Molecules, morphology, and ecology indicate a recent, amphibious ancestry for echidnas 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, Australia; bSchool of Earth and Environmental Sciences, University of Adelaide, Adelaide, SA 5005, Australia; and cEarth Sciences Section, South Australian Museum, 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 platypus and terrestrial echidnas (spiny anteaters) Fossil echidnas do not appear until the mid-Miocene (Ϸ13 are the only living egg-laying mammals (monotremes). The fossil Ma) (13), despite excellent late Oligocene–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 Cretaceous Teinolophos and Steropodon to the platy- lacking teeth (14), which are the most common fossil remains pus lineage implies that platypuses 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 fossils. 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 monotreme crown group (platypuses and echidnas) specif- general mandibular reduction). A multigene evaluation of the EVOLUTION echidna–platypus divergence using both a relaxed molecular clock 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 (Monotrematum) predate of the three mammalian crown groups (Monotremata, Marsu- this divergence, indicating that echidnas had aquatically foraging pialia, and Placentalia). 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). marsupials 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 basal 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 mammal species 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 molar 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. South America via Antarctica 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, Obdurodon (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 www.pnas.org/cgi/content/full/ sparseness of earlier Tertiary mammal-bearing sites in Australia. 0904649106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.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 (Theria 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 vertebrates (amniotes). 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