Population Studies on Dodecaceria Kauhanen 2 INTRODUCTION One of the cirratulid polychaetes found in Monterey Bay, Dodecaceria fewkesi, is a significant reef builder. It is known to exist in thick mats of coralline algae and also in massive limestone tube networks (Hartman, 1944) both inter- tidally and subtidally. These two forms differ greatly in appearance when viewed in the field. Variation in tube open- ing, as noted by Berkeley (1932; 1954) is present, along with differing amounts of tube protrusion. Moore (1909), Berkeley (1932; 1952; 1954), and Hartman (1944) described the general morphology of Dodecaceria yet little is known of the ability of these polychaetes to withstand environmental stresses. Investigation of tolerances of Dodecaceria fewkesi from various habitats to salinity, decreased oxygen tension, and tempera¬ ture were carried out at Hopkins Marine Station, Pacific Grove, California, during April and May 1976. The purpose of these experiments was to examine the variation in stress tolerances of spatially separated populations as an indication of reproductive isolation. Results of these experiments and a discussion of certain morphological variations among dis- tinct Dodecaceria populations are presented here. MATERIALS & METHODS Parts of the colonies were removed using a hammer and chisel while on SCUBA. These clumps were kept in the labora¬ tory submerged in running seawater at 12°0. Prior to any of Population Studies on Dodecaceria Kauhanen 3 the stress experiments, each group remained under identical temperature, light, salinity, oxygen tension, and feeding con- ditions for a period of three weeks. Animals were collected from the following locations: Intertidal; east of the Monterey Boat Works at the 0.O to -1.0 foot tidal height on granite rock surrounded by a slow sloping sand beach of protected surf exposure. in calm water on Wharf #2 pilings in Monterey, Wharf; at a depth of 5 to 15 feet. Hopkins Reef in 65 to 80 feet of water on granite Deep Reef; outcroppings protruding upward from the sandy bottom. Surge and strong currents are infrequent. rocky edge of the Carmel Canyon in 20 to 40 feet Monastery; of water. The dive spot was on the north end of San Jose Creek Beach where surge is often great. foot of Macrocystis bed growing from a depth of Pinnacles; 75 to 90 feet on granite outcroppings rising out of the sand. It is located midway between Lone Cypress and Cypress Point on the 17 Mile Drive and a half mile out from shore. Surge conditions are often strong here also. Portions of the colonies were cracked with a chisel and worms were removed for experimentation. By placing the worm tubes in a MgCl, solution isotonic with seawater for about one hour prior to cracking, a higher percentage of uninjured Kauhanen 4 Population Studies on Dodecaceria worms was obtained. Only uninjured subjects were used for experiments. All observations were made with a dissecting microscope. For the osmotic stress experiments, Instant Ocean (Aqua¬ rium Systems, Inc.) was added to 12°0 distilled water. These solutions were glass-fiber filtered and salinity was checked with a refractometer. Ten ml of 14% solution, approximating 50% normal seawater concentration, was added to twelve 25 ml shell vials. This was repeated for the 45%0 solution or roughly 150% seawater. Eight vials were run with a single worm per vial and four vials with three worms per vial. All vials were kept in a 12°0 water bath and the experimental sub¬ jects were checked for mortality. If no movement occurred when the animals were disturbed with a probe, viewed at 20X magnification with a dissecting microscope, they were presumed to be dead. Oxygen was depleted from seawater by bubbling N, through a large jug of cold seawater for 25 minutes. Twenty ml were then carefully siphoned into 25 ml shell vials, a worm added, and a 5 mm layer of mineral oil poured over the surface. This method for the exclusion of oxygen has been shown to be effec¬ tive (Bahr, 1974). The oxygen content of the depleted sea¬ water was found to be 0.3 ml/liter by the Winkler test modi- fied by Carritt and Carpenter (1966). Inspection for mortal- ity was done through the tube without disrupting the surface. Heat stress was accomplished by placing twelve 25 ml Population Studies on Dodecaceria Kauhanen 5 shell vials for each of the five populations in a 29°0 constant temperature oven. Each vial contained one worm and 20 ml of seawater. Examinations for mortality were made periodically using a dissecting microscope. Some worms were gently folded in half to simulate their natural position inside their tubes and carefully placed in 2.5 mm internal diameter glass tubes about 2 cm in length with one end melted shut. These were kept in running seawater. RESULTS Collection sites for Dodecaceria yielded different morphs of the worm. Body length, number of segments, and number of branchial tentacles raanged from 42.1 mm, 160 segments, and 7 pairsof tentacles for the Deep Reef morph to 13.8 mm, 85 seg- ments, and 3 pair of tentacles for the Pinnacle variety. Values for segment number and tentacle number of all worms examined form a continuum between these extremes. Table 1 shows these variations and variations in the colony morphology. Two major colony types occurred, those in 1 to 2 cm thick coralline algal mats found where surge was prevalent (Fig. 1 & 2) and those growing in massive clusters 6 to 30 cm thick. The massive type can be further divided into those building cement-like boulders such as the Intertidal morph (Fig. 3) and those of a more individual, less densely packed tube form, found on the Deep Reef and Wharf (Fig. 4 & 5, respectively). The tube openings of Intertidal, Deep Reef, and Wharf Dodecaceria, all Population Studies on Dodecaceria Kauhanen 6 have tube protrusions, whereas the coralline type is flush with the colony surface. Tube orifices are solitary in the coralline and Wharf areas, but fused to contain two or three worms in the Deep Reef and Intertidal colonies. The general form of the worms was consistent with the descriptions of D. fewkesi by Fewkes (1889, Sabella pacifica) and Berkeley (1952, D. pacifica). The results of osmotic stress experiments are contained in Figures 6 and 7. Intertidal and Wharf individuals survived considerably longer at 14% than did the Deep Reef or Pinnacle polychaetes. The Intertidal, Deep Reef, and Wharf worms with¬ stood 45%0 salinity longer than Monastery or Pinnacle subjects. The experiment was terminated after eight days so as to limit the effect of accumulated waste products, 0, depletion, or other extraneous factors influencing mortality. When worms were exposed to either 25% seawater or distilled water, im- mediate release of a yellow-green pigment was observed and death resulted. In 175% and 200% seawater, color was again released with subsequent death, but it was darker green to black in hue. Oxygen depletion experiments resulted in no mortality within three days to Dodecaceria from any of the collection areas. After eight days, 5 of the 6 initial worms from each location were still alive (see Table 2). All of the Dodeca¬ eria from the Pinnacles were active until the experiment was terminated after eight days. No major differences in tolerance Population Studies on Dodecaceria Kauhanen 7 were noticed among groups. Preliminary temperature stress experiments showed that Dodecaceria kept at 31°0 could survive more than six hours although the worms did release their gametes. Figure 8 shows survival time at 29°0. The Wharf and Intertidal groups not only survived significantly longer, by a 2 X 2 independence test (G-statistic), but were the most active when introduced to this stress. (p£.00) Tube building was observed in D. fewkesi placed in glass tubes. After ten days, the inside of the glass was definitely hazed by a white chalky substance and beginning signs of tube extending were evident. After 25 days, the majority of the worms in glass tubes had added up to 2.5 cm of this substance to their now curving tubes (Fig. 9). This whitish substance reacted in the same manner as the CacO, tube masses found in nature when treated with 5% HCl. It was similar in appearance except it lacked the sand grains found in the natural matrices. DISCUSSION One explanation for the variation in colony morphology is the effect of habitat. It is possible that strong surge could keep tube protrusion reduced, slowing vertical growth of the colonies at Monastery and Pinnacles. In areas of less surge, such as the Wharf and Deep Reef, tubes may not be kept cropped, allowing detritus and sand to collect in between the tubes and become cemented into the matrix. Intertidal tube Population Studies on Dodecaceria Kauhanen 8 projections are not obvious unless the tube openings are pro¬ tected from wave shock. It appears possible that a transplanted larva from any colony might forma tube, influenced by the environment, which resembled the local colonies of that area. This I was unable to verify since the worms were slow to recon¬ struct tubes, and larvae were not available. However, Inter- tidal forms in glass tubes made tube extensions indistinguish- able from those of the Deep Reef. In this case, the habitat seemed to control the architecture of the colony. Glass tube colony transplants among all five areas could yield conclusive results as to the question of environment or genetic control of colony morphology. Individual body variation seems to coincide with size: the more segments, the greater the length, and the more branchial tentacles present. If all groups are considered one popula¬ tion, a continuum in characteristics is evident with the ex- ception of length. Perhaps scouring by surge in areas of coralline algae dwarfs the worms realized length by limiting colony thickness. All the worm variations noted can be easily influenced by environmental conditions and thus cannot be used in evidence for or against the existence of a single or multi¬ ple species. Osmotic stress does not seem to be an environmental problem for D. fewkesi since in the lab they tolerated salinity concentrations beyond extremes to which they are exposed in the field. Intertidal Dodecaceria are exposed to greater Population Studies on Dodecaceria Kauhanen 9 environmental fluctuation so one would expect them to exhibit greater tolerance to stressful conditions than subtidal worms. In the field, exposure to air is limited to a few hours daily by their location at the zero tidal level. This limits the extent of potential osmotic stress resulting from exposure, local fresh water input, and evaporative water loss. The Wharf population of Dodecaceria also inhabits a fluctuating and potentially hostile environment and may consequently ex¬ hibit greater salinity tolerance than other subtidal forms. Even when removed from their tubes, all worms are quite able to withstand a large range of salinity. It is assumed that in the field, this ability is enhanced by the colony morphology Reduced oxygen tension had little effect on Dodecaceria over several days. This could be adaptive if the worms should become buried by shifting sand. It is interesting that no noticeable differences resulted among populations. D. fewkesi can tolerate a wider range of temperatures than it might encounter naturally. They are never exposed long enough that temperatures could reach their lethal level. The worms from the Wharf and Intertidal, areas which underge the greatest temperature fluctuations, withstood exposure to 29°0 the longest and remained the most active of the worms examined under these conditions. It is not clear wether long-term acclimitization or inherent population differences account for the variation in high temperature tolerance. Population Studies on Dodecaceria Kauhanen 10 SUMMARY 1. The Dodecaceria collected at five various habitats varied in appearance yet none of these variations indicate cer¬ tain genetic isolation over environmental effects. 2. Habitat may play the significant role in colonial morph- ology, with surge possibly being a key factor. 3. No individual morphological evidence was found to support a single or multiple species as within colony variation is adequate to form a continuum of values if all popula¬ tions are treated as one. 4. Tube extending is identical between Intertidal and Deep Reef populations in the lab. All populations tested were well adapted to handle any naturally occurring osmotic stress. 6. Dodecaceria can live for more than eight days in reduced O, tension of 0.3ml/liter. 7. Temperature must be extreme to be lethal as even at 31°0 a time interval of over six hours was needed to cause death to the worms. ACKNOWLEDGEMENTS My earnest appreciation goes to Jim Watanabe as my ad¬ visor, photographer, and mentor through this project. Also my thanks go to Larry Harding and Chuck Baxter for their time and concern in editing. e Population Studies on Dodecaceria Kauhanen 11 LITERATURE CITED Bahr, L. M. 1974. Energetic aspects of the intertidal oyster reef community at Sapelo Island, Georgia. Ecology 57(1). 121-131. Berkeley, E. & C. 1932. On a collection of littoral Poly- chaeta from the west coast of Vancouver Island. Contrib to Canadian Biol. & Fisheries, NSZ (f21):311-318. Berkeley, E. & C. 1952. Canada Pac. Fauna. Fish. Res. Bd. of Canada 9b(2):33-34. Berkeley, E. & C. 1954. Notes on the life history of the Polychaeta Dodecaceria fewkesi. Journal Fish. Res. Bd. of Canada 11(3):326-334. Carritt, D. E. & J. H. Carpenter. 1966. Comparison and eval- uation of currently employed modifications of the Winkler Method for determining dissolved 0, in sea water. A NASCO Report, Journal Marine Research 24(3):286-318. Fewkes. 1889. New invertebrata from the coast of California, Bull. Essex Instit. 21:99-146. Hartman, Olga. 1944. Polychaeteus Annelids from California. Allan Hancock Pacific Exped. 10:239-310. Moore, J. P. 1909. Polychaeteus Annelids from Monterey Bay and San Diego. Proc. Acd. Nat. Sci., Philadelphia 61:235. 294. Population Studies on Dodecaceria Kauhanen 12 Figure Captions Figure 1. Pinnacle Dodecaceria, shown as they occur with coralline algae. The scale is in cm. Note the flushness of the colony. Figure 2. Monastery Dodecaceria in coralline algae. Figure 3. Intertidal Dodecaceria from the zero tidal level. Figure 4. Deep Reef Dodecaceria with tube protrusions ap- parent. Note fused tubes by the 11 cm mark. Figure 5. Wharf Dodecaceria with widely varying topography to the colony. Figure 6. Dodecaceria tolerance to osmotic stress at 11%a Note that even the first to die are considerably tolerant. Figure 7. Osmotic stress at 45%.. Many of the subjects were alive at the end of 8 days. It is interesting to note that Deep Reef worms survived 45%much more successfully in relation to the other populations than it did in 14%. Mortality plot at 29°0 for Dodecaceria. Note the Figure 8. Intertidal LD50 at this temperature is greater than 48 hours. Temperatures of this duration and degree are not found in the field. Artificial tubes with CacO, extentions inhabited Figure 9. by Dodecaceria. Note size and bending of tube. Population Studies on Doded Kauhanen 13 TABLE CAPTIONS Table 1. Morphological characteristics of Dodecaceria fewkesi which vary in the field between populations. Length is given in mm one standard error. The sample size is given in parenthesis. Branchial tentacular stubs refer to stump-like tentacles less than imm in length. Variztion on segment number was roughly t 10%. Sex is for the population sampled only, not for all populations in the area of collection. Table 2 Time of death under reduced 0, tension; 0, is O.3m1/1. It appears Dodecaceria can tolerate anerobic condi¬ tions. Population Studies on Dodecaceria FIGURE 1 FIGURE 2 Kauhanen 14 opulation Studies on Dodecaceria Kauhanen 15 FIGURE 3 LNTTU75 Soet O Att vou. Population Studies on Dodecacer: FIGURE 4 FIGURE 5 Kauhanen 16 Population Studies on Dodecaceria FIG URE 6 —+. 26 4 — O 48 72 24 OURS W 6 Kauhanen 17 120 Population Studies on Dodecaceri Kauhanen 18 NO. SURVIVING c. O S 0 N 82 Population Studies on Dodecaceria Kauhanen 19 FIGURE 8 12 10 14 2 8 YG O WV 4 Z 10 20 30 40 50 HOURS Population Studies on Dodecaceria FIGURE 9 Kauhanen 20 Populati TABLE 1 LENGTH (mm) NUMBER BRANCHIAL TENTACLES S NUMBER SEGMENTS SEX TUBE OPENING (worm / hole) TUBE MASS THICKNESS INTERTIDAL 175t.63 (13) 3 p 1.0-3.5mm 2pr stubs 110 volcano-like 2mm (2) reef-like 15-30 WHARF 29.641.18 (14) 4-6p Cmm 4 6 stubs not always in pr. 135 tube projection 8mm (1) 6 DEEP REEF MONASTERY PINNACLES 42112.0 13.61.51 13.84.54 (5) (22) (9) 5-7p 4 p 3-4p 1-7mm 2mm 2mm 5 7 pr stubs 1pr stubs 160 85 90 O tlush with tlush with tube projection Amm coralline coralline algae algae (23) encrusts encrusts 10 Population Studies on Dodecaceria TABLE 2 02 NTERTIDAL WHARE DEEP REEF MONASTERY PINNACLES O Kauhanen 22 DAYS 4 5 6 8 O 0