ORIGINAL RESEARCH published: 15 March 2021 doi: 10.3389/fevo.2021.642814 Tempo and Mode: Evidence on a Protracted Split From a Dense Fossil Record Yuri Kimura 1,2,3† , Lawrence J. Flynn 4* † and Louis L. Jacobs 5† 1 National Museum of Nature and Science, Tsukuba, Japan, 2 Department of Geology and Paleontology, National Museum of Nature and Science, Tsukuba, Japan, 3 Institut Català de Paleontologia Miquel Crusafont, ICTA-ICP, Edifici Z. Carrer de les Columnes, s/n., Campus de la Universitat Autònoma de Barcelona, Barcelona, Spain, 4 Department of Human Evolutionary Biology, Harvard University, Cambridge, MA, United States, 5 Roy M. Huffington Department of Earth Sciences, Southern Methodist University, Dallas, TX, United States Fossil records generally inform paleobiologists about extinct taxa and rates of evolution measured at the scale of millions of years. Good records that are densely sampled through time can reveal species level details such as longevity in local sections. Yet fossil data normally do not address details of lineage microevolution because the density Edited by: through time of lineage sampling is insufficient to perceive patterns at a precision finer Irina Ruf, than 106 years in most cases. This study concerns details of a splitting event in the Senckenberg Research Institute and Natural History Museum evolution of murine rodents, an event for which multiple fossil samples dated to a Frankfurt, Germany precision of 105 years fortuitously document the tempo and mode of origin of sister Reviewed by: species, the stems of two extant tribes of mice. Evolution of early Murinae in the northern Paloma López-Guerrero, Universidad Complutense de Madrid, part of the biogeographically restricted Indian subcontinent between 11.6 and 10.5 Ma Spain involved cladogenesis of two crown taxa, the extant tribes Murini and Arvicanthini. Large Wilma Wessels, samples of fossil rodent teeth document their divergence from a common morphological Utrecht University, Netherlands pool. Definitive basal Murini and Arvicanthini at 10.5 Ma are similar in size and differ by *Correspondence: Lawrence J. Flynn subtle features of the dentition. Those features occur sporadically in the common pool of ljflynn@fas.harvard.edu older fossil teeth at 11.2, 11.4, and 11.6 Ma as inconsistent polymorphisms. Interpreted † These authors have contributed as a single lineage in the 11.6–11.2 Ma interval, variability of this abundant murine equally to this work and share first authorship incorporated the roots of the two crown tribes. The pattern through time suggests morphological stasis for several hundred thousand years prior to splitting. This special Specialty section: case informs us on one example of evolution and shows that the tempo of splitting This article was submitted to Paleontology, evolution in some cases may be measured in hundreds of thousands of years, followed a section of the journal by stasis once daughter species have differentiated morphologically. Frontiers in Ecology and Evolution Keywords: speciation, evolution, Murinae, Miocene, Siwaliks, morphology Received: 16 December 2020 Accepted: 19 February 2021 Published: 15 March 2021 INTRODUCTION Citation: Kimura Y, Flynn LJ and Jacobs LL Gould (1985) and Reznick and Ricklefs (2009) discussed the importance of the fossil record (2021) Tempo and Mode: Evidence on a Protracted Split From a Dense for evolutionary biology in unveiling major events, especially those under the category of Fossil Record. macroevolution. Generally, paleontology does not address details on how evolution proceeds at the Front. Ecol. Evol. 9:642814. population level because relevant data are not sampled densely enough in space and time. In special doi: 10.3389/fevo.2021.642814 circumstances, however, the fossil record may be detailed enough to address aspects of evolution Frontiers in Ecology and Evolution | www.frontiersin.org 1 March 2021 | Volume 9 | Article 642814 Kimura et al. Tempo and Mode that lie between macro- and microevolution, such as species of mammals in existence, the true mice or Murinae. This group is longevity, changes in abundance, and rates of morphological native to the Old World, including Africa, Australia, and Pacific change. The potential for approaching microevolutionary islands, and global as commensals. It contains the laboratory patterns through the fossil record is great for muroid rodents mouse (Mus) and laboratory rat (Rattus), long the subject of in cases where stratigraphy and dating are well resolved, and innumerable studies (da Costa et al., 2019), the former with more multiple large samples of individuals are available. than forty living wild species (Suzuki, 2020) and the latter with a Resolution of muroid rodent phylogenetics has had a quantum confusingly large number of cryptic species (Pages et al., 2010; advance with the application of mitochondrial and nuclear Rowe et al., 2011). Because fossil murines are known mainly genetic analyses. Steppan and Schenk (2017) resolved a massive from isolated teeth, the spectrum of morphological characters is amount of data to develop a well-supported hypothesis on limited, which restricts the application of standard morphology- relationships of over 900 species. This and previous works based cladistic analysis. However, the backbone provided by (e.g., Lecompte et al., 2008) distinguished major clades of molecular phylogenies of the vast number of living species allows muroids at the family, subfamily, and tribe levels, including fossil mice to be related confidently. tribes Arvicanthini and Murini, both of which have fossil The fossil mice from Pakistan have long been recognized members in South Asia. as relevant to calibrating molecular evolutionary hypotheses Here we set out to interpret a unique fossil record for its (Jacobs and Pilbeam, 1980; Jacobs and Flynn, 2005), but earlier relevance to a splitting event that marked the origin of the extant studies have been much improved upon by more comprehensive murine rodent tribes Arvicanthini and Murini. Sediments from work (e.g., Kimura et al., 2015; Aghová et al., 2018) and the the Siwalik Group of Pakistan yield a well dated and resolved addition of newly studied fossil samples (e.g., Kimura et al., series of fossil mouse assemblages. We have developed the alpha 2017). Divergence dates from Pakistan have been useful in taxonomy of these species in a series of papers (Jacobs, 1977, molecular sequence studies applied to biogeography, ecology, 1978; Cheema et al., 2000; Kimura et al., 2015, 2017) and specified and evolutionary patterns (e.g., Rowe et al., 2016, 2019; Bryja the timing of morphological divergence and the emergence of et al., 2017), as well as studies of pathogens and genetic features involved in that divergence (Kimura et al., 2013a,b; mechanisms (e.g., Casola, 2018; Ma et al., 2018), and many Flynn et al., 2020). Early late Miocene Siwalik species assigned other topics. This study contributes by providing a more explicit to the stem genera Karnimata and Progonomys, respectively, description of a fossil-based cladogenic event in a precise fortuitously capture the rise of distinctive morphological features chronological framework. that by 10.5–10 million years ago (Ma) characterize dentitions of members of the tribes Arvicanthini and Murini. These features occur in fossil samples from the same geographic area before MATERIALS AND METHODS 11 Ma (Flynn et al., 2020), but inconsistently so that species level taxonomy at that age is problematic. The source of the fossil data is a biostratigraphy of successive We contend that the common origin of the Arvicanthini and rodent sites from the Miocene formations of the Siwalik Group Murini was recorded in South Asia in Siwalik fossil samples of South Asia. Here we focus on Miocene age sites that are between 12 and 11 Ma. The distinct morphologies of the tribes dated 12.5–10 Ma. Each site produces a sample of isolated expressed clearly in fossil species of Karnimata and Progonomys rodent teeth concentrated most likely by avian predators of the dating to 10.5 Ma and younger occur across a mix of individuals time. Sites contain remains of individuals that died (and are in the 11.6–11.2 Ma interval that are otherwise inseparable. These time-averaged) over a decadal to centennial scale. The fossil appear to be polymorphisms that were stable for hundreds of rodent fauna is represented by squirrels, primitive burrowing thousands of years and later characterized the extant tribes. rats, hamsters, gerbils, gundis, dormice, and dominant true mice. Our purpose is to document the simultaneous existence of The series of sites that captures the cladogenic event spans at least arvicanthin and murinin morphologies in a series of samples 400,000 years from 11.6 to 11.2 Ma. Fossil sites are concentrated from single localities of early late Miocene age, discuss the in a relatively small area of 200 km2 near Chinji and Nagri relevance of their splitting origin within a refined chronology villages on the Potwar Plateau of northern Pakistan (area B in of mouse evolution, and thereby address the tempo and mode Barry et al., 2002: their Figure 1). of this special event recorded in the fossil record. We interpret these fossils to represent the common ancestral lineage for the Geology two living mouse tribes. Possibly, they represent individuals of The Potwar Plateau in northern Pakistan presents an closely related cryptic species that did not differ in any other extraordinarily continuous record of Siwalik sediments shed features preserved in the fossils. In either case the time frame from deformed highlands to the north, the present day mountain for morphological separation of the two tribes is measured ranges bordering the Tibetan Plateau. Drainages carrying in hundreds of thousands of years. detritus southward continue to the present day as components The significance of understanding the tempo and mode of the of the mighty Indus River system. The highlands, principally specific cladogenic event we are studying is that it provides a more related to the Himalayan and Hindu Kush mountain ranges, confident and precise date for an evolutionary divergence that have been uplifted continuously throughout the Cenozoic Era is relevant for calibrating the molecular phylogeny of essentially by the tectonic forces that thrust the Indian Plate northward every living species of one of the most diverse and speciose groups under the southern margin of Asia. Currently at 33◦ North Frontiers in Ecology and Evolution | www.frontiersin.org 2 March 2021 | Volume 9 | Article 642814 Kimura et al. Tempo and Mode FIGURE 1 | Age and stratigraphic sequence of Potwar Plateau rodent localities of the middle and late Miocene with a time scale in millions of years (Ma); key localities plotted on left. Relative abundances of each taxon and morphotype shown for each stratigraphic level. Pattern reflects evolutionary divergence and diversification of Siwalik Murinae based on species distributions through time. Stem Murinae are succeeded after 11 Ma by crown Murini represented on the one hand by Progonomys and its more derived sister clades (Aghová et al., 2018) and on the other hand by crown Arvicanthini represented by Karnimata and its more derived sister clades. Columns indicate relative abundances among all contemporary murines. The color scheme is maintained in later figures. latitude, the area has moved northward with the Indian plate This analysis crucially depends on dated fossil sites with by about 4◦ since 11 Ma (Tauxe and Opdyke, 1982). Erosional resolution up to 100,000 year precision (see Barry et al., 2002). detritus carried southward and accumulating on pre-Indus The dating method involves careful stratigraphic correlation floodplains (over 3000 m thick on the Potwar Plateau; Tauxe of fossil sites to a master stratigraphic section, in which and Opdyke, 1982) comprise layers of rock of varied lithology, the paleomagnetic properties of successive samples of rock formations of the Siwalik Group. The layers of rock of most throughout the stratigraphic sequence build a composite interest here, mainly Miocene in age, occur in a broad belt magnetostratigraphy for the Siwaliks. Due to the completeness across Indo-Pakistan and as exposed on the Potwar Plateau of the Potwar record, the magnetostratigraphy can be matched are ∼18–6 million years old (18–6 Ma). The Siwaliks are to the dated global geomagnetic polarity time scale, yielding important for evolutionary biology because the sediments age estimates for the rocks (Barry et al., 2013). Estimates are entomb rich samples of the vertebrate biota that populated not direct dates, but the dates are relative to one another and the region through the Miocene. Fossil bones and teeth are distinguishable at a scale of 105 years. Because Siwalik strata are preserved at many levels in the sequence, with the potential that generally well exposed, individual fossil localities can be dated remains of populations in single lineages can be sequentially in this fashion. This method is widely applied globally (e.g., sampled through time. Garcés et al., 2003; Tedford et al., 2013; Van Dam et al., 2014; The dating of terrestrial fossils through magnetic polarity García-Paredes et al., 2016). stratigraphy began in the early 1970’s. At that time a consortium of universities began paleontological, stratigraphic, Siwalik Rodent Fossils and paleomagnetic studies on the Potwar Plateau because of Small mammal fossils accumulated under special conditions at a its fossil abundance known since the 19th Century, its very number of Potwar localities. They are dominantly dental remains long fossiliferous stratigraphic section related to the uplift of presumably accumulated by raptors (mostly owls) in riparian the Himalaya, and as a focus to develop and validate magnetic settings. Such conditions did not occur everywhere, so fossil sites polarity stratigraphy in developing a global polarity timescale. are not uniformly distributed through the composite geological From those beginnings, the tradition of precise dating applied to section (Figure 1)–but they occur densely enough in time to terrestrial paleontology, evolution, and paleoecological studies in sample details of lineage evolution. Fossils were recovered by the Siwaliks has continued (Keller et al., 1977; Tauxe and Opdyke, wet screening large quantities of fossiliferous sediment from 1982; Johnson et al., 1985). promising localities that usually showed small bone fragments Frontiers in Ecology and Evolution | www.frontiersin.org 3 March 2021 | Volume 9 | Article 642814 Kimura et al. Tempo and Mode TABLE 1 | Potwar Plateau rodent localities utilized in this study, their ages, and fossil sample sizes. Age (Ma) Locality Number Total Rodent Fossils Murine Fossils 10.1 Y311 283 205 10.2 Y450 91 41 10.5 Y259 607 395 11.2 Y797 321 108 11.2 Y791 67 48 11.4 Y076 377 142 11.4 Y809 127 67 11.6 Y083 51 17 11.6 Y504 146 52 12.3 Y634 85 32 12.4 Y496 218 88 on the ground surface. After sieving, the concentrate was sorted under magnification and isolated teeth were retrieved. Samples of individual species range from a few to several tens of teeth per site. For the stratigraphic interval capturing the time of divergence of major murine groups, Table 1 shows the ages of localities, the number of rodent fossils recovered from each, and the number of those rodents that are murines. With fossils, FIGURE 2 | Diagrams of typical murine first molars, upper (above, M1) and more complete is always better than less complete, but our use lower (below, m1), with cusp nomenclature. Major cusps are labeled, anterior of isolated teeth was necessitated because that is the nature is to the left. Upper molar shows cusps arranged in transverse chevrons; lower molar shows six major cusps in opposed pairs. The axis of the upper of the samples and is mitigated by the observation that living molar metacone-hypocone complex is termed a “metaloph.” murine rodent taxa are commonly characterized by molars (e.g., Misonne, 1969). Beginning with samples at 11.6 Ma, the debut of the Morphology late Miocene, we observe sporadic occurrence of advanced Siwalik fossil teeth recovered by screening represent mostly M1 features (as below) that characterize crown mouse rodents, but include hedgehogs, shrews, bats, and a few other tribes. Yet these traits occur in samples of teeth that are mammals. The most abundant rodents retrieved are murine otherwise homogeneous. Characteristic features of Murini and mice, and these, like all crown muroids, have only molars in each Arvicanthini appear to be randomly distributed among the quadrant behind the enlarged procumbent incisor. The largest specimens. These mouse teeth represent either a single lineage and best represented of the molars is the first upper molar (M1). with considerable variation, or two lineages that are cryptic as far Because it is large (by mouse standards) and relatively complex, as fossil teeth are concerned. this tooth locus presents diagnostic characters and is the most informative of the molars for systematic study. In some cases, the Murini first lower molar (m1) is also useful. The key feature of the M1 that characterizes the Murini Middle Miocene fossils represent stem murines, but exhibit concerns the anterostyle (Jacobs, 1978). This lingual cusp the apomorphic characteristics of the subfamily, namely extra is associated with the first chevron. In Murini the cusp cusps in M1 and a twinned anteroconid plus labial cingulum is shifted posteriorly, its crest running anterolabially to in m1 (Figure 2). These features are derived, absent in muroid the double anterocone. The anterior part of the M1 is outgroups. The extra cusps in M1 (anterostyle and enterostyle therefore transversely narrow. In addition to its posterior behind it) are lingual in position and constitute, with neighboring position, the anterostyle is not a circular cusp, but cusps, transverse sectorial chevrons. In middle Miocene taxa, relatively laterally compressed. Molar cusps are distinctly the chevrons are poorly developed: most Potwarmus (bottom of inclined posteriorly. Figure 1) lack the anterostyle, and in Antemus the enterostyle This combination of features appears in some teeth as is unconnected with its chevron. Jacobs et al. (1989) described a variation in fossil samples of 11.6–11.2 Ma. By 10.5– the considerable morphological variation in Antemus M1 for a 10.1 Ma, it is stable. Samples of that age and younger are large sample from locality Y491 (13.8 Ma). Toward the end of readily assigned to the genus Progonomys. By the end of the the middle Miocene (12.3 Ma) the early murine lineage, by then Miocene, the derivative living genus Mus shows a strongly advanced over Antemus, exhibited full chevrons on M1, but with shifted anterostyle, a narrow anterior shelf surmounted by low ridges weakly connecting neighboring cusps. In none of these the double anterocone, and a reduced posterior cingulum samples do we observe definitive features of crown murine tribes. (Jacobs, 1978). Frontiers in Ecology and Evolution | www.frontiersin.org 4 March 2021 | Volume 9 | Article 642814 Kimura et al. Tempo and Mode Arvicanthini the bootstrap permutation, 9999 replicates were resampled. The This group conserved the primitive anterostyle position, not bootstrap confidence intervals were calculated in the R package strongly repositioned posterolingual to the double anterocone. boot (Canty and Ripley, 2020) using the boot.ci function. All data However, arvicanthins developed progressively a separately are plotted in Figure 3. Due to updates in taxonomic assignments, derived feature; this concerns the metacone cusp (Kimura the different bootstrap calculation method, and the selection of et al., 2015). A large metacone that is posteriorly inclined, groups plotted in Figure 3, some details differ slightly from that similar to the paracone, is a primitive condition for mice. The of Kimura et al. (2013b, Figure 9). posterior inclination is most obvious in lateral view. Arvicanthins progressively fix a derived condition of the metacone, which Stratigraphic Age and the Molecular is vertical and diminished in size. Most specimens older than Clock 11 Ma show the primitive metacone condition, but some A time-calibrated tree (discussed below) was generated using have a modified metacone, smaller than the paracone. By “Stratigraphic Tree Analysis for Paleontology” (strap) in R 9.2 Ma, approximately 25% of Karnimata darwini show the fully package for proposed relationships of Siwalik murines. We transformed metacone. Progressive development of the derived constructed a tree file (.tre) with a single tree as our hypothesis by metacone in the Karnimata lineage without similar change in manually editing tree shape in Mesquite version 3.04 (Maddison the posterostyle, led Kimura et al. (2015) to recognize that and Maddison, 2017) based against an updated review of ranges the genus represents a stem in the Arvicanthini-Otomyini- of Siwalik fossils. For time calibration, first and last appearances Millardiini clade. were compiled as the lowermost and uppermost stratigraphic occurrences of the taxa. In the R package “strap,” the equal Relative Abundances of Siwalik Murines scaling method was used in the DatePhylo function with a root Temporal change in the relative abundances of the major length (i.e., length of the base of the tree) of 1 million. Although components of the Siwalik murine rodents in Figure 1 is the time-scaling algorithm of the “equal” method is tuned to based on the Harvard University-Geological Survey of Pakistan avoid zero-length branches instead of generating best estimates database (e.g., Barry et al., 2013), which is continuously updated. of divergence times, the model ages of the fossil calibration Although the master database includes fossils representing all points are consistent with our previous proposals and molecular tooth positions, we analyzed a subset (extracted December, 2020) estimates (Lecompte et al., 2008; Aghová et al., 2018), and of only upper first molars (M1) for those taxa with total number support the proposition that the density of sampled Siwalik fossils of specimens of ten or more. Complexity of M1 maximizes may be sufficient to capture transitional morphological changes precision of taxonomic assignment for that tooth. The relative near divergences. abundance of M1 was calculated as the number of fossils in a species (taxonomic unit) relative to the total number of murine fossils. The stratigraphic diagrams were generated with 0.1 m.y. RESULTS (105 year) time bins by the strat.plot function in the package rioja (Juggins, 2020) implemented in R (R Core Team, 2020). Previously, we (Flynn et al., 2020) found a good match between the stratigraphic record of clade appearances and the node age Crown Morphology in Transition estimates from the molecular time tree of Lecompte et al. (2008). To visualize change through time of dental characters as Nevertheless, the Siwalik murine record has obtained more detail they were fixed in the Mus-Arvicanthis divergence (Figure 3 in recent years from review and analysis of carefully controlled and Tables 2–5), we obtained the measurements of four data for over 3600 specimens and therefore the evolution of morphometric traits of M1 from time-ordered and sequential murines can be traced in more detail. In samples of 9.2 Ma samples: the ratio of the major and minor axes of the anterostyle; (locality Y182), Jacobs (1978) recognized two clearly separate the acute angle of the anterostyle relative to the longitudinal tooth species, Progonomys debruijni and K. darwini, now recognized axis; the acute angle between a line connecting the protocone- to represent Murini in the former case and Arvicanthini in the enterocone and the longitudinal tooth axis; and the acute angle latter (Figure 4). P. debruijni had the murine anterostyle shifted of the metaloph (line through the major axis of the metacone) and laterally compressed. All cusps showed posterior inclination relative to the longitudinal tooth axis. The datasets of Kimura and P. debruijni was the smaller of the two. The other mouse et al. (2013b) are combined with the latter feature, which is newly K. darwini was clearly larger with a plesiomorphic anterostyle. obtained for this study. Taxonomic assignments of Kimura et al. Kimura et al. (2013b, 2015) recognized the significance of the (2013b) are updated following Kimura et al. (2015, 2016, 2017) morphological distinctions between Progonomys and Karnimata and Flynn et al. (2020). Previously, in Kimura et al. (2013b), and distinguished a number of other dental features, including we calculated the 95% bias-corrected and accelerated bootstrap isotopic differences (Kimura et al., 2013a), that set the lineages (BCa) confidence intervals for groups with more than three apart especially after 9 Ma. entries (i.e., taxa in different time slices are treated as separate In older samples dating to 10.5–10.1 Ma (localities Y259, 450, groups), and only these data are plotted in Figure 9 of Kimura 311), the murines resembled the 9.2 Ma samples, but represented et al. (2013b). In this study, we calculated both 95% bootstrap more primitive species, which Kimura et al. (2017) recognized confidence intervals for groups with more than two entries. The as Progonomys hussaini and Karnimata fejfari (Figure 5). choice of the bootstrap method did not affect our conclusions. In These earlier representatives of the Progonomys and Karnimata Frontiers in Ecology and Evolution | www.frontiersin.org 5 March 2021 | Volume 9 | Article 642814 Kimura et al. Tempo and Mode FIGURE 3 | Temporal change and variation in four morphometric characters in Siwalik murines. (A) Axis ratio of anterostyle shape (length of the minor axis relative to that of the major axis). (B) Acute angle between the anterostyle and the longitudinal axis of the tooth. (C) Acute angle formed by a line connecting the centers of protocone and enterostyle with the longitudinal axis of the tooth. (D) Acute angle between the major axis of the metacone (forms a metaloph with the hypocone) and the longitudinal axis of the tooth. Error bars indicate 95% bootstrap confidence intervals. The specimen illustrated is Progonomys debruijni (YGSP 7740) in early wear. Versions of panels (A–C) initially in Kimura et al. (2013b), updated here with revised taxonomy; panel (D) is new. Error bars represent 95% confidence intervals. lineages, the stems of Murini and Arvicanthini, were less distinct, If the older P. hussaini and K. fejfari were more similar showed lower frequency of derived character states, and were to each other than younger members of these genera (at least much closer in size. in features that could be perceived in the dentition), would Frontiers in Ecology and Evolution | www.frontiersin.org 6 March 2021 | Volume 9 | Article 642814 Kimura et al. Tempo and Mode TABLE 2 | Summary statistics for anterostyle ratio in Siwalik murine rodents, corresponding to Figure 3A. Taxonomic Unit N Average Mean Min Max SD Lower CI Upper CI Lower CI Upper CI Age (Ma) basic basic BCa BCa GTS2012 Karnimata huxleyi 10 6.52 0.78 0.60 0.90 0.09 0.73 0.84 0.72 0.83 Mus auctor 12 6.52 0.48 0.40 0.58 0.05 0.45 0.50 0.45 0.51 Parapelomys robertsi 5 6.52 0.93 0.87 0.98 0.04 0.89 0.97 0.89 0.96 Mus sp. nov. 2 7.34 0.42 0.40 0.44 0.03 0.40 0.44 0.40 0.44 Karnimata huxleyi 20 7.34 0.75 0.45 0.94 0.15 0.68 0.82 0.67 0.81 Progonomys sp. at 11 7.34 0.49 0.37 0.58 0.08 0.44 0.54 0.44 0.53 Y581 locality Parapelomys cf. 2 7.37 0.89 0.85 0.93 0.06 0.85 0.93 0.85 0.93 robertsi Karnimata sp. (large) 5 8.13 0.91 0.80 1.02 0.10 0.83 1.02 0.81 1.00 Progonomys debruijni 12 8.18 0.56 0.41 0.87 0.12 0.49 0.62 0.51 0.66 Karnimata darwini 12 8.27 0.88 0.81 0.97 0.05 0.85 0.91 0.85 0.91 Progonomys debruijni 16 8.74 0.56 0.45 0.68 0.07 0.52 0.59 0.52 0.60 Karnimata sp. (large) 3 8.75 1.04 1.00 1.10 0.05 0.99 1.09 1.00 1.10 Progonomys debruijni 3 9.02 0.60 0.53 0.68 0.08 0.52 0.67 0.53 0.68 Karnimata sp. (large) 3 9.10 0.79 0.73 0.83 0.05 0.74 0.84 0.73 0.83 Karnimata darwini 31 9.24 0.76 0.56 0.95 0.10 0.73 0.80 0.73 0.80 Parapodemus hariensis 2 9.24 0.82 0.69 0.95 0.18 0.69 0.95 0.69 0.95 Progonomys debruijni 15 9.24 0.46 0.26 0.61 0.11 0.41 0.52 0.40 0.52 Parapodemys cf. 1 10.06 0.86 0.86 0.86 NA NA NA NA NA badgleyae Progonomys hussaini 5 10.09 0.64 0.57 0.76 0.09 0.55 0.71 0.58 0.74 Karnimata fejfari 9 10.12 0.77 0.58 0.97 0.12 0.69 0.86 0.69 0.85 Parapodemus 3 10.35 0.73 0.73 0.74 0.01 0.73 0.74 0.73 0.74 badgleyae Karnimata fejfari 9 10.49 0.75 0.62 0.91 0.10 0.68 0.82 0.68 0.82 Gen. et sp. nov. C 1 11.18 0.62 0.62 0.62 NA NA NA NA NA “Indeterminate 7 11.20 0.61 0.46 0.81 0.13 0.52 0.71 0.52 0.72 Progonomys-Karnimata grade” (Progonomys-like morphotype) “Indeterminate 6 11.20 0.58 0.50 0.68 0.07 0.52 0.64 0.52 0.64 Progonomys-Karnimata grade” (Karnimata-like morphotype) “Progonomys” 1 11.23 0.59 0.59 0.59 NA NA NA NA NA morganae “Pre-Progonomys” 21 11.41 0.60 0.49 0.83 0.09 0.56 0.64 0.57 0.65 Gen. et sp. nov. D 1 11.41 0.53 0.53 0.53 NA NA NA NA NA “Pre-Progonomys” 3 11.63 0.61 0.54 0.67 0.07 0.54 0.67 0.54 0.67 “Post-Antemus” 7 12.36 0.62 0.46 0.79 0.11 0.55 0.70 0.55 0.71 Antemus chinjiensis 7 13.05 0.67 0.60 0.75 0.06 0.62 0.71 0.63 0.72 Antemus chinjiensis 14 13.62 0.48 0.31 0.74 0.14 0.40 0.55 0.41 0.56 Antemus chinjiensis 12 13.80 0.57 0.40 0.69 0.10 0.52 0.63 0.51 0.63 CI: 95% confidence interval. BCa: bias-corrected and accelerated bootstrap. SD: standard deviation. still older samples (those available to us being 11.6–11.2 Ma) and two unnamed mice; Flynn et al., 2020). Most individual show more subtle differences, or appear to merge? Localities specimens appear comparable to P. hussaini and K. fejfari, but Y797 and Y791, dating to 11.2 Ma, have abundant murines, all the collections, taken as biological samples, do not sort readily about the same size and resembling Progonomys and Karnimata into more than one species. These fossils were considered a (plus a few teeth representing small “Progonomys” morganae single population also in model-based Bayesian cluster analysis Frontiers in Ecology and Evolution | www.frontiersin.org 7 March 2021 | Volume 9 | Article 642814 Kimura et al. Tempo and Mode TABLE 3 | Summary statistics for anterostyle angle in Siwalik murine rodents, corresponding to Figure 3B. Taxonomic Unit N Average Mean Min Max SD Lower CI Upper CI Lower CI Upper CI Age (Ma) basic basic BCa BCa GTS2012 Karnimata huxleyi 10 6.52 53 43 62 7.0 48.7 57.6 48.4 57.3 Mus auctor 12 6.52 41 34 49 4.1 38.6 43.3 38.5 43.2 Parapelomys robertsi 5 6.52 61 50 70 10.0 53.8 72.3 50.3 69.0 Mus sp. nov. 2 7.34 41 40 42 1.4 40.0 42.0 40.0 42.0 Karnimata huxleyi 20 7.34 55 37 72 10.6 49.9 59.4 50.0 59.4 Progonomys sp. at 11 7.34 40 34 45 4.3 37.3 42.3 37.3 42.3 Y581 locality Parapelomys cf. 2 7.37 63 54 72 12.7 69.0 81.0 69.0 81.0 robertsi Karnimata sp. (large) 5 8.13 54 45 81 15.4 37.5 61.4 46.3 81.0 Progonomys debruijni 12 8.18 42 34 53 5.7 38.8 45.3 39.2 45.8 Karnimata darwini 12 8.27 58 40 73 8.5 52.9 62.6 52.7 62.4 Progonomys debruijni 16 8.74 43 32 57 6.5 39.3 45.6 39.8 46.2 Karnimata sp. (large) 3 8.75 60 58 61 1.5 58.3 61.3 58.0 61.0 Progonomys debruijni 3 9.02 44 37 48 5.9 39.3 50.3 37.0 48.0 Karnimata sp. (large) 3 9.10 58 52 66 7.4 49.3 63.3 52.0 66.0 Karnimata darwini 31 9.24 47 40 64 6.4 44.9 49.4 45.2 49.8 Parapodemus hariensis 2 9.24 53 45 60 10.6 45.0 60.0 45.0 60.0 Progonomys debruijni 15 9.24 37 31 45 4.5 35.0 39.5 35.1 39.6 Parapodemys cf. 1 10.06 54 54 54 NA NA NA NA NA badgleyae Progonomys hussaini 5 10.09 44 36 53 6.9 37.2 50.2 37.0 50.0 Karnimata fejfari 9 10.12 53 40 62 6.6 48.5 57.1 47.6 56.5 Parapodemus 3 10.35 45 42 48 3.1 42.7 48.7 42.0 48.0 badgleyae Karnimata fejfari 9 10.49 40 31 51 6.0 35.4 43.4 36.1 44.4 Gen. et sp. nov. C 1 11.18 39 39 39 NA NA NA NA NA “Indeterminate 7 11.20 38 31 43 4.1 35.3 41.4 34.2 40.8 Progonomys-Karnimata grade” (Progonomys-like morphotype) “Indeterminate 6 11.20 39 30 43 4.6 35.7 42.9 31.8 41.0 Progonomys-Karnimata grade” (Karnimata-like morphotype) “Progonomys” 1 11.23 23 23 23 NA NA NA NA NA morganae ”Pre-Progonomys” 21 11.41 40 28 54 7.3 36.5 42.7 36.7 43.0 Gen. et sp. nov. D 1 11.41 44 44 44 NA NA NA NA NA “Pre-Progonomys” 3 11.63 38 38 38 0.0 36.5 42.7 36.7 43.0 “Post-Antemus” 7 12.36 37 26 51 8.9 30.4 43.9 31.0 44.6 Antemus chinjiensis 7 13.05 38 33 43 3.3 35.1 40.0 35.2 40.2 Antemus chinjiensis 14 13.62 36 22 50 7.9 31.9 40.2 31.4 39.7 Antemus chinjiensis 12 13.80 33 21 50 9.5 27.2 38.0 27.7 38.6 CI: 95% confidence interval. BCa: bias-corrected and accelerated bootstrap. SD: standard deviation. (Kimura et al., 2016). Some M1’s have the murinin (Progonomys- samples that show no consistent differentiation and no pattern like) morphology and some present the arvicanthin morphology for taxonomic association with M1. For the 11.2 Ma samples (Figures 1, 3, 6). The array of morphologies observed among Flynn et al. (2020) could not defend two species and opted for an the M1 is not bimodal–there are intermediates. Further, other informal designation of “indeterminate Progonomys–Karnimata molars in the dentition (m1, second and third molars) comprise grade.” There is no record of P. hussaini and K. fejfari on Frontiers in Ecology and Evolution | www.frontiersin.org 8 March 2021 | Volume 9 | Article 642814 Kimura et al. Tempo and Mode TABLE 4 | Summary statistics for angle formed by the protocone and enterostyle in Siwalik murine rodents, corresponding to Figure 3C. Taxonomic Unit N Average Mean Min Max SD Lower CI Upper CI Lower CI Upper CI Age (Ma) basic basic BCa BCa GTS2012 Karnimata huxleyi 10 6.52 66 60 72 4.2 63.8 69.0 63.5 68.7 Mus auctor 12 6.52 60 52 69 5.3 56.5 62.6 56.8 63.0 Parapelomys robertsi 5 6.52 75 63 81 7.5 69.9 82.4 63.0 80.0 Mus sp. nov. 2 7.34 58 55 60 3.5 55.0 60.0 55.0 60.0 Karnimata huxleyi 20 7.34 68 53 77 5.6 65.9 70.8 65.3 70.2 Progonomys sp. at 11 7.34 63 55 70 4.8 60.0 65.6 60.0 65.8 Y581 locality Parapelomys cf. 2 7.37 75 69 81 8.5 69.0 81.0 69.0 81.0 robertsi Karnimata sp. (large) 5 8.13 69 63 73 4.0 66.3 73.3 63.0 72.0 Progonomys debruijni 12 8.18 61 55 66 3.7 58.6 62.7 58.3 62.5 Karnimata darwini 12 8.27 74 67 81 4.8 71.4 76.9 71.4 76.8 Progonomys debruijni 16 8.74 63 55 75 5.7 60.5 66.1 60.4 66.1 Karnimata sp. (large) 3 8.75 67 65 71 3.2 63.7 69.7 65.0 71.0 Progonomys debruijni 3 9.02 64 61 67 3.1 61.7 67.7 61.0 67.0 Karnimata sp. (large) 3 9.10 69 63 76 6.6 62.0 75.0 63.0 76.0 Karnimata darwini 31 9.24 73 59 87 6.0 70.7 75.0 70.8 75.1 Parapodemus hariensis 2 9.24 68 66 70 2.8 66.0 70.0 66.0 70.0 Progonomys debruijni 15 9.24 62 51 75 6.8 58.4 65.4 58.4 65.4 Parapodemys cf. 1 10.06 55 55 55 NA NA NA NA NA badgleyae Progonomys hussaini 5 10.09 70 64 74 4.0 67.3 74.3 64.0 73.0 Karnimata fejfari 9 10.12 70 61 83 6.2 65.7 73.8 66.6 74.9 Parapodemus 3 10.35 60 50 69 9.6 51.7 70.7 50.0 69.0 badgleyae Karnimata fejfari 9 10.49 64 50 82 8.9 57.6 69.5 58.8 70.8 Gen. et sp. nov. C 1 11.18 67 67 67 NA NA NA NA NA “Indeterminate 7 11.20 71 59 82 7.1 65.9 76.9 65.3 76.3 Progonomys-Karnimata grade” (Progonomys-like morphotype) “Indeterminate 6 11.20 66 56 80 9.3 58.0 73.5 58.7 74.2 Progonomys-Karnimata grade” (Karnimata-like morphotype) “Progonomys” 1 11.23 76 76 76 NA NA NA NA NA morganae “Pre-Progonomys” 21 11.41 67 51 83 9.3 63.0 71.0 62.9 70.9 Gen. et sp. nov. D 1 11.41 61 61 61 NA NA NA NA NA “Pre-Progonomys” 3 11.63 73 69 77 4.0 69.0 77.0 69.0 75.7 “Post-Antemus” 7 12.36 65 53 70 6.6 60.1 70.1 57.4 68.5 Antemus chinjiensis 7 13.05 68 48 84 12.4 59.6 78.6 57.6 76.6 Antemus chinjiensis 14 13.62 69 54 93 9.8 63.6 73.6 64.5 74.7 Antemus chinjiensis 12 13.80 64 57 71 4.5 61.4 66.6 61.3 66.5 CI: 95% confidence interval. BCa: bias-corrected and accelerated bootstrap. SD: standard deviation. the Potwar Plateau prior to 10.5 Ma. The roots of distinct not show one or the other derived morphologies consistently. Progonomys and of Karnimata lineages appear to lie in samples Morphologies observed from somewhat older localities were captured at 11.2 Ma. yet more variable, but with inconsistently derived morphologies The mice from Y791 and Y797, 11.2 Ma, were very close to (Figure 7). Our field surveys in subjacent strata located fossil the crown murine genera Progonomys and Karnimata but did localities Y76 and Y809 at 11.4 Ma, and below these (therefore Frontiers in Ecology and Evolution | www.frontiersin.org 9 March 2021 | Volume 9 | Article 642814 Kimura et al. Tempo and Mode TABLE 5 | Summary statistics for axis angle through metacone in Siwalik murine rodents, corresponding to Figure 3D. Taxonomic Unit N Average Mean Min Max SD Lower CI Upper CI Lower CI Upper CI Age (Ma) basic basic BCa BCa GTS2012 Karnimata huxleyi 10 6.52 66 47 81 9.5 60.2 72.1 59.0 71.3 Mus auctor 12 6.52 69 56 80 7.6 64.9 73.6 64.3 73.0 Parapelomys robertsi 5 6.52 60 41 73 12.1 49.2 69.7 46.3 68.1 Mus sp. nov. 2 7.34 86 84 87 2.1 84.0 87.0 84.0 87.0 Karnimata huxleyi 20 7.34 72 59 97 10.4 67.2 76.1 67.8 76.8 Progonomys sp. at 11 7.34 80 64 91 10.8 73.2 86.1 72.9 85.8 Y581 locality Parapelomys cf. 2 7.37 66 64 68 2.8 64.0 68.0 64.0 68.0 robertsi Karnimata sp. (large) 5 8.13 69 56 79 9.3 60.6 78.1 58.3 76.3 Progonomys debruijni 12 8.18 83 71 91 7.3 79.3 87.4 78.6 86.9 Karnimata darwini 12 8.27 70 55 80 6.7 66.0 73.5 65.4 73.1 Progonomys debruijni 16 8.74 84 75 91 5.4 81.2 86.5 81.0 86.3 Karnimata sp. (large) 3 8.75 58 57 58 0.6 57.3 58.3 57.0 58.0 Progonomys debruijni 3 9.02 87 84 89 2.5 84.3 89.3 84.0 89.0 Karnimata sp. (large) 3 9.10 69 64 78 7.8 60.0 74.0 64.0 78.0 Karnimata darwini 31 9.24 72 55 89 9.2 69.0 75.4 69.2 75.7 Parapodemus hariensis 2 9.24 61 58 63 3.5 58.0 63.0 58.0 63.0 Progonomys debruijni 15 9.24 78 67 87 5.6 75.6 81.3 75.4 81.2 Parapodemys cf. 1 10.06 76 76 76 NA NA NA NA NA badgleyae Progonomys hussaini 5 10.09 79 70 90 9.3 70.3 88.8 70.0 88.0 Karnimata fejfari 9 10.12 74 53 91 11.4 66.6 81.7 65.4 80.8 Parapodemus 3 10.35 83 78 89 5.5 77.7 88.7 78.0 89.0 badgleyae Karnimata fejfari 9 10.49 80 74 86 4.7 77.3 83.5 77.0 83.2 Gen. et sp. nov. C 1 11.18 83 83 83 NA NA NA NA NA “Indeterminate 7 11.20 81 66 87 7.0 76.3 86.5 71.3 84.0 Progonomys-Karnimata grade” (Progonomys-like morphotype) “Indeterminate 6 11.20 83 72 90 7.6 77.0 89.2 74.0 87.7 Progonomys-Karnimata grade” (Karnimata-like morphotype) “Progonomys” 1 11.23 81 81 81 NA NA NA NA NA morganae “Pre-Progonomys” 21 11.41 88 81 96 3.7 86.5 89.7 86.6 89.8 Gen. et sp. nov. D 1 11.41 78 78 78 NA NA NA NA NA “Pre-Progonomys” 3 11.63 89 88 90 1.0 88.0 90.0 88.0 89.7 “Post-Antemus” 7 12.36 86 78 97 7.0 81.1 91.9 81.0 91.8 Antemus chinjiensis 7 13.05 93 86 100 5.0 88.8 96.4 89.2 96.8 Antemus chinjiensis 14 13.62 87 74 92 5.3 85.0 90.5 83.2 89.5 Antemus chinjiensis 12 13.80 88 76 98 5.9 84.5 91.3 84.0 90.8 CI: 95% confidence interval. BCa: bias-corrected and accelerated bootstrap. SD: standard deviation. older) sites Y83 and Y504 at 11.6 Ma. The collections presented M1 resembled their “post-Antemus” predecessors of 12.3 Ma, a medley of features, some specimens showing a displaced, many with anterocones not elongated antero-posteriorly, and pinched anterocone, others showing variably inclined cusps. connections between adjacent cusps generally undeveloped. Individual M1 were occasionally reminiscent of Progonomys in These “pre-Progonomys” mice were basal crown murines (stem having the anterostyle displaced backward (Figure 7C), but most species) containing the roots of later tribe-level radiations, but molars lacked derived conditions of extant mouse tribes. The themselves undifferentiated along modern lines. Frontiers in Ecology and Evolution | www.frontiersin.org 10 March 2021 | Volume 9 | Article 642814 Kimura et al. Tempo and Mode FIGURE 6 | Indeterminate Karnimata–Progonomys grade molars from FIGURE 4 | Upper first molars of the dominant murines present at locality locality Y797, 11.2 Ma. (A) Karnimata-like left M1 YGSP 36826 and Y182, 9.2 Ma. (A) Right M1 of Karnimata darwini, specimen YGSP 7720, and (B) Progonomys-like left M1 YGSP 33878. Both at same 1 mm scale, anterior (B) smaller left M1 of Progonomys debruijni, YGSP 7736. Both at same 1 mm is upward. scale, anterior is upward. DISCUSSION It is well worth emphasizing the unique set of conditions that allowed this evaluation of tempo and mode in evolution using the fossil record. Siwalik Group rocks or their equivalents span a vast area along the Himalaya front from Pakistan to Myanmar. Correlative fossils from India largely complement and corroborate the Siwalik murine record in Pakistan (Patnaik, 2014, 2020). Older fossils are not relevant to this study because they are phylogenetically distant from the problem; younger fossil taxa are more derived members of Murini and Arvicanthini (e.g., Jacobs, 1978; Musser, 1987; Patnaik et al., 1993, 2018; Kotlia, 2008; Patnaik, 2020). Although fossil murines and other fossil rodents have been known in Europe (Schaub, 1938; Michaux, 1971) and globally for many years, no murine fossil known is older than the oldest murines of Pakistan. Flynn et al. (2020) have discussed this restricted biogeographic setting of the Siwaliks as the “murine cradle.” FIGURE 5 | Early Karnimata and Progonomys from locality Y311, 10.1 Ma. Moreover, Siwalik murines and other small mammals were (A) Karnimata fejfari left M1 YGSP 54147 and (B) Progonomys hussaini left linked with the remainder of the fauna as part of a large, evolving M1 YGSP 541443. Progonomys is slender with more inclined cusps but is ecosystem that responded to global conditions (Barry et al., 1985; near the size of Karnimata. Both at same 1 mm scale, anterior is upward. Blois and Hadley, 2009; Figueirido et al., 2012) and to more regional abiotic and climatic changes (Flynn and Jacobs, 1982; Badgley et al., 2008; López-Antoñanzas et al., 2015). Stable isotopes have been used to track changes in Siwalik murine diets The documentation of multiple, sequentially progressive and tooth morphology has been correlated with dietary changes transitional samples, whether of mixed cryptic species or (Kimura et al., 2013a,b, 2016). These aspects of paleobiology, all of consecutive individually variable species, provides a of which have a temporal component, reiterate the importance timescale for this cladistic event and a view of its mode. In of high-quality geologic dating and dense sampling of fossils in either case, the 11 Ma divergence provided by “strap”-based the Siwaliks. Finally, the group of animals being studied, with stratigraphic age estimation (Figure 8) is congruent with the an enormous modern diversity and broad geographic range, most recent molecular estimates of the Murini-Arvicanthini split renders the study of their deep roots both multidisciplinary and provided by Aghová et al. (2018). highly significant. Frontiers in Ecology and Evolution | www.frontiersin.org 11 March 2021 | Volume 9 | Article 642814 Kimura et al. Tempo and Mode through time (Figure 3). We selected four features (see section “Materials and Methods”) and quantified change from 14 to 6 Ma. These features show considerable variation until about 11 Ma, when they diverge, slowly at first, as Murini and Arvicanthini (and other minor lineages). We contend that this faithfully reflects growing divergence in morphology consistent with the scenario of a split of the two lineages from a common ancestor, with clear separation by 10.5 Ma. Fossil samples at 9.2 Ma and younger securely represent early Murini and Arvicanthini in morphological features and dietary preferences (Kimura et al., 2015, 2016). Key species at that age are the distinct P. debruijni and K. darwini. Predecessor species, P. hussaini and K. fejfari, occur in rocks one million years older. These species were morphologically closer to each other than their successors were to their contemporaries, but still distinct, and are closer in time to the origins of the tribes. The oldest records of P. hussaini and K. fejfari are from locality Y259 at 10.5 Ma; they may well have been distinct before that. However, at 11.2 Ma we find that while the species were not yet present, samples are consistent with the hypothetical origin of Murini and Arvicanthini. Fossil samples at 11.2 Ma comprise morphological variants that resemble Progonomys and Karnimata but appear to represent a single lineage of rodents. The samples do not present a bimodal distribution of shape or size. Hypothetical cryptic species may be a possibility but cannot be FIGURE 7 | Four left M1 from locality Y76, 11.4 Ma, showing stem distinguished morphologically. murinin-arvicanthin morphology. (A) YGSP 33586, (B) 33571, (C) 33591, and These data speak to the mode of evolution in these murines. (D) 33572. Of these four, panel (C) is Progonomys-like; the others resemble Karnimata. Same 1 mm scale, anterior upward. The split of the modern tribes was incremental. It began in a common pool of morphological variants. Extremes in variation, e.g., the murinin anterostyle, became hallmarks of mice exploiting one part of the range of variation, in contrast The middle and late Miocene age murine fossil record to the arvicanthin strategy of retained vertical cusps and is rich throughout the Old World, but the oldest fossils broadened molars. Absolute change in M1 morphology is greater of the subfamily are from the early Miocene of South in arvicanthins, which later exploited preferentially a C4 diet. Asia (Flynn et al., 2020). Significant events in early Our data suggest such a scenario but are not sufficient to murine evolution appear to be centered in southern claim that the tribes originated at 11.2 Ma exactly. Indeed, Asia, and the best fossil record that captures part of that the lineages may have been close, but not yet morphologically radiation is being developed in the Siwaliks of the Indian distinct at that age in the features we can perceive. The fossils Subcontinent (Jacobs and Downs, 1994; Jacobs and Flynn, at 11.2 Ma may sample two cryptic species, Progonomys and 2005; Patnaik, 2014). This exceptional fossil record bears on Karnimata ancestors. the origin of two extant tribes, Murini and Arvicanthini. The Siwalik fossil record also addresses the tempo of the We contend that while the fossil record does not address origin of Murini and Arvicanthini. Prior to 11.2 Ma, fossil population microevolution, it is relevant to fine scale samples show similar variation embracing early murinin and evolutionary phenomena between the microevolutionary arvicanthin morphology. This suggests a very long interval and macroevolutionary levels. of time during which basal crown murines experimented The Siwalik deposits of the Potwar Plateau contain a record with diversification as Murini and Arvicanthini. Hundreds of of superposed fossil samples that is continuous and well thousands of years passed before the definitive tribe-equivalent dated on the scale of 100,000 years especially for the time morphologies emerged. Even if Progonomys and Karnimata had span discussed here, 14–8 Ma. We have applied this precise diverged by 11.2 Ma, the completion of their splitting process was biostratigraphy to a special episode in murine evolution. The protracted over a very long time, at least 400,000 years during relative ages of fossils are consistent with the pattern of evolution this interval. of crown clades (Lecompte et al., 2008; Aghová et al., 2018; What may have gone on during these 100,000 years? While Flynn et al., 2020). the clear separation of crown tribes may be arbitrary within Major morphological features are in this case an insufficient tight limits, it is quite feasible that gene flow, even if limited, basis for tracing microevolution so we dissected molar crown continued between their earliest species members for some morphology into distinct, definable traits and traced change time after their genetic divergence. The phenomenon of genetic Frontiers in Ecology and Evolution | www.frontiersin.org 12 March 2021 | Volume 9 | Article 642814 Kimura et al. Tempo and Mode FIGURE 8 | Time-tree for proposed phylogenetic relationships of major Siwalik murine rodents with well-defined time calibration points on the standard time scale, modeled with the “strap” package in R (Bell and Lloyd, 2015). Stem murines Potwarmus primitivus and Antemus chinjiensis are succeeded on the tree by basal crown murines, followed by a grade labeled “indeterminate Progonomys–Karnimata” (the cf. Progonomys hussaini and cf. Karnimata fejfari of Flynn et al., 2020). These are succeeded by the Progonomys and Karnimata clades, which are basal to subclades Murini-Praomyini-Apodemini and Arvicanthini-Otomyini-Millardiini, respectively. The bold lines connect range-through occurrences of the taxa in the Potwar record. The light blue node indicates a fossil calibration point evidenced by an initial split into two clades. The orange nodes indicate calibration points defined by the earliest occurrence of stem species of the clade. Horizontal arrows show the earliest occurrences of stem fossils. “Strap”-based ages of fossil calibration points are consistent with those previously proposed by Bayesian-based molecular divergence estimates in Aghová et al. (2018). Considering that the “equal” time-scaling algorithm in “strap” is tuned to avoid zero-length branches and that the molecular phylogeny is based on a framework of Bayesian statistics, the consistent age estimates between the two approaches indicate that Siwalik fossil sampling is dense enough to capture transitional morphological changes. Best practices for the use of fossils in molecular phylogenetics proposed by Parham et al. (2012) and followed by Aghová et al. (2018) yield credible divergence estimates for the Murinae tree. MRCA: most recent common ancestor. introgression within a temporal murine metacommunity over DATA AVAILABILITY STATEMENT a long interval before complete isolation of daughter species (Presgraves and Yi, 2009) has important implications for The original contributions presented in the interpreting age of lineage splitting and therefore the molecular study are included in the article/supplementary clock. The timing of cladogenesis as perceived by paleontologists material, further inquiries can be directed to the from preserved morphology may post-date significantly a corresponding author/s. protracted interval of gene exchange at the base of the split. We have been fortunate to be able to view the results of one cladogenic event in detail, the split of Tribes Murini and AUTHOR CONTRIBUTIONS Arvicanthini. That process may unfold quickly or take hundreds of thousands of years for significant morphological change All authors contributed equally to this work and each agrees to accumulate. Judging from our case, morphological change to be accountable for the content of the work. YK developed following sporadic genetic divergence may be a significantly much of the original comparative data and developed the protracted process. temporo-stratigraphic analyses. LJ integrated the morphological Frontiers in Ecology and Evolution | www.frontiersin.org 13 March 2021 | Volume 9 | Article 642814 Kimura et al. Tempo and Mode and molecular patterns into a coherent whole, and ACKNOWLEDGMENTS organized specimen imaging. LF applied this special case for the fossil record to current molecular Many collaborators in the field worked to build the fossil records and revised specimen identifications and collections on which his study is based. Scientists from the locality dating. Geological Survey of Pakistan and the Pakistan Museum of Natural History, and from various institutions in the United States, especially our small mammal colleague, Will FUNDING Downs, made this study possible. Roy Beavers of Southern Methodist University skillfully produced SEM images. David JSPS KAKENHI Grant Number JP15H06884 (Grant-in-Aid for Pilbeam, John Barry, and Michèle Morgan supplied both Young Scientists Start-up), JSPS KAKENHI Grant Number feedback and data, reviewers stimulated improvements, and 18K13650, ISEM at SMU, and the American School of we thank the editors and the series editors for their patience Prehistoric Research. and encouragement. REFERENCES Flynn, L. J., and Jacobs, L. L. (1982). Effects of changing environments on Siwalik rodent faunas of northern Pakistan. Palaeogeogr. Palaeoclimatol. Palaeoecol. 38, Aghová, T., Kimura, Y., Bryja, J., Dobigny, G., Granjon, L., and 129–139. doi: 10.1016/0031-0182(82)90067-0 Kergoat, G. J. (2018). Fossils know it best: using a new set of fossil Flynn, L. J., Kimura, Y., and Jacobs, L. L. (2020). “The murine cradle,” in Biological calibrations to improve the temporal phylogenetic framework of murid Consequences of Plate Tectonics: New Perspectives on Post-Gondwanaland rodents (Rodentia: muridae). Mol. 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A comparative analysis No use, distribution or reproduction is permitted which does not comply with of the molars of Mus booduga, Mus dunni and fossil Mus of the Indian these terms. Frontiers in Ecology and Evolution | www.frontiersin.org 15 March 2021 | Volume 9 | Article 642814