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Alfaro et al. 10.1073/pnas.0811087106 SI Text Age of the Root Between and Outgroups. The sister Appendix 1: Comparison of Divergence Times from Earlier Studies. group of the Osteichthyes (bony ) is currently unknown. It Our inferred dates for the splits among the major gnathostome may be represented by the (cartilaginous fishes) lineages (Table S5) are largely consistent with those from or by the Acanthodii. The record of the Chondrichthyes recently published studies (Table S6) (1–9). Among the sarcop- dates to at least the early-middle Caradoc of the Harding terygians, the mean age of the split between amniotes and Sandstone, Colorado (, 456–454 Myr) (13). The is 350 million years (Myr) compared with 354 Myr acanthodian Onchus clintonii dates to the Sheinwoodian and inferred by Hugall (4). Within the amphibians, we date the split Homerian (, 436–428 Myr) (14). Because of the uncer- between the and the batrachia ϩ urodela at 320 tainty in the relationships among Osteichthyes, Myr and between and salamanders at 233 Myr compared Acanthodii, and Chondrichthyes, we assigned to the root a with Hugall, et al. (4), who estimated means of 292 to 322 Myr minimum age of 428 Myr. Placing an upper limit on the root is and 266 to 274 Myr. problematic because of uncertain relationships of many worm- We date the split between mammals and the remaining like that have been tentatively referred to as amniotes (Fig. S2, node 31) at 320 Myr, which falls within the . Pikaia, from the Burgess Shale fossil deposits of the 95% credible interval obtained by Hugall et al. (4). Similarly, our late Cambrian of British Columbia, Canada (505 Myr), is a estimated age for the splits between monotremes and therians prevertebrate that has been identified as probably being related and between marsupials and eutherians (201 and 164 Myr, to cephalochordates (15). Our prior uses the age of these respectively) is similar to ages estimated by Hugall et al. (207 or deposits to set the 95% confidence interval. 227 Myr and 182 or 195 Myr, depending on the choice of (Lamnidae vs. Scyliorhinidae) (Fig. S2, node 1). Al- constraints). Our mean estimates are older than dates inferred though Elasmobranchii () have a fossil record that spans by Bininda-Emonds (2), but our 95% credible interval contains back to the Ordovician, crown sharks are much more recent, the ages they estimated (166 Myr for eutherians/marsupials and having appeared in the fossil record during the . The 148 Myr for montremes/therians). earliest relevant fossil is an unnamed and undescribed Our mean age for the split between ϩ and from deposits of the Bathonian (, 167.7–164.7 Myr), lepidosaurs (270 Myr) is slightly younger than the 285–289 Myr which was assigned to the Scyliorhinidae (16). Our prior inferred by Hugall et al. but in closer agreement with the fossil assumed a minimum age of 165 Myr and placed 95% of the record. Within turtles, we estimate a mean age of the split weight on divergence times within the time span marked by the between cryptodires and pleurodires, node 34, at 215 Myr, which first Elasmobranchii (415 Myr) (16). is very close to the dates inferred by 2 recent studies (4, 7). Within the archosaurs, the mean age of the split between crocodilians Most Recent Common Ancestor (MRCA) of Osteichthyes ( and birds, node 33, is dated at 249 Myr, slightly older than the vs. ) (Fig. S2, node 2). The oldest known fossil from 245 Myr of the fossil calibration by Muller and Reisz (10). The this clade is the stem actinopteygian Andreolepis hedei from the split between and is 87 Myr, which is Lludlowian of Gotland, Sweden (Silurian, 422–418 Myr) (17). significantly older than the 42 Myr inferred by Hugall et al. but Our prior assumed a minimum age of 418 and used the age of in much better agreement with both the crocodilian fossil record the Burgess Shale deposits (505 Myr) for the 95% confidence (10) and other molecular timescale studies based on mitog- interval. enomes (11). Within birds, the mean age of the split between ratites and the neognathans is dated at 118 Myr, in very good MRCA of Sarcopterygii (Dipnotetrapodomorpha vs. Coelacanthimor- agreement with Hugall et al. and Slack et al. (12), but younger pha) (Fig. S2, node 3). The oldest sarcopterygian is Psarolepis than the 139 Myr inferred by Pereira and Baker (8). romeri from the Lludlow-Pridoli of (Silurian, 420–418 Among the Actinopterygii, the crown ray-finned fishes (Fig. Myr), but this is likely a stem taxon. The oldest taxon that marks S2, node 4) have a mean age of 298 Myr. This is Ϸ100 Myr older the split between coelacanths, lungfishes, and tetrapods is Eo- than the oldest fossil but also Ϸ100 Myr younger than recent actinistia foreyi, the oldest coelacanthimorph from the Pragian mitogenomic studies (1, 6). Similarly, our estimated ages for (, 409–407 Myr) (18). The prior thus assumed 407 Myr splits within the are younger than Azuma et al. (1) but in as the minimum age of the split and a 95% upper bound of 505 close agreement with dates provided by time-calibrated nuclear Myr (reflecting the upper age of the Burgess shale deposit). gene divergences (5). This discrepancy might be due to an overall higher rate of evolution in mitochondrial genomes as discussed MRCA of Actinopterygii (Actinistia vs. vs. Neote- by Hurley et al. (5). Our estimates for actinopterygian divergence leostei) (Fig. S2, node 4). The oldest fossil known to belong to this times incorporate the largest number of fossil calibrations in clade is the stem actinopteygian Andreolepis hedei from the a study to date and also cover a very broad phylogenetic scope. Lludlowian of Gotland, Sweden (Silurian, 422–418 Myr) (17). Thus our estimated ages should be more reliable than those with The oldest taxon that can be assigned to the crown is the fewer fossils or those based on a smaller number of represen- neopterygian Brachydegma caelatum, from the Artkinsian of tative fish lineages. Texas (early , 284 Myr) (5). Our prior thus assumed 284 Myr as the minimum age and 418 Myr for the upper bound. Appendix 2: Description of Timetree Calibrations. The priors for the Tetrapoda were obtained from Hugall et al. (4), and readers are MRCA of ( vs. Lepisosteiformes vs. Teleostei) referred to that paper or the literature therein cited, for a more (Fig. S2, node 5). The oldest crown taxon is the stem teleost detailed discussion. Here we list only minimum age and upper Pholidophoretes salvus (19). The oldest stem is Brachydegma boundaries. caelatum, from the Artkinsian of Texas (early Permian, 284 Myr)

Alfaro et al. www.pnas.org/cgi/content/short/0811087106 1of20 (5). Our prior thus assumed 225 Myr as the minimum age and materials from the Adamantina Formation of 284 Myr for the upper bound. , Turonian-Santonian (Late , 93.6–83.5 MyrR) (28). Our prior thus assumed 73 Myr as the minimum age, and MRCA of Teleostei ( vs. vs. Clupeo- 83.5 MyrR as the upper boundary. cephala) (Fig. S2, node 6). The oldest taxon assigned to this clade is Anaethalion, from the late Kimmeridgian lithographic lime- MRCA of (Fig. S2, node 14). The oldest bagrid fossils, stone of Nusblingen, Germany, and Cerin, France (Jurassic, 152 Eomacrones wilsoni, Nigerium gadense, Nigerium wurnoe¨nse, have Myr) (20). The oldest stem teleost is Pholidophoretes salvus all been assigned to deposits in Niger of age (Ϸ59 (Pholidophoridae), from the early Carnian (Julian) of Polzberg MyrR) (27). Our prior assumed 59 Myr as the minimum age, and bei Lunz, Austria (, 228–225 Myr) (19). Our prior 73 Myr, age assigned to the crown siluriforms, as the upper assumed 152 Myr as the minimum age and 225 Myr for the upper boundary (27). bound. MRCA of (Fig. S2, node 15). The oldest callichthyid, MRCA of Osteoglossomorpha (Fig. S2, node 7). The oldest crown Corydoras revelatus, dates from the of osteoglossomorphs are several taxa from the assem- (Paleocene, Ϸ55 Myr) (27). Our prior assumed 55 Myr as the blage of the Barremian of China (, 130 Myr), minimum age, and 73 Myr, the age assigned to the crown such as the Hiodontidae Yanbiania wangqingica (21). To estab- siluriforms, as the upper boundary (27). lish an upper boundary, we used the elopomorph Anaethalion, from the late Kimmeridgian lithographic (Jurassic, MRCA of (Fig. S2, node 16). The argentiniform 152 Myr) (22). Our prior assumed 130 Myr as the minimum age Nybelinoides brevis from the Barremian- of Bernissart, and 152 Myr as the upper bound. Belgium (Early Cretaceous, 127–124 Myr) marks the earliest appearance of the crown argentiniforms (29). Leptolepides sprat- MRCA of Elopomorpha (Fig. S2, node 8). The oldest crown elopo- tiformis (Orthogonikleithridae) from the late Kimmeridgian morph is the Albulidae Albuloideorum ventralis, from the Early lithographic limestone of Cerin, France (Jurassic, 152 Myr) is the Hauterivian (Jurassic/Cretaceous border, 135 Myr) (23). The oldest euteleost (20). Our prior assumes 124 Myr as the mini- oldest stem elopomorph is Anaethalion, from the late Kimmer- mum age, and 152 Myr as the upper boundary. idgian lithographic limestone of Nusblingen, Germany, and Cerin, France (Jurassic, 152 Myr) (22). Our prior assumed 135 MRCA of (Fig. S2, node 17). Stomporia rogersmithi from Myr as the minimum age and 152 Myr for the upper bound. the Maastrichthian of South Africa (, 70 Myr) (30) indicates the appearance of the crown galaxiids. The MRCA of Ostarioclupeomorpha (Ostariophysii vs. Clupeomorpha) (Fig. argentiniform Nybelinoides brevis from the Barremian-Aptian of S2, node 9). The oldest known crown ostarioclupeomorph fossil is Belgium (Early Cretaceous, 127–124 Myr) is used to establish Tischlingerichthys viohli, upper Tithonian of Muhlheim, Bavaria, the upper boundary (29). The prior thus assumed 70 Myr as the Germany (Jurassic, 149 Myr) (20). To establish a 95% upper minimum age, and 124 Myr for the upper boundary. boundary, we used the elopomorph Anaethalion, from the late Kimmeridgian lithographic limestone of Nusblingen, Germany, MRCA of (Fig. S2, node 18). The oldest esociform is and Cerin, France (Jurassic, 152 Myr) (22). Estesesox foxi from the of the Milk River Fomation, Alberta, Canada (Late Cretaceous, 85 Myr) (31). Leptolepides MRCA of Ostariophysii (Fig. S2, node 10). The oldest crown ostari- sprattiformis (Orthogonikleithridae) from the late Kimmeridgian ophysans are the Chanidae Gordichthys and Rubiesichthys from lithographic limestone of Cerin, France (Jurassic, 152 Myr) is the the Berriasian/Barremian (Jurassic/Cretaceous, 145–125 Myr) oldest euteleost (20). Our prior assumes 85 Myr as the minimum (24). The ostarioclupeomorph Tischlingerichthys viohli, from age, and 152 Myr as the upper boundary. upper Tithonian of Muhlheim, Germany (Jurassic, 149 Myr), was used to establish an upper boundary (20). Our prior thus MRCA of Salmoniformes (Fig. S2, node 19). The origin of Salmoni- assumed 125 Myr as the minimum age and 149 Myr as the 95% formes is indicated by Helgolandichthys schmidi, from the early upper boundary. Aptian of Tock, Helgolang, Germany (Early Cretaceous, 125 Myr) (32). Because of uncertainty on the placement of this taxon MRCA of (Fig. S2, node 11). Santanichthys diasii from we assign its age to the upper boundary. The minimum age of the the Santana Formation in Brazil, (Early Cretaceous, crown salmoniforms in our prior is that of Eosalmo driftwood- 112–100 Myr) is the oldest stem characiform (25). The oldest ensis from the Lutetian (, 48 Myr) (33). crown characiforms are various Serrasalminae indeterminate. and Tetraogonopterinae indet. from the middle of MRCA of (Fig. S2, node 20). An undeterminate alepi- the El Molino Formation, (Late Cretaceous, 69–68 Myr) sauroid from the early Barremian of Alcaine, Oliete subbasin, (25). Our prior assumed 68 Myr as the minimum age and 100 NE Spain (Early Cretaceous, 130–128 Myr) (33) is used to mark Myr as the 95% upper boundary. the appearance of the earliest aulopiforms. This taxon is a stem to the crown alepisauriforms in our study, so we assign its age to MRCA of (Fig. S2, node 12). Parabarbus spp. from the the upper boundary. The minimum age of the crown alepisau- of the Obailinskaya Formation, Zaissan Basin, Kaza- riforms is assigned on the basis of the fossils Nematonotus spp. khstan (early-middle Eocene, 51–49 Myr) is the oldest crown (Aulopididae) and Acrognathus dodgei (Chlorophthtalmidae) cyprinid (26) and provided the minimum age for the cyprinid from the of Hakel, Lebanon (Late Cretaceous, calibration. The stem characiform Santanichthys diasii from the 98–96 Myr) (34). The prior assumed 96 Myr as the minimum age, Santana Formation in Brazil, Albian (Early Cretaceous, 112–100 and 128 Myr as the upper boundary. Myr) was used to set the 95% upper boundary (25). MRCA of (Fig. S2, node 21). The fossil otoliths MRCA of Siluriformes (Fig. S2, node 13). The oldest fossil that have assigned to the ‘‘Acanthomorphorum’’ forcallensis from the been assigned to the crown Siluriformes are incomplete remains early Aptian of the Maestrazgo, Castellon Province, Spain (Early of from the Campanian-Maastrichtian of Argentina Cretaceous, 124–122 Myr) (35) indicate the first appearance in (Late Cretaceous, 73 Myr) (27). The oldest siluriforms are the fossil record of acanthomorphs, although it is not known

Alfaro et al. www.pnas.org/cgi/content/short/0811087106 2of20 whether this was a stem or crown taxon. Various non (Cretaceous, 98 Myr) (46). We thus assigned a lower (e.g., , Trachichthyoides) from the Cenomanian of boundary of 59 Myr and an upper boundary of 98 Myr to this the Lower Chalk, SE England (Late Cretaceous, 99 Myr) calibration. represent the oldest record of crown acanthomorphs (36). The prior assumed a minimum age of 99 Myr and an upper boundary MRCA of Balistoidea (Balistidae vs. Monacanthidae) (Fig. S2, node 29). of 122 Myr. The stem balistids Balistomorphus, which includes the B. orbiculatus, B. ovalis, and B. spinosus, and Oligobalistes robustus MRCA of Beryciformes (Fig. S2, node 22). Various Beryciformes are all from the early of, respectively, Switzerland and (e.g., Hoplopteryx, Trachichthyoides) from the Cenomanian of Caucasus (Ϸ35 Myr) (41). They provide a minimum age estimate the Lower Chalk, Southeast England (Late Cretaceous, 99 Myr) for the split between balistids and monacanthids. Our prior represent the appearance of the crown (36). The age of fossil assigns a minimum age of 35 Myr to this calibration, and an otoliths assigned to the genus ‘‘Acanthomorphorum’’ forcallensis upper boundary of 50 Myr to this calibration (reflecting the from the early Aptian of the Maestrazgo, Castellon Province, appearance of several other tetraodontiform families in Monte Spain (Early Cretaceous, 124–122 Myr) (35) is used as an upper ) (42). boundary. The prior assumed a minimum age of 99 Myr and an upper boundary of 122 Myr. MRCA of (Fig. S2, node 30). Cretazeus rinaldii from the Late Campanian of Nardo´, Italy (Late Cretaceous, 72 Myr) MRCA of Lampridiformes (Fig. S2, node 23). Nardovelifer altipinnis is marks the appearance of the crown zeiforms (47, 48). The the oldest crown lampridiform currently known, from the Late Beryciformes (e.g., Hoplopteryx, Trachichthyoides) from the Cen- Campanian of Nardo´, Italy (Late Cretaceous, 75–70 Myr) (37). omanian of the Lower Chalk, Southeast England (Late Creta- Undescribed taxa from the Cenomanian of Hakel, Lebanon ceous, 99 Myr) are used to establish an upper boundary (36). The (Late Cretaceous, 97–98 Myr) (38) represent the earliest ap- prior assumed a minimum age of 72 Myr and an upper boundary pearance of lampridiforms in the fossil record. The prior as- of 99 Myr. sumed 70 Myr as the minimum age, and 98 Myr as the upper boundary. MRCA of Amniota (Fig. S2, node 31). The bird–mammal split, dated at 315 Myr, is used for the minimum age (49) based on the Nova MRCA of Moronidae (Fig. S2, node 24). Morone sp. from the Late Scotian Middle Paleothyris, the oldest known Campanian of Coffee Sand, Northeastern Mississippi (Late amniote. The upper boundary is based on the Eucritta melano- Cretaceous, 74–73 Myr) is the oldest fossil assigned to moronids limnetes, a crown tetrapod from the Lower of (39). Because of the uncertainty of the phylogenetic relation- Scotland (50). ships within the , it is not currently known which is the sister group of the Moronidae. The otoliths assigned to the MRCA of Reptilia (Fig. S2, node 32). The lepidosaur- split genus ‘‘Epigonidarum’’ weinbergi from the Coniacian of Tiefe was dated at 255 Myr and is used for the minimum age (10). The Gosau, Ennstaler Alpen, Austria (Late Cretaceous, 89–84 Myr), upper boundary is set at 282 Myr and it is obtained using the however, indicate the earliest appearance of taxa belonging to Earliest known Mesosaurid that dates the parareptilia (51). the clade of Perciformes that likely includes the Moronidae, and are thus used to establish an upper boundary (40). The prior MRCA of Archosauria (Fig. S2, node 33). The bird– split has assumed 74 Myr as the minimum age, and 84 Myr as the upper been dated at 245 Myr and is used for the minimum age (10). boundary. This is based on the age of the Triassic , Euparkeria capensis. The upper boundary is obtained from the age of 280 MRCA of Ostracioidea ( vs. ) (Fig. S2, node 25). Myr indicated by Hugall et al. (4) as the minimum age for the Eolactoria sorbinii is a stem ostraciid, and Proaracana dubia is a split between Archosauria and Lepidosauria in their analysis. stem aracanid (41). Both are from the Ypresian of Monte Bolca, Italy (middle Eocene, 50 Myr). We assigned to this calibration MRCA of Chelonia (Fig. S2, node 34). The split between pleurodire a lower bound of 50 Myr and an upper bound of 70 Myr, the and cryptodire turtles, dated at 210 Myr, indicates the minimum likely age of crown tetraodontiforms (42). age of chelonians and is based on the age of Proterochersis (52). The upper boundary was set to 245 Myr and is derived from the MRCA of Tetraodontoidea ( vs. Diodontidae) (Fig. S2, age for Euparkeria capensis, the age for the Archosaur crown age node 26). Several stem diodontids, Prodiodon erinaceus, Prodi- (4). odon tenuispiis, Heptadion echinus, and Zignodon fornasieroae (S41), and the stem tetraodontid, Eotetraodon pygmaeus (41, 43) MRCA of Trionychoidea (Fig. S2, node 35). The split between Lisse- are known from the Ypresian of Monte Bolca, Italy (middle mys and based on the Mongolian fossil Aspideretes Eocene, 50 Myr). We used this date as a lower bound and maartrensis (53) dated at 100 Myr, provides the minimum age for assigned an upper bound of 70 Myr, the likely age of crown trionychoid turtles (7). The upper boundary was derived from tetraodontiforms, as derived from Alfaro et al. (42). Near et al. (7) estimate for the lower bound of their node, Ϸ143 Myr. MRCA of Tetraodontidae (Fig. S2, node 27). The fossil Archaeotetra- odon winterbottomi (44) from the Oligocene of Caucasus, Russia, vs. (Fig. S2, node 36). The split between has been assigned to the crown Tetraodontidae, and provides a scincomorphs and anguimorphs is dated at 168 Myr and indicates minimum age estimate of 35 Myr for the MRCA of the family the minimum age (54). This is based on the fossil unequivocal (45). We assigned an upper bound of 50 Myr (age of stem Paramacellodids (55) from the Middle Jurassic. The upper tetraodontids; see above) to this calibration. boundary was derived from the age of 190 Myr indicated by Hugall et al. (4) as the minimum age for the radiation of the MRCA of (Fig. S2, node 28). This oldest fossil that crown squamates in their analysis. can be assigned to the crown tetraodontiforms is the stem balistoid Moclaybalistes danekrus, from the Moclay deposits of vs. Elgaria (Fig. S2, node 37). The split Heloderma– Denmark (Palaeocene, 59 Myr) (41). The oldest tetraodontiform Elgaria is dated at 99 Myr and indicates the minimum age (56). fossil is Plectocretacicus clarae, from the Cenomanian of Leba- This is based on the oldest known terrestrial platynotan, Prima-

Alfaro et al. www.pnas.org/cgi/content/short/0811087106 3of20 derma nessovi, was derived from the oldest serpent, Lapparen- MRCA of (Fig. S2, node 41). The elephant–pig split (extant tophis defrennei, from the Lower Cretaceous (57). eutherians) was dated at 98 Myr and is used for the minimum age (59). The upper boundary is set to 125 Myr based on the stem Varanus vs. Shinisaurus (Fig. S2, node 38). The split Varanus– eutherian Eomaia scansoria (60). Shinisaurus is dated at 85 Myr and indicates the minimum age (4). The upper boundary was derived from Wiens et al. (58) using Appendix 3. MEDUSA Analyses with Varying AIC Thresholds. We used a date that is 5 Myr from their estimate of the split of Shinisaurus ϩ a set of 3 alternative thresholds in determing what level of from the clade formed by Lanthonotus Veranus, 104 MyrA. support is necessary to add additional parameters to MEDUSA. First, we used both a liberal 2 and strict 10 cutoff value for AIC; MRCA of Alligatorinae (Fig. S2, node 39). The – split models where the AIC score improved less than the cutoff were is dated at 68 Myr and indicates the minimum age (10). This is based on the alligatorine fossil Stangerochampsa mccabei. The not accepted. Second, we implemented corrected AIC scores upper boundary is dated to 116 Myr and was derived from the [AICc; (61)], which account for small sample sizes. This correc- Early Cretaceous crocodylomorph, Hylaeochampsa vectiana the tion requires inputting the sample size used for an analysis but oldest known Eusuchian (50). such a measure is difficult to interpret in the case of data in the form of a with incompletely resolved tip . Sphenicus vs. Grus (Fig. S2, node 40). The penguin–crane split is We conservatively used a value of k ϭ 2nϪ1, which represents dated at 62 Myr and provides the minimum age (12). The The the total number of branches in the tree (nϪ1) plus the diver- upper boundary is dated to 87 Myr based on the age of the sities of the tips (n). In Table S7, an x indicates that each rate shift maximum constraint for the Palaeognathae–Neognathae split was supported by the final MEDUSA model. (Neornithes) given in Benton and Donoghue (59).

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Alfaro et al. www.pnas.org/cgi/content/short/0811087106 5of20 BeryciformesB and SteStephanoberyciformes

Ophidiiformes

Scombridae Polymixiiformes MyctophiformesLampriformes

Zeiforms ArgentiniformesAulopiformes

Osmeriformes

Stomiiformes Galaxiiformes Esociformes Salmoniformes

Ostariophysi

Sharks Latimeridae Dipnoi Clupeomorpha Caudata Osteoglossomorpha Anura

Elopomorpha Amiiformes Monotremes Polypteriformes Marsupials

Afrotheria + Xenarthrans

Boreoutheria

Non-Gekkonid Squamates Alli CrocodCamininae gatorinae TinamiforStruthioniformes Anseriformes

Gekkos Gal

Sphenodon Neoaves y liformes lin ae mes

Fig. S1. Radial timetree for gnathostomes. Fifty percent majority rule consensus chronogram, highlighting clade names used in this study. Red lineages represent Sarcopterygii, black lineages represent Actinopterygii. Dotted circles represent time increments of 100 Myr.

Alfaro et al. www.pnas.org/cgi/content/short/0811087106 6of20 Paleozoic Mesozoic Cambrian Ordovician Silurian Devonian Carboniferous Permian Triassic Jurassic Cretaceous Paleogene Neog.

Mustelus asterias 01 Carcharodon carcharias Sharks Polypterus Polypteriformes 04 Polyodon spathul albus Chondrostei calva Amiiformes Megalops atlanticus 08 vulpes Elopomorpha Ophichthus gomesii bicirrhos 05 07 Notopterus notopterus Osteoglossomorpha Pantodon puchholzi Anchoa delicatissima Engraulis japonicus 06 Clupea pallasii Clupeomorpha Alosa pseudoharengus 09 Chanos chanos Carassius auratus 12 Hesperoleucus symmetri exilicauda Pimephales promelas Danio renio Gymnotus maculosus Sternopygus Distichodus notospilus Gnathocharax steindach 11 10 Catoprion mento Hepsetus odoe nattereri Hypostomus latifrons Callichthys callichthy 15 Corydoras trilineatus 02 13 mesembrinu Bagrus docmak 42 14 To Panel C Leiocassis poecilopter Phractocephalus hemiol Pimelodus ornatus Ictalurus punctatus 43 Ameiurus nebulosus Bagre marinus peruvianus Heterobranchus longifi Prosopium williamsoni 19 Oncorhynchus mykiss Salmoniformes pygmae 18 Esox lucius Esociformes bullock 17 Galaxias fasciatus Galaxiiformes bathyphilum Retropinna tasmanica Plecoglossus altivelis To Panel B olidus

Overview Map

500 450 400 350 300 250 200 150 100 50 present

Fig. S2. (A–D) Detailed view of gnathostome phylogeny (Fig. S1). Bars around nodes represent the 95% HPD. Orange 95% HPD bars indicate posterior probabilities greater than 0.95, gray indicates support values less than 0.95. Black 95% HPD bars indicate clades constrained to be monophyletic. Highlighted lineages mirror lineages in text and Fig. S1.

Alfaro et al. www.pnas.org/cgi/content/short/0811087106 7of20 Mesozoic Cenozoic Jurassic Cretaceous Paleogene Neog.

Argentina sialis 16 Bathylagus ochotensis Argentiniformes

To Panel A Panel To Chlorophthalmus 20 Synodus intermedius Aulopiformes kroyeri japonica Polymixiiformes 23 Regalecus glesne transmontana Percopsiformes verrucosus 30 Zenopsis conchifer Zeiformes 21 mediterra 22 Sargocentron punctatis Beryciformes and splendens Neobythites stigmosus Petrotyx sanguineus Scomberomorus commerso Scombridae Anabas testdineus Ctenopoma pellegrini Centropomus medius Ophisternon aenigmatic Synbranchus marmoratus Gadus morhua 44 albidus Solea solea 45 Hippoglossus hippoglos Pseudopleuronectes ame Mugil cephalus Abudefduf saxatilis 46 Pomacentrus pavo Astronotus ocellatus 47 Oreochromis tanganicae Fundulus heteroclitus Xiphophorus gordoni Bedotia geayi Oryzias latipas saurus Menidia menidia Labrus bergylta 48 Scarus psittacus Lepomis macrochirus Elassoma evergladei Sparus aurata Lutjanus analis Percomorpha Aruma histrio 49 Riso ruber Etheostoma caeruleum Perca flavescens Holanthias chrysostict Peristedion miniatum Scorpaena onaria Gasterosteus aculeatus Lates calcarifer 24 Morone chrysops Dicentrarchus labrax Chaetodon semilarvatus Zebrasoma scopas Scatophagus argus 50 capros americanus Siganus doliatus Balistes capriscus 51 29 ciliatus ornata 25 Tetrasomus concatenatus 28 Diodon holocanthus Chilomycterus schoepfi Overview Map 26 Takifugu rubripes 27 Sphoeroides dorsalis

150 1001 50 present

Fig. S2. continued

Alfaro et al. www.pnas.org/cgi/content/short/0811087106 8of20 Paleozoic Mesozoic Cenozoic Ordovician Silurian Devonian Carboniferous Permian Triassic Jurassic Cretaceous Paleogene Neog.

Latimeria menadoensis Latimeridae Lepidosiren paradoxa Dipnoi Protopterus dolloi 54 Ambystoma mexicanum Pleurodeles walti Caudata Litoria ewingii 55 53 Xenopus laevis Anura glutinosus To Panel A Panel To 56 bivittatum Gymnophiona Typhlonectes natans 03 Hypogeophis rostratus Ornithorhynchus anatinus 57 Tachyglossus aculeatus Monotremes Monodelphis domestica 60 58 Notoryctes typhlops 59 Cercartetus concinnus Marsupials Sarcophilus harrisii 61 Elephas maximus Afrotheria and 52 Chaetophractus villosus Myrmecophaga tridactyla Xenarthrans 41 Bradypus tridactylus Lama glama 62 Sus scrofa 63 Homo sapiens 64 Oryctolagus cuniculus Boreoeutheria 65 Rattus norvegicus 66 Mus musculus Apomys hylocoetes latisternum 67 expansa Pleurodira williamsi 31 34 Carettochelys insculpta Apalone spinifera 35 punctata 68 pardalis Cryptodira Platysternon megacephalum Dermatemys mawii 33 Chelonia mydas Alligator mississipiensis Alligatorinae 39 Caiman latirostris Caminae 69 cataphractus Crocodylinae Tomistoma schlegelii Gavialis gangeticus Gavialidae 70 Struthio camelus Struthioniformes Tinamus guttatus Tinamiformes Anas strepera Anseriformes 71 Megapodius treycinet Galliformes 332 Gallus gallus Gavia immer To Panel D Charadrius vociferus 72 40 Spheniscus humboldti Grus canadensis Neoaves Passer montanus Coracias caudata Overview Map

450 400 350 300 250 200 150 100 50 present

Fig. S2. continued

Alfaro et al. www.pnas.org/cgi/content/short/0811087106 9of20 Paleo. Mesozoic Cenozoic Perm. Triassic Jurassic Cretaceous Paleogene Neog.

Sphenodon punctatus Sphenodon Gekko gekko

To Panel C Panel To Lialis jicari Gekkos 73 lindneri Dibamus sp. vigilis

Zonosaurus sp. Typhlosaurus lomii

Ctenotus robustus Asymblepharus sikimmensis

Eumeces anthracinus Euprepis auratus 36 Aspidoscelis tigris Leposoma parietale Eremias sp. floridana 74 Bipes biporus Ramphotyphlops braminus

75 Dinodon sp. 76 ruffus Non-Gekko Xenosaurus grandis Squamates Elgaria panamintina 37 Heloderma suspectum

Shinisaurus crocodilurus 38 Lanthanotus borneensis Varanus griseus

Basiliscus plumifrons Leiocephalus carinatus Anolis paternus

77 Leiolepis belliana Chamaeleo rudis Brookesia thieli Ctenphorus salinarum Physignathus cocincinus

Overview Map calotes Japalura tricarinata

250 200 150 100 50 present

Fig. S2. continued

Alfaro et al. www.pnas.org/cgi/content/short/0811087106 10 of 20 Table S1. List of taxa and GenBank accession nos Lineage RAG1 GenBank they belong sequence no. Taxon Common name Family Order to in Fig.1

AF135482 Carcharodon carcharias Great white shark Lamnidae Lamniformes Elasmobranchii AY462188 Mustelus asterias Starry smooth-hound shark Triakidae Charcariniformes AF369055 Polypterus sp. Polypteridae Polypteriformes Polypteriformes AY430199 Amia calva Bowfin Amiidae Amiiformes AF369056 sp. Acipenseridae Acipenseriformes AY430198 Scaphirhynchus albus U15613 Polyodon spathula Mississippi paddlefish Polyodontidae Chondrostei AF369057 AF369063 Notopterus notopterus Osteoglossomorpha Osteoglossomorpha AY430201 Osteoglossum bicirrhosum Arawana Osteoglossidae AF369061 Pantodon buchholzi Freshwater butterflyfish Pantodontidae AY430202 Albula vulpes Bonefish Albulidae Albuliformes Elopomorpha AY430204 Megalops atlanticus Megalopidae AY430203 Ophichthus gomesii Ophichthidae DQ912115 Alosa pseudoharengus Alewife Clupeidae Clupeiformes Clupeomorpha DQ912118 Clupea pallasii Pacific DQ912108 Anchoa delicatissima Slough anchovy Engraulidae AY430205 Engraulis japonicus Japanese anchovy AY430212 Catoprion mento Wimple Characidae Characiformes Ostariophysi AY430211 Gnathocharax steindachneri DQ492425 Distichodus notospilus Citharinidae DQ912097 Hepsetus odoe Kafue pike Hepsetidae U15614 Carassius auratus Goldfish Cyprinidae U71093 Danio rerio Zebrafish AY059468 Hesperoleucus symmetricus AY059469 Lavinia exilicauda Hitch AY430210 Pimephales promelas Fathead minnow AY430207 Chanos chanos Milkfish Chanidae AY359225 Gymnotus sp. Knifefish Gymnotidae DQ492426 Sternopygus sp. Glass knifefishes Sternopygidae DQ492524 Bagre marinus Gafftopsail sea catfish Ariidae Siluriformes DQ492527 Peruvian sea catfish DQ492458 Bagrus docmak Semutundu Bagridae DQ492457 Leiocassis poecilopterus DQ492436 Cascarudo Callichthyidae DQ492437 Corydoras trilineatus Threestripe corydoras DQ492520 Heterobranchus longifi Vundu Clariidae DQ492428 Diplomystes mesembrinus Diplomystidae AY430209 Ameiurus nebulosus Brown bullhead Ictaluridae DQ492511 Ictalurus punctatus Channel catfish DQ492441 Farlowella nattereri Armored catfishes Loricaridae AY552038 Hypostomus latifrons Armored catfishes DQ492476 Phractocephalus Redtail catfish Pimelodidae hemioliopterus DQ492475 Pimelodus ornatus AY380542 Esox lucius Northern pike Esocidae Esociformes Esociformes AY380549 Umbra pygmae Eastern mudminnow AY430228 Argentina sialis North-Pacific argentine Argentiniformes Argentiniformes AY443564 Bathylagus ochotensis Eared blacksmelt Bathylagidae Galaxiiformes AY430219 Galaxias Galaxiidae Galaxiiformes AY430218 Galaxias fasciatus Banded kokopu AY380538 Hypomesus olidus Pond Osmeridae Osmeriformes Osmeriformes AY380536 Plecoglossus altivelis Ayu Plecoglossidae AY430216 Retropinna tasmanica Tasmanian smelt Retropinnidae U15663 Oncorhynchus mykiss Salmoniformes Salmoniformes AY430213 Prosopium williamsoni Mountain whitefish AY438703 Gonostoma bathyphilum Bristlemouths Stomiiformes Stomiiformes AY442363 Vinciguerria sp. Lightfishes AY430220* Chlorophthalmus agassizi Greeneyes Chlorophthalmidae Aulopiformes Aulopiformes AY308763 Synodus intermedius Sand diver Synodontidae AY430221 Notoscopelus kroyeri Lancet fish Myctophidae Myctophiformes Myctophiformes

Alfaro et al. www.pnas.org/cgi/content/short/0811087106 11 of 20 Lineage RAG1 GenBank they belong sequence no. Taxon Common name Family Order to in Fig.1

AY308764 Lampris guttatus Lampridae Lampriformes Lampriformes AY430222 Regalecus glesne King of Regalecidae AY308765 Polymixia japonica Silver eye Polymixiidae Polymixiiformes Polymixiiformes EF095636 Beryx splendens Splendid Beryciformes Beryciformes AY430223 Sargocentron Speckled squirrelfish punctatissimum EF095635 Hoplostethus Mediterranean Trachichthyidae mediterraneus EF033043 Neobythites stigmosus Ophidiidae Ophidiiformes Ophidiiformes AY308782 Petrotyx sanguineus Redfin brotula EF095676 Scomberomorus Narrow-barred Spanish Scombridae Perciformes Scombridae commerson mackerel AF369064 Gadus morhua Atlantic Gadidae Percomorpha AY308787 Merluccius albidus Offshore hake AY308786 Lophius americanus American angler Lophiidae Lophiiformes AY308766 Sand roller Percopsiformes EF095640 Bedotia geayi Red-Tailed Silverside Bedotiidae AY430225 Menidia menidia Atlantic silverside Atherinopsidae AB120889 Oryzias latipes Japanese rice fish Adrianichthyidae EF095641 AY308771 Scomberesox saurus Atlantic saury EF033040 Fundulus heteroclitus Mummichog Fundulidae DQ235866 Xiphophorus gordoni Southern platyfish Poeciliidae AF369065 Mugil cephalus Flathead Mugilidae Mugiliformes EF033039 Gasterosteus aculeatus Threespine stickleback Gasterosteidae AY308776 Zebrasoma scopas Twotone tang Acanthuridae Perciformes AY763773 Anabas testudineus Climbing perch Anabantidae AY763776 Ctenopoma pellegrini ctenopoma AY308785 Antigonia capros Deepbody boarfish EF095638 Capros aper Boarfish AY430227 Lepomis macrochirus Bluegill sunfish Centrarchidae EF095649* Centropomus medius Blackfin snook Centropomidae EF095655 Chaetodon semilarvatus Bluecheek butterflyfish Chaetodontidae EF095671 Astronotus ocellatus Oscar Cichlidae DQ012223 Oreochromis tanganicae Cichlid AY308772 Drepane punctata Spotted sicklefish Drepaneidae AY308784 Elassoma evergladei Everglades pygmy sunfish Elassomatidae EF095670 Embiotoca jacksoni Black perch Embiotocidae AY846564 Aruma histrio Slow goby Gobiidae AY846561 Risor ruber Tusked goby EF095669 Labrus bergylta Ballan wrasse Labridae AF369066 Lates calcarifer Barramundi Latidae EF033042 Lutjanus analis Mutton snapper Lutjanidae EF095659 Mene maculata Moonfish Menidae EF095651 Dicentrarchus labrax European seabass Moronidae AY308767 Morone chrysops White bass AY430226 Etheostoma caeruleum Rainbow darter Percidae AY308768 Perca flavescens Yellow perch AY208624 Abudefduf saxatilis Sergeant major Pomacentridae EF095673 Pomacentrus pavo EF095675 Scarus psittacus Common parrotfish Scaridae EF095668 Scatophagus argus Spotted scat Scatophagidae EF095645 Holanthias chrysostictus Serranidae AY308777 Siganus doliatus Barred spinefoot Siganidae EF095657 Sparus aurata Gilthead seabream Sparidae AY454396 Hippoglossus hippoglossus Atlantic halibut Pleuronectidae Pleuronectiformes AF369067 Pseudopleuronectes Winter flounder americanus EF095644 Solea solea Common sole Soleidae AY308774 Peristedion miniatum Armored searobin Peristediidae EF095642 Scorpaena onaria Scorpaenidae AY359218 Synbranchus marmoratus Marbled swamp eel Synbranchidae AY700348 Ornate cowfish Aracanidae Tetraodontiformes

Alfaro et al. www.pnas.org/cgi/content/short/0811087106 12 of 20 Lineage RAG1 GenBank they belong sequence no. Taxon Common name Family Order to in Fig.1

AY700308 Balistes capriscus Grey triggerfish Balistidae AY700325 Diodon holocanthus Long-spine porcupinefish Diodontidae AY700326 Chilomycterus schoepfi Striped burrfish Diodontidae AY700334 Monacanthus ciliatus Fringed filefish Monacanthidae AY308794 Tetrosomus concatenatus Triangular boxfish Ostraciidae AY308795 Sphoeroides dorsalis Marbled puffer Tetraodontidae AF108420 Takifugu rubripes Torafugu AY308781 Allocyttus verrucosus Zeiformes AY308778 Zenopsis conchifer Silvery John dory AY442926 Lepidosiren paradoxa South American lungfish Lepidosirenidae Lepidosireniformes Dipnoi AY442928 Protopterus dolloi African lungfish Protopteridae AY442925 Latimeria menadoensis Coelacanth Latimeriidae Coelacanthiformes Latimeridae EF551562 Litoria ewingii Brown tree Hylidae Anura Batrachia L19324 Xenopus laevis African clawed frog Pipidae EF551561 AY323752 Ambystoma mexicanum Axolotl Ambystomatidae Caudata Caudata AJ010258 Pleurodeles waltl Iberian Ribbed Newt Salamandridae EF551565 Hypogeophis rostratus Frigate Island Caeciliidae Gymnophiona Gymnophiona EF551566 Typhlonectes natans Rubber Eel EF551563 AY456256 EF551564 AY456257 Two-lined Caecilian EF551559 AF303974 Ornithorhynchus anatinus Platypus Ornithorhynchidae Monotremata Monotremata EF551558 AF303971 Tachyglossus aculeatus Short-beaked Echidna Tachyglossidae EF551556 AY125037 Sarcophilus harrisii Tasmanian Devil Dasyuridae Dasyuromorphia Marsupialia U51897 Monodelphis domestica Gray Short-tailed Opossum Didelphidae Didelphimorphia EF551557 AY125036 Cercartetus concinnus Southwestern Pygmy Burramyidae Diprotodontia Possum EF551555 AY125040 Notoryctes typhlops Southern Marsupial Mole Notoryctidae Notoryctemorphia AF305953 Lama glama Llama Camelidae Artiodactyla Eutheria AB091392 Sus scrofa wild boar Suidae M77666 Oryctolagus cuniculus European Rabbit Leporidae Lagomorpha AY243401 Bradypus tridactylus Pale-throated Three-toed Bradypodidae Pilosa Sloth AY011870 Myrmecophaga tridactyla Giant anteater Myrmecophagidae AY011868 Chaetophra villosus Large Hairy Armadillo M29474 Homo sapiens Human Hominidae Primates EF551560 AY125021 Elephas maximus Asian Elephant Elephantidae Proboscidea AY294942 Apomys hylocoetes Mt. Apo Mouse Muridae Rodentia NM009019 Mus musculus House Mouse XM230375 Rattus norvegicus Norway rat AY662576 Sphenodon punctatus Tuatara Sphenodontidae Sphenodontia Sphenodontidae AY662627 Crenadactylus ocellatus Clawless AY662625 Gekko gecko Tokay Gecko AY662626 Pseudothecadactylus lindneri AY662628 Lialis jicari New Guinea Lizard AY662584 Calotes calotes Common Green Forest Scincomorpha- Lizard Anguimorpha AY662580 salinarum Saltpan Ground Dragon AY662585 Japalura tricarinata Three Keeled Mountain Lizard AY662587 Leiolepis belliana Common Butterfly Lizard AY662582 Physignathus cocincinus Chinese Water Dragons AY662603 Elgaria panamintina Panamint Alligator Lizard AY662616 Bipes biporus Mexican mole lizard AY662577 Brookesia thieli Leaf Chamaeleonidae AY662578 Chamaeleo rudis Rudi’s Chameleon AY662611 Dinodon sp. Big-toothed snakes AY662599 Basiliscus plumifrons Green basilisk AY662613 Red-tailed pipe snake Cylindrophiidae AY662645 Dibamus sp. Blind lizards AY662644 Zonosaurus sp Plated lizards AY662621 Leposoma parietale Spectacled lizards AY662606 Heloderma suspectum Helodermatidae

Alfaro et al. www.pnas.org/cgi/content/short/0811087106 13 of 20 Lineage RAG1 GenBank they belong sequence no. Taxon Common name Family Order to in Fig.1

AY662589 Anolis paternus Anole AY662615 Eremias sp. Wall Lizards AY662609 Lanthanotus borneensis Earless Lanthanotidae AY662598 Leiocephalus carinatus Cuban Curly-tailed Lizard Leiocephalidae AY662618 Rhineura floridana North American worm lizard AY662631 Asymblepharus Scincidae sikimmensis AY662630 robustus Eastern Striped AY662634 Eumeces anthracinus Coal Skink AY662629 Euprepis auratus Levant Skink AY662641 Typhlosaurus lomii Lomi’s Blind Legless Skink AY662620 Aspidoscelis tigris Western whiptail AY662612 Ramphotyphlops braminus Brahminy blind snake AY662608 Varanus griseus AY662642 Xantusia vigilis Desert Xantusiidae AY662610 Shinisaurus crocodilurus Chinese crocodile lizard AY662607 Xenosaurus grandis Knob-scaled Lizard AY687902 Lissemys punctata Indian flap-shelled turtle Testudines Criptodira AY687904 Carettochelys insculpta Pig-nosed turtle Carettochelyidae AY687907 Chelonia mydas Green turtle Chelonidae AY687910 Dermatemys mawii Mesoamerican river turtle AY687905 Platysternon Big-headed turtle Platysternidae megacephalum AY687912 Geochelone pardalis Leopard Testudinidae AY687901 Apalone spinifera Spiny softshell turtle AY687902 Lissemys punctata Indian flap-shelled turtle AY687920 Elseya latisternum Australian snapping turtle Testudines Pleurodira AY687923 Pelusios williamsi African mud turtles AY687924 Podocnemis expansa Arrau turtle AY239174 Crocodylus cataphractus Slender-snouted crocodile Crocodylidae Crocodylinae AF143724 Alligator mississipiensis Alligatorinae AY239167 Caiman latirostris Broad-snouted caiman Camininae AF143725 Gavialis gangeticus Indian gavial Gavialidae Gavialidae AY239176 Tomistoma schlegelii AF143726 Tinamus guttatus White-throated Tinamou Tinamidae Tinamiformes Tinamiformes AF143727 Struthio camelus Ostrich Struthionidae Struthioniformes Struthioniformes AF143736 Charadrius vociferus Killdeer Charadriidae Charadriiformes Neoaves AF143737 Coracias caudata Lilac-breasted roller Coraciidae Coraciiformes AF143733 Gavia immer Common loon Gaviidae Gaviiformes AF143732 Grus canadensis Sandhill crane Gruidae Gruiformes AF143738 Passer montanus Tree sparrow Passeridae Passeriformes AF143734 Spheniscus humboldti Spheniscidae Sphenisciformes AF143731 Megapodius freycinet Dusky scrubfowl Megapodiidae Galliformes Galliformes M58530 Gallus gallus Chicken Phasianidae AF143729 Anas strepera Gadwall Anatidae Anseriformes Anseriformes

Alfaro et al. www.pnas.org/cgi/content/short/0811087106 14 of 20 Table S2. Priors used in divergence time analysis Node Clade or split Min (Myr) Upper 95% (Myr)

Root 428 505 1 Elasmobranchii (Lamnidae vs. Scyliorhinidae) 165 415 2 MRCA of Osteichthyes 418 505 3 MRCA of Sarcopterygii 407 505 4 MRCA of Actinopterygii 284 420 5 MRCA of Neopterygii 225 284 6 MRCA of Teleostei 152 228 7 MRCA of Osteoglossomorpha 130 152 8 MRCA of Elopomorpha 135 152 9 MRCA of Ostarioclupeomorpha 149 152 10 MRCA of Ostariophysii 125 140 11 MRCA of Characiformes 68 100 12 MRCA of Cyprinidae 49 100 13 MRCA of Siluriformes 73 83.5 14 MRCA of Bagridae 59 73 15 MRCA of Callichthyidae 55 73 16 MRCA of Argentiniformes 127 152 17 MRCA of Galaxiidae 70 124 18 MRCA of Esociformes 85 152 19 MRCA of Salmoniformes 48.6 125 20 MRCA of Aulopiformes 96 128 21 MRCA of Acanthomorpha 99 122 22 MRCA of Beryciformes 99 122 23 MRCA of Lampridiformes 70 98 24 MRCA of Moronidae 74 84 25 MRCA of Ostracioidea 50 70 26 MRCA of Tetraodontoidea 50 70 27 MRCA of Tetraodontidae 35 50 28 MRCA of Tetraodontiformes 59 98 29 MRCA of Balistoidea 35 50 30 MRCA of Zeiformes 72 98 31 MRCA of Amniota 315 353 32 MRCA of Reptilia 255 282 33 MRCA of Archosauria 245 280 34 MRCA of Chelonia 210 245 35 MRCA of Trionychoidea 100 143 36 Scincomorpha vs Anguimorpha 168 190 37 Heloderma vs Elgaria 99 126 38 Varanus vs Shinisaurus 85 104 39 MRCA of Alligatorinae 68 116 40 Sphenicus vs Grus 62 87 41 MRCA of Eutheria 98 125

Alfaro et al. www.pnas.org/cgi/content/short/0811087106 15 of 20 Table S3. Constrained taxonomic groups Node Clade Membership Ref.

42 Clupeocephala ϩ 62 43 Ictaluridae Ictalurus ϩ Ameiurus 63 44 Gadiformes Gadus ϩ Merluccius 62 45 Pleuronectiformes Solea ϩ Hipoglossus ϩ Pseudopleuronectes 64 46 Pomacentridae Abudefduf ϩ Pomacentrus 65 47 Cichlidae Astronotus ϩ Oreochromis 65 48 Labridae Labrus ϩ Scarus 65 49 Gobiidae Aruma ϩ Riso 66 50 Caproidae Capros ϩ Antigonia 48 51 Tetraodontiformes Siganidae ϩ Lophiiformes Lophius ϩ Siganus ϩ Tetraodontiformes 64 52 Tetrapoda Amphibia ϩ Amniota 4 53 Amphibia Caudata ϩ Anura ϩ Gymnophiona 4 54 Caudata Pleurodeles ϩ Ambystoma 4 55 Anura Xenopus ϩ Litoria 4 56 Gymnophiona Typhlonectes ϩ Hypogeophis ϩ Rhinatrema ϩ Ichthyophis 4 57 Monotremata Onithorhynchus ϩ Tachyglossus 4 58 Marsupialia Monodelphis ϩ Notoryctes ϩ Cercartetes ϩ Sarcophilus 4 59 Notoryctomorpha ϩ Dasyuromorpha ϩ Sarcophilus ϩ Cercartetes ϩ Notoryctes 4 Diprotodontia 60 Mammalia Monotremata ϩ Marsupialia ϩ Eutheria 4 61 Theria Marsupialia ϩ Eutheria 4 62 Artiodactyla Lama ϩ Sus 4 63 Laurasiatheria ϩ Euarchontaglires Artiodactyla ϩ Euarchontaglires 4 64 Euarchontaglires Homo ϩ Glires 4 65 Glires Rodentia ϩ Oryctolagus 4 66 Rodentia Rattus ϩ Mus ϩ Apomys 4 67 Pleurodira Podocnemis ϩ Pelusios ϩ Elseya 4 68 Cryptodira Chelonia ϩ Dermatemys ϩ Geochelone ϩ Platysternon ϩ Lyssemys ϩ 67 Apalone ϩ Carettochelys 69 Crocodilia Crocodylus ϩ Gavialidae ϩ Alligatoridae 4 70 Archosauria Crocodilia ϩ Aves 4 71 Aves Struthio ϩ Tinamus ϩ Anas ϩ Gavia ϩ Megapodius ϩ Gallus ϩ Neoaves 4 72 Neoaves Coracias ϩ Passer ϩ Grus ϩ Spheniscus ϩ Charadrius ϩ Gavia 4 73 Lepidosauria Sphenodon ϩ Squamata 4 74 Rhineura ϩ Bipes 68 75 Serpentes Dinodon ϩ Cylindrophis ϩ Ramphotyphlops 4 76 Dinodon ϩ Cylindrophis 4 77 Iguania Basiliscus ϩ Leiocephalus ϩ Anolis ϩ Leiolepis ϩ Chamaeleo ϩ 68 Brookesia ϩ Ctenophorus ϩ Physignathus ϩ Calotes ϩ Japalura

Alfaro et al. www.pnas.org/cgi/content/short/0811087106 16 of 20 Table S4. Gnathostome species richness Clade Richness Ref.

Dipnoi 369 Elopomorpha 856 69 Percomorpha 15493 69 Salmoniformes 66 69 Latimeriidae 269 Elasmobranchi 970 69 Osteoglossomorpha 218 69 Osmeriformes 36 69 Galaxiiformes 52 69 Ostariophysi 7931 69 Aulopiformes 236 69 Stomiiformes 391 69 Myctophiformes 246 69 Argentiniformes 182 69 Esociformes 10 69 Polypteriformes 15 69 Holostei 869 Chondrostei 27 69 Clupeomorpha 364 69 Polymixiiformes 10 69 Zeiformes 32 69 Lampriformes 21 69 Beryciformes ϩ Stephanoberyciformes 219 69 Ophidiiformes 385 69 Scombridae 126 69 Percopsiformes 969 Tinamiformes 47 70 Struthioniformes 12 70 Galliformes 290 70 Anseriformes 162 70 Neoaves 9191 70 Anura 5503 71 Caudata 563 71 Gymnophiona 173 71 Monotremes 572 Marsupials 331 72 Afrotheria ϩ Xenarthra 110 72 Boreoeutheria 4967 72 Cryptodira 228 73 Pleurodira 79 73 Gavialidae 273 Camininae 673 Alligatorinae 273 Crocodylinae 13 73 Sphenodon 273 Gekkos 1076 73 Nongekko Squamates 6831 73

Alfaro et al. www.pnas.org/cgi/content/short/0811087106 17 of 20 Table S5. Divergence time estimates of focal gnathostome nodes Clade or split Mean (95% HPD) Myr

1. Elasmobranchii (Lamnidae vs. Scyliorhinidae) 168 (165 - 203) 2. MRCA of Osteichthyes 428 (418 - 463) 3. MRCA of Sarcopterygii 409 (407 - 418) 4. MRCA of Actinopterygii 298 (284 - 362) 5. MRCA of Neopterygii 231 (225 - 268) 6. MRCA of Teleostei 204 (179 - 230) 7. MRCA of Osteoglossomorpha 133 (130 - 150) 8. MRCA of Elopomorpha 137 (135 - 161) 9. MRCA of Ostarioclupeomorpha 150 (149 - 153) 10. MRCA of Ostariophysii 100 (88 - 113) 11. MRCA of Characiformes 72 (68 - 85) 12. MRCA of Cyprinidae 104 (75 - 127) 13. MRCA of Siluriformes 85 (74 - 97) 14. MRCA of Bagridae 61 (59 - 65) 15. MRCA of Callichthyidae 57 (55 - 62) 16. MRCA of Argentiniformes 129 (127 - 143) 17. MRCA of Galaxiidae 72 (70 - 88) 18. MRCA of Esociformes 89 (85 - 108) 19. MRCA of Salmoniformes 53 (49 - 80) 20. MRCA of Aulopiformes 102 (96 - 138) 21. MRCA of Acanthomorpha 141 (123 - 159) 22. MRCA of Beryciformes 104 (99 - 119) 23. MRCA of Lampridiformes 72 (71 - 78) 24. MRCA of Moronidae 75 (74 - 79) 25. MRCA of Ostracioidea 51 (50 - 54) 26. MRCA of Tetraodontoidea 53 (50 - 59) 27. MRCA of Tetraodontidae 37 (35 - 41) 28. MRCA of Tetraodontiformes 66 (59 - 76) 29. MRCA of Balistoidea 37 (35 - 44) 30. MRCA of Zeiformes 74 (72 - 83) 31. MRCA of Amniota 320 (315 - 335) 32. MRCA of Reptilia 270 (257 - 292) 33. MRCA of Archosauria 260 (249 - 277) 34. MRCA of Chelonia 215 (210 - 228) 35. MRCA of Trionychoidea 105 (100 - 122) 36. Scincomorpha vs Anguimorpha 173 (169 - 184) 37. Heloderma vs Elgaria 103 (100 - 110) 38. Varanus vs Shinisaurus 95 (87 - 109) 39. MRCA of Alligatorinae 79 (71 - 92) 40. Sphenicus vs Grus 66 (63 - 74) 41. MRCA of Eutheria 105 (99 - 120)

Alfaro et al. www.pnas.org/cgi/content/short/0811087106 18 of 20 Table S6. Comparison of divergence times Clade or split Mean Age (Myr) 95% HPD (Myr) Previous estimates

Chondrichthyes vs. Osteichthyes 466 MRCA of Neoselaceii 168 165 to 203 MRCA of Osteichthyes 428 418 to 463 415 to 524 (74) MRCA of Sarcopterygii 409 407 to 418 MRCA of Tetrapoda 351 326 to 386 341 to 365 (4); 341 to 369 (8) MRCA of Amphibia 320 234 to 378 292 to 322 (4) Batrachia vs Caudata 233 132 to 325 266 to 274 (4) MRCA of Amniota 320 315 to 335 305 to 342 (8) MRCA of Reptilia 270 257 to 292 285 to 289 (4) Archosauria vs. Chelonia 260 249 to 277 252 to 278 (4) MRCA of Archosauria 249 245 to 257 238 to 279 (8) MRCA of Chelonia 215 210 to 228 196 to 239 (8) MRCA of Crocodilia 87 74 to 110 42 MY (4) MRCA of Aves 118 80 to 174 115 to 136 (4); 127 to 154 (8); 125 to 141 (3) MRCA of Neoaves 92 72 to 128 92 to 113 (8) MRCA of Squamata 246 208 to 275 190 to 201 (4); ϳ 179 (51) MRCA of Mammalia 209 149 to 286 207 to 227 (4); 187 to 228 (8); 166 (2) MRCA of Monotremata 45 8 to 112 37 to 48 (4) MRCA of Metatheria 164 121 to 229 182 to 195 (4); 172 to 212 (8); 148 (2). MRCA of Marsupialia 78 33 to 131 66 to 73 (4); MRCA of Eutheria 105 99 to 120 102 to 107 (4) MRCA of Actinopterygii 298 284 to 362 397 to 478 mit (4); 374 to 448 (6) MRCA of 270 238 to 321 348 to 391 nuc (4); 346 to 391 mit (4); 337 to 413 (6) MRCA of Neopterygii 231 225 to 268 295 to 372 nuc (4); 327 to 378 mit (4); 340 to 442 (74) MRCA of Teleostei 204 179 to 230 268 to 326 mit (4); 295 to 372 (6) MRCA of Osteoglossomorpha 133 130 to 150 221 to 283 mit (4) MRCA of Elopomorpha 137 135 to 161 210 to 272 mit (4) MRCA of Ostarioclupeomorpha 150 149 to 153 192 to 255 mit (4); 242 to 332 (74); 204 to 275 (6) MRCA of Euteleostei 174 154 to 195 182 to 244 mit (4); 240 to 326 (74); 197 to 267 (6) MRCA of Acanthomorpha 141 123 to 159 125 to 186 mit (4); 191 to 264 (74); 130 to 191 (6) MRCA of Tetraodontiformes 66 59 to 76 124 to 184 (74) MRCA of Tetraodontidae 37 35 to 41 55 to 86 (1); 57 to 94 (74) MRCA of Balistoidea 62 56 to 72 95 to 146 (74) MRCA of Cichlidae 36 11 to 68 72 to 108 (1)

Alfaro et al. www.pnas.org/cgi/content/short/0811087106 19 of 20 Table S7. Supported rate shifts under varying ⌬AIC cut-off values AIC cutoff AICc

Number Clade 2 4 10 4

1 Percomorphs X x x x 2 Euteleosts X x x x 3 Latimeridae ϩ Dipnoans X x x x 4 Neoaves x x x x 5 Crocodilians x x x x 6 Tuatara x x 7 Boreoeutherians x x 8 Ostariophysans x x 9 Nongekko squamates x x 10 Anurans x 11 Galliformes ϩ Anseriformes x

Alfaro et al. www.pnas.org/cgi/content/short/0811087106 20 of 20