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Nova Acta Cientíca Compostelana (Bioloxía), 28: 1-33 (2021) - ISSN 2340-0021
Artículo de investigAción
Doris adrianae sp. nov. (Heterobranchia; Nudibranchia; Doridina)
from the Galician coasts (NW Iberian Peninsula)
Doris adrianae sp. nov. (Heterobranchia; Nudibranchia; Doridina)
de las costas de Galicia (NW Península Iberica)
*urgorri, v.1, señArís, M.P. 1, díAz-AgrAs, g.1, cAndás, M.1, & góMez-rodríguez, c.2
1 Estación de Bioloxía Mariña da Graña (REBUSC), Universidade de Santiago de Compostela,
rúa da Ribeira 1-4 (A Graña), 15590 Ferrol (Galicia), Spain.
2 Departamento de Bioloxía Funcional, Instituto CRETUS, Universidade de Santiago de
Compostela, rúa Lope Gómez de Marzoa, s/n. Campus Vida.
15782 Santiago de Compostela (Galicia), Spain.
*Corresponding author: vituco.urgorri@usc.es
(Recibido: 15/02/2021; Aceptado: 02/03/2021)
Abstract
A new species of dorid nudibranch, Doris adrianae sp. nov. is described, from the Ría de Ferrol (NW Iberian
Peninsula) on rocky bottoms, between 11 and 20 m deep, where its prey, the sponge Polymastia boletiformis (La-
marck, 1815) is common. The new species is oval-shaped and yellow to yellow-orange in colour, with the dorsum
covered by rounded tubercles of various sizes, reinforced by tegumentary spicules. This new species is characterised
by having numerous integumentary and fusiform calcareous spicules, mainly grouped in multispicular bundles re-
sulting in a complex and very dense skeletal structure, giving the animal great consistence without losing flexibility.
In addition, it differs from other known species of the genus Doris Linnaeus, 1758 by various external and internal
characters, mainly by the coloration and the shape of the tubercles and the morphology of the radula, the digestive
and reproductive systems. Doris adrianae sp. nov. also presents a marked genetic distance in the barcode fragment
(cox1-5’) with other species of the genus Doris.
Keywords: Gastropoda, Ría de Ferrol, anatomy, habitat, feeding, micro computed tomography.
Resumen
Se describe una nueva especie de un nudibranquio doridáceo, Doris adrianae sp. nov., recolectada en la
Ría de Ferrol (NW Península Ibérica) en fondos de roca, entre 11 y 20 m de profundidad, donde es frecuente
el porífero Polymastia boletiformis (Lamarck, 1815) del que se alimenta. La nueva especie tiene forma ova-
lada y de color amarillo a amarillo-anaranjado, con el dorso cubierto por tubérculos redondeados de varios
tamaños, reforzados por espículas tegumentarias. Se caracteriza por presentar numerosas espículas calcáreas
tegumentarias fusiformes, principalmente agrupadas en haces multiespiculares que le coneren una estructura
esquelética compleja y muy densa, dándole al animal una gran consistencia sin perder exibilidad. Además,
se diferencia de otras especies conocidas del género Doris Linnaeus, 1758 por diversos caracteres externos e
internos, principalmente por la coloración y tubérculos del cuerpo, caracteres de la rádula y de los aparatos
Nova Acta Cientíca Compostelana (Bioloxía), 28 (2021) 2
digestivo y reproductor. Doris adrianae sp. nov. también presenta una marcada distancia genética en el frag-
mento “barcode” (cox1-5’) con otras especies del género Doris.
Palabras clave: Gastropoda, Ría de Ferrol, anatomía, hábitat, alimentación, microtomografía compu-
tarizada.
This paper is registered in Zoobank under:
http://zoobank.org/urn:lsid:zoobank.org:pub:51C800B8-6F45-4F56-8E68-ED6D53ECF248
Abbreviations
a: anus
am: ampulla
ao: aorta
ar: adhesive region
au: auricle
ba: anterior blood gland
bb: buccal bulb
bc: bursa copulatrix
bg: buccal ganglion
bp: posterior blood gland
c: cerebral nerves
ca: caecum
cg: cerebral ganglion
cgl: capsule gland
d: deferent duct
dg: digestive gland
e: oesophagus
ey: eye
fg: female glands
g: branchial leaves
ga: genital atrium
go: genital openings
gpo: postampullar gonoduct
gpr: preampullar gonoduct
hdg: hermaphrodite and digestive glands
hg: hermaphrodite gland
i: intestine
m: mouth
mbb: buccal bulb muscle
megl: membrane gland
mugl: mucous gland
mot: retractor muscles
og: gastro-oesophageal ganglion
ot: oral tube
ovp: proximal oviduct
ovd: distal oviduct
p: pedal nerves
pc: pedal commissure
pe: penis
pg: pedal ganglion
pl: pleural nerves
plg: pleural ganglion
ppc: parapedal commissure
pr: prostate
ps: penial sheath
pu: pericardium
r: rhinophoral nerves
rs: radular sac
sg: salivary gland
sr: seminal receptacle
st: stomach
sy: syrinx
ud: uterine duct
va: vagina
ve: ventricle
vgl: vestibular gland
vl: visceral loop
INTRODUCTION
The coasts of the Iberian Peninsula re-
present a zoogeographic enclave comprising
two regions: Lusitanian and Mediterranean
(gofAs, 2011). A total of 715 species of
heterobranchs sea slugs are known from
the Iberian Peninsula and the Balearic and
Canary Islands, of which 279 belong to the
order Nudibranchia (gofAs, et al. 2017;
cerverA, et al. 2004). Galicia (NW Iberian
Peninsula) represent more than a third of the
total coastline of the Iberian Peninsula, as it is
heavily indented, with exposed environments
Urgorri, V., Señarís, M.P. , Díaz-Agras, G., Candás, M. & Gómez-Rodríguez, C.: Doris adrianae sp. nov. 3
open to the beating of the ocean and with deep
coastal indentations called rías (richthofen,
1886). The latter were formed by marine oo-
ding of a terminal river course caused by the
subsidence of the coastal edge and the rise
in sea level. The rías vary greatly in size and
orography, which determines the existence of
a great diversity of habitats, from very rough
to very sheltered environments, with a high
diversity of ora and fauna (BAñón, 2017).
The heterobranchs sea slugs of Galicia
were poorly known until the last quarter of
the 20th century, when urgorri (1981) wrote
his doctoral thesis and the rst records and
new species were published (orteA & urgo-
rri, 1978, 1979a, 1979b, 1981a, 1981b; among
others). urgorri & Besteiro (1983) published
the inventory of the “opisthobranchs” of
Galicia, in which they compiled the data from
the doctoral thesis of urgorri (1981) and all
previous data, cataloguing 148 species from
coastal and bathyal bottoms down to 3000
m depth. In subsequent years, the inventory
was increased with new species and records,
mainly of nudibranchs, by various authors (see
urgorri, et al. 2011). Currently, 110 species
of the order Nudibranchia are known from
Galicia, of which 62 are classied in the su-
border Cladobranchia and 48 in the suborder
Doridina (trigo, et al. 2018).
Three species of the genus Doris Linnaeus,
1758 are known in Galicia. These include
Doris verrucosa Linnaeus, 1758 (urgorri &
Besteiro, 1983, 1984; rolán, 1983; trigo &
otero, 1987; trigo, et al. 2018). Doris pseu-
doargus Rapp, 1827 (urgorri & Besteiro,
1983, 1984, both as Archidoris pseudoargus;
rolán (1983), as A. pseudoragus; trigo, et
al. 2018) and Doris ocelligera (Bergh, 1881 in
Bergh 1881a) (urgorri & Besteiro, 1983,
1984; rolán, 1983; trigo, et al. 2018). All
three species are well known from the Ría de
Ferrol and found on rocky and gravel substrata
in both the lower mesolittoral and infralittoral.
Twenty-two specimens of a dorid were
collected at the Ría de Ferrol during the last
34 years, between 1986 and 2019, on a rocky
bottom between 11 and 20 m deep, mostly
under a gorgonian forest of Leptogorgia
lusitanica (Stiasny, 1937). After its study, it
was classied within the genus Doris according
to vAldés (2002) and was found to be a new
species described here as Doris adrianae sp. nov.
MATERIAL AND METHODS
Collection: The 22 specimens studied
were collected by SCUBA diving, from roc-
ky bottoms between 11 and 20 m deep in
the localities of Punta Fornelos, Castelo de
San Felipe and Punta Barbeira, at the Ría
de Ferrol (NW Iberian Peninsula) (Fig. 1).
They were collected mostly one specimen
per sampling; only 6 times 2 specimens were
collected together. In some samplings, in situ
photographs of the animal, habitat and ac-
companying species were taken. Samples of
porifera that could potentially be preyed upon
by the dorid were also collected, in the same
habitat. All specimens were observed in vivo
and all characters that were lost after xation
were described. Some specimens were photo-
graphed in vivo in their habitat, although most
were photographed in the laboratory. Both the
general appearance of the animal and details
of its external anatomy were captured, using
Figure 1. Map showing the type locality of Doris adria-
nae sp. nov. in Punta Fornelos, Castelo de San Felipe and
Punta Barbeira at the Ría de Ferrol (NW Iberian Penin-
sula) where all specimens were collected.
Figura 1. Mapa que muestra la localidad tipo de Doris
adrianae sp. nov. en Punta Fornelos, Castelo de San Fe-
lipe y Punta Barbeira en la Ría de Ferrol (NW Península
Ibérica) donde se recolectaron todos los ejemplares.
Nova Acta Cientíca Compostelana (Bioloxía), 28 (2021) 4
an Olympus SCX12 stereo microscope with an
Olympus E-330 digital camera. In addition,
the animals were kept alive for 2 or 3 days in
individualised aquaria for the collection of
faeces and spawning.
Anaesthesia, xation and preservation: All
specimens were anaesthetised with 7% magne-
sium chloride (MgCl2), diluted in equal parts
(50%) in seawater and fresh water (urgorri,
1981). After anaesthesing, the xation process
was carried out gradually, adding drops of the
diluted xative liquid with a Pasteur pipette,
observing under the stereoscopic microscope
if any reaction was produced in the animal,
until complete xation. The xative liquid
used was 70% EtOH in 7 specimens, 100%
EtOH in 6 specimens and Bouin’s uid in 10
specimens; specimens xed in 70% EtOH and
Bouin’s uid were preserved in 70% EtOH
neutralized with Borax to avoid degradation
of the calcareous structures, while those xed
in EtOH 100º were preserved in EtOH 100º
(for details see Type material and deposition).
Dissection, radula and labial disc prepara-
tions: For the study of the internal anatomy,
paratypes 1, 7, 10, 12 and 17 were dissected
under an Olympus SCX12 stereo microscope
with an Olympus E-330 digital camera. After
dissection, preparations of the radula and la-
bial disc were made for light microscopy (LM)
and scanning electron microscopy (SEM), by
macerating the buccal bulb with 5% KOH. For
LM the radula and labial disc were mounted
in Hoyer’s liquid, while for SEM, the radula
was mounted in distilled water between two
coverslips and the labial disc in water in a
small container, frozen in liquid nitrogen and
lyophilised; Thus the radula was perfectly
stretched and the lip disc was not wrinkled,
glued to the SEM stub with DHMF resin
(dimethyl-hydantoin formaldehyde), sputter
coated with gold-palladium on a Bio-Rad
e5000 coated and examined under a LEO-
435VP SEM with Microanalysis (EDX, Oxford
300) Leica Microsystems (Cambridge, U. K.).
Spicule preparations: The study of the spi-
cules and spicular skeletal arrangement pre-
sented numerous problems, as the traditional
methods of isolation: 4% NaOH, 4% KOH,
5% NaClOH and H2O2, resulted in spicules
decay. Thus, alternative methods were used:
Cl2CH4, CaO, 0’25% trypsin, protease K at
15 ppm, natural bacterial decomposition in
seawater and incineration of the organic matter
in a mufe at 500°C for 48 hours, the latter
being the one with which the best results were
obtained. Spicule preparations for OM were
mounted in Hoyer’s liquid, synthetic resin
and Canada balsam, while for SEM they were
mounted on a circular glass coverslip glued
with a carbon adhesive to the SEM stub, sput-
ter coated with gold-palladium. The study of
the skeletal arrangement was performed by
X-ray computed microtomography (Micro-CT
Skyscan 1172).
Serial sectioning: Serial sections 15 µm thick
were taken from two specimens (Paratypes
4 and 15 and from parts of the dissection
of paratypes 10 and 17) which were xed
in Bouin’s uid, decalcied with ethylene-
diaminotetraacetic acid (EDTA), embedded
in parafn, sectioned with a Microm HM310
rotary microtome and stained with Mallory’s
trichrome (gil-MAnsillA, et al. 2008).
Micro-CT: The study of the anatomical
and skeletal structure was carried out using
micro-computed tomography, the specimens
used (Paratypes 6 and 16) were preserved in
70% ethanol. Subsequently, they were dehydra-
ted in successive baths of 80%, 90% and 96%
ethanol. The specimens were scanned with a
Skyscan 1172 microtomograph.
Paratype 6 was scanned three times with
the following treatments and parameters:
1.- The specimen was stained with 1% iodine
in 96º ethanol during one week before dehydra-
tion with hexamethyldisilazane (HMDS) for
four hours, and subsequently air dried over-
night (AlBA-tercedor & sAnchez-tocino,
2011; fAulwetter, et al. 2013; cAndás, et al.
2016). The following parameters were used: 55
kv, 165 µA, no lter and pixel size of 6.78 um.
The sample was rotated 360º and the projec-
tion images were obtained at 0.25° intervals.
2.- The specimen was placed inside a closed
polypropylene tube with a paper moistened with
96º EtOH at the bottom to maintain a moist
atmosphere inside. The scanning para
meters
Urgorri, V., Señarís, M.P. , Díaz-Agras, G., Candás, M. & Gómez-Rodríguez, C.: Doris adrianae sp. nov. 5
were: 55 kv, 165 µA, no lter and pixel size
of 13.57 um. The sample was rotated 360º
and the projection images were obtained at
0.30° intervals.
3.- The specimen was submerged in 5.5 %
EDTA in 10% formaldehyde for four days.
Then it was dehydrated in successive baths
of 80%, 90% and 96% ethanol. Subsequently,
it was stained again with 1% iodine in 96º
ethanol during ve days before dehydration
with HMDS for four hours, prior to scanning.
Scanning was performed at: 55 kv, 165 µA, no
lter and pixel size of 13.57 um. The sample
was rotated 360º and the projection images
were obtained at 0.20° intervals.
Paratype 16 was scanned twice with the
following treatments and parameters:
1.- The specimen was stained with 1%
iodine in 96º ethanol during one week before
scanning. It was placed inside a closed poly-
propylene tube with a paper moistened with
96º ethanol at the bottom to maintain a moist
atmosphere inside. The scanning parameters
were as follows: 60 kv, 167 µA, no lter and
pixel size of 13.57 um. It was rotated 360º
and the projection images were obtained at
0.30° intervals.
2.- The specimen was stained with 1% io-
dine in 96º ethanol during two weeks before
scanning. It was placed inside a closed poly-
propylene tube with a paper moistened with
96º ethanol at the bottom to maintain a moist
atmosphere inside. The scanning parameters
were as follows: 60 kv, 167 µA, no lter and
pixel size of 13.57 um. It was rotated 360º
and the projection images were obtained at
0.25° intervals.
The images were reconstructed with the
NRecon software (Bruker, Belgium) and
the obtained sections were cleaned with the
CTAnalyzer software (Bruker, Belgium). The
software CTVox and DataViewer (Bruker, Bel-
gium) were used for the correct visualization of
the data. After studying the obtained sections,
3D reconstruction was carried out using the
program AVIZO 6.4 (Thermo Fisher Scientic)
which allows three-dimensional anatomical
models to be obtained from the two-dimen-
sional images of the sections. After choosing
the pixel size and loading the cross-sectional
images into AVIZO, they were aligned, drawing
the different structures manually in the form of
overlapping colour layers and then smoothed,
to eliminate imperfections in order to obtain
a sharp three-dimensional image.
DNA analysis: For molecular analysis, a
fragment of the foot of Paratype 20 was soaked
in water for 30 minutes before genomic DNA
extraction with DNeasy Blood & Tissue Kit
(Qiagen, Germany). A 657 base pair region
from the 5’ end of mitochondrial cox1 (“bar-
code region”) was amplied with standard
LCO / HCO primers (folMer, et al. 1994).
Amplication was performed with Bioline-
MyTaq and the following cycling: 94ºC for 2
min 30 sec, 40 cycles of 94ºC for 30 s, 47ºC for
45s and 72ºC for 1 min 15 sec, and nal exten-
sion of 72ºC for 10 min. PCR products were
sent to StabVida (Portugal) for purication
with magnetic beads and sequencing in both
directions using ABI 3730xl DNA Analyzer.
Sequence chromatograms were assembled and
manually edited using Geneious 5.6.
A total of 177 cox1-5' sequences within
the genera Doris, Archidoris and Austrodoris
were downloaded from GenBank in December
2020. The following species were represen-
ted, according to GenBank identication: 1
sequence of Archidoris montereyensis, 1 se-
quence of Archidoris pseudoargus, 1 sequence
of Archidoris wellingtonensis, 93 sequences
of Austrodoris kerguelenensis, 64 sequences
of Doris kerguelenensis; 9 sequences of Doris
montereyensis, 1 sequence of Doris nobilis, 6
sequences of Doris pseudoargus, 1 sequence
of Doris sp. (this sequence was discarded in
further analysis as it was not identied to
species level). The full list of downloaded
sequences is provided in Supplementary ma-
terial Appendix 1. Unique haplotypes were
identied (n= 132; including the barcode
sequence of Doris adrianae sp. nov.) and an
alignment of 627 bp was produced with Muscle
in Geneious 5.6 after sequence trimming. One
sequence per species (according to GenBank
identity) was selected and an uncorrected
pairwise identity (i.e., proportion of identical
sites) was computed as a distance matrix in
Nova Acta Cientíca Compostelana (Bioloxía), 28 (2021) 6
Geneious 5.6. This preliminary analysis con-
rmed that Archidoris, Austrodoris and Doris
were synonymous genera. Thus, one sequence
per non-synonimous species of Doris as well
as an outgroup sequence (Hexabranchus sp.,
GenBank MN224071.1) were included in a
phylogenetic tree run in Beast2 2.6 (BouckAert
et al. 2019). Model priors were set in BEAUti
as a GTR + G + I and uncorrelated relaxed
clock with mcmc = 10 000 000 and log every 1
000. In TreeAnnotator, 5000 (50%) trees were
discarded and a maximum clade credibility
tree with median heights was built.
RESULTS
Class Gasteropoda Cuvier, 1795
Subclass Heterobranchia Burmeister, 1837
Orden Nudibranchia Cuvier, 1814 in Bla-
inville, 1814
Suborden Doridina Bouchet et al, 2017
Superfamily Doridoidea Ranesque, 1815
Family Dorididae Ranesque, 1815
Genus Doris Linnaeus, 1758
Type species: Doris verrucosa Linnaeus,
1758, by subsequent designation: Bouchet
& vAldés (2000).
Doris adrianae Urgorri & Señarís sp. nov.
http://zoobank.org/urn:lsid:zoobank.org:-
pub:51C800B8-6F45-4F56-8E68-ED-
6D53ECF248
Diagnosis: Oval-shaped and orange-yellow
in colour with ne black dorsal stippling,
denser in the mid-lateral areas, 32-76 mm long
and 18-55 mm wide. Dorsum covered with
rounded tubercles of various sizes, slightly
pointed, with a rough surface and a small
basal stalk. Lamellar rhinophores and 7-8
tripinnate branchial leaves, surrounded at their
base by wide sheaths with small tubercles on
their surface. Foot orange, frontally bilaminate
without notch. Head with two lateral oral
lobes and without tentacles. Radular formula
0:33-53:3-4 x 38-50. Rachidian tooth absent,
lateral teeth simple and curved with prominent
ridge, with rst central lateral teeth strongly
curved and marginal teeth with plumose edge.
Labial disc peltate in shape with soft rugosi
ties.
Gastric caecum near the opening to the di-
gestive gland. Reproductive system triaulic,
ampulla long, tubular and thick, penis fusiform
and unarmed, prostate large and granular in
appearance. Vagina short and wide, bursa
copulatrix large and oval, three times larger
than the oval seminal receptacle. Vestibular
gland at the end of the distal oviduct, near
its opening in the genital atrium.
Derivatio nominis. The species is dedicated
to Adriana Álvarez Urgorri, granddaughter
of the senior author.
Type locality: Ría de Ferrol (Galicia, NW
Iberian Peninsula): Punta Fornelos (43º 28’
02” N; 008º 18’ 50” W) all animals (Holotype
and Paratype 1-16) were collected between 16
and 20 m deep on rocky bottoms with muddy
sand sedimentation; Castelo de San Felipe
(43º 27’ 47” N; 008º 16’ 57” W) all animals
(Paratype 17-20) were collected between 11
and 18 m deep on rocky bottoms with sand
on the surface; Punta Barbeira (43° 28’ 3.19”
N; 008° 19’ 11.52” W) all animals (Paratype
21-22) were collected between 12 and 18 m
deep on rocky bottoms with muddy sand
sedimentation (Fig. 1).
Other localities: 01010204: 3 specimens
and 01250404: 1 specimen in O Grelle (Ría
da Coruña, NW Iberian Peninsula) (43° 22’
53” N; 008° 23’ 30” W). The 4 specimens were
not collected but were photographed in situ,
on rocky bottoms with small red algae and
incrusting sponges at a depth between 12-
16 m. 01141190: 1 specimen 50 mm long at
Sálvora (Ría de Arousa, NW Iberian Penin-
sula): (42° 25’ 38” N; 009° 0.478’ W). It was
collected on maërl bottoms with Veretillum
cynomorium (Pallas, 1766) and Pteroeides
griseum (Bohadsch, 1761), at a depth of 60
m. Orig. xative and preserved in 70% EtOH
neutralized with Borax.
TYPE MATERIAL AND DEPOSITION:
Holotype: 02020892 (MHN-USC 10119).
1 specimen, complete, 65 mm long in vivo.
Punta Fornelos (Ría de Ferrol) 02/08/1992.
Orig. xed in Bouin’s solution and preserved
in 70% EtOH neutralized with Borax.
Urgorri, V., Señarís, M.P. , Díaz-Agras, G., Candás, M. & Gómez-Rodríguez, C.: Doris adrianae sp. nov. 7
Paratype 1: 01060786-01 (MHN-USC
10119-01). 1 specimen, dissected, 50 mm
long in vivo. Punta Fornelos (Ría de Ferrol)
06/07/1986. Orig. xed in Bouin’s solution
and preserved in 70% EtOH neutralized with
Borax.
Paratype 2: 01060786-02 (MHN-USC
10119-02). 1 specimen, complete, 40 mm
long in vivo. Punta Fornelos (Ría de Ferrol)
06/07/1986. Orig. xed in Bouin’s solution
and preserved in 70% EtOH neutralized with
Borax.
Paratype 3: 01160591 (MCUC.2021.1.1).
1 specimen, complete, 32 mm long in vivo.
Punta Fornelos (Ría de Ferrol) 16/05/1991.
Orig. xed in Bouin’s solution and preserved
in 70% EtOH neutralized with Borax.
Paratype 4: 02030592 (MHN-USC 10119-
04). 1 specimen, histological sections, 48 mm
long in vivo. Punta Fornelos (Ría de Ferrol)
03/05/1992. Orig. xed in Bouin’s solution and
preserved in 70% EtOH neutralized with Bo-
rax. The anterior and posterior portions of the
specimen are still embedded in parafn block.
Paratype 5: 02300596 (ZSM Mol 20210004).
1 specimen, complete, 37 mm long in vivo.
Punta Fornelos (Ría de Ferrol) 30/05/1996.
Orig. xed and preserved in 70% EtOH neu-
tralized with Borax.
Paratype 6: 05050896 (MHN-USC 10119-
06). 1 specimen, complete, 40 mm long in vivo,
scanned in the Micro-CT. Punta Fornelos (Ría
de Ferrol) 05/08/1996. Orig. xed and preser-
ved in 70% EtOH neutralized with Borax.
Paratype 7: 05130597-01 (MHN-USC
10119-07). 1 specimen, dissected, 60 mm
long in vivo. Punta Fornelos (Ría de Ferrol)
13/05/1997. Orig. xed and preserved in 70%
EtOH neutralized with Borax.
Paratype 8: 05130597-02 (MNHN-
IM-2012-25572). 1 specimen, complete, in
45 mm long in vivo. Punta Fornelos (Ría de
Ferrol) 13/05/1997. Orig. xed and preserved
in 70% EtOH neutralized with Borax.
Paratype 9: 01080598-01 (MCUC.2021.1.2).
1 specimen, complete, 50 mm long in vivo. Pun-
ta Fornelos (Ría de Ferrol) 08/05/1998. Orig.
xed and preserved in 70% EtOH neutralized
with Borax.
Paratype 10: 01080598-02 (MHN-USC
10119-10). 1 specimen, dissected, 45 mm
long in vivo. Punta Fornelos (Ría de Ferrol)
08/05/1998. Orig. xed and preserved in 70%
EtOH neutralized with Borax.
Paratype 11: 02101199 (MHN-USC 10119-
11). 1 specimen, complete, 40 mm long in vivo.
Punta Fornelos (Ría de Ferrol) 10/11/1999.
Orig. xed in Bouin’s solution and preserved
in 70% EtOH neutralized with Borax.
Paratype 12: 01240505 (MHN-USC 10119-
12). 1 specimen, dissected, 40 mm long in vivo.
Punta Fornelos (Ría de Ferrol) 24/05/2005.
Orig. xed and preserved in 100% EtOH.
Paratype 13: 03220705 (MHN-USC 10119-
13). 1 specimen, complete, 40 mm long in vivo.
Punta Fornelos (Ría de Ferrol) 22/07/2005.
Orig. xed and preserved in 100% EtOH.
Paratype 14: 04190805 (MHN-USC 10119-
14). 1 specimen, dissected, 50 mm long in vivo.
Punta Fornelos (Ría de Ferrol) 19/08/2005.
Orig. xed and preserved in 70% EtOH neu-
tralized with Borax.
Paratype 15: 03040106 (MHN-USC 10119-
15). 1 specimen, histological sections, 51 mm
long in vivo. Punta Fornelos (Ría de Ferrol)
04/01/2006. Orig. fixed in Bouin’s solution and
preserved in 70% EtOH neutralized with Borax.
Paratype 16: 01240510 (MHN-USC 10119-
16). 1 specimen, complete, 41 mm long in vivo,
scanned in the Micro-CT. Punta Fornelos (Ría
de Ferrol) 24/05/2010. Orig. xed in Bouin’s
solution and preserved in 70% EtOH neutra-
lized with Borax.
Paratype 17: 01290806-01 (MHN-USC
10119-17). 1 specimen, dissected and cut in
histological sections, 71 mm long in vivo. Cas-
telo de San Felipe (Ría de Ferrol) 29/08/2006.
Orig. xed and preserved in 100% EtOH.
Paratype 18: 01290806-02 (MHN-USC
10119-18). 1 specimen, dissected, 63 mm long
in vivo. Castelo de San Felipe (Ría de Ferrol)
29/08/2006. Orig. xed and preserved in 100%
EtOH. Spawning sample for DNA.
Paratype 19: 01220807-01 (MHN-USC
10119-19). 1 specimen, complete, 76 mm long
in vivo. Castelo de San Felipe (Ría de Ferrol)
22/08/2007. Orig. xed and preserved in 100%
EtOH.
Nova Acta Cientíca Compostelana (Bioloxía), 28 (2021) 8
Paratype 20: 01220807-02 (MHN-USC
10119-20). 1 specimen, complete, 55 mm long
in vivo. Castelo de San Felipe (Ría de Ferrol)
22/08/2007. Orig. xed and preserved in 100%
EtOH. Spawning sample for DNA.
Paratype 21: 01020519-01 (MHN-USC
10119-21). 1 specimen, complete, 40 mm
long in vivo. Punta Barbeira (Ría de Ferrol)
02/05/2019. Orig. fixed in Bouin’s solution and
preserved in 70% EtOH neutralized with Borax.
Paratype 22: 01020519-02 (MHN-USC
10119-22). 1 specimen, complete, 55 mm
long in vivo. Punta Barbeira (Ría de Ferrol)
02/05/2019. Orig. fixed in Bouin’s solution and
preserved in 70% EtOH neutralized with Borax.
The Holotype and the Paratypes 1, 2, 4, 6,
7, 10-20 have been deposited at the Museo de
Historia Natural of the Universidade de San-
tiago de Compostela, Galicia, Spain (MHN-
USC). Paratypes 3 and 9 have been deposited
at the Museu da Ciência da Universidade de
Coimbra, Portugal (MCUC), Paratype 5 at
the Zoologische Staatssammlung München,
Germany (ZSM) and Paratype 8 at the Mu-
seum National d’Histoire Naturelle, Paris,
France (MNHN).
Faecal pellets, spawning and preparations: Sam-
ples of faecal pellets of the Holotype and Para-
types 4, 6, 8, 9, 13, 17, 18, 19, 20, 21 and 22
are preserved in 70% EtOH neutralized with
Borax. One spawning collected at sea with
Paratypes 17 and 18 and several egg masses
made in the laboratory aquarium, larvae and
eggs of Paratypes 17, 18, 21 and 22 are also
preserved. In addition, 92 SEM preparations
are preserved of: radula, spicules, labial disc,
eggs, larval shell, faecal pellets and spicules
of Polymastia boletiformis (Lamarck, 1815)
and 79 LM preparations: radula, spicules of
the foot, dorsal and ventral mantle, tubercles
and rhinophores, faecal pellets and spicules of
the sponge Polymastia boletiformis (Lamarck,
1815). Also preserved are 383 preparations of
histological sections of Paratype 4, 941 prepa-
rations of histological sections of Paratype 15,
81 preparations of histological sections of the
genital system of Paratype 17 and 6 prepara-
tions of histological sections of the penis of
Paratype 10. All this material has also been
deposited at the Museo de Historia Natural of
the Universidade de Santiago de Compostela,
Galicia, Spain (MHN-USC 10119).
DESCRIPTION
External Anatomy
Habitus: The general colour of live animals
varies from yellow to yellow-orange, although
yellow specimens predominate (Fig. 2). The
entire dorsal surface is nely dotted with black
dots, which are only visible under the stereos-
copic microscope. These dots increase their
density in the mid-lateral areas of the notum,
forming two somewhat darker bands running
from the rhinophores to the gill (Figs. 2E,F).
The outline of the mantle is oval to oblong,
dorsally strongly convex, elevated, with a very
hard consistency covering the whole animal,
even when it is moving; the semicircular me-
tapodium never protrudes (Figs. 2G,H). It
possesses two small eyes, located between the
two lobes of the blood gland, but they are not
visible externally (Figs. 2A-G, 8B). The size
of collected specimens varies between 32 and
76 mm in length and 18 and 55 mm in width,
although the most frequent size uctuates
between 40-55 mm in length and 30-40 mm
in width; the length/width ratio ranges from
1.3 to 1.8.
The entire dorsum is covered with rounded,
slightly pointed, rough-surfaced tubercles with
a small, broad basal stalk (Figs. 2A-G, 3G,H).
In lateral view the tubercles have a conical
appearance (Fig. 3H) and their colour is the
same as that of the mantle with the same black
stippling. The rough surface of the tubercles
has an orange-peel appearance. The lattice of
radial spicules giving shape to the tubercle can
be seen through transparency, with the tips of
the spicules directed towards the surface but
not protruding outwards.
The tubercles range in size from large to
very small, with various sizes in between so
they cannot be grouped into any particular
category, and with no consistent distribution.
All sizes are present throughout the dorsum
of the mantle, although the larger ones are
much more abundant in the central part of
Urgorri, V., Señarís, M.P. , Díaz-Agras, G., Candás, M. & Gómez-Rodríguez, C.: Doris adrianae sp. nov. 9
Figure 2. Doris adrianae sp. nov. Habitus and external appearance in vivo. A: Holotype 02020892, dorsal view in habitat,
65 mm. B: O Grelle 01250404, lateral view in habitat, 40 mm. C: Paratypes 17 and 18 01290806, top view of the two
specimens copulating in habitat, 71-63 mm. D: Paratype 2, 01060786, dorsal view in aquarium, 40 mm. E: Paratype 19,
01220807, dorsal view in aquarium, 76 mm. F: Paratype 19, 01220807, lateral view in aquarium, 76 mm. G: Paratype 15,
03040106, lateral view in aquarium, 51 mm. H: Paratype 2, 01060786, ventral view in aquarium, 40 mm.
Figura 2. Doris adrianae sp. nov. Habitus y aspecto externo in vivo. A: Holotipo 02020892, vista dorsal en hábitat, 65 mm.
B: O Grelle 01250404, vista lateral en hábitat, 40 mm. C: Paratipos 17 y 18 01290806, vista superior de dos ejemplares
copulando en hábitat, 71-63 mm. D: Paratipo 2, 01060786, vista dorsal en acuario, 40 mm. E: Paratipo 19, 01220807,
vista dorsal en acuario, 76 mm. F: Paratipo 19, 01220807, vista lateral en acuario, 76 mm. G: Paratipo 15, 03040106, vista
lateral en acuario, 51 mm. H: Paratipo 2, 01060786, vista ventral en acuario, 40 mm.
Nova Acta Cientíca Compostelana (Bioloxía), 28 (2021) 10
Figure 3. Doris adrianae sp. nov. Details of the external appearance. A: Rhinophore in vivo, Paratype 19, 01220807-01, 76
mm. B: Branchial leaves, Paratype 19, 01220807-01, 76 mm. C: Rhinophoral sheath, Paratype 19, 01220807-01, 76 mm.
D: Rhinophoral sheath, Paratype 16, 01240510, 41 mm. E: Branchial leaves and branchial sheath, Paratype 2, 01060786-
02, 40 mm. F: Branchial sheath, Paratype 16, 01240510, 41 mm. G: Dorsal tubercles, Paratype 19, 01220807-01, 76 mm.
H: Dorsal tubercles and detail of a tubercle, Paratype 6, 05050896, 40 mm. I: Oral lobes and anterior edge of bilaminated
foot, Paratype 15, 03040106, 51 mm. J: External genital atrium, Paratype 19, 01220807-01, 76 mm. K: Ventral view of the
anterior third of the animal, Paratype, 16 01240510, 41 mm. (A,B,C,E,G,I,J in vivo. D,F,H,K Micro-CT).
Figura 3. Doris adrianae sp. nov. Detalles del aspecto externo. A: Rinóforo in vivo, Paratipo 19, 01220807-01, 76 mm. B:
Branquias, Paratipo 19, 01220807-01, 76 mm. C: Vaina rinofórica, Paratipo 19, 01220807-01, 76 mm. D: Vaina rinofóri-
ca, Paratipo 16, 01240510, 41 mm. E: Branquias y vaina branquial, Paratipo 2, 01060786-02, 40 mm. F: Vaina branquial,
Paratipo 16, 01240510, 41 mm. G: Tubérculos dorsales, Paratipo 19, 01220807-01, 76 mm. H: Tubérculos dorsales y
detalle de un tubérculo, Paratipo 6, 05050896, 40 mm. I: Lóbulos orales y borde anterior del pie bilaminado, Paratipo
15, 03040106, 51 mm. J: Atrio genital externo, Paratipo 19, 01220807-01, 76 mm. K: Vista ventral del tercio anterior del
animal, Paratipo, 16 01240510, 41 mm. (A,B,C,E,G,I,J in vivo. D,F,H,K Micro-CT).
Urgorri, V., Señarís, M.P. , Díaz-Agras, G., Candás, M. & Gómez-Rodríguez, C.: Doris adrianae sp. nov. 11
the notum and in the two darker lateral bands
(black dots) from the rhinophores to the gills.
In general, the size of the large tubercles de-
creases towards the edges of the mantle, where
the smaller ones are much more abundant
(Figs. 2A,B,D,F,G, 3G,H). Very small tuber-
cles are scattered over the entire surface of the
notum, including the rhinophoral sheaths and
gill sheath, being slightly more abundant on
the dorsal half and on the edge of the notum
(Figs. 2C-H).
The rhinophores are elongate, narrow,
slightly curved backwards with 29-34 lamellae
in the upper two thirds of their length and
with a slightly truncated conical basal stalk
occupying the lower third (Figs. 2F,G, 3A).
Yellow lamellae with slight black stippling,
with the spicules arranged radially; they are
arched and arranged alternately to right and
left in a zigzagging frontal line. The tip of the
rhinophore is a small truncated cylinder and
the rhinophore stalk is white.
Gill with 6-8 tripinnate leaves, which appear
to be bipinnate, as the tertiary branching is
only visible at high magnication (Figs. 2A-G,
3B,E). In some specimens it has been obser-
ved that the two posterior leaves on each side
each arise from a single basal bifurcation. The
colour of the gill is quite variable, as in most
specimens they is yellow to yellow-orange like
the mantle (Figs. 2F, 3B), while in others it is
whitish (Figs. 2G, 3E). All leaves show black
dots like those on the dorsum, less dense on
the rachis, but the rest of the leaf has diffe-
rent densities that give it a more or less dark
colour, especially on the whitish leaves (Figs.
2G, 3B,E). The anus opens at the end of the
anal papilla in the centre of the gills and is
yellowish with few dots.
The rhinophoral and branchial sheaths
are complete, raised, yellow with small to
medium-sized tubercles on the edge (Figs. 3A-
F). The two sheaths are of the same height,
but the branchial sheath is much wider; the
rhinophoral sheath may be as long as one-
third the length of the rhinophore. When the
rhinophores and gill retract, the sheaths close
completely, bringing the edges closer together
like a sphincter (Figs. 3C-F). The rhinophores
are retracted by the action of three pairs of
small muscles inserted at the base of the stalk
and diverging forwards, medially and poste-
riorly; the gill is also retracted by the action
of three pairs of muscles, but of larger size
than those of the rhinophores.
The mantle is ventrally slightly paler in
colour than the dorsum, being the dense net
of spicules visible through transparency (Figs.
2G-H). The foot, head and epipodium are
more orange in colour than the rest of the
animal, which is more yellow, with very few
black dots. On the head, on the sides of the
mouth, there are two attened lobes without
oral tentacles. The anterior edge of the foot
is complete, without any notch and grooved
on the anterior quarter, the upper lamella
protruding more than the lower one (Figs.
2H, 3I,K). At rest and during movement the
foot does not protrude from the edge of the
mantle. However, very exceptionally, when the
movement is very fast the posterior part of the
metapodium protrudes slightly (Figs. 2G,H).
Internal Anatomy
Skeletal structure: Doris adrianae sp. nov.
has numerous calcareous spicules throughout
its body, except in the visceral organs, which
are arranged in a dense network, forming an
armoured skeletal structure (Fig. 6A). This
gives the animal a very compact consistency,
clearly perceptible to the touch when specimens
are collected.
Doris adrianae sp. nov. has two types of
spicules, some monoaxonic fusiform, irregu-
lar, elongated and with blunt ends (Fig. 4A),
ranging in size from 70-770 µm in length, with
sizes between 111-320 µm being more abun-
dant (Fig. 5A). A second type of spicules are
globose spherules, practically spherical, with a
smooth surface and ranging in size from 5-15
µm in diameter (Fig. 4B).
Fusiform spicules are more or less circular
but irregular in section, with the thickest part
located more or less in the centre of the spicule
and with blunt ends, almost never acuminate.
Although they are generally rectilinear, almost
all show some degree of greater or lesser cur-
vature, some being very curved and even with
one of the ends bent at 45º (Fig. 4A). Their
Nova Acta Cientíca Compostelana (Bioloxía), 28 (2021) 12
Figure 4. Doris adrianae sp. nov. Skeletal structure. A: Different shapes and sizes of the fusiform spicules. B: Spherules.
C: Spicules in a rhinophore lamina, Paratype 10 01080598-02, 45 mm. D: Skeletal structure of ventral mantle. E: Sagittal
skeletal structure of lateral mantle. F: Radial spicular arrangement in a tubercle. G: Spicular arrangement of tubercle
stalk. H: Spicular arrangement of foot. (D-H: Paratype 16 01240510, 41 mm). (A,B,D,E,F,G,H SEM. C LM).
Figura 4. Doris adrianae sp. nov. Estructura esquelética. A: Diferentes formas y tamaños de las espículas fusiformes.
B: Esférulas. C: Espículas en una lámina del rinóforo, Paratipo 10 01080598-02, 45 mm. D: Estructura esquelética del
manto ventral. E: Estructura esquelética sagital del manto lateral. F: Disposición espicular radial en un tubérculo. G:
Disposición espicular del tallo del tubérculo. H: Disposición espicular del pie. (D-H: Paratipo 16 01240510, 41 mm).
(A,B,D,E,F,G,H SEM. C MO).
Urgorri, V., Señarís, M.P. , Díaz-Agras, G., Candás, M. & Gómez-Rodríguez, C.: Doris adrianae sp. nov. 13
Figure 5. Doris adrianae sp. nov. Size distribution of fusiform spicules. A: Lengths of total fusiform spicules (n = 1400).
B: Lengths of spicules of notum (n = 350). C. Lengths of spicules of subnotum (n = 175). D: Lengths of spicules of
tubercles (n = 175). E: Lengths of gill spicules (n = 175). F: Lengths of foot spicules (n = 350). G: Lengths of rhinophore
spicules (n = 175).
Figura 5. Doris adrianae sp. nov. Distribución de tamaños de las espículas fusiformes. A: Longitudes de las espículas fusi-
formes totales (n = 1400). B: Longitudes de las espículas del notum (n = 350). C. Longitudes de las espículas del subno-
tum (n = 175). D: Longitudes de las espículas de los tubérculos (n = 175). E: Longitudes de las espículas de las branquias
(n = 175). F: Longitudes de las espículas del pie (n = 350). G: Longitudes de las espículas del rinóforo (n = 175).
Nova Acta Cientíca Compostelana (Bioloxía), 28 (2021) 14
surface is smooth, without extensions or pro-
tuberances; however, some spicules, especially
the larger ones, may have a series of small,
irregularly distributed protuberances at their
apices (Fig. 4A).
SEM microanalysis of the fusiform spicules
showed that they are composed of: Mg, Ca, O,
C and F, elements that crystallise amorphously,
although when subjected to 400 ºC they recrys-
tallise in the form of calcite, brucite, uorite
and uorapatite. SEM microanalysis of the
spherules showed that they are composed of
Ca, O and C.
Fusiform spicules are found on the mantle
(notum and subnotum), tubercles, gills, foot
and rhinophores. Their size distribution is
more or less similar to all others, except for
the rhinophore spicules which show a diffe-
rent size distribution (Figs. 5A-G). Globular-
shaped spherules are found only on the mantle,
rhinophores and at the base of the tubercles.
In the different body areas where these
spicules are present, they are not arranged in
the same way. For instance, in the mantle, the
fusiform spicules form a network composed
of a series of multispicular bundles that form
a skeleton which is exclusively made up of
agglutinated fusiform spicules, mostly arran-
ged in the same direction. The crisscrossed
arrangement of these bundles in the ventral
mantle or subnotum delimits small spaces
of different shapes, forming a triangular or
polygonal reticulation (Figs. 4D, 6G). The
spicules of the subnotum have a smaller size
range, 86% of which are restricted to a range
of 111-260 µm in length (Fig. 5C).
In transversal section of the mantle (Fig.
6C) the multispicular ascending tracts branch
and intertwine more densely at the mantle
margins (Fig. 4E). In the dorsal mantle or
notum where the tubercles are located, the
ascending multispicular bundles show larger
gaps between them, as they converge at the base
of the tubercles. The greater the number and
size of the converging tracts are, the larger the
tubercle (Figs. 6C,F). This originates a thick
spicular stem of the tubercle (Fig. 4G), from
the upper end of the stem, a broad bundle of
spicules is arranged radially with their ends
outwards but without crossing the epidermis,
forming a hemispherical set (Figs. 4F, 6F).
The mantle spherules are arranged in an ap-
parently irregular shape and are distributed
throughout the mantle up to the base of the
tubercles where there are no spherules. The
spicules of the notum have the largest size
range between 70-770 µm in length, of which
93% are restricted to a range of 111-470 µm
in length (Fig. 5B).
In the mantle tubercles there are only fusi-
form spicules that have a reduced size range,
very similar to those of the foot, as 85% of
the spicules are circumscribed to a range of
141-320 µm in length (Fig. 5D). In the foot
there are only fusiform spicules which form a
less complex but denser net than in the mantle
(Fig. 4h, 6G), because the spicules do not form
multispicular bundles but an irregular and
complex net of abundant spicules. The spicules
of the foot have a smaller size range than the
dorsal mantle, 93% of which are restricted to
a range of 110-350 µm in length (Fig. 5F).
Both types of spicules are present in the rhi-
nophores, the fusiform spicules are irregularly
arranged parallel to the axis of the rhinophore
stalk while in the lamellae they are less numer-
ous but arranged horizontally radially (Fig.
4C, 6E); the spherules in the rhinophore are
only irregularly placed on the stalk, there are
none in the lamellae. The rhinophore spicules
show a different size distribution, as 99% of
the spicules are distributed in a size range
between 180-650 µm in length, with no more
than 2 spicules outside this range (Fig. 5G).
Finally, only fusiform spicules are found on
the branchial leaves and they are irregularly
placed, from the base to the middle of the
rachis, being less numerous than in the other
parts of the animal. In the gills, the size range
is small, as 94% of the spicules are conned
to a size range between 111-350 µm in length
(Fig. 5E).
As can be seen in gure 6 (Figs. 6A-G) Do-
ris adrianae sp. nov. presents a very complex
skeletal structure, constituting a very charac-
teristic armature in the different body areas.
In the images of this gure only the skeletal
structure is shown, as the organic matter has
Urgorri, V., Señarís, M.P. , Díaz-Agras, G., Candás, M. & Gómez-Rodríguez, C.: Doris adrianae sp. nov. 15
Figure 6. Doris adrianae sp. nov. Skeletal structure. A: Skeletal structure of the dorsal mantle and sheaths of the rhino-
phores and branchial leaves. B: Skeletal structure of ventral mantle and foot. C: Skeletal structure in cross-section: c1
at the level of the rhinophores, c2 at the level of the genital atrium and c3 at the level of the branchial leaves. D: Spicule
density on branchial leaves. E: Density of spicules in the right rhinophore. F: Detail of the skeletal structure of the dorsal
mantle. G: Detail of the skeletal structure of the ventral mantle and the edge of the foot. A-G: Paratype 16 01240510, 41
mm. (A-G Micro-CT).
Figura 6. Doris adrianae sp. nov. Estructura esquelética. A: Estructura esquelética del manto dorsal y vainas de rinóforos
y branquias. B: Estructura esquelética del manto ventral y del pie. C: Estructura esquelética en sección transversal: c1 a
nivel de los rinóforos, c2 a nivel de la atrio genital y c3 a nivel de las branquias. D: Densidad de espículas en las branquias.
E: Densidad de espículas en el rinóforo derecho. F: Detalle de la estructura esquelética del manto dorsal. G: Detalle de
la estructura esquelética del manto ventral y del borde del pie. A-G: Paratipo 16 01240510, 41 mm. (A-G Micro-CT).
Nova Acta Cientíca Compostelana (Bioloxía), 28 (2021) 16
been removed, except in the visceral cavity.
Despite this complex structure, it neither lim-
its nor hinders the animal’s ability to move,
its body shape or adaptation to the substrate
surface (Figs. 2A-G).
Digestive system: The digestive system
occupies a large part of the visceral cavity
(Figs. 9A-D) and begins in a mouth anked
by two oral lobes (Figs. 3I,K). The oral mass
is composed of a rounded buccal bulb and
a shorter glandular oral tube. The posterior
part of the oral tube has six strong retractor
muscles (Fig. 7K), three on each side, which
attach to the body wall at the posterior part
of the buccal bulb. The intermediate pair of
muscles bifurcates almost at the beginning into
two divergent muscles (mot2 in Fig. 7K). The
buccal bulb is rounded in shape, almost twice
as large as the oral tube, with a radular sac
emerging from the posterior ventral surface
and recurved posteriorly (Fig. 7K). From the
posterior-lower half of the buccal bulb arise
a pair of long, strong retractor muscles, one
on each side, which insert into the body wall,
roughly towards the middle of the visceral
cavity where the hermaphrodite and digestive
glands begin (Fig. 7K).
At the anterior end of the pharynx, towards
the oral tube, there is a chitinous labial cuti-
cle of pelted shape, the labial disc (Fig. 7G),
whose frontal surface is smoothly roughened,
while the surface of the central canal is much
rougher (Fig. 7G). In the posterior part of the
pharynx, the oesophagus opens at the upper
end (Figs. 7I,K). Two long salivary glands
connect with the pharynx on either side of
the oesophageal junction, between the vis
ceral
loop and the oesophagus, on either side of
the buccal ganglia (Figs. 7H-K); they are rib-
bon-shaped, wider in their initial section, and
extend ventrally, running under the visceral
mass to the mid-height of the stomach.
It has a multidenticulate radula (Fig. 7B),
whose radular formula is 38-50 x 3-4;33-
53;0;33-53;3-4. The radula lacks a rachidial
tooth The lateral teeth are of simple hooked
shape, with a strong ridge more marked
the larger the tooth, the tip of the tooth is
slightly blunt and its surface is smooth (Figs.
7A,C). The rst central lateral teeth are more
curved, being more hooked than the others
(Figs. 7D,E). The marginal teeth are similar
to the lateral ones but the hooked edge is not
smooth but feathery, the rst ones having a
slightly hooked shape, while the outermost
ones acquire a feathery spade-like appearance
(Fig. 7F).
The oesophagus is long and curved and
opens laterally to the stomach which is large,
curved and of variable diameter, opening pos-
teriorly to the digestive gland. There is a gastric
caecum that opens into the stomach very close
to where it connects to the oesophagus and
digestive gland. From the left anterior part of
the stomach a long intestine emerges dorsally,
it crosses across the visceral mass dorsally
and runs along the right side of the animal,
until it enters laterally into the gill crown, in
the centre of which the anus is located (Figs.
7I, 9C,D). The digestive gland is interspersed
with the hermaphrodite gland, although most
of the gonad surrounds the digestive gland
supercially (Fig. 8G).
Nervous system: In the central nervous sys-
tem (Fig. 8A) the cerebral and pleural ganglia
are fused and distinct from the pedal ganglia.
There are four cerebral nerves emerging from
each cerebral ganglion and three pleural nerves
emerging from each pleural ganglion. From
each of the pedal ganglia there are 5 pedal
nerves. The rhinophore nerves emerge one
from each cerebral ganglion, from a position
more centred in the ganglion. The buccal
ganglia (Figs. 7H, 8A) are located posterior
to the buccal bulb, below the beginning of the
oesophagus and are connected to the cerebral
ganglia by two narrow, relatively short nerves.
Very close to the buccal ganglia are the gas-
tro-oesophageal ganglia. The optic ganglion is
close to the insertion of the rhinophore nerve.
The pedal and the parapedal commissures are
together in the visceral loop.
Circulatory system: The circulatory system
consists of a heart situated in the posterior part
of the animal. The pericardial cavity is more
or less circular, anterior to the branchial leaves
ring and dorsal to the hermaphrodite and
digestive glands. The auricle is funnel-shaped
Urgorri, V., Señarís, M.P. , Díaz-Agras, G., Candás, M. & Gómez-Rodríguez, C.: Doris adrianae sp. nov. 17
Figure 7. Doris adrianae sp. nov. Digestive system, radula and labial cuticle. A: Radula tooth, Paratype 10, 01080598-02,
45 mm. B-C: Complete radula and detail of the mid-lateral teeth of the radula, Paratype 17, 01220807-01, 71 mm. D-E:
Inner lateral teeth hooked, Paratype 7, 05030597, 60 mm. F: Outer marginal teeth plumose, Paratype 17, 01220807, 71
mm. G: Labial cuticle, Paratype 1, 01060786-01, 50 mm. H: Salivary glands, buccal ganglia and gastro-oesophageal gan-
glia, Paratype 7, 05130597-01, 60 mm. I: Schematic drawing of the regionalization of the digestive system. K: Schematic
drawing of oral tube and buccal bulb. (A,H LM. B-G SEM).
Figura 7. Doris adrianae sp. nov. Aparato digestivo, rádula y cutícula labial. A: Diente de la rádula, Paratipo 10, 01080598-
02, 45 mm. B-C: Rádula completa y detalle de los dientes laterales medios de la rádula, Paratipo 17, 01220807-01, 71
mm. D-E: Dientes laterales internos ganchudos, Paratipo 7, 05030597, 60 mm. F: Dientes marginales externos plumosos,
Paratipo 17, 01220807, 71 mm. G: Cutícula labial, Paratipo 1, 01060786-01, 50 mm. H: Glándulas salivales, ganglios
bucales y ganglios gastroesofágicos, Paratipo 7, 05130597-01, 60 mm. I: Dibujo esquemático de la regionalización del
aparato digestivo. K: Dibujo esquemático del tubo oral y del bulbo bucal. (A,H OM. B-G SEM).
Nova Acta Cientíca Compostelana (Bioloxía), 28 (2021) 18
Figure 8. Doris adrianae sp. nov. Internal anatomy. A: Central nervous system, Paratype 7, 05130597-01, 60 mm. B:
Circulatory system, Paratype 7, 05130597-01, 60 mm. C: Conuent ducts in the distal oviduct. D: Bursa copulatrix and
seminal receptacle, Paratype 7, 05130597-01, 60 mm. E: Penis, Paratype 17, 01220807-01, 71 mm. F: Openings in the ge-
nital atrium, Paratype 16 01240510, 41 mm. G: Schematic drawing of the regionalization of the reproductive system. H:
Vestibular gland, Paratype 16 01240510, 41 mm. (A,B,D,E LM. C 3D reconstruction with AVIZO 6.4. F,H Micro-CT).
Figura 8. Doris adrianae sp. nov. Anatomía interna. A: Sistema nervioso central, Paratipo 7, 05130597-01, 60 mm. B:
Aparato circulatorio, Paratipo 7, 05130597-01, 60 mm. C: Conductos conuentes en el oviducto distal. D: Bursa copula-
triz y receptáculo seminal, Paratipo 7, 05130597-01, 60 mm. E: Pene, Paratipo 17, 01220807-01, 71 mm. F: Aberturas en
el atrio genital, Paratipo 16 01240510, 41 mm. G: Dibujo esquemático de la regionalización del aparato reproductor. H:
Glándula vestibular, Paratipo 16 01240510, 41 mm. (A,B,D,E OM. C Reconstrucción 3D con AVIZO 6.4. F,H Micro-CT).
Urgorri, V., Señarís, M.P. , Díaz-Agras, G., Candás, M. & Gómez-Rodríguez, C.: Doris adrianae sp. nov. 19
and provided with numerous small vessels
(Fig. 8B); ventricle below the auricle, elongat-
ed in shape. In a 32 mm specimen, 68 beats
per minute were counted in the heart. From
the heart, a dorsal vessel runs through the
animal towards the anterior region, where a
large blood gland is located above the central
nervous system; the blood gland consists of
two parts, an anterior and a posterior part,
which is the largest (Fig. 8B), revealing the
eyes between them. The renal syrinx is ellip-
tical and located on the right dorsal side of
the pericardium, near the base of the auricle
(Fig. 8B).
Reproductive system: The reproductive
system is triaulic and is located in a right lat-
ero-dorsal position between the buccal bulb
and the hermaphrodite and digestive glands
(Fig. 9B). The genital opening is located in the
subnotum (Fig. 3J), close to the epipodium,
in the anterior third of the animal’s length,
through which the genital atrium is accessed,
into which the penis, vagina and distal oviduct
open (Fig. 8F). The hermaphrodite gland is
interspersed with the digestive gland which it
wraps supercially (Fig. 8G). The preampullar
gonoduct is short and narrow and joins the
ampulla above the stomach and below the
intestine. The ampulla is long, tubular and
thick, and folds in on itself before narrowing
into the postampullar gonoduct which is
short and narrow connecting with the proxi-
mal oviduct (Figs. 8C,G, 9E,F,G). The penis
is small, elongated, fusiform and unarmed
(Fig. 8E), covered by a muscular penial sheath
which has some fusiform spicules in its distal
part. A narrow, long and sinuous deferent
duct, emerges from the penis and opens into
a large, tubular, granular prostate that folds
back on itself, nally connecting through a
short deferent duct to the proximal oviduct
(Figs. 8C,G, 9 E,F,G).
The vagina is short and wide and the prox-
imal end of the vaginal duct joins the bursa
copulatrix. From the bursa copulatrix another
duct leads to the uterine duct and the seminal
receptacle (Figs. 8C,D,G, 9E,F,G). The bursa
copulatrix is large, globose and oval in shape,
about three times larger than the seminal
receptacle which is also globose and oval. Both
have smooth surfaces with slight bulges (Fig.
8D). The short uterine duct together with the
deferent duct and the postampullary gonoduct
open into the proximal oviduct which after
a short section penetrates the female gland
through the capsule gland (Fig. 8G).
The capsule gland is centrally located and
of medium size; the membrane gland, the
smallest, is located in the ventral anterior
area; the mucus gland, the largest, surrounds
the others except in the anterior region (Fig.
8G). In the nal section of the distal oviduct,
near its opening in the genital atrium, there is
a globular structure of glandular appearance
which could be a vestibular gland, most likely
as an adhesive region (Figs. 8G,H, 9H).
DNA sequence data and phylogenetic analy-
sis: The cox1-5’ sequence for Paratype 20, DNA
is available under GenBank accession number
MW602531.The genetic distance between Do-
ris adrianae sp. nov. and other Doris species
was large (mean proportion of identical sites =
80.9% ± 1.5 (S.D.)) although similar to the ones
among Doris sequences available in GenBank
(see Table 1).The bayesian phylogenetic tree
shows Doris adrianae sp. nov. as sister species
of a clade with Doris wellingtonensis, Doris
montereyensis, Doris pseudoargus and Doris
nobilis, with a relatively high support for the
node (Bayesian posterior probability=0.709,
Fig. 10).
Spawning: The spawning is hyaline white,
slightly greyish and with the upper edge of
the band brown, while the lower edge is hya-
line. The spawning is in the form of a festoon
ribbon with 2 to 2.5 turns and 26 to 31 waves
(Fig. 9K). The height of the ribbon is 15 mm.
The egg capsules have a linear arrangement
from the upper to the lower edge, but oblique
or arched across the width of the ribbon, al-
though this tendency is more evident towards
the upper edge and less noticeable in the
lower half (Fig. 9I). The capsules have 1, 2,
3 or 4 eggs, but mostly two (Fig. 9I). On the
upper edge of the ribbon the egg capsules are
arranged almost close to the margin, while on
the lower edge there is a small hyaline strip
devoid of eggs which is the line of attachment
Nova Acta Cientíca Compostelana (Bioloxía), 28 (2021) 20
Figure 9. Doris adrianae sp. nov. 3D reconstruction of internal anatomy, adhesive region, feeding and spawning. A-B:
Organs in situ, left (A) and right (B) sides. C-D: Digestive system in lateral view, left (C) and right (D). E-G: Reproductive
system in dorsal (E), right anterior-lateral (F) and right posterior-lateral views (G). H: Adhesive region. I: Arrangement of
eggs in the spawn and egg capsules in vivo. J: Larval shell (protoconch type I). K: Spawning in type locality (Castelo de San
Felipe), brown upper edge (arrow) and fusiform spicules of the brown edge. L: The sponge Polymastia boletiformis (La-
marck, 1815) in type locality (Punta Fornelos). M: Faecal pellet full of spicules of P. boletiformis and small (133 µm) and
large (586 µm) tylostyles of the faecal pellet. (A-G 3D reconstruction with AVIZO 6.4. H LM. J,M SEM. I,K,L in vivo).
Figura 9. Doris adrianae sp. nov. Reconstrucción en 3D de la anatomía interna, región adhesiva, alimentación y desove.
A-B: Órganos in situ, lateral izquierdo (A) y derecho (A). C-D: Aparato digestivo en vista lateral, izquierda (C) y derecha
(D). E-G: Aparato reproductor en vista dorsal (E), antero-lateral derecha (F) y postero-lateral derecha (G). H: Región
adhesiva. I: Disposición de los huevos en la puesta y cápsulas con huevos in vivo. J: Concha larvaria (protoconcha tipo
I). K: Puesta en la localidad tipo (Castelo de San Felipe), borde superior marrón (echa) y espículas fusiformes del borde
marrón. L: La esponja Polymastia boletiformis (Lamarck, 1815) en la localidad tipo (Punta Fornelos). M: Pelota fecal
repleta de espículas de P. boletiformis y tilostilos pequeños (133 µm) y grandes (586 µm) de las pelotas fecales. (A-G Re-
construcción 3D con AVIZO 6.4. H OM. J,M SEM. I,K,L in vivo).
Urgorri, V., Señarís, M.P. , Díaz-Agras, G., Candás, M. & Gómez-Rodríguez, C.: Doris adrianae sp. nov. 21
to the substratum. A preparation under light
microscopic of the upper edge of the ribbon
revealed that the brown colour of the edge
was due to the presence of fusiform spicules
like those found in adult specimens on the
mantle and foot (Fig. 9K). The larval shell is
a smooth type I protoconch (thoMPson, 1961)
with a large turn of the spire and a wide oval
opening with a smooth edge (Fig. 9J).
Habitat and associated fauna: The three
localities of the Ría de Ferrol where the
specimens were collected between 11 and 20
m deep, have a very similar habitat, charac-
terised by the existence of extensive, at and
low rocky outcrops of granodiorites, where a
forest of the gorgonian Leptogorgia lusitanica
(Stiasny, 1937) is located. By its proximity
to a sandy-muddy sedimentary bottom, the
rock surface is covered with a thin layer of
ne sediment. The community beneath the
gorgonian forest is devoid of large algae, but
shows a wide diversity of sessile invertebrate
species where certain species of sponges
Polymastia boletiformis (Lamarck, 1815),
Mycale (Aegogropila) antiae Urgorri & Díaz-
Agras, 2019, Cliona celata Grant, 1826, Adreus
fascicularis (Bowerbank, 1866), Desmacidon
fruticosum (Montagu, 1814), Ciocalypta
penicillus Bowerbank, 1862 and Haliclona
(Haliclona) oculata (Linnaeus, 1759), the
cnidarians Dynamena pumila (Linnaeus,
1758), Sertularella gayi (Lamouroux, 1821),
Aglaophenia acacia Allman, 1883, Actinothoe
sphyrodeta (Gosse, 1858), Epizoanthus arena-
ceus (Delle Chiaje, 1823), Caryophyllia smithii
Stokes & Broderip, 1828, Eunicella verrucosa
(Pallas, 1766) and Alcyonium digitatum Lin-
naeus, 1758, the bivalve Mimachlamys varia
(Linnaeus, 1758), the polychaete Sabellaria
alcocki Gravier, 1906 and the ascidian Sto-
lonica socialis Hartmeyer, 1903 and also other
species of vagile invertebrates, such as the
molluscs Calliostoma zizyphinum (Linnaeus,
1758), Turritella communis Risso, 1826, Tritia
reticulata (Linnaeus, 1
758), Trapania tartanella
(Ihering, 1886 in ihering, 1886b) and Cadlina
Figure 10. Doris adrianae sp. nov. Phylogenetic tree resulting from BEAST analysis of Doris adrianae sp. nov. and cox1-5'
sequences of Doris sequences downloaded from GenBank One GenBank sequence has been selected for each species.
Node values represent Bayesian posterior probabilities.
Figura 10. Doris adrianae sp. nov. Árbol logenético resultante del análisis BEAST de Doris adrianae sp. nov. y de las
secuencias cox1-5' de Doris descargadas de GenBank Se ha seleccionado una secuencia de GenBank para cada especie.
Los valores de los nodos representan las probabilidades posteriores bayesianas.
Nova Acta Cientíca Compostelana (Bioloxía), 28 (2021) 22
laevis (Linnae
us, 1767) and the echinoderms
Marthasterias glacialis (Linnaeus, 1758), Echi-
nus esculentus Linnaeus, 1758 and Holothuria
forskali Delle Chiaje, 1823.
Feeding behaviour: The study of the contents
of the faecal pellets obtained from live animals
of Doris adrianae sp. nov. (Fig. 9M), as well
as the digestive contents of the 4 dissected
animals and that of the serial sections of two
specimens (paratypes 4 and 15), revealed a
spicular composition of tylostyles, identical
to that of the sponge Polymastia boletiformis
(Lamarck, 1815). This sponge has two types
of tylostyles, some large (500-700 µm) fusi-
form, rectilinear, with a tylo at one end and
the other ending in a point, and others small
(125-190 µm) fusiform, slightly curved and
with a scarcely marked tylo (Fig. 9M). The
exclusive presence of Polymastia boletiformis
(Lamarck, 1815) spicules in all the samples
studied, without having found any other type
of spicule, makes it possible to ensure that
Doris adrianae sp. nov. feeds exclusively on
the sponge Polymastia boletiformis (Lamarck,
1815), which is very abundant in the three
type localities of the Ría de Ferrol (Fig. 9L)
and that both species coincide remarkably in
their colouring.
DISCUSSION
According to the diagnosis of the genus
Doris Linnaeus, 1758 established by vAldés
(2002), Doris adrianae sp. nov. clearly ts
within the genus Doris on the basis of the
absence of a notch on the anterior margin of
the foot, the labial cuticle lacking rodlets, a
radula composed of simple, hamate teeth, a
penis and vagina devoid of hooks and also on
the fact that it matches the other diagnostic
characters of the genus (vAldés, 2002).
Furthermore, according to some diagnostic
characters of the other genera that comprise
the family Dorididae Ranesque, 1815, it
could not be classied in any of them, as
there are clear differences with Doris adrianae
sp. nov. For example, the genus Aphelodoris
Bergh, 1879 differs in that it does not have
integumentary spicules and has a smooth
dorsum without tubercles (vAldés, 2002).
Doris adrianae sp. nov. could not be classi-
ed within the genus Conualevia Collier &
Farmer, 1964 because this genus has mantle
glands and rhinophores almost smooth, with
several irregular and inconspicuous lamellae
(vAldés, 2002). The genus Goslineria Valdés,
2001 has several large sacs sacs in the genital
atrium, each containing a long, simple, exible
spine (vAldés, 2001), which are absent in D.
adrianae sp. nov. It also does not belong in the
genus Artachaea Bergh, 1881 in Bergh, 1881b,
because Artachaea has oral tentacles and the
penis is armed with spines (vAldés, 2002),
which Doris adrianae sp. nov. lacks (vAldés,
2002). The genus Pharadoris Valdés, 2001, has
a genital atrium with two large glands, each
containing a long, bid, rigid spine, which
Doris adrianae sp. nov. does not have. The
genus Doriopsis Pease, 1860 is characterised
by a wide semicircular crown of gill leaves very
different from those of Doris adrianae sp. nov.
Undoubtedly, the most reliable diagnostic
character of Doris adrianae sp. nov. that clearly
differentiates it from the rest of the species of
the genus Doris, is the skeletal structure that
forms a dense net of spicules, in a complex
network of multispicular bundles.
Dorids are known to have tegumentary
spicules, although most papers describe their
presence as spicules radiating from the base
of the tubercles and other vertical spicu-
les supporting the tubercles (kress, 1981;
cAttAneo-vietti, et al. 1993; Brodie, 2004;
ehrlich, 2019; orteA & esPinosA, 2017;
among others). Some authors make more de-
tailed descriptions of the skeletal structure of
dorids (gArcíA, et al. 1986; Penney, 2006), in
some use micro-computed tomography (AlBA-
tercedor & sAnchez-tocino, 2011; Penney,
et al. 2018; Penney, et al. 2020) and include
species of the genus Doris (Penney, 2008) such
as Doris montereyensis Cooper, 1863, but none
of these studies describe a skeletal structure
as complex as that of Doris adrianae sp. nov.
This complex skeletal structure could initially
be interpreted as having a primarily defensive
or at least deterrent function for potential
predators (thoMPson, 1960; ros, 1976), but,
probably in D. adrianae sp. nov. they also
Urgorri, V., Señarís, M.P. , Díaz-Agras, G., Candás, M. & Gómez-Rodríguez, C.: Doris adrianae sp. nov. 23
have a structural function, a function that was
described by cAttAneo-vietti, et al. (1993) in
Peltodoris atromaculata Bergh, 1880, stating
that they have an important role in determi-
ning the rmness, structure and architecture
of the notum.
Of the genus Doris Linnaeus, 1758, 51 spe-
cies are currently recognised as valid (WoRMS,
2021), generally with an external appearance
different from that of Doris adrianae sp. nov.
Of these, 13 are distributed along the Nor-
theastern Atlantic which ranges from Sweden
to Cape Verde. All these species, in addition
to differing in skeletal structure, they exhibit
other characters different from Doris adrianae
sp. nov. For example, Doris atypica (Eliot,
1906), Doris bicolor (Bergh, 1884 in Bergh,
1884a), Doris ocelligera (Bergh, 1881 in Bergh,
1881a) and Doris verrucosa Linnaeus, 1758,
have unipinnate branchial leaves and Doris
marmorata Risso, 1818 and Doris pseudoverru-
cosa (Ihering, 1886 in ihering, 1886a) show
bipinnate branching and in addition these six
species have rhinophoral sheaths with two
lateral tubercles that close the rhinophoral
cavity like two valves (eliot, 1906; orteA
& Moro, 2017; Bergh, 1884a; Pruvot-fol,
1954; orteA, et al. 2014; cAttAneo-vietti, et
al. 1990; vAldés, 2002; liMA & siMone, 2015;
ihering, 1886a; schMekel, 1968; schMekel
& PortMAnn, 1982). Doris hayeki Ortea, 1998
and Doris morenoi Ortea, 1989 also have uni-
pinnate leaves, both exhibit hooked marginal
teeth without denticles and their bursa copu-
latrix is slightly larger (x1.5) than the seminal
receptacle (orteA, 1989, 1998). The remai-
ning ve species have tripinnate or bipinnate
leaves, but Doris nobilis Odhner, 1907, Doris
sticta
(Iredale & O’Donoghue, 1923) and Doris
bertheloti (d’Orbigny, 1839) have rhinopho
ral
sheaths with two lateral tubercles and the
marginal teeth are hooked without denticles
(odhner, 1907, 1926; thoMPson & Brown,
1984; hunnAM & Brown, 1975; gAvAiA, et al.
2003; d’orBigny, 1839; orteA & BAcAllAdo,
1981; Perrone, 1989), while Doris alboranica
Bouchet, 1977 also has hooked marginal teeth
without denticles and a bursa copulatrix globo-
se and much larger than the seminal receptacle
which is pyriform (Bouchet, 1977). In Doris
pseudoargus Rapp, 1827 the bursa copulatrix
is irregular in shape, about 10 times larger
than the seminal receptacle and shows a single
large blood gland (vAldés, 2002). All of the
above characters clearly differentiate these
thirteen eastern North Atlantic species from
Doris adrianae sp. nov., which corroborates
the validity of this new species.
In general, the internal anatomy of Do-
ris adrianae sp. nov. matches the diagnostic
characters of the genus Doris established by
vAldés (2002). However, in the description
of the reproductive apparatus (vide supra) it
is explained that in the nal section of the
distal oviduct, close to its opening in the ge-
nital atrium, there is a globular structure of
glandular appearance that could be a vestibu-
lar gland, most likely as an adhesive region
(Figs. 8G,H, 9H). This is the only character
that does not coincide with the diagnosis of
the genus Doris, in which vAldés (2002) states
that vestibular or accessory glands absent. It
is not a vestibular gland in its usual sense,
which is very diverse, as it is usually described,
sometimes as a clearly visible and prominent
pouch, and sometimes as a small swelling, of
diverse location in the genital opening, in the
opening of the oviduct, on one side or at the
end of the female glandular mass and with
lobulated, branched, blind tubule-like forms,
among others. This type of vestibular gland
was widely mentioned in species of several
f
amilies of the Infraorder Doridoidei, but it
is considered that these glands are not homo-
logous among these families (rudMAn, 1985,
1987; gosliner, 1994; Johnson & gosliner,
1998; vAldés & cAMPillo, 2000; vAldés, 2002).
wägele (1985; 1989) described a specia-
lised glandular tissue near the distal oviduct
opening in Phyllidia pulitzeri Pruvot-Fol,
1962 and Austrodoris kerguelenensis (Bergh,
1884 in Bergh 1884b) which she refers to as
the adhesive region or “Kleberegion”, a term
already used by schMekel (1971). wägele, et
al. (1999) consider in Dendrodoris nigra that
the function of the vestibular gland has more
to do with egg laying than receipt of sperm
and klussMAnn-kolB & Brodie (1999) who
studied it histologically and ultrastructurally,
observed that internally the gland itself pre
sents
Nova Acta Cientíca Compostelana (Bioloxía), 28 (2021) 24
a characteristic convoluted appearance and
that the position of the gland seems to reect
a specic function in the nal stages of egg
mass production.
klussMAnn-kolB (1999; 2001a; 2001b)
did histological and ultrastructural research
on the nidamental glandular system in seve-
ral heterobranchs sea slugs (Cephalaspidea,
Aplysiida, Sacoglossa and Nudibranchia),
describing in detail a specialised glandular
epithelium lining the most distal part of the
oviduct, consisting of columnar glandular cells
with short to moderately long cilia, which he
named adhesive region. The vestibular gland
located in the distal oviduct of the genital
tract of Doris adrianae sp. nov. with ciliated
columnar cells (Fig. 9H) has remarkable simi-
larities with the adhesive region described by
klussMAnn-kolB (2001b). Thus, considering
that the adhesive region is frequent in several
dorid species, because it is the part where the
secretion of an adhesive substance that binds
the eggmasses to the substratum takes place,
it must also be present in many other dorid
species (wägele, personal communication).
However, a detailed histological and ultras-
tructural study of the nidamental gland of
Doris adrianae sp. nov. will be necessary to
determine with certainty that it is undoubtedly
an adhesive region.
As explained, the egg mass of Doris adria-
nae sp. nov. is a festoon ribbon of 2 to 2.5
turns with one edge adhering to the substratum
and with the upper edge brown due to the
presence of fusiform spicules. In the scientic
literature on nudibranchs (fernAndez-ovies
& orteA, 1981; klussMAnn-kolB & wägele,
2001; wilson, 2002; among others) no refe-
rence was found describing in any species the
presence of spicules on the egg ribbon, which
reveals another very specic character in Doris
adrianae sp. nov.
As far as feeding is concerned, it is a known
fact that poriferans are the basis of the diet of
dorids. Apparently, the diet does not seem to
be very selective, because in many occasions
the sponges on which the animals are placed
are considered as food. This fact should not
be taken for granted, the most reliable way is
to analyse the spicules of the faecal pellets,
which even allows us to obtain proportions
in the case that the dorid feeds on more
than one species of sponge. Thus, urgorri
& Besteiro (1984) by faecal pellet analysis
obtained a proportion of spicules of 70% of
Hymeniacidon perlevis (Montagu, 1814) and
30% of Halichondria panicea (Pallas, 1766) in
Doris verrucosa. In Doris pseudoargus 80% of
the spicules of Hymeniacidon perlevis (Mon-
tagu, 1814) and 20% of Halichondria panicea
(Pallas, 1766) were obtained in one occasion
and 100% of Halichondria panicea (Pallas,
1766) in another occasion.
Consequently, dorids do not feed on the
species that are available in their environment,
but rather they are located in those habitats
where the species they prey on are present. To
verify this fact, all the sponge species of large
size present in the type locality were collected
and identied (vide supra Habitat and associa-
ted fauna), in order to check the availability of
potential food, including Polymastia boletifor-
mis (Lamarck, 1815). Additionally, the faecal
pellets of the holotype and 11 paratypes (4,
6, 8, 9, 13, 17, 18, 18, 19, 20, 21 and 22), the
digestive contents of the dissected paratypes
(1, 7, 10, 12 and 17) and the histological sec-
tions of paratype 15 were analysed. In all of
them the result was the same, they contained
only the tylostyles of Polymastia boletiformis.
In conclusion, it can be stated that Doris
adrianae sp. nov. feeds exclusively on Poly-
mastia boletiformis despite the presence of
six other sponge species of good size in the
environment in which it lives. It is also dedu-
ced that Doris adrianae sp. nov. is only found
in habitats where Polymastia boletiformis is
present, due to a monospecic predator-prey
relationship, not appearing in other infralitto-
ral bottoms of the Ría de Ferrol where several
species of porifera are very abundant but where
Polymastia boletiformis is not present, which
determines the habitat where Doris adrianae
sp. nov. is found.
Finally, as explained in the material and
methods (vide supra DNA analysis), a total of
177 cox1-5' sequences with in the genera Doris,
Archidoris and Austrodoris were downloaded
Urgorri, V., Señarís, M.P. , Díaz-Agras, G., Candás, M. & Gómez-Rodríguez, C.: Doris adrianae sp. nov. 25
from GenBank in December 2020, whose full
list of downloaded sequences is provided in
Supplementary material Appendix 1. Doris
adrianae sp. nov. has a high genetic distance to
other species of the genus Doris whose sequen-
ces are available in GenBank. However, this
high genetic distance (approx. 20% of different
nucleotides in the barcode fragment) is simi-
lar to that among other Doris species (except
for the species pair Doris nobilis and Doris
pseudoargus). The Bayesian tree constructed
with Beast (Fig. 10), shows Doris adrianae sp.
nov. as a sister species to the clade composed
of Doris wellingtonensis, Doris montereyensis,
Doris pseudoargus and Doris nobilis, although
the support for this relationship is not high
(Bayesian posterior probability=0.709). Howe-
ver, it should be noted that this phylogenetic
analysis has been carried out on the basis
of the sequences available in GenBank and,
therefore, the diversity of the genus Doris,
for which 51 species are recognised (WoRMS,
2021), is under-represented. Therefore, genetic
distances could be smaller with other species
that are not currently represented in GenBank.
ACKNOWLEDGEMENTS
The authors would like to thank Dr.
Gonçalo Calado, Dr. Ramiro R. Tato, Dra.
Xela Cunha and Dr. Marcos Abad for their
collaboration in collecting the samples by
SCUBA diving. We would like to thank Gon-
zalo Montoto for the data and photographs of
the specimens from O Grelle (Ría da Coruña).
We thank Prof. Dr. Heike Wägele for her bi-
bliographical assistance and wise comments.
We would also like to thank Raquel Antón
and Ramiro Barreiro from the Electronic
Microscopy Service of the USC, for their
help in making the images at SEM. We are
grateful for the support of the infrastructure
PRISC - Portuguese Research Infrastructure
for Scientic Collections through the Science
Museum of the University of Coimbra. We
thank Miquel Pontes (OPK) for his biblio-
graphical assistance. In addition, we are very
grateful to Julia García who kindly checked
the English version of the paper. We are also
grateful to three anonymous reviewers for
their comments which have helped to improve
this paper.
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php?p=taxdetails&id=137914 on 2021-01-28
Table 1. Genetic distance matrix showing the uncorrected pairwise identity (i.e. proportion of identical sites in the bar-
code fragment) between sequences of Doris adrianae sp. nov and other Doris species. Sequences have been downloaded
from GenBank and one sequence has been selected per species (according to GenBank identity). GenBank accession
numbers are also provided.
Tabla 1. Matriz de distancia genética que muestra la identidad por pares no corregida (es decir, la proporción de sitios
idénticos en el fragmento del “barcode”) entre las secuencias de Doris adrianae sp. nov. y otras especies de Doris. Las
secuencias se han descargado de GenBank y se ha seleccionado una secuencia por especie (según la identidad de Gen-
Bank). También se proporcionan los números de acceso de GenBank.
Doris adrianae sp. nov.
Doris sp.
EU823145.1
Austrodoris kerguelenensis
JX680585.1
Doris kerguelenensis
AJ223256.1
Archidoris pseudoargus
KR084616.1
Doris pseudoargus
GQ292034.1
Archidoris wellingtonensis
GQ292041.1
Archidoris montereyensis
KF643613.1
Doris montereyensis
MG935354.1
Doris nobilis
Doris adrianae sp. nov. 100 78.3 78.9 80.2 82 82.6 80.3 81 80.9 82.8
Doris sp. 78.3 99.8 79.3 79.9 76.7 77 77.4 76.8 76.7 77.3
EU823145.1
Austrodoris kerguelenensis 78.9 79.3 100 95.1 81.8 81.8 79.7 80.4 80.5 82.3
JX680585.1
Doris kerguelenensis 80.2 79.9 95.1 100 82.3 82.1 80.2 81.3 81.3 82.6
AJ223256.1
Archidoris pseudoargus 82 76.7 81.8 82.3 99.2 98.5 84 86.6 86.2 99
KR084616.1
Doris pseudoargus 82.6 77 81.8 82.1 98.5 100 84.1 87.3 86.8 99.4
GQ292034.1
Archidoris wellingtonensis 80.3 77.4 79.7 80.2 84 84.1 100 84.6 83.3 84.6
GQ292041.1
Archidoris montereyensis 81 76.8 80.4 81.3 86.6 87.3 84.6 100 99.3 87.3
KF643613.1
Doris montereyensis 80.9 76.7 80.5 81.3 86.2 86.8 83.3 99.3 100 86.6
MG935354.1
Doris nobilis 82.8 77.3 82.3 82.6 99 99.4 84.6 87.3 86.6 100
Nova Acta Cientíca Compostelana (Bioloxía), 28 (2021) 32
Appendix 1. Accession number and identication of sequences downloaded from GenBank on December 2020.
Apéndice 1. Número de acceso e identicación de las secuencias descargadas de GenBank en diciembre de 2020.
GenBank
Accession
Number
Species
GenBank
Accession
Number
Species
GenBank
Accession
Number
Species
AJ223256.1 Archidoris pseudoargus EU823185.1 Austrodoris kerguelenensis JX680547.1 Doris kerguelenensis
EU823127.1 Austrodoris kerguelenensis EU823186.1 Austrodoris kerguelenensis JX680548.1 Doris kerguelenensis
EU823128.1 Austrodoris kerguelenensis EU823187.1 Austrodoris kerguelenensis JX680549.1 Doris kerguelenensis
EU823129.1 Austrodoris kerguelenensis EU823188.1 Austrodoris kerguelenensis JX680550.1 Doris kerguelenensis
EU823130.1 Austrodoris kerguelenensis EU823189.1 Austrodoris kerguelenensis JX680551.1 Doris kerguelenensis
EU823131.1 Austrodoris kerguelenensis EU823190.1 Austrodoris kerguelenensis JX680552.1 Doris kerguelenensis
EU823132.1 Austrodoris kerguelenensis EU823191.1 Austrodoris kerguelenensis JX680553.1 Doris kerguelenensis
EU823133.1 Austrodoris kerguelenensis EU823192.1 Austrodoris kerguelenensis JX680554.1 Doris kerguelenensis
EU823134.1 Austrodoris kerguelenensis EU823193.1 Austrodoris kerguelenensis JX680555.1 Doris kerguelenensis
EU823135.1 Austrodoris kerguelenensis EU823194.1 Austrodoris kerguelenensis JX680556.1 Doris kerguelenensis
EU823136.1 Austrodoris kerguelenensis EU823195.1 Austrodoris kerguelenensis JX680557.1 Doris kerguelenensis
EU823137.1 Austrodoris kerguelenensis EU823196.1 Austrodoris kerguelenensis JX680558.1 Doris kerguelenensis
EU823138.1 Austrodoris kerguelenensis EU823197.1 Austrodoris kerguelenensis JX680559.1 Doris kerguelenensis
EU823139.1 Austrodoris kerguelenensis EU823198.1 Austrodoris kerguelenensis JX680560.1 Doris kerguelenensis
EU823140.1 Austrodoris kerguelenensis EU823199.1 Austrodoris kerguelenensis JX680561.1 Doris kerguelenensis
EU823141.1 Austrodoris kerguelenensis EU823200.1 Austrodoris kerguelenensis JX680562.1 Doris kerguelenensis
EU823142.1 Austrodoris kerguelenensis EU823201.1 Austrodoris kerguelenensis JX680563.1 Doris kerguelenensis
EU823143.1 Austrodoris kerguelenensis EU823202.1 Austrodoris kerguelenensis JX680564.1 Doris kerguelenensis
EU823144.1 Austrodoris kerguelenensis EU823203.1 Austrodoris kerguelenensis JX680565.1 Doris kerguelenensis
EU823145.1 Austrodoris kerguelenensis EU823204.1 Austrodoris kerguelenensis JX680566.1 Doris kerguelenensis
EU823146.1 Austrodoris kerguelenensis EU823205.1 Austrodoris kerguelenensis JX680567.1 Doris kerguelenensis
EU823147.1 Austrodoris kerguelenensis EU823206.1 Austrodoris kerguelenensis JX680568.1 Doris kerguelenensis
EU823148.1 Austrodoris kerguelenensis EU823207.1 Austrodoris kerguelenensis JX680569.1 Doris kerguelenensis
EU823149.1 Austrodoris kerguelenensis EU823208.1 Austrodoris kerguelenensis JX680570.1 Doris kerguelenensis
EU823150.1 Austrodoris kerguelenensis EU823209.1 Austrodoris kerguelenensis JX680571.1 Doris kerguelenensis
EU823151.1 Austrodoris kerguelenensis EU823210.1 Austrodoris kerguelenensis JX680572.1 Doris kerguelenensis
EU823152.1 Austrodoris kerguelenensis EU823211.1 Austrodoris kerguelenensis JX680573.1 Doris kerguelenensis
EU823153.1 Austrodoris kerguelenensis EU823212.1 Austrodoris kerguelenensis JX680574.1 Doris kerguelenensis
EU823154.1 Austrodoris kerguelenensis EU823213.1 Austrodoris kerguelenensis JX680575.1 Doris kerguelenensis
EU823155.1 Austrodoris kerguelenensis EU823214.1 Austrodoris kerguelenensis JX680576.1 Doris kerguelenensis
EU823156.1 Austrodoris kerguelenensis EU823215.1 Austrodoris kerguelenensis JX680577.1 Doris kerguelenensis
EU823157.1 Austrodoris kerguelenensis EU823216.1 Austrodoris kerguelenensis JX680578.1 Doris kerguelenensis
EU823158.1 Austrodoris kerguelenensis EU823217.1 Austrodoris kerguelenensis JX680579.1 Doris kerguelenensis
EU823159.1 Austrodoris kerguelenensis EU823218.1 Austrodoris kerguelenensis JX680580.1 Doris kerguelenensis
EU823160.1 Austrodoris kerguelenensis GQ292034.1 Archidoris wellingtonensis JX680581.1 Doris kerguelenensis
EU823161.1 Austrodoris kerguelenensis GQ292035.1 Doris kerguelenensis JX680582.1 Doris kerguelenensis
EU823162.1 Austrodoris kerguelenensis GQ292036.1 Doris kerguelenensis JX680583.1 Doris kerguelenensis
EU823163.1 Austrodoris kerguelenensis GQ292037.1 Doris kerguelenensis JX680584.1 Doris kerguelenensis
EU823164.1 Austrodoris kerguelenensis GQ292038.1 Doris kerguelenensis JX680585.1 Doris kerguelenensis
EU823165.1 Austrodoris kerguelenensis GQ292039.1 Doris kerguelenensis JX680586.1 Doris kerguelenensis
EU823166.1 Austrodoris kerguelenensis GQ292041.1 Archidoris montereyensis JX680587.1 Doris kerguelenensis
Urgorri, V., Señarís, M.P. , Díaz-Agras, G., Candás, M. & Gómez-Rodríguez, C.: Doris adrianae sp. nov. 33
EU823167.1 Austrodoris kerguelenensis GQ292046.1 Doris sp. JX680588.1 Doris kerguelenensis
EU823168.1 Austrodoris kerguelenensis GU227115.1 Austrodoris kerguelenensis JX680589.1 Doris kerguelenensis
EU823169.1 Austrodoris kerguelenensis JX680531.1 Doris kerguelenensis KC153022.1 Doris montereyensis
EU823170.1 Austrodoris kerguelenensis JX680532.1 Doris kerguelenensis KF643435.1 Doris montereyensis
EU823171.1 Austrodoris kerguelenensis JX680533.1 Doris kerguelenensis KF643446.1 Doris montereyensis
EU823172.1 Austrodoris kerguelenensis JX680534.1 Doris kerguelenensis KF643613.1 Doris montereyensis
EU823173.1 Austrodoris kerguelenensis JX680535.1 Doris kerguelenensis KF643914.1 Doris montereyensis
EU823174.1 Austrodoris kerguelenensis JX680536.1 Doris kerguelenensis KF644212.1 Doris montereyensis
EU823175.1 Austrodoris kerguelenensis JX680537.1 Doris kerguelenensis KR084378.1 Doris pseudoargus
EU823176.1 Austrodoris kerguelenensis JX680538.1 Doris kerguelenensis KR084586.1 Doris pseudoargus
EU823177.1 Austrodoris kerguelenensis JX680539.1 Doris kerguelenensis KR084616.1 Doris pseudoargus
EU823178.1 Austrodoris kerguelenensis JX680540.1 Doris kerguelenensis KR084907.1 Doris pseudoargus
EU823179.1 Austrodoris kerguelenensis JX680541.1 Doris kerguelenensis MF958425.1 Doris montereyensis
EU823180.1 Austrodoris kerguelenensis JX680542.1 Doris kerguelenensis MG422353.1 Doris montereyensis
EU823181.1 Austrodoris kerguelenensis JX680543.1 Doris kerguelenensis MG935320.1 Doris pseudoargus
EU823182.1 Austrodoris kerguelenensis JX680544.1 Doris kerguelenensis MG935354.1 Doris nobilis
EU823183.1 Austrodoris kerguelenensis JX680545.1 Doris kerguelenensis MG935407.1 Doris pseudoargus
EU823184.1 Austrodoris kerguelenensis JX680546.1 Doris kerguelenensis MH242734.1 Doris montereyensis
Apéndice 1. Continuación
