Carlon, HCRI Y.9 Final Report
CARLON- YEAR 9
HCRI FINAL REPORT
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: March 5, 2008
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) soft-sediment habitats provide a refuge for urchin recruits. This 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 intriguing hypothesis that recruitment of the keystone urchins is occurring in deep-water, soft sediment habitat with secondary migration to shallowreef habitat. If true, this pattern has important implications for the inclusion of these habitats into
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Carlon, HCRI Y.9 Final Report management strategy that aims to promote diversity and function on coral reefs.
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.
3.
To integrate data on population structure and nursery areas to design MPAs for keystone reef organisms.
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Approach
A.
list individuals and organizations actually performing the work
Carlon, HCRI Y.9 Final Report
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 listed in Table 1.
Table 1.
Islands, localities, sample sizes, and collection partners for samples of the ember parrotfish
Scarus rubrioviolaceus used in this study.
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
Skippy Hau, DLNR Maui Pacific Fish Market and Oki’s Seafood
Corner
Big Island, Hawaii Ho’okena
24
6 Fitzpatrick, noninvasive night diving
Tanya Bearn Big Island, Hawaii Hilo 14
Since we did not detect genetic structure in urchins or parrotfish within the Main
Hawaiian Islands (MHIs), nor in comparisons between the MHIs and the more distant Northwest
Hawaiian Islands and Johnston Atoll, we obtained additional tissue samples of both species from populations that occur throughout each biogegeographic range. Collaborative institutions and people include the Hawaiian Institute of Marine Biology (Dr. Rob Toonen), Smithsonian
Tropical Research Institute, Panama (Drs. Haris Lessios and Ross Robertson); James Cook
University, Australia (Dr. J. Howard Choat); and the Tokyo Institute of Technology (Dr. Nina
Yasuda). We have sequenced and genotyped these samples to determine the spatial scale at which gene flow declines.
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Carlon, HCRI Y.9 Final Report
E.
data collection procedures
Question 1. What localities/habitats represent either sources or sinks for parrotfish and urchin larvae?
Mitochondrial sequence data (mtDNA)
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) in Tripneustes and the control region (also called the D-loop) in Scarus . The PCR recipe for a 25 µl reaction was as follows: 1.0 µl (10-20 ng µl
-1
) of DNA, 18 µl H
2
0, 1 µl MgCl
2
(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
Microsatellite markers and PCR conditions are described completely in Carlon and Lippé
(2007a) for Tripneustes and Carlon and Lippé (2007b) for Scarus . Briefly, 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 (NH
4
)
2
SO
4
; 67 mM Tris-
HCl (pH 8.8 at 25°C), 0.01% Tween-20, 2.0 mM MgCl
2
, 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, T a
°C (depending on locus) for 40 s and 72°C for 1 min, followed by a final elongation at 72°C for 10 min. PCR products were scored using fluorescently labeled primers and genotyped on ABI 3730 XL automated sequencer. The resulting electropherograms were scored with GeneMapper software (Applied Biosystems Inc.). For genotyping we used multiplex PCR reactions involving 3-4 primer sets, and optimized scoring of 5-7 loci on a single capillary lane.
F.
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, F
ST
was calculated between localities, islands, and biogeographic regions. We used the software packages ARLEQUIN
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Carlon, HCRI Y.9 Final Report
(Schneider et al. 2000) for mitochondrial sequence data (mtDNA) and MSA Anaylzer (Dieringer and Schlotterer 2003) for microsatellite genotype data.
Second, we used a Bayesian model called STRUCTURE (Pritchard et al. 2000) to estimate the best fitting model of K populations represented by our entire T. gratilla and S. rubroviolaceus sample sets. Samples are input into the model with no apriori information on geographic location, and the algorithm assigns samples to K populations which minimize departures from
Hardy-Weinberg expectations.
G.
contact information for companies used to purchase items unique to your project (if applicable)
H.
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 in general that small juveniles first emerge onto exposed benthic surfaces in the late summer and fall at various sites on Oahu and Maui. However, the specific localities in which we could observe benthic recruitment was not predictable an a yearly basis. This recruitment stochasticity makes answering questions 2 and 3 in the time-frame of a 1-year funding cycle impractical, and unlikely to produce robust answers to these important ecological questions.
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 Islands (MHIs), Kure
Atoll, and Johnston Atoll. Both F
ST
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.
At larger spatial scales, our collaborative work reveals an enormous population that spans the tropics of the Central, North, and Western Pacific (Fig. 1). Genotypes from this population
(indicated by red and green bars in Fig. 1) extend into the Indian Ocean, but we do find some significant population structure (+ F
ST
values) between the Pacific and Indian Ocean (IO), as indicated by the higher assignments into the green population for IO samples. The most significant structure is found at the periphery of the range. In the Eastern Pacific a unique genetic population (blue) corresponds to the subspecies T. gratilla depressus . In the Indian
Ocean, a unique genetic population (yellow) corresponds to the subspecies T. gratilla elatensis.
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Carlon, HCRI Y.9 Final Report
Figure 1.
Assignment of individual urchin samples ( Tripneustes gratilla ) into four populations (K- 4) by
STRUCTURE. Each sample is represented by a single bar on the upper plot, and each color represents a unique population. Assignment values on the Y-axis are the probability that the sample is a member of the population indicated by color. Bars with mixed colors, suggest population mixture.
Colors are mapped onto geographic sampling region nodded lines. Analysis based on microsatellite data.
Parrotfish ( Scarus rubrioviolaceus )- We have completed mtDNA sequencing and genotyping of all 94 S. rubrioviolaceus samples among the MHIs listed in Table 1 (Section IV.D
above). Both
F
ST
analysis and population modeling indicate no genetic structure within or between the MHIs.
Our interpretation is that planktonic parrotfish larvae are regularly dispersing among these islands.
At larger spatial scales, our collaborative work finds that the Hawaiian Islands form a unique subpopulation within the biogeographic range (Fig. 2). In fact the Hawaiian Islands are exchanging migrants with the Central Pacific at very low rates, as indicated by the small number of mixed assignments between Hawaii and the other populations in Fig. 3.
Figure 2.
Assignments of parrotfish samples ( Scarus rubroviolaceus ) into four populations (K-4) by STRUCTURE.
Each sample is represented by a single bar on the upper plot, and each color represents a unique population. Note unique subpopulation (pink) in the
Hawaiian Islands. Assignment values on the Y-axis are the probability that the sample is a member of the population indicated by color. Bars with mixed colors, suggest population admixture.
Colors are mapped onto geographic sampling region nodded lines. Analysis based on microsatellite data.
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 within the Hawaiian
Islands. 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
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Carlon, HCRI Y.9 Final Report emphasize a need to manage reef systems within the state of Hawaii as a collective network, anticipating that local management strategies can, and will, have effects on distant reef systems.
B.
Site specific results for each location (Can place in an appendix as electronic file).
Resource Management Implications
A.
Given the results from VI, what are the implications for resource managers?
There are three key management implications from our research. First, these two herbivores are dispersing broadly across a scale encompassing the Main and Northwest Hawaiian Islands.
This lack of structure is indicated in the homogeneity of gene frequencies across the Hawaiian
Archipelago. Our combined nuclear (microsatellite loci) and mitochondrial (COI and D-loop genes) markers have more than sufficient power to detect even weak structure. We are confident that the Hawaiian Islands are connected by high rates of larval dispersal in these two grazing species. These results are likely to also apply to other reef fish and urchins. The majority of
Hawaiian reef fish and echinoids have feeding planktotrophic larvae with developmental periods greater than several weeks, often extending over one month. The main implication for resource managers is that local populations will act as sources of larvae for distant populations- within and between islands. The best management strategy should focus on a network of parks (MPAs or MLCDs) distributed equitably throughout the Hawaiian Islands. A second prediction from this population structure is that the local effects of protection of a single park may not be apparent for many years after policy implimination. This is because the park is likely to receive recruits from other islands, and the inherent stochastic nature of larval dispersal may delay the arrival of new recruits for many years following protection.
Second, the absence of genetic structure within or between islands implies that the management strategy of moving urchins between islands to control algae at heavily impacted sites is not likely to have negative effects on local populations. We do not anticipate that hybridization between native and non-native urchins to have negative effects on larval survivorship, nor benthic survivorship or fecundity.
Lastly, the third major finding is that the isolation of Hawaii from other regions of the Pacific is taxon dependent. Urchins regularly traverse between the Hawaiian Islands and other regions of the Central and West Pacific Ocean (e.g. Fig. 1). By comparison, the parrotfish species shows little evidence for any connectivity to other regions (Fig. 2). The implication is that the Hawaii parrotfish population is an evolutionary significant unit (ESU, analogous to a unique run of
Salmon) that if not conserved properly, could reduce the total diversity and evolutionary potential of the species globally. In contrast Hawaiian urchins share evolutionary similarity to many other Pacific regions, and it is likely that the islands are continually colonized and subsidized by outside sources.
How do these implications and results help to address the resource management problem(s) identified in III(A)?
A recent paper (Botsford et al. 2001) indicates that when dispersal distances are much larger than the size of individual MPAs, the total area of an MPA network should be around 35% of available habitat, and individual management units should be evenly spaced along the range of habitat. This fraction of habitat in protection guarantees that a similar proportion of brood stock of target species is maintained within the total population. Recent estimates of Ivor Williams of
Hawaii’s Department of Aquatic Resources shows that ~1% of the Hawaiian coastline of the
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Carlon, HCRI Y.9 Final Report
Main Hawaiian Islands is protected. Moreover, MPAs are not distributed evenly across the Main
Islands. Assuming that many important reef herbivores have similar dispersal dynamics as
Tripneustes urchins and Scarus parrotfish, then this small percentage of protected habitat (with unrepresentative spatial distributions) is not likely to be effective either as a fisheries management tool, or as a mechanism to indirectly benefit biodiversity by protecting keystone species from overfishing.
B.
What recommendations for resource managers can be made based on the implications and results?
The people of Hawaii need to think seriously about how they want to protect natural marine environments from the imminent acceleration of habitat degradation. The economy here remains tourism based and tourists place high value on esthetics. A second reason to improve management of natural resources is to protect the high cultural value of fishing for future generations. The majority of nearshore fishing in Hawaii is not commercial in any “mainland” sense, yet local fishing effort probably accounts for the majority of reef fish landings. Two management options which essentially have the same effect are (i) to enforce more restrictive catch and size limits on a wider range of reef invertebrates and fish that have demonstrated keystone effects, or (ii) to create a larger Network of larger MPAs with complete “no-take” restrictions. The success of MPAs is now well established in a variety of marine habitats and geographic regions. Our genetic data can aid in the design of this conservation strategy, but ultimately this is a political issue that will need wide cultural acceptance to succeed in Hawaii.
The MPA is a better solution for tropical reefs because it is an ecosystem approach that protects not only individual species (that may, or may not have commercial value), but the interactions between species. We know from a long history of ecological research that complex interactions between diverse species can have numerous indirect effects that result in the “natural” systems that are often favored culturally. Our study has focused on the larval dispersal of two key species within this ecosystem, but it is likely that there are many other species within the reef ecosystem that have similar scales of dispersal. On the other hand, there are clearly other organisms whose larval biology tells us they disperse over much shorter distances (some corals, algae, and the newer work of R. Toonen’s group on Opehi). This means that we need parallel studies that examine population genetics of the ecosystem, all conducted at the same spatial scale. Ultimately, the effective design of an MPA network will need to consider different scales of dispersal, and trade-offs in design are likely. These trade-offs reflect the complex reproductive biology of tropical marine organisms.
Evaluation
A.
Was the objective attained? How? If not, why?
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.
We succeeded in this objective by showing in genetic terms (using microsatellite and
DNA sequence-based molecular markers) that rates of migration among the Northwest and Main
Hawaiian Islands are very high for the collector urchin Tripneustes gratilla . We have shown similar high rates of migration among the MHIs in the parrotfish Scarus rubroviolaceus.
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Carlon, HCRI Y.9 Final Report
Were modifications made to objective? If so, explain.
No modifications to objective.
If significant problems developed, resulting in less than satisfactory or negative results, discuss.
No significant problems.
Description of need, if any, for additional work
There is a management need to examine genetic structure at regional scales that include the biogeographic boundaries of target organisms. Our preliminary results with collaborators indicate significant genetic differentiation between Hawaii and the Eastern Pacific, as well as
Hawaii and the Central Pacific. Such scales span the political boundaries of Pacific nations, territories, and protectorates.
What performance measures are used to evaluate how well the project met the stated objective?
The robustness of statistical estimates of genetic structure are our main measure of project performance. Large samples sizes and high genetic polymorphism lead to high confidence in population parameters. We also see congruence between mitochondrial and microsatellite data sets; and between individual microsatellite loci, each potentially sampling a different part of the genome.
Objective 2. To determine if deep-water, soft-sediment habitats act as nurseries for Tripneustes gratilla on coral reefs.
This objective was not met because the episodic nature of Tripneustes recruitment made it logistically impracticable to carry out recruitment experiments and tracking studies. Our observations of spatial variability in recruitment suggest that this goal can only be met with significant long-term funding of a research project that specifically targets the dynamics of recruitment, post-recruitment survivorship, and movement. A reasonable study period is five years, but 10 would be far better.
Objective 3. To integrate data on population structure and nursery areas to design MPAs for keystone reef herbivores.
Our data aids design of a MPA network as discussed under “Resource Management
Implications- A”
Were modifications made to objective? If so, explain.
Since dispersal is so high in these systems, specific localities within individual Islands are not an important variable in terms of the biology of these species that support overall ecosystem health. However, decisions to allocate new parks or MPAs will obviously be dictated by other social-political considerations.
Description of need, if any, for additional work
More species, representing other functional groups could be assayed in the same way to understand if population structure varies among the most important functional groups.
What performance measures are used to evaluate how well the project met the stated objective?
Performance measures for this Objective are the same for those described under
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Carlon, HCRI Y.9 Final Report
Objective 1. See discussion of the robustness of our statistical estimates of genetic structure.
Dissemination of Project results
A.
Explain, in detail, how the projects results have been, and will be, disseminated.
The PI and graduate student (Fitzpatrick) each presented papers at the 2007 Western Society of
Naturalist Meeting in Ventura, CA (titles below). A third paper will be presented by John
Fitzpatrick at the 11 th
International Coral Reef Symposium in Ft. Lauderdale this summer.
We are currently working on two publications resulting from HCRI sponsored research (Years 8 and 9). 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.
Oral presentations:
1. 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.
2. 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
D.
Partnerships established with agencies or organizations

Hawaiian Institute of Marine Biology

DLNR

Cooperative Fisheries Unit, University of Hawaii

Alii Holo Kai dive club

Smithsonian Tropical Research Institute, Panama

James Cook University, Townsville, Australia

Tokyo Institute of Technology
References
Botsford, L. W., A. Hastings, and S. D. Gaines. 2001. Dependence of sustainability on the configuration of marine reserves and larval dispersal distance. Ecology Letters 4:144-
150.
Carlon, D. B., and C. Lippe. 2007a. Eleven new microsatellite markers for the tropical sea urchin
Tripneustes gratilla and cross-amplification in Tripneustes ventricosa. Molecular Ecology
Notes 7:1002-1004.
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Carlon, HCRI Y.9 Final Report
Carlon, D. B., and C. Lippe. 2007b. Isolation and characterization of 17 new microsatellite markers for the ember parrotfish Scarus rubroviolaceus, and cross-amplification in four other parrotfish species. Molecular Ecology Notes 7:613-616.
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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