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2003

A Genetic Investigation of Population Structure and Phylogenetics of the Benthic Polychaete Paraprionospio pinnata in Chesapeake Bay

Julie Beck Stubbs College of William and Mary - Virginia Institute of Marine Science

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Recommended Citation Stubbs, Julie Beck, "A Genetic Investigation of Population Structure and Phylogenetics of the Benthic Polychaete Paraprionospio pinnata in Chesapeake Bay" (2003). Dissertations, Theses, and Masters Projects. Paper 1539617807. https://dx.doi.org/doi:10.25773/v5-m0sk-v651

This Thesis is brought to you for free and open access by the Theses, Dissertations, & Master Projects at W&M ScholarWorks. It has been accepted for inclusion in Dissertations, Theses, and Masters Projects by an authorized administrator of W&M ScholarWorks. For more information, please contact [email protected]. A GENETIC INVESTIGATION OF POPULATION STRUCTURE AND

PHYLOGENETICS OF THE BENTHIC POLYCHAETE

PARAPRIONOSPIO PINNATA IN CHESAPEAKE BAY

A Thesis

Presented to

The Faculty of the School of Marine Science

The College of William and Mary in Virginia

In Partial Fulfillment

Of the Requirements for the Degree of

Master of Science

by

Julie Beck Stubbs

2003 APPROVAL SHEET

This thesis is submitted in partial fulfillment of the requirements for the degree of

Master of Science

Julie Beck Stubbs

Approved, July 2003

Kimberly S. Reece, Ph.D. Co-Committee Chairperson/Advisor

IjVfrMJt- U <*y(------Robert J. Di4z, Ph.D . f j Co-Committee Chairperson/Advisor

mmett Duffy, Ph.IF /( ) TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS...... iv LIST OF TABLES...... v LIST OF FIGURES...... vi ABSTRACT...... ix INTRODUCTION...... 2 Effects of environment on population genetic structure ...... 2 Hypoxia in Chesapeake Bay and its tributaries ...... 3 Paraprionospio pinnata...... 5 Genetic markers ...... 11 Phylogeny of the Class Polychaeta ...... 14 Study obj ectives ...... 16 MATERIALS AND METHODS...... 18 Study area...... 18 Sample collection and preparation ...... 18 Morphological measurements ...... 23 DNA extraction ...... 23 PCR amplification ...... 24 Cloning and sequencing ...... 27 RFLP analysis ...... 28 Phylogenetic analysis ...... 28 RESULTS...... 32 Sample collection ...... 32 Morphological measurements ...... 32 RFLP analysis ...... 39 Sequencing ...... 46 Phylogenetic analysis ...... 61 DISCUSSION...... 81 Population study ...... 81 Phylogenetic study ...... 83 LITERATURE CITED...... 91

Hi Acknowledgments

I would like to thank my advisors, Dr. Robert J. Diaz, for guiding me towards this project, and Dr. Kimberly S. Reece for all of her help and support. I am also grateful to the other members of my committee, Dr. J. Emmett Duffy, and Dr. Peter van Veld for their review of and suggestions on this thesis.

I owe a special debt of gratitude to Karen Hudson and Wendi Ribeiro for all of the time they put in helping me to troubleshoot. Thanks are also due to all my labmates, Seon-sook An, Corinne Audemard, Gwynne Brown, Ryan Carnegie, Lidia Sandoval, Gail Scott, and Qian Zang for all their support and encouragement. George Pongonis and Sharon Miller went above and beyond the call of duty in helping me to obtain my field samples.

Most especially, I would like to thank my husband and all my family for the wonderful gift of love and support.

iv List of Tables page 1. Sample sites and the number of individuals collected from each site. Latitude and longitude of each site are given, along with the depth of the station. The number of individuals of Paraprionospio pinnata collected from each site is reported in the final column ...... 21

2. PCR primers used in this study. Three sets of previously published primers were used for this study. Two primers were designed for this study. Reaction conditions are provided for each set of primers. Amplification was performed on a MJ thermocycler ...... 25

3. Restriction enzymes screened at each gene for polymorphisms. A panel of twelve Paraprionospio pinnata individuals was screened with a variety of restriction enzymes at each gene ...... 29

4. P values for Cochran-Mantel-Haenszel test of fifth setiger width. Above the diagonal are the P values (adjusted for ties) for a pair wise Cochran-Mantel- Haenszel test of the fifth setiger distribution of four sites. Site abbreviations are UCB= upper Chesapeake Bay main-stem, RAP= Rappahannock River, YRK= York River, and LCB= lower Chesapeake Bay main-stem. Below the diagonal, significant differences are marked...... 35

5. P values for exact test of sample differentiation based on allele frequency. Above the diagonal are the P values of and exact test of sample differentiation. Below the diagonal significant differences are marked ...... 44

6. COI BLAST Results. Sequences were BLASTed against the Entrez database. BLAST results from BrY6_3 are reported here ...... 49

V List of Figures page 1. Paraprionospio pinnata (Ehlers) 6

2. Sample Stations. Four stations, chosen on the basis of historical presence of Paraprionospio pinnata. Two of the sites, in the upper main-stem and in the Rappahannock River, are subject to annual hypoxia. The station in the York River is above the depth at which moderate hypoxia occurs in that river and the station in the lower main-stem is never subject to hypoxic conditions ...... 19

3. Frequency of 5th setiger width and branchiae length categories in each of the four stations. Because of the difference in sample size, the data is presented as proportions of individuals in the size class to the total number of individuals at that site. For 5th setiger data N for the Upper Chesapeake Bay, Rappahannock River, York River and Lower Chesapeake Bay are 27, 47, 48, and 31 respectively. The N for the branchia length data for the same stations are 14, 21, 36, and 22 ...... 33

4. Comparison of size frequency distribution in samples from the York River in years 1996 and 2001. Because of the difference in sample size, the data is presented as proportions of individuals in the size class to the total number of individuals collected that year...... 37

5. Historical abundance of P. pinnata in the York River. Data is part of an unpublished monitoring program at a site in the York River with the coordinates Latitude = 37°14.64’, Longitude = -76 °29.67’. Samples are collected quarterly with a Smith-Mac sediment grab. * indicate missing data ...... 40

6. Allelic variation in the COI gene. Proportion of each allelic variation of the COI gene, as determined by RFLP analysis with the restriction enzyme Bbs I, in four samples from Chesapeake Bay and its tributaries ...... 42

7. Alignment of COI gene. An alignment of four individuals from the York River site generated using the CLUSTAL-W algorithm (Thompson et al. 1994) in the Mac Vector 7.0 sequence analysis software package (Oxford Molecular) using an open-gap penalty of 20 and an extend-gap penalty of 10 ...... 47

8. Alignment of ND4 gene using Bielawski/Gold primers. An alignment of 2 individuals from the lower Chesapeake Bay main-stem site generated using the CLUSTAL-W algorithm (Thompson et al. 1994) in the MacVector 7.0 sequence analysis software package (Oxford Molecular) using an open-gap penalty of 20 and an extend-gap penalty oflO ...... 51

vi 9. Alignment of ND4 gene using primers designed for this study. An alignment of 4 individuals from the lower Chesapeake Bay main-stem and upper Chesapeake Bay main-stem sites generated using the CLUSTAL-W algorithm (Thompson et al. 1994) in the Mac Vector 7.0 sequence analysis software package (Oxford Molecular) using an open-gap penalty of 20 and an extend-gap penalty of 10 ...... 53

10. Alignment of AL1 gene. An alignment of 4 individuals from the lower Chesapeake Bay main-stem and one from the upper main-stem generated using the CLUSTAL-W algorithm (Thompson et al. 1994) in the Mac Vector 7.0 sequence analysis software package (Oxford Molecular) using an open-gap penalty of 20 and an extend-gap penalty of 10. One clone from one individual from the lower main- stem site shows significant differences in the 58-82 bp region. This clone contains an EcoR I restriction enzyme cut site. The others do not ...... 56

11. Alignment of ITS gene. An alignment of 5 individuals from the upper Chesapeake Bay main-stem and the York River generated using the CLUSTAL- W algorithm (Thompson et al. 1994) in the MacVector 7.0 sequence analysis software package (Oxford Molecular) using an open-gap penalty of 20 and an extend-gap penalty of 10 ...... 58

12. Trimmed multi-species alignment of the COI gene. An alignment of 30 species in the Class Polychaeta. The alignment was generated using the CLUSTAL-W algorithm (Thompson et al. 1994) in the MacVector 7.0 sequence analysis software package (Oxford Molecular) using an open-gap penalty of 20 and an extend-gap penalty of 10. Two Orders, four Sub-order, and thirteen Families. The cephalopod Opisthoteuthis sp. was used as an out-group ...... 61

13. Neighbor-Joining tree of partial COI sequence. The Neighbor-Joining tree generated using uncorrected “p” genetic distances of a portion of the COI gene following sequence alignment using CLUSTALW (Thompson, 1994). Opisthoteuthis sp. was used as an outgroup ...... 65

14. Parsimony tree of partial COI sequence. The parsimony tree generated using of a portion of the COI gene following sequence alignment using CLUSTALW (Thompson, 1994). Opisthoteuthis sp. was used as an outgroup. Jackknife support values above 50 are shown on branches that had support. Twenty percent character deletion was used for the Jackknife analysis ...... 67

15. Alignment of COI DNA sequences translated to amino acid sequence. COI DNA sequence was translated to an amino acid sequence using the invertebrate mitochondrial genetic code in MacVector 7.0 sequence analysis software package (Oxford Molecular) and aligned in the same program using an open-gap penalty of 10 and an extend-gap penalty of0.05 ...... 70

vii 16. Neighbor-Joining tree of partial COI sequence with the third codon position removed. The Neighbor-Joining tree generated using uncorrected “p”values for genetic distances of a portion of the COI gene following sequence alignment using CLUSTALW (Thompson, 1994). Opisthoteuthis sp. was used as an outgroup ...... 72

17. Two most parsimonious trees of partial COI sequence with the third position removed. The parsimony tree generated using a portion of the COI gene following sequence alignment using CLUSTALW (Thompson, 1994). Opisthoteuthis sp. was used as an outgroup. Jackknife support values above 50 are shown on branches that had support. Twenty percent character deletion was used for the Jackknife analysis ...... 74

18. Neighbor-Joining tree of partial COI amino acid sequence. The Neighbor- Joining tree generated using uncorrected “p” genetic distances of a portion of the COI gene following sequence alignment using CLUSTALW (Thompson, 1994). Opisthoteuthis sp. was used as an outgroup ...... 77

19. Two most parsimonious trees of partial COI amino acid sequence. The parsimony tree generated using of a portion of the COI gene following sequence alignment using CLUSTALW (Thompson, 1994). Opisthoteuthis sp. was used as an outgroup. Jackknife support values above 50 are shown on branches that had support. Twenty percent character deletion was used for the Jackknife 79

20. Basic classification of the taxa used by Rouse and Fauchald (1997). This phylogenetic tree is based on a parsimony analysis of weighted morphological characteristics, (Same as Fig 2 in Rouse and Fauchald, 1998) ...... 84

21. Minimum-length tree for combined H3, U2 snRNA, and 28S rDNA sequence data. This phylogenetic tree is based on a parsimony analysis combining sequence for the three genes. Percentages greater than 50 for bootstrap pseudo­ sampling are shown above the clade branch. Bremer decay indices of three or more are shown below the branch. (Same as Fig. 5 in Brown et al. 1999) ...... 87 Abstract

Communities of organisms that live in estuarine environments such as Chesapeake Bay and its tributaries often exhibit symptoms of stress, such as low species diversity and high abundance of “opportunistic” species, associated with fluctuating environmental conditions. In such cases it becomes difficult, using traditional methods, to determine when organisms are reacting to stress from additional factors. One way to measure sub-lethal responses to stress is to measure genetic diversity. Using the benthic polychaete Paraprionospio pinnata as a model organism and hypoxia as the stressor, a combination of morphological and genetic characters were to look for differentiation between sites that are exposed to hypoxic and normoxic regimes. Four sites were chosen. Two sites, one in the upper main-stem of Chesapeake Bay and one lower Rappahannock River, are subjected to severe hypoxia, lasting weeks in the Bay and days in the Rappahannock River, that leads to defaunation of the benthic habitat. The sites in the lower York River and the lower Chesapeake Bay main-stem are not generally subject to defaunation or severe hypoxia. Worm sizes and branchia lengths of Paraprionospio pinnata were measured for all worms at the four sites. Branchiae surface area has been linked to survival in hypoxic conditions (Lamont and Gage 2000). Some differences in size distribution occurred between sites. The largest worms were only present at the hypoxic stations. There was no significant difference in branchia length distribution between the sites. Two mitochondrial and two nuclear markers were developed to examine genetic differentiation between sites. The mitochondrial markers, ND4 and COI, were highly conserved, and sequences were almost identical between all individuals examined. The COI gene contained one polymorphism suitable for RFLP analysis with the Bbs I restriction enzyme. No differences in allele proportions were observed between sites. The nuclear markers, ITS and the anonymous locus 1 (AL1), developed for this study, were confounded by multiple copies of each region within individuals These sequences were also highly conserved among individual genomes. Very little genetic variation for Paraprionospio pinnata in Chesapeake Bay was observed using these markers. COI sequences generated for this study were also used, along with sequences downloaded from GenBank, to construct a phylogenetic tree for the Polychaeta Class. COI proved to be robust at recovering family groupings, but suggested that several higher classifications based on morphology need to be re-examined.

ix POPULATION STRUCTURE AND PHYLOGENETICS OF

PARAPRIONOSPIO PINNA TA Introduction

Effects of environment on genetic population structure

Traditional indicators of stress on benthic communities, such as lowered species diversity, and the presence or absence of indicator species, have been shown to problematic in estuarine systems (Dauer et al. 1993) when environmental conditions are not severe. Natural stress due to salinity and physical fluxes often are related to low species diversity. In the 1993 study by Dauer et al., no communities in the Chesapeake

Bay estuary were consistently classified as unstressed.

Stress also has been linked to lowered genetic diversity. In a study by Street and

Montagna (1996), it was shown that populations of six benthic copepod species near oil rig platforms in the Gulf of Mexico demonstrated lower genetic diversity in their mtDNA than did reference populations more distant from the rigs. Genetic diversity of populations might be used as an alternate indicator of the environmental health of a habitat.

Decreased genetic diversity and increased homozygosity can be indicators of small population sizes and/or a high level of inbreeding (Avise 1994). Alleles particular to individual sites are an indication that gene flow is limited. Conversely, in populations where wide dispersal and high gene flow occur, genetic divergence will be greatly reduced. 3 Genetic differentiation between sites usually occurs in two ways. First, there can be limited dispersal of individuals or gametes between sites. This can occur at several life stages. If gametes have no mobility independent of the adult then matings are more likely to occur between adults that live in the same environment. If the larval and adult stages also have limited mobility, then populations may become differentiated as genetic drift and different selection pressures drive the population genetics in different directions.

If, on the other hand, any life stage has a high potential for mobility, populations can remain homogeneous. In the marine environment even those species which are not sedentary can be contained by currents, temperature and salinity clines, and increased risk of predation.

The other major cause of population differentiation is natural selection. Migration between sites may occur, but if individuals with particular genotypes fail to mature and reproduce in some habitats then they will not contribute to the gene pool in those location. Over time, the gene pool at particular sites may reflect only the genes of those individuals well adapted to those environments.

Hypoxia in Chesapeake Bay and its tributaries

Low dissolved oxygen has occurred in Chesapeake Bay since at least 1938

(Newcombe and Horne 1938), and has become an increasing problem as the Bay's watershed has become more developed. Hypoxia is defined as the condition where the dissolved oxygen concentration drops below 2ml/L. Such events are detrimental to benthic communities by reducing species diversity and abundance. Total biomass of demersal fish is reduced during hypoxia as well (Pihl 1989). The severity of detrimental 4 ecological events is related to how low dissolved oxygen concentrations become, and the length of exposure to hypoxic conditions (Diaz and Rosenberg, 2001). Additional stress is put on the organisms by the release of H 2 S from the sediments in severely hypoxic bottom water (<0.2 ml O 2 /I).

The principle cause of hypoxia/anoxia in Chesapeake Bay is stratification of the water column caused by the spring freshet combined with the settlement of organic matter from the spring phytoplankton bloom. A large inflow of lighter, fresh water flows over the denser, more saline water, creating a steep halocline and isolating the two water masses. The magnitude of stratification depends upon the magnitude of the freshet. In

Chesapeake Bay this stratification usually begins in March. Oxygen is depleted in isolated bottom water faster than it is replaced by diffusion from above. Water oxygen levels reach a hypoxic state in May and peak in August. Only the deepest parts of the upper and mid Bay are affected by oxygen depletion. By November the bay is once again well mixed (Rennie and Neilson, 1994). Defaunation of affected areas results from this annual event (Dauer and Alden, 1995).

The lower Rappahannock, a tributary of Chesapeake Bay, is also affected by summer anoxia/hypoxia (Dauer et al., 1989, 1992). Dissolved oxygen concentrations are at a minimum around 42 km upstream of the mouth. Hypoxia from the adjacent main stem of the Chesapeake Bay has some effect on the severity of the conditions, though it does not control formation of the hypoxic bottom waters (Park et al., 1996). The hypoxia and anoxia in the area is intermittent (on the order of days), but has been related to defaunation of the macrobenthic community (Llanso, 1992). 5 Stratification and hypoxia in the York River, another Chesapeake Bay tributary,

in contrast, are driven by a spring-neap tidal cycle (Haas, 1977). It experiences moderate

to severe hypoxic events that last on the order of two to 12 days. These events usually

occur approximately five times in the summer months (Diaz et al., 1992). Only areas at

greater than 9m depth are affected by hypoxia in the York River. The hypoxia is

generally not severe enough, or of long enough duration, to cause defaunation (Neubauer,

1993).

Paraprionospio pinnata

The numerically dominant macro-infaunal species of the York River, and one of the most numerically dominant species throughout mud bottom habitats in all of

Chesapeake Bay, is the spionid deposit feeding polychaete Paraprionospio pinnata

(Ehlers) (Figure 1). It is found in coastal and estuarine waters all over the world at mesohaline and polyhaline salinities in areas with high nutrient inputs (Holland et al.

1977; Harper et al. 1991; Carrasco and Gallardo 1983; Vargas 1988; Yokoyama and

Tamai, 1981). This polychaete is known to survive in areas of low dissolved oxygen, though the actual degree of tolerance is not known (Tamai, 1985).

Paraprionospio pinnata is a convenient model animal for examining intra- specific population variation. The organism’s life history and feeding behavior has previously been examined in the Chesapeake Bay system (Hinchey, 1996, Dauer, 1985).

It is also abundant, relatively large (15-30 mm) in size and easy to identify.

Paraprionospio pinnata has been found to be tolerant of very low oxygen conditions (Llanso 1992), which it likely survives through a variety of mechanisms, such 6

Figure 1: Adult Paraprionospio pinnata (Ehlers) approximately 30 mm in length (adapted from Lippson and Lippson 1984). 7

bronchia

1 mm as behavioral and ventilatory adaptations, and improved hemolymph pigments. In

Paraprionospio pinnata oxygen exchange takes place across the membranes of its pinnate branchiae. Increased surface area of the oxygen exchange organs can increase the rate of oxygen absorption. Lamont and Gage (2000) were able to demonstrate a significant correlation between depth, a proxy for oxygen depletion, and branchiae surface area in species in the Paraprionospio genus. Their study location, the Arabian

Sea, is subject to much greater variation in depth and oxygenation than is Chesapeake

Bay, but it might be possible to see a such a trend among samples taken in Chesapeake

Bay and its tributaries.

A number of physio-chemical pathways of varying efficiencies are utilized by organisms in order to survive hypoxic events. Gonzalez and Quinones (2000) found that

P. pinnata utilized four enzymes, lactate dehydrogenase (LDH), octopine dehydrogenase

(OPDH), alanopine dehydrogenase (ALPDH), and strombine dehydrogenase (STRDH) in anaerobic metabolism. In the area in which that study was conducted, P. pinnata dominated the infauna, especially in areas of reduced oxygen. Only one other polychaete studied by Gonzalez and Quinones utilized this number of enzymes. Very few species used LDH or OPDH. This metabolic versatility may give P. pinnata its edge in hypoxic waters. Mangum (1994) showed that different hemocyanin monomers in the blue crab

Callinecties sapidus, were associated with hypoxic and normoxic habitats in the wild.

Metabolic diversity of this sort can be caused either by differential expression of certain genes or by separation of genetically differentiated individuals or species based on the form of enzyme that they produce. In the first case, all individuals or species may be capable of producing the same enzymes, but only those living in certain environments are 9 induced to express particular forms. In the second case, some species or individuals are able to produce certain enzymes or forms of enzymes required for survival in that environment more efficiently that others, and therefore survive preferentially.

Extensive study of P. pinnata's morphological and physiological variation has been undertaken in Japan, the Gulf of Mexico and in Chesapeake Bay. The species

Paraprionospio pinnata in Japan was divided into four sibling species (A, B, Cl, CII) on the basis of morphology, distribution and habitat preference (Tamai, 1985). Among the characteristics used for morphological classification in that study were the filament at the base of the third branchia, pigment spots on the peristomium, transverse dorsal crests, a ventral bi-lobed ridge on setiger 8, and adult body size. Because of the variation in maturation rates observed in the Gulf of Mexico and the bimodal over wintering population, Mayfield (1988) proposed that there may be more than one Paraprionospio species in the Gulf as well. No evidence has been found to suggest that there are more than one species of the genusParaprionospio in Chesapeake Bay (Hinchey, personal communication).

The spawning season for P. pinnata lasts approximately two months beginning in mid-June in Japan. The pelagic stage is thought to last one to two months, with recruitment beginning in July. Paraprionospio pinnata produces pelagic, planktotrophic larvae that settle at the 32- to 37- setiger stage, which is approximately 1-2 months after spawning, usually in August through October in Chesapeake Bay (Hinchey, 1996).

Mortality in the benthic juvenile stage is high. (Yokoyama, 1981).

All four sibling species of P. pinnata in Japan are thought to have pelagic development (Tamai, 1985). Type A, the most common and most extensively studied 10 species in Japan is characterized by summer recruitment, rapid growth and high tolerance to low dissolved oxygen concentrations. The settlement period for type A larvae in late summer coincides with a decrease in abundance in other benthic infauna due to low oxygen levels. Large numbers of Type A larvae were collected from stations in low oxygen waters where adults were scarce (Yokoyama, 1995). Yokoyama concluded that the occurrence of P. pinnata larvae was associated with low oxygen water. Those individuals, however, that spawned in late spring and early summer failed to recruit; possibly because the benthic juveniles are less tolerant of the absence of oxygen. He supports this argument with the observation that "many juveniles found in July at stations

B and C [areas of low oxygen] disappeared in August.” Experimental data showed that juveniles were more sensitive to variations in temperature than other life stages

(Yokoyama, 1988). He suggests that the larvae may accumulate in low oxygen areas in order to decrease losses to predation. In addition, adaptation to stagnant bottom waters may keep larvae from being swept out into unsuitable ocean waters, reducing dispersal, and therefore leading to genetic differentiation among populations (Yokoyama, 1988).

There has been no work done to confirm a genetic separation in this case.

Mayfield (1988) found that in the Gulf of Mexico recruitment pulses occurred during September and October. Mayfield recorded growth rates of 3.9 microns per day in the period between December and March. Higher growth rates were recorded for the summer period. The stage at which worms become sexually mature varies with conditions and location. Yokoyama (1990) found that worms in stable habitats grew more rapidly than did those in stressed, hypoxic areas, and were consequently larger (had more setigers) when they became sexually mature. Fecundity also depends on worm 11 size so that the worms in stressed areas are smaller and less fertile than their counterparts in more stable habitats. Most adults expire after spawning, a process that occurs about a year after settlement (Tamai, 1985). Yokoyama (1990), using cohort studies, found evidence that some adults survive spawning and live for more than one year.

Genetic Markers

Genetic markers, in animals, can fall into one of two categories, either they are part of the mitochondrial genome or part of the nuclear genome.

Mitochondrial DNA (mtDNA) is maternally inherited in most organisms. This means that only one copy of each gene is present within each individual. Typically animal mtDNA is a composed of approximately 37 genes, with a total length of 15-20 kilobases. Of the 37 or so genes, there are around 22 tRNAs, 2 rRNAs and 13 mRNAs that code for proteins involved in electron transport and oxidative phosphorylation.

Introns are very rare in the mitochondrial genome, and almost all of the sequence is involved in coding function. This puts an evolutionary constraint on most of the sequence. On the other hand, most of the metabolic activity occurring within the cell is occurring in the mitochondria and there is a lot of free energy generated in these organelles. Mechanisms present in the nucleus to repair damaged DNA are lacking in the mitochondria. (Wilson et al. 1985). This combination is thought to lead to a much higher mutation rate in the mitochondrial genome. The net result of these opposing forces is that the third position in amino acid coding regions is highly variable while the second and to a lesser extent the first position are relatively conserved for many mitochondrial genes.

Changes in the third position frequently do not change the resulting amino acid sequence, 12 and so there is believed to be little evolutionary constraint on the third position. MtDNA, unlike nuclear DNA, does not recombine, so for the purposes of phylogenetic and population studies, the entire mtDNA molecule is one genealogical unit with multiple alleles.

The cytocrome oxidase I (COI) protein is used in oxidative posphorylation as the final proton pump in the electron transport chain of the mitochondria. (Brzezinski, 1996)

It is encoded by a single copy gene of the mitochondrial genome frequently used in the phylogeographic study of several estuarine and many other taxa including the study of intraspecific relationships in Asterinidae (Hart et al. 1997), mollusks (Foighil et al. 1998) and most importantly for the purposes of this study, in an examination of the evolution of poecilogony of polychaetes in the genus Streblospio (Schulze et al. 2000), a member of the same family as Paraprionospio. Their study was able to confirm the division of

Streblospio benedicti into two species, Streblospio benedicti, and Streblospio gynobranchiata, that was originally based on brooding behavior. Individuals that were pouch brooders were classified as S. benedicti, while those which had a branchiate brooding form were classified as S. gynobranchiata. They found that the pouched brooding species, Streblospio benedicti, was a paraphyletic species because populations of the pouched breeding species in Florida showed a strong genetic affinity with populations of S. gynobranchiata in the Gulf of Mexico.

The COI gene has often been used in studies examining the relationships between closely related species as the study described above by Schulze et al. Its application is limited, however, when examining closely related populations of the same species because as a translated region it is relatively conserved. 13 The nicotinamide adenine dinucleotide dehydrogenase subunit 4 (ND4) locus of the mitochondrial genome has been used in the past for elucidating the phylogenetic relationships between several groups of organisms including shrews of the Sorex cinereus group (Demboski and Cook, 2003), and for examining the intraspecific phylogeography of the slender madtom (Hardy et al., 2002). Gorrochotegui-Escalante et al. (2000) detected seven ND4 haplotypes among ten populations of Aedes aegypti, the yellow fever mosquito, along the coast of Mexico using single strand conformation polymorphism

(SSCP) analysis. These haplotypes resolved the populations into two distinct clades, and indicated that the populations were isolated by distance.

Nuclear loci differ from mitochondrial loci in several important ways. Most nuclear loci, in diploid organisms, are bi-parentally inherited, meaning that each individual carries two copies of each gene. Unlike mitochondrial DNA, nuclear DNA contains a large amount of non-coding, non-functional DNA, sometimes as introns within gene regions. Introns are generally not thought to be under selective pressure and mutations may accumulate within them. Nuclear DNA is also recombinant, making each locus more or less independent from others. Recombination can also lead to gene duplication. Multiple copies of a gene are then able to evolve independently of each other.

Two nuclear loci amplified strongly enough to be examined in this study. The first was an unintentional by-product of the PCR amplification using the Bielawski/Gold

(1996) ND4 primers. After sequences of this secondary product and the P. pinnata ND4 gene were determined, new primers were developed to target the P. pinnata ND4 gene and another set of primers were designed to target the non-specific region. One of these products was clearly the intended target (ND4 gene) of the Bielawski/Gold primers, but the other had very little sequence similarity to anything in GenBank. I called this initially non-specific region anonymous locus 1 (AL1).

The other nuclear locus examined was the ITS (internal transcribed spacer) region of the ribosomal RNA gene complex. The ITS region actually contains three regions,

ITS 1, the 5.8s ribosomal RNA gene and ITS 2. The regions flanking this locus, coding for the small and large subunit ribosomal RNA genes, are highly conserved. The ITS 1 and the ITS 2 regions, because of their lack of coding function, are highly variable. ITS sequences have been used successfully in examining the phylogenies of closely related species of Perinereis polychaetes (Annelida; Polychaeta; Nereididae) (Chen et al., 2002).

ITS 2 was also used by Patti and Gambi (2001) to examine the relationships between different populations of the invasive polychaete species Sabella spallanzanii (Gmelin).

Twelve populations throughout southern Australia and in the Mediterranean were examined, and a clear delineation between the Australian and European populations was observed. A reduced variability in the Australian populations also suggested a founder effect at this site.

Phylogeny of the Class Polychaeta

The class Polychaeta is a large and ecologically important group for which there is little fossil record to help elucidate the evolutionary relationships of modern groups.

What little record exists is composed largely of tube and trace fossils which points to the increasing diversity of the taxa (Robison 1987). The homology of some modern morphological characteristics causes difficulty in the analysis of relationships based on 15 morphology. There has also been a great deal of argument within the literature of the appropriate methods of scoring character states that are dependent on other character states (Eernisse 1997; Rouse and Fauchald 1995). Rouse and Fauchald (1997) was the first to create a phylogeny based on a parsimony analysis of morphological characters.

A number of studies have been published using molecular markers to examine the phylogenetics of the Annelida (see McFlugh 2000 for a thorough review of the literature), but most of these have focused on the relationships of such groups of the clitellates, siboglinids, echiurids and sipunculids to the polychaetes. There is strong evidence to suggest that these groups should be included within class Polychaeta as more derived forms. Many of these studies only addressed the question of relationships within the

Polychaeta as a secondary objective, if they addressed it at all. Brown et. al. (1999) used three nuclear markers and included 24 polychaetes, but there were very few families represented by more than one species. Those that were did show monophyly, but many of the higher relationships (proposed by Rause and Fauchald 1997) were not supported by this analysis. Winnepeninckx et al. (1998) included 15 polychaetes in their analysis of the 18S rDNA sequences. They also found that several of the groups proposed by Rouse and Fauchald (1997) were poly- or para-phyletic, as did Kojima (1998) where 13 polychaetes in nine orders were analyzed using EF-la sequences. In none of these studies, however, were the groupings that conflicted with Rouse and Fauchald (1997) supported with high bootstrap support. 16

Study Objectives

This study attempts to determine if the presence of hypoxia could be a factor in the genetic structure of populations of Paraprionospio pinnata in Chesapeake Bay. The second objective of this work is to examine the phylogenetic relationship of the class

Polychaeta, using DNA sequences from a mitochondrial locus, COI.

Since 1985, the Chesapeake Bay EPA program has been collecting quarterly benthic samples from a variety of locations throughout Chesapeake Bay and its tributaries. From these data it was possible to locate areas with a history of hypoxia and to identify which of these were likely to provide populations of P. pinnata in sufficient numbers for my analysis. Between 25 and 50 individuals were needed at each site. I chose sites in the lower York, and Rappahannock Rivers, and two areas in the

Chesapeake Bay main stem. The York, Rappahannock and upper main stem stations represent varying degrees of hypoxia. Mortality of the sessile benthic community is frequent in the Rappahannock and main stem, but not in the York. The site chosen in the

York River is too shallow to experience hypoxia and the site in the lower Chesapeake

Bay is not affected by hypoxia. These will be used as reference sites.

If there is a substantial genetic flow then we may expect that the populations will be more or less homogeneous over geographical distances. This is the null hypothesis, that there is no differentiation between sites and that all sites are part of a single population. Yokoyama's (1988) research may lead us to believe that the populations may be isolated from each other and that we may see genetic differentiation. If the hypoxic 17 populations are more similar to each other than to populations geographically nearer to them that are not affected by hypoxia, then this would suggest that selection is acting. Materials and Methods

Study Area

Four sites were selected for this study (Figure 2). Two of them were near the mouths of Chesapeake Bay tributaries and two were in the main stem off the Bay itself

Of the main stem stations, UCB is subject to severe hypoxia/anoxia, LCB does not experience hypoxia. In the tributaries, the Rappahannock frequently experiences moderate to severe hypoxia, while the York River only experiences light to moderate hypoxia in regions deeper than 9m. The site selected in the York River is shallower than

9 m (Table 1) and does not experience frequent or prolonged hypoxia (Diaz et al. 1992).

Sample Collection and Preparation

Fifteen to twenty gallons of sediment were collected from each of the four sites on

November 11 or 14, 2001 and sieved on a 0.5mm sieve. The organisms retained on the sieve were returned to ambient water and were examined under a microscope to identify

Paraprionospio pinnata.

Paraprionospio pinnata were then removed to a dish of sea water, filtered on a

500 pm filter to remove any potential food substances, and placed at 4°C for 36 to 48 hours. This was done in order to clear out the contents of the guts and minimize the occurrence of contaminating DNA for the genetic work. 19

Figure 2: Sample Stations. Four stations, chosen on the basis of historical presence of Paraprionospio pinnata. Two of the sites, in the upper main-stem and in the Rappahannock River, are subj ect to annual hypoxia. The station in the York River is above the depth at which moderate hypoxia occurs in that river, and the station in the lower main-stem is never subject to hypoxic conditions. 2 0

• Hypoxic • Normoxic 21

Table 1: Sample sites and the number of individuals collected from each site. Latitude and longitude of each site are given, along with the depth of the station. The number of individuals of Paraprionospio pinnata collected from each site is reported in the final column. 22

1—1 i i o cl n '■+ */■. CCJ

cK (m) r r ^+ 39 39 it

D eptli D r\ cl

’-f 1 1 O crj r\ •o O ri 00 O'] i-H cl cl r-H ’O rVO 'Or r r■o

Longitude ■ ■ i ■

'O O', o r\ Cl ••o >r i —1" 00 O 7+ 1—1 p r 1 1 f

Latitude <*Cj rr> cn Station York York River Upper Upper Chesapeake Bay Rappahannock River Lower Chesapeake Bay 23 Morphological Measurements

After a period of starvation, the worms were placed under a dissecting scope.

Because many worms will shed setigers when exposed to stress, the fifth setiger width was measured, as a proxy for body size (Maxemchuk-Daly, 1998), under a dissecting microscope fitted with an ocular micrometer. The second branchiae length was measured to approximate branchiae surface area.

Fifth setiger width was subjected to pair-wise Cochran-Mantel-Haenszel test at an alpha = 0.05 to determine if there were any differences in size distribution between sites.

DNA Extraction

After measurement the worms were washed again with filtered sea water and placed in 95% ethanol and stored at 4 C until DNA extraction. The entire worm was used for extraction, either by a CTAB- phenol:chloroform extraction (Sambrook et al. 1989) or using a Qiagen tissue extraction kit (Qiagen, Valencia, CA) following the manufacture's protocol. For the CTAB extraction: The entire worm was added to 500pl of isolation buffer (50mM EDTA, 50mM Tris, NaCl 150 mM pH 8.0), 60pl of SDS 10%, lOpl of

RNAse (lOmg/mL) and lOpl of proteinase K (25 mg/mL) (Invitrogen Corporation,

Carlsbad, CA). Cell lysis mixture was incubated overnight in 37°C water bath. lOpl of

CTAB solution (0.4 lg NaCl, lg CTAB in 10 mL of volume) was added and the solution incubated at 65°C for 20 minutes. Cell pellet was precipitated by adding 350pl of a saturated NaCl solution and vortexing for 15 minutes followed by a 4°C, 30 minute centrifugation at 14,000. DNA was extracted with an equal volume of phenol, followed by an extraction using equal volumes of phenol: chloroform: isoamyl alcohol (25:24:1) 24 and another equal volume extraction using chloroform:isoamyl alcohol (24:1). DNA was precipitated with an equal volume of isopropanol and collected by centrifugation at 4°C for 20 minutes. Reconstitution of the DNA pellet was in 50pl of TE (pH 8.0). Method of extraction did not seem to affect DNA quality.

PCR amplification

Samples were amplified using a variety of primers listed with the amplification reaction conditions in Table 2. Approximately 0.25pl of DNA was used in a 25pl reaction containing 15.375pi of DNA-free water, 5pi of bovine serum albumin (BSA) (1 mg/ml), 2.5pl of 10X reaction buffer (100 mM Tris, pH 8.3, 0.5 M KCL), 1.5 mM

MgCf, 0.25 pmole of each primer and 0.625 U of Taq polymerase. Amplifications were done using a MJ Thermal Cycler. Cycling parameters are listed in Table 1. After amplification, samples were visualized on a 1 % agarose gel stained with ethidium bromide.

Upon visualization on a 2% agarose gel stained with ethidium bromide, I found that the ND4 primers were actually amplifying two fragments, one approximately 950 bp in length and the other, about 1050 bp. Since ND4 is a mitochondrial gene and not likely to be multi-copy except in the very rare case of heteroplasmy (bi-parental inheritance of the mitochondria), I decided that it was necessary to sequence the fragments to discover what regions were being amplified with these primers. 25

Table 2: PCR primers used in this study. Three sets of previously published primers were used for this study. Two primers were designed for this study. Reaction conditions are provided for each set of primers. Amplification was performed on a MJ thermocycler. 26

o & & O o o

ar -3

P CO P CO C CD c CD - -3 - -3 S | SI o o ,r o .r o E

cd " O CD" O of O CD" O cd " O - 3 Sn ^3 i n ■S ■ C “ tc E ts ts E ■E P “1c a ) *- a) 3 CO , 0 co £ o ■ £ £ (D * = •e £ 05 ** >* 0 '■3* ^ O 0 ■3 O o ° ■g o o = to o *. I E S> s. I I s I a5 oj 05 -« .Q TC e o 5 O S' — O & _ O S - P S' ■= p 27 Cloning and Sequencing

DNA from four individuals was amplified using the COI gene primers, and the amplicons were cloned into the TA cloning vector pCR 2.1 (Invitrogen Corporation,

Carlsbad, CA) and transformed into Escherichia coli INFaF’ following the manufacturer's protocol. The bacteria were plated onto agar plates treated with ampicillin to prevent the growth of bacteria without plasmid, and X-gal to differentiate colonies with plasmid insert from those without by blue/white selection. Selected colonies were cultured overnight in 2X Yeast Tryptone (YT) media (0.6% w/v tryptone, 0.1% w/v yeast extract, 85 mM NaCl) with 50 mg/L ampicillin. Plasmid DNA was extracted using the

Plasmid MiniPrep kit (Qiagen, Valencia, CA) following the manufacturer’s protocol and sequenced using the dideoxy-chain termination method (Sanger et al., 1977) using the thermo sequenase infrared labeled primer cycle sequencing kit with 7- deaza-dGTP

(Amersham Corporation, Arlington fleights, IL) in conjunction with M l3 forward and reverse primers (LI-COR, Inc., Lincoln, NE). Sequencing reactions were run on a polyacrylamide gel on a LI-COR 4200 automated sequencer. Sequences were read using the e-seq software (LI-COR, Inc., Lincoln, NE). Virtual digests were performed on the sequence using MacVector 7.0 (Oxford Molecular). One enzyme(5^ I) was identified as producing a polymorphism. Resulting sequences were subjected to BLAST (Basic Local

Alignment Search Tool) searches (Altschul et al. 1990) of the National Center for

Biotechnology Information (NCBI) GenBank database and compared to COI sequences from other members of the Polychaete class.

Amplification, cloning and sequencing were repeated for each of the other loci.

DNA from two individuals was amplified using "universal primers" for ND4 (Bielawski 28 and Gold 1996). New primers were designed for both the ND4 locus and the alternate fragment that I have designated anonymous locus 1 (AL1). The new ND4 primers were used to amplify for sequencing DNA from two more individuals and sequences for four individuals generated at the AL1 locus by the "universal" ND4 primers were also determined.

Restriction Fragment Length Polymorphism Analysis

Table 3 shows a list of the enzymes screened at each locus for polymorphisms.

Digestions were performed overnight at 37°C (except where noted). 5 pi of amplified

DNA, 1 pi of enzyme and 2pl of the appropriate manufacturer supplied buffer was used in each 20pl digestion reaction. Restriction fragments were run on a 1.5% agarose gel and visualized with ethidium bromide. Exact test of population differentiation was performed on allele frequencies with Arlequin (Schneider et al. 2000).

Phylogenetic Analysis

COI sequences for other members of the class Polychaeta were downloaded from the Entrez database and aligned to the COI sequence from P. pinnata. Accession numbers and primary references for GenBank sequences are as follows: Opisthoteuthis sp.,

AF377961 (Carlini et al. 2001); Amphiglena terebro, AF342670, Cirratulus sp.,

AF342672, Dodecaceria sp., AF342673, Trichobranchus sp., AF342674, Amphitritides harpa, AF342676, Isolda sp. AF342677, Amphicteis dalmatica, AF342678,

Rhinothelepus lobatus, AF342679, Amaeana trilobata, AF342680, Lysillapacifwa,

AF342681, Amphitritides sp., AF342683, Reterebella queenslandica, AF342684, Loimia 29

Table 3: Restriction enzymes screened at each gene for polymorphisms. A panel of twelve Paraprionospio pinnata individuals was screened with a variety of restriction enzymes at each gene. 30

Locus Enzymes Company Locus EnzymesCompany COI Alu 1 Invitrogen AL1 Bam HI Invitrogen Bam HI Invitrogen Ban II New England Biolabs Ban II New England Biolabs Bbs 1 New England Biolabs Bbs 1 New England Biolabs Dde 1 Fisher Bfa 1 New England Biolabs Eco Rl Takara Bgl II Takara Hinf 1 New England Biolabs Cla 1 Invitrogen ITS Alu 1 Invitrogen Dra 1 Invitrogen Bam HI Invitrogen Eco Rl Takara Ban II New England Biolabs Hae II New England Biolabs Bbs 1 New England Biolabs Hha 1 Invitrogen Bfa 1 New England Biolabs Hinc II Invitrogen Bgl II Takara Hind III Takara Dde 1 Fisher Hinf 1 New England Biolabs Eco Rl Takara Mbo 1 New England Biolabs Hae II New England Biolabs N e il New England Biolabs Hha 1 Invitrogen Pvu II Invitrogen Hinc II Invitrogen Rsa 1 Fisher Hind III Takara ND4 Alu 1 Invitrogen Hinf 1 New England Biolabs Bam HI Invitrogen Hpa 1 Gibco Ban II New England Biolabs Hpa II Fisher Bbs 1 New England Biolabs Kpn 1 Takara Bfa 1 New England Biolabs Mbo 1 New England Biolabs Bgl II Takara Mse 1 New England Biolabs Dde 1 Fisher Msp 1 Invitrogen Eco Rl Takara Nae 1 Takara Eco RV New England Biolabs Nci 1 New England Biolabs Hae II New England Biolabs Nde 1 New England Biolabs Hha 1 Invitrogen Not 1 Takara Hind III Takara P stl Takara Hinf 1 New England Biolabs Rsa 1 Fisher Hpa 1 Gibco Tag 1 Promega Hpa II Fisher Mbo 1 New England Biolabs Mse 1 New England Biolabs Msp 1 Invitrogen Nae 1 Takara Nci 1 New England Biolabs Nde 1 New England Biolabs Not 1 Takara Nsp 1 New England Biolabs Pvu II Invitrogen Rsa 1 Fisher Sal 1 Takara Ssp 1 Takara S stl Gibco Sst II Invitrogen 31 ingens, AF342685 (Colgan, Hutchings, and Brown direct submission to GenBank);

Chysopetalum debile, AF221567, Dysponetus caecus, AF221568, Hesionides arenaria,

AF221569, Heteropodarke formalis, AF221570, Microphthalmus listensis, AF221571,

Neanthes virens, AF221572, Ophiodromus flexuosus, AF221573, Sigambra tentaculata,

AF221574, Pisione remota, AF221575 (Dahlgren et al. 2000); Paralvinellapalmiformis,

U74070 (Black et al. 1997); Manayunkia zenkewitschii, AJ428405 (Pudovkina direct submission to GenBank); Prionospio steenstrupi, AF138955, Scololepis squamata,

A F138956, Streblospio gynobranchiata, AF13 8933, Streblospio benedicti A F138954

(Schulze et al. 2000); and Branchipolynoe sp., AF399963 (Vrijenhoek and Hurtado direct submission to GenBank). Sequences were aligned for phylogenetic analysis using the

CLUSTAL-W algorithm (Thompson et al. 1994) in the MacVector 7.0 sequence analysis software package (Oxford Molecular) using the program default open-gap penalty of 20 and an extend-gap penalty of 10.

Phylogenetic trees were generated using three models, and two methods of tree generation. The methods of tree generation were Neighbor-Joining (Saitou and Nei,

1987) and parsimony analysis. Parsimony jackknife analysis (Montvay and Munster

1994) was performed with 20% replacement and 100 jackknife replications. All trees were generated using PAUP*4b8.0 (Swofford 2001).

Three alternate phylogenetic models were explored, one including all nucleotide sites, one that removed the third position from the analysis, and another that translated the

DNA sequence to its amino acid sequence. Results

Sample Collection

The number of individuals collected at each site is reported in Table 1. Since the objective in sampling was to collect sufficient numbers of P. pinnata, and not to quantify the density of the organism in the sediment, the number of individuals collected cannot be used as a quantitative measurement. Qualitatively, more P. pinnata were present in the river samples than in the bay main-stem samples.

Morphological Measurements

Fifth setiger width varied between 0.4 and 1.0mm (Figure 3). The largest worms were only found in the upper main-stem and Rappahannock stations. A Cochran-Mantel-

Haenszel statistic (SAS) showed that the distribution of the size of the Rappahannock P. pinnata was significantly different from that at any other station (Table 4).

In the 1996 study of P. pinnata's autecology by Hinchey, samples were taken from roughly the same station as the York River samples collected for this study. In

November of 1996 the abundance of P. pinnata at the York River station was 956 individuals/m2, and was a month of high abundance for the species throughout the year of that study. The size distribution of individuals at the York River station collected in 1996 and 2001 were similar (Figure 4) with 1996 having a higher proportion of large animals. 33

Figure 3: Frequency of 5th setiger width and branchae length categories in each of the four stations. Because of the difference in sample size, the data is presented as proportions of individuals in the size class to the total number of individuals at that site. For 5th setiger data N for the Upper Chesapeake Bay, Rappahannock River, York River and Lower Chesapeake Bay are 27, 47,48, and 31 respectively. The N for the branchia length data for the same stations are 14, 21, 36, and 22. Frequency 0.2 .3 0 0.4 .5 0 0.6 0.1 0.2 0.3 0.4 0.5 0.6 0.2 0.5 0.3 0.4 0.6 . 05 . 07 . 09 1 0.9 0.8 0.7 0.6 0.5 0.4 N=31 N=48 N=27 N=47 t Sei rWit ( m) (m idth W er etig S 5th oe hspae Bay Chesapeake Lower pe hspae Bay Chesapeake Upper Rappahannock River Rappahannock York River York 0.05 0.15 0.25 0.35 0.45 0.05 0.15 0.25 0.35 0.45 0.1 0.2 0.3 0.4 0.1 0.2 0.3 0.4 0 0 . 0 0 07 . O 1 . 1 13 . 15 1.6 1.5 1.4 1.3 12 1.1 1 OS 0.8 0.7 05 05 0.4 n N=22 N=21 N=36 N=14 - IPI PI j-i - Branchae Length (mm) Length Branchae - - - — — - Lower Chesapeake Bay Chesapeake Lower Upper Chesapeake Bay Chesapeake Upper Rappahannock River Rappahannock n. . York River York 34 35

Table 4: P values for Mann-Whitney test of fifth setiger width. Above the diagonal are the P values (adjusted for ties) for a pair wise Mann-Whitney test of the fifth setiger distribution of four sites. Site abbreviations are UCB= upper Chesapeake Bay main-stem, RAP= Rappahannock River, YRK= York River, and LCB= lower Chesapeake Bay main-stem. Below the diagonal, significant differences are marked. 36

UCB RAP YRK LCB UCB - 0.0169 0.8436 0.1103 RAP * - 0.0012 <0.0001 YRK * - 0.0659 LCB 37

Figure 4: Comparison of size frequency distribution in samples from the York River in years 1996 and 2001. Because of the difference in sample size, the data are presented as proportions of individuals in the size class to the total number of individuals at that year. Animals in 1996 were larger than those in 2001. Size Frequency Comparison O LO LO O LO LO 0 . ^ 0 s s b io 3Z |s jo jo |s 3Z io b s s O CO LO O CO > > cn CM o o □ □ > 0) > d) o QC CD CD CD j j d b n o u o A O CN LO O O CN O LO CD O LO 1= A 7 % *

Fifth Setiger Width (mm) 38 39 Examination of historical data from the ongoing monitoring of the York River station (1960-present)(Data presented through 1988 in Figure 5) (Boesch et al. 1976) shows that before 1973 densities of P. pinnata were much lower that was recorded in the

70's and 80's.

Collection of the branchiae length data was problematic because the P. pinnata often sheds one or more pairs of branchiae when under stress. Not all worms whose fifth setiger were measured had all of their branchiae. Branchiae length varied between 0.4 and 1.6 mm. Sample sizes in this case were insufficient to run a statistical analysis.

Restriction Fragment Length Polymorphism Analysis

COI was the only gene region in which a restriction site polymorphism was identified among Chesapeake Bay P. pinnata. Virtual digests of the AL1 locus indicated that EcoR I would show a polymorphism at the AL1 locus, but further screening of AL1 with the EcoR I enzyme showed that all individuals were heterozygous.

The proportion of individuals bearing each allele at the COI gene in each population are reported in Figure 6. An exact test of population differentiation (Raymond and Rousset 1995, Goudet et al. 1996) performed in Arlequin (Schneider et al. 2000) showed that there was no significant partitioning of alleles between populations (see

Table 5 for P values).

The ND4 amplicons generated, using the newly designed species specific primers, were screened with 29 restriction enzymes. None of these displayed any polymorphisms.

Three enzymes were chosen to screen 92 individuals in order to show that the likelihood of rare alleles being present in the population was low. 40

Figure 5: Historical abundance of P. pinnata in the York River. Data from a site in the York River with the coordinates Latitude= 37°14.64’, Longitude= -76 °29.67\ Samples are collected quarterly with a Smith-Mac sediment grab. * indicate missing data. Historical Abundance of P. pinnata in York River {f>ii!|(lun>s/$|LMi|>!A!|>uj) doui?|>im(|v > □ □ □ a) GO cn Q. GO E F CD = j _ □ Ll_ m % % > 41 42

Figure 6: Allelic variation in the COI gene. Proportion of each allelic variation of the COI gene, as determined by RFLP analysis with the restriction enzyme Bbs I, in four samples from Chesapeake Bay and its tributaries. Proportion of Each COI Allele Within Samples £ CO 00 \D CO

CL O CD CL O < □_ 3 CD 43 44

Table 5: P values for exact test of sample differentiation based on allele frequency. Above the diagonal are the P values of and exact test of sample differentiation. Below the diagonal significant differences are marked. 45

UCB RAP YRK LCB UCB - 0.3019+/-0.134 0.7940 +/- 0.0052 1.0000 +/- 0.0000 RAP - 0.5079 +/- 0.0055 0.3063 +/- 0.0092 YRK - 0.8090 +/- 0.0059 LCB 46 ITS amplicons were screened with 23 restriction enzymes. It rapidly became apparent that the multi-copy nature of ITS was confounding gel scoring. The problem was compounded by the variable nature of the amplified product. An individual sample amplified in two separate reactions might not have the same number of amplicons. There appeared to be non-specific amplification in many cases. Sequence analysis of multiple copies was needed to determine the degree of sequence difference among copies of ITS.

Sequencing

A sequence alignment of 4 individuals generated from the COI locus is shown in

Figure 7. Sequences were BLASTed against the Entrez database and the results are

given in Table 6. Based on the BLAST results, these sequences from P. pinnata were

most similar to Opisthoteuthis, a cephalopod.

Sequence alignments forND4 are displayed in Figure 8. One segment BLASTed to an ND5 sequence in Drosophila madeirensis. The other sequence had no likely match in GenBank. Because of differences between sequences within a single individual, and the lack of BLAST data that would suggest that it was a mitochondrial region, I presumed this to be an anonymous region of the nuclear genome and named it Anonymous Locus 1

(AL1). I proceeded to treat it as an independent locus.

Of the three sequences gathered for the true ND4 locus, two were separate clones of the same individual and therefore they were expected to be identical. Between those two sequences and the third, there were no site differences in 1022 bases. Further sequencing using the species specific ND4 primers designed for this study (Figure 9) show that in the shorter amplicon, four individuals showed no site differences in 393 47

Figure 7: Alignment of COI gene. An alignment of COI DNA sequences from four individuals from the York River site generated using the CLUSTAL-W algorithm (Thompson et al. 1994) in the Mac Vector 7.0 sequence analysis software package (Oxford Molecular) using an open-gap penalty of 20 and an extend-gap penalty of 10. Ambiguities are marked in white and are coded as follows:B= not A; D= not C; H= not G; K= GT; M=AC; N=ACGT; R=AG; S=CG; V= not T; W=AT; Y=CT. 48 49

Table 6: COI BLAST Results. Sequences were BLASTed against the Entrez database. One of them is reported here. 50

BLAST 2.2.5 Results Description List

BLAST Analysis for Sequence: BrY6_3consensus Search from 1 to 658 Program: BLASTN Expect: 10 Low complexity filter: on Matrix: n/a Genetic Code: n/a Gapped search: on Open cost: 11 Extend cost: 1 X-Dropoff: 50

Database: All GenBank+EMBL+DDBJ+PDB sequences (but no EST, STS, GSS, or phase 0, 1 or 2 HTGS sequences) (1711089 sequences) Build Date: Mar 26, 2003 10:25 PM

Smallest Sum High Probability Sequences producing high-scoring segment pairs: Score P(N)

1 . gi 15421815 Opisthoteuthis sp. CYV-2001 cytochrome cE 73 0 2 . gi 19850195 Rhinothelepus lobatus cytochrome oxidaseE 71 0 3. gi 3243207 Fridericia tuberosa cytochrome c oxidaseE 70 0 4 . gi 7008040 Cincinnatia winkleyi cytochrome c oxidasE 69 0 5 . gi 19850201 Pista australis cytochrome oxidase subunE 67 0 6. gi 19850187 Trichobranchus sp. AMW24401 cytochrome oE 67 0 7 . gi 2267265 Urechis s p . 'California' cytochrome c oxE 67 0 8 . gi 21745181 Floridobia winkleyi cytochrome c oxidaseE 65 0 9. gi 21745205 Pyrgulopsis pecosensis cytochrome c oxidE 62 0 10 . gi 6684012 Fundulus zebrinus isolate CIM112 cytochrE 62 0 11 . gi 27802102 Brotia pagodula ZMB 200208 cytochrome oxE 59 0 12 . gi 15986589 Allothereua maculata cytochrome oxidase E 59 0 13 . gi 21745249 Pyrgulopsis neritella cytochrome c oxidaE 58 0 14 . gi 5006525 Streblospio benedicti strain BS3 cytochrE 58 0 15. gi 29171016 Clostera albosigma cytochrome c oxidase E 57 0 16. gi 5123682 Baicalia carinata partial mitochondrial E 57 0 17 . gi 28882778 Monetaria caputdraconis isolate BJLinnSoE 55 0 18 . gi 6684015 Fundulus zebrinus isolate CAN2 cytochromE 55 0 19. gi 6684011 Fundulus zebrinus isolate SFNIN2 cytochrE 55 0 20 . gi 6684010 Fundulus zebrinus isolate CHIK1 cytochroE 55 0 21. gi 6684009 Fundulus zebrinus isolate TBll cytochromE 55 0 22 . gi 6684008 Fundulus zebrinus isolate WH2 cytochromeE 55 0 23. gi 6684007 Fundulus zebrinus isolate WH1 cytochromeE 55 0 24 . gi 6684006 Fundulus zebrinus isolate SH4 cytochromeE 55 0 25. gi 6684005 Fundulus zebrinus isolate SHI cytochromeE 55 0 26. gi 6684004 Fundulus zebrinus isolate WYLA1 cytochroE 55 0 27 . gi 6684003 Fundulus zebrinus isolate ARIK1 cytochroE 55 0 28 . gi 6684002 Fundulus zebrinus isolate SPNP1 cytochroE 55 0 29. gi 2760016 Lumbricus rubellus mitochondrial mRNA foE 55 0 30 . gi 28207581 Sepia latimanus cytochrome oxidase subunE 54 0 31. gi 23320961 Haementeria gracilis cytochrome c oxidasE 54 0 32 . gi 6684014 Fundulus zebrinus isolate CAN1 cytochromE 54 0 33. gi 6684013 Fundulus zebrinus isolate BEAVER1 cytochE 54 0 34 . gi 13991776 Sepiella maindroni cytochrome c oxidase E 54 0 35. gi 13752531 Sepia esculenta cytochrome c oxidase subE 54 0 36. gi 9754701 Erynnis tristis cytochrome c oxidase I (E 54 0 37 . gi 13378048 Sepiella maindroni cytochrome c oxidase E 54 0 38. gi 2352339 Haementeria gracilis cytochrome c oxidasE 54 0 39. gi 29170956 Ochropleura implecta cytochrome c oxidasE 53 0 40. gi 29170748 Nemoria rubrifrontaria cytochrome c oxidE 53 0 51

Figure 8: Alignment of ND4 gene using Bielawski/Gold primers. An alignment of ND4 sequences from two individuals from the lower Chesapeake Bay main-stem site generated using the CLUSTAL-W algorithm (Thompson et al. 1994) in the MacVector 7.0 sequence analysis software package (Oxford Molecular) using an open-gap penalty of 20 and an extend-gap penalty of 10. Ambiguities are marked in white and are coded as follows:B= not A; D= not C; H= not G; K= GT; M=AC; N=ACGT; R=AG; S=CG; V= not T; W=AT; Y=CT. 52

70 3 0 9 0 ( m 1 ( 0 RGRAOCTGTTGMHGTMGTTTAARARflfiR

Hhfl iHHHHqrTqrClic[rTGT^rTGGTTCTTTGTTTGTOTHhHC HfiTHGRt|GTTAAHSrCGTC[rfifiGRHCC|rGTTGRfiGTRGTTTAAflfiflRfifi Rflfl iflflHftqrTqrt3Fi4rTGT*:frTGGTTt;lTTTG'TTTGT flflTRGR§3TTRRfl|rpGTqrflRGRfl^|rGTTGflHGTRGTTHHRRflRRRR

| (70 (30 (90\ L 10 -1 c o n 2 Hfl CtTTRT jrdrTTRCctrcfrGHTTTi TTGRG TGGTTTORfiRAR ••T cdrT i t c t t c i a ■ TTRCTCcb'cirTTRCCjrc(rGflTTTCCGCirTTHflTi RT Th Th Cfr Th TH T T T DCn CT T T TTGRG TGGTTTCAHfihA RfiTCCTTfiTOTTQifiCAH TTR^eqrqTTTReC|Tc|rGflTTT»|3C|rTTRRTI §WTfiTfic|rTflTflTTTCCnO!rTT

■Hf f f IBM HpgTRTfiTTTT TTGTRRRCfoTTRCCirctacARTTfifiCRTfifiClTflTTRTTGTCGflCCCCjrfiOGGflfi AGRTGRG TH#rgrrBrTi^T iHcCrrRTRTTTT TTGT1 ' h O-TTI■CGTCfTGOIfITTI•>.Q 'T. .nCOT 'TTr TTGTCIg *lftCCSrH|GGt^ RGRTCRGT

3 3 0 3 7 0 3 3 0 3 9 0 400 4 1 0 ... 77,} ;-r0 7 70 frRH^R»frRR|*^RMW frB^iifenvv|l>vWlf TRHTRTTRHTTTarRTHHHTGTRHTRRTTTTTGdrOidrGrrTRO=lTflfiGTGHGGRCCCC|T-TTRTTHfifl^GGTTclRTtRTTTTRGTTTTRTTRTTTGTTGSltflTRflflSTT^rTR TflRrfiTTRRTTT^rBrRRRTGTflRTRflTTTTTGi^rlR^rfcjrTHSGTRRGTGHGGRCiCCClr-TTRTTfiH^pGTTflTTRTTTTfiGTTTTRTTRTTTGTTGGCtrSHflrHHflC^rTCfrTR

4 3 0 4 9 0 3 0 0 5 ( 0 520 530 540 550 SSI? 5 7 0 5 3 0 5 9 0 AMflflTGCHfifli TTTTT A TCCCQA H HTTR GT0R OTfflTG faTTR TTGG GGflTTRGGflCjmR(a:[rqTTTTTTG^TTGTTHTTTflTTHCO1flRflTGD:flfiflT0H|rCiGGTGdiGGRHrHHTTR L 3 _ 0 4 CON TTTTTflrCCCCRHqrTHGTOHC(rC[rGqTTHTTGG bGRnRGoigrRFiferclrTTTTTGlrrTGTTRTTTRTTRBRRRRatog^RRRjMiirtjjGTGyGGRRffiRTTR I ■ _ L 10 -1 c o n 2 TTTRACCM.ee. CCCfrRTCjTTCCHH L3 0 2 CON TTTRflC»RCCBRflT(|sGGGH0GD-iCTO1TCtCTTRTTTCTHTTG0!RTGRHOTTTGTCRHTfiGGCCHTTGRRRpCTTRTRRTCCCCRC|rGHHTc}TCCctrRTCrTC(|RHTTTTHTCCRTT TTTflfl^fl|^flRT§jGGGflfj§Hfr§RT^fflTTTfrflTTG|RTGRH|rTTGTjiHTRGG^.TTGRHR^rTRTRRT^^H|rGRHTfr@^(l>1TfrT^RRTTTTRT|OiTT

10 720 73 0 740 75 0 75 0 770 j W 790 3 0 0 3 ( 0 3 2 0 iCTCRTCTflCCCjT jo T R fl TT R»flHRflGRGCrrCRGHTG TTTT RCfrCCTGTTT TCf-CTO• rCT. -CCCTCGTl •• • TTTMR1RG h B hh RRRGMGCTCAGh TG TTTT CiTCclrGTTT ID CTD TCT. C C C fD .ri

GTR TTTTTRTTR T T J f c

9 5 0 9 50 0 0 L 1 0 -1 c o n 2 AGfifiAi

L3 0 2 CON O^i CRGRGTGTGRC mTGr !HGfiHRHTTRTTGCfrqr! TThh CGGTGTTRTGRTRGTRRGR J|raGTGTGA§flTGMlRGRRRflTTRTTGBtlfr! h CTTTRh DGGTGTTRTGRTflGTRRGRBnjRR Figure 9: Alignment of ND4 gene using primers designed for this study. An alignment of ND4 DNA sequences from four individuals from the lower Chesapeake Bay main-stem and upper Chesapeake Bay main-stem sites generated using the CLUSTAL-W algorithm (Thompson et al. 1994) in the MacVector 7.0 sequence analysis software package (Oxford Molecular) using an open-gap penalty of 20 and an extend-gap penalty of 10. Ambiguities are marked in white and are coded as follows:B= not A; D= not C; H= not G; K= GT; M=AC; N=ACGT; R=AG; S=CG; V= not T; W=AT; Y=CT. 54

U 36 R5-1 piCCTC;TTTTTTGCTTGTTRTTTRTf3feRflRTG|RRfifiTj|RCpi^GTG|RGGfifiTflfiT - h h CC h h CCG h h T i

R5-2 RTTRGGIXTflRfCTCTTTTTTGcjrTGTTfiTTTRTTHlXRRfiRTGBTRRfiTCHCTCGGTGQRGGRRThRTTRECGC ■ r r H r h Ccgrrtcggggr | R5-3 RTTHGGCCTflHlcTclTTTTTTGCtTGTTHTTTHTTHCC^nfiflTGfflHfiflTChCTCGGTGQHGGFIflTHHTTfij^ TT-RRCCRRCCGRRTCGGGGRC R18-1 HTTRGGOTrflflCCTcjrTTTTTGCTTGTTfiTTTfiTTfiCCTfififlTG^RfifiTCRCTCGGTGC^GGflRTfifiTTfiCri TT-flH(JCRRCCGRRTCGGGGfli R18-2 RTTRGGCCTRfliCTCTTTTTTGGTTGTTR TTTHTTHCCRRflflTGSiRfiHTCHCTCGGTGE^RGGHHTHflTTfl^GE TT-flfi»flK fiRr|G G G G Rj L 10-1 RTTRGGCC|rRRCCTC!TTTTTTGCTTGTTfiTTTRTTRCCRRfifiTGQRRflRTCRCTCGGTGGjRGGflfiTflflTTfiCCGCn 'TTTHRCdRfiBsRRTCGGGGRC

L3_02 RTTRGGCCTfiflCCTCTTTTTTGCTTGTTflTTTRTTfiCCARflfiTGj^RRfiTCRCTCGGTGCRGGfifiTfifiTTfljXlGCn TT-flfiCCRRCCGRRTpGGGGRC L3 04 fl^GGBrHfltcrrc[rTTTTTGCrrTGTTHTTTHTTfl^ RflRi0|1flflflT^CfrCGGTG|HGGHRTHHTTHCCGC^TT-RRCCfifiCCGfiflTCGGGGRC

45!? UG | !2G !3G MG !5G !6G 176 tSG !9G R5-1 f«TTG|HTGflR|rTTGir|RflTfiGGBRTTGRfiRBrTfirfiflTCCCCHCTGMnTCTCCC|rRTCjrTSRfiTTTTnTHRTTTG R5-2 G0HC!r9^rCCtrTRTTTeJrRTTG®=lTGRRCtTTGTCnRTRGGCCflTTGfifiRMrTRTRRTCCCCnCrGRfiTCTCCCTRTC^TCCfiRTTTTflTfcCHTTTG

R5-3 GCRCYCHTCCiTTHTTTCh'HTTGCflTGHRCtrTTGTCHHTHGGCCRTTGHflHCCn’TRTHHTCCCCHC TGRRTCTCCCT h T c(r TCCR R TU TTh T CCR TTTG R18-1 HclrCRTCClrTRTTTcfrflTTGOiTGflRclrTTGTOHRTRGGCclHTTGflfiRa^rTRTRRTCCCCRCTGhRTokcCcjrRTCfrTCaiflTTTTRrCChTTTG R18-2 GCucVOirCetrTRTTTlrHTTGiiTGRRlrTTGT^RTHGGBHTTGRRHBrTRTHnrCCCCnC TGRRTCjTCCCTRTcjTTCCflflTTTTRTCCRTTTG L I 0-1 GCRCTCRTCCTTATTTCTRTTGCRTGRRCTTTGTC iCGCCRTTGRRRCC TTATflRTCCCCR^GRRTC^TCCcWcjTTCCRRTTTTRTCCRTTTG L3_02 G(5tc|rCSlTCCTTRTTTcJrnTTG&irGHHBrTTGTt!'HTHGG®iTTGRRH|cTTRTHRTCCCCl!CTGHRTCTCCC|rHTCTTCClRTTTTHTCCHTTTG L3 04 Grl r4qTCCTTflTTT4rflTTGi^TGRflt^TTGTw,RTRGGwHTTGflfifl|ckTfiTflRT(Mw^FGfiRT|^ttx|rfl^T^fif’TT^rpoRTTTG

2GG 2!G 2 S & b h h b b236 h h h R5-1 GtlRCCRRCjrCCfrGTTTl R5-2 TTTRRTRGTRGI RRRRGRGCTCRGRTGC TTTTCCRGRTGRTTRCCRi vTGCtTflTRGCRGCflCCRRCTCc|TGTTTCTGCR^rRGTTC R5-3 TTTRfiTRGTRGfiTGfrfiTPF HRflRRGRGCjTCpGfiTGCCjTTTTTCfTRGRTGRTTRCCAG CjTG CjTR Tfi G CP G CR CCR fi i TCCTGTTTCjTGCRCjTRGTTC R18-1 TTTRRTRGTflGl HHfiRfiGRGCTCRGflTGCCjTTTTTcirfiGfiTGRTTfiCCRF CTGGTHTRGCRGCRCCRfl! TCCTGTTTCTGCRDTRGTTC R18-2 TTTRRTRGTRGI iHflfifiGfiGCTCjHGRTGCc|rTTTTC|THGRTGHTTRCCRi-q r c c T R ih g c h g c r c c h R! TCCpTTTCTGCRCjTRGTg t t | L10-1 TTTRRTRGTRGilTGiTRTRRj lHRRflGfiGCTGpGRTGMl|rTTTTqTRGRTGHTTRCOiGCfrGCTHTRGCRGCRCCHRi TCCTCTTTCTGCRClTRGTTiGTTC L3_02 TTTiR R GT R GETG oT R ^ i fi J iHRRRGRG^OiGRTGCdTTTTTDTfiGRTGRTTflCCRC CTGC|rRTRGCRGCRO^iRCSTCC'rGTTTCJGCflCjTHGTTiG T T l L3 04 TTTRRTRGTfiGiTGCfrRTRR WfiRRGfiGCTCpGRTGlcTTTTTCrrflGfiTGRTW^iC drcCfTRTRGCpGWCciHfiOTCCTOTTTCTGCRCTRi GTTC 3GG StG 32G 336 346 356 366 37G 3SG 336 \ R5-1 HCTOHTCTHCKCTdGTHfiCepiGGGRGThTTTTTfiTTHTTCcjGHTTCTRfcci&iTTTTTGTCClTCTTTCHlHHHCTTTTHHTTCTTGCTTGSrRGTTRT R5-2 RClTCfiTC|TRCCC^CGTRflCCOCGGGflGTRTTTTTfiTTRTTCCGRTTDtMCCCHTTTTTGTCCTCTTTCRRfifiCTTTTflflTTCTTGC|TTGCjTUGTTflTT R5-3 CflTqrfiGCCjrCGTRflCCGCGGGRGTflTTTTTflTTfiTTCCGHTTCTRtCCHTRTTTGTCCTCTRTCRfifiRCrrTTTfiRTTCTRGaiTGCn-flGTTHTT R18-1 CRTCtRCCCTCGTRflCCuCGGGfiGTRTTTTTRTTflTTCCjGfiTTdlrHCCCHTTTTTGTCCTcVTTCHflfiRCTTTTHflTTCtTGC^TGCnrfiGTTH R18-2 rCRTCTRCCCVCGTflfiCCGCGGGRGTRTTTTTflTTRTTCCGRTTCTRCCCRTTTTTGTCCrrCTTTCRRRRCTTTTfiHTTclTTGCh-TGclrfiGTTH L10-1 rCHTCjTHCCCTp3THfiCCGqGGGHGTflTTTTTflTTRTTCCGRTTC[rRCCCRTTTTTGTCCTCTTTCRHRHCTTTTfiHTTtjTTGCTTGCTHGTTHTT L3_02 rCRTqTRCCCTCGTflRCCGD3GGF;GTflTTTTTRTTflTTCCGRTTCtRCCCRTTTTTGTCCiTClTTTCflfiflRdrTTTRHTTCTTGCrTTGCtRGTTRTT L3 04 rchTarR|CCfTCbTflR|CGQGGGRGTHTTTTTRTTRTTCCGRTTc(rflCCCRTTTTTGTCCTCfTTTCRfiRR|TTTTRRTTCfrTGCTTCfrfiGTTRTT 55 bases. As discussed above, none of the restriction enzymes surveyed on ND4 amplicons revealed any polymorphisms either, suggesting that there is very little variation at this locus.

The sequence alignment for the AL1 locus is shown in Figure 10. A BLAST search of GenBank returned no significant similarities. The closest potential match was a ribonuclease in Camelus bactrianus and Lama pacos. Comparison of the DNA sequence for the AL1 region to a database of restriction enzyme recognition sequences suggested that EcoR I would reveal polymorphisms at this locus. As discussed above, all 103 individuals screened with EcoR I were heterozygous. The sequences revealed a significant difference in sequence, in the 58-82 base pair region, between the one clone sequenced, which did not have the EcoR I cut site and the other 5 individuals which did.

Otherwise, the sequences are very similar, with only 11 fixed differences out of 949 base pairs of sequence.

An alignment of the ITS sequences is presented in Figure 11. There were 32 variable sites in 1068 aligned bases. The sequence was not particularly close to any sequences available in GenBank except in the highly conserved 5.8S region. All of the 32 variable sites were observed among various DNA clones from the same individual, making the intra-individual variation as great as the inter-individual variation.

Phylogenetic Analysis

The COI alignment of 30 species from the Polychaeta Class and one molluscan out-group, trimmed to the region shared by all sequences, is shown in Figure 12. There are 578 of which 402 were parsimony informative. 56

Figure 10: Alignment of the AL1 region. An alignment of AL1 DNA sequences from four individuals from the lower Chesapeake Bay main-stem and one from the upper main-stem generated using the CLUSTAL-W algorithm (Thompson et al. 1994) in the MacVector 7.0 sequence analysis software package (Oxford Molecular) using an open-gap penalty of 20 and an extend-gap penalty of 10. One clone from one individual from the lower main-stem site shows significant differences in the 58- 82 bp region. This clone contains an Eco RI restriction enzyme cut site. The others do not. Ambiguities are marked in white and are coded as follows:B= not A; D= not C; H= not G; K= GT; M=AC; N=ACGT; R=AG; S=CG; V= not T; W=AT; Y=CT. 57

L1_4con C B 5_3con L9_02C O N L 4_05C O N L 1_02C O N LI _0 3 CON

L 1_4con C B 5_3con L 9_02C O N L4_0 5 CON L1_02C O N L 1_03C O N

t1 _ 4 c o n C B 5_3con L9_02CON L 4_05C O N iDRflflf L I_ 0 2 C O N L I_ 0 3 C O N Iflflflflt

L 1_4con Iflflflfll C B 5_3c on ififlfifiQflfil

L 9_02C O N tflflflfi R h ) L 4_05C O N tfiRRRQFIR L 1 _0 2 CON IflRRRORR L 1 _0 3 CON iflflflfl RR

e/e L 1_4con C B 5_3con L 9_02C O N L 4_05C O N L l_ 0 2 C O N L 1_03C O N

L 1_4con C B 5_3con L 9_02C O N L 4_05C O N L 1_02C O N L 1_03C O N 58

Figure 11: Alignment of ITS region. An alignment of ITS region DNA sequences from five individuals from the upper Chesapeake Bay main-stem and the York River generated using the CLUSTAL-W algorithm (Thompson et al. 1994) in the MacVector 7.0 sequence analysis software package (Oxford Molecular) using an open-gap penalty of 20 and an extend-gap penalty of 10. Ambiguities are marked in white and are coded as follows:B= not A; D= not C; H= not G; K= GT; M=AC; N=ACGT; R=AG; S=CG; V= not T; W=AT; Y=CT. 120 138 MO CO , OO Oxl I CM (fl (O (O (O (O (fl CM Oxl I 33 33 33 OO CO , > - > - >u - . CM, | I — \ C I CO C\l *— I | | | , M .C — U o O C o o o •»— o o o CO : — CO — O 3 I o t I I w o o o oo o o O o ir> o t ir> I I | | I I 0 0 f r m m 33 3 3 3 t t. H i ■ Btttt.B H B ■ i U o o o o o o o o C O ' : a c E C E C d i i r ( E'BCESSSE! ■frfrf+frfrg- 59 60

m i i i

S- i - i - o o o c c C O1 O O O o o o o o o O o _ T— CO o o o <-> o “■— CO O O I I I •»— C\J CO I | LO LO LO LO , I m lo lo I | m m m m i i i CO 0 0 ■»— :» — CM C J (D CO CO CO CO ; ■»— CM C O C O - * — ■*— C\l C\J CO CO CO =>=>=>Z>=>=>>->->-u- I> Z> => => Z>Z>Z>=>3=>>->->- 61

Figure 12: Trimmed multi-species alignment of the COI gene. An alignment of 30 species in the Class Polychaeta. The alignment was generated using the CLUSTAL-W algorithm (Thompson et al. 1994) in the Mac Vector 7.0 sequence analysis software package (Oxford Molecular) using an open-gap penalty of 20 and an extend-gap penalty of 10. Sequences from two Orders, four Sub-orders, and thirteen Families were included. The cephalopod Opisthoteuthis sp. was used as an out-group. Ambiguities are marked in white and are coded as follows:B= not A; D= not C; H= not G; K= GT; M=AC; N=ACGT; R=AG; S=CG; V= not T; W=AT; Y=CT. 62

Opisthoteuthi3 3p Amphicteis dalmatica Amphiglenaterebro Amphitritides harpa rTHGTTRTH Amphitritides sp Cirratulussp HR TRRTTTTOTTHgrRGTTR 1 Dodecaceriaap ______rT»TRG-TTF'tr ip c f tJ^tttgtr GGH GG|Tt t | Isoldasp i! IB IIRRTHHTTTI rBTTGTTtiTH( 5TTTTTR1 iGGRTTj Loimiaingens rTRTHRTTTTTTTTi tTTTThh TTGGTGGGTn Lysilla pacific* ______rTTPGTTRTft ^TTGGRGGHl Ret erebella queenslandica iggtrgrtt B rTRRTRRTTTTirTTTTRGTRRTh Rhinothelepuslobalus fifl 30dRRC i t t r t h r h rm iTRGTTRTu iTTGGRGGTH^TfiRTTTTi Trichobranchussp 3TRTTTRTTGGRGGRn Amaeanatrilobata rTTTTTTTR Paraprionospio pinneta TTGTRf rTRTiRRTTTTBTTTTTRGTR R TR«F GTTT1 Prion os pio steenstrupi 3TPRTTTTfrTTTTRGTR R T R » GtTTTTR 1 'TRTRGCftl Si gem bra tent acute* a ihlpnl iRTPRTTTTTTI Scololepissquamala 5TRTJRR1 TTGTTR fG | iTRRI I I ifll 18 1 1 IGTTi Tfj TTCGR Pisioneremota 3TRRTTTTC|rTTTTROTTR TRC HTTTTRGTRGCGGGRTTTGC TRGGRC Paralvinefla palmif oimis i«R«RRl R WflRflF 3jpr|RTHGGflGGR TTGRTTGG1 O p hi o drom us fl exu os us rRTFRI N eanthes virena -rttgtrr tg | iTTTTTR-Th RTTI Microphthalmus listensis rPTPTlRRTP ^RTTGTTR 3G( ifRTTRGC Manayunkia z enk evritsc hii rTTRTRR 3 RR TGTTR iC rTRTRTTRTTTGTRC Heteropodaikefotmalis TGTTt-CjTGO d i iTGRTTTI rGTTR TPR TTGGH GGGTTJ3C Hesionidesarenaria rCTRTRRl TTGTRR GG( iTPRTTTI [TRGTTRT h | j3irTRTRRTTGGGGGGT TO.-': Dysponetuscaecus rpTRTPRTR hrttgtrr rc| rTTGTPRTPRTTTTj ITRGTRRl rGTTRTHR TTGGGGGRTTTGj ChrysopetaJum debile IrI r pI HRTPRTTTTTTTBrTPC 3TTPTPR TTGGPGGGTTTGC Bronchipolynoesp rTTTTRRTPRTn STT^TRGTRGGGGGGTTTGC ^RTPTTPGGl Streblospio gynobranchiata rTTTTRRTPRTTTTn rPGTHRTC 3TTTT§GTPGGTGGGTTTGC h GPTPI Streblospio benedicti TGTTPCfTGC rTTTTTPRTPRTTl ) | rq ( | | r) r n|rrTGTTGGGGGG‘rTTG( BGRTPl

22* 230 240 250 see 278 288 Opisthoteuthissp f 0 ^ROfRRTTTRR Amphicteis daJmatica rr 3 " iRGWRiRTTp H I f Amphiglenaterebro ETS f iRG§HR|TTRGi Amphitritides harpa ft * iRGRRRTRTRGI Amphitritides sp 2? Cirratulussp Dodecaceria3p Isoldasp wJpRftOfifTTIpOi Loimiaingens __ BROTH 3RfTTTTR{ rTGTTRGTT Ly3iHa pacifica Relerebellaqueenslan

Opisthoteuthissp Amphicteis dalmatica Amphiglenaterebro Amphitritides harpa RRTTTTgrdrTTHOHTC Amphitritides sp ______Cirratulussp " " f lf *TTN HT1 Dodecaceriasp GRTfTTfrarrfiRi Isoldasp TR TTTTTTflrJrTI = il Loimiaingens HHT^TTTf^qrCO^rfl Lysilla pacifica Ret erebella queenslandica Rhinothelepus lobatus Trichobranchus3p Amaeanatrilobata h TTTTTT6TTh 6 h T1 Paraprionospio pinnata Pri on os pi o st e enstru pi 1TTTTTH Sigambratentaculata rr^ H Scololepissquamata Pisione remota ____ Paralvinella palmiformis rrM O p hi o drom us fl exu os us Neanthes virens Micro p ht halm us list ensis RRWTTTCtrTTt° ' TT Manayunkia z enk evritsc hii ITTTTTTCO TI H et ero p o dark e f ormali3 iTTTTSrc(rTTF( Hesionides arenaria Dysponetus caecus f f j •

Opisthoteuthissp Amphicteis dalmatica Amphiglenaterebro Amphitritides harpa Amphitritides sp Cirratulussp ______Dodecaceriasp Isoldasp ITTTTGf Loimiaingens rTTTTTTGRTCC Lysilla pacifica ^ttStttghccc Ret erebella queenslandica 'Trq|rT§DHCcc Rhinothelepus lobatus HTTTTTTGf Trichobranchussp Amaeanatrilobata Paraprionospio pinnata Pri onospio steenstrupi Sigambratentaculata ■■ n m hi ■ a * Scololepissquamata Pisione remota ______TTTTTTGnr( Paralvinella palmiformi3 O p hi o drom us fl exu os us Neanthes virens Microphthalmus listensis h G h TTTTTTG h S&J Manayunkia z enk evit sc hii H et ero podarke f ormalis rTTTT|GH Hesionides arenaria Dysponetus caecus ChrysopetaJum debile Branchipolynoesp Streblospio gynobranchiata Streblospio benedicti 64 A Neighbor-Joining (NJ) tree, and parsimony tree are shown in Figures 13 and

14. Jack-knife support values above 50 are shown on the parsimony tree. Most support values were very low, and the branch lengths on the NJ tree are very long. The NJ tree generated using all the characters does not agree very well with the polychaete phylogeny based on parsimony analysis of morphological characteristics as proposed by Rouse and

Fauchald (1997). They proposed a tree (Figure 20) based on a weighted presence/ absence character matrix. This included the orders Aciculata and Canalipalpata which are in common usage. The Aciculata order was strongly supported, and is defined by the presence of aciculae. The other order, Canalipalpata, which is defined by the presence of grooved palps, is not as well supported by the morphological data. The Aciculata is divided into two sub-orders, the Phyllodocida and the Eunicida, only the first of which is represented in the current study. The Canalipalpata is divided into three groups, the

Sabellida, the Terebellida and the Spionida.

The order Aciculata groups within the order Canalipalpata, with the exception of a group containing Sigambra tentaculata and Microphthalmus listensis that is less derived than the rest of its order. The species Manayunkia zenkewitschii falls basal on the tree. Family groupings in this tree are generally consistent with traditional morphological phylogenies. The Spionidae family falls out. The family Terebellidae contains the Sabellida Amphiglena terebro and is missing only Rhinothelepus lobatus to be a monophyletic group.

The parsimony tree was very different from the NJ tree. In this tree Paralvinella palmiformis appeared to be the most basal, while Manayunkia zenkewitschii grouped fairly closely with Microphthalmus listensis, a polychaete in the Aciculata order. The 65

Figure 13: Neighbor-Joining tree of partial COI sequence. The Neighbor-Joining tree generated using uncorrected “p” genetic distances of a portion of the COI gene following sequence alignment using CLUSTALW (Thompson, 1994). Opisthoteuthis sp. was used as an outgroup. 6 6

OpisffvoJeaffws sp -N Pataptfonospto pmnata > ------Priortospto steens&upiSpionidae Scololepis squamata - gynobrandmta — Strebtospia henedicH &n I 3 Arnphictels dalmatic? V - Ampharetidae B- fsofda sp ►5' Sabellidae B- - Amphigiena tenebto ^ PO Pr-t- AmphiMdes harpa Lysiffa padtica Amaea/ia tritobata Terebellidae - AmphtWdes sp Retetebelfa queenstatictica - Lcttmiaingens Trichobtandws sp Paiafvindta patmifonnis Cirratulidae ------Cttmktfus sp ------Docfecacsria sp• > Rmofaetepsjs iobatus ^ J Pisione temoia — Brandvpotynoe sp o_> o' ------Ophiodronius ftexuosus ET <—*■ Heieropodatke foimatis• > pa Nsantfies metis Hesionidae Dysponetus caecus '[Chrysopetalidac Chrysopetedum debtie^ ■ Heskantdes aienaiia Stgatnbta tent&utata ------Maophfoalmus Sstensis _ J Sabellidae Manayunkia zenkemtschfi g 0.05 changes 67

Figure 14: Parsimony tree of partial COI sequence. The parsimony tree generated using of a portion of the COI gene following sequence alignment using CLUSTALW (Thompson, 1994). Opisthoteuthis sp. was used as an outgroup. Jackknife support values above 50 are shown on branches that had support Twenty percent character deletion was used for the Jackknife analysis. Gpisttioieutiiss sp Ampharetidae 100 AmpNctets dabrtabca 68 Isofda sp } Sabellidae Ampfvgfena terebro ------Pataprionospio pinnate Spionidae ------Prionospio steenstupi Sculolepss squdmata < ffefbne iBmota > o _ Bsmciiipoiynoe sp co '

— Cftty’sopetetum debile r-t-ET Cbrysopetalidae ca Dysponekis caecus m " > Hesionidae Hesiofxdes arenaria ^

90 Ophiodtumus ftexuusus 54 Heteropodarke formatis Neanihes ittierts } < Sbebtospio gynobranchiata Spionidae 98 n Sbebfospio benedicb pa } 3 — Amphiffides harpa Si >3' Si 100 Lysiffa parities Terebellidae 3T Amasaira biSobata r-K

90 Amphrbibdessp Cirratulidae Reterebella queenslandica

100 ------CmaMis sp ------Doriecacstia sp} Triehobtanehus sp Loimia ingens } Terebellidae Rmotoelepus iobatus Sigatnbra benbacuiata Hesionidae fttbaophffiafmus tistensis * 63 Sabellidae Manayunkiazenkemtschii < — Patalmeba pabnitiamTis 50 changes 69 Terebellidae family was monophyletic, again with the exception of Rhinothelepus

Iobatus, and the inclusion of Amphiglena terebro. Most of the Aciculata order fell together within a group containing the Spionidae family and members of the Sabellidae and Ampharetidae families. The Spionidae family was split with the Streblospio genus being removed from the others. Jackknife values were high for several pairs of species, but there was very little support for any higher groupings.

A translation of the DNA sequence using the invertebrate mitochondrial code

(Figure 15) shows that the amino acid sequence is fairly conserved. The homoplasy of third position substitutions can be dealt with in the analysis in two ways. The third position can be removed from the analysis, or the DNA sequence can be translated into an amino acid sequence, removing all synonymous substitutions at the DNA level. When the third position is removed from the analysis a very different tree topology emerges. In the NJ analysis (Figure 16), the Aciculata order is separated into three groups, one that falls with the Spionidae family, and another with the Ampharetidae family and

Amphiglena terebro. The final species in the Aciculata order, Dysponetus caecus is a basal branch. As in the previous trees, Amphiglena terebro does not group with its family member Manayunkia zenkewitschii. That polychaete is in a group with the members of the Cirratulidae family. Again, Rhinothelepus Iobatus is more basal than the rest of the Terebellidae family.

The trees generated by parsimony analysis, still excluding the third position,

(Figure 17) are similar to the tree generated by NJ analysis with all characters included in that the order Canalipalpata is a derived group and members of the Aciculata order are more ancestral. Again, Manayunkia zenkewitschii falls external to the other members of 70

Figure 15: Alignment of COI gene translated to amino acid sequence. COI DNA sequence was translated to an amino acid sequence using the invertebrate mitochondrial genetic code in MacVector 7.0 sequence analysis software package (Oxford Molecular) and aligned in the same program using an open-gap penalty of 10 and an extend-gap penalty of 0.05. 71

Translation of Opisthoteuthiss - g s l l W l v I v VM 1 G -G g | m L I L M L G fl— |^f1SFWLLPp§L?LLLTSAAV ►PLSSNLM

Translati on of S b en e dicti T S4 - g s l l g s w l v ^ t VG-G g n w LI L M L G fl— MAFP L(^nSFMMLpPSLtLLVA|AAV •PLSSNLR

Translation of Sgynobranchiate - g r l l g s H l v ! VFVG-G chwLI LMLGfl-- M AFP l S n m SFULLWSLTLLVASAAV| ■P LS3N LH Tranalati on of Pri on oa pi o 3t e en - g s l l g JH l v j M lp LVM V F 1 G -G GflW LV L M L f l f l - - MAFF LNNtlSFULLPPSLTLLI •P L S R flL fl Translation of BfY 6_3consensus - g s l l g J d q l v n t i V MIBFLVM V F 1 G -G g S wLL LMLflfl-- MAFP L|ltjnSFWLLPHAL§LLvj •PLRSMLH

Translation of Trichobranchus3 GRFLGSDQ ■M I B F L V H VF 1 G-G GNULL LMLGfl-- M A fP m B m SFUL I . B a l LLLV RRVB ^ P L S S fj I fl

Translation of Sigam bratentacu - gsllgsoqlvh MIBFLVM VM i g - g F g n u LV L M L G fl- - m a f p l IH msfullppsltll I RRVB •p l s s H i r

Translation of R etereb ellaq u e e - g s f l g s H l v n t I V MIBFLVM I L I G - d g | u L I L M L G fl- - MRFP F S F U . I H l r s b i R Translation of Manayunkia zenke -GSLLGSDjdl VW /IV M L g V V V L

Translation of Loim iaingens - g s f l g s H i V N T I V m i I f l v m I L I G -G B g I LM LG fl- MRFP mNMMSFULLPhALLLLV V VPPLSSl^MR Translation of R hinothelepus lo ULGSjdLVMTI V i | i g - g | g I LMLSA MRFP m B m xfhllppsllllI PPLSSNMR Translation of Pi3ione rem ote LLGSMLVNTI V i l v g - g | g | LM LG fl MRFP lBmSFULlI>P"SL I L L L ^ S S A Vi P P L H S M IH Translation of Paralvinella pal g sf l g WB ol v ni i t I I BLL—MLFFFVT i f m g - g f g | L M IG fl MRFP m B lgfull^m lvllva|aav| PPLRG NM H

Translation of O phiodrom usflex g r l l g s | o l v n t ?i V LVM VMMG-GF g IWLV L M L G $ - - MLVP Jm B m sfU llB sltlllasa I V PPLSSWXX Translation o fN e an th e3 viren3 GSLLGSDQLVMTIV v m i g - gI gB l v LM LG fl MRFP lnm m sfw llPpsltlll! PP L R S ^ IR Translation of Micro pht halm us I GSLLGSiQLVMTIV VMIG-GfGp4LV LM LG fl MVFP L M IiM S F U F L P P S L L L L L G S A L V GAG PP L S S N lR Translation of Lysilla pacifica G A F L G S lQ V«T I L I G - ( LM LG fl MRFP tlpnSFULLpSBLLLLL! RRV VVPPLRs|lR

Translation of teoldasp LGSMLVMYHT v | l G - ( l m l g | MRFP . W l SFHl.LpPALTI.Lt. RRV VPPLRSMIH

Translation ofHeteropodaikefo ■G flLLGS L V M T I V vm ig-gFg|hlv l m l g | - :iisfw llppsltllla S a v V P P L S S H IR Translation of H es ionidesarena -GSLLGS LVNTIV -G F G f LM LG fl MSFMLL^SLTLLVASSAVl Translation of D ys p onetuscaecu ■g s l l I s VMIG-GFANUFL LMLGV MRFP !l s f w l l p p ;a l t l l l I Translation of D odecaceriasp i|vg-g|gB l L M L flfl MRFP Lg^LSLHLLPRALTLLVLSAL

Translation of Cirratulussp l l g - g I g B l L M L 3 C -- m B l s f u l l B s l l l l v ! V P P LSGRTS

Translation of Chry30 petal urn de -G S LLG S J L V H | I V VM I G - g | g H l LM LG fl ^ m s f u l l B s l t m l l a s s a vj

Translation of Amphitritides ha -Gfl|LGS |M vi|lV i l i g - g | g B l i LM LG fl Im r f p Smsfullmblllll !

Translation of A m phiglenatereb X-GXLLjjsOQLVNTVX LVM _ IG -G F G M U L L L M L f lj- MRFP l « | m s f u f l B s l | l l I V P P LR

Translation of Am phicteis dalma |-GSFLGsljLVNTlV i g - g | g H l l LMLGS- MRFP L&t|LSFWLLlfl§ALTLLL| VYPPLRSI Translation of Am aeanatrilobat j-GflgLG SM LV ST 1 V ILIG- g I g I LMLGA—B| mAFP i imhmsfullppialllllssitini iotwll VVPPLRS Translation of Branchipolynoe s p I m t m s t s s p s w I m p t i H S C VSJ PI C I F(J|FLPSVfl|sSSGVI _ ||C P U V S F G vM K 1!t p p | i |> v p s s f | i Ic l H i s s I Translation of Scololepis squam |L|LSLA-|TN!cf1MLLLLLMLi|L| |l — l J l l l l ar| l v S § - ^ | l L«| [l g l m m

Translation of Opisthoteuthi3 3 jGiSV LflllSL LflGV ILGAIlFX ISLLI flILLLL VLRGRI Translation of 5 bene dicti T S 4 AGBSV LAIFSL LAG i § I L G A l I f M IflL I RILLLL VLRGGI Translation of Sgynobranchiate AGRSV LAIFSL LAG I ILGALBFN IRM I R L L L L L VLRGGI

Translation of Prionospio steen AGRSV LAIFSL LAGA ILG A lIfi flvfeI RILLLL V L flG R I ------

Translation of BrY6_3con3ensus AG| V LAIFSL LAGV ILG AlIfI flV I RILLLL V L flG R I rM L L T H | l Nt s f f u p r g g g I f f l L V i

Translation of Trie ho branch us s AGP V LAIFSL LAG I IL g |||F I RV I | IL L L L V L f lG f l1 niLLToft i | t s f f | p s g g g | Translation of Sigam bratentacu AGR V LAIFSL LAGV ILGAViFI GV I RVLLLL V L flG R I rMLLTllft|Lfi|TSFF jpflGGGl d | : Translation of R e tereb ellaq u e e AGG V LAIFSL LAG I ILGAljFI RV I VI LLLL) V L f lG f l1 rMLLTHvNTSFFqP3XGG| R 1 L X |

Translation of M anayunkia z enfce AGP M LTIFSM LAGX ILAS aJ f G SIVV GVMXILfl V L f lG f l1 Translation of A m phitritide3sp AGP V LAIFSL LAG I ILGAV|FI RV I VI LLLL! V L f lG f l1 MLLljBjvNTSFFciPRGGG

Translation of Loimia in gens AGP V LAIFSL LAG I ILG A ll'l RV I VVLLLL V L flG R I m l l t i B i n t s f f H p r g g g

Translation of R hinothelepus lo AGPSV LR||SL LAGAS IMASViFI V L flG R I M L L T D * 1 t s f f q p s g g g 1 L F

Translation of Pisione rem ote agIsv la iE l iagvs ilgalJfi V L flG R I m l l t H l t r f f J p r g g g I L V LF Translation of Paralvinella pal A G $S V LAlIsM LAGV ILG$l| I v | a g a i m l l t d B im t s f f o p s g g g PVLVL- Translation of O phiodrom u3flex AGRSV LAIFSL IAGVSSI la s v If j /MS I I RILLL V L flG R I M L L T r » * L TRFFBPRGGG PVLVL

Translation of N ea nthesviren3 AGffsV LAIFSL LAGVSSIMGBlIrI' /MSVMI RILLL V L f lG f l1 M L L T ^ B l TRFFBPRGGG 1 L V i L

Translation of Micro pht halm us I SGPSV MAIFSL LAGVSSILASvIfI- iB lIl RVLLL V L f lG f l1 m l l t o B l TSFFBPSGGG B l LVQ L

Translation of Lysilla pacifica SGPSV LAIFSL LAG ISS ILGAINF11 /|flV |l VVLLL V L flG R I MLLT b H v TSFFBPSGGG IL V j

Translation of Isoldasp SGPSV FAIFSL LA G I»ILG A lH |] /Hflvlv VILLL V L f lG f l1 MLL t H . TSFFBPSGGG 1 L V

Translation of H eterop o d arke fo AGpSV LAIFSL LAGVS^ I L A 8vH I j /MSV|| RILLL V L flG R I MLL t K m l TRFFBPRGGG PVLV

Translation of H esionide3 arena IGASV LAIFSL LAGVraiLGAvHl /H v l i R IL L L V L f lG f l1 m l l t IH l I t r f f I p r g g g 1 LV^ L

Translation of D ysponetus caecu GPSV LAIFSL LAG ISSILGALBFI " IMSv|I VILLL V L R G R 1 m l l t ^ B i t r f f | p s g g g 1 LVQ L

Translati on of D o d ecac eria s p SGPSV LAIFSL LAGASS ILGSLaFL' /USLVV RILLV V L f lG f l1 m l l t H l SSFFIP§GGG p v l y q LF Translalion of Cirralulus sp SGRSV LAIFSL LAGVSSILGSliFr /USLVVTTILLL V L R G R 1 m l l t L ^ m l I t s f f I p r g g g 1 LV^ LF

Translation of Chryso petal urn de AGjSV FAIFSL LAGASSILGALlFI' /M SvIlTRILLL V L R G R 1 m l l t IH l I t s f f I p r g g g BVLVCLF

Translation of Amphitritides ha t g p s v l a iE l l a g i s | i l g a i| f i ' /MRVBI TV ILLL V L flG R I M L L T O U N V N T S F F B P S G G G P V L V i LF Translation of A m phiglenatereb S G PSV l a iE l LAGVSS ILG ALIFI' /|flvll|w LLLL V L f lG f l1 m l l t JA f n t s f f B p t g g g P I LVQ LF Translation of Amphicteis dalma SGRSV FAIFSL LAG ISSILGALBFIf /MflVFVTVILLL V L flG R I MLLTfcft.NTSFFdpSGGG i L v i LF

Translation of Am aeanatrilobat SGPSV LAIFSL LAG I SSI LGA I |p r /M fiv|lfVVLLL V L flG R I M L L T J | v | t S F FOP S G G G 1 LVl LF

Translation of Branchipolynoe s c Ma l c s P c v P l l a | | 'CPI ic m v N t v | g 5S SS FP PC 3Ssc | v v t f n s s | fJ | c llw p lu U S S S V F

Translation of Scololepis squam l l c l | l ( E | v | a| g] »l v t | p m i - q g f p q f l | l - l i — l - m v r l | v s v l | v | l | l f | | l l l - | l p 72

Figure 16: Neighbor-Joining tree of partial COI sequence with the third codon position removed. The Neighbor-Joining tree generated using uncorrected “p” genetic distances of a portion of the COI gene following sequence alignment using CLUSTALW (Thompson, 1994). Opisthoteuthis sp. was used as an outgroup. 73

Opfsffioteufftis sp Ampharetidae Amphrcteis cfafmaSca i tsdda sp Sabellidae Amphigtena terebro

Opfvoetoomus tfexuosus Hesionidae Hefetopodatke futmafis Mfcrophtha/rmis fistensis Amphiiriticles harpa r r LysHta pacrSca Terebellidae Amaeana trilobate ReterebeSa queensfandica COn Ampfiitritfctes sp — Loimia ingens £L Ttichobrartcftus sp r—t co ------Paratvrrrefte paimitermis Rrirroifreiepus iobatus ------CirraMjssp Cirratulidae

Doc/ecacetiap sJ p Sabellidae Manayunkia zenkewiteciw Pataptiorrospfo pinnate ------Prionospso steensfrupt Spionidae Scotaiepis squamata Strebfospte gynobranctmte ------Stoebfospio benecEcS J Sigambra tentaculata o Chrysopetalidae c — Chrysopetaium cfebtfe Co r-t-CO — Pisione remote — Neanthes virens ------Branchipolynoe sp Hesionidae Hesionictes arermia Chrysopetalidae Dysponetus caecus □.□1 changes 74

Figure 17: Two most parsimonious trees of partial COI sequence with the third position removed. The parsimony tree generated using of a portion of the COI gene following sequence alignment using CLUSTALW (Thompson, 1994). Opisthoteuthis sp. was used as an outgroup. Jackknife support values above 50 are shown on branches that had support. Twenty percent character deletion was used for the Jackknife. Opisthotsufas sp v , Opisffioteulfiis sp Canalipalpata Canalipalpata | 5 « « 5 | b a a - a £ Aciculata n Dl I I 1 75 76 Canalipalpata. Rhinothelepus Iobatus and the Cirratulidaes do not group with the rest of the Terebellida sub-order. They fall within the Spionida group. The rest of the

Terebellidae family is monophyletic. Amphiglena terebro falls within the Terebellida sub-order. A second, equally parsimonious tree moves the clade containing

Ophiodromus flexuosus and Heteropodarke formalis to a more internal branch.

Figure 18 depicts the NJ tree produced using the protein sequences. Much like the

NJ tree for the full DNA analysis, this tree had very long branch lengths. The Aciculata order, with the exception of Sigambra tentaculata and Microphthalmus listensis grouped as an within the complete Spionidae family, but outside to the remaining Canalipalpata and Aciculata. Manayunkia zenkewitschii is external to all the other branches.

The parsimony trees generated using the amino acid sequence (Figure 19) had the highest jackknife support of all of the parsimony trees. As in several of the other trees, the Canalipalpata order was not separate from the Aciculata order. There was good support for the Spionidae and Terrebellidae family groupings, but not for the Terrebellida sub-order. Dysponetus caecus fell within the Canalipalpata order while Amphiglena terebro fell outside it. There was not good support for the Hesionidae family group. 77

Figure 18: Neighbor-Joining tree of partial COI amino acid sequence. The Neighbor-Joining tree generated using uncorrected “p” genetic distances of a portion of the COI gene following sequence alignment using CLUSTALW (Thompson, 1994). Opisthoteuthis sp. was used as an outgroup. 78

Opisihoteutiiis sp Amplvcieis datmatca Ampharetidae^ isoida sp t } Sabellidae ------Atnphigiena terebro nco a ' AmpNtritides harpa £L ■ Lysiifa padfica T erebe 11 i dae co Amaeana iriiobata r—f- — Amphiffides sp Retetehella queensiancSca Lamia ingens Ttichobranchus sp ParstlmeHa palmitotmis ------Cirratutussp Cirratu idae ------Dodecaceriasp Rhinofoeiepus Iobatus J Pisione remote o> — Branchipolynoe sp o' ------Qp/iioatamus jfe^jost/s CO Cof—► Hetetupodarke famaiis} Ne.anthes wotens - Dysponetus caecus Chrysopetalidt e - Chrysopetektm debile Hesionidae - He&(onides at&naria ^ Faraprionospio pinnata - < ------Prionospio steerrsirupiSpionidae Scololepis squamata - Sbeblospio gynobtanchiate — Strebiospio benedict Sigambia tentacufata ------Mioophtealmus (istensis ------Manayunkia zenkewtsdvi ell idae 0.05 changes 79

Figure 19: Two most parsimonious trees of partial COI amino acid sequence. The parsimony tree generated using of a portion of the COI gene following sequence alignment using CLUSTALW (Thompson, 1994). Opisthoteuthis sp. was used as an outgroup. Jackknife support values above 50 are shown on branches that had support. Twenty percent character deletion was used for the Jackknife analysis. 80

iculata

T3 Terebellidae

Q.

Q- T3 CL

Q. 00 T3

^^Canalipalpata iculata W

Terebellidae

Cs3 N

I a Discussion

Population Study

There were significant differences between the size distributions of P. pinnata among the four sites. Most of those differences were between the Rappahannock River site and the other three sites. The individuals from Rappahannock River site and the upper Chesapeake Bay site tended to be a little larger than those in the York River and lower main-stem.

The relationship between fifth setiger width and branchiae length was weak

R =0.22. This lack of correlation may be an indication that branchia length is a trait independent from size, and therefore potentially under a different selection pressure. The results from this study do not indicate that hypoxic conditions have any effect on the length of branchia at each site.

None of the genes surveyed in this study displayed much variation. The mitochondrial genes in particular were almost identical in all the organisms sequenced.

The nuclear genes, both of them apparently multi-copy, showed as much variation within individuals as between them. The COI polymorphism alone is not sufficient to distinguish any of the populations from one another. Each allele accounted for roughly half of each population. This is what we would expect from a common and neutral mutation. A more extensive mitochondrial survey might be more informative, but with 82 only the single difference in the COI gene, it was impossible to separate the four populations. This supports the null hypothesis of no population structure.

The lack of variation at the ND4 locus seems to also support the null hypothesis of no differentiation between populations. ND4 has been used successfully in past studies to distinguish between populations of the same species (Hardy et al., 2002;

Gorrochotegui-Escalante et al, 2002; Hauser et al, 2001). If the power were higher and more individuals had been successfully sequenced the low degree of variation indicated in this study could be strong evidence that there is no population structure.

It is highly unlikely that every individual would be heterozygous at the ATI locus. Such an event would most likely happen if this locus were a coding region and both the homozygous states were lethal. This is very unlikely. More probable is that the re-designed primers are still amplifying at least two loci. The sequence analysis showed that there were 21 total unique changes, out of the 949 sites surveyed between the single

DNA clone that contained the EcoR I cut site and the 5 DNA clones that did not. Ten of those changes are concentrated in a 20 base pair stretch. Redesigning a forward primer to that region may allow independent PCR amplification of the two loci.

Taken together, the genetic evidence indicates that there is little to no genetic variation between populations of P. pinnata at the surveyed sites. No genetic differentiation was revealed between sites, and no correlation of any factor, genetic or morphological, with the presence or absence of hypoxia was observed. Nothing can lead us to reject the null hypothesis of no differentiation between the sites. In fact, the extreme mitochondrial similarity may indicate that the population has recently undergone a severe bottleneck. There appeared to have been a population crash in the York River 83 (Figure 5) in the winter of 1984 that may explain the lack of variation. Alternately, abundances before 1973 were very low. It is possible that Paraprionospio pinnata was introduced into Chesapeake Bay only sometime in the last fifty years and that it did not gain a strong foot hold until the early 1970's. The introduction of a species and the presence of a founder effect can lead to a lack of variation similar to what was observed in this study. A larger number of sequences from several individuals from multiple sites would help to confirm this.

Phylogenetic Study

Although the COI gene by itself is not ideal for determining phylogenetic relationships, as is shown by the long branch lengths in the NJ trees and the low jackknife support values, these analyses did provide some valuable insights. The analyses provide strong support for several of the taxonomic family groupings proposed by Rouse and

Fauchald (1997)(Figure 20) among the Canallipalpata order, namely Terebellidae and

Spionidae. There was some indication that the inclusion of Rhinothelepus Iobatus in the family Terebellidae may be inappropriate. The two species representing the

Ampharetidae family were also very strongly supported as a single grouping and appeared to be more closely related than even the members of the Streblospio genus are to one another. Of the three sub-orders in the Canalipalpata, there was strong support for the Terebellida sub-order. The Spionida group was also well supported, but this study only presented one family group in the Spionida, and so it was not particularly strong evidence that the sub-order would hold up if more taxa are added to the analysis. The

Sabellida group was very poorly represented in this study (only two species), but there 84

Figure 20: Basic classification of the taxa used by Rouse and Fauchald (1997). Phylogenetic tree based on a parsimony analysis of weighted morphological characteristics, as proposed in Fig. 2 of Rouse and Fauchald, 1998). 85

E ch iu ra C Euarthropoda OnychophoraOnvi'hnnnnr.1 Clitcllata

Siboglimdac Sabcllariidac Sabellidae SABELLIDA Serpulidac O w eniidae _____

A erocim dac Flabclligcridae Cirratulidac Alvincllidac TEREBELLIDA Ampharetidae Pcctinariidae Terebellidae Trichobranchidae

Apistobranchidae Spionidae Trochochactidac SPIONIDA Longosomatidae M agclonidae Poccilochaetidae Chaetopteridae P A A coetidac o Aphroditidae N Eulepethidac L N Polynoidac Y Sigalionidac E Pholoidae C Chrysopetalidae H L G lyccndac G oniadidac A I Paralacvdoniidac PHYLLODOCIDA E D Pisionidac T Lacydoniidac A Phyllodocidac A N cphtyidac Nercitlidac H esionidae Pilargidae Sphaerodondac Syllidae ------Ainphinomidae Eupnrosinidac Dorvilleidac ELfNCIDA Lumbrineridae Eunicidae Onuphidae _____ Arcnicolidae M aldanidac Capitcllidae O pneliidae Scalibrcgmatidae Orbiniiaac Paraonidac Questidae Cossuridac _____ 1 S ip u n cu la 86 was no evidence to support that either one of these species should be grouped together, or separated from other groups, though there was very little consensus on where they do belong. There was very little evidence to support a division of the orders Aciculata and

Canalipalpata, but that may be as much due to the locus being unsuitable to examine that level of divergence as it was due to any real lack of differentiation between the orders.

Manayunkia zenkewitschii never grouped with its nominal family member,

Amphiglena, and frequently fell outside the grouping including the rest of the

Canalipalpatas. Manayunkia zenkewitschii is native to Lake Bikal, and as it has likely been isolated from marine polychaetes for a very long time in a significantly different environment. Based on this, we may expect it to be divergent from the rest.

The family groups within the Aciculata order were not as strongly supported as those within the Canalipalpata order. None of the analyses contained groups that encompassed more than two of the Hesionidae family, and the Chrysopetalidae family had no strong support either.

Brown et al. (1999) reviewed Rouse and Fauchald's relationships using several genetic markers including, the histone H3 gene, U2 small nuclear RNA gene and two segments of the 28S ribosomal DNA cistrons. The tree representing their combined data is shown in Figure 21. They found that they were unable to support monophyly of any of the clades for which more than one sequence was available (Phyllodocida, Spionida or

Sabellida). Only families that were represented by multiple members in their analysis had those family groupings supported (Cirratulidae and Terebellidae). As in the current study, the Aciculata species did not form a clade, though in the Brown study the presence 87

Figure 21: Minimum-length tree for combined H3, U2 snRNA, and 28S rDNA sequence data. This phylogenetic tree based on a parsimony analysis of three genes was proposed by (Brown et al. 1999, Fig. 5). Percentages greater than 50 for bootstrap pseudo-sampling are shown above the clade branch. Bremer decay indices of three or more are shown below the branch. Abbreviations of the families are as follows: Sbi=Sabellariidae; Sab= Sabellidae; Ser= Serpulidae; Owe= Oweniidae; Fav= Fauveliopsidae; Ste= Stemaspidae; Sib= Siboglinidae; Cir= Cirratulidae; Pec= Pectinariidae; Ter= Terebellidae; Spi= Spionidae; Cha= Chaetopteridae; Pol= Polynoidae; Sig= Sigalionidae; Gly= Flyceridae; Nep= Nephtyidae; Amp= Amphinomidae; Lum= Lumbrineridae; Eun= Eunicidae; Mal= Maldanidae; Cap= Capitellidae; Oph= Opheliidae; Orb= Orbiniidae. Idanthyrsus (Sbi) FauveSiopsis ( Fla)

1 00 Dodecaceria (Cir) 1 8 — Cirratulus (C\r) Sternaspis (Ste)

76 ------Owenia(Owe) Mesochaetopterus (Cha) Nep/ifys(Nep) Glycera (Gty) W196835 Eurythoe (Amp) rC ...... - Sipunculan 1 oo Paralepidonotus (Pol) 89 1 9 — Slgalion (Sig) Ceratonereia (Ner) Lumbrineris (Lum) — Eumce(Eun) — Lamellibrachia (Sib) ------Clitellate

68 - Amphitritlna© (Ter) 56 — Amphitritldca (Ter)

Piata (Ter)

S3 Euclymene(Ma\) Polyophthalmua (Oph) Amphiglena (Sab) ------Galeofaria {Ser) ------Maiacoceroa (Spi) Echiuran Turbellarian Nematode 89 of aciculae could not even be interpreted as an ancestral character, as several of the trees in my study suggest.

A 2000 study by Dalgren et al. examined the monophyly of the Chrysopetalidae and Hesionidae families using morphological characteristics and COI sequences. I utilized the sequences generated by Dalgren et al. in my analysis, and though I did not include morphological characters, my analysis supports their conclusions, that

Chrysopetalidae was not monophyletic, and that the genuses Microphthalmus and

Hesionides, do not belong in the Hesionidae family.

McHugh (2000) indicates that none of the studies examining polychaete relationships strongly conflict with the results of Rouse and Fauchald (1997) because none of the groupings that contradict Rouse and Fauchald are supported by bootstrap values >50. Low bootstrap values were an issue in my analysis as well. Jackknife support was rather higher, though in only one tree, the parsimony analysis of DNA sequences with the third position removed, was a grouping higher than family supported with a Jackknife value >50. This group included all polychaetes in the analysis with the exception of Dysponetus caecus, and the Jackknife value was only moderate (74).

More molecular data, or a more thorough complement of taxa, added to the analysis might prove enlightening as the molecular data to date has conflicted with much of the morphological data. The cytochrome b gene has proven to be a robust marker for examining the phylogenetic relationships among fish (Orrell 2000, Cantatore et al. 1994) because it is less conserved than COI and therefore better for examining more closely related species, though COI did seem to recover most family groupings in this study.

Brown et. al provided a fairly broad range of nuclear markers, but seldom more than one 90 species representing each family. If one of the species chosen to represent a family were found to be misclassified, it would cause considerable problems with their interpretation of their results. Their phylogenetic tree would likely be strengthened by a larger taxonomic sampling. 91

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VITA

Julie Beck Stubbs

Bom Albuquerque, New Mexico on 18 February 1977. Graduated from James Madison High School, San Antonio, Texas, in 1994. Earned a B.S. in Biology and a B.A. in Liberal Arts from the University of Texas at Austin, Austin, Texas, in May 1998. Worked as a laboratory assistant to Jonathan Allen at The Southwest Foundation of Biomedical Research 1998-1999. Entered the Master of Science program at the College of William & Mary, School of Marine Science in August, 1999.