ArticlePDF Available

A new species from the Mediterranean Sea and North-Eastern Atlantic Ocean: Knoutsodonta pictoni n. sp. (Gastropoda Heterobranchia Nudibranchia)

Authors:

Abstract and Figures

Knoutsodonta pictoni n. sp. (Gastropoda Heterobranchia Nudibranchia) is described here based on morphological and molecular analyses of specimens from Mediterranean Sea and North Atlantic Ocean. Ecological notes on the egg development and new species distribution range are also presented. COI DNA barcoding was used to molecularly identify this species and to assess one sequence present in Genbank but for which identification was not provided. Furthermore, the intraspecific genetic divergence was explored for specimens belonging to different populations.
No caption available
… 
Content may be subject to copyright.
Biodiversity Journal, 2017, 8 (2): 725–738
A new species from the Mediterranean Sea and North-
Eastern Atlantic Ocean: Knoutsodonta pictoni n. sp. (Gastro-
poda Heterobranchia Nudibranchia)
Giulia Furfaro1,* & Egidio Trainito2
1Department of Science, University of “Roma Tre”, Viale G. Marconi 446, I-00146 Rome, Italy; email: giulia.furfaro@uniroma3.it
2Villaggio I Fari, Loiri Porto San Paolo, I-07020 Olbia-Tempio, Italy.
*Corresponding author
ABSTRACT
Received 19.04.2017; accepted 23.05.2017; printed 30.06.2017
Knoutsodonta pictoni n. sp. (Gastropoda Heterobranchia Nudibranchia) is described here based
on morphological and molecular analyses of specimens from Mediterranean Sea and North
Atlantic Ocean. Ecological notes on the egg development and new species distribution range
are also presented. COI DNA barcoding was used to molecularly identify this species and
to assess one sequence present in Genbank but for which identification was not provided.
Furthermore, the intraspecific genetic divergence was explored for specimens belonging to
different populations.
IN TR ODUCTIO N
At the beginning of 2015 the genera placed
within the family Onchidorididae (Gastropoda Het-
erobranchia Nudibranchia) were: Acanthodoris Gray,
1850, Adalaria Bergh, 1878, Calycidoris Abraham,
1876, Corambe Bergh, 1869, Diaphorodoris Iredale
et O’Donoghue, 1923, Onchidoris Blainville, 1816
and Onchimira Martynov, Korshunova, Sanamyan
et Sanamyan, 2009. Later on, Hallas & Gosliner
(2015) based on the results of molecular and
morphological analyses reestablished the families
Corambidae, with the genus Corambe, and Ca-
lycidorididae including the genera Calycidoris and
Diaphorodoris. The remaining genera were main-
tained as genus as in the case of the Acanthodoris,
or divided into two different genera as happened for
Onchidoris and Adalaria. In particular, the species
of Onchidoris and Adalaria with a rachidian tooth
were grouped into the genus Onchidoris whereas
species with no rachidian tooth were placed in
Knoutsodonta Hallas et Gosliner, 2015. In 2015, al-
most concurrently, specimens of the Onchidoris-
like group were photographed and collected in
Sardinia (Italy), in Catalunya (Spain) and in Ireland.
Some photographed individuals, displayed in social
media, showed a strong similarity with specimens
from Mediterranean Sea and North-Eastern Atlantic
Ocean.
In Trainito & Doneddu (2015) the Sardinian find-
ings were reported as Onchidoris sp. and in the dis-
cussion the possibility was expressed that they
should be assigned to a new species, considering
that their external morphology did not match any of
the described species of the family Onchidorididae.
One specimen was found at East Wall Loch Nevis,
Scotland on 22nd August 2015 and afterwards pub-
lished by J. Anderson on the web as Knoutsodonta
KEY WORDS
DNA-Barcoding; new species; Nudibranchs; Onchidorididae; Knoutsodonta.
GIULIA FURFARO & EGIDIOTRAINITO
Trainito & Doneddu (2015). Furthermore, other
specimens with the same phenotype have been re-
ported as Onchidoris pusilla from Ensenada de los
Berengueles (Granada, Spain), Estartit, Blanes
and Tossa de Mar (Catalunya, Spain) (GROC,
http://www.opistobranquis.org/en/guia/10).
In this paper we describe this species as new to
science, through morphological and molecular ana-
lyses on individuals collected from Central Tyrrhe-
nian (North-Eastern Sardinia) and Adriatic (Trieste)
Seas and from the North-eastern Atlantic Ocean
(Ireland), discussing their taxonomic position with
a focus on other Mediterranean species of the
genus.
MATERIAL AND METHODS
Specimens of the new species were collected by
scuba diving from different localities (Table 1). In-
dividuals, egg spawns and the bryozoans on which
726
sp. A. (http://www.nudibranch.org/Scottish%20Nud-
ibranchs/html/knoutsodonta-spA-01.html).
A similar specimen was photographed in 2011
by G. Brown at Loch Sween, Scotland and pub-
lished by M. Faasse on the Facebook group page of
NE Atlantic Nudibranchs. Subsequently, in Balles-
teros et al. (2016), one photograph of an individual
was reported showing the same external features of
the samples found in 2015. In the discussion, it was
described as Knoutsodonta sp. A, based on a pre-
liminary analysis of the radula that lacked the
rachidian tooth, and the dataset of observations was
increased, dating back to 1992. In 2016 other indi-
viduals with the same external morphology were
photographed and collected in Northern Adriatic
Sea (Sistiana, Trieste, Italy). One individual with
the characteristic egg coils, was found at Cape Noli
(Liguria, Italy) and preliminarily identified as
Onchidoris pusilla (Alder et Hancock, 1845) (Betti
et al., 2017), but the external morphology revealed
this to be more similar to the specimens reported in
Table 1. List of the species names, vouchers, collection localities, COI GenBank accession numbers
and references of the species of Knoutsodonta and Onchidoris genera and the out-group.
Figures 1–4. In situ photographs of the living specimensof Knoutsodonta pictoni n. sp. Fig. 1: holotype (MNHN-IM-2000-
33333) (red arrow) and paratype (MNHN-IM-2000-33333) (light blue arrow) and the eggs (on the right side). Fig. 2: lateral
view of the K. pictoni n. sp. Sardinian specimen (MNHN-IM-2000-33333). Fig. 3: three adults specimens from Ireland.
Fig. 4: the encrusting bryozoan Reptadeonella violacea on which the new nudibranch species feed on. Figures 5–8. Holotype
of the Knoutsodonta pictoni n. sp. (MNHN-IM-2000-33333). Fig. 5: dorsal view of the living adult. Fig. 6: ventral view.
Fig. 7: a particular of the shape of the rhinophores. Fig. 8: a particular of the gills. Scale bar = 1 mm.
A new species from the Mediterranean Sea and N-E Atlantic Ocean: Knoutsodonta pictoni n. sp. (Nudibranchia) 727
Automatic Barcode Gap Discovery (ABGD, avail-
able at http://wwwabi.snv.jussieu.fr/public/abgd/),
a distance-based method designed to detect the so
called “barcode gap” in the distribution of pairwise
distances calculated in a sequence alignment (Puil-
landre et al., 2012a, b) to test species delimitation
in the newly produced COI dataset and produce
primary species hypotheses based on DNA dis-
tances. Alignment of the COI sequences was sub-
mitted and processed in ABGD using the Kimura
two parameters (K2p) nucleotide substitution model
and the following settings: a prior for the maximum
value of intraspecific divergence between 0.001 and
0.1, 30 recursive steps within the primary partitions
defined by the first estimated gap, and a gap width
of 0.1. The uncorrected pairwise genetic distances
(p-distance) among COI sequences of the putative
new species were also calculated with MEGA 6.0,
and the maximum intraspecific distance estimated.
Phylogenetic analyses on the COI dataset, were
then performed basing on Bayesian Inference (BI)
and Maximum Likelihood (ML) methods to test
whether the primary species hypotheses of ABGD
proved also monophyletic. In particular, BI was per-
formed using MrBayes 3.2.6 (Ronquist et al., 2012)
with four Markov-chains of five million generations
each, sampled every 1000 generations. Consensus
trees were calculated on trees sampled after a
burnin of 25%. ML searches were performed using
MEGA 6.0 with a starting tree topology generated
by a Neighbour Joining algorithm (Zwickl, 2006).
Nodal support was assessed by means of 1000
bootstrap replicates. The model of evolution was
selected in JModel Test 0.1 (Posada, 2008) accord-
ing to the Bayesian Information Criterion (BIC).
Diaphorodoris luteocincta (Voucher BAU2754)
was used as out-group since it shows a basal place-
ment within Onchidoridoidea according to Hallas
& Gosliner (2015).
RESULTS AND DISCUSSION
Ten individuals were observed in situ (Figs. 1–
4) and, within these, six were collected, from dif-
ferent localities (Table 1), and studied in an
aquarium (Figs. 5–8) before anatomical analysis.
The egg development was documented until the
veliger stage (Figs. 9, 10).
they were feeding were documented in situ with
high definition photographs. Sardinian specimens
were kept in an aquarium where both egg depos-
ition and development were documented up to the
veliger stage. The holotype and the paratype of the
new species were preserved in Ethanol 95% and de-
posited at the Muséum national d'histoire naturelle
(MNHN). All other collected individuals were stored
in the malacological collection at the Department
of Biology and Biotechnologies “Charles Darwin”
(“Sapienza” University of Rome, Italy) (Table 1).
Anatomy of the reproductive system was stud-
ied under a dissecting microscope from at least two
individuals. The buccal mass was placed in a 10%
NaOH solution to isolate the radula, which was de-
hydrated to 100% ethanol, critical point-dried, gold
coated, and examined by a Dualbeam SEM. The re-
productive system was observed under a dissecting
optical microscope and photographed at different
stages of dissection.
Morphological analyses of the radula structure
were performed using both SEM and optical micro-
scope techniques.
Molecular identity was tested by using a partial
sequence of the molecular marker mostly used for
DNA barcoding of nudibranchs, the mitochondrial
cytochrome c oxidase subunit I (COI) (see Table 1
for full list of samples, localities, and voucher refer-
ences). A piece of tissue was cut from the foot for
DNA extraction. Total genomic DNA was extracted
using a standard proteinase K phenol/chloroform
method with ethanol precipitation, as reported in
Oliverio & Mariottini (2001). Partial sequences of
COI were amplified by polymerase chain reaction
(PCR) using the primers LCO1490(5’-GGTCAA-
CAAATCATAAAGATATTGG-3’) and HCO2198
(5’-TAAACTTCAGGGTGACCAAAAATCA-3’)
(Folmer et al., 1994) (PCR profile: 5 min denatura-
tion step at 94°C; 35 cycles of 94°C/30 s, 48°C/60
s, 72°C/60 s; 7 min final extension at 72°C). Amp-
licons were sequenced by European Division of
Macrogen Inc. (Amsterdam, The Nederland), using
the same PCR primers. Sequences from each DNA
strain were assembled and edited with Staden
Package 2.0.0b9 (Staden et al., 2000). BLASTN
(Altschul et al., 1990) search was conducted to ex-
clude contamination. Sequences obtained were
aligned together with those already present in Gen-
Bank using Muscle algorithm implemented in
MEGA 6.0 (Tamura et al., 2013). We employed the
GIULIA FURFARO & EGIDIOTRAINITO
728
Diagnostic anatomical features were compiled
from at least two specimens (Figs. 11, 12; Figs.
13–18).
DNA-barcoding. The COI sequences were de-
posited at the European Nucleotide Archive
(http://www.ebi.ac.uk/ena) and the information on
voucher, accession numbers and collection local-
ities are listed in Table 1. The BLAST search found
a high similarity (99%) between the new species se-
quences and one sequence present in GenBank
(Accession number: KP340411, voucher: CASIZ
208194) identified as Knoutsodonta sp. A. The pair-
wise genetic distances calculated on the COI se-
quences revealed 2.0% of maximum intraspecific
divergence of the putative new species (Table 2). A
final COI dataset, excluding the out-group, con-
sisted of 29 sequences from ten different species
belonging to Knoutsodonta and Onchidoris. The
COI final alignment consisted of 609 nucleotide
positions with 198 polymorphic sites.
Results from the ABGD analysis returned 11
Preliminary Species Hypothesis (PSH) with O. bil-
amellata sequences split in two different PSH. All
the recursive steps in the ABGD analysis resulted
in the same sequence repartition, with all the se-
quences of the putative new species grouped in a
single PSH (Fig. 19) including Knoutsodonta sp. A
(KP340411).
The phylogenetic analyses resulted in mono-
phyletic clades (Fig. 20) that were congruent with
the PSH obtained with the ABGD analysis. The
phylogenetic inference confirmed that all the speci-
mens belonging to the putative new species grouped
in one monophyletic clade together with the Gen-
Bank sequence of Knoutsodonta sp. A (KP340411)
with high support values (BI=1, ML=100). The
729
Figures 9–12. Knoutsodonta pictoni n. sp. Fig. 9: in aquarium eggs deposition. Fig. 10: eggs at different developmental
stages. One veliger is indicated with a red circle. Figs. 11, 12: reproductive system: bc = bursa copulatrix, ro = reproductive
opening, dd = deferent duct, am = ampulla, fg = female gland, rs = receptaculum seminis. Scale bar = 1 mm.
A new species from the Mediterranean Sea and N-E Atlantic Ocean: Knoutsodonta pictoni n. sp. (Nudibranchia)
COI resulting topology showed also that the se-
quences ascribed to O. bilamellata split in two di-
vergent clades congruently with the ABGD species
delimitation results. In particular, the specimen of
O. bilamellata with COI accession number
KR084801, was the sister to the new species. with
high statistical support (BI=1, ML=99).
TAXONO MY
Familia ONCHIDORIDIDAE Gray, 1827
Genus Knoutsodonta Hallas et Gosliner, 2015
Knoutsodonta pictoni n. sp. (Figs. 1–8)
TYPE MATERIAL. Holotype. Voucher MNHN-IM-
2000-33333, 11 mm in length alive, Tavolara-Punta
Coda Cavallo Marine Protected Area (MPA), Porto
San Paolo, North Eastern Sardinia, Central Tyrrhe-
nian Sea, Mediterranean Sea, Italy, (40.881635°N,
9.637065°E), 31.III.2015 (Figs. 1, 2, 5–8).
Paratype. Voucher MNHN-IM-2000-33334, one
specimen, dissected, 10 mm in length alive, same
data as the holotype (Fig. 1).
OTHER EXAMINED MATERIAL. BAU02982, one
specimen, dissected, 8 mm in length alive, South of
Inishgalloon, Purteen, Keel, Achill Island, Ireland,
Atlantic Ocean, (53.9556°N, 10.1023°W), 05
April 2015 (Fig. 3). BAU02985, one specimen, 9
mm in length alive, 6 m depth, Sistiana, Trieste,
North Adriatic Sea, Mediterranean Sea, Italy,
(45.7728726°N,13.6292581°E), 31 December
2016. BAU02983, one specimen, 6 mm in length
alive, South of Inishgalloon, Purteen, Keel, Achill
Island, Ireland, Atlantic Ocean, (53.9556°N,
10.1023°W), 05 April 2015 (Fig. 3). BAU02984,
one specimen, 6 mm in length alive, South of
Inishgalloon, Purteen, Keel, Achill Island, Ireland,
Atlantic Ocean, (53.9556°N, 10.1023°W), 05 April
2015 (Fig. 3).
DESCRIPTION OF HOLOTYPE. External morpho-
logy: body elliptical, depressed, equally rounded
both in anterior and posterior edges. Body colour
uniform dark brown, almost black with small bluish
white speckles scattered along the mantle (Figs. 1,
5). Body colour of ventral part light grey with dif-
fuse spots dark brown as mantle (Figs. 5, 6). Long
and slender rhinophores, totally white, retractable,
lamellate (10 lamellae) (Fig. 7). Foot not projected
beyond notum: anterior margin indented forming
two large lobes, posterior part forms a rounded tail
(Fig. 6). Mantle structure very hard, with strong re-
ticulation formed by translucent spicules. Almost
globular tubercles, evenly spaced, covering mantle
and surrounding anus opening and rhinophores
sheaths. Mouth surrounded by large oral veil with
just outlined lateral lobes (Fig. 6). Non-retractile
gills with 9 bipinnate branchial leaves, larger an-
teriorly than posteriorly, forming an almost complete
semicircle around the anus: one tubercle may be
present just behind the anus (Fig. 8). Several narrow
tubercles of variable height inside the gill circlet.
VARIABILITY
. Body length ranging from 9 mm to
12 mm (10 specimens examined). Rhinophores
with 9–11 lamellae. Gills with 9 or 10 bipinnate
branchial leaves.
Paratype internal anatomy: Rachidian tooth ab-
sent. Radular formula 25–28 x 1.1.0.1.1. Radular
teeth almost colourless (Figs. 13, 14). First lateral
teeth with long, wide base and strong, almost
straight beak-shaped cusp, denticulate with 10–13
small denticles along the internal surface (Figs. 15–
18). Second lateral teeth in shape of rectangular
plates, with downward directed cusp on lower out-
side corner (Figs. 15, 16). Swollen tube of ampulla
connected through a short duct to bursa copulatrix
and seminal male duct. Bursa copulatrix leads to
distal part of female duct, in connection with sem-
inal receptaculum ending into vagina and female
opening. Relatively long loop of prostatic part of
vas deferens adjacent to bursa copulatrix (Figs. 11,
12). Prostate smooth, not granulated, first narrow,
then rapidly wide into a long swollen penial sheath
with several folds of ejaculatory duct. (Figs. 11, 12).
Bean-shaped bursa copulatrix, slightly yellowish
(Figs. 11, 12), enters distal part of vagina through a
relatively long stalk. At its base, a duct leads to
ovoid seminal receptacle (Figs. 11, 12). Vagina
wide and long (Figs. 11, 12).
ETYMOLOGY. The new species is named after
Bernard Picton ((National Museums Northern Ire-
land, 153 Bangor Road, Cultra, Holywood, BT18
0PE, UK)) who kindly presented us specimens from
Ireland and whose prominent work on North At-
lantic nudibranchs is a mandatory reference for re-
searchers and nudibranch enthusiasts all over the
world.
730 GIULIA FURFARO & EGIDIOTRAINITO
Figures 13–18. Radula of the Knoutsodonta pictoni n. sp. Figs. 13, 15, 17: Optical microscope images.
Figs. 14, 16, 18: SEM images at different magnification levels.
A new species from the Mediterranean Sea and N-E Atlantic Ocean: Knoutsodonta pictoni n. sp. (Nudibranchia) 731
Figures 19, 20. DNA Barcoding analyses. Fig. 19: the ABGD histogram of the COI barcoding region shows the distribution
of the pairwise estimated genetic distances (K2p) in intraspecific (left, light grey) and interspecific (right, dark grey) com-
parisons. Fig. 20: Bayesian resulting tree of the COI dataset. Numbers at nodes are Bayesian posterior probability and ML
bootstrap support, respectively.
GIULIA FURFARO & EGIDIOTRAINITO
732
Figure 21. Distribution of Knoutsodonta pictoni n. sp. The black numbers correspond to the sites indicated in Table 3. Spe-
cimens examined in the present paper, red star; Atlantic specimen identified as Knoutsodonta sp. A (KP340411), green loz-
enge; Atlantic specimen identified as Onchidoris sp., yellow lozenge; Mediterranean specimens identified as Onchidoris
pusilla, orange triangle; Mediterranean specimens identified as Knoutsodonta sp. A, blue circle. Figure 22. Distribution of
the species belonging to the family Onchidorididae. The numbers correspond to the number of species with the same distri-
bution, the size of the circles is correlated to the number of species.
A new species from the Mediterranean Sea and N-E Atlantic Ocean: Knoutsodonta pictoni n. sp. (Nudibranchia) 733
734
Table 3. List of the map sites, localities and reference sources of the records of Knoutsodonta pictoni n. sp.
Mediterranean Sea, green lines; North Atlantic Ocean, blue lines.
Table 2. Pairwise distances (p-distance) between specimens belonging to Knoutsodonta pictoni n. sp.
GIULIA FURFARO & EGIDIOTRAINITO
735
Table 4. Comparison between external morphology, radular formula and distribution of Knoutsodonta pictoni n. sp.
with those of the other 14 Knoutsodonta species (Mediterranean species in grey lines).
A new species from the Mediterranean Sea and N-E Atlantic Ocean: Knoutsodonta pictoni n. sp. (Nudibranchia)
DISTRIBUTION. The species is distributed in West-
ern and Central Mediterranean and in North East
Atlantic Ocean. In addition to the localities listed in
Table 1 this species has been also recorded under
the name of Knoutsodonta sp. in different localities
along the coast of Catalunya (Ballesteros et al.,
2016); it is also recorded in the coast of Granada,
in Estartit, in Tossa de Mar and Blanes (Spain) and
in Cape Noli (Italy) as Onchidoris pusilla (Alder et
Hancock, 1845) (GROC, www.opistobranquis.org,
and Betti et al., 2017). The species distribution is
summarized in figure 21 and Table 3.
REMARKS. At present, there are 15 Onchidor-
ididae species that can be ascribed to the genus
Knoutsodonta, based on their radular formula (Hal-
las & Gosliner, 2015) and among them, five have a
Mediterranean distribution (Table 4).
The main morphological features that diagnose
K. pictoni n. sp. are the dark brown background
body colour, the white rhinophores and the dark
gills. None of the known Onchidorididae displays
these three combined external morphological char-
acters (Table 4). The confusion that has occurred
with O. pusilla can be easily resolved mainly be-
cause this species has translucent white gills and
secondly for the body colour whose apparent dark-
ness is due to the presence, on the mantle, of dense
dark brown pigmented spots. A comparison of the
main morphological features between correlated spe-
cies of the genus Knoutsodonta is reported in Table 4.
All the specimens collected were found feeding
and spawning on the encrusting bryozoan Rept-
adeonella violacea (Johnston, 1847) (Gymno-
laemata: Chelostomatida: Adeonidae) (Fig. 4), a
species of warm temperate waters, distributed in the
Mediterranean, and from the North East Atlantic to
West Africa waters. The species is also present in
West Atlantic (North America and Gulf of Mexico)
and along the Pacific Coast of North America (Hay-
ward & McKinney, 2002). When the nudibranchs
are upon their bryozoan prey they are very cryptic,
and in fact they can be detected only for the pres-
ence of a discoloured area of the bryozoan where
they have fed (or are feeding) or for the presence of
the egg coils. The egg spawn of K. pictoni n. sp. is
very distinctive among those of the Onchidorididae:
it is a flat mucous ribbon forming an almost perfect
Archimedean spiral containing egg capsules (Figs.
9, 10). Sometimes two different individuals spawn
in the same place and it may happen that part of the
last laid ribbon surrounds the other one (Fig. 1). In
aquarium, at 18 °C, one specimen was observed
spawning on 6th of April 2015 at 7.00 PM (Fig. 9).
On 8th of April at 10.00 AM the coil was full of
9248 egg capsules, while 3 veligers were swimming
nearby (Fig. 10). At 8.30 PM of the same day there
was only a slight trace of the coil and a large num-
ber of veligers was swimming in the entire mass of
water.
DISCUSSION
A recent phylogenetic reassessment of the fam-
ily Onchidorididae by Hallas & Gosliner (2015)
proposed some systematic changes regarding the
genera Adalaria, Onchidoris and Knoutsodonta.
According to this revision, we described a new spe-
cies of this group through an integrative approach.
Morphological evidences revealed the absence of
the rachidian tooth in the new species, positioning
it in the genus Knoutsodonta, while all the mo-
lecular analyses performed confirmed the assign-
ment of the Mediterranean and Atlantic specimens
to the new species K. pictoni n. sp. Additionally,
the COI DNA barcoding allowed to identify one
sequence present in GenBank (COI accession num-
ber KP340411) and corresponding to the new spe-
cies here described. Interestingly, phylogenetic
analyses here proposed (although based on the
single COI marker), revealed K. pictoni n. sp. as
sister to (yet not conspecific with) a specimen (COI
accession number KR084801) previously ascribed
to O. bilamellata that needs further analyses. The
study by Hallas & Gosliner (2015) could not
provide a complete definition of the genus Knouts-
odonta and, furthermore, it included only two out
of the five Mediterranean species of this genus.
The species of Onchidorididae are mainly distrib-
uted in the northern hemisphere, and, as depicted
in figure 7, for the total of 54 accepted species the
highest diversity area is the North Atlantic with the
presence of 25 species from the NW Atlantic to the
Mediterranean Sea. Twelve species are distributed
in the NE Pacific, while eight are in the NW Pa-
cific. Four species are distributed in the SW Pa-
cific, one in Central W Pacific, three in SW
Atlantic and one in SE Atlantic-S Indian Ocean.
736 GIULIA FURFARO & EGIDIOTRAINITO
With the description of K. pictoni n. sp., the
Onchidorididae of the Mediterranean Sea raise to 8
species, two of which endemic, whose generic at-
tribution needs further investigation. For all these
reasons a further integrative study is desirable to
understand the systematic position of some critical
Onchidorididae taxa and to investigate on the valid-
ity of the genus Knoutsodonta.
ACKNOWLEDGMENTS
The authors gratefully thank Bernard Picton and
the late Barbara Camassa for the specimens respect-
ively from North Ireland and North Adriatic Sea.
We are in debt to Prof. Paolo Mariottini (Depart-
ment of Sciences, University Roma Tre, Rome) for
his critical suggestions that helped us to improve
the manuscript. The authors are also very grateful
to Prof. Andrea Di Giulio (Department of Sciences,
University Roma Tre, Rome) for the SEM photo-
graphs carried out at the Interdepartmental Labor-
atory of Electron Microscopy. Authors also wish to
thank Prof. Marco Oliverio who has reviewed the
Manuscript helping in improving it. GF wishes to
thank University of Roma Tre for financial support.
The authors wish to thank MPA Tavolara Punta
Coda Cavallo for the permission for collecting
samples.
REFERENCES
Altschul S.F., Gish W., Miller W., Myers E.W. & Lipman
D.J., 1990. Basic local alignment search tool. Journal
of Molecular Biology, 215: 403–410.
Anderson J., 1999–2017 accessed on 14/04/2017 in
ht tp://ww w.n udibr an ch.org/Sc ot tish%20 Nu d-
ibranchs/html/knoutsodonta-spA-01.html.
Ballesteros M., Madrenas E. & Pontes M., 2016. Actual-
ización del catálogo de los moluscos opistobranquios
(Gastropoda: Heterobranchia) de las costas catalanas.
Spira 6: 1–28.
Barco A., Raupach M.J., Laakmann S., Neumann H. &
Knebelsberger T., 2016. Identification of North Sea
molluscs with DNA barcoding. Molecular ecology
resources, 16: 288–297.
Bhave V., Salunkhe R.C., Shouche Y.S. & Apte D., un-
published. Onchidoris konkanensis sp. nov. from Rat-
nagiri, Maharashtra with first record of the Genus
Onchidoris from Arabian sea.
Betti F., Bava S. & Cattaneo Vietti R., 2017. Hetero-
branch assemblage composition and seasonality in a
Mediterranean sublittoral unconsolidated wave-
disturbed community. Journal of Molluscan Studies,
83: 325–332. https://doi.org/10.1093/mollus/eyx019.
Folmer O., Black M., Hoeh W., Lutz R. & Vrijenhoek R.,
1994. DNA primers for amplification of mitochon-
drial cytochrome c oxidase subunit I from diverse
metazoan invertebrates. Molecular Marine Biology
and Biotechnology, 3: 294–299.
Furfaro G., Picton B., Martynov A. & Mariottini P., 2016.
Diaphorodoris alba Portmann & Sandmeier, 1960 is
a valid species: molecular and morphological com-
parison with D. luteocincta (M. Sars, 1870) (Gastro-
poda: Nudibranchia). Zootaxa, 4193: 304–316.
Grande C., Templado J., Cervera J.L. & Zardoya R.,
2004. Phylogenetic relationships among Opistho-
branchia (Mollusca: Gastropoda) based on mito-
chondrial cox 1, trnV, and rrnL genes. Molecular
phylogenetics and evolution, 33: 378–388.
GROC, 2017. http://www.opistobranquis.org/en/guia/
100.
Hallas J.M. & Gosliner T.M., 2015. Family matters: the
first molecular phylogeny of the Onchidorididae
Gray, 1827 (Mollusca, Gastropoda, Nudibranchia).
Molecular phylogenetics and evolution, 88: 16–27.
Hayward P.J. & McKinney F.K., 2002. Northern Adriatic
Bryozoa form the vicinity of Rovinj, Croatia. Bulletin
of the American Museum of Natural History, 270,
139 pp., 63 figures, 1 table.
Layton K.K., Martel A.L. & Hebert P.D., 2014. Patterns
of DNA barcode variation in Canadian marine mol-
luscs. PLoS One, 9 (4): e95003.
Oliverio M. & Mariottini P., 2001. A molecular frame-
work for the phylogeny of Coralliophila and related
muricoids. Journal of Molluscan Studies, 67: 215–
224.
Posada D., 2008. jModelTest: Phylogenetic Model Aver-
aging. Molecular Biology and Evolution, 25: 1253–
1256.
Puillandre N., Lambert A., Brouillet S. & Achaz G.,
2012a. ABGD, Automatic Barcode Gap Discovery
for primary species delimitation. Molecular Ecology,
21: 1864–1877.
Puillandre N., Modica M.V., Zhang Y., Sirovich L., Bois-
selier M.C., Cruaud C., Holford M. & Samadi S.,
2012b. Large-scale species delimitation method for
hyperdiverse groups. Molecular Ecology, 21: 2671–
2691.
Ronquist F., Teslenko M., Van der Mark P., Ayres D.L.,
Darling A., Höhna S., Larget B., Liu L., Suchard
M.A. & Huelsenbeck J.P., 2012. MrBayes 3.2: Effi-
cient Bayesian Phylogenetic Inference and Model
Choice Across a Large Model Space. Systematic Bio-
logy, 61: 539–542.
Staden R., Beal K.F. & Bonfield J.K., 2000. The Staden
package, 1998. Methods in Molecular Biology, 132:
115–130.
737
A new species from the Mediterranean Sea and N-E Atlantic Ocean: Knoutsodonta pictoni n. sp. (Nudibranchia)
Tamura K., Stecher G., Peterson D., Filipski A. & Kumar
S., 2013. Mega6: Molecular Evolutionary Genetics
Analysis version 6.0. Molecular Biology and Evolu-
tion, 30: 2725–2729.
Thollesson M., 2000. Increasing fidelity in parsimony
analysis of dorid nudibranchs by differential weight-
ing, or a tale of two genes. Molecular Phylogenetics
and Evolution, 16: 161–172.
Trainito E. & Doneddu M., 2015. Contribution to the
knowledge of the molluscan fauna in the Marine Pro-
tected Area Tavolara-Punta Coda Cavallo: Ordo Nud-
ibranchia. Bollettino Malacologico, 51: 54–70.
Zwickl D.J., 2006. Genetic algorithm approaches for the
phylogenetic analysis of large biological sequence
datasets under the maximum likelihood criterion.
Ph.D. dissertation, The University of Texas at Austin.
738 GIULIA FURFARO & EGIDIOTRAINITO
... Mediterranean marine biodiversity is still underestimated and new cryptic species continue to be identified, described, and added to our inventory of marine fauna (Calvo et al., 2009;Uriz et al., 2017;González-Castellano et al., 2020;Furfaro et al., 2021). Our knowledge on diversity of groups such as nudibranchs is recently increasing as demonstrated by the number of studies published in the last years (Cella et al., 2016;Furfaro and Trainito, 2017;Korshunova et al., 2017Korshunova et al., , 2019Furfaro et al., 2018;Chimienti et al., 2020;Furfaro and Mariottini, 2020). In nudibranchs, few morphological characters are available, and the same chromatic pattern is often shared between closely related species, thereby making difficult the species identification based on morphology alone (Johnson and Gosliner, 2012;Furfaro et al., 2021). ...
... Our inventory of Mediterranean nudibranchs is rapidly growing as new species are continuously added, including cryptic species within historically accepted species (Furfaro and Trainito, 2017;Korshunova et al., 2019;Chimienti et al., 2020;Furfaro and Mariottini, 2020). Nowadays, the integrative approach combining morphological and molecular characters is commonly used in nudibranch taxonomy. ...
Article
Full-text available
Mediterranean marine biodiversity is still underestimated especially for groups such as nudibranchs. The identification of nudibranchs taxa is challenging because few morphological characters are available and among them chromatic patterns often do not align with species delimitation. Molecular assessments helped unveiling cryptic diversity within nudibranchs and have been mostly based on mitochondrial markers. Fast evolving nuclear markers are much needed to complement phylogenetic and systematic assessments at the species and genus levels. Here, we assess the utility of the nuclear Internal Transcribed Spacer 2 (ITS2) to delimit species in the eolid nudibranchs using both primary and secondary structures. Comparisons between the variation observed at the ITS2 and at the two commonly used mitochondrial markers (COI and 16S) on 14 eolid taxa from 10 genera demonstrate the ability of ITS2 to detect congeneric, closely related, species. While ITS2 has been fruitfully used in several other mollusc taxa, this study represents the first application of this nuclear marker in nudibranchs.
... The first images of M. gemmii in the Mediterranean Sea are dated back to 2009: this is a further evidence of the presence of unknown nudibranch diversity (i.e. cryptic, pseudo cryptic or at least neglected species) in this semi closed basin (Furfaro et al., 2016a & b;Furfaro & Trainito, 2017;Furfaro & Mariottini, 2020) and an enforcement of the need for further in-depth studies on Mediterranean Nudibranchia species. The future discovery of new specimens of M. gemmii and their collection will allow to compare the Mediterranean populations with the one from the type locality in order to investigate their genetic variability. ...
Article
Full-text available
Marionia gemmii Almón, Pérez & Caballer, 2018 has been described as a new species from six localities in Ría de Arousa, Galicia (North-Western Spain) and in the Golfe de Cádiz (Southern Spain), both within Atlantic waters. Old and new findings of this species from the North-Western Mediterranean radically expand its distribution and an in-depth study of the images available on the web dates its first documented record in this area back to 2009.
... In this context, Mollusca is one of the more represented group with about 2,200 accepted species (Coll et al. 2010), approximately 550 of which belonging to the Heterobranchia subclass (Trainito & Doneddu 2014). The study of Mediterranean Heterobranchia diversity is particularly interesting due to the continuous discovery of new and/or cryptic species, often endemic of this semi-closed marine basin (Furfaro & Trainito 2017;Martín-Hervás et al. 2019;Furfaro & Mariottini 2020). Within this group, the order Nudibranchia includes organisms with soft bodies and lacking shells, whose identification can be often difficult or not possible based on images or external anatomy. ...
Article
Full-text available
The Mediterranean Sea is a hot spot for marine biodiversity, and this is particularly evident taking into consideration the diversity observed in many animal groups, among them the Molluscs. In the last decade, several works have revealed a high rate of cryptic diversity characterizing the Molluscan fauna of the Mediterranean Sea and an increasing number of endemic and/or new species inhabiting this semi-enclosed basin have been recorded or described. The DNA-barcoding method is considered an essential step in the integrative taxonomy applications, to unravel cryptic diversity and for species identification. Here we report the case of DNA-barcoding technique applied to identify a nudibranch (Heterobranchia) collected from the Adriatic Sea, in the Bay of Kotor (Montenegro), for which a standard morphological identification was not possible. Mediterranean specimen belonging to Pruvotfolia pselliotes (Labbé, 1923) is for the first time molecularly identified and its COI DNA sequence compared with the one of an individual collected from the type locality. In addition, this is the first verified report of this species from the Adriatic Sea. Finally, the potential of using DNA-barcoding is here discussed, together with the habitat and the geographical distribution of this uncommon species.
... cons., a 0-6 m, bajo piedra en pradera de P. oceanica. Furfaro & Trainito (2017), a partir del estudio de varios ejemplares recolectados en Irlanda y en las costas italianas (Trieste e isla de Cerdeña), describieron como nueva esta especie, y la separaron claramente a nivel morfológico y molecular de Onchidoris pusilla y de otras especies de Onchidoris y Knoutsodonta. Con los registros conocidos hasta el momento, Knoutsodonta pictoni resulta tener una distribución atlanto-mediterránea. La presente constituye la primera cita de la especie después de su descripción original e implica su adición a la lista de las especies catalanas y de la Península Ibérica. ...
Article
Full-text available
The “opisthobranchs” from the Lluís Dantart collection, deposited at the Animal Biodiversity Resource Center (CRBA) of the Universitat de Barcelona, are studied. The studied samples belong to the orders Acteonacea, Ringiculida, Pleurobranchomorpha, Nudibranchia, Cephalaspidea, Anaspidea, Thecosomata, Umbraculida and Sacoglossa. For each lot, its taxonomic identification has been verified (correcting it when it was considered erroneous), and its nomenclature has been adapted to current knowledge. In total, 1,095 specimens have been studied, belonging to 68 different species and totaling 265 records. The specimens mostly belong to samples collected on the Catalan coasts, but also from different points in the Iberian Peninsula, the Balearic Islands, and the Bissagos Archipelago (off the coast of Guinea-Bissau, in West Africa). Of the 68 species, 2 belong to Acteonacea, 3 to Ringiculida, 17 to Nudibranchia, 3 to Pleurobranchomorpha, 1 to Umbraculida, 1 to Anaspidea, 31 to Cephalaspidea, 9 to Thecosomata and 1 to Sacoglossa. For each of the studied species, all the data of their records in the collection are provided: locality, date of collection, number of specimens, whether they are dry (shells) or preserved in alcohol, depth at which the specimen was found and, occasionally, some details of the substrate, as well as the sample collector. For some of the species, interesting taxonomic or biogeographic observations are added. Of all the studied species, Retusa robagliana and Atys brocchii had not been previously reported from the Catalan coasts; the latter and Ringicula buccinea were neither included in the most recent species catalogs of opisthobranchs or mollusks in general for the Iberian Peninsula. The nudibranch Knoutsodonta pictoni is reported for the first time after its original description and therefore represents the first record of the species for Catalan waters and for those of the Iberian Peninsula. The importance of natural history collections present in museums or research centers for the scientific knowledge of biodiversity and its formative aspect for the general public is highlighted.
Article
Full-text available
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.
Article
Full-text available
98 species of nudibranchs (Mollusca, Gastropoda) were recorded from the Marine Protected Area of Tavolara- Punta Coda Cavallo (North-eastern Sardinia) and its vicinities during the years 1989-2015. The most interesting species were annotated and shown in colour photos.
Article
Full-text available
Molluscs are the most diverse marine phylum and this high diversity has resulted in considerable taxonomic problems. Because the number of species in Canadian oceans remains uncertain, there is a need to incorporate molecular methods into species identifications. A 648 base pair segment of the cytochrome c oxidase subunit I gene has proven useful for the identification and discovery of species in many animal lineages. While the utility of DNA barcoding in molluscs has been demonstrated in other studies, this is the first effort to construct a DNA barcode registry for marine molluscs across such a large geographic area. This study examines patterns of DNA barcode variation in 227 species of Canadian marine molluscs. Intraspecific sequence divergences ranged from 0-26.4% and a barcode gap existed for most taxa. Eleven cases of relatively deep (>2%) intraspecific divergence were detected, suggesting the possible presence of overlooked species. Structural variation was detected in COI with indels found in 37 species, mostly bivalves. Some indels were present in divergent lineages, primarily in the region of the first external loop, suggesting certain areas are hotspots for change. Lastly, mean GC content varied substantially among orders (24.5%-46.5%), and showed a significant positive correlation with nearest neighbour distances. DNA barcoding is an effective tool for the identification of Canadian marine molluscs and for revealing possible cases of overlooked species. Some species with deep intraspecific divergence showed a biogeographic partition between lineages on the Atlantic, Arctic and Pacific coasts, suggesting the role of Pleistocene glaciations in the subdivision of their populations. Indels were prevalent in the barcode region of the COI gene in bivalves and gastropods. This study highlights the efficacy of DNA barcoding for providing insights into sequence variation across a broad taxonomic group on a large geographic scale.
Article
Full-text available
We announce the release of an advanced version of the Molecular Evolutionary Genetics Analysis (MEGA) software, which currently contains facilities for building sequence alignments, inferring phylogenetic histories, and conducting molecular evolutionary analysis. In version 6.0, MEGA now enables the inference of timetrees, as it implements our RelTime method for estimating divergence times for all branching points in a phylogeny. A new Timetree Wizard in MEGA6 facilitates this timetree inference by providing a graphical user interface (GUI) to specify the phylogeny and calibration constraints step-by-step. This version also contains enhanced algorithms to search for the optimal trees under evolutionary criteria and implements a more advanced memory management that can double the size of sequence data sets to which MEGA can be applied. Both GUI and command-line versions of MEGA6 can be downloaded from www.megasoftware.net free of charge.
Article
Full-text available
Since its introduction in 2001, MrBayes has grown in popularity as a software package for Bayesian phylogenetic inference using Markov chain Monte Carlo (MCMC) methods. With this note, we announce the release of version 3.2, a major upgrade to the latest official release presented in 2003. The new version provides convergence diagnostics and allows multiple analyses to be run in parallel with convergence progress monitored on the fly. The introduction of new proposals and automatic optimization of tuning parameters has improved convergence for many problems. The new version also sports significantly faster likelihood calculations through streaming single-instruction-multiple-data extensions (SSE) and support of the BEAGLE library, allowing likelihood calculations to be delegated to graphics processing units (GPUs) on compatible hardware. Speedup factors range from around 2 with SSE code to more than 50 with BEAGLE for codon problems. Checkpointing across all models allows long runs to be completed even when an analysis is prematurely terminated. New models include relaxed clocks, dating, model averaging across time-reversible substitution models, and support for hard, negative, and partial (backbone) tree constraints. Inference of species trees from gene trees is supported by full incorporation of the Bayesian estimation of species trees (BEST) algorithms. Marginal model likelihoods for Bayes factor tests can be estimated accurately across the entire model space using the stepping stone method. The new version provides more output options than previously, including samples of ancestral states, site rates, site d(N)/d(S) rations, branch rates, and node dates. A wide range of statistics on tree parameters can also be output for visualization in FigTree and compatible software.
Article
The nudibranch Diaphorodoris luteocincta (M. Sars, 1870) shows two colour morphotypes defined as D. luteocincta var. alba and D. luteocincta var. reticulata, which are easy to identify and which share an overlapping distribution in the Mediterranean Sea and the North-Eastern Atlantic Ocean. Their systematics has long been discussed by several authors until recently when a molecular study proposed the two varieties as intraspecific colour variability occurring within D. luteocincta species. In order to solve their ranking status, we have carried out a morphological study on anatomical characters and molecular analyses on the mitochondrial markers (COI and 16S rDNA) and the nuclear H3 gene. Results proved the usefulness of the integrative taxonomy approach in assessing species delimitation; in fact Diaphorodoris alba stat. nov. and D. luteocincta were revealed to be two different species. D. luteocincta var. reticulata is confirmed as synonym of D. luteocincta s.str. A hypothesis on phylogenetic relationship among most of the currently recognised species of the genus Diaphorodoris Iredale & O'Donoghue, 1923 is also here presented.
Article
One hundred six species of Bryozoa collected from the northern Adriatic in the vicinity of Rovinj, Croatia, are distributed among the orders Ctenostomata (8 species), Cheilostomata (79 species), and Cyclostomata (19 species). Ctenostomes are underrepresented in the collections relative to the two orders with calcified colonies. Five of the cheilostome species are new: Hagiosynodos hadros n. sp., Schizomavella subsolana n. sp., Cellepora adriatica n. sp., Celleporina siphuncula n. sp., and Rhynchozoon revelatus n. sp. (previously referred to as Rhynchozoon sp. II Hayward). Seven species named by Heller (1867) are stabilized by selection of lectotypes (Beania hirtissima, Adeonella pallasii, Hagiosynodos kirchenpaueri, Exidmonea triforis, Crisia recurva) and neotypes (Mollia circumcincta, Schizomavella cornuta) from Heller's collection in the University of Innsbruck Institute of Zoology. Lectotypes are designated for the Adriatic species Hippoporina lineolifera (Hincks, 1886) and for Schizomavella mamillata (Hincks, 1880). Beania cylindrica (Hincks, 1886) and Schizoporella asymetrica (Calvet, 1927) are recognized as species rather than as subspecific units. The species-rich cheilostome genus Schizoporella Hincks, 1877, which contains some of the most widely known fouling bryozoans, is designated a nomen protectum. The species name Smittina cheilostoma (Manzoni, 1869) is preserved as established usage.
Article
Sequence-based specimen identification, known as DNA barcoding, is a common method complementing traditional morphology-based taxonomic assignments. The fundamental resource in DNA barcoding is the availability of a taxonomically reliable sequence database to use as a reference for sequence comparisons. Here we provide a reference library including 579 sequences of the mitochondrial cytochrome c oxidase subunit I (COI) for 113 North Sea mollusc species. We tested the efficacy of this library by simulating a sequence-based specimen identification scenario using Best Match (BM), Best Close Match (BCM) and All Species Barcode (ASB) criteria with three different threshold values. Each identification result was compared with our prior morphology-based taxonomic assignments. Our simulation resulted in 87.7% congruent identifications (93.8% when excluding singletons). The highest number of congruent identifications was obtained with BCM and ASB and a 0.05 threshold. We also compared identifications with genetic clustering (BINs) computed by the Barcode of Life Datasystem (BOLD). About 68% of our morphological identifications were congruent with BINs created by BOLD. Forty-nine sequences were clustered in 16 discordant BINs, and these were divided in two classes: sequences from different species clustered in a single BIN; and conspecific sequences divided in more BINs. Whereas former incongruences were likely caused by BOLD entries in need of a taxonomic update, the latter incongruences regarded taxa requiring further investigations. These include species with amphi-Atlantic distribution, whose genetic structure should be evaluated over their entire range to produce a reliable sequence-based identification system. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
Article
Accelerating the description of biodiversity is a major challenge as extinction rates increase. Integrative taxonomy combining molecular, morphological, ecological and geographical data is seen as the best route to reliably identify species. Classic molluscan taxonomic methodology proposes primary species hypotheses (PSHs) based on shell morphology. However, in hyperdiverse groups, such as the molluscan family Turridae, where most of the species remain unknown and for which homoplasy and plasticity of morphological characters is common, shell-based PSHs can be arduous. A four-pronged approach was employed to generate robust species hypotheses of a 1000 specimen South-West Pacific Turridae data set in which: (i) analysis of COI DNA Barcode gene is coupled with (ii) species delimitation tools GMYC (General Mixed Yule Coalescence Method) and ABGD (Automatic Barcode Gap Discovery) to propose PSHs that are then (iii) visualized using Klee diagrams and (iv) evaluated with additional evidence, such as nuclear gene rRNA 28S, morphological characters, geographical and bathymetrical distribution to determine conclusive secondary species hypotheses (SSHs). The integrative taxonomy approach applied identified 87 Turridae species, more than doubling the amount previously known in the Gemmula genus. In contrast to a predominantly shell-based morphological approach, which over the last 30 years proposed only 13 new species names for the Turridae genus Gemmula, the integrative approach described here identified 27 novel species hypotheses not linked to available species names in the literature. The formalized strategy applied here outlines an effective and reproducible protocol for large-scale species delimitation of hyperdiverse groups.