Phylogenetic Analysis of 18S rRNA and the Mitochondrial Genomes of the Wombat, Vombatus ursinus, and the Spiny Anteater, Tachyglossus aculeatus: Increased Support for the Marsupionta Hypothesis Axel Janke, 1 Ola Magnell, 1 Georg Wieczorek, 1 Michael Westerman, 2 Ulfur Arnason 1 1 Department of Genetics, Division of Evolutionary Molecular Systematics, University of Lund, So ¨ lvegatan 29, S-223 62 Lund, Sweden 2 Department of Genetics, La Trobe University, Bundoora, Victoria 3083, Australia Received: 28 October 2000 / Accepted: 23 March 2001 Abstract. The monotremes, the duck-billed platypus and the echidnas, are characterized by a number of unique morphological characteristics, which have led to the common belief that they represent the living survi- vors of an ancestral stock of mammals. Analysis of new data from the complete mitochondrial (mt) genomes of a second monotreme, the spiny anteater, and another mar- supial, the wombat, yielded clear support for the Marsu- pionta hypothesis. According to this hypothesis marsu- pials are more closely related to monotremes than to eutherians, consistent with a basal split between euthe- rians and marsupials/monotremes among extant mam- mals. This finding was also supported by analysis of new sequences from a nuclear gene—18S rRNA. The mt ge- nome of the wombat shares some unique features with previously described marsupial mtDNAs (tRNA rear- rangement, a missing tRNA Lys , and evidence for RNA editing of the tRNA Asp ). Molecular estimates of genetic divergence suggest that the divergence between the platypus and the spiny anteater took place ≈34 million years before present (MYBP), and that between South American and Australian marsupials ≈72 MYBP. Key words: Phylogenetics — Marsupionta — Theria — Spiny anteater — Wombat — Mitochondrial DNA — 18S rDNA Introduction Monotremes are a unique group of mammals. As “egg- laying” mammals and with a number of apparently ple- siomorphic anatomical characters, they play a crucial role in the study of mammalian evolution. A clearer un- derstanding of monotreme relationships vis-a `-vis other mammals would render invaluable insight into the origin of modern mammals in general. Unfortunately there are only three extant and highly specialized monotreme spe- cies living in Australia and New Guinea, though previ- ously they had a wider distribution. Thus Cretaceous monotreme fossils have been found not only in Australia (Musser 1999) but also in South America (Pascual et al. 1992). However, the generally poor fossil record of Monotremes (Carroll 1988) essentially limits analysis of their phylogeny to recent species. Even the relationships between, and time of origin of the two extant monotreme families remains somewhat enigmatic, despite new fos- sils findings from extinct monotreme families having been described (Flannery et al. 1995; Musser 1999). Paleontology of Early Cretaceous mammals has been limited to a few fragmentary findings, mostly teeth, but recently nearly complete skeletons have been discovered (Hu et al. 1997; Rich et al. 1997; Qiang et al. 1999). The mosaic assembly of ancestral and modern characters in Jeholodens (Qiang et al. 1999) demonstrates that homo- plasies in skeleton characters are common in the mor- phology of early mammals (Qiang et al. 1999; Zimmer 1999; Rowe 1999). Despite the relatively poor early mammalian fossil record, homoplasies, and the highly Correspondence to: Axel Janke; email: axel.janke@gen.lu.se J Mol Evol (2002) 54:71–80 DOI: 10.1007/s00239-001-0019-8 © Springer-Verlag New York Inc. 2002