Accepted on 3 March 2015
J Zoolog Syst Evol Res doi: 10.1111/jzs.12101
© 2015 Blackwell Verlag GmbH
1
Museo di Storia Naturale di Venezia, Venezia, Italy; 2Dipartimento di Bioscienze, Universit
a degli Studi di Milano, Milano, Italy;
Dipartimento di Biologia, Universita degli Studi di Padova, Padova, Italy
3
Morphological evidence that the molecularly determined Ciona intestinalis type A
and type B are different species: Ciona robusta and Ciona intestinalis
R ICCARDO B RUNETTI 1 , C ARMELA G ISSI 2 , R OBERTA P ENNATI 2 , F EDERICO C AICCI 3 , F ABIO G ASPARINI 3 and L UCIA M ANNI 3
Abstract
Ciona intestinalis is considered a widespread and easily recognizable tunicate, the sister group of vertebrates. In recent years, molecular studies suggested that C. intestinalis includes at least two cryptic species, named ‘type A’ and ‘type B’, morphologically indistinguishable. It is dramatic to certify
that two different species may be hidden under the name of a species widely used as a model species in biological researches. This raised the problem
of identifying diagnostic morphological characters capable of distinguishing these types. We compared the morphology of specimens belonging to the
two types and found that only type A specimens possess tunic tubercular prominences, allowing unambiguous discrimination. Remarkably, these structures were already described as distinctive of the Japanese species Ciona robusta, Hoshino and Tokioka, 1967; later synonymized under C. intestinalis
(sensu Millar, 1953). In this study, we have confirmed that C. intestinalis type A corresponds to C. robusta. Based on the geographic distribution of
C. intestinalis type B, and considering that the original C. intestinalis species was described from North European waters, we determined that C. intestinalis type B corresponds to C. intestinalis as described by Millar in 1953 and possibly to Linnaeus’ Ascidia intestinalis L., 1767 for which we have
deposited a neotype (from Roscoff, France) and for which we retain the name Ciona intestinalis (Linnaeus, 1767).
Key words: Tunicata – Ciona robusta – Ciona intestinalis – cryptic species – neotype – topotypes
Introduction
In the last decades, Ciona intestinalis, a putatively cosmopolitan
representative of the tunicates – likely, the sister group of vertebrates – has become a model chordate in various fields of biology, from comparative genomics to evo-devo and developmental
biology (Satoh 2003). The nuclear genome draft of an individual
sampled in California was published in 2002. This promoted the
study of this organism and helped to clarify the evolutionary origin of chordate novelties (Dehal et al. 2002).
C. intestinalis is a shallow water species often present in harbours or semi-enclosed basins in extensive communities. The
species has been recorded in all oceans and from high to low latitudes. The original, very short description made by Linnaeus –
as Ascidia intestinalis Linnaeus 1767 – was extended by Roule
(1884) and subsequently by Millar (1953). Despite the large
number of articles devoted to specimens collected in different
localities, populations with morphological characters substantially
deviating from Millar’s description (1953) have not been
described. In general, the wide geographic distribution of C. intestinalis might be explained by its adaptability to environmental
variation as well as by the effect of natural spread of the larvae
and of human-related transportation of adults (e.g. on vessel
keels) (Darbyson et al. 2009; Davidson et al. 2010; Kanary et al.
2011). Indeed, experiments showed that populations from different Scandinavian sites have different ranges of salinity tolerance
for the development of fertilized eggs and larvae (Dybern 1967).
A similar experimental study on a population from the Lagoon
of Venice, testing various combinations of salinity and temperatures on different stages of the biological cycle, also suggested
that populations from different localities might be somewhat
genetically separated among them (Marin et al. 1987). More
recently, another work on Scandinavian C. intestinalis popula-
Corresponding author: Fabio Gasparini (fabio.gasparini@unipd.it)
Contributing authors: Riccardo Brunetti (ric.brunetti@gmail.com),
Carmela Gissi (carmela.gissi@unimi.it), Roberta Pennati (roberta.
pennati@unimi.it), Federico Caicci (federico.caicci@unipd.it), Lucia
Manni (lucia.manni@unipd.it)
tions found that differences in salinity tolerance at larval metamorphosis were primarily plastic and driven by parental
experience (Renborg et al. 2014).
Recent molecular studies of specimens sampled in several
localities around the world have shown that different, cryptic,
species might be grouped under the name C. intestinalis, leading
to the hypothesis that C. intestinalis constitutes a complex of
species (Suzuki et al. 2005; Caputi et al. 2007; Iannelli et al.
2007; Nydam and Harrison 2007, 2011; Zhan et al. 2010; Sato
et al. 2012). In particular, these studies have identified two
forms, named ‘type A’ and ‘type B’, as highly genetically divergent and with disjoint global distributions (Suzuki et al. 2005;
Caputi et al. 2007; Nydam and Harrison 2007; Zhan et al.
2010). Type A specimens have been found primarily in the Mediterranean Sea, the Pacific Ocean (Australia, Japan, New Zealand,
South Korea, and West coast of North America), and the Atlantic
coasts of South Africa (Caputi et al. 2007; Zhan et al. 2010).
Type B individuals have been found on the coasts of both the
NE and NW North Atlantic Ocean, as well as in the Bohai and
Yellow Seas (China) (Zhan et al. 2010). Remarkably, type A
and type B coexist in the English Channel and in some localities
of the French Atlantic coasts (i.e. in Plymouth, UK, and in Brest,
France) which, accordingly, represent sympatric areas (Sato et al.
2012).
The reproductive isolation of type A and type B has been
investigated in the context of the biological species concept
(Mayr 1963), using experiments of in vitro fertilization between
individuals coming from different localities (Suzuki et al. 2005;
Caputi et al. 2007; Sato et al. 2012, 2014). However, available
data are currently insufficient to clarify this issue and the problem of the existence of distinct species under this species concept. Indeed, fertilization experiments have sometimes produced
partially contradictory results. This was probably due to the
usage of different experimental conditions (i.e. developmental
temperature-salinity) and of specimens coming from different
localities/populations (see also the discussion in Sato et al.
2014). The most comprehensive study, performed on sympatric
type A and type B populations of the English Channel, indicates
that hybrids can mature and produce viable gametes. Yet,
2
naturally introgressed animals are present only at low percentage,
probably as consequence of several pre- and postzygotic mechanisms of reproductive isolation (Sato et al. 2014).
In spite of the wealth of molecular data, only two studies have
compared the morphology of Ciona intestinalis type A versus
type B (Caputi et al. 2007; Sato et al. 2012). Caputi et al.
(2007) found that, with some exceptions, the two C. intestinalis
types can be told apart by the sperm duct pigmentation, as type
A individuals have orange genital papillae and an uncoloured
duct, while type B shows pigmentation only in the duct. Subsequently, Sato et al. (2012) described three external inherited morphological characters as useful markers to distinguish types A
and B ‘in the field’ and within the region of sympatry. The identified characters, all observable with the unaided eye and without
the help of any laboratory facilities, are as follows: (1) body colour, (2) presence/absence of tubercular prominences on the
siphons and (3) yellow/orange pigments at the distal end of
siphons. However, none of these three characters was found
exclusively in a particular C. intestinalis type, and the existence
of a number of exceptions makes them unreliable indicators of
the specific type. For example, in the sympatric area of Plymouth, 92% of the specimens with yellow or orange pigmentation
at the distal end of siphons were type B and only 78% of all
specimens without pigmentation at the distal end of siphons were
type A (Sato et al. 2012). In conclusion, although outwardly useful in the field, all the identified morphological differences
between type A and type B appear devoid of taxonomic value
(Caputi et al. 2007; Sato et al. 2012). That is a critical situation
for scientists, because C. intestinalis is used as a model organism
in biological research (Satoh and Jeffery 1995; Corbo et al.
2001; Satoh 2003; Satoh et al. 2003).
The genus Ciona includes deep water and shallow water species. The two arctic taxa C. longissima Hartmeyer 1899 (a
poorly described species) and C. gelatinosa Bonnevie 1896 (redescribed by Sanamyan and Sanamyan 2007) range from subtidal
zone down to bathyal–abyssal depths; both species are characterized by the presence of a posterior abdomen into which the lateral muscle bands extend. Other deep water species are as
follows: C. antarctica Hartmeyer 1911 (redescribed by Monniot
and Monniot 1983); the abyssal C. mollis Ritter 1907 (redescribed by Monniot 1998); C. imperfecta Monniot and Monniot
1977 (whose belonging to the genus Ciona is doubtful); and
C. pomponiae Monniot and Monniot, 1989 (redescribed by Sanamyan and Sanamyan 2007).
The six shallow water species are as follows:
1 C. intestinalis (Linnaeus 1767) sensu Millar 1953 (not including C. intestinalis sensu Hoshino and Tokioka 1967 and Pisano et al. 1972, both of which correspond to C. savignyi);
2 C. edwardsi (Roule 1886) redescribed by Copello et al. (1981);
3 C. savignyi Herdman 1882 (C. intestinalis sensu Hoshino and
Tokioka 1967 and Pisano et al. 1972);
4 C. roulii Lahille 1890 of which only the original description
is available, eventually transcribed by Harant and Vernieres
(1933) in the Faune de France volume on ascidians, who
unjustifiably changed the original name into roulei which is
an incorrect subsequent spelling (ICZN 1999, art 33.3), and
therefore, it must be corrected (art 32.5);
5 C. robusta Hoshino and Tokioka 1967; described also by Pisano et al. (1972), later regarded as a junior synonym of
C. intestinalis (Hoshino and Nishikawa 1985); and
6 C. sheikoi Sanamyan 1998 (a shallow subtidal species collected at 80 m from north-west Pacific Ocean).
Hoshino and Nishikawa (1985) suggested a classification of
the Ciona species based essentially on the presence/absence of
the endostylar appendage and on the arrangement of the pharyndoi: 10.1111/jzs.12101
© 2015 Blackwell Verlag GmbH
BRUNETTI, GISSI, PENNATI, CAICCI, GASPARINI and MANNI
go-epicardiac openings, two characters often ignored by previous
authors. In our opinion, this classification had probably overestimated these two characters to the detriment of other morphological considerations. Based on this new classification, these authors
then synonymized C. robusta and C. edwardsi under C. intestinalis, undervaluing the careful description previously given for
both these species (Hoshino and Tokioka 1967; Copello et al.
1981). As a proof of this overestimation, later experiments of
hybridization performed by Lambert et al. (1990) showed that
C. intestinalis, C. roulii and C. edwardsi are distinct species.
Here, we perform detailed morphological comparisons
between C. intestinalis type A and type B specimens from different localities, with the aim of identifying significant morphological differences that unambiguously distinguish the two types.
Material and methods
Specimen collection and analyses
The source localities and the sampling dates of the analysed 51 specimens are reported in Table 1. For morphological analyses, the specimens
were anaesthetized in seawater saturated with menthol, fixed in 10% seawater formalin and then dissected. Organs were stained with Mayer’s
haemalum and dehydrated with ethanol at crescent concentrations. Morphological observations were made on dissected whole animals, on
stained organs, and also on specimens before dissection according to the
‘in field’ morphological characterization developed by Sato et al. (2012).
Specimens were analysed with a dissecting stereomicroscope using
reflected light as well as transmitted light. Ciona intestinalis type A and
type B specimens were identified based on the analysis of the mitochondrial genome, according to the two PCR-based screening tests described
in the Supplementary Materials of Iannelli et al. (2007). These tests take
into account the translocation of the trnC and the presence/absence of a
85-bp-long non-coding region to discriminate type A from type B individuals. Specimens coming from the sympatric zone (Plymouth) were
also analysed according to the genotyping test and the ‘in field’ morphological characters described in Sato et al. (2012). In particular, the used
genotyping test is based on restriction enzyme digestion at one mitochondrial (cox1) and three nuclear loci (vAChTP, CiCesA and patched)
according to Sato et al. (2012) and Nydam and Harrison (2010).
Histology
Specimens of Ciona intestinalis type A from Plymouth and Venice, and
type B from Plymouth and Roscoff were anaesthetized with menthol,
fixed in 4% paraformaldehyde in phosphate-buffered saline (pH 7.4). A
total of ten specimens were then dehydrated with ethanol at crescent concentrations, embedded in paraplast, dissected, and serial sections 7 lm
thick were cut and counterstained with haematoxylin–eosin.
For 3D reconstructions, 84 histological sections 7 lm thick belonging
to an anterior part of one of the ten sectioned C. intestinalis type A specimens were recorded with a digital camera (Leica DFC 480) mounted on
a Leica DMR compound microscope. Images were aligned using Adobe
Photoshop CS on a Windows 7 computer. Based on the resulting stack
of images, 3D models of the anatomy of the tunic corresponding to the
area near the oral siphon were created in AMIRA 5.3.3 software (Mercury
Computer Systems, Berlin, Germany).
Results
We first analysed six individuals from Plymouth (the region of
sympatry): three classified as Ciona intestinalis type A and three
as type B. Then, we compared 8 ‘type B’ from Roscoff and 30
‘type A’ from the Venetian lagoon. Both specimen sets ranged
from 2 to 12 cm in height (Fig. 1). All analysed individuals were
molecularly classified as described in Material and Methods, and
the type identification was found in accordance with their source
locality. In each animal, we carefully inspected the tunic and some
internal characters described by Millar (1953) as characteristics of
Ciona intestinalis types A and B are two species
3
Table 1. Source and date of sampling of the analysed Ciona intestinalis (formerly known as C. intestinalis type B) and C. robusta (formerly known
as C. intestinalis type A) specimens. Latitude and longitude geographic coordinates expressed in decimal degrees (DD)
Date
C. robusta
(formerly ‘type A’)
C. intestinalis
(formerly ‘type B’)
Plymouth, UK
Autumn 2011
3
3
Lagoon of Venice, IT
Autumn 2013
20
0
Lagoon of Venice, IT
Roscoff, FR
Roscoff, FR
Autumn 2013
Autumn 2013
Summer 2014
10
0
0
0
8
7
Locality
(a)
(b)
Fig. 1. (a) Individual of Ciona robusta (formerly known as C. intestinalis type A) from the Lagoon of Venice (arrows: tubercular prominences).
(b) Individuals of C. intestinalis (formerly known as C. intestinalis type
B) from Roscoff. As, atrial siphon; Os, oral siphon. Scale bars: 1 cm
Ciona intestinalis. These characters are all detailed below, but only
the tunic allowed us to clearly distinguish type A from type B individuals.
Tunic
In all type A specimens, the tunic presented tubercular prominences, papilla shaped or elongate, distributed along the whole
body and prevalently arranged around the siphons, in more or
less longitudinal rows (Fig. 2). These tubercular prominences are
present in all type A specimens, also in young and small individuals, although in these cases, they are invisible to the unaided
eye and their identification requires the usage of a dissecting stereomicroscope equipped with transmitted light. These structures
are present also in the thin layer of tunic lining the internal surface of the oral siphon (Fig. 2c–e), where they can be lobed; in
Ciona robusta, they were named by Hoshino and Tokioka
(1967) ‘endocarps’, a term commonly used to describe internal
features of the parietal body wall in the atrium of stolidobranch
ascidians; see, for example Kott 1985, p 11.
Histological analyses confirmed the existence of the tubercular
prominences along the whole body and showed that these structures are not due to pathologic conditions. No signs of cell-mediated events, such as encapsulation or tissue injury, were observed.
Details
Panels to the Queen Anne’s Battery Marina
(DD: 50.364, 4.131)
Piers to the south-west of Chioggia centre
(DD: 45.2138, 12.2732; 45.2135, 12.2738)
Floats to the West of Sottomarina (DD: 45.2180, 12.2898)
Marina of Roscoff (DD: 48.7274, 3.9872)
Marina of Roscoff (DD: 48.7274, 3.9872)
The oral siphons of type A individuals (three from Plymouth and
three from Venice) were transversely cut and analysed by light
microscopy (Fig. 3). In general, the tunic around the oral siphon
was thin except in tubercular prominences, which were outgrowths
of both the firm outer layer and the gelatinous inner layer of the
tunic (Fig. 3a,b; Fig. 4). The tunic was crossed by numerous
dense fibres, prevalently oriented parallel to the epidermis. The
cuticle limiting the tunic was dense and irregular in profile
(Fig. 3c,d). Both in tubercular prominences and in surrounding
tunic regions, tunic cells were particularly dense in the vicinity of
the cuticle.
In all Ciona intestinalis type B specimens, the tunic was quite
soft, its colour was pale, translucent and greyish (Millar’s colour
type 3; Millar 1953), and it was completely transparent in young
individuals. The tunic around the siphons of large individuals
could sometimes be lightly wrinkled. Remarkably, the tubercular
prominences were absent in all type B specimens, both in small
and in large individuals.
In conclusion, we found that the presence of tubercular prominences in the tunic is a character exclusive to Ciona intestinalis
type A specimens and can unambiguously discriminate type A from
type B samples. Remarkably, these tubercular prominences are a
characteristic of Ciona robusta Hoshino and Tokioka 1967; the species identified in Japanese waters, and later synonymized to C. intestinalis (Hoshino and Nishikawa 1985) (see Introduction). Indeed,
Hoshino and Nishikawa (1985) observed that C. robusta does not
differ in any other characteristic from C. intestinalis (L.; not Hoshino and Tokioka 1967) except for the aspect of the tunic. Thus, we
conclude that C. intestinalis type A corresponds to C. robusta.
Siphons
No differences in length and arrangement of the siphons were
detected between Ciona intestinalis types A and B specimens,
both corresponding to Millar’s description (1953).
Body muscles
In both types, there were six longitudinal bands on each side:
four extending up to the oral siphon and two to the atrial one.
This is essentially the situation described by Millar (1953),
although the latter description was highly schematic and did not
consider intraspecific variability, which is present in the arrangement of muscles.
Tentacles and ciliated funnel
Tentacles were simple, of different lengths, and variable in numbers in relation to the size of individuals (Fig. 2c). In both types
A and B, the aperture of the ciliated funnel (=dorsal tubercle)
was horseshoe shaped, opened upward, with the ends curled
inwards as described by Millar (1953).
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BRUNETTI, GISSI, PENNATI, CAICCI, GASPARINI and MANNI
(a)
(b)
(c)
(d)
(e)
Fig. 2. Ciona robusta (formerly known as C. intestinalis type A). (a, b) Siphons belonging to the same individual; many tubercular prominences
(arrows) are evident, arranged also in longitudinal row. (a) Oral siphon. (b) Atrial siphon. (c–e) Oral siphon of a dissected individual (inner view) to
show tubercular prominences (asterisks) in the thin layer of tunic lining the internal siphon surface. (c) Siphon rim is visible at top, tentacles at bottom
(arrowheads); also tubercular prominences are visible (arrows). (d) Enlarged view of area marked by white rectangle in c. (e) Enlarged view of area
marked by black rectangle in c. Cm, circular muscle fibres; Lm, longitudinal muscle fibres. Scale bars: a, 5 mm; b, 7 mm; c, 2 mm; d, 0.25 mm; e,
0.20 mm
(a)
(c)
(b)
(d)
Fig. 3. Histological sections of the tunic of Ciona robusta (formerly known as C. intestinalis type A); haematoxylin–eosin. (a) Cross section of oral
siphon showing a number of tubercular prominences. (b) A tubercular prominence. Note that tunic fibres (arrows) are parallel to each other. The prominence is an outgrowth of both the firm outer layer (two asterisks) and the gelatinous inner layer (one asterisk) of the tunic. The cuticle is irregular in
profile. The dense tunic line indicated by the arrowhead is an artefact due to fixation. (c, d) Tunic cells in a tubercular prominence. Note that numerous
tunic cells are close to the cuticle. (d) Enlarged view of area marked by rectangle in c. c, cuticle; ep, epidermis; Os, oral siphon; t, tunic; tb, tubercular
prominences; Tc, tunic cell. Scale bars: a, 500 lm; b, 100 lm; c, 50 lm; d, 25 lm
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Ciona intestinalis types A and B are two species
(a)
5
(a)
(b)
(b)
Fig. 4. (a, b) 3D reconstruction of the tunic corresponding to an area
near the oral siphon of an individual of Ciona robusta (formerly known
as C. intestinalis type A). Note that the prominences (arrows) are
arranged in longitudinal rows and are outgrowths of both the firm outer
layer (lighter grey) and the gelatinous inner layer (medium grey) of the
tunic. Darker grey: body wall. ep, epidermis. Scale bars: 1 mm
Structure of the branchial wall
Both A- and B-type individuals presented the same structure figured by Brunetti (1979, Pl. IV fig. A; Pl V fig. C) and Copello
et al. (1981, fig. 4A); that is the branchial wall is horizontally
pleated as in an accordion (Fig. 5). This important morphological
character distinguishes the typical Ciona intestinalis from Ciona
edwardsi (Copello et al. 1981) and was already noted by Roule
(1884, Pl 7 fig 66) but not by other authors, probably because it
is clearly visible only in well-anaesthetized animals.
Fig. 5. (a, b) Bottom of the branchial basket of a dissected individual of
C. intestinalis (formerly known as C. intestinalis type B) stained with
Mayer’s Haemalum. The branchial wall is horizontally pleated (arrowheads). The retropharyngeal groove extends between the oesophageal
opening and the endostylar appendage. The pharyngo-epicardiac opening
(arrows) is close to the latter. (b) Enlarged view of area marked by rectangle in a. b, branchial wall; Ea, endostylar appendage; en, endostyle;
Eo, oesophageal opening; Lb, longitudinal bars; p: papillae; Rg, retropharyngeal groove. Scale bars: a, 2.5 mm; b, 250 lm
(Fig. 5). Their size and number (one or two) were variable, as
previously observed by Millar (1953).
Stomach
Again, no difference was detected between the two types. In general, the stomach had a very delicate wall, with a large number
of longitudinal folds in its internal surface.
Discussion
Anus and genital openings
No difference was detected between types A and B. In both
types, the anus was situated about halfway along the branchial
sac. Its edge was irregularly lobed (in the examined individuals,
there were 13–15 lobes). The gonoducts ended forward of the
anus almost at the base of the atrial siphon; their ends were
orange pigmented in both type A and type B specimens.
Endostylar appendix and pharyngo-epicardiac openings
These two structures were present in both types. The pharyngoepicardiac openings were situated near the endostylar appendix
Our morphological analyses show that Ciona intestinalis type A
and type B individuals are indistinguishable according to the
characters proposed by Millar (1953) as characteristics of C. intestinalis and also according to the pigmentation of the distal
ends of the siphons (Sato et al. 2012). Indeed, a survey of the
siphon pigmentation on molecularly identified C. intestinalis type
A and type B was recently performed by Sato et al. (2012), on a
large number of individuals collected at Plymouth, where they
occur in sympatry. However, this study concluded that this character does not provide an absolute distinction between the types.
Thus, the siphon pigmentation is able to discriminate type A
from type B in the sympatric area in the majority of specimens
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(Sato et al. 2012), but there are some exceptions. In general, the
ascidian taxonomic literature reports a very large number of
polychromatic species in which the colorations change according
to the environmental parameters (e.g. Mukai 1974). This fact led
taxonomists to doubt the taxonomic value of animals’ coloration
(‘. . .there is often so much variation in colour in different individuals of the same species that in some genera little reliance
can be placed on colour as a diagnostic character’. Van Name
1945, p. 19). In ascidians, the colorations may be due to tunic
cells (Endean 1961), epithelial cells (Ishii et al. 1993), tunic spicules and symbionts (Monniot et al. 1991), but the best known
cause of colour is the blood cell containing pigments (Millar
1953; Wright 1981; Hirose et al. 1998). Moreover, different proportions of the same chromocytes can be the cause of ascidian
different colour. Finally, fixative liquids usually change or completely extract colours; therefore, Sato’s suggested method of
identification based on siphon colours is not verifiable on fixed
specimens.
Ciona intestinalis types A and B can best be discriminated
morphologically by the presence/absence of tubercular prominences in the tunic. This is the first time that the presence of
these tubercular prominences is described as a character exclusive to type A and therefore as a character able to unequivocally discriminate type A from type B individuals. In their
article, Sato et al. (2012) also described the tubercular prominences as ‘small raised regions on the surface of the tunic of
the siphons’ present mainly in C. intestinalis type A. However,
they reported that 80% of the specimens with tubercular prominences were type A, and 81% of specimens without tubercular
prominences were type B. Therefore, Sato et al. (2012) concluded that this character is just a ‘rough indicator’ of the type
identity. The different result of our study is ascribable to the
methodology employed for the detection of tubercular prominences. Sato et al. (2012) worked without magnification (Sato
A., personal communication), as they aimed to find a method
of ‘field identification’ of the two types defined by molecular
data. On the contrary, we analysed our specimens in detail at
the dissecting stereomicroscope, using reflected light as well as
transmitted light: the most numerous and large tubercular prominences are visible with the reflected light system, while transmitted light is necessary to identify tubercular prominences of
smaller size present in younger individuals. Unlike Sato et al.
(2012), we also found that tubercular prominences are dispersed
in the tunic along the whole body, although they are particularly abundant around the siphons.
Histological analyses of the tunic (in both type A and type
B), and specifically of the tubercular prominences, also demonstrated that these structures are not due to pathologic conditions.
No signs of cell-mediated events, such as encapsulation or tissue
injury, typical of inflammatory responses already described in
Ciona species, were recognized (Parrinello 1981; Parrinello and
Patricolo 1984; Parrinello et al. 1984, 2007; Cammarata et al.
2008).
In this study, we also showed that the presence/absence of
tubercular prominences is a distinctive character not only of the
type A/type B specimens sampled in the region of sympatry but
also of specimens from Roscoff (only type B) and Venice (only
type A). Thus, this character does not seem to be related to the
environmental conditions of the region of sympatry.
The absence of any mention of the presence of tubercular
prominences in type A and, in general, in C. intestinalis specimens, in previous molecular, taxonomic, faunistic and ecological
studies (except for Sato et al. 2012), is surprising. This is probably due to the widespread belief that C. intestinalis is the only
abundant shallow water species of the genus, easy to recognize
doi: 10.1111/jzs.12101
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BRUNETTI, GISSI, PENNATI, CAICCI, GASPARINI and MANNI
without particular attention to the morphological details and
without dissecting stereomicroscope analyses.
It is striking to note that the tubercular structures here
described and found in each of the type A specimens correspond
to those described and figured by Hoshino and Tokioka (1967)
in the Japanese C. robusta. Probably, Ciona individuals with
large tubercular prominences were collected also in other localities, but based on the authoritative taxonomy of Hoshino and
Nishikawa (1985), which synonymized C. robusta with C. intestinalis, they were not considered further; therefore, specimens
were described merely as C. intestinalis. For example, individuals of Ciona with large tubercular prominences corresponding to
those described in C. robusta collected by one of us, in the
lagoon of Venice previously, were considered abnormal or
affected by parasites, as had been initially supposed by Pisano
and Rengel (1972). Outside Japan, a single record of C. robusta
in Mar del Plata (Argentina) was reported (Pisano et al. 1972).
These authors reported that this species is able to reproduce also
in strongly polluted water. Moreover, it presents self-fertility (Pisano and Rengel 1972). Caputi et al. (2007) and Nydam and
Harrison (2010) have performed molecular analyses on Japanese
individuals from Onagawa (Miyagi Prefecture), a locality only
50–60 km south of the type locality of C. robusta (Mone Inlet,
Karakuwa cho, Miyagi Prefecture) (Hoshino and Tokioka 1967):
all these individuals belong to type A. Therefore, although the
syntypes and paratypes of C. robusta deposited by Hoshino and
Tokioka (1967) at the Seto Marine Biological Laboratory are not
suitable for molecular analyses (i.e. they were not fixed in
ethanol) and have not been seen by us, we hypothesize that the
original C. robusta specimens should be molecularly indistinguishable from type A.
Ciona intestinalis type B is common on North Atlantic coasts
(Suzuki et al. 2005; Caputi et al. 2007; Nydam and Harrison
2007), as well as in the Bohai and Yellow Seas (Zhan et al.
2010). Ciona intestinalis (L.) was first described – as Ascidia intestinalis – from the northern European Seas (Linnaeus’ ‘Oceano
europaeo’ Syst. Nat. 1791. Gmelin edition pag 3123), which
may be assumed as its original type locality. Type B is common
in the same ocean region of C. intestinalis (L.) type locality. As
a consequence, because C. intestinalis (L.) is universally recognized to correspond to Millar’s description (1953), we assume
this to be the detailed description of the Linnaean species and
assign type B to the valid Linnaean species. Moreover, as a
deposited type of the C. intestinalis (L.) is lacking, and being
faced with an important zoological problem, we have deposited a
neotype from Roscoff (where the numerous samplings and
molecular screening testify the presence of only type B) at the
Natural History Museum in Venice and some topotypes at both
the same museum and the Zoological Museum, University of Padova. Data on deposited specimens are reported in Table 2. As a
further important step in the specimens deposit, prior to fixation
in formalin, a small piece of each sample was fixed and stored in
95% ethanol for future possible molecular analyses. A similar
operation should be performed even for Ciona robusta Hoshino
and Tokioka 1967, even if syntypes and paratypes are still available for this species.
In conclusion, our morphological analyses provide a taxonomically valid character able to discriminate Ciona intestinalis type
A from type B specimens and show that the two types are two
distinct species, from both the genetic (Suzuki et al. 2005; Caputi et al. 2007; Iannelli et al. 2007; Nydam and Harrison 2007,
2010) and morphological perspectives. Our results are also confirmed by the identification of additional morphological differences at the larval stage (Pennati et al., 2015). Indeed, the larvae
of the two types significantly differ in shape. In particular, late
Ciona intestinalis types A and B are two species
7
Table 2. Data on the deposited neotype and topotypes of Ciona intestinalis (L.) (formerly known as C. intestinalis type B). Appearance of the specimens in fixative: tunic soft, smooth, transparent and colourless. Latitude and longitude geographic coordinates expressed in decimal degrees (DD)
Locality
Date
Size (cm high)
Depth (m)
Sex condition
Location1
Catalogue No.
MSNV-23282
neotype
MSNV-23283
topotype
TU26
topotype
TU27
topotype
Roscoff, DD: 48.7274,
3.9872
21.08.2014
5
0–2
Mature
VE
Roscoff, DD: 48.7274,
3.9872
21.08.2014
5
0–2
Mature
VE
Roscoff, DD: 48.7274,
3.9872
21.08.2014
6
0–2
Mature
PD
Roscoff, DD: 48.7274,
3.9872
21.08.2014
2.5
0–2
Mature
PD
1
PD: Zoological Museum, University of Padova (Italy); VE: Natural History Museum in Venice (Italy).
larvae of type B exhibit a longer pre-oral lobe, longer and relatively narrower total body length, and lower ocellus–tail distance
than type A. Therefore, all together, our adult and larval morphological data support the conclusion that Ciona intestinalis type B
is a different species from the molecularly defined type A, and
we identify C. intestinalis type A as corresponding to
C. robusta.
The results here illustrated permit to unambiguously distinguish
the two formerly known Ciona intestinalis types A and B as the
two species C. robusta and C. intestinalis, respectively. Therefore, we invite, encourage and advocate the use of the specific
names C. robusta and C. intestinalis for types A and B, to clearly
distinguish the individuals in future research and publications.
Considering the relevance of C. intestinalis in the study of
chordate evolution and developmental biology, and the number
of researchers working with this species all over the world, we
expect that the impact on the scientific community of the easy
morphological discrimination of type A and type B and finally
the assigning of correct species names to both will be welcome.
Acknowledgements
The authors thank Atsuko Sato, John DD Bishop, Alessandro Minelli
and Teruaki Nishikawa for helpful discussions. We also thank John DD
Bishop (Marine Biological Association of the UK, Plymouth, UK) for
kindly providing specimens from Plymouth. This work was supported
by: MIUR PRIN Projects 2009 to LM and CG (http://www.istruzione.it,
grant numbers 2009XF7TYT and 2009NWXMXX_003, respectively),
University of Padova Senior postdoc 2012 Project to FG (http://www.unipd.it, grant number GRIC120LSZ); and by grant n. 2013-0752 from
CARIPLO Foundation to RP. Authors have no conflict of interests.
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Lucia Manni