View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by University of Salford Institutional Repository JChemEcol DOI 10.1007/s10886-016-0784-x Species-Specific Cuticular Hydrocarbon Stability within European Myrmica Ants Rhian M. Guillem1,2 & Falko P. Drijfhout3 & Stephen J. Martin4 Received: 1 December 2015 /Revised: 15 May 2016 /Accepted: 4 July 2016 # The Author(s) 2016. This article is published with open access at Springerlink.com Abstract Recognition is a fundamental process on which all Despite the morphological similarity that occurs within the subsequent behaviors are based at every organizational level, genus Myrmica, their CHCs were highly diverse but remark- from the gene up to the super-organism. At the whole organ- ably species-specific and stable across wide geographical ism level, visual recognition is the best understood. However, areas. This indicates a genetic mechanism under strong selec- chemical communication is far more widespread than visual tion that produces these species-specific chemical profiles, communication, but despite its importance is much less un- despite each species encountering different environmental derstood. Ants provide an excellent model system for chemi- conditions across its range. cal ecology studies as it is well established that compounds known as cuticular hydrocarbons (CHCs) are used as recog- Keywords Chemotaxonomy . Cuticular hydrocarbons . nition cues in ants. Therefore, stable species-specific odors Myrmica . Chemical recognition should exist, irrespective of geographic locality. We tested this hypothesis by comparing the CHC profiles of workers of twelve species of Myrmica ants from four countries across Introduction Europe, from Iberia to the Balkans and from the Mediterranean to Fennoscandia. CHCs remained qualitatively As the species category is a fundamental unit in biology, the stable within each species, right down to the isomer level. central goal of species delimitation is to develop robust and highly replicable measures for identification. This has focused mainly on morphology-based delimitation, utilizing visual characteristics found in study species. However, in insects, the primary recognition system between individuals is via Electronic supplementary material The online version of this article (doi:10.1007/s10886-016-0784-x) contains supplementary material, chemical cues, so it is predicted that species-specific chemical which is available to authorized users. recognition should be stable across a species’ entire geograph- ical range due to strong selection pressures. It is well * Rhian M. Guillem established that cuticular hydrocarbons (CHCs) function ex- [email protected] tensively in chemical communication within insects and serve as recognition signals (e.g., Blomquist and Bagnères 2010). 1 Department of Animal and Plant Sciences, University of Sheffield, Despite this, chemical recognition remains less understood S10 2TN, Sheffield, UK than visual recognition. 2 Department of Earth & Life Sciences, Gibraltar Botanic Gardens It has long been known that CHCs are species-specific Campus, University of Gibraltar, Gibraltar GX11 1AA, Gibraltar (Blomquist and Bagnères 2010;Martinetal.2008a) and can 3 Chemical Ecology Group, School of Physical and Geographical thus be readily exploited for use in chemotaxonomy (Berville Sciences, Lennard-Jones Laboratory, Keele University, Keele ST5 et al. 2013;Pageetal.2002; Takematsu and Yamaoka 1999). 5BG, UK However, in order to effectively exploit chemistry as a taxo- 4 School of Environment & Life Sciences, University of Salford, nomic tool we need to ensure that a species’ CHC profile is Manchester M5 4WT, UK stable throughout its geographical distribution. A review of JChemEcol the CHCs of 78 species of ant found that no two species had multiple countries (see Table 1), so these were used in a de- the same combination of compounds, despite almost 1000 tailed statistical analysis to test for species-specific CHC sta- CHCs described (Martin and Drijfhout 2009). Despite this, bility. The sampling localities represent a good range of hab- few comparative studies exploring the CHCs of species across itats and climates, and within Europe cover some of the most their geographical ranges exist (Berville et al. 2013; Bonelli northerly (Finland), southerly (Spain and Greece), western et al. 2015;Martinetal.2008a; Steiner et al. 2002), and none (Great Britain, Spain), and eastern (Finland, Greece) distribu- specifically investigates intra-specific variability across many tions. Ants were collected over a period of two years from geographic localities. If, as predicted, species-specific CHC 2010 to 2012, from a number of localities within each country profiles are stable across their entire range, then this will have (Table 1, Online Resource 1). The localities in Greece and benefits in identifying cryptic or sibling species (Akino et al. Spain were chosen as they represent two different glacial 2002; Lucas et al. 2002; Steiner et al. 2002). Since chemotax- refugia - the Balkans and Iberia, respectively - thus maximiz- onomy can outperform other taxonomic tools (e.g., Seppä ing any potential chemical diversity within species profiles. et al. 2011) its value in the taxonomic and ecological world We used previous data from Guillem et al. (2012)for could potentially be beneficial, especially as current tax- M. sabuleti and M. scabrinodis from Great Britain, but includ- onomic methods such as morphological characters and ed an additional six colonies of M. scabrinodis. All samples mitochondrial genes can sometimes fail in delineating were morphologically identified prior to chemical analysis, species (Seppä et al. 2011). In cases such as these, a using keys in Radchenko and Elmes (2010). Where possible, multi-disciplinary approach is best (Schlick-Steiner et al. we selected colonies from as many different sites/localities for 2010; Seifert et al. 2013). maximum CHC diversity. The ant genus Myrmica makes an ideal model system for testing ecological and evolutionary hypotheses, as species are Chemical Analyses Chemical analyses were conducted on abundant throughout Europe and have been well studied bio- one sample from each colony, comprising a pooled sample of logically, taxonomically and phylogeographically (Jansen five workers (non-callows). The five ants were placed in a glass et al. 2010; Radchenko and Elmes 2010; Seifert 1988). In vial and immersed in 50 μl of high-performance liquid chroma- addition, earlier studies on ant chemistry often used species tography grade hexane that contained 1 mg 100 ml−1 of an in- from this genus (Attygalle et al. 1983;Evershedetal.1982; ternal standard (docosane, C20 alkane) for 10 min. Ants were Morgan et al. 1979). Limited studies on Myrmica have shown removed, and hexane evaporated to dryness. Prior to analysis, CHC profiles to be species-specific, at least within a small 30 μl of hexane were added to the vials. Samples were analyzed geographical range for a limited number of species (Elmes on an HP 6890 gas chromatograph (GC) connected to an HP et al. 2002; Guillem et al. 2012). The Myrmica species used 5973 MSD (quadrupole) mass spectrometer (MS: −70 eV, elec- in this study were chosen because they are common and abun- tron impact ionization). The GC was equipped with an HP-5MS dant, have extensive and continuous temperate-boreal column (length 30 m; ID 0.25 mm; film thickness 0.25 μm), and Palaearctic distributions, and have wide ecological tolerances, the oven temperature was programmed from 70 °C to 200 °C at occurring in a diverse array of habitats and climates. Species 40 °C min−1 and then from 200 °C to 380 °C at 25 °C min−1. boundaries have been thoroughly tested morphologically and Samples were injected in splitless mode, with helium as the genetically, and all are currently considered to be good species carrier gas, at a constant flow rate of 1.0 ml min−1.CHCswere (Radchenko and Elmes 2010). Their chemical profiles are identified by their mass spectra, diagnostic ions, and corroborated relatively simple compared to some other genera, making by Kovats indices (Carlson et al. 1998). Ten extracts, three for them good candidates for a large-scale CHC study. M. sabuleti,threeforM. scabrinodis, and four for M. ruginodis This study investigated the stability of CHC profiles of 12 were subjected to dimethyl disulphide (DMDS) derivatization in species of Myrmica across large geographic distances order to determine the alkene double bond positions within the (countries) and a wide range of habitats, with the aim of test- alkenes (Carlson et al. 1989). These were then re-analyzed on the ing if stable species-specific CHC profiles exist irrespective of GC-MS under the same conditions as the non-derivatized sam- a variable environment, which is believed to play a major role ples. Each DMDS sample comprised a pool of 15–20 individual in determining the production of CHCs. ants from each country. For each sample, the CHC abundance of each compound was determined (area under the peak) and converted to rela- Methods and Materials tive abundances by dividing by the total abundance in each sample. Hydrocarbons with chain lengths less than C21 were Source Material This study analyzed the CHC profiles of 12 excluded from the analysis to avoid possible contamination of species of Myrmica from across Great Britain, Spain, Finland, volatile compounds (i.e., pheromones) that may have derived and Greece, across 219 colonies (Fig.1;Table1; Online from the Dufour’s gland. We analyzed two sets of data; first Resource 1). Five species of Myrmica are represented from using the relative compound abundances and second using JChemEcol Fig. 1 Geographic localities of the 12 Myrmica species collected from Great Britain, Spain, M. rub Finland, and Greece. See Online Resource 1 for locality details. M. rug rub = M. rubra; rug = M. rug M. scab M. sab M. ruginodis; sab = M. sabuleti; M. sch M. scab scab = M. scabrinodis; M. lob sch = M. schencki;alo= M. aloba; lob = M. lobicornis; lobu = M. lobulicornis;spec= M. specioides;sulc= M. sulcinodis;van=M. vandeli; wes = M. wesmaeli. Species in M. rub bold were used in a more detailed M.