genetic population structure of the hawaiian alien ...223 genetic population structure of the...

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Genetic Population Structure of the Hawaiian Alien Invasive SeaweedAcanthophora spicifera (Rhodophyta) as Revealed by DNA Sequencing

and ISSR Analyses1

Daniel C. O’Doherty2 and Alison R. Sherwood2,3

Abstract: Acanthophora spicifera (Vahl) Børgesen is the most widespread and in-vasive alien macroalga on coral reefs throughout the main Hawaiian Islands.This alga disperses from harbors and ports to coral reefs throughout the state,producing high quantities of biomass that affect a wide range of reef flora andfauna. Population samples of A. spicifera from across the main Hawaiian Islandswere collected and compared through two kinds of analyses: DNA sequencing( based on a variable region of the nuclear large subunit ribosomal RNA gene,and the mitochondrial cox 2-3 spacer region) and fragment techniques (Inter-Simple Sequence Repeats [ISSRs]). DNA sequencing revealed no variation forthe two markers, even when collections from other areas of the Pacific and Aus-tralia were included. In contrast, ISSR analyses revealed highly structured Ha-waiian populations of A. spicifera with a substantial range of both within- andamong-population variation, with individual plants forming discrete clusterscorresponding to geographic locality.

The invasion of coral reef ecosystems bynonnative species can alter ecosystem struc-ture and reduce indigenous biodiversity (Pan-dolfi et al. 2005). In Hawai‘i, the number ofdocumented alien algal species has increasedfrom 18 in 1992 (Russell and Balazs 1994) to24 species in 2003 (Godwin 2003). Of the fivenotably invasive macroalgae, Acanthophoraspicifera (Vahl) Børgesen is the most commonspecies throughout the main Hawaiian Is-lands (Smith et al. 2002). Successful invaderssuch as A. spicifera have substantial impactson Hawai‘i’s coral reef ecosystems (Godwin2003). In addition to damaging native ecosys-tems, invasive algal blooms have causedeconomic losses to the state exceeding 20 mil-lion dollars (Smith et al. 2004). The wide-

spread distribution and rapid growth of A.spicifera make it a particularly dangerousthreat to Hawai‘i’s coral reefs and a priorityspecies for control efforts.

The vegetative characteristics of A. spici-fera contribute to it being a successful colo-nizer that is capable of attaching to, andcompeting with, native algal species (Russelland Balazs 1994). The spiny thalli of this brit-tle alga are easily fragmented by wave action(Kilar and McLachlan 1986) and readily snagon suitable substrates, including other macro-algae. Kilar and McLachlan (1986) found thatplants growing on exposed reef in Panamawere able to generate up to 74 kg of vegeta-tive fragments per month, and each fragmenthad the potential to reattach after only 2 days.Although the reproductive phenology of alienalgae in Hawaiian waters has not been thor-oughly investigated, A. spicifera is the onlyspecies in this category that has been consis-tently observed in a sexually reproductivestate (Smith et al. 2002). Genetic recombina-tion may result in increased population-levelvariation that allows A. spicifera to be moreresistant to control efforts than clonal inva-sive species. Reproductive species may posean additional threat through the release ofcopious quantities of microscopic spores that

Pacific Science (2007), vol. 61, no. 2:223–233: 2007 by University of Hawai‘i PressAll rights reserved

1 This research was funded by the Hawai‘i Division ofAquatic Resources and the University of Hawai‘i. Manu-script accepted 12 June 2006.

2 Department of Botany, 3190 Maile Way, Universityof Hawai‘i at Manoa, Honolulu, Hawai‘i 96822 (e-mail:[email protected]).

3 Corresponding author.

disperse over great distances. As with manyinvasive organisms, the physical and repro-ductive characteristics of A. spicifera fosterwidespread dispersal and enable successfulcompetition for resources within native reefcommunities.

Algal invasions are often related to anthro-pogenic factors, including aquaculture (Ab-bott 1999), eutrophication (Stimson et al.2001), hull fouling (Smith et al. 2002), ballastwater discharge (Godwin 2003), and overfish-ing (McClanahan et al. 2001, Pandolfi et al.2005). The major phase shifts involving pri-mary producers on coral reefs associatedwith these factors have substantial and oftencatastrophic impacts on macroalgal, coral, andfish assemblages (Littler and Littler 1985,Hughes 1994, Russell and Balazs 1994, Stim-son et al. 2001, Pandolfi et al. 2005). Acantho-phora spicifera is implicated in major phaseshifts on Hawaiian reefs because it has beenfound to produce high biomass throughoutthe main Hawaiian Islands (Russell and Ba-lazs 1994). In Hawai‘i, alien algae have beendocumented to compete with native speciesof algae (Russell and Balazs 1994) and corals(Stimson et al. 2001). However, the ecologicalinteractions involving A. spicifera on Hawaiianreefs may be complex. For example, the reg-ular consumption of A. spicifera by Hawaiiansea turtles and herbivorous fish has beendocumented by Russell and Balazs (1994). Al-though the effects of alien algae ingestion onthe diets of native herbivores are unknown(Smith et al. 2004), a large increase in ediblealgal biomass at a particular locale would beexpected to have numerous impacts through-out the reef community.

Although research concerning the effectsof invasive macroalgae on tropical reefs is stilllimited (e.g., Coles and Eldredge 2002, Smithet al. 2004), even fewer studies have examinedthe genetic structure of invasive algae in trop-ical waters. In temperate latitudes, DNA se-quencing and fragment techniques have beenused to investigate invasions of green and redseaweeds. These molecular tools have beenused to determine the possible geographicsources and the genetic diversity of notableinvasive algae such as Caulerpa taxifolia,

Grateloupia doryphora, and Asparagopsis taxifor-mis (Fama et al. 2002, Marston and Villalard-Bohnsack 2002, Chualain et al. 2004). In thestudy reported here, DNA sequence analysesof a region of the nuclear ribosomal largesubunit gene and the mitochondrial cox 2-3spacer region, and Inter-Simple SequenceRepeats (ISSRs) were used to investigate thepopulation structure of A. spicifera through-out the main Hawaiian Islands. ISSRs are acost-effective method for the resolution ofintraspecific genetic variation and have beenused to study a wide variety of plant taxa (Liand Ge 2001, Sica et al. 2005), as well asother red algae (Vis 1999, Sherwood et al.2002). Here we illustrate the utility of ISSRanalyses for revealing the degree of geneticstructure within and between populations,and use these data to provide insight and rec-ommendations for management of this alieninvasive seaweed.

materials and methods

Sample Collection

Specimens were collected from tide pools andreef flats by snorkeling or wading. Individualsof A. spicifera were collected from O‘ahu(n ¼ 30), Moloka‘i (n ¼ 7), Maui (n ¼ 15),and Hawai‘i (n ¼ 15) (Figure 1, Table 1).For each population, individuals were col-lected from an area no more than 30 m onthe longest dimension. Freshly collected sam-ples from O‘ahu were immediately cooledand transported to the laboratory for process-ing. Samples from other Hawaiian islandswere transported to O‘ahu frozen in seawateror desiccated in silica gel.

DNA Extraction and Morphological VoucherPreparation

Before DNA extraction, 10 of the cleanest in-dividuals from each location were examinedunder a stereomicroscope to determine re-productive status and to remove visibleepiphytes. When reproductive status (tetra-sporangial, spermatangial, or carpogonial)was not discernible using stereomicroscopy,

224 PACIFIC SCIENCE . April 2007

specimens were mounted, sectioned, stainedin 1% aniline blue, and examined with a com-pound microscope (Olympus BX51). Totalgenomic DNA was extracted by freezing inliquid nitrogen, grinding using a mortar andpestle, and processing approximately 50 mgof fresh material or 10 mg of silica-dried ma-terial using the Qiagen DNeasy Plant MiniKit (Valencia, California). All DNA extractswere stored in labeled screw-cap vials at�20�C. Remaining plants were processed asmorphological vouchers, either stored in 4%formalin/seawater or pressed onto herbariumsheets. Voucher specimens of each popula-tion are maintained in the Sherwood Labora-tory at the University of Hawai‘i at Manoa(DCO001–DCO009).

DNA Sequence Analyses

Genetic regions representing the nuclear andmitochondrial genomes were investigatedthrough DNA sequencing for a subset of theA. spicifera collections from the main Hawai-ian Islands. The same DNA extracts wereused as for the ISSR study. The mitochon-drial cox 2-3 spacer region was sequencedfor 43 individuals (20 from O‘ahu, two fromMoloka‘i, 11 from Maui, and 10 from Ha-wai‘i) using previously published reactionand cycling conditions (Zuccarello et al.1999). Several additional A. spicifera collec-tions from other parts of the Pacific wereadded to the DNA sequencing study as acomparison across a broader geographical

Figure 1. Map of collection localities for Acanthophora spicifera samples used in this study.

Acanthophora spicifera Population Structure . O’Doherty and Sherwood 225

range. Samples from Guam (Pago Bay), Aus-tralia (Townsville), and Japan (Uken Beach,Okinawa) were sequenced for the cox 2-3spacer and the LSU-Y fragment to provide acontext for comparing the DNA sequencevariation in the Hawaiian collections. Thisspacer region has been successfully used todiscern population structure for red algae(Zuccarello et al. 1999) and thus was a logicalcandidate for our study. A variable region ofthe nuclear ribosomal large subunit gene(‘‘LSU-Y,’’ according to the terminology ofHarper and Saunders [2001]) was sequencedfor seven A. spicifera individuals (five fromO‘ahu and two from Hawai‘i), again usingpreviously published reaction and cyclingconditions (Harper and Saunders 2001). Al-though in entirety the LSU gene is quite con-served and is useful for distinguishing speciesin red algae (Harper and Saunders 2001), thisvariable region has the potential to detect in-traspecific variation.

Polymerase chain reaction (PCR) productswere verified for size and concentration bygel electrophoresis. PCR products were di-rect purified using the Qiagen PCR Purifica-tion Kit (Valencia, California) and sequenced

on an automated DNA sequencer (ABI377XL). Resulting sequence chromatogramswere examined and consensus sequences as-sembled from both forward and reverse readsusing BioEdit v.7.0.4.1 (Hall 1999), and con-sensus sequences were aligned by eye. DNAsequence data are available via GenBank(accession numbers DQ500955 for cox 2-3spacer and DQ500956 for LSU-Y).

ISSR Analyses

Fourteen ISSR primers were screened forsuccessful amplification. Five of these 14 pri-mers produced clear and well-separated bands,and were used to generate the populationdata set (see Table 2). Because some DNA ex-tracts were too dilute or degraded for repro-ducible ISSR amplification, only seven oreight individuals from each population wereincluded in the final analyses. To account forvariable extract concentration, 0.1–1.0 ml ofDNA extract was added to each 25-ml reac-tion containing 2.5 ml of buffer (Promega),2.0 ml 2.0 mM magnesium chloride (Pro-mega), 1.5 ml of 1.0% bovine serum albumin,1.0 ml (0.4 mM) of primer, 0.25 ml (20 mM)

TABLE 1

Collection Details for Populations of Acanthophora spicifera Analyzed in ISSR and DNA Sequence Analyses

Collection LocationaCollection

Date CollectorReproductive

Status Exposure

Richardson’s BP,Hawai‘i

13 Jan. 2005 R. Okano Unknown Partially protected cove:windward

Kahala BP, O‘ahu 16 Jan. 2005 A.R.S. & G. Presting Unknown Exposed reef: leewardKualoa BP, O‘ahu 05 Feb. 2005 D.C.O. Sterile Exposed reef: windwardWaiehu BP, Maui 21 May 2005 A.R.S. & G. Presting Sterile Exposed reef: windwardAla Moana BP, O‘ahu 08 July 2005 V. Nestor Tetrasporangial Sheltered/exposed reef:

leewardOnekahakaha BP,

Hawai‘i13 Jan. 2005 R. Okano Unknown Sheltered tide pool:

windwardMaili BP, O‘ahu 26 May 2005 D.C.O. Tetrasporangial Exposed reef: leewardD. T. Fleming BP, Maui 21 May 2005 A.R.S. & G. Presting Sterile Exposed reef: leewardKamiloloa BP, Moloka‘i 28 Apr. 2005 B. Puleloa Unknown Exposed reef: leeward*Uken Beach, Okinawa

Is., Japan08 Mar. 2005 R. Terada Unknown Unknown

*Pago Bay, Guam 03 Oct. 2004 R. Tsuda Unknown Unknown*Bangi Is., Guam 01 Apr. 2005 K. Peyton & L. Basch Unknown Unknown*Townsville, Australia 24 Mar. 2005 P. Skelton Unknown Unknown

a BP ¼ beach park.* Collections used only for DNA sequencing.


of each dNTP, 0.1 ml of Promega Taq Poly-merase (Madison, Wisconsin), and 12.8 ml au-toclaved nanopure water. DNA amplificationwas carried out in a thermocycler (EppendorfGradientS, Stuttgart, Germany) with the fol-lowing parameters: 94�C for 2 min, followedby 45 cycles of 94�C for 45 sec, 37�C for 45sec, 72�C for 2 min, and a final extension of72�C for 5 min. PCR products and 150 basepair ( bp) DNA ladders were resolved electro-phoretically on 1.5% agarose gels run at 100V for 2.5 hr in 0.5X TBE buffer. DNA frag-ments were visualized by staining with ethi-dium bromide, and photographed under UVlight. ISSR bands were scored as present (1)or absent (0) for each DNA sample, and thesebinary data were entered into a spreadsheet.Fragments of the same molecular weightwere assumed to be homologous. The Multi-Variate Statistical Package (MVSP) was em-ployed to evaluate the genetic similaritybetween all accessions (Kovach ComputingServices 1986–1999). MVSP was used to cre-ate a similarity matrix based on Jaccard’s coef-ficient and to conduct a cluster analysis withthe Unweighted Pair Group Method usingArithmetic Averages (UPGMA) algorithmand a Principal Co-Ordinates (PCO) analysisbased on the similarity matrix. Popgene soft-ware v.1.32 (Yeh et al. 1997) was used to cal-culate Nei’s genetic distance and geneticdiversity within each population as Percent-age Polymorphic Bands (PPB) and Shannon’sIndex. GenAlEx was used to conduct an Anal-ysis of Molecular Variance (AMOVA) andestimate population differentiation (FST) asmeasures of genetic relationships within andamong populations.

results

DNA Sequencing Analyses

No sequence-level variation was observed ineither the Hawaiian collections of Acantho-phora spicifera or the more broadly distributedPacific collections of the alga. A single haplo-type for the cox 2-3 spacer (305 nucleotides[nt]) was observed for all sequenced collec-tions from the main Hawaiian Islands(n ¼ 43 samples), as well as collections from

Guam (n ¼ 5), Australia (n ¼ 3), and Japan(n ¼ 2). Similar results were obtained for se-quence data of the LSU-Y region. Again, asingle haplotype (531 nt) was recovered forall A. spicifera collections from the main Ha-waiian Islands (n ¼ 7). In addition, this samehaplotype was sequenced for the samplesfrom Guam (n ¼ 1), Australia (n ¼ 1), and Ja-pan (n ¼ 1).

ISSR Fragment Polymorphism and GeneticStructure

Of the 14 ISSR primers tested, five generateda total of 45 bright and reproducible bandsranging in size from approximately 375 to1,600 bp (Table 2). The vast majority(91.1%) of fragments produced from the 67individuals in eight populations were poly-morphic. Analyses of PPB and Shannon’s di-versity index ranged from 4.4% and 0.030 (D.T. Fleming Beach, Maui) to 44.4% and 0.278(Onekahakaha Beach, Hawai‘i), indicating awide range of genetic diversity within popu-lations (see Table 4).

Cluster analysis with Jaccard’s coefficientwas used to visualize the degree of similaritybetween accessions of A. spicifera (Figure 2).The cluster dendrogram exhibits a high de-gree of population structure and clearlyshows the distinct grouping of individualscollected at each location. When all individu-als are included in the comparison, the aver-age genetic similarity measured by Jaccard’scoefficient is 0.493. The coefficient amongpopulations ranged from a low of 0.424 (AlaMoana Beach, O‘ahu, and Richardson’s

TABLE 2

ISSR Oligonucleotide Primers That Produced Clear andReproducible Bands for Population-Level Analyses of the

Red Alga Acanthophora spicifera

Primer Name Sequence Total No. of Bands

ISSR 1 (CA)6GG 8ISSR 2 (CT)8AC 7ISSR 5 (CT)8GC 6ISSR 10 (GAG)3CC 13ISSR 12 (CAC)3GC 11


Figure 2. UPGMA dendrogram ( Jaccard’s coefficient) of Acanthophora spicifera collections from the main Hawaiian Islands.

Beach, Hawai‘i) to a high of 0.769 (Kamilo-loa Beach, Moloka‘i, and D. T. FlemingBeach, Maui) (Table 3). Nei’s measure of ge-netic distance revealed similar trends of ge-netic relationships among populations (Table3). The PCO analysis generated a biplot visu-ally representing the associations of individu-als within and between populations. Therelationships among individuals in the PCOanalysis were similar to those revealed byboth cluster analysis and Nei’s measure of ge-netic distance, and only the cluster dendro-gram based on Jaccard’s coefficient is shown(Figure 2). All measures of genetic distanceplaced the population samples from theisland of Hawai‘i at a considerable distancefrom populations collected from all otherislands. The AMOVA yielded high values(FST ¼ 0:609) that were significantly differ-ent (P ¼ :001) from zero with 999 randompermutations, indicating a substantial degreeof differentiation among populations. Al-though individuals within populations fromall islands formed discrete clusters, for somepopulations we observed a substantial amountof within-population variation.

Seasonality of Reproduction of A. spicifera

Acanthophora spicifera samples from all shoresof O‘ahu were regularly collected and ob-served for reproductive status between Feb-ruary 2005 and late October 2005. Noreproduction was visible for any of these col-lections until the final week of May 2005.

Many of these tetrasporophyte thalli con-tained spores in various stages of develop-ment. Nearly every subsequent collectioncontained at least one tetrasporangial plant.Ongoing collections have revealed tetrasporeproduction into October 2005.

The isomorphic life history of many redalgae, including A. spicifera, makes it im-possible to visually distinguish sterile tetra-sporophytes and gametophytes. Althoughcarpogonial specimens have been observed inthe past (Smith et al. 2002), no spermatangialor carpogonial plants were observed in ourcollections, and tetrasporangia represent theonly reproductive cells detected in this study.

discussion

Although the cox 2-3 spacer has been used toresolve genetic variation at the intraspecificlevel (Zuccarello et al. 1999), no sequencevariation was found for samples from evendistant locations (Hawai‘i, Guam, Australia,and Japan). In contrast, our ISSR analysesdemonstrate a substantial degree of popula-tion structure that is related to geographicdistance, with populations forming discretegroups. This high degree of genetic structuresuggests that currently used population-levelDNA sequence markers (e.g., cox 2-3 spacer,LSU-Y) do not possess resolution highenough to detect population structure in allalgal species.

Patterns of genetic variation in A. spiciferaare likely influenced by a myriad of natural

TABLE 3

Genetic Distance Presented as Jaccard’s Coefficient of Distance (above Diagonal) and Nei’s Genetic Distance (belowDiagonal) among Eight Hawaiian Populations of Acanthophora spicifera Used in ISSR Analyses


and anthropogenic variables. The degree ofgenetic similarity found both within andamong populations in our current studyranged widely (Figure 2, Tables 3 and 4).The genetic differentiation and relationshipsamong populations suggest that a limitedamount of genetic exchange takes placewithin the main Hawaiian Islands. Popula-tions collected near Hilo, Hawai‘i (Onekaha-kaha Beach and Richardson’s Beach), are themost geographically and genetically distantfrom populations surrounding the suspectedsite of introduction (Pearl Harbor, O‘ahu)(Smith et al. 2002). The highest within-population variation was observed for well-established populations (e.g., Ala MoanaBeach, O‘ahu, and Onekahakaha Beach, Ha-wai‘i) that are proximal to intense boat traffic(Honolulu Harbor, O‘ahu, and Hilo Bay, Ha-wai‘i) and are thus likely to be exposed to in-coming propagules from hull-fouled vesselsand ballast water discharge.

Acanthophora spicifera is a hardy species thatis capable of flourishing under a wide rangeof environmental conditions. The naturaland anthropogenic conditions that varyamong collection sites likely affect the quan-tity of sexual and asexual propagules pro-duced, as well as the likelihood of dispersalto other sites. Occasional events such as se-vere weather systems and large ground swellswould certainly generate unusually high num-bers of fragments. Anthropogenic influencessuch as boat traffic are implicated in the ini-

tial introduction of A. spicifera to Hawai‘i(Doty 1961), and it is likely that hull-fouledinterisland vessels act as occasional dispersalvectors. Due to an abundance of dispersalmechanisms, this species is widely distributedacross Hawai‘i. Our analyses indicate that itpossesses a surprising amount of populationstructure. The discrete population groups re-vealed by cluster analysis and AMOVA sup-port the hypothesis of Smith et al. (2002)that A. spicifera is occasionally introduced tonew localities by hull fouling, where it readilyspreads to nearby reefs through fragmenta-tion or spore production.

Tetrasporangia are typically produced bysexually reproducing populations of red algaeas one of the three life history stages, but in-stances are known in which the gametophytestages are omitted and the algae live continu-ously as tetrasporophytes (e.g., the marinespecies of the red alga Hildenbrandia [Sher-wood et al. 2002]). Because gametophyteswere never observed in our own collectionsof A. spicifera in the main Hawaiian Islands,it is possible that this species also has, or istransitioning to, such a truncated life historyin Hawai‘i. However, detailed collectionsfrom throughout the year are necessary to de-termine this possibility. This scenario wouldaccount for the abundance of tetrasporo-phytic plants in our collections and the lackof gametophytic plants. Thus, although evi-dence of sexual reproduction has been re-ported in the past (Smith et al. 2002), we

TABLE 4

Percentage of Polymorphic Bands (PPB) and Shannon Index (I ) for Nine Populations of Acanthophora spicifera Used inISSR Analysis

Collection Location No. of Polymorphic Bands PPB I (SD)

Richardson’s Beach, Hawai‘i 12 26.67 0.1676 (0.2846)Kahala Beach, O‘ahu 13 28.89 0.1496 (0.2571)Kualoa Beach, O‘ahu 18 40.00 0.2320 (0.2981)Waiehu Beach, Maui 19 42.22 0.2478 (0.3048)Ala Moana Beach, O‘ahu 20 44.44 0.2556 (0.3045)Onekahakaha Beach, Hawai‘i 20 44.44 0.2779 (0.3116)Maili Beach, O‘ahu 16 35.56 0.2134 (0.2967)D. T. Fleming Beach, Maui 2 4.44 0.0301 (0.1411)Kamiloloa Beach, Moloka‘i 5 11.11 0.0704 (0.2042)

SD, standard deviation.


cannot confirm these data based on our ownobservations. The molecular data are onlypartially congruent with our observations ofreproductive status. A large amount of ge-netic variation was recovered based on theISSR analyses (Figure 2), but no variationwas observed in the cox 2-3 spacer or LSU-Y DNA sequences of A. spicifera across thePacific. Thus, it is not clear whether A. spici-fera is commonly employing sexual reproduc-tion as a strategy in the main HawaiianIslands. Population-level sequence markershave not always revealed variation withinpopulations of invasive seaweeds (Marstonand Villalard-Bohnsack 2002). However,dominant genetic markers, including ISSRand Random Amplified Polymorphic DNA(RAPD), have been used to confirm sus-pected clonal invasions (Hollingsworth et al.1998) and reveal population structure in inva-sive species (Meekins et al. 2001, Marstonand Villalard-Bohnsack 2002, Sun et al.2005). The high degree of genetic structurefound with our ISSR analyses suggests thatA. spicifera in Hawai‘i is reproducing sexually,but that gene flow between geographicallydistant populations is rather limited.

Management Recommendations

Of the many invasive organisms in Hawai‘i,introduced red algae appear to be the mostpressing threat to reef habitats (Coles and El-dredge 2002). Among all the invasive sea-weeds, A. spicifera is the most widespread andsuccessful (Smith et al. 2002). Therefore, thisspecies should be considered a priority for in-vasive species managers. Although completeeradication of this alien alga is highly un-likely, the prevention or reduction of furtherspread is a feasible goal.

prevention of spread via boat traf-

fic. Because boat traffic is the primary vec-tor involved in the spread of invasive marinespecies in Hawai‘i (Godwin 2003), increasedpublic awareness and more stringent regula-tions regarding hull fouling and ballast waterdischarge are necessary to prevent or slow therecruitment of new populations.

site-specific control efforts. Theresults of our research should help invasive

species managers target control efforts to spe-cific sites. Populations that are not yet wellestablished may lack ‘‘genetic flexibility’’ (i.e.,abundant genetic variation) and thereby beless likely to resist control measures such aslarge-scale removal or introduced herbivores.Control efforts should thus target locationsthat have strong potential to recruit new pop-ulations through the frequent production anddispersal of propagules. Populations on ex-posed reefs, when compared with those inprotected bays, lagoons, and tide pools, wouldbe expected to produce and disperse a greaternumber of propagules because of higherwave action. Efforts at particular sites shouldbe focused on outer areas of the reef that ex-perience relatively high wave action andtherefore likely generate a greater quantityof fragments. Being much smaller propagulesthan vegetative fragments, tetraspores wouldbe expected to be carried farther by oceancurrents. Thus, removal efforts should betimed to predate the release of tetraspores(likely in the late spring, although detailedyear-round observations are needed to con-firm this). Other local conditions associatedwith increased dispersal, such as proximity toboat harbors and strong currents, need to beinvestigated to best select these target local-ities around the state.

conclusions

Although molecular tools have been used toinvestigate invasive algae in the North Atlan-tic (Chualain et al. 2004, Provan et al. 2005)and Mediterranean Sea (Fama et al. 2002,Meusnier et al. 2002, Piazza and Cinelli2003), this study is the first to examine thepopulation genetics and biogeography of analien seaweed in Hawai‘i. Our analyses indi-cate a surprising amount of within- andamong-population variation based on ISSRmarkers, but DNA sequence analyses of themitochondrial cox 2-3 spacer and a fragmentof the nuclear ribosomal large subunit genefailed to reveal any variation. This geneticanalysis provides a ‘‘first look’’ at the geneticstructure of A. spicifera and a basis for futureanalyses to monitor dispersal and gene flowover time. The current research also presents


a foundation upon which to develop furtherresearch regarding the sexual strategies anddispersal mechanisms that influence thespread of other alien algae in Hawai‘i.

acknowledgments

We gratefully acknowledge the followingpeople for their assistance with field collec-tions or for obtaining access to collections:Larry Basch, Sally Beavers, Skippy Hau, Re-becca Most, Victor Nestor, Ryan Okano,Kim Peyton, Gernot Presting, Bill Puleloa,Posa Skelton, Cheryl Squair, Ryuta Terada,Roy Tsuda, and Bill Walsh. We thank TonyMontgomery (Hawai‘i Division of AquaticResources) for his support of this research,and Isabella Abbott, Celia Smith, and JenniferSmith (Botany Department, University ofHawai‘i) for their helpful discussions andideas. The National Park Service graciouslyallowed us to use collections of Acanthophoraspicifera from Guam and the island of Ha-wai‘i.

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