Turkish Journal of Zoology Turk J Zool (2017) 41: 1010-1023 http://journals.tubitak.gov.tr/zoology/ © TÜBİTAK Research Article doi:10.3906/zoo-1701-28 Mating behaviour and its relationship with morphological features in the millipede Pachyiulus hungaricus (Karsch, 1881) (Myriapoda, Diplopoda, Julida) 1, 2 1 2 Zvezdana JOVANOVIĆ *, Sofija PAVKOVIĆ-LUČIĆ , Bojan ILIĆ , Vukica VUJIĆ , 1 1 1 1 Boris DUDIĆ , Slobodan MAKAROV , Luka LUČIĆ , Vladimir TOMIĆ 1 Department of Animal Development, Faculty of Biology, University of Belgrade, Belgrade, Serbia 2 Department of Genetics and Evolution, Faculty of Biology, University of Belgrade, Belgrade, Serbia Received: 24.01.2017 Accepted/Published Online: 12.07.2017 Final Version: 21.11.2017 Abstract: Although millipedes (Diplopoda) represent one of the most diverse classes of arthropods, data concerning details of their mating behaviour are very scarce. In this work, we explored mating behaviour of the European millipede Pachyiulus hungaricus under laboratory conditions, and its relationship with the size and shape of certain morphological traits. We conducted 3 types of behavioural tests: a mating arena test, a female choice test, and a male choice test. Premating behaviour was “sequenced” in 5 behavioural steps and, together with the duration of copulation, scored in all mating assays. Males were the more active sex in searching for mates, while females were the “choosier” sex. Furthermore, in the choice tests, previous mating partners had significantly more copulations than new ones, thus raising questions about postcopulatory sexual selection in this species. On the other hand, our results indicate that size and/or shape of the tested morphological traits, except for the shape of the male walking legs, were not subject to precopulatory sexual selection. Other sensory domains known to influence courtship behaviour need to be investigated in this regard in P. hungaricus. Key words: Millipedes, Pachyiulus hungaricus, sexual selection, mating behaviour, mating system, morphology 1. Introduction Cator and Zanti, 2016) correlates to male mating and fer- Mating systems exist in different forms and gradations tilisation success. Additionally, it has been shown that (Dugatkin, 2004). Shuster and Wade (2003) defined a mat- variation in characteristics of mating sequences, such as ing system as the species-specific pattern of associations copulation duration, may be a factor that influences male between sexes. Although establishment of the number of fertilisation success (Simmons and Parker, 1992; Arnqvist mating partners can be viewed as a milestone for defin- and Danielsson, 1999a; Andrés and Cordero Rivera, 2000; ing a mating system (Dugatkin, 2004), the definition of a Jones et al., 2006). Apart from differences in the aforemen- mating system in the broadest sense should also include tioned aspects of morphology and behaviour, which are information about how copulatory partners are acquired, often subject to precopulatory sexual selection (Arnqvist characteristics of the mates, and patterns of parental care and Danielsson, 1999b), variation in male genital mor- (if present) by males and/or females (Davies, 1991). phology or courtship behavioural sequences may arise Inter- and intrasexual variation in morphology can af- due to postcopulatory sexual selection (Eberhard, 1985). fect all components that define a mating system. For ex- In light of the correlation that exists between male genital ample, males of many insect species are characterised by morphology and fertilisation success, it is presumed that variability in body colour, ornamentation, and size and/ this relationship can arise due to sperm competition and/ or shape of some morphological traits (e.g., Moczek and or cryptic female choice (Eberhard, 1985, 1996). Addi- Emlen, 1999; Kawano, 2000; Okada and Hasegawa, 2005). tionally, the intersexual coevolutionary arms race over the The existence of such patterns of morphological variabil- control of fertilisation, i.e. sexual conflict, can be viewed ity may lead to intrasexual variation in mating tactics and/ as a mechanism driving variation in genital morphology or mating success. Indeed, many studies have shown that (Arnqvist and Rowe, 1995). In general, postcopulatory overall body size (Lewis and Austad, 1990, 1994; Wedell, sexual selection is possible in animal taxa with internal 1991; Simmons et al., 1996; Wenninger and Averill, 2006; fertilisation where females are engaged in multiple copula- Pavković-Lučić et al., 2009; Helinski and Harrington, 2011; tions during the breeding season. Furthermore, it can be * Correspondence: [email protected] 1010 JOVANOVIĆ et al. / Turk J Zool pointed out that this mode of sexual selection operates in mainly on the Balkan Peninsula (Makarov et al., 2004). taxa where females store sperm from different males in The aims of this study were as follows: (1) to describe the their receptive organs for an extended period (Wojcieszek mating system of P. hungaricus; (2) to determine whether and Simmons, 2011). males and females of P. hungaricus would repeatedly mate One of the potentially most interesting and underrep- under laboratory conditions; (3) if so, to determine the resented animal groups in studies of mating behaviour are type of mating partner in successive matings (i.e. the same members of the class Diplopoda. Diplopods (millipedes) or different from the partner in previous mating); (4) to represent one of the most diverse and ancient groups of compare behavioural sequences between tests; and (5) terrestrial animals (Blower, 1985; Wilson and Anderson, to examine whether differences in certain morphological 2004; Sierwald and Bond, 2007). With respect to repro- traits exist between mated and nonmated individuals. ductive behaviour, it is known that millipedes are po- lygynandrous, i.e. both sexes mate repeatedly during the 2. Materials and methods breeding season (Hopkin and Read, 1992; Rowe, 2010; 2.1. Sampling and handling of millipedes under Wojcieszek and Simmons, 2011). Males of the infraclass laboratory conditions Helminthomorpha possess secondary sexual structures A total of 95 adult millipedes (44 males and 51 females) in the form of gonopods, which represent modified legs were sampled from Mt. Avala, near Belgrade (Čarapićev of the seventh and/or eighth body ring. Gonopods are Brest, village of Beli Potok; 44°41′32.18″N; 20°31′06.23″E), used for sperm transfer from male gonopores to female in May 2016. During field investigations, we observed that receptacula and are linked with sperm removal or sperm the abundance of specimens was greatest from mid- to displacement in female receptive organs (Minelli and Mi- late spring, under warm and rainy weather conditions. chalik, 2015, and references therein). Gonopod morphol- We usually found groups of specimens in the kinds of ogy is often very complex and variable, and is the character places favoured by them, e.g., on or under moist rotten that is the most reliable for species identification (Sierwald logs covered with moss, on thick bunches of branches, and Bond, 2007; Koch, 2015). During the breeding sea- or on tree bark (much more often on beech bark than on oak bark). Solitary individuals were rarely found, son, females mate many times with the same and/or dif- and then mostly when they were sitting several metres ferent males (Carey and Bull, 1986; Tadler, 1993; Telford up on a tree. More often they were found in groups with and Dangerfield, 1993a; Rowe, 2010; Wojcieszek and Sim- other conspecifics. The sex of specimens can be easily mons, 2011). They may store the sperm of multiple males distinguished at first sight: adult females of P. hungaricus for a prolonged period (Rowe, 2010; Wojcieszek and Sim- are approximately twice the size of adult males. To avoid mons, 2011). Because of the type of mating system in mil- possible mistakes, millipedes were immediately checked lipedes, and due to sperm storage in female receptacula, for the presence of gonopods, which are present only in there is a possibility that postcopulatory sexual selection males. After sex determination, males and females were drives evolution and variability of gonopod morphology placed in separate boxes filled with ground cover from the (Barnett et al., 1993, 1995; Telford and Dangerfield, 1993a; sampling site. The boxes were regularly sprayed with water Tadler, 1996). Consequently, one would expect differences to maintain high humidity under laboratory conditions. in gonopod morphology between mated and nonmated Before the experiments were conducted, individuals were males. kept separately by sex in the laboratory for 7 days under Most of the previous studies on mating systems in mil- room temperature conditions, relative humidity of about lipedes focused on members of the superorder Juliformia 60%, and a 12 h:12 h light:dark cycle. (Haacker, 1969; Carey and Bull, 1986; Mathews and Bult- 2.2. Mating assays man, 1993; Telford and Dangerfield, 1993a, 1993b; Barnett Three types of mating assays were performed: a mating et al., 1995; Cooper and Telford, 2000). However, the rela- arena test, a male choice test, and a female choice test. All tionship between morphology and fertilisation success has behavioural tests were conducted from 0800 to 2000. In been insufficiently studied not only within this taxonomic the mating arena test, where multiple mating combina- unit, but within other taxa of Diplopoda as well. Generally tions may occur, males and females were placed in plastic speaking, many behavioural