Supplementary Material for – Geomolecular Dating and the Origin of Placental Mammals Matthew J. Phillips [email protected] Fossil calibration sources 1 Figure S1 3 Alternative Phylogenetic trees 5 Analysis conditions and supplemental results 10 Table S1 10 Table S2 11 Figure S2 16 References 18 Fossil calibration sources Fossil calibrations used in this study draw upon two primary sources. First, the 82 calibration scheme that Meredith et al. (2011) employed for their 26 gene, 169 taxon DNA sequence analyses. Second, the 27 calibration scheme that dos Reis et al. (2012) employed for their genome-scale, 36 taxon analyses. I refer here to these schemes as Mer82a and dR27a, respectively. Variations on these fossil calibration schemes are detailed below. Mer82b Mer82a typically uses the most precise minimum age that can be attributed to a reference fossil (e.g. from a radiometric date), but when this was not possible, the scheme defaults to the younger, minimum geochronological relative age of the strata. This was not the case for four calibrations, for which more precise fossil ages are available and yield older minimum bounds. Mer82b incorporates the following four variations. 1. The Messel bat, Tachypteron franzeni (calibrating Emballonuroidea) is known from radiometric dating to be at least 47.8 +/- 0.2 Ma (Mertz et al. 2004). Mer82a instead used the top of the Middle Eocene, at 40.2 Ma. The appropriate minimum used in Mer82b is 47.6 Ma. 1 2. The Wind River Formation bat, Honrovits tsuwape (calibrating both Yangochiroptera and Natalidae/Vespertilionidae) is known to be of Wasatchian age (Beard et al., 1992), at least 50.3 Ma. Mer82a instead used the top of the Early Eocene, at 48.4 Ma. The appropriate minimum used in Mer82b is 50.3 Ma. 3. The Vastan ankle bones (calibrating Lagomorpha) are regarded as ~53 Ma (Rose et al. 2008), based on associated, diagnostic foraminifera. Mer82a instead used the top of the Early Eocene, at 48.4 Ma. The appropriate minimum used in Mer82b is 52.0 Ma, which covers minor uncertainties in dating (Sahni et al., 2006). 4. A further uncertainty that Meredith et al. (2011) consider, is that mesonychids may be crown artiodactyls. If this is the case, then their usage of stratigraphic bounding would move the maximum bound for Artiodactyla to the base of the Maastrichtian (71.2 Ma), which is used in Mer82b. Mer82c As an alternative to providing new minimum dates for the above four bat and lagomorph calibrations, Mer82c instead excludes these calibrations. This is justified, because in each case the placement of the reference fossil is highly speculative. 1. The Messel bat, Tachypteron franzeni (calibrating Emballonuroidea) is regarded as an emballonurid (Storch, 2002) on the basis of similarities, and under an evolutionary framework that considered emballonurids as sister to rhinolophoids. These two groups are now shown to fall on opposite sides of the chriropteran tree (Teeling et al., 2005), and some other Eocene European bats previously assigned to both Emballonuridae and Rhinolophoidea have since been placed in a new family of uncertain affinities (Maitre et al., 2008). Tachypteron was not considered in that study and has yet to be published in any formal phylogenetic analysis of which I am aware. 2. The Wind River Formation bat, Honrovits tsuwape (calibrating both Yangochiroptera and Natalidae/Vespertilionidae) has been regarded as a member of the morphology-based clade Nataloidea (Morgan and Czaplewski, 2003). Molecular studies (e.g. Teeling et al. 2005) have since rejected Nataloidea at very high significance levels, and distribute its constituent clades widely across Yangochiroptera. Moreover, Honrovits is now considered to be an onychonycterid, falling outside the chiropteran crown altogether (Smith and Habersetzer, 2012). 2 3. The Vastan ankle bones (calibrating Lagomorpha) were found by Rose et al. (2008) to group with leporids, and more specifically, with Oryctolagus to the exclusion of other rabbits and hares. If true, the ankle bones would pre-date molecular dating expectations for the Oryctolagus-Sylvilagus divergence by ~5-fold (Matthee et al. 2004). Rose et al. (2008) noted that the phylogenetic signal may be confounded by functional similarities. Whether any such convergence also falsely promotes the more general crown lagomorph placement for the Vastan material remains to be tested with independent characters. Moreover, the analyses did not consider sampling error, presumably because this was not computationally practical. I have replicated the analysis, with bootstrapping enabled by excluding non-lagomorph taxa (Fig. S1). The relationship of the Vastan ankle bones to crown lagomorphs is unresolved. FIGURE S1. Majority-rule bootstrap consensus tree from 500 replicates based on the lagomorph data of Rose et al. (2008). Minimum crown clade inclusion is shown. Bootstrapping was run in PAUP* 4.0b10 (Swofford, 2002) and employed 20 random-addition heuristic searches per replicate. 3 dR27b dos Reis et al.’s (2012) 27-calibration scheme (dR27a) was also employed, and varied as justified in the main text. dR27b adds maximum bounds for Primates and Chiroptera, and shifts the maximum bound for Rodentia. I follow Barnett et al. (2005) in defining maximum bounds. These cover the time back until relatively well sampled fossil assemblages in potential geographic regions of origin that contain no putative crown group members, but contain stem members or ecological equivalents. Where applicable, numerical conversion of stratigraphic ages follows Ogg et al. (2008). Rodents, Primates and bats provide arguably the best opportunities for tight, yet appropriately conservative maximum bounds to buffer against rate model errors. In each case their earliest crown members are closely preceded by stem members from well-sampled faunas in regions of origin that match molecular biogeographic predictions (Springer et al. 2011, 2012). The first crown rodents appear in Asia (Meng et al. 2003), close to the Paleocene/Eocene boundary. Earlier rodents of Thanetian or possibly late Selandian age (Paramys, Cocomys and Tribosphenomys) are placed among first and second (or third) outgroups to Rodentia by recent cladistic analyses (Asher at al. 2005; Wible et al. 2005; O’Leary et al. 2013). I use the base of the Selandian (61.1. Ma) as a soft maximum bound. Crown Primates appear close to the Paleocene/Eocene boundary in Asia and North America (Smith et al., 2006), or about one million year earlier, if the fragmentary Altiatlasius from Morocco is a primate (Williams et al., 2010). Older mixodectids and Plesiadapiformes of earlier Thanetian and late Selandian age are placed among first and second outgroups to Primates by recent cladistic analyses (Ni et al., 2013). I use the base of the Selandian (61.1 Ma) as a soft maximum bound. At the ordinal level, the global bat fossil record is exceptional among mammals in its potential for providing a tight maximum bound. As flying mammals that filled new ecological space, bats first appear almost simultaneously in North America, Eurasia, Australia, Africa and South America, early in the Ypresian. From their first appearance, bats occur in every subsequent sub-epoch in the fossil records of North America, Eurasia and in at least one of the Gondwanan continents (Gunnell and Simmons, 2005). In all cases (except for fragmentary dental material that cannot be assigned) the first appearances have been assigned stem, rather than crown placements (Simmons et al., 2008, Tabuce et al., 2009; Ravel et al., 2011; Smith et al., 2007). Hence, it is likely that crown Chiroptera 4 originated in the Ypresian. However, I use the base of the next stage (Thanetian) at 58.9 Ma as a more conservative soft maximum bound. Consistent with Mer82c, the dR27b set excludes the questionable lagomorph calibration based on the Vastan ankle bones. The non-mammal and marsupial calibrations from Meredith et al. (2011) are also retained. dos Reis et al. (2012) included only a single marsupial calibration. One further alteration in dR27b is the minimum bound for the divergence of shrews and hedgehogs is based on Litolestes (57.8 Ma, O’Leary et al., 2013), rather than Adunator (61.1 Ma), as employed in the Mer82 sets. Several authors have questioned whether Adunator and similar early Paleocene taxa assigned to Erinaceomorpha instead fall outside Lipotyphla. For example, Novacek et al. (1985) states that Adunator is in “limbo between primitive insectivorans and primitive condylarths”, while Hooker and Russell (2012) surprisingly place Adunator with elephant shrews. Litolestes provides a more conservative bound, although may also require confirmation. Alternative phylogenetic trees MerDNA_tree All divergence analyses were run in MCMCTREE following the method and models used by Meredith et al. (2011) on their most comprehensive (26 nuclear gene, DNA) dataset, with calibration priors soft bound (2.5% prior probability tails). I employed the phylogeny that Meredith et al. (2011) used for their DNA timetrees when direct comparison was appropriate. I refer to this phylogeny as MerDNA_tree. Con_tree Two relationships within the MerDNA_tree are contradicted by the majority of recent molecular studies (e.g. Murphy et al. 2001; Janecka et al. 2007; Phillips et al. 2009; Morgan et al. 2013; Romiguier et a. 2013; Lin et al. 2014), and the most comprehensive, recent morphological studies (e.g. Wible et al. 2009; O’Leary et al. 2013). These are the placements of Scandentia (tree shrews) with rodents and lagomorphs, and sciuromorphs