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Biologia 64/6: 1206—1211, 2009 Section Zoology DOI: 10.2478/s11756-009-0207-3

Sphragis in Parnassius mnemosyne (Lepidoptera: Papilionidae): male-derived insemination plugs loose efficiency with progress of female flight Petr Vlasanek1,2 & Martin Konvicka1,2 1

Faculty of Sciences, University South Bohemia, Branišovská 31, CZ-37005 České Budějovice, Czech Republic Institute of Entomology, Biological Centre of the Czech Academy of Sciences, Branišovská 31, CZ-37005 České Budějovice, Czech Republic; e-mail: [email protected]

2

Abstract: During a mark-recapture study of Parnassius mnemosyne butterfly, we recorded incidence of females bearing or not bearing sphragis, a waxy structure delivered by males during copulation and preventing subsequent inseminations by other males. Despite the common belief that sphragis ensures monopolisation of females, we observed that 74 out of 579 marked females did not bear sphragis at a time of capture. We also recorded five instances of sphragis loss and one female that lost and subsequently re-acquired the structure. A sphragis thus does not represent a reliable indicator of females’ mating status in P. mnemosyne. Repeated inseminations occur in this species and might buffer some extremely small populations against loss of within-population genetic variation. However, the proportion of sphragis-free females increased with season, and it remains to be ascertained to what extend is this caused by sphragis loss in previously inseminated females, and to which this is an outcome of earlier reproductive asynchrony, or early emergence of males that renders some of the late-emerging females unmated. Key words: Apollo butterflies; insect behaviour; Lepidoptera ecology; mating access; sexual selection

Introduction In butterflies with multiple female inseminations, the last male to obtain mating inseminates the majority of subsequent eggs (Ae 1962; Clarke & Sheppard 1962; Labine 1966; Sims 1979; Boggs & Watt 1981). This sperm precedence generates a selection pressure in males for mechanisms preventing subsequent inseminations of once-mated females, and hence monopolising paternity. The mechanisms involve various types of gonopore plugs, produced by the male bodies and delivered to the females during copulation (Labine 1964; Ehrlich & Ehrlich 1978; Orr 1995). A female mated by such monopolising male has few opportunities left. All of her offspring are fathered by a single male, which would considerably reduce her fitness if the monopolist is otherwise low quality, or if a better male appeared subsequently. It follows that any mechanisms that would allow females of such monopolising species to get rid of the gonopore plug left from previous mates would enhance females’ fitness in the evolutionary mating games (Fox & Rauter 2003). Apollo butterflies (genus Parnassius Latreille, 1804) (Lepidoptera: Papilionidae) employ a particularly conspicuous type of mating plug, called sphragis. It is a firm waxy structure derived from male tissues during copulation, attached to ventral side of female’s abdomen and externally preventing other males from apc 2009 Institute of Zoology, Slovak Academy of Sciences 

proaching female’s genital orifices (Orr 1995). Beyond Parnassius, a sphragis also occurs in related Papilionidae genera, e.g., Cressida (Swainson, 1832); Luehdorfia Cr¨ uger, 1878, and in the Nymphalidae genus Acraea F., 1807 (Pierre 1985; Matsumoto 1987). It is generally believed that whereas males of these butterflies can inseminate multiple females during their lifespan, females can copulate only once, except for rare occasions when sphragis is lost during female life (Petersen 1928) or a mating does not result in complete sphragis formation (Matsumoto 1987). How rare these “rare occasions” are has never been quantified with field-collected data. Recently, Calabrese et al. (2008) used data on sphragis incidence in two North American Parnassius species, P. clodius Ménétriés, 1855 and P. smintheus (Doubleday, 1847), to quantify the effect of reproductive asynchrony on population dynamics of butterflies. Many butterflies with discrete generation display conspicuous protandry, emergence of males preceding that of females (e.g., Fagerstr¨om & Wiklund 1982; Néve & Singer 2008). If males live too shortly to inseminate lately appearing females, some females must die unmated. Calabrese et al. (2008) showed that such a “temporal Allee effect” may affect 10–20% of female cohort, diminishing the genetically effective population sizes in small populations of declining species. The Clouded Apollo, Parnassius mnemosyne (L., 1758), is a vulnerable European butterfly depending

Multiple inseminations in Parnassius

1207

161

160

0

159

0 1

158

156

1

157

155

153

152

151

150

149

148

0 1 0 0 0

154

1

147

146

145

1

1 1

144

1

143

0

142

141

139

138

137

136

135

134

133

215 284 410 613 627 570 709 783 867 832 960

140

h p p p p h h p h p h

132

Butterfly code /Julian date

131

Table 1. Observations of the eleven handled Parnassius mnemosyne females that changed their sphragis status during the markrecapture study.

1

1 1 0

1 0

1 1

0 1

0

Explanations: 1 – female bears sphragis; 0 – female without sphragis. Regular letters – relatively fresh female, wing wear 1–2 on a 1–4 scale. Bold letters – worn female, wing wear 3–4.

on open structures within deciduous forests (Konvicka & Kuras 1999; Meier et al. 2005). It is becoming a model species in ecology (Valimaki & Itamies 2005; Ovaskainen et al. 2008) and biogeography (e.g., Luoto et al. 2001; Araújo & Luoto 2007; Gratton et al. 2008). Hoping that incidence of sphragis could provide a reliable marker of females’ mating status, we recorded this information during an intensive mark-recapture study of the largest P. mnemosyne population in the Czech Republic. In agreement with Calabrese et al. (2008), we detected an increasing proportion of sphragis-free females with progressing season. In addition, we detected several instances of sphragis loss. In this paper, we focus on the latter finding, discuss its underlying causes and possible consequences for demographic structure and long-term survival of this species.

Material and methods As a butterfly depending on woodland openings, P. mnemosyne is threatened across C. Europe by forestry intensification. The egg is the overwintering stage; larvae feed in spring on several species of Corydalis spp. The protandrous males appear about a week before females, and seek fresh females with whom they mate for dozens of minutes to a few hours (e.g., Wiklund & Solbreck 1982; Zonneveld & Metz 1991; Zonneveld 1996; Konvicka & Kuras 1999; Konvicka et al. 2001). The sphragis is formed towards the end of mating. We carried out a mark-recapture study between 11 May and 10 June, 2005, in Milovicky wood, SE Czech Republic (48◦ 49 N, 16◦ 42 E, altitude 200–250 m a.s.l.). For each individual handled, we recorded information on locality, time of day, behaviour prior to capture, wing wear, and incidence of a sphragis in females. We used logistic regression to ascertain that sex ratio of females to males increased with marking days. We computed two such regressions, for daily numbers of captured individuals and for daily sex-specific estimates of population sizes. The population size estimates were obtained using the POPAN method for open populations with births and deaths, as provided in program MARK (White & Burnham 1999). Details of the estimates will appear elsewhere (Vlasanek et al., in review); the estimated total numbers for the entire population were 2494 ± 70 (males; N ± SE) and 1585 ± 100 (females; N ± SE).

We then computed the probability of each female to bear a sphragis in successive sampling days, using logistic regression with sphragis presence/absence as the dependent variable and marking day as continuous predictor. We used glm function with binomial distribution of errors (i.e., logit link) in R 2.8.0 (R Development Core Team 2008). We used the Akaike information criterion (AIC) to select between linear and quadratic response of the probability to marking day. We repeated this computation twice, (1) with all marking days and (2) after exclusion of days with incomplete marking effort, defined as days with < 6 females handled, mainly due to unstable weather.

Results We captured and marked 579 females, 74 (12.8%) of which did not bear a sphragis, during 31 marking days; the respective numbers of female handlings were 841 and 100. Out of 180 females captured more than once, the sphragis status changed in eleven (6.1%) (Table 1). Five females (2.8% of total) lost sphragis between captures; the same number attained it (sphragis absent at first capture, present subsequently); and one female lost sphragis between first and second capture and reattained it subsequently. We thus observed sphragis losses in 3.3% of all recaptured females. As expected in a protandrous species, the ratio of females to males increased with time, for both numbers of handled butterflies and population size estimates (Fig. 1). The proportion of sphragis-free females increased with season (Fig. 2). For both all marking days and days with > 5 females handled, both linear and quadratic functions describing the increase fitted the data well, in terms of both deviance explained (D2 ) and the Akaike information criterion (AIC ) (Table 2). The concave quadratic functions, allowing for a high proportion of unmated females early in flight period, a low proportion in a middle flight, and an increase towards end of flight, attained slightly better fit than linear functions, but the ∆AIC between quadratic and linear functions were < 2.0, rendering the fits practically identical. The functions indicated that 30% (linear response function)

P. Vlasanek & M. Konvicka

1208 1.0

s

Females : males ratio

0.8

Capture sex ratio y = exp (-15.821 +0.1015x) / (1+exp(-15.818 +0.1015x))

0.6

0.4

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Estimated sex ratio y = exp(-15.094+ 0.1023x) / (1+exp(-15.094 +0.1023x) 0.0 130

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Julian day Fig. 1. Fitted logistic regression showing changes of daily females : males ratio in a Parnassius mnemosyne population with progression of season. The capture sex ratio is based on individuals handled during a mark – recapture campaign, whereas estimated sex ratio is based on daily population size estimates. Both logistic regressions were highly significant (capture: χ2 = 233.7, df = 1, P < 0.0001; estimated: χ2 = 1442.0, df = 1, P < 0.0001).

Females handled Linear fit 80

Quadratic fit

1

Percentage without sphragis 0.9

70

0.7 50

0.6 0.5

40

0.4

30

0.3

% without sphragis

Number females handled

0.8 60

20 0.2 10

0.1

0

0 131 133 135 137 139 141 143 145 147 149 151 153 155 157 159 161 Julian date

Fig. 2. Numbers of females captured each day (grey columns), percentages of females not bearing sphragis from all females all females handled (black diamonds), and linear and quadratic trend lines, obtained using logistic regression of numbers of handled females bearing and not bearing sphragis against marking days. See Table 2 for the regression equations.

to 50% (quadratic response function) of females present in the population at the end of the flight period did not bear sphragis. Discussion Prior to conducting this mark-recapture study, we expected that the occurrence of females without sphragis would follow a concave pattern generated by a presence of not yet inseminated females early in flight period, plus by a lack of males that would inseminate latelyemerging females. Plus, we expected sphragis-free females to be rather rare, because (i) fresh females remain virgins only for a short time and are rapidly discovered by patrolling males (e.g., Konvicka et al. 2001), and

(ii) as females are less active than males, observing any rare conditions would be near impossible. In line with former expectation, we detected an increase of sphragisfree females lately in the season (as in Calabrese et al. 2008), but we did not detect a clear indication of a concave pattern, probably because fresh females are mated shortly after emergence at the beginning of flight period. Still, we found that over ten per cent of females did not bear sphragis while handled, and that a minimum of 3.3% recaptured females lost this structure during their lifetimes. The latter observation casts a doubt on the reliability of sphragis as an indication of female mating status. The proportion of sphragis-free females was close

Multiple inseminations in Parnassius

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Table 2. Regression models relating numbers of handled P. mnemosyne females not bearing a sphragis to serial number of marking days. Model

Equation (∼ intercept + ax + bx2 ) (± confid. intervals)

Complete data Null −2.00 (±0.209) Linear −18.76 (±5.537) +0.11 (±0.037)x Quadratic −2.16 (±0.273) + 17.26 (±6.721)x + 5.94 (± 6.497)x2 Excluding four days with < 6 handled females Null −2.03 (±0.213) Linear −19.78 (±5.852) + 0.12 (±0.039)x Quadratic −2.20 (±0.242) + 17.47 (±6.796)x + 6.34 (±6.641)x2

df

D2

∆ AIC

840 1,839 2,838

0.064 0.069

38.3 0.9 0.0

825 1,824 2,823

0.067 0.072

39.0 1.1 0.0

Explanations: D 2 – Deviance in the data explained by the model; ∆ AIC – the difference in value of the Akaike information criterion between best-fitting and alternative models.

to 0.5 towards the termination of flight period, exceeding the proportion of 0.3 observed by Calabrese et al. (2008) for Parnassius smintheus. The authors attributed the high proportion of sphragis-free (putatively uninseminated) females to the conspicuous protandry in Parnassius populations (e.g., Scott 1973; Matsumoto 1985; Konvicka & Kuras 1999; Auckland et al. 2004; Vlasanek et al., in review), due to which some late emerging females never encounter a mate. In our population, females indeed prevailed over males during last days of flight (Fig. 1), and some of them could indeed had been unmated. Due to sphragis losses, however, the proportion of sphragis-free females combines uninseminated females with inseminated females that lost their sphragises. The relative representation of the two groups remains unknown. For sphragis losses, the observed 3.3% of recaptured individuals is certainly an underestimate, because the duration of handling of each captured female is extremely short relative to her lifespan, whereas sphragis loss can occur any time. In this moment, we have no indication of a true magnitude of this phenomenon. How and when sphragis loses occur remains to be investigated, but in one female (code “p 410” in Table 1), we observed gradual deterioration of the structure. We do not know whether females actively assist in sphragis removal, or whether the structure simply wears out in time. The effect on female, however, is identical: sphragis loss opens new mating opportunity. The single female that lost its sphragis and reattained it subsequently documents the existence of multiple insemination in P. mnemosyne. It supports the conjecture of Petersen (1928) that sphragis does completely prevent subsequent inseminations in butterflies. Petersen discovered multiple spermatophores in female copulatory organs of two Parnasssius spp., the European P. mnemosyne and Central Asian P. delphius Eversmann, 1843. In contrast, Matsumoto (1987) studied sphragis incidence in the Parnassinae species Luehdorfia japonica Leech, 1889, finding only single spermatophores (i.e., single mating) in 31 dissected females. He was cautious about his conclusions, however, noting that it was based on a low number of individuals. Similarly, Scott (1973) dissected female genitalia of “about twenty” females of “Parnassius phoebus (F., 1793)” [in fact P. smintheus (Doubleday, 1847); cf.

DeChaine & Martin (2005)], also detecting just single spermatophores. Multiple matings can increase female fitness either by allowing her to produce more eggs or by increasing her longevity (Wiklund et al 1993, 1998; LaMunyon 1997; Karlsson 1998). It also enhances the genetic diversity of populations, which can have positive fitness consequences (Adamski & Witkowski 1999; Hanski & Saccheri 2006; Vandewoestijne et al. 2008). In contrast, Kawagoe et al. (2001) observed harmful effects of multiple mating on females of Asian papilionid butterfly Atrophaneura alcinous (Klug, 1836). A second mating, which occurs shortly after the first one and involves penetration through not yet hardened sphragis, significantly decreased female longevity, probably due to a physiological damage to the female. Lifetime numbers of eggs laid per female remained identical as in females mated just once, however, perhaps owing to a slight increase of daily eggs intake. The authors concluded that second mating may be harmful to butterflies if it occurs shortly after the first mating, but becomes beneficial if some time elapses between copulations. Instances of second mating shortly after the first one are unlikely in P. mnemosyne, because it takes days before a fully-formed sphragis disintegrates. Hence, the beneficial effects of (postponed) multiple mating likely prevail. Alternatively, the males that survive towards end of adult flight may have depleted their body resources, producing incompletely developed sphragis during belated mating (Pierre 1985; Matsumoto & Suzuki 1992; M.S. Fred, personal communication). Matsumoto (1987) observed a Luehdorfia japonica Leech, 1889 female bearing two sphragises, one normal and one incompletely developed. Pierre (1985) reported, for Acraea butterflies (Nymphalidae), that if a female mates immediately after her first copulation, when the first sphragis has not solidified yet, the second male can push the sphragis away from her genital apertures. The situation in Acraeinae nymphalids may differ greatly from Parnasssiinae papilionids, as the use of sphragis likely evolved independently in these two clades, but it is possible that active removal of a sphragis by males could occur more easily if the earlier sphragis was incompletely developed. In any case, multiple insemination occurs in P. mnemosyne, and is probably more likely later in the

1210 flight season. It seems to be quite rare, though, because males become scarcer with season’s progression. Another factor in play is female longevity. The estimated average female longevity in our population was 5.5 days (Vlasanek et al., in review), similar to other Parnassinae species (e.g., Scott 1973; Matsumoto 1985; Kawagoe et al. 2001), but our re-mated female “h 215” needed a minimum of eleven days to loose and re-acquire its sphragis (Table 1). Even if rare, multiple inseminations likely contribute to the maintenance of within-population genetic diversity, compared to strict monogamy. Although the population genetics effects may be minuscule in large and apparently viable populations such as the one discussed here, they may contribute to persistence of P. mnemosyne populations that are small, isolated and genetically impoverished (Kudrna & Seufert 1991; Descimon & Napolitano 1993; Meglecz et al. 1997, 1999). In addition, the fact that some sphragises are lost during female lifetimes suggest that although the Parnassius protandry leaves some late females mateless, the proportions of actually uninseminated females may be less dramatic than estimated by Calabrese et al. (2008). Further research should focus on quantifying the relative importance of sphragis absence, sphraigs loss and reproductive asynchrony. Acknowledgements We thank D. Hauck and J. Benes for help in field, and to Zidlochovice Forest Enterprise, part of the Czech National Forests Inc., for access to Milovicky wood and permission to use a hunting lodge as our field base. The research was supported by Czech Ministry of Education (MSM6007665801, LC06073) and the Grant Agency of the Czech Republic (206/08/H044).

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1211 Sims S.R. 1979. Aspects of mating frequency and reproductive maturity in Papilio zelicaon. Am. Midl. Nat. 102: 36–50. Valimaki P. & Itamies J. 2005. Effects of canopy coverage on the immature stages of the Clouded Apollo butterfly [Parnassius mnemosyne (L.)] with observations on larval behaviour. Entomol. Fenn. 16: 117–123. Vandewoestijne S., Schtickzelle N. & Baguette M. 2008. Positive correlation between genetic diversity and fitness in a large, well-connected metapopulation. BMC Biol. 6: 46. DOI 10.1186/1741-7007-6-46. White G.C. & Burnham K.P. 1999. Program Mark: survival estimation from populations of marked animals. Bird Study 46 (Suppl.): 120–139. Wiklund C. & Solbreck C. 1982. Adaptive versus incidental explanations for the occurrence of protandry in a butterfly, Leptidea sinapis L. Evolution 36: 56–62. Wiklund C., Kaitala A., Lindfors V. & Abenius J. 1993. Polyandry and its effect on female reproduction in the greenveined white butterfly (Pieris napi L.). Behav. Ecol. Sociobiol. 33: 25–33. DOI 10.1007/BF00164343 Wiklund C., Kaitala A. & Wedell N. 1998. Decoupling of reproductive rates and parental expenditure in a polyandrous butterfly. Behav. Ecol. 9: 20–25. DOI 10.1093/beheco/9.1.20 Zonneveld C. 1996. Being big or emerging early? Polyandry and the trade-off between size and emergence in male butterflies. Am. Nat. 147: 946–965. DOI 10.1086/285887 Zonneveld C. & Metz J.A.J. 1991. Models on butterfly protandry – virgin females are at risk to die. Theor. Popul. Biol. 40: 308–321. DOI 10.1016/0040-5809(91)90058-N Received February 4, 2009 Accepted July 12, 2009