DNA sequence evidence supports a superordinal clade of mammals that comprises elephants, sea cows, hyraxes, aardvarks, elephant shrews, golden moles, and tenrecs, which all have their origins in Africa, and therefore are dubbed Afrotheria. Morphologically, this appears an unlikely assemblage, which challenges-by including golden moles and tenrecs-the monophyly of the order Lipotyphla (Insectivora). We here identify in three proteins unique combinations of apomorphous amino acid replacements that support this clade. The statistical support for such ''sequence signatures'' as unambiguous synapomorphic evidence for the naturalness of the Afrotherian clade is reported. Using likelihood, combinatorial, and Bayesian methods we show that the posterior probability of the mammalian tree containing the Afrotherian clade is effectively 1.0, based on conservative assumptions. Presenting sequence data for another African insectivore, the otter shrew Micropotamogale lamottei, we demonstrate that such signatures are diagnostic for including newly investigated species in the Afrotheria. Sequence signatures provide ''protein-morphological'' synapomorphies that may aid in visualizing monophyletic groupings.

Shared insertions or deletions (indels) in protein-coding DNA can be strong indicators of the monophyly of a taxon. A three-amino acid deletion had previously been noted in the eye lens protein alpha A-crystallin of two species of sloths and two species of anteaters, which represent the Pilosa, one of the two infraorders of Xenarthra (Edentata). This deletion has not been observed in 55 species from 16 other eutherian orders, or in 2 species of marsupials, or in 34 nonmammalian vertebrates, from birds to shark. At the genomic level, we have now detected this deletion in two species of armadillos of the second xenarthran infraorder, Cingulata, as well as in an additional species of anteater. Phylogenetic trees were constructed from a 145-bp sequence of the alpha A-crystallin gene of 39 tetrapod species, supporting xenarthran monophyly with values from 76% to 90%. To quantify the additional support for xenarthran monophyly, as given by the three-residue deletion, we computed the probabilities for the occurrence of this deletion per evolutionary time unit for alternative hypothetical tree topologies. In the estimates obtained, the six trees in which the xenarthran subgroups are unresolved or paraphyletic give an increasingly lower likelihood than do the two trees that assume xenarthran monophyly. For the monophyletic trees, the probability that the deletion observed in the xenarthrans is due to a single event is > 0.99. Thus, this deletion in alpha A-crystallin gives strong molecular support for the monophyly of this old and diverse order.

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