A molecular phylogeny and historical biogeography of the marine sponge genus Placospongia (Phylum Porifera) indicate low

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A molecular phylogeny and historical biogeography of the marine sponge genus Placospongia (Phylum Porifera) indicate low

dispersal capabilities and widespread crypsis

Scott A. Nichols

^a,

T , Penelope A.G. Barnes

^b,1

aUniversity of California, Department of Integrative Biology, 3060 Valley Life Sciences Building, Berkeley, CA 94123, USA

bSmithsonian Tropical Research Institute, P.O. Box 2072, Balboa, Republic of Panama Received 14 September 2004; received in revised form 21 January 2005; accepted 20 February 2005

Abstract

Very little is known about the dispersal of marine sponges but hypotheses based upon observations of larval life suggest limited dispersal ability. Until recently, the diversity of dispersal mechanisms employed by different species, coupled with the putative cosmopolitan distribution of some species, suggested that short larval life may not limit sponge dispersal. However, recent studies have found thatbcosmopolitan speciesQfrequently represent morphologically cryptic species complexes. Here, the historical biogeography of the circum-tropical marine sponge genusPlacospongiais investigated using sequence data from the internal transcribed spacer region (ITS). ITS is the predominant marker used for fine-scale investigations in sponges (and many other taxa), but problems with paralogy (divergent copies within individual genomes) have brought the utility of this marker into question. Ninety-eight ITS subclones were sequenced from twenty-eight individuals ofPlacospongiasampled from twelve localities worldwide. Divergent ITS paralogs were detected but the data still reflected geographic (and presumably phylogenetic) structure. Placospongia populations sampled were found to harbor at least 9 unique evolutionary lineages, several of which are sympatrically distributed but morphologically indistinguishable. Most of the relationships reconstructed are consistent with the hypothesis that marine sponges do not disperse very far (by any means). Nevertheless, phylogenetic divergences between lineages were not clearly consistent with modern, or historical, geological or oceanographic processes. No component of the phylogeny presented clearly reflected the geological event of the rising of this Isthmus of Panama and populations from marine lakes in Indonesia were found to be indistinguishable from populations in the Seychelles. These biogeographically anomalous results highlight that ITS is useful as a tool to identify interesting preliminary patterns in widespread groups, but that these patterns should be tested using multiple independent loci.

Keywords:Biogeography; Cosmopolitanism; Crypsis; Dispersal; Phylogeny; Porifera

doi:10.1016/j.jembe.2005.02.012

TCorresponding author. Tel.: +1 510 643 9417; fax: +1 510 642 1822.

E-mail addresses:[email protected] (S.A. Nichols), [email protected] (P.A.G. Barnes).

1 Present address: Centre for Shellfish Research, Malaspina University, 900 Fifth Street, Nanaimo, B.C., Canada V9R 5S5. Tel.: +1 250 740 6302; fax: +1 250 740 6482.

www.elsevier.com/locate/jembe

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1. Introduction

The dispersal curve of most marine organisms is poorly characterized (Kinlan and Gaines, 2003), and this is particularly true of sponges. There is widespread opinion that sponges do not disperse far (Maldonado and Young, 1996; Lindquist et al., 1997; Uriz et al., 1998; Maldonado and Bergquist, 2002), despite the fact that different sponge species exhibit a range of reproductive strategies (Maldonado and Bergquist, 2002) and life histories that possibly result in a range of dispersal outcomes. The existence of sponge species with cosmopolitan distributions has stood in apparent contradiction to the prediction that sponges are capable of only short-distance dispersal but a number of studies have demonstrated that cosmopolitanism is often just a case of bover- conservative systematicsQ due to the paucity of morphological characters useful for fine-scale species discrimination (Sole-Cava and Thorpe, 1986; Boury- Esnault et al., 1992; Sole-Cava et al., 1992; Klautau et al., 1994, 1999; Muricy et al., 1996; Wo¨rheide et al., 2003). Most cosmopolitan species [with notable exceptions as given in Lazoski et al. (2001)], when evaluated using molecular genetic techniques, are found to encompass cryptic species diversity and high levels of population genetic structure on geographic scales more compatible with hypotheses of limited dispersal ability (Sole-Cava and Thorpe, 1986; Boury- Esnault et al., 1992; Sole-Cava et al., 1992; Klautau et al., 1994, 1999; Muricy et al., 1996; Wo¨rheide et al., 2003). In fact, morphologically cryptic species are hypothesized to be more prevalent among sponges than other taxa due to the simplicity and plasticity of their morphological characters (Klautau et al., 1999).

Phylogeographic and population genetic methods that utilize molecular sequence data offer a promising means to address questions about dispersal potential and cryptic speciation in marine invertebrate taxa, including sponges (for review, see van Oppen et al., 2003). However, the successful application of molecular methods to population and species-level questions in sponges requires markers that evolve at fast enough rates to reflect recent divergences. Mitochon- drial genes frequently are used for such studies in other taxa because they typically are maternally inherited and are expected to undergo lineage sorting three times faster than nuclear markers that evolve at

similar rates (Palumbi et al., 1998). However, several studies indicate that the standard mitochondrial markers are too conserved to be very useful for fine- scale analyses in sponges (Erpenbeck et al., 2002;

Shearer et al., 2002; van Oppen et al., 2003; Duran et al., 2004). Although several potentially useful single- copy nuclear markers have been published for other metazoan taxa (e.g., Jarmon et al., 2002), these markers have not been identified in sponges nor has their phylogenetic utility been tested. The best markers that have emerged for fine-scale analyses in sponges, and many other taxa, are the internal transcribed spacer regions (ITS) of tandemly-repeated nuclear ribosomal clusters (van Oppen et al., 2003).

The present study uses sequence data from the internal transcribed spacer regions (ITS) as a preliminary means to make inferences about the dispersal potential and evolutionary history of the genus Placospongia. Placospongia is a marine sponge genus with a circum-global distribution in tropical and some subtropical environments. A number of distinct species have been named from different regions globally, but it recently has been suggested that most of these species are synonyms and perhaps as few as three valid species names should remain (Ru¨tzler, 2002). Indeed, standard morphological examination reveals few fixed differences between taxa from very distant geographic regions. There is no direct evidence for the mode of reproduction or larval development in Placospongia, but, based upon its phylogenetic position (Nichols, 2005), we predict that members of this genus are oviparous (although eggs may already be fertilized upon release) and that they produce a clavablastula larva that has either a crawling or planktonic dispersal stage of unknown duration (see Maldonado and Bergquist, 2002). In general, most planktonic sponge larvae settle within a few days (Maldonado and Bergquist, 2002). In terms of dispersal potential, therefore, it is impossible to predict where on the dispersal spectrum that Placo- spongia falls. However, the morphological homoge- neity of samples collected globally supports a high- dispersal scenario. The specific aims of this study are to, 1) test for the presence of morphologically cryptic lineages within and between geographic populations ofPlacospongia; 2) infer the historical biogeographic patterns ofPlacospongiain the context of a molecular phylogenetic hypothesis; 3) evaluate ITS as a pre-

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liminary tool for framing phylogenetic and historical biogeographic hypotheses; 4) speculate about the dispersal ability of members ofPlacospongia.

2. Materials and methods

2.1. Specimen collection and preservation

Twenty-eight individuals ofPlacospongiasp., two individuals ofSpirastrella hartmani, one individual of Spirastrella sabogae, and one individual ofPolymas- tiasp. were sampled for this study. Samples collected by the authors were fixed in 90–100% EtOH upon collection and voucher specimens have been deposited at the University of California, Museum of Paleontology (see Table 1). Samples acquired from other localities were borrowed from the institutions indicated in Table 1. These samples are deposited at the Zoological Museum Amsterdam (POR numbers), Queensland Museum, Brisbane (G numbers), the Coral Reef Research Foundation (OCDN numbers), or the Western Australian Museum, Perth (WAMZ numbers) (Table 1). Furthermore, genomic DNA for all samples is available upon request to one of us (S.A. Nichols).

2.2. DNA extraction, PCR, sub-cloning, sequencing DNA was extracted using the bAnimal TissuesQ protocol included with the DNEasyRTissue Kit from QIAGEN, Inc. Because sponge spicules clogged mini- column filters, a single centrifugation step was added after the initial incubation/tissue lysis period in order to remove sponge spicules.

A fragment of nuclear DNA including partial 18S, ITS1, 5.8S, ITS2, and partial 28S rDNA was amplified for all taxa (Table 1) using RA2/ITS2.2 PCR primers (Wo¨rheide, 1998). PCR reactions (25 AL) consisted of 2.5AL 10PCR Buffer II (Applied Biosystems), 1.25AL MgCl2(25 mM), 1AL DMSO, 1.25 AL dNTP mix (2.5 mM each), 0.25 Ag of each primer, 0.625 Units of Amplitaq GoldR (Applied Biosystems), and varying concentrations of water and template depending upon the taxa being amplified.

PCR reactions were performed on a PTC-200 DNA EngineR (MJ ResearchR) under the following conditions: initial denaturation at 958C for 10 min; 35

cycles of 958C for 1 min, 458C for 1 min, 728C for 1 min; a final extension step of 728C for 7 min; storage at 4 8C. Products were cloned into pCRR4Blunt- TOPORvector using the Zero BluntRTOPORPCR Cloning Kit for Sequencing (Invitrogen). Colonies were picked for direct PCR following manufacturer specifications, amplified products were purified using ExoSAP-ITR (USB Corporation) and sequenced using dye-labeled dideoxy terminator cycle-sequencing (Applied Biosystems). Cycle-sequenced products were purified using isopropanol precipitation (75%) and analyzed using an ABI PRISMR 377 automated DNA sequencer and associated software programs (Applied Biosystems).

2.3. Sequence alignment

Sequences were aligned manually in Se-Al (Ram- baut, 1996) and because different taxa exhibited variation in gene length, insertion/deletion events had to be inferred in the sequence alignment. Some regions were too divergent to confidently align between all taxa but that were informative about relationships between more closely related taxa. These regions were included in the analysis because the error in homology assign- ment was not systematic meaning that the phylogenetic bnoiseQ should be swamped by non-random dsignalT from unambiguously aligned regions.

Because no repeatable criteria were invoked in the determination of which regions were divergent or ambiguously aligned in the manual alignment using Se-Al, the alignment used for phylogenetic analyses was tested for its sensitivity to misaligned regions identified using the program Gblocks (Castresana, 2000). Gblocks parameters were defined as follows:

minimum number of sequences for a conserved position: 56; minimum number of sequences for a flanking position: 94; maximum number of contig- uous non-conserved positions: 8; minimum length of a block: 10; allowed gap positions: with half.

All sequences are available on Genbank (Table 2) and the alignment is available upon request from one of us (S.A. Nichols).

2.4. Analysis of intragenomic variation

Because the ITS regions of nuclear ribosomal operons can exhibit intragenomic variation in sponges

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and other organisms (see Alvarez and Wendel, 2003) multiple sub-clones were sequenced for each individual included in this study (Table 1). All unique sub-clones from each individual were divided into five partitions corresponding to the sampled 18S, ITS1, 5.8S, ITS2, and 28S regions of the ribosomal repeats. Nucleotide diversities were calculated using all clones for each of these

divisions per individual using the software program Matrix. The analysis of nucleotide diversities will allow these data to be related to a recent study (Wo¨rheide et al., 2004) that surveys diverse sponge lineages for their levels of intragenomic variation and makes recommendations for dealing with such variation when ITS is used for phylogenetic studies.

Table 1

Specimen locality and accession information

Loc. Museum accession 18sp ITS1p 5.8sp ITS2p 28sp Clones Sequence-type groups Placospongiasp.

2 UCMPWC860 0 0.0027 0 0 0 3 A: 1, 2

2 UCMPWC495 0 0.0030 0.0011 0.0010 0 11 B: 64, 65, 70, 72, 76

2 UCMPWC866 0 0.0014 0 0.0018 0.0064 6 C: 65, 67, 69, 73

2 UCMPWC873 N/A N/A N/A N/A N/A 1 D: 37

2 UCMPWC896 0 0.0029 0.0042 0.0107 0 3 E: 66, 68, 71

3 UCMPWC863 0.0046 0.0082 0.0018 0.0061 0 7 F: 41, 42, 43, 44, 45, 46, 54

3 UCMPWC864 0.0020 0.0103 0.0021 0.0082 0 6 G: 48, 49, 50, 51, 61

3 UCMPWC865 0 0.0057 0.0083 0 0 3 H: 74, 75

3 UCMPWC498 0.0024 0.0026 0.0083 0.0064 0 10 I: 36, 47, 55, 56, 57, 58, 62, 63

3 UCMPWC872 0.0060 0 0 0 0 2 J: 59, 60

3 UCMPWC874 0 0.0053 0 0.0020 0 7 K: 37, 38, 39, 40, 52, 53

12 POR07643 0 0 0 0 0.0077 2 L: 65

4 POR11818 0 0 0 0 0 6 M: 77

4 POR11367 0.0060 0.0126 0 0 0 2 N: 78, 79

1 UCMPWC548 0.0030 0.0102 0.0015 0.0049 0 16 O: 4, 5, 6, 7, 8, 9, 15, 16, 17,

18, 19, 20, 21

1 UCMPWC899 N/A N/A N/A N/A N/A 1 P: 15

1 UCMPWC900 0.0080 0.0110 0 0.0066 0 6 Q: 3, 10, 11, 12, 13, 14

1 UCMPWC902 0.0069 0.0054 0 0 0.0055 7 R: 22, 23, 24, 25, 26, 27

9 UCMPWC963 0.0020 0 0 0 0 6 S: 28, 29, 30, 31

9 POR13097 0.0050 0 0 0 0.0096 4 T: 32, 33, 34, 35

7 UCMPWC1093 0 0.0145 0.0083 0.0277 0 3 U: 93, 94, 95

8 UCMPWC1092 N/A N/A N/A N/A N/A 1 V: 80

8 UCMPWC1094 0 0 0 0 0 2 W: 81

11 QM317896 0 0.0084 0 0.0118 0 7 X: 96, 97, 98

6 QM303439 0 0.0057 0 0 0.0128 3 Y: 82, 83, 84

5 UCMPWC1001 0 0.0060 0 0.0031 0 5 Z: 85, 86, 87

5 UCMPWC1002 0.0602 0.0791 0.0025 0.1217 0 4 AA: 87, 88, 89, 90

10 OCDN8188K 0 0.0087 0 0.0052 0 2 AB: 91, 92

Spirastrella hartmani

N/A UCMPWC871 0.0060 0 0 0 0 2 107, 108

N/A UCMPWC879 0 0.0029 0.0051 0.0105 0.0077 5 102, 103, 104, 105, 106

Spirastrella sabogae

N/A UCMPWC861 0 0.0064 0 0.0106 0 3 99, 100, 101

Polymastiasp.

N/A WAMZ3927 0 0 0 0.0263 0.0128 3 109, 110, 111

Nucleotide diversities (p) are shown for each region sequenced per individual. Sequence-types are arranged into groups labeled A–AB so that the individual they are derived from can be referenced inFig. 2.

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2.5. Phylogenetic analyses

In all phylogenetic analyses, gaps were treated as missing data. A maximum parsimony analysis was conducted for the full alignment of all unique sequence-types and the Gblocks alignment with equal character weighting invoked. Outgroup taxa were chosen based upon the results of a broader- scale phylogenetic analysis that included exemplars from the genus Placospongia (Nichols, 2005). Data were analyzed using PAUP’s (4.0 beta 10,Swofford, 2001) heuristic search algorithm, TBR branch-swap-

ping, and 1000 random addition sequence replicates for the complete alignment and 100 random addition sequence replicates for the Gblocks alignment.

Swapping was constrained to a maximum of 100 trees per replicate due to computational limitations resulting from the inclusion of many very similar sequence-types. It was considered more important to increase the number of random starting seeds than to swap on every most-parsimonious tree found during any single replicate. The resulting most-parsimonious trees were used to calculate a strict consensus tree.

Table 2

Genbank accession numbers for all unique sequence-types presented herein

Sequence-type ID Genbank accession Sequence-type ID Genbank accession Sequence-type ID Genbank accession

1 AY561988 38 AY562025 75 AY562062

2 AY561989 39 AY562026 76 AY562063

3 AY561990 40 AY562027 77 AY562064

4 AY561991 41 AY562028 78 AY562065

5 AY561992 42 AY562029 79 AY562066

6 AY561993 43 AY562030 80 AY562067

7 AY561994 44 AY562031 81 AY562068

8 AY561995 45 AY562032 82 AY562069

9 AY561996 46 AY562033 83 AY562070

10 AY561997 47 AY562034 84 AY562071

11 AY561998 48 AY562035 85 AY562072

12 AY561999 49 AY562036 86 AY562073

13 AY562000 50 AY562037 87 AY562074

14 AY562001 51 AY562038 88 AY562075

15 AY562002 52 AY562039 89 AY562076

16 AY562003 53 AY562040 90 AY562077

17 AY562004 54 AY562041 91 AY562078

18 AY562005 55 AY562042 92 AY562079

19 AY562006 56 AY562043 93 AY562080

20 AY562007 57 AY562044 94 AY562081

21 AY562008 58 AY562045 95 AY562082

22 AY562009 59 AY562046 96 AY562083

23 AY562010 60 AY562047 97 AY562084

24 AY562011 61 AY562048 98 AY562085

25 AY562012 62 AY562049 99 AY562086

26 AY562013 63 AY562050 100 AY562087

27 AY562014 64 AY562051 101 AY562088

28 AY562015 65 AY562052 102 AY562089

29 AY562016 66 AY562053 103 AY562090

30 AY562017 67 AY562054 104 AY562091

31 AY562018 68 AY562055 105 AY562092

32 AY562019 69 AY562056 106 AY562093

33 AY562020 70 AY562057 107 AY562094

34 AY562021 71 AY562058 108 AY562095

35 AY562022 72 AY562059 109 AY562096

36 AY562023 73 AY562060 110 AY562097

37 AY562024 74 AY562061 111 AY562098

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Bootstrap support values were obtained using PAUP’s dfast-bootstrapT option with 10,000 pseudoreplicates.

These values have been shown to conservatively approximate the values obtained from a full nonpara- metric bootstrap analysis (Mort et al., 2000), which could not be completed due to computational constraints.

The alignment was evaluated further for its fit to different models of nucleotide substitution using Mod- eltest v.3.06 (Posada and Crandall, 1998), and analyzed using Mr. Bayes 3.0 (Huelsenbeck and Ronquist, 2001).

Two nucleotide substitution types were specified, allowing transitions and transversions to have potentially different rates. A gamma (C) distribution of among-site rate heterogeneity was specified to have four rate categories. No initial values were assigned to the model parameters and empirical nucleotide frequencies were used. Four Markov chains were run for a million generations and sampled every 100 generations to yield a posterior probability distribution of 10,001 trees.

Posterior probabilities were calculated by constructing a 50% majority-rule consensus of the trees retained after dburn-inTtrees were excluded.

Biogeography was mapped onto the cladogram using MacClade v.4.06 OS X (Maddison and Maddi- son, 2000). Character state transformations were evaluated under both ACCTRAN and DELTRAN

models of maximum parsimony character state reconstruction. When the nodal conditions predicted under these models were in conflict, the reconstructed character state was deemed equivocal.

3. Results

3.1. Levels of intragenomic variation

Nucleotide diversities (p), calculated per region sequenced for all individuals, are shown in Table 1.

Most individuals exhibit intragenomic variation in either the 18S, ITS1, 5.8S, ITS2, or 28S rDNA regions. WithinPlacospongia, each region exhibits an average nucleotide diversity of 0.0042, 0.0082, 0.0015, 0.0087, and 0.0017 respectively. Both ITS regions exhibit the highest levels of intragenomic variation with ITS2 showing slightly more variation, on average, than ITS1. Sequence-type diversity is very high, with 136 sequenced sub-clones yielding 98 unique sequence-types withinPlacospongia. Most of the sequence-types from any one individual or within individuals from any one geographic region are nearly identical, exhibiting only one to several absolute differences (Fig. 1).

1 2

3

1. Baja, Mexico 2. Panama, Pacific 3. Panama, Caribbean 4. Seychelles 5. New Brittain, PNG 6. Brisbane, Australia 7. Berau, Indonesia (reef) 8. Kakaban & Maratua lakes 9. Sulawesi, Indonesia 10. Palau

11. Solomon Islands 12. Grenada

7, 8 4

5 11 10

6 9

12

Fig. 1. Locality Map.

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3.2. Phylogenetic analyses

Results of both the maximum parsimony and Bayesian analyses of the complete alignment are summarized inFig. 2and the result of the maximum parsimony analysis of the Gblocks alignment is shown inFig. 3. In the maximum parsimony analysis of the complete alignment, 318 out of 926 aligned base pairs were parsimony informative and 100,000 most-parsimonious trees of length 653 were found. In the maximum parsimony analysis of the Gblocks alignment, 151 out of 644 aligned base pairs (69% of the original dataset) were parsimony informative and 10,000 most-parsimonious trees of length 312 were found. A 50% majority-rule consensus was con- structed for 8000 trees obtained from the Bayesian analysis after 2001dburn-inTtrees were excluded.

3.2.1. General tree features

When alignment regions characterized as bambi- guously alignedQ using the criteria designated in Gblocks were excluded, the topology remained unchanged (the position of clade C2 changed slightly, but could be further resolved to agree) but support values diminished slightly as compared to the complete alignment with no positions excluded (see Figs. 2 and 3).

Analyses of the complete alignment using both parsimony and Bayesian optimality criteria resulted in very similar topologies with concordant bootstrap and posterior probability values. In fact, no conflicting relationships were reconstructed using these methods, although the strict consensus tree obtained using maximum parsimony is slightly less resolved at the tips (not shown). In general, most sequence-types form monophyletic groups that correlate well to the geographic regions in which they were sampled.

However, divergent sequence-types sampled from within each individual do not cluster phylogenetically, rather, there is very little resolution between sequence- types sampled from different individuals in any single geographic area. The exceptions to this are cryptic lineages that are co-distributed.

3.2.2. Number of discrete evolutionary lineages Using the criterion of monophyly to identify discrete evolutionary lineages, at least 9 independent lineages are identified (Fig. 2). These lineages are not

interpreted as distinct species because independent corroborative data are lacking. Also, the discrete lineages identified are interpreted as minimum esti- mates of diversity because too few samples were collected from most localities to suggest that all unique lineages were sampled.

As per Fig. 2, one discrete lineage is identified along the Pacific coast of Panama (C1), one lineage is identified in the Caribbean Sea of Panama (C7), and one lineage is found in both oceans (C8). One lineage is present in each of the Sea of Cortez, Baja California Mexico (C2) and Sulawesi, Indonesia (C9), and sequence-types from the individual sampled from Brisbane, Australia form a monophyletic clade (C6).

Samples from Papua New Guinea, Palau, and Berau Indonesia (reef) represent at least one lineage (C5), as do samples from the Seychelles and Kakaban and Maratua marine lakes, Indonesia (C4). Sequence- types from the single individual sampled from the Solomon Islands form a single, divergent clade (C3).

Lineage boundaries are difficult to distinguish when two reciprocally monophyletic groups are reconstructed within a single geographic region (e.g., C7 and C8) or when geographically disjunct populations form a single clade with little divergence observed between regions (e.g., C5 and C6).

Despite the fact that only the sequence-types from a single individual from the Caribbean side of Panama were found to be divergent from (and reciprocally monophyletic with) sequence-types in the larger dPanamicT clade, high bootstrap and posterior probability values support this division.

Furthermore, sequence-types within the Panamic clade were found to be 0.38% divergent from each other (on average), the two sequence-types from the Caribbean Placospongia lineage (UCMPWC872) were found to be 0.11% divergent from each other, whereas comparisons between these two clades revealed sequence-type divergences that averaged 2.59%. These data are interpreted as support for the hypothesis that there are two unique lineages of Placospongia in the Caribbean.

Sequence-types from the individual sampled from the reef environment of Berau Indonesia (as opposed to the Indonesian marine lakes, Kakaban and Maratua) are reconstructed as paraphyletic with respect to samples from Papua New Guinea and Palau, but pair-wise sequence-type divergences are

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A-1 PAN PAC A-2 PAN PAC Q-3 BAJA

O-7 BAJA O-16 BAJA O-4 BAJA O-9 BAJA O-5 BAJA O-6 BAJA O-8 BAJA Q-10 BAJA Q-11 BAJA Q-12 BAJA

Q-13 BAJA Q-14 BAJA O-15 BAJA

O-17 BAJA O-18 BAJA

O-19 BAJA O-20 BAJA O-21 BAJA R-22 BAJA

R-23 BAJA R-24 BAJA R-25 BAJA R-26 BAJA R-27 BAJA

S-28 SUL IND S-29 SUL IND S-30 SUL IND S-31 SUL IND T-32 SUL IND T-33 SUL IND

T-34 SUL IND T-35 SUL IND

I-36 PAN CAR F-46 PAN CAR

I-55 PAN CAR I-56 PAN CAR

I-57 PAN CAR I-58 PAN CAR I-62 PAN CAR I-63 PAN CAR K-37 PAN CAR K-38 PAN CAR K-39 PAN CAR F-41 PAN CAR F-42 PAN CAR F-44 PAN CAR F-45 PAN CAR K-52 PAN CAR E-66 PAN CAR K-40 PAN CAR

I-47PAN CAR K-53 PAN CAR

G-61 PAN CAR H-74 PAN CAR F-43 PAN CAR

F-54PAN CAR B-64 PAN PAC B,C,L-65 PAN PAC / GREN

C-67 PAN PAC E-68 PAN PAC E-71 PAN PAC C-69 PAN PAC B-70 PAN PAC B-72 PAN PAC C-73 PAN PAC B-76 PAN PAC G-48 PAN CAR G-49 PAN CAR G-50 PAN CAR H-75 PAN CAR

G-51 PAN CAR J-59 PAN CAR J-60 PAN CAR Y-82 BRI AUS

Y-83 BRI AUS Y-84 BRI AUS

Z-85 PNG Z-86 PNG Z,AA-87 PNG

AA-89 PNG AA-90 PNG

AB-91 PALAU AB-92 PALAU AA-88 PNG

U-94 BER IND U-93 BER IND U-95 BER IND M-77SEY

N-78 SEY N-79 SEY V-80 LAK IND

V-81 LAK IND X-96 SOL X-97 SOL X98 SOL 99

100 101 102

103 104 105 106 107 108

109 110 5 changes 111

}

Spirastrella sabogae

Spirastrella hartmani

}

Polymastia sp.

100 100

100 87

100 100 97 70

99

73 100

96

96 60 100

93 100

98

98 100 71

99 82 51

100 100 59

51 100

74 100

100

C1

C2

C8

C7 C6

C5

C4 C3

C9

Fig. 2. Summary Phylogram of 8001 post-dburn-inT tree obtained from Bayesian analysis. Values shown above branches are posterior probabilities. Bootstrap values for nodes that were present in the Strict Consensus tree obtained using Maximum Parsimony are indicated below branches. If the node was not reconstructed using maximum parsimony no bootstrap value is shown (–). Taxon labels correspond to the information listed inTable 1and are organized as follows: Specimen–Sequence-type–Locality.

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A-1 PAN PAC A-2 PAN PAC Q-3 BAJA O-4 BAJA O-5 BAJA O-6 BAJA O-8 BAJA O-7 BAJA O-9 BAJA Q-10 BAJA Q-11 BAJA Q-12 BAJA Q-13 BAJA Q-14 BAJA O-15 BAJA O-16 BAJA O-17 BAJA O-18 BAJA O-19 BAJA O-20 BAJA O-21 BAJA R-22 BAJA R-23 BAJA R-24 BAJA R-25 BAJA R-26 BAJA R-27 BAJA S-28 SUL IND S-29 SUL IND S-30 SUL IND S-31 SUL IND T-32 SUL IND T-33 SUL IND T-34 SUL IND T-35 SUL IND I-36 PAN CAR K-37 PAN CAR K-38 PAN CAR K-39 PAN CAR K-40 PAN CAR F-41 PAN CAR F-42 PAN CAR F-44 PAN CAR F-45 PAN CAR F-43 PAN CAR F-54 PAN CAR F-46 PAN CAR I-47 PAN CAR G-48 PAN CAR G-49 PAN CAR G-50 PAN CAR G-51 PAN CAR K-52 PAN CAR K-53 PAN CAR I-55 PAN CAR I-56 PAN CAR I-57 PAN CAR I-58 PAN CAR G-61 PAN CAR I-62 PAN CAR I-63 PAN CAR B-64 PAN PAC

B,C,L-65 PAN PAC / GREN E-66 PAN CAR

C-67 PAN PAC E-68 PAN PAC C-69 PAN PAC B-70 PAN PAC E-71 PAN PAC B-72 PAN PAC C-73 PAN PAC H-74 PAN PAC H-75 PAN CAR B-76 PAN PAC J-59 PAN CAR J-60 PAN CAR Y-82 BRI AUS Y-83 BRI AUS Y-84 BRI AUS Z-85 PNG Z-86 PNG Z,AA-87 PNG AA-88 PNG AA-89 PNG AA-90 PNG AB-91 PALAU AB-92 PALAU U-93 BER IND U-94 BER IND U-95 BER IND M-77 SEY N-78 SEY N-79 SEY V-80 LAK IND V-81 LAK IND X-96 SOL X-97 SOL X-98 SOL 99 100 101 102 103 104 105 106 107 108 109 110 111 100

100

99

<50

65 62

65

90 54

94 99

100

<50

Outgroups

C3 C4 C5 C7

C8 C9

C2 C1

Fig. 3. Strict Consensus topology from a Maximum Parsimony analysis of the Gblocks alignment.bFast-bootstrapQvalues are shown above branches.

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greater between the two groups than within (values averaged 0.19% within the PNG/Palau clade, 0.86%

within the Berau sequence-types, and 1.06% between the two groups). Without better sampling and data from independent loci, samples from these regions are nevertheless interpreted as representative of a single lineage.

Similarly, samples from the Kakaban and Maratua marine lakes of Indonesia are unresolved with respect to their relationship to samples from the Seychelles. In this instance, measurements of sequence-type divergences indicate that these samples represent a single lineage (the average divergence within the marine lakes sequence-types is 0.54%, sequence-types from the Seychelles average 0.29% divergence, and comparisons between the two regions average 0.66%).

Samples from Baja California, Mexico form a basal polytomy along with two sequence-types from a single individual from the Pacific of Panama. Reso- lution of this polytomy could indicate that these clades are sister taxa, in which case their evolutionary independence would not be evident. However, pair- wise comparisons of the sequence-types within and between these clades are interpreted as clear evidence that they are sufficiently divergent as to represent separate lineages (0.47% average divergence within the Baja clade, 0.11% divergence in the Pacific clade, and 7.37% average divergence between the two clades).

3.3. Biogeography

Two localities were sampled in each of the Eastern Pacific Ocean (Baja California, Mexico and the Pacific of Panama) and the Western Atlantic Ocean (the Caribbean of Panama and Grenada). Sixteen individuals were sequenced from these localities, yielding as many as four discrete evolutionary lineages. Likewise, nine localities were sampled in the Indo-Pacific (Seychelles; Solomon Islands; Papua New Guinea; Brisbane, Australia; Maratua and Kakaban marine lakes, Indonesia; Berau, Indonesia (reef); and Sulawesi, Indonesia). Of the twelve individuals sequenced from these localities, at least five discrete evolutionary lineages are apparent.

Three biogeographic patterns are apparent in the data. First, no tree component clearly corresponds to the geological event of the rising of the Isthmus of

Panama. Even if the divergence between clades C7 and C8 does correspond to this event, further explanation is necessary to explain why clade C8 is present in both oceans. Second, specimens collected from Indonesian marine lakes that have been isolated from surrounding marine environments since the Pleistocene are undifferentiated from individuals collected from the Seychelles indicating that populations from these geographically disparate regions are, or have recently been, connected by gene flow despite the lack of evidence for connectivity between these lakes and nearby reefs. Finally, most of the biodiversity sampled can be reconstructed as Indo-Pacific in origin (Fig. 4). Due to the unresolved relationships between basal Placospongia lineages, no dcenter of originT hypothesis can be proposed for the entire clade.

4. Discussion

4.1. Features of ITS in Placospongia

Alvarez and Wendel (2003)point to a number of processes that impact ITS sequences in phylogenetically misleading ways. For example, intragenomic variation has been detected in many lineages as a result of duplication events, the presence of pseudogenes in various states of decay, and incomplete array homogenization. The biological factors that cause divergent ITS paralogs to be maintained within individual genomes are unknown, but several hypotheses have been proposed (Harris and Cran- dall, 2000; van Oppen et al., 2002; Marquez et al., 2003). Regardless of the cause of incomplete chromosomal homogenization, the data presented here are consistent with the conclusions of Wo¨rheide et al. (2004); if all divergent ITS paralogs are included in phylogenetic/phylogeographic analyses, tree structure corresponds to geography in a non- random way indicating that the historical signal of the marker is not obscured by paralogy. However, paralogy does interfere with the reconstruction of spatial patterns of variation within geographic populations because the divergent ITS sequence- types from any single individual do not sort mono- phyletically [a result of this study and Wo¨rheide et al. (2004)].

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Even though the ITS dataset of Placospongia seems to be phylogenetically structured, most of the different ITS sequence-types included must be paralogous. Therefore, the phylogenetic structure in the dataset may reflect duplication events rather than organismal relationships. The presence of paralogous copies in modern genomes also cautions that paralogy may have been present in ancestral populations. The sequence-types that remain might represent a mixture of orthologs and paralogs whose history of duplication is lost due to concerted evolution (Alvarez and Wendel, 2003). In this case, the direction of sequence homogenization in geographically/genetically isolated lineages may have been such that ITS is indicative of which populations are isolated from one another, but the relationships between different areas is misleading.

To date no other markers have been discovered in sponges that evolve at rates comparable to ITS (van Oppen et al., 2003). Mitochondrial genes, such as cytochrome oxidase subunit I (Cox1), that are useful population-level markers in some invertebrates, are highly conserved in sponges (Shearer et al., 2002; van Oppen et al., 2003; Duran et al., 2004; Nichols, 2005).

Furthermore, sequence data from the complete mitochondrial genome of three demosponges has revealed that most regions are highly conserved (F. Lang and D.

Lavrov, personal communication). Nuclear introns

found in single-copy nuclear genes (Jarmon et al., 2002) presently offer the greatest hope for fine-scale phylogenetic/phylogeographic analyses in sponges.

Despite the lack of independent data to corrobo- rate our ITS results, and despite incomplete sampling between and within some of the localities where Placospongia is reportedly distributed, the data presented here are sufficient to formulate preliminary hypotheses about the biodiversity, dispersal ability and historical biogeography of Placospongia. For example, only one individual was sampled from the Solomon Islands, one from Brisbane, two from Sulawesi, two from the Seychelles, one from each of the Indonesian marine lakes, three from Papua New Guinea, and one from Palau. These sample sizes are insufficient to say that all lineages are not present at all localities, but reciprocal monophyly between most of these localities is suggestive that these geographic localities are not well connected and that they are diverging. Better sampling is required in order to determine the actual geographic range of the clades that are reconstructed.

4.2. Systematics

The taxonomy of the genusPlacospongiahas been recently reviewed (Ru¨tzler, 2002) and so will not be discussed here in detail. Eight species ofPlacospongia

Distribution unordered

Eastern Pacific Western Atlantic Indo-Pacific equivocal

C1 C2 C9 C3 C4 C5 C6 C7 C8

Fig. 4. Summary cladogram where O.T.U.s correspond to the clades identified inFig. 2. Ancestral distributions were mapped under both ACCTRAN and DELTRAN models of character optimization in MacClade.

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have been described, butRu¨tzler (2002) reviews the literature and suggests that only three of these species may be valid,P. decorticans,P. melobesioidesand P.

carinata. A fourth species, P. labrynthica, described from South Africa (Kirkpatrick, 1904), is not reviewed byRu¨tzler (2002). Within the samples examined in the present study, spicule composition and morphology were variable within and between clades reconstructed using ITS data. For example, some individuals within thedPanamicTclade (distributed on both sides of the Isthmus of Panama,Fig. 2) contain a spicule type called dspirastersTand others do not. Spirasters are considered to be useful for distinguishing betweenP. carinataand P. melobesiodes (Ru¨tzler, 2002). Very little is known about the heritable basis of variation in spicule morphology, but because spicule characters can exhibit such eco-phenotypic plasticity (to the extent of their presence/absence) (Klautau et al., 1999; Maldonado et al., 1999), it is likely that the characters used for species-level taxonomy ofPlacospongiaare misleading. In fact, our results (Fig. 2) suggest that diversity is higher than morphology reflects. In fact, samples from 12 geographic locations (several of which are very close to one another) are found to minimally represent 7 evolutionarily independent lineages, and possibly as many as 10 [particularly if percent sequence divergence is interpreted as indication of phylogenetic divergence in the cases of Berau (reef) and Panama, Caribbean (UCMPWC872) specimens]. This result is consistent with the hypothesis that sponges do not disperse very far (by any means) and that geographically distant populations are probably not connected by gene flow.

However, geography alone is not a suitable indication of species boundaries, both because it conflicts with character-based systematic methods and because geographic criteria for species recognition would be misleading in several instances withPlacospongia:

1) A clade (C8) contains a mixture of individuals found on both sides of the Isthmus of Panama.

2) Landlocked specimens collected in Kakaban and Maratua marine lakes of Indonesia are phylogenetically indistinguishable from geographically distant specimens from the Seychelles (C4) but not closely related to nearby reef populations (C5 and C9).

3) Cryptic, non-sister lineages are co-distributed in the Pacific of Panama (C1 and C8) and reef environments of Indonesia (C5 and C9), and

cryptic, putative sister lineages are distributed in the Caribbean of Panama (C7 and C8).

4) Clades present in the Solomon Islands, Palau, Papua New Guinea, Indonesia, and Brisbane cannot be predicted based upon the distance between sampling localities. For example, New Brittain, PNG is geographically closer to the Solomon Islands than to Palau, yet samples from PNG are very divergent from the Solomon Islands and indistinguishable from Palau.

It is not surprising that geography does not an accurately reflect species boundaries because it is well known that history, geology, current patterns, and dispersal potential interplay to determine how lineages are distributed and how faunas from different geographic regions are connected. Nevertheless, if species cannot be recognized using morphological or geo- graphical criteria, what are the consequences for ecological studies and biodiversity surveys of sponges?

Sponge species are typically identified using gross and ultrastructural morphological characteristics.

Recently,Bell and Barnes (2002)have even proposed that within most habitats, gross morphology can serve as a rough estimate of species diversity for the practical purposes of biotic surveys (in the context of conservation biology). While this may be the only practical approach, our study and others (Klautau et al., 1994; Sole-Cava et al., 1991a,b; Wo¨rheide et al., 2003) indicates that molecular data may be the only accurate measure of sponge biodiversity and sponge species boundaries. In the case ofPlacospongiain the Pacific of Panama, two very divergent lineages cannot be distinguished from one another, but ITS data indicate that they have been evolving independently as long as any other two lineages sampled globally.

Because such phylogenetically divergent lineages are presumably ecologically divergent as well, it is as important to distinguish between them as it is important to distinguish between any two morphologically recognizable sponge species.

4.3. Biogeography 4.3.1. Isthmus of Panama

Given the data presented here it is not possible to decide whether most Panamanian samples of Placo- spongia are comprised of a Caribbean-only lineage

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and a transisthmian lineage, or whether they represent a single transisthmian lineage (not considering the non-sister cryptic lineage found in the Pacific, clade C1). The first scenario implies that the Pacific geminate pair diverged following the closure of the isthmus and has recently been re-introduced—perhaps via human-mediated dispersal—into the Caribbean, or that both lineages were present before the closure of the Isthmus and one went extinct in the Pacific. The second hypothesis implies that the populations on either side of the isthmus contain ancestral poly- morphisms that have not yet sorted and that ITS diverges at rates too slow to reflect a vicariant event that separated populations at least 3.1–3.5 mya (Keigwin, 1982; Duque-Caro, 1990a,b; Coates and Obando, 1996; Collins, 1996). A study conducted by Wo¨rheide et al. (2003) on Astrosclera supports possibility that ITS may evolve so slowly in some sponge taxa as to not reflect long periods of genetic isolation between populations.

Boury-Esnault et al. (1999), investigated putative transisthmian populations ofSpirastrellasp. cf.mollis using allozyme, detailed morphological, and cytological data, and determined that these putative geminate populations are as divergent as taxa classified as belonging to separate sponge genera. These authors subsequently distinguished between these populations, naming the Caribbean lineageS. hartmani and the Pacific lineageS. sabogae. ITS has recently been investigated for these putative transisthmian geminates, as well as for populations of Prosuberites laughlini found in both oceans. Preliminary results indicate that ITS can be used to reconstruct transisthmian geminates as reciprocally monophyletic in both cases, with similar, but low, levels of sequence divergence (Wo¨rheide et al., 2004; Nichols, unpub- lished). Either ITS evolves/sorts at slower rates in Placospongia, or Prosuberites and Spirastrella were isolated long before the final closure of the isthmus, a hypothesis that finds support from studies of other transisthmian pairs (Marko, 2002; Dick et al., 2003).

Either scenario requires that the allozyme loci studied by Boury-Esnault et al. (1999) evolve at rates significantly faster than ITS inSpirastrella.

4.3.2. Seychelles–Indonesian marine lakes

It is surprising that samples from Indonesian marine lakes are undifferentiated from samples from reef en-

vironments around the Seychelles. These two regions are separated by geographic distances that would seem to preclude gene flow based upon predictions and molecular phylogenetic inferences about the dispersal ability of Placospongia. Furthermore, marine lake specimens have likely been isolated from the marine environment since the last global sea-level high-stands (i.e., Pleistocene). We cannot dismiss the possibility that this same lineage is actually continuously distributed and that further sampling of the marine environment around Indonesia, and at intermediate localities such as India and Oman, will reveal that distant populations are connected by adjacency. This biogeographic problem highlights the utility of ITS as a preliminary guide to the most pertinent questions that could not otherwise be anticipated.

5. Conclusions

ITS data from Placospongia support the proposi- tion that ITS is suitable for phylogenetic analyses despite the presence of divergent paralogs within individuals. However, when intragenomic variation is detected, ITS is not useful for analyses at the population-level. These data suggest that Placospon- giais heterogeneous on all spatial scales globally, and is further diversified than morphology reflects. Cryp- tic lineages are found to be co-occurring within geographic regions (e.g., Pacific of Panama) and single lineages are isolated by great distances and seemingly impermeable barriers to dispersal (e.g., specimens from marine lakes in Indonesia are identical to samples from the Seychelles). Most lineages show geographic coherence with a primary division between Central American and Indo-Pacific lineages, but no component of the phylogeny clearly reflects the known geological event of the rising of the Isthmus of Panama. No systematic revisions are suggested until better geographic sampling is conducted and the relationships presented here are tested using multiple independent molecular markers.

Nevertheless, these data are consistent with the generally accepted hypotheses that sponges are more diverse than either their morphology or taxonomy suggests and that they have limited dispersal capabilities. Most of the divergences reconstructed within Placospongia are consistent with geographic isola-

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tion, but other processes must be responsible for sympatrically distributed lineages.Placospongia is a group that is ripe for more detailed geographic sampling and ITS adequately points to the historical and biogeographic questions of interest.

Acknowledgements

We would like to acknowledge Gabriel Jacome, Emina Begovic, Nicole De Voogd, Rob van Soest, Elly Beglinger, Jane Fromont, John Hooper, Stephen Cook, and Pat and Lori Colins and Mike Dawson (Coral Reef Research Foundation, under contract to the National Cancer Institute) for either their direct help in collecting specimens or for making otherwise unobtainable specimens available. Financial support for this study was provided by the University of California’s Department of Integrative Biology and Museum of Paleontology and the Smithsonian Trop- ical Research Institute. Thanks to Carole Hickman, David Lindberg, George Roderick and Emina Begovic for their helpful comments on earlier drafts of this manuscript. All experiments conducted for this research comply with the laws of the countries where the research took place.[SS]

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