STUDY OF GE NETIC DISCO NTI N UITY IN A CO N'II N UO USLY DISTRIB UTED SPECIES BASED O N MOLEC ULAR EVIDE NCE

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PHYLOGEOGRAPHY OF

GRACILARIA TIKVAHIA E

(GRACILARI ACE AE, RHODOPHYTA):

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STUDY OF GE NETIC DISCO NTI N UITY IN A CO N'II N UO USLY DISTRIB UTED SPECIES BASED O N MOLEC ULAR EVIDE NCE

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Carlos Fr ederico Deluqui Gurgel", Suzanne F redericq

Deparun c nt or Bio\ o h'!',Cnivc rsit y or Louisia n aat l.al a yc n c,l.a la veu e,Louisiana 7(),j()4-~4 ;,I,USA

and

[tunes N.Norris

Dcp.mmeut of'Botany :--.!H B I ()(), National Museum orNat u ra l Hist ory, Smithsonian l usriruti o n,l~(). Box :\7 ()J ~,\\'ashin~ton , DC

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Graci/aria tikvahiae, a highly morphologically vari- able red alga, is one of the most common species of Gracilariaceae inhabiting Atlantic estuarine envi- ronments and the Intracoastal Waterway of eastern North America. Populations of G. tihuahiae at the extremes of their geographic range (Canada and southern Mexico) are subjected to verydifferent en- vironmental regimes. In this study, we used two types of genetic markers, the chloroplast-encoded rbcL and the nuclear internal transcribed spacer (ITS) region, to examine the genetic variability with- in G. tikvahiae, for inferring the taxonomic and phylogenetic relationships between geographically isolated populations, and to discuss its distribution- al information in a phylogeographic framework.

Based on rbcL and ITS phylogenies, specimens from populations collected at the extreme distribu- tional ranges reported for G. tikvahiae are indeed part of the same species; however,rbcL- but not ITS- based phylogenies detected phylogenetic structure among the ten G. tihuahiae different haplotypes found in this study. The four distinctrbcL lineages were identified as 1) a Canadian-northeast U.S. lin- eage, 2) a southeast Florida lineage, 3) an eastern Gulf of Mexico lineage, and 4) a western Gulf of Mexico lineage. We found no evidence for the oc- currence of G. tihuahiae in the Caribbean Sea. Ob- served phylogeographic patterns match patterns of genetic structures reported for marine animal taxa with continuous and quasicontinuous geographic distribution along the same geographic ranges.

Key index words: biogeography; Gracilaria tikua- hiae; Gracilariaceae; ITS; phylogeography; rbcL;

rDNA; Rhodophyta

Abbreviations: indels,nucleotide insertions and de- letionsevents; ITS, internal transcribed spacer; ML, maximum likelihood; MP, maximum parsimony;

IReceived 10'\ l' l'il:ZOO:I.,\cccl'ted" .vpri !:Z004.

:!:\ l ll !lor !()r("( I I T CS ) ( )Il c le ll ct': l'-m a ill~g llrg'('I((l y ;tIH )( ) . ("(Hll.

74 S

rbcL, large subunit of the RUBISCO gene; SPN, statistical parsimony network

Crurilari« tikoahia« is one or the most common or seven speciesolC rac ila riaceae(Grac ila r iales)cu r re nt ly recogn ized in the easte rn North Ame r ica n benthic alga l [lora. It is a curyi hc rma l and eury ha line red alga (tolera nce range , 8-60psu) (Bir d and Mcl.achl an 1986) typi cal or bays, inlet s, and estua r ine environ - mern s (Bir d etal, 1979, Mcl.achl an 1979). Morpho- logical var ia tio n within th isspec ies rangesfromteret e to com p letely flat tha lli,an d this phe no typ icvaria tio n resul ted in much ta xonomic con fu sio n,The ext ent to whic h taxa have been misid eutilied with thisspeciesis still unknown. Specim ens or (;. lil:va!li{/(' ihat ex h ib it som ewh at sim ilar exte r na l morphol ogies have been report ed under differentnam esthrou ghoutthenorth- wester n Atla n tic as "C;. uerrucosa" or "C;..Iii/ii/iTa val'. ungnstissima" (Taylor 19:')7, 1960,Gan csan 1989,Sch n- eide r and Searles 1991, BelJorin etal. 20(2). Because or the morphological diversity an d taxonomic conlu- sion an d the range or the ge ographic distribution or (;. tikoahiae,especially its south ernmostlimit, the num- ber or cryp tic species passing under this name is still unresolved .Several locales hav ebeen proposed as the sou thern limit of this species: the mid-Atlantic region or the FIOI'id a Penin sul a (i.c . Palm Bea ch County;

Mclachl an 1979), the so u the rn Gu ll' of Me xico (Ed- wards 1970), the southern Car ibb ean (l. ittle r and Lit- tier 2( 00), an d Brazil (Sch nei de r and Searl es 1991, p.326,Olive ira -Filho 1977,as"G..Iii/ii/i:m").Howev e r, Can esan (1989) noted that becau se or the presen ce of morp ho log ica l characters that arc SGII'Ce or ambigu- ous, record s or this specie s (or the Carib bea n an d Sout h America need to be considered with cau tio n and requi re criticalexaminat io n. Outsi de the weste rn Atla n tic,G.tikoaliiar has been recorded as an invasive species in Hawaii (Ab bo tt 1999 ).

At the distributional extremesor well-kn own popu- lati ons orC;. tikoahute,specime ns arc su b jec te d to dil- ferent temperatures and pho to regimes. For instance , insu bho re al Canada (;.tikualtin«ove rwin te rs under ice

PIIYLOCl,n ( ;R,\PIlY OFc: n;':I;,III//IF 749 shee ts (Mclach la n 197~) , whe reas in Me xico the spe-

cies ex pe r ie nces war m tropical temperatures year arou nd.The distinct enviro n me n ta lcon d itio ns inhab- ite d by northern and southern populations of G. tikuahia« raise the quest ion wheth er these popula- tion s are part of the same spec ies or present eno ugh genetic dille renee to place the m in distinct taxa. Fo r some warm- te m pe ra te estuarinespecies, thesouth ern tip 0['the Florida Penin sula corres po nd sto a place of genetic disco n tinui tyisolat in g the Cu lt'0['Mexico pop- ulatio ns fro m those of the Atla nt ic and pnmlOting speciatio n, pos sibl y due to historical (and ma yb e pre- sen t) barri e rs to gene flow (Collin 200 I, Cold ctal.

2(02) . Marine eco syste ms nt the sou the rn tip 0[' the Flor id a Peninsulaand the Flo ri da Ke ys bcing the At- lantic Ocean and the Caribb ean Sea arc distinct and ch arac te r ized by a marine 1I0ra that is different Irorn that of the warm-tempera te Carolin ia n environments (H u rn rn 1969, Searles 19H4 ). The d'ket of historical

l~tctors (e,g . Pleistocene glac iatio ns) and the Florida Peni n sula working as a biogeog raphic barrier on nca r-sh ore marin e org a n ism s has been studied and confir me d usin g mol ecul a r techn iqu es (Avise 2( 00).

Molecu lar stu d ies have also re vea led 1he presence of othergenetic discont inuit iesalong the easte r n co ast of the Flori da Peninsul a that influence the disrri bution of not on ly marine (Reeb andAvisc 1990, Ad a rn kc wicz an d I larasewych IDDo,Collin 200I)butalso tert-e strial species (Risingan d Avise 199 :~),

Mol ecular DNA seq uence dat a ha ve be en used to study ta xonom ic an d geograph ic rela tio ns h ips in red algae for wh ich morphol og ical cha rac ters ar c scarce and/or ambiguous (Brodie elal. 199H, Broom etal.

1999 ,Zuccarello et al, 2000, Mci vor etal.200 I, Zuc- carcllo an d \Vest 2002, Lind str om and Fred ericq

20(J:~). Howe ver, few intras pecific molecul ar studies existth a texp lo re the genet icdistr ib u tio n and discon- iinuity in the economically import ant order Cracilari- ales(C o ff and Coleman 19HH, Can d ia ct al. f99~). In this study we used two typ es ofge n eLic markers to test whether the tropical populati ons or (;, lilcoaluae (Irorn Mexico)pertain to the same speciesas temperate pop- ulati onslrom Canada,10 inves tiga terhebiogeography ofth isspeci es,and to investig a tewheth er morphol og- ica lly diffe ren t spec ime ns are also gene lica lly distinct.

The ma rk ers used were the ch loro p last-e nco ded rhc):

genean d thenuclearinternal tran scribed spacer(ITS) reg ion sI(IT S I) and 2 (ITS2) an d theintervening 5.HS rDNA region.

\I.\TERL \ I.SA:\1l\IETI I O ]) S

SlIlIIlllillg, 'I"Tn ly spe cim en s of(;,/i1:1I1I1Iilll'wcre obtai ne d from 17 dist inc t lo cati on s ('l;thle I, Fig. I), Collcctions Wl'J'C

mad\:' fro m spec ime ns growing altadwd in Ihe shallow snb- tid al or fn>lll drili in g mat s in eSlu a r ine e('()S\'Sle m Ssuc h as salt ma r sh e s,Iag o ou s'.pn ll cct ed bays,illlracoa~'tal^{wa te rways.}

jellies, and mau gnl H's. re g a rd le ss orsex or life -cycle slage and based onoppo n u n ity,On ly thalli 12 cm or lon g erwe n~

considered fill' both molecu lar and Illorph ol o gi cal analyses, an d the !'ange of morph ol o gical \'ariat io n report cd Ii ,,'

(;, tiltvohiu« was com p ile d. For mo lecular work . thal li wcr«

silicageldr ied or liquid pre ser ved in 70'Icisopro py lalco ho l.

Vouc her specimens were liquid preserv ed in :i 'lc lill'mal in/

scaw.ue r, kepi an d sto re d in silicagel pr e ssed an d/orpresse d on herbari um sheets, an d rhendepo sited in the He rbruium ofthe Lrrive rsityofI.oui sia n a at l.a taycu e(L\ F)an dihe:\ 1·

galCollen ion ofthe C.S,:'\alio na lHcrba ri u m, Sm it hson ian Inst ituti on (L S) , Habitsofthe recentlycollecte d specimens were co m p a re d wirh rollcrtions of (;, li/:"lIltilll'deposit cd in

the t\\'Onu-nrion cdhe r ha r ia.

/).\'/1extrnrtions, PC/{ IIII/jJlilil'l)/illll", and ,\(''1I1I'II( ilig. Il:si:\

s.un pk-s \\TI T prep a red usin g the IlN eas )' Planl :'.liu i Ki:

(Qiagen, Vale nci a.c:.\, USA) from fresh,silica gel dried.or 70';' isop ro p ylalcoh ol fixed specimen s, Th e rlu),g\:' nt' was am p lifie d using the primer comhi n.u io ns F!'h cI.sl al'l -R 7":\ , F;",7-R 7:i:\. F,,77 - R 1:-IXI.an d F9 9:\-RrhcSstal'landseq ue nced wirh pr imers Frbrlst.ui , F7 , F:i 7, F;~~J2 , F,,77, F7:i :\, F9 ~J:\, R7":\. RIIO:i, Rl:IXI, an d Rrb rxsr art (Frc s h w.u cr and Rue - ne ss 1(94), peR amplilic.ui ous ofthe ITSI. ",XS rll:sJA, and ITS2 re g io n s wert' te-sted usiu g all ITS primers lis led in Lindstrom el al. (ILJLJli) and C;olf elal.(I~)r)4), I'(:R prim e r romb in. u io u s tluu produced ihc he st result s and thcrci or«

.ulo p ted to amplify the IT S cl.ua in this study were CI - Red".XR an d Red:i, XF-AB 2 X. ITS seq uencing primers used we r e CI. ITS I. ITS2, IT S:\ (Linds tro m etal. 1996).TWX I,

Red;"I.HI'~ Red".XR, IT S2· XO O

+,

ITS2- 700-i-", and IT S2-

1100- (Coil' etal. I!J~)4). I'rol o col s Ii II' douhlc-st ra ndcd I'CR arn p lifir.u ion and automated D:'s1:\ se</Ile nc ing used fill'both genetic ma rkers are id e m ical to Iho se give n in I.in etal. (20 0I),

/'hyl()gl'lIl'1ir (IIIalssrs, The gene rated sequence da ta were compiledan d alig ne d withScq ucnrhcr(C ene CodesCorp..

.\ n n Arbor, :-''11, LS.\) and \lacCla de 4,0 (Ma d dison and Mad di so n 2(00) an d expo rte d fi,r phylo g en et ic analvsis . The rbct. alig n me nt inc luded two ou tgro u p sequenr'es,

c.aIr tlmn ttrcoru is an d

c ..

larin ulut« Crable I). sel eCle d he -

cause or thei r closest phylo ge netic rela tio nshi p with (;.

/i/limhi,,!' «;u l'gei an d Frcdcricq 20()4), The fir st 10 0 hp or the 14( j7-b p rl«I.seq ue ll(Ts wereremoved fr om the ana lyses du e to missin g datu at ihis re g ion on the alig n me n l. The fla n ki n g re gi ons of IT SI an d IT S2 wlTes po lHling 10 the:r

end or the small subunit rD:si:\ an d thc ,,' end ofihc la r g e subuni t rD;\'\ gene s \\TI T remo ved Irorn the unal vs es , rc- strirtiru; Ihc data sel 10 ihr- ITSI.:i, XS rD\l,'\.and iT S2 rc-- gio n s, ITS phylogeneti c analyses we r e performed unroo tcd because sequence divergen ce belwe en dilfcrcnt but closel y relate d sp e cie s of Crarilaria did not provide re-liable align- mcn ts. Sequence inlornuuion Ii, I'rbc),and ITS makers an d (;enBa n kacce ss io nnumburs arelistedinTahle-I,Alignm ents were depo site d inTre clias e (stu dyaccess io n# = S9(7 ),

Ma ximum parsim on y(:'.11')and maxim u m likel ih o od(:'vI I.) ana lyses as implem enl ;'d in P.\l:1"" \A.Obe la 10 (Swolford

200I)\\TIT cond ucted ril l'bothrhd.an d ITS data sets, Pa rs i-

mon )'trees \\'CI T obtai ned under Ih(' Filch criu-rion ofe'lua l weig ht s Ior all su hstit uuons (Fitch I_~)71), :-',IP aual )'Ses we r e inferre-d hased ou a t\\'o-Slr' p ap p ro ach. Fil'st. we co n d urte d h(,u risticsealThesor5 000ran do mrep lica tio n s,holdi ng 20tn'es aleach step . TBRswap pi ng algo r it h m with Ihe :vILI TR E ES an d STEEPES T IJESC E\lT options,excludi ng uninl orm at i,'c' charaetel's,Secon d. mo st parsimon io us tre es save r!on Ihe fir st ana lysis lI'er(' swa p ped 10 com p le tio n in a secon d slC'p :-',1P an alvsis.\11. Iree sIi,I' both dat asetswe rt'inferred based on a h('m:isticsea rc hor 1000 ran d om rep lica tions,hold in g 10 trce s al eac h stcp. TIIR swap p ing algo l'it hm \\,jlh :VILU 'REES and STEEPEST DESC E\lT opt io ns.The oJll im a l models of sequc' ll<'e el'll iu tio n to fit Ihe dala alig u llle n lS were estim a led by hin a n:hical likelih o od ra lio tests periill'lJle d hy \Iod el- tesl ,',:Ull) wilh alp ha=0.01 (Posa da an d Cr a n da ll 19SJIl).

Th(: o[1 lima l Illodel used lor the rhrL data se l was the

7;-iO C.\ RI.OS FRED ERI CO DEI.L<-2LI(; U U ; EI. ET,\L

T \ HI.I' I. LiSI or spe ci es st u d ie d:identifi cat ion, colle cti on information,Grarilari« tikvahiae lo c ation numbers on the map or

Figure I,rlwl .,ITS I,5.8S, ITS2GenBank acce ss io n numbers, an d fraction sequenc ed.

,\Y04~ 1:\'2 (i(1OOll )

ITS.,\l'( l lIl 'IH T

idcl1tilicaricHls

AYIl4!I448(IOO'if) 0·1

'"

,\\,04 9 4 ;j9(IOO'ii) g'2

AY04 9449 (IOO'l) HI,~

.W 04!H:, 8 (IOO'if ) g:1

AY04!)4;jI (!)~) .4'ii) g;')

..\YO'! 9·'1:">7 (H9.0'if) g4

.\\'0 4 94:">:1 (I OO'k)

gl~

,\ Y04 94 ;jO(IOO'ii) ,,6

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AY(W 14fi:\(IOO'lr) gH

.\ Y0494 5'2(~)6.7(ii)

gi

AY0494 (i 1(100'1<) g9

AY04 94 fi'2(10 0'1<.) ,,10

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AY0494 (iO(IOO'Ir) gil

,-hd.hap lolyp(~n.mu-s

- -- - - -

(~cnlb ll kaccessio nno..',!,sCfl uenccdrbrl,haplot Yl w names. ITSSl'CjlleIH"l"idl'lI l ifKaliolls

AY(H9:I(i'2(!);">.'!'if) hlO

AY04 9 447(0:'),(;'ii) h!)

AY049:>4 9(Y8.4'I<) h:',

AY4 '2(i74 9 (0H.4(I<) h;,)

AYIH~1440 (9H'(i'ii) It;')

,\\'04 9441(07.8'l) h:,

AY04!U 44(97.1'Ir.)

,\ Y04!J.!84 (97.0'l) hfi

'2 .W4'2 (;7;",O(!)7.'!(l) h7

:1 AY04944:\ (97.:>'Ir ) h7

4 AY(W148fi(9H.7'1<) h7

;; AYIWH4 '2 (!17.4'A.) rh7

(i AY04 94 :\;') (94.1'if) h7

7 !\\' 0494 ,1'2(99'1<) rhH

H ,\ Y04 9,"'(;(9:).'2'if.)

h7

II 9

lU

l;j 1'2

1:1

14 (;.til:iVd,itll>

ivnlan- numlu-r Specim encollection data

Harbor Branch Oce an og raphi c lnsritution northjetty. Indian River, Fort Pierce. Florida. L:SA.'27'8 '2'0 0 "

N x HO''20 ' :19'' \V,mil.C.ED.(;urgd , I:IJuly 199 8

l.a Enc ruc ijuda,Peninsula l'a ra gu un.i, Falco n Stat e,Ve nezuela;coil.C.ED.

(;urgel .J E. Con de.

c.

^{Car mona:}

#10'(;-1 9 , 1:I.lu1 y 1999

Can ad a:spec im e n #I,MOITe l pond.

l'omqu ct Harbor,An lig'lm ishCo.,Nova Scotia.mil. CJ Bird;#10'(;-99. :1July 1!J!J9

Cana da: spe cimen #'2,Monet Pond. Pomqu c tHarbor.Antig onish Co.• Nova Scotia,4:'):HI':"Jx 6J';jO'\\,. mil.C.J Bir d,#F(;- IO I.0:1.1uly I!19!J RhodeIsland:Charl est onbrearh wa v, uSA,co11.C.E D. (;urg el. #F(;-I:')9.'11 Aug us t'20 0 0

:--JewJerse y:I.akonubea ch, Bar ne gat Bay. l'SA,mil.C T Frankovish ,#]0'(;- 109, O;j April I!)99

De laware:Reh obothBa y.Dewe yBea ch. USA, mil.CEll .(;u rg cl, #F{;-I4 6, 1'2 Aug us t'200 0

NorthCaro lin a: Nort h Ma sonboro in let jell)'.Wrighlsville Be a ch.Ne w Hanover Co.•LSA.coli.\0\'.n.Freshwat er. #F<;- 10'2,'28 ""la y 1998

SE Florida:Sebastian Inlet ,Vero Beach Co.• LSA. '2T5I''20''N x HO'27'Oo"\\', coil. C.ED.{;urgel , 10 October 100 8 SEFlorida: Indian River,close tothe Mcl.arrv'Ircasurc M uscum , ;'\. J-1utchi,;s on Is..USA,

'2T;';O'['2":"Jx 80''2(;'1I"\\'.coil.CYD . {;ul·gel. 10October 19 98

SEFIOI'ida:Harbor Branch

Oceanograph iclustiuuion northjcuv, Indian River. Fort Pierce, St. Lucie Co., USA, '27:1'2'00" N x HO 20',\9"\\', coil.

C.ED,ClIl'gel. UJuly 199H

SE Florida:Cu lt u red Gracilariast rai n, varie ty br own ,HarborBrauc hOcean o- gra p h ic Ins tituti on.Fo rt Pierce, St.

Lu cie Co..LSA,leg.;"1.D. Han isak, vii.199 H

WFlo r ida:'Jarn p a Ba y,specim en #1- drill Iorm, close to the mouth or Coc kr o a c h Bay.USA,coiL C.

J.

Dawes.

#F<;-94 . '2(jOctober 1l)l)9

V\·FI01'ida:Tampa Ba y,speci me n #'2- la x form ,close tothe mou th or Coc kroac h Bay.USA,coil.c..I.Dawcx,

#F(;-9;'),'2(j Oct ob er 19 9 9

WFlor ida:TampaBay,northsideof719'2 Gand vBlvd.,USA, coil.C.Are good,

#F(;-'14 ,1,'29 No vc-m he r I9l)!j

\VFlorida:TampaBay,drifting in a mangrovechan n el, so uthsideor {;;m d v Blvd'., USA. co il.C. Areg ood.#F<;.1 4 4, '2~1Xovcrnber 19 9 9

Mississ ipp i:Gu ll'Isla nd Nat io na l Seash o re. Hom Island-PetitBo is. L'SA, Spc.:dl"....01"(;mriltll71(

t.,lacinulat«

(Va hl)Ho we

(;.liI:1IIIhi(/f' Mcl.achlan

c.alLdn marrorn is

J

^{,\ga n l h}

'I; lh leI. (continued)

PJ-lY!.O(;[':O(;J{'\I'HYOF c.nf.;J~·IHJAJ;· 7:>1

(;("111\; 111" acn:ssioll 110.,',"Sl"ljlll"IICl'd rlnLhaplutype 1l00 Il h:S.IT S'i('«III ("I1("("idcllr it icali o J)s

Spcrin u-nrolh-rtiondata coil.Carte l',#F<;-14 1.06Ju ne 2000 Te X,IS:specime n#Icylin d rica l,Rcdf ish Bay,Port Aransas, LSA,coll. S.

FITd el'icq&C. E D. (;nl'gd,18Ma y199H Tcxns:specimen #2 flat, Rcdlish Ba y,

1'01'1Ara ns as,USA,co il.S.Fredeiicq&

eED.(;u rge l, 18 \lay 19!18 Me xico:Alvarado La goon, Alva ra do, Pucrta Prieta area, 18 47.80':-.J x !):i 48.8:1'IV,coil.C.E D. (;lIrgd,l:i February I!)98,

Mexico:SanAgustin, Santana Citr,Vera (:ruz Slat e ,19' ;):>.2:1' :-.J x % ':11.8 Y IV, coli.C.F.Il.(;urgcl, !l February 1908 Mexico:Man gro ve, Paraiso town. Porto Ccibas, 18 2:> .9H':-.J x 9:\ 09.8Y II', coil. C.ED.(;lIrgel, 14 February 1998

(;.tiltra hiar isolan- n umlx-r

Hi

17

18

I!)

20

rhd. haplotypenames

.\Y04944:) (9:1.7'k) hI

A1'04 9 444 (!)(i.2';'.) h2

,\ 1'0494:1:1 (9HA';') hi

.W 04 94 :17 (99.1'It) h:1

AY0494:18 (9HA';') h4

ITSsequence id e-ntifirmiu us

i\Y0494:i4 (10 0';') g8

AY0494:ili (I OO'ln) 0' 1'1

"

i\1'0494:>;) (I OO'J,) gl:-\

1-IKnF>

+

I

+ (;

(Ha scgawa -Kas h ino -Yano mod el [H asega wa (,Ial. HlH:i] with invariahk-sue sand gam m a diS\l'ibulion )and lhe (:TR

+

I

+ (;

lorthe ITSd.uaset.Theparametersestima t- ed lortherhd.dala sciwere asloll o ws:nucleotide I'n:quen cies

..\ 0=O.:IO!l:i; C=O.lliOl; (;=0.21:,·1;T =(UI :,O;substitut ion mod el Ii/IV⁰⁼ 1.92 0I: proport ion orinva riah lesites= 0.82 90, anda gam ma disl rihunon withsha peparam e tel'= 0.81[:>.The parame lLTs estima ted ()I' the ITS data sci \\TI-e as loll o w»:

7 Atlantic

/ Ocean

~8 15

0 ~9-IO

11-14 ....

~~\ .

~~". _~

....

~ ~

, . . . - ,. .

J~

^{e, •}

• . \ - - o t ' . """.

~

FIt;. I. Map showing distri- burien or(;m,.ilaria tik,'alli{/(! col- lecti on s.Arrows point to regiolls from where SI'CCilllCIiS were col- lcrt cd. Numbers coincide with locat ion numln-rx in-b bl" I.The geogra phicdisui b u rion range ol (;. tihvah ia«exte n ds from Canada down 10 Ihe Yu ra ta n Pen in sul a, Mexico,

CAR!.OS FREDERICO DEl.U2lil CeRCE!. ET AI..

1'1(;. 2. Grarilorio ukvalnor compressed alignment or 10rbr), haplotypcs (only the variable sites arc shown). Haplotype names wincide with those rin-d in 'Jahlc I (h I-h 10). Substitutions at first and second codon positions marked as underlined and hold- faced characters, respectively.

nucleotide lrcquenric-s .'\=O.2(i:i:i; C=0.2006; C;=O.22')!);

T =0.:\040; substitution model: A-C=I.H I D4,.\-C;=H.44:l7, .\-T=1.921 D,C-C; =1.9:\27,C-T=I H.H'i2(i,C-T=1.0; pro- portion invariable sill'S= O..i:iOH,and gamma distribution with shape parameter= 1.4:iH4. Nonparamenic bootstrap values Ior nodes in :\H' and M L phylograms were calculated based on ,'.000 and 1000 n-samplings, respectively.

Phylogenetic relationships amongrbl"!.haplotvpes and ITS genotypes were also estimated using the sr.uistica] pnrsimony procedure ofTcmpkton ct al. (1992) developed specilicall)'to reconstruct within-species gene networks. l nihiscase, the term parsimony refers to the minimum number or d illcruno-s sep- arating two individual sequences ruther than a global mini- mu m tree length based on shared derived characters. The statistical parsimouy procedure was pe rforrucd using the com- puter package TCS version 1.1:> (Clement el al. 2(00). D"J.'\

sequeuces with pairwise absolute distance values equal10zero were considered the same and were abo collapsed iuto h.iplo- types (lbeL) or genotypes (ITS) by TCS.

Pairwise sequence divergence values cited in this study arc based on uncOlTecled /J dist.mrex, which is equivalent to the number or segregating sin-s per nucleotide site. Pairwise/idis- tance values were calculated hy PAC 1'*

hI h2

h3

h4

h5 h6

h7

h8

h9 hIO

CGACGCTTACTT

- - -

CGACGCTTACTG

_{- - -}

CGACGCTGCCTT

- - -

CGACGCTTATTT

_{- - -}

TAACGCTTACTT

_{- - -}

CAACACTTACTT

_{- -}

-

CAACGCTTACTT

_{- - -}

CAGCGCTTACTT

_{- - -}

CAGTGCTTACCT

_{- - -}

CAGTGTATACTT

RI',SLl:IS

rbc/. data set, In the J:{67-bp rbcl . alignment, in- cluding the two outgroup sequences, GO bp vary and 22 bp were parsimony informative. When the two outgroup sequences were excluded from the align- ment, there were 12 variable and Iour parsimony- informative sites. Ten haploi.ypes (hl-hIO) were obtained among the 19 (;. tikoahiae sequenced sam- ples. A com pressed rbci .alignment including details of substitution type and codon position are given in Figure 2. The overall genetic diversity among rheL haplotypcs ranged between 0.(17,{1;{ and 0.312^cx' Ce- ographically, most distant haplotypes (Canada vs.

Mexico) presented a smaller rbcL pairwise genetic divergence that ranged from O.07;{C;{ (h 1 vs. h7) to 0.29:\% (hf vs. h;{) when compared with samples from geographically closer localities. The haplotype Irorn a specimen cultured at the Harbor Branch Oceanographic Institution, hI 0, was ranked the most divergenl in pairwise comparisons, with sequence distances ranging from 0.219 C;{ (h I 0 vs. hH-h9) to 0.:')12% (Ill 0 vs, h'\).

The lwo-step ivIPrbci.. phylogenetic analysis resulted in a single most parsimonious Iree, and all G.tilroaluae rbci: sequences formed a monophyletic group (Fig. '{).

The ML analysis resulted in three trees of similar to- pology and same likelihood score (-In= I H41.:Hi44H;

data not shown). I nternal nodes in the (;. tikoalua«

clade that were not resolved in the MP tree were also not resolved in the ML result, rbc),phylograrns recog- nizcclIour genetically and geographically distinct line- ages: I) a western Cull' olMcxico lineage, 2) an eastern Gulf of Mexico lineage, ,{) a southeastern Florida lin- eage, and 4) an eastern Canada-northern U .S. lineage (Fig. :\). In allr!JeL phylograms, eastern GuJfof Mexico haplotypes always appeared at the most basal position, whereas haplotypcs from the western Gul(" of Mexico and southeastern Florida appeared as two derived line-

ages. Further phylogenetic relationships among rbci.

lineages are still not clear and received low bootstrap support. Haplotypes were not shared among the four geographic locations, with the exception of h7 that was colleclecl in southeast Florida hui pertains to the Canadian lineage (Figs. 2 and ,I).

The rbci.statistical parsimony network (SPN) does not exhibit a single star-shaped topology but instead comprises lour distinct internal nodes composed 0("

Ihree actual haplotypes (h I, h7, and hH) and a (HIrth hypothetical haplotype (Fig. 4A). These four internal nodes are connected to the other seven derived sam- pled haplorypes. Haplotypcs from the western Gulf of Mexico lineage (h l-h4) form a separate group char- acterized by a G at position Fi8:) that distinguishes them clearly from the remaining haplotypes that have a synapornorphic A at this position. The eastern Gulf of Mexico haplotype (h.i) is recognized in therbci: SPN by a T at position ,\81 (Fig. 4A).

FrSdata.II'!. The orig'inal PCR amplified fragment

consisted 0(" 12,\5 bp. Aller removing the flanking regions0("the ITS I and ITS2 that correspond to the :\' end ofthe small subunit rDNA (= 120 bp) and the

;)" endofthelarge subunit rDNA genes(=2H bp), the ITS sequence data set included in the analyses was IOH7 bp long. The length of the ITS I region ranged from 292 to 295 bp, 0(" the :).HS rDNA from 209 to 214 bp, and the ITS2 region from :')77 to 57H bp. ITS regions were completely sequenced with the excep- tion of three samples that had missing data in the :).HS region, not exceeding I IcXof the entire number of sequenced bp (Table I, C;{ sequenced). No different ITS genotypes were observed within the same spec- imen, and only one ITS genotype was obtained from each isolate with the protocols implemented in this study. The ITS alignment (no outgroups) presented 1,\ distinct indels composedofone to Iwo nucleotides in length. When indels were considered as a fifth character slate, there were 4,{ variable and nine

I'IlYLOG!'O(;RAI'IIY OF C.nKJlAJlI/1F

Isolate 16. Texas Isolate 17. Texas

63

Isolate 18.Mexico Isolate 19. Mexico Isolate 20. Mexico

Western GulfofMexico

lineage

- 0.5 changes

100

Gracilaria tikvahiae Clade

64

^{Isolate 10.}Fort Pierce Isolate9.Fort Pierce Isolate 7.Sebastian Inlet

64

Isolate I. Canada Isolate2.Canada Isolate3.Rhode Island

Isolate4.New Jersey Isolate 5. Delaware Isolate6.North Carolina Isolate 8. SE Florida Isolate II.Tampa Bay

SE Florida lineage

Eastern Canada Northern USA

lineage

Gracilaria lacinulata Graci/aria damaecornis

Isolate 12.Tampa Bay Isolate 13. Tampa Bay Isolate 14. Tampa Bay

Eastern Gulf of Mexico

lineage

Fit;.:>. Single most parsimonious tree based on a data set ol' I~) Crillillll'ill tilumhiar rbrL sequences. Tree length= 2:1 steps,COJl-

sistcncv index

=

0.9:;(;, retention index

=

0.9(;7. I'lu-numbers above Ihe branches represenl noup.uamet rir bootstrap value s (:;0% or higher') hased(1) :3000 replicates.

parsimony-informative nucleotide sites (Fig. Pi). The presence of indels occurred in all distinct sequences and ranged between O.O~JC!c and 1.2% of the total number of base pairs in the alignment. When indels were not considered as a fifth character state, there were 30 variable and seven parsimony-inforrnativc nucleotide sites. Fourteen distinct ITS genotypes were found among the 16 samples (gl-gI4), and a compressed alignment is given in Figure ;J. The overall sequence divergence among ITS genotypes

ranged from 0.04% to 1.47^r!c(considering indels as a fifth character state). The most divergent genotype was g12 from Mississippi, with pairwise genetic dis- tances ranging from O.t);)'!r (g12 vs. g7) to 1.47^f!c (e.g.

g12 vs. g9), followed by g2 from Rhode Island (0.4()f!c.-1.24%).

The :VII' analyses or the ITS data produced three most parsimonious trees, with and without considering gaps as a hlth character state. A consensus Irorn any of the two sets of three most parsimonious trees did not

754

B

C\ RI. OS FRED ERI CO m:I.l:<'2c1CL:!{( ; EI. ETo'\!..

trees obta in e d in the ITSM P anal ysesand similar val- ues or bootstrap su p p o rt (Fig. 6). The ITS SPN presented a netlike topology with one internal hypo- thet icalsequence (Fig. 4B). No phylogeographi c str uc- ture was found in the ITS SPN. Genotypesg4, g8,and the hypothetical genotype (square) were the most in- ternal sequences in the network and presented the highest number or con n ectio ns to the remaining gen -

otype s:six, live ,and six branch es ,respectivel y.Thes e three seq uences, together with g14, comprised the cen te roft he ITS phylo genet icne twork.The geno type Irorn Mississippi, g12, was iden tified as the most di- verge n t from its closest sampled gen o ty p e (= nine evo lu tio na ry steps or eig h t interm ediate states).

For both clat a s ets(rbc), and ITS ) and reganllessor the Iradiiional method or phylo genetic analysis used (1vIPor Ml), specimens Irorn Canada, Rhode Island, New Jersey, and Ta m p a Bay, Florida were always found in a distinct clade (Figs. gand 6). Morpholog- ical com p a r iso ns within and among C.tiltoaluaepopu- lations included in this studydispl a yed high lev el s or intra- an d inte rp opulation phenotypi c variation (Fig.

7) with com p letely flat to co m p le te lycylin d r ica lspec- imens. ofte n inhabitin g the same location. No mor- phological syna p o rno r p hy could be distinguish ed lor any parti cular lineage Ihat ot herwise co u ld defin e a particularintraspecific taxon. Howe ver, thalli or drift- ing specimens among the Me xican populations were typicall y Hatter, thinner, narrower.and more densely branch ed (Fig. 7, B and C), a phenomenon hitherto on lyobse rv ed in Mexicancollecti ons.

I'lL.4. Mai n ne two rkor relatio ns hi ps to r(;w l"il l/l'il/ lil:i!IIltiw' DNA sequeuces based on the sta tistica l pa rsimou y me th od of Temple ton cral.(199~ )an dcons idering ind cls as a lifthcha rac- ter sta te.Lin esindicatethenuuati onal con nertio ns . All bran ch es an'onecha nge lon g (= parsimon ybased evolu t io naryste ps thai ar e equal ro thenumberorsubst itu tionssepara tin g two connect- edseq uence s) unless anuotat edas numbe rsnextto each branch . Circles cor re spo nd to actual sampled seq uc nces identified Ill' their nam es(quoted insid e circles). Squarescorrespond to hy- potheti cal DNA sequen ces. rh,.L haplotyp e (h I-h I 0) and ITS gcnotyp e (gl -g I4) names coinc id e with those mentioned inTa- ble I and Fig u res ~-().respecti vel y.Black .gray.an d whilecircles corres pond . respecti vel y,10 a Tampa Bay, a weste r n Cull'or Me xir'o, and an At la n tic or ig in for the seq ue nces obtai ned . (A) Sta tistica l pa r sim o n y netwo r k based on 10

1"""1.

hapl o type».

(H)Sta tistica l parsim on y ne two rk based on 1'1 IT S1-:;.HS-I T S2 nuclearrDNA geno types.Ceno ty pegHwas samp ledinbot hrhe western Gu lfof Me xico anr lthe Atla n tic.

produce the same topology as found in the rbci:anal- yses, but both recognizedtwo cladeswith low bootstrap support (d a ta not shown).One cladewas compos ed01' north Atlant icspecimens (fro m Canad a,Rhode Island, and NewJersey,boot strap va lue s= 6~(l),and the sec - on d was com p ose d or theTampa gay specime n s(boo t- strap values= 65'/(.).The ]\vI L analysis of the ITSdat a set also resulted in three phylo grams with eq u a l like- lihoods(- In= I780.76HR~).Aconsensusor thes ethree MI. trees had the same topolog y as the two co nse ns u s

IJISC C SSIO"

Even with a relatively low number or phylogen eti- ca llyinformati vecharacte rs and a sm a ll sampl e size, a dramati c result was the striking sim ila r ity in the pat- tern or cp DN A divergence in (;. tikoahut e to that or other previouslyreported marine an d nonmarinespe- cies (Avise 20(0). The geographic location or the geneti c disjunction sep a r a tin g different G. tikualuae phylogroups ca n no t he precisel y determined with our limited sampling,an d in ad d itio n, regions orge- net ic disju nctio ns along the Flo rid a Peninsula diller amon gdistin ctta xa . Amore robust sa m p lingalo ng the en tire geogra p h ic distri bution ofG.tikuulna cis cr uc ia l to further test the hypoth esis or gene tic disjunct ion between differentpopulati on s, How e ver,the lour rbcL phylo geneticlineagesfoundin thisstudy(Fig,3)arein agreementwith somespecific marin ephylogeographic studies an d are suggestive of three regions where the disjunction or G. tikuahiae haplot yp es might occur:

I) north eastern Florida, 2) the sou th e r n tip or the Florida Peninsula,and :~) some place in northern Cull"

or Mexico.

Northeastern Florida. A sepa ra tio n betwe en the sou the as te r n Florid a an d north east ern U.S. haplo- typ e grou ps has also be en observed for the marine oyst erCrassostreauirginica(Ree ban d Avise 1990 );the horsesh o e cra b Limulus jJo!-Y!J}lI'IflIlS (Riska 1981 ,

gl g2

g3

g4 g5 g6 g7 g8 g9 g10 gIl g12

g13

g14

1'1-[YLOGEOGJ{,\I'JIY OF C. rIKV/llflAF

TCATTTCTGAAAAGTT-GGTGTATGG-CGGTTTACGAAAGGTC TCATTTCTGAAATGTT-GGTGTATGG-AGGTTTACGTAAGGTC TCATTTCTGAAAAGTT-GGTGTATGGCCGGTTTACGACAAGTC TCAT--CTGAAA?????TGGGTATGG-CGGTTTACGA??????

TAAT--CTGAAAAG-T-GGGGCATGG-CGGTTTACTAAAGGTC TCAT--CTGAAAAGTT-GCGGTATGG-CGGTTTACGAATGGTC -CAT--C--A--A?TT-GGGGTATGG-CGGTTTACGAAAGGTC TCAT--CTGAAAAGTT-GGGGTATGG-CGGTTTACGAATGGTC TCAT--CTGAAAAGTT-GGGGTATGG-CGACTTATGAATGTTG TCAT--CTG-AAAGTT-GGGGT-AGG-CGGCTTACGAATGGTC TCAT--CTGAAAAGTTGGGGGTATAG-CGACTTATGAAAGTTG TCGA--ATGAAAATTT-GGGCTATGG-CAGTCCGCGAAAGGCC TCAT--CTGAAAAGTT-GGGGTATGC-CGGTTTACGAAAGGTC TCAT--CTGAAAAGT--GGGGTATGG-CGGTTTACGAATGGTC

755

Fn.. ;i. Cmcilnna tikvahiae com- pressed alignment01"14ITS 1-:i.HS- ITS2 nuclear rDNA genotypes (only variable sites arc shown, con- sidering indels as a filth character state). Genotype names (gl-g14) coincide with those cited in 'Iable I and Figures4and6,respenively. -, missingbase (indel); ~,missing data (not sequenced).

Saunders et al. 1986); marine toadfishes, Ojisanus tau and 0. beta (Avisc et al. 1987); and the seaside spar- row Amnuulramus maritimus (Avise and Nelson 1989).

The geographic isolation of haplotypes observed be- tween northern and southern coastal marine popu- lations along the eastern coast of Florida is usually not abrupt, and haplotypes with higher frequencies in one population may be found with lower frequen- cies in other geographically close but distinct popu-

lations (lack of complete genetic isolation). However, as samples are drawn far from the proposed genetic disjunction locations, the genetic distance bet ween populations tend to increase sharply (Fischer 19GO, Reeb and Avise 1990,Aviso 1992, 1994, Collin2(01).

Southern tiji ojtlie Florida Peninsula. On the basis of DNA sequence analysis, divergence between Atlantic and Gulf of Mexico haplotype groups have also been documented for sea anemones (McCommas 1982),

Isolate 10 (g8) SE Florida

Isolate 18 (g13)

Mexico Isolate 19 (g8) Mexico Isolate 9 (g7)

SE Florida Isolate 8 (g6)

SE Florida

Isolate 7 (g5)

SE Florida - - - 1

Isolate 6 (g4) North Carolina

Isolate 12 (gIO) Tampa Bay

Isolate 13 (gIl)

Tampa Bay IsolateII(g9) Tampa Bay

Isolate 20 (gI4) Mexico

Isolate 15 (g12) Mississippi

Isolate I (gl) Canada

Isolate 2 (gl) Canada Isolate 3 (g2) Rhode Island Isolate 4 (g3)

New Jersey

Fu.. (i. Stric! consensus 01"

three 'VII. trees with equal likeli- hood scores (-In= IH70.7(;8H;i) based on a data set composed 01"

1(;(;mriti/rii/ li/cui/hilll' ITS rONA sequences. The numbers inside circles on the branches represent nonparame! ric boot st rap values (;i()l/r or higher) based on IOO() replicates.

756 C.\ RI.OS FREDERICO DEI.LQU C;UU;J·:1.ETA!..

Fie. 7. Cmrilarin lilil'llhifWphe- norypir variation. (A) Specimen lrom Paraiso, Porto Ccibas, Me xi- co. Scale bar, 2 ern. (1\) Specimen (i'OIllSani\ g u srin lagoon, Santana, VeraCruz Stale,Mexico. Scale bar, 4 em. (C) Specimen from Pori Aran sus, 'Jexas, USA Scale bar.

(inn. (D) Specimen from Morrcr

pond.l'omqu et Harbor, Ant ig o n ish

Co..Nova Scotia.Ca na d a.Scale hal'.

4em .(1-:-1')CysIOC;IIV icspe cimens co llecte dgr o wing attachedandco- occurr ing in Se bas tia n Inlet,cas t- ern Florida . USA. (1-:) Cylindrica l spec ime n . Scale bar. n.;)un. (F) Flat specimen . Scale bar,Icm.

mussels (Sarver et al. 1992), marsh and stone crabs (Ben and Harrison 191)1), Feld er and Staton 1994), mosquitolish (Wo o te n et al. 19H8),and Ihe black sea bass (Bo we n and Avise 1990 ).Courernporary ge ne tic disjunctions often persist acro ss the southern lip of the Florida Peninsula ,a region known as a geograph- ic barri er to co m m o n estuarine species (Fel d e r and Staron 1994).

Northern (;/11/ of Mexico, rbc) , results suggest the ex iste nce of a uniqueeaste r n Gulfof 1\'1cxico linea g e.

II' this is the ca se , places 1<11' possiblegenetic discon- tinuitie s in co n tin u o u sly and quasi continuou sly dis- tributed benthic species along the north ern Gulf of Mexico include the mouth of the Mississippi River (Adarnkcwi cz and Harasewych 199B) and/or the

Chenier Plain, a large marsh/estuarine system , be- tween so u t h easte r n Louisian a an d the northeastern coas t of Texas. Very few hard su bst rata for intertidal and su b tid a l species are located in the later inshore region ,which is char act erizedbyshallow muddy su b- strata (Sta to n and Feld er 1995). De spite seve r a l at- temptstoobtain moleculardata Irorn specimens Irorn the northern Gull' of Mexi co , effo rt s were unsuccess- Iu], In the northern co as t of the Cull' of Mexico, (;.tikuahiaepopulation s are most likel ypatchy,oppor- tunistic, an d transient (Ka p ra u n 1974 , as (;. [oliifera val'.angustissnna),

The application ofrLJrL DNAsequences to identity so u rc es of marine macroalgal species invasions has been successfully applied (Mcivor et al, 2(01). If re-