Amphibia-Reptilia 28 (2007): 467-474 Can microsatellite markers resolve phylogenetic relationships between closely related crested newt species (Triturus cristatus superspecies)? Peter Mikulíˇ cek 1,2 , Jelka Crnobrnja-Isailovi´ c 3 , Jaroslav Piálek 4 Abstract. Theoretical and empirical studies revealed that fast evolving microsatellite markers might be successfully employed in phylogenetic reconstruction. In this study we used seven highly polymorphic microsatellite loci and six different genetic distances to infer the performance of microsatellites in estimation of phylogenetic relationships between closely related crested newt species (Triturus cristatus superspecies). Many intraspecific distances exceeded interspecific values likely due to loss of genetic distance linearity in time, revealing that none of them was able to estimate divergence between the species. The Neighbor-joining trees constructed on the basis of genetic distances showed trichotomies (three-way polychotomies) and low bootstrap support at the species level. This study thus revealed limits of microsatellites to estimate phylogenetic relationships between the crested newt taxa. Introduction Investigation of evolutionary relationships be- tween closely related species requires analyz- ing fast evolving genetic markers. Microsatel- lites may be good candidates for such studies due to their high mutation rate and polymor- phism (Jarne and Lagoda, 1996; Estoup and Cornuet, 2000; Goldstein and Schlötterer, 2000; Ellegren, 2004). Many empirical studies have shown that microsatellites might be success- fully employed in phylogenetic reconstruction (Estoup et al., 1995; Harr et al., 1998; Petren et al., 1999, 2005; Richard and Thorpe, 2001; Peréz et al., 2002; Orsini et al., 2004; Chirhart et al., 2005; Hughes et al., 2005; Kankare et al., 2005; Knaden et al., 2005), although in some cases they did not give a phylogeny congruent with other genetic markers or theoretical expec- 1 - Department of Zoology, Comenius University, Mlynská dolina B-1, SK-84215 Bratislava, Slovak Republic e-mail: pmikulicek@fns.uniba.sk 2 - Department of Zoology, Biodiversity Research Group, Charles University, Viniˇ cná 7, CZ-12844 Prague 2, Czech Republic 3 - Department of Evolutionary Biology, Institute for Bi- ological Research, Boulevard Despota Stefana 142, 11060 Belgrade, Serbia & Montenegro 4 - Institute of Vertebrate Biology, Academy of Sciences of the Czech Republic, Department of Population Biology, CZ-67502 Studenec 122, Czech Republic tations (Peatkau et al., 1997). The number of studies successfully applying microsatellites ex- ceeds the number of studies showing their lim- its in phylogenetic reconstruction, although this shift might be caused by failure to publish neg- ative results. In this paper we aim to show lim- itations of microsatellites to resolve phyloge- netic relationships in the case of closely related crested newt species. The crested newts (Triturus cristatus super- species) form a monophyletic group of four currently recognized species (Arntzen and Spar- reboom, 1989; Wallis and Arntzen, 1989; Mac- gregor et al., 1990; Arntzen and Borkin, 1997; Arntzen, 2003): T. cristatus (Laurenti, 1768), T. dobrogicus (Kiritzescu, 1903), T. carnifex (Lau- renti, 1768) and T. karelinii (Strauch, 1870). Divergence times between the species was es- timated at 2.5-3 My based on mtDNA RFLP data only (Wallis and Arntzen, 1989), but ex- tended up to 5-7 My when paleontological and allozyme genetic data were taken into ac- count (Kalezi´ c and Hedgecock, 1980; Macgre- gor and Sessions, 1986; Macgregor et al., 1990; Arntzen, 2003). All the species are supposed to have originated in the central Balkans, where they could evolve by allopatric speciation (Wal- lis and Arntzen, 1989; Crnobrnja-Isailovi´ c et al., 1997). Despite numerous studies focused on © Koninklijke Brill NV, Leiden, 2007. Also available online - www.brill.nl/amre