A Molecular Phylogeny for the Order Clathrinida Rekindles and Refines Haeckel's Taxonomic Proposal for Calcareous Sponges

Abstract

Most biological groups are still longing for a phylogenetically sound taxonomic organization. In this article, we aimed to verify the consistency of morphological characters in calcarean sponges of the well-known non-monophyletic order Clathrinida using a molecular phylogeny. For this we included 50 species, including six type species, currently assigned to eight different genera. A maximum likelihood topology was generated for the nuclear ITS marker using the General Time Reversible model and the bootstrap reliability test. Our topology indicated 10 clathrinid clades that included species with consistent morphological characters. In the present study, we defined nine of these clades as clathrinid genera, including four newly described and two newly diagnosed genera. Recent studies have indicated that not much phylogenetic information may be found in morphology, but our findings contradict this general assertion. Our study confirms the suitability of skeleton and body anastomosis as valid characters in a phylogenetically sound taxonomy for the order. Interestingly, we have also found that, apart from the Calcinea/Calcaronea split and a few minor details, Haeckel's original proposal is remarkably similar to our own, which was based on a molecular phylogeny 140 years later.

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SYMPOSIUM
A Molecular Phylogeny for the Order Clathrinida Rekindles and
Refines Haeckel’s Taxonomic Proposal for Calcareous Sponges
Michelle Klautau,
1,
* Fernanda Azevedo,* Ba
´slavi Co
´ndor-Luja
´n,* Hans Tore Rapp,
†
Allen Collins
‡
and Claudia Augusta de Moraes Russo
§
*Universidade Federal do Rio de Janeiro, Instituto de Biologia, Departamento de Zoologia, Rio de Janeiro, RJ, 21941-902,
Brazil;
†
University of Bergen, Department of Biology and Centre for Geobiology, Thormøhlensgate 53A, N-5020, Bergen,
Norway;
‡
National Systematics Laboratory of NOAA Fisheries Service and Department of Invertebrate Zoology,
National Museum of Natural History, Smithsonian Institution, Washington, DC 20560, USA;
§
Universidade Federal
do Rio de Janeiro, Instituto de Biologia, Departamento de Gene
´tica, Rio de Janeiro, RJ, 21941-902, Brazil
From the symposium ‘‘Assembling the Poriferan Tree of Life’’ presented at the annual meeting of the Society for
Integrative and Comparative Biology, January 3–7, 2013 at San Francisco, California.
1
E-mail: mklautau@biologia.ufrj.br
Synopsis Most biological groups are still longing for a phylogenetically sound taxonomic organization. In this article, we
aimed to verify the consistency of morphological characters in calcarean sponges of the well-known non-monophyletic
order Clathrinida using a molecular phylogeny. For this we included 50 species, including six type species, currently
assigned to eight different genera. A maximum likelihood topology was generated for the nuclear ITS marker using the
General Time Reversible model and the bootstrap reliability test. Our topology indicated 10 clathrinid clades that
included species with consistent morphological characters. In the present study, we defined nine of these clades as
clathrinid genera, including four newly described and two newly diagnosed genera. Recent studies have indicated that
not much phylogenetic information may be found in morphology, but our findings contradict this general assertion.
Our study confirms the suitability of skeleton and body anastomosis as valid characters in a phylogenetically sound
taxonomy for the order. Interestingly, we have also found that, apart from the Calcinea/Calcaronea split and a few minor
details, Haeckel’s original proposal is remarkably similar to our own, which was based on a molecular phylogeny
140 years later.
Introduction
The central biological tenet of evolution may be
finely tuned with classification to compose a phylo-
genetically sound taxonomy (de Queiroz and
Gaultier 1992). The first step towards a phylogenetic
proposal for the taxonomy of calcareous sponges was
tailored by Ernst Haeckel (1872). His detailed taxo-
nomic scheme for the group was based on the com-
position of spicules and on the aquiferous system.
Nevertheless, his proposal met strong criticism and
was deemed unnatural by fellow taxonomists
(Pole
´jaeff 1883;Dendy 1891,1893;Minchin 1896).
After Haeckel’s pioneering study, other researchers
came forward with different suggestions for the tax-
onomy of Calcarea.
Among those, Pole
´jaeff (1883) suggested that a more
natural taxonomy would take the aquiferous system
into account but not the composition of spicules as
proposed by Haeckel. A decade later, Minchin (1896)
proposed that the first major distinction of Calcarea
should be between two large groups, later named
Calcinea and Calcaronea (Bidder 1898). His proposal
was strongly supported by several cytological features
and it was a crucial step towards a phylogenetic taxon-
omy for the calcareans.
By the turn of the 20th century, the first formal
phylogenetic tree for the Calcarea became available
(Dendy and Row 1913). In that study, the authors
acknowledged the clear-cut division between Calcinea
and Calcaronea, as evidenced by several cytological
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observations, but claimed that it would be impractical
to use such laborious techniques in taxonomy. Hence,
their phylogenetic proposal was based on aspects of the
architecture of the skeleton.
Half a century later, Hartman (1958) confirmed di-
vision into the two subclasses Calcinea and Calcaronea
(Bidder 1898), and these have been in use ever since.
Furthermore, he added features related to the body
cortex (corticalization) as key characters for lower
taxonomical levels, proposing the orders Leucettida
and Clathrinida for Calcinea. In the most recent
review, Borojevic et al. (1990,2002) claimed that
corticalization and the aquiferous systems evolved in
several lineages and extinguished the order Leucettida
sensu Hartman (1958).
When the results of the first molecular studies
became available, they unquestionably supported
the division of the class Calcarea into Calcinea and
Calcaronea (Manuel et al. 2003,2004), but not of the
lower taxonomic ranks (Dohrmann et al. 2006;Voigt
et al. 2012). Most of these molecular studies
indicated that the aquiferous system might not be
phylogenetically informative (Manuel et al. 2003,
2004;Dohrmann et al. 2006), but a more recent
analysis showed otherwise (Voigt et al. 2012). Due
to poor taxon sampling, however, other morpholog-
ical characters have never been properly tested in a
molecular phylogenetic study and the taxonomy of
Calcarea remains mainly typological.
In a recent study, our research group found a
surprisingly strong phylogenetic signal for spicule
composition and body anastomosis when many
species of Clathrina were analyzed (Rossi et al.
2011). Hence, a phylogenetic systematics for this
group might be within reach if such characters are
considered. It remains to be tested, however, whether
the consistency of these characters remains in a
broader taxonomic perspective.
In this study, our aim was to propose a phylogenet-
ically sound scenario for the classification of the order
Clathrinida, the most speciose order in the subclass
Calcinea. For this, we have gathered an unprecedented
taxon sampling with 50 clathrinid species, including
six type species, currently assigned to eight genera,
so as to evaluate the consistency of morphological
characters with a well-resolved molecular phylogeny.
Furthermore, we included samples from different
geographical regions in order to test the consistency
of current diagnoses of species (Klautau et al. 1999;
Manuel et al. 2003).
Materials and methods
Specimens
The subclass Calcinea is monophyletic and it is
currently divided into two orders, Clathrinida and
Murrayonida. Nevertheless, it has been shown that
genera of Murrayonida cluster within Clathrinida
(Voigt et al. 2012), revealing that the presence of a
hyper-calcified skeleton, as in Murrayonida, is not
a valid taxonomic character. For this study, we are
considering order Clathrinida sensu Borojevic et al.
(2002), since we were unable to include species of
Murrayonida in our dataset.
Our dataset comprises 50 species currently assigned
to eight genera of Clathrinida, making this the most ex-
tensive dataset analyzed to date in the order (Table 1).
Furthermore, due to a reported plasticity of the mor-
phological characters of sponges (Cavalcanti et al.
Table 1 Analyzed specimens with collection sites, voucher numbers, and GenBank accession numbers
Species Collection site Voucher number GenBank (ITS)
Calcinea
Ascandra falcata Mediterranean Sea UFRJPOR 5856 HQ588962
Clathrina antofagastensis Chile MNRJ 9289 HQ588985
Clathrina aspina Brazil UFRJPor 5245 HQ588998
Clathrina aurea Brazil MNRJ 8998 HQ588968
Clathrina brasiliensis Brazil UFRJPor 5214 HQ588978
Clathrina cerebrum Mediterranean Sea UFRJPor 6322 HQ588964
Clathrina clathrus Mediterranean Sea UFRJPOR 6315 HQ588974
Clathrina conifera Brazil MNRJ 8991 HQ588959
Clathrina contorta Mediterranean Sea UFRJPor 6327 HQ588970
Clathrina corallicola Norway UFRJPor 6329 HQ588994
Clathrina coriacea Norway UFRJPor 6330 HQ588986
Clathrina cylindractina Brazil UFRJPor 5206 HQ588979
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Table 1 Continued
Species Collection site Voucher number GenBank (ITS)
Clathrina fjordica Chile MNRJ 8143 HQ588984
Clathrina helveola Australia QMG313680 HQ588988
Clathrina hirsuta Cabo Verde ZMAPOR07061 KC843431
Clathrina hispanica Mediterranean Sea UFRJPOR6305 KC843432
Clathrina luteoculcitella Australia QMG313684 HQ588989
Clathrina nanseni Greenland UFRJPor 6332 HQ588982
Clathrina reticulum Mediterranean Sea UFRJPOR 6258 HQ588973
Clathrina tetractina Brazil UFRJPor 5183 HQ589000
Clathrina wistariensis Australia QMG313663 HQ588987
Clathrina sp. nov. 1 Brazil UFRJPOR6621 KC843433
Clathrina sp. nov. 2 Brazil UFRJPOR6617 KC843434
Clathrina sp. nov. 3 Caribbean, Curac¸ao UFJPOR6737a KC843435
Clathrina sp. nov. 4 Caribbean, Curac¸ao UFRJPor 6733 KC843436
Clathrina sp. nov. 4 Caribbean, Curac¸ao UFRJPor 6741 KC843437
Clathrina sp. nov. 5 French Polynesia, Moorea UF:Porifera:1600 KC843438
Clathrina sp. nov. 5 French Polynesia UFRJPOR6461 KC843439
Clathrina sp. nov. 6 New Zealand UFRJPOR6839 KC843440
Clathrina sp. nov. 7 New Zealand UFRJPOR6843 KC843441
Clathrina sp. nov. 8 Brazil UFRJPOR6545 KC843442
Clathrina sp. nov. 8 USA, Florida UFRJPOR5818 KC843443
Clathrina sp. nov. 8 Caribbean, Virgin Islands ZMAPOR08344 KC843444
Clathrina sp. nov. 8 Caribbean, Curac¸ao UFRJPOR6761 KC843445
Clathrina sp. nov. 9 French Polynesia BMOO16290 KC843446
Clathrina sp. nov. 10 Caribbean UFRJPOR6945 KC843447
Clathrina sp. nov. 11 Brazil UFRJPOR6084 KC843448
Clathrina sp. nov. 11 Caribbean P10x13 KC843449
Clathrina sp. nov. 12 Azores UFRJPOR5627 KC843450
Clathrina sp. nov. 13 Indonesia ZMAPOR08390 KC843451
Clathrina sp. nov. 14 Antarctica, Weddell Sea SMF11866 KC874655
Guancha lacunosa Norway UFRJPor 6334 HQ588991
Guancha ramosa Chile MNRJ 10313 HQ588990
Guancha aff. blanca Norwegian Sea ZMBN90440 KC874656
Leucaltis clathria Australia, DJ’s Reef QMG316022 AJ633861
Leucaltis clathria Caribbean, Panama P10x28T KC843452
Leucaltis nuda Chile MNRJ 10804 KC843453
Leucascus simplex French Polynesia, Moorea BMOO16283 KC843454
Leucetta chagosensis French Polynesia, Moorea BMOO16210 KC843455
Leucetta floridana Caribbean, Panama PTL09.P100 KC843456
Leucetta microraphis Australia, Wistari Reef QMG313659 AJ633874
Leucetta potiguar Brazil UFPEPor547 EU781986
Leucetta cf. pyriformis Antarctic MNRJ13843 KC843457
Leucetta sp. Antarctic, Weddell Sea SMF 11868 KC874654
Leucetta sp. Antarctic, Weddell Sea MNRJ 13798 KC849700
Leucettusa sp. New Zealand OCDN6676-Q KC843458
Pericharax heteroraphis Australia QMG313657 AF479062.1
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2007), we decided to include samples from different
localities to provide a consistency check for taxonomic
assignments at the species level. Species, collection
sites, and voucher and GenBank accession numbers
for all sequences are provided in Table 1.
DNA sequencing
In our dataset, we included sequences of the internal
transcribed spacer (ITS) since it appears to be suit-
able for Calcarea phylogeny (Wo
¨rheide et al. 2004;
Rossi et al. 2011). Genomic DNA was extracted from
ethanol-preserved specimens with the guanidine/
phenol-chloroform protocol (Lo
ˆbo-Hajdu et al.
2004) or with a QIAampÕDNA MiniKit (Qiagen).
The entire region comprising the two spacers
(ITS1 and ITS2) and the 5.8S ribosomal DNA was
amplified by PCR with the following primers: 18S
(50-TCATTTAGAGGAAGTAAAAGTCG-30) and 28S
(50-GTTAGTTTCTTTTCCTCCGCTT-30)(Lo
ˆbo-
Hajdu et al. 2004). PCR mixes contained buffer
(75 mM Tris–HCl, pH 8.8, 20 mM (NH
4
)
2
SO
4
,
0.01% Tween 20), 50 mg/mL bovine serum albumin,
0.4 mM dNTPs, 0.5 pmol mL
1
of each primer, 1 mM
MgCl
2
, and one unit of Taq DNA-polymerase
(Fermentas or Bioline).
PCR steps included 5 min at 958C, 35 cycles of
1 min at 928C, 1 min at 50–558C, and 1 min at
728C, followed by 5 min at 728C. Forward and
reverse strands were automatically sequenced in
ABI 3500 (Applied Biosystems). The sequences ob-
tained were edited using the programs Chromas Lite
2.01, DNASTAR (SeqMan) or Geneious, and BLAST
searches (http://www.ncbi.nlm.nih.gov/blast/) were
performed to confirm their biological source.
Alignment and phylogenetic analyses
ITS sequences were aligned using the Q-INS-i option
of the MAFFT program (Katoh and Standley 2013),
with Scoring matrix 200 PAM/k¼2, gap penalty 1.53
and offset value ¼0. This step was critical for obtain-
ing a reliable alignment for the ingroup sequences,
because the option takes the secondary structure into
consideration. Final alignments were 1407 bp for
ITS1, 5.8S, and ITS2 and were visually inspected.
Furthermore, due to their high variability, most of
the ITS sequences from calcaronean species did not
align properly with the ingroup sequences and a suit-
able outgroup is not available. Therefore, we decided
to root our tree using the mid-point rooting method
that has been shown to be remarkably efficient in
obtaining the root (see Hess and Russo 2007).
A maximum likelihood tree was generated using
the MEGA 5.0 platform (Tamura et al. 2011). The
substitution model was selected by that option in
MEGA, which indicated general time reversal
(GTR) with four gamma categories. The ML algo-
rithm also requires an input tree, and a BIONJ tree
(Gascuel 1997) was used. A heuristic tree bisection
and reconnection algorithm was applied on the
BIONJ tree to find the ML tree. Gap sites were main-
tained for the phylogenetic analyses. One thousand
bootstrap pseudo-replicates (Felsenstein 1985;Russo
1997) were performed on the ML tree.
Results and discussion
Since sequences from geographically distant sponges
identified by morphological characters as a nominal
species clustered in our tree, our results demonstrate
that these morphological characters are reliable for
determining actual biological entities in the order
Clathrinida. In our tree, 10 distinct lineages are
evident (Fig. 1). Of those, nine may be clearly
defined with morphological characters (Table 2)and
a high bootstrap support and, thus, we are formally
designating them as distinct calcinean genera.
The first lineage includes the major cluster of 20 cla-
throid species that we designate as the genus Clathrina
(100 BP). The Clathrina cluster is distinct from all
remaining lineages since it includes Clathrina clathrus,
the type species, and other species all devoid of tetra-
ctines. This lineage has been recovered previously
(Rossi et al. 2011) but in the present article we
have included many additional species and the same
morphological pattern remained. Based on our tree,
we are now formally proposing a new diagnosis for
the genus Clathrina (see Diagnoses section).
Apart from the skeleton, a large clade, formed by
yellow sponges with only triactines, has been previ-
ously reported (Rossi et al. 2011). In that article, the
authors showed a second lineage of yellow sponges
with tetractines, indicating that the yellow color
appeared at least twice in Clathrinida. In the present
study, we have included more yellow Clathrina
species to our analysis and they also clustered.
Nevertheless, two yellow Clathrina species from
New Zealand grouped separately from the yellow
Clathrina clade. Curiously, these yellow species
from New Zealand are the only true clathrinas that
also possess tripods. This result shows that the yellow
color appeared twice in the genus Clathrina and that
the presence of tripods marks the latter clade.
Furthermore, some species of Guancha, with no
tetractines, are grouped within the clade Clathrina
and must be transferred to Clathrina, as previously
indicated (Rossi et al. 2011). In the present article,
however, we have also included a specimen for which
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the morphological pattern confers with that of the
type species of Guancha,G. aff. blanca. Since G.
blanca has triactines only, we are formally synony-
mizing Guancha with Clathrina. The morphological
distinction between Guancha and Clathrina is limited
to the presence of peduncle and the presence of
parasagittal spicules in Guancha and species of this
genus appear scattered in the Clathrina portion of
the tree. Therefore, all Guancha species with triactine
spicules only must be assigned from now on to
Clathrina. On the other hand, Guancha species
with tetractines must be assigned to the other
genera we are proposing, according to the composi-
tion of their skeleton.
The second lineage (90 BP) comprises four cla-
throid species with triactines and tetractines. In this
group, tetractines are, at least, as abundant as triac-
tines but frequently surpass their proportion. We are
ranking this lineage as a new genus, named Ernstia.
The apical actine of the tetractines of Ernstia gen.
nov. is remarkably long, thin, and needle-like, a
feature that is also found in the sister group, the
genus Ascandra. The third clade, Ascandra
(100 BP), presents seven species, including the type
species, Ascandra falcata. Based on their morphology,
we provide a new diagnosis for this genus.
The main difference between the two genera is
that Ernstia gen. nov. has a regular clathroid body
quite similar to that of Clathrina, but in Ascandra
the body anastomosis is loose with free tubes at least
at the apical region of the cormus. The similarity
between species formerly known as Clathrina and
Ascandra has been reported earlier in morphological
analyses (Borojevic 1971). In that study, the author
discussed this point when he originally described
Clathrina ascandroides. Indeed, according to the pre-
sent study, this particular species must be transferred
to the genus Ascandra along with other species that
conform to the diagnostic features of free tubes,
abundant tetractines, and very thin apical actines.
Fig. 1 Maximum likelihood tree built with the GTR plus gamma correction, with 1407 bp of the nuclear ITS marker for 50 clathrinid
species, assigned to 12 genera. Black spicules represent the most abundant categories.
Taxonomic proposal for Clathrinida 5
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Table 2 Morphology table
Species Spicules
Apical actine
Body Cortex
Aquiferous
system ColorSpines/shape
Arthuria hirsuta Tri
a
, tetra (rare), di No/conical Clathroid, irreg, loose No Asconoid ?
Ascaltis reticulum Tri
a
, tetra, di No/conical Clathroid, reg, tight No Asconoid, pseud. White
Ascandra sp. nov. 14 Tri, tetra
a
No/needle Clathroid, irreg, loose (no apical anastomosis) No Asconoid Beige
Ascandra contorta Tri, tetra
a
, di, trich No/needle Clathroid, irreg, tight (no apical anastomosis) No Asconoid White
Ascandra corallicola Tri I, tri II, tetra I
a
, tetra II
a
No/needle Clathroid, irreg, loose (no apical anastomosis) No Asconoid White (transp)
Ascandra falcata Tri, tetra
a
, di No/needle Clathroid, irreg, loose (no apical anastomosis) No Asconoid White
Ascandra sp. nov. 10 Tri (r), tetra I
a
(r), tetra II
a
(r/s), di No/needle Clathroid, irreg, loose (no apical anastomosis) No Asconoid White
Ascandra sp. nov. 11 Tri (r), tetra I
a
(r), tetra II
a
(r/s), di No/needle Clathroid, irreg, loose (no apical anastomosis) No Asconoid White
Ascandra sp. nov. 9 Tri, tetra I
a
(r/s), tetra II
a
(r/s) No/needle Clathroid, irreg, loose (no apical anastomosis) No Asconoid White
Borojevia aspina Tri
a
, tetra, trip Yes/conical Clathroid, reg, tight Rud Asconoid White
Borojevia brasiliensis Tri
a
, tetra, trip Yes/conical Clathroid, reg, tight Rud Asconoid White
Borojevia cerebrum Tri
a
, tetra, trip Yes/conical Clathroid, reg, tight Rud Asconoid White
Borojevia sp.nov.12 Tri
a
, tetra, trip Yes/conical Clathroid, irreg, tight Rud Asconoid White
Brattegardia nanseni Tri, tetra I, tetra II No/conical Clathroid, reg, tight Rud Asconoid White
Clathrina aff. blanca Tri, tri (p) No Clathroid, irreg, loose, peduncle No Asconoid White
Clathrina antofagastensis Tri I, tri II No Clathroid, irreg, tight No Asconoid White
Clathrina aurea Tri No Clathroid, irreg, loose No Asconoid Yellow
Clathrina clathrus Tri No Clathroid, irreg, loose No Asconoid Yellow
Clathrina conifera Tri No Clathroid, irreg, loose No Asconoid White
Clathrina coriacea Tri No Clathroid, irreg, loose No Asconoid White
Clathrina cylindractina Tri No Clathroid, irreg, loose No Asconoid White
Clathrina fjordica Tri No Clathroid, irreg, loose No Asconoid White
Clathrina helveola Tri No Clathroid, irreg, loose No Asconoid White
Clathrina hispanica Tri No Clathroid, irreg, loose No Asconoid ?
Clathrina lacunosa Tri, tri (p), di No Clathroid, irreg, tight, peduncle No Asconoid White
Clathrina luteoculcitella Tri, di No Clathroid, irreg, tight No Asconoid Yellow
Clathrina ramosa Tri, tri (p) No Clathroid, irreg, loose, peduncle No Asconoid White
Clathrina sp. nov. 3 Tri I, tri II No Clathroid, irreg, loose No Asconoid White
Clathrina sp. nov. 4 Tri I, tri II, tri III No Clathroid, irreg, loose No Asconoid Yellow
(continued)
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Table 2 Continued
Species Spicules
Apical actine
Body Cortex
Aquiferous
system ColorSpines/shape
Clathrina sp. nov. 5 Tri No Clathroid, irreg, loose No Asconoid Yellow
Clathrina sp. nov. 6 Tri, trip No Clathroid, reg, tigth No Asconoid Yellow
Clathrina sp. nov. 7 Tri, trip No Clathroid, reg, tigth No Asconoid Yellow
Clathrina sp. nov. 8 Tri No Clathroid, reg, tight No Asconoid Yellow
Clathrina wistariensis Tri No Clathroid, irreg, loose No Asconoid White
Ernstia sp.nov.1 Tri
a
, tetra, trich No/needle Clathroid, reg, tight, globose No Asconoid Yellow
Ernstia sp. nov. 13 Tri I
a
, tri II
a
, tetra No/needle Clathroid, irreg, loose No Asconoid ?
Ernstia sp. nov. 2 Tri, tetra
a
No/needle Clathroid, irreg, loose No Asconoid Yellow
Ernstia tetractina Tri, tetra
a
No/needle Clathroid, irreg, loose No Asconoid White
Leucaltis clathria Tri I, tri II (r/s), tetra I, tetra II (r/s) No/conical Anastomosed tubes Yes Leuconoid Pink
Leucascus simplex Tri, tetra No/conical Clathroid, reg, tight, globose Yes Solenoid Beige
Leucetta cf. pyriformis Tri I, tri II, tetra (rare) No/conical Massive, globose, surface smooth, no oscular crown Yes/very
thin
Leuconoid,
reduced atrium
Leucetta chagosensis Tri I (r/s), tri II, tetra No/conical Massive, globose, surface smooth, subdermal cavities, no oscular crown Yes/thin Leuconoid, large
atrium
Bright yellow
Leucetta floridana Tri I, tri II, tetra I, tetra II No/conical Massive, lobate, surface with ridges, no oscular crown Yes Leuconoid, large
atrium
Light blue
Leucetta microraphis Tri I (r), tri II (r/s), tetra (r/s) No/conical Massive, lobate, surface smooth, subdermal cavities, no oscular crown Yes Leuconoid,
reduced atrium
Dark yellow
Leucetta potiguar Tri I, tri II, tetra I, tetra II No/conical Massive, lobate, surface smooth, no oscular crown Yes Leuconoid,
reduced atrium
Light pink
Leucetta sp. Tri, tetra (rare) No/conical Massive and ovoid tube, no oscular crown Yes/very
thin
Leuconoid Beige
Leucettusa sp. Tri I, tri II, tetra (rare) No/conical Massive, tubular Yes Leuconoid ?
Leucettusa nuda Tri I, tri II, tetra I, tetra II No/conical Ramified tubes Yes Leuconoid White
Pericharax heteroraphis Tri I, tri II, tetra, trip (r/s) No/conical Massive, folded, no oscular crown, subdermal cavities Yes Leuconoid, large
atrium
Dark yellow
a
Most abundant spicule. tri, triactines; tetra, tetractines; trip, tripods; di, diactines; trich, trichoxeas; r, regular (equiangular and equiradiate); p, parasagittal; s, sagittal; reg, regularly anastomosed; irreg,
irregularly anastomosed; tight, tightly anastomosed; rud: rudimentary; pseud, pseudoatrium; trans, transparent.
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The next clade includes the fourth and fifth line-
ages. Leucascus is represented by the type species,
Leucascus simplex, and the species Clathrina reticu-
lum, which is neither a Clathrina nor a Leucascus.
The cormi of C. reticulum and L. simplex are well-
defined, with tightly anastomosed tubes and their
sequences form a fairly well-supported clade (85
BP), but our tree indicates a large distance between
them. Additionally, a long array of morphological
characters would easily permit their clear-cut distinc-
tion into two different genera. For instance, C. retic-
ulum possesses a pseudo-atrium, a distinct cavity
with no pinacoderm, while L. simplex has a true
atrium, with a pinacoderm, apical actines with
spines, and a solenoid aquiferous system
(Cavalcanti and Klautau 2011;Cavalcanti et al.
2013). Therefore, we consider that these two species
should be assigned to two different genera.
In fact, Haeckel (1872) originally described
C. reticulum as an Ascaltis. Nevertheless, he based
his assertion on the asconoid aquiferous system of
this species and on the presence of triactines, tetra-
ctines, and diactines in the skeleton, which were for-
merly diagnostic characters for Ascaltis. The current
diagnosis for this genus is quite different as it is
characterized by the presence of a pseudoatrium
and a thin cortex. Clathrina reticulum does not ex-
hibit a cortex, but a well-defined cormus is evident.
The sponge body is composed of tightly anastomosed
tubes forming an external structure that does resem-
ble a cortex. Therefore, species such as C. reticulum,
Clathrina gardineri, and Clathrina panis, with a well-
defined cormus and a pseudoatrium, should be tem-
porarily transferred to Ascaltis until the type species,
Ascaltis lamarcki, is analyzed under an integrative
molecular framework.
The sixth lineage has a single species, Clathrina
hirsuta. This species is characterized by the clathroid
body and by the presence of diactines, triactines, and
tetractines, the latter being very rare. We are propos-
ing here that species with triactines and rare tetra-
ctines should be included in the new genus Arthuria.
Our tree includes only one species of Arthuria but
the species (C. hirsuta) is well separated from all the
others and it easily may be characterized in morpho-
logical terms. We believe that other species that bear
such characteristics, previously assigned to Clathrina,
will group under this new genus such as Clathrina
africana.
The seventh lineage (59 BP) included four cla-
throid sponges with tightly anastomosed tubes and
a skeleton composed of triactines, tripods, and tetra-
ctines with spines. We are calling this new genus
Borojevia. Tripods and tetractines with spines also
appeared in other clades of our tree. Tripods, for in-
stance, are present in a Clathrina clade, whereas all
Leucascus also present spines on the apical actine
of tetractines (Cavalcanti et al. 2013). Therefore,
the new genus Borojevia is characterized by the
well-defined cormus with tripods on the external
tubes, triactines, and tetractines with spines on the
apical actines. The clade that reunites Clathrina
brasiliensis,Clathrina sp. nov. 12, and Clathrina
cerebrum had a strong support (100 BP), but
Clathrina aspina joined this clade with a low sup-
port. We are including C. aspina in Borojevia gen.
nov. since the group is well defined on the basis
of morphological characters although spines in
C. aspina have a different shape.
The eighth lineage (97 BP) contained two Leucaltis
specimens that fit the current diagnosis for Leucaltis
clathria, one from Australia and another from the
Caribbean. The genus Leucaltis comprises sponges
with a body of very large anastomosed tubes.
Differently from Clathrina, however, in Leucaltis
each tube has a distinct cortex with large spicules.
Also, the aquiferous system is considered leuconoid,
but may be composed of elongated and ramified
choanocyte chambers. A true atrium is present and
the choanosome is full of small triactines and tetra-
ctines. Leucaltis was previously assigned to the family
Leucaltidae, along with Leucettusa. In fact, the only
difference between these genera is that Leucaltis
has anastomosed tubes, but Leucettusa does not.
According to our results, Leucaltis is a valid genus,
but it is more closely related to Borojevia gen. nov.
than to Leucettusa (see also Voigt et al. 2012 for the
same result). Since L. clathria is the type species of
the genus and the type locality is the Caribbean Sea
(Florida), we suggest that L. clathria from Australia
is a distinct species and must receive a new name. In
this sense, the diagnosis for type species of Leucaltis
must be revised to avoid lumping of distinct biolog-
ical species into a single name.
Our ninth lineage includes a single species, Clathrina
nanseni. This is a clathroid species with a single oscu-
lum and a cormus surrounded by a membrane, at least
in the young forms. A stalk may be present. The skel-
eton is composed of triactines and two categories
of tetractines, one with normal apical actine and the
other with a rudimentary knob-like apical actine.
Parasagittal spicules may be found at the base of
some specimens (Rapp 2006). We propose a new
genus for this lineage: Brattegardia gen. nov.
The tenth lineage includes nine species assigned
to Leucetta,Pericharax,Leucaltis, and Leucettusa
(88 BP). It must be noted, however, that the species
originally described as Leucaltis nuda (Azevedo et al.
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2009) is present in this clade as Leucettusa nuda. The
species was later found to be a Leucettusa after fur-
ther detailed morphological examination and we are
formally assigning the species to Leucettusa with a
new name, Leucettusa nuda.
In a recent article, Voigt et al. (2012) suggested
that Leucetta is not monophyletic, which is consistent
with our results. The monophyletic status of
Leucettusa and Pericharax was not broken and their
diagnostic characters remain consistent after our mo-
lecular analyses. In order to maintain phylogenetic
consistency, however, the former genus Leucetta
should be split into three genera: (1) Leucetta flori-
dana,Leucetta potiguar, and Leucetta microraphis
(100 BP); (2) Leucetta chagosensis; and (3) Leucetta
cf. pyriformis and Leucetta sp. (99 BP).
Unfortunately, however, we do not have the type
species, Leucetta primigenia, in our tree. Thus, it
would be unclear at this point which lineage would
retain the generic name. Additionally, our prelimi-
nary morphological analysis showed no obvious di-
agnostic characters for the three lineages. Hence, a
formal revision (Alencar, Rapp, and Klautau, unpub-
lished results) and a detailed morphological analysis
are required before the split of Leucetta.
Final remarks
Recent publications have stated that not much phy-
logenetic information is contained in the morpholog-
ical characters of calcareous sponges (Manuel et al.
2003,2004;Dohrmann et al. 2006;Voigt et al. 2012).
Our results contradict this general assertion and, in-
stead, revealed a rather strong phylogenetic signal
in carefully selected morphological characters
within the order Clathrinida. Our systematic pro-
posal for Clathrinida is based on spicule composi-
tion, body anastomosis, and aquiferous system.
Using these morphological characters, we were able
to recognize 11 genera that agree with our molecular
phylogenetic pattern. Considering our dataset, only
the genus Leucetta still requires further analyses.
It is surprising to perceive how our new systematic
proposal is similar to that proposed by Haeckel in
the 19th century (Haeckel 1872). Haeckel’s taxo-
nomic proposal was based mainly on the aquiferous
system and on composition of spicules. According to
him, Calcarea should be divided into three families
according to the aquiferous system: Ascones,
Sycones, and Leucones. The genera in those families
would all use the prefixes Asc, Syc, and Leuc, respec-
tively. In order to complete the generic name, each
prefix would receive a suffix that would make refer-
ence to the presence of spicule types. Thus, asconoid
species with only triactines would be included in
genus Ascetta, whereas asconoid species with only
tetractines would be Ascilla, and so on.
Comparing Haeckel’s system to our proposal, it
becomes obvious that he selected, more than
140 years ago, the same morphological characters
that are disclosed as clade markers in our tree. In
fact, he would be surprisingly close to a phylogenetic
classification of Calcarea apart from three points.
The most important is that the major split between
Calcinea and Calcaronea was not clear to him.
Additionally, he used diactines as markers and he
also neglected the importance of the relative abun-
dance of spicule types. Still, many of Haeckel’s
genera may be well compared with those revealed
in our tree. The exclusive presence of triactines, for
instance, would be diagnostic for his genus Ascetta,
as it is to our Clathrina, a genus of asconoid calci-
nean sponges. Species with only tetractines were
associated with the Ascilla in his monograph. His
taxon Ascilla would be comparable to our Ernstia
and Ascandra clade, asconoid calcinean with a
much larger proportion of tetractine spicules.
In the past few years, a more detailed and finer pic-
ture of the evolution of morphological characters is
beginning to unfold in calcareous sponges. It is our
expectation that the availability of additional variable
molecular markers (Lavrov et al. 2013) allied to a truly
comprehensive taxon sampling may well reclaim the
importance of selected morphological characters as
diagnostic markers for even higher taxa, such as fam-
ilies and orders, even in groups with a particularly
simple morphology such as Calcarea.
Diagnoses
Descriptions of genera
Asterisks designate species that were tested in our
molecular phylogeny. Names between brackets are
the original genus of the listed species.
Genus Clathrina Gray, 1867
Type species: Grantia clathrus Schmidt, 1864 currently
accepted as Clathrina clathrus (Figs. 2A and 3A, B).
Diagnosis: Calcinea in which the cormus comprises
anastomosed tubes. A stalk may be present. The skel-
eton contains regular (equiangular and equiradiate)
and/or parasagittal triactines, to which diactines and
tripods may be added. Asconoid aquiferous system.
Species: C. angraensis (Azevedo and Klautau
2007); C. antofagastensis* (Azevedo et al. 2009);
C. (Guancha)arnesenae (Rapp 2006); C. aurea* Sole
´-
(Cava et al. 1991); C. (Guancha)blanca* (Miklucho-
Maclay 1868); C. broendstedi (Rapp, Janussen, and
Tendal 2011); C. (Guancha)camura (Rapp 2006);
C. ceylonensis (Dendy 1905); C. chrysea (Borojevic
Taxonomic proposal for Clathrinida 9
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Fig. 2 Photographs of specimens of the new and rediagnosed genera of Clathrinida. (A)Clathrina aurea (photo in situ: Andre
´Padua).
(B)Ernstia sp. nov. 2 (photo in situ: Andre
´Padua). (C) Ascandra sp. nov. 9 (photo in situ: Cristina Diaz and Belinda Alvarez). (D)Arthuria
hirsuta (photo in vitro: Fernanda Azevedo). (E)Borojevia brasiliensis (photo in situ: Eduardo Hajdu). (F)Brattegardia nanseni (photo in situ:
Bjørn Gulliksen; this photo was previously published in Rapp 2006).
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Fig. 3 Photographs of spicules and skeleton of the new and rediagnosed genera of Clathrinida. (A,B)Clathrina aurea.(C,D)Ernstia sp.
nov. 2. (E,F,G)Ascandra sp. nov. 9. (a) apical actine.
Taxonomic proposal for Clathrinida 11
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and Klautau 2000); C. clara (Klautau and Valentine
2003); C. clathrus* (Schmidt 1864); C. conifera*
(Klautau and Borojevic 2001); C. coriacea* (Montagu
1818); C. cribrata (Rapp, Klautau, and Valentine 2001);
C. cylindractina* (Klautau, Sole
´-Cava, and Borojevic
1994); C. fjordica* (Azevedo et al. 2009); C. hispanica*
(Klautau and Valentine 2003); C. hondurensis (Klautau
and Valentine 2003); C. jorunnae (Rapp 2006); C.
(Guancha)lacunosa* (Johnston 1842); C. laminocla-
thrata (Carter 1886); C. luteoculcitella* (Wo
¨rheide
and Hooper 1999); C. heronensis (Wo
¨rheide and
Hooper 1999); C. parva (Wo
¨rheide and Hooper
1999); C. (Guancha)pellucida (Rapp 2006); C. primor-
dialis (Haeckel 1872); C. procumbens (Von Lendenfeld
1885); C. (Guancha)ramosa* (Azevedo et al. 2009);
C. rotunda (Klautau and Valentine 2003); C. sinusara-
bica (Klautau and Valentine 2003); C. tendali (Rapp,
submitted for publication); and C. wistariensis*
(Wo
¨rheide and Hooper 1999) (¼C. helveola*
Wo
¨rheide and Hooper 1999).
Genus Ernstia gen. nov.
Etymology: For Ernst Haeckel in recognition of his
building of a tentative phylogenetic classification for
Calcarea (Figs. 2B and 3C, D).
Type species: Clathrina tetractina (Klautau and
Borojevic 2001).
Diagnosis: Calcinea in which the cormus com-
prises a typical clathroid body. A stalk may be pre-
sent. The skeleton contains regular (equiangular and
equiradiate) and/or sagittal triactines and tetractines.
Tetractines are the most abundant spicules or occur
at least in the same proportion as the triactines.
Tetractines frequently have very thin (needle-like)
apical actines. Diactines may be added. Asconoid
aquiferous system.
Species: E. (Clathrina)adusta (Wo
¨rheide and
Hooper 1999); E. (Clathrina)quadriradiata (Klautau
and Borojevic 2001); E. (Clathrina)sagamiana
(Ho
ˆzawa 1929); E. (Clathrina)septentrionalis (Rapp
et al. 2001); E.(Clathrina)tetractina* (Klautau and
Borojevic 2001).
Genus Ascandra Haeckel 1872
Type species: Ascandra falcata Haeckel 1872 (Figs. 2C
and 3E–G).
Proposed neotype: UFRJPOR 5856 (Universidade
Federal do Rio de Janeiro, Instituto de Biologia)
Diagnosis: Calcinea with loosely anastomosed
tubes. Tubes are free, at least in the apical region.
The skeleton contains regular (equiangular and
equiradiate) or sagittal triactines and tetractines.
Tetractines are the main spicules, occurring at least
in the same proportion as the triactines. They have
very thin (needle-like) apical actines. Diactines may
be added. Asconoid aquiferous system.
Species: A. (Clathrina)ascandroides (Borojevic
1971); A. (Clathrina)atlantica (Thacker 1908); A.
(Clathrina)biscayae (Borojevic and Boury-Esnault
1987); A. (Clathrina)contorta* (Minchin 1905); A.
(Clathrina)corallicola* (Rapp 2006); A. (Leucosolenia)
depressa (Dendy 1891); A. falcata* (Haeckel 1872); A.
(Leucosolenia)loculosa (Dendy 1891); A. minchini
(Borojevic 1966); A. (Clathrina)osculum (Carter 1886).
Arthuria gen. nov.
Etymology: For Arthur Dendy, in recognition of all
his precise and detailed work on the taxonomy of
Calcarea (Figs. 2D and 4A, B).
Type species: Clathrina hirsuta (Klautau and
Valentine 2003).
Diagnosis: Calcinea in which the cormus com-
prises a typical clathroid body. A stalk may be pre-
sent. The skeleton contains regular (equiangular
and equiradiate) triactines and tetractines. However,
tetractines are more rare. Diactines may be added.
Asconoid aquiferous system.
Species: A. (Clathrina)africana (Klautau and
Valentine 2003); A. (Clathrina)alcatraziensis (Lanna
et al. 2007); A. (Clathrina)canariensis (Miklucho-
Maclay 1868); A. (Clathrina)dubia (Dendy 1868); A.
(Clathrina)hirsuta* (Klautau and Valentine 2003);
A. (Clathrina)sueziana (Klautau and Valentine
2003); A. (Clathrina)tenuipilosa (Dendy 1905).
Borojevia gen. nov.
Etymology: For Radovan Borojevic, in gratitude for
teaching his deep knowledge on calcareous sponges
and in recognition for all his scientific works
(Figs. 2E and 4C, D).
Type species: Ascaltis cerebrum Haeckel 1872 cur-
rently accepted as Clathrina cerebrum.
Diagnosis: Calcinea in which the cormus com-
prises tightly anastomosed tubes. The skeleton con-
tains regular (equiangular and equiradiate) triactines,
tetractines, and tripods. The apical actine of the tet-
ractines has spines. Aquiferous system asconoid.
Species: B. (Clathrina)aspina* (Klautau, Sole
´-Cava,
and Borojevic 1994); B. (Clathrina)brasiliensis* (Sole
´-
Cava et al. 1991); B. (Clathrina)cerebrum* (Haeckel
1872); B. (Clathrina)paracerebrum (Austin 1996); and
B. (Clathrina)tetrapodifera (Klautau and Valentine
2003).
Brattegardia gen. nov.
Etymology: After the Norwegian marine zoologist
Torleiv Brattegard for his tremendous effort on ex-
ploring and sampling the deeper parts of the
12 M. Klautau et al.
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Fig. 4 Photographs of spicules and skeleton of the new and rediagnosed genera of Clathrinida. (A,B)Arthuria hirsuta.(C,D)Borojevia
brasiliensis. In detail, spines on the apical actine of a tetractine. (E,F)Brattegardia nanseni.
Taxonomic proposal for Clathrinida 13
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Norwegian-Greenland-Iceland (GIN) Seas. His col-
lections include numerous new calcareous sponges
from abyssal depths, among them a new species of
Brattegardia (Rapp and Tendal, unpublished results)
(Figs. 2F and 4E, F).
Type species: Leucosolenia nanseni (Breitfuss 1896)
currently accepted as Clathrina nanseni.
Diagnosis: Calcinea in which the cormus is formed
by anastomosed tubes covered by a thin membra-
nous layer, at least in young specimens. Cormus is
massive/globular with or without a stalk. The skele-
ton contains regular (equiangular and equiradiate)
triactines and tetractines, but parasagittal triactines
may be present. Triactines are the most numerous
spicules. Aquiferous system asconoid.
Species: B. (Clathrina)nanseni* (Breitfuss 1896).
Acknowledgments
We are indebted to Andre
´Padua and Pedro
Leocorny for invaluable assistance in morphological
and molecular analyses. Annelise Fraza
˜o and
Alexandre Selvatti provided important help with
the phylogenetic analysis and Carolina Voloch for
figures. We thank the Zoological Journal of the
Linnean Society for allowing the reproduction of
figure 2F. We would also like to thank Cesar
Cardenas, Dirk Shories, Eduardo Hajdu, Gisele
Lo
ˆbo-Hajdu, and Rob van Soest for collecting and/
or sending specimens.
Funding
The Moorea Biocode project, funded by the Gordon
and Betty Moore Foundation, is gratefully acknowl-
edged for supporting the use and collection of
calcarean specimens. Part of this work was supported
by NSF’s Porifera Tree of Life project (DEB 0829986
awarded to R. Thacker, P. Bangalore, and AGC).
H.T.R. was supported by the Research Council of
Norway (through the Centre for Geobiology) and the
Norwegian Biodiversity Information Centre. M.K. and
C.A.d.M.R. are funded by fellowships and research
grants from the Brazilian National Research Council
(CNPq) and the Rio de Janeiro State Research
Foundation (Fundac¸a
˜o Carlos Chagas Filho de
Amparo a
`Pesquisa do Estado do Rio de Janeiro -
FAPERJ). B.C.L. and F.A. received scholarships from
the CNPq and FAPERJ, respectively.
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