Towards a Molecular Phylogeny of the Fungus Gnat Genus Boletina (Diptera: Mycetophilidae)
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Zoologica Scripta Towards a molecular phylogeny of the fungus gnat genus Boletina (Diptera: Mycetophilidae) SVANTE MARTINSSON,JOSTEIN KJÆRANDSEN &PER SUNDBERG Submitted: 17 November 2010 Martinsson, S., Kjærandsen, J. & Sundberg, P. (2011). Towards a molecular phylogeny of Accepted: 6 February 2011 the fungus gnat genus Boletina (Diptera: Mycetophilidae). — Zoologica Scripta, 40, 272–281. doi:10.1111/j.1463-6409.2011.00474.x Boletina is a species rich genus of fungus gnats (Diptera: Mycetophilidae) with a mainly Holarctic distribution. The systematics within the genus has gained little attention and this is a first attempt to shed some light over the systematics of Boletina and to test the segrega- tion of the genera Saigusaia and Aglaomyia from Boletina. The nuclear marker 28S and mitochondrial 16S, COI and CytB were amplified and sequenced for 23 taxa that were analysed separately and together with a broad sample of outgroup taxa obtained from Gen- Bank, where also 18S sequences were added. Phylogenies were estimated using maximum likelihood, Bayesian inference and parsimony. We strengthen the hypothesized sister-group relationship between Docosia and Boletina, but the genus Boletina as currently delimited appears to be paraphyletic and nested in a clade together with Aglaomyia, Coelosia and Gnoriste. The genus Saigusaia, on the other hand, seems to be well separated from Boletina. The Boletina erythropyga species group is consistently found as a distinct basal clade within Boletina s.l. The results obtained are otherwise ambiguous both for the taxa in focus and in some analyses globally with a statistically supported total breakdown of the traditional higher classification into tribes, subfamilies and even families. Interestingly, this breakdown almost disappeared when additional 18S sequences were added. Corresponding author: Svante Martinsson, Department of Zoology, University of Gothenburg, Box 463, SE-405 30 Go¨teborg, Sweden. E-mail: [email protected] Jostein Kjærandsen, Museum of Zoology, Lund University, Helgonava¨gen 3, SE-223 62 Lund, Sweden. E-mail: [email protected] Per Sundberg, Department of Zoology, University of Gothenburg, Box 463, SE-405 30 Go¨teborg, Sweden. E-mail: [email protected] Introduction and window trap samples (Russell-Smith 1979; Økland The fungus gnat genus Boletina Staeger, family Myceto- 1994; Jakovlev 1995). As such the genus may be a suitable philidae, consists of about 160 known species (Evenhuis model group to study the effects of climatic change in the et al. 2008) mainly confined to the Holarctic region, but northern hemisphere and arctic, but both its systematics representatives of the genus are found in the Oriental and biology remain insufficiently outlined. A number of region as well (Bechev 2000). Larvae of Boletina are con- new Boletina species have recently been described from the sidered mainly saproxylobointic (Jakovlev 1995) and larval Nordic region (Zaitzev & Polevoi 1995; Polevoi & Hed- habitats include decaying wood and fruiting bodies of mark 2004; Jakovlev & Penttinen 2007) and more species fungi (Økland 1999; Jakovlev et al. 2008). There are also a awaits description (Kjærandsen et al. 2007), but so far only few records of exposed mineral soil, mosses and liverworts a few subgroups within the genus have been revised (Za- as larval habitats (Edwards 1925; Økland 1999), but it is itzev & Polevoi 2001; Zaitzev et al. 2005). not clear whether they were really larval macrohabitats or The genus Boletina was traditionally placed in the sub- just pupation places. Adults of Boletina are found in a family Gnoristinae, but the classification and delimitations wider range of habitats than most species in other genera of higher taxa in the family has been debated for decades of the family (Hutson et al. 1980), including alpine and (e.g. Tuomikoski 1966; Va¨isa¨nen 1986; Søli et al. 2000; arctic environments where some species can be very abun- see Gammelmo 2004 for a overview of the traditional clas- dant (JK unpublished data). Other species of Boletina are sifications). All recent phylogenetic studies indicate that abundant both in boreal and nemoral forests where adults the subfamilial and tribal classification of fungus gnats, as can make up a considerable proportion of Malaise trap traditionally accepted, includes non-monophyletic groups 272 ª 2011 The Authors d Zoologica Scripta ª 2011 The Norwegian Academy of Science and Letters, 40, 3, May 2011, pp 272–281 S. Martinsson et al. d Molecular phylogeny of Boletina and the higher phylogeny of fungus gnats remains both ‘‘SPM–’’ in a BIOTA 2.04 DATABASE (Colwell 2007). A largely unresolved and contentious (Søli 1997a; Baxter large amount of material was studied and specimens 1999; Hippa & Vilkamaa 2005, 2006; Amorim & Rindal within Boletina were chosen to obtain a broad sample for 2007; Rindal et al. 2009a). Recent studies agree, however, sequencing. Samples of 16 species of Boletina, two species to retain Boletina in subfamily Gnoristinae where it is of Aglaomyia, and two species of Saigusaia were included placed in a clade together with Gnoriste Meigen (Søli as the ingroup. Based on previous studies (Søli 1997a; 1997a; Baxter 1999; Rindal et al. 2009a). Baxter 1999; Rindal et al. 2009a) Gnoriste longirostris, A close relationship between Docosia Winnertz (tradi- Coelosia truncata and one undescribed species of Docosia tionally placed in subfamily Leiinae) and Boletina was first were added as potential outgroups. Table 1 lists speci- suggested by Baxter (1999) and further supported by mens sequenced in this study, together with GenBank Rindal et al. (2009a). Baxter (1999) studied the phylogeny accession numbers. Additional collection data and deposi- of the Sciaroidea based on 12S rRNA sequences, and tory for the voucher specimens can be found in Appendix included three species of Boletina that appeared to be non- S1. monophyletic nested together with Gnoriste. He found that the Boletina–Gnoriste group formed a clade together with DNA extraction and amplification Docosia, but well separated from Coelosia Winnertz. Rindal Numerous DNA sequence markers have been used to infer et al. (2009a), who analysed the 16S, 18S and 28S markers, phylogenetic relationships in insects. The genes COI, 16S, found that Docosia together with Palaeodocosia Meunier may 18S and EF-1a have been proposed as a standard for form the sister-group of a clade consisting of Boletina, molecular insect phylogenies (Catrino et al. 2000). The Gnoriste and Coelosia. genes 28S, EF-1a and opsin may be useful for resolving Vockeroth (1980) segregated the genera Saigusaia within family-level relationships whereas mitochondrial Vockeroth and Aglaomyia Vockeroth from Boletina based loci and ITS seem to be a better choice for lower level on morphological characters such as wing venation, seta- phylogenies (Rokas et al. 2002). Based on these recom- tion of thoracic and abdominal sclerites and characters mendations and previous molecular works on fungus gnats in the male and female terminalia. In the morphological (Rindal et al. 2007, 2009a,b) we settled on the nuclear phylogeny by Søli (1997a) Saigusaia appeared in a clade marker 28S and the mitochondrial markers 16S, COI and together with Synapha Meigen and Palaeodocosia, well added CytB to be tested for the first time. separated from Boletina. The genus Aglaomyia has not The abdomen except terminalia were used for DNA been included and tested in any phylogenetic studies as extraction, the rest of the specimens were stored in alcohol yet. as hologenophore vouchers [following the terminology The aim of this study was to estimate phylogenies with proposed by Pleijel et al. (2008)] deposited in MZLU. focus on Boletina based on multiple molecular markers, DNA was extracted using the Dneasy Blood & Tissue Kit and to test the monophyly of the genus and also whether (Qiagen, Hilden, Germany), following the included proto- Saigusaia and Aglaomyia deserve their status as separate col ‘Purification of Total DNA from Animal Tissues genera. In addition, we aimed at testing the placement of (Spine-Column Protocol)’. PCR amplification of segments the Boletina erythropyga group that in our view seems mor- from the nuclear 28S and the mitochondrial 16S, CytB phological as well separated from the rest of the genus as and COI genes follows standard procedures, using the is Aglaomyia and Saigusaia. primers listed in (Table S1). The amplification programme for the 28S gene was 95 °C for 5 min, followed by 35 Material and methods cycles of 95 °C for 40 s, 52 °C for 40 s and 72 °C for Material 1 min and a final extension step at 72 °C for 8 min. The The study is based on specimens from the collections at amplification programme for the 16S gene was 95 °C for the Museum of Zoology, Lund University (MZLU) 5 min, followed by 35 cycles of 95 °C for 30 s, 45 °C for (mainly from the Nordic region), and some Aglaomyia 30 s and 72 °C for 1 min and a final extension step at specimens that were obtained on the courtesy of T. Sai- 72 °C for 8 min. The amplification programme for the gusa (Fukuoka, Japan) and from Hokkaido University COI gene was 95 °C for 5 min, followed by 35 cycles of Museum, Japan. Fresh specimens were obtained from 95 °C for 40 s, 45 °C for 45 s and 72 °C for 1 min and a recent Malaise trap and sweep net collections. In some final extension step at 72 °C for 8 min. The amplification cases, where fresh material was unavailable, older programme for the CytB gene was 94 °C for 3 min, fol- museum material from MZLU both dry pined and stored lowed by 35 cycles of 94 °C for 45 s, 40 °C for 45 s and in alcohol, were tried. All specimens examined were 72 °C for 1 min and a final extension step at 72 °C for recorded with unique identification codes prefixed by 10 min.