Size-advantage in nest-holding tactics in the dusky frillgoby Bathygobius fuscus males: mating success and parental care success but not fertilization success

Size-advantage in nest-holding tactics in the dusky frillgoby Bathygobius fuscus males: mating success and parental care success but not fertilization success | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Size-advantage in nest-holding tactics in the dusky frillgoby Bathygobius fuscus males: mating success and parental care success but not fertilization success Shoma Kawase, Noriyosi Sato, Masa-aki Yoshida, Takeshi Takegaki This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8818992/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Body size is a critical trait influencing reproductive success through mate competition, female choice, and parental care ability. In fish species with alternative reproductive tactics, large males typically adopt dominant tactics, while smaller males employ parasitic tactics, such as sneaking tactics. However, the reproductive advantage of larger males can be compromised by intense sperm competition with small males. We investigated the effects of body size on the reproductive success of nest-holding males in the dusky frillgoby Bathygobius fuscus , a species where small males frequently engage in sneaking tactics. Field investigations using artificial nests revealed that larger nest-holding males had significantly higher mating success (number of eggs acquired) and parental care success (egg survival rate). In contrast, DNA paternity analysis showed that fertilization success (paternity rate) was not correlated with male body size. This is because of the presence of males with extremely low paternity rate, including two cases of zero paternity, suggesting high sneaking pressure and replacement of nest-holding males (i.e., alloparental care). Consequently, the cumulative reproductive success of nest-holding males showed no significant relationship with body size. These results showed that the size-advantages of nest-holding males in attracting females and tending eggs are offset by the sperm competition with sneaker males. The lack of a clear size advantage in reproductive success may explain the plastic nature of reproductive tactics in this species, where large males may occasionally adopt sneaking tactics or abandon nests. alternative reproductive tactics size-advantage body size paternity Figures Figure 1 Introduction Body size is an important trait that affects many aspects of life history in animal, usually favoring larger individuals (Peters 1983 ; Roff 2002 ; Blanckenhorn 2000 ; Ahti et al. 2020 ). In reproduction, larger body size confers advantages in gamete production for females and in competition over reproductive resources and females for males (Lande 1980 ; Hedrick and Temeles 1989 ; Parker 1992 ; Blanckenhorn 2000 ; Rollinson and Rowe 2016 ). Moreover, in species exhibiting parental care, larger individuals obtain higher parental care success through their ability to defend, feed, and more (Clutton-Brock 1991 ; Gross 2005 ; Royle et al. 2012 ). These body size-related advantages could also benefit their mates, thus body size is likely to be an indicator trait for mate choice (Andersson, 1994 ). The evolution of sexual size dimorphism, particularly male-biased, is considered largely influenced by female mate choice (insect, Blanckenhorn et al. 2007 ; fish, Horne et al. 2020 ; bird, Fairbairn et al. 2007 ; reptile, Shine 1994 ; mammal, Clutton-Brock and McAuliffe 2009 ). Male size advantages produce individual differences in reproductive success among males within a breeding population, thereby influencing the evolution of male life history strategies, reproductive tactics, and reproductive traits. The evolution of alternative reproductive tactics by small, subordinate males is one such example. Alternative reproductive tactics refer to different reproductive phenotypes within a species (Gross, 1996 ), which are maintained to contribute to maximizing individual fitness (Koprowski, 1993 ; Kempenaers et al., 1995 ; Gross, 1996 ; Shuster and Wade, 2003 ). The difference in reproductive phenotype is usually observed in males. While different tactics coexist as genetic polymorphisms (i.e., strategy), most are tactics that evolved to increase fitness under the conditions faced by socially subordinate males (Tomkins and Hazel 2007 ). Such subordinate males often have tactic-specific traits—such as morphology, coloration, or behavior—to increase their success. For example, small males of the beetle species Podischnus agenor adopt a tactic of widely searching for females, avoiding combat with larger males without developing horns as weapons (Eberhard, 1979 ). Furthermore, the change to dominant tactics occurs in response to changes in social status (Gross 1996 ; Neff and Svensson 2013 ). Since social status of individuals strongly depends on body size, to understand what tactics individuals adopt and when they change tactics, it is essential to clarify how body size affects the reproductive success of each tactic. Parasitic tactics such as sneaking and satellite tactics, in which a subordinate male exploits opportunity in dominant male to mate with a female, are primary alternative reproductive tactics in many taxa (Oliveira et al. 2008 ; Dougherty et al. 2022 ). Particularly in fish, where many species exhibit external fertilization, this is the most common alternative tactic (Taborsky 1994 ; Oliveira et al. 2008 ), and consequently results in intense sperm competition (Taborsky 1998 ; Kustra and Alonzo 2020 ; Dougherty et al. 2022 ). Dominant males aggressively attack and drive away sneaker males attempting to participate in reproduction to defend their paternity (fertilization success). On the other hand, in some species, sneaker males display female-like coloration and behavior to avoid attacks from dominant males (Oliveira et al. 2008 ). Since sneaker males always face sperm competition with dominant males, they generally have larger testes than dominant males. They increase their fertilization success by producing and storing more sperm to achieve higher ejaculate volumes and more frequent sperm release (Parker 1998 ; Petersen and Warner 1998 ; Taborsky 1998 ; Parker and Pizzari 2010 ; Dougherty et al. 2022 ). Furthermore, some species are known to have evolved sperm characteristics favorable for fertilization, such as high swimming speed and long lifespan (Birkhead and Møller 1998 ; Snook 2005 ; Montgomerie and Fitzpatrick 2009 ; Kustra and Alonzo 2020 ). Despite having these tactic-specific traits, sneaker males in fish usually have extremely low fertilization success (Avise et al. 2002 ; Coleman and Jones 2011 ), and are considered to be at a disadvantage in reproduction (Oliveira et al. 2008 ). The relatively small males of the dusky frillgoby Bathygobius fuscus , a small coastal marine fish, adopt a sneaking tactic where they intrude into the spawning pairs of a large nest-holding male and a female to release their sperm (Taru et al. 2002 ; Takegaki et al. 2012 ). Sneaker males are not only attacked by the nest-holding males during spawning, but also compete with other sneaker males for sneaking opportunities. Consequently, their chances and time for intrusion and fertilization are highly limited, and their reproductive success is predicted to be lower than that of nest-holding males (Takegaki et al. 2012 ). This is also indicated by the fact that sneaker males change tactics to nest-holding during the breeding season (Taru et al. 2002 ; Takegaki et al. 2013 ). However, on the other hand, in both field observations and aquarium experiments, sneaker males do not always change tactics to nest-holding when nests and females are available (Takegaki et al. 2013 ). This decision-making by sneaker males is thought to be due to the possibility that participating in reproduction as small nest-holding males may not obtain high reproductive success: for example, because of their low mating success, limited parental care capacity, and poor defense against sneaker males (Takegaki et al. 2013 ). To investigate these possibilities, this study aims to demonstrate the effects of body size on mating success, parental care success, fertilization success, and reproductive success in nest-holding males, as well as to determine extent to which sneaker males ensure fertilization success. Materials and methods Study sites Field investigations were conducted in two tidepools on the Miezaki Coast (32°48' N; 129°44' E) of Nagasaki Prefecture, Japan (surface areas at low tide: about 44.0 and 84.4 m 2 ; depths at low tide: about 0.3 and 0.5 m). Breeding season for dusky frillgoby at study site is from late May to mid-September (Takegaki et al. 2013 ; Ishibashi and Takegaki 2023 ), spawning cycle is synchronized with semi-luna cycle (Taru et al. 2002 ). Investigations for this study were conducted for 57 days at low tide during three days before and after high tide from May 29 to September 8, including high tide during day. Artificial nests (n = 30) were placed in study sites to measure mating and parental care success of nest-holding males of this species. Artificial nests consisted of a PVC pipe (internal diameter 40 mm, length 120 mm) and two covers (Ishibashi and Takegaki 2023 ), one cover was perforated to provide nest opening. In this species, larger nest-holding males preferentially use nests with larger nest opening (Takegaki et al. 2013 ), three different size of nest openings were set up (10 mm, 15 mm, and 20 mm; each 10 nests) to induce nesting in nest-holding males of various body sizes. In order to monitor presence and number of eggs caring, and to collect eggs for paternity analysis, transparent plastic sheets (120 mm × 180 mm), which could be freely inserted and removed, were rolled up and inserted into artificial nests. Mating success ໿Spawning in this species occurs in synchronization with semilunar periods, and nest-holding males repeat reproduction (i.e., spawning and egg care) several times during the breeding season. In this study, mating success of each male was evaluated based on the number of eggs obtained in a single reproductive cycle (n = 29 males). Males usually mate with multiple females during the egg acquisition period (ca. 3 days) of a single breeding cycle (Nagase and Takegaki 2017 ), during which the number of eggs increase but sometimes decrease due to predation by egg predators. Therefore, in this study, the number of eggs that reached its maximum during the egg acquisition period was considered as mating success of the male. The presence and the number of eggs were observed every day during the reproductive cycle, by withdrawing the plastic sheets in the artificial nests. If males have eggs, the egg sheet was photographed using a digital camera to count the number of eggs. Then, the egg sheet was returned to the nest, and the nest-holding male was confirmed to continue egg care. These series of procedures were repeated until the day before hatching of the eggs, and the eggs were collected for DNA paternity analysis; they were hatched in the laboratory and ໿the newly hatched larvae were anaesthetized with quinaldine (600 ppm) and fixed with 99% ethanol for DNA analysis. The digital images of eggs were uploaded onto a personal computer, and they were counted using counting software (Kachikachi counter 2.6; GT). To examine the effects of traits of the nest-holding males on their reproductive success, they were collected and sacrificed with a lethal dose of quinaldine (1250 ppm). The total length (TL, 0.1 mm), body weight (0.01 g) and testis weight (0.0001 g) were measured, and condition factor, as an index of body condition was calculated from body weight and TL, and gonadosomatic index (GSI), as an index of testis investment was calculated from body weight and testis weight (Kawase et al. 2017 ). For DNA analysis, ໿a piece of second dorsal fin was obtained and fixed in 99% ethanol. Parental care success Parental care success of the males was evaluated by the mean daily survival rate of eggs from the day reaching maximum number of eggs until the day before hatching. Fertilization success Fertilization success of the nest-holding males was measured for 13 nest-holding males randomly selected from the 29 individuals for which mating and parental care success were measured. For genetic analysis, we used five DNA markers (kumohaze2, kumohaze13, kumohaze15, kumohaze16, kumohaze17), and genotyped 13 datasets consisting of 13 nest-holding males and 1169 embryos (84–94 embryos per nest) (electronic supplementary material, Table S1 ). DNA was extracted using a commercial kit (KAPA Express Extract, Roche Diagnostics, Basel, Switzerland) according to the manufacturer’s protocol. Polymerase chain reaction (PCR) was performed using fluorescent primers 5'-labelled with NED, HEX, PET, or FAM (Applied Biosystems), and PCR fragment sizes were determined using a genetic analyser (ABI Prism 3730xl DNA Analyzer, Applied Biosystems, USA). The PCR mixture contained 1.0 µL Taq buffer, 0.8 µL of each dNTP, 1 unit of bioTaq DNA polymerase (Bioline), 0.25 µL of each primer, 3.0 µL of template DNA, and distilled water to a final volume of 10 µL. Thermal cycling conditions consisted of an initial denaturation at 94°C for 60 s, followed by 22–25 cycles of 94°C for 30 s, primer-specific annealing temperature for 30 s, and 72°C for 60 s, with a final extension at 72°C for 5 min. Paternity was inferred using an exclusion method (Jones and Wang, 2010). The combined non-exclusion probability for the first parent across the five microsatellite loci was 0.003, as estimated using CERVUS 3.0 (Kalinowski et al., 2007 ), indicating high power for paternity assignment. Reproductive success Reproductive success of the nest-holding males (n = 13) was calculated by multiplying the number of eggs survived until the day before hatching—derived from their mating success and parental care success—by the paternity rate. Statistical analysis In this study, statistical analyses were conducted using generalized linear models (GLMs) to evaluate the multivariate effects of potentially relevant traits (body size, body condition, GSI, and nest opening size) of nest-holding males on mating success, parental care success, fertilization success, and reproductive success. For each response variable, no more than three explanatory variables were included in the model, given the sample size. Explanatory variables were selected after assessing multicollinearity among candidate variables using variance inflation factors (VIFs), with all selected variables showing VIF values < 3. The explanatory variables included for each response variable were male body size, body condition, and nest opening size for mating success, parental care success and reproductive success, and male body size, GSI, and nest opening size for fertilization success. In this study, two nest-holding males with zero paternity were confirmed (see Results). Based on previous reports (Wisenden 1999 ; Avise et al. 2002 ; Coleman and Jones 2011 ), this strongly suggested the occurrence of replacement of the male parent during the egg-tending period. Therefore, analyses excluding these two cases were also conducted. Specifically, the effects of body size (total length), body condition, and nest opening size on the number of eggs acquired by nest-holding males were analyzed using a GLM with a quasipoisson error distribution and a log link function. The effects of body size, body condition, and nest opening size on the survival rate of cared eggs were analyzed using a GLM with a quasibinomial error distribution and a logit link function. The effects of body size, nest opening size, and GSI on the paternity rate of nest-holding males were analyzed using a GLM with a quasibinomial error distribution and a logit link function. The effects of body size, body condition, and nest opening size on the reproductive success were analyzed using a GLM with a quasipoisson error distribution and a log link function. The significance of explanatory variables in these analyses was assessed using F-tests. All statistical analyses were performed using R (version 4.5.2; R Development Core Team, 2025 ). Results A significant effect on the number of eggs acquired by nest-holding males (mean ± SD = 24878 ± 13320 eggs, range = 2733–49689 eggs, n = 29) was detected only for size: i.e., larger males acquired more eggs, whereas body condition and nest entrance size tended to be higher in larger males but did not significantly affect the number of eggs acquired (Table 1, Fig. 1 a). Similarly, the survival rate of eggs (mean ± SD = 73.6 ± 15.4%, range = 35.8–95.6%, n = 29) was significantly higher for larger males, but body condition and nest opening size did not affect the survival rate of eggs (Table 1, Fig. 1 b). The paternity rate of nest-holding males (mean ± SD = 43.2 ± 27.8%, range = 0.0–90.5%, n = 13) was not affected by their body size, nest opening size and testis size (GSI) (Table 1, Fig. 1 c). The reproductive success (mean ± SD = 6904 ± 7123 eggs, range = 0–22085 eggs, n = 13) was not affected by their body size, body condition and nest entrance size (Table 1, Fig. 1 d). In analyses excluding the two males in which paternity was not detected, there were no changes in any variable effects except for the effect of nest-entrance size on reproductive success (Table 1). Discussion This study demonstrated that larger body size of Bathygobius fuscus nest-holding males confers advantages in mating success and parental care success. However, this size-advantage was not observed in fertilization success, and consequently, there was no significant relationship between body size and reproductive success. Mating success There are two possible mechanisms for the higher mating success of larger B. fuscus nest-holding males: i.e., intra-sexual selection and inter-sexual selection. In general, large body size is advantageous for male-male competition over reproductive resources, such as females and territory (Wong and Candolin 2005 ; Hunt et al. 2009 ; Arnott and Elwood 2009 ), and thus male size dimorphism is more likely to evolve in such species (Shine 1989 ; Fairbairn 1997 ; Fairbairn et al. 2007 ). In B. fuscus , competition among nest-holding males over females has not been observed; however, they show preferences for the entrance size of spawning nests (Takegaki et al. 2013 ) and frequently compete over nests (Taru et al. 2002 ). Because larger males have an advantage in the competition (Taru et al. 2002 ), if females prefer to lay eggs in such nests, the size advantage in mating success detected in this study may be partially influenced by intra-sexual selection. Another possibility is female preference for larger males: i.e., inter-sexual selection. Male body size is one of the primary traits in female mate choice (Andersson 1994 ; Roff 2002 ), and larger males are preferred in almost all cases (but see, Hakkarainen et al. 1996 ; Voight et al. 2005; Sato et al. 2014 ). Females gain direct benefits from mating with larger males such as through the higher quality of reproductive resources and higher ability of parental care (Møller and Jennions 2001 ; Jones and Ratterman 2009 ), or indirect genetic benefits such as higher offspring growth rates and survival rates (Jennions and Petrie 2000 ; Jones and Ratterman 2009 ; Crean and Bonduriansky 2014 ). For example, in the smallmouth bass Micropterus dolomieu , larger males are preferred by females due to their higher ability to care for offspring, resulting in greater mating success. (Wiegmann and Baylis 1995 ). Also in the Trinidadian guppy Poecilia reticulata , larger males are preferred by females, and their daughters grow well and are larger in body size, thus having larger reproductive output (Reynolds and Gross 1992 ). The higher ability of parental care of larger B. fuscus nest-holding males demonstrated in this study (see below) suggests that females may prefer to mate with larger males. In addition, since it has been suggested that females of this species may prefer males already tending eggs in the nests (Nagase and Takegaki 2017 ), if larger males can acquire eggs earlier during each reproductive cycle, the size-advantage in mating is expected to be greater. On the other hand, regardless of female preference for larger males, there may be constraints on the mating success of smaller males. Since nest-holding males of this species prefer to use nests with entrance sizes assortative to their own body size (Takegaki et al. 2013 ), smaller males using nests with smaller entrances are physically unable to accept larger females. Large females cannot enter the small-entrance artificial nests used in this study, suggesting that this unavoidable constraint may influence the size-dependent mating success of nest-holding males in this species. Parental care success Survival rate of eggs until the day before hatching was higher in larger nest-holding males. In many taxa including fish, parents with larger body size generally exhibit higher ability to care for their offspring (Clutton-Brock 1991 ; Svensson and Kvarnemo 2023 ). Potential causes of mortality of eggs tended by B. fuscus nest-holding males are predation by egg predators and other individuals of B. fuscus (Hishida 2002 ; Nagase and Takegaki 2017 ), filial cannibalism by caregiving males (Hishida 2002 ; Nagase and Takegaki 2017 ), oxygen deprivation due to lack of parental care (Takegaki et al. 2013 ). In this study, carnivorous snails ( Ergalatax contractus and Thais clavigera ) preying on the eggs of this species (Hishida 2002 ; Nagase and Takegaki 2017 ) were observed in the nests where eggs had disappeared before hatching. Since snails were observed forcibly intruding into the tight nests of a blenny Neoclinus bryope , driving out the defending males to eat the eggs (Murase and Sunobe 2011 ), larger males would likely have an advantage in egg defense. Males of this species are known to eat a part of their own eggs during the period of egg care (Hishida 2002 ), effect of male body size on the intensity of filial cannibalism has not been reported. The partial filial-cannibalism is generally considered as a strategy for poor-condition parents to continue providing egg care by obtaining nutrients; consequently, in many fish species, the intensity of partial filial-cannibalism strongly depends on male body condition rather than body size (Manica 2002 ; Bose 2022 ). In this study, there was no significant effect of male body condition on egg survival rate (Table 1), the proportion of eggs lost due to cannibalism may not be high. In species that provide egg care in enclosed nests similar to B. fuscus , water exchange in the nest to supply oxygen to the eggs is critically important for egg survival (Kramer 1987 ). Fanning behavior to exchange nest water is known to be more effective in large males with large pectoral fins in three-spined stickleback fish (Künzler and Bakker 2000 ) and gobiid fishes (Meunier et al. 2013 ; Wantola et al. 2013 ), resulting in higher survival rate of eggs (Künzler and Bakker 2000 ). Because nest-holding males of B. fuscus tend to have specifically large pectoral fins compared to sneaker males (Takegaki, unpublished data), larger males may perform more effective water exchange through fanning. Fertilization success In all egg masses tended by nest-holding males, the paternity of other males was detected. Paternity of nest-holding males in this species are considerably lower than that of fishes with the breeding system involving sneaking tactics (Avise et al. 2002 ; Coleman and Jones 2011 ), surprisingly several egg masses with zero paternity of nest-holding males were also confirmed (see below). Paternity analysis in this study could not detect how many males other than nest-holding males succeeded in fertilization, but it is assumed that multiple sneaker males were involved in fertilization in many cases. This is because, at the same study site, nest-holding males usually mate with multiple females during a tidal cycle (Nagase and Takegaki 2017 ), and intrusions by sneaker males have been observed an average of 2.6 times per spawning event (Takegaki et al. 2012 ). Fertilization success of nest-holding males was not correlated with the body size, though their mating and parental care success were higher in larger individuals. These results remained unchanged even after excluding data with zero paternity, where replacement of nest-holding males was likely to have occurred. The fertilization success of nest-holding males would strongly depend on presence and intensity of sperm competition with sneaker males, so they should try to avoid or minimize the competition. For example, the high paternity rate of nest-holding males in the grass goby Zosterisessor ophiocephalus is thought to be due to their high defensive ability against sneaker males exhibited by larger males (Pujolar et al. 2012 ). But, in the case of B. fuscus nest-holding males, their aggressive ability dependent on body size may not contribute much to defending against sneaker males, because intrusions by sneaker males mostly occur during moments when nest-holding males are absent from the nest entrance—either chasing away other sneaker males or entering inside the nest probably to release sperm. Conversely, it is known that sneaker males of this species attempt more intrusions into the nests of larger nest-holding males (Ishibashi and Takegaki 2023 ). This is presumed to occur because larger nest-holding males obtain more eggs, i.e., larger females. This preference of sneaker males for larger nest-holding males may be the primary reason for the loss of the size-advantage in fertilization success. The accessibility to the nest by sneaker males is also influenced by the nest entrance width. For example, sneaker males of the cichlid fish Lamprologus lemairii more easily intrude into nests with wider entrance and achieve higher fertilization success (Ota et al., 2014 ). However, the paternity rate of B. fuscus nest-holding males was not affected by the size of the nest entrance. This is probably because, as mentioned above, sneaking in this species mostly occurs when nest-holding males are absent from the entrance (Takegaki et al. 2012 ). In cases where sperm competition among males occurs, fertilization success generally depends on the relative number of one's own sperm (Parker and Pizzari 2010 ). In fish, higher sperm competition risk or intensity predicts larger testis size, and larger testis size tends to result in higher fertilization success (Parker and Pizzari 2010 ). In B. fuscus , nest-holding males repeatedly release sperm during the repeated egg-laying by females over several hours (Nakanishi et al. 2017 ). Therefore, larger testis size, equipped with more sperm, would be expected to be advantageous. However, testis size did not have a significant effect on fertilization success in this species. The sperm longevity of nest-holding males in this species is long, with half surviving even 90 minutes after activation (Nakanishi and Takegaki 2019 ), suggesting they may not need to produce large quantities of sperm for additional release. Similarly, no association was found between testis size and fertilization success in nest-holding males of the grass goby, which also release sperm for extended periods within the nest (Pujolar et al. 2012 ). The number of sperm and sperm proportion within the nest for B. fuscus males are significantly influenced by the sperm removal behavior of nest-holding males. When nest-holding males detect the semen of sneaker males within the nest, they fan their fins to exchange the seawater in the nest, expelling the sperm and thereby increasing their own fertilization success (Takegaki et al. 2020 ; Kikuyama and Takegaki, 2023 ). This sperm-removal behavior results in the discharge of an average of over 80% of the sperm within the nest, including nest-holding males' own sperm. Therefore, it is considered to have a significant influence on the outcome of sperm competition in this species. The fact that the intensity of this sperm removal behavior is unrelated to the body size of nest-holding males might be a critical factor explaining the absence of correlation between fertilization success and body size. Possibility of alloparental care The extremely low paternity rate of nest-holding males observed in this study is unlikely to be due to issues with DNA analysis or sample bias because it has been confirmed in several cases. One possible mechanism is that the fertilization success was stolen by many sneaker males. Previous study at the same site confirmed that more than 20 sneaker males attempted to intrude into a nest during spawning, and that five sneaker males successively intruded into a nest (Ishibashi and Takegaki 2023 ). However, since two cases of zero paternity have been confirmed, this phenomenon cannot be explained solely by the sneak fertilization. Another possibility is the replacement of nest-holding males during the egg-tending period. The replacement of the male parent has been documented in many fish species exhibiting paternal care (Wisenden 1999 ; Avise et al. 2002 ; Coleman and Jones 2011 ), and some of these studies are based on extremely low paternity rates between attending males and eggs, similar to the present study (e.g., Dewoody et al. 2000 ; Withler et al. 2004 ; Bose et al., 2019 ). This replacement occurs either when a new male inherits the role following the abandonment of nest and eggs by original male or through nest takeover. In fathead minnow Pimephales promelas , the higher frequency of male replacement in population with lower density of spawning nests suggests the possibility of nest takeover due to nest scarcity (Bessert et al. 2007 ). In any case, the noteworthy aspect of the replacement is that the care of non-related eggs by new males, though in many species the new males partially consume the eggs. The most plausible reason for this is that females prefer to lay eggs in nests that already contain eggs, thus making it easy for males to acquire new females. In this study, B. fuscus nest-holding males were not individually identified, so it is unclear whether replacement of the males occurred during the egg-tending period. However, since nest-holding males of this species readily mate with new females when they have eggs in their nests (Nagase and Takegaki 2017 ), an adaptive explanation is possible for the care of eggs with extremely low paternity rates, including zero paternity, by new males. Reproductive success and conclusion Reproductive success, calculated from mating success, parental care success, and fertilization success, showed no significant relationship with the body size of nest-holding males. This result undoubtedly reflects the absence of the size-advantage in fertilization success, particularly influenced by the presence of individuals with extremely low paternity. If such low paternity is caused, as mentioned above, by sneaker males' preference for large nest-holding males, then the situation where large nest-holding males often exhibit low reproductive success is not particularly unexpected. When nest-holding males perceive that they have allowed many sneaker males to intrude and severely lost their own paternity, they may abandon parental care because the reproductive success obtained does not meet the costs of parental care, as demonstrated in some fishes (e.g., Neff and Gross 2001 ; Manica, 2004 ). This may trigger the replacement of nest-holding males described above. Moreover, the instability of size advantages in reproductive success of nest-holding males may lead larger males to abandon nest-holding tactics as not worth the effort. In fact, at our study site, unexplained disappearances of the largest nest-holding males very early in the breeding season have been observed (Takegaki et al. 2013 ). Although the fate of these large males could not be followed, sneaking by large males, including neighboring nest-holding males, was observed, and their fertilization success was confirmed (Takegaki unpublished data). It is possible that they are changing tactics again, reversely from nest-holding to sneaking. Detailed field studies with individual identification are needed going forward. Declarations Supplementary Information The online version contains supplementary material available at XXXX Acknowledgements We thank the members of the Evolutionary and Behavioral Ecology Laboratory, Nagasaki University for their generous help in field and laboratory work. We also thank S. Muko for statistical advice. Author contributions Conceptualisation, S.K., and T.T.; Methodology, S.K., M.Y., N.S., and T.T.; Investigation, S.K., and N.S.; Writing – Original Draft, Y.K., N.S., and T.T.; Writing – Review and Editing, T.T.; Project Administration and Funding Acquisition, S.K. and T.T. Fundings This research was partially supported by the Mikimoto Fund for Marine Ecology to S.K. and the Sumitomo Foundation grant for Basic Science Research Projects (grant no. 130127) to TT. Data availability Data supporting the results can be found in the electronic supplementary material. Declarations Conflict of interest We declare no competing interests. Ethical approval These experiments and observations were approved by the Animal Care and Use Committee of the Faculty of Fisheries, Nagasaki University (permission no. NF-0008), in accordance with the Guidelines for Animal Experimentation of Faculty of Fisheries (fish, amphibians, and invertebrates), Nagasaki University. References Ahti PA, Kuparinen A, Uusi-Heikkilä S (2020) Size does matter — the eco-evolutionary effects of changing body size in fish. Environ Rev 28:311–324. https://doi.org/10.1139/er-2019-0076 Andersson MB (1994) Sexual selection. Princeton University Press, Princeton Arnott G, Elwood RW (2009) Assessment of fighting ability in animal contests. Anim Behav 77:991–1004. https://doi.org/10.1016/j.anbehav.2009.02.010 Avise JC, Jones AG, Walker D, DeWoody JA (2002) Genetic mating systems and reproductive natural histories of fishes: lessons for ecology and evolution. Annu Rev Genet 36:19–45. https://doi.org/10.1146/annurev.genet.36.030602.090831 Bessert ML, Brozek J, Orti G (2007) Impact of nest substrate limitations on patterns of illegitimacy in the fathead minnow Pimephales promelas . J Hered 98:716–722. https://doi.org/10.1093/jhered/esm092 Birkhead TR, Møller AP (1998) Sperm competition and sexual selection. Academic Press, New York Blanckenhorn WU (2000) The evolution of body size: what keeps organisms small? Q Rev Biol 75:385–407. https://doi.org/10.1086/393620 Blanckenhorn WU, Dixon AFG, Fairbairn DJ, Foellmer MW, Gibert P, van der Linde K, Meier R, Nylin S, Pitnick S, Schoff C, Signorelli M, Teder T, Wiklund C (2007) Proximate causes of Rensch’s rule: does sexual size dimorphism in arthropods result from sex differences in development time? Am Nat 169:245–257. https://doi.org/10.1086/510597 Bose APH (2022) Parent–offspring cannibalism throughout the animal kingdom: a review of adaptive hypotheses. Biol Rev 97:1868–1885. https://doi.org/10.1111/brv.12868 Bose APH, Lau MJ, Cogliati KM, Neff BD, Balshine S (2019) Cannibalism of young is related to low paternity and nest take-overs in an intertidal fish. Anim Behav 153:41–48. https://doi.org/10.1016/j.anbehav.2019.04.018 Clutton-Brock T, McAuliffe K (2009) Female mate choice in mammals. Q Rev Biol 84:3–27. https://doi.org/10.1086/596461 Clutton-Brock TH (1991) The evolution of parental care. Princeton University Press, Princeton Coleman SW, Jones AG (2011) Patterns of multiple paternity and maternity in fishes. Biol J Linn Soc 103:735–760. https://doi.org/10.1111/j.1095-8312.2011.01673.x Crean AJ, Bonduriansky R (2014) What is a paternal effect? Trends Ecol Evol 29:554–559. https://doi.org/10.1016/j.tree.2014.07.009 Dewoody JA, Fletcher DE, Mackiewicz M, Wilkins SD, Avise JC (2000) The genetic mating system of spotted sunfish Lepomis punctatus . Mol Ecol 9:2119–2128. https://doi.org/10.1046/j.1365-294X.2000.01123.x Dougherty LR, Skirrow MJA, Jennions MD, Simmons LW (2022) Male alternative reproductive tactics and sperm competition: a meta-analysis. Biol Rev 97:1365–1388. https://doi.org/10.1111/brv.12846 Eberhard WG (1979) The function of horns in Podischnus agenor (Dynastinae) and other beetles. In: Blum MS, Blum NA (eds) Sexual selection and reproductive competition. Academic Press, New York, pp 231–258 Fairbairn DJ (1997) Allometry for sexual size dimorphism. Annu Rev Ecol Syst 28:659–687. https://doi.org/10.1146/annurev.ecolsys.28.1.659 Fairbairn DJ, Blanckenhorn WU, Székely T (2007) Sex, size and gender roles. Oxford University Press, Oxford Gross MR (1996) Alternative reproductive strategies and tactics: diversity within sexes. Trends Ecol Evol 11:92–98. https://doi.org/10.1016/01695347(96)81050-0 Gross MR (2005) The evolution of parental care. Q Rev Biol 80:37–45. https://doi.org/10.1086/431023 Hakkarainen H, Huhta E, Lahti K, Lundvall P, Mappes T, Tolonen P, Wiehn J (1996) A test of male mating and hunting success in the kestrel: the advantages of smallness? Behav Ecol Sociobiol 39:375–380. https://doi.org/10.1007/s002650050303 Hedrick AV, Temeles EJ (1989) The evolution of sexual dimorphism in animals: hypotheses and tests. Trends Ecol Evol 4:136–138. https://doi.org/10.1016/0169-5347(89)90212-7 Hishida Y (2002) Egg consumption by the female in the paternal brooding goby Bathygobius fuscus . Fish Sci 68:449–451. https://doi.org/10.1046/j.1444-2906.2002.00445.x Horne CR, Hirst AG, Atkinson D (2020) Selection for increased male size predicts variation in sexual size dimorphism among fish species. Proc R Soc B 287:20192640. https://doi.org/10.1098/rspb.2019.2640 Hunt J, Breuker CJ, Sadowski JA, Moore AJ (2009) Male–male competition, female mate choice and their interaction: determining total sexual selection. J Evol Biol 22:13–26. https://doi.org/10.1111/j.1420-9101.2008.01633.x Ishibashi R, Takegaki T (2023) Timing of sneaking behavior in the dusky frillgoby Bathygobius fuscus . Environ Biol Fish 106:93–100. https://doi.org/10.1007/s10641-022-01383-w Jennions MD, Petrie M (2000) Why do females mate multiply? A review of the genetic benefits. Biol Rev 75:21–64. https://doi.org/10.1017/S0006323199005423 Jones AG, Ratterman NL (2009) Mate choice and sexual selection: what have we learned since Darwin? Proc Natl Acad Sci USA 106:10001–10008. https://doi.org/10.1073/pnas.0901129106 Kalinowski ST, Taper ML, Marshall TC (2007) Revising how the computer program CERVUS accommodates genotyping error increases success in paternity assignment. Mol Ecol 16:1099–1106. https://doi.org/10.1111/j.1365-294X.2007.03089.x Kawase S, Hayashi T, Matsumoto Y, Takegaki T (2017) Testis size variation within sneaker males of the dusky frillgoby Bathygobius fuscus (Gobiidae): effects of within-tactic competition. Biol J Linn Soc 122:394–399. https://doi.org/10.1093/biolinnean/blx075 Kempenaers B, Verheyen GR, Dhondt AA (1995) Mate guarding and copulation behaviour in monogamous and polygynous blue tits: do males follow a best-of-a-badjob strategy? Behav Ecol Sociobiol 36:33–42. https://doi.org/10.1007/BF00175726 Kikuyama S, Takegaki T (2023) Sperm removal behavior by the nest-holding males of dusky frillgoby Bathygobius fuscus : a test against other nest-holding males. Bull Fac Fish Nagasaki Univ 103:1–4 (in Japanese with English abstract) Koprowski JL (1993) Alternative reproductive tactics in male eastern gray squirrels: “making the best of a bad job”. Behav Ecol 4:165–171. https://doi.org/10.1093/beheco/4.2.165 Kramer DL (1987) Dissolved oxygen and fish behavior. Environ Biol Fish 18:81–92. https://doi.org/10.1007/BF00002597 Künzler R, Bakker TCM (2000) Pectoral fins and paternal quality in sticklebacks. Proc R Soc B 267:999–1004. https://doi.org/10.1098/rspb.2000.1102 Kustra MC, Alonzo SH (2020) Sperm and alternative reproductive tactics: a review of existing theory and empirical data. Phil Trans R Soc B 375:20200075. https://doi.org/10.1098/rstb.2020.0075 Lande R (1980) Sexual dimorphism, sexual selection, and adaptation in polygenic characters. Evolution 34:292–305. https://doi.org/10.2307/2407393 Manica A (2002) Filial cannibalism in teleost fish. Biol Rev 77:261–277. https://doi.org/10.1017/S1464793101005905 Manica A (2004) Parental fish change their cannibalistic behaviour in response to the cost-to-benefit ratio of parental care. Anim Behav 67:1015–1021. https://doi.org/10.1016/j.anbehav.2003.09.011 Meunier B, White B, Corkum LD (2013) The role of fanning behavior in water exchange by a nest-guarding benthic fish before spawning. Limnol Oceanogr Fluids Environ 3:198–209. https://doi.org/10.1215/21573689-2413017 Møller A, Jennions M (2001) How important are direct fitness benefits of sexual selection? Naturwissenschaften 88:401–415. https://doi.org/10.1007/s001140100255 Montgomerie R, Fitzpatrick JL (2009) Testis, sperm and sperm competition. In: Jamieson BGM (ed) Reproductive biology and phylogeny of fishes. Science Publishers, New Hampshire, pp 1–53 Murase A, Sunobe T (2011) Interspecific territoriality in males of the tube blenny Neoclinus bryope (Actinopterygii: Chaenopsidae). J Ethol 29:467–472. https://doi.org/10.1007/s10164-011-0276-y Nagase S, Takegaki T (2017) Effect of the presence of eggs in the nests on additional mating of the dusky frillgoby Bathygobius fuscus . Bull Fac Fish Nagasaki Univ 98:1–4 (in Japanese with English abstract) Nakanishi Y, Kanatani Y, Takegaki T (2017) Prespawning sperm release behavior by the nest-holding males of dusky frillgoby Bathygobius fuscus . Bull Fac Fish Nagasaki Univ 98:5–9 (in Japanese with English abstract) Nakanishi A, Takegaki T (2019) Tactic‐specific sperm traits in the dusky frillgoby ( Bathygobius fuscus ). J Zool 307:71–77. https://doi.org/10.1111/jzo.12619 Neff BD, Gross MR (2001) Dynamic adjustment of parental care in response to perceived paternity. Proc R Soc B 268:1559–1565. https://doi.org/10.1098/rspb.2001.1678 Neff BD, Svensson EI (2013) Polyandry and alternative mating tactics. Phil Trans R Soc B 368:20120045. https://doi.org/10.1098/rstb.2012.0045 Oliveira RF, Taborsky M, Brockmann HJ (2008) Alternative reproductive tactics: an integrative approach. Cambridge University Press, Cambridge Ota K, Awata S, Morita M, Yokoyama R, Kohda M (2014) Territorial males can sire more offspring in nests with smaller doors in the cichlid Lamprologus lemairii . J Hered 105:416–422. https://doi.org/10.1093/jhered/esu010 Parker GA (1992) The evolution of sexual size dimorphism in fish. J Fish Biol 41:1–20. https://doi.org/10.1111/j.1095-8649.1992.tb03864.x Parker GA (1998) Sperm competition and the evolution of ejaculates: towards a theory base. In: Birkhead TR, Møller AP (eds) Sperm competition and sexual selection. Academic Press, New York, pp 3–54 Parker GA, Pizzari T (2010) Sperm competition and ejaculate economics. Biol Rev 85:897–934.https://doi.org/10.1111/j.1469-185X.2010.00140.x Peters RH (1983) The ecological implications of body size. Cambridge University Press, Cambridge Petersen CW, Warner RR (1998) Sperm competition in fishes. In: Birkhead TR, Møller AP (eds) Sperm competition and sexual selection. Academic Press, New York, pp 436–463 Porter BA, Fiumera AC, Avise JC (2002) Egg mimicry and allopaternal care: two mate-attracting tactics by which nesting striped darter ( Etheostoma virgatum ) males enhance reproductive success. Behav Ecol Sociobiol 51:350–359.https://doi.org/10.1007/s00265-002-0456-4 Pujolar JM, Locatello L, Zane L, Mazzoldi C (2012) Body size correlates with fertilization success but not gonad size in grass goby territorial males. PLOS ONE 7:e46711. https://doi.org/10.1371/journal.pone.0046711 R Development Core Team (2025) R: a language and environment for statistical computing, version 4.5.2. R Foundation for Statistical Computing, Vienna Reynolds DJ, Gross RM (1992) Female mate preference enhances offspring growth and reproduction in a fish, Poecilia reticulata . Proc R Soc Lond B 250:57–62. https://doi.org/10.1098/rspb.1992.0130 Roff D (1993) Evolution of life histories: theory and analysis. Springer, New York Roff DA (2002) Life history evolution. Sinauer, Sunderland Rollinson N, Rowe L (2016) The positive correlation between maternal size and offspring size: fitting pieces of a life-history puzzle. Biol Rev 91:1134–1148. https://doi.org/10.1111/brv.12214 Rowley AG, Daly-Engel TS, Fitzpatrick JL (2019) Testes size increases with sperm competition risk and intensity in bony fish and sharks. Behav Ecol 30:364–371. https://doi.org/10.1093/beheco/ary174 Royle NJ, Smiseth PT, Kölliker M (2012) The evolution of parental care. Oxford University Press, Oxford Sato N, Kasugai T, Munehara H (2014) Female pygmy squid cryptically favour small males and fast copulation as observed by removal of spermatangia. Evol Biol 41:221–228. https://doi.org/10.1007/s11692-013-9261-4 Shine R (1989) Ecological causes for the evolution of sexual dimorphism: a review of the evidence. Q Rev Biol 64:419–461. https://doi.org/10.1086/416458 Shine R (1994) Sexual size dimorphism in snakes revisited. Copeia 1994:326–346. https://doi.org/10.2307/1446982 Shuster SM, Wade MJ (2003) Mating systems and strategies. Princeton University Press, Princeton Snook RR (2005) Sperm in competition: not playing by the numbers. Trends Ecol Evol 20:46–53.https://doi.org/10.1016/j.tree.2004.10.011 Stiver KA, Alonzo SH (2011) Alloparental care increases mating success. Behav Ecol 22:206–211.https://doi.org/10.1093/beheco/arq186 Svensson O, Kvarnemo C (2023) How sexual and natural selection interact and shape the evolution of nests and nesting behaviour in fishes. Phil Trans R Soc B 378:20220139. https://doi.org/10.1098/rstb.2022.0139 Taborsky M (1994) Alternative life-history patterns. In: Thompson NS (ed) Advances in the study of behavior, vol 23.Academic Press, San Diego, pp 1–111 Taborsky M (1998) Sperm competition in fish: 'bourgeois' males and parasitic spawning. Trends Ecol Evol 13:222–227.https://doi.org/10.1016/S0169-5347(97)01318-9 Takegaki T, Kaneko T, Matsumoto Y (2012) Large- and small-size advantages in sneaking behaviour in the dusky frillgoby Bathygobius fuscus . Naturwissenschaften 99:285–289. https://doi.org/10.1007/s00114-012-0899-z Takegaki T, Kaneko T, Matsumoto Y (2013) Tactic changes in dusky frillgoby Bathygobius fuscus sneaker males. J Fish Biol 82:475–491. https://doi.org/10.1111/j.1095-8649.2012.03496.x Takegaki T, Nakanishi A, Kanatani Y, Kawase S, Yoshida M, Sato N (2020) Evidence of sperm removal behaviour in an externally fertilizing species. Proc R Soc B 287:20202004. https://doi.org/10.1098/rspb.2020.2004 Taru M, Kanda T, Sunobe T (2002) Alternative mating tactics of the gobiid fish Bathygobius fuscus . J Ethol 20:9–12.https://doi.org/10.1007/s10164-002-0047-x Tomkins JL, Hazel W (2007) The status of the conditional evolutionarily stable strategy. Trends Ecol Evol 22:522–528.https://doi.org/10.1016/j.tree.2007.09.002 Voigt CC, Heckel G, Mayer F (2005) Sexual selection favours small and symmetric males in the polygynous greater sac-winged bat Saccopteryx bilineata (Emballonuridae, Chiroptera). Behav Ecol Sociobiol 57:457–464.https://doi.org/10.1007/s00265-004-0874-6 Wantola K, Clelland E, Grande G, Corkum LD (2013) Fin size and associated fanning behaviour as indicators of reproductive status in male round gobies ( Neogobius melanostomus ). Behaviour 150:1623–1639.https://doi.org/10.1163/1568539X-00003113 Wiegmann DD, Baylis JR (1995) Male body size and paternal behaviour in smallmouth bass Micropterus dolomieui .Anim Behav 50:1543–1555. https://doi.org/10.1016/0003-3472(95)80010-7 Wisenden BD (1999) Alloparental care in fishes. Rev Fish Biol Fish 9:45–70. https://doi.org/10.1023/A:1008865801329 Withler RE, King JR, Marliave JB, Beaith B, Li S, Supernault KJ, Miller KM (2004) Polygamous mating and high levels of genetic variation in lingcod, Ophiodon elongatus , of the Strait of Georgia, British Columbia. Environ Biol Fish 69:345–357. https://doi.org/10.1023/B:EBFI.0000022898.49007.bc Wong BBM, Candolin U (2005) How is female mate choice affected by male competition? Biol Rev 80:559–571.https://doi.org/10.1017/S1464793105006809 Table Tables 1 is available in the supplementary files section Supplementary Files Table1.pdf KawaseTableS1.pdf Kawaseanalysis.docx Kawasedata.csv Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 14 Feb, 2026 Reviewers invited by journal 10 Feb, 2026 Editor assigned by journal 10 Feb, 2026 First submitted to journal 07 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8818992","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":589242591,"identity":"5550e66a-7162-4143-a0f1-70f37442d93c","order_by":0,"name":"Shoma Kawase","email":"","orcid":"","institution":"Nagasaki Daigaku - Bunkyo Chiku Campus: Nagasaki Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Shoma","middleName":"","lastName":"Kawase","suffix":""},{"id":589242592,"identity":"806f5ab5-657d-4661-a8cc-65cc356fc0e2","order_by":1,"name":"Noriyosi Sato","email":"","orcid":"","institution":"Tokai University - Shimizu Campus: Tokai Daigaku - 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In reproduction, larger body size confers advantages in gamete production for females and in competition over reproductive resources and females for males (Lande \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1980\u003c/span\u003e; Hedrick and Temeles \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Parker \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Blanckenhorn \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Rollinson and Rowe \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Moreover, in species exhibiting parental care, larger individuals obtain higher parental care success through their ability to defend, feed, and more (Clutton-Brock \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Gross \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Royle et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). These body size-related advantages could also benefit their mates, thus body size is likely to be an indicator trait for mate choice (Andersson, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). The evolution of sexual size dimorphism, particularly male-biased, is considered largely influenced by female mate choice (insect, Blanckenhorn et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; fish, Horne et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; bird, Fairbairn et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; reptile, Shine \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; mammal, Clutton-Brock and McAuliffe \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Male size advantages produce individual differences in reproductive success among males within a breeding population, thereby influencing the evolution of male life history strategies, reproductive tactics, and reproductive traits. The evolution of alternative reproductive tactics by small, subordinate males is one such example.\u003c/p\u003e \u003cp\u003eAlternative reproductive tactics refer to different reproductive phenotypes within a species (Gross, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1996\u003c/span\u003e), which are maintained to contribute to maximizing individual fitness (Koprowski, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Kempenaers et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Gross, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Shuster and Wade, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The difference in reproductive phenotype is usually observed in males. While different tactics coexist as genetic polymorphisms (i.e., strategy), most are tactics that evolved to increase fitness under the conditions faced by socially subordinate males (Tomkins and Hazel \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Such subordinate males often have tactic-specific traits\u0026mdash;such as morphology, coloration, or behavior\u0026mdash;to increase their success. For example, small males of the beetle species \u003cem\u003ePodischnus agenor\u003c/em\u003e adopt a tactic of widely searching for females, avoiding combat with larger males without developing horns as weapons\u003c/p\u003e \u003cp\u003e(Eberhard, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1979\u003c/span\u003e). Furthermore, the change to dominant tactics occurs in response to changes in social status (Gross \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Neff and Svensson \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Since social status of individuals strongly depends on body size, to understand what tactics individuals adopt and when they change tactics, it is essential to clarify how body size affects the reproductive success of each tactic.\u003c/p\u003e \u003cp\u003eParasitic tactics such as sneaking and satellite tactics, in which a subordinate male exploits opportunity in dominant male to mate with a female, are primary alternative reproductive tactics in many taxa (Oliveira et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Dougherty et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Particularly in fish, where many species exhibit external fertilization, this is the most common alternative tactic (Taborsky \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Oliveira et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), and consequently results in intense sperm competition (Taborsky \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Kustra and Alonzo \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Dougherty et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Dominant males aggressively attack and drive away sneaker males attempting to participate in reproduction to defend their paternity (fertilization success). On the other hand, in some species, sneaker males display female-like coloration and behavior to avoid attacks from dominant males (Oliveira et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Since sneaker males always face sperm competition with dominant males, they generally have larger testes than dominant males. They increase their fertilization success by producing and storing more sperm to achieve higher ejaculate volumes and more frequent sperm release (Parker \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Petersen and Warner \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Taborsky \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Parker and Pizzari \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Dougherty et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Furthermore, some species are known to have evolved sperm characteristics favorable for fertilization, such as high swimming speed and long lifespan (Birkhead and M\u0026oslash;ller \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Snook \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Montgomerie and Fitzpatrick \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Kustra and Alonzo \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Despite having these tactic-specific traits, sneaker males in fish usually have extremely low fertilization success (Avise et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Coleman and Jones \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), and are considered to be at a disadvantage in reproduction (Oliveira et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe relatively small males of the dusky frillgoby \u003cem\u003eBathygobius fuscus\u003c/em\u003e, a small coastal marine fish, adopt a sneaking tactic where they intrude into the spawning pairs of a large nest-holding male and a female to release their sperm (Taru et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Takegaki et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Sneaker males are not only attacked by the nest-holding males during spawning, but also compete with other sneaker males for sneaking opportunities. Consequently, their chances and time for intrusion and fertilization are highly limited, and their reproductive success is predicted to be lower than that of nest-holding males (Takegaki et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). This is also indicated by the fact that sneaker males change tactics to nest-holding during the breeding season (Taru et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Takegaki et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). However, on the other hand, in both field observations and aquarium experiments, sneaker males do not always change tactics to nest-holding when nests and females are available (Takegaki et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). This decision-making by sneaker males is thought to be due to the possibility that participating in reproduction as small nest-holding males may not obtain high reproductive success: for example, because of their low mating success, limited parental care capacity, and poor defense against sneaker males (Takegaki et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). To investigate these possibilities, this study aims to demonstrate the effects of body size on mating success, parental care success, fertilization success, and reproductive success in nest-holding males, as well as to determine extent to which sneaker males ensure fertilization success.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy sites\u003c/h2\u003e \u003cp\u003eField investigations were conducted in two tidepools on the Miezaki Coast (32\u0026deg;48' N; 129\u0026deg;44' E) of Nagasaki Prefecture, Japan (surface areas at low tide: about 44.0 and 84.4 m\u003csup\u003e2\u003c/sup\u003e; depths at low tide: about 0.3 and 0.5 m). Breeding season for dusky frillgoby at study site is from late May to mid-September (Takegaki et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Ishibashi and Takegaki \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), spawning cycle is synchronized with semi-luna cycle (Taru et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Investigations for this study were conducted for 57 days at low tide during three days before and after high tide from May 29 to September 8, including high tide during day.\u003c/p\u003e \u003cp\u003eArtificial nests (n\u0026thinsp;=\u0026thinsp;30) were placed in study sites to measure mating and parental care success of nest-holding males of this species. Artificial nests consisted of a PVC pipe (internal diameter 40 mm, length 120 mm) and two covers (Ishibashi and Takegaki \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), one cover was perforated to provide nest opening. In this species, larger nest-holding males preferentially use nests with larger nest opening (Takegaki et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), three different size of nest openings were set up (10 mm, 15 mm, and 20 mm; each 10 nests) to induce nesting in nest-holding males of various body sizes. In order to monitor presence and number of eggs caring, and to collect eggs for paternity analysis, transparent plastic sheets (120 mm \u0026times; 180 mm), which could be freely inserted and removed, were rolled up and inserted into artificial nests.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMating success\u003c/h3\u003e\n\u003cp\u003e໿Spawning in this species occurs in synchronization with semilunar periods, and nest-holding males repeat reproduction (i.e., spawning and egg care) several times during the breeding season. In this study, mating success of each male was evaluated based on the number of eggs obtained in a single reproductive cycle (n\u0026thinsp;=\u0026thinsp;29 males). Males usually mate with multiple females during the egg acquisition period (ca. 3 days) of a single breeding cycle (Nagase and Takegaki \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), during which the number of eggs increase but sometimes decrease due to predation by egg predators. Therefore, in this study, the number of eggs that reached its maximum during the egg acquisition period was considered as mating success of the male. The presence and the number of eggs were observed every day during the reproductive cycle, by withdrawing the plastic sheets in the artificial nests. If males have eggs, the egg sheet was photographed using a digital camera to count the number of eggs. Then, the egg sheet was returned to the nest, and the nest-holding male was confirmed to continue egg care. These series of procedures were repeated until the day before hatching of the eggs, and the eggs were collected for DNA paternity analysis; they were hatched in the laboratory and ໿the newly hatched larvae were anaesthetized with quinaldine (600 ppm) and fixed with 99% ethanol for DNA analysis. The digital images of eggs were uploaded onto a personal computer, and they were counted using counting software (Kachikachi counter 2.6; GT).\u003c/p\u003e \u003cp\u003eTo examine the effects of traits of the nest-holding males on their reproductive success, they were collected and sacrificed with a lethal dose of quinaldine (1250 ppm). The total length (TL, 0.1 mm), body weight (0.01 g) and testis weight (0.0001 g) were measured, and condition factor, as an index of body condition was calculated from body weight and TL, and gonadosomatic index (GSI), as an index of testis investment was calculated from body weight and testis weight (Kawase et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). For DNA analysis, ໿a piece of second dorsal fin was obtained and fixed in 99% ethanol.\u003c/p\u003e\n\u003ch3\u003eParental care success\u003c/h3\u003e\n\u003cp\u003eParental care success of the males was evaluated by the mean daily survival rate of eggs from the day reaching maximum number of eggs until the day before hatching.\u003c/p\u003e\n\u003ch3\u003eFertilization success\u003c/h3\u003e\n\u003cp\u003eFertilization success of the nest-holding males was measured for 13 nest-holding males randomly selected from the 29 individuals for which mating and parental care success were measured. For genetic analysis, we used five DNA markers (kumohaze2, kumohaze13, kumohaze15, kumohaze16, kumohaze17), and genotyped 13 datasets consisting of 13 nest-holding males and 1169 embryos (84\u0026ndash;94 embryos per nest) (electronic supplementary material, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDNA was extracted using a commercial kit (KAPA Express Extract, Roche Diagnostics, Basel, Switzerland) according to the manufacturer\u0026rsquo;s protocol. Polymerase chain reaction (PCR) was performed using fluorescent primers 5'-labelled with NED, HEX, PET, or FAM (Applied Biosystems), and PCR fragment sizes were determined using a genetic analyser (ABI Prism 3730xl DNA Analyzer, Applied Biosystems, USA). The PCR mixture contained 1.0 \u0026micro;L Taq buffer, 0.8 \u0026micro;L of each dNTP, 1 unit of bioTaq DNA polymerase (Bioline), 0.25 \u0026micro;L of each primer, 3.0 \u0026micro;L of template DNA, and distilled water to a final volume of 10 \u0026micro;L. Thermal cycling conditions consisted of an initial denaturation at 94\u0026deg;C for 60 s, followed by 22\u0026ndash;25 cycles of 94\u0026deg;C for 30 s, primer-specific annealing temperature for 30 s, and 72\u0026deg;C for 60 s, with a final extension at 72\u0026deg;C for 5 min.\u003c/p\u003e \u003cp\u003ePaternity was inferred using an exclusion method (Jones and Wang, 2010). The combined non-exclusion probability for the first parent across the five microsatellite loci was 0.003, as estimated using CERVUS 3.0 (Kalinowski et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), indicating high power for paternity assignment.\u003c/p\u003e\n\u003ch3\u003eReproductive success\u003c/h3\u003e\n\u003cp\u003eReproductive success of the nest-holding males (n\u0026thinsp;=\u0026thinsp;13) was calculated by multiplying the number of eggs survived until the day before hatching\u0026mdash;derived from their mating success and parental care success\u0026mdash;by the paternity rate.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eIn this study, statistical analyses were conducted using generalized linear models (GLMs) to evaluate the multivariate effects of potentially relevant traits (body size, body condition, GSI, and nest opening size) of nest-holding males on mating success, parental care success, fertilization success, and reproductive success. For each response variable, no more than three explanatory variables were included in the model, given the sample size. Explanatory variables were selected after assessing multicollinearity among candidate variables using variance inflation factors (VIFs), with all selected variables showing VIF values\u0026thinsp;\u0026lt;\u0026thinsp;3. The explanatory variables included for each response variable were male body size, body condition, and nest opening size for mating success, parental care success and reproductive success, and male body size, GSI, and nest opening size for fertilization success. In this study, two nest-holding males with zero paternity were confirmed (see Results). Based on previous reports (Wisenden \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Avise et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Coleman and Jones \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), this strongly suggested the occurrence of replacement of the male parent during the egg-tending period. Therefore, analyses excluding these two cases were also conducted.\u003c/p\u003e \u003cp\u003eSpecifically, the effects of body size (total length), body condition, and nest opening size on the number of eggs acquired by nest-holding males were analyzed using a GLM with a quasipoisson error distribution and a log link function. The effects of body size, body condition, and nest opening size on the survival rate of cared eggs were analyzed using a GLM with a quasibinomial error distribution and a logit link function. The effects of body size, nest opening size, and GSI on the paternity rate of nest-holding males were analyzed using a GLM with a quasibinomial error distribution and a logit link function. The effects of body size, body condition, and nest opening size on the reproductive success were analyzed using a GLM with a quasipoisson error distribution and a log link function. The significance of explanatory variables in these analyses was assessed using F-tests.\u003c/p\u003e \u003cp\u003eAll statistical analyses were performed using R (version 4.5.2; R Development Core Team, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eA significant effect on the number of eggs acquired by nest-holding males (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u0026thinsp;=\u0026thinsp;24878\u0026thinsp;\u0026plusmn;\u0026thinsp;13320 eggs, range\u0026thinsp;=\u0026thinsp;2733\u0026ndash;49689 eggs, n\u0026thinsp;=\u0026thinsp;29) was detected only for size: i.e., larger males acquired more eggs, whereas body condition and nest entrance size tended to be higher in larger males but did not significantly affect the number of eggs acquired (Table\u0026nbsp;1, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Similarly, the survival rate of eggs (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u0026thinsp;=\u0026thinsp;73.6\u0026thinsp;\u0026plusmn;\u0026thinsp;15.4%, range\u0026thinsp;=\u0026thinsp;35.8\u0026ndash;95.6%, n\u0026thinsp;=\u0026thinsp;29) was significantly higher for larger males, but body condition and nest opening size did not affect the survival rate of eggs (Table\u0026nbsp;1, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The paternity rate of nest-holding males (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u0026thinsp;=\u0026thinsp;43.2\u0026thinsp;\u0026plusmn;\u0026thinsp;27.8%, range\u0026thinsp;=\u0026thinsp;0.0\u0026ndash;90.5%, n\u0026thinsp;=\u0026thinsp;13) was not affected by their body size, nest opening size and testis size (GSI) (Table\u0026nbsp;1, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). The reproductive success (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u0026thinsp;=\u0026thinsp;6904\u0026thinsp;\u0026plusmn;\u0026thinsp;7123 eggs, range\u0026thinsp;=\u0026thinsp;0\u0026ndash;22085 eggs, n\u0026thinsp;=\u0026thinsp;13) was not affected by their body size, body condition and nest entrance size (Table\u0026nbsp;1, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). In analyses excluding the two males in which paternity was not detected, there were no changes in any variable effects except for the effect of nest-entrance size on reproductive success (Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study demonstrated that larger body size of \u003cem\u003eBathygobius fuscus\u003c/em\u003e nest-holding males confers advantages in mating success and parental care success. However, this size-advantage was not observed in fertilization success, and consequently, there was no significant relationship between body size and reproductive success.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMating success\u003c/h2\u003e \u003cp\u003eThere are two possible mechanisms for the higher mating success of larger \u003cem\u003eB. fuscus\u003c/em\u003e nest-holding males: i.e., intra-sexual selection and inter-sexual selection. In general, large body size is advantageous for male-male competition over reproductive resources, such as females and territory (Wong and Candolin \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Hunt et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Arnott and Elwood \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), and thus male size dimorphism is more likely to evolve in such species (Shine \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Fairbairn \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Fairbairn et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). In \u003cem\u003eB. fuscus\u003c/em\u003e, competition among nest-holding males over females has not been observed; however, they show preferences for the entrance size of spawning nests (Takegaki et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and frequently compete over nests (Taru et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Because larger males have an advantage in the competition (Taru et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), if females prefer to lay eggs in such nests, the size advantage in mating success detected in this study may be partially influenced by intra-sexual selection.\u003c/p\u003e \u003cp\u003eAnother possibility is female preference for larger males: i.e., inter-sexual selection. Male body size is one of the primary traits in female mate choice (Andersson \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Roff \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), and larger males are preferred in almost all cases (but see, Hakkarainen et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Voight et al. 2005; Sato et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Females gain direct benefits from mating with larger males such as through the higher quality of reproductive resources and higher ability of parental care (M\u0026oslash;ller and Jennions \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Jones and Ratterman \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), or indirect genetic benefits such as higher offspring growth rates and survival rates (Jennions and Petrie \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Jones and Ratterman \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Crean and Bonduriansky \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). For example, in the smallmouth bass \u003cem\u003eMicropterus dolomieu\u003c/em\u003e, larger males are preferred by females due to their higher ability to care for offspring, resulting in greater mating success. (Wiegmann and Baylis \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Also in the Trinidadian guppy \u003cem\u003ePoecilia reticulata\u003c/em\u003e, larger males are preferred by females, and their daughters grow well and are larger in body size, thus having larger reproductive output (Reynolds and Gross \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). The higher ability of parental care of larger \u003cem\u003eB. fuscus\u003c/em\u003e nest-holding males demonstrated in this study (see below) suggests that females may prefer to mate with larger males. In addition, since it has been suggested that females of this species may prefer males already tending eggs in the nests (Nagase and Takegaki \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), if larger males can acquire eggs earlier during each reproductive cycle, the size-advantage in mating is expected to be greater.\u003c/p\u003e \u003cp\u003eOn the other hand, regardless of female preference for larger males, there may be constraints on the mating success of smaller males. Since nest-holding males of this species prefer to use nests with entrance sizes assortative to their own body size (Takegaki et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), smaller males using nests with smaller entrances are physically unable to accept larger females. Large females cannot enter the small-entrance artificial nests used in this study, suggesting that this unavoidable constraint may influence the size-dependent mating success of nest-holding males in this species.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eParental care success\u003c/h2\u003e \u003cp\u003eSurvival rate of eggs until the day before hatching was higher in larger nest-holding males. In many taxa including fish, parents with larger body size generally exhibit higher ability to care for their offspring (Clutton-Brock \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Svensson and Kvarnemo \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Potential causes of mortality of eggs tended by \u003cem\u003eB. fuscus\u003c/em\u003e nest-holding males are predation by egg predators and other individuals of \u003cem\u003eB. fuscus\u003c/em\u003e (Hishida \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Nagase and Takegaki \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), filial cannibalism by caregiving males (Hishida \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Nagase and Takegaki \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), oxygen deprivation due to lack of parental care (Takegaki et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In this study, carnivorous snails (\u003cem\u003eErgalatax contractus\u003c/em\u003e and \u003cem\u003eThais clavigera\u003c/em\u003e) preying on the eggs of this species (Hishida \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Nagase and Takegaki \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) were observed in the nests where eggs had disappeared before hatching. Since snails were observed forcibly intruding into the tight nests of a blenny \u003cem\u003eNeoclinus bryope\u003c/em\u003e, driving out the defending males to eat the eggs (Murase and Sunobe \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), larger males would likely have an advantage in egg defense.\u003c/p\u003e \u003cp\u003eMales of this species are known to eat a part of their own eggs during the period of egg care (Hishida \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), effect of male body size on the intensity of filial cannibalism has not been reported. The partial filial-cannibalism is generally considered as a strategy for poor-condition parents to continue providing egg care by obtaining nutrients; consequently, in many fish species, the intensity of partial filial-cannibalism strongly depends on male body condition rather than body size (Manica \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Bose \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In this study, there was no significant effect of male body condition on egg survival rate (Table\u0026nbsp;1), the proportion of eggs lost due to cannibalism may not be high.\u003c/p\u003e \u003cp\u003eIn species that provide egg care in enclosed nests similar to \u003cem\u003eB. fuscus\u003c/em\u003e, water exchange in the nest to supply oxygen to the eggs is critically important for egg survival (Kramer \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). Fanning behavior to exchange nest water is known to be more effective in large males with large pectoral fins in three-spined stickleback fish (K\u0026uuml;nzler and Bakker \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) and gobiid fishes (Meunier et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Wantola et al. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), resulting in higher survival rate of eggs (K\u0026uuml;nzler and Bakker \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Because nest-holding males of \u003cem\u003eB. fuscus\u003c/em\u003e tend to have specifically large pectoral fins compared to sneaker males (Takegaki, unpublished data), larger males may perform more effective water exchange through fanning.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFertilization success\u003c/h2\u003e \u003cp\u003eIn all egg masses tended by nest-holding males, the paternity of other males was detected. Paternity of nest-holding males in this species are considerably lower than that of fishes with the breeding system involving sneaking tactics (Avise et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Coleman and Jones \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), surprisingly several egg masses with zero paternity of nest-holding males were also confirmed (see below). Paternity analysis in this study could not detect how many males other than nest-holding males succeeded in fertilization, but it is assumed that multiple sneaker males were involved in fertilization in many cases. This is because, at the same study site, nest-holding males usually mate with multiple females during a tidal cycle (Nagase and Takegaki \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), and intrusions by sneaker males have been observed an average of 2.6 times per spawning event (Takegaki et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFertilization success of nest-holding males was not correlated with the body size, though their mating and parental care success were higher in larger individuals. These results remained unchanged even after excluding data with zero paternity, where replacement of nest-holding males was likely to have occurred. The fertilization success of nest-holding males would strongly depend on presence and intensity of sperm competition with sneaker males, so they should try to avoid or minimize the competition. For example, the high paternity rate of nest-holding males in the grass goby \u003cem\u003eZosterisessor ophiocephalus\u003c/em\u003e is thought to be due to their high defensive ability against sneaker males exhibited by larger males (Pujolar et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). But, in the case of \u003cem\u003eB. fuscus\u003c/em\u003e nest-holding males, their aggressive ability dependent on body size may not contribute much to defending against sneaker males, because intrusions by sneaker males mostly occur during moments when nest-holding males are absent from the nest entrance\u0026mdash;either chasing away other sneaker males or entering inside the nest probably to release sperm. Conversely, it is known that sneaker males of this species attempt more intrusions into the nests of larger nest-holding males (Ishibashi and Takegaki \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This is presumed to occur because larger nest-holding males obtain more eggs, i.e., larger females. This preference of sneaker males for larger nest-holding males may be the primary reason for the loss of the size-advantage in fertilization success.\u003c/p\u003e \u003cp\u003eThe accessibility to the nest by sneaker males is also influenced by the nest entrance width. For example, sneaker males of the cichlid fish \u003cem\u003eLamprologus lemairii\u003c/em\u003e more easily intrude into nests with wider entrance and achieve higher fertilization success (Ota et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). However, the paternity rate of \u003cem\u003eB. fuscus\u003c/em\u003e nest-holding males was not affected by the size of the nest entrance. This is probably because, as mentioned above, sneaking in this species mostly occurs when nest-holding males are absent from the entrance (Takegaki et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn cases where sperm competition among males occurs, fertilization success generally depends on the relative number of one's own sperm (Parker and Pizzari \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In fish, higher sperm competition risk or intensity predicts larger testis size, and larger testis size tends to result in higher fertilization success (Parker and Pizzari \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In \u003cem\u003eB. fuscus\u003c/em\u003e, nest-holding males repeatedly release sperm during the repeated egg-laying by females over several hours (Nakanishi et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Therefore, larger testis size, equipped with more sperm, would be expected to be advantageous. However, testis size did not have a significant effect on fertilization success in this species. The sperm longevity of nest-holding males in this species is long, with half surviving even 90 minutes after activation (Nakanishi and Takegaki \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), suggesting they may not need to produce large quantities of sperm for additional release. Similarly, no association was found between testis size and fertilization success in nest-holding males of the grass goby, which also release sperm for extended periods within the nest (Pujolar et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe number of sperm and sperm proportion within the nest for \u003cem\u003eB. fuscus\u003c/em\u003e males are significantly influenced by the sperm removal behavior of nest-holding males. When nest-holding males detect the semen of sneaker males within the nest, they fan their fins to exchange the seawater in the nest, expelling the sperm and thereby increasing their own fertilization success (Takegaki et al. \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kikuyama and Takegaki, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This sperm-removal behavior results in the discharge of an average of over 80% of the sperm within the nest, including nest-holding males' own sperm. Therefore, it is considered to have a significant influence on the outcome of sperm competition in this species. The fact that the intensity of this sperm removal behavior is unrelated to the body size of nest-holding males might be a critical factor explaining the absence of correlation between fertilization success and body size.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePossibility of alloparental care\u003c/h2\u003e \u003cp\u003eThe extremely low paternity rate of nest-holding males observed in this study is unlikely to be due to issues with DNA analysis or sample bias because it has been confirmed in several cases. One possible mechanism is that the fertilization success was stolen by many sneaker males. Previous study at the same site confirmed that more than 20 sneaker males attempted to intrude into a nest during spawning, and that five sneaker males successively intruded into a nest (Ishibashi and Takegaki \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, since two cases of zero paternity have been confirmed, this phenomenon cannot be explained solely by the sneak fertilization. Another possibility is the replacement of nest-holding males during the egg-tending period. The replacement of the male parent has been documented in many fish species exhibiting paternal care (Wisenden \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Avise et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Coleman and Jones \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), and some of these studies are based on extremely low paternity rates between attending males and eggs, similar to the present study (e.g., Dewoody et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Withler et al. \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Bose et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This replacement occurs either when a new male inherits the role following the abandonment of nest and eggs by original male or through nest takeover. In fathead minnow \u003cem\u003ePimephales promelas\u003c/em\u003e, the higher frequency of male replacement in population with lower density of spawning nests suggests the possibility of nest takeover due to nest scarcity (Bessert et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). In any case, the noteworthy aspect of the replacement is that the care of non-related eggs by new males, though in many species the new males partially consume the eggs. The most plausible reason for this is that females prefer to lay eggs in nests that already contain eggs, thus making it easy for males to acquire new females. In this study, \u003cem\u003eB. fuscus\u003c/em\u003e nest-holding males were not individually identified, so it is unclear whether replacement of the males occurred during the egg-tending period. However, since nest-holding males of this species readily mate with new females when they have eggs in their nests (Nagase and Takegaki \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), an adaptive explanation is possible for the care of eggs with extremely low paternity rates, including zero paternity, by new males.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eReproductive success and conclusion\u003c/h2\u003e \u003cp\u003eReproductive success, calculated from mating success, parental care success, and fertilization success, showed no significant relationship with the body size of nest-holding males. This result undoubtedly reflects the absence of the size-advantage in fertilization success, particularly influenced by the presence of individuals with extremely low paternity. If such low paternity is caused, as mentioned above, by sneaker males' preference for large nest-holding males, then the situation where large nest-holding males often exhibit low reproductive success is not particularly unexpected.\u003c/p\u003e \u003cp\u003eWhen nest-holding males perceive that they have allowed many sneaker males to intrude and severely lost their own paternity, they may abandon parental care because the reproductive success obtained does not meet the costs of parental care, as demonstrated in some fishes (e.g., Neff and Gross \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Manica, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). This may trigger the replacement of nest-holding males described above. Moreover, the instability of size advantages in reproductive success of nest-holding males may lead larger males to abandon nest-holding tactics as not worth the effort. In fact, at our study site, unexplained disappearances of the largest nest-holding males very early in the breeding season have been observed (Takegaki et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Although the fate of these large males could not be followed, sneaking by large males, including neighboring nest-holding males, was observed, and their fertilization success was confirmed (Takegaki unpublished data). It is possible that they are changing tactics again, reversely from nest-holding to sneaking. Detailed field studies with individual identification are needed going forward.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe online version contains supplementary material available at XXXX\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the members of the Evolutionary and Behavioral Ecology Laboratory, Nagasaki University for their generous help in field and laboratory work. We also thank S. Muko for statistical advice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualisation, S.K., and T.T.; Methodology, S.K., M.Y., N.S., and T.T.; Investigation, S.K., and N.S.; Writing – Original Draft, Y.K., N.S., and T.T.; Writing – Review and Editing, T.T.; Project Administration and Funding Acquisition, S.K. and T.T.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFundings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was partially supported by the\u0026nbsp;Mikimoto\u0026nbsp;Fund for Marine Ecology to S.K. and the Sumitomo\u0026nbsp;Foundation grant for Basic\u0026nbsp;Science\u0026nbsp;Research\u0026nbsp;Projects (grant no.\u003cbr\u003e130127) to TT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData supporting the results can be found in the electronic supplementary material.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThese experiments and observations were approved by the Animal Care and Use Committee of the Faculty of Fisheries, Nagasaki University (permission no. NF-0008), in accordance with the Guidelines for Animal Experimentation of Faculty of Fisheries (fish, amphibians, and invertebrates), Nagasaki University.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAhti PA, Kuparinen A, Uusi-Heikkil\u0026auml; S (2020) Size does matter \u0026mdash; the eco-evolutionary effects of changing body size in fish. Environ Rev 28:311\u0026ndash;324. https://doi.org/10.1139/er-2019-0076\u003c/li\u003e\n \u003cli\u003eAndersson MB (1994) Sexual selection. Princeton University Press, Princeton\u003c/li\u003e\n \u003cli\u003eArnott G, Elwood RW (2009) Assessment of fighting ability in animal contests. Anim Behav 77:991\u0026ndash;1004. https://doi.org/10.1016/j.anbehav.2009.02.010\u003c/li\u003e\n \u003cli\u003eAvise JC, Jones AG, Walker D, DeWoody JA (2002) Genetic mating systems and reproductive natural histories of fishes: lessons for ecology and evolution. Annu Rev Genet 36:19\u0026ndash;45. https://doi.org/10.1146/annurev.genet.36.030602.090831\u003c/li\u003e\n \u003cli\u003eBessert ML, Brozek J, Orti G (2007) Impact of nest substrate limitations on patterns of illegitimacy in the fathead minnow \u003cem\u003ePimephales promelas\u003c/em\u003e. J Hered 98:716\u0026ndash;722. https://doi.org/10.1093/jhered/esm092\u003c/li\u003e\n \u003cli\u003eBirkhead TR, M\u0026oslash;ller AP (1998) Sperm competition and sexual selection. Academic Press, New York\u003c/li\u003e\n \u003cli\u003eBlanckenhorn WU (2000) The evolution of body size: what keeps organisms small? Q Rev Biol 75:385\u0026ndash;407. https://doi.org/10.1086/393620\u003c/li\u003e\n \u003cli\u003eBlanckenhorn WU, Dixon AFG, Fairbairn DJ, Foellmer MW, Gibert P, van der Linde K, Meier R, Nylin S, Pitnick S, Schoff C, Signorelli M, Teder T, Wiklund C (2007) Proximate causes of Rensch\u0026rsquo;s rule: does sexual size dimorphism in arthropods result from sex differences in development time? Am Nat 169:245\u0026ndash;257. https://doi.org/10.1086/510597\u003c/li\u003e\n \u003cli\u003eBose APH (2022) Parent\u0026ndash;offspring cannibalism throughout the animal kingdom: a review of adaptive hypotheses. Biol Rev 97:1868\u0026ndash;1885. https://doi.org/10.1111/brv.12868\u003c/li\u003e\n \u003cli\u003eBose APH, Lau MJ, Cogliati KM, Neff BD, Balshine S (2019) Cannibalism of young is related to low paternity and nest take-overs in an intertidal fish. Anim Behav 153:41\u0026ndash;48. https://doi.org/10.1016/j.anbehav.2019.04.018\u003c/li\u003e\n \u003cli\u003eClutton-Brock T, McAuliffe K (2009) Female mate choice in mammals. Q Rev Biol 84:3\u0026ndash;27. https://doi.org/10.1086/596461\u003c/li\u003e\n \u003cli\u003eClutton-Brock TH (1991) The evolution of parental care. Princeton University Press, Princeton\u003c/li\u003e\n \u003cli\u003eColeman SW, Jones AG (2011) Patterns of multiple paternity and maternity in fishes. Biol J Linn Soc 103:735\u0026ndash;760. https://doi.org/10.1111/j.1095-8312.2011.01673.x\u003c/li\u003e\n \u003cli\u003eCrean AJ, Bonduriansky R (2014) What is a paternal effect? Trends Ecol Evol 29:554\u0026ndash;559. https://doi.org/10.1016/j.tree.2014.07.009\u003c/li\u003e\n \u003cli\u003eDewoody JA, Fletcher DE, Mackiewicz M, Wilkins SD, Avise JC (2000) The genetic mating system of spotted sunfish \u003cem\u003eLepomis punctatus\u003c/em\u003e. Mol Ecol 9:2119\u0026ndash;2128. https://doi.org/10.1046/j.1365-294X.2000.01123.x\u003c/li\u003e\n \u003cli\u003eDougherty LR, Skirrow MJA, Jennions MD, Simmons LW (2022) Male alternative reproductive tactics and sperm competition: a meta-analysis. Biol Rev 97:1365\u0026ndash;1388. https://doi.org/10.1111/brv.12846\u003c/li\u003e\n \u003cli\u003eEberhard WG (1979) The function of horns in \u003cem\u003ePodischnus agenor\u003c/em\u003e (Dynastinae) and other beetles. In: Blum MS, Blum NA (eds) Sexual selection and reproductive competition. Academic Press, New York, pp 231\u0026ndash;258\u003c/li\u003e\n \u003cli\u003eFairbairn DJ (1997) Allometry for sexual size dimorphism. Annu Rev Ecol Syst 28:659\u0026ndash;687. https://doi.org/10.1146/annurev.ecolsys.28.1.659\u003c/li\u003e\n \u003cli\u003eFairbairn DJ, Blanckenhorn WU, Sz\u0026eacute;kely T (2007) Sex, size and gender roles. Oxford University Press, Oxford\u003c/li\u003e\n \u003cli\u003eGross MR (1996) Alternative reproductive strategies and tactics: diversity within sexes. Trends Ecol Evol 11:92\u0026ndash;98. https://doi.org/10.1016/01695347(96)81050-0\u003c/li\u003e\n \u003cli\u003eGross MR (2005) The evolution of parental care. Q Rev Biol 80:37\u0026ndash;45. https://doi.org/10.1086/431023\u003c/li\u003e\n \u003cli\u003eHakkarainen H, Huhta E, Lahti K, Lundvall P, Mappes T, Tolonen P, Wiehn J (1996) A test of male mating and hunting success in the kestrel: the advantages of smallness? Behav Ecol Sociobiol 39:375\u0026ndash;380. https://doi.org/10.1007/s002650050303\u003c/li\u003e\n \u003cli\u003eHedrick AV, Temeles EJ (1989) The evolution of sexual dimorphism in animals: hypotheses and tests. Trends Ecol Evol 4:136\u0026ndash;138. https://doi.org/10.1016/0169-5347(89)90212-7\u003c/li\u003e\n \u003cli\u003eHishida Y (2002) Egg consumption by the female in the paternal brooding goby \u003cem\u003eBathygobius fuscus\u003c/em\u003e. Fish Sci 68:449\u0026ndash;451. https://doi.org/10.1046/j.1444-2906.2002.00445.x\u003c/li\u003e\n \u003cli\u003eHorne CR, Hirst AG, Atkinson D (2020) Selection for increased male size predicts variation in sexual size dimorphism among fish species. Proc R Soc B 287:20192640. https://doi.org/10.1098/rspb.2019.2640\u003c/li\u003e\n \u003cli\u003eHunt J, Breuker CJ, Sadowski JA, Moore AJ (2009) Male\u0026ndash;male competition, female mate choice and their interaction: determining total sexual selection. J Evol Biol 22:13\u0026ndash;26. https://doi.org/10.1111/j.1420-9101.2008.01633.x\u003c/li\u003e\n \u003cli\u003eIshibashi R, Takegaki T (2023) Timing of sneaking behavior in the dusky frillgoby \u003cem\u003eBathygobius fuscus\u003c/em\u003e. Environ Biol Fish 106:93\u0026ndash;100. https://doi.org/10.1007/s10641-022-01383-w\u003c/li\u003e\n \u003cli\u003eJennions MD, Petrie M (2000) Why do females mate multiply? A review of the genetic benefits. Biol Rev 75:21\u0026ndash;64. https://doi.org/10.1017/S0006323199005423\u003c/li\u003e\n \u003cli\u003eJones AG, Ratterman NL (2009) Mate choice and sexual selection: what have we learned since Darwin? Proc Natl Acad Sci USA 106:10001\u0026ndash;10008. https://doi.org/10.1073/pnas.0901129106\u003c/li\u003e\n \u003cli\u003eKalinowski ST, Taper ML, Marshall TC (2007) Revising how the computer program CERVUS accommodates genotyping error increases success in paternity assignment. Mol Ecol 16:1099\u0026ndash;1106. https://doi.org/10.1111/j.1365-294X.2007.03089.x\u003c/li\u003e\n \u003cli\u003eKawase S, Hayashi T, Matsumoto Y, Takegaki T (2017) Testis size variation within sneaker males of the dusky frillgoby \u003cem\u003eBathygobius fuscus\u003c/em\u003e (Gobiidae): effects of within-tactic competition. Biol J Linn Soc 122:394\u0026ndash;399. https://doi.org/10.1093/biolinnean/blx075\u003c/li\u003e\n \u003cli\u003eKempenaers B, Verheyen GR, Dhondt AA (1995) Mate guarding and copulation behaviour in monogamous and polygynous blue tits: do males follow a best-of-a-badjob strategy? Behav Ecol Sociobiol 36:33\u0026ndash;42. https://doi.org/10.1007/BF00175726\u003c/li\u003e\n \u003cli\u003eKikuyama S, Takegaki T (2023) Sperm removal behavior by the nest-holding males of dusky frillgoby \u003cem\u003eBathygobius fuscus\u003c/em\u003e: a test against other nest-holding males. Bull Fac Fish Nagasaki Univ 103:1\u0026ndash;4 (in Japanese with English abstract)\u003c/li\u003e\n \u003cli\u003eKoprowski JL (1993) Alternative reproductive tactics in male eastern gray squirrels: \u0026ldquo;making the best of a bad job\u0026rdquo;. Behav Ecol 4:165\u0026ndash;171. https://doi.org/10.1093/beheco/4.2.165\u003c/li\u003e\n \u003cli\u003eKramer DL (1987) Dissolved oxygen and fish behavior. Environ Biol Fish 18:81\u0026ndash;92. https://doi.org/10.1007/BF00002597\u003c/li\u003e\n \u003cli\u003eK\u0026uuml;nzler R, Bakker TCM (2000) Pectoral fins and paternal quality in sticklebacks. Proc R Soc B 267:999\u0026ndash;1004. https://doi.org/10.1098/rspb.2000.1102\u003c/li\u003e\n \u003cli\u003eKustra MC, Alonzo SH (2020) Sperm and alternative reproductive tactics: a review of existing theory and empirical data. Phil Trans R Soc B 375:20200075. https://doi.org/10.1098/rstb.2020.0075\u003c/li\u003e\n \u003cli\u003eLande R (1980) Sexual dimorphism, sexual selection, and adaptation in polygenic characters. Evolution 34:292\u0026ndash;305. https://doi.org/10.2307/2407393\u003c/li\u003e\n \u003cli\u003eManica A (2002) Filial cannibalism in teleost fish. Biol Rev 77:261\u0026ndash;277. https://doi.org/10.1017/S1464793101005905\u003c/li\u003e\n \u003cli\u003eManica A (2004) Parental fish change their cannibalistic behaviour in response to the cost-to-benefit ratio of parental care. Anim Behav 67:1015\u0026ndash;1021. https://doi.org/10.1016/j.anbehav.2003.09.011\u003c/li\u003e\n \u003cli\u003eMeunier B, White B, Corkum LD (2013) The role of fanning behavior in water exchange by a nest-guarding benthic fish before spawning. Limnol Oceanogr Fluids Environ 3:198\u0026ndash;209. https://doi.org/10.1215/21573689-2413017\u003c/li\u003e\n \u003cli\u003eM\u0026oslash;ller A, Jennions M (2001) How important are direct fitness benefits of sexual selection? Naturwissenschaften 88:401\u0026ndash;415. https://doi.org/10.1007/s001140100255\u003c/li\u003e\n \u003cli\u003eMontgomerie R, Fitzpatrick JL (2009) Testis, sperm and sperm competition. In: Jamieson BGM (ed) Reproductive biology and phylogeny of fishes. Science Publishers, New Hampshire, pp 1\u0026ndash;53\u003c/li\u003e\n \u003cli\u003eMurase A, Sunobe T (2011) Interspecific territoriality in males of the tube blenny \u003cem\u003eNeoclinus bryope\u003c/em\u003e (Actinopterygii: Chaenopsidae). J Ethol 29:467\u0026ndash;472. https://doi.org/10.1007/s10164-011-0276-y\u003c/li\u003e\n \u003cli\u003eNagase S, Takegaki T (2017) Effect of the presence of eggs in the nests on additional mating of the dusky frillgoby \u003cem\u003eBathygobius fuscus\u003c/em\u003e. Bull Fac Fish Nagasaki Univ 98:1\u0026ndash;4 (in Japanese with English abstract)\u003c/li\u003e\n \u003cli\u003eNakanishi Y, Kanatani Y, Takegaki T (2017) Prespawning sperm release behavior by the nest-holding males of dusky frillgoby \u003cem\u003eBathygobius fuscus\u003c/em\u003e. Bull Fac Fish Nagasaki Univ 98:5\u0026ndash;9 (in Japanese with English abstract)\u003c/li\u003e\n \u003cli\u003eNakanishi A, Takegaki T (2019) Tactic‐specific sperm traits in the dusky frillgoby (\u003cem\u003eBathygobius fuscus\u003c/em\u003e). J Zool 307:71\u0026ndash;77. https://doi.org/10.1111/jzo.12619\u003c/li\u003e\n \u003cli\u003eNeff BD, Gross MR (2001) Dynamic adjustment of parental care in response to perceived paternity. Proc R Soc B 268:1559\u0026ndash;1565. https://doi.org/10.1098/rspb.2001.1678\u003c/li\u003e\n \u003cli\u003eNeff BD, Svensson EI (2013) Polyandry and alternative mating tactics. Phil Trans R Soc B 368:20120045. https://doi.org/10.1098/rstb.2012.0045\u003c/li\u003e\n \u003cli\u003eOliveira RF, Taborsky M, Brockmann HJ (2008) Alternative reproductive tactics: an integrative approach. Cambridge University Press, Cambridge\u003c/li\u003e\n \u003cli\u003eOta K, Awata S, Morita M, Yokoyama R, Kohda M (2014) Territorial males can sire more offspring in nests with smaller doors in the cichlid \u003cem\u003eLamprologus lemairii\u003c/em\u003e. J Hered 105:416\u0026ndash;422. https://doi.org/10.1093/jhered/esu010\u003c/li\u003e\n \u003cli\u003eParker GA (1992) The evolution of sexual size dimorphism in fish. J Fish Biol 41:1\u0026ndash;20. https://doi.org/10.1111/j.1095-8649.1992.tb03864.x\u003c/li\u003e\n \u003cli\u003eParker GA (1998) Sperm competition and the evolution of ejaculates: towards a theory base. In: Birkhead TR, M\u0026oslash;ller AP (eds) Sperm competition and sexual selection. Academic Press, New York, pp 3\u0026ndash;54\u003c/li\u003e\n \u003cli\u003eParker GA, Pizzari T (2010) Sperm competition and ejaculate economics. Biol Rev 85:897\u0026ndash;934.https://doi.org/10.1111/j.1469-185X.2010.00140.x\u003c/li\u003e\n \u003cli\u003ePeters RH (1983) The ecological implications of body size. Cambridge University Press, Cambridge\u003c/li\u003e\n \u003cli\u003ePetersen CW, Warner RR (1998) Sperm competition in fishes. In: Birkhead TR, M\u0026oslash;ller AP (eds) Sperm competition and sexual selection. Academic Press, New York, pp 436\u0026ndash;463\u003c/li\u003e\n \u003cli\u003ePorter BA, Fiumera AC, Avise JC (2002) Egg mimicry and allopaternal care: two mate-attracting tactics by which nesting striped darter (\u003cem\u003eEtheostoma virgatum\u003c/em\u003e) males enhance reproductive success. Behav Ecol Sociobiol 51:350\u0026ndash;359.https://doi.org/10.1007/s00265-002-0456-4\u003c/li\u003e\n \u003cli\u003ePujolar JM, Locatello L, Zane L, Mazzoldi C (2012) Body size correlates with fertilization success but not gonad size in grass goby territorial males. PLOS ONE 7:e46711. https://doi.org/10.1371/journal.pone.0046711\u003c/li\u003e\n \u003cli\u003eR Development Core Team (2025) R: a language and environment for statistical computing, version 4.5.2. R Foundation for Statistical Computing, Vienna\u003c/li\u003e\n \u003cli\u003eReynolds DJ, Gross RM (1992) Female mate preference enhances offspring growth and reproduction in a fish, \u003cem\u003ePoecilia reticulata\u003c/em\u003e. Proc R Soc Lond B 250:57\u0026ndash;62. https://doi.org/10.1098/rspb.1992.0130\u003c/li\u003e\n \u003cli\u003eRoff D (1993) Evolution of life histories: theory and analysis. Springer, New York\u003c/li\u003e\n \u003cli\u003eRoff DA (2002) Life history evolution. Sinauer, Sunderland\u003c/li\u003e\n \u003cli\u003eRollinson N, Rowe L (2016) The positive correlation between maternal size and offspring size: fitting pieces of a life-history puzzle. Biol Rev 91:1134\u0026ndash;1148. https://doi.org/10.1111/brv.12214\u003c/li\u003e\n \u003cli\u003eRowley AG, Daly-Engel TS, Fitzpatrick JL (2019) Testes size increases with sperm competition risk and intensity in bony fish and sharks. Behav Ecol 30:364\u0026ndash;371. https://doi.org/10.1093/beheco/ary174\u003c/li\u003e\n \u003cli\u003eRoyle NJ, Smiseth PT, K\u0026ouml;lliker M (2012) The evolution of parental care. Oxford University Press, Oxford\u003c/li\u003e\n \u003cli\u003eSato N, Kasugai T, Munehara H (2014) Female pygmy squid cryptically favour small males and fast copulation as observed by removal of spermatangia. Evol Biol 41:221\u0026ndash;228. https://doi.org/10.1007/s11692-013-9261-4\u003c/li\u003e\n \u003cli\u003eShine R (1989) Ecological causes for the evolution of sexual dimorphism: a review of the evidence. Q Rev Biol 64:419\u0026ndash;461. https://doi.org/10.1086/416458\u003c/li\u003e\n \u003cli\u003eShine R (1994) Sexual size dimorphism in snakes revisited. Copeia 1994:326\u0026ndash;346. https://doi.org/10.2307/1446982\u003c/li\u003e\n \u003cli\u003eShuster SM, Wade MJ (2003) Mating systems and strategies. Princeton University Press, Princeton\u003c/li\u003e\n \u003cli\u003eSnook RR (2005) Sperm in competition: not playing by the numbers. Trends Ecol Evol 20:46\u0026ndash;53.https://doi.org/10.1016/j.tree.2004.10.011\u003c/li\u003e\n \u003cli\u003eStiver KA, Alonzo SH (2011) Alloparental care increases mating success. Behav Ecol 22:206\u0026ndash;211.https://doi.org/10.1093/beheco/arq186\u003c/li\u003e\n \u003cli\u003eSvensson O, Kvarnemo C (2023) How sexual and natural selection interact and shape the evolution of nests and nesting behaviour in fishes. Phil Trans R Soc B 378:20220139. https://doi.org/10.1098/rstb.2022.0139\u003c/li\u003e\n \u003cli\u003eTaborsky M (1994) Alternative life-history patterns. In: Thompson NS (ed) Advances in the study of behavior, vol 23.Academic Press, San Diego, pp 1\u0026ndash;111\u003c/li\u003e\n \u003cli\u003eTaborsky M (1998) Sperm competition in fish: \u0026apos;bourgeois\u0026apos; males and parasitic spawning. Trends Ecol Evol 13:222\u0026ndash;227.https://doi.org/10.1016/S0169-5347(97)01318-9\u003c/li\u003e\n \u003cli\u003eTakegaki T, Kaneko T, Matsumoto Y (2012) Large- and small-size advantages in sneaking behaviour in the dusky frillgoby \u003cem\u003eBathygobius fuscus\u003c/em\u003e. Naturwissenschaften 99:285\u0026ndash;289. https://doi.org/10.1007/s00114-012-0899-z\u003c/li\u003e\n \u003cli\u003eTakegaki T, Kaneko T, Matsumoto Y (2013) Tactic changes in dusky frillgoby \u003cem\u003eBathygobius fuscus\u003c/em\u003e sneaker males. J Fish Biol 82:475\u0026ndash;491. https://doi.org/10.1111/j.1095-8649.2012.03496.x\u003c/li\u003e\n \u003cli\u003eTakegaki T, Nakanishi A, Kanatani Y, Kawase S, Yoshida M, Sato N (2020) Evidence of sperm removal behaviour in an externally fertilizing species. Proc R Soc B 287:20202004. https://doi.org/10.1098/rspb.2020.2004\u003c/li\u003e\n \u003cli\u003eTaru M, Kanda T, Sunobe T (2002) Alternative mating tactics of the gobiid fish \u003cem\u003eBathygobius fuscus\u003c/em\u003e. J Ethol 20:9\u0026ndash;12.https://doi.org/10.1007/s10164-002-0047-x\u003c/li\u003e\n \u003cli\u003eTomkins JL, Hazel W (2007) The status of the conditional evolutionarily stable strategy. Trends Ecol Evol 22:522\u0026ndash;528.https://doi.org/10.1016/j.tree.2007.09.002\u003c/li\u003e\n \u003cli\u003eVoigt CC, Heckel G, Mayer F (2005) Sexual selection favours small and symmetric males in the polygynous greater sac-winged bat \u003cem\u003eSaccopteryx bilineata\u003c/em\u003e (Emballonuridae, Chiroptera). Behav Ecol Sociobiol 57:457\u0026ndash;464.https://doi.org/10.1007/s00265-004-0874-6\u003c/li\u003e\n \u003cli\u003eWantola K, Clelland E, Grande G, Corkum LD (2013) Fin size and associated fanning behaviour as indicators of reproductive status in male round gobies (\u003cem\u003eNeogobius melanostomus\u003c/em\u003e). Behaviour 150:1623\u0026ndash;1639.https://doi.org/10.1163/1568539X-00003113\u003c/li\u003e\n \u003cli\u003eWiegmann DD, Baylis JR (1995) Male body size and paternal behaviour in smallmouth bass \u003cem\u003eMicropterus dolomieui\u003c/em\u003e.Anim Behav 50:1543\u0026ndash;1555. https://doi.org/10.1016/0003-3472(95)80010-7\u003c/li\u003e\n \u003cli\u003eWisenden BD (1999) Alloparental care in fishes. Rev Fish Biol Fish 9:45\u0026ndash;70. https://doi.org/10.1023/A:1008865801329\u003c/li\u003e\n \u003cli\u003eWithler RE, King JR, Marliave JB, Beaith B, Li S, Supernault KJ, Miller KM (2004) Polygamous mating and high levels of genetic variation in lingcod, \u003cem\u003eOphiodon elongatus\u003c/em\u003e, of the Strait of Georgia, British Columbia. Environ Biol Fish 69:345\u0026ndash;357. https://doi.org/10.1023/B:EBFI.0000022898.49007.bc\u003c/li\u003e\n \u003cli\u003eWong BBM, Candolin U (2005) How is female mate choice affected by male competition? Biol Rev 80:559\u0026ndash;571.https://doi.org/10.1017/S1464793105006809\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTables 1 is available in the supplementary files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"marine-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mabi","sideBox":"Learn more about [Marine Biology](https://www.springer.com/journal/227)","snPcode":"227","submissionUrl":"https://submission.nature.com/new-submission/227/3","title":"Marine Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"alternative reproductive tactics, size-advantage, body size, paternity","lastPublishedDoi":"10.21203/rs.3.rs-8818992/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8818992/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBody size is a critical trait influencing reproductive success through mate competition, female choice, and parental care ability. In fish species with alternative reproductive tactics, large males typically adopt dominant tactics, while smaller males employ parasitic tactics, such as sneaking tactics. However, the reproductive advantage of larger males can be compromised by intense sperm competition with small males. We investigated the effects of body size on the reproductive success of nest-holding males in the dusky frillgoby \u003cem\u003eBathygobius fuscus\u003c/em\u003e, a species where small males frequently engage in sneaking tactics. Field investigations using artificial nests revealed that larger nest-holding males had significantly higher mating success (number of eggs acquired) and parental care success (egg survival rate). In contrast, DNA paternity analysis showed that fertilization success (paternity rate) was not correlated with male body size. This is because of the presence of males with extremely low paternity rate, including two cases of zero paternity, suggesting high sneaking pressure and replacement of nest-holding males (i.e., alloparental care). Consequently, the cumulative reproductive success of nest-holding males showed no significant relationship with body size. These results showed that the size-advantages of nest-holding males in attracting females and tending eggs are offset by the sperm competition with sneaker males. The lack of a clear size advantage in reproductive success may explain the plastic nature of reproductive tactics in this species, where large males may occasionally adopt sneaking tactics or abandon nests.\u003c/p\u003e","manuscriptTitle":"Size-advantage in nest-holding tactics in the dusky frillgoby Bathygobius fuscus males: mating success and parental care success but not fertilization success","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-16 17:18:10","doi":"10.21203/rs.3.rs-8818992/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-02-15T02:54:59+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-10T22:43:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-10T05:27:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"Marine Biology","date":"2026-02-07T23:01:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"marine-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mabi","sideBox":"Learn more about [Marine Biology](https://www.springer.com/journal/227)","snPcode":"227","submissionUrl":"https://submission.nature.com/new-submission/227/3","title":"Marine Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"65161a37-cb72-41b2-83ce-b9ac85d57f8c","owner":[],"postedDate":"February 16th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-28T09:41:24+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-16 17:18:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8818992","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8818992","identity":"rs-8818992","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}