Table Of ContentReference:Biol. Bull. 193:359-367.(December,
Polyphyly of "Sclerosponges'
(Porifera, Demospongiae) Supported
by 28S Ribosomal Sequences
CATHERIANNENICEHTOIMLBLIAERRD1.1A*,NNDICJOELAEN BVOAUCREYL-EETS2NAULT .
' Laboratoire de biologic desInvertebresMarins etMalacologie (CNRS URA 699) etSen'icede
SystematiqueMoleculaire (CNRS GDR 1005), MuseumNationald'Histoire Naturelle, 57, rue
Cuvier, 75005Paris, France: and-Centre d'Oceanologie deMarseille (CNRS UMR 6540),
Universitede la Mediterranee. Station Marined'Endoume, 13007Marseille, France
Abstract. To test the competing hypotheses of poly- during the Paleozoic and Mesozoic eras and that were
phyly and monophyly of "sclerosponges," sequences long thought to be extinct (Hartman and Goreau, 1970;
from the 5' end of 28S ribosomal RNA were obtained Vacelet, f983; Wood, 1990).
forAstrosclera willeyana, Acanthochaetetes wellsi, and Since the discovery ofthese living coralline sponges,
sixotherdemospongespecies.Phylogeneticrelationships theyhavebeenclassifiedaccordingtothreesystems,each
deduced from parsimony and neighbor-joining analyses reflecting a different belief in the number of times that
suggest that these sclerosponges belong to two different sponges have inventeda massivecalcareous skeleton. In
orders of Demospongiae: Astrosclera willevana, being the first, all ofthe coralline sponges are included in the
closely related to the Agelasidae, belongs to the Agela- class Ischyrospongiae (Termier and Termier, 1973). In
sida,Acanthochaeteteswellsi, beingcloselyrelatedtothe thesecond,themassivecalcareousskeletonisbelievedto
Spirastrellidae,belongstotheHadromerida.Theseresults haveevolvedatleasttwice,onceamongcorallinesponges
contradict the hypothesis that sclerosponges are mono- withsimilaritiestotheCalcareaandonceamongcoralline
phyleticandimplythatamassivecalcareousskeletonhas sponges that more closely resemble the Demospongiae:
evolved independently in several lineages ofsponges. the latter group is assigned to the class Sclerospongiae
(Hartman and Goreau, 1970). This second interpretation
Introduction hasbeenthemostwidelyused,appearinginmanyrecent
treatisesonzoology(Parker, 1982;Riedl, 1983)andpale-
Recentspongesgenerallyhaveaskeletonmadeofspic- ontology (Rigby and Stearn. 1983). A third system (Va-
ules that are either siliceous (classes Demospongiae and celet, 1979, 1985) reflects the assertion that the massive
Hexactinellida) orcalcareous (class Calcarea). However, calcareous skeleton is more plastic and has evolved in
16livingspeciesbuildanunusualsolidcalcareousskele- several different lineages within the Demospongiae and
ton, which bears a striking similarity to that of various Calcarea. Underthis system, living and, where possible,
Cnidaria, in addition to this spicular skeleton. These fossil coralline sponges are classified within the various
"coralline sponges" are believed to be the survivors of taxa of Demospongiae and Calcarea with which they
the stromatoporoids, sphinctozoans, and chaetetids, im- share derived characters.
portant ancient reefbuilders that were highly diversified Three coralline sponges are included in this study:
Acanthochaetetes wellsi Hartman and Goreau, 1975;
Received 12March 1997;accepted29August 1997. Astrosclerawilleyana Lister, 1900; and Petrobionamas-
*To whom correspondence should be addressed. E-mail: gdretudi siliana Vacelet and Levi, 1958. Acanthochaetetes wellsi
(S'mnhn.fr andAstrosclerawilleyanaareofspecial interest,because
359
360 C. CHOMBARD ETAL
theyareconsideredtobelivingrepresentativesofchaete- was crushed in a sterile mortar after total dehydration
tidsandstromatoporoids,twogroupsofgreatimportance (overnightair-dry at +4C orspeedvac) forthe alcohol-
in the fossil record. The affinities ofthese groups were preserved samples and in liquid nitrogen for the frozen
previously uncertain, but they were most oftenclassified samples.Thepowderwasgentlymixedforafewminutes
in the Cnidaria (Lecompte, 1956: Fischer, 1970). The with500ij\oflysisbuffer(Palumbietal.. 1991).Spicules
separationofthetwogroupsimpliesanindependentderi- andcellularremainswerethenremovedbycentrifugation
vation of the massive calcareous skeleton. The spicular for2 minat 13.000rpm. Digestedtissuewaspurifiedsuc-
andcytologicalcharactersofbothspeciesstronglyresem- cessively in phenol, phenol-chloroform-isoamylalcohol.
ble those found in well-defined families ofnon-calcified andchloroform-isoamylalcoholextractions.Nucleicacids
demosponges. The choanocytes of Acanthochaetetes were precipitated with ammonium acetate-isopropanol,
wellsi possess a periflagellar sleeve; a central cell at the followedby a70% ethanol wash. Total DNA was resus-
apopyleofthechoanocytechambers,asintheHadromer- pendedin steriledistilledwateranditsconcentrationde-
ida;andaspiculecomplementsimilartothatofthefamily termined by optic density at 260nm.
Spirastrellidae in the order Hadromerida (Hartman and Polvmerase chain reaction. Two overlapping frag-
Goreau, 1975: Vacelet and Garrone, 1985; Reitner and ments ofthe ribosomal RNA gene were amplified using
Engeser, 1987; Boury-Esnault et ai, 1990). Astrosclera auniversal primerand a sponge-specificprimer. Primers
willeyana has smallchoanocyte chambers, flattenedcho- usedwereasfollows(specificity,orientation,andposition
anocytes. verticillate acanthostyles. and chemical affini- of primers in the aligned sequences of Figure 1 follow
tieswiththeorderAgelasida(HartmanandGoreau, 1970; each sequence): ITS3 5'-GTCGATGAAGAACGCAGC-
Vacelet. 1981; Boury-Esnault etal, 1990; Williams and 3'. universal, forward, external 5'; Eplb' 5'-GTGGCC-
Faulkner, 1996). Petrobionamassiliana has morphologi- GGGAGAGGCAGC-3',partofDemospongiaenotTetrac-
cal affinities with the class Calcarea. tinellida,forward.257-274;Ep25'-CTYYGACGTGCC-
In this work we generate a new. independent data set TTTCCAGGT-3'. Demospongiae. reverse. 303-323; D2
basedonDNAsequences,useittoconstructphylogenies. 5'-TCCGTGTTTCAAGACGGG-3', universal, reverse.
and compare these with morphological ones. Our main external 3'.
objectiveisthentodeterminewhichofthethreehypothe- The fragment 'TTS3-Ep2" contains apartofthe 5.8S
ses are consistent with the molecularphylogeny. rRNAgene,the ITS2, the Cl domain andhalfofthe Dl
domainofthe28SrRNAgene;the "Eplb'-D2"fragment
Materials and Methods contains theotherhalfofthe Dl domain, the C2 andthe
D2 domains ofthe 28S rRNA gene.
Material: selection andpreservation A 50p] double-stranded PCR reaction mix contains
lisTtehdeinspTeacbileesI.anSaolmyseeddeamnodsptohenigresistpeescioefscwoelrleectsieolnectaered 0d.N3TAP/,g 3t0emppmloatleeaDcNhAp.rob2e..5^/11.5DUMTSaOq,D0N.A16p5omlAy/mereaasche
as representatives ofthe various taxa supposedly related (Bioprobe). This reaction mix wasoverlaid with mineral
toAstrosclera andAcanthochaetetes. Otherspecies with oil and placed in aTrio-thermoblock thermocycler (Bio-
vthaersieousinlcelvuedlesorefpdriessteanntcaetifvresomotfheotihne-rgrdoeumpowseproengcehosseunb:- mteetmrpae)r.atCuyrceli(nTga)c:onrdeistpieoctnisvealrye v6a0riCablaendfo6r3thCe afonrneIaTlSi3n-g
classes one Ceractinomorpha species (Halichondria Ep2 and Eplb'-D2 primer pair. The first cycle is 4min
panicea) and two Tetractinellida species (Cinachyrella at94C, 2min atTa. and 2minat72C; this is followed
s(pC.luatnhdriDniascceordeebrrmuima),powliytdhiswchuisc)hPaetnrdoobfiocnlaasmsaCsasliclairaneaa Tbay,3a0ndcyc1lmeisneaatch72coCn;sisttheingreaocfti1onmiins faitni9s4hCed,b1ym4inmiant
has affinities. For further convenience, all demosponge at 72C.
csoplelceicetsivtehattearrme n"omtoTneatcrtaicnteilnleilldisd.a"arAellgrsopuepceidmeunnsderwetrhee agaArfotseer vgeils,ualtihzeatrieomnaionfi5ng^/l45offj\theofrePaCctRionproonducat1w.a5%s
eniitthreorgepnreasnedrvtehdeninke7pt0%ate-t8ha0nCol,odrepdeenedpi-nfrgooznencoilnlelcitqiunigd ppuarniofli,edfoblylopwreedcipbiytaati7on0%witehthaanmomlownaisuh.mTahceetapteel-liestopwraos-
conditions. then resuspended in 6/A ofsterile distilled water.
The approximateconcentration wasevaluatedvisually
DNA byelectrophoresisof 1 ^1ofthepurified PCR productin
processing a 1.5%agarosegel,andcomparisonto 1.5p\oftheDNA
Extraction. The total genomic DNA extraction tech- molecularweight marker VI (Boehringer Mannheim).
nique was modified from the Simple Fool's Guide to Cloning and sequencing. Each PCR fragment was
PCR (Palumbi et al., 1991). Less than 0.5g of tissue cloned into PCR-Script SK(+) cloning vector (PCR-
POLYPHYLY OF "SCLEROSPONGES' 361
TableI
iigespeciessequencedforanalysisofphylogeneticrelationshipsamongsclerosponges
Classification Species Collectionlocality
DEMOSPONGIAE
Tetractinellida
Tetillidae Cinachyrellasp.* NewCaledonia
Theonellidae DiscodermiapolydixciisBocage, 1870* Mediterraneansea.3PPcave.LaCiotat
"monactinellids"
Axinellidae Axinelladamicurnis(Esper. 1794)* Mediterraneansea.LaCiotat
Agelasidae Agelasoroides(Schmidt, 1864)** Mediterraneansea,LaCiotat
Astroscleridae AstroscleralwilleyanaLister, 1900 NewCaledonia 1992
Astrosclera2willeyanaLister, 1900 NewCaledonia 1994
Clionidae Clionarirulis(Schmidt. 1862)* Mediterraneansea.LaVesse
ASpciarnatshtorcelhlaiedtacetidae AScpiarnatshtorcehllaaetcef.tecsocwceilnlseiaH(aDrutcmhaasnsa&inGgor&eaMui,ch1e9lo7t5ti, 1874) NPaenwamCaa.leAdtolnaintaic19c9o2astSanBiasIsland
Halichondriidae Halichondriapanicea(Pallas, 1766)* SouthWestChannel,AberWrac'h
CALCAREA
Clathrinidae Clathrinacerebrum(Haeckel, 1872)** Mediterraneansea.LaVesse
Petrobionidae PetrobionamassilianaVacelet&Levi. 1958** Mediterraneansea.AnsedesCuivres
*SequencesfromChombardetal. (Inpress).
**SequencesfromLafayetal. (1992).
ScriptSK(+)cloningkit,Stratagene)andsequencedwith matrices (with NET or from NJ trees), distance calcula-
the T7 Sequencing kit (Pharmacia Biotech) using [33P]- tions andconstruction oftrees with the neighborjoining
dATP and adding DMSO in the annealing reaction. The algorithm (with NJ), matrix comparison (with COMP-
internal probe C2' is used to obtain the middle of the MAT), and calculation of bootstrap proportions from
"Eplb'-D2" fragments, in addition tothe vectorprobes neighborjoining trees (with NJBOOT). Wherever likely
Ks and T3 <C2' 5'-GAAAAGAACTTTGRARAG- secondary structures were detected, sequences were
AGAGT-3',universalspecificity,forwardorientation,po- aligned according to supposed conservation ofhelices.
sition 483-505 on the aligned sequences ofFigure 1). PAUP, version 3.1.1 (Swofford. 1991), was also used
Each PCRproductwassequencedfromaminimumof forconstructionoftreesandcalculationofbootstrappro-
twoclones; whencontradictions in the sequencesofsev- portions,discussedbelow. Inbootstrapcalculations,non-
eralclonescouldnotberesolved,thecorrespondingposi- majority nodes were compared in order to explore the
tions werecodedaccording tothe UPIACcode. Thetwo robustness ofalternative topologies.
strandsweresequencedforthe mainpartofthe sequence ThefinalalignmentpresentedinFigure 1 wasobtained
length, with special attention to the D2 domain where by eye using the editor of MUST (ED). The ITS2 (not
strong secondary structures of the molecule cause com- presented in Fig. 1) and part of the 5' extremity of the
pressions inthe sequence migration. From the twoover- D2 domain (corresponding to positions 575-640, Fig. 1)
lappingPCRproducts,thefinal sequencewas 1104bpto are very divergentand cannot be aligned in all oursam-
1197bp in length, depending on the species. This frag- ples, thus these regions were not used in the sequence
ment corresponds to the 3' extremity ofthe 5.8S rRNA analysis.
(about 108bp), the InternalTranscribe Spacer ITS2 (be-
tween 167bp and 224bp), and the fourfirst domains of Results
the 5' extremity ofthe 28S rRNA: Cl, Dl, C2, and D2
(between 816 and 866bp). Becausepreviouslypublishedsequencesof28S rRNA
(Lafayetal.. 1992) are shorterthanours, twosuccessive
Sequence managementandalignment analyses were made. The first grouped all species and
correspondstothelengthpublishedbyLafayetal. (Table
TheMUSTpackage(Philippe. 1993)wasusedtoman- I): inthesecond, Clathrina. Petrobiona. andAgelaswere
age sequences, including registration (with ENTRYSEQ removed so that we could use ourtotal alignable length.
program), alignment (with ED), construction ofdistance The first analysis included 12 species and 374bp of
362 C. CHOMBARD ETAL.
1 10 20 30 40 50 60
460
CCGATAGCAAACAJ
364 C. CHOMBARD ETAL.
21 *
a;
CD 20-
CO-
"o'~^-
^ ">
-CEQD CCcDJ
^ CD
CD!^
CO73
CD
CL
31 62 93 124
Pairwise numberofinferred substitutions
inthe mostparsimonioustree
Figure2. Global saturation curve for 12 species and short-length
aligned sequences (374bp). CC0.98. Whitecircles are distancesbe-
tween pairs monactinellids-monactinellids. Dark circles are distances
betweenpairsTetractinellida-monactinellids.Darksquaresaredistances
between pairs Calcarea-Demospongiae. White squares are distances
withinTetractinellidaandwithinCalcarea.
ofPAUPprovidedonesingleshortesttreewith268steps,
aconsistency index (CD of0.795, aretention index (RI)
of0.767,andaGl of 1.50.Thetreewasrootedusingthe
out-groupmethodonbothspeciesofCalcarea(Clathrina
cerebrum andPetrobiona massiliana). Theresulting sin-
gle topology is presented in Figure 3. The Branch and
Boundsearchoptionwasusedtoprovideabootstrapwith
1000 replicates in PAUP. The majority-rule consensus
99
P01.YPHYLY OF --.SCLEROSPONGES" 365
28- .2.00. 100
-o
0>
Q) O
8
o
iff
to-o
*
05
CL
0-O
86 172 258 124
Pairwise numberofinferred substitutions
inthe mostparsimonioustree
Figure 5. Global saturation curve for 9 species and full-length
aligned sequences (914bp). CC0.97. Whitecirclesaredistancesbe-
tween pairs monactinellids-monactinellids. Dark circles are distances
between pairs Tetractinellida-monactinellids. White squares are dis-
tanceswithinTetractinellida.
tive for parsimony. No global saturation is evident (CC
0.97. Fig. 5). The exhaustive search algorithm ofPAUP
provided one single shortest tree with 640 steps. CI =
0.839,RI = 0.783,andGl = -1.01.Thetreewasrooted
using the out-group method on the tetractinellids (Cina-
chyrella sp. and Discodermiapolydiscus). The resulting
singletopology ispresented in Figure6. The Branchand
Boundsearchoptionwasusedtoprovideabootstrapwith
1000 replicates in PAUP. The majority-rule consensus
treeexhibitsthesametopologyasthe shortesttreefound
byexhaustivesearch(BPreportedonFig. 6).The neigh-
366 C. CHOMBARD ETAL.
1985; Reitner and Engeser, 1987; Boury-Esnault et a/.. biius.andananonymousreviewerforcommentsandsug-
1990). These results support the interpretation that the gestions.
capacitytosecreteamassiveskeletonofcalciumcarbon-
ate has developed several times during the course ofthe Literature Cited
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