Reproduction of marble-mouth frogfish Lophiocharon lithinostomus (Lophiiformes, Antennariidae) and the evolution of parental care among frogfishes

19 Here, we observed the reproductive behavior of marble -mouth frogfish ( Lophiocharon 20 lithinostomus) and the morphology of newly hatched juveniles under captive conditions. 21 Adult males showed pursuit behavior towards females approximately seven days before 22 spawning; spawning and ejaculation took place almost simultaneously. An adult female 23 cared for a fertilized egg mass adhered to their right of the left side of the flank. The 24 position of the adhered eggs on the flank was left-biased (3 on the right and 25 on the 25 left). The females exhibited a proactive fanning behavior towards the egg masses using 26 their dorsal fins; the fanning frequency increased over time after spawning. Meanwhile, 27 the males did not display any form of parental care behavior. The eggs had hook-like 28 structures that might enable the eggs to attach to the flank of the parent. Hatching 29 occurred between 22 and 28 days after spawning. Newly hatched juveniles already had 30 a full set of fin rays ; their morphology was similar to that of adult fish. Moreover, we 31 reviewed the evolution of parental care behaviors and egg types among frogfish family. 32 33

34 Antennariidae, aquarium experiment, early ontogeny, egg care, parental care35 Author-formatted, not peer-reviewed document posted on 06/04/2022. DOI:  https://doi.org/10.3897/arphapreprints.e84895

36 37 Antennariidae, the frogfish, belongs to the order Lophiiformes and consists of two 38 subfamilies: Antennariinae and Histiophryninae, with 14 genera (Pietsch and Arnold 39 2020). Frogfishes are found in all tropical and subtropical oceans and seas except for the 40 Mediterranean (Jordan and Richardson 1908; Pietsch 2004; Pietsch and Arnold 2020), 41 and they are predatory fishes (Arnold et al. 2014). Almost all frogfishes resemble 42 certain environmental objects, such as sponges, rocks, and coral (Arnold et al. 2014). 43 Cryptic colorations can help frogfishes avoid predation and misidentification by prey 44 (Arnold et al. 2014). Although the fishes in the family Antennariidae have diverse egg 45 types and reproductive behaviors (Pietsch and Grobecker 1980; Pietsch and Grobecker 46 1987; Kuiter 1993; Liem 1998; Pietsch et al. 2009; Arnold et al. 2014; Arnold and 47 Pietsch 2018; Pietsch and Arnold 2020; Bray and Gomon 2020), there are few detailed 48 studies on their reproduction. According to the few reports in the literature (Mito 1960; 49 Fries 1973; Pietsch and Grobecker 1987), the fishes in the family Antennariidae are 50 divided into two types of species that lay an aggregated pelagic egg (e.g., Antennarius 51 spp. and Histrio spp.) and demersal eggs (e.g., Histiophryne spp. and Lophiocharon 52 spp.) (Friese 1974; Molter 1983; Fujita and Uchida 1985; Arnold et al. 2014). 53 Author-formatted, not peer-reviewed document posted on 06/04/2022. DOI:  https://doi.org/10.3897/arphapreprints.e84895 54 Some species of Antennariidae demonstrate egg care behavior. For instance, 55 Lophiocharon spp. cares for a demersal egg mass adhered to the flank of the parents 56 (Pietsch and Grobecker, 1980). Nevertheless, the sex of the parent engaging in egg care 57 remains unclear. For example, Pietsch and Grobecker (1980) reported that only male L. 58 trisignatus could care for their eggs; however, Pietsch and Arnold (2020) later observed 59 that Lophiocharon trisignatus females cared for their eggs. This confusion is likely due 60 to the difficulty in determining the sex of frogfishes based on their external 61 morphologies, although the females are usually are bigger than the males (Pietsch et al. 62 2013). Therefore, detailed studies of reproduction are needed to fully understand the 63 early-life history of frogfishes exhibiting various reproductive strategies. 64 65 Here, we report the sequences of spawning and egg care behavior of marble-mouth 66 frogfish (L. lithinostomus). This species inhabits coastal reefs of Indonesia and the 67 Philippines and resembles algae-covered rocks (Arnold and Pietsch 2012; Arnold et al. 68 2014). There have been no reports on the species’ reproductive behavior. In addition to 69 the analysis of the reproductive behavior, we report the unique egg structure adapting to 70 the egg care behavior by female parents and the morphology of newly hatched juveniles. 71 Author-formatted, not peer-reviewed document posted on 06/04/2022. DOI:  https://doi.org/10.3897/arphapreprints.e84895 Moreover, we review the evolution of parental care behaviors and egg types, pelagic or 72 demersal eggs, among frogfish families using limited available reports. 73 74

75 Studied Fish 76 77 Ten adult L. lithinostomus, including four males with a standard length (SL) of 105.7 ± 78 15.6 mm and six females with an SL of 120.0 ± 10.1 mm, were obtained from the 79 ornamental fish company (Kamihata Fish Industries LTD, Hyogo, Japan) and identified 80 according to Pietsch (2004). The sexes of these individuals were determined by 81 dissection when they died. Moreover, we used one female with an SL of 89.5mm bred 82 by the Marine Science Museum, Fukushima. Thus, a total of 11 L. lithinostomus 83 individuals were used in this study. They were individually identified by observers 84 using their unique coloration and body morphology. The individuals used here have 85 been registered as voucher specimens in the Marine Science Museum, Fukushima 86 (registration number: AMF0053 and 0115). 87 88 Fishkeeping and reproduction 89 Author-formatted, not peer-reviewed document posted on 06/04/2022. DOI:  https://doi.org/10.3897/arphapreprints.e84895 90 We used two water tanks in this study. Four fish (two males and two females) were held 91 in a 1-m3 water tank (1.0 × 1.0 × 1.0 m) at 26.0℃ from 2016 to 2019, and six fish (two 92 males and five females) were held in a 0.45-m3 water tank (0.7 × 1.0 × 0.6 m) at 93 25.0 ℃ from 2018 to 2019. The fish were fed silver-stripe round herrings 94 (Spratelloides gracilis) once daily. Between 2016 and 2019, 28 spawning events were 95 observed. 96 97 Behavioral observation 98 99 When tracking behavior by the males toward the females was observed, we videotaped 100 their reproductive behaviors and continued throughout the day until spawning was 101 observed. Then, we recorded individuals that cared for eggs and the lateral surface of 102 the body to which the eggs were attached. 103 104 We quantified the investment in offspring by parents by conducting detailed behavioral 105 observation of four reproductive events (n = four females). Female parents attached 106 spawned eggs to the left or right flank and exhibited fanning behavior toward the eggs 107 Author-formatted, not peer-reviewed document posted on 06/04/2022. DOI:  https://doi.org/10.3897/arphapreprints.e84895 using their dorsal fin. We count the fanning behavior for 10 min per day. This 108 behavioral observation was conducted between 8:00 and 17:00. Lastly, statistical 109 analysis was performed using R (R development core team 2020). 110 111 Morphology of Eggs and newly hatched juvenile 112 113 Because one female (137.8 mm SL) renounced an egg mass twice during observation, 114 we used these the egg masses for measurements of egg morphology. The number of 115 eggs per clutch was estimated by comparing the weight of fifty eggs and the weight of 116 egg mass. Egg size was measured using a stereomicroscope (ZEISS SteREO 117 Discovery.V12, ZEISS Research Microscopy Solution, Ltd., Germany). The egg mass 118 was not consumed by other fish because we sampled them right after it was renounced. 119 120 We observed the morphology of newly hatched juveniles by fixing them in 10% 121 formalin (n = 10). Their standard lengths were measured to the nearest 0.1 mm under a 122 microscope, according to Hubbs and Lagler (1958), after they were overdosed with an 123 anesthetic (MS-222, Wako Pure Chemical Industries). The number of fin ray of all fins 124 of the newly hatched juveniles were counted and compared with those of adult fish. The 125 Author-formatted, not peer-reviewed document posted on 06/04/2022. DOI:  https://doi.org/10.3897/arphapreprints.e84895 observed individuals were deposited as voucher specimens (registration number: 126 AMF0127). We classified the newly hatched fish as "juveniles," according to the 127 developmental classification by Kendall et al. (1984) because their fin rays already 128 attained the full complement right after hatching. 129 130 Review of the evolution of egg care in the frogfish family 131 132 To understand the functional role of egg care behaviors and morphology of eggs in L. 133 lithinostomus, we performed a phylogenic analysis to examine the evolution of egg care 134 behaviors in the frogfish family. The data on the reproductive characteristics, such as 135 the size and number of eggs, egg types such as pelagic or demersal, and egg care 136 behaviors, were collected from the primary literature. Eventually, we found a total of 14 137 reports on the reproduction of 14 frogfish species. The egg care behaviors were 138 classified into four types according to Arnold et al. (2014): 1) Attaching to the body of 139 the parent (A-type); 2) carrying within a pocket of parent (P-type); 3) guarding in the 140 nest (N-type); 4) no egg care. In the species with A-type behavior, an egg mass is 141 attached to the lateral side of the parent’s body and nurtured (Pietsch and Grobecker 142 1980; Pietsch and Arnold 2020). In the species with P-type behavior, the parents 143 Author-formatted, not peer-reviewed document posted on 06/04/2022. DOI:  https://doi.org/10.3897/arphapreprints.e84895 embrace their egg masses by curling their dorsal and anal fins (Pietsch and Grobecker 144 1987; Pietsch et al. 2009; Arnold and Pietsch 2018; Pietsch and Arnold 2020). Finally, 145 in species with N-type behavior, the parents spawn specific substrates and care for their 146 egg mass (Kuiter 1993; Liem 1998; Arnold et al. 2014; Bray and Gomon 2021). We also 147 recorded the flank that the A-type and P-type parents carried their egg mass because we 148 found a left-bias in egg caring on the flank in L. lithinostomus. 149 150 In addition, the presence or absence of egg care and egg types (pelagic or demersal) in 151 major species relative to frogfishes were recorded. Reports on the presence or absence 152 of egg care and egg types for coffinfish Chaunax abei (Mimori 2015), anglerfish 153 Lophius litulon (Ishikawa et al. 2022), and batfish Ogcocephalus nasutus (Christie 154 2016) were obtained. Although footballfishes (e.g., Himantolophus appelii) are also a 155 major species relative to frogfishes (Rabosky et al. 2018), we could not find reports on 156 their reproduction because they inhabit the deep sea. 157 158 For the phylogenetic analyses, the phylogenetic tree of frogfishes and related species 159 was obtained or modified from a previously published tree (Rabosky et al. 2018). This 160 tree was estimated using RAxML and dated using treePL, and non-target species were 161 Author-formatted, not peer-reviewed document posted on 06/04/2022. DOI:  https://doi.org/10.3897/arphapreprints.e84895 excluded using the “droptip” function in R ver. 4.0.3 (R Core Team. 2014), package 162 “ape” (Paradis et al. 2004). Ancestral states of the egg were assessed by using Mesquite 163 Version 3.61 (Maddison and Maddison 2019) with the likelihood method in a Markov, 164 k-status, 1-parameter model, using the modified tree. The reconstruction of ancestral 165 states was conducted using 11 frogfish species and their related species because of their 166 clear phylogenetic relationships (Rabosky et al. 2018). 167 168

169 Spawning behavior 170 171 During observation, we successfully videotaped three spawning behaviors 172 (Supplementary S1). Adult males and females were usually solitary in the water tank; 173 however, multiple males began to follow females approximately seven days before 174 spawning. When spawning began, a male ejaculated when a female released an egg 175 mass from its gonopore. Before the egg mass was attached to the side of the female, the 176 male left the female (Fig. 1a). A female attached the spawned egg mass to its side using 177 its caudal fin to wrap it around the lateral surface of its body. The egg mass was not 178 released into the water but transferred from the gonopore to the lateral surface of the 179 Author-formatted, not peer-reviewed document posted on 06/04/2022. DOI:  https://doi.org/10.3897/arphapreprints.e84895 body (Fig. 1b). A female protected an egg mass by covering it with her dorsal, anal, and 180 caudal fins and occasionally fanned it with her dorsal fin (Supplementary S1). 181 182 Egg care and laterality of female parents 183 184 All egg care behaviors (n = 28) were performed only by the female parents, and no 185 males cared for the eggs, based on the spawning events by seven females. While one of 186 the females cared for its eggs on its right flank three times, 89.2 % of the egg masses 187 (25 out of 28 spawning events) were cared for on the left flank of the parent, suggesting 188 a significant bias towards egg care on the left flank (Pearson’s chi-squared test χ21 189 =37.605; p < .001). The number of fanning behaviors towards attached eggs varied 190 considerably, ranging from 0 to 537 per 10 min with a mean of 201.9 ± 148.6; it 191 increased from the day after spawning until fifteen days after spawning. However, after 192 that, the number of fanning behaviors remained unchanged (Fig. 2). Hatching began 22 193 to 23 days after spawning; almost all juveniles hatched by days 27 to 28. The juveniles 194 were scattered throughout the tank after hatching. 195 196 Morphological characteristics of eggs and newly hatched juveniles 197 Author-formatted, not peer-reviewed document posted on 06/04/2022. DOI:  https://doi.org/10.3897/arphapreprints.e84895 198 The egg mass had an elliptic shape with a mean major axis of 91.2 mm and a mean 199 minor axis of 62.5 mm. The eggs were round; their average length was 2.9 ± 0.3 mm, 200 ranging from 2.4 to 4.1 mm (n = 50; from two egg masses from one female). The 201 coloration of eggs was semi-transparent white just after spawning. Eye pigmentation 202 was confirmed 11 days after spawning. Interestingly, demersal eggs had a unique 203 structure, an s-shaped hook (Fig. 3). Although juveniles (n = 10) had yolk sacs just after 204 hatching, all their fin rays already attained the full complement compared with the 205 adults (Fig. 4). The standard length (SL) of the newly hatched juveniles (n = 10) was 206 5.70 ± 0.20 mm, ranging from 5.40 to 6.09 mm. They already had esca, which was the 207 distinctive protuberance of this group used for predation as a lure; in addition, they 208 could swim freely and often they could attach to substrates in the water tank. 209 210 Evolution of egg care among frogfish family 211 212 We obtained 14 anecdotal or qualitative reports in the field associated with egg care 213 behaviors of frogfishes (Table 1). The species in Antennariinae do not display egg care 214 behaviors and have pelagic eggs. In contrast, the species in Histiophryninae have three 215 Author-formatted, not peer-reviewed document posted on 06/04/2022. DOI:  https://doi.org/10.3897/arphapreprints.e84895 types of egg care behaviors, i.e., attaching to the body of the parent (A-type), carrying 216 within a pocket (P-type), and guarding at the nest (N-type). The species in 217 Histiophryninae have demersal eggs. Any form of egg care behavior has not been 218 reported from the species related to frogfishes. Ancestral state reconstruction revealed 219 that P-type and N-type egg care behaviors had been evolved independently from the 220 no-care species in Histiophryninae (Fig. 5). In addition, A-type care had been evolved 221 from a group within the P-type egg care (Fig. 5). 222 223

224 225 In this study, we investigated the reproductive behavior of marble-mouth frogfish 226 (Lophiocharon lithinostomus). We found that multiple males stalked breeding females 227 before spawning, although only one male could participate in the spawning event. 228 Similar behavior by adult males before spawning has been observed in several fishes 229 and functions as mate guarding (Yokoi et al. 2016). Moreover, female filefish (Rudarius 230 ercodes) move around and choose any male following them as their reproductive 231 partners (Akagawa et al. 1998). Thus, the behavior of L. lithinostomus will be regarded 232 as a process of mate choice or mate guarding. For L. lithinostomus, fertilization 233 Author-formatted, not peer-reviewed document posted on 06/04/2022. DOI:  https://doi.org/10.3897/arphapreprints.e84895 occurred immediately after the onset of spawning, and the male left the female before 234 the entire egg mass was laid. The time difference between fertilization and spawning did 235 not affect the fertilization rate, and most of the eggs were fertilized (Mori personal 236 observation). Fertilized eggs were covered with a gelatinous curtain. Since the activity 237 time of the sperms in the ovarian cavity fluid of females plays an important role in 238 fertilization, the gelatinous membrane covering the eggs may play a role similar to the 239 ovarian cavity fluid (Hayakawa and Munehara 2001). 240 241 We found that only females attached egg masses to their flanks and began to fan the egg 242 masses after spawning. Also, regarding L. trisignatus, a related species to L. 243 lithinostomus, only female parents care for egg masses (Pietsch and Arnold 2020). In 244 many species in the frogfish family (Antennariidae), the females are larger and have 245 larger abdomens than the males (Pietsch et al. 2013). Moreover, according to the reports 246 on the sexual dimorphism in frogfishes, there are sexual differences in the number of 247 dermal spinules in striated frogfish (Antennarius scaber) (Breder and Rosen 1966). 248 However, no definitive sex differences have been found among frogfishes. Therefore, 249 the study of the sex of the frogfishes caring for egg mass should be reconsidered. 250 251 Author-formatted, not peer-reviewed document posted on 06/04/2022. DOI:  https://doi.org/10.3897/arphapreprints.e84895 More generally, the morphologies of newly hatched and developing teleost fishes are 252 incomplete compared with adults (Mosher 1954; Ehrlich et al. 1976; Martin and Drewry 253 1978; Kendall et al. 1984; Satoh et al. 2017). In some species, the mouth and anus of 254 the newly hatched larvae do not open, making feeding impossible (Satoh et al. 2017). 255 Even with an open mouth, it would be difficult for the larvae to catch small prey with 256 fin folds due to their low swimming ability (Satoh et al. 2017). Therefore, 257 morphological ontogeny can highly influence early-life history, especially foraging 258 ecology (Satoh et al. 2017). For example, the newly hatched larvae of frogfishes in the 259 Antennariinae with pelagic eggs, such as Histrio histrio, do not have advanced 260 organogenesis (Mosher 1954). In contrast, the newly hatched juveniles of L. 261 lithinostomus already have open mouths, and all their fin rays attain the full 262 complement, suggesting that they already have had robust foraging abilities 263 immediately after hatching. 264 265 The dispersal of many marine fishes is commonly concentrated in egg or early larval 266 stages (Barlow 1981; James et al. 2002). However, the dispersal ability of L. 267 lithinostomus would be even lower than the pelagic egg group, such as Histrio histro 268 and Antennarius striatus, because L. lithinostomus eggs do not float and are cared for by 269 Author-formatted, not peer-reviewed document posted on 06/04/2022. DOI:  https://doi.org/10.3897/arphapreprints.e84895 the female parents. Indeed, frogfishes in Histiophryninae have a smaller distribution 270 area than those in Antennariinae and highly depend on the specific local area (Arnold 271 and Pietsch 2012). In general, species with a low dispersal ability are more susceptible 272 to local environmental changes (Baguette et al. 2012; Gonçalves et al. 2016). Recently, 273 the deterioration of the coastal environment, including coral reefs, has been reported 274 (Kennish 2002). Trends in species such as L. lithinostomus, which are presumed to be 275 less tolerant of environmental changes, should be all eyes. 276 277 According to the ancestral state reconstruction, three types of egg care had evolved only 278 among the species of Histiophryninae, which have demersal eggs, from non-egg care 279 species. These findings suggested that egg care behaviors had co-evolved with demersal 280 eggs. Demersal eggs are generally larger than pelagic eggs, and the fry can hatch with 281 advanced organogenesis (Sargent et al. 1987; Araujo‐Lima 1994). Indeed, we found 282 newly hatched juveniles of L. lithinostomus had highly advanced organogenesis. 283 However, demersal eggs stick to substrates and thus, cannot change location. Therefore, 284 demersal eggs will be exposed to high predation pressure unless they have a chemical 285 defense or camouflage coloration (Eisner et al. 2000; Ruxton et al. 2004; Skelhorn 286 Author-formatted, not peer-reviewed document posted on 06/04/2022. DOI:  https://doi.org/10.3897/arphapreprints.e84895 2015). The frogfishes of Histiophryninae may have evolved egg care as a 287 counter-strategy to avoid the high predation pressure applied on demersal eggs. 288 289 The N-type egg care is thought to have evolved independently of these lineages. Also, 290 we found that A-type egg care may have evolved from P-type egg care. It would be very 291 unusual for diverse modes of egg care to evolve among closely related species. In 292 species with P-type egg care, female parents embrace their egg masses by curling dorsal 293 and anal fins into a pocket-like structure (Arnold et al. 2014). In contrast, egg masses 294 are adhered to the flank for fishes with A-type egg care, such as L. lithinostomus. We 295 found an s-shaped hook structure in the egg masses of L. lithinostomus. This structure 296 may function as adhesives to attach the egg masses to the flank of female parents so that 297 the eggs are tightly glued to the parental body. Indeed, when a female parent abandoned 298 the glued eggs, she shook her body vigorously to remove the eggs. (Mori personal 299 observation). 300 301 The number of eggs varies between egg care types. For example, speceis with N-type 302 has the highest number of eggs at approximately 5000 eggs (n = 1 species), followed by 303 A-type with 320 to 1311 eggs (n = 2 species) and P-type with 105 to 220 eggs (n = 3 304 Author-formatted, not peer-reviewed document posted on 06/04/2022. DOI:  https://doi.org/10.3897/arphapreprints.e84895 species) (Table 1). The number of eggs by N-type fish will not be limited because the 305 fish spawn egg masses on a substrate (Arnold et al. 2014). However, in A-type and 306 P-type, the size of egg masses will be restricted by the flank of the parents because the 307 fish attach or entrap their eggs to the body (Arnold et al. 2014). Although they are 308 constrained by the number of eggs they can spawn in one reproductive event, the 309 parents with A- and P-type egg care can migrate while raising their eggs. Additionally, 310 the species with A-type egg care have more eggs than those with P-type egg care (Table 311 1). In general, oxygen demand, hence the frequency of fanning, increases with 312 embryonic development (Abe and Munehara 2005). Unlike a P-type species, an A-type 313 species can use its dorsal fins for fanning behavior, likely allowing it to hold more eggs, 314 even though the number of eggs is limited by body size (Bagenal and Braum 1978; 315 Warner 1984). Although these comparisons are constrained by the simplicity of the 316 evolution of egg care modes among frogfishes, they suggest that the number of eggs 317 will vary depending on the egg care mode. 318 319 Finally, we found that L. lithinostomus females preferred to use the left side of their 320 bodies for egg care. Interestingly, they also appeared able to use their right flank for egg 321 care because one female used the right flank. Lateralized (left-right asymmetry) 322 Author-formatted, not peer-reviewed document posted on 06/04/2022. DOI:  https://doi.org/10.3897/arphapreprints.e84895 morphology and behavior among fishes have been reported in many species (Takeuchi 323 et al. 2016; Hori et al. 2017). Because left-biased egg care was not detected in species 324 with A-type egg care (L: R = 3:2 from 5 reports for 3 species, Table 1) but L. trisignatus 325 also displayed left-biased egg care similar to L. lithinostomus (Table 1), this tendency 326 may have co-evolved with A-type egg care. However, we could not determine why L. 327 lithinostomus females used their left side for egg care; this interesting phenomenon 328 requires further morphological or evolutionary validation. 329 330 Acknowledgments 331 332 We are grateful to Mr. Yoshitaka Abe and Mr. Takeshi Furukawa of Aquamarine 333 Fukushima for cooperation for fishkeeping. We would also like to thank Dr. Toshiro 334 Saruwatari and Dr. Toru Miura of University of Tokyo, Dr. Takashi Asahida and Dr. Go 335 Katayose of Kitasato University, and Dr. Atsushi Sogabe of Hirosaki University for the 336 comments on our study. We would also like to thank anonymous reviewers for their 337 helpful comments. 338 339

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Comparison of reproduction modes for frogfish family Antennaridae. 515 Species Egg size (mm) The number of eggs Type of egg Parental care mode Laterality of body side used for egg care Reference(s) Antennariinae Antennarius striatus 0.6 89635 pelagic No care － Fujita and Uchida 1959 Antennarius nummifer 0.7 73000–288000 pelagic No care － Piestch and Grobecker 1987 Histrio histro 0.7 48800 pelagic No care － Ray 1961 Histiophryninae Histiophryne cryptacanthus 3.6–4.2 115 demersal P-type L:R = 1:0 (n = 1 report) Piestch and Grobecker 1987 Histiophryne bouganivilli 3.2–3.9 105 demersal P-type L:R = 1:1 (n = 2 reports) Piestch and Arnold 2020 Histiophryne psychedelica 3.0–4.0 220 demersal P-type L:R = 1:1 (n = 2 reports) Piestch et al. 2009; Piestch and Arnold 2020 Histiophryne narungga No data No data demersal P-type No data Arnold and Piestch 2018 Lophiocharon trisignatus 3.2–3.6 650 demersal A-type L:R = 4:0 (n = 4 reports) Piestch and Grobecker 1980; Piestch and Arnold 2020 Lophiocharon lithinostomus 2.1–4.1 320–1311 demersal A-type L:R = 26:3 (n = 3 reports) I.O.P Diving News 2001; Piestch and Arnold 2020; Present study Echinophryne crassispina No data 150 demersal N-type － Liem 1998; Bray and Gomon 2021 Echioophryne reynoldsi No data No data demersal N-type － Bray and Gomon 2021 Rhycherus filamentosus 5.0 5000 demersal N-type － Kuiter 1993; Bray and Gomon 2021 Porophryne erythrodactylus No data No data demersal N-type － Arnold et al. 2014 Phyllophryne scortea No data No data demersal N-type － Bray and Gomon 2021 516 A-type: attaching to the body of the parent, P-type: carrying within a pocket of parent, N-type: guarding in the nest. 517 Author-formatted, not peer-reviewed document posted on 06/04/2022. DOI:  https://doi.org/10.3897/arphapreprints.e84895 Figure and legends 518 519 Figure 1. A Photograph of a gravid female (dark brown color) marble-mouth frogfish 520 Lophiocharon lithinostomus) surrounded by two courting males (green color). B Female 521 marble-mouth frogfish with egg mass attached on the left side of bodies. 522 523 Author-formatted, not peer-reviewed document posted on 06/04/2022. DOI:  https://doi.org/10.3897/arphapreprints.e84895 Figure 2. Relationship between days after spawning and the frequency of fanning 524 behavior by female marble-mouth frogfish Lophiocharon lithinostomus. A solid line 525 and a grey area indicate the loess regression and 95% Confidence interval, respectively. 526 527 Figure 3. Photograph of fertilized eggs and gelatinous curtains with S-shaped hooks 528 surrounding the eggs. White scale bar: 2 mm. 529 Author-formatted, not peer-reviewed document posted on 06/04/2022. DOI:  https://doi.org/10.3897/arphapreprints.e84895 530 Figure 4. Photographs of a marble-mouth frogfish Lophiocharon lithinostomus juvenile 531 just after hatching. A Overhead view. B Lateral view. White scale bar: 1 mm. 532 Author-formatted, not peer-reviewed document posted on 06/04/2022. DOI:  https://doi.org/10.3897/arphapreprints.e84895 533 Figure 5. Evolution and phylogenetic distribution of the forms egg care in 534 Antennariidae and related species. The ultrametric tree was modified from Rabosky et 535 al. (2018). Egg care behaviors were classified into four types: 1) Attaching to the body 536 of the parent (blue); 2) carrying within a pocket of a parent (green); 3) guarding in the 537 nest (black); 4) no egg care (white). 538 539 ESM S1 540 Spawning and fanning behavior of marble-mouth frogfish Lophiocharon lithinostomus. 541 542 Author-formatted, not peer-reviewed document posted on 06/04/2022. DOI:  https://doi.org/10.3897/arphapreprints.e84895