Conflicting molecular phylogenies of European long-eared bats (Plecotus) can be explained by cryptic diversity A. Kiefer, a, * F. Mayer, b J. Kosuch, a O. von Helversen, b and M. Veith a a Department of Ecology, Institute of Zoology, Mainz University, Saarstraße 21, D-55099 Mainz, Germany b Institute of Zoology II, Erlangen University, Staudtstr. 5, Erlangen D-91058, Germany Received 13 March 2002; received in revised form 14 June 2002 Abstract Conflicting phylogenetic signals of two data sets that analyse different portions of the same molecule are unexpected and require an explanation. In the present paper we test whether (i) differential evolution of two mitochondrial genes or (ii) cryptic diversity can better explain conflicting results of two recently published molecular phylogenies on the same set of species of long-eared bats (genus Plecotus). We sequenced 1714 bp of three mitochondrial regions (16S, ND1, and D-loop) of 35 Plecotus populations from 10 European countries. A likelihood ratio test revealed congruent phylogenetic signals of the three data partitions. Our phylogenetic analyses demonstrated that the existence of a previously undetected Plecotus lineage caused the incongruities of previous studies. This lineage is differentiated on the species level and lives in sympatry with its sister lineage, Plecotus auritus, in Switzerland and Northern Italy. A molecular clock indicates that all European Plecotus species are of mid or late Pliocene origin. Plecotus indet. was previously described as an intergrade between P. auritus and Plecotus austriacus since it shares morphological characters with both. It is currently known from elevations above 800 m a.s.l. in the Alps, the Dinarian Alps and the Pindos mountains in Greece. Since we could demonstrate that incongruities of two molecular analyses simply arose from the mis-identification of one lineage, we conclude that molecular phylogenetic analyses do not free systematists from a thorough inclusion of morphological and ecological data. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Phylogeny; Partition homogeneity; Cryptic diversity; Molecular clock; Plecotus; Altitudinal niche separation 1. Introduction The application of molecular methods has added new insights into organismic evolution. Recently, some spectacular cases drastically changed long-held beliefs of taxa affiliations like the paraphyly of crustaceans with respect to insects (Burmester, 2001; Garc  ıa-Machado et al., 1999) or the phylogenetic position of turtles as a sister group of the Archosauria (crocodiles and birds; Zardoya and Meyer, 1998). Conflicts between molecular data and classical taxonomy often result from conver- gent morphological evolution during a speciesÕ radiation into vacant ecological niches. This produces similar phenotypes among non-related lineages (Schluter, 2000; W€ agele et al., 1999). Beak morphology of Darwin fin- ches is a classic example (Grant, 1986). In bats, Ruedi and Mayer (2001) showed that similar ecomorphs evolved convergently among unrelated Palearctic and Nearctic species of the genus Myotis. Morphological similarity among species that occupy similar ecological niches complicates the recognition of species on the basis of morphological characters. Since differences accumulate with time in neutrally evolving genomic regions, DNA sequence analysis is a powerful tool to discover morphologically cryptic species diver- sity. Within European bats, several morphologically near-indistinguishable pairs of species are known, al- though genetically they are very distinct. In some cases they even do not group as sister taxa in phylogenetic analyses and thus have to be considered as similar eco- morphs that occupy similar ecological niches (Mayer and von Helversen, 2001). In 2001, two studies on mitochondrial gene sequences revealed inconsistent phylogenetic relationships among European Plecotus lineages. Mayer and von Helversen Molecular Phylogenetics and Evolution 25 (2002) 557–566 MOLECULAR PHYLOGENETICS AND EVOLUTION www.academicpress.com * Corresponding author. Fax: +49-6131-3923731. E-mail address: kiwi@oekologie.biologie.uni-mainz.de (A. Kiefer). 1055-7903/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII:S1055-7903(02)00299-3