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Molgula pugetiensis is a pacific tailless ascidian within the Roscovita clade of molgulids.

The molgulid ascidians are a monophyletic group of tunicates that share specific morphological features, including a heart/kidney complex and hermaphroditic gonads found on both sides of the adult. The larval body plan, however, is quite plastic, with tailless species evolving at least four times independently within the molgulids. The Roscovita clade of molgulid ascidians includes three species that are found sympatrically in Roscoff, France, and includes both tailed and tailless species. Another species in this clade, Molgula pacifica, is a direct developing tailless molgulid ascidian within the Roscovita clade found on the outer coast of Vancouver Island. M. pacifica "larvae" never hatch, and they undergo metamorphosis within the chorion. Molgula pugetiensis is a molgulid ascidian found in the San Juan Islands, but its development has not been reported. Here, we show that this species, belonging to the Roscovita clade, is a tailless ascidian species with "larvae" that hatch from the chorion before metamorphosis, similar to the well-described tailless Molgula occulta from Roscoff France. Immediately after hatching, the tailless larvae develop ampullae and proceed to metamorphose into juveniles.

Tunicates in the Molgulidae are a monophyletic group of solitary ascidians (Ascidiacea: Stolidobranchia) that are sister group to the families Styelidae and Pyuridae (1-3). Evidence from 18S rDNA shows that the stolidobranch tunicates may also be closely related to the appendicularians, which include the pelagic oikopleurid tunicates that build mucous houses (2-4). Molgulidae is an interesting group for research on the evolution of body plans because it contains species that have urodele, tailed, chordate larvae with notochord and muscle as well as closely related species with anural, tailless larvae that completely lack larval structures, including the sensory otolith and muscle and notochord in the tail (5). Anural species can develop indirectly by hatching from the chorion before metamorphosis or directly by hatching from the chorion after metamorphosis. This loss of a tailed larva is likely to have happened multiple times evolutionarily (1, 3, 4, 6) and is tractable at a molecular level (7, 8).

Within the molgulid ascidians, there are two monophyletic clades of the species that have been extensively studied, the "Woods Hole" clade and the "Roscovita" clade (1). The Woods Hole clade includes three sympatric free-spawning species that can be found near Woods Hole, Massachusetts: M. mahattensis (tailed), M. provisionalis (tailless), and M. arenata (tailless). The Roscovita clade within the Molgulidae includes six species, three of which can be found in the mud flats off Roscoff, France: M. oculata (tailed), M. occulta (tailless), and M. bleizi (tailless) (1, 2). Two of the other species, M. pacifica and M, pugetiensis, are found within the Northwest Pacific coast of the United States and Canada, while the tailed, brooding M. citrina is found on the Atlantic coast and in Europe (1). M. citrina and M. echinosiphonica have recently been synonymized to M. citrina (9). The recent discovery of M. citrina in Alaska also suggests either that there has always been a circumpolar distribution or that it may be a recent invasion (10). Here, we show that Molgula pugetiensis, first described by Herdman (11) near Victoria, British Columbia, in 1898, is a tailless ascidian species found in the U.S. Pacific Northwest that hatches from the chorion before metamorphosing, similar to what has been reported for M. occulta (5) and M. arenata (12).

A single individual of Molgula pugetiensis was collected in a dredge near Friday Harbor, Washington, in 2003 by the "Evolution and Development of Metazoans" course given at the University of Washington's Friday Harbor Laboratories (FHL). Dr. Billie J. Swalla and Gretchen Lambert examined this adult specimen and found seven folds in the branchial basket; genomic DNA was then extracted and sequenced in the Swalla laboratory (4) (GenBank accession number AY903920.1 for 18S rDNA). Taxonomic and sequencing results showed that M. pugetiensis belongs in the Roscovita clade of seven-fold molgulid ascidians (2) first described in Huber et al., 2000 (1). Even though the adult animal was gravid, with viable eggs and sperm, it did not self-fertilize. Three years later, in 2006, Dr. Bruno Pernet found a pair of M. pugetiensis in a dredge sample collected during "Marine Invertebrate Zoology," another FHL summer course. The animals were returned to FHL, cross-fertilized by Dr. Swalla, and found to be another tailless species, like M. arenata and M. occulta, that hatches from its chorion before metamorphosis, in an anterior-to-posterior manner.

M. pugetiensis was dissected and cultured at 12 [degrees]C in sea tables at FHL. M. occulta and M. oculata were dissected, cultured at room temperature (20 [degrees]C), and photographed in Roscoff, France, in 1999. In all three species, gonads were dissected with forceps from animals after the tunic had been removed: one gonad was kept for DNA analysis, and the other was dissected and fertilized. Eggs and sperm were loosened from the gonad by gentle pipetting with a glass Pasteur pipet. Sperm was removed from one dish and used to fertilize eggs of a second individual, and vice versa. About 50 embryos of M. pugetiensis were left to develop in 12 [degrees]C seawater until they hatched from the chorion. Embryos and hatched "larvae" of M. pugetiensis were photographed at FHL with a Nikon Eclipse E600 microscope with a Q Imaging camera from Photometrics.

M. pugetiensis was collected at a depth of about 15-30 m in sand and shell hash. This environment is similar to that of other described indirect developing anural species (13, 14). The type of larval development, habitat, and distribution of all known anural molgulids and some of the sympatric urodele species is summarized in Table 1. Molgulids with anural indirect development, like M. pugetiensis, have been found primarily in the northern Pacific and the northern Atlantic (Table 1). There is a single specimen of an arctic species, M. kolaensis, which was found brooding anural, indirect developing embryos (13; Table 1).
Table 1

Selected anural and Pacific urodele mogulid characteristics

Species              Tail?    Larval       Adult habitat
                              development

M. tubifera          Urodele  Indirect     Algae, wood: attached

M. cooperi           Urodele  Indirect     Sand, gravel: attached

M. verrucifera       Urodele  Indirect     Intertidal rock: attached

M. oculata           Urodele  Indirect     Sand: unattached

M. occulta           Anural   Indirect     Sand: unattached

M. solenota          Anural   Indirect     Sand: unattached

M. macrosiphonica    Anural   Indirect     Sand: unattached

M. arenata           Anural   Indirect     Sand: unattached

M. robusta           Anural   Indirect     Sand: unattached

Bostrichobranchus    Anural   Indirect     Sand: unattached
digonas

B. pilularis         Anural   Indirect     Sand: unattached

Eugyra arenosa       Anural   Indirect     Sand: unattached

Molgula pugetiensis  Anural   Indirect     Sand: unattached

M. retortiformis     Anural   Direct       Rock: attached

M. bleizi            Anural   Direct       Rock: attached

M. kolaensis         Anural   Indirect     Sand: unattached (?)

M. pacifica          Anural   Direct       Rock: attached

M. tectiformis       Anural   Direct       Rock: attached

M. provisionalis     Anural   Direct       Rock: attached

M. oregonia          ?        ?            ?: ?

M. regularis         ?        ?            Sand: unattached

Species              Distribution       Reference *

M. tubifera          E. Atlantic wide   Berrill 1931 (13)

M. cooperi           N.E. Pacific       Berrill 1931 (13)

M. verrucifera       N.E. Pacific       Berrill 1931 (13)

M. oculata           E. Atlantic wide   Berrill 1931 (13)

M. occulta           E. Atlantic wide   Lacaze-Duthiers 1877 (19)

M. solenota          E. Atlantic local  Berrill 1931 (13)

M. macrosiphonica    E. Atlantic wide   Lacaze-Duthiers 1877 (19)

M. arenata           W. Atlantic local  Berrill 1931 (13)

M. robusta           W. Atlantic local  Berrill 1931 (13)

Bostrichobranchus    W. Atlantic wide   Swalla and Jeffery 1992 (20);
digonas                                 da Rocha 2002 (21)

B. pilularis         W. Atlantic wide   Berrill 1931 (13)

Eugyra arenosa       E. Atlantic wide   Berrill 1931 (13)

Molgula pugetiensis  N.E. Pacific       This study

M. retortiformis     Circumpolar        Bates 1995 (22)

M. bleizi            E. Atlantic local  Berrill 1931 (13)

M. kolaensis         E. Arctic local    Berrill 1931 (13)

M. pacifica          E. Pacific         Young et al. 1988 (17)

M. tectiformis       N.W. Pacific       Nishikawa 1991 (23)

M. provisionalis     N.W. Atlantic      Bates 1991 (24)

M. oregonia          N.E. Pacific       Ritter 1913 (25)

M. regularis         N.E. Pacific       Van Name 1945 (14)

* Original description.


Figure 1 shows the external morphology of one of the adult Molgula pugetiensis recently collected near Friday Harbor Laboratories (University of Washington) in the U.S. Pacific Northwest. The adult is shown from the right side, where the gut loop is seen as a darkened line forming an S-shape before the anus loops out the atrial siphon. The elongated hermaphroditic gonad lies within the gut loop. This molgulid species looks remarkably similar to published photographs of M. citrina, one of the tailed, brooding species found in the Roscovita clade that is common to the eastern and western Atlantic and has been recently found off the Alaskan coast (1, 10).

[FIGURE 1 OMITTED]

Tailless molgulid ascidians frequently lack the extracellular space normally found between the egg and the follicle cells in tailed ascidian species, where the test cells reside as shown in Figure 2 (5). Molgula occulta, a tailless molgulid ascidian that is found off the coast of Roscoff, France, has tightly flattened follicle cells, as seen in a hatching larva (Fig. 2A), and had been reported earlier (5). M. pugetiensis also lacks extracellular space in the chorion and has flattened follicle cells (Fig. 2C). Figure 2 also compares hatching in M. occulta, which emerges from the chorion after 12 h (Fig. 2A) and then develops four ampullae before immediately metamorphosing into a juvenile (Fig. 2B). This species develops well at room temperature, or 20 [degrees]C. In M. pugetiensis, hatching also occurs in an anterior-to-posterior manner from the chorion, but time to hatching was 34 h at a much cooler temperature, 12 [degrees]C (Fig. 2C). The follicle cells are flattened against the chorion (Fig. 2C), similar to M. occulta (Fig. 2A). M. pugetiensis also develops four ampullae before immediately metamorphosing into the adult form (Fig. 2D). This is in stark contrast to M. oculata, a closely related tailed ascidian that has an otolith, a tail, and contains notochord and muscle cells (Fig. 2E, F), also reported previously (5). Note that the follicle cells are rounded up on the chorion (Fig. 2E).

M. pugetiensis development is described here for the first time as having anural, indirect larval development. Follicle and test cell differentiation, hatching from the chorion, adult habitat, development of four ampullae immediately after hatching, and the relatively short timing of larval development are characteristics similar to those of the well-studied tailless molgulid M. occulta (5; Table 1). Both of these species hatch from the chorion before metamorphosis and then, shortly after hatching, develop ampullae and metamorphose directly into the adult form. This is in contrast to direct anural larval development as seen in M. pacifica and M. provisionalis, where the larvae do not hatch out of the chorion before metamorphosis. Instead, the fertilized eggs attach to substrate immediately and develop through to metamorphosis as sessile embryos and juveniles (5, 24). Although the eggs, embryos, and juveniles looked remarkably similar to those of M. occulta found in France, the timing of hatching was delayed, with developmental timing more similar to the Pacific Northwest M. pacifica. This is likely due to the water temperature, with the seawater in the Pacific Northwest at 9-12 [degrees]C in contrast to the room temperature (20 [degrees]C) development for M. occulta. Both of the Pacific species, M. pugetiensis and M. pacifica, are members of the Roscovita clade and would probably develop much more quickly if the eggs and embryos were heat-tolerant.

Phylogenetic analyses of the Molgulidae show that urodele development is likely to have been lost at least four times independently (1, 2, 6). The tailed urodele larva is probably the ancestral mode in Molgulidae, because tailed urodele larval development is highly conserved across the Ascidiacea and the tadpole larva has been lost in only two other non-mogulid taxa, in another stolidobranch ascidian family, the Styelidae (15, 16). Therefore, outgroup analyses strongly support the existence of an ancestral tailed tadpole larva for the Molgulidae. Within the Roscovita clade, at least two independent origins of tail loss have been documented in closely related species (8). Evidence of larval muscle actin pseudogenes forming independently from the muscle actin genes commonly expressed in ascidian urodele larvae has been found in both of the anural developing species M. occulta and M. bleizi (8). However, the insertions and deletions leading to pseudogene formation were not shared between the two closely related tailless species, supporting the hypothesis that they evolved independently from a tailed ancestor. Table 1 also lists the larval development (if known) and distributions of all the described molgulid taxa on the Pacific coast of North America. M. pugetiensis has been only sporadically reported since first being described in 1898. Several molgulid species have few documented locations along the Pacific coast; these include M. cooperi, M. oregonia, and M. regularis. Although none of these species have yet been sequenced, the six folds in their branchial baskets would suggest that all of them may be more closely related to the Woods Hole Molgula clade. The urodele, or tailed, species M. verrucifera that has been found in southern California has seven folds and will likely be another member of the Roscovita clade (13). Therefore, future work examining the molecular phylogenetic and developmental status of these species will shed light on the evolution of larval development in the Molgulidae and speciation in the Pacific Ocean.

Anural larvae are unable to swim because of their tail loss. They lack the gravity-sensing sensory otolith, notochord, and larval muscle actin, so after hatching out of the chorion, these larvae are unable to swim or orient themselves and are thus completely ineffective at any active dispersal (5). It is a conundrum, then, how these species, which would seem incapable of dispersal, are able to establish stable breeding populations, sometimes even distantly located from their sister species. Most of the described anural species are found in sandy habitats unattached to any substratum as adults (Table 1). However, two anural direct developing species (M. bleizi and M. pacifica; both in the Roscovita clade) can be found attached to hard substrates, including the roof of rock caves inside blowholes formed by wave action and hollowed out rocks in the very low inter-tidal (13, 17).

Since sandflats contain both urodele and anural species, this environment has been proposed to allow for the independent loss of the urodele larva in molgulids (13). A novel hypothesis to account for the loss of active dispersal potential is that many of these species have been found in northern latitudes with relatively large tidal exchanges that allow for enough passive dispersal to relax selection on the genes that control active swimming traits (1). This hypothesis could be tested by comparing the gene flow of sympatric closely related species like the urodele M. oculata and the anural, indirect M. occulta.

Larval development has been described for only a small fraction of the nearly 150 known species of Molgula (18). A total of 14 species have been reported with anural larval development, and we report the 15th anural Molgula species (Table 1). The questions of how speciation occurred in the Roscovita clade and how many times anural development evolved in Molgulidae may become clearer as knowledge on the development of more species becomes available.

Acknowledgments

We especially thank Dr. Bruno Pernet of California State University, Long Beach, who discovered the Molgula pugetiensis adults in a dredge at FHL in 2006, recognized them as molgulid ascidians, and gave them to BJS. We thank Gretchen Lambert of Friday Harbor Laboratories, University of Washington, who examined the single adult specimen found in 2003 and confirmed that it was Molgula pugetiensis. We also thank Hazel Lozano, an undergraduate in the Swalla lab, who worked on this project for one quarter doing undergraduate research. This research was partly funded by an NSF OACIS GK12 fellowship to Max E. Maliska. This material is based in part upon work supported by the National Science Foundation under Cooperative Agreement No. DBI-0939454. Any opinions, findings, conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation.

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MAX E. MALISKA (1), (2), (3) AND BILLIE J. SWALLA (1), (2), (3), *

(1) Biology Department, University of Washington, Seattle, Washington 98195; (2) Friday Harbor Laboratories, University of Washington, Friday Harbor, Washington 98250; and (3) BEACON Center for the Study of Evolution in Action

Received 6 September 2010; accepted 14 November 2010.

* To whom correspondence should be addressed. Email: bjswalla@u.washington.edu
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Author:Maliska, Max E.; Swalla, Billie J.
Publication:The Biological Bulletin
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Geographic Code:4EUFR
Date:Dec 1, 2010
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