Phylogenetic Relationships in the Subfamily Psychodinae (Diptera, Psychodidae)

Zoologica Scripta Phylogenetic relationships in the subfamily Psychodinae (Diptera, Psychodidae) ANAHI´ ESPIŃDOLA,SVEN BUERKI,ANOUCHKA JACQUIER,JAN JEZˇ EK &NADIR ALVAREZ Submitted: 21 December 2011 Espıńdola, A., Buerki, S., Jacquier, A., Jezˇek, J. & Alvarez, N. (2012). Phylogenetic rela- Accepted: 9 March 2012 tionships in the subfamily Psychodinae (Diptera, Psychodidae). —Zoologica Scripta, 00, 000–000. doi:10.1111/j.1463-6409.2012.00544.x Thanks to recent advances in molecular systematics, our knowledge of phylogenetic relationships within the order Diptera has dramatically improved. However, relationships at lower taxonomic levels remain poorly investigated in several neglected groups, such as the highly diversified moth-fly subfamily Psychodinae (Lower Diptera), which occurs in numerous terrestrial ecosystems. In this study, we aimed to understand the phylogenetic relationships among 52 Palearctic taxa from all currently known Palearctic tribes and sub- tribes of this subfamily, based on mitochondrial DNA. Our results demonstrate that in light of the classical systematics of Psychodinae, none of the tribes sensu Jezˇek or sensu Vaillant is monophyletic, whereas at least five of the 12 sampled genera were not monophyletic. The results presented in this study provide a valuable backbone for future work aiming at identifying morphological synapomorphies to propose a new tribal classification. Corresponding author: Anahı´ Espıńdola, Laboratory of Evolutionary Entomology, Institute of Biology, University of Neuchaˆtel. Emile-Argand 11, 2000 Neuchaˆtel, Switzerland. E-mail: [email protected] Present address for Anahı´ Espıńdola, Department of Ecology and Evolution, Biophore Building, University of Lausanne, 1015 Lausanne, Switzerland Sven Buerki, Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, UK. E-mail: [email protected] Anouchka Jacquier, Laboratory of Evolutionary Entomology, Institute of Biology, University of Neuchaˆtel. Emile-Argand 11, 2000 Neuchaˆtel, Switzerland. E-mail: anouchka.jacquier@ gmail.com Jan Jezˇek, Department of Entomology, National Museum of Prague (Natural History), Praha 4, Czech Republic. E-mail: [email protected] Nadir Alvarez, Department of Ecology and Evolution, Biophore Building, University of Lausanne, 1015 Lausanne, Switzerland. E-mail: [email protected] Introduction Because Diptera comprise ca. 180 families, of which The insect order Diptera (true flies) includes over 150 000 many are poorly known in terms of number and distribu- species and is considered one of the largest and most tion of species, the inference of the evolutionary history of diverse groups of organisms (Yeates et al. 2007). Despite many lineages has been difficult. Psychodidae (Lower Dip- the extreme abundance and species richness of Diptera, tera) is one of the worldwide families currently lacking the systematics of flies remains poorly understood. such phylogenetic studies. It comprises ca. 3000 species Recently, the popularization of molecular methods has (Jezˇek & Barta´k 2000) and is divided into six subfamilies triggered the reconstruction of phylogenetic relationships (Bruchomyiinae, Trichomyiinae, Horaiellinae, Phlebo- for this group. These methods have allowed testing previ- tominae, Psychodinae and Sycoracinae). Current molecular ous classifications and have enabled using phylogenies as chronograms estimate the origin of the family during the guidelines to establish new taxonomies when necessary Cretaceous (ca. 95 Mya, Bertone & Wiegmann 2009), (e.g. Friedrich & Tautz 1997; Bertone et al. 2008), provid- whereas fossil evidence [Blagoderov et al. (2007); not taken ing, for instance, the first complete, resolved and dated into account for the dating of Diptera by Wiegmann et al. phylogenetic inference of the order at the family level (2011)] suggests an earlier origin during the Late Triassic- (Wiegmann et al. 2011). Early Jurassic (ca. 201 Mya). Phylogenetic relationships ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters 1 Phylogenetic relationships in Psychodinae d A. Espıńdola et al. within the family are only known for the subfamily on amplification success of samples). These were obtained Phlebotominae, a well-studied group because of its ability from the personal collection of Dr. J. Jezˇek. Samples were to transmit human diseases (Depaquit et al. 1998; Beati collected during several field trips, from years 1995 to et al. 2004). In this study, to contribute to the knowledge 2009 in Eastern Europe, and were preserved in 70% etha- of the evolution of the family, we investigated the phylog- nol. Outgroup species were added to the dataset based on eny of the cosmopolitan and species-rich subfamily Psych- recent phylogenetic studies on Diptera (see e.g. Bertone odinae (Vaillant 1971) that is frequently found in et al. 2008; Wiegmann et al. 2011). The outgroup decomposing trophic networks (Jung 1956). sequences were obtained from GenBank (Table S1) and The Psychodinae show broad morphological and eco- consisted of eight samples from seven species belonging to logical diversity, allowing them to disperse worldwide and the infraorder Culicomorpha (Culicidae: Anopheles funestus, to colonize and survive in remote places such as oceanic A. gambiae, A. quadrimaculatus, A. darlingi, Aedes albopictus islands and subantarctic regions (Withers 1988). In the last and A. aegypti; Ceratopogonidae: Culicoides arakawae) and 40 years, Vaillant (1971) and Jezˇek (1984a) have provided one species belonging to the Psychodidae subfamily a species-level taxonomic framework for this subfamily, Phlebotominae (Phlebotomus riouxi). especially for Palearctic taxa. For instance, Vaillant (1971) attempted to describe and normalize characters for classifi- DNA extraction, amplification and sequencing cation of larvae and proposed identification keys to relate Insect DNA was extracted using the QIAgen DNEasy them to their respective imagoes, whereas Jezˇek (1984a) Animal tissue extraction kit (Qiagen, Hombrechtikon, solved nomenclatural problems and described species from Switzerland). Two mitochondrial (hereafter mtDNA) frag- poorly investigated geographic regions. Supra-specific ments were amplified with two primer pairs described in entities have, however, remained subject to controversy, Simon et al. (1994): CB-J-11338 –CAC ATT CAA CCA and most of them contradicted the first classification of GAA TGA TAT TT– and N1-N-12051 –GAT TTT Enderlein (1936) (see Table 1 for a summary of the classi- GCT GAA GGT GAA TCA GA– for the first fragment fication of Psychodinae). For instance, Vaillant (1971) rec- and N1-J-12248 –AAG CTA ATC TAA CTT CAT ommended dividing the Palearctic Psychodinae into four AAG– and LR-N-12866 –ACA TGA TCT GAG TTC tribes: Telmatoscopini, Psychodini, Pericomini and Bru- AAA CCG G– for the second fragment. While the first nettiini. Unfortunately, most of Vaillant’s work lacked fragment amplified portions of genes Cytochrome B (cytB) nomenclatural (several types were wrongly cited) and cla- and NADH1, the second partially covered genes NADH1 distic rigour (relationships among species and genera were and 16S (see below and Fig. S1, for more details). Amplifi- not clarified), and Jezˇek (1983, 1990a,b) thus proposed an cations were performed in a 20 lL master mix consisting alternative classification based on a cladistic approach of 0.5· buffer, 100 mM dNTP, 0.5 lM of each primer, (Table 1). 0.12 mM MgCl2, 1 unit of GoTaq Taq-polymerase (Pro- Although the circumscription of suprageneric levels of mega, Du¨bendorf, Switzerland) and 4 lL DNA. Reactions classification within the subfamily remains a challenging were run in a Biometra Thermocycler (Biometra, Goettin- task, the strong taxonomic background available for Pale- gen, Germany) with an initial denaturation of 1:30 min at arctic species (as well as the cladistic hypotheses provided 95 °C, followed by 35 cycles of 1 min of denaturation at by Jezˇek 1983, 1990a,b) allows testing the current Psych- 95 °C, 1 min of annealing at temperatures comprised odinae systematics with a molecular approach. In this between 42 °C and 52 °C, depending on samples and study, based on a sampling covering all Palearctic tribes of primers, and 30 s of elongation at 72 °C. Cycles were fol- Psychodinae, we used molecular sequence data of two lowed by 5 min of final elongation at 72 °C. Purification mitochondrial regions to infer phylogenetic relationships of PCR products and Sanger sequencing were done by within the subfamily. The aims of this survey were (i) to Macrogen Inc. (Seoul, South Korea) and Fasteris SA (Gen- evaluate the validity of Palaearctic tribes sensu Jezˇek and eva, Switzerland) with the same primers as used for the PCR sensu Vaillant; (ii) when possible, to test the monophyly of amplification. genera; (iii) to propose guidelines for future systematics within Psychodinae. Sequence alignment and phylogenetic reconstruction The program CHROMASPRO 1.41 (Technelysium Pty Ltd., Material and methods Brisbane, Australia) was used to assemble complementary Sampling strands and verify software base-calling. The two amplified We sampled 52 species [88 specimens, distributed in 26 mtDNA fragments were initially aligned applying the genera, representing all tribes in the European Psychodi- Clustal-Wallis algorithm (implemented in BioEdit 7.0.4.1; nae (Jezˇek 2009)] (Table 1; see Table S1 for information Hall 1999) and further visually adjusted. Fragments from 2 ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of

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