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The complete mitochondrial genomes of two microbats, the horseshoe bat Rhinolophus pumilus, and the Japanese pipistrelle Pipistrellus abramus, and that of an insectivore, the long-clawed shrew Sorex unguiculatus, were sequenced and analyzed phylogenetically by a maximum likelihood method in an effort to enhance our understanding of mammalian evolution. Our analysis suggested that (1) a sister relationship exists between moles and shrews, which form an eulipotyphlan clade; (2) chiropterans have a sister-relationship with eulipotyphlans; and (3) the Eulipotyphla/Chiroptera clade is closely related to fereuungulates (Cetartiodactyla, Perissodactyla and Carnivora). Divergence times on the mammalian tree were estimated from consideration of a relaxed molecular clock, the amino acid sequences of 12 concatenated mitochondrial proteins and multiple reference criteria. Moles and shrews were estimated to have diverged approximately 48 MyrBP, and bats and eulipotyphlans to have diverged 68 MyrBP. Recent phylogenetic controversy over the polyphyly of microbats, the monophyly of rodents, and the position of hedgehogs is also examined.

Four tRNA-related SINE families were isolated from the genome of the shrew Sorex araneus (SOR element), mole Mogera robusta (TAL element), and hedgehog Mesechinus dauuricus (ERI-1 and ERI-2 elements). Each of these SINEs families is specific for a single Insectivora family: SOR, for Soricidae (shrews); TAL, for Talpidae (moles and desmans); ERI-1 and ERI-2, for Erinaceidae (hedgehogs). There is a long polypyrimidine region (TC-motif) in TAL, ERI-1, and ERI-2 elements located immediately upstream of an A-rich tail with polyadenylation signals (AATAAA) and an RNA polymerase III terminator (T(4-6)) or TCT(3-4)). Ten out of 14 analyzed mammalian tRNA-related SINE families have an A-rich tail similar to that of TAL, ERI-1, and ERI-2 elements. These elements were assigned to class T+. The other four SINEs including SOR element have no polyadenylation signal and transcription terminator in their A-rich tail and were assigned to class T-. Class T+ SINEs occur only in mammals, and most of them have a long polypyrimidine region. Possible models of retroposition of class T+ and T- SINEs are discussed.

Multiple band patterns of DNA repeats in the 20-500-nucleotide range can be detected by digesting genomic DNA with short-cutting restriction endonucleases, followed by end labeling of the restriction fragments and fractionation in nondenaturing polyacrylamide gels. We call such band patterns obtained from genomic DNA "taxonprints" (Fedorov et al. 1992). Here we show that taxonprints for the taxonomic groups studied (mammals, reptiles, fish, insects-altogether more than 50 species) have the following properties: (1) All individuals from the same species have identical taxonprints. (2) Taxonprint bands can be subdivided into those specific for a single species and those specific for groups of closely related species, genera, and even families. (3) Each restriction endonuclease produces unique band patterns; thus, five to ten restriction enzymes (about 100 bands) may be sufficient for a statistical treatment of phylogenetic relationships based on polymorphisms of restriction endinuclease sites. We demonstrate that taxonprint analysis allows one to distinguish closely related species and to establish the degree of similarity among species and among genera. These characteristics make taxonprint analysis a valuable tool for taxonomic and phylogenetic studies.

The sequence of the mitochondrial DNA (mtDNA) molecule of the European hedgehog (Erinaceus europaeus) was determined. The length of the sequence presented is 17,442 nucleotides (nt). The molecule is thus the largest eutherian mtDNA molecule so far reported. The organization of the molecule conforms with that of other eutherians, but the control region of the molecule is exceptionally long, 1,988 nt, due to the presence of repeated motifs at two different positions in the 3' part of the control region. The length of the control region is not absolute due to pronounced heteroplasmy caused by variable numbers of the motif TACGCA in one of the repetitive regions. The sequence presented includes 46 repeats of this type. The other repeated region is composed of different AT-rich repeats. This region was identical among four clones studied. Comparison of mitochondrial peptide-coding genes identified a separate position of the hedgehog among several mammalian orders. The concatenated protein sequence of the 13 peptide-coding genes was used in a phylogenetic study using the opossum as outgroup. The position of the hedgehog sequence was basal among the other eutherian sequences included: human, rat, mouse, cow, blue whale, harbor seal, and horse. The analysis did not resolve the relationship among carnivores, perissodactyls, and artiodactyls/cetaceans, suggesting a closer relationship among these orders than acknowledged by classical approaches.