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On the Phylogeny of Halichondrid

Erpenbeck, D.J.G.

Publication date 2004

Link to publication

Citation for published version (APA): Erpenbeck, D. J. G. (2004). On the Phylogeny of Halichondrid Demosponges. Universiteit van Amsterdam.

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Download date:30 Sep 2021 Chapterr 3

Phylogeneticc relationships of halichondrid demosponges Implicationss from a 28SrDNA tree

Erpenbeckk D., Breeuwer J.A.J, and R.W.M. van Soest

Abstract t

Halichondridaa is a pivotal order of which the classification underwent major changes inn the recent history. The monophyly of Halichondrida and its intra-ordinal phylogeny cannot be reliablyy determined on the basis of morphology. Here, we present a 28SrDNA gene tree of selected halichondrids,, which supports the hypothesis of halichondrid non-monophyly and elucidates furtherr inter-ordinal relationships: Most Halichondrid families (Axinellidae, and Halichondriidae)) are polyphyletic and the molecular classification of certain genera such as andd do not agree with the current (morphological) system.

47 7 3:: Phylogcnetic relationships of halichondrid demosponges - Implications from a 28SrDNA tree

Introduction n

Thee phylogenetic systematics of the demosponge order Halichondrida is pivotal in demosponge systematicss as its composition and affinities in history reflect the major changes in demosponge systematicss that were proposed in the second half of the 20th century. Grayy (1867) erected the taxon "Halichondriadae". Its composition was modified based onn morphological features by Vosmaer (1886 [1887]), Ridley and Dendy (1887) Topsent (1928) andd de Laubenfels (1936). Later, a new demosponge classification was founded by Levi (1953), entirelyy based on reproductive features. Demosponges were divided in two major subclasses 'Tetractinomorpha'' (oviparous taxa) and 'Ceractionomorpha' (viviparous taxa) as these features matchedd with large demosponge groups and resulted in a reallocation of formerly recognized orders andd families in separate clades. The order Halichondrida sensu Laubenfels (1936) was split up in the ceractinomorphh "Halichondrida s.s." (including the currently recognized families Halichondriidae andd Dictyonellidae) and the tetractinomorph "Axinellida" (including the currently recognized familiess Axinellidae, Desmoxyidae and Bubaridae). Levi's classification was elaborated by Bergquist (e.g.,, 1980) and Hartman (1982). It formed the backbone of demosponge systematics in the second halff of the 20th century. Withh the introduction of cladistic character analyses in systematics (Van Soest, 1985) parsimony-inconsistenciess of Levi's classification were criticized (Van Soest, 1987; Hooper, 1990). Evidencee for a paraphyletic nature of the two major demosponge subclasses could be shown and an alternativee classification was suggested (Van Soest, 1991) which gained broader acceptance in the recentt years (Levi, 1997). The taxon Axinellida sensu Levi was abandoned and partially (Axinellidae andd Desmoxyidae) merged with Halichondrida (Van Soest et al, 1990). Van Soest and Hooper (2002)) currently assign the families Halichondriidae, Dictyonellidae, Desmoxyidae, Bubaridae, andd Axinellidae to this order. However, until now not a single morphological synapomorphy, whichh could define the families as monophyletic units, has been described, nor synapomorphies, whichh could unite the families to a monophyletic taxon Halichondrida. The order is still defined by characterr combinations and underlying synapomorphies, which results in the fact, that intra-ordinal phylogeneticc relationships of this pivotal demosponge taxon are not properly resolved. In particular the familyy Dictyonellidae sensu Van Soest et al., (2002) is rather based on assumed secondary losses than onn observable characters. Van Soest et al. (1990) proposed a phylogeny for the order and the family Halichondriidaee based on distribution of morphological characters, but did not present statistical support.. The erection of a comprehensive cladistically supported phylogeny of the Halichondrida is hamperedd (as for many other demosponge taxa) by either the lack of phylogenetically informative characterss and diverging views of their interpretation (Carballo et al., 1996). To gain additional characterss in sponge systematics, cytological features (Boury-Esnault et al., 1994) as well as chemical compounds,, e.g., Van Soest and Braekman (1999) have been recruited but these approaches suffer untill now from a taxon bias and ambiguities in pathway homology and metabolite origin (sponge or symbiont?). . Thee main progress in recent sponge systematics lies in the gain of DNA sequence data. The largee subunit of the cytoplasmatic ribosome (28SrDNA) frequently has been used to reconstruct phylogeneticc trees (Alvarez et al., 2002; McCormack and Kelly, 2002) although several other sponge geness have recently been sequenced (an overview in Borchiellini et al., 2000). Somee of these 28SrDNA analyses yielded evidence against a monophyletic relationship of thee halichondrids sensu Van Soest et al. (1990). One analysis of the C1-D2 region found the family

48 8 3:: Phylogenetic relationships of halichondrid demosponges - Implications from a 28SrDNA tree Halichondriidaee in a shared clade with the hadromerid family Suberitidae (Chombard and Boury- Esnault,, 1999). The resulting taxon, called "Suberitina" still lacks acceptance as it fails to show unambiguouss morphological synapomorphies notably regarding the spicule geometry. McCormack andd Kelly (2002) analyzed the D3-D5 region of the 28SrDNA molecule to unravel the phylogenetic positionn of the hermit-crab sponge Spongosorites suberitoides. Their resulting tree favored a close relationshipp of the halichondrid genus (Halichondriidae) with the hadromerid genus Tethya (Tethyidae)) and additionally (Halichondrida: Halichondriidae) with Aaptos and SuberitesSuberites (Hadromerida: Suberitidae), which increased molecular evidence for close Halichondrid- Hadromeridd relationships. Furtherr 28SrDNA studies (Lafay et al., 1992; Chombard et al., 1997; Alvarez et al., 2000) foundd a close relationship of species of the order Agelasida {Agelas) with axinellid halichondrids {Axinella).{Axinella). Such an Agelas I Axinella clade can only be explained morphologically with multiple aa priori assumed analogies, but it finds support in chemical analyses by the common possession of chemicall compounds of the pyrrole-2-carboxylic acid family (Braekman et al., 1992; Van Soest and Braekman,, 1999) and glycosyl ceramides (Costantino et al., 1996). Alll these analyses comprised only a subset of halichondrids and made a comprehensive studyy of halichondrids of all families necessary to elucidate its composition and relationships to non-halichondrids.. In mis study we present a subsequent comprehensive 28SrDNA study from the Halichondridd perspective. Erpenbeck et al. {submitted, Chapter 2) observed that the 28S gene structure inn underlies differences on order level. Based on those findings we could narrow down the taxonn set to a homogeneous and comparable character set without long branches which might mask phylogeneticc information and lead to an erroneous signal. Here, we present the results of a combined taxonn set based on the 28 S D3-D5 region, which unites representative Halichondrida with Agelasida andd certain Hadromerida. In order to test in an enlarged taxon set for the monophyly of Halichondrida sensuu Van Soest et al. (1990) and to get insight in phylogenetic relationships between and inside their families.. We test the existence of a "Suberitina" clade as proposed by Chombard et al. (1997) in this enlargedd taxon set with a different 28SrDNA fragment.

Materiall and Methods

Thee sponge tissue was either freshly collected with SCUBA diving, or sampled during the SYMBIOSPONGEE project (EU-MAS3CT 97-0144). A complete list of species studied is given in Tab.. 1. Up to three specimens per species were examined. DNA extraction and PCR setup, cloning andd sequencing was carried out as described in Erpenbeck et al. (2002). PCR primers employed weree taken from McCormack and Kelly, (2002, primers: RD3A: GACCCGTCTTGAAACACGA andd RD5B2: (ACACACTCCTTAGCGGA, temperature regime: 94° 3min during which the Taq polymerasee is added, 35x (94° 30s; 50° 20s; 72° 60s), 72° lOmin). The new sequences are submitted too Genbank. Sequencee management was performed using MacClade 4.03 (Maddison and Maddison, 1992).. DAMBE (Xia and Xie, 2001) was used to fetch and splice the sequences from Genbank (http://www.ncbi.nlm.nih.gov/)) which were included in an enlarged data set for comparison reasons. Wee received a first alignment by Clustal x (1.8, Jeanmougin et al., 1998) under default settings andd optimised it by eye under usage of secondary structure information. This secondary structure informationn was obtained by annealing to 23S-like secondary structures published by Hancock et al., (1988)) and Alvarez et al., (2000) under comparison with the common eukaryote model (Schnare et al.,, 1996). Priorr to phylogenetic reconstructions we performed several a priori analyses to check the qualityy of character and taxon set. In sponges more than in most other organisms the true sponge

49 9 3:: Phylogenetic relationshipso f halichondrid demosponges - Implications from a 28SrDNA tree

Class s Order r Family y Taxon n Acc.Nr. . Authorr or location & Voucher Nr Demospongiae e Agelasida a Ageiasidae e AgetasAgetas oroides AY319311 1 Erpenbeckk et al. submitted Demospongiae e Agelasida a Ageiasidae e AgelasAgelas mauritana AF051429 9 Alvarezz et al. 2000 Demospongiae e Agelasida a Ageiasidae e AgelasAgelas nakamurai Sulawesi/Indonesia;; POR17662 Demospongiae e Agelasida a Astroscterida a AstrosderaAstrosdera wüleyana AF051430 0 Alvarezz et al. 2000 Demospongiae e "Uthistida" " CoraHistidae e CoraHistesCoraHistes sp. AJ005913 3 Mclnemeyy et 31.1999 Demospongiae e "Lithistkla" " TheoneWdae e DiscodermiaDiscodermia dissolutaAJ00591 4 4 Mclnemeyy et al.1999 Demospongiae e "Lithistkia" " Theoneltidae e TheoneHasp. TheoneHasp. AJ005917 7 Mclnemeyy et al.1999 Demospongiae e "Uthistida* * Vetulinidae e Vetulinasp. Vetulinasp. AJJ 00591S Mclnemeyy et al.1999 Demospongiae e Chondrosida a Chondrillidae e ChondrosiaChondrosia sp. AJJ 005916 Mclnemeyy et al.1999 Demospongiae e Hadromerida a Clionaidae e SphedospongJaSphedospongJa AY31931vagabunda.0 0 Erpenbeckk et al. submitted Demospongiae e Hadromerida a Suberitidae e AaptosAaptos suberitoktes AY319308 8 Erpenbeckk et al. submitted Demospongiae e Hadromerida a Suberitidae e SuberitesSuberites massa Sulawesi/Indonesia;; POR17663 Demospongiae e Hadromerida a Suberitidae e SuberitesSuberites suberia 7 AY319309 9 Erpenbeckk et al. submitted Demospongiae e Hadromerida a Suberitidae e SuberitesSuberites suberia 2 Roscoff/F F Demospongiae e Hadromerida a Suberitidae e SuberitesSuberites virgultosa Northh Sea; POR12969 Demospongiae e Halichondrida a Axinellidae e AxineHaAxineHa aniensis AF051433 3 Alvarezz et al. 2000 Demospongiae e Halichondrida a Axinellidae e AxinetlaAxinetla damicornisl AY319314 4 Erpenbeckk et al. submitted Demospongiae e Halichondrida a Axinellidae e AxineHaAxineHa damicomis 2 AF051436 6 Alvarezz et al. 2000 Demospongiae e Halichondrida a Axinellidae e AxinetlaAxinetla pofypoides Roscoff/F;; POR14093 Demospongiae e Halichondrida a Axinellidae e AxineHaAxineHa verrucosa 7 AY319312 2 Erpenbeckk et al. submitted Demospongiae e Halichondrida a Axinellidae e AxineHaAxineHa verrucosa 2 Turkey;; POR9187 Demospongiae e Halichondrida a Axinellidae e CymbastetaCymbasteta coralliophUaAF05143 9 9 Alvarezz et al. 2000 Demospongiae e Halichondrida a Axinellidae e CymbastetaCymbasteta stipitata AF0S1440 0 Alvarezz et al. 2000 Demospongiae e Halichondrida a Axinellidae e CymbastetaCymbasteta vespertmaAF0S144 1 1 Alvarezz et al. 2000 Demospongiae e Halichondrida a Axinellidae e DragmaddonDragmaddon australeAF05144 3 3 Alvarezz et al. 2000 Demospongiae e Halichondrida a AxineHidae e DragmacidonDragmacidon durissimumAF05144 4 4 Alvarezz et al. 2000 Demospongiae e Halichondrida a Axinellidae e DragmaddonDragmaddon reticulatumAF05144 5 5 Alvarezz et al. 2000 Demospongiae e Halichondrida a Axinellidae e DragmacidonDragmacidon sp. AF051446 6 Alvarezz et ai. 2000 Demospongiae e Halichondrida a Axinellidae e PtHocauHsPtHocauHs sp. AF051447 7 Alvarezz et al. 2000 Demospongiae e Halichondrida a Axinellidae e PtilocaulisPtilocaulis walpersii AF0S1448 8 Alvarezz et al. 2000 Demospongiae e Halichondrida a Axinellidae e ReniochalinaReniochalina sp. AF051449 9 Alvarezz et al. 2000 Demospongiae e Halichondrida a Axinellidae e RenhchatmaRenhchatma stalagmitisAF0514S 0 0 Alvarezz et al. 2000 Demospongiae e Halichondrida a Desmoxyidae e DidiscusDidiscus anisodiscus Sulawesi/Indonesia;; POR17679 Demospongiae e Halichondrida a Desmoxyidae e DidiscusDidiscus oxeata 1 AY319320 0 Erpenbeckk et al. submitted Demospongiae e Halichondrida a Desmoxyidae e DidiscusDidiscus oxeata 2 Jamaica;; POR13539 Demospongiae e Halichondrida a Desmoxyidae e HigginsiaHigginsia mixta Sulawesi/Indonesia;; POR14495 Demospongiae e Halichondrida a Desmoxyidae e MyrmekiodermaMyrmekioderma AY31931granulata9 9 Erpenbeckk et al. submitted Demospongiae e Halichondrida a Dictyoneflidae e AcantheHaAcantheHa acuta 1 AY319322 2 Erpenbeckk et al. submitted Demospongiae e Halichondrida a Dictyonellidae e AcantheHaAcantheHa acuta 2 AF051431 1 Alvarezz et al. 2000 Demospongiae e Halichondrida a Dictyonellidae e AcantheHaAcantheHa cavernosa 1 Sulawesi/Indonesia;; POR17663 Demospongiae e Halichondrida a Dictyonellidae e AcantheHaAcantheHa cavernosa AF051432 2 2 Alvarezz et al. 2000 Demospongiae e Halichondrida a Dictyonellidae e AcantheHaAcantheHa pukherrimaAF0S143 3 3 Alvarezz et al. 2000 Demospongiae e Halichondrida a Dictyonellidae e DictyonellaDictyonella sp. AY319325 5 Erpenbeckk et al. submitted Demospongiae e Halichondrida a Dictyonellidae e LiosinaLiosina paradoxa 1 AY319318 8 Erpenbeckk et al. submitted Demospongiae e HaKchondrida a Dictyonellidae e LiosinaLiosina paradoxa 2 Sulawesi/Indonesia;; POR 17664 Demospongiae e Halichondrida a Dictyonelltdae e LiosinaLiosina paradoxa 3 Sulawesi/Indonesia;; POR17665 Demospongiae e Halichondrida a Dictyonellidae e ScopalinaScopalina lophyropodaAY31932 3 3 Erpenbeckk et al. submitted Demospongiae e Haüchondrida a Dictyonellidae e ScopalinaScopalina ruetzleri AF051451 1 Alvarezz et al. 2000 Demospongiae e Halichondrida a Dictyonellidae e StyiissaStyiissa carter! 1 Sulawesi/Indonesia;; POR17666 Demospongiae e Halichondrida a Dictyonellidae e StylissaStylissa carteri 2 Sulawesi/Indonesia:: POR17667 Demospongiae e Halichondrida a Dictyonellidae e StyiissaStyiissa carters 3 AF051434 4 Alvarezz et al. 2000 (as AxineHa) Demospongiae e Halichondrida a Dictyonelltdae e StylissaStylissa carteri 4 AF051435 5 Alvarezz et al. 2000 (as AxineHa) Demospongiae e Halichondrida a Dictyonellidae e StyiissaStyiissa Habeiliformis 1 AY319316 6 Erpenbeckk et al. submitted Demospongiae e Halichondrida a Dictyonellidae e StylissaStylissa HabeHfformis 2 Sulawesi/Indonesia;; POR17666 Demospongiae e Halichondrida a Dictyonellidae e StylissaStylissa massa 1 Sulawesi/Indonesia;; POR17669 Demospongiae e Halichondrida a Dictyonellidae e StylissaStylissa massa 2 Sulawesi/Indonesia;; POR17670 Demospongiae e Halichondrida a Dictyonellidae e SvenzeaSvenzea devoogdae Sulawesi/Indonesia;; POR17671 Demospongiae e Halichondrida a Dictyonellidae e SvenzeaSvenzea zeai AF441349 9 McCormackk et al. 2002 Demospongiae e Halichondrida a Halichondriidae e AmorphinopsisAmorphinopsis excavansAY31931 3 3 Erpenbeckk et al. submitted Demospongiae e Halichondrida a Halichondriidae e AmorphinopsisAmorphinopsis siamensis Gulff of Siam; POR17672 Demospongiae e Halichondrida a Halichondriidae e AxinyssaAxinyssa ambrosia Curacao;; POR14311 Demospongiae e Halichondrida a Halichondriidae e AxinyssaAxinyssa sp. Sulawesi/lndonesia;POR14501 1 Demospongiae e Halichondrida a Halichondriidae e AxinyssaAxinyssa sp. "oranje" Sulawesi/Indonesia;; POR17673 Demospongiae e Halichondrida a Halichondriidae e AxinyssaAxinyssa aplysnotdes AY319324 4 Erpenbeckk et al. submitted Demospongiae e Halichondrida a Halichondriidae e CiocalyptaCiocalypta confössa AF051453 3 Alvarezz et al. 2000 Demospongiae e Halichondrida a Halichondriidae e CiocalyptaCiocalypta penicillus Roscoff/France;; POR 14111 Demospongiae e Halichondrida a Halichondriidae e CiocalyptaCiocalypta tyteri Seychelles;; POR11683 Demospongiae e Halichondrida a Halichondriidae e HaHchondriaHaHchondria bowerbanki 1 Netherlands;; POR17674 Demospongiae e Halichondrida a Halichondriidae e HaHchondriaHaHchondria bowerbanki 2 Netherlands;; POR1767S Demospongiae e Halichondrida a Halichondriidae e HaHchondriaHaHchondria paniceaAY31931 5 5 Erpenbeckk et al. submitted Demospongiae e Halichondrida a Halichondriidae e HaHchondriaHaHchondria phakelloidesAF0514S 5 5 Alvarezz et al. 2000 Demospongiae e Halichondrida a Halichondriidae e HymeniaddonHymeniaddon perlevisAY31931 1 7 7 Erpenbeckk et al. submitted Demospongiae e Halichondrida a Halichondriidae e HymeniacidonHymeniacidon perlevis 2 Netherlands;; POR 17676 Demospongiae e Halichondrida a Halichondriidae e TopsentiaTopsentia haHchondroides Srii Lanka; POR8450 Demospongiae e Poecilosclerida a Mycalidae e MycateMycate fibrexiHs AY026376 6 Medinaa et al. 2001 Demospongiae e Poedlosdenda a Mycafidae e MycaleMycale HageKifera AY319321 1 Erpenbeckk et al. submitted Calcarea a Calcaronea a Leucosoteniidae e LeucosoiemaLeucosoiema sp. AY026372 2 Medinaa et al. 2001 Tab.1:: Samples used in this analysis with genbank accession numbers, sampling location, collection number off the Zoological Museum Amsterdam (POR) and authors. 50 0 3:: Phylogenetic relationships of halichondrid demosponges - Implications from a 28SrDNA tree originn of PCR amplification products should be verified as contaminations by symbionts and food particless can occur. We performed a BLAST search (Altschul et al., 1990) against Genbank sequences andd additionally checked the phylogenetic position of our sequences against other eukaryotes in a phylogeneticc tree as in Erpenbeck et al. (2002). This approach was used to verify that our sequences clusterr monophyletically and in close relationship to other diploblastic Metazoa. Wee employed a PTP test (Faith, 1991) as implemented in PAUP*4.0blO (Swofford, 2002) withh 100 replicates based on heuristic search to determine whether the information of our data sets arosee by chance. The base frequencies have been tested for homogeneity in a x2 test of all taxa as implementedd in PAUP*. Severall sponge sequences fetched from Genbank were shorter than our own sequences. The combinationn would have resulted in a data matrix with a majority of missing characters. To reduce thee resulting noise we performed two different approaches on two different data sets: a)) A Long (D3-D5) data set of taxa with sequences of the full length (including D3 + D4 + D5 subdomains)) to receive the strongest possible phylogenetic signal. It resulted in a data set of 53 taxaa and 760 characters. b)) An Extended taxon set (D3) of all taxa including several shorter sequences from Alvarez et al., (2000).. It resulted in a data set of 75 taxa and 455 characters of the D3 region only.

Phylogeneticc reconstructions were based on Bayesian analyses performed by MrBayes v3b4 (Huelsenbeckk and Ronquist, 2001). The likelihood and prior probability parameters were estimated usingg MrModeltest vl. 1 (J. Nylander) under PAUP*. All bayesian analyses were performed with four Metropolis-Coupledd Markow Chains and two million generations. The chains were "burned in" until thee posterior probabilities reached a stable plateau. Differentt approaches were analyzed regarding application of the relatively best-fitting evolutionaryy model. The data set was split in several partitions: Partition "stem" contained all helix regionss sequenced. Partition "loop" contained all not pairing nucleotides. Furthermore partition "excluded"" comprised ambiguous alignable positions. Characters of this partition were not included inn the analysis. We performed the bayesian analyses as a two-partition ("stem" and "loop") approach andd compared the results with a maximum-likelihood reconstruction. We did not apply a nucleotide substitutionn model based on a 16x16 nucleotide doublet matrix (Schoniger and vonHaeseler, 1995) forr the helices, because it reduced the resolution power in smaller character sets as observed in previouss analyses (Erpenbeck et al. submitted, chapter 2). Forr all analyses we chose a non-demosponge outgroup. As phylogenetic relationships of demospongess are still mostly unresolved a calcareous sponge outgroup seemed more suitable than a demospongee that might lead to uncertain tree polarity. We chose the calcareous sponge Leucosolenia sp.. as sequenced by Medina et al. (2001). Branch length differences to other demosponges were previouslyy regarded to be moderate (Erpenbeck et al. submitted, chapter 2).

Results s

LongLong (D3-D5) data set:

Somee 190 of the 760 characters were excluded from the long (D3-D5) data set due to alignment ambiguities.. Sequence length varied from 574 to 591 base pairs. The alignment revealed no exceptionall secondary structural features as found among other demosponge orders (Erpenbeck et al.. submitted, chapter 2). MrModeltest estimated based on the likelihood-ratio-test the following nucleotidee substitition models as the relative best-fitting for the included characters of the partitions: Partitionn "stem": HKY+I+G (Hasegawa et al., 1985), partition "loop" GTR+G (Rodriguez et al.,

51 1 3:: Phylogenetic relationships of halichondrid demosponges - Implications from a 28SrDNA tree

99 9 HAL AX I Axinella verrucosa 7 98^^— — HAL AX I Axinella verrucosa 2 ,HALL DC! Stylissa caneri 1 .. HAL DCT Sfyfesa carter; ,? 99 9 HAL DCT SO'fesa flabelliformis 1 HAL DCT Sfyfesa flabelliformis 2 Dictyonellidae e 98 8 ,, HAL DCT S^fesa massa 7 ,HALL DCT Stylissa massa 2 HAL DCT 3 100 0 .. HAL DCT Stylissa carteri 4 .. HAL AX I Axinella aruensis ii HAL AX I Axinella damicornis 1 98 8 .. HAL AX I Axinella damicornis 2 Axinellidae e ,, HAL DES Higginsia mixta Desmoxyidae e .AGEE KL Agelas nakamurai 81 1 .AGEE bGL Agelas oroides 100 0 .AGEE KL Agelas mauretania 94 4 -AGEE ASC Astrosclera willeyana HAL AX I Axinella polypoides 97 7 iHALL DCT Liosina paradoxa 1 96 6 __ _ 97 7 ii HAL DCT Liosina paradoxa 2 Dictyonellidae e 1000 .HALL DCT Liosina paradoxa 3 .. HAD CLI Spheciospongia vagabunda HAD SUB Suberites suberia 1 r— —HAD SUB Suberites suberia 2 99 9 HAD SUB Suberites virgultosa ^^ ^ 1— —HAD SUB Suberites massa — —ii HAD SUB /laptos suberitoides HAL HLC siamensis 94 4 100 0 HAL HLC tyleri HAL HLC Topsentia halichondroides 100 0 — — HAL HLC Hymeniacidon perlevis 7 90 0 HAL HLC Hymeniacidon perlevis 2 HAL HLC Amorphinopsis excavans 92 2 ~ ~ HAL HLC bowerbanki 1 Halichondriidae e 100 0 95 5 ^__ _ HA L HLC 2 HAL HLC -HALL HLC Halichondriaphakellioides .. HAL HLC Ciocalypta confossa ,, HAL HLC Ciocalypta penicillus P0E MYC Myca/e (Carmia) fibrexilis .POEE MYC Mpcafe (Naviculina) flagellifera 100 0_. —-HA —LL AX I Cymbastela stipitata HAL AX I Cymbastela vespenina 90 0 1 1 HAL DCT cavernosa 1 .. HAL DCT Acanthella cavernosa 2 HAL DCT Acanthella pulcherrima 98 9 8 _ _ HAL DCT Acanthella acuta 1 Dictyonellidae e 100 0 .. HAL DCT Acanthella acuta 2 1 310 0 100 0 .HALL DCT Dictyonellasp. T9~ ~ C C HA L HLC Axinyssa ambrosia HAL HLC Axinyssa sp. 6 I Halichondriidae e 10MM00 I J— J— HA L HLC Axinyssa aplysinoides 96 6 .HALL HLC Axinyssa sp. /4 t t HAL DES Didiscus oxeata 1 800 | 1— — HA L DES Didiscus oxeata 2 100 0 T4™ ™ HAL DES Didiscus anisodiscus Desmoxyidae e 977 | L_ii HA_L DES Myrmekioderma granulata HAL AX I Reniochalina sp. 97 7 -- HAL AX I Reniochalina stalagmitis 100 0 i— — LL AX I Ptilocaulis sp. ^— — HA L AX I Ptilocaulis walpersi 98 8 HAL AX I Dragmacidon durissimum 96 6 ~i.—— —-HA— LL AX I Dragmacidon so. 10 10 0 HAL AX I Dragmacidon australe HAL AX I Dragmacidon reticulatum HAL DCT Svenzea devoogdae 100 0 T9™ ™C C HA L DCT Svenzea zeai 100 0 HAL DCT Scopalina lophyropoda Dictyonellidae e -= = HA L DCT Scopalina ruetileri 110 10 0 LIT THE Discodermia dissoluta 100 0 T?sr r C .LICTT THE Theonellasp. 100 0 11 1 LIT COR Corallistessp. LIT VET Vetulinasp. HAL AX I Cymbastela coralliophila .CH00 CHN Chondroma sp. .. Leucosolenia sp.

52 2 3:: Phylogenetic relationships of halichondrid demosponges - Implications from a 28SrDNA tree Fig.. 1: (previous page) Combined tree of the long (D3-D5) data set (bold lines, posterior probabilities in bold letterss below the branches) and the extended taxon set (D3) (bold lines and thin lines, posterior probabilities abovee the branches). Clades with posterior probabilities lower than 80% are collapsed. Dashes indicate a posteriorr probability < 80% in that particular analysis. Abbreviations: Orders: AGE = Agelasida, CHO == Chondrosida, HAD = Hadromerida, HAL = Halichondrida, LIT = "Lithistida", POE = Poecilosclerida. Families:: AGL = Agelasidae, ASC = Astroscleridae, AXI = Axinellidae, CHN = Chondrillidae, CLI = Clionaidae,, COR = Corallistidae, DCT = Dictyonellidae, DES = Desmoxyidae, HLC = Halichondriidae, MYC == Mycalidae, SUB = Suberitidae, THE = TheoneUidae, VET = Vetulinidae.

1990).. Maximum-likelihood (one partition): SYM+G+I (Zharkikh, 1994).

ExtendedExtended taxon set (D3):

Somee 167 characters of this data set were excluded from the phylogenetic analyses because they couldd not be aligned unambiguously. Based on the likelihood-ratio-test we used the following models forr the partitions: Stem: HKY+I+G (Hasegawa et al., 1985), loop: GTR+I+G (Rodriguez et al., 1990).. Maximum-likelihood (one partition): TrN+I+G with even frequencies (=TrNef+I+G, Tamura andd Nei, 1993).

TheThe resulting gene tree:

Wee retained the gene trees of both analyses with clades of less than 80% posterior probability collapsed. Thee trees are in agreement with their particular maximum-likelihood trees. We reconstructed the "combinablee component"-consensus of both trees (figure 1). The tree of the 'Long (D3-D5) data set' consistss of a subsett of taxa and is plotted with bold branches on the 'Extended taxon set (D3)' tree. Thee two trees differed in the relationships between Liosina paradoxa and Axinella polypoides: Thee (shorter) 'extended taxon set (D3)' favoured a sister-group relationship and the 'Long (D3-D5) dataa set' a paraphyletic and more derived position of Axinella polypoides.

PhylogeneticPhylogenetic implications:

Figuree 1 displays a polyphyletic order Halichondrida (sensu Hooper and Van Soest, 2002) because sampless of the orders Agelasida and Hadromerida cluster among the halichondrids. None of the halichondridd families clusters monophyletic. The Halichondriidae with the exception of Axinyssa sp. formm a clade with the Suberitidae (Suberites and Aaptos, order Hadromerida). Thee Axinellidae are split up in three clades: (a) an Axinella-ctede which is formed with alll species of Stylissa (Dictyonellidae), (b) a paraphyletic clade which consists of Ptilocaulis and ReniochalinaReniochalina together with the Desmoxyidae samples, (c) a Dragmacidon clade and (d) a Cymbastela spp.. clade clustering with some Dictyonellidae. Axinella is not monopyletic as Axinella damicornis clusterss basal to Stylissa and A. verrucosa in a sister-group relationship to the Agelasida samples (Agelas(Agelas and Astrosclera). Axinella polypoides is even more basal. Thee Agelasida group together. The Caribbean Agelas oroides and the Indian Ocean species AgelasAgelas nakamurai form a clade with Astrosclera willeyana in a well-supported sister group to the Halichondriidaee / Suberitidae clade. The clustering of one sequence of the desmoxyid Higginsia mixtamixta appears odd and should be verified by additional Higginsia sequences. Thee Dictyonellidae split up in multiple clades: Stylissa groups separately, Acanthella and DictyonellaDictyonella cluster with Axinyssa (Halichondriidae) and Cymbastela (Axinellidae). Affinities of ScopalinaScopalina and Svenzea to other Dictyonellids are not supported as well as the position of Liosina paradoxa.paradoxa. They are distant from other Dictyonellidae of the taxon set. 53 3 3:: Phylogenetic relationships of halichondrid demosponges - Implications from a 28SrDNAtree

Discussion: :

Wee restrict ourselves to regard bayesian results supported by posterior probabilities of 80 and higherr as bayesian posterior probabilities in general tend to be higher than bootstrap support values (Huelsenbeckk et al. 2002). This stringency affects the resolution power negatively but that is certainly moree acceptable than a higher resolved tree under erroneous phylogenetic signal. Here we combine thee consensus trees of different approaches to keep the amount of ambiguities as low as possible. Thee tree topologies retained are comparable with exception of the Axinella polypoides I LiosinaLiosina paradoxa relationships in the long (D3-D5) data set and in agreement with the maximum- likelihoodd results. Thiss analysis can be regarded as an investigation on the influence of the taxon set on the views off phylogenetic relationships in a difficult systematic taxon such as the demosponges. The overall topologiess of the thorough morphological and molecular studies on Axinellidae by Alvarez ( 1996) andd Alvarez et al. (2000) can be re-found in the halichondrid gene tree of figure 1, but the inclusion off additional taxa splits up clades recognized by those authors. Inn our tree, taxa of the order Hadromerida mingle with Halichondrida. With Suberites, Aaptos (bothh Suberitidae) and Spheciospongia (Clionaidae) representatives of two hadromerid families clusterr with Halichondrida. These results are similar to the molecular findings of Chombard and Boury-Esnaultt (1999) and McCormack and Kelly (2002) in which the sequences of the hadromerid generaa Suberites, Tethya and Spheciospongia group with the halichondrid Axinella, Topsentia andd Hymeniacidon. Further Halichondrida / Hadromerida clusters might be detected at least on 28SrDNA-genee fragment level. Nevertheless, only if further independent data confirm these findings, thee "Suberitina" (Chombard and Boury-Esnault 1999) should be expanded. Similarly,, more Halichondrida / Poecilosclerida clusters might be expected with 28SrDNA. Poecilosclerida,, the taxon-richest sponge order, is clearly underrepresented in our current taxon set. Inn our analysis the sequences of Mycale flagellifera and Mycale fibrexilis do not form a supported sisterr group and remain at uncertain positions in the gene tree but both cluster with Halichondrida. MycaleMycale fibrexilisi s assigned to the subgenus Carmia, Mycale flagellifera (Vacelet and Vasseur, 1971) too the subgenus Naviculina. Their morphological differences, such as the different architecture of the skeletonn and shape of the anisochelae, are reflected in their different positions in the 28SrDNA gene tree. . Thee monophyly of the family Axinellidae is not supported in this enlarged taxon set. Polyphylyy of the morphologically variable genus Axinella was previously shown in 28SrDNA analysess by Alvarez et al. (2000). The genus Axinella is in our analysis not distinguishable from the dictyonellidd genus Stylissa, with the exception of Axinella damicornis. Several Stylissa species such ass S. massa and S. carteri, both included in our data set, were previously regarded as Axinella because off the vaguely reticulate skeleton of styles, lacking a distinct surface specialization. The distant clusteringg of the type species Axinella polypoides is in congruence with the shorter data of Alvarez ett al. (2000). With the inclusion of Alvarez et al.'s sequences we provide evidence that the axinellid taxaa Reniochalina, Ptilocaulis and Cymbastela are more distant. The phylogenetic position of the CymbastelaCymbastela coralliophila sequence as published by Alvarez et al. (2000) still remains ambiguous. C. coralliophilacoralliophila is atypical for its genus as it possesses a paratangential ectosomal skeleton. Theree is only one genus of the family Halichondriidae not clustering with the other memberss of that family: Axinyssa species display closer affinities to and Acanthella (Dictyonellidae)) in this gene tree. Morphological evidence for a closer relationship between Axinyssa andd Dictyonellidae may be the shared absence of a special ectosomal spicule skeleton, which is part off the definition of Dictyonellidae (Van Soest et al., 2002).

54 4 3:: Phylogenetic relationships of halichondrid demosponges - Implications from a 28SrDNA tree Dictyonellidaee sensu (Van Soest et al., 2002) are polyphyletic in our results. Next to Stylissa, whichh is discussed above, Liosina is distant from the type genus Dictyonella. This configuration impliess upcoming difficulties to keep an integral Dictyonellidae valid in the future. On the other hand, thee genus Acanthella (Dictyonellidae) confirms its recent placement in this family. It was previously regardedd as axinellid in morphological (Bergquist, 1970) and molecular studies (Alvarez et al., 2000). Here,, it clusters close to Dictyonella, separate from Axinella. The recent morphological assignment off Svenzea to Dictyonellidae (Alvarez et al., 2002) finds molecular support as both the Carribean S. zeaizeai and the Pacific S. devoogdae cluster as a sister group to Scopalina. MyrmekiodermaMyrmekioderma and Didiscus were both previously regarded as Halichondriidae (Van Soestt et al., 1990) from which they are clearly distant in our gene tree. Their placement with other Desmoxyidaee (Van Soest and Lehnert, 1997; Hooper, 2002) has to be verified by additional molecular data.. The single sequence of Higginsia mixta clusters with Agelasida in an morphologically odd position.. Its correctness should be verified prior to further assumptions on the phylogeny of the Desmoxyidae. . Moree taxa of Chondrosida and the "lithistid" species are needed to resolve their phylogenetic affinitiess to the Halichondrida. Their in- or exclusion of these taxa had no major influence on the topologies. . Ourr data elucidates a different perspective of halichondrid phylogeny. We do not intend to comee up with a new phylogeny of halichondrid demosponges at this stage, or a proposal to abandon thiss taxon, but present an alternative view on halichondrid relationships. The current analysis is based onn only a fragment of one single gene, and the topology should, like every gene tree, be compared andd validated with other gene trees, preferably from independent genes such as mitochondrial genes orr nuclear proteins. The suitability of 28SrDNA to resolve demosponge relationships appears to be acceptedd (Mclnerney et al., 1999), but inconsistencies have been shown in Haplosclerida (McCormack ett al., 2002) which supports our view that 28SrDNA data should be compared with other genes. A thoroughh comparison between independent phylogenetic data sets of molecular, morphological and biochemicall data will be necessary. Nevertheless,, the current analyses provides evidence for alternative relationships in demosponges.. It elucidates that one should be aware that in such a morphologically rather "simple" taxonn as the Porifera character-analogies should never be disregarded. Phylogenies remain ambiguous ass long as monophyly is not assessed and not all taxa are investigated (Graybeal, 1998).

Acknowledgements s

Wee would like to thank Matthijs van Couwelaar, Raquel Gomez, Bert W. Hoeksema, Mario J. dee Kluijver, Peter Kuperus, Frederick R. Schram, Jan J. Vermeulen and Nicole J de Voogd for contributionss in various ways to this paper. Part of the material was freshly collected during thee SYMBIOSPONGE project (EU-MAS3CT 97-0144). This work was financed by the Dutch organisationn for scientific research (NWO) Nr: ALW 809.34.003. All experiments comply with the currentt laws of the Netherlands and the European Union.

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