Cuticular hydrocarbons distinguish cryptic sibling in orchid bees Tamara Pokorny, Klaus Lunau, J. Quezada-Euan, Thomas Eltz

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Tamara Pokorny, Klaus Lunau, J. Quezada-Euan, Thomas Eltz. Cuticular hydrocarbons distinguish cryptic sibling species in Euglossa orchid bees. Apidologie, Springer Verlag, 2014, 45 (2), pp.276-283. ￿10.1007/s13592-013-0250-5￿. ￿hal-01234729￿

HAL Id: hal-01234729 https://hal.archives-ouvertes.fr/hal-01234729 Submitted on 27 Nov 2015

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Apidologie (2014) 45:276–283 Original article * INRA, DIB and Springer-Verlag France, 2013 DOI: 10.1007/s13592-013-0250-5

Cuticular hydrocarbons distinguish cryptic sibling species in Euglossa orchid bees

1,2 2 3 1 Tamara POKORNY , Klaus LUNAU , J. Javier G. QUEZADA-EUAN , Thomas ELTZ

1Department of Ecology, Evolution and Biodiversity, University of Bochum, 44780 Bochum, Germany 2Institute of Sensory Ecology, University of Düsseldorf, 40225 Düsseldorf, Germany 3Departamento de Apicultura, Universidad Autónoma de Yucatán, Mérida, Yucatán 97100, Mexico

Received 1 July 2013 – Revised 18 September 2013 – Accepted 8 October 2013

Abstract – Cuticular lipid profiles have been shown to be species specific within many genera, allowing a chemotaxonomic classification of individuals. In this study, we analysed the cuticular lipids of the orchid bees Euglossa viridissima Friese and Euglossa dilemma Bembé & Eltz, cryptic sibling species whose taxonomic status has only recently been clarified. Male individuals of both species were obtained from two locations on the Yucatán Peninsula, Mexico, both in the dry and wet seasons. Their hydrocarbon profiles proved to be species specific, irrespective of location and season. They also allowed a correct assignment of a rare morph of E. viridissima which had long obscured the distinction of the two species. Our results suggest that cuticular hydrocarbon profiles may be suited for chemical of orchid bees and might provide an additional clue for difficult species distinctions. cuticular hydrocarbons / / Euglossa / orchid bee / chemotaxonomy

1. INTRODUCTION 2005;Zimmermannetal.2009). However, since the perfume is gradually accumulated from the The orchid bees (, Apidae, environment, its composition is variable between ), neotropical, often conspicuously individuals and may not always allow coloured bees, are well known for the males' unambiguous species assignment. Analyses based behaviour of collecting volatiles. They have on molecular markers may also present evolved specialized structures for the difficulties, especially when concerning closely manipulation of those chemicals, for example related species. In orchid bees base changes in hind tibial pouches and tufts of hair on the middle cytochrome c oxidase subunit 1 were tibia (Dressler 1982). These structures, together uninformative among the sibling species Euglossa with other morphological characteristics as well as viridissima Friese and Euglossa dilemma Bembé coloration, are used for identification (see Roubik & Eltz (Eltz et al. 2011), while microsatellite and Hanson 2004). Analyses of the males' markers showed too much overlap in allele sizes perfume composition have provided chemical to be diagnostic for every single individual bee information for species distinction (Eltz et al. (Eltz et al. 2008). A similar case of nuclear data yielding ambiguous results has been reported for two closely related species of ants. The species could nevertheless be distinguished based, among Corresponding author: T. Pokorny, others, on cuticular hydrocarbon profiles (Seppä [email protected] et al. 2011). Considering these points, we aimed to Manuscript editor: Klaus Hartfelder determine whether cuticular lipids, which can be Hydrocarbons distinguish orchid bee species 277

used for chemotaxonomic purposes (i.e., are Bees were killed by freezing, and all legs were species specific) within various insect families removed to avoid contamination with perfume (see Howard 1993; Howard and Blomquist 2005; substances. The body was then extracted in 500 μL Bagnères and Wicker-Thomas 2010), might also of hexane for 1 min. Extracts were analysed using a present a reliable option for orchid bees. To test HP 5890 II gas chromatograph coupled with a HP this, we focused on the sibling species E. 5972 mass spectrometer. Injection was splitless onto viridissima and E. dilemma.Theyare a DB-5MS column (30 m×0.25 mm×0.25 μm). The morphologically very similar except for the oven was programmed from 60 to 300 °C at 5 °C/ position and the number of mandibular teeth in min. The final temperature was kept constant for males, of which all E. dilemma have three. In E. 15 min. Components were characterised by their viridissima, the majority of males have two, mass spectra and retention indices, and considered while, confusing matters, a rare morph (~10 % being identical in any two samples if both aspects of the male population) expresses a third clearly matched. Tentative compound identification mandibular tooth (Eltz et al. 2011). This had long was accomplished by comparison with synthetic prevented the recognition of the two distinct standards or, when none were available, by mass species (thus, the rare morph will hence be spectral interpretation and comparison with entries in described as exhibiting an ‘obscuring’ commercially available mass spectral libraries characteristic). However, since the middle tooth (Adams 2001 and Wiley 275). Double-bond position of the rare morph is always slightly dislocated for alkenes was evaluated by mass spectral towards the tip of the mandible, it can be interpretation of dimethyl disulfide (DMDS) distinguished from E. dilemma based on the derivatives (see Carlson et al. 1989). Integrated ion resulting differences of inter-tooth distances. This currents (peak areas) were standardized by total to finding, combined with analyses of perfume yield percentage contributions of each compound to composition and genetic characters, could finally an individual profile. Analyses were carried out using settle the classification (Eltz et al. 2011). In our all components (all) or subgroups consisting of either study we analysed the cuticular lipid profiles of only aliphatic hydrocarbons (hc) or solely acetates males of both species, including individuals of E. and alcohols (aa). Intraspecific and interspecific viridissima with the obscuring mandibular similarity was evaluated by non-metric characteristic, in order to assess their potential multidimensional scaling (MDS) and one-way for species distinction. As cuticular profiles can analysis of similarities (ANOSIM). ANOSIM is used differ among populations (Dapporto 2007)or to test for compositional differences between sample might depend on food or seasonal factors (Liang groups and is based on average ranked values of and Silverman 2000; Liu et al. 2001;Buczkowski Bray–Curtis measures of dissimilarity for multivariate et al. 2005;Martinetal.2012), our samples data sets (Clarke and Gorley 2006). Resulting R covered two locations and both seasons, rainy and values range from −1 to 1 and are a comparative dry. measure of the degree of separation, rendering them at least as important as their statistical significance. 2. METHODS AND MATERIALS Negative values are rare, resulting from higher dissimilarity within sample groups than between Bees were captured at chemical baits placed at the them. R=0 indicates that no differences are present locations Tecoh and Chicbul, situated 266 km apart, between sample groups, while R=1 results when on the western side of the Yucatán Peninsula, groups differ completely. Values in between indicate Mexico. Collection took place in March (dry season) the degree of differentiation of the groups. Substance and September (rainy season) of 2010. Per season contribution to dissimilarity of species was calculated and location, seven individuals of E. dilemma and E. using one-way Bray–Curtis similarity percentage viridissima and, always additionally, an eighth (SIMPER) analyses. All analyses were done with individual of the latter species exhibiting the the software Primer v6 (Clarke 1993; Clarke and obscuring mandibular characteristic were captured. Gorley 2006). 278 T. Pokorny et al.

3. RESULTS 9-heptacosene (9-C27:1) and 9-pentacosene (9- C25:1), as well as, to a much lesser extent, Cuticular extracts contained up to 68 (11Z)-eicosene-1-yl acetate and (9Z)-eicosene- compounds, mainly aliphatic hydrocarbons (on 1,20-diyl diacetate. Of these, the average average 59.65 % of total compounds), as well as contribution of the first two substances lies over acetates and alcohols. Seven substances could 25 % each to the dissimilarity observed, while not be assigned to any of these compound the next highest contribution is below 13 %. classes. DMDS derivatives allowed the When examining only the data subset of evaluation of double-bond position in alkenes, aliphatic hydrocarbons, the average contribution but not in alkadienes. In all ANOSIM analyses, to the dissimilarity of species rises to over 38 % there was a marginal distinction within species for 9-C27:1 and to 37 % for 9-C25:1. A concerning season (all: R<0.11, E. dilemma: comparison of the relative compound n.s., E. viridissima: P<0.05; hc: R<0.19, P< contributions to the cuticular aliphatic 0.05 (both species); aa: R<0.12, E. dilemma: hydrocarbon profile clearly shows the n.s., E. viridissima: P<0.05). A distinction differentiation of the two species. Each of the between the locations from which the extracts two compounds features prominently in the had been obtained was only found for aa of E. respective species' hydrocarbon profile with dilemma (all: R<0.08, n.s.; hc: R<0.04, n.s.; aa: the relative amounts of 9-C27:1 and 9-C25:1 R<0.16, E. dilemma: P<0.01, E. viridissima: differing by about 30 % between species n.s.). The slight seasonal and local differences (Table I, Figure 1). Relative abundances for within each species had no influence on the more than half of the other aliphatic overall species specificity of the cuticular hydrocarbons differed as well (pairwise profiles, which was based predominantly on comparisons, t test with Bonferroni correction; differences in the relative proportions of see Table I), though to a much lesser extent. compounds, rather than on exclusive compounds, as almost all were present in both 4. DISCUSSION species (Figure 1). MDS analyses using the subset of acetates and alcohols did not allow A low intraspecific differentiation of the unambiguous species classification owing to a cuticular extracts was found between seasons slight overlap of clusters (Figure 2c), while and only once between the two sampled analyses using all components (Figure 2a) or the locations. Since the trend of changes was not subset of aliphatic hydrocarbons (Figure 2b) identical for both species, it seems unlikely that produced distinct, species-specific clusters, the changes were a response to the dryer and clearly placing the individuals with the hotter or, respectively, wetter and colder obscuring mandibular characteristic within E. conditions. The differences might instead be viridissima (ANOSIM differentiation