Dimorphic ejaculates and sperm release strategies associated with alternative mating behaviors in the squid

Sperm competition is a powerful postcopulatory selective force influencing male adaptations associated with increasing fertilization success, and it is usually related to the evolution of different strategies of ejaculate expenditure between individuals. Ejaculates may also be influenced by additional selective pressures associated with sperm competition, such as timing between insemination and fertilization, female reproductive tract morphology, and fertilization environment. Also, males that adopt alternative mating tactics may face distinct sperm competition pressures, which may lead to the evolution of intraspecific diversity in ejaculates. In loliginid squids, males with alternative reproductive tactics (sneakers and consorts) differ not only in mating behavior, but also transfer spermatophores into two distinct sites within the female. Here, we compared structure and functioning of spermatophores between sneakers and consorts in the squid Doryteuthis plei applying microscopy techniques and in vitro experiments. Sneakers and consorts exhibit differences in spermatophore structure that lead to distinct spermatophoric reactions and spermatangium morphologies. Moreover, in sneakers, sperm release lasts longer and their sperm show an aggregative behavior not detected in consorts. Slow sperm release may be a strategy to guarantee longer sperm provision, given the wide interval between sneaker mating and egg release. For consorts, in turn, intense and quick sperm discharge may be advantageous, as timing between mating and egg‐laying is relatively short. Within the complex squid mating system, factors such as (i) different fertilization sites and (ii) interval between mating and egg release may also influence sperm competition, and ultimately shape the evolution of divergent ejaculates between dimorphic males.     

Alternative male mating behaviors dependent on relative body size in captive oval squid Sepioteuthis lessoniana (Cephalopoda, Loliginidae)

We observed the reproductive behavior of the oval squid Sepioteuthis lessoniana in captivity. The male used three different mating behaviors: male-parallel (MP), male-upturned (MU) and sneaking. Male competition over females frequently occurred before and during the female egg-laying period, and the outcome of most fights depended on male body size. Larger males guarded their partners from other males and performed MP mating during the egg-laying period of the paired females. In contrast, there was no pairing and mate guarding in MU mating and sneaking, which were adopted by smaller subordinate males as alternative tactics outside female egg-laying period and during the period, respectively. MP matings were 95% successful, but more than half of MU matings were unsuccessful. Higher mating success in MP mating was achieved through pairing, whereas males in MU mating were less successful because mating attempts without pair formation were often foiled by escape of the female. Sneaking was successful in all cases but occurred less frequently. Spermatophores were attached at the opening of the oviduct in MP mating, whereas they were attached around the female buccal membrane in MU mating and sneaking. Considering the route of egg transportation, higher fertilization success can be expected in MP mating because of the advantageous location of the attached spermatophores. Our results suggest that MP mating is used by larger, paired males during the female egg-laying period, and that MU mating and sneaking are alternative tactics adopted by smaller, subordinate males. These alternative mating behaviors would be conditional strategy dependent on relative body size, because some individual males displayed both MP and MU mating behaviors.     

Fitness advantage from nuptial gifts in female fireflies

Abstract 1. In many insects, males provide nuptial gifts to females in the form of spermatophores, sperm-containing structures produced by male accessory glands.
2. The work reported here examined the influence of both spermatophore number and spermatophore size on female reproductive output in two related firefly beetles, Photinus ignitus and Ellychnia corrusca (Coleoptera: Lampyridae). Based on differences in adult diet, male spermatophores were predicted to increase female reproductive output to a greater extent in P. ignitus than in E. corrusca .
3. Female fecundity was significantly higher in triply mated females than in singly mated females in both species, with no difference between mating treatments in female lifespan or egg hatching success. No effects of second male spermatophore size on fecundity, lifespan, or egg hatching success were detected in either species.
4. These results suggest a direct fitness advantage from multiple mating for females in both species, although enhanced fecundity may be due either to allocation of spermatophore nutrients to eggs or to other substances transferred within the spermatophore acting as oviposition stimulants.     

Male Condition, Female Choice, and Extreme Variation in Repeated Mating in a Scaly Cricket, Ornebius aperta (Orthoptera: Gryllidae: Mogoplistinae)

Mating in the scaly cricket Ornebius aperta often includes the transfer of many spermatophores to individual females during extended copulatory interactions. We manipulated male condition in staged matings to determine whether this could explain variation in the number of repeated copulations seen across pairs. Males on a high nutrient diet were in good condition, were more likely to mate repeatedly, and transferred more spermatophores on average than low-diet males (in poor condition). High-diet males were more likely to produce a vibratory signal that increased female receptivity to repeated mating attempts. Courtship and copulatory interactions were always terminated by females, and in every case males had already formed a spermatophore when deserted by females. We conclude that variation in male repeated mating success may be due to female choice rather than an inability or unwillingness of low-diet males to produce spermatophores.     

Mating system of an exceptional primate, the olive colobus (Procolobus verus)

In the olive colobus (Procolobus verus), many groups have multiple males and the males have large testes. This indicates that even though this species lives in small groups, single males do not monopolize the groups. We investigated the strategies employed by males to secure their mating success, and sought to determine whether the lack of male monopolization was a result of female mating strategies, as indicated by the exaggerated sexual swellings of the females. Four study groups were monitored for demographic changes, and group composition was determined in six additional groups in Taï National Park, Ivory Coast, between 1994 and 1999. Social behavior was recorded by scan and focal sampling in the study groups. The almost permanent association of olive colobus with Diana monkeys (Cercopithecus diana) in effect provided males a resource at which they could expect females to visit and sometimes even permanently join them, as well as protection from predators. As alternative strategies for obtaining females, one male took over the group of another male and one male immigrated into a bisexual group. Within bi‐male groups, dominant males mated most frequently and males defended their groups during intergroup interactions. Lone females that visited groups or solitary males had a swelling more often than expected, and generally mated with the males they visited. Females had long receptive periods, several consecutive receptive cycles, and some overlap in receptive periods within groups. Females mated with extragroup males, and during infertile periods. We concluded that the males used the Diana monkeys for safety reasons and to obtain mating partners, and that female reproductive biology and behavior prevented the monopolization of groups of females by single males. Our data were inconclusive as regards the benefits to females of avoiding monopolization by males. Am. J. Primatol. 62:261–273, 2004. © 2004 Wiley‐Liss, Inc.     

Linking larval nutrition to adult reproductive traits in the European corn borer Ostrinia nubilalis

Throughout an organism's lifetime, resources are strategically allocated to many different functions, including reproduction. Reproduction can be costly for both sexes; females produce nutrient‐rich eggs, whereas males of many species produce large and complex ejaculates. In capital breeding insects, nutrients are mainly acquired during the larval period, yet allocation decisions impact the reproductive fitness of adults. The present study examines the effect of larval dietary nitrogen on both male and female reproductive traits in the European corn borer moth Ostrinia nubilalis Hübner, whose adults do not feed and whose males transfer a large, nitrogen‐rich spermatophore. One day post‐eclosion, O. nubilalis adults reared on one of three different diets (3.0%, 1.6%, or 1.1% nitrogen) are mated and two experiments are undertaken: one to measure nitrogen and carbon content of male ejaculates, and the other to determine female fecundity and fertility. Although male larval diet does not alter the percentage nitrogen content of adult somatic tissue, males reared on the higher nitrogen diet (3.0%) produce spermatophores with increased nitrogen relative to somatic nitrogen. Furthermore, females raised on the 3.0% nitrogen diet receive spermatophores with lower carbon : nitrogen ratios and thus more nitrogen. Overall, females lay more eggs as their larval dietary nitrogen increases, although they lay fewer eggs when their mates are raised on the higher (3.0%) nitrogen diet. This suggests that O. nubilalis females may use male‐derived nitrogen not to supplement egg production, but rather for somatic maintenance. Overall, the present study furthers our understanding of how larval diet can affect adult fitness in Lepidoptera.     

Social Behavior,Reproductive Strategies,and Population Genetic Structure of <Emphasis Type="Italic">Lagothrix poeppigii</Emphasis>

For species of primates in which females emigrate, we would expect males within groups to be related to one another. Kin selection theory suggests that these males should associate preferentially with one another, be more affiliative and cooperative with one another than females are, and compete less overtly with one another over reproductive opportunities than males in female philopatric taxa do. Precisely these patterns of social behavior characterize well-studied populations of 2 of the 3 atelin primate genera: spider monkeys (Ateles) and muriquis (Brachyteles). For the third atelin genus, Lagothrix, patterns of intragroup social behavior have been less well-documented. We studied the social and reproductive behavior of lowland woolly monkeys (Lagothrix lagotricha poeppigii) in Ecuador during a one-year observational study and subsequently used molecular techniques to investigate population genetic structure and dispersal patterns for this taxon. Among adult male woolly monkeys, both affiliative and agonistic interactions were rare, and males were seldom in close proximity to one another. Relationships among male woolly monkeys are best characterized as tolerant, especially in the context of mating wherein direct competition among males was minimal despite the fact that females mated with multiple males. Relationships among females were likewise generally tolerant but nonaffiliative, though females often directed harassment towards copulating pairs. Affiliative interactions that did occur among woolly monkeys tended to be directed either between the sexes—primarily from female to male—or from younger towards older males, and the proximity partners of females tended to be members of the opposite sex. These results suggest that bonds between the sexes may be more important than same-sex social relationships and that direct female-female competition is an important feature of woolly monkey reproductive biology. Our genetic results indicate that, as in other atelins, dispersal by females is common, but some male dispersal likely occurs as well. In some but not all groups we studied, nonjuvenile males within social groups were more closely related to one another on average than females were, which is consistent with greater male than female philopatry. However, differences in these patterns among our study groups may reflect local variation in dispersal behavior.     

Male mating strategies and reproductive constraints in a group of wild tufted capuchin monkeys (Cebus apella nigritus)

Tufted capuchin monkeys (Cebus apella) provide an extreme example of active female sexual solicitation of males. In spite of being targeted by females for sex, males may delay copulation for hours or days. Data were collected on the sexual interactions in one wild capuchin group at the Estação Biológica de Caratinga in Brazil from September 1996 to August 1997. All successful conceptions during this year occurred in the dry season, yet sexual behavior was observed during 9 months of the year. This study tested whether male sexual response to female proceptivity was seasonally‐mediated. Male consortship participation, solicitation of females, latency to copulation, and copulation frequency were compared between fertile and nonconceptive females. Seasonal patterns in copulation interference, mating style, and alternative mating strategies were also examined. Thirty‐two copulations were observed. The alpha male was solicited for significantly more consortship days per female, but his mating success, in terms of copulation frequency, did not differ from that of two other adult males in the group. In the dry season, when the females were fertile, the males showed increased contest competition for mates, a higher frequency of alternative mating strategies against copulation interference, and increased monitoring of the females' condition. However, contrary to expectations, the alpha male's latency to copulation was significantly longer in the fertile season than in the nonconceptive months, and no males were observed to mate more than one time per day, even at the conceptive peak. Male mating strategies were affected by both season and rank, and there was evidence for reproductive constraints on males throughout the year. Limited male ejaculatory capacity and male choice in the timing of copulations within female proceptive phases may both be important factors in driving the sexual dynamics of this species. Am. J. Primatol. 67:313–328, 2005. © 2005 Wiley‐Liss, Inc.     

Multiple mating and its effect on the reproductive success of female Epiphyas postvittana (Lepidoptera: Tortricidae)

Abstract.

• 1 Multiple mating and its effect on reproductive performance of female Epiphyas postvittana (Walker) moths were studied under controlled conditions.
• 2 The age at which the moths mated for the first time ranged from the first to the tenth day after emergence, but 71% of first matings were during the first 3 days.
• 3 The majority (63%) of females had one or two spermatophores in the bursa copulatrix. Some (24%) were found with three to five spermatophores, whereas no successful mating occurred among 13% of individuals. The number of matings was partly dependent on the number of mates available to the female. Between the range of sex ratios of one male to one female and four males to one female maximal mating success occurred at the ratio of three males to one female.
• 4 Virgin females were capable of egg-laying, but mating stimulated and accelerated oviposition. Mated individuals laid twice as many eggs as unmated ones.
• 5 The level of copulatory activity did not influence the longevity of females irrespective of the number of males available to them.
• 6 Sex ratios with greater than one male to a female improved the reproductive success by marginally increasing fecundity and fertility.
• 7 It is concluded that multiple mating would enhance population growth, and is of particular benefit to populations with a preponderance of females, as is known to occur naturally in this species.

     

Squid egg mops provide sensory cues for increased agonistic behaviour between male squid

Males of many species compete for access to females. In order to avoid performing potentially costly agonistic behaviour for their entire adult lives, many group-living males use environmental cues to limit agonistic behaviour to times when it will be of most benefit. Long-finned squid, Loligo pealeii, live less than a year and aggregate in mixed- and single-sex schools. Adults participate in several spawning events, then die. During spawning events, males actively compete for females. Winning males pair with females, which subsequently lay eggs in communal sites on the ocean floor (‘egg mops’). To determine whether males use sensory cues provided by egg mops to regulate agonistic behaviour, we conducted four laboratory experiments. We measured the agonistic responses of pairs of adult males before, during and after exposure to conspecific egg mops. In three experiments, egg mops were manipulated to provide differing sensory stimuli (tactile, water-borne, visual). The addition of conspecific egg mops to tanks of paired male squid dramatically increased agonistic behaviour above control levels within minutes. Male squid were first attracted to the egg mops visually, but contact with the capsules was necessary to increase agonistic behaviour. After initial contact, agonistic behaviour was almost continuous as long as egg mops remained present, even when squid touched the egg mops infrequently. Visual stimuli seemed important in maintaining elevated agonistic behaviour between egg mop touches. When egg mops were removed from the tank, measured agonistic behaviour declined within minutes. When egg mops were added to the tank while covered by an opaque and porous cover that allowed water-borne stimuli to circulate into the tank, squid did not approach the covered egg mop or show increased agonistic behaviour. This result suggests that water-borne stimuli are not sufficient to increase agonistic behaviour. It is unusual for male agonistic behaviour in any species to be increased by contact with fertilized eggs. In this species, however, egg capsules might signal that sexually mature, receptive females are about to lay eggs. Indirect evidence suggests that mating with a female immediately before she lays eggs increases male paternity. If this prediction is true, the presence of egg mops may indicate the optimal time for male squid to establish mating precedence through agonistic bouts. Copyright 2003 Published by Elsevier Science Ltd on behalf of The Association for the Study of Animal Behaviour.      

Old‐male paternity advantage is a function of accumulating sperm and last‐male precedence in a butterfly

Old‐male mating advantage has been convincingly demonstrated in Bicyclus anynana butterflies. This intriguing pattern may be explained by two alternative hypotheses: (i) an increased aggressiveness and persistence of older males during courtship, being caused by the older males' low residual reproductive value; and (ii) an active preference of females towards older males what reflects a good genes hypothesis. Against this background, we here investigate postcopulatory sexual selection by double‐mating Bicyclus anynana females to older and younger males, thus allowing for sperm competition and cryptic mate choice, and by genotyping the resulting offspring. Virgin females were mated with a younger virgin (2–3 days old) and afterwards an older virgin male (12–13 days old) or vice versa. Older males had a higher paternity success than younger ones, but only when being the second (=last) mating partner, while paternity success was equal among older and younger males when older males were the first mating partner. Older males produced larger spermatophores with much higher numbers of fertile sperm than younger males. Thus, we found no evidence for cryptic female mate choice. Rather, the findings reported here seem to result from a combination of last‐male precedence and the number of sperm transferred upon mating, both increasing paternity success.     

Movement and reproductive behavior of solitary male redtail guenons (Cercopithecus ascanius)

Adult male redtail guenons (Cercopithecus ascanius) live as resident males in heterosexual groups or as nonresident males outside groups. Data are reported here on movement and temporary intrusions by nonresident males in heterosexual groups. These data further elucidate the reproductive strategies of this class of individual. Observations of three radiocollared males provided data on the details of their movement relative to groups. Regular sampling of three heterosexual groups provided data on patterns of nonresident males' intrusions. During peak mating months, nonresident males formed loose affiliations with groups. The males affiliated with one group at a time, with each affiliation lasting several weeks. Nonresident male intrusions in groups were unevenly distributed and were not correlated with the number of potentially fertile females in each group. Estimates of male density indicate that there were about two nonresident males per group, which suggests an adult sex ratio of three females per male within the study area. Nonresident male redtail guenons apparently do not spread their reproductive efforts over several groups at once, but instead each male tends to direct his attention toward the group offering the best opportunity for reproductive payoffs. Factors that determine which groups they attempt to infiltrate may include the characteristics of resident males, female choice, or both, but the number of fertile females probably has no effect. The male low density and female-biased sex ratio among adults should facilitate resident males' attempts to monopolize fertilizations in groups; however, resident males were unable to prevent copulations by nonresident males even when only one nonresident was intruding in a group.     

Correlated responses to selection on female egg size in male reproductive traits in a butterfly

Genetic correlations between male and female traits can act as evolutionary constraints and, if involving reproductive traits, potentially influence sexual selection. Artificial selection on egg size in the tropical butterfly Bicyclus anynana has yielded highly divergent lines. Here we report evidence for correlated evolution in male traits. Males from the large-egg selected lines produced significantly heavier spermatophores independent of body size and tended to have more fertile sperm stored in their reproductive tracts than those from the small-egg selected lines. This may be due to an underlying genetic correlation in reproductive effort between the sexes. However, non-fertile sperm number and testis size remained unaffected by selection on egg size. Phenotypic correlations within an unselected population revealed that spermatophore mass and fertile sperm number, but not testis size and non-fertile sperm number, were positively related to male body size, and that larger spermatophores contained more fertile, but not non-fertile sperm. In addition, males provided larger females with bigger spermatophores and more fertile sperm, indicating males may be exercising mate choice during copulation.     

Testing the role of male–male competition in the evolution of sexual dimorphism: a comparison between two species of porcelain crabs

Theory predicts marked sexual dimorphism in terms of body size and body structures used as weapons (e.g. chelipeds) in gonochoric species with intense male sexual competition for receptive females and reduced or no sexual dimorphism in species where competition among males is trivial. We tested this hypothesis using a pair of closely‐related species of symbiotic porcelain crabs as a model. In one species that inhabits sea anemones solitarily, competition among males for receptive females is unimportant. In a second species that dwells as dense aggregations on sea urchins, male–male competition for sexual partners is recurrent. We expected considerable sexual dimorphism in body size and weaponry in the urchin‐dwelling crab and reduced sexual dimorphism in the anemone‐dwelling crab. In agreement with expectations, in the urchin‐dwelling crab, male body size was, on average, larger than that of females and males invested considerably more to cheliped length than females. Also supporting theoretical considerations, in the anemone‐dwelling crab, sexual dimorphism in terms of body size was not detected and differences between the sexes in investment to cheliped length were minor. Interestingly, chelipeds were more developed both in males and females of the anemone‐dwelling crab than in the urchin‐dwelling crab as a result of the importance of these structures for monopolization of their naturally scarce anemone hosts. Another difference between the studied species was the existence of two clearly distinguishable ontogenetic phases in males of the urchin‐dwelling crab but not in males of the anemone‐dwelling crab. Whether the two different male morphs display different male reproductive strategies in the urchin‐dwelling crab remains to be addressed. Other conditions that might additionally explain the observed differences in sexual dimorphism (e.g. female mate choice) between the studied species remain to be explored. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 548–558.     

Experimental evolution exposes female and male responses to sexual selection and conflict in Tribolium castaneum

Between-individual variance in potential reproductive rate theoretically creates a load in reproducing populations by driving sexual selection of male traits for winning competitions, and female traits for resisting the costs of multiple mating. Here, using replicated experimental evolution under divergent operational sex ratios (OSR, 9:1 or 1:6 ♀:♂) we empirically identified the parallel reproductive fitness consequences for females and males in the promiscuous flour beetle Tribolium castaneum. Our results revealed clear evidence that sexual conflict resides within the T. castaneum mating system. After 20 generations of selection, females from female-biased OSRs became vulnerable to multiple mating, and showed a steep decrease in reproductive fitness with an increasing number of control males. In contrast, females from male-biased OSRs showed no change in reproductive fitness, irrespective of male numbers. The divergence in reproductive output was not explained by variation in female mortality. Parallel assays revealed that males also responded to experimental evolution: individuals from male-biased OSRs obtained 27% greater reproductive success across 7-day competition for females with a control male rival, compared to males from the female-biased lines. Subsequent assays suggest that these differences were not due to postcopulatory sperm competitiveness, but to precopulatory/copulatory competitive male mating behavior.     

Multiple mating,sperm transfer and oviposition pattern in the giant sperm species,Drosophila bifurca

Many diverse traits are involved in gamete systems, and several models have analysed sperm length variation in terms of the intensity of sperm competition. This study investigates mating, sperm transfer and oviposition patterns in Drosophila bifurca, which possesses the longest sperm in the animal kingdom (about 6 cm). The prediction is that sperm gigantism should prevent male–male interaction. In this study, we examine how sperm transfer varies as males mate with a series of females, and how female receptivity changes with time after mating. As predicted, we found an extremely limited overlap of ejaculates owing to (1) reduced sperm transfer to females that had already mated, and (2) female remating depended both on the amount of sperm transferred and the modes of egg laying. The amount of sperm transferred to the female is discussed in relation to the peculiar morphology of the male reproductive tract and to sexual dimorphism and ecological hypotheses.     

Mating tactics in the sub-Antarctic deep-sea squid <Emphasis Type="Italic">Onykia ingens</Emphasis> (Cephalopoda: Onychoteuthidae)

The behavior of polar deep-sea nekton is very poorly known. To obtain insight into mating behavior of the abundant and ecologically important sub-Antarctic deep-sea squid Onykia ingens, our goals were to (1) quantify spermatophore production; (2) determine the preferred location for spermatangia deposition; (3) investigate whether male and female O. ingens mate with multiple mates; and (4) discuss the location of implanted spermatangia in light of mating behavior and egg fertilization. Toward this end, we examined male and female O. ingens specimens from Falkland Island and New Zealand waters. Male O. ingens store up to 198 spermatophores (mean 103 ± 61; n = 12) in their reproductive system, which are produced over a period of considerable somatic growth (200–400 mm ML), and which may have a considerable size range. Males insert their long extendible terminal organ in the mantle cavity of the female, potentially through the funnel, to deposit spermatophores in one or more of four regions on the female’s body. Most implanted spermatangia (52.5 %) were found in the funnel region, but many were also found inside the mantle cavity closer to the oviducts. Males with longer terminal organs therefore may be able to position closer to the oviducts where fertilization chances are higher than for spermatangia located in the funnel region. The number of implanted spermatangia per individual female (4–60, mean 29 ± 20; n = 24), the multiregional spermatangia deposition, and the different outer appearance of spermatangia, suggested that females have multiple mating events. Since males produce more spermatophores (up to 200) than the number of spermatangia in one region (<60), it is likely that males too mate with more than one female. We show how quantitative assessment of reproductive characteristics can provide insight into the reproductive behavior of deep-sea species for which in situ observations are currently lacking.     

The effect of the presence of quiescent female nymphs, males and their spermatophores on spermatophore placement in two species of eriophyoid mites

Under sex dissociated sperm transfer, females seek spermatophores and pick up sperm without male assistance. In several species males adjust spermatophore deposition rate to the presence of conspecifics. It is not known, however, which factors could favor such elasticity in non-pairing males. In this paper, we compare male response towards conspecifics between the sex dissociated eriophyoid mites Aculus fockeui (Nalepa and Trouessart) and Aculops allotrichus (Nalepa). The species differ significantly in male reproductive strategies and, consequently, the intensity of male–male-competition. Aculus fockeui males deposit spematophores all over the leaves and occasionally leave single spermatophores beside quiescent female nymphs (QFNs). In contrast, A. allotrichus males guard QFNs and encircle them with spermatophores. In this study, males of both species deposited spermatophores close to and apart from the rival spermatophores. Aculops allotrichus males had similar spermatophore output whether they were kept alone or in a group of seven males. They did not change spermatophore output in the presence of five rival spermatophores, a QFN or a QFN and varying number of rivals, either. In contrast, A. fockeui males increased spermatophore output in the presence of rival spermatophores or when on the arena with a QFN the male number increased to eight males. They did not respond, however, to the presence of a QFN and one rival or a QFN alone. The possible effect of the species-specific intensity of male–male competition, population density, the availability of receptive females and the rate of spermatophore output on the flexibility of eriophyoid spermatophore deposition is discussed.     

How many daddies: microsatellite genotyping reveals polyandry in a live‐bearing clinid fish Muraenoclinus dorsalis

Fishes belonging to the family Clinidae in South Africa display super‐embryonation, a rare reproductive mode were females gestate broods at different gestational stages, but little is known regarding the mating systems of this family. Here we tested the hypothesis that multiple males would contribute not only to the offspring of each female, but that several males would contribute to each brood, by sampling Muraenoclinus dorsalis from three sampling locations along the west and south‐west coast of South Africa. Larval (n = 97) and maternal (n = 14) genotpyes, generated with newly developed microsatellites, were used to estimate the number of potential mates per female. Our results show that up to 78% of females displayed multiple mating with an average of 2·1–2·2 males. In addition, 39–42% of females displayed polyandry with an average of 1·5–1·6 sires per brood. This study provides the evidence for multiple mating and polyandry within a clinid fish characterized by super‐embryonation that offers important baseline information regarding rare reproductive strategies, highlighting several gaps in our knowledge concerning clinid reproduction and mating systems.