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Life-History Consequences of Artificial Selection for Increased Egg Size in Hydroides elegans (Polychaeta: Serpulidae)

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PAGE 1

LIFE-HISTORY CONSEQUENCES OF ARTIFICIAL SELECTION FOR INCREASED EGG SIZE IN Hydroides elegans (POLYCHAETA: SERPULIDAE) By CECELIA MARIE MILES A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2006

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Copyright 2006 by Cecelia Marie Miles

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This work is dedicated to Dr. Larry McEdwar d. Larry’s ability to distill complex topics down to their essential elements served to focu s my efforts as a new graduate student. As I have advanced in my career, I have come to respect this skill immensely and it is one that I aspire to emulate. This project was conceived and planned under his guidance and his influence is present in every aspect.

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iv ACKNOWLEDGMENTS For insight, guidance, encouragement, a nd especially for leading by example, I thank Dr. Marta L. Wayne. I also thank committee members Dr. Colette St. Mary, Dr. Shirley Baker, and Dr. Gustav Paulay. I am es pecially grateful to Dr. Ben Bolker for his clear thinking and concis e statistical advice. I thank Dr. Michael G. Hadfield for his generosity and advice, and everyone at Kewalo Marine Laboratory in Honolulu, Hawaii for their kokua. I could never have completed this project without my fellow “w orm wrangler” at Kewa lo, Sharon Kelly. I thank the Zoology Department at the Un iversity of Florida for financial, emotional, and spiritual support. I would al so like to acknowledge the Auzenne Graduate Scholars Fellowship from the University of Fl orida, and the Grinter Fellowship from the Graduate School. I wish to thank my family and especia lly my husband, Myron G. Miles, for his infinite support.

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v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES...........................................................................................................ix ABSTRACT....................................................................................................................... ..x CHAPTER 1 GENERAL INTRODUCTION....................................................................................1 2 ESTIMATES OF HERITABILITY FOR EGG SIZE IN THE SERPULID POLYCHAETE Hydroides elegans .............................................................................8 Introduction................................................................................................................... 8 Material and Methods.................................................................................................11 Collection and Spawning.....................................................................................11 Half Sib Breeding Design....................................................................................14 Selection..............................................................................................................16 Results........................................................................................................................ .17 Discussion...................................................................................................................18 3 DIRECT AND INDIRECT RESULT S OF ARTIFICIAL SELECTION FOR INCREASED EGG SIZE IN TH E SERPULID POLYCHAETE Hydroides elegans ........................................................................................................................24 Introduction.................................................................................................................24 Methods......................................................................................................................29 Collection, Spawning, and Selection...................................................................29 Larval Size...........................................................................................................31 Juvenile Tube Length..........................................................................................32 Total Egg Energy.................................................................................................32 Fecundity.............................................................................................................34 Adult Dry Weight................................................................................................35 Data Analysis.......................................................................................................35 Results........................................................................................................................ .36 Direct Response to Selection...............................................................................36

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vi Correlated Responses to Selection......................................................................36 Spontaneous Spawning and Sex Ratios...............................................................38 Discussion...................................................................................................................39 More + Bigger = Better… or Does It?.................................................................39 Conclusions and Future Directions.....................................................................44 4 CONCLUSIONS........................................................................................................49 LIST OF REFERENCES...................................................................................................53 BIOGRAPHICAL SKETCH.............................................................................................67

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vii LIST OF TABLES Table page 2-1 Number of females used to establis h six individual lines in Generation 7..............21 2-2 Half-sib breeding design; Fully nested ANOVA and variance component analysis for egg diameter in Hydroides elegans .......................................................22 3-1 Egg diameter ( m) in replicate selected vs. control lines of Hydroides elegans after 4 generations of selection.................................................................................46 3-2 Total egg energy in replicate selected vs. control lines of Hydroides elegans (calculated as g C egg-1).........................................................................................46 3-3 Absolute fecundity in replicat e selected vs. control lines of Hydroides elegans (eggs female-1)..........................................................................................................47 3-4 Relative fecundity based on dry weight of the female in replicate selected vs. control lines of Hydroides elegans (eggs mg-1).......................................................47 3-5 Adult female dry weight (mg) in re plicate selected vs. control lines of Hydroides elegans ......................................................................................................................47 3-6 Larval volume ( L) at 18 to 20 h post-fertilizat ion in replicate selected vs. control lines of Hydroides elegans ...........................................................................47 3-7 Volume ( L) of competent (5 d) larvae in repl icate selected vs. control lines of Hydroides elegans ....................................................................................................47 3-8 Juvenile tube length (mm) at 21 d in replicate selected vs control lines of Hydroides elegans ....................................................................................................47 3-9 Summary of direct and correlated re sponses to selecti on for increased egg diameter in replicate selected v controls lines of Hydroides elegans ......................48 3-10 Effect of selection (treatment) on the relative numbers of males and females in Generations 7 to 11..................................................................................................48 3-11 Mean percent females present at sp awning (6 weeks post-fertilization) in replicate selected vs control lines of Hydroides elegans .........................................48

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viii 4-1 Phenotypic correlations between the tra it of egg size and a suite of life-history traits in replicate selected a nd control lines in the polychaete Hydroides elegans ..52

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ix LIST OF FIGURES Figure page 2-1 Response to direct selection on egg di ameter for four generations of in the polychaete Hydroides elegans ..................................................................................22 2-2 Mean egg diameter ( SE) before and after direct selection for increased egg diameter in replicate selected (1,3,5) and control (2,4,6) lines of Hydroides elegans ......................................................................................................................23 3-1 Cumulative response to selecti on for increased egg diameter in H. elegans was positive through Generation 11................................................................................46

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x Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy LIFE-HISTORY CONSEQUENCES OF ARTIFICIAL SELECTION FOR INCREASED EGG SIZE IN Hydroides elegans (POLYCHAETA: SERPULIDAE) By Cecelia M. Miles May 2006 Chair: Marta L. Wayne Department: Zoology Goals of this study were to esti mate narrow-sense heritability ( h2) for egg size in the polychaete worm Hydroides elegans to examine direct and correlated responses to selection on increased egg size in a suite of life-history ch aracters, and to explore how these correlations ch anged as egg size was increase d by artificial selection. Narrow-sense heritability is a predictor of short-term response to selection. Many quantitative models have invoke d selective response in e gg size as a key transitional element in the evolution of life histories in marine invertebrates, assuming that egg size can and does respond to selecti on. I tested this assumpti on using a half-sib breeding design and obtained an estimate for h2 of 0.45 for egg size in a Pearl Harbor, HI population. I performed four gene rations of artificial selectio n for increased egg size that allowed me to estimate cumulative realized he ritability in the same population as 0.58. Artificial selection resulted in a direct response of 2.5 P in egg size relative to the common base population. Though I predicted negative correlated responses in some

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xi traits under selection, none was measured. A positive correlated response to selection was observed in fecundity, total egg energy, a nd larval size at comp etence, relative to control lines. No significan t correlated response was obser ved in early larval size, juvenile tube length, or adult dry weight. H. elegans is a protandrous hermaphrodite, and sex ratio data across generations indicated that large-egg sele cted lines were changing sex earlier than control lin es. I propose that this earlier se x change resulted in selected individuals spending less time as males relative to controls, conseque ntly decreasing their overall fitness. Phenotypic correlations between egg diameter and the traits outlined above were estimated, and control line means showed no significant correlation with egg size, with the exception of the positive correlation for la rval size at competence. Unexpectedly, phenotypic correlations among selected line means were significantly negative for fecundity, but positive for female dry weight. The positive correlation for larval size at competence also persisted after selection. This may indicate where further work could be useful in examining trade-offs between egg size and other lifehistory traits.

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1 CHAPTER 1 GENERAL INTRODUCTION The diversity of larval form and fu nction among marine invertebrates has fascinated researchers for decades. Thors on (1950) was among the first to recognize broad-scale patterns in developmental mode and their strong correlation with specific life-history traits (egg size and fecundity). A useful classification of larval types has been based on this observation that numerous mari ne invertebrates produce high numbers of relatively small, energy-poor eggs, while many others produce fewer, larger, energy-rich eggs. A closely associated dichotomy invol ves the coexistence of planktotrophic and lecithotrophic larval development. Many ma rine invertebrates produce pelagic, feeding (planktotrophic) larvae, and these are generally hatched in large numbers from small eggs. Other, often closely related, species produce lecit hotrophic larvae that do not depend on exogenous food in order to reach me tamorphosis, and these hatch in smaller numbers from larger eggs. Phylogenetic an alysis suggests that the transition between these life-history modes has occurred ma ny times (Wray 1996, Duda and Palumbi 1997, Cunningham 1999, Rouse 2000, McEdward and Miner 2001). The adaptive significance of developmen tal mode has received considerable attention. Selection pressure s such as predation, starvati on, and dispersal have been proposed for developmental mode evol ution (Thorson 1950, Chia 1974, Strathmann 1985), and increases in egg size are often cite d as important transi tional states between modes (Wray and Raff 1991, Hart 1996, Miner et al. 2005). The suite of characteristics that defines a larval type has not evolve d in isolation from post-larval and adult

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2 characteristics. Egg size and fecundity ar e two members of a much larger complex of life-history traits that co-evolved, presumabl y, under the influence of natural selection (Havenhand 1995, Ramirez Llodra 2002). E gg size and fecundity are both directly related to parental fitness (Roff 1992, Bern ardo 1996) and many authors have mentioned the importance of interactions between larval characteristics and ot her parts of the life cycle (e.g., Vance 1973a,b, Christiansen and Fenchel 1979, Todd and Doyle 1981, Strathmann 1985, Moran 1994, Pechenik et al. 1998, Gimnez et al. 2004). Few efforts have been made to examine the implicatio ns over the entire life cycle of selective pressures on these important and in ter-related fitness components. Many studies of marine ecology have used a comparative appr oach to assess the influence of one life-history stage on anothe r. For instance, maternal environmental conditions influence larval performance in various groups (e.g., Laughlin and French 1989 for the crab Rhithropanopeus harrisii George 1999 for the seastar Pisaster ochraceus Qiu and Qian 1997 for the polychaete Hydroides elegans Gimnez and Anger 2003 for the crab Chasmagnathus granulata ). Studies have demonstrated that larval experience is linked to juvenile quality (e .g., Jarrett and Pechenik 1997 for the barnacle Semibalanus balanoides Wendt 1998 for the bryozoan Bugula neritina Phillips 2002 for the bivalve mollusk Mytilus galloprovincialis reviewed in Pechenik et al. 1998). Size at hatching affects growth, time to maturity and survivorship in the gastropod Nucella ostrina (Moran and Emlet 2001). Size at settlement influences survival in the ascidian Botrylloides violaceus (Marshall et al. 2006). Hilbish et al. (1993) discussed the common assumption that variation in larval and j uvenile growth rates have a common genetic basis, and found that significant genetic variat ion for larval shell growth in the bivalve

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3 Mercenaria mercenaria is not related to the genetic va riation in shell growth during the juvenile period. Pechenik et al. (1996) found no correlation of growth rates between larval and juvenile stages in two species of the mollusk Crepidula Delayed metamorphosis is associated with reduced j uvenile or adult performance most often in non-feeding larval forms (e.g., Wendt 1996 for the bryozoan Bugula neritina Pechenik and Cerulli 1991 for the polychaete Capitella sp. I, Pechenik et al. 1993 for the barnacle Balanus amphitrite and Maldonado and Young 1999 for the sponge Sigmadocia caerulea ). Delaying metamorphosis in the feeding larvae of polychaete Hydroides elegans negatively affects juvenile survival and growth rates whether or not they are fed during the delay (Qian and Pechenik 1998). Delayed metamorphosis in the feeding larvae of the sipunculan Apionsoma misakianum results in reduced juvenile growth whether or not larvae are fed, but does not a ffect juvenile survival (Pechenik and Rice 2001). Empirical studies have examined the re lationship between egg size and larval characters such as rate of development and larval size at metamorphosis in a range of taxa. For example, in opisthobranch mollu sks, Havenhand (1993) reports a negative relationship between egg size and development time. Gimnez (2002) reports that the crustacean Chasmagnathus granulata hatching from smaller eggs takes longer to develop, as does the seastar Pisaster ochraceus (George 1999). This negative relationship between egg size and development time is also reported for echinoids and asteroids combined (Emlet et al. 1987). Le vitan (2000) further refi ned this relationship by showing that it is negative curvilinear usi ng data from a large group of echinoids. For echinoids and asteroids combined the correla tion between size of egg and the size of

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4 juvenile at metamorphosis is positive overall (Emlet et al. 1987), but Levitan (2000) shows these two traits to be independent of one another in echinoids, after controlling for phylogeny. Many fewer studies have used an experiment al approach to directly manipulate egg size. Sinervo and McEdward (1988) used blas tomere separation to show the relationship between reduced parental investment and resul ting larval characters in two species of the echinoid Strongylocentrotus They found that larvae that develop from experimentally reduced zygotes are smaller and take longer to reach metamorphosis than those from whole eggs. Hart (1995) used the same t echnique and found reduced feeding rates in larvae developing from experi mentally reduced zygotes of S. droebachiensis Emlet and Hoegh-Guldberg (1997) experimentally reduced the lipid content of early zygotes in another urchin, Heliocidaris erythrogramma and showed a link between reduced parental investment and juvenile performance. Another approach to evaluating the influe nce of egg size on life history is to examine a single species that shows both developmental modes (poecilogony). Levin et al. (1987) examined the demographic conseque nces of divergent life-history patterns in a single species of polychaete, Streblospio benedicti with both planktotrophic and lecithotrophic developmental types. They found that both types achieve similar population growth rates. This overview represents a sa mple of the diversity of ta xa, traits, and methods that have been employed to demonstrate to what extent one stage of life can influence another. Thus, while a large and dive rse body of empirical, comparative, and experimental work has demonstrated links be tween one life-history stage and another, a

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5 unified examination of egg, larval, juvenile, and adult traits, and how these correlations influence fitness over the entire life cycle ha s not been undertaken. As shifts in egg size are often invoked as important transitional states between developmental modes (Wray and Raff 1991, Hart 1996, Miner et al. 2005), and egg size plays a critical role in many of the quantitative modeling approaches that have been used to try and identify selection pressures and processes that influence de velopmental mode evolution (Vance 1973a,b, Smith and Fretwell 1974, Christiansen and Fenchel 1979, Perron and Carrier 1981, Roughgarden 1989, Podolsky and Strathmann 1996, McEdward 1997, Levitan 2000, Hendry et al. 2001, Luttikhuizen et al. 2004), I focused this study on egg size. Though it is often reported as a fixed parame ter, egg size within species displays a large amount of variation (Jaeckle 1995, Ha dfield and Strathmann 1996). Considering marine invertebrates, intraspecific variation in mean egg size has been reported for the barnacle Balanus balanoides (Barnes and Barnes 1965). Turner and Lawrence (1979) found significant differences in egg size with in species when examining 11 echinoderms, and Lessios (1987) found this same result in a comparison of 13 echinoid species sampled across the Panamanian isthmus. The presence of significant variation in egg size has been reported within species for the starfishes Pteraster tessalatus (McEdward and Coulter 1987), and Solaster stimpsoni (McEdward and Carson 1987), for the polar shrimps Chorismus antarcticus and Notocrangon antarcticus (Clarke 1993), for the dorid nudibranch Adalaria proxima (Jones et al. 1996), for the ascidian Pyura stolonifera (Marshall et al. 2002), an d for the euphausiid Thysanoessa raschii (Timofeev 2004). Within and between-species variation in egg size was reviewed for serpulimorph polychaetes by Kupr iyanova (2001).

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6 Certainly there is abundant phenotypic variation in egg size, but only that portion that has a genetic basis is accessible to se lection in order to influence life-history evolution. In order for a trait to respond to natural selection it must have heritable variation, and that va riability must have fitness cons equences. What component of phenotypic variation in egg size is available to natural selection; what is the heritability of this trait? There is evidence of negative phenotypic trade-offs between fecundity and egg size in unmanipulated populations of a vari ety of species (Roff 1992). Examples are numerous among arthropods and include crustacean s, lepidopterans, dipterans, and others (Fox and Czesak 2000). The same trade-off ha s been recorded for treefrogs (Lips 2001); and Sinervo (1990) demonstrated a negativ e trade-off in phenotypically manipulated lizards. But, there is a limitation to conclusions about evolutionary response to natural selection that can be drawn from phenotypi c correlations. If the observed phenotypic trade-off does not represent some underlying genetic antagonism among traits, then it is not revealing the set of options available for selection on thes e traits. One way to detect this relationship is by using artificial selec tion experiments that can reveal short-term evolutionary responses for the selected trait. Using both th e selection and the manipulation approaches, Schwarzkopf et al. (1999) found no negative genetic correlation between egg size and fecundity in Drosophila melanogaster Azevedo et al. (1996) also found no evidence for a trade-off between egg size and fecundity in their laboratory-select ed lines of D. melanogaster. Blanckenhorn and Heyland (2004) found no evidence of a genetic tradeoff between egg size and number in the yellow dung fly Scathophaga stercoraria But, in another arthr opod, the cladoceran water flea Daphnia

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7 a genetic basis for this trade-off was shown (Lynch 1984, Ebert 1993). Levin et al. (1991) also confirmed a negativ e genetic correlation between fecundity and egg size in the poecilogonous polychaete Streblospio benedicti supporting the assumption of trade-offs between these traits. Goals of this study were to investigate the evolvability of the tr ait of egg size, to examine the direct and correlated responses to selection on increas ed egg size in a specified set of life-history characters that sp an the life cycle, and to explore how these correlations change as egg size is increased by artificial selection in the polychaete worm Hydroides elegans Haswell, 1883. Using adult H. elegans collected from the wild, spawned in the laboratory, and raised under common environmental conditions, variation in egg size was documented and heritability for this trait was estimated using a sib analysis (Falconer and Mackay 1996). Usi ng artificial selection for increased egg diameter, realized her itability was estimated (Hill 1972) a nd the results of selection were documented in a suite of life-history characters as they are reflected through the larval, juvenile, and adult stages. Life-history trai ts examined included egg diameter, fecundity, and total egg energy (at 6 w eeks post-fertilization), larv al size at 18 to 20 h post-fertilization, larval size at competence (5 d), tube length at 21 d, and adult dry weight (at 6 weeks post-fertilization).

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8 CHAPTER 2 ESTIMATES OF HERITA BILITY FOR EGG SIZE IN THE SERPULID POLYCHAETE Hydroides elegans Introduction Life histories represent compromises between selec tion on indivi dual fitness components and constraints acting on the orga nism as an integrated whole. Stearns (1992) suggested that the anal ysis of life-histor y strategies must be based on, and consistent with, the evolution of the individu al life-history traits. Few individual traits are more fundamental to life history than re source allocation to the egg. Egg size is directly related to parental fitness, and can also have substantial fitness effects for progeny (Roff 1992). Life histories in marine invertebrates are very diverse, and the adaptive significance of developmental mode has received consider able attention. One co mmon feature in the life histories of many invertebrates is an oblig ate feeding (planktotrophic) larval stage. Paradoxically, it can also often be observed within closely related groups that some members lack this dependence on exogenous food and exhibit lecithotrophic development. It has been hypothesized that nonfeeding larvae evolved as a response to either direct or indirect selection for increased egg size (Jgersten 1972, Strathmann 1978, 1985, Raff 1987, Hart 1996). Selection on egg size has also been linked to developmental mode (Hadfield and Miller 1987), larval developm ent time (Sinervo and McEdward 1988), larval form (Strathmann 2000) size at settlement (Strathmann 1985), survivorship (Bridges and Heppell 1996), a nd fertilization success (Levitan 2000).

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9 Vance’s (1973a,b) fecundity-time model rega rding the evolution of life histories in marine invertebrates began a series of quantitative modeling efforts focused on egg size as a key trait (Christiansen and Fenc hel 1979, Caswell 1981, Perron and Carrier 1981, Grant 1983, Roughgarden 1989, McEdward 1997, Levitan 2000, Luttikhuizen et al. 2004). While the quantitative modeling efforts regarding evolution of life histories in marine invertebrates have become increasingly refined, there has been little research into the genetic architecture of the important trai t of egg size in marine invertebrates. In order for a trait to res pond to natural selection it must have heritable variation, and that variability must have fitness cons equences. Numerous studies have found that egg size typically exhibits phe notypic variability across a wide range of taxa (Hadfield and Strathmann 1996). Ph enotypic variation ( VP) has several components including the genetic ( VG), the environmental ( VE), and the interaction between them ( VGXE). Unless phenotypic variation represents some underlying genetic varia tion, it is not heritable and is consequently unavailable to natural (or artificial) selection. VG can be further partitioned; the component that accounts fo r the resemblance between offspring and parents in sexually reproducing organism s is called additive genetic variance ( VA). The proportion of VP that is made up of VA is a quantity called narrow-sense heritability ( h2 = VA/ VP). This value is a measure of the re semblance between parents and offspring after sexual recombination and, therefore, is a predictor of short-term response to selection (Falconer and Mackay 1996). Among protostome invertebrates, mo st of the investigations into h2 of egg size have been focused on the Arthropoda (reviewed by Fox and Czesak 2000): especially the Insecta, a mostly terrestrial group that includes Diptera (flie s and mosquitoes),

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10 Lepidoptera (moths and butterflies), and Coleoptera (beetles), among others. Many experiments examined egg size in Drosophila melanogaster and demonstrated that there is a significant genetic com ponent to variation for this trait (Warren 1924, Bell et al. 1955, Azevedo et al. 1997, Schwarzkopf et al. 1999). Among lepidopterans, Fischer et al. (2004) estimates h2 for egg size of ca. 0.4 in the butterfly Bicyclus anynana and Harvey (1983) reports h2 for egg weight in the budworm Choristoneura fumiferana at 0.75. Fox (1993) estimates h2 of egg size for the seed beetle Callosobruchus maculatus at 0.43 to 0.59 and 0.60 to 0.74 using two diffe rent designs. Czesak and Fox (2003) calculate realized h2 for egg size in the seed beetle Stator limbatus of 0.36 to 0.55. Thus, there is abundant evidence of VA for egg size in insects, and estimates of h2 are generally moderate to large. The only marine invertebrate to receive similar attention was the poecilogonous polychaete Streblospio benedicti where a reciprocal mating design used to examine the genetic components of life-his tory traits gave a herita bility estimate of 0.75 for egg diameter (Levin et al. 1991). With the excep tion of this one study on a species with an unusual life history that incl udes producing both planktotroph ic and lecithotrophic larvae, empirical support for theoretical assumptions concerning the heritability of egg size in marine invertebrates has not been examined. The serpulid polychaete Hydroides elegans Haswell, 1883 is an excellent choice as a model organism for estimating h2 for egg size in a marine invertebrate for several reasons. It produces a large number of plankt otrophic larvae that can be cultured in the laboratory with relative ease. The larvae will settle and metamorphose in culture in the presence of natural biofilms (Carpizio-Itua rte and Hadfield 1998). The generation time is

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11 reasonably short, 16 to 28 days at optimal te mperature, salinity, and food concentrations (Qiu and Qian 1998). Both larvae and adult worms can be fed the chrysophyte alga Isochrysis galbana which is easily culture d in the laboratory. H. elegans develops in the water column with no maternal care. The objective of this study was to obtain es timates of narrow sense heritability for the trait of egg size in Hydroides elegans I calculated h2 using three different techniques. First, I constructed a half-sib breeding design and used a nested ANOVA for analysis (Falconer and Mackay 1996). Next Restricted Maximum Likelihood Analysis (Quercus Quantitative Genetics Software, Un iversity of Minnesota, USA) was performed on the same data set. Last, artificial sele ction on egg size was performed and cumulative realized heritability was cal culated according to the me thods of Hill (1972). Material and Methods Collection and Spawning This project was completed at Kewalo Marine Laboratory, Honolulu, Hawaii using protocols developed there by various workers. Wild worms were collected in November 2002 from Vexar screens suspended for 1 month from a floating dock at Ford Island, Pearl Harbor, Hawaii. The sex of each worm was determined and gametes secured by agitation of the calcareous tube surrounding each adult, resulting in release of gametes (Unabia and Hadfield, 1999). Gametes from 270 female worms were used to establish the initial laboratory population. At least 120 males were used, but the exact number of males was not determined. I used a Nikon Coolpix 990 camera mounted on an Olympus compound light microscope to obtain digital images of eggs from each female immediately after their

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12 release. Mean egg diameter for each mother ( n = 10 eggs mother-1) was obtained from these images using NIH Image software. Larval cultures were establis hed at densities of 5 to10 larvae mL-1 in 1L plastic tripour beakers and raised in pooled cultures (4 to 5 mothers and 2 to 3 fathers beaker-1) for Generations 1 through 3. Each beaker had different and unique parents. Cultures were maintained at 25 C, and fed 6 X 104 cells mL-1 day-1of the chrysophyte alga Isochrysis galbana (Tahitian strain). Isochrysis galbana was cultured in the lab at room temper ature and constant li ght, using f/2 media (Guillard 1975) and used duri ng exponential growth phase. All embryos were allowed to hatch in FSW (0.22 m filtered seawater) and swimming larvae were counted approximately 12 h post-fertiizat ion. At that time larval cu ltures were established at maximum densities of 10 larvae mL-1 and larvae were fed for the first time. Larvae were fed on Day 2 by adding the appropriate volume of algae to the larval culture beaker without changing water or beakers. On Da y 3 and Day 4 larvae were fed and both water and beakers were changed. Day 5 larvae we re considered competent to settle and metamorphose. Small transparent plastic chips (2.5 X 1.5 X 0.08 cm; K&S Engineering, Chicago IL, #1306) were placed in fl ow-through seawater tables for 10 days in order to accumulate natural biofilm, which has been shown to induce settlement in H. elegans (Hadfield et al. 1994). One biofilmed chip was placed in each well of a standard ice cube tray along with 15 mL FSW, I. galbana (6 X 104 cells mL-1), and 20 to 25 competent larvae. Larvae and newly settled juveniles were fe d daily at this food density without changing the water for 5 days. This proce dure allowed a wide window of opportunity for

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13 settlement and avoided inadvertent selection fo r early settling individuals. Ice trays were kept at room temperature (~24 C) in a Plexiglas rack on the bench top with ambient illumination. The position of trays within the rack was randomized daily. On Day 10 (post-fertilization), each chip was removed fr om the tray and all animals but one were removed. The survivor was determined by position on the chip, with that individual closest to the center selected to be the survivor. Thus, no inadvertent selection for early or late settling individuals, or for body size, t ook place during this process. At this point, the food level was increased 6-fold (36 X 104 cells mL-1) and worms were fed with the daily water change (FSW) until animals had reached 6 weeks of age. At maturity (here defined as 6 weeks), individual worms were plac ed in Petri dishes and spawned as before. H. elegans is a protandrous hermaphrodite. Sin ce I was measuring a maternal trait (egg size), I chose a generation time of 6 weeks in order to assu re that 50 to 60% of the spawning population would be female. Th ree generations were so raised under laboratory conditions to minimize variance due to maternal effects. Two hundred or more females were spawned to produce each of these two subsequent generations (217 in Generation 2 and 236 in Generation 3). Within Generation 3 a subset of individua l crosses was established to setup the breeding design experiment de scribed below. Alongside th e pooled cultures of mixed parentage described above, indi vidual larval cultures consis ting only of offspring from a unique mother crossed with a known father were raised under conditions identical to those described above.

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14 Half Sib Breeding Design A subset of the worms in Generation 3 was used as parents for the half-sib design experiment (Falconer and Mackay 1996) that was analyzed in Generation 4. Ten males were arbitrarily selected and their sperm wa s split between two females each (3 females in one case). The result of this mating sc heme is to produce offspring related in two different ways, that is, full-si blings (within females, or da ms) and half-siblings (within males, or sires). The advantage of this design is to allow the partitioning of the phenotypic variance according to its sour ce (the progeny of different males ( 2 SIRE), the progeny of different females mated to the same male ( 2 DAM), and individual offspring of the same female ( 2 WITHIN). The component of total vari ance attributable to sires is 1/4 VA, therefore the most straightforward estimate of h2 = 4 2 SIRE/ 2 TOTAL (Falconer and Mackay 1996). Parents and offspring involved in this breeding design were raised in individual and not pooled cultures, but otherwis e all conditions were as descri bed above. A fully nested Random Model ANOVA was then used to partitio n variance in egg diameter according to its source. Type III sums of squares were calculated using the general linear model of SAS Version 6.12 (PROC GLM, with RANDOM statement and TEST option). The variance component data were also us ed to calculate the additive genetic and residual coefficients of variation ( CVA = 100 VA / X and CVR = 100 VP VA / X where X = trait mean) as recommended by Houle (1992). Evolvability is de fined as the ability of a trait to respond to sel ection (Houle 1992) and, using the assumption of directional selection, can be calculated as IA = VA / X 2.

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15 I also used Restricted Maximum Likeli hood (REML) to analyze these same data, using the nf3 program in Quercus of R.G. Sh aw and F.H. Shaw (University of Minnesota, USA; downloaded November, 2004; www.cbs.umn.edu/eeb/events/quercus.shtml ). This program estimates the likelihood of observi ng the data given a set of parameters. Iterative methods are then used to find the set of parameters that maximize this likelihood (Shaw 1987). I used the program to perfor m REML on a two-genera tion pedigree with mean egg diameter of each Generation 4 daughter as the single character. The sources of total phenotypic variance were analyzed using the model VP = VA + VD + VE. In this model the genetic variance mentioned above (VG) is partitioned into two components, VA (additive) and VD (dominance). Log likelihood ratio tests were used to compare the fit of the model for significant differences ( 2 1) as successive variance components were constrained to zero. Technicall y, this violates the assumpti on of the likelihood ratio test because the null value of the parameter (i.e., variance = 0) lies on the boundary of the feasible range. However, the test is belie ved to be conservative under these conditions (Pinheiro and Bates 2000). Linear regression of mid-offspring egg di ameters onto maternal egg diameters was not used because, while each daughter had an independent mother, they were not independent with respect to the father. Therefore, the assumption of independence necessary for the linear regres sion could not be met. The family based design described he re for Generation 4 was repeated in Generations 5 and 6 in an attemp t to increase sample size.

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16 Selection In Generation 7 the common laboratory popul ation was split into six lines. Three were established as selected lines and thr ee as control lines. Between 12 and 16 females were used to establish each line (Table 2-1). In subsequent generati ons (Generations 8 to 10), 16 mothers were kept in each line. With in selected lines, the eggs from a specific mother were sub-sampled as described above Measurements were made immediately and eggs were fertilized only if the mean di ameter of the eggs was greater than one phenotypic standard deviation (P) over the Generation 6 population mean. In subsequent generations, selection proceeded in the same manner, with mothers remaining in the line only if the mean di ameter of their eggs exceeded 1 P over the mean of the selected mothers from the previous genera tion. If the mothers meeting this criterion happened to include multiple si sters I retained no more than two sisters in a line within a generation. The same rule, that no more than two sisters remain in a line, was also followed for control lines. In control lines, ev ery third or fourth female was fertilized without regard to the size of her eggs. Othe rwise, eggs were handled exactly the same as for selected lines. All crosses were between one male and one female with no shared parentage. Males were chosen haphazardly w ith respect to the mean egg size of the clutch from which they came. Care was taken not to use siblings of either selected or control females as fathers. Larval culture a nd settlement proceeded as before. Selection continued for four genera tions (7 through 10). Generally, realized heritability is the change in mean trait value over each generation of selection (offspring mean – parental population mean) divided by the selection differential (trait mean for selected parents – parental population mean). As

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17 selection was carried out in one direction onl y, and the selected a nd control lines came from the same base population, the realized heritability was calcul ated according to the specific methods described by Hill (1972). Cumulative realized heritability was calculated as the difference between selected and control values at each generation (Xi) multiplied by the cumulative selection differential for that generation (Si), summed over generations ( XiSi). This quantity is divided by the sum over the square of the cumulative selection differential for each generation ( Si 2). Results Expected mean squares and variance co mponents for the half-sib breeding design in Generation 4 are listed in Table 2-2. Na rrow-sense heritability calculated using the sire component of variance (P = 0.069) was h2 = 4 2 SIRE/ 2 TOTAL = 0.45. Confidence intervals were calc ulated based on the F-distribution, according to Knapp (1986), as recommended for small sample sizes (Hohls 199 8). The 95% confidence intervals were large (-0.36 to 0.90) as is often the case fo r CI for heritability from a breeding design (Koots and Gibson 1996, Markow and Clarke 1 997). The mean egg size of Generation 4 daughters used in the nested ANOVA, grouped by mother, was 45.68 m ( 0.21 m SE), CVA = 3.22 and CVR = 3.59. Evolvability for the trait of egg size for the Pearl Harbor population of H. elegans was calculated at IA = 10.35 X 10-4. Results from the breeding design experiments in Generations 5 and 6 did not result in an increase in sample size and in both cases the sire com ponent of variance was not significant. Results from the REML analysis on the Generation 4 data were not significant using likelihood ratio test s. The full model (VA, VD, VE unconstrained) gave a negative estimate for VD. Since this is not biologically meaningful, VD was constrained to zero.

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18 This produced a result not significantly di fferent than the unconstrained model (2 1 = 0.735; P = 0.45). The estimate of h2 based on this model was VA / (VA + VE) = 0.38. However, when VA was constrained to zero the l og likelihood ratios were still not significantly different (2 1 = 2.519; P = 0.13). Mean egg diameter ( m SE) before selection (G eneration 6) was 44.78 (0.15), and after selection the mean of the three selected lines was 49.23 (0.35). Direct selection on egg diameter for four gene rations produced a shift of 4.45 m or 2.5 P from the common base population mean (Figure 2-1). Figu re 2-2 illustrates the mean egg diameter ( m SE) in the base population before selec tion and among lines af ter selection began. Egg diameter data for selected Line 1 in Generation 7 were lost due to a corrupted computer file and therefore were not include d in calculations of r ealized heritability. Cumulative realized heritability was calculated using the method of Hill (1972) as 0.58. Bootstrapping based on re-sam pling with replacement, and stratified by generation and line, was used to calculate 95% confidence intervals around this es timate. Confidence intervals based on 1000 replicates were 0.51 to 0.66. Discussion There has been considerable interest in the evolution of life histories in marine invertebrates, and egg size has played a critic al role in many of the quantitative modeling approaches that have been used to try to identify selection pressu res and processes that influence the evolution of deve lopmental mode in this group. In order for evolution to occur, some component of phenotypic variati on in egg size must be additive genetic variation. My objective was to determine if the assumption of many models, that egg size can and does respond to selecti on, is reasonable by estimating h2 for a marine

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19 invertebrate with a wide ge ographic range, small egg, and obligate planktotrophic larvae. All estimates of heritability carry the constraint that they only apply to the population measured under those environmental conditions and are not specifically applicable to other populations (Falconer and Mackay 1996). I have shown that there is si gnificant additive genetic variation for egg size in the Pearl Harbor population of H. elegans using various analytical techniques. The response of 2.5 P to direct selection on egg size indicates th at there is substantial potential for egg size to respond to varying selective pressures, at least in the direction of an increase. It is apparent in Figure 2-2 that va riation within selected lines was not reduced relative to either values before selec tion, or to control lines. In a half-sib breeding design, offspring ha ve one parent in common and the other parent different. The degree of resemblance among half-sibs (the covariance of half-sibs) represents half the variance of the common parent or a quarter of the additive genetic variance (Falconer and Mackay 1996). The estim ate of heritability reported here (0.45) was based on the sire component of variance. One of the advantages of using the sire component for this estimate is that it doe s not include maternal or dominance effects (Falconer and Mackay 1996). It is possible to use this br eeding design to estimate these effects by examining the variance within dams ( 2 DAM), since it includes both maternal (common environment) and dominance effects, and comparing it to the estimate based on the sire component. However, since my estimate of 2 DAM was not statistically significant (P = 0.667), and the proportion of VP explained by this estimate was essentially zero (Table 2-2), further extrap olation based on this estimate cannot provide useful information. Maternal effects are wi dely recognized as important components of

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20 Vp (Mousseau and Fox 1998), although this is more common in mammals and animals with longer periods of maternal care. Hydroides elegans has external fertilization. All development up to a competent larva takes pl ace in the water column and there is no evidence of parental care. However, it is possible that the small sample size in my breeding design (10 sires, 21 dams) resulted in an overestimate of 2 SIRE, obscuring my ability to detect 2 DAM and estimate maternal effects. Therefore, based on these data, I cannot say whether maternal effects are contri buting significantly to the total phenotypic variance. Cumulative realized heritability is consid ered the most precise of the various methods for determining narrow-sense heritabi lity (Hill 1971). The cumulative realized heritability estimate does include maternal effects, since it is based on response to selection, but passing six genera tions under laboratory conditi ons prior to the beginning of selection minimized the phenotypic expression of any maternal effects. The half-sib breeding design gave only a slightly lower estimate of h2 (0.45) compared to the cumulative realized heritability (0.58). The f act that two estimates are similar lends some support to the idea that matern al and dominance effects are not large in this population with respect to this trait. It is thought that additive ge netic variation for traits clos ely related to fitness, such as egg size, should be under consta nt directional selection, eroding VA and resulting in low heritabilities (Gustafsson 1986, Roff and Mousseau 1987, Falconer and Mackay 1996). This trend is genera lly supported for life-history traits as compared to morphological traits (Mousseau and Roff 1987). However, many exceptions can be found (e.g., Levin et al. 1991, Gibson 1993, Fox and Csezak 2000, Edmands 2003),

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21 including this work. Houle (1992) pointed out that because h2 is a ratio of VA to VP, the magnitude of VP greatly influences the outcome: low h2 could be the result of low VA or high VP. Therefore, it is a biased estimate of additive genetic variance and potentially a misleading indicator of the abil ity of a particular trait to respond to selection. Using coefficients of variation, that is VA standardized by the tr ait mean rather than VP, is recommended as a more useful approach. My estimates of CVA and CVR give very similar values (3.22 and 3.59 respec tively) implying that nearly half of the total variation present is additive genetic. This is consistent with my estimate of h2. Using the traditional methods of estimating h2, and also by calculating CVA and IA, I have found significant levels of additive gene tic variance for the tra it of egg size in the Pearl Harbor population of H. elegans. Several hypotheses have been offered to explain the apparent paradox of finding VA for a life-history trait that is closely linked to fitness and, presumably, under directional selection. One such hypothesis is the presence of microevolutionary trade-offs. Microevolutiona ry trade-offs occur when a change in one trait that acts to increase fitness is linked to a change in another trait that decreases fitness (Stearns 1992). These relati onships can act to constrain the simultaneous evolution of suites of fitness-related traits and thereby maintain additive genetic variance in a population. Examination of the correlated res ponses of other fitne ss-related traits to artificial selection for incr eased egg size is one way to elucidate this relationship. Table 2-1. Number of females used to esta blish six individual lines in Generation 7 Line Number of Females 1 12 2 12 3 15 4 15 5 16 6 16

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22 Table 2-2. Half-sib breeding design; Fu lly nested ANOVA and variance component analysis for egg diameter in Hydroides elegans (number of observations 550, s = 10, d = 2.1, k = 2.6) Source df EMS (X 10-5) F P Proportion VP explained Sire 9 4.149 16.54 0.069 11.13 Dam (Sire) 11 1.575 6.28 0.667 0 Daughter (Dam (Sire)) 34 2.047 8.16 0.0001 37.11 Error 495 0.251 --51.75 0 5 10 15 20 25 30 35 40 45 40414243444546474849505152535455 Egg diameter (m) Generation 6 Generation 11 Figure 2-1. Response to direct selection on egg diameter for four generations of in the polychaete Hydroides elegans. Selection began from a common base population in Generation 6 and conti nued from Generation 7 through 10

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23 43 44 45 46 47 48 49 50 51 Gen 3Gen 4Gen 5Gen 6Gen 7Gen 8Gen 9Gen 10Gen 11 Generation Selected Line 1 Selected Line 3 Selected Line 5 Controls Line 2 Controls Line 4 Controls Line 6 Common Population Figure 2-2. Mean egg diameter ( SE) before and after direct sele ction for increased egg diameter in replicate selected (1,3,5) and control (2,4,6) lines of Hydroides elegans

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24 CHAPTER 3 DIRECT AND INDIRECT RESULTS OF ARTIFICIAL SELECTION FOR INCREASED EGG SIZE IN TH E SERPULID POLYCHAETE Hydroides elegans Introduction Marine invertebrates exhibit a wide diversity of developm ental strategies and their mode of development is thought to have im portant effects on dispersal and population structure (e.g., Hedgecock 1982, Levin and Huggett 1990, McMillian et al. 1992, Hoskin 1997, Collin 2001), geographic range (Scheltema 1989, Kohn and Perron 1994, but see Emlet 1995), and rates of speciation and extinction (Strathma nn 1985, Jablonski 1986, 1987). Egg size has been the focus of a series of quantitative modeling efforts to analytically assess the patterns we observe in marine invertebrate life histories. Beginning with Vance’s influential fec undity-time model (Vance 1973a,b), numerous authors have examined trade-offs between egg size, fecundity, mortality and their relationship with the evolution of different reproductive strategies (Christiansen and Fenchel 1979, Caswell 1981, Perron and Carrier 1981, Grant 1983, Roughgarden 1989, McEdward 1997, Levitan 2000). Another set of models examines fertilization kinetics, which also can pose selective pressures on egg size and thus developmental mode (Levitan 1993, 1996, Luttikjizen 2004). Many authors have emphasized that the suite of characteristics that defines a larval type has evolved in conjunction with post-larval and adult characteristics, rather than in isolation. Egg size and fecundity are two members of a much larger complex of life-history traits that has co -evolved, presumably under the in fluence of natural selection

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25 (Havenhand 1995, Ramirez Llodra 2002). E gg size and fecundity are both directly related to parental fitness (Roff 1992, Bern ardo 1996) and many authors have mentioned the importance of interactions between larval characteristics and ot her parts of the life cycle (e.g., Vance 1973a,b, Christiansen and Fenchel 1979, Todd and Doyle 1981, Strathmann 1985, Moran 1994, Pechenik et al. 1998, Gimnez et al. 2004). Few efforts have been made to examine the implicatio ns over the entire life cycle of selective pressures on these important and inter-related fitness components. Much of life history theory is based on the assumption of trade-offs among traits that contribute to fitness, that is, th at natural selection cannot produce unlimited simultaneous increases in individual fitness components. This could result in creating theoretical optima based on trade-offs between fitness components (Roff 1992, Stearns 1992). Fitness components, and therefore trad e-offs between them, must have a genetic basis for selection to act. Alleles that increase fitness w ithout a cost are expected to achieve a frequency of one very quickly; therefore genetic correlations between fitness-related traits that ha ve been subject to simultaneous selection are expected to become negative. Several methods are in use to examine th e genetic architecture of fitness-related traits. Genetic correlations may be estimated by examining the resemblance between relatives using designs similar to those for estimating heritabilities (Falconer and Mackay 1996, Lynch and Walsh 1998). These correlations are caused by pleiotropy, where traits of interest are influenced by common gene s; however, they may also be caused by linkage disequilibrium between distinct loci, each influencing a different trait. But genetic correlations between life-history tra its can be difficult to extrapolate from

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26 breeding designs. Correlations estimated in this manner are dependent on linkage equilibrium and other requirements (Rose et al. 1990), and standard errors around these estimates are often very la rge (Koots and Gibson 1996). A second, and more direct method that can be used to look for evidence of evolutionary trade-offs is ar tificial selection. Artificial selection experiments allow workers to manipulate the frequency of allele s associated with a phenotypic value of a selected character by controlling the parental genetic contribution to the next generation in laboratory populations, thereby producing di vergent phenotypes. If selected lines differ from control lines in traits other than the selected trait (and environmental variation has been kept constant across generations), this response indicates an additive genetic correlation between the traits (Cheverud 1984). Repeated episodes of selection over multiple generations are more likely to reveal potential interactions between fitness components that may be subtle or difficult to measure across a single generation. Artificial selection experiments are a partic ularly powerful way to explore trade-offs (Reznick 1985, 2000, Brakefield 2003). However, selection experiments are subject to experimental or procedural ar tifacts that may be difficult to control, and the complexity and interactive nature of response to sele ction can produce varying outcomes (Harshman and Hoffman 2000). Selection experiments on life-history tr aits have a long history, with the preponderance of work in invertebrates involvi ng insects. For example, Englert and Bell (1969, 1970) selected on larval deve lopment time in the flour beetle Tribolium casteneum, Roff (1990) selected on wing dimorphism (an indicator of development time) in the sand cricket Gryllus firmus, Bradshaw and Holzapfel (1996) selected on

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27 development time in the pitcher-plant mosquito Wyeomyia smithii, and Palmer and Dingle (1986) selected on wing length (an indicat or of dispersal ability) in the milkweed bug Oncopeltus fasciatus. Focusing on correlated responses in egg size, Tucic et al. (1998) and Seslija and Tucic ( 2003) found that bean weevils, Acanthoscelides obtectus, selected for faster development times lay sign ificantly larger eggs. A similar correlated response in egg size to selection on development time is reported for Drosophila melanogaster by Bakker (1969). Selection experime nts involving life-history traits in D. melanogaster are numerous, especially focusing on trade-off between longevity, fecundity, and larval development time (e .g., Tantawy and El-Helw 1966, Partridge and Fowler 1992, Zwaan et al. 1995, Nunney 1996). Several studies have examined response to selection on egg size. A significant response to selection toward both large and small eggs has been reported in Drosophila melanogaster in several studies (Be ll et al. 1955, Parsons 1964, Sc hwarzkopf et al. 1999). Azevedo et al. (1997) found evidence of a selective advantage to larger eggs in D. melanogaster, including increased embryonic viabilit y, hatchling weight, and larval and pre-adult development rates. Czesak and Fox (2003) selected directly on egg size in the seed beetle Stator limbatus and report correlated respons es in lifetime fecundity and female body mass. Among marine invertebrates genetic correlati ons for selected life-history characters have been reported using breeding designs. In mollusks, Ernande et al. (2003) report a positive correlation between reproductive plasticity, growth and survival for the bivalve Crassostrea gigas. Hilbish et al. (1993) discuss th e common assumption that variation in larval and juvenile growth rates have a common genetic basis, but find no significant

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28 correlation between variation for larval and juvenile shell growth in the bivalve Mercenaria mercenaria. Gibson (1993) explored gene tic correlations among life-history traits in the poecilogonous opisthobranch Haminoea callidegenita and found that many reproductive traits showed significant genetic correlations while exhibiting little or no phenotypic correlations. In crustaceans, Arcos et al. (2004) estimate the genetic basis for a series of reproductive tr aits in the white shrimp Penaeus vannamei and report no correlation between egg diameter and other traits, but did find a positive correlation between egg number and egg total protein. Le vin et al. (1991) inve stigated the genetic architecture of the poecilogonous polychaete Streblospio bendicti using a diallel design and confirmed a negative genetic correlati on between egg size and fecundity, as well as the expected positive genetic correlation be tween fecundity and female body size. Ernande et al. (2004) used a nested half-sib mating design to examine five traits in Crassostrea gigas: larval shell length, size at set tlement, weight after metamorphosis, juvenile weight, adult weight, but did not ex amine egg size. They report that larval development rate is positively genetically correlated with size at settlement, but negatively correlated with both metamorphic suc cess and juvenile survival. Little work has been done to examine the consequences acro ss the life cycle of shifts in egg size. The purpose of this work was to examine the direct and correlated results of artificial selection to increase egg size in Hydroides elegans Haswell, 1883. H. elegans is a marine, serpulid polychaete worm that produces numerous plankt otrophic larvae and can be cultured in the laboratory over multiple generations. Larvae will readily settle and metamorphose in culture in the presence of natural biofilms (Carpizio-Ituarte and Hadfield 1998). Fertilization and larval de velopment takes place in the water column

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29 with no maternal care and H. elegans develops as a protandrous hermaphrodite (Ranzoli 1962). The generation time is 16 to 28 days at optimal temperature, salinity, and food concentrations (Qiu and Qian 1998). Both larvae and adult worms can be fed the chrysophyte alga Isochrysis galbana, easily cultured in the la boratory. Using artificial selection for increased egg diameter, correlate d responses were documented in a suite of life-history characters: egg diameter, fecundity, total egg energy (at 6 weeks post-fertilization), larval si ze at 18 to 20 h post-fertiliza tion, and at competence (5 d), juvenile tube length at 21 d, and adult dry weight (at 6 weeks). Methods Collection, Spawning, and Selection Wild Hydroides elegans were collected at Ford Is land, Pearl Harbor, Hawaii in November 2002. Details of the methods for spawning, larval cultu re, measurement of egg diameter and selection are described in detail in Chapter 2. Field collected worms were raised under controlled environmenta l conditions for six generations before selection began in Generation 7. Between 12 an d 16 females were used to establish each line in Generation 7 (Table 2-1). In subse quent generations (Generations 8 to 10), 16 mothers were kept in each line. All crosse s were unique and the fathers were chosen haphazardly without regard to the mean egg size of the clutch from which they hatched. No crosses were permitted between brothers and sisters within lines. Three replicate control and three replicate selected lines we re established with 128 worms in each line, for a total of ~768 worms in each generati on. All females were spawned at 6 weeks post-fertilization and females were retained in the selected lines if the mean diameter of their eggs was 1 than that of the previous genera tion. For control lines, every third female spawned was retained in wi thout regard to egg diameter.

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30 Larval culture was performed according to methods described by Unabia and Hadfield (1999). Cultures were maintained at 25 C, and fed 6 X 104 cells mL-1 day-1of the chrysophyte alga Isochrysis galbana (Tahitian strain). La rvae were competent to metamorphose at 5 d post-fert ilization, when I exposed 20 to 25 larvae to individual small plastic chips with natural biofilm as the se ttlement cue. Each chip was isolated in a single well of a standard ice cube tray filled with 15 mL of 0.22 m filtered seawater (FSW) and algal food was maintained at 6 X 104 cells mL-1 day-1. On Day 10 all juveniles but the one nearest th e center of the chip were killed by destroying the tube, so that growth from that point took place at a density of 1 worm well-1. Food density was increased 6-fold and water, food, and contai ner (ice tray) were all changed daily until adults were spawned. I observed that some female worms in both selected and control lines spontaneously released a few eggs (from 10 to a few hundred eggs) shortly after the daily food and water change. I coll ected all the early-spawned e ggs that were produced after the daily food and water change over a fourday period (October 10 to 14 2003) prior to the scheduled spawning of Generation 9 using a Pasteur pipet. There were 25 selected and 7 control females (n = 32 females) that produced early-spawned eggs over this four-day period. I began the scheduled spawning of these same females two days later. Therefore, the timing of the sample of early-s pawned eggs ranged 6 to 12 d prior to the scheduled spawning date for these individuals. I used digital imagery (Chapter 2) to measure mean egg diameter of these early-spawned eggs (n = 10 eggs female-1) and compared this to mean egg d measured at the time of induced sp awning (later-spawned eggs) of the same female.

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31 Selection continued for four generations with lines terminated at Generation 11, when adult dry weight, total egg energy, fecundity and final egg diameter were determined. Larval size at two different ti me points, and juvenile tube length were measured in Generation 10, as no larvae or juve niles were available after termination of the lines. Larval Size At 18 to 20 h post-fertilization sub-samp les of trochophore-stage larvae were taken from nearly all of the crosses (between 11 and 16 of the 16 crosses in each line) in Generation 10. Any crosses for which I ha d not obtained video by the time they had developed to 20 h were not sampled. Larvae from a single cross were placed on a clean slide and excess water was removed with a micropipette. A cover slip was placed on each slide using Scotch tape to elevate the slip above the slide. Using a technique similar to that used by Leonardos and Lucas (2 000) for bivalve larvae, videotapes of the swimming larvae were obtained using a S ony Digital Video Camera Recorder (Model DCR-TRV30) mounted on an Olympus light mi croscope. A stage micrometer was used as a scale for each series of shots. Videos were downloaded to a Macintosh G4 where iMovie software was used to grab still photos of larvae (n = 10 larvae cross-1). NIH Image software was used to measure the wi dth across the prototroch, and the distances from the prototroch to the apex of the episphere and hypos phere, respectively. Larval size was then calculated as the volume of two circular cones (V = r2h/3; r = 1/2 prototroch width and h = distan ce to apex of episphere or hyposphere). The same process was repeated for competent larvae at 5 d pos t-fertilization, includi ng between 9 and 15 of the 16 crosses in each line (n = 10 larvae cross-1) in Generation 10.

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32 Juvenile Tube Length Shortly after metamorphosis a calcareous t ube is constructed by the juvenile. The tube lengthens as the worm grows, and tube length is a reasonably reliable indicator of juvenile size (Qian and Pechenik 1998, Qiu and Qian 1998). This indirect method of measuring juvenile size was chosen to minimi ze loss of individuals from laboratory lines, since removal from the tube results in deat h. Images of juvenile tubes at 21 d were obtained using the Nikon Coolpix 990 described above, attached to an Olympus dissecting microscope. Each i ndividual chip was removed from the ice tray and placed in a plastic Petri dish along w ith a 1cm length of ruler. Images were obtained for all offspring (n = 8 juveniles cross-1) from between 10 and 16 crosses from each line in Generation 10. The images were analyzed fo r tube length using NIH Image software. Total Egg Energy In Generation 11 each sub-sample that had been used to measure egg diameter was placed in a cryotube (FisherBrand 0.5 mL screw-top tube) and frozen at -20 C. Frozen samples from each line were later an alyzed for organic content using a micro-modification of the dichromate oxida tion technique (Jarrett and Pechenik 1997, Gosselin and Qian 1999) against a glucose standard (0 to 20 g C). Eggs were thawed at room temperature a nd distilled water was added to raise the volume to 200 L. Four replicate sub-samples of 10 L were obtained and counted on a Petri dish using a dissecting microscope and a hand-counter. This al lowed calculation of the total number in the sample that was needed to complete the fecundity estimate (see below). The counted samples were removed fr om the Petri dish using a micropipette and placed in a 13mm glass test tube. Between 300 and 500 eggs were placed in each of

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33 three replicate tubes from a particular cros s. Sixteen samples, each corresponding to a different randomly selected cross from each lin e, were prepared. Tubes were allowed to stand until the eggs had settled to the bo ttom and excess water was removed using a micropipette. The eggs were incubated in 1 mL concentr ated (85.5%) phosphoric acid (15 min at 105 C) to remove excess chloride. Tubes were cooled to room temperature and 1 mL of 0.04% acid dichromate was added to each tube to oxidize the sample. Tubes were mixed on a vortex mixer followed by incubation at 105 C for 15 min. Reduction in dichromate concentration indicates the amount of organic carbon oxidized. Samples were diluted by adding 5 mL dist illed water followed by vortex mixing. A 0.5 mL aliquot was removed from each tube and combined with 4.5 mL of cadmium iodide starch reagent (Parsons et al. 1984) a nd allowed to stand at room temperature for 20 min. Each sample was then diluted again by adding 5 mL distilled water and examined spectrophotometrically (Kontron In struments, Model Uvikon 930) at 575 nm. Total energy was estimated as g C equivalents in glucose and converted to mJ using the relationship 1 g C = 39 mJ (McEdward and Carson 1987). Though widely used in comparative studies of egg energy in marine invertebrates, the dichromate oxidation t echnique is known to underest imate energy contributed by proteins (Gosselin and Qian 1999). Pernet and Jaeckle (2004) analyzed this technique across species within an nelids and echinoderms and conclude that the likely cause is that different proteins oxidize to different degrees in the aci d dichromate reaction. Their comparison points out that across a wide range of species, developmental modes, and egg sizes there is probably a wide range of differential alloca tion of various proteins and

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34 because of this the absolute value of energy egg-1 may be underestimated using this assay. Here I used the acid dichromate tech nique for a relative comparison of mean total energy egg-1 within species, over a small range of egg sizes after short-term selection. Data presented by Pernet and Jaeckle (2004) fo r planktotrophic annelids show that error of total energy estimates based on this techni que within species is generally small (e.g., estimates for serpulid polychaetes include Serpula columbiana with 0.65 mJ egg–1 0.015 SE, or 2.3% and Hydroides sanctaecrucis with 0.40 mJ egg–1 0.023 SE, or 5.8%) and therefore unlikely to be important over the small changes in egg size considered here. There are two possible ways that an increase in er ror could affect my results. First, in the unlikely event that short-term selection on e gg size resulted in a change in the type of protein allocated to eggs of selected lines relative to control lines, error could be increased. Second, if selection produced increas ed densities of a particular protein in larger eggs, this could affect the error. E ither of these would be an interesting outcome and should be considered by future workers us ing techniques specific to these questions. Absolute values of energy estimates based on this technique may be underestimates. Fecundity In Generation 11, after the sub-sample of eggs for measurement and total energy oxidation were removed, the remaining eggs from each female (n = 435) were fixed in 5% formalin in FSW (~2% formaldehyde). These samples were set aside and eggs were counted later. The samples were diluted to 30 mL using filtered seawater and a standard 50 mL graduated cylinder. The sample wa s mixed by inversion and 4 sub-samples of 250 L each were placed on a Petri dish and counted using a dissecting scope and a hand counter. The number of eggs in the fixed sa mple was then calculated. The number of

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35 eggs from the frozen samples (see above) was ad ded to this number to obtain the absolute number of eggs produced by each female. Th e relative fecundity was also calculated by dividing the absolute number of eggs per fema le by her dry weight (s ee below) to give eggs mg-1. Adult Dry Weight The dry weight of each adult in Generation 11 (n = 685) was determined. Small squares of aluminum foil were numbered and pre-tared on a Cahn (Model C-35) micro-balance. Immediately after spawni ng sex of the worm was recorded, and each individual was removed from the calcareous t ube using forceps to crack the tube. Worms were rinsed in distilled water and placed on aluminum foil squares. Specimens were placed in a drying oven at 75 C for 48 h. Worms were re-weighed on the Cahn balance to the nearest 0.1 g and the weight of the foil was subtracted providing a post-spawn weight for each individual. Data Analysis I used a fully nested mixed model ANOV A with fixed effects at the level of treatment only with lines nested within treatm ents and crosses nested within lines for the traits egg diameter, total egg energy, larval sizes at 18 to 20 h and at 5 d, and juvenile tube length (all nested at all levels between selected and control lines). Data were visually checked for radical departures from normality and heteroscedasticity and none were found. For the analysis of adult dry we ight and fecundity the same fully nested mixed model ANOVA was used with fixed effect s at the level of treatment only and lines nested within treatments. In order to test for an effect of se lection on the relative numbers of males and females across generati ons I used a model I ANOVA. In all cases

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36 Type III sums of squares were calculated using the general linear model of SAS Version 6.12 (PROC GLM, with RANDOM statement and TEST option), = 0.05 unless otherwise stated. To compare mean egg d be tween earlyand later-spawned eggs within mothers I used a paired t-test in JMP 5.1 (SAS Institute, Inc.). Results Direct Response to Selection Mean egg diameter in the base population (Generation 6) was 44.78 m ( 0.15 SE); the mean egg diameter in the selected lines after selecti on (Generation 11) was 49.23 m ( 0.35 SE). Direct selection for four gene rations for increased egg size resulted in a shift of 4.45 m or 2.5 P from the common base populati on mean (Figures 2-1, 2-2). Figure 3-1 illustrates the continued positive slope of the cumulative response to selection through Generation 11 as the difference between the mean egg diameter of the replicate selected and control lines. Egg diameters between selected and control lines were significantly different at Generation 11 (P = 0.0006, Table 3-1). Correlated Responses to Selection Replicate lines selected for larger egg di ameter were found to have a significantly higher mean value for total energy relative to the control lines (P = 0.0350, Table 3-2). The mean value (mJ egg-1 SE) for the selected lines wa s 0.37 (0.17) and for the control lines the mean was 0.29 (0.18). Fecundity, calculated as the absolute numb er of eggs produced by a female ( SE), was significantly higher in the large-egg selected lines relative to controls (P = 0.0304, Table 3-3). Female worms in the large-e gg lines produced a mean of 10071 (481) eggs and females in control lines produced a m ean of 7936 (418). When egg number was

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37 standardized by the dry weight of the mother to give eggs mg-1 the difference between the selected and control line means became less apparent, significant at = 0.10 (P = 0.0679, Table 3-4). Both of these analyses were also performed on datasets omitting all mothers that had produced eggs prior to the i nduced spawning at 6 weeks (see Spontaneous Spawning below) and the outcome was not changed. No significant difference was found in th e dry weight of adult females from selected lines relative to control lines (P = 0.6567, Table 3-5). The mean dry weight (mg SE) for females in the selected lines was 0.80 (0.019) and for controls was 0.82 (0.026). The same result was found if males and females were combined within treatments (P = 0.5791), and for males alone (P = 0.8526). The Gene ration 11 mean dry weight of adults of both sexes from the selected lines was 0.81 (0.025) and from the control lines this value was 0.83 (0.020). Males could not be distinguished from females on the basis of dry weight w ithin either the control (P = 0.1974) or selected (P = 0.1698) lines. No significant difference was detected in larval size (volume) at 18 to 20 h between selected and control lines (P = 0.3392, Table 3-6). The mean value for larval size ( L SE) at 18 to 20 h for the largeegg selected lines was 1.12 X10-4 (0.064 X 10-4). The control lines had a mean value of 1.14 X10-4 (0.047 X 10-4). Larval size (volume) at comp etence was significantly larger in the selected than in the control lines at = 0.10 (P = 0.0894, Table 3-7). The mean size at competence in the selected lines size ( L SE) was 1.29 X 10-3 (0.041 X 10-3) and for the control lines it was 1.16 X 10-3 (0.036 X 10-3).

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38 Selected lines did not significantly differ fr om control lines in tube length at 21 d (P = 0.1249, Table 3-8). M ean tube length (mm SE) was 8.08 (0.31) in the selected lines and 8.60 (0.10) in the control lines. Direct and correlated responses to sel ection for increasing egg diameter are summarized in Table 3-9. Spontaneous Spawning and Sex Ratios In an effort to explore the relationship between egg diameter and time of spawning, I compared the mean egg diameter from spont aneously or early-spawned females to that of eggs produced at 6 weeks by the same female (n = 32) in Generation 9. The later-spawned eggs were significantly larger than the early-spawned within individual mothers (paired t-test, t = 9.8582, P < 0.0001). In an unexpected development, I observed th at females in the selected lines tended to spawn spontaneously more frequently and earlier than control females. Timing and frequency of these events was not recorded since it was not part of the experimental design. Within this design I controlled the absolute age of indivi duals at spawning (6 weeks), but not the time to sex change. At 6 weeks an individual female might have been producing eggs for variable periods of tim e. Considering that within mothers later-spawned eggs are significantly larger th an earlier-spawned eggs was it possible that large-egg selected mothers had been produci ng eggs longer than control mothers? If selected lines were changing sex earlier than control line s, one prediction would be a higher proportion of females in se lected lines relative to cont rols at 6 weeks. Since the sex of each individual was record ed at the time of spawning fo r all generations I was able to document an effect of selection on sex rati os, showing that the proportion of females at

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39 6 weeks increased in the select ed lines, relative to controls across Generations 7 through 11 (P = 0.0122, Table 3-10). The mean percentage of females in selected lines ranged from 64.70% to 74.30% and for control lines the range was 59.21% to 64.21% (Table 3-11). H. elegans has been described as a sequential hermaphrodite in all accounts since Ranzoli (1962) clarified their status as herm aphrodites, and I also found this condition in all control lines (as well as in thousands of individuals ra ised under labora tory conditions prior to selection). However, in all 3 replicate selected lin es on at least one occasion I observed simultaneous eggs and sperm bei ng produced by the same individual (again, spawned at 6 weeks). Discussion More + Bigger = Better… or Does It? I predicted that there would be negative co rrelations between at least some fitness related traits, because we exp ect alleles underlying advantag eous genetic correlations to be rapidly fixed in natural populations. Ye t I found that large-egg selected females produced more eggs, with higher energy cont ent, that grew into larger larvae at competence. Increased size at settlement is considered a selective advantage (reviewed by Pechenik 1999). No trade-off with this apparently advantageous condition (the production of more energy-rich eggs that grow into larger competent larvae) was immediately evident by evaluating the initial trai ts of interest. This set of apparently advantageous correlated traits raises the question what other traits offset the selective advantage of larger egg si ze in natural populations? One of the advantages of using a selec tion experiment to explore evolutionary processes is that correlated re sponses to selection can reve al unexpected relationships

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40 between traits that were not predicted at the start of the experiment (Harshman and Hoffman 2000). The observation that sel ected worms appeared to produce eggs spontaneously both earlier and more frequen tly than control lines prompted me to examine the relative numbers of males and females across generations and I found that direct selection for increased egg diameter produced significantly higher proportion of females at 6 weeks of age. Considering that I also found that egg diameters are significantly larger in females that spawn late r than those that spaw n earlier, I propose that selection for increased egg size was manifest through selection for earlier sex change. This earlier sex change resulted in selected individuals spending less time as males relative to controls, consequently re ducing the number of offspring they could potentially father. Sex allocation theory predicts that sex change (sequential hermaphroditism) is favored when an individual experiences diffe rential reproductive su ccess in relation to size (or age). According to the size-adva ntage hypothesis sele ction should favor protandry if male reproductive success does not increase as indivi duals grow larger, while female reproductive success does (Ghi selin1969). There has been broad empirical support for this hypothesis (e.g., Charnov 1979a for the protandrous shrimp Pandalus, Warner 1984a,b for protogynous coral reef fi shes, Berglund 1990 for the protandrous polychaete Ophryotrocha puerilis). The optimal timing of sex change within individuals is also predicted by the m odel (Charnov 1979b, 1982). Sex cha nge should theoretically occur when an individual’s reproductive va lue (future expecta tion of reproductive success weighted by the probability of survival) as a member of its present sex declines below the reproductive value of the opposite sex. Alterations between male versus

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41 female investment should only be favored if the gain in reproductive value in one sex exceeds the loss in the other sex (Leigh et al. 1976). An individual should change sex if it can increase its lifet ime reproductive value by doing so, an d should change at that point where the sum of the male and female components is maximized. The earlier sex change that resulted as a correlat ed response to selection in H. elegans could reduce the contribution of the male component to lifetime reproductive value to such an extent that the female component cannot compensate for th e loss. This assumes that males are not able to shift their own sexual maturity earlier, or at least not sufficiently earlier to make up for the loss in reproductive fitness re sulting from terminating sperm production earlier. Earlier sex change could occur as a result of male and female gametogenic processes being controlled by different and unlin ked genes, that is, a response that shifts the timing of investment in oogenesis manipulates a different suite of genes entirely than those that control the timing of spermatogene sis. It is likely that the spermatogenic pathway has already been selected to functi on at the earliest possibl e age, and therefore smallest size, because H. elegans is an opportunistic poly chaete, and among the first settlers into disturbed environments. Maternal biosynthesis, mobilization and bioaccumulation of nutrients during oogenesis is an energy intensive activity and it is reasonable to assume that there is a cost invo lved in pushing this activity to an earlier time in the life cycle until it nearly, or actually, overlaps with sperm production. Therefore, I propose that the constraint on in creased egg diameter (at 6 weeks) in this population is selective pressure to maintain the optimum male c ontribution to lifetime reproduction. One question that follows on this reasoning is, if it is co stly to change sex early, then how is variation for the trait of “t ime to female” maintained in the population?

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42 One possible explanation is frequency-dependent selection, that it is very profitable to be an early sex-changing female when the trait is rare (and everyone else is male), but becomes less profitable, even costl y, as the frequency increases. Though it is widely reported that sequentia l hermaphrodites change sex as their size increases, field studies commonly report extens ive overlap between male and female size classes (Wright 1989, Sewell 1994, Collin 1995). Ranzoli (1962) surveyed field populations of H. elegans (as H. norvegica) over a three year period using segment number as an indicator of size and also f ound considerable overlap between the sexes. Population studies necessarily include overla pping generations; however, even with the discrete generations raised here I observed no significant difference in somatic dry weight between the sexes. This variation in size at 6 weeks may indicate significant genetic variation in maximum body size, or possibly in growth rate in this population. Many of the males that remained in both selected and control lines at 6 weeks were quite large and the mean dry weight for males was larger (although not signif icantly so) in both treatments. Given the positive correlation between egg size and egg number reported here, one may ask how often, and under wh at conditions the predicted trade-off between egg size and egg number has been empirically demons trated. Varying environmental conditions can result in an interaction between genotype and environm ent (G X E) and reveal a trade-off. Czesak and Fox (2003) found that the magnitude of the trade-off between egg size and fecundity varied between e nvironments in the seed beetle Stator limbatus. Varying food levels revealed a trade-off betw een increased fecundity and mortality in the polychaete Dinophiilus gyrociliatus (Prevedelli and Zunarell i Vandini 1999, Prevedelli

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43 and Simonini 2000). Tatar et al. (1993) nicely demons trate the trade-off between increased egg production and increased age-specific mortality in the beetle Callosobruchus maculatus. A longstanding idea concerni ng the evolution of offspring number is the “Lack clutch size” hypothesis in birds, that is, that clutch size has evolved towards the size that will ma ximize the number of successful fledglings. Empirical tests of the “Lack clutch”, and deviations from it, continue to produce mixed results. For example VanderWerf (1992) used meta-analysi s on data from 77 different ornithological studies and found that the hypothe sis was not supported. Frequently cited examples of the egg size-egg number trade-off in marine invertebrates are poecilogonous species such as the polychaetes Streblospio benedicti (Levin and Creed 1986, Levin et al. 1987) and Capitella sp. (Qian and Chia 1991) that have symp atric populations cap able of producing either planktotrophic or lecithotrophic larvae. The reproductive output of both strategies is similar but lecithotrophs produce fewer, larger eggs while pl anktotrophic populations produce smaller, more numerous eggs. The negative genetic correlation between egg size and fecundity was confirmed for Streblospio benedicti by Levin et al. (1991). However, poecilogony is an extremely unusual developm ental strategy (Hoa gland and Robertson 1988, Bouchet 1989) and may not represent a ge neral pattern. In examining the more typical reproductive strategies the genetic evidence of a trade-off within species regarding egg size and egg number is often mixed or absent altogether (e. g., Czesak and Fox 2003 for the seed beetle Stator limbatus, Schwarzkopf et al. 1999 for Drosophila melanogaster, Allan 1984 for the copepod Mesocyclops, Stearns 1983 for the mosquito fish Gambusia affinis, Reznick 1982 for the guppy Poecilia reticulata).

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44 Conclusions and Future Directions The constraint on increased egg diameter (at 6 weeks) in the Pearl Harbor population of Hydroides elegans appears to be selective pressure to maintain the optimum male contribution to lifetime reproduction by delayi ng the shift to the female sex. Considerable genetic variation for egg si ze, fecundity, and the time to sex change is present in this population and this is not surprising give n that this population likely includes substantial genetic diversity. Pearl Harbor is an extremely busy international port, and H. elegans is a cosmopolitan member of the biofouling community that is commonly found on ship and boat hulls, water in takes, and other underwater structures, the population in Pearl Harbor likely ha s been assembled from multiple founding populations. One way to test the hypothesis whether the hermaphroditic life style is constraining egg size is to perform a phylogenetic analysis that maps egg size and sex system onto a phylogeny of family Serpulidae and reconstructi ng ancestral transitions This type of cladistic analysis was used by Rouse and Fitzhugh (1994) to test whether broadcast spawning and planktonic larvae are pleisiomorphic for the polychaete family Sabellidae. Another set of questions that arise from these results concerns the traits of sperm that are produced by larger-egg selected hermaphrodites. It has been suggested (Levitan 1993, 1998) that sexual selection acts on gamete traits to produce suites of co-adapted traits that include egg size, egg number, sperm velocity, and sperm longevity. Do sperm traits vary with egg traits as selection fo r increased egg size proceeds? How correlated are sperm traits and egg traits within individuals, within the population? The observed variation in size of males s uggests additional work on sex change in this animal. It is reported that all indivi duals change sex, but many males remaining in

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45 the population at 6 weeks were considerably larger than females. Allsop and West (2003) showed that the relative timing of sex change was invariant across broad taxonomic groups and largely predicted by maxi mum body size, that is, that a sex change should occur at 72% of maximum body size. Da ta collected in this study do not provide a way to test this prediction but population level studies woul d be a useful approach to this question. This same data set could al so inform us concerning the genetic variation for maximum body size and the trade-off between growth and fecund ity at the population level. Reznick et al. (2000) pointed out that studies evaluating life-history traits (and the trade-offs between them) necessarily focus on specific characters. The choice of what components of fitness are included in a st udy can make the difference between detecting a fitness trade-off or not, since the influence of correlations between unmeasured traits is not known. As efforts to examine the genetic basis of trade-offs broaden to include marine invertebrate life-history evolution, methods that are the mo st integrative across the life cycle (including various developmen tal stages/ages/sizes) and across the breadth of traits that define a life hi story may be the most effective. Large sample sizes are often required to detect genetic co rrelations using breeding designs and this is reasonable in insect systems. Frequently in marine i nvertebrate systems this is a logistical impossibility because culture of the larvae is time-, space-, and labor-intensive. It is difficult to detect complex or unexpected pa tterns in studies that examine correlations across a single generation when sample si zes become limiting. These patterns may be more apparent against a dynamic backgr ound where observations are made across multiple life-history stages and generations.

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46 0 0.5 1 1.5 2 2.5 3 3.5 4 Gen 6Gen 7Gen 8Gen 9Gen 10Gen 11 Generation Figure 3-1. Cumulative response to sele ction for increased egg diameter in H. elegans was positive through Generation 11. No e rror bars were calculated because this is the difference between the mean of 3 selected lines and the mean of 3 control lines at each generation beginni ng in the base population (Generation 6) and ending at the end of selection (Generation 11). Table 3-1. Egg diameter ( m) in replicate selected vs. control lines of Hydroides elegans after 4 generations of sel ection; Fully nested ANOVA Source df EMS (X 10-4) F P Treatment 1 147.0604 95.7328 0.0006 Line (Treat) 4 01.5686 6.1680 0.0001 Cross (Line (Treat)) 428 0.2545 18.1511 0.0001 Error 3916 0.0140 --Table 3-2. Total egg energy in repli cate selected vs. control lines of Hydroides elegans (calculated as g C egg-1); Fully nested ANOVA Source df EMS (X 10-4) F P Treatment 1 2.535578 9.8169 0.0350 Line (Treat) 4 0.258603 2.3369 0.0611 Cross (Line (Treat)) 90 0.111605 1.6279 0.0032 Error 174 0.0686 --

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47 Table 3-3. Absolute fecundity in rep licate selected vs. control lines of Hydroides elegans (eggs female-1); Fully nested ANOVA Source df EMS (X 106) F P Treatment 1 443.82396 10.5232 0.0304 Line (Treat) 4 42.662385 1.9105 0.1078 Error 403 22.330157 --Table 3-4. Relative fecundity based on dry weight of the female in replicate selected vs. control lines of Hydroides elegans (eggs mg-1); Fully nested ANOVA Source df EMS (X 106) F P Treatment 1 795.873456 6.0931 0.0679 Line (Treat) 4 132.548182 2.5540 0.0386 Error 403 51.897613 --Table 3-5. Adult female dry weight (mg) in replicate selected vs. control lines of Hydroides elegans; Fully nested ANOVA Source df EMS F P Treatment 1 0.025218 0.2290 0.6567 Line (Treat) 4 0.112206 2.8075 0.0253 Error 432 0.0399669 --Table 3-6. Larval volume ( L) at 18 to 20 h post-fertilizat ion in replicate selected vs. control lines of Hydroides elegans; Fully nested ANOVA Source df EMS (X 10-4) F P Treatment 1 0.650836 1.1703 0.3392 Line (Treat) 4 0.558838 1.5227 0.2048 Cross (Line (Treat)) 69 0.366555 0.9246 0.6495 Error 648 0.3964 --Table 3-7. Volume ( L) of competent (5 d) larvae in re plicate selected vs. control lines of Hydroides elegans; Fully nested ANOVA Source df EMS (X 10-6) F P Treatment 1 2.44511 4.9009 0.0894 Line (Treat) 4 0.50505 1.8355 0.1324 Cross (Line (Treat)) 66 0.276535 4.9538 0.0001 Error 641 0.0006 --Table 3-8. Juvenile tube lengt h (mm) at 21 d in replicate se lected vs. control lines of Hydroides elegans; Fully nested ANOVA Source df EMS F P Treatment 1 47.0398 3.7050 0.1249 Line (Treat) 4 12.6558 0.6682 0.6162 Cross (Line (Treat)) 69 19.16195 4.7420 0.0001 Error 558 4.0409 --

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48 Table 3-9. Summary of direct and correlated responses to selection fo r increased egg diameter in replicate selected v controls lines of Hydroides elegans. All significant differences represen t positive correlations Trait Selected Mean ( SE) Control Mean ( SE) Egg d ( m) 49.23 (0.35) 45.46 (0.083) Total Energy (mJ egg-1) 0.37 (0.17) 0.29 (0.18) 18 to 20 h larval Vol ( L) 1.12 X10-4 (0.064 X 10-4) 1.14 X10-4 (0.047 X 10-4) NS 5 d larval Vol ( L) 1.29 X 10-3 (0.041 X 10-3) 1. 16 X 10-3 (0.036 X 10-3) 21 d Juvenile tube length (mm) 8.08 (0.31) 8.60 (0.10) NS Adult female dry weight (mg) 0.80 (0.019) 0.82 (0.026) NS Fecundity (eggs) 10071 (481) 7936 (418) Relative Fecundity (eggs mg-1) 13451 (834) 10462 (798) P < 0.05. P < 0.10. NS P > 0.10 Table 3-10. Effect of selec tion (treatment) on the relative numbers of males and females in Generations 7 to 11; Model I ANOVA Source df EMS F P Treatment 1 1.3958 6.28 0.0122 Gen 4 0.3852 1.73 0.1395 Gen*treat 4 0.1717 0.77 0.5427 Error 3461 0.2221 --Table 3-11. Mean percent females present at spawning (6 weeks post-fertilization) in replicate selected vs control lines of Hydroides elegans in Generations 7 to 11 Mean Percent Females in Selected Lines (SE) Mean Percent Females in Control Lines (SE) Generation 7 64.70 (9.036) 61.86 (2.281) Generation 8 64.88 (2.299) 59.21 (2.291) Generation 9 72.11 (5.564) 61.72 (0.877 Generation 10 70.45 (9.022) 62.61 (6.820 Generation 11 74.30 (8.661) 64.21 (3.071 Grand Mean 69.29 (1.935) 61.92 (0.809

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49 CHAPTER 4 CONCLUSIONS The goal of this study was to investigate the evolvability of the tr ait of egg size, to examine the direct and correlated responses to selection on incr eased egg size in a specified set of life-history characters, a nd to explore how these correlations might change as egg size was increased by artifi cial selection in the polychaete worm Hydroides elegans. Life-history traits examined included egg diameter, fecundity, relative fecundity, total egg energy (at 6 weeks) larval size at 18 to 20 h, larval size at competence (5 d), tube length at 21 d, and a dult dry weight (at 6 weeks). I have used various analytical techniques to show that th ere is significant addi tive genetic variation for egg size in the Pearl Harbor population of H. elegans. The response of 2.5 P to direct selection on egg size indi cates that there is substant ial potential for egg size to increase in response to selective pressures (C hapter 2). I predicte d that there would be negative correlations between at least some fitness-related traits, because we expect alleles underlying positive genetic correlations to be rapidly fixed in natural populations. Yet, I found that large-egg selected female s produced more eggs, with higher energy content, that grew into larger larvae at co mpetence. I have proposed that the constraint on increased egg diameter (at 6 weeks) in the Pearl Harbor population of Hydroides elegans appears to be selective pressure to ma intain the optimum male contribution to lifetime reproduction by delaying the shift to the female sex (Chapter 3). Phenotypic correlations between the tra it of egg diameter and the suite of fitness-related traits outlined above were estimated using bi variate analysis on replicate

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50 line means (n = 3 lines treatment-1) in JMP 5.1 (SAS Institute, Inc.). The results comparing control with selected line mean s can be seen as Pearson product moment correlations (r) in Table 4-1. Among lines the mean values for cont rols showed no phenotypic correlation between egg diameter and any of the traits of interest, with the exception of larval size at competence (5 d) where a significant pos itive correlation (0.98) was found. This relationship persisted in selected line means (0.99). However, despite the analysis at the level of treatment showing positive correlati ons between egg diameter and fecundity, examination of individual se lected lines showed a significant negative correlation between egg diameter and both measures of fecundity (-0.95 and -0.99 respectively). The selected lines also showed a significant pos itive correlation between egg diameter and female dry weight (0.99). A pattern is sugge sted in the differences between control and selected lines with respect to these phenotypic correlations; the replicat e selected line that achieved the highest mean egg diameter also had the highest female dry weight and the lowest fecundity. This pattern (the larges t mothers producing the largest eggs in the fewest numbers) is reminiscent of the pred icted trade-off between growth, egg size, and fecundity that I did not detect between treatmen ts (Chapter 3). This relationship emerges at the extreme end of the continuum of repr oductive investment (large-egg selected lines) and is not detectable among the control lin es. While this observation cannot be considered quantitative (n = 3 is the minimum sample size for a correlation), it suggests that further study into the influence of trad e-offs in defining reproductive investment might be most fruitful at the extreme ends of the investment continuum in this species.

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51 Within polychaetes, fecundity within species can vary with nutri tional state of the mother, population density, and adult age and size (Eckelbarger 1986). In this experiment all animals were fed ad libidum at all stages of development, while density and adult age were contro lled. Varying any of these parameters or inducing environmental stress at different developmen tal stages might also reveal evidence of trade-offs influencing reproduc tive effort. Qiu and Qian (1998) looked at effects of varying salinity and temperat ure on juvenile traits in H. elegans, and found that fecundity was affected by the stress of low salinit y. Czesak and Fox (2003) found while working with the seed beetle Stator limbatus that the magnitude of the tradeoff between egg size and fecundity varied between environments. Another possible cost associated with in creased reproductive effort is higher mortality, as was found in the polychaete Dinophiilus gyrociliatus by Prevedelli and coworkers (Prevedelli and Zunarelli Vandini 1999, Prevedelli and Simonini 2000). They found that higher food led to increased fec undity but was accompanied by greater and earlier mortality. Tatar et al. (1993) nicely demonstrated the tradeoff between increased egg production and increased age-specific mortality in the beetle Callosobruchus maculatus. Many avenues of inquiry can be pursued ba sed on this study. A large body of work has been compiled investigating the adaptiv e significance of developmental mode in marine invertebrates using comparative, empi rical, and manipulative techniques. A great deal of effort has also been invested in qua ntitative modeling approaches that have been used to try and identify selection pressure s and processes that influence developmental mode evolution. The suite of characteris tics that defines a developmental type has

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52 co-evolved in concert and compromise with a wider complex of life-history traits under the influence of natural selection. The mo st rewarding efforts to pursue these dynamic and sometimes subtle relationships will be me thods that are the most integrative across the stages that define a life cycle, and across th e breadth of traits that define a life history. Understanding the nature of the variation in fitness-related traits and how it is partitioned in a particular population has th e potential to reveal the insights we seek concerning the evolution of the diverse life histories among mari ne invertebrates. Table 4-1. Phenotypic correlations between the trait of egg size and a suite of life-history traits in replicate selected a nd control lines in the polychaete Hydroides elegans, based on line means (n = 3 lines treatment-1). Pearson product moment correlations (r) with absolute value > 0.95 indicated with Correlation coefficient (r) among Control Line Means (n = 3) Correlation coefficient (r) among Selected Line Means (n = 3) Egg d Fecundity 0.09 Egg d Fecundity -0.95* Egg d Relative Fecundity 0.24 Egg d Relative Fecundity -0.99* Egg d Female Dry Weight -0.05 Egg d Female Dry Weight 0.99* Egg d Total energy egg-1 -0.48 Egg d Total energy egg-1 -0.28 Egg d Larval size at 18 to 20 h -0.13 Egg d Larval size at 18 to 20 h -0.63 Egg d Larval size at 5 d 0.98* Egg d Larval size at 5 d 0.99* Egg d Tube length at 21 d -0.86 Egg d Tube length at 21 d -0.91

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67 BIOGRAPHICAL SKETCH I was born in North Carolina and raised in south Florida. I have traveled extensively as an adult, and settled in Texas where I renewe d my pursuit of education and received an Associate of Arts degree from Texas Southmost College in Brownsville, Texas and a Bachelor of Science degree, summa cum laude, from Texas A&M University. I earned my Master of Science degree from Florida In stitute of Technology in 2000 and continued to earn my Ph.D. in 2006 from the University of Florida with a major in zoology.


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LIFE-HISTORY CONSEQUENCES OF ARTIFICIAL SELECTION FOR
INCREASED EGG SIZE IN Hydroides elegans (POLYCHAETA: SERPULIDAE)















By

CECELIA MARIE MILES


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2006

































Copyright 2006

by

Cecelia Marie Miles




























This work is dedicated to Dr. Larry McEdward. Larry's ability to distill complex topics
down to their essential elements served to focus my efforts as a new graduate student. As
I have advanced in my career, I have come to respect this skill immensely and it is one
that I aspire to emulate. This project was conceived and planned under his guidance and
his influence is present in every aspect.















ACKNOWLEDGMENTS

For insight, guidance, encouragement, and especially for leading by example, I

thank Dr. Marta L. Wayne. I also thank committee members Dr. Colette St. Mary, Dr.

Shirley Baker, and Dr. Gustav Paulay. I am especially grateful to Dr. Ben Bolker for his

clear thinking and concise statistical advice.

I thank Dr. Michael G. Hadfield for his generosity and advice, and everyone at

Kewalo Marine Laboratory in Honolulu, Hawaii for their kokua. I could never have

completed this project without my fellow "worm wrangler" at Kewalo, Sharon Kelly.

I thank the Zoology Department at the University of Florida for financial,

emotional, and spiritual support. I would also like to acknowledge the Auzenne Graduate

Scholars Fellowship from the University of Florida, and the Grinter Fellowship from the

Graduate School.

I wish to thank my family and especially my husband, Myron G. Miles, for his

infinite support.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF TA BLE S ......... .... ........ .... .... ...... ....................... .... vii

LIST OF FIGURES ......... ............................... ........ ............ ix

A B STR A C T ................................................. ..................................... .. x

CHAPTER

1 G EN ER A L IN TR O D U C TIO N ......................................................... .....................1

2 ESTIMATES OF HERITABILITY FOR EGG SIZE IN THE SERPULID
POLYCHAETE Hydroides elegans ........................................ ......... ............... 8

Introduction ............... ....... ..................................................... ................. 8
M material and M methods ......... .. .. ......... .. .. .. ............................ ....11
Collection and Spaw ning........................ .. ................................ ............... 11
H alf Sib B reeding D esign............................. ......................... ............... 14
S election n ........................................... .............................................. 16
R esu lts ......... ...... ................................................................................. 17
D isc u ssio n ........................................... ................................................................. 1 8

3 DIRECT AND INDIRECT RESULTS OF ARTIFICIAL SELECTION FOR
INCREASED EGG SIZE IN THE SERPULID POLYCHAETE Hydroides
e le g a n s .................................................................................................................. 2 4

Intro du action .................................................................................................... 2 4
M eth o d s ................................................................................................... 2 9
Collection, Spawning, and Selection .......................................29
L arval Size......................................................... 3 1
Juvenile Tube Length ............................................... ............... 32
Total Egg Energy ...... ...... ............. .................32
F ecu n d ity ....................................................... 3 4
A dult D ry W eight .............. ......................... ... ..........................................35
D ata A n a ly sis ................................................................................................. 3 5
Results .............. .......................... ...................................... 36
D direct R response to Selection .................................... ................. 36









C orrelated R responses to Selection ........................................... .....................36
Spontaneous Spawning and Sex Ratios.................................. ...............38
D iscu ssio n ................................ ............................................................................. 3 9
More + Bigger = Better... or Does It?....................... ....... ...............39
Conclusions and Future Directions ........................................ ............... 44

4 CON CLU SION S .................................. .. .......... .. .............49

L IST O F R E FE R E N C E S .......... .................... .......................................... .........................53

B IO G R A PH IC A L SK E TCH ..................................................................... ..................67















LIST OF TABLES


Table pge

2-1 Number of females used to establish six individual lines in Generation 7 .............21

2-2 Half-sib breeding design; Fully nested ANOVA and variance component
analysis for egg diameter in Hydroides elegans........ ......... .................................22

3-1 Egg diameter ([tm) in replicate selected vs. control lines of Hydroides elegans
after 4 generations of selection ......... ............................................. ..... ......... 46

3-2 Total egg energy in replicate selected vs. control lines of Hydroides elegans
(calculated as |tg C egg )......... ............................................ .......... ............. 46

3-3 Absolute fecundity in replicate selected vs. control lines ofHydroides elegans
(eg g s fem ale-1).................................................. ................ 4 7

3-4 Relative fecundity based on dry weight of the female in replicate selected vs.
control lines of Hydroides elegans (eggs mg ) ...................................... ........... 47

3-5 Adult female dry weight (mg) in replicate selected vs. control lines of Hydroides
elegans ........... ........................................ ....................... .. 47

3-6 Larval volume ([tL) at 18 to 20 h post-fertilization in replicate selected vs.
control lines of Hydroides elegans.................................................... 47

3-7 Volume ([tL) of competent (5 d) larvae in replicate selected vs. control lines of
Hydroides elegans ......... ..... ........... .... .................... .. ...... 47

3-8 Juvenile tube length (mm) at 21 d in replicate selected vs. control lines of
Hydroides elegans ......... ..... ........... .... .................... .. ...... 47

3-9 Summary of direct and correlated responses to selection for increased egg
diameter in replicate selected v controls lines of Hydroides elegans ......................48

3-10 Effect of selection (treatment) on the relative numbers of males and females in
Generations 7 to 11 ................... ................... ................. .. 48

3-11 Mean percent females present at spawning (6 weeks post-fertilization) in
replicate selected vs. control lines of Hydroides elegans.............................48









4-1 Phenotypic correlations between the trait of egg size and a suite of life-history
traits in replicate selected and control lines in the polychaete Hydroides elegans ..52















LIST OF FIGURES


Figure pge

2-1 Response to direct selection on egg diameter for four generations of in the
polychaete Hydroides elegans ......... .............................................. ............... 22

2-2 Mean egg diameter (+ SE) before and after direct selection for increased egg
diameter in replicate selected (1,3,5) and control (2,4,6) lines of Hydroides
elegans ........... ........................................ ........................ 23

3-1 Cumulative response to selection for increased egg diameter in H. elegans was
positive through G generation 11 ........................................ .......................... 46















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

LIFE-HISTORY CONSEQUENCES OF ARTIFICIAL SELECTION FOR
INCREASED EGG SIZE IN Hydroides elegans (POLYCHAETA: SERPULIDAE)

By

Cecelia M. Miles

May 2006

Chair: Marta L. Wayne
Department: Zoology

Goals of this study were to estimate narrow-sense heritability (h2) for egg size in

the polychaete worm Hydroides elegans, to examine direct and correlated responses to

selection on increased egg size in a suite of life-history characters, and to explore how

these correlations changed as egg size was increased by artificial selection.

Narrow-sense heritability is a predictor of short-term response to selection. Many

quantitative models have invoked selective response in egg size as a key transitional

element in the evolution of life histories in marine invertebrates, assuming that egg size

can and does respond to selection. I tested this assumption using a half-sib breeding

design and obtained an estimate for h2 of 0.45 for egg size in a Pearl Harbor, HI

population. I performed four generations of artificial selection for increased egg size that

allowed me to estimate cumulative realized heritability in the same population as 0.58.

Artificial selection resulted in a direct response of 2.5 op in egg size relative to the

common base population. Though I predicted negative correlated responses in some









traits under selection, none was measured. A positive correlated response to selection

was observed in fecundity, total egg energy, and larval size at competence, relative to

control lines. No significant correlated response was observed in early larval size,

juvenile tube length, or adult dry weight. H. elegans is a protandrous hermaphrodite, and

sex ratio data across generations indicated that large-egg selected lines were changing sex

earlier than control lines. I propose that this earlier sex change resulted in selected

individuals spending less time as males relative to controls, consequently decreasing their

overall fitness.

Phenotypic correlations between egg diameter and the traits outlined above were

estimated, and control line means showed no significant correlation with egg size, with

the exception of the positive correlation for larval size at competence. Unexpectedly,

phenotypic correlations among selected line means were significantly negative for

fecundity, but positive for female dry weight. The positive correlation for larval size at

competence also persisted after selection. This may indicate where further work could be

useful in examining trade-offs between egg size and other life-history traits.














CHAPTER 1
GENERAL INTRODUCTION

The diversity of larval form and function among marine invertebrates has

fascinated researchers for decades. Thorson (1950) was among the first to recognize

broad-scale patterns in developmental mode and their strong correlation with specific

life-history traits (egg size and fecundity). A useful classification of larval types has been

based on this observation that numerous marine invertebrates produce high numbers of

relatively small, energy-poor eggs, while many others produce fewer, larger, energy-rich

eggs. A closely associated dichotomy involves the coexistence of planktotrophic and

lecithotrophic larval development. Many marine invertebrates produce pelagic, feeding

(planktotrophic) larvae, and these are generally hatched in large numbers from small

eggs. Other, often closely related, species produce lecithotrophic larvae that do not

depend on exogenous food in order to reach metamorphosis, and these hatch in smaller

numbers from larger eggs. Phylogenetic analysis suggests that the transition between

these life-history modes has occurred many times (Wray 1996, Duda and Palumbi 1997,

Cunningham 1999, Rouse 2000, McEdward and Miner 2001).

The adaptive significance of developmental mode has received considerable

attention. Selection pressures such as predation, starvation, and dispersal have been

proposed for developmental mode evolution (Thorson 1950, Chia 1974, Strathmann

1985), and increases in egg size are often cited as important transitional states between

modes (Wray and Raff 1991, Hart 1996, Miner et al. 2005). The suite of characteristics

that defines a larval type has not evolved in isolation from post-larval and adult









characteristics. Egg size and fecundity are two members of a much larger complex of

life-history traits that co-evolved, presumably, under the influence of natural selection

(Havenhand 1995, Ramirez Llodra 2002). Egg size and fecundity are both directly

related to parental fitness (Roff 1992, Bernardo 1996) and many authors have mentioned

the importance of interactions between larval characteristics and other parts of the life

cycle (e.g., Vance 1973a,b, Christiansen and Fenchel 1979, Todd and Doyle 1981,

Strathmann 1985, Moran 1994, Pechenik et al. 1998, Gimenez et al. 2004). Few efforts

have been made to examine the implications over the entire life cycle of selective

pressures on these important and inter-related fitness components.

Many studies of marine ecology have used a comparative approach to assess the

influence of one life-history stage on another. For instance, maternal environmental

conditions influence larval performance in various groups (e.g., Laughlin and French

1989 for the crab Rhithropanopeus harrisii, George 1999 for the seastar Pisaster

ochraceus, Qiu and Qian 1997 for the polychaete Hydroides elegans, Gimenez and Anger

2003 for the crab Chasmagnathus granulata). Studies have demonstrated that larval

experience is linked to juvenile quality (e.g., Jarrett and Pechenik 1997 for the barnacle

Semibalanus balanoides, Wendt 1998 for the bryozoan Bugula neritina, Phillips 2002 for

the bivalve mollusk Mytilus galloprovincialis, reviewed in Pechenik et al. 1998). Size at

hatching affects growth, time to maturity and survivorship in the gastropod Nucella

ostrina (Moran and Emlet 2001). Size at settlement influences survival in the ascidian

Botrylloides violaceus (Marshall et al. 2006). Hilbish et al. (1993) discussed the common

assumption that variation in larval and juvenile growth rates have a common genetic

basis, and found that significant genetic variation for larval shell growth in the bivalve









Mercenaria mercenaria is not related to the genetic variation in shell growth during the

juvenile period. Pechenik et al. (1996) found no correlation of growth rates between

larval and juvenile stages in two species of the mollusk Crepidula. Delayed

metamorphosis is associated with reduced juvenile or adult performance most often in

non-feeding larval forms (e.g., Wendt 1996 for the bryozoan Bugula neritina, Pechenik

and Cerulli 1991 for the polychaete Capitella sp. I, Pechenik et al. 1993 for the barnacle

Balanus amphitrite, and Maldonado and Young 1999 for the sponge Sigmadocia

caerulea). Delaying metamorphosis in the feeding larvae of polychaete Hydroides

elegans negatively affects juvenile survival and growth rates whether or not they are fed

during the delay (Qian and Pechenik 1998). Delayed metamorphosis in the feeding

larvae of the sipunculan Apionsoma misakianum results in reduced juvenile growth

whether or not larvae are fed, but does not affect juvenile survival (Pechenik and Rice

2001).

Empirical studies have examined the relationship between egg size and larval

characters such as rate of development and larval size at metamorphosis in a range of

taxa. For example, in opisthobranch mollusks, Havenhand (1993) reports a negative

relationship between egg size and development time. Gimenez (2002) reports that the

crustacean Chasmagnathus granulata hatching from smaller eggs takes longer to

develop, as does the seastar Pisaster ochraceus (George 1999). This negative

relationship between egg size and development time is also reported for echinoids and

asteroids combined (Emlet et al. 1987). Levitan (2000) further refined this relationship

by showing that it is negative curvilinear using data from a large group of echinoids. For

echinoids and asteroids combined the correlation between size of egg and the size of









juvenile at metamorphosis is positive overall (Emlet et al. 1987), but Levitan (2000)

shows these two traits to be independent of one another in echinoids, after controlling for

phylogeny.

Many fewer studies have used an experimental approach to directly manipulate egg

size. Sinervo and McEdward (1988) used blastomere separation to show the relationship

between reduced parental investment and resulting larval characters in two species of the

echinoid S.'i/t ii'o. l, etllf ,itn They found that larvae that develop from experimentally

reduced zygotes are smaller and take longer to reach metamorphosis than those from

whole eggs. Hart (1995) used the same technique and found reduced feeding rates in

larvae developing from experimentally reduced zygotes of S. droebachiensis. Emlet and

Hoegh-Guldberg (1997) experimentally reduced the lipid content of early zygotes in

another urchin, Heliocidaris erythrogramma, and showed a link between reduced

parental investment and juvenile performance.

Another approach to evaluating the influence of egg size on life history is to

examine a single species that shows both developmental modes (poecilogony). Levin

et al. (1987) examined the demographic consequences of divergent life-history patterns in

a single species of polychaete, Streblospio benedicti, with both planktotrophic and

lecithotrophic developmental types. They found that both types achieve similar

population growth rates.

This overview represents a sample of the diversity of taxa, traits, and methods that

have been employed to demonstrate to what extent one stage of life can influence

another. Thus, while a large and diverse body of empirical, comparative, and

experimental work has demonstrated links between one life-history stage and another, a









unified examination of egg, larval, juvenile, and adult traits, and how these correlations

influence fitness over the entire life cycle has not been undertaken. As shifts in egg size

are often invoked as important transitional states between developmental modes (Wray

and Raff 1991, Hart 1996, Miner et al. 2005), and egg size plays a critical role in many of

the quantitative modeling approaches that have been used to try and identify selection

pressures and processes that influence developmental mode evolution (Vance 1973a,b,

Smith and Fretwell 1974, Christiansen and Fenchel 1979, Perron and Carrier 1981,

Roughgarden 1989, Podolsky and Strathmann 1996, McEdward 1997, Levitan 2000,

Hendry et al. 2001, Luttikhuizen et al. 2004), I focused this study on egg size.

Though it is often reported as a fixed parameter, egg size within species displays a

large amount of variation (Jaeckle 1995, Hadfield and Strathmann 1996). Considering

marine invertebrates, intraspecific variation in mean egg size has been reported for the

barnacle Balanus balanoides (Barnes and Barnes 1965). Turner and Lawrence (1979)

found significant differences in egg size within species when examining 11 echinoderms,

and Lessios (1987) found this same result in a comparison of 13 echinoid species

sampled across the Panamanian isthmus. The presence of significant variation in egg size

has been reported within species for the starfishes Pteraster tessalatus (McEdward and

Coulter 1987), and Solaster stimpsoni (McEdward and Carson 1987), for the polar

shrimps Chorismus antarcticus and Notocrangon antarcticus (Clarke 1993), for the dorid

nudibranch Adalaria proxima (Jones et al. 1996), for the ascidian Pyura stolonifera

(Marshall et al. 2002), and for the euphausiid Thysanoessa raschii (Timofeev 2004).

Within and between-species variation in egg size was reviewed for serpulimorph

polychaetes by Kupriyanova (2001).









Certainly there is abundant phenotypic variation in egg size, but only that portion

that has a genetic basis is accessible to selection in order to influence life-history

evolution. In order for a trait to respond to natural selection it must have heritable

variation, and that variability must have fitness consequences. What component of

phenotypic variation in egg size is available to natural selection; what is the heritability of

this trait? There is evidence of negative phenotypic trade-offs between fecundity and egg

size in unmanipulated populations of a variety of species (Roff 1992). Examples are

numerous among arthropods and include crustaceans, lepidopterans, dipterans, and others

(Fox and Czesak 2000). The same trade-off has been recorded for treefrogs (Lips 2001);

and Sinervo (1990) demonstrated a negative trade-off in phenotypically manipulated

lizards.

But, there is a limitation to conclusions about evolutionary response to natural

selection that can be drawn from phenotypic correlations. If the observed phenotypic

trade-off does not represent some underlying genetic antagonism among traits, then it is

not revealing the set of options available for selection on these traits. One way to detect

this relationship is by using artificial selection experiments that can reveal short-term

evolutionary responses for the selected trait. Using both the selection and the

manipulation approaches, Schwarzkopf et al. (1999) found no negative genetic

correlation between egg size and fecundity in Drosophila melanogaster. Azevedo et al.

(1996) also found no evidence for a trade-off between egg size and fecundity in their

laboratory-selected lines ofD. melanogaster. Blanckenhorn and Heyland (2004) found

no evidence of a genetic tradeoff between egg size and number in the yellow dung fly

,N tth//ll ihgll/ stercoraria. But, in another arthropod, the cladoceran water flea Daphnia,









a genetic basis for this trade-off was shown (Lynch 1984, Ebert 1993). Levin et al.

(1991) also confirmed a negative genetic correlation between fecundity and egg size in

the poecilogonous polychaete Streblospio benedicti, supporting the assumption of

trade-offs between these traits.

Goals of this study were to investigate the evolvability of the trait of egg size, to

examine the direct and correlated responses to selection on increased egg size in a

specified set of life-history characters that span the life cycle, and to explore how these

correlations change as egg size is increased by artificial selection in the polychaete worm

Hydroides elegans Haswell, 1883. Using adult H. elegans collected from the wild,

spawned in the laboratory, and raised under common environmental conditions, variation

in egg size was documented and heritability for this trait was estimated using a sib

analysis (Falconer and Mackay 1996). Using artificial selection for increased egg

diameter, realized heritability was estimated (Hill 1972) and the results of selection were

documented in a suite of life-history characters as they are reflected through the larval,

juvenile, and adult stages. Life-history traits examined included egg diameter, fecundity,

and total egg energy (at 6 weeks post-fertilization), larval size at 18 to 20 h

post-fertilization, larval size at competence (5 d), tube length at 21 d, and adult dry

weight (at 6 weeks post-fertilization).














CHAPTER 2
ESTIMATES OF HERITABILITY FOR EGG SIZE IN THE SERPULID
POLYCHAETE Hydroides elegans

Introduction

Life histories represent compromises between selection on individual fitness

components and constraints acting on the organism as an integrated whole. Steams

(1992) suggested that the analysis of life-history strategies must be based on, and

consistent with, the evolution of the individual life-history traits. Few individual traits

are more fundamental to life history than resource allocation to the egg. Egg size is

directly related to parental fitness, and can also have substantial fitness effects for

progeny (Roff 1992).

Life histories in marine invertebrates are very diverse, and the adaptive significance

of developmental mode has received considerable attention. One common feature in the

life histories of many invertebrates is an obligate feeding (planktotrophic) larval stage.

Paradoxically, it can also often be observed within closely related groups that some

members lack this dependence on exogenous food and exhibit lecithotrophic

development. It has been hypothesized that nonfeeding larvae evolved as a response to

either direct or indirect selection for increased egg size (Jagersten 1972, Strathmann

1978, 1985, Raff 1987, Hart 1996). Selection on egg size has also been linked to

developmental mode (Hadfield and Miller 1987), larval development time (Sinervo and

McEdward 1988), larval form (Strathmann 2000), size at settlement (Strathmann 1985),

survivorship (Bridges and Heppell 1996), and fertilization success (Levitan 2000).









Vance's (1973a,b) fecundity-time model regarding the evolution of life histories in

marine invertebrates began a series of quantitative modeling efforts focused on egg size

as a key trait (Christiansen and Fenchel 1979, Caswell 1981, Perron and Carrier 1981,

Grant 1983, Roughgarden 1989, McEdward 1997, Levitan 2000, Luttikhuizen et al.

2004). While the quantitative modeling efforts regarding evolution of life histories in

marine invertebrates have become increasingly refined, there has been little research into

the genetic architecture of the important trait of egg size in marine invertebrates.

In order for a trait to respond to natural selection it must have heritable variation,

and that variability must have fitness consequences. Numerous studies have found that

egg size typically exhibits phenotypic variability across a wide range of taxa (Hadfield

and Strathmann 1996). Phenotypic variation (Vp) has several components including the

genetic (VG), the environmental (VE), and the interaction between them (VGXE). Unless

phenotypic variation represents some underlying genetic variation, it is not heritable and

is consequently unavailable to natural (or artificial) selection. VG can be further

partitioned; the component that accounts for the resemblance between offspring and

parents in sexually reproducing organisms is called additive genetic variance (VA). The

proportion of Vp that is made up of VA is a quantity called narrow-sense heritability

(h2 = VA Vp). This value is a measure of the resemblance between parents and offspring

after sexual recombination and, therefore, is a predictor of short-term response to

selection (Falconer and Mackay 1996).

Among protostome invertebrates, most of the investigations into h2 of egg size have

been focused on the Arthropoda (reviewed by Fox and Czesak 2000): especially the

Insecta, a mostly terrestrial group that includes Diptera (flies and mosquitoes),









Lepidoptera (moths and butterflies), and Coleoptera (beetles), among others. Many

experiments examined egg size in Drosophila melanogaster and demonstrated that there

is a significant genetic component to variation for this trait (Warren 1924, Bell et al.

1955, Azevedo et al. 1997, Schwarzkopf et al. 1999). Among lepidopterans, Fischer et

al. (2004) estimates h2 for egg size of ca. 0.4 in the butterfly Bicyclus anynana and

Harvey (1983) reports h2 for egg weight in the budworm Choristoneurafumiferana at

0.75. Fox (1993) estimates h2 of egg size for the seed beetle Callosobruchus maculatus

at 0.43 to 0.59 and 0.60 to 0.74 using two different designs. Czesak and Fox (2003)

calculate realized h2 for egg size in the seed beetle Stator limbatus of 0.36 to 0.55. Thus,

there is abundant evidence of VA for egg size in insects, and estimates of h2 are generally

moderate to large.

The only marine invertebrate to receive similar attention was the poecilogonous

polychaete Streblospio benedicti, where a reciprocal mating design used to examine the

genetic components of life-history traits gave a heritability estimate of 0.75 for egg

diameter (Levin et al. 1991). With the exception of this one study on a species with an

unusual life history that includes producing both planktotrophic and lecithotrophic larvae,

empirical support for theoretical assumptions concerning the heritability of egg size in

marine invertebrates has not been examined.

The serpulid polychaete Hydroides elegans Haswell, 1883 is an excellent choice as

a model organism for estimating h2 for egg size in a marine invertebrate for several

reasons. It produces a large number of planktotrophic larvae that can be cultured in the

laboratory with relative ease. The larvae will settle and metamorphose in culture in the

presence of natural biofilms (Carpizio-Ituarte and Hadfield 1998). The generation time is









reasonably short, 16 to 28 days at optimal temperature, salinity, and food concentrations

(Qiu and Qian 1998). Both larvae and adult worms can be fed the chrysophyte alga

Isochrysis galbana, which is easily cultured in the laboratory. H. elegans develops in the

water column with no maternal care.

The objective of this study was to obtain estimates of narrow sense heritability for

the trait of egg size in Hydroides elegans. I calculated h2 using three different

techniques. First, I constructed a half-sib breeding design and used a nested ANOVA for

analysis (Falconer and Mackay 1996). Next, Restricted Maximum Likelihood Analysis

(Quercus Quantitative Genetics Software, University of Minnesota, USA) was performed

on the same data set. Last, artificial selection on egg size was performed and cumulative

realized heritability was calculated according to the methods of Hill (1972).

Material and Methods

Collection and Spawning

This project was completed at Kewalo Marine Laboratory, Honolulu, Hawaii using

protocols developed there by various workers. Wild worms were collected in November

2002 from VexarTM screens suspended for 1 month from a floating dock at Ford Island,

Pearl Harbor, Hawaii. The sex of each worm was determined and gametes secured by

agitation of the calcareous tube surrounding each adult, resulting in release of gametes

(Unabia and Hadfield, 1999). Gametes from 270 female worms were used to establish

the initial laboratory population. At least 120 males were used, but the exact number of

males was not determined.

I used a Nikon CoolpixTM 990 camera mounted on an Olympus compound light

microscope to obtain digital images of eggs from each female immediately after their









release. Mean egg diameter for each mother (n = 10 eggs mother-) was obtained from

these images using NIH Image software. Larval cultures were established at densities of

5 tolO larvae mL-1 in 1L plastic tripour beakers and raised in pooled cultures (4 to 5

mothers and 2 to 3 fathers beaker-) for Generations 1 through 3. Each beaker had

different and unique parents. Cultures were maintained at 250C, and fed 6 X 104

cells mL-1 day-'of the chrysophyte alga Isochrysis galbana (Tahitian strain). Isochrysis

galbana was cultured in the lab at room temperature and constant light, using f/2 media

(Guillard 1975) and used during exponential growth phase. All embryos were allowed to

hatch in FSW (0.22 |tm filtered seawater) and swimming larvae were counted

approximately 12 h post-fertiization. At that time larval cultures were established at

maximum densities of 10 larvae mL-1 and larvae were fed for the first time. Larvae were

fed on Day 2 by adding the appropriate volume of algae to the larval culture beaker

without changing water or beakers. On Day 3 and Day 4 larvae were fed and both water

and beakers were changed. Day 5 larvae were considered competent to settle and

metamorphose.

Small transparent plastic chips (2.5 X 1.5 X 0.08 cm; K&S Engineering, Chicago

IL, #1306) were placed in flow-through seawater tables for 10 days in order to

accumulate natural biofilm, which has been shown to induce settlement in H. elegans

(Hadfield et al. 1994). One biofilmed chip was placed in each well of a standard ice cube

tray along with 15 mL FSW, I. galbana (6 X 104 cells mL-1), and 20 to 25 competent

larvae.

Larvae and newly settled juveniles were fed daily at this food density without

changing the water for 5 days. This procedure allowed a wide window of opportunity for









settlement and avoided inadvertent selection for early settling individuals. Ice trays were

kept at room temperature (-240C) in a PlexiglasTM rack on the bench top with ambient

illumination. The position of trays within the rack was randomized daily. On Day 10

(post-fertilization), each chip was removed from the tray and all animals but one were

removed. The survivor was determined by position on the chip, with that individual

closest to the center selected to be the survivor. Thus, no inadvertent selection for early

or late settling individuals, or for body size, took place during this process. At this point,

the food level was increased 6-fold (36 X 104 cells mL1) and worms were fed with the

daily water change (FSW) until animals had reached 6 weeks of age. At maturity (here

defined as 6 weeks), individual worms were placed in Petri dishes and spawned as before.

H. elegans is a protandrous hermaphrodite. Since I was measuring a maternal trait (egg

size), I chose a generation time of 6 weeks in order to assure that 50 to 60% of the

spawning population would be female. Three generations were so raised under

laboratory conditions to minimize variance due to maternal effects. Two hundred or

more females were spawned to produce each of these two subsequent generations (217 in

Generation 2 and 236 in Generation 3).

Within Generation 3 a subset of individual crosses was established to setup the

breeding design experiment described below. Alongside the pooled cultures of mixed

parentage described above, individual larval cultures consisting only of offspring from a

unique mother crossed with a known father were raised under conditions identical to

those described above.









Half Sib Breeding Design

A subset of the worms in Generation 3 was used as parents for the half-sib design

experiment (Falconer and Mackay 1996) that was analyzed in Generation 4. Ten males

were arbitrarily selected and their sperm was split between two females each (3 females

in one case). The result of this mating scheme is to produce offspring related in two

different ways, that is, full-siblings (within females, or dams) and half-siblings (within

males, or sires). The advantage of this design is to allow the partitioning of the

phenotypic variance according to its source (the progeny of different males (2 SIRE), the

progeny of different females mated to the same male (C2DAM), and individual offspring of

the same female withinHIN. The component of total variance attributable to sires is

1/4 VA, therefore the most straightforward estimate of h2 = 4C2sIRE/ C TOTAL (Falconer and

Mackay 1996).

Parents and offspring involved in this breeding design were raised in individual and

not pooled cultures, but otherwise all conditions were as described above. A fully nested

Random Model ANOVA was then used to partition variance in egg diameter according to

its source. Type III sums of squares were calculated using the general linear model of

SAS Version 6.12 (PROC GLM, with RANDOM statement and TEST option).

The variance component data were also used to calculate the additive genetic and

residual coefficients of variation (CVA = 100 VA IX and CVR = 100 V- VA IX, where

X = trait mean) as recommended by Houle (1992). Evolvability is defined as the ability

of a trait to respond to selection (Houle 1992) and, using the assumption of directional

selection, can be calculated as IA = VA/X2.









I also used Restricted Maximum Likelihood (REML) to analyze these same data,

using the nf3 program in Quercus of R.G. Shaw and F.H. Shaw (University of Minnesota,

USA; downloaded November, 2004; www.cbs.umn.edu/eeb/events/quercus.shtml). This

program estimates the likelihood of observing the data given a set of parameters.

Iterative methods are then used to find the set of parameters that maximize this likelihood

(Shaw 1987). I used the program to perform REML on a two-generation pedigree with

mean egg diameter of each Generation 4 daughter as the single character. The sources of

total phenotypic variance were analyzed using the model Vp = VA + VD + VE. In this

model the genetic variance mentioned above (VG) is partitioned into two components, VA

(additive) and VD (dominance). Log likelihood ratio tests were used to compare the fit of

the model for significant differences (2 21) as successive variance components were

constrained to zero. Technically, this violates the assumption of the likelihood ratio test

because the null value of the parameter (i.e., variance = 0) lies on the boundary of the

feasible range. However, the test is believed to be conservative under these conditions

(Pinheiro and Bates 2000).

Linear regression of mid-offspring egg diameters onto maternal egg diameters was

not used because, while each daughter had an independent mother, they were not

independent with respect to the father. Therefore, the assumption of independence

necessary for the linear regression could not be met.

The family based design described here for Generation 4 was repeated in

Generations 5 and 6 in an attempt to increase sample size.









Selection

In Generation 7 the common laboratory population was split into six lines. Three

were established as selected lines and three as control lines. Between 12 and 16 females

were used to establish each line (Table 2-1). In subsequent generations (Generations 8 to

10), 16 mothers were kept in each line. Within selected lines, the eggs from a specific

mother were sub-sampled as described above. Measurements were made immediately

and eggs were fertilized only if the mean diameter of the eggs was greater than one

phenotypic standard deviation (op) over the Generation 6 population mean. In

subsequent generations, selection proceeded in the same manner, with mothers remaining

in the line only if the mean diameter of their eggs exceeded 1 op over the mean of the

selected mothers from the previous generation. If the mothers meeting this criterion

happened to include multiple sisters I retained no more than two sisters in a line within a

generation. The same rule, that no more than two sisters remain in a line, was also

followed for control lines. In control lines, every third or fourth female was fertilized

without regard to the size of her eggs. Otherwise, eggs were handled exactly the same as

for selected lines. All crosses were between one male and one female with no shared

parentage. Males were chosen haphazardly with respect to the mean egg size of the

clutch from which they came. Care was taken not to use siblings of either selected or

control females as fathers. Larval culture and settlement proceeded as before. Selection

continued for four generations (7 through 10).

Generally, realized heritability is the change in mean trait value over each

generation of selection (offspring mean parental population mean) divided by the

selection differential (trait mean for selected parents parental population mean). As









selection was carried out in one direction only, and the selected and control lines came

from the same base population, the realized heritability was calculated according to the

specific methods described by Hill (1972). Cumulative realized heritability was

calculated as the difference between selected and control values at each generation (X,)

multiplied by the cumulative selection differential for that generation (S,), summed over

generations (YXYS,). This quantity is divided by the sum over the square of the

cumulative selection differential for each generation (yS,2).

Results

Expected mean squares and variance components for the half-sib breeding design

in Generation 4 are listed in Table 2-2. Narrow-sense heritability calculated using the

sire component of variance (P = 0.069) was h2 = 4C2sIRE/ 2TOTAL =0.45. Confidence

intervals were calculated based on the F-distribution, according to Knapp (1986), as

recommended for small sample sizes (Hohls 1998). The 95% confidence intervals were

large (-0.36 to 0.90) as is often the case for CI for heritability from a breeding design

(Koots and Gibson 1996, Markow and Clarke 1997). The mean egg size of Generation 4

daughters used in the nested ANOVA, grouped by mother, was 45.68 ptm

(+ 0.21 |tm SE), CVA = 3.22 and CVR = 3.59. Evolvability for the trait of egg size for the

Pearl Harbor population ofH. elegans was calculated at I = 10.35 X 10-4. Results from

the breeding design experiments in Generations 5 and 6 did not result in an increase in

sample size and in both cases the sire component of variance was not significant.

Results from the REML analysis on the Generation 4 data were not significant

using likelihood ratio tests. The full model (VA, VD, VE unconstrained) gave a negative

estimate for VD. Since this is not biologically meaningful, VD was constrained to zero.









This produced a result not significantly different than the unconstrained model

(2i = 0.735; P = 0.45). The estimate ofh2 based on this model was VA / (VA +

VE) = 0.38. However, when VA was constrained to zero the log likelihood ratios were still

not significantly different (,i = 2.519; P = 0.13).

Mean egg diameter ([tm + SE) before selection (Generation 6) was 44.78 (0.15),

and after selection the mean of the three selected lines was 49.23 (0.35). Direct selection

on egg diameter for four generations produced a shift of 4.45 |tm or 2.5 op from the

common base population mean (Figure 2-1). Figure 2-2 illustrates the mean egg diameter

([tm SE) in the base population before selection and among lines after selection began.

Egg diameter data for selected Line 1 in Generation 7 were lost due to a corrupted

computer file and therefore were not included in calculations of realized heritability.

Cumulative realized heritability was calculated using the method of Hill (1972) as 0.58.

Bootstrapping based on re-sampling with replacement, and stratified by generation and

line, was used to calculate 95% confidence intervals around this estimate. Confidence

intervals based on 1000 replicates were 0.51 to 0.66.

Discussion

There has been considerable interest in the evolution of life histories in marine

invertebrates, and egg size has played a critical role in many of the quantitative modeling

approaches that have been used to try to identify selection pressures and processes that

influence the evolution of developmental mode in this group. In order for evolution to

occur, some component of phenotypic variation in egg size must be additive genetic

variation. My objective was to determine if the assumption of many models, that egg size

can and does respond to selection, is reasonable by estimating h2 for a marine









invertebrate with a wide geographic range, small egg, and obligate planktotrophic larvae.

All estimates of heritability carry the constraint that they only apply to the population

measured under those environmental conditions and are not specifically applicable to

other populations (Falconer and Mackay 1996).

I have shown that there is significant additive genetic variation for egg size in the

Pearl Harbor population ofH. elegans using various analytical techniques. The response

of 2.5 op to direct selection on egg size indicates that there is substantial potential for egg

size to respond to varying selective pressures, at least in the direction of an increase. It is

apparent in Figure 2-2 that variation within selected lines was not reduced relative to

either values before selection, or to control lines.

In a half-sib breeding design, offspring have one parent in common and the other

parent different. The degree of resemblance among half-sibs (the covariance of half-sibs)

represents half the variance of the common parent or a quarter of the additive genetic

variance (Falconer and Mackay 1996). The estimate of heritability reported here (0.45)

was based on the sire component of variance. One of the advantages of using the sire

component for this estimate is that it does not include maternal or dominance effects

(Falconer and Mackay 1996). It is possible to use this breeding design to estimate these

effects by examining the variance within dams (C2DAM), since it includes both maternal

(common environment) and dominance effects, and comparing it to the estimate based on

the sire component. However, since my estimate of G2DAM was not statistically

significant (P = 0.667), and the proportion of Vp explained by this estimate was

essentially zero (Table 2-2), further extrapolation based on this estimate cannot provide

useful information. Maternal effects are widely recognized as important components of









Vp (Mousseau and Fox 1998), although this is more common in mammals and animals

with longer periods of maternal care. Hydroides elegans has external fertilization. All

development up to a competent larva takes place in the water column and there is no

evidence of parental care. However, it is possible that the small sample size in my

breeding design (10 sires, 21 dams) resulted in an overestimate of o2SIRE, obscuring my

ability to detect C2DAM and estimate maternal effects. Therefore, based on these data, I

cannot say whether maternal effects are contributing significantly to the total phenotypic

variance.

Cumulative realized heritability is considered the most precise of the various

methods for determining narrow-sense heritability (Hill 1971). The cumulative realized

heritability estimate does include maternal effects, since it is based on response to

selection, but passing six generations under laboratory conditions prior to the beginning

of selection minimized the phenotypic expression of any maternal effects. The half-sib

breeding design gave only a slightly lower estimate of h2 (0.45) compared to the

cumulative realized heritability (0.58). The fact that two estimates are similar lends some

support to the idea that maternal and dominance effects are not large in this population

with respect to this trait.

It is thought that additive genetic variation for traits closely related to fitness, such

as egg size, should be under constant directional selection, eroding VA and resulting in

low heritabilities (Gustafsson 1986, Roff and Mousseau 1987, Falconer and Mackay

1996). This trend is generally supported for life-history traits as compared to

morphological traits (Mousseau and Roff 1987). However, many exceptions can be

found (e.g., Levin et al. 1991, Gibson 1993, Fox and Csezak 2000, Edmands 2003),









including this work. Houle (1992) pointed out that because h2 is a ratio of VA to Vp, the

magnitude of Vp greatly influences the outcome: low h2 could be the result of low VA or

high Vp. Therefore, it is a biased estimate of additive genetic variance and potentially a

misleading indicator of the ability of a particular trait to respond to selection. Using

coefficients of variation, that is VA standardized by the trait mean rather than Vp, is

recommended as a more useful approach. My estimates of CVA and CVR give very

similar values (3.22 and 3.59 respectively) implying that nearly half of the total variation

present is additive genetic. This is consistent with my estimate of h2.

Using the traditional methods of estimating h2, and also by calculating CVA and IA, I

have found significant levels of additive genetic variance for the trait of egg size in the

Pearl Harbor population of H. elegans. Several hypotheses have been offered to explain

the apparent paradox of finding VA for a life-history trait that is closely linked to fitness

and, presumably, under directional selection. One such hypothesis is the presence of

microevolutionary trade-offs. Microevolutionary trade-offs occur when a change in one

trait that acts to increase fitness is linked to a change in another trait that decreases fitness

(Stears 1992). These relationships can act to constrain the simultaneous evolution of

suites of fitness-related traits and thereby maintain additive genetic variance in a

population. Examination of the correlated responses of other fitness-related traits to

artificial selection for increased egg size is one way to elucidate this relationship.

Table 2-1. Number of females used to establish six individual lines in Generation 7
Line Number of Females
1 12
2 12
3 15
4 15
5 16
6 16









Table 2-2. Half-sib breeding design; Fully nested ANOVA and variance component
analysis for egg diameter in Hydroides elegans (number of observations 550,
s = 10, d= 2.1, k= 2.6)
Source df EMS F P Proportion


(X 10-5) Vp explained


Sire
Dam (Sire)
Daughter (Dam (Sire))
Error






45

40

35

30

25

20

15

10

5

0 _


9
11
34
495


4.149
1.575
2.047
0.251


16.54
6.28
8.16


0.069
0.667
0.0001


11.13
0
37.11
51.75


* Generation 6
l Generation 11


40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55
Egg diameter ([tm)
Figure 2-1. Response to direct selection on egg diameter for four generations of in the
polychaete Hydroides elegans. Selection began from a common base
population in Generation 6 and continued from Generation 7 through 10
















- Selected Line 1
--- Selected Line 3
A Selected Line 5
- -X- Controls Line 2
- -X- Controls Line 4
- -0- Controls Line 6
Common Population






V %


Gen 3 Gen 4 Gen 5 Gen 6 Gen 7 Gen 8 Gen 9 Gen 10 Gen 11

Generation

Figure 2-2. Mean egg diameter (+ SE) before and after direct selection for increased egg diameter in replicate selected (1,3,5) and
control (2,4,6) lines ofHydroides elegans















CHAPTER 3
DIRECT AND INDIRECT RESULTS OF ARTIFICIAL SELECTION FOR
INCREASED EGG SIZE IN THE SERPULID POLYCHAETE Hydroides elegans

Introduction

Marine invertebrates exhibit a wide diversity of developmental strategies and their

mode of development is thought to have important effects on dispersal and population

structure (e.g., Hedgecock 1982, Levin and Huggett 1990, McMillian et al. 1992, Hoskin

1997, Collin 2001), geographic range (Scheltema 1989, Kohn and Perron 1994, but see

Emlet 1995), and rates of speciation and extinction (Strathmann 1985, Jablonski 1986,

1987). Egg size has been the focus of a series of quantitative modeling efforts to

analytically assess the patterns we observe in marine invertebrate life histories.

Beginning with Vance's influential fecundity-time model (Vance 1973a,b), numerous

authors have examined trade-offs between egg size, fecundity, mortality and their

relationship with the evolution of different reproductive strategies (Christiansen and

Fenchel 1979, Caswell 1981, Perron and Carrier 1981, Grant 1983, Roughgarden 1989,

McEdward 1997, Levitan 2000). Another set of models examines fertilization kinetics,

which also can pose selective pressures on egg size and thus developmental mode

(Levitan 1993, 1996, Luttikjizen 2004).

Many authors have emphasized that the suite of characteristics that defines a larval

type has evolved in conjunction with post-larval and adult characteristics, rather than in

isolation. Egg size and fecundity are two members of a much larger complex of

life-history traits that has co-evolved, presumably under the influence of natural selection









(Havenhand 1995, Ramirez Llodra 2002). Egg size and fecundity are both directly

related to parental fitness (Roff 1992, Bernardo 1996) and many authors have mentioned

the importance of interactions between larval characteristics and other parts of the life

cycle (e.g., Vance 1973a,b, Christiansen and Fenchel 1979, Todd and Doyle 1981,

Strathmann 1985, Moran 1994, Pechenik et al. 1998, Gimenez et al. 2004). Few efforts

have been made to examine the implications over the entire life cycle of selective

pressures on these important and inter-related fitness components.

Much of life history theory is based on the assumption of trade-offs among traits

that contribute to fitness, that is, that natural selection cannot produce unlimited

simultaneous increases in individual fitness components. This could result in creating

theoretical optima based on trade-offs between fitness components (Roff 1992, Steams

1992). Fitness components, and therefore trade-offs between them, must have a genetic

basis for selection to act. Alleles that increase fitness without a cost are expected to

achieve a frequency of one very quickly; therefore genetic correlations between

fitness-related traits that have been subject to simultaneous selection are expected to

become negative.

Several methods are in use to examine the genetic architecture of fitness-related

traits. Genetic correlations may be estimated by examining the resemblance between

relatives using designs similar to those for estimating heritabilities (Falconer and Mackay

1996, Lynch and Walsh 1998). These correlations are caused by pleiotropy, where traits

of interest are influenced by common genes; however, they may also be caused by

linkage disequilibrium between distinct loci, each influencing a different trait. But

genetic correlations between life-history traits can be difficult to extrapolate from









breeding designs. Correlations estimated in this manner are dependent on linkage

equilibrium and other requirements (Rose et al. 1990), and standard errors around these

estimates are often very large (Koots and Gibson 1996).

A second, and more direct method that can be used to look for evidence of

evolutionary trade-offs is artificial selection. Artificial selection experiments allow

workers to manipulate the frequency of alleles associated with a phenotypic value of a

selected character by controlling the parental genetic contribution to the next generation

in laboratory populations, thereby producing divergent phenotypes. If selected lines

differ from control lines in traits other than the selected trait (and environmental variation

has been kept constant across generations), this response indicates an additive genetic

correlation between the traits (Cheverud 1984). Repeated episodes of selection over

multiple generations are more likely to reveal potential interactions between fitness

components that may be subtle or difficult to measure across a single generation.

Artificial selection experiments are a particularly powerful way to explore trade-offs

(Reznick 1985, 2000, Brakefield 2003). However, selection experiments are subject to

experimental or procedural artifacts that may be difficult to control, and the complexity

and interactive nature of response to selection can produce varying outcomes (Harshman

and Hoffman 2000).

Selection experiments on life-history traits have a long history, with the

preponderance of work in invertebrates involving insects. For example, Englert and Bell

(1969, 1970) selected on larval development time in the flour beetle Tribolium

casteneum, Roff (1990) selected on wing dimorphism (an indicator of development time)

in the sand cricket Gryllusfirmus, Bradshaw and Holzapfel (1996) selected on









development time in the pitcher-plant mosquito Wyeomyia smithii, and Palmer and

Dingle (1986) selected on wing length (an indicator of dispersal ability) in the milkweed

bug Oncopeltusfasciatus. Focusing on correlated responses in egg size, Tucic et al.

(1998) and Seslija and Tucic (2003) found that bean weevils, A.,luilvteli Ie'\ obtectus,

selected for faster development times lay significantly larger eggs. A similar correlated

response in egg size to selection on development time is reported for Drosophila

melanogaster by Bakker (1969). Selection experiments involving life-history traits in D.

melanogaster are numerous, especially focusing on trade-off between longevity,

fecundity, and larval development time (e.g., Tantawy and El-Helw 1966, Partridge and

Fowler 1992, Zwaan et al. 1995, Nunney 1996).

Several studies have examined response to selection on egg size. A significant

response to selection toward both large and small eggs has been reported in Drosophila

melanogaster in several studies (Bell et al. 1955, Parsons 1964, Schwarzkopf et al. 1999).

Azevedo et al. (1997) found evidence of a selective advantage to larger eggs in D.

melanogaster, including increased embryonic viability, hatchling weight, and larval and

pre-adult development rates. Czesak and Fox (2003) selected directly on egg size in the

seed beetle Stator limbatus and report correlated responses in lifetime fecundity and

female body mass.

Among marine invertebrates genetic correlations for selected life-history characters

have been reported using breeding designs. In mollusks, Ernande et al. (2003) report a

positive correlation between reproductive plasticity, growth and survival for the bivalve

Crassostrea gigas. Hilbish et al. (1993) discuss the common assumption that variation in

larval and juvenile growth rates have a common genetic basis, but find no significant









correlation between variation for larval and juvenile shell growth in the bivalve

Mercenaria mercenaria. Gibson (1993) explored genetic correlations among life-history

traits in the poecilogonous opisthobranch Haminoea callidegenita and found that many

reproductive traits showed significant genetic correlations while exhibiting little or no

phenotypic correlations. In crustaceans, Arcos et al. (2004) estimate the genetic basis for

a series of reproductive traits in the white shrimp Penaeus vannamei and report no

correlation between egg diameter and other traits, but did find a positive correlation

between egg number and egg total protein. Levin et al. (1991) investigated the genetic

architecture of the poecilogonous polychaete Streblospio bendicti using a diallel design

and confirmed a negative genetic correlation between egg size and fecundity, as well as

the expected positive genetic correlation between fecundity and female body size.

Ernande et al. (2004) used a nested half-sib mating design to examine five traits in

Crassostrea gigas: larval shell length, size at settlement, weight after metamorphosis,

juvenile weight, adult weight, but did not examine egg size. They report that larval

development rate is positively genetically correlated with size at settlement, but

negatively correlated with both metamorphic success and juvenile survival. Little work

has been done to examine the consequences across the life cycle of shifts in egg size.

The purpose of this work was to examine the direct and correlated results of

artificial selection to increase egg size in Hydroides elegans Haswell, 1883. H. elegans is

a marine, serpulid polychaete worm that produces numerous planktotrophic larvae and

can be cultured in the laboratory over multiple generations. Larvae will readily settle and

metamorphose in culture in the presence of natural biofilms (Carpizio-Ituarte and

Hadfield 1998). Fertilization and larval development takes place in the water column









with no maternal care and H. elegans develops as a protandrous hermaphrodite (Ranzoli

1962). The generation time is 16 to 28 days at optimal temperature, salinity, and food

concentrations (Qiu and Qian 1998). Both larvae and adult worms can be fed the

chrysophyte alga Isochrysis galbana, easily cultured in the laboratory. Using artificial

selection for increased egg diameter, correlated responses were documented in a suite of

life-history characters: egg diameter, fecundity, total egg energy (at 6 weeks

post-fertilization), larval size at 18 to 20 h post-fertilization, and at competence (5 d),

juvenile tube length at 21 d, and adult dry weight (at 6 weeks).

Methods

Collection, Spawning, and Selection

Wild Hydroides elegans were collected at Ford Island, Pearl Harbor, Hawaii in

November 2002. Details of the methods for spawning, larval culture, measurement of

egg diameter and selection are described in detail in Chapter 2. Field collected worms

were raised under controlled environmental conditions for six generations before

selection began in Generation 7. Between 12 and 16 females were used to establish each

line in Generation 7 (Table 2-1). In subsequent generations (Generations 8 to 10), 16

mothers were kept in each line. All crosses were unique and the fathers were chosen

haphazardly without regard to the mean egg size of the clutch from which they hatched.

No crosses were permitted between brothers and sisters within lines. Three replicate

control and three replicate selected lines were established with 128 worms in each line,

for a total of -768 worms in each generation. All females were spawned at 6 weeks

post-fertilization and females were retained in the selected lines if the mean diameter of

their eggs was > 1o than that of the previous generation. For control lines, every third

female spawned was retained in without regard to egg diameter.









Larval culture was performed according to methods described by Unabia and

Hadfield (1999). Cultures were maintained at 250C, and fed 6 X 104 cells mL-1 day-of

the chrysophyte alga Isochrysis galbana (Tahitian strain). Larvae were competent to

metamorphose at 5 d post-fertilization, when I exposed 20 to 25 larvae to individual

small plastic chips with natural biofilm as the settlement cue. Each chip was isolated in a

single well of a standard ice cube tray filled with 15 mL of 0.22[tm filtered seawater

(FSW) and algal food was maintained at 6 X 104 cells mL-1 day-. On Day 10 all

juveniles but the one nearest the center of the chip were killed by destroying the tube, so

that growth from that point took place at a density of 1 worm well-'. Food density was

increased 6-fold and water, food, and container (ice tray) were all changed daily until

adults were spawned.

I observed that some female worms in both selected and control lines

spontaneously released a few eggs (from 10 to a few hundred eggs) shortly after the daily

food and water change. I collected all the early-spawned eggs that were produced after

the daily food and water change over a four- day period (October 10 to 14 2003) prior to

the scheduled spawning of Generation 9 using a Pasteur pipet. There were 25 selected

and 7 control females (n = 32 females) that produced early-spawned eggs over this

four-day period. I began the scheduled spawning of these same females two days later.

Therefore, the timing of the sample of early-spawned eggs ranged 6 to 12 d prior to the

scheduled spawning date for these individuals. I used digital imagery (Chapter 2) to

measure mean egg diameter of these early-spawned eggs (n = 10 eggs female-) and

compared this to mean egg d measured at the time of induced spawning (later-spawned

eggs) of the same female.









Selection continued for four generations with lines terminated at Generation 11,

when adult dry weight, total egg energy, fecundity and final egg diameter were

determined. Larval size at two different time points, and juvenile tube length were

measured in Generation 10, as no larvae or juveniles were available after termination of

the lines.

Larval Size

At 18 to 20 h post-fertilization sub-samples of trochophore-stage larvae were taken

from nearly all of the crosses (between 11 and 16 of the 16 crosses in each line) in

Generation 10. Any crosses for which I had not obtained video by the time they had

developed to 20 h were not sampled. Larvae from a single cross were placed on a clean

slide and excess water was removed with a micropipette. A cover slip was placed on

each slide using ScotchTM tape to elevate the slip above the slide. Using a technique

similar to that used by Leonardos and Lucas (2000) for bivalve larvae, videotapes of the

swimming larvae were obtained using a Sony Digital Video Camera Recorder (Model

DCR-TRV30) mounted on an Olympus light microscope. A stage micrometer was used

as a scale for each series of shots. Videos were downloaded to a Macintosh G4 where

iMovie software was used to grab still photos of larvae (n = 10 larvae cross-1). NIH

Image software was used to measure the width across the prototroch, and the distances

from the prototroch to the apex of the episphere and hyposphere, respectively. Larval

size was then calculated as the volume of two circular cones (V = 7 r2h/3; r = 1/2

prototroch width and h = distance to apex of episphere or hyposphere). The same process

was repeated for competent larvae at 5 d post-fertilization, including between 9 and 15 of

the 16 crosses in each line (n = 10 larvae cross-1) in Generation 10.









Juvenile Tube Length

Shortly after metamorphosis a calcareous tube is constructed by the juvenile. The

tube lengthens as the worm grows, and tube length is a reasonably reliable indicator of

juvenile size (Qian and Pechenik 1998, Qiu and Qian 1998). This indirect method of

measuring juvenile size was chosen to minimize loss of individuals from laboratory lines,

since removal from the tube results in death. Images of juvenile tubes at 21 d were

obtained using the Nikon CoolpixTM 990 described above, attached to an Olympus

dissecting microscope. Each individual chip was removed from the ice tray and placed in

a plastic Petri dish along with a 1cm length of ruler. Images were obtained for all

offspring (n = 8 juveniles cross-) from between 10 and 16 crosses from each line in

Generation 10. The images were analyzed for tube length using NIH Image software.

Total Egg Energy

In Generation 11 each sub-sample that had been used to measure egg diameter was

placed in a cryotube (FisherBrand 0.5 mL screw-top tube) and frozen at -200C. Frozen

samples from each line were later analyzed for organic content using a

micro-modification of the dichromate oxidation technique (Jarrett and Pechenik 1997,

Gosselin and Qian 1999) against a glucose standard (0 to 20 |tg C).

Eggs were thawed at room temperature and distilled water was added to raise the

volume to 200 [LL. Four replicate sub-samples of 10 pL were obtained and counted on a

Petri dish using a dissecting microscope and a hand-counter. This allowed calculation of

the total number in the sample that was needed to complete the fecundity estimate (see

below). The counted samples were removed from the Petri dish using a micropipette and

placed in a 13mm glass test tube. Between 300 and 500 eggs were placed in each of









three replicate tubes from a particular cross. Sixteen samples, each corresponding to a

different randomly selected cross from each line, were prepared. Tubes were allowed to

stand until the eggs had settled to the bottom and excess water was removed using a

micropipette.

The eggs were incubated in 1 mL concentrated (85.5%) phosphoric acid (15 min at

1050C) to remove excess chloride. Tubes were cooled to room temperature and 1 mL of

0.04% acid dichromate was added to each tube to oxidize the sample. Tubes were mixed

on a vortex mixer followed by incubation at 1050C for 15 min. Reduction in dichromate

concentration indicates the amount of organic carbon oxidized.

Samples were diluted by adding 5 mL distilled water followed by vortex mixing. A

0.5 mL aliquot was removed from each tube and combined with 4.5 mL of cadmium

iodide starch reagent (Parsons et al. 1984) and allowed to stand at room temperature for

20 min. Each sample was then diluted again by adding 5 mL distilled water and

examined spectrophotometrically (Kontron Instruments, Model Uvikon 930) at 575 nm.

Total energy was estimated as |tg C equivalents in glucose and converted to mJ using the

relationship 1 |tg C = 39 mJ (McEdward and Carson 1987).

Though widely used in comparative studies of egg energy in marine invertebrates,

the dichromate oxidation technique is known to underestimate energy contributed by

proteins (Gosselin and Qian 1999). Pernet and Jaeckle (2004) analyzed this technique

across species within annelids and echinoderms and conclude that the likely cause is that

different proteins oxidize to different degrees in the acid dichromate reaction. Their

comparison points out that across a wide range of species, developmental modes, and egg

sizes there is probably a wide range of differential allocation of various proteins and









because of this the absolute value of energy egg-1 may be underestimated using this

assay. Here I used the acid dichromate technique for a relative comparison of mean total

energy egg- within species, over a small range of egg sizes after short-term selection.

Data presented by Pemet and Jaeckle (2004) for planktotrophic annelids show that error

of total energy estimates based on this technique within species is generally small (e.g.,

estimates for serpulid polychaetes include Serpula columbiana with 0.65 mJ egg 1

0.015 SE, or 2.3% and Hydroides sanctaecrucis with 0.40 mJ egg 1 0.023 SE, or 5.8%)

and therefore unlikely to be important over the small changes in egg size considered here.

There are two possible ways that an increase in error could affect my results. First, in the

unlikely event that short-term selection on egg size resulted in a change in the type of

protein allocated to eggs of selected lines relative to control lines, error could be

increased. Second, if selection produced increased densities of a particular protein in

larger eggs, this could affect the error. Either of these would be an interesting outcome

and should be considered by future workers using techniques specific to these questions.

Absolute values of energy estimates based on this technique may be underestimates.

Fecundity

In Generation 11, after the sub-sample of eggs for measurement and total energy

oxidation were removed, the remaining eggs from each female (n = 435) were fixed in

5% formalin in FSW (-2% formaldehyde). These samples were set aside and eggs were

counted later. The samples were diluted to 30 mL using filtered seawater and a standard

50 mL graduated cylinder. The sample was mixed by inversion and 4 sub-samples of

250 p.L each were placed on a Petri dish and counted using a dissecting scope and a hand

counter. The number of eggs in the fixed sample was then calculated. The number of









eggs from the frozen samples (see above) was added to this number to obtain the absolute

number of eggs produced by each female. The relative fecundity was also calculated by

dividing the absolute number of eggs per female by her dry weight (see below) to give

-1
eggs mg.

Adult Dry Weight

The dry weight of each adult in Generation 11 (n = 685) was determined. Small

squares of aluminum foil were numbered and pre-tared on a Cahn (Model C-35)

micro-balance. Immediately after spawning sex of the worm was recorded, and each

individual was removed from the calcareous tube using forceps to crack the tube. Worms

were rinsed in distilled water and placed on aluminum foil squares. Specimens were

placed in a drying oven at 750C for 48 h. Worms were re-weighed on the Cahn balance

to the nearest 0.1 |tg and the weight of the foil was subtracted providing a post-spawn

weight for each individual.

Data Analysis

I used a fully nested mixed model ANOVA with fixed effects at the level of

treatment only with lines nested within treatments and crosses nested within lines for the

traits egg diameter, total egg energy, larval sizes at 18 to 20 h and at 5 d, and juvenile

tube length (all nested at all levels between selected and control lines). Data were

visually checked for radical departures from normality and heteroscedasticity and none

were found. For the analysis of adult dry weight and fecundity the same fully nested

mixed model ANOVA was used with fixed effects at the level of treatment only and lines

nested within treatments. In order to test for an effect of selection on the relative

numbers of males and females across generations I used a model I ANOVA. In all cases









Type III sums of squares were calculated using the general linear model of SAS Version

6.12 (PROC GLM, with RANDOM statement and TEST option), c = 0.05 unless

otherwise stated. To compare mean egg d between early- and later-spawned eggs within

mothers I used a paired t-test in JMP 5.1 (SAS Institute, Inc.).

Results

Direct Response to Selection

Mean egg diameter in the base population (Generation 6) was 44.78[tm (+ 0.15

SE); the mean egg diameter in the selected lines after selection (Generation 11) was

49.23 tm (0.35 SE). Direct selection for four generations for increased egg size resulted

in a shift of 4.45 |jm or 2.5 op from the common base population mean (Figures 2-1, 2-2).

Figure 3-1 illustrates the continued positive slope of the cumulative response to selection

through Generation 11 as the difference between the mean egg diameter of the replicate

selected and control lines. Egg diameters between selected and control lines were

significantly different at Generation 11 (P = 0.0006, Table 3-1).

Correlated Responses to Selection

Replicate lines selected for larger egg diameter were found to have a significantly

higher mean value for total energy relative to the control lines (P = 0.0350, Table 3-2).

The mean value (mJ egg'1 SE) for the selected lines was 0.37 (0.17) and for the control

lines the mean was 0.29 (0.18).

Fecundity, calculated as the absolute number of eggs produced by a female (+ SE),

was significantly higher in the large-egg selected lines relative to controls (P = 0.0304,

Table 3-3). Female worms in the large-egg lines produced a mean of 10071 (481) eggs

and females in control lines produced a mean of 7936 (418). When egg number was









standardized by the dry weight of the mother to give eggs mg-1 the difference between the

selected and control line means became less apparent, significant at a = 0.10 (P = 0.0679,

Table 3-4). Both of these analyses were also performed on datasets omitting all mothers

that had produced eggs prior to the induced spawning at 6 weeks (see Spontaneous

Spawning below) and the outcome was not changed.

No significant difference was found in the dry weight of adult females from

selected lines relative to control lines (P = 0.6567, Table 3-5). The mean dry weight

(mg SE) for females in the selected lines was 0.80 (0.019) and for controls was 0.82

(0.026). The same result was found if males and females were combined within

treatments (P = 0.5791), and for males alone (P = 0.8526). The Generation 11 mean dry

weight of adults of both sexes from the selected lines was 0.81 (0.025) and from the

control lines this value was 0.83 (0.020). Males could not be distinguished from females

on the basis of dry weight within either the control (P = 0.1974) or selected (P = 0.1698)

lines.

No significant difference was detected in larval size (volume) at 18 to 20 h between

selected and control lines (P = 0.3392, Table 3-6). The mean value for larval size

([tL SE) at 18 to 20 h for the large-egg selected lines was 1.12 X10-4 (0.064 X 10-4).

The control lines had a mean value of 1.14 X10-4 (0.047 X 10-4).

Larval size (volume) at competence was significantly larger in the selected than in

the control lines at a = 0.10 (P = 0.0894, Table 3-7). The mean size at competence in the

selected lines size ([tL SE) was 1.29 X 10-3 (0.041 X 10-3) and for the control lines it

was 1.16 X 10-3 (0.036 X 10-3).









Selected lines did not significantly differ from control lines in tube length at 21 d

(P = 0.1249, Table 3-8). Mean tube length (mm + SE) was 8.08 (0.31) in the selected

lines and 8.60 (0.10) in the control lines.

Direct and correlated responses to selection for increasing egg diameter are

summarized in Table 3-9.

Spontaneous Spawning and Sex Ratios

In an effort to explore the relationship between egg diameter and time of spawning,

I compared the mean egg diameter from spontaneously or early-spawned females to that

of eggs produced at 6 weeks by the same female (n = 32) in Generation 9. The

later-spawned eggs were significantly larger than the early-spawned within individual

mothers (paired t-test, t = 9.8582, P < 0.0001).

In an unexpected development, I observed that females in the selected lines tended

to spawn spontaneously more frequently and earlier than control females. Timing and

frequency of these events was not recorded since it was not part of the experimental

design. Within this design I controlled the absolute age of individuals at spawning (6

weeks), but not the time to sex change. At 6 weeks an individual female might have been

producing eggs for variable periods of time. Considering that within mothers

later-spawned eggs are significantly larger than earlier-spawned eggs, was it possible that

large-egg selected mothers had been producing eggs longer than control mothers? If

selected lines were changing sex earlier than control lines, one prediction would be a

higher proportion of females in selected lines relative to controls at 6 weeks. Since the

sex of each individual was recorded at the time of spawning for all generations I was able

to document an effect of selection on sex ratios, showing that the proportion of females at









6 weeks increased in the selected lines, relative to controls, across Generations 7 through

11 (P = 0.0122, Table 3-10). The mean percentage of females in selected lines ranged

from 64.70% to 74.30% and for control lines the range was 59.21% to 64.21% (Table

3-11).

H. elegans has been described as a sequential hermaphrodite in all accounts since

Ranzoli (1962) clarified their status as hermaphrodites, and I also found this condition in

all control lines (as well as in thousands of individuals raised under laboratory conditions

prior to selection). However, in all 3 replicate selected lines on at least one occasion I

observed simultaneous eggs and sperm being produced by the same individual (again,

spawned at 6 weeks).

Discussion

More + Bigger = Better... or Does It?

I predicted that there would be negative correlations between at least some fitness

related traits, because we expect alleles underlying advantageous genetic correlations to

be rapidly fixed in natural populations. Yet I found that large-egg selected females

produced more eggs, with higher energy content, that grew into larger larvae at

competence. Increased size at settlement is considered a selective advantage (reviewed

by Pechenik 1999). No trade-off with this apparently advantageous condition (the

production of more energy-rich eggs that grow into larger competent larvae) was

immediately evident by evaluating the initial traits of interest. This set of apparently

advantageous correlated traits raises the question what other traits offset the selective

advantage of larger egg size in natural populations?

One of the advantages of using a selection experiment to explore evolutionary

processes is that correlated responses to selection can reveal unexpected relationships









between traits that were not predicted at the start of the experiment (Harshman and

Hoffman 2000). The observation that selected worms appeared to produce eggs

spontaneously both earlier and more frequently than control lines prompted me to

examine the relative numbers of males and females across generations and I found that

direct selection for increased egg diameter produced significantly higher proportion of

females at 6 weeks of age. Considering that I also found that egg diameters are

significantly larger in females that spawn later than those that spawn earlier, I propose

that selection for increased egg size was manifest through selection for earlier sex

change. This earlier sex change resulted in selected individuals spending less time as

males relative to controls, consequently reducing the number of offspring they could

potentially father.

Sex allocation theory predicts that sex change (sequential hermaphroditism) is

favored when an individual experiences differential reproductive success in relation to

size (or age). According to the size-advantage hypothesis selection should favor

protandry if male reproductive success does not increase as individuals grow larger,

while female reproductive success does (Ghiselinl969). There has been broad empirical

support for this hypothesis (e.g., Charnov 1979a for the protandrous shrimp Pandalus,

Warner 1984a,b for protogynous coral reef fishes, Berglund 1990 for the protandrous

polychaete Ophryotrochapuerilis). The optimal timing of sex change within individuals

is also predicted by the model (Charnov 1979b, 1982). Sex change should theoretically

occur when an individual's reproductive value (future expectation of reproductive

success weighted by the probability of survival) as a member of its present sex declines

below the reproductive value of the opposite sex. Alterations between male versus









female investment should only be favored if the gain in reproductive value in one sex

exceeds the loss in the other sex (Leigh et al. 1976). An individual should change sex if

it can increase its lifetime reproductive value by doing so, and should change at that point

where the sum of the male and female components is maximized. The earlier sex change

that resulted as a correlated response to selection in H. elegans could reduce the

contribution of the male component to lifetime reproductive value to such an extent that

the female component cannot compensate for the loss. This assumes that males are not

able to shift their own sexual maturity earlier, or at least not sufficiently earlier to make

up for the loss in reproductive fitness resulting from terminating sperm production

earlier. Earlier sex change could occur as a result of male and female gametogenic

processes being controlled by different and unlinked genes, that is, a response that shifts

the timing of investment in oogenesis manipulates a different suite of genes entirely than

those that control the timing of spermatogenesis. It is likely that the spermatogenic

pathway has already been selected to function at the earliest possible age, and therefore

smallest size, because H. elegans is an opportunistic polychaete, and among the first

settlers into disturbed environments. Maternal biosynthesis, mobilization and

bioaccumulation of nutrients during oogenesis is an energy intensive activity and it is

reasonable to assume that there is a cost involved in pushing this activity to an earlier

time in the life cycle until it nearly, or actually, overlaps with sperm production.

Therefore, I propose that the constraint on increased egg diameter (at 6 weeks) in this

population is selective pressure to maintain the optimum male contribution to lifetime

reproduction. One question that follows on this reasoning is, if it is costly to change sex

early, then how is variation for the trait of "time to female" maintained in the population?









One possible explanation is frequency-dependent selection, that it is very profitable to be

an early sex-changing female when the trait is rare (and everyone else is male), but

becomes less profitable, even costly, as the frequency increases.

Though it is widely reported that sequential hermaphrodites change sex as their size

increases, field studies commonly report extensive overlap between male and female size

classes (Wright 1989, Sewell 1994, Collin 1995). Ranzoli (1962) surveyed field

populations ofH. elegans (as H. norvegica) over a three year period using segment

number as an indicator of size and also found considerable overlap between the sexes.

Population studies necessarily include overlapping generations; however, even with the

discrete generations raised here I observed no significant difference in somatic dry weight

between the sexes. This variation in size at 6 weeks may indicate significant genetic

variation in maximum body size, or possibly in growth rate in this population. Many of

the males that remained in both selected and control lines at 6 weeks were quite large and

the mean dry weight for males was larger (although not significantly so) in both

treatments.

Given the positive correlation between egg size and egg number reported here, one

may ask how often, and under what conditions the predicted trade-off between egg size

and egg number has been empirically demonstrated. Varying environmental conditions

can result in an interaction between genotype and environment (G X E) and reveal a

trade-off. Czesak and Fox (2003) found that the magnitude of the trade-off between egg

size and fecundity varied between environments in the seed beetle Stator limbatus.

Varying food levels revealed a trade-off between increased fecundity and mortality in the

polychaete Dinophiilus gyrociliatus (Prevedelli and Zunarelli Vandini 1999, Prevedelli









and Simonini 2000). Tatar et al. (1993) nicely demonstrate the trade-off between

increased egg production and increased age-specific mortality in the beetle

Callosobruchus maculatus. A longstanding idea concerning the evolution of offspring

number is the "Lack clutch size" hypothesis in birds, that is, that clutch size has evolved

towards the size that will maximize the number of successful fledglings. Empirical tests

of the "Lack clutch", and deviations from it, continue to produce mixed results. For

example VanderWerf (1992) used meta-analysis on data from 77 different ornithological

studies and found that the hypothesis was not supported. Frequently cited examples of

the egg size-egg number trade-off in marine invertebrates are poecilogonous species such

as the polychaetes Streblospio benedicti (Levin and Creed 1986, Levin et al. 1987) and

Capitella sp. (Qian and Chia 1991) that have sympatric populations capable of producing

either planktotrophic or lecithotrophic larvae. The reproductive output of both strategies

is similar but lecithotrophs produce fewer, larger eggs while planktotrophic populations

produce smaller, more numerous eggs. The negative genetic correlation between egg size

and fecundity was confirmed for Streblospio benedicti by Levin et al. (1991). However,

poecilogony is an extremely unusual developmental strategy (Hoagland and Robertson

1988, Bouchet 1989) and may not represent a general pattern. In examining the more

typical reproductive strategies the genetic evidence of a trade-off within species

regarding egg size and egg number is often mixed or absent altogether (e.g., Czesak and

Fox 2003 for the seed beetle Stator limbatus, Schwarzkopf et al. 1999 for Drosophila

melanogaster, Allan 1984 for the copepod Mesocyclops, Stearns 1983 for the mosquito

fish Gambusia affinis, Reznick 1982 for the guppy Poecilia reticulata).









Conclusions and Future Directions

The constraint on increased egg diameter (at 6 weeks) in the Pearl Harbor

population of Hydroides elegans appears to be selective pressure to maintain the

optimum male contribution to lifetime reproduction by delaying the shift to the female

sex. Considerable genetic variation for egg size, fecundity, and the time to sex change is

present in this population and this is not surprising given that this population likely

includes substantial genetic diversity. Pearl Harbor is an extremely busy international

port, and H. elegans is a cosmopolitan member of the biofouling community that is

commonly found on ship and boat hulls, water intakes, and other underwater structures,

the population in Pearl Harbor likely has been assembled from multiple founding

populations.

One way to test the hypothesis whether the hermaphroditic life style is constraining

egg size is to perform a phylogenetic analysis that maps egg size and sex system onto a

phylogeny of family Serpulidae and reconstructing ancestral transitions. This type of

cladistic analysis was used by Rouse and Fitzhugh (1994) to test whether broadcast

spawning and planktonic larvae are pleisiomorphic for the polychaete family Sabellidae.

Another set of questions that arise from these results concerns the traits of sperm

that are produced by larger-egg selected hermaphrodites. It has been suggested (Levitan

1993, 1998) that sexual selection acts on gamete traits to produce suites of co-adapted

traits that include egg size, egg number, sperm velocity, and sperm longevity. Do sperm

traits vary with egg traits as selection for increased egg size proceeds? How correlated

are sperm traits and egg traits within individuals, within the population?

The observed variation in size of males suggests additional work on sex change in

this animal. It is reported that all individuals change sex, but many males remaining in









the population at 6 weeks were considerably larger than females. Allsop and West

(2003) showed that the relative timing of sex change was invariant across broad

taxonomic groups and largely predicted by maximum body size, that is, that a sex change

should occur at 72% of maximum body size. Data collected in this study do not provide

a way to test this prediction but population level studies would be a useful approach to

this question. This same data set could also inform us concerning the genetic variation

for maximum body size and the trade-off between growth and fecundity at the population

level.

Reznick et al. (2000) pointed out that studies evaluating life-history traits (and the

trade-offs between them) necessarily focus on specific characters. The choice of what

components of fitness are included in a study can make the difference between detecting

a fitness trade-off or not, since the influence of correlations between unmeasured traits is

not known. As efforts to examine the genetic basis of trade-offs broaden to include

marine invertebrate life-history evolution, methods that are the most integrative across

the life cycle (including various developmental stages/ages/sizes) and across the breadth

of traits that define a life history may be the most effective. Large sample sizes are often

required to detect genetic correlations using breeding designs and this is reasonable in

insect systems. Frequently in marine invertebrate systems this is a logistical

impossibility because culture of the larvae is time-, space-, and labor-intensive. It is

difficult to detect complex or unexpected patterns in studies that examine correlations

across a single generation when sample sizes become limiting. These patterns may be

more apparent against a dynamic background where observations are made across

multiple life-history stages and generations.















3

2.5

2

1.5

1

0.5

0
Gen 6 Gen 7 Gen 8 Gen 9 Gen 10 Gen 11
Generation

Figure 3-1. Cumulative response to selection for increased egg diameter in H. elegans
was positive through Generation 11. No error bars were calculated because
this is the difference between the mean of 3 selected lines and the mean of 3
control lines at each generation beginning in the base population (Generation
6) and ending at the end of selection (Generation 11).

Table 3-1. Egg diameter ([tm) in replicate selected vs. control lines ofHydroides elegans
after 4 generations of selection; Fully nested ANOVA
Source df EMS (X 10-4) F P
Treatment 1 147.0604 95.7328 0.0006
Line (Treat) 4 01.5686 6.1680 0.0001
Cross (Line (Treat)) 428 0.2545 18.1511 0.0001
Error 3916 0.0140 -- --


Table 3-2. Total egg energy in replicate selected vs. control lines ofHydroides elegans
(calculated as |tg C egg-'); Fully nested ANOVA
Source df EMS (X 10-4) F P
Treatment 1 2.535578 9.8169 0.0350
Line (Treat) 4 0.258603 2.3369 0.0611
Cross (Line (Treat)) 90 0.111605 1.6279 0.0032
Error 174 0.0686 -- --









Table 3-3. Absolute fecundity in replicate selected vs. control lines ofHydroides elegans
(eggs female-'); Fully nested ANOVA
Source df EMS (X 106) F P
Treatment 1 443.82396 10.5232 0.0304
Line (Treat) 4 42.662385 1.9105 0.1078
Error 403 22.330157 ---

Table 3-4. Relative fecundity based on dry weight of the female in replicate selected vs.
control lines ofHydroides elegans (eggs mg-1); Fully nested ANOVA
Source df EMS (X 106) F P
Treatment 1 795.873456 6.0931 0.0679
Line (Treat) 4 132.548182 2.5540 0.0386
Error 403 51.897613 -- -

Table 3-5. Adult female dry weight (mg) in replicate selected vs. control lines of
Hydroides elegans; Fully nested ANOVA
Source df EMS F P
Treatment 1 0.025218 0.2290 0.6567
Line (Treat) 4 0.112206 2.8075 0.0253
Error 432 0.0399669 -- -

Table 3-6. Larval volume ([tL) at 18 to 20 h post-fertilization in replicate selected vs.
control lines ofHydroides elegans; Fully nested ANOVA
Source df EMS (X 10-4) F P
Treatment 1 0.650836 1.1703 0.3392
Line (Treat) 4 0.558838 1.5227 0.2048
Cross (Line (Treat)) 69 0.366555 0.9246 0.6495
Error 648 0.3964 -- -

Table 3-7. Volume ([tL) of competent (5 d) larvae in replicate selected vs. control lines
ofHydroides elegans; Fully nested ANOVA
Source df EMS (X 10-6) F P
Treatment 1 2.44511 4.9009 0.0894
Line (Treat) 4 0.50505 1.8355 0.1324
Cross (Line (Treat)) 66 0.276535 4.9538 0.0001
Error 641 0.0006 .

Table 3-8. Juvenile tube length (mm) at 21 d in replicate selected vs. control lines of
Hydroides elegans; Fully nested ANOVA
Source df EMS F P
Treatment 1 47.0398 3.7050 0.1249
Line (Treat) 4 12.6558 0.6682 0.6162
Cross (Line (Treat)) 69 19.16195 4.7420 0.0001
Error 558 4.0409









Table 3-9. Summary of direct and correlated responses to selection for increased egg
diameter in replicate selected v controls lines ofHydroides elegans. All
significant differences represent positive correlations
Trait Selected Mean (+ SE) Control Mean (+ SE)
Egg d (im) 49.23 (0.35) 45.46 (0.083) *
Total Energy 0.37 (0.17) 0.29 (0.18) *


(mJ egg-1)
18 to 20 h larval
Vol ([LL)
5 d larval Vol
(2 L)
21 d Juvenile tube


1.12 X10-4 (0.064 X 10-4)

1.29 X 10-3 (0.041 X 10-3)


8.08(0.31)


length (mm)
Adult female dry 0.80 (0.019)
weight (mg)
Fecundity (eggs) 10071 (481)
Relative 13451 (834)
Fecundity
(eggs mg-1)
* P < 0.05.+P < 0.10. NSP>0.10


1.14 X10-4 (0.047 X 10-4)

1. 16 X 10-3 (0.036 X 10-3)


8.60 (0.10)

0.82 (0.026)


7936(418)
10462(798)


Table 3-10. Effect of selection (treatment) on the relative numbers of males and females
in Generations 7 to 11; Model I ANOVA
Source df EMS F P
Treatment 1 1.3958 6.28 0.0122
Gen 4 0.3852 1.73 0.1395
Gen*treat 4 0.1717 0.77 0.5427
Error 3461 0.2221 -- -

Table 3-11. Mean percent females present at spawning (6 weeks post-fertilization) in
replicate selected vs. control lines ofHydroides elegans in Generations 7 to 11
Mean Percent Females in Mean Percent Females in
Selected Lines (SE) Control Lines (SE)
Generation 7 64.70 (9.036) 61.86 (2.281)
Generation 8 64.88 (2.299) 59.21 (2.291)
Generation 9 72.11 (5.564) 61.72 (0.877
Generation 10 70.45 (9.022) 62.61 (6.820
Generation 11 74.30 (8.661) 64.21 (3.071
Grand Mean 69.29 (1.935) 61.92 (0.809














CHAPTER 4
CONCLUSIONS

The goal of this study was to investigate the evolvability of the trait of egg size, to

examine the direct and correlated responses to selection on increased egg size in a

specified set of life-history characters, and to explore how these correlations might

change as egg size was increased by artificial selection in the polychaete worm

Hydroides elegans. Life-history traits examined included egg diameter, fecundity,

relative fecundity, total egg energy (at 6 weeks), larval size at 18 to 20 h, larval size at

competence (5 d), tube length at 21 d, and adult dry weight (at 6 weeks). I have used

various analytical techniques to show that there is significant additive genetic variation

for egg size in the Pearl Harbor population ofH. elegans. The response of 2.5 op to

direct selection on egg size indicates that there is substantial potential for egg size to

increase in response to selective pressures (Chapter 2). I predicted that there would be

negative correlations between at least some fitness-related traits, because we expect

alleles underlying positive genetic correlations to be rapidly fixed in natural populations.

Yet, I found that large-egg selected females produced more eggs, with higher energy

content, that grew into larger larvae at competence. I have proposed that the constraint

on increased egg diameter (at 6 weeks) in the Pearl Harbor population of Hydroides

elegans appears to be selective pressure to maintain the optimum male contribution to

lifetime reproduction by delaying the shift to the female sex (Chapter 3).

Phenotypic correlations between the trait of egg diameter and the suite of

fitness-related traits outlined above were estimated using bivariate analysis on replicate









line means (n = 3 lines treatment-) in JMP 5.1 (SAS Institute, Inc.). The results

comparing control with selected line means can be seen as Pearson product moment

correlations (r) in Table 4-1.

Among lines the mean values for controls showed no phenotypic correlation

between egg diameter and any of the traits of interest, with the exception of larval size at

competence (5 d) where a significant positive correlation (0.98) was found. This

relationship persisted in selected line means (0.99). However, despite the analysis at the

level of treatment showing positive correlations between egg diameter and fecundity,

examination of individual selected lines showed a significant negative correlation

between egg diameter and both measures of fecundity (-0.95 and -0.99 respectively). The

selected lines also showed a significant positive correlation between egg diameter and

female dry weight (0.99). A pattern is suggested in the differences between control and

selected lines with respect to these phenotypic correlations; the replicate selected line that

achieved the highest mean egg diameter also had the highest female dry weight and the

lowest fecundity. This pattern (the largest mothers producing the largest eggs in the

fewest numbers) is reminiscent of the predicted trade-off between growth, egg size, and

fecundity that I did not detect between treatments (Chapter 3). This relationship emerges

at the extreme end of the continuum of reproductive investment (large-egg selected lines)

and is not detectable among the control lines. While this observation cannot be

considered quantitative (n = 3 is the minimum sample size for a correlation), it suggests

that further study into the influence of trade-offs in defining reproductive investment

might be most fruitful at the extreme ends of the investment continuum in this species.









Within polychaetes, fecundity within species can vary with nutritional state of the

mother, population density, and adult age and size (Eckelbarger 1986). In this

experiment all animals were fed ad libidum at all stages of development, while density

and adult age were controlled. Varying any of these parameters or inducing

environmental stress at different developmental stages might also reveal evidence of

trade-offs influencing reproductive effort. Qiu and Qian (1998) looked at effects of

varying salinity and temperature on juvenile traits in H. elegans, and found that fecundity

was affected by the stress of low salinity. Czesak and Fox (2003) found while working

with the seed beetle Stator limbatus that the magnitude of the tradeoff between egg size

and fecundity varied between environments.

Another possible cost associated with increased reproductive effort is higher

mortality, as was found in the polychaete Dinophiilus gyrociliatus by Prevedelli and

coworkers (Prevedelli and Zunarelli Vandini 1999, Prevedelli and Simonini 2000). They

found that higher food led to increased fecundity but was accompanied by greater and

earlier mortality. Tatar et al. (1993) nicely demonstrated the tradeoff between increased

egg production and increased age-specific mortality in the beetle Callosobruchus

maculatus.

Many avenues of inquiry can be pursued based on this study. A large body of work

has been compiled investigating the adaptive significance of developmental mode in

marine invertebrates using comparative, empirical, and manipulative techniques. A great

deal of effort has also been invested in quantitative modeling approaches that have been

used to try and identify selection pressures and processes that influence developmental

mode evolution. The suite of characteristics that defines a developmental type has









co-evolved in concert and compromise with a wider complex of life-history traits under

the influence of natural selection. The most rewarding efforts to pursue these dynamic

and sometimes subtle relationships will be methods that are the most integrative across

the stages that define a life cycle, and across the breadth of traits that define a life history.

Understanding the nature of the variation in fitness-related traits and how it is partitioned

in a particular population has the potential to reveal the insights we seek concerning the

evolution of the diverse life histories among marine invertebrates.

Table 4-1. Phenotypic correlations between the trait of egg size and a suite of life-history
traits in replicate selected and control lines in the polychaete Hydroides
elegans, based on line means (n = 3 lines treatment-1). Pearson product
moment correlations (r) with absolute value > 0.95 indicated with *
Correlation coefficient (r) among Correlation coefficient (r) among
Control Line Means (n = 3) Selected Line Means (n = 3)
Egg d Fecundity 0.09 Egg d Fecundity -0.95*
Relative Relative -0.99*
Egg d 0.24 Egg d
Fecundity Fecundity
Egg d Female Dry -0.05 Egg Female Dry 0.99*
Weight Weight
Egg d Total energy -0.48 Egg d Total energy -
Egg d 1 -0.48 Egg d 1 -0.28
egg egg
Larval size Larval size
gg at 18 to 20 h ggat 18 to 20 h
Sd Larval size .* Larval size 0.
Eggd at 5 d 0.98* Egg d at5d 0.99
at 5d at 5d
Sd Tube length 6 E Tube length -
d at21d -86 d at 21 d -
















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BIOGRAPHICAL SKETCH

I was born in North Carolina and raised in south Florida. I have traveled

extensively as an adult, and settled in Texas where I renewed my pursuit of education and

received an Associate of Arts degree from Texas Southmost College in Brownsville,

Texas and a Bachelor of Science degree, summa cum laude, from Texas A&M

University. I earned my Master of Science degree from Florida Institute of Technology

in 2000 and continued to earn my Ph.D. in 2006 from the University of Florida with a

major in zoology.