig. 3C, D) with distally

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open barbs comparable to some secondary wing plumes of enantiornithines (Xing et al.,

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2016b; Xing et al., 2017). Both NMV P186979 and NMV P165474 have truncated apices like

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the head feathers of Confuciusornis (Li et al., 2018), and an isolated contour feather reported

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by Vinther et al. (2008) from the Crato Formation of Brazil. NMV P186979 additionally

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incorporates what appear to be originally flexible afterfeathers that are clearly differentiated

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from the distal vane as in advanced birds (Prum, 1999). These include at least 10 elongate

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darkly toned barbs that extend perpendicularly from either side of the rachis (Fig. 3C).

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NMV P32192 and NMV P26059 are asymmetrical pennaceous feathers. NMV P32192 is

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partially covered by matrix (Fig. 3E), but appears to have had a rounded tip like the wing

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feathers of Archaeopteryx (Carney et al., 2012; Longrich et al., 2012; Foth et al., 2014; Kaye

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et al., 2019). In contrast, NMV P26059 is distally tapered (similar to the alula feathers of

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enantiornithines: Xing et al., 2017) with ‘open-vaned’ barbs (Fig. 4A, B) that resemble the

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remiges of Serikornis and Anchiornis (Lefèvre et al., 2017; Saitta et al., 2018), as well as the

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short secondary coverts of Confuciusornis (Li et al., 2018), enantiornithines and modern birds

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(Xing et al., 2016b); the ventral coverts of Archaeopteryx are proportionately much longer

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(Longrich et al., 2012).

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The calamus on the part specimen NMV P26059A is robust like the isolated covert of

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Archaeopteryx (Carney et al., 2012; Longrich et al., 2012; Kaye et al., 2019), and the rachis

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(split longitudinally on the counterpart NMV P26059B: see Fig. 4B) is basally broad and

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distally tapered, similar to the flight feathers of both Archaeopteryx (Longrich et al., 2012;

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Foth et al., 2014) and enantiornithines (Xing et al., 2017). The vanes of both NMV P26059,

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and NMV P32192 (Fig. 3E) consist of high-angled barbs on the leading (around 20º) and

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trailing edges (between 30º and 40º) that are commensurate with the rigid aerodynamic

271

feathers of advanced enantiornithines and ornithuromorphs (Feo et al., 2015). However,

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NMV P26059 is only 14.3 mm long (measured from the part specimen NMV P26059A) and

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thus of equivalent size to the wing plumage of hatchling enantiornithines, which are known to

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have possessed well-developed remiges (Chiappe et al., 2007; Xing et al., 2016b; Xing et al.,

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2017).

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NMV P26059 additionally preserves microscopic barbicel-like structures, which seem to

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represent mineralized remnants of the original keratinaceous material, and retain a regular

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branching sequence at intervals between the barbules close to the rachis (Fig. 4C; see also

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Appendix A). Most of these barbicel-like structures are angled perpendicularly relative to the

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barbules (Fig. 4D); however, others project obliquely, and are morphologically comparable to

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the spine-like processes observed on the barbules of enantiornithine wing feathers (Xing et

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al., 2019), and in some modern ratites (see McGowan, 1989).

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Barbicels are usually not recorded in lithified Mesozoic feathers (Zhang and Zhou, 2000;

284

Kellner, 2002), and are thought to have been absent in basal paravians (Lefèvre et al., 2017;

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Saitta et al., 2018). Nevertheless, functional hooklets have been identified on enantiornithine-

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like feathers preserved in amber (Kellner, 2002; Xing et al., 2016b; Xing et al., 2019), and

287

have also been inferred in Confuciusornis (Li et al., 2018) and Archaeopteryx (McGowan,

288

1989; Carney et al., 2012) on the basis of their closed pennaceous vanes.

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3.2. Possible melanosome traces

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Our FEG-SEM analyses revealed largely homogeneous aggregations of elongate

293

microbodies and corresponding imprints embedded into the dark surfaces of the

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‘protofeather’ NMV P160550A (Fig. 5A), the degraded filamentous feather NMV P250594A

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(Fig. 5B, C), and the contour feathers NMV P165474B (see Appendix A) and NMV

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P186979A (Fig. 5D). Their ellipsoidal form, localized distribution, and dense packing is

297

almost identical to eumelanosome traces previously identified from other fossil feathers (e.g.,

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Clarke et al., 2010; Zhang et al., 2010; Li et al., 2010; Zhang et al., 2010; Barden et al., 2011;

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Wogelius et al., 2011; Carney et al., 2012; Li et al., 2012; Li et al., 2014; Lindgren et al.,

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2015b; Wang et al., 2017; Hu et al., 2018; Li et al., 2018). However, melanosome

301

impressions have also been ascribed to bacterial cells associated with decompositional

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biofilms (Moyer et al., 2014). In addition, despite the Koonwarra Fossil Bed melanosome

303

imprints lacking obvious binary fission, which is usually preclusive of a microbial origin

304

(Lindgren et al., 2015a), some were clearly overlapping or connected at their terminal ends

305

(Fig. 5C) as has been illustrated in bacterial cultures (Moyer et al., 2014), and others have

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deformed shapes (Fig. 5A).

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In contrast, the solid microbodies on the barbules of NMV P186979A (Fig. 5D) and

308

NMV P250594A (Appendix A) are more sparsely distributed and devoid of recognizable

309

internal features, as well as being conspicuously rod-shaped and integrated into the fabric of

310

the barbule, as would be expected for preserved eumelanosomes (Li et al., 2010; McNamarra,

311

2013; Moyer et al., 2014; Lindgren et al., 2015a). Notably, they lack the regular ‘end-to-end’

312

organisation (which is evident in the corresponding imprints: Fig. 5A–C) reported in

313

melanosome traces from the glossy black feathers of Microraptor (Li et al., 2012), together

314

with the compressed platelet-like arrangement associated with iridescent plumage in basal

315

paravians, such as Caihong (Hu et al., 2018). Nevertheless, their elongate (~1010–1445 nm)

316

and narrow (~281–327 nm) proportions yield high aspect ratios (~4.3) that are consistent with

317

iridescent feathers in modern birds (Carney et al., 2012; Li et al., 2012). Admittedly, the

318

shape and size of fossil melanosomes is demonstrably equivocal for assigning discrete

319

colours (McNamara, 2013; McNamara et al., 2013; Lindgren et al., 2015a), yet the

320

Koonwarra Fossil Bed melanosome traces compare well with those considered indicative of

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dark plumage in Anchiornis (Li et al., 2010), Microraptor (Li et al., 2012), Archaeopteryx

322

(Carney et al., 2012) and Confuciusornis (Li et al., 2018).

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Interestingly, some residual patterning is discernible as depigmented bands on the vane

324

of NMV P165474B (Fig. 3B), and distal tip of the barbs in NMV P160550A (Fig. 2A). This

325

could represent differential melanisation (Vinther et al., 2008; Clarke et al., 2010;

326

McNamara, 2013), and/or microbial overgrowth localized to the more nutrient-rich decay

327

resistant melanin-containing regions (Moyer et al., 2014). Similar dark and light coloured

328

banding has been described in other isolated Mesozoic dinosaur feathers (Vinther et al., 2008;

329

Kellner, 2002), together with the retrices of Caudipteryx (Ji et al., 1998) and various feathers

330

attributed to enantiornithine birds (Xing et al., 2016b; Xing et al., 2017). The darkened tip on

331

NMV P165474 also resembles the head and neck feathers of Confuciusornis (Li et al., 2018).

332 333

3.3. Biomolecular investigation

334 335

ToF-SIMS data from the barb and barbule surfaces of NMV P165474B produced

336

positive ion spectra that were dominated by secondary inorganic peaks corresponding to the

337

embedding matrix, specifically Na+, Mg+, Al+, Si+, K+, Ca+, Fe+ and CaOH+ (Fig. 6A, B). In

338

addition, organic ions were observed at m/z 149 and 245; however, these most likely originate

339

from environmental contaminants, as well as weak signals from organic fragment ions in the

340

mass range up to m/z 200. No positive ions were found to localize on the barb and barbules

341

surfaces of NMV P165474B, and characteristic polycyclic aromatic hydrocarbon (PAH) ions

342

(e.g., at m/z 115, 128, 141, 152, 165, 178, 189 and 202: Stephan et al., 2003) were not

343

detected, suggesting that if present, these must only occur in insignificant amounts.

344

Likewise, the negative ion spectra from NMV P165474B (Fig. 7A, B) were dominated

345

by inorganic peaks corresponding to clay minerals in the sediment, especially SiO2-, SiO3-,

346

AlSiO4- at m/z 60, 76 and 119, respectively. Substantial signal intensities were also identified

347

from organic ions, including CnH- and CnN-. Nevertheless, further analysis revealed

15

348

comparable peak distributions in the matrix (Fig. 7C). We therefore conclude that no

349

molecular components are specifically restricted to the barb and barbules surfaces of NMV

350

P165474B, nor are there any obvious similarities to previously published eumelanin spectra

351

(e.g., Lindgren et al., 2012; Lindgren et al., 2015a; Lindgren et al., 2015b).

352

Our failure to detect melanin-related compounds, as well as any other organic matter

353

indicative of proteins, such as keratin, probably reflects their loss via hydrolytic and oxidative

354

reactions during diagenesis and/or subsequent weathering (Lindgren et al., 2015a; Saitta et

355

al., 2017). Because PAHs have been recorded in untreated Koonwarra Fossil Bed matrix

356

samples (Tuite et al., 2016), we suspect that the biomolecular degradation of NMV

357

P165474B might have occurred during post-excavation treatments of the host rock, which

358

historically employed prolonged subaerial exposure to solar radiation and repeated wet-dry

359

cycles intended to split the sediment bedding planes and reveal fossils (Talent et al., 1966).

360 361

3.4. Conclusions

362 363

Like most assemblages of isolated dinosaur feathers (e.g., Davis and Briggs, 1995;

364

Kellner, 2002; Knight et al., 2011; Prado et al., 2016a), the Koonwarra Fossil Bed feather

365

specimens are associated with a lacustrine setting, and mainly include examples of contour

366

plumage. These may have been dispersed from decaying or dismembered carcasses (e.g.

367

Davis and Briggs, 1995), or been shed from living animals during moulting, or flight in the

368

case of volant paravians. Irrespectively, the depositional environment of the Koonwarra

369

Fossil Bed suggests that the feathers accumulated close to the shoreline (Waldman, 1971; Jell

370

and Duncan, 1986), and probably underwent limited aeolian or water-borne transport. Indeed,

371

this accords with the exceptional quality of the accompanying invertebrate, bony fish and

372

terrestrial plant fossils (Waldman 1971, Drinnan and Chambers 1986, Jell and Duncan 1986),

16

373

which are considered a predominantly autochthonous biota inhabiting either the waterbody,

374

or immediately surrounding forested setting (Jell and Duncan 1986).

375

The high-fidelity preservation of the Koonwarra Fossil Bed feathers is comparable with

376

existing records of ‘carbonized’ dinosaur plumage, and indicates that they were rapidly

377

interred in the lake bed sediments under anaerobic conditions (Foth, 2012). Decay may have

378

been further inhibited by melanic pigmentation, which would have increased their resistance

379

to chemical decomposition (Vinther et al., 2008). In life, such melanization could have

380

assisted in crypsis, species recognition, intraspecific communication, and possibly heat

381

regulation and radiant energy absorbance, as it does in birds today (Wolf and Walsberg,

382

2000; Galván and Solano, 2016). Nevertheless, post-burial diagenesis, perhaps including

383

thermal maturation (see McNamara et al., 2013), coupled with hydrolysis and oxidization

384

during prolonged surface weathering (Talent et al., 1966) has clearly broken down the

385

original biomolecules. We therefore recommend that any future sampling of the host

386

Koonwarra Fossil Bed deposits, which have considerable potential to produce more complete

387

feathered dinosaur remains (Rich et al. 2012), must incorporate preparation techniques that

388

are better conducive to the survival of residual organics.

389

Finally, the Koonwarra Fossil Bed feathers document a range of recognizable

390

morphologies (Perrichot et al., 2008; Knight et al., 2011; McKellar et al., 2011; Prado et al.,

391

2016a) that reveal a cryptic biodiversity of polar paravian taxa hitherto not well represented

392

in the corresponding skeletal fossil record. The very small size of some of these feather

393

specimens additionally suggests the presence of ontogenetically immature individuals,

394

especially NMV P26059, which exhibits a robust rachis, asymmetrical pennaceous vane, and

395

barbicel-like structures that are characteristic of powered flight in what might have been a

396

precocial infant bird (see Zhou and Zhang, 2004; Xing et al., 2016b; Xing et al., 2017).

397

Moreover, our identification of possible barbicels in NMV P26059 is amongst the oldest

17

398

documented examples worldwide — these range from Hauterivian (Kellner, 2002) to late

399

Campanian (McKellar et al., 2011; Xing et al., 2016b) in age — and implies that advanced

400

‘closed-vaned’ plumage was widely acquired in stem avians by at least the mid-Early

401

Cretaceous (potentially with an earlier independent development in Archaeopteryx: Carney et

402

al., 2012; Wang et al., 2015); this also coincides chronostratigraphically with the earliest

403

appearances of basally branching enantiornithines and ornithuromorphs by around 130 Myr

404

ago (Wang et al., 2015; Chiappe and Meng, 2016; Wang et al., 2017).

405

Ultimately, the Koonwarra Fossil Bed feathers evince the first dinosaurian integumentary

406

structures recovered from the Mesozoic polar circle, and hint at a global distribution of

407

feathered paravians, including advanced bird antecedents, during the Early Cretaceous.

408 409

Acknowledgements

410 411

We thank Tim Ziegler (NMV) for assistance with information and photography, and

412

VicRoads for facilitating on-going field surveys. Peter Trusler (Melbourne) generously

413

contributed his copyrighted artwork commissioned for the DinoQuest exhibition managed by

414

the Singapore Science Centre. Our manuscript benefited from constructive comments made

415

by the Editor and reviewers. Financial support was provided by UNESCO IGCP Projects 608

416

and 609 involving P.V.-R. and T.H.R., the Slovak Research and Development Agency

417

(APVV-18-0251) and Scientific Grant Agency VEGA of the Slovak Ministry of the

418

Education, Science, Research and Sport and Slovak Academy of Sciences (1/0853/17) to

419

M.K., and a Swedish Research Council grant (642-2014-3773) to J.L.

420 421 422

Author contributions

18

423

M.K. and B.P.K., designed the project, T.H.R., and P.V-R., conducted fieldwork and

424

collected the stratigraphic data, M.K., B.P.K., P.S., and J.L., performed the research, and

425

B.P.K. and M.K. wrote the manuscript with contributions from L.M.C., J.L., P.S., P.V-R.,

426

and T.H.R.

427 428

Appendix A. Supplementary data

429 430

Additional photographs of part and counterpart specimens, as well as FEG-SEM

431

micrographs are archived under Project 3403 on Morphobank

432

(http://morphobank.org/permalink/?P3403).

433 434

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717 718

Fig. 1. Schematic stratigraphic column of Koonwarra Fossil Bed sediments at the NMV 2013 sample locality;

719

adapted from data compiled for NMV by David Flannery (QUT). Inset map shows geographic location of the

720

Koonwarra Fossil Bed locality and distribution of associated Cretaceous strata (Strzelecki Group). Graphically

721

depicted fossil biota includes: (1) paravian dinosaur feathers; (2) freshwater teleosts and ceratodont lungfish; (3)

722

various insects, arachnids and other terrestrial invertebrates; (4) aquatic insect larvae, hydrophilid beetles, and

723

horseshoe crabs; (5) mosses, liverworts, fern-like plants, Ginkgo, and conifers (see Poropat et al., 2018 for

724

summary).

725 726

Fig. 2. Polar dinosaur feathers from the Koonwarra Fossil Bed. A. NMV P160550A, basal paravian-like

727

compound ‘protofeather’. B. NMV P250594A, decomposed basal paravian-like body feather. C. NMV

728

P250595, decomposed basal paravian-like body feather with rachis. D. NMV P231783, basal paravian-like

729

contour feather with preserved barbules.

730 731

Fig. 3. Polar dinosaur feathers from the Koonwarra Fossil Bed. A. NMV P162963A, paravian-like contour or

732

limb feather. B. NMV P250624, incomplete avian-like contour feather. C. NMV P186979A, avian-like contour

733

plume with afterfeathers. D. NMV P165474B, avian-like contour feather with residual patterning. E. NMV

734

P32192, asymmetrical avian-like wing feather partly covered by matrix (arrows).

735 736

Fig. 4. Polar avian wing feather NMV P26059 from the Koonwarra Fossil Bed. A. NMV P26059A, part

737

specimen with calamus. B. NMV P26059B, counterpart specimen showing intact distally tapered tip. C. FEG-

738

SEM micrograph of the exposed proximal barb and barbule surfaces (dashed lines) in NMV P26059B (inset box

739

indicates enlargement in D). D. FEG-SEM micrograph depicting remnant barbicel-like structures preserved

740

between the barbules of NMV P26059B.

741 742

Fig. 5. FEG-SEM micrographs of feather melanosome-like traces from the Koonwarra Fossil Bed. A.

743

Microbody imprints with deformed outlines (arrow) in NMV P160550A. B. Densely packed microbody

744

imprints in NMV P250594A (inset box indicates enlargement in C). C. Microbody imprints showing

745

overlapping (arrow) and connected terminal ends (arrow) in NMV P250594A. D. Solid rod-shaped microbodies

746

in NMV P186979A.

30

747 748

Fig. 6. ToF-SIMS analysis of NMV P165474B. A. Positive ion spectra showing matrix peaks and (+) potential

749

environmental contaminants. B. Positive ion images of the barb/barbule surfaces showing signal intensity

750

distributions from organic fragments (top left), matrix (top right), environmental contaminants (bottom left), and

751

a total ion image with the selected region of interest (ROI) used to generate the barb/barbule spectrum (blue fill).

752 753

Fig. 7. ToF-SIMS analysis of NMV P165474B. A. Negative ion spectra showing (o) matrix peaks. B. Negative

754

ion images of the barb/barbule surfaces showing signal intensity distributions from organic CnH- ions (top left),

755

matrix (top right), organic CnN- ions (bottom left), and a total ion image with the selected ROI used to generate

756

the barb/barbule spectrum (green fill). C. ToF-SIMS intensities of organic negative ions in spectra from the

757

barb/barbules and matrix surfaces.

1

Table 1. List of fossil feather specimens recovered from the Koonwarra Fossil Bed in southeastern Australia. Specimen

Accession (collection)

Description

Views

Reference

NMV P26059

1970 (1966)

Avian wing feather

part/counterpart

Waldman (1970)

NMV P32192

1974 (1973)

Avian wing feather

part only

this paper

NMV P160550

1981 (1981)

Tufted paravian ‘protofeather’

part/counterpart

this paper

NMV P162963

1982 (1981)

Possible avian feather

part/counterpart

this paper

NMV P165474

1982 (1981)

Avian contour feather

part/counterpart

Kellner (2002)

NMV P186879

1987 (1962)

Avian contour feather

part/counterpart

Talent et al. (1966)

NMV P231783

2012 (1985)

Paravian body feather

part only

this paper

NMV P250594

2014 (no data)

Decomposed body feather

part/counterpart

this paper

NMV P250595

2014 (1962)

Paravian body feather

part only

Talent et al. (1966)

NMV P250624

2014 (no data)

Possible avian feather

part only

this paper

1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

A polar dinosaur feather assemblage from Australia Highlights •

Fossil feathers from the Koonwarra Fossil Bed in southeastern Australia record the first demonstrable dinosaur (including birds) integumentary structures described from the Mesozoic polar regions.

•

This diverse range of non-avian theropod (paravian) and bird feathers more than doubles the number of Mesozoic fossil feather specimens and morphologies recovered from the Gondwanan landmasses to date.

•

Possible traces of eumelanosomes imply original dark colouration and patterning. Some of the geologically oldest barbicel-like structures also evince advanced avian-grade flight feather morphologies in the Early Cretaceous.