BULLETIN OF MARINE SCIENCE, 65(3): 745–754, 1999

DOES EARLY DEVELOPMENT OF THE CHILEAN PRAEPUTIALIS (HELLER, 1878) EXPLAIN THE RESTRICTED DISTRIBUTION OF THE SPECIES?

Marcela Clarke, Verónica Ortiz and Juan Carlos Castilla

ABSTRACT Relationships between the rate of larval development and dispersal of a species can have a large influence on observed patterns of its distribution and abundance. Distribu- tion patterns of the dense belt-forming rocky intertidal ascidians Pyura praeputialis and P. stolonifera (Urochordata: : ) show drastic differences. P. stolonifera extends along the coast of for 1000s of kilometers and P. praeputialis shows a similar range in (New South Wales, Victoria and ). On the contrary, in Chile P. praeputialis is characterized by a population which occurs along less than 70 km of coastline in and around the Bay of Antofagasta. The larval and development char- acteristics of the South African and Australian species have been studied, while those of the population from Chile has not been described. This study describes the early embry- ology and larval development of the Chilean species and shows that the development times and the free-swimming larval stage of P. praeputialis in Chile and Australia are similar. Therefore, it is suggested that the differences in the extension of their range of distribution in Australia and Chile cannot be accounted for due to differences in the de- velopmental biology, but it may be due to a recent invasion of the species to Chile. Com- parison between the development biology of P. praeputialis and P. chilensis are also made.

Geographic patterns of the distribution of species result from the combination of his- toric and regional environmental factors and local processes, such as perturbation, com- petition and predation, which determine the survival of a species in a given habitat (Ricklefs, 1987; Cornell and Lawton, 1992; Ricklefs and Schulter, 1993). On the other hand, devel- opment, larval biology and dispersal capabilities of a given species play important roles in determining distribution patterns over geographic scales; whereby understanding a species development and larval biology may provide insights into how species occupy wide-spread, restricted, or disjunct geographic patterns. Knowledge of the larval develop- ment and dispersal capabilities of organisms is also important to determine population dynamics and has implications for the management of natural populations, including the design of nature and fisheries reserves and forecasting the response of populations to regional environmental changes. Biogeographic patterns in the Southern Hemisphere have revealed the importance of historic and tectonic processes in determining the distribution of both marine and terres- trial species, indicated by the existence of common genera among the southern continents which were once part of Gondwana (Keast ,1971; Knox, 1980; Stevens, 1980). One ma- rine example is the ascidian fauna, which in the southern hemisphere is dominated by the genus Pyura (Kott, 1985). Although several of these species form dense intertidal belts along rocky shores of the southern continents (Fairweather, 1991; Paine and Suchanek, 1983; Castilla, 1998), they are still taxonomically poorly understood. In South Africa, (Heller, 1878) extends along 1000s of kilometers from Cape Town to Natal (Day, 1974), and P. praeputialis (see Fairweather, 1991) [= P. stolonifera, see Kott, 1985] in Australia is also found along 1000s of kilometers along the coast of New South

745 746 BULLETIN OF MARINE SCIENCE, VOL. 65, NO. 3, 1999

Wales, Victoria and Tasmania (Kott, 1985). On the contrary, in South America P. praeputialis (Heller, 1878) [= P. bradleyi Van Name, 1931] shows a restricted geographic distribution, where it is found exclusively along approximately 60–70 km of coastline in and around the Bay of Antofagasta, Chile (Cerda and Oliva, 1997; Castilla 1997, 1998). Nevertheless, Van Name (1945), based on samples collected for the Peabody Museum of Yale University, reported the presence of P. bradleyi (used as a synonym of P. praeputialis, Kott, 1985) in Peru, at Zorritos (3°40'S, 80°40'W). These ascidians inhabit exposed rocky shores where they form beds of densely aggre- gated individuals permanently attached to the rocks, at mid and low intertidal fringes and extend to the shallow subtidal (Guiler, 1959; Paine and Suchanek, 1983; Fairweather, 1991; Fielding et al., 1994; Castilla, 1998). The population of P. praeputialis in Antofagasta Bay forms extensive intertidal beds up to 10–12 m wide (Castilla 1997, 1998), with indi- viduals reaching up to 35–40 cm in height. The species attains a pseudo-coloniality, which imparts upon them distinct competitive advantages (Paine and Suchanek, 1983). The embryonic development, metamorphosis and post-metamorphosis have been reported for P. praeputialis from Sydney, Australia (Anderson et al., 1975), P. stolonifera, from Cape Town, South Africa (Griffiths, 1976), and for P. chilensis from Concepción, Chile (Cea, 1969/1970). Nevertheless, P. praeputialis embryonic development, from Antofagasta, has not been described. The disjunct geographical distribution of these species make inter- continental comparative studies attractive, since the species of these locations could be characterized by different larval developments. Furthermore, it has been hypothesized that the local population of P. praeputialis at Antofagasta could represent a relic group of the fauna of Gondwanaland (Kott, 1985). The aims of this paper, that form part of a long-term study on the biology and ecology of the Chilean P. praeputialis, are to describe its embryonic, metamorphic and post-meta- morphic stages, and develop comparisons with the other southern hemisphere pyurid species, in particular with P. praeputialis from Australia.

MATERIALS AND METHODS

Adult specimens of P. praeputialis were collected during low tides, from May 1997 to January 1998, from two mid-intertidal rocky platforms at Quebrada El Way (23º45'S, 70º26'W), approxi- mately 10 km south from the city of Antofagasta, and at Las Conchitas (23º31'S, 70º32'W), ap- proximately 40 km north of Antofagasta (Fig. 1). Specimens were knife-detached from the base of their stolon, transported to the laboratory and kept in an open sea water circulating system at the University of Antofagasta. Temperatures ranged between 19–21ºC. Each specimen was removed from its test and opened to expose the gonads. The state of maturity was assessed by visual estimates following the scale proposed by Gutiérrez and Lay (1965) and confirmed with microscopic observations. These authors reported that in P. praeputialis (wrongly identified in the paper as P. chilensis) the gonadal lobes are packed with sperm and ova from March to August. However, unpublished results (J. C. Castilla) indicate that this period extends from March to December–January. Olive-green turgescent or distended ovaries, and mammillated while-yel- lowish testes were considered as evidence for mature stages. The ovaries and testes were removed with a scalpel to separate off pieces of egg and sperm slices, which were then lightly pressed between two microscope slides. Eggs and sperm were suspended with filtered sea water (1 µm AMETEX filter) and washed three times with filtered sea water. Eggs were dispensed into glass Petri dishes (9 cm diam) in sufficient number to provide an evenly spaced distribution on the bottom. The sperm suspension was prepared in a similar dish with filtered sea CLARKE ET AL.: EARLY DEVELOPMENT OF PYURA PRAEPUTIALIS 747

Figure 1. Map of the Mejillones Peninsula and Bay of Antofagasta, showing in black the extension of the intertidal Pyura praeputialis beds. Main sites of collection are shown. water until a white suspension was obtained. Finally, 500 cc of solution of eggs and 200 cc of sperm were mixed in a 1-L glass beaker under continuous aeration provided by a diaphragmatic pump (1.4 W), at 19–21ºC (according to Stern and Holland, 1993, ascidians resist polyspermy under these conditions). After 30 min the water was changed three times to remove excess sperm. Following the decanting of fertilized eggs, the supernatant was carefully eliminated. No mesh sieve was used in the operation. Thereafter, filtered sea water was changed every 24 h. Twenty six artificial cross fertilizations, from individual specimens (n = 52), were conducted. Because Pyura feeding occurs after metamorphosis, embryonic development stages advance with- out providing food. The development was checked several times during the day by collecting em- bryonic stages with a 10 cc pipette and examining them under a Zeiss stereomicroscope and an Olympus CH2 microscope. The drawings of the different stages were made by direct observation of the embryos and the use of photographs and video recording. To facilitate the observation of tad- pole larvae they were transferred to a 1-L glass flask (18 cm in diameter) with four Petri dishes (4 cm in diameter) placed on the bottom. Embryos were cultured up to 17 d.

RESULTS

Normal development was observed in 21 of the laboratory fertilizations. Five fertiliza- tions produced larvae which did not metamorphose and died before hatching. In these cases the Pyura specimens were collected after a storm in August 1997, and showed mature, although flaccid, gonads. Gamets.—Mature eggs were spherical, olive-green with reddish pigments scattered over the surface and 240–300 µm in diameter, including the inner follicle cells (Fig. 2A). The egg pigment crescent is absent. After immersion in sea water an external vitelline coat (chorion) lifts off with small inner follicle cells in the outer surface and test cells 748 BULLETIN OF MARINE SCIENCE, VOL. 65, NO. 3, 1999

Figure 2. Embryonic development of Pyura praeputialis from Antofagasta, Chile: A = Unfertilized egg. B = 2 cell stage. C, D, E = 4, 8 and 16 cell stages. F = Later gastrula. G = Early neurula. H = Later neurula. I = Early embryo. J = Later embryo. (opposite page) K = Metamorphosis: tadpole larvae showing the trunk, tail and leaf-like caudal fin. L, M = Tadpole attachment and tail resorption. N, O = Ampullae development. P = Final development of ampullae and early siphons. Q = Young P. praeputialis 16 d after fertilization. within the 10–20 µm wide perivitelline space between the egg surface and the chorion (see Satoh, 1994). The sperm varied in length, from 38 to 51 µm, and have a narrow rod-like head ranging from 7 to 10.8 µm and a tail 31–43 µm long. The sperm become active when sea water is added and remain so for 7–8 h. Embryonic Development.—P. praeputialis development schedule is shown in Table 1. The first division cuts the egg along its longitudinal axis (Fig. 2B) and the second is CLARKE ET AL.: EARLY DEVELOPMENT OF PYURA PRAEPUTIALIS 749

meridional. The direction of this division determines the anterior and posterior poles of the embryo. The inequality of the blastomeres is shown in Figure 2C. The third division results in four dorsal and four ventral blastomeres (Fig. 2D). Following cleavages raise the number of cells to 16; 32 and then 64 (Fig. 2E). The blastula has no cilia and the segmentation cavity is not recognizable. Blastulas are spherical and range from 230 to 260 µm in diameter. The early gastrula is bowl-shaped and gastrulation is achieved by invagination and involvement of the wall cells at the vegetal pole (Satoh, 1994). Later gastrula (Fig. 2F) are spherical and their size is similar to the blastula. The neurula varies 750 BULLETIN OF MARINE SCIENCE, VOL. 65, NO. 3, 1999

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Tf)setunim:sruoh(emi Numbero SntageMmeaMmaximuMsinimuObservation F6irstcleavage00:510:300:43 S9econdcleavage15:010:400:43 89-cell 10:110:500:53 156-cell 10:327:410:03 392-cell 25:021:410:43 B5lastula 25:335:520:13 E2arlygastrula35:130:320:43 L9atergastrula40:150:340:03 N0eurula 55:275:148:13 L6aterembryo150:5105:478:33 H7atchinglarvae151:3108:1190:05 S8ettlementlarvae155:0225:4132:27 Admpullae2 tnempolev 159:1256:4115:23 S2iphonsdifferentiation650:1658:0527:22 O3paquetunic3033:03084:02516:01 from 240 to 290 µm long and 160–260 µm wide, and the pear-shaped tail bud elongation is approximately 100–170 µm long (Fig. 2G). Early embryos show the tail rolled (Fig. 2I) and exhibit convulsion movements. At this stage the notochord, otolith (static organ) and ocelli are present. Later embryos (Fig. 2J) hatch out of the chorionic membrane on average 11:37 h after fertilization (Table 1). Tadpole Larvae.—The early hatched tadpole larva are slender and range from 1080 to 1380 µm long (Fig. 2K). They are completely covered by an extracellular tunic (Katz, 1983), impeding direct feeding (Conn, 1993). The trunk (anterior part) varies from 180 to 300 µm long and from 125 to 220 µm wide. The tail (middle portion) ranges from 600 to 800 µm long and 60 to 93 µm wide, excluding the fins. The leaf-shaped caudal fin (pos- terior part) ranges from 130 to 360 µm long and 80 to 110 µm wide. In the front of the trunk there are three adhesive papillae arranged in a triangle. They consist of a stalk and a terminal rod-like organ and range from 10 to 30 µm in length. The static organ is about 10 µm in diameter and the ocelli are approximately 5 µm diameter; they are approxi- mately 30 µm apart. Tadpole larva actively swim and show negative phototropic behavior. Before settlement the tadpoles faced the bottom of the culture flasks in an downward position. Metamorphosis.—In the laboratory the mean active free-swimming period for tadpoles was 2:38 h (SD = 1:03 h, 52 observations). Larva became attached to the Petri dishes (Fig. 2L,M) by the anterior end. The initial stages of metamorphosis were characterized by the resorption of the tip of the caudal fin and the attachment of the adhesive papillae to the substrate, followed by an expansion of the tunic in contact with the substrate which spread approximately 70 µm and the development of the ampullae. The full process takes on average 3:49 h (SD = 2:52, 27 observations). At first 6 to 8 ampullae are extended onto the substrate and later the number increases up to 15 (Fig. 2 N,O,P) reaching up to 180 µm in length. In eight specimens we observed a delay in larval settlement (metamorphosis), CLARKE ET AL.: EARLY DEVELOPMENT OF PYURA PRAEPUTIALIS 751 and the free-swimming larva remained in such stage between 7–24 h after hatching. We seldom observed the initiation of metamorphosis in unattached free-swimming larvae. Final Stages.—Approximately 57–68 h after fertilization (Table 1) juvenile Pyura bod- ies range from 260 to 330 µm in diameter and the siphons differentiate. Between the third and fourth days after fertilization, the endostyle and one pair of protostigma, as well as ciliary activity, were observed. At the same time inside the ventral gut vesicular cells (crescent form) and longitudinal and circular muscle fibers can be seen. Half a day later, the heart beats in irregular intervals and the vesicular cells have diminished. By the fifth day after fertilization, the oral tentacles are about 20 µm in length and were seen inside the oral siphon. Pyuridae spicules, both aciculate and triangular, can be seen arranged in circles around the siphons (Monniot, 1971; Lowestam, 1989). Seven days after fertiliza- tion, the pigmented circle around the tip of the siphons appears. Between 9–17 d (highly variable) after fertilization, the tunic turns opaque, and the young, filtering ascidians, measure from 875 to 1200 µm in length (Fig. 2Q).

DISCUSSION

According to laboratory results the early development biology of P. praeputialis from Antofagasta, Chile is characterized by similar embryonic and larval elements to P. praeputialis (= P. stolonifera) from Australia (Anderson et al., 1976). This include the ovum diameter, tadpole trunk and tail length and the absence of pigmented crescent cells in the eggs (Table 1). The timing of the embryonic stages of P. praeputialis from Sydney and Antofagasta are also similar (Table 2). P. praeputialis from these two geographic regions show free-swimming larval period of approximately 1–3 h; while the reported free-swimming larval period for P. stolonifera from Cape Town lasts “a few hours after hatching” (Griffiths, 1976). Development time differences regarding the siphon differen- tiation between the South African (shorter time) and the Chilean (longer time) species are reported (Table 2). On the other hand, comparison of the embryonic, metamorphic and post-metamorphic development of P. praeputialis from Antofagasta with its sympatric subtidal species, P. chilensis (Cea, 1969/1970) show large differences, including a free- swimming larva span in P. chilensis lasting over 7 h (Table 2). In the case of Chile the short span of the free-swimming larva of P. praeputialis may partially help to understand its restricted geographic distribution in and around the Bay of Antofagasta, particularly if associations with water circulation retention mechanisms present in the Bay are made (J. C. Castilla, pers. observ.). However, P. praeputialis (= P. stolonifera) from Australia, with a geographical distribution along 1000s of kilometers of rocky shore shows similar embryonic, free-swimming larval and metamorphic times. Therefore, the short free-swimming larval span of the species can not account in itself for its restricted range of distribution in Chile. The prevailing hypothesis is that the Antofagasta P. praeputialis is a Gondwana relic population (Kott, 1985), once showing a more exten- sive geographical range. In such case, according to laboratory results, the Australian and Chilean populations of P. praeputialis, yet probably disconnected since the late Meso- zoic, still remain very similar in their development biology. Nevertheless, another possi- bility is to consider that the disparity between the reduced distribution of P. praeputialis in Chile vs Australia may be due to a recent invasion of the species to Antofagasta (i.e., via ships), and that the water circulation retention mechanisms inside the Bay has so far restrained its dispersion. In spite of collecting efforts we have found no evidence of the 752 BULLETIN OF MARINE SCIENCE, VOL. 65, NO. 3, 1999 ) r e p a m i p t s 0 i 0 s s e 3 n i t 7 : n / a h u ° 1 e 3 t e c 9 0 0 l : n 1 ( 5 0 0 0 i i p 6 5 0 0 3 1 3 3 3 m 0 0 : : r h e e 9 4 : : : : : − l m . : 8 8 c o c 1 i 3 0 6 : d 8 4 c 0 p 8 5 0 n s h 1 1 1 n , 3 2 p a 0 2 r 2 2 o : a a r m e 3 C u : e 2 e c u Cón.elihC, , e o 7 y n T C a g h ( t o m P ( a n s o c C a a r (ón)elihC, fi g R , a f s i o s t n n e l A i a h n c m i c i m o r i h . t r r f 7 c s P f; t e e 7 f l A n a t i silaitupearp.P 0 9 a a . : h u 0 ° n 1 e d 2 S n 0 0 i o 0 0 r i : , t l 7 , 2 m 0 e 0 0 s 2 : t b o : : − n m . t h f 1 : 8 u d 6 2 t 4 s p a s i w s 1 n 9 7 2 a r f o : a s a m f c d u r i r T 8 e n r e o u u 20 TC a e g h o y G () p () n a h Ps a a c i C Re r w laditretniehtfotnempolevedsisohpromatem-tsopdnasisohpromatem,cinoyrbmeevitarapmoC.2elbaT (i f e” * A “g h t u o a S i l , i a l 6 n C r s a t 7 i e w °C s t t 9 m 0 0 0 o 3 u i : 0 : : u u 1 0 t 0 0 : 0 2 T 4 : : 4 4 p 2F n 0 A , 0 0 f i : − e 1 e 6 1 3 2 : n , 1 o 2 0 a p − s − − − − 3 o m − 1 r 2 0 : a e s 0 e 0 0 0 0 : l s 0 r a p 0 g 0 3 0 0 a r C 1 a e c : 0 : : : : p c0 n : u a d 3 5 00 8 0 a a r W 80 o m m n 10 c-00: 2 2 u . h o e Re () r y A S T f () . Pa , N a () r e f i n o l o ) t s s e i l . ) l i C 0 s e P a h 5 5 5 5 0 i . e ) °. l 5 : t t r m C 1 4 4 5 0 5 0 n 12−91 a i 00 : : : 4 : 4 u u e t , 4 1 3 o : d 8 6 5 : 8 : : 8 p i n p a i 5 t f i t t 1 2 2 e 1 6 7 a 5 3 a 1 r o s a − − − p i − − − − − m e a − r : t 0 5 2 v 0 5 5 e 0 0 s g s 0 p S .p e i n 0 2 2 4 2 1 g 4 0 r ( a i : : : 3 : : : : : d h f n : u a t 0 5 2 ; 00 7 40 2 6 5 a r o 70 () o meT a d 1 10 10 t 50 1 3 i u r h Re : l n 2 () y a a 2=)95: d A r Ps t () n s a u t s r A e , p y a e DS.noitazilitrefggemorfdetnuocemi p n l d a y n i S g i r m o o r f m e o a s r i v f l r t e a ) a e i l g r t r e a e p u t t g n g r v p a a n e n o a l t t i e i e e t ) n a l S i d a m 1 r m c i n ( t o p p m t a d n i o e t a l e n l a y r ) w r e l o u v s o c e u r 2 v c c r i f ( i y y a e f e e a f n r e i t l f s P d e ( u i b d n r t l d g a d e a F a m l s a n l e n e l l e d i n ) m u u a i g u u h t r o e 2 r r n q t t p l r ( c e h a t a s t u s e t t r ) p t m a e a p R a i i e 1 n A T= i H L N F G O (D S S * CLARKE ET AL.: EARLY DEVELOPMENT OF PYURA PRAEPUTIALIS 753 presence of the species outside the range of distribution showed in Figure 1. They include two large scale rocky shore Chile-Peru (3°–33°S) expeditions between 1996–1998 (J. Alvarado, pers. comm.), which contemplated collections at Zorritos in Peru (see Van Name, 1945). Colonial ascidians and those presenting zooids vascularly connected, with asexual or internal fertilization and producing free-swimming larvae that are brooded and with swim- ming periods that can be measured in minutes, have been associated with restricted popu- lation dispersion and gene flow (Berril, 1950; Grosberg, 1987; Davis and Butler, 1989; Ayre et al., 1997). While solitary broadcaster ascidians with free-swimming larvae aver- aging approximately 12 h, show wide dispersal abilities (Ayre et al., 1997). According to development laboratory results in Australia and Chile (Anderson et al., 1976; this paper) the solitary P. praeputialis ascidia presents a larvae with swimming periods from 1 to 3 h. In nature the dispersion capabilities of P. praeputialis larvae are unknown; but based on reported larvae swimming periods for other ascidians (i.e., Ayre et al., 1997) we can assume that they may be relatively restricted. This hypothesis, the water circulation reten- tion mechanism inside the Bay of Antofagasta and the taxonomic status of P.praeputialis from Australia and Chile, are presently under study.

ACKNOWLEDGMENTS

J. C. Castilla acknowledges financial support from the Presidential Chair in Sciences, Chile, granted in 1998. Minera Escondida Ltd. (MEL) provided an extra research grant support through the Pontificia Universidad Católica de Chile. We sincerely thank A. Camaño from MEL. We ac- knowledge support from the Universidad de Antofagasta. We thank J. Alvarado, C. Pacheco, M. Varas and R. Guiñez for assistance during field work. We also thanks the help of F. Smith, Estación Costera de Investigaciones Marinas, Las Cruces, Chile and the comments made by two reviewers.

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DATE SUBMITTED: September 30, 1998. DATE ACCEPTED: June 7, 1999.

ADDRESSES: (M.C.) Departamento de Acuicultura, Facultad de Recursos del Mar, Universidad de Antofagasta, Chile. (V.O., J.C.C.) Departamento de Ecología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Casilla 114-D, Santiago, C.P. 6513677, Chile. CORRESPONDING A UTHOR (J.C.C.) E-mail: .