HCRI Project Final Report Format
I.
Report Title: Characterizing green algal biodiversity of Hawaiian reef and estuarine communities:
expansion of the sequence diversity assessment framework to distinguish native from alien genotypes
Author: Alison R. Sherwood and Gernot G. Presting
Organization: University of Hawaii
Grant Number: NOA06NOS4260200
Date: 30 November 2007
II.
Abstract
We have modified an existing DNA sequence diversity assessment procedure that is currently being
applied to Hawaiian red algae to characterize the coral reef and estuarine green algal diversity of Hawai‘i.
Such a system has strong potential for rapid and accurate characterization of algae that may be difficult or
impossible to identify based on available morphological characters. This well-rounded DNA-based
assessment procedure overcomes many of the difficulties associated with traditional taxonomic work and
builds on the strengths of modern DNA-barcoding (see Approach and Methodology for more details). The
acquired data have the potential to allow detection of different strains within a species, which may be of
importance in monitoring invasive strains of some green algae, such as Caulerpa taxifolia. The results of
this study – a strongly populated sequence diversity framework for Hawaiian green algae, will be invaluable
in rapid assessments of native versus alien genotypes, and will serve as a baseline profile for current coral
reef and estuarine species composition. These data, which will profile shallow native reef habitats and
estuarine areas, are a direct response to Priority 1.
III.
Executive Summary
To be provided with the Final Report.
IV.
Purpose
A.
Detailed description of the HCRI research priority addressed.
This project responds to Priority 1 - Native biotic reef and closely related aquatic habitats (profiling native
biotic reef habitats in shallow nearshore areas and estuarine habitats).
B. Objectives of the project.
The goal of the proposed research is to use DNA barcode-based taxonomic techniques to develop a
contextual framework for rapid identification of Hawaiian green algae. The benchmark for achieving this
goal will be inclusion of most or all known Hawaiian marine and estuarine green algae in the framework,
and evidence that the framework can be used for accurate species identification. Specific objectives of the
study are:
a) to tailor an existing DNA sequence diversity procedure for red algae (using two gene regions) so
it can be applied to Hawaiian green algae,
b) to populate the resulting sequence framework with all available marine and estuarine Hawaiian
green algal species using both archived material (Bishop Museum) and new field collections, and
c) as the sequence framework becomes populated, to evaluate the limits of the framework as an
identification tool by comparing morphological identifications with DNA sequences for species represented
by multiple accessions.
V.
Approach
A. Detailed description of the work that was performed.
To date (November 2007), all four inter-island collecting trips have been completed (one each to
Kauai and Maui, and two to Hawaii), and a number of collecting trips have also been made around the
island of Oahu. Additional trips to Lanai and Molokai were made by students and researchers in the
Sherwood lab for other projects, and green algal samples for the current study were also obtained to
support the current project. Approximately 250 collections of green algae have been collected from marine
and estuarine habitats, and morphological vouchers (herbarium sheets and / or formalin samples) have
been prepared for all collections. Approximately 60 collections from Bishop Museum have been sampled by
A. Sherwood to date. All green algal samples that have been extracted have had PCR attempted at least
once.
We originally proposed to amplify and sequence two markers – UPA (a plastid 23S rRNA marker)
and COI (mitochondrial cytochrome oxidase subunit I, which is used as the DNA barcode for most groups
of organisms). Initial PCR success rates in June 2007 for the 23S rRNA plastid marker were 20-25%, which
was much lower than anticipated based on preliminary data collection. In total, we have attempted PCR at
least once for every DNA extract using the UPA protocols, but have been unable to increase success for
the green algae. As a result, we have focused attention in recent months on identifying and testing
additional markers to use for the sequence assessment. The mitochondrial COI gene was initially proposed
as the second marker, but subsequent literature reviews have indicated that this region is inappropriate for
our purposes given the widespread occurrence of introns in the COI gene for green algae. Additionally, the
Consortium for the Barcode of Life has yet to endorse specific region(s) for land plant barcoding, which
further illustrates the difficulty in developing such a protocol for closely related organisms to the green
algae. Nonetheless, we have attempted PCR and sequencing of several candidate second markers, with
complex results.
We have investigated several “second markers” since June 2007, including the nuclear ITS regions
(which are commonly used for taxonomic purposes for at least some green algae), a fragment of the
nuclear LSU gene (again, also used commonly for some green algal groups), and approximately 50% of
the plastid rbcL gene (generally used at or above the species level in green algae). Sequencing results vary
depending on the green algal order to which samples belong. For example, the nuclear ITS region can be
successfully sequenced from members of the Ulvales, and possibly also the Dasycladales and
Bryopsidales (although resulting sequences match more closely to the Ulvales), while the nuclear LSU
region is much more successful, and can be sequenced from basal chlorophytes, Ulvales, Bryopsidales
and Cladophorales. The plastid rbcL gene sequencing is not successful with the order Cladophorales, but
works well for the Bryopsidales and Ulvales. As a result, we are not employing the same second marker for
all green algal taxa, but are dividing them so that some are sequenced for ITS, some for LSU, and some for
rbcL.
The most successful order of green algae in our analyses has been the Ulvales, for which we are
able to sequence all markers tested. Additionally, this group of algae contains species of concern, species
that are very difficult to discern morphologically, and species that have not been previously recognized as
members of the Hawaiian green algal flora. Thus, we have identified this order as one that deserves
detailed attention. To date, we have collected 81 samples that can be identified as members of Ulva or
“Enteromorpha” (currently a form genus), and are planning a separate analysis of these samples that
should result in a peer-reviewed publication sometime in 2008.
The community outreach component of the project is twofold: 1) a public presentation explaining
the concepts behind using DNA technologies for identification purposes, and 2) construction and release of
a publicly accessible database that houses the data collected for the project. The first component will be
presented in early 2008 by Tara Chandrasekharan. She has been working with a professor in the
Philosophy Department at UH to identify classrooms that would be interested in hearing her speak about
the project, and although this could not be included in the fall curriculum for the schools, it will be completed
in early 2008. The second component (the project database) is explained under section IX, dissemination
of results.
In late summer 2007 we contacted state resource managers and explained the nature of project,
and sent instructions on how to send samples for analysis and inclusion in the project. These requests
resulted in a number of collections from the island of Maui, which were a great help to the study. The
informational brochure that was constructed and distributed is appended to this report.
B. Project management: List individuals and/or organizations actually performing the work
and how it was done.
Dr. Alison R. Sherwood, Assistant Professor (Botany Department, University of Hawaii) – P.I.
Dr. Gernot G. Presting, Assistant Professor (MBBE, University of Hawaii) – co-P.I.
Tara Chandrasekharan, undergraduate student research assistant (U. of Hawaii)
The P.I. (Alison Sherwood) is responsible for overseeing the spending of funds as outlined in the
budget and justification, and for the hiring of student assistance to assist with sample processing. Co-P.I.
Gernot Presting assists with training, and oversees the management of the large amount of DNA sequence
data that are being generated through the study, and advises the design and maintenance of the project
database. Tara Chandrasekharan (student research assistant) undertakes the majority of the field work to
collect specimens (in coordination with other researchers in the Sherwood lab), and sample processing
(DNA extraction and making vouchers, PCR, sequencing and data assembly). Sherwood and Presting
analyze the data and lead troubleshooting efforts. Sherwood will undertake the final data analyses and will
take the lead on publishing the results from the study.
VI.
Findings To be included in final report.
A. Actual accomplishments and findings.

Answers to the stated Management Question(s)in your proposal
B. Site specific results
VII.
Evaluation To be included in final report.
A. Describe the extent to which the project goals and objectives were attained. This
description should address the following:
1. Were the goals and objectives attained? How? If not, why?
2. Were modifications made to the goals and objectives? If so, explain.
3. If significant problems developed which resulted in less than satisfactory or negative
results, they should be discussed.
4. Description of need, if any, for additional work.
B. Indicators of success s (What performance measures were used to evaluate how well the
project met its stated goals and objectives?)
VIII.
Recommendations to managers
To be included in final report.
IX.
Dissemination of Project results:
A. Explain, in detail, how the projects results have been, and will be, disseminated.
We have constructed a relational database to house all forms of data generated by studies such as
this HCRI green algal biodiversity study. The database was under construction from August 2006 – April
2007 (funded by the National Science Foundation), and is now being used on a daily basis by the
Sherwood lab as a central repository for data. Over the remainder of the project we will be adding in
photographs of herbarium sheets and microscope slide vouchers, nomenclatural information, collecting site
information, and DNA sequences for all collections made for this study. These data will be eventually
available via an internet-accessible website (currently projected for release in fall 2008).
We also expect that two peer-reviewed journal articles will result from the research (outlining the
construction of the DNA barcode framework for green algae and presenting comparisons between the
frameworks based on the markers, and analyzing the Ulva specimens), and there is also strong potential
for descriptions of new taxa that may be revealed through the barcode comparisons. These manuscripts
will be in preparation in early 2008.
B. List of publications and presentations
None to date. Anticipated for end of 2007 / beginning of 2008.