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2013 Family Matters: An Analysis of Genetic Relatedness of rubescens (The inkP Volcano ) Over Several Spatial Scales at Monterey and Bodega Bay, California Kelly N. Chang Scripps College

Recommended Citation Chang, Kelly N., "Family Matters: An Analysis of Genetic Relatedness of (The inkP Volcano Barnacle) Over Several Spatial Scales at Monterey and Bodega Bay, California" (2013). Scripps Senior Theses. Paper 298. http://scholarship.claremont.edu/scripps_theses/298

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Family Matters: An analysis of genetic relatedness of Tetraclita Rubescens (the Pink volcano barnacle) over several spatial scales at Monterey and Bodega Bay, California

A Thesis Presented

By

Kelly N. Chang

To the Keck Science Department

Of The Claremont Colleges

In partial fulfillment of

The degree of Bachelor of Arts

Senior Thesis in Biology December 10, 2012

Table of Contents Abstract...... 3 Introduction...... 4 Materials and Methods...... 10 Sampling ...... 10 Molecular Analysis ...... 12 Statistical Analysis...... 13 Results...... 14 Pairwise Relatedness ...... 14 Geographic Distance vs. Genetic Relatedness ...... 17 Size vs. Genetic Relatedness ...... 19 Discussion ...... 19 Conclusion ...... 24 Acknowledgements ...... 26 References...... 27

2 Abstract

Inbreeding involves the mating of closely related individuals at a higher frequency than at random; this can decrease the average fitness of populations and individuals by reducing the presence of heterozygotes and augmenting the expression of deleterious genes. Since marine invertebrates exhibit widespread dispersal, their potential for inbreeding is often disregarded. The adult sessile state of creates the potential for inbreeding as a result of necessary copulation between neighboring individuals. Depending on the degree of mixing that occurs during dispersal, closely related individuals or siblings may settle in close proximity, generating the possibility of kin aggregation and consequent inbreeding. Despite the high probability of closely related or sibling barnacles to settle contiguously, their genetic relatedness and potential for inbreeding remain relatively understudied. We examined genetic relatedness between individuals of Tetraclita rubescens at Monterey and Bodega Bay to elucidate the potential for nonrandom dispersal and subsequent inbreeding. Genetic relatedness was assessed through microsatellite analysis, and correlated with geographic distance, and size. There was a significant association between geographic distance and genetic relatedness at Bodega Bay, which may be attributed to lower densities of individuals, frequency of settlement events and oceanographic conditions. The results of this study demonstrate high genetic relatedness at small spatial scales, bolstering the potential for nonrandom dispersal and subsequent inbreeding of T. rubescens.

3 Introduction

Inbreeding involves the mating of closely related individuals, or kin possessing alleles identical by descent from a common ancestor or ancestors at a higher frequency than at random

(Waser, 1993). As a consequence, inbreeding reduces the presence of heterozygotes and augments the expression of deleterious genes (Roughgarden, 1979). The loss of heterozygotes, in turn, produces individuals with more extreme morphologies or behaviors in comparison to a randomly mating population, and increases the appearance of homozygous recessives

(Roughgarden, 1979). Therefore, inbreeding can have a profound effect on an individual’s offspring fitness, the evolution of populations through changes in allele frequencies, and on mating systems.

Marine invertebrates exhibit a diversity of lifestyles in terms of mating mechanisms, life histories, and modes of dispersal, which may greatly affect their potential for inbreeding.

Dispersal patterns and mechanisms, which necessarily depend on life history can have a profound impact on settlement and genetic structure (Grosberg, 1987). Individual dispersal of marine invertebrates occurs through various modes at different stages in the life cycle. The unique planktonic developmental stage of marine invertebrates contributes to widespread dispersal, which in turn affects the geographical range, genetic structure of populations and the overall landscape of species’ distributions (Selkoe et al., 2006; Veliz et al., 2006; Dawson et al.,

2010). Previous studies suggest random larval associations during the planktonic period, where larvae from distinct locations and/or parents become thoroughly mixed and randomly distributed at settlement, provide sufficient gene flow and genetic variation among distinct populations

(Knowlton & Jackson, 1993; Veliz et al., 2006). Since marine invertebrates exhibit widespread dispersal, they are assumed to undergo sufficient random mixing before settlement such that

4 inbreeding is unlikely. However, the findings of more recent research indicate discrete patches of larvae may remain aggregated within the water column, bolstering the possibility of nonrandom dispersal of larval groups (Selkoe et al., 2006; Veliz et al., 2006; Miller-Sims et al., 2008;

Buston et al., 2009). Under certain conditions larvae from the same family group could be transported in the same water mass, therefore settling in close proximity (Veliz et al., 2006).

Furthermore, changes in oceanographic processes (i.e. currents) as a result of climate change may alter or provide new avenues for dispersal thus modifying marine larvae distribution.

In addition to dispersal, the presence of chemical inducers and a “sweepstakes effect,” in which only a few individuals within a population reproduce, increasing the potential for relatedness in larvae (Hedgecock & Puvodkin, 2011), have been found to influence genetic structure in various marine species. Only the most highly fecund individual contributes to the future pool of reproductively mature adults, which can greatly decrease genetic variation

(Hedgecock & Puvodkin, 2011). Gregarious settlement has also been observed in marine invertebrates (Pawlik, 1992), where individuals’ and/or populations demonstrate differing responses to settlement inducing stimuli (Young & Braithwaite, 1980; Raimondi & Keough,

1990; Kamel & Grosberg, 2012). Toonen and Pawlik (1994, 1996, 2001a, b) found that larvae of the tubeworm, Hydroides dianthus, concurrently settled on substrate absent of conspecifics, providing evidence for highly specified settlement behavior of larvae. The potential for larvae to exhibit disparate behavior in response to various environmental factors may contribute to a

“social environment” and influence settlement patterns (Veliz et al., 2006). In addition cyprid- cyprid associations have been found to stimulate settlement in several species of barnacles, affecting overall distribution and genetic structure (Veliz et al., 2006).

5 The unique life history of barnacles encompasses a pelagic planktonic larval phase and a benthic sessile adult phase, which affects their dispersive and reproductive capacities, as well as creates the potential for inbreeding (Veliz et al., 2006). As adults, barnacles exhibit a benthic sessile phase, during which they are permanently attached to bottom substrate. During this sessile stage they are limited to copulation with adjacent partners, increasing the probability of fertilization between gametes originating from nearby sources (Knowlton & Jackson, 1993;

Kelly & Sanford, 2011). Since by nature barnacles must reproduce with other contiguous partners, they maintain extraordinarily long penises to take advantage of the limited reproductive opportunities (Knowlton and Jackson, 1993). Because barnacles are sessile copulators, their mating groups must be small and spatially localized compared to most other marine invertebrates

(Kelly & Sanford, 2011; Kamel & Grosberg, 2012). This paradigm creates the potential for inbreeding depending on whether their neighbors are close relatives.

Based on the mating behavior and mechanism of barnacles, the proximity of individuals, or density may determine reproductive success or at least capacity. Kelly & Sanford (2011) found that Tetraclita rubescens broods could sire over distances greater than the penis length under certain environmental conditions, but proximity to the sperm recipient greatly increased the probability of siring success. Therefore a greater density of individuals may result in a higher reproductive success rate, affecting overall genetic structure. Subsequently, a high density of closely related individuals would increase the potential for inbreeding. In extreme low-density cases, isolated barnacles have demonstrated the capacity to self-fertilize, a severe form of inbreeding (Knowlton & Jackson, 1993).

During early development, barnacles undergo two nektonic larval stages, a nauplius and cyprid phase during which they engage in 21 days of pelagic activity followed by a complex

6 search for a suitable settlement location, and metamorphose into a sessile juvenile barnacle. The pelagic nauplius developmental phase generates the potential for widespread and random dispersal, bolstering traditional theory of random dispersal and settlement of marine invertebrates. However, research on marine fish demonstrated kin remain associated for substantial periods of time following recruitment (Selkoe et al., 2006; Buston et al., 2009; Kamel

& Grosberg, 2012). Furthermore, Veliz et al. (2006) provided evidence of genetic relatedness in the acorn barnacle, Semibalanus balanoides at the individual spatial scale despite their great potential for admixture during the planktonic period. Therefore, the probability of closely related individuals settling in close proximity and inbreeding depends on various parts on their life cycle.

The dispersal of larvae, reproductive behavior and sessile lifestyle of adult barnacles increases the possibility of closely related species or siblings settling in contiguous proximity.

This pattern of siblings congregating adjacent to one another is referred to as kin aggregation.

Buston et al. (2007, 2009) demonstrated kin aggregation in the clownfish, Amphiprion percula, and the humbug damselfish, Dascyllus aruanus. As noted, Veliz et al. (2006) observed a possible association between genetic relatedness and settlement of Semibalanus balanoides, bolstering the potential for nonrandom dispersal. However, the probability and efficacy of kin aggregation depends greatly on dispersal patterns with the assumption that limited dispersal forms groups composed of kin, increasing the degree of relatedness, which in turn affects individual behavior within these groups (Buston et al., 2009). Since marine invertebrates exhibit such widespread modes of dispersal, they are often excluded from kin aggregation theory (Buston et al., 2009).

Therefore, the social evolution and organization of marine species as a result of kin aggregation and nonrandom dispersal remain relatively understudied.

7 In addition to dispersal, individual size may be a useful indicator of kin aggregation and subsequent genetic relatedness. Previous studies have found that relatedness is greater in similarly sized humbug damselfish, Dascyllus aruanus individuals than in individuals of dissimilar sizes (Buston et al., 2009). Furthermore, food availability, temperature and other environmental conditions may affect growth and development of larval stages, as well as young adults (Skinner et al., 2009). Individuals of similar size most likely experience similar environmental conditions including food availability and temperature conditions during the larval stage, and presumably close in age. Therefore similar sized individuals could be from analogous larval dispersal groups, further supporting nonrandom dispersal. Subsequently, variance in individual size may suggest distinct generations, and a decrease in likeliness of genetic relatedness. Understanding the effect of relatedness on social structures of marine invertebrate populations and whether this relates to dispersal may elucidate the potential for less complex organisms to display social hierarchies and have important ramifications for how climate change may alter the landscape of marine invertebrate distribution and genetic structure.

The dispersal patterns, reproductive capacities, and life history of barnacles provide an ideal system for examining the genetic relatedness of individuals and its implications for kin aggregation, as well as the role of dispersal in shaping geographic range, structure and distribution. In this study Tetraclita rubescens, more commonly known as the pink volcano barnacle, was analyzed. T. rubescens is a southern species of barnacle prevalent throughout the

California coast, with an approximate range from Baja, Mexico (24025’0N, 111050’0W) to Cape

Mendocino (400 24.42’ 0N, 124023.42’0W; Dawson et al., 2010; Figure 1). Dawson et al. (2010) observed a northern range expansion of T.rubescens using microsatellite analysis, which has had profound effects on the species distribution, abundance, genetic variation and environment. In

8 addition, Dawson et al. (2010) discovered an increase in self-recruitment at northern peripheral locations, which suggests a decrease in gene flow between peripheral populations. Therefore the potential for closely related T.rubescens individuals to aggregate and inbreed might be higher within the expanded northern range. Consequently, this recent range expansion has interesting dispersal and population structure implications as well as provides known microsatellite primers for T. rubescens, which will be used in this research.

Figure 1. The distribution of T.rubescens across the historic and newly expanded range (Kamel et al, grant in review)

The aim of this study was to correlate genetic relatedness of individuals within clusters with their respective geographic distances and sizes. The degree of genetic relatedness among clusters of T. rubescens individuals at Monterey and Bodega Bay in relation to geographic distance and size may help elucidate the effect of dispersal patterns on kin aggregation and inbreeding potential. Since adult T.rubescens are sessile, they must propagate with their adjacent

9 neighbors. Therefore there is a higher chance for contiguous individuals to exhibit genetic relatedness, such that as distance increases, genetic relatedness decreases. Furthermore, differences in size may provide good indicators of age and settlement events. Individuals closer in size may be from corresponding settlement events and therefore more likely to be closely related. Conversely, dissimilarly sized individuals may be from distinct settlement events and exhibit no relatedness. The degree and distribution of genetic relatedness among clusters of individuals was determined using microsatellite analysis, which was correlated with geographic distance and size using a Mantel test.

Material and Methods

Sampling

Individuals were sampled from rocky jetties at Monterey (360 26.86 N’, 1210 55.73 W’) and Bodega Bay (380 18.921 N’, 1230 03.174 W’), which mark the mid to upper limit of

T.rubescens’ range. Monterey Bay encompasses the middle range of T.rubescens, with the highest density of individuals (Figure 1 and 2B). In contrast, Bodega Bay occurs at the upper/expanded range, with relatively low density of individuals (Figure 1 and 2A). Eighty individual T. rubescens were collected from Monterey Bay, CA and sixty from Bodega Bay, CA.

At both sampling sites, only closely grouped individuals were considered for collection. At

Bodega Bay, individuals were arranged in clusters of 10-20 individuals, with one individual arbitrarily chosen as a reference point (Figure 3A). The physical distance between each individual in the cluster, and the reference point was measured in inches and converted to centimeters (Figure 3A). Each individual marked on the spatial map was then collected, and placed on ice. This process was repeated for the 3 sampling clusters within the Bodega Bay sampling site. Each sampling cluster was chosen within a minimum 5 meter distance.

10 Individuals at Monterey Bay occurred in much higher densities and abundances, however a similar sampling method was used. At Monterey Bay clusters of 30 or more individuals were chosen for sampling and spatial analysis. When a cluster of 30 individuals or more in a given area was identified, one individual was selected as a reference point (Figure 3B). Subsequently, the relative distance between individuals and from the reference point was measured and spatial maps were sketched. Since Monterey Bay individuals occurred in such high densities, clusters were organized even further, where individuals settled within roughly 5 cm of each other were given sub labels (Figure 3B). For example if 5 individuals were found at about the same distance from the reference point and were at the third distance measured, they would be marked as

3a,3b,3c,3d, and 3e. After each relative distance was measured, the individuals were collected and their approximate locations were marked on the spatial map. This process was repeated for the 2 sampling clusters within the Monterey Bay sampling site. Again sampling clusters were chosen within a minimum distance of 5 meters. All individuals were placed on ice during transportation from sample site to the lab. In the lab, the girth of the widest region of the individual shell was measured to the nearest 0.1mm using calipers, and the dissected placed in a labeled tube with 95% ethanol.

A. B.

Figure 2. Density of clusters of T.rubescens at (A) Bodega Bay and (B) Monterey Bay collection sites (Kamel et al, grant in review).

11 A. B.

Figure 3. Spatial map of T. rubescens clusters at (A) Monterey Bay site 1 and (B) Bodega Bay site 1.

Molecular Analysis Two feeding appendages with some prosoma tissue were used for DNA extractions.

DNA extractions were performed using the Puregene DNA purification Kit for marine invertebrates (Gentra Systems, MN, USA 158388) and following Dawson et al. (2010).

Extracted DNA was then amplified via the Qiagen Multiplex PCR kit (QIAGEN, CA, USA

206143) with pre-designed microsatellite marker primers (Dawson et al. 2010; Table 1). PCR samples were prepared and run under PCR temperature conditions found in Dawson et al.

(2010). The PCR products were denatured using a formamide/liz500 mixture and sequenced by the UC Davis DNA sequencing facility. The PCR products were genotyped in genescan, ABI

3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA), which identifies the marked alleles, determines the size and number of repeats. Data from the ABI 3130 Genetic

Analyzer was examined in STRand (Toonen & Hughes, 2001), which then reads the size of the marked alleles and illustrates the number of repeats in the form of peaks. The data in STRand was edited by eye to remove artificial peaks (stutter) and prevent the use of falsely identified

12 allele, and exported into Excel. In Excel, a list of each individual with its corresponding allele sizes for each of the 10 loci was compiled and prepared for statistical analysis.

Table 1. A summary of microsatellite locus primers used (Dawson et al., 2010).

Statistical Analysis

Once the allele sizes for each individual were determined, the data was imported into an excel spreadsheet and input into Genepop (version 4.1; Raymond & Rousset, 1995; Roussett,

2008), an online population genetics program that calculates allele frequencies. Population allele frequencies for Bodega Bay and Monterey Bay were calculated and individuals were sorted into their respective sampling groups. Allele frequency files were then input into STORM (Frasier,

2008), a program used to calculate individual pairwise relatedness and within group relatedness.

Pairwise relatedness values between each individual within a sampling group and average pairwise relatedness values for each sampling group were calculated. Calculations were based on methods from Li et al. (1993). Pairwise relatedness values occurred in a range from -1 to 1, with -

1 indicating individuals were less related than random, and +1 indicating maximal relatedness

(i.e. individuals are clones). Geographic distances between individuals were correlated with pairwise relatedness values using a Mantel test, which examines the relationship between two

13 matrices, in this case geographic distance and pairwise relatedness. Size was also correlated with genetic distance using a Mantel test. The mantel calculations followed those of Fortin and

Gurevitch (1993).

Results

Pairwise Relatedness

Individuals within each sampling group at Monterey and Bodega Bay were less related than at random, with mean pairwise relatedness values falling below 0 (Table 2).

Table 2. Mean pairwise relatedness values for each sampling group at Monterey and Bodega Bay (mean±SE; n=140). Population Mean pairwise relatedness value

MB 1 -0.033437 ± 0.140522

MB 2 -0.42678 ± 0.090068

BB 1 -0.017869 ± 0.106638

BB 2 -0.038598 ± 0.083384

BB 3 -0.02622 ± 0.089145

14 At both Monterey Bay sampling locations, there were more individuals that were less related than they would be randomly, with the majority of values occurring less than 0 (Figure

4). Group 1 demonstrates a negative relationship between the number of individuals and subsequent relatedness. Most individuals were less related than at random with pairwise values in the -0.1 to -0.2 range (Figure 4). Group 2 demonstrates a slightly different pattern with the most individuals displaying pairwise relatedness values in the -0.1 to 0 range (Figure 4).

Between groups 1 and 2, group 2 overall had more closely related individuals than group 1.

250

200

150

100 MB1

comparisons MB2 50 # of individual pairwise

0 -0.1 to -0.2 -0.1 to 0 0 to 0.1 0.1 to 0.2 Intervals of pairwise relatedness values

Figure 4. The distribution of individual pairwise relatedness values for Monterey sampling groups 1 (n=25; ) and 2 (n=31; ). The “n” indicates sample size, however there were 300 individual comparisons for group 1 and 457 comparisons for group 2.

15 All three groups at Bodega Bay show a similar pattern in distribution of pairwise relatedness values (Figure 5). There were more individuals that were less related than at random since most of the values occurred at less than 0. For each group, the greatest number of individuals displayed pairwise related values in the -0.1 to 0 range (Figure 5). The Bodega Bay groups were more related than the Monterey Bay groups as indicated by their pairwise relatedness values, with the majority of individuals exhibiting values in the -0.1 to 0 range compared to the -0.2 to -0.1 range in Monterey Bay.

120

100

80

60 BB1 BB2

comparisons 40 BB3

# of individual pairwise 20

0 -0.2 to -0.1 -0.1 to 0 0 to 0.1 0.1 to 0.2 Intervals of pairwise relatedness

Figure 5. The distribution of individual pairwise relatedness values for Bodega Bay sampling groups 1 (n=15), 2 (n=21), and 3 (n=9). The “n” indicates sample size, however there were 105 individual comparisons for groups 1, 231 comparisons for group 2, and 36 comparisons for group 3.

16 Geographic Distance vs. Pairwise Relatedness

There was no significant relationship between pairwise relatedness values and geographic distance between individuals at the Monterey Bay sampling sites (r2=0.003, p=0.39; Figure 6).

Figure 6. The relationship between individual pairwise relatedness values and geographic distances less than 20cm for all individuals at Monterey Bay (n=80).

17 There is a decreasing relationship between individual pairwise values and geographic distance between individuals at Bodega Bay (Figure 7). Pairwise relatedness decreases as geographic distance between individuals’ increases. This inverse relationship between geographic distance and genetic relatedness was significant (r2=0.14, p=0.05).

Figure 7. The relationship between individual pairwise relatedness values and geographic distances less than 20cm for all individuals at Bodega Bay (n=60).

18 Size vs. Pairwise Relatedness

A mantel test revealed no significant relationship between size of individuals and genetic relatedness for groups at Bodega Bay (p=0.93) and Monterey Bay (p=0.91) (Table 3). There was also no significant correlation between size and genetic relatedness between individual clusters at

Bodega and Monterey Bay (Table 3).

Table 3. A correlation of geographic distance and size difference between individuals within each sampling group and overall at Monterey and Bodega Bay Population R2 value P-value BB1 0.003 0.86 BB2 0.04 0.39 BB3 0.002 0.91 All Bodega 0.0002 0.93 MB1 0.02 0.35 MB2 0.002 0.56 All Monterey 0.00006 0.91

Discussion

The results of this study provide evidence for kin aggregation at lower densities in

Bodega Bay as compared to higher densities in Monterey Bay. There were significantly fewer negative pairwise relatedness values for clusters BB1 and BB3 (Figure 5), suggesting a higher occurrence of relatedness at Bodega Bay. Furthermore, individuals occurring in lower densities at Bodega Bay exhibited a significant correlation between geographic distance and genetic relatedness (Figure 7). The higher density individuals at Monterey Bay demonstrated no relationship between geographic distance and relatedness (Figure 6). Size of individuals demonstrated no significant correlation for sampling clusters at either location (Table 3). These results support the possibility of nonrandom dispersal and kin aggregation at small spatial scales

19 and low densities, as indicated by Veliz et al. (2006) in their study of genetic relatedness of

Semibalanus balanoides.

The higher genetic relatedness and significant correlation between geographic distance and genetic relatedness among individuals at Bodega Bay may be attributed to two main factors.

First, since T.rubescens individuals must settle within penile distance in order to successfully copulate, selective pressures at lower density locations may create a tendency to aggregate (Veliz et al., 2006). Dawson et al. (2010) reported an increase in self-recruitment at the northern periphery of T.rubescens’ range. These patterns correspond with the higher frequency of genetic relatedness at Bodega Bay, which is in the northern portion of T.rubescens’ range. Therefore density may result in closely related individuals necessarily settling in close proximity to one another to maximize reproductive success in low-density settings (Kelly & Sanford, 2010), and have a considerable effect on settlement patterns and genetic structure (Veliz et al., 2006).

Conversely, there may be less of a propensity for individuals, and potentially siblings to aggregate in high-density locations, such as Monterey Bay because of the increased chance of settling contiguously with other individuals.

Second, genetic relatedness among individuals may have been obscured by differences in settlement events. Monterey Bay occurs at the center of T. rubescens’ range, and therefore maintains the greatest density of individuals, and experiences the highest settlement frequency.

Contrarily, Bodega Bay, which lies near the northern limit of T. rubescens’ range has less individuals and therefore experiences fewer settlement events. The lower number of individuals at Bodega Bay may promote noticeable patterns of genetic relatedness as opposed to high- density locations such as Monterey Bay. Therefore, low densities of individuals may make relatedness patterns more detectable as opposed to high densities of individuals. An assessment

20 of genetic relatedness among individual T.rubescens at multiple sites between Monterey and

Bodega Bay to determine a possible location at which there becomes a higher proportion of closely related individuals, may help to clarify whether settlement events affect relatedness patterns.

Oceanographic conditions such as currents may also be limiting the extent of dispersal of

T.rubescens larvae, which in turn may affect the density and distribution of individuals. The variation in density of individuals-higher densities at Monterey Bay and lower densities at

Bodega Bay- may reflect the geographic patterns of retention and recruitment facilitated by eddies, relaxation of upwelling, or other oceanographic features occurring along eastern boundary currents (Dawson et al., 2010). A study regarding oceanographic currents around Cape

Mendocino observed offshore flow patterns, suggesting that topography of the cape may be deflecting currents seaward. Therefore, patterns of oceanic currents at Cape Mendocino may be restricting dispersal northward and preventing settlement of T.rubescens larvae at Bodega Bay.

In addition, the oceanographic conditions where the species occurs may be conducive to limited larval mixing despite high dispersal potential (Veliz et al., 2006). Roughgarden et al. (1988) demonstrated the effect of variation in upwelling along the coast of California and Oregon on distance larvae travel from shore. They found that strong upwelling forces transport larvae far from shore, resulting in high larval loss (Roughgarden et al., 1988). This pattern of larvae dispersal was demonstrated in the extent of larval displacement off the California and Oregon shores. Because of the different degrees of upwelling, larvae of Balanus glandula (acorn barnacle) occurred 50 miles out from the California coast, whereas Merluccius productus (hake larvae) were found only 10 miles offshore the Oregon coast (Roughgarden et al., 1988).

Therefore depending on the strength of upwelling forces, larvae may be displaced far out or close

21 to shore, thereby affecting the degree of admixture, settlement patterns and probability of kin aggregation.

The potential role of oceanographic conditions in driving marine larval dispersal and settlement has important implications for climate change. Shifts in oceanographic currents as a result of changes in sea surface temperature may enable new patterns of dispersal, which could alter the overall distribution and density of individuals, as well as the propensity for siblings to settle adjacently and inbreed. King et al. (2011) observed changes in species distribution, more specifically a northward extension of southern species as a result of changes in sea surface temperature. Northward shifts in T. rubescens’ range have been demonstrated by (Dawson et al.,

2010), and are likely to continue with the projected changes in ocean surface temperature. The range extension of T. rubescens could contribute to more low-density environments of individuals, increased kin aggregation and subsequent inbreeding. Furthermore, Snyder et al.

(2003) demonstrated increases in CO2 and ocean surface temperatures augmented intensity of upwelling along the California coast, which could induce retention of organisms and food sources to the same area. The retention of organisms and food within the same area may result in less admixture, and greater probability of kin aggregation and inbreeding. Therefore, the effect of climate change on oceanographic conditions may have critical consequences for dispersal and settlement patterns of T. rubescens and other marine invertebrates, potentially intensifying kin aggregation and incidence of inbreeding.

“Sweepstakes effects” and chemical communication at early stages may have also contributed to high genetic relatedness at Bodega Bay. A “sweepstakes effect”, in which a small proportion of the available gene pool successfully replenishes an entire population, may contribute to reduced genetic diversity, and a higher potential for inbreeding in lower density

22 locations (Christie et al., 2010; Hedgecock & Pudovkin, 2011). Furthermore the possibility of closely related larvae necessarily aggregating in low-density areas as a consequence of mating mechanics, may promote chemical communication between sibling larvae. The possibility of chemical communication at early larval stages may provide an additional avenue for kin recognition during planktonic development, and promote kin-related larvae associations prior to settlement (Veliz et al., 2006). However, whether a “ sweepstakes effect” or chemical communication actually occurs and plays any significant role in kin aggregation and settlement remains ambiguous and requires further research.

Size did not seem to have a significant effect on genetic relatedness between individuals

(Table 3). However, previous studies have found a correlation between size and relatedness in

Dascyllus aruanus (humbug damselfish), with closely related individuals maintaining similar overall sizes (Buston et al., 2009). Therefore, further research with larger sample sizes of individuals may help reveal whether there is any relationship between size and relatedness among barnacles specifically T. rubescens.

Potential experimental errors which may have affected the results for this study, include sample size and quantity of geographic distance data. In Veliz et al. (2006), genetic relatedness was observed in a sample of 1250 individuals of Semibalanus balanoides, compared to 140 total individuals over both locations for this study. A larger sample size would have a provided a more comprehensive and accurate representation of T.rubescens populations, as well as more definitive patterns of relatedness across clusters. Each individual T.rubescens was compared to all other individuals within the sample cluster to give pairwise relatedness values. However, measurements of geographic distance were only made between adjacent individuals and sometimes in relation to the reference individual. The lack of geographic distance data may have

23 skewed distance versus relatedness results. Therefore, more geographic distance data would have provided a more accurate correlation between relatedness and geographic distance and potentially yielded a different outcome.

To further elucidate the effect of dispersal, life history and mating behavior on kin aggregation, genetic structure and subsequently inbreeding, additional research regarding larval behavior, oceanographic conditions, reproductive mechanisms and possible chemical signals among kin remain necessary. Examining how oceanographic conditions vary with season and how this may contribute to larval dispersal may prove informative. Assessing whether chemical communication occurs at the larval stage and the extent to which this affects kin aggregation and settlement may also be useful. It may also be interesting to identify singular T.rubescens individuals at the very upper limit of their range and examine whether selfing occurs. Lastly, observing possible philopatric behavior T. rubescens larvae may provide insight into dispersal patterns.

Conclusion

The aim of this study was to identify evidence of genetic relatedness among clusters of individuals at Bodega and Monterey Bay, and whether this correlated with geographic distance and size. Our results suggest that the potential for inbreeding may be higher in low-density locations. There was greater genetic relatedness among individuals at Bodega Bay, and a significant correlation between geographic distance and pairwise relatedness values. The significant pattern of relatedness and geographic distance at Bodega Bay could be attributed to lower density of individuals. T. rubescens individuals may be demonstrating a greater tendency to aggregate due to selective pressures, or fewer settlement events may be promoting a more noticeable pattern of relatedness. Furthermore, differences in oceanographic currents at each

24 collection location could account for dissimilar admixture and displacement of larval groups resulting in subsequent discrepancies in abundance/density at Bodega and Monterey Bay. There was no relationship between size and pairwise relatedness values, suggesting age or differences in settlement events may not be good indicators of genetic relatedness and kin aggregation. The results of this study suggest a higher inbreeding potential in decreased abundance areas, however the relative effects of oceanographic processes, sweepstakes effects, and larval behavior on settlement and kin aggregation require further investigation. In addition, more in-depth studies of reproductive mechanisms may help elucidate the potential for inbreeding among closely related individuals of T.rubescens. Understanding how the degree of genetic relatedness among individuals relates to geographic distance and size, and whether this is a product of irregular dispersal patterns or other means, has important implications for kin aggregation and the potential for inbreeding to determine individual and population fitness. Moreover, T. rubescens’ potential for inbreeding as a result of various environment factors, may have important ramifications for speciation and evolution. In addition, recognizing the disparate processes driving species distribution, and genetic structure can provide valuable and compelling indicators of the possible effects of climate change.

25 Acknowledgements

This research was supported by an NSF research grant to Dr. Richard Grosberg at the

University of California Davis (NSF ANB041673 and NSF OCE 090907). I first want to thank

Dr. Richard Grosberg for providing me the opportunity to intern in his lab and conduct research.

The Grosberg Lab (Brenda Cameron, Rachel Brooks and Lauren Holtz) provided invaluable help and support throughout the entire research/experimental process. Dr. Stephanie Kamel’s knowledge and guidance of the subject area and statistical analysis were instrumental and crucial to the completion of this project. Furthermore, I am grateful for the opportunity to collect samples from their natural sites and gain experience with fieldwork. Finally, Dr. Sarah Gilman and Dr. Stephanie Kamel provided feedback and helped develop my ideas throughout the entire thesis writing period.

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