Eur. J. Entomol. 109: 435–444, 2012 http://www.eje.cz/scripts/viewabstract.php?abstract=1728 ISSN 1210-5759 (print), 1802-8829 (online) Molecular identity of Stomaphis quercus (Hemiptera: Aphidoidea: Lachnidae) and description of a new species àUKASZ DEPA1, EWA MRÓZ1 and KAROL SZAWARYN 2 1 Department of Zoology, Faculty of Biology and Environmental Protection, University of Silesia, Bankowa 9, 40-007 Katowice, Poland; e-mails: [email protected]; [email protected] 2 Museum & Institute of Zoology, Polish Academy of Sciences, Wilcza 64, 00-679 Warsaw, Poland; e-mail: [email protected] Key words. Hemiptera, Aphidoidea, Lachnidae, Stomaphis, COXI, COXII, molecular systematics Abstract. The two species of the genus Stomaphis feeding on oak and birch, respectively, although morphologically similar, are considered to be separate species. However, the birch-feeding S. betulae Mamontova is considered to be a synonym of the oak and birch feeding S. quercus (L.) by some authors. The purpose of this study was to determine whether the birch feeding and oak feeding populations attributed to S. quercus belong to the same species. The mitochondrial cytochrome-c oxidase I (COXI) and II (COXII) were used to determine whether these two populations differ. There are no significant differences in these markers from oak and birch feeding individuals, indicating that these populations are conspecific. However, morphologically and ecologically distinct populations of Stomaphis were discovered feeding on oak. The molecular analysis confirmed that these populations are distinct, which resulted in the description of the new oak-feeding species, Stomaphis wojciechowskii Depa, sp. n. This new species previously remained unrecognized due to its very cryptic mode of life. INTRODUCTION Klimaszewski & Wojciechowski (1996) conducted a The genus Stomaphis Walker 1870 (Aphidoidea: Lach- series of biochemical analyses of the hemolymph of rep- resentatives of Polish populations, both on birches and nidae) comprises over 25 species of Palaearctic distribu- oaks, and found no discontinuity between the oak and tion, associated predominately with deciduous trees but birch feeding populations. There is also morphological also recorded from conifers. Among them, Stomaphis support for the thesis that S. quercus and S. betulae are quercus (Linnaeus, 1758) is the most widely distributed, synonomous. recorded throughout the whole of the Western Palae- Thus, the need arose for detailed studies of these spe- arctic. Host plants are various species of Quercus and cies taxa, including extensive field research and mole- Betula (Blackman & Eastop, 1994). However, since the cular analysis of the material collected. Two mito- description of Stomaphis betulae Mamontova, 1969, the chondrial genes, COXI and COXII, were used as mole- distribution of S. quercus is debatable. In Mamontova’s cular markers in this study. opinion, only S. betulae lives on birches in the Ukraine Mitochondrial genes are widely used in studies on the and reports of the occurrence of S. quercus on birches in molecular systematics of insects (Simon et al., 1994). As other countries most probably refer also to S. betulae these genes are maternally inherited (Hoy, 1994), mito- (Mamontova, 1969, 1972). chondrial sequences are suitable for reconstructing phylo- Mamontova’s (1969, 1972) description and the general genetic relationships of parthenogenetic organisms like consensus that Stomaphis species are monophagous has aphids (von Dohlen & Moran, 2000). Mitochondrial resulted in many authors regarding S. betulae as a good COXI and COXII have been used in phylogenetic studies species (Eastop & Hille Ris Lambers, 1976; Czylok & of aphid families, e.g., Hormaphididae (Stern 1994, 1998; Blackman, 1991). However, some authors question its Stern et al., 1997), Lachnidae (Normark, 1999, 2000) and taxonomical status based on the intra-specific variation in Aphididae (von Dohlen et al., 2006). These genes appear morphological features of S. quercus (Klimaszewski & to be very useful for clarifying phylogenetic relationships Páachta, 1977; Klimaszewski & Wojciechowski, 1996). at the tribal to generic level in aphids (Normark, 2000; The questionable status of S. betulae results mainly from Stern, 1994), as it was in the case of the tribe Fordini a lack of distinctive features not only of the specimens (Zhang & Qiao, 2007) and the genera Uroleucon Mord- collected from Central European populations, but also vilko (Moran et al., 1999), Megoura Buckton (Kim & those from the type locality (Klimaszewski & Woj- Lee, 2007) and Toxoptera Koch (Wang & Qiao, 2009). ciechowski, 1996). Populations feeding on oaks and on The mitochondrial genes were also used to resolve prob- birches in Poland and the Ukraine do not clearly differ lems with species identity as in the case of a few species morphologically (Klimaszewski & Wojciechowski, 1996) in the genus Macrosiphum Passerini (Turþinaviþienơ & and the specimens described by Mamontova are the only Rakauskas, 2009) and the subgenus Bursaphis McVicar ones attributed to another species of Stomaphis feeding Baker (Rakauskas et al., 2011), or general systematic on birches in Ukraine. Blackman & Eastop (1994) also relations within the genus Aphis L. (Coeur d’acier et al., report this species as occurring only in the Ukraine. 2007). The aim of this study was to check the molecular 435 TABLE 1. Localities where the material studied was collected, their voucher numbers and GenBank accession numbers of COXI and COXII. Voucher Collection Associated ant GenBank Accession No. Species Collection site / Province Host plant number date species (Lasius) COXI COXII Bobrowniki A2 12/06/10 Betula verrucosa L. fuliginosus JN944537 JQ302807 50°22´39.07˝N, 18°59´50.49˝E Rudy Wielkie A3 9/07/10 Betula verrucosa L. fuliginosus JN944538 — 50°12´47.99˝N, 18°27´26.59˝E Rudy Wielkie A4 9/07/10 Quercus robur L. fuliginosus JN944539 — 50°12´35.66˝N, 18°27´27.38˝E Piekary ĝląskie – Szarlej A5 5/07/10 Betula verrucosa L. fuliginosus JN944540 — 50°22´14.87˝N, 18°57´43.32˝E ĝwierklaniec A6 24/09/10 Quercus robur L. fuliginosus JN944541 — 50°26´27.16˝N, 18°57´25.43˝E Kalinowice A7 8/07/10 Quercus robur L. fuliginosus JN944542 — 50°29´58.71˝N, 18°10´37.02˝E Stomaphis MiĊdzyzdroje S1 20/08/10 Quercus petraea L. fuliginosus JN944543 JQ302806 quercus 53°56´2.29˝N, 14°27´31.92˝E Bratkowice S4 21/09/10 Quercus robur L. fuliginosus JN944544 — 50°7´27.02˝N, 21°50´59.96˝E Sandomierz S7 22/09/10 Betula verrucosa L. fuliginosus JN944545 JQ302808 50°41´12.00˝N, 21°47´12.54˝E Piekary ĝląskie S11 24/09/10 Betula verrucosa L. fuliginosus JN944546 — 50°24´2.75˝N, 18°57´22.84˝E Piekary ĝląskie S13 18/09/10 Betula verrucosa L. fuliginosus JN944547 JQ302809 50°24´9.36˝N, 18°56´35.70˝E Piekary ĝląskie S14 1/07/10 Quercus robur L. fuliginosus JN944548 JQ302810 50°24´9.36˝N, 18°56´35.70˝E Kotulin S25 28/09/10 Betula verrucosa L. fuliginosus JN944549 — 50°28´6.83˝N, 18°26´7.42˝E Sieniawa Dobra S8 24/09/10 Quercus robur L. brunneus JQ302794 — 50°11´24.6˝N, 22°39´23.9˝E Sieniawa Dobra S9 24/09/10 Quercus robur L. platythorax JQ302795 — 50°11´24.6˝N, 22°39´23.9˝E Lubaczów S10 23/09/10 Quercus robur L. umbratus JQ302796 — 50°7´9.79˝N, 22°55´54.05˝E Lubaczów S15 23/09/10 Quercus robur L. platythorax JQ302797 JQ302811 50°7´6.85˝N, 22°55´54.96˝E Nowa DĊba S17 22/09/10 Quercus robur L. brunneus JQ302798 — 50°28´48.49˝N, 21°41´55.99˝E Stomaphis Lubaczów S18 23/09/10 Quercus robur L. brunneus JQ302799 — wojciechowskii 50°7´8.74˝N, 22°55´55.04˝E Bratkowice S19 21/09/10 Quercus robur L. brunneus JQ302800 JQ302812 50°6´3.42˝N, 21°49´33.63˝E Bratkowice S20 21/09/10 Quercus robur L. brunneus JQ302801 JQ302813 50°6´3.42˝N, 21°49´33.63˝E S23 Piekary ĝląskie 23/10/10 Quercus robur L. brunneus JQ302802 JQ302814 type series 50°24´11.48˝N, 18°57´47.87˝E Piekary ĝląskie Kalwaria S26 25/10/10 Quercus robur L. brunneus JQ302803 JQ302815 50°22´54.96˝N, 18°56´44.44˝E ĝwierklaniec S27 2/11/10 Quercus robur L. brunneus JQ302804 — 50°25´4.76˝N, 18°57´30.23˝E Stomaphis Kalinowice S24 28/09/10 Acer platanoides L. brunneus JQ302805 JQ812049 graffii 50°28´6.83˝N, 18°26´7.42˝E identity of oak and birch feeding aphids of the genus Sto- mens feeding on birches (12 specimens) the only morphological maphis (supposedly S. quercus and S. betulae). features studied were those diagnostic of S. betulae: hair length on abdominal tergites VI–VII and the presence of sclerites at the MATERIAL AND METHODS base of the abdominal hairs (Mamontova, 1972). We did not Morphological analysis carry out a detailed morphological study of typical oak feeding specimens as this information is available for Central European The morphological features of the material collected for populations (Klimaszewski & Wojciechowski, 1996). Only the molecular analyses were also studied. In the case of the speci- morphology of oak feeding specimens that were distinctly dif- 436 TABLE 2. Accession numbers of GenBank aphid sequences Primers used for amplification are listed in Table 3. PCR con- used in this study. ditions for each DNA fragment are given in Table 4. DNA was visualized using 1–2% agarose gel electrophoresis with ethi- Genus Species COXI COXII dium-bromide staining. PCR products were purified using a Rhopalosiphum nymphaeae GU457794 EU358855 QIAquick® PCR purification kit (Qiagen) and sequenced Trama rara EU701939 AF156221 directly using an automated sequencer (Genome Sequencer GS Stomaphis longirostris — AF156213 FLX Roche). Complete sequences were deposited in GenBank Stomaphis fagi — AF156210 under accession numbers given in Table 1.