Discovery of a new mode of oviparous reproduction in sharks and its evolutionary implications

Two modes of oviparity are known in cartilaginous fishes, (1) single oviparity where one egg case is retained in an oviduct for a short period and then deposited, quickly followed by another egg case, and (2) multiple oviparity where multiple egg cases are retained in an oviduct for a substantial period and deposited later when the embryo has developed to a large size in each case. Sarawak swellshark Cephaloscyllium sarawakensis of the family Scyliorhinidae from the South China Sea performs a new mode of oviparity, which is named “sustained single oviparity”, characterized by a lengthy retention of a single egg case in an oviduct until the embryo attains a sizable length. The resulting fecundity of the Sarawak swellshark within a season is quite low, but this disadvantage is balanced by smaller body, larger neonates and quicker maturation. The Sarawak swellshark is further uniquely characterized by having glassy transparent egg cases, and this is correlated with a vivid polka-dot pattern of the embryos. Five modes of lecithotrophic (yolk-dependent) reproduction, i.e. short single oviparity, sustained single oviparity, multiple oviparity, yolk-sac viviparity of single pregnancy and yolk-sac viviparity of multiple pregnancy were discussed from an evolutionary point of view.

, and occupied most of the available space of the oviduct. Egg cases had a smooth surface and with tough, thick shell, roughly quadrangular in shape and rather flattened, with a truncate anterior end and a rounded posterior end ( Fig. 1b-d). The egg case was completely transparent, and the yolk and embryo (when present) were clearly visible through the casing. A very long and strong tendril was present on the horn at four corners of the egg case. There were four openings, or corner slits, one at the base of each horn. Corner slits at anterior horns were linear and long, and those at posterior horns were curved. Corner slits were closed in the egg cases without developing embryo, and were open in the egg cases with 43 mm TL and larger embryos. Eight egg cases from four females had only yolk, without developing embryos. The egg cases from nine females each contained a developing embryo. The largest embryo observed in the egg case (77.0 mm ECL) was 102 mm TL, with many polka-dot markings (Fig. 1d) and the smallest one was 43 mm TL without markings. External yolk was present on all these embryos.
Two already deposited egg cases were collected from trawl catch landings (Fig. 2a). One egg case (NMMB-P32979A, 80.0 mm ECL, Fig. 2a1) contained an embryo of 94.5 mm TL, and the other (NMMB-P32979B, 80.0 mm ECL, Fig. 2a2) had an embryo of 65.0 mm TL. Both embryos had dark polka-dot markings and external yolk that were clearly visible through the transparent casing. These egg cases were firmly tied to the tube of a tubeworm Paradiopatra sp. (family Onuphidae, order Eunicida) by posterior tendrils. The anterior tendrils of one egg case (NMMB-P32979A) were loosely entangled with the tube, and those of the other egg case (NMMB-P32979A) were broken and missing.
Juveniles. The smallest, free-swimming specimen was a 125 mm TL female (NMMB-P22719) with a remnant of the external yolk sac (Fig. 2b). The body is light brownish with many dark polka-dot markings on lateral and dorsal surfaces of body. The second and third smallest juveniles were a 134 mm TL male (NMMB-P24872) and a 143 mm TL male (NMMB-P17143), both polka-dotted and with a reduced external yolk sac and a distinct scar.

Discussion
Two modes of oviparity, i.e. single and multiple oviparity, are currently recognized in chondrichthyans 1-3,5-12 . Single oviparity (Fig. 3a) is a mode where each oviduct in pregnant females contains one egg case, i.e. a pair of egg cases in a pregnant female. These egg cases are retained in the oviduct only for a short time and deposited immediately before the embryo has begun developing. The embryos are not recognizable at oviposition and www.nature.com/scientificreports/ become visible in a few weeks. Oviposition is repeatedly performed, and each mature female can deposit tens of egg cases over the course of a spawning season 6 ("Short single" oviparity in Fig. 4). Multiple oviparity (Fig. 3b) is a mode where several egg cases accumulate in each oviduct and are retained for several months before oviposition, in which time embryos begin development in the oviduct and the egg cases are deposited later when the embryos grow large to a certain developmental stage ("Multiple" oviparity in Fig. 4). Cephaloscyllium sarawakensis does not fit the classic single oviparity, nor multiple oviparity. Pregnant females of this species always have a single egg case, never two or more, in each oviduct (Fig. 3c), and keep it until embryo attains a certain developmental stage ("Sustained single" oviparity in Fig. 4). These facts indicate that reproduction of C. sarawakensis represents a new mode of oviparity, which is herein termed "sustained single oviparity". A 450 mm TL female of Cephaloscyllium silasi from the Indian Ocean had one egg case with a well-developed embryo in each oviduct 13 . Although they reported only one female specimen, this species possibly also displays the sustained single oviparity.
Various technical terms have been used for oviparity. Single oviparity has at least three alternative names, "extended" oviparity 3,4,6,12,14 , "external" oviparity 5,15 , and "simple" oviparity 5 . Multiple oviparity has been also termed "retained" oviparity [3][4][5][6]12,14,16 . These terms are used for the same reproductive mode, and this could lead to confusion and misunderstanding about the oviparity. Therefore, we herein summarized the terms of oviparity and proposed a new set of technical terms as follows: (1) "short single oviparity" for the single oviparity previously known; (2) "sustained single oviparity" for the new type of oviparity reported in this study; and (3) "multiple oviparity" instead of the former "retained" oviparity. The new definition of oviparity in the cartilaginous fishes was summarized, with additions of two modes of yolk-sac viviparity in catsharks (Table 1).
Typically in the catsharks displaying short single oviparity, the shell of egg case is tough and thick, with two pairs of long strong tendrils on its anterior and posterior ends ( Fig. 5a-c,e,f), although tendrils are very short or replaced by fine silky materials (Fig. 5d) or absent in some species. The posterior pair of tendrils is used to attach the egg case to the substrate on the sea bottom, to pull it out from the oviduct, and coil it around the substrate, together with anterior pair of tendrils. The egg case is firmly secured on the substrate until juvenile hatches. The tendrils in the multiple oviparous species (Fig. 5f) tend to be thinner and shorter than those of the short single oviparous species. Cephaloscyllium sarawakensis of the sustained single oviparity has a thick shell and long tendrils (Fig. 1b,c,d1). The two egg cases (Fig. 2a) collected from fishery landings can be inferred to have been deposited intentionally on the tube of a tubeworm Paradiopatra sp. based on the fact that the posterior tendrils are firmly twined around it. www.nature.com/scientificreports/ The egg cases in the species of sustained single oviparity are very large, with its length (ECL) ranging 16.5-20.1% TL in Cephaloscyllium sarawakensis and 18.9-19.2% TL in C. silasi 13 , while the egg cases of short single oviparous C. umbratile are 10.6-15.1% TL 2,17,18 . The egg cases of other short single oviparous species are also small, with lengths 8.6-9.0% TL in Holohalaelurus regani 19 30 . As seen in Fig. 3, the egg cases of C. sarawakensis (Fig. 3c1) are far larger than those of G. sauteri (Fig. 3a) and H. buergeri (Fig. 3b), occupying most of the available space of oviduct and abdominal cavity. Thus, the two species of sustained single oviparity have much larger egg cases than short single oviparous and multiple oviparous species, suggesting larger neonates at hatching in C. sarawakensis and C. silasi.
The embryos in the egg cases recorded from the oviduct indicate that Cephaloscyllium sarawakensis retains the egg case until the embryo attains about 102 mm TL ( Fig. 1d; largest embryo in the egg inside the oviduct), and then oviposition occurs later. However, the two egg cases (both 80.0 mm ECL) from the seabed contained a 65 mm TL and a 94.5 mm TL embryo each (Fig. 2a). These facts suggest the timing of oviposition is rather wide in this species, and the egg cases are laid when the embryo grows roughly 6-10 + cm TL, stimulated by some internal or external factors. The smallest free-swimming juvenile collected was 125 mm TL with a remnant of external yolk-sac (Fig. 2b), suggesting the hatching size from egg case being around 120 mm TL in this species. These evidences indicate C. sarawakensis keeps the egg case in the oviduct until embryo has developed to 50 ~ 80+ % of its hatching size. Therefore, the retention of egg case in the oviduct continues for an extended period, perhaps several months or more, in C. sarawakensis and probably also in C. silasi.
The other remarkable characteristic of Cephaloscyllium sarawakensis is the glassy transparent egg cases ( Fig. 1b-d), and the transparency is completely maintained even after oviposition (Fig. 2a). The egg cases of www.nature.com/scientificreports/ oviparous cartilaginous fishes are opaque, usually yellowish to dark brownish (Fig. 5), and sometimes with longitudinal or transverse ridges (Fig. 5a). The functional role of egg case is to protect the embryo from physical, physiological and biological hazards from the environment. The colored egg cases can be also effective to conceal the embryo in it. However, the egg case of C. sarawakensis is transparent and never cryptic that the orange yellow yolk would clearly be recognizable through transparent egg case, if the egg case is deposited immediately after egg case being formed. As shown in this study, C. sarawakensis retains an egg case in each oviduct until the embryo is developed with a distinct dark polka-dot color pattern on light brownish body (Figs. 1d, 2a), typically seen in the juveniles (Fig. 2b). One of the reasons for transparent egg case may be related to their vivid body color patterns. Benthic and reef-dwelling sharks have complicated color patterns, which are effective to blend the body into their background or for camouflage 31 . The present egg cases of C. sarawakensis (Fig. 2a) were deposited around the tube of a tubeworm sticking out from the seabed. Their vivid polka-dots and light brownish body coloration could function as more effective camouflage against the complex and dark background through the transparent egg case. Thus, the long retention of transparent egg cases and vivid embryonic coloration could suggest a new method of reproductive tactics in cartilaginous fishes.
The oviparity is advantageous as a method to increase the fecundity in small elasmobranchs that have limited space in body cavity for care and storage of the embryos 6 . The species of short single oviparity (Fig. 3a) repeatedly deposits two egg cases immediately after the cases are completed and laid, resulting in 20-100 eggs per season 6 . The captive Cephaloscyllium umbratile was recorded as depositing two egg cases at intervals of 11-38 days (20 days in average) for whole year 32 , which means a single female deposited about 36 egg cases a year. Similarly, the captive C. laticeps laid two egg cases at intervals of up to 28 days throughout whole year 11 , equating more than 26 egg cases being deposited annually.
The multiple oviparous species retains a number of egg cases in each oviduct (Fig. 3b) for several months until the embryos have developed to a certain stage. All the species of Halaelurus are multiple oviparous, and H. buergeri has been recorded to deposit 8 egg cases one by one at a stage when the embryos inside have attained 70 mm TL 33 , or 10 egg cases at one time 34 . One specimen of H. buergeri we (KN) collected (Fig. 3b) 35 .
The maternal environment offers the best protection to the developing embryos and can shorten the exposed period of time on the substrate. Therefore, the survival rate could be expected to be much higher in the sustained single and multiple oviparous species than the short single oviparous species. The species of the sustained single oviparity (Fig. 3c) and those of the multiple oviparity (Fig. 3b) are the same in that the egg cases have long maternal protection, but the number of the egg cases deposited at a time is considerably less in the sustained single www.nature.com/scientificreports/ oviparous species, i.e. 2 eggs vs. 4-16 eggs per mother, respectively. Hence, the fecundity of Cephaloscyllium sarawakensis could be very low, 1/8-1/2 of the multiple oviparous species (see "Oviparity" in Fig. 4). It is crucial to produce a certain number of offspring to maintain a sustainable population, and the very low fecundity in C. sarawakensis could be decisively disadvantageous for the species. Similar issues exist in the yolksac viviparous species. The yolk-sac viviparous catsharks, such as Bythaelurus clevai, B. hispidus, B. lutarius, B. stewarti and Cephalurus cephalus retain only one embryo per uterus or per mother 1,28,36-38,KN pers.obs , which is hence termed here "single pregnancy". These species of single pregnancy would have also lower fecundity than the species of "multiple pregnancy" seen in Galeus polli (see "Yolk-sac viviparity" in Fig. 4).
Species of the genus Cephaloscyllium are generally large in body sizes, mostly growing to more than 70 cm TL and some species (C. isabellum, C. laticeps, and C. umbratile) attain more than 100 cm TL 39 , whereas C. sarawakensis and C. silasi are dwarf species within the genus. Cephaloscyllium sarawakensis attains a maximum of only 39.7 cm TL in males and 49.5 cm TL in females 40,present study , and matures at the sizes less than 32.5 cm TL and 35.4 cm TL in males and females, respectively 41 . Similarly, Cephaloscyllium silasi attains only 50 cm TL 13 and reaches its maturity at less than 36.8 cm TL in males 1 and less than 45 cm TL in females 13 . Bythaelurus clevai, B. hispidus and B. lutarius attain 42 cm TL, 36 cm TL and 39 cm TL, respectively 37,39 and these species are also the smallest species for the genus. Cephalurus cephalus reaches 30 cm TL 36,39 , and this is known as one of the smallest sharks 1 .
The ratio of length at maturity to the largest total length was reported at around 0.73 for elasmobranchs 42 , and this indicates the smaller species could reach their maturity at smaller sizes than the larger species. Actually, the captive Cephaloscyllium umbratile which grows up to 118 cm TL hatches at lengths of 16-22 cm TL 32 and attains its full maturity at 96-104 cm TL 17 . In contrast, C. sarawakensis which produces very large egg cases relative to the mother size is expected to hatch at about 12 cm TL and mature at less than 35 cm TL. Therefore, C. sarawakensis grows only about 23 cm in length until maturity is attained, whereas C. umbratile needs to grow 75-90 cm to its maturity. Cephalurus cephalus produces 7-9 cm TL neonates and attains maturity at 18-22 cm TL 43 , thus www.nature.com/scientificreports/ 9-15 cm in length to grow to attain maturity. Eridacnis radcliffei gives birth 10.5-12.8 cm TL neonates 36 , and attains maturity at 18.3 cm TL, only 5.5-7.8 cm in length to the maturity. Hence, these dwarf sharks of the sustained single oviparity and the yolk-sac viviparity of single pregnancy likely attain their maturity in a shorter time frame than the larger species do, enabling them to reproduce at an earlier age and keep their life-time fecundity high. Other factors to increase their lifetime fecundity include quick repetition of reproduction, longer lifetime reproduction and higher proportion of females, but these will not be referred to here. Figure 6a shows five modes of lecithotrophic (yolk-dependent) reproduction in the cartilaginous fishes, and Fig. 6b1-6 denote six combinations of these reproductive modes in the catsharks. Figure 6b1 (short single oviparity) represents ten genera such as Apristurus, Asymbolus, Atelomycterus, Figaro, Haploblepharus, Holohalaelurus, Parmaturus, Poroderma, Scyliorhinus and Schroederichthys. Figure 6b3 (multiple oviparity) and Fig. 6b6 (yolk-sac viviparity of single pregnancy) are Halaelurus and Cephalurus, respectively. However, Fig. 6b2,b4,b5 include two or three modes of reproduction. Cephaloscyllium (Fig. 6b2) performs short single oviparity + sustained single oviparity, Bythaelurus (Fig. 6b4) involves short single oviparity + yolk-sac viviparity of single pregnancy, and Galeus (Fig. 6b5) performs short single oviparity + multiple oviparity + yolk-sac viviparity of multiple pregnancy. These facts could suggest rather facile diversification of reproductive mode in closely related species, or may suggest necessity of some taxonomic reconsideration of them, as indicated for Bythaelurus 38 .
Oviparity has been suggested to be the ancestral mode of reproduction for vertebrates 7,44 , and it has also traditionally been believed as the ancestral mode for chondrichthyan fishes 3,14,45 . However, recent studies 5,6,12,46,47 suggest that viviparity is ancestral for all chondrichthyans, with many reversions to oviparity and secondary reversions to viviparity. Phylogenetic interrelationships for the Galeomorphi (orders Heterodontiformes, Orectolobiformes, Lamniformes and Carcharhiniformes) 4,45,[48][49][50] show that short single oviparity is the ancestral mode for the Galeomorphi, and also for the orders Heterodontiformes, Orectolobiformes and Carcharhiniformes. Multiple oviparity is generally considered to have evolved from short single oviparity 5 , or evolved intermediately between the short single oviparity and the yolk-sac viviparity 1,7,14,51 .
The catsharks (now Pentanchidae and Scyliorhinidae) in the Carcharhiniformes are separated into a few isolated groups, based on genetic works 49,50,52 . Mapping of the reproductive modes on their phylogenetic www.nature.com/scientificreports/ relationships suggests that the short single oviparity is ancestral for each group. According to the phylogenetic result 50 which covers more catshark taxa than the other works, their Scyliorhinidae I 50 (Fig. 7a)    www.nature.com/scientificreports/ the groups of short single oviparous catsharks, and the relationships suggest the multiple oviparity has derived from the short single oviparity. The genus Bythaelurus, which is also deeply merged in groups of short single oviparous species in the Scyliorhinidae I 50 (Fig. 7a) (Fig. 7a). These facts suggest that the short single oviparity is ancestral for Bythaelurus and the yolk-sac viviparity of single pregnancy could have derived from the short single oviparity 1 , maybe via sustained single oviparity.
The genus Galeus contains 18 species with three reproductive modes (Fig. 6b5), i.e. short single oviparity (G. antillensis and seven other species), multiple oviparity (G. atlanticus, G. melastomus and G. piperatus) and yolksac viviparity of multiple pregnancy (G. polli). The genus Galeus is deeply embedded within groups of short single oviparous species in the Scyliorhinidae I 50 (Fig. 7a), and the short single oviparity is considered to be ancestral for the genus Galeus, and the yolk-sac viviparity of multiple pregnancy in G. polli could have derived from short single oviparity via multiple oviparity.
Their Scyliorhinidae II 50 includes three genera Atelomycterus, Schroederichthys of short single oviparity and oviparous Aulohalaelurus 55 . Scyliorhinidae III 50 includes three genera Cephaloscyllium, Scyliorhinus and Poroderma, and they all display short single oviparity. Cephaloscyllium sarawakensis and C. silasi of sustained single oviparity were not treated in their analysis 50 , but the relationships of Cephaloscyllium in the Scyliorhinidae III 50 that is composed of short single oviparous species could suggest derivation of the sustained single oviparity directly from short single oviparity by longer retention of one egg case in an oviduct.
The modes of reproduction in the catsharks were summarized in Table 2, and the phylogenetic evidences mentioned above suggest: (1) short single oviparity is ancestral for the catsharks; (2) more diverse modes of reproduction evolved in the family Pentanchidae than family Scyliorhinidae; (3) sustained single oviparity in Cephaloscyllium was derived directly from short single oviparity; (4) multiple oviparity in Halaelurus was derived from short single oviparity; (5) multiple oviparity in Galeus was derived from short single oviparity, and originated yolk-sac viviparity of multiple pregnancy; (6) yolk-sac viviparity of single pregnancy in Bythaelurus was derived from short single oviparity, possibly via sustained single oviparity; and (7) yolk-sac viviparity of single pregnancy in Cephalurus was derived possibly from short single oviparity via sustained single oviparity.

Materials and methods.
All specimens of Cephaloscyllium sarawakensis examined were bycatch from commercial bottom trawlers operating in the South China Sea off southwest Taiwan, and were collected at Hsinda port (HD) and Ke-tzu-liao (KTL) in Kaohsiung. Specimens were fixed in 4% formalin and then transferred to 70% Ethanol or 50% Isopropanol ethanol. All specimens were deposited at Pisces collection of the National Museum of Marine Biology & Aquarium, Pingtung, Taiwan (NMMB-P). Total length (TL) was measured using a ruler or digital caliper, to nearest 1 or 0.1 mm, respectively.
Egg cases (Table 3): a total of 8 egg cases without visible embryo on the yolk, and 15 egg cases with a developed embryo each were collected from the oviduct of thirteen specimens of C. sarawakensis. Two egg cases with a developed embryo tied on the tube of a tubeworm Paradiopatra sp. (family Onuphidae, order Eunicida) were collected from the fishery landings by fishers, and were kept frozen. Egg cases were deposited and catalogued in NMMB-P collection. Length (excluding tendrils, ECL) and width (ECW) of the egg case were measured by a ruler or digital caliper. Juveniles