Fine structure of the ascidian tunic cuticle was surveyed for minute protrusions in 30 species of the suborder Aplousobranchia, 10 species of Phlebobranchia and 23 species of Stolidobranchia (Ascidiacea, Chordata). The present results, combined with previously published data, describe the fine structure of the tunic cuticle in 116 species of ascidians covering all the families and subfamilies of the class Ascidiacea except for the phlebobranch families Octacnemidae and Plurellidae. These data confirm the general stability of the character-state distribution (presence or absence) of the tunic cuticular protrusions within families or subfamilies. Cuticular protrusions occur only in Polyclininae and Clavelininae of the order Enterogona (Aplousobranchia and Phlebobranchia) and in all families of the order Pleurogona (Stolidobranchia and Aspiraculata). A few exceptions were the occurrence of cuticular protrusions in Polycitor proliferus (Polycitorinae), Distaplia dubia (Holozoinae), Pterygascidia longa (Cionidae) and Adagnesia vesiculiphora (Agneziidae), and the absence in some styelids and pyurids that have hard tunics. Possible character-state changes in the ascidian phytogenies are discussed briefly.
INTRODUCTION
Urochordates (tunicates) constitute one of the three sub-phyla in the phylum Chordata, and are characterized by the presence of the tunic, an integumentary tissue wholly covering the epidermis. The tunic contains cellulose fibers linked with proteins (De Leo et al., 1977; Van Daele et al., 1992), which makes it unique among metazoan tissue. All ascidians have this integumentary tissue but it varies greatly in structure between the various groups and thus may be important to study comparatively from the viewpoint of ascidian phylogeny.
The outermost layer of the tunic is called the tunic cuticle, underlain by the fibrous matrix furnished densely with mesenchymal free cells (tunic cells). Under an electron microscope, the tunic cuticle is revealed to be a continuous layer composed of electron dense materials. The cuticle is often furnished with minute protrusions over the surface, and/or with semi-electron dense layers (subcuticle) beneath it. In some species, the cuticular surface has numerous protrusions (Katow and Watanabe, 1978; Milanesi et al., 1978). We have examined these fine structures from the viewpoint of ascidian taxonomy and phylogeny. On the basis of a survey of 51 species covering 13 families out of the 15 recognized ones in all suborders of the class Ascidiacea (i.e., Aplousobranchia, Phlebobranchia, Stolidobranchia and Aspiraculata), Hirose et al. (1992) recognized the following patterns in the occurrence of cuticular protrusions: (1) Cuticular protrusions occur in only a few aplousobranch families; (2) In the Phlebobranchia, protrusions occur only in Pterygascidia longa and Adagnesia vesiculiphora among the 17 species examined; (3) In the Stolidobranchia, most of the examined species have protrusions except a few styelid species. These patterns suggest that the character-state distribution (presence or absence of the protrusions) in the tunic cuticle may be a valid clue to ascidian phylogeny. To verify this, however, further extensive surveys of additional species were necessary.
In the present study, we examined 30 additional aplousobranch species, including those belonging to two subfamilies not previously studied, and two subfamilies so far represented only by a single species. Similarly, we also investigated the cuticular fine structures in 10 additional phlebobranchs and 23 stolidobranchs. As a result we now have a perspective of the character-state distribution of tunic cuticular protrusions in 116 species covering all the families and subfamilies included in a traditional classification (see Table 1), except for the phlebobranch families Octacnemidae and Plurellidae.
Table 1
Occurrence and height of tunic cuticular protrusions in ascidians
MATERIALS AND METHODS
Animals
Thirty species of aplousobranchs, 10 phlebobranchs and 23 stolidobranchs were examined. Collection sites are listed in Table 1. The specimens collected in Australia and Hawaii were generous gifts from Dr. Hiromichi Koyama (Yokohama City University).
Electron microscopy
Tunic pieces were prefixed and stored in (A) 2.5% glutaraldehyde-0.1 M cacodylate-0.45 M sucrose (pH 7.4), (B) 2.5% glutaralde-hyde-0.14 M NaCI-0.2 M Millonig's phosphate buffer (pH 7.4), (C) 2% paraformaldehyde plus fix B, or (D) 10% formalin-seawater. In some cases, the specimens were anesthetized with menthol-seawater or 6% MgCI2-menthol for several hours at 12–14°C, before fixation. After brief rinsing through 0.1 M cacodylate-0.45 M sucrose (pH 7.4), the specimens were postfixed in 1% osmium tetroxide-0.1 M cacodylate (pH 7.4) for 1.5 hr, and dehydrated through an ethanol series. The specimens were cleared with n-butyl glycidyl ether and embedded in low viscosity epoxy resins. Thin sections were stained with uranyl acetate and lead citrate, and examined in a Hitachi HS-9 transmission electron microscope.
In some didemnid and polycitorid specimens, the tunic was impregnated densely with spicules of calcium carbonate. Such specimens were decalcified before postfixation by incubating in 5% EDTA-2Na (ethylenediamine tetra acetic acid, disodium salt) for 2–3 days. The EDTA treatment had no detectable effects on the fine structure of the tunic in control experiments applied to the spicule-free Botryllus schlosseri (Fig 1) and Botrylloides fuscus with cuticular protrusions of 100 nm height (Hirose et al., 1990), and to the spicule-bearing Didemnum moseleyi and Leptoclinides echinatus which had been shown to lack any tunic protrusions using SEM without the EDTA treatment (Hirose et al., 1990,1992). Approximate height of cuticular protrusions was directly measured from the negatives of electron micrographs.
Phylogenetic considerations
These data were tabulated on a traditional ascidian classification based on the systems given by Berrill (1950), Monniot and Monniot (1972), Kott (1985) and Nishikawa (1986). Due attention was paid to the newly proposed system of Kott (1990, 1992).
RESULTS
Our present results are shown in Table 1 in combination with the previously published data (Hirose et al., 1990, 1992; Cloney, 1994; Turon and Vázquez, 1996).
Aplousobranchia
In the family Polyclinidae, all the examined Aplidium species (subfamily Polyclininae) have cuticular protrusions of 30–60 nm (Fig 2, Table 1). On the other hand, 3 species from 3 different genera in the subfamily Euherdmaniinae have only a very thin cuticular layer lacking the protrusions completely (Fig 3).
In the Didemnidae, the currently examined 7 species and 3 previously observed species from 5 genera invariably lacked protrusions, whether they were spicule-bearing or not. These 10 species were quite similar to one another in their tunic ultrastructure.
The family Polycitoridae includes three subfamilies: Polycitorinae, Clavelininae and Holozoinae. In the Polycitorinae, only Polycitor proliferus had been previously examined and was found to have distinct protrusions of 40 nm. The 7 additional species used here were revealed to lack them. It follows that the presence of cuticular protrusions in P. proliferus may be unusual in the Polycitorinae. Eudistoma gilboviride has a thicker cuticle than the other 2 congeners (Figs. 4 and 5), though all lack protrusions. In the Clavelininae, the 3 Clavelina spp. examined here were very similar in the size of protrusions (30–40 nm) to the previously observed species, but their embossed cuticle was corrugated densely at an amplitude of 100–200 nm (Fig 6). In the Holozoinae, Distaplia dubia has 20–30 nm high protrusions (Fig 7), while the other 5 examined species including its two congeners lack them (Fig 8).
Phlebobranchia
All 10 newly examined species have a flat cuticle without any protrusions, consistent with most of the previous results from phlebobranchs (Table 1). As the information about cuticle structure in the family Agneziidae had been limited only to the single species Adagnesia vesicufiphora, we examined another species, Agnesia himeboja, but unsuccessfully; the tunic was not well-preserved. Further, we failed to examine well-preserved material from a sand impregnated species of Plurellidae and a deep water species of Octacnemidae. In the suborder Phlebobranchia, the tunic of many species is gelatinous, and its cuticle is thinner with less electron density (Fig 9) than ascidians of the other suborders. In Chelyosoma, however, the tunic is rather thick and hard, and its cuticle is thicker than usual for phlebobranchs (Fig 10).
Stolidobranchia
Character-state distributions of the cuticular protrusions in the newly examined 23 stolidobranchs were consistent with those in the previous results (Table 1) for Botryllidae, Polyzoinae (Styelidae) and Molgulidae (Figs. 11,15,16). Further, the previously detected occurrence of both character states in the Styelinae (i.e., present in some species, absent in others) was also true of the newly examined 6 species (Fig 12). The only exception concerned the Pyuridae, in which Microcosmus curvus lacked protrusions while all the other previously or presently analyzed species had them.
Leathery or cartilaginous tunic in some species tended to have a thicker and more electron-dense cuticular layer than the gelatinous tunic of other species. Even in the same individual, the cuticular layer was often thicker where the tunic was harder, and the mode of occurrence of the protrusions varied in some cases. In Halocynthia igaboja, the cuticular protrusions of the tunic proper were larger than those of the somewhat hardened tunic spines (Fig 13). In Pyura spinifera, the protrusions were detectable on the tunic of the body proper, while completely absent on that of the much hardened stalk (Fig 14).
DISCUSSION
Stability of the character-state
Table 1 supports our previous conclusion that the presence of cuticular protrusions appears to have a phylogenetic significance in many cases (Hirose et al., 1992), because of the general stability of the character-state (presence or absence) within the families or subfamilies in the traditional classification adopted here. A few exceptions are Distaplia dubia (Holozoinae), Polycitor proliferus (Polycitorinae), Pterygascidia longa (Cionidae), Microcosmus curvus (Pyuridae), and the subfamily Styelinae. The cases of D. dubia and M. curvus are difficult to explain, and further studies are needed to document the peculiarity of these species in other characters. The observed heterogeneity in character-state distribution in the Styelinae and even within its genera Cnemidocarpa and Styela might be due to a certain variability in this feature in the concerned clades, as discussed below.
In the Phlebobranchia, cuticular protrusions are absent in most species, but there are two major exceptions; Pterygascidia longa in the Cionidae and Adagnesia vesiculiphora in the Agneziidae. This fact may support Kott's opinion that “Ciallusiinae”, represented by the genus Pterygascidia, should be included as a subfamily of the Agneziidae (Kott, 1969, 1985).
The case of Polycitor proliferus is worthy of consideration in relation to its somewhat controversial taxonomic position. Kott (1990,1992) made a major modification in the traditional aplousobranch classification by erecting several new families and raising several subfamilies to family rank. Although she has not yet presented a conclusive phylogenetic hypothesis supporting her new classification, it is relevant to apply our data to it. First of all, Kott (1992) divided the family Polyclinidae into 6 families: Polyclinidae sensu strict (containing Aplidium spp. in the present study), Placentelidae (containing Placentela crystallina examined here), Protopolyclinidae (no examined species here), Ritterellidae (Ritterella pulchra and Polycitor proliferus), Euherdmaniidae (no species here) and Pseudodistomidae (Pseudodistoma kanoko). According to this system, Polyclinidae is distinguishable from the remaining 5 families by the atrial aperture not 6-lobed and having well-developed cloacal systems. These differences were used by other taxonomists (Nishikawa, 1986; Monniot and Monniot, 1972) to distinguish the subfamily Polyclininae from Euherdmaniinae in the traditional Polyclinidae. Significantly, these character-states parallel the presence or absence of cuticular protrusions, with the single exception of P. proliferus which is discussed below. We agree with Kott's delimitation of the family Didemnidae which treats it in the traditional manner.
If we were to follow Kott's (1990) scheme, all of the polycitorid species we examined (Table 1), except P. proliferus (see below), of the subfamily Polycitorinae would be included in her family Polycitoridae, all of our Clavelininae would be moved to her Clavelinidae, and all of our Holozoinae and Sigillina signifera of our Polycitorinae to her Holozoidae. So far as S. signifera (without cuticular protrusions) is concerned, her new system is consistent with our present results, because the absence of protrusions is a consistent trait in our Polycitorinae and Holozoinae with the exception of Distaplia dubia. At present, character-state distribution of cuticular protrusions, however, can not completely support Kott's system, because it does not cover all of her aplousobranch families. Therefore, we still prefer to use the traditional phylogenetic system until a comprehensive study is developed in this group.
Polycitor proliferus (Oka, 1933), thus far recorded from Japanese warm waters on the Pacific coasts (Nishikawa, 1995), was transferred to the genus Ritterella of the family Ritterellidae by Kott (1969, 1992). This judgment is based on the presence of a short postabdominal extension of P. proliferus which she believed to be a true postabdomen because the gonads are situated within it (see Tokioka, 1953, pl. 25, fig. 6). On the other hand, Tokioka (1953) and Nishikawa (1986, 1995) regarded this species as a member of Polycitor of the family Polycitoridae because they believe the postabdominal extension is not a true postabdomen. Oka (1942) clarified in his original description of Polycitor mutabilis, now regarded as a junior synonym of P. proliferus (see Tokioka, 1953), that the postabdominal extension is markedly detectable only during the breeding season, and includes the ovary and testicular follicles but “does not contain the heart” (p. 159). The heart remains in its usual position along the rear end of the intestinal loop (Ebara, 1951). P. proliferus seems unusual among its congeners, in which the gonads are located within or along the posterior part of intestinal loop (see Kott, 1990)
Thus, the taxonomic position of P. proliferus remains an open question in terms of morphology. In so far as the cuticular protrusions are concerned, as shown in Table 1, their presence in P. proliferus is not shared by Kott's Ritterellidae or Placentelidae or our Polycitorinae, but is shared by our Polyclininae, Clavelininae and Distaplia dubia in the Holozoinae. We hope that the taxonomic significance of this similarity will be verified by molecular phylogenetics.
Brief phylogenetic considerations
Only a few attempts have been made to present phylogenetic trees of ascidians exclusively on a morphological basis (see Millar, 1966; Kott, 1969). We still have no detailed trees in terms of modern phylogenetics. However, the genus Ciona (which lacks protrusions) is generally regarded as the most primitive among extant ascidians, while Stolidobranchia are believed to be monophyletic and to have arisen more recently. If these assumptions are accepted, stolidobranchs obtained cuticular protrusions during their derivation from the common ancestor of ascidians, which would have lacked protrusions as in the modern Ciona. In the stolidobranch clade, all families have species with cuticular protrusions.
It follows, then, that the above mentioned heterogeneity in the character-state distribution seen in Styelinae can be attributed to a reversal of this character-state within the clade. Loss of the protrusions may be related to tunic thickening and/or hardening. And the apomorphic state (presence of protrusions) seen in many non-stolidobranchs may have occurred several times in different lineages. Some may well ask whether the tunic protrusions of stolidobranchs are identical to those in the other ascidians. So far, we have no ideas against the homoplasy of cuticular protrusions in the Ascidiacea. However, we must take this possibility into account especially in determining their functional aspects, which are as yet unknown.
Plate I
Transmission electron micrographs of the tunic cuticle in some aplousobranchs. Fig. 1, Botryilus schlosseri (treated with EDTA); Fig. 2, Aplidium solidum; Fig. 3, Placentela crystallina; Fig. 4, Eudistoma glaucus; Fig. 5, Eudistoma gilboviride; Fig. 6, Clavelina cyclus; Fig. 7, Distaplia dubia; Fig. 8, Distaplia occidentalis. Arrowheads in Fig. 4 and 5 indicate the epibiont layer covering the outside of cuticle. Small arrowheads in Fig. 6 show the protrusions over the corrugated cuticle (for details see the text). Magnifications are the same in all figures. Scale bar (Fig. 1)=0.5 μm.
Plate II
Transmission electron micrographs of the tunic cuticle in some phlebobranchs and stolidobranchs. Fig. 9, Ascidia paratropa; Fig. 10, Chelyosoma productum; Fig. 11, Stolonica socialis; Fig. 12, Cnemidocarpa bicornuta; Fig. 13, Halocynthia igaboja (A, body tunic; B, tunic spine); Fig. 14, Pyura spinifera (A, body tunic; B, stalk tunic); Fig. 15, Molgula bleizi; Fig. 16, Eugyra arenosa. Arrowheads in Fig. 12 indicate projections of uneven surface of the tunic cuticle. Magnifications are the same in all figures. Scale bar (Fig. 1)=0.5 μm.
Acknowledgments
We wish to thank Prof. C. Lambert (California State University), Dr. B. J. Swalla (Vanderbilt University) and Dr. H. Koyama (Yokohama City University) for helping with material collections. We also thank Dr. Y. Saito and other staff members of Shimoda Marine Research Center (SMRC), University of Tsukuba for providing the facilities for electron microscopy and for their hospitality. The present study includes contributions from SMRC #609.
