THE FLORIDA STATE UNIVERSITY COLLEGE OF ARTS AND SCIENCES

REPRODUCTION IN CARD- FJ,OmANA (CONRAD) (BIVALVIA CARDITIDAE)

BY MARGARET C. HARVEY

A Thesis submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Master of Science

Degree Awarded Summer Semester, 1995 The members of the Committee approve the thesis of Margaret C. Harvey defended on 19 August 1994.

Professor Directing Thesis

Committee Member

Committee Member

Appproved:

__L______I______of Biological Science ACKNOWLEDGEMENTS

I would like to thank the following people for their help with various aspects of my project: S. Cook, here at the University of South Florida in Tampa, for her unlimited patience and technical expertise in formatting this manuscript; Dr. F. James, for help with the morphornetrics, and M.B. Kotrla, K. Riddle, K. Womble and also B. Barfield, of the Panama City Shell Club, for introducing me to my research site. A. Black and D. Watson were most gen- erous in their assistance with the histology. I appreciate the guidance and ad- vice of my committee members, Drs. W. Heard, R. Mariscal, L. Keller, and W. Krebs, and Dr. Heard for some financial support. I also wish to thank Dr. Krebs for both the insightful statistical consultations and his friendship. I am thankful for the $500 grant awarded to me by the Conchological Society of America and to R. Tucker Abbott for his helpful advice. I would like to thank the personnel of Tyndall Air Force Base for allowing me access to the site. Finally, I am deeply grateful for assistance in the field and especially for the unlimited patience and support of M. Palotisi Harvey.

iii TABLE OF CONTENTS

EXe LIST OF TABLES vi LIST OF FIGURES vii ABSTRACT ix INTRODUCTION 1 Terminology 1 Bivalvia 6 Carditacea: Carditidae: Carditamera floridana 7 MATERIALS AND METHODS 11 Individual Sexuality, Sex Ratio, Sexual Dimorphism in She11 Shape 11 Reproductive System Structure 14 Reproductive Periodicity 14 Histochemistry of Reproductive System 16 Egg Mass and Developmental Stages 17 RESULTS 18 Individual Sexuality, Sex Ratio, Sexual Dimorphism of Shell Shape 18 lndlvldualSexualitvdbW.. 18 Sexual DimorDhism in Shell Shape 18 Reproductive Periodicity 21 AnnualPeriodicitv 21 Svnchronicity Amon2 Jndividuals 25 Tcmucrature and Annual Periodicity 25 Histochemistry of the Reproductive System 31 Structure &&g Reproductive&Ska 31 Vitellov- 31 SourcesdNaturepfMucins 31 Egg Mass and Developmental Stages 35 Counts of Developinv Individuals 40 Develoomental- 40

iv DISCUSSION 43 Oviparity in the Bivalvia 43 Oviparity in Carditamera floridana and Possible Adaptive Significance 44 Individual Sexuality, Sex Ratio, Sexual Dimorphism.. in Shell Shape 47 IndlYKuSexualitvd%BiltiQ 47 Sexual Dimorphism in Shell Shape 49 Reproductive Periodicity 49 Annual- 49 Synchronicity Amone Individuals 51 Temperature and Annual Periodicity 53 Histochemistry of the Reproductive System 53 Structure of Reuroductive Svstem 53 Vitelloeenesis 54 Sourcesan8mQfMucins 55 Egg Mass and Developmental Stages 57 counts. Develouine Individuals 58 DeveloDmental- 59

APPENDIX I Compilation of Oviparous Bivalves Exhibiting Extended Care: Adult Size, Habitat, and Distribution 61

APPENDIX 11 Compilation of Oviparous Bivalves Exhibiting Extended Care: Oocyte Diameters, Notes on Reproduction and Development, and References 65 LITERAWRE CITED 71 BIOGRAPHICAL SKETCH 79

V LIST OF TABLES rn w 1. Comparison of Gonadal States of Male and 23 Female c. floridana 2. Shell Lengths of Juvenile c. floridana 24

3. Kolmogorov-Smirnov s-Sample Test; Column 28 Totals

4. Kolmogorov-Smirnov s-Sample Test 29

5. Histochemical Test Results 34

vi LIST OF FIGURES

Eik3m u 1. Map of Study Site 12 2. Size-frequencies of Male and Female c. florldana 19 3. Shape Variables of Male and Female c. foridana 20 4. Reproductive Periodicity 22 5. Synchronicity of Reproduction 26 6. Mean Oocyte Diameters and Seawater Temperatures 30

7. Photo of Histologic Slide of Ovary 33

8. Photo of Histologic Slide of Ovary 33

9. Photo of Unfixed Oocyte 33

10. Photo of Histologic Slide of Ovary 33

11. Photo of Histologic Slide of Kidney 37

12. Photo of Histologic Slide of Kidney 37

13. Photo of Histologic Slide of Ovary 37

14. Photo of Histologic Slide of Ovary 37

15. Photo of C floridam with Egg Mass 39

16. Photo of G. floridana with Egg Mass 39 17. Photo of c. floridana with Egg Mass 39 18. Photo of Live c.floridma Embryo 39 vii A Eiw=

19. Adult Size and Number of Embryos 43

viii ABSTRACT

Oviparous reproduction in Carditamera floridana was studied in St. Andrew's Sound, near Mexico Beach, Florida, between May 1992 and February 1994.

This species is gonochoristic; the sex ratio is highly skewed toward males in the lowest size range. Shell height plays a greater role in larger fe- males' shape than in males. The reproductive cycle was monophasic between January 1993 and February 1994. Females were spent between 6 April and 6 June. Mean oocyte diameters were greatest between February and March. Synchronicity among females within a sampling period was greatest prior to the spawning event and less evident at other times. There is no evidence of a distinct vitellogenic mechanism. Mucous se- cretions from various tissues are noted and characterized. The egg mass was a sticky, mucilagenous secretion issuing from the right suprabranchial chamber. The right inner ctenidium distends so that the filaments of the ascending and descending lamellae were widely separated. The egg mass passed through the exhalant chamber to the exhalant siphon, to which it remains attached. Development is direct, with the release of crawl- away juvcniles. Density of embryos within an egg mass ranged to a high of approximately 17,000 and appeared to be correlated to some extent with the size of the adult. The egg mass deteriorates over a period of 14 days.

ix This oviparous mode of deveIopment is unique in the number of ju- veniles produced, the extent of parental care involved, the subtropical range of the species, and the lack of a distinct vitellogenic mechanism.

X INTRODUCTION Terminology Many marine benthic invertebrates are external fertilizers, although representatives of diverse phyla have varying strategies of oviparity, ovo- viviparity, and viviparity. Oviparity is the production of eggs that undergo development after deposition by the parent on the substrate or other firm ob- ject. Ovoviviparity is the production of eggs that undergo development within the body of the parent without a vascular connection between the par- ent and the developing young. Viviparity is production of eggs that undergo development within the body of the parent with a vascular (placental) con- nection between the parent and the developing young (Houghton, 1869). Young may be released either as larvae or juveniles (Mackie, 1984). Several classifications of different modes of larval development of ma- rine benthic invertebrates have been devised to present an integrated and useful descriptive scheme. Thorson's (1946,1950) proposal was broadIy based on the location of larval development, &., pelagic and non-pelagic. Pelagic larvae remain in the plankton throughout development. Thorson also cre- ated terms to make a distinction between larval feeding types within this group. Those larvae that rely on yolk reserves exclusively are lecitllotrophic, whereas planktotrophic larvae take nourishment from planktonic food ex- clusively. Thorson believed that the time spent feeding in the plankton is a further significant distinction. The term planktotrophy was reserved for those planktonic larvae that feed for a relatively long period of time. Nan-

1 pelagic larval developmental types included viviparous forms that were de- fined as undergoing development entirely within the parent, the young being released as juveniles (Sellmer, 1967). Also included in this groups are those larvae that develop completely within an egg-mass, feeding on either yolk re- serves of nurse eggs, and that are released in a juvenile benthic phase. Ockelmann (1965) revised the terminology and presented a classifica- tion that was based in part on studies of over 200 species of marine bivalves. He listed planktotrophic, lecithotrophic, and direct larval developmental types. Planktotrophy referred to those larvae for which feeding on plankton was imperative in order to achieve metamorphosis. Lecithotrophic larvae were stated to rely either solely on yolk reserves up until metamorphosis or to feed on plankton immediately prior to this event, although not by neces- sity. Direct development implied lecithotrophy and the lack of pelagic larval phase. Milcikovsky (1974) recognized four categories of larval development among marine benthic invertebrates: (1)pelagic (occurring in the open sea); (2) demersal (larvae capable of swimming to a limited extent and in which development occurs within the near-substrate water layer or on the substrate itself); (3) direct, and (4)viviparity, The pelagic developmental type was ex- tended to include seven subtypes based on the ecology of various species. Due to the innumerable permutations that exist, Mileikovsky acknowledged the near impossibility of establishing clear distinctions between the four types de- fined. Poccilogony, a polymorphism in development in which there is pre- sent than one distinctive kind of development within the Same sexu- ally reproducing species (Hoagland & Robertson, 1988)~comprises Yet another

n L dimension in the classification of larval types. very few cases have been veri- fied, all in Polychaeta. The presence of cryptic species was the most frequent source of error. In most of the more credible studies, small eggs develop planktotrophically and the large eggs develop directly. This phenomenon might occur as a geographical variation between populations, as a seasonal variation within a population, or as environmentally determined. Bouchet (1989) presented another definition of poecilogony as intraspecific variation involving planktotrophy and non-planktotrophy. Patterns of larval development have been analyzed for tlicir adaptive significance. Energetics is an important component because the amount do-

cated to reproduction comprises a considerablc portion of the total energy budget. Sources of mortality, length of prefeeding and feeding developmental times, total developmental time, dispersaI, and mutation rates are among the variables that must also be considered (Vance, 1973; Chia, 1974; Crisp, 1974). A selection pressure for efficiency must exist. Vance (1973) noted that non- pelagic development might be the mast costly in terms of energy reserves in- vested in developing embryos PIUSthe energy required to produce the spawn

or provide extended care by brooding. The assumption of a limited amount of energy available for gamete production together with limited dispersal and a decreased mutation rate could prove lecithotrophy to bc the inferior mode of development in long term evolution (Chia, 1974). The nonpelagic strategy might be the most advantageous one in habitats that are stable in time and where energy supplies are low, planktonic mortality rates arc high, and dc- velopment times are long (Vance, 1973; Crisp, 1974). Havenhand (1993) prc-

sented a model that predicted the existcncc of selection pressures for non- feeding larvae in marine invertebrates due to an increase in fitness from a re-

3 duced egg-to-juvenile period. Christiansen & Fenchel (1979) developed a model that predicted the relative unimportance of reproductive effort as a primary determinant of both larval developmental type or number of eggs produced per individual. They cited the inadequacies of models of r- and K- selection to explain the adaptive nature of the reproductive patterns found. Vitellogenesis is the synthesis of yolk during oocyte production in in- vertebrates &.e;., Sastry, 1979; Jong-Brink, dd.,1983; Wourms, 1987). It is an energy-requiring process, and it is often the most time-consuming. As a rate- limiting step in egg growth, the various mechanisms by which vitellogenesis achieved might have implications for the evolution of various life history patterns (Eckelbarger, 1994). Dispersal has long been considered to be a distinct advantage of pelagic development. Selection for dispersal might not be the driving force main- taining a feeding larval stage in life histories; feeding and safety in the plank- ton with extensive dispersal, as an accidental byproduct, could be the more critical factors (Strathmann, 1985). Physical and biological barriers to larval dispersal, such as abundance of prey items, temperature, behavior, and swimming abilities, might exist, Pelagic larvae could experience reduced via- bility or fecundity when reaching distant populations (Hedgecock, 1986). Modeling based on game theory that seeks what is "optimal" might instead be better focused in exploring what is "good enough' under prevailing condi- tions (Schcltema, 1986). Knowledge of the movements of waters and the nat- ural history of species are essential in assessing the complex role of dispersal in life-history theory. A number of somewhat counterintuitive examples from studies of molIuscs exist. Congeners with different developmental types have proved

4 to have greater or lesser geographic ranges than predicted Oohannesson, 1988; O'Foighil, 1989). Furthermore, additional dispersal mechanisms have been discovered for those species once thought to be restricted in range. These in- clude byssus-drifting, mucous-drifting, Aoating, algal drifting, swimming (Bayne, 1964; Sigurdsson, d.,1976; Highsmith, 1985; Sorlin, 1988; Beukema & Was, 1989). Several of these authors have also considered these methods to be opportunities for secondary dispersal. The existence of migratory rhythms in some species is yet another factor to consider in assessing the relative vagility of a species (Armonies, 1992). In descriptions of reproductive behavior, the concept of the protection of young has acquired a plethora of vaguely-defined terms that are often syn- onymous, not useful, and perhaps incorrect. Thorson (1946) Ioosely defined the expression "extended care" as protection, more or less, during develop- ment and "brood protection" as "the many types of egg spawn". Ockelmann (1965) suggested that the latter expression could be used in association with any of the three developmental types he had not outlined. SeIlmer (1967), in his extensive study of the ovoviviparous marine clam Gemma eemma.reex- amined the terminology and the variety of criteria used. He defined "ex- tended care" as applying to cases of the retention of young either internally or externally. Usage of the term "brood protection" Thorson (1946) and Ockelmann (1965) was redefined as "all types of maternal solicitude, used in its most general sense". Sellmer noted that the term "incubation" was first used in a 1935 treatise by Pelseneer and had been subdivided into internal and external forms and that Pelseneer (1906) earlier denied the existence of viviparity in lamellibranchs. Thorson (1946) used the latter term but made no mention of sustenance from the adult. The term "incubation" was used

5 by Mackie (1984) to comprise one of the two categories of larval development, oviparity being the second. "Incubation" included both ovoviviparity and egg-retention. TIie term "egg retention" was defined for species that hold their young for a short time in the mantle cavity, releasing them as veligers or pediveligers. For clarity, I will use the terms "extended care" and "brood protection" Sellmer.

Bivalvia The majority of marine bivalves are external fertilizers (Mileikovsky, 1974). Gametes are spawned by broadcasting into the surrounding waters. Development involves a feeding larval stage that is pelagic (Thorson, 1950); after a period of dispersal, settlement and metamorphosis into juveniles oc- cur. Continuous reproduction is the general rule for bivalves inhabiting tropical climates due in part to the combined effects of increased temperature and increased food abundance (Thorson, 1946; Mackie, 1984). Development is mostly pelagic shallow-waters. Direct development is relatively uncommon, occurring in 10-15% of species from the Atlantic coast and shelf of Europe. Extended care in the Bivalvia has arisen independently numerous times, occurring in almost half of the eulamellibranch families (Jones, 1963; Sellmer, 1967; Yonge, 1969). Examples include more than one of the devel- opmental types listed in the schemes above. Nonfeeding larvae have evolved from feeding larvae more frequently than the reverse (Day & McEdward, 1984, who hypothesized that this pattern might be due to the often complex morphology of the structures involved in feeding). The various forms that extended care have taken in the Bivalvia were tabulated by Sellmer (1967). It may vary greatly in time, stage released, in the number of

6 developing young involved, locations inside and outside the animal, amount of yolk invested in the eggs, and nourishment by the production of maternal secretions. Eggs can be anchored to the substrate individually, allowed to float in a mass, or be laid in a nest or in strings. They can be attached to the adult via a special inpocketing of the shell, an expansion of the mantle over the shell, or to byssaI threads. There must be selective advantages to the retention of the developing individual during the most vulnerable stage in its life cycle. These could in- clude timing until optimal feeding conditions prevail, a reduction in preda- tor-induced mortality, and decreased dispersal in an unstable environment (Chia, 1974; Crisp, 1974; Christiansen & Fenchel, 1979; Strathmann, 1985). Sublethal effects due to rate changes during critical periods of morphogenesis can disrupt development and alter energy balances over evolutionary time (Day & McEdward, 1984).

Carditoidea: Carditidae: arditamera floridana The Carditoidea is a superfamily within the Order Eulamellibranchia. It and the Astartoidea are considered to be the most primitive superfamilies within the order. The shell hinge type appeared in the middle Ordovician. Characters uniting the superfamily include the absence of siphons, widely separate lobes of the mantle, very small labial palps, extensive pallial mucous glands, and the presence of hemoglobin (Yonge, 1969). The gills are composed of homorhabdic and non-plicate ctenidia, the condition in which the fila- ments are identical and free or only slightly united to one another at distant intervals (Ridewood, 1903). They display a sedentary, primitively infaunal habit, with the majority of families having a byssus. Dall (1902) made the ob-

7 servation that the young are retained within the female Carditoidea in "many, if not all, cases". Extended care occurs with the greatest frequency in this group, with the possible exception of the Erycinoidea (Yonge, 1969). The family Carditidae Fleming comprises 16 genera with approxi- mately 200 species worldwide; in North America, there are 7 genera and 22 species. They are a benthic group, inhabiting waters up to 8 m in depth and range in shell length from 1 to 15.9 cm (Rehder, 1981). The family contains the largest species in the superfamily; they are often epifaunal and byssally at- tached (Yonge, 1969) and display a wide range of morphological variability (Coan, 1977). The subfamily Carditamerinae Chavan contains the species mfloridana(Conrad, 1838). It is the largest species in the subfamily, and the only living. The type-species CvDricardla' &Conrad, 1832 (= c. floridana) by original designation (Moore, 1969) is known from the Miocene of the Atlantic coast of southern Florida (Olsson, &d., 1953). The common name for the species is the "broad-ribbed Cardita", and it has a distribution from the southern half of Florida and Mexico (Abbott, 1974). Specimens as large as 4 cm have been recorded. Extended care might be part of the reproductive habits of all members of the Carditidae (Dall, 1902; Yonge, 1969; Coan, 1977). Internal retention of the developing young occurs in either the demibranchs, as in aviculina Lamarck (Schneider, 1993), or in one of two highly unusual modes in the pallial chamber. Females of the species Milneriakghqi Dall (6.4 mm shell length) possess a shell modified by a ventral infolding and covered with periostracum such that a brood pouch or marsupium is formed in which up to 50 young are held (Dall, 1902; Abbott, 1974). A variation of this phenom-

8 enon is found in Thecaliaconcamerata Brugueire. The females of this much larger species (20 mm shell length, fide Kennelly, 1964) retain their young well beyond the prodissoconch stage in a completely internal marsupium ere- ated by a "funicular infold of each valve. All members of the Carditidae are known to produce crawl-away young. Carditamera floridana is highly unusual within the Eulamellibranchia in that it produces a buoyant, translucent, mudlagenous egg mass that re- mains attached to the adult animal and is often larger than the adult itself. Development is direct, without a free-swimming larval stage, and large num- bers of crawl-away young are released from the egg mass. Oviparity in the form of production of an egg spawn is an uncommon reproductive mode in the Bivalvia; when associated with extended care, it is a rare phenomenon (Drew, 1901; Oldfield, 1955). There are no known cases to date, other than the present study, of oviparity in the Carditidae. This represents a third mode of parental care within this family, Very little is known of the biology of c. floridana. Lloyd (1962) studied oxygen and carbon isotope ratios in populations located in Florida Bay, find- ing that they paralleled ambient conditions. In 1963, Manwell studied vari- ous aspects of the hemoglobin in this species from a population in Alligator

Harbor, Florida, discovering an unusual relationship between heme units and their affinity for oxygen. It is a negative heme-heme interaction in which oxygen is bound non-cooperatively. The resulting effect is that as oxygen ten- sion in the water increases, the affinity of the individual heme units for this molecule decreases. The hemoglobin in c.floridana is of the extracehlar type; according to the lack of sequence similarity with intracellular herno- globins, the divergence took place a long time ago (Beintema, 1990). Fkmo-

9 globins are known to possess a broad spectrum of functional properties in in- vertebrates (Eggs, 1990). The results of a study of the confamilial - hd&t Conrad from a population in the northeast Gulf of Mexico include finding hemoglobin free in the plasma, in the cells of the mantle and the cy- toplasm of eggs (Harry, 1966). Interestingly, the hemoglobin of the confamil- ial ~affinisSowerby is one of the largest hemoglobins known (Pfaffenbacli & Riggs, 1990). Nothing is known of the reproductive of c. floridana beyond the un- published groundwork of former Florida State University students in the early 1970s on a population from Alligator Point, Florida. John Jensen origi- nally made the connection between the egg masses, which are superficially similar to those of a common species of polychaete [Arenicola-) and the bivalve. Notes were taken on various aspects of the natural history of the animal, the production of egg masses and early developmental stages, and histochemical tests were done to determine the chemical composition of the mucilagenous matrix of the masses, Robert Cardenas worked briefly on the histology of the gonads and the ctenidia. The objectives of the present study of reproduction in c.floridana were to describe the structure of the reproductive system, describe and define re- productive periodicity, to describe the nature of the egg sac and to record ob- servations of developing individuals.

10 MATERIALS AND METHODS

This study was conducted from the end of May 1992 through February 1994. The study site was located in St. Andrew's Sound, a shallow embay- ment approximately 14 km west of the city of Mexico Beach, Bay County, Florida (Fig. 1). Approximate coordinates are 85"W and 30.5"N. Samples were taken from an alongshore-distance of approximately 25 m during a low tide. The tides are mostly diurnal, with a range of approximately 0.4 m. Seawater temperature was taken in the near-bottom layer using a mercury

thermometer at a depth of approximately 1 m.

Individual Sexuality, Sex Ratio, Sexual Dimorphism in Shell Shape Twenty individuals and a few juveniles (when present) were taken monthly throughout the duration of the study and biweekly during the re- productive period for a total of 24 collections. Animals were chosen in a size series; those animals not selected were return-ed to grass beds in a region away from the study site. The grass beds are composed of Halodule, Svrinrzodium. Thalassia and intergradations. Attempts were made to sample throughout all types of grass beds. Grass beds containing only Halodule were found closest to shore, followed by intergradations of I&&&& and

Surineodium. intergradations of Svrinrodlum' and Thalassia. followed by pure stands of Thalassia. found in the deepest sections. Animals were pro- cessed on the day of collection. A 1%formalin-seawater solution was used

11 Figure 1. Map of the study site in St. Andrew's Sound, Bay County, Florida (approximate coordinates: 85OW,30.5ON). S indicates collection site.

12 as an anaesthetic during dissections. The general condition of the specimen, presence of associated organisms, and sex (when possible to determine) were noted. The animals were left whole, were bisected through the gonad, or had the gonad removed. Tissues were preserved in a 10% formalin-seawater so- lution for under 24 hours at room temperature. Shells were measured using metric vernier calipers with an accuracy of plus or minus 0.1 mm for length, width, and height as per Abbott (1974). A number of protocols for paraffin-embedding using light microscopy were compared. The following protocol, following fixation, was employed for most of the study (M.B. Kotrla, pers. commun.); variations in duration were made to accommodate the sizes of the tissues: two washes of 1- 1-1/2 hours duration in distilled water; a dehydration series in alcohol, each step 1 hour; overnight or longer in methyl salicyIate; 6 changes, 20 minutes each, in xy- lene, room temperature; 1 hour in xylene; 6-12 hours in a Paraplast-saturated solution of xylene at room temperature; repeat; 6 changes, 6-8 hours each, in Paraplast in 60 C paraffin oven; and embed. Delafield's hematoxylin-alcoholic eosin staining of deparaffin ized sec- tions was used for routine survey work. Parasitized animaIs were not used. Size at sexual maturity was determined by the pre- sence of gametes. Viability was not investigated. The statistical package SYSTAT (SYSTAT, Inc.) was used for most statistical analyses. Based on measurements of shell length, sexed animals were binned into 26 categories of 1mm increments for graphi- cal comparisons using size-frequency plots. A test for trend in proportions was done (Armitage, 1955), using 13 categories based on increments in shell length of 2 mm. Because the question being addressed was whether there was

13 a specific departure that changed systematically from equality of proportions, this statistic was preferred over the Chi-square test (W. B. Krebs, pers. comm.). Allometry of growth was examined using morphometrics of she11 shape. Animals were collected between 31 May 1992 and 23 May 1993; only those animals that could be sexed were used for this analysis (N=162 males and 148 females). Standardized shape variabtes were obtained by log-trans- forming shell measurements and standardizing these for overall size (=length) (Kotrla & James 1987). Graphical comparisons using the LOESS smoothing function and regression analyses were done.

Reproductive System Structure Serial sections of both 6 pm and 8 pm thicknesses using Delafield's hematoxylin-alcoholic eosin stains were used to determine gross morphology of the reproductive system and to examine the cellular composition of the acini in the gonad.

Reproductive Periodicity Five females from each of 17 collections taken between 24 January 1993 and 24 February 1994 (23.8 mm * 2.6 mm shell length) were selected for quan- titative determinations of oocyte diameters and qualitative assessments of gonadal stages. Transverse, sagittal, and longitudinal sections of 8 pm thick- nesses were made and stained with Delafield's hematoxylin-alcoholic eosin. Two hundred oocytes were first measured for each of two females (taken by transverse section) within a collection and the same number of oocytes for a female from the successive collection in the series. The first 50 eosinophilic

14 oocytes With the nUChs clearly visible were measured at 200~using an OCU- lar micrometer fitted on a Wild compound microscope. A video image anal- ysis system UAVA; Jandel Scientific) could not be used in these determina- tions due to the ubiquitous presence of the matrix surrounding the oocytes, which precluded the establishment of a clear foreground/background distinc- tion. Variances of units of 50 oocytes were compared in sections proceeding from anterior to posterior within animals and found to be sufficiently similar such that heterogeneity of development within the portion of the gonad ex- amined could be eliminated as a source of possible error. Variances of units of 50 oocytes were compared between animals in successive collections, and it was determined that the number was sufficient to perceive changes over time. Calculations of the standard error of the mean supported this choice. Mean oocyte diameters (50 oocytes from each female) were plotted against time in days, and a nested Kruskal-Wallis test was done to determine repro- ductive periodicity. Fewer than five females had enough oocytes to measure on 29 June (N=4 used), 14 July (N=3), 25 August (N=3), 9 September (N=O), and 10 October (N=4). Analyses of gonadal states were done for females and males. Five males per colIection (19.3mm f 5.2 mm shell length) were chosen. Assign- ments were made to one of 4 stages, as follow: ripe, spending, spent, and recovery. Mean oocyte diameters (N=50) for each female with enough oocytes to measure were plotted for each sampling period. To examine more closely the variability bemeen animals within collecting periods, an S-samPle Kolmogo- rov-smirnov test (fiefer, 1959) was done comparing mean oocyte diameters. fienull hypothesis of no significant difference between mean Oocyte

15 eters among individuals within a sampling period was tested. Oocytes were binned in 5 pm diameter increments. Initially, column totals were taken for descriptive purposes in order to provide a broader perspective.

Temperature ys. mean oocyte diameter per collection (N=250) was plot- ted, and a regression analysis was done.

Histochemistry of the Reproductive System Serial transverse sections of 6 pm thickness were taken from a female fixed in 10% seawater-formalin during the production of an egg mass. The protocol differed from the one outlined above with regard to the steps fol- lowing dehydration, as follows: overnight in methyl salicylate followed by two changes of two-hour duration each under vacuum; four changes of heptane for one-hour durations under vacuum; two changes of 56'-58" paraffin ("Histo- Prep"); and one change into 60'-62" paraffin ("Peel-Away") followed by embed- ding in the same paraffin. The egg mass was fixed for one hour at room temperature in a 10% for- malin-seawater solution, placed in a sucrose gradient and sectioned at lOum thicknesses using an Ames cryostat (Miles Corporation). Sections were stained with 0.5% Toluidine Blue in borate (aqueous). To determine the chemical composition of various tissues in the fe- male, the following series of 10 histochemical tests (see Ghiselin, 1965) were done: Alcian Blue (pH 2.5, 1.0, and 0.4); Dahl's test for calcium; Azure A; Delafield's Hematoxylin and alcoholic Eosin; Lasky's mucihaematin; Mercury-Bromphenol Blue; Alcian Blue (pH 2.5) - Periodic acid-Schiff, and Sudan Black. Histochemical protocols of Luna (1967), Pearse (1989) and

Bancroft & Stevens (1990) were used. 16 TO determine the chemical composition of the egg mass, 0.5% Toluidine Blue in borate (aqueous) and Alcian Blue (pH 2.5) - Periodic acid-Schiff tests were done; tests were limited to these two due to difficulties encountered in sectioning this material.

Egg Mass and Developmental Stages The computer video analysis system mentioned above was used to de- termine approximate counts of developing embryos for each of 10 egg masses collected from the field site. Approximately 0.5 ml was sampled from the cen- ter of each mass. The volume of the mass was estimated by measurement within a 25 ml graduated cylinder. A regression analysis was done on adult size (=she11 length) and number of developing embryos in the egg mass. Re- peated samples of embryos were taken from an egg mass and developmental stages and behaviors noted over a period of 14 days. Observations of newly emerged juveniles were made as well. Whole mounts were made of juven- iles following the method of Clement and Cather (1957). Voucher specimens have been deposited in the Florida State Museum in Gainesville, Florida.

17 RESULTS

Individual Sexuality, Sex Ratio, Sexual Dimorphism in She11 Shape .. -. -. The animals of C floridana from the St. Andrew's Sound collection site are gonochoristic. Forty-six of the 365 specimens were parasitized by an unidentified digenetic trematode and all of them had been parasitically castrated. a w. A size-frequency plot of males and females is shown in Fig. 2. The sex ratio is highly skewed towards males at the lower end of the size distribu- tion. The result of the test for trends in proportions was highly significant (S* = -9.00). The final sex ratio was 172 males to 193 females.

sexldDlmorDhlsm' hwb. The log-transformed measurements of shell height, standardized for overall size (=length), were plotted against shell width for 162 males and 148 females (Fig. 3). There is an indication of some difference between the sexes in regard to the ratio of height to width as they grow larger. The trend in females is for both height and width to increase to a similar degree as length increases. Males increased disproportionately greater in width rather than height as size in length increased. A regression analysis produced an F-statistic of 153.4 for females and 56.1 for males. Because the samples were not selected in a random manner, no p-values are attached to these.

18 50 * + F /\I! 40 .. S*=9.00 m .-.- Males '8 30 _. 6 & F4E! 20 ..

Shell kngh (mm)

Fig. 2. Size-frequencies for 172 male and 193 female C. floridana.

19 -0.1 F=56.1 162 males

' -0.2 - .' Shell Height, .. Standardized

-0.3 -

-0.4 I I 10

-0.1 F=153.4 148 females

-0.2 - Shell Height, Standardized

-0.3 -

Shell Width, Standardized

Fig. 3. Shell shape variables of 310 sexually identifiable c,floridam.

20 Reproductive Periodicity Annual-. Beginning 24 January 1993, five females were selected from each sample, and the mean of 50 oocyte diameters was calculated for each animal. During the mid- to late summer and early fall sampling peri- ods, fewer than five females had enough oocytes to measure on June 29 (N4used), July 14 (N=3), August 25 (N=3), September 19 (N=O), and October 10 (N=4). The mean for each sampling period was plotted against time. AC- cording to the findings shown in Fig. 4, there was one reproductive cycle in 1993, The results of a Kruskal-Wallis test were highly significant {H=64.43, p<0.0001, df=16), indicating that there might have been seasonality of repro- duction in the population under study. Egg size increased to a maximum during March. A sharp decline in egg size occurred at this time, perhaps marking a spawning event. Table 1 displays male and female gonadal states throughout the sam- pling period. Males were mostly ripe throughout the year. Females generally followed the trend in mean oocyte diameters throughout the year. The presence or absence of juveniles in the field may be an indication of the presence or absence of more than one spawning event in a year. Table 2 lists the sizes of all small individuals (42 mm long) collected during the period 31 May 1992 through 24 February 1994. The cut-off of 12 mm was cho- sen for two reasons. It is a reasonable size for an individual to have achieved over the course of a year's growth and there is a peak at this size in the size- frequency distribution curve in Fig. 2. Egg masses noted in the field include a total of 4 on 31 May 1992 and 10 June of the same year (preliminary observa- tions for this project took place at this time). In 1993, a massive tropical storm, accompanied by record cold temperatures, occurred during mid-March.

21 135

130

125

120

115

110

105

100

Fig. 4. Reproductive periodicity in &. floridana: Mean oocyte diameters (pm) over time. Error bars signify standard deviations. Collection dates corresponding to the first letter of the months of the year are listed in se- quence as follow: J = 24 January 1993 F =21February M = 8March M = 21 March A = 6 April A = 18 April M = 4May M =23May J = 6 June J = 29 June J =14 July A = 25 August S = 19 September 0 =loOctober N = 14 November J = 2 January 1994 F =24February 22 Table 1. Comparison of gonadal states in 5 male and 5 female c. fIoridana selected from each collection from 24 January 1993 to 24 February 1994. I, spent; 11, recovering; III, ripe; IV, spending.

MaIes Females Gonadal- PateDI 11 111 V 1/24 100% 100% 2/21 100% 100% 318 100% 100% 3/21 100% 20% 80% 416 100% 100% 4/18 100% 100% 514 lOQ% 100% 5/23 100% 100% 616 100% 100% 6/29 80% 20% 20% 80% 7/14 30% 70% 40% 60% 8/25 10% 90% 40% 60% 9/19 60% 40% 60% 40% loll0 50% 50% 20% 80% 11/14 100% 100% 112 80% 2076 80% 20% 2/24 60% 40% 100%

23 It most likely perturbed the population to such an extent that no egg masses were fomd in the field that entire year. Yet, Table 2 indicates the presence of juveniles. In 1994, during biweekly sampling periods, a major spawning event was observed on 20 May,

Records of spawning events occurring within a seawater tank can indi- cate the possibility of reproductive windows other than that observed in the field. An egg mass was found 28 October 1992 after a water change; three egg masses were produced after a 6" C decrease in temperature during a water change 26 February 1993. Egg masses were also produced by aquarium indi- viduals on 2 April 1993,5 May 1993, and 12 April 1994.

.. SvnchronlcltvAmoneIndlvlduals. Mean oocyte diameters for each female with enough oocytes to measure (see page 22) were plotted over time. The re- sults are shown in Fig. 5. Variability between animals within collecting peri- ods was also examined with an s-sample Kolmogorov-Smirnov test. Column totals, the results of an exploratory data analysis, are displayed in Table 3. T values and associated p values are given in Table 4.

d Annuid -. Temperature as a possible environ- mental correlate with reproductive periodicity was examined. Fig. 6 shows the mean oocyte diameter per collection ys. temperature at the time of collec- tion. There was a trend of decreased oocyte diameters during periods of higher water temperature (above 25T to about 34OC). The results of a regres- sion analysis are highly significant (F=35.17, with an associated p<.OOOl). The following model was used Oocyte Diameter = A + B (Temperature). The res-

25 140

130

1.

I. 120 'I I.. 8 I. I I I 110 I :

I 100

90 I I1 IIIIII I! I 1 I I I I J FMMAAMMJ J J A S 0 N J F Months of Collections

Fig. 5. Synchronicity among individual c.floridana within a collection. 50 oocytes from each female with enough oocytes to count (N=5, maximum).

26 Table 3. Column totals, Kolmogorov-Smirnov s-sample test: exploratory data analysis for examining synchronicity in reproduction among female c. flondana within a sample during the period 1993-94. Females with enough oocytes to measure are numbered 1-5.

lf 74 7/71 318 3/21 416 1 1.67 1.89 15.37 0.64 4.55 2 0.42 4.18 4.44 9.96 1.41 3 2.92 0.96 2.10 0.35 0.71 4 0.79 0.48 1.34 3.82 4.08 5 0.51 0.41 1.03 4.14 2.72

4/18 514 5/73 616 6/79 1 1.96 0.66 0.76 5.08 13.22 2 6.59 0.30 1.68 9.70 8.63 3 0.59 0.14 0.71 1.07 2.71 4 0.27 0.71 1.39 1.36 5.57 5 20.37 0.53 0.54 2.38

7/14 8125 10110 11 111 1I? 194 1 4.28 1.10 2.99 3.90 0.68 2 3.62 0.44 0.76 0.63 2.91 3 0.69 1.62 4.18 0.92 1.74 4 0.60 0.72 6.33 5 2.92 2.62

2124/94 1 0.62 2 4.15 3 2.85 4 2.17 5 0.22

28 TabIes 3 and 4. The Kolmogorov-Smirnov s-sample test (Kiefer, 1955) for examining synchronicity in reproduction among female c.floridana.

5 2 T= maximum C ni [Fi(x) - F(x)] i=l where Fi(x) = # (observations in sample i 5 x)/ni where ni=50 F(x) = # (observations in all samples x)/n. 5 n, = C ni, or 250. I

T is the point at which the weighted sum of the squared differences between the individual cumulative distribution frequencies and the average cumu- lative distribution frequency reaches its maximum. The square root of this value is taken and the p-value determined df4from Table 1 provided by the author. This test provides a single comparison rather than making 10 pairwise comparisons.

27 Table 3. Column totals, Kolmogorov-Smirnov s-sample test: exploratory data analysis for examining synchronicity in reproduction among female c. flondana within a sample during the period 1993-94. FemaIes with enough oocytes to measure are numbered 1-5.

1/24 3/71 318 3/31 416 1 1.67 1.89 15.37 0.64 4.55 2 0.42 4.18 4.44 9.96 1.41 3 2.92 0.96 2.10 0.35 0.71 4 0.79 0.48 1.34 3.82 4.08 5 0.51 0.41 1.03 4.14 2.72

41 18 5/4 5 /23 6/6 6/79 1 1.96 0.66 0.76 5.08 13.22 2 6.59 0.30 1.68 9.70 8.63 3 0.59 0.14 0.71 1.07 2.71 4 0.27 0.71 1.39 1.36 5.57 5 20.37 0.53 0.54 2.38

71 14 8/35 10110 11 111 1/2/94 1 4.28 1.10 2.99 3.90 0.68 2 3.62 0.44 0.76 0.63 2.91 3 0.69 1.62 4.18 0.92 1.74 4 0.60 0.72 6.33 5 2.92 2.62

2/24/94 1 0.62 2 4.15 3 2.85 4 2.17 5 0.22

28 Table 4. Results of the Kolmogorov-Smirnov s-sample test for examining synchronicity in reproduction in female C. floridana: T values and associated p values (* indicates value is too high and thus beyond the limit of the table).

Date 1/24 2/21 3 18 3/21 416

T 1.54 3.08 6.98 6.74 3.74 P 0.68 0.10 * * 0.04

Date 4/18 514 5/23 616 6/79 T 11.92 0.90 1.30 4.34 10.08 * * P 0.97 0.81 0.01

Date 7/14 8/25 loll0 11/11 117 /94 T 2.28 1.21 3.62 2.94 4.86 P 0.07 0.44 0.02 0.13 0.01 u T 2.84 P 0.14

29 130

120

110 I 33 33 1

34 27 F=35.17 100 L p<<.oo1 27

90 ~~~~~~~ ~~~ ~ JFMMAAMMJJ J AS0 N J F Months of Collections

Fig. 6 Temperature (“C) vs. oocyte diameters (pm)from 24 January, 1993 to February, 1994. The one letter abbreviations represent the months of collection, as listed on p. 22. There were no females with enough oocy- tes to measure on 19 September, 1993; the water temperature on that day was 32 ‘C. Bars are the standard error of the mean.

30 iduals were plotted against predicted values and against temperature; there were no striking outliers, and residuals were independent of the estimate.

Histochemistry of the Reproductive System s3l.uawQfh- ' -. The gonad is distributed throughout the visceral mass in both male and female I=. floridana (Fig. 7). Acini can be found both within and bordering the digestive gland, adjacent to the stomach and intestine and within the foot. There do not appear to be any follicular cells within the acinus; the germinal epithelium is surrounded by a layer of muscle (Fig. 8: W). The gonad increasingly invades tissues surrounding the organs in the visceral mass as the reproductive period approaches. Oogenesis is a continuous process; oogonia are present year-round, as are oocytes. Fig. 9 is a photograph of an unfixed, stalked oocyte in the ovary of a live aquarium specimen. The ovary contains a considerable amount of a mucilagenous sub- stance that I refer to as matrix. This matrix becomes increasingly prominent in the ovary during the onset of the reproductive period (Fig. 10: MA).

-. -. Although the mechanism of vitellogenesis, is., the actual production and/ or uptake of eosinophilic granules, was not examined di- rectly, it is presumed to be a process of contributions from various sources, considering the simple morphology of the acini and the juxtaposition of the gonad with organs of the digestive system.

Sources & Nature d-. Table 5 displays histochemical test results for I=. florldana. Oogonia were basophilic (results not shown) and oocytes were

31 Figs. 7-10. Photomicrographs of 8 pm-thick sections of the ovary of C. floridana.

Fig. 7. Delafield's hematoxyIin-alcoholic eosin stain of a 24.1 mm-long animal collected on 24 January 1993. D, digestive gland; 0, ovarian tissue. Scale bar = 100 pm.

Fig. 8. Delafield's hematoxylin-alcoholic eosin stain of a 24.6 mm-long animal collected on 10 October 1993. A, acinus; D, digestive gland; Mu, muscle. Scale bar = 10 pm.

Fig. 9. An unfixed, unstained oocyte from an aquarium specimen, as viewed with differential interference (Nomarski) optics. Scale bar = 100 pm.

Fig. 10. Delafield's hematoxylin-alcoholic eosin stain of a 22.9 mm-long animal collected on 21 February 1993. MA, matrix; 0,oocyte. Scale bar = 100 urn.

32

LI + +++ + + 24 + + + 2 + +:: + + 3$ +++++ + nu +

-.cn -.cn + +++ + + +++ + +:: 22 + +++ + + ++ my + + 40 + +:+ + + ++ + +

+ ++ +++ ::+ ++ :+ + +

w ++ ++ \ + ++ ++ ++ 3: : ++ ++ ++

34 acidophilic (eosinophilic); a basophilic nucleus was observed in a number of developing oocytes during the reproductive period. The egg mass matrix is possibly a contribution from numerous sources. The most important source is likely the glandular distal portion of the kidney referred to in Table 5 as se- cretory material. Fig. 11-12 show sections of the kidney of a female main- tained in an aquarium and fixed during the production of an egg mass. Both sections stained strongly for the presence of strongly acidic sulfated muco- polysaccharides. Concretions in the digestive gland (Fig. 13) stained strongly for the presence of calcium, perhaps to be invested for shell production in de- veloping young. Fig. 14 is a photograph of an ovarian section from the same female showing the strong reaction of these concretions when stained for the presence of mucins.

Egg Mass and Developmental Stages Fig. 15 shows a female C. florldana with an egg mass. The translucent egg mass is attached to the posterior portion of the shell and was frequentIy found to be as large as 30 X 50 mm. It is composed of a very strong, adhesive material; the adult can remain attached to the mass even when suspended by it out of water. The egg mass is permeable to a wide variety of organisms, c.g., protozoa, copepods, nematodes and crustacean larvae. Egg masses that be- came detached from the adult due to transport from the collection site were maintained in an aquarium and sampled daily for a period of approximately 14 days; viability did not seem to be compromised, and development of the embryos continued at the same rate as those in egg masses that had remained attached to the adult. It is not likely, therefore, that continually-produced ma- ternal sustenance is a factor in embryonic development.

35 ~i~~,11-14. Photomicrographs of 6 km-thick sections of a 24.3 rnm-long c. floridanp,maintained in an aquarium and fixed during the production of an egg mass. Scale bars = 100 km.

Fig. 11. Delafield's hematoxylin-alcoholic eosin stain of a kidney. K, kidney; S, secretory material.

Fig. 12. Alcian Blue (pH 0.4)-NeutralRed stain of a kidney. The blue stain confirms the location of a secretory tissue shown in Fig. 11. K, kidney; 0, ovary; S, secretory material.

Fig. 13. Alizarin Red S (Dahl's test for calcium) of ovarian tissue. C, concretions; D, digestive gland; 0, oocyte; W, wall of the visceral mass.

Fig. 14. Lasky's Mucihaematin (for mucins) of ovarian tissue. C, concretions; D, digestive gland; 0, oocyte; W, wall of the visceral mass.

36

Figs. 15-18. Photomicrographs of aquarium-maintained specimens of c. floridana.

Fig. 15. Female c. floridana (21.7 mm shell length) collected 31 May 1992. The egg mass was produced sometime prior to 10 June and was in a deteriorating state.

Fig. 16. Female c. floridana (24.3 mm shell length). The egg mass was pro- duced 3 April 1993. E, egg mass; F, foot; V, visceral mass.

Fig. 17. Same female as in Fig. 16, but at higher magnification.

Fig. 18. An unfixed and unstained bivalved embryo (D stage) removed from an egg mass. The membrane and funiculus are visible. Scale bar = 100 vm.

38

The egg mass emerges between the gill lamellae in the interlamellar space in the right inner ctenidium (Figs. 16-17). In many of the histologic sec- tions of females taken during the reproductive period, this ctenidium was observed to be greatly distended in comparison to the corresponding ctenid- ium on the left side of the animal. Cryosectioning of the egg mass was prob- lematic, due to the presence of embedded sand grains which caused ripping. Both eggs and matrix in the few sections that were obtained stained strongly with ToIuidine Blue. The presence of both neutral and acid glycoproteins in the matrix was indicated by the results of the ABIPAS test.

.. Cwnts pf Developiug Indlvlduals. Fig. 19 displays the results of a plot of the shell lengths of 10 adults and the numbers of embryos present in the corre- sponding egg mass. The correlation between adult size and number of em- bryos per mass was significant (F=5.01, p=0.055).

Develop- m.Fertilized eggs ranged in size from 138-144 pm and 210-230 pm including the outer membrane. Morula, gastruIa, modified tro- chophore, and straight-hinge stages were observed. Embryos developed for the most part in synchrony and unfertilized eggs were rarely observed. Em- bryos in the modified trochophore stage measured 195 pm, on average, and 235 pm together with the outer membrane. It was during this stage that the embryos were observed to spin. Most (>500/0) of the developing embryos were associated with spherical masses within the confines of the membrane; an in- dividual embryos was observed with approximately 25 such spheres, the largest sphere being 28 pm in diameter. These spheres are possibly pseudo- feces and/or discards from shell production. They were moved about within

40 17000

h 0 d 2 13600

M wM !+ .. 6800 ln

P; 6 3400 w

0 I I I I I I I I I I I 18.2 21.4 23.2 24.4 24.5 24.7 24.8 25.7 27.8 29.7 Shell Length in mm

Fig. 19. Number of embryos per 10 egg masses and she11 lengths in mm of the 10 corresponding adult c.floridana.

41 the membrane by the ciliary currents produced by the embryos. Although actual emergence of a juvenile crawl-away from the egg mass was not ob- served, the smaIlest shell measurements recorded at this time were 221 pm shell length, 209 pm shell width, and 147 pm shell height. The valves of these juveniles were frequently observed to be covered with a “fuzzy coating” perhaps consisting of algae; the protozoan Vorticella was observed on occa- sion attached to the shell. It is not known if this coating is merely a product of the aquarium environment, although it could serve to provide a cryptic covering for the juvenile. Recently emerged juveniles were very vigorous and were observed on several occasions to engage in a type of movement similar to that of a gastropod, i.e.,the bivalved shell was elevated to a posi- tion directly above the foot and carried. about for short distances. The foot is highIy extensible. Juveniles were often observed engaging in what appeared to be feeding behavior. The highly ciliated foot would be rapidly withdrawn a, .. into the shell and wiped across an area in the anterior portion of the animal; the adults possess labial palps and it is not known at what stage they develop in the juvenile. Such activities could be observed because the shells were translucent prior to the establishment of the epibiota. The beating of the heart and the rhythmic beating of the cilia within the two large, central cili- ated chambers and the one smaller chamber anterior to these, along with the frequent pauses and reversals of direction of these cilia, could be observed as well. Within each of the two large ciliated chambers, what appeared to be in- dividuaI gill filaments were observed to periodically extend toward and be- tween the filaments opposite them and then withdraw. It is not known if this is related to feeding behavior.

42 DISCUSSION

Oviparity in the Bivalvia Carditamera floridana is one of only 17 species of oviparous marine bi- valves (Appendices I and II). Of the nine genera that are oviparous, six are eulamellibranchs, one is a protobranch, and two are filibranchs. Only five species are listed as undergoing direct development. The appendices are adapted partly from Sellmer (1967); corrections were made where necessary. Cyamium Jninutum was listed twice, as belonging to both the family Cyami- idae and Leptonidae, and listed together with Turtonia minuta. These are not separate species; the species in question is Turtonia minuta (Cyamioidea: ..a* r- Turtoniidae) (Appendix II). Mackie (1984) listed three species of &Iacomt\ as ... 1; 1; oviparous, as follow: M. lovani. M. moesta, and M.m. The reference cited was Ockelmann (1958), in which these species are noted only as having a "pelagic stage assumed to be very short or lacking". boripes lacteus (Lucin- oidea) was cited as well by Mackie (1984) as oviparous. The reference cited (Miyazaki, 1938) contained no mention of this species, although another paper published by Miyazaki in 1938 (referenced by Oldfield, 1955) contained a note on the lack of agreement between investigators as to whether or not the gel-like material surrounding the eggs of L. Iacteus had an adhesive quality. The literature is often quite vague or nonexistent for many aspects of reproduction in marine bivalves; the evidence for reproductive types is un- clear and many references are anecdotal in nature.

43 Oviparity in <3arditamera floridana and possible adaptive significance In the literature dealing with developmental types of marine inverte- brates, useage of the term "larvae" is unclear; this term is more properly asso- ciated with organisms that undergo a metamorphosis (W. Heard, pers. comm.). I will refer to developing young of S, florldana while contained within the egg mass as "embryos". The embryos develop entirely within the egg mass, possibly feeding on yolk reserves and material that can diffuse through from the environment, to emerge as crawl-away juveniles. No evi- dence for a separate and distinct developmenta1 type (poecilogony) was found during the course of this study. The adaptive significance of the reproductive mode of C. floridana must be considered (Vance, 1973; Chia, 1974; Crisp, 1974; Christiansen & Fenchel, 1979; Havenhand, 1993). Fertilization accomplished by the broadcast- ing of gametes into open waters (the most primitive mode of reproduction, & Jong-Brink, d.,1983) leads inevitably to great Iosses and the risk of the gametes not meeting. Large numbers of eggs and sperm are consumed by pro- tists (Galvao, & d., 1989). Internal fertilization is a method to ensure a greater probability of reproductive success. Direct development might lead to de- creased mortality. Time spent by the larvae of species undergoing indirect development in the open sea can range from a few weeks to a few months; this is the most vulnerable stage in the life cycle. Not only do adequate sup- plies of food need to be found by pelagic larvae, but predator-induced mortal- ity prior to recruitment to the juvenile benthic phase is high. Direct devel- opment is correlated with a reduced generation time (Thorson, 1950); it might have a "balancing" effect in that there are fewer losses, although fewer off-

44 spring are produced (however, see below). The egg mass of c. floridana de- generates after an average maximum period of 14 days, accompanied by the release of juveniles. Contrary to the model of Thorson (1950), in which the presence of incubatory species is correlated with boreal climes and/or deep waters, c. floridana is found in shallow, subtropical waters. It is a relatively large species, as compared to other species that exhibit extended parental care (Appendix I). C. floridana is also unusual among oviparous bivalves in producing large quantities of young (as many as 17,000 eggs were found in a single egg mass) (Fig. 19; Appendix 11). Although not examined in this study, the repro- ductive effort would appear to be high in this species; the egg mass is most of- ten several times the volume of the adult that produced it. Additionally, the egg mass remains attached to the adult during the development of the young to the crawl-away juvenile stage. This extended care might be a source of predator-induced mortality, both for the adult and the developing young, es- pecialIy because the production of egg masses by adults appears to be a syn- chronous event and would seem to draw visual and other types of predators. The Haladule beds, located close to shore, were observed to be densely inhab- ited by adults with egg masses during the reproductive period. This might present an easy target for shorebirds, because the grassbeds are often exposed during low tides. Highsmith (1985) predicted that intense predation pressure at low latitudes would restrict forms of extended care such as brooding in fa- vor of planktonic forms. Alternatively, the retention of the developing young within the egg mass must be considered as a means to ensure increased survivorship. The material of which the egg mass is composed is strongly adhesive in nature; this feature might serve to protect the young from the ef-

45 fects of wave action and keep the juveniles in the same environment as the adults. Reduced dispersal abilities are traditionally associated with direct devel- opment (Strathmann, 1985; Hedgecock, 1986; Johannesson, 1986; Scheltema, 1986; O'Foighil, 1989). However, species of A a genus of brooding bivalve (OFoighiI, 1989), have restricted geographic ranges. Dispersal without a larval stage might not be uncommon among bivalves due to the phenomenon of byssus-drifting (Beauchamp, 1986). This mode of dispersal might be applicable to the case of c. floridana. Juveniles have a byssus but it is not known at this time if they are capable of byssus-drifting. Juveniles and adults have been ob- served attached to sponges, urchins, tunicates, and gastropods, and are capable of climbing and attaching themseIves to the sides of an aquarium. It is possi- ble that some degree of dispersal could be attained by the fragmentation of sponges and wave action on these and species of colonial tunicates, by a sym- biosis with various species of gastropods, and by rafting. The population of c. floridana examined in this study might be inhab- iting what is for them a stressful environment, Although St. Andrew's Sound is a protected embayment, the wave action produced by winter storms can result in great losses, such as those observed during the storm of March 1993. Grassbeds consisting of monospecific stands of (the species preferred by c. floridana.* pers. obv.) in the sampling area are limited in size and fragmented; intraspecific and interspecific competition for sites might be intense. Various species of tunicates plis&, Amarouclum'stelIatum. m),a number of which were observed to be seasonally abundant, and cockles (eg., Trachvcardlum ermontianum ) are likely to be ca- pable of filtering large numbers of immature invertebrates from the sur-

46 rounding waters. There are a number of species of carnivorous polychaetes, gastropods, crabs, batfish, and rays inhabiting the area that might prey on all stages of the life cycle of this species. It might therefore be advantageous to mature early and reproduce in such an unstable environment where preda- tion pressure is high. The life span of this species is unknown. The mode of reproduction exhibited by c.foridana could also be a specialization in which a low metabolic rate has an adaptive advantage (Blacknell & AnseII, 1974); planktotrophic species were noted as having a higher metabolic rate than lecithotrophic species. Nothing is known of the dietary requirements of c. floridana. Fragments of various unidentified diatoms were observed in the stomachs of a number of specimens; the species might be limited in its the range of acceptable food items. The retention of young until such a time that they are capable of independent life as juveniles might serve to retain the next generation in an area in which the adults were successful.

Individual Sexuality, Sex Ratio, Sexual Dimorphism in Shell Shape .. Indlvldual Sexualitv %w. Although the overall sex ratio in c. florldana is approximately 1:l (172 male, 193 female), the juvenile age-classes are male-biased. Histological examinations ruled out protandry. A possible explanation could be differential mortality, la., males do not live as long as females and/or mortality is high for young females. The age-class structure might be the result of differential production. Rudimentary germ cells might respond to the level of food reserves; if these are low, sexual development could be biased toward the production of males whereas if the reserves are high, a bias toward femaleness could result (Sastry, 1979). Morton (1991) noted the sex ratio of Mercenaria mercenaria as being skewed toward males

47 when individuals are < 1 year old (9.51) and changing to 1:1 as they aged. The preferential production of males could be a strategy to compensate for the loss of sperm in the sublittoral zone and the bias towards females as the popula- tion ages might select for increased productivity. Another possibility is the differential segregation of the sexes by habitat preference. The population of G. floridana studied was infected with a digenetic trematode (12.6% of the population). Castration was the result in all but a very few cases. The mean shell length of infected individuals (N46) was 22.3 mm k 3.2 ; the mean shell length of all females was 23.8 mm f 2.6 and for all males it was 19.3 mm k 5.2. The overall shell size is known to become en- larged in individuals that bear a parasite load (M.B. Kotrla, pers. comm.). Females might become more resistant to the trematode than males as they age; this could provide one explanation for the relative absence of small fe- males in the population. However, Moore & L6pez (1969), in a study of a population of in Biscayne Bay, Florida, found 7.7% of their specimens infected with trematode parasites; although the sex ratio was 1:1, the largest size group contained more males than females, leading the au- thors to speculate that large females were more frequently affected by parasitic castration than males. Although no evidence was found, the population might also be infected with cestodes; these are known to use bivalves as in- termediate hosts before attaching to their final host (numerous species of elasmobranchs) in the Gulf of Mexico (Cake, 1975).

hwa.Sexual dimorphism of bivalve shells s.m&Ll- ’ has been reported in the families Astartidae (Saleuddin, 1965), Galeommati- dae (Morton, 1976), Montacutidae (Chanley & Chanley, 1970), and Teredinidae

48 (Turner & Yakovlev, 1983). As previously stated on p. 8, there is a prece- dence for sexual dimorphism in shell shape in the Carditidae. This character is rare in bivalves and is considered to be a direct consequence of larviparity

(Sastry, 1979). Kotrla &K James (1987) conducted a morphometric examination of shell shape in two species of freshwater bivalves; although neither species was found to exhibit sexual dimorphism in size, shape sexual dimorphism was found to be constant during growth for (Wright) but changed with growth in Elliotio (Conrad). The results of the regression analysis in this study indicated a signifi- cant difference between male and female c.floridana in shell shape indepen- dent of size. Shell height was found to increase in females as they grew larger to a disproportionate extent as compared to males. This might be understood as an adaptation in females related to the gonadal volume achieved during the reproductive period and the production of the egg mass. Alternatively, the presence of some degree of shell sexual dimorphism might be indicative of past ovoviviparity.

Reproductive Periodicity Wperiodlcitv. Giese (1959) listed five methods by which to determine the reproductive periodicity of a marine invertebrate, as follow: field obser- vations of spawning, aquarium and laboratory observations, the occurrence of larvae in the plankton, calculation of the gonad index, and histological inves- tigations. In this study, field observations of spawning, aquarium and labora- tory observations, and histological investigations were used as evidence to es- tablish periodicity in c.flondana. Qualitative assessments of gonadal stages can be considered as arbitrary (Seed, 1977), dependent upon the density and

49 development of the gametes; the number of stages depends on the rate at which the reproductive cycle proceeds. The criteria employed in this study were after W. Heard (pers. comm.) and Nott (1980). The statistical analyses were based partly on the studies of Grant & Tyler (1983). Oogenesis was found to be continuous in c.floridana. Although the statistical analysis of oocyte diameters in females over the period of one year indicate seasonality of reproduction (one reproductive cycle per year), egg masses were found in the field 20 March 1994,31 May 1992, and 10 June 1992. Gross dissections indicated a distinct increase in the adhesive quality of the mucous in the filll months as well, particularly in October. Fig. 4 shows an increase in mean oocyte diameter one month later. Records of young in the field suggest the existence of another reproductive event. Aquarium observa- Y tions indicated that reproduction could be induced by a water change (28 't October 1992) and by an increase in water temperature of 6" C and/or a cue contained within the water during a water change (26 February 1993). Egg mass production was observed in an aquarium on 3 March, 10 March, 19 March, 2 April, and 5 May 1993 and 12 April 1994. Undocumented aquarium observations by former students on another population of this species indi- cated a single individual female could produce more than one egg-spawn in a year. Males are mostly ripe with sperm year-round; this is not unknown, as some members of the family Astartidae have ripe gametes in both sexes months before the reproductive season (Giese, 1959). Other members of the family Carditidae are both seasonal and continuous reproducers (Tones, 1963; Yonge, 1969). In support of the hypothesis of one reproductive event per year was the observation that from 29 June to 10 October 1993, five females with

50 sufficient oocytes to measure could not be found; the 19 September collection contained no females with this qualification. The sampling site was checked on a monthly basis close to the same date each month during fall and winter; sampling might not have been fre- quent enough and a source of error could have been introduced by missing a spawning event. During determinations of gonadal stages in females, incor- rect assessments might have been made in some cases due to an inability to distinguish between oogonia and oocytes (free) because the stalk was not al- ways visible, although the egg perimeter was distinct (clearly resolved) from the background material. Also, "empty" acini (=spent) might have been arti- factual in some cases due to mechanical stress during tissue processing. According to Giese (1959), oocytes can either be retained or undergo au- tolysis. There was no indication of phagocytosis of relict oocytes. A number of other species of bivalves at the sampling site were col- lected when available and processed for histological examination. Several specimens of the following species were found in either a ripe or partly spent condition around the time that egg masses of c.floridana were found in the field: sp., rancellata. Anadara a dlxE&Trachvcardiumeemontianum. and AeauiDecten irradians concentri- cus.

.. SvnchronlcihrAmone- I The precedence for synchronicity in re- production in marine invertebrates has been well established, although the environmental cues remain to be elucidated. The appearance of egg masses of c,florldana in the field were observed to co-occur with the appearance of egg masses of the polychaete Arenicolacristata. with which they are cryptic.

51 An advantage to synchrony in reproduction among female c. floridana dur- ing this event might be the concept of safety in numbers; the event might provide too large a target for some visual predators. The ubiquitous presence of an unidentified species of brown alga was observed also at the same time as the spawning event. This might provide protection from ultraviolet rays for the developing young within the translucent masses, serve as a cryptic cover, as a source of nourishment from organisms associated with the algal bloom, and as a possible means of dispersal for the crawl-away juveniles. The cou- pling of marine invertebrate spawning with phytoplankton blooms has been well documented (Lewis, 1986; Starr, 1990). Although, as mentioned previously, it is not known what the dietary habits of c.floridana are, it might be the case that one of the cues involved in the initiation of the spawn- ing event is the appearance of a food source. The Kolmogorov-Smirnov statistic T is a test statistic for the null hy- pothesis Ho, ia., all females within a sampling period have the same distribu- tion of egg sizes. Small values of T indicate that the distributions are similar (Table 4). The T value for the females sampled in the collection of 4 May and 23 May 1993 are the lowest for any collection. This corresponds to the sharp decrease in mean oocyte diameters calculated for those collections (Fig. 4) and to the tightly clustered values in the graph of mean oocyte diameters for each female within a collection (Fig. 5). Most regrettably, a spawning event in 1993 was not observed.

TemDerature & Annual -. Temperature might be an environ- mental variable related to reproductive periodicity (Orton, 1920). The in- crease of 6Tin water temperature during a change of water in an aquarium

52 might have induced the production of an observed egg mass, The tides in the sampling area are lowest during the late afternoon and early evening, gener- alIy, in the summer months; in winter, they are early in the morning. The

exposure of egg masses to potentially damaging ultraviolet rays might need to be minimized, because numerous egg masses are produced in seagrass beds that are often above water levels or in very shallow water. The F-statistic from a regression analysis is highly significant. The co-occurrence of slowly rising temperatures (Fig. 6) in May 1993 and a sharp reduction in the mean oocyte diameters might be indicative of a relationship between these two variables.

Histochemistry of the Reproductive System

.shlLduQfhXeDroductlve’ m. The gonad in most molluscs origi- nates as a paired organ, is located in the posterior part of the animal and is closely associated with the digestive gland Uong-Brink, &d.,1983). In the Bivalvia, the gonads become fused and the number of gonoducts generally corresponds to the number of gonads. The anatomy of the gonoducts and their locations are unknown for C.floridana. The oviduct must open in to the interlamellar space between the ascending and descending lamellae of the inner right demibranch because it was from this region that the egg mass was observed to issue (Figs. 16 & 17). Inasmuch as this event was recorded with some detail for only one female, it is not known if this was the only way in which the egg spawn could be produced. Nevertheless, young can be brooded in either the inner or outer or both pairs of demibranchs in various members of the superfamily Carditoidea (Jones, 1963; Yonge, 1969); in one specimen of

53 the carditid Cardita ventricosa. larvae were present on only the left side of the animal (inner and outer demibranchs) (Jones, 1963). The gonad of c.floridana is "diffuse" in nature i.E., it consists of a number of acini which might be found scattered throughout the visceral mass (Fig. 7); the diameter of the acini increased in size as the reproductive period approached. The walls of acini containing differentiating oocytes werc thick. A layer of muscle tissue could be discerned (Fig. 8: MU); accessory/ auxiliary cells could not be clearly distinguished. As oogenesis proceeded, no follicular cells were found in close association with the developing oocytes in the manner described by Jong-Brink, dill. (1983); however, it seems reason- able to assume that some type of somatic cell must exist. It could not be de- termined if a delimiting cell type (acinar epithelial cells -Kotrla, 1988) comprised the peripheral layer of each acinus. Oocytes were surrounded by a double membrane (Fig. 9); as the reproductive period approached, oocytes were found complexed with strands of basophilic material (Fig. lo), and the acini were increasingly fragile in appearance; it is not known is this was an ar- tifact of processing the tissue.

-. -. Oogonia were acidophilic; the cytoplasm became increasingly basophilic due to the synthesis and accumulation of RNA, followed by in- creasing acidophilia as vitellogenesis proceeded (see Jong-Brink, & d.,1983). This process is usually the result of the accumulation of lipoproteins from in- side the developing oocyte and from extracellular sources. Because an ultra- structural analysis was not included in this study, the exact details of this de- velopmental phase in not known. It would appear reasonable that contribu- tions are likely to occur, perhaps by diffusion, from the digestive glands, con-

54 sidering their proximal locations and the nature of the staining of the inclu- sions discovered within (Table 5). The inclusions within the digestive gland might be related to the mineral concretions located in the spermatogenic and oogenic acini referred to by Kotrla (1988) in a study of freshwater bivalves. The inclusions in G. floridana were found in close proximity to oogenic acini but not inside; they were as large as approximately 20 pm in diameter, whereas the largest in the freshwater species was 0.5 pm in diameter. None of the types of follicular cells described by Jong-Brink, &d.,(1983) appear to correspond to the anatomy of the acini of c. floridana.

Sources & af Mucins. Harry (1966) found a "peculiar mantle gland" in his study of a population of Crassinella lunulata (Conrad) (Carditoidea) in the NE Gulf of Mexico; eosinophilic granules were found in histological sec- tions. Tissue staining strongly with hematoxylin was found in the anterior, distal portion of the mantle margin in both male and female C. floridana (not shown). As there were no figures to accompany the description of this struc- ture in c. it is not known at this time if it is related to the structure in c.florldana. Morton (1977) described the hypobranchial gland in the BivaIvia; its primitive function was the production of mucus to expedite the elimination of waste materials. This function has been retained in some filibranchs, whereas in a number of other bivalves this gland is involved in reproduc- tion. Inm- the hypobranchial gland secretes a brood pouch and in the eulamellibranchs Corblrula' flumineaand- fimbriata, it serves as a source of nutrition for developing young. The loca- tion of this organ varies among different species. In the former species, the

55 hypobranchial gland lines the interlamellar and interfilamentar junctions of the inner demibranch of both the left and right ctenidia, whereas in E. fimbriata it lines the upper surface of the suprabranchial chamber posteriorly. A comparative cytological study of the hypobranchial glands in these species and of the tissue located in/near the kidney in c. floridana. which stained strongly with both hematoxylin and Alcian blue (pH 0.4), might point to an homology . Oviparity could have arisen in the Bivalvia in a number of ways to in- clude the following: the presence of mucous-secreting tissue; peristalsis, or some other means to control transport; the storage of sperm; a fluid medium, and an increase in the amount of yolk invested in the eggs (Ockelmann, 1965; Runham, 1983). The histochemical tests conducted in this study (Table 5) were based in part on the 1965 study of GhiseIin on opisthobranchs. Material staining strongly for the presence of mucins (Lasky's mucihaernatin stain) was found in the wall of the visceral mass, in the acini, the digestive gland inclusions, the gill secretions and in the suprabranchial chambers. Mucins are an ubiqui- tous substance in molluscs (Prezant, 1985), playing a role in a number of di- verse functions. It is not known which of the above sources, if any, con- tribute to the production of the egg mass in C. -. The inner lining of the demibranchs of c.fuminea is composed of mucocytes (Morton, 1977); al- though these are likely involved in internal brood protection, it seems rea- sonable that the secretions of the gill in c. might perform an analo- gous function by contributing to the production- of the mucilagenous matrix of which the egg mass is composed. The tissue listed as "secretory material" in Table 5 is the area located in the proximal regions of the kidney on both

56 sides of the female. The strong staining reaction to Alcian blue (pH 0.4) is an indication of the presence of strongly acidic sulfated mucopolysaccharides; this material also reacted strongly with hematoxylin. The matrix material (within the ovary) was shown to stain strongly with a combination of Alcian blue (pH 2.5) and periodic acid-Schiff's reagent, which is an indication of the presence of carbohydrate-containing proteins. The egg mass matrix stained strongly for this dye as well (results not shown). It is likely that the material of which the egg mass is composed is a contribution from a number of sources. Further analytical procedures, such as geI electrophoresis, might prove useful in localizing the mucins.

Egg Mass and Developmental Stages Strathmann & Chaffee (1984) reasoned that, because animals without a circulatory system are limited in size or form, so should masses of embryos. Constraints on the thickness of egg masses might include the following fac- tors: a sufficient pressure head to ventilate the embryos; oxygen supply per embryo increases in proportion to the radius of a spherical mass, and thin- walled capsules within a mass present less of a constraint than do gelatinous masses. They speculated that brooding might be correlated with small adult size in many taxa, because large adults cannot ventilate all the eggs that they produce. Strathmann & Strathmann (1988) experimented with the produc- tion of artificial egg masses (agarose matrix) in a normally planktonic echi- noid eratilla (Linnaeus); normal development was achieved by creating egg masses in which the developing embryos were evenly spaced and in which the egg masses were made thin. The authors hypothesized that ad-

57 ditional matrix would interfere with fertilization and hatching and require a large site for deposition. The egg mass produced by C. floridana is not composed of a gelatinous matrix but rather of a mucilagenous matrix. Efficient exchange with the en- vironment might be achieved by this less dense material and through the re- tention of the mass by the adult together with its disposition in a vertical di- rection, raised above the substrate and into the water column, as opposed to lying on its side. Although it was not demonstrated in this study, some ven- tilation of the mass directly by the adult might occur, inasmuch as the mass is attached to the exhalant aperture. The mass is somewhat fluid in form; it is capable of changing shape with the water currents while remaining attached to the adult. Therefore, the egg mass is not strictly spherical in shape and em- bryos located near the center are likely to be as well ventilated as those occu- pying a more peripheral area. The Strathmanns' concern for the potential for interference of the matrix with fertilization would seem irrelevant for the species that have internal fertilization and most likely store sperm. Further- more, the matrix might be composed of a materia1 that naturally undergoes decomposition after a period of 2 weeks to coincide with the development of the juveniles to the crawl-away stage.

.. !&un.ts Qf Developing Indmduh, Analyses of more egg masses are neces- say given the possibility of: correlation between adult size and the number of embryos within an egg mass. Although all animals with the egg mass at- tached had been collected on the same day, the possibility for error exists in that the date of egg mass production was not known with certainty. None of the egg masses collected was found to be in a deteriorating condition, and all

58 embryos in each of the 10 egg masses were at a similar stage of development. Upwards of 17,000 developing embryos were found to be contained within.the egg mass of a female measuring 24.7 mm shell length. This is the largest number of embryos per egg mass reported for any species of oviparous bi- valve to date (Appendix 11).

Develogme&d&g.g&. Very little is known of development in oviparous bi- valves (Appendix 11). Development might not be direct in all cases, gg., in in which a shelled veliger emerges from the egg-spawn. fie duration of the developmental period of hm (2-3 weeks) is simi- lar to that off, floridana (2 weeks), whereas that of gaulrli appears to be protracted (2 months).

The presence of populations of Carditamera florldana in the northern Gulf of Mexico might represent an expansion of range for the species; it is uniformly listed as occurring from southern Florida to southern Mexico. A shift in the breeding period could be taking place, as evidenced by the pres- ence of ripe egg and sperm for prolonged periods of time (Sastry, 1979). The coastline in the area of St. Andrew's Sound has undergone changes over the years. A severe storm a few years prior to the initiation of this study has al- lowed the introduction of a number of previously unknown species of gas- tropods into the area (B. Barfield, pers. comm.); it is not known how many of these are potential predators of c.floridana. Seagrass beds of Halodule. which appear to be preferred by c,floridana. are extremely fragmented in the Sam- pling site; indeed, the seagrass is a pioneer species that is under stress in a number of sites in Florida (D. Camp, pers. comm.). Future studies of habitat

59 preference of 5=. florldana might provide useful information on the stability of populations located in such areas. Other carditids inhabiting the Florida area include: dominguensis Orbigny; Fleuromerls' hidmi& Say, and Fteromerls.iw=?Lm Conrad (Abbott, 1974). Nothing is known of the reproductive mode of these species. It would be most interesting to have such information available in order to conduct a comparative study.

60 APPENDIX I COMPILATION OF OVIPAROUS BIVALVES EXHIBITING EXTENDED CARE: ADULT SIZE, HABITAT, AND SIZE DISTRIBUTION

61 h -H W V V 64

62 *h

63 64 APPENDIX II COMPILATION OF OVIPAROUS BIVALVES EXHIBITING EXTENDED CARE: OOCYTE DIAMETERS, NOTES ON REPRODUCTION AND DEVELOPMENT, AND REFERENCES

65 Y 5 E a 0 -o' 2 a Y U *W 4 ;n' %w 3 'r m

66 0 0 0 p.1 m Ln x x 0 0 N \D m N

67 68 69 70 LITERATURE CITED

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BIOGRAPHICAL SKETCH

Margaret C. Harvey was born in Brooklyn, New York, on March 14, 1953, to parents from Bremerhaven, Germany, and Budapest, Hungary. She received her B.S. degree in Biology from Eckerd College, St. Petersburg, Florida in 1977. After a number of years of travel both within the US. and abroad, she attended the University of Oregon in Eugene from 1987 until 1988 in a non-degree-seeking status. She continued at Florida State University and entered graduate school in 1991. She is currently in the Ph.D. program at the University of South Florida in Tampa, engaged in research both in the Department of Biology and the Department of Anatomy in the College of Medicine on Sertoli cells in crustacean testes.

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