OCS Study MMS 2001-078

Coastal Marine Institute

How Does Produced Water Cause a Reduction in the Genetic Diversity of Harpacticoid Copepods?

Final Report

U.S . Department of the Interior Cooperative Agreement Minerals 11Aanagement Service Coastal Marine Institute Gulf of Mexico OCS Region IR Louisiana State University

OCS Study MMS 2001-078

Coastal Marine Institute

How Does Produced Water Cause a Reduction in the Genetic Diversity of Harpacticoid Copepods?

Final Report

Authors

John W. Fleeger David W. Foltz Axayacatl Rocha-Olivares

September 2001

Prepared under MMS Contract 14-35-0001-30660-19949 by Coastal Studies Institute Louisiana State University Baton Rouge, Louisiana 70801

Published by

U.S. Department of the Interior Cooperative Agreement Minerals Management Service Coastal Marine Institute Gulf of Mexico OCS Region Louisiana State University

DISCLAIMER

This report was prepared under contract between the Minerals Management Service (MMS) and Louisiana State University and A&M College. This report has been technically reviewed by the MMS and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Service, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. It is, however, exempt from review and compliance with MMS editorial standards.

REPORT AVAILABILITY

Extra copies of the report may be obtained from the Public Information Office (Mail Stop 5034) at the following address:

U.S . Department of the Interior Minerals Management Service Gulf of Mexico OCS Region Public Information Office (MS 5034) 1201 Elmwood Park Boulevard New Orleans, LA 70123-2394

Telephone Number: (504) 736-2519 or 1-800-200-GULF

CITATION

Suggested citation :

Fleeger, J .W., D.W. Foltz, and A. Rocha-Olivares . 2001 . How does produced water cause a reduction in the genetic diversity of harpacticoid copepods? Final report . OCS Study MMS 2001-078 . U .S . Dept . of the Interior, Minerals Management Service, Gulf of Mexico OCS Region, New Orleans, Louisiana. x + 35 pp.

ACKNOWLEDGMENT

We thank John McCall, Markus Wetzel, Debra Dexter, Samuel Gomez, Ana Puello-Cruz, Mark Ohman and Annie Townsend for specimen collection or donation and Mary Martin, Deb Taranik, and Nannette Crochet for laboratory assistance .

ABOUT THE COVER

The third swimming leg of a specimen of Cletocamptus deitersi (genotyped as type II) collected near Port Fourchon, LA.

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TABLE OF CONTENTS

List of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

List of Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ., . . . . . ix

1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1

2 How Does Produced Water Cause a Reduction in the Genetic Diversity of Harpacticoid Copepods? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3

2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3

2.2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

2.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8

2.3 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12

3 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17

4 Figures and Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

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LIST OF FIGURES

Figure 2 .1 Multiple haplotype-specific PCR (MHS-PCR) used to genotype individual Cletocamptus deitersi based on COX-I amplification . A) A pair of non-specific primers is used as positive control and an additional pair of haplotype-specific primers yields amplicons of diagnostic size used for genotyping. B) Negative gel image of the results obtained with C. deitersi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Figure 2 .2 Clustal W DNA multiple alignment of part of the nuclear rDNA comprising the entire internal transcribed spacers (ITS 1 and ITS2) and the intervening 5.85 ribosomal RNA gene. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24

Figure 2 .3 DNA multiple alignment of representative sequences of the mitochondrial cytochrome oxidase I gene bounded by Folmer et al. (1994) LCOI and HCOI primers. 25

Figure 2 .4 Neighbor-Joining (NJ) phylogenetic reconstructions based on genetic distances of distinct mitochondrial cytochrome oxidase subunit I (COX-I, K81 of+I+G distances) and 16S ribosomal RNA (165, TIM+I+G distances) haplotypes of the harpacticoid copepods Cletocamptus deitersi, C. helobius, Coullana sp., and the calanoid copepod Calanus pacificus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26

Figure 2.5 Neighbor-Joining (NJ) phylogenetic reconstruction based on Jukes-Cantor genetic distances of distinct nuclear 5 .8S ribosomal RNA alleles of the harpacticoid copepods Cletocamptus deitersi, C. helobius, and the calanoid copepod Calanus pacifrcus . . . . . . 27

Figure 2 .6 . Percent survival of type I and II Cletocamptus deitersi for individuals exposed and unexposed (control) to saturated phenanthrene for 96 hours . . . . . . . . . . . . . . . . . . . . . . 28

Figure 2 .7 Percent survival of type I and II Cletocamptus deitersi for individuals exposed and unexposed (control) to a mix of heavy metals (see text for composition) for 96 hours. . 29

Figure 2.8 Survivorship curves of type I and II Cletocamptus deitersi in 96-hour exposures to heavy metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .30

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LIST OF TABLES

Table 2.1 Number of copepods (n), number of distinct mtDNA haplotypes or nuclear gene copies (h), and accession numbers of representative sequences submitted to Genbank (GB), of each copepod species genotyped by direct sequencing (COX-I, 165, n rDNA) or multiplex haplotype-specific PCR of the COX-I gene (MHS-PCR) . . . . . . . . . . . . . . . . 31

Table 2 .2 Mean pairwise genetic distances in the COX-1 (above diagonal) and 16S (below diagonal)a mitochondria) genes in four lineages of Cletocamptus deitersi (types I, II, IIIC, and HIM) and in C. helobius (CH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Table 2 .3 Mean uncorrected genetic distances (%) in the ITS 1, 5 .8S, and ITS2 genes regions in four lineages of Cletocamptus deitersi (types I, II, IIIC, and HIM) and in C. helobius (CH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .33

Table 2 .4 Parameters of the models of nucleotide substitution fit to mitochondria) and nuclear genes of C. deitersi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .34

Table 2 .5 . Ranges of divergence since common ancestor based on corrected sequence divergence and crustacean molecular clock calibrations . . . . . . . . . . . . . . . . . . . . . . . . . 35

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1 . ABSTRACT

Cryptic species (morphologically-similar but genetically-distinct sibling species) may contribute to observed reductions in genetic diversity at contaminated sites (a) if some ta}ca among the groups studied form a cryptic-species complex in which cryptic species co-occur at uncontaminated sites, and (b) if cryptic species exhibit different responses to contaminants such that differential mortality among cryptic species occurs at contaminated sites. We studied the common and wide-spread harpacticoid copepod Cletocamptus deitersi to determine if it is comprised of morphologically-similar species and if these cryptic species are sympatric. We also conducted toxicity bioassays with C. deitersi from two locations with both poly-nuclear aromatic hydrocarbon and metal contamination. Our molecular and limited morphological analyses suggest that at least four putative species of C. deitersi are found in North America and that two of these co-occur at two intensively-sampled locations. Furthermore, these molecular types display different toxic responses to heavy metals, suggesting different tolerances at contaminated sites . These data suggest losses of genetic diversity at contaminated sites may be associated with losses in species diversity due to a reduction in the number of cryptic species caused by contamination. Future studies using genetic diversity as a marker of contaminant effects should consider the possibility that cryptic species may contribute to losses in genetic diversity.

2 . How Does Produced Water Cause a Reduction in the Genetic Diversity of Harpacticoid Copepods?

2 .1 Introduction

Oil and gas exploration and production activities in the marine environment produce and release large quantities of organic, hydrophobic compounds and heavy metals . Unfortunately, the effects of these toxicants and their causal linkages with benthic and pelagic biota are difficult to identify and are frequently poorly understood . Research associated with the GOOMEX project (Kennicutt et al ., 1996; Peterson et al., 1996) identified an intriguing phenomenon : a loss of genetic diversity in meiobenthic copepods was found in close proximity to production platforms (Street and Montagna, 1996). The magnitude of this genetic change appears to be consistent with contamination gradients, and is equal to or greater than the response seen with several traditional indicators (e.g ., body burdens, biomarker activation, community structure and toxicity) based on species- and community-level attributes . Specifically, Street and Montagna (1996) analyzed the 16S rRNA gene of the mitochondrial genome in several meiobenthic harpacticoid copepod species by polymerise chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis . They found a reduction in genotypic (haplotype) diversity in harpacticoid species collected in close proximity to production platforms .

More generally, the effects of contaminants on genetic variation in marine organisms have been studied for about two decades (reviewed by Hebert and Luiker, 1996) . Previous studies of genetically-controlled protein variation (reviewed by Hummel and Patarnello, 1994) have produced inconsistent and even contradictory results in that exposure to contaminants (heavy metals have been most frequently studied) has led to increases and decreases in genetic diversity compared to unexposed populations. Genetic adaptation to contaminants may occur quickly under strong selective pressures imposed by moderate to high levels of contamination (Brown, 1976; Klerks and Weis, 1987; Klerks and Levinton, 1989a, 1989b; Klerks, 1990; Mulvey and Diamond, 1991 ; Posthuma and Van Straalen, 1993) and lead to increased tolerance of contaminants . Thus, the relationship between genetic structure and contaminants is likely to be complex, and cause and effect may be difficult to assign .

A loss of genetic diversity associated with studies such as Street and Montagna (1996) may have several causes . A reduction in genetic diversity may be the direct result of exposure to contamination via the process of genotypic selection . Selection may favor some genotypes/haplotypes over others causing genetic change. Sediment contamination is not a likely cause of selection for or against the rRNA gene used by Street and Montagna (1996) because its function is unrelated to any known detoxification mechanism. Linkage with other genes in the mitochondria) genome that may be under selection is presumably responsible for the loss in diversity of rRNA genotypes (the mitochondria) genome is completely non-recombining) if selection is the cause. Linked gene effects are not limited to the mitochondria) genome, and are potentially important for understanding the effects of contaminants on nuclear-encoded proteins (Gillespie and Guttman, 1993) . Heavy metals and poly-nuclear aromatic hydrocarbons (PAH) are elevated in close proximity to production platforms, and either or both could provide the selective pressure necessary to reduce genetic diversity . Analysis of sediments along

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distance gradients around the GOOMEX platforms suggests, however, that heavy metals are more likely to occur at concentrations that may cause mortality or chronic reductions in reproduction than are PAH (Peterson et al ., 1996). In addition, a population that is reduced in abundance by exposure to a contaminant may experience a population bottleneck . If so, genetic diversity may be lost through the process of genetic drift. Street et al . (1998) conducted a laboratory selection experiment on Nitocra lacustris, an estuarine harpacticoid copepod. They found that under strong selection from phenanthrene (a compound frequently used as a model PAH), loss of genetic diversity of the 16S rRNA gene occurred . They concluded that the observed reduction was not associated with resulting small population size, and their work suggests that selection may cause the reduction in genetic diversity associated with Street and Montagna's observation. Therefore genetic diversity has been proposed as a new marker for contaminant effects.

Alternatively, it is possible that some of the mitochondria) DNA genotypes found in the GOOMEX study represent interspecific variation in the form of cryptic species rather than within- species variants . Cryptic species are morphologically-similar but genetically-distinct sibling species . Such complexes are known to occur within numerous cosmopolitan and previously well-studied (based on classical morphologically-based systematics) marine invertebrate taxa (Knowlton, 1993). Furthermore, Warwick and Clarke (1995) point out that many colonizing species frequently found in polluted habitats are actually complexes of cryptic species . Although the possible existence of cryptic species has been mentioned in some previous studies of pollution effects (e.g ., Pearson and Rosenberg, 1978), few investigators have explicitly examined this possibility . Cryptic species have been found to occur in widely-studied pollution indicator species (e.g ., Grassle and Grassle, 1976; Lobel et al ., 1990), and in harpacticoids (Bergmans, 1979 ; Battaglia, 1982; Ganz and Burton, 1995) and other meiofaunal taxa (Todaro et al., 1996). If cryptic species with differing levels of pollution tolerance occur among harpacticoids, the apparent reduction in genetic diversity near platforms could represent a loss of species diversity rather than genotypic selection . The frequency with which cryptic species complexes are being discovered with molecular techniques suggests that this explanation is possible, and that caution should be applied in future work that utilizes genetic diversity as a marker for contaminant effects .

Meiobenthic harpacticoids are small crustaceans that range from about 0.4 - 1 .5 mm in length and average about 1-3 [ig in dry body mass as adults . This small size may inhibit the detection of cryptic species (Costello et al ., 1996). Harpacticoid taxonomy is based on morphological characters that must be visualized with high-power microscopy after whole-body dissection . Furthermore, closely-related species that require dissection for identification may be sympatric among harpacticoids (Gee, 1988). The advent of sophisticated optics (e.g., differential interference microscopy) now allows the use of detailed observation of body ornamentation and traditional features important to crustacean taxonomy such as mouthpart structure that has produced extremely rigorous taxonomic criteria (Huys et al ., 1996). Modern taxonomic standards suggest that apparent cryptic species can be resolved morphologically (Lee and Huys, 1999), and many cryptic species are likely to become pseudo-cryptic species complexes. However the dissection required for detailed morphological observation destroys tissue, precluding DNA extraction and intense taxonomic analysis on the same individuals. Thus,

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individuals for which DNA extraction is to be conducted can only be examined cursorily at low magnification; cryptic or closely-related pseudo-cryptic species may be missed. Large numbers of specimens (over 300 per species) were processed for genetic analysis by Street and Montagna (1996), increasing the chance of encounters with rare species and thus the chance that mis- identifications . If species mis-identifications are common, resulting estimates of genetic diversity will be inflated . If species diversity is reduced near contaminated areas, an apparent reduction in genotypic diversity will result . Studies using RFLP may be especially prone to this criticism, because restriction enzyme digestion samples only a portion of the nucleotide site diversity present in a particular gene region and may underestimate the true amount of divergence between sequences (DeGiorgi et al ., 1990) . Also, sequence data allow comparison to a large body of previous studies on molecular divergence of invertebrate taxa at widely-varying taxonomic levels for numerous mitochondrial genes . For these reasons, in the present research project we used direct sequencing of PCR products as our primary molecular technique .

A key question regarding species complexes has to do with tolerances to contaminants . If cryptic species have different tolerances, then a community exposed to this contaminant may selectively lose species in contaminated areas . Previous work has suggested that cryptic species have unique tolerances to contaminants (Hogg et al., 1998; Sturmbauer et al., 1999; Linke-Gamenick et al ., 2000), but no similar study has been conducted with harpacticoids, although (Kovatch et al., 2000) reports that intra-specific variation in tolerance to contaminants exists .

Cletocamptus deitersi (Richard 1897) is a canthocamptid meiobenthic copepod with a highly cosmopolitan distribution reported from inland brines (seeps, streams, and lakes), as well as coastal estuaries and mangroves in all continents except Europe (Mielke 2000) . This species has been shown to be morphologically polymorphic within and between populations (e .g ., Fleeger 1980; Mielke 2000). For example, Fleeger (1980) reported consistent variation in setal patterns of the third and fourth swimming legs among specimens from a single location ; these differences could not be discerned without dissections. C. deitersi is similar to many harpacticoids in that it lacks a planktonic dispersing larval stage (free-living larval stages develop in sediments), and adults and juveniles are capable of short-distance (meter-scale) dispersal through the water column (Sun and Fleeger 1994). This species is unusually tolerant of salinity variation (Simpson et al ., 1998; Dexter, 1995) and has been found to be highly resistant to hydrocarbon contaminants (Cayman et al ., 1997) and disturbance (DeLaune et al ., 1984) . The geographical isolation of inland brines suggests that long-distance migration and colonization events are rare and that gene flow should be limited among inland populations. Morphological variability and potential for geographic differentiation make C. deitersi a good candidate for analyses of geographic genetic variation aimed at determining the existence of cryptic (or pseudo-cryptic) species.

Using a multilocus genetic approach and limited morphological observations, we analyzed Cletocamptus deitersi from four localities in North America to determine if separate populations are represented by morphologically and/or genetically differentiated species. Two sites were intensively sampled to determine if cryptic species live in sympatry. We also obtained genetic data from C. helobius, a readily identifiable and morphologically differentiated congeneric species, to establish a reference level of indisputably interspecific genetic

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differentiation. An undescribed harpacticoid (Canuellidae : Coullana sp.) and a planktonic calanoid (Calanus pacificus Brodsky 1948) were used as outgroups in copepod phylogenetic reconstructions. In addition, tests for tolerance to PAH and metal exposure were conducted on C. deitersi from two locations .

2.2 Materials and Methods

Copepod samples and DNA extraction

Samples were collected at an inland brine seep near Jackson, Alabama (June 1998, 31 °23'N 87°53'V) and at two locations on the coast of Louisiana (Port Fourchon, December 1998, 29°05 .4' 90°05 .8'W and Cocodrie, January 1999, 29°15 .2'N, 90°39 .8'W). Cletocamptus deitersi was also obtained from laboratory cultures established from collections made in the Salton Sea, California (July 1997, 33°13'N 115°52'W), and at the mouth of the "Estero del Yugo" estuary (January 2000, 23°18 .14'N 106°29'W), in Mazatlan, Sinaloa, Mexico . These environments are very different . For example, salinity at the Alabama brine seep reaches 45%0 close to the source, and the stream is located in a bottomland forest subject to annual flood and drought cycles, some 83 km from the coast. The Louisiana salt marsh is tidally flooded and salinity ranges from 0-28 %o. The Salton Sea, where salinity has recently increased to > 45 %o, was created in 1905 and is more than 100 km inland from the Pacific Ocean and from the Colorado River Delta in the upper Gulf of California.

Field-collected copepods were removed from sieved sediment and fixed in 95% ethanol. All harpacticoids were carefully identified by one of us (J.W .F.) . We extracted total genomic DNA from individual copepods based on Schizas et al . (1997) . Ethanol-preserved specimens of Calanus pacifrcus from southern California (32°25'N 119°58'W) were provided and identified by Annie Townsend (Scripps Institution of Oceanography). DNA extraction for these copepods was done by standard proteinase-K digestion and phenol-chloroform-isoamyl organic extractions, followed by ethanol precipitation .

PCR amplification and sequencing

All copepods were subject to an initial PCR amplification of the mitochondria) DNA (mtDNA) cytochrome oxidase subunit I (COX-I) gene using universal primers (Folmer et al . 1994, 0 .72 mM total dNTP, 2 .5 MM MgC12, 1 unit of Promega Taq in manufacturer's "A" buffer ; Perkin Elmer 480 thermal cycling was l Omin at 95°C, followed by 40 cycles of 15s 95°C; 30s 40°C ; 60s 72°C, and a final extension time of Smin at 72°C) . We sequenced two additional gene regions in copepods representative of the major lineages identified on the basis of COX-I sequences (except for the Mexican copepods for which all gene regions were sequenced in all individuals) : (1) part of the mitochondria) large subunit ribosomal DNA (LSU rDNA 16S), amplified using Palumbi's (1996) 16SAR and 16SBR universal primers, and (2) part of the nuclear rDNA, comprising the entire ITS 1, ITS2 and the intervening 5.85 rDNA, amplified using Heath's (1995) ITS primers (cf. Table 1 in Heath et al. 1995). We designed Cletocamptus-specific internal primers for re-amplification of weak initial reactions (COX-I : CLEintF TTTTGATTTTCTYATCCAGC and CLEintR CCTAGTAANGARGAAATTCC), for

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amplification when universal primers failed (165: 16SciF YTAAGGTAGCATAGTAA and 16SciR TTAATTCAACATCGANGTC), and for sequencing of long PCR products (nuclear rDNA: 5.8SciF GGGGTCGATGAAGAACG and 5 .8SciR CCCTGAGCCAGACATGG). Target PCR products were electrophoresed, excised from gels (2% agarose), purified using columns (QiaQuick, Promega), and sequenced using Applied BioSystems ABI-Prism Big-Dye terminator chemistry (Perkin-Elmer) in scaled-down reactions and run on an ABI-377 Gene Analyzer . Representative nucleotide sequences have been submitted to GenBank (AF315001-AF315033).

Multiplex haplorype-specific PCR

We designed a rapid, cost-effective method of large-scale genotype screening of individual copepods, using a multiplex haplotype-specific PCR reaction (MHS-PCR, Rocha-Olivares 1998), to assign copepods to one of the first two COX-I mtDNA major lineages . Initial LCOI-HCOI amplicons were used as template of the MHS-PCR, in which the size of the products, visualized in an agarose gel, could be used for PCR-based genotyping . Multiplexing with nested primers produced a positive control amplicon in all haplotypes and a second haplotype-specific amplicon, whose size would be diagnostic (Fig . 2 .1) . All MHS-PCR batches included experimental positive and negative controls . We sequenced a number of copepods genotyped with MHS-PCR to verify the accuracy of the method.

Data analyses

Orthologous nucleotide sequences were aligned with the Clustal W algorithm (Thompson et al . 1994) with default parameters . In mitochondrial and nuclear rDNAs, inferred indels were further adjusted by eye to minimize those separated by one or very few bases. Aligned nucleotide sequences, with the exception of ITS regions, were used to reconstruct gene trees using maximum parsimony (MP) and neighbor joining (NJ) with the program PAUP* 4.Ob4a (Swofford 1998). NJ searches were made with distances computed using a best-fit model of nucleotide substitution . Models were fit to each data set (COX-I, 16S, and 5 .85) under a maximum likelihood framework and tested for significance (overall a = 0.05 after Bonferoni correction) with likelihood ratio tests (LRT) using the program Modeltest 3 .04 (Posada and Crandall 1998). Branch support was assessed by 1000 non-parametric bootstrap replicates . Phylogenetic analyses of the mtDNA 16S rDNA were performed on the data set bounded by 16SciF-R primers (313 base pairs [bp]), not the entire 16SAR-BR alignment (466 bp) that was incomplete for the Mexican C. deitersi and C. helobius . Competing phylogenetic hypotheses and the existence of a molecular clock were tested under a likelihood framework with LRTs. The program Seq-Gen (Rambaut and Grassly 1997) was used for parametric bootstrapping of datasets . Rates of synonymous and non-synonymous susbstitutions were computed following Yang and Nielsen (1998) with the program PAML 3.Ob (Yang 1997).

Morphological-genetic comparisons

The number of inner setae on the distal segment of the third swimming-leg exopod of both sexes is known to differ within and among C. deitersi populations (Fleeger 1980) and in related species (Mielke 2000). To determine if morphological and genetic patterns correlate in

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C. deitersi, 51 specimens from Louisiana and 23 from Alabama were examined morphologically and genetically . The third leg was removed with a fine probe, mounted on a microscope slide, and examined with phase microscopy . The remaining copepod tissue was used for DNA extraction and genotyping by MHS-PCR. Specimens were given an identification number upon dissection, and genetic analysis was conducted without knowledge of their origin . In addition, the third leg exopod of several specimens from California and Mexico was examined and compared to specimens from the Gulf of Mexico .

Toxicity Tests

Phenanthrene and metal toxicity tests were conducted on C. deitersi collected from Port Fourchon, LA and from the Jackson, AL brine seep from November, 1999 - April, 2000 . Copepods at both sites were collected by skimming the upper 1 cm of surficial sediments by hand. Collected sediments were placed in buckets and water from the collection site was added. Buckets were transported to Baton Rouge where the overlying water was replaced with 25%0 artificial seawater, and the green alga Selanastrum capricornicutum was added as a food source . After a five-day acclimation period at room temperature, copepods were removed from the sediment surface and overlying water by aspiration and sorted under a dissection microscope to be available for bioassays . Bioassays measured acute toxicity . C. deitersi was exposed to contaminants by aqueous exposure in 20 ml of 25%o artificial seawater in 35X50 mm crystallizing dishes without food . During the experiments, copepods were maintained in a temperature-controlled incubator (at 25° C) in constant darkness . Bioassays with phenanthrene were conducted in triplicate with 10 C. deitersi per dish under saturated conditions (about 1200 ~ig phenanthrene/1) in 1 g/1 acetone . The number of living C. deitersi after 96 h were tallied . Metal toxicity was tested with a mixture of three metals based on proportions representative of produced water (Zn 100: Pb 10 : Cd 0.5) . After a range finding test, 20 C. deitersi per dish were exposed to a solution of 6249 ~Lg Zn/l, 685 ~Lg Pb/1 and 62 ~Lg Cd/1 (verified by ICAP). The number of living C. deitersi in eight replicate dishes (and two control dishes) were tallied every three h for 96 h. Dead copepods from both experiments were placed in 95% ethanol and later genotyped using the multiplex haplotype-specific PCR reaction to assign individuals to a COX-I mtDNA major lineage . C. deitersi survivorship measured in toxicity tests after 96 h in each lineage was analyzed by pooling all replicates and utilizing goodness of fit tests . These tests include the z test and Fisher's Exact Test and were used to examine differences in survivorship frequencies between control and exposed populations and between type I and type II copepods (expected frequencies were equality) . Sigma Stat software was used . Finally, the Cox-Mantel test was used to compare survivorship curves generated by types I and II individuals exposed to metals using Statistica software.

2.3 Results

Genorypic variability

A total of 135 copepods (121 Cletocamptus deitersi, 7 C. helobius, 2 Coullana sp., and 5 Calanus pacificus) was initially sequenced for one or more gene regions. The number of distinct COX-I haplotypes per locality ranged from one (Mazatlan) to 10 (Alabama) (Table 2.1) . COX-I

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sequences fell in one of three major types, which will be hitherto referred to as "major types" or "major lineages" . Type I was found among copepods from Alabama, type II predominantly among organisms from Louisiana but also in Alabama, whereas type III was exclusively present in all copepods from California (IIIC) and Mazatlan (IIIM) . A small fraction of copepods from Louisiana also harbored type I sequences.

Of all the C.deitersi genotyped of types I and II (sequencing + MHS-PCR, total N = 240), 11 (7.7%) from Louisiana harbored type I and 132 (92 .3%) type II COX-I sequences . Conversely, 90 (92 .8%) C. deitersi from Alabama had type I whereas only seven (7.2%) had type II . All COX-I sequences obtained from 15 haphazardly selected copepods genotyped via MHS-PCR (both type I and type II) corroborated the identifications obtained with the PCR-based method. Eleven distinct 16S sequences were found among 47 C. deitersi analyzed (Table 2 .1) . Overall the 16S rDNA followed the same pattern as the COX-I, in that sequences fell in major types analogous to those identified earlier.

We obtained nuclear rDNA sequences from 23 Cletocamptus deitersi, four C. helobius and one Calanus pacificus (Table 2 .1) . The nuclear gene regions showed different levels of variation, ITS being the highest and 5 .85 the lowest. Except for the Mexican copepods that shared two distinct ITS 1 alleles but only one ITS2, the rest of copepods had the same number of ITS 1 and ITS2 distinct alleles . In sharp contrast, the 5 .8S rDNA showed very low variability with only 4 distinct alleles among the 23 C. deitersi and two among the four C. helobius (Table 2.1) . However, no nuclear alleles were shared between the two congeneric species . In both ITS regions, alleles assorted into three extremely divergent groups, each matching the major mitochondrial types (I, II, and III) previously identified on the basis of the mtDNA sequences . On the other hand, one of the four 5 .85 alleles was shared by individuals with different mtDNA types, notwithstanding the fact that in each organism the same 5 .8S allele was flanked by drastically different ITS alleles falling in the divergent groups described above .

Intra- and interspecifrc levels ofgenetic differentiation

Levels of genetic differentiation within each major type of C. deitersi were small and typical of those found among conspecific organisms, leading to the assumption that each type represents a putative species . Mitochondrial genes were less variable (0.2% to 1 .7%, diagonal of Table 2 .2) than non-coding nuclear ITS regions (2 .7 - 8.5% in faster-evolving ITS 1 and 0 .7 -2.6% in ITS2, Table 2 .3) . As expected, intra-lineage variation in the slowly-evolving 5 .85 rRNA gene was virtually absent . Although copepods from Mazatlan are unequivocally most closely related to those from California, they are quite differentiated in both mitochondria) (11% in COX-I and 5% in 16S rRNA) and nuclear (12% ITS 1 and 2.8% ITS2) genes (Table 2 .3) . Thus, four species may be represented within our three major lineages of C. deitersi.

Further evidence that our nominal C. deitersi is composed of more than one species comes from a comparison with its morphologically differentiable congener C. helobius. Organisms of the three major lineages of C. deitersi were as genetically differentiated from each other as they were from C. helobius . Corrected levels of mtDNA sequence divergence among the three major lineages were at least one if not two orders of magnitude higher than intra-lineage

9

divergences, values ranging from 48 .7 to 88.3% (20.8 - 26 .4% uncorrected) in the COX-I gene and from 29 .4 to 82.5% (20.3 - 36.0% uncorrected) in the 16S rRNA (Table 2.2). The corresponding values of indisputably interspecific (i .e . C. deitersi vs . C. helobius) differentiation were 71 .9 - 91 .7% (23.7-26.8% uncorrected) for the COX-I gene and 63 .1 - 81 .4% (31 .7-35 .8% uncorrected) for the 16S rRNA (Table 2.2). Data from nuclear genes were consistent with mitochondrial results. In the case of the widely divergent non-coding ITS regions, nucleotide sequences of alleles from different lineages are so different that their alignment is dubious (Fig . 2 .2). Conspicuous size differences (ITS I lengths in each type were 359-376 in I, 292-300 by in II, 288-289 in IIIC and 284 in HIM ; whereas ITS2 lengths were 221-228 in I, 204-206 in II, 215-219 in IIIC, and 216 in HIM) and a large degree of sequence differentiation are indicative of a thorough randomization of the nucleotide sequences via mutation and recombination, such that statements of sequence homology can no longer be established confidently by alignment . Thus no quantitative appraisal of divergence is given (Table 2 .3); instead, we present the imposed multiple alignment as qualitative evidence of the extent of differentiation (Fig . 2 .2) . The alignment shows that aside from the well conserved 18S and 5 .8S rDNAs, the alignability of the sequences decays rapidly in the ITS regions but not to the same extent among the lineages of Cletocamptus since types II and III share more structural elements . Finally, in the slowly evolving 5.85 rRNA gene, mean levels of intraspecific variation in the nominal C. deitersi were smaller (0.6-1 .5%) than the interspecific (3 .7 .6%).

Molecular evolution and phylogenetic analyses

The 16S amplicons in C. deitersi were either 462 by (type I) or 467-468 by (types II and III) in length . The amplified mitochondrial COX-I fragment was 658 by long in all organisms. Almost a third of the nucleotides were variable (228 total variable sites, ratio of variable 1 :2:3 codon positions was 43 :8 :177). Inferred COX-I amino acid sequences revealed that all nucleotide substitutions within types I and II were silent (details not shown). Most amino acid replacements were conservative with respect to hydrophobicity (20 out of 23). Rates of synonymous and non-synonymous substitutions along the lineages leading to types I, II, and III were ds = 11 .19 dN = 0.04 (I), ds = 1 .56 dN = 0.006 (II), and ds = 1 .87 dN = 0.007 (III). Different models of nucleotide substitution were fit to each data set. For the COX-I gene, the best-fit model was Kimura 81 (Kimura 1981) with unequal base frequencies corrected for proportion of invariable sites (I) and for rate heterogeneity among sites (G) with a Gamma distribution (Yang 1993). For the 16S rRNA gene, the best-fit model was TIM (TIM+I+G Rodriguez et al . 1990); whereas for the 5 .85 rRNA gene, the best-fit model was Jukes-Cantor (Jukes and Cantor 1969) (Table 2 .4). The COX-I (Fig . 2 .3) and 16S rDNA alignments including all taxa had, respectively, 309 and 215 variable sites (treating indels as missing data) of which 275 and 180 were parsimony-informative . The strict consensus tree of the eight COX-I most-parsimonious reconstructions obtained via branch-and-bound (length = 670 steps, CI = 0.72, RI = 0.91) was completely congruent with the NJ tree (Fig . 2.4) . For the 165, the NJ tree differed from the three branch-and-bound most-parsimonious trees (length = 419 steps, CI = 0.79, RI = 0.87) in that Cletocamptus was not monophyletic . Instead, C. helobius was sister to type I sequences and both were sister group to the rest of C. deitersi sequences (Fig . 2.4). A NJ tree constrained by monophyly of C. deitersi was not significantly different from the unconstrained tree (LRT, S = 2.48, p = 0.43) . In the MP analyses, the monophyly of C. deitersi had a non-parametric bootstrap

10

branch support of 64%. Trees featured extensive unresolved polytomies within each major type (I, II, and IIIC) but were otherwise completely resolved . Both mtDNA gene-trees featured long branches, in excess of 30% corrected sequence divergence, separating the C. deitersi types I, II, and III, and the other congeneric and non-congeneric species. All bootstrap replicates recovered the monophyly of the three major mitochondria) types in both genes. Within type III sequences, the Californian haplotypes formed a reciprocally monophyletic assemblage relative to the Mexican with very high bootstrap support . Despite overall topological congruence of the gene trees, considering that a 16S NJ tree constrained by the monophyly of C. deitersi is not significantly worse than the one shown (Fig 2 .4), the two mtDNA genes were not equally powerful in resolving the relationships of the major lineages within Cletocamptus. For example, the sister-taxon relationship between types II and III was very strongly supported by the NJ analysis of the 16S data set but less so by the corresponding MP analysis or by the COX-I data set (bootstrap 55 - 57%). Monophyly of Cletocamptus and Harpacticoida, however, was very well supported by the mtDNA data (bootstrap > 93%) . The nuclear 5 .8S rDNA alignment was 166 by long with 32 variable sites, of which only 6 were parsimony-informative . An exhaustive search produced a single most parsimonious reconstruction (length = 33, CI = 1 .0, RI = 1 .0) and the NJ reconstruction was completely congruent (Fig . 2 .5) . Unlike the mitochondria) gene trees, the nuclear 5 .8S rDNA proved more powerful for supporting the monophyly of C. deitersi (Fig. 2.5) . Variation within C. deitersi was too small to gain any insight into the phylogenetic relationships among mitochondria) lineages . Branch-and-bound maximum likelihood analyses using best-fit models of substitution and one representative sequence of each Cletocamptus lineage, including C. helobius, indicated that the existence of a molecular clock could not be rejected in the mtDNA genes (COX-I : x2 = 2.89, p = 0 .41 and 165 : x2= 6.04, p = 0.11) .

Morphological-genetic comparisons

All specimens of C. deitersi examined in this study had indistinguishable body plans (i.e ., body length and width, rostrum shape, caudal rami shape and leg segmentation were essentially identical) and none could be distinguished by inspection with a dissection microscope and without examining dissected body parts such as swimming legs . Small variations in leg setation and body ornamentation have been described in species closely related to C. deitersi . For example, the number of inner setae on the distal segment of the third leg exopod of C. axi (one seta) differs from that of C. schmidti (two setae) in both sexes (Mielke 2000). Based on examination of the third leg exopod after dissection, all specimens from Louisiana, California and Mexico had one inner seta, whereas those from Alabama had two on the distal segment. MHS-PCR was successful in 70 of the 74 specimens in which the third leg was examined . Copepods with one inner seta on the third leg exopod distal segment, all from Louisiana, were of mtDNA COX-I major type II whereas those with two inner setae, all from Alabama, were of type I .

Toxicity Tests

C. deitersi has previously been shown to be very tolerant to PAH contamination (Cayman et al ., 1997), and our research with two major lineages suggests that both are highly tolerant to phenanthrene (Fig . 2.6). About 80% of type I and type II individuals survived exposure to

11

phenanthrene under saturated conditions for 96 h in aqueous solution. Goodness of fit tests demonstrated no difference in survivorship between exposed and unexposed individuals of both lineages (type I, z = 0.8 and p = 0 .424 and type II, z = 0 .716 and p = 0 .474) and between type I and type II (Fisher's Exact Test, p = 0.759) individuals .

The two C. deitersi lineages tested were also both highly tolerant to metals ; however, survivorship patterns differed among lineages (Figs. 2.7 and 2 .8). Survivorship in both lineages to metal exposure was high until about 70 h. After 70 h, survivorship in type I individuals (all collected from the AL brine seep) decreased markedly to slightly less than 50% of the total while survivorship in the type II individuals (all collected from a LA salt marsh) remained above 75%. Goodness of fit tests suggested that survivorship after 96 h in type II copepods did not differ between metal exposed and unexposed individuals (z = 1 .134, p = 0.257), while survivorship in metal exposed populations of lineage I was significantly decreased by metal exposure (z = 5.021, p < 0.001) . Type I and type II survivorship differed after 96 h; fewer type I individuals survived (Fisher's Exact Test, p < 0.001). Furthermore, the Cox-Mantel demonstrated that survival pattern of type I and II lineages varied over time ; the shape of the survivorship curves differed significantly (p < 0.001) (Fig . 2 .8) .

2.3 Discussion

For cryptic species to contribute to losses in genetic diversity associated with contaminated sites, three observations must be true . Firstly, some taxa among the groups studied must form a cryptic-species complex or be easily mis-identified (to act as cryptic species) . One of the goals of this research project was to determine if a common harpacticoid copepod frequently found at contaminated sites is comprised of morphologically-similar species . The harpacticoid copepod Cletocamptus deitersi was selected for intensive study because it is known to be morphologically variable and highly cosmopolitan in distribution . In addition, C. deitersi in Louisiana consistently responds favorably to contamination by increasing in absolute and relative abundance compared to other harpacticoid copepods (Cayman et al ., 1997). Secondly, cryptic species must co-occur at uncontaminated sites . Our research examined this possibility with C. deitersi at two locations . Thirdly, cryptic species must exhibit different responses to contaminants such that differential mortality occurs at contaminated sites . We examined this possibility by conducting toxicity bioassays with C. deitersi from two locations with both PAH and metal contamination. If these three conditions are met, it is possible that losses in genetic diversity at contaminated sites may be associated with losses in species diversity due to a reduction in the number of cryptic species at contaminated sites .

Our genetic analyses and limited morphological comparisons strongly suggest that C. deitersi in North America is not mono-specific (see Rocha-Olivares et al ., 2001 for a detailed discussion). Our analysis identified four putative species among specimens obtained from four widely-separated locations; additional collections may well yield more species. This conclusion is strongly supported by three lines of evidence . Firstly, molecular sequences revealed four extremely differentiated lineages with unalignable nuclear intergenic spacers and mitochondrial uncorrected divergences reaching 25% (cytochrome oxidase) and 36% (16S rDNA). These levels of divergence are greater than those reported previously for congeneric species in diverse

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invertebrate taxa including crustaceans . Secondly, the intraspecific divergence among the lineages of C. deitersi equaled or exceeded the corresponding divergence from a known congener (C. helobius) with pronounced morphological divergence from the C. deitersi body plan . Finally, although all specimens of C. deitersi examined in this study had body plans (i .e ., body length and width, rostrum shape, caudal rami shape and leg segmentation were essentially identical) that could not be distinguished at magnifications attainable with a dissection microscope, small differences in leg setation were detected between molecular lineages . Specimens from Louisiana, California and Mexico all had one inner seta on the distal segment of the third leg exopod, whereas those from Alabama had two at the same location . Furthermore, MHS-PCR was successful in genotyping 70 specimens in which setation from one leg was also examined . Copepods with one inner seta on the third leg exopod, all from Louisiana, were of mtDNA COX-I major type II whereas those with two inner setae, all from Alabama, were of type I . Even though leg setation could be environmentally determined (e.g., by higher salinity at the brine stream), Fleeger (1980) found that both third-leg morphotypes co-occur in Louisiana, suggesting that leg setation is not a non-genetic response to environmental differences between coastal and brine-seep habitats . This suggests that setation is not an rapidly evolving, adaptive character under strong habitat-specific selection. Work in progress (Gomez et al., unpublished) has identified differences in body ornamentation among all four putative species that are consistent with separate-species status using morphological criteria currently applied in harpacticoid taxonomy, and plans have been formulated to publish formal descriptions .

Based on our molecular data, two species within the nominal C. deitersi co-occurred at each of two intensively-studied field sites . Overall, 11 (7.7%) of the specimens we examined from Louisiana harbored type I and 132 (92 .3%) type II COX-I sequences. Conversely, 90 (92 .8%) C. deitersi from the Alabama brine seep were of type I whereas seven (7.2%) were of type II . Similarly Fleeger (1980) found two morphological patterns in a collection of C. deitersi from Louisiana corresponding to type I and type II designations, suggesting that co-existence of sympatric species may occur over long periods of time. If a study of genetic structure in C. deitersi using RFLP was conducted from either site and if a large number of individuals was processed for genetic analysis without examination of relevant morphological features (e.g . the third leg), both species would likely be included and coded as a single species.

To test how such a mis-identification would influence RFLP results with C. deitersi, restriction sites for 186 commercially-available restriction enzymes in the 460 by region of the 16S gene were inferred from the primary sequence data for type I and II C. deitersi . The number of restriction enzymes surveyed exceeds the number employed in a typical screening experiment by 10-20 times . This large number, although impractical for use with actual PCR products, was used to reduce the sampling variation inherent in using a small number of restriction enzymes to assay restriction site differences . Types I and II differed from each other at 36% of the restriction sites found in one or both types. The corresponding frequency of within-type restriction-site differences was 2%. We conclude that the rare within-type restriction-site polymorphisms have a greater than 80% chance of going undetected in routine laboratory surveys employing 10 different restriction enzymes, whereas the more abundant between-type polymorphisms have a less than 1 % chance of going undetected under similar conditions. Therefore, if both species had been present in a collection of C. deitersi analyzed

13

using only RFLP techniques, the apparent genetic diversity within a "single" taxon would very likely have been inflated .

Our research suggests that type I and type II C. deitersi from two locations have similar tolerances to poly-cyclic aromatic hydrocarbons but different tolerances to heavy metals . The results of 96-h bioassays reveal that both type I and type II are very resistant to phenanthrene . Mortality in control populations did not differ from those exposed to concentrations of phenanthrene at the limit of solubility in aqueous solution. C. deitersi from Louisiana appears to be more tolerant than other estuarine species . Phenanthrene causes mortality at sediment concentrations below saturation in other benthic harpacticoids (Lotufo and Fleeger, 1997; Lotufo, 1997), and Carman et al . (1995 ; 1997) found that C. deitersi survives diesel contamination when most other harpacticoids suffer severe mortality . Type I and type II C. deitersi are also both relatively tolerant to a mix of Zn, Pb and Cd (at 6249 ~tg Zn/1, 685 ~Lg Pb/1 and 62 ~Lg Cd/l, based on ratios present in produced water) . Mortality in exposed groups was significantly greater than in controls for both species although mortalities were not high enough to calculate an LTSO value . However, significant differences between type I and type II were identified ; type I (from Alabama) was found to be more tolerant than type II (from Louisiana) . Metal resistance often varies with exposure history within species (Klerks and Levinton, 1989b), but inter-specific differences may be associated with the ability to sequester metals (Wallace and Lopez, 1997). Our data suggest that type I and type II would be impacted differently if exposed to metals and that there can be a metal exposure regime in which type II is eliminated from a contaminated site while type II survives .

The large observed genetic differentiation among the lineages of C. deitersi suggests long times since initial divergence . Assuming that the rate of molecular evolution in the COX-I gene calibrated in other crustaceans can be applied to C. deitersi, copepods from the Salton Sea and Mazatlan appear to have shared a common ancestor between the late Miocene and early Pliocene. The molecular ancestor of lineages II and III dates back to the early and middle Miocene and the oldest divergence between lineages I and II and lineage III to a time between the late Eocene and early Miocene (Table 2.5). These dates are in close agreement with the fossil record . Fossilized specimens assignable to Cletocamptus have been found in sedimentary deposits dating to the middle and late Miocene (Palmer 1960). The fossils are similar to an extant species, C. albuquerquensis (Herrick), but identifications cannot be confirmed due to a lack of preservation of key characters . However, close resemblance between the fossil and recent Cletocamptus suggests a morphological conservatism that has lasted for more than ten million years, a time-scale comparable to the one suggested by molecular data on cryptic species of C. deitersi . This long time of divergence, particularly if much of the time since divergence of lineages was spent in contrasting environments, could facilitate the development of differences in tolerances between morphologically-cryptic species. Furthermore, we do not know how common this kind of morphological stasis is among harpacticoids or other meiofaunal taxa . If it is common, the frequency of cryptic species may be higher in meiofauna than in other groups such as small macrofauna . Although it seems unlikely that true cryptic harpacticoids species exist, it is possible that many collections will contain close relatives that are difficult if not impossible to separate without dissection . Gee (1988) found closely-related harpacticoids in sympatry in the Celtic Sea, however no systematic survey has been conducted to determine how often close

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relatives co-occur in marine habitats . Furthermore, close relatives of common species may be rare within a collection further decreasing the likelihood of detection. Investigators employing harpacticoids for genetic analysis in the future may wish to consider taking the extra precaution of dissecting and archiving a single swimming leg (as was done in this study) to allow limited morphological comparisons among individuals used in genetic analysis . In addition, DNA sequence data offer many advantages over RFLP, and sequencing is replacing RFLP as the mainstay of genetic studies as sequencing cost decreases and the availability of instrumentation increases. Sequence data facilitate the identification of cryptic species or mis-identified individuals by examining all of the nucleotide diversity of the amplified gene regions .

Our research suggests that cryptic species may contribute to the losses of genetic diversity at contaminated sites . This potential reduction occurs by a loss of species at contaminated sites, and a reduction in species diversity is a generally acknowledged consequence of contamination in benthic communities (Peterson et al ., 1996). A loss of genetic diversity associated with a reduction in species diversity at contaminated sites is fundamentally different than adaptation-related changes in genetic diversity within species and has an ecological rather than an evolutionary basis. If genetic diversity is used as a marker for contamination effects in the future, the possible contributions of cryptic species should be considered .

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21

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22

MHS-PCR

DNA template (CaX- I gene}

~ AmPlicons : Positive control Haplotype-specific 1 Haplotype-specific 2

Legend : --> non-specific primer -o- haplotype-specific primer

1 Now +r 1000 by

750 by

500 by 00, 400 b fait--fait--

300 by

200 by

Figure 2 .1 Multiple haplotype-specific PCR (MHS-PCR) used to genotype individual Cletocamptus deitersi based on COX-I amplification. A) A pair of non-specific primers is used as positive control and an additional pair of haplotype-specific primers yields amplicons of diagnostic size used for genotyping . B) Negative gel image of the results obtained with C. deitersi . Lanes 1 and 8 are molecular markers; lane 2 is a type I copepod verified by direct sequencing (427 and 349 by bands) ; lanes 3 and 4 are unknown copepods from Louisiana; lanes 5 and 6 are unknown copepods from Alabama; and lane 7 is a type II copepod verified by direct sequencing (427 and 253 by bands) .

23

10 20 30 .0 50 60 70 80 90 100 110 120 130 190 150 160 170 180 190 200

HF315016 G7TTCCG7/lG G7G1ACC7GC GGIUI4GATU 7.TR7<GG7TN G7UWC777 r1CG7CG("7A AGCTW7TM MCC7CGGG11 CG7GGCCGGC TAP.CGCTGGC CTGT7RPGC LG7UGGGCC 7GCCTGCCCT CGnCCTGAU CGTT7TGTGC CGG4CGlQ7G 47AGTi7GTC WT7GCCiGT GCQ7W<GG QCGn67AC AF315017 . . . . . . . . . . -A

--- _______ _____ ____ __________ __ _.______________ GT.MA.GT7 G777~7QC . GCGT . . . . . G 7. . . . G7 . .G CAGCCC74 . . MGCC7 . .C7

AF 315018 . . . . . . . . . . . . . . . . . . . . .. . . . . . .C ..w-- - G7.MA. .71 GTTTw7RC . GCGT . . . . . A T. . . .G7. .G CAGUCi4. . MGUT . .R "F315019 . . . . . . . . . . . . . . . . . . . . . . . . . . . . .c ..w- IT .awA . .R Gl'rrA7c7c . GfG7 . . . . .G r. . . .c1. .G UGCCCrc. . u~cccl . .Q

4F315020 . . . . . . . . . . . . . . . . . . . . . . .c . .w______ ._____ _______ Gi.awn . .ii cTTlniRC . GCCi . . . . .c CA :

CnGC<RG. . nAGCC7 . .ci MGAGnGnGn GTCWG7CG . GTCG .G6C . IT . . .qG-U .hC .nL. . .T .CA.T . .R . .AG-G.RCC ATTC7 .r--~ nF315021 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .G C .iCITf . .c i7T. .ACT .c .RCi .n .TT 4.M-ii . .C A.GCMGRG A. . .AAACTG nwL .GGTTi i.A .GCCCiA

CT7 wF315022 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .G C .7C7T~ . .C iTT. .wCi .G .CTG7 .n .7T G.wn-TT . .C w.GCnnGCTG n. . .nnnCTG nnC . .GG7TT T.n .G .CCTa WGnCwL4W G7CG(G7CL . GTCG .G .G<G GT . . .wG- . . .L7 .nG.7G7 7.4 .7 . .CT . wGGGir~Ci .T7CTTT

A.. .---- G7CG .G .GCG IT . . .NG- . . .GT ./~G .TGT .,w.7 . .C7 . .wG.WGG . . T7TC7 .7---"F315023 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .G C .TCITf . .c TT7. .ACT .G .QGi .A .ii c.AMii . .c MGCMGRC 7c A4C . .ccTTi i.P .c .cciA GAWGAWW GiCGCA7cc .

AF315024 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .G C .7CTTT . .C T7T. .NCT .G .f7G7 .A .7T G.M-T7. .C N.GCMGQG A.. .7GwCCG wnC . .GG4"rt T.n .G .CCTn WWWWGA 47CG .G7CG . GTCG .GUCG GCw.T .--w . .7 .AGG7A7 7.A .7 . .R . .AGGG7TTCi ,iT<TTTC7 . AF315025 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .G C.7C7TT . .C IT7. .ACT .G .CiGT .q .7T G.AA -7-C .UGCM4CTG A .. .----7L MC . .GGTT7 T.A .G .CCTn WWWG4GP G7CGCATCG . GTCG .G .G<G IT . . .wG- . . .G7 .AG.7Gf . .".i . .Ci . ,AG.WGG . . G7'i<7 .7--nF315026 . . . . . . . . . . . . . . . . . . . .

... ... . .. . 7GC7T .A T7GTT .TT CJ .WGCA77 .Cn7n7 . .C 7CG .WG .G . i7LGnG_--- .TT. .CT .C7 7G .-7C .77

AF315027 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .--- ----- -- ----- --------- - ------- -------- - ---------- ----- - - - ---------- - -7GC77 .n T7GTT.7GA7 LA .LNGUTT GC TPT.G .C TCG .GwG .G . T7GGAG--7T . .Ci .C7 iG .-~TC.7T C.t7 w1315028 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . --- --- 77GTr .iGni W.GwGCwi7' GCniwi .G .C

C iC G .G.W GCG C .c . R i.

iTGG4c cGAGn

- c n . AAA

.Cr iC .

.<C ii iG .

tG . i-.Ti< .T7 -315 "29 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ---

-- ------- ---------- _________ ______ c.crus.r-G T. . T7 ./~in.0. T. . . . .

nF315030 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..A___ .. ._____ . -------

TA . . G .CT~wnT-G .T . .AGCaGC

..wn M ii .AT4

.G .G.C 7

.G .L.G .G.GG .CTci 7.GGAGn . .C .ann .TC .CC TG .C .T7.T7

.wiw.C i. . i.ccaW. .c ANA.rc .C~ ic .i iL Ti G .wGC .ww n Aa'315031 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .w---- -- - --- - - -- L .cTMA7- t

nF315008 . .. . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . A- :: _____---- _ __________ ------- TA . G .CiMnT-G .r . .AGC .M n .47".G .c c.c .GC4 .c . 7.GCA~w . .c .Mn.ic .CC TG .c .rc.r7

AF315033 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : : : : ::A .......... __________ __________ __ _____. rn . GclTf .wrwG .M . .w.M .ACM-4 .c T.G . .-G .c7 r.cWW . .c .M . .rc .cr rc .r .r<.__

w1315033 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..w___ ---------- -------- __ _______iA

/ . GCTTT .ATGC .i .AGCMM ACT. CG .CC i.G .G .G .R T.GAPCJ . .C .M . .iC .Ci iG .i .7<.77

/////////

210 220 230 340 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 .00 CCCT7GTT L GW7GTGCAG wF315016 GwGG7nCTG7 wGCCCGT7W 7CC64MMG GC7GAMGGC RI CiCUGG nGLG7w7nGC 77CGG74wnC wG77CMAGA ,CTQCGU7 C7CG7CGGGT nG7WTC7C C C6TMGTTG w77WA7CGG 7GGCTTGC7G CC(tM7CGC7 CA WCTCTwC GTTTWCJ~w7 CCAAn7A-

AF315017 .CTT .U . .G T. .TGMC .G CG7CATRC TT .AC.C . .A G. .ACG .C . . CR .AG.CT7 GGG7 .CAGG7 C7C .7CTfC7 7. .7CTCTTG 4W .GA .T .A AAC .:CC . : M AGTW .r .CA CCGTGRCA G4TAT .T . .n TC . .C7. .TT GC .A .GCG .T IT . 7TA6CT . TTTTT.TCTT TWM. .T7. W.C7.GT7T

AF315018 .CTT .U . .G T. .TGAAC .G CGTC .TTCTC TT .AC.C . .A G. ."CG .C . . CCT.AG.C77 GG47 .UGG . CTC .TCTTR T. .--I GW.W .T .A ML M AGTGA.T .CA CCC .7GCTCA GG7A7 .T . .A TC . .R . .R GC .A .LCG .T IT . TTAGR. 7TTTl.TCTf TGAAA. .T7. W.CT.GTTT

AF315019 .CTT .CA. .G 7. .7GqAC .G CG7CATTC7C 77 .AC.C . .A G. .PCG .L . CCT.AG.C~T GGGT .CAGQ RC .7C77'C7 T. .TC7CTTG GW.W .,n nAC ..C . .AA nG7W .7 .Cn CCG TGC7U GG7AT .7 . .n TC . .CT. .TT GC .A .GCG .T 47 .7TAGR . 777T7.TCTf 7GrlM. .TT. W.CT.GTT7

wF315020 .crr .U . .G r. .rGwwC .4 CGrC .riQC ii .nC.C . .e G. .nCC .c . . cci .wG.Ri GGGT .UCCr CrG iCrrCr T. .rcrCiiG cW.W .T .n AA'

.c . .!..4 ncTw .r .Cn CCC .rGCrU GGiwr .T . .w rG .c7 . .Tr GC .w .GCG .i GT .iinGCT. 7iii7.iC7T TGnnn. .iT. W.Cr.cCri P.F315021 TfTT .CT-C 7fT .TTCC.T C.G .CTTCCC T.C-CT7C7 . TGC7GG .G . CCG ~GC7"1"f GGGTCGTCC . TT .U . .. . . 77 .7 .7 .77 . T.T .ATT . .C GACA. .MCA GTT.CCT .GC TCGG . .TT7 . . .7 .7GCGO 7CGTGCCW . CTCTGC" . M. . .AGAM AWTTGTCG . A. . .7C MT

M. .---AAA 4W77G7CG . A. . .TT .T7. . . . .CG. MC n1315022 TcT7 .Q . .c T7 . .rACC .7 Crr.CT7ccc rrGCTlc7 . TGCiG4 .C . . <CC.ACCrT7 GGC7CG7CC . rr .U . .. . . Q.7 .7 .7r . r.7 .All . .C cACA . .MCn c7r.cCT .cc r6 G. .777 . . .T .TGCGO TCC7GCCW . <7TrcCMA . 4F315023 rCT7 .Ci-C iT7 .T7CC.T Cii.CTTCCC TiPC77C7 . 7GCTG4 .G . CCC.AGCiT7 LGG7CGiCC . i7 .C4. . . . . Ai .7Ciiii . i .i .Aii . .C G4CA . .4ACA cii.CCT .GC iCGG . .1Ti . . .i .lGCGP iCCiGCCGA. CTRGCHM . M. . .ALMA wWTTGICG, w, . .TC M7

wF31502. 7C7T .C7. .C 77 . . .ACG7 C77.C77<CC T7 .7C7TR . 7LQW.G . GCC.AGCTTT 4GGTWTCC . TT .CA. . . . . R.T4T .TT . iCT.wTT, .C GaCn . .A.:Cn GT7.CCT .GC 7CGG .GC7G . .7 .7GCGG 7CGiGfCG4. CTTTGCwW . M. .--wwG wWT7GTCG . A. . .T7 .T7. . . . .CGCMC AF315025 TCTT .CT-C TTT.TTCC.T C1T.ClTCCC TTC-CTTCT. TGCTGG .C . . CCLAGC7T7 -G-C . -CA . .. . . AT .7CT .77 . 7.7 .A7T . .C WU. .AKA G77.CCT .GC TCGG . .T7T . . .T .TGCGC- TCG7GCCW . CTC7GCMA . M. . .AGMA AWTTGTCG . A. . .TO ------M7 AF315026 CT- -GCfWG .RG.TGCAT CTf.TTfCCT . . .77.7 .A7 7CCTC.7T.C 7TUCCG .CG GC .T .A .TCT 7. . .TTGCQ C. . T.TTAT. T.GMG .ATG NAAWCNGCT T.7TGCT . .C .CC<7, . .7C GG . .7Tf . .7 T.GTGCCTG G.- . . . . TW . .M--- -GTT7W .77 T. . .CUT7. . .T77GCCT7

7. . .TTGCCT C. .T .TTnT . 7 .GMG .A7G nMGAWGCT 7.7TGC7 .CC .CCCT. . .TC GG . .7"T7. .T T.GTGCCfC . G .AAAA. . . . TW . .M--G77TW.7T 7. . .CUT7. -.TTTGCCTG AF315027 CT--GRW. .C70.TGGCf C77 777CC7 . 7.T ./~T TCCTC[Ti .C TTUCCG .TG AC .T .ATTCT .C7G .TGGCf C7T.7TfCR C.T . .T 7CRC477.C .TQCCG .CG GC .L "77R 7. . .T7GCR C. .T .TTw7 . i.GqwG.aTG AMWGnGCT T,Ti4CT,CC .CCR . . .7C GG . .777 . .7 7-G7GCC7O G.AAM . . . . TGI. . .M--- -GA. T. . .CU77. .TT7GCQG wF315028 C--

GA . ~ . . . .

iUT7 . i."lii.CTwG AF315029 C7TI .ii-AG .CiGGAG4'1'7 Qi . .77CCi .G . . .G . .i ii.GGG .C 7"ffTCGGGCG GC .1 . . .iCT T .GGTTGCQ CTCT .T .n-G G6nMGAMG ~u.MG.i .7 .i7' .GCCCCi .CCU4.if. -ii .TG7 .-- -GTGCRC. . .MW. . . . iGi . .qqpww wGi-----t l .. . . 7 . .MW. . . . TG7 ..-- .UTi . i.17i .CiwG ~F315030 mi .ii..~G .CiGW4G1"f .ii. .T7CCi .G . .C .G . .r iCC7 .GGG .C iiiKGGiCG GC .i .GRC7 i .WT1GCCi RR .i .A- GR.i .A- G.A-G G6AMGr1MG 4c .AAC .i .i .Ii.GCC<Cf .CCUC.ifi wTi .iG7 .--

: : GiGCCiC. MMA -G7 -7 T.

AF315031 CTTT .T7-AG .CTGGAGGCT CTf. .TTCQ .G . .C .G . .T 7CCT .GGG .C 7TTTCGG7CG GC .T .G .TC7 7,GG77GCR C7C7 .T .w-0 LGWALnanG 4A.MG.7 .T .77.GCCCQ .CCCAG .7T. -TT.TG. .-- G7GCCTC. . .MW. . . . GG7. .AMM nG7--7 7. . .TUTT. 7.TTT .RAG nF315008 QCT.TT-AG .CT6WGGCf CTT. .TTCCT .G . .C .G . .i iCC . .GGG .C QT7CGGiCG GC .7 .G .iQ i.GGiicCC7 RR .i .A-c cGGM6AAAG AA .rlAC.i .T .i7.GQQi .UCAG.TT. -TT.iG---- --GiGCC7C. -GA: 4Gi. .MMA wGi----r i. . .iUTT. i.TTT .CiwG

.ir GCCC .i .CGCnG .TTT nTT.7G7 . - --G7GCC7C. . .MU. . ., iL7. .~nN.A -Gi---- --i T: : :1 A- 7.TT7 .C7AL . . .C .G . .7 7 G.C i TiCLG7CG GC .L . .TR 7.GG7TGCR CTCT .7 .A-G CAGa .GnuG nG .nnG .T,i nF315032 R7T.77-AG .RG,ALGC7 CT7. .77CC7 7 CC7. . .PAW . . . 7 G7 .7 .MAM AG7---~~--T 7. . .TUTT. T.TTT .C7qG hf315033 CTTT .T--AG .RGGAGGCT CTT. .T7CCT .T . .C .G . .T TCCT .GG7

7 7G .C TTfTCGG7CG GC .7 . . .7CT 7.GG7TGCCT RCT.T .G-G GGGA .GMM M.AAG .T .T .TT. .CfC .T .CGUG.TTt TIT.TGT .-G7GCCTC.

410 42O X30 4 0 450 a60 470 <80 C90 500 $10 520 530 540 550 560 570 580 590 600

AF315016 --------- --------- ----CMGIC TNTPCAG7GG FiCA7T7GGC iCGGGGG7CG wiGAnGAACG WGCinwC7C TG7W7GUG iGiC.MCiGC 4LGACAUiG MCA7cWCA CT7iGMCCC AiATiGCGW MiMGCTiG iCTTG77iCC nin7CiWR CAGGGi<W7 iMA--Ui ccATCnWCA

A"1 15017 <M/~7M7U 74qWWAM qAn. .7 . . . . . . . . . . . . . . . . . .C . . . . . . . . . . . . . . . . . . . . . . . . .

CC : <.,.7G 7C.4M t

T . . . . . . . . . .C . . . . . . . . CCG7G. . . . . .UC .G . . . . .G . . . .G . . . . . .A . . .A 4G . .MG. . . 1. . . . .a77G

AT 3 150 18 C wwn 7U .C . . . . . . . . CCGTG. . . .U< .G . . . . .G . . . .G . . . . . . .A . . . . 4G . .4PG . . . T� . . .w77G . . . . . . . . . . G . . . . . . . WA . . . . . . . . . . . . inn iGnUGI..un aU . .i . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CAA/~7 . . . . .

C . . . . . C . C . . . . . . . . . . . nF31S019 MT iGAWGMA~ . .T . . . . . . . . . . . . . . . . . .c . . . . . . . . . . . . . . . . . . . . . . . L . . . . . . .G c .G ic .GM . . . . . . . i. . . . . . . . . .C . . . . . cccic. . . . . .ca . . . . nG . .nnG . . . .nTc

nF315020 C4AnTwwTU U rGnUGHAAw nna. .r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c . c. . 'G : : : :' :

. .a . . . . . i. . .

. . . . . . . . . . . . .C . . . . . . . . . . . . . . . . . . . . . . . . . C. . . . . . . .G C.L 7C .Oaw . . . . . . . . . . . 7. . . . . . . . . .C . . . . . . . . KGTG . . . . . . .G< .G . . . . A. . .~ wG . .MG . . . i. . . . .wTTG AF315021 GCAiARiCM iciw . . . . . . . . . .i .c . . . . . . . . . . . . . . . ..c . . . . . . . . . . . . . . . . . . . . . . . . . c C . . . . . . . .c C.G TC .GM . . . . . . . . . . . . . . . . . CTCic. . . . . . .UUG . . . . .G . . . .G . . . . . . . . . . . . .G . . . . . . . u.TT . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. . . .

nF315022 AAATMTCM 7GTq . . . . . . . . . .T . . . . . . . . . . . . . . . . . . . C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G C.G 7G GM . . . . . . . . . . . . . . . . . . . . .C . . . . . . . . RGTG . . . . . . .UUG . . . .G . . . ..: : . . . . . . . . . . .G . . . . . . . . T. . . .U .7T

iGTw . . . . . . T. : . . . . . . . . . . . . . : : : :~C: : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G C.C .TC.GM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .c . . . . . . . . RGTG . . . . . . .UUG . . . . .G . . . .G . . .c . . . . . . . . T. . . .U .TT nF315023 ccwi~wicaw . . . .r _ . . . . . . . . . . . . . . c. . . . . . .c c.c .rc.wn .c . . . . . . . . crc,c. . _ . . .cncnc . . . _G . . . .. . . . . . . . .c . . . . . . . . r . . . .u .rr nc3isoza cnnrnnrcw~ rc,w . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . .C CTGTG . . . . . . .UUG . . . . .G . . . . G . . . . . . . . . . . . .G . . . . . . . . T . . . .U .T7 <csisozs ccnl~wruw rc1A . . . . . . . . . .t . . . . . . . . . . . . . . . . . . .c . . . . . . . . . . . . . c . . . . . . . . c c .c .rc .wn nF315026 TGGGCMMG MTMWGM AT . . . . . . . . . . . . . . . . . . . . . . . . . . . C. . .C ..I . .G C.C .TC.C~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C . . . . . . . . CT4T4. . . . . . .GCAG. . . . .G . . . .G . . . . . . . . . . . . .G . . . . . . . . 7. . . .U .-G . . .C . . . . . . . . . . . . . . .

U .-. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C . . . . . . . . CTG7G. . . . . . .UUG . . . . .G . . . .G . . . . . . . . . . . . .L . . . . . . . . T. . . .G 4F315027 TGCGCA4AAG MTMG/~C ATU. . . . . . . . . . . . . . . . C . . .C . . . . . . . . . . . . . . . C. . .L . . .G C.C .T<.6M . . . . . . . . . . nF315038 7GCGCIIAAAG M7A74.4M r17GA. . . . . . . . . . . . . . . . C . ..C . . . . . . . . . . . . . . . . . . . . . . . . .

.:~ :C C.: : : :' G C.TC.G4w . . . . . . . . . . . . . . . . . . . . .f . . . . . . . . CTGiG. . . . . . .CaUG. . . . .G . . . .G . . . . . . . . . . . . .G . . . . . . . . 7. . . .U .-L

nF315029 TMMT4G.U AGA"AGA6U 4T .A . . . . . . . . ..C . . . . . . . . . . . . . . . C . . .C . . . .G C.- .7C.GM . . . . . . . . . . . . . . . . . ., . . . . . . . . ., . . . . . . . . . . . .C . . . . . . . . C7GTG. . . . . . .UUG . . . . .G . . . .G . . . . . . . . . . . . .L . . . . . . . . T. . . .U .AG AF315030 TNUATFGM AGMAUC r~T.P . . . . . .

. . . . . .. . . .

. . . .C . . . . . . . . . . . . . . . . . . . . . . . . . C. . .C . . . .G C.C .7C.GAA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C . . . . . . . . CTGTG. . . . . . .CAUG. . . . .G . . . .G . . . . . . . . . . . . .G . . . . . . . . T. . . .U .nG -3158331 TMRATAGM AC.MAWGM AT .A . . . . . . . . . . . . . . . . . . ..C . . . . . . . . . . . . . . . . . . . . . . . . . C. . .C . . . .G C.C .TC.WA . . . . . . . . . . . . . . . . . . . . .C . . . . . . . . CTGTG. . . . . . .UUG . . . . .G . . . .G . . . . . . . . . . . . .G . . . . . . . . T. . . .U .AG

. . . . . . . . . . . . ..C . . . . . . . . . . . . . . . . . . . . . . . . . C. . .C . . . .G C.C .TC.C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C .. . . . . . .

CT GTG. . . . . . .fAUG. . . . .G . . . .G . . . . . . . . . . . . .G . . . . . . . . 7. . . .G .RG nF315aoB rwuwrnCJ~A wC MGwG~A w7 .w . . . . . . . " . . . . . . "" . . . . . . . . . . NF315032 TMMTAGM AGRMWC AT .R . . . . . . . . . . . . . . . . . . ..C . . . . . . . . . . . . . . . . . . . . . . . . . < . . .C . . . . G C.C .7C.WA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C . . . . . . . . RG7G . . . . . . .CAUG. . . . .G . . . .G . . . . . . . . . . . . .G . . . . . . . . T. . . .U .4G

nv315a33 rNUninGAA nWMGnWw nr .n . . . . . . . . . . G . . . . . . . . . . . . . . c . . .c . . . . G C.C .iC.CWh . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C . . . . . . . . RciG . . . . . . .UUG . . . . .G . . . . G. . . . . . . . . . . . .G . . . . . . . . 7. . . .U .wG

///// //////~~ .. . . .

.-e~~ ~ . . . . . . . . . .

610 620 630 6.0 650 660 670 680 690 700 710 770 730 7.0 750 760 770 780 790 800 AF315016 GCT7GRGA iGU1T7GGGC GGiCGC7wiG CC"l7GGCAU 4CGQLClCC --------- ---------- ---------- --------- MGG7wrGCC GiaCGWGiT CAC ------- -wWC7CaAC KT7.CA--- MC7TGi-Gi CQGGLTiGT CC GTGLCC GiGiGiG--- -------- -UiGGiGC r1F315017 T6WT .GT. . .T .W .T7. . AA.-A- ([GC .7?-CTG AGAW .ACAt TRRC7TR GTTCTTTCTC AGTGGCTTTA MATGUTGT .- .A .LA.GG AGCACTG7 . . A.A . . .WGC TUCGG. . . . . . .C. . .C . . T.AG.TA. . . . . . .4 . . . .C .ACT .MCTT .GTGT.T . . . . . . . . . . . . . . . .TGM .R AF315018 TGG"T .GT. . .T .W .TT. . M.7 . .AC. . GGC.TT-QG "4PQA .AGl1T TUTTCAATT CTTCTTiCTC AGTGGCTTTA 4MTGU7GT .- .F .W .GG AGCACTGf . . A.rI . . .WGC TUCGG. . . . . . .C . . .C .. T.AG.T" . . . . . . ./~ . . . .C .ACT .MCTT .GTGT.T . . . . . . . . . . . . . . . .T6M .C7 Af315019 T6WT .GT. . .T .-.TT. . M.7 . .AC. . 6GC.TT-CTG AGPW .IIGAG AWTACTTTF CTTCTTTCTC AGTGGCfTTF M."TGCATGT .-.A .W.W AGCGCTG7 . . A.w ., .WGC TUCGG. . . . . . .C . . .C . . 7.AG.711 . . . . . . .11 . . . .C ."CT .MCTT .GTGT.1 . . . . . . . . . . . . . . . .7C .R AF315030 TGCWT .GT. . .T .fW.77. . M.7 . .K . . GGG.TT-CTG /~I W.AGhL l~UTTUTTT GTTCTTTCTC AGT6GCTT7A MATGU7Gf .-.--- "GCAQG T. .A .A . . .C WGCTUCGG . . . . . . .C . . . C. .T .wG.7A . . . . . . .". . . .C .AQ.M CTT .GTGT.T . . . . . . . . . . . . . . . .TCNA

.C7~F315 021TG4A.IU~ ~--------- ----T.G . .AT . .GGC .TTT CTGCGACN.MM . . . . . .TCTG TTTCTTTTGC AGTGGCTTU --AT TTC. . .P .AG A.CAATTUC AGf7GGCTT7 CTT .G"AG . . . . . . . . .A . . TTi.MAf .U T.CT. .G .L TTRTGT--G M. . .AC.GC . . .7677G M.7WT . .G nF315022 7C w.AAA-- ---------- -T .G . .A7. . GGC.7T7CTG CGACN.Nwn . . . . . .7C7G 7T"LC7TTTGC wGTGGCTTCw AGATTTTTw7 TTC. . .w .AG A.CAATTCAC nGT7G6C7C7 C77 .6MG . . . . . . . . .A . . TI"f~M<.U 7.Q . .G .C . TTCT7G7--G w- . . .wCCGC . . . T-1 M.7Wi . .G ~F315073 TWA.A4~-- --- ----- -T .G . .AT. . GGC.TTTRG CGF6A.AAAF . . . . . .TCTG TT7CTTTfGC AGTGGCTTCA AGA7llTTPT TTC. . .n .AG N.CAAT7UC AG7T6GC7CT CT7 .GnAG. . . . . . . . .A . . TTT.M~C .U T.CT. .G .L TTCTTGT--G A- . . .wCCGC . . .7OGAT7G M.TLAT. .4

wG31502/ 7WA.AM-- ------- -T-G--

AT . . GGC.7TfC7G CCN.AAA~ . . . . . .7C7G 777C77TfGC AGTGGCTTCA AGA7'71TL17 T7C. . .A .AG w.CAA77CqC AGTTCF.C7f7 CTT .GML . . . . . . . . .A . . 7TTMAC .U 7.C7. .G .C . T7CT7GT--G M. . .ACCGC . . .7GGATTG M.TWT, .q NF315a25 rGM,AM-- ------- -r .c . .w7. . GGC.7TfC7G CGA.AAM . . . . . .TRG r7TCiTT~GC AcTCGCT7Ca AGr177777Ar TTC. . .rl.rlc A.cAA77CAC AGirGGCTC7 CTT .G~AG. . ITfAA"LU 7.Cr. .G .C . TTC77Gr-c *- : : :A <cGC . . .TGGwT7G ~w .7W7 . .N

.M7 W. M7 . GG NF315026 .AC.TT. . .- --<. .T .. . .~7.4 .GCl .7 .TGC7TTQC A---GTMnG . . . . . . . . . . . . . . . . . . .C AGTGGC77U AGG7"ITM7A TC-- -.GUCTC . G.w7CiCIGT 7CiCfTG4 AG6A/~7 -. . . p. . G.AM7

.

. ..CC

.

.TCT.,.G .C

.

. T< . GC W----GG A A- - .

~GGTTfMTA -- .A .TO-- .GCnCTC . . TGAC7CT7GG AG T G .AMT CC TCT. G . C TC .GC W --..TCTC M GI.GTCTG

TAAW7TCTG TGI.GTCTGT T

. GG .W.MT AF315027 I~ Q . 77 . . .- ---< . .7 .. . AT .G .OR.7 .T4CTTfCTC A---G7AAAG . . . . . . . . . . . . . . . . . . . C AGT6GCTTU -- CPRC . G.n7<PTC7CTGTTCTGT TWC7C7TGG AG(" AT-. . . G.M."7. .CC .TR. .G .L TC .GCGA--G ". . . . .. . TC MG~IG7CTGT .GG.W.M7 4F315028 .l~C .TT---- --C. .T . . . P.T .G .GCT .T .T4C77TQC A--4TAAAG . . . . . . . . . . . . . . . . . . .C P.GTGG7U AGGI"I7W7A P..TC--- -- .G

G

4F315029 iiuTT. . . . G~GC . .i . . . Ai .G .4CTGi .iKiTTCGi iMAC-AAnc AGTGGC7i7A AGGiccGMA .- . . .iclc. TG . A. .CTC . G.~1RRGi 1W<r<TiGG . . . . . . . . . . . . . . . . . . .C wGGCTT- . . . T;--.T :

.7C7 . .G .C . 1C .GCGM-G A- . . .G- .'C 7MCCTT . . . .GG.W.MT 4F315030 TTUTT. . . . WGC. .T .. . AT . G.GCTGT .TGCTTTCGT TTMG ..WAG . . . . . . . . . . . . . . . . . . . C AGTGGCTTTI. AGGTGCAAPA .- . . .TCAG. 7. . . . . CTC . G.ATCTC7G7 7WCTCT7GG AGGC7T- . . . TTGCWG .T . .TCT . .G .C . 7C .GC6AP-G 4- . . .G- .7C 7MCCT7 . . . .GL.GA."AT

. . . . . . . . .C "G7GGC7T7A wGGTGC6AM .- . . .7C7G~ nGG--QC . G.ATCiC7G7 7WC7fT7GG "GGCTT- . . . TTUWG.T . .7CT, . .C . 7C .GCGnA-G A- . . .G- .iC 7MCCTT . . . .GG.Gw,ww7 qF315031 TTUT7. . . . WGC. .T .. . AT .G .GI GT .TGCTTfCGT TMwG-AAAG . . . . . . . . . . AGT6GCTTTn AG4TGCGAM .- . . .TCTW nGG--C7C . G.nTRC7G7 7WRCT7GG AG4C77- . . . 7TLCG4G .7 . .iCT . .L .C . TC .GCW~-G w-, . .4- .7C 7MCf7i . . . .GG.GA.Mi wF315008 TTC4TT . . . . GAGC . .T .. . "T.G .GCTGT .TGC7"ITCGT TAG-AAnG . . . . . . . . . . . . . . . . . . .C wG7GGCT7Tn wGG7G<GnaA . . . .TCTW 4GL--C7C . G.4TRCTG7 7GPRCT7GG nLGCTTT. . . T7GCGaG .C . .7R . .G .C . 7C .GGGM -4 n- . . .G- .7f TMCCTT . . . .GG.Gn.M7 AF315032 77CATT . . . . WGC. .T .. . <T .G.G- G7 .7GCT77CGT 7AAAG-nnnG . . . . . . . . . . . . . . . . . . .C

AF315033 iiUTT. . . . wGC. .i .. . AT .G .C,CTCi .iGCi77cci irUNC-MAC . . . . . . . . . . . . . . . . . . .c AGTGGCT77A AGGiccGAM .- . . .icTW ncc--RC . G.n1CiQLi iWCTCTTGC nGGCTTf. . . TTGCC4G .c . .rCi . .c .c . rC .ccWn-G n- . .,c- .iC innCC77 . . . .GL.U .sAr

010 020 830 840 850 860 868 AF315016 CGnGNQCCN QGFCiGiR CGiMGCGCC M TTwRrrC GnC<TCACWi UWCCwG _315 017 .C ,..G.Gn G.rG . .- . ., * . .*~.. .r *.,T~R - - F: . .*I.CA . . . . . . . . . . . . . . . . . _ Legend' AF315018 .CTw . WGTT c.TC. .- ../~ i. .icU . .i i.iiGCT . . . f . . .rG.Cn. . . . . . . . . . . . . . . . . . . aF315019 .ciA .WCT c.i0..- . .w i. .icU . .i i.iiGR. . . C . . .ic.Ca. . . . . . . . . . . . . . . . . . . w[315020 .Qa.WGTT G.TG. .- . .w i, .TGU .,r i.iicCT . . . < . . .iG.U . . . . . . . . . . . . . . . . . . . wF315021 MGT7TC'TT7 w.TTf .7c-c T7 .C .w7C7T G.TT- . . . . . .UTTGG . . NF315022 MGf7TG7Tf A .TTf .TG-C TT .C .hTCI"'! G. *,-. . . . . .Ca,~_ . . : : : : : : : : : : : ::»» «« Forward and reverse primer -315223 .u.GrTrGrrr w.7rr .r .rc rr .c .wTCrr G. T7 . .'rWr . .UrrGG . . . . . . . . . . . . . . . . . . . . ,F31 5o~, ,~.~rrr~�T ,.,T, .r~-C �.C .,*C� G .�- . . . . . .C�n

TTGG .~. . 18S SSU rRNA gene 4F315025 MGTTTGTT7 i.TIT .7 .-C TT . G AiCTT G.ii- . . . . . .U . . . . . . . . . . . . . . . . . .

AF315026 G .77CT.-- . anC7T. .G C TC .CTC .i .w . .A .CG. . . . . i,G.C . . . . . . . . . . . ITS 1 AF3150T7 G .iiCT.-- . MCTT . .G C iC .RC.i .w . .A .CG. . . . . . . .i .G .L . . . . . . . . . .

wG315028 G .iiCi.T7 . MC77 . .C .C iC .CiC .i .n . .A .cc. . . . . . . .i .G .C . . . . . . . . . . . . . . . . . . . AFSisozs G .,« .C - .T w~,T . .C .C ,C .RC ., .A C*** .,T . . . C .A . . . . . . . . . . . . . . . . . . . . . . . . . ________= 5.SS rRNA gene AF335030 G .TCG C- .T GAC7T. .C .C TG Cfc.T .A Q7 . .T7. . . C .A . . . . . . . . . . . . . . . . . . . . . . . . . �F315031 G .,« .C-., wC�. .C .C ,C .C.C ., . . C,-r . .,T . . . ' ., . . . . . . . . . . . . . . . . . . . . . . . . . \\\\\\\\\\\\\\\\\\\ ITS2 hF315008 G .7CG0 .7 WCTf . .C .C TC .RC.T ." Ri . .TT. . . C .P . . . . . . . . . . . . . . . . . . . . . . . . . wF315032 G .7CQC- .T G4C77. .G C 7C .C7< .7 .n CT77 .T7. . . C .A . . . . . . . . . . . . . . . . . . . . . . . . . AF315033 G.7CR0.T WCTf . .G C TC .CTC .7,A CTTT .TT. . . C .n . . . . . . . . . . . . . . . . . . . . . . . . .

\\\\\\\\\\ \\\\\\\\\\ \\\\\\\\\\ \\\\~\\\\\ \\\\\\\\\< ««c<a«r <acc««

Figure 2 .2 Clustal W DNA multiple alignment of part of the nuclear rDNA comprising the entire internal transcribed spacers (ITS 1 and ITS2) and the intervening 5 .85 ribosomal RNA gene. Gene regions are indicated at the bottom of the alignment . Labels refer to Genbank accession numbers presented in Table 2.1 . Dots represent identity with the majority rule consensus sequence on top and dashes represent inferred indels .

5 T w H K O I G T L V L M 5 G A W 5 G N V G T A L 5 M L I R V J31 ] AF31SOO9 AAT CAN AAA CAT A AR CR TAC TfA ATA I1GA GGG GU TGG TCT GGA ATA GTG GG4 ACC GR Cff AGA ATA TTA An CW GTG [9S]

. . . . . . . . . . . . . . . . . .A AF315012 . , .A T .G . .T . . . . .7 . .G . .A .7 . . . A.C . .G . .A . .C T.G . .G G.C ... AF 315O11 . . .C T .G . . . C . . . .T . . . . . . . . . A .T . . . . .G . .A . .C . . . G .T . . . . .C . . . A .C .T . . . . . . . . . AF315010

LCOI ' ' " C T .G . . . C . . . .T . .G . . . . .T . . . . .A

. . .G . . . A.7 . .7 . .G . .A . .G . . . G.T C. . . . . . . . A. T

A F31501{ . . . . . . .T . . . T. . . . . . .T T.T . .A . .A . .G T. . . .A . . . . .A . .. T:A . .G T.T C.T . . . . .T T .A AF315O1S . . . . T.A . .T . . . T.G GC . . .A . .C . .A G .G . .G . . . . . . . .G . .A .GA . . . . . . . . . A.T . . . . .T T.A AF315013 , .C T .A . .T . . . T.G GCC . . . . .7 .AC . . . . .C . . . A.C A GG . .C . . . . . . A.T . . . . . . C.A

E L 4 Q P G 5 L I G N D Q I Y N V I V T A H A F Z M I F F M V M [63] 7 AF3SS009 GAA CfA GGN CAA CR GGC AGA TTG ATC GGG MC GAT CAA A7T TAT MT GfC An GTC AR GR CAT GG7 T7T ATT ATA An 7T7 TTf ATG GfA ATA[191]

. . . . . . . . . . . .. . . . . . . . . . . AF315012 . . . T. . . .C . . . . . . . .G .CG A .T . .T . .T . .T . .C . . . . . . . . . . .G G.A . . . . .G . .A A . .T . .A . .G . .G AF315011 . .G T. . . . . . .G . . . . . . .CC AT . . . . .A . .T . .C . .G . .. . .C . . . . .A G.C . .T . .G . . . . . . . .C . . . . .C . .G . .T . . . . .C . .A . . . . .G AF315010 . .G T. . . . . . .G . . . . . . .CC A .T . . . . .A . .T . . . . . . . . . . .C . . . . .A G.C . .T . .G . . . . . . . .A . . . ..C . .G . .T . . . . .C . .A . .G . .G

.G . . . . . . . .A . .. . .T . .G . . . . . . . . . . . . . . . . .A . .T . . . AF315011 ., . T . . . .i . . . . .G . . . TCf . .T G.A . .A .GA . . . . . . . " . . G.G . . . . .7 AF315015 . .G , .G . .T . . . . . . . .A . . . C .7 . .T . . . G,T . .C . .G . .. . . . . . . . .A G.C . .T . .C . . . G.A . .G . .7 . . . . .C .,A . .7 . .G

. .A . .G . .G . . . . . . . . . .. . . . . . .T . .C . .C . . . . . . . AF315013 . .G ..G . .T . .G G .G . .G TCC . .A . .T . .A G .T . . . . . . G.A . . . . . . . .A 4. . .G O O O

P I L I G G F G N V L V P L N L G A P D N A F P R l N N N 5 F Y [95] AF315009 CR An TfA An GGC GGA TTC GGA MT TGA TTG GTG CCf TfA ATA TfA GGG GCC CR WT ATG GCC m CCC CGT RA MT MT ATA AfA TfT TW[287]

. . . . . . . . . . . . . . . . . .T ..A T . . . . .G . .C . . . . . . AF315012 . . . .. . . . . . . . . .A . .G . . . . . . . . . . . . G.A ..C . .C . . . . . . C.G .A . . . . . . . . AF315011 G . . . . . .. . C. . . . . .C C. C . .G . . . . .T . .G . .C . . . . . . . . . . .C . .T ..A T. . . ., . .C . . . .,G . .C . . . . . . . . . . .7 . .C . . . . . . . . . . Af31SOSO . . . . .C C. . . . . . .A T . .C . .C . . . C. . . . . . .C C.T . .G C. . . .T . .A . .C . . . . . . . .T ..C . .T . .G T. . . . . . .C . . . . . . . .C . . . AF315031 . . . .. . . . . . . . . .G . .G . .T . .G . . . . .G . .A . .T . . . . . . . . . C.T . . . . .A . .A . .T T ..A T. . . . . AF315015 . . . . . . . .G . . . . .G . . . . .T . .4 . .C . .G . .A . .T . . . . . . . . . C . . . . . . . . . . . . .C . . . . .T . .C . .T . . . T. . . . . . . . . . . . . . . . . . . .

. . . . . . . .C . . . . . . . . AF315013 . .C . .C . . . . . . . .A . .G . .T . .T . .C . . . C.A . . . . . . . . . . . . C .G . . . . .T G. . . .T . .C . .A . .A A. . . . . 101

F L I P A l I M L L L 5 S L V E A G V G T G w T V V P D L $ 5 N [127] AF315009 m RC ATC CU GU TTA An ATG RC Cff Cff AG4 AGC TTG GTG GAG GCC GGG GTf GGG AR GGT 7GG AR GTC TAT CCC CCC RA AGG TR MT[383] Af31SO12 . . . . .T . . . . . . . .T . .G G . . . .A . .G . . . T .A . .G G .A A .A . .A . .A ATA . .A .CA . .C . .A . . . . .A . . . . . . . .C . .G . .T T . . . .T G .C . . .

. . . . . . .A . .G . .G . . . . .T . .T T . . . . . G . . . .C AFIISOII . . . T .A . .T . .T . .C C .T . . . . . . . .T T .A T .A . . . G .A A . . . .C . .A ATA . .A .CA . .C A AFI1SOlO . . . T .G . . . . .T . .T C . T . . . . . . . .T T :A T :A . . . G .A A . . . .C . .A ATA . . . .G . .C . . . . .A . .A . .G . .A . .C . .T . .G T . . . . . G . . . .C AF315014 . . . . .T T .G . .T . .7 . . . .GA . .A . .T . . . T .A . . . . .A A ., . .7 . .A 7 .G . . . .C . . .T . . . .,G . .A . .A .,T . .C . .G . .T T . . . .A GGG GGA AF31SO15 . . . T .A T .A . . . . .G C . . G .G T .A . .G T .G TCG . .G T CT . . . . .T . .A AGA . . . .CG . . . . .A . .G . .A . .A . .G . . . . .T . .T T .G T C . . .A . . . AF3SSO13 . . . T :A . .G . . . . .C . .G . . . . .A . .T T .A TCC . . . TC . . .A . .A . . . AW . . . .CC . . . . .G . . . . .A . .A . .T . .C . .T . .T T . . TU AGN . . .

O O O O O I A H 5 G K 5 V D C A I F 5 L N L n G I S 5 L l G A V N F I S T[1591

AF31S009 An GCC UT AGG GGA MG TCf GfA WT TGT GCf An m TR CTf UT 7TA GCC GG4 An TCC TCG TfA RA GGA GCT GfA MT m ATC AGA AR[479] . .T . .T .. . . . . . .T . .G . . . . . . . . . . .T . .G . . . AF31S012 . . . . . . . . . . .T . . . . .A . . . . .G . .C . . . . .A .. . . . . . . . T.G . .C . .G . .A . . . . . .

(,/1 AF315O1S . .C . .A . .C . .A . . . . .A . . . . .G . . . . . . . .A . . . . . . . . . . . . . . . . . . . . . . .T G. . . .T . .T .. . .T . .G . . . . . . . . . . .T . . . . .A AF315010 . .C . .A . .C . .A . . . . .A . . . . .G . . . . . . . .A . .C . . . . . . . .C . .. . . . . .7 . .T . . . . . . . .7 .. . T.G . . . . . . . .T . . . . . . . .T . . . . .A AF3SSO1/ . . . .. . . . . . . . . . . . .A . . . . . . . . . . . . . . . . . . . . . . . . T.A . .. . . . . .A . .G . . . . .T . .A .. . . .G . .G . . . . . . . . . . . . . .T . . . .A

. . . . . . . .T . .G . . . AF315015 . . . . . . . . . GCT . . . .G . AGG . . . . . . .T . . .C . . . . . . . . . T:A . . . . .G . .A . .T . . . . .A . .T .. . T. . . .7 . . . . . . . .. . . . . .C . .C . .G . . . . . . . .T . . . . .C AF31SO13 . .C . . . . . . GCT . .C GC . . . . . . . . . . .T . . .A C . . . . . .. . . . . . .A . . . G.A AG . . . . A.C T. .

O I F N L a C F G M L l E Q M P L F A w 5 V L I T A I L L L l 5 L(1911

AF315009 An m MT CTT CGG TGf m GGG ATA RA RA GM CAA ATG CR TTA m GU TW TR 6TT RC A7C ACT GCf ATC TTA Cff RC Cff TCf TfGE515] AF315012 T .A GCC T :A .C . . . . . .T . . . . .C . . . ..G . . . . .A . . . . .G . . . . .C . .G . . . . . . T:A ..T . .G . . . . .T . .G . .A . .T . . . . . . . .A . . .A AF315011 T.G GCA . . . . . . . .T .C . . . . . .T . .G . .T . . . ..G . . . . .A . . . C.T . . . . . . . . . . .G . .A T .A ..T . .C ..C . .T LT . . . T.A T.G . . . . . . AF315010 T:A GCA . . . " . . . .T .C . . . . . .C . .G ..T T. . ..G . .G . .A . . . C.T . . . . .G . . . . .A . . . T:A ..T . .C . . . . .T C.T . . . T:A T.G . . . . . .

. .A . . . A.G G .C .. . . .A . . . C.. . . . TGT . .G . .G . . . T .G ..T . . . . .A . .T . . . T.G . .T T.A . . . C.A . . . . AF315014 T .A A.A T A . C. .GG . .A . . AF315015 T.G GW . .C T.G . .A ACC .AC . . . T.G T. . A.G ..G .G . . .A . .C C.T . .C . .G . . . . . . . . . T:A ..T . .A . . . . . . . .G T.6 . .T T.G . .C C.A AF315013 T.A GGA . . . . .G . .A GTA . . . . .A . . . A .T . ., ..T .GT . . . . .C C .G . .C . . . . .G G.A . . . ..A ..T . .G . . . G.A C . . 7.A . . . . .C . .C . . .

O O O P V l A G A I T M L L 5 D R N L N 5 T F F D A A G G G D P V L Y(223]

AF315009 CCG GTA RT GCA GGG GR ATC AU ATG RA RC TR GAT CGT AAC TTA MT AGA AU T7T 777 GAT GCA GCC GGA GGG GGG CAC CR GTG CiT TATE671] AF315012 . .T . . . T.G . .C . .A . . . . .T . .T . .A T. . . . . .. A . .C . .A . . . . . . .CC T.T . . . . . . . .C .. . .GG . . . . .A . . . . . . . . . . .T . .A . .C

. .A . .A . . . . . . . AF315011 . .A . . . T:A . .T . .A . .A . .T . .T . . . . .T . .T .. . . . . . .A . .T C.T . . . .C . T.G . . . . . . ..C .. . .GA . .G .A T:A . .C AF3SSOlO . .A . . . TA . .T . .A . .C . . . . .T . . . . .T . .T .. . . . . . .A . .T CT . .C .CC T.T . . . . . . . .C ..C .GA . . . . .A . .A . .T . .C . . . TA . .C AF31501" . .A . .T T.A . . . . .A . . . . .T . . . . .A . . . . .T .G . . . . . .A . .T C.. . . . . .G 7. . . . . . . . . . . .n . .A . .T . .A . .A . .T . . . . .T T.A . . . AF315O15 . . . . .G T.A . . . . .A . .G . .T . .T . . . T.G T.A A.G . . . . .A . .T . .G . . . .CG T .T . . . .A . . .C T.T AGA . .G . .C . .A . . . . .A A.T . . . . . . AF31SO13 . .A . .T T.G . . . . . . . . . . . . . . . . .A ..T . . . A. . . .C . . . . . . . .. . . . .R . . . . . . .AC . . . .TG .GG ..G . .T . .A . .T . . . A.T . .G . . .

0 0 0 Q H L F [2J5]

AF315009 UG UT TTA Tff TW TTT TTT GGf CAC CR GAA GTT TA [709 AF315012 . .A . . . C .G . . AF315011 . .A . . . . . . . . . AF315010 . . . . . . . .G . . . H COI AF31501t . . . . . . . .c . . . AF315015 . . . . . . C .7 . . . AF)1SOS3 . .A . . . . . . . . .

(A)

Figure 2 .3 DNA multiple alignment of representative sequences of the mitochondrial cytochrome oxidase I gene bounded by Folmer et al. (1994) LCOI and HCOI primers. A translation of the reading frame is provided for the first reference sequence, dots indicate identity with it . Symbols below the nucleotides refer to conservative (11) and non-conservative ( " ) amino acid substitutions. Symbols in parentheses indicate that the amino acid substitution was found in a sequence not included in the alignment

C. deitersi

MA COX-1 MA M

,oo ~ ~ ,oo goo

64 ~ " ioo

ioo

.30 55 98 ~ " 98

A/ 86

sa

0 " <50 100

00 100 Type 111

0 100

65 : Q 100

100 100 .

T I f~l 100

ype " 100 85 96 A

100 100

C. helobius Coullana sp .

Calanus pacificu s

16S

0.30 50 64

100

95 I

x,100 I 100

Localities : A Alabama /Louisiana 0 California O Mexico

Figure 2 .4 Neighbor-Joining (NJ) phylogenetic reconstructions based on genetic distances of distinct mitochondrial cytochrome oxidase subunit I (COX-I, K81uf+I+G distances) and 16S ribosomal RNA (165, TIM+I+G distances) haplotypes of the harpacticoid copepods Cletocamptus deitersi, C. helobius, Coullana sp., and the calanoid copepod Calanus pacifrcus . Branch lengths are proportional to genetic distance according to scales shown. Numbers on branches are non-parametric bootstrap support values (1000 replications) obtained with NJ (above) and with maximum parsimony (below). Localities where haplotypes were found are indicated by symbols, numbers in symbols are the number of copepods sharing a particular haplotype .

26

5.8S 0

0.05 ;~ C. deitersi D

68 FO 60

EEO

2

98 C. helobius 97

Calanus pacificus

Mitochondria) (COX-I) haplotypes " Type I / Type II 0 Type III

Figure 2 .5 Neighbor-Joining (NJ) phylogenetic reconstruction based on Jukes-Cantor genetic distances of distinct nuclear 5 .85 ribosomal RNA alleles of the harpacticoid copepods Cletocamptus deitersi, C. helobius, and the calanoid copepod Calanus pacificus . Branch lengths represent genetic distances according to the scale shown. Numbers on branches represent nonparametric bootstrap support values (1000 replicates) obtained with NJ (above) and with maximum parsimony (below). The corresponding mitochondria) haplotype found in the same copepods is indicated by the symbols . Numbers in symbols represent the individuals sharing the same 5 .8S rRNA gene copy .

27

D Control _ Exposed

1 .20

0.90

a c

Z

0.60 0 U

L

0 .30

0.00

Figure 2.6 . Percent survival of type I and II Cletocamptus deitersi for individuals exposed and unexposed (control) to saturated phenanthrene for 96 hours. Error bars show f one standard deviation . There was no variation among replicates for the type II control individuals.

28

Type l Type II

O Control - Exposed

1 .20

0.90

c .Z

0 .60 0 U (0 L

0 .30

0.00

Figure 2 .7 Percent survival of type I and II Cletocamptus deitersi for individuals exposed and unexposed (control) to a mix of heavy metals (see text for composition) for 96 hours. Error bars show ± one standard deviation.

29

Type l Type II

II ex II con I ex I con

--~ 100

8 0 -

60 .Z

v, 0 0

40 c cv

20

0 0 9 18 27 36 45 54 63 72 81 90

Tim e (h)

Figure 2.8 Survivorship curves of type I and II Cletocamptus deitersi in 96-hour exposures to heavy metals . I ex = type I exposed to metals, I con = type I not exposed, 11 ex = type 11 exposed to metals and 11 con = type II not exposed to metals .

Table 2 .1 .

w

Number of copepods (n), number of distinct mtDNA haplotypes or nuclear gene copies (h), and accession numbers of representative sequences submitted to Genbank (GB), of each copepod species genotyped by direct sequencing (COX-I, 165, n rDNA) or multiplex

haplotype-specific PCR of the COX-I gene (MHS).

Species (sample) X-Ie 1 n rDNA` MHS

n h GB n h GB n hd GB n

Cletocamptus deitersi (AL)' 42 10 AF315009 12 2 AF315001 5 5/2/5 AF315021-25 55

Cletocamptus deitersi (LA) 17 6 AF315012 20 5 AF315002 3 3/2/3 AF315026-28 126

Cletocamptus deitersi (CA) 33 4 AF315011 4 3 AF315003 4 4/1/4 AF315008+29-31 0

Cletocamptus deitersi (MX) 11 1 AF315010 11 1 AF315004 11 2/1/1 AF315033-34 0

Cletocamptus helobius 6 1 AF315014 3 1 AF315005 4 4/2/4 AF315017-20 0

Coullana sp . 2 1 AF315015 1 1 AF315007 0 -- --- 0

Calanus pacificus 3 3 AF315013 4 3 AF315006 1 1 AF315016 0

a mitochondrial cytochrome oxidase subunit I b mitochondrial LSU rDNA (16S) nuclear rDNA consisting of ITS 1, 5.85 rRNA gene, and ITS2

d number of distinct ITS 1/5.8S rRNA/ITS2 gene copies 'AL= Alabama, LA= Louisiana, CA= California, MX= Mazatlan, Mexico

Table 2 .2

w N

Mean pairwise genetic distances in the COX-I (above diagonal) and 16S (below diagonal)a mitochondrial genes in four lineages of Cletocamptus deitersi (types I, II, IIIC, and HIM) and in C. helobius (CH).

Type I Type II Type IIIC Type HIM CH

Type I b0.27 (0.2-0.5) n=28 23 .15 (22.5-25.3) 24.68 (24.0-26.1) 26.27 (26.1-26.4) 26.77 (24.5-26 .8)

0.35 n=1 61 .59 (58 .5-74.4) 68.44 (62.2-82.0) 76.70 (73 .9-88 .3) 79.67 (77.3-91 .7)

`n=64 n=32 n=8 n=8

Type II 32.15 (31 .4-32 .4) 0 .33 (0 .2-0.6) n=28 21 .49 (20.8-22.0) 22.13 (22.0-22 .3) 23 .82 (23 .7-24 .0)

58 .03 (56.0-58 .9) 0.69 (0 .3-1 .0) n=10 51 .28 (48 .7-53.4) 52.02 (51 .2-53 .0) 72.80 (71 .9-74 .1)

n=10 n=32 n=8 n=8

Type IIIC 35 .77 (35.6-36.0) 20.92(20.3-21 .3) 1 .19 (0.2-1 .7) n=6 8.66 (8.4-8 .8) 26.37 (26.1-26 .6)

80.76 (79.1-82.5) 30.54(29.4-31 .2) 0.69 (0.3-1 .0) n=3 11 .04 (10.6-11 .3) 88.45 (80.5-82 .0)

n=6 n=15 n=4 n=4

ype HIM 35 .68 (35.7-35-7) 21 .07 (20.7-21 .1) 4.48 (4.1-4 .8) d n.a . 25 .84

82.16 (81 .8-82.5) 31 .67 (30.9-32 .2) 4.64 (4.3-5 .0) 79.26

n=2 n=5 n=3 n=1

H 35 .77 (35.8-35 .8) 31 .96 (31 .7-32 .1) 32.89 (32.6-33 .0) 32.28 n.a .

81 .12 (80.8-81 .4) 63 .57 (63 .1-63 .9) 70.17 (69.9-70 .5) 68.51

n=2 n=5 n=3 n=1

a 16S distances computed on 16SAR-16SBR fragment corrected for missing data for type HIM and CH b In diagonal. line 1 = COX-I, line 2 = 16S (mean uncorrected %p, n=number of comparisons)

Off diagonal . line 1 = uncorrected %p, line 2 = %K81uf+I+G (COX-I) and %TIM+I+G (16S) (range in parentheses), Line 3 n = number of comparisons .

d Not applicable

Table 2.3

Mean uncorrected genetic distances (%) in the ITS 1, 5.8S, and ITS2 genes regions in four lineages of Cletocamptus deitersi (types I, II, IIIC, and HIM) and in C. helobius (CH) .

Gene Comparison Mean na Range

ITS-1 type I x type I 7 .63 10 0.6-12.6

type II x type II 2 .73 3 2.1-3 .4

type IIIC x type IIIC 4.06 6 2.4-5.9

type HIM x type HIM 8.50 1 n .a .b

type IIIC x type HIM 11 .95 8 8.5-15 .7

ITS-2 type I x type I 0 .73 10 0.0-1 .4

type II x type II 0.65 3 0.5-1 .0

type IIIC x type IIIC 2.60 3 1 .8-3 .2

type HIM x type HIM n.a . 0 n.a .

type IIIC x type HIM 2.80 3 1 .4-4 .6

5 .85 type I x type I 0.61 1 n .a . type II x type II 0.61 1 n .a .

type III x type III n.a . 0 n .a .

type I x type II 1 .53 2 1 .2-1 .8

type I x type III 0.92 2 0.6-1 .2

type II x type III 0.61 1 n .a .

CH x type I 3 .70 4 3.1-4.3

CH x type II 4.62 2 4.3-4.9

CH x type III 3 .99 2 3 .7-4.3

'Number of pair-wise comparisons averaged b Not applicable

All type III copepods from California (IIIC) and Mazatlan (IIIM) shared the same 5 .8S gene copy .

33

Table 2 .4

Parameters of the models of nucleotide substitution fit to mitochondria) and nuclear genes of C. deitersi .

Gene Models Base frequencies (ACG) Rate matrix' I` Gd

COX-1 Kimura 0.2689, 0.1688, 0.2051 r(A G) = 9.778 0.435 1 .141 81 r(AT) = 2.014

16S rDNA TIM 0.4024, 0.1518, 0.1443 r(A G) = 8 .327 0.222 4.235 r(AT) = 3 .047 r(C T) = 11 .053

5 .85 rDNA JC 0 .25, 0.25, 0 .25 r(N notN) = 1 .0 n.a .e n.a .

a Kimura 81 (Kimura 1981), TIM (Rodriguez et al. 1990), and JC (Jukes and Cantor 1969) b relative rates to r(A C) fixed at 1 .0, only distinct elements of the matrix are shown proportion of invariable sites

d shape parameter of the Gamma distribution assumed to describe rate heterogeneity among sites e not applicable

34

Table 2 .5

Ranges of divergence since common ancestor based on corrected sequence divergence and crustacean molecular clock calibrations a .

111 111 11 1111 111 111 11111 16S COX-1

Common ancestor of

Mean corrected distance (%)b

Divergence range (Ma)b

Mean corrected distance (%)b

Divergence range (Ma)'

HIM and IIIC 4.66K S.1K - 12.3K 9.35K - 11 .11H 39K - 7.9H

II and III 24.52K 27.2K - 64.SK 26 .20K - 40.67H 10.9K - 29.1H

I and II, III 46.83K 52.OK - 123 .2K 30 .56K - 51 .17H 12.7K - 36.6H

a Rates of molecular evolution used for the 16S rRNA gene include 0.38% Kimura 2-parameter/Ma for anomurans (Cunningham et al. 1992) and 0.90% Kimura 2-parameter/Ma for fiddler crabs (Sturmbauer et al. 1996). A second calibration of 0.65-0.88% Kimura 2-parameter/Ma (Schubart et a1.1998) obtained from grapsid crabs gives intermediate values . COX-I calibrations of geminate species of alpheid shrimps include 1 .4% maximum likelihood-I'/Ma (Knowlton and Weigt 1998) and 2.4% Kimura 2-parameter/Ma (Knowlton et al . 1993) . A third calibration of 1 .66% Kimura 2-parameter/Ma (Schubart et al. 1998) obtained from grapsid crabs also gives intermediate values .

b Letters after values refer to the correction using the HKY85-I' (H) or the Kimura 2-parameter model (K). These models were used in order to match those used in the calibrations, best fit models would produce older estimates.

35

Q~N,EN7 OF~y The Department of the Interior Mission j QP ~'

o`` ' y9 As the Nation's principal conservation agency, the Department of the Interior has responsibility for most of our nationally owned public lands and natural resources . This includes fostering sound use of our land and water resources; protecting our fish, wildlife, and biological diversity ; preserving the environmental and cultural values of our national parks and historical places ;

qqCH 3 18x9 and providing for the enjoyment of life through outdoor recreation . The Department assesses our energy and mineral resources and works to ensure that their development is in the best interests of all our people by encouraging stewardship and citizen participation in their care . The Department also has a major responsibility for American Indian reservation communities and for people who live in island territories under U .S . administration .

The Minerals Management Service Mission

As a bureau of the Department of the Interior, the Minerals Management Service's (MMS) primary responsibilities are to manage the mineral resources located on the Nation's Outer

1A Continental Shelf (OCS), collect revenue from the Federal OCS and onshore Federal and Indian

'°~~ h.��,am ~~' lands, and distribute those revenues .

Moreover, in working to meet its responsibilities, the Offshore Minerals Management Program administers the OCS competitive leasing program and oversees the safe and environmentally sound exploration and production of our Nation's offshore natural gas, oil and other mineral resources. The MMS Minerals Revenue Management meets its responsibilities by ensuring the efficient, timely and accurate collection and disbursement of revenue from mineral leasing and production due to Indian tribes and allottees, States and the U .S . Treasury .

The MMS strives to fulfill its responsibilities through the general guiding principles of: (1) being responsive to the public's concerns and interests by maintaining a dialogue with all potentially affected parties and (2) carrying out its programs with an emphasis on working to enhance the quality of life for all Americans by lending MMS assistance and expertise to economic development and environmental protection .