Tolerance to salinity and thermal stress by larvae and adults of the serpulid annelid Ficopomatus enigmaticus

Jessica Peria and Bruno Pernet*

Department of Biological Sciences, California State University, Long Beach, 1250 Bellflower Blvd, Long Beach, CA 90840, USA

*Corresponding author E-mail: [email protected] Tel: 562-985-5378

Additional key words: invasive; non-indigenous species; Serpulidae

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Abstract. The serpulid annelid Ficopomatus enigmaticus is a widely distributed invader of shallow-water, brackish habitats in subtropical and temperate regions, where it has numerous damaging ecological and economic effects. Its distributional pattern suggests that temperature and salinity play important roles in limiting its distribution, but because other factors often covary with these, drawing strong conclusions from these patterns is difficult. In an effort to more clearly identify the effects of these factors, we examined tolerance to acute thermal (16-

28°C) and salinity (0-35 psu) stress by larvae (5 day exposure, unfed) and adults (14 day exposure, unfed) of F. enigmaticus in laboratory experiments. Larvae showed higher mortality at the highest temperature tested 28°C; adult survival was unaffected by temperature. Neither larvae nor adults survived exposure to pure freshwater (0 psu), but survived well at salinities ranging from 3.5-35 psu. In addition, high salinity did not slow tube growth in adults. These results suggest that salinity stress, in particular, is not directly responsible for the frequent limitation of F. enigmaticus to low-salinity habitats. Experimental work on the distribution of F. enigmaticus is uncommon in the literature, but is likely needed to identify the abiotic or biotic factors that limit the distribution of this frequently invasive species.

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1. INTRODUCTION

The distribution of a species is partly determined by its interaction with a host of

environmental factors, both abiotic and biotic. A fundamental goal of ecology is to identify

which of these are important in limiting species distributions, and how they do so. Identification of limiting factors is particularly important in studies of the rapidly changing distributions of

non-indigenous species, both in terms of understanding existing patterns in site occupancy and in

making predictions about the establishment of new populations (e.g., Compton et al., 2010;

Franklin, 2010; Newcomer et al., 2018).

One such species is the serpulid annelid Ficopomatus enigmaticus (FAUVEL 1923). This

annelid, whose native range has not been conclusively identified, is a widely distributed invader

of shallow-water, brackish habitats in subtropical and temperate regions (reviewed by Dittman et

al., 2009). Because the worms build calcareous tubes, and because they often occur in very large

aggregations, they may dramatically alter native benthic habitat and communities (Schwindt et

al., 2001; Heiman & Micheli, 2010; McQuaid & Griffiths, 2014). Adults are active suspension

feeders, and where abundant can have major effects on phytoplankton and zooplankton

communities (Bruschetti et al., 2008, 2018; Pan & Marcoval, 2014). In addition to these

ecological effects, aggregations of F. enigmaticus on human-produced structures – for example,

boat hulls, tide gates, or cooling water intake pipes for power plants – may cause economic harm

(e.g., Tebble, 1953; Read & Gordon, 2010). Because of the severity of these impacts, identifying

factors that control the establishment of new populations of this species is of great interest.

Two abiotic factors that may be important in this regard are salinity and temperature. As

noted above, F. enigmaticus is typically found in brackish water habitats in subtropical or

temperate regions. Adults of this species are thought to grow most rapidly and reproduce most 4 effectively at salinities between 10-30 psu and temperatures higher than 10°C (reviewed by

Dittmann et al., 2009). However, adults of F. enigmaticus in some populations can apparently tolerate wider ranges of salinity, at least for short periods of time. Living adults have been observed, for example, in full-strength seawater (Britain: Harris, 1970), and even in hypersaline waters up to 67 psu (South Australia: Geddes & Butler, 1984). The apparent tolerance of this species to wide variation in salinity suggests that this factor may not be directly involved in limiting the range of F. enigmaticus. The role of temperature in limiting its distribution is not clear, though Dittmann et al. (2009) note that F. enigmaticus has only rarely become established in the tropics; this suggests the possibility that high temperatures exclude them from these habitats.

Several issues complicate these conclusions, however. First, most of our information on tolerance to stressful salinity or temperature conditions is derived from studies of adults; we have nearly no information about how other life history stages (e.g., larvae) respond to these factors.

Second, most of our evidence is derived from field surveys, not experiments (reviewed by

Dittmann et al., 2009). Because abiotic and biotic factors often covary in the field, it is difficult to distinguish which factors are most important in shaping the distribution of F. enigmaticus from these kinds of data. Finally, our knowledge of the systematics and population genetic structure of Ficopomatus spp. is limited, such that differences in results among studies or regions, or sometimes even within studies or regions, may in fact be attributable to workers studying genetically different entities. Prior to ten Hove and Weerdenburg’s (1978) revision of the genus, for example, F. enigmaticus and the closely related F. uschakovi were frequently confused in the literature, so it is sometimes not clear if studies on “F. enigmaticus” were in fact carried out on individuals of that species (Dittmann et al., 2009). More recently, Styan et al. 5

(2017) discovered that there was surprisingly high genetic differentiation within F. enigmaticus in southern Australia, suggesting that worms we identify using morphological characters as F. enigmaticus today may in fact be members of one of two cryptic species. Yee et al. (2019) found a similar pattern on the coast of California, where members of two genetically distinct clades of

F. enigmaticus (the same two found in southern Australia) cooccurred at all sites surveyed.

Though morphologically indistinguishable, members of these two clades may of course be physiologically distinct. All of these complications mean that prior generalizations about the biology of this species should be viewed with caution.

Here we explore the tolerance of both larvae and adults of F. enigmaticus to salinity, temperature, and their interaction in the context of the ongoing spread of this non-indigenous species on the coast of California. Established populations of F. enigmaticus were first noticed in central California, in San Francisco Bay, in the early 1920s. To the best of our knowledge the species remained localized in San Francisco Bay for ~70 years. Since the mid-1990s, however, populations have been observed to the south of San Francisco Bay, as far as San Diego Bay

(Pernet et al., 2015; Obaza & Williams, 2018; Yee et al., 2019). Across this range, populations are often (but not always) associated with brackish water habitats (Yee et al., 2019). Information on the tolerances of larvae and adults to variation in salinity and temperature will be useful in determining if their apparent absence from other sites is due to physiological limitations, or to alternative factors. Because of their success in invading brackish water habitats, we hypothesized that larvae and adults would show low survival in high salinities, and that adults would grow most rapidly at brackish salinities. We also hypothesized that both larvae and adults would experience low survival at high temperatures.

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2. METHODS

2.1 Collection

Adults of Ficopomatus enigmaticus were collected from the intertidal zone near the mouth of the Los Angeles River (33.7627, -118.2023) from October 2016 through May 2017, in the form of small chunks of tube aggregations broken off from larger aggregations. A 2018 survey of this population showed that it includes members of two genetically very distinct clades of F. enigmaticus: of 31 genotyped individuals, 38% were members of one clade, and 62% were members of the other (Yee et al., 2019). In this study, however, we did not identify studied worms to clade, as this genetic diversity was only discovered after the current work was carried out. Based on the results of Yee et al. (2019), however, it is very likely that the current study included members of both clades.

At each collection, water salinity and temperature at the site were measured using a calibrated handheld refractometer (±1 psu) and a thermometer (±0.5°C). Salinity and temperature at each of these collections are shown in Table 1. Prior to experiments with either larvae or adults, field-collected adults were held for up to 3 d in field-collected seawater at ~20°C with aeration, but no added food.

2.2 Establishment of salinity and temperature treatments

Larvae and adults were exposed to water of seven salinities ranging from 0 psu (deionized water) to 35 psu (full-strength seawater). Treatments of intermediate salinity (3.5, 7, 14, 21, 28 psu) were prepared by addition of deionized water to 0.2 µm filtered 35 psu seawater (FSW).

Salinity of each experimental solution was checked using a calibrated handheld refractometer. 7

Larvae and adults were exposed to three temperature regimes: 16, 22, and 28°C. These were

chosen to roughly bracket the range of temperatures observed at coastal sites in southern

California across the year. All experiments were carried out in three insulated coolers held in a

walk-in environmental chamber whose temperature was set at 16°C. Each cooler was partly filled with freshwater, into which was placed a rack to support vials (for experiments with larvae) or beakers (for experiments with adults). The depth of the freshwater was adjusted so that vials or beakers were partially submerged in it. One cooler was not manipulated further, and its freshwater rapidly equilibrated to 16°C. Each of the other two coolers was equipped with a water circulation pump and an aquarium heater set at 22°C or 28°C, as appropriate. Freshwater temperature was monitored in each cooler using an Onset HOBO datalogger set to record temperature once per hour. Summaries of temperatures recorded during each experiment are shown in Table 1.

We unfortunately did not rotate temperature treatments among coolers; each temperature treatment was associated with the same insulated cooler throughout the series of experiments. Thus cooler is not independent of temperature, and effects we attribute to temperature may actually be caused by the specific cooler in question. This does, however, seem unlikely, since coolers were made of food-safe plastic; coolers were purchased new specifically for these experiments; and neither larvae nor adults were ever in direct contact with any component of the cooler, but instead were housed in covered glass vials or beakers partly immersed in water in the coolers. Further, temperature had no effect on adult survival, suggesting that there was nothing associated with the high temperature cooler (other than high temperature) that would have affected larval survival.

2.3 Larval survival 8

Experiments with larvae were carried out from April-May 2017 (Table 1). The day before an experiment began, we removed living adult worms from their tubes and isolated each of them in

35 psu FSW in a well of a tissue culture plate. This induced spawning in most individuals. Eggs and sperm from a single female and a single male were mixed in 35 psu FSW, yielding a unique family of full-sib embryos. These were held for ~24 h at room temperature (~21°C), at which point they had developed into swimming trochophore larvae. Swimming larvae were then concentrated on to a 20 µm mesh, and resuspended in a smaller volume of 35 psu FSW.

To establish treatments, three 20 ml glass vials were filled with water of one of the treatment salinities at room temperature; this was repeated for the remaining six salinity treatments (total of

21 vials). Approximately 500 µl of the suspension of concentrated larvae was placed into each of the 21 vials, yielding ~50-100 larvae per vial. Vials were loosely capped. Each set of seven vials

(including one of each salinity treatment) was then partly submerged in one of the three temperature-controlled coolers. During each of the next five days, vials were briefly removed from their coolers and the presence or absence of swimming larvae determined using a dissection microscope. Vials that contained swimming larvae were considered “alive”, and those that contained no swimming larvae “dead”. Larvae were not fed during the experiment, and the water in each vial was not changed over the five day experimental period. Scoring survival in this way was easy and rapid, but low in resolution. It did not, for example, allow us to distinguish between vials in which larvae had experienced very low mortality, and vials in which larvae had experienced high but not complete mortality; using our method, both vials were simply scored as

“alive”. It is likely that more subtle patterns in larval survival could be identified using other methods of scoring survival. 9

The experiment was replicated over five different dates (Table 1). Each replicate used a unique full-sib family.

2.4 Adult growth and survival

Experiments with adults were carried out from Oct 2016-Mar 2017 (Table 1). Two days prior to each experiment, we used forceps to break aggregations of adults into groups of 5-10 adult worms, with each adult in a group having a tube substantially longer than the worm it contained.

To mark the tube opening for tube growth measurements, we placed these groups of adults into

35 psu seawater containing 0.1 mg mL-1 calcein, a fluorescent dye that is incorporated into calcium carbonate structures as they are formed (Moran 2000). Adults were incubated in this solution (without added food) at room temperature for the next two days before the experiment began. Calcein was incorporated into any new tube formed in these two days.

To establish treatments, three 250 ml glass beakers were filled with water of one of the treatment salinities at room temperature; this was repeated for the remaining six salinity treatments (total of 21 beakers). Groups of worms were removed from the calcein, briefly rinsed in 35 psu seawater, then placed in beakers (one group per beaker). Beakers were loosely covered with plastic food wrap to prevent condensed water on cooler lids from dripping into them. Each set of seven beakers (including one of each salinity treatment) was then partly submerged in one of the three temperature-controlled coolers. During each of the next 14 d, each beaker was examined daily to determine if any of the adults in the group were moving (usually detected by withdrawal of the feeding crown into the tube). Clumps that included moving adults were considered “alive”, and those that contained no moving adults “dead”. Every other day, water was completely decanted from each beaker, and replaced with clean water of the appropriate 10 salinity and temperature. Adults were not fed during the experiment. As in the larval survival experiment, scoring survival in this way was potentially low in resolution. In practice, however, dead adults were quite easy to identify (as the adult tissue decayed rapidly), and we saw no dead adults in any clump considered “alive”.

After 14 d, tubes were removed from their beakers and tube growth over the experimental period was measured. Tubes illuminated with 490-515 nm light were viewed with a dissection microscope equipped with a 550 nm long-pass emission filter (using a NIGHTSEA Stereo

Microscope Fluorescence Adapter with the “Cyan” light head). Under these conditions the calcein mark in the tube fluoresced green. In each group of tubes, the tube with the largest growth increment was measured (to the nearest 0.1 mm, measured with a plastic ruler). The inner diameter of that tube was also estimated.

The experiment was replicated over five different dates (Table 1).

2.5 Analyses

For both larval and adult experiments, survival at the end of the experiment (days 5 and 14, respectively) was assessed by two-way ANOVA (with salinity and temperature as fixed factors).

For the larval experiments, we included date as a random factor in order to assess variation among experimental dates. For adult experiments, there was no within-treatment variation among experimental dates (each replicate yielded exactly the same results), so we did not include date as a factor . The effect of temperature on the number of days adults survived at 0 psu was assessed with a one-way ANOVA. Analyses were carried out using Minitab 19 and GraphPad

Prism 6.0f.

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3. RESULTS

Salinity and temperature each had significant effects on larval survivorship to day five, but their interaction did not; in addition, date had no significant effect on larval survivorship to day five (Fig. 1; Table 2). All larvae in the 0 psu treatment died within 24 h, but in all other salinity treatments larval survival was high for the length of the experiment. Tukey’s pairwise comparisions bore this out, showing that larval survival in the 0 psu treatment was significantly lower than in all other salinity treatments; the only other significant pairwise difference in larval survival among salinities was between the 3.5 and 14 psu treatments. In terms of temperature,

Tukey’s pairwise comparisons showed that larvae held at the warmest temperature (28°C) had lower survival to day five than did larvae held at the two cooler temperatures, which did not differ in larval survival.

Adult survivorship was affected by salinity, but not temperature or the interaction between salinity and temperature (Fig. 2A; Table 2). All adults held at 0 psu died by day 14, regardless of temperature, but adults in all other salinity/temperature combinations survived to day 14. As is clear from Fig. 2A, the only significant pairwise difference in adult survival is between the 0 psu treatment and all other salinity treatments. For adults held at 0 psu, there was a suggestive relationship between temperature and the mean number of days adults survived, with length of survival decreasing with increasing temperature (Fig. 2B). This relationship was not statistically significant, however (one-way ANOVA: F=3.351, p=0.0698).

Because we had marked the initial tube with calcein, and because adults in all salinity treatments except 0 psu survived to day 14, we were able to compare tube growth over two weeks among salinities (3.5-35 psu) and temperatures (16, 22, and 28°C). Note that adults were not fed during this time period. Under these conditions, tube growth appeared to be positively 12 related to salinity, but unaffected by temperature (Fig. 3). These results were not an artifact of body size variation among salinity treatments, as mean tube diameters were similar among salinity treatments (ranging from 0.9-1.2 mm; one-way ANOVA: F=1.247, p=.2949).

4. DISCUSSION

The goal of this study was to examine how salinity and temperature affect short-term survival of larvae and adults of Ficopomatus engimaticus, in hopes that this information might help us to understand the current distribution of this non-indigenous species on the coast of California, as well as to predict where it might establish new populations in future. Prior work assessing the performance of F. enigmaticus as a function of these abiotic parameters has mostly been correlative (reviewed by Dittmann et al., 2009); experimental analyses, like those carried out here, allow us to better isolate effects of temperature and salinity on this important species.

Our results suggest that across the ranges tested, temperature and salinity hardly constrain short-term survival of either larvae or adults of F. enigmaticus. This is particularly true for temperature, which had no effect at all on survival of adults, and only a minor effect on survival of larvae; larvae showed reduced survival at the highest temperature tested, 28°C (Fig. 1). It is not clear that this is a direct lethal effect of temperature, though. Larvae of F. enigmaticus develop from tiny eggs (~43 µm in diameter: B. Pernet, unpubl. data) which very likely have few energy reserves (Pernet & Jaeckle, 2004). As in other marine invertebrate larvae (e.g., Collis &

Walker, 1995; Zhang et al., 2014), temperature likely has a strong positive effect on larval metabolic rate. Since larvae were not fed over the course of this experiment, it is possible that many larvae held at the highest temperature simply ran out of energy. If this indirect effect of temperature is the cause of our experimental result, then larvae of F. enigmaticus may in fact 13

survive well at 28°C in the field, where there is likely always food present. We suspect this is the

case, and that to identify a high temperature that is directly lethal to F. enigmaticus we would have test temperatures higher than 28°C. As additional suggestive evidence that this is correct,

Yee et al. (2019) found a dense and apparently thriving population of adults of F. enigmaticus in a lagoon in Foster City, California, at a temperature of 27.9°C.

There is one major exception to the generalization that these two abiotic factors had little effect on F. enigmaticus – survival of both life history stages was low at the lowest salinity tested, 0 psu (deionized water). For both larvae and adults, immersion in deionized water was fatal within 24 h (for larvae; Fig. 1) or a few days (for adults; Fig. 2), but survival was high at salinities of 3.5-35 psu. The longer survival time of adults vs. larvae in deionized water might be a consequence of lower surface area to volume ratios in adults compared to larvae, or the fact that much of the surface area of adults was protected by a calcareous tube, slowing exchange with the medium, while the larval epidermis was fully exposed to the surrounding medium. In addition, when adults were placed into deionized water, 35 psu water held within the tubes undoubtedly mixed with the treatment medium, raising its actual salinity to slightly above 0 psu and ameliorating salinity stress slightly. Though Dittman et al. (2009) state that “all species of the genus Ficopomatus are extremely euryhaline”, we know of no records of populations of F. enigmaticus in habitats with very low, stable salinity (e.g., rivers and lakes, which typically have salinities <0.3 psu; Wetzel 2001). Our experiments suggest that F. enigmaticus may be excluded from such freshwater habitats because neither larvae nor adults can tolerate very low salinity for very long.

Thus both larvae and adults of F. enigmaticus appear to tolerate a broad range of water temperatures and salinities, at least in the short-term experiments carried out here. This result 14 contrasts strongly with our initial predictions, but is consistent with the generalization that non- indigenous marine invertebrates are often very tolerant of environmental stressors (Lenz et al.,

2011). This broad tolerance suggests that F. enigmaticus should be able to establish populations at many more coastal sites than they currently occupy (e.g., Pernet et al., 2016; Yee et al., 2019).

In particular, both larvae and adults exhibited excellent survival (Figs. 1, 2) in full-strength seawater, and adults also showed high tube growth in full-strength seawater (Fig. 3). Why, then, are adults of this species often found in brackish water habitats but only rarely at sites with full- strength seawater (Dittmann et al., 2009)?

One possibility is that life history stages or processes that we did not examine are more sensitive to extreme abiotic parameters (such as high temperature or salinity) than the two stages we examined here, larvae and adults. For example, Pineda et al. (2012) showed that fertilization and early larval developmental stages were much more negatively affected by high temperature and low salinity than the processes of settlement and metamorphosis (or the survival of juveniles and adults) in the ascidian Styela plicata introduced to South Africa. Oliva et al. (2018) studied the effects of salinity (5-35 psu) on fertilization and the first 24 h of development in F. enigmaticus from Italy. They found results similar to ours – rates of successful fertilization and early development were low at 5 psu, slightly submaximal at 10 psu, and maximal at 15 psu and higher salinities. Fertilization and early embryonic development do not appear to be reduced in full-strength seawater. It is possible that the processes of settlement or metamorphosis, or the juvenile stages that immediately follow those processes, are more sensitive to high salinity, but our preliminary results suggest that they are not (A. Yee, J. Peria, and B. Pernet, unpubl. data).

Thus direct lethal effects of high salinity seem unlikely to play a role in limiting the distribution of populations of F. enigmaticus. It is possible, however, that indirect physiological 15 effects of high salinity may be important. For example, perhaps juveniles or adults of F. enigmaticus grow more slowly in full-strength seawater than they do in brackish water, reducing their effectiveness as competitors in full-strength seawater habitats. There is no clear support for this hypothesis, however. Correlative data on growth rate as a function of salinity are sparse and ambiguous (reviewed by Dittmann et al., 2009). Our experimental data on tube growth as a function of salinity suggest, if anything, that tube growth rates are positively related to salinity

(Fig. 3).

Another possible explanation for the exclusion of F. enigmaticus from full-strength salinity habitats is even more indirect. The tubes of F. enigmaticus are quite delicate and easily damaged; this is due to the fact that relative to the tubes of some other serpulids, they are both thin and low in bulk density (Bianchi & Morri, 2001). Because they have weak tubes, members of this species may be excluded from habitats with even moderate wave exposure. Protected habitats are more frequently brackish than exposed habitats. Thus, restriction to protected habitats may lead to an apparent (but imperfect) restriction of F. enigmaticus to low salinity habitats. Of course, causality might be reversed here; that is, it is possible that the tubes of F. enigmaticus are weak because they are produced in low salinity habitats. These alternatives should be easily distinguished by experiments.

Finally, as suggested above already, explanations of the distribution of F. enigmaticus may need to look beyond the organismal level to the community level. Members of this species may simply be poor competitors, or (perhaps because of their weak tubes) be unusually susceptible to predation. If so, then perhaps they are restricted to low salinity habitats simply because these habitats are free of fully marine species that otherwise might outcompete or prey on them. Low 16 salinity habitats, in this view, allow F. enigmaticus to escape from biotic contests that they are likely to lose.

We suggest that two practices will be particularly helpful in making efficient progress in testing the hypotheses above. First, it is important that future studies of the biology of “F. enigmaticus” clearly define the taxon or taxa being studied, as the discovery of members of two very distinct clades living in sympatry in two regions – Australia and California – opens the possibility that variation within and among studies may be due to variation in the taxa studied.

Unfortunately, the current study was carried out before we knew that this genetic diversity existed, in either Australia or California. There was relatively little within-treatment variation in our experiments, however, suggesting that members of the two clades do not differ much in their responses to temperature or salinity stress. Second, though prior work on understanding the distribution of F. enigmaticus has almost exclusively been correlative, most of the hypotheses mentioned above should also be tested by simple laboratory experimentation, where the effects of specific factors can be isolated much more cleanly. The results of laboratory experimentation on carefully identified individuals will aid greatly in the interpretation of results derived from field surveys.

Acknowledgments. We thank Alison Yee for help with collections, Valerie Langland for help designing and carrying out preliminary versions of these experiments, and Dr. Bengt Allen for assistance with statistical analyses. This work was supported by National Science Foundation grants DEB-1257355 and OCE-1756531 to B.P.

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Table 1. Water salinity and temperature at the collection site, and temperature conditions during experiments. Conditions at the collection site were measured 1-4 days before each experiment start date. On a few occasions salinity or temperature were not measured; those dates are indicated by “--“. Temperatures in experimental coolers were logged hourly with Onset HOBO dataloggers.

Conditions at collection site Mean (min-max) temperature in coolers (°C) Salinity (psu) Temp. (°C) 16°C (nominal) 22°C (nominal) 28°C (nominal) Larval experiment start date 13 Apr 2017 26 15 15.7 (15.5-17.9) 21.0 (20.6-21.9) 27.4 (26.5-28.0) 24 Apr 2017 -- -- 15.7 (15.6-15.8) 21.3 (21.1-21.7) 27.5 (27.2-27.8) 30 Apr 2017 -- -- 15.7 (15.6-15.8) 21.3 (21.2-21.7) 27.4 (27.2-27.7) 8 May 2017 28 19 15.7 (15.6-15.9) 21.3 (21.1-21.8) 27.7 (27.1-28.0) 13 May 2017 28 17 15.7 (15.6-16.0) 21.6 (21.2-22.0) 27.9 (27.3-28.3)

Adult experiment start date 21 Oct 2016 20 22 16.1 (15.7-17.5) 23.2 (21.9-24.3) 26.9 (25.5-28.6) 6 Nov 2016 29 20 15.7 (15.5-17.1) 21.6 (20.0-22.8) 25.1 (20.4-26.8) 29 Jan 2017 24 -- 15.9 (15.7-20.9) 21.9 (20.9-22.6) 26.2 (25.2-26.7) 13 Feb 2017 27 17 15.9 (15.5-17.2) 22.3 (21.2-22.8) 27.5 (26.3-28.1) 3 Mar 2017 26 -- 15.9 (15.5-17.1) 22.0 (21.3-22.7) 27.6 (25.3-28.0)

Table 2. ANOVA table showing effects of salinity, temperature, their interaction, and date (as a random factor) on survival of larvae of Ficopomatus enigmaticus. The same information is shown for survival of adults of F. enigmaticus, except that since there was no variation among dates in results, date was not included as a factor.

Larval survival after five days SS DF MS F P value Date 0.610 4 0.1524 1.97 0.107 Salinity 309.733 6 51.6222 667.12 < 0.001 Temperature 1.086 2 0.5429 7.02 0.002 Salinity*Temperature 1.181 12 0.0984 1.27 0.252 Residual 6.190 80 0.0774

Adult survival after 14 days SS DF MS F P value Salinity 12.86 6 2.143 2.533e+016 < 0.0001 Temperature 0 2 0 0.0 > 0.9999 Salinity*Temperature 0 12 0 0.0 > 0.9999 Residual 7.11E-15 84 8.46E-17

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Figure legends Fig. 1. Survival of larvae of Ficopomatus enigmaticus after five days at experimental salinities and temperatures. Error bars represent standard errors.

Fig. 2. Survival of adults of Ficopomatus enigmaticus at experimental salinities and temperatures. A. Survival of adults after 14 days. B. The mean (and standard error) length of survival of adults in the 0 psu treatment, as a function of temperature.

Fig. 3. Mean tube growth increment (mm) of adults of Ficopomatus enigmaticus at experimental salinities and temperatures. Error bars represent standard errors.

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