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J Mol Evol (1995) 40:413-427
jou.. o, MOLEEULAR IEVOLUTII:IN
© Springer-Verlag New York Inc. 1995
Multiple Origins of Anural Development in Ascidians Inferred from rDNA Sequences
Kristen A. Hadfield,* Billie J. Swalla,** William R. Jeffery
Bodega Marine Laboratory and Section of Molecular and Cellular Biology, University of California, Davis, P.O. Box 247, Bodega Bay, CA 94923, USA Station Biologique, Roscoff 29680, France
Received: 14 February 1994 / Accepted: 8 June 1994
Abstract. Ascidians exhibit two different modes of development. A tadpole larva is formed during urodele development, whereas the larval phase is modified or absent during anural development. Anural development is restricted to a small number of species in one or pos- sibly two ascidian families and is probably derived from ancestors with urodele development. Anural and urodele ascidians constitute a model system in which to study the evolution of development, but the phylogeny of anural development has not been resolved. Classification based on larval characters suggests that anural species are monophyletic, whereas classification according to adult morphology suggests they are polyphyletic. In the present study, we have inferred the origin of anural de- velopment using rDNA sequences. The central region of 18S rDNA and the hypervariable D2 loop of 28S rDNA were amplified from the genomic DNA of anural and urodele ascidian species by the polymerase chain reac- tion and sequenced. Phylogenetic trees inferred from 18S rDNA sequences of 21 species placed anural developers into two discrete groups corresponding to the Styelidae and Molgulidae, suggesting that anural development evolved independently in these families. Furthermore, the 18S rDNA trees inferred at least four independent origins of anural development in the family Molgulidae.
* Present address: Department of Vegetable Crops, University of Cal- ifornia, Davis, CA 95616 ** Present address: Department of Biology, Vanderbilt University, Nashville, TN 37235 Correspondence to: W.R. Jeffery
Phylogenetic trees inferred from the D2 loop sequences of 13 molgulid species confirmed the 18S rDNA phy- logeny. Anural development appears to have evolved rapidly because some anural species are placed as closely related sister groups to urodele species. The phylogeny inferred from rDNA sequences is consistent with mol- gulid systematics according to adult morphology and supports the polyphyletic origin of anural development in ascidians.
Key words: Ascidians - - Larval development - - 18S rDNA sequences - - 28S rDNA sequences - - Molecular phylogeny - - Polyphyly
Introduction
Ascidians exhibit two different modes of development (Berrill 1931; Jeffery and Swalla 1992a). Urodele devel- opment involves the formation of a tadpole larva con- sisting of a head and tail (Katz 1983). The head contains the brain, with one or more sensory cells, and an endo- dermal mass, which forms most of the adult structures after metamorphosis. The tail consists of a notochord flanked by the spinal cord, the endodermal strand, and lateral bands of muscle cells. The tail muscle cells are specified by determinants localized in the egg (Swalla 1992), whereas other larval tissues are specified by in- ductive cell interactions during embryogenesis (Venuti and Jeffery 1989; Nishida, 1992). The larval tail is formed after gastrulation by concerted movements of the
414
prospective notochord, spinal cord, muscle, and posterior epidermal cells. Later, a pigmented sensory organ(s) ap- pears in the brain, and striated muscle cells differentiate in the tail. After dispersal by swimming, the tadpole settles on a substrate and undergoes metamorphosis into the adult. During metamorphosis most of the larval struc- tures are destroyed and replaced by adult specific tissues and organs. Urodele development is ubiquitous in all 14 ascidian families.
Anural development is an alternate mode of develop- ment which has been described in only 16 species, mostly assigned to the family Molgulidae (Berrill 1931; Jeffery and Swalla 1990). Anural developers have exten- sively modified or eliminated the tadpole larva. This mode of development involves dramatic changes in or- ganization of the myoplasm (Swalla et al. 1991; Jeffery and Swalla 1993), an egg cytoskeletal domain responsi- ble for specifying the tail muscle cells and other urodele features (Jeffery and Meier 1983; Jeffery 1990), embry- onic cell lineages (Jeffery and Swalla 1991), morphoge- netic movements of notochord cells (Damas 1902), and brain sensory organ and muscle cell differentiation (Whittaker 1979; Swalla and Jeffery 1990, 1992; Bates and Mallet 1991a,b). Remarkably, notochord, brain sen- sory organ, and some aspects of muscle cell differentia- tion can be restored when the genome of the urodele developer Molgula oculata is introduced into the egg of the anural developer MoIgula occuIta by interspecific fertilization (Swalla and Jeffery 1990; Jeffery and Swalla 1991, 1992b). The restoration of urodele features in these hybrids suggests that anural development is mediated by loss of function mutations in zygotic genes. Recently, genes encoding potential regulatory factors have been identified with altered expression patterns in M. oculata and M. occulta (Swalla et al. 1993).
It is important to determine the mechanisms respon- sible for the evolution of anural development in ascidi- ans. At present, this question cannot be properly ad- dressed because the phylogeny of anural development is unresolved. Because of their restriction to only one or two ascidian families and the presence of vestigial urodele structures (Damas 1902; Whittaker 1979; Swalla and Jeffery 1990, 1992; Jeffery and Swalla 1991; Bates and Mallet, 1991a), anural species are likely to have evolved from ancestors with urodele development. How- ever, it is uncertain based on morphological characters whether anural development evolved only once or many times from different urodele ancestors. In the first de- scription of anural development, Lacaze-Duthiers (1874, 1877) proposed a monophyletic origin of anural species and created the genus Anurella for all ascidian taxa lack- ing the tadpole larva. Monophyly is supported by reduc- tion or loss of the same suite of larval cells and tissues in different anural species. In contrast, )krnb~ick-Christie- Linde (1928) and Berrill (1931) later proposed that anural development is polyphyletic. Their conclusions
were based primarily on differences in adult branchial sac morphology, which is currently used as a primary character in ascidian systematics (Van Name 1945; Mon- niot 1969). Polyphyly gained credibility after the discov- ery of anural development in two ascidian species tradi- tionally assigned to the family Styelidae rather than to the Molgulidae (Millar 1954, 1962). However, the struc- ture of the branchial sac is aberrant in one of these spe- cies, making its classification questionable. Furthermore, branchial sac morphology is sometimes variable within a single species and possibly subject to convergence in the molgulids (Huntsman 1922; Berrill 1931). Consequently, the earlier molgulid classification scheme based on larval morphology still influences some taxonomic treatments of this group (Plough 1978).
Although phylogenetic inference is necessary to use anural and urodele ascidians to study the evolution of development (Jeffery and Swalla 1992a), the evolutionary history of anural development is still uncertain. Here we describe phylogenetic analysis of anural development based on 18S and 28S rDNA sequences. The results strongly support the polyphyletic origin of anural devel- opment.
Materials and Methods
Biological Materials. The ascidians Bostrichobranchus digonas and Molgula occidentalis were purchased from Gulf Specimens, Inc., Pan- acea, FL; Molgula manhattensis, Molgula provisionalis, and Molgula citrina were purchased from the Supply Department, Marine Biological Laboratory, Woods Hole, MA; Styela plicata was purchased from Marinus Inc., Long Beach, CA; Cnemidocarpafinmarkiensis was pur- chased from Westwind Sealab Supplies, Victoria, BC, Canada; and Pelonaia corrugata was purchased from The University Marine Sta- tion, Millport, Scotland, U.K. Eugyra arenosa, Molgula complanata, Molgula bleizi, Molgula occulta, Molgula oeulata, MoIgula echinosi- phonica, and Dendrodoa grossularia were collected at Roscoff, France. Molgula socialis was collected at Trtguier, France. Polycarpa pomaria was collected at Banyuls sur Met, France. Styela clava was collected at Woods Hole, MA, and Styela montereyensis and Aseidia ceratodes were collected at Bodega Bay, CA. An additional collection of M. manhattensis was made at Lorient, France. The animals were main- tained in tanks of running seawater.
DNA Isolation. DNA was isolated from whole animals dissected free of their tunics or from isolated gonads using the procedure of Davis et al. (1986) or the following method. Fifty to 100 mg of tissue was homogenized in 150 gl of preheated 50 mM glucose, 100 mM EDTA, 25 mM Tris (pH 8.0), 0.5 mg/ml proteinase K and incubated for 3 min at 65°C. The homogenates were brought to room temperature, and 300 gl of a freshly prepared solution containing 1% SDS in 0.2 M NaOH was added to each sample. After the samples were incubated on ice for 5 min, 225 pl of ice-cold 3 M KC1, 5 M Na acetate was added, the incubation on ice was continued for an additional 5 rain, and then the samples were centrifuged for 10 rain at 13,000 g. The superuatants were extracted once with phenol-chloroform-isoamyl alcohol (50:48: 2), and the aqueous phases were precipitated with 100% ethanol over- night at -70°C. The DNA was collected by centrifugation (13,000 g; 10 min), washed with 80% ethanol, and dissolved in sterile water. Sperm DNA from Molgula tectiformis collected at Otsuchi, Japan was pro- vided by H. Wada and N. Satoh.
415
Table 1. The ascidian species used in this analysis showing their taxonomy, mode of development, and the Genbank accession number or source
of their rDNA sequences
Accession numbers or source
Species and taxonomy a Development b 18S rDNA 28S rDNA
Order Enterogona Suborder Phlebobranchiata
Family Ascidiidae Ascidia ceratodes Urodele L12378 - -
Order Pleurogona Suborder Stolidobranchiata
Family Pyuridae Herdmania momus Urodele - - Degnan et al 1990
Family Styelidae Cnemidocarpa finmarkiensis Urodele L 12413 - - Dendrodoa grossularia Urodele L12416 - - Pelonaia corrugata Anural L12440 - - Polycarpa pomaria Urodele L12441 - - Styela clava Urodele L12442 - - Styela montereyensis Urodele L12443 - - Styela plicata Urodele L 12444 - -
Family Molgulidae Subfamily Eugyrinae
Bostrichobranchus digonas Anural L12379 L12415 Eugyra arenosa Anural L12414 L12417
Subfamily Molgulinae Molgula bleizi Anural L12418 L12419 Molgula citrina Urodele L12420 L12421 Molgula complanata Urodele L12422 L12423 Molgula echinosiphonica Urodele L12424 L12425 Molgula manhattensis Urodele L12426 L12427 Molgula provisionalis Anural L12434 L12435 Molgula occidentalis Urodele L12428 L12429 Molgula occulta Anural L12430 L12431 Molgula oculata Urodele L12432 L12433 Molgula socialis Urodele L12436 L12437 Molgula tectiformis Anural L12438 L12439
a Taxonomic designations according to Van Name (1945) b Anural development was described in P. corrugata by Millar (1954), E. arenosa by Berrill (1931), B. digonas by Swalla and Jeffery (1992), M. provisionalis by Bates and Mallet (1991a), M. bleizi by Lacaze-Duthiers (1874) and Damas (1902), and M. occulta by Lacaze-Duthiers (1874), Berrill (1931), and Swalla and Jeffery (1990). Unpublished observations show M. tectiformis to be an anural developer
Polymerase Chain Reaction Amplification of DNA and Direct Se- quencing. A region of about 1,000 bp in the central region of the 18S rDNA was amplified by the polymerase chain reaction (PCR) using the oligodeoxynucleotide primers 18SA 5'-CAGCAGCCGCGGTAATTC- CAGCTC-3' and 18SB 5'-AAAGGGCAGGGACGTAATCAACG-3' (Wada et al. 1992). A region of about 400 bp in the D2 loop region of the 28S rDNA was amplified by PCR using the oligodeoxynucleotide primers 28SA 5'-CGAGACCGATAGTAAACAAGTA-3' and 28SB 5'-CTTGGTCCGTGTTTCAAGA-3' (Hassouna et al. 1984). The PCR reaction conditions were 100 ng template DNA, 50 mM KC1, 10 mM Tris-HC1 (pH 8.3), 0.001% (w/v) gelatin, 1-3 mM MgC12, 200 gM each of dATP, dTTP, dCTP, and dGTP, 0.8 ~M each of the primers 18SA and 18SB or 28SA and 28SB, and 1.5 units of AmpliTaq DNA polymerase (Perkin-Elmer, Norwalk, CT). The PCR reactions were performed in a Perkin-Elmer thermal cycler for 35 cycles consisting of denaturation for 1 min at 94°C, annealing for 1 min at 50°C, a 1-min ramp transition to 72°C, and extension for 1 rain at 72°C. After the final cycle, the samples were incubated at 72°C for 10 min and then chilled to 4°C.
The double-stranded PCR products were purified using the Seph- aglas Band Prep Kit (Phannacia, Piscataway, NJ), and both DNA strands were sequenced directly using the Sequenase Kit (US Biochem-
icals, Cleveland, OH) with the following modifications (M.J. Smith, pers. commun.). Two micrograms template DNA was mixed with 2.5 pmol of the appropriate primer pairs and 1 gl dimethylsulfoxide. The mixture was brought to a total volume of 8 gl with distilled water, heated for 3 min at 95°C, quick frozen at -70°C for 5 rain, and then thawed. Two microliters of reaction buffer was added, and the template with annealed primers was carried through sequencing reactions ac- cording to the Sequenase protocol. The 18S and 28S (D2 loop) rDNA sequences are deposited in the GenBank DNA sequence database under the accession numbers indicated in Table 1. The sequence of the D2 loop region of Herdmania momus was obtained from Degnan et al. (1990).
Sequence Alignment and Phylogenetic Inferences. The 18S and 28S rDNA sequences were aligned using the CLUSTAL V program (Hig- gins et al. 1992) and trimmed to 973 and 399 nucleotides, respectively. Phylogenetic trees were inferred from the aligned sequences using the maximum parsimony (MP) and neighbor-joining (NJ) (Saitoh and Nei 1987) algorithms. The MP method was implemented using the PAUP package (version 3.0; Swofford 1990). The 18S and 28S rDNA se- quences were analyzed by a heuristic search with 100 replicates under
416
the random addition option to increase the likelihood of finding the most parsimonious trees. The NJ method was implemented using the NEIGHBOR program in the PHYLIP 3.4 package (Felsenstein 1991). The distance matrices were created using the DNADIST program and the Kimura two-parameter model of nucleotide substitution. The degree of support for internal branches of the trees inferred by the MP and NJ methods was assessed in 1,000 bootstrap pseudoreplicates (Felsenstein 1985).
Results
A total of 22 ascidian taxa were analyzed in this study (Table 1). Among these were seven anural species, which represent about half the described taxa exhibiting this mode of development (Jeffery and Swalla 1990). The central region of the 18S rDNA was amplified by PCR from genomic DNA of 21 species in the families Ascidiidae, Styelidae, and Molgulidae and sequenced. The alignment of the 18S rDNA sequences is shown in Fig. 1. Three or four different M. ocuIata, M. occulta, M. bleizi, M. provisionalis, and M. manhattensis (including members of the North American and European popula- tions) individuals showed identical 18S rDNA se- quences; therefore, sequences for the other species were usually obtained from a single animal. M. citrina and M. echinosiphonica, which may be varieties of a single spe- cies (Berrill 1931), also showed the same 18S rDNA sequences. The aligned 973-nucleotide region contains 189 variable sites, of which 149 are informative (Li and Graur 1991). Most of the variable sites are positioned at the ends of the aligned sequences (Fig. 1), as shown previously in 18S rDNA of other ascidian species (Wada et al. 1992).
The 18S rDNA sequences were subjected to phyloge- netic analysis using the MP and NJ algorithms. The MP method generated two minimal trees of 322 steps with consistency indices (CI) of 0.688. The topology of one of the shortest trees is presented in Fig. 2. The alternate tree inferred the urodele developer M. socialis, rather than the urodele developer M. manhattensis, as the sister species of the anural developer M. provisionalis. The tree shown in Fig. 2 has the same topology as the tree inferred from the distance matrix shown in Table 2 using the NJ method. The MP and NJ trees place the anural develop- ers in two discrete clades. The anural developer P. cor- rugata was grouped with six urodele styelid species, sup- porting its taxonomic assignment to the family Styelidae (Van Name 1945; Millar 1970). The inferred sister group of P. corrugata is an assemblage of three Styela species. The six other anural species were grouped with seven urodele molgulids. The styelid and molgulid clades are supported in 99% or more of the bootstrap pseudorepli- cates. In addition, the MP and NJ trees distinguish four clades within the molgulids, each containing both urodele and anural developers (Fig. 2). The clades con- raining (1) the urodele developer M. complanata and the
anural developer M. tectiformis, (2) the urodele develop- ers M. manhattensis and M. socialis and the anural de- veloper M. provisionalis, and (3) the urodele developers M. citrina, M. echinosiphonica, and M. oculata and the anural developers M. bleizi and M. occulta are supported in 96% or more of the bootstrap pseudoreplicates. The fourth clade, which consists of the urodele developer M. occidentalis and the anural developers B. digonas and E. arenosa, has less bootstrap support but is inferred in both MP and NJ analysis (Fig. 2). As in the case of P. cor- rugata and Styela spp., some of the anural and urodele molgulid species (e.g., M. provisionalis and M. manhat- tensis or M. socialis; M. occulta or M. bleizi and M. oculata) are placed as closely related sister groups with short branch lengths, suggesting that anural development can evolve rapidly. The close relationship inferred be- tween M. occulta and M. oculata is consistent with lab- oratory studies showing that these species are capable of interspecific hybridization (Swalla and Jeffery 1990; Jef- fery and Swalla 1991, 1992b). It is unlikely that inter- specific hybridization has an impact on the phylogeny, however, because hybrids between these species have not been observed in nature (Swalla and Jeffery unpub- lished). The 18S rDNA phylogeny suggests that anural development evolved independently in styelid and mol- gulid ascidians and is also polyphyletic in the Molgul- idae.
The relationship between anural and urodele mol- gulids was also inferred from 28S rDNA sequences. The D2 loop in the 5' region of the 28S rRNA molecule was sequenced because it is highly divergent between species and likely to provide higher resolution for phylogenetic analysis (Larson 1991). About 400 bp of the D2 loop region was amplified by PCR from 13 molgulid species, sequenced, and aligned (Fig. 3). The D2 loop sequence of the pyurid ascidian H. momus was used as the out- group (Degnan et al. 1990). Several different individuals of M. oculata, M. occulta, M. bleizi, M. provisionalis, and North American and European M. manhattensis showed the same D2 loop sequences. In addition, M. citrina and M. echinosiphonica had the same sequences, providing further evidence of their close relationship (Berrill 1931). There are 294 variable and 196 informa- tive sites within the aligned 28S rDNA sequences. The variable sites are distributed throughout the D2 loop (Fig. 3). Tree-building procedures were the same as those used for the 18S rDNA sequences. The MP method generated two minimal trees of 605 steps with CIs of 0.584. One of the most parsimonious trees is shown in Fig. 4. The alternate tree differed in placing the anural developer M. bleizi, rather than the anural developer M. occulta, as the sister species of the urodele developer M. oculata. The NJ tree inferred from the distance matrix shown in Table 3 has the same topology as the MP tree presented in Fig. 4. The trees inferred from 28S rDNA sequences placed the molgulid species in the same four clades as those
417
B.dlgonas
C.finmarkiensis
D.grossularla
E.arenosa
M.bleizl
M.citrlna
M.complanata
M.echlnosiphonlca
M.manhattensls
M.occidentalls
M.occulta
M.oculata
M.provlslonalis
M.socialls
M.tectlformis
P~corrugata
P.pemarla
S.clava
S.montereyensis
S.pli~ata
A.ceratodes
B.di~onas
C.finmarkiensis
D.grossularia
E.arenosa
M.blelzl
M,citrina
M.complanata
M.echlnosiphonlca
M.manhattensls
M.occidentalis
M.occulta
M.oculata
M.provisionalis
M. soclalis
M.tectiformls
P.corrugata
P.pomarla
S.clava
S.montereyensis
S.plicata
A.ceratodes
B.dlgonas
C.finmarkiensis
D.grossularia
E.arenosa
M.blelzl
M.cltrina
M.complanata
M.echinosiphonica
M.manhattensis
M.occidentalis
M.occulta
M.oculata
M.provlslonalis
M.soclalls
M.tectlformis
P.corrugata
P.pomarla
S.clava
S.montereyensis
S.plicata
A.ceratodes
1 80
GCTCGTAGTT GGATCTAGGG CGCCGGTCGG TGGTCCGCCG CAAGGCGTGT -ACTG---CT GGCCGGGGTC TTACCTCTGG
.............. T.T ..... GGC.CA.C C ........... G ...... C C .... GTTGC .CT..--C-. .CG...TC.
.............. T.T ..... GGC.CT.C C ...... T .... G ....... C .... GCAGC .TT..--C-. .CG...TC.
..................................... T ........... C -....---TC ...... A ............
.................. T. T ........ C C .................. C -....---GC .TT...CC.. .CTTTG.C.
.................. T. T ..... G..C C .................. C -....---GC .TT..ACC.. .CTTTGTC.
.................... TAT...CT.. C ...... T ............ - .... ---T. ..T,..TC ..... T.A...
.................. T. T ..... G..C C .................. C -....---GC .TT..ACC.. .CTTTGTC.
.................... T..T ..................... T .... - .... ---TC ...... TC .... TTT ....
.................... T..T .......................... - .... ---TC ...T..CC ............
.................. T. T ..... G..C C .................. C -....---GC .TT...CC.. .CTTTG.C..
.................. T. T ..... G..A C .................. C -....---TC .TT...CC..
....................... T ..................... T .... - .... ---TC ...... TC..
....................... T ..................... T .... - .... ---TC ...... TC..
.................... T ..... G ....................... - .... ---.C .CT...CC..
.............. T.T ..... GGC.CAAC C ..... T .... G ....... C .... GTTGC .TT..--C-.
.............. T.T ..... GGC.CA.C C ..... T .... G ....... T .... GTTGC .TT..--C-.
.............. T.T ..... AGC.C..C C ..... T ............ C .... GTTGC .TT..--C-.
.............. T.T ..... GGC.CA.C C ..... T .... G ....... T .... GTTGC .TTT.--C-.
.............. T.T ..... AGC.CA.C C ..... T .... G ....... T .... GTTGC .TT..--C-.
.............. T.T ...... A..CG.C C ..... T ............ - .... GCTGC AC.T..CC-.
.CTTTG.C..
..TTT .....
..TTT .....
...TGA...
.C .... TC.
.C .... TC.
.C .... TC.
.C .... TC.
.C .... TC.
.C...C.C.
81 160
TTCTCTGCCG GTGCTCTTAA CTGAGTGTCG GTGGTGGCCA GAAATTTTAC TTTGAAAAAA TTAGAGTGTT CAAAGCAGGC
..... C.T .......... G ............ C ....... G AT..G ...................................
..... C.T .......... G ............ C ....... G AG..G ...................................
............................... C ................................. . ..............
..... G.T .......... G ............ CC ...... G .CGG ....................................
..... G.T .......... G .......... T .CC ...... G .CGG .......................... T .........
..T..C .............. TC.G .............. TG .T..A ......................... T .........
..... G.T .......... G .......... T .CC ...... G .CGG .......................... T .........
..T..C.G .... T ................. TC ......... TGCG ......................... T .........
....... T .......... G ............ C ......... T..G ...................................
..... G.T .......... G ............ CC ...... G .CGG ....................................
..... G.T .......... G ........... TCC ...... G .CGG ....................................
..T..C.T ....................... C ......... TGCG ......................... T .........
..T..C.T ....................... C ......... TGCG ......................... T .........
..T..C ............... C.G ............ A.TG .T ............................ T .........
..... C.T .......... G ............ C ....... G AT..G ...................................
..... C.T .......... G ............ C ....... G AG..G ...................................
..... C.T .......... G ............ C ....... G AT..G ...................................
..... C.T .......... G ............ C ....... G AT..G ...................................
..... C.T .......... G ............ C ....... G AT..G ...................................
CA...C ........ C...G ............ C..C .... G .G.CG ...................................
161 240
TTTTTGCCTG AATATTCGTG CATGGAATAA TGGAATAGGA CCTCTGTTCT ATTTT-GTTG GTTTTTGGAA TGTGAGGTAA
.GC.C ..... C...G.GT .......................... G .......... - ......... C...G C.C .......
.GC.C ..... T...G.GT .......................... G .......... - .......... C...G C.C .......
....................................................... - ......... C ..............
C.G.C .... T ................................. GTG... G .... - ....... A.-A.G.C.C ......
.AG.C .... T ................................. GAG... G .... T ..... G...-ATG.
.G ....... T .... CGA ............. A ..... A ...... GTAA ....... - ............ T.
.AG.C .... T ................................. GAG... G .... T ..... G...-ATG.
.G ............ CG ........................... GAG ........ - ......... C...
.A ..... T. T .... CG ........................... G .......... - ......... C...
C.G.C .... T...G ............................. GAG... G .... - ......... -A.G.
C.G.C .... T...G ............................. GAG... G .... - ......... -A.G.
.G ............. CG ........................... GAG ........ - ......... C ....
.G ............. CG ........................... GAG ........ - ......... C ....
.AA ....... G...GCGA ............. A ............ GT.CT ...... - ......... C.AG.
.GC.C ..... C...G.GT .......................... G .......... - ......... C...G
.GC.C ..... T...G.GT .......................... G .......... - ......... C...G
.G..C ..... C...G.GT .......................... G .......... - ......... C...G
.GC.C ..... C...G.GT .......................... G .......... - ......... C...G
.GC.C ..... C...G.GT .......................... G .......... - .......... C...G
GG..C ......... A.G ........................... G .... ...... - ......... C...G
CTC ......
-AC ......
CTC ......
• -.C ......
• ..-. .....
C.C ......
C.C ......
-.C ......
-.C ......
-AC ......
C.C ......
C.C ......
CAC ......
CAC ......
CAC ......
C.C ......
Fig. 1. The aligned 18S rDNA sequences of 21 ascidian species. The 973-nucleotide sequence is shown for the molgulid B. digonas on thefirst line of each row. The dots below the first sequence represent identical nucleotide positions, the letters represent variable positions, and the dashes represent gaps in the 18S rDNA sequences of the other ascidian species.
inferred from 18S rDNA sequences (compare Figs. 2 and 4), supporting a polyphyletic origin of anural develop- ment in the Molgulidae.
The family Molgulidae consists of two subfamilies, the Molgulinae and Eugyrinae, which are distinguished by the presence and number of folds in the adult bran- chial sac (Van Name 1945; Monniot 1969; Plough 1978). The eugyrinids lack these folds but have evolved large
longitudinal blood vessels and spiral infundibula in their place (Berrill 1950), whereas the molgulinids usually ex- hibit either six or seven branchial folds. The eugyrinids B. digonas and E. arenosa were placed as a sister group in the 18S and 28S rDNA trees; however, the urodele de- veloper M. occidentalis, a molgulinid with six branchial sac folds, was grouped in the same clade as the eugyrinid species (Figs. 2 and 4). If this grouping is of historical
418
B.digonas
C.finmarkiensis
D.grossularia
E.arenosa
M.bleizi
M.citrina
M.complanata
M.echinoslphonica
M.manhattensis
M.occldentalis
M.occulta
M.oculata
M.provisionalls
M.socialis
M.tectiformis
P.corrugata
P.pomaria
S.clava
S.montereyensis
S.pllcata
A.ceratodes
B.digonas
C.finmarkiensis
D.grossularia
E.arenosa
M.blelzl
M.citrina
M.complanata
M.echinosiphonica
M.manhattensis
M.occidentalls
M.occulta
M.oculata
M.provisionalis
M.soclalis
M.tectiformls
P.corrugata
P.pomaria
S.clava
S.montereyensis S.plicata
A.ceratodes
B.dlgonas
C.finmarklensls
D.grossularia
E.arenosa
M.bleizl
M.citrina
M.complanata
M.echinosiphonica
M.manhattensis
M.occidentalis
M.occulta
M.oculata
M.provisionalls
M.socialls
M.tectiformis
P.corrugata
P.pomarla
S.clava
S.montereyensls
S.plicata
A.ceratodes
Fig. 1. Continued.
241 320
TGATT~GAG GGACAGACGG GGGCATCCGT ACTCTGCCGT TAGAGGTGAA ATTCTTGGAT CGGCGGAAGA CG~CTATTG
........................ G .................................................... C.o
........................ G. ................................................... C..
.......... ° .....................................................................
............................................................................. C..
........................ G .................................. . ................. C.o
........................ G .................................................... C.o
........................ G .................................................... C°.
.............. G ........... T ...... G ............................... C ......... G.C..
321 400 CGAAAGCATT TGCC~G~T GTTTTCTTTA ATCAAGAGCG A~GTCAGAG GTTCGAAGAC GATCAGATAC CGTCCTAGTT
.......... C .....................................................................
.......... C .....................................................................
[[[[[[[[[[ ~[[[[[[[[[ [[[[[[~[i[ [[[[[[[[[[ [i[[[[~[[[ [[[[[[[[[[ [[[[[[[[[[ [[[[[[[[~i . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
401 480 CTGACCATAA ACGATGCC~ CTAGCGATCC GTCGGCGTTA CCATGACGAC TCGACGGGGA GCTCCCGGGA ~CCAAAGTC
..... T ....................... G .GA ................. CTTC.C..C .... T ................
..... T ....................... G .GA ................. CTTC.C..C .... T ................
............................... C..A .................. S ..........................
............................... C..A .................. S ..........................
..................................................... G ..........................
............................... C..A .................. S ..........................
............................... C ..................... G .... T .....................
..................................................... S ..........................
............................... C..A .................. S ..........................
............................... C..A .................. S ..........................
............................... C ..................... S .... T .....................
............................... C ..................... S .... T .....................
..................................................... G ..........................
..... T ....................... G .GA ................. CTTT.C..C .... T ................
..... T ....................... G .GA ................. CTTC.C..C .... T ................
..... T ....................... G .GA ................. CTTT.C..C .... T ................
..... T ....................... G .GA ................. CTTT.C..C .... T ................
..... T ....................... G .GA ................. CTTT.C..C .... T ............. ...
..... T ............... C ....... G .GG ....... GTT ....... CTCC.C..C .... TG ...............
significance, it suggests that the Molgulinae are not a natural assemblage. Further distinction among the Mol- gulinae is based on the number of branchial sac folds (Monniot 1969). The 18S and 28S rDNA trees group M. manhattensis, M. provisionalis, and M. socialis, which have six folds, and M. citrina, M. echinosiphonica, M. bleizi, M. oculata, and M. occulta, which have seven folds, into distinct clades with high bootstrap support (Figs. 2 and 4). Unfortunately, the clade containing the
anural developer M. tectiformis and the urodele devel- oper M. complanata cannot be evaluated by these criteria because the number of branchial sac folds is variable in M. complanata (Berrill 1931, 1950; Van Name 1945; Millar 1970). In general, however, the molecular phy- logeny is consistent with molgulid systematics based on the presence and number of branchial sac folds. Finally, evolutionary distances based on rDNA sequences sug- gest either a more ancient divergence than previously
B.digonas
C.finmarkiensis
D.grossularla
E.arenosa
M.blelzl
M.citrina
M.complanata
M.echlnosiphonica
M.manhattensis
M.occidentalis
M.oeculta
M.oculata
M.provisionalls
M. socialis
M.tectlformis
P.corrugata
P.pomarla
S.clava
S.montereyensis
S.pllcata
A.ceratodes
B.dlgonas
C.finmarklensis
D.grossularia
E.arenosa
M.blelzl
M. citrina
M.complanata
M.echinoslphonica
M.manhattensis
M,occidentalls
M.occulta
M.oculata
M.provislonalis
M.soclalis
M.tectiformis
P.corrugata
P.pomarla
S.clava
S.montereyensis
S.plicata
A,ceratodes
B.digonas
C.finmarkiensis
D,grossular±a
E.arenosa
M.blelzl
M.cltrina
M.complanata
M.eehinosiphonica
M.manhattensls
M.oecidentalls
M.occulta
M.oculata
M.provlslonalis
M. socialis
M.tectlformi~
P.corrugata
P.pomarla
S.clava
S.montereyensls
S.plicata
A.ceratodes
Fig. 1. Continued.
481 560
TTTGGGTTCC GGGGGAAGTA TGGTTGCAAA GCTGAAACTT A~GG~TTG ACGG~GGGC ACCACCAGGA GTGGAGCCTG
............. , ............. o ....................................................
........................................... . ............................... . ....
........................... o ..... o ..............................................
..................................................................... C ..........
561 640
CGGCTT~TT TGACTC~CA CGGGGAAACT CACCCGGCCC GGACACAGGA AGGATTGACA GATTGAGAGC TCTTTCTTGA
................................................. T ..............................
................................................. T ..............................
................................................. T ..............................
........... . ..................................... T ................. G ............
................................................. T ..............................
................................................. T ................. G ............
................................................. T ..............................
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . G . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . T ~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
641 720
TTCTGTGGGT GGTGGTGCAT GGCCGTTCTT AGTTGGTGGA GCGATTTGTC TGGTTAATTC CGAT~CG~ CGAGACTTTG
............................................................................. CO.
................................................................. . ........... C..
............................................................................. C.A
............................................................................. C.A
............................................................................. C,A
............................................................................. C.A
............................................................................. C.A
............................................................................. C.A
............................................................................. C.A
............................................................................. CoA
............................................................................. C.A
............................................................................. C.A
............................................................................... A
............................................................................. C.o
............................................................................. Co.
.................................................................. • .......... Coo
............................................................................. C..
............................................................................. C..
............................................................................. Coo
419
recognized or a fast molecular clock in the molgulid ascidians.
Discussion
We conclude that anural development evolved multiple times in ascidians. The phylogeny inferred from 18S rDNA sequences strongly supports independent origins of anural development in the families Styelidae and Mol- gulidae. The 18S rDNA phylogeny and additional phy-
logenetic analysis using the hypervariable D2 loop se- quences of 28S rDNA support at least four independent origins of anural species in the Molgulidae. The rDNA phylogeny is consistent in general with molgulid system- atics based on adult morphological characters. However, the rDNA sequences imply that molgulid ascidians are either more divergent than apparent from morphological characters or are evolving with a fast molecular clock.
Anural species are considered to be derived from urodele ancestors because anural development is re-
420
B.digonas
C.finmarkiensis
D.grossularia
E.arenosa
M.blelzi
M.cltrlna
M.complanata
M.echinosiphonlca
M.manhattensls
M.occidentalls
M.occulta
M.oculata
M.provislonalis
M.soclalls
M.tectlformis
P.corrugata
P.pomaria
S.clava
S.montereyensis
S.pllcata
A.ceratodes
B.digonas
C.finmarklensls
D.grossularia
E.arenosa
M.bleizi
M. cltrlna
M.complanata
M.echlnosiphonlca
M.manhattensis
M.occldentalis
M.occulta
M.oculata
M.provislonalls
M.socialis
M.tectiformis
P.corrugata
P.pomarla
S.clava
S.montereyensis
S.pllcata
A.ceratodes
B.dlgonas
C.finmarkiensls
D.grossularia
E.arenosa
M.blelzi
M.citrina
M.complanata
M.echinosiphonlca
M.manhattensis
M.occldentalis
M.oceulta
M.oculata
M.provlslonalis
M.socialis
M.tectiformis
P.corrugata
P.pomaria
S.clava
S.montereyensis
S.plicata
A.ceratodes
Fig. 1. Continued.
721 800
ATCTGCTAAA TAGTTACGCG ACCTTTTCGG TTGGCGTC-- -AAACTTCTT AGAGAGACTC GTGGCGTTTA GCCACACGAG
GCA ...... C ............... C ..... C ..... TT- --. ........... G .... A ....................
GCA ...................... C ..... C ...... T- --. ........... G .... A ....................
................... A ............ A ..... -- -. ......................................
T ...................... C.G ..... C ..... TGT TG ................. A ....................
T ...................... C,G,,..
.C ............................
T ...................... C.G ....
..A ...................... A ....
.C ....................... A ....
T ...................... C.G ....
T ...................... C.G ....
..A ...................... A ....
. .G ............... GC ..... A ....
.C ..... TGT T .................. A ....................
.C ..... TAA G ............. G .... A ....................
.C ..... TGT T .................. A ....................
.C ..... TTT G ............ T ..... A ................... A
.C ...... TAG ............. G .... A ....................
.C ..... TGT TG ................. A ....................
.C ..... TGT TG ................. A ....................
.C ..... TTA G ............ T ..... A ....... A ........... A
.C ..... TTG G ............ T ..... A ...... C ............ A
T .................. A ............ A ..... AA TT ................. A ....................
GCA ...................... C ..... C ...... T- --. ........... G .... A ....................
GCA ...................... C ..... C ...... T- --. ........... G .... A ....................
GCA ...................... C ..... C ...... T- --. ........... G .... A ....................
GCA ...................... C ..... C ...... T- --. ........... G .... A ....................
GCA ...................... C ..... C ...... T- --. ........... G .... A ....................
-CT ...... C ............. CGA ........... TT- --. ........... G .... A ....................
801 880
AATGAGCAAT AACAGGTCTG TGATGCCCTT AGATGTCCGG GGCCGCACGC GCGCTACAAT GAATAAAGCA GCGTGTACTA
.T ........................................................ C ..... G..T ..... A..G.CT
.T .................................. T ..................... C ..... G..T ..... A..TTCT
.T ........................................................................... T.T
.T ......................................... T ................................ CTGT
.T ......................................... T ................................ CTGT
.T ......................................... T ....................... A ........ CG..
.T ......................................... T ................................ CTGT
.G ......................................... T ................... C..GA ........ CG.T
............................................................................ -T.T
.T ......................................... T ................................ CT.T
.T ......................................... T ................................ CT.T
.G ......................................... T ................... C..GA ........ CG.T
.G ......................................... T ...................... GA ........ CG.T
.T ......................................... T ...................... CT ....... GT..
.T ........................................................ C. ..G.G ....... A..GTCT
.T .................................. T ..................... C ..... GG.T .... A..TTCT
.T ........................................................ C. . .G.G ....... A..GTCT
.T ........................................................ C. ..G.G ....... A..GTCT
.T ........................................................ C. ..G.G ....... A..GTCT
.T .................................. T ..................... C. ..CCGG.T ....... G-CT
881 960
ACCCTTGGCC GAAAGGTCTG GGA~TCCCC TT~ATTTAT TCGTGATTGG GATAGAGATC TGG~TC--C TCGCTTGAAC
.A...A ............ C. ..T..C.,GT .G..CC.C ................. G..CT ..C...TGTT ..C .......
CA...A ............ C. ..T..C..GT .G..CC.C ................. G..CT ..C...TGTT ..C .......
.T ............... T ................................................. --. ..........
TA ....................... - .... G .............. C.A ................... --. ..T ......
TA ....................... - .... GA ............. C.A ................... --. ..T ......
TA ............................ A .................................... --T ..T ......
TA ....................... - .... GA ............. C.A ................... --. ..T ......
.A ....................... - .... G ....... G ...... C .................... T--T..T ......
.T ....................... C .... G .................................. A.--. .,T .....
TA ....................... - .... G .............. C.A ................... --. ..T ......
TA ....................... - .... G .............. C.A ................... --. ..T ......
.A ....................... - .... G ....... G ...... C .................... T--T ..T ..A..,
.A ....................... - .... G .............. C .................... T--T ..T..A...
TA ................................................................. --T ..T. .A...
.A...A ............ C. ..T..C..GT .G..CC.C ................. G..CT ..C...TGTT ..C .....
.A...A ............ C. ..T..C..GT .G..CC.C ................. G..CT ..C...TGTT ..C .....
.A...A ............ C. ..T..C..GT .G..CC.C ................. G..CT ..C...TGTT ..C .....
.A...A ............ C. ..T..C..GT .G..CC.C ................. G..CT ..C...TGTT ..C .....
.A...A ............ C. ,.T..C..GT .G..CC.C ................. G..CT ,.C...TGTT ..C .....
CG ................ C. ..T..C..G. .G..CCCCGG ..... C.A ....... G..AT ..C...TGTT .,C .....
stricted to a limited number of taxa in only one or two of 14 ascidian families (Berrill 1931; Plough 1978) and vestigial urodele features are present in embryos of some anural species (Damas 1902; Whittaker 1979; Swalla and Jeffery 1990, 1992; Jeffery and Swalla 1991; Bates and Mallet 1991 a). Our molecular phylogenetic analysis pro- vides further support for the derived nature of anural development. First, each of the clades with anural spe- cies also contains one or more urodele species. This re- lationship was observed in the styelid assemblage and in each of the four molgulid clades. Second, the branches
leading to anural species are generally short and/or pre- ceded by one or more nodes leading to urodele species. There is phylogenetic evidence that reversion from direct to indirect development has occurred during the diversi- fication of marsupial hylid frogs in the Andes (Wasser- sug and Duellman 1984; Duellman and Hillis 1987). Likewise, a pelagic feeding larva may have reevolved from an ancestor with a brooded nonfeeding larva in starfish (McEdwards 1992). It is possible that a similar reversion has occurred in ascidians? The only potential case for reversal of anural development in the species we
961 973
B.digonas GAGGAATTCC CAG
C.finmarklensis .............
D.grossularla .............
E.arenosa .............
M.bleizi .............
M.citrina .............
M.complanata .............
M.echinoslphonlca .............
M,manhattensis .............
M.occldentalis .............
M.occulta .............
M.oculata .............
M.provlslonalls .............
M.soclalls .............
M.tectlformis .............
P.corrugata .............
P.pomaria .............
S.clava .............
S.montereyensis .............
S.plicata
A.ceratodes ::[[~[[G[[ i[:
Fig. 1. Continued.
have examined is in the molgulid clade containing M. citrina, M. echinosiphonica, M. bleizi, M. occulta, and M. oculata. The rDNA phylogenies suggest that the urodele developer M. oculata shared recent common an- cestors with the anural developers M. bleizi and M. oc- culta, and is more distantly related to the urodele devel-
421
opers M. citrina and M. echinosiphonica. This placement is consistent with M. oculata being derived from either an anural or a urodele ancestor. Further studies (see be- low), including analysis of additional anural and urodele species in this clade, will be necessary to determine the evolutionary history of M. oculata.
The rDNA phylogeny supports independent origins of anural species in the Styelidae and Molgulidae. The anural developer P. corrugata was placed in a clade con- taining the urodele styelids, whereas six other anural spe- cies were placed in a different clade containing the urodele molgulids. Thus, despite uncertainties due to branchial sac morphology (Berrill 1950), the molecular analysis is consistent with the conventional taxonomic assignment of P. corrugata to the family Styelidae. The placement of this anural developer as the sister group of urodele Styela species is an important inference because the latter are frequently used in developmental studies (Jeffery 1992). Therefore, the mechanisms underlying the origin of anural development in the Styelidae could be examined by comparing development of P. corrugata and Styela, as they are being determined for the Molgu- lidae by comparing development of M. occulta and M.
Ascidia ceratodes
76 84
67 40 f 58 36 •
' ~ , ,~
94
100 69 81
10
59
I 6.
[ Pelonala corrugata [
Styela clava
Styela montereyensis
Styela plicata
Polycarpa pomarla
Dendrodoa grossularla
Cnemidocarpa flnmarklensis
I Bostrichobranchus digonas [
I Eugyra arenosa [
Molgula occidentalis
Molgula complanata
I Molgula tectiformis I
Molgula manhattensis
Molgula provlsionalis [
Molgula socialis
Molgula bleizil
Molgula occultal
Molgula oculata
Molgula eitrina
Molgula echinosiphonica
Fig. 2. Phylogenetic relationships of urodele and anural ascidians inferred from 18S rDNA sequences. The figure shows one of the two shortest trees inferred by the MP method. The branch lengths are pro- portional to evolutionary distances. A tree of the same topology was inferred by the NJ method. The numbers above and below the branches
Moigulidae
Styelldae
leading to the nodes represent the percentage of 1,000 bootstrap pseu- doreplications which support the node in the MP and MJ analyses, respectively. Species exhibiting anural development are boxe& other taxa exhibit urodele development. Conventional taxonomic assign- ments according to Van Name (1945) are shown on the right.
422
Table 2. Distance matrix for 18S rDNA sequences of 21 ascidian species a
(1) (2) 1,235 - - (3) 730 1,026 - - (4) 708 1,026 157 - - (5) 1,210 147 1,014 990 - - (6) 1,225 698 1,006 993 675 (7) 1,310 790 1,042 1,029 767 (8) 1,320 616 1,049 1,012 672 (9) 1,310 790 1,042 1,029 767
(10) 1,211 618 1,027 1,050 618 (11) 1,113 362 896 873 351 (12) 1,211 698 981 968 675 (13) 1,249 709 1,018 1,005 674 (14) 1,197 606 1,003 1,026 606 (15) 1,258 618 1,026 1,050 607 (16) 1,378 594 1,107 1,106 594 (17) 753 1,002 125 136 966 (18) 697 1,014 146 73 978 (19) 731 966 167 167 931 (20) 742 1,014 157 167 978 (21) 753 1,002 146 147 966
(1) (2) (3) (4) (5)
157 739 786 - - 157 0 786 - - 640 686 592 686 - - 618 686 558 686 449 52 147 740 147 628 84 179 739 179 605
651 697 592 697 73 662 708 614 708 136 717 741 448 741 696
1,005 1,041 1,024 1,041 1,038 1,005 1,041 1,025 1,041 1,050
981 1,005 977 1,005 1,002 1,017 1,041 1,013 1,041 1,050 1,005 1,029 1,001 1,029 1,038 (6) (7) (8) (9) (10)
a The distance matrix was created using the Kimura two-parameter model in the DNADIST program of the PHYLIP application. The numbers in the matrix represent P distance x 104. The numbers in parentheses represent the following taxa: (1) A. ceratodes, (2) B. di- gonas, (3) C. finmarkiensis, (4) D. grossularia, (5) E. arenosa, (6) M.
bleizi, (7) M. citrina, (8) M. complanata, (9) M. echinosiphonica, (10) M. manhattensis, (11) M. occidentalis, (12) M. occulta, (13) M. ocu- lata, (14) M. provisionalis, (15) M. socialis, (16) M. tectiformis, (17) P. corrugata, (18) P. pomaria, (19) S. clava, (20) S. montereyensis, and (21) S. plicata.
oculata (Swalla and Jeffery, 1990; Swalla et al. 1991; Jeffery and Swalla 1992a), The only other anural devel- oper assigned to the Styelidae is Polycarpa t inctor
(Millar 1962). Although this species could not be ob- tained for our analysis, we did examine the urodele de- veloper Polycarpa pomaria, which was placed in a dif- ferent styelid subgroup than P. corrugata and Styela.
Therefore, anural development may be diphyletic in the Styelidae. The grouping of anural molgulids in four clades containing one or more urodele species suggests that anural development evolved at least four times in the Molgulidae. This conclusion supports that of Arnb~ick- Christie-Linde (1928) and Berrill (1931), who suggested that anural development is polyphyletic in the Molgul- idae, rather than that of Lacaze-Duthiers (1874, 1877), who proposed a monophyletic origin of anural species. Multiple origins of direct development have also been inferred in a variety of marine invertebrate phyla (Strath- mann 1978; Emlet 1990; Raft 1992) and frogs (Duell- man and Trueb 1986).
The rDNA phylogeny suggests that anural develop- ment can evolve rapidly in ascidians. This possibility is supported by the relatively small evolutionary distances between some of the urodele and anural species inferred as sister groups. We have already discussed the sister- group status of P. corrugata and Styela in the styelid clade. Similar relationships were inferred between the anural developer M. provis ional is and the urodele devel- oper M. manhat tensis (or M. socialis) and the anural
developer M. occulta (or M. bleizi) and the urodele de- veloper M. oculata in the molgulid clade. The close re- lationship between M. occulta and M. oculata is of par- ticular interest because these species are capable of interspecific hybridization in the laboratory (Swalla and Jeffery 1990). The relationship inferred between M. oc-
uIata, M. occulta, and M. bleizi is also consistent with biogeographic data (Monniot 1969) showing that these species overlap in distribution in the Roscoff (France) region, where they probably evolved (Berrill 1931). Un- fortunately, interspecific hybridization cannot be used to test the inferred relationship between M. oculata, M. oc-
culta, and M. bleizi because the latter species is vivipa- rous with internal fertilization. Therefore, further assess- ment of the evolutionary history of these species will require sequence data from more variable regions in nu- clear or mitochondrial DNA. Closely related species ex- hibiting different developmental modes also occur in sea urchins (Smith et al. 1990; Wray and Raft 1991), sug- gesting that rapid evolution of developmental modes is a general phenomenon.
If direct development can appear so readily, why has it evolved in only two ascidian families? Although anural developers may still be described in other ascidian fam- ilies, it is striking that they are relatively numerous only in the Molgulidae. The molgulids may be preadapted for modification and loss of the larval stage. The tadpole larvae of all urodele developers in this family are defi- cient in one of the two brain sensory organs typical of
Table 2. Continued
423
m
617 617 31 - - 438 639 639 - - 460 651 650 94 636 683 694 673 849 980 1,017 1,013 861 981 1,017 1,024 814 957 993 978 838 992 1,029 1,025 849 980 1,017 1,013 (11) (12) (13) (14)
m
684 1,037 1,095 - - 1,050 1,107 104 - - 1,002 1,047 62 14 - - 1,049 1,083 52 115 52 - - 1,037 1,071 41 104 42 10 (t5) (16) (17) (18) (19) (20) (21)
urodele species in other families (Berrill 1950). The ma- ture eggs of molgulids also lack ankryin, a protein that may link the egg cytoskeleton and myoplasm to the plasma membrane in eggs of urodele developers (Jeffery and Swalla 1993). An explanation for the elimination of the larval phase may be the adaptation of some anural molgulids (and the styelid P. corrugata) to subtidal sand and mud flats where there is no selective advantage for a swimming larva (Berrill 1931; Whittaker 1979). Indeed, the swimming larva may be under negative selective pressure in these restricted habitats because dispersal might land juveniles in places where they would not be able to survive. Developmental studies on more ascidian species, particularly those adapted to sea-bottom habi- tats, will be necessary to determine whether modification of the larval stage is related to habitat selection and/or preadaptation.
A primary character used in the classification of mol- gulid ascidians is the presence and number of folds in the adult branchial sac (Van Name 1945; Monniot 1969, Plough 1978). The molgulinids usually contain six or seven folds, whereas the folds are replaced by large blood vessels and spiral infundibula in the eugyrinids (Berrill 1950). However, branchial sac folding can vary within species and may be convergent in the molgulids (Huntsman 1922; Krnsb~ck-Christie-Linde 1928; Ben'ill 1931). Nevertheless, the molecular phylogeny is consis- tent in general with molgulid systematics based on bran- chial sac structure. The anural developers B. digonas and E. arenosa, which are assigned to the Eugyrinae, were inferred as a sister group. In the subfamily Molgulinae, the manhattensis group (M. manhattensis, M. socialis, and M. provisionalis), which exhibits six folds, and the
roscovita group (M. occulta, M. oculata, M. ble&i, M. citrina, and M. echinosiphonica), which exhibits seven folds, were placed as discrete assemblages with high bootstrap support. These inferences support the use of branchial sac folds as a taxonomic character. However, the molgulinid M. occidentalis, which has six folds, was placed in the same subgroup as the eugyrinids B. digonas and E. arenosa. If this grouping is correct, then the Molgulinae is not a natural assemblage, and branchial sac folding could be misleading as a taxonomic charac- ter.
Although both molecular and morphological studies suggest that anural development is polyphyletic, em- bryos of distantly related anural species exhibit the same modifications in urodele development. First, p58, a pro- tein localized in the myoplasm in eggs of all urodele species, is markedly reduced in titer or absent in the eggs of anural developers in at least three molgulid subgroups inferred from the rDNA phylogeny (Swalla et al. 1991). Second, the styelid P. corrugata and every anural devel- oper in the inferred molgulid subgroups have lost the notochord (Millar 1962; Damas 1902; Berrill 1931; Swalla and Jeffery 1990, 1992; Bates and Mallet 1991a,b). Third, terminal differentiation of tail muscle cells has been eliminated in every anural species that has been carefully investigated (Whittaker 1979; Swalla and Jeffery 1990, 1992; Jeffery and Swalla 1991, Bates and Mallet 1991a,b). While these biochemical and morpho- logical similarities may be a result of convergence, it is also possible that anural development could be mediated by a conserved switch which is triggered in different evolutionary lineages. Genes encoding potential regula- tory factors expressed in the urodele developer M. ocu-
424
B.digonas
E.arenosa
M,blelzl
M.citrlna
M°complanata
M.echinoslphonlca
M.manhattensls
M.oceidentalls
M.occulta
M.oculata
M.provislonalls
M. soeialls
M.teetiformis
H.momus
B.digonas
E.arenosa
M.blelzl
M.citrina
M.complanata
M.echlnosiphonlea
M.manhattensls
M.occldentalis
M.occulta
M.oculata
M.provisionalls
M.soeialls
M.tectlformis
H.momus
B.digonas
E.arenosa
M.bleizl
M.citrina
M.complanata
M.echlnoslphonlea
M.manhattensls
M. occidentalis
M.occulta
M.oculata
M.provlsionalls
M.soolalls
M.tectlformis
H.momus
B.dlgonas
E.arenosa
M.bleizl
M.citrina
M.complanata
M.echinosiphonica
M.manhattensis
M.occldentalis
M.occulta
M.oculata
M.provisionalis
M.soclalis
M.tectiformls
H.momus
1
CCGTTGTGAG GTAAACGGAG GGGGCCCGGC GGTCGTTTTG ATGCTTTCAG TTG-GATGCG
....................... C..G.TG ..... G..C. GC ........... -.T...A
.................... A..C..G.CG ..... C..C. GC ........... -.GC.GC
.................... A..C..G.CG ..... C..C. G ........... C-.GC.GC
..... A .............. A..C..G.CG ..... C .... G ........... C-.GC.AT
.................... A..C..G.CG ..... C..C. G ........... C-.GC.GC
.................... A..C..G.CG ..... C..C. G ............ -.G..G-
................ T... A..C..G.CG A .... C..C. GC .......... T-.GC.T.
.................... A..C..G.CG ..... C.CC. GC ........... -.GC.GC
.................... A..C..G.CG ..... C.CC. GC ........... -.GC.GC
.................... A..C..G.CG ..... C..C. G ............ -.G..G-
.................... A..C..GACG .~.. .C..C, G ............ -.G..GC
..... A .............. A..C..G.CG ..... C .... G ............ -.GC.G-
...... A .... C ..... AGA ...C..GTC- A .... CCCG. CC ........... A.GC.GC
80
--CCGT---C GCGTAGCGT-
--,...---. ...CGT..CC
---G..---. .... TCGC,C
---G..---T ...CTTGC.T
--TT..---G AA.ACTG-.T
---G..---T ...CTTGC.T
--T...---T TG.CTCGCCC
--T...---G .TT.TCG-.T
---G..---? ...CTCGC.C ---G..---. ...CTCGC.C
--TT.C---. .GACTCGCCC
--TT..---T .ATCTCGCCC
--TG..---G .A,A.TGCCG
GG.G..GCG. .GCACCG..C
81 160
GTACG-GGAT GCCT-TTGGC ACTCAGAAAG CGTGACGTGG TG-AGCAGCC A-GCGCACTA GTGTCGATTC GAACGCCGCG
..... -A .... T..- ......... G ........ TG ..... C.- ........ - .............. GG ........ T...
..T..A-. .... TAAC--. ..... G.GCG. GTGTTT.C.. CC-GT.GT.T T-. .......... C..T.G ...........
.CTT.G-. .... G.A--A ...... G.GCG.
,CTGTC .... ATTAA..TA. TTG...TG.T
.CTT.G-. .... G.A--A ..... G.GCG.
-GC..GA .... TG.~.-A.
..CG.T ...... T.TAC..
..T..G-. ...... A.CA.
..T..G-. .... T-A.CA.
-GT..AA .... TG.G.-A.
TGT..GA .... TGCA.-A.
T.G..-A .... TTCAC--.
CGC..C...C ,.TCACGC.
.... T.GCG.
...GCTGTC.
.... G.GCG.
.... G.GCG.
...TT,GCG.
...TC.TCG.
.... GTTTC.
CGG.TCCGG.
GC.TGT.C.. CT-GTTGC.T T-. .... T ..... CT.T.G ....... T...
GTGT-T.AC. C,-.TTGCTT T-. .... A ..... C.AT.G ....... TA..
GC.TGT.C.. CT-GTTGC.T T-. .... T ..... CT.T.G ....... T...
GTGTC.CG.. C.-GC.GC.T T-.-. ...... C.C..T.G ...........
GTGTTGACT. C.-TT.GC.G .-A .......... CT.T.G. ..GT.A ....
GCGTTT.C.. CC-GT.GT.T T-. ........ C.C..T.G ...........
GCGTTT.C.. CC-GT.GT.T T-. ........ C.C..T.G ...........
GTGTCTC... C.-.T.GC.T T-. ........ C.C.,T.G ...........
GTGATTT... CA-GT.GC.T T--....T.. .C.C..T.G ...........
GTGTTTAAC. CT-GCTGC.T T-. .... T ..... C.AT.GT ..........
TCG.C.AAC. C.C.C.GC.T CA ......... CAC..G-G. ..GA..-A..
161
ACCGCTTGTT GGGCGACCAG AAGCCTTGTG CGAAGATAGC ATCTAT-G-C -CTTCGGGTG TAGGTGTTTT
.... T .... G ................ C.C .............. GG.- ........... CT ........
.... G...GG C..G ........ ATTCC ........ G .... T.GG.C-.G. -T.C-..CCT G.C.A..G..
.T..G...GG C..GA..T .... ATTC..C ...... G .... TC.G.A-.G. -T.CT..C.C G.A.A..G..
.... G..CGG T ........... AG..C ..... C..G .... TCTGC.-.T. CT..T...CA GT..A..GA.
.T..G...GG C..GA..T .... ATTC..C ...... G .... TC.G.A-.G. -T.CT..C.C G.A.A..G.
...G .... G T ............ T ........... G .... C.GGC.-.-. -TC ....... GCC.G..G.
...T...GA .............. TCCTG ....... C..T C.-G..-AA. -.C ...... T GTT.G.CA.
• ..G...GA C..G ........ ATT.C...
• ..G...GG C..G ........ ATT.C...
...G .... G T ............ T..C.C.
• ..G .... G T...T ........ T .... C.
...TC.TGG T ........... A.TC..C.
..... CCGG CC-...AG.C CG...GCTC.
241
G-AGTGTTGA TGGCTTGCTT GGCGAGTGGG
.- .... C...C ...... T.CC.T...C...
.-GT-C.G.. C ..... C... CC...TC...
.-GA-A.G.. C ..... T..G CCT..TC...
.-G.-AAA ........... G CTG.GCC...
.-GA-A.G. C ..... T..G CCT..TC...
C ........ G T .... TC.A.
C ...... T.C TT...AC.TC
C ..... C... TC...TC...
C ..... C... CC...TC...
C ........ G C .... TC.A.
C ..... CG.G C .... TC.A.
..... G...
..... S...
..... T...
..... G...
T.C..G...
.C...G...
TCGG.C-.G. -T.C-..C.? G.A.A..G.
TCGG.C-.G. -T.C-..CCC ..C.A..G.
C.GGG.-.-. - .......... CC.G..G.
CCTA.C-.-T -. ......... T..G..G.
CCGGCA-.-. -.C ..... A. GCT.G..G.
TC.GCCC.G. AG.GTTACA. G.C.G.GCG.
• -G. -..A.
• -GA-TGG.
• -GA-T.G.
.-GA-T.G.
.-G.-, .A.
• -G.-, ,A.
GAAT-CCTGG CCGCGTCTGC CCTTTAGGGC GAGTCTCGCG
.T..- .......... C.A.. .T..CG ....... CG.T.GT
.T--A .... - ....... GA. .G.--.. .T ...CGC..T.
.T--A .... - ....... GA. .G.--.. .T .... G .....
.T--A ................ T..CG ....... GGT..T
.T--A .... - ....... GA. ,G.--.. .T .... G .....
.TG.A .......... C ..... T..CG ....... GGT.TC
.T.-A .... - ..... C.C.. .T..CG ..... T.GCT.TT
.T--A .... - ....... GA. .G.--....T .... GC..T.
.T--A .... - ....... GA. .G.--....T ...CGC.-..
.T..A .......... C ..... T..CG ........ GGT.TC
.T..G .......... C ..... T..CG ........ GGT.TC
.T--A ............... T...CG ..... G..G.T.GC
..G.CGT..T .---...G.. .GG-CG..CT .C.CG-.A.T
.-GA-AG.. C ...... T.G CTG.G.C...
.CGAC..GA. A.A..GTGCG C.TCCA.T..
321
B.digonas GTCTGT-AGC GATCTGTCTT CTGTTCTCAG AGGGCGGGAC GAGGCGGGCT TTCG---TG- CTGCGTTGT-
E.arenosa ..T.T.-C...GGTC ...................... A...C ....... C .... ---..- .GCT.CCA.-
M.bleizi ..T..C-T.T .TGT .... CA ................. CG..CA.T .... G .,.-GTC-.- TGCT.CG..-
M.cltrlna ..... G-T.T TTGT .... CA ..... T .......... ACTT .CAT ..... G ...-GTC-.- TGCT.CGA.-
M. complanata T.T..AAC.A ....... TCG .......... T..AT ...... GA.TTT.A .... CATTC.G TG.TA..C.G
M.echlnoslphonica ..... G-T.T TTGT .... CA ..... T .......... ACTT .CAT ..... G ...-GTC-.- TGCT.CGA.-
M.manhattensis A..GT,- .... G..C.CTC ............... T.,TTT .C-TG.CTT. CG..-TC..- -GCG...CG-
M.occidentalis ..TA.C-TT...C...CTCA ..A .... T ...... TT ..... TT..T...- .GTCGTC..- -GCG.CG..-
M.occulta ..... C-T.T .TGT .... CA ................. CG..CA.T.?.TG ...CGTC..- ?GCT.CG.A-
M.oculata .CTC.C-T.T .TGT .... CA ................. C-. .CA ..... TG ...-GTC .... GC..CG..-
M.provisionalis A..GC,-T...G..C.CTCA ..... T ........... TTT .C-TG.CTT. CG..-TC..- -GCG...CG-
M. sociali~ T..GT.-C ..... TC.CTCA ..... T .......... TTTT .C-.T.C.T. CG..-TC..- -GCG.C.CG-
M.tectiformis ..TGTG-C.T .T.T..CTCA .................. G..C-.T.CTGC .... GTCC.- -GCG-.C.G-
H.momus CA.GC.G-A..C.TC..--G .C.CA.GAT. C.AC.TCCT..C.A..C--- CGG.GTC..T GGT.ACGTCG
240
ACAGAGCG-C
.T ...... -.
.... C...-.
.... C.T.-.
.... T.T.-T
.... C.T.-.
.... C...-.
G ...... T-G
.... C...-.
.... C...-.
.T..C...-.
.... C.T.-.
.... T...-.
..GCC...T.
320
T-TGCCGTTG
.S ........
GTGCG .... C
ATGCG .... T
GTC.T..GGA
ATGCG .... T
GTC.GTCG..
GT.AG..GC.
GTGCG .... C
.GGTG .... C
GTC.GTCG..
GTC.GTCG..
GT..GTCG..
GT.CT.AG..
399
TCTCATAGG
G...-.C..
C.CTC.C..
C.CTC.C..
CTCTT.G.A
C.CTC.C,
CTCTCCG.
.... GAG.
C.CTC.C,
C.CTC.C.
CTCTCCG.
CTCTC.G.
CGCTC.T,A
G.CACCC--
Fig. 3. The aligned 28S rDNA (D2 loop region) sequences of 14 ascidian species. The 399-nucleotide sequence is shown for the molgulid B. digonas on the f irst line o f each row followed by the sequences of 13 molgulid species and the pyurid H. momus. The other details are the same as described in Fig. 1. The D2 loop sequence of 1-1. momus was obtained from Degnan et al. (1990).
Herdmanla momus
100
75 67
52
98 9O
[Molgula blelzl[
[ Bostrlchobranchus dlgonas ]
[ Eugyra arensosa[
Molgula occidentalis
Molgula complanata
[ Molgula tecUformls[
Molgula manhattensis
[Molgula provisionalis [
Molgula soclalls
Molgula oculata
100 I Molgula o¢culta[
99
6 1 6 1 1 0 0 1 [ 00 M o l g u l a c l tr ina
Molgula echlnoslphonica
Fig. 4. Phylogenetic relationships of urodele and anural ascidians inferred from 28S (D2 loop) rDNA sequences. The figure shows one of the two shortest trees inferred by the MP method. A tree of similar topology was inferred by the NJ method. Other details are the same as
425
Folds
'1 o
6
6 o r 7
7
6
6
6
7
7
7
7
7
Eugyrlnae
Molgullnae
described in Fig 2. The numbers between the taxa and subfamily disig- nations represent the number of branchial sac folds in adults of each species (Van Name 1945; Berrill 1950; Monniot 1969; Plough 1978; Nishikawa 1991).
Table 3. Distance matrix for 28S rDNA sequences of 14 ascidian species a
(1) - - (2) 9,272 - - (3) 8,304 2,081 - - (4) 7,148 4,352 3,962 - - (5) 7,611 4,935 4,370 1,550 - - (6) 8,727 5,456 5,349 4,216 4,485 - - (7) 7,611 4,935 4,370 1,550 0 4,485 (8) 7,398 4,519 4,001 3,569 3,913 4,295 (9) 9,062 5,522 4,987 4,158 5,281 5,396
(10) 7,101 4,339 4,125 566 1,399 4,475 (11) 6,870 4,329 3,974 597 1,569 4,606 (12) 7,444 4,542 3,713 3,292 3,709 4,267 (13) 7,380 4,987 4,213 3,396 3,432 4,230 (14) 7,948 5,461 4,502 3,380 3,745 3,585
(1) (2) (3) (4) (5) (6)
m
3,915 - - 5,281 4,517 - - 1,399 3,227 4,271 - - 1,569 3,607 4,610 482 3,709 816 4,686 3,159 3,432 1,508 5,056 3,053 3,745 3,117 4,695 3,582 (7) (8) (9) (10)
3,317 - - 3,199 1,248 - - 3,731 3,303 3,14l (11) (12) (13)
m (14)
The distance matrix was created using the Kimura two-parameter model in the DNADIST program of the PHYLIP application. The numbers in the matrix represent P distance x 10 4. The numbers in parentheses represent the following taxa: (1) 11. momus, (2) B. digonas, (3) E. arenosa, (4) M. bleizi, (5)M. citrina, (6)M. complanata, (7)M. echinosiphonica, (8)M. manhattensis, (9)M. occidentalis, (10)M. occulta, (11)M. oculata, (12) M. provisionalis, (13) M. socialis, and (14) M. tectiformis
lata but not in the anural developer M. occulta have recently been identified and characterized (Swalla et al. 1993). The rDNA phylogeny of anural and urodele as- cidians will serve as a framework to determine whether these genes or other regulatory factors are involved in a
conserved evolutionary switch leading to anural devel- opment.
Acknowledgments. We thank M. Just for technical assistance, the staff at the Station Biologique, Roscoff, France, for their help in collecting
426
E. arenosa, M. occulta, and M. oculata, J. Marthy and the staff at the Laboratoire Arago, Banyuls sur Met, France, for their hospitality and assistance in collecting P. pomaria, H. Wada and N. Satoh for provid- ing samples of M. tectiformis DNA, C. Young for identification of B. digonas, C. Monniot for information on collecting sites and identifi- cation ofM. socialis and European M. manhattensis, and M.J. Smith for sharing direct sequencing methods. This work was supported by a NIH postdoctoral fellowship (HD-07493) and NSF (IBN-9304958) grant to B.J.S. and NSF (DCB-9115543) and NIH (HD-13970) grants to W.R.J.
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