Supplementary data for the article: Bodner, M.; Vagalinski, B.; Makarov, S. E.; Antić, D. Ž.; Vujisić, L. V.; Leis, H.-J.; Raspotnig, G. “Quinone Millipedes” Reconsidered: Evidence for a Mosaic-Like Taxonomic Distribution of Phenol-Based Secretions across the Julidae. Journal of Chemical Ecology 2016, 42 (3), 249–258. https://doi.org/10.1007/s10886-016-0680-4

Supplementary material for: [https://doi.org/10.1007/s10886-016-0680-4]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/1924

Alkaloids from millipedes: a re-evaluation of defensive exudates from Polyzonium germanicum

Millipedes are known to produce various sets of chemical compounds in exocrine defensive glands to protect themselves against predators and microorganisms. Here, we reanalyzed the gland secretion of Polyzonium germanicum, a millipede of the order Polyzoniida, by using a combination of analytical techniques such as GC-MS, LC-HRMS and high field 1D and 2D NMR spectroscopy. Previously only one compound (polyzonimine, 1) had been described, but our approach allowed us to add six compounds to the defensive chemistry of this species. Besides polyzonimine (1), we found nitropolyzonamine (2) and five new compounds: 3 (2,3-dimethyl-7’-nitro-2’,3’,5’,6’,7’,7a’-hexahydrospiro[cyclopent-2-ene-1,1’-pyrrolizine]), 4 (2,3-dimethyl-7’-nitro-2’,3’,5’,6’,7’,7a’-hexahydrospiro[cyclopentane-1,1’-pyrrolizin]-2-ene), 5 ((1Z)-8,9-dimethyl-1-(nitromethylidene)-2-azaspiro[4.4]non-8-en-7-one), and not fully identified compounds A, B. For compounds 3–5 we were able to determine the molecular constitution, for two of them (4, 5) we were able to give relative configurations. Overall, the combination of advanced analytical techniques applied herein allowed detailed insights into the defensive chemistry of P. germanicum with a low number of individuals needed for analysis and without prior compound isolation

"Quinone Millipedes" Reconsidered: Evidence for a Mosaic-Like Taxonomic Distribution of Phenol-Based Secretions across the Julidae

The defensive chemistry of juliformian millipedes is characterized mainly by benzoquinones ("quinone millipedes"), whereas the secretions of the putative close outgroup Callipodida are considered to be exclusively phenolic. We conducted a chemical screening of julid secretions for phenolic content. Most species from tribes Cylindroiulini (15 species examined), Brachyiulini (5 species examined), Leptoiulini (15 species examined), Uncigerini (2 species examined), Pachyiulini (3 species examined), and Ommatoiulini (2 species examined) had non-phenolic, in most cases exclusively benzoquinonic secretions. In contrast, tribes Cylindroiulini, Brachyiulini, and Leptoiulini also contained representatives with predominantly phenol-based exudates. In detail, p-cresol was a major compound in the secretions of the cylindroiulines Styrioiulus pelidnus and S. styricus (p-cresol content 93 %) and an undetermined Cylindroiulus species (p-cresol content 51 %), in the brachyiulines Brachyiulus lusitanus (p-cresol content 21 %) and Megaphyllum fagorum (p-cresol content 92 %), as well as in an undescribed Typhloiulus species (p-cresol content 32 %, Leptoiulini). In all species, p-cresol was accompanied by small amounts of phenol. The secretion of M. fagorum was exclusively phenolic, whereas phenols were accompanied by benzoquinones in all other species. This is the first incidence of clearly phenol-dominated secretions in the Julidae. We hypothesize a shared biosynthetic route to phenols and benzoquinones, with benzoquinones being produced from phenolic precursors. The patchy taxonomic distribution of phenols documented herein supports multiple independent regression events in a common pathway of benzoquinone synthesis rather than multiple independent incidences of phenol biosynthesis.Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3323

Supplementary data for the article: Bodner, M.; Vagalinski, B.; Makarov, S. E.; Antić, D. Ž.; Vujisić, L. V.; Leis, H.-J.; Raspotnig, G. “Quinone Millipedes” Reconsidered: Evidence for a Mosaic-Like Taxonomic Distribution of Phenol-Based Secretions across the Julidae. Journal of Chemical Ecology 2016, 42 (3), 249–258. https://doi.org/10.1007/s10886-016-0680-4

Supplementary material for: [https://doi.org/10.1007/s10886-016-0680-4]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/1924

Chemical Ecology of Cave-Dwelling Millipedes: Defensive Secretions of the Typhloiulini (Diplopoda, Julida, Julidae)

Cave animals live under highly constant ecological conditions and in permanent darkness, and many evolutionary adaptations of cave-dwellers have been triggered by their specific environment. A similar "cave effect" leading to pronounced chemical interactions under such conditions may be assumed, but the chemoecology of troglobionts is mostly unknown. We investigated the defensive chemistry of a largely cave-dwelling julid group, the controversial tribe "Typhloiulini", and we included some cave-dwelling and some endogean representatives. While chemical defense in juliform diplopods is known to be highly uniform, and mainly based on methyl- and methoxy-substituted benzoquinones, the defensive secretions of typhloiulines contained ethyl-benzoquinones and related compounds. Interestingly, ethyl-benzoquinones were found in some, but not all cave-dwelling typhloiulines, and some non-cave dwellers also contained these compounds. On the other hand, ethyl-benzoquinones were not detected in troglobiont nor in endogean typhloiuline outgroups. In order to explain the taxonomic pattern of ethyl-benzoquinone occurrence, and to unravel whether a cave-effect triggered ethyl-benzoquinone evolution, we classed the "Typhloiulini" investigated here within a phylogenetic framework of julid taxa, and traced the evolutionary history of ethyl-benzoquinones in typhloiulines in relation to cave-dwelling. The results indicated a cave-independent evolution of ethyl-substituted benzoquinones, indicating the absence of a "cave effect" on the secretions of troglobiont Typhloiulini. Ethyl-benzoquinones probably evolved early in an epi- or endogean ancestor of a clade including several, but not all Typhloiulus (basically comprising a taxonomic entity known as "Typhloiulus sensu stricto") and Serboiulus. Ethyl-benzoquinones are proposed as novel and valuable chemical characters for julid systematics.Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3085

Supplementary data for article: Makarov, S. E.; Bodner, M.; Reineke, D.; Vujisić, L. V.; Todosijević, M. M.; Antić, D. Ž.; Vagalinski, B.; Lučić, L. R.; Mitić, B. M.; Mitov, P.; et al. Chemical Ecology of Cave-Dwelling Millipedes: Defensive Secretions of the Typhloiulini (Diplopoda, Julida, Julidae). Journal of Chemical Ecology 2017, 43 (4), 317–326. https://doi.org/10.1007/s10886-017-0832-1

Supplementary material for: [https://doi.org/10.1007/s10886-017-0832-1]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2450

Genetic diversity and landscape genetic structure of otter (Lutra lutra) populations in Europe

Eurasian otter populations strongly declined and partially disappeared due to global and local causes (habitat destruction, water pollution, human persecution) in parts of their continental range. Conservation strategies, based on reintroduction projects or restoration of dispersal corridors, should rely on sound knowledge of the historical or recent consequences of population genetic structuring. Here we present the results of a survey performed on 616 samples, collected from 19 European countries, geno- typed at the mtDNA control-region and 11 autosomal microsatellites. The mtDNA variability was low (nucleo- tide diversity = 0.0014; average number of pairwise dif- ferences = 2.25), suggesting that extant otter mtDNA lineages originated recently. A star-shaped mtDNA net- work did not allow outlining any phylogeographic infer- ence. Microsatellites were only moderately variable (Ho = 0.50; He = 0.58, on average across populations), the average allele number was low (observed Ao = 4.9, range 2.5–6.8; effective Ae = 2.8; range 1.6–3.7), sug- gesting small historical effective population sizeotters likely originated from the expansion of a single refugial population. Bayesian clustering and landscape genetic analyses however indicate that local populations are genetically differentiated, perhaps as consequence of post-glacial demographic fluctuations and recent isolation. These results delineate a framework that should be used for implementing conservation programs in Europe, particu- larly if they are based on the reintroduction of wild or captive-reproduced otters.Peer reviewe

Phenol-based millipede defence: antimicrobial activity of secretions from the Balkan endemic millipede Apfelbeckia insculpta (L. Koch, 1867) (Diplopoda: Callipodida).

Millipedes use an array of chemical compounds to defend themselves from predator attack. These chemical substances can have additional roles, i.e. defence against various pathogens. We evaluated the efficacy of the defensive secretion of Apfelbeckia insculpta (L. Koch, 1867) against bacteria, yeasts, and filamentous fungi. The tested secretion consisted of two compounds, p-cresol and phenol, and showed antibacterial, antibiofilm, and antifungal potential against all selected microorganisms. The most sensitive bacterium in our study was Pseudomonas aeruginosa, while the tested defensive secretion manifested the lowest activity against Escherichia coli. The defensive secretion of A. insculpta also showed an ability, albeit mild, to suppress biofilm formation by P. aeruginosa. Among the tested yeasts, Candida albicans and C. krusei were the most susceptible and most resistant species, respectively. Finally, the concentration of extracts obtained from the tested defensive secretion needed to achieve an antifungal effect was lowest in the case of Cladosporium cladosporioides. Fusarium verticillioides and Penicillium rubens were the micromycetes most resistant to the tested secretion. Our results indicate that antibacterial activity of the defensive secretion of A. insculpta is similar to or slightly weaker than that of streptomycin, while comparison with antimycotics showed that the tested millipede secretion has stronger activity than fluconazole, but weaker activity than nystatin and ketoconazole. The present study corroborates previous findings indicating that the defensive secretions of millipedes can have different roles apart from antipredator protection and are effective against pathogenic microorganisms