Claremont CollegesScholarship @ Claremont

Scripps Senior Theses Scripps Student Scholarship

2013

Family Matters: An Analysis of Genetic Relatednessof Tetraclita rubescens (The Pink VolcanoBarnacle) Over Several Spatial Scales at Montereyand Bodega Bay, CaliforniaKelly N. ChangScripps College

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Recommended CitationChang, Kelly N., "Family Matters: An Analysis of Genetic Relatedness of Tetraclita rubescens (The Pink Volcano Barnacle) OverSeveral Spatial Scales at Monterey and Bodega Bay, California" (2013). Scripps Senior Theses. Paper 298.http://scholarship.claremont.edu/scripps_theses/298

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

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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

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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 barnacles 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.

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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

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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).

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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

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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.

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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

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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

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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.

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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 animal

placed in a labeled tube with 95% ethanol.

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

B. A.

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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

B. A.

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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

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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

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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.

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.

0  

50  

100  

150  

200  

250  

-­‐0.1  to  -­‐0.2   -­‐0.1  to  0   0  to  0.1   0.1  to  0.2    

#  of  individual  pairwise  

comparisons  

Intervals  of  pairwise  relatedness  values  

MB1  

MB2  

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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.

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.

0  

20  

40  

60  

80  

100  

120  

-­‐0.2  to  -­‐0.1   -­‐0.1  to  0   0  to  0.1   0.1  to  0.2    

#  of  individual  pairwise  

comparisons  

Intervals  of  pairwise  relatedness  

BB1  

BB2  

BB3  

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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).

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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).

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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

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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

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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

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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

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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

24

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

25

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.

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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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