361Porifera research: Biodiversity, innovation and sustainaBility - 2007

Introduction

Sponges of the genus Aplysina, Nardo 1834 are abundant in the subtropical and tropical waters of the Mediterranean Sea (Boury-Esnault 1971, Kreuter et al. 1992), Pacific Ocean (Carney and Rinehart 1995, Betancourt-Lozano et al. 1998), and Atlantic Ocean (Pinheiro and Hajdu 2001, Saeki et al. 2002).

Only two species of this genus are described for the Mediterranean Sea: A. aerophoba (Schmidt, 1862) and A. cavernicola (Vacelet 1959). A. aerophoba lives in shallow water from 1 – 30 m on rocks and tolerates high variation of temperature, insolation and density (Kreuter et al. 1992, Vacelet 1971). In contrast A. cavernicola favours caves and shadowy areas in depth from 7 – 130 m (Vacelet 1959, 1969). A. aerophoba and A. cavernicola can be differentiated in the Western Mediterranean Sea in morphology and biochemistry (Vacelet 1959, Ciminiello 1997, Heim 2003). In the Limski kanal north of Rovinj/Croatia, both species occur in the same

habitat. In addition, species differentiation is difficult due to high intra-specific variation of form and colour (Heim 2003; Fig. 1). The same problem was reported from the Aegean Sea, which gave rise to the question whether or not A. cavernicola is a true species of its own or just an ecological variant of A. aerophoba, living in caves (Voultsiadou-Koukoura 1987).

In addition to morphological characters, the biochemistry of secondary metabolites was recently investigated in order to differentiate the two Mediterranean species. However, it turned out that biochemical profiles are no valuable character since specimens were found in the Limski kanal in Croatia, which combine the secondary metabolite profile of A. aerophoba and A. cavernicola or do not display any of the characteristic substances at all (Heim 2003, Thoms 2004).

For this reason, a molecular marker for species discrimination would be useful in the case of the Mediterranean Aplysina species. Especially for the differentiation of the species found in the Adriatic Sea. For this purpose different regions from the nuclear or mitochondrial genome are potential markers.

In the last few years, several studies were performed on sponges and corals using the one or the other candidate marker. The first and the second internal transcribed spacer regions (ITS-1 and ITS-2) between the 18S, 5,8S and 28S ribosomal

Molecular markers for species discrimination in poriferans: a case study on species of the genus AplysinaIsabel Heim(*), Michael Nickel, Franz Brümmer

Universität Stuttgart, Biologisches Institut, Abteilung Zoologie, Pfaffenwaldring 57, 70569 Stuttgart, Germany.(†) isabel.heim@bio.uni-stuttgart.de, m.nickel@uni-jena.de, franz.bruemmer@bio.uni-stuttgart.de

Abstract: We tested different molecular markers for their utility as species discriminators in Porifera: internal transcribed spacer-1 and -2 (ITS-1, ITS-2) rDNA, mitochondrial 12S, 16S, and cytochrome oxidase subunit I (COI). The study was performed on specimens of the genus Aplysina from different locations in the Mediterranean Sea, East, and West Atlantic. This genus is widespread in tropical and subtropical waters and rich in natural substances. From the Mediterranean Sea, two different species are known: A. aerophoba (Schmidt, 1862) and A. cavernicola (Vacelet, 1959). We intended to find an adequate molecular marker to differentiate the Mediterranean species A. aerophoba and A. cavernicola. However, there was a high degree of intra-individual polymorphism within the markers ITS-1 and ITS-2. Consequently, these markers were not adequate for species differentiation of A. aerophoba and A. cavernicola for technical reasons discussed here. In contrast, the mitochondrial 12S and 16S are highly conserved and no differences among species were observed. Only the COI showed a low variability in the seven analysed Aplysina species. Based on COI, it was possible to classify the specimens COI in two clades. One clade is represented by A. aerophoba and A. cavernicola which could be distinguished from the Caribbean Aplysina species. However, the resolution was low in the second clade consisting of Western Atlantic Aplysina species, suggesting a recent radiation event. Our results suggest that a general molecular markers for species discrimination does not exist. Hence, the choice of a suitable marker strongly depends on the evolutionary context of each single taxon and will have to be tested accordingly.

Keywords: Aplysina, COI, ITS, mt-rDNA, species discrimination

(†) M. Nickel present address:Friedrich-Schiller-Universität Jena, Institut für Spezielle Zoologie und Evolutionsbiologie mit Phyletischem Museum, Erbertstr. 1, 07743 Jena, Germany

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RNA genes are preferably used for intra- and interspecific phylogeographic relationships (Wörheide 1998, King et al. 1999, van Oppen et al. 2002, Wörheide et al. 2002b, Duran et al. 2004a, Schmitt et al. 2005). The ITS-regions evolve rapidly and, hence, are useable as “high resolution marker” in populations’ genetics (van Oppen et al. 2002, Wörheide et al. 2002a). However, in some cases polymorphisms have been detected in these non-coding regions (Wörheide et al. 2004, Nichols and Barnes 2005). When intragenomic variations are found, the ITS region is not useful for analyses at population-level (Nichols and Barnes 2005). On this account it is necessary to screen the investigated taxon for the presence and extent of intragenomic polymorphisms to adapt the molecular methods (Wörheide et al. 2004).

In other invertebrate taxa the mitochondrial 12S rDNA as well as the 16S rDNA were used for phylogeny and phylogeography analysis, including scleractinian corals and hydrozoans (Chen et al. 2002, Govindarajan et al. 2005). Both studies reported a slow divergence rate in Cnidaria which is probably triggered by a mismatch repair system homologue to the bacterial MutSLH system (Pont-Kingdon et al. 1998). The same could be occurring in sponges because Duran et al. (2004b) reported about a low genetic variation in the cytochrome oxidase subunit I for the species Crambe crambe, but there are no studies yet on mitochondrial 12S and 16S in sponges.

Another region of the mitochondrial genome is often used in marine invertebrates. For population genetics the cytochrome c oxidase subunit I (COI) was used for mussels and ascidians (Avise et al. 1987, King et al. 1999, Tarjuelo et al. 2001). In Crambe crambe as well as in Astrosclera willeyana the COI sequences were tested for its use as an intraspecific genetic marker among populations of sponge species (Duran et al. 2004b, Wörheide 2005), but in both cases the marker was too conserved for population studies, as also shown for cnidarians (Shearer et al. 2002, van Oppen et al. 2002). Although the COI has been found to be too conserved for population studies in cnidarians and sponges, it could be possible to use the marker for species discrimination in sponges. Recent studies demonstrated that the COI is a valuable marker in higher taxa like birds, fishes and butterflies to differentiate species (Hebert et al. 2004, Ward et al. 2005).

In the present study we tested five different molecular markers (ITS-1, ITS-2, 12S, 16S and COI) for their usefulness in species discrimination within the genus Aplysina.

Material and methods

SamplingLive sample organisms of the genus Aplysina were collected

during fieldwork within the research project BioteCmarin: Aplysina aerophoba (CRO), (FRA), (SPA) and (POR); A. cavernicola (CRO), (FRA) and (ITA); Aplysina. sp. (CRO); A. fistularis (Pallas, 1766) from Cat Island/Bahamas; A. archeri (Higgin, 1875), A. cauliformis (Carter, 1882), A. insularis (Duchassing and Michelotti, 1864) from Little San Salvador/Bahamas and A. fulva (Pallas, 1766) from Sweetings Cay/Bahamas (Fig. 2). In Table 1 the processed sponge specimens are listed with their abbreviations used in this

paper. The Aplysina sp. specimen has secondary metabolites that are present in A. aerophoba as well as in A. cavernicola (Thoms 2004). Sampling was undertaken by SCUBA diving. Pseudocertina sp. was taken from the public aquarium of the zoological and botanical gardens Stuttgart (Wilhelma) and used as outgroup. The cortex of the samples was cut off before frozen in liquid nitrogen for storage.

DNA extraction, amplification and sequencingWhole cellular DNA extraction for all organisms was

performed with Guanidinethiocyanate-buffer (25 mg tissue per ml; 5 M Guanidinethiocyanate, 20 mM EDTA dissolved at 65°C, cooled down and than add 10% N-Laurylasarcosinin) followed by phenol - chloroform extraction.

Polymerase chain reaction (PCR) amplifications were performed in a total volume of 50 µl using Genaxxon polymerase (Genaxxon Biosciences, Biberach, Germany).

For the PCR of the ITS-region (including ITS-1, 5.8S and ITS-2), the primers ITS1 and RA2 (Wörheide 1998) were used. After an initial denaturation step at 96°C for 3 min, rDNA was amplified during 30 cycles of 95°C for 1min, 55°C for 30 s and 72°C for 1 min and a final extension at 72°C for 7 min.

In the case of the mitochondrial 12S region, the primers 12S-For and 12S-Rev (Chen and Yu 2000) were utilised. The amplification started with an initial denaturation at 95°C for 4 min, rDNA was amplified during 4 cycles of 94°C for 1 min, 50°C for 30 s and 72°C for 3 min and 30 cycles of 94°C for 30 s, 60°C for 1 min and 72°C for 3 min.

For the mitochondrial 16S region, the primers 16S1 and 16S2 were used (Bridge et al. 1995). After an initial denaturation step at 95°C for 4 min, the rDNA was amplified during 4 cycles of 94°C for 1 min, 50°C for 30 s and 72°C for 3 min and 30 cycles of 94°C for 30 s, 52°C for 1 min and 72°C for 3 min.

A part of the cytochrome oxidase subunit I (COI) was amplified using the primers COI-For and COI-Rev (Folmer et al. 1994). The amplification started with an initial denaturation step at 94°C for 2 min followed by 35 cycles of 94°C for 50 s, 40°C for 55 s and 72°C for 1 min and a final extension at 72°C for 7 min.

All of the obtained PCR-products (ITS-1, ITS-2, 12S, 16S and COI) were cleaned with NucleoSpin Extract Kit (Machery-Nagel, Düren, Germany) before they were ligated into pCR®4-TOPO® vector (Invitrogen, Carlsbad, Canada) and transformed by heat-shock into competent e. coli One Shot®TOP10 (Invitrogen, Carlsbad, Canada). Plasmid DNA was isolated with NucleoSpin Plasmid Quick Pure (Machery-

Fig. 1: Different form and colour variations of Aplysina specimens in the Mediterranean Sea. A. A. cavernicola (Giglio, Secca II, Italy); B. A. cavernicola, arrow tags colour variation within one individual (Giglio, Fenaio, Italy); C. A. aerophoba (Banyuls-sur-mer, France); D. A. aerophoba (Banyuls-sur-mer, France); E. Different variations of A. aerophoba and A. cavernicola (Limski kanal, Rovinj, Croatia); F. A. cavernicola (Limski kanal, Rovinj, Croatia); G. A. aerophoba (San Giovanni, Rovinj, Croatia); H. A. aerophoba (Rovinj, Croatia) and I. Aplysina sp. (Limski kanal, Rovinj, Croatia).

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Nagel). The correct insert size was verified by using agarose gel electrophoresis following an insert check with the internal primer and the M13 forward.

The sequencing was performed by AGOWA GmbH (Berlin, Germany). The sequencing reactions were done with the M13 forward or M13 reverse primers. Three clones per individual were picked and sequenced to make a consensus sequence expect of the 12S and 16S cloning. An overview of the tested molecular markers and the Aplysina species we used is given in Table 2.

Sequences were edited and manipulated using BioEdit (Hall 1999). Sequence alignment was performed using the multiple sequence alignment program CLUSTAL X (Thompson et al. 1997).

A similarity search (BLAST) was performed to confirm that sequences were from sponge origin and not from other possible contaminants such as symbionts. The nucleotide

sequence data reported in this paper have been deposited in the GenBank nucleotide sequence database with accession numbers EF043343 to EF043378.

Phylogenetic analysis

For the COI we obtained 654 bp of four A. aerophoba specimens (SPA, POR, FRA and CRO), three A. cavernicola specimens (FRA, CRO and ITA), one Aplysina sp. (CRO), one specimen respectively of A. fulva, A. archeri, A. cauliformis, A. fistularis, A. insularis and Pseudoceratina sp. from the public aquarium in Stuttgart. These sequences were utilised for the following phylogenetic analysis. Aiolochroia crassa (AJ843885) from GenBank and Pseudoceratina sp. were used as outgroup in the calculations.

Fig. 2: Map showing the localities localities in Europe (large map) and the Bahamas area (inset map) where Aplysina individuals were sampled (1 = A. aerophoba, Madeira; 2 = A. aerophoba, Cadaqués, Spain; 3 = A. aerophoba, Banyuls-sur-mer, France; 4 = A. cavernicola, Marseille, France; 5 = A. cavernicola, Fenaio, Giglio, Italy; 6 = A. aerophoba, A. cavernicola and Aplysina sp., Rovinj, Croatia; 7 = A. fulva, Sweetings Cay, Bahamas; 8 = A. archeri, A. cauliformis and A. insularis, Little San Salvador Island, Bahamas; 9 = A. fistularis, Cat Island, Bahamas).

Species Origin Abbreviation

A. aerophoba Limski kanal, Croatia A. aerophoba (CRO)A. aerophoba Banyuls-sur-mer, France A. aerophoba (FRA)A. aerophoba Cadaquéz, Spain A. aerophoba (SPA)A. aerophoba Madeira, Portugal A. aerophoba (POR)A. cavernicola Limski kanal, Croatia A. cavernicola (CRO)A. cavernicola Marseille, France A. cavernicola (FRA)A. cavernicola Isola del Giglio, Italy A. cavernicola (ITA)Aplysina sp. Limski kanal, Croatia Aplysina sp. (CRO)A. fistularis Cat Island, Bahamas A. fistularisA. archeri Little San Salvador, Bahamas A. archeriA. cauliformis Little San Salvador, Bahamas A. cauliformisA. insularis Little San Salvador, Bahamas A. insularisA. fulva Sweetings Cay, Bahamas A. fulva

Table 1: Listing of the analysed Aplysina species with origin and the abbreviations used in this paper.

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Bayesian analysis

The hierarchical Akaike information criterion (AIC), which is implemented in MrModeltest 2.2, was used for calculation of models of nucleotide substitution (Posada and Crandall 1998). The AIC was chosen instead of the hierarchical likelihood ratio test (hLRT) because it imposes a disadvantage for model complexity resulting in models with better predictive accuracy (Sober 2002).

The HKY model was chosen as the best fit model and the parameters were used for the calculation in MrBayes 3.1.2. Four Markov chains were run for one million generations and sampled every 100 generations to generate a posterior probability distribution of 10.001 trees. Posterior probabilities were calculated by constructing a 50% majority rule consensus tree of the stationary trees (i.e. trees saved after “burn-in” trees are excluded).

Neighbour-Joining analysisAccessorily to the Bayesian approach we carried out a

neighbour-joining analysis (NJ) using PAUP*4.0b10. To find the best model of DNA substitution we used Modeltest 3.7. The HKY+I was the best-fit model and the parameters of this model were used for the subsequent neighbour-joining analysis.

All trees were rooted by the outgroup Pseudoceratina sp. and displayed by using TreeView 1.6.6 (Page 1996).

Results

itS-1For the ITS-1 region we obtained a total of 270 bp. Two

deletions are present in clone 2 between position 42 to 47 with six bp as well as in position 133 to 163 with 31 bp and one insertion in position 216 to 219 in clone three. Altogether 8 bp are exchanged in all three clones (Fig. 3).

itS-2For the ITS-2 region we obtained a sequences length

between 209 and 274 bp for A. aerophoba (CRO), A. aerophoba (FRA), A. cavernicola (CRO) and A. cavernicola (FRA) specimens. In the case of A. aerophoba (FRA), A. cavernicola (FRA) and A. cavernicola (CRO), we detected intra-individual polymorphism at the ITS-2 region within one individual. Clone 2 of A. cavernicola (FRA) displays an insert of 61 bp at position 124 to 184 (Fig. 4A), which does not occur in the other two clones. In the case of A. aerophoba (FRA) a deletion of 25 bp is present (Fig. 4B). Furthermore, clone 3 shows another deletion of 6 bp at position 117 to 123 (Fig. 4B). For A. cavernicola (CRO), we found a total of 12 base pair exchanges and one deletion of six base pairs in the poly-C region at position 152 to 157 in the clone 3 (Fig. 4C). The specimen A. aerophoba (CRO) displays only one deletion of two base pairs in the poly-C region of the clone 3 (Fig. 4D).

Fig. 3: Alignment of the three sequenced ITS-1 clones of A. cavernicola, Giglio.

Table 2: Molecular markers analysed in the present study for species of the genus Aplysina.

Sample/Origin ITS-1 ITS-2 12S 16S COI

A. aerophoba (CRO) ● ● ● ●Aplysina sp. (CRO) ●A. cavernicola (CRO) ● ● ● ●A. cavernicola (ITA) ● ●A. cavernicola (FRA) ● ●A. aerophoba (FRA) ● ● ● ●A. aerophoba (SPA) ●A. fistularis ●A. archeri ●A. cauliformis ●A. insularis ●A. fulva ●Pseudoceratina sp. ●

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12SIn case of the mitochondrial 12S rRNA we obtained a

partial sequence with 966 bp for A. aerophoba (FRA), A. aerophoba (CRO) and A. cavernicola (CRO). No base pair exchanges are present in this region (data not shown, see GenBank entries).

16SAdditionally, we analysed the mitochondrial 16S rRNA

and obtained a partial sequence of 707 bp for A. aerophoba (FRA), A. aerophoba (CRO) and A. cavernicola (CRO). All three sequences are identical (data not shown, see GenBank entry).

CoiA BLAST search for the COI sequences in GenBank

revealed A. fistularis, Aiolochroia crassa, Axos cliftoni and Chondrosia sp. sequences as closest matches. No contaminating sequences were found.

Analysis of sequence variations within the COI gene among the seven Aplysina species displayed six phylogenetically informative sites. The transition to transversion substitution ratio was 1.0 (3/3). All six informative sites represent substitutions in the third position of the respective codon. None of these base pair exchanges resulted in an amino acid substitution (Table 3: upper section). All samples analysed of the genus Aplysina, regardless of their species have the same amino acid sequence (alignment not shown).

Table 3 (lower section) displays pairwise base pair exchanges between all the species (including pairwise comparisons between specimens of the same species but from different locations). The most exchanges are present between A. archeri and A. cavernicola (CRO, ITA, and FRA) with 5 bp. This represents an exchange rate of 0.75% between these species. The lowest substitution rate is found between the Caribbean species A. fistularis, A. cauliformis, A. fulva and A. insularis with 0 bp. The exchange rate between the Mediterranean species A. aerophoba (CRO, SPA) and A. cavernicola (CRO, FRA, ITA) is 1 bp with a substitution rate of 0.15%. Between the A. aerophoba specimens from Madeira and Banyuls-sur-mer occurs 1 bp respectively 2 bp differences to the A. aerophoba individuals from Croatia and Spain. This conforms an exchange rate of 0.15% and 0.30%.

The phylogenetic trees calculated with the Bayesian approach and the neighbour-joining analyse display identical topologies (Fig.5A, B). A. archeri represents a basal branch within the genus and is separated from the other Caribbean species A. fistularis, A. fulva, A. insularis and A. cauliformis. There are no sequence differences between the later four species. In the case of the Mediterranean species A. aerophoba and A. cavernicola, the situation is complicated. All three A. cavernicola specimens (CRO, ITA, FRA) form a single clade

in contrast to the A. aerophoba individuals analysed here: The specimens of A. aerophoba from Croatia and Spain group together, while the specimens of A. aerophoba from Madeira and Banyuls-sur-mer are separated from the others and even display differences among each other.

Discussion

This is the first time that different molecular markers are analysed for their usefulness in species discrimination of sponges within a genus. For this purpose, seven species of the genus Aplysina were collected in the Mediterranean Sea, East, and West Atlantic Ocean spanning a distance of around 9.000 km apart.

In the case of the internal transcribed spacer regions (ITS-1 and ITS-2) our results for the genus Aplysina showed a high intra-individual variability. The ITS-1 and ITS-2 region has been frequently used for intra- and interspecific relationships in corals and sponges (van Oppen et al. 2002, Wörheide et al. 2002b, Duran et al. 2004a). For the Mediterranean species of the genus Aplysina (A. aerophoba and A. cavernicola) we found in both regions intragenomic variations like Wörheide et al. (2004) have described. Inserts as well as deletions occur in both species. The largest insert was found in A. cavernicola from Marseille with 61 bp. In the specimen from Marseille, a deletion with 21 bp occurred. There is no general pattern behind the inserts and deletions, so it is not possible to differentiate the Mediterranean Aplysina species with the ITS-1 and ITS-2 region. Principally, this polymorphism does not exclude ITS sequences from the list of suitable genetic markers for species discrimination. However, in order to accurately use such a polymorphic marker, it would be necessary to sequence a high number of clones from every single specimen, in order to reach a saturation of all present sequences. Only then, the corresponding sequences could be used for phylogenetic analyses. This is a very expensive and timely endeavour.

We also tested part of the mitochondrial 12S and 16S rDNA for the purpose of species discrimination. In contrast to the ITS sequences, both coding rDNA regions are highly conserved and no basepair exchanges have been found in the two Aplysina species analysed. Therefore, as shown the mitochondrial 12S and 16S regions are also not adequate for species differentiation. However, there could be the possibility for using these markers at the family or genus level like it has been used in damselfishes and shrimps (Jang-Liaw et al. 2002, Quan et al. 2004, respectively).

A more promising marker for species discrimination in sponges seems to be COI. Its usefulness for population and biogeography studies had tested only in two sponge species so far: Crambe crambe and Astrosclera willeyana. In both cases, the variability was not sufficiently high enough for population and phylogeographical analyses (Duran et al. 2004b, Wörheide 2005). Recent studies in higher taxa like birds, fishes and butterflies show that the differences between closely related species is 18 time higher than within the species (Hebert et al. 2004, Ward et al. 2005, Hajibabaei et al. 2006). In addition, a recent study on the sponge genus tethya, provided promising results with clear species discrimination (Heim et al. 2007).

Fig. 4: Alignment of the three sequenced ITS-2 clones of A. aerophoba, Limski kanal, Croatia (A), A. aerophoba, Banyuls-sur-mer, France (B), A. cavernicola, Limski kanal, Croatia (C) and A. cavernicola, Marseille, France (D).

368

A. c

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RA

A. c

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la, I

TAA

. cav

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, CR

O

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, CR

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CR

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. aer

opho

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PA

A. a

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, FR

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A. c

aulif

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A. i

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aris

A. a

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A. cavernicola, FRAA. cavernicola, ITAA. cavernicola, CRO

0 0 0 0 0 0 0 0

Am

ino

acid

exc

hang

esA. aerophoba, CROAplysina sp., CROA. aerophoba, SPA

1 0 0 0 0 0 0 0

A. aerophoba, FRA 3 2 0 0 0 0 0 0

A. aerophoba, POR 2 1 1 0 0 0 0 0

A. fistularis 3 2 1 1 0 0 0 0

A. fulva 3 2 1 1 0 0 0 0

A. cauliformis 3 2 1 1 0 0 0 0

A. insularis 3 2 1 1 0 0 0 0

A. archeri 5 4 4 3 2 2 2 2

Nucleotide base pair exchanges

Table 3: Amino acid exchanges (upper section) and base pair exchanges (lower section) of the COI between two different Aplysina species.

Clades within the phylogenetic tree of Aplysina specimens partly correspond to geographical locations A. archeri is basal to all other species analysed here. The Western Atlantic species A. insularis, A. cauliformis, A. fulva and A. fistularis display identical sequences, even though they represent, based on morphological characters, clearly identifiable species. In the context of the clear species discrimination in the genus tethya, we may conclude that the radiation of these four Aplysina species started only recently. As a consequence, the morphology changed, but no base pair exchange in the COI occurred up to now. The West Atlantic Aplysina species are relatively young and seem to represent sexually compatible taxa (Schmitt et al. 2005). They also mentioned a low resolution in the COI within the genus Aplysina. In contrast, the situation is more difficult amongst he European specimens of Aplysina. Despite all sequence similarities, the present differences in the COI sequence are sufficient to separate at least A. cavernicola from specimens which are regarded as A. aerophoba. This is maintained through sequencing different clones from every individual and we can find the same base pair exchange in the position 607 in different geographical localities (Rovinj/Croatia, Marseille/France, and Giglio/Italy). They also show all the same transversion. This supports the hypothesis that A. cavernicola is a true species (Vacelet 1959) and not an ecological variant of A. aerophoba (Voultsiadou-Koukoura 1987).

Both trees support the possibility to identify A. cavernicola regardless of geographic origin using COI sequences.

However, this seems not to be the case for specimens regarded as A. aerophoba. This species came out to be problematic. The species A. aerophoba sensu morphology does not form its own genetic clade of itself, but two separate groups, of which the specimens from Croatia and Spain form a branch together with A. cavernicola. The A. aerophoba specimens from France and Portugal branch out more basally. There are two possible scenarios: either all A. aerophoba analysed here represent one species. In this case, A. cavernicola would consequently be no valid species as suggested earlier in the literature (Voultsiadou-Koukoura 1987). However, due to the clear identity of A. cavernicola sensu morphology and sensu COI, a second scenario seems to be more likely: A. aerophoba sensu morphology is not a single valid species, but a cluster of species. Aplysina sp. (CRO), which displays secondary substances both from A. aerophoba and A. cavernicola, shares its complete COI sequence with the A. aerophoba CRO and SPA. It had been speculated before whether it is a hybrid of both species because in the Limski kanal near Rovinj/Croatia both A. aerophoba and A. cavernicola overlap in the inhabiting environment. However, this is not verifiable with the COI sequence. This group of three identical genotypes would represent a sister species of A. cavernicola.

In contrast, the samples identified as A. aerophoba POR and FRA most likely represent one or even two distinct species. This cannot be decided from the present dataset. By any means, our result calls for a complete revision of the European Aplysina species, to resolve the emerging

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problem of A. aerophoba. Beside careful morphological and histological studies, the application of bulk ITS-sequencing might help, if a saturation of the intra-individual alleles is reached as discussed above. The same might be true for the West Atlantic group.

All base pair exchanges found in COI took place at the third codon position. Consequently, no mutations are present in the amino acid sequences of any species we analysed from West and East Atlantic Ocean and Mediterranean Sea. It is not clear yet why most sponge genera display such a low mutation rate in the COI. Contrary to the Aplysina species we have found for the genus tethya a good resolution for species discrimination with the COI (Heim et al. 2007). Studies on octocorals show also a low mutation rate in the mitochondrial DNA (van Oppen et al. 1999) but this is probably caused by a mismatch repair system homologue to the bacterial system (Pont-Kingdon et al. 1998). Although possible, until now there is no evidence for such a mismatch repair system in sponges

(Lavrov et al. 2005, Lavrov and Lang 2005). Furthermore the low mutation rate could be avowed through the variable environmental conditions Aplysina species living in, because mitochondrial evolution is advantaged by relaxed selection pressure (Quesada et al. 1998).

To conclude, it was not possible to differentiate the Mediterranean Aplysina species with the ITS’s-regions without massively increasing the number of sequencings per individual. Also, for the 12S and 16S, a species differentiation is not probable, because of a high genetic similarity between the species. But maybe they are useful at the family or genus level. Nevertheless, for COI the discrimination between A. aerophoba and A. cavernicola is feasible, despite lower differences in their sequences in comparison to those between the species of the genus tethya (Heim et al. 2007). It is always necessary to check the COI for its usefulness in species discrimination. Additionally, the same is to be considered for the other tested molecular markers.

Fig. 5: A. 50% majority rule consensus phylogram of the stationary trees obtained from the Bayesian analysis of COI sequences. Posterior probabilities are shown below branches. B. Neighbour-joining tree calculated with PAUP*4.0b10. Bootstrap values higher than 50% are plotted above branches.

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Acknowledgements

We would like to thank Renato Batel (Institut Ruđder Bošković, Rovinj), Ute Hentschel (University Würzburg), Isabel Koch (Wilhelma Stuttgart) and Wolfgang Zucht (University Stuttgart) for providing sponge specimens. Gisela Fritz (Universität Stuttgart, Germany) for critical reading of our manuscript and two anonymous reviewers greatly helped to improve the manuscript. This study was supported by the project BioteCmarin (03F0414D) funded by the German Federal Ministry of Education and Research and the University of Stuttgart.

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