Molecular Determination of Species Boundaries in Corals: Genetic Analysis of the Montastraea annularis Complex Using Amplified Fragment Length Polymorphisms and a Microsatellite Marker

14 Pages·1999·5.6 MB·English

by  J V Lopez

Reference: Bio/. Bull. 196: 80-93. (February, 1999)
Molecular Determination of Species Boundaries in
Corals: Genetic Analysis of the Montastraea annularis
Complex Using Amplified Fragment Length
Polymorphisms and a Microsatellite Marker
JOSE V. LOPEZ', RALF KERSANACH, STEPHEN A. REHNER2 AND NANCY KNOWLTON3
,
Smithsonian Tropical Research Institute, Apartado 2072, Balboa, Republic ofPanama
Abstract. Analyses ofDNA have not been widely used to on somatic tissues may be feasible, particularly after diag-
distinguish coral sibling species. The three members of the nostic differences have been established using sperm DNA.
Montastraea annularis complex represent an important test
case: they are widely studied and dominate Caribbean reefs. Introduction
yet their taxonomic status remains unclear. Analysis of The recognition of species boundaries in sympatry is
amimcprloisfaiteedllfitreaglmoecnuts,luesnignthg DpoNlAymofrrpohmissmpserm(,AFsLhPosw)edantdhaat bstrreaeidgihntgfoirmwpalridesinthepreixnciisptleen,cebeocfaautsleeatshtesoabmseenfcixeedofgeinnetteirc-
Montastraea faveolata is genetically distinct. One AFLP differences between taxa (Avise and Ball, 1990). However,
primeryielded adiagnostic product (880bp inM.faveolata, the number of such differences may be very small if the
920 bp in M. franksi and M. annularis) whose homology isolation oftaxa is recent or the rate ofevolution is slow. If
was established by DNA sequencing. A second primer in addition sporadic hybridization occurs, the problem of
revealed a 630 bp band that was fixed in M.faveolata. and defining species becomes particularly difficult (e.g.,
rare in M.franksi and M. annularis: in this case homologies Howard et al., 1997).
were confirmed by Southern hybridizations. A tetranucle- Closely related coral species appear to be especially
otide microsatellite locus with several alleles exhibited challenging in this regard (Veron. 1995; Knowlton and
strong frequency differences between M. faveolata and the Weigt, 1997). Speciesboundariesare in flux foranumberof
other two taxa. We did not detect comparable differences well-studied groups (e.g., Miller and Babcock, 1997; Miller
between M. annularisandM.franksi with eitherAFLPs (12 and Benzie, 1997; Odorico and Miller, 1997; Willis et al..
primers screened) or the microsatellite locus. Comparisons 1997; Knowlton and Budd, unpubl.), and it is unclear
of AFLP patterns obtained from DNA from sperm, somatic whether these controversies are due to the technical chal-
tissues, and zooxanthellae suggest that the technique rou- lenge of finding diagnostic characters between generally
tinely amplifies coral (animal) DNA. Thus analyses based similar but reproductively isolated taxa, or alternatively, to
the blurring ofspecies boundaries by hybridization (Veron,
1995; Knowlton and Weigt, 1997; Willis et al., 1997).
Received 5 June 1998; accepted 1 December 1998. Molecular methods have great potential to resolve the na-
Oce1aCnuorgrreanpthiacddrIenssst:ituDtiiovni.sio5n60o0fUB.iSo.med1icNoarlthR.esFeta.rcPhi,ercHea.rbFoLr B3r4a9n4c6h; ture of species boundaries because of the large number of
E-mail: [email protected] unambiguous characters they provide (Avise. 1994).
2Current address: Department ofBiology, P.O. Box 23360, University A clear example of these issues is presented by the
ofPuerto Rico, Rio Piedras, San Juan, Puerto Rico, 00931. proposed members of the Montastraea annularis species
O02c0e12aA;nlosEgo-rmaaatpihlMy:a,[e-mail]nonDi0e2g0o2,.LScariJpolplsa,InCstAitu9t2i0on93o-f cmoomrpplh)e.x:MM..faanvneuollaartias((ffoorrmmeerrllyymmoorrpphhoottyyppee IIIororcomlausmsniaver
Abbreviations: AFLP - amplified fragment length polymorphism, a morph). and M.franksi (formerly morphotype III or bumpy
registered trademark of Keygene. morph) (Knowlton etal., 1992; Van Veghel and Bak, 1993:
80
GENETIC ANALYSIS OF MONTASTRAEA 81
Weil and Knovvlton, 1994). In sympatry, these taxa differ in Materials and Methods
colony morphology, growth rate, stable isotope chemistry,
aggressive behavior, allozymes. corallite structure, and life Sample acquisition and DNA preparation
history (Tomascik. 1990: Van Veghel and Bak, 1993. 1994:
Van Veghel, 1994; Van Veghel and Kahmami. 1994; Weil All corals were collected from the San Bias Islands,
and Knovvlton, 1994; Van Veghel and Bosscher, 1995; Van Panama. Colonies were identified to species in the field,
Veghel c/ al.. 1996; Szmant et al.. 1997; Knowlton and basedoncolony morphology, andbrought to waters nearthe
Budd. unpubl.). Such concordance of suites of independent laboratory shortly before the anticipated date of spawning
characters in sympatric taxa strongly suggests reproductive (Knowlton etai. 1997). At dusk, each colony was placed in
isolation (Avise and Ball. 1990). and differences in the a separate container: spawning generally occurred 2-4 h
timing of spawning and apparent barriers to interspecific after sunset, and the gamete bundles were collected imme-
fertilization (Knowlton et al.. 1997) also support this inter- diately after release. The gamete bundles from each con-
pretation (but see Szmant et al.. 1997). Overall, these data tainer were washed separately over plankton netting. The
support separate species status regardless of the species eggs were retained on the netting, while the sperm passed
concept used (Templeton, 1989: Cracraft. 1989; Mallet, through with the wash water, which was collected and
199?; Knowlton and Weigt, 1997). centrifuged. The pelleted sperm were quick frozen (details
Nevertheless, a preliminary molecularsurvey revealed no in Lopez and Knowlton, 1997). Abundant DNA (hundreds
fixed DNA sequence differences among these taxa in two of micrograms) was extracted from 1-2 ml of highly con-
regions that might, a priori, be expected to have them: the centrated sperm solution using standard techniques (Sam-
ITS regions of rDNA and an intron in a /3-tubulin gene brooks et al.. 1989), as previously described (Lopez and
(Lopez and Knowlton. 1997). Sequence-based methods can Knowlton. 1997).
DNA
only be used toexamine alimited stretch ofDNA, however, Sperm provide an ideal source ofcoral (McMillan
and methods that screen a larger proportion of the genome et al.. 1988), but they cannot be collected routinely. How-
appear to offer greater promise (Lopez and Knowlton, ever, high molecularweight DNA is difficult toextract from
1997). One such approach is analysis ofamplified fragment somatic tissues (McMillan et al., 1988) and may be con-
DNA
length polymorphisms (AFLPs), which screens for poly- taminated by from symbiotic dinoflagellates (zooxan-
morphisms at. or adjacent to. restriction endonudease sites thellae), which the gametes of Montastraea lack (Szmant,
(Zabeau and Vos, 1995). In a preliminary survey, we found 1991). To determine whether DNA extracted from somatic
evidence for potentially diagnostic differences between M. tissues is of sufficient quality for AFLP analyses, we com-
faveolata and M. franksi using two AFLP primers (Lopez pared the analyses of DNA from sperm with those ofDNA
and Knowlton. 1997). from somatic tissues from the same colonies. DNA from
In the present study we wished to ( 1) determine whether somatic samples was extracted according to the protocol of
these apparently diagnostic AFLPdifferences hold up when Rowan and Powers (1991 ), except that tissue was removed
sample sizes are increased. (2) screen additional AFLP from 25-50cm2 ofcoral with an airbrush at 75-100psi and
mM mM
primers to see if any show promise for distinguishing M. suspended in 5-20 ml of L buffer (100 EDTA, 10
anniilurix and M. franksi, (3) determine, using Southern Tris-Cl. pH 7.6). To enrich for coral (animal) DNA within
hybridization and DNA sequencing, whether apparently somatic tissues, frozen samples were ground in a glass
similar AFLP bands are indeed homologous, and (4) assess homogenizer 5-10 times and centrifuged in an RT6000B
whether diagnostic polymorphisms detected using high- Sorvall centrifuge at 50-100 X g for 10 min at room
quality DNA derived from spermcould alsobe seen in more temperature. This spin was repeated one or two times for
readily collected, but potentially less pure, somatic tissue samples especially rich in zooxanthellae. The animal-en-
samples. riched DNA was then incubated for 3 h in 20-50 /j.g/ml
During earlier work on tubulin introns (Lope/ and proteinase K with \7c SDS (final concentration), followed
Knowlton, 1997), we also uncovered a tetranucleotide mi- by successive phenol:chloroform extractions (Sambrook et
DNA
crosatellite locus (here called Mfra-gtttl) in a genomic al.. 1989). that remained resistant to restriction diges-
clone derived from M. franksi. Microsatellite or simple tions was further purified by the GeneClean (Bio 101)
repeat loci have become increasingly important tools in protocol. Toclarify furtherthe potentially confounding con-
evolutionary and population studies because of their high tribution ofzooxanthellae in coral somatic samples, we also
levels of polymorphism and codominant inheritance (Jarne analyzed zooxanthella DNA provided by Rob Rowan. This
and Lagoda, 1996). Here we report on evidence for allelic DNA came from other colonies of Montastraea (primarily
frequency differences at this locus among the Montastraea M.faveolata) from the same region, andwas not necessarily
taxa. entirely free of coral (animal) DNA.
82 J. V. LOPEZ ET AL.
AFLP-PCR possibility of incomplete restriction enzyme digestion.
RFLPscan (Scanalytics) convertedbandpatterns intobinary
ePtstTahIle.adAa1Fp9t9Le6Pr).smyeGstethneoomdmhiaacnvdeDpbrNeeepAnardawetasisconricobuftedtaeitmnpsldpeaettcaeiisfliuc(sMiu6ne-glblateshree pdsirhseatsraiennncgcee(/eNasebtisiemananctdeesLcihf.oarr1ap9ca7ti9er)rw.sisfoerceoamcpharsiasmopnlsebaansdedcoomnpubtaendd
rtehecno^ganistyinotnhestiecq,ue2n1cbesp abdyapttheerPwsatsIlriegsattreidcttioontheeneznydmse,ofatnhde Cloning, Southern hybridisation, ami sequencing
fragments. The polymerase chain reaction (PCR) was then Both gel-purified and cloned AFLP fragments were used
used to amplify these restriction fragments, using primers as hybridization probes and as sequencing templates. Spe-
matching the adapter sequence. To limit the number of cific AFLP bands were dissected from low melting temper-
different"fragments that are amplified (and hence improve ature aearose (NuSieve/Metaphor, FMC) gels, addedto 200
tahrbeitcrlaarriiltyychofosethnebraesseusltwiengrepraoddduecdtst)o,tsheevePrCalRapdrdiimteiornsala,t w,11a!sofusdeidstialsleadteHm:pOl.ataenidn mae"lrtee-damaptli6f5icCa.tioWnh"ePnCRthiwsitDhNthAe
their 3' ends. These additional bases (by which primerDsNarAe original primer to obtain more material, PCR products were
identified, e.g., ATG orGGAG) overlapwithgenomic visualizedon an agarose minigel to verify that a single band
beyond the restriction site, and amplify the subset of frag- ofthe correct size had been amplified. Alternatively, AFLP
ments that contain the additional nucleotides. fragments were cloned directly into pGEM-T vectors (Pro-
We used the same methods and extension primers (ATG. mega). Probes for Southern hybridizations were labeled
GGAG) aspreviously reported(Lopezand KnGowGlAtoGn, 1997; with a[32P]-dCTP via nick-translation to a specific activity
note, however, that in our earlier report, the primer of 10"-107 cpm//ag (Sambrook et al., 1989).
was incorrectly listed as GAG). We also used primers with After separation on agarose gels. AFLP products were
the following 3' extensions: ATT, GAC, GTG, ATC. TGT, blotted onto nylon membranes (Duralon, Stratagene) ac-
ACT.TTG, AGC,TAG. and ACGC. The PCR profile forall cording to standard procedures (Southern, 1975; Sambrook
AFLP extension reactions was 94C/45 s, 60C/60 s, and et al.. 1989). Higher molecular weight fragments (> 1 kb)
72C/90 s for 30 cycles, using an MJ Research PTC-100 or appeared to be transferred to membranes less efficiently
PTC-200 thermocycler. Typically, a "preamplification" than smaller fragments, probably because of the relatively
oPnCeRadwdaistiopnearlfboarsmeed(Aw,itCh, GanoerxTt)ens(iVoons pelriamt.e.r1p9o9s5s)e.sTshiinsg huisegdh.gAelftceorncheynbtrriaditziaotnisonofaangdarsotsreing(e1n.t2%w-a1sh.i4n%g) t(ihnat0.w1erXe
reaction enriches for the subset of amplifiable templates SSC and0.5% SDS at 50C) (Sambrook etal.. 1989), filters
possessing the extra nucleotide, improves the targeted band were exposed to Kodak XAR-2 X-ray film, generally for
signal, and reduces background. The preamplification PCR 2-3 days.
was run with the same AFLP profile as above, but with Informative fragments that were generated using the
fewer (20) cycles. However, this preamplification prGoGtoAcoGl GGAG primer were eitherdirectly sequenced aftercleaning
did not improve the clarity of the patterns for the the PCR product with QIAquick PCR purification kits
primer. The best electrophoretic resolution ofPCR products (Quiagen), or sequenced from plasmid-cloned fragments
winagsaotblteaaistne5d0w%itMhet1a.p2h%o-r1.a4ga%roasgea,rFosMeC/)TBrEungealts5(.c4onVt/acimn.- prueraicftiieodnswiwtehreWizruanrdonminaiuptroempatkeitds D(PNroAmesgeaq)u.enSceeqrusen(cAiBnIg
The agarose-based technique used here and in our previous 373A or 377. Perkin Elmer), initially using primers com-
study does not require radioactive nucleotides, is less toxic, plementary to T7 or SP6 promoter regions, following the
and is relatively easy to perform (Mueller et al.. 1996; standard cycle sequencing protocols (ABI, Perkin Elmer)
Lopez and Knowlton. 1997). although it yields fewer dis- used previously (Lopez and Knowlton. 1997). The follow-
crete bands per lane (6-12) than the original polyaery1- ing primers were then designed and usedto obtain complete
amide gel electrophoresis (PAGE) method (Vos et al.. sequences for the GGAG 880 and 920 bp fragments; 5'
1995) due to poorerresolution offragmentsofless than400 CCCTGATCAGTATTTTGGG 3' (880i), 5' TTGGAATA-
bp. All AFLP analyses shown here were performed more TTTGCCTTACCG 3' (880f), and 5' GGAGGGCTCTGT-
than once to ensure reproducibility, and AFLP-PCRs with TATTCTATC 3' (880r). The 880fprimer (slightly internal to
no DNA added served as controls for contamination. 880i) matches available Montastraea sequences, and when
AFLP banding patterns and DNA sequences (see below) used with primer 880r yielded products of 837 or 804 bp.
BLAST
were analyzed with RFLPscan (Scanalytics), (Alt-
schul et al.. 1990). and MAPD (Yuhki and O'Brien. 1990; Microsatellite analyses
Srwitanenceiogpgnehhsetiwnsbestarenenetdtcsaaolmni.s.pslimid1ofe9ri9rce2ea)dt.d,iifoOsfninincloucfyletlbvtaaaorrngiiedansttDieroiNpnnrAeittnhfedhruia0egg.hm3te-eor1n.ptmo6sotlekanebntcdiuasliltazlhreye iwnaTsahcreleocmnoiecvreo(rsteaudtbe2wl9lhiAit)leedleworeciuvsweedMrtferra-osgcmtrteMte.lnifwnragasnfkosirniitt(ianaxol.olyn4od2me6it)ceactlthleaydt
GENETIC ANALYSIS OF MONTASTRAEA 83
informative /3-tubulin introns (Lope/, and Knowlton, 1997). and among samples ofM. franksi (22%) were very similar
Its occurrence and polymorphism in other Montastraea to the value calculated for interspecific comparisons be-
species were determined by designing the following 2 oli- tween these taxa (27%).
gonucleotide primers, which are complementary to the
AAACA
genomic sequences flanking Mt'ra-gtttl: Sputlf-5'
TACGG CCAGT GCTGG 3' and Sput2rc - 5' GAAAA Homology ofbands
GAGCA ATCTT TTGTA TGGTG 3'. The PCR profile
used for Mfra-gtttl amplification from genomic DNA was Southern hybridization with DNA probes derived from
94C/40 s. 60C/45 s, and 72C/60 s for 30 cycles. All the 630 bp ATG bands provided further insights into the
PCRs shown here were reproducible and included negative nature of genetic differences between M. faveolata and M.
controls. The resolution of PCR products was better when franksi. In general, results were better when the hybridiza-
4.0% agarose (Metaphor. FMC) TBE gel electrophoresis tion probes were derived from DNA clones. Multiple bands
awcarsyluasmeidd,eagnedl (b1an0d%i)ngelepcattrtoeprhnosrewseirseuscionngfitrhmeeedntbiyrepPolCyR- wAeTrGe blaanbdelse,dsuwghgeenstipnrgobtehsatwtehree63d0erbipvebdanfdrsomwegreel-ciosonltaatme-d
product (approximately 1 /u.g DNA). inated with fragments of different molecular weight.
Southern hybridizations showed that the 630 bp ATG
Results
bands found in all M.faveolata and one M.franksi (no. 19)
AFLP handpatterns (Fig. 3A) are homologous (Fig. 3B). Moreover, another M.
a cBlaeanrddpiaatgtneorsntsicprdoidfufecreedncuesibnegttwheeenGGM.AGfavperoilmaetrasahnodwMe.d fmreanntksitha(tno.al2so0)hypborsisdeiszseedd taohtihgehe6r30molbepcuAlTarGwperiogbhet f(rFaigg-.
ftraainnkesdiw(iFtihg.s1mAa)l.lewrhiscahmpcloenfsiirzmess o(uLropperezviaonuds rKensoulwtlstoonb,- 3FBi)g.urTeh3isA,laarngderprforbaagbmleyntarmoaseybbyeotnheeosrammoeroenDeNviAsibilneseri-n
1997). The 920 bp GGAG band was absent from, and the tions at the 630 bp locus. Unfortunately, similar Southern
GGAG
880 bp band was present in. all 16 M. faveolata tested hybridizations using the diagnostic 880 fragment as
(including 6 previously analyzed), while the reciprocal pat- a probe were unsuccessful due to our inability to use the
tern occurred in 15 M. franksi (including 7 previously preamplification protocol.
analyzed). A third band, migrating at around 850 bp, may We therefore used DNA sequences to evaluate the ho-
also occur at significantly different frequencies in the two mologies ofthe 880 and 920 bp GGAG bands. Partial DNA
taxa, but our sample sizes are too limited to test for this. sequences were initially obtained from both 5' and 3' ter-
The ATG primer also provided evidence of genetic dif- mini ofthe GGAG 880 (M.faveolata) and GGAG 920 (M.
ference between M. faveolata andM.franksi: as previously franksi) fragments. These preliminary sequences permitted
reported (Lopez and Knowlton, 1997), the 630 band was the design of PCR primers by which the corresponding
characteristically present in the former and absent from the locus was amplified from genomic DNA. The GGAG 880
latter(Fig. IB). In this case, however, increasing the sample locus was amplified consistently from samples ofM.faveo-
size indicated that this difference between the species is not lata (Fig. 4A), but a larger band appeared for M. franksi
fixed: the 630 bp band was present in all 16 individuals of (Fig. 4A) andM. annularis (data not shown) when the same
M. faveolata tested (including 7 from the previous study). primers were used. DNA sequencing confirmed the homol-
but it also appeared in one individual ofM.franksi (no. 19, ogy of PCR products for 9 M. franksi, 6 M. anmdaris,
lane 9). The remaining 14 M. franksi (including 6 previ- and 7 M. faveolata (sequences deposited in Genbank
ously studied) lacked this band. AF1 101 14-AF1 10129, API 12346-API 12351). Three inser-
In contrast to the clear differences separating M. faveo-
lata from M. franksi, no diagnostic bands separated M. tions or deletions (22, 8, and 3 bp) constituted the primary
franksi and M. anmdaris. This was true, not only for the differences between M. faveolata and the other two taxa
ATG (Fig. IB) and GGAG primers (five M. annularis (Fig. 4B), as would be expected from the estimated size
analyzed, data not shown), but also for 10 additional prim- differences in the 880 and 920 bp bands. There were also 5
ers that were screened (data not shown). The ATT primer nucleotide substitutions [4 transitions, 1 transversion in 837
yielded band patterns with the strongest quantitative differ- bp within the GGAG 920 fragment (see methods); data not
ences (Fig. 2). but the differences are not statistically sig- shown] that distinguished M. faveolata from M. annularis
nificant by a chi-square test, once a Bonferroni correction and M.franksi. The sequences exhibited d(AT) contents of
(Rice. 1989) for the total number of bands examined is 58%-64%, and did not resemble sequences in current da-
applied (see legend Fig. 2). Moreover, mean average per- tabases (GenBank and EMBL; April 1998). The lack of
cent difference (MAPD; Yuhki and O'Brien, 1990) in ATT significant open reading frames in the sequences suggests
band-sharing values among samples ofM. anmdaris (28%) that they do not represent protein-encoding regions.
84 J. V. LOPEZ ET AL.
-GGAG
A.
M.faveolata M.franksi
bp
880 920
-ATG
B.
A/, faveolata M. franksi M. annularis
bp
630
Figure 1. APLP hand patterns. Samples are grouped by species, and individual sample numbers are
indicatedaboveeach lane. A 1.0kbladder(Gibco/BRL, Bethesda) was used as the molecularweight standard.
(A) AFLP patterns derived with theGGAG primer. Species-specific bands at 880and 920bpare indicated by
arrows. (B) AFLP patterns derived with the ATG primer. The 630 bp band is indicated by an arrow.
GENETIC ANALYSIS OF MONTASTRAEA 85
-ATT
M. annularis M. franksi
Figure 2. Comparison ot Montastraeafranksi and M. annularis AFLP patterns obtained with the ATT
primer. Polymorphic bands showing the greatest frequency differences between the species are marked by
asterisks.Themostextremefrequencydifference(bandindicatedbyupperasterisk,presentin5of13versus 12
of 13 individuals of M. annularis and M. franksi, respectively; not all samples shown) was individually
significant(chi-square = 6.1.P<0.02).However,thisdifferenceisnotsignificantwhenaBonferronicorrection
(Rice, 1989) forthe total numberofbands (18) is applied (P must be less than 0.003).
Comparisons ofbandpatternsfrom gametes, somatic somatic tissues, we obtained AFLP bands from DNA
tissues, and zooxanthellae purified from zooxanthella types A, B, and C from Mon-
iscoamTlalhtyeiicAnftToiGrssmuapetaitwvteeerr6nes3g0feonbreprDabNlalAnyddcwoenarsisivsectdoennftsr,poiamcnusdoputeshremintaaannxdaolnfyorsmoe-ms bmntoaaassnyttdirscabeef6ar3s(o0osmembeezpoRopobxowataneanndtnthieiaanlnlldMaf.oKtrnyfopcaweovlsnetfooAulnsa.tiaaon1nd9a9bnB5de,)twsa(ielFmietighnl.oaut6rg)hl.hey-Ttdshhiieeazrsgeee-d
oibfilsiotymaftoircthteisGsuGesAfGropmriMm.erfawvaesolpaotoare(rFidgu.e5tAo).ouRreipnGraobGdilAuictG-y zcoonotxaamnitnhaetliloan.banIndsgemnaeyrali,n ftahcetsbiemidlaureittyo ocofratlhe(agnaimmeatle)
tboanudsseatth9e2p0reaanmdpl8i8f0icbaptiwoenrperovtioscioblle,ibnutandailaygzneodstsiacmplesof and somatic tissue samples (Fig. 5) and the difference
DNA from somatic tissues from M. franksi and M. faveo- between zooxanthella-enriched and zooxanthella-absent
lata, respectively (Fig. 5B). This suggests that AFLP anal- (AsFpLerPm)tescahmnpilqeuse(pFriigm.ar6)i,lysuagmgpelsitfitehsatcfoorralth(easneimcaorla)lsD,NthAe
yses can be informative with somatic tissues, especially from somatic tissue samples.
once diagnostic patterns have been established with sperm
samples. The general lackofhighermolecularweight AFLP
Microsatellite locus
bands from analyses ofsomatic tissue compared to those of
gamete samples may be due to degradation during DNA Analysis of a clone from M. franksi derived from a
purification ofsomatic samples (e.g., McMillan elai, 1998) PCR amplification product using primers for (3-tubulin
or to the presence of contaminants that interfered with the revealed a microsatellite locus (Mfra-gtttl) whose core
reactions, but these bands were typically not scored. Some repeat sequence (GTTT) was perfectly repeated 9 times
differences between somatic and gamete samples (e.g., for (EMBL accession number AJ223626). It is similar (but
M. franksi no. 467 in Fig. 5A) cannot currently be ex- not identical) to simple repeats in other scleractinian
plained. corals (McMillan et ai, 1991). Analysis of additional
To determine how zooxanthella-derived bands in par- samples using the same primers revealed that a smaller
ticular might confuse the interpretation of analyses of allele (approximately 160 bp, its size presumably due to
86 J. V. LOPEZ ET AL
A.
M. M.
faveolata franksi
B.
M. M.
faveolata franksi
Figure 3. Southern hybridi/ution experiments with the ATG 630 bp fragment to determine band homolo-
gies. (A) Ethidium bromide-stained agarose gel used for Southern blotting, showing typical AFLP patterns
obtained using the ATG primer. (B) Autoradiograph produced with probe for the ATG 630 bp fragment. A
cloned 630bp ATG fragment was radiolabeled and used forprobing the filterofthe gel in (A). This fragment
also hybridized to the 1.6 kb fragment in the marker lane (M).
a loss of 2-3 repeat units) is the most common allele in shown). One sample (from M. anniiluris no. 27, Fig. 7)
both M. franksi and M. annultiris (Fig. 7; Table I). Two yielded three bands ( 160 bp, 169 bp, and an intermediate
individuals appeared to be heterozygous for the 160 bp band migrating between them); this pattern suggests the
and 169 bp alleles (i.e., two bands amplified; data not presence of an additional locus, although it could be a
GENETIC ANALYSIS OF MONTASTRAEA 87
A.
M. M.
faveolata franksi
kh
l.o
B.
131 141
M. franksi ACCTATITTCCCTAAA ATTTCTCGC
M. franksi
M. franksi
M. annularis
M. annularis
M. faveolata
. .
M. faveolata
. .
M. faveolata
. .
M. faveolata
. .
M. faveolata
. .
II
361 371 501 511
M. franksi GIAGTTTAACAACT AACCTTCTGCGITT
M. franksi A
M. franksi A
M. annularis A
M. annularis A
M. faveolata .... ....
.
M. faveolata .... ....
.
M. faveolata .... ....
.
Af. faveolata . .... ....
M. faveolata .... ....
.
Figure4. Agarosegelandsequencealignmentsshowingthedifferencesbetweenthe880and920bpGGAG
fragments. (A) Fragments amplified from genomic DNA ofMonlustraeafaveolata and M. franksi using the
Montastraea-biasedprimers. (B)Sequencealignment showingtheregionsthatgeneratethedifferenceinsizeof
the 880 and 920 bp fragments.
PCR artifact. In contrast, most samples of M. ftnenlutu Overall, this microsatellite locus suggests that genetic
yielded a higher molecular weight smear above 220 bp, differences exist between M.faveolata and the other two
rather than discrete 160 or 169 bp bands, when using the taxa. but determining the precise nature of these differ-
same primers and PCR conditions ("null" alleles. Fig. 7). ences would require further analyses.
J. V. LOPEZ ET AL
A. -ATG
M.
M.faveolata franksi
bp
630
G Gamete DNA
-
DNA
S - Somatic Tissue
-GGAG
B.
M. M.
faveolata franksi
Figure 5. Comparison of AFLP patterns from gametic (G) and somatic (S) tissue samples. DNA derived
from sperm and from somatic tissue ofthe same Montaxtraea colony were analyzed in parallel AFLP-PCRs.
usingidenticalconditions.(A)ResultsfromATGprimer. (B)ResultsfromGGAGprimer. Diagnosticbandsare
identified by arrows.
GENETIC ANALYSIS OF MONTASTKAEA 89
ZoAoxaBntheCllae Table I
Mfra-gtttl alli'lc distributions in members ofthe Montastraea annularis
Fav complex
bp
630
Figure6. AFLPassayofzooxanthella samples. TheATG primerwas
used after PCR preamplification ofzooxanthella templates (see methods).
Identical conditions for AFLP analyses were used on both Zooxanthellae
andcoral (Montaxtraeafaveolala) DNA samples. Three faintbands (indi-
catedbyarrows)obtainedfromTypeAZooxanthellaeappearsimilartothe
threedominantAFLPbandsobtainedfromM.faveolata(550,630.750bp)
shown in Fig. IB;thesemaybeduetocoral(animal)contaminationofthe
zooxanthella DNA.