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Deposited in DRO: 19 June 2019 Version of attached le: Accepted Version Peer-review status of attached le: Peer-reviewed Citation for published item: Asher, R. and Smith, M. R. and Rankin, A. and Emry, R. (2019) 'Congruence, fossils, and the evolutionary tree of rodents and lagomorphs.', Royal Society open science., 6 (7). p. 190387. Further information on publisher's website: http://doi.org/10.1098/rsos.190387

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1 2 3 1 Congruence, fossils, and the evolutionary tree of rodents and lagomorphs 4 5 6 2 Robert J. Asher1, Martin R. Smith2, Aime Rankin1, Robert J. Emry3. 7 8 9 3 1Department of Zoology, University of Cambridge UK, 2Department of Earth Sciences, University of Durham UK, 10 11 4 3Department of Paleobiology, Smithsonian Institution, Washington DC USA 12 13 14 5 Keywords: Phylogenetics, Paleontology, Mammalia, Geomyoidea, Sciuromorpha 15 16 17 18 6 Author for correspondence: Robert J. Asher, r.asher-at-cam.ac.uk 19 20 21 7 1. Summary 22 23 24 8 Given an evolutionary process, we expect distinct categories of heritable data, sampled in ever 25 26 27 9 larger amounts, to converge on a single tree of historical relationships. We tested this assertion 28 29 10 by undertaking phylogenetic analyses of a new morphology-DNA dataset for mammals, 30 31 11 focusing on Glires and including the oldest known skeletons of geomyoid and Ischyromys 32 33 12 rodents. Our results support geomyoids in the mouse-related clade (Myomorpha) and a 34 35 36 13 ricochetal locomotor pattern for the common ancestor of geomyoid rodents. They also support 37 38 14 Ischyromys in the squirrel-related clade (Sciuromorpha) and the evolution of sciurids and 39 40 15 Aplodontia from extinct, "protrogomorph"-grade rodents. Moreover, ever larger samples of 41 42 43 16 characters from our dataset increased congruence with an independent, well-corroborated tree. 44 45 17 Addition of morphology from fossils increased congruence to a greater extent than addition of 46 47 18 morphology from extant taxa, consistent with fossils' temporal proximity to the common 48 49 50 19 ancestors of living species, reflecting the historical, phylogenetic signal present in our data, 51 52 20 particularly in morphological characters from fossils. Our results support the widely held but 53 54 21 poorly tested intuition that fossils resemble the common ancestors shared by living species, and 55 56 57 Asher et al. Congruence, Evolution, and Glires 1 58 59 60 https://mc.manuscriptcentral.com/rsos Royal Society Open Science: For review only Page 4 of 73

1 2 3 22 that fossilizable hard tissues (i.e., bones and teeth) help to reconstruct the evolutionary tree of 4 5 23 life. 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 Asher et al. Congruence, Evolution, and Glires 2 58 59 60 https://mc.manuscriptcentral.com/rsos Page 5 of 73 Royal Society Open Science: For review only

1 2 3 25 2. Introduction 4 5 6 26 The theory of biological evolution leads to many hypotheses about the history of life. Given an 7 8 9 27 evolutionary mechanism in which heritable variation yields biodiversity over geological time, 10 11 28 we expect patterns of relationship derived from independent sources of data to exhibit 12 13 29 similarity, and for that similarity to increase as more data are analyzed with methods capable of 14 15 30 measuring historical signal (1,2). Data typically used for phylogenetic purposes include 16 17 18 31 genotypes and phenotypes, and many subcategories within each, including (but not limited to) 19 20 32 mitochondrial and nuclear DNA exons, introns, retrotransposons and short interspersed nuclear 21 22 33 elements (SINEs), rare genomic changes (RGCs), ultraconserved elements (UCEs), patterns of 23 24 25 34 insertions and deletions (indels), as well as anatomical patterns of development, soft tissues, 26 27 35 bones and teeth. Fossilization almost always reduces data to hard tissues only, but we still 28 29 36 expect that overall, increasing amounts of such phenotypic data should yield patterns of 30 31 32 37 relationship that converge on those based on soft tissues and genotype. Tests of evolution as the 33 34 38 primary mechanism behind biodiversity are numerous and compelling (3). However, the extent 35 36 39 to which anatomical data in general, and fossilizable hard tissues in particular, help or hinder 37 38 40 phylogenetic reconstruction remains contentious. Estimates of morphology's phylogenetic 39 40 41 41 information content range from limited (4) to substantial (5), and that of morphological hard 42 43 42 tissues from biased (6) to statistically consistent (7), at least for some groups. 44 45 46 43 Here, we use biologically and paleontologically well-documented rodents and lagomorphs 47 48 49 44 (collectively known as Glires) to test predictions derived from evolutionary theory. We 50 51 45 assembled a morphological matrix sampling extant and fossil mammals, including the oldest 52 53 46 and most complete skeletons yet known of an Eocene geomyoid (Fig. 1, Heliscomys (8)) and 54 55 56 47 "ischyromyid" (Fig. 2, Ischryomys (9)), as well as previously undescribed cranial fossils of 57 Asher et al. Congruence, Evolution, and Glires 3 58 59 60 https://mc.manuscriptcentral.com/rsos Royal Society Open Science: For review only Page 6 of 73

1 2 3 48 Douglassciurus (10). We combined hard tissue data from 42 fossils with morphological and 4 5 49 molecular characters from 60 extant genera in order to measure, firstly, congruence of 6 7 8 50 topologies derived from increasingly large samples from our dataset to a well-corroborated tree 9 10 51 (Fig. 3), derived from SINEs (11), microRNAs (12: their fig. S5), introns (13), and rare 11 12 52 genomic changes (14), data which played no role in our own original dataset. This tree is 13 14 53 furthermore consistent with an analysis of ultraconserved elements from over 3700 nuclear loci 15 16 17 54 (15), which shows less than 0.002% (8/3700) overlap with the eight nuclear genes in our 18 19 55 original alignment. By "well-corroborated" we mean that one or an extremely small number of 20 21 56 mutually consistent topologies, out of an astronomically huge number of possibilities, result 22 23 24 57 from phylogenetic optimality criteria applied to these datasets. Secondly, we measure the 25 26 58 phylogenetic impact of fossils, extant taxa, and hypothetical ancestors (16) on congruence with 27 28 59 this well-corroborated topology of extant taxa. We hypothesize that if evolution left a historical 29 30 31 60 signal in bones and teeth, then ever-increasing samples from our dataset should converge on the 32 33 61 well-corroborated species tree of mammals in general and Glires in particular. 34 35 36 62 3. Materials and Methods 37 38 39 63 We combined 14 genes (8 nuclear, 6 mitochondrial) with their patterns of insertions and 40 41 42 64 deletions (indels) in a matrix for 60 living genera, yielding an alignment of 15407 nucleotides 43 44 65 and 188 binary indels. Our morphology partition consists of 219 hard tissue characters coded 45 46 66 for those 60 taxa plus another 42 fossils. We coded all taxa for morphological characters 47 48 49 67 (17,18) and incorporated further improvements based on Wible (19), von Koenigswald (20,21) 50 51 68 and digital reconstructions from new specimens based on CT scans taken primarily at the 52 53 69 Cambridge Biotomography Centre (UK) and rendered in 3D with Drishti 2.6.4 (22). Graphic 54 55 56 70 documentation of all character states are available in project 2769 on morphobank.org (23). 57 Asher et al. Congruence, Evolution, and Glires 4 58 59 60 https://mc.manuscriptcentral.com/rsos Page 7 of 73 Royal Society Open Science: For review only

1 2 3 71 DNA+indel characters were unavailable for fossils. Species were chosen to maximize overlap 4 5 72 of morphological data with available DNA sequences. Our sample of 60 extant mammals 6 7 8 73 included a core of 41 genera with known phylogenetic affinities based on SINEs (11), 9 10 74 microRNAs (12), introns (13), and RGCs (14), all of which are independent of the novel 11 12 75 morphological and DNA data in our dataset and are furthermore consistent with analysis of 13 14 76 UCEs noted above (15). The consensus of these studies defines a well-corroborated tree of 15 16 17 77 Mammalia, focusing on rodents and lagomorphs (Fig. 3). Supplementary Information provides 18 19 78 further details on taxon and character sampling, DNA alignment, modeling and character 20 21 79 partitioning, phylogenetic search strategies, Figures S1-S7 and Tables S1-S5. Our 22 23 24 80 morphological matrix and DNA+indel alignments are available in supplementary electronic 25 26 81 appendix S1; appendix S2 provides a summary of character coding edits relative to past 27 28 82 phylogenetic analyses of fossils and morphology (e.g., 17,18); appendix S3 provides species- 29 30 31 83 level taxonomy, museum accession numbers for specimens used to code morphological data 32 33 84 and Genbank accession numbers; appendix S4 provides our optimal topologies in nexus format. 34 35 85 These electronic appendices are available at datadrad.org (24). Tables S1 and S4 summarize our 36 37 86 DNA partitioning scheme, model choice, and indices of convergence for our Bayesian analyses. 38 39 40 41 87 Beck & Baillie (16) used a novel method to incorporate data from living taxa in studies of 42 43 88 fossils where genomic data are generally unavailable. Using the topology from Meredith et al. 44 45 89 (25), they optimized morphological characters at internal nodes and re-included these nodes as 46 47 48 90 taxa for additional phylogenetic analysis. Inclusion of such hypothetical ancestors resulted in a 49 50 91 substantial increase in congruence of topologies generated by morphology alone with that 51 52 92 generated by the 26 nuclear gene fragments from Meredith et al. (25). Ancestors inferred using 53 54 55 93 maximum parsimony (MP) were "somewhat more successful in recovering" (16) well- 56 57 Asher et al. Congruence, Evolution, and Glires 5 58 59 60 https://mc.manuscriptcentral.com/rsos Royal Society Open Science: For review only Page 8 of 73

1 2 3 94 corroborated clades compared to likelihood-inferred ancestors. They assumed the phylogeny of 4 5 95 Meredith et al. (25) to be accurate and regarded hypothetical common ancestors inferred from 6 7 8 96 that phylogeny to represent a "best case scenario" in paleontology; i.e., such ancestors represent 9 10 97 hypotheses of the morphology of real common ancestors from which modern clades have 11 12 98 evolved. Their study was not a test of evolution per se, or a test of the topology proposed by 13 14 99 Meredith et al (25), but it did provide a means by which to increase congruence of topologies 15 16 17 100 derived from fossils with topologies based on living taxa sampled for DNA. The method of 18 19 101 Beck & Baille (16) thus comprises a means to include the phylogenetic information content 20 21 102 from modern taxa in an analysis of fossils, without directly having to sample non-fossilizable 22 23 24 103 data, such as DNA. 25 26 27 104 Here, we used the "describetrees /xout=internal" command in PAUP 4.0a (26) to estimate states 28 29 105 for our 219 morphological characters for each of the 29 internal nodes in the well-corroborated 30 31 32 106 topology (Fig. 3). This tree was based on data noted above (11,12,13,14), independent of the 33 34 107 DNA and morphological data used in our own phylogenetic analyses. We generated character 35 36 108 states assuming both accelerated ("acctran") and delayed ("deltran") transformations and added 37 38 109 these hypothetical terminals to our morphology matrix, and manually ensured that inapplicable 39 40 41 110 character optimizations were coded as such in our hypothetical ancestors. Acctran and deltran 42 43 111 generated similar results (Fig. S1) and we used deltran-optimized ancestral states for the 44 45 112 majority of our analyses (available in nexus format in supplementary appendix S1). We 46 47 48 113 acknowledge that optimizing characters on a topology with polytomies (as evident in our well- 49 50 114 corroborated tree, Fig. 3) may underestimate any uncertainty associated with non-bifurcating 51 52 115 nodes (27). 53 54 55 56 57 Asher et al. Congruence, Evolution, and Glires 6 58 59 60 https://mc.manuscriptcentral.com/rsos Page 9 of 73 Royal Society Open Science: For review only

1 2 3 116 We relied primarily on two quartet metrics to quantify the similarity (amount of information in 4 5 117 common) between the well-corroborated tree (Fig. 3) and each test tree: (a) a symmetric 6 7 8 118 difference measure (28) normalized against the maximum information attainable (function 9 10 119 QuartetDivergence in the Quartet R package (29)) and (b) a shared information measure, which 11 12 120 calculated the number of shared quartets normalized against the number of quartets resolved in 13 14 121 the independent tree, using the function SharedQuartetStatus in the Quartet package. Where 15 16 17 122 taxon samples differed, taxa not held in common were pruned using drop.tip (in ape (30)) 18 19 123 before calculating consensus trees (R script available in supplementary Table S5). We also 20 21 124 made comparisons using two further metrics: (a) Robinson-Foulds distance (RF.dist in the 22 23 24 125 Phangorn package (31)) scaled by proportion resolved, RF.dist (testtree,reftree) / 25 26 126 (Nnode(testtree)/length(testtree$tip.label)-1), and (b) counts of shared partitions using either 27 28 127 Mesquite (32) or with prop.clades in the ape package, sum(prop.clades(testtree,reftree)). All 29 30 31 128 calculations of congruence are relative to the well-corroborated tree (Fig. 3) and derive from 32 33 129 newick-formatted, strict consensus topologies rooted on Didelphis. 34 35 36 130 We evaluated our hypotheses on the phylogenetic information content of hard-tissue data and 37 38 131 fossils through a series of resampling experiments. These iteratively increased the number of 39 40 41 132 sampled DNA characters with and without morphology, and also iteratively increased the 42 43 133 number of sampled fossils, hypothetical ancestors, and sub-sampled living taxa, as detailed 44 45 134 below. 46 47 48 49 135 4. Results 50 51 52 136 Topologies derived from Bayesian (Fig. 4) and parsimony (MP; Fig. S2) searches applied to 53 54 137 our combined dataset, with or without fossil taxa, are highly congruent with the independent, 55 56 57 Asher et al. Congruence, Evolution, and Glires 7 58 59 60 https://mc.manuscriptcentral.com/rsos Royal Society Open Science: For review only Page 10 of 73

1 2 3 138 well-corroborated tree (Fig. 3), and to only a slightly lesser extent to previous paleontological 4 5 139 estimates based on dental data (33). Bayesian analysis (Fig. 4) shows higher congruence by 6 7 8 140 reconstructing primates together to the exclusion of Dermoptera, whereas most MP analyses 9 10 141 with DNA+indels favor Dermoptera-Papio to the exclusion of strepsirhines. 11 12 13 142 In order to explore the extent to which increasingly large subsamples of our dataset converged 14 15 143 on the well-corroborated tree of Mammalia, we undertook a series of resampling experiments 16 17 18 144 that gradually increased the number of sites from our alignment used in phylogenetic analyses. 19 20 145 We ran 50 MP analyses each for 11 different levels of sampling, from 0.2% to 90% coverage of 21 22 146 our alignment. For each level we ran 25 MP analyses with and 25 without morphology and 23 24 25 147 calculated the mean and standard deviation of similarity metrics relative to the well- 26 27 148 corroborated tree (Fig. 3). Both metrics exhibit a clear asymptote towards congruence (Fig. 5). 28 29 149 As more DNA+indel sites are added, the amount of conflict falls and information in common 30 31 32 150 increases. This is limited by the inability of MP analyses without morphology to recover 33 34 151 monophyly of primates relative to Dermoptera, a limitation which is overcome in some 35 36 152 instances with the addition of morphology and in other studies which have a better sample of 37 38 153 primates (7). 39 40 41 42 154 Our morphological dataset consists of 219 characters and cannot be subsampled as extensively 43 44 155 as our DNA+indel alignment. However, it can be subsampled by taxon (below), and we can 45 46 156 also compare how our subsampled DNA+indel analyses perform with and without 47 48 49 157 morphological data. For each of our 11 categories of resampled data, addition of morphology 50 51 158 decreases conflict and increases information in common between a given test tree and the well- 52 53 159 corroborated tree. This improvement can be substantial; randomly subsampled DNA+indel 54 55 56 160 analyses recovered significantly fewer shared quartets with the well-corroborated tree (Fig. 3) 57 Asher et al. Congruence, Evolution, and Glires 8 58 59 60 https://mc.manuscriptcentral.com/rsos Page 11 of 73 Royal Society Open Science: For review only

1 2 3 161 than those same analyses combined with the 219 morphological characters, although 4 5 162 significance varies depending on the test used (Table S2). Mean congruence values improved 6 7 8 163 with the addition of morphological data for all of our subsampled analyses (Fig. 5), and it is 9 10 164 only with the addition of morphology that some MP analyses recovered (for example) a 11 12 165 monophyletic primates to the exclusion of Dermoptera. This well-corroborated signal appears 13 14 166 in our optimal Bayesian trees (Figs. 4, S3A) but not in the largest MP analyses (Figs. S2, S3B). 15 16 17 18 167 4.1 Fossils and hypothetical ancestors improve congruence 19 20 21 168 Gradually increasing the number of sampled fossils and hypothetical ancestors to our dataset of 22 23 169 60 living taxa, using morphological characters alone, increased similarity and reduced conflict 24 25 26 170 (Fig. 6) relative to the well-corroborated tree. The correlation between increased congruence 27 28 171 and increased sampling of taxa known for morphology was highly significant both for fossils 29 30 172 and hypothetical ancestors. In agreement with Beck & Baillie (16), addition of hypothetical 31 32 33 173 ancestors was particularly effective and exhibited increased congruence with a higher slope 34 35 174 (i.e., more congruence per added taxon) compared to fossils (Fig. 6). Although these 36 37 175 morphology-only analyses exhibited increased congruence with added fossils and hypothetical 38 39 176 ancestors, they still show more conflict than topologies derived from the combined or 40 41 42 177 DNA+indel datasets. However, unlike the MP subsampled analyses of our DNA+indel dataset 43 44 178 (Fig. 5), the slope of increased similarity and reduced conflict with the addition of fossils does 45 46 179 not appear to asymptotically approach a limit (Fig. 6), implying that, at least for our dataset, 47 48 49 180 congruence would continue to increase with further addition of fossils. 50 51 52 181 In order to compare the addition of fossils and hypothetical ancestors to addition of living taxa, 53 54 182 we subsampled from our set of 60 extant taxa. The core of 41 taxa in our sample with well 55 56 57 Asher et al. Congruence, Evolution, and Glires 9 58 59 60 https://mc.manuscriptcentral.com/rsos Royal Society Open Science: For review only Page 12 of 73

1 2 3 183 corroborated affinities (Fig. 3) allowed us to divide our dataset into those 41 taxa plus 18 4 5 184 unique sets of one to 18 additional extant taxa (given that there is only one set of all 60 extant 6 7 8 185 taxa, 41+19, in our dataset). We therefore subsampled random combinations (18 iterations 9 10 186 each) of up to 18 other extant taxa, hypothetical ancestors, and fossils in order to further 11 12 187 explore congruence with the well-corroborated tree. Again, this smaller series of added 13 14 188 hypothetical ancestors significantly decreased conflict in terms of normalized symmetric 15 16 17 189 differences (Fig. 7); addition of fossils did not monotonically do so, but addition of 18 distinct 18 19 190 sets of 18 fossils each did result in a higher overall mean number of shared partitions than 18 20 21 191 distinct sets of 1-17 added fossils (Fig. S4). In contrast, successive addition of randomized sets 22 23 24 192 of up to 18 other extant taxa to the 41-taxon set from our well-corroborated tree significantly 25 26 193 increased conflict as measured by either normalized symmetric difference (Fig. 7) or raw 27 28 194 number of shared partitions (Fig. S4) with the well-corroborated tree. Living taxa not among 29 30 31 195 the 41 taxa in the well-corroborated tree (Fig. 3) include both relatively long- (e.g., Cricetomys, 32 33 196 Hystrix, Napaeozapus, Platacanthomys, and Ratufa) and short- (e.g., Arvicanthis, Dipus, 34 35 197 Geomys, and Tamiasciurus) branched taxa, according to our optimal Bayesian tree (Fig. 4). 36 37 38 198 Although morphological characters significantly improved congruence when combined with 39 40 41 199 our DNA+indel dataset, the extent to which they do so in isolation depends greatly on taxon 42 43 200 sample (Figs. S5, S7), character weighting (Fig. S6, S7), and optimality criterion (Figs. S7; 44 45 201 Table S3). Equally weighted MP applied to extant taxa recovers only six out of 28 possible 46 47 48 202 well-corroborated groups (calculated with "shared partitions" in Mesquite (32)). This increases 49 50 203 to 12 with a Bayesian analysis of morphology only, 13 with MP and fossils, and 17 with either 51 52 204 MP and hypothetical ancestors or Bayesian analysis of morphology including fossils. Implied 53 54 55 205 weights in TNT (34,35) improved congruence substantially for morphology of extant taxa 56 57 Asher et al. Congruence, Evolution, and Glires 10 58 59 60 https://mc.manuscriptcentral.com/rsos Page 13 of 73 Royal Society Open Science: For review only

1 2 3 206 alone, slightly for the combined dataset, and not at all for DNA+indels for extant taxa alone 4 5 207 (Fig. S6). Using just morphological data from extant taxa, the number of shared partitions 6 7 8 208 increased from six to 13 with implied weighting concavity (k) constants of 2, 4, 6, or 8 and 9 10 209 dropped off to seven with k from 32 to 512. For weighted MP analyses including fossils, the 11 12 210 number of shared partitions increased from 13 to 16 when k = 24 and returned to 13 with k at 13 14 211 128, 256, or 512. Weighted MP analyses of the combined dataset of living and fossil taxa 15 16 17 212 increased number of shared partitions from 26 to 27 for all k values we explored from 2 to 512. 18 19 213 Unlike the equally weighted analysis, implied weights consistently placed the fossils Anagale, 20 21 214 Pseudictops, and Anagalopsis with Rhynchocyon outside Euarchontoglires, resulting in 27 22 23 24 215 rather than 26 shared partitions with the well-corroborated tree. For DNA+indels of extant taxa 25 26 216 alone, MP recovered 27 of 28 possible shared partitions under equal weighting and all k values 27 28 217 we explored (Fig. S6). 29 30 31 32 218 The use of hypothetical ancestors as terminals in our dataset improved congruence, nearly 33 34 219 tripling the number of well-corroborated groups from six in the topology based on extant taxa 35 36 220 only to 17 (Fig. S5). Inclusion of hypothetical ancestors in our morphology-only analysis 37 38 221 recovers such groups as diprotodonts, placentals, euarchontoglires, glirids, sciuroids and 39 40 41 222 Aplodontia, myodonts, and geomyoids. MP applied to morphological data including fossils also 42 43 223 recovered diprotodonts and Eutheria (showing some Cretaceous non-placental eutherians in a 44 45 224 polytomy with extant placentals), along with a monophyletic Glires, Rodentia, and 46 47 48 225 Ctenohystrica, but did not recover myomorphs, sciuromorphs, or major groups within the 49 50 226 former such as myodonts or geomyoids. 51 52 53 227 4.2 The evolution of rodents and lagomorphs 54 55 56 57 Asher et al. Congruence, Evolution, and Glires 11 58 59 60 https://mc.manuscriptcentral.com/rsos Royal Society Open Science: For review only Page 14 of 73

1 2 3 228 Our dataset supports the same basic phylogenetic structure for Mammalia and Glires (Figs. 4, 4 5 229 S2) as do independent studies of phylogenetic data that we did not sample (Fig. 3). For 6 7 8 230 example, Placentalia is monophyletic; Glires, primates, and Tupaia share a common ancestor to 9 10 231 the exclusion of Rhynchocyon; rodents and lagomorphs are sister taxa; Ochotona is the sister 11 12 232 taxon to Leporidae within Lagomorpha. Extant lagomorphs, in turn, comprise the crown 13 14 233 radiation within Duplicidentata, which also includes early Eocene fossils such as Gomphos. The 15 16 17 234 total clade including Rodentia is Simplicidentata and includes taxa such as Rhombomylus 18 19 235 (unresolved in equally weighted MP; Fig. S2), followed by fossils such as Tribosphenomys and 20 21 236 Paramys, then crown Rodentia. Our Bayesian topology weakly supports the sciuromorph clade 22 23 24 237 at the rodent root (Fig. 4). 25 26 27 238 The articulated skeleton of Heliscomys (Fig. 1) sheds light on the early evolution of the 28 29 239 myomorph (mouse-related) clade. Cope (36) first described Heliscomys vetus based on a lower 30 31 32 240 jaw with four teeth from the Oligocene Cedar Creek beds of Colorado, USA. Until now, the 33 34 241 most complete, published material of Heliscomys consisted of cranial fragments (8). Our 35 36 242 skeleton (USNM PAL 720183) represents H. ostranderi and is preserved in a nodule collected 37 38 243 from the Chadronian (late Eocene) locality of Jenny's Pocket at Flagstaff Rim, Wyoming (8). 39 40 41 244 MicroCT scanning reveals a skull and jaws, associated vertebral column and proximal ribs, 42 43 245 forelimbs and articulated manus, displaced but associated hindlimbs with articulated pes, a 44 45 246 partial femur, and an innominate, and enabled us to code 182 of 219 morphological characters 46 47 48 247 in our matrix. 49 50 51 248 Our analysis is the first based on a combined DNA+morphology matrix to place Heliscomys 52 53 249 with extant geomyoid rodents near the base of the myomorph (mouse-related) radiation. It also 54 55 56 250 confirms previous interpretations that Heliscomys is related to geomyoids (8,37). Our data 57 Asher et al. Congruence, Evolution, and Glires 12 58 59 60 https://mc.manuscriptcentral.com/rsos Page 15 of 73 Royal Society Open Science: For review only

1 2 3 251 further render "heteromyids" paraphyletic, a possibility implied by the interpretation of 4 5 252 Heliscomys as ancestral to all living geomyoids (37) and by patterns of cranial vasculature in 6 7 8 253 extant species (38). Our most congruent phylogenetic analysis (Fig. 4) shows Heliscomys and 9 10 254 the extant Dipodomys outside the remaining "heteromyid"-grade taxa in our sample 11 12 255 (Heteromys, Perognathus, and Chaetodipus). All of our Bayesian and MP analyses support 13 14 256 "heteromyid" paraphyly as well as a terrestrial, ricochetal ancestral morphology for the two 15 16 17 257 ecologically very different geomyoid groups alive today: fossorial geomyids and terrestrial 18 19 258 "heteromyids". The ricochetal locomotion in extant "heteromyids" is in fact a phylogenetically 20 21 259 ancient feature of the geomyoid clade. In our taxon sample, Heliscomys is the earliest-diverging 22 23 24 260 geomyoid, but shows an elongate metatarsus and some reduction of the lateral digits, 25 26 261 particularly digit I which shows a proximal phalanx terminating before the distal end of the 27 28 262 adjacent metatarsal II (Fig. 1). Our results also support the placement of Castor with 29 30 31 263 geomyoids, consistent with Castorimorpha (39) and supported by (among other features) shared 32 33 264 possession of an elongate infraorbital canal (Fig. 1). 34 35 36 265 Our study also includes data from the oldest articulated skeleton yet known for Ischryomys 37 38 266 (Fig. 2; USNM PAL 617532), from the Duchesnean (Late Eocene) of West Canyon Creek, 39 40 41 267 Wyoming (9). Ischyromys is related to sciuromorphs. None of our Bayesian or MP topologies 42 43 268 recovered a monophyletic "Ischyromyoidea", a grade of fossil rodents sometimes classified 44 45 269 together in the literature (40). Instead, early Eocene "ischyromyoids" such as Paramys fall 46 47 48 270 outside crown Rodentia (Fig. 4). CT scanned petrosals of the late Eocene Douglassciurus along 49 50 271 with data from its skull and skeleton (10) place this taxon close to Ischyromys among extant 51 52 272 sciuromorphs. As originally described (10), Douglassciurus (then known as "Protosciurus" 53 54 55 273 (41)) is indeed "the oldest fossil squirrel", reconstructed at the base of Sciuridae in a polytomy 56 57 Asher et al. Congruence, Evolution, and Glires 13 58 59 60 https://mc.manuscriptcentral.com/rsos Royal Society Open Science: For review only Page 16 of 73

1 2 3 274 with Aplodontia, sister to sciurids. Both Ischyromys and Douglassciurus resemble most extant 4 5 275 sciurids, and differ from Paramys, in possessing features such as a lower molar entoconid 6 7 8 276 anterior to the hypoconid, prominent medial curvature to the angular process of the jaw, and 9 10 277 septae within the auditory bulla (Fig. 2). Contra previous literature (42), all Ischryomys crania 11 12 278 sampled with microCT for this study (Fig. 2, appendices S2, S3) exhibit a promontory groove 13 14 279 for the stapedial artery traversing the middle ear. 15 16 17 18 280 5. Discussion 19 20 21 281 An evolutionary mechanism behind life's biodiversity predicts that patterns of relationship 22 23 282 derived from independent sources of data should exhibit similarity (2) and that ever-larger 24 25 26 283 samples analyzed with appropriate methods should increase confidence in that similarity (1). 27 28 284 Our results bear out both expectations; optimal topologies derived from independent data are 29 30 285 highly congruent with the well-corroborated tree, and congruence increases with larger samples 31 32 33 286 from our DNA+indel+morphology dataset (Fig. 5) or more fossil taxa (Fig. 6). It is worth 34 35 287 underscoring that the well-corroborated tree (Fig. 3), approximated by increased sampling from 36 37 288 our DNA+indel+morphology dataset, is one of an astronomically huge number of possible 38 39 289 topologies and was reconstructed from data (11,12,13,14) not used in any of our phylogenetic 40 41 42 290 analyses (Figs. 4-7, Figs. S2, S3, S5). 43 44 45 291 Compared to extant taxa, fossils exist closer in geological time to the common ancestors 46 47 292 hypothesized to exist by evolutionary theory. Therefore, fossils potentially have a greater 48 49 50 293 resemblance than living taxa to these common ancestors (43,44). When added to a given 51 52 294 phylogenetic study in ever-increasing numbers, and assuming a general, positive correlation 53 54 295 between time and the probability of character change (45), one would expect that the addition 55 56 57 Asher et al. Congruence, Evolution, and Glires 14 58 59 60 https://mc.manuscriptcentral.com/rsos Page 17 of 73 Royal Society Open Science: For review only

1 2 3 296 of fossil phenotypes to a given taxon sample will increase similarity to the well-corroborated 4 5 297 tree to a greater extent than addition of living phenotypes. This is similar to the observation that 6 7 8 298 most methods reconstruct phylogenetic history more accurately with an abundance of short- 9 10 299 rather than long-branched taxa (1,46,47). Fossils can succeed in improving phylogenetic 11 12 300 accuracy due to their capacity to break up long branches (43,44). Stated differently, the living 13 14 301 mammals sampled here are removed from the Mesozoic and Paleogene common ancestors 15 16 17 302 shared with other living taxa by many millions of years, meaning that there has been 18 19 303 substantially more time for patterns of historical relationship to have been obscured by 20 21 304 evolutionary change among living taxa than among Paleogene fossils. 22 23 24 25 305 The morphology of hypothetical ancestors optimized on a well-corroborated tree derived from 26 27 306 genomic data (Fig. 3) is independent of whatever one might find in the fossil record. Such 28 29 307 ancestors comprise a novel means to incorporate an otherwise inaccessible, genomic signal into 30 31 32 308 phylogenetic studies of fossils (16). An optimal topology based on a morphological dataset that 33 34 309 includes hypothetical ancestors represents a minimum path of change for anatomical characters 35 36 310 given the well-corroborated topology by which such ancestors were estimated. Therefore, while 37 38 311 of substantial utility to incorporate a genomic signal into studies of fossils that would otherwise 39 40 41 312 lack such a signal (16), increased congruence with the addition of such hypothetical ancestors is 42 43 313 in some sense trivial. We expect to observe increased resemblance towards that topology as 44 45 314 more hypothetical ancestors are sampled. What is not trivial, and indeed a key finding of this 46 47 48 315 study, is that increased sampling of fossils results in more topological congruence than 49 50 316 increased sampling of extant taxa (Figs. 7, S4). The morphology observed in fossils is 51 52 317 independent of morphology of hypothetical ancestors estimated by optimizing characters on a 53 54 55 318 well-corroborated tree of living taxa (Fig. 3). Addition of fossils and hypothetical ancestors 56 57 Asher et al. Congruence, Evolution, and Glires 15 58 59 60 https://mc.manuscriptcentral.com/rsos Royal Society Open Science: For review only Page 18 of 73

1 2 3 319 known for just morphological characters has a net positive effect on congruence (Figs. 6, 7), 4 5 320 whereas addition of extant taxa decreases congruence (Fig. 7). We interpret this as evidence 6 7 8 321 that paleontology supports the morphological reconstructions of common ancestors 9 10 322 hypothesized based on a well-corroborated topology. This is a quantitative example of the 11 12 323 qualitative observation (48) that fossils mix phenotypic features intermediate between daughter 13 14 324 species and the common ancestors they share. Accordingly, fossils help to constrain the 15 16 17 325 morphological transformation series through which extant taxa have passed relative to that 18 19 326 common ancestor (49). 20 21 22 327 Addition of living taxa generally has the effect of improving phylogenetic accuracy (50), not 23 24 25 328 least because of the huge amounts of data typically available for them. However, with the 26 27 329 important qualification that evolutionary rate can vary between and within lineages, living taxa 28 29 330 known for morphology alone potentially have a greater handicap in phylogenetic reconstruction 30 31 32 331 than most fossils or hypothetical ancestors; they are neither temporally close to geologically 33 34 332 distant branching events, nor are they defined by morphological character states optimized on a 35 36 333 well-corroborated tree. We do not claim that addition of morphological data from extant taxa 37 38 334 necessarily decreases congruence, as indeed there is evidence that such addition can improve it 39 40 41 335 (44). Rather, our expectation is that, assuming some consistency of evolutionary rate across 42 43 336 branches and over time, addition of morphological data from fossils to a given phylogenetic 44 45 337 study should generally result in more congruence compared to the addition of morphological 46 47 48 338 data from living taxa. Our results bear out this expectation (Fig. 7) 49 50 51 339 6. Conclusions 52 53 54 55 56 57 Asher et al. Congruence, Evolution, and Glires 16 58 59 60 https://mc.manuscriptcentral.com/rsos Page 19 of 73 Royal Society Open Science: For review only

1 2 3 340 Our data support the interpretation, originally made based on much more limited and 4 5 341 fragmentary fossil remains, that Heliscomys is a geomyoid, that "heteromyids" are paraphyletic, 6 7 8 342 and that extant geomyoids share a ricochetal common ancestor and are part of the myomorph 9 10 343 radiation of Rodentia. Our analysis also supports the association of Ischyromys and 11 12 344 Douglassciurus with the clade of extant sciuromorphs (including Aplodontia) and the paraphyly 13 14 345 of "ischyromyoids". Less decisively, our data also support the sciuromorph root favored by an 15 16 17 346 independent SINE dataset (11). In this study, genomic data such as SINEs (11), microRNAs 18 19 347 (12), introns (13), and RGCs (14) independently defined the well-corroborated tree and thus 20 21 348 needed to be kept separate in order to be able to measure congruence in our dataset without 22 23 24 349 circularity. However, an obvious step for future work would be to combine these datasets and 25 26 350 thereby resolve further, high-level questions about mammalian phylogeny with a yet-larger 27 28 351 pool of data, one which would further enable phylogenetic reconstructions of both living and 29 30 31 352 fossil species (7,51). More generally, and as predicted by evolution, our study confirms that 32 33 353 distinct bodies of heritable data, such as morphology and DNA, converge towards a single 34 35 354 mammalian tree of life. Even when reduced to hard tissues via fossilization, morphology 36 37 355 positively contributes to identifying the tree-like pattern by which life has evolved. 38 39 40 41 356 Acknowledgements 42 43 44 357 We have many individuals to thank, in alphabetical order by institution, for helping to support 45 46 358 our research. For access to key living and fossil specimens we thank Ewan St. John Smith 47 48 49 359 (Cambridge), Doug Boyer and Arianna Harrington (Durham USA), Pip Brewer, Jerry Hooker, 50 51 360 and Roberto Portela Miguez (London), Pierre-Henri Fabre and Lionel Hautier (Montpellier), 52 53 361 Meng Jin (New York), Roger Benson (Oxford), Anthony Herrel (Paris), Brian Kraatz 54 55 56 362 (Pomona), Ornella Bertrand and Mary Silcox (Toronto), Cathrin Pfaff (Vienna), and Jennifer 57 Asher et al. Congruence, Evolution, and Glires 17 58 59 60 https://mc.manuscriptcentral.com/rsos Royal Society Open Science: For review only Page 20 of 73

1 2 3 363 Strotman (Washington). We are also grateful to the online resources at digimorph.org (Austin) 4 5 364 and morphosource.org (Durham USA). For discussion of phylogenetic methods and software 6 7 8 365 we thank Seraina Klopfstein (Basel), Joe Keating and Robert Sansom (Manchester), and Robin 9 10 366 Beck (Salford). We thank Stuart Rankin, Jeffrey Salmond (Cambridge) and Caroline Willich 11 12 367 (Cambridge, Ulm) for assistance with the University of Cambridge HPC cluster. We are 13 14 368 grateful to Robert Sansom and two anonymous reviewers for their constructive comments on 15 16 17 369 the manuscript. 18 19 20 370 Data Accessibility 21 22 23 371 Our supplementary data section includes two additional files. "Asher_openSci- 24 25 26 372 suppdata_V2.doc" contains details on systematic and phylogenetic methods and Table S1 27 28 373 (optimal Bayesian partition scheme), Table S2 (proportion shared quartets for subsampled 29 30 374 DNA+indel+morphology datasets), Table S3 (summary of optimal MP topologies), Table S4 31 32 33 375 (summary of burnin values and indices of convergence for our Bayesian analyses), and Table 34 35 376 S5 (R script to calculate tree similarity using Quartet package). "Asher-openSci-suppFigs.pdf" 36 37 377 contains Fig. S1 (acctran & deltran shared partitions), Fig. S2 (combined data strict consensus 38 39 378 MPT), Fig. S3 (optimal Bayesian & MP topologies for extant taxa), Fig. S4 (shared partitions 40 41 42 379 with well-corroborated tree resulting from one to 18 added hypothetical ancestors, extant, and 43 44 380 fossil taxa), Fig. S5 (optimal MP topologies from morphology), Fig. S6 (shared partitions with 45 46 381 well-corroborated tree resulting from MP implied weights), and Fig. S7 (shared partitions with 47 48 49 382 well-corroborated tree across datasets and analysis parameters). 50 51 52 383 A downloadable morphological matrix and graphic documentation of all morphological 53 54 384 character states are available in project 2769 on morphobank.org/permalink/?P2769 (23). 55 56 57 Asher et al. Congruence, Evolution, and Glires 18 58 59 60 https://mc.manuscriptcentral.com/rsos Page 21 of 73 Royal Society Open Science: For review only

1 2 3 385 Four appendices are available via Dryad doi.org/10.5061/dryad.3840vd7 (24), and review link 4 5 386 here: https://datadryad.org/review?doi=doi:10.5061/dryad.3840vd7. Appendix S1 is an archive 6 7 8 387 with DNA alignment, indels, combined data matrix, and hypothetical ancestors in nexus format. 9 10 388 Appendix S2 is a spreadsheet with morphological character edits. Appendix S3 is a spreadsheet 11 12 389 with genus-species names, museum numbers, & DNA accession numbers. Appendix S4 13 14 390 provides optimal trees in nexus format. 15 16 17 18 391 Competing Interests 19 20 21 392 We have no competing interests. 22 23 24 393 Authors' Contributions 25 26 27 28 394 RJA conceived the project. The text was written by RJA and MS, with additional edits to text 29 30 395 and figures from AR and RJE. RJA, RJE and AR collected the data; RJE found and collected 31 32 396 USNM 617532 and USNM 720183 and made available further USNM specimens; RJA 33 34 35 397 undertook CT scans; RJA and MS analyzed the data and made the figures. 36 37 38 398 Funding 39 40 41 399 Our work has been supported by the Department of Zoology, University of Cambridge, the 42 43 400 University of Durham, and the Smithsonian Institution. 44 45 46 47 401 Research Ethics 48 49 50 402 Our work involved no live animals and we were not required to complete an ethical assessment 51 52 403 prior to conducting our research. Paleontological fieldwork was carried out in accordance to 53 54 55 404 national and regional laws. 56 57 Asher et al. Congruence, Evolution, and Glires 19 58 59 60 https://mc.manuscriptcentral.com/rsos Royal Society Open Science: For review only Page 22 of 73

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Figure 1. Heliscomys ostranderi (USNM PAL 720183) from the Chadronian (latest Eocene) of Jenny’s Pocket, 46 Flagstaff Rim, Wyoming. Virtually dissected, CT-scanned images are (clockwise from upper left) block in 47 which specimen was found with elements colored therein; left hindlimb in medial view; skull in ventral, lateral and dorsal views; left jaw in lateral, occlusal and medial views; right forelimb in medial view; partial 48 scapula in dorsal, ventral and lateral views. ioc = anterior entrance of infraorbital canal, p1 = proximal 49 phalanx of pedal digit I, p5 = proximal phalanx of pedal digit V. Scalebars = 5mm. 50 51 209x296mm (300 x 300 DPI) 52 53 54 55 56 57 58 59 60 https://mc.manuscriptcentral.com/rsos Royal Society Open Science: For review only Page 40 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Figure 2. Ischyromys sp. USNM PAL 617732 from the Duchesnean (late Eocene) of West Canyon Creek, 46 Wyoming. Virtually dissected skeleton in original matrix (right), skull in ventral view with R & L dP4-M3 and 47 cutaway into right auditory bulla (top left), left jaw in occlusal view with dp4-m3 (bottom left). Ec = entoconid, hc = hypoconid, ja = angle of the jaw, s = intrabullar septae, sg = stapedial groove. Scalebars = 48 10mm. 49 50 209x296mm (300 x 300 DPI) 51 52 53 54 55 56 57 58 59 60 https://mc.manuscriptcentral.com/rsos Page 41 of 73 Royal Society Open Science: For review only

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Figure 3. Well-corroborated mammalian phylogenetic tree, focusing on Glires, based on consensus from 46 SINEs (11), microRNAs (12), introns (13), and RGCs (14). Ct = Ctenohystrica, Di = Diprotodontia, Eu = 47 Euarchontoglires, Ge = Geomyoidea, Gl = Glires, La = Lagomorpha, My = Myomorpha, Pl = Placentalia, Pr = Primates, Ro = Rodentia, Sc = Sciuromorpha. Primates-Dermoptera are shown in light gray, Lagomorpha in 48 dark gray, Sciuromorpha in green, Myomorpha in orange, and Ctenohystrica in red. Numbers represent 49 hypothetical ancestors onto which morphological characters were optimized (see Appendix S1). Branch 50 lengths are arbitrary. 51 52 209x296mm (300 x 300 DPI) 53 54 55 56 57 58 59 60 https://mc.manuscriptcentral.com/rsos Royal Society Open Science: For review only Page 42 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Figure 4. Majority rule consensus of 17,500 post-burnin (50% of 35G generations sampling every 1000) 46 Bayesian topologies based on DNA+indel+morphology dataset with posterior probabilities shown as 47 percentages adjacent to each node. Resolved, internal nodes without a number have a posterior probability of 1.0 (= 100%). Bold indicates fossils; branch lengths within Placentalia (demarcated with "PL") correspond 48 to scale at bottom. Colors and high-level taxon abbreviations are as in Fig. 3. Burn-in values defined in 49 Table S4 yield the same topology and similar support values, except for collapsing the "50" node within the 50 mouse-related clade identified with the asterisk. 51 52 209x296mm (300 x 300 DPI) 53 54 55 56 57 58 59 60 https://mc.manuscriptcentral.com/rsos Page 43 of 73 Royal Society Open Science: For review only

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Figure 5. Proportion of shared quartets (orange squares), and normalized symmetric difference (blue 46 circles), between strict-consensus MP topologies of extant taxa relative to the well-corroborated tree (Fig. 47 3). X-axis shows 2%, 5%, 10%, 20%, ... 90% resamples of 15596 DNA+indel characters (each consisting of 25 sets of randomly chosen sites) with (dark shades) and without (light shades) 219 morphological 48 characters. Y-axis error bars show one standard deviation on either side of the mean (polygons). 49 50 209x296mm (300 x 300 DPI) 51 52 53 54 55 56 57 58 59 60 https://mc.manuscriptcentral.com/rsos Royal Society Open Science: For review only Page 44 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Figure 6. Shared quartets (top) and normalized symmetric difference (bottom) between the well- 46 corroborated tree and strict consensuses generated by equal weights MP analysis of morphological data from 47 60 extant taxa, with 25 analyses per N added fossils (circles, pale) or hypothetical ancestors (squares, dark) on X-axis. Vertical error bars represent one standard deviation on either side of the mean (polygons). 48 49 209x296mm (300 x 300 DPI) 50 51 52 53 54 55 56 57 58 59 60 https://mc.manuscriptcentral.com/rsos Page 45 of 73 Royal Society Open Science: For review only

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Figure 7. Normalized symmetric difference between the well-corroborated tree (Fig. 3) and strict 46 consensuses generated by equal weights MP analysis of morphological data for 41 taxa in well-corroborated 47 tree plus random combinations of 1–18 added extant species (triangles), fossils (circles), or hypothetical ancestors (squares), each sampling 18 replicates per number of added taxa on X-axis. Vertical error bars 48 represent one standard deviation on either side of the mean (polygons). Adjusted R-squared and 49 significance values based on linear models (lm) calculated for each pair of x & y axes in R. 50 51 215x279mm (300 x 300 DPI) 52 53 54 55 56 57 58 59 60 https://mc.manuscriptcentral.com/rsos