ACCESS NUMBER: 30660-19949

STUDY TITLE: How does produced water cause a reduction in the genetic diversity of 

harpacticoid copepods? 

REPORT TITLE: How does produced water cause a reduction in the genetic diversity 

of harpacticoid copepods? 

CONTRACT NUMBER: 14-35-001-30660-19949 

SPONSORING OCS REGION: Gulf of Mexico OCS Region 

APPLICABLE PLANNING AREA: Central Gulf of Mexico 

FISCAL YEARS OF PROJECT FUNDING: 1998-2001 

COMPLETION DATE OF REPORT: September 2001 

COSTS: FY 1998 $88,250; FY 1999 $63,369; FY 2000 $34,704; CUMULATIVE 

PROJECT COST: $186,323 

PROJECT MANAGERS: John W. Fleeger and David W. Foltz 

AFFILIATION: Louisiana State University 

ADDRESS: Department of Biological Sciences, 508 Life Science Building, Baton 

Rouge, LA 70803 

PRINCIPAL INVESTIGATORS*: John W. Fleeger and David W. Foltz 

KEYWORDS: cryptic species, meiofauna, harpacticoid copepods, genetic diversity, 

toxicity tests 

BACKGROUND: Research associated with the GOOMEX project found a reduction in 

genetic diversity in meiobenthic copepods found in close proximity to production 

platforms. Specifically, the16S rRNA gene of the mitochondrial genome in several 

meiobenthic harpacticoid copepod species was analyzed by polymerase chain reaction 

(PCR) and restriction fragment length polymorphism (RFLP) analysis. Such a loss of 

genetic diversity may have several causes. A reduction in genetic diversity may be the 

direct result of exposure to contamination via the process of genotypic selection. 

Alternatively, it is possible that some of the mitochondrial DNA genotypes found in the 

GOOMEX study represent interspecific variation in the form of cryptic species rather 

than within- species variants. If cryptic species with differing levels of tolerance to 

sediment contaminants occur among harpacticoids, the apparent reduction in genetic 

diversity near platforms could represent a loss of species diversity rather than genotype 

selection. 
ACCESS NUMBER: 30660-19949

OBJECTIVES: Using a multilocus genetic approach and limited morphological 

observations, we analyzed Cletocamptus deitersi (a common harpacticoid copepod 

frequently found at contaminated sites with known morphological variability and highly 

cosmopolitan in distribution) 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 level of 

interspecific genetic differentiation. An undescribed harpacticoid and a planktonic 

calanoid 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. 

DESCRIPTION: C. deitersi was collected at an inland brine seep near Jackson, 

Alabama and at two locations on the coast of Louisiana. C. deitersi was also obtained 

from laboratory cultures established from collections made in the Salton Sea, California 

and at the mouth of the "Estero del Yugo" estuary in Mazatlán, Sinaloa, Mexico. All 

copepods were subject to an initial PCR amplification of the mitochondrial DNA 

(mtDNA) cytochrome oxidase subunit I (COX-I) gene. We sequenced two additional 

gene regions in copepods representative of the major lineages identified on the basis of 

COX-I sequences (1) part of the mitochondrial large subunit ribosomal DNA (LSU rDNA 

16S) and (2) part of the nuclear rDNA, comprising the entire ITS1, ITS2 and the 

intervening 5.8S rDNA. Limited morphological observations were made on the third 

swimming leg of specimens from all locations. Toxicity bioassays, over 96 hours, were 

conducted on specimens from Alabama and Louisiana for tolerance to metals and 

phenanthrene. 

SIGNIFICANT CONCLUSIONS: Our genetic analyses and limited morphological 

comparisons strongly suggest that C. deitersi in North America is not mono-specific. 

Our analysis identified four putative species among specimens obtained from four 

widely separated locations. Based on our molecular data, two species (referred to as 

type I and II below) within 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. Furthermore, 

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. Type I and type II C. deitersi are also both relatively tolerant 

to a mix of Zn, Pb and Cd (at 6249 g Zn/l, 685 g Pb/l and 62 g Cd/l, based on ratios 

present in produced water). Mortality in exposed groups was significantly greater than 

in controls for both species. 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). 
ACCESS NUMBER: 30660-19949

STUDY RESULTS: 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. The harpacticoid 

copepod Cletocamptus deitersi is not mono-specific in North America but exists as a 

complex of at least four species. Secondly, cryptic species must co-occur at 

uncontaminated sites. Our research found that cryptic species of C. deitersi are 

sympatric at two locations. Thirdly, cryptic species must exhibit different responses to 

contaminants such that differential mortality occurs at contaminated sites. Toxicity 

bioassays with C. deitersi from two locations found a difference in tolerance to metals. 

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. 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 primarily by facilitating the identification of 

cryptic species by examining all of the nucleotide diversity of gene region. 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. 

STUDY PRODUCTS: Fleeger, J.W., D.W. Foltz and A. Rocha-Olivares. 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, 48 pP. 

*P.I.’s affiliation may be different than that listed for Project Managers.