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 Pachyiulus hungaricus (Karsch, 1881) (, Diplopoda, )

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 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 (Diplopoda) represent one of the most diverse classes of , 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 (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 traits of millipedes are still in- boxes (38 cm × 25 cm) filled with leaf-litter, pieces of bark, sufficiently known, probably because their long-term rear- and soil from the sampling site. All individuals were left to ing under laboratory conditions is more difficult than in mate for ~30 min. After copulation started, each mating the case of more traditional model organisms. pair was carefully transferred to a separate plastic box. Fol- In the present study, we investigated mating behaviour lowing the experiment in the mating arena test, millipedes in relation to variations in some morphological traits in were individually kept in plastic boxes filled with leaf litter the European millipede Pachyiulus hungaricus (Karsch, for 48 h. After this period, they were tested again in male 1881), one of the largest European diplopods, distributed and female choice tests.

1011 JOVANOVIĆ et al. / Turk J Zool

In the female choice test, 1 female and 2 males (the Apart from linear measurements, we analysed size and previous partner from the mating arena test and a new shape of the walking legs and gonopod promeres using one) per replication were placed in a plastic box (20 cm geometric morphometrics, a method for examination of × 13 cm) also filled with leaf-litter, pieces of bark, and soil morphological variability (e.g., Drake and Klingenberg, from the sampling site. The same principle was used in the 2008; Jojić et al., 2012; Sherratt et al., 2014; Lazić et al., male choice test (i.e. 1 male and 2 females per replication). 2015). For this procedure, we used leg pairs from the 31st For distinguishing mating partners, we used UV dust, one body ring in 28 mating pairs and 21 nonmated individuals of the most commonly employed agents for insect mark- (11 females and 10 males); and gonopods of 26 mated ing (Hagler and Jackson, 2001): the previous partner was males and 8 nonmated males. Gonopod promeres were marked with red dust, while green dust was used for new used for assessment of variation between mated and partners. This marking method can be used because UV nonmated males. Both structures of each millipede with a dust does not modify mating behaviour (Terzić et al., reference scale were photographed using a Nikon DS-Fi2 1994). A small amount of UV dust was applied to the tel- camera with a Nikon DS-L3 camera controller attached son of each millipede, and its colour was later identified to a Nikon SMZ 1270 binocular stereomicroscope. using a UV lamp. We placed 26 landmarks on digitalised leg pictures and Several behavioural traits were scored in the mating 9 landmarks on digitalised gonopod pictures in TpsDig experiments. We observed mating latency (ML), defined software (Rohlf, 2008) (Figure 1). The landmarks’ as the time elapsed from placing millipedes in the mating positions of the legs (Figure 1A) were as follows: 1 – the arena until the beginning of copulation; and the duration most proximal point of the coxa (dorsal side); 2 – the most of copulation (DC), defined as the time from the begin- distal point of the coxa (dorsal side); 3 – the most distal ning to the end of mating. In addition, we examined the point of the trochanter (dorsal side); 4 – the most proximal contact to copulation time (CC); the time from entry of point of the prefemur (dorsal side); 5 – the most distal individuals into plastic boxes to contact resulting in copu- point of the prefemur (dorsal side); 6 – the most proximal lation (ECC); the time from entry of individuals into plas- point of the femur (dorsal side); 7 – the most distal point tic boxes to contact without copulation (ECWC); and the of the femur (dorsal side); 8 – the most proximal point of duration of contact without copulation (CWC). the postfemur (dorsal side); 9 – the most distal point of The differences in DC, ECC, and CC values between the postfemur (dorsal side); 10 – the most proximal point the first copulation (in the mating arena) and the second of the tibia (dorsal side); 11 – the most distal point of the tibia (dorsal side); 12 – the most proximal point of the copulation (in the female or male choice tests) were tested tarsus (dorsal side); 13 – the most distal point of the tarsus using the t-test. Mating behaviour was also quantified us- (dorsal side); 14 – tip of the tarsal claw; 15 – the most distal ing descriptive statistics and the χ2 test. All statistical pro- point of the tarsus (ventral side); 16 – the most proximal cedures were performed in Statistica 7 (StatSoft, Tulsa, point of the tarsus (ventral side); 17 – the most distal point OK, USA). of the tibia (ventral side); 18 – the most proximal point of 2.3. Morphological analyses the tibia (ventral side); 19 – the most distal point of the To investigate whether significant morphological differ- postfemur (ventral side); 20 – the most proximal point of ences exist between mated and nonmated individuals, the postfemur (ventral side); 21 – the most distal point of the following 6 morphological characters were measured: the femur (ventral side); 22 – the most proximal point of body length (BL), body mass (M), antennal length (AL), the femur (ventral side); 23 – the most distal point of the trunk height (TH), trunk width (TW), and leg length (LL) prefemur (ventral side); 24 – the most proximal point of the (see Ilić et al. [2016] for descriptions of linear measure- prefemur (ventral side); 25 – the most distal point of the ments). In this analysis, trunk measurements were taken trochanter (ventral side); 26 – the most distal point of the from the 30th body ring. As an indicator of leg length, the coxa (ventral side); 27 and 28 – scale line. The landmarks’ anterior pair of legs of the following (31st) body ring was positions on the gonopods (Figure 1B) were: 1 – external measured. All measurements were performed in the Im- side of the base of the mesal prominence; 2 – mesal ageJ program (Abràmoff et al., 2004). For morphological denticle; 3 – apical tip of the promere; 4 – apical tip of the analyses, 28 mating pairs and 21 nonmated millipedes (11 median ridge; 5 – external side of the base of the median females and 10 males) were used. Data were log-trans- ridge; 6 – internal side of the base of the median ridge; 7 formed to meet the assumptions for parametric statistical – lateral denticle; 8 – projection of the mesal prominence; procedures. Differences in mean values of the aforemen- 9 – internal side of the base of the mesal prominence; 10 tioned traits between mated and nonmated females and and 11 – scale line. between mated and nonmated males were tested using the The CoordGen6 program (Sheets, 2003) was used to t-test. calculate centroid size (CS) of the mentioned traits, while

1012 JOVANOVIĆ et al. / Turk J Zool

Figure 1. A – position of landmarks on the walking leg in P. hungaricus. B – position of landmarks on the gonopodal promere, mesocaudal view. principal component analysis (PCA) and canonical variate species. The male actively taps the dorsal side of the female analysis (CVA) in the MorphoJ program (Klingenberg, with its antennae and moves forward to the head. After 2011) were used to analyse the shape variation of these reaching the female`s head, the male bends the anterior traits. The difference in CS of legs or gonopods in relation part of its body below the female, and, if the female is to the mating status of the millipedes was tested using receptive and ready to mate, it will uncoil the anterior part ANOVA. Statistical analyses of linear measurements and of its body and expose the gonopores. The male then ejects variation in CS of the observed morphological traits were its gonopods, and the next step in achieving copulation conducted in Statistica 7. is extrusion of the female’s vulvae. During copulation, animals can be found in a parallel position (Figure 3A) or 3. Results the male can coil around the female (Figures 3B and 3C). 3.1. Mating behaviour of P. hungaricus 3.1.2. Female and male choice tests 3.1.1. Mating arena In the female choice test, 66% of all contacts resulted in In the mating arena test, 47% of all contacts resulted copulation (78% of them were achieved with the previous in copulation, while 53% of contacts ended with no partner and 22% with a new partner). In the male choice copulation. Descriptive statistics of the observed test, 54% of all contacts resulted in copulation (64% of behavioural traits (ML, DC, CC, ECC, ECWC, and CWC) them were with the previous partner and 36% with a new are shown in Table 1. partner) (Figure 4). The resultsalso indicated that indi- Our direct observations of mating behaviour indicated viduals of both sexes achieved significantly more copula- that a pattern of daily dynamics is present in the number of tion with the previous partner than with a new partner. copulations. To be specific, the most matings erew observed In the female choice test, 68% of all observed contacts between 1000 and 1200 (21 copulations) and between (with and without copulation) were with the previous 1400 and 1600 (10 copulations). Watching millipedes in partner, while 32% were with a new partner. In the male the arena, we observed that males are the more active choice test, 53% of all contacts (with and without copu- sex during the search for mates, while females are the lation) were with the previous partner, while 47% were “choosier” sex. We also recorded successive behavioural with a new partner. A higher percentage of all contacts sequences that may define precopulatory behaviour in this ending in copulation was observed in both the female

1013 JOVANOVIĆ et al. / Turk J Zool

Table 1. Descriptive statistics of behavioural traits observed in seconds. Abbreviations: as noted in Materials and methods.

Behavioural traits N Min. Max. Median Variance Std. dev. Quartiles 25% Quartiles 75% ML 42 180.0 2640.0 600.0 296,649 544.6 413.0 900.0 DC 42 180.0 13,270.0 3215.0 5,296,711 2301.4 1860.0 3780.0 CC 42 30.0 1440.0 180.0 68,702 262.1 120.0 240.0 ECC 42 60.0 2520.0 360.0 276,715 526.0 209.0 660.0 ECWC 47 27.0 4260.0 350.0 477,057.7 690.7 180.0 720.0 CWC 47 5.0 1200.0 120.0 75,748.7 275.2 50.0 300.0

(71% vs. 29%) and the male (69% vs. 31%) choice tests grid deformation, size of the vectors, and their direction (Table 2). illustrate the pattern of leg shape variation in mated and Additionally, the highest percentage of copulations was nonmated individuals (Figures 5 and 6). detected after 1 contact in both the female and the male We found no significant difference in CS of gonopods choice tests (63% and 77%, respectively), while smaller between mated and nonmated males (F1,32 = 0.06, P = percentages of copulations was recorded after 2 or 3 con- 0.8043). The pattern of gonopod shape variation in mated tacts (19% and 9%, respectively). The smallest number of and nonmated males is illustrated by deformation grids copulations (5%) was noted after 5 contacts in the male with vectors (Figures 7 and 8). The shape of the observed choice test. gonopod part did not significantly differ between the 2 Results of the χ2 test indicated that the previous groups of males (P = 0.0662) (Figure 8). partners achieved significantly more copulations than the 2 new partners in both tests (male choice test: χ = 7.44, df = 4. Discussion 2 1, P < 0.01; female choice test: χ = 30.86, df = 1, P < 0.01). This study was conducted in order to contribute to a better The number of all contacts (with and without copulation) understanding of the mating strategies of millipedes, using with the previous and new partners was significantly the polygynandrous species P. hungaricus as a model- 2 different only in the female choice test (χ = 13.58, df = 1, system. We sequenced the species’ mating behaviour and P < 0.01). gave a quantified description of it based on a mating arena We also tested differences of behavioural traits in test followed by male and female choice tests. Courtship millipedes that mated twice (in the mating arena and in display is performed by males; they are the ones which the female or male choice test). No significant differences initiate contact. Precopulatory steps in a number of other of DC and ECC were found between 2 successive species are quite similar to the ones we observed: the copulations, but CC was significantly different (Table 3). julid Ommatoiulus moreletii (Lucas, 1860) (Carey and 3.2. Morphological variation in P. hungaricus Bull, 1986), the parajulid Aniulus bollmani Causey, 1952 3.2.1. Analyses of linear measurements (Mathews and Bultman, 1993), the spirostreptid Alloporus No significant differences in mean values of 6 uncinatus Attems, 1914 (Telford and Dangerfield, 1993a; morphological traits were observed between mated and Barnett et al., 1995), 6 tropical spirostreptidans (Telford nonmated females, or between mated and nonmated and Dangerfield, 1993b), and the spirobolid Centrobolus males (Table 4). (Cook, 1897) sp. (Cooper and Telford, 2000), which 3.2.2. Analyses of leg and gonopod size and shape belong to the superorder Juliformia; the craspedosomatid variation Craspedosoma Leach, 1814 sp. (Tadler, 1993); the The centroid size of legs did not differ between mated paradoxosomatid Parafontaria Verhoeff, 1936 sp. (Tanabe and Sota, 2008); and the paradoxosomatid Cladethosoma and nonmated individuals (F3,150 = 1.614, P = 0.1885). The first principal component (PC1) explained 81.03% clarum (Chamberlin, 1920) (Rowe, 2010). The male of the morphological variability of walking legs (Figure contacts the female and exhibits interest, then moves from 5). Canonical variate analysis revealed that leg shape in the posterior to anterior end of the dorsal side of the female, males significantly varied as a function of mating status tapping with its antennae, after which the pair assumes the (P = 0.0174), but that was not the case in females (P = ventral to ventral position. In the case of Craspedosoma, 0.7503). The first axis in CVA explained 82.26% of shape uncoiling of the female has to happen prior to achieving variation of the analysed trait (Figure 6). The degree of the copulating position. The initiation of contact and

1014 JOVANOVIĆ et al. / Turk J Zool

Figure 2. Mating in P. hungaricus. A – male; B – female; C, D – contact; E, F – extrusion of gonopods (arrowheads); G – copulation (arrowhead) (photo: B. Ilić).

1015 JOVANOVIĆ et al. / Turk J Zool

Figure 3. Positions of mating pairs of P. hungaricus. A – male and female in parallel position; B, C – male coiling around female (photo: B. Ilić).

100 90 80 70 60 50 40 30

Percentage of copulat ons 20 10 0 Female cho ce test Male cho ce test

Figure 4. Percentage of copulations achieved with the previous partner (white bars) and with a new partner (black bars) in female and male choice tests. copulation position are different in Julus scandinavius occur if the female responds positively. In that species, Latzel, 1884 (Haacker, 1969). With the forepart of its body glandular secretion is also included in the courtship ritual: lifted, the male approaches the female, and copulation will the male offers the coxal glands’ secretion to the female.

1016 JOVANOVIĆ et al. / Turk J Zool

Table 2. Percentage of contacts realised between males and females of P. hungaricus.

All contacts (ended with or without copulation) (%) All contacts ended with copulation (%) Type of test Previous partner New partner Contacts ended with copulation Contacts ended without copulation

Female choice test 68 32 71 29

Male choice test 53 47 69 31

Table 3. DC, ECC, and CC of males and females in the mating arena and male/female choice tests, expressed in seconds.

Males Females N Mating arena Male choice test P value N Mating arena Female choice test P value DC 22 3122.54 ± 442.90 3509.23 ± 250.61 0.452 27 3670.93 ± 498.67 3623.00 ± 261.05 0.932 ECC 22 480.04 ± 97.53 835.64 ± 174.88 0.083 27 535.41 ± 106.43 438.07 ± 76.63 0.461 CC 22 221.32 ± 47.36 502.68 ± 102.74 0.017* 27 185.11 ± 19.46 492.70 ± 93.06 0.002*

* Significant P-value.

Table 4. Descriptive statistics of morphological traits and t-tests.

Females Males Morphological FP FN MP MN traits t / P t / P (N = 28) (N = 11) (N = 28) (N = 10) 1.968 ± 0.005 1.966 ± 0.008 1.856 ± 0.005 1.833 ± 0.008 BL (mm) 0.295 / 0.770 0.312 / 0.694 (1.915–2.018) (1.909–2.009) (1.793–1.899) (1.823–1.841) 0.394 ± 0.010 0.369 ± 0.016 0.043 ± 0.010 0.031 ± 0.017 M (g) 1.309 / 0.199 0.588 / 0.560 (0.313–0.487) (0.249–0.446) (–0.032–0.176) (–0.024–0.110) 0.712 ± 0.006 0.700 ± 0.006 0.708 ± 0.004 0.702 ± 0.007 AL (mm) 1.888 / 0.070 1.431 / 0.162 (0.677–0.736) (0.682–0.726) (0.685–0.736) (0.683–0.727) 0.802 ± 0.006 0.791 ± 0.007 0.675 ± 0.005 0.685 ± 0.008 TH (mm) 1.894 / 0.066 0.541 / 0.591 (0.786–0.846) (0.762–0.837) (0.635–0.700) (0.632–0.699) 0.792 ± 0.006 0.780 ± 0.007 0.678 ± 0.006 0.687 ± 0.009 TW (mm) 1.771 / 0.085 0.516 / 0.608 (0.772–0.842) (0.754–0.823) (0.632–0.706) (0.637–0.690) 0.625 ± 0.007 0.630 ± 0.008 0.604 ± 0.006 0.596 ± 0.010 LL (mm) 0.521 / 0.605 1.377 / 0.177 (0.580–0.679) (0.608–0.664) (0.567–0.628) (0.538–0.633)

The data are presented as means ± SE; minimum and maximum values are given in parentheses. Abbreviations: FP = mated females, FN = nonmated females, MP = mated males, MN = nonmated males.

Secretion may also be present when the copulation has Millipedes in the mating arena and choice tests already begun, as in Centrobolus males, where mandibular displayed different levels of activity and willingness to secretion is then observed (Cooper and Telford, 2000). In mate, depending on the time of day. Since our experiments our experiment, secretion during the courtship ritual or were conducted in the laboratory, it is questionable during copulation was not found. whether they reflect the real daily dynamics in nature.

1017 JOVANOVIĆ et al. / Turk J Zool

0.10

14

15 13 25 26 24 23 4 22 1716 2 3 12 11 1 6 5 21 20 1918

78 910

0.00 PC 2 (12%) 14

15 13 25 26 24 23 4 22 1716 2 3 12 1 6 11 5 21 20 1918 78 910 -0.10 -0.30 -0.20 -0.10 -0.00 0.10 0.20 0.30 PC 1 (81.03%)

14

15 13 14 25 26 24 23 15 13 4 22 1716 25 26 2 3 12 24 11 1 6 5 21 23 4 22 1716 20 1918 2 3 12 11 1 6 78 910 5 21 20 1918

78 910

Figure 5. Principal component analysis (PCA) of leg shape in relation with mating status (rectangle: black – nonmated females, grey – mated females; circle: black – nonmated males, grey – mated males).

4 14 1513 252624 4 2223 1716 2 23 12 1 6 21 11 5 20 1918 78 910 0

-2

14

1513 CV2 (13.03%) 252624 -4 4 2223 1716 1 23 6 1112 5 2120 1918 78 910 -6

-8 -6 -4 -2 0 2 4 6 8

CV1 (82.26%)

14 14 1513 25 1513 2624 252624 4 2223 1716 2223 23 12 234 171612 1 6 21 11 1 6 21 11 5 20 1918 5 20 1918 78 910 78 910

Figure 6. Canonical variate analysis (CVA) of leg shape in relation with mating status (rectangle: black – nonmated females, grey – mated females; circle: black – nonmated males, grey – mated males).

Thus far, our field work indicates that these animals are in Millipede mating strategy has been described as a fact active during the daytime, but for the time being, we kind of scrambled competitive polygyny (Telford and have no precise information about the period when they Dangerfield, 1993a; Rowe, 2010; Holwell et al., 2016). are most inclined toward the opposite sex. This strategy designates males as the more active sex.

1018 JOVANOVIĆ et al. / Turk J Zool

3 2 0.06 7

4

8 0.03 )

1 9 6 5

3 2 7

4 0.00 PC2 (18.15 %

8

1 9 6 5 -0.03 -0.06 -0.03 0.00 0.03 0.06 0.09 0.12 PC1 (49.24%) 3 3 2 2 7 7

4 4

8 8

1 9 6 5 1 9 6 5

Figure 7. Principal component analysis (PCA) of gonopod shape in relation with mating status (circle: black – nonmated males; grey – mated males).

Males of P. hungaricus have to spend most of their time in fertilisation. Those authors also stated an alternative searching for mates, while females have to spend most explanation about the evolution of longer copulation, of their time feeding, because they require more energy taking into account the possible advantages gained by for egg production. In the zephroniid Zephronia cf. females in the form of increased reproductive success or viridescens Attems, 1936, feeding was recorded as the fitness. Since we currently have no information regarding main activity of females, while walking was observed as time spent solely in sperm transfer, the relatively long the main male activity (Wongthamwanich et al., 2012). copulation observed in P. hungaricus could be due to some The goal of the males would be to reach as many females kind of mate guarding. as possible (Rowe, 2010), or to ensure the precedence The female and male choice tests, conceived as a first of their sperm by prolonging copulation (Telford and step in finding the basis of mate attractiveness, showed that Dangerfield, 1993a). The observations of P. hungaricus having been a previous partner was an advantage, both in in our study indicate that active searching for mates by regard to the number of initiated contacts and with respect males is present. We observed that contacts leading to to the number of achieved copulations. Female choice copulation were twice as long in both choice tests as in the experiments conducted on the spirostreptid A. uncinatus mating arena. Furthermore, investment in the duration of also revealed more copulations achieved with the first mate copulation could be linked with a possible mate-guarding (Telford and Dangerfield, 1993a). According to data in the strategy (Telford and Dangerfield, 1990; Rowe, 2010). literature, preferences for previous or new mates in both Barnett and Telford (1994) pointed out that this tactic also types of choice tests are different in various arthropods serves the purpose of bringing the male`s sperm closer to (Zeh et al., 1998; Li et al., 2014), and are possibly linked the part of the oviduct where fertilisation occurs—i.e. to with their different mating demands and strategies. the distal connection of the oviduct with the spermatheca, In our study, newly introduced males were often found which would enhance the chance of paternity. Harari et coiled under a leaf or moss and did not respond to the al. (2003) postulated that guarding of the female happens presence of either a female or a male. This kind of behaviour only in species where the last male’s sperm has priority was not exhibited by all new males: on the contrary,

1019 JOVANOVIĆ et al. / Turk J Zool

3.0

2.0 Frequency

1.0

0.0 -4 -2 0 2 4 CV1

3 3 2 2 7 7

4 4

8 8

1 9 1 9 6 5 6 5

Figure 8. Canonical variate analysis (CVA) of gonopod shape in relation with mating status (circle: black – nonmated males; grey – mated males). approximately one-third of all contacts were initiated by hungaricus, significant differences in the number of all an introduced male. Previously mated males exhibited a contacts were detected only in the female choice test. This higher level of activity in both choice tests. In those tests, suggests that the female determines the final decision females accepted the male after one attempt in most cases. about the accomplished copulation. Thus, we can consider the tapping of the female’s head as A question that arises from our study is how males the only clearly distinctive sequence of male courtship. and/or females can discriminate between previous and As what they considered courtship behaviour, Cooper new mates. Previous partners were more successful in and Telford (2000) described 3 sequences of behaviour achieving copulations, but the basis of their higher mating after a male of Spirobolida contacted a female: tapping success remains unknown. In this connection, we need to the head of the female around her ocelli, a mandibular examine multiple stimuli involved in the mating behaviour secretion of the male, and a hydropneumatic rhythm of of P. hungaricus, including olfactory/gustatory profiles or the genital collar. In Typhloblaniulus lorifer consoranensis mechanical/acoustic/tactile stimulation. (= Blaniulus lorifer) (Brölemann, 1921), mating is initiated Morphological characteristics of the body and genital as in P. hungaricus (Mauriès, 1969). On the other hand, structures influence mating and fertilisation success in Rowe (2010) did not recognise any particular courtship insects and millipedes (Telford and Dangerfield, 1993a, behaviour in the polydesmid C. clarum. Courtship 1993b; Adolph and Geber, 1995; Arnquist and Danielsson, behaviour certainly requires various male efforts. The 1999a, 1999b; Burns, 2005; Wenninger and Averill, 2006; strategies used by males to acquire a mate do not have Tanabe and Sota, 2008; Wojcieszek and Simmons, 2011). to be based only on their higher activity and courtship Furthermore, sperm precedence has been previously displays; male persistence during courtship may also be studied in Juliformia (Barnett et al., 1993, 1995), with important. In some insects, males that invest more in emphasis on morphological structures of males that enable courtship display are more successful in mating (Okada them to redistribute their sperm in the female’s oviduct and Hasegawa, 2005; Puniamoorthy et al., 2012). In P. and on utilisation of the received sperm by the female.

1020 JOVANOVIĆ et al. / Turk J Zool

To define relationships between some morphological of information established after the male begins tapping traits and the mating status of the studied individuals the female with its legs also presumably occurs in some (mated or nonmated), we used linear and geometric Opiliones (Machado et al., 2015). morphometrics. These analyses should also give us a basis Gonopod morphology has an impact on mating for understanding female choice, if the morphological success in millipedes (Tanabe and Sota, 2008). Male body traits under study are associated with mating success. size can be important if it is correlated with gonopod size However, analyses of linear measurements showed (Telford and Dangerfield, 1993b). In our sample, it was not no significant differences in morphological traits relating confirmed that gonopod shape and size influence mating to body size (BL and M) or body appendages (AL and success; we can only assume that genital structure affects LL) between mated and nonmated individuals. It was paternity success in our case, as was previously noted in previously noted that nonrandom mating with respect to A. variabilis (Wojcieszek and Simmons, 2011). Certain body size was observed in the spirostreptid A. uncinatus, distinct features of the gonopods might be important for with larger males achieving about 60% of copulations success of males in stimulation of females and/or sperm under laboratory conditions (Telford and Dangerfield, competition. 1993a). This was also observed in the polydesmid In conclusion, this study confirms that the millipede Nyssodesmus python (Peters, 1864) (Burns, 2005; but see species P. hungaricus is characterised by a polygynandrous Adolph and Geber, 1995). Body size, along with genital mating system in which the males are the pursuers, while morphology, determined the success of insemination in the females determine the accomplishment of copulation. the genus Parafontaria Verhoeff, 1936 (Tanabe and Sota, Size and/or shape of the morphological traits (except the 2008). shape of male walking legs) used in our study were not By applying geometric morphometrics, we were able to subject to precopulatory sexual selection. Other sensory separately analyse differences in size and shape of walking domains known to influence courtship behaviour legs and gonopods in mated and nonmated individuals. (olfactory, gustatory, acoustic, mechanical, tactile) need to Our results showed that the size of both traits does not affect be investigated in this connection. Since previously mated mating success in P. hungaricus. Significant difference was partners were more successful in mating than new ones, observed only in the shape of walking legs between mated postcopulatory sexual selection should also be explored in and nonmated males. The length of walking legs could be this millipede species. important in male mate-search efforts, particularly under scramble competition polygyny (Telford and Dangerfield, Acknowledgements 1993b; Rowe, 2010; Baena and Macías-Ordóñez, 2015). This work was supported by the Serbian Ministry of Our results corroborate patterns established in previous Education, Science, and Technological Development studies focused on different insect species (Yang and (Grant No. 173038). We thank the three anonymous Wang, 2004; Lu et al., 2013, and references therein). reviewers for critically reading the manuscript and Considering that the male approaches the female from providing useful comments that improved its content. The the dorsal side, we assume that, during the following male authors are also highly grateful to Mr Raymond Dooley for movements, the female receives and processes some tactile his help in preparing the English version of the manuscript. information about the male’s qualities. This exchange

References

Abràmoff MD, Magalhães PJ, Ram SJ (2004). Image processing with Arnqvist G, Danielsson I (1999a). Postmating sexual selection: the ImageJ. Biophotonics International 11: 36-42. effects of male body size and recovery period on paternity and egg production rate in a water strider. Behav Ecol 10: 358-365. Adolph SC, Geber MA (1995). Mate guarding, mating success and body size in the tropical millipede Nyssodesmus python (Peters) Arnqvist G, Danielsson I (1999b). Copulatory behavior, genital (: Platyrhacidae). Southwest Nat 40: 56-61. morphology, and male fertilization success in water striders. Evolution 53: 147-156. Andrés JA, Cordero Rivera A (2000). Copulation duration and fertilization success in a damselfly: an example of cryptic Barnett M, Telford SR, De Villiers CJ (1993). Sperm displacement female choice? Anim Behav 59: 695-703. in a millipede? An investigation into the genital morphology of the southern African spirostreptid millipede Orthoporus Arnqvist G, Rowe L (1995). Sexual conflict and arms races between pyrhocephalus. J Zool 231: 511-522. the sexes: a morphological adaptation for control of mating in a female insect. Proc R Soc Lond B Biol Sci 261: 123-127. Baena ML, Macías-Ordóñez R (2015). Mobility and mating frequency in the scramble competition polygyny of a chrysomelid beetle. Behav Ecol 26: 416-424.

1021 JOVANOVIĆ et al. / Turk J Zool

Barnett M, Telford SR (1994). The timing of insemination and Ilić BS, Mitić BM, Makarov SE (2016). Sexual dimorphism in its implications for sperm competition in a millipede with Apfelbeckia insculpta (L. Koch, 1867) (Myriapoda: Diplopoda: prolonged copulation. Anim Behav 48: 482-484. Callipodida). Arch Biol Sci: doi:10.2298/ABS160229060I. Barnett M, Telford SR, Tibbies BJ (1995). Female mediation of sperm Jojić V, Nenadović J, Blagojević J, Paunović M, Cvetković D, Vujošević competition in the millipede Alloporus uncinatus (Diplopoda: M (2012). Phenetic relationships among four Apodemus Spirostreptidae). Behav Ecol Sociobiol 36: 413-419. species (Rodentia, Muridae) inferred from skull variation. Zool Anz 251: 26-37. Blower JG (1985). Millipedes. In: Kermack DM, Barnes RSK, editors. Linnean Society Synopses of the British Fauna (New Series), Jones TM, McNamara KB, Colvin PGR, Featherston R, Elgar MA No 35. London, UK: E. J. Brill/Dr. W. Backhuys. (2006). Mating frequency, fecundity and fertilization success in the hide beetle, Dermestes maculatus. J Insect Behav 19: 357- Burns M (2005). The role of size and defensive compounds in mate 371. choice by Nyssodesmus python (Polydesmida: Platyrhacidae). Report. Tampa, FL, USA: University of South Florida. http:// Kawano K (2000). Genera and allometry in the stag beetle family digital.lib.usf.edu/SFS0001374/00001. Lucanidae, Coleoptera. Ann Entomol Soc Am 93: 198-207. Carey CJ, Bull CM (1986). Recognition of mates in the Portuguese Klingenberg CP (2011). MorphoJ: an integrated software package for millipede Ommatoiulus moreletii. Austr J Zool 34: 837-842. geometric morphometrics. Mol Ecol Resour 11: 353-357. Cator LJ, Zanti Z (2016). Size, sounds and sex: interactions between Koch M (2015). Diplopoda: general morphology. In: Minelli A, body size and harmonic convergence signals determine mating editor. The Myriapoda. Vol. 2. Leiden, the Netherlands: Brill, success in Aedes aegypti. Parasite Vectors. doi: 10.1186/s13071- pp. 7-67. 016-1914-6. Lazić MM, Carretero MA, Crnobrnja-Isailović J, Kaliontzopoulou Cooper MI, Telford SR (2000). Copulatory sequences and sexual A (2015). Effects of environmental disturbance on phenotypic struggles in millipedes. J Insect Behav 13: 217-230. variation: an integrated assessment of canalization, developmental stability, modularity, and allometry in lizard Davies NB (1991). Mating systems. In: Krebs JR, Davies NB, editors. head shape. Am Nat 185: 44-58. Behavioural Ecology: An Evolutionary Approach. 3rd ed. Lewis SM, Austad SN (1990). Sources of intraspecific variation in Oxford, UK: Blackwell Scientific Publications, pp. 263-294. sperm precedence in red flour beetles. Am Nat 135: 351-359. Drake AG, Klingenberg CP (2008). The pace of morphological Lewis SM, Austad SN (1994). Sexual selection in flour beetles: the change: historical transformation of skull shape in St Bernard relationship between sperm precedence and male olfactory dogs. Proc R Soc Lond B Biol Sci 275: 71-76. attractiveness. Behav Ecol 5: 219-224. Dugatkin LA (2004). Principles of Animal Behavior. 1st ed. New Li YY, Yu JF, Lu Q, Xu J, Ye H (2014). Female and male moths display York, NY, USA: WW Norton & Company, Inc. different reproductive behavior when facing new versus Eberhard WG (1985). Sexual Selection and Animal Genitalia. previous mates. Plos One 9: e109564. Cambridge, MA, USA: Harvard University Press. Lu W, Wang Q, Tian M, Xu J, Lv J, Qin A (2013). Mating behavior and Eberhard WG (1996). Female Control: Sexual Selection by Cryptic sexual selection in a polygamous beetle. Curr Zool 59: 257-264. Female Choice. Princeton, NJ, USA: Princeton University Machado G, Requena GS, Gadea CT, Stanley E, Macías-Ordóñez R Press. (2015). Male and female mate choice in harvestmen: general Haacker U (1969). An attractive secretion in the mating behaviour of patterns and inferences on the underlying processes. In: a millipede. Z Tierpsychol 26: 988-990. Peretti AV, Aisenberg A, editors. Cryptic Female Choice in Arthropods: Patterns, Mechanisms and Prospects. Cham, Hagler JR, Jackson CG (2001). Methods for marking insects: current Switzerland: Springer, International Publishing, pp. 169-201. techniques and future prospects. Annu Rev Entomol 46: 511- 543. Makarov SE, Ćurčić BPM, Tomić VT, Legakis A (2004). The Diplopods of Serbia, Montenegro and the Republic of Harari AR, Landolt PJ, O’Brien CW, Brockmann HJ (2003). Macedonia. Monographs, Volume IX. Institute of Zoology- Prolonged mate guarding and sperm competition in the weevil Faculty of Biology, University of Belgrade; Hellenic Zoological Diaprepes abbreviatus (L.). Behav Ecol 14: 89-96. Society, Committee for Karst and Speleology–Serbian Helinski MEH, Harrington LC (2011). Male mating history and body Academy of Sciences and Arts: Belgrade–Athens. size influence female fecundity and longevity of the dengue Mathews PL, Bultman TL (1993). Mating behaviour of a parajulid vector Aedes aegypti. J Med Entomol 48: 202-211. millipede, Aniulus bollmani. Can J Zool 71: 2297-2300. Holwell GI, Allen PJD, Goudie F, Duckett PE, Painting CJ (2016). Mauriès, JP (1969). Observations sur la biologie (sexualité, Male density influences mate searching speed and copulation périodomorphose) de Typhloblaniulus lorifer consoranensis duration in millipedes (Polydesmida: Gigantowales chisholmi). Brölemann (Diplopoda, Blaniulidae). Ann Spéléol 24: 495-504. Behav Ecol Sociobiol 70: 1381-1388. Minelli A, Michalik P (2015). Diplopoda: reproduction. In: Minelli Hopkin SP, Read HJ (1992). The Biology of Millipedes. Oxford, UK: A, editor. The Myriapoda. Vol. 2. Leiden, the Netherlands: Oxford University Press. Brill, pp. 237-265.

1022 JOVANOVIĆ et al. / Turk J Zool

Moczek AP, Emlen DJ (1999). Proximate determination of male horn Tadler A (1996). Functional morphology of genitalia of four species dimorphism in the beetle Onthophagus taurus (Coleoptera: of julidan millipedes (Diplopoda: Nemasomatidae: ). Scarabaeidae). J Evol Biol 12: 27-37. Zool J Linn Soc 118: 83-97. Okada Y, Hasegawa E (2005) Size-dependent precopulatory behavior Tanabe T, Sota T (2008). Complex copulatory behavior and the as mate-securing tactic in the Japanese stag beetle, Prosopocoilus proximate effect of genital and body size differences on inclinatus (Coleoptera; Lucanidae). J Ethol 23: 99-102. mechanical reproductive isolation in the millipede genus Parafontaria. Am Nat 171: 692-699. Pavković-Lučić S, Kekić V, Čvoro A (2009). Larger male mating advantage depends on sex ratio in Drosophila melanogaster. Telford SR, Dangerfield JM (1990). Manipulation of the sex ratio Ethol Ecol Evol 21: 155-160. and duration of copulation in the tropical millipede Alloporus uncinatus: a test of the copulatory guarding hypothesis. Anim Puniamoorthy N, Blackenhorn WU, Schäfer MA (2012). Behav 40: 984-986. Differential investment in pre- vs. post-copulatory sexual selection reinforces a cross-continental reversal of sexual size Telford SR, Dangerfield JM (1993a). Mating tactics in the tropical dimorphism in Sepsis punctum (Diptera: Sepsidae). J Evolution millipede Alloporus uncinatus (Diplopoda: Spirostreptidae). Biol 25: 2253-2263. Behaviour 124: 45-56. Rohlf FJ (2008). TpsDig, Version 2.12. Stony Brook, NY, USA: SUNY Telford SR, Dangerfield JM (1993b). Mating behavior and mate at Stony Brook. http://life.bio.sunysb.edu/morph/soft-dataacq. choice experiments in some tropical millipedes (Diplopoda: html. Spirostreptidae). S Afr J Zool 28: 155-160. Rowe M (2010). Copulation, mating system and sexual dimorphism Terzić T, Anđelković M, Stamenković-Radak M (1994). Effect of in an Australian millipede, Cladethosoma clarum. Aust J Zool fluorescent dust, as a marker, on the frequency of mating in 58: 127-132. Drosophila melanogaster. Dros Inf Serv 75: 164-165. Sierwald P, Bond JE (2007). Current status of the myriapod class Wedell N (1991). Sperm competition selects for nuptial feeding in a Diplopoda (millipedes): taxonomic diversity and phylogeny. bushcricket. Evolution 45: 1975-1978. Annu Rev Entomol 52: 401-420. Wenninger EJ, Averill AL (2006). Influence of body and genital Simmons LW, Parker GA (1992). Individual variation in sperm morphology on relative male fertilization success in oriental competition success of yellow dung flies Scatophaga stercoraria. beetle. Behav Ecol 17: 656-663. Evolution 46: 366-375. Wilson HM, Anderson LI (2004). Morphology and Simmons LW, Stockley P, Jackson RL, Parker GA (1996). Sperm of Paleozoic millipedes (Diplopoda: Chilobognatha: competition or sperm selection: no evidence for female Archipolypoda) from Scotland. J Paleontol 78: 169-184. influence over paternity in yellow dung flies Scatophaga Wojcieszek JM, Simmons LW (2011). Male genital morphology stercoraria. Behav Ecol Sociobiol 38: 199-206. influences paternity success in the millipede Antichiropus Sheets HD (2003). IMP: Integrated Morphometrics Package. Buffalo, variabilis. Behav Ecol Sociobiol 65: 1843-1856. NY, USA: Department of Physics, Canisius College. http:// Wongthamwanich N, Panha S, Sitthicharoenchai D, Pradatsundarasar www3.canisius.edu/~sheets/morphsoft.html. A, Seelanan T, Enghoff H, Thirakhupt K (2012). Daily activities Sherratt E, Klingenberg CP, Wilkinson M, Gower DJ (2014). Evolution of the giant pill-millipede Zephronia cf. viridescens Attems, of cranial shape in caecilians (Amphibia: Gymnophiona). Evol 1936 (Diplopoda: Sphaerotheriida: Zephroniidae) in a Biol 41: 528-545. deciduous forest in northern Thailand. Zool Stud 51: 913-926. Shuster S, Wade M (2003). Mating Systems and Strategies. Princeton, Yang LH, Wang Q (2004). Premating sexual selection in Nysius NJ, USA: Princeton University Press. huttoni White (Heteroptera: Lygaeidae) in relation to morphometric traits. J Insect Behav 17: 695-707. Tadler A (1993). Genitalia fitting, mating-behavior and possible hybridization in millipedes of the genus Craspedosoma Zeh JA, Newcomer SD, Zeh DW (1998). Polyandrous females (Diplopoda, , ). Acta Zool discriminate against previous mates. P Natl Acad Sci USA 95: 74: 215-225. 13732-13736.

1023