Marine Biology (2004) 145: 681–692 DOI 10.1007/s00227-004-1368-9 R ES E AR C H A RT I C L E Marı́a Soledad Romero Æ Carlos S. Gallardo Gilda Bellolio Egg laying and embryonic-larval development in the snail Thais (Stramonita) chocolata (Duclos, 1832) with observations on its evolutionary relationships within the Muricidae Received: 16 September 2003 / Accepted: 24 March 2004 / Published online: 18 May 2004 Ó Springer-Verlag 2004 Abstract Oviposition and embryonic-larval development are described for the muricacean snail Thais (Stramonita) chocolata from the Southeast Pacific coast. As with numerous other muricacean snails, this species engages in communal egg laying, with females depositing egg capsules in clusters on subtidal rocks. Each cluster of capsules contains 100–150 pedunculate, ampulliform egg capsules, with each capsule containing an average of 2,600 small (130 lm) eggs. Intracapsular development was followed using light and scanning electron microscopy to describe the successive embryonic stages of the species. Free-swimming veliger larvae of about 225 lm length were released from capsules after 49 days incubation at 13.6°C. The planktotrophic larvae were cultured in seawater aquaria by feeding with pure cultures of phytoplankton, recording growth and form of the larvae. Larvae reached competence after 4 months at 22°C, at 1,450–1,740 lm in size, and a few larvae were observed through metamorphosis and early definitive growth. The embryonic-larval development of T. chocolata coincides with the general characteristics of the ontogeny observed in other Thais species as well as of other genera of the Rapaninae such as Concholepas. This lent support to grouping these genera into a single clade. The lack of knowledge of the development of free larvae of Thais spp. means that we do not know whether these similarities also include an extensive larval phase as generally characteristic of other members of the clade. The mode of development may be useful in Communicated by O. Kinne, Oldendorf/Luhe M. Soledad Romero (&) Departamento de Biologı́a Marina, Universidad Católica del Norte, Casilla 117, Coquimbo, Chile E-mail: msromero@nevados.ucn.cl Fax: +56-51209791 C. S. Gallardo Instituto de Zoologı́a, Universidad Austral de Chile, Casilla 567, Valdivia, Chile G. Bellolio PO Box 3016, Concepción, Chile characterizing some clades of this family. Thus for example, the transference of some Thais to the genus Nucella (Subfamily Ocenebrinae) is supported by differences in the mode of embryonic development, which differentiates these subfamilies. Paleobiological data reported for Neogastropoda allow postulation of primitiveness in planktotrophic larval development compared to more recent developmental strategies such as direct development of different types, which characterize various clades of this family. Introduction The Family Muricidae is a group of carnivorous gastropods, which form a very diverse and important component of marine communities, with over 1,150 species grouped into eight subfamilies (Vokes 1996). However, available knowledge of the embryoniclarval development in this family is still very limited. Material available in this area consists primarily of descriptions of egg masses, and some basic parameters that describe the mode or pattern of development, such as the size and number of eggs, and, less frequently, the stage at hatching. In a few cases there is a descriptive record of development in sufficient detail to make a comparative definition of the pattern observed, but less on the morphological changes undergone by the free larval phase until reaching metamorphosis. This objective is particularly impeded in species whose larval phase is relatively long and is difficult to culture for extended periods in the laboratory. One of the few cases in which there has been special interest in obtaining this information has been with the rapanine Concholepas concholepas, which is commercially exploited, and for which there is basic knowledge of intracapsular and larval development (Gallardo 1973, 1979; Ramorino 1975; DiSalvo 1988, DiSalvo and Carriker 1994). The development of small and numerous eggs observed in their spawnings is linked to a long planktotrophic larval 682 phase lasting 3–4 months, which is probably one of the longest for a member of this family. The preceding may help explain the geological history of the genus (Beu 1970) as well as the present day behavior of the population in relation to recruitment events on the Chilean coast (Moreno et al. 1993a, b; Stotz et al. 1991). Background data on embryonic-larval development in other muricaceans from the Chilean coast, including species of interest to fisheries, has demonstrated diversity in patterns to be expected in Southeast Pacific waters. Among these is a short-term larval phase, which in some cases may be very brief and lecithotrophic, as well as species which demonstrate direct development supported by nutritive eggs; here may be included representatives of the subfamilies Ocenebrinae (Chorus giganteus, Acanthina monodon), Ergalataxinae (Xanthochorus cassidiformis) and Trophoninae (Trophon geversianus) (Gallardo 1979, 1980, 1981; Gallardo and González 1994). The present study investigates the embryonic-larval development in the rapanine Thais (Stramonita) chocolata, together with a description of its oviposition, and reproductive behavior of the snails in the laboratory. This species is a fisheries resource whose exploitation began in 1978, following imposition of a strict limit on the collection of Concholepas concholepas, another highly valued human food with which it shares many organoleptic characteristics. Harvesting of the ‘‘locate’’ increased gradually, reaching a maximum of 8,244 t in 1986. In spite of increases in fisheries efforts, the total catch declined to 1,174 t in 2000 (SERNAPESCA 2001). This demonstrated the inability of the resource to compensate for the heavy fishing pressure. At present T. chocolata is subject to two periods of total restriction on harvesting during the year, which total 8 months (SERNAPESCA 2001). The Genus Thais is widely represented among the Rapaninae, such that comparison with data on other congeneric species discussed here may allow definition of a dominant pattern among members of this subfamily. It may also present the first background on the relative extension reached by the development of the free larval phase in these muricids. Previous studies (D’Asaro 1966; Roller and Stickle 1988), working with congeneric species (T. haemastoma floridana and T. haemastoma canaliculata), have observed free larvae whose early morphology is similar to that observed in C. concholepas, although the total duration of this larval development was not directly determined in the studies cited. Most of the discussion given in the conclusion of the present study attempts to explore in what measure the assignation of muricids to some subfamilies is or is not congruent with the developmental pattern characterized by their evolution and by the potential usefulness of this type of ontogenetic data in phylogenetic relations among the Muricidae. For this, a review is made of information on the embryonic-larval development in various of the Muricidae scattered through the current literature, particularly that treating subfamilies evolutionarily related or similar to the species studied here. Materials and methods Broodstock of T. chocolata were collected by diving at Pisagua, (19° 36¢ 36¢¢ S, 70° 14¢ 30¢¢ W), Chile and studied in laboratories of the Coastal Center of the Universidad Católica del Norte, Coquimbo. Thirty five individuals of between 100 and 120 mm in length were maintained in 2,000-l seawater tanks having a through-flow of raw seawater (15 l/min) from La Herradura Bay at ambient temperature (12.8–18.7°C), with continuous aeration. The specimens were fed mainly with local scallops (Argopecten purpuratus), although various other bivalves were occasionally included in the diet. The individuals copulated en masse in October 1992 and deposited their egg capsules on the walls of the tank. Intracapsular development was described by selecting recently deposited capsules from four different females selected randomly. About 50 capsules from each female were tied to microscope slides and maintained in 1 l of 1-micron filtered, UV-treated seawater, which was changed every other day. One capsule from each of the four egg layings was dissected daily, and the larval stages observed were measured and photographed using a Nikon Biophot light microscope. A portion of each larval sample was fixed in 2% glutaraldehyde for later observation in a scanning electron microscope (SEM) (Turner and Boyle 1974). Six mature egg capsules from which larvae were hatching were employed in obtaining specimens for observing the developmental stages of free larvae. Larvae hatched over a period of 24 h were maintained in a 170-l seawater tank using filtered, UV-treated water as above, at an initial density of one larva per ml. Larvae were fed with a 1:1 mixture of artificially cultured Isochrysis galbana, and Chaetoceros calcitrans or C. gracilis at 40,000 cells per ml of the larval culture water. The culture was maintained with constant aeration at a thermostatically regulated temperature of 22±1°C. These conditions, although different than those that occur in nature, have been described as the most appropriate for the culture of veliger larvae of Concholepas concholepas under controlled conditions (Lara and Montes 1989). Maximal length of the protoconch, width of the velum, and lengths of tentacles and foot were measured using an ocular micrometer. Embryos and larval shells were observed and photographed using a model JSMT300 JEOL brand SEM. To evaluate induction of settlement by natural substrates, competent larvae were exposed to stones of 4–10 cm in length collected from the subtidal habitat at 5-m depth in Herradura Bay, which were encrusted with the barnacles Balanus laevis and the polychaete Romanchella pustulata. Recruits of the snails Crassilabrum crassilabrum, Tegula spp., and Calypraea trochiformis, as well as unidentified polychaetes were manually removed from the substrates prior to their use with the larvae. 683 Results Reproductive behavior and spawning characteristics Reproduction of Thais (Stramonita) chocolata is of the community type, with males and females piled on one another during copulation and deposition of gametes. Males position themselves over the females with shell borders aligned, and insert the penis below the mantle edge of the female. Copulation occurs as the females deposit egg capsules on the walls of the aquarium, or upon shells of other snails (Fig. 1a). The capsules of different females are deposited in irregular groups in close proximity, and are difficult to distinguish as to individual female of origin. The capsules are ampulliform (D’Asaro 1970, 1991; Cañete, 1992). The capsular body measures between 7 and 15 mm in length (x=10.8±2.0 mm, n=20), with a breadth of 3.0 mm (n=38) (Table 1). The peduncle is narrow and flat, measuring 2.5–4 mm in length, and is attached to the substrate by an adhesive secretion (Fig. 1b, c). Intracapsular development Soon after copulation, zygotes and a few spermatozoa were found in the capsules, within the intracapsular albumin. An average of 2,579±848 zygotes was counted in 36 capsules analyzed. Non-viable eggs, which could have been interpreted as nutritive eggs, were not observed. Fertilized eggs were encapsulated, and showed either no extrusion of polocytes, or only extrusion of the first polocyte. The zygotes were spherical, measuring 130±5.3 lm in diameter (n=100), and were opaque white due to contained vitelline material (Fig. 2a). The vitelline coat was granular in appearance, and distributed in the form of a network at the animal pole (Fig. 2b). Intracapsular development to the hatching stage required a total of 49 days incubation at 13.6±0.4°C. Early development was characterized by three polar lobes. The first lobe was formed before ejection of the second polocyte (Fig. 3a, b), the second is formed prior to the first division (Fig. 3c, d) and the third prior to the second division (Fig. 3e, f). Gastrulation occurred by epiboly (Fig. 4a, b); the trocophore (Fig. 4c), the preveliger (Fig 4d) and the intracapsular veliger (Fig. 4e, f) agree with the pattern described for other muricacean Gastropods having indirect development (D’Asaro 1966; LeBoeuf 1972; Gallardo 1973; Gallardo and González 1994). The SEM technique allowed clear observation of ciliated areas and their distribution in the intracapsular larval stages, as well as the type of sculpture on the surface of the larval shell (Amio 1963). A chronology of ontogenetic events considered here, as well as characterization of the embryonic and larval stages in the intracapsular period, are summarized in Table 2. Development of the free larvae Fig. 1 a Communal spawning group of Thais (Stramonita) chocolata depositing capsules in the aquaria. Bar=5 cm. b Group of ampulliform egg capsules. c Convex side of an egg capsule. Bar=4 mm Veliger larvae at hatching measure 220±10 lm in length (n=20). The embryonic protoconch is ornamented with granules scattered uniformly (Fig. 4e). After hatching, the larvae swim upward to the water surface, forming small aggregations. Recently hatched larvae fed on either I. galbana or Chaetoceros sp. showed intestines full of the microalgae after 30 min, demonstrating their readiness to feed at the moment of hatching (typical planktotrophy). Table 1 Capsular dimensions and mean number of embryos of Thais (Stramonita) chocolata Length of capsular body (cm) Length of capsular peduncle (cm) Mean no. of embryos Deposition I Deposition II Deposition III Deposition IV 1.02±0.04 0.44±0.05 1,832±161 (n=6) 1.04±0.05 0.44±0.07 2,267±69 (n=6) 1.28±0.04 0.6±0.07 3,181±103 (n=6) 1.3±0.0 0.24±0.07 1,691±113 (n=6) 684 Fig. 2 a Fertilized oocyte with first polocyte extruded. Arrow indicates the polocyte. Bar=50 lm. b Detail of surface of the zygote to show vitelline coat. Bar=1 lm; vc vitelline coat, pm plasma membrane Larval development continued over a period of 4 months at 22±1°C, during which there was notable development and increase in size of the larval structures. At the end of the first week post-eclosion, the velum is mildly asymmetrical, with the right lobe measuring about 170 lm in width, while the left lobe measures about 150 lm (Fig. 5a). Beginning in the second week post-hatching, a mucoid filament appeared at the extreme end of the metapodium, which remained until the larva reached the competent stage (Fig. 5b). Each velar lobe divided slowly over a period of 6 weeks, to produce a tetralobulate velum, with each lobe measuring approximately 1,595 lm in length (n=10) in larvae that had completed their growth (Fig. 5c, d). An extensible and motile propodium developed, with a propodial sulcus on its anterior portion (Fig. 5c). The tentacles grow to equal lengths, reaching about 250 lm (Fig. 5d). The protoconch II grows to its maximum length (1,450–1,740 lm) in helicoidal form with 3.25 turns around the embryonic (intracapsular) protoconch I (Fig. 5e, f). At competence, the veliger larva of T. chocolata demonstrates formation of a premetamorphic lip (Fig. 5f) as described for competent larvae of Concholepas concholepas by DiSalvo (1988). Growth of the shell does not proceed after formation of this structure. The shell of the competent veliger bears a beak over the central portion of the aperture coinciding with the central line of the body anfract, and a similar smaller projection on the internal border of the siphonal canal (Fig. 5d, f). The chronological development of free larvae with corresponding morphological changes are summarized in Table 3. Settlement and metamorphosis Competent larvae settled on the substrate crawl on the foot, actively employing the propodium in exploration of the substrate as they advance. They pause on reaching the tops of barnacles (B. laevis) and make slow semi-gyratory motions. Then, the first morphological event observed is the detachment of cilia from the velum about 5–15 min post-settlement. At 24 h after initiation of metamorphosis, the remnant of the velum is observed as a stump Fig. 3 a First polar lobe. b Zygote with second polocyte extruded. c Second polar lobe (‘‘cloverleaf’’ stage). d Blastomeres AB and CD. e Third polar lobe. f Blastomeres A, B, C, and D. Bars=50 lm; pl polar lobe, b blastomeres being formed; AB, CD, A, B, C, D blastomeres. Arrow indicates polocytes 685 Fig. 4 a Blastula. Bar=40 lm. b Gastrula. Note formation of the blastopore around quadrant D and the development of first ciliated regions. Bar=50 lm. c Trocophore, ventral view. Bar=40 lm. d Preveliger at 26-days development. Bar=50 lm. e Veliger at hatching, side view showing granular ornamentation of the protoconch. f Veliger larva, cephalic region. D Quadrant D, b blastomeres, e stomodeum, c cilia of shell gland, epc external preoral cilia, ipc internal preoral cilia, pc protoconch, pt prototroch, lk larval kidneys, t tentacle, v velum. Arrow indicates expansion of the prototroch 100–200 lm in length bearing a few remaining cilia. The remnant of the velum is observable up to 3 days postmetamorphosis (Fig. 6a). Another notable change is observed in the direction of the main axis between larvae and postlarvae. Precompetent and competent larvae have the cephalic region directly anterior to the central line of the body anfract below the beak of the shell between the tentacles (Fig. 5c, d). After completion of metamorphosis the cephalic region is turned about 45° to its left, and located beneath the siphonal canal and siphon (Fig. 6b). Another metamorphic change is observed in the buccal region. The larval mouth is a semi-circular ciliated aperture located near the base of the foot. At 24–48 h after initiation of metamorphosis continuous eversion of the proboscis may be noted. This structure may be rapidly retracted when larvae are disturbed. At the beginning of metamorphosis the protoconch is translucent brown in color, which changes to an opaque whitish color 24 h after initiation of metamorphosis. Discussion Developmental patterns This is the first report detailing the complete intracapsular and extracapsular developmental stages of a Thais species, facilitated by observation under laboratory conditions. Clearly the mode of indirect development shown by T. chocolata showed hatching of pelagic larvae Pre-trocophore Trochophore Trochophore Pre-veliger Veliger Hatched veliger 16 19 21 26 30 49 Emission of primary and secondary polocytes. Cleavage. Formation of the 1st, 2nd and 3rd polar lobes (Fig. 4a–c). Blastomeres group at the animal pole of quadrant D (Fig. 5a). Blastomeres are compacted in the animal region. Cilia develop in the animal region of the embryo. Major cell proliferation on dorsum. Formation of larval kidneys initiated. Formation of the gastrula by epiboly. Formation of blastopore. Four ciliated zones develop around the blastopore. Polocytes are released (Fig. 5b). The embryo lengthens in the antero-posterior dimension. The blastopore moves to the upper apical third and the stomodeum is formed. Cilia are formed in the region of the head vesicle. The shell gland is formed. Formation of the prototroch is initiated. Apical sensorial organ is formed. Ciliation increases in the ventral region. Beginning of formation of foot and operculum. Formation of larval prototroch, larval protoconch, statocysts and anal gland (Fig. 5c). Protoconch is oriented dorsally and torsion begins. Prototroch expands laterally. The anal gland has rotated through 90°. Musculature becomes functional (Fig. 5d). Bilobulate velum is formed with compound preoral cilia. Eyes develop pigmentation. Anal gland has rotated 180°. Larvae begin to swim within the capsule. (Fig. 5e). The velum develops a second band of simple preoral cilia and the food groove. Heart and stomach become visible. The foot has developed mesopodial and metapodial lobes. The protoconch has a punctiform ornamentation and measures 220 lm (Fig. 5f). Early embryo Blastula Blastula Stereoblastula Gastrula Post-gastrula 1 2 3 5 14 15 Morphological changes in the embryos Developmental stage Days after spawning Table 2 Chronology of intracapsular development under laboratory conditions at 13±0.4°C, and principal morphological changes characteristic of the different stages 686 which were planktotrophic and numerous, with a free larval period which, according to results obtained in laboratory culture, may be described as very long. Although there have been few equally exhaustive studies for other species of this extensive family, the comparison of some key traits in ontogeny (compiled in Table 4) allow analysis as to how far congeneric species in general, and the Rapaninae in particular, share or do not share a common embryonic-larval development pattern, and to what extent this also provides a basis for the present generic composition proposed for this clade. Some species of Thais have been transferred to Nucella and thus to the Ocenebrinae, implying an evolutionarily important change in the mode of embryonic development of these, and a trait which until now has not been sufficiently evaluated for use in phylogenetic considerations for the family. A comparison of these two subfamilies, considered to be ‘‘sister clades’’ within the Muricidae (Collins et al. 1996) permits an initial approximation in this sense. Parallel elements in the early ontogeny of Thais species and their relation to other members of the Rapaninae As a result of recent studies, the Genus Thais has been phylogenetically associated with other genera (for examples Rapana, Purpura, Concholepas, Dicathais, Plicopurpura and others) forming the subfamily Rapaninae (Kool 1993a; Vokes 1996). Kool (op cit) proposed a new phylogenetic classification of the genera within this subfamily of muricids based on anatomical characteristics, as well as radular, opercular, protoconch, and shell ultrastructural morphology. The cladistic analysis of 18 characters resulted in a synonymization of the Thaidinae of previous authors with the Rapaninae. Furthermore, using evidence obtained from protoconch morphology, he concluded that all members of the Rapaninae he studied probably have planktonic larvae, suggesting that this ontogenetic trait is a general characteristic of members of this subfamily. In fact, the cladistic analysis of the morphological features determined that species of Nucella (snails with direct development) should be excluded from the Rapaninae and included in the Ocenebrinae (Kool 1993a, b). All indications from the cladogram presented by Kool (1993a) are that two subfamilies of the Muricidae are recognizable, and that the species within each differ markedly from each other in their mode of development. In order to evaluate this situation, we have gathered data from a significant number of muricids as regards their patterns of development (Table 4). This table allows clear corroboration of the preceding conclusion, clearly showing that systematic ordering of the Muricidae should also include background data on comparative embryoniclarval development. As can be observed, Thais species have a development pattern that is more or less generally shared among the other genera of Rapaninae, providing support for including this genus in a common clade. The 687 Fig. 5 a Larva 1 week after hatching. b Larva 2 weeks after hatching showing the mucoid filament. c Larva 16 weeks after hatching; detail of cephalic region and ventral region of foot. d Shell aperture, anterior lateral view to show position of central beak of the larval protoconch in reference to the tentacles and anterior extreme of propodium. Note propodial sulcus. e Larval protoconch. Bar=300 lm. f Larval protoconch in apical view. =500 lm. c cilia, e eyes, f foot, m mouth, mf mucoid filament (protoconch), pg pedal gland, pr propodium, ps propodial sulcus, rvl right velar lobes, lvl left velar lobes, op operculum, t tentacles predominant pattern includes pelagic larvae derived from numerous, relatively small to medium-sized eggs without nutritive eggs, characteristic of planktotrophic larvae. Very little is known about the extent of the larval period in other species of Thais, except for data on T. haemastoma (D’Asaro 1966; Scheltema 1986; Butler 1954) describing a long-lived planktotrophic larva, and an individual production of about 500,000 larvae per year. The developmental pattern apparently coincides with that presently given for T. chocolata. There are strict evolutionary relationships among the Ocenebrinae as inferred by the comparisons of intracapsular developmental traits, also supported by the cladistic analysis made by Kool (1993a). This is the case of the clade proposed by this author where a strict evolutionary relationship is shown between Nucella, Acanthina, and Forreria (this last closely related to the genus Chorus from the Chilean coast). Species in this group whose development is known have in common lecithotrophic development, with nutritive eggs having very similar morphological characteristics. This similarity is very strict in the case of the Chilean species Acanthina monodon and Chorus giganteus, suggesting ancestral traits of common origin (Gallardo, in preparation). Vermeij and Carlson (2000) reviewed the evolutionary relationships of the Rapaninae as deduced by Kool (1993a), taking into consideration a larger number of taxa (45 genus-level taxa, and five muricid outgroups) and incorporating functional (shell characters), 688 Table 3 Development of the free larva, showing size and morphological characteristics of the veliger larva in its different stages Weeks after Morphological features hatching Mean length (lm) 1 2 3 220±10 296±0.2 320±23 4 6 8 10 12 16 Initiation of formation of protoconch II or larval protoconch. Velum slightly asymmetrical. Projection over the cephalic region becomes apparent. Pigmentation increases in the base and borders of foot. Secretion of byssal thread begins. The larval shell completes its first revolution. Axials are formed at the base of the first revolution of protoconch II. Formation of the central line and siphonal canal begin. The larval shell has completed 1.5 revolutions. Division of velar lobes is initiated. Velum in ‘‘butterfly’’ form. Larval shell almost completes two revolutions. Development of siphonal canal is marked. Primordium of left tentacle appears. Tetralobulate velum in the form of an ‘‘X’’. Increase in pigmentation of foot and border of mouth Primordium of the propodium appears. The larval shell passes 2.5 revolutions around the embryonic protoconch. Siphonal projection is formed. Relaxed propodium measures 200 lm in length. The protoconch shows 3.25 revolutions around the embryonic protoconch. Growth stops. ‘‘Premetamorphic lip’’ is formed. 395±50 492±55 556±65 878±52 1,450–1,750 Fig. 6a, b Process of metamorphosis in T. chocolata. a Resorption of velar lobes. Bar=200 lm. b Postlarva, 48 h after initiation of metamorphosis; note eversion of proboscis. Bar=300 lm. f Foot, pb proboscis, lp larval protoconch, t tentacles, v remnant of velar lobe ecological, and fossil evidence in their cladistic analyses. They used their phylogenetic results to probe aspects of the ecological history of this subfamily, including evolution of antipredatory shell defenses, methods of predation and specialization for life in upper-shore habitats offering opportunities for refuge. They conducted five separate analyses on matrices with varying numbers of Table 4 Comparison of developmental characteristics in Rapaninae and Ocenebrinae muricacean snails Subfamily Species Rapaninae Rapana venosa No. of eggs Egg diameter or embryos (lm) per capsule 790–1,300 eggs; few embryos Time for Mode of developmenta intracapsular development (days) PD Rapana bulbosa Rapana venosa Rapana thomasiana Purpura patula Concholepas concholepas Thais haemastoma Thais chocolata Thais dubia Thais hippocastaneum Thais rustica PD PD 260 PD 240 PD 158–169 2,600–13,200 eggs. PD Thais Thais Thais Thais Thais Thais Thais 125 190 200–230 140 142 1,094 252 800–1,400 embryos coronata clavigera carinifera rudolphi bufo deltoidea tissoti Dicathais aegrota Plicopurpura pansa Ocenebrinae Ocenebra japonica Ocenebra interfossa Ocenebra lurida Ocenebra aciculata Ocenebra erinacea Ocenebra inermicosta Ocenebra sp. Pteropurpura festiva Ceratostoma burnetti Nucella lapillus Nucella emarginata Nucella lamellosa Nucella canaliculata Time for Hatching larval size (lm) development (days) 420 790–1,300 eggs 500–900; 4,000 1,700–3,200 eggs 80 1,070 eggs; 400 embryos 215 149 19–32 embryos, 255–295 730–7,180 eggs 95–1,092 embryos 170–200 3–5 embryos 5–12 juveniles 450 225–250 782–870 300 187 500–1,000 eggs 180–210 300–1,000 eggs; 20–33 embryos 590–638 19–81 eggs 375–620 13–25 embryos 240 Acanthina monodon 240 249 500–900 eggs 12–49 juveniles 10–17 juveniles 710–2,120 eggs; 45–143 embryos 134–1,116 eggs PD PD PD PD PD PD PD 36–50 (17–18°C) 15 (24°C) 49 (13.6°C) 90–120 120 19 17 10–11 (24°C) 19 PD PD 36–65 (21–23°C) (n.e.) 410 400 260 130–160 225 1,320 >700 300–320 340–400 224 334–367 360 231 Spight 1976 Spight 1976 Spight 1976; Barkati and Ahmed 1983 Barkati and Ahmed 1983 D’Asaro 1991; Barkati and Ahmed 1983 Spight 1976 D’Asaro 1991; Barkati and Ahmed 1983 240 188 Phillips 1969; Spight 1976 L .Naegel (personal communication) 1,400–1,760 Spight 1976 D’Asaro 1991 D’Asaro 1991 Spight 1976 Spight 1976 D’Asaro 1991 Spight 1976 D’Asaro 1991 Spight 1976 Spight 1976; Costello et al. 1957 Spight 1976; D’Asaro 1991 (n.e.) 960 (n.e.) 775–2,025 21–28 (n.e.) (n.e.) (n.e.) DD DD (n.e.) DD (n.e.) DD DD PD (n.e.), veliconch DD (n.e.) 120 72 (9–11°C) 80 (8–10°C) 29 (11.5–17°C) 90–150 60–72 (15.5°C) 2–3 70–80 (9.7–10.6°C) Spight 1976 D’Asaro 1991 Spight 1976 Spight 1976 1,500–1,700 Gallardo 1973, 1979; DiSalvo 1988; DiSalvo and Carriker 1994 D’Asaro 1966; Butler 1954 1,450–1,750 Present study Spight 1976 Spight 1976 D’Asaro 1970; Spight 1976 1,000 1,150–1,190 1,000 1,300 Spight 1976; Strathmann 1987 Strathmann 1987; Lyons and Spight 1973; D’Asaro 1991 Strathmann 1987; Pechenik et al. 1984 D’Asaro 1991 D’Asaro 1991 1,042–1,226 1,042–1,226 Gallardo 1981; González and Gallardo 1999 825–1,300 Gallardo 1979 689 Nucella lima Nucella cingulata Nucella dubia Chorus giganteus PD PD DD (n.e.) DD (n.e.) PD DD DD DD DD DD DD DD PD DD DD DD Source D’Asaro 1991 280–320 107 130 Size at settlement (lm) 1,500 700–1,100 402 DD DD DD (n.e.) DD DD PD 800 800–1,000 900 D’Asaro 1991 Spight 1976 Spight 1976; Costello et al. 1957; Hancock 1956 Spight 1976; D’Asaro 1991 DD (n.e.) DD D’Asaro 1991 Spight 1976 D’Asaro 1991 Spight 1976 Gallardo and González 1994 D’Asaro 1991 DD (n.e.) D’Asaro 1991; Spight 1976 670 a DD direct development, PD planktonic development, n.e. nurse eggs. PD 1,250–1,500 2embryos 60–220 15–20 embryos 300 Ceratostoma rorifluum Ceratostoma burnetti Ceratostoma nuttalli Eupleura caudata Crassilabrum crassilabrum Forreria belckeri Ceratostoma foliatum 340–390 230 47–80 embryos; 19–35 juveniles 7–12 embryos 60 28–50 eggs Acanthina lapilloides Urosalpinx cinerea Acanthina paucilirata 240, 300–400 720 40–140 eggs; 20–39 embryos 153 eggs; 8 embryos 275 Acanthina spirata DD (n.e.) No. of eggs or embryos per capsule Egg diameter (lm) Species Subfamily Table 4 (Contd.) Mode of developmenta Time for intracapsular development (days) Time for larval development (days) Hatching size (lm) Size at settlement (lm) Source 690 taxa and characters in each. Although differing in several respects, the hypothesis of the rapanine phylogenetic relationships favored by these authors (that resulting from the analysis including a combined shell and anatomical data matrix) is largely in agreement with relationships previously postulated by Kool (1993a). A notable point here is that the results confirm that Concholepas concholepas, Rapana, and Cymia are among the most primitive members of the Rapanine clade, consistent with Kool’s analysis and their relatively early appearance in the fossil record. An outstanding fact in comparing the Rapaninae is also the strict developmental similarity we can see between T. chocolata and Concholepas concholepas. The developmental pattern shared by these two species (planktotrophic pelagic larvae derived from small, numerous eggs) is particularly similar in intracapsular development (Gallardo 1973; Ramorino 1975), the relative duration of this under controlled temperature conditions (Gallardo 1973, 1994) and above all the morphological development occurring during free larval life (DiSalvo 1988; DiSalvo and Carriker 1994) until reaching metamorphosis over a comparable time period. Common developmental traits and larvae reflect a reproductive pattern and structural organization common in species with long planktotrophic lives according to standard morphological criteria (Scheltema 1971, 1986) for this type of larva. These include (a) quite high fecundity, (b) marked morphological development and growth during the larval phase, and important development of a multilobulate velum and, (c) the well defined apertural beak and beak line (a feature also shared with T. hemastoma floridana) suggested as a characteristic trait of most long-term planktotrophic prosobranch veligers (D’Asaro 1966). The extended free larval phase which clearly differentiates these muricids from the rest of the Chilean species in the family raises a question concerning the high potential for larval dispersion which this implies, and its effect on recruitment and population dynamics of these muricids which are exposed to a high degree of commercial exploitation (Castilla 1988; Castilla and Camus 1992). Interpopulational genetic studies have been postulated as being an indirect estimator of potential larval dispersal among species differing in extension of their larval lives (Scheltema 1971; Palumbi 1995). Genetic data obtained for Chilean muricaceans (C. concholepas, Chorus giganteus), the dispersion potentials of which are very different from one another, are in agreement with the postulated dispersion potentials (Gallardo and Carrasco 1996; Gajardo et al. 2002) and are an interesting avenue for examining this type of relation. Other evolutionary implications Based on paleobiological data, which support conclusions reached for the Neogastropoda (Jablonski and Lutz 1983; Hansen 1983) we postulate that the planktotrophic 691 larval development is probably a primitive evolutionary condition in the Muricidae compared with the development of lecithotrophy and/or direct development predominant in other clades, a conclusion which is also derived from the cladistic analysis made by Kool (1993a). In order to test this hypothesis it is of interest to observe the relative times of appearance of the different clades in the fossil record. The importance of considering the mode of larval development in the paleontological history of muricaceans has been considered by some authors (Beu 1970), postulating possible implications of an extended larval phase in the capacity of Concholepas species to survive extinction events in the past. The two subfamilies of the Muricidae considered here show clear differences in developmental patterns. While pelagic, probably planktotrophic, larvae predominate in the Rapaninae, with planktotrophy considered a primary condition, the Ocenebrinae show developmental modes considered to be evolutionarily derived, such as direct lecithotrophic development. Here, retention of some species of Thais that have direct development and nutritive eggs, and are classified within the family Rapaninae (Table 4) raises some doubt. There are other species with this type of development which, on the basis of other taxonomic considerations, were changed from the Genus Thais to Genus Nucella (e.g. T. emerginata, T. lamellosa, T. canaliculata), and thus to the subfamily Ocenebrinae. Almost all of these show direct development (hatching size 1–1.4 mm) and to a lesser degree, short lived lecithotrophic larvae as also suggested by Kool (1993a) in characterizing this subfamily, all of which support the generic change indicated. Direct development in the Ocenebrinae is obtained by means of large eggs (400–700 lm) and in some cases (more commonly) by means of nutritive eggs with intermediate egg size as in many Acanthina and Nucella spp. with a secondary loss of the nutritive egg habit in species of Nucella (Collins et al. 1996). Egg capsule morphology The ampuliform egg capsule type shown for T. chocolata is one of the morphological patterns described for the genus by D’Asaro (1991), and has species-specific characters which permit recognizing them among those of other congeneric species due to structural details of the capsular body and peduncle. As suggested by D’Asaro (1991) the macroscopic morphology of the capsule deposition does not in and of itself permit establishing evolutionary connections among the muricids, and must be accompanied by other comparative characteristics. Furthermore, a better comparative description of the egg capsules among different taxa may be obtained by examining the microscopic structure of their walls (D’Asaro 1988; Garrido and Gallardo 1993), which together with other traits in embryonic-larval development provide ontogenetic and reproductive data potentially useful in phylogenetic determinations. 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