Supplementary Information for Quantitative assessment of tarsal morphology illuminates locomotor behaviour in Paleocene mammals following the end-Cretaceous mass extinction Sarah L. Shelleya,b,1, Stephen L. Brusattea, Thomas E. Williamsonc aSchool of GeoSciences, Grant Institute, University of Edinburgh, James Hutton Road, King's Buildings, Edinburgh, EH9 3FE, United Kingdom. bSection of Mammals, Carnegie Museum of Natural History, 5800 Baum Blvd, Pittsburgh, Pennsylvania, 15206, United States of America (previous address). cNew Mexico Museum of Natural History and Science, 1801 Mountain Rd NW, Albuquerque, New Mexico, 87104, United States of America. 1To whom correspondence should be addressed. Email: [email protected] (SLS) This PDF file includes: Supplementary text Figs. S1 to S24 Tables S1 to S16 References for SI Other supplementary materials for this manuscript include the following: Datasets S1 to S5 1 Supplementary Information Text Methods Datasets Tarsal data. Measurements. 29 tarsal measurements (Fig. S1) were taken from the astragalus and calcaneum for a sample of extant and fossil mammals (Dataset S1). Note we refer to these measurements as tarsal measurements for ease of reading but recognise that they are from only two tarsal bones. Measurements were taken with the tarsals orientated as if the taxon being measured were plantigrade to maintain consistency in terminology. The following list of measurements is based on personal observation of tarsal osteology and compiled to describe the morphological and functional variation of the bones thoroughly, whilst also remaining measurable for as many taxa as possible. Note that there are some zero values in our measurements: zero values indicate the absence or loss of a given feature in the view measured which in itself is a character. In some instances, extinct taxon measurements are composited. This was done with care and only where the specimens closely approximate each other in size (often times the specimens are found in close association but cannot be determined to be from the same individual or have been accessioned separately). Specimen numbers are listed in Dataset S1. List of measurements used in this study corresponding to those illustrated in Fig. S1b: 17 measurements were taken from the astragalus: A1 maximum anteroposterior length of the astragalus A2 maximum anteroposterior length of the astragalar body A3 maximum anteroposterior length of the medial trochlear keel (highest edge) A4 maximum anteroposterior length of the lateral trochlear keel (highest edge) A5 maximum mediolateral width of astragalar body A6 maximum width or length of posteromedial expansion 2 A7 maximum width of fibial facet in dorsal view A8 maximum mediolateral width of trochlea at anterior border A9 maximum mediolateral width of astragalar head A10 maximum length of ectal facet A11 maximum anteroposterior length of sustentacular facet A12 maximum mediolateral width of sustentacular facet A13 maximum dorsoplantar depth of astragalar body A14 maximum dorsoplantar depth of astragalar head A15 maximum dorsoplantar height of lateral trochlear keel A16 maximum dorsoplantar height of medial trochlear keel A17 maximum anteroposterior length between anterior medial rim of trochlear and anterior margin of astragalar head 12 measurements were taken from the calcaneum: C1 maximum anteroposterior length of the calcaneum C2 maximum anteroposterior length of the calcaneal tuber C3 maximum mediolateral width of the posterior end of the calcaneum C4 maximum mediolateral width of the calcaneal body C5 minimum mediolateral width of the calcaneal tuber C6 maximum anteroposterior length of the sustentacular facet C7 maximum mediolateral width of the sustentacular facet C8 maximum length of the ectal facet C9 maximum width of the ectal facet C10 maximum dorsoplantar depth of the calcaneal tuber C11 maximum dorsoplantar depth of the cuboid facet C12 maximum mediolateral width of the cuboid facet Taxon sample. Extant taxa. We sampled a total of 85 extant therian taxa. Given the improbability of measuring tarsals for every extant mammal species, we aimed to build a proxy dataset by 3 selecting taxa that were relatively straightforward to assign to a given locomotor group, covered all extant orders that locomote primarily on land, and covered a broad range of locomotor behaviours that occur on land. This number includes representatives for every pertinent placental order with the exception of Chiroptera, Dermoptera, Pholidota and Sirenia. We did not include extant representatives for Chiroptera given they do not use their hindlimbs for sustained locomotion on land (although we recognise the unusual exception of mystacinid bats). We also chose to not include dermopterans in this study given their extreme morphologies and outlier position in morphospace associated with gliding behaviour (e.g. see [1] but also [2]) and the fact we were not able to obtain enough measurements for ostensible Paleocene dermopterans to include in our dataset (e.g. Mixodectes) [3]. We did not include Pholidota given they possess highly unusual tarsal bones (i.e. extreme reduction of the sustentacular facet) making meaningful inclusion into this dataset difficult. We also excluded Sirenia given that they lack tarsal bones. We note that 85 out of 6490 extant eutherian mammals species or 72 out of 1311 genera [4] is sampling slightly over 1% of extant mammal species and 5.5% of genera. This value is diminutive, however, consider that how species are defined does likely not directly correlate with tarsal diversity. Furthermore, when breaking down the taxonomic composition of extant species, 2552 of extant species are rodents (many of which are recognised by genotypic rather than phenotypic features) and we have sampled across a range of rodents exhibiting an exemplary range of morphologies and locomotor behaviours; 1386 species are bats which are not pertinent to this current study; 551 are artiodactyls which although relevant are relatively constrained in their tarsal morphology and locomotor behaviours (with the exception of extant cetaceans which lack tarsals and were therefore not included in our dataset). This is the same rationale commonly used in phylogenetic analyses using morphological data; for example, the Mammal Tree of Life project used 46 taxa to represent the diversity of extant mammals [5]. Furthermore, we utilise discriminant analyses to test the validity of our data sample at predicting locomotor behaviour—the results of this analysis show that tarsals can be used to predict locomotor behaviour accurately in our dataset, and thus this dataset is useful for our purposes. 4 Paleocene taxa. We were able to measure a total of 40 Paleocene taxa. A core aim of this project was to incorporate as many Paleocene mammals as possible. These mostly belonged to so-called ‘archaic’ groups but also included species considered to be members (or stem members) of extant orders. In our sample we have chosen to include species of Eocene age known for tarsal bones which have a generic temporal range that extends back into the Paleocene. Taxon age information is provided in Dataset S1. Cretaceous taxa. We sampled a total of five Cretaceous species. We chose to not extend our sample outside of Cladotheria as taking homologous measurements would have become difficult. To this end, our sample includes cladotherians known from three- dimensionally preserved tarsal specimens for which we were able to obtain the necessary measurements. Notable exceptions include Zalambdalestes [6] and Asioryctes which are known for tarsal bones but for which we could not obtain enough measurements to include in this study. Regression data. Five measurements were taken for a sample of 79 extant therian taxa and four extinct Paleocene taxa (Dataset S5). Our fossil sample was limited to specimens that allowed for body weight estimates to be calculated using associated postcranial material (see below) List of measurements used for regression analyses: Maximum proximodistal length of the humerus Minimum midshaft circumference of the humerus Maximum mediolateral width of the humerus Maximum proximodistal length of the femur Minimum midshaft circumference of the femur Institutional abbreviations AMNH, American Museum of Natural History, New York, New York, USA CM, Carnegie Museum of Natural History, Pittsburgh, Pennsylvania, USA 5 CNHM, Chicago Natural History Museum, Chicago, Illinois, USA DGM, Divisão Nacional de Producão Mineral, Rio de Janeiro, Brazil IMM, Inner Mongolian Museum, Hohhot, Inner Mongolia, China IPPAS, Institute of Palaeozoology, Polish Academy of Sciences, Warsaw, Poland IRSNB, Royal Belgian Institute of Natural Sciences, Brussels, Belgium. ISEZ, Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Kraków, Poland IVPP, Institute of Vertebrate Paleontology and Paleoanthropology, Beijing, China; MACN, Museo Argentino de Ciencias Naturales, Buenos Aires, Argentina MHNC, Museo de Historia Natural “Alcide d’Orbigny”, Cochabamba, Bolivia; MNHN, Muséum national d’Histoire naturelle, Paris, France NMMNH, New Mexico Museum of Natural History and Science, Albuquerque, New Mexico, USA NMS, National Museum of Scotland, Edinburgh, UK PSS-MAE, Paleontological and Stratigraphy Branch (Geological Institute), Mongolian Academy of Sciences – American Museum of Natural History Expeditions, Ulaanbaatar, Mongolia UCMP, Museum of Paleontology, University of California, Berkeley, California, USA; UF, Vertebrate Paleontology collection, Florida