Microsatellite‐based species identification method for Drosophila virilis group species

摘要

The Drosophila virilis group species have been extensively used in studies concerning genome structure, speciation and evolution of species-specific behavioural characters (Patterson and Stone 1952; Throckmorton 1982; Hoikkala et al. 2005; Routtu et al. 2007). The group consists of 11-14 species or subspecies, depending on whether D. canadiana is included, whether D. borealis is separated into eastern and western forms and whether D. americana americana and D. americana texana are classified as subspecies. Spicer and Bell (2002) have divided the group into four phylads based on the phylogeny of mitochondrial 12S and 16S rRNA genes: the virilis phylad (D. virilis, D. americana americana, D. a. texana, D. novamexicana and D. lummei), the montana phylad (D. montana, D. lacicola, D. borealis eastern and D. borealis western and D. flavomontana), the littoralis phylad (D. littoralis, D. canadiana and D. ezoana) and the kanekoi phylad represented by D. kanekoi.The species of D. virilis group are especially suitable for speciation studies as they represent different levels of divergence with an estimated time of 9.0±0.7 mya between virilis and montana phylad species and 2.6±0.4 mya between D. virilis and the other species in the virilis phylad (Nurminsky et al. 1996). Many of these species can be crossed with each other to study the genetic basis of interspecific differences (Throckmorton 1982). Also, geographically isolated populations of some species of the group offer a good opportunity to study the effects of ecological and behavioural factors on population divergence, and to trace the genetic mechanisms underlying the process. For example the divergence time between the D. montana populations from different continents is estimated to be from 0.45 to 0.9 mya (P??llysaho et al. 2005; Mirol et al. 2007).Species of the D. virilis group share some morphological characters such as a relatively large size for Drosophilidae and a dark shade around the longest cross-vein of the wing, which make identification at the group level easy. The species are, however, difficult to distinguish from each other. Researchers have used different methods for identifying the species of wild-collected flies, such as classifying the flies on the basis of external male genitalia (Lakovaara and Hackman 1973; Kulikov et al. 2004) female spermathecae (Pitnick et al. 1999), mating flies with laboratory-reared flies of known species (Liimatainen and Hoikkala 1998) and recording and analyzing the songs of wild-caught males (Huttunen et al. 2002). Species-identification has also been made by using protein gel electrophoresis (Lakovaara et al. 1976; Spicer 1991) and RAPD fingerprinting (Mikhailovsky et al. 2007). Unfortunately, these methods are not very accurate and/or they are very time consuming and require special expertise.Microsatellite markers have been used earlier to explore the phylogeny of the D. virilis group species (Orsini et al. 2004) and to map genes that affect male song traits (Huttunen et al. 2004). Orsini et al. (2004) focused their study on the species of the virilis phylad. We have used here a similar approach with a special emphasis on north-European species. The main aim of this study was to develop a fast and easy species identification method for the D. virilis group species occurring sympatrically in Northern Europe (D. montana, D. littoralis, D. ezoana and D. lummei; Aspi et al. 1993; B?chli et al. 2005). For this purpose the microsatellite locus Vir72ms appeared to be especially informative. The 14 polymorphic microsatellite loci used in the study amplified in all D. virilis group species, which gave us an opportunity also to check how well they cluster the fly strains of different species throughout the group and to identify possible misclassifications.