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Fossils of the family Vespertilionidae are rare in Florida Oligocene fossil deposits, represented by small samples from three localities: two teeth and a partial radius from I-75 (Whitneyan), seven isolated teeth representing two species from White Springs (early Arikareean), and an isolated lower molar from SB-1A/Live Oak (early Arikareean). Vespertilionids are the only bats known from the White Springs and SB-1A/Live Oak faunas, whereas vespertilionids are either rare (I-75) or absent (Brooksville 2) in the two most diverse Florida Oligocene bat faunas. Major changes occurred in the Florida chiropteran fauna between the late Oligocene (about 25 Ma) and the early Miocene (about 18 Ma) because the early Miocene Thomas Farm bat fauna is dominated by vespertilionids, including at least five species, and representatives of the principally Neotropical families Emballonuridae, Natalidae, and Molossidae are rare. The Eptesicus-like vespertilionid Suaptenos whitei is by far the most common bat at Thomas Farm (Lawrence 1943). The other described vespertilionids from this fauna are Miomyotis floridanus and Karstala silva (Lawrence, 1943; Czaplewski and Morgan 2000). None of the vespertilionid mandibles from Thomas Farm have the same dental formula as Myotis, and thus Miomyotis is probably unrelated to Myotis, although Lawrence (1943) suggested the two genera were closely related in her original description of Miomyotis floridanus. Karstala silva is a very large vespertilionid similar in size to some of the largest living members in this family (Czaplewski and Morgan 2000). Undescribed vespertilionids from Thomas Farm include two new genera similar to Corynorhinus and Lasiurus. A single upper molar of a vespertilionid from the early Hemingfordian Seaboard LF and two isolated teeth from the late Hemingfordian Brooks Sink LF complete the Florida early Miocene chiropteran record.

From the middle Miocene to the late Pliocene (~16.5-2.0 Ma) the fossil record of bats in Florida is poor, and all known specimens are vespertilionids, including: several teeth from the middle Miocene (early Barstovian NALMA) Bird Branch LF, a tooth and a proximal radius from the late Miocene Love Bone Bed LF (late Clarendonian NALMA), a partial humerus from the late Miocene (early Hemphillian NALMA) McGehee Farm LF, and a partial humerus from the early Hemphillian Tyner Farm LF. The rarity of bats in Florida middle to late Miocene and early Pliocene faunas is almost certainly related to the absence of paleokarst deposits during this time interval. Paleokarst deposits once again become common in Florida during the late Pliocene and throughout the Pleistocene, and as a consequence fossil bats are common in Florida during the last 2 Ma. Two Florida late Pliocene sites contain bats (Morgan 1991), the Inglis 1A LF and De Soto Shell Pit LF, both of which are latest Blancan in age (about 2 Ma). Inglis 1A has an abundant and diverse chiropteran fauna composed of seven species including: the earliest record of the vampire bat Desmodus archaeodaptes and six genera of vespertilionids Antrozous, Corynorhinus, Eptesicus, Lasiurus, Myotis, and Pipistrellus. The De Soto Shell Pit fauna was deposited in a nearshore marine environment, not a cave or similar karst deposit; nonetheless this site contains a small sample of teeth and postcranial elements of the tree bat Lasiurus.

Outside of the Florida peninsula, members of Vespertilionidae dominate the Oligocene through Pleistocene chiropteran record of North America. The North American fossil record of vespertilionids exclusive of Florida includes the following six extinct genera (from oldest to youngest): Oligomyotis from the early Oligocene of Colorado (Galbreath 1962); Ancenycteris from the middle Miocene of Montana (Sutton and Genoways, 1974); Potamonycteris from the middle Miocene of Nebraska (Czaplewski 1991); Plionycteris from the early Pliocene of northern Mexico (Lindsay and Jacobs 1985); Anzanycteris from the late Pliocene of California (White 1969); and Simonycteris from the late Pliocene of Arizona (Stirton 1931). Many extant genera of vespertilionids appear in North American vertebrate faunas during the middle to late Miocene (Antrozous, Eptesicus, Lasiurus, and Myotis) and Pliocene (Corynorhinus, Lasionycteris, and Pipistrellus).