Phylogenetic relationships of the family Axinellidae (Porifera: Demospongiae) using morphological...

Phylogenetic relationships of the family Axinellidae (Porifera:Demospongiae) using morphological and molecular dataBELINDA ALVAREZ, MICHAEL D. CRISP, FELICE DRIVER, JOHN N. A. HOOPER & ROB W. M. VAN SOEST

Accepted: June 1999 Alvarez, B., Crisp, M.D., Driver, F., Hooper, J.N.A. & Van Soest, R.W.M. (2000).

Phylogenetic relationships of the family Axinellidae (Porifera: Demospongiae) using

morphological and molecular data. Ð Zoologica Scripta, 29, 169±198.

Twenty-seven species of marine sponges belonging to Axinellidae and related groups

(Halichondriidae, Dictyonellidae, Agelasida) were selected to test the monophyly of

Axinellidae and investigate their phylogenetic relationships using parsimony and maximum

likelihood methods. Partial 28S rDNA sequences, including the D3 domain, and traditional

morphological characters (mainly skeletal ones) were used independently to construct

phylogenetic trees. Sequences were aligned using the appropriate model of secondary

structure of the RNA and compared to that produced by the multiple sequence alignment

program, ClustalW. The alignment using secondary structure constraints produced a better

estimate of the phylogeny and was demonstrated to be an effective and objective method.

Results of the cladistic analyses of the molecular and morphological data sets were not

fully congruent; the morphological data suggest that Axinellidae is monophyletic, however,

the molecular data suggest that it is nonmonophyletic. The single most-parsimonious tree

derived from the molecular data showed that species of Axinella (except A. polypoides) are

united in a clade that is more closely related to members of Agelasida than to other species

of Axinellidae; the remaining members of Axinellidae form a monophyletic group that is

closely related to the families Dictyonellidae and Halichondriidae. The consensus tree of

20 most-parsimonious trees from the morphological analysis, on the other hand, showed

that all the sampled species of Axinellidae belong to a monophyletic group which is closely

related to the species of Dictyonellidae and Halichondriidae. Only two branches were

identical in both cladograms, the one uniting the species of Ptilocaulis and Reniochalina and

the one with the species of Dictyonellidae.

The robustness of the molecular and morphological trees (or parts of the trees), was tested

using bootstrap, jack-knife, PTP and T-PTP tests. The results of the PTP test were

significant indicating significant cladistic structure in both data sets. The bootstrap and

jack-knife values indicate that the molecular tree is in general better supported than the

morphological one. The lack of morphological characters and the homoplastic nature of

some may explain the weak support of the morphological tree. A T-PTP test of

nonmonophyly showed that the nonmonophyly of Axinellidae, as indicated by the results

of the molecular analysis, is not significant; however, a T-PTP test of monophyly of

Axinellidae, as indicated by the morphological tree, produced significant results. This

indicates that the monophyly of Axinellidae based on morphological data cannot be

rejected; the family however, cannot be defined in terms of a unique diagnostic character

common to all members of the ingroup.

Tests of heterogeneity (reciprocal T-PTP and partition homogeneity test) indicated that

the data partitions are heterogeneous, which could be due to sampling errors (in either

data set) or differences in the underlying phylogenies; therefore data were not combined in

a single analysis. Further, both data sets are unequally sized (95 informative molecular

characters vs. 16 informative morphological characters), which means that the molecular

signal could swamp the morphological signal if the data is combined.

Nonmonophyly of Axinellidae is supported by chemical and genetic evidence available in

the literature and DNA sequences data of axinellid species from New Zealand. However,

this needs to be confirmed using independent evidence from different genes (or gene

regions), biochemistry, histology or cell ultrastructure. Therefore, no changes to the

taxonomic position of the family in the higher classification are proposed at this stage.

Q The Norwegian Academy of Science and Letters . Zoologica Scripta, 29, 2, April 2000, pp169±198 169

Belinda Alvarez, Division of Botany and Zoology, Australian National University, Canberra, ACT

0200, Australia. Present address: National Institute of Water and Atmospheric Research, PO Box

14±901, Kilbirnie, Wellington, New Zealand. E-mail: [email protected]

Michael. D. Crisp, Division of Botany and Zoology, Australian National University, Canberra,

ACT 0200, Australia.

Felice Driver, Division of Entomology, CSIRO, Canberra, ACT 2601, Australia.

John N.A. Hooper, Queensland Museum, PO Box 3300, South Brisbane, QLD, 4101, Australia.

Rob. W.M. Van Soest, Institute for Systematics and Ecology, University of Amsterdam, PO Box

94766±1090 GT Amsterdam, The Netherlands.

IntroductionThe Axinellidae (Porifera: Demospongiae) is a group of

sponges characterized by some of the simplest morpholo-

gical characters currently used in sponge taxonomy; the

group is without a single synapomorphy to de®ne it. As

currently de®ned by Hooper & Wiedenmayer (1994),

Axinellidae includes sponges of diverse growth forms

(encrusting, massive, branching, fan shaped and tubular)

with the surface usually hispid due to projecting spicules;

with a skeleton typically divided into axial and extra-axial

components; with the main skeletal tracts generally

condensed in the axial component and organized in a

plumose or plumoreticulate fashion in the extra-axial

portion; the megascleres are combination of styles, oxeas

and strongyles, usually smooth, sometimes tuberculate,

spined, ¯exuous or vermiform; the microscleres are usually

absent, although few genera have raphides and trichodrag-

mata. This de®nition is based on homoplastic characters

(i.e. axial condensation of the skeleton, plumose spicule

tracts, combination of oxeas and styles) that are present in

other sponge taxa and raises the possibility that the family

might not be monophyletic (Van Soest et al. 1990; Hooper

& Bergquist 1992; Hooper & LeÂvi 1993; Alvarez & Crisp

1994). As a consequence, approximately 92 nominal genera

have been included in the family at one time or other.

Studies to determine the actual generic content of the

family and to revise the de®nitions and diagnoses of each

genus in order to rede®ne the concept of the Axinellidae

are currently in progress (Alvarez & Hooper, unpublished

data).

The position of the Axinellidae at higher levels of classi-

®cation is also controversial and uncertain. The subdivi-

sion of the class Demospongiae (LeÂvi 1953, 1955, 1957,

1973) into three subclasses (Ceractinomorpha, Tetractino-

morpha and Homoscleromorpha), mainly on the basis of

reproductive strategies, affected the higher taxonomic posi-

tion of this and other sponge families. After this subdivi-

sion the Axinellidae was allocated to the order Axinellida,

in the subclass Tetractinomorpha, along with other

families having axial condensation of the skeleton (i.e.

Raspailiidae, Hemiasterellidae). A phylogenetic analysis by

Van Soest et al. (1991) showed that the axial condensation

of the skeleton was a homoplastic character, therefore the

families included in Axinellida did not comprise a mono-

phyletic group.

Van Soest et al. (1990) also indicated the morphological

af®nities of the Axinellidae with other members of their

rede®ned order Halichondrida. According to these authors

the Axinellidae is more closely related, in terms of skeletal

structure and spicule geometry, to the halichondrid

families Desmoxyidae, Dictyonellidae and Halichondriidae,

than to other members of Axinellida. This has not been

completely accepted and in recent publications (Hooper &

Bergquist 1992; Hooper & LeÂvi 1993; Carballo et al.

1996), the Axinellidae is still allocated to the order Axinel-

lida. Hooper et al. (1992) found Axinellida to be clearly

polyphyletic in their biochemical pro®les, but despite this

evidence they were unable to propose an alternative

system.

The phylogenetic analysis of Van Soest et al. (1990) is

based on morphological characters and few taxa. Some of

the families included in their analysis, such as the Desmox-

yidae, Dictyonellidae and Axinellidae, have not been

recently revised and their generic content is poorly known.

Therefore doubts must exist regarding the monophyly of

these families and the synapomorphies on which they are

based. Consequently the position of these families in the

classi®cation of Demospongiae is debatable at this stage.

The question of whether or not the Axinellidae is mono-

phyletic needs to be answered before any attempt to place

the family in a higher classi®cation scheme.

In recent years, phylogenetic systematics has been used

to study different groups of the class Demospongiae (Van

Soest 1987b; Weerdt 1989; Van Soest et al. 1990; Volk-

mer-Ribeiro 1990; Bergquist & Kelly Borges 1991;

Hooper 1991; Van Soest 1991; Van Soest et al. 1991;

Hajdu 1993; Van Soest & Hooper 1993; Alvarez & Crisp

1994; Hajdu et al. 1994; SaraÁ & Burlando 1994; Bergquist

& Kelly-Borges 1995; Hajdu 1995; Rosell & Uriz 1997).

Studies that include Axinellidae in particular, are the one

by Van Soest et al. (1990), who explored the position of

the family in relation to other demosponge families (as

mentioned above) and the one by Alvarez & Crisp (1994),

who studied relationships of a group of axinellid species

Phylogenetic relationships of axinellidae . Alvarez et al.

170 Zoologica Scripta, 29, 2, April 2000, pp169±198 . Q The Norwegian Academy of Science and Letters

from the Central-West Atlantic. All of these phylogenies

are based on morphological characters traditionally used in

taxonomic descriptions and related to features such as

shape, colour, surface, consistency, skeletal architecture

and spicule composition. Some of these characters may be

phylogenetically informative, but are subjective (such as

colour or consistency), and therefore could be interpreted

or scored in different ways (e.g. Alvarez & Crisp 1994;

SaraÁ & Burlando 1994). Bergquist & Kelly-Borges (1991),

for example, categorize morphological characters a priori

into `well-de®ned' (those for which characters states can be

easily assigned) or `questionable' (those for which there is a

possibility of ambiguity in their assignment of character

states). According to these authors such a categorization

provides a qualitative con®dence value for subsequent

phylogenetic analyses.

Of the morphological characters commonly used for the

study of sponge systematics, skeletal architecture and

spicule composition or geometry are thought to be the best

indicators of phylogenetic relationships (Hajdu & Van

Soest 1996); however, results are always dependent on the

initial assessment of homology. Morphometric and continu-

ous characters such as length, width, ratio (length/width)

and size categories of the different types of spicules, are also

commonly used in these studies. In general, they are coded

in the form of ranges of values separated by gaps, but in

many cases there is overlap of the scores and the de®nition

of the gaps becomes ambiguous. It is not known whether

these types of continuous characters are variable at the

intraspeci®c level or whether the differences among taxa are

statistically signi®cant or are heritable characters useful in

reconstructing phylogeny. Further, many sponge taxa

included in the above mentioned studies are polymorphic

for some characters (multiple character states present in the

same taxon), which imposes additional complications for

their use in phylogenetic studies. This is common when

considering macroscopic characters such as habit, surface,

colour and consistency. Due to the dif®culties arizing from

character interpretation, subjectivity and high phenotypic

plasticity, homology assessments in sponge phylogeny are

problematic and therefore the data sets of (even) closely

related groups become dif®cult to compare.

The current knowledge of the systematics of the Axinel-

lidae and other sponge taxa points to the need for a search

for new characters and re-interpretation of traditional ones

to produce more robust phylogenies. Molecular data,

biochemical characters, cell ultrastructure, anatomical

characters (i.e. size and shape of the choanocyte chambers,

ratio of aquiferous cavities/mesohyl) and developmental

features are all examples of the types of non-traditional

characters that could be used to study phylogenetic rela-

tionships of this and other groups of sponges.

Biochemical characters, in particular, seem to be useful

taxonomic markers and good indicators of sponge phylo-

geny (Van Soest et al. 1996a). The diversity of biochemical

properties of sponges has been demonstrated by the contin-

uous ®nding of novel compounds which have pharmacologi-

cal properties (Sarma et al. 1993; Daloze & Braekman 1994

and references within; Garson 1994). These and other

compounds such as free amino acids, sterols, carotenoids,

terpenoids, fatty acids, sterols, brominated phenols, bromo-

pyrroles, alkaloids dibromotyrosine-derived and bromotyra-

mine compounds, to mention some, appear to be species-

speci®c, or characteristic of higher taxa. These types of

chemical characters have been used already in the study of

sponge systematics or some aspects of it (Bergquist & Hart-

man 1969; Bergquist & Hogg 1969; Bergquist et al. 1980;

Bergquist & Wells 1983; Braekman et al. 1992; Hooper

et al. 1992; Braekman et al. 1994; Fromont et al. 1994; Kelly-

Borges et al. 1994; Van Soest et al. 1996a; Van Soest et al.

1996b; Williams & Faulkner 1996). According to Van Soest

et al. (1996a) the chemistry of groups with easily recognizable

and unequivocal morphological characters re¯ects different

levels of morphological similarities but they recognize as

well, that in many cases no apparent congruence between

chemical and morphological similarity has been found.

Several researchers have used molecular data to study

different aspects of sponge evolution. Kelly-Borges et al.

(1991) and Kelly-Borges & Pomponi (1994) were the ®rst

to use partial RNA sequences of the small subunit (18S) of

the ribosomal RNA molecule to study phylogenetic rela-

tionships within the orders Hadromerida and Lithistida

(Demospongiae), respectively. Other studies have used

nucleic acid sequences to study sponge phylogeny in rela-

tion to other eukaryote groups. Lafay et al. (1992) for

example, analysed partial sequences of 28S ribosomal RNA

of 11 sponge species (including one species of Axinellidae,

Axinella damicornis, and one species of its sister family

Dictyonellidae, Dictyonella incisa) and other invertebrates to

study phylogenetic relationships of lower metazoans.

Rodrigo et al. (1994) used partial sequences of 18S riboso-

mal RNA from several organisms, including sponges, to

demonstrate the apparent absence of phylogenetic signal in

that particular region of the molecule for the set of taxa

investigated. West & Powers (1993) and Cavalier-Smith et

al. (1996) used complete sequences of the 18S ribosomal

gene to test the monophyly of sponges and their position

in relation to other eukaryotes. MuÈ ller (1997 and refer-

ences within, 1998) analysed genes encoding several

proteins of the sponge Geodia cydonium, and other Protozoa

and Metazoa, to demonstrate the monophyletic origin of

the Metazoa, including sponges. More recently, the 50 end

of 28S ribosomal RNA has been used to study the phylo-

genetic position of several species of coralline sponges in

Alvarez et al. . Phylogenetic relationships of axinellidae


relation to demosponges (Chombard et al. 1997) and to

assess the homology of some morphological characters

used in the classi®cation of tetractinellid sponges (Chom-

bard et al. 1998). The latter authors also indicate that

different regions of the large ribosomal subunit are appro-

priate to resolve phylogenetic relationships of sponges at

different levels, from genera to the class level.

In the present study, a region of approximately 300 bases

from the large subunit (28S) of the nuclear ribosomal RNA

gene was selected to study phylogenetic relationships of a

representative group of taxa of the family Axinellidae and

related groups. This region includes the D3 domain, one of

the 12 variable subunits of the 28S rRNA, which exhibits a

moderate size variation during evolution (54±174 nucleo-

tides among the species analysed by Michot et al. 1990);

however, Nunn et al. (1996) showed that this region varied

from 180 to 518 nucleotides among 11 species of Isopoda

(Crustacea). Based on the study of the secondary structure

(folded con®guration of a rRNA sequence) of several

prokaryotes and eukaryotes, both Michot et al. (1990) and

Nunn et al. (1996) showed that the D3 domain contains a

subset of universally conserved structural features, inter-

spersed with four variable subdomains (despite the high size

variability in the D3 of Isopoda). Such features make this

section of the molecule well suited for phylogenetic studies,

because the presence of this subset of conservative regions

helps in the identi®cation and alignment of homologous

sites among the sequences.

Although the use of skeletal characters in sponge

systematics has been challenged, especially for groups such

as Axinellidae (Wiedenmayer 1989; Hooper & Bergquist

1992; Hooper et al. 1992; Hooper & LeÂvi 1993), there

have been no studies to date that compare the performance

of such characters against other types of data using the

same set of taxa. This paper presents a study of the phylo-

genetic relationships of selected taxa of Axinellidae and

other closely related groups, using data derived from

partial sequences of 28S rDNA and traditional morpholo-

gical characters, to test whether or not the family constitu-

tes a monophyletic group and evaluate its current

taxonomic position within the current classi®cation.

Materials and MethodsCollection and preservation of samples

A total of 27 species (Table 1) belonging to the Axinelli-

dae, Halichondriidae, Dictyonellidae, Agelasidae and

Table 1 Species selected for this study, locality of collection, current classi®cation at the family level and GeneBank accession number (seeAppendix for more details).

Taxon

code

Species Locality Family GeneBank

Accession No.

Agma Agelas mauritiana (Carter) Seychelles, Indian Ocean Agelasidae AF051429

Aswi Astroclera willeyana Lister Seychelles, Indian Ocean Astroscleridae AF051430

Acac Acanthella acuta Schmidt La Ciotat, Bec de l Aiyle, France Axinellidae AF051431

Acca Acanthella cavernosa Dendy Seychelles, Indian Ocean Axinellidae AF051432

Acpu Acanthella pulcherrima Ridley & Dendy Darwin, Northern Territory, Australia Axinellidae AF051433

Axar Axinella aruenis (Hentschel) Seychelles, Indian Ocean Axinellidae AF051434

Axca Axinella carteri (Dendy) Great Barrier Reef, Queensland, Australia Axinellidae AF051435

Axcar Axinella carteri (Dendy) Seychelles, Indian Ocean Axinellidae AF051436

Axda Axinella damicornis (Esper) La Ciotat, Bec de l Aiyle, France Axinellidae AF051437

Axpo Axinella polypoides Schmidt La Ciotat, Bec de l Aiyle, France Axinellidae AF051438

Cyco Cymbastela coralliophila Hooper & Bergquist Great Barrier Reef, Queensland, Australia Axinellidae AF051439

Cyst Cymbastela stipitata (Bergquist & Tizard) Darwin, Northern Territory, Australia Axinellidae AF051440

Cyve Cymbastela vespertina Hooper & Bergquist Darwin, Northern Territory, Australia Axinellidae AF051441

Phsp Phakellia sp Darwin, Northern Territory, Australia Axinellidae AF051442

Psau Pseudaxinella australis Bergquist Darwin, Northern Territory, Australia Axinellidae AF051443

Psdu Pseudaxinella durissima Dendy Seychelles, Indian Ocean Axinellidae AF051444

Psre Pseudaxinella reticulata (Ridley & Dendy) Carrie Bow Cay, Belize Axinellidae AF051445

Pssp Pseudaxinella sp Darwin, Northern Territory, Australia Axinellidae AF051446

Ptsp Ptilocaulis sp Seychelles, Indian Ocean Axinellidae AF051447

Ptwa Ptilocaulis walpersii (Duchassaing & Michelotti) Carrie Bow Cay, Belize Axinellidae AF051448

Resp Reniochalina sp Great Barrier Reef, Queensland, Australia Axinellidae AF051449

Rest Reniochalina stalagmitis Lendenfeld Darwin, Northern Territory, Australia Axinellidae AF051450

Sru Scopalina ruetzleri (Wiedenmayer) Carrie Bow Cay, Belize Dictyonellidae AF051451

Ulsp Ulosa sp Darwin, Northern Territory, Australia Dictyonellidae AF051452

Cicon Ciocalypta confossa Hooper et al. Darwin, Northern Territory, Australia Halichondriidae AF051453

Hapa Halichondria panicea (Pallas) Oosterchelde, SW Netherlands Halichondriidae AF051454

Haph Halichondria phakellioides Dendy & Frederick Darwin, Northern Territory, Australia Halichondriidae AF051455



Astroscleridae were selected for the present study. Most of

the species selected have been described. The author and

date of the original description as well as the references

that include re-descriptions or additional records of these

species are provided in Appendix I. Details of the material

collected for this study and taxonomic descriptions of

those species that have not been previously described are

also included in this appendix.

Samples of these species were collected mainly in inter-

tidal and shallow subtidal areas (using SCUBA) in the

vicinity of Darwin Harbour, Northern Territory (NT),

Australia. Colour photographs were taken in situ and

immediately after collection to record morphological

characters such as shape and colour. Additional macro-

scopic features related to the surface, consistency and

oscules were also registered after collection. Additional

samples of species were collected from the Great Barrier

Reef (Australia), Seychelles Islands (Indian Ocean), Neth-

erlands, Mediterranean and Carrie Bow Cay (Belize,

Caribbean Sea).

Approximately 5 mm3 of each sample was chopped

®nely and preserved in an Eppendorf tube with silica gel

(particle size 0.00630.004 mm) for DNA extraction. This

method of tissue preservation for extraction of genomic

DNA gives good results for sponges (M. C. DõÂaz, Univer-

sity of Santa Cruz, California, pers. comm.). The rest of

each sample was preserved in 70% alcohol for preparation

of thick sections and spicule slides, following the methods

described by RuÈ tzler (1978), and study of skeletal compo-

nents using light microscopy and scanning electron micro-

scopy (SEM). Samples collected were registered and

deposited in the Queensland Museum, Australia (QM)

The National Museum of Natural History, Smithsonian

Institution, Washington DC (USNM) and Zoologisch

Museum, University of Amsterdam (ZMA).

Molecular methods

Extraction of genomic DNA. Approximately 0.05 g of sponge

tissue preserved in silica gel was ground to a ®ne powder

with liquid nitrogen, added to 500 mL of proteinase K

buffer (2 mg/mL proteinase K, 0.1 m Tris-HCl ph 8.5,

0.05 M EDTA, 0.2 M NaCl; 1% SDS) and incubated for

30 min at 65 8C. Total genomic DNA was then extracted in

three stages with equal volumes of phenol (saturated with

1�TE, 10 mm Tris-HCl,1 mm EDTA ph8), phenol/

chloroform and chloroform/isoamyl alcohol (24%). The

DNA was precipitated by addition of 2.5 volumes of etha-

nol, pelleted, washed with 75% ethanol, dried under a

vacuum and re-suspended in 1�TE containing 20 mg/mL

of RNase. The DNA was then incubated at 37 8C for

30 min and extracted again in two stages with equal volumes

of phenol/chloroform and chloroform/isoamyl alcohol with

subsequent precipitation in ethanol, and washing and

drying as described above. DNA was re-suspended in 10±

100 mL of 0.5�TE depending on the size of the pellet.

Extraction of DNA with a chelating resin, Chelex 100

(Walsh et al. 1991) was used also as an alternative method

with a few of the samples preserved in silica gel and for

one preserved in 70% alcohol (see Table 2).

PCR ampli®cation. A region corresponding to the D3 domain

of the 28S rDNA and a highly conserved region of approxi-

mately 150 bases adjacent to the 30 end of this domain was

ampli®ed using the Polymerase Chain Reaction (PCR)

(Saiki et al. 1988) (primer sequences in Nunn et al. 1996; Al-

Banna et al. 1997). The PCR ampli®cations were performed

in 50 mL reaction volumes containing 2±20 ng of genomic

DNA, 10 mM Tris pH 8.4, 50 mM KCl, 1.5 mm MgCl2,

0.05% Tween 20, 0.05% Nonidet P40, 0.4 mM each of each

primer, 25 mM each dATP, dCTP, dGTP and dTTP. The

DNA was denatured at 94 8C for 5 min, followed by 30

cycles of ampli®cation using the following denaturation,

annealing and extension conditions: 1 min at 94 8C, 90 s at

55 8C, 2 min at 72 8C with a ®nal extension for 5 min at

Table 2 Methods of DNA extraction, ampli®cation and cloningfor the taxa used in this study.

Species extraction ampli®cation cloning vector

Agelas mauritiana PKW STD pGEM-T

Astrosclera willeyana CHEL STD pUC 18

Acanthella acuta CHEL STD pUC 18

Acanthella cavernosa PK STD pUC 119

Acanthella pulcherrima PKW STD pGEM-T

Axinella aruensis PKW OPT pGEM-T

Axinella carteri PK STD pGEM-T

Axinella carteri PKW OPT pGEM-T

Axinella damicornis PKW OPT pGEM-T

Axinella polypoides CHEL STD pUC 18

Cymbastela coralliophila PK STD pUC 119

Cymbastela stipitata PKW STD pUC 119

Cymbastela vespertina PK STD pUC 119

Phakellia sp PKW STD pGEM-T

Pseudaxinella australis PKW OPT pGEM-T

Pseudaxinella durissima PKW OPT pGEM-T

Pseudaxinella reticulata PKW STD pGEM-T

Pseudaxinella sp PKW OPT pGEM-T

Ptilocaulis sp PKW OPT pGEM-T

Ptilocaulis walpersii PKW STD pGEM-T

Reniochalina sp PKW STD pUC 119

Reniochalina stalagmitis PK STD pGEM-T

Scopalina ruetzleri PKW STD pGEM-T

Ulosa sp PKW STD pGEM-T

Ciocalypta confossa PK STD pUC 119

Halichondria panicea CHEL STD pUC 18

Halichondria phakellioicdes PK STD pUC 119

CHEL: Chelex; OPT: Opti-prime PCR optimization kit used; PK: Proteinase K; PKW:

Proteinase K�puri®cation with `Wizard columns'; STD: standard PCR described in

the text.



72 8C. 2.5 units of the enzyme Taq polymerase was added

following the initial 5 min denaturation.

When ampli®ed products were not obtained using the

method described above (see Table 2), the genomic DNA

was puri®ed using the `Wizard DNA-cleanup' system of

PROMEGA (Catalogue no. A7280) and PCR ampli®cation

was tried again. Alternatively, the buffer concentration and

components of the PCR reaction were altered using `Opti-

prime PCR optimization kit' from STRATAGENE (Cata-

logue no. 200422).

Cloning of PCR products. The ampli®ed DNA was treated

with T4 DNA polymerase then puri®ed by phenol/chloro-

form (1 : 1) extraction, and precipitated with ethanol prior to

cloning into the SmaI site of vector pUC 18 or pUC 119

(Table 2). Plasmid DNA was subsequently introduced into

competent cells of Escherichia coli by a transformation process

(Maniatis et al. 1982). Alternatively, a pGEM-T cloning kit

from PROMEGA (Catalogue no. A3610) was used to clone

the PCR products. Bacteria were grown on agar plates with

ampicillin, X-galactoside and IPTG to select colonies with

recombinant plasmids (i.e. bacterial colonies that contain

nonrecombinant plasmids are blue, whereas colonies with

recombinant plasmids are white). A maximum of 14 white

colonies were selected and grown individually with the

appropriate media. The plasmid DNA was puri®ed using an

alkaline lysis, resuspended in 20±50 mL of TE/RNase. An

aliquot was checked by electrophoresis in an agarose gel

using the appropriate plasmid vector as standard, to con®rm

the presence of inserted PCR amplicons and to estimate the

concentration of the plasmid DNA. One ml of the media

culture with the recombinant plasmids was preserved in a

75% glycerol solution at ÿ20 8C.

Sequencing. Sequencing of clones was done with: 1) Manual

sequencing using the Sanger Dideoxy method (described

in Hillis et al. 1990) and a Pharmacia T7 DNA sequencing

kit according to manufacturers recommendations. Both

DNA strands of three to six clones (depending on the ef®-

ciency of the transformation) of each species were

sequenced. 2) Automatic sequencing using an Applied

Biosystems 373 A DNA Sequencer. Both dye primer and

dye terminator cycle reactions using Taq were used to

obtain labelled extension products that were loaded in the

automatic DNA sequencer.

Sequence alignment

Sequences were edited and manipulated using SeqApp/Pup

for Macintosh (D.G. Gilbert, Biology Department, Indiana

University; ftp://iubio.bio.indiana.edu/molbio/seqpup/).

Bases were con®rmed using both strands of the sequenced

clones. Sequence alignment was performed using the

multiple sequence alignment program CLUSTAL W

version 1.5 (Thompson et al. 1994) with the default para-

meters.

In addition, sequences were aligned manually based on

secondary structure. The secondary structure of the D3

domain (®rst 180 bases) was drawn for each sequence,

following the models proposed by Michot et al. (1990),

Gutell et al. (1993) and de Rijk et al. (1994). The secondary

structure for the conservative region adjacent to the 3Â end

of the D3 domain was not drawn as there is almost no

variation in that region of the molecule. The sequences

were realigned following the nomenclature and series of

steps proposed by Kjer (1995) and the predicted secondary

structure models obtained. The helices (base pairing

region separated by other hydrogen-bonded stems) were

indicated by square brackets, the stems (nucleotide

sequences separated from their complementary sequences

by single stranded loops) were separated by parentheses

and nucleotides in single-strand loops were indicated by

small letters. Using SeqApp (see above) all the brackets

and parentheses were aligned as well as the nucleotides of

the stems and single stranded loops; gaps were introduced

in the loop regions.

To check whether the alignment using secondary struc-

ture constraints could be further improved, all the indels

in the previous alignment were replaced by an `N'. The

sequences were then realigned using Clustal W with the

default parameters. No new gaps were introduced in this

second pass.

Selection of morphological characters

A total of 16 characters (53 character states) (Table 3), was

selected to study the phylogenetic relationships based on

morphology, and scored for the same set of taxa for which

sequence data were obtained. The scoring of the morpho-

logical characters was based mainly on the examination of

the voucher specimens. Taxonomic descriptions available

from the literature were used also to complement and

verify the assignment of the character states. Terminology

used in the character list follows that of Boury-Esnault &

RuÈ tzler (1997). The characters selected for this analysis are

related to features of the choanosomal skeleton at the

surface level (character 1), presence or absence of ectoso-

mal skeleton (character 2), general architecture of the

choanosomal skeleton (characters 3 & 4), features of the

individual components of the skeleton (characters 6 & 7)

and spicule geometry (characters 8±16). Each type of

spicule (i.e. oxeas, styles, anisoxeas, anisoxeas terminally

spined, strongyles, verticillate acanthostyles and raphides

and trichodragmata) were treated as separate characters

and coded presence/absence. Spicules of the same type but

located in different parts of the choanosomal skeleton

(axial and extra-axial regions), were considered nonhomo-



logous and thus treated as different characters (e.g. charac-

ter 8±9, 12±13, Table 3).

For each taxon the length and width of 25 spicules were

measured for each spicule type found in a slide of

40 � 22 mm. Student's T-tests were performed to deter-

mine whether the length and/or ratio (length/width) of the

different spicule types were signi®cantly different among

the taxa and therefore could be included in the cladistic

analysis, following the recommendations of Thiele (1993)

on coding quantitative morphometric characters. The

results of Student's T-tests indicated that the differences

among the taxa were not signi®cant and therefore they

were excluded from this analysis; however, different size

categories of some spicules (characters 8 and 12, Table 3)

were identi®ed as different character states.

The morphological characters used here have been rein-

terpreted from previous works (Alvarez & Crisp 1994;

Alvarez 1996) and evaluated in preliminary cladistic

analyses (not presented here), which allowed a reassess-

ment of the initial homology statements. Some characters,

such as habit (generally massive, massive-encrusting,

massive-lobate, branching-erect, lamellate, fan-shaped or

cup-shaped), colour (generally orange, yellow, red brown

or within that range), and oscule shape, included in these

preliminary analyses, were excluded as they were found to

be polymorphic for some of the taxa and made the assign-

ment of the states questionable.

The presence or absence of three different types of

secondary metabolites (terpenoids, pyrroles and isocya-

nides), were also evaluated in preliminary analyses. These

data were taken from Braekman et al. (1992), Braekman

et al. (1994) and Williams & Faulkner (1996). However,

the lack of information for most of the taxa (19 taxa out of

27 had to be coded as missing `?') could introduce an unac-

ceptable level of uncertainty; therefore those characters

were excluded from the ®nal analysis. It could be argued

Table 3 List of morphological character and character states used to establish the phylogenetic relationships of some axinellid sponges.

1. Projections of the choanosomal skeleton at surface 7. Secondary (connecting) tracts

0 Absent 0 Absent

1 Single spicules projecting slightly 1 Single spicules/paucispicular

2 Brushes of spicules projecting slightly 2 Multispicular (plurispicular)

3 Short conules or tubercles 8. Oxeas in the primary skeleton

4 Conules 0 Absent

5 ``Scopiform'' process 1 One size category

2. Specialised ectosomal skeleton 2 Two sizecategories

0 Absent 9. Oxeas in the axial skeleton

1 Tangential 0 Absent

2 Pratangential 1 Present

3. Choanosomal (primary/extra-axial) skeleton 10. Anisoxeas

0 Stromatoporoid 0 Absent

1 Reticulate 1 Present

2 Irregularly reticulate 11. Anisoxeas terminally spined

3 Plumose 0 Absent

4 Plumoreticulate 1 Present

5 Plumose-radiate 12. Styles in the primary skeleton

6 Dendritic 0 Absent

7 Halichondrioid 1 One size category

4. Axial skeleton 2 Two size categories

0 Absent 13. Styles in the axial skeleton

1 Condensed 0 Absent

2 Plumoreticulate 1 Present

3 Vaguely plumose 14. Strongyles

4 Close-set reticulation of irregularly anastomosing ®bres 0 Absent

5. Spongin ®bres 1 Present

0 Absent 15. Verticillated acanthostyles

1 Cored and echinated with spicules 0 Present

2 Cored with spicules 1 Absent

3 Lightly investing (cementing) spicule tracts 16. Raphides and trichodragmata

6. Primary (ascending) spicule tracts 0 Absent

0 Absent 1 Present

1 Directionless bundles

2 Plumose

3 Columns of sinuous strongyles echinated by styles



also that chemical characters being a different class of data,

should not be combined in an analysis with molecular or

morphological data (e.g. Bull et al. 1993).

Scores for each of the 27 species and 16 morphological

characters are shown in the Table 4.

Phylogenetic analysis

Maximum-parsimony analyses of the aligned sequences,

and the morphological data set, were performed using

PAUP 4.0* (test version 4.0d64; used with the permission

of D. Swofford). The heuristic search option was used to

®nd minimum-length trees; starting trees were obtained by

stepwise addition of the closest taxon at each step; tree

bisection-reconnection (TBR) was selected as the branch

swapping algorithm. Gaps were treated as missing data in

the molecular set. Characters in both the morphological

and molecular data were unordered and uniformly

weighted in all of the analyses. Maximum-parsimony

analyses of the data were also performed using Henning 86

version 1.5 (J.S Farris, NY, USA). MacClade version 3.04

(Maddison & Maddison 1992) was used for tree manipula-

tions.

The sequence data also were analysed using the maxi-

mum-likelihood optimality criterion using PAUP 4.0* with

the default parameters (all sites evolving at the same rate;

transition/transversion ratio� 2; molecular clock not

enforced).

To assess the robustness of the branching sequence and

signi®cance of the phylogenetic signal of the cladograms

(or part of the cladograms) obtained from maximum parsi-

mony analyses, the following techniques were used:

(a) Permutation Tail Probability (PTP) test (Faith 1991;

Faith & Cranston 1991) included in PAUP 4.0*; 1000

randomizations; all taxa randomised. The NO-PTP

(i.e. excluding designated outgroups) test suggested by

Trueman (1996) was not applicable because all the

analyses in this study were unconstrained.

(b) T-PTP (Topology-dependent PTP) test (Faith 1991),

included in PAUP 4.0*; 1000 randomizations; all taxa

randomised. NO-TPTP suggested by Trueman

(1996) was not applicable for the same reason given

above.

(c) Bootstrap (Felsenstein 1985) and single-order Jack-

knife of characters (Siddall 1995 and references

within) of 1000 heuristic searches (with the same

settings mentioned above) using PAUP 4.0*.

Rooting and outgroup selection. Trees were rooted using the

outgroup method (see Nixon & Carpenter 1993 for

review). In this method the ingroup and outgroup taxa are

Table 4 Taxon/character data matrix. Character numbers according to Table 3.

Taxa\Characters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Agelas mauritiana 0 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0

Astrosclera willeyana 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Acanthella acuta 4 0 6 0 3 3 0 0 0 0 0 1 0 1 1 0

Acanthella cavernosa 4 0 5 1 3 3 0 0 1 1 0 1 0 1 1 0

Acanthella pulcherrima 4 0 5 1 3 3 0 0 1 1 0 1 0 1 1 0

Axinella aruensis 4 0 3 3 3 2 0 1 1 0 0 1 1 0 1 0

Axinella carteri 4 0 3 3 3 2 0 0 0 0 0 1 1 0 1 0

Axinella carteri 4 0 3 3 3 2 0 0 0 0 0 1 1 0 1 0

Axinella damicornis 1 0 4 2 3 2 1 1 1 0 0 1 1 0 1 0

Axinella polypoides 1 0 4 2 3 2 1 1 1 0 0 1 1 0 1 1

Cymbastela coralliophila 2 2 4 2 3 2 1 2 1 0 0 0 0 0 1 0

Cymbastela stipitata 2 0 4 2 3 2 1 1 1 0 0 0 0 0 1 0

Cymbastela vespertina 2 0 4 2 3 2 1 1 1 0 0 0 0 0 1 0

Phakellia sp 1 0 5 1 3 3 0 0 0 0 0 2 0 1 1 0

Pseudaxinella australis 3 0 4 0 3 2 2 1 0 0 0 1 0 0 1 1

Pseudaxinella durissima 3 0 4 0 3 2 2 2 0 0 0 1 0 0 1 1

Pseudaxinella reticulata 3 0 4 0 3 2 2 1 0 0 0 1 0 0 1 0

Pseudaxinella sp 3 0 4 0 3 2 2 1 0 0 0 1 0 0 1 0

Ptilocaulis sp 5 0 3 4 2 2 1 0 0 0 0 2 1 0 1 0

Ptilocaulis walpersii 5 0 3 4 2 2 1 0 0 0 0 2 1 0 1 0

Reniochalina sp 5 0 3 4 2 2 1 0 0 0 1 0 0 0 1 0

Reniochalina stalagmitis 5 0 3 4 2 2 1 0 0 0 1 0 0 0 1 0

Scopalina ruetzleri 4 0 2 0 2 0 0 0 0 1 0 1 0 0 1 0

Ulosa sp 4 0 2 0 2 0 0 0 0 1 0 1 0 0 1 0

Ciocalypta confossa 0 1 7 0 0 1 0 0 0 0 0 1 0 0 1 0

Halichondria panicea 0 1 7 0 0 0 0 1 0 0 0 1 0 0 1 0

Halichondria phakellioides 0 1 7 0 0 1 0 2 0 0 0 0 0 0 1 0



included in a simultaneous and unconstrained analysis (e.g.

outgroup relationships are unspeci®ed prior to the analy-

sis). Data for the outgroup and ingroup taxa are collected

and analysed globally in a single matrix and the resulting

cladogram is rooted a posteriori between the outgroup and

the ingroup (Smith 1994; Kitching et al. 1998).

Three different groups of species included in this data

set are suitable as outgroups to root the trees. These

groups are the species of Agelasidae and Astroscleridae

(Astrosclera willeyana and Agelas mauritiana), the species of

Halichondriidae (Halichondria panicea, H. phakellioides and

Ciocalypta confossa) and the species of Dictyonellidae (Ulosa

sp. and Scopalina ruetzleri). Astroscleridae and Agelasidae

have been included in the order Agelasida based on chemi-

cal and molecular evidence (Williams & Faulkner 1996;

Chombard et al. 1997; WoÈ rheide 1998 and references

within); both families also share the presence of verticillate

acanthostyles. Members of this order are morphologically

very different to Axinellidae, however, chemical (Braekman

et al. 1992) and molecular evidence (Lafay et al. 1992;

Chombard et al. 1997; Chombard et al. 1998) indicates

that members of Agelasida are closely related to some

species currently included in the family Axinellidae. The

families Halichondriidae and Dictyonellidae on the other

hand, have also been shown to be closely related to Axinel-

lidae by Van Soest et al. (1990) based on morphology. As

there is not evidence available to establish unequivocally

which of these three groups is the actual sister group of

Axinellidae, all were included in an unconstrained parsi-

mony analysis. Alternative rooting using the three possible

outgroups was explored after the unrooted trees were

obtained.

Analyses of data heterogeneity. In this study the `conditional'

approach of Huelsenbeck (1996) and Bull et al. (1993) was

followed to determine whether both data sets (molecular

and morphological) should be combined into one analysis.

With this approach the data are combined only if data

partitions are not signi®cantly different. If the result of the

test shows that the two data sets are heterogeneous, the

data should not be combined (Bull et al. 1993). The causes

of data heterogeneity may be due to sampling errors, e.g.

misinterpretation of homology, errors in alignment, site

saturation (`multiple hits'), codon-usage bias, paralogy,

etc., or to differences in the underlying phylogenies, e.g.

reticulated evolution and lineage sorting (Doyle 1997;

Maddison 1997). Tests for heterogeneity, were conducted

using the partition homogeneity test included in PAUP

4.0* and the reciprocal T-PTP test as described by Thiele

(1993).

The partition homogeneity test has the null hypothesis

that a given character partition of a data set into two or

more subsets (two subsets in the present study: molecular

and morphological characters) represents a random parti-

tion of the data (Swofford, personal communication).

Therefore a signi®cant result (P < 0.05) means that the

data partitions are different. In the reciprocal T-PTP test,

each minimum tree, or consensus tree, was used as a

constraint for the other data set (i.e. the morphological

tree was constrained in the molecular data set and vice

versa). If the tree length difference, with and without the

constraint, matches or betters the differences obtained in

50 of the 1000 randomised matrices (in this case), then the

null hypothesis is rejected at the 5% level, and the data

should not be combined. If the null hypothesis cannot be

rejected both types of data re¯ect the same underlying

phylogeny and a phylogenetic analysis using all the

evidence is justi®ed (Rodrigo et al. 1993; Huelsenbeck

et al. 1996)

ResultsSequence data and alignment

Partial sequences of 28S rDNA from the selected taxa

were deposited in GenBank (See Table 1 for Accession

numbers).

The predicted secondary structure of the D3 domain of

28S rRNA for Axinella polypoides is presented in Fig. 1.

Secondary structures for the remaining taxa are basically

the same but the length of the subdomains is variable

(range of variation, in nucleotide numbers, is indicated in

Fig. 1). In general, the secondary structure for the present

taxa agrees with the models presented by Michot et al.

(1990), Gutell et al. (1993), de Rijk et al. (1994) and Nunn

et al. (1996). A consensus secondary structure of the D3

domain for eukaryotes derived by Michot et al. (1990) is

presented as an inset in Fig. 1 for comparative purposes.

Helix H14 and the stems A, B, C and E are present in the

secondary structure derived from the sponge taxa in this

study. Stem D is absent only in the species Ulosa sp.; this

stem, as indicated by Michot et al. (1990), is the most vari-

able region of the D3 domain and is present only in some

metazoan. The alignment of homologous positions in this

stem, and in the region III of subdomain C, was dif®cult

as these regions exhibit the largest variation in nucleotide

numbers.

Figure 2 shows the aligned sequences using secondary

structure constraints. The alignment of homologous posi-

tions using this method proved to be more accurate than

the alignment produced by Clustal W, especially in the

variable regions. To demonstrate this, a portion of the

Clustal W alignment of the subdomains D and C (posi-

tions 75±155) is shown in Fig. 3. Parentheses were

included in this portion of the alignment to indicate the

beginning and the end of both subdomains considering



Fig. 1 Predicted secondary structure of the D3 domain of the 28S rRNA for Axinella polypoides. Inset represents the consensus secondarystructure of the D3 domain derived for eukaryotes by Michot et al. (1990). Stems and variable regions are identi®ed in boldface using thesame nomenclature as Michot et al. (1990). Range of variation in nucleotide numbers for the stems are indicated in parentheses.



Figure 2Ðcontinued overleaf.



structural information. The Clustal W alignment fails to

identify homologous positions within those subdomains,

especially for those taxa in the upper (Cymbastela corallio-

phila) and lower (Scopalina ruetzler and Ulosa sp.) range of

the nucleotide variation. The alignment of other regions of

the molecule, using the multisequence alignment program

Clustal W, however, was not different from the alignment

using secondary structure information.

Phylogenetic estimates using the molecular data

The alignment presented in Fig. 2 resulted in 344 nucleo-

tide positions. Of these, 95 positions were phylogenetically

informative under the parsimony criterion and 214 were

constant.

The phylogenetic analysis of this data set produced a

single most parsimonious tree (Fig. 4) of 309 steps, consis-

tency index (CI) (excluding uninformative characters) of

0.608 and a retention index (RI) of 0.754. The PTP test

was signi®cant (P� 0.001) indicating that the cladistic

structure did not arise by chance alone. Bootstrap and

jack-knife values, as well as branch lengths, are indicated in

Fig. 4. Most relationships are supported by bootstrap and

jack-knife values higher than 50% and several clades have

bootstrap values higher than 85% (equivalent to a 95%

con®dence limit according to Hillis & Bull 1993) which

indicates that the relationships presented in this cladogram

are relatively well supported.

According to the parsimony analysis the family Axinelli-

dae comprises two clades, labelled Axinellidae I and II in

Fig. 4. Axinellidae I is comprised of all species of Axinella

except A. polypoides. This is the sister-group to a clade

comprising species of Halichondriidae, Dictyonellidae and

those included in Axinellidae II. A T-PTP for nonmono-

phyly of Axinellidae (search constrained to ®nd trees that

included Axinellidae I and II as part of a monophyletic

group) was not signi®cant (T-PTP� 0.785), which indi-

cates that monophyly of Axinellidae cannot be rejected.

This phylogenetic analysis indicates that some genera

are nonmonophyletic (i.e. Acanthella, Axinella, Cymbastela,

Halichondria). T-PTP tests of non-monophyly suggest

these data can reject the null hypothesis that Acanthella and

Axinella are monophyletic (T-PTP� 0.002 and 0.034,

respectively), however, cannot reject the hypothesis that

Cymbastela and Halichondria are monophyletic (T-

PTP� 0.739 and 0.600, respectively). Other genera (i.e.

Ptilocaulis, Reniochalina and Pseudaxinella) are monophyletic,

showing that there is some congruence between the mole-

cular data used in this study and the morphological data

currently used to de®ne these genera.

Maximum parsimony analyses of the same data set, but

excluding those regions of variable length indicated as I, II

and III in Fig. 2 (seven possible combinations), were

performed in order to examine how such regions affect the

topology of the tree presented in Fig. 4. As shown in

Table 5, the exclusion of these regions (especially III)

affects the resolution of several branches (indicated with

bold numbers in brackets in Fig. 4), generally below the

genus level, and the topology of the clade `[4]' that unites

species of Acanthella and Cymbastela. An alternative topol-

ogy [(Cyco (Cyve,(Cyst,(Acac,(Acca,Acpu)))) see Fig. 4 for

Fig. 2 Aligned sequences of partial 28S rDNA using secondary structure constraints (see text for explanation). The ®rst 209 bases corre-spond to the D3 domain of the 28S rDNA. Bases within square brackets correspond to the helix denoted as H14 in Figure 1; H140, down-stream complementary counterpart of H14; bases within parentheses correspond to the stems A, B, C, E in the same ®gure. Most variableregions numbered as I.1 (and its downstream counterpart I.1Â ), I.2, II and III. Nucleotides in single-strand loops are indicated by smallletters. Informative positions in the phylogenetic analysis using parsimony are indicated with ^. Dashes indicate gaps. Taxon codes areaccording to Table 1. Sequences are sorted in phyletic order.



taxon codes] is found when analyses 5-7 of Table 5 are

performed. These results suggest that even though these

regions of variable length may have higher levels of homo-

plasy, they appear to be appropriate for reconstruction of

phylogenetic relationships among closely related taxa.

The alignment produced by Clustal W resulted in 321

nucleotide positions, of which only 89 were phylogeneti-

cally informative under the parsimony criterion. Phyloge-

netic analysis of this alignment produced two most-

parsimonious trees of 303 steps and CI� 0.584 (excluding

uninformative characters). The majority consensus rule

tree of this analysis, shows similar topology to the tree

presented in Fig. 4; however, there is poor resolution in

the clade that unites the species of Axinella carteri and A.

aruensis (Fig. 4, branches [1] and [2]) and Cymbastela coral-

liophila is placed as the sister group that unites the species

of Dictyonellidae.

The maximum likelihood analysis of the data set

presented in Fig. 2 produced a very similar topology to the

one obtained with parsimony (Fig. 5). Cymbastela corallio-

phila, in this case is placed as the sister group of the Axinelli-

dae II branch. The tree obtained is only 2 steps longer

(measured by parsimony) than the one presented in Fig. 4.

Phylogenetic relationships using the morphological characters.

The results of the parsimony analysis of the morphological

data set (Table 4) produced 20 most-parsimonious trees of

53 steps, consistency index of 0.698 and retention index of

0.850. The PTP test was signi®cant (PTP� 0.001) indicat-

ing that the cladistic structure did not arise by chance alone.

The majority-rule consensus tree of the 20 most parsi-

monious trees is presented in Fig. 6. In this tree, the taxa

of Axinellidae are united in a single clade. This arrange-

ment indicates that the family is monophyletic group,

rather than nonmonophyletic as shown by the molecular

data. The monophyly of Axinellidae, in this case, is

supported by a T-PTP test (T-PTP� 0.003).

Fig. 3 Portion of the aligned sequences using the multiple sequence alignment program, Clustal W. Bases within parentheses correspondto the stems D and C of Fig. 1. Note that the alignment fails to identify homologous positions in these regions of the D3 domain of the28S rDNA. Base numbers in the bottom correspond to the aligned positions of Fig. 2.

Table 5 Results of parsimony analyses excluding regions ofvariable length (I,II,III) in the alignment presented in Fig. 2.Branches affected by the exclusion of these regions are indicatedwith numbers in brackets in Fig. 4.

No. of parsimonious trees

Analyses Excluded regions Tree length; CI, RI Branches affected

1 I 4; 278; 0.612; 0.759 4±6

2 II 12; 285; 0.611; 0.764 4±6, 8

3 III 56; 209; 0.641; 0.786 1±8

4 I� II 9; 253; 9.617; 0.773 4, 6, 8

5 I� III 24; 177; 0.655; 0.801 1±2, 7±8

6 II� III 32; 183; 0.656; 0.809 1±3, 7±8

7 I� II� III 16; 151; 0.675; 0.828 1±2, 7±8



In general there is little congruence between this phylo-

geny and the one obtained with the molecular data set.

However, relationships among Halichondriidae and

Dictyonellidae species are similar in both data sets with

the species of Halichondriidae, Dictyonellidae and most

taxa of Axinellidae (except for the group labelled as Axinel-

lidae I in the molecular phylogeny) united in a single clade

and with Halichondriidae as the sister group of Dictyonel-

lidae and Axinellidae.

All of the species of Axinellidae in the consensus tree

presented in Fig. 6 are united in a clade with the species of

Ptilocaulis and Reniochalina as the sister group of the

remaining axinellids. In the molecular tree the relation-

ships among the species of Ptilocaulis and Reniochalina are

identical to those indicated in Fig. 6, but the position of

the clade uniting the species of both genera is different in

relation to the other groups.

The remaining taxa of Axinellidae are grouped in a

polytomy with one clade uniting Axinella carteri and A.

aruensis, another uniting Acanthella and Phakellia sp., and a

dichotomous clade with the remaining species of Axinelli-

dae. The latter branch includes the clade that unites the

species of Pseudaxinella to another dichotomous branch

with A. damicornis and A. polypoides in one clade and the

species of Cymbastela in the other. These relationships

differ to those indicated in Fig. 4.

Fig. 4 Most-parsimonious tree (unrooted) derived from the aligned sequences presented in Fig. 2. Branch length is indicated aboveeach branch and bootstrap/jack-knife values below. Branches with bootstrap values less than 50% have poor support and should beregarded with scepticism. Numbers in brackets indicate those branches that collapse when regions of variable length are excluded (seeTable 5).



The arrangement of the species of Axinella, according to

the morphological data, indicates that the genus is nonmo-

nophyletic. In the phylogeny derived from sequencing data

all the species of Axinella, with the exception of A. poly-

poides, are included in a single clade. A T-PTP test for

nonmonophyly, using a constraint tree in which all the

species of Axinella are included in a single monophyletic

group, was non signi®cant (T-PTP = 0.993) indicating

that these data cannot reject the hypothesis that Axinella is

monophyletic.

The placement of Phakellia sp. also differs from the

molecular tree. In this case (Fig. 6) the species is united in

a clade with the species of Acanthella, rather than as the

sister species of the clade containing the Pseudaxinella spp.

(Fig. 4). The relationships of Phakellia and the species of

Acanthella are therefore ambiguous, and thus the mono-

phyly of the genus Acanthella is questionable; however, a

T-PTP test for nonmonophyly of Acanthella was not

signi®cant (T-PTP� 0.954) and indicates that the mono-

phyly of the genus cannot be rejected.

The bootstrap and jack-knife values of the tree

presented in Fig. 6 range from 33 to 80%; branches with

values less than 50% re¯ect no support.

The character-state changes were mapped onto one of

the 20 most-parsimonious trees (Fig. 7). Some characters

appear to be good indicators of the phylogeny of the

group with consistency indices (CI) greater than, or equal

to, 0.5 (characters 1±7, 10, 11, 14±16). The best characters,

with CI� 1, in this phylogenetic analysis are as follows:

A specialized ectosomal skeleton is absent 2(0) in most

species of Axinellidae (except for Cymbastela coralliophila which

has a paratangential ectosomal skeleton 2(2)) and present as a

tangential crust 2(1) in the species of Halichondriidae.

Fig. 5 Maximum likelihood tree. Only the part of the topologythat differs from the tree in Fig. 4 is shown. Relationships withinthe different groups (Agelasida, Halichondriidae, Agelasida, andAxinellidae I-II) are the same in both trees.

Fig. 6 Majority consensus tree (unrooted) derived from the morphological character set (Table 4). Bootstrap/jack-knife values are indi-cated below the branches. Branches with bootstrap values less than 50% have poor support and should be viewed cautiously.



Stromatoporid choanosomal skeletons, 3(0) and reticu-

lated ones, 3(1) are autapomorphies of Astrosclera willeyana

and Agelas mauritiana, respectively. Irregularly reticulated

skeletons 3(2) are present in Scopalina ruetzleri and Ulosa

sp.; plumose ones 3(3) are present in species of Axinella

(carteri and aruensis), Reniochalina and Ptilocaulis; plumoreti-

culate skeletons 3(4) are a synapomorphy for species of

Axinella (polypoides and damicornis), Cymbastela, and Pseudax-

inella; plumose-radiate skeletons 3(5) de®nes the clade that

unites species of Acanthella (except A. acuta) and Phakellia

sp.; dendritic skeleton 3(6) is an autapomorphy for

Acanthella acuta.

An axial skeleton is absent 4(0) in all non-axinellid

species, in Acanthella acuta and in the species of Pseudaxi-

nella; the axial skeleton is condensed in species of

Acanthella (except A. acuta) and Phakellia sp. 4(1); plumore-

ticulate in the species of Cymbastela, Axinella polypoides and

A. damicornis 4(2); vaguely plumose in the rest of Axinella

spp. 4(3) and a close set reticulation of irregularly anasto-

mosing ®bres in the species of Ptilocaulis and Reniochalina,

4(4).

Spongin ®bres are absent 5(0) in Astrosclera willeyana,

cored and echinated with spicules 5(1) in Agelas mauritiana,

cored (but not echinated), with columns or tracts of

spicules 5(2) in the species of Dictyonellidae, Ptilocaulis and

Reniochalina; and lightly investing spicule tracts 5(3) in the

rest of Axinellidae.

Primary (ascending) spicule tracts are absent in the

species of Agelasida, Dictyonellidae and in Halichondria

panicea 6(0) and present as directionless bundles 6(1), in

the other two species of Halichondriidae (H. phakellioides

and Ciocalypta confossa). The plumose condition 6(2), is a

synapomorphy of all taxa of Axinellidae but changes to the

state columns of sinuous strongyles echinated by styles

6(3) at the node that unites species of Acanthella and

Phakellia sp.

Terminally spined anisoxeas 11(1) is a synapomorphy

for species of Reniochalina.

The presence of strongyles 14(1) is a synapomorphy for

the species of Acanthella (A.cavernosa and A. pulcherrima)

and Phakellia sp.

Verticillate acanthostyles 15(0) are present in Agelas

mauritiana and Astrosclera willeyana.

Rooting the trees

The cladograms presented above (Figs 4,5,6,7) are

unrooted. Three alternative outgroups can be used to root

these trees. Figure 8a-c shows the different topologies

obtained when the tree derived from molecular data is

rooted using the members of Agelasida (e.g. Astrosclera

willeyana and Agelas mauritiana) (Fig. 8a), Halichondriidae

(Fig. 8b) and Dictyonellidae (Fig. 8c) as outgroups. The

molecular tree cannot be rooted using all three groups, or

a combination of any two, without constraining the

ingroup to be monophyletic and thus resulting in a less

parsimonious tree. The three different topologies

presented in Fig. 8a-c also indicate that Axinellidae I and

II are members of different groups and con®rm that the

family is nonmonophyletic independently of the outgroup

selection. In Fig. 8d-f the morphological tree is rooted

with the same outgroups. In this case however, the tree

can be rooted using all three groups, or a combination of

any two; that means Axinellidae is a monophyletic group

independently of the outgroup selection.

Analyses of data heterogeneity

The results of the partition homogeneity test were signif-

icant (P� 0.001) indicating that the two data partitions

of this study (molecular and morphological) are signi®-

cantly different, and therefore should not be combined.

Results of reciprocal T-PTP tests were inconclusive;

differences in the trees estimated from both data sets

were not signi®cant (T-PTP� 0.987) when the molecular

tree was constrained onto the morphological data set (i.e.

either tree could be supported by the morphological

data) but signi®cant (T-PTP� 0.019) when the majority

rule consensus tree derived from the morphological data

was constrained onto the molecular data set (i.e. the

morphological tree cannot be supported by molecular

data).

Discussion and ConclusionsThe main purpose of this study was to test whether or not

Axinellidae is a monophyletic group and to evaluate the

position of the family in the higher classi®cation scheme.

The phylogenetic relationships inferred using molecular

and morphological data are not fully congruent; the mole-

cular data set shows the Axinellidae to be non-monophy-

letic whereas the morphological data shows it as

monophyletic. Only two branches were identical in both

cladograms, the one uniting the species of Ptilocaulis and

Reniochalina and the one with the species of Dictyonellidae;

the branch uniting the species of Pseudaxinella was compa-

tible in both trees.

The analysis of data heterogeneity indicated that the

two data sets are signi®cantly different and therefore

should not be combined under the conditional approach

(Bull et al. 1993; Huelsenbeck et al. 1996). Further, the

two data sets are unequally sized (95 informative molecular

characters vs. 16 informative morphological characters) so

that the molecular signal, which is different, will swamp

the morphological signal. Therefore, a simultaneous analy-

sis (e.g. one including both types of data) was considered

inappropriate.



With these incongruent results the central question of

this paper cannot be answered unambiguously; however,

several aspects of these con¯icting results can be discussed.

Selection of taxa. The objective representative of a genus is

its type species. Thus in a comparative analysis of molecu-

lar and morphological data sets, for each genus at least its

type species should be included. Additional species of a

genus should preferably be included to con®rm mono-

phyly. If the type species is not included, or if species with

dubious generic af®nity are included, discrepancies that

might confound the conclusions could be expected. In this

study, seven of 13 genera are represented by the type

species. Agelas, Scopalina and Pseudaxinella are represented

by species that are not types, but their membership in

those genera is not contested. Phakellia, Ciocalypta and

Ulosa are the only genera represented by species whose

generic position could conceivably be questioned; however,

voucher material is available to con®rm the identity of

these species and to resolve any future discrepancies that

could arise from this study. Some valid genera of Axinelli-

dae such as Bubaris and Auletta were not included in this

study mainly because of dif®culties in obtaining material

suitable for DNA extraction. Overall, the sample of taxa is

considered appropriate to test the monophyly of Axinelli-

dae as it includes a balanced set of species that are

currently considered unequivocal members of the family.

The phylogenetic relationships depicted by both data sets

can be tested in future studies by the addition of taxa that

are not represented here.

Selection of outgroups. Representatives of the families Hali-

chondriidae, Dictyonellidae and Agelasida were selected as

outgroups because it has been suggested that they are

closely related to the Axinellidae based on morphology,

molecular and chemical evidence. Members of other

families (e.g. Raspailiidae and Hemiasterellidae), considered

related to Axinellidae by some authors (e.g. LeÂvi 1973;

Fig. 7 Character distribution in one of the 20 most parsimonious trees derived from the morphological character set (Table 4); solid bar,synapomorphy; stippled bur, parallelism; slanted lines, reversal



Bergquist 1980), were not included in this analysis because

they are currently allocated to different orders (Poecilo-

sclerida and Hadromerida, respectively) and are considered

here more distantly related to Axinellidae than the selected

outgroups. The Desmoxyidae, proposed as one of the sister

groups of Axinellidae by Van Soest et al. (1990), has not

been revised lately and its monophyly is questionable;

therefore, it was considered unsuitable as an outgroup too.

Different topologies were obtained when each of the

selected outgroups was used to root the trees (see Fig. 8)

However, the answer to the question under study remained

unaffected; the molecular tree shows Axinellidae to be

nonmonophyletic and the morphological tree as monophy-

letic, independently of the position of the root or the

outgroup selection. Determination of the actual sister

group of Axinellidae relies on accepting the hypothesis

that Axinellidae is monophyletic. If further evidence is

found to support or reject the monophyly of this family,

then the inclusion of additional monophyletic groups more

distantly related to the ones selected here, will be necessary

to establish the phylogenetic relationships of Axinellidae

(as a monophyletic group) or of the two different groups

of `Axinellidae' (e.g. Axinellidae I and II) in relation to

their sister taxa.

The position of the family in the higher taxonomic clas-

si®cation also depends on whether one accepts or not

monophyly of Axinellidae. The order Halichondrida sensu

Van Soest et al. (1990), is currently accepted as the best

taxon in which to place the Axinellidae; however, the

results derived from the present study cannot be used to

challenge or con®rm the phylogenetic relationships within

that order. A more balanced data set, including other

members of the families Halichondriidae, Dictyonellidae,

and also Desmoxyidae, would be necessary to study phylo-

genetic relationships within the order Halichondrida

(assuming that Halichondrida is monophyletic).

Selection of the DNA region. The region of the 28S rDNA

selected here, has not been used to date to study sponge

phylogeny. It includes the D3 domain, which exhibits a

size variation of 115±146 bp among the species analysed

here and is within the range reported for other eukaryotes

(see Michot et al. 1990), plus a conservative region of

approximately 135 bp with only 7 phylogenetic informative

positions under parsimony criterion (in the alignment

presented in Fig. 2). The remaining 88 phylogenetically

informative positions are all included in the D3 region.

The D3 domain also includes several subdomains with

Fig. 8 Molecular (a-c) and morphological (d-f ) tree, rooted with three different outgroups (Agelasida, Halichondriidae and Dictyonelli-dae). Taxon labels correspond to those in Fig. 4.



large size variation in nucleotide numbers (especially

subdomain D and C indicated in Fig. 1). Subdomain D in

particular is absent in Ulosa sp. These variable subdomains

are dif®cult to align and may have higher levels of homo-

plasy but their exclusion affects the resolution (generally

below the genus level) of at least eight branches in the

phylogenetic tree presented in Fig. 4. These results

suggest that although these regions of variable length may

have higher levels of homoplasy, they seem to be appro-

priated for reconstruction of the phylogenetic relationships

among closely related taxa. Similar ®ndings are reported

by Titus & Larson (1995) when regions of variable length

of mitochondrial rDNA are excluded from a phylogenetic

analysis of a salamander family.

Detailed study of the divergent domain D3 by Michot

et al. (1990) shows that the region is suitable for phyloge-

netic studies across archaebacteria, eubacteria, eukaryotes,

or within subgroups of metazoans, as it includes conserva-

tive regions that increase the accuracy in the alignment of

homologous sites among the sequences and enough varia-

tion interspersed within the universally conserved subdo-

mains. These ®ndings are con®rmed from the sequences of

sponges obtained here. Phylogenetic relationships of other

invertebrates such as species of the crustacean order

Isopoda (Nunn et al. 1996) and species of the nematode

genus Pratylenchus (Al-Banna et al. 1997) have also been

investigated using this region, showing that the D3

domain presents enough variation within these groups. For

sponges, the region selected seems to have an appropriate

amount of phylogenetic signal to study relationships

within the family level and especially at the genus level (see

below under taxonomic considerations).

Analysis of molecular data. The use of secondary structure

constraints has been demonstrated here to be an effective

and objective method to align homologous positions of

DNA sequences and is recommended as a method for

general use. The alignment presented in Fig. 2 which was

used to produce the phylogenetic trees (Figs 4,5) is consid-

ered accurate and free from potential problems derived

from the misalignment of homologous positions. The

aligned sequences were used to construct trees using two

different optimality criteria, parsimony and maximum like-

lihood. The parsimony method might be sensitive to the

phenomenon described as `long-branch attraction'; the

method of maximum likelihood, which has a less chance to

be affected by this problem, was used as an alternative

method (for review on this topic see Morrison 1996; Swof-

ford et al. 1996; Huelsenbeck 1998). The tree topologies

obtained with both methods are nearly identical, differing

only in the position of Cymbastela coralliophila, which is the

terminal taxon in the longest branch in the tree obtained

by parsimony. This result suggests that the phylogenetic

relationships of C. coralliophila in relation to the other taxa

might be debatable (see also below under taxonomic

comments).

Selection of morphological characters. Members of Axinellidae

and related groups are characterized by a relative small

number of very simple characters, mostly skeletal ones and

those are generally homoplastic. A phylogenetic analysis

based on this type and quantity of data is likely to be less

than satisfactory. This is re¯ected in the trees resulting

from the morphological analysis (Figs 6,7) in which most

terminal branches have zero length, and some are collapsed

leaving some groups as unresolved polytomies (e.g. species

of Cymbastela, Pseudaxinella). This indicates that there are

insuf®cient morphological differences to distinguish

amongst the species included in the present study.

Although skeletal characters seem to be good indicators

of the phylogeny for several sponge groups (Bergquist &

Kelly-Borges 1991; Hajdu 1993; Hajdu et al. 1994; Hajdu

& Van Soest 1996; Rosell & Uriz 1997 for example), they

seem to be insuf®cient to study the phylogeny of groups

like Axinellidae. Other type of characters related to the

sponge anatomy, such as the features of the aquiferous

system or the ultrastructure of cells, need to be considered

to complement the present morphological characters in

future studies. Such characters were not included here, as

special methods for the preparation of the sponge tissue

are required, and they were not suitable to use.

Robustness of the trees. The results of the PTP tests for both

data sets were signi®cant, indicating that the cladistic

structure of the trees presented here did not arise by

chance alone. Most of the branches of the phylogeny based

on molecular data (Fig. 4), are supported by low bootstrap

values (28±85%) indicating that the relationships among

some of the taxa included in the analysis cannot be con®-

dently resolved and might have arisen by chance alone.

The inclusion of more characters (i.e. longer sequences)

might increase the support of individual branches but this

is not always the case (see Gee 1995 and references within).

The phylogenetic relationships using morphological

data (Fig. 6) are not supported by high bootstrap values

(33-80%) either and should be viewed cautiously. The lack

of morphological characters suitable for this morphological

analysis and the simplicity of the only ones available are

possible reasons for the poorly supported relationships.

Is Axinellidae monophyletic? The results obtained in this

study are incongruent and leave the question without a

de®nitive answer. According to the morphological data set

the family Axinellidae is monophyletic, which is not unex-

pected, since the family was recognized on the basis of

morphology. The evidence provided by the morphological



analysis is weak. Monophyly of Axinellidae is supported by

the presence of plumose ascending spicule tracts 6(2)

which later changes to the character state 6(3) (columns of

strongyles echinated by styles), in the node that unites the

species of Acanthella and Phakellia. The presence of

plumose ascending tracts does not seems to be a good

diagnostic character of the family as this type of spicule

tracts are present in other sponge families (i.e. Raspailiidae

and Microcionidae). Note also that the presence of an axial

skeleton (character 4), which has been used by some

authors in the diagnosis of some genera as well as for the

family Axinellidae itself (Vosmaer 1912; Bergquist 1970;

Wiedenmayer 1989; Van Soest et al. 1990; Hooper &

Bergquist 1992; Hooper & LeÂvi 1993; Hooper & Wieden-

mayer 1994), is not a synapomorphic character for the

family. An axial skeleton might be also dependent on the

growth habit; therefore, it will be present in branching or

stalked species such as many of the ones represented in

this data set. As suggested by Van Soest (1991) the

condensation of the axial skeleton in this type of growth

form might be a functional adaptation to provide rigidity.

The results obtained from this analysis demonstrate that

the homology assessment of morphological characters in

the study of phylogenetic relationships of sponges is

problematic. Therefore the use of skeletal characters in

this type of studies should be considered carefully.

The results from the molecular analysis on the other

hand, seem to be more robust and indicate nonmonophyly

of Axinellidae. This has been suggested previously from

biochemical evidence as follows.

Based on pro®les of free amino acids, carotenoids and

general proteins, from species of sponge families including

Hooper & Bergquist (1992) found evidence to indicate that

the family Axinellidae `is clearly heterogeneous'. Differ-

ences in the pro®les of 27 species of six genera of Axinelli-

dae identi®ed two disparate groups of `Axinellidae': one (i.e.

Phakellia and Reniochalina) showing af®nities with members

of families Raspailiidae, Microcionidae and Myxillidae, and

a second group (i.e. Acanthella, Pseudaxinyssa [�Cymbastela]

and Axinella) showing af®nities with members of the

families Desmoxyidae and Hemiasterellidae.

Braekman et al. (1992) on the other hand, found that the

distribution of secondary (� species-speci®c) metabolites,

in particular terpenoid derivatives, pyrroles and isocya-

nides, among several species of Agelasidae, Axinellidae and

Halichondriidae indicated that the Axinellidae do not form

an homogeneous group and suggested that the family

should be divided. The authors presented two alternative

explanations for the relationships exhibited: that the

chemicals were homoplastic; or that the Axinellidae

included two groups, with one of them (Axinellidae I),

closely related to species of Agelasida. The detection of N-

methylated ageliferins (a group of terpenoid derivatives

common to Agelas spp.) in Astrosclera willeyana by Williams

& Faulkner (1996) further supports the suggested relation-

ship between the hypercalci®ed sponges (previously allo-

cated to Sclerospongiae), and members of the Agelasidae.

It also supports the inclusion of Astroscleridae in the order

Agelasida (van Soest 1984; Hooper & Wiedenmayer 1994;

WoÈ rheide et al. 1996; WoÈ rheide 1998). In this study, the

Agelasida and some of the Axinellidae (Axinellidae I) were

found to be closely related based on molecular characteris-

tics (see Fig. 8b,c). Such an arrangement is supported also

by the presence of pyrroles in both groups, and coincides

with one of the alternative explanations of the relationships

of these two groups proposed by Braekman et al. (1992).

Additional support for a close relationship between some

species of Agelasida and species of Axinella has been found

by Bergquist et al. (1980), who detected a dominance of

saturated stanols in the sterol mixture of Axinella damicornis

and Agelas oroides, and by Lafay et al. (1992) and Chombard

et al. (1997), who showed, based on partial 28S rDNA

sequences, that Axinella damicornis is the sister species of

Astrosclera willeyana and Agelas oroides. From the morpholo-

gical point of view however, these two groups are very

different and no morphological synapomorphy is currently

available to support such a grouping.

It should be mentioned also that some of the secondary

metabolites detected in sponges might be the products of

biosynthesis by symbionts commonly associated with the

sponge-host; this possibility should be investigated as it

would confound phylogenetic analysis of the taxa involved.

Some members of Agelasidae (such as Agelas oroides) and

Axinellidae (i.e. Acanthella acuta, Axinella polypoides and A.

damicornis) have shown associated bacteria and at least in

Agelas they can occur in large numbers (Vacelet & Dona-

dey 1977). The question remains open; if more molecular

and chemical evidence is found to support relationships

such as the one between Agelasidae and Axinellidae I, the

value of the morphological characters currently used in

phylogeny of these sponges will be seriously challenged.

Additional evidence from the sequences of the same

region of 28S rDNA studied here, and axinellid species

from the New Zealand area (Alvarez, unpublished data)

further support the nonmonophyly of the Axinellidae and

encourage further investigation of the topic. No changes

to the taxonomic position of the family in the higher clas-

si®cation should be carried out without additional evidence

from a different gene(s) (or gene regions), biochemical

characters and morphological characters other than skeletal

ones.

Clari®cation of some taxonomic problems. Based on the

present results, several comments can be made regarding



the taxonomy of some of the species and genera included

here:

(a) Axinella carteri (Dendy). This species has been

included in Acanthella by some authors (Dendy 1889;

LeÂvi 1979; Van Soest 1989) or in Axinella by others

(Burton 1959; LeÂvi 1979; Hooper & LeÂvi 1993). The

species shows some morphological features of

Acanthella, such as shape and ectosome. Other

features, such as spicule geometry and plumose skele-

ton and presence of an axial skeleton, are in common

with Axinella spp. The present study shows that both

the specimen from the Seychelles and the one from

Great Barrier Reef are, genetically and morphologi-

cally, more closely related to other species of Axinella

such as A. damicornis and A. aruensis than to the

species of Acanthella, thus supporting the inclusion of

A. carteri in Axinella.

(b) Acanthella pulcherrima Ridley & Dendy. This species

was transferred to Phakellia by Hooper & LeÂvi (1993),

based on features described from material collected

from New Caledonia which lacked the anisoxeas seen

in previous records of the species (Ridley & Dendy

1886; Dendy 1922), and in the specimen included in

this study. Although anisoxeas are common in

Acanthella species, such as A. cavernosa, this is not an

important diagnostic character differentiating

Acanthella from Phakellia. The features of the material

described by Hooper & LeÂvi (1993) under Phakellia

are in agreement with the concept of Acanthella

adopted in this and previous (Alvarez et al. 1998)

studies and the species is therefore returned to this

genus. This decision is further supported here by the

results of both phylogenetic analyses, which show that

the species is closely related to Acanthella cavernosa.

(c) Reniochalina and Ptilocaulis are closely related genera

from the morphological point of view, as has already

been established (Hooper & LeÂvi 1993). Both have

similar skeletal architecture, spicules and external

morphology, including colour and surface processes

(called here `scopiform', character state 1(4) which

represents a synapomorphy for both genera), and

might even be confused in the ®eld. Species of Renio-

chalina are characterized by the possession of termin-

ally spined anisoxeas (minute in the case of

Reniochalina sp.).

(d) The position of the single specimen of Phakellia sp. is

ambiguous. Based on the molecular data this species is

closely related to Pseudaxinella, but there are no

obvious morphological characters to support this rela-

tionship. Based on morphology, this species is closely

related to Acanthella pulcherrima and A. cavernosa and

might be better placed in Acanthella. The close rela-

tionships between Phakellia sp. and Pseudaxinella

species shown in the analysis derived from the mole-

cular data is intriguing.

(e) The monophyly of the genus Cymbastela and its rela-

tionships with other Acanthella species need further

investigation. The molecular data showed that species

of Acanthella and Cymbastela (except C. coralliophila) are

part of a monophyletic group. This branch is well

supported by a bootstrap value of 94% and a T-PTP

test (P� 0.002). The two genera from the morpholo-

gical point however, are not closely related. Further

evidence is required to con®rm that they are as closely

related as shown by the molecular data, perhaps from

additional morphological characters not obvious at

present. The position of Cymbastela coralliophila is also

debatable. The species is rather atypical of the genus,

as it contains two distinct sizes of oxeas, with the

smallest ones forming a specialized dermal skeleton, a

feature not seen in other species of Axinellidae. The

results of the maximum likelihood analysis suggest

that the species might not be a member of the group

labelled here as Axinellidae II. The placement of the

species within Cymbastela should be reconsidered.

(f ) The relationships among the species of Halichondria

and Ciocalypta confossa are incongruent in the two

analyses. In the molecular tree the latter is the sister

species of H. panicea and in the morphological tree

appears as the sister species of H. phakelloides. From

the morphological point of view, Ciocalypta and Hali-

chondria are closely related taxa with similar skeletal

architectures (Van Soest et al. 1990; Hooper et al.

1997). Additional species of Halichondria, Hymeniacidon

or species of the Halichondria-Hymeniacidon and Cioca-

lypta-Amorphinopsis groups in the sense of Van Soest

et al. (1990) should be added to this analysis to test

whether or not Halichondria and Ciocalypta are mono-

phyletic groups.

AcknowledgementsThis study was made possible by an Australian National

University (ANU) graduate scholarship. Additional ®nan-

cial and logistic support was also provided by Division of

Botany and Zoology, Canberra (ANU), Queensland

Museum, Brisbane (QM); CSIRO, Division of Entomol-

ogy, Canberra (CSIRO, Entomology), Electron Micro-

scopy Unit, Research School of Biological Sciences

(EMU-ANU), National Institute of Water and Atmo-

spheric Research, Wellington (NIWA) and New Zealand

Lottery Grants Board.

We would like to thank Dr Christopher J. Glasby, Dr

Dennis Gordon, Dr Chris Battershill (NIWA) and Dr



Penny Gullan (ANU), for their comments on the manu-

script. Dr Dave Swofford, Laboratory of Molecular

Systematics, Smithsonian Institution, for allowed the use

of his test version of the phylogenetic program paup 4.0*;

Dr M. Cristina DõÂaz, University of California, Santa Cruz

and Dr Nicole Boury-Esnault, Centre d' OceÂanologie de

Marseille, for providing specimens for DNA extraction; Dr

John Trueman, Research School of Biological Sciences

(ANU) for his assistance with some of the cladistic

analyses. Lisa Goudy (University of Melbourne, Australia),

Steve Cook and John Kennedy (QM) for their assistance

with the sampling and SEM work.

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AppendixList of species and material examined in this study with a

brief taxonomic description of those species that have not

been previously recorded in the literature. Spicule sizes are

in micrometres presented as ranges of length and width

with the average and standard deviation indicated in

parentheses. The number of spicules measured is indicated

in square brackets.

Family ASTROSCLERIDAE Lister, 1900

Astrosclera willeyana Lister, 1900

Astrosclera willeyana Lister 1900: 459; Ayling, 1982: 100;

Hooper & Wiedenmayer, 1994 : 68; WoÈ rheide et al.,

1996: 150

Material. ZMA Por. 11342, Seychelles, Ile Bijoutier/

Alphonse Atoll, SW part of lagoon, 7820S 528430E, coll.

R.W.M Van Soest, 3 JAN 93, Indian Ocean Program-

Theme E expedition [IOP-E], station 792/01, 6±18 m.

Family AGELASIDAE Verrill (1907)

Agelas mauritiana Carter, 1883

Ectyon mauritianus Carter, 1883: 310.

Agelas mauritiana Ridley, 1881: 164; Dendy, 1905: 174,

with additional synonyms; Burton, 1959: 243; Hooper &

Wiedenmayer, 1994: 47.

Material. ZMA Por. 10991, Seychelles, La Digue, S coast,

0423S 5549E, coll. H.A. 10 Hove, 23 DEC 92, IOP-E

station 735/12, 12 m.

Family AXINELLIDAE Carter, 1875

Acanthella acuta Schmidt, 1862

Acanthella acuta Schmidt 1862: 65; Gray, 1867: 512;

Schmidt, 1868: 10; Topsent, 1902: 349; Babic, 1922: 243;



Topsent, 1934: 26; SaraÁ , 1958:223; Vacelet, 1969: 177;

Boury-Esnault, 1971:304; Pulitzer-Finali, 1977: 36; Pulit-

zer-Finali, 1983: 519.

Material. QM G 313206, La Ciotat; Bec de l Aiyle; France,

coll. Nicole Boury-Esnault, 10 OCT 94, 15 m.

Acanthella cavernosa Dendy, 1922

Acanthella cavernosa Dendy, 1922: 120; Burton, 1934: 565;

Van Soest 1989: 226; Hooper & LeÂvi, 1993 : 1415 [as

Phakellia]; Hooper & Wiedenmayer, 1994: 71.

Material. ZMA Por. 10989, Seychelles, Ile Desroches,

W rim, 058410S 538350E, coll. R.W.M Van Soest, 31 DEC

92, IOP-E station, 772/07, 10±11 m.

Acanthella pulcherrima Ridley & Dendy, 1886

Acanthella pulcherrima Ridley & Dendy, 1886 : 479; Ridley,

1884: 463 [as Acanthella]; Ridley & Dendy, 1887: 177;

Vosmaer, 1912: 316

Phakellia pulcherrima Hooper & LeÂvi, 1993 : 1415

Material. QM G303386, Stephen's Rock, West Arm,

Darwin Harbour NT, Australia, 12.29.20S; 130.47.00E,

coll. Hooper, JNA & Hobbs, LJ, station, JH-93±024, coral

pinnacle, SCUBA, 19 m

Axinella aruensis (Hentschel, 1912 : 420)

Phakellia aruensis Hentschel, 1912: 420; Pulitzer-Finali,

1993: 283

Axinella aruensis Hooper & Wiedenmayer, 1994: 72

Material. QM G 303332, East Point Bommies, Darwin

Harbour NT, Australia, 12.24.50S; 130.48.80E, coll.

Hooper, JNA & Hobbs, LJ, 23 SEP 93, station JH-93±

023, coral heads, Junceela, SCUBA, 10 m.

Axinella carteri (Dendy, 1889)

Acanthella carteri Dendy, 1889: 93; Dendy, 1905: 193; Dendy,

1922: 119; Vacelet et al., 1976:43; Van Soest 1989: 228

Axinella carteri Burton, 1959: 258; Hooper & LeÂvi,

1993 : 1410; Hooper & Wiedenmayer 1994: 72

Material. QM G 304294, N. tip of Day Reef, Cormorant

Pass Section, GBR QLD, Australia, 14.28.40S; 145.31.20E,

coll. Hooper, J.N.A; Hobbs, L.J.; Kennedy, J. & Cook,

S.D., 7 APR 94, station, JH-94±01, barrier coral reef, drop

off near outer barrier, sheer coral slope, caverns, rubble at

base, SCUBA, 25 m. ZMA Por. 10992 [now registered as

Stylissa carteri], Seychelles, Bird Isl., coll. R.W.M Van Soest,

038430S 558120E, 22 DEC 92, IOP-E station 723/13, 7 m.

Axinella damicornis Esper, 1794 Schmidt 1862

Spongia damicornis Esper, 1794 ®de Schmidt 1862

Axinella damicornis Schmidt 1862: 61; Topsent, 1891: 529;

Vosmaer, 1912: 308, 315; Vosmaer, 1932±35: 723;

Topsent 1934: 33; SaraÁ 1958: 221; SaraÁ, 1960: 452; Siri-

belli, 1961:122; Cabioch, 1968: 222; Vacelet 1969: 175;

Pulitzer-Finali 1977: 35; Pansini, 1982,83: 87; Pulitzer-

Finali 1983: 514

Material. QM G 313204, La Ciotat; Bec de l'Aiyle, France,

coll. Nicole Boury-Esnault, 8 AUG 94, 15 m.

Axinella polypoides Schmidt, 1862

Axinella polypoides Gray 1867: 514; Schmidt 1868: 9;

Schmidt, 1870: 60; Carter, 1884: 205; Topsent 1902: 348;

Babic 1922: 238; Topsent, 1928: 173; Vosmaer 1932±35:

723; Topsent 1934: 34; SaraÁ 1960: 452; Vacelet

1969 : 175; Boury-Esnault 1971: 303; Pulitzer-Finali 1983:

516

Material. QM G 313205, La Ciotat; Bec de l'Aiyle, France,

coll. Nicole Boury-Esnault, 8 AUG 94, 20 m.

Cymbastela stipitata Bergquist & Tizard, 1967

Pseudaxinyssa stipitata Bergquist & Tizard, 1967: 189±191

Cymbastella stipitata Hooper & Bergquist, 1992: 106;

Hooper & Wiedenmayer, 1994: 75

Material. QM G 303262, East Arm, Darwin, reef N of

boat ramp, S of S Shell, NT, Australia, 12.29.80S;

130.53.50E, coll. Hooper, JNA & Hobbs, LJ, 19 SEP 93,

station JH-93±021, intertidal reef, by hand.

Cymbastela coralliophila Hooper & Bergquist, 1992

Cymbastela coralliophila Hooper & Bergquist, 1992: 120

Material. QM G 304173, off Turtle beach, Lizard Island,

headland N. side Bay; QLD; Australia, 14.39.10S;

145.27.00E, coll. Hooper, J.N.A; Hobbs, L.J.; Kennedy, J.

& Cook, S.D, 4 APR 94, station 94±006, fringing coral

reef, sand at base, SCUBA, 15 m.

Cymbastela vespertina Hooper & Bergquist 1992

Cymbastela vespertina Hooper & Bergquist 1992: 110

Material. QM G 303341, East Point Bommies, Darwin

Harbour NT, Australia, 12.24.5S; 130.48.8E, coll.

Hooper, JNA & Hobbs, LJ, 23 SEP 93, station JH-93±

023, coral heads, Junceela, SCUBA, 10 m.

Phakellia sp.

Material. QM G 303365, Stephen's Rock, West Arm,

Darwin Harbour NT,

Australia, 12.29.20S; 130.47.00E, coll. Hooper, JNA &



Hobbs, LJ, 24 SEP 93, station JH-93±024, coral pinnacle,

SCUBA, 19 m.

Description. Shape. Lamellate, 3 mm thick, 10 cm tall, with

undulate margins, partially folded.

Surface. Smooth to velvety, due to projections of spicules

through ectosome, with small tubercles, not higher than

1 mm, distributed unevenly or in small rows on each side

of lamella; without visible oscules or pores.

Consistency. Hard in alcohol.

Colour. Ectosome pale yellow; choanosome pale orange.

Skeleton. Without specialized ectosomal skeleton. Choano-

somal skeleton as dense mass of interwoven strongyles,

occupying whole thickness of sponge, and styles projecting

perpendicular to surface, protruding through ectosome.

Spicules. Strongyles, length 200.5±611.2 mm (435.6 � 100.8)

[25], width 5.6±11.5 mm (8.8 � 1.8) [25]. Styles in two size

categories: I. length 231.6±483.4 mm (330.7 � 74.7), width

9.3±18.8 mm (13.1 � 2.4) [25] and II. length 142.8±259.4 mm

(192.7 � 39.1), width 3.7±11.8 mm (7.6 � 1.7) [25].

Remarks. The description of the species does not agree

completely with the de®nition of Phakellia sensu Alvarez

et al. (1998). The choanosomal skeleton is strongly

compressed, without a reticulation or multiple axes of

thick spicule tracts as seen in other Phakellia spp. Other

characters, such as habit, the styles echinating the mass of

interwoven strongyles of the choanosome, the composi-

tion, distribution and the type of other megascleres agree

with the de®nition of Phakellia.

Pseudaxinella australis Bergquist, 1970

Pseudaxinella australis Bergquist, 1970: 20; Hooper ,& LeÂvi

1993: 1439; Hooper & Wiedenmayer, 1994: 80

Material. QM G 303444, Fish Reef, W side near Brisbane,

Bynoe Harbour, NT, Australia, 12.26.10S; 130.26.50E, coll.

Hooper, JNA & Hobbs, LJ, station JH-93±027, coral

slope, SCUBA, 11 m.

Pseudaxinella durissima Dendy, 1905 new combina-

tion.

Thrinacophora durissima Dendy, 1905: 187.

Sigmaxinella durissima Dendy, 1922: 113.

Axinella durissima Burton, 1959 : 259, with additional

synonyms.

Material. ZMA Por. 10990, Seychelles, St. Joseph Atoll,

NW rim, 058250S 538190E, coll. R.W.M Van Soest, 26

DEC 92, IOP-E station 753/15, 5±22 m.

Remarks. The species is assigned here to Pseudaxinella

based on its similarities in habit, skeleton architecture,

spicule composition and geometry, with other species of

Pseudaxinella. It is in accord with the de®nition of Pseudaxi-

nella of recent authors Bergquist, 1970 : 20; LeÂvi, 1973;

Wiedenmayer, 1977: 152, 155; Wiedenmayer, 1989: 47;

Hooper & LeÂvi, 1993: 1436; Alvarez et al., 1998.

Pseudaxinella reticulata Ridley & Dendy, 1886.

Axinella reticulata -Ridley & Dendy, 1886:481; Ridley &

Dendy 1887:184; Wilson, 1902:400 [Non Wells and

Wells, in Wells et al., 1960:221 � Axinella polycapella].

Pseudaxinella reticulata -Wiedenmayer, 1977:159; Alvarez

et al. 1998 with additional synonyms

Material. USNM51469, Carrie Bow Cay, Belize, 27 m,

coll. M.C. Diaz, SCUBA, 4 APR 94.

Pseudaxinella sp.

Material. QM 313203, Dudley Point Reef, East Point,

Darwin NT, Australia, 12.25.30S; 130.49.00E, coll. Alvarez,

B, by hand, 20 SEP 93, station, JH-93±022, intertidal reef

Description.

Shape. Lamella of asymmetric shape found detached from

original substrate, approximately 5 mm thick, 8 cm in

maximum length and 6 cm in maximum width; with holes

of irregular shape, up to 1 cm in maximum length.

Surface. Small conules or tubercules, less than 1 mm long,

distributed uniformly in narrowly separated parallel rows,

that run lengthwise in both faces of lamella and give a reti-

culate aspect to surface. No visible oscules.

Consistency. Firm

Colour. Orange alive; beige in alcohol.

Skeleton. Without specialized ectosomal skeleton. Choano-

some skeleton plumoreticulated, nearly isotropic, forming

meshes up to 1200 m in diameter; primary (ascending)

plumose spicule tracts of oxeas and styles, 500±700 mm

connected by secondary tracts, also plumose and of similar

thickness. Primary tracts ending in small conules at surface.

Spicules: Styles, length, 202.2±257.8 mm (230.6 � 14.5),

width, 11.3±18.7 mm (14.9 � 2.3). Oxeas, length, 170.3±

247.5 mm (198.8 � 18.6), width, 10.8±17.6 mm

(14.3 � 1.9) [25].

Remarks. The specimen or fragment collected, was

detached from its original substrate. It might had been a

part of a bigger specimen, thus little can be said about the

actual shape of this species; the shape of the described

fragment does not agree with the typical massive-encrust-

ing shapes observed in other Pseudaxinella spp.

Ptilocaulis sp.

Material. ZMA Por. 10993 (exchange), Seychelles,

Alphonse Atoll, coll. R.W.M Van Soest, 3 JAN 93, station

788/12, 6±8 m.



Description. Shape. Globular to lobate, on broad base,

12 cm in maximum height, 15 cm in maximum width.

Surface. With ¯attened, spatula-like `scopiform' processes,

up to 5 mm long, uniformly distributed giving a brush-like

appearance. Oscules, 5 mm in diameter, aggregated in

groups of two or three, rounded, ¯ushed, with contractile

membrane. Smaller pores or ostia visible among the

surface processes.

Consistency. Firm.

Colour. Orange, alive. Beige in alcohol.

Skeleton. Ectosomal skeleton not specialized. Choanosomal

skeleton, plumose, without clear differentiation of axial

and extra-axial region; with ascending plumose tracts of

spicules, 50±70 mm thick, connected by single spicules at

all angles, anastomosing irregularly and diverging to

surface where they form scopiform processes.

Spicules. Styles in two size categories: I, length, 510±620 mm

(573.3 � 45.0), width, 8±13 m (10.3 � 2.3) [6]; II, length,

270±380 mm (325.6 � 32.2), width, 5±13 mm (9.6 � 2.6)

[25]. Type I less frequent and located at the surface

Ptilocaulis walpersii Duchassaing & Michelotti, 1864

Pandaros walpersi Duchassaing de Fonbressin & Michelotti

1864 : 90.

Ptilocaulis walpersi Zea, 1987:187; Alvarez & Crisp,

1994 : 119; Alvarez et al., 1998 with additional synonyms

Material. Carrie Bow Cay, Belize, 27 m, coll. M.C. Diaz,

SCUBA, 4 APR 94. [No voucher material available.

Morphological characters were coded using other speci-

mens collected from the same area, descriptions from the

literature and ®eld notes].

Reniochalina stalagmitis von Lendenfeld 1888

Reniochalina stalagmitis von Lendenfeld 1888: 83; White-

legge, 1902: 283; Hallmann, 1914a: 346

Reniochalina lamella von Lendenfeld 1888: 83; Whitelegge

1902: 283; Hallmann 1914a: 346; Hooper & Wieden-

mayer, 1994: 81

Axiamon folium Hallmann, 1914b: 441.

Material QM G 303362, Stephen's Rock, West Arm,

Darwin Harbour NT, Australia, 12.29.20S; 130.47.00E,

coll. Hooper, JNA & Hobbs, LJ, JH-93±024, coral pinna-

cle, SCUBA, 19 m

Reniochalina sp.

Material. QM G 304472, off North Point, Lizard Island,

Queensland, Australia, 14.48.40S; 145.26.30E, coll. Hooper,

J.N.A; Hobbs, L.J.; Kennedy, J. & Cook, S.D, 12 APR 94,

station, JH-94±030, on Halimeda beds, SCUBA, 24 m.

Description. Shape. Branching, erect; with 1±2 cylindrical

branches, semiburied into substrate, up to 1 cm in

diameter and 10±15 cm long, dichotomously divided near

the tips.

Surface. With long processes, 1±2 mm in length and

uniformly distributed, sometimes joined. No visible oscules

or pores.

Colour. Red-orange alive; brown in alcohol.

Consistency. Flexible but ®rm.

Skeleton. Ectosomal skeleton non specialized. Choanosomal

skeleton, plumose to halichondrioid, slightly compressed

in axial region; with plumose ascending spicule tracts, 50±

70 mm, enforced by spongin, connected by single spicules

or short and thinner tracts, at all angles, or anastomosing

and forming oval to round meshes. Ascending tracts, diver-

ging to periphery, anastomosing and forming thick

plumose tracts that correspond with surface processes.

Spicules. Megascleres are oxeas to anisoxeas, length, 165.8±

274.2 mm (211.1 � 29.7), width, 2.4±7.9 mm (5.0 � 1.4)

[25] with microspined tips not visible with light micro-

scopy.

Remarks. The species is very similar to Reniochalina stalag-

mitis but it is regarded here as a different species based on

the differences in habit, the presence of microspines on the

oxeas, which are much smaller than the ones in R. stalagmi-

tis, and the results of the DNA sequencing (see Discussion

and Conclusions).

Family DICTYONELLIDAE Van Soest, Diaz &

Pomponi ,1990

Ulosa sp.

Material. QM G 303402, S Shell I., rock on N side of

island, Darwin Harbour, Northern Territory, Australia,

12.29.80S; 130.52.90E, coll. Hooper, JNA & Hobbs, LJ,

station, JH-93±025, coral pinnacle, SCUBA, 14 m.

Description. Shape. Massive-lobate on broad base.

Surface. Uniformly covered with conules up to 2 mm long;

¯eshy; collapsed in preserved state.

Consistency. Soft

Colour. Yellow alive; yellow brown in alcohol.

Skeleton. Ectosomal skeleton nonspecialized. Choanoso-

mal skeleton composed of spongin ®bres, 200±250 mm

thick, cored by styles, forming reticulation of rectangu-

lar to rounded meshes, 500±900 mm in maximum

diameter. Reticulation becomes looser and halichon-

drioid close to periphery; with short tracts of styles,

cemented with spongin forming conules. Skeleton

partially obscured by granular pigmentation, probably

cyanobacterias.



Spicules. Styles, some with blunt ends, length, 146.4±535.0 mm

(219.9 � 93.5), width, 3±12.5 mm (6.7 � 2.1) [24]; anisoxeas,

less frequent, length, 203.3±444.1 mm (333.5 � 72.3) [7],

width 8.6±12.0 mm (10.2 � 1.4) [7]; occasionally strongyles.

Remarks. The specimen is assigned here to the genus Ulosa

and agrees with the description of Ulosa angulosa (Lamarck)

provided by Van Soest 1987a: 23, but this needs to be

con®rmed.

Scopalina ruetzleri Wiedenmayer, 1977

Ulosa ruetzleri Wiedenmayer, 1977: 145; Pulitzer-Finali,

1986:118.

Dictyonella ruetzleri Van Soest 1987a: 23

Scopalina ruetzleri Lehnert & Van Soest (1996): 59.

Material. Carrie Bow Cay, Belize, 27 m, coll. M.C. Diaz,

SCUBA, 4 APR 94.

[No voucher material available. Morphological characters

were coded using other specimens collected from the same

area, descriptions from the literature and ®eld notes].

Family HALICHONDRIIDAE Van Soest, DõÂaz &

Pomponi 1990

Halichondria panicea Pallas, 1766

Spongia panicea Pallas, 1766

Halichondria panicea Gray 1867: 519; Carter, 1887: 69;

Dendy 1887: 157; Topsent 1891: 527; Whitelegge, 1901:

68; Dendy, 1922: 37; Burton, 1932: 199; Vosmaer, 1932±

35: 523; SaraÁ , 1958 : 226; Pulitzer-Finali, 1977: 67; DõÂaz

et al., 1991: 139

Material. ZMA Por. 14290. Oosterschelde, S.W. Nether-

lands, 27.3.1998, 3 m, coll. M. de Kluijver.

Halichondria phakellioides Dendy & Frederick

Halichondria phakellioides Dendy & Frederick, 1924: 498;

Hooper et al., 1997.

Material: QM G 303261, East Arm, Darwin, reef N of

boatramp, S of S Shell, NT, Australia, 12.29.80S;

130.53.50E, coll. Hooper, JNA & Hobbs, LJ, 19 SEP 93,

station JH-93±021, intertidal reef, by hand.



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