Journal of Ethology (2019) 37:21–29 https://doi.org/10.1007/s10164-018-0562-z

ARTICLE

A nudibranch removes rival sperm with a disposable spiny penis

Ayami Sekizawa1,2 · Shin G. Goto1 · Yasuhiro Nakashima3

Received: 23 January 2018 / Accepted: 21 September 2018 / Published online: 27 October 2018 © The Author(s) 2019, corrected publication 2019

Abstract Simultaneous hermaphroditism is, at least initially, favoured by selection under low density — and therefore it can be assumed that sperm competition has little importance in this sexual system. However, many simultaneously hermaphroditic nudibranchs have both an allo-sperm storage organ (the seminal receptacle) and an allo-sperm digesting organ (the copu- latory bursa), suggesting the possibility of the occurrence of sperm competition. A nudibranch, Chromodoris reticulata, autotomizes its penis after every and replenishes it within about 24 h to perform another copulation. We observed that the surface of the autotomized penis was covered with many backward-pointing spines and that a sperm mass was often entangled on the spines. This suggests that the nudibranch removes sperm that is already stored in a mating partner’s sperm storage organ(s) with its thorny penis. Using six microsatellite markers, we determined that the sperm mass attached to the penis were allo-sperm originating from individual(s) that had participated in prior copulations. We revealed that C. reticulata performed sperm removal using the thorny penis. These results suggest that competition in fertilization is quite intense and mating frequency in the wild is relatively high in this species.

Keywords Sexual selection · Sperm removal · Sperm competition · Nudibranch · Simultaneous hermaphrodite

Introduction et al. 2005; Kupfernagel et al. 2010), suggesting that sperm competition occurred in these hermaphroditic animals. It was originally thought that simultaneous hermaphrodit- These fndings indicate that sexual selection functions in ism was favoured by selection under low density, such as simultaneous hermaphrodites as strongly as in gonochorists, in deep-sea fshes, or low mobility, such as snails, and that if not more strongly. Furthermore, mating behaviours, such sperm competition had little importance in simultaneous as penis fencing in fatworms (Michiels and Newman 1998), hermaphrodites (Ghiselin 1969). However, some simul- cephalo-traumatic secretion transfer in head-shielded slugs taneous hermaphrodites are known to live in high density (Lange et al. 2013, 2014), and disposable penises in nudi- populations and show complicated mating behaviour (Fis- branchs (Sekizawa et al. 2013), show that some simultane- cher 1980, 1981: egg trading in hamlets; Leonard et al. 2007: ous hermaphrodites undergo severe competition to acquire phally polymorphism in banana slugs). Moreover, multiple the chance to inseminate, and suggest that they expose them- paternity was confrmed in some simultaneously hermaph- selves to sperm competition. Additionally, some simultane- roditic molluscs (Baur 1994; Angeloni et al. 2003; Evanno ously hermaphroditic invertebrates have an organ(s) to digest and absorb sperm received from mating partners (Baur 1998; Michiels 1998). Together, these characteristics indicate the * Ayami Sekizawa existence of intense sperm competition among simultaneous aym‑[email protected] hermaphrodites as well as in gonochorists. All nudibranchs are simultaneous hermaphrodites. Dur- 1 Department of Biology and Geosciences, Graduate School of Science, Osaka City University, 3‑3‑138 Sugimoto ing mating, they simultaneously play “male roles” as sperm Sumiyoshi‑ku, Osaka‑shi, Osaka 558‑8585, Japan donors and “female roles” as sperm recipients, in princi- 2 Present Address: Graduate School of Agricultural Science, ple. The morphology of their female is Tohoku University, 468‑1 Aramaki Aza Aoba, Aoba‑ku, complicated, with two sperm storage organs, the copulatory Sendai 980‑0845, Japan bursa and the seminal receptacle. Hermaphroditic gastro- 3 Nihon University, College of Economics, 3‑2‑1 pods are generally thought to be able to digest allo-sperm Kanda‑Misakicho, Chiyoda‑ku, Tokyo 101‑8360, Japan

Vol.:(0123456789)1 3 22 Journal of Ethology (2019) 37:21–29 stored in the copulatory bursae (Brandrif and Beemen 1973; studies in insects. Subsequently, this showed direct evidence Dillen et al. 2009; Lind 1973). The morphology, arrange- of sperm competition by sperm removal. We propose that ment, and connectivity of the copulatory bursa and seminal sexual selection in this simultaneously hermaphroditic nudi- receptacle are highly variable among nudibranch species branch functions as strongly as that in gonochorists. (Rudman 1984; Valdés et al. 2010). This rich diversity in female reproductive systems implies that mating strategies vary among species and intense sexual selection operates, Materials and methods for example, in the form of sperm competition. Although reproductive system morphology is relatively well studied in Study animals nudibranchs, the function of each reproductive organ is not entirely clear. Additionally, direct evidence of sperm compe- The study animal, C. reticulata (Quoy and Gaimard 1832) tition has not yet been obtained in nudibranchs (Baur 1998). (Opisthobranchia, Nudibranchia, Chromodoridae), is dis- Chromodoris reticulata is reported to autotomize its penis tributed in the Indo-West Pacifc. In Japan, C. reticulata after every copulation and replenish it within about 24 h in occurs on the rocky coast from central to southern Hon- order to perform another copulation (Sekizawa et al. 2013). shu and around the Ryukyu Islands. This nudibranch has a The fact that this nudibranch has a mechanism to repeat reticulated network of red lines over the surface of its man- copulation within a short period of time with disposable tle, reaches a length of 60 mm, and feeds on sponges (Gos- penises indicates their high mating frequency. The autoto- liner et al. 2008). The reproductive season of this species mized penis is covered with many backward-pointing spines ranges from spring to early summer, and individuals on its surface. It is known that simultaneously hermaphro- ribbon-shaped, yellow egg masses. As is typical of most ditic nudibranchs have a wide variety of penis morphologies, opisthobranchs (Baur 1998), C. reticulata is a non-selfng, including hooks at the tip and spines on the surface (Valdés simultaneously hermaphroditic nudibranch. The allo-sperm et al. 2010). These hooks and spines are supposed to provide received from the mating partner is stored in the copulatory anchorage to prevent the penis from being unplugged from bursa and seminal receptacle (Rudman 1984). C. reticulata the mating partner during copulation (Valdés et al. 2010). exchanges sperm reciprocally during copulation in principle. However, the exact function of these projections on the penis has not been understood. Since a sperm mass was often Collection and maintenance entangled in the spines on the surface of an autotomized penis after copulation in C. reticulata (Sekizawa et al. 2013), The animals used in this study were collected using SCUBA this nudibranch has been proposed to scrape out allo-sperm equipment during their reproductive season, between April already stored in the allo-sperm storage organ(s) of the mat- and May of 2012, in the shallow coral reefs (2–7 m depth) ing partner with its thorny penis. in the vicinity of the Sesoko Station of the University of the The genetic diversity of stored sperm in female sperm Ryukyus, Okinawa, Japan (26°38′N, 127°52′E). Collected storage organs (Siva-Jothy and Hooper 1995), multiple animals were kept individually in a perforated small plastic paternity of a clutch of ofspring, and last male precedence case sunk in a seawater tank. The tank was submerged in in reproductive success (Cooper et al. 1996) have been stud- running seawater and aerated. We did not feed the nudi- ied in insects using DNA analysis. These results suggest branchs, as their food consumption decreases substantially many insects perform sperm displacement by sperm removal during their reproductive season (Thompson 1966). (Córdoba-Aguilar et al. 2003; Kamimura 2000, 2003). Fur- thermore, Takami (2007) showed that spermatophore dis- Repeated copulation experiment placement was highly probable by DNA analysis of a sper- matophore placed in the female genitalia of a ground beetle. We designated fve copulatory groups, each consisting of However, verifcation of sperm removal by DNA analysis of four individuals, and put two individuals into a clear, acrylic scraped out sperm has never been carried out in any animal frame experimental tank measuring 27 × 9 × 6 cm (length, to our best knowledge, including gastropods and the well- breadth, height: 1458 ml) for copulation. The other pair of studied damselfies. each group was also permitted to copulate. The mating tri- In this study, we clarifed, by DNA analysis using micro- als were performed 3 times per animal with each of three satellite markers, that the sperm mass attached to the autoto- diferent partners within the mating group, with an interval mized penis in C. reticulata originated, at least in part, from of 2–3 days between each mating trial. We observed a total allo-sperm removed from the copulatory pouch(es) of the of 30 copulations. After each copulation, animals were kept sperm recipient, and certifed that the nudibranch performed individually in small plastic cases until the next trial, and the sperm removal. We compared the traits and mechanisms of seawater in the experimental tank was replaced to prevent sperm displacement in this nudibranch to those of previous sperm contamination. We succeeded in collecting 36 out

1 3 Journal of Ethology (2019) 37:21–29 23 of the 40 sperm mass samples after the second and third mating partner(s). Among them, we classifed the same alleles copulations. We graded the amount of sperm attached to as those detected from experimentally copulated individuals the autotomized penis into three ranks (Fig. 1a), collected (recipient’s ex-partner or ex-ex-partner) as E alleles, and those each sperm mass with tweezers, and preserved them in 90% alleles not detected from all four individuals within each ethanol. We also cut of a part of the mantle (approximately experimental group as W alleles. W alleles have a possibility 20 mg) of each individual and preserved it in 90% ethanol, of being derived from individuals with which the study ani- after all of the copulation trials. mals copulated in the wild before capture. In case only some but not all the alleles of the donor/recipient were detected, DNA analysis these were not assigned to either D, R, E, or W alleles.

We compared the genotypes of sperm donors and recipients and alleles of sperm samples with six microsatellite markers: Results sara 10, 18, 20, 44, 45, and 59 (Sato et al. 2011). We extracted DNA from the mantle of each adult animal and from each We identifed the alleles of the sperm mass attached to the sperm mass following the Sato et al. (2011) method. We iden- backward-pointing spines on the surface of autotomized tifed the alleles of the six microsatellite markers in the DNA penises after the second and third copulations of the repeated samples (Sato et al. 2011) using a 3130 Genetic Analyzer copulation experiments. We also identifed the alleles of (Applied Biosystems) and Gene Mapper 4.0 (Applied Bio- the sperm donor and recipient, respectively. The number systems), and used CERVUS 3.0 (Kalinowski et al. 2007) to of alleles, observed and expected heterozygosities, and null calculate the observed and expected heterozygosities and null allele frequencies of the donors and recipients are shown allele frequencies. When all the donor’s alleles were found in Table 1. Negative values for the null allele frequency in a sperm mass in all of the six markers, these alleles were estimates imply that the observed heterozygote genotype judged to be derived from the donor (D). When all the recipi- exceeded the statistical expectation (Marshall et al. 1998). ent’s alleles were found in a sperm mass, these alleles were Six microsatellite markers successfully distinguished each judged to be derived from the recipient (R). If the alleles do individual from the others. not correspond to the alleles of the recipient or donor, they are It is noteworthy that the number of alleles detected in the possibly derived from the sperms of the recipient’s previous sperm samples was generally larger than that detected in the donors and recipients (Table 1). Moreover, we detected three or more alleles in each locus from each of the sperm samples a (Table 2). That is, the sperm mass was a mixture of sperm from multiple individuals. In 24 samples, the same alleles as s p the donor were detected at all of six loci used in the experi- ments, and thus, the alleles were possibly derived from the donor (Table 2). Similarly, the same alleles as the recipient ++++++ b were detected in three samples, and thus, the alleles were 16 possibly derived from the recipient (Table 2). In 21 samples, 14 the same alleles as the ex- or ex-ex-partner were detected at some, but not all of the six loci (allele­ E, Table 2). For exam- 12 ple, the sperm mass sample of Fsp2 was collected from the 10 penis of individual F after the second copulation trial. In donor 8 this second copulation trial, F’s mating partner was B that case s donor + recipient

. of had copulated with A in the frst copulation trial. Therefore, 6 no previous + donor + recipient there is some possibility that B received and stored sperm 4 previous + donor from A in its allo-sperm storage organs in the frst copula-

2 previous tion trial, and then F removed the sperm originally from A from B’s allo-sperm storage organs in the second copulation 0 ++++++ trial. We assumed that allele 149 in locus sara 20 of ‘Fsp2’ grades of amount of sperm mass originated from A and thus indicated it as ­alleleE (previously copulated individual). However, it is also possible that the Fig. 1 Amount and origin of sperm mass attached to an autotomized allele is derived from an unknown wild individual. There- penis after copulation. a Schematic presentation of three grades of the fore, we could not specify the origin of the sperm. In 23 amount of sperm mass. p penis, s sperm mass. b Detected allele pat- terns in each sperm mass. “Previous” indicates the sperm mass con- samples, some alleles were not identical with any alleles of tained ­alleleE or ­alleleW (see Table 2) individuals of the experimental group. We thought that those

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Table 1 Characterization of Locus Individuals (n = 20) No. of alleles H H Null allele six microsatellite loci from O E n frequency Chromodoris reticulata Sperm masses ( = 36) sara10 Individuals 9 0.850 0.860 − 0.008 Sperm masses 11 sara18 Individuals 19 0.650 0.958 0.179 Sperm masses 21 sara20 Individuals 18 0.750 0.947 0.102 Sperm masses 20 sara44 Individuals 22 0.450 0.972 0.355 Sperm masses 23 sara45 Individuals (n = 19)a 17 0.474 0.954 0.327 Sperm masses 18 sara59 Individuals 5 0.350 0.579 0.249 Sperm masses 5

CERVUS 3.0 (Kalinowski et al. 2007) was used to calculate the observed and expected heterozygosities and null allele frequencies. Negative values for null allele frequency estimates imply an observed heterozy- gote genotype in excess of statistical expectation (Marshall et al. 1998)

HO observed heterozygosity, HE expected heterozygosity of individuals a In the locus of sara45, we could analyse only 19 samples samples with alleles diferent from the donor or the recipient mass attached to the autotomized penis, we detected alleles had a possibility of originating from the recipient’s previous that originated from the recipient’s previous partner(s) partner prior to collection ­(alleleW, Table 2). (Fig. 1) This result demonstrates sperm removal in C. retic- In the results of the genotype and allele comparison of ulata, in which allo-sperm already stored in the reproduc- sperm masses, donors and recipients, alleles from neither tive pouch(s) of a mating partner was scraped out using the donors nor recipients were detected in 28 sperm masses thorny penis. It is well known that some damselfies remove [sperm masses including alleles of the recipient’s previous sperm from the copulatory pouch of mating partners (Cór- partner(s) only (previous), n = 12; alleles of recipient’s pre- doba-Aguilar et al. 2003). However, sperm displacement in vious partner(s) and alleles of donor, n = 15 (donor + previ- damselfies was evaluated based on increases and decreases ous); alleles of the recipient’s previous partner(s) and alleles in the amount of stored sperm (Córdoba-Aguilar et al. of donor and recipient (donor + recipient + previous), n = 1; 2003). Direct evidence of sperm removal by DNA analysis Table 2]. By contrast, the alleles of the recipient’s previous of scraped-out sperm has not yet been shown in animals partner(s) were not detected in eight sperm masses [sperm reported to perform sperm displacement (Kamimura 2005). mass including alleles of donor only (donor), n = 6, donor The manner of sperm removal in C. reticulata difered and recipient (donor + recipient), n = 2; Table 2]. from that in damselfies. Some damselfies such as Calop- Some samples contained sperm of the recipient’s previous teryx maculata are known to use a hook-shaped structure at partner(s), irrespective of the grade of the amount of sperm the tip of the appendage of male copulatory organ to remove attached to the autotomized penis (n = 28, Fig. 1b). Sperm allo-sperm already stored in the copulatory pouch (Waage from the recipient’s previous partner(s) was detected from all 1979; Cordero and Miller 1992; Miller 1987). Damselfies the sperm mass samples of grade ‘+’ (n = 15, Fig. 1b). Some removed allo-sperm with a voluntary movement by the sperm mass samples of grade ‘++’ and ‘+++’ did not contain tergo-sternal muscle that is associated with a hook-shaped sperm of the recipient’s previous partner(s) (n = 8, Fig. 1b). appendage (Córdoba-Aguilar et al. 2003). Unlike the hook- All of the samples that lacked the sperm of the recipient’s shaped appendage in damselfies, the disposable penis in previous partner(s) contained the sperm of the donor. C. reticulata does not have a retractor muscle (Sekizawa et al. 2013), so it appears impossible to move each spine in a controlled manner. It is plausible that they insert their Discussion penis deeply into the copulatory pouch of the mating partner, and ejaculate their own sperm at the innermost part, then In this study, we obtained the frst proof of sperm removal entangle allo-sperm with backward-pointing spines on the from the reproductive or copulatory organ by DNA analysis swollen tip of the penis, pull out the penis entangled with of scraped-out sperm. In 28 out of the 36 samples of sperm allo-sperm, and fnally autotomize the penis after copulation.

1 3 Journal of Ethology (2019) 37:21–29 25 ss

previous previous previous ma r r r r m evious rvious evious evious evious evious evious evious Origin of sper Dono Donor + Dono Donor + Donor + Dono Dono Pr Pe Pr Pr Pr Pr Pr Pr D D D 23 23 23 21 12 3 12 3 12 3 12 3 12 3 12 3 12 3 12 3 D D D D W D W D D 19 19 23 01 19 sara59 12 3 12 3 11 91 10 11 11 9 11 9 10 1 12 11 12 3 12 3 12 3 12 3 12 3 12 3 12 1 11 91 12 3 12 3 30 21 W 27 6 D D E W D D -1 42 46 02 76 62 54 28 01 28 01 28 0 28 0 28 0 28 01 30 2 30 2 D D D W D D W -- -- 44 42 42 32 62 46 54 sara45 23 22 23 42 24 63 26 22 24 4 24 4 24 02 24 2 23 8 24 6 24 62 24 8 24 6 W 52 27 W .2 23 7 22 32 21 22 D W W D D E W 19 31 19 19 41 20 6 22 3 21 92 22 7- 19 6 24 1 24 1 22 7 20 9 21 92 D W D D E D W E D D W E 94 87 96 01 87 sara44 22 32 22 3 20 1 23 1 23 1 18 7 20 1 20 1 19 6 20 22 23 12 19 42 20 22 18 72 20 12 19 4 22 72 21 22 W D D W 16 7 16 11 16 71 16 31 19 0 15 3 16 7 15 62 16 71 D D D D D W 149 167 153 149 149 167 149 149 153 143 153 153 153 153 163 163 149 161 161 140 D D D W E D D W E sara20 143 161 143 136 143 153 156 140 149 149 149 136 153 143 143 143 136 143 149 156 156 136 136 W 340 D 320 325 D D D D D D W E 325 320 317 325 335 345 325 297 317 317 304 345 345 301 320 320 276 276 D W D D W D D D W W -- sara18 297 304 276 335 297 308 320 297 286 276 276 320 320 320 282 320 297 265 304 265 286 308 D D D W 212 212 212 208 208 208 212 200 W W W D W W 200 200 208 200 204 200 194 208 198 212 D D D W W W W E D D E 212 208 208 194 212 196 208 194 194 192 200 208 194 194 194 194 192 194 194 208 212 s D W D D W W W W E E D D W Locu sara10 196 196 208 188 190 192 190 212 194 188 204 192 192 192 192 190 192 194 192 192 182 190 194 me le na sp3 sp2 Asp2 D Fsp3 B Bsp3 I J Asp3 Dsp3 F Csp3 I Csp2 Bsp2 Dsp2 Fsp2 I Jsp3 Lsp2 Lsp3 L mp Sa ×F ×F ×D ×J ×L

J× L 䠥 JI FB BD LI DB 1A 2C Alleles of individuals and sperm masses C× C× A× A× C× A× iring of each grou p Pa Group 1 1 3 2 2 3 Group 2 Table

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previous previous previous previous previous previous previous previous previous ma + r m evious evious evious evious Origin of sper Donor + Donor + Donor + Donor + Donor + Donor + Pr Pr Pr Donor + Donor + Dono Pr Donor D 123 D D 25 12 3 12 3 12 3 12 5 D 123 123 123 121 123 D E D D 01 01 23 23 12 3 10 1 12 3 10 1 12 3 12 3 12 31 12 3 D D D D D 121 123 sara59 123 121 121 119 123 119 123 119 W 266 D E D D 254 92 92 66 26 61 28 0 28 0 26 6 29 21 28 01 D D D D 304 240 304 304 241 254 D D D W D -- 56 56 28 42 25 4 26 6 25 6 25 6 28 42 25 62 26 61 26 6 D D E D D E 240 234 sara45 254 234 234 240 254 234 254 241 W W D 235 289 289 22 7 W E W W D D D D D 17 17 06 17 29 289 235 209 231 225 235 27 26 6 22 9 22 7 D D D D D E W D D 198 225 sara44 225 198 198 215 215 198 215 235 20 12 20 12 19 62 21 7 19 62 19 6 20 2 26 62 20 12 19 62 20 62 20 6 D D 165 165 E D D 147 147 147 147 W D D D D D D E 163 167 161 167 161 141 165 147 147 130 136 140 147 140 147 D D E D W D D D E D 145 145 147 147 138 141 147 147 145 138 141 126 128 130 130 128 128 136 136 136 sara20 140 136 D 335 D 325 325 317 345 D D D D D D E E D 294 297 325 294 294 325 294 297 297 335 317 317 335 317 302 333 302 345 D D W D D D D D D E 282 276 286 282 280 280 325 280 282 325 276 276 302 276 sara18 276 276 302 286 286 317 286 333 D D 208 208 208 208 E W D W 200 200 200 200 208 E W E E W 200 200 208 194 196 200 194 194 200 D D W W D E W D D D D D E 192 192 196 194 194 200 194 196 208 200 192 192 194 208 192 208 194 194 194 192 208 208 D D E E D s D D D D D D 188 192 190 190 188 192 188 190 190 190 190 190 192 192 Locu 192 192 188 188 sara10 192 192 192 188 me le na Ksp2 Msp2 Nsp2 Msp3 Nsp3 Ksp3 K N M Gsp3 Gsp2 Psp2 P Osp2 Qsp2 Rsp3 Psp3 Q Qsp3 R mp Sa ×K ×M ×M ×P ×Q ×Q

KN MN NK PR RP QR 3G (continued) 4O G× G× G× O× O× O× iring of each 3 2 grou p Pa Group 1 3 2 Group 1 2 Table

1 3 Journal of Ethology (2019) 37:21–29 27 previous + t t

ass previous previous previous m m Origin of sper Donor + recipient Donor + recipien Donor + Donor + Donor + recipien Donor Donor + D 12 3 represent the represent same alleles as the E R DR D D 12 3 11 9 12 3 11 9 11 9 11 9 represents a sperm represents the to mass attached 2 DR D D D DR D D 01 23 01 sara5 9 10 1 10 1 10 1 10 1 12 3 12 3 12 3 12 3 D R D 24 8 30 4 30 4 30 41 R D D D R D D 44 04 24 81 sara45 24 0 24 8 24 0 24 0 24 4 30 4 24 4 24 01 R D D R 25 2 25 2 25 2 24 1 E R E E R D 17 21 9 21 7 21 7 21 9 24 1 24 12 D D E D D D 02 19 52 sara44 20 2 20 2 20 22 20 2 21 7 21 7 21 9 21 73 R D 16 1 16 1 R D 15 6 15 6 DR D R D D 61 14 72 14 7 14 7 15 1 16 1 15 1 15 12 represents a sperma represents aftermass theindicate the DNA; amplifed of pairs base third Numbers copulation. 3 D D D D R D D 43 47 32 sara20 14 3 14 3 14 7 13 2 15 6 13 2 14 72 15 61 D D 33 1 32 3 D D E R D 33 11 32 3 33 1 32 31 32 3 30 4 30 4 30 41 R DR D D R D D 23 94 90 23 29 4 32 3 29 4 29 4 29 0 32 3 29 0 sara18 D represents the represents same allele as the donor in the sperm the in all six loci. same allele pattern detected mass in which as the donor was 08 D R D 21 2 21 2 20 02 represents alleles seen in none of the four individuals in a group of successive copulation experiments. “Previous” indicates the sperm “Previous” copulation experiments. mass individuals in a group of successive alleles seen in none of the represents four W R R R D 20 8 21 2 20 8 19 6 E R D D D D W 20 0 20 0 21 2 21 2 20 0 21 2 19 4 21 22 20 82 21 23 s D D D D R D W 08 96 00 Locu 19 03 19 0 20 8 19 6 19 2 sara10 20 8 20 0 19 0 me W le na Ssp2 Tsp2 Usp2 Wsp2 T2 W1 Tsp3 U2 Usp3 Ssp3 mp Sa allele or ­ E ×U ×U ×W

­ allele represents the represents same allele as the in the recipient sperm in all six loci. Allele(s) the detected same allele pattern mass in which as the was recipient TW UT WT R (continued) 5S S× S× S× iring of each grou p Pa Group 1 3 2 – indicates that the sample was not amplifed. Allele(s) amplifed. not – indicates that the was sample 2 Table group the is surrounded Pairings experiments. of each - sp with a square. name indicate 20 individuals used for sample on each characters Alphabetic autotomized penis after the second copulation of successive copulation experiment, - sp experiment, copulation afterpenis the autotomized successive of copulation second Allele(s) contained recipient’s ex-partner or ex-ex-partner. Allele(s) ex-partner or ex-ex-partner. recipient’s

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No alleles of the recipient’s previous partner(s) detected and functional inconveniency may have made C. reticulata in the removed sperm mass was coincident with that of the develop a cheap and fragile penis and dispose of it, rather recipient’s ex- or ex-ex-partner used in the mating experi- than a robust but expensive one and reuse it. ments in all six loci. Alleles that did not match with any The mating behaviour of C. reticulata suggests that sex- of the four individuals in each group were detected from ual selection functions intensely in simultaneous hermaph- 23 sperm masses ­(alleleW, Table 2). This suggests that C. rodites. In C. reticulata, we clarifed that sperm donors reticulata removed sperm originating from the mating removed allo-sperm stored from a third or more preceding partner(s) of preceding copulations that occurred in the wild copulation, and that sperm originating from 2 or more indi- prior to collection. There are a few possible reasons why the viduals was stored in the seminal receptacle of recipients. same alleles as the recipient’s ex- and ex-ex-partner were The ability to displace allo-sperm with a thorny penis and not detected from the removed sperm mass. The primary the ability to copulate repeatedly at intervals of 24 h by rapid possibility is that the amount of sperm originating from replenishment of the disposed penis (Sekizawa et al. 2013) the ex- and ex-ex-partner was too little to detect the same in C. reticulata suggest that competition to fertilize is quite alleles at all of the six loci necessary to determine genotype, intense and mating frequency in the wild is not so low in because the detection sensitivity of alleles varied among this species. As the disposable penis in C. reticulata has a markers (loci). The other possibility is that the amount similar function as the accessory structure of the copulatory of the sperm of the recipient’s ex- and ex-ex-partner was organ of damselfies, we suppose that sexual selection may greatly reduced in (the removable area of) the copulatory work as intensely in simultaneously hermaphroditic animals pouches of the sperm recipient at the time of fxation, owing as it does in gonochorists. Additionally, we propose that the to sperm movement and transport, or sperm digestion in the low-density model, which states that simultaneous hermaph- copulatory pouches. The sperm of the recipient’s previous roditism evolved to fully utilize scarce mating opportuni- partner(s) was detected from some sperm mass samples of ties caused by low density or low mobility (Ghiselin 1969), each grade of the amount of sperm. This shows that sperm should be re-evaluated, at least in the case of C. reticulata. displacement succeeded irrespective of the amount of sperm attached to the autotomized penis. Some sperm mass sam- Acknowledgements We are grateful to K. Sakai of the Sesoko Station of the University of Ryukyus, for his help during the feld survey in ples of grade ‘++’ and ‘+++’ did not contain sperm of the Okinawa. We are also thankful to A. Shimizu and M. Okubo of the recipient’s previous partner(s) but sperm of the donor itself. National Research Institute of Fisheries Science, Fisheries Research; N. In these cases, the sperm donor may ejaculate in an inappro- Sato of Shimane University; and S. Nanami of Osaka City University, priate place (for example the diverticulums connecting the for their help performing DNA analysis. We thank S. Shiga for her help and encouragement. This work was fnancially supported by a Grant- copulatory bursa and seminal receptacle) before inserting its in-Aid (#22570029) for Scientifc Research to Y. N. from the Japanese penis into the correct position. This may cause the attach- Ministry of Education, Culture, Sports, Science and Technology. ment of auto-sperm to the surface of the penis to prevent it from scraping out allo-sperm. There are possible causes for Compliance with ethical standards the detection of the DNA of the recipient from the sample of sperm mass: (1) cells of the recipient’s female reproductive Conflict of interest The authors declare that they have no confict of organ were scraped and attached to the penis of the sperm interest. donor at copulation, (2) sperm that leaked out from the penis Ethical approval This article does not contain any studies with human of the recipient was attached to the penis of the donor when participants performed by any of the authors. All applicable interna- both partners pulled out their respective penises at the end tional, national, and/or institutional guidelines for the care and use of of copulation. animals were followed. C. reticulata autotomizes its penis after every copulation Informed consent Irrelevant. (Sekizawa et al. 2013) and it is thought that the autotomy of the penis evolved to remove allo-sperm from the mat- Open Access This article is licensed under a Creative Commons Attribu- ing partner efciently. We clarifed in this study that sperm tion 4.0 International License, which permits use, sharing, adaptation, donors removed allo-sperm already stored in the copula- distribution and reproduction in any medium or format, as long as you tory pouch(es) of sperm recipients with backward-pointing give appropriate credit to the original author(s) and the source, provide a spines on the penis as the fnal process of their copulation. link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included Though a long and thorny penis is advantageous in scrap- in the article’s Creative Commons licence, unless indicated otherwise ing out allo-sperm at copulation, such a penis is difcult in a credit line to the material. If material is not included in the article’s to pull back into the body again after copulation. And the Creative Commons licence and your intended use is not permitted by backward-pointing spines on the penis covered with sperm statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. at copulation will not remove allo-sperm efciently at the TM To view a copy of this licence, visit http://creat​iveco​mmons​.org/ next copulation, like a Velcro tape. Such morphological licen​ses/by/4.0/.

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