CARLON- YEAR 9
HCRI Project Progress Report #2
I.
Project Title: Of Urchins and parrotfish – sources and sinks of keystone herbivores
Principle Investigator: David B. Carlon,
Project Staff: Joanna Bince (Technician), John Fitzpatrick (Graduate Student)
Organization: Department of Zoology, University of Hawaii
Grant Number: NOA06NOS4260200
Date: November 30, 2007
II.
Executive Summary
The astounding biological diversity on coral reefs support thriving fishing and
tourist economies, yet in the last 20 years it has become increasingly clear that the
structure, diversity, and function of coral reef ecosystems is strongly influenced by the
effects of fish and invertebrate consumers on tropical seaweeds (macroalgae). In the
absence of herbivorous fish and invertebrate grazers, tropical coral reefs can undergo
“phase shifts” in community structure from coral dominated to macroalgae dominated
systems. In the main Hawaiian Islands, alien species of macroalgae are dominating many
reefs once characterized by high coral, and low algal abundance. Comparisons between
the Northwest and Main Hawaiian Islands strongly suggest that historical and present
levels of fishing have reduced the herbivore community on the densely populated Main
Islands to the point where top-down control of macroalgae is lacking. It follows, that
management of reefs to maximize biodiversity and function should concentrate on
maintaining healthy stocks of keystone grazers. To design Marine Protected Areas
(MPAs) and implement new fishing policies, the state of Hawaii needs information on the
movement of larvae of keystone species within and between islands. Our approach is to
use high resolution genetic data to understand whether islands or populations are isolated
or connected. This year, we also propose to test the idea that deep water (>20 m) softsediment habitats provide a refuge for urchin recruits. Our 2006-07 proposal has three
objectives:
1. To describe the population structure of two keystone species: the collector urchin
Tripneustes gratilla and the ember parrotfish Scarus rubroviolaceus within and among
the main Hawaiian Islands.
2. To determine if deep-water, soft-sediment habitats act as nurseries for Tripneustes
gratilla on coral reefs.
3. To integrate data on population structure and nursery areas to design MPAs for
keystone reef organisms.
This 2006-07 proposal builds on, and is a significant extension of research funded in
2005-06. First, we are using our preliminary genetic data to guide choice of additional
sampling sites on Hawaii and Kauai, and to resolve fine-scale structure that will be useful
to DAR in designing and implementing management strategy. Second, we are adding a
second, charismatic reef fish (Scarus rubroviolaceous) with a similar free-spawning life
history that provides an independent test of patterns and general hypotheses of the
oceanographic features controlling population structure. Third, we are investigating the
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intriguing hypothesis that recruitment of the keystone urchins is occurring in deep-water,
soft sediment habitat with secondary migration to shallow-reef habitat. If true, this
pattern has important implications for the inclusion of these habitats into management
strategy that aims to promote diversity and function on coral reefs.
III.
Purpose
A. Detailed description of the resource management problem(s) to be addressed.
“In order to design ecologically effective marine protected areas, basic information on
important species is critical. At present, the basic knowledge of reef organisms'
population structure is inadequate to design a management regime to improve the
sustainability of species of concern, such as: Gracilaria parvispora, Gracilaria
coronopifolia, Grateloupia filicina, Ahnfeltiopsis concinna, Laurencia nidifica, edible sea
cucumber, Achilles tang, spiny lobster, parrotfish, weke, octopus, Tripnuestes gratilla,
Echinothrix calamaris, Echinometra mathaei, Echinostrephus acciculatus, cowries and
other commonly collected limu and mollusk species.”
“The Management Committee is seeking proposals for projects that will: identify
specific problems; propose recommendations for design of management options to ensure
their sustainability; provide tools for measuring the effectiveness of recommended
management options for species of concern; and produce objective criteria for
determining, assessing, and comparing sites for possible management action for species
of concern…”
This proposal will produce objective criteria based on genetic estimates of migration
within and among islands and the ecological function of soft-sediment habitats as nursery
areas for keystone species. Sites can be evaluated in terms of their local (sinks) or
regional (sources) contributions of larval recruits, as well as whether they harbor unique
genetic diversity that may prevent population reduction and local extinction in the face of
environmental change.
B. Detailed description of the question(s) asked to answer the resource management
problem(s)
1. What localities/habitats represent either sources or sinks for parrotfish and urchin
larvae?
2. Are recruitment rates are higher in deep-water soft-sediment habitats compared to
shallow soft sediment and reef habitats?
3. Do juveniles migrate from deep soft-sediment habitat to shallow reef areas?
C. Objectives to answer each question.
1. To describe the population structure of two keystone species: the collector urchin
Tripneustes gratilla and the ember parrotfish Scarus rubroviolaceus within and
among the main Hawaiian Islands.
2. To determine if deep-water, soft-sediment habitats act as nurseries for Tripneustes
gratilla on coral reefs.
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3. To integrate data on population structure and nursery areas to design MPAs for
keystone reef organisms.
IV.
Approach
Detailed description of the work performed for each objective from III(C), including
(but not limited to):
A. list individuals and organizations actually performing the work
David B. Carlon (PI), University of Hawaii at Manoa
Joanna Bince (Technician), University of Hawaii
John Fitzpatrick (Graduate Student), University of Hawaii at Manoa
B. material list (See D and E)
C. construction instructions for anything used to accomplish the III(C)
objectives (NA)
D. deployment steps
Question 1. What localities/habitats represent either sources or sinks for parrotfish
and urchin larvae?
We have collected Scarus rubroviolaceus tissue samples from the following islands
and sites.
Table 1. Collection islands and localities for individual tissue samples of ember
parrotfish Scarus rubrioviolaceus
Hawaiian island
Locality
n
Partners
Oahu
North Shore,
Haleiwa
23
George Matsuda,
Alii Holo Kai dive
club
North Shore,
Waiale'e
27
George Matsuda,
Alii Holo Kai dive
club
Maui
Pacific Fish Market
and Oki’s Seafood
Corner
24
Skippy Hau, DLNR
Big Island, Hawaii
Ho’okena
6
Fitzpatrick, noninvasive night
diving
Big Island, Hawaii
Hilo
14
Tanya Bearn
Since we are not detecting evidence for genetic structure in urchins or parrotfish
within the main Hawaiian Islands, nor with comparisons between the more distant
Northwest Hawaiian Islands and Johnston Atoll, we have collaboratively obtained
additional tissue samples of both species from populations that occur throughout the
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biogegeographic ranges. Collaborative institutions include the Hawaiian Institute of
Marine Biology, Smithsonian Tropical Research Institute, Panama; James Cook
University, Australia; and the Tokyo Institute of Technology. We are sequencing and
genotyping these samples to determine the spatial scale at which gene flow declines.
data collection procedures
Question 1. What localities/habitats represent either sources or sinks for parrotfish
and urchin larvae?
Genotyping and analyses of population structure
From each genetic sample from tube feet (urchins) or fin clips (fish), genomic
DNA was extracted with QIAGEN DNeasy tissue kits. DNA was quantified on a
Nanodrop spectrophotometer and diluted to 10-20 ng µl-1. We used the Polymerase
Chain Reaction (PCR) to amplify a portion of the mitochondrial gene cytochrome
oxidase I (COI) using primers designed specifically for Tripneustes. The PCR recipe
for a 25 µl reaction was as follows: 1.0 µl (10-20 ng µl-1) of DNA, 18 µl H20, 1 µl
MgCl2 (25 mM), 1.0 µl dNTP mix (8 mM), forward primer 0.3 µl (10 μm), reverse
primer 0.3 µl (10 μm), 1 µl Taq Biolase DNA Polymerase.
PCR was conducted in a TC-200 MJ Peltier thermocycler (MJ Research). The PCR
temperature profile consisted of denaturation at 94°C for 10 min, then 35 cycles at
94°C for 30 s, 56 for 40 s, 72°C for 1 min. We used a final elongation at 72°C for 10
min. PCR Products were directly sequenced after first incubating with the ExoSAPit
Kit (USB Corporation). We used the ASGPB Sequencing Facility (Snyder Hall,
University of Hawaii at Manoa) for all sequencing and genotyping related to this
project. This facility uses Applied Biosystems XL 3730 Capillary Sequencers, and
BigDye sequencing chemistry.
Microsatellite genotyping
Loci were divided into sets for muliplex PCR. Polymerase chain reactions (PCR)
were carried out in a 11-µl reactions containing: 1 µl (25-50 ng) of total genomic
DNA, 16 mM (NH4)2SO4; 67 mM Tris-HCl (pH 8.8 at 25°C), 0.01% Tween-20, 2.0
mM MgCl2, 0.08 mM of each dNTPs, 0.18 mM of each primer, and 0.5 U of Taq
Biolase DNA Polymerase (Bioline USA, Randolph, Massachusetts). Amplifications
were performed with a PTC-200 MJ Peltier thermocycler (MJ Research), and PCR
temperature profile consisted of denaturation at 94°C for 10 min, then 35 cycles at
94°C for 30 s, Ta°C (depending on locus) for 40 s and 72°C for 1 min, followed by a
final elongation at 72°C for 10 min. Primers for eleven polymorphic microsatellite
loci were fluorescently labeled, the products genotyped on an ABI 3730 XL
sequencer, and scored with GeneMapper software (Applied Biosystems Inc.).
We extract DNA using Qiagen DnEasy columns, then use PCR and fluorescently
labeled primers to amplify microsatellite products that are scored on ABI 3730XL
capillary sequencers housed in The Center for Genomics, Proteomics, and
Bioinformatics “CGPBRI”(http://cgpbr.hawaii.edu/) located in Snyder Hall. We use
multiplex PCR reactions involving 3-4 primer sets, and optimize scoring of 5-7 loci
on a single capillary lane. Motifs are identified with ABI’s GENEMAPPER
software, and alleles are sized with ALLELOGRAM.
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E. data analysis techniques
Question 1. What localities/habitats represent either sources or sinks for parrotfish
and urchin larvae?
The first step is to determine the scale of genetic structure which indicates the scale at
which dispersal declines. A standardized measure of genetic structure, FST is
calculated between localities, islands, and biogeographic regions. We use the
software packages ARLEQUIN (Schneider et al. 2000) for mitochondrial sequence
data (mtDNA) and MSA Anaylzer (Dieringer and Schlotterer 2003) for microsatellite
genotype data.
Second, we use a variety of Bayesian statistical tools (Beaumont and Rannala 2004)
to analyze population structure in more complex and realistic models that do not
require equilibrium assumptions. These algorithms use likelihood functions to find
the optimal model of K populations. For example, we can estimate the total number
of populations in the sample by using the model STRUCTURE (Pritchard et al.
2000). Samples are input into the model with no apriori information on geographic
location, and the algorithm assembles populations based on the best fit without
violating Hardy-Weinberg expectations.
photos from research during each stage (construction, in situ, lab)
F. contact information for companies used to purchase items unique to your
project (if applicable)
G. Modification of project scope with regards to Questions 2 and 3.
2. Are recruitment rates are higher in deep-water soft-sediment habitats compared to
shallow soft sediment and reef habitats?
3. Do juveniles migrate from deep soft-sediment habitat to shallow reef areas?
Our natural history observations of recruitment of juvenile Tripneustes gratilla
reveal that recruitment is extremely episodic in time and space in the Hawaiian
Islands. We find that small juveniles start emerging onto exposed benthic surfaces in
the late summer and fall, but exactly where they do this within islands does is not
predictable on Oahu and Maui among years. This biological reality makes answering
questions 2 and 3 in the time-frame of a 1-year funding cycle impractical and unlikely
to produce answers to these important questions.
For the remainder of this year we will focus on completing the genetic data analyses
of all urchin and fish samples, to get a more complete picture of connectivity between
Hawaii and her neighboring territories and countries in the Pacific.
V.
Results
A. Findings for each III(C) objective.
Objective 1- To describe the population structure of two keystone species: the
collector urchin Tripneustes gratilla and the ember parrotfish Scarus rubroviolaceus
within and among the main Hawaiian Islands.
Urchins (Tripneustes gratilla)- We have completed all mtDNA sequencing and
microsatellite genotyping for over 400 samples of T. gratilla from the main Hawaiian
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Islands (MHIs), Kure Atoll, and Johnston Atoll. Both FST analysis and population
modeling indicate no genetic structure at these spatial scales. Our interpretation is that
planktonic urchin larvae are regularly dispersing among the MHIs; and among the
MHIs and the Northwest Islands and Johnston Atoll.
Parrotfish (Scarus rubrioviolaceus)- We have completed mtDNA sequencing and
genotyping off all S. rubrioviolaceus samples among the MHIs listed in Table 1
(Section IV.D above). Both FST analysis and population modeling indicate no genetic
structure at these spatial scales. Our interpretation is that planktonic parrotfish larvae
are regularly dispersing among the MHIs.
B. Answers to III(B) each resource management question(s).
B.1.What localities/habitats represent either sources or sinks for parrotfish and urchin
larvae?
Our data indicate dispersal capabilities by of urchin and parrotfish are broad. There
are strong larval connections between all Main Hawaiian Islands (MHIs), as well as
between the MHIs and the Northwest Hawaiian Islands and Johnston Atoll. These
results emphasize a need to manage reef systems within the state of Hawaii as a
collective network, anticipating that local management strategies can have effects on
distant reef systems.
C. Site specific results for each location (Can place in an appendix as electronic
file).
VI.
Resource Management Implications (NA for Progress Reports)
VII.
Evaluation (NA for Progress Reports)
VIII. Dissemination of Project results
A. Explain, in detail, how the projects results have been, and will be, disseminated.
We have presented two oral papers (titles below) this Fall from HCRI sponsored
research at the annual Western Society of Naturalist Meeting in Ventura, CA. We are
working on 2 publications from HCRI sponsored research. The first describes
population structure in Hawaii and elsewhere of the urchin Tripneustes gratilla, the
second describes population structure of the parrotfish Scarus rubroviolaceus.
B. List of publications, workshops, and presentations

Fitzpatrick, J., Lippé, C., and D. B. Carlon. How many populations? The
genetic structure of the ember parrotfish (Scarus rubrioviolaceus) throughout the
Indian and Pacific oceans. Oral presentation at the Western Society of
Naturalists, Ventura, CA.

Carlon, D. B. and C. Lippé. How many populations too? The genetic structure of
the urchin Tripneustes gratilla from Panama to the Red Sea. Oral presentation at
the Western Society of Naturalists, Ventura, CA.
C. Data or information products
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D. Partnerships established with agencies or organizations

DLNR

Cooperative Fisheries Unit, University of Hawaii

Alii Holo Kai dive club
References
Beaumont, M. A., and B. Rannala. 2004. The Bayesian revolution in genetics. Nature Reviews
Genetics 5:251-261.
Dieringer, D., and C. Schlotterer. 2003. MICROSATELLITE ANALYSER (MSA): a platform
independent analysis tool for large microsatellite data sets. Molecular Ecology Notes
3:167-169.
Pritchard, J. K., M. Stephens, and P. Donnelly. 2000. Inference of population structure using
multilocus genotype data. Genetics 155:945-959.
Schneider, S., J. D. Roessli, and L. Excoffier. 2000. Arlequin: a computer program for
population genetics data analysis.
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