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Assessing the systematics of Tylodinidae in the Mediterranean Sea and Eastern Atlantic Ocean: resurrecting Tylodina rafinesquii Philippi, 1836 (Gastropoda, Heterobranchia, Umbraculida)


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The systematics of the gastropod clade Umbraculida, particularly the family Tylodinidae, has been a matter of debate in the Mediterranean Sea, with no comprehensive molecular assessment of its diversity until now. Several species and genera have been erected and synonymized in the course of the last two centuries and only two single species belonging to the genera Tylodina and Anidolyta are considered present in these waters. In order to shed light into the controversial taxonomy of the group we carried out both morpho-anatomical study and molecular analyses, using fragments of the mitochondrial cytochrome c oxidase subunit I and the 16S rRNA and the nuclear gene histone H3. Phylogenetic analyses and species delimitation tests clearly recovered two independent lineages of Tylodina in the Mediterranean and Eastern Atlantic coast, the type species T. perversa and the resurrected T. rafinesquii. We found clear differences in the shell and radular morphology between both species, as well as differences in their habitat and food prey. Interestingly, T. rafinesquii is more closely related to T. fungina from the Eastern Pacific than to the sympatric T. perversa. Furthermore, the new morphological data strongly encourage the suppression of the genus Anidolyta considering it a junior synonym with Tylodina.
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Journal of The Malacological Society of London
Molluscan Studies
Journal of Molluscan Studies (2020) 0: 1–17. doi:10.1093/mollus/eyaa031
Published online 27 November 2020
Assessing the systematics of Tylodinidae in the Mediterranean Sea and Eastern Atlantic
Ocean: resurrecting Tylodina rafinesquii Philippi, 1836 (Heterobranchia: Umbraculida)
Robert Fernández-Vilert1,2, Gonzalo Giribet2, Xavi Salvador1and Juan Moles1,2,3,4
1Catalan Opisthobranch Research Group (GROC), Mas Castellar, 17773 Pontós, Catalonia, Spain;
2Museum of Comparative Zoology and Department of Organismic and Evolutionary Biology, Harvard University, 26 Oxford Street, Cambridge, MA 02138, USA;
3SNSB-Bavarian State Collection of Zoology, Section Mollusca, Münchhausenstrasse 21, D-81247 Munich, Germany; and
4Biozentrum Ludwig Maximilians University and GeoBio-Center LMU Munich, Munich, Germany
Correspondence: R. Fernández-Vilert; e-mail:
(Received 17 February 2020; editorial decision 22 May 2020)
The systematics of the gastropod clade Umbraculida, particularly the family Tylodinidae, has been a matter
of debate. The Tylodinidae of the Mediterranean Sea are a case in point, with no comprehensive molecular
assessment of diversity having been carried out to date. Several species and genera have been erected and
synonymized in the course of the last two centuries and only a single species from each of the genera Tylodina
and Anidolyta are considered to be present in these waters. In order to shed light on the controversial taxon-
omy of the group, we carried out both morpho-anatomical study and molecular analyses using fragments
of two mitochondrial genes, cytochrome coxidase subunit I and 16S rRNA, and the nuclear gene his-
tone H3. Phylogenetic analyses and species delimitation tests clearly recovered two independent lineages of
Tylodina from the Mediterranean and Eastern Atlantic coast, the type species T. p e r v e r s a and the resurrected
T. rafinesquii. We found clear differences in shell and radular morphology between both species, as well as
differences in their habitat and food preferences. Interestingly, we found strong evidence that T. rafinesquii is
sister to T. fungina from the Eastern Pacific rather than to the sympatric T. p e r v e r s a. Furthermore, the new
morphological data strongly encourage the suppression of the genus Anidolyta, which should be considered
a junior synonym of Tylodina.
A great number of researchers have contributed substantially to the
study of Mediterranean heterobranch taxonomy (Vayssière, 1885;
Schmekel & Portmann, 1982;Gosliner, Cervera & Ghiselin, 2008),
with the use of molecular tools having increased our knowledge on
species diversity in recent years (e.g. Carmona et al., 2014;Furfaro
et al., 2016). A total of 537 species of heterobranchs are recognized
as naturally occurring within the Mediterranean Sea (Gosliner
et al., 2008), and around 523 species are recognized for the Iberian
Peninsula (including the Atlantic coastline; Cervera et al., 2004),
among which 257 are found off the Catalan coast (i.e. NE Spain;
Ballesteros, Madrenas & Pontes, 2016;Salvador, 2020). Although
the malacofauna of the Iberian Peninsula is one of the most di-
verse in the Mediterranean, new species continue to be discov-
ered, with many cryptic lineages requiring systematic evaluation.
Mediterranean marine biodiversity is now considered to be higher
than earlier estimated, cryptic speciation representing an impor-
tant source of hidden diversity in marine taxa (Lee & Foighil, 2004;
Mathews, 2006;Calvo et al., 2009), including heterobranch mol-
luscs (e.g. Furfaro et al., 2016;Korshunova et al., 2019).
Among Heterobranchia, Umbraculida are commonly known as
false limpets and were once considered to be related to the side-
gilled pleurobranchids in a group named Notaspidea. Following
recent phylogenetic assessments, Umbraculida are now treated as
an independent lineage within Tectipleura, a group character-
ized by two main synapomorphies, a monaulic reproductive sys-
tem and a cuticularized oesophagus (Wägele et al., 2014;Zapata
et al., 2014). Umbraculids are usually found in shallow and deep
waters, and occur widely in both temperate and tropical waters
(Pilsbry, 1895–1896). Although originally described in 1827 (Dall,
1889), Umbraculida comprises only ten currently accepted species
(Valdés, 2001), and more species remain to be discovered (Willan,
1987). This taxon is divided into two families, Umbraculidae and
Tylodinidae, each of which are presented by two genera. Within
Umbraculidae, the genus Umbraculum Schumacher, 1817 consists of
two large-bodied species that are exclusively spongivorous but not
restricted to a single Demospongiae species (Willan, 1984). Wägele,
Vonnemann & Rudman (2006) re-evaluated the status of Umbrac-
ulum umbraculum (Lightfoot, 1786), which was synonymized with U.
mediterraneum (Lamarck, 1819). Although some workers treat many
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of the described species as synonyms (Burn, 1959), other authors
disagree (Marcus & Marcus, 1967;Guangyu, 1981;Marcus, 1985).
The second umbraculid genus is Spiricella Rang, 1828. This includes
a species originally described from fossil material, namely Spiricella
unguiculus Rang, 1828, for which Recent shells in good condition
were found in the Mediterranean and Eastern Atlantic (Carrozza
& Rocchini, 1987), and S. redferni da Silva & Landau, 2008 from the
Caribbean. Apart from limited data on the taxonomy of the two
umbraculid genera, virtually nothing is known about the ecology of
these molluscs nor has a living animal ever been recorded (da Silva
& Landau, 2008).
Tylodinidae consists of six species in two genera, Tylodina
Rafinesque, 1814 (four species) and Anidolyta Willan, 1987 (two
species), of which the type species are Tylodina perversa (Gmelin,
1791)andAnidolyta duebenii (Lovén, 1846). Anidolyta species are rare
and inhabit deep waters. Anidolyta duebenii (Lovén, 1846) is found
in Norway, Portugal and Corsica (Warén & di Paco, 1996), and
A. spongotheras (Bertsch, 1980) occurs in British Columbia (Austin,
2000). Anidolyta is the most enigmatic genus of the order (Willan,
1987). The type species was originally described as Tylodina duebenii
Lovén, 1846 with Anidolyta being later erected (based on shell and
radular characters) to accommodate this species (Willan, 1987), al-
though Odhner (1939) considered that there was no basis for split-
ting Tylodina into two genera. Based on a comparison of the type
species of the two genera, Romani (2014) indicated that Tylodina
and Anidolyta differed in the colour of the protoconch and its promi-
nence. Two other characters that have been traditionally used to
differentiate the two genera are that in Anidolyta the shell muscle scar
is incomplete and the intermediate muscle scar is absent (Bertsch,
1980;Marcus, 1985); later studies have found the muscle scar to be
complete but faint (Warén & di Paco, 1996;Romani, 2014). Finally,
in the descriptions provided by Marcus (1985) and Bertsch (1980),
Anidolyta was shown to lack rachidian teeth (a character present in
Tylodina). However, it is plausible that the teeth may have been lost
during manipulation of the specimen, as shown by Marcus (1985)
and Warén & di Paco (1996) for material described by Willan (1983)
and Mazzarelli (1897), respectively.Romani (2014) stated that most
of the specimens identified as A. duebenii were in fact T. p e r v e r s a from
deep water; thus, the validity of the genus Anidolyta remains uncer-
Tylodina perversa (Gmelin, 1791), the type species of Tylodina,was
first mentioned by Adanson (1757) under the name Lepas “Le liri”
from the Senegalese coast. The same specimen was cited by Martini
(1769) as Lepas exigua cornea, and in 1791 it was formally described
by J.F. Gmelin as Patella perversa Gmelin, 1791. The genus Tylod-
ina was subsequently introduced by Rafinesque (1814) for T. punctu-
lata Rafinesque, 1814,withT. citrina Joannis, 1834 and T. rafinesquii
Philippi, 1836 being described later. The last two species were syn-
onymized with T. punctulata by Gray (1856), who also erected T.
atlantica Gray, 1856, which was later synonymized with T. p e r v e r s a
by Pilsbry (1895–1896).Mazzarelli (1897) described the new genus
Tylodinella for Tylodinella trinchesii Mazzarelli, 1897,butPruvot-Fol &
Fischer-Piette (1934) found no justification for the new genus and
it was synonymized with Tylodina. Finally, Fischer-Piette, Germain
& Pallary (1942) transferred Patella perversa to Tylodina, declaring it
the senior synonym of T. citrina, a name ultimately erroneously used
for the Mediterranean species T. punctulata (see Valdés, 2001); T. p e r -
versa is currently only known from the Mediterranean and Eastern
Atlantic. Until now, no studies have investigated the molecular phy-
logenetics of the species in the Mediterranean and Eastern Atlantic
and, thus, we do not know whether there is a single species of Tylod-
ina (Pruvot-Fol, 1954;Thompson, 1970;Willan, 1987) in this region
or multiple co-occurring ones (Pilsbry, 1895–1896;Watson, 1897;
Marcus, 1985). In this study, our main goal is to provide molecular
systematic and morpho-anatomical evidence to resolve the prob-
lematic taxonomy of the Tylodinidae of the Mediterranean and
Eastern Atlantic coasts.
Tylodina specimens are difficult to observe and are not often en-
countered by divers. A total of 34 specimens were collected by
hand during scuba diving and snorkelling in shallow water in
the Mediterranean Sea (NE Spain) and off the Canary Islands
(NE Atlantic Ocean); additional specimens were obtained from mu-
seum collections. Images of living specimens were taken underwa-
ter with a Nikon D90 camera coupled with a 60-mm macro lens.
The specimens were fixed in 95% ethanol and have been deposited
in the Museum of Comparative Zoology (MCZ), Harvard Univer-
sity. Voucher numbers and collection data are reported in Tabl e 1.
Permits to collect samples were issued by the Catalan Government
(permit no. SF/0589/2018).
DNA extraction, amplification and sequencing
Total genomic DNA was extracted from a small piece of the
foot using the Qiagen DNeasy Blood and Tissue Kit following
the manufacturer’s protocol. Three genes were amplified: the
mitochondrial genes cytochrome coxidase subunit I (COI; primers:
LCO1490, HCO2198; Folmer et al., 1994) and 16S rRNA (primers:
16S ar-L, 16S br-H; Palumbi et al., 1991), and the nuclear gene
histone H3 (primers: H3AD53, H3BD53;Colgan et al., 1998).
PCR reactions were carried out in 25-µl volumes with 5 µl 5X
Green GoTaq®Buffer, 0.5 µl dNTPs, 1 µl of each primer and 0.25
µl GoTaq®DNA Polymerase (Promega, WI, USA). For the mito-
chondrial markers, PCR conditions were as follows: an initial hot
start step of 3 min at 94°C; 35 cycles of 30 s at 94°C (denaturation),
30 s at 50°C (annealing) and 2 min at 72°C (extension); and a final
extension for 10 min at 72°C. PCR conditions were the same for
H3 apart from the annealing temperature, which was 53°C. PCR
failure and contamination were tested using gel electrophoresis.
Successful amplifications were purified using 1 µl ExoSAP-IT
(Affymetrix, CA, USA); sequencing was done in both directions on
an ABI 3730xl DNA Analyzer (Applied Biosystems Inc., CA, USA).
Phylogenetic analyses
Sequence contamination was checked for each sequence against
the GenBank nucleotide database, using the BLAST algorithm
(Altschul et al., 1997); all sequences were confirmed as belonging
to one of two umbraculid superfamilies, Tylodinoidea and Um-
braculoidea. Sequences were visualized, edited and assembled in
Geneious Pro 8.1.5 (Kearse et al., 2012). All new sequences have
been deposited in GenBank (see Table 1for acc. nos). Sequences of
each gene were individually aligned using the MUSCLE algorithm
(Edgar, 2004) implemented in Geneious; missing positions at the
ends of sequences were coded as missing data. We decided to avoid
filtering of the 16S rRNA alignment since this has been shown to
impact tree accuracy (Tan et al., 2015); in any case, our data for
this gene did not contain much variation. Saturation was tested for
the first, second and third codon positions of the protein-coding
genes COI and H3 using MEGA7 (Kumar, Stecher & Tamura,
2016); this was done by plotting GTR pairwise distances against
total substitutions (transitions +transversions).
Forty-nine specimens in total were used in the phylogenetic
analyses and the outgroup comprised two species each of Aplysi-
ida and Cephalaspidea. Phylogenetic analyses involved maximum
likelihood (ML) and Bayesian approaches and were run on the
CIPRES Science Gateway v. 3.3 ( ML
analysis was performed using IQ-TREE v. 1.6.9 (Trifinopoulos
et al., 2016); model selection for the concatenated dataset was car-
ried using ModelFinder (Kalyaanamoorthy et al., 2017) and the best
substitution models selected were TIM +F+I+G4 for COI,
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Tabl e 1 . Material used in molecular phylogenetic and species delimitation analyses, with GenBank acc. nos (by gene region) and relevant references.
Voucher Species Date/source Locality COI 16S rRNA H3 Reference
MCZ 371810 Tylodina perversa 11 September 1993 Port Lligat, Girona,
MN900754 MN902262 MN901054 This study
MCZ 392595 Tylodina perversa 10 August 2013 Morro de Potala,
Canary Islands
MN900755 MN902263 MN901055 This study
MCZ 392596 Tylodina perversa 10 August 2013 Morro de Potala,
Canary Islands
MN900756 MN902264 MN901076 This study
MCZ 392597 Tylodina perversa 10 August 2013 Morro de Potala,
Canary Islands
MN900757 MN902265 – This study
MCZ 392598 Tylodina perversa 10 August 2013 Morro de Potala,
Canary Islands
MN900758 MN902266 MN901056 This study
MCZ 392599 Tylodina perversa 10 August 2013 Morro de Potala,
Canary Islands
MN900759 MN902267 MN901057 This study
MCZ 392600 Tylodina perversa 10 August 2013 Morro de Potala,
Canary Islands
MN900760 MN902268 MN901058 This study
MCZ 392601 Tylodina perversa 10 August 2013 Morro de Potala,
Canary Islands
MN900761 MN902269 MN901059 This study
MCZ 392602 Tylodina perversa 10 August 2013 Morro de Potala,
Canary Islands
MN900762 MN902270 MN901060 This study
MCZ 392603 Tylodina perversa 10 August 2013 Morro de Potala,
Canary Islands
MN900763 MN902271 MN901061 This study
MCZ 392604 Tylodina perversa 10 August 2013 Morro de Potala,
Canary Islands
MN900764 MN902272 MN901062 This study
MCZ 392605 Tylodina perversa 10 August 2013 Caleta Caballo,
Canary Islands
MN900765 MN902273 MN901063 This study
MCZ 392606 Tylodina perversa 10 August 2013 Caleta Caballo,
Canary Islands
MN900766 MN902274 MN901064 This study
MCZ 392607 Tylodina perversa 10 August 2013 Caleta Caballo,
Canary Islands
MN900767 MN902275 MN901065 This study
MCZ 392608 Tylodina perversa 10 August 2013 Caleta Caballo,
Canary Islands
MN900768 MN902276 MN901066 This study
MCZ 392609 Tylodina perversa 10 August 2013 Caleta Caballo,
Canary Islands
MN900769 MN902277 MN901077 This study
MCZ 392610 Tylodina perversa 10 August 2013 Caleta Caballo,
Canary Islands
MN900770 MN902278 MN901067 This study
MCZ 392611 Tylodina perversa 10 August 2013 Caleta Caballo,
Canary Islands
MN900771 MN902279 MN901068 This study
MCZ 392612 Tylodina perversa 10 August 2013 Caleta Caballo,
Canary Islands
MN900772 MN902280 MN901069 This study
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Tabl e 1 . Continued
Voucher Species Date/source Locality COI 16S rRNA H3 Reference
MCZ 392613 Tylodina perversa 10 August 2013 Caleta Caballo,
Canary Islands
MN900773 MN902281 MN901070 This study
MCZ 392614 Tylodina perversa 10 August 2013 Caleta Caballo,
Canary Islands
MN900774 MN902282 MN901071 This study
MCZ 392615 Tylodina perversa 10 August 2013 Caleta Caballo,
Canary Islands
MN900775 MN902283 MN901072 This study
MCZ 392616 Tylodina perversa 10 August 2013 Caleta Caballo,
Canary Islands
MN900776 MN902284 MN901073 This study
MCZ 392617 Tylodina perversa 10 August 2013 Caleta Caballo,
Canary Islands
MN900777 MN902285 MN901074 This study
MCZ 392618 Tylodina perversa 10 August 2013 Caleta Caballo,
Canary Islands
MN900778 MN902286 MN901075 This study
MCZ 392666 Tylodina perversa 18 September 2018 Es Caials,
Girona, Spain
MN900782 This study
MCZ 392667 Tylodina perversa 18 September 2018 Es Caials,
Girona, Spain
MN900783 This study
MCZ 392623 Tylodina perversa 18 September 2018 Punta d’en Bosc,
Sant Feliu de
Guíxols, Girona,
MN900779 This study
MCZ 392626 Tylodina perversa 18 September 2018 Punta d’en Bosc,
Sant Feliu de
Guíxols, Girona,
MN900781 This study
MCZ 392624 Tylodina perversa 18 September 2018 Punta d’en Bosc,
Sant Feliu de
Guíxols, Girona,
MN900780 This study
MCZ 392625 Tylodina rafinesquii 19 January 2018 Punta d’en Bosc,
Sant Feliu de
Guíxols, Girona,
MN900786 MN902289 MN901080 This study
MCZ 392619 Tylodina rafinesquii 10 August 2013 Caleta Caballo,
Canary Islands
MN900784 MN902287 MN901078 This study
MCZ 392620 Tylodina rafinesquii 10 August 2013 Caleta Caballo,
Canary Islands
MN900785 MN902288 MN901079 This study
MCZ 392627 Tylodina rafinesquii 20 September 2018 Punta d’en Bosc,
Sant Feliu de
Guíxols, Girona,
MN900787 This study
Tylodina fungina GenBank Panama,
Caribbean Sea
GU213060 GU213046 – Göbbeler &
Tylodina perversa GenBank Spain,
AF249809 – Wollscheid-
Lengeling et al.
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Tabl e 1 . Continued
Voucher Species Date/source Locality COI 16S rRNA H3 Reference
Tylodina perversa GenBank Tenerife, Canary
KF992172 KJ022803 KJ022917 Oskars, Bouchet &
Malaquias (2015)
Tylodina perversa GenBank Porto Santo, Madeira AY345024 Grande et al. (2004)
EED-Phy-740 Tylodina rafinesquii A GenBank France, Mediterranean
GU213059 GU213045 – Göbbeler &
Tylodina rafinesquii B GenBank France, Mediterranean
FJ917424 – Göbbeler &
Akera bullata GenBank Algoleran, Sweden, NE
AF156143 AF156127 EF133474 Medina & Walsh (2000)
Aplysia parvula GenBank Spain, Mediterranean
AF249822 AF192291 JX560158 Wollscheid-Lengeling
et al. (2001)
GenBank Girona, NE Spain AY345023 Grande et al. (2004)
Umbraculum cf.
umbraculum A
GenBank Australia DQ256200 DQ256203 Wägele et al., 2006
Umbraculum cf.
umbraculum B
GenBank Mediterranean Sea DQ256201 DQ256204 Wägele et al., 2006
Umbraculum cf.
umbraculum C
GenBank Azores, Atlantic Ocean DQ256202 DQ256205 Wägele et al., 2006
EED-Phy-661 Umbraculum sp. GenBank Mediterranean Sea GU213058 GU213044 Göbbeler &
Haminoea hydatis GenBank France, Atlantic DQ238004 KJ022796 KJ022925 Klussmann-Kolb &
Dinapoli (2006)
Aglaja tricolorata GenBank Giglio, Italy AM421902 AM421854 KJ022932 Anthes, Schulenburg &
Michiels (2008)
EED-Phy-51 Umbraculum
GenBank Australia, NSW EF489322 Klussmann-Kolb et al.
The third column shows the date of collection for material collected for this study or the source (GenBank) of sequence data generated by other studies.
TVM +F+G4 for 16S rRNA and TN +F+G4 for H3. Branch
support was estimated via ultrafast bootstrap with 1,000 replicates
(Hoang et al., 2017). Separate analyses were carried out for individ-
ual gene regions and the concatenated alignment. The nucleotide
substitution model used for the Bayesian analysis of the concate-
nated dataset was the GTR +Gmodel(Yang, 1996). Bayesian
analysis was performed using MrBayes v. 3.2 (Ronquist et al., 2012)
with four parallel runs of 20 million generations, sampling every
1,000 generations and a burn-in of 25%; stationarity was assessed
using Tracer v. 1.7 (Rambaut et al., 2018). Trees were visualized in
FigTree v. 1.4.4 (Fig. 1).
Species delimitation tests were conducted on the aligned COI
dataset to further investigate the monophyly of the studied
species. An automatic barcode gap discovery (ABGD; Puillandre
et al., 2012) analysis was run using the web interface at http://
K80 and JC69 TS/TV distance matrices with default parameters
(Pmin =0.001, Pmax =0.1, 10 steps, 20 Nb bins and relative gap
width =1.5). Species delimitation analyses were also carried using
the Poisson Tree Processes (PTP) and the Bayesian implementation
of the PTP model (bPTP; Zhang et al., 2013). Analyses were run on
the web server ( using 500,000 gen-
erations on a rooted tree with specified outgroups.
Anatomy and scanning electron microscopy
Preserved specimens were photographed with a Keyence
VHX-6000 Digital Microscope; ventral, dorsal and lateral
views were taken as well as details of the protoconch and the gills.
Total lengt h ( L), width (W) and height (H) of the specimens were
measured with a Vernier calliper before dissection with the aid
of fine forceps. The buccal mass and the shell were extracted,
immersed in a 50% bleach solution for up to 1 h (to dissolve
the organic tissues) and then rinsed with distilled water in an
ultrasonic bath. The radula, shell and crop contents were mounted
on metallic stubs with carbon sticky tabs and coated with platinum
and palladium for scanning electron microscopy (SEM). The crop
and the penis were critical point dried before SEM with a Tousimis
931 GL critical point dryer. Samples were imaged using a Zeiss
Ultra55 field emission scanning electron microscope in the Center
for Nanoscale Systems (CNS), Harvard University.
Phylogenetic analyses
The final sequence dataset for the 49 taxa totalled 1,564 bp
(c. 690 bp for COI with the third codon position, c. 498 bp for 16S
rRNA and c. 376 bp for H3). ML and Bayesian analyses of the con-
catenated alignment (COI +16S +H3) were largely congruent
with maximal support for the monophyly of both Tylodina perversa
and T. rafinesquii (Fig. 1and Supplementary Material Figs S1–S3).
We found strong support in both sets of analyses for the sister group
relationship of T. rafinesquii to the Eastern Pacific T. fungina and the
clade comprising these two species was strongly supported as sister
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Figure 1. ML and Bayesian trees for Umbraculida based on the concatenated COI, 16S rRNA and H3 genes (1,564 bp). Support values are shown on
branches with ML bootstrap support values on the left and Bayesian posterior probabilities on the right; red dots at nodes indicate maximal branch support
in both analyses. The outgroup consists of two species each of Anaspidea and Cephalaspidea. Specimens in bold font are those sequenced for this study.
The results of the ABGD, PTP and bPTP species delimitation analyses based on COI are represented by the vertical bars to the right of the tree. Scale bar
indicates substitutions per site.
to the Mediterranean T. p e r v e r s a . The ABGD analysis recognized T.
rafinesquii,T. fungina and T. p e r v e rs a as distinct species, with intraspe-
cific distances of 0–1.4% for T. p e r v e r s a and 0.1–1% for T. rafinesquii.
Interspecific distances ranged from 18.1% to 20.2% between T. per -
versa and T. rafinesquii, from 19.2% to 20% between T. p e r v e r s a and
T. fungina and from 14.4% to 14.8% between T. rafinesquii and T.
fungina. Both the PTP and bPTP analyses yielded the same result,
with Bayesian support values for T. p e r v e r s a of 0.91 and 0.89 for T.
rafinesquii, being recovered as distinct species.
Order UMBRACULIDA Odhner, 1939
Family TYLODINIDAE Gray, 1847
Genus Tylodina Rafinesque, 1814
Type species:Patella perversa Gmelin, 1791 (by original designation).
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Figure 2. Tylodina perversa. A, B. In life. A. Specimen crawling on top of the alga Padina pavonica.B. Close-up of the head of a specimen laying eggs (black
arrow); note the threads of mucus (with secondary metabolites; white arrows) extending between eggs and mantle. C–E. Preserved specimen. C. Dorsal view.
D. Ventral view. E. Lateral view. F–J. Shell. F. Dorsal view. G. Ventral view. H. Lateral view. I. SEM micrograph of the protoconch. J. SEM micrograph of
the muscle scar. 7
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Figure 3. SEM micrographs of Tylodina perversa. A. Detail of rachidian and first lateral teeth. B. Detail of the outer lateral teeth. C. Crop and close-up of
papillae. D. Spermatozoa found in the bursa copulatrix and close-up showing the head of one.
Tylodina perversa (Gmelin, 1791)
(Figs 2A–J, 3A–D, 4A–C, 5A–D)
Patella perversa Gmelin, 1791: 3714–3715.
Tylodina perversaVayssière, 1885: 151–162, figs 130–136. Pilsbry, 1895–
1896: 175–189, pl. 73: figs 77–83. Fischer-Piette et al., 1942: 103–374.
Pruvot-Fol, 1954: 207–210, fig. 80. Willan, 1987: 215–241, fig. 1. Va ldés,
2001: 29–34. Romani, 2014: 515–520, figs 4–6, 14, 16, 19.
Tylodina punctulata Rafinesque, 1814: 161–166.
Tylodina citrina Joannis, 1834: 266, pl. 36: figs 1–5.
Tylodina atlantica Gray J.E., 1856: 45–46.
Tylodinella trinchesii Mazzarelli, 1897: 597–605, pl. 23–24: figs 1–21.
Type locality: African coast (Senegal, West Africa).
Diagnosis: Shell conical, depressed, covered by periostracum with ra-
dial brown stripes. Body yellow; elevated with foot large in relation
to shell. Radular formula: 150–130 ×130–65.1.65–130. Rachidian
tooth small, with faint serration on each side. Lateral teeth hook-
shaped, thick, short and blunt, with one to two denticles.
Material examined: One spec. (dissected and sequenced; L=32
mm, W=27 mm, H=16 mm), Port Lligat, Girona, NE
Spain, 42°1737.06N, 3°1717.93E, 12 m depth, coll. G.
Giribet, 11 Sep. 1993, MCZ 371810. Two specs (sequenced;
L=13–20 mm, W=10–17 mm, H=6–13 mm), Es Caials,
Cadaqués, NE Spain, 42°176.14N, 3°1747.68E, 7 m depth,
coll. X. Salvador, 18 Sep. 2018, MCZ 392666–392667. Three
specs (sequenced; L=10–17 mm, W=8–11 mm, H=5–9 mm),
Punta d’en Bosc, Sant Feliu de Guíxols, NE Spain, 41°4558.1 N,
3°010.65E, 4 m depth, coll. X. Salvador, 20 Sep. 2018, MCZ
392623–392624, 392626. Ten specs (MCZ 392601–392602 dis-
sected and sequenced, the rest only sequenced; L=6–16 mm,
W=4–14 mm, H=4–7 mm), Morro de Potala, Fuerteventura,
Canary Islands, 28°425.82N, 14°2940.2W, 0.5 m depth, coll.
J. Moles, 8 Oct. 2013, MCZ 392595–392604. Fourteen specs (se-
quenced; L=5–19 mm, W=3–10 mm, H=3–7 mm), Caleta Ca-
ballo, Lanzarote, Canary Islands, 29°713.61N, 13°3824.41W, 3
m depth, coll. J. Moles, 8 Oct. 2013, MCZ 392605–392618.
External morphology (Fig. 2A–E): Body 5–32 mm in length, elevated,
bright yellow in colour. Mantle narrow, not completely covering
shell, slightly grooved on edge. Foot large, thick, rounded, larger in
relation to shell, truncated anteriorly. Head extending forward with
a pair of short, conical oral tentacles. Rhinophores long, cylindrical,
folded longitudinally, wider at base. Eyes anteriorly placed at base
of rhinophores, slightly delimited by unpigmented area. Gill found
at right side of body, protected by shell, with 7–14 plumes per side.
Shell (Fig. 2F–J): Patelliform, oval, conical, depressed; yellowish
and shiny in colour. Shell sculpture consists of concentric growth
lines; periostracum displays some thick, brown radial stripes that
are absent on apex. Apex slightly extended backwards and towards
left due to presence of protoconch. Protoconch globose (Fig. 2I).
Muscle scar circular, complete, with faint sinus in gill region
(Fig. 2J).
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Figure 4. Anatomy of Tylodina perversa.A. Digestive system. B. Reproductive system. C. Nervous system. Abbreviations: a, anus; amp, ampulla; bc, bursa
copulatrix; ceg, cerebral ganglia; cr, crop; dgl, digestive gland; gon, gonad intersection (gonad not represented); int, intestine; m, mouth; nidgl1, mucous
gland; nidgl2, membrane gland; nidgl3, albumen gland; nr, nerve ring; oes, oesophagus; org, oral ganglia; pdg, pedal ganglia; pha, pharynx; po, penial
opening; sgl, salivary glands; sto, stomach; vag, vagina; and visg, visceral ganglia.
Radula (Fig. 3A, B): Radular formula 150–130 ×75–65.1.65–75.
Rachidian teeth small, thinner than lateral teeth, with some small
faint serrations on both sides. Inner lateral teeth hook-shaped, thick,
short, with one or two pointy denticles on inner side and blunt tip.
Outer lateral teeth hook-shaped, thick, short, blunt and decreasing
in size towards radula edge; tip rounded, sometimes without denti-
cles (Fig. 3B).
Digestive system (Figs 3C, 4A): Mouth subventral, leading into short
oral tube. Oral disc cuticularized with ornaments. Pharyngeal bulb
rather rounded, very muscled, connecting with oesophagus pos-
terodorsally. Salivary glands fusiform, located close to pharynx–
oesophagus connection, passing through nerve ring. Oesopha-
gus leaving pharynx with an anterior loop after passing through
nerve ring, widening until reaching crop ventrally. Crop saccu-
lar, strongly folded longitudinally, with cuticularized spines on fold
ridges (Fig. 3C). Stomach small, elongated; followed by thin intes-
tine curved to left side. Digestive gland occupying most of posterior
part of viscera. Intestine bending dorsally to right side, leading to
anal papilla and situated posterior to gills.
Reproductive system (Figs 3D, 4B): Monaulic. Gonad purple coloured,
granulose; covering most of visceral cavity and lying in front of di-
gestive gland. Ampulla large, sausage shaped. Receptaculum sem-
inis present at ampulla–female gland mass connection (not de-
picted in Fig. 4B). Oviduct short, proximally connecting to large
nidamental glands. Albumen gland reddish, relatively small, gran-
ulated, composed of narrow coils. Capsule gland pinkish, tubular,
soft, fragile. Mucous gland massive, whitish, hardened. Bursa copu-
latrix globulose, white, translucid, thin and smooth, with long duct
opening between right tentacle and rhinophore. Internally bursa
copulatrix has velvety appearance due to presence of cilia; sperm
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Figure 5. Tylodina perversa. A. Egg masses. B. Live individual on the sponge Aplysina aerophoba.C, D. SEM micrographs of crop contents, showing detail of
style-type spicules (C) and spongin fibres of A. aerophoba (D).
found intermingled in cilia, strongly reddish purple in colour inter-
nally. Penis non-protractile. Male and female apparatus connected
by short sperm groove.
Nervous system (Fig. 4C): Circumoesophageal, postpharyngeal nerve
ring composed of four ganglia (two cerebral and two pedal). Cere-
bral ganglia oval, connected by a relatively long, thick commissure;
statocysts can be seen within it. Pedal ganglia variable in shape,
attached to each other; situated just below cerebral ganglia. Oral
ganglia interconnected by a short commissure just above oral bulb.
Visceral loop short, attached to circumoesophageal ring and com-
posed of three bulky, visceral ganglia.
Ecology (Fig. 5A–D): Specimens from the Mediterranean Sea and
the Atlantic Ocean have been observed both in daytime and at
night on rocky surfaces, where their main food, the sponges Aplysina
aerophoba or A. cavernicola, is found. The slugs perforate the sponge
surface while feeding and are found—sometimes at high density—
in these holes, which resemble sponge oscula (Val d é s & L o z u e t,
2000). Numerous Aplysina spongin fibres and small amounts of
diatoms, foraminiferans and sponge tylostyle spicules (probably
from Pseudosuberites sp.) were found in the crop contents of the
two specimens studied here. The narrow diet may be linked to
the fact that these snails sequester brominated alkaloid metabolites
from their prey (Teeyapant et al., 1993) to protect themselves and
their egg masses against predation (Ebel, Marin & Proksch, 1999).
Tylodina perversa prefers shallow waters, most likely due to the higher
densities of cyanobacterial symbionts present on the external tis-
sues of the sponge that T. p e r v e r s a prefers to graze on, A. aerophoba
(Becerro et al., 2003). The mucous secretions and egg masses of T.
perversa contain alkaloids derived from A. aerophoba and these are
thought to act as chemical protection (Ebel et al., 1999). Individual
egg masses of T. p e r v e r s a consist of a flat spiral ribbon that is yel-
lowish in colour due to the sponge-derived secondary metabolites
secreted by the adult; this secretion is glued with the mucus for egg
mass protection against predation (Fig. 5B).
Distribution: 0–40 m depth (this study; Vayssière, 1885). Portugal,
Strait of Gibraltar, Southern and Eastern Mediterranean Iberian
coast, Balearic Islands, Canary and Selvagens Islands, Madeira and
the Azores (reviewed in Cervera et al., 2004); Cape Verde (Rolán,
2005); Atlantic coast of France (Valdés & Lozouet, 2000); Mediter-
ranean coast of France (Köhler, 2020); Greece (Koukouras, 2010);
Turkey, Israel and Croatia (Rudman, 1999–2010); Malta (Sammut
& Perrone, 1998); and Italy (Doneddu & Manunza, 1990).
Remarks:Tylodina perversa was described by Gmelin (1791) based
on a specimen from Senegal and, because of its external resem-
blance with Patella, it was assigned to the latter genus. A few years
later in Italy, Rafinesque described the genus Tylodina (based on
T. punctulata Rafinesque, 1814 as the type species), with T. cit-
rina Joannis, 1834 being described subsequently. Pruvot-Fol (1954)
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identified the different species of Mediterranean Tylodina that had
been described up to that time as the species P. p e r v e r s a Gmelin,
1791; he synonymized them with T. p e r v e r s a (i.e. on the basis of over-
lapping of morphological characters), which thus became the only
species of the genus present in the Mediterranean Sea. Our spec-
imens broadly agree with the historical descriptions of T. p e r v e r s a .
The only difference is that the material described by Pilsbry (1895–
1896),Pruvot-Fol (1954) and Vayssière (1885) had a greater number
of lateral teeth (80–130); this is most likely due to differences in on-
togeny. In addition, Romani (2014) mentioned that the longitudinal
bands in the periostracum of the shell may occasionally be absent;
this may be an artefact of shell surface erosion and/or algal growth
on the shell. In our dissections of the reproductive system, we did
not observe the receptaculum seminis, so the details provided here
are from the descriptions of T. americana by Gosliner (1981) and T.
perversa by Klussmann-Kolb (2001).
Tylodina rafinesquii Philippi, 1836
(Figs 6A–J, 7A–D, 8A–D)
Tylodina rafinesquii Philippi, 1836: 114, pl. 7: fig. 8a, b. Pilsbry, 1895–1896:
175–189, pl. 73: figs 84, 85. Watson, 1897: 325. Pruvot-Fol, 1954: 207–
210, fig. 80. Marcus, 1985: 1–15.
Tylodinella trinchesii Mazzarelli, 1897: 597–605, pl. 23, 24: figs 1–21.
Type locality: Sicily, Italy.
Diagnosis: Shell conical, highly elevated and covered by an in-
distinct periostracum lacking radial brown stripes. Body yellow,
foot narrower than shell and concealed by it. Radular formula:
215–220 ×110–95.1.95–110. Rachidian teeth hook-shaped, thin,
small, without denticles. Lateral teeth hook-shaped, thin, large; with
pointed tip and one denticle on inner side. Distal teeth lacking den-
Material examined: Two specs, Caleta Caballo, Lanzarote, Canary
Islands, 29°713.61N, 13°3824.41W, 3 m depth, MCZ 392619–
392620; MCZ 392619 dissected and sequenced (L=19 mm,
W=12 mm, H=7 mm); MCZ 392620 sequenced (L=14 mm,
W=8 mm, H=9 mm), coll. J. Moles, 8 Oct. 2013. Two specs,
Punta d’en Bosc, Sant Feliu de Guíxols, NE Spain, 41°4558.1 N,
3°010.65E, 4 m depth, MCZ 392625, MCZ 392627; MCZ
392625 dissected and sequenced (L=17 mm, W=13 mm, H=10
mm), 19 Jan. 2018; MCZ 392627 sequenced (L=16 mm, W=12
mm, H=12 mm), coll. X. Salvador, 20 Sep. 2018.
External morphology (Fig. 6A–E): Body yellow, oval, elevated and
not as wide as shell. Mantle narrow, not extending over shell.
Rhinophores long, cylindrical, folded longitudinally and thicker at
base. Eyes placed anteriorly at base of rhinophore; slightly delim-
ited by unpigmented area. Head extended forward, with a pair of
triangular oral tentacles. Gill on right side, protected by shell and
with 14–16 plumes per side. Foot large, oval.
Shell (Fig. 6F–J): Patelliform, oval, elevated and conical; surface
shiny whitish-yellowish with concentric growth lines. Apex ex-
tending slightly backwards and towards left side due to proto-
conch (Fig. 6I); whitish-yellowish, shiny with concentric growth
lines. Periostracum extending beyond shell margin and without ra-
dial brown stripes. Muscle scar circular, complete and with sinus
(Fig. 6J).
Radula (Fig. 7A, B): Radular formula 215–220 ×110–95.1.95–
110. Rachidian tooth hook-shaped, smooth, thin and small; den-
ticles absent. Inner lateral teeth hook-shaped, thin and large, with
one denticle on inner side pointed tip. Outer lateral teeth de-
creasing in size towards radula margin and generally without
Digestive system:AsinT. p e r v e r s a.
Reproductive system:AsinT. p e r v e r s a .
Nervous system:AsinT. p e r v e r s a .
Ecology (Fig. 8A–D and Supplementary Material Fig. S4A–E): Spec-
imens from the Mediterranean Sea and the Atlantic Ocean were
found active during both day and night, and were found at shal-
low depths of 0–4 m. The Atlantic Ocean specimens were col-
lected in an area with abundant Aplysina aerophoba. The individ-
uals from the Mediterranean were observed at the entrance of a
cave and were feeding on a species of Aplysina, which was dis-
tinct from the commonly found Aplysina cavernicola (also found in
caves) and is possibly an undescribed species (M.J. Uriz, personal
communication). Diatoms, foraminiferans, sponge spongin fibres
of Aplysina sp., tylostyle spicules of Pseudosuberites sp.and bivalves
were found in the crop contents (Supplementary Material Fig.
S4A–E) of T. rafinesquii. The egg mass consists of a flat, spiral and
yellowish ribbon with white eggs that are smaller than those of
T. p e r v e r s a .
Distribution:Madeira(Watson, 1897); Mediterranean coast of France
(as T. p e r v e r s a ;Göbbeler & Klussmann-Kolb, 2010,2011); Italy
(as Tylodinella trinchesii;Philippi, 1836;Mazzarelli, 1897); Eastern
Iberian Peninsula and Lanzarote, Canary Islands (this study).
Remarks: The name Tylodina rafinesquii Philippi, 1836 is the earliest
available name for this species. Tylodina rafinesquii was first described
by Philippi (1836) and later synonymized with T. punctulata by Gray
(1856) who, in our opinion, did not note the morphological differ-
ences between the specimens examined by Philippi (e.g. the shell of
T. rafinesquii is rounder, more elevated and lacks radial brown bands).
Gray (1856) also described the species T. atlantica from the Canary
Islands, but Pruvot-Fol (1954) later concluded that Gray’s speci-
men was a small Umbrella sp. (accepted as Umbraculum). Mazzarelli
(1897) described the new genus Tylodinella, with the single species
Tylodinella trinchesii. He characterized this genus as having a more
conical and elevated shell that is larger than the body and lacks
brown stripes; he also indicated that the radula lacked rachidian
teeth. Marcus (1985) suggested that the lack of rachidian teeth was
due to loss during preparation. We consider, however, that these
teeth were not recognized due to their small size and slender ap-
pearance (Fig. 7A). On the basis of morphological similarities in the
descriptions and illustrations of Philippi and Mazzarelli, we suggest
that T. rafinesquii and Tylodinella trinchesii are in fact the same species.
We, therefore, treat Tylodinella trinchesii as a junior synonym of T.
Our specimens of T. rafinesquii have a shell (resembling that of T.
fungina Gabb, 1865; Valdés, 2019) that is highly elevated and lacks
conspicuous brown radial stripes on the periostracum. Pilsbry’s
(1895–1896) description broadly agrees with ours. Watson (1897)
did not agree with Vayssière’s (1885) view that T. rafinesquii should
be synonymized with T. pe r v e r s a on the basis of the form of the
shell of the former species, as depicted in Philippi’s (1836) figure.
In contrast, Willan (1987) accepted the opinion of Pruvot-Fol &
Fishcer-Piettte (1934) that all Tylodina species based on Mediter-
ranean specimens are synonyms of T. p e r v e rs a . In our dissections of
the reproductive system, we did not find the receptaculum seminis,
as was also the case in Valdés, Gosliner & Ghiselin’s (2010) study
of T. fungina. Nonetheless, we suspect that a receptaculum seminis
similar to those described for T. americana (Gosliner, 1981)andT.
perversa (Klussmann-Kolb, 2001) may be present in both T. rafinesquii
and T. fungina.
The taxonomy of Tylodinidae has been subject of controversy
for the past two centuries. Until we did our study, no molecular
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Figure 6. Tylodina rafinesquii. A,B. Living specimens resting next to the sponge Aplysina sp.C–E. Preserved specimen. C. Dorsal view. D. Ventral view. E.
Lateral view. F–J. Shell. F. Dorsal view. G. Ventral view. H. Lateral view. I. SEM micrograph of the protoconch. J. SEM micrograph of the muscle scar.
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Figure 7. SEM micrographs of Tylodina rafinesquii. A. Detail of rachidian (white arrows) and first lateral teeth. B. Detail of the outer lateral teeth. C. Crop
and close-up of papillae. D. Spermatozoa found in the bursa copulatrix.
phylogenetic analyses of Tylodina using multiple specimens had
been carried out. Although we did not study samples from the
type locality or studied the type material of the two species we
investigated, the wide distributional range of our Tylodina species
(i.e. ranging from the Eastern Atlantic to the eastern Mediter-
ranean) and the fact that morphotypes similar to the type material
can be found in the localities sampled by us allow us to draw the
conclusions presented in this study. Here, we provide detailed SEM
micrographs and anatomical descriptions for the type species of
the genus and for a second species from the Mediterranean and
Eastern Atlantic. In our ML and Bayesian trees of the 40 Tylodina
specimens, the monophyly of T. p e r v e r s a and T. rafinesquii were max-
imally supported. Tylodina rafinesquii is considered to be the earliest
available name for the species described by Philippi (1836) and later
synonymized with T. p e r v e r s a (Pruvot-Fol, 1954;Gray, 1856). Fur-
ther support for our resurrection of T. rafinesquii is provided by the
results from the species delimitation tests and clear differences be-
tween T. p e r v e rs a and T. rafinesquii in the shell and radula. We found
no differences in the anatomy of the reproductive, digestive and
nervous systems, but fine histological studies may reveal otherwise.
We also found ecological differences were between the two species.
Tylodina perversa is usually found feeding on the sponge Aplysina
aerophoba (rarely also on A. cavernicola at cave entrances in shallow
waters). In contrast, in the Mediterranean, we found T. rafinesquii
at the entrance of shallow-water caves, feeding on an undescribed
species of Aplysina (recently described as a miniaturized form of
A. aerophoba on the basis of flawed molecular systematic analyses;
Costa et al., 2020); in the Atlantic, this species was found to occur
at high density on A. aerophoba. Our findings indicate that T. p e r v e r s a
and T. rafinesquii are sympatric and that both feed on Aplysina
spp.; the gut contents of both species also contained tylostyle
spicules (possibly from Pseudosuberites spp.) and additional biofouling
Here, we provide strong evidence for the recognition of T.
perversa and T. rafinesquii as distinct species. Whereas T. rafinesquii
has an elevated shell with an indistinct periostracum lacking brown
radial stripes, T. p e r v e r s a has a depressed shell with a well-developed
periostracum marked by brown radial bands. This clear difference
in shell morphology between T. p e r v e r s a and T. rafinesquii is consistent
with the descriptions of Pilsbry (1895–1896) and Watson (1897);
the latter, in fact, disagreed with Vayssière’s (1885) view that the
species should be synonymized. Contrarily, Pruvot-Fol & Fishcer-
Piettte (1934) and Willan (1987) alleged that all Tylodina species
from the Mediterranean should be synonymized, T. p e r v e r s a being
the only species occurring in the Mediterranean. However, as we
have shown here, two species are present in the Mediterranean (T.
perversa and T. rafinesquii) and the morphological differences between
these species also include differences in the radula. Our specimens
of T. p e r v e rs a have thick, short and blunt lateral teeth, often without
denticles, and a small faintly serrated rachidian tooth. In contrast,
T. rafinesquii has thin, large and pointed lateral teeth with one
denticle on their inner side; the rachidian tooth is smaller than in
T. pe r v e r s a and is smooth and hook-shaped. Therefore, we agree
with Marcus (1985) that the teeth of these two Tylodina species are
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Figure 8. Tylodina rafinesquii. A. Egg mass. B. The sponge Aplysina sp. in life. C, D. SEM micrographs of crop contents, showing details of tylostyle spicule
(possibly from a Pseudosuberites sp.) (C) and spongin fibres of Aplysina sp. (D).
remarkably different and we thus reject the view that T. rafinesquii
is a junior synonym of T. p er v e r s a . Among the species described
from the Mediterranean, we consider T. punctulata and T. citrina as
synonyms of T. p e r v e r s a and Tylodinella trinchesii to be a synonym of
T. rafinesquii.
Numerous similarities between T. rafinesquii and Anidolyta duebenii
(originally described as T. duebenii Lovén, 1846) can now be with-
drawn from our new morpho-anatomical description. In both
species, the shell takes a high, conical form (Warén & di Paco,
1996;Romani, 2014), and both taxa have a hook-shaped rachidian
tooth with a single large cusp, and thin, large lateral teeth with
pointed tips (Warén & di Paco, 1996). A faint yet complete muscle
scar is also found on the shell of A. duebenii (Warén & di Paco,
1996;Romani, 2014), although some authors have indicated that
the muscle scar is incomplete (Marcus, 1985). The synonymy of
A. duebenii with T. rafinesquii is not consistent with the differences
observed in the radula: T. rafinesquii has a single denticle at the base
of the main cusp, whereas A. duebenii has two or three denticles
between the cusp and the base (Willan, 1987;Warén & di Paco,
1996). Also, T. rafinesquii has been recorded in shallow waters
(0–4 m) in contrast to A. duebenii, which can be found at depths
>100 m (Romani, 2014). However, based on all the similarities
found in the literature, Romani’s (2014) view that “most A. duebenii
records in collections are actually T. p e r v e r s a from deep waters” and
the lack of synapomorphies for the genus Anidolyta,wepropose
that Anidolyta should be treated as a junior synonym of Tylodina.
Consequently, we reinstate T. duebenii Lovén, 1846 and introduce
the new combination T. sp o n go t h era s (Bertsch, 1980).
The first species of Umbraculida described was T. p e r v e r s a in the
late 18th century, but relatively few species have been described
since then (Sabelli, Giannuzzi-Savelli & Bedulli, 1992;Val d é s ,
2001). Only two fossil and nine extant species are currently ac-
cepted. Within the genus Umbraculum, many described species are
currently considered synonyms (Burn, 1959;Wägele et al., 2006).
Similarly, within Tylodina, eight species with restricted ranges are
presently accepted: T. p e r v e r s a and T. rafinesquii from the Mediter-
ranean Sea and Eastern Atlantic; T. americana Dall, 1889 from the
Western Atlantic; T. fungina from the Eastern Pacific; T. corticalis
(Tate, 1889) from Australia (Willan, 1987); T. duebenii from deep
water in the Mediterranean Sea and Eastern Atlantic (Wa n &
di Paco, 1996); and T. spon g o th e r as n. comb. from British Columbia
(Austin, 2000). The findings of this study of Mediterranean and At-
lantic Tylodina coupled with the fact that this group occurs in trop-
ical and temperate waters characterized by high molluscan diver-
sity (Valentine & Jablonski, 2015) suggest that Umbraculida contain
much hidden diversity.
Supplementary material is available at Journal of Molluscan Studies
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Assistance from the CNS and the Harvard Center for Biological
Imaging made this study possible. We are grateful to M.J. Uriz
[Centre d’Estudis Avançats de Blanes (CEAB), Catalonia] for help
with sponge identification. Lab and analytical work was supported
by funding to G.G. from the Faculty of Arts and Sciences, Harvard
University. J.M. was supported by a Fundación Ramón Areces
postdoctoral fellowship. R.F.-V. was supported by an Early Career
Research Grant from the Malacological Society of London and
GROC. This is the study no. 6 of GROC. Associate Editor Manuel
Malaquias and an anonymous reviewer provided comments that
helped improve this paper.
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... In the dynamically changing Mediterranean system, we use community or citizen science platforms to track down rare or new species (e.g. Trainito et al. 2017;Fernández-Vilert et al. 2021), distribution patterns (see GROC 2009GROC -2021 and the spread of alien species (e.g. Fernández-Vilert et al. 2018;Kleitou et al. 2019). ...
... These correspond to 23 new records for the Catalan coast (NE Spain; see Table 1). Our new data, together with the recently described Trinchesia morrowae (Korshunova et al. 2019) and Tylodina rafinesquii (Fernández-Vilert et al. 2021), increase the overall diversity of marine heterobranchs in this region by 10%, to a total of 230 species. We also provide a new species record from the southern Spanish Mediterranean coast for the sacoglossan Elysia flava. ...
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Citizen (or community) science has provided copious and valuable information about charismatic marine taxa such as heterobranch gastropods, thus contributing enormously to the known geographic distribution of many sea slug species. This study reports new records of elusive sea slugs in the coastal western Mediterranean (especially on the Catalan and French Mediterranean coasts) and contributes to new ecological information regarding their phenology, diet and behaviour. Out of 39 species reported here, 23 are new records for the Catalan coast (NE Spain), three are new records of pelagic pteropods for the Spanish Iberian coast, and eight are new records for the French Mediterranean coast. With 25 species found active at night, this study highlights the importance of sampling at night and in shallow, often under-sampled waters with high species diversity. Shallow waters usually have less diving activity and are harder to survey with heavy scuba equipment. We believe that the high-quality photos herein and the related species information will enable researchers, divers and the community to find and recognise these rare species in the Mediterranean basin.
... Integrative taxonomy applied to marine Heterobranchia is proving to be a fruitful field of study. In fact, in the last few years, many papers have been published, which included information derived from several biological points of view, including morphological, molecular, chemical, ecological and behavioural characters [1][2][3][4][5][6][7]. Regarding the molecular approach, there is a continuous search in the genome for new informative DNA coding and non-coding regions, consequentially resulting in molecules such as proteins and RNA [7][8][9]. ...
... This approach has been applied to marine Heterobranchia (Mollusca, Gastropoda) whose evolutionary history, when studied at family level, is reconstructed using mainly three molecular markers, the two mitochondrial genes, part of Cytochrome oxidase subunit I (COI) and part of the ribosomal subunit 16S (16S), as well as the nuclear gene histone 3 (H3) which is well known to be poorly or not quite informative at lower taxonomic levels [7,17,18]. By means of these markers, the systematics of several heterobranchs families has been clarified and erroneous outcomes derived from previous morphological studies have been resolved and corrected [2,3,5]. ...
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Integrative taxonomy is an evolving field of multidisciplinary studies often utilised to elucidate phylogenetic reconstructions that were poorly understood in the past. The systematics of many taxa have been resolved by combining data from different research approaches, i.e., molecular, ecological, behavioural, morphological and chemical. Regarding molecular analysis, there is currently a search for new genetic markers that could be diagnostic at different taxonomic levels and that can be added to the canonical ones. In marine Heterobranchia, the most widely used mitochondrial markers, COI and 16S, are usually analysed by comparing the primary sequence. The 16S rRNA molecule can be folded into a 2D secondary structure that has been poorly exploited in the past study of heterobranchs, despite 2D molecular analyses being sources of possible diagnostic characters. Comparison of the results from the phylogenetic analyses of a concatenated (the nuclear H3 and the mitochondrial COI and 16S markers) dataset (including 30 species belonging to eight accepted genera) and from the 2D folding structure analyses of the 16S rRNA from the type species of the genera investigated demonstrated the diagnostic power of this RNA molecule to reveal the systematics of four genera belonging to the family Myrrhinidae (Gastropoda, Heterobranchia). The “molecular morphological” approach to the 16S rRNA revealed to be a powerful tool to delimit at both species and genus taxonomic levels and to be a useful way of recovering information that is usually lost in phylogenetic analyses. While the validity of the genera Godiva, Hermissenda and Phyllodesmium are confirmed, a new genus is necessary and introduced for Dondice banyulensis, Nemesis gen. nov. and the monospecific genus Nanuca is here synonymised with Dondice, with Nanuca sebastiani transferred into Dondice as Dondice sebastiani comb. nov.
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We present the latest version of the Molecular Evolutionary Genetics Analysis (MEGA) software, which contains many sophisticated methods and tools for phylogenomics and phylomedicine. In this major upgrade, MEGA has been optimized for use on 64-bit computing systems for analyzing bigger datasets. Researchers can now explore and analyze tens of thousands of sequences in MEGA. The new version also provides an advanced wizard for building timetrees and includes a new functionality to automatically predict gene duplication events in gene family trees. The 64-bit MEGA is made available in two interfaces: graphical and command line. The graphical user interface (GUI) is a native Microsoft Windows application that can also be used on Mac OSX. The command line MEGA is available as native applications for Windows, Linux, and Mac OSX. They are intended for use in high-throughput and scripted analysis. Both versions are available from free of charge.
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In the Mediterranean Sea, the two sponges of the genus Aplysina (A. aerophoba and A. cavernicola) are identified on the basis of their external morphology and the environment in which they live. During a research program on the sponge fauna in semi-submerged caves of the Italian coasts, we have sampled an abundant very small yellow sponge, often living in the tidal zone, which were attributed to the genus Aplysina. Failing to assign the samples to a species through classical taxonomic methodologies (growth form and skeleton arrangement) and for the particular environment where this sponge lives, we have decided to use the COI analysis to solve the taxonomic problem offered by these miniaturized specimens. The analysis indicated that, in spite of the morphological differences, they belong to A. aerophoba. During old detailed surveys, conducted in the ’60 years in some of the studied caves, this species was not recorded. It is possible that its abundant presence is related to the modifications occurred in the Mediterranean sponge communities occurred in the last decades in relation to global warming.
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‘Cryptic’ species are an emerging biological problem that is broadly discussed in the present study. Recently, a cryptic species definition was suggested for those species which manifest low morphological, but considerable genetic, disparity. As a case study we present unique material from a charismatic group of nudibranch molluscs of the genus Trinchesia from European waters to reveal three new species and demonstrate that they show a dual nature: on one hand, they can be considered a ‘cryptic’ species complex due to their overall similarity, but on the other hand, stable morphological differences as well as molecular differences are demonstrated for every species in that complex. Thus, this species complex can equally be named ‘cryptic’, ‘pseudocryptic’ or ‘non-cryptic’. We also present evidence for an extremely rapid speciation rate in this species complex and link the species problem with epigenetics. Available metazoan-wide data, which are broadly discussed in the present study, show the unsuitability of a ‘cryptic’ species concept because the degree of crypticity represents a continuum when a finer multilevel morphological and molecular scale is applied to uncover more narrowly defined species making the ‘cryptic’ addition to ‘species’ redundant. Morphological and molecular methods should be applied in concordance to form a fine-scale multilevel taxonomic framework, and not necessarily implying only an a posteriori transformation of exclusively molecular-based ‘cryptic’ species into morphologically-defined ‘pseudocryptic’ ones. Implications of the present study have importance for many fields, including conservation biology and fine-scale biodiversity assessments.
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Bayesian inference of phylogeny using Markov chain Monte Carlo (MCMC) (Drummond et al., 2002; Mau et al., 1999; Rannala and Yang, 1996) flourishes as a popular approach to uncover the evolutionary relationships among taxa, such as genes, genomes, individuals or species. MCMC approaches generate samples of model parameter values - including the phylogenetic tree -drawn from their posterior distribution given molecular sequence data and a selection of evolutionary models. Visualising, tabulating and marginalising these samples is critical for approximating the posterior quantities of interest that one reports as the outcome of a Bayesian phylogenetic analysis. To facilitate this task, we have developed the Tracer (version 1.7) software package to process MCMC trace files containing parameter samples and to interactively explore the high-dimensional posterior distribution. Tracer works automatically with sample output from BEAST (Drummond et al., 2012), BEAST2 (Bouckaert et al., 2014), LAMARC (Kuhner, 2006), Migrate (Beerli, 2006), MrBayes (Ronquist et al., 2012), RevBayes (Höhna et al., 2016) and possibly other MCMC programs from other domains.
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The standard bootstrap (SBS), despite being computationally intensive, is widely used in maximum likelihood phylogenetic analyses. We recently proposed the ultrafast bootstrap approximation (UFBoot) to reduce computing time while achieving more unbiased branch supports than SBS under mild model violations. UFBoot has been steadily adopted as an efficient alternative to SBS and other bootstrap approaches. Here, we present UFBoot2, which substantially accelerates UFBoot and reduces the risk of overestimating branch supports due to polytomies or severe model violations. Additionally, UFBoot2 provides suitable bootstrap resampling strategies for phylogenomic data. UFBoot2 is 778 times (median) faster than SBS and 8.4 times (median) faster than RAxML rapid bootstrap on tested datasets. UFBoot2 is implemented in the IQ-TREE software package version 1.6 and freely available at
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Update of the catalog of opisthobranch mollusks (Gastropoda: Heterobranchia) from the Catalan Coasts.—An extension of the checklist of opisthobranch species (Gastropoda: Heterobranchia) known from the Catalan Coasts is presented, based on numerous unpublished findings by the authors and reports confirmed with pictures posted in several Internet platforms. A total of 53 species are added to the previous catalog: 4 Cephalaspidea, 9 Runcinacea, 3 Anaspidea, 8 Sacoglossa and 29 Nudibranchia (11 Doridacea, 10 Aeolidacea, 2 Dendronotacea and 6 Cladobranchia incertae sedis). Data for the different reports of each of these species are provided, along with some biological, distribution or taxonomical remarks of interest. Finally, an updated and taxonomically sorted list of all opisthobranch species known for Catalonia is provided, including a total of 257 species, of which 9 are basal Heterobranchia, 35 Cephalaspidea, 13 Runcinacea, 10 Anaspidea, 22 Sacoglossa, 8 Pleurobranchomorpha, 2 Umbraculida, 3 Gymnosomata, 11 Thecosomata and 144 Nudibranchia (66 Doridacea, 51 Aeolidida, 12 Dendronotida, 12 Cladobranchia incertae sedis and 3 Euarminida). With all the new data reported in this paper, and regarding opisthobranch mollusks, the Catalan Coast becomes the biologically most diverse geographical region in the Iberian Peninsula.
A compendium of the northeast Pacific benthic shelled sea slugs formerly classified in the paraphyletic group "Opisthobranchia" is provided. These include organisms with internal and/or reduced shells. Shell-less groups such as Nudibranchia or closely related benthic shelled clades such as the Pyramidelloidea and the Siphonarioidea are excluded. The Sacoglossa is not represented by any shelled forms in the northeast Pacific and therefore is also excluded. Descriptions include diagnostic characteristics, species abundance information, geographic and bathymetric ranges, and ecological data (if available). Short remarks for most species and higher taxa provide additional information published elsewhere and/or address outstanding taxonomic or nomenclatural issues. Illustrations of the shells and-in some cases-the live animals are provided. Species are arranged based on current classification schemes and a full list of primary synonyms, location of type material (if known), and type localities of all synonyms are provided for each species. Three new species are described in this paper: Microglyphis michelleae new species, Microglyphis sabrinae new species and Bogasonia jennyae new species. Oscaniella purpurea Bergh, 1897 is here designated the type species of Oscaniella Bergh, 1897.
Model-based molecular phylogenetics plays an important role in comparisons of genomic data, and model selection is a key step in all such analyses. We present ModelFinder, a fast model-selection method that greatly improves the accuracy of phylogenetic estimates by incorporating a model of rate heterogeneity across sites not previously considered in this context and by allowing concurrent searches of model space and tree space.
Os Umbraculacea do Atlântico Ocidental são Umbracuturn plioatulum v. Martens, 1881, e Tylodina americana Dall, 1890. Recebi cinco exemplares do Umbraculum, dois da Flórida, um de Cayenne, e dois do Brasil, como também alguns exemplares de Umbraculum mediterraneum para comparação. De Tylodina americana tive três exemplares mais duas conchas.