Molecules, morphology, and ecology indicate a recent, amphibious ancestry for

Matthew J. Phillipsa,1, Thomas H. Bennetta, and Michael S. Y. Leeb,c

aCentre for Macroevolution and Macroecology, Research School of Biology, Australian National University, Canberra, ACT 0200, ; bSchool of Earth and Environmental Sciences, University of Adelaide, Adelaide, SA 5005, Australia; and cEarth Sciences Section, South , Adelaide, SA 5000, Australia

Edited by David B. Wake, University of California, Berkeley, CA, and approved August 14, 2009 (received for review April 28, 2009) The semiaquatic and terrestrial echidnas (spiny anteaters) echidnas do not appear until the mid- (Ϸ13 are the only living -laying (). The fossil Ma) (13), despite excellent late –Early Miocene mam- record has provided few clues as to their origins and the evolution mal fossil records in both northern and southern Australia. This of their ecological specializations; however, recent reassignment absence has tentatively been attributed in part to echidnas of the Early Teinolophos and Steropodon to the platy- lacking teeth (14), which are the most common fossil remains pus lineage implies that and echidnas diverged >112.5 from mammals. Alternatively, if the molecular dating studies million years ago, reinforcing the notion of monotremes as living that estimate the divergence of echidnas from platypuses at . This placement is based primarily on characters related to 17–35 Ma (15–22) are correct, then characters that clearly ally a single feature, the enlarged mandibular canal, which supplies fossil taxa with echidnas would not be expected to have evolved blood vessels and dense electrosensory receptors to the platypus until even more recently. bill. Our reevaluation of the morphological data instead groups Molecular dating can play a pivotal role in inferring the platypus and echidnas to the exclusion of Teinolophos and Stero- evolutionary history of taxa with a sparse fossil record, such as podon and suggests that an enlarged mandibular canal is ancestral monotremes (14, 23, 24). Recent reassignment of the 112.5–121 Ϸ for monotremes (partly reversed in echidnas, in association with Ma Teinolophos and the 105 Ma Steropodon from outside of the crown group (platypuses and echidnas) specif- general mandibular reduction). A multigene evaluation of the EVOLUTION –platypus divergence using both a relaxed ically to the platypus lineage has profound implications (25). and direct fossil calibrations reveals a recent split of 19–48 million Teinolophos would be the oldest fossil unequivocally within any years ago. Platypus-like monotremes () predate of the three mammalian crown groups (Monotremata, Marsu- this divergence, indicating that echidnas had aquatically foraging pialia, and ). The fundamental morphological and ancestors that reinvaded terrestrial ecosystems. This ecological ecological differences between platypuses and echidnas also would date back over 100 Ma, reinforcing the notion of shift and the associated radiation of echidnas represent a recent monotremes being ‘‘living fossils,’’ a term that Darwin (26) first expansion of niche space despite potential competition from mar- coined with reference to the platypus. Furthermore, it implies supials. Monotremes might have survived the invasion of marsu- slow molecular evolution within monotremes that challenges pials into Australasia by exploiting ecological niches in which current views of molecular evolutionary rates (25). are restricted by their reproductive mode. Morphology, Upon revising Luo and Wible’s (27) data set, Rowe et al. (25) ecology, and molecular biology together indicate that Teinolophos assigned Teinolophos to the platypus lineage, based primarily on and Steropodon are monotremes rather than platypus rela- characters related to the mandibular canal. In the platypus, tives, and that living monotremes are a relatively recent radiation. Teinolophos, and certain other fossil monotremes, the canal is enlarged; in the platypus it contains the hypertrophied mandib- ͉ ͉ ͉ ͉ calibration molecular dating Monotremata niche phylogeny ular branch of the trigeminal nerve, which supports an extensive mechanosensory–electrosensory system (28). The mandibular ore than 99% of the Ϸ5,400 extant are canal is narrower in echidnas than in other monotremes, al- Mtherian (marsupials and placentals) (1). Monotremes, the though still relatively larger than in most other mammals. But only egg-laying mammals, are their living sister group and whether the condition in echidnas is primitive for monotremes comprise just 5 extant species. One of these species is the or a partial reversal correlated with the reduction of the man- semiaquatic, invertebrate feeding platypus (Ornithorhynchus dible to little more than elongate splints of bone remains unclear. anatinus) of eastern and southern Australia; the others are the All support for the platypus affinities of Teinolophos derives terrestrial echidnas (Tachyglossidae), the short-beaked echidna from mandibular characters. This is surprising given that the or spiny anteater, Tachyglossus aculeatus of Australia and New initial description of Teinolophos (29), based largely on a man- Guinea, and three species of New Guinean long-beaked echidnas dible, aligned it with stem therians rather than with monotremes, (Zaglossus bruijni, Z. attenboroughi, and Z. bartoni), which feed let alone platypuses. Full exposure of the revealed on worms and arthropod larvae. Fossil monotremes, such as monotreme affinities (30) but suggested that Teinolophos was a Teinolophos trusleri and Steropodon galmani (2), along with their stem monotreme, diverging before the common ancestor of putative relatives, the insectivore-like ausktribosphenids (3–6), platypus and echidnas underwent a size increase and a dietary make up the bulk of the known Australian Cretaceous mammal shift that required relatively weaker bite forces. fauna. Known monotreme diversity then contracts to only platypus-like taxa, subsequent to the arrival of marsupials from Ϸ Author contributions: M.J.P. and M.S.Y.L. designed research; M.J.P., T.H.B., and M.S.Y.L. via 71–54.6 million years ago (Ma) performed research; M.J.P. and M.S.Y.L. analyzed data; and M.J.P. and M.S.Y.L. wrote the (7, 8). Monotrematum sudamericanum (9, 10) from the Palaeo- paper. cene (Ϸ61 Ma) of South America is known from two platypus- The authors declare no conflict of interest. like distal femora and several molar teeth that closely match This article is a PNAS Direct Submission. those of the extinct Australian platypus, (11, 12). The 1To whom correspondence should be addressed. E-mail: [email protected]. Ϸ later appearance ( 25 Ma) of Obdurodon probably reflects the This article contains supporting information online at sparseness of earlier Tertiary mammal-bearing sites in Australia. 0904649106/DCSupplemental.͞cgi͞doi͞10.1073͞pnas.0904649106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 28, 2021 Table 1. Divergence age estimates from the BEAST analyses for dating studies, a practice that is problematic (37). An additional Monotremata (platypus vs. echidnas), Tachyglossidae (Zaglossus concern is that the upper estimate reported for Monotremata of vs. Tachyglossus), and Mammalia ( vs. Monotremata) 130.8 Ma (25) cannot be considered in isolation from the upper Median 95% HPD estimate of 322.8 Ma for the adjacent monotreme–therian divergence. There often is a strong correlation between credi- Monotremata bility intervals for adjacent nodes on trees (38), and the 322.4 Ma mtnuc14 32.1 18.5–47.8 upper estimate for crown mammals appears implausible, being nuc14 37.8 14.8–73.8 twice as old as the oldest unequivocal fossils (39) and predating mt88 27.7 13.3–47.1 even the earliest fully terrestrial (). Tachyglossidae With a view to increasing the precision of molecular dating mt88 5.5 1.8–10.6 estimates for the echidna–platypus divergence, we add long Mammalia sequences from 2 other nuclear genes (Rag1 and apob) to make mtnuc14 186.5 160.8–216.9 a 7–nuclear gene data set (7,137 nucleotides) and analyze this nuc14 203.4 163.5–252.2 alongside complete mitochondrial (mt) genome sequences. We mt88 168.8 145.8–196.2 use relaxed-clock dating methods, including up to 20 prior distributions for calibrations derived directly from the fossil record. We first estimate the age of the echidna–platypus One concern with the morphological data set of Rowe et al. divergence with no age constraints on this node, then examine (25) is that one character (enlarged mandibular canal) overlaps how bounding the age of this node with Teinolophos (i.e., with others (hypertrophy of the mandibular canal; characters 425 assuming that it is a stem platypus) effects estimates of molecular and 440) and is also used to infer states for unfossilized char- evolutionary rates. acters (presence of a bill and electrosensory capability; charac- ters 423 and 424). We address this nonindependence issue by Results removing redundant characters and indirect inferences [see Molecular divergence dates were derived using relaxed clocks supporting information (SI) Text]. The support for grouping and three alternative calibration schemes. The discussion that Teinolophos and Steropodon with platypuses disappears when follows focuses on the scheme that incorporates the most these changes are made. Furthermore, with the addition of 2 calibrations (Table 1; see Materials and Methods), but dates other characters discussed in the original description of Teinolo- derived using the other two schemes were very similar (Tables S3 phos as a monotreme but not included in the data set Rowe et and S4). All analyses indicate a mid-Tertiary origin for living al. (25), Teinolophos and Steropodon fall outside the monotreme Monotremata, the divergence between platypuses and echidnas. crown . The 14-taxon combined nuclear (7,137 sites) plus mitochondrial Most previous molecular dating (15–22, 31–36) places the (10,452 sites) data set, referred to herein as mtnuc14, provides a divergence between living Monotremata (echidna/platypus)— median estimate of 32.1 Ma, with a 95% highest posterior and thus the platypus lineage—within the Tertiary (Ͻ65.5 Ma), distribution (HPD) of 18.5–47.8 Ma. As expected, the variances contradicting placement of Teinolophos (112.5–121 Ma) and associated with the estimates for the individual nuclear and Steropodon (Ϸ105 Ma) along the platypus lineage. But all of the mitochondrial components are slightly larger, although the aforementioned studies either assumed a constant-rate molec- median estimates are similar. For nuc14, this is 37.8 Ma. A ular clock or were based on a single gene (34). Applying a median estimate of 27.7 Ma was provided by an expanded relaxed-clock approach to a data set including 5 nuclear genes 88-taxon mitochondrial data set (mt88), which also incorporates (2,793 nucleotides) (23), Rowe et al. (25) recovered broad another 13 calibration prior distributions beyond the 7 used for credibility intervals of 51.6–130.8 Ma, with the uppermost the 14-taxon analyses. With both echidna genera included in extreme overlapping the age of Teinolophos; however, this mt88, the median divergence estimate for Zaglossus–Tachyglossus consistency has its foundations in imprecision rather than in is 5.5 Ma (95% HPD, 1.8–10.6 Ma). signal, and is equally consistent with a Tertiary divergence. The The divergence times shown in Table 1 and Fig. 1 were molecular dating estimates from the 5–nuclear gene data set inferred using BEAST (40). This Bayesian inference program were derived using calibrations taken from previous molecular has a number of desirable properties, including simultaneous

Fig. 1. Time scale of mammalian evolution (in Ma) as median (and 95% HPD) estimates from BEAST analyses for the combined nuclear and mitochondrial sequences (mtnuc14). The (shaded) temporal window for the migration of marsupials into Australasia is 71–54.6 Ma. All nodes received Bayesian posterior probabilities of 1.00. Fossil calibrations were used at nodes indicated with an asterisk.

2of6 ͉͞cgi͞doi͞10.1073͞pnas.0904649106 Phillips et al. Downloaded by guest on September 28, 2021 Table 2. Maximum parsimony (shortest) trees that group the living platypus with echidna, Teinolophos (with or without Steropodon), and Steropodon (with or without Teinolophos) Platypus Platypus with Platypus with with echidna Teinolophos Steropodon

Morphol439 Unconstrained 1,7911 1,7902 1,7903 - 1,7944 1,7985 1,7946 constrained

Morphol441 Unconstrained 1,8061 1,8082 1,8073 Australosphenida- 1,8094 1,8155 1,8106 constrained

Backbone constraints were used to define these alternatives for morphol439 and morphol441, both with the remainder of the tree topology unconstrained or with monotremes constrained to group within Australosphenida (ЉAustra- losphenida-constrainedЉ). Shortest trees for each of these 4 analyses are in Fig. 2. Substitution rates in substitutions/1,000 sites/Ma for the platypus bold; note that the first analysis placed both Teinolophos and Steropodon (black circles), echidna (dotted circles), and monotreme stem (white circles) along the platypus lineage, to the exclusion of the echidna. Superscript lineages, estimated using BEAST, for mt88 (a and c) and nuc14 (b and d). The age numbers indicate the relevant constraint on relationships within monotremes of monotremes was either unconstrained (a and b) or bounded by the age of satisfied by each tree (see SI Text). Teinolophos (c and d). The substitution rates for each monotreme branch are compared with ranges for therian mammals (right vertical bar), sauropsids (left vertical bar), the overall mean (horizontal bar), and central 90% distri- and 1.81), similar to those for murid and at approximately bution (shaded). The y-axis is log-scaled. the upper 5th percentile for the distribution of rates among all of the branches (Fig. 2c and d). The extraordinary (4.5- to 12.5-fold)

rate decelerations from the monotreme stem lineage to the platypus EVOLUTION phylogeny–branch-length–dating coestimation with incorpora- and echidna lineages in the bound analyses are approximately tion of stochastic error, flexible models for calibration prior double to triple the magnitude of the next-steepest deceleration distributions, and relaxation of the assumption of rate correla- across the entire tree, for both nuc14 and mt88. tion between adjacent branches. Nevertheless, BEAST assumes The 63-taxon morphological data sets include modern (Ornitho- that rate variation follows a specific (appropriately plastic) rhynchus, Tachyglossus) and fossil monotreme taxa from the Ter- lognormal distribution (41). Our results are not methodologi- tiary (Obdurodon) and the Cretaceous (Teinolophos, Steropodon). cally sensitive, however. The semiparametric, penalized likeli- Modifications to the data set used by Rowe et al. (25), and the hood method implemented by a different program, r8s (42), analyses that follow herein, are fully described in SI Text. These provided similar dates, with a best estimate of 31.3 Ma for the modifications involve the elimination of redundant characters and combined data set, mtnuc14. Variance estimation in r8s is speculative (unfossilized) character-state codings (related to the problematic (43), but even with a generous 4-lnL unit cutoff, the width of the mandibular foramen/canal) from among characters 95% confidence interval was 28.6–34.1 Ma. 423, 424, 425, and 440. In doing so, the data set is reduced to 439 The analyses were repeated with the echidna–platypus diver- characters, and is referred to as morphol439. The addition of 2 gence, enforced to be Ն 112.5 Ma. This assessed the impact of characters derived from the original description of Teinolophos as assuming platypus affinities for Teinolophos (25) on rates of mo- a monotreme (30), body size and mandibular aspect ratio, provides lecular evolution and echoes a recent study of substitution rates an alternative data set referred to as morphol441. Alternative among reptiles (44). Fig. 2 shows substitution rates for platypus, resolutions for Teinolophos and Steropodon relative to the modern echidna, and their (Monotremata) stem lineage relative to the platypus and echidna were tested using backbone constraints, as ranges of rate estimates across sauropsids (reptiles and birds) and well as bootstrapping in PAUP*4.0b10 (47). therian mammals, as well as the central 90% distribution across the Morphol439 produced 9 shortest trees (L ϭ 1,790), all with both tree (n ϭ 26 for nuc14, n ϭ 174 for mt88). The mitochondrial and Teinolophos and Steropodon along the platypus lineage (Table 2); nuclear patterns are similar. When the age of crown Monotremata however, trees only a single step longer placed both Teinolophos and is not constrained (Fig. 2a and b), rates for all three monotreme Steropodon outside an echidna–platypus clade (i.e., outside living branches fall well within both the therian range and the central 90% monotremes). With the more character-inclusive morphol441, sup- distribution, with the monotreme stem lineage rate close to the port for the latter phylogeny increased; 20 shortest trees (L ϭ 1,806) overall mean rate for both mt88 and nuc14. all placed both Teinolophos and Steropodon outside an echidna– Applying a hard bound of Ն 112.5 Ma to the age of crown platypus clade (Table 2). Two extra steps were required to place Monotremata forces the substitution rates on the monotreme Teinolophos along the platypus lineage, and trees one step longer branches to opposite extremes of the distributions (Fig. 2c and d). placed Steropodon along this lineage. For both mt88 and nuc14, the rate estimates (substitutions/1,000 Furthermore, all of the aforementioned trees united the Chinese sites/Ma) for the echidna (0.44 and 0.41) and platypus (0.33 and ‘‘primitive triconodont’’ Hadrocodium with monotremes, contrary 0.26) branches are the lowest in the trees, well below the lowest to the majority opinion (5, 6, 24, 27, 48) that monotreme affinities among all other mammals (0.78 and 0.50), including those with lie with the Gondwanan clade, Australosphenida. The original similar putative life history correlates of evolutionary rates, such as incarnation of the present morphological data sets (27) favored body size, generation time, and mass-specific metabolism (e.g., uniting monotremes with Australosphenida, specifically with aus- xenarthrans, strepsirrhine , and many marsupials) (45, 46). ktribosphenids, an Australian group contemporaneous with They are even below those in the reptiles examined here, and even Teinolophos. It is notable, then, that even without any modification, further below the mean rates for the tree (2.00 and 0.99). Con- reanalysis of Rowe et al. ’s original matrix (25), constraining versely, the mt88 and nuc14 substitution rate estimates for the Australosphenida, causes Teinolophos to fall outside the echidna– monotreme (echidna ϩ platypus) stem lineage are very high (4.11 platypus clade. Reanalysis of morphol439 with monotremes con-

Phillips et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on September 28, 2021 monotremes, consistent with its age and general morphology. Teinolophos resembles typical small insectivorous basal mam- mals (from which monotremes presumably evolved) in its small adult body size (Ϸ40–180 g), deep twin-rooted molars, and deep mandibles with substantial coronoid and angular processes for strong muscle attachments. In contrast, platypuses and echidnas are derived with respect to this morphotype, being relatively large (Ϸ700 g–18 kg) specialists on soft-bodied invertebrates, with shallow multiple-rooted molars (absent in adults of extant species) and shallow mandibles with small/vestigial coronoid and angular processes. These inferred ecological shifts correspond well with the stratigraphic record and the phylogeny provided by our ex- panded morphological data set, which places Teinolophos basal to all other monotremes (Fig. 3). A Ͼ5-fold size (mass) increase occurs along the branch leading to Steropodon plus crown monotremes, and a further shift to relatively weak bite forces can be inferred along the branch leading to crown monotremes Fig. 3. Maximum parsimony bootstrap trees for extant (echidna and platy- (platypuses and echidnas). pus) and extinct [Teinolophos (Ͼ112.5 Ma), Steropodon (Ϸ105 Ma), and Our molecular clock analyses (Fig. 1) combine relaxed-clock Obdurodon (from Ϸ25 Ma)] monotremes, based on analyses in which methods, direct fossil calibration priors, and multiple genes for monotremes were either unconstrained phylogenetically or constrained to monotremes and are in full agreement with the morphological group within Australosphenida. Bootstrap percentages are shown for 4 anal- phylogeny and the foregoing ecological inferences. When the age yses: morphol unconstrained/morphol unconstrained/morphol con- 439 441 439 of the monotreme crown (echidna–platypus divergence) is un- strained/morphol441 constrained. (A) The reduced bootstrap consensus after Steropodon is pruned. (B) The bootstrap consensus for all taxa. An asterisk constrained, each of the highly parametric (BEAST) and semi- indicates that the clade was enforced. A total of 58 nonmonotreme taxa are parametric (r8s) molecular divergence best estimates place this not shown. divergence between 27 and 38 Ma, with a 95% HPD for the combined data of 19–48 Ma (Table 1). These dates strongly exclude the possibility that the Ն 112.5 Ma Teinolophos and strained to group with australosphenidans revealed 24 shortest trees the Ն 105 Ma Steropodon belong within crown monotremes. The (L ϭ 1,793), 12 of which excluded both Teinolophos and Steropodon estimate for the monotreme–therian divergence from the com- from the echidna–platypus clade and 12 of which placed Steropodon bined data (186 Ma; 95% HPD, 161–217 Ma) falls squarely on the platypus lineage (Table 2). Teinolophos was outside the within the Early and is highly consistent with the echidna–platypus clade in all of these trees; the shortest trees that stratigraphic record. Candidate stem monotremes and stem placed it on the platypus lineage were 4 steps longer. Similar therians are both known from Ϸ166 Ma (39), whereas unques- ϭ analysis of morphol441 also revealed 24 shortest trees (L 1,808), tioned outgroups to crown mammals, such as the morganuc- all of which placed both Teinolophos and Steropodon outside an odontans, had diverged before the end of the (200 Ma). echidna–platypus clade. Seven extra steps were required to place Furthermore, the rates of both mt and nuclear evolution in the Teinolophos along the platypus lineage, while trees a single step monotreme stem and crown lineages fall comfortably within the longer placed Steropodon on this lineage (Table 2). variability across other land vertebrates, including therian mam- Bootstrapping also reveals a consistent signal that Teinolophos mals (Fig. 2a and b). is basal rather than a crown monotreme. Analyses of morphol439 When the analyses are repeated with the proposed 112.5 Ma and morphol441, with and without constraining Australos- echidna–platypus divergence imposed, the extensive amount of phenida all produce bootstrap consensus trees placing Teinolo- molecular change on the monotreme stem needs to be com- phos outside an echidna–platypus clade (Fig. 3A). Support for pressed into a short time, whereas the small molecular diver- this arrangement ranges from 61% to 99%, being greatest with gence between the platypus and echidna needs to be reconciled the additional characters and the Australosphenida constraint. with a very lengthy interval. This results in inferred mitochon- Teinolophos falls outside the echidna–platypus clade in all 4 drial and nuclear evolutionary rates in stem monotremes that are analyses; Steropodon is similarly positioned in 3 of the 4 analyses among the highest in land vertebrates (Fig. 2c and d), compa- (Fig. 3B). Bootstrap support for all nodes increases when the rable to the extreme rates in murid rodents, which have com- phylogenetically unstable Steropodon is pruned from the primary promised mutational repair activity (49). Conversely, the in- bootstrap trees. Thus, there is clear evidence that Teinolophos is ferred rates in branches leading to the platypus and to the a basal monotreme, and weaker evidence for a similar position echidna are extraordinarily slow, below those of all other am- for Steropodon. It is notable that Steropodon is the least complete niotes considered (including reptiles). Slower rates have been taxon and shares Ͻ3% of the characters in the matrices with both associated with large body mass, long generation times, and low the echidna and either Ornithorhynchus or Obdurodon. As such, metabolism, although their effects typically explain only a small more complete material from Steropodon is needed to confi- proportion of the variability (50–52). Living monotremes are not dently resolve its position among other monotremes. large and do not have long life cycles for mammals, and their Overall, the molecular divergence dating strongly indicates mass-specific metabolism is similar to many of the other included that the Cretaceous Teinolophos and Steropodon lie outside mammals (45, 46) and indeed is higher than that of ectothermic crown monotremes. The morphological data also favor this view, reptiles. but less decisively. It was previously recognized that a crown position for monotremes implies extraordinarily slow molecular Discussion evolution within living monotremes (25). But that study did not Teinolophos trusleri is the oldest known monotreme (121–112.5 discuss how that phylogenetic hypothesis simultaneously (and Ma), as indicated by its distinctive molar structure (30). Al- paradoxically) also implies extremely rapid rates along the though recently aligned with platypuses (25), earlier studies (6, monotreme stem lineage. The implied rate decrease (fast in stem 24, 27, 30) considered Teinolophos to be basal to crown (living) monotremes, slow in crown monotremes) for both the nuclear

4of6 ͉͞cgi͞doi͞10.1073͞pnas.0904649106 Phillips et al. Downloaded by guest on September 28, 2021 and mitochondrial sequences is 2- to 3-fold greater than de- the evolution of an anteater-style ‘‘’’ (62). The only (remotely) creases across any other node in the tree. A recent study of ecologically analogous marsupials to the platypus and echidnas, the substitution rates among placental mammals reveals that dra- semiaquatic yapok and termite-eating numbat, are far more re- matic rate decreases are far less likely than dramatic rate stricted in their aquatic and fossorial activities compared with their increases (53), perhaps because the mechanisms involved in monotreme counterparts. DNA copying and repair are far easier to break than to fix. Monotremes and therian mammals diverged by the Early The consilience of evidence from molecular dating, morphology, Jurassic; however, the popular notion of monotremes as ‘‘living ecology, and the temporal order of monotreme fossils indicates that fossils’’ is belied by such morphologically and ecologically dis- hypertrophy of the mandibular canal in Teinolophos (25) does not tinct forms as platypuses and echidnas diverging only in the reflect close affinities with platypuses, but rather is a primitive mid-Tertiary, more recently than the earliest divergences within monotreme feature, secondarily reduced in echidnas. This reduc- most placental and orders (35). The semiaquatic-to- tion could be correlated with functional constraints (e.g., stress terrestrial niche shift and associated adaptive radiation of echid- tolerance) associated with the general reduction in echidnas of the nas represents a recent expansion of niche space despite poten- mandibles to little more than elongate splints. In echidnas, the tial competition from marsupials. This contradicts the beak-like rostrum is used to lever soil (54), and the number of assumption of arrested morphological and molecular evolution specialized electroreceptors is at least 10-fold fewer compared with that continues to be associated with monotremes (25). It further the platypus (55). The relatively wider mandibular canal of Teinolo- suggests that in certain niches, oviparous reproduction in phos is consistent with extensive mechanoreception–electrorecep- monotremes confers advantages over marsupials, a view consis- tion capabilities, but further evidence is required to confirm this and tent with present ecological partitioning between monotremes also to infer whether it is associated with platypus-like, -like, and marsupials. or other behavior. The 61 Ma Monotrematum is the oldest known Tertiary Materials and Methods monotreme, with teeth and femora very similar to those of The primary molecular data set consists of 7 nuclear genes and complete undoubted fossil and living platypuses. The mid-Tertiary origin mitochondrial (mt) genome protein-coding and RNA-coding DNA sequences for crown monotremes inferred in all of our molecular analyses for 7 placental mammals, 3 marsupials, both the platypus and short-beaked indicates that Monotrematum is a late stem monotreme, and thus echidna, and 2 outgroup representatives (lizard and chicken). The nuclear that the immediate ancestors of living monotremes already genes are acetylcholinergic receptor M4 (chrm4), dopamine receptor type 1A (drd1a), ␣-2B adrenergic receptor (adra2b), proto-oncogene C-MOS (c-mos), EVOLUTION exhibited a platypus-like morphology (9–11, 56). Therefore, -determining transcription factor SOX-9 (sox9), recombination-activating echidnas essentially would be derived, terrestrial platypuses. gene RAG1 (rag1), and apolipoprotein B (apob). The mitochondrial data set Compelling evidence for secondary derivation of terrestrial was expanded to include 88 taxa for analyses in which sampling was not habits from semiaquatic ancestors has been offered previously limited to taxa available for the nuclear data set. All sequences were aligned only for the evolution of elephants (57). in Se-Al 2.0a9 (63), and sites of ambiguous homology were excluded. GenBank A number of aspects of echidna biology are consistent with an accession numbers are provided in Table S1 and Fig. S1, and alignments are origin from a platypus-like ancestor with such traits as aquady- available online at TreeBASE. namic streamlining (58), dorsally projecting hind limbs acting as Evolutionary models for the analyses of the combined data set (mtnuc14) rudders (59), and locomotion founded on hypertrophied hu- are partitioned to reflect the primary sources of genomic and functional variation among the sequences: mt first and second codon positions, mt third meral long-axis rotation, which provides a very efficient swim- codon positions, mt RNAstems,mtRNAloops, nuclear first and second codon ming stroke (60). In echidnas, traits that are potentially homol- positions, and nuclear third codon positions. Within the nuclear data, 3 ogous with these are dorso-ventral compression, reversed gene-wise partitions were used to ensure that genes with differing taxon sets hind-foot posture, and ‘‘front-wheel drive’’ locomotion based on were modeled separately. These were rag1 (complete taxon sampling), apob humeral long-axis rotation. Each of these traits would be highly (lizard missing), and chrm4/drd1a/adra2b/c-mos/sox9 (elephant missing), anomalous if derived directly from a more generalized terrestrial within each of which codon position partitioning is maintained. insectivore morphotype (typical of basal mammals). The em- In accord with concerns for signal loss in mt sequences (64), we tested the bryologic presence in echidnas of the marginal cartilage that relative ability of likelihood models to appropriately correct for multiple 3 3 contours the bill of platypuses (58) similarly suggests that a bill substitutions at sites (saturation). RY coding (A,G R; C,T Y) was required to appropriately correct for saturation among the mt protein-coding first and (rather than a beak or snout) is ancestral for crown monotremes. second positions and RNAloops (see SI Text and Table S2). After RY coding, Other features of echidnas also suggest a substantial, relatively saturation remained problematic for the mt protein-coding third positions, recent ecological shift. Despite now lacking teeth, relaxation of and so these were excluded from our analyses. Standard nucleotide coding selection has yet to result in degraded sequences for the tooth was retained for the nuclear sequences and the mt RNAstems. matrix protein amelogenin (61). Similarly, the ankle spurs, which Model selection for the molecular data sets used ModelTest 3.7 (65), with are venomous only in platypuses, are retained in many echidnas, the most general available model suggested by either the Akaike information despite their derived hind-limb morphology that ensures that criterion or likelihood ratio test used. The strict molecular clock hypothesis was they are nonfunctional (54). Finally, the absence of echidna-like rejected at P Ͻ .01 for each data set, under likelihood ratio clock tests fossils before 13 Ma is consistent with mid-Tertiary origins. (performed in PAUP*4.0b10) (47). Relaxed-clock divergence times and rates of evolution were estimated under Bayesian inference in BEAST 1.4.8 (40) and From the diversity of monotremes and other mammals (e.g., under penalized likelihood in r8s 1.70 (44); see SI Text for full details. ausktribosphenids) that composed the Australian Early Cretaceous To provide temporal calibration, we used prior age distributions (in Ma) for faunas, only the lineage leading to platypuses and echidnas demon- 7 nodes on the 14-taxon nuclear and combined-data trees: Amniota (305– strably survived the marsupial invasion (Fig. 1). Competitive dis- 330.4), (255.9–299.8), Theria (124.6–167), Marsupialia (58.5–84), placement by marsupials is consistent with surviving monotremes Australasian marsupials (25.5–71.2), Tethytheria (52–71.2), and Cow-pig occupying ecological niches that may have been subject to the least (52.4–65.8). Prior age distributions were used for a further 13 nodes on the competition from marsupials. Ausktribosphenids, which did not 88-taxon mitochondrial trees: (58.5–71.2), Primates (55–71.2), Ro- survive, appear to have been generalized insectivores (4, 29) dentia (55.6–71.2), Shrew- (61.5–101.5), Perissodactyla (54–58.7), ecologically similar to many Early Tertiary marsupials. Conversely, (39.7–65.8), Whippomorpha (49–61), (23–45.6), Galloanserae (66–86.5), Penguin-albatross (61–74), Petauroidea (25.5–54.6), platypuses and echidnas exploit ecological niches in which marsu- Alligatoridae (64–80), and Archosauria (239–250.4). Three alternative cali- pials are restricted by their reproductive mode. After birth, mar- bration schemes were used. The above scheme, which uses the most calibra- supial young begin an extended period of fixation onto a . tions, was devised by M.J.P. To test the robustness of the dating estimates to This both compromises the ability of the mother to forage in aquatic calibration variability, two additional calibration schemes were used, one environments and provides an early developmental constraint on devised by M.S.Y.L. and the other taken directly from a recent review of

Phillips et al. PNAS Early Edition ͉ 5of6 Downloaded by guest on September 28, 2021 calibration points by Benton et al. (66). Fossil record justifications and refer- Maximum parsimony trees from the morphological data sets (morphol439 ences for the primary and alternative calibration schemes are provided in SI and morphol441) were inferred under 100 random addition heuristic searches Text. Notably, median estimates among the 3 calibration schemes for the in PAUP 4.0b10. Backbone constraint trees used for evaluating alternative echidna/platypus divergence differ by only 0.1–3.5 Ma across the mtnuc14, hypotheses of relationships are provided in SI Text. Bootstrap support for mt88, and nuc14 data sets (see SI Text and Table S4). was based on 1,000 replicates. Analyses in which Teinolophos was used for calibration adopted the results of Rowe et al. (25) for timing. The minimum and maximum hard bounds were ACKNOWLEDGMENTS. We thank Tom Rich, Renae Pratt, and 2 anonymous the minimum stratigraphic age of Teinolophos (112.5 Ma) and the upper reviewers for constructive suggestions and the Australian Research Council for confidence interval of their molecular estimate (130.8 Ma). financial support.

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