Journal of Insect Science (2016) 16(1): 38; 1–14 doi: 10.1093/jisesa/iew017 Research article Morphological and Molecular Identification of Anagrus ‘atomus’ Group (Hymenoptera: Mymaridae) Individuals from Different Geographic Areas and Plant Hosts in Europe P. Zanolli,1 M. Martini,1 L. Mazzon,2 and F. Pavan1,3 1Department of Agricultural, Food, Environmental and Animal Sciences, University of Udine, Via delle Scienze 206, 33100 Udine, Italy; 2Department of Agronomy Food Natural Resources Animals and Environment, University of Padua, Agripolis, Viale dell’Universita 16, 36020 Legnaro (Padova), Italy and 3Corresponding author, email: [email protected] Subject Editor: Daniela Takiya Received 24 December 2015; Accepted 16 February 2016 Abstract Morphological identification and molecular study on the COI gene were simultaneously conducted on Anagrus Haliday ‘atomus’ group individuals collected in the field in Italy or supplied from a UK biofactory. Females were morphologically identified as A. atomus L. and A. parvus Soyka sensu Viggiani (¼A. ustulatus sensu Chiappini). Alignment of COI gene sequences from this study permitted recognition of a total of 34 haplotypes. Phylogenetic and network analyses of molecular data not only confirmed that A. atomus is a species distinct from A. parvus, but also suggested that two species may be included within morphologically identified A. par- vus. Different geographical distribution and frequency of haplotypes were also evidenced. For males consid- ered in this study, morphometric analyses revealed a character that could be useful to discriminate A. atomus from A. parvus. Both species were found in vineyards and surrounding vegetation, confirming the potential role of spontaneous vegetation as a source of parasitoids for leafhopper control in vineyards. Key words: mymaridae; identification; COI gene; molecular analysis; morphometry Anagrus Haliday (Hymenoptera: Mymaridae) includes extremely (Chiappini et al. 1999) or be influenced both by the host parasitized small egg parasitoids mostly associated with leafhoppers (Huber and Rajakulendran 1988) and by the host plant from which (Hemiptera: Cicadellidae) (Huber 1986; Wallof and Jervis 1987; individuals emerge (Nugnes and Viggiani 2014). In general, identifi- Arno et al. 1988; Matteucig and Viggiani 2008; Triapitsyn et al. cation at species level of individuals belonging to Anagrus is often 2010). Within the genus, two species groups (i.e., ‘incarnatus’and difficult due to the paucity of diagnostic characters and morphologi- ‘atomus’) have been recognized morphologically (Chiappini et al. cal variability within species (Triapitsyn et al. 2010). In the case of 1996). Until a short time ago, inside the ‘atomus’ group only A. atomus and A. parvus males, are morphologically indistinguish- Anagrus atomus (L.) and A. parvus Soyka sensu Viggiani (2014) able (Viggiani 1970, 1989; Chiappini and Mazzoni 2000). (¼A. ustulatus sensu Chiappini 1989) (hereafter A. parvus) had been Regarding A. ustulatus there is also a misidentification problem, described for Italy (Chiappini et al. 1996; Floreani et al. 2006; since Viggiani (2014) recently proposed to refer A. ustulatus sensu Viggiani et al. 2006), but recently also Anagrus lindberginae Nugnes Chiappini et al. (1996) to A. parvus Soyka (Soyka 1955). and Viggiani was described in Nugnes and Viggiani (2014). The lat- Sibling species are particularly frequent in extremely small ter species is exclusively associated with Quercus ilex (L.) and eggs Hymenoptera parasitoids (Masner 1975; Stouthamer et al. 1999; of the leafhopper Lindbergina aurovittata (Douglas), whereas Borghuis et al. 2004). For example, Anagrus epos Girault has been A. atomus and A. parvus emerge from other plant species (e.g., grape- sub-divided into several different species, grouped in the A. epos spe- vines and Rubus L. spp.). Females of these latter species were distin- cies complex (Triapitsyn 1998; Triapitsyn et al. 2010). In biological guished on the basis of a morphological character (Chiappini et al. control, the characterization of natural enemies is essential because 1996) which, in most cases, is combined with a morphometric one cryptic species may have different biological performances leading (Chiappini 1987; Floreani et al. 2006). However, morphological and to a variable ability to control a specific pest (DeBach and Rosen morphometric identifications of mymarid species can fail because 1991; Nugnes and Viggiani 2014). For example, both A. atomus discriminant characters can be intermediate in some individuals and A. parvus can emerge from grapevine and bramble leaves VC The Author 2016. Published by Oxford University Press on behalf of the Entomological Society of America. 1 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] 2 Journal of Insect Science, 2016, Vol. 16, No. 1 (Chiappini 1987; Floreani et al. 2006; Viggiani et al. 2006), but in 12 open field Italian sites. The remaining 10 individuals were A. Northern Italy on grapevines only A. atomus was associated with atomus that emerged in the laboratory from leaf portions of Primula the eggs of Empoasca vitis (Gothe)€ (Chiappini 1987; Zanolli and L. sp. containing parasitized eggs of the leafhopper Hauptidia mar- Pavan 2011, 2013), whereas A. parvus emerged only from the eggs occana (Melichar), supplied by Biowise (Petworth, West Sussex, of Zygina rhamni Ferrari (Zanolli and Pavan 2011). However, the UK). Another 10 males that emerged from grapevine leaves collected number of potential hosts for Anagrus spp. in Europe is large in Friuli Venezia Giulia (FVG) were used exclusively for morpho- (Huber 1986; Arno et al. 1988; Matteucig and Viggiani 2008). In metric analysis. These individuals were identified as A. atomus on particular, on Rubus spp., considered an important source of the basis of cuticular hydrocarbons (Floreani et al. 2006) or because Anagrus spp. for biological control of leafhoppers in vineyards they emerged from E. vitis eggs, known to be parasitized only by (Arno et al. 1988; Cerutti et al. 1991; Ponti et al. 2005; Matteucig this species (Zanolli and Pavan 2013). All individuals were frozen as and Viggiani 2008; Zanolli and Pavan 2011), the leafhoppers soon as they emerged and stored at À80C until used. Once removed Zygina Fieber spp., Ribautiana (Zachvatkin) spp., Edwadrsiana from the freezer, the parasitoids were soaked in ethanol at 95C. rosae (L.), and Arboridia parvula (Boheman) were found to lay eggs. Under a dissecting microscope, the head of females and the genitalia Therefore, species limits based only on morphology and mor- of males were dissected with fine pins from the rest of the body. All phometry may be difficult to assess without supporting data from instruments used for dissection were disinfected in alcohol and their biology, and from molecular and biochemical analyses. When flamed before processing each individual. Female head and male two sibling species are identified with molecular analysis, the molec- genitalia were mounted on slides in Berlese’s medium and used for ular results have a key role in making correct identifications, to- morphological and morphometric analyses. The rest of the body gether with species identification based on morphological characters was processed for DNA extraction. that may be of variable taxonomic value (Triapitsyn et al. 2010). In order to discriminate A. atomus and A. parvus, several enzymes Morphological and Morphometric Analyses were tested, but loci with alternative fixed alleles have not yet been To establish with certainty that Anagrus females belonged to the detected (Cargnus and Pavan 2007). Taxonomic study of the two ‘atomus’ group the presence of three multiporous plate sensilla (mps) (¼ Anagrus species on the basis of cuticular hydrocarbons showed two sensory ridges, of authors) on the antennal club was checked (Chiappini distinct profiles that were largely, but not always, consistent with et al. 1996). Females were also identified to species by the presence (A. classification based on morphology (Floreani et al. 2006). Among atomus)orabsence(A. parvus)ofonempsonF4(Chiappini et al. the alternatives to morphological taxonomy, barcoding can help 1996). Anagrus ‘atomus’ group males were separated from ‘incarnatus’ identify the amount of biodiversity (Hebert et al. 2003) and how to group males according to Chiappini and Mazzoni (2000). differentiate the species found (Kruse and Sperling 2002; Lin and For individuals belonging to the ‘atomus’ group morphometric Wood 2002, MacDonald and Loxdale 2004). As mitochondrial analyses were also conducted. For females, the length of club as well genes are weakly subject to recombination and are inherited mater- as length of funicle segments F3 and F4 was measured and the ratio nally with independent replication (Saccone et al. 1999), primers for between antennal club length and the combined length of F3 and F4 conserved regions of mitochondrial DNA (mtDNA) are valuable was calculated (Chiappini 1987; Floreani et al. 2006). For males, tools for molecular systematic studies (Folmer et al. 1994; Simon lengths of the entire genitalia (Fig. 1a), phallobase (Fig. 1b) and digi- et al. 1994). For Chalcidoidea, the COI gene has been successfully tus (Fig.