Quinone Millipedes^ Reconsidered: Evidence for a Mosaic-Like Taxonomic Distribution of Phenol-Based Secretions Across the Julidae
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J Chem Ecol (2016) 42:249–258 DOI 10.1007/s10886-016-0680-4 BQuinone Millipedes^ Reconsidered: Evidence for a Mosaic-Like Taxonomic Distribution of Phenol-Based Secretions Across the Julidae Michaela Bodner1 & Boyan Vagalinski2 & Slobodan E. Makarov3 & Dragan Ž.Antić3 & Ljubodrag V. Vujisić4 & Hans-Jörg Leis5 & Günther Raspotnig1,5 Received: 15 July 2015 /Revised: 18 February 2016 /Accepted: 29 February 2016 /Published online: 14 March 2016 # The Author(s) 2016. This article is published with open access at Springerlink.com Abstract The defensive chemistry of juliformian millipedes cresol content 93 %) and an undetermined Cylindroiulus spe- is characterized mainly by benzoquinones (^quinone cies (p-cresol content 51 %), in the brachyiulines Brachyiulus millipedes^), whereas the secretions of the putative close lusitanus (p-cresol content 21 %) and Megaphyllum fagorum outgroup Callipodida are considered to be exclusively pheno- (p-cresol content 92 %), as well as in an undescribed lic. We conducted a chemical screening of julid secretions for Typhloiulus species (p-cresol content 32 %, Leptoiulini). In phenolic content. Most species from tribes Cylindroiulini (15 all species, p-cresol was accompanied by small amounts of species examined), Brachyiulini (5 species examined), phenol. The secretion of M. fagorum was exclusively pheno- Leptoiulini (15 species examined), Uncigerini (2 species ex- lic, whereas phenols were accompanied by benzoquinones in amined), Pachyiulini (3 species examined), and Ommatoiulini all other species. This is the first incidence of clearly phenol- (2 species examined) had non-phenolic, in most cases exclu- dominated secretions in the Julidae. We hypothesize a shared sively benzoquinonic secretions. In contrast, tribes biosynthetic route to phenols and benzoquinones, with Cylindroiulini, Brachyiulini, and Leptoiulini also contained benzoquinones being produced from phenolic precursors. representatives with predominantly phenol-based exudates. The patchy taxonomic distribution of phenols documented In detail, p-cresol was a major compound in the secretions of herein supports multiple independent regression events in a the cylindroiulines Styrioiulus pelidnus and S. styricus (p- common pathway of benzoquinone synthesis rather than mul- tiple independent incidences of phenol biosynthesis. Electronic supplementary material The online version of this article Keywords Chemical defense . Quinone millipedes . (doi:10.1007/s10886-016-0680-4) contains supplementary material, . which is available to authorized users. Phenols Julida Julidae Cylindroiulini Brachyiulini Leptoiulini . Diplopoda * Günther Raspotnig [email protected] Introduction 1 Institute of Zoology, University of Graz, Universitätsplatz 2, 8010 Graz, Austria Millipedes are well protected against predators. Apart from hardened cuticles, disturbance-related coiling behaviors, or 2 Institute of Biodiversity and Ecosystem Research, Department of Animal Diversity and Resources, Bulgarian Academy of Sciences, 2 defensive bristles (Polyxenida), the majority of millipedes Gagarin Street, 1113 Sofia, Bulgaria are known to release repellent and noxious fluids from serial 3 Institute of Zoology, Faculty of Biology, University of Belgrade, exocrine glands, serving as an effective means of active de- Studentski Trg 16, Belgrade 11000, Serbia fense. Depending on the taxonomic group, diplopod defensive 4 Faculty of Chemistry, University of Belgrade, Studentski Trg 12-16, secretions show considerable chemical diversity as compre- Belgrade 11000, Serbia hensively reviewed recently (Shear 2015). Briefly, Glomerida 5 Research Unit of Osteology and Analytical Mass Spectrometry, and Polyzoniida produce unique alkaloids (e.g., Meinwald Medical University, University Children’s Hospital, et al. 1966, 1975;Schildknechtetal.1966; Shear et al. Auenbruggerplatz 30, 8036 Graz, Austria 2011;Woodetal.2000), Polydesmida produce cyanogenic 250 J Chem Ecol (2016) 42:249–258 compounds (Makarov et al. 2011; Shear et al. 2007), whereas Methods and Materials all groups of Juliformia mainly secrete quinones (e.g., Eisner et al. 1978; Vujisić et al. 2011). Indeed, the latter group - the Collection of Species Individuals, mostly adults, from 42 Juliformia - have been known as Bquinone millipedes^ (Eisner species belonging to 17 genera and 6 tribes of Julidae and 2 et al. 1978), implying that this superorder almost exclusively species of Blaniulidae (see supplemental Table S1)werecol- produces quinone-based secretions. Interestingly, within the lected by hand, either from the leaf litter layer or from deeper putative juliformian sister group BPolydesmida soil and breakstone beneath the leaf litter layer at various (Chordeumatida (Stemmiulida + Callipodida))^ several taxa locations in Austria, Germany, Italy, France, Spain, Bulgaria, rely on phenolic secretions (e.g., Blanke and Wesener 2014; Serbia, and Azerbaijan. Vouchers were deposited at the Shear 2015). Callipodids, for instance, appear to exclusively Natural History Museum of Vienna (NHMW), Natural produce phenols (e.g., Makarov et al. 2011), which give them History Museum of Denmark (ZMUC), Hungarian Natural a characteristic and obtrusive odor, easily noticeable by History Museum (HNHM), National Museum of Natural humans over distances of several meters. Additionally, History Sofia (NMNHS), and at the Faculty of Biology, stemmiulidans (Shear 2015) and several polydesmidans have Institute of Zoology, University of Belgrade (FBIZO). been reported to discharge phenolics, and in both cases the most frequently occurring component was p-cresol (Duffey Extraction and Analysis of Defensive Secretions Secretions et al. 1977;Morietal.1994; Noguchi et al. 1997;Shear were obtained by whole body extraction of single individuals et al. 2007; Taira et al. 2003). in hexane for 15 min. The defensive secretions were In many Arthropoda, the biosynthesis of phenols and discharged directly into the solvent. Aliquots of diluted ex- benzoquinones is thought to be related. There are good exam- tracts (1.5 μl) were analyzed by gas chromatography– mass ples of arthropods that utlize both phenolic and benzoquinonic spectrometry (GC-MS), using a trace gas chromatograph compounds (e.g., Rocha et al. 2013). In laniatorean harvest- coupled to a DSQ I mass spectrometer (MS), both from men, for instance, the secretions of representatives of basal Thermo (Vienna, Austria). GC and MS conditions were the grassatorean families rely on phenolics, whereas the secretions same as previously described (Bodner and Raspotnig 2012). of derived grassatoreans may contain both phenols and Gas chromatographic retention indices (RI) of extract compo- benzoquinones or may even be purely benzoquinonic nents were calculated using an alkane standard mixture (Van (Föttinger et al. 2010; Raspotnig et al. 2015). In laniatoreans, den Dool and Kratz 1963). benzoquinones appear to present a derived character state, that High-resolution mass spectrometry was carried out on a can be produced from phenols by para-oxidation, and the pro- Q-exactive high-resolution Orbitrap MS from Thermo duction of benzoquinones may be considered an extension of (Vienna, Austria). Samples containing secretion in hexane the pathway to phenols (Raspotnig et al. 2015; Rocha et al. were gently reduced under nitrogen, then dissolved in metha- 2013). nol: water (1:1) with 1 % formic acid, and analyzed by direct In the scope of our studies on the evolutionary history of infusion ESI-MS and HPLC-MS, respectively. Components secretion chemistry in diplopods, it is essential to pinpoint were observed as [M + H]+.-ionsaswellasNa-andK- basic correlations between the chemistry of different taxa. adducts. We here hypothesize that benzoquinones in juliformians evolved from the ancestral state of phenolic secretions, as still present in juliformian outgroups (Raspotnig and Bodner Reference Compounds and Derivatization For comparison 2014). To test our idea, we conducted extensive chemical of GC-MS data to authentic reference compounds 1,4-benzo- screening of julid secretions for their phenolic content. So quinone, 2,3-dimethoxy-5-methyl-1,4-benzoquinone, p-cre- far, phenolics in juliformians have been regarded to be an sol, o-cresol, and m-cresol, methyl-paraben, and 2- exception or even completely missing: only one represen- phenylphenol were purchased from Sigma (Vienna, Austria). tative of Julida, namely a member of the basal julidan 2,3,5,6-Tetramethoxy-1,4-benzoquinone was from family Parajulidae, Oriulus venustus (Wood 1864), was MicroCombiChem (Wiesbaden, Germany), and 2,3- reported to discharge a phenol-rich secretion (Kluge and dimethoxy-5-methyl-1,4-hydroquinone from abcr GmbH & Eisner 1971). There are, however, an increasing number Co KG (Karlsruhe, Germany). As reference for other com- of reports on julid phenolics as minor, trace, or by- pounds, particularly 2-methyl-1,4-benzoquinone, 2-hydroxy- products of the quinone-rich secretions of certain species 3-methyl-1,4-benzoquinone, 2-methoxy-3-methyl-1,4-benzo- (e.g., Sekulić et al. 2014). quinone, 2,3-dimethoxy-1,4-benzoquinone, 2- We provide evidence that phenol-based secretions are more methylhydroquinone, we used natural sources from which widespread in the Julidae, and that phenolics even prevail over these compounds had already been identified (Allajulus quinones in some species of the tribes Cylindroiulini, dicentrus: Bodner and Raspotnig 2012; Cylindroiulus boleti: Brachyiulini, and Leptoiulini. Vujisić et al. 2011). J Chem Ecol (2016) 42:249–258