Table Of ContentZOOLOGICAL SCIENCE 9: 405-412 (1992) 1992Zoological SocietyofJapan
Minute Protrusions of Ascidian Tunic Cuticle: Some
Implications for Ascidian Phylogeny
Euichi Hirose1 Teruaki Nishikawa2 Yasunori Saito3
, ,
and Hiroshi Watanabe4
1,3,4Shimoda Marine Research Center, University of Tsukuba, Shimoda,
Shizuoka 415, and 2Biological Laboratory, College ofGeneral
Education, Nagoya University, Chikusa-ku,
Nagoya 464-01, Japan
—
ABSTRACT Fine structure of the ascidian tunic cuticle was studied by transmission and scanning
electron microscopy in 26 species belonging to 10 families. In 11 species of 8 families, the cuticular
surface is ornamented with numerous minute protrusions that are papillate in shape and usuallyup to
100nm in height. The present results in addition to the previous ones [1] gave us more information
aboutmorphologyofprotrusionsandtheirdistributionin51 speciesrangingover 13familiesoutofthe
15onesofascidians. Itrevealsageneraltendencythattheprotrusionsoccurinthelimitedfamiliesand
relatedspecieshavetheprotrusionsofsimilarsize. Thus,theprotrusionsprovedtohavesomefeatures
of certain phylogenetic significance in many cases. Brief references were made to the taxonomic
positionofsuchproblematicgeneraasPterygascidiaintheCionidaeandSorberaintheHexacrobylidae
on the basis ofthe present study.
ascidian phylogeny.
INTRODUCTION
To examine this suggestion closely, we should
The tunic is a gelatinous or leathery integument have more information. In this study, fine struc-
which is peculiar to the animals belonging to the ture of tunic cuticle was newly described in 26
subphylum Urochordata (=Tunicata). The asci- species of 10 families. Thus, we now get informa-
dian tunic is composed ofground substance (tunic tion about the protrusions from 51 species ranging
matrix) overlaid by a thin cuticle. The surface of over 13 families, although not yet examined speci-
the tunic cuticle is sometimes, though never al- mensofthetworemainingfamilies (Octacnemidae
ways, specializedtohave numerousminuteprotru- and Plurellidae). On the basis ofthe information,
sions, which are mostly papillate in shape and up we will give a comment on the phylogenetic sig-
to 100nm high. Such a structure was firstly re- nificance of the stated structure.
portedbyKatowandWatanabe [2] andMilanesiet
al. [3] in two species of the family Botryllidae. MATERIALS AND METHODS
Then, Hirose etal. [1] detected the protrusions in
18 species ofascidians belonging to 5 families, out
of the 25 species of9 families, and they suggested Animals andprefixation
that the protrusions have some implications for Specimens of Leptoclinides echinatus, Clavelina
viola, Ascidia ahodori, Ascidia zara, Ascidia sp.
Accepted November25, 1991 (cf. tapni) and Pyura mirabilis were collected in
Received October 2, 1991 Shimoda, Shizuoka Pref., and fixed in a solution
Present address: Department of Biology, Keio Uni- containing 2.5% glutaraldehyde, 0.45 M sucrose
versity, Yokohama, Kanagawa 223, Japan. M
Present address: Tokyo Kasei Gakuin Tsukuba Col- and 0.1 cacodylate (pH 7.4). Ascidia gemmata
lege, Tsukuba, Ibaraki 305, Japan. and Chelyosoma siboja were collected in Asa-
To whom requests for reprints should be addressed. mushi, Aomori Pref., and fixed in 2.5% glutar-
406 E. Hirose, T. Nishikawa et al.
aldehyde-seawater. Specimens of the following
RESULTS
species were fixed in 10% formol-seawateror 10%
formol-water: Syndiazona grandis collected off Tunic cuticle is an electron dense layer covering
Minabe, Wakayama Pref.; Adagnesia vesicu- tunic matrix, and its structure varies from species
liphora offFukushima Pref.; Corella sp. (cf. japo- to species. In some species, the cuticle forms
nica) in Kuroshima, Okinawa Pref.; Eugyrioides minute protrusions. Sometimes there is a layer of
glutinans off Oga Pen.; Molgula manhattensis in moderate electron densityor azone ofthe mixture
the Nagoya Harbor, Aichi Pref.; Molgula tectifor- of tunic matrix and cuticular material under the
mis in the Mutsu Bay; Chelyosoma yezoense and cuticle, and it is called "subcuticle" by De Leo et
Cnemidocarpa clara from Otsuchi, Iwate Pref.; al. [6]. Subcuticle is almost indistinguishable from
Cnemidocarpa Irene, Polycarpa cryptocarpa kro- the cuticle, when they are both of high electron
boja and Polycarpa maculata taken off the Oki density and thick, as seen in some solitary species
Island, Shimane Pref.; Ciona edwardsi and Ciona in the Corellidae and Styelidae. Thickness of the
roulei [cf. 4] collected around Banyuls-sur-Mer cuticle and subcuticle is revealedvariable in differ-
(France); Ciona intestinalis in Napoli (Italy); and ent species, and in different parts of tunic in a
Sorbera unigonas during the Iucal cruise from N. specimen. When the cuticle and/or subcuticle are
Atlantic (47°30'N, 9°35'W; 4217-4366m deep). thick, the tunic is usually hard, and leathery or
Pterygascidia longa was collected off Zamami, cartilaginous. The protrusions shown in the pre-
Okinawa, andfixedin 70% ethanol. The paratype sent study are papillate in shape without excep-
specimens of Polyandrocarpa stolonifera, fixed in tion.
70% ethanol, aredepositedinthe ShimodaMarine In the suborder Aplousobranchia, two species
Research Center. are studied. In Leptoclinides echinatus the cuticle
is flat and thin, about 10 nm thick, and its surface
Electron microscopy
shows a fuzzy appearance (Fig. 1A). In Clavelina
Samples of tunic, several millimeters square in viola the cuticular surface has minute protrusions,
size, were cut offfrom the above-listedspecimens. about 30-40nm high (Fig. IB).
Samples from the specimens in the fixative con- In the suborder Phlebobranchia, only 2 species
M
taining cacodylate were washed with 0.45 su- out of the 13 studied species have minute protru-
M
crose solution buffered with 0.1 cacodylate (pH sions. In the family Cionidae, Syndiazonagrandis
7.4), while samples from the specimens in the and three Ciona species have a flat cuticle accom-
fixatives without cacodylate were washed with panied with a subcuticle, but no protrusions (Fig.
filtrated seawater. Then, the samples were post- 1C). Thickness of the subcuticle ranges from
fixed with 0.1% osmium tetroxide solution buf- about 30nm (in C. edwardsi) to more than 300 nm
M
fered with 0.1 cacodylate (pH7.4) for 1.5 (in C. roulei). On the other hand, Pterygascidia
hours, andafterward, dehydratedthrough anetha- longa of the same family has minute cuticular
nol series. For scanning electron microscopy protrusions, about 60 nm in height (Fig. ID). In
(SEM), they were dried in a critical point dryer, the Ascidiidae, the four species ofAscidia have a
sputter-coated with Au-Pd, and examined by flat cuticle without any protrusions; its thickness is
Hitachi S-570 scanning electron microscope at 15 about 20-30nm (in A. ahodori, A. sp. (cf. tapni)
to 20 kV. For transmission electron microscopy and A. zara) or more than 100nm (in A. gemma-
(TEM), they were cleared in n-butyl glycidyl ta). In the Agnesiidae, Adagnesia vesiculiphora
ether, and embedded in a low viscosity resin [5]. has minutecuticularprotrusions, onlyabout20nm
Thin sections were doubly stained, and examined high (Fig. IE). In the Corellidae, all the three
by Hitachi HS-9 transmission electron microscope species examined here have no minute protru-
at 75 kV. sions. Corella sp. (cf. japonica) has a thin cuticle,
only about 30 nm, while the two species of Chely-
osomahave athickone, indistinguishable from the
underlying subcuticle (Fig. IF).
Minute Cuticular Protrusions of Ascidian Tunic 407
•-
B
;;*V; *4yJ?&*<
i?h '
?%
'
Fig. 1. FinestructureofthetuniccuticleintheorderEnterogona. Tunicmatrixpositionsinlowerpartofeachfigure
in TEM. (A) Leptoclinides echinatus (TEM). (B) Clavelina viola (TEM). (C) Ciona roulei (TEM). (D)
Pterygascidia longa (SEM). (E) Adagnesia vesiculiphora (TEM). (F) Chelyosomasiboja (TEM). Arrowheads
indicate minute protrusions. Scale bars, 0.2/um.
In the suborder Stolidobranchia, the minute Cnemidocarpa clara has the protrusions about 100
cuticularprotrusions are found in allthe examined nm (Fig. 2A, B), and Styelaclava has smallerones
species excluding some solitary ones in the Styeli- (about 30nm high) and a thick subcuticle under-
A
dae. strange styelid, Polyandrocarpa stolo- lying the cuticle. The other four solitary styelids
nifera, which propagates by stolonic budding but lacktheprotrusions;thetunicisleatheryorcartila-
without any functional connection among the ginous, with the thick cuticle and subcuticle (Fig.
zooids [see 7], has the protrusions, 100nm or less 2C). In Pyura mirabilis of the Pyuridae, minute
in height. Amongthe6examinedsolitarystyelids, protrusions are about 100nm high, and the sub-
408 E. Hirose, T. Nishikawa et al.
E
i
Minute Cuticular Protrusions of Ascidian Tunic 409
cuticle has a complex structure (Fig. 2D). In the height (Fig. 2G, H). The protrusionsaresimilarin
Molgulidae, the protrusions are found in all ofthe size and shape to those usually seen in the Stoli-
3 examined species. They are about 40-50nm dobranchia.
high (in Molgula manhattensis and M. tectiformis)
or 30nm (in Eugyrioides glutinans) (Fig. 2E, F), DISCUSSION
and they are rather lower than those seen in the
other species in the Stolidobranchia. Table 1 represents a summation of the present
In the suborderAspiraculata, we could examine results and the previous ones [1] in terms of the
only a signle species, Sorbera unigonas. The presence or absence and height of the minute
cuticle has minute protrusions, about 80nm in cuticular protrusions. So far as these results are
Table 1. Presence and height of minute cuticular protrusions of ascidian tunic
Solitary (S) or Approx. height of
Species
Colonial (C) protrusions (nm)
Order Enterogona
Suborder Aplousobranchia
Family Polyclinidae
Aplidium pliciferum 30
A. yamazii 60
Family Didemnidae
Diplosoma mitsukurii absent
Didemnum moseleyi absent
*Leptoclinides echinatus absent
Family Polycitoridae
Clavelina miniata 30
*C. viola 30-40
Polycitor proliferus 40
Suborder Phlebobranchia
Family Cionidae
*Syndizona grandis absent
Ciona savignyi absent
*C. edwardsi absent
*C. intestinalis absent
*C. roulei absent
*Pterygascidia longa 60
Family Perophoridae
Perophora japonica absent
P. multiclathrata absent
Family Ascidiidae
Ascidia sydneiensis absent
*A. ahodori absent
*A. gemmata absent
*A. zara absent
Fig. 2. Finestructureofthe tuniccuticleintheorderPleurogona. (A, B) Cnemidocarpaclara (A: TEM, B: SEM).
(C) Cnemidocarpa irene (TEM). (D) Pyura mirabilis (TEM). (E) Molgula manhattensis (TEM). (F) Molgula
tectiformis (SEM). (G, H) Sorbera unigonas (G: TEM, H: SEM). Arrowheads indicate minute protrusions,
asterisks debris. Scale bars, 0.2jum.
410 E. Hirose, T. Nishikawa et al.
Table 1. (continued)
Solitary (S) or Approx. height of
Species
Colonial (C) protrusions (nm)
M. sp. (cf. tapni) S absent
Family Agnesiidae
*Adagnesia vesiculiphora S 20
Family Corellidae
*Chelyosoma siboja S absent
*C. yezoense S absent
*Corella sp. (cf. japonica) s absent
Order Pleurogona
Suborder Stolidobranchia
Family Botryllidae
Botryllus primigenus c 100
B. scalaris c 100
B. sexiens c 100
B. schlosseri c 100
Botrylloides fuscus c 100
B. lentus c 100
B. simodensis c 100
B. violaceus c 100
Family Styelidae
Metandrocarpa uedai c 100
Polyandrocarpa misakiensis c 100
*Polyandrocarpa stolonifera c 100
Polyzoa vesiculiphora c 100
Symplegma reptans c 100
*Cnemidocarpa clara s 100
*C. irene s absent
*Polycarpa cryptocarpa kro- s absent
boja
*P. maculata s absent
Styela plicata s absent
*S. clava s 30
Family Pyuridae
Halocynthia roretzi (Type A) s 50a
Herdmania momus s 100
*Pyura mirabilis s 100b
Family Molgulidae
*Eugyrioides glutinans s 30
*Molgula manhattensis s 40-50
*M. tectiformis s 40-50
Suborder Aspiraculata
Family Hexacrobylidae
*Sorbera unigonas s 80
* species firstly examined for the protrusions in this study.
a protrusions are not papillate.
b subcuticle has complex structure.
Minute Cuticular Protrusions of Ascidian Tunic 411
concerned, the shape of the protrusions is always significance of these exceptions at present.
papillate only except in Halocynthia roretzi, which There is a hot controversy on the systematic
has irregular-shaped ones [1]. This table shows position of the family Hexacrobylidae in the sub-
clearly such a general tendency that the protru- order Aspiraculata. Monniot et al. [11] separated
sions occur in the limited families and the related this family from the class Ascidiacea and estab-
species have the protrusions ofsimilar size. Thus, lished for it a new class Sorberacea ofthe subphy-
we may reasonably conclude that the mentioned lum Urochordata (=Tunicata). The reason for
features are of certain phylogenetic significance. this treatment was that they considered such fea-
Ontheotherhand,wecannotmake anyreferences tures of this family to be quite unique among
to the function of the protuberances. tunicates, as the virtual lack of the branchial sac,
In the suborder Aplousobranchia, the protru- the existence ofthe "cordon nerveux dorsal", and
sions are found in the Polyclinidae and Polycitor- the histological peculiarities of the gut [also see
idae. The protrusions are about 50 nm or less in 12]. On the contrary, Kott has been holding the
height. Incontrast, noprotrusionsarefoundinthe opinion that the family Hexacrobylidae should be
Didemnidae. regarded as very closely related (or possibly even
In the suborder Phlebobranchia, most species joinable) to the family Molgulidae ofthe suborder
lack the protrusions. Exceptionally, however, we Stolidobranchiaandthereforenevermertisadiffer-
could find them in Pterygascidia longa in the ent suborder. Kott [13] regarded the unique
Cionidae and Adagnesia vesiculiphora in the features ofthis group mentioned by Monniotetal.
Agnesiidae. This fact reminds us of Kott's long- as due merely to a high adaptation to its deep-sea
heldopinionthat "Ciallusiinae" representedbythe life, and claimed that its basic morphological fea-
genus Pterygascidia should be included as a sub- tureswere sharedwith the Molgulidae, such as the
family in the family Agnesiidae, which contains presence of a kidney, the arrangement of gonads,
Adagnesia as a member of the other subfamily the thin but tough and fibrous tunic, etc. On the
Agnesiinae [ref., 8]. Kott [9] paid attention to the other hand, Nishikawa [14] supported the tradi-
similarityinthe mantle musculature between these tional treatment of the Hexacrobylidae as the
two subfamilies delimited by herself (for discus- single constituent of the suborder Aspiraculata,
sions on the taxonomicpositionofPterygascidia in although acknowledging the uniqueness of this
terms of macroscopical morphology, see Tokioka group strongly claimed by Monniot et al. The
[10]). Further examinations of Kott's opinion are present study revealed that the protrusions found
expected on the basis of more information about in Sorbera unigonas were similar in shape and size
the protrusions from the related species. tothoseofthestolidobranchianspeciesratherthan
Amongthe 25 examined species ofthe suborder of the aplousobranchian and phlebobranchian
Stolidobranchia, only the four solitary species in ones. This fact may favor Kott's view, but it does
the Styelidae lack the protrusions. It seems to be not necessarily exclude the other two taxonomic
curious that Cnemidocarpa clara and Styela clava treatmentsifwe regardthe mentionedsimilarityas
have the protrusions, while the respective conge- a convergent nature. On the other hand, a closer
ners C. ireneandS. plicatalackthem. Itshouldbe examination shows that the protrusions of S. un-
noted that such inconsistencies are limited to the igonas are muchhigherthanthoseoftheexamined
subfamily Styelinae in the Styelidae. The lack of species in the Molgulidae (about 80nm in the
the protrusions in the four species may be related former, instead of 30-50nm in the latter). This
tothe factthat the cuticle isratherthickerin these might suggest the somewhat distance of phylo-
species than the others in this suborder. In the genetic position between Sorbera and the Molgu-
other stolidobranchians, the protrusions are papil- lidae. Accordingly, we prefer the tranditional
late and about 100nm high. Exceptionally, they systematic treatment of this group at present.
are of smaller size in Styela clava and the three More information should be gained as to the fine
molgulid species, or are not papillate in Halocy- structure of tunic in some other species of the
nthia roretzi. It is unclear about the phylogenetic Molgulidae and Hexacrobylidae.
412 E. Hirose, T. Nishikawa et al.
As shown above, some features seen in the fine 293-295.
structure oftunic cuticle have certain phylogenetic 5 Kushida, H. (1980) An improved embedding
significance. We expect that more attentions will methodusingERL4206and Quetol653. J. Electron
Microsc, 29: 193-194.
be paid to the fine structures in terms of the
6 De Leo, G., Patricolo, E. and Frittitta, G. (1981)
plylogeny of not only ascidians but also the other
FinestructureofthetunicofCionaintestinalisL. II.
tunicates. Tunic morphology, cell distribution and their func-
tional importance. Acta Zool. (Stockh.), 62: 259-
271.
ACKNOWLEDGMENTS
7 Kawamura, K. andWatanabe, H. (1981) Studiesof
We wish to thank Drs. T. Hirata, K. Ito, C. & F. Japanese compound styelid ascidians. III. A new,
Monniot, F. Lafargue, T. Numakunai, T. Ohtsuka and possibly asexual Polyandrocarpa from Shimoda
H. Uchida forkindlyproviding specimens. Thisstudyis Bay. Publ. Seto mar. Biol. Lab., 26: 425-436.
supported by Grant-in-Aid for Co-operative Research 8 Kott, P. (1985) The Australian Ascidiacea Part I,
(A) #01304007from the MinistryofEducation, Science Phlebobranchia and Stolidobranchia. Mem. Qd
and CultureofJapan. Thisreportincludescontributions Mus.,23: 1-440.
from the Shimoda Marine Research Center, No. 538. 9 Kott, P. (1969) AntarcticAscidiacea. Antarct. Res.
Ser. Washington, 13: 1-239.
10 Tokioka, T. (1971) The ascidian genera Pterygasci-
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