Sponsoring Institution
National Institute of Food and Agriculture
Project Status
Funding Source
Reporting Frequency
Accession No.
Grant No.
Project No.
Proposal No.
Multistate No.
Program Code
Project Start Date
Oct 1, 2007
Project End Date
Sep 30, 2010
Grant Year
Project Director
Klein, A. S.
Recipient Organization
Performing Department
Biological Sciences
Non Technical Summary
Global commerce on the oceans has led to the introduction of nonindigenous organisms into coastal ecosystems. Sometimes the introduced species may alter local ecosystems by disrupting juvenile fish habitats or harming shellfish beds, or may foul beaches as large mats of algae wash ashore after storms. The purpose of this project is use molecular genetic tools to examine how Neosphonia harveyi(doughball) is spreading in the Gulf of Maine and assess whether another invasive algal species, Codium fragile ssp. tomentosoides (Dead man's fingers), is an important vector for increased dispersion of N. harveyi.
Animal Health Component
Research Effort Categories

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
Goals / Objectives
The objectives of this project are to: 1) develop molecular markers for higher resolution population structure of the invasive algal species Neoshiponia harveyi (doughball) 2) survey new and extant collections of the algae from Long Island Sound to the Gulf of Maine for genetic variation. The resulting genetic haplotypes of New England accessions will be compared to those in Europe and Japan by exchange of herbarium specimens or DNAs with investigators in Ireland and Japan in order to: a) determine if there has been a single or multiple introductions of Neosiphonia harveyi in the coastal waters of New England. b) determine if the genetic profile of N. harveyi in New England has remained stable since the mid 20th century or has changed in parallel with the expansion of the invasive alga in recent decades. 3) test the hypothesis that C. fragile ssp. tomentosoides is the principal vector for N. harveyi in New England by comparing the distributions of genotypes of the two taxa. Results from this AES project on N. harveyi will be contrasted with the outcomes of an ongoing Sea Grant project on C. fragile ssp. tomentosoides. 4) prepare outreach materials to enable students and members of the general public to recognize and report new populations of Neosiphonia harveyi. The materials will be made available through existing web sites and through brochures available at National Estuary Program locations in New England.
Project Methods
Hyper-variable genetic markers will be identified to distinquish haplotypes by amplification and sequencing of the nuclear and organelle genes with the the polymerase chain reaction. A preliminary geographic survey will be used to identify markers that distinguish populations. Subsequently larger surveys of the population structure and phylogeographic reconstructions of historical and extant populations of N. harveryi along coastal New England. The haplotypes of archival (herbarium) and extant populations in New England will be compared to samples from the sea of Japan to determine whether there has been a single or multiple introduction of the doughball to coastal New England. The patterns of N. harveyi and C. fragile ssp. tomentosoides haplotypes will be compared to test the hypothesis that the latter is a vector for dispersion of N. harveyi. Outreach materials materials, with pictures of the algae, a description of its habitat, and describing the ecological consequences will be distributed as a pamphlet at the NH Seacoast Science Center and the Great Bay Sandy Point Discover Center. The pamphlet will also be distributed through similar National Estuary Sites along the coast of New England. This pamphlet will be tied to a web site, hosted at UNH, which will provide maps showing historical and current distributions of the alga. A project web site will provide a reporting mechanism for the public to record new populations of N. harveyi.

Progress 10/01/07 to 09/30/10

OUTPUTS: The current distribution of C. fragile in the NW Atlantic (from Long Island Sound to Atlantic Canada) was surveyed over two summers ( The distribution of both C. fragile and N. harveyi from NY to Maine were also surveyed during the summer of 2010 as part of the MIT Sea Grant's Rapid Assessment program (; note that the results of the 2010 Rapid Assessment have not yet been posted to the web site). We used chloroplast haplotype determination for the rps3-rpl16 locus to confirm that C. fragile subsp. fragile is the predominant introduced Codium taxon in the NW Atlantic (GenBank FJ754465-FJ754478l; GQ177974-178005). Further we determined that NW Atlantic accessions had the same haplotype at the trnG locus (GenBank accessions GQ274899-GQ274924) indicating a common source as the introduced C. fragile subsp. fragile populations in the NE Atlantic, and with indigenous alga from Kochi, Sagami Bay and Nakagi Japan (cf. Provan et al. 2005). PARTICIPANTS: collaborators: Prof. Arthur Mathieson, University of New Hampshire Prof. David Garbary, St. Frances Xavier College Antigonish Nova Scotia Dr. Judith Pederson, MIT Sea Grant Training and professional development: Lucy E. Pleticha, MS degree UNH Plant Biology 2009 Christopher S. Benton, ongoing Ph.D. student UNH Biochemistry Renee Eriksen, ongoing Ph.D. student UNH Plant Biology Stephen McKernan, undergraduate Molecular Cellular and Developmental Biology UNH TARGET AUDIENCES: The target audiences for this research included other Phycologists (especially those studying invasive species), students, members of the general public, fishermen, and managers of National Estuary Program in locations throughout New England. The C. fragile project was featured in a NH SeaGrant publication ( This account was highlighted on Maine Public radio in December 2009 ( temId/10304/Default.aspx); the report noted the public has a role in preventing further spread of invasives by removing Codium from boats and fishing gear. Project outcomes have been disseminated through genetic databases (GenBank), brochures on C. fragile, web sites (; and through manuscripts planned for submission to professional journals. PROJECT MODIFICATIONS: The overall goal of the project was to test the hypothesis that C. fragile is serving as the principal vector for recent dispersal of N. harveyi in the NW Atlantic. We have not yet been able to develop robust and highly variable markers for a phylogeographic study of the spread of C. fragile within the NW Atlantic. Surveys of extant populations of both species have been conducted; tissue and/or DNA samples of these collections are available when better markers become available. A partial transcriptome of C. fragile was produced at the very end of this project; this may provide new markers for future studies.

The overall goal of the project was to use phylogenetic approaches to determine whether the recent range expansion of the Neosiphonia harveyi in the NW Atlantic was linked to the spread of another invasive species, Codium fragile. Progress was toward the answering this question was limited because of the difficulty encountered in developing robust genetic markers sufficiently variable to track the dispersal of the proposed vector with the NW Atlantic. Previous work by several investigators (Hubbard and Garbary 2002, Kusakina et al. 2006) suggested that the C. fragile populations from Prince Edward Island Canada (PEI) represented a separate introduction or second invasive subsp. atlanticum. We recently discovered a novel haplotype for four samples of C. fragile collected from PEI by Garbary and Benton in the summer of 2009. The haplotype of the PEI accessions is most closely related to C. fragile subsp. fragile, not subsp. atlanticum. We concluded from a detailed morphometric survey of NW Atlantic C. fragile subsp. fragile that there is considerable phenotypic plasticity in the utricle features typically used to differentiate the two subsp. Chloroplast haplotype differences are therefore more accurate for diagnostic differentiation of the subspecies; similar conclusions were drawn by Provan and coworkers (2007) from a broader geographic survey of extant and herbarium C. fragile subsp. accessions.


  • Klein,A.S., Pleticha,L.E., Benton, C.S. and Mathieson,A.C. Morphological variation within Codium fragile subsp. fragile in the NW Atlantic. Botanica Marina, 2011 (pending)

Progress 10/01/08 to 09/30/09

OUTPUTS: Range wide collections of Codium fragile from Long Island Sound to the Canadian Maritimes were evaluated for morphological variation and for chloroplast haplotype. Morphological variation was observed for `diagnostic' characters of C. fragile subspecies: utricle length, narrowest and widest diameter, and mucron length. DNAs were isolated from representative Codium fragile from populations in Long Island Sound, the Gulf of Maine and the Canadian Maritimes. The haplotypes were determined for the chloroplast regions rps3-rpl16, trnG, and the rbcL intron. A limited number of herbarium samples dating from the early 1970's were evaluate for the rps3-rpl16 locus; these archival materials appear to have the same haplotype as Codium fragile subsp. fragile. New molecular markers are being developed for Codium nuclear loci;it is hoped that these markers will be more variable and therefore useful to examine population structure of the introduced species. Neosiphonia harveyii populations were collected from twelve locations in southern New England and in the Gulf of Maine. These populations will be screened for genetic structure in the last year of the project. One scientific presentation was provided: Christopher S. Benton, Lucy E. Pleticha, Anita S. Klein, and Arthur C. Mathieson Molecular and morphological variation in Codium fragile in the NW Atlantic;Joint meeting American Society of Plant Biology and Phycological Society of America July 2009 P04034; Plant Biology 2009 Final Program pg.120 PARTICIPANTS: PI Klein supervised the research activities of three graduate students, assisting them with analyses, preparation of posters presentations and in the case of one, a M.S. thesis. Three graduate students contributed to this project: Lucy Pleticha carried out a morphometric comparison of NW Atlantic Codium fragile for her MS thesis. Chris Benton is characterizing the chloroplast haplotypes of Codium fragile and is developing nuclear markers for a comparative population genetic survey of Codium that will be part of his Ph.D. research. Renee Eriksen, a second Ph.D. student,initiated collections of a second Japanese invasive species Neosiphonia harveyi,and will carry out genetic studies. The dispersal of this Neosiphonia has been linked to drifting populations of Codium. Professors Arthur Mathieson, and Larry Harris, also in the Department of Biological Sciences are collaborating in this project. Mathieson is the principal resource for all field collections. Mathieson and Harris have monitored the spread of Codium fragile in the Gulf of Maine for the last 35 years. TARGET AUDIENCES: The information gained in these studies are important for managers of estuarine reserves and coastal resources in the Gulf of Maine. PROJECT MODIFICATIONS: We have not yet developed molecular markers for higher resolution studies of population structure in Codium fragile. Delays in this objective (1) will limit our ability to complete object 3, to test the hypothesis that Codium fragile is the principal vector for the dispersion of Neosiphonia in the Gulf of Maine.

Statistical analysis suggested that C. fragile populations from Long Island Sound differed from more northern C. fragile populations (Gulf of Maine, Canadian Maritimes). All DNAs have chloroplast haplotypes consistent with the C. fragile subsp. fragile haplotype, which is common to Kochi, Sagami Bay, and Nakagi in Japan, and to invasive populations from the Northeast Atlantic. One of the experimental objectives of this project was to verify the existence of two subspecies of Codium fragile in Atlantic Canada. Graduate student Pleticha carried out an in depth morphometric analysis of NW Atlantic C. fragile populations. She observed that the distribution of utricle morphologies spanned the ranges previously reported for both subsp. atlanticum and subsp. fragile. Mucron size is the primary dichotomy between subsp. atlanticum and subsp. fragile. Plants with very small mucrons were observed in Long Island Sound. However, Graduate Student Christopher Benton showed that these plants had the chloroplast haplotype associated with C. fragile subsp. fragile. Pleticha (2009) concluded that neither overall utricle size nor mucron length was adequate to distinguish between Codium fragile subspecies.


  • (1)Lucy Pleticha Morphological variation in Codium fragile in the Northwest Atlantic. M.S. thesis, University of New Hampshire December 2009, 112 pgs. (2)Lucy E. Pleticha, Christopher S. Benton, Anita S. Klein, and Arthur C. Mathieson Molecular and morphological variation in Codium fragile in the NW Atlantic Lucy : P12, Northeast Algal Society Symposium, April 2009. (

Progress 10/01/07 to 09/30/08

OUTPUTS: The Japanese invasive green algae Codium fragile is thought to be a vector for the spread of another introduced species the red algae Neosiphonia harveyi. During the first year of this project activities centered on establishing the current distribution of Codium fragile in the Western North Atlantic from Long Island Sound North to the Canadian Maritimes. Collections were made in August, September and October, at 19 sites. A representative plant from each site was prepared as a herbarium specimens, and was deposited in the University of New Hampshire Albion Hodgdon Herbarium. Growing tissue from each sample was preserved for morphometric analysis of the utricles, a specialized tissue that is used to differentiate subspecies. Tissue samples were dried in silica gel, for DNA extraction and haplotype analysis. A database of collections was established in the lab. A web site ( was prepared that describes the problem of Codium fragile as an invasive species in the Gulf of Maine. The web site maps all known populations of C. fragile and invites the public to report new sightings (populations) of Codium. The preliminary version of the web site was demonstrated at the Northeast Algal Symposium, in April 2008, at the University of New Hampshire. PARTICIPANTS: Anita Klein, PI. Laboratory instruction, supervision and planning Collaborator: Arthur Mathieson. Advised on survey and collection work. Training: Lucy Pleticha, MS Student, Graduate Program in Plant Biology: Codium survey and collection, morphometric analysis, web site development, DNA extractions. Christopher Benton, Ph.D. Student, Graduate Program in Biochemistry. Codium survey and collection, DNA extraction, haplotype analysis Mathew R. MacKenzie, Undergraduate, Laboratory Maintenance, DNA extraction and amplification Allison Baldio, Undergraduate, DNA extraction and amplification TARGET AUDIENCES: A web site ( was prepared that describes the problem of Codium fragile as an invasive species in the Gulf of Maine. This web site is being publicized to the general public, K-12 science organizations, and to Fisheries cooperatives in the Gulf of Maine, to alert the public to the impact of invasive algal species on the ecosystem health. PROJECT MODIFICATIONS: Not relevant to this project.

The first year of this project has focused on the putative vector (Codium fragile) for distribution of Neosiphonia harveyi in the Gulf of Maine. Many of the Codium samples collected were found to harbor N. harveyi. In the second year of the project activities will be initiated specifically related to new marker development for N. harveyi, survey work and preparing outreach material for the public on the spread of this invasive red alga.


  • Lucy E. Pleticha, Anita S. Klein and Arthur C. Mathieson AN INTERACTIVE WEB SITE TO TRACK NEW POPULATIONS OF CODIUM FRAGILES SSP TOMENTOSOIDES IN THE GULF OF MAINE. Northeast Algal Society, Symposium, Durham, NH April 2008.