Effects of crowding and wave exposure on penis morphology of the acorn barnacle, Semibalanus balanoides
- First Online:
- Cite this article as:
- Hoch, J.M. Mar Biol (2010) 157: 2783. doi:10.1007/s00227-010-1536-z
- 291 Views
Wave action and low population density can strongly reduce the ability of sessile acorn barnacles to find mates and copulate. For Semibalanus balanoides, penis morphology varies with wave exposure and with characteristics of the mating neighborhood. Field experiments were conducted at five intertidal sites on Long Island, New York, USA from July to December 2005 to determine how wave exposure and aggregation structure influence the length, diameter, mass, and number of annulated folds of the penis. Sparsely crowded barnacles had more annulations in the penis and are inferred to have greater ability to stretch. At higher wave exposure, the diameter of the penis was greater, but the mass was not. This study identifies density of crowding as the most important cue that barnacles respond to when perceiving their mating group and details how penis morphology varies in response to wave exposure.
Acorn barnacles are obligate-outcrossing, simultaneous hermaphrodites that reproduce by copulating (or more accurately, pseudo-copulating) with their neighbors. Owing to their sessile lifestyle, they accomplish this with a long, flexible penis (Darwin 1854), which also may act as a sensory organ in the search for mates (Munn et al. 1974). The penis has an annulated exoskeleton with accordion-like folds, allowing it to stretch to many times its relaxed length (Klepal et al. 1972). In Long Island, New York, the North Atlantic acorn barnacle, Semibalanus balanoides (Fig. S1 in “Electronic supplementary material”), develops reproductive tissues, including the penis, between August and November, mates in mid-November, and broods its larvae until their release in mid-winter (Crisp and Patel 1958; Stubbings 1975; Barnes 1992; Hoch 2008).
Individuals in densely crowded groups of S. balanoides (defined as physical contact between tests of neighbors) have penises with significantly fewer annulations. They are inferred to have a reduced capacity to stretch than those from sparsely crowded groups (aggregations where tests of neighbors do not touch) (Hoch 2008; Yuen and Hoch 2010). S. balanoides from wave-exposed sites develop penises with a greater basal diameter than those from sites protected from waves (Hoch 2008, 2009). This pattern is also present in the Pacific acorn barnacle, Balanus glandula, which has a shorter, thicker, more massive penis in wave-exposed conditions (Neufeld and Palmer 2008). Experimental work has shown that this is the result of phenotypic plasticity in response to wave action and that the observed morphologies match the environments in which they are found (Neufeld and Palmer 2008; Hoch 2009). These patterns of variation in penis morphology are similar to the patterns of variation observed in the feeding cirri of B. glandula and several other species of barnacles: barnacles in sites exposed to greater wave action grow shorter, but stouter, feeding cirri than those from areas protected from waves (Arsenault et al. 2001; Marchinko 2003; Marchinko and Palmer 2003; Li and Denny 2004; Chan and Hung 2005).
Questions remain on the factors influencing morphological variation of the penis in S. balanoides. Hoch (2008) found a distinct difference in penis annulation number between densely crowded and sparsely crowded groups of barnacles. However, that study only took crowding into account; the numbers of available mates and distances to nearby mates were not accounted for, although such factors may be important in determining penis morphology. Physical contact with neighbors, cues related to the number of neighbors (such as the concentration of dissolved chemicals), or some other signal may allow barnacles to sense their mating neighborhood. Understanding the barnacle’s ability to perceive its mating neighborhood is critical to the tests of sex allocation theory. Classic sex allocation theory for simultaneous hermaphrodites includes the assumption that reproductive individuals sense the magnitude of competition for mates (Charnov 1982; Schärer 2009). Despite the fact that acorn barnacles were one of the first simultaneous hermaphroditic species for which optimal sex allocation in response to mating group size was predicted (Charnov 1980), a few studies have investigated their ability to perceive the mating neighborhood.
I used field experiments to test several hypotheses concerning the functional morphology of the penis of the North Atlantic acorn barnacle, S. balanoides. Variation in penis form, in response to wave exposure, has been documented in S. balanoides (Hoch 2008, 2009), but each of these studies only compared two sites. I set up factorial experiments in five sites, creating manipulated mating groups with varying numbers of mates available and differing levels of crowding to test the hypotheses that the factors were significantly related to penis length and annulation number. At the same time, I measured the distances between potential mates to test the hypothesis that the barnacle penis increases in length or annulation number with nearest neighbor distance. The sites were distributed over a range of wave exposure, so I tested the hypothesis that penis morphology responds to greater wave action with increased diameter and mass or reduced length and annulation number.
Materials and methods
In July 2005, I created experimental mating aggregations in the intertidal zone at five sites on Long Island (New York, USA): Stony Brook Harbor (40°55′17.20″N, 73° 8′59.06″W), Port Jefferson Harbor (40°56′46.96″N, 73° 4′19.29″W), Flax Pond Inlet Jetty (40°58′1.59″N, 73° 8′16.01″W), Shinnecock Inlet Jetty (40°50′18.73″N, 72° 28′29.44″W), and Robert Moses Beach Jetty (40°37′13.11″N, 73° 18′23.23″W). I located naturally occurring patches of barnacles in the middle third of their vertical range in the intertidal zone to hold tidal height and immersion time constant among sites. Within these patches, I set up plots (used as blocks in later analysis) by selectively removing adult individuals. I manipulated two factors: the number of individuals and the level of crowding. High-number treatments had greater than ten individuals; low-number treatments had fewer than four individuals. Barnacles in the densely crowded treatments made physical contact with the tests of neighbors, that is, the test of each barnacle directly abutted the tests of its neighbors. In sparsely crowded groups, each individual’s test did not contact any other barnacle. This resulted in four treatments (Fig. S2 in “Electronic supplementary material”) in each plot: high numbers, densely crowded (the HD treatment), high numbers, sparsely crowded (the HS treatment), low numbers, densely crowded (the LD treatment), and low numbers, sparsely crowded (the LS treatment). S. balanoides does not begin development of the penis or other reproductive tissue until mid-September, so there was ample time for treatment-specific phenotypes to develop (Barnes 1992; Hoch 2008).
The sites used in this experiment are located over a gradient of wave exposures. Robert Moses Beach Jetty is on the exposed shore of the Atlantic Ocean and has nearly constant wave exposure. Shinnecock Inlet Jetty is also on the exposed shore of the Atlantic Ocean; the site is located on the inlet side of the jetty and is exposed to oceanic waves as well as tidal exchange from Shinnecock Bay. Flax Pond Inlet Jetty is exposed to the waves of the main body of Long Island Sound and is of intermediate wave exposure. The Stony Brook Harbor site is in a protected channel between Long Island Sound and Stony Brook Harbor and is rarely exposed to large or constant waves; however, it experiences tidal flow when Stony Brook Harbor ebbs and floods. Port Jefferson Harbor is in the interior of a protected, blind harbor and experiences almost no perceptible wave action or tidal flow. I attempted to quantify relative wave exposure (actually, relative water motion) by comparing the dissolution rates of plaster spheres. Mass loss is positively correlated with water motion (Thompson and Glenn 1994), but can also be affected by temperature, sediment scour, and water chemistry. The sites in this experiment are all located on rock or concrete structures surrounded by sandy bottoms, so differential sediment scour among sites was a concern. I assumed that the sediment profiles at each site were similar and that any consequent scour was proportional to the strength of the water flow. To ensure that submergence time was approximately equal among sites, I chose points within the middle third of the vertical range of barnacles. Immediately prior to the mating season (penises and egg masses were fully developed), I bolted two 3-cm-diameter plaster spheres to the rocks among the barnacle treatments at each of the sites. Plaster spheres were left in the intertidal zone for six high tides (submerged for approximately 36 h), after which they were dried and weighed to determine the percent mass of plaster lost from each. I repeated this three times at each site, between 29 October and 7 November 2005, for a total of six measurements per site.
Number of blocks from each site and total number of individuals collected from each treatment in each site
Number of blocks
Individuals from HD
Individuals from HS
Individuals from LD
Individuals from LS
In the laboratory, I measured the height of each barnacle’s test and the radii of the aperture and base. Test volume was estimated by considering it as a truncated cone (following Hoch 2008). I dissected the penis from each barnacle, wet-mounted them on glass slides, and photographed them with a compound microscope at 40× magnification (Fig. S1). I removed the testes, seminal vesicles, and other male-specific tissues from each barnacle. Penises and the male reproductive tissues were dried at 50° C for 24 h, and masses were determined with a microbalance (accurate to 0.001 mg). Photographs were analyzed with ImageJ (Rasband 1997–2007). I measured the diameter of each penis at its base (at the annulation nearest to the body), and the length along the center axis of each penis. For images that were of high enough quality (N = 203; 58% of those photographed), I counted the number of annulations as an alternate measure of length. Preservation, dissection, and handling can shrivel, damage, or stretch the penis; the number of annulations represents an estimation of morphological variation of the penis and its ability to extend that is not affected by preparation (Barnes 1992; Hoch 2008).
All morphological data (including test volume, penis length, annulation number, and penis diameter) were log-transformed to meet standard assumptions prior to analysis in SAS (SAS 9.2; SAS Institute 2003–2008). I used a split-plot ANOVA (Gotelli and Ellison 2004) to test the hypothesis that site (the whole-plot factor), numbers of individuals (a sub-plot factor), crowding (a sub-plot factor), and block (treated as a random factor) with test volume as a covariate (to remove the effect of body size), affected the length of the penis (mm). I repeated the analysis using the number of annulations in the penis as the dependent variable. A Tukey–Kramer post hoc test was used to determine which treatments had significantly different size-adjusted means. I used linear regression to determine the relationship between penis annulation number and nearest neighbor distance. Densities of crowding and nearest neighbor distances were strongly confounded and very different in range (densely crowded barnacles ranged from 0.0 to 1.2 cm in nearest neighbor distance, sparsely crowded barnacles ranged from 0.5 to 8.8 cm), so I performed separate tests for densely crowded barnacles and sparsely crowded barnacles. I performed a second set of regressions (keeping the density treatments separated) to determine the effect of nearest neighbor distance on the residual variation in annulation number after removing the effect of body size (as measured by test volume).
I used a general linear model and Tukey HSD test to compare the percent mass lost from each plaster sphere in each site (using the arcsine-square root transformation, as suggested for percentage data by Sokal and Rohlf 1995). I used a split-plot ANOVA with the factors of site (the whole-plot factor), test volume, their interaction, and block (a random factor) to test the hypothesis that they affected penis diameter. I used a Tukey–Kramer post hoc test to determine which sites had penis diameters (in mm, log-transformed) with significantly different means (adjusted for body size). I then used regression to test whether relative wave exposure (the independent variable, measured by mass loss of plaster spheres, arcsine-square root transformed) affected the size-adjusted means of penis diameter (the dependent variable). Slopes of penis mass against body volume were the same among sites, so I used ANCOVA, with test volume as a covariate, to test for differences in penis mass among sites and blocks (a random factor, nested within sites). I used Tukey–Kramer post hoc tests to identify pairs of sites with significantly different size-adjusted means and used linear regression to determine the effect of relative water motion (transformed mass lost from the plaster spheres, the independent variable) on those means (the dependent variable).
Penis mass (ln mg, N = 294; Fig. S3 in “Electronic supplementary material”, Table S1) varied significantly among sites (P = 0.0004) and with the covariate, test volume (P < 0.0001). Penises from Robert Moses Beach Jetty (N = 51) were significantly less massive than those from Flax Pond (N = 71; Tukey–Kramer, P = 0.0449) and Shinnecock Inlet Jetty (N = 63, P = 0.0008). Penises from Stony Brook Harbor (N = 59) were less massive than those from Shinnecock Inlet Jetty (P = 0.0102). Size-adjusted means of penis mass from all other pairs of sites did not significantly differ from each other (P > 0.05). Regression showed no significant variation of mean penis mass with relative water motion (P = 0.9508, slope = −0.01567, R2 = 0.0015; Table S2).
Crowded barnacles had fewer annulations in the cuticle of their penises although they were no shorter. Penises from barnacles growing in crowded conditions probably have a reduced ability to stretch owing to fewer folded annulations in the cuticle. Penis length and annulation number did not change in response to the number of neighbors or in response to nearest neighbor distance. Barnacles must perceive something related to the density of crowding and physical contact with neighbors as a cue for estimating the distance over which the penis must reach. Barnacles whose tests do not touch those of their neighbors will almost always have a greater distance over which to reach, so density of crowding is likely a reliable cue. The number of potential mates or the distance of near neighbors may not be as useful as cues, or may not be detected. Increasing the number of annulations of the penis most likely allows these mating barnacles to reach more distant neighbors, allowing greater opportunity for mating. Densely crowded barnacles are more likely to have many mates within a short distance, so increasing their ability to reach more mates may not be worth costs such as increased risk of damage or greater metabolic effort. Diminishing returns to investments in penis structure may restrain densely crowded barnacles from growing penises with more annulations. At extreme levels of dense settlement, barnacles form hummocks; tests of neighbors grow together and into tall columns rather than short cones, eventually growing wider at the aperture, forming trumpet-like shapes (Barnes and Powell 1950). Hummocked barnacles have different proportions of soft tissue to test and different growth rates, owing to neighbors sharing skeletal elements and growth in test height outstripping somatic growth (Wu et al. 1977; Wethey 1984; Bertness et al. 1998). For this reason, I intentionally avoided barnacles showing the hummock growth form. Hummocked barnacles would have an even greater number of neighbors within a very short distance, far more than could be fertilized by a single barnacle, so I predict even fewer penis annulations would be found in individuals from such aggregations. Penis length and annulation number did not change with wave exposure, confirming the results of previous studies that were carried out at only two sites (Hoch 2008, 2009). It is known from those studies that ability to fertilize the broods of neighbors is reduced by greater wave exposure.
The results of this experiment confirm prior studies on S. balanoides showing significant differences in penis basal diameter between sites with different water motion, a significant effect of the interaction between test volume and water motion but no variation in length or annulation number (Hoch 2008, 2009). Because neither exact height above the tidal datum nor possible variation in sediment characteristics were measured, I must include a caveat as to the robustness of quantitative estimates of water motion provided by dissolution rates of plaster spheres. Despite this potential source of error, observations of wave action over the course of the experiment corroborate the qualitative rankings of each site’s wave exposure. As barnacles in a wave-exposed site get larger, they develop penises that increase in diameter more rapidly than those from a protected site. In areas protected from wave activity, barnacles produce thinner penises that are able to reach more mates (Hoch 2009). For another barnacle, B. glandula, penises are shorter, but more massive in wave-exposed sites than in protected sites (Neufeld and Palmer 2008). S. balanoides exposed to greater water motion did not produce penises with increased mass than those from protected sites or with differences in length. The additional support provided by the thicker structure must be enough to reduce risk of breaking or maintain function in rough water without additional mass (which may be provided by something like extra muscle). Bending beam theory suggests that increasing the diameter of such a structure may help it to resist deformation (Vogel 2003; Neufeld and Palmer 2008). Other factors, such as the timing of wave impacts or the length of calm periods, may vary among sites and be important for variation in penis mass. For example, sites with longer calm intervals or with longer wave periods will allow more opportunity for barnacles to attempt feeding and mating while free of risk.
Barnacles are important model organisms for the studies of sex allocation in hermaphrodites (Charnov 1980, 1982; Raimondi and Martin 1991; Schärer 2009). This research highlights phenotypically plastic allocation to a male-specific, fixed cost, the penis (in the sense of Heath 1977: a cost which must be paid for reproduction, but only once, in contrast to renewable costs such as sperm). Variation in penis structure and function must be accounted for when studying sex allocation in barnacles. Penises from this study were on average 9.8% of the mass of the total male reproductive system (sum of sperm, testes, penis, etc.). Penis mass may trade off with investments into sperm or with total investment in both sexes. Further, penises define the limits of mating groups for barnacles. That is, all neighbors within reach of a focal barnacle’s penis (and all of those that can reach it) form its mating group. Relative allocation to male function in simultaneously hermaphroditic acorn barnacles is predicted to be driven by sperm competition with other members of the mating group. As mating group size increases, barnacles are predicted to allocate a greater proportion of resources to male function, toward an asymptote at 50% (Charnov 1980, 1982). Variation in functional penis length (in the form of annulation number) or reach (as constrained by wave action) has a direct impact on the size of the mating group by limiting the distance over which mating is possible and thus the number of potential mates. In calm sites, a mating group will contain neighbors that are a greater distance away from a focal barnacle than those in wave-exposed sites. Phenotypic plasticity of penis traits allows individual barnacles some control over the size of their mating group and to increase the likelihood of successfully mating. The presence of the penis may hinder other aspects of the barnacle’s functions. Opportunity costs such as reduced feeding efficiency or reduced scope for growth are likely to be imposed by the development of the penis. With more research on metabolic and physical constraints imposed by the penis, these costs of reproduction could be fruitfully incorporated into the models of the costs of sex and costs paid for male-specific function in hermaphrodites.
Valuable ideas were incorporated into this manuscript deriving from conversations with J. Levinton, D. Padilla, D. Rand, R. Strathmann, J. True, and B. Yuen. This manuscript was meaningfully improved thanks to comments from the associate editor and anonymous reviewers. Help in the field was provided by B. Allen, P. Bourdeau, L. Brown, A. Ehmer, M. Fung, R. Junkins, C. McGlynn, B. Rodgers and E. Woo. Laboratory assistance was provided by E. O’Donnell, W. Wang and B. Yuen. Laboratory work was carried out in the Levinton Laboratory at Stony Brook University and the Functional Ecology Research and Training Lab (FERTL) in the department of Ecology and Evolution at Stony Brook University. Logistical support was provided by M. Doall. Assistance with statistical analysis was provided by Paulette Johnson at the FIU Statistical Consulting Center. Funding was provided by a Grant-in-aid-of-research from the Society for Integrative and Comparative Biology, a Student Research Fellowship from the Crustacean Society and a Doctoral Dissertation Improvement Grant from the National Science Foundation. An earlier version of this manuscript appears as a chapter in the author’s PhD dissertation.