Morphological descriptions of the eggcases of skates (Rajidae) from the central‑Western Mediterranean, with notes on their distribution

Abstract

Eggcases of eight rajiform skates (Dipturus nidarosiensis, D. oxyrinchus, Leucoraja melitensis, Raja asterias, R. brachyura, R. clavata, R. miraletus and R. polystigma) present in the central-western Mediterranean are described, based on specimens obtained from fishery surveys. Eggcase features such as dimensions, horns and apron lengths, and presence/absence of lateral keels were crucial to discriminate the eggcases of the various species. Morphological and morphometric data, confirmed by the multivariate analysis, indicated that the eggcase of R. miraletus and L. melitensis were distinct from those of the other species for being unkeeled. Within the species having keeled eggcases, those of the genus Dipturus and R. brachyura were discriminated from the remaining group by having the largest dimensions and aprons. Sandy bottoms (<100–150 m depth) were identified as egg-laying sites (i.e. sites with females bearing eggcases in uteri) for many species belonging to genus Raja Raja asterias, R. brachyura, R. miraletus and R. polystigma). The finding of R. asterias and R. miraletus carrying eggcases yearly on the same sites, seems to confirm the theory that many rajid species demonstrate site fidelity, returning to the same depositional area on an annual basis. Some remarks on reproductive biology of these skates are also provided. The eggcase identification key reported here represents the first for the Mediterranean and may be useful, in the future, to identify egg-laying grounds of skates with a nonlethal method.

Full text

Porcu et al. Helgol Mar Res (2017) 71:10
DOI 10.1186/s10152-017-0490-2
ORIGINAL ARTICLE
Morphological descriptions ofthe
eggcases ofskates (Rajidae) fromthe
central-western Mediterranean, withnotes
ontheir distribution
Cristina Porcu1*, Martina F. Marongiu1, Andrea Bellodi1, Rita Cannas1, Alessandro Cau1,2, Riccardo Melis1,
Antonello Mulas1, Giuditta Soldovilla1, Laura Vacca1 and Maria C. Follesa1
Abstract
Eggcases of eight rajiform skates (Dipturus nidarosiensis, D. oxyrinchus, Leucoraja melitensis, Raja asterias, R. brachyura, R.
clavata, R. miraletus and R. polystigma) present in the central-western Mediterranean are described, based on speci-
mens obtained from fishery surveys. Eggcase features such as dimensions, horns and apron lengths, and presence/
absence of lateral keels were crucial to discriminate the eggcases of the various species. Morphological and morpho-
metric data, confirmed by the multivariate analysis, indicated that the eggcase of R. miraletus and L. melitensis were
distinct from those of the other species for being unkeeled. Within the species having keeled eggcases, those of the
genus Dipturus and R. brachyura were discriminated from the remaining group by having the largest dimensions
and aprons. Sandy bottoms (<100–150 m depth) were identified as egg-laying sites (i.e. sites with females bearing
eggcases in uteri) for many species belonging to genus Raja Raja asterias, R. brachyura, R. miraletus and R. polystigma).
The finding of R. asterias and R. miraletus carrying eggcases yearly on the same sites, seems to confirm the theory
that many rajid species demonstrate site fidelity, returning to the same depositional area on an annual basis. Some
remarks on reproductive biology of these skates are also provided. The eggcase identification key reported here repre-
sents the first for the Mediterranean and may be useful, in the future, to identify egg-laying grounds of skates with a
nonlethal method.
Keywords: Rajidae, Mediterranean Sea, Eggcases, Identification key, Distribution
© The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made.
Background
e Rajidae family represents the most species-rich
group among cartilaginous fish, having 30 genera and 245
valid species [1]. ese demersal elasmobranchs often
have limited, well-defined distributions, presumed low
natural mortality [2] and are an important component
of marine biodiversity. Many species live close to shore,
generally at depths <100m [3]. Some species are known
to undertake seasonal migrations towards egg-laying
grounds which maybe located close to shore [4], or over
continental slopes, and usually discovered during explor-
atory surveys [5].
Among elasmobranchs, skates, together with the
Scyliorhinidae family and the Heterodontiformes order,
are the only strictly oviparous group [6] producing a
tough eggcase that preserves the embryo development,
ranging from months to years depending on the species
[7, 8]. Most rajids show single oviparity (i.e., one embryo
per eggcase), with pairs of eggcases (one from each ovi-
duct) deposited during the spawning season [9]. e
few exceptions include Raja pulchra [10] and R. binocu-
lata [11], which can produce multiple embryos in each
eggcase.
One of the greatest issues characterizing this family is
historically linked to their taxonomy, often problematical,
Open Access
Helgoland Marine Research
*Correspondence: cporcu@unica.it
1 Department of Life and Environmental Sciences (DISVA), University
of Cagliari, Via Fiorelli 1, 09126 Cagliari, Italy
Full list of author information is available at the end of the article

Page 2 of 14
Porcu et al. Helgol Mar Res (2017) 71:10
due to phenotypic similarity between some taxa and
individual variability in others, and are responsible for
their misidentification, related probably to biological
and environmental characteristics. Another tool to dis-
tinguish skate species from each other, after examining
their external morphology, is looking at their eggcases
morphology. is feature is unique to these species and
can be used for their identification [10, 12–17]. Moreo-
ver, the finding of sites where demersal eggcases are laid
on the bottom gives information concerning distribution
and reproductive ecology [16, 18], such as the spawning
habitats.
Four genera of Rajidae live in the Mediterranean basin
(Dipturus, Leucoraja, Raja and Rostroraja) with 16 pos-
sible valid species [19]. In spite of the fact that chondrich-
thyan reported landings in the Mediterranean Sea have
considerably increased in recent years [20], many aspects
of their reproductive biology are still unknown. Eggcases
have been described for some skates in the Mediterra-
nean basin [15, 22, 23], but there are few published data
on their lengths and widths. Furthermore, regional iden-
tification keys based on eggcase morphology and com-
parative studies are lacking.
e aim of this work is to provide a detailed morpho-
metric and morphological description of eight Mediterra-
nean skate species eggcases, providing also comparisons
with previously published data. In particular, seven of
these were caught around Sardinian waters, central-west-
ern Mediterranean (Dipturus nidarosiensis recently
reported for the Mediterranean [24, 25], D. oxyrinchus,
Raja asterias, R. brachyura, R. clavata, R. miraletus and
R. polystigma) and only one (Leucoraja melitensis) was
found in the Sentinelle Bank (Sardinian Channel, off the
Sardinian waters). Moreover, a specific key that could be
useful in the identification of eggcases found on the sea
bed, information on the distribution of active females per
species and a brief description of the bottom biocenosis,
useful to identify possible nursery sites for these vulner-
able organisms, were provided.
Furthermore, given the common pattern for which
an intraspecific latitudinal cline in elasmobranch sizes
between Atlantic and Mediterranean species (e.g. [26])
exists, it could be hypothesized that the same dynamic
could be reflected in the eggcase dimensions [27, 28]. For
this reason, we investigated in this way, considering also
the interspecific variability of eggcases, that may repre-
sent an adaptation to several kind of bottoms in order to
minimize the competition for nursery grounds.
Methods
Active females (i.e. with eggcases in the uteri or extrud-
ing from the cloaca; henceforth referred to as active) of
seven rajids present in Sardinian waters (central-western
Mediterranean Sea), were collected between 2005 and
2016 during seasonal experimental surveys and com-
mercial hauls (trammels and trawlings) at depths from
28 to 1700m. Additional active individuals belonging to
L. melitensis were caught in 2017 (February and March)
during commercial trawlings in the “Sentinelle Bank” at
200m of depth from a vessel registered in a Sardinian
district (Cagliari).
For each individual, the total length (TL) was recorded
in millimeters (mm).
For an accurate identification, eggcases were removed
directly from the uteri of active females, except those of
D. nidarosiensis specimens, which were collected from
the sea bed. Eggcases were photographed using a Canon
EOS 1100D, preserved in 80% ethanol and deposited in
the collection at the Department of Life and Environ-
mental Sciences, University of Cagliari, Italy. To assure
a proper identification of a fully formed embryo found
within an eggcase on the sea floor, presumably belonging
to D. nidarosiensis, a piece of muscle was collected from
the embryo and stored in absolute ethanol at −20°C in
order to proceed to the genetic identification. e COI-3
primers cocktail and PCR conditions from Ivanova etal.
[29] were used to amplify the cytochrome oxidase I gene
(COI).
As reported by Gordon etal. [30], the term “anterior”
used in this work is considered to refer to the part that
forms first in the oviducal gland. Ten morphometric
measures (Fig. 1) were recorded in millimeters using a
caliper following Concha etal. [31]: eggcase length (ECL,
measured longitudinally between the anterior and pos-
terior apron borders); eggcase width (ECW, the trans-
verse width of the eggcase in its lateral plane included
the keels); anterior and posterior apron length (AAL and
PAL, the distance from the central body eggcase to the
apron border); left and right keel width (LKW and RKW,
the transverse width of the case from the eggcase keel
junction to the keel edge); left and right anterior horn
length (LAHL and RAHL, the distance from the anterior
apron border to the horn tips); left and right posterior
horn length (LPHL and RPHL, the distance from the pos-
terior apron border to the horn tips). All measurements
were then expressed in % of ECL and ECW in order to
easily compare them to the others. e measurements
in millimeters were also reported to contrast with those
present in literature. In addition, the general morphology
(shape and other features), fresh color (using the Pantone
Matching System, PMS, Carlstadt, NJ, USA), texture and
presence and position of adhesion fibres were recorded.
Statistical analysis
e PRIMER (v6) package [32] was used to analyze the
morphometric measures matrix. e outcomes were

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Porcu et al. Helgol Mar Res (2017) 71:10
subjected to a Principal Component Analysis (PCA). e
components that mostly contributed to the variance were
identified. In addition, the SIMPER procedure (SIMilarity
PERcentage Analysis) was used to identify those meas-
ures responsible for discriminating between groups. e
observed differences between groups were tested using an
analysis of Similarity Randomization Test (ANOSIM) [33].
Moreover, for each species, the geographical coordinates
where females carried eggcases were visualized were pro-
vided using Mapsource software version 6.16.3 (Garmin).
During fishing activities, the benthic community, associ-
ated with the bottom at each site, found in the net, was
recorded. For each haul, all taxa were identified following
the taxonomic guide for the Mediterranean [34] and in
addition number and weight of every species (if possible)
were registered in order to determine their abundance.
Results
A total of 177 eggcases of eight rajid species were exam-
ined and measured.
Eggcase description
Genus: Dipturus
Dipturus nidarosiensis (Storm, 1881) (n=5)
During the sampling period, only one female carrying
eggcases (not yet fully formed with only anterior horns
and the apron visible) was caught in September 2015 at a
depth of 991m (Table1). However, one eggcase contain-
ing a well-developed embryo and four empty eggcases
were trawled at depths of 974–1212m.
e embryo was a male (TL 193 mm) showing the
typical features of this species: medium brown to grey-
brownish dorsal surface and dark brown ventral surface,
darker than the dorsal surface. e embryo’s sequence
(658bp long) was compared to 38 COI sequences of the
genus Dipturus from Cariani etal. [35] and resulted iden-
tical to the specimens of D. nidarosiensis from the Medi-
terranean Sea (GenBank accession id: KT307210).
e eggcase appeared very large (Fig. 2a) reaching a
maximum of 177mm ECL (Table2). e width was 43.9%
of the length, giving a long rectangular shape (Table3).
Anterior and posterior aprons were well developed (25.1
Fig. 1 Morphometric measurements performed in the eggcases. AAL
anterior apron length, ECL eggcase length, ECW eggcase width, LAHL
left anterior horn length, LKW left keel width, LPHL left posterior horn
length, PAL posterior apron length, RAHL right anterior horn length,
RKW right keel width, RPHL right posterior horn length
Table 1 Females carrying eggcases analyzed from2005 to2017, represented byspecies, total length (TL) range, depth-
range andmean (mean±SD) andsampling time
In italics, the number of females carrying eggcases per month
Species TL range (mm) Depth range (m) Sampling time
J F M A M J J A S O N D
Dipturus nidarosiensis 1376 991 1
Dipturus oxyrinchus 1010–1070 100–620 (500 ± 144) 524 4411 433
Leucoraja melitensis 335–382 200 1 3
Raja asterias 517–725 28–92 (44 ± 13) 1 37 34 7
Raja brachyura 730–1060 30–62 (47 ± 9) 3763
Raja clavata 610–871 42–473 (144 ± 118) 31 843
Raja miraletus 346–481 32–158 (69 ± 24) 3 2 39 13 17
Raja polystigma 512–595 38–126 (56 ± 24) 741

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Porcu et al. Helgol Mar Res (2017) 71:10
and 20.2% of ECL, respectively): the first was straight, on
the contrary, the latter was rounded and slightly shorter
(Table3). Both anterior and posterior horns were short
(presumably damaged) and lateral keels were present and
well developed (9.0% of ECW) (Table 3). It possessed
adhesion fibrils attached to the keels, and in the dorsal
and ventral surfaces.
Dipturus oxyrinchus (Linnaeus, 1758) (n=37)
Active females were found during all months of the year,
except in April and September, at a mean depth of 500m
(Table1).
is species (Fig.2b) had a smaller eggcase than its con-
generic D. nidarosiensis with a maximum of 116.2mm
ECL (Table 2). e eggcases had a rectangular shape
(Table3), since the ECW was 54.9% of ECL. Anterior and
posterior aprons were well developed (25.1 and 14.2% of
ECL, respectively), and the anterior one was longer and
straighter in contrast to the posterior, which was more
rounded and shorter (Table3). Horns were short with
anteriors slightly longer and tapered then the posterior
ones (33.7 and 31.3% of ECL, respectively). Lateral keels
were pronounced (9.6% of ECW) and had adhesive fibres.
e fresh color varied from brown shades (PMS 4485,
1535) to greenish ones (PMS 3975) (Fig.2b; Table3).
Genus: Leucoraja
Leucoraja melitensis (Clarke, 1926) (n=6)
Females with eggcases were caught in the Sentinelle Bank
(N38° 03′ 314′′ E9° 41′ 998′′) in February and March at a
depth of 200m (Table1). L. melitensis had small eggcases
(45.0mm ECL maximum, Table2), rectangular in shape
(ECW 54.0% of ECL). Anterior apron (13.5% of ECL) was
longer then the posterior one (1.7% of ECL). e posterior
horns were moderate and thin, differently from the anteri-
ors, very long, thin and intersecting (103.9% of ECL). No
lateral keels were observed and the surface was smooth
and semi-transparent with no external fibres covering the
surface (Fig.2c). e fresh color was yellowish (PMS 110)
with brown shades (PMS 125) (Table3).
Genus: Raja
Raja asterias Delaroche, 1809 (n=59)
Active females were found in late spring and summer
(May to August) exclusively on the continental shelf
(28–92 m depth) (Table1). e eggcases (Fig. 2d) were
Fig. 2 Eggcases belonging to the eight rajid species present in central-western Mediterranean. a D. nidarosiensis; b D. oxyrinchus; c L. melitensis; d R.
asterias; e R. brachyura; f R. clavata; g R. miraletus; h R. polystigma

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Porcu et al. Helgol Mar Res (2017) 71:10
Table 2 Eggcase morphometric measurements ofthe eight rajid species analyzed
Species N ECL ECW AAL PAL RKW LKW RAHL LAHL RPHL LPHL
D. nidarosiensis 5 Range 153–177 61.8–80.5 38.2–49.7 26.2–40.9 6.5–7.7 6.9–7.8 9–14.6 9–12.4 18.1–25 18.1–35.0
Mean ± SD 170.2 ± 9.8 74.8 ± 7.5 42.6 ± 4.4 34.3 ± 5.4 7.2 ± 0.5 7.3 ± 0.4 10.9 ± 2.1 10.5 ± 1.2 21.2 ± 2.5 23.2 ± 6.7
D. oxyrinchus 37 Range 91.6–116.2 48.9–67.5 20.3–31.7 7.7–20.8 3.2–8 3.2–8 27.1–47.4 27.1–47.4 26.1–40.4 26.1–40.4
Mean ± SD 103.8 ± 5.3 57.0 ± 4.0 26.0 ± 2.5 14.9 ± 3.0 5.4 ± 1.1 5.4 ± 1.1 35.0 ± 6.1 35.0 ± 6.1 32.5 ± 5.1 32.5 ± 5.1
L. melitensis 6 Range 42.3–45 22.4–26 5.6–6.4 0.4–1.3 – – 42.5–52.5 38.9–52.0 16.5–20.8 13.1–20.2
Mean ± SD 44.1 ± 1.0 23.8 ± 1.4 6.0 ± 0.4 0.7 ± 0.4 – – 47.0 ± 4.0 44.7 ± 4.8 18.2 ± 1.9 16.6 ± 2.6
R. asterias 59 Range 39.7–55.4 22.4–40.5 4.5–8.9 1–3.8 1.5–4.3 1.5–4.3 18.8–30.5 18.8–30.5 12.1–27.3 12.1–27.3
Mean ± SD 48.3 ± 3.0 33.7 ± 2.6 6.9 ± 1.0 2.1 ± 0.7 2.4 ± 0.7 2.4 ± 0.7 24 ± 3.3 24 ± 3.3 22.3 ± 3.1 22.3 ± 3.1
R. brachyura 12 Range 113–133 64.7–72.2 18.5–25.7 17.5–26.6 3.5–7.4 3.5–7.4 55.6–80.7 55.2–80.7 30.4–59.2 30.4–59.2
Mean ± SD 119.3 ± 4.9 69.5 ± 2.3 21.9 ± 2.5 21.7 ± 2.6 5.5 ± 1.4 5.7 ± 1.4 69.7 ± 10.0 69.5 ± 10.3 43.7 ± 11.0 44. ± 10.7
R. clavata 10 Range 62.5–67.5 41.7–47.4 9.5–15.9 6–8.4 3.1–4.8 3.1–4.8 25–40.6 25–40.6 19.2–24.5 19.2–24.5
Mean ± SD 64.1 ± 1.9 43.8 ± 2.0 11.4 ± 1.8 6.8 ± 0.9 3.8 ± 0.6 3.8 ± 0.6 32.5 ± 5.0 32.5 ± 5.0 22.7 ± 1.6 22.7 ± 1.6
R. miraletus 34 Range 41.8–56.5 22.8–29.2 3–9.9 1.3–4.2 – – 8.8–27.9 8.8–27.9 10.2–21.7 10.2–21.7
Mean ± SD 47.4 ± 4.0 24.7 ± 1.7 7.2 ± 1.7 2.7 ± 0.8 18.1 ± 4.7 18.1 ± 4.7 16.3 ± 3.3 16.3 ± 3.3
R. polystigma 14 Range 56.6–69.2 33.9–44.3 4.7–13.4 1–2.8 0.8–2.7 0.8–2.3 18.9–36.0 18.4–35.2 12–26.5 12–26.6
Mean ± SD 62.3 ± 4.3 37.9 ± 2.9 9.4 ± 2.4 2.0 ± 0.6 1.7 ± 0.6 1.6 ± 0.5 27.5 ± 5.2 27.3 ± 5.1 22.4 ± 4.8 22.3 ± 4.8

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Porcu et al. Helgol Mar Res (2017) 71:10
small (55.4mm ECL maximum, Table2) with a rectangu-
lar shape (ECW 70.0% of ECL). Aprons were different to
each other: the posterior was rounded and narrow (4.3%
of ECL), and the anterior was straight and long (14.2% of
ECL) (Table3). Anterior and posterior horns were mod-
erate in length and thin (49.6 and 46.0% of ECL, respec-
tively). Lateral keels were pronounced (2.4% of ECW) with
adhesive fibres (Table3). e fresh color was yellowish
(PMS 110) with brown shades (PMS 125) (Fig.2d; Table3).
Raja brachyura LaFont, 1873 (n=12)
Active females were caught from May to August in shal-
low waters (30–62m depth) (Table1). e eggcases had
a rectangular shape (ECW about 58.3% of ECL) and
were large in size (maximum ECL=133 mm) (Fig.2e;
Tables 2, 3). Posterior apron was rounded and pro-
nounced and the anterior one was slightly longer and
straighter (Table3). e posterior horns were short and
sturdy, while the anterior ones were thin and long (18.2%
of ECL). Keels were developed (8.3% of ECW) and had
attachment fibres. e fresh color was reddish brown in
the eggcase body (PMS 469) and greenish (PMS 399) in
the edges along the keels (Fig.2e; Table3).
Raja clavata (Linnaeus, 1758) (n=10)
Active females were sampled during the summer (from
June to August) and winter months (January and Febru-
ary) displaying a broad bathymetric distribution (42–
473m depth) (Table1).
is skate had a medium size eggcase (maximum
ECL=67.5mm, Table2) with a rectangular shape (ECW
68.3% of ECL) (Fig.2f). e eggcases had a rounded ante-
rior apron and a straight posterior one (Table3); the for-
mer was longer than the posterior one (17.8 and 10.6% of
ECL respectively). Posterior horns were short and sturdy
(35.3% of ECL), instead the anterior ones were thin and
moderately long (50.4% of ECL). Lateral keels were pro-
nounced (8.7% of ECW) and presented adhesive fibres
(Table3). To the naked eye, the eggcase surface appeared
covered by several fibres; the fresh color varied from red-
dish brown (PMS 1395, 139) in the eggcase body, to yel-
lowish in the edges (PMS 119) (Fig.2f; Table3).
Raja miraletus Linnaeus, 1758 (n=34)
Active females were caught mostly in late spring and
summer (May to August) and only a few times in winter
(January), exclusively in the continental shelf (32–158m
depth) (Table1).
e eggcases were small (Fig.2g) with a maximum of
56.5mm ECL (Table2) and rectangular in shape (ECW
52.3% of ECL). e posterior apron was rounded and
short (5.5% of ECL), and the anterior one was straight
and three times the length of the anterior (15.2% of ECL).
Posterior horns were short (34.5% of ECL) and sturdy,
while the anteriors were thinner and slightly longer
(38.3% of ECL) (Tables2, 3). Lateral keels were totally
absent and the adhesion fibres were attached to the horns
(Table3). e eggcase surface was covered with visible
Table 3 General description ofrajid eggcases
Species Shape Anterior
horns Posterior horns Anterior apron Posterior apron Keels Fresh color
D. nidarosiensis Long rectangular Short Short and sturdy Straight and well
developed Rounded and well
developed Well evident –
D. oxyrinchus Rectangular Short Short and sturdy Straight and well
developed Rounded and well
developed Well evident Shades of brown (PMS
4485, 1535), and
greenish (PMS 3975)
L. melitensis Rectangular Long, thin and
intersecting Moderate and thin Rounded and
moderate Rounded and
narrow Absent Yellowish (PMS 110)
with brown shades
(PMS 125)
R. asterias Rectangular Moderate and
thin Moderate and thin Straight Rounded and
narrow Evident Yellowish (PMS 110)
with brown shades
(PMS 125)
R. brachyura Rectangular Long and thin Short and sturdy Straight and well
developed Rounded and well
developed Well evident Reddish brown (PMS
469) and greenish
(PMS 399)
R. clavata Rectangular almost
square Moderate and
thin Short and sturdy Straight and mod-
erate Rounded and
moderate Well evident Reddish brown (PMS
1395, 139), yellow-
ish brown (PMS 119)
R. miraletus Rectangular Short and thin Short and sturdy Straight Rounded and
narrow Absent Brown (PMS 161) and
amber (PMS 138)
R. polystigma Rectangular Moderate and
thin Moderate and
sturdy Straight and mod-
erate Rounded and
narrow Narrow Brown (PMS 133, 161)
and yellowish (PMS
126)

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Porcu et al. Helgol Mar Res (2017) 71:10
fibres; the fresh color varied from brown (PMS 161) to
amber (PMS 138) (Fig.2g; Table3).
Raja polystigma Regan, 1923 (n=14)
As with R. asterias and R. brachyura, active females were
found only during the summer season (June to August)
on the continental shelf (38–126 m depth) (Table 1).
is skate presented a medium size eggcase (maximum
ECL = 69.2 mm, Table2) with rectangular shape (ECW
60.9% of ECL) (Fig.2h). e eggcases had a rounded and
narrow posterior apron and a moderately developed straight
anterior one (3.2 and 15.0% of ECL, respectively) (Table2).
Posterior horns were moderate and sturdy (35.8% of ECL),
instead the anterior ones were thin and had a moderate
length (43.9% of ECL). Lateral keels were narrow (4.4% of
ECW) and had adhesion fibres (Table3). e fresh color
had shades of brown (PMS 133, 161) in the eggcase body,
and yellowish in the edges (PMS 126) (Fig.2h; Table3).
Statistical analysis
Eggcase measurements of D. nidarosiensis (all found on
the sea bed) were excluded from this analysis because
they could have been damaged during the recovery in
fishing operations (especially the horns) and the meas-
urements could have been different with respect to those
found in uteri.
e results of the PCA (Fig.3) highlighted the existence
of the four groups with high dissimilarity values. In particu-
lar, group A (composed of eggcases of D. oxyrinchus and
R. brachyura) was different from groups B (composed of
eggcases of R. asterias, R. clavata and R. polystigma) princi-
pally in the eggcase dimension (ECL) and the anterior and
posterior aprons length (AAL and PAL) and from group
D (L. melitensis) in ECL and PAL. Group C (composed by
eggcases of R. miraletus) was dissimilar to group B essen-
tially for the lack of the keels (RKW and LKW) and to the
other group (A) for eggcase length (ECL), width (ECW)
and anterior horn length RAHL and LAHL). Finally, group
D has discriminated from groups B and C especially for the
anterior horn lengths (RAHL and LAHL) as confirmed also
by the SIMPER routine (Table4).
Based on the main eggcase characteristics of each
species and their differences with the other ones and
considering statistical analysis, we provided a species
identification key based on the eggcase descriptions
(Table5).
Eggcase distribution
Females carrying eggcases of R. asterias and R. brachyura
(Fig.4) were caught mainly around the west coast in shal-
low waters (within a depth of 100m) characterized by
sandy bottoms in association with the seagrass Posidonia
oceanica (Linnaeus) Delile 1813, the sea star Astropecten
aranciacus (Linnaeus, 1758) and the seaweed Codium
bursa Agardh, 1817.
Raja miraletus active females (Fig.4) were distrib-
uted all around Sardinia with the exception of the
south-eastern part. The specimens preferred sandy
substrata with low algal cover populated mainly by
the irregular sea-urchins Spatangus purpureus (Mül-
ler, 1776) and the holothuroid Parastichopus regalis
(Cuvier, 1817). The highest occurrence of females car-
rying eggcases was observed in the central western
side, with more specimens caught on the same sites
(N40° 11′131′′ E8° 23′155′′ at a depth of 80m) over
several years of sampling. Also R. polystigma females
were sampled in sandy bottoms within the continen-
tal shelf (38–126 m), but given the few specimens
collected (Fig. 4), it was difficult to establish if this
species had a geographical preference. The same pat-
tern was observed for R. clavata females (Fig. 4),
poorly sampled over the years and at a wide depth
range (42–473m). D. oxyrinchus females carrying egg-
cases were observed around all the Sardinian Island
(Fig.4) at a mean depth of 500m where the bottoms
were mainly muddy and constituted by the bathyal
biocoenosis as Axinella cannabina (Esper, 1794), Echi-
nus melo Lamarck, 1816 and Gryphus vitreus (Born,
1778). Also the congeneric D. nidarosiensis seemed to
prefer muddy substrata; the eggcases and the only one
female carrying developing eggcases were found in
deep-waters (>750m depth) in the south-eastern part
of the Island (Fig.4).
No data about the benthic community associated to L.
melitensis active were available, but preliminary informa-
tion on their distribution was given (Fig.4).
Fig. 3 Principal components analysis (PCA) conducted of eggcase
morphometric measurements

Page 8 of 14
Porcu et al. Helgol Mar Res (2017) 71:10
Discussion
Eggcase reports and information on the egg-laying rate
for the overwhelming majority of rajids have received lit-
tle attention in the Mediterranean context. In this regard,
the present study provides updated eggcase descrip-
tions and quantitative data on their sizes for developing
regional and specific identification keys, useful in the
recognition of eggcases found over sea beds, providing
useful information on the distribution and reproductive
biology of skates living in the central-western Mediterra-
nean. We do report for the first time information on egg-
cases of the endemic L. melitensis, considered Critically
Endangered in the Mediterranean [36].
e eggcases from the eight analyzed species exhibited
peculiar morphologies between genera and even species.
Despite some overlap in size between species, features
such as horn and apron lengths, and presence/absence of
lateral keels were crucial to discriminate the eggcase at
species level. In particular, from morphological and mor-
phometric analysis, actually corroborated by multivariate
analyses, R. miraletus and L. melitensis stood out from all
the others for having eggcases without keels. Moreover,
within the species having keeled eggcases, those belong-
ing to D. oxyrinchus and R. brachyura were discriminated
to the remaining group for having the longest eggcases
and aprons (ECL and AAL-PAL).
Considering the ECL (without horns), females of D.
nidarosiensis possessed, overall, the biggest eggcases
(153–177mm ECL). Our results represent the first avail-
able qualitative and quantitative characterization for egg-
cases from this species in the Mediterranean basin.
Table 4 Results ofthe SIMPER analysis routine considering
the morphometric measures inthe dierent groups
Only highest contributing measures are shown
Group A, D. oxyrinchus and R. brachyura; Group B, R. asterias, R. clavata, R.
polystigma; Group C, R. miraletus; Group D, L. melitensis; SD, standard deviation
Morphometric
measures Average
squared
distance
Squared
distance/SD Contribution %
Group D versus A 60.61
ECL 14.00 4.51 23.16
PAL 10.50 3.02 17.36
ECW 8.33 3.28 13.74
Group D versus B 15.45
RAHL 3.56 1.89 23.02
LAHL 3.06 1.62 19.78
RKW 2.47 2.64 15.97
Group A versus B 38.32
ECL 10.20 2.59 26.71
PAL 6.57 2.04 17.14
AAL 5.12 2.55 13.37
Group D versus C 15.26
RAHL 7.21 2.28 47.26
LAHL 6.46 2.00 42.34
Group A versus C 64.75
ECL 12.40 3.56 19.12
ECW 7.91 3.12 12.21
LAHL 6.81 0.94 10.52
Group B versus C 10.88
RKW 2.47 2.64 22.69
LKW 2.46 2.89 22.60
ECW 1.17 0.82 10.79
Table 5 Identication key forrajid eggcases ofcentral-western Mediterranean
1a Eggcase large >90 mm ECL, with well-developed aprons 2
1b Eggcase small-medium in length 40 < ECL < 70 mm, with moderate or narrow aprons 3
2a Eggcase rectangular: ECL approximately two times the ECW (ECL range = 153–177 mm; ECW range = 61.8–80.5 mm) with well-
developed aprons (AAL range = 38.2–49.7 mm; PAL range = 26.2–40.9 mm) D. nidarosiensis
2b Eggcase rectangular: ECL approximately two times the width (ECL range = 91.6–116.2 mm; ECW range = 48.9–67.5 mm). Devel-
oped aprons (AAL range = 20.3–31.7 mm PAL; range = 7.7–20.8 mm). Short anterior and posterior horns D. oxyrinchus
2c Eggcase rectangular: ECL approximately two times the ECW (ECL range = 113–133 mm; ECW range = 64.7–72.2 mm). Developed
aprons (AAL range = 18.5–25.7 mm; PAL range = 17.5–26.6 mm). Long and thin horns, particularly the anterior (about 50% of
ECL)
R. brachyura
3a Eggcase small-medium in length 40 < ECL < 70 mm with evident or narrow keels 4
3b Eggcase small and rectangular (ECL range = 41.8–56.5 mm) without keels 5
4a Eggcase moderate in size (ECL range = 62.5–67.5 mm), rectangular/almost square in shape (ECL approximately 1.4 times the
ECW). Aprons and keels developed. Anterior horns moderate and thin, posterior horns short and sturdy R. clavata
4b Eggcase small (ECL range = 39.7–55.4 mm) rectangular/almost square in shape (ECL approximately 1.4 times the ECW). Evident
keels and both anterior and posterior horns moderate and thin R. asterias
4c Eggcase moderate in size (ECL range = 56.6–69.2 mm) with rectangular shape (ECL approximately two times the ECW). Narrow
keels. Anterior horns moderate and thin, posterior horns moderate and sturdy R. polystigma
5a Anterior and posterior horns short R. miraletus
5b Moderate posterior horns and very long, thin and curved inwards L. melitensis

Page 9 of 14
Porcu et al. Helgol Mar Res (2017) 71:10
Fig. 4 Map of the study area where active females (filled circle) of the eight investigated species and eggcases (filled triangle) laid on the bottom
were indicated

Page 10 of 14
Porcu et al. Helgol Mar Res (2017) 71:10
In addition, considering the two distinct eggcase forms
described in literature for the genus Dipturus [17], on
the basis of our observations, it was possible to insert D.
nidarosiensis and D. oxyrinchus in the D-II phylogenetic
type. e latter, matching with other Dipturus species
(e.g. D. gigas [13], D. trachiderma [16], D. batis [17]) was
characterized by well-developed aprons and lateral keel
united with the main portion rectangular in shape, differ-
ently from the D–I type showing close resemblance from
other genera of the family Rajidae [17].
Generally, the length of the horns shows rather large
intraspecific variations with the anterior horns longer
than the posteriors [12, 37, 38]. In our specific case,
eggcases of L. melitensis presented the longest anterior
horns (intertwining) with a ratio anterior/posterior horns
of 2.6, followed by R. brachyura egg cases with a ratio of
1.6, that enable them to be fixed to various structures as
seagrasses, algae or debris. In other studies, similar, or
even higher horn proportions were found in other spe-
cies belonging to the family Rajidae as Atlantoraja cyclo-
phora and A. platana [18], Leucoraja naevus [38], Rioraja
agassizi [39] and Psammobatis scobina [37].
Almost all the analyzed eggcases were covered by adhe-
sive fibers providing their camouflage and anchoring to
marine debris, plants, mud and rock and may function
as a barrier to predation [40]. As a general rule, eggcases
covered by fibers were produced by mixed gland tubules
(mucous and serous) situated deep in the terminal zone of
the oviducal gland as reported for many Rajidae species (e.g.
D. oxyrinchus, R. montagui, R. brachyura, R. clavata and R.
undulata) by Maia et al. [38] and Marongiu et al. [41]. L.
melitensis differed from the remaining species, because its
eggcases were smooth, devoid of fibres on their surface.
is feature seems to be common to other species belong-
ing to the genus Leucoraja, as L. naevus, marked by the
mucous gland tubules secretions (suphated acid mucins) of
the terminal zone that could work as an important chemical
defense against predation and pathogens [42, 43].
Furthermore, D. nidarosiensis, D. oxyrinchus and R.
brachyura eggcases were the most robust. is feature
was certainly related to their oviducal gland microarchi-
tecture, characterized by a higher number of lamellae of
the baffle zone [38, 41], if compared with the eggcases
from R. miraletus characterized by a lower number of
lamellae [41]. From an ecological perspective, these fea-
tures, probably due to the length of the developmental
period, may represent an advantage against predation
and water turbulence [38] considering that their big
dimensions did not allow a proper camouflage.
Sandy bottoms (<100–150 m depth) were identified as
egg-laying sites (i.e. sites with females bearing eggcases in
uteri) of many species belonging to genus Raja (e.g. R. aste-
rias, R. brachyura, R. miraletus and R. polystigma). A similar
behavior was observed for R. brachyura females in Portugal
[38] and around the British Isles [44] as well as for R. mirale-
tus in the south west of India where eggcases were found on
the soft sea bed at a depth range of 112–123m [45].
With the exception of R. polystigma, all other species
showed a clear distribution in the western part of Sardinia,
which is characterized by a wide continental shelf (tens of
kilometres) and a continental margin dwelling between
a depth of 150 and 200m depth, contrarily to other sides
(especially the eastern side), where the shelf is narrower
and steeper [46]. Regarding R. clavata, it presented a high
diversity of egg-laying habitats with a wide bathymetric
range, which may differ in terms of bottom topography or
sediment composition, as reported also by other authors
in the Atlantic Ocean [38, 44]. Finally, D. oxyrinchus active
females showed a clear preference for deep muddy bot-
toms (mean depth of 500 m) around Sardinia, confirm-
ing their bathyal habits as reported for the same waters by
Mulas etal. [47]. e occurrence of D. nidarosiensis, exclu-
sively in the south eastern part, was related to the deep
trawl-surveys performed only in that area.
roughout the analyzed period (12years), R. asterias
and R. miraletus active skates were found on the same
sites every year, located in the central western coast of
Sardinia, confirming the theory that many rajid species
demonstrate site fidelity, returning to the same depo-
sitional area or nursery ground on an annual basis as
reported for Bathyraja aleutica, B. interrupta and B. par-
mifera in eastern Bearing Sea [48]. Moreover, this finding
could also suggest the existence of possible nursery areas,
as recently observed for oviparous species as R. clavata
and Galeus melastomus [49] and Scyliorhinus canicula
[50] around Sardinian waters.
Because of their inherent low fecundity and slow
growth rates, skates may reproduce with distinct seasonal
pulses, over protracted periods, or in some cases contin-
uously throughout the year [51, 52]. In this regard, our
results suggested a continuous reproduction throughout
the year for the deeper D. oxyrinchus confirmed by the
presence of spawning capable females during all seasons
with a predominance of active individuals in autumn
and winter months (late September to late March) [53].
A reproduction restricted mainly to summertime was
described, instead, for the other species (except for L.
melitensis for which no sufficient reproductive data was
available), as also confirmed by other studies in Italian
waters [54, 55]. Year-round reproduction may be a strat-
egy to compensate the high maternal energy investment
and late maturity typical to the elasmobranchs. Given
the generalized high biological productivity in the sum-
mer period, a distinct pulse of egg deposition during this
period could represent an advantage for those species
with shallower habits in order to maximize the fitness.

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Porcu et al. Helgol Mar Res (2017) 71:10
Table 6 Eggcase dimensions reported inseveral geographical areas bylatitudinal clines
Species Eggcase length (mm) Eggcase width (mm) Area References
D. nidarosiensis 182–260 95–120 Atlantic (Norwegian Sea) [56]
182–260 92–113 Atlantic (North Sea) [57]
153–177 (170.2 ± 9.8) 61.8–80.5 (74.8 ± 7.5) Mediterranean (Sardinia) Present study
D. oxyrinchus 133 79.5 Atlantic (British waters) [58]
128–133 74–101 Atlantic (British waters) [59]
120–235 58–120 Atlantic (North Sea) Bor [57]
102–126 (112 ± 10.4) 60–66 (63.8 ± 2.6) Atlantic (British waters) [30]
100–150 – Mediterranean/Atlantic [3]
91.6–116.2 (103.8 ± 5.3) 48.9–67.5 (57 ± 4.0) Mediterranean (Sardinia) Present study
35–46 (38.4 ± 0.7) 17–28 (24.2 ± 0.2) Mediterranean (Tunisia) [23]
140 – Mediterranean (Naples) [21]
L. melitensis 42.3–45 (44.1 ± 1.0) 22.4–26.0 (23.9 ± 1.4) Mediterranean (Sentinelle Bank) Present study
R. asterias 30–45 – Mediterranean [3]
39.7–55.4 (48.3 ± 3.0) 22.4–40.5 (33.7 ± 2.6) Mediterranean (Sardinia) Present study
45 – Mediterranean (Naples) [21]
R. brachyura 122 ± 5 68 ± 4 Atlantic (Portuguese waters) [38]
136 76 Atlantic (British waters) [60]
128.4 78.5 Atlantic (British waters) [58]
115–143 72–90 Atlantic (British waters) [59]
82–132.2 (108.6 ± 10.1) 32.7–86.4 (65.4 ± 11.7) Atlantic (British waters) [30]
121 79 Atlantic (North Sea) [61]
115 70 Atlantic (North Sea) [62]
120 – Mediterranean/Atlantic [3]
115–143 – Mediterranean [15]
113–133 (119.3 ± 4.9) 64.7–72.2 (69.5 ± 2.3) Mediterranean (Sardinia) Present study
105 – Mediterranean (Naples) [21]
R. clavata 65 ± 5 48 ± 5 Atlantic (Portuguese waters) [38]
80 50 Atlantic (France) [63]
63–90 (74.9) 49–68.5 (67.1) Atlantic (British waters) [58]
60–90 50–70 Atlantic (British waters) [59]
32.7–83.3 (67.2 ± 10) 32.7–69 (37 ± 8.2) Atlantic (British waters) [30]
70 50 Atlantic (North Sea) [62]
60–90 – Mediterranean/Atlantic [3]
61–66 – Mediterranean (France) [22]
62.5–67.5 (64.1 ± 1.9) 41.7–47.4 (43.8 ± 2.0) Mediterranean (Sardinia) Present study
70–78 (75) 50–54 (52) Mediterranean (Tunisia) [64]
60 – Mediterranean (Naples) [21]
R. miraletus 48–52 – Atlantic (Senegal) [65]
46 ± 3 26 ± 2 Atlantic (Portuguese waters) [38]
<50 – Atlantic (South Africa) [66]
45–52 – Mediterranean/Atlantic [3]
41.8–56.5 (47.4 ± 4.0) 22.8–29.2 (24.7 ± 1.7) Mediterranean (Sardinia) Present study
42–47 – Mediterranean ( Tunisia) [67]
59 – Indian Ocean [45]
R. polystigma 35–46 – Mediterranean [3]
56.6–69.2 (62.3 ± 4.3) 33.9–44.3 (37.9 ± 2.9) Mediterranean (Sardinia) Present study

Page 12 of 14
Porcu et al. Helgol Mar Res (2017) 71:10
We also observed that eggcase dimensions seem to
change in relation to geographical area. From a compari-
son with the available literature from the Mediterranean
(Table6), our samples were similar in sizes (ECL and ECW)
to those observed in other areas of the basin. Instead,
eggcases analysed here (excluding the endemic species)
seemed to be smaller than those described in the Atlantic
studies (Table6). e only exception was represented by R.
brachyura, showing eggcases slightly larger than the Atlan-
tic ones, probably due to similar body dimensions between
the two areas [55]. is pattern was reported also for other
chondrichthyan eggcases (e.g. G. melastomus, [28]) and it
could probably be due to the intraspecific latitudinal cline
in elasmobranchs size in which Atlantic specimens reach a
bigger body size than Mediterranean ones [26, 39, 68].
In conclusion, given the absence of Mediterranean egg-
case descriptions, the present study should shed light on
the taxonomy, distribution and reproductive habits of
many Mediterranean skates. Indeed, this identification key
could act as useful tool for non-invasive identification of
eggcases through image analysis (i.e., ROV imaging), which
have proven to be an efficient tool for identifying nursery
ground and delineate ecological traits of species. In addi-
tion, since knowledge of the location of nursery grounds for
elasmobranchs is practically nonexistent [50] and given the
most recent policy approaches to the protection of marine
ecosystems (e.g. the Marine Strategy Framework Directive,
MSFD; 2008/56/EC currently in force in European seas),
the identification of essential fishing habitats such as nurs-
ery grounds of sensitive species like skates represent an
indispensable component to protect and manage.
Abbreviations
n: number of eggcase analyzed; TL: total length; ECL: eggcase length; ECW:
eggcase width; AAL: anterior apron length; PAL: posterior apron length;
LKW: left keel width; RKW: right keel width; LAHL: left anterior horn length;
RAHL: right anterior horn length; LPHL: left posterior horn length; RPHL: right
posterior horn length; PCA: Principal Component Analysis; SIMPER procedure:
SIMilarity PERcentage Analysis; ANOSIM: Similarity Randomization Test.
Authors’ contributions
CP, MFM and MCF conceived the study; CP wrote the manuscript with signifi-
cant input of MFM and MCF. MFM and CP analyzed, described and measured
all specimens’ samples and wrote the morphometric and morphological
part of the manuscript. AC performed statistical analysis; RM performed
molecular analysis to genetic identification; CP, MFM, MCF, AB, AM, RC, LV and
GS performed sampling and all the analyses and data handling. All authors
contributed to data interpretation. All authors read and approved the final
manuscript.
Author details
1 Department of Life and Environmental Sciences (DISVA), University of Cagli-
ari, Via Fiorelli 1, 09126 Cagliari, Italy. 2 Department of Architecture, Design
and Urban Development, University of Sassari, Palazzo Pou Salit, Piazza Duomo
6, 07041 Alghero, Italy.
Competing interests
The authors declare that they have no competing interests.
Availability of data and materials
The datasets used and/or analyzed during the current study available from the
corresponding author on reasonable request.
Ethics approval and consent to participate
All applicable international, national and/or institutional guidelines for the
care and use of animals were followed.
Funding
This study was financed by Autonomous Region of Sardinia within the frame
of the research project ‘Approccio multidisciplinare per la conservazione e
gestione della selacofauna del Mediterraneo’ (LR7 CRP-25321) and carried out
within the Data Collection Regulation and Framework—module trawl surveys
MEDITS (Mediterranean International Trawl Surveys).
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in pub-
lished maps and institutional affiliations.
Received: 8 November 2016 Accepted: 15 June 2017
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