European Journal of Taxonomy 178: 1-52

http://dx.doi.org/10.5852/ejt.2016.178

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This work is licensed under a Creative Commons Attribution 3.0 License.

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2016 • Klautau M. et al.

Monograph

urn:lsid:zoobank.org:pub:E70C7637-C476-46CA-BAA7-BA959E0E64F5

Adriatic calcarean sponges (Porifera, Calcarea),

with the description of six new species and a richness analysis

Michelle KLAUTAU 1 * a , Mirna IMESEK 2 * a , Fernanda AZEVEDO 3 , Bruna PLESE 4 ,

Vedran NIKOLIC 5 & Helena CETKOVIC 6

13 Universidade Federal do Rio de Janeiro, Instituto de Biologia, Departamento de Zoologia,

Av. Carlos Chagas Filho 373, Cidade Universitaria, 21941-902 Rio de Janeiro (RJ), Brazil.

2 4 6 Ruder Boskovic Institute, Division for Molecular Biology,

Bijenicka cesta 54, 10002 Zagreb, Croatia,

institute of Oceanography and Fisheries, Laboratory for Benthos,

RO. Box 500, 21000, Split, Croatia.

a Equally contributed

* Corresponding authors: [email protected]; [email protected]

3 E-mail: [email protected]

4 E-mail: [email protected]

5 E-mail: niko1ic@izorhr

6 E-mail: [email protected]

1 urn: lsid: zoobank, org: author: C6BB3D65-7166-4A2A-AF2B-7F13EE94F485

2 urn:lsid:zoobank.org:author:5461D38C-ElB5-48Bl-A41B-0134F337A143

3 urn:lsid:zoobank.org:author:DC0BE6B4-F24F-4765-BA2B-ED17D774B2B7

4 urn:lsid:zoobank.org:author:C9E3BEBl-01E6-44A4-807B-4800E9393C09

5 urn:lsid:zoobank.org:author:84828CF3-6BA3-4541-AEF2-24422CEA0179

6 urn:lsid:zoobank.org:author:BD5D084E-8AAE-4CBA-991B-0901EB6C8DEl

Abstract. In this study we analyze the calcarean sponge diversity of the Adriatic Sea, the type locality

of some of the first described species of calcarean sponges. Morphological and molecular approaches

are combined for the taxonomic identification. Our results reveal six species new to science and

provisionally endemic to the Adriatic Sea ( Ascandra spalatensis sp. nov., Borojevia croatica sp. nov.,

Leucandra falakra sp. nov., L. spinifera sp. nov., Paraleucilla dalmatica sp. nov., and Sycon ancora

sp. nov.), one species previously known only from the Southwestern Atlantic ( Clathrina conifera),

and three already known from the Adriatic Sea ( Ascaltis reticulum , Borojevia cerebrum , and Clathrina

primordialis). We confirm the presence of the alien species Paraleucilla magna in the Adriatic and again

record Clathrina blanca , C. clathrus , and C. rubra. We emend the description of the genus Ascaltis ,

propose a lectotype for Borojevia cerebrum and synonymise B. decipiens with B. cerebrum. A checklist

of all calcarean species previously and currently known from the Adriatic Sea (39 species) is given. The

Central Adriatic is indicated as the richest calcarean sponge fauna sector; however, the biodiversity of

this class is underestimated in the whole Adriatic Sea and new systematic surveys are desirable.

Keywords. Porifera, Calcarea, Adriatic Sea, molecular taxonomy, morphological taxonomy.

1

European Journal of Taxonomy 178: 1-52 (2016)

Klautau M., Imesek M., Azevedo F., Plese B., Nikolic V. & Cetkovic H. 2016. Adriatic calcarean sponges (Porifera,

Calcarea), with the description of six new species and a richness analysis. European Journal of Taxonomy 178:

1-52. http://dx.doi.org/10.5852/eit.2016.178

Introduction

Although the biodiversity of sponges of the Mediterranean Sea has been studied for a long time (e.g.,

Schmidt 1862, 1864; Haeckel 1872), some regions and sponge taxa have remained rather neglected.

The Adriatic Sea is one of the seven eco-regions of the Mediterranean Province (Spalding et al. 2007)

and is considered a biodiversity hotspot (Bianchi et al. 2012) of major ecological importance. It forms

a very narrow, semi-enclosed basin in the northernmost part of the Mediterranean Sea, subdivided into

three sectors: Northern Adriatic, Central Adriatic and Southern Adriatic (Bianchi & Morri 2000). The

karst limestone is known for forming unique habitats such as caves, overhangs and pits, which are

rather inaccessible and often inhabited by a number of invertebrate groups, including calcarean sponges.

They are generally less investigated, mainly because of a smaller number of experts involved in their

taxonomy, and consequently a large number of species is still unknown. Several calcarean species are

known from the Mediterranean, including the Adriatic (see Pansini & Longo 2008), but literature data

on these Adriatic species is very scarce or difficult to access, moreover lacking a comprehensive and

detailed morphological and molecular descriptions.

Dohrmann et al. (2006), Rossi et al. 2011 and Voigt et al. (2012) demonstrated that modern methods of

DNA taxonomy are necessary to highlight the phylogenetic signals in morphological features of, mostly

ambiguous, calcarean sponges. Thus, expanding the number of analysed species helps in defining

the species-specific morphological features related to certain phylogenetic traits (Rossi et al. 2011). A

clear and precise taxonomy, based on molecular analyses, represents a strong foundation for accurate

systematics changes on different taxonomic levels (Klautau et al. 2013).

Some of the first studies on the class Calcarea were done along the Dalmatian coast (e.g., Schmidt

1862, 1864; Haeckel 1870, 1872). Therefore, the Adriatic Sea is the type locality of many of the first

known species of calcarean sponges (Haeckel 1870, 1872). Nevertheless, a large number of the original

descriptions are fragmentary and many type specimens were lost over time. Hence, in addition to the

importance of species diversity records for this eco-region, it is crucial to establish new collections

and descriptions of Adriatic calcarean sponges, preferably with detailed morphological and molecular

analyses.

In the present work, we study the calcarean sponge diversity of the Adriatic Sea and re-describe some

of Haeckel’s species using morphological and DNA taxonomy. Furthermore, we performed an analysis

of species richness and compiled a checklist comprising all species previously known from the Adriatic

Sea (32 species), together with the new data provided here (7 species). Our results indicate that the

calcarean sponge fauna of the Adriatic Sea is still underestimated and new systematic surveys including

molecular markers are very welcome.

Materials and methods

The material studied is preserved in the following collections:

BMNH = The Natural History Museum, Tondon, UK

GW = Gert Worheide

IRB = Institut Ruder Boskovic, Zagreb, Croatia

MNRJ = Museu Nacional do Rio de Janeiro, Brazil

PMJ = Phyletisches Museum Jena, Germany

PMR = Prirodoslovni Muzej Rijeka, Croatia

2

KLAUTAU M. et al ., Adriatic calcarean sponges (Porifera, Calcarea)

QM = Queensland Museum, Australia

SAM = South Australian Museum, Australia

UFRJPOR = Sponge collection of the Biology Institute of the Universidade Federal do Rio de

Janeiro, Brazil

ZMAPOR = Zoologisch Museum, Instituut voor Systematiek en Populatiebiologie, Amsterdam, The

Netherlands

ZMB = Museum fur Naturkunde and der Universitat Humboldt zu Berlin, Germany

Whenever possible, specimens were divided among two different collections. In such cases the specimens

received two register numbers. Both numbers are indicated in the text.

Fig. 1. Map of the Croatian coast. Studied locations along the coast are marked with gray circles

and numbers. 1. Near Selce. 2. Island of Pag. 3. Near Zadar. 4. Island of Blitvenica. 5. Near Split.

6a-b. Island of Ciovo. 7. Island of Brae. 8. Vrulja Cove. 9. Port of Ploce. 10. Prapratno Cove. 11. Near

Dubrovnik. 12. Island of Lokrum.

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European Journal of Taxonomy 178: 1-52 (2016)

Sampling and morphological analyses

Calcarean sponges were collected by SCUBA in 12 localities along the Croatian coastline (Fig. 1).

Sponges were fixed and preserved in 96% ethanol. Spicule preparations and sections followed standard

procedures (Worheide & Hooper 1999; Klautau & Valentine 2003).

Length and width at the base of each actine of the spicules were measured. The results are presented

in tabular form, featuring length (min im um [min], mean, standard deviation [sd] and maximum

[max]), width (minimum [min], mean, standard deviation [sd] and maximum [max]) and sample size

(n). Photomicrographs were taken with a digital camera mounted on a Zeiss Axioskop microscope.

Micrographs were taken with a JEOL, JSM-6510 scanning electron microscope (SEM) at the Biology

Institute (Universidade Federal do Rio de Janeiro).

Specimens are deposited in the sponge collections of the Biology Institute / Universidade Federal do Rio

de Janeiro, Brazil, the Natural History Museum in Rijeka, Croatia, and at the Ruder Boskovic Institute,

Croatia (Table 1).

DNA isolation, amplification, cloning and sequencing

Total DNA was extracted from 0.05-0.3 g of tissue, using the E.Z.N.A. Forensic DNA Kit (Omega

Bio-telc) or G-spin Genomic DNA Extraction Kit (Intron) following the manufacturer’s protocol. The

ITS1-5.8S-ITS2 rDNA region was amplified by polymerase chain reaction (PCR) using primers 18SF

(5 f -TCATTTAGAGGAAGTAAAAGTCG-3') plus 5.8SR (5'-GCGTTCAAAGACTCGATGATTC-3')

(Lobo-Hajdu et al. 2004) and ITS2F (5'-CGGCTCGTGCGTCGATGAAGAAC-3') plus ITS2R

(5'-CGCCGTTACTGGGGGAATCCCTGTTG-3') (Harcet et al. 2010). Partial 28SrDNA gene

sequences were amplified with two pairs of primers, NL4F (5'-GACCCGAAAGATGGTGAACTA-3')

plus NL4R (5'-ACCTTGGAGACCTGATGCG-3') (Nichols 2005), and primers CAL-28SFW

(5 '-GKCGGATCCGAAYGGACCG-3') plus CAL28SRV (5'-CCTCTAATCATTCGCTTTACC-3')

designed at the Laboratory of Molecular Genetics of the Ruder Boskovic Institute, based on multiple

alignments with the sequences available from GenBank.

PCR reactions were performed under the following conditions: 3 min/95 °C, 30 cycles (30 s/94 °C, 45

s/55 °C, 90 s/70 °C) and final elongation 10 min/72 °C. Reaction mixtures containing 2.5 pi of lOxPCR

buffer, 3 pi MgCl 0 (25 mM), 0.8 pi of each primer (10 mM), 0.5 pi dNTPs (10 mM each), 1 unit of

Taq-DNA polymerase and 100-150 ng template. The quality and quantity of the amplified DNA were

estimated by agarose gel electrophoresis. PCR products were purified from gel using QIAquick Gel

Extraction Kit (Qiagen) and directly sequenced using the ABI PRISM 3100 automatic sequencer and

ABI PRISM BigDye Terminator v. 3.1 Ready Reaction Cycle Sequencing Kit (Applied Biosystems).

In a few cases, the outcomes of the sequencing reaction were mixed peaks caused by non-target DNA

contamination, such as bacteria or algae, which were identified using BLAST network service ( http://

www.ncbi.nlm.nih.gov/ ). These regions were ligated with the pGEM-T Vector Kit (Promega), cloned

into XL 1-Blue competent cells and up to three clones were purfied using the QIAprep Spin Miniprep

Kit (Qiagen) and sequenced.

Sequence alignment and phylogenetic reconstruction

Cloned sequences were assembled using Lasergene processing software (DNASTAR Inc., Madison,

WI, USA) and checked manually for sequencing errors. The BLAST network service ( http://www.ncbi.

nhn.nih.gov/ ) was used for sequence homology searches. Multiple alignments were performed with the

Q-INS-i option of the MAFFT program (Katoh & Standley 2013), using score matrix 200 PAM/k=2,

gap penalty 1.53 and offset value 0. Alignments were run through a Gblocks v. 0.91b server under less

stringent parameters (Castresana 2000) to exclude poorly aligned regions from further analyses. Two

4

KLAUTAU M. et al., Adriatic calcarean sponges (Porifera, Calcarea)

Table 1. Specimens included in the phylogenetic analyses with collection sites, voucher numbers and

GenBank accession numbers. ^Specimens with newly generated DNA sequences.

Species

Collection site

Voucher number

GenBank accession

number

CALCINEA

ITS

28S

Arthuria hirsuta

Cape Verde

ZMAPOR07061

KC843431

-

Arthuria hirsuta

Cape Verde

ZMAPOR 07103

KC985143

-

Arthuria spirallata

Peru

MNRJ 13652

KC985140

-

Arthuria spirallata

Peru

MNRJ 11414

KC985142

-

Ascaltis reticulum

Mediterranean Sea

UFRJPOR6258

HQ588973

-

Ascaltis reticulum

Mediterranean Sea

UFRJPOR6260

HQ588977

-

Ascaltis reticulum *

Adriatic Sea

PMR-13739 =UFRJPOR6870

KP740022

KP739998

Ascandra contorta

Mediterranean Sea

UFRJPOR6327

HQ588970

-

Ascandra corallicola

Norway

UFRJPOR6329

HQ588994

-

Ascandra falcata

Mediterranean Sea

UFRJPOR5856

HQ588962

-

Ascandra falcata

Mediterranean Sea

UFRJPOR6320

HQ588963

-

Ascandra spalatensis sp. now*

Adriatic Sea

PMR-17806 =UFRJPOR7540

KP740024

KP740003

Borojevia cf. aspina

Brazil

UFRJPOR5211

HQ588969

-

Borojevia cf. aspina

Brazil

UFRJPOR5245

HQ588998

-

Borojevia brasiliensis

Brazil

UFRJPOR5214

HQ588978

-

Borojevia brasiliensis

Brazil

UFRJPOR5230

HQ588999

-

Borojevia cerebrum

Mediterranean Sea

UFRJPOR6322

HQ588964

-

Borojevia cerebrum

Mediterranean Sea

UFRJPOR6323

HQ588971

-

Borojevia cerebrum

Mediterranean Sea

UFRJPOR6324

HQ588975

-

Borojevia cerebrum*

Adriatic Sea

IRB-CLB26

KP740029

KP740008

Borojevia cerebrum- |:

Adriatic Sea

IRB-CLB32

KP740031

KP740010

Borojevia cerebrum*

Adriatic Sea

PMR-17808

KP740030

KP740009

Borojevia cerebrum -*

Adriatic Sea

IRB-CLB33 = UFRJPOR7539

KP740032

KP740011

Borojevia croatica sp. now*

Adriatic Sea

PMR-13740 = UFRJPOR6864

KP740020

KP739995

Borojevia croatica sp. now*

Adriatic Sea

PMR-13741 = UFRJPOR6865

KP740021

KP739997

Borojevia croatica sp. now*

Adriatic Sea

IRB-CLB6

KP740023

KP740002

Borojevia croatica sp. now*

Adriatic Sea

IRB-CLB17

KP740026

KP740005

Borojevia croatica sp. now*

Adriatic Sea

IRB-CLB18

KP740027

KP740006

Borojevia croatica sp. now*

Adriatic Sea

IRB-CLB19

KP740028

KP740007

Clathrina adusta

GBR, Wistari Reef

QM G313665

-

JQ272288

Clathrina aphrodita

Peru

MNRJ 14180

KC985137

-

Clathrina aphrodita

Peru

MNRJ 12994

KC985138

-

Clathrina aurea

Brazil

MNRJ 8998

HQ588968

-

Clathrina aurea

Brazil

MNRJ 8990

HQ588958

-

Clathrina antofagastensis

Chile

MNRJ 9289

HQ588985

-

Clathrina antofagastensis

Peru

MNRJ 11294

KF002722

-

Clathrina blanca

Adriatic Sea

PMR-14307

KC479087

KC479085

Clathrina blanca*

Adriatic Sea

PMR-13744

KP740017

KP740000

Clathrina clathrus

Mediterranean Sea

UFRJPOR6315

HQ588974

-

Clathrina clathrus

Mediterranean Sea

UFRJPOR6325

HQ588965

-

Clathrina clathrus

Mediterranean Sea

UFRJPOR6326

HQ588972

-

Clathrina clathrus

Adriatic Sea

PMR-14308

KC479089

KC479083

Clathrina clathrus*

Adriatic Sea

IRB-CLB12

KP740025

KP740004

Clathrina clathrus*

Adriatic Sea

PMR-13745

KP740015

KP740001

Clathrina conifer a

Brazil

MNRJ 8997

HQ588957

-

Clathrina conifer a

Brazil

MNRJ 8991

HQ588959

-

Clathrina conifer a*

Adriatic Sea

PMR-1373 8 = UFRJPOR6869

KP740019

KP739994

Clathrina conifer a*

Adriatic Sea

PMR-17807

KP740033

KP740012

Clathrina conifer a*

Adriatic Sea

IRB-S2 = UFRJPOR7541

KP740034

KP740013

Clathrina conifer a*

Adriatic Sea

IRB-S3 = UFRJPOR7542

KP740035

KP740014

Clathrina coriacea

Norway

UFRJPOR6330

HQ588986

-

Clathrina fjordica

Chile

MNRJ 8143

HQ588984

-

Clathrina helveola

Australia

QMG313680

HQ588988

AM 180987.1

Clathrina liispanica

Mediterranean Sea

UFRJPOR6305

KC843432

-

Clathrina lacunosa

Norway

UFRJPOR6334

HQ588991

-

Clathrina lacunosa

Norway

UFRJPOR6335

HQ588992

-

Clathrina luteoculcitella

Australia

QMG313684

-

AM 180988.1

Clathrina peruana

Peru

MNRJ 13144

KC985134

-

Clathrina peruana

Peru

MNRJ 12839

KC985135

-

Clathrina primordialis

Adriatic Sea

PMR-14305

KC479086

KC479084

Clathrina primordialis*

Adriatic Sea

IRB-CLB3 = UFRJPOR6863

KP740016

KP739996

5

European Journal of Taxonomy 178: 1-52 (2016)

Clathrina ramosa

Chile

MNRJ 10313

HQ588990

Clathrina rubra

Adriatic Sea

PMR-14306

KC479088

KC479082

Clathrina rubra *

Adriatic Sea

IRB-CLC2

KP740018

KP739999

Clathrina wistariensis

Australia

QMG313663

-

AMI 80990

Lelapiella incrustans

Vanuatu

QM G313914

-

JQ272306

Leucaltis clathria

GBR, DJ’s reef

QM G316022

-

JQ272302

Levinella prolifera

GBR, Hook Reef

QM G313818

-

JQ272292

Murrayona phanolepis

Coral Sea, Osprey Reef

QMG313992

-

JQ272304

Murrayona phanolepis

Coral Sea, Bougainville Reef

QM G316290

-

AMI 80998

Soleneiscus radovani

GBR, Wistari Reef

QMG313661

-

JQ272289

Soleneiscus stolonifer

GBR, Wistari Reef

QM G313668

-

JQ272290

CALCARONEA

ITS

28S

Grantia compressa

-

AY563538

Grantiopsis cylindrica

GBR, Lizard Island

GW 973

-

JQ272263

Grantiopsis heroni

GBR, Wistari Reef

QM G313670

-

JQ272261

Leucandra aspera

-

AY563535

Leucandra falakra sp. now*

Adriatic Sea

PMR-13748/UFRJPOR8349

KT447551

KT447560

Leucandra nicolae

-

JQ272268

Leucandra spinifera sp.now*

Adriatic Sea

PMR-13742/UFRJPOR6861

KT447552

KT447562

Leucandra spinifera sp.nov.*

Adriatic Sea

IRB-SG3/UFRJPOR8348

KT447553

KT447561

Leucascandra caveolata

GBR

QMG316057

-

JQ272259

Paraleucilla dalmatica sp. now*

Adriatic Sea

PMR-13747

KT447556

KT447565

Paraleucilla dalmatica sp. now*

Adriatic Sea

IRB - SD5/UFRJPOR8346

KT447557

KT447566

Paraleucilla magna

Brazil

GW 824

-

JQ272267

Paraleucilla magna

South Atlantic

-

AM181005

Paraleucilla magna*

Adriatic Sea

PMR-13743

KT447554

KT447563

Paraleucilla magna*

Adriatic Sea

IRB-PI 4

KT447555

KT447564

Sycettusa aff. hastifera

Red Sea

GW 893

-

JQ272282

Sycettusa cf. simplex

Western Indian Ocean

ZMA PORI 1566

-

JQ272279

Sycettusa tenuis

GBR, Heron Reef

QM G313685

-

JQ272281

Sycon ancora sp. now*

Adriatic Sea

PMR-17809/UFRJPOR8345

KT447558

KT447567

Sycon ancora sp. now*

Adriatic Sea

IRB - SD12/UFRJPOR8347

KT447559

KT447568

Sycon capricorn

-

QM G316025

AJ633889

-

Sycon capricorn

GBR, Ribbon Reef 3

QM G316187

-

JQ272272

Sycon carteri

Australia

SAM PS 0142

-

JQ272260

Sycon ciliatum

-

AJ627187

AY563532

Sycon raphanus

-

AY563537

Syconessa panicula

GBR, Wistari Reef

QM G313671

-

JQ272276

Syconessa panicula

GBR, Wistari Reef

QM G313672

-

AM181007

Teichonopsis labyrinthica

Australia

SAM PS 0228

-

JQ272264

Ute ampullaeea

GBR, Wistari Reef

QM G313669

-

JQ272266

Ute aff. syconoides

GBR, Yonge Reef

QM G313694

-

JQ272271

Ute aff. syconoides

Tasmania

QM G323233

-

JQ272269

different methods were applied for phylogenetic reconstruction: maximum likelihood (ML) and Bayesian

inference (BI). The Alcaike Information Criterion (AIC) implemented in jModeltest 3.7 (Guindon &

Gascuel 2003; Darriba et al. 2012) was used to select the best-fit models of sequence evolution. The

models were chosen for each dataset as follows: for 28S analysis, GTR+I+G and TrN+I+G models

were chosen for Calcinea and Calcaronea, respectively; for ITS analysis, the TrN+G model was chosen

for both datasets. Phylogenetic analyses were performed in PhyML 3.0 (Guindon et al. 2010), where

datasets were analysed by the ML method. Bootstrap tests of phylogeny were performed with 1000

replicates. Bayesian MCMC analyses were performed in MrBayes v. 3.1.2. (Huelsenbeck & Ronquist

2001), considering the same models for given datasets. Two parallel runs each comprising four Markov

chains were run for 1,000,000 generations with a sampling frequency of one in every 100 trees; a

consensus tree was constructed based on the trees sampled after burn-in of 100,000. Phylogenetic trees

were generated separately for each dataset, rooted at midpoint and displayed in FigTree v. 1.4.2 ( http://

6

KLAUTAU M. et al, Adriatic calcarean sponges (Porifera, Calcarea)

tree.bio.ed.ac.uk/software/figtree/ ). Obtained sequences were submitted to GenBank under the accession

numbers KP739994-KP740035 and KT447551-KT447568 (Table 1).

Analysis of species richness

A richness analysis of all calcarean species present in the Adriatic Sea was performed using DIVA-GIS

version 7.5, a free computer program for mapping and analyzing biodiversity data ( http ://www. diva-gis.

org/) (Hijmans et al. 2012). Menu options chosen for this analysis were: species (parameters), 0.5 x 0.5

degrees (cell size) and simple (point to grid procedure).

Results

Species list

Ascaltis reticulum (Schmidt, 1862)

Ascandra spalatensis sp. nov.

Borojevia cerebrum (Haeckel, 1872)

Borojevia croatica sp. nov.

Clathrina conifera Klautau & Borojevic, 2001

Clathrinaprimordialis (Haeckel, 1872)

Leucandra falakra sp. nov.

Leucandra spinifera sp. nov.

Paraleucilla dalmatica sp. nov.

Sycon ancora sp. nov.

Taxonomy

Class Calcarea Bowerbank, 1864

Subclass Calcinea Bidder, 1898

Genus Ascaltis Haeckel, 1872

Ascaltis reticulum (Schmidt, 1862)

Fig. 2; Table 2

Nardo a reticulum Schmidt, 1862: 18.

Tarrus reticulatus Haeckel, 1870: 244.

Nardopsis reticulum Haeckel, 1870: 247.

Ascandra reticulum Haeckel, 1872: 87.

Olynthus reticulum Haeckel, 1872: 88.

Clistolynthus reticulum Haeckel, 1872: 88.

Soleniscus reticulum Haeckel, 1872: 88.

Nardorus reticulum Haeckel, 1872: 88.

Tarrus reticulum : Haeckel, 1872: 88.

Auloplegma reticulum Haeckel, 1872: 88.

Ascometra reticulum Haeckel, 1872: 88.

Ascandra retiformis Haeckel, 1872: 88.

Ascandra reticulata Haeckel, 1872: 88.

Clathrina reticulum Minchin, 1896: 359.

Ascandra hermesi Breitfuss, 1897a: 39.

Leucosolenia hermesi Dendy & Row, 1913: 722.

Nardoa reticulum — Schmidt 1869: 91; 1870: 73.

Ascandra reticulum - Vosmaer 1881: 5. —Lendenfeld 1891: 39. —Breitfuss 1897b: 214; 1898a: 23;

1898b: 92. —Brondsted 1914: 530.

7

European Journal of Taxonomy 178 : 1-52 ( 2016 )

Leucosolenia reticulata -Dendy & Row 1913: 723. —Breitfuss 1932: 243.

Leucosolenia reticulum - Dendy & Row 1913: 723. —Breitfuss 1930: 275; 1932: 243; 1935: 14. —

Topsent 1934: 9; 1936: 22. —Hozawa 1940: 32. —Arndt 1941: 4. —Tanita 1942: 82; 1943: 386. —

Burton 1963: 200.

Ascandra retiformis -Breitfuss 1932: 243.

Leucosolenia hermesi - Tanita 1942: 82.

Clathrina reticulum -Borojevic 1967: 189. —Borojevic & Peixinho 1976: 993. —Borojevic & Boury-

Esnault 1987: 12. — Klautau & Valentine 2003: 36. — Longo & Pronzato 2011: 230. — Muricy et al.

2011: 34.

Ascaltis reticulum - Klautau et al. 2013: 452.

Original type locality

ADRIATIC SEA: Zara (Croatian: Zadar) and Sebenico (Croatian: Sibenik).

Fig. 2. Ascaltis reticulum (PMR 13739 = UFRJPOR 6870). A. Specimen in situ. B. Section showing the

perpendicular arrangement of diactines. C. Regular triactines. D. Regular tetractine. E. Apical actine of

a tetractine covered with short spines. F. Diactine.

8

KLAUTAU M. et al., Adriatic calcarean sponges (Porifera, Calcarea)

Table 2. Spicule measurements of Ascaltis reticulum (Schmidt, 1862) (PMR-13739 = UFRJPOR 6870).

length (jam)width (jam)

Spicule

Actine

min

mean

sd

max

min

mean

sd

max

n

Diactine

-

60.0

106.3

26.0

142.5

3.8

4.9

0.5

6.3

20

Triactine

Basal

56.7

88.2

11.7

108.0

6.8

9.5

1.5

10.8

21

Tetractine

Basal

60.0

79.1

12.4

107.5

7.5

8.8

1.3

10.0

30

Apical

27.5

41.9

9.7

62.5

2.5

3.2

0.6

3.8

17

Type specimen

FRANCE: Banyuls-sur-Mer, Pyrenees, E.A. Min chin Collection (BMNH 1896.9.15.13, neotype

proposed by Klautau & Valentine 2003).

Material examined

ADRIATIC SEA: near the Island of Ciovo, 43°28'58.5" N, 16°21'25.6" E, 5 m, 5 Nov. 2010, collected

by B. Plese and V. Nikolic (PMR-13739 = UFRJPOR 6870).

Colour

White in life and white in ethanol.

Description

Cormus is composed of regular and tightly anastomosed tubes. Water-collecting tubes are present

(Fig. 2A). As the specimen was fragmented, it was not possible to observe the pseudoatrium. The skeleton

is composed of one category of triactines, one of tetractines and diactines. Diactines are organised in

tufts of two to five spicules, perpendicularly disposed in the tubes (Fig. 2B). Triactines are the most

abundant spicules.

Spicules (Table 2)

Triactines. Regular (equiangular and equiradiate). Actines are slightly conical to cylindrical, with sharp

tips (Fig. 2C). Size: 88.2/9.5 pm.

Tetractines. Regular (equiangular and equiradiate). Actines are slightly conical to cylindrical, with sharp

tips (Fig. 2D). The apical actine is very thin and shorter than the basal ones. It is cylindrical and blunt,

covered by abundant tiny spines (Fig. 2E). Size: 79.1/8.8 pm (basal actine); 41.9/3.2 pm (apical actine).

Diactines. Slightly curved. The tip that protrudes through the surface is lanceolated (Fig. 2F). Trichoxeas

are also present on the surface of the tubes. Size: 106.3/4.9 pm.

Ecology

Specimens were collected on a vertical, shaded hard limestone bottom.

Remarks

Klautau etal. (2013) proposed to transfer this species to the genus Ascaltis based mainly on morphological,

but also on molecular data. Although the type species of this genus (A. lamarcki Haeckel, 1870) was not

included in the molecular dataset, A. reticulum did not group with any of the included genera (Fig. 16).

Besides, morphologically it is more similar to Ascaltis than to any other genus. Therefore, although the

classification of A. reticulum in the genus Ascaltis must still be verified regarding the type species of the

9

European Journal of Taxonomy 178: 1-52 (2016)

genus, it was morphologically and molecularly proved that it cannot be included in the genus Clathrina.

Hence, we keep the proposition of Klautau et al. (2013) and name this species A. reticulum.

This is the first time that spines were observed on the apical actine of the tetractines of A. reticulum. For

that reason, we examined the neotype of this species and detected spines as well. They are abundant and

very small. We also observed a great variation in the size of the diactines, which are much larger in the

neotype (102.0-212.2 (±54.1)-306.0 /14.3 (±5.1) pm).

Genus Ascandra Haeckel, 1872

Ascandra spalatensis sp. nov.

urn:lsid:zoobank.org:act:A5DC68F2-D856-4492-AF50-E5F6AlA7FD8A

Fig. 3; Table 3

Etymology

From the type locality. Spalato is an Italian name for Split, the largest city of the Dalmatian region.

Fig. 3. Ascandra spalatensis sp. nov., holotype (PMR 17806 = UFRJPOR 7540). A. Specimen in ethanol.

B. Tangential section. C. Triactines. D. Tetractines. E. Apical actine of a tetractine.

10

KLAUTAU M. et al, Adriatic calcarean sponges (Porifera, Calcarea)

Table 3. Spicule measurements of the holotype of Ascandra spalatensis sp. nov. (PMR-17806 =

UFRJPOR 7540).

length (pm)width (jam)

Spicule

Actine

min

mean

sd

max

min

mean

sd

max

n

Triactine

Basal

43.2

90.5

17.2

113.4

6.8

8.0

0.8

9.5

20

Tetractine

Basal

51.3

99.4

16.9

135.0

8.1

12.0

1.6

14.9

21

Apical

72.9

74.3

1.9

75.6

10.8

0

10.8

2

Material examined

Holotype

ADRIATIC SEA: near Zadar, 44°08T4.8" N, 15°12'38.2" E, 1 m, collected by V. Nikolic, 13 Feb. 2011

(PMR-17806 = UFRJPOR 7540, in ethanol).

Colour

White in ethanol.

Description

The sponge is small, only a fragment, but it is possible to recognise large and loosely anastomosed tubes,

typical of Ascandra (Fig. 3A). The skeleton is composed of triactines and a few tetractines (Fig. 3B).

Spicules (Table 3)

Triactines. Regular (equiangular and equiradiate), but there are also subregular (sagittal) spicules.

Actines are cylindrical to slightly conical with sharp tips (Fig. 3C). Size: 90.5/8.0 pm.

Tetractines. Regular (equiangular and equiradiate) or subregular. Actines are strongly conical with

sharp tips (Fig. 3D). The apical actine is shorter than the basal ones, thick, conical, sharp and smooth

(Fig. 3E). Size (basal actine): 99.4/12.0 pm; 74.3/10.8 pm (apical actine).

Ecology

The specimen was collected on a shaded, vertical hard bottom.

Remarks

The genus Ascandra is so far composed of 13 species: A. falcata Haeckel, 1872; A. ascandroides

(Borojevic, 1971); A. atlantica (Thacker, 1908); A. biscayae (Borojevic & Boury-Esnault, 1987);

A. brandtae (Rapp et al. , 2013); A. contorta (Bowerbank, 1866); A. corallicola (Rapp, 2006); A. crewsi

Van Soest & De Voogd, 2015; A. densa Haeckel, 1872; A. kakaban Van Soest & De Voogd, 2015;

A. loculosa (Dendy, 1891); A. minchini Borojevic, 1966; and A sertularia Haeckel, 1872.

In 2013, Klautau et al. proposed the following diagnosis for Ascandra: “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.”

After the discovery of A spalatensis sp. nov., we propose here an emendation to this 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. The apical actine is very thin

(needle-like) or very thick at the base. Diactines may be added. Asconoid aquiferous system.”

11

European Journal of Taxonomy 178: 1-52 (2016)

Our new species is a very typical Ascandra , with apically free, loosely anastomosed tubes. Its skeleton is

very similar to that of A. ascandroides, i.e., composed of triactines and tetractines, the former being more

abundant than the latter and the apical actine of the tetractines being very thick at the base. Both species,

however, can be differentiated by the size of the spicules (A. ascandroides - triactines: 90-130(±20)-163

/13(±2); small tetractines: 107.5-164.5(±35)-260/16.5(±2.8); large tetractines: 193.8-313.1 (±63.2)-

418.2/39.8(±8.2)). Moreover, A. ascandroides has two categories of tetractines and A. spalatensis sp.

nov. only one. In our ITS tree (Fig. 16) this species is well nested within the Ascandra clade, with high

support values in both, Bayesian and ML analyses.

Genus Borojevia Klautau et al., 2013

Borojevia cerebrum (Haeckel, 1872)

Fig. 4; Table 4

Ascaltis cere brum Haeckel, 1872: 54.

Auloplegma cerebrum Haeckel, 1872: 55.

Ascaltis decipiens Haeckel, 1872: 55.

Ascaltis gyrosa Haeckel, 1872: 55.

Fig. 4. Borojevia cerebrum (IRB-CLB33 = UFRJPOR 7539). A. Specimen in ethanol. B. Tangential

section. C. Tripods. D. Triactines. E. Small tetractine. F. Large tetractine. G. Apical actine of a tetractine

ornamented with spines.

12

KLAUTAU M. et al, Adriatic calcarean sponges (Porifera, Calcarea)

Table 4. Spicule measurements of Borojevia cerebrum (IRB-CLB33 = UFRJPOR 7539).

length (pm)width (jam)

Spicule

Actine

min

mean

sd

max

min

mean

sd

max

n

Tripod

Basal

72.9

91.8

9.9

108.0

8.1

11.2

1.5

13.5

20

Triactine

Basal

54.0

84.6

11.8

105.3

5.4

8.9

1.3

10.8

20

Tetractine

Basal

64.8

81.9

10.0

105.3

6.8

8.5

1.5

12.2

20

Apical

35.1

46.8

8.4

64.8

5.4

0.0

5.4

20

Ascetta cerebrum -Bianco 1888: 386. —Lendenfeld 1891: 206 — Bidder 1891: 628.

Clathrina cerebrum - Minchin 1896: 359.—Borojevic 1967: 192. —Borojevic et al. 1968:31. — Sole-

Cava etal. 1991: 382. —Klautau & Valentine 2003: 14. —Longo & Pronzato 2011: 219.

Leucosolenia cerebrum - Kirk 1896: 207. —Breitfiiss 1897b: 210; 1898c: 172; 1935: 8. —Dendy &

Row 1913: 724. — Burton 1933: 236; 1963: 186. — Topsent 1934: 7; 1936: 17.

Leucosolenia decipiens - Dendy & Row 1913: 725. — Ferrer 1918: 9.— Breitfiiss 1935: 9. — Tanita

1943: 78.

Borojevia cerebrum - Klautau et al. 2013: 452.

non Clathrina cerebrum - Borojevic 1971: 526 {non Clathrina cerebrum).

Type specimen

ADRIATIC SEA: Lesina (Croatian: Island of Hvar), Haeckel collection (PMJ-Inv. Nr. Porif. 156,

syntype/ethanol).

Material examined

ADRIATIC SEA: Vrulja Cove, 43°24'01.3" N, 16°53T0.9" E, 10 m, collected by V. Nikolic, 24 Aug.

2011 (PMR-17808; IRB-CTB33 = UFRJPOR 7539).

Colour

Light yellow in life and in ethanol.

Description

Cormus is composed of regular and tightly anastomosed tubes (Fig. 4A). Large water-collecting tubes

are present. The skeleton consists of triactines, a few tetractines and tripods, which in fact are large

triactines. It has no special organisation (Fig. 4B).

Spicules (Table 4)

Tripods. Regular (equiangular and equiradiate). The tripods of analysed specimens are more similar to

large triactines than to true tripods with an elevated centre. Actines are conical, straight, with sharp tips

(Fig. 4C). Size: 91.8/11.2 pm.

Triactines. Regular (equiangular and equiradiate). Actines are slightly conical to conical, straight, with

sharp tips. Sometimes they are slightly undulated near the tips (Fig. 4D). Size: 84.6/8.9 pm.

Tetractines. Regular (equiangular and equiradiate). Actines are slightly conical to conical, straight,

with sharp tips. Sometimes they are slightly undulated near the tips. It is possible to recognise two

types of tetractines: small (Fig. 4E) and large (Fig. 4F). Large tetractines are the same size as tripods.

The apical actine of the tetractines is shorter than the basal ones, slightly conical, sharp and frequently

curved only at the tip. It is ornamented with few (ca. six) spines, which are large, conical and cover only

the last third of the apical actine. (Fig. 4G). Size: 81.9/8.5 pm (basal actine); 46.8/5.4 pm (apical actine).

13

European Journal of Taxonomy 178: 1-52 (2016)

Ecology

The specimen was collected on a semi-vertical hard limestone bottom.

Remarks

Similar to other species of Borojevia , B. cerebrum has thin, regular and tightly anastomosed tubes forming

the cormus. The oscula are present at the end of water-collecting tubes. The skeleton is composed of

tripods (with the characteristic elevated centre or similar to large triactines), triactines and tetractines.

Individuals of B. cerebrum always have spines on the apical actine of their tetractines; however, in the

same individual some tetractines may be smooth. In B. cerebrum , the spines are not very abundant;

they are large and scattered, only near the tip of the apical actine. The Adriatic and Mediterranean

specimens of B. cerebrum formed a well supported clade in the ITS tree (Fig. 16), separated from the

clade comprising B. brasiliensis (Sole-Cava, Klautau, Boury-Esnault, Borojevic & Thorpe, 1991).

Borojevia cerebrum is the type species of the genus. Its type locality is Tesina (Island of Hvar) and

it co mm only occurs in the Mediterranean and the Adriatic Sea. The type specimen of B. cerebrum

(PMJ-Inv. Nr. Porif. 156) is not very well preserved (Klautau & Valentine 2003); thus, we got a great

opportunity to redescribe this species from near its type locality.

Analyses of other individuals of B. cerebrum from several sites in the Adriatic and Mediterranean Seas

verily that the shape of the tripods is very variable. It varies from the characteristic shape of tripods,

with stout actines and elevated centre, to only large triactines. This kind of variability may be assigned to

polymorphism or plasticity. Indeed, Haeckel (1872) proposed two varieties of B. cerebrum (as Ascaltis

cerebrum ), based on the presence of either characteristic tripods or large triactines. The first variety he

called B. cerebrum var. gyrosa , while the other one he considered B. cerebrum var. decipiens. Dendy &

Row (1913) elevated B. cerebrum var. decipiens to species level (as Leucosolenia decipiens ) and kept

B. cerebrum (as L. cerebrum) as a valid species. The variety gyrosa had not been oficially elevated to the

status of species; however, it was mentioned as Ascaltis gyrosa in a synonym list of B. cerebrum made

by Burton (1963: 186).

Considering that both varieties were proposed only to differentiate specimens with characteristic

tripods from those with only large triactines and that we found this morphological variation inside

individuals and among specimens placed within the same species, we propose here the synonymisation

of B. decipiens with B. cerebrum.

Borojevia croatica sp. nov.

urn:lsid:zoobank.org:act:A9F84084-E033-43A9-AB71-83670090C7Cl

Fig. 5; Table 5

Etymology

From the type locality.

Material examined

Holotype

ADRIATIC SEA: near the Island of Ciovo, 43°28'58.5" N, 16°21'25.6" E, 5 m, collected by B. Plese and

V. Nikolic, 5 Nov. 2010 (PMR-13740 = UFRJPOR 6864, in ethanol).

Paratype

ADRIATIC SEA: same data as holotype (PMR-13741 = UFRJPOR 6865, in ethanol).

14

KLAUTAU M. et al., Adriatic calcarean sponges (Porifera, Calcarea)

Colour

White in life and in ethanol.

Description

Cormus composed of regular and tightly anastomosed tubes (Fig. 5A). Water-collecting tubes are present

and form a single apical osculum. The skeleton is composed of tripods, triactines and rare tetractines. It

has no special organisation (Fig. 5B).

Spicules (Table 5)

Tripods. Regular (equiangular and equiradiate) or sagittal. Some of them have an elevated centre, but

most appear like large regular triactines. Actines are conical, straight, with sharp tips (Fig. 5C). Size:

102.6/11.9 pm.

Triactines. Regular (equiangular and equiradiate). Actines are conical, straight, with sharp tips (Fig. 5D).

Size: 66.6/7.5 pm.

Fig. 5. Borojevia croatica sp. nov., holotype (PMR 13740 = UFRJPOR 6864). A. Specimen in situ.

B. Tangential section. C. Tripod. D. Triactines. E. Tetractine. F. Apical actine of a tetractine ornamented

with spines.

15

European Journal of Taxonomy 178: 1-52 (2016)

Table 5. Spicule measurements of Borojevia croatica sp. nov. Holotype (PMR-13740 = UFRJPOR

6864) and paratype (PMR-13741 = UFRJPOR 6865).

length (^im)width (tun)

min

mean

sd

max

min

mean

sd

max

n

Holotype

Tripod

Basal

85.0

102.6

10.0

115.0

10.0

11.9

1.5

15.0

20

Triactine

Basal

57.5

66.6

6.7

82.5

7.5

0.0

7.5

20

Tetractine

Basal

60.0

70.0

6.3

77.5

7.5

8.3

1.2

11.3

10

Apical

-

20

-

5.0

-

1

Paratype

Tripod

Basal

50.0

78.8

19.1

115.0

7.5

10.8

1.8

15.0

13

Triactine

Basal

50.0

66.1

8.6

80.0

7.5

7.8

0.7

10.0

20

Tetractine

Basal

62.5

71.0

5.1

80.0

7.5

8.3

1.1

10.0

20

Tetractines. Regular (equiangular and equiradiate). Actines are conical, straight, with sharp tips

(Fig. 5E). The apical actine has very short and abundant spines organised in parallel rows. These spines

cover the first 2 A of the apical actine (Fig. 5F). Size: 70.0/8.3 pm (basal actine); 20.0/5.0 pm (apical

actine).

Ecology

Specimens were collected on a shaded, vertical, hard limestone bottom.

Remarks

The genus Borojevia is currently composed of five species: B. aspina (Klautau, Sole-Cava & Borojevic,

1994), B. brasiliensis , B. cerebrum , B. paracerebrum (Austin, 1996) and B. tetrapodifera (Klautau &

Valentine, 2003). All of them show a very well defined cormus, with regular and tightly anastomosed

tubes and water-collecting tubes. The skeleton is always composed of tripods, triactines and tetractines

with spines on the apical actines. Tetrapods may also be present (. B . tetrapodifera ). The sixth species of

the genus, B. croatica sp. nov., is closer to B. cf. aspina in our ITS tree (Fig. 16). Both species have short

spines; however, B. croatica sp. nov. has numerous spines, while in B. cf. aspina there are few.

Given that B. cerebrum is also present in the Adriatic Sea, the best way to differentiate it from B. croatica

sp. nov. is by the shape and location of spines. They are shorter, more abundant and distributed along

most of the actine length in B. croatica sp. nov., and larger, fewer and scattered only near the tip of the

apical actine in B. cerebrum.

Genus Clathrina Gray, 1867

Clathrina conifera Klautau & Borojevic, 2001

Fig. 6; Table 6

Clathrina conifera Klautau & Borojevic, 2001: 404.

Clathrina primordialis {non C. primordialis (Haeckel, 1872)) - Borojevic 1971: 527. — Borojevic &

Peixinho 1976: 992. — Mothes de Moraes 1985: 228. — Klautau et al. 1994: 372. — Muricy & Silva

1999: 160.

16

KLAUTAU M. et al ., Adriatic calcarean sponges (Porifera, Calcarea)

Clathrina conifera - Klautau & Valentine 2003: 18. — Monteiro & Muricy 2004: 682. — Muricy &

Hajdu 2006: 86. — Lanna et al. 2007: 1554. — Custodio & Hajdu 2011: 4. — Muricy et al. 2011: 33.

— Bouzon et al. 2012: 42.

Material examined

Holotype

BRAZIL: Arraial do Cabo, Rio de Janeiro (BMNH 1999.9.16.19, in ethanol).

Other material

ADRIATIC SEA: near the Island ofLokrum, 42°37 , 55.6” N, 18 o 06’49.4” E; 1-3 m deep, collected by V.

Nikolic, 8 Oct. 2010 (PMR-13738 = UERJPOR 6869); near Dubrovnik, 42°38'26.5" N, 18°06'14.2" E;

1 m, collected by V. Nikolic, 24 Sep. 2011 (PMR-17807, IRB-S2 = UFRJPOR 7541, IRB-S3 = UERJPOR

7542).

Colour

White in life and white or brown in ethanol.

Fig. 6. Clathrina conifera (PMR 13738 = UERJPOR 6869). A. Specimen in ethanol. B. Tangential

section. C. Triactines with variable sizes and shapes.

17

European Journal of Taxonomy 178: 1-52 (2016)

Table 6. Spicule measurements (triactines) of Clathrina conifera Klautau & Borojevic, 2001 (PMR-

13738 = UFRJPOR 6869; IRB-S2 = UFRJPOR 7541; IRB-S3 = UFRJPOR 7542).

length (pm)width (pm)

min

mean

sd

max

min

mean

sd

max

n

PMR-13738 = UFRJPOR6869

57.5

88.5

11.8

122.5

7.5

10.2

1.6

15.0

30

IRB-S2 = UFRJPOR7541

59.4

86.3

9.6

97.2

8.1

9.5

1.4

10.8

20

IRB-S3 = UFRJPOR7542

64.8

82.5

8.9

102.6

6.8

8.8

1.3

10.8

20

Description

Cormus composed of irregular and loosely anastomosed tubes (Fig. 6A). Water-collecting tubes are not

present. The skeleton consists of triactines without organisation (Fig. 6B).

Spicules (Table 6)

Triactines. Regular (equiangular and equiradiate). Their size is very variable. Actines are slightly

conical to conical, straight, with blunt tips. Sometimes they are slightly undulated near the tips (Fig. 6C).

Size: 88.5/10.2 pm.

Ecology

Specimens were collected on a semi-shaded, vertical hard limestone bottom under overhangs. They

were often found in association with the macroalga Ellisolandia elongata (J. Ellis & Solander, 1786).

Remarks

Until now, this species was considered endemic to Brazil (Borojevic 1971; Mothes de Moraes 1985;

Klautau et al. 1994; Monteiro & Muricy 2004; Muricy & Hajdu 2006; Tanna et al. 2007). Originally,

it was identified as C. primordialis (Haeckel, 1872) (Borojevic 1971; Mothes de Moraes 1985; Klautau

et al. 1994). However, considering differences in the size of the actines (holotype of C. conifera :

62.5-77.3(±9.3)-97.5/9(±1.0) pm; Haeckel measurements of C. primordialis'. 100-150/8-12 pm), it

was described as a new species: C. conifera. In the present work, we confirmed by morphological and

molecular analyses (Fig. 16) that C. conifera is really distinct from C. primordialis and that it occurs in

the Adriatic Sea.

Clathrina primordialis (Haeckel, 1872)

Fig. 7; Table 7

1 Grand a pulchra Schmidt, 1862:18.

ILeucosoleniapidchra Schmidt, 1866: 8.

Prosycumprimordiale Haeckel, 1870: 237.

Olynthus simplex Haeckel, 1870: 237.

Nardoa arabica Miklucho in Haeckel, 1872: 16.

Ascettaprimordialis Haeckel, 1872: 16.

Olynthus primordialis Haeckel, 1872: 16.

Cl istolynthus primordial is FI aecke1, 1872: 16.

Soleniscusprimordialis Haeckel, 1872: 16.

Ascometraprimordialis Haeckel, 1872: 16.

1 Ascaids primordialis Haeckel, 1872: 17.

lAscordsprimordialis Haeckel, 1872: 17.

18

KLAUTAU M. et al, Adriatic calcarean sponges (Porifera, Calcarea)

lAscandra primordialis Haeckel, 1872: 18.

Ascettaprimordialis Lendenfeld, 1885: 897.

L. primordialis var. apicalis Brondsted, 1931: 9.

Clathrina cf. hondurensis - Imesek et al. 2014: 25.

Ascetta primordialis - Lendenfeld 1891: 11.—Arnesen 1901: 12.

Clathrina primordialis - Carter 1886: 510. —Minchin 1896: 359. —Jenlcin 1908: 436. —Row 1909:

184. — Klautau & Valentine 2003: 32. — Longo & Pronzato 2011: 229.

Leucosolenia primordialis - Lackschewitsch 1886: 299. —Breitfiiss 1898a: 12; 1898b: 91. —Dendy

& Row 1913: 726. —Ferrer Hernandez 1918: 10. —Burton 1926: 71; 1963: 197. —Row & Hozawa

1931: 736. —Breitfuss 1932: 242; 1935: 12. — Arndt 1941: 45. — Tanita 1942: 73; 1943: 370.

non C. primordialis - Borojevic 1971: 527. — Borojevic & Peixinho 1976: 992. — Mothes de Moraes

1985: 228. —Klautau et al. 1994: 372. —Muricy & Silva 1999: 160.

Fig. 7. Clathrina primordialis (IRB-CLB3 = UFRJPOR 6863). A. Specimen in situ. B. Tangential

section. C. Triactines with variable sizes and shapes.

19

European Journal of Taxonomy 178: 1-52 (2016)

Table 7. Spicule (triactine) measurements of Clathrina primordialis (Haeckel, 1872). IRB-CLB3 =

UFRJPOR 6863 (present work); PMJ 154 (suggested lectotype); PMR-14305 (C. cf. hondurensis in

Imesek et al. 2014); BMNH 1938.3.28.4 (holotype of C. hondurensis).

length (jim) width (pm)

min

mean

sd

max

min

mean

sd

max

n

IRB-CLB3 = UFRJPOR 6863

47.5

121.5

27.9

157.5

7.5

12.2

2.4

15.0

20

Haeckel, 1872

100.0

-

150.0

8.0

-

12.0

-

PMJ 154 (lectotype)

97.5

134.0

16.3

157.5

10.0

13.0

2.2

17.5

30

PMR-14305

101.8

128.0

9.6

151.5

13.3

15.2

1.3

19.0

33

BMNH 1938.3.28.4

105.6

133.4

17.0

156.0

12.0

15.6

1.7

19.2

20

Material examined

Syntype

ADRIATIC SEA: Lesina (Croatian: Island of Hvar), E. Haeckel collection (PMJ-Inv. Nr. Porif. 154, in

ethanol).

Other material

ADRIATIC SEA: near the Island of Ciovo, Croatia, 43°28'58.5" N, 16°21'25.6" E; 5 m, collected by B.

Plese and V. Nikolic, 5 Nov. 2010 (IRB-CLB3 = UERJPOR 6863).

Colour

White in life and in ethanol.

Description

Cormus is formed by large and loosely anastomosed tubes. Water-collecting tubes are absent (Fig. 7A).

The skeleton is composed of one category of triactines (Fig. 7B). The size of the spicules is very variable

and it is therefore not possible to categorize them.

Spicules (Table 7)

Triactines. Regular (equiangular and equiradiate). Actines are conical to slightly conical with sharp tips

(Fig. 1C). Their size is very variable. Size: 121.5/12.2 pm.

Ecology

The specimen was collected on a shaded, vertical hard limestone bottom.

Remarks

Haeckel (1872) assigned the name Ascettaprimordialis to a group of different species, and even genera,

whose skeleton comprised only triactines, but, unfortunately, did not select a holotype. In 2003, Klautau

& Valentine revised the genus Clathrina and analysed two specimens of C. primordialis , one from the

Adriatic Sea (PMJ 154) and another one from Naples (ZMB 1306). Both specimens clearly represented

different species and the authors suggested the specimen ZMB 1306 was the true C. primordialis , because

C. primordialis (originally Prosycum primordiale Haeckel, 1870) was first described from Naples.

However, analysing the present specimen and re-analysing the slides of the specimens PMJ 154 and

ZMB 1306 and the catalogue from the ZMB, we now have a different opinion. On the specimen’s

label and in the catalogue of the ZMB it is not noted that ZMB 1306 is a syntype of C. primordialis.

Consequently, Klautau & Valentine (2003) should not have designated the specimen ZMB 1306 as a

lectotype of C. primordialis. On the other hand, the label of the specimen PMJ 154 mentions it is a

20

KLAUTAU M. et al, Adriatic calcarean sponges (Porifera, Calcarea)

syntype of C. primordialis. Therefore, in our opinion, the specimen PMJ 154 is more reliably a true

representative of this species and should be considered the lectotype of C. primordialis.

Considering the morphology of PMJ 154, the specimen IRB-CLB3 = UFRJPOR 6863 represents

C. primordialis , as well as the specimen PMR 14305, recently published as C. cf. hondurensis Klautau

& Valentine, 2003 (Imesek et al. 2014). The similarities between C. primordialis and C. hondurensis

made us ponder on the possibility of synonymy between these two species. However, as we could not

obtain DNA sequences of C. hondurensis from the type locality (Honduras) to verily this, we prefer to

keep C. hondurensis as a valid species restricted to the Caribbean Sea, until further analyses are done.

Subclass Calcaronea Bidder, 1898

Genus Leucandra Haeckel, 1872

Leucandra falakra sp. nov.

urn:lsid:zoobank.org:act:3DEBFC2C-2E7E-4D8E-A7FA-7FE971CFEDB2

Figs 8-9; Table 8

Etymology

From the Greek falakra (cpoAdKpa), meaning bald, for the absence of diactines.

Material examined

Holotype

ADRIATIC SEA: near the Island of Blitvenica, 43°37'31.96" N, 15°34'25.94" E; 5 m, collected by V.

Nikolic, 10 Oct. 2012 (PMR-13748 = UFRJPOR 8349, in ethanol).

Colour

White in life and in ethanol.

Description

The sponge is massive and vase-shaped, with one apical osculum without crown. The atrium is central

and large. The aquiferous system is leuconoid (Fig. 8A). The sponge surface is smooth, but harsh. The

cortical skeleton is composed of small, tangentially arranged triactines. The choanosomal skeleton has

no organisation (Fig. 8B). It is composed of two categories of triactines (giant triactines and triactines

larger than those of the cortex) (Fig. 8C). There are also tetractines and some triactines surrounding the

canals (Fig. 8D). The atrial skeleton is smooth, composed mainly of triactines, with a few tetractines

also present (Fig. 8E).

Spicules (Table 8)

Cortical triactines. Subregular to sagittal, equiradiate and small. Actines are cylindrical, blunt and

curved (Fig. 9A-B). Size: 136.4/11.1 pm (paired actine); 106.0/11.4 pm. (unpaired actine).

Choanosomal small triactines. Subregular to sagittal. Actines are conical and sharp (Fig. 9C-D). Size:

214.2/18.3 pm (paired actine); 189.7/19.8 pm. (unpaired actine).

Choanosomal giant triactines. Subregular to sagittal, equiradiate. Actines are conical and sharp

(Fig. 9E-F). Size: 624.5/81.5 pm.

Choanosomal tetractines. Sagittal. These spicules are present only surrounding the canals. Actines

are cylindrical, sharp and curved. The unpaired actine is a little shorter than the paired ones. The apical

21

European Journal of Taxonomy 178: 1-52 (2016)

Fig. 8. Leucandra falakra sp. nov., holotype (PMR-13748 = UFRJPOR 8349). A. Specimen in ethanol.

B. Cross section. C. Detail of the cortex. D. Detail of a canal in the choanosome. E. Atrial skeleton.

Abbreviations: at = atrium; ex = cortex; c = canal.

22

KLAUTAU M. et al , Adriatic calcarean sponges (Porifera, Calcarea)

actine is straight, short, conical and sharp (Fig. 9G-H). Size: 154.0/12.4 pm (paired actine); 143.0/12.4

pm (unpaired actine); 80.6/9.6 pm (apical actine).

Atrial triactines and tetractines. Strongly sagittal. Triactines are the most abundant spicules (Fig. 91).

Actines are cylindrical and blunt. The unpaired actine is shorter than the paired ones. The apical actine

of the tetractines is conical, straight, sharp and short. Frequently they are longer and thicker than the

apical actine of the choanosomal tetractines (Fig. 9J). Size (triactines): 222.7/15.1 pm (paired actine);

111.2/12.3 pm (unpaired actine). Size (tetractines): 191.4/14.9 pm (paired actine); 92.0/13.1 pm

(unpaired actine); 110.3/11.9 pm (apical actine).

Ecology

The specimen was collected on a shaded, semi-vertical, hard limestone bottom.

Remarks

To our knowledge, there are only three described species of Leucandra without diactines and with

triactines being the main atrial spicules: L. consolida Tanita, 1943, L. glabra Hozawa, 1940 and

Fig. 9. Leucandra falakra sp. nov., holotype (PMR-13748 = UFRJPOR 8349). A-B. Cortical triactines.

C-D. Small choanosomal triactines. E-F. Giant choanosomal triactines. G-H. Tetractines of the canals.

I. Atrial triactine. J. Atrial tetractine. Scale bar A-J =100 pm.

23

European Journal of Taxonomy 178: 1-52 (2016)

Table 8. Spicule measurements of Leacandra falakra sp. nov. (PMR-13748 = UFRJPOR 8349).

length (pm)

width (jam)

sd

max

mm mean

sd

max

n

Cortical

Paired

mm

94.5

mean

136.4

triactine

Unpaired

70.2

106.0

Cortical and

choanosomal

324.0

624.5

triactine

Choanosomal

Paired

162.0

214.2

triactine

Unpaired

108.0

189.7

Tetractine

Paired

99.9

154.0

(canals)

Unpaired

45.9

143.0

Apical

50.0

80.6

Atrial triactine

Paired

140.4

222.7

Unpaired

78.3

111.2

Atrial

Paired

145.8

191.4

tetractine

Unpaired

59.4

92.0

Apical

67.5

110.3

24.0

180.9

8.1

11.1

1.9

13.5

20

18.8

143.1

8.1

11.4

2.4

16.2

20

192.3

1047.6

48.6

81.5

20.6

118.8

23

39.8

288.9

13.5

18.3

4.0

27.0

20

58.9

351.0

13.5

19.8

4.1

29.7

20

26.4

199.8

8.1

12.4

2.4

16.2

19

56.5

288.9

9.5

12.4

1.9

16.2

19

24.4

137.5

7.5

9.6

1.5

12.5

20

33.7

294.3

9.5

15.1

2.5

20.3

30

24.4

159.3

8.1

12.3

1.7

16.2

30

26.0

256.5

10.8

14.9

2.6

18.9

16

22.1

126.9

10.8

13.1

1.7

16.2

16

30.3

162.0

8.1

11.9

2.8

16.2

15

L. okinoseana Hozawa, 1929, all three from Japan. Leucandra falakra sp. nov. can be differentiated

from L. consolida by the presence of an oscular crown in the latter (although “feebly developed”), by

the absence of tetractines in the choanosome, and by the absence of the large triactines in the cortex.

Moreover, the size of some spicules is different (cortical triactines: 240-350/20-25 pm; choanosomal

triactines - paired actines: 590-740/60-86 pm, unpaired actine: 550-720/60-86 pm; atrial triactines -

paired actines: 220-270/15-18 pm, unpaired actine: 250-300/15-18 pm; atrial tetractines: same size of

the atrial triactines but with an apical actine of 80/14 pm).

Leucandra glabra has a different external morphology, with several oscula in a single individual.

Besides, the size of some spicules is different (cortical triactines: 120-240/14-28 pm; small choanosomal

triactines: 100-200/10-20 pm; large choanosomal triactines: 400-950/42-110 pm; choanosomal

tetractines: similar to the small choanosomal triactines but with an apical actine of 80/10 pm; atrial

triactines: 90-200/12-20 pm).

Leucandra okinoseana can be differentiated from L. falakra sp. nov. by the presence of “small

protuberances for attachment” in L. okinoseana and by the size of some spicules, which are larger in the

Japanese species (cortical triactines - paired actines: 120-250/16-24 pm, unpaired actine: 150—350/14—

16 pm; cortical and choanosomal large triactines: 400-1400/32-120 pm; tetractines of the canals -

paired actines: 150-200/16-20 pm, unpaired actine: 120-570/12-16 pm, apical actine: 70-200/8-12

pm; atrial triactines - paired actines: 190-370/20-32 pm, unpaired actine: 70-270/16-24 pm; atrial

tetractines - same size as the atrial triactines but with an apical actine of 50-110/8-16 pm).

24

KLAUTAU M. et al., Adriatic calcarean sponges (Porifera, Calcarea)

Fig. 10. Leucandra spinifera sp. nov., holotype (IRB-SG3 = UFRJPOR 8348). A. Specimen in ethanol.

B. Cross section. Detail: cortical microdiactine (arrow). C. Detail of the cortex. D. Tangential section of

the cortex. E. Choanosome. F. Atrial skeleton. Abbreviations: at = atrium; ex = cortex.

25

European Journal of Taxonomy 178: 1-52 (2016)

Leucandrci spinifera sp. nov.

um:lsid:zoobank.org:act:280369B2-48FF-4F3D-88E3-73317D5919A5

Figs 10-11; Table 9

Etymology

From the Latin spinifer , meaning prickly, for the presence of numerous diactines.

Material examined

Holotype

ADRIATIC SEA: Vrulja Cove, 43°24'01.3" N, 16°53T0.9" E, 10 m, collected by Vedran Nikolic, 24

Aug. 2011 (IRB-SG3 = UFRJPOR 8348, in ethanol).

Paratype

ADRIATIC SEA: Island of Ciovo, 43°28'58.5" N, 16°21'25.6" E, 5 m, collected by B. Plese and V.

Nikolic, 6 Nov. 2010 (PMR-13742 = UFRJPOR 6861, in ethanol).

Fig. 11 . Leucandra spinifera sp. nov., holotype (IRB-SG3 = UFRJPOR 8348). A-B. Cortical diactines

(scale bar = 200 pm). C. Microdiactine (scale bar = 20 pm). D. Detail of the spines of a microdiactine

(scale bar =10 pm). E-F. Cortical triactines. G. Choanosomal triactine. H-I. Choanosomal tetractines.

J-L. Atrial triactines. M. Atrial tetractine. Scale bar E-M =100 pm.

26

KLAUTAU M. et al., Adriatic calcarean sponges (Porifera, Calcarea)

Table 9. Spicule measurements of Leiicandra spinifera sp. nov. (IRB-SG3 = UFRJPOR 8348).

length (]iim)width (jam)

Diactine

min

430.0

mean

866.5

sd

217.6

max

1400.0

min

20.0

mean

54.4

sd

16.0

max

90.0

n

17

Micro diactine

70.0

100.4

29.9

180.0

2.5

4.2

1.2

7.5

20

Cortical

triactine

Paired

110.0

189.5

45.2

260.0

7.5

12.9

2.8

17.5

20

Unpaired

105.0

150.8

29.0

195.0

10.0

13.5

2.5

20.0

20

Choanosomal

triactine

Paired

140.0

192.8

35.5

300.0

10.0

12.8

1.4

15.0

20

Unpaired

115.0

188.8

36.6

260.0

10.0

14.4

1.6

17.5

20

Atrial triactine

Paired

230.0

305.3

69.8

500.0

7.5

7.9

0.9

10.0

14

Unpaired

110.0

211.4

52.7

325.0

7.5

9.8

0.7

10.0

14

Atrial

tetractine

Paired

165.0

276.8

63.3

362.5

7.5

8.4

1.1

10.0

15

Unpaired

137.5

222.0

62.6

350.0

7.5

9.7

0.9

10.0

15

Apical

32.5

42.5

16.8

67.5

5.0

6.9

1.6

8.8

4

Colour

White in life and in ethanol.

Description

The body has the shape of a vase (0.8 x 0.4 cm), with a single apical osculum surrounded by a membrane

and a crown of a few, or even no trichoxeas (Fig. 10A). The osculum is supported by sagittal tetractines,

but a few triactines are also present. They are organised in parallel and point their apical actines to

the osculum. They become disorganized, smaller, thinner and less sagittal farther from the osculum.

They are also substituted by triactines. Numerous diactines on the surface make it very hispid. The

aquiferous system is leuconoid and the atrium is large (Fig. 10A). The cortical skeleton is composed

of tangential triactines, perpendicular giant diactines, microdiactines and rare trichoxeas (Fig. 10B-E).

The giant diactines frequently cross the entire choanosome (Fig. 10B). The choanosomal skeleton has

no organisation. It is composed mainly of subregular triactines, with curved paired actines. Tetractines

are also present, but only surrounding canals. The atrial skeleton has triactines and a few tetractines that

project their apical actines into the atrium (Fig. 10F). Microdiactines are also present in the atrium.

Spicules (Table 9)

Oscular triactines (very few) and tetractines (abundant). Sagittal. Actines are cylindrical and blunt to

sharp. The unpaired actine is thinner than the paired ones. The apical actine of the tetractines is conical,

sharp, smooth and strongly curved towards the osculum aperture.

Trichoxeas. Very thin, long and straight. They are frequently broken. These spicules are rare, but can be

found in the cortex and atrium.

Diactines. Almost fusiform. The tip that penetrates the choanosome is a little larger and more rounded

(Fig. 11A-B). Size: 866.5/54.4 pm.

27

European Journal of Taxonomy 178 : 1-52 ( 2016 )

Microdiactines. Fusiform (Fig. 11C). They are present in the cortex and atrium. They frequently have

microspines (Fig. 11D), but smooth spicules are also present. Size: 100.4/4.2 pm.

Cortical triactines. Sagittal. Actines are slightly conical, with blunt tips. The unpaired actine is shorter

than the paired ones, which are curved. One of the paired actines is frequently shorter than the other

(Fig. 11E-F). Size: 189.5/12.9 pm (paired actine); 150.8/13.5 pm (unpaired actine).

Choanosomal triactines. Subregular to sagittal. The paired actines are curved, consequently the

unpaired angle is smaller than the paired angles. Actines are slightly conical with blunt tips. They are

almost the same length (Fig. 11G). These spicules are spread in the choanosome and surrounding the

canals. Size: 192.8/12.8 pm (paired actine); 188.8/14.4 pm (unpaired actine).

Choanosomal tetractines. Sagittal. The paired actines are curved, consequently the unpaired angle is

smaller than the paired angles. Actines are slightly conical with blunt tips. The apical actine is straight or

curved, conical, smooth and sharp (Fig. 11H-I). These spicules are present only surrounding the canals.

Atrial triactines and tetractines. Triactines are much more abundant. These spicules are strongly

sagittal. The paired actines are curved and much longer than the unpaired one. Actines are slightly conical

and blunt (Fig. 11J-L). The apical actine of the tetractines is straight or slightly curved near the end,

conical, smooth and sharp (Fig. 11M). These tetractines are very similar to those of the choanosome. Size

(triactine): 305.3/7.9 pm (paired actine); 211.4/9.8 pm (unpaired actine). Size (tetractine): 276.8/8.4 pm

(paired actine); 222.0/9.7 pm (unpaired actine); 42.5/6.9 pm (apical actine).

Ecology

Specimens were collected on a cliff in a shaded area.

Remarks

This species differs from all other species of Leucandra mainly by the composition of the skeleton,

particularly by the presence of mainly triactines in the atrial skeleton, with very long and slender

paired actines and few spiny microdiactines in the cortex. The most similar species is the Californian

L. heathi Urban, 1906. However, this species has no tetractines, while L. spinifera sp. nov. has a few

tetractines. Besides, microdiactines are not abundant in L. spinifera sp. nov., while in L. heathi they form

a continuous palisade in the cortex.

We found 10 species of Leucandra recorded from the Mediterranean until now, and L. spinifera sp. nov.

can be differentiated from all of them: L. aspera (Schmidt, 1862) has no microdiactines; L. balearica

(Lackschewitz, 1886) has only tetractines in the atrium and its microdiactines are much smaller (12-

24/1 pm); L. globosa (Sara, 1951) has different microdiactines; L. bolivari Ferrer-Hemandez, 1916 has

no diactines; L. crambessa Haeckel, 1872 has no microdiactines and has tetractines only in the atrium;

L. nausicaae (Schuffner, 1877) has no diactines and the atrial skeleton comprises only tetractines; L.

riojai Ferrez-Hernandez, 1918 has only tetractines in the atrium; L. rodriguezii (Lackschewitz, 1886)

has shorter microdiactines (12-14/1 pm) which occur only in the atrium and the atrium is also composed

of only tetractines; L. sulcata Ferrer-Hemandez, 1918 has microdiactines of a different shape, which are

present abundantly only in the cortex, while the atrium is composed mainly of tetractines.

28

KLAUTAU M. et al, Adriatic calcarean sponges (Porifera, Calcarea)

Fig. 12. Paraleucilla dalmatica sp. nov., holotype (IRB-SD5 = UFRJPOR 8346). A. Specimen in

ethanol. B. Cross section. C. Cortex. D. Detail of the cortex showing the tufts of diactines (white arrow =

trichoxeas; black arrow = diactine). E. Choanosome with the outer and inner regions. F. Atrial skeleton.

Abbreviations: at = atrium; ex = cortex.

29

European Journal of Taxonomy 178 : 1-52 ( 2016 )

Genus Paraleucilla Dendy, 1892

Paraleu cilia dalmatica sp. nov.

um:lsid:zoobank.org:act:D066F56A-lFD7-4742-98E2-2CEE390DlE21

Figs 12-13; Table 10

Etymology

From the type locality. Dalmatia is one of the four historical regions of Croatia.

Material examined

Holotype

ADRIATIC SEA: near the Island of Ciovo, 43°29'02.0" N, 16°22T0.9" E, 5 m, collected by B. Plese and

V. Nikolic, 5 Nov. 2010 (IRB-SD5 = UFRJPOR 8346, in ethanol).

Paratype

ADRIATIC SEA: same data as holotype (PMR-13747, in ethanol).

Fig. 13. Paraleucilla dalmatica sp. nov., holotype (IRB-SD5 = UFRJPOR 8346). A. Cortical diactine

(scale bar = 50 pm). B. Cortical microdiactine (scale bar = 20 pm). C-D. Cortical tetractines. E. Cortical

triactine. F-H. Subatrial triactines. I-K. Subatrial tetractines. L-M. Atrial tetractines. Scale bar C-M =

100 pm.

30

KLAUTAU M. et al., Adriatic calcarean sponges (Porifera, Calcarea)

Table 10. Spicule measurements of Paraleucilla dalmatica sp. nov. (IRB-SD5 = UFRJPOR 8346).

length (pm)width (pm)

min

mean

sd

max

min

mean

sd

max

n

Diactine

1000.0

25.0

50.0

-

Trichoxea

>330.0

2.5

5.0

-

Micro diactine

57.5

95.0

23.5

142.5

2.5

0

2.5

15

Cortical

triactine

Paired

85.0

142.8

35.8

190.0

5.0

12.4

4.3

20.0

20

Unpaired

65.0

149.3

48.0

230.0

5.0

12.9

4.7

20.0

20

Cortical

tetractine

Paired

120.0

159.1

19.2

195.0

10.0

13.4

2.0

17.5

16

Apical

75.0

133.1

33.6

190.0

7.5

13.4

2.6

17.5

16

Subatrial

tetractine and

Paired

170.0

180.0

7.1

190.0

12.5

13.2

1.2

15.0

7

triactine

Unpaired

155.0

205.8

26.7

245.0

10.0

12.7

1.1

15.0

15

Apical

23.8

37.8

11.9

50.0

7.5

8.8

1.4

10.0

4

Atrial

tetractine

Paired

105.0

157.9

32.0

197.5

5.0

10.5

3.0

17.5

21

Unpaired

75.0

157.0

35.8

212.5

7.5

11.4

2.1

15.0

20

Apical

57.5

115.7

55.4

245.0

5.0

7.3

0.6

7.5

25

Colour

Beige or light brown in life and white in ethanol.

Description

The body has the shape of a vase with a single apical osculum surrounded by a crown of trichoxeas

(Fig. 12A). Surface is very hispid. The aquiferous system is leuconoid (Fig. 12B). The cortical skeleton

is composed of the basal system of large tangential tetractines and few triactines (Fig. 12C). Giant

diactines cross the surface, penetrating deeply into the choanosome. They are present from the osculum

to the base of the sponge. Among these giant diactines there are also very thin and long trichoxeas,

organised in tufts, and very few microdiactines (Fig. 12D). The choanosomal skeleton is characteristic

of Paraleucilla , with an inarticulate region (outer region) and a zone without organisation (inner region)

(Fig. 12E). The outer region is formed by the apical actine of the cortical tetractines, the unpaired

actine of subatrial tetractines and very few triactines. The paired actines of these subatrial spicules are

frequently curved, resembling a hook. The inner region is formed by scattered subatrial tetractines and

very few triactines. The atrial skeleton is composed of tetractines only (Fig. 12F). In some parts of the

sponge the inarticulate skeleton seems not to exist and it becomes more similar to Leucandrilla.

Spicules (Table 10)

Oscular triactines. Strongly sagittal. Actines are conical and sharp. The unpaired actine is longer and

thinner than the paired ones and basipetally directed.

Diactines. Giant. They are present in the oscular crown and cortex. They are almost fusiform but slightly

curved, with a thicker tip outside the sponge (Fig. 13A). The size is very variable. Many diatoms are

attached to the diactines surrounding the osculum. Size: 1000.0/25.0-50.0 pm.

Trichoxeas. Present in the oscular crown and cortex. They are thin, straight and most of them are

broken. Size: > 330.0/2.5-5.0 pm.

31

European Journal of Taxonomy 178 : 1-52 ( 2016 )

Microdiactines. Very rare, fusiform or arrow-headed. Sometimes one of the tips has small spines while

the other one is thicker (Fig. 13B). They are present in the cortex. Size: 95.0/2.5 pm.

Cortical tetractines. Sagittal. Actines are conical with sharp tips. The apical actine is longer than the

basal ones, conical, straight and sharp (Fig. 13C-D). Size: 159.1/13.4 pm (paired actine); 133.1/13.4 pm

(apical actine).

Cortical triactines. There are very few, subregular to regular. Actines are slightly conical with sharp

tips (Fig. 13E). Size: 142.8/12.4 pm (paired actine); 149.3/12.9 pm (unpaired actine).

Subatrial triactines and tetractines. The triactines are rare. Actines are conical and sharp. The

unpaired actine is longer than the paired ones. The paired actines are frequently strongly curved. One of

them is often shorter than the other. The apical actine of the tetractines is very short, thin, smooth and

strongly curved (Fig. 13F-K). Size: 180.0/13.2 pm (paired actine); 205.8/12.7 pm (unpaired actine);

37.8/8.8 pm (apical actine).

Atrial tetractines. Sagittal. Actines are slightly conical and sharp. The apical actine is slightly

conical, smooth, thinner than the basal ones and straight or only slightly curved (Fig. 13T-M). Size:

157.9/10.5 pm (paired actine); 157.0/11.4 pm (unpaired actine); 115.7/7.3 pm (apical actine).

Ecology

Specimens were collected on a cliff in a shaded area.

Remarks

Currently there are 11 known species of Paraleucilla , and P. magna Klautau et al. , 2004 is the only

one that has been recorded in the Mediterranean Sea up to now. Both the external morphology and

spicule composition differ in these two species. The most similar species to P. dalmatica sp. nov. are

P. perlucida Azevedo & Klautau, 2007, from Brazil, and P. princeps (Row & Hozawa, 1931), from

Australia. Nonetheless, P. dalmatica sp. nov. can be differentiated from P. perlucida mainly by the

absence of diactine I and trichoxea in the latter. Paraleucilla princeps also differs by the absence of

diactine I and microdiactines. Therefore, P. dalmatica sp. nov. is the second species of Paraleucilla

recorded from the Mediterranean Sea.

Genus Sycon Risso, 1826

Sycon ancora sp. nov.

urn:lsid:zoobank.org:act:F39F5C07-44BF-4AC4-822D-77AA1155B018

Figs 14-15; Table 11

Etymology

From the Tatin ancora , meaning anchor, for the presence of anchor-like spicules for attachment.

Material examined

Holotype

ADRIATIC SEA: Island of Pag, 44°28'34.96" N, 15°02'39.74" E, 1 m, collected by V. Nikolic, 14 Feb.

2011 (PMR 17809 = UFRJPOR 8345, in ethanol).

Paratype

ADRIATIC SEA: near Split, 43°30'27.57" N, 16°23'20.55" E, 5-10 m, collected by V. Nikolic, 15 Aug.

2011 (IRB-SD12 = UFRJPOR 8347, in ethanol).

32

KLAUTAU M. et al., Adriatic calcarean sponges (Porifera, Calcarea)

Fig. 14. Sycon ancora sp. nov., holotype (PMR 17809 = UFRJPOR 8345). A. Specimen in ethanol.

B. Cross section. C. Detail of the distal cone. D. Tubar and subatrial skeletons. E. Atrial skeleton (white

arrows = subatrial triactines). F. Tangential section of the atrial skeleton. Abbreviation: at = atrium.

33

European Journal of Taxonomy 178 : 1-52 ( 2016 )

Colour

White in life and in ethanol.

Description

The body is vase-shaped (1.1 x 0.8 cm), with a single apical osculum surrounded by a crown of trichoxeas

(Fig. 14A) and diactines supported by sagittal tetractines. These tetractines are arranged parallel to each

other and their unpaired actines are basipetally directed. The unpaired actine is longer and thinner than

the paired ones and the apical actine is curved towards the osculum aperture. The paired actines are

slightly curved. There is no suboscular region. The aquiferous system is syconoid and the atrium is

central. The radial tubes are coalescent (Fig. 14B). Diactines and trichoxeas protrude through the distal

cones; consequently, the surface is very hispid. These diactines (ca 10 to 15) penetrate only a little into

the sponge surface (Fig. 14C). The unpaired actine of some triactines also protrudes through the cones.

The tubar skeleton is articulated, but not so well organised as in most sycons (Fig. 14D). It is composed

of rows of sagittal triactines that point their unpaired actines to the surface. These tubar triactines are

Fig. 15. Sycon ancora sp. nov., holotype (PMR 17809 = UFRJPOR 8345). A. Cortical diactine.

B-C. Anchor-like tetractines. D-E. Triactines of the cones. F-H. Tubar triactines. I. Subatrial triactine.

J. Atrial triactine. K. Atrial tetractine. Scale bar D-K =100 pm.

34

KLAUTAU M. et al., Adriatic calcarean sponges (Porifera, Calcarea)

Table 11. Spicule measurements of Sycon ancora sp. nov. (PMR 17809 = UFRJPOR 8345).

length (pm)width (pm)

min

mean

sd

max

min

mean

sd

max

n

Diactine

378.0

537.8

180.1

800.0

10.8

16.1

4.5

20

6

Anchor-like

tetractine

-

>1000

25.0

37.5

2

Triactine

(distal cone)

Paired

59.4

112.3

27.2

148.5

5.4

6.9

1.4

10.8

20

Tubar

triactine

Unpaired

54.0

78.6

18.7

124.2

5.4

7.0

1.5

10.8

20

Paired

116.1

168.2

25.8

216.0

8.1

13.0

2.7

16.2

20

Unpaired

143.1

188.1

29.6

259.2

8.1

12.4

2.8

18.9

20

Subatrial

triactine and

Paired

67.5

97.9

41.6

159.3

4.1

5.4

1.1

6.8

4

tetractine

Unpaired

108.0

212.4

36.5

264.6

4.1

6.0

1.5

8.1

21

Atrial

tetractine I

94.5

153.5

31.7

202.5

7.6

10.8

2.1

16.2

17

Paired

Unpaired

55.1

219.4

75.8

332.1

7.6

10.7

1.5

13.5

17

Atrial

Apical

97.5

123.8

21.9

177.5

8.8

11.4

1.4

12.5

20

Paired

-

162.5

-

6.3

-

1

tetractine II

Unpaired

-

137.5

-

6.3

-

1

Apical

50.0

77.1

14.5

112.5

5.0

5.6

1.0

7.5

20

larger than those of the distal cones and the paired actines are frequently curved. The subatrial skeleton

is composed of sagittal triactines and tetractines (Fig. 14E) with very thin actines. The unpaired actine

is much longer than the paired ones and the longest ones are frequently localized among the choanocyte

chambers. They point their unpaired actines towards the distal cones. Some of the subatrial triactines

are similar to pseudosagittal spicules. The atrial skeleton is composed of two categories of tetractines

tangentially organized (Fig. 14E). They frequently have long, unpaired and short, paired actines. One

of the paired actines is commonly shorter than the other; however, the three basal actines can have the

same size (Fig. 14F). When one of the paired actines is shorter than the other, it frequently penetrates

an exhalant canal. The main difference between the two categories of atrial tetractines is in the apical

actine. Tetractines with thinner apical actines project these actines mainly into the canals, while thicker

and curved apical actines penetrate into the atrium (Fig. 14E). Few anchor-lilce tetractines are present at

the sponge base and project their basal actines into the substrate.

Spicules (Table 11)

Diactines. Almost fusiform, but the tip outside the sponge is a little thicker (Fig. 15A). Size:

537.8/16.1 pm.

Trichoxeas. Very thin, long and straight. They were always broken.

Anchor-like tetractines. The basal actines are very short and curved, whil e the apical one is very long.

Frequently there are spines on the apical actine, but near the basal ones. They vary from four to seven,

but seven spines are more common (Fig. 15B). Size: > 1000.0/25.0 pm.

35

European Journal of Taxonomy 178 : 1-52 ( 2016 )

Triactines of the cones. They are smaller than the tubar triactines. The unpaired actine protrudes through

the cones and it is shorter than the paired ones, which are curved. Actines are slightly conical and sharp

(Fig. 15C-D). Size: 112.3/6.9 pm (paired actine); 78.6/7.0 pm (unpaired actine).

Triactines of the tubes. Subregular to sagittal. The unpaired actine is a little longer or has the same

length of the paired ones. The paired actines are straight or slightly curved. Actines are slightly conical

and sharp (Fig. 15E-G). Size: 168.2/13.0 pm (paired actine); 188.1/12.4 pm (unpaired actine).

Sub atrial triactines and TETRACTiNES. The subatrial spicules are very thin. They are sagittal or,

sometimes, similar to pseudosagittal spicules. Actines are slightly conical and sharp. The unpaired

actine is longer than the paired ones (Fig. 15H). The apical actine of the tetractines is conical, sharp,

smooth, shorter than the basal ones and curved in the direction of the atrium. Size: 97.9/5.4 pm (paired

actine); 212.4/6.0 pm (unpaired actine).

Atrial tetractines I and II and triactines. There are two categories of atrial tetractines and the

triactines are very rare. They are sagittal or subregular. The unpaired actine is frequently longer than the

paired ones (Fig. 151). It is also common to find one of the paired actines shorter than the other (63.5-

109.3(±64.7)-155.0/10-11.3(±1.8)-12.5 pm (n=2); Fig. 15J). This shorter, paired actine is frequently

projected inside the exhalant canal. Actines are cylindrical and sharp. Sometimes, the tip of the unpaired

actine is thicker (Fig. 151). The main difference between the two categories of tetractines is in the

shape and size of the apical actines, which are straight and thinner in one and curved and thicker in the

other. Size (tetractine I): 153.5/10.8 pm (paired actine); 219.4/10.7 pm (unpaired actine); 123.8/11.4

pm (apical actine). Size (tetractine II): 162.5/6.3 pm (paired actine); 137.5/6.3 pm (unpaired actine);

77.1/5.6 pm (apical actine).

Ecology

Specimens were collected on a semi-vertical hard limestone bottom. They were found among Cystoseira

macroalgae.

Remarks

Currently there are 12 accepted species of Sycon in the Mediterranean Sea, 10 of which have already

been reported for the Adriatic. We compared our specimens to all known species of Sycon and even more

carefully to the Mediterranean ones, yet we could not find a perfect match.

The main characteristic discerning Sycon ancora sp. nov. from other species is the shape of the atrial

triactines and the presence of anchor-lilce tetractines at the base. If we exclude these characteristics, this

species would be mostly comparable to S. raphanus ; however, there are several important differences

between them.

Sycon raphanus was originally described from the Adriatic Sea by Schmidt (1862). Unfortunately, his

description was not detailed enough. According to him, S. raphanus has a bulb shape and a peduncle.

He even considered these characteristics to distinguish S. raphanus from S. ciliatum (Fabricius, 1780), a

species from the English Channel which he believed to be present in the Adriatic Sea.

Haeckel (1872) disagreed with the possibility of S. ciliatum occurring in the Mediterranean Sea and

considered that all specimens called S. ciliatum were, in fact, S. raphanus. He also mentioned that he

analysed all the specimens from Schmidt’s collection identified as S. raphanus and found a potpourri

of species, including Leucandra aspera, Sycon humboldti , Sycon setosum and “the real S. raphanus ”.

Therefore, he made a detailed description of this species, which has since then been considered as the

official description of S. raphanus. According to his description, S. raphanus is morphologically very

variable, solitary or not, with or without peduncle. The skeleton is composed of tufts of 5-10 cylindrical

36

KLAUTAU M. et al., Adriatic calcarean sponges (Porifera, Calcarea)

diactines (var. tergestina) to 20-50 diactines (var. procumbens) and the size of the diactines varies from

400-800/20-30 pm up to 1000-2000/20-40 pm, rarely attaining 3000 pm. Analyzed specimens of

S. ancora sp. nov. have tufts of 10-15 diactines measuring 378—>1500/10.8—18.9 pm. Sycon raphanus

has triactines with curved paired actines in the distal cones and in the tubar skeleton. The tubar triactines

are 100-180/10-12 pm (paired) and 150-250/10-12 pm (unpaired), which are thinner than in S. ancora

sp. nov. The subatrial skeleton of S. raphanus has triactines (paired: 100-180/5-8 pm; unpaired: 150—

250/5—8 pm), while S. ancora sp. nov. has triactines and tetractines. The atrial skeleton of S. raphanus

shows subregular to regular (rarely sagittal) triactines and tetractines (basal: 150-250/8-10 pm; apical:

60-120 pm), while our species has tetractines with two types of apical actines (there is a variation in the

thickness and position), a long unpaired actine and paired actines with different sizes. Haeckel (1872)

also mentioned the presence of only triactines supporting the oscular crown, while S. ancora sp. nov.

has only tetractines.

Although we believe the entire genus Sycon is in urgent need of revision, the characteristics we found in

our specimens strongly indicate the presence of a new species.

Other calcarean species from the Adriatic Sea

Apart from the species described here, we also recorded and molecularly analyzed specimens of Clathrina

blanca (Milducho-Maclay, 1868), C. clathrus (Schmidt, 1864), C. rubra Sara, 1958 and Par ale uc ilia

1.00/99

- Ascandra contorts

wiomr Ascandra corallicola

0.86/77 1

0.73/67'

C?

I/67H

Ascandra falcata

-Ascandra spaiatensis sp. nov."

1.00/100

I Ascaltis reticulum*

] Ascaltis reticulum

I I

0.91/56

0.52

1.00/98

... . . ,, , Ascaltis reticulum

1.00/99 r Arthuna spiral ata

Arthuna spirallata

_ r-Arthuria hirsute

'Arthuria hirsute

1.00/98

0.95/82. Borojevia brasiliensis

. 00/991 -1 Borojevia brasiliensis

1 1.00/95 Borojevia cerebrum

- Borojevia cerebrum

Borojevia cerebrum**

Borojevia cerebrum

Borojevia cerebrum*

1,00/92

Borojevia cerebrum,,

Borojevia cerebrum

1- Borojevia aspina

1 .oq/99 1 - Borojevia aspina

1.00/98

0.79/-

1 , 00/88

0.85/-

0.99/65

1.00/98

Clathrina fjordica

- Clathrina coriacea

1 —Borojevia croatica sp, nov.

CBorojevia croatica sp. nov.*

Borojevia croatica sp. nov.*

—Borojevia croatica sp. nov.*

Clathrina conifera*

Clathrina conifera***

Clathrina conifera*

Clathrina conifera***

j Clathrina peruana

1 r'l'h+hrirr'i namono

1.00/100 1 Clathrina peruana

1.00/100

1.00/79

0.88/-

1.00/98

1 00/100 1 —Clathrina aurea

' Clathrina aurea

Clathrina clathrus

Clathrina clathrus*

Clathrina clathrus

Clathrina clathrus*

1.00/100

0.98/66

_i Clathrina primordialis

51 Cttathrirra rtrimnrriialic*

1 , 00 / 100 ' Clathrina primordialis ’

1 Clathrina antofagastensis

0.99/84

1.00/100 1 Clathrina antofagastensis

Clathrina lacunosa

).58/-r

-Clathrina helveola

1.00/100 'Clathrina lacunosa

0.94/88, clathrina blanca

1.00/100 1 < Clathrina blanca*

L- Clathrina hispanica

0.78/58 1 - Clathrina ramosa

1.00/100

I Clathrina aphrodita

\Clathrina aphrodita

_I Clathrina rubra

i.oo/i 00 1 Clathrina rubra*

0.06

Fig. 16. Maximum likelihood (ML) tree based on ITS1-5.8S-ITS2 rDNA sequences of Calcinea.

Bayesian posterior probabilities (PP) and bootstrap values (BS) are given near the branches (PP/BS;

when >0.50). Adriatic specimens are written in bold; * Adriatic specimens obtained during this study;

'""Mediterranean specimens of Borojevia cerebrum ; '"'"'"Brazilian specimens of Clathrina conifera.

37

European Journal of Taxonomy 178 : 1-52 ( 2016 )

magna Klautau, Monteiro & Borojevic, 2004. These species are not redescribed here, since specimens

from the Adriatic Sea have already been recorded and described in earlier works (Cvitkovic et al. 2013;

Imesek et al. 2014). In the present study, C. blanca was recorded near Selce (45°09'07.8" N, 14°43T5.0"

E ), about 1 m deep and C. rubra was recorded near the Island of Ciovo (43°28'58.5" N, 16°21'25.6"

E), about 5m deep on a shaded hard bottom. In August and November 2010 they were quite abundant,

always only a few millimeters in size and often found on bryozoans. C. clathrus was found in numerous

locations along the coast (e.g., Prapratno Cove, 42°48'36.8" N, 17°40'38.4" E; near the Island of Ciovo,

43°28'58.5" N, 16°21'25.6" E) and the cryptogenic species P. magna was found in large numbers in on

aquaculture installations in Grslca Cove on the Island of Brae and in the Port of Ploce.

Molecular analysis

The number of sites used for the final alignments (gaps included) was as follows: 513 for ITS Calcinea,

1434 for 28S Calcinea, 734 for ITS Calcaronea and 846 for 28S Calcaronea. Both markers revealed

the same tree topology in both analyses (but see Fig. 19), yet the Bayesian analysis rendered much

better support values than ML in all cases. However, the Adriatic species nested within the respective

genera with high bootstrap (BS) and posterior probability (PP) values, thereby confirming the results of

morphological analysis (Figs 16-19).

Once more the presence of diactines did not show any phylogenetic signal (Rossi et al. 2011; Klautau

et al. 2013). Furthermore, we found former guanchas with only triactines reunited in a monophyletic

clade in the ITS analysis, with high support values inside the Clathrina group (0.99 PP and 0.84 BS;

Fig. 16). In the 28S calcinean tree (Fig. 17) we recovered a clade where Levinella represents a sister

1 . 00/68

1 , 00/92

1 . 00/78

Lelapiella incrustans

— Leucaltis clathria

1 . 00/100

1 . 00/99

Borojevia croatica sp. nov.*

1 . 00/99

Borojevia cerebrum *

Borojevia cerebrum*

0 . 71/54

1 . 00/99 j— Murrayona phanolepis

Murrayona phanolepis

Ascaltis reticulum*

1 . 00/99

1 . 00/88

1 . 00/92

Clathrina rubra*

Clathrina rubra

0 . 99/51

1 . 00 / 99 1 Clathrina helveola

1 . 00/100

Clathrina wistariensis

Clathrina conifera*

1 . 00/100

1 . 00/94

0 . 50/62

1 . 00/100

Clathrina blanca

' Clathrina blanca*

Clathrina primordialis

Clathrina primordialis*

0 . 96/77

Clathrina luteoculcitella

1 . 00/99

0 . 97/67

0 . 87/51

1 . 00/99

Clathrina clathrus

I Clathrina clathrus*

^— Clathrina clathrus*

- Clathrina adusta

- Soleneiscus radovani

- Soleneiscus stolonifer

1 . 00/99

-Levinella prolifera

Ascandra spalatensis sp. nov.’

0.06

Fig. 17. Maximum likelihood tree (ML) based on partial 28S rDNA sequences of Calcinea. Bayesian

posterior probabilities (PP) and bootstrap values (BS) are given near the branches (PP/BS; when >0.50).

Adriatic species are written in bold. Adriatic specimens obtained in this study are marked with an asterisk.

38

KLAUTAU M. et al., Adriatic calcarean sponges (Porifera, Calcarea)

group to Ascandra with high support values (1.00 PP and 0.99 BS), which confirms the results of

Voigt et al. (2012). We also recovered a clade comprising the genera Murrayona and Ascaltis in both

analyses; however, the support values were less good (0.71 PP and 0.54 BS). The molecular analyses

also confirmed the presence of P. magna in the Adriatic Sea (Figs 18-19). Besides, we recovered a

calcaronean clade with high support (1.00 PP and 0.99 BS in ITS analysis; 0.95 PP and 0.64 BS in 28S

analysis) formed only by Paraleucilla species. The genus Paraleucilla formed a highly supported clade

with Leucandra nicolae , while Leucandra spinifera sp. nov. is a sister species of L. aspera (Fig. 19).

Sycon ancora sp. nov. represents a sister species of S. raphanus (Fig. 19). We confirmed the paraphyly

of the genera Sycon and Leucandra (Voigt et al. 2012).

Species richness

Considering previous data, together with our present results based on morphological and molecular

analyses, we found a total of 13 species of Calcinea (Table 12) and 26 of Calcaronea in the Adriatic Sea

(Table 13). Taking into account the species richness by sectors (Fig. 20), the richest sector is the Central

Adriatic, where 34 species were found, followed by the Northern Adriatic with 18, and the Southern

Adriatic with only 5 species. Most of the species present in the Adriatic Sea are also present in other

Mediterranean areas, yet, altogether we recorded six species provisionally endemic for the Adriatic, two

calcinean and four calcaronean.

Discussion

Since some of the first studies on the class Calcarea were mainly done along the Dalmatian coast by

Schmidt and Haeckel in the 19 th century (e.g., Schmidt 1862, 1864; Haeckel 1870, 1872), the knowledge

of the current species diversity and distribution certainly awakes taxonomic interest. Analysing previous

1 . 00/100

Sycon ciliatum

Sycon capricorn

1 . 00/100

1 . 00/99

1 . 00/100

Paraleucilla magna *

1 . 00/99

Paraleucilla dalmatica sp. nov.*

Leucandra falakra sp. nov.*

1 . 00/90

0.05

1 . 00/100

Sycon ancora sp. nov.*

1 . 00/99

Leucandra spinifera sp. nov.

*

Fig. 18. Maximum likelihood tree based on ITS1-5.8S-ITS2 rDNA sequences of Calcaronea. Bayesian

posterior probabilities (PP) and bootstrap values (BS) are given near the branches (PP/BS; when >0.50).

Adriatic species are written in bold. Adriatic specimens obtained in this study are marked with an asterisk.

39

European Journal of Taxonomy 178 : 1-52 ( 2016 )

results with ours, we found a total of 39 species of calcarean sponges in the Adriatic Sea (Tables 12-13).

In this species list we do not consider the records of Clathrina coriacea (Montagu, 1814), Sycon ciliatum

(Fabricius, 1780) or S. proboscideum (Haeckel, 1870). The occurrence of C. coriacea was not considered

because, analysing the descriptions of this species for the Adriatic Sea, we think that they most probably

represent C. conifera or C. primordialis. Sycon ciliatum seems to be restricted to the North Atlantic and

was probably mistaken for S. raphanus (Haeckel, 1872). Sycon proboscideum is a species from the Red

Sea and its occurrence in the Adriatic Sea was mentioned only by Breitfuss (1935), which suggested that

some specimens previously identified as S. raphanus could in fact be S. proboscideum. He did not give

a description or any further clues. Therefore, the occurrence of this species in the Adriatic Sea has to be

verified (Burton 1963; Longo & Pronzato 2011).

0.96

0.61

-Sycon carteri

-Leucascandra caveolata

o.oi

1.00

- Leucandra nicolae

0.95

1 .oo f~ Paraleucilla dalmatica sp. nov.

1.00

Different topology in Bayesian analysis

Paraleucilla dalmatica sp. nov.

—Paraleucilla magna

'—Paraleucilla magna

-1 53

1 . 00/100

0.93/-

- Sycon carteri

■ Leucascandra caveolata

1 . 00/100

. Leucandra nicolae

0.95/64

1.00/100 | Paraleucilla dalmatica sp. nov.*

I Paraleucilla dalmatica sp. nov.*

1.00/99

Paraleucilla magna

Paraleucilla magna*

1 . 00/66

- Ute ampullacea

0.96/58

- Teichonopsis labyrinthica

1.00/100 1 Grantiopsis cylindrica

' Grantiopsis heroni

1 . 00/100

- Syconraphanus

1.00/97| Sycon ancora sp. nov.*

"1 Sycon ancora sp. nov.*

1 00/99 Leucandra aspera

I Leucandra spinifera sp. nov.*

0.92/751 Leucandra spinifera sp. nov.

Leucandra falakra sp. nov.*

- Grantia compressa

1 . 00/100

0.92/-

■ Sycon capricorn

1.00/73J— Lite aff. syconoides

1- Ute aff. syconoides

1.00/87 1 Syconessa panicuia

' Syconessa panicuia

1.00/99

0.95/-

1.00/99 | Sycettusa cf. simplex

'— Sycettusa tenuis

0.71/-

• Sycettusa aff. hastifera

— Sycon ciliatum

0.04

Fig. 19. Maximum likelihood tree based on partial 28S rDNA sequences of Calcaronea. Bayesian

posterior probabilities (PP) and bootstrap values (BS) are given near the branches (PP/BS; when >0.50).

Adriatic specimens are written in bold. Adriatic specimens obtained in this study are marked with an

asterisk. The detached tree shows the only difference in the topology of the MT and Bayesian analyses.

40

KLAUTAU M. et al, Adriatic calcarean sponges (Porifera, Calcarea)

Table 12. Calcinean species reported from the Adriatic Sea and their distribution. *Type locality.

** Probably Clathrina conifera or C. primordialis.

Species

Longitude Latitude

(N) (E)

Locality

Source

Ascaltis reticulum (Schmidt, 1862)

15.22

44.10

Zadar = Zara*

Schmidt 1862

Ascaltis reticulum (Schmidt, 1862)

15.92

43.73

Sibenik = Sebenico*

Schmidt 1862

Schmidt 1862 (according to

Ascaltis reticulum (Schmidt, 1862)

16.73

43.13

Hvar = Lesina

Haeckel 1872); Heller 1864

{apud Haeckel 1872); Haec¬

kel 1872

Ascaltis reticulum (Schmidt, 1862)

13.63

45.08

Rovinj

Longo & Pronzato 2011

Ascaltis reticulum (Schmidt, 1862)

16.36

43.48

V

Island of Ciovo

Present work

Ascandra contorta Bowerbank,

15.5

42.12

Tremiti Island

Lendenfeld 1891 {asAscetta

1866 {sensu Minchin 1905)

spinosa ); Sara 1961

Ascandra contorta Bowerbank,

13.80

45.63

Trieste

Lendenfeld 1891 (as Ascetta

1866 {sensu Minchin 1905)

spinosa)

Ascandra contorta Bowerbank,

13.77

45.60

Muggio

Lendenfeld 1891 {as Ascetta

1866 {sensu Minchin 1905)

spinosa)

Ascandra contorta Bowerbank,

16.22

43.01

Island of Vis = Lissa

Lendenfeld 1891 {as Ascetta

1866 {sensu Minchin 1905)

spinosa)

Ascandra contorta Bowerbank,

16.73

43.13

Hvar = Lesina

Lendenfeld 1891 {as Ho-

1866 {sensu Minchin 1905)

mandra falcata)

Ascandra falcata Haeckel, 1872

13.63

45.08

Rovinj

Longo & Pronzato 2011

Ascandra falcata Haeckel, 1872

13.80

45.63

Trieste

Lendenfeld 1891

Ascandra falcata Haeckel, 1872

16.73

43.13

Hvar = Lesina*

Haeckel 1872; Lendenfeld

1891 (as Homandra falcata)

Ascandra falcata Haeckel, 1872

15.50

42.12

Tremiti Island

Sara 1961; Longo & Pron¬

zato 2011

Ascandra spalatensis sp. nov.

15.21

44.14

Zadar = Zara*

Present work

Borojevia cerebrum

(Haeckel, 1872)

16.73

43.13

Hvar = Lesina*

Haeckel 1872; Lendenfeld

1891; Imesek et al. 2014

Borojevia cerebrum

(Haeckel, 1872)

16.89

43.40

Vrulja Cove

Present work

Borojevia cerebrum

(Haeckel, 1872)

13.63

45.08

Rovinj

Lendenfeld 1891

Borojevia croatica sp. nov.

16.37

43.48

Island of Ciovo*

Present work

Clathrina blanca

13.62

45.05

Island of St. Giovanni

Imesek et al. 2014

(Miklucho-Maclay, 1868)

(near Rovinj)

Clathrina blanca

(Miklucho-Maclay, 1868)

13.63

45.08

Rovinj

Longo & Pronzato 2011

Clathrina blanca

(Miklucho-Maclay, 1868)

16.73

43.13

Lesina=Hvar

Lendenfeld 1891

Clathrina blanca

(Miklucho-Maclay, 1868)

14.72

45.15

Selce

Imesek et al. 2014

Clathrina blanca

(Miklucho-Maclay, 1868)

16.37

43.48

Island of Ciovo

Imesek et al. 2014

41

European Journal of Taxonomy 178 : 1-52 ( 2016 )

Clathrina lacunosa

(Johnston, 1842)

13.63

45

Clathrina lacunosa

(Johnston, 1842)

16.73

43

Clathrina conifera

18.1

42

Klautau & Borojevic, 2001

Clathrina conifera

18.11

42

Klautau & Borojevic, 2001

Clathrina clathrus (Schmidt, 1864)

15.92

43

Clathrina clathrus (Schmidt, 1864)

16.73

43

Clathrina clathrus (Schmidt, 1864)

16.22

43

Clathrina clathrus (Schmidt, 1864)

13.63

45

Clathrina clathrus ( Schmidt, 1864)

13.62

45

Clathrina clathrus (Schmidt, 1864)

16.22

43

Clathrina clathrus (Schmidt, 1864)

17.68

42

Clathrina clathrus (Schmidt, 1864)

16.36

43

Clathrina coriacea

(Montagu, 1814)**

13.63

45

Clathrina coriacea

(Montagu, 1814)**

15.5

42

Clathrina coriacea

(Montagu, 1814)**

16.87

41

Clathrina primordialis

16.73

43

(Haeckel, 1872)

Clathrina primordialis

13.80

45

(Haeckel, 1872)

Clathrina primordialis

13.77

45

(Haeckel, 1872)

Clathrina primordialis

15.22

44

(Haeckel, 1872)

Clathrina primordialis

15.92

43

(Haeckel, 1872)

Clathrina primordialis

16.87

42

(Haeckel, 1872)

Clathrina primordialis

16.22

43

(Haeckel, 1872)

Rovinj

Lendenfeld 1891 (as Ascan-

dra angulata)

Lendenfeld 1891 (as Ascan-

dra angulata)

Hvar = Lesina

Dubrovnik

Present work

Island of Lokrum

Present work

Sibenik = Sebenico*

Schmidt 1864

Hvar = Lesina

Schmidt (according to

Haeckel 1872); Heller 1864

{apud Haeckel 1872);

Haeckel 1872

Island of Vis = Lissa*

Heller 1864 {apud Haeckel

1872)

Rovinj

Imesek et al. 2014

Island of St. Giovanni

(near Rovinj)

Present work

Island of Vis = Lissa*

Present work

Prapratno Cove

Present work

V

Island of Ciovo

Present work

Rovinj

Longo & Pronzato 2011

Tremiti Island

Sara 1961; Longo & Pron¬

zato 2011

Bari

Longo & Pronzato 2011

Schmidt (according to

Haeckel 1872); Haeckel

Hvar = Lesina*

1872; Heller 1864 {apud

Haeckel 1872); Lendenfeld

1891

Trieste

Lendenfeld 1891

Muggio

Lendenfeld 1891

Schmidt (according to

Zadar = Zara

Haeckel 1872); Lendenfeld

1891

V"

Sibenik = Sebenico

Schmidt (according to

Haeckel 1872); Lendenfeld

1891

Schmidt (according to

Lastovo = Lagosta

Haeckel 1872); Lendenfeld

1891

Island of Vis = Lissa

Lendenfeld 1891

08

13

64

63

73

13

01

08

05

01

81

48

08

12

13

63

60

10

73

01

42

KLAUTAU M. et al ., Adriatic calcarean sponges (Porifera, Calcarea)

Clathrina primordialis

(Haeckel, 1872)

13.63

45.08

Rovinj

Lendenfeld 1891; Imesek

et al. 2014 (as Clathrina cf.

hondurensis)

Clathrina primordialis

(Haeckel, 1872)

16.36

43.48

V

Island of Ciovo

Present work

Clathrina rubra Sara, 1958

13.63

45.08

Rovinj

Imesek et al. 2014

Clathrina rubra Sara, 1958

16.36

43.48

\s

Island of Ciovo

Present work

Leucetta solida (Schmidt, 1862)

15.92

43.73

Sibenik = Sebenico*

Schmidt 1862; Lendenfeld

1891

Schmidt (according to

Leucetta solida (Schmidt, 1862)

16.87

42.73

Lastovo = Lagosta

Haeckel 1872); Lendenfeld

1891

Schmidt (according to

Leucetta solida (Schmidt, 1862)

16.73

43.13

Hvar = Lesina

Haeckel 1872); Haeckel

1872; Lendenfeld 1891

Lastovo = Lagosta

Leucetta solida (Schmidt, 1862)

16.87

42.73

(Zaklopatica = Porto

Chiave)*

Schmidt 1864

Leucetta solida (Schmidt, 1862)

15.5

42.12

Tremiti Island

Sara 1961; Longo & Pron-

zato 2011

Leucetta solida (Schmidt, 1862)

16.87

41.13

Bari

Longo & Pronzato 2011

47 "N

Fig. 20. Species richness in the Adriatic Sea divided by sectors. □: 1-9 species; □: 10-17 species; □:

18-26 species.

43

European Journal of Taxonomy 178 : 1-52 ( 2016 )

Table 13. Calcaronean species reported from the Adriatic Sea and their distribution. * = Type locality.

Species

Longitude

(N)

Latitude

(E)

Locality

Source

Amphoriscus chrysalis

(Schmidt, 1864)

16.73

43.13

Hvar = Lesina*

Schmidt 1864; Haeckel

1872

Amphoriscus chrysalis

(Schmidt, 1864)

16.22

43.01

Island of Vis = Lissa*

Schmidt, 1864

Amphoriscus cylindrus

(Haeckel, 1872)

16.73

43.13

Hvar = Lesina*

Haeckel 1872; Lendenfeld

1891

Amphoriscus gregori

(Lendenfeld, 1891)

16.73

43.13

Hvar = Lesina*

Lendenfeld 1891

Aphroceras corticata

(Lendenfeld, 1891)

16.73

43.13

Hvar = Lesina*

Lendenfeld 1891

Grantia capillosa

(Schmidt, 1862)

15.92

43.73

Sibenik = Sebenico*

Schmidt 1862

Grantia capillosa

(Schmidt, 1862)

13.63

45.08

Rovinj

Lendenfeld 1891

Grantia capillosa

(Schmidt, 1862)

13.77

45.60

Muggio

Lendenfeld 1891

Grantia capillosa

(Schmidt, 1862)

13.57

45.53

Pirano

Lendenfeld 1891

Grantia capillosa

(Schmidt, 1862)

16.73

43.13

Hvar = Lesina

Schmidt 1864; Haeckel

1872; Lendenfeld 1891

Grantia capillosa

(Schmidt, 1862)

15.92

43.73

V

Sibenik = Sebenico

Lendenfeld 1891

Leucandra aspera

(Schmidt, 1862)

15.22

44.10

Zadar = Zara*

Schmidt 1862; Lendenfeld

1891

Leucandra aspera

(Schmidt, 1862)

15.50

42.12

Tremiti Island

Sara, 1961; Longo & Pron-

zato 2011

Leucandra aspera

(Schmidt, 1862)

16.22

43.01

Island of Vis = Lissa*

Heller 1864 ( apud Haeckel

1872); Lendenfeld 1891

Leucandra aspera

15.92

43.73

V

Sibenik = Sebenico

Schmidt 1862; Lendenfeld

(Schmidt, 1862)

1891

Leucandra aspera

16.87

42.73

Lastovo = Lagosta

Schmidt 1862; Lendenfeld

(Schmidt, 1862)

1891

Leucandra aspera

(Schmidt, 1862)

16.73

43.13

Hvar = Lesina

Schmidt 1862; Haeckel

1872; Lendenfeld 1891

Leucandra falaki*a sp. nov.

15.57

43.63

Blitvenica*

Present work

Leucandra spinifera sp. nov.

16.36

43.48

V

Island of Ciovo

Present work

Leucandra spinifera sp. nov.

16.89

43.40

Vrulja Cove*

Present work

Leucosolenia goethei

Haeckel, 1870

13.63

45.08

Rovinj

Lendenfeld 1891

Leucosolenia variabilis Haec¬

kel, 1870

13.63

45.08

Rovinj

Longo & Pronzato 2011

Paraleucilla dalmatica sp. nov.

16.36

43.48

's/

Island of Ciovo

Present work

Paraleucilla magna Klautau,

Monteiro & Borojevic, 2004

17.43

43.05

Port of Ploce

Cvitkovic etal. 2013

Paraleucilla magna Klautau,

Monte iro & Borojevic, 2004

16.39

43.51

Port of Ploce

Present work

44

KLAUTAU M. et al., Adriatic calcarean sponges (Porifera, Calcarea)

Paraleucilla magna Klautau,

Monteiro & Borojevic, 2004

16.48

43.29

Island of Brae

Present work

Polejaevia telum

(Lendenfeld, 1891)

16.73

43.13

Hvar = Lesina

Lendenfeld 1891

Sycantha tenella

Lendenfeld, 1891

13.8

45.63

Trieste

Lendenfeld 1891

Sycetta conifera

(Haeckel, 1872)

16.73

43.13

Hvar = Lesina

Haeckel 1872; Lendenfeld

1891

Sycon ancora sp. nov.

15.04

44.48

Island of Pag*

Present work

Sycon ancora sp. nov.

16.39

43.51

Split

Present work

Sycon elegans

(Bowerbank, 1845)

15.50

42.12

Tremiti Island

Sara 1961

Sycon helleri

(Lendenfeld, 1891)

16.73

43.13

Hvar = Lesina

Lendenfeld 1891

Sycon humboldti Risso, 1826

42.75

16.87

Lastovo = Lagosta

Schmidt 1862

Sycon humboldti Risso, 1826

16.22

43.01

Island of Vis = Lissa

Heller 1864 {apud Haeckel

1872); Schmidt 1862

Sycon humboldti Risso, 1826

16.73

43.13

Hvar = Lesina

Schmidt 1862; Haeckel

1872

Sycon humboldti Risso, 1826

12.34

45.44

Venice

Martens 1824

{apud Haeckel 1872)

Sycon humboldti Risso, 1826

15.10

43.93

Dugi otok = Isola Grossa

Martens 1824 {apud Haek-

kel 1872)

Sycon quadrangulatum

(Schmidt, 1868)

13.61

45.68

Dalmatia, Gulf of Trieste

Schmidt, 1868

Sycon quadrangulatum

(Schmidt, 1868)

16.73

43.13

Hvar = Lesina

Haeckel 1872

Sycon quadrangulatum

(Schmidt, 1868)

16.22

43.01

Island of Vis = Lissa

Heller 1864 {apud Haeckel

1872)

Sycon raphanus Schmidt, 1862

13.63

45.08

Rovinj

Longo & Pronzato 2011

Sycon raphanus Schmidt, 1862

16.73

43.13

Hvar = Lesina

Schmidt 1862; Haeckel

1872

Sycon raphanus Schmidt, 1862

16.87

41.13

Bari

Longo & Pronzato 2011

Lieberkuhn 1859

Sycon raphanus Schmidt, 1862

13.8

45.63

Trieste

{apud Haeckel 1872);

Schmidt, 1862 (as S. cilia-

tum ); Haeckel 1872

Sycon raphanus Schmidt, 1862

15.22

44.10

Zadar = Zara

Schmidt 1862

Sycon raphanus Schmidt, 1862

15.92

43.73

Sibenik = Sebenico

Schmidt 1862

Sycon schmidt (Haeckel, 1872)

42.75

16.87

Lastovo = Lagosta

Schmidt 1862 {apud Haek-

kel 1872); Haeckel 1872

Sycon schmidt (Haeckel, 1872)

16.73

43.13

Hvar = Lesina*

Haeckel 1872

Sycon setosum Schmidt, 1862

16.73

43.13

Hvar = Lesina

Heller 1864 {apud Haeckel

1872); Haeckel, 1872

Sycon setosum Schmidt, 1862

16.22

43.01

Island of Vis = Lissa

Heller 1864 {apud Haeckel

1872)

45

European Journal of Taxonomy 178 : 1-52 ( 2016 )

Sycon setosum Schmidt, 1862

13.63

45.08

Rovinj

Longo & Pronzato 2011

Sycon tuba (Lendenfeld, 1891)

13.8

45.63

Trieste*

Lendenfeld 1891

Sycyssa huxleyi Haeckel, 1872

16.73

43.13

Hvar = Lesina*

Haeckel 1872

Ute glabra Schmidt, 1864

16.73

43.13

Hvar = Lesina

Haeckel 1872; Lendenfeld

1891

Ute glabra Schmidt, 1864

16.22

43.01

Island of Vis = Lissa

Heller 1864 (apiid Haeckel

1872)

Lastovo = Lagosta

Ute glabra Schmidt, 1864

16.87

42.73

(Zaklopatica = Porto

Chiave)*

Schmidt 1864

Our results indicate Sycon to be the most diverse genus, with nine species, followed by Clathrina with six

species. However, it is important to consider that Sycon is not a monophyletic genus. It is very difficult

to identify Sycon species unequivocally, as most of them have a similar spicule composition - diactines,

trichoxeas and triactines in the distal cones, tubar triactines, subatrial triactines and tetractines, and atrial

tetractines. To date there have been no studies on the intraspecific morphological variability of Sycon. In

addition, most species were poorly described and insufficiently analyzed on the molecular level, which

also applies to the calcaronean genera Paraleucilla , Leucandrilla, Leucandra and Leucilla. Molecular

phylogenetic studies including as many species as possible would be very desirable to evaluate the limits

between these genera. Hence, new calcaronean species are welcome to facilitate more thorough revision

of their systematics and to link the molecular traits to the phylogenetically important morphological

traits.

We have also confirmed the presence of a few species known so far only from the Atlantic. It was

unexpected to find Clathrina conifera in the Adriatic Sea, as this species was first described along

the Brazilian coast and was considered endemic (Klautau et al. 1994). Our finding raises the question

whether this species was ever truly endemic for Brazil. Since Adriatic calcarean sponges are vastly

unexplored and C. conifera is part of the C. primordialis species complex, it is possible that it has been

recorded previously as C. primordialis (or C. coriacea ). In 2010, a specimen of Clathrina conifera was

observed for the first time in the Southern Adriatic, near the Island of Lokrum, and a year later, more

than 20 specimens were recorded near the city of Dubrovnik. As both locations are close to the area in

Dubrovnik frequently visited by cruise ships, it is possible that this species has been introduced into the

Adriatic. However, if this species arrived by anthropogenic means, we cannot state whether it arrived

from the Western Atlantic to the Adriatic or vice-versa. It is important to mention that Paraleucilla

magna is also present in the Southern Adriatic (Cvitlcovic et al. 2013). It was first recorded in Brazil

in the 1980’s; however, the origin of this species is unknown. It seems to have been introduced by

anthropogenic means into the Mediterranean (Longo et al. 2007) and to have spread into the Eastern

Mediterranean, including the Adriatic Sea. Here, we molecularly confirm the presence of P. magna near

the Port of Ploce and at a new location, near the Island of Brae (Table 13).

The molecular analyses revealed some interesting taxonomic traits. At the generic level, the monophyletic

clade of former guanchas indicates that the development of a peduncle and of parasagittal spicules

probably appeared only once in the evolution of Clathrina. Clathrina hispanica was nested within this

group, although in the original description of this species neither peduncle nor parasagittal spicules were

mentioned (Klautau & Valentine 2003). The type specimen of this species is fragmented, resulting in the

impossibility of confirming if a peduncle was present or not; however, we re-analysed the slides of the

holotype and found some parasagittal spicules. Another interesting result indicated the close relationship

46

KLAUTAU M. et al, Adriatic calcarean sponges (Porifera, Calcarea)

among Ascandra , Soleneiscus and Levinella revealed in the 28S analysis. Voigt et al. (2012) showed

that the genus Ascandra is closely related to Soleneiscus and Levinella , which is now confirmed by

our results. This implies that in the future the genera Levinella and Soleneiscus might be synonymised

with the genus Ascandra ; nonetheless, more detailed molecular and morphological analyses on a larger

number of specimens and species are needed to confirm this action. At the species level, the molecularly

confirmed presence of Clathrina conifera in the Adriatic raises a doubt of the earlier identification of

the C. primordialis syntype, allowing the selection of a true lectotype of this species. Additionally, the

re-description of Borojevia cerebrum , based on a molecular analysis of specimens discovered near its

type locality (Lesina - Island of Hvar), confirmed the presence of this species in the Mediterranean Sea

(Table 1; Fig. 16). Observing the morphological variations within a single, molecularly verified species,

enabled the synonymization of two “ cerebrum ” varieties. Step by step, the “ cerebrum complex” is being

solved. All this again confirms that molecular verification of morphological traits is very important for

a proper species assignment. It goes hand in hand with morphological confirmation relying on the type

specimens, which often become deteriorated or even lost, without detailed descriptions. This review

of some of the first species of calcarean sponges, that were last recorded and described by Haeckel

in the 19 th century, allowed validation of their taxonomic status at the molecular and morphological

levels. It bears a significant weight in reviving museum collections, which would be of a great help for

systematics research of calcarean sponges in the future.

Acknowledgements

r

M.I., B.P. and H.C. were funded by the Croatian Ministry of Science, Education and Sports (MSES) grant

098-0982913-2874. V.N. was funded by the Croatian MSES grant 001-0000000-3203. M.K. is funded

by fellowships and research grants from the Brazilian National Research Council (CNPq), the Rio de

Janeiro State Research Foundation (Funda 9 ao Carlos Chagas Filho de Amparo a Pesquisa do Estado

do Rio de Janeiro - FAPERJ) and Coordination for the Improvement of Higher Education Perso nn el

(CAPES). F.A. has a fellowship from CAPES. We are grateful to Anne Frijsinger and Mat Vestjens for

collecting and Diana Tmski for bringing in the samples of C. blanca. Jelena Godrijan is acknowledged

for helping with the preliminary SEM analysis of C. cerebrum.

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Manuscript received. 17 September 2015

Manuscript accepted. 17 November 2015

Published on: 2 March 2016

Topic editor. Rudy Jocque

Desk editor. Kristiaan Hoedemakers

Printed versions of all papers are also deposited in the libraries of the institutes that are members of the

EJT consortium: Museum national d’Histoire naturelle, Paris, France; Botanic Garden Meise, Belgium;

Royal Museum for Central Africa, Tervuren, Belgium; Natural History Museum, London, United

Kingdom; Royal Belgian Institute of Natural Sciences, Brussels, Belgium; Natural History Museum of

Denmark, Copenhagen, De nm ark

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