BULLETIN OF MARINE SCIENCE, 43(1): 41~O, 1988

LARVAL SETTLEMENT AND METAMORPHOSIS OF SABELLARIID POL YCHAETES, WITH SPECIAL REFERENCE TO PHRAGMATOPOMA LA PIDOSA, A REEF-BUILDING SPECIES, AND FLORIDENSIS, A NON-GREGARIOUS SPECIES

Joseph R. Pawlik

ABSTRACT The naturally-occurring inducers of larval settlement and metamorphosis have recently been isolated and identified for the northeast Pacific reef-building sabellariid , Phragmatopoma californica, and the larval responses of this species compared, in reciprocal laboratory settlement assays, to those of its European counterpart, Sabel/aria alveolata, The present study includes the larval behavior of two additional sabellariids from the western Atlantic, P. lapidasa, a reef-building species, and S. jlaridensis, a non-gregarious species. Larval responses of P. lapidosa were very similar to those of P. californica. In reciprocal laboratory assays of both species, greater metamorphosis occurred on conspecific than on heterospecific tube sand, but both metamorphosed more frequently on heterospecific tube sand than on control sand. Organic solvent extraction of the sand/cement matrix of tubes of P. lapidosa removed its capacity to induce conspecific metamorphosis. The capacity was retained in the lipid-soluble extract and was recovered as a single fraction by high-performance liquid chromatography. The inducers were identified by gas chromatography as a mixture of free fatty acids (FFAs) ranging from 14 to 22 carbons in length. The mixture contained the same component FFAs as the inductive fraction from the natural tube sand of P. californica, but the relative proportions were different. Of the FFAs found in the naturally-occurring mixture, larval metamorphosis was greatest in response to 16: I, 18:2, 20:4 and 20:5, with a significant response to 16: I at as low as I /l-g!gsand (surface area;;! 36 cm2). Metamorphosis- inductive FFAs were isolated from natural tube sand of P. lapidosa at approximately 4-5 lLg!gsand, although extraction was believed to be incomplete. In assays of 37 FF A standards and 9 FFA derivatives, metamorphosis of P. lapidosa was dependent on the length and conformation of the acyl chain length and on the presence of a carboxylic acid functional group, as had previously been demonstrated for P. cali/ornica. Reciprocal cross-fertilization of gametes of P. lapidosa and P. californica resulted in larvae that developed and metamor- phosed normally. Results of this and previous investigations suggest that P. lapidasa and P. cali/arnica are geographic races of the same species, and the trinomials Phragmatapoma lapidosa lapidosa and P. I. cali/ornica are proposed for each subspecies. Larval responses of Sabel/ariajloridensis resembled those of S. alveo/ata in three respects: (I) upon reaching maturity, a large percentage of the larvae metamorphosed spontaneously in culture vessels, (2) in laboratory settlement assays, a large proportion of the larvae meta- morphosed on control sand, and (3) metamorphosis was not enhanced upon exposure to the FF As that induce metamorphosis of Phragmatopoma larvae. Unlike the larvae of S. a/veo/ata, which settle gregariously, larvae of the non-gregarious S. jloridensis did not metamorphose to any greater extent on con specific tube sand than on control sand. Among sabellariid polychactes, enhanced settlement on conspecific tube sand may be the only requirement for reef formation.

The is a family of marine that constructs tubes of cemented grains of sand and shell fragments. There are at least six extant genera encompassing more than 50 species, of which approximately two-thirds build solitary tubes at intertidal to abyssal depths worldwide. About 20 species, pre- dominantly of the genera Phragmatopoma, Sabella ria, and including the mono-

41 42 BULLETIN OF MARINE SCIENCE, YOLo 43, NO. I, 1988

specific genus Gunnerea, construct colonies and reefs of aggregated tubes in the intertidal and subtidal of temperate and tropical coasts in many parts of the world (Achari, 1972; Kirtley, 1974; Pawlik and Faulkner, 1988). Phragmalopoma lapidosa, a gregarious species, occurs along the east coast of Florida from Cape Canaveral south, throughout the Caribbean, to the southern coast of Brazil (Kirtley, 1974). Reefs of the sand tubes of P. lapidosa extend for hundreds of kilometers of coastline (Kirtley and Tanner, 1968), and are thought to be of considerable importance in the sorting, deposition and stabilization of beach sand (Kirtley, 1967; Multer and Milliman, 1967; Gram, 1968; Kirtley and Tanner, 1968; Mehta, 1973; Gore, 1986). Like coral reefs, colonies of P.lapidosa support large and diverse assemblages of associated invertebrates and fishes (No- nato and Peres, 1961; Narchi and Rodrigues, 1965; Fanta, 1968; Gore et aI., 1978). Sabella ria j/oridensis is sympatric over much of the range of P. lapidosa, occurring along the coast of the southeastern United States and throughout the Caribbean (Kirtley, 1974). Unlike P. lapidosa, S. j/oridensis is a non-gregarious species, usually building solitary or paired tubes on shells and stones resting on sandy or muddy bottoms. The present account is the fourth in a series describing studies of the chemical basis for the initiation oflarval settlement among sabellariid polychaetes (Pawlik, 1986; 1988; Pawlik and Faulkner, 1986). Larvae of gregarious sabellariids settle and metamorphose with a high degree of specificity on the cemented sand tubes of the adult worms (reviewed in Pawlik, 1986; 1988). Organic solvent extraction of the tube sand of P. californica, a reef-building species from the west coast of North America, greatly diminished the capacity of the tube sand to induce set- tlement of con specific larvae in laboratory assays. The inductive capacity was retained, however, in the lipid-soluble extract, and from it, a single, highly active fraction was isolated. Analysis of this fraction revealed a mixture of free fatty acids (FFAs) ranging from 14 to 22 carbons in length, some of which were effective at inducing llirval settlement and metamorphosis at naturally-occurring concen- trations. Subsequent assays of 37 FFA standards and 9 FFA derivatives dem- onstrated that the metamorphic response was highly specific, dependent upon the length and conformation of the acyl chain and on the presence ofa free carboxylic acid functional group. Surprisingly, Sabella ria alveolala, a reef-building species from European waters, exhibited very different settlement behavior than did P. californica (Pawlik, 1988). In reciprocal assays, metamorphosis of both species occurred to a greater extent on con specific tube sand than on heterospecific tube sand. Extraction of the tube sand of S. alveolala diminished its capacity to induce metamorphosis of conspe- cific larvae, but the capacity was not transferred to the organic extracts, and a lipophilic inducer could not be isolated or identified. The FFAs that induced metamorphosis of the larvae of P. calif arnica were either ineffective at enhancing metamorphosis of S. alveolala or inhibited the response. Moreover, FFAs were present in the natural tube sand of S. alveolala at less than 1;;0 the concentration at which they were found in natural tube sand of P. californica. Larvae of S. alveolala metamorphosed with much less discrimination in their choice of sub- strata than did those of P. californica, and a high percentage settled spontaneously in culture vessels. While the larval settlement behavior of P. californica appeared well suited for colony and reef formation, this was much less apparent for S. alveolala. The investigation reported herein is an extension of the previous comparisons of sabellariid larval settlement behavior to include those of P. lapidasa and S. j/oridensis. Although the larval development of both species was described in detail by Eckelbarger (1976; 1977; 1978), no experimental assessments were made PAWLIK: SETILEMENT OF SABELLARIID POLYCHAETES 43

of substrate preferences at the time of settlement. The settlement responses of the two species were hypothesized to show greater similarity to those of their re- spective congeners. It was additionally hoped that differences in the larval behavior of the non-gregarious S. jloridensis. as compared with that of S. alveolala, might better explain reef formation by the latter.

MATERIALS AND METHODS

Blocks ofIiving colonies of P. lapidosa were collected intertidally from Seminole Shores, S1. Lucie Inlet, Martin County, Florida, April 1985, and from the north jetty of Fort Pierce Inlet, St. Lucie County, Florida and Sailfish Point, Martin County, Florida, September 1985. Colonies were collected subtidally from Pointe Dunkerque, south coast of Martinique, West Indies, August 1985 and June 1986. Worm-free tube sand was obtained from crushed blocks of red reef from the Fort Pierce Inlet and Sailfish Point collections of September 1985 and from the Martinique collection of August 1985. Collection, transport and maintenance of reef blocks and production of worm-free tube sand were the same for P. /apidosa as for P. californica and S. alveolala (Pawlik, 1986; 1988). Specimens of S. jloridensis were obtained, in individual or paired tubes on bivalve shells or sand dollar tests, from the Gulf Specimen Company, Panacea, Florida, May 1985 and June 1986. Cross-fertilization experiments were performed as a simple assessment of relatedness between species. Female worms, bearing lavender or pink abdominal setigers, and male worms, with white to yellow abdominal setigers, were removed from their tubes (2-4 of each), segregated and rinsed twice in flowing seawater. Females were allowed to spawn their gametes into dishes containing 200 ml of 1-lLm-filtered seawater (FSW) at 20°C. Males placed on a glass surface shed dry (undiluted) sperm. For each species in a given experiment, 2,000-3,000 unfertilized eggs were added to 50 ml of FSW in 8-cm diameter covered dishes. One dish was set aside without addition of sperm to serve as a control. Five drops of dry sperm were added to 10 ml of FSW and five drops of this suspension were added to a dish containing conspecific eggs. The dish was lightly stirred and addition of sperm repeated twice after 10-min intervals. The seawater overlaying the eggs in the dish was then decanted and replaced with 50-ml FSW. At the same time as self-fertilizations were performed, cross-fertilizations were undertaken in the same fashion. Dishes were placed on a rocking platform set at 28 cycles/min at 20°C. After 24 h, unfertilized eggs of P. californica and P. lapidosa remained stuck to the bottom of the glass dish, while the hatched, swimming trochophores were in suspension. The trochophores were decanted into a separate dish and fixed with several drops of 10% formalin in FSW. Each dish was then placed on a transparent grid of I-cm squares, and the number of either eggs or trochophores counted in each of 10 squares chosen haphazardly. Percentage fertilization was calculated from the mean values of 10 counts. Unfertilized eggs of S. jloridensis and S. alveo/ala did not adhere to glass. After 24 h, the entire contents of each dish were transferred to a 100-ml graduated cylinder and the contents brought up to 100 ml. After 10 min 60 cm beneath two 40 watt fluorescent lamps, swimming trochophores were distributed near the top of the water-filled cylinder and unfertilized eggs sank to the bottom. Trochophores were pipetted into one dish and the unfertilized eggs transferred to another. Percentage fertilization was determined as previously described. Methods for larval culture, for determination oflarval growth, maturation and metamorphosis and for production of the types of sand used in assays were those detailed in Pawlik (1988). Inasmuch as S. jloridensis is non-gregarious, assays of the tube sand of this species employed natural tube sand from whole, occupied tubes. Tube sand was rinsed in deionized water, frozen, lyophilized and sieved (retained on mesh sizes 25 and 40) before use in larval assays. Extraction, isolation and identification of lipid soluble components of the natural tube sand of P. /apidosa were undertaken as described in Pawlik (1986; 1988). The diethyl ether: methanol extract was separated by flash chromatography and high-performance liquid chromatography (HPLC) to yield six components: one fraction comprising the material adsorbed onto the flash column and subsequently eluted, four HPLC fractions, and one fraction composed of waste material not collected during HPLC fractionation. Larval assays were performed as in Pawlik (1988). Larvae used in assays were cultured at 20°C and assayed at the same temperature. All extracts, fractions and standards were water-insoluble and were adsorbed onto sand grains by spreading organic solvent solutions containing known concentrations of each substance onto clean sand and removing the volatile solvent under a vacuum. Unless otherwise indicated, all assays were run with 5 replicates (30 ± 2 larvae per replicate) and the mean percentage of larval response for each assay determined. Differences in mean larval metamorphosis were tested with one-way analysis of variance (ANOYA) performed on arcsin transformed data. Tukey's honestly significant difference method (T-Method) was employed a posteriori to determine which treatments resulted in different mean larval responses at the 0.05 level of significance (Sokal and Rohlf, 1981). For situations in which the conditions for parametric testing were not met (e.g., zero variance for one or more treatments), the non-parametric Kruskal- Wallis test (KWT) was performed in lieu of a one- way ANOY A (Conover, 1980). 44 BULLETIN OF MARINE SCIENCE, VOL. 43, NO. I, 1988 A. D. Pc PI Pc Sf Sa 0 0 0 0 0 Pc 98.4 72.1 Pc 13.7 0 -* PI 57.4 4.2 Sf 0 7.3 0 Sa -* 19.1 89.7 B. ~ Pc PI 1.0 1.2 a Pc 93.8 14.9 E. PI 72.4 91.4 Pc Sf Sa 0.8 0 0 c. ~ Pc 99.0 0.6 -* Sf 0.4 92.3 0 Pc PI Sa 96.0 99.0 0 0 -* a Pc 48.3 48.0 Pi 54.6 98.7 Figure 1. Percentage fertilization in reciprocal crosses of the gametes of Phragmatopoma californica and P. lapidosa (A, 8, C), and Sabel/ariafloridensis, P. calif arnica and S. alveolata (D, E). ·Cross not performed in this study; P. californica and S. alveo/ata were previously found not to be interfertile (Pawlik, 1988).

RESULTS Cross-fertilization Experiments. -Interfertility was employed as a simple indi- cator of the relatedness of Phragmatopoma lapidosa to P. californica and of Sabel/aria jloridensis to P. californica and to S. alveolata. Phragmatopoma ca/i- fornica and S. alveolata were previously found not to be interfertile (Pawlik, 1988). Phragmatopoma californica and P. lapidosa were fully interfertile (Fig. lA, B, C) and hybrids resulting from both reciprocal crosses swam normally and were cul- tured through metamorphosis. Sabel/aria jloridensis and P. californica were not interfertile (Fig. ID, E). Trochophores that swam normally were produced from fertilization of eggs of S. jloridensis with sperm of S. alveolata, but the reciprocal cross did not result in fertilized eggs. Low percentage fertilization of conspecific crosses occurred in some instances (Fig. ID); this was not considered unusual and may reflect immaturity of the spawned gametes. Growth, Maturation and Metamorphosis of Larvae in Culture. -Data on larval growth, the onset of metamorphic competence and larval metamorphosis in cul- ture for both P. lapidosa and S. jloridensis cultured at 15°C and 20°C are presented in Figure 2 and Table I. Effect of Organic Solvent Extraction of Tube Sand of P. lapidosa on Conspecific Larval Metamorphosis. - The percentage metamorphosis of larvae of P. lapidosa in response to microbially-filmed control sand, conspecific tube sand extracted in organic solvents and unextracted conspecific tube sand is presented in Figure 3. There were significant differences in mean larval responses among treatments (ANOVA, F2,12 = 81.57, P < 0.001). A posteriori analysis (T-method, a = 0.05) PAWLIK: SETTLEMENT OF SABELLARIID POLYCHAETES 45

BOO A. P./apldosa, 1S'C 800 B. P. lapldoss, 20'C 100 100

90 90 700 700 80 80 600 600 70 70 500 60 500 60

400 50 400 50 40 40 300 300 30 30 200 200 20 20 100 100 10 10 o . o . o 10 20 30 40 o 10 20 30 40

C. S. floridensls, 1S'C 800 D. S. florldensls, 20'C BOO 100 100 90 700 90 700 80 80 600 600 70 70 500 500 60 60 400 50 400 50 40 40 300 300 30 30 200 200 20 20 100 100 10 10 o o o . o 10 20 30 40 o 10 20 30 40 DAYS ARER HATCHING DAYS AFTER HATCHING

Figure 2. Growth, maturation, and cumulative percentage metamorphosis of larvae of Phragrnato- pOrna lapidosa and Sabellaria j/oridensis in culture. Vertical bars represent one standard deviation about the mean larval length (N = 10). The arrow indicates the day on which 50% or more of the larvae metamorphosed in assays of con specific tube sand. (A) Larvae of P. lapidosa cultured at IS·C. (B) P. lapidosa cultured at 20·C. (C) Larvae of S. j/oridensis cultured at 15·C. (0) S. j/oridensis cultured at 20·C. indicated that the mean percentage of metamorphosed larvae was significantly greater in response to unextracted tube sand than to control or to extracted tube sand. There was no difference in larval response to control sand versus extracted tube sand. Reciprocal Assays oj the Larvae oj P. lapidosa and P. californica. - There were significant differences in mean larval metamorphosis among treatments in recip- rocal assays of both P. lapidosa and P. californica (Fig. 4; F2 12 = 59.05, P < 0.001 for P. lapidosa; F2,I2 = 137.19, P < 0.001 for P. californica). For both species, there was greater metamorphosis on con specific tube sand than on either micro- bially-filmed control sand or heterospecific tube sand, and there was greater meta- morphosis on heterospecific tube sand than on control sand. Responses oj the Larvae oj P. lapidosa to Lipophilic Isolates oj Natural Tube Sand. - The percentage larval response of P. lapidosa to organic solvent extracts of natural conspecific tube sand (Sailfish Point collection) is shown in Figure 5. A mean of 93.1% of the larvae metamorphosed in response to the 1:1 diethyl 46 BULLETIN OF MARINE SCIENCE. VOL. 43. NO. I, 1988

Table I. Data on the growth, maturation and metamorphosis of larvae of Phragmatopoma lapidosa and Sabel/aria jloridensis in culture at 15·C and 20·e. *Larvae of S. jloridensis did not reach meta- morphic competence at 15·e within the 40-day time-course

P.lapidosa S. floridensis lS"C 20'C IS'C 20'C

Maximum mean length 651 I'm 710 I'm 560 I'm 643 I'm Time to max. mean length 40 days 28 days 40 days 26 days Time to metamorphic competence 27 days 16 days * 17 days Percent metamorphosis in culture 4.3% 32.3% 0%* 49.3% Percent accounted for at end of time-course 20% 22% 23% 41%

ether: methanol extract. A small percentage of larvae responded abnormally to the diethyl ether: methanol extract; these larvae rotated their larval tentacles and opercular cirri anteriorly, shed their provisional setae or held them forward, and moved sluggishly. There were no significant differences between larval responses to control sand and any of the other extracts (KWT, H4 = 7.4, P > 0.05). HPLC traces of the diethyl ether: methanol extract of the natural tube sand of P. lapidosa exhibited the same peaks as the trace presented in Pawlik (1986: fig. Ib) for fractionation of the extracts of the tube sand of P. californica. Mean larval responses to the fractionated components of the extract are shown in Figure 6. Assay of fraction 2 resulted in a mean of 85.3% metamorphosis and 12.0% abnormal response, the latter described previously. There were no significant differences in mean larval response to control sand and sand treated with any of the other fractions. The larvae of P. lapidosa and P. californica were exposed to I mg extract/g sand of fraction 2 from extracts of the natural tube sand of each species in recip- rocal assays (Fig. 7). The proportion oflarvae undergoing normal versus abnormal metamorphosis was very similar for larvae of P. lapidosa in response to both the con specific fraction and the heterospecific fraction. The same was not true for larvae of P. californica; there was greater mean abnormal response on exposure to fraction 2 from extracts of tubes of P. lapidosa than in response to the conspecific fraction. Composition oj Fraction 2 Jrom Extracts oj the Tube Sand oj P. lapidosa.- The results of gas chromatographic analysis of fraction 2 derived from extracts of five samples of natural tube sand of P. lapidosa are presented in Table 2. The yield of fraction 2 ranged from 10.7 to 18.3 J.Lg/gtube sand with a mean of 13.8 J.Lg/gtube sand (N = 3). Responses oJthe Larvae oJP. lapidosa to FFA Standards and FFA Derivatives.- Results of assays of the larvae of P. lapidosa in response to FFA standards of variable chain length and un saturation at six concentrations are presented in Figure 8. At I mg FFNg sand (Fig. 8A), larval response to sand treated with fully saturated FFAs was not different than response to control sand. The cis isomer of 16:1 (palmitoleic acid) induced 72.3% metamorphosis, while the trans isomer (palmitelaidic acid) induced no response at all. Assay of 18:3g, 18:4,20:4,20:5, 22:4 and 22:6 at I mg/g sand resulted in 100% larval mortality. Abnormal re- sponses were otherwise as previously described. For assays ofFFAs at 300 J.Lg/gsand (Fig. 8B), there were significant differences in mean larval metamorphosis among the four treatments in which there were no abnormal responses (KWT, HI\ = 55.28, P < 0.001). There was greater PAWLIK: SETTLEMENT OF SABELLARIID POLYCHAETES 47

100

90

80

Iol 70 1/1 Z 0 0.. 60 1/1 Iol Ill:: 50 I- Z Iol 0 40 Ill:: Iol 0.. 30

20

10 -

0 PI/C PI/PI-X PI/PI

Figure 3. Mean percentage metamorphosis (±SE, N = 5) of larvae of P. lapidasa in response to microbially-filmed control sand (PI/C), extracted con specific tube sand (PI/PI-X) and conspecific tube sand (PI/PI). Larvae cultured and assayed at 20°C.

100

90

80

Iol 70 1/1 Z 0 IL 60 1/1 Iol Ill:: 50 I- Z Iol 0 40 Ill:: Iol IL 30

20

10

0 PI/C PI/PI PI/Pc Pc/C Pc/Pc Pc/PI

Figure 4. Mean percentage metamorphosis (±SE, N = 5) of larvae of P. lapidasa in response to microbially-filmed control sand (PI/C), conspecific tube sand (PI/PI) and tube sand of P. califarnica (PI/Pc) and oflarvae of P. califarnica in response to microbially-filmed control sand (Pc/C), conspecific tube sand (Pc/Pc) and tube sand of P. lapidasa (Pc/PI). 48 BULLETIN OF MARINE SCIENCE, VOL. 43, NO.1, 1988

100 lZZI "'ETA"'ORPHOSED 90 ~ ABNOR"'AL

80

70 ••••III Z 0 60 II. ....III II: 50 I- ....Z 0 -40 ....II: II. 30

20

10

0 Control Hexane Ether Ether/"'eOH ••••OH

Figure 5, Mean percentage metamorphosis (±SE, N = 5) ofIarvae of P. lapidosa in response to clean sand treated with solvent alone (Control) and clean sand treated with 1 mglg of the hexane extract (Hexane), diethyl ether extract (Ether), 1:I diethyl ether: methanol extract (Ether/MeOH), methanol extract (MeOH) and distilled water extract (H20) of natural conspecific tube sand.

100 IZZl METAMORPHOSED 90 ~ ABNORMAL

80

70 ••••III Z 0 II. 60 ....III II: 50 I- Z ••••0 40 0:: w II. 30

20

10

0 C F"2 F"3 F"-4 Waste Adsorb

Figure 6. Mean percentage response (±SE, N = 5) oflarvae of P. lapidosa to clean sand treated with solvent alone (C) and clean sand treated with I mglg of HPLC-fractions 1-4, waste from HPLC fractionation (Waste) and material adsorbed onto flash column (Adsorb). PAWLIK: SETTLEMENT OF SABELLARIlD POLYCHAETES 49

100 ~ETA~ORPHOSED 90 ABNORMAL

80

10.1 70 1Il Z 0 a.. 60 1Il 10.1 0:: 50 •.... Z 10.1 0 .40 0:: 10.1 a.. 30

20

10

0 PVC PVPlf2 PVPcf2 Pc/C Pc/Pcf2 Pc/Plf2 N= 7 7 7 7 4 7 Figure 7. Mean percentage metamorphosis (±SE) oflarvae of P. lapidasa in response to clean sand treated with solvent alone (PI/C), clean sand treated with I mg/g of conspecific HPLC-fraction 2 (Pl/ PIF2) and clean sand treated with I mg/g of fraction 2 from the tube sand of P. calif arnica (PI/PcF2), and of larvae of P. calif arnica in response to clean sand treated with solvent alone (Pc/C), clean sand treated with I mg/g of conspecific fraction 2 (Pc/PcF2) and clean sand treated with I mg/g of fraction 2 from the tube sand of P. lapidosa (Pc/PIF2). metamorphosis in response to 14:1, 15:1, 16:1, 18:2, 18:3, 18:3g, 18:4 and 20:4 than to control sand. The same was true for assays ofFFAs at 100 ~g/g sand (Fig.

8C), which resulted in no abnormal larval responses (ANOVA, F1S•64 = 10.97, P < 0.001). Mean larval metamorphosis in response to 15:1, 16:1, 18:3, 18:3 g, 20:4 and 20:5 was greater than in response to control sand. At 50 ~glg sand (Fig.

Table 2. Percentage composition of free fatty acid (FFA) fractions isolated from extracts of the natural tube sand of P. lapidosa as determined by gas chromatography. For each FFA, the number of carbon atoms precedes the colon, the number of double bonds follows (br = branch- and straight-chain FFAs present)

Fort Pierce Sailfish I Sailfish 2 Martinique I Martinique 2 FF As detected 14:0 4.6 4.6 6.1 2.5 5.2 4.6 15:0 br 5.0 2.1 3.9 3.6 2.1 3.3 16:2,16:1 6.8 3.7 7.4 11.7 12.0 8.3 16:0 26.7 16.5 28.1 34.6 36.1 28.4 17:0 br 10.0 5.5 3.6 5.6 1.7 5.3 18:3, 18:2, 18: I 7.8 7.7 9.6 8.3 10.5 8.8 18:0 14.5 9.8 12.7 10.4 10.8 11.6 19:0 2.1 0 1.8 1.9 0 0.8 20:5 5.1 5.0 6.3 6.8 6.3 5.9 20:4, 20:3, 20:2 8.1 6.4 8.6 6.8 5.9 7.2 22:4, 22:3, 22:2 8.7 38.0 11.7 7.1 9.3 15.0 50 BULLETIN OF MARINE SCIENCE, VOL. 43, NO.1, 1988

A. 1 mg/g PERCENT B. 300 p,g/g PERCENT ABNORMAL "'ETAMORPHOSED PERCENT RESPONSE co N g ~ ~ ~ ~ g t ~~(;0 F"F'A o co .. oo~~~g~c:~~g "o '"o , "o o "o o o 8 T C 3 , 3:1 C - 3 14:0 14:1 14: ,-pa. 3• l~:O 15:1 3• le:o 15:1 3 16:1. 1&:1 c 16:1 3• 17:0 3 17:1 17:1 "!:l 3 18:0 - ~ 3 1S:1eS o 18:2 " 3 18:1c9 .. 3 lB:lcl1 :J: 18:2 18:3 o • 18,3 In •3 l1h3g '"o 18:3 g 3 18:4 3 19:0 3 '9~1 18:4 3 20:0 3 20:1 20:2 20:2 • 20:3 3• 20:4 20:3 3 20:' 3 22:0 20: ..•. 3 22:1t 3 22:\c 20:~ 22:2 • 22:3 3• 22:. 22:3 3 22:8 3 23:0 22:4 3 23:1 3 24:0 3 2.:1 22:6

C. 100 p,g/g D. 50 p,g/g E. 10 p,g/g F. 1p,g/g •. co •. ;; ;; ;; 0 ;; 0 0" 0'" 0 0" 0 0" 0 0" 0'" 0 0" 0 0" 0'" ~ " 0 "0 ~

C C C

'4:1 C

15:1 15:1 15:1

16:1

16:1 16:1 17:1

18:2 16:1 18:2 18:2 18:3

18:3 18:3 18:4

20:2 18:3 20:3

20:4 20:. 20:4

20:'

22:3 20,5 20:'

22:" 20:5 22,8 22:8 22:6

Figure 8. Mean percentage response (±SE, N = 5 unless otherwise indicated) of]arvae of P. lapidosa to clean sand treated with solvent alone (C) and clean sand treated with free fatty acids (FFAs). The num ber of carbon atoms in the FF A molecu]e precedes the colon, the number of double bonds follows. A. 1 mg FFAIg sand. B. 300 ~g/g. C. 100 ~g/g. D. 50 ~g/g. E. ]0 ~g/g. F. 1 ~g/g.

8D), there was greater mean larval metamorphosis in response to 16: I, 18:2, 18:3, 20:4 and 20:5 than to control sand (F8 36 = 6.62, P < 0.001). For assays ofFFAs at 10 J.Lglg sand (Fig. 8E) there were significant differences in larval metamorphosis PAWLIK: SETTLEMENT OF SABELLARIID POLYCHAETES 51

100 METAMORPHOSED

90 ABNORMAL

80 w 70 VI z 0 Q. 60 VI w a: 50 t- Z w u 40 a: w Q. 30

20

10

0 « w •.... Cl w ...J U « Cl ...J u z U •.... ~ ~ 0 « ~ ~ o « w 16:1 18:2 ------N= 5 5 3 3 3 3 5 3 3 3 3 3 Figure 9. Mean percentage response (±SE) oflarvae of P. lapidosa to clean sand treated with solvent alone (C) and clean sand treated with the FFA, monoglyceride (MG), methyl ester (ME), alcohol (OL) and alcohol acetate ester (AC) of 16: I and these five plus the n-alkene of 18:2 (ENE) at I mg/g sand.

(KWT, Hs = 22.09, P < 0.01), with greater metamorphosis in response to 16:1, 18:2, 18:3,20:4,20:5 and 22:6 than in response to control sand. At 1 IJ-g/gsand (Fig. 8F), 16: 1 induced greater larval metamorphosis than did control sand (H3 = 7.12, P < 0.05). The results of assays of 16:1 and 18:2 and derivatives of these FFAs at 1 mg/g sand are presented in Figure 9. Percentage metamorphosis was much greater in response to the FF As. There were significant differences in mean larval meta- morphosis among the 10 treatments in which there were no abnormal responses (H9 = 18.02, P < 0.05). Larval metamorphosis was greater in response to the monoglyceride of 18:2 and to the alcohols of both 16: 1and 18:2 (cis-9-hexadecen- 1-01and cis-9, l2-octadecadiene-l-ol) than in response to control sand. Effect of Organic Solvent Extraction of Tube Sand of Sabel/aria jloridensis on Conspecific Larval Metamorphosis. - The percentage metamorphosis of larvae of S. jloridensis in response to microbially-filmed control sand, extracted natural con specific tube sand and unextracted natural conspecific tube sand is shown in Figure 10. There were no significant differences in mean larval responses among

the three treatments (ANOYA, F2,12 = 2.63, P > 0.05). ReCiprocal Assays of the Larvae of s. jloridensis with P. californica and S. al- veolata. - There were no significant differences in assays of the larvae of S. jlo- ridensis on microbially-filmed control sand, natural con specific tube sand and 52 BULLETIN OF MARINE SCIENCE, VOL. 43, NO.1, 1988

100

90

80

L.I 70 III Z 0 Q. 60 III L.I 0: 50 I- Z L.I 0 40 0: L.I Q. 30

20

10

0 Sf/e Sf/Sf-X Sf/Sf

Figure 10. Mean percentage metamorphosis (± SE, N = 5) of larvae of S. jIoridensis in response to microbially-filmed control sand (Sf/C), extracted con specific tube sand (Sf/Sf-X) and conspecific tube sand (Sf/Sf).

heterospecific tube sand (Fig. 11; ANOV A, F4,20 = 0.205, P > 0.05). For larvae of both P. californica and S. alveolata, there was greater metamorphosis on con- specific tube sand and on natural tube sand of S. jloridensis than on control sand (F2,8= 64.31, P < 0.00 1 for P. californica; F2,8= 8.10, P < 0.05 for S. alveolata). There was no difference in mean larval metamorphosis on con specific tube sand versus tube sand of S. jloridensis for larvae of either P. californica or S. alveolata.

Responses of the Larvae of S. jloridensis to FFA Standards. - The responses of the larvae of S. jloridensis to FFAs of variable chain length and unsaturation at three concentrations are presented in Figure 12. At 1 mg FFNg sand (Fig. l2A), there were no differences in mean larval metamorphosis in response to control sand versus any of the FFAs that did not elicit an abnormal response (Fs 18 = 0.0 1, P > 0.05). The same was true for assays of FF As at both 300 and 100 '/lglg sand(Fig. 12B, F3,8= 0.64, P > 0.05; Fig. 12C, F7,16 = 0.77, P > 0.05, respectively). Assays of 18:2, 18:3, 20:4 and 20:5 at 1 mglg sand resulted in larval mortality, and at 300 /lglg sand, abnormal responses to these FFAs were manifested as sluggish movements and some loss of provisional setae, but without any other components of normal metamorphosis.

DISCUSSION Phragmatopoma lapidosa and P. californica both Metamorphose in Response to Naturally-occurring FFAs. -Overall, the larval responses of Phragmatopoma lap- PAWLIK: SETrLEMENT OF SABELLARIID POLYCHAETES 53

100

90

80

Iol 70 III Z 0 II. 60 III Iol 0: 50 t- Z Iol U 40 0: Iol II. 30

20

10

0 Sf/C Sf/Sf Sf/Pc Sf/Sa Pc/C Pc/Pc Pc/Sf Sa/C Sa/Sa Sa/Sf N= 5 5 5 5 .3 .3 5 .3 .3 5 Figure II. Mean percentage metamorphosis (±SE) oflarvae of S. j/oridensis in response to micro- bially-filmed control sand (Sf/C), conspecific tube sand (Sf/Sf), tube sand of P. cali/ornica (Sf/Pc) and tube sand of S. a/yeo/ala (Sf/Sa), of larvae of P. ca/i/ornica in response to microbially-filmed control sand (Pc/C), conspecific tube sand (Pc/Pc) and tube sand of S. j/oridensis (Pc/Sf), and of larvae of S. a/yeo/ala in response to microbially-filmed control sand (Sa/C), conspecific tube sand (Sa/Sa) and tube sand of S. j/oridensis (Sa/Sf).

idosa at the time of settlement and metamorphosis were very similar to those of P. califarnica (Pawlik, 1986; 1988). Larvae of P. lapidosa, like those of P. ca/i- jornica, were highly discriminatory in choosing a substrate upon which to meta- morphose: although 40-50% of the larvae of P. lapidosa metamorphosed in re- sponse to con specific tube sand (Figs. 3,4), for all assays performed in the present study, the mean of means was 1.0 ± 0.8% metamorphosis onto clean or micro- bially-filmed control sand (N = 11). This compares with 60-90% metamorphosis of larvae of P. californica in response to con specific tube sand and 1.5 ± 1.7% metamorphosis in response to control sand (N = II; Pawlik, 1986; 1988). Organic solvent extraction of the tube sand of P. lapidosa, like that of P. californica, completely eliminated its capacity to induce con specific larval meta- morphosis. The inductive capacity was retained in the lipophilic extract, and a mixture ofFFAs was isolated as the component that induced metamorphosis. As with P. californica (Pawlik and Faulkner, 1986), larvae of P. lapidosa responded with a high degree of specificity to FFA standards: larval response was dependent on the presence of at least one cis double bond in the molecule, conservation of molecular shape with increasing acyl chain length by the addition of cis double bonds, and the presence ofa free carboxyl group. Larvae of both species responded abnormally to high concentrations of some FFAs (particularly polyenoic acids), but these responses included many of the behavioral components of normal meta- 54 BULLETIN OF MARINE SCIENCE, VOL. 43, NO. I, 1988

tOO

90

80

70 z'"Ul 0 ... 60 Ul '"II: •... 50 z U II:'" 40 '"... 30 .•...•.be bll 20 ~ Q 10 Q ••• 0 U

tOO

90 IZZI METAMORPHOSED ~ ABNORMAL 80

70 zUl'" 0 ... 60 Ul '"II: •... 50 z U '"II: 40 '"... 30 .•...•.be bll 20 ::l.. Q 10 Q M 0 a:l

tOO

90

80

'" 70 Ulz ~ 60 Ul '"II: •... 50 z y ~ 1/ y 1/ '" 1/ 1/ ~ 40 ~ 1/ >' '"... V 1/ 30 V V 1/ 1/ 1/ 1/ be 20 / V 1/ .•...•. / ,/ bll V 1/ E 10 V 1/ ~ ~ ~ o f

o o II) u 9- o N o ... ., < ~ .;; •.. oii oii cD o o o o '" N N N PAWLIK: SETTLEMENT OF SABELLARIID POLYCHAETES 55

morphosis, including loss of provisional setae, anterior rotation oflarval tentacles and opercular cirri, and reduction of the episphere. The differences in the larval responses of P. lapidosa and P. californica were more intriguing than the similarities. By the end of 40 days of development, over 30% of the larvae of P. lapidasa had metamorphosed in culture at 20°C as com- pared with only 5% of the larvae of P. califarnica, suggesting that P. lapidosa is less able to postpone metamorphosis than is P. californica (Pawlik, 1988). Yet, both species were equally discriminating in their choice of settlement substrata in laboratory assays (Fig. 4). There were further discrepancies between the two species in the lowest effective concentration of FF As required to induce larval metamorphosis, in the mean concentration of FFAs isolated from natural tube sand, and in the relative com- position of the natural FFA mixtures. In assays of FFA standards, larvae of P. lapidosa metamorphosed on sand treated with 16:1to a significantly greater extent than on control sand at as little as 1 J.Lglgsand, whereas the lowest effective FFA concentration for inducing metamorphosis of P. califarnica (also on 16: 1) was 10 J.Lglgsand (Pawlik, 1986). This increased sensitivity to FF As appears to be balanced by both lower overall concentrations of FF As in the tube sand of P. lapidosa and lower proportions of the FFAs that induce metamorphosis, relative to non-stim- ulatory FFAs, in the natural FFA mixture. The mean concentration ofFFAs from the tube sand of P. califarnica was 35 J.Lglg sand (Pawlik, 1986); the mean con- centration isolated from tube sand of P. lapidosa was 13.8 J.Lglg sand. The com- position of fraction 2 isolated from natural tube sand of P. lapidosa had lower proportions of the most abundant inducers identified from fraction 2 of P. callfornica, 16:1, 18:2 and 20:5 (Pawlik, 1986), although the FFA mixture from tube sand of P. lapidosa had a higher proportion of stimulatory 22-carbon poly- enoic acids (Table 2). Ifinductive FFAs are secreted by the adult worms onto tube sand, the observed variability in the composition of the FFA mixture may reflect differences in the lipid composition of the phytoplankton upon which the adult polychaetes feed (cf. see Sargent and Whittle, 1981). Despite these differences, stimulatory FFAs were isolated from the natural tube sand of P. lapidosa at sufficient concentrations to induce metamorphosis: approximately 1/3 of the mixture was composed of stimulatory FFAs, or approximately 4-5 J.Lglg sand. As with the extraction and isolation ofFFAs from the tube sand of P. califarnica, the FFA concentration values determined in the present study are probably low estimates of the actual concentrations ofFFAs perceived by larvae in nature. The proteinaceous cement secreted by the adult worms to form sand tubes may bind FFAs in a manner analogous to the binding of FFAs and other lipid-soluble compounds within the kerogen matrix of sedimentary rocks (Hoering and Abelson, 1965; Huxley et al., 1984). The likelihood of underestimating the actual concen- tration of stimulatory FFAs present on the surface of the sand tubes is further discussed in Pawlik (1986). If the larvae of P. lapidasa and P. califarnica both metamorphose in response

Figure 12. Mean percentage response (± SE, N = 3) of larvae of S.j/oridensis to clean sand treated with solvent alone (C) and clean sand treated with free fatty acids (FFAs). The number of carbon atoms in the FFA molecule precedes the colon, the number of double bonds follows. A. I mg FFA/g sand. B. 300 /JogFFNg sand. C. 100 /JogFFNg sand. 56 BULLETIN OF MARINE SCIENCE, VOL. 43, NO. I, 1988 to the same suite ofFFAs, and these FFAs can be isolated from natural tube sand at effective concentrations, why was there greater metamorphosis in response to conspecific tube sand than to heterospecific tube sand for larvae of both species? There are several plausible explanations of the observed specificity that are neither mutually exclusive nor exhaustive. The demonstrated differences in the FFA compositions from extracts of the sand/cement matrix of the two species may account for the specificity. Subtle changes in the proportions of volatile phero- mones have been shown to elicit very different behavioral responses in many insect species (Prestwich, 1985). It is also possible that chemical cues other than FFAs playa role in the larval settlement of these two species, and variability in these signals are responsible for greater metamorphosis on conspecific tube sand. In the present study, larval growth of P. lapidosa in culture was considerably faster than that reported by Mauro (1975; 3-4 weeks at 22-25°C), and within the range reported by Eckelbarger (1976; 14-30 days at 2 I-23°C). Eckelbarger (1976) found that less than 50% of the eggs of P. lapidosa fertilized at 15°e developed into trochophores that were alive 48 h later, although it remains unclear whether low temperature adversely affected fertilization or subsequent development. As reported herein, development of P. lapidosa at 15°e took approximately 70% longer than at 200e (Table 1; Fig. 2A, B), but the percentage of larvae accounted for at the end of the time-course was nearly the same at both temperatures (Table 1). Mauro (1975: 392) attributed the wide gap in the development rates he ob- served for P. lapidosa (3-4 weeks at 22-25°C) and those of S. alveolata reported by Wilson (1968; 6 weeks or more at ~ 15°C) to culture temperature differences or to "species differences unrelated to the temperature of the medium." However, standardized procedures employed in the culture of both species (Pawlik, 1988 and present study) have revealed similar maturation rates between species at both 15°e and 20°e. Lower culture temperature affected larval growth and development of S. jloridensis to a greater degree than any of the other sabellariids studied. Larvae of S. jloridensis grew very slowly at 15°e and did not attain metamorphic competence within the 40 day time-course, but grew more than twice as fast at 200e and 50% or more were competent to metamorphose by day 17. Although the distributions of S. jloridensis and P. lapidosa overlap to a considerable extent, the effect of temperature on the development of the latter was much less re- markable (Fig. 2A, B). A comparison of the rates oflarval development in culture for the 4 species discussed herein is compiled in Table 3 from data present here and in previous studies. The wide disparities in development rates reported in these studies are further discussed in Pawlik (1988). Settlement of s. jloridensis is Nonspecific; Larval Behavior Otherwise Similar to that of S. alveolata. - The results of this study indicate that S. jloridensis and S. alveolata are closely related species, although certainly not as closely related as P, lapidosa and P. cali/ornica. Eggs of S. alveolata were not fertilized by sperm of S. jloridensis, but the reciprocal cross resulted in normally-swimming trocho- ph ores (Fig. 1D, E). Both S. jloridensis and S. alveolata exhibited high levels of non-specific metamorphosis: for all assays performed in the present study, the mean of means was 54.4 ± 8.5% metamorphosis of S. jloridensis in response to clean or microbially-filmed control sand (N = 5), as compared to 43.7 ± 8.3% metamorphosis of S. alveolata (N = 12; Pawlik, 1988). Levels of spontaneous metamorphosis of S. jloridensis in culture at 200e were intermediate between those of S. alveolata and P. lapidosa under identical conditions (Fig. 2B, D and Pawlik, 1988). Metamorphosis was not enhanced in the presence of FFAs for PAWLIK: SETTLEMENT OF SABELLARIID POL YCHAETES 57

Table 3. Summary of reported values for the time oflarval development from fertilization to meta- morphosis of sabellariids cultured in the laboratory

Culture temperature Species ("C) Development time Reference

Phragmatopoma lapidosa 22-25 (?) 3-4 wks Mauro, 1975 21-23 14-30 days Eckelbarger, 1976 20 16 days present study 15 27 days present study Phragmatopoma californica 21-23 18-25 days Eckelbarger, 1977 17-18 34-39 days Eckelbarger, 1977 20 16 days Pawlik, 1988 15 23 days Pawlik, 1988 Sabellaria alveolata ? 12 wks Cazaux, 1964 15 6-32 wks Wilson, 1968 20 14 days Pawlik, 1988 15 25 days Pawlik, 1988 Sabellaria j/oridensis 21-23 18-27 days Eckelbarger, 1977 20 17 days present study

either species of Sabel/aria (Fig. 12; Pawlik, 1988). The abnormal responses of larvae of S.jloridensis upon exposure to polyenoic FF As, like those of S. alveolata, lacked any similarity to normal metamorphosis, in contrast to the abnormal responses of larvae of both species of Phragmatopoma. In agreement with the present investigation, Eckelbarger (1977) reported that more than half of the larvae of S.jloridensis in some of his cultures metamorphosed in the absence of tube-building materials. He also observed that some recently metamorphosed larvae, in the presence of sand grains, would crawl around culture dishes for several days before starting tube construction. Once sand tubes were built, some of these juveniles continued to crawl on their tentacles, bearing sand tubes detached from the substrate. In the present study, post-larval behavior of this kind was occasionally observed in assay dishes, but it occurred no more frequently than in assays of S. alveolata. It is not clear why high percentages of larvae of both P. californica and S. alveolata metamorphosed in response to the natural tube sand of S. jloridensis (Fig. 11). Adult S.jloridensis may secrete substances along with their tube cement (e.g., FFAs) that induce metamorphosis of these other species, but to which con- specific larvae are insensitive. Perhaps the more advanced, reef-forming sabel- lariids have appropriated compounds present in the tubes oftheir non-gregarious predecessors for use as chemical cues of gregarious larval settlement. Unfortu- nately, because S.jloridensis is non-gregarious, insufficient natural tube sand could be obtained to attempt the isolation of whatever compounds might be present that induce metamorphosis of P. californica and S. alveolata. Inasmuch as most of their responses are so similar, why do larvae of S. alveolata settle gregariously and form reefs, while larvae of S.jloridensis do not? Comparison of the larval behavior of two reef-building species demonstrated that P. californica is well adapted for gregarious settlement, but the same could not be said for S. alveolata (Pawlik, 1988). Larvae of P. californica responded with nearly absolute specificity to con specific tube sand and, in its absence, could delay metamorphosis for an indefinite period of time. Larvae of S. alveolata preferred to metamorphose on conspecific tube sand, but a large portion would metamorphose on control sand or on the walls of culture vessels. Unlike the larvae of either ofthese colony- 58 BULLETIN OF MARINE SCIENCE, VOL. 43, NO. I, 1988

Table 4. Comparison of the larval settlement behavior of four sabellariid species studied under identical culture and assay conditions (present study and Pawlik, 1988). Pc = Phragmatopoma cali- jomica. PI = P. lapidosa. Sa = Sabellaria alveolata, Sf = S. floridensis

Condition Pc PI Sa Sf Gregarious settlement, forms reefs yes yes yes no Indefinite delay of metamorphosis in absence of suitable substrate yes no no no Low percentage metamorphosis on control sand yes yes no no Enhanced metamorphosis on conspecific tube sand over control sand yes yes yes no

formers, larvae of S. jloridensis showed no preference for conspecific tube sand (Figs. 10, 11), which may explain the lack of gregarious settlement in this species. Wilson (1977) similarly found that the larvae of another non-gregarious sabel- lariid, Lygdamis muratus, displayed no greater tendency to metamorphose on conspecific tube sand than on control sand. Reef formation by sabellariid polychaetes is apt to be influenced by several intrinsic and extrinsic factors (see discussion in Pawlik, 1988; Pawlik and Faulk- ner, 1988). Substrate specificity and delay of metamorphosis would be likely intrinsic factors to affect gregarious settlement. Among the four species of sabel- lariids studied, substrate specificity appears to be the most important factor con- tributing to gregarious settlement (Table 4). Although three of the four species studied formed aggregations, only the larvae of P. californica indefinitely delayed metamorphosis in culture. Larvae of both species of Phragmatopoma exhibited an "all or nothing" response in settlement assays; there were high levels of meta- morphosis on con specific tube sand or sand treated with inductive FFAs, but almost no metamorphosis on control sand. A significant proportion of the larvae of both species of Sabel/aria metamorphosed on control sand, but while meta- morphosis was enhanced on conspecific tube sand for larvae of the reef-building species, S. alveolata, it was not for the non-gregarious species, S. jloridensis. Therefore, preferential settlement and metamorphosis on con specific tube sand may be all that is required for reef formation in the Sabellariidae. Synonomy within the Genus Phragmatopoma. - Evidence from this and previous investigations indicate that P. lapidosa and P. californica are geographic races of the same species: (1) Adult morphology of the two forms is identical, except for slight differences in the ornamentation of the opercular setae (Hartman, 1944). In his key to the species of Phragmatopoma, Kirtley (1974: 52) differentiated P. lapidosa from P. californica solely on the basis of the intensity of body pigmen- tation. (2) Larval morphology of the two forms is identical. Eckelbarger (1977: 248) remarked that " ... the larvae of P. californica are virtually impossible to distinguish from those of P. lapidosa" and concluded that the two forms were very closely related based on his comparison of gamete morphology and larval development. (3) In reciprocal assays, larvae of both forms metamorphosed on conspecific and heterospecific tube sand; only negligible metamorphosis occurred on control sand. Moreover, both forms metamorphosed in response to the same suite of FFAs isolated from the natural sand tubes of their respective adults. (4) Reefs formed by the two taxa are indistinguishable and occur in similar habitats and over the same vertical range (inter- to subtidal). (5) The two forms are interfertile. Reciprocal cross-fertilizations resulted in healthy hybrid larvae that developed and metamorphosed normally. In light of these similarities, the tri- nomial P. lapidosa lapidosa Kinberg, 1867 is proposed for the western Atlantic, and P. lapidosa californica (Fewkes, 1889) for the northeast Pacific subspecies. PAWLIK: SEITLEMENT OF SABELLARIID POLYCHAETES 59

It seems likely that the two subspecies of P. lapidosa descended from a contin- uous population of a single species that existed prior to the formation of the isthmus of Panama, 3.1-3.6 million years ago (Keigwin, 1978). Separation of the two populations since that time has probably resulted in the slight differences in adult morphology, larval development and metamorphic specificity observed for the two forms. The remaining two species in the genus Phragmatopoma, P. at- tenuata and P. moerchi (and synonyms of the latter, including P. virgini and P. peruensis; Hartman, 1944; Kirtley, 1974), both from the Pacific coast of South America, probably share the same ancestry and fall within the same species com- plex. Both P. attenuata and P. moerchi settle gregariously to form reefs and differ from each other and from the two subspecies of P. lapidosa only in minor aspects of the morphology of their opercular setae (Hartman, 1944; Kirtley, 1974). Ph rag- matopoma may be a monospecific genus, but further consolidation awaits com- parison of the interfertility, larval development and settlement behavior of P. lapidosa with P. attenuata and P. moerchi.

ACKNOWLEDGMENTS

I thank K. J. Eckelbarger, D. J. Faulkner, W. Fenical, M. Burch and M. Kernan for providing collections of living sabellariids. The Graduate Department of Scripps Institution of Oceanography contributed travel funds. N. D. Holland, D. J. Faulkner and W. A. Newman read and commented on the manuscript. This study was supported in part by a pre-doctoral fellowship from the National Science Foundation to the author and by NSF grant CHE86-03091 to D. J. Faulkner.

LITERATURE CITED

Achari, G. P. K. 1972. Polychaetes of the family Sabellariidae with special reference to their intertidal habitat. Proc. Indian Nat. Sci. Acad. Pt. BioI. Sci. 38: 442-455. Cazaux, C. 1964. Developpement larvaire de Sabella ria alveolala (Linne). Bull. Inst. Oceanogr. Monaco 62(1296): 1-15. Conover, W. J. 1980. Practical non parametric statistics, 2nd ed. John Wiley and Sons, New York. 493 pp. Eckelbarger, K. 1. 1976. Larval development and population aspects of the reef-building polychaete Phragmalopoma lapidosa from the east coast of Florida. Bull. Mar. Sci. 26: 117-132. --. 1977. Larval development of Sabellariajloridensis from Aorida and Phragmatopoma cal- ifornica from southern California (Polychaeta: Sabellariidae), with a key to the sabellariid larvae of Florida and a review of development in the family. Bull. Mar. Sci. 27: 241-255. --. 1978. Metamorphosis and settlement in the Sabellariidae. Pages 145-164 in F.-S. Chia and M. Rice, eds. Settlement and metamorphosis of marine invertebrate larvae. Elsevier, New York. Fanta, E. S. 1968. Sabre a biologia de Phragmalopoma lapidosa (Sabellariidae, Polychaeta). Ciencia e Cultura 20: 327-328. Gore, R. H. 1986. Anastasia and the reefs of sand. Sea Frontiers 32: 204-213. --, L. E. Scotto and L. J. Becker. 1978. Community composition, stability, and trophic parti- tioning in decapod crustaceans inhabiting some subtropical sabellariid worm reefs. Bull. Mar. Sci. 28: 221-248. Gram, R. 1968. A Florida Sabellariidae reef and its effect on sediment distribution. 1. Sedim. Petrol. 38: 863-868. Hartman, O. 1944. Polychaetous , family Sabellariidae. Allan Hancock Pacif. Exped. 10: 323-389. Hoering, T. C. and P. H. Abelson. 1965. Fatty acids from the oxidation of kerogen. Yb. Carnegie Inst. Wash. 64: 218-223. Huxley, R., D. L. Holland, D. J. Crisp and R. S. L. Smith. 1984. Influence of oil shale on intertidal organisms: effect of oil shale surface roughness on settlement of the barnacle Balanus balanoides (L.). J. Exp. Mar. BioI. Ecol. 82: 231-237. Keigwin, L. D. 1978. Pliocene closing of the isthmus of Panama, based on biostratigraphic evidence from nearby Pacific Ocean and Caribbean Sea cores. Geology 6: 630-634. Kirtley, D. W. 1967. Worm reefs as related to beach stabilization. Shore and Beach 35: 31-34. --. 1974. Geological significance of the polychaetous family Sabellariidae. Ph.D. Dis- sertation, Florida State University. 270 pp. 60 BULLETINOFMARINESCIENCE,VOL.43, NO.1, 1988

--- and W. F. Tanner. 1968. Sabellariid worms: builders ofa major reef type. J. Sedim. Petrol. 38: 73-78. Mauro, N. A. 1975. The premetamorphic developmental rate of Phragmatopoma lapidosa Kinberg, 1867, compared with that in temperate sabellariids (Polychaeta: Sabellariidae). Bull. Mar. Sci. 25: 387-392. Mehta, A. J, 1973. Coastal engineering study ofsabellariid reefs: report ofa hydraulic model study to the Harbor Branch Foundation Laboratory, Ft. Pierce, Horida. Coastal and Oceanographic Engineering Laboratory, University of Horida, Gainesville. 67 pp. Multer, H. G. and J. D. Milliman. 1967. Geologic aspects ofsabellarian reefs, southeast~m Horida. Bull. Mar. Sci. 17: 257-267. Narchi, W. and S. A. Rodrigues. 1965. Observacoes ecol6gicas sabre Phragmatopoma lapidosa Kinberg. Ciencia e Cultura 17: 228. Nonato, E. and J.-M. Peres. 1961. Observations sur quelques peuplements intertidaux de substrat dur dans la region d'Ubatuba (Etat de Sao Paulo). Cah. BioI. Mar. 2: 263-270. Pawlik, J. R. 1986. Chemical induction of larval settlement and metamorphosis in the reef-building tube worm Phragmatopoma californica (polychaeta: Sabellariidae). Mar. BioI. 91: 59-68. ---. 1988. Larval settlement and metamorphosis of two gregarious sabellariid polychaetes: Sab- el/aria alveolata compared with Phragmatopoma californica. J. Mar. BioI. Ass. U,K. 68: 101- 124. --- and D. J. Faulkner. 1986. Specific free fatty acids induce larval settlement and metamorphosis of the reef-building tube worm Phragmatopoma californica (Fewkes). J. Exp. Mar. BioI. Ecol. 102: 301-310. --- and ---. 1988. The gregarious settlement of sabellariid polychaetes: new perspectives on chemical cues. Pages 475-487 in M.-F. Thompson and R. Nagabhushanam, eds. Marine bio- deterioration. Oxford and IBH Publishing Co., New Delhi. Prestwich, G. D. 1985. Communication in insects: II. Molecular communication in insects. Quart. Rev. BioI. 60: 437-456. Sargent J. R. and K. J. Whittle. 1981. Lipids and hydrocarbons in the marine food web. Pages 491- 533 in A. R. Longhurst, ed. Analysis of marine ecosystems. Academic Press, New York. Sokal, R. R. and F. J. Rohlf. 1981. Biometry, 2nd ed. W. H. Freeman and Co., San Francisco. 859 pp. Wilson, D. P. 1968. Some aspects of the development of eggs and larvae of Sabella ria alveolata (L.). J. Mar. BioI. Ass. U.K. 48: 367-386. ---. 1977. The distribution, development and settlement of the sabellarian polychaete Lygdamis muratus (Allen) near Plymouth. J. Mar. BioI. Ass. U.K. 57: 761-792.

DATEACCEPTED: September 29, 1987.

ADDRESS: Scripps Institution ojOceanography, A-OOB. University oj California, San Diego, La Jolla, California 92093.